Download neuropathology of dopamine systems in schizophrenia

NEUROPATHOLOGY OF DOPAMINE SYSTEMS IN SCHIZOPHRENIA:
REGIONAL DOPAMINE PATHOLOGIES IN THE SUBSTANTIA
NIGRA/VENTRAL TEGMENTAL AREA COMPLEX
by
MATTHEW W. RICE
MIGUEL MELENDEZ-FERRO, COMMITTEE CO-CHAIR
EMMA PEREZ-COSTAS, COMMITTEE CO-CHAIR
FRANKLIN R. AMTHOR
EDWIN W. COOK III
LINDA OVERSTREET-WADICHE
ROSALINDA C. ROBERTS
DIANE C. TUCKER
A DISSERTATION
Submitted to the graduate school faculty of The University of Alabama at Birmingham,
in partial fulfillment of the requirements for the degree of
Doctor of Philosophy
BIRMINGHAM, ALABAMA
2014
NEUROPATHOLOGY OF DOPAMINE SYSTEMS IN SCHIZOPHRENIA:
REGIONAL DOPAMINE PATHOLOGIES IN THE SUBSTANTIA
NIGRA/VENTRAL TEGMENTAL AREA COMPLEX
MATTHEW W. RICE
BEHAVIORAL NEUROSCIENCE GRADUATE PROGRAM
ABSTRACT
Dopaminergic neurotransmission anomalies are a well-established aspect of
schizophrenia, a devastating mental disorder that affects approximately 1% of the world
population. The substantia nigra/ventral tegmental area (SN/VTA), which extends from
diencephalic to mesencephalic territories, provides the largest dopaminergic input to the
brain. The SN/VTA contains subpopulations of dopaminergic neurons that project
preferentially through different pathways, providing dopamine input to brain regions
where dopaminergic anomalies have been reported in schizophrenia. We hypothesized
that anomalies in the synthesis of tyrosine hydroxylase (TH), the rate-limiting enzyme for
the production of dopamine, could contribute to the dopaminergic anomalies observed in
this disorder. We first tested the expression of TH mRNA and protein in schizophrenia
and matched control cases. In our schizophrenia cases we found significant deficits in TH
protein expression that were driven by the rostral SN/VTA, which preferentially provides
dopaminergic input to cortical areas. Next, we assessed if metabolic anomalies in the
SN/VTA could contribute to this deficit by examining cytochrome c oxidase (COX)
activity and subunit composition. We found a decrease in specific key subunits for the
assembly and function of the enzyme. Interestingly, these deficits were not accompanied
by alterations in COX activity. These findings can be explained by the fact that
measurements of COX activity in postmortem tissue provided only a “snapshot” of the
ii
basal activity at time of death, while changes in subunit protein expression are related to
long-term regulation of the enzyme, affecting the “reserve capacity” of the mitochondria
to respond to higher energy demands. Finally, we performed neuronal counts of
dopaminergic and total number of neurons, as well as a qualitative analysis of TH
expression
in
the
SN/VTA
of
schizophrenia
and
matched
controls
using
immunohistochemistry. We found that, while neuronal counts were unaltered, TH protein
expression was markedly reduced within rostral areas of the SN/VTA in schizophrenia.
These studies support that presynaptic dopaminergic deficits are concentrated in cell
populations that preferentially project through the mesolimbic and mesocortical
pathways. Thus, our results indicate that presynaptic dopaminergic anomalies contribute
to the hypodopaminergia of schizophrenia neuropathology, which is directly related to
the negative symptomology and cognitive impairments of this disorder.
Keywords: cytochrome c oxidase, immunohistochemistry, mesocortical pathway,
mesolimbic pathway, metabolism, tyrosine hydroxylase
iii
ACKNOWLEDGEMENTS
First, I wish to express my gratitude and appreciation to my mentors, Drs. Emma
Perez-Costas and Miguel Melendez-Ferro for their patience, guidance, and support
throughout my graduate career. I would further like to acknowledge the members of my
dissertation committee, Drs. Franklin Amthor, Edwin Cook, Linda Overstreet-Wadiche,
Rosalinda Roberts, and Diane Tucker for their comments and critical evaluations.
Furthermore, I am grateful for the remaining members of the laboratory, Kristen Smith,
Lesley McCollum, and Joy Roche for their assistance and support. Last but not least, I
want to thank the members of my family and my friends who were always willing to
provide encouragement. Thank you all.
iv
TABLE OF CONTENTS
Page
ABSTRACT ........................................................................................................................ ii
ACKNOWLEDGEMENTS ............................................................................................... iv
LIST OF TABLES ............................................................................................................ vii
LIST OF FIGURES ........................................................................................................... ix
INTRODUCTION ...............................................................................................................1
The Mesodiencephalic Dopaminergic System.........................................................1
Neuroanatomy ..............................................................................................2
Efferent Projections of the SN/VTA ............................................................3
Afferent Projections to the SN/VTA............................................................7
Dopamine .....................................................................................................9
Developmental Origins of the Mesodiencephalic Dopaminergic System .............14
Connecting the Dopaminergic System.......................................................18
Schizophrenia .........................................................................................................22
Schizophrenia Pathology ...........................................................................23
Dopamine Neurotransmission in Schizophrenia ........................................26
Metabolic Anomalies in Schizophrenia .....................................................30
DOPAMINE PATHOLOGY IN SCHIZOPHRENIA: ANALYSIS OF TOTAL
AND PHOSPHORYLATED TYROSINE HYDROXYLASE IN
THE SUBSTANTIA NIGRA ............................................................................................46
AN ACCURATE METHOD FOR THE QUANTIFICATION OF
CYTOCHROME C OXIDASE IN TISSUE SECTIONS .................................................88
ASSESSMENT OF CYTOCHROME C OXIDASE DYSFUNCTION IN THE
SUBSTANTIA NIGRA/VENTRAL TEGMENTAL AREA IN SCHIZOPHRENIA ....113
MAPPING DOPAMINERGIC DEFICIENCIES IN THE SUBSTANTIA NIGRA/
VENTRAL TEGMENTAL AREA IN SCHIZOPHRENIA............................................162
v
DISCUSSION ..................................................................................................................211
LIST OF GENERAL REFERENCES .............................................................................225
APPENDIX: IRB APPROVAL FORM...........................................................................262
vi
LIST OF TABLES
Table
Page
DOPAMINE PATHOLOGY IN SCHIZOPHRENIA: ANALYSIS OF TOTAL AND
PHOSPHORYLATED TYROSINE HYDROXYLASE IN THE SUBSTANTIA NIGRA
1
Pilot study: demographic table and clinical data in schizophrenia and controls .........81
2
Demographic table and clinical data second postmortem study ..................................82
AN ACCURATE METHOD FOR THE QUANTIFICATION OF CYTOCHROME C
OXIDASE IN TISSUE SECTIONS
1
Final concentrations of pure COX in standards and detailed chart for their
preparation .................................................................................................................108
ASSESSMENT OF CYTOCHROME C OXIDASE DYSFUNCTION IN THE
SUBSTANTIA NIGRA/VENTRAL TEGMENTAL AREA IN SCHIZOPHRENIA
1
Demographic and clinical data of the cases used for the study .................................148
2
Effect of demographic and sample variables on COX activity and COX subunit
expression ..................................................................................................................149
MAPPING DOPAMINERGIC DEFICIENCIES IN THE SUBSTANTIA NIGRA/
VENTRAL TEGMENTAL AREA IN SCHIZOPHRENIA
1
Demographic and clinical data of the cases used for the study .................................198
2
Neuronal counts obtained as the average of the neuronal counts of two different
control cases ...............................................................................................................199
vii
3
Effect of demographic variables (i.e. age, gender,race) on neuronal counts .............200
4
Effect of sample quality variables (i.e. postmortem interval, brain pH) on
neuronal counts ..........................................................................................................201
viii
LIST OF FIGURES
Figure
Page
INTRODUCTION
1
Organization of nuclei within the mesodiencephalic dopamine system ......................35
2
Sagittal image of the human brain showing the three main dopaminergic
pathways and their projection sites ..............................................................................36
3
Schematic of the afferent and efferent connections of the SN/VTA ...........................37
4
In vivo biosynthesis of dopamine .................................................................................38
5
Diagram of a dopaminergic synapse ............................................................................39
6
Primary pathways of dopamine catabolism .................................................................40
7
Development of the mesodiencephalic dopaminergic system .....................................41
8
Progression of axons from the developing mesodiencephalic dopaminergic
complex ........................................................................................................................42
9
Schematic of the electron transport chain of the mitochondria ...................................43
10 Three-dimensional representation of cytochrome c oxidase .......................................44
11 Model for cytochrome c oxidase (COX) assembly in cultured human cells ...............45
DOPAMINE PATHOLOGY IN SCHIZOPHRENIA: ANALYSIS OF TOTAL AND
PHOSPHORYLATED TYROSINE HYDROXYLASE IN THE SUBSTANTIA NIGRA
1
Pilot study: TH mRNA and protein levels in schizophrenia and control cases ...........83
2
Animal study: antipsychotic treatment effect on TH protein levels ............................85
3
Second postmortem study: regional distribution of TH and pTH................................86
ix
AN ACCURATE METHOD FOR THE QUANTIFICATION OF CYTOCHROME C
OXIDASE IN TISSUE SECTIONS
1
Preparation of standards and Image J ........................................................................109
2
Relation between micrograms of COX, optical density and units of activity ...........110
3
Creation of a standard curve in Image J.....................................................................111
4
Measurement of COX activity in brain sections ........................................................112
ASSESSMENT OF CYTOCHROME C OXIDASE DYSFUNCTION IN THE
SUBSTANTIA NIGRA/VENTRAL TEGMENTAL AREA IN SCHIZOPHRENIA
1
Analysis of COX activity in samples containing the entire rostro-caudal extent of the
human SN/VTA .........................................................................................................150
2
Analysis of COX activity in samples containing only the mid-caudal regions of the
human SN/VTA .........................................................................................................152
3
Analysis of COX subunits I, II, and III in samples containing the entire rostro-caudal
extent of the human SN/VTA ....................................................................................153
4
Analysis of COX subunits IV-I and IV-II in samples containing the entire rostrocaudal extent of the human SN/VTA .........................................................................155
5
Analysis of COX subunits I, II, and III protein expression in the mid-caudal human
SN/VTA .....................................................................................................................156
6
Analysis of COX subunits IV-I and IV-II protein expression in the mid-caudal human
SN/VTA .....................................................................................................................158
7
Analysis of COX subunits I, II, and III in the SN/VTA of animals treated with
antipsychotic drugs ....................................................................................................159
x
8
Analysis of COX subunits IV-I and IV-II in the SN/VTA of animals treated with
antipsychotic drugs ....................................................................................................161
MAPPING DOPAMINERGIC DEFICIENCIES IN THE SUBSTANTIA NIGRA/
VENTRAL TEGMENTAL AREA IN SCHIZOPHRENIA
1
Neuronal counts for the entire SN/VTA ....................................................................202
2
Neuronal counts in the diencephalic (rostral) SN/VTA .............................................203
3
Neuronal counts in the mesencephalic (mid-caudal) SN/VTA..................................204
4
Tyrosine hydroxylase labeling in the diencephalic SN/VTA ....................................205
5
Tyrosine hydroxylase labeling of cell and processes in the diencephalic
SN/VTA .....................................................................................................................207
6
Tyrosine hydroxylase labeling in the mesencephalic SN/VTA .................................209
DISCUSSION
1
Measurement of oxygen consumption rate from isolated neonatal rat ventricular
myocytes ....................................................................................................................224
xi
INTRODUCTION
The Mesodiencephalic Dopaminergic System
The substantia nigra/ventral tegmental area (SN/VTA) is a cellular complex
located within the mesencephalon that contains the largest group of dopaminergic
neurons in the brain (Fallon et al., 1978a,b; Gibb and Lees, 1991; Gaspar et al., 1992;
Gibb, 1992; McRitchie et al., 1995; Hardman et al., 1996; see also as reviews van
Domburg and ten Donkelaar, 1991; Haber and Fudge, 1997). The SN/VTA, along with
the retrorubral area (RRA), are collectively known as the mesodiencephalic dopaminergic
(mdDA) complex. The mdDA complex contains 400,000-600,000 neurons in the human
brain (Van den Heuvel and Pasterkamp, 2008) and is composed of a diverse group of
dopaminergic subpopulations with unique developmental origins (Puelles and Verney,
1998). The mdDA complex is also the origin of several major dopaminergic pathways in
the brain (i.e. nigrostriatal, mesolimbic, and mesocortical pathways) and is an important
complex in regard to movement, learning, cognition, goal-driven behaviors, emotion, and
attention (see as reviews DeLong and Georgopoulos, 1981; Horvitz, 2000; Floresco and
Magyar, 2006; Berridge, 2007; Cousins et al., 2009; Roze et al., 2010; Subramaniam and
Snyder, 2011).
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Neuroanatomy
The “substantia nigra” or “black substance” derives its name from the strong
black/brown pigmentation that accumulates in the cytoplasm of mesodiencephalic
dopaminergic neurons. This pigmentation is due to the presence of neuromelanin, which
is a byproduct of the metabolism of dopamine, giving the SN its distinct dark color
(Double et al., 2000; Zecca et al., 2008). Within this mesodiencephalic dopaminergic
complex, the VTA is located medial to the SN, with both nuclei primarily rostral to the
RRA [Figure 1]. Based on morphology, chemical characterization, and cellular
organization, the SN can be divided in two distinct subareas: the pars reticulata (SNr) and
the pars compacta (SNc) (see as a review Bjorklund and Dunnett, 2007).
The SNr is located in the most ventro-lateral area of the SN and is composed
primarily of GABAergic neurons that project to the thalamus, superior colliculus,
reticular formation, and SNc (Hopkins and Niessen, 1976; Beckstead et al., 1979; Menke
et al., 2010) [Figure 1]. The SNc is located medial and dorsal to the SNr and has a denser
cell population, consisting primarily of dopaminergic neurons (Yelnik et al., 1987;
Hardman et al., 2002; see as a review Haber and Gdowski, 2004) [Figure 1]. The SNc can
be further divided in a “dorsal tier” and a “ventral tier,” which will be described in detail
in subsequent paragraphs. Projections from the SNc merge with dopaminergic axonal
processes from the VTA forming a continuum of dopamine input, which also includes
projections from neurons of the retrorubral area and the hypothalamus (Beckstead et al.,
1979; Veazey et al., 1982; Loughlin and Fallon, 1984; Sanchez-Gonzalez et al., 2005;
Ferreira et al., 2008) [Figure 2]. This continuum is responsible for the dopaminergic
efferent projections to the striatum (nigrostriatal pathway), limbic regions (mesolimbic
2
pathway), frontal and prefrontal cortex (mesocortical pathway), hypothalamus
(tuberoinfundibular pathway) and thalamus (Porrino and Goldman-Rakic, 1982; Veazey
et al., 1982; Sadikot and Parent, 1990; Gaspar et al., 1992; Sanchez-Gonzalez et al.,
2005; Menke et al., 2010) [Figure 2].
Efferent Projections of the SN/VTA
The striatum, the target region for the dopaminergic projections of the
nigrostriatal and mesolimbic pathways, is the principal receptive region of the basal
ganglia, which are a group of functionally related brain areas that include the striatum,
SN, globus pallidus, and subthalamic nucleus. In the brain of primates and other higher
mammals, the striatum can be divided into dorsal and ventral regions. The functional role
of the dorsal striatum has long been associated with the control of movement (see as
reviews DeLong and Georgopoulos, 1981; Obeso et al., 2008; Cousins et al., 2009; Roze
et al., 2010; Subramaniam and Snyder, 2011). More recently, the dorsal striatum has been
associated with cognition and emotion, as well as being connected to psychiatric and
psychological disorders (Middleton and Strick, 1994; Roberts et al., 1996, 2005; see also
as reviews Dunlop and Nemeroff, 2007; Wanat et al., 2009; Perez-Costas et al., 2010).
The dorsal striatum includes the caudate nucleus and putamen, which in higher mammals
are separated by the internal capsule, while the ventral striatum includes the nucleus
accumbens and the olfactory tubercle (see as a review Nieuwenhuys et al., 2007). The
distinction between these two areas of the striatum, dorsal and ventral, becomes
important when discussing the synaptic connections of the mdDA complex.
3
Two distinct cytoarchitectonic components can be distinguished in the dorsal
striatum. The first is referred to as the “matrix” and constitutes the majority of the dorsal
striatum; the second is the “striosomes” (also known as “patches”) that lie within the
matrix (Graybiel and Ragsdale, 1983; Donoghue and Herkenham, 1986; Bolam et al.,
1988; Ragsdale and Graybiel, 1988; Hirsch et al., 1989; Graybiel, 1990; Gerfen, 1992;
Kubota and Kawaguchi, 1993; Holt et al., 1997; Roberts and Knickman, 2002; see as a
review Nieuwenhuys et al., 2007). These two distinct compartments receive preferential
projections, with limbic regions synapsing primarily onto the striosomes, while motor
and somatosensory cortex predominantly synapse onto the matrix (Graybiel and
Ragsdale, 1983; Donoghue and Herkenham, 1986; Ragsdale and Graybiel, 1988;
Graybiel, 1990; Gerfen, 1992; Kubota and Kawaguchi, 1993; see as reviews Crittenden
and Graybiel, 2011; Buot and Yelnik, 2012). The dorsal striatum has also been shown to
receive topographically organized connections from the SN/VTA, which constitute the
nigrostriatal pathway (Fallon et al., 1978a,b; Beckstead et al., 1979; Kubota et al.,
1986; Gaspar et al., 1992; McRitchie et al., 1995; see also as reviews Iversen, 1984;
Haber and Fudge, 1997; Joel and Weiner, 2000) [Figure 2, see also Figure 3]. Projections
of the nigrostriatal pathway have a medio-lateral (i.e. the medial SNc sends projections to
the medial caudate and putamen) and a rostrocaudal (i.e. the rostral SNc projects to the
rostral caudate and putamen) organization (Carpenter and Peter, 1972; Szabo, 1979,
1980; see as a review Haber and Fudge, 1997). The majority of the connections of the
nigrostriatal pathway arise from neurons of the ventral portion of the SNc (Fallon and
Moore, 1978a,b; Fallon et al., 1978b; McRitchie et al., 1995; Haber and Fudge, 1997;
Joel and Weiner, 2000; Halliday, 2004). This area of the SNc is known as the “ventral
4
tier” and is located primarily in the mid to caudal portions of the substantia nigra (Fallon
and Moore, 1978a,b; Haber and Fudge, 1997; Joel and Weiner, 2000) [Figure 1C].
The “dorsal tier” of the SNc extends from diencephalic to mesencephalic
territories, and is located primarily within the rostral to mid portions of the SN/VTA
complex (Crosby and Woodburne, 1943; see as reviews Haber and Fudge, 1997; Marin et
al., 1998; Joel and Weiner, 2000; Augustine et al., 2008) [Figure 1C]. Projections of the
dorsal tier of the SNc merge medially with projections of the VTA dopaminergic neurons
that are located at the same rostrocaudal level, forming a common dopaminergic output
(see as reviews Haber and Gdowski, 2004; Nieuwenhuys et al., 2007). This dorsal tier is
responsible for the two remaining dopaminergic projections that arise from the SN/VTA,
the mesolimbic and mesocortical pathways (Fallon and Moore, 1978a,b; Fallon et al.,
1978a,b; Porrino and Goldman-Rakic, 1982; Gaspar et al., 1992; McRitchie et al., 1995;
Haber and Fudge, 1997; Halliday, 2004). The mesolimbic pathway [Figure 2] sends
dopaminergic projections to the nucleus accumbens (ventral striatum), amygdala,
hippocampus, and the bed nucleus of the stria terminalis (Fallon and Moore, 1978a,b;
Fallon et al., 1978a,b; Aggleton et al., 1980; Mehler, 1980; Norita and Kawamura, 1980;
Porrino and Goldman-Rakic, 1982; Gaspar et al., 1992; McRitchie et al., 1995; Haber and
Fudge, 1997; Halliday, 2004; see as a review Nieuwenhuys et al., 2007). The
dopaminergic connections of the mesolimbic pathway that terminate onto the nucleus
accumbens are commonly referred to as the “reward pathway.” Increased activity in the
mesolimbic pathway, which would yield increased dopamine output, has been
demonstrated during times of reward, adverse stimuli, and addiction (see also as reviews
Schultz, 2002; Wise, 2002; Gonzales et al, 2004; Laviolette, 2007). Due to its
5
connections with the amygdala and hippocampus, the mesolimbic pathway is also
associated with learning, emotion, motivation, and salience/novelty seeking behavior
(Ballard et al., 2011; see also as reviews Di Chiara, 1998, Horvitz, 2000; Berridge, 2007).
The mesocortical pathway [Figure 2] is the third major dopaminergic pathway
originating from the SN/VTA, and is responsible for the large number of dopaminergic
terminals found within the cortex (Porrino and Goldman-Rakic, 1982; Veazey et al.,
1982; Sadikot and Parent, 1990; Gaspar et al., 1992; Sanchez-Gonzalez et al., 2005;
Menke et al., 2010). Dopaminergic projections of the mesocortical pathway innervate
several regions of the cortex, although the majority of these projections proceed to the
frontal cortex, innervating the orbital, medial, and dorso-lateral prefrontal cortex (see as a
review Haber and Gdowski, 2004). As opposed to the nigrostriatal pathway, axonal
processes of the mesocortical pathway are diffusely arranged, in such manner that a
single neuron of the SN/VTA is capable of projecting to several different areas of the
cortex (Gaspar et al., 1992). Due to its strong projection to the frontal cortex, the
dopaminergic mesocortical pathway is involved in a variety of aspects of higher
cognition such as communication, executive function, planning, learning, and memory
(Weinberger and Berman, 1988; see as a review Floresco and Magyar, 2006).
In summary, it has been shown that the SN/VTA dopaminergic projections are
highly heterogeneous with specialized subgroups of dopaminergic neurons providing
dopamine input to different brain regions, using different pathways. The nigrostriatal
pathway that projects to the dorsal striatum originates from the ventral tier of the SNc,
which is located in the mid to caudal portions of the substantia nigra pars compacta
(Fallon and Moore, 1978a,b; Haber and Fudge, 1997; Joel and Weiner, 2000). Rostral to
6
mid regions of the SNc contain most of what is known as the dorsal tier (Fallon and
Moore, 1978a,b; Haber and Fudge, 1997; Joel and Weiner, 2000) and along with the
VTA, projections from this area preferentially synapse onto the cortex, ventral striatum,
and limbic regions. These projections constitute the mesocortical and mesolimbic
pathways (Fallon and Moore, 1978a,b; Fallon et al., 1978a,b; Porrino and GoldmanRakic, 1982; Gaspar et al., 1992; McRitchie et al., 1995; Haber and Fudge, 1997;
Halliday, 2004).
Afferent Projections to the SN/VTA
Dopaminergic neurons of the SN/VTA receive modulatory input from other
nuclei of the basal ganglia as well as from other brain regions [Figure 3]. Neurons from
the prefrontal cortex and subthalamic nucleus send excitatory glutamatergic and
cholinergic afferents that synapse onto the SN/VTA (Lavoie and Parent, 1994; Charara et
al., 1996; Frankle et al., 2006). There are also inhibitory GABAergic projections, and
these projections account for at least 50-70% of the afferents that synapse onto the
dopaminergic neurons of the substantia nigra (Bolam and Smith, 1990). The majority of
these GABAergic projections originate from nuclei within the basal ganglia, including
the dorsal striatum (Grofova and Rinvik, 1970; Somogyi et al., 1981; Bolam and Smith,
1990), the external segment of the globus pallidus (GPe) (Grofova, 1975, Smith and
Bolam, 1990), and the substantia nigra pars reticulata (Grace and Bunney, 1979, 1985;
Nitsch and Riesengerg, 1988; Hajos and Greenfield, 1993, 1994; Tepper et al., 1995;
Celada et al., 1999; Mailly et al., 2003; see as a review Tepper and Lee, 2007).
Additional GABAergic input arises from areas outside the basal ganglia such as the
7
superior colliculus, lateral habenula, and central nucleus of the amygdala (Gao et al.,
1996; Coizet et al., 2003; Comoli et al., 2003).
The location of GABAergic neurons in the dorsal striatum determines the
preferential target of their projections in the substantia nigra. Projections from neurons
located in the matrix synapse primarily onto the GABAergic neurons of the SNr, while
projections from neurons located in the striosomes terminate primarily onto the
dopaminergic neurons of the SNc (Gerfen, 1985; Gerfen et al., 1987). In contrast to
striatal afferents, GABAergic projections from GPe neurons have been shown to synapse
onto both GABAergic and dopaminergic neurons of the SN without preference (Bolam
and Smith, 1990). Inhibitory input from the SNr onto the SNc arises from local collaterals
of SNr GABAergic projection neurons, rather than locally projecting interneurons
(Matsuda et al., 1987; Tepper et al., 1995, 2002; Mailly et al., 2003; see as a review
Tepper and Lee, 2007). When stimulated, these SNr GABAergic neurons inhibit the
activity of dopaminergic neurons located within the SNc (Hajos and Greenfield, 1994).
Additionally, it has been shown that SNr inhibitory input has a greater influence in the
firing characteristics of the dopaminergic neurons of the SNc than inhibitory input
originating from either the GPe or striatum (see as review Tepper and Lee, 2007). For
example, when the GABAergic projections of the GPe or striatum are stimulated, an
increase in the firing of dopaminergic neurons of the SNc is observed. This increase in
dopaminergic neuronal firing is due to the inhibition of the GABAergic neurons of the
SNr, which leads to a disinhibition of the dopaminergic neurons of the SNc. This
connection has a greater impact than the direct inhibition of the SNc dopaminergic
neurons by the striatal and pallidal afferents (Grace and Bunney, 1985; Lee et al., 2004).
8
In brief, the excitatory and inhibitory afferents described above regulate the firing of
dopaminergic neurons located in the SN/VTA, modulating the release of dopamine.
Dopamine
Dopamine is an organic chemical messenger and a member of the catecholamine
and phenethylamine families. In vivo, the synthesis of dopamine is carried out through a
series of enzymatic reactions [Figure 4], beginning with the non-essential amino acid
tyrosine, which is converted to L-3,4-dihydroxyphenylalanine (L-DOPA) by the enzyme
tyrosine hydroxylase (Pickel et al., 1975; Lindgren et al., 2001; Dunkley et al., 2004; see
as a review Miyake et al., 2011). L-DOPA is then converted to dopamine by the enzyme
DOPA decarboxylase, also known as aromatic L-amino acid decarboxylase (see as
reviews Elsworth and Roth, 1997; Miyake et al., 2011). Dopamine can be further
metabolized into the neurotransmitters noradrenaline (norepinephrine), by the enzyme
dopamine β-hydroxylase (DBH), and adrenaline (epinephrine) by the enzyme
phenylethanolamine N-methyltransferase (Hartman et al., 1972; Vincent, 1988; Bailhache
and Balthazart, 1993). Importantly, DBH is not expressed in the mdDA complex,
ensuring that neurons are dopaminergic rather than noradrenergic or adrenergic (Hartman
et al., 1972; Vincent, 1988; Bailhache and Balthazart, 1993).
Tyrosine hydroxylase (TH) is the rate-limiting enzyme for the production of
dopamine, and measurements of this enzyme are often used in human post-mortem and
animal studies in order to determine the capability of neurons to produce dopamine
(Pickel et al., 1975; Lindgren et al., 2001; Dunkley et al., 2004). Among mammalian
species the number of TH isoforms synthesized differ, with a single isoform identified in
9
rodents and non-primates, two in non-human primates, and four TH isoforms in humans.
All these different isoforms are the result of alternative mRNA splicing from a single
gene (Le Bourdelles et al., 1991; Haycock, 2002; Gordon et al., 2009). This TH gene
encodes for tyrosine hydroxylase and it has been shown that the regulation of the
production of this enzyme is unique within the mdDA complex, such that, TH mRNA
expression levels do not necessarily correspond to TH protein levels (Wong and Tank,
2007; Chen et al., 2008; Tank et al., 2008; Perez-Costas et al., 2012). This is due to the
posttranscriptional regulation mechanism that stabilizes TH mRNA in neurons of the
mdDA complex (Tank et al., 2008; Lenartowski and Goc, 2011). Tank et al. (2008)
postulated that Poly(C)-binding proteins bind to TH mRNA, which results in the
stabilization of the mRNA molecule and an increased efficacy of its translation into
protein. It is also important to note that TH becomes active when it is phosphorylated,
with Ser40 being the most physiologically active phosphorylation site in the SN/VTA
(Lindgren et al., 2001; Dunkley et al., 2004; Nakashima et al., 2009). The advantages of
using TH as a marker for dopaminergic neurons instead of measuring dopamine directly
include: 1) Dopamine has been demonstrated to degrade at a faster rate than TH, thus
limiting the usefulness of directly studying dopamine in post-mortem cases (Blank et al.,
1979), 2) TH is located throughout the cytoplasm of the neurons, as opposed to dopamine
which is sequestered into vesicles, thus the detection of TH allows for the visualization of
the entire cell (Pickel et al., 1975; 1980), and 3) the analysis of TH levels allows for the
study of the ability of the cell to produce dopamine, rather than its ability to store
dopamine.
10
Once synthesized, dopamine is transported by vesicular monoamine transporter 2
(VMAT2) from the cytosol into specialized storage vesicles located primarily at axon
terminals (Eiden et al., 2004; Sun et al., 2012; Hu et al., 2013) [Figure 5]. In addition,
dopamine can also be released from the dendrites where it can be stored both in vesicles
and within the endoplasmic reticulum (Geffen et al., 1976; Kerwin and Pycock, 1978;
Araneda and Bustos, 1989; Cragg and Greenfield, 1997; Patel et al., 2009). Upon the
arrival of an action potential, dopamine is released into the synaptic cleft where it may
bind to one of two families of dopamine receptors, D1 or D2 [Figure 5]. The D1-like
family of dopamine receptors includes the D1 and D5 dopamine receptor subtypes, and is
coupled to the G protein Gsα (Nestler et al., 1990; Tepper et al., 1997; Chien et al., 2010).
When activated, D1-like receptors activate the adenylate cyclase enzyme, resulting in an
increase of cyclic adenosine monophosphate (cAMP) production (Neves et al., 2002; see
as a review Jose et al., 2003). The D2-like family of dopamine receptors include the
D2(short), D2(long), D3, and D4 dopamine receptor subtypes, and are coupled to the G protein
Gi/oα. Activation of Gi/oα results in the inhibition of adenylate cyclase and a reduction in
the amount of cAMP produced (Nestler et al., 1990; Tepper et al., 1997; Neves et al.,
2002; Chien et al., 2010).
In addition to the postsynaptic dopamine receptors, there are also presynaptic
receptors (autoreceptors), that have been identified as D2(short) dopamine receptors, and
extrasynaptic receptors (Meador-Woodruff et al., 1991; Cooper and Stanford, 2001; Blasi
et al., 2009; see as reviews De Mei et al., 2009; Fuxe et al., 2012). Dopamine
autoreceptors are located in the vicinity of synapses throughout the structure of the
neuron, including the soma, dendrites, and axon terminals (Meador-Woodruff et al.,
11
1991; Cragg and Greenfield, 1997; see as reviews Elsworth and Roth, 1997; Adell and
Artigas, 2004) [Figure 5]. When activated, autoreceptors located in the soma and
dendrites function to slow the rate of firing of the dopaminergic neurons, whereas
autoreceptors located in terminals inhibit the release and/or synthesis of dopamine (see as
reviews Elsworth and Roth, 1997; Adell and Artigas, 2004). Supporting the existence of
functional differences among subpopulations of the mdDA complex, it has been shown
that neurons located within ventral portions of the SNc express high levels of dopamine
transporter (DAT) and D2 receptor mRNA, while neurons contributing to the
mesocortical and mesolimbic pathways express low levels of DAT and D2 receptor
mRNA (Hurd et al., 1994). Another mechanism regulating the firing of dopaminergic
neurons is through reuptake of dopamine by either the high-affinity DAT or low-affinity
norepinephrine transporter (NET) (Moron et al., 2002; see as a review Torres et al.,
2003). These transporters serve to recycle dopamine that has been released into the
synaptic cleft by pumping extracellular dopamine back into the presynaptic neuron
[Figure 5]. The activities of dopamine and norepinephrine transporters increase the
intracellular concentration of dopamine within the presynaptic neuron, and are critical in
terminating neurotransmission and maintaining transmitter homeostasis (see as reviews
Elsworth and Roth, 1997; Torres et al., 2003). In some dopaminergic synapses the rate of
dopamine release supersedes the actions of DAT, resulting in the formation of a “cloud”
of dopamine. This dopaminergic “cloud” can activate neurons outside the synaptic cleft
via extrasynaptic receptors in a process called “volume transmission” (Rice and Cragg,
2008; see as a review Fuxe et al., 2012).
12
Catabolism of dopamine [Figures 5, 6] is mediated by a set of enzymes consisting
of catechol-O-methyltransferase (COMT), monoamine oxidase (MAO), aldose reductase
(ALD), aldehyde dehydrogenase (ALD-D), and alcohol dehydrogenase (ADH) (see as
reviews Kopin, 1985; Elsworth and Roth, 1997; Eisenhofer et al., 2004). These enzymes
act in concert to form a number of intermediate metabolites [Figure 6] including 3,4dihydroxyphenylacetic acid (DOPAC), 3,4-dihydroxyphenylacetaldehyde (DOPAL), 3,4dihydroxyphenylethanol
(DOPET),
3-Methoxytyramine
(3-MT),
3-methoxy-4-
hydroxyphenylethanol (MOPET; also known as homovanillyl alcohol) and homovanillic
acid (HVA) (see as reviews Kopin, 1985; Elsworth and Roth, 1997; Eisenhofer et al.,
2004). These metabolic pathways can also result in the formation of neuromelanin within
the neuronal somas of the mdDA neurons, the accumulation of which results in the
characteristic dark pigmentation of the SN/VTA (Double et al., 2000; Zecca et al., 2008).
In most areas of the brain, dopamine is metabolized by MAO into DOPAC after the
uptake of dopamine by dopamine transporters from the synaptic cleft. However, in the
prefrontal cortex DATs are only present in restricted areas, while low-affinity NETs are
pervasive throughout the entire region (Lewis et al., 2001; Moron et al., 2002).
Additionally, COMT mRNA and protein are more strongly expressed in cortical areas
than in subcortical areas such as the striatum (Matsumoto et al., 2003; see as a review
Mannisto et al., 1999). This results in an increased catabolism of dopamine by COMT
into 3-MT, which is then further catabolized by MAO into HVA. Dopamine that is not
fully metabolized can be repackaged into vesicles for future release (Michael et al., 1987;
Ungless, 2004).
13
Developmental Origins of the Mesodiencephalic Dopaminergic System
According to the prosomeric model of brain development, the early vertebrate
brain is a segmented structure that consists of a continuous sequence of transverse
divisions within the neural tube (see as reviews Puelles, 2001; 2009; Puelles and
Rubenstein, 2003). These divisions are known as “neuromeres” [Figure 7] and from
rostral to caudal include a large forebrain structure (i.e. secondary prosencephalon)
comprising the telencephalon, hypothalamus, and developing eyes; the diencephalic
neuromeres (i.e. prosomeres 1-3); the mesomere (i.e. the developing mesencephalon); the
rhombencephalon consisting of eleven rhombomeres; and spinal myelomeres (Bergquist,
1932; Bergquist and Kallen, 1953; Lauter et al., 2013; see as reviews Rubenstein et al.,
1994; Puelles and Rubenstein, 2003; Puelles, 2009; Martinez-Ferre and Martinez, 2012).
MdDA progenitor cells originate specifically within the mesomere and the diencephalic
prosomeres 1-3 (Puelles and Verney, 1998; see as reviews Smits et al, 2006; Smidt and
Burbach, 2007). Along with the anterior-posterior organization of the neuromeres, there
is also a dorsoventral organization of the neural tube consisting of the roof, alar, basal,
and floor plates (see as a review Smits et al., 2006) [Figure 7]. Molecular signaling from
the roof plate and floor plate divide the developing brain into dorsal and ventral
components, and these same molecular signals influence the development and
differentiation of the mdDA neurons (Lin and Rosenthal, 2003; Placzek and Briscoe,
2005; see as reviews Smits et al., 2006; Van den Heuvel and Pasterkamp, 2008).
During development, the neuronal progenitors of the mesodiencephalic
dopaminergic complex are situated in areas of the neural tube that develop into the
mesencephalon and caudal diencephalon (see as a review Jacobs et al., 2006; Smits et al.,
14
2006; Smidt and Burbach, 2007; Van den Heuvel and Pasterkamp, 2008; Hegarty et al.,
2013) [Figure 7]. The diverse neurodevelopmental origins of the retrorubral area, ventral
tegmental area, and substantia nigra result in a heterogeneous collection of THexpressing neuronal subpopulations that differ in regard to their molecular profiles,
electrophysiological properties, genetic expression, neurochemical characterization, and
susceptibility to disease (Gibb and Lees, 1991; Chung et al., 2005a,b; Greene et al., 2005;
Van den Heuvel and Pasterkamp, 2008).
A number of molecular signals are involved in the differentiation and final
positioning of mdDA neurons. Sonic hedgehog (Shh) is a glycoprotein that is secreted by
the floor plate of the neural tube and is essential for the development of the ventral
portion of the mesencephalon and diencephalon, as well as for the induction and
differentiation of dopaminergic neurons (Hynes et al., 1995a,b; Hynes and Rosenthal,
1999; Placzek and Briscoe, 2005). In fact, ectopic expression of dopaminergic neurons
can be achieved by overexpression of Gli-1, a downstream effector of Shh (Hynes et al.,
1997). Fibroblast growth factor 8 (Fgf8) is secreted from the isthmus (the region between
the rhombencephalon and mesencephalon), and is responsible for confining the
differentiation of mdDA neurons to ventral portions of the neural tube (Hynes and
Rosenthal, 1999; Lin and Rosenthal, 2003). In addition, many other transcription factors
are required for the correct development and maturation of mdDA neurons. Otx2 is
required for neuronal specification of mdDA progenitors (Puelles et al., 2003, 2004;
Vernay et al., 2005), Msx1 and Ngn2 are needed for neuronal differentiation (Andersson
et al, 2006a,b; Kele et al., 2006), Lmx1b participates in the maintenance of mature mdDA
neurons (Smidt et al., 2000), En1/2 are required for both the generation and survival of
15
mature neurons (Simon et al., 2001; Alberi et al., 2004), Nurr1 is essential for the
transcriptional activation of TH (Zetterstrom et al., 1997; Saucedo-Cardenas et al., 1998;
Wallen et al., 1999, 2001; Smits et al., 2003), and Pitx3 is involved in the development
and survival of mdDA neurons (Hwang et al., 2003; Nunes et al., 2003; van den
Munckhof et al., 2003; Smidt et al., 2004; Maxwell et al., 2005). Pitx3 is a bicoid-related,
homeodomain-containing transcription factor that is expressed by all TH-positive
neurons of the mdDA complex (see as a review Hegarty et al., 2013). However, the
absence of Pitx3 expression only affects the development of the dopaminergic neurons
that contribute to the nigrostriatal pathway (i.e. ventral tier), while those dopaminergic
neurons contributing to the mesocortical and mesolimbic pathways remain unaffected
(Hwang et al, 2003; Nunes et al., 2003; van den Munckhof et al., 2003; Smidt et al.,
2004a,b). It is believed that impaired Pitx-3-dependent functions within these neurons
contribute directly to this susceptibility, although the exact mechanism is not fully
understood (Luk et al., 2013). In Parkinson’s disease the neurons of the nigrostriatal
pathway are most heavily impacted (see as a review Feve, 2012), and it has been
demonstrated that those neurons that express Pitx3 are more susceptible to this neuronal
death pathology (Fuchs et al., 2009; Bergman et al., 2010; Luk et al., 2013). The
difference in the susceptibility of dopaminergic neurons to the absence of Pitx3 further
supports the existence of different dopaminergic subpopulations within the mdDA
complex.
The exact origin site of the different nuclei of the mdDA complex within the
developing brain has not been fully elucidated. It has been demonstrated that different
subgroups of dopaminergic neurons of the SN and VTA originate from several locations
16
within the neural tube, from the most caudal limit of the developing mesencephalon (i.e.
the isthmus) to the most rostral part of prosomere 3 within the developing diencephalon
(Puelles and Verney, 1998; see as reviews Smits et al, 2006; Smidt and Burbach, 2007).
The retrorubral area is believed to originate entirely from neuronal progenitors that are
present only in the developing mesencephalon, or mesomere (Puelles and Verney, 1998).
Smits et al. (2006) postulated that in the mouse there might be as many as eight different
subsets or groups of neurons that constitute the developing mdDA complex. The
identification of these eight subgroups is based on their location within the anteriorposterior axis (mesomere and prosomeres 1-3) and on their place of origin within the
dorsoventral territories of the neural tube (i.e. floor plate or basal plate). For the
developing human brain, Puelles and Verney (1998) have shown that there is a
plurisegmental origin for the SN/VTA mdDA neurons, which only later fuse into one
complex. Further evidence of the existence of distinct mdDA subpopulations comes from
electrophysiological, genetic, and neurochemical studies. For example, unique firing
characteristics have been reported between the dopaminergic populations of the
substantia nigra and ventral tegmental area in rodents (Grimm et al., 2004; Korotkova et
al., 2004; Thuret et al., 2004; Chung et al., 2005a,b; Greene et al., 2005). Also, it has
been demonstrated that there are subpopulations of the mdDA complex that present
different genetic profiles, which may help explain the different susceptibility of
dopaminergic neurons in Parkinson’s disease (Chung et al., 2005a; Greene et al., 2005).
Van den Heuvel and Pasterkamp (2008) state that the axon guidance receptor “deleted in
colorectal cancer” (DCC) is strongly expressed in dopaminergic neurons of the ventral
portion of the SNc, while it is almost absent in the dorsal SNc and VTA. In contrast,
17
dopaminergic neurons of the dorsal tier of the SNc and VTA express the calcium binding
protein calbindin, while dopaminergic neurons of the ventral tier of the SNc do not (Gibb
and Lees, 1991; Haber et al., 1995; Nemoto et al., 1999; see as a review Smith and
Kieval, 2000; Cebrian and Prensa, 2010; Luk et al., 2013).
In summary, the mdDA progenitors of the developing brain arise from areas
stretching from the most caudal part of the mesomere to the most rostral diencephalic
prosomere. Despite the fact that these dopaminergic subpopulations later coalesce to form
the mdDA complex and share a common TH+ phenotype, these subpopulations differ in
many aspects. In short, it is important to realize that, regardless of their spatial proximity
and shared dopaminergic nature, the different developmental origins and molecular
“make-up” of these subpopulations have important implications for the functioning of the
mdDA system as a whole.
Connecting the Dopaminergic System
The dopaminergic neurons of the mdDA complex start to send out their
projections shortly after acquiring their dopaminergic phenotype. These projections
initially proceed dorsally from their mesodiencephalic origin, but are soon redirected
towards a more rostral direction by chemical signals of the midbrain-hindbrain boundary
(Nakamura et al., 2000; Gates et al., 2004; Yamauchi et al., 2009) [Figure 8]. Rostrallydirected projections proceed through the ventral portion of the developing brain towards
the diencephalon and telencephalon (Nakamura et al., 2000; Gates et al., 2004) and form
two large bundles of axons called the medial forebrain bundles (MFBs). The MFBs
proceed to striatal and cortical areas, forming the basis of the major dopaminergic
18
pathways of the mdDA complex (Specht et al., 1981a; Verney, 1999; Zhao et al., 2004)
[Figure 8].
Synaptic connections of the nigrostriatal pathway form earlier than connections of
the remaining two pathways, with the most mature neurons of the mdDA complex
synapsing onto the most mature neurons of the striatum (Specht et al., 1981b; Voorn et
al., 1988). Connections between the mdDA complex and cortex are established later,
partially due to molecular signals originating from unidentified regions of the developing
cortex and striatum that result in a “waiting period” for the development of dopaminergic
cortical connections (Hemmendinger et al., 1981, Gates et al., 2004; see as a review Van
den Heuvel and Pasterkamp, 2008). These molecular signals eventually dissipate or are
overcome by more powerful attractant signals, with mdDA fibers only synapsing onto
differentiated layers of the developing cortex (Verney et al., 1982; Berger et al., 1985; see
as a review Van den Heuvel and Pasterkamp, 2008). After these initial connections are
established, axonal pruning becomes a critical step in the final maturation of the mdDA
pathways. Pruning within the nigrostriatal, mesolimbic, and mesocortical pathways is
necessary to remove excess synaptic contacts that would otherwise disrupt the correct
functioning of the mdDA system (Hu et al., 2004; Lou and O’Leary, 2005). It is
important to note that all the nuclei of the mdDA complex (i.e. RFF, SN, and VTA)
provide projections that will innervate the developing striatum and cortex (Lindvall and
Bjorklund, 1974; Fallon and Moore, 1978b; Roffler-Tarlov and Graybiel, 1984; see as a
review Van den Heuvel and Pasterkamp, 2008).
Studies on the development of these pathways have provided further evidence
supporting the existence of distinct subpopulations within each of the nuclei of the mdDA
19
complex. For example, it has been demonstrated that two distinct regions of the VTA
give rise to projections that terminate onto the PFC and nucleus accumbens in rodents
(Fallon, 1981; Swanson, 1982; Margolis et al., 2006). Interestingly, in paired box 6
(PAX6) knockout mice the majority of the mdDA projections that form the MFBs do not
fasciculate properly, although there is a small subset of axons whose path to the forebrain
is not disrupted (Vitalis et al., 2000). In addition, Eells et al. (2002) demonstrated that
nuclear receptor related-1 (Nurr1) heterozygous mice have significantly reduced levels of
dopamine and of the dopamine metabolite DOPAC in the projection areas of the
mesolimbic and mesocortical pathways (e.g. nucleus accumbens and prefrontal cortex,
respectively), whereas no significant change in dopamine or DOPAC was observed
within the striatum. Finally, in the developing mdDA complex of the rat brain, areas
corresponding to both the SN and VTA express mRNA for the axon guidance factor
robo1 receptor, while robo2 receptor mRNA is expressed only within the SN (Marillat et
al., 2002; Lin et al., 2005). All these findings support that distinct neuronal
subpopulations exist within the mdDA complex, which respond differently to pathwayfinding cues during development.
Molecules transiently expressed during development act as chemoattractants and
chemorepellents and contribute to the formation of the dopaminergic pathways, ensuring
proper axon guidance (see as a review Prasad and Pasterkamp, 2009). The generally
rostral orientation of mdDA projections is determined by chemorepellent molecules
expressed along the midbrain-hindbrain boundary of the developing brain, in conjunction
with chemoattractant molecules expressed in the forebrain (Nakamura et al., 2000; Gates
et al., 2004; Yamauchi et al., 2009) [Figure 8]. However, the chemical signals that guide
20
the initial dorsal movement of the dopaminergic fibers have yet to be discovered (see as a
review Van den Heuvel and Pasterkamp, 2008). Chemoattractant and chemorepellent
factors act in concert to guide the developing dopaminergic axons into the MFBs and
subsequently into the striatum and cortex (see as a review Van den Heuvel and
Pasterkamp, 2008) [Figure 8]. Within the developing striatum and cortex, chemorepellent
molecules act to halt the rostral projections of a subset of the axons, resulting in the
differentiation of the nigrostriatal and mesolimbic/mesocortical pathways (Gates et al.,
2004; see as reviews Van den Heuvel, 2008; Prestoz et al., 2012). One of these guidance
molecules, Ephrin, which serves as the ligand to the Ephrin receptor (Maisonpierre et al.,
1993; Yue et al. 1999; Willson et al., 2006), has been shown to play a direct role in the
determination of the synaptic connections of the axons proceeding from the mdDA
complex. Ephrin-A5 is a member of the ephrin ligand family, and is found primarily
within the ventral midbrain. Studies have demonstrated that the degree to which
developing axons of the mdDA complex respond to ephrin-A5 correlates with their final
target. That is, low-responding ephrin-A5 mdDA axons connect primarily to the ventral
striatum, while high-responding ephrin-A5 mdDA axons are repelled towards more
dorsal areas of the striatum. MdDA axons that do not respond in any way to ephrin-A5
proceed to cortical regions (Deschamps et al., 2009; see as a review Prestoz et al., 2012).
Other specific chemoattractant and chemorepellent molecules as well as their
receptors have been identified and include netrins/DCC (Livesey and Hunt, 1997; Vitalis
et al., 2000; Lin et al., 2005), semaphorins/plexins/neuropilins (Kawano et al., 2003;
Hernandez-Montiel et al., 2008), and Robo/Slits (Dugan et al., 2011; see as a review
Hegarty et al., 2013). Many of these chemical signals can act as both attractants and
21
repellents in the developing brain, depending on the neurochemical characteristics of the
growth cone (Kennedy et al., 1994; Colamarino and Tessier-Lavigne, 1995; VarelaEchavarria et al., 1997; Alcantara et al., 2000; Zhou et al., 2008; see as a review Masuda
and Shiga, 2005). In fact, midbrain neurons from rodent models of Parkinson’s disease
and from individuals who suffer from Parkinson’s disease react differently from their
respective controls when exposed to axon guidance cues, demonstrating again the
heterogeneity of the mesodiencephalic dopaminergic system (Grunblatt et al., 2001,
2004; Miller et al., 2004; Hauser et al., 2005).
Schizophrenia
Estimates indicate that as much as 1% of the world population may be afflicted
with schizophrenia, with this mental illness producing a “marked social or occupational
dysfunction” (DSM-IV-TR, 2000). In the United States there are approximately 3.2
million individuals that suffer from this disorder, incurring an estimated cost of $62.7
billion in 2002 (Wu et al., 2005). In the clinic, patients are diagnosed with schizophrenia
based on the presence of symptoms that are commonly grouped into three distinct
categories: positive symptoms, negative symptoms, and cognitive impairments. Positive
symptoms “appear to reflect an excess or distortion of normal functions” (DSM-IV-TR,
2000). Manifestations of these symptoms include hallucinations, disorganized speech,
and delusions, which normally appear in early adulthood. Negative symptoms “appear to
reflect a diminution or loss of normal function” (DSM-IV-TR, 2000; Stauss et al., 2013).
Avolition, affective flattening, and anhedonia are all examples of the negative
symptomatology of schizophrenia. Cognitive impairments found in schizophrenia include
22
deficits in communication, executive function, and memory (Pietrzak et al., 2009). None
of these symptoms are exclusive of schizophrenia, and different aspects of the disease
(e.g. psychotic behavior, anhedonia, cognitive deficits, etc.) can be found in other mental
disorders such as paranoid personality disorder, bipolar disorder, major depression, and
delusional disorders (DSM-IV-TR, 2000). In addition, the clinical diagnosis of
schizophrenia allows for the presence of a wide array of symptoms (DSM-IV-TR, 2000).
Schizophrenia Pathology
Numerous studies have demonstrated that genetic inheritance is a major
underlying factor in the development of schizophrenia (Kallmann, 1946; Sullivan et al.,
2003; see as reviews Tsuang, 2000; Shih et al., 2004; Sullivan, 2005). Genetic inheritance
of schizophrenia does not depend on a single gene, rather it is due to a medley of genes
acting in concert (see as a review Harrison and Weinberger, 2005), supporting the
concept of schizophrenia as a heterogenetic disorder. A non-exhaustive list of genes
shown to be altered in schizophrenia include Dystrobrevin-binding protein 1, Neuregulin
1, D-amino acid oxidase activator, disrupted in schizophrenia 1, Proline dehydrogenase,
COMT, and D2 and D4 receptor genes (Fan et al., 2003; Kirov et al., 2004; Corvin et al.,
2007; Liu et al., 2007; Pletnikov et al., 2008). Additionally, these genetic anomalies
differ within the schizophrenia population, such that not all of these genes are affected in
all individuals diagnosed with this disorder (Gardner et al., 2006; Pletnikov et al., 2008;
see as a review Harrison and Weinberger, 2005). However, studies have consistently
found that genes involved in dopamine production, transmission, and metabolism are
altered in schizophrenia (Wiesel et al., 1987; Cleghorn et al., 1989; Wiesel, 1992;
23
Sommer et al., 1993; Arinami et al., 1997; Akil et al., 2003; Prabakaran et al., 2004; see
as a review Harrison and Weinberger, 2005). One of the most replicated genetic
anomalies found in schizophrenia is that of COMT, which is involved in monoamine
catabolism [Figures 5, 6]. In addition, anomalies in the expression of the dopamine D2
receptor and dopamine D4 receptor genes have also been demonstrated, highlighting the
importance of abnormal dopamine function in the pathology of this illness (Sommer et
al., 1993; Arinami et al., 1997; Akil et al., 2003; see as a review Harrison and
Weinberger, 2005).
Another important contributor to the elevated risk of developing schizophrenia is
environmental factors. These include season and geographic location of birth, substance
abuse by the mother, infection and illness, malnutrition during pregnancy, and obstetric
complications (Mortensen et al., 1999; Willinger, 2001; Henquet et al., 2005; Schwarcz
and Hunter, 2007; see as reviews Leask, 2004; Clarke et al., 2006; Jenkins, 2013). More
important than a specific environmental factor is the fact that all these environmental
conditions result in some degree of stress being placed on the individual. Tsuang (2000)
remarked on the possibility that it might be that these stressful events affect genome
expression, contributing to the development of schizophrenia.
The examination of the gross neuroanatomy of brains from individuals that
suffered from schizophrenia yields few differences when compared to non-psychiatric
controls. The only consistently reported changes are mild enlargements of the lateral and
third ventricles, and decreased cerebral volume (Suddath et al., 1990; Van Horn and
McManus, 1992; Goldman et al., 2008, 2009; Rimol et al., 2010; Schultz et al., 2010; see
also as reviews Harrison, 1999; Shenton et al., 2001). This increase in ventricular size
24
and reduction in cerebral volume is believed to stem from a reduction in cortical neuronal
size and/or neuropil without neuronal death (Garey et al., 1998; Pierri et al., 2001). These
gross neuroanatomical changes have also been shown to be present in drug-naïve
patients, indicating that these anomalies are intrinsic to the disorder rather than the result
of antipsychotic medications (Bertolino et al., 1998; Cahn et al., 2002; Chua et al. 2007).
Differences between the brains of individuals with schizophrenia and brains from
non-psychiatric individuals are more apparent at the microscopic level. There are subtle
changes in many areas of the brain including the basal ganglia, prefrontal cortex,
thalamus, and limbic regions (Woo et al., 2008; Sodhi et al., 2011; Kimoto et al., 2014;
see as reviews Harrison, 1999; Powers, 1999; DeLisi et al., 2006; Perez-Costas, 2010;
Haukvik et al., 2013). In addition, these anomalies affect several neurotransmitter
systems including the dopaminergic, serotonergic, cholinergic, glutamatergic, and
GABAergic systems (Gonzalez-Maeso et al., 2008; Lewis et al., 2008; Roberts et al.,
2009; Perez-Costas et al., 2012; see also as reviews Carlsson et al., 1999; Tandon, 1999;
Kristiansen et al., 2006, 2007; Raedler et al., 2007; Stone et al., 2007; Howes and Kapur,
2009; Perez-Costas et al., 2010; Noetzel et al., 2012; de Bartolomeis et al., 2013; Eggers,
2013). Examples of this include hyperdopaminergia in subcortical regions and
hypodopaminergia in cortical regions (Akil et al., 1999; see also as reviews Davis et al.,
1991; Howes and Kapur, 2009; Perez-Costas et al., 2010; Howes et al., 2012; Kuepper et
al., 2012; Laruelle, 2013), alterations in cholinergic receptor density and binding in the
striatum and frontal cortex respectively (Tandon, 1999), changes in GABA receptors,
glutamic acid decarboxylase (GAD) mRNA and protein levels, and GABA transporter-1
mRNA levels throughout the brain (Simpson et al., 1989; Impagnatiello et al., 1998;
25
Guidotti et al., 2000; Volk et al., 2001; Fatemi et al., 2005; Bullock et al., 2008;
Thompson et al., 2009; see also as reviews Guidotti et al., 2000; Costa et al., 2004;
Lisman et al., 2008; Costa et al., 2009; Daskalakis and George, 2009), as well as NMDA
receptor hypofunction (see as reviews Jentsch and Roth, 1999; Olney et al., 1999;
Kristiansen et al., 2006, 2007; Stone et al., 2007; Lisman et al., 2008; Gordon, 2010;
McCullumsmith et al., 2012).
Dopamine Neurotransmission in Schizophrenia
The involvement of dopamine pathology in schizophrenia was first elucidated by
the observation that phenothiazines (a group of drugs that decrease dopaminergic
neurotransmission) could be used to treat psychotic symptoms, while amphetamines
(drugs that increase dopaminergic neurotransmission) could produce a state resembling
psychosis (Woodward et al., 2011; see as a review Ohlow and Moosmann, 2011).
Experimentally, the seminal work of Carlsson and Lindqvist (1963) revealed that
antipsychotic medication, specifically chlorpromazine and haloperidol, increased the
accumulation of catecholamine (e.g. dopamine) metabolites in in the brains of mice. It
was speculated that these increased metabolites were the result of a compensatory
increase in the activation of monoaminergic neurons due to a proposed blocking of
monoamine receptors by chlorpromazine and haloperidol. In addition, earlier work had
demonstrated that the antipsychotic reserpine blocked the reuptake of dopamine into
presynaptic neurons (Carlsson et al., 1957). Further studies on antipsychotic medications
revealed that their effectiveness could be directly linked to their ability to bind to
dopamine receptors (Seeman and Lee, 1975; Seeman et al., 1976; Creese et al., 1976; see
26
also as a review Frankle and Laruelle, 2002). Taking this information into account,
version I of the dopamine hypothesis of schizophrenia was formulated in the 1970s. This
early version stated that schizophrenia was caused by an excess of dopaminergic
transmission throughout the brain (see as a review Howes and Kapur, 2009). However,
studies in subsequent years yielded information on the contrary. For instance, positron
emission tomography and functional magnetic resonance imaging studies revealed a
decrease in dopaminergic activity in the frontal cortex of individuals with schizophrenia
(Hazlett et al., 2000; Riehemann et al., 2001). Also, studies examining the dopamine
metabolite HVA found reduced levels of this metabolite in cerebrospinal fluid, which is
indicative of decreased dopamine levels in cortical regions (Davidson and Davis, 1988;
see as a review Davis et al., 1991). Taking this new information into account, Davis et al.
(1991) postulated the version II of the dopamine hypothesis of schizophrenia.
Version II of the dopamine hypothesis of schizophrenia states that
hyperdopaminergia is associated with subcortical structures, in particular the striatum,
while hypodopaminergia is associated with cortical areas such as the prefrontal cortex
(see as reviews Davis et al., 1991; Howes and Kapur, 2009). Furthermore, version II
states that the subcortical hyperdopaminergia is the root of psychotic symptoms, while
negative symptoms and cognitive deficits could be linked to the cortical
hypodopaminergia (see as reviews Davis et al., 1991; Howes and Kapur, 2009). This
hypothesis is supported by studies showing an increase in dopamine neurotransmission in
the striatum (Kegeles et al., 2010; see also as reviews Toda and Abi-Dargham, 2007;
Meyer-Lindenberg, 2010), and a decrease in dopaminergic axonal length and overall
activation in the prefrontal cortex of individuals with schizophrenia (Akil et al., 1999, see
27
also as reviews Toda and Abi-Dargham, 2007; Meyer-Lindenberg, 2010). Also
supporting this hypothesis is the fact that all antipsychotic medications act to block
dopamine receptors, and that these medications normally relieve only psychotic
symptoms, which are associated with the hyperdopaminergia observed in subcortical
areas. An increase in subcortical dopamine release was also observed when healthy
individuals were subjected to an amphetamine challenge, resulting in a state that
resembled psychosis (Klawans and Margolin, 1975; Flaum and Schultz, 1996; Woodward
et al., 2011). In addition, it has been reported that individuals with schizophrenia release a
comparatively greater amount of dopamine than non-psychiatric controls when
administered an amphetamine challenge, reflecting the presence of increased basal levels
of subcortical dopamine (Abi-Dargham et al., 1998). This increase in dopamine release
has been shown to be present in schizophrenia patients who are experiencing an episode
of clinical deterioration, but not in clinically stable patients (see as a review Laruelle,
2000). Recent findings demonstrate that altered dopamine transmission is not a result of
medication, but rather an intrinsic feature of the disease (see as a review Perez-Costas et
al., 2010). Additionally, altered dopamine transmission predates the onset of prodromal
psychotic symptoms and may be a risk factor that could be used as a predictor for the
development of schizophrenia (Howes et al., 2009; see also as reviews Howes et al.,
2007; Perez-Costas et al., 2010).
Disturbances within the nigrostriatal, mesolimbic, and mesocortical dopaminergic
pathways are also correlated with the phenotypic symptomology of schizophrenia.
Through projections to the dorsal striatum, the nigrostriatal pathway is involved in the
alleviation of psychosis in schizophrenia with the use of antipsychotic medications
28
(Howes et al., 2009). The nigrostriatal pathway is also associated with tardive dyskinesia,
a potential side effect of the administration of first generation antipsychotic medications
(see as reviews Newman-Tancredi and Kleven, 2010; Jafari et al., 2012). The mesolimbic
pathway proceeds from the SN/VTA and projects to areas of the brain that are associated
with emotion and motivation, e.g. nucleus accumbens and amygdala (Fallon and Moore,
1978a,b; Fallon et al., 1978a,b; Porrino and Goldman-Rakic, 1982; Gaspar et al., 1992;
McRitchie et al., 1995; Haber and Fudge, 1997; Halliday, 2004), and disruptions within
this pathway are associated with emotional and motivational disturbances. This type of
pathology is present in schizophrenia, and is grouped under the category of negative
symptomology (DSM-IV-TR, 2000). Anomalies in the mesocortical dopaminergic
pathway are most closely related to the cognitive impairments, e.g. deficits in
communication, executive function, and memory, which also take place in schizophrenia
(Weinberger and Berman, 1988; see as a review Floresco and Magyar, 2006). The reason
for this association is due to the fact that the mesocortical pathway contains the
projections that arise from the mesodiencephalic dopaminergic neurons of the SN/VTA
and proceed to the cortex, particularly the frontal cortex (see as a review Haber and
Gdowski, 2004). Since the frontal cortex, and more specifically the prefrontal cortex, is
closely associated with these aspects of human functioning (e.g. cognition, etc.),
disruptions in this pathway could lead to deficits commonly seen in schizophrenia. In
fact, it has been demonstrated that individuals who carry the Val variant of the gene that
encodes for COMT present a four-fold increase in activity of the enzyme, and thus an
increase in the rate of dopamine catabolism within the cortex. This COMT variant and its
subsequent effects have been connected with deficits in executive function (Egan et al.,
29
2001). Furthermore, low levels of homovanillic acid, which are indicative of decreased
dopamine activity in the prefrontal cortex, correlate with poor performances on working
memory tasks (see as a review Toda and Abi-Dargham, 2007). In summary, the
nigrostriatal pathway is most closely associated with positive symptoms, the mesolimbic
pathway is involved in negative symptomology, and the mesocortical pathway is closely
related to the cognitive impairments that have been described in schizophrenia.
Metabolic Anomalies in Schizophrenia
Changes in cellular metabolism have also been implicated in the pathology of
schizophrenia, and previous studies have reported anomalies in the metabolic rate of
cortical and subcortical regions of brains from individuals with schizophrenia when
compared to non-psychiatric controls (Wiesel et al., 1987; Cleghorn et al., 1989; Wiesel,
1992; Prabakaran et al., 2004). One possible explanation for these observed differences is
the existence of anomalies in mitochondrial function. In support of this possibility,
mitochondrial density and mitochondrial morphological anomalies have been observed in
both anterior limbic cortex and the caudate nucleus in postmortem schizophrenia brain
tissue (Uranova and Aganova, 1989; Kung and Roberts, 1999; Somerville et al.,
2011a,b). In addition, Prabakaran et al. (2004) reported that transcriptional alterations of
genes involved in glucose, fatty acid, and oxidative phosphorylation metabolism, as well
as genes involved in oxidative stress were present in approximately 90% of the
postmortem brain tissue collected from schizophrenia patients.
The most well documented abnormalities of metabolism in schizophrenia revolve
around the study of perturbations in oxidative phosphorylation. This pathway is studied
30
extensively because of its role in the production of adenosine triphosphate (ATP), which
is used to power the metabolism of cells (see as a review Campbell et al., 2006).
Reductions in ATP concentrations have been observed in the frontal lobe of
schizophrenia subjects, which implies either a reduced number or dysfunctional
mitochondria (Volz et al., 2000). The production of ATP is dependent on a proton
gradient that is established across the inner mitochondrial membrane by the electron
transport chain (ETC) [Figure 9]. The ETC is composed of four enzymes that are located
in the inner membrane of the mitochondria consisting of NADH dehydrogenase (complex
I), succinate dehydrogenase (complex II), cytochrome bc1 (complex III), and cytochrome
c oxidase (complex IV) (Saddar et al., 2008; see as a review Manatt and Chandra, 2011),
with each enzyme performing a specific task along the chain. The proton gradient
established by the ETC is then used to power ATP synthase, resulting in the synthesis of
ATP from surrounding ADP and inorganic phosphate (see as reviews Michel et al, 1998;
Campbell et al., 2006; Rezin et al., 2009) [Figure 9]. It has been shown that anomalies
within the individual complexes can be sufficient to disrupt cellular metabolism
(Barksdale et al., 2010; Lee et al., 2013), although disruptions in one complex of the ETC
does not necessarily affect other complexes downstream (Prince et al., 1997; Barksdale et
al., 2010).
Cytochrome c oxidase (complex IV or COX) is one of the most relevant enzymes
of the ETC, and has been referred to as the rate-limiting enzyme of ATP production (Li et
al., 2006). Studies on the structure of COX using x-ray crystallography have revealed that
this enzyme is composed of 13 subunits [Figure 10]. Three of these subunits (COX I, II,
and III) are encoded by the mitochondrial genome, while the remaining subunits (COX
31
IV, Va, Vb, VIa, VIb, VIc, VIIa, VIIb, VIIc, and VIII) originate from nuclear DNA,
requiring the bidirectional communication of the two genomes for the coordinated
synthesis of the different subunits (Tsukihara et al., 1996, Nijtmans et al., 1998).
Mitochondrial-encoded subunits constitute the “catalytic core” of the enzyme, which is
responsible for substrate binding, electron transfer, oxidation, and eventual expulsion of
H+ ions from the matrix into the intermembrane space (see as a review Taanman, 1997).
Of the nuclear-encoded subunits, COX IV is the most functionally relevant due to its
roles in electron transfer and protection of the catalytic core from reactive oxygen species
(Nijtman et al., 1998; Li et al., 2006). In addition, COX IV plays an important role in the
assembly of the enzyme. A model for the assembly of COX has been proposed that
consists of two assembly intermediates. The first intermediate is formed by the binding of
COX I to COX IV, along with the insertion of heme A and heme A3 groups. This is
followed by the insertion of the other two subunits of the catalytic core (COX II, COX
III), together with COX Va, COX Vb, COX VII a or b, COX VIIc, and COX VIII to form
the second intermediate. The addition of COX VIa and COX VII a or b finishes the
formation of the complete COX holoenzyme, which will then form a dimer resulting in
the active form of COX (Nijtmans et al., 1998; Li et al., 2006; see as a review Fontanesi
et al., 2006) [Figure 11].
Several studies have shown that within the schizophrenia population COX activity
is differently affected depending on the region of the brain being tested. For example,
reduced activity has been noted within the frontal cortex, temporal cortex, and the
caudate nucleus (Cavelier et al., 1995; Maurer et al., 2001), with an increase in activity
within the putamen (Prince et al., 1999), and no changes in COX activity in the nucleus
32
accumbens, globus pallidus, thalamus, cerebellum, or mesencephalon (Prince et al., 1999;
Maurer et al., 2001). Although antipsychotic medications have been reported to cause
increases (Prince et al. 1997; 1998) and decreases (Maurer and Moller, 1997) in COX
activity, the majority of studies have established that COX activity is not affected by
antipsychotic medication (Burkhardt et al., 1993; Whatley et al., 1998; Balijepalli et al.,
1999; 2001; Streck et al., 2007). These findings suggest that reduced COX activity is
intrinsic to the pathology of schizophrenia. COX subunits have not been extensively
studied in schizophrenia, with only one paper reporting increased levels of COX-II
mRNA in the frontal cortex (Whatley et al., 1996). Mitochondrial DNA (mtDNA)
deletions have also been suggested to play a role in the COX anomalies observed in
schizophrenia (see as a review Kato, 2001), although no significant changes have been
found in the number of mtDNA deletions within the hippocampus, thalamus, inferior
temporal and superior temporal gyrus, cingulate gyrus, caudate nucleus, as well as
prefrontal, occipital, and parietal cortices of individuals with schizophrenia, when
compared to non-psychiatric controls (Cavelier et al., 1995; Lindholm et al., 1997;
Kakiuchi et al., 2005; Sabunciyan et al., 2007).
Despite of the fact that the SN/VTA houses the majority of the dopaminergic
neurons of the brain, this brain region is the least studied when it comes to the pathology
of schizophrenia. To this end, the present study was conducted to determine the
contribution of regional anomalies in the SN/VTA to the pathology of this disorder.
Specifically, our study of the pathology of the SN/VTA in schizophrenia was focused in
three main aspects: 1) Study of regional differences of TH mRNA and protein expression
in the SN/VTA in schizophrenia compared to non-psychiatric controls. 2) Assessment of
33
metabolic anomalies linked to COX dysfunction and their contribution to the pathology
of the SN/VTA in schizophrenia. This part of the study required the development of an
improved method for the measurement of COX activity, which was then used for the
assessment of COX activity in the SN/VTA. A second aspect of the analysis of COX
dysfunction was to assess if alterations in the expression of critical subunits for the
functioning and assembly of the COX enzyme could contribute to schizophrenia
pathology in the SN/VTA. 3) Taking into account our findings on TH protein expression
anomalies in the SN/VTA in schizophrenia, we wanted to assess if TH pathology could
be linked to a significant reduction in the number of neurons in the SN/VTA (i.e.
reduction of the total number of neurons or specifically dopaminergic neurons). To test
this, we performed stereological counts of dopaminergic and total number of neurons in
the SN/VTA of schizophrenia and control cases. As an alternative hypothesis, we also
wanted to test the possibility of a reduction of TH protein expression without significant
neuronal loss, and the possibility that reductions in TH expression could be assigned to
specific neuronal groups within the SN/VTA. To test this, we performed a qualitative
analysis of TH protein expression to pinpoint the specific areas and subcellular location
of TH deficits within the SN/VTA.
34
Figure 1. Organization of nuclei within the mesodiencephalic dopaminergic system. (A)
Sagittal view of the location of the substantia nigra within the human brain. (B) Coronal
slice of the human brain showing the location of the substantia nigra. (C) Coronal section
through the human brainstem immunohistochemically stained for tyrosine hydroxylase
showing the distribution of dopaminergic nuclei at the intermediate level of the substantia
nigra. The retrorubral area is located caudal to this section. (D) Coronal section of the rat
brainstem immunostained for tyrosine hydroxylase showing the distribution of the
dopaminergic cell groups at the intermediate level of the substantia nigra. The retrorubral
area is located caudal to this section. 3n: third cranial nerve, Aq: cerebral aqueduct, cp:
cerebral peduncle, PAG: periaqueductal gray, PBP: parabrachial pigmented nucleus, PN:
paranigral nucleus, R: red nucleus, RLi: rostral linear nucleus, SNC: substantia nigra pars
compacta SND: dorsal tier of the substantia nigra pars compacta, SNL: substantia nigra
pars lateralis, SNM: substantia nigra pars medialis, SNR: substantia nigra pars reticulata,
SNV: ventral tier of the substantia nigra pars compacta, VTA: ventral tegmental area.
Figures A & B reproduced with permission from Warner, J. J. (2001). Atlas of
neuroanatomy: With systems orgaization and case correlations. (Oxford: ButterworthHeinemann). Figure C reproduced with permission from Reyes, S., Fu, Y., Double, K.,
Thompson, L., Kirik, D., Paxinos, G., & Halliday, G. M. (2012). GIRK2 expression in
dopamine neurons of the substantia nigra and ventral tegmental area. J Comp Neurol,
520(12), 2591-2607. Figure D from Drs. Miguel Melendez-Ferro and Emma PerezCostas (unpublished).
35
Figure 2. Sagittal image of the human brain showing the three main dopaminergic
pathways and their projections sites: Nigrostriatal pathway (red), mesolimbic pathway
(green), and mesocortical pathway (blue). Note the intermingling of the pathways and
their initial overlapping origin among the individual nuclei of the mdDA complex. SNc:
substantia nigra pars compacta, VTA: ventral tegmental area. Reproduced with
permission from Arias-Carrion, O., Stamelou, M., Murillo-Rodriguez, E., MenendezGonzalez, M., & Poppel, E. (2010). Dopaminergic reward system: a short integrative
review. Int Arch Med, 3, 24.
36
Figure 3. Schematic of the afferent and efferent connections of the SN/VTA. Pathways
colored in red indicate GABAergic connections; pathways in green indicate
glutamatergic connections. C: caudate nucleus, Ctx: cortex, DA: dopamine, Enk:
enkephalin, GPe: globus pallidus external segment, GPi: globus pallidus internal
segment, NStrP: nigrostriatal pathway, P: putamen, SNc: substantia nigra pars compacta,
SNr: substantia nigra pars reticulata, SP: substance P, STN: subthalamic nucleus, StrNP:
striatonigral projections, VA: ventral anterior thalamic nucleus, VLa: anterior part of the
ventral lateral thalamic nucleus. D1-like family of dopamine receptors and D2-like family
of dopamine receptors are indicated by empty and filled small rectangles, respectively.
The efferent projection of the SNc to the striatum (nigrostriatal pathway; NStrP) is shown
in black. Reproduced with permission from Nieuwenhuys R., Voogd J., and van Huijzen
C. (2007). Telencephalon: Basal Ganglia. In: The Human Central Nervous System: A
Synopsis and Atlas, Fourth Edition (New York: Springer), 427-489.
37
Figure 4. In vivo biosynthesis of dopamine. Reproduced with permission from Purves,
D., Augustine, G. J., Fitzpatrick, D., et al. (2001). Neuroscience, 2nd edition.
(Sunderland: Sinauer Associates).
38
Figure 5. Diagram of a dopaminergic synapse. The presynaptic terminal is located at the
top and the postsynaptic neuron is located at the bottom of the image. COMT: catecholO-methyltransferase,
DOPA:
3,4-dihydroxyphenylalanine,
DOPAC:
3,4dihydroxyphenylacetic acid, D1R: D1-like family of dopamine receptors, D2R: D2-like
family of dopamine receptors, HVA: homovanillic acid, MAO: monoamine oxidase,
VMAT2: vesicular monoamine transporter 2.
39
Figure 6. Primary pathways of dopamine catabolism. Enzymes (in bold italics) are ADH:
alcohol dehydrogenase, ALD-D: aldehyde dehydrogenase, ALR: aldehyde reductase,
COMT: catechol-O-methyltransferase, and MAO monoamine oxidase. Cofactors (in
parenthesis) are NAD+: nicotinamide adenine dinucleotide, NADP+: nicotinamide
adenine dinucleotide phosphate, O2: molecular oxygen, and SAM: S-adenosyl-Lmethionine. Metabolites include 3-MT: 3-Methoxytyramine, DOPAC: 3,4dihydroxyphenylacetic acid, DOPAL: 3,4-dihydroxyphenylacetaldehyde, DOPET: 3,4dihydroxyphenylethanol, HVA: homovanillic acid, and MOPET: 3-methoxy-4hydroxyphenylethanol. Starred enzymes indicate pathways that have been shown to be
altered in schizophrenia pathology. Underlined metabolites indicated the most common
dopamine metabolite of the striatum (DOPAC) and the cortex (HVA). Reproduced with
permission from Elsworth, J. D., & Roth, R. H. (1997). Dopamine synthesis, uptake,
metabolism, and receptors: relevance to gene therapy of Parkinson's disease. Exp Neurol,
144(1), 4-9.
40
Figure 7. Development of the mesodiencephalic dopaminergic system. Top: Sagittal
view of an archetypical mammalian brain during development. Dopaminergic neuronal
precursors originate in ventral portions of the mesomere and prosomeres 1-3 (area shown
in red). The isthmus is indicated in blue. Bottom: Cross-section through the developing
mesencephalon at the level indicated by the dashed line. Dopaminergic neuronal
precursors originate from the basal and floor plates of the developing brain. AP: alar
plate, Aq: cerebral aqueduct, BP: basal plate, FP: floor plate, IZ: intermediate zone, M:
mesomere, MZ: marginal zone, P1: prosomere 1, P2: prosomere 2, P3: prosomere 3, RD:
rostral diencephalon, Rhom: rhombencephalon, SNc: substantia nigra pars compacta,
Tele: telencephalon, VTA: ventral tegmental area, VZ: ventricular zone.
41
Figure 8. Progression of axons from the developing mesodiencephalic dopaminergic
complex. (a) Chemorepulsive signals from caudal areas of the developing brain direct
axons towards a dorsal direction. (b) Dorsal progression of dopaminergic axons is halted,
and axons are directed towards a more rostral direction. (c) Axon guidance cues from the
thalamus result in the development of the medial forebrain bundles (MFBs). (d)
Dopaminergic axons of the nigrostriatal and mesolimbic/mesocortical pathways begin to
differentiate due to guidance cues emanating from the striatum and cortex, respectively.
(e) Guidance cues from the cortex help to establish the mesolimbic and mesocortical
pathways, while having a chemorepulsive effect on axons of the nigrostriatal pathway. (f)
Chemoattractants within the striatum help to finalize the synaptic connections of the
nigrostriatal pathway. OB: olfactory bulb, DM: dorsal midbrain, MFB: medial forebrain
bundles, SNc: substantia nigra pars compacta, VTA: ventral tegmental area. Reproduced
with permission from Van den Heuvel, D. M., & Pasterkamp, R. J. (2008). Getting
connected in the dopamine system. Prog Neurobiol, 85(1), 75-93.
42
Figure 9. Schematic of the electron transport chain of the mitochondria. Image showing
flow and direction of electrons (e-) and hydrogen ions (H+). ADP: adenosine diphosphate,
ATP: adenosine triphosphate, Cyt c: cytochrome c, Pi: inorganic phosphate, ROS:
reactive oxygen species, UQ: ubiquinone. Reproduced with permission from Al Ghouleh,
I., Khoo, N. K., Knaus, U. G., Griendling, K. K., Touyz, R. M., Thannickal, V. J.,
Barchowsky, A., Nauseef, W. M., Kelley, E. E., Bauer, P. M., Darley-Usmar, V., Shiva,
S., Cifuentes-Pagano, E., Freeman, B. A., Gladwin, M. T., & Pagano, P. J. (2011).
Oxidases and peroxidases in cardiovascular and lung disease: new concepts in reactive
oxygen species signaling. Free Radic Biol Med, 51(7), 1271-1288.
43
Figure 10. Three-dimensional representation of cytochrome c oxidase. The flux of H+
from the matrix to the intermembrane space is indicated by the red arrow. COX I, II, and
III (circled) constitute the catalytic core of the enzyme. COX IV (boxed) is fundamental
for the assembly and short-term regulation of the enzyme. Reproduced with permission
from Tsukihara, T., Aoyama, H., Yamashita, E., Tomizaki, T., Yamaguchi, H.,
Shinzawa-Itoh, K., Nakashima, R., Yaono, R., & Yoshikawa, S. (1996). The whole
structure of the 13-subunit oxidized cytochrome c oxidase at 2.8 A. Science, 272(5265),
1136-1144.
44
Figure 11. Model for cytochrome c oxidase (COX) assembly in cultured human cells.
Note the particular importance of COX IV, as well as COX II, in the initial assembly of
the enzyme. S1: stage 1, S2: stage 2, S3: stage 3, S4: stage 4. Reproduced with
permission from Nijtmans, L. G., Taanman, J. W., Muijsers, A. O., Speijer, D., & Van
den Bogert, C. (1998). Assembly of cytochrome-c oxidase in cultured human cells. Eur J
Biochem, 254(2), 389-394.
45
DOPAMINE PATHOLOGY IN SCHIZOPHRENIA: ANALYSIS OF TOTAL AND
PHOSPHORYLATED TYROSINE HYDROXYLASE IN THE SUBSTANTIA NIGRA
by
Emma Perez-Costas, Miguel Melendez-Ferro, Matthew W. Rice, Robert R. Conley, &
Rosalinda C. Roberts.
Frontiers in Psychiatry
Copyright
2012
by
Frontiers
Used with permission
Format adapted and errata corrected for dissertation
46
Abstract
Introduction: Despite the importance of dopamine neurotransmission in
schizophrenia, very few studies have addressed anomalies in the mesencephalic
dopaminergic neurons of the substantia nigra/ventral tegmental area (SN/VTA). Tyrosine
hydroxylase (TH) is the rate-limiting enzyme for the production of dopamine, and a
possible contributor to the anomalies in the dopaminergic neurotransmission observed in
schizophrenia. Objectives: In this study, we had three objectives: (1) Compare TH
expression (mRNA and protein) in the SN/VTA of schizophrenia and control postmortem
samples. (2) Assess the effect of antipsychotic medications on the expression of TH in
the SN/VTA. (3) Examine possible regional differences in TH expression anomalies
within the SN/VTA. Methods: To achieve these objectives three independent studies
were conducted: (1) A pilot study to compare TH mRNA and TH protein levels in the
SN/VTA of postmortem samples from schizophrenia and controls. (2) A chronic
treatment study was performed in rodents to assess the effect of antipsychotic
medications in TH protein levels in the SN/VTA. (3) A second postmortem study was
performed to assess TH and phosphorylated TH protein levels in two types of samples:
schizophrenia and control samples containing the entire rostro-caudal extent of the
SN/VTA, and schizophrenia and control samples containing only mid-caudal regions of
the SN/VTA. Results and Conclusions: Our studies showed impairment in the
dopaminergic system in schizophrenia that could be mainly (or exclusively) located in the
rostral region of the SN/VTA. Our studies also showed that TH protein levels were
significantly abnormal in schizophrenia, while mRNA expression levels were not
affected, indicating that TH pathology in this region may occur posttranscriptionally.
47
Lastly, our antipsychotic animal treatment study showed that TH protein levels were not
significantly affected by antipsychotic treatment, indicating that these anomalies are an
intrinsic pathology rather than a treatment effect.
Keywords: neuropsychiatric disorders, postmortem studies, human brain, rat brain,
western-blot
48
Introduction
Tyrosine hydroxylase (TH) is a crucial enzyme in the production of dopamine and
other catecholamines. In the substantia nigra/ventral tegmental area (SN/VTA), dopamine
is the final product of cells that express TH and these cells are the largest source of
dopamine in the brain. Studies of TH in the SN/VTA of schizophrenia subjects are almost
anecdotal, with just one study that addressed enzymatic activity (Toru et al., 1988), and
two studies that addressed mRNA levels (Ichinose et al., 1994; Mueller et al., 2004).
However, no studies have assessed TH protein levels in the SN/VTA in schizophrenia. In
addition, recent studies on the synthesis of TH have shown that the regulation of the
production of TH is very different in the SN/VTA than in other catecholaminergic
neurons (Wong and Tank, 2007; Chen et al., 2008; Tank et al., 2008). These studies also
showed that posttranscriptional regulations have a crucial role in the synthesis of TH in
the SN/VTA (Chen et al., 2008; Tank et al., 2008). Posttranscriptional modulation occurs
after the production of mRNA is completed, and includes the stabilization of the mRNA,
the attachment to polyribosomes, and the regulation of the efficacy of translation into
protein (Tank et al., 2008; Lenartowski and Goc, 2011). For all these reasons, we believe
that is necessary to study TH protein levels in the SN/VTA in schizophrenia, to assess the
possibility of pathologies that could be located in posttranscriptional steps in the
synthesis of TH, rather than at the transcription level.
In addition to its production, TH protein activity in the SN/VTA is modulated by
the phosphorylation of certain serine sites in the first forty amino acids of the N-terminus
of the TH protein (Haycock, 1990; Nakashima et al., 2009). Of these phosphorylation
sites, serine 40 has been shown to be the most important in the regulation of TH activity
49
in the brain (Nakashima et al., 2009). The phosphorylation of this site is a complex
process that includes proper conservation of the amino acid residues flanking this serine
residue, and phosphorylation of this site produces the disinhibition of the catalytic
subunit of TH, thus allowing the production of dopamine (Nakashima et al., 2009).
Genetic association studies in schizophrenia have produced somewhat
inconclusive results (Meloni et al., 1995; Wei et al., 1995; Jonsson et al., 1996, 1998;
Thibaut et al., 1997; Burgert et al., 1998; Ishiguro et al., 1998; Kunugi et al., 1998;
Kurumaji et al., 2001; Ota et al., 2001; Chao and Richardson, 2002; Pae et al., 2003;
Hoogendoorn et al., 2005; Jacewicz et al., 2008; Talkowski et al., 2008; Andreou et al.,
2009). However, some of these studies have shown consistent data in different cohorts of
patients, indicating an association of certain mutations in the first intron of the TH gene
with a higher risk of schizophrenia (Meloni et al., 1995; Wei et al., 1995; Kurumaji et al.,
2001; Pae et al., 2003; Jacewicz et al., 2008). Interestingly this first intron of the TH gene
has been postulated to be a modulatory region for the synthesis of TH (Pae et al., 2003;
Jacewicz et al., 2008).
Previous studies assessing anomalies of the dopaminergic system in schizophrenia
have heavily focused their attention on different aspects of the dopamine
neurotransmission at the postsynaptic level, and studies of molecules involved in the
transport and degradation of dopamine (see as reviews, Abi-Dargham et al., 2010; PerezCostas et al., 2010; Tost et al., 2010). Another important aspect of the dopaminergic
neurotransmission, which originates in neurons in the SN/VTA, is the topographical
organization of the dopamine producing cells in this complex, which defines the
preferential terminal target of their projections (e.g. the mesolimbic, mesocortical or
50
nigro-striatal pathways; Fallon and Moore, 1978a,b; Fallon et al., 1978a,b; Porrino and
Goldman-Rakic, 1982; Gaspar et al., 1992; McRitchie et al., 1995; Haber and Fudge,
1997; Haber and Gdowski, 2004; Halliday, 2004). Some studies have found anomalies in
TH in the terminals of the nigro-striatal pathway in schizophrenia (Roberts et al., 2009)
and in the terminal fields of the mesocortical pathway (Akil et al., 1999; 2000), but none
of theses studies have assessed the cells of origin of the dopaminergic inputs to these
brain regions. This brought our attention to the importance of assessing regional
anomalies in TH synthesis within the SN/VTA in schizophrenia.
Materials and Methods
Ethics statement
All the human brain samples used in this study were obtained from the Maryland
Brain Collection (Maryland Psychiatric Research Center, University of Maryland School
of Medicine) with permission from the Maryland Brain Collection Steering Committee.
All experimental procedures were also approved by the University of Alabama at
Birmingham Institutional Review Board (protocol # N110505002) and in accordance
with The Code of Ethics of the World Medical Association.
All the animal experiments were conducted at the University of Maryland
following a protocol approved by the Institutional Animal Care and Use Committee
(protocol #0705010), in accordance with National Institutes of Health guidelines
regarding the care and use of animals for experimental procedures.
51
Postmortem human brain samples
For all the postmortem human cases used in this study, two independent senior
psychiatrists established DSM-IV diagnoses based on the review of all available medical
records and the data obtained from the Structured Clinical Interview for DSM-IIIR
(Spitzer et al., 1992) and DSM-IV Axis I disorders with the next of kin. Control cases
had enough information from next of kin and medical records to discard any major
neurological or neuropsychiatric disorders.
Two types of samples were used in this study: samples containing the entire
rostro-caudal extent of the SN/VTA (pilot study and second postmortem study), and
samples containing only mid-caudal SN/VTA (second postmortem study). In both cases
samples were frozen on dry ice and stored at -800C until use. Samples were sectioned on
a cryostat at -200C obtaining five parallel series of 16 μm thick sections. Four parallel
series were collected on superfrost slides, while the fifth series was collected in a tube for
protein extraction. In all cases, series #1 was stained with thionin (Nissl stain) to assess
possible morphological anomalies. Only cases that presented good preservation of the
SN/VTA region were included in the study. Rostral and caudal limits for the SN/VTA
were determined using the Paxinos and Huang (1995) brainstem atlas. For samples
containing only mid-caudal regions the rostral limit was set at the starting point of the
substantia nigra compacta pars ventralis as defined by McRitchie et al. (1995).
Two different sets of experiments were performed with human brain samples: a) a
pilot study in which TH mRNA and TH protein levels were assessed in samples from the
same cases (see Table 1); b) a second postmortem study of total TH protein levels and
52
phosphorylated TH levels in samples containing the entire extent or only mid-caudal
regions of the SN/VTA (see Table 2).
Animal samples
A total of 27 adult male Sprague-Dawley rats (Charles River Laboratories,
Wilmington, MA, USA) were used in this study to test the effect of chronic treatment
with antipsychotic drugs on TH protein levels. Animals were allowed to adapt to the
animal room for 1 week prior to starting any treatment, and water consumption was
monitored to calculate initial treatment doses. Animals were then randomly assigned to
one of the 3 treatment groups (n=9 per group): haloperidol (1.5 mg/kg/day), olanzapine
(6 mg/kg/day), or control. Antipsychotics were administered for 3 weeks in drinking
water, adjusting the doses to body weight and daily liquid consumption as previously
described in Perez-Costas et al. (2008). At endpoint, animals were decapitated, and trunk
blood was collected to monitor plasma drug levels. Brains were immediately removed
from the skull, frozen in dry ice, and stored at -800C until use. Four parallel series at the
level of the SN/VTA were collected on superfrost slides, while the 5th series was
collected in a tube for protein extraction. For all animals, series #1 was stained with
thionin to assess possible morphological anomalies. No morphological anomalies were
found in any of the samples used.
In situ hybridization
In situ hybridization was performed for the postmortem SN/VTA samples
included in the pilot study (Table 1). Slides containing sections at the level of the rostral,
53
medial and caudal SN/VTA were selected from each case to perform this technique. To
select the sections, series #1 (stained with thionin) was used to accurately identify
matching rostral, medial and caudal areas of the SN/VTA for each case. A 48-mer
antisense oligonucleotide probe that hybridizes to nucleotides 32-79 of the human TH
mRNA sequence was used. This sequence is common to all three mRNA transcript
variants identified for the human TH (GeneBank accession numbers: NM_199292;
NM_000360; NM_199293). Probe labeling, hybridization, and post-hybridization rinses,
were performed as described in Gao et al. (1998). Briefly, oligonucleotide probes were
labeled with
35
S-dATP at the 3’-end using terminal transferase. The sections were
hybridized overnight at 37°C with an hybridization buffer containing 50% deionized
formamide, 4X sodium saline citrate (SSC), Denhardt’s solution, 10X dextran sulfate,
yeast tRNA (250 μg/ml), single strand salmon DNA (500 μg/ml) and 100mM
dithiothreitol (DTT). After hybridization, the sections were sequentially rinsed in 1X SSC
containing 10 mM DTT at 550C, and 1X SSC at room temperature. Finally, the sections
were gradually dehydrated in ethanol, air dried and autoradiograms were generated by
exposing Kodak BioMax MR films (Kodak, Rochester, NY) along with [ 14C] microscale
standards (Amersham Biosciences, Little Chalfont, UK) for 14 days. Films were
developed using Kodak D-19 developer and fixer, scanned at 600 dpi, and optical density
was measured using Image Pro-Plus 6.2 software (Media Cybernetics, Bethesda, MD).
Western-blot
Western-blot assays were performed for human postmortem SN/VTA protein samples
(pilot study and second postmortem study; see Tables 1 and 2), and for rat SN/VTA
54
protein samples (animal study). In all cases the protein extraction procedure and sample
handling were identical. The only exceptions were the antibodies used for the detection of
TH. For the human postmortem studies a mouse monoclonal anti-TH antibody (SigmaAldrich, St. Louis, MO, USA) diluted 1:10,000 was used, while for the animal study a
mouse monoclonal anti-TH antibody (Millipore, Billerica, MA, USA) diluted 1:20,000
was used. In addition, a rabbit polyclonal anti-phospho-Ser40 TH antibody
(PhosphoSolutions, Aurora, CO, USA) diluted 1:4,000 was used in human samples
(second postmortem study) for the detection of phosphorylated TH (pTH). Finally, reblots for actin were performed in all the western-blot experiments (human and rat) using
a mouse monoclonal anti-actin antibody (Millipore) diluted 1: 40,000.
Antibodies specificity
In this work, we used two different types of antibodies to detect TH, one type to
detect total content of tyrosine hydroxylase, and another type to detect the active
(phosphorylated) form of this enzyme. In addition, an antibody against actin was used as
an internal control for the accuracy of the experiments performed (e.g. protein loading
and tissue preservation control).
For the detection of total tyrosine hydroxylase, two different antibodies were used:
For the human studies, a mouse monoclonal anti-TH manufactured by Sigma-Aldrich
(clone TH-16, catalog # T2928) was used. As per information provided in the data sheet
by the manufacturer, this antibody is raised against purified rat tyrosine hydroxylase and
recognizes an epitope in the N-terminal region between aminoacids 40-152 of human TH.
For the rat study, we used an anti-TH antibody manufactured by Millipore (clone
55
2/40/15, catalog # MAB5280). As per information provided by the manufacturer, this
antibody is raised against purified tyrosine hydroxylase from a rat pheochromocytoma
and its specificity is routinely assessed by western-blot by the manufacturer.
For the detection of phosphorylated (active) tyrosine hydroxylase, a rabbit
polyclonal anti-phospho-ser40 antibody by Phosphosolutions (catalog# p1580-40) was
used. As per information provided by the manufacturer, this antibody is raised against a
phosphopeptide corresponding to aminoacid residues surrounding the phosphor-ser40 of
rat tyrosine hydroxylase. The antibody is purified by affinity purification via sequential
chromatography on phospho- and dephosphopeptide affinity columns. The antibody
specificity was also tested by western-blot, and has been shown to recognize exclusively
phosphorylated TH.
For the detection of actin, a mouse monoclonal anti-actin antibody by Millipore
(catalog# MAB1501, clone C4) was used. As per information provided by the
manufacturer, this antibody is raised against purified chicken gizzard actin, and
recognizes an epitope located at the N-terminal two thirds of the protein near aminoacids
50-70. This antibody reacts against actin from all vertebrates.
Protein extraction
Tissue collected for western-blot was diluted 1:5 in lysis buffer containing TrisHCl pH 8.0, sodium chloride, sodium dodecyl sulphate, disodium EDTA, and a protease
inhibitor cocktail (Sigma-Aldrich; P8340). Samples were homogenized with a sonicator,
and the homogenate was centrifuged at 13,000 rpm for 15 minutes at 40C. The
56
supernatant was recovered, and the total protein concentration was measured using a
modified Lowry kit (Bio-Rad; 500-0113, 500-0114).
Gel electrophoresis and western-blot
Protein extracts with total protein concentrations of 30μg (rat), or 60μg (human),
were loaded onto 10% polyacrylamide gels along with a molecular weight marker (BioRad, Hercules, CA; 161-0324). Proteins were resolved at 150 volts, and then transferred
at 30 volts overnight onto PVDF membranes (Bio-Rad; 162-0174). The following day,
the membranes were blocked in Tris-buffered saline (TBS) containing 5% non-fat
powdered milk and 0.1% Tween-20 for 1 hour at room temperature. TH or pTH were
detected by incubating the membranes with the appropriate primary antibodies (see
above) diluted in TBS containing 1% non-fat powdered milk and 0.1% Tween-20
(TBSTm) for 21 hours at 40C. After rinsing in TBSTm, the membranes were incubated
for 1 hour at room temperature with a goat anti-mouse alkaline phosphatase-coupled
secondary antibody (Millipore; AP124A) diluted 1:15,000, or a goat anti-rabbit alkaline
phosphatase-coupled secondary antibody (Vector Laboratories, Burlingame, CA; AP1000) diluted 1:1,000. The membranes were then rinsed in TBS, and the bands were
visualized using a chemiluminescent system (Bio-Rad; 170-5018), exposing Kodak
Biomax XAR films. In all cases, the membranes were reblotted and re-incubated with an
antibody against actin (see details above). Films were scanned at 600 dpi using a flatbed
scanner, and optical density was measured using Image Pro Plus 6.2 software (Media
Cybernetics).
57
Statistical analysis
All data sets (outcome measures and possible covariates) were assessed for
normality using the Kolmogorov-Smirnov test. For this and subsequent statistical tests,
categorical variables (i.e. gender and race) were assessed using a “dummy coding”
scheme. Power analyses were performed entering the outcome measures (i.e. TH protein
levels in schizophrenia and control groups) from the first postmortem study (pilot study)
in G power 3.1 software (Faul et al. 2007). For subsequent postmortem studies (i.e.
regional expression of TH and pTH protein) an “a priori” test to compute sample size for
t-test comparison was used. For the animal study, an “a priori” test to compute sample
size for one-way ANOVA was used. The effect of possible covariates in the outcome
measures of interest (i.e. mRNA TH levels, TH protein, and pTH protein levels) for
postmortem studies was assessed using a multiple regression model, in which the effect
of the possible covariates was tested independently for each of the outcome measures of
interest. The covariates included in the multiple regression analyses were carefully
selected based on their possible influence on the expression of TH (mRNA or protein) or
pTH (protein). The tested covariates included age, race, gender, postmortem interval
(PMI), pH, and actin levels. PMI and pH are directly related with the quality of the tissue
preservation (Stan et al., 2006), while actin levels are directly related with the accuracy
on the processing of the samples (i.e. loading of the samples on the electrophoresis gels).
The model of multiple regression analysis used also included the assessment of possible
multicollinearity. For the animal study, the only covariate assessed was actin levels, and
this was done by linear regression analysis. For postmortem human studies the statistical
analysis of the outcome measures was performed by using the appropriate t-test (i.e.
58
Student’s t-test, or Welch’s t-test) for data that were normally distributed, or a nonparametric test (Mann-Whitney U-test), when one or both of the data sets were not
normally distributed. One-way ANOVA was used in the case of animals treated with
antipsychotics. The statistical analysis was performed using InStat 3.0 software
(GraphPad Software Inc., La Jolla, CA).
Results
Demographic features
Human postmortem cases were carefully selected for each study, trying to match
as much as possible the cases in the control and schizophrenia groups by their age,
gender, race, PMI and brain pH (see Tables 1 and 2). In addition, statistical analysis was
performed for each of these demographic features to ensure that the control and
schizophrenia groups were not significantly different.
Pilot study
Age, gender, race, and PMI were not normally distributed (Kolmogorov-Smirnov
Normality test) for one or both groups, therefore a Mann-Whitney U-test was used to
compare each of these demographic features between the control and schizophrenia
groups (Age: U=13.00; P=0.79. Race: U=11.00; P=0.46. Gender: U=14.00; P=0.91;
PMI: U=14.50; P>0.99). Brain pH data were normally distributed for both groups,
therefore a standard unpaired t-test was performed (t(9)=0.72; P=0.49). In summary, all
the demographic features were not significantly different between the control and
schizophrenia groups for the pilot study (Table 1).
59
Regional expression of TH and pTH study
This study was performed in two different types of samples that were analyzed
independently: Cases containing all the rostro-caudal extent of the SN/VTA, and cases
containing only the mid-caudal region of the SN/VTA. The demographic features for
control and schizophrenia cases for each of these two types of samples were statistically
analyzed. In rostro-caudal samples, age, PMI, and brain pH were normally distributed for
the control and schizophrenia groups, therefore an unpaired t-test was performed (Age:
t(12)=0.15; P=0.89. PMI: t(12)=0.40; P=0.70. Brain pH: t(12)=1.79; P=0.1). On the other
hand, race and gender for the rostro-caudal samples were not normally distributed at least
for one of the groups, therefore a Mann-Whitney U-test was performed (Race: U=18.00;
P=0.39. Gender: U=22.00; P=0.79). In summary, no demographic features were
significantly different between the control and schizophrenia groups for the cases
containing all the rostro-caudal extent of the SN/VTA. In mid-caudal samples, age, PMI,
and pH were normally distributed for the control and schizophrenia groups, therefore an
unpaired t-test was performed (Age: t(12)=1.08; P=0.30. PMI: t(12)=0.50; P=0.63; Brain
pH: t(12)=1.70; P=0.11). For mid-caudal samples race and gender were not normally
distributed and a Mann-Whitney U-test was performed (Race: U=10.00; P=0.045.
Gender: U=18.00; P=0.39). In summary, race was the only demographic feature that
presented significant differences between the schizophrenia and control groups in midcaudal samples. This significant difference was further assessed (see below, second
postmortem study results) as a possible predictor for the main outcome measures (TH and
pTH protein levels; Table 2).
60
Pilot study: TH mRNA and protein expression in human postmortem schizophrenia and
control cases
TH mRNA and TH protein levels were tested in SN/VTA samples from the same
cases (see Table 1). In this pilot study a total of 11 cases were used (n=5 controls; n=6
schizophrenia).
TH mRNA levels
Multiple regression analysis with the main outcome measure (TH mRNA levels)
as the dependent variable, and the possible covariates as independent variables was used
to test if the covariates (i.e. age, race, gender, PMI, pH) significantly affected the TH
mRNA levels regardless of diagnoses (schizophrenia or control). The results of this test
were non-significant (R2=0.38; P=0.70). After that, TH mRNA data in the schizophrenia
and control groups were tested for normality using the Kolmogorov-Smirnov test. For
both groups the TH mRNA data were normally distributed (Control group: KS=0.23;
P>0.10. Schizophrenia group: KS=0.23; P>0.10). Since the data were normally
distributed, an unpaired two-tailed t-test assuming equal variances was performed to test
for differences in TH mRNA levels between the controls and schizophrenia groups,
which yielded a non-significant result (t(9)=1.74; P=0.12). See also Figure 1A.
TH protein levels
Multiple regression analysis with TH protein as the main outcome measure
(dependent variable) and age, race, gender, PMI, pH, and actin levels as possible
covariates (independent variables) revealed a non-significant result (R2=0.60; P=0.52).
61
Kolmogorov-Smirnov normality test revealed that TH protein levels data in the control
and schizophrenia groups were normally distributed (Control group: KS=0.21; P>0.10.
Schizophrenia group: KS=0.29; P>0.10). An unpaired two-tailed t-test assuming unequal
variances (Welch’s correction) was used since the standard deviations between the two
groups were significantly different (F=72.75; P=0.001). The t-test with Welch’s
correction revealed significant differences in TH protein levels between the control and
schizophrenia groups (t(5)=2.96; P=0.031). See also Figure 1B.
In summary, this pilot study showed that TH protein levels were significantly
different between the schizophrenia and control groups, while TH mRNA levels did not
differ between the two groups. These results prompted us to design new experiments to
test: a) if chronic antipsychotic treatment could significantly affect the levels of TH
protein (animal study), and b) if the differences observed in TH protein expression were
regionally specific within the SN/VTA, and if these differences were also present in the
active form of TH (pTH), which were tested in a subsequent postmortem human study.
Animal study: Antipsychotic treatment effect on TH protein levels
Power analysis
For this study, an “a priori” power analysis was performed to determine the
minimum adequate sample size for a one-way ANOVA with a minimum power of 0.80.
Taking into account the results of the pilot study for TH protein levels in postmortem
human tissue, we used the following assumption: if changes in TH protein levels can be
explained as an effect of the antipsychotic medication, these changes have to be large to
explain the differences observed in the pilot study between the control and schizophrenia
62
groups. For this reason, we modeled the study to expect a large effect size (0.80). With
these assumptions, the power analysis determined that a minimum sample size of n= 21
(n=7 per group) was necessary to obtain the minimum power required. Since in our study
the sample size was n=27 animals (n=9 per group), a post-hoc power analysis using the
actual effect size obtained (f=0.64) revealed that the actual power of our study was 0.804
(critical F=3.40; α=0.05).
TH protein levels in animals chronically treated with APDs
The only covariate to be assessed for this study was actin protein levels, which is
a measurement of the accuracy of the experiment. The effect of this possible covariate on
TH protein levels was analyzed by linear regression, which revealed a non-significant
result (r=0.15; P=0.44). The Kolmogorov-Smirnov test showed that TH protein levels
were normally distributed for the three groups (Control group: KS=0.22; P>0.10;
Haloperidol group: KS=0.17; P>0.10. Olanzapine group: KS=0.26; P=0.08). However, a
Kruskal-Wallis non-parametric ANOVA was used, since a Bartlett’s test for homogeneity
of variances showed very significant differences among the three groups (Bartlett
statistic=10.37; P=0.006). The Kruskal-Wallis ANOVA revealed non-significant
differences among the three groups for TH protein levels (KW=1.79; P=0.41). See also
Figure 2.
In summary, this study showed that chronic treatment with antipsychotic
medications (typical or atypical) did not produce significant differences in TH protein
levels when compared with untreated controls.
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Second postmortem study: Regional expression of TH and phosphorylated TH
Power analysis
For this study, an “a priori” power analysis was performed to determine the
minimum adequate sample size for a two-tailed t-test analysis with a minimum power of
0.80. To calculate this, data obtained from the TH protein levels in the pilot study were
entered to calculate the effect size. This analysis yielded that a sample size of n=14 cases
(schizophrenia n=8; control n=6) would produce an actual power of 0.83 (t critical =2.18;
α=0.05). In mid-caudal samples, an outlier was present in the schizophrenia group (case
SZ11). This case was detected as an outlier using a combination of different factors: first,
the pH of this sample was highly basic (pH=7.80, see Table 2); second, TH levels were
highly different from any other case in the schizophrenia or control groups, and a
Grubb’s test was performed using Quick Calcs Online calculator (Graphpad Software
Inc.). This test showed that case SZ11 was a significant outlier for TH levels (Z=2.94;
P<0.05). For these reasons, this case was removed from the subsequent statistical
analysis of mid-caudal TH and pTH expression. After removing this case for the analysis,
a two-tailed t-test “compromise” power was calculated, entering the data obtained for TH
protein levels in the pilot study to calculate the effect size. This analysis yielded a power
of 0.80 (critical t=2.20; α=0.05) for the mid-caudal analysis with a sample size of n=13
(control n=6; schizophrenia n=7).
TH protein levels in rostro-caudal SN/VTA samples
Multiple regression analysis with TH protein as the main outcome measure
(dependent variable) and age, race, gender, PMI, pH, and actin levels as possible
64
covariates (independent variables) revealed a non-significant result (R2=0.74; P=0.06).
Kolmogorov-Smirnov normality test revealed that TH protein levels in the control and
schizophrenia groups were normally distributed (Control group: KS=0.31; P=0.07.
Schizophrenia group: KS=0.17; P>0.10). An unpaired two-tailed t-test assuming unequal
variances (Welch’s correction) was used since the standard deviations between the two
groups were significantly different (F=32.97; P=0.001). The t-test with Welch’s
correction revealed significant differences in TH protein levels between the control and
schizophrenia groups (t(7)=2.59; P=0.036). See also Figure 3A.
TH protein levels in mid-caudal SN/VTA samples
Multiple regression analysis with TH protein as the main outcome measure and
age, race, gender, PMI, pH, and actin levels as possible covariates revealed a nonsignificant result (R2=0.75; P=0.11). In addition, the multiple regression analysis showed
that, even when race was detected as a significantly different demographic feature
between the schizophrenia and control groups for mid-caudal cases (see demographic
features analysis above), this was not a significant contributor (predictor) of TH levels
outcome (t ratio=0.75; P=0.48). Kolmogorov-Smirnov Normality test revealed that TH
protein levels were normally distributed for the control group but not for the
schizophrenia group (Control group: KS=0.27; P>0.1. Schizophrenia group: KS=0.31;
P=0.047). Since data were not normally distributed for one of the groups, a nonparametric Mann-Whitney U-test was used. This test revealed non-significant differences
in TH protein levels between the control and schizophrenia groups (U=13.0; P=0.29). See
also Figure 3A.
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Phosphorylated TH protein levels in rostro-caudal SN/VTA samples
Multiple regression analysis with pTH protein levels as the main outcome
measure, and age, race, gender, PMI, pH, and actin levels as possible covariates revealed
a non-significant result (R2=0.51; P=0.39). Kolmogorov-Smirnov normality test revealed
that pTH protein levels data in the control and schizophrenia groups were normally
distributed (Control group: KS=0.27; P>0.10. Schizophrenia group: KS=0.27; P=0.09).
An unpaired two-tailed t-test assuming unequal variances yielded no significant
differences (t(12)=2.12; P=0.056), although the P value was low enough to indicate a trend
towards significance. See also Figure 3B.
Phosphorylated TH protein levels in mid-caudal SN/VTA samples
Multiple regression analysis with pTH protein as the main outcome measure and
age, race, gender, PMI, pH, and actin levels as possible covariates revealed a nonsignificant result (R2=0.73; P=0.13). In addition, the multiple regression analysis showed
that, even when race was detected as a significantly different demographic feature
between the schizophrenia and control groups for mid-caudal cases (see demographic
features analysis above), this was not a significant contributor (predictor) of pTH levels
outcome (t ratio=0.36; P=0.73). Kolmogorov-Smirnov normality test revealed that pTH
protein levels were normally distributed for the control group but not for the
schizophrenia group (Control group: KS=0.24; P>0.1. Schizophrenia group: KS=0.35;
P=0.01). Since data were not normally distributed for one of the groups, a non-parametric
Mann-Whitney U-test was used. This test revealed non-significant differences in TH
66
protein levels between the control and schizophrenia groups (U=12.5; P=0.25). See also
Figure 3B.
In summary, this regional postmortem study showed that TH protein levels in
samples containing the entire rostro-caudal extent of the SN/VTA were significantly
different between the schizophrenia and control groups, while no significant differences
were detected in samples that only contained mid-caudal regions. In addition, no
significant differences were detected for pTH protein levels for samples containing either
the entire rostro-caudal, or only mid-caudal regions of the SN/VTA. However, a trend
towards significance (P=0.056) was observed for pTH levels in samples containing the
entire rostro-caudal extent of the SN/VTA.
Discussion
As far as we are aware, this is the first study to analyze TH protein levels in the
SN/VTA of schizophrenia compared to controls, and only two previous studies have
assessed TH mRNA levels in schizophrenia (Ichinose et al., 1994; Mueller et al., 2004).
Pilot study: TH mRNA and protein expression in human postmortem schizophrenia and
control cases
The most remarkable finding of our first postmortem study (pilot study) was the
fact that in some of the schizophrenia cases (three out of the six cases studied) TH protein
levels were profoundly altered, while in the same brain samples TH mRNA levels were
similar to control cases. It was also remarkable the large variability presented in the
schizophrenia group, with some cases presenting levels in the same range than controls,
67
while others presented very low TH protein levels. The regulation of the transcription and
translation of the TH gene is a highly complex process that is also specific to the type of
TH expressing cell (i.e. dopaminergic versus adrenergic or noradrenergic neurons). Tank
and collaborators have shown that while in adrenergic and noradrenergic cell populations
of the locus coeruleus and adrenal medulla TH is highly regulated at the transcription
level, this type of regulation is minimal in the case of dopaminergic cells of the SN/VTA
(Chen et al., 2008; Tan et al., 2008). In the locus coeruleus and adrenal medulla, most
types of stress can induce short-term and long-term upregulation of TH mRNA
transcription, that is well correlated with upregulation of TH protein levels (Alterio et al.,
2001; Wong and Tank, 2007; Tank et al., 2008). This is also correlated with changes in
transcription factors that are known to regulate the TH gene promoter (Wong and Tank,
2007; Tank et al., 2008). However, in the case of the TH expressing cells of the SN/VTA
it has been shown that induction of the transcription for TH mRNA does not always lead
to an upregulation of TH protein levels (Chen et al., 2008; Tan et al., 2008). The
application of drugs that are known to affect the transcription rate of TH mRNA in the
adrenal medulla and the locus coeruleus (Alterio et al., 2001; Wong and Tank, 2007;
Tank et al., 2008), does not produce any effect in TH mRNA levels in the SN/VTA,
while they produce an upregulation of TH protein levels (Chen et al., 2008; Tan et al.,
2008). In midbrain slice explants containing the SN/VTA, these authors demonstrated
that forskolin and cAMP analogs increase TH protein levels in a dose dependent manner,
while TH mRNA levels are unchanged. These authors proposed that in the midbrain there
is a cAMP-mediated induction of TH protein by a translational mechanism that includes
the formation of binding complexes with Poly(C)-binding proteins, producing the
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stabilization of TH mRNA, and/or increase of its association with polysomes, finally
leading to increased translation (Tank et al., 2008).
The only two previous studies addressing TH mRNA levels in schizophrenia
reported opposite results. One of these studies (Ichinose et al., 1994) showed that, as in
our study, TH mRNA levels in schizophrenia were unchanged compared to controls. A
second study (Mueller et al., 2004) showed significantly elevated levels of TH mRNA in
schizophrenia. Several factors can contribute to this difference in the results obtained,
including differences in the cohort of patients as well as methodological differences.
Animal study: Antipsychotic treatment effect on TH protein levels
In our study, we also addressed the possibility that the observed changes in TH
protein in the SN/VTA of schizophrenia patients could be an effect of antipsychotic drug
treatments. Our results showed that chronic treatment with antipsychotic medication did
not produce significant changes in TH protein expression in the SN/VTA of rodents.
Supporting our findings, a previous study in rodents using a different atypical
antipsychotic (clozapine) has shown similar results for the SN/VTA dopaminergic cells,
with no changes in TH protein levels or TH immunolabeling (Tejedor-Real et al., 2003).
In addition, it has been shown that chronic treatment with haloperidol does not produce
changes in TH axonal density in the prefrontal and entorhinal cortex of monkeys, while
lamina specific changes in TH axonal density are present in schizophrenia patients on
antipsychotic medication (Akil et al., 1999, 2000). Two laboratories have reported
changes in cell morphology and TH immunolabeling in the SN/VTA after chronic
treatment with haloperidol (Levinson et al., 1998; Mazurek et al., 1998; Marchese et al.,
69
2002). However, in these studies drugs were administered in very high doses. Mazurek
and collaborators performed two studies using intraperitoneal injections of haloperidol
once a day in doses ranging from 1 to 10/mg/kg/day (Levinson et al., 1998; Mazurek et
al., 1998). The other group found changes in animals that were subcutaneously
administered haloperidol twice a day at a dose of 1mg/kg (Marchese et al., 2002). The
doses used by these investigators are between 10- and 100-fold the dose necessary to
achieve receptor occupancies equivalent to therapeutic conditions (Kapur et al., 2003),
which can lead to cellular toxicity. Finally, another chronic rodent study using
haloperidol or combinations of haloperidol and quetiapine did not find any morphological
anomalies in the dopaminergic neurons of the SN/VTA (Zhang et al., 2007).
Second postmortem study: Regional expression of TH and phosphorylated TH
Our second postmortem study addressed the regional expression of TH and pTH
protein using two different types of samples: One set of cases contained the entire rostrocaudal extent of the SN/VTA, and another set contained only mid-caudal regions.
Compared to controls, schizophrenia rostro-caudal samples presented significant
differences in TH protein levels, while for pTH there was a trend towards significance.
However, in the case of samples containing only mid-caudal regions there were no
differences detected between schizophrenia and controls for TH or pTH protein levels.
These results are especially interesting because there is a regional segregation on the
projections from the SN/VTA complex to cortical and subcortical regions. The rostral to
mid-regions of the SN/VTA complex contain the rostral part of the dorsal tier of the SNc
and the adjacent VTA. Both of these neuronal groups project preferentially to the
70
prefrontal, entorhinal and piriform cortex, as well the ventral striatum and the amygdala
(Fallon and Moore, 1978a,b; Fallon et al., 1978a,b; Porrino and Goldman-Rakic, 1982;
Gaspar et al., 1992; McRitchie et al., 1995; Haber and Fudge, 1997; Halliday, 2004). The
mid to caudal regions of the SN/VTA complex contain the remainder of the dorsal tier, as
well as the entire ventral tier of the SNc that projects preferentially to the dorsal striatum
(Fallon and Moore, 1978a,b; Fallon et al., 1978b; McRitchie et al., 1995; Haber and
Fudge, 1997; Joel and Weiner, 2000; Halliday, 2004). Our findings indicate that the
tyrosine hydroxylase anomalies are located preferentially (or exclusively) in the rostral
areas of the SN/VTA complex. This finding suggests that TH protein anomalies could be
preferentially located in the mesocortical and mesolimbic pathways. Supporting this,
anomalies in TH immunolabeling have been found in specific layers of the prefrontal and
entorhinal cortex in schizophrenia (Akil et al., 1999, 2000; Lewis and Gonzalez-Burgos,
2006).
Although TH protein levels have not been assessed before, a previous study in a
cohort of Japanese patients addressed TH enzymatic activity in the SN/VTA of
schizophrenia compared to controls (Toru et al., 1988). TH enzymatic activity is linked
with the portion of the TH protein that is active, which in our study we assessed by
measuring the levels of pTH. In brain tissue, it has been shown that TH can be
phosphorylated at several serine residues, including serine 19, 31, and 40, all located in
the first 40 amino acids of the N-terminus (Haycock, 1990). Among these serine
phosphorylation sites, serine 40 appears to be the most relevant for the activation of TH
(Haycock, 1990; Nakashima et al., 2009), and the positively charged amino acids
flanking this serine play an important role in the proper phosphorylation of this site
71
(Nakashima et al., 2009). This serine phosphorylation area is considered a modulatory
region for the activity of the catalytic subunit of the TH protein, since phosphorylation in
this area produces an enhancement of the activity of TH (Haycock, 1990; Nakashima et
al., 2009). Our study of pTH levels in schizophrenia showed a trend toward a significant
difference compared to controls in the samples containing the entire rostro-caudal extent
of the SN/VTA, while no differences where observed for the samples containing midcaudal regions. Toru et al. (1988) in their work assessing TH enzymatic activity reported
a significant increase in TH activity in the SN/VTA of schizophrenia samples. In our
study, pTH levels presented a very heterogeneous profile: For the samples containing the
entire rostro-caudal extent of the SN/VTA, pTH levels were lower or equal than in
control samples, while for the cases containing only mid-caudal regions, pTH levels
ranged from higher than controls to lower (see scatter plot graphs in Figure 3B). The
discrepancy between our results and the previous study by Toru et al. (1988) may be due
to several factors. This includes differences in the area selected for study (e.g. their
dissection included only the substantia nigra, while we included in our study the
substantia nigra and the VTA as a dopaminergic producing complex), differences in the
cohort of patients, and the use of different techniques.
Another interesting finding was that TH protein levels were very variable in
schizophrenia, while they were rather constant in the control group, which suggests that
the schizophrenia group has a large heterogeneity of TH expression anomalies. Based on
previous studies concerning the regulation of TH expression, genetic studies of the TH
gene in schizophrenia, and in our data, we hypothesize that different anomalies in the TH
gene and modulatory elements in the translation of the protein may account for this
72
heterogeneity. A large number of genetic association studies for the TH gene have found
very diverse results. The most consistent association was found for intron 1 of the TH
gene (TH01 locus), an area that has been postulated as a modulatory element for TH
transcription (Pae et al., 2003; Jacewicz et al., 2008). In the TH01 locus, certain alleles of
a DNA microsatellite sequence repeat have been linked with a significant higher risk for
schizophrenia. However, this association was found in some cohorts of patients (Meloni
et al., 1995; Wei et al., 1995; Kurumaji et al., 2001; Pae et al., 2003; Jacewicz et al.,
2008), but not in others (Burgert et al., 1998; Jonsson et al., 1998). In addition, TH
protein synthesis is a highly regulated process, with epigenetic, transcriptional and
posttranscriptional regulations (Lenartowski and Goc, 2011) that vary depending on the
class of catecholaminergic neuron (i.e. dopaminergic versus adrenergic or noradrenergic;
Tank et al., 2008). Our data strongly support that TH anomalies in schizophrenia occur at
the posttranscriptional level, which may include anomalies in the stabilization of the
mRNA, the proper attachment of mRNA to polysomes, and/or the efficacy of translation
of the mRNA into protein.
Conclusions of the study
In summary, our study shows for the first time that an anomaly in TH protein
synthesis is present in the SN/VTA in schizophrenia. Since this pathology is present in
the protein, but not in the mRNA, it strongly suggests that the anomalies occur during
posttranscriptional processes, but this will require further investigation to identify the
exact processes that are affected. Finally, our study provides the first data indicating that
within the SN/VTA, the anomalies in the synthesis of TH may be restricted to specific
73
dopaminergic neuronal populations, mostly located in the rostral regions of this complex.
This suggests that anomalies in TH synthesis may be linked with pathologies in the
mesocortical and/or mesolimbic dopamine pathways, but further studies will be needed to
pinpoint the specific populations affected.
Conflict of interest statement
Dr. Robert R, Conley is currently a Distinguished Lilly Scholar at Eli Lilly and
Co. However, his involvement in this work is independent from his current contractual
relation with Eli Lilly and Co. None of the work reported here has been financially
supported by Eli Lilly and Co., nor has this company had any role in the study design, in
the collection, analysis and interpretation of data, writing of the report and submission of
the paper. All the other authors have no conflict of interest to declare.
Acknowledgements
The authors wish to thank the staff of the Maryland Brain Collection, University
of Maryland School of Medicine for their help to obtain the samples used in this study.
We also wish to thank Dr. Xue-Min Gao for her assistance with the in situ hybridization
experiments. This work was supported by the National Institutes of Health (USA), grant
RO1MH066123 to Miguel Melendez-Ferro, Emma Perez-Costas, and Rosalinda C.
Roberts.
74
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Table 1. Pilot study: demographic table and clinical data in schizophrenia and controls.
Abbreviations: A, atypical antipsychotic; AA, African American; APD, antipsychotic
drug; ASCVD, arteriosclerotic cardiovascular disease; C, Caucasian; COD, cause of
death; DVT, deep vein thrombosis; F, female; M, male; MI, myocardial infarction; NOS,
not otherwise specified; PMI, postmortem interval; SD, standard deviation; SZ subtype,
schizophrenia subtype; T, typical antipsychotic; TD, tardive dyskinesia; un, unknown.
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Table 2. Demographic table and clinical data second postmortem study. Abbreviations:
A, atypical antipsychotic; AA, African American; APD, antipsychotic drug; ASCVD,
arteriosclerotic cardiovascular disease; C, Caucasian; COD, cause of death; CUT, chronic
undifferentiated type; DVT, deep vein thrombosis; F, female; M, male; MI, myocardial
infarction; NOS, not otherwise specified; MVA, motor vehicle accident; PMI,
postmortem interval; SD, standard deviation; SZ subtype, schizophrenia subtype; T,
typical antipsychotic; TD, tardive dyskinesia; un, unknown.
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Figure 1. Pilot study: TH mRNA and protein levels in schizophrenia and control cases.
*P<0.05. (A) TH mRNA levels in control (n=5) and schizophrenia (n=6) cases. The top
panel shows representative images of TH mRNA in situ hybridization in rostral (R),
medial (M), and caudal (C) sections of the SN/VTA of a control case (C2), and a
schizophrenia case (SZ2). Comparison of the mean values for TH mRNA OD is shown in
83
the bar graph (middle panel). Mean optical density (OD) values for TH mRNA levels in
control and schizophrenia (SZ) cases did not differ significantly (P=0.12; OD mean±SD:
control=0.187±0.027, schizophrenia=0.161±0.023). The bottom panel shows a scatter
plot for the individual TH mRNA OD values. The horizontal bar in the scatter plot
indicates the mean OD value for each group. Note the dense clustering of all the OD
values for both groups. (B) TH protein levels in the same control and schizophrenia cases
used for the mRNA study. The top panel shows representative images of the same
western-blot membrane, first blotted for TH protein, and then reblotted for actin. This
membrane contained all the control (C1-C5) and schizophrenia (SZ1-SZ6) cases used in
this pilot study. Note the conspicuous difference in blot signal among the different
samples in the TH western-blot image, while actin expression is uniform among all cases.
The comparison of the means for TH protein levels in controls and schizophrenia is
shown in the mid panel (bar graph). OD values for TH protein were significantly
different (*) between the two groups (P=0.031; OD mean±SD: control= 0.912±0.046,
schizophrenia= 0.433±0.393). The bottom panel shows a scatter plot for the individual
TH protein OD values. The horizontal bar in the scatter plot indicates the mean OD value
for each group. Note the dense clustering of the OD values for the control group, while in
the schizophrenia group there is a large scattering of the data. However, note that in the
lower TH expression cluster, two cases presented the same exact TH OD value and are
represented in the scatter plot as a single circle.
84
Figure 2. Animal study: antipsychotic treatment effect on TH protein levels. (A)
Western-blot images of TH protein for all the animals of the three treatment groups. All
the samples (n=9 per treatment group) were incubated and developed together on the
same film to accurately measure OD. Note the homogeneity of expression of TH for all
cases independently of the treatment group. (B) The same western-blot membrane shown
in A was reblotted for the detection of actin protein. (C) Comparison of the mean OD for
TH protein levels showed no significant differences among the three groups (P=0.41; OD
mean±SD: control=0.824±0.181, haloperidol=0.917±0.049, olanzapine=0.973±0.136).
(D) Scatter plot graph showing the individual TH protein OD values for the 3 groups. The
horizontal bar in the scatter plot indicates the mean OD value for each group.
85
Figure 3. Second postmortem study: regional distribution of TH and pTH. *P<0.05;
#
P>0.05<0.06. (A) The top panel shows images of 2 western-blots for TH that were
developed on the same film, containing all the cases used in this study. Note that rostrocaudal and mid-caudal cases were run together in the same gels. In addition, reblots for
actin of the same membranes are shown immediately below. The graph bar (mid panel)
shows the comparison of the mean OD values for TH protein in rostro-caudal and midcaudal samples for controls and schizophrenia. Only the rostro-caudal samples showed
86
significant differences (*) between schizophrenia and control cases (P=0.036; OD
mean±SD: control rostro-caudal=0.749±0.044, schizophrenia rostro-caudal=0.513±0.252;
control mid-caudal=0.743±0.107, schizophrenia mid-caudal=0.712±0.261). The bottom
panel shows a scatter plot for the individual TH OD data. The horizontal bar in the scatter
plot indicates the mean OD value for each group. (B) The same membranes in A were
reblotted for the detection of pTH (top panel). In addition, reblots for actin of the same
membranes are shown immediately below. The graph bar in the mid panel shows the
comparison of the mean OD values for pTH in rostro-caudal and mid-caudal samples.
Even when there were no significant differences in pTH OD values between controls and
schizophrenia in rostro-caudal samples, a trend (#) towards significance was observed
(P=0.056; OD mean± SD: control rostro-caudal=0.405±0.120, schizophrenia rostrocaudal=0.203±0.208;
control
mid-caudal=0.421±0.168,
schizophrenia
midcaudal=0.419±0.246). For comparison, the dashed line on the top of each of the
individual bars indicates the mean level of total TH for each group. The ratio of pTH
versus total TH for each groups was as follows: control rostro-caudal=54.07%,
schizophrenia rostro-caudal=39.57%; control mid-caudal=56.66%, schizophrenia midcaudal=58.85%. The bottom panel shows a scatter plot for the individual pTH OD data.
The horizontal bar in the scatter plot indicates the mean OD value for each group.
87
AN ACCURATE METHOD FOR THE QUANTIFICATION OF CYTOCHROME C
OXIDASE IN TISSUE SECTIONS
by
Miguel Melendez-Ferro, Matthew W. Rice, Rosalinda C. Roberts, & Emma Perez-Costas
Journal of Neuroscience Methods
Copyright
2013
by
Elsevier
Used with permission
Format adapted and errata corrected for dissertation
88
Abstract
Cytochrome oxidase (COX) is the enzyme that constitutes the last step of the
mitochondrial electron transport chain for the production of ATP. Measurement of COX
activity can be achieved by histochemistry, thus providing information about the
metabolic status of the brain. Brain regions with high metabolism will present high COX
activity in histochemistry assays and vice versa. Using histochemistry versus
biochemistry to assess COX activity presents the advantage of providing a map of the
differences in metabolism in discrete brain regions. Moreover, COX histochemistry
allows quantifying the activity of a particular brain region, by converting units of optical
density into units of activity. In the present work we have devised a methodology that
allows not only quantifying differences in COX activity between groups, but also
quantifying the amount of COX present in brain tissue sections, by directly relating
optical density (OD) measurements to cytochrome C oxidase concentration, something
that traditionally is achieved by the use of western blot. For this purpose we created a set
of standards of known concentration of COX that were affixed to a nitrocellulose
membrane, and this membrane was incubated together with the tissue sections in which
COX activity was assessed. A standard curve was created using a gradient of different
concentrations of purified bovine heart cytochrome oxidase (from 2 micrograms to 0.1
micrograms in intervals of 0.25 micrograms). This standard curve allowed us to detect
changes in optical density as low as 5%, and relate these OD differences with known
concentrations of cytochrome C oxidase.
Keywords: Optical density; histochemistry; human brain; substantia nigra; ATP;
electron transport chain; image analysis
89
Introduction
Metabolism has the ultimate goal of producing the energy necessary to maintain
all the body functions, and this energy is stored in the form of ATP. The brain is one of
the most metabolically demanding organs, requiring high levels of ATP production to
maintain its functions, and different brain regions utilize varying amounts of ATP
depending on their metabolic status (Hevner and Wong-Riley, 1991). Within the same
brain region these differences in metabolic activity can be found at the cellular and
subcellular levels (Hevner and Wong-Riley, 1989; 1991; Wong-Riley, 2012). ATP is
synthesized in mitochondria by oxidative phosphorylation, a process that takes place at
the inner mitochondrial membrane with participation of the electron transport chain. The
electron transport chain is composed of a series of enzymes or complexes, of which
cytochrome C oxidase (COX or Complex IV) is the terminal enzyme. Cytochrome C
oxidase catalyzes the oxidation of its substrate cytochrome C, and the transfer of
electrons for the reduction of molecular oxygen. This ultimately produces the
electrochemical proton gradient that culminates in the production of ATP, by pumping
protons from the matrix to the cytosolic side of the inner mitochondrial membrane (see as
a review Wong-Riley, 2012).
Measurement of the metabolic activity of a particular brain region can be reliably
achieved by measuring COX activity by histochemistry. Metabolically active brain
regions (e.g. with high firing rates) show increased COX histochemistry ratios compared
with other regions in which neuronal activity is lower (Hevner and Wong-Riley, 1989;
1991). The advantage of COX histochemistry is that it can provide an accurate mapping
90
of the differences in metabolism in a certain brain region, something that cannot be
achieved by the biochemical measurement of COX (see Hevner et al., 1993). The
histochemical assay for the detection of COX activity in tissue has been used for decades,
however very variable methods have been applied for the conversion of optical density
measurements to enzyme amount or to enzymatic activity. When standards have been
used, the most commonly reported technique to create these consisted of the preparation
of homogenates from tissue that was known to contain a high concentration of COX (i.e.
heart tissue; Hess and Pope, 1953), or homogenates from tissue of the same brain area to
be studied (Gonzalez-Lima and Garrosa, 1991; Gonzalez-Lima and Jones, 1994). These
homogenates are shaped in blocks and sliced using various methodologies. Then, these
standards are used to directly compare tissue samples, or their OD values are compared to
spectrophotometric measurements of COX activity for the same standards, so they can be
transformed into “units of activity” (Gonzalez-Lima and Cada, 1994; Gonzalez-Lima and
Jones, 1994). However, none of these methods is able to provide exact measurements of
COX expression in the tissue sample, since the concentration of COX in the standards is
unknown.
In the present study we show a technique that allows obtaining optical density
(OD) measurements of COX histochemistry, and directly convert those to measurements
of COX expression in tissue, to then compare COX activity among samples. Our
technique has enough resolution to accurately detect changes in COX as low as 5%
between samples, in the lower end of the scale. In addition, we show how to analyze the
optical data measurements and convert directly to micrograms of COX present in the
tissue using NIH Image J software.
91
Materials and Methods
Brain tissue samples
We chose the human substantia nigra/ventral tegmental area (SN/VTA) to test this
technique due to our long-term interest in studying SN/VTA pathology in
neuropsychiatric and neurological disorders (Perez-Costas et al., 2010, 2012). However,
this technique would be equally useful to assess COX expression and activity in other
brain areas.
Postmortem human SN/VTA tissue samples (University of Alabama at
Birmingham, IRB protocol # N110505002) were obtained from the Alabama Brain
Collection (ABC, University of Alabama at Birmingham). These samples were from a 69
year old Caucasian male without any neuropsychiatric or neurological disorder (i.e.
control case), with a brain pH of 6.10, and a postmortem interval of 15 hours. The
brainstem containing the SN/VTA was dissected immediately upon reception of the brain
and frozen in dry ice and stored in a -800C freezer. The frozen block containing the entire
rostro-caudal extent of the SN/VTA was sectioned coronally in five parallel series on a
cryostat obtaining 14 micron-thick sections. Four of the series were collected on charged
slides, while the fifth series was collected in a vial for protein determination. All the
series were preserved at -800C until use. The first series was stained with thionin (Nissl
stain) to assess proper morphological preservation. Using this series for landmark
determination, several slides containing the SN/VTA from a parallel series were
randomly chosen for COX histochemistry.
92
Preparation of standards
Cytochrome C oxidase from bovine heart (Sigma-Aldrich, St. Louis, MO, catalog
#C5499) was used to create the standards. The concentration of COX in the vial was 5
mg/ml, and this purified COX had 20 units of activity per mg of enzyme.
To prepare the standards it was necessary to determine the highest reasonable
concentration of COX to be expected in brain tissue. This was necessary to create an
array of standards spanning the high and low ends of COX expression in the brain. We
used some assumptions to do this calculation: Since COX is a very abundant protein in
brain tissue we assumed that it could represent at least 1% of the total protein content in
the brain. Then, we used series #5 to calculate total protein concentration in this brain
area, using a spectrophotometer (Bio-Rad SmartSpec Plus, Bio-Rad, Hercules, CA) and
the DC protein assay (Bio-Rad, catalog #500-0113 and #500-0114). Since we knew the
number of sections collected per series, we were able to estimate the total protein
concentration in a single 14 micron-thick section of the SN/VTA, dividing the total
protein concentration of series #5 (13,970µg) by the number of sections contained in a
series (n=127). Our calculations yielded a total protein concentration of 110µg per
section. Using our assumption that COX could represent as much as 1% of the total
protein content in the tissue, we estimated that each SN/VTA section could contain 1.1
µg of COX. Since we wanted to guarantee that none of the COX present in the brain
could be out of range of our standards, we set the highest concentration of our standards
as 2 µg of pure COX. From this highest concentration we created an array of 16 standards
(8 of them ranging from 2µg to 0.25µg in decreasing steps of 0.25µg, and 8 additional
standards ranging from 0.1µg to 0.03µg, in decreasing steps of 0.01µg).
93
Once the concentrations of pure COX to be used as standards were determined,
the next step was to design a methodology that would allow us to reliably obtain optical
density measurements of known concentrations of pure COX. To achieve this objective,
we used a dot-blot apparatus (Bio-Dot SF microfiltration apparatus, Bio-Rad, catalog
#170-6542) to affix the desired concentrations of pure COX to a nitrocellulose
membrane. Table 1 presents the concentrations of COX that were used to create the
standards in the nitrocellulose membranes.
To create the standards, two stock solutions of bovine heart COX were prepared
in Tris-Buffered saline (TBS, Bio-Rad, catalog #170-6435). A 1:500 dilution stock
(0.01µg/µl of COX) was prepared to create the 2.0 to 0.10µg standards. A second dilution
(1:10,000; 0.0005µg/µl of COX) was prepared to create the 0.09 to 0.03µg standards.
From these stock solutions the appropriate volumes were taken to create the standards by
further diluting the stocks in TBS (Table 1).
Following the instructions on the manufacturer’s manual, the dot-blot apparatus
was assembled using the optional slot-blot insert. This allowed applying the samples in
rectangular wells instead of the traditional round-shaped wells. Within the apparatus,
filter paper (Bio-Dot SF, Bio-Rad, catalog #162-0161) and the nitrocellulose membrane
(Bio-Rad, catalog #162-0117) were sandwiched between the top and the bottom parts of
the dot-blot apparatus. After proper assemblage, the system was connected to an in-house
vacuum system using a vacuum trap. The nitrocellulose membrane was rinsed 2 times
using 100µl of TBS per well. For each rinse vacuum was applied to force the TBS
through the membrane. Then, 200µl per well of freshly prepared dilutions of the pure
COX in TBS (table 1) were applied (from most concentrated to most diluted) to two rows
94
of the dot-blot apparatus. The unused wells were filled with 200µl of TBS. To affix the
COX complex to the membrane, vacuum was applied, and immediately after this, 2
consecutive rinses with TBS were applied using the same vacuum method. These steps
allowed the immobilization of COX complex into the nitrocellulose membrane and thus,
obtaining an easy to handle and durable support for the standards. Care was taken to
uniformly load the wells of the dot-blot apparatus to avoid within-well side-to-side
gradients of COX. After this process was completed, the membrane was placed in TBS to
be immediately incubated for COX histochemistry together with the sections to be tested
for COX activity.
Preparation of negative controls
These controls serve the purpose of monitoring the specificity of the COX
reaction by inactivating the activity of the enzyme prior to the histochemical reaction
using sodium azide as the inactivating agent (Seligman et al, 1968). Negative controls
were prepared by randomly selecting some of the SN/VTA sections from the same case
and pretreating them to inactivate the COX enzyme present in the tissue. The control
sections were removed from the -800C freezer and immediately fan-dried for 25 minutes
at room temperature (RT). Sections were immersed in 4% paraformaldehyde in 0.1M
phosphate buffer (PB) pH 7.4 for 60 minutes at RT. After five rinses in PB (5 minute
each), the slides were incubated in a 10mM sodium azide solution in PB for 17 hours at
RT to inactivate COX. After this, sections were rinsed in PB (5 times, 5 minute each) and
they were ready to be incubated together with the standards and the sections to be
assayed for COX activity.
95
COX histochemistry assay
We used a previously published COX histochemistry assay that produces reliable
and consistent results in frozen brain samples (Divac et al., 1995; Poeggeler et al., 1998).
Prior to starting the COX assay, SN/VTA sections were removed from the -800C freezer
and fan-dried as described for the negative controls (see above). After that, these sections,
together with the negative controls and COX standards were placed in the same cuvette
for incubation. The incubation medium contained the following components: 22.4 mg
cytochrome C (Sigma-Aldrich, catalog #C3131), 115.23 mg diaminobenzidine (DAB,
Sigma-Aldrich, catalog #D5637), 4.5 g sucrose (Sigma-Aldrich, catalog #S0389), 12.51
ml of a 1% nickel ammonium sulfate hexahydrate (Aldrich, catalog #A1827) solution, all
mixed in 100 ml of 0.1M Hepes buffer (Sigma-Aldrich, catalog #H3375), pH 7.4. This
solution was prepared in an aluminum foil-covered beaker under constant stirring adding
the cytochrome C, DAB and the sucrose to the Hepes buffer first, and then adding the
nickel ammonium sulfate solution dropwise under constant stirring. The sections and the
standards were incubated with this solution for 2 hours in an incubator at 370C in the
dark. The COX enzymatic reaction was terminated by immersing the sections and
standards in 4% paraformaldehyde in PB for 1 hour at RT. After that, sections and
standards were rinsed in PB (4 times, 5 minute each). Sections were then dehydrated in
ethanol, cleared in xylene and coverslipped with Eukitt (Electron Microscopy Sciences,
catalog #15322). The nitrocellulose membrane containing the standards was rinsed in
distilled water and dried at RT between two sheets of filter paper.
96
Image acquisition and analysis
Images from SN/VTA sections and standards were acquired using a flatbed
scanner at a resolution of 600 dpi and 16 bits grayscale. For optical density analysis all
images were saved as uncompressed TIFF files, and then imported into NIH Image J
software version 1.46r (http://rsbweb.nih.gov/ij/). In Image J, prior to measuring optical
density (OD), background was subtracted for all images first (process>subtract
background), using a “rolling ball radius” of 25 pixels for light background. After this,
images were inverted to negative (edit>invert) in order to obtain OD values.
A standard curve was created by measuring the OD values for the standards in
Image J and then inputting the known values of COX protein concentration in
micrograms (table 1) for each standard. To obtain the OD values for each standard, a
rectangular selection tool was used to define the “region of interest” (ROI). Once all the
OD
values
for
the
standards
were
obtained,
Image
J
calibration
tool
(image>measure>calibrate) was used to input manually the COX microgram values for
each standard versus their OD values. The appropriate type of function (equation) that
relates OD versus COX micrograms was selected (i.e. exponential), and the standard
curve was plotted, bringing the standard curve graph and the goodness of fit of the curve
into view. Once the calibration curve was created, images for analysis were opened in
image J, and OD values for ROIs were measured. Rectangular ROIs were used to select
several areas within the section, and using the measurement tools (analyze>tools>ROI
manager, and then, analyze>measure), OD values were measured and immediately
converted by the software to micrograms of COX. Afterwards, micrograms of COX were
converted manually to COX activity units, using the information provided by the
97
manufacturer regarding the COX activity units present in the COX used for our standards
(i.e. 20 activity units per microgram of COX).
Statistical analysis
Graphpad Prism 5.04 software (Graphpad Inc., San Diego, CA, USA) was used to
perform a regression analysis of optical density values versus COX concentration loaded
for the standards. A paired t-test was also performed for the analysis of the interpolated
values of COX amount obtained with Image J.
Results and Discussion
In the present work we tested a set of 16 different concentrations of pure COX for
the creation of our standards (from 2 µg as the highest value to 0.03 µg as the lowest, see
Table 1). Our tests showed that in the low end of the curve, the limit of resolution for
reliable measurement of COX optical density values was 0.1 µg of COX, since below this
value the measurement of OD levels using Image J software became unreliable for
reproducibility. Thus, a set of 9 different COX concentrations ranging from 2 to 0.1 µg
was the optimal range to build our standard curve to assess COX expression in brain
tissue sections (Figure 1), which was enough to cover the whole range of COX amount
present in our brain tissue sections. Traditionally, COX standards were created using
brain homogenates sectioned at different thicknesses that were incubated in the same
conditions as the brain sections to be analyzed, thus allowing a quantitative analysis of
COX activity (Gonzalez-Lima and Cada, 1994; Gonzalez-Lima and Jones, 1994). Since
sample preparation can significantly influence enzymatic activity of COX (Hevner et al.,
98
1993) the use of pure COX enzyme that is affixed to an inert matrix such as nitrocellulose
(thus requiring a minimal amount of manipulation) can eliminate those issues. However,
in our method it is important to avoid gradients in the distribution of COX within the well
when creating the standards. The uniformity in the amount of COX in the well allows the
use of different voxel sizes for the measurement of COX amount in the standards. In our
case, the use of different voxel size within the same sample gave identical results.
Our COX histochemical experiments show that the concentration of our standards
and their corresponding OD values fit well a linear relation (R2 value=0.929 Figure 2A).
Since the amount of histochemical reaction for COX is mainly dependent on the amount
of enzyme (Hevner and Wong-Riley, 1989), a linear relation is usually predicted for the
histochemical reaction of standards, as it occurs for the spectrophotometric measurement
of COX activity (Hevner et al., 1993). However, with our experimental conditions, and
for the range of COX enzyme contained in our standards, the mathematical analysis of
our results reveals that the predicted relation between the amount of COX in the
standards and their OD values also fits an exponential curve as revealed by the R2 value
(see figures 2B and 3). For the range of our standards, an analysis of the interpolated
values of COX amount obtained from the two curves using Image J revealed no
significant differences (paired t-test assuming equal variances; n=27 values per curve;
P=0.3516, df=26). For the two curves, the interpolated data were normally distributed, as
revealed by the Kolmogorov-Smirnov normality test (KS=0.15, P>0.10). Finally, a point
by point assessment of the interpolated values revealed a high correlation between the
two curves (r=0.9662, P<0.0001). Differences in turnover of individual enzymes may
account for the exponential COX reaction observed for our standards in our experimental
99
conditions. Total COX activity is equal to the product of COX amount and COX turnover
(Hevner and Wong-Riley, 1989). Although a minor role for enzyme turnover has been
postulated (Hevner and Wong-Riley, 1989), this cannot be discarded when we consider a
reaction taking place in a biological material (see the differences between ROI #5 and
ROI #8 in figure 4).
Several agents have been shown to inhibit COX activity. Among these are
chemical fixatives (Chalmers and Edgerton, 1989), as well as sodium azide (Seligman et
al., 1968). In our experiments, we used a combination of both methods to ensure a
complete abolition of COX activity in our controls (see above). Our negative controls
performed this way showed no residual activity of COX in sections of the SN/VTA (not
shown).
The use of COX OD measurements together with standards created using known
amounts of enzyme allows the quantification of the amount of enzyme present in brain
sections (Figure 4). Our methodology allows for the detection of differences as low as
5% in COX concentration, and hence activity, in the lower end of the standard scale. This
is important because subtle differences in COX amount in different subregions can thus
be quantified, since there is a close relation between COX OD measured by
histochemistry in sections, and the amount of enzyme present in those sections (Hevner
and Wong-Riley, 1989). As it can be seen in figure 4, different ROIs within the SN/VTA
show different amounts of COX in micrograms (compare the value of micrograms of
COX for adjacent ROIs # 4 and 5, with ROI #8 located in the cerebellar peduncle), which
indicates the existence of differences in the metabolic status of different subnuclei and
between different regions (Hevner and Wong-Riley, 1989). The fact that these
100
differences can be measured with our methodology allows sampling in brain subregions
to detect differences in COX activity that otherwise would be masked by the use of
biochemical assays for its measurement (Hevner et al., 1993). In addition, with our
methodology the amount of COX present in the tissue is easily converted to units of
activity using the information provided by the manufacturer, and does not require the use
of any biochemical assay.
Our methodology employs metal enhancement to increase the contrast between
gray and white matter in the histochemical reaction, something that is not achieved using
DAB alone (Divac et al., 1995). The use of metal enhancement can negatively affect the
COX reaction by making regions with low amount of COX appear stained qualitatively
equal to regions with high COX activity, due to the saturation of the system (GonzalezLima and Cada, 1994; Gonzalez-Lima and Jones, 1994). Our quantitative analysis shows
that this is not the case for our samples, since different ROIs within the same section
present different amounts of COX OD (compare ROI #5 and ROI #4 with ROI #8; Figure
4), which allows quantifying COX amount and activity in different subregions of the
SN/VTA.
Quantification of proteins is important, since the expression of a particular protein
in the brain can reflect a pathological status in a disease (see e.g. Perez-Costas et al.,
2012). Proteins are usually quantified by their immunological detection in western blot
techniques. However, western blots can mask the subtle differences in protein expression
in a particular neuronal subpopulation in a brain region, and at the same time does not
provide the anatomical distribution of that protein in the tissue. The use of a method that
can give an accurate measurement of protein quantity and activity and at the same time
101
provides a map of the expression of that protein is desirable, especially if regional
differences are expected or observed (Hevner and Wong-Riley, 1993). That said, there is
a series of assumptions that have to be taken into account when quantifying OD with
histochemistry for COX. This type of analysis works with the assumption that the entire
COX present in the tissue is functional, thus the differences observed by histochemistry
are based on amount of enzyme. In the case of a structural deficiency of COX that
impairs the functioning of the complex, histochemistry will underestimate the amount of
COX present in the tissue, and it will only determine the amount of functional COX
present based on its ability to produce a reaction product that is measurable by OD.
Another important parameter to take into account when quantifying enzymatic
activity is the fact that the enzyme has to be active for the reaction to take place. The
method of preservation of the tissue has to ensure that the full activity of the enzyme is
kept. Our samples were all fresh-frozen human postmortem brain samples, not subjected
to chemical fixation. Chemical fixation has been shown to diminish the enzymatic
activity of COX at various degrees and a reduction of up to 10.5% has been reported,
with some neurons being more affected than others (Chalmers and Edgerton, 1989).
Although some authors have described a small decrease of COX activity after chemical
fixation of the sections (Gonzalez-Lima and Jones, 1994), this small reduction in activity
can mask subtle differences in COX activity within and between samples, especially if
differential effects are produced. Fixation is usually utilized to avoid detachment of
sections from slides through the histochemical procedure (Gonzalez-Lima and Jones,
1994). In our case, our samples were all frozen, and no detachment of sections from the
slides occurred. In our protocol fixation was only used as the last step of the
102
histochemical reaction to stop the enzymatic activity of COX. Moreover, freezing of the
samples has been shown not to have an effect in COX activity (Hevner at al., 1993).
A variety of other methods have been used to assess cell metabolism. These
include studying the expression of mRNA levels for specific subunits of the COX
enzyme (Vila et al., 1997; Perier et al., 2000; Bacci et al., 2002; Rolland et al., 2007),
assessing the activity of the enzyme succinate dehydrogenase that participates both in the
electron transport chain and in the tricarboxylic acid cycle (Bertoni-Freddari et al., 2001;
Bubber et al., 2011), the measurement of 2-deoxyglucose levels (see e.g. Porrino et al.,
1987; Mitchell et al., 1989, 1992), and the analysis of c-fos expression by
immunohistochemistry (see e.g. Joh and Weiser, 1993; Schulte et al., 2006; Reyes and
Mitrofanis, 2008). Among these methods, the measurement of COX subunit I mRNA
levels has been shown to be a very reliable marker that correlates well with cytoplasmic
COX activity, although it requires the use of radioactive mRNA probes and dedicated
laboratory space. Succinate dehydrogenase assays provide a good measurement of
general metabolic status, but do not assess specifically the health of the electron transport
chain. On the other hand, 2-deoxyglucose measurements have been used reliably to
measure short term changes in metabolism, but COX measurements are more reliable for
the assessment of long term metabolic changes (Vila et al., 1997). Finally, c-fos
immunohistochemistry has been used to assess neuronal activity. However, the use of cfos as a metabolic marker presents several problems (Dragunow and Faull, 1989), and is
only reliable for the study of acute metabolic changes (Hoffman et al., 1993; Hoffman
and Lyo, 2002).
103
Conclusions
Here we have devised a method to accurately measure the amount of COX
enzyme present in brain tissue sections, using standards of known COX concentration.
The amount of COX can be easily converted into activity units without the need of
complex biochemical assays. Our methodology can detect differences as low as 5% in
COX amount. This is useful to analyze and compare the relative abundance of COX
between different regions within a brain, or between subregions within a brain region.
Acknowledgments
The authors wish to thank the Alabama Brain Collection (University of Alabama
at Birmingham) for providing the human brain tissue used in this work. This work was
supported by NIMH RO1 grant MH066123 to MMF, EPC and RCR.
104
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Table 1. Final concentrations of pure COX in standards and detailed chart for their
preparation.
108
Figure 1. Preparation of standards and Image J. After opening Image J (A), the scanned
image of the nitrocellulose membrane containing the COX standards is imported into the
software, and background is subtracted. B) Scanned image of the nitrocellulose
membrane after background subtraction. The numbers indicate the amount of pure COX
loaded for each standard, in micrograms. C) Image of the standards after it has been
inverted in Image J, ready to be sampled. Note that bands of 0.09 to 0.03 micrograms of
COX are also visible, but they are not reliably detected by the software. D) The ROI
manager records the location of each measurement within the image, thus allowing
having all the ROIs visible in the image.
109
Figure 2. Relation between micrograms of COX, optical density and units of activity. In
our experimental conditions and for the values of the standards, the analysis of the best fit
equation for the relation between micrograms of COX loaded in the standards and OD
values (as a surrogate of the reaction product) showed that both a linear (A) and an
exponential (B) curve provided a good fit for the data, as shown by the R2 values. For
both graphs, the equivalence of COX micrograms to COX activity units is shown in the
right Y axis.
110
Figure 3. Creation of a standard curve in Image J. A) OD measurements are obtained for
each standard (see box). B) Using the calibration tools of Image J, the appropriate
function (equation) is selected (i.e. exponential). The OD values for each standard
obtained in A are inputted in the table together with the amount of COX that was loaded
for each standard, in micrograms. C) The curve is then plotted and ready to start
measuring COX activity in the brain tissue samples.
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Figure 4. Measurement of COX activity in brain sections. A) A digitized image of COX
histochemistry in a section through the human brainstem containing the SN/VTA and the
red nucleus (RN) is opened in Image J. This image has already been processed for
background subtraction and inverted. A total of eight ROIs were randomly selected, 1-5
in the SN/VTA, 6 and 7 in the RN, and 8 in the cerebellar peduncle (CP), a tissue area
that presented little to no COX activity. B) The ROI manager records the location of each
ROI selected for measurement. For informative purposes we have noted the number of
the ROI next to each location. C) The results of the measurements of OD in the 8 ROIs
are converted directly to micrograms of COX (boxed area). Note the big difference in
COX amount in micrograms between ROI #5 and ROI #8, calculated by Image J. This
can also be inferred qualitatively by the difference in “brightness” between those two
areas. Other data in the results include the area of the ROIs, maximum and minimum
pixel intensity, and integrated density (IntDen), which is the result of multiplying the area
by the mean intensity. D: dorsal; L: lateral; M: medial; V: ventral.
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ASSESSMENT OF CYTOCHROME C OXIDASE DYSFUNCTION IN THE
SUBSTANTIA NIGRA/ VENTRAL TEGMENTAL AREA IN SCHIZOPHRENIA
by
Matthew W. Rice, Kristen L. Smith, Rosalinda C. Roberts, Emma Perez-Costas, &
Miguel Melendez-Ferro
PLOS ONE
Copyright
2014
by
PLOS ONE
Used with permission
Format adapted and errata corrected for dissertation
113
Abstract
Perturbations in metabolism are a well-documented but complex facet of
schizophrenia pathology. Optimal cellular performance requires the proper functioning of
the electron transport chain, which is constituted by four enzymes located within the
inner membrane of mitochondria. These enzymes create a proton gradient that is used to
power the enzyme ATP synthase, producing ATP, which is crucial for the maintenance of
cellular functioning. Anomalies in a single enzyme of the electron transport chain are
sufficient to cause disruption of cellular metabolism. The last of these complexes is the
cytochrome c oxidase (COX) enzyme, which is composed of thirteen different subunits.
COX is a major site for oxidative phosphorylation, and anomalies in this enzyme are one
of the most frequent causes of mitochondrial pathology. The objective of the present
report was to assess if metabolic anomalies linked to COX dysfunction may contribute to
substantia nigra/ventral tegmental area (SN/VTA) pathology in schizophrenia. We tested
COX activity in postmortem SN/VTA from schizophrenia and non-psychiatric controls.
We also tested the protein expression of key subunits for the assembly and activity of the
enzyme, and the effect of antipsychotic medication on subunit expression. COX activity
was not significantly different between schizophrenia and non-psychiatric controls.
However, we found significant decreases in the expression of subunits II and IV-I of
COX in schizophrenia. Interestingly, these decreases were observed in samples
containing the entire rostro-caudal extent of the SN/VTA, while no significant differences
were observed for samples containing only mid-caudal regions of the SN/VTA. Finally,
rats chronically treated with antipsychotic drugs did not show significant changes in COX
subunit expression. These findings suggest that COX subunit expression may be
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compromised in specific sub-regions of the SN/VTA (i.e. rostral regions), which may
lead to a faulty assembly of the enzyme and a greater vulnerability to metabolic insult.
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Introduction
Schizophrenia is a devastating mental illness that affects approximately 1% of the
world population [1]. Currently, most studies on schizophrenia concentrate on the study
of pathologies of either neuronal circuitry or molecular mechanisms at the cellular and
subcellular levels. This includes the assessment of cellular metabolism and mitochondrial
function.
One of the first studies that implicated mitochondrial dysfunction in the pathology
of schizophrenia was performed by Takahashi and Ogushi [2], which revealed reduced
aerobic glycolysis in schizophrenia post-mortem brain tissue. Since then, perturbations in
metabolism have become a well-documented, if complex, facet of schizophrenia
pathology. This is also supported by studies showing changes in mitochondrial density
and increased mitochondrial morphological anomalies in several brain regions, including
the prefrontal and limbic cortex, the striatum and the substantia nigra [3-8].
Some of the most thoroughly studied metabolic anomalies in schizophrenia are
related to disruptions in oxidative phosphorylation. The brain is a high energy-demanding
organ, which obtains the majority of its energy from oxidative phosphorylation [9-11]
and disruptions of this pathway could account for some of the metabolic anomalies
observed in schizophrenia. As an example, decreased concentrations of ATP have been
observed in the frontal lobe of schizophrenia subjects [12], which is indicative of a deficit
in oxidative phosphorylation. The synthesis of ATP requires proper functioning of the
electron transport chain (ETC), which consists of a series of four enzyme complexes
located within the inner membrane of the mitochondria [13,14]. These enzymes transfer
electrons between electron donors and acceptors, establishing a proton gradient that is
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ultimately used to power the enzyme ATP synthase [15-18]. Adequate production of ATP
is crucial for neuronal plasticity, intracellular signaling, calcium buffering, and
neurotransmission [19-23]. Anomalies in any single individual complex of the ETC can
be sufficient to cause a disruption in cellular metabolism [24, 25]. However, the activity
of a given complex is not contingent on the proper functioning of the other complexes
[24, 26].
In schizophrenia, anomalies have been reported in individual components of the
ETC, including complexes I, III, and IV [14, 27-34]. Complex IV or cytochrome c
oxidase (COX) is the terminal enzyme of the ETC, and its role is to catalyze the
oxidation of cytochrome c, transferring electrons to molecular oxygen in order to produce
a molecule of H2O [35-37]. COX has been recognized as a major regulation site for
oxidative phosphorylation, and anomalies in this enzyme are some of the most frequent
causes of mitochondrial pathology [38-40]. X-ray crystallography has shown that COX is
composed of 13 subunits, three of which (COX I, II, and III) are encoded by the
mitochondrial genome, with the remaining subunits (COX IV, Va, Vb, VIa, VIb, VIc,
VIIa, VIIb, VIIc, and VIII) encoded by the nuclear DNA [41-42]. Mitochondrial DNAencoded subunits are commonly known as the “catalytic core” of the enzyme due to their
role in electron transfer and proton expulsion [43]. Mutations within subunits of the
catalytic core result in disruptions of both structure and function of the entire COX
enzyme [40, 44-47]. Among the nuclear DNA-encoded subunits, COX subunit IV is
especially important for the assembly and proper functioning of the enzyme [37]. COX
subunit IV is the largest of the nuclear DNA-encoded subunits and may be involved in
the modulation of electron transfer between components of the catalytic core [36-37].
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Suppression of subunit IV has been linked to a reduced function in overall COX activity
and an increased susceptibility to apoptosis [36-37].
In schizophrenia COX activity is differently affected depending on the region of
the brain studied. Reduced COX activity has been reported in the frontal and temporal
cortex, as well as in the caudate nucleus [27, 30], while an increase in activity was
observed in the putamen [31], and no changes in activity have been reported in brain
tissue from the nucleus accumbens, globus pallidus, thalamus, cerebellum, or
mesencephalon [27, 31]. On the other hand, Whatley et al. [48] did not find changes in
COX activity within the frontal cortex, adding to the complexity of the pathology of
COX within the schizophrenia brain. Although some studies have reported increases [26,
31] and decreases [49] in COX activity due to the use of antipsychotic medication, the
majority of studies have concluded that COX activity is not affected by antipsychotic
drugs [50-54]. Taken together, these findings suggest that COX anomalies are likely an
intrinsic feature of schizophrenia.
The substantia nigra/ventral tegmental area complex (SN/VTA) contains the
largest group of dopaminergic neurons in the brain, and is the origin for several major
dopamine pathways [55-56]. Dopaminergic anomalies are a well-known component of
schizophrenia pathology [57], and previous findings from our laboratory have shown
significant reductions in tyrosine hydroxylase (TH) protein levels within rostral regions
of the SN/VTA in schizophrenia [58].
The aim of this study was to assess if metabolic anomalies linked to COX
dysfunction might contribute to SN/VTA pathology in schizophrenia. To accomplish this
we tested for differences in COX activity within the SN/VTA in human postmortem
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samples from schizophrenia subjects compared to non-psychiatric controls. In addition,
we tested the hypothesis that alterations in the subunit composition of COX could
contribute to schizophrenia pathology in the SN/VTA. To test this we analyzed protein
expression for all the COX subunits that constitute the catalytic core, as well as for
subunit IV, which is critical for the assembly and function of the enzyme.
Materials and Methods
Ethics statement
Human postmortem brain samples used in this study were obtained from the
Maryland Brain Collection (Maryland Psychiatric Research Center, University of
Maryland School of Medicine) with permission from the Maryland Brain Collection
Steering Committee. The Maryland Brain Collection obtains written consent from the
next-of-kin for the use in research of all human brain samples collected. This brain
collection has full approval from the Institutional Review Board (IRB) at the University
of Maryland School of Medicine (DHMH protocol # 07-23). In addition, all postmortem
human brain experimental procedures were conducted following a protocol approved by
the University of Alabama at Birmingham Institutional Review Board (protocol #
N110505002), and in accordance with the principles expressed in the Declaration of
Helsinki.
No animals were handled, treated or sacrificed for this study. However, brain
protein extracts from adult rats from a stock maintained in our laboratory were used. In
strict accordance with the National Institutes of Health Policy on Humane Care and Use
of Laboratory Animals, the samples used in this study are considered cadaveric tissues,
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and were not prepared or collected specifically for this study. This protein stock was the
remnant from a previously published study [58] performed by some of the authors of the
present work at the University of Maryland School of Medicine, and on that previous
study, all research was conducted following an approved IACUC protocol (IACUC
protocol # 0705010). All animal housing, care, and experimental procedures were done in
strict accordance with National Institutes of Health Guidelines regarding the care and use
of animals for experimental procedures.
Postmortem human brain samples
All human brain samples were obtained from the Maryland Brain Collection
(Maryland Psychiatric Research Center, University of Maryland School of Medicine).
For all postmortem human cases, two independent psychiatrists established DSM-IV
diagnoses based on the review of available medical and autopsy records, and data
obtained from the Structured Clinical Interview for DSM-IIIR [59] and DSM-IV Axis I
disorders with the next-of-kin. Table 1 contains demographic data, as well as information
on antipsychotic use, cause of death, and other relevant parameters for the samples used
in this study.
Human brain samples were dissected in two different ways: 1) samples that
contained the entire rostro-caudal extent of the SN/VTA (n=15; Table 1) or 2) samples
that contained only the mid-caudal regions of the SN/VTA (n=14; Table 1). Cases from
these two different dissections were analyzed separately due to the heterogeneous nature
of the subnuclei within the SN/VTA. All samples were frozen in dry ice and stored at
-80°C prior to sectioning. Using a cryostat, five parallel (i.e. adjacent) series of 16 µm
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thick sections were obtained in the coronal plane following the longitudinal axis of the
brainstem. Sample blocks were trimmed to reduce the presence of cell groups not related
to our study. All samples were sectioned in their entirety. Four series were collected on
charged slides, while the fifth series was collected in a vial for protein extraction. Series
#1 was stained with thionin to assess proper morphological preservation, and only those
cases that presented good preservation of the SN/VTA were included in the study.
Exclusion criteria included the presence of extensive autolysis of cells, cell shrinkage and
poor morphological preservation. These criteria were equally applied to select the
schizophrenia and control cases included in the study. Rostral and caudal boundaries for
samples containing the entire extent of the SN/VTA were determined using the Paxinos
and Huang [60] atlas of the human brainstem. Mid-Caudal SN/VTA samples were
defined as those transversally dissected caudal to the transition between the red nucleus
parvocellularis and magnocellularis.
Animal brain protein samples
A stock of protein extracts maintained in the laboratory, which was obtained from
rats previously treated with antipsychotics [58], was used to assess the effects of
antipsychotic medication on COX subunit protein expression. Treatment procedures for
these animals have been previously described in detail in [58]. Briefly, animals were
randomly assigned to one of three treatment groups (n=9 per group) consisting of
haloperidol (1.5mg/kg/day), olanzapine (6mg/kg/day), or control (i.e. water).
Antipsychotics were administered for three weeks in drinking water, adjusting the doses
to body weight and daily water consumption as described in Perez-Costas et al. [61].
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Animals were euthanized and brains were immediately removed from the skull, frozen in
dry ice, and stored at -80°C. Brains were sectioned on a cryostat at -20°C in the coronal
plane, and 16m thick sections through the SN/VTA were collected in five parallel
series. Sample blocks were trimmed to reduce the presence of cell groups not related to
our study. Four series were collected on charged slides, while the fifth series was
collected in a vial for protein extraction. Series #1 was stained with thionin to assess
morphology and general quality of the tissue collected. No morphological anomalies
were found in any of the samples used in this study.
Cytochrome c oxidase histochemistry in human SN/VTA
Slides containing human SN/VTA sections were removed from -80°C storage and
fan-dried for 25 minutes at room temperature. Sections were then incubated in the dark
for two hours at 37°C in the COX incubation medium, which consisted in a solution of
22.4 mg cytochrome c (Sigma-Aldrich, St. Louis, MO, USA; C3131), 115.23 mg
diaminobenzidine (DAB, Sigma-Aldrich, D5637), 4.5 g sucrose (Sigma-Aldrich, S0389),
12.51 ml of a 1% nickel ammonium sulfate solution (Sigma-Aldrich, A1827), all mixed
in 100 ml of 0.1M Hepes buffer (Sigma-Aldrich, H3375) pH 7.4 [62, 63]. To terminate
the COX enzymatic reaction, sections were immersed in 4% paraformaldehyde in 0.1M
phosphate buffer (PB) pH 7.4 for one hour at room temperature. Sections were then
rinsed in PB, dehydrated in ethanol, cleared in xylene, and coverslipped using Eukitt
(Electron Microscopy Science, Hatfield, PA, USA; 15322). Negative controls were done
in adjacent tissue sections by prior immersion in a solution of 4% paraformaldehyde in
0.1M PB pH 7.4 for one hour at room temperature, followed by rinses in PB, and then
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immersion in a solution of 10mM sodium azide in PB for 17 hours at room temperature.
The aim of the negative controls is to subtract from the analysis the presence of any
background not specifically due to COX activity (e.g. possible random precipitation of
DAB or nickel ammonium sulfate). Thus, the prior treatment with paraformaldehyde and
sodium azide was used to irreversibly inactivate COX in the negative control sections.
After inactivating the COX enzyme in the negative control, these sections were incubated
simultaneously with the samples in the COX incubation medium.
In order to more accurately assess the differences in COX activity among our
groups, we applied a novel methodology developed in our laboratory [64]. Briefly,
cytochrome c oxidase from bovine heart (Sigma-Aldrich, C5499) was diluted in TrisBuffered-Saline (TBS) to create a series of standards that contained a known quantity of
pure COX. Each of these known standards was loaded onto a nitrocellulose membrane,
using vacuum and a slot-blot microfiltration apparatus (Bio-Rad, Hercules, CA, USA;
170-6542). For all COX histochemical assays, negative controls and COX standards were
incubated together with sections to be analyzed for COX activity.
Protein extracts for COX subunit analysis in human and rat SN/VTA
Human and rat brain protein extracts were obtained by sonication of tissue in a
cold lysis buffer mixture (1:5 w:v) containing 50mM Tris pH 8.0, 5mM EDTA, 150mM
NaCl, 1% SDS, and 10l/ml of a protease inhibitor cocktail (Sigma-Aldrich; P8340).
Homogenates were then centrifuged at 15000g for 15 minutes at 4°C, the supernatant was
collected, and total protein concentration was measured using a modified Lowry
technique (Bio-Rad, DC Protein Assay; 500-0113 and 500-0114). Known concentrations
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of bovine serum albumin were used as standards. Aliquots of both rat and human
SN/VTA protein extracts were stored at -80°C until use.
Gel electrophoresis and western-blot
Western-blot assays were used to test for differences in COX subunit protein
expression in both animal and human SN/VTA samples. For gel electrophoresis, samples
were diluted 1:1 in loading buffer (62.5mM Tris-HCl pH 6.8, 25% glycerol, 2% SDS,
0.01% bromophenol blue, and 5% β-mercaptoethanol) and heated to 95°C in a dry bath
for 5 minutes before loading on a 4-20% polyacrylamide gradient gel (Lonza, Basel,
Switzerland; 58505). For rat samples, 25g of SN/VTA total protein extract were loaded
in each lane, while for human samples 60g of SN/VTA total protein extract were
loaded. In addition, a molecular weight marker (Lonza; 50550) was loaded for molecular
weight reference. Proteins were resolved using a constant 150V current and transferred
onto polyvinylidene fluoride (PVDF) membranes (Bio-Rad; 162-0714) under a constant
current of 30V for 21 hours at 4°C.
For western-blot assays, membranes were initially rinsed in 0.05M TBS, followed
by blocking in a solution of 5% non-fat dry milk (Bio-Rad; 170-6404) in TBS containing
0.1% Tween 20 (TBS-T) for one hour at room temperature. Membranes were then
incubated with the appropriate primary antibody at the concentrations listed below, in a
solution of 1% non-fat dry milk in TBS-T for 19 hours at 4°C. Following that,
membranes were rinsed in a solution of 1% non-fat dry milk in TBS-T prior to incubation
with the appropriate secondary antibody at the concentrations listed below, in a solution
of 1% non-fat dry milk in TBS-T for one hour at room temperature. After incubation with
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the secondary antibody, membranes were rinsed first with TBS-T and then with TBS.
Membranes were developed using an Immun-Star alkaline phosphatase substrate
chemiluminescence kit (Bio-Rad; 170-5018), exposing Kodak Biomax XAR films
(Kodak, Rochester, NY, USA; 166-0760).
For each subunit tested a minimum of two separate western-blot experiments
were carried out. For human SN/VTA, rostro-caudal and mid-caudal samples were run in
the same experiment, which required the simultaneous incubation and development of 3
western-blot membranes per experiment. Three western-blot membranes were also
necessary to accommodate all rat samples in a single experiment.
Antibodies
The following primary antibodies were used for western-blot assays: mouse
monoclonal anti-COX subunit I (MitoSciences, Eugene, OR, USA; ab14705, diluted
1:1000 for rat samples), rabbit polyclonal anti-COX subunit I (Abcam, Cambridge,
England; ab90668, diluted 1:1000 for human samples), mouse monoclonal anti-COX
subunit II (MitoSciences; ab110258, diluted 1:1000 for human samples), goat polyclonal
anti-COX subunit II (Santa Cruz Biotechnology, Dallas, TX, USA; sc-23984, diluted
1:1000 for rat samples), rabbit polyclonal anti-COX subunit III (Mitosciences; ab138956,
diluted 1:2000 for rat samples and 1:1000 for human samples), rabbit polyclonal antiCOX subunit IV-I (Sigma-Aldrich; HPA002485, diluted 1:2000 for rat and human
samples), rabbit polyclonal anti-COX subunit IV-II (Sigma-Aldrich; SAB4503384,
diluted 1:1000 for rat samples), and rabbit polyclonal anti-COX subunit IV-II (SigmaAldrich, HPA029307, diluted 1:2000 for human samples). For all western-blot
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experiments membranes were reblotted for actin using a mouse monoclonal anti-actin
antibody (Millipore, Billerica, MA, USA; MAB1501, diluted 1:40,000 for both rat and
human samples). All primary antibodies used in the study were previously tested for
specificity by the manufacturers and produced consistent bands at the expected molecular
weight.
Secondary antibodies used included alkaline phosphatase-conjugated goat antimouse (Millipore; AP124A, diluted 1:15,000), alkaline phosphatase-conjugated goat antirabbit (Vector Laboratories, Burlingame, CA, USA; AP-1000, diluted 1:1000), and
alkaline phosphatase-conjugated donkey anti-goat (Millipore, AP180A, diluted
1:15,000).
Image acquisition and analysis
Complete series of slides containing SN/VTA sections that were assessed using
COX histochemistry, membranes containing COX standards, and western-blot films,
were digitized using a flatbed scanner (Epson Perfection V750-M Pro; Seiko Epson
Suwa, Japan), and saved at a resolution of 600 dpi as uncompressed TIFF files. Images of
sections assessed by COX histochemistry were acquired as 16 bit grayscale, while images
of western blot films were obtained as 8 bit grayscale. NIH Image J software (version
1.46r; http://rsbweb.nih.gov/ij/) was used to measure optical density (OD) for all
experiments.
For COX activity measurements, masks defining the region of interest were
created by delineating the SN/VTA on the corresponding scanned Nissl-stained section
within each case. A standard curve was created using OD values from the COX standards
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[64]. In addition, as an internal control for the validity of the assay, COX activity was
also measured in the red nucleus within the same section, since no studies have reported
changes in COX activity for the red nucleus in schizophrenia. Using a random number
generator (random.org), twelve slides (for rostro-caudal cases) or eight slides (for midcaudal cases) representing different sub-regions of the SN/VTA were randomly chosen
for analysis. For each section measured, using Image J rectangle tool, five nonoverlapping boxes of 0.19 x 0.19 cm were randomly placed within the mask delineating
the region of interest (i.e. SN/VTA or red nucleus).
For western-blot analysis of human and rat samples, an OD standard curve was
created using a step calibration tablet of known calibrated OD (Stouffer Industries Inc.,
Mishawaka, IN, USA; T2120, series #130501). After calibration, OD measurements were
obtained using Image J rectangle tool to delineate each band.
In all cases, measurements of digitized images were performed by two different
researchers blind to diagnosis or treatment group. Each researcher performed three
measurements for each sample assessed. Measures obtained from the same experiment
were averaged for analysis.
Statistical analysis
Schizophrenia and non-psychiatric control cases were matched for demographic
variables (age, gender, race), and sample variables (postmortem interval, brain pH). We
used multiple regression to assess the possible influence of demographic and sample
variables on our outcome measures (i.e. COX activity and subunits expression)
independent of disease status. Demographic variables (i.e. age, gender, race) and sample
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variables (i.e. postmortem interval and pH) where tested in separated multiple regression
analyses. For the analysis of demographic variables a dummy coding was used to
numerically interpret gender and race. For protein measures, actin values were
standardized by dividing the observed actin value of each case by the average actin value
of each individual film. COX subunit protein measures were then normalized over actin
by dividing the observed “raw” calibrated OD value of each measure by its respective
standardized actin calibrated OD value.
Human postmortem samples containing the entire rostro-caudal extent of the
SN/VTA and those containing only mid-caudal regions were analyzed separately. In both
cases, outcome measures obtained from schizophrenia and non-psychiatric control
samples were assessed using unpaired Student’s t-test or Mann-Whitney (non-parametric)
tests after assessing normality and the possible lognormal distribution of the data. Since
COX activity has been previously shown to best fit an exponential curve [64], all COX
activity data were transformed to their common logarithm prior to analysis.
For COX subunit expression, the data were assessed using the following scheme:
Data sets were assessed for normality, and when samples presented significant deviations
from normality, the data were transformed to their common logarithms in order to test if
they were a good fit for a lognormal distribution. If the lack of normality persisted after
logarithmic transformation, the original (untransformed) data were tested for the presence
of outliers and used for analysis. One-way ANOVA was used to assess the outcome
measures on protein samples from animals treated with antipsychotic medications.
Assumptions of the parametric tests were addressed in all analyses, including normal
distribution of the residuals in the dependent variable, homogeneity of variance among
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the groups, and independence of errors. The presence of outliers was determined using
the “Robust regression and OUtlier Removal” (ROUT) test for outlier detection [65],
with the coefficient Q set to 1%. When detected, outliers were removed from the analysis
and graphical representations, but are still present in the western-blot images. ShapiroWilk and Kolmogorov-Smirnov normality tests were used to assess normality in all data
sets. Significant deviations from normality, and the presence of outliers, when present,
are reported in the results section.
The experimental design was done considering each subunit outcome independent
(i.e. not corrected for multiple comparisons). It has been shown that specific subunits of
the COX enzyme can be independently regulated and targeted by specific
pharmacological treatments [70-74]. For example, mutations in subunit I [74] as well as
specific decreases in the expression of subunit II [72] or subunit III [73] affect the
activity of the complex without altering significantly the expression of the other subunits
of the complex [70-74]. On the other hand, it has been shown that multiple comparison
corrections reduce the risk of type I errors, but greatly increase the risk of type II errors,
that is, not detecting relevant differences due to the reduction of power produced by the
correction [66, 67]. However, due to the lack of agreement in the field of statistics on the
use (or not) of corrections for multiple comparisons [67] data were also analyzed using
multiple t-test correction (Holm-Sidak correction with alpha 0.05). Thus, results using
independent t-test (i.e. uncorrected) as well as corrected for multiple comparisons, are
both reported (see results). For the multiple corrections approach, the COX subunits that
form the catalytic core (i.e. subunits I, II, III) were analyzed together. Subunits IV-I and
IV-II were also analyzed using this correction. Subunits I, II and III constitute the
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catalytic core of the enzyme, are encoded by the mitochondrial genome, and their
transcription and translation occur within the mitochondria, while subunits IV-I and IV-II
are encoded by the nuclear genome and their synthesis occurs in the cytoplasm [43, 6869]. Thus, mitochondrial and nuclear subunits were analyzed separately.
Mean and standard deviation data are reported in the figure legends. Statistical
analysis and graphical representation of the results obtained were done using GraphPad
Prism 6.0 software (La Jolla, CA, USA).
Results and Discussion
Anomalies in metabolism are a well-established aspect of schizophrenia
pathology. Reported anomalies include decreased overall metabolic activity in the cortex,
changes in mitochondrial density within the caudate nucleus and putamen, and reductions
in ATP concentration within the frontal lobe [3-7, 12]. Despite the potential role of
metabolic anomalies in the pathology of the dopamine system in schizophrenia, few
studies have addressed the metabolic status of the SN/VTA in this disorder. Interestingly,
there have been reports of mitochondrial hyperplasia within presynaptic neurons that
synapse onto dopamine neurons in the substantia nigra compacta [8]. In the present study
we assessed the metabolic health of the SN/VTA complex by quantifying COX activity,
and by measuring protein expression for key subunits of the COX enzyme. Activity
measures provided a “snapshot” of the functional status of the SN/VTA, while assessing
the protein expression of key subunits of the COX enzyme allowed us to infer
information about the subunit composition of the enzyme in the SN/VTA in
schizophrenia.
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Demographic and sample features
Postmortem human samples selected for this study were carefully matched
between schizophrenia and non-psychiatric control groups for 1) demographic (age,
gender, race) and 2) sample variables (brain pH and PMI) (see Table 1). In addition,
multiple regression was used to assess if 1) demographic or 2) sample variables, had a
significant contribution to our dependent measures. The effect of these demographic and
sample variables was tested independently for COX activity, as well as for the expression
of each COX subunit assessed in our study (i.e. COX subunits I, II, III, IV-1, IV-II). In all
cases, multiple regression analysis showed that these variables did not have significant
predictive value. Table 2 summarizes the results obtained from the multiple regression
tests performed independently for the rostro-caudal and mid-caudal SN/VTA cases, and
reports the exact p value and adjusted R2 for each independently tested outcome measure
(see Table 2). In addition we did not find any significant colinearity among the
demographic or sample variables.
Cytochrome c oxidase activity in schizophrenia versus non-psychiatric controls
COX activity was measured independently for samples containing the entire
rostro-caudal extent of the SN/VTA and those containing only mid-caudal regions. For
both types of dissections COX activity was also measured for the red nucleus for each
case as an internal control for the validity of the assay (see methods). As stated in
methods, COX activity has been shown to best fit an exponential curve [64], thus, all OD
data for COX activity were transformed to their common logarithm [y=-1*log (y)], prior
to analysis. COX activity is shown in all graphs as the logarithm of OD units, and the
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mean value reflects the geometrical mean of the lognormal distribution. After logarithmic
transformation, all COX activity data were normally distributed (Shapiro-Wilk and
Kolmogorov-Smirnov tests in all cases p>0.1), which further supports the exponential
curve fit of COX activity.
COX activity in Rostro-Caudal samples
COX activity measurements for the entire rostro-caudal extent of the SN/VTA
were assessed using unpaired t-test to compare the geometric means of the schizophrenia
and control groups. This analysis yielded non-significant differences in COX activity
between schizophrenia and controls for the rostro-caudal SN/VTA (p=0.6001, t=0.5373,
df=13). The geometric mean for schizophrenia group was 1.389 (95% CI: 1.212, 1.592)
and for the control group was 1.328 (95% CI: 1.130, 1.561) [Figure 1]. For the red
nucleus, unpaired t-test revealed non-significant differences for COX activity (p=0.2011,
t=1.347, df=13). The geometric mean for the red nucleus for the schizophrenia group was
1.513 (95% CI: 1.426, 1.605) and for the control group was 1.409 (95% CI: 1.255, 1.581)
[Figure 1].
In addition, analysis of COX activity just for the rostral region of the SN/VTA in
these same cases also yielded non-significant differences in activity (unpaired t-test
p=0.5816, t=0.5652, df=13). For this sub-analysis of the rostral region, COX activity was
measured at 4 different rostro-caudal levels within the rostral region. The geometric mean
for COX activity in the rostral SN/VTA for the schizophrenia group was 1.435 (95% CI:
1.259, 1.635) and for the control group was 1.340 (95% CI: 1.060, 1.695).
132
COX activity in Mid-Caudal samples
In samples containing only mid-caudal regions of the SN/VTA unpaired t-test
revealed non-significant differences between schizophrenia and control cases (p=0.8737,
t= 0.1624, df= 11) [Figure 2]. The geometric mean for the schizophrenia group was 1.083
(95% CI: 0.9102, 1.290) and for the control group was 1.076 (95% CI: 0.9021, 1.285).
For the red nucleus, unpaired t-test revealed non-significant differences for COX activity
(p=0.5145, t=0.7011, df=5). The geometric mean for the red nucleus for the
schizophrenia group was 1.252 (95% CI: 0.8047, 1.947) and for the control group was
1.127 (95% CI: 0.8010, 1.585) [Figure 2].
Anomalies of COX activity in schizophrenia have been studied in various regions
of the brain including frontal and temporal cortex, caudate nucleus, putamen, nucleus
accumbens, globus pallidus, thalamus, and cerebellum [27, 30, 31, 48]. These studies
revealed that COX activity is differently affected depending on the region examined. In
the present study we tested for differences in COX activity in two different types of
samples from the SN/VTA (i.e. rostro-caudal and mid-caudal). Our analysis of COX
activity did not yield any significant differences for either region of the SN/VTA. This is
in agreement with a previous study by Prince et al. [31] that also found no changes in
COX activity within the mesencephalon. An alternative explanation for our findings is
that metabolic pathology, and more specifically anomalies in COX activity, may affect
discrete subpopulations of dopaminergic neurons within the SN/VTA.
133
Cytochrome c oxidase subunit analysis in the SN/VTA
Subunit composition of the COX enzyme was assessed in schizophrenia and nonpsychiatric control cases, as well as in animals chronically treated with antipsychotic
medications, by measuring protein expression. Subunits I, II, and III of the COX enzyme
were selected for study because these mitochondrial DNA-encoded subunits constitute
the catalytic core of the enzyme. In addition, COX subunit IV, which is encoded by
nuclear DNA, was selected for study due to its essential role in the assembly of the
enzyme and in respiratory function [37]. COX subunit IV has two isoforms (i.e. COX IVI and COX IV-II) in the human, rat, and mouse [36], thus, protein expression of both
isoforms was assessed in our study.
COX subunits in Rostro-Caudal SN/VTA samples
COX subunit I expression did not differ significantly between schizophrenia and
non-psychiatric control samples (unpaired t-test, p=0.6895, t= 0.4086, df= 13) [Figure
3A]. Interestingly, COX subunit II was significantly decreased in schizophrenia subjects
versus non-psychiatric controls (unpaired t-test with Welch’s correction, p=0.0332,
adjusted t= 2.716, adjusted df=6.305). Welch’s correction was used to address the
significant differences in variance between the two groups (F-test p=0.0007). Two
outliers were identified in the schizophrenia group (SZ1 and SZ6). Notably, the mean
expression of COX subunit II in schizophrenia compared to the control group presented a
42.77% reduction [Figure 3B]. COX subunit III did not present significant differences
between the two groups (unpaired t-test with Welch’s correction, p=0.0925, adjusted t=
1.884, adjusted df= 8.92). Welch’s correction was used to address the significant
134
differences in variance between the two groups (F-test p=0.0267). One outlier (C2) was
identified for COX subunit III in the control group [Figure 3C]. COX subunit IV-I
expression data were not normally distributed for the schizophrenia group (Shapiro-Wilk,
p=0.0018; Kolmogorov-Smirnov p=0.0141), and one outlier was identified in the
schizophrenia group (SZ6). There were significant differences in COX subunit IV-I
expression between schizophrenia and non-psychiatric controls (Mann-Whitney test,
p=0.048, U= 9) [Figure 4A]. However, no significant differences were observed between
the two groups for COX subunit IV-II (unpaired t-test, p= 0.9234, t= 0.0981, df= 13)
[Figure 4B].
Multiple t-test Holm-Sidak correction for the analysis of subunits I, II, III (i.e.
catalytic core) produced similar results to the independent analysis of these subunits,
yielding significant differences in the expression of subunit II between the schizophrenia
and control samples (p=0.0291, t= 2.5082, df=11), while subunits I and III did not present
significant differences between the two groups (subunit I: p=0.6904, t= 0.4074, df=13;
subunit III: p=0.1218, t= 1.6649, df=12). For subunits IV-I and IV-II (i.e. nuclear genome
encoded) Holm-Sidak corrected t-test yielded non-significant differences between
schizophrenia and control groups (subunit IV-I: p=0.9212, t= 0.0386, df=12; subunit IVII: p=0.0291, t= 0.1008, df=13).
COX subunits in Mid-Caudal SN/VTA samples
COX subunit I expression did not differ significantly between schizophrenia and
control groups for mid-caudal SN/VTA samples (unpaired t-test, p=0.6736, t= 0.4327,
df=11) [Figure 5A]. Despite the significant differences found in rostro-caudal SN/VTA
135
samples for COX subunit II protein expression, in mid-caudal samples no significant
differences were found between the two groups (unpaired t-test, p=0.2093, t=1.334,
df=11) [Figure 5B]. For COX subunit III, an outlier was detected in the schizophrenia
group (SZ14), and no significant differences were found between the two groups for this
subunit (unpaired t-test with Welch’s correction, p=0.2057, t=1.443, df=5). Welch’s
correction was used to address the significant differences in variance between the two
groups (F-test p=0.0013). [Figure 5C]. No significant differences were found for either of
the two isoforms of COX subunit IV (subunit IV-I, unpaired t-test, p=0.0963, t=1.818,
df= 11; subunit IV-II unpaired t-test, p=0.3249, t=1.031, df=11) [Figure 6A-B].
Multiple t-test Holm-Sidak correction also yielded non-significant results for the
analysis of the catalytic core subunits (subunit I: p=0.6742, t= 0.4318, df=11; subunit II:
p=0.2096, t= 1.3327, df=11; subunit III: p=0.1796, t= 1.4429, df=10), as well as for the
nuclear genome encoded subunits (subunit IV-I: p=0.0960, t= 1.8203, df=11; subunit IVII: p=0.3232, t= 1.0343, df=11).
Effect of antipsychotic medication on COX subunit expression
Since all schizophrenia samples included in our study were obtained from
individuals that were medicated with antipsychotic drugs at time of death, we tested in
rats if antipsychotic treatment could have an impact in the expression of the COX
subunits included in our study. All rat samples included the entire rostro-caudal extent of
the SN/VTA.
One-way analysis of variance (ANOVA) was used to compare the expression of
each subunit among the 3 treatment groups. We did not find any statistically significant
136
differences among the three treatment groups for the expression of any of the COX
subunits tested. COX subunit I [one-way ANOVA p= 0.5971, F (2,24)= 0.5269] [Figure
7A], COX subunit II [one-way ANOVA p=0.2668, F (2,24)= 1.397] [Figure 7B], COX
subunit III [one-way ANOVA p= 0.6903, F (2, 24)= 0.3764] [Figure 7C], COX subunit
IV-I [one-way ANOVA p= 0.8636, F(2,24)= 0.1475] [Figure 8A], and COX subunit IVII [one-way ANOVA p= 0.1403, F (2,24)= 2.134] [Figure 8B]. These data support that
antipsychotic drugs do not affect COX subunits expression in the SN/VTA.
Unlike COX activity, little research has been conducted on the subunit
composition of cytochrome c oxidase in regard to schizophrenia pathology. Furthermore,
to the authors’ knowledge, no studies have examined how antipsychotic medication may
affect the individual subunits of this enzyme. For this study we concentrated our efforts
on examining COX subunits that contribute directly to electron transfer, proton pumping,
and enzyme stability. To this end, we studied COX subunits I, II, and III (i.e. the
“catalytic core”) and the two isoforms of COX subunit IV (i.e. COX IV-I and COX IVII). Our study revealed that COX subunit II presented robust significant reductions (i.e.
regardless of the type of analysis used) of its expression in schizophrenia when the entire
rostro-caudal SN/VTA region was present. In addition, a significant reduction of subunit
IV-I was also found for SN/VTA containing the entire SN/VTA in the independent
analysis, but not when the Holm-Sidak correction for multiple t-test comparisons was
applied. All the other subunits assessed (i.e. subunits I, III and IV-II) did not yield any
significant differences between the two groups for samples containing the entire
SN/VTA. For samples containing only mid-caudal SN/VTA regions, none of the subunits
assessed presented significant differences between schizophrenia and non-psychiatric
137
controls. Even though all the samples used in the study were trimmed to reduce the
presence of other cell groups, we cannot discard small contributions of surrounding cell
groups to our results.
Although we did not find significant changes in COX activity, the alterations in
the expression of key subunits of the COX enzyme could still have important functional
implications. It has been demonstrated that mitochondria have a “reserve capacity” of
energy production that is critical in determining cellular metabolic response to insults
such as metabolic or xenobiotic stress (e.g. oxidative stress and hypoxia) [75-76]. It is
highly plausible that disruptions in subunit composition could affect the proper assembly
of the COX enzyme, thus lowering the reserve capacity of mitochondria, and making
them more vulnerable to insult. In other words, a non-optimally assembled COX enzyme
may be able to maintain proper basal COX activity and ATP production, but may have an
impaired capacity to respond to higher energy demands. Supporting this idea, it has been
shown that specific mutations in subunits of the catalytic core produce severe impairment
of mitochondrial function [44-47].
Subunits II and IV of the COX enzyme are crucial for the proper functioning of
the COX complex as a whole [42, 45-46]. COX subunit II is responsible for the binding
of cytochrome c and the subsequent electron transfer to subunit I of the COX enzyme
[43]. Interestingly, anomalies in COX subunit II mRNA expression have been previously
reported in the frontal cortex in schizophrenia without significant changes in COX
activity [45]. In addition, reduced expression of COX subunit II has been shown to
correlate with a higher susceptibility to neuronal loss, and with an increased susceptibility
to kainic acid-induced epilepsy in mice deficient in DNA mismatch repair [77]. COX
138
subunit IV is encoded by nuclear DNA and is not a component of the catalytic core,
although several studies support that subunit IV is necessary for proper electron transfer
and enzyme stability [36-37, 42]. In addition, subunit IV has a crucial role in the correct
assembly of the COX enzyme, and the binding of subunit IV to subunit I is the first step
for the assembly of the entire COX complex [15, 37, 42]. Furthermore, suppression of
COX subunit IV expression has been shown to increase cell susceptibility to apoptosis
[37]. It is important to note that subunits encoded by both the mitochondria and nuclear
genomes were affected in our study. As far as we are aware, mutations in the genes
encoding these subunits have not been reported in schizophrenia. Deficiencies in the
expression of these subunits in the SN/VTA in schizophrenia could be due to a faulty
transcription or translation, although anomalies in mRNA expression for subunit II have
been previously reported in the cortex for this disorder [48].
Another interesting finding of our study is that significant decreases in protein
expression for subunits II and IV-I of the COX enzyme were found only in schizophrenia
samples that contained the entire rostro-caudal extent of the SN/VTA. These findings
suggest that anomalies in the expression of key subunits of the COX enzyme could affect
specifically rostral regions of the SN/VTA, which is in strong agreement with our
previous report on deficits in tyrosine hydroxylase (i.e. the rate limiting enzyme for the
production of dopamine) in the same rostral region of the SN/VTA [58]. This holds
important neuroanatomical and clinical significance since rostral areas of the SN/VTA
mainly contain dopaminergic neurons that contribute to the mesolimbic and mesocortical
pathways [56, 78-81]. Interestingly, anomalies in the expression of tyrosine hydroxylase
[82] and subunit II of the COX enzyme [48] have also been reported in the prefrontal and
139
frontal cortex in schizophrenia, which are some of the main projection areas for the
neurons of the rostral SN/VTA. Decreases in the expression of these subunits could
produce a faulty stoichiometry in the assembly of the COX enzyme, leading to a greater
vulnerability to metabolic insults. Additional studies will be needed to fully elucidate the
meaning of these findings, including if the anomalies observed in subunits II and IV-I of
the COX enzyme occur at the level of transcription or translation, and if other
components of the metabolic machinery may be compromised within the rostral
SN/VTA.
Acknowledgements
The authors wish to thank the staff of the Maryland Brain Collection, University
of Maryland School of Medicine for their help in obtaining the samples used in this
study. Drs. Emma Perez-Costas and Miguel Melendez-Ferro contributed equally to this
work.
140
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Press. pp. 49-68.
82. Akil M, Pierri JN, Whitehead RE, Edgar CL, Mohila C, et al. (1999) Lamina-specific
alterations in the dopamine innervation of the prefrontal cortex in schizophrenic
subjects. Am J Psychiatry 156: 1580–1589.
147
Table 1. Demographic and clinical data of the cases used for the study. Abbreviations: A,
atypical antipsychotic; AA, African American; APD, antipsychotic drug; ASCVD,
arteriosclerotic cardiovascular disease; COD, cause of death; CUT, chronic
undifferentiated type; DVT, deep vein thrombosis; F, female; M, male; MI, myocardial
infarction; NOS, not otherwise specified; MVA, motor vehicle accident; PMI,
postmortem interval; SD, standard deviation; SZ subtype, schizophrenia subtype; T,
typical antipsychotic; TD, tardive dyskinesia; un, unknown; UNK, unknown medication
at time of death; W, white.
148
Table 2. Effect of demographic and sample variables on COX activity and COX subunit
expression. Note that experiments were run independently for each outcome measure,
thus, independent multiple regression tests were run for each outcome measure. Analyses
were carried independently for rostro-caudal and mid-caudal samples. Demographic and
sample variables were tested in separated tests. Abbreviations: R2a, adjusted R squared.
pH, brain pH. PMI, postmortem interval.
149
Figure 1. Analysis of COX activity in samples containing the entire rostro-caudal extent
of the human SN/VTA. A) Representative images of thionin-stained sections and COX
histochemistry in the SN/VTA and red nucleus from schizophrenia and control cases.
Dashed lines in the thionin-stained sections show the masks used for the delineation of
the region of interest. B) Top panel shows the logarithm of optical density (log OD) for
150
COX activity in the SN/VTA (geometric mean with bar representing the 95% confidence
interval). The mid panel shows a scatter plot of the individual data. The horizontal bar in
each scatter plot indicates the geometric mean OD. The bottom panel shows COX
activity in the red nucleus.
151
Figure 2. Analysis COX activity in samples containing only the mid-caudal regions of
the human SN/VTA. A) Representative images of thionin-stained sections and COX
histochemistry in the SN/VTA and red nucleus from schizophrenia and control cases.
Dashed lines in the thionin-stained sections show the masks used for the delineation of
the region of interest. B) Top panel shows the logarithm of optical density (log OD) for
COX activity in the SN/VTA (geometric mean with bar representing the 95% confidence
interval). The mid panel shows a scatter plot of the individual data. The horizontal bar in
each scatter plot indicates the geometric mean OD. The bottom panel shows COX
activity in the red nucleus.
152
Figure 3. Analysis of COX subunits I, II, and III in samples containing the entire rostrocaudal extent of the human SN/VTA. For each subunit, western blot images of all the
samples assessed in schizophrenia and control cases are shown at the top of each panel,
153
together with an image of the same western blot membrane reincubated for the detection
of actin. Outliers are indicated with a red box surrounding the case number. Bar graphs
indicate the mean calibrated optical density (OD) and standard deviation. Scatter plots are
shown on the right with a horizontal bar indicating the mean. A) Analysis of COX
Subunit I expression. Subunit I protein expression did not differ significantly between
schizophrenia and control cases (OD mean ± SD: schizophrenia: 0.6182 ± 0.07451;
control: 0.6014 ± 0.08532). B) Analysis of COX Subunit II expression. There were
significant (*) differences for subunit II expression between schizophrenia and controls
(OD mean ± SD: schizophrenia: 0.21719 ± 0.02887; control: 0.4751± 0.1954). C)
Analysis of COX Subunit III expression. Subunit III protein expression did not differ
significantly between schizophrenia and control cases (OD mean ± SD: schizophrenia:
0.8153 ± 0.3878; control: 0.5388 ± 0.1281).
154
Figure 4. Analysis of COX subunits IV-I and IV-II in samples containing the entire
rostro-caudal extent of the human SN/VTA. For each subunit, western blot images of all
the samples assessed in schizophrenia and control cases are shown at the top of each
panel, together with an image of the same western blot membrane reincubated for the
detection of actin. An outlier is indicated with a red box surrounding the case number.
Bar graphs indicate the mean calibrated optical density (OD) and standard deviation.
Scatter plots are shown on the right with a horizontal bar indicating the mean. A)
Analysis of COX Subunit IV-I expression. Subunit IV-I protein expression showed
significant (*) differences between the two groups (OD mean ± SD: schizophrenia:
0.43541 ± 0.20501; control: 0.84416 ± 0.56772). B) Analysis of COX Subunit IV-II
protein expression. Subunit IV-II protein expression did not differ significantly between
schizophrenia and control cases (OD mean ± SD: schizophrenia: 1.340 ± 0.2261; control:
1.354 ± 0.3104).
155
Figure 5. Analysis of COX subunits I, II, and III protein expression in the mid-caudal
human SN/VTA. For each subunit, western blot images of all the samples assessed in
schizophrenia and control cases are shown at the top of each panel, together with an
156
image of the same western blot membrane reincubated for the detection of actin. Outliers
are indicated with a red box surrounding the case number. Bar graphs indicate the mean
calibrated optical density (OD) and standard deviation. Scatter plots are shown on the
right with a horizontal bar indicating the mean. A) Analysis of COX Subunit I. Subunit I
protein expression did not differ significantly between schizophrenia and control cases
(OD mean ± SD: schizophrenia: 0.6355 ± 0.1680; control: 0.5998 ± 0.1213). B) Analysis
of COX Subunit II. Subunit II protein expression did not differ significantly between
schizophrenia and control cases (OD mean ± SD: schizophrenia: 0.9332 ± 0.3837;
control: 1.352 ± 0.7247). C) Analysis of COX Subunit III. Subunit III protein expression
did not differ significantly between schizophrenia and control cases (OD mean ± SD:
schizophrenia: 0.5228 ± 0.08916; control: 0.8408 ± 0.5324).
157
Figure 6. Analysis of COX subunits IV-I and IV-II protein expression in the mid-caudal
human SN/VTA. For each subunit, western blot images of all the samples assessed in
schizophrenia and control cases are shown at the top of each panel, together with an
image of the same western blot membrane reincubated for the detection of actin. Bar
graphs indicate the mean calibrated optical density (OD) and standard deviation. Scatter
plots are shown on the right with a horizontal bar indicating the mean. A) Analysis of
COX Subunit IV-I. Subunit IV-I protein expression did not differ significantly between
schizophrenia and control cases (OD mean ± SD: schizophrenia: 1.336 ± 0.4237; control:
1.789 ± 0.4741). B) Analysis of COX Subunit IV-II. Subunit IV-II protein expression
did not differ significantly between schizophrenia and control cases (OD mean ± SD:
schizophrenia: 1.279 ± 0.3895; control: 1.097 ± 0.1950).
158
Figure 7. Analysis of COX subunits I, II, and III in the SN/VTA of animals treated with
antipsychotic drugs. Western blot images of the expression of each COX subunit in all
159
animals from the three treatment groups are shown at the top of each panel. Each western
blot membrane was reincubated for the detection of actin. Bar graphs indicate the mean
calibrated optical density (OD) and standard deviation. Scatter plots are shown on the
right with a horizontal bar indicating the mean. A) Analysis of COX subunit I. Subunit I
protein expression did not differ significantly among the three treatment groups (OD
mean ± SD: control: 0.4753 ± 0.06210; haloperidol: 0.5145 ± 0.1158; olanzapine: 0.4731
± 0.1025). B) Analysis of COX subunit II. Subunit II protein expression did not differ
significantly among the three treatment groups (OD mean ± SD: control: 0.4740 ±
0.1118; haloperidol: 0.5535 ± 0.1312; olanzapine: 0.5564 ± 0.1121). C) Analysis of COX
subunit III. Subunit III protein expression did not differ significantly among the three
treatment groups (OD mean ± SD: control: 1.480 ± 0.4543; haloperidol: 1.609 ± 0.2989;
olanzapine: 1.455 ± 0.4438).
160
Figure 8. Analysis of COX subunits IV-I and IV-II in the SN/VTA of animals treated
with antipsychotic drugs. Western blot images of the expression of each COX subunit in
all animals from the three treatment groups are shown at the top of each panel. Each
western blot membrane was reincubated for the detection of actin. The bar graphs
indicate the mean calibrated optical density (OD) and standard deviation. Scatter plots are
shown on the right with a horizontal bar indicating the mean. A) Analysis of COX
subunit IV-I. Subunit IV-I protein expression did not differ significantly among the three
treatment groups (OD mean ± SD: control: 1.084 ± 0.2609; haloperidol: 1.021 ± 0.3270;
olanzapine: 1.098 ± 0.3559). B) Analysis of COX subunit IV-II. Subunit IV-II protein
expression did not differ significantly among the three treatment groups (OD mean ± SD:
control: 0.6829 ± 0.1375; haloperidol: 0.8100 ± 0.1611; olanzapine: 0.7551 ± 0.08140).
161
MAPPING DOPAMINERGIC DEFICIENCIES IN THE SUBSTANTIA NIGRA/
VENTRAL TEGMENTAL AREA IN SCHIZOPHRENIA
by
Matthew W. Rice, Rosalinda C. Roberts, Miguel Melendez-Ferro, & Emma Perez-Costas
Under Review in Brain Structure and Function
Format adapted and errata corrected for dissertation
162
Abstract
Previous work from our laboratory showed deficits in tyrosine hydroxylase
protein expression within the substantia nigra/ventral tegmental area (SN/VTA) in
schizophrenia. However, little is known about the nature and specific location of these
deficits within the SN/VTA.
The present study had two aims: 1) test if tyrosine hydroxylase deficits could be
explained as the result of neuronal loss; 2) assess if deficits in tyrosine hydroxylase are
sub-region specific within the SN/VTA, and thus, could affect specific dopaminergic
pathways.
To achieve these objectives: 1) we obtained estimates of the number of
dopaminergic neurons, total number of neurons and their ratio in matched SN/VTA
schizophrenia and control samples; 2) we performed a qualitative assessment in SN/VTA
schizophrenia and control matched samples that were processed simultaneously for
tyrosine hydroxylase immunohistochemistry.
We did not find any significant differences in the total number of neurons,
dopaminergic neurons, or their ratio. Our qualitative study of TH expression showed a
conspicuous decrease in labeling of neuronal processes and cell bodies within the
SN/VTA, which was sub-region specific. Dorsal diencephalic dopaminergic populations
of the SN/VTA presented the most conspicuous decrease in TH labeling. These data
support the existence of pathway-specific dopaminergic deficits that would affect the
dopamine input to the cortex without significant neuronal loss. Interestingly, these
findings support earlier reports of decreases in tyrosine hydroxylase labeling in the target
areas for this dopaminergic input in the prefrontal and entorhinal cortex. Finally, our
163
findings support that tyrosine hydroxylase deficits could contribute to the
hypodopaminergic state observed in cortical areas in schizophrenia.
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Introduction
In schizophrenia, anomalies within the dopaminergic system are an intrinsic
pathological feature of the illness (see as reviews van Os and Kapur 2009; Abi-Dargham
et al. 2010; Perez-Costas et al. 2010; Tost et al. 2010). In addition, the effectiveness of
antipsychotic medication is directly linked to their action on the dopaminergic system
(Carlsson et al. 1957; Carlsson and Lindqvist 1963; Seeman and Lee 1975; Seeman et al.
1976; Creese et al. 1976; see also as reviews Frankle and Laruelle 2002; Howes and
Kapur 2009; Perez-Costas et al. 2010). Despite the importance of dopaminergic
pathologies in schizophrenia, only a few studies have examined the substantia
nigra/ventral tegmental area (Bogerts et al. 1983; Ichinose et al.1994; Mueller et al. 2004;
Perez-Costas et al. 2012; Howes et al. 2013; Williams et al. 2013), which is the most
prominent source for dopamine in the brain.
The substantia nigra/ventral tegmental area (SN/VTA) complex is a
heterogeneous collection of dopaminergic cell groups that extends from diencephalic to
mesencephalic territories within the brain of rodents, non-human primates, and humans
(Puelles and Verney 1998; see as reviews Smits et al. 2006; Smidt and Burbach 2007;
Smits et al. 2013). The SN/VTA contains the largest number of dopaminergic neurons in
the brain (see as reviews van Domburg and ten Donkelaar 1991; Haber and Fudge 1997;
Nieuwenhuys et al. 2008), with average estimates ranging in most studies from
approximately 250,000-440,000 dopaminergic neurons within the complex (Bogerts et al.
1983; Hirsch et al. 1988; van Domburg and ten Donkelaar 1991; Damier et al. 1999a;
Kubis et al. 2000).
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The human SN/VTA presents high complexity and is composed of several
dopaminergic neuronal subpopulations that differ in their neurodevelopmental origin
(Zecevic and Verney 1995; Puelles and Verney 1998; Verney et al. 2001), genetic and
neurochemical profile (Haber et al. 1995; Grimm et al. 2004; Thuret et al. 2004; Chung et
al. 2005; Luk et al. 2013), projection sites (van Domburg and ten Donkelaar 1991;
Damier et al. 1999a; Nieuwenhuys et al. 2008), and susceptibility to disease (Gibb and
Lees 1991; Damier et al. 1999b; Hauser et al. 2005; Fuchs et al. 2009; Bergman et al.
2010). In fact, developmental studies of the human brain have clearly demonstrated the
existence of well-defined rostro-caudal (i.e. anterior-posterior) sub-regions within the
SN/VTA, which contain subgroups of dopaminergic cells with different genetic profiles
and developmental schedule (Puelles and Verney 1998; Verney et al. 2001; van den
Heuvel and Pasterkamp 2008; Nelander et al. 2009). For example, TH-expressing
dopaminergic neurons located in the caudal sub-region (i.e. immature mesencephalon) of
the SN/VTA are already detected by the 6th gestational week, while in the rostral
SN/VTA sub-region (i.e. immature diencephalon or prosomeres 1-3 of the neural tube)
these cells are not clearly observed until the 10th gestational week (Zecevic and Verney,
1995; Verney, 1999; Puelles and Verney 1998; Verney et al. 2001). In the adult brain,
this diencephalic sub-region includes approximately all SN/VTA neuronal populations
located rostral to the exit of the 3rd cranial nerve and to the transition between the
parvocellular and magnocellular parts of the red nucleus (see for anatomical details van
Domburg and ten Donkelaar 1991; Damier et al. 1999a; Nieuwenhuys et al. 2008). In
addition, detailed neuropathological studies in Parkinson’s disease have shown that
diencephalic SN/VTA dopaminergic neurons are significantly more resilient to neuronal
166
death than the mesencephalic ones (Damier et al. 1999b), thus supporting the validity of
using developmental criteria to define SN/VTA sub-regions for neuropathological studies
in the adult brain.
Detailed studies on the projection targets of the dopaminergic neurons of the
SN/VTA have also demonstrated the existence of neuronal subgroups within the complex
that preferentially project to cortical or subcortical regions (see as reviews van Domburg
and ten Donkelaar 1991; Haber and Gdowski 2004; Nieuwenhuys et al. 2008). For
example, dorsal neuronal groups of the SN/VTA in primates preferentially provide
dopaminergic input to cortical areas through the mesocortical pathway, although they
also partially contribute to the mesolimbic pathway (Gaspar et al. 1992; Haber and Fudge
1997; see also as reviews Haber and Godowski 2004; Nieuwenhuys et al. 2008).
Interestingly, dopaminergic deficits in these pathways have been associated with the
hypodopaminergic state observed in individuals diagnosed with schizophrenia (Akil et al.
1999, 2000; Finlay 2001; Seamans and Yang 2004; Toda and Abi-Dargham 2007), and
are most closely related to the cognitive deficits that are a core feature of the illness
(Finlay 2001; Seamans and Yang 2004; Tanaka 2006; Toda and Abi-Dargham 2007).
Ventrally-located neurons of the SN/VTA complex preferentially project to subcortical
regions such as the dorsal and ventral striatum through the nigrostriatal, and in part,
through the mesolimbic pathway (see as reviews Haber and Godowski 2004;
Nieuwenhuys et al. 2008). Disturbances of dopamine neurotransmission within the
nigrostriatal pathway in schizophrenia are associated with a hyperdopaminergic state, and
are most closely linked to the positive symptoms (e.g. psychosis) seen in this illness
167
(Seeman 1987; Davis et al. 1991; Toda and Abi-Dargham 2007; Howes and Kapur 2009;
Meyer-Lindenberg 2010).
Dopamine plays a key role in the modulation of cognitive processes in the
prefrontal cortex, and it is known that low dopamine levels reduce working memory
performance in humans (Seamans and Yang 2004; Floresco and Magyar 2006; Tanaka
2006). Several different molecular pathologies that produce, as a net result, a deficit of
dopaminergic neurotransmission in the prefrontal cortex have been identified in
schizophrenia subjects (Akil et al. 1999; Seamans and Yang 2004; Guillin et al. 2007;
Tan et al. 2007; Raznahan et al. 2011; Perez-Costas et al. 2012a; Kaalund et al. 2013;
Lopez-Garcia et al. 2013). Among these pathologies, work from our laboratory has
revealed deficits in TH protein expression in the SN/VTA in schizophrenia subjects,
although these deficits were only observed in cases that contained the rostral
(diencephalic) region of the SN/VTA complex (Perez-Costas et al. 2012a), which
indicates a possible sub-region specific deficit within the complex. In this study we
sought to elucidate if these TH deficits could be related to overall and/or subregionspecific reductions in neuronal number, by examining both total and dopaminergic
neuronal counts in schizophrenia subjects compared to non-psychiatric controls. In
addition, we performed a qualitative assessment on the specific location of these TH
expression deficits.
168
Materials and Methods
Ethics statement
All human brain samples were obtained from the Maryland Brain Collection
(Maryland Psychiatric Research Center, University of Maryland School of Medicine)
with approval from the Maryland Brain Collection Steering Committee. All experimental
procedures were approved by the University of Alabama at Birmingham Institutional
Review Board (protocols #N110505002 and #N131015007) and were in accordance with
The Code of Ethics of the World Medical Association.
Postmortem human brain samples
For all postmortem human cases, two independent psychiatrists established DSMIV diagnoses based on the review of available medical records and data obtained from the
Structured Clinical Interview for DSM-IIIR (Spitzer et al., 1992) and DSM-IV Axis I
disorders with the next-of-kin. Non-psychiatric control cases had enough information
from medical records and the next-of-kin to discard any major neurological or
neuropsychiatric disorders. Table 1 contains demographic data, as well as information on
the use of antipsychotic treatment, cause of death, and other relevant parameters for the
samples used in this study.
All postmortem SN/VTA samples were dissected as described by Damier et al
(1999a) and preserved in 4% paraformaldehyde prepared in 0.1M phosphate buffer pH
7.4 (PB), at 4°C. Prior to sectioning, the tissue was thoroughly rinsed in PB, and
immersed in a solution containing 30% sucrose in PB at 4°C, for a minimum of two
weeks. Tissue samples were frozen using dry ice and sectioned on a freezing microtome.
169
Eight adjacent (i.e. parallel) series were obtained perpendicular to the longitudinal axis of
the brainstem at a thickness of 50m. All samples were sectioned in this manner to allow
for replication of the experiments, and to maintain backup series for future studies. All
series were equivalent and contained sections of the entire rostro-caudal extent of the
SN/VTA.
Series #1 and 2 were collected in PB containing 0.02% sodium azide and stored at
4°C until use. Series #3 through 8 were collected in a cryoprotectant solution prepared as
described in Watson et al. (1986) and stored at -20°C. For all cases, series #1 was stained
using thionin (FD Neurotechnologies, MD, USA; PS101-01) to assess for any
morphological anomalies related to poor preservation of the tissue, and only those cases
that presented good preservation of the SN/VTA were included in the study. Exclusion
criteria were used in the same manner for schizophrenia and control samples as
previously described (Perez-Costas et al, 2012a).
Immunohistochemistry
Complete
series
immunohistochemistry.
preserved
in
cryoprotectant
solution
were
used
for
Sections were rinsed multiple times in 0.01M phosphate
buffered saline (PBS) at room temperature (RT) to remove the cryoprotectant solution.
Sections were then transferred to citrate buffer (Vector Laboratories, Burlingame, CA,
USA; H-3300) for ten minutes at RT, prior to a 30 minute citrate pretreatment at 80°C.
Following pretreatment, sections were rinsed in citrate buffer at RT and transferred to
PBS. After that, endogenous peroxidase was blocked with 5% H202 in PBS for 30
minutes at RT, followed by several rinses in PBS. Sections were then incubated with 10%
170
normal horse serum (NHS; Vector Laboratories, S2000) in PBS containing 0.5% Triton
X-100 (PBS-T) for 1 hour at RT. Immediately after, sections were incubated for 66 hours
at 4°C with an anti-TH antibody (Millipore, Billerica, MA, USA; MAB318, diluted
1:5000) prepared in a solution containing 3% NHS in PBS-T. Next, sections were rinsed
in PBS and incubated with a biotinylated horse anti-mouse secondary antibody (Vector
Laboratories, BA-2001, diluted 1:400) in a solution containing 3% NHS in PBS-T for 45
minutes at RT. After this, sections were rinsed in PBS and incubated with a peroxidasecoupled avidin-biotin-complex (Vector Laboratories PK6100, diluted 1:100) for 45
minutes at RT. Finally, sections were rinsed in PBS and developed using a 3,3'diaminobenzidine peroxidase kit (Vector Laboratories, SK4100). A negative control,
incubated in a solution lacking the primary antibody and consisting of 3% NHS in PBST, was also performed to ensure specificity of the secondary antibody. In addition, the
specificity of the primary antibody has been previously determined by the manufacturer.
For each case, a minimum of two complete series representing the entire rostrocaudal extent of the SN/VTA were immunolabeled for TH. One of these series was
immunolabeled for TH without counterstaining, while the other series was counterstained
using thionin (FD Neurotechnologies) after immunolabeling. In all cases, sections were
dehydrated in ethanol, cleared in xylene and coverslipped using Eukitt (Electron
Microscopy Sciences, Hatfield, PA, USA; 15322).
Schizophrenia and matched control cases were processed together for
immunohistochemistry, carefully maintaining the exact same incubation and developing
conditions across cases.
171
Photography and image processing
Images were obtained using a Nikon Eclipse 50i microscope equipped with a
Nikon DS-Fi1 color digital camera ((Nikon, Tokyo, Japan). Images were saved as
uncompressed TIFF files at a 2560x1920 resolution. Adjustments of brightness/contrast
were done using the same parameters for all images. These adjustments as well as
photomontage and lettering of figure plates were done using Corel DRAW X5 (Corel
Corporation, Ottawa, Canada).
Neuronal counts and mapping of TH deficits
Oversampling test for study design
A pilot study was conducted to determine the optimal number of sections, and the
number of counting frames to be analyzed from each section. A complete series
(approximately 30-32 sections) from each of two control cases (C3 and C6) were used to
perform this preliminary assessment. Each section was photographed using a 10X
objective and setting randomly a fixed point in the Z axis within the 50μm thickness of
the section. Ten non-overlapping images were taken from each section to cover the entire
SN/VTA complex within the section. Each image was considered a “counting frame”
with the bottom and right margins of the image as the forbidden lines of the counting
frame. Neurons were selected for counting only if the entire cell body and the nucleus
were clearly defined in the image. Neurons touching the right and bottom borders of the
frame were excluded from counting. A “complete series estimate” (CSE) for the number
of total and dopaminergic neurons was obtained as the mean of the counts of all the
neurons (dopaminergic or total) for one entire series for the two cases. The neuronal
172
counts accuracy was calculated as the “percentage of variation” from the CSE using the
following formula:
% of variation= |[(Partial count estimate – CSE)/CSE] x 100|
A summary of the results of this oversampling test is reported in Table 2 (see also
results).
Neuronal counts in schizophrenia and control cases
Following the oversampling test, the optimal counting scheme was set as follows:
1) For each case, using a complete SN/VTA series labeled for TH and counterstained
with thionin, the rostral and caudal boundaries of the SN/VTA were determined using the
Paxinos and Huang (1995) atlas of the human brainstem as well as the neuroanatomical
study of van Domburg and ten Donkelaar (1991). The boundary between the diencephalic
(i.e. rostral) and mesencephalic (i.e. mid-caudal) sub-regions of the SN/VTA was defined
as the transition between the parvocellular and magnocellular parts of the red nucleus, at
the level of the exit of the oculomotor nerve (see e.g. Damier et al. 1999a). 2) The first
section to be counted was selected randomly within 1200μm of the rostral boundary of
the SN/VTA using a random number generator (i.e. random.org). 3) From this randomly
chosen rostral starting point, every third section of the series, and up to the caudal limit of
the SN/VTA, was selected for neuronal counts (i.e. approximately 10-11 sections per
case). 4) A random focal point in the Z axis was chosen within the 50μm thickness of the
section. 5) Using the 10X objective, five images representing 5 randomly placed and nonoverlapping “counting frames” were taken for each section within the SN/VTA. 6) The
173
“cell counter” plug-in of NIH Image J software version 1.46r (http://rsbweb.nih.gov/ij/)
was used for the quantification of dopaminergic and non-dopaminergic neuronal counts.
To avoid investigator bias on data collection, all cases were coded, and the
researcher performing the neuronal counts was blind for the case diagnoses. In addition,
our experimental design prevented any risk of double-counting neurons since each
section selected for neuronal counts was 1200μm apart (i.e. one series was used for
counts out of 8 series collected, and from this series, 1 out of every 3 sections was
counted). Dopaminergic neurons were identified by the presence of TH immunolabeling,
and non-dopaminergic neurons identified by their purple-blue thionin staining and lack of
TH immunostaining.
Qualitative mapping of dopaminergic deficits
Three schizophrenia cases and their most closely matched control cases were used
for this qualitative analysis. Two entire SN/VTA series per case labeled for TH were used
for this assessment. For this part of the study we selected schizophrenia cases SZ1, SZ3
and SZ6, for which we previously assessed TH protein expression by western-blot in the
opposite hemisphere (Perez-Costas et al. 2012; cases SZ1, SZ4, SZ6). The matched
controls cases were C2, C5 and C6, from which C6 was also included in the previous
protein expression study (i.e. case C4 in Perez-Costas et al. 2012).
174
Statistical analysis of neuronal counts
Effect of demographic and sample quality variables on outcome measures
Schizophrenia and non-psychiatric control cases were matched as much as
possible for demographic (i.e. age, gender, race), and sample (i.e. postmortem interval,
brain pH) variables. We used multiple regression to assess the possible influence of
demographic and sample variables on our outcome measures (i.e. cell counts)
independent of disease status. Demographic and sample variables (i.e. postmortem
interval and pH) were tested in separated analyses. For the analysis of demographic
variables a “dummy coding” was used to numerically interpret gender and race. The
effect of demographic and sample variables was assessed independently for each brain
region/sub-region (i.e. entire SN/VTA, diencephalic region, mesencephalic region).
However, the effect of these variables on all cell counts of a given region/sub-region was
assessed in the same analysis, correcting for multiple comparisons, and the adjusted R 2 is
reported to eliminate the add-on effect of testing for multiple outcome variables.
Neuronal counts in schizophrenia versus controls
All data collected on neuronal counts were assessed for the presence of significant
deviations from normality. In addition, the presence of outliers was determined using the
“Robust regression and OUtlier Removal” (ROUT) test for outlier detection (Motulski
and Brown 2006), with the coefficient Q set to 1%. Unpaired t-tests were corrected for
multiple comparisons using the False Discovery Rate (FDR) with Q set at 1% (Motulsky
2010). Multiple comparisons corrections were applied for all parameters tested in the
same specific region/sub-region (e.g. total number of neurons, dopamine neurons and
175
ratio in the diencephalic SN/VTA were treated as multiple comparisons). Statistical
analysis and graphical representation of the results obtained were done using GraphPad
Prism 6.0 software (Graphpad Software Inc., La Jolla, CA, USA).
Results
Oversampling test for study design
In order to obtain accurate estimates of the number of dopaminergic and total
neurons, we performed an oversampling study using two control cases (C3 and C6), in
which neuronal counts were performed for the entire rostro-caudal extent of the SN/VTA.
A summary of the results obtained is shown in Table 2. From this assessment we
concluded that we can obtain highly accurate cell count estimates by starting at a random
section within the most rostral 1200µm of the SN/VTA, and counting cells on every 3rd
section. We also showed that reducing the number of counting frames to one-half (i.e. 5
randomly selected frames counted per section) did not have a significant impact on the
accuracy of the counts.
Demographic and sample features
The effect of demographic (i.e. age, gender, race) and sample (i.e. brain pH and
postmortem interval) variables on neuronal counts (i.e. total neurons and dopamine
neurons) was assessed by performing independent multiple regression analyses for these
two types of variables. Data obtained are summarized in Tables 3 and 4. None of these
variables had any significant effect on cell counts (total or dopamine neurons) for any of
the regions/sub-regions (i.e. rostro-caudal, rostral, mid-caudal SN/VTA) analyzed.
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Dopaminergic and total neuronal counts
Rostral and caudal limits of the SN/VTA were defined as described in previous
neuroanatomical studies and atlases that used the same sectioning orientation used in the
present study (van Domburg and ten Donkelaar 1991; Paxinos and Huang 1995; Damier
et al. 1999a; Nieuwenhuys et al. 2008). The rostral sub-region was defined as the
diencephalic territories of the SN/VTA, while mid-caudal sub-region consisted in the
mesencephalic territories of the complex. These subdivisions were included in the study
design (see methods), and were based on previous developmental studies showing clearly
demarcated diencephalic and mesencephalic SN/VTA sub-regions in the human (Zecevic
and Verney 1995; Puelles and Verney 1998; Verney et al. 2001), and conspicuous
differences in the response to pathological processes of these two sub-regions (Gibb and
Lees 1991; Damier et al. 1999b; Hauser et al. 2005).
Neuronal counts in the entire SN/VTA
We obtained estimates of the total number of neurons and dopaminergic neurons
for the entire SN/VTA. Unpaired t-test corrected for multiple comparisons were used to
compare these measurements in schizophrenia versus controls. There were no significant
differences in
the total number of neurons (schizophrenia=603,160±165,990;
control=656,537±128,964; p=0.5272, t=0.6529, df=11), number of dopaminergic neurons
(schizophrenia=218,528±70,563; control=249,161±44,353; p=0.3997, t=0.8760, df=11),
or
the
ratio
of
dopaminergic/total
neurons
(schizophrenia=0.3610±0.0735;
control=0.3819±0.0328; p=0.5099, t=0.6811, df=11) [Figure 1].
177
Neuronal counts in the diencephalic SN/VTA
Unpaired t-test corrected for multiple comparisons yielded non-significant
differences
in
the
total
number
of
neurons
(schizophrenia=224,064±28,903;
control=202,519±61,180; p=0.4479, t=0.787, df=11), number of dopaminergic neurons
(schizophrenia=70,640±25,495; control=70,512±22,552; p=0.9925, t=0.0096, df=11), or
the
ratio
of
dopaminergic/total
neurons
(schizophrenia=0.3198±0.1168;
control=0.3554±0.0748; p=0.5189, t=0.6664, df=11) in the diencephalic (rostral) subregion of the SN/VTA [Figure 2].
Neuronal counts in the mesencephalic SN/VTA
Unpaired t-test corrected for multiple comparisons for the mesencephalic (midcaudal) sub-region also yielded non-significant differences in the total number of neurons
(schizophrenia=379,096±174,111; control=454,018±93,937; p=0.3445, t=0.9876, df=11),
number
of
dopaminergic
neurons
(schizophrenia=147,888±52,813;
control=178,649±50,190; p=0.3051, t=1.0757, df=11), or the ratio of dopaminergic/total
neurons (schizophrenia=0.4165±0.1389; control=0.3898±0.0410; p=0.6354, t=0.4876,
df=11) [Figure 3].
Qualitative mapping of dopaminergic deficits in the SN/VTA
There is great variability in the sectioning orientation and nomenclature of
subdivisions of the human SN/VTA in anatomical studies, creating great complexity for
comparative assessments of neuropathology. In the present study all samples were
sectioned in a plane perpendicular to the main axis of the brainstem, which is the same
178
sectioning orientation previously used by van Domburg and ten Donkelaar (1991),
Damier et al (1999a) and Nieuwenhuys et al (2008) detailed anatomical studies of the
human SN/VTA. For the qualitative assessment of TH expression we also followed the
major sub-regions within the SN/VTA (i.e. diencephalic versus mesencephalic subregions) previously used for the neuronal counts. In addition, we also took into account
the dorso-ventral and medio-lateral location of the dopaminergic cell groups, defining
three sub-areas for our qualitative assessment: dorsal SN/VTA, ventro-lateral SN/VTA,
and ventro-medial SN/VTA [Figures 4-6].
Diencephalic SN/VTA
The dorsal area of the diencephalic SN/VTA included the nuclei identified by van
Domburg and ten Donkelaar (1991) as the pars lateralis of the substantia nigra, the
anterolateral nucleus of the substantia nigra compacta, and the diencephalic part of the
parabrachial nucleus pigmentosus. The ventrolateral area corresponded with the
anterointermediate nucleus of the substantia nigra compacta, while the ventro-medial area
included the anteromedial nucleus of the substantia nigra compacta and the diencephalic
part of the paranigral nucleus of van Domburg and ten Donkelaar (1991) [Figures 4A-B].
The diencephalic sub-region of the SN/VTA presented an overall conspicuous
reduction of TH labeling in schizophrenia compared to matched controls [Figure 4].
Interestingly, there was also a clear dorso-ventral pattern of TH deficits in schizophrenia
compared to controls [Figures 4-5]. In schizophrenia, dorsal areas of the SN/VTA
showed the lowest TH labeling, while ventral areas (ventro-lateral and ventro-medial),
although also presenting clearly decreased labeling, consistently showed stronger TH
179
expression than dorsal SN/VTA dopaminergic neuronal groups [Figures 4 and 5].
Immunolabeling for TH was clearly decreased in cell bodies and also in neuronal
processes [Figure 5].
Mesencephalic SN/VTA
The dorsal area of the mesencephalic SN/VTA included the postero-superior and
postero-lateral subnuclei of the substantia nigra compacta, as well as the mesencephalic
part of the parabrachial nucleus pigmentosus of van Domburg and Donkelaar (1991),
while the ventrolateral and ventromedial areas corresponded with the posteromedial
nucleus of the substantia nigra compacta and the mesencephalic part of the paranigral
nucleus, respectively.
In the mesencephalic region of the SN/VTA differences in TH labeling between
schizophrenia and controls were subtle [Figure 6]. Dorsal SN/VTA presented moderate to
subtle reductions in overall labeling [Figures 6C-D, F-G]. Ventrolateral and ventromedial
SN/VTA areas did not present clear differences in TH immunolabeling between
schizophrenia and their matched control cases [Figures 6C, 6E, 6F, 6H].
Discussion
The present study shows evidence of conspicuous sub-region specific deficits in
TH protein expression within the diencephalic sub-region of the SN/VTA, without
neuronal loss.
Neuropathological changes in the SN/VTA, and more specifically, in the
dopaminergic cell groups of the SN/VTA in schizophrenia subjects have been scarcely
180
studied. An early report by Bogerts et al. (1983) reported deficits in volume and neuronal
size in specific subnuclei of the SN/VTA, while a recent work has reported increases in
the volume of dopaminergic cells as well as glial anomalies in the mesencephalic portion
of the SN/VTA (Williams et al. 2013). However, most of the few postmortem studies
performed have focused on the expression of tyrosine hydroxylase (Ichinose et al. 1994;
Mueller et al. 2004; Perez-Costas et al. 2012a; Howes et al. 2013), which is the ratelimiting enzyme for the production of dopamine. Tyrosine hydroxylase expression
studies have reported different results, from increases in TH mRNA (Mueller et al. 2004)
and TH protein immunolabeling (Howes et al. 2013), to no changes in TH mRNA
(Ichinose et al. 1994; Perez-Costas et al. 2012a), and deficits in TH protein expression
(Perez-Costas et al. 2012a). The differences in the data obtained in these studies could be
due to several factors including the heterogeneity of the disease (see as reviews Keshavan
et al. 2011; Nasrallah et al. 2011), methodological differences in the detection of TH
mRNA and protein expression, and of special importance, differences in the sampling
area within the SN/VTA. Our results will be discussed taking into account developmental
and neuroanatomical considerations and addressing potential functional implications.
Neuronal counts
Neuronal count estimates obtained in the present study for total neurons as well as
dopaminergic neurons, in schizophrenia and control samples, were comparable to
previous reports in which estimates were obtained from the entire SN/VTA (Bogerts et al.
1983; Hirsch et al, 1988; van Domburg and ten Donkelaar 1991; Damier et al. 1999a;
Rudow et al. 2008). Our analysis of the effect of demographic variables on the number of
181
dopaminergic neurons showed no significant effect of any of these variables, including
age. While progressive cell loss related to normal aging has been reported in the SN/VTA
(Fearnley and Lees 1991; Kubis et al. 2000; Rudow et al. 2008), most of the cases
included in the present study were in the “middle age” group, ranging from 40-65 years
of age, and only two cases were above this age (see Table 1). Previous studies assessing
total and dopaminergic neuronal cell number in the entire SN/VTA have shown similar
results for dopaminergic neuronal estimates in their “middle age” groups (Kubis et al.
2000; Rudow et al. 2008). It is worth noting that although Rudow et al. (2008) reported
significant reductions in neuronal number with age in their study, their differences were
reported by comparing individuals with an average age for their groups of 19.9±1.21
(young) vs 50.1±5.01 (middle age) vs 87.4±7.87 (elderly), thus large gaps of age (e.g. >
17 years between the “young” and “middle age”, and >20 years between the “middle
age” and “elderly”) were present among these groups.
Our comparison of neuronal counts in the schizophrenia and control cases did not
reveal significant differences in the total number of neurons or dopaminergic neurons,
both, when the entire SN/VTA was analyzed as a whole, and when the diencephalic and
mesencephalic sub-regions were assessed independently. These results confirm the early
report by Bogerts et al. (1983) who also found a lack of significant differences in
neuronal number in schizophrenia subjects compared to controls in the SN/VTA.
Qualitative mapping of TH immunolabeling
The qualitative mapping of TH labeling in the SN/VTA was done in three of the
schizophrenia cases and their most closely matched control cases (see methods). In this
182
qualitative assessment we found conspicuous deficits in TH immunolabeling in
diencephalic (rostral) sub-region, while TH deficits in the mesencephalic sub-region were
weak or not found (see Figure 4-5 versus Figure 6). These data are in accordance with
our previous western-blot study, in which we found that TH protein deficits in
schizophrenia cases were only present in protein homogenates that contained the rostral
sub-region of the SN/VTA and could not be explained as an effect of antipsychotic
medication (Perez-Costas et al. 2012a). In addition, in the present study we found that
TH deficits affected very conspicuously not only the cell bodies, but also the neuronal
processes of these dopaminergic neurons (see detail images in Figure 5), which suggests
that these deficits should also be present in the target areas of these projections.
Interestingly, Akil et al. (1999, 2000) have reported layer-specific deficits in
dopaminergic processes in the prefrontal and entorhinal cortex of schizophrenia subjects.
In earlier neuroanatomical studies of dopaminergic afferents of the human SN/VTA, it
was already described that approximately the rostral third of the SN/VTA preferentially
provided dopaminergic input to cortical areas (see as a review van Domburg and ten
Donkelaar 1991). Supporting this idea, detailed developmental studies have shown that in
the human embryonic brain, the rostral (i.e. diencephalic) dopaminergic neurons of the
SN/VTA differentiate later, and that there is a caudo-rostral gradient of maturation for
these cells and their projections (Zecevic and Verney 1995; Verney 1999; Verney et al.
2001). The dopaminergic SN/VTA neurons of the mesencephalic sub-region are clearly
observed at gestational week 6, and their projections reach subcortical areas such as the
dorsal striatum by the 7th-8th week of embryonic development. Meanwhile the
dopaminergic neurons of the diencephalic subregion are not clearly observed until the 10-
183
11th week (Zecevic and Verney 1995; Verney 1999; Puelles and Verney 1998; Verney et
al. 2001). Dopaminergic projections reach cortical regions by week 13th (Zecevic and
Verney 1995; Verney 1999; Verney et al. 2001), correlating with the maturation of the
diencephalic SN/VTA neurons.
Developmental and neuropathological studies on the primate SN/VTA support the
existence of fundamental differences between the diencephalic and mesencephalic
SN/VTA dopaminergic neurons. In the adult brain, dopaminergic neurons of these two
sub-regions present different neurochemical profiles (Haber et al. 1995; Grimm et al.
2004; Thuret et al. 2004; Chung et al. 2005; Luk et al. 2013) and susceptibility to
pathology (Gibb and Lees 1991; Damier et al. 1999b; Hauser et al. 2005; Fuchs et al.
2009; Bergman et al. 2010). It has been shown that these neuronal populations are
segregated from early on in the development of the embryonic human brain (Zecevic and
Verney 1995; Verney 1999; Puelles and Verney 1998; Verney et al. 2001). Gene
expression boundaries between the diencephalic and mesencephalic sub-regions are
developed very early on, thus separating the immature dopaminergic cell precursors of
both sub-regions, which are exposed to different neurochemical factors (Smits et al.
2006; Smidt and Burbach 2007; Smits et al. 2013). This argues in favor of the existence
of diencephalic-specific deficits of TH protein expression in the SN/VTA of
schizophrenia subjects. Also supporting the existence of fundamental differences between
diencephalic and mesencephalic sub-regions, the detailed study by Damier et al (1999b)
showed that dopaminergic SN/VTA neurons located in the mesencephalic sub-region
degenerate at much earlier stages in Parkinson’s disease than diencephalic SN/VTA
neurons.
184
Taking into account all the evidence presented above, we hypothesize that TH
deficits in the SN/VTA of schizophrenia subjects will preferentially (or perhaps
exclusively) affect dopaminergic cortical projections of the mesocortical and mesolimbic
pathways, thus connecting the present findings with the hypodopaminergic state observed
in the cortex in schizophrenia patients (Finlay 2001; Remington et al. 2011), and with
postmortem studies showing dopaminergic deficits in the cortex (Akil et al. 1999; 2000).
Although Howes et al. (2013) have reported increases in TH labeling, a careful review of
their study shows that there are fundamental differences in their sampling methods and
postmortem interval, as well as in the area sampled. While in our study we have mapped
the entire SN/VTA, Howes et al. (2013) performed an analysis of an area located at the
level of the superior colliculus, limiting their study to a small portion of the
mesencephalic SN/VTA sub-region. In addition, in the supplementary data section of
Howes et al. (2013) it is reported that their time from death to sample preservation ranged
from 24 to 126 hours for the schizophrenia group, and between 24 and 68 hours for the
control group, while in the present study all cases were below 32 hours from death to
preservation (see Table 1). In our qualitative assessment of the mesencephalic SN/VTA,
we only found subtle differences in TH immunolabeling restricted to the dorsal portion of
the mesencephalic SN/VTA between schizophrenia and control subjects, while
ventrolateral and ventromedial areas did not present any differences (see Figure 6). The
lack of conspicuous differences in mesencephalic SN/VTA is also in agreement with our
previous quantitative study showing no significant differences in TH protein expression
in this sub-region (Perez-Costas et al. 2012a).
185
Another feature clearly observed in our qualitative assessment of the rostral
SN/VTA of the schizophrenia samples was the presence of a dorso-ventral pattern of TH
deficits, in which the dorsal region was the most affected (see Figure 4). It has been
consistently reported that dopaminergic neurons located in the “dorsal tier” of the
SN/VTA (see as a review Haber and Godowski 2004), which approximately correspond
with the dorsal sub-area of the present study, preferentially project to cortical areas
(Fallon and Loughlin 1987; Gaspar et al. 1992; Haber et al. 1995; Haber and Godowski
2004; Nieuwenhuys et al. 2008), further supporting a direct link between the tyrosine
hydroxylase deficits reported here and the reports by Akil et al. (1999, 2000) on TH
deficits in specific layers of the entorhinal and prefrontal cortex.
It is important to note that for most of the schizophrenia cases included in this
study we have previously assessed TH mRNA and protein expression in the opposite
hemisphere (i.e. SZ2-SZ6 of the present study were cases SZ1 and SZ3-SZ6 of PerezCostas et al. 2012a), in which we found TH protein deficits without a decrease in TH
mRNA expression. Those data suggested that TH deficits in schizophrenia must occur
after mRNA transcripts are successfully produced. This is also supported by a
preliminary study in which we found that deficits in TH protein expression in
schizophrenia significantly correlate with deficits in poly-c-binding protein 4, which is
involved in the stabilization of TH mRNA for its translation into protein (Perez-Costas et
al. 2012b). In addition, we have found deficits in the expression of specific subunits of
cytochrome c oxidase that are also linked to the diencephalic sub-region of the SN/VTA
in schizophrenia (Rice et al. 2014), which may also contribute to a lack of proper TH
protein synthesis.
186
In summary, the present study confirms our previous findings of dopaminergic
deficits in the SN/VTA in schizophrenia subjects (Perez-Costas et al. 2012a), mapping
these deficits to the rostral SN/VTA, and supporting the existence of pathway-specific
deficits in dopaminergic neurotransmission that would preferentially or exclusively affect
cortical areas. In addition, we show here that these deficits cannot be attributed to
significant neuronal loss but rather to a deficit in TH protein synthesis. Dopamine is
essential for the proper performance of tasks controlled by the prefrontal cortex, which
include working memory, behavioral flexibility and attention (Goldman-Rakic 1996;
Mizoguchi et al. 2009; Winter et al. 2009). Future studies should further address the
mechanisms involved in these dopaminergic deficits, and the relation of these deficits
with specific symptoms observed in the clinic in schizophrenia.
Acknowledgements
The authors wish to thank the Maryland Brain Collection, University of Maryland
School of Medicine for providing the samples used in this study. This work was
supported by the National Institutes of Health (USA) grant RO1MH066123 awarded to
MMF, EPC and RCR. None of the authors have any conflict of interest to disclose.
Ethical Standards
The manuscript does not contain clinical studies or patient data.
Conflict of Interest
The authors do not have any conflict of interest to report.
187
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Table 1. Demographic and clinical data of the cases used for the study
Schizophrenia samples (n=6)
Case
#
Age
(years)
race
gender
COD
PMI
(hours)
pH
SZ subtype
APD type
Chlorpromazine +
Clozapine +
Fluphenazine +
Quetiapine (T + A)
SZ1
45
W
M
ASCVD
31
6.78
CUT
SZ2
53
W
M
ASCVD
14
6.01
Undifferentiated
Quetiapine (A)
46
W
F
DVT
24
6.18
NOS
Clozapine (A)
SZ4
54
W
M
Suicide (bleeding)
19
6.58
NOS
Quetiapine (A)
SZ5
83
W
M
Electrocution
18
6.98
NOS
Quetiapine (A)
SZ6
42
AA
F
ASCVD
13
5.86
Chronic
paranoid
Fluphenazine +
Quetiapine (T + A)
Mean
53.80
19.83
6.40
SD
15.04
6.74
0.45
COD
PMI
(hours)
pH
SZ3
Control samples (n=7)
Case
#
Age
(years)
race
C1
49
AA
M
Cardiac arrest
19
6.76
C2
45
W
M
Cardiac arrest
18
6.97
C3
51
AA
M
ASCVD
19
C4
63
W
M
ASCVD
11
C5
37
W
F
DVT
10
6.49
C6
62
W
M
Cardiac arrest
19
6.48
C7
67
AA
M
ASCVD
17
6.68
Mean
53.43
16.14
6.75
SD
10.92
3.93
0.25
gender
7.18
6.71
Table 1. Demographic and clinical data of the cases used for the study. Abbreviations: A,
atypical antipsychotic; AA, African American; APD, antipsychotic drug; ASCVD,
arteriosclerotic cardiovascular disease; COD, cause of death; CUT, chronic undifferentiated type;
DVT, deep vein thrombosis; F, female; M, male; NOS, not otherwise specified; PMI,
postmortem interval; SD, standard deviation; SZ subtype, schizophrenia subtype; T, typical
antipsychotic; W, white.
198
Table 2: Oversampling data summary
Number of
Number
sections per
of
Estimated total
series used
counting
neurons
for counts
frames
per
section
All sections
10
635,240
All sections
5
635,568
1/3rd
10
651,624
rd
1/3
5
638,832
%
variation
from CSE
Estimated
dopamine
neurons
%
variation
from CSE
0
0.05%
2.58%
0.56%
361,464
357,248
370,416
354,048
0
1.17%
2.48%
2.05%
Table 2. Neuronal counts obtained as the average of the neuronal counts of two different
control cases. Neuronal count estimates of the SN/VTA (both hemispheres) were performed
by counting neurons in a entire series. Each counting frame corresponds to the area of the
SN/VTA fitting in an image taken with the 10X magnification objective. Abbreviations:
CSE, complete series estimate; 1/3rd, one out of each 3 sections.
199
Table 3: Effect of demographic variables on neuronal counts
OUTCOME
MEASURES
ENTIRE SN/VTA DIENCEPHALIC
SN/VTA
MESENCEPHALIC
SN/VTA
Total neurons
p=0.2141
R2a=0.1697
p=0.5490
R2a=-0.0665
p=0.4145
/
2
R a=0.0138
/
Dopaminergic
neurons
p=0.2283
R2a=0.1561
p=0.1520
R2a=0.2377
p=0.4597
/
2
R a=-0.0145
/
Table 3. Effect of demographic variables (i.e. age, gender,race) on neuronal counts. These
analyses were done testing the effect of these variables on cell counts regardless of disease
status. For all analyses n=13, df=9. None of the analyses yielded significant results.
Abbreviations: R2a= adjusted R2 for multiple comparisons.
200
Table 4: Effect of sample quality variables on neuronal counts
OUTCOME
MEASURES
ENTIRE SN/VTA
DIENCEPHALIC
SN/VTA
MESENCEPHALIC
SN/VTA
Total neurons
p=0.2055
R2a=0.1256
p=0.8659
R2a=-0.1659
p=0.1947
/
2
R a=0.1349
/
Dopaminergic
neurons
p=0.1710
R2a=0.1571
p=0.9619
R2a=-0.1907
p=0.1423
/
2
R a=-0.1875
/
Table 4. Effect of sample quality variables (i.e. postmortem interval, brain pH) on neuronal
counts. These analyses were done testing the effect of these variables on cell counts
regardless of disease status. For all analyses n=13, df=10. None of the analyses yielded
significant results. Abbreviations: R2a= adjusted R2 for multiple comparisons.
201
Figure 1. Neuronal counts for the entire SN/VTA. A-B) Number of total and
dopaminergic neurons in the schizophrenia and control groups. In (A), data are presented
as the mean neuronal count for each group with their standard deviation. In (B),
individual data for each subject is plotted, with the horizontal bar indicating the mean for
the group. C-D) Ratio of dopaminergic/total neurons. The bar graph in (C) represents the
mean ratio for each group and their standard deviations. The scatter plot in (D) represents
the ratio values for each individual subject, with the horizontal bar indicating the mean
ratio for each group. No significant differences were observed for any of these measures
between the two groups for the SN/VTA as a whole. Abbreviations: CTRL: control; SZ:
schizophrenia
202
Figure 2. Neuronal counts in the diencephalic (rostral) SN/VTA. A-B) Number of total
and dopaminergic neurons in the schizophrenia and control groups for the diencephalic
sub-region of the SN/VTA. In (A), data are presented as the mean neuronal count for
each group with their standard deviation. In (B), individual data for each subject is
plotted, with the horizontal bar indicating the mean for the group. C-D) Ratio of
dopaminergic/total neurons. The bar graph in (C) represents the mean ratio for each
group and their standard deviations. The scatter plot in (D) represents the ratio values for
each individual subject, with the horizontal bar indicating the mean ratio for each group.
No significant differences were observed for any of these measures between the two
groups in the diencephalic SN/VTA. Abbreviations: CTRL: control; SZ: schizophrenia
203
Figure 3. Neuronal counts in the mesencephalic (mid-caudal) SN/VTA. A-B) Number of
total and dopaminergic neurons in the schizophrenia and control groups for the
mesencephalic sub-region of the SN/VTA. In (A), data are presented as the mean
neuronal count for each group with their standard deviation. In (B), individual data for
each subject is plotted, with the horizontal bar indicating the mean for the group. C-D)
Ratio of dopaminergic/total neurons. The bar graph in (C) represents the mean ratio for
each group and their standard deviations. The scatter plot in (D) represents the ratio
values for each individual subject, with the horizontal bar indicating the mean ratio for
each group. No significant differences were observed for any of these measures between
the two groups in the mesencephalic SN/VTA. Abbreviations: CTRL: control; SZ:
schizophrenia
204
Figure 4. Tyrosine hydroxylase labeling in the diencephalic SN/VTA. A) Sagittal
schematic drawing indicating the rostro-caudal level of sectioning (red line) of the images
shown in this figure. B) Representative schematic drawing of a transverse section at the
level shown in A. Three SN/VTA subareas defined by the dorso-ventral and mediolateral location of the dopaminergic neurons are indicated (dashed lines in gray-shaded
205
area). There was a conspicuous qualitative difference in overall immunolabeling between
matched schizophrenia (C-E) and control (F-H) samples in the diencephalic sub-region of
the SN/VTA. Note also that within this sub-region, the dorsal area of the SN/VTA
presented the weakest immunolabeling in the schizophrenia samples (C), while was very
intensely labeled in the controls (F). Ventro-lateral areas of the SN/VTA presented also
notably weaker immunolabeling in schizophrenia samples (D) compared to the controls
(G). Differences in labeling were less apparent when comparing the ventro-medial area of
matched schizophrenia (E) and control samples (H). The white asterisks indicate a similar
level within the section for comparison of TH labeling between the schizophrenia and
control samples. Coordinates indicate the orientation of the sections: C: caudal; D: dorsal;
L: lateral; R: rostral. Dashed lines in D, G indicate the approximate transition between the
dorsal (d) and ventro-lateral (vl) regions of the SN/VTA. Abbreviations: cp: cerebral
peduncle; CTRL: control; d: dorsal SN/VTA; III: oculomotor nerve exit; m: mammillary
body; pg: periaqueductal grey; RN: red nucleus; SN/VTA: Substantia nigra/ventral
tegmental area; SZ: schizophrenia; Thal: thalamus; vl: ventro-lateral SN/VTA; vm:
ventro-medial SN/VTA. Calibration bar: 1 mm in C-H.
206
Figure 5. Tyrosine hydroxylase labeling of cell and processes in the diencephalic
SN/VTA. A, D) Dorsal subarea of the diencephalic SN/VTA: In schizophrenia (A, A’)
dopaminergic cell bodies presented the typical neuromelanin pigmentation (n), but TH
labeling was very weak. Dopaminergic processes also presented weak labeling for TH
(white arrowheads). The matched control (D, D’) displayed very intense immunostaining
for TH both in cell bodies and processes. B, E) The ventro-lateral subarea of the
diencephalic SN/VTA also presented conspicuous deficits in TH immunolabeling in
schizophrenia (B, B’) compared with a matched control (E, E’). Note also that there is a
gradient of TH immunolabeling deficit, with the areas close to the transition between the
207
ventro-lateral (vl) and dorsal (d) subareas presenting even weaker TH labeling in the
schizophrenia (B) but not in the control (E). Boxed areas in A, D, B, E are shown at
higher magnification in A’, D’, B’, E’, respectively. C, F) The ventro-medial subarea of
the SN/VTA presented scarce dopaminergic neurons (arrows) sparsely distributed among
a dense net of thin TH labeled processes both in the schizophrenia (C) and control (F)
samples. The differences in TH immunolabeling between schizophrenia and matched
controls for this ventro-medial subarea were more subtle, displaying overall moderate
qualitative deficits of TH labeling in their dense net of thin dopaminergic processes, and
no clear qualitative reductions of labeling in dopaminergic cell bodies. The white
asterisks indicate a similar level within the section for comparison of TH labeling
between the schizophrenia and control samples. Coordinates indicate the orientation of
the sections: D: dorsal; L: lateral. Abbreviations: CTRL: control; SZ: schizophrenia.
Calibration bar: 250 µm in A-F.
208
Figure 6. Tyrosine hydroxylase labeling in the mesencephalic SN/VTA. A) Sagittal
schematic drawing indicating the rostro-caudal level of sectioning (red line) of the images
shown in this figure. B) Representative schematic drawing of a transverse section at the
level shown in A. Three SN/VTA subareas defined by the dorso-ventral and mediolateral location of the dopaminergic neurons are indicated (dashed lines in gray-shaded
209
area). C, F) These images show the overall pattern of TH labeling in the mesencephalic
sub-region of matched schizophrenia (C) and control (F) samples. In these low
magnification images the differences in labeling between schizophrenia and controls
were very subtle. D, G) At higher magnification, in the dorsal (d) subarea there were
moderate-to-subtle deficits in labeling in schizophrenia (D) compared with matched
control (G) samples. E, H) In the ventro-lateral (vl) subarea there were no obvious
differences between schizophrenia (E) and matched control (H) samples. Coordinates
indicate the orientation of the sections: C: caudal; D: dorsal; L: lateral; R: rostral. The
white asterisks in C and F indicate a similar level within the section for comparison of
TH labeling between the schizophrenia and control samples Dashed lines in C, F indicate
the approximate transition between the dorsal (d) and ventro-lateral (vl) regions of the
SN/VTA. The areas marked with dashed boxes in C and F are shown at high
magnification in D, E, G, H. Arrowheads in D-E and G-H indicate some examples of TH
immunolabeling in thin processes. Abbreviations: cp: cerebral peduncle; CTRL: control;
d: dorsal SN/VTA; III: oculomotor nerve exit; m: mammillary body; pg: periaqueductal
grey; RN: red nucleus; SN/VTA: Substantia nigra/ventral tegmental area; SZ:
schizophrenia; Thal: thalamus; vl: ventro-lateral SN/VTA; vm: ventro-medial SN/VTA.
Calibration bars: 1 mm in C, F; 500 µm in D, E, G, H.
210
DISCUSSION
Anomalies in the dopaminergic system are a core feature of schizophrenia, and a
large body of data has been generated concerning pathologies associated with the main
cortical and subcortical areas that receive dopaminergic input (Akil et al., 1999, 2000; see
also as reviews Davis et al., 1991; Harrison, 1999; Powers, 1999; DeLisi et al., 2006;
Howes and Kapur, 2009; Perez-Costas et al., 2010; Howes et al., 2012; Kuepper et al.,
2012; Haukvik et al., 2013; Laruelle, 2013). Surprisingly, when the present work was
started, few studies had addressed possible pathologies associated with the region of
origin of the dopaminergic innervation (Bogerts et al., 1983; Toru et al., 1988; Ichinose et
al., 1994; Mueller et al., 2004). Even though dopaminergic cell groups are present in all
main brain areas, the main source of dopamine for the cortical and subcortical areas
affected in schizophrenia is the substantia nigra/ventral tegmental area (SN/VTA), which
stretches from diencephalic to mesencephalic regions within the human brain (Puelles
and Verney, 1998; Damier et al. 1999a; Verney, 1999; Nieuwenhuys et al. 2008). The
SN/VTA has a complex embryonic development, and since early development there are
fundamental differences between diencephalic (i.e. rostral) and mesencephalic (i.e. midcaudal) SN/VTA dopaminergic neurons (Zecevic and Verney, 1995; Verney, 1999;
Puelles and Verney 1998; Verney et al. 2001). Although all dopaminergic neurons of the
SN/VTA contribute to some degree to all main pathways (i.e. mesocortical, mesolimbic,
and nigrostriatal), neurons located in the diencephalic sub-region of the SN/VTA
preferentially provide dopamine to cortical areas, while mesencephalic neurons
preferentially project to subcortical brain areas (van Domburg and ten Donkelaar, 1991;
211
see as reviews Haber and Gdowski, 2004; Halliday, 2004; Nieuwenhuys et al., 2007).
Interestingly, morphological studies often divide the SN/VTA in a so-called “dorsal tier,”
in which cells are primarily located in the rostral, diencephalic region of the SN/VTA,
and a “ventral tier” that starts more caudally and is most prevalent in the mid-caudal,
mesencephalic region of SN/VTA (Crosby and Woodburne, 1943; see as reviews Haber
and Fudge, 1997; Marin et al., 1998; Joel and Weiner, 2000; Augustine et al., 2008). In
summary, the SN/VTA presents great complexity from early on in development, and
different subsets of dopaminergic neurons within the complex preferentially provide
input to different brain areas. In addition, studies in Parkinson’s disease have shown that
there are significant differences in the resilience to Parkinson’s pathology for different
sub-regions of the SN/VTA complex (Damier et al 1999b; see as a review Feve, 2012).
The overall goal of this dissertation was to assess the existence of pathologies in
dopaminergic neurons of the SN/VTA that could affect the dopaminergic input to
particular brain areas in schizophrenia. Specifically, our study focused on three main
aspects: 1) The examination of regional differences of TH mRNA and protein expression
in the SN/VTA in schizophrenia compared to non-psychiatric controls. 2) Assessment of
metabolic anomalies linked to COX dysfunction and their contribution to the pathology
of the SN/VTA in this disorder. A second aspect of the analysis of COX dysfunction was
to assess if alterations in the expression of critical subunits for the assembly and
functioning of the COX enzyme could contribute to pathology in the SN/VTA in
schizophrenia. 3) Taking into account our findings on TH protein expression anomalies
in the SN/VTA in schizophrenia, we wanted to assess if TH pathology could be linked to
a significant reduction in the number of neurons in the SN/VTA (i.e. a reduction in the
212
total number of neurons, or specifically in dopaminergic neurons). As an alternative
hypothesis, we also wanted to test the possibility of a reduction in TH protein expression
without significant neuronal loss, as well as the possibility that the reduction in TH
expression could be assigned to specific neuronal groups within the SN/VTA.
1) Study of regional differences of TH mRNA and protein expression in the
SN/VTA in schizophrenia compared to non-psychiatric controls
Our first study (Perez-Costas et al., 2012) revealed significant TH protein
expression deficits in schizophrenia, without alterations in TH mRNA expression. This
was found by analyzing TH mRNA and protein expression in the same schizophrenia and
non-psychiatric control cases. We also found that TH protein deficits in the schizophrenia
samples were only present if protein expression was assessed for the entire SN/VTA
complex, while the analysis of TH protein expression for the mesencephalic sub-region
of the SN/VTA did not yield any significant differences between schizophrenia and nonpsychiatric control samples. These findings supported the existence of a TH protein
expression pathology that preferentially affected the diencephalic sub-region (i.e. rostral
portion) of the SN/VTA. Taking into account that the diencephalic SN/VTA
preferentially provides dopaminergic input to cortical areas, our study supports earlier
work showing TH deficits in the entorhinal and prefrontal cortex in postmortem human
brains from schizophrenia subjects (Akil et al, 1999, 2000). These data are also in
agreement with the current hypothesis of dopamine pathology in schizophrenia, which
includes a hypodopaminergic state in cortical areas (see as a review Davis et al., 1991).
This led us to hypothesize that TH pathology may be concentrated in the dopamine cell
213
populations that preferentially project through the mesolimbic and mesocortical
pathways.
To ensure that these findings were not a result of the use of antipsychotic
medication by the subject, TH protein levels were also assessed in animals treated with
both typical (Haloperidol) and atypical (Olanzapine) antipsychotics. These antipsychotics
were administered in doses that matched effective clinical dosing in humans, based upon
dopamine receptor occupancy (Perez-Costas et al., 2008). The result of this analysis
showed that antipsychotics do not affect TH protein levels in the SN/VTA complex,
supporting that the TH anomalies observed in the SN/VTA of schizophrenia brains is
intrinsic to the pathology of the illness.
2) Assessment of metabolic anomalies in the SN/VTA in schizophrenia
A second aspect of our study of the pathology of the SN/VTA in schizophrenia
was to assess if metabolic anomalies could contribute to the dopaminergic deficits
identified in the first part of our study. In schizophrenia, metabolic anomalies are a welldocumented aspect of the pathology of the disorder (Wiesel et al., 1987; Cleghorn et al.,
1989; Wiesel, 1992; Prabakaran et al., 2004). We decided to examine the activity of
cytochrome c oxidase (COX) within the SN/VTA complex as an indicator of metabolic
health. The reason behind choosing COX activity as a marker of metabolic health was
three-fold. First, COX is one of the major regulation sites of oxidative phosphorylation
that leads to the production of ATP (Li et al., 2006). Second, regional anomalies in COX
activity, both increases and decreases, have been reported in schizophrenia (Cavelier et
al., 1995; Prince et al., 1999; Maurer et al., 2001), although no studies have specifically
214
examined COX activity within the SN/VTA complex. Finally, it has been shown that the
activity of the COX enzyme is not affected by antipsychotic medication (Burkhardt et al.,
1993; Whatley et al., 1998; Balijepalli et al., 1999; 2001; Streck et al., 2007).
At the time that our study was planned, the available methodologies for the
detection of COX activity in tissue sections were not sensitive enough for our measures,
nor did they allow for the quantity of COX enzyme in the tissue to be realized. Thus, we
developed an improved methodology for the measurement of COX activity (MelendezFerro et al., 2013). This new methodology involved the application of a known amount of
pure COX enzyme onto a nitrocellulose membrane, which was then incubated alongside
the tissue sections. Not only did this improve the accuracy of our measures (being able to
detect changes as small as 5%, compared to 20% with previous methods), but also
allowed for the determination of the amount of COX enzyme present in the tissue.
Utilizing this methodology, we tested for differences in COX activity within the
SN/VTA between schizophrenia and non-psychiatric control cases (Rice et al., 2014).
Our study revealed that COX activity did not differ significantly between the two groups.
This lack of difference in COX activity was observed for the entire SN/VTA, as well as
when the diencephalic (rostral) and mesencephalic (mid-caudal) sub-regions were
analyzed independently. However, it should kept in mind that the analysis of COX
activity in postmortem human brain sections only provides a “snapshot” of metabolic
health at time of death. Thus, to assess if long-term COX function is compromised in
schizophrenia, we also examined protein expression of key subunits for the functioning
and assembly of the COX enzyme.
215
Of the subunits chosen for analysis, subunits I, II and III of the COX enzyme
constitute the “catalytic core”, and are encoded exclusively by the mitochondrial genome.
These enzymes are responsible for substrate binding, electron transfer, and oxidation that
ultimately lead to the expulsion of H+ ions, creating the proton gradient necessary for the
production of ATP (see as a review Taanman, 1997). Subunit IV of the COX enzyme is
encoded by the nuclear genome and is critical for the proper assembly of the complex,
protects the catalytic core from reactive oxygen species, and aids in electron transfer (Li
et al., 2006; Nijtman et al., 1998) [see figure 11 in Introduction]. Additionally, there are
two isoforms of subunit IV in the mouse, rat, and primate, which are referred to as
subunits IV-1 and IV-2 (Huttemann et al., 2001).
Western-blot analysis of COX subunits within the SN/VTA revealed significant
decreases in the expression of subunits II and IV-1 in schizophrenia cases compared to
non-psychiatric controls. Furthermore, these decreases were observed only in those
schizophrenia cases that contained the entire rostral-caudal extent of the SN/VTA
complex. In cases that contained only the mesencephalic sub-region (i.e. mid-caudal
portion) of the SN/VTA, no significant changes were observed in any COX subunit
analyzed. Importantly, we also tested the effect of antipsychotic medications on the
expression of the different COX subunits. Our analysis revealed that neither firstgeneration/typical
(Haloperidol)
nor
second-generation/atypical
(Olanzapine)
antipsychotics administered in clinically relevant doses had any significant effect on
protein expression for any of the COX subunits analyzed in this study. Our data indicate
that the observed deficits in subunits II and IV-1 are driven by the diencephalic (i.e.
216
rostral) sub-region of the SN/VTA complex, and that these anomalies in COX subunit
expression are intrinsic to the pathology of schizophrenia.
Subunit II of the COX enzyme is responsible for the binding of the cytochrome c
substrate and subsequent electron transfer to subunit I within the complex (see as a
review Taanman, 1997). Furthermore, reductions in subunit II expression are associated
with a higher susceptibility to neuronal loss (Francisconi et al., 2006). Subunit IV, as
mentioned previously, is critical for the proper assembly of the COX enzyme. In fact, the
binding of subunit IV to subunit I forms the first intermediary step in the assembly of the
enzyme (Nijtmans et al., 1998; Fontanesi et al., 2006; Li et al., 2006). Additionally,
subunit IV has a role in the modulation of the enzyme kinetics, so that the ATP/ADP
ratio is maintained (Napiwotzki and Kadenbach, 1998). Finally, the suppression of
subunit IV has been linked to an overall reduction of COX activity and an increased
susceptibility to apoptosis (Li et al., 2006).
In summary, our findings indicate that COX activity within the SN/VTA does not
significantly differ between schizophrenia and non-psychiatric controls. However, the
expression of specific subunits encoded by both mitochondrial (i.e. subunit II) and
nuclear (i.e. subunit IV-I) genomes is significantly decreased, with these deficits linked to
the rostral sub-region of the SN/VTA complex. The explanation of how subunit
composition of COX is perturbed without affecting activity may lie in the innate
functioning of the mitochondria. In physiologically “normal” conditions, the
mitochondria function well below their maximum capacity, and the difference between
basal activity and maximum respiratory capacity is known as “reserve capacity” (Perez et
al., 2010; see as a review Mitchell et al., 2013) [Figure 1]. We hypothesize that in
217
schizophrenia this reserve capacity could be decreased in the SN/VTA, thus making
mitochondria more vulnerable to insult. In other words, a non-optimally assembled COX
enzyme may be able to maintain proper basal COX activity and ATP production, but
have an impaired capacity to respond to higher energy demands. Supporting this idea, it
has been shown that specific mutations in subunits of the catalytic core produce severe
impairment of mitochondrial function (Bruno et al, 1999; Clark et al, 1999; Rahman et al,
1999; Tiranti et al., 2000)
3) Assessing changes in the number of neurons, and mapping of dopaminergic
deficits in the SN/VTA
This part of the study was designed to test if the TH deficits identified in our
previous study (Perez-Costas et al, 2012) could be linked to alterations in neuron number
and/or changes in TH immunolabeling within sub-regions of the SN/VTA complex. This
was accomplished, in part, by performing neuronal counts to test if there was a significant
difference in the total number of neurons, in the number of dopaminergic neurons, and in
the ratio of dopaminergic neurons to total neurons in the SN/VTA between schizophrenia
and non-psychiatric control cases. Neuronal counts were analyzed independently for the
diencephalic and mesencephalic sub-regions, and also for the entire rostro-caudal extent
of the SN/VTA complex.
The comparison of neuronal counts between schizophrenia and non-psychiatric
control cases did not reveal significant differences in the total number of neurons, number
of dopaminergic neurons, or the ratio between the two. This was true when the entire
SN/VTA was analyzed as a whole, and also when the diencephalic and mesencephalic
218
sub-regions were assessed independently. These results confirm an earlier report by
Bogerts et al. (1983) that also found a lack of significant differences in neuronal number
in schizophrenia subjects compared to non-psychiatric controls in the SN/VTA. These
data support that the dopaminergic deficits previously reported by our group (PerezCostas et al., 2012) cannot be explained by a reduction in the number of neurons.
Our qualitative immunohistochemical study of TH expression showed
conspicuous deficits in TH immunolabeling in the diencephalic (rostral) sub-region,
while TH deficits in the mesencephalic (mid-caudal) sub-region were weak or not found.
These data are in accordance with our previous western-blot study, in which we found
that TH protein deficits in schizophrenia cases were only present in protein homogenates
that contained the rostral sub-region of the SN/VTA (Perez-Costas et al. 2012). In
addition, we found that TH deficits affected not only the cell bodies, but also the neuronal
processes of these dopaminergic neurons, which suggests that these deficits could also be
present in the target areas of these projections. Interestingly, Akil et al. (1999, 2000) have
reported layer-specific deficits in dopaminergic processes in the prefrontal and entorhinal
cortices of schizophrenia subjects, which are target areas for the dopaminergic neurons of
the diencephalic sub-region of the SN/VTA (van Domburg and ten Donkelaar, 1991; see
as reviews Haber and Gdowski, 2004; Nieuwenhuys et al., 2008).
In summary, the present study shows evidence of conspicuous sub-region specific
deficits in TH protein expression within the diencephalic portion of the SN/VTA, without
neuronal loss. Taking this evidence into account, we hypothesize that TH deficits in the
SN/VTA of schizophrenia subjects will preferentially (or perhaps exclusively) affect
dopaminergic cortical projections of the mesocortical and mesolimbic pathways. This
219
establishes a connection between our present findings and the hypodopaminergic state
observed in the cortex in schizophrenia patients (Finlay 2001; Remington et al. 2011),
and with postmortem human brain studies showing dopaminergic deficits in the cortex
(Akil et al. 1999; 2000). The lack of differences in the mesencephalic SN/VTA is also in
agreement with our previous quantitative study showing no significant differences in TH
protein expression in this sub-region (Perez-Costas et al. 2012).
Future studies should further address the mechanisms involved in these
dopaminergic deficits, and the relation of these deficits with specific symptoms observed
in the clinic in schizophrenia.
Conclusions
The primary goal of this work was to assess if dopaminergic impairments
described previously in target areas for dopamine input in schizophrenia (e.g. the striatum
and prefrontal cortex) could also be present in the neurons of origin for those
dopaminergic inputs, which are located in the substantia nigra/ventral tegmental area.
From this work we drew the following conclusions:

Examination of TH protein and mRNA revealed that deficits in tyrosine
hydroxylase protein expression (but not mRNA) are present in the SN/VTA
complex. This examination also suggested that TH deficits were concentrated in
the diencephalic (i.e. rostral) sub-region of the SN/VTA, which contains cell
populations that preferentially contribute to the mesolimbic and mesocortical
pathways.
220

The preferential presence of this TH protein deficit in the diencephalic sub-region
of the SN/VTA was confirmed by our immunohistochemical study of TH
expression in schizophrenia and matched non-psychiatric control cases. This
study also revealed that these deficits occurred without significant neuronal loss,
and that decreases in TH immunolabeling affected the cell bodies and processes.
This linked our findings with previous studies reporting decreased TH
immunolabeling in the prefrontal and entorhinal cortex, which are target areas for
the dopaminergic projections of the diencephalic sub-region of the SN/VTA.

Finally, we found deficits in key subunits of the cytochrome c oxidase complex
(i.e. subunits II and IV-1), which are involved in long-term regulation and
assembly of the enzyme. These deficits were also linked to the rostral sub-region
of the SN/VTA.

Overall, the results of the present work show the existence of sub-region specific
deficits in dopamine synthesis in the SN/VTA in schizophrenia, which mostly
affect cortical dopaminergic input. In addition, metabolic anomalies related to
deficits in the assembly and/or long-term regulation of the COX enzyme may also
contribute to these deficits.
These findings provide a framework for the understanding of pathway-specific
presynaptic alterations within the dopaminergic system in schizophrenia pathology.
Importantly, our findings can be directly applied to the current view of the dopamine
pathology of schizophrenia. In fact, version II of the dopamine hypothesis of
schizophrenia states that there is a hypodopaminergic state within cortical regions (see as
221
a review Davis et al., 1991). It is plausible that our observed presynaptic reductions in TH
protein expression and increase in metabolic vulnerability within the rostral SN/VTA
contribute to the hypodopaminergic state observed in schizophrenia pathology.
Additionally, this can be related back to the phenotypic symptoms observed in the clinic
that are used for the diagnosis of schizophrenia. Specifically, perturbations within the
mesolimbic and mesocortical pathways are most closely associated with the negative
symptomology and cognitive impairments of schizophrenia, respectively (see as a review
Howes and Kapur, 2009).
Our findings can also have relevance for other neuropsychiatric disorders, since
cortical hypodopaminergia is not exclusive an exclusive feature of schizophrenia. Several
other neuropsychiatric disorders including attention-deficit/hyperactivity disorder, bipolar
disorder, and major depression have also been hypothesized to present deficits in cortical
dopaminergic neurotransmission (Rossetti et al., 1993; Berk et al., 2007; see as reviews
Russell, 2002; Nestler and Carlezon, 2006; Prince, 2008; Yadid and Friedman, 2008;
Gowrishankar et al., 2013). Dopaminergic deficits have also been postulated to play a
role in addiction, likely through abnormalities of dopamine signaling within the
mesolimbic pathway, and also in perturbations in working memory and attention, which
are most closely related to anomalies of the dopaminergic mesocortical pathway (Egan et
al., 2001; see as reviews Toda and Abi-Dargham, 2007; Li and Gao, 2011; Gowrishankar
et al., 2013). In fact, it is now recognized that many neuropsychiatric disorders share a
common pathological background. Recently, the National Institute of Mental Health
(NIMH) has proposed a strategic plan referred to as The Research Domain Criteria
project (RDoC), which aims to “define basic dimensions of functioning … cutting across
222
disorders as traditionally defined.” (NIMH, 2014). Essentially, the goal of the NIMH
RDoC strategy is to analyze aspects of the pathologies of neuropsychiatric illness
regardless of psychiatric diagnosis in order to discover shared abnormalities among the
neuropsychiatric disorders currently diagnosed in the clinic. This new strategy is
proposed as a research framework that will eventually be able to produce better
diagnostic and treatment tools for the clinic.
Taking into account our data and the existence of cortical dopaminergic
anomalies in several neuropsychiatric disorders, it would be plausible that deficits in
tyrosine hydroxylase cross current diagnosis categories. In this scenario, using the RDoC
approach could lead to the development of treatments for TH deficits that would be
symptom-specific rather than disorder-specific
In summary, the work presented here contributes to the understanding of
localized, presynaptic alterations in the dopamine pathology of schizophrenia, which can
be related directly to schizophrenia symptomology. Furthermore, as stated above, the
findings of this dissertation may be applicable to other neuropsychiatric disorders, in
which deficits of cortical dopamine neurotransmission have been observed.
223
Figure 1. Measurement of oxygen consumption rate from isolated neonatal rat
ventricular myocytes. OCR: oxygen consumption rate. Reproduced with permission,
from Hill, B. G., Dranka, B. P., Zou, L., Chatham, J. C., & Darley-Usmar, V. M. (2009).
Importance of the bioenergetic reserve capacity in response to cardiomyocyte stress
induced by 4-hydroxynonenal. Biochem J, 424(1), 99-107. © the Biochemical Society.
224
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