Download Viral vector-based tools advance knowledge of basal ganglia

J Neurophysiol 115: 2124 –2146, 2016.
First published February 17, 2016; doi:10.1152/jn.01131.2015.
Review
CALL FOR PAPERS
Methods to Understand Brain Connections and Neural Function
Viral vector-based tools advance knowledge of basal ganglia anatomy
and physiology
Rachel J. Sizemore,1* Sonja Seeger-Armbruster,2* Stephanie M. Hughes,3 and X Louise C. Parr-Brownlie1
1
Submitted 21 December 2015; accepted in final form 16 February 2016
Sizemore RJ, Seeger-Armbruster S, Hughes SM, Parr-Brownlie LC.
Viral vector-based tools advance knowledge of basal ganglia anatomy and
physiology. J Neurophysiol 115: 2124 –2146, 2016. First published February
17, 2016; doi:10.1152/jn.01131.2015.—Viral vectors were originally developed
to deliver genes into host cells for therapeutic potential. However, viral vector
use in neuroscience research has increased because they enhance interpretation
of the anatomy and physiology of brain circuits compared with conventional
tract tracing or electrical stimulation techniques. Viral vectors enable neuronal
or glial subpopulations to be labeled or stimulated, which can be spatially
restricted to a single target nucleus or pathway. Here we review the use of viral
vectors to examine the structure and function of motor and limbic basal ganglia
(BG) networks in normal and pathological states. We outline the use of viral
vectors, particularly lentivirus and adeno-associated virus, in circuit tracing,
optogenetic stimulation, and designer drug stimulation experiments. Key studies that have used viral vectors to trace and image pathways and connectivity at
gross or ultrastructural levels are reviewed. We explain how optogenetic
stimulation and designer drugs used to modulate a distinct pathway and
neuronal subpopulation have enhanced our mechanistic understanding of BG
function in health and pathophysiology in disease. Finally, we outline how viral
vector technology may be applied to neurological and psychiatric conditions to
offer new treatments with enhanced outcomes for patients.
adeno-associated virus; lentivirus; basal ganglia neuronal phenotypes; optogenetics; DREADDs; confocal and electron microscopy
to increase the knowledge
gained in basic neuroscience experiments and in preclinical
and clinical studies to test potential treatments for neurological
and psychiatric disorders. Vectors are often combined with
cutting-edge technologies such as advanced tracing and microscopy techniques and optogenetic and chemogenetic stimulation and can be used for gene therapy strategies to alter the
production of therapeutic or defective proteins. This review
discusses how viral vectors have been used in basal ganglia
(BG) studies and outlines how their use has advanced the
anatomical and physiological knowledge gained. To aid readers from different fields, we provide a short summary of BG
VIRAL VECTORS HAVE BEEN USED
* R. J. Sizemore and S. Seeger-Armbruster contributed equally to this work.
Address for reprint requests and other correspondence: L. C. Parr-Brownlie,
Dept. of Anatomy, PO Box 913, Dunedin 9054, New Zealand (e-mail:
[email protected]).
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gross anatomy and the viral vectors used in neuroscience
research. Within these sections we provide tables that list the
neuronal phenotypes in each BG nucleus and the generic and
specific promoters used in BG research as resources for readers. We then explain how viral vector use has enabled BG
anatomy to be investigated at the gross level in vivo and also
at pathway and ultrastructural levels in vitro. We clarify how
specific BG subpathway functions have been elucidated by
using optogenetic and chemogenetic stimulation combined
with electrophysiology, electrochemistry, or behavior. We describe viral vectors used to create or treat animal models of BG
disorders because they advance understanding of disease
mechanisms or provide preclinical studies of potential therapies. Finally, we outline how viral vectors and new technologies may be used in the future for preclinical and clinical
treatment of BG disorders.
0022-3077/16 Copyright © 2016 the American Physiological Society
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Department of Anatomy, Otago School of Medical Sciences, Brain Health Research Centre, Brain Research New Zealand,
University of Otago, Dunedin, New Zealand; 2Department of Physiology, Otago School of Medical Sciences, Brain Health
Research Centre, Brain Research New Zealand, University of Otago, Dunedin, New Zealand; and 3Department of
Biochemistry, Otago School of Medical Sciences, Brain Health Research Centre, Brain Research New Zealand, University of
Otago, Dunedin, New Zealand
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VIRAL VECTOR USE IN BASAL GANGLIA
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Introduction to Basal Ganglia Structure and Function
Fig. 1. Schematic diagram showing parallel motor and limbic pathways
between the cortex, BG, and thalamus. Motor pathway (A) with differentiated
direct (solid gray lines), indirect (dashed gray lines), and hyperdirect (dotted
gray lines) pathways and the limbic pathway (B) receive information from the
cortex and project it to the thalamus and cortex (solid black lines). A: for the
direct pathway, information flows from the cortex through the striatum to
the internal segment of the globus pallidus (GPi) and substantia nigra pars
reticulata (SNr). The indirect pathway sends information from the cortex to the
striatum and then onto the external globus pallidus (GPe) and subthalamic
nucleus (STN) before it reaches the BG output nuclei, the SNr and GPi. For the
hyperdirect pathway, information goes from the cortex to the STN, then onto
the GPi and SNr. The striatum receives dopamine input from the substantia
nigra pars compacta (SNc; light gray arrow). B: for the limbic pathway, the
nucleus accumbens (NAc) receives input from the cortex, amygdala, and
hippocampus and innervates ventral pallidus (VP) and ventral tegmental area
(VTA). The NAc receives dopamine input from the VTA (light gray arrow),
and only VP projects to the thalamus and cortex (black lines). The neurotransmitters released at each nucleus in the motor and limbic pathways include
glutamate (solid arrowheads), GABA (blunt ends), and dopamine (open gray
arrowheads).
Terman et al. 2001) and send information to the GPe, which
then projects to the STN before information reaches the GPi
and SNr. The indirect pathway has the longest latency for
information to pass from the cortex to the BG output nuclei
(Nambu et al. 2000). For all of these pathways, information is
sent from the GPi and SNr to the ventral motor and intralaminar thalamic nuclei before it is returned to associative and
motor cortical areas or to the brain stem and spinal cord
(Bosch-Bouju et al. 2013; Fisher and Reynolds 2014).
The ventral division of the BG controls limbic functions and
includes the nucleus accumbens (NAc) (or ventral striatum),
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BG are a group of interconnected subcortical nuclei in the
forebrain and midbrain of birds, reptiles, and mammals (Deschêne et al. 1996; Wilson 1998) involved in cognitive, motor,
associative, memory, and learning processes (Bolam et al.
2000), including reward-related learning, behavioral reinforcement, addiction, arousal, selection, and initiation of movement
(Albin et al. 1989; Bolam et al. 2000). BG nuclei receive inputs
from the cerebral cortex and the thalamus (Wilson 1998) and
then process information through multiple parallel loops
(Graybiel et al. 1994) before feeding it back to selected regions
of the cerebral cortex via the thalamus (Alexander et al. 1986;
Alexander and Crutcher 1990).
It is widely recognized that BG nuclei are highly conserved
across animal species, indicating their key role in controlling
behavior. In mammals, dorsal BG nuclei are important for
controlling motor and associative functions and include the
caudate nucleus, putamen, globus pallidus [external (GPe) and
internal (GPi) segments], substantia nigra [pars compacta
(SNc) and pars reticulata (SNr)], and the subthalamic nucleus
(STN) (Tepper et al. 2007) (Fig. 1A). The caudate nucleus and
putamen are collectively called the dorsal striatum, and in
some lower mammals (e.g., rodents) these nuclei are indistinguishable (Wilson 1998). The caudate-putamen and GPi are
known as the lentiform or lenticular nucleus in humans, which
is the collective target site for treating Parkinson’s disease (PD)
and other movement disorders (Carpenter and Sutin 1983;
Russmann et al. 2003). In cats and rodents, the GPi is known
as the entopeduncular nucleus (Gerfen and Wilson 1996; Wilson 1998). Information primarily enters the dorsal BG at the
striatum, which receives extensive inputs from the cortex and
some information from the intralaminar and ventral motor
thalamic nuclei (Bolam et al. 2000; Villalba et al. 2015; Young
and Penney 2002), whereas the STN represents a secondary
input nucleus (Villalba et al. 2015). The dorsal striatum also
receives dopaminergic input from the SNc, which is important
in selecting the motor programs to promote or suppress, a
process called action selection.
Information is processed through motor-related BG in the
hyperdirect, direct, and indirect pathways (Fig. 1A), with the
names based on the number of connections and the latency of
information transmission between the cortex and BG output
nuclei (GPi and SNr) (Albin et al. 1989; Alexander and
Crutcher 1990; Nambu et al. 2002). The hyperdirect pathway
contains the fewest connections; cortical information enters the
BG at the STN (Kita 1994), where it is projected to the GPi and
SNr with the shortest latency (Bosch et al. 2011, 2012). For
both the direct and indirect pathways, cortical information
enters the BG at the dorsal striatum. Medium spiny neurons
(MSNs) in the dorsal striatum were once thought to be morphologically and electrophysiologically similar. More recent
studies show that MSNs have different membrane properties
and that indirect pathway MSNs are more excitable (Galvan et
al. 2012). MSNs contain neurochemicals that are distinct to
each pathway (Tepper et al. 2007), and they preferentially
express different types of dopamine receptors (Gerfen and
Wilson 1996; Kravitz et al. 2010). Direct-pathway MSNs
express D1-like receptors (Gerfen 2000) and project information to the output nuclei. In contrast, indirect-pathway MSNs
express D2-like receptors (Albin et al. 1989; Gerfen 2000;
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VIRAL VECTOR USE IN BASAL GANGLIA
impairments occur later in the disease and are thought to be due
to neuronal degeneration (Estrada-Sánchez and Rebec 2013).
HD causes selective degeneration of striatal MSNs due to
nuclear inclusions and cytosolic aggregation of the huntingtin
protein (Ramaswamy et al. 2007; Southwell and Patterson
2011). Nonmovement disorders involve the limbic BG nuclei
and include attention deficit hyperactivity disorder (ADHD)
and obsessive-compulsive disorder (OCD) (Delong and Wichmann 2007). Psychiatric disorders arising from BG dysfunction include Tourette’s syndrome, schizophrenia, depression,
OCD, and drug addiction (Ring and Serra-Mestress 2002).
Dorsal and ventral BG nuclei contain GABAergic, glutamatergic, cholinergic, and dopaminergic neurons, with GABAergic neurons being the dominant phenotype in the majority of
nuclei (Fig. 1). In addition, there are many subtypes of
GABAergic neurons in the BG, each with different electrophysiological characteristics and calcium binding proteins,
which will also affect how these neurons respond to inputs
(Bolam et al. 2000; Ellens and Leventhal 2013). Furthermore,
some of these GABAergic neurons corelease a peptide, for
example, somatostatin, which affects responses at postsynaptic
neurons (Gittis et al. 2014; Lévesque and Parent 2005; Smith
and Bolam 1990; Tepper and Bolam 2004; Yelnik et al. 1984).
This review does not focus on these differences; however, for
clarity we present the neuronal phenotypes and subtypes in
each BG nucleus (motor and limbic) in Table 1.
Much of our understanding of BG circuitry comes from
historical tracing technology. The advent of viral vectors has
rapidly advanced our knowledge of the anatomical and functional intricacies of these pathways, including the role of
individual neuronal phenotypes within these nuclei. Next, we
outline the viral vectors used in the BG and explain how their
use has added to our knowledge of BG circuitry.
Viral Vector Use in Neuroscience
Background. Since the concept of gene therapy was first
described in the 1970s (Friedmann and Roblin 1972), their use
and development in the brain for both network discovery and
therapy has exploded. Viral vectors utilize the ability of a
wild-type virus to infect target cells, hijacking the cellular
machinery to express their genome. However, in most cases
viral vectors differ in that they are rendered replication incompetent; they cannot replicate their genome or produce a productive infection. Vectors are a delivery vehicle for expression
of reporter and/or functional proteins under the control of
constitutive, inducible, or cell-type promoters. Here we describe the general characteristics of the major viral vectors used
in neuroscience and their advantages and disadvantages. Each
virus has characteristic tropism (targeting of cells) and spread
from injection sites, in some cases via retrograde or anterograde transport of viral particles, which are important to
consider when designing experiments.
Lentivirus. Lentiviruses, a group including the immunodeficiency viruses (human, HIV; feline, FIV; equine infectious
anemia, EIAV), are part of the Retroviridae family of RNA
viruses. Lentiviruses, in contrast to many members of the
family, are capable of transducing nondividing cells and hence
are more commonly used in the adult brain. Wild-type HIV, the
basis of most lentiviral vectors, is modified to improve tropism
and safety. The HIV1 genome is deleted of all viral encoded
genes (gag, pol, and env), with only the viral long-terminal
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ventral pallidum (VP), and ventral tegmental area (VTA)
(Alexander et al. 1986; Bolam et al. 2000; Galvan et al. 2015)
(Fig. 1B). Here, the NAc is the major input nucleus, receiving
glutamatergic input from the prefrontal cortex (PFC), hippocampus, and amygdala and dopaminergic innervation from
the VTA (Russo and Nestler 2013). It also receives serotonergic input from the dorsal raphe and noradrenergic input from
the locus coeruleus (Lorrain et al. 1999; Unemoto et al. 1985;
Young and Penney 2002). In addition, the VTA receives
glutamatergic input from the lateral habenula (Balcita-Pedicino
et al. 2011; Brinschwitz et al. 2010; Omelchenko et al. 2009)
and dorsal raphe (Qi et al. 2014) and GABAergic inputs from
the rostromedial tegmental nucleus (Jhou et al. 2009; Kaufling
et al. 2010; Russo and Nestler 2013) and the NAc (Russo and
Nestler 2013). The ventral BG output nuclei include the VP
and rostromedial GPi, which project to thalamic nuclei [mediodorsal, rostral parafascicular nucleus, the magnocellular part
of the ventral anterior (VA) nucleus, and the medial part of the
ventral lateral (VL) nucleus; Galvan et al. 2015; Swanson et al.
1987] before information reaches the PFC and anterior cingulate cortex (Alexander and Crutcher 1990; Bolam et al. 2000;
Galvan et al. 2015; Haber and Knutson 2009; Ligorio et al.
2009). The role of VTA dopamine release in the ventral BG is
to provide context and motivational significance to behaviorally important signals, particularly in relation to rewarding,
motivational, and cognitive functions, and it also has a role in
drug addiction and psychiatric disorders (Bourdy and Barrot
2012; Wang and Tsien 2011).
Initially, knowledge about the functions of BG nuclei was
based on observations and symptoms associated with neurodegenerative disorders (Wilson 1998). Many disorders are implicated consistent with its complex, highly interconnected circuitry (Wilson 1998), signifying that the BG have multiple
functions (Gerfen and Wilson 1996). Generally, these degenerative disorders are characterized by abnormal movements,
ranging from hypokinesia to hyperkinesia (Young and Penney
2002). Hypokinetic disorders are characterized by paucity of
movement such as the symptoms observed with PD, where
patients have difficulty initiating movement (akinesia), movements are slower (bradykinesia), and patients experience postural rigidity and resting tremor (Albin et al. 1989). The
primary pathology of PD is loss of midbrain dopamine neurons. Although PD is considered a motor disorder, many
patients experience cognitive decline and depression later in
the disease process (Starkstein et al. 1989). The most noted
example of a hyperkinetic disorder is Huntington’s disease
(HD), a progressive genetic neurological disorder caused by an
autosomal dominant mutation resulting from expansion of
CAG repeats in exon 1 of the huntingtin gene. The symptoms
most commonly occur in adults during and after child-bearing
age (30 –50 yr) and are characterized by excessive uncontrollable movements known as chorea (from its dancelike appearance) (Young and Penney 2002). The exact sequence of HD
pathogenesis is not fully understood but involves dysfunction
and degeneration within the corticostriatal circuit (Ross and
Tabrizi 2011). A decrease in striatal volume occurs before
symptom-based diagnosis of HD and is associated with subtle
behavioral changes (Ross et al. 2014). Cortical dysfunction
occurs early and is associated with cognitive, psychiatric, and
metabolic decline. The earliest motor symptom is chorea,
which occurs because of striatal dysfunction, while other motor
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VIRAL VECTOR USE IN BASAL GANGLIA
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Table 1. Neuronal phonotypes in basal ganglia nuclei
Nucleus
Striatum (dorsal and
ventral)
Subthalamic nucleus
Globus pallidus,
external
Neurotransmitter
Medium spiny projection
Cholinergic interneuron
Parvalbumin interneuron
Somatostatin interneuron
GABA
Acetylcholine
GABA
GABA
Calretinin interneuron
Fast-adapting interneuron
Tyrosine hydroxylase-immunoreactive
(primates, mice)
Glutamatergic
GABAergic (humans)
GABAergic
Subtypes:
Lhx6-GPe
Parvalbumin
Arkypallidal-GPe
GABAergic
Parvalbumin (primates)
Dopaminergic
GABA
GABA
Dopamine and GABA
GABA
GABA
Dopamine
GABAergic
Dopaminergic
GABAergic
Dopaminergic
Glutamatergic
GABA
Dopamine
GABA
Dopamine
Glutamate
Neuropeptides
Parvalbumin
Somatostatin,
neuropeptide Y
Calretinin
Glutamate
GABA
GABA
Parvalbumin
Preproenkephalin
repeats (LTR) and packaging signal remaining. Third-generation vectors also have a deletion in the 3=-untranslated region
(UTR) preventing transcription from the integrated viral promoter and a recombinant 5=-UTR, which is not reliant on the
viral tat protein (Dull et al. 1998). The major contributing
factor to tropism and transduction of lentiviral vectors is the
envelope used. Traditionally, the wild-type envelope from HIV
is replaced by the glycoprotein from the vesicular stomatitis
virus (VSVg). This envelope mediates wide tropism to the
Parvalbumin
Reference
Smith and Bolam 1990
Kawaguchi et al. 1995
Tepper and Bolam 2004
Tepper and Bolam 2004
Tepper and Bolam 2004
Faust et al. 2015
Betarbet et al. 1997;
Xenias et al. 2015
Lévesque and Parent 2005
Lévesque and Parent 2005
Parent et al. 1999
Gittis et al. 2014
Nambu et al. 2002
Yelnik et al. 1984
Nair-Roberts et al. 2008
Nair-Roberts et al. 2008
Gerfen and Wilson 1996
Nair-Roberts et al. 2008
Nair-Roberts et al. 2008
Nair-Roberts et al. 2008
majority of cell types via the low-density lipoprotein receptor
(Finkelshtein et al. 2013). The overall size of the vector
particles and high affinity for receptors limit spread of lentivirusVSVg vectors to a maximum of ⬍1 mm in most brain regions
(Linterman et al. 2011). LentivirusVSVg has no retrograde or
anterograde transport properties (Fig. 2). However, using glycoproteins from other viruses or VSVg chimerics with rabies
glycoproteins can allow strong retrograde uptake (Kato et al.
2011, 2014; Schoderboeck et al. 2015). Additional envelopes
Fig. 2. Schematic diagram illustrating the differences between viral vectors exemplified for striatal or GPe injections. Viral vector injections from a micropipette
(green triangles) are shown for anterograde tracing (A), anterograde transport (B), and retrograde transport (C). Pale green shaded circular areas indicate diffusion
of the viral vector within the striatum or GPe, which are outlined in brown boxes. A: injection of a lentiviral (LV) vector containing the GAD67 promoter
transduces GABAergic (red outline) neurons by anterograde tracing of green fluorescent protein (GFP, green filled neurons). GABAergic medium spiny neurons
(MSNs) and interneurons (e.g., fast-spiking interneurons) with their somata in the striatum, but not other neuronal phenotypes (e.g., cholinergic interneurons,
purple outline), express GFP along the length of the neuron, including at axon terminals of MSNs in the GPe, GPi, and SNr. B: injection of an adeno-associated
virus serotype 2 (AAV2) vector containing the CaMKII␣ promoter in the GPe transduces glutamatergic (black outline) neurons in the STN. The viral particles
are anterogradely transported by GPe neurons and subsequently transduce glutamatergic neurons in the STN. STN neurons that are not in contact with the GPe
are not transduced. C: viral particles from injection of AAV9 containing the tyrosine hydroxylase (TH) enzyme promoter in the striatum will be retrogradely
transported and selectively transduce and express GFP in dopamine (blue outline) neurons with terminals in the striatum and soma in the SNc.
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Globus pallidus,
internal
Substantia nigra, pars
compacta
Substantia nigra, pars
reticulata
Ventral tegmental
area
Neuron Type
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VIRAL VECTOR USE IN BASAL GANGLIA
1995; Papp et al. 2012; Tomioka and Rockland 2007). The
recombinant adenoviral genome can also be packaged into one
of several capsids, the most common based on adenovirus 5.
The serotype (group A-F) determines transduction efficiency
(Chillon et al. 1999). Tropism of adenovirus is mediated by the
fiber knob, which binds coxsackie and adenovirus receptor
(CAR) and integrin ␣V␤5 (Arnberg 2009). Adenovirus today
is most commonly used as a retrograde neuronal tracer (Kuo et
al. 1995; Papp et al. 2012; Tomioka and Rockland 2007). The
genome remains episomal in both wild-type virus and recombinant vectors, and expression is limited by a high immunogenicity against the virus (Lowenstein et al. 2007).
Transsynaptic viruses. Transsynaptic viruses include the
alpha-herpes viruses, e.g., pseudorabies and HSV, and the
Rhabdoviridae family member, rabies, that have been used
extensively as retrograde tracers. Each have double-stranded
DNA genomes with large packaging capacity as viral vectors,
up to 150 kb. These viruses can enter the CNS from the
periphery, for example, via a muscle infection, and “jump”
synapses after replication (sometimes referred to as self-replicating tracers). While these vectors have been invaluable for
identifying microcircuit structure and connectivity in the brain,
their replication eventually kills infected neurons, preventing
their use for longer-term functional studies. Newer-generation
vectors have been developed to limit transsynaptic spread to a
single synapse, eliminating the toxicity. These self-limiting
vectors have also been used for traditional gene therapy studies
(HSV-1, Goins et al. 2014; Neve 2012) and monosynaptic
tracing (rabies and pseudorabies; Arenkiel and Ehlers 2009).
Neuronal specificity of viral vectors. In neuroscience research, viral vectors were initially used to dissect the anatomy
of brain circuits. However, viral vector use has continued to
increase substantially in the last decade with the development
of tools to examine the function of circuits with light (optogenetic) and designer drug (chemogenetic) stimulation. Modifying the innate tropism of viruses has enabled viral vectors to
effectively transduce neurons and glia. A further advancement
has been the addition of a promoter sequence to the transgene
to target a specific population or subpopulation of neurons or
glia in the brain. The promoter transgene encodes a protein that
is uniquely expressed in the target cell population [e.g., glutamate decarboxylase (GAD) for GABAergic neurons] and limits transduction of nonnative proteins, such as expression of a
reporter fluorophore, to the target population. Here we do not
focus on the function of each promoter, but a list of promoters
that have been used in BG research is provided in Table 2.
Comparison of neuronal phenotypes in BG nuclei in Table 1
and available promoters that have been used in the BG in Table
2 may be useful for designing future viral vector BG
experiments.
Tracing and Imaging
Background. Historically, traditional neuronal tracers have
been used to investigate brain circuits such as the cortico-BGthalamocortical pathway (Lanciego et al. 2012; Seeger-Armbruster et al. 2015). These neuronal tracers include biotinylated
dextran amine, Phaseolus vulgaris leucoagglutinin, wheat
germ agglutinin, horseradish peroxidase, and cholera toxin
subunit B. They will trace anterograde, retrograde, or bidirectionally depending on their uptake mechanism and molecular
weight (Callaway 2008; Huh et al. 2010; Parr-Brownlie et al.
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have been used to select specific neural types, e.g., the lymphocytic choriomeningitis virus (LCMV) glycoprotein results
in preferential transduction of astrocytes in the rat substantia
nigra (Cannon et al. 2011), or rabies to confer retrograde
transport (Kato et al. 2011, 2014; Schoderboeck et al. 2015).
The packaging capacity of lentiviruses is ⬃10 kb, allowing
relatively large and complex constructs to be introduced. Unless specifically packaged with an integrase-deficient system
(Wanisch and Yanez-Munoz 2009), lentiviral vectors will
integrate into the host genome, but there are no reports of
integration-related pathologies.
Adeno-associated virus. Adeno-associated viruses (AAVs)
are members of the Parvoviridae family of single-stranded
DNA nonenveloped viruses. AAV is a dependovirus, meaning
that alone it cannot generate a productive infection; it requires
coinfection with adenovirus or herpes simplex virus (HSV).
The AAV genome is packaged in a proteinaceous capsid that
differs in amino acid sequence and tropism depending on
serotype. AAVs are widespread in mammals; many serotypes
have been isolated from humans and monkeys and developed
as viral vectors (reviewed in Castle et al. 2016). Historically,
the AAV2 serotype has been used in viral vectors and gene
therapy trials. In the brain, it shows minimal spread (though
further than lentiviruses) and limited anterograde transduction,
moving out from local soma down axons to transduce synaptically connected soma, without retrograde transport (Fig. 2)
(Murlidharan et al. 2014; Salegio et al. 2013). AAV5 is a
retrograde tracer in some situations (Aschauer et al. 2013),
although it is mainly restricted to the site of injection within the
BG with anterograde spread of the transgene-encoded protein
(Salegio et al. 2013). AAV8 and 9 serotypes will trace neurons
in both directions (Aschauer et al. 2013; Castle et al. 2014;
Ciesielska et al. 2011; Hutson et al. 2012; Salegio et al. 2013).
AAV9 and AAVrh10 (rhesus monkey-derived serotype 10) can
also be used to deliver genetic material to the brain via
systemic administration (Schuster et al. 2014).
Packaging of AAV1, 4, 5, and 6, and more recently 8, 9, and
rh10, generally incorporate the AAV2-based genome into capsids from the other serotypes. In many studies these are
denoted as AAV2/1, AAV2/rh10, etc. to indicate their chimeric
nature. The change to other serotypes has had a significant
impact on both tropism and targeting of neurons via anterograde and retrograde transport (reviewed in Castle et al. 2016).
Note that transduction patterns can also vary between brain
structures targeted and promoters, even when constitutive promoters are used to drive transgene expression. AAV genetic
constructs are limited to 4.7 kb by the smaller capacity for
effective packaging. This limits applications for some larger
genes and more complex multipart regulators. Trans-splicing
and minigenes have been used to overcome this (reviewed in
Hirsch et al. 2016). Notably, recombinant AAV, unlike their
wild-type counterparts, do not routinely integrate into the host
genome, instead remaining as an episome (extragenomic circular DNA). In nondividing cells this can still result in longterm transgene expression (at least 6 yr has been demonstrated
in monkey BG; Rivera et al. 2005).
Adenovirus. Adenovirus was the first to be used as a viral
vector in the early 1990s. With a large capacity (at least 10 kb,
but up to 36 kb if completely gutted), adenovirus has been
widely used in proof-of-principle gene therapy studies (Bemelmans et al. 1999; Ikari et al. 1995) and for tracing (Kuo et al.
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VIRAL VECTOR USE IN BASAL GANGLIA
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Table 2. Promoters used in basal ganglia
Abbreviation
Promoter Origin
Use
References
Creed et al. 2015; Guo et al.
2015; Stefanik et al. 2013
Basic science
EF1␣#
Mammalian elongation factor 1␣
Basic science
NSE*
PGK
Syn*
Mammalian neuron-specific enolase promoter
Phosphoglycerate kinase I
Synapsin
Basic science
Basic science
Basic science ⫹ preclinical
hSyn
〈-Syn*
CaM*
CMV
Basic science
Preclinical
Preclinical
Preclinical
CNTF*
Dlx5/6*
Drd1a*
GFAP*
Human synapsin
Alpha synuclein
Calmodulin
Human cytomegalovirus immediate-early
enhancer promoter
Ciliary neurotrophic factor
369-bp murine homeobox Dlx5/6
Dopamine receptor 1
Mammalian glial fibrillary acidic protein
GluR1*
GluR2*
NGF*
TH*
Tac1*
Glutamate receptor 1
Glutamate receptor 2
Nerve growth factor
Tyrosine hydroxylase
Preprotachykinin 1
Preclinical
Preclinical
Preclinical
Preclinical
Preclinical ⫹ animal model
VEGF*
Vector expressing vascular endothelial
growth factor
Aromatic amino acid decarboxylase
Brain-derived growth factor
Glutamic acid decarboxylase 65 and 67
Preclinical
Chiarlone et al. 2014; Friedman et
al. 2015; Gradinaru et al. 2009
Bass et al. 2010; Galvan et al.
2012
Betley and Sternson 2011
Delzor et al. 2012
Airan et al. 2009; Chen et al.
2014; Mahler et al. 2014
Stefanik et al. 2013
Koprich et al. 2011
Southwell and Patterson 2011
Lee et al. 2005; Lerchner et al.
2007; Stefanik et al. 2013
Southwell and Patterson 2011
Delzor et al. 2012
Delzor et al. 2012
Bull et al. 2014; Scofield et al.
2015; Shevtsova et al. 2004
Delzor et al. 2012
Kelz et al. 1999
Southwell and Patterson 2011
Muramatsu et al. 2002
Delzor et al. 2012; Ena et al.
2011
Shen et al. 2013
Rate-limiting enzyme in the synthesis of a
cofactor for TH called tetrahydrobiopterin
Glial cell line-derived neurotrophic factor
Neurturin neurotrophic factor
Clinical (ProSavin)
Clinical
pENK*
Three genes: aromatic amino acid
decarboxylase, tyrosine hydroxylase, GTPcyclohydrolase 1
Protein at presynaptic terminals
Mutant huntingtin’s protein (82 CAG
repeats)
Leucine-rich repeat kinase 2
Parkin-associated endothelin receptor-like
receptor
A substrate aminoacyl-tRNA synthetase
cofactor
Enkephalin
Pdyn*
Dynorphin
Animal model ⫹ basic science
Synphilin-1*
Synphilin 1, a neuronal cytoplasmic protein
Animal model
AADC*
BDNF*
GAD*
GCH-1*
GDNF*
NTN*
ProSavin*
CDCrel*
Huntingtin*
LRRK-2*
Pael-R*
P38/JTV*
Preclinical
Preclinical
Preclinical
Preclinical ⫹ basic science
Clinical
Clinical
Clinical
Clinical
Clinical
Denyer and Douglas 2012
Denyer and Douglas 2012
Denyer and Douglas 2012; Feigin
et al. 2007; Kaplitt et al. 2007
Denyer and Douglas 2012
Denyer and Douglas 2012
Denyer and Douglas 2012; Marks
et al. 2010; Olanow et al. 2015
Denyer and Douglas 2012; Palfi et
al. 2014
Animal model
Animal model
Löw and Aebischer 2012
Ramaswamy et al. 2007
Animal model
Animal model
Löw and Aebischer 2012
Löw and Aebischer 2012
Animal model
Löw and Aebischer 2012
Animal model ⫹ basic science
Ena et al. 2011; Ferguson et al.
2011, 2013; Michaelides et al.
2013
Ena et al. 2011; Ferguson et al.
2011, 2013; Michaelides et al.
2013
Löw and Aebischer 2012
#
*Promoter is specific; promoter can be specific or nonspecific depending on the viral vector.
2015). Like neuronal tracers, some viral vectors can travel
retrogradely or anterogradely along the axon (see Fig. 2), and
vectors can be pseudotyped with different glycoprotein envelopes (lentiviruses) or chimeras created (lentiviruses and
AAVs) to control the direction of transport (Castle et al. 2016;
Mazarakis et al. 2001). Viral vector technologies are starting to
replace neuronal tracers for mapping neuronal circuits because
they have the advantages of selectively labeling specific neu-
ronal phenotypes and pathways (Betley and Sternson 2011;
Oztas 2003). Using a specific promoter sequence that controls
gene expression allows targeting of proteins for a specific
neuronal population, phenotype, or component in the cell
membrane, nucleus, or cytoplasm in a specific brain region,
pathway, or multisynaptic circuit. Consistent with traditional
tracers, the number of neurons transduced and the spread of the
virus can also be controlled by the volume injected and by the
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Basic science
CaMKII␣*
CMV coupled with chicken ␤-actin promoter
and first intron and rabbit ␤-globin splice
acceptor
Ca2⫹/calmodulin-dependent kinase II␣
CAG
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VIRAL VECTOR USE IN BASAL GANGLIA
Neural circuits can be investigated at gross and ultrastructural levels depending on how the tissue is processed. At the
gross level, improved microscope optics and refined tissue
clearing techniques (Chung et al. 2013; Deisseroth and
Schnitzer 2013; Hama et al. 2011; Ke et al. 2013; Richardson
and Lichtman 2015; Tomer et al. 2014) enable visualization of
circuits in large blocks of tissue; depth of tissue is only limited
by the working distance of the microscope’s objective lens.
This offers the advantage that one fluorescently labeled population of cells can be imaged along its entire length and the
branch points and targets of collaterals can be determined. This
can be undertaken in intact brains, with little damage done to
the tissue because sectioning is not required (Chung et al.
2013), compared with mapping pathways by examining 5- to
50-␮m sections for expression of the fluorophores at injection
and target sites (Kuo et al. 1995). To increase the knowledge
gained about neural circuits, it is becoming more common to
combine viral vector neural tracing with other methods to
visualize subcellular components (i.e., synapses) or examine
the location of proteins within transduced neurons to either
confirm the specificity of the vector or delineate a circuit
(Galvan et al. 2012; Konermann et al. 2013; Kuramoto et al.
2009; Lobbestael et al. 2010; Pastrana 2010; Shu et al. 2011).
Fluorophores expressed by virally transduced neurons can
be immunolabeled with anti-fluorophore antibodies and in turn
labeled with an electron-dense chromogen such as diaminobenzidine (DAB) or immunogold particles, which can be
visualized at the ultrastructural level by electron microscopy
(Dautan et al. 2014; Galvan et al. 2012; Scotto-Lomassese et
al. 2011; Sosinsky et al. 2007). Here, technical advances in
electron microscopy and imaging in the last decade enable
greater visualization of cellular components. Combining fluorescent viral vector technology with electron microscopy reconstruction techniques such as serial section transmission
electron microscopy, serial block face scanning electron microscopy, serial section electron tomography, array tomography, and correlative light and electron microscopy allows
ultrastructure visualization in intact blocks of tissues (Arenkiel
and Ehlers 2009). However, to date, not all of these electron
microscopy techniques have been combined with viral vector
technology. Viral vector-induced labeling of mini-singlet oxygen generator (mini-SOG) allows polymerization of DAB
after exposure of the transduced neurons to blue light, thus
producing cell phenotype- and pathway-specific DAB labeling
(Pollock et al. 2014; Shu et al. 2011). Furthermore, by combining light activation of neuronal activity (optogenetic stimulation, see Optogenetics), high-pressure freezing, and electron
microscopy, functional changes to circuitry can be explored at
the subcellular level (Konermann et al. 2013; Pastrana 2010;
Watanabe et al. 2014).
Noninvasive gross anatomical circuitry can be investigated
in situ with bioluminescence or functional magnetic resonance
imaging (fMRI) by transducing specific cell populations with
viral vectors. For bioluminescence imaging, a reporter gene
encoding an enzyme that generates light is detected by a
biosensor (Massoud et al. 2008; Shah et al. 2008). This lowresolution imaging method shows gross structural circuitry and
has been used to investigate differences in spread and transduction efficiency of AAV, adenovirus, and lentiviral vectors
(Cho et al. 2005; Contag and Bachmann 2002; Deroose et al.
2006; Massoud et al. 2008). Furthermore, optogenetic stimu-
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vector used (Callaway 2008; Huh et al. 2010; Oztas 2003). In
addition, pseudorabies virus enables transsynaptic tracing
across sequential synapses within a circuit, and the number of
synapses crossed is determined by the time the virus is left to
transduce neurons (Callaway 2008). It is also possible to
combine traditional tracers with viral vectors so that a specific
transsynaptic pathway can be labeled. This has been achieved
by inserting cDNA for a transsynaptic protein, like wheat germ
agglutinin, as a transgene into viruses (Huh et al. 2010).
Currently, combining viral vectors with imaging techniques
permits anatomical analysis of circuits and functional manipulation; therefore, consequential changes in anatomy that arise
from changes in physiology or pathology can be investigated in
the same tissue. The next section reviews both gross and
ultrastructural imaging techniques that have been combined
with viral vector technology to investigate BG circuits.
Gross and ultrastructural imaging of circuits. Combining
viral vector technology with imaging techniques enhances
knowledge gained from neuroanatomical studies. Traditionally, cell phenotypes were determined by immunohistochemically staining for proteins that are selectively expressed within
the cells of interest. While phenotype was established, the
pathway (inputs or targets) remained unknown. For example,
using GAD immunostaining to label GABAergic neurons in
the GPe of rats would show which somata and axons are
GABAergic, but we would not know whether the axons were
from the striatum or from local neurons. Additional neural
circuitry information is gained by examining the target innervations of, or inputs onto, transduced neurons. If GABAergic
inputs from the striatum to the GPe were to be investigated,
injection of a viral vector under the control of the GAD67
promoter will enable transduced axon terminals from the striatum to be identified (Fig. 2A). To do this, the vector contains
the genetic code to express a reporter fluorophore (e.g., green
fluorescent protein, yellow fluorescent protein, or mCherry) in
virally transduced neurons, which is then visualized with
fluorescence or confocal microscopy. Neuron phenotype-specific tracing has been achieved on small (Rah et al. 2015) and
large (Beyer et al. 2013; Kasthuri et al. 2015; Lakadamyali et
al. 2012) connectome scales by combining brainbow mice and
superresolution imaging of neural cell cultures or large blocks
of brain, respectively.
When viral vectors are used, transduction specificity is
verified by immunohistochemically staining for a neuronal or
glial phenotype-specific protein and also showing that markers
for other types of neurons and glia are not coexpressed.
Nonspecific promoters used in the brain, including the BG, for
neuronal tracing studies include the human cytomegalovirus
immediate-early enhancer promoter (CMV), CMV coupled
with chicken ␤-actin promoter and first intron and rabbit
␤-globin splice acceptor (CAG), phosphoglycerate kinase I
(PGK), and mammalian elongation factor 1␣ (EF1␣) promoters (see Table 2). Phenotype-specific promoters include
GAD67, parvalbumin (PV), Ca2⫹/calmodulin-dependent kinase II␣ (CaMKII␣), choline acetyltransferase (ChAT), tyrosine hydroxylase (TH), and glial fibrillary acidic protein
(GFAP) to label GABAergic neurons, GABAergic neurons
with the PV calcium binding protein, glutamatergic, cholinergic, and dopaminergic neurons and astrocytes, respectively
(Huh et al. 2010; Oh et al. 2009).
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VIRAL VECTOR USE IN BASAL GANGLIA
topography differs: cholinergic neurons from the pedunculopontine nucleus innervate the dorsolateral striatum, whereas
neurons in the laterodorsal tegmental nucleus innervate medial
striatum and NAc and also send collaterals to the thalamus, and
SNc, and VTA in the midbrain (Dautan et al. 2014). Circuit
tracing was taken a step further by preparing the tissue for
electron microscopy to verify that these cholinergic innervations formed asymmetric (excitatory) synapses (Dautan et al.
2014). The use of ChAT-Cre mice and a modified rabies virus
revealed that cholinergic interneurons and MSNs in dorsolateral striatum receive similar extrastriatal inputs; however, cholinergic interneurons receive more extensive intrastriatal cholinergic inputs (Guo et al. 2015).
Future anatomical viral vector studies will likely continue to
show greater complexity within the BG and between the BG
and other areas of the brain. In addition, the use of viral vectors
will enable greater understanding of how changes in structure
affect function (and vice versa) because spatially and temporally specific physiological and anatomical techniques can be
conducted in the same animals.
Optogenetics
The term optogenetics describes technology in which genes
encoding light-sensitive proteins are expressed in a target cell
population to modulate excitability using light stimulation of
specific wavelengths (see, e.g., Yizhar et al. 2011). Optogenetics provides scientists with tools to control neuronal activity
and glial function in a temporal, spatial, and phenotype-specific
manner relevant for physiologically intact tissue and behavioral tests (Fenno et al. 2011), which could not be achieved
previously with other neuroscience methods such as deep brain
stimulation (DBS) or drug administration. Since the first report
of optogenetics (Boyden et al. 2005), application of this technique has significantly increased the understanding of physiological networks and pathways in mammalian brains.
Optogenetics is frequently used in combination with neuronal cell recordings or electrochemistry ex vivo (e.g., Cepeda et
al. 2013; Tsai et al. 2009; Zhang et al. 2015) or in vivo (e.g.,
Bass et al. 2010; Galvan et al. 2012; Gradinaru et al. 2009).
One of the aims of neuroscience is to understand the mechanisms and circuits involved in generating and controlling
behavior, and optogenetics increases knowledge about distinct
behaviorally relevant circuits in healthy and pathological states
(Aquili et al. 2014; Chaudhury et al. 2013; Kravitz et al. 2010;
Seeger-Armbruster et al. 2015). A key advantage of optogenetic stimulation is that a specific neuronal subpopulation
and/or pathway can be selectively targeted by using viral
vectors with neuron phenotype-specific promoters (see Table
2) and/or transgenic animals and appropriate placement of light
probes. Below we discuss the impact optogenetics has had on
the understanding of BG function.
Background. Viral vectors represent the most popular means
of introducing microbial opsin (light-sensitive protein) genes
into neurons. The first article about optogenetics in neuroscience (Boyden et al. 2005) and some recent studies used
lentiviruses as the viral vector of choice (see Parr-Brownlie et
al. 2015). However, the majority of optogenetic studies use
AAVs and occasionally HSVs (see Yizhar et al. 2011; Zhang
et al. 2010). Most optogenetic studies in the BG have introduced genes for transmembrane channel or carrier proteins.
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lation and gross anatomical imaging techniques have been
combined to look at functional changes in circuits. For example, fMRI has been used to image blood oxygen level changes
when neurons are stimulated (Adriani et al. 2010; Desai et al.
2011; Lee et al. 2010; Schmid et al. 2016) and positron
emission tomography (PET) has been combined with viral
vector technology to investigate changes in brain glucose
metabolism (Thanos et al. 2013).
To date, not all viral vectors have been combined with all in
situ imaging techniques, but there are no limitations preventing
this. In the future, enhanced in situ imaging resolution by fMRI
or bioluminescence would greatly improve circuitry knowledge in health and disease. Viral vectors could be combined
with tissue clearing techniques, gene therapy, optogenetic stimulation, tissue processing, and imaging (Galvan et al. 2012;
Watanabe et al. 2014; Weber 2012) to address unanswered
questions relating to brain circuitry, particularly in the BG.
Investigating BG circuitry. The use of viral vectors in basic
research has revealed that BG circuitry is more complex than
previously thought based on traditional neuronal tracers (Akkal
et al. 2007; Dautan et al. 2014; Dum and Strick 2013; Fujiyama
et al. 2011; Hoover and Strick 1999; Hoshi et al. 2005; Kelly
and Strick 2004; Kuramoto et al. 2009; Middleton and Strick
2002; Oztas 2003). Vectors that have been commonly used to
investigate afferent and efferent BG connections are HSV,
rabies, and sindbis virus, which transsynaptically label pathways. Rabies virus has shown the existence of a closed-loop
circuit between the BG and cerebral cortex as well as a
bidirectional circuit between the BG and cerebellum (Dum and
Strick 2013; Hoshi et al. 2005). Using rabies and HSV, Akkal
et al. (2007) revealed that there is a disynaptic connection from
GPi to the supplementary motor cortex (SMA), with rostral
GPi conveying associative information to pre-SMA whereas
sensorimotor information goes from caudal GPi to SMA
proper. GPi, SNr, STN, GPe, and striatum bi- or trisynaptically
innervate the primary motor cortex and PFC, but information is
conveyed by distinct parallel pathways (Hoover and Strick
1999; Kelly and Strick 2004). GPi and SNr innervate specific
and different parts of PFC, and these connections are segregated from GPi and SNr neurons that innervate the motor
cortex (Middleton and Strick 2002). Sindbis virus has been
used in single-cell tracing studies to confirm that thalamocortical neurons from the rostromedial region of VA-VL also
innervate the striatum and striatofugal neurons arising in striatal striosomes project to the SNc and send collaterals to GPi,
entopeduncular nucleus, and SNr (Fujiyama et al. 2011;
Kuramoto et al. 2009).
More recently, AAV and lentiviral vectors have revealed the
complexity of inputs to, and microcircuits within, the BG.
Acetylcholine is released into the striatum and NAc from local
cholinergic interneurons and also from projection neurons with
somata in brain stem nuclei. The Cre-lox system is an example
of site-specific recombination that uses viral vector technology
to deliver either the Cre-recombinase enzyme or a pair of short
target sequences, known as the lox sequence, to direct sitespecific deletion or insertion of genes or carry out gene translocations or inversions (Sauer and Henderson 1988). Combining ChAT-Cre rats with AAV2 encoding Cre-dependent ChR2
constructs, Dautan et al. (2014) found that cholinergic neurons
innervate the striatum and NAc directly but also indirectly
through thalamic nuclei, SNc, and VTA. Furthermore, the
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protein-coupled receptor chimeras, Opto-XRs, green light (500
nm) stimulation modulates cellular signaling by affecting intracellular G protein-coupled pathways (Fenno et al. 2011;
Yizhar et al. 2011; Zhang et al. 2010), and this was first
described by Airan et al. (2009) in the BG of mice. The
advantage of Opto-XRs is that they permit cell signaling
pathway-derived effects to be separated from changes in receptor activation and the neuron’s membrane potential, and
genetic adaptations would increase their use in more brain
regions. Other opsins [light oxygen voltage (LOV) domains,
cryptochromes, and phytochromes] are fused to effector proteins (e.g., Rac) and located intracellularly (Pastrana 2013;
Zhang and Cui 2015). Here, light of appropriate wavelength
activates the effector protein, altering its function and those of
downstream mediators, resulting in polymerization of actin,
protein dimerization, altering DNA binding proteins, or other
changes in cellular function, which can be detected by including a reporter fluorophore in the genetic construct or measured
as changes in cell activity, accumulation of a downstream
protein, or changes in behavior. Optogenetics can also be used
to alter DNA binding and mRNA expression by using lightinducible transcription effectors (LITEs) that interact with
transcription activator-like effector (TALE) DNA binding domains (Kennedy et al. 2010; Konermann et al. 2013; Schindler
et al. 2015). To date, such in vivo experiments have examined
effects on gene expression but have not been combined with
behavior and other physiological measures (cell recordings,
electrochemistry, or microdialysis), but this is critical to un-
Fig. 3. Schematic diagram of optogenetic and chemogenetic tool families currently used in the BG. A: activation wavelengths (nm), transported ions, and
signaling pathways are indicated for depolarizing channelrhodopsin-2 (ChR2) and C1V1, hyperpolarizing halorhodopsin (NpHR) and archaerhodopsin (ArchT),
and internal signal pathway-altering [Opto-XRs, rhodopsin-G protein-coupled receptor (GCPR) chimeras] microbial opsins. B: designer receptors exclusively
activated by designer drug (DREADDs) with their pharmacologically inert ligands clozapine-N-oxide (CNO) or salvinorin B (SalB) and G protein-coupling
properties. hM3Dq and hM4Di are point-mutated human M3 and M4 muscarinic DREADDs, coupled to Gq and Gi signaling, respectively. rM3Ds consists of
a chimeric point-mutated rat M3 muscarinic DREADD with intracellular loops from the turkey ␤1-adenoceptor (dashed intracellular loops), coupled to Gs
signaling. CNO stimulation of hM3Dq and hM3Ds activates neurons. CNO stimulation of hM4Di and SalB stimulation of the Gi-coupled kappa-opioid receptor
DREADD (KORD) inhibit neurons. cAMP, cyclic adenosine monophosphate.
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The two most widely used opsins are channelrhodopsin-2
(ChR2) and halorhodopsin (NpHR), or mutated variants of
these, which were cloned from Chlamydomonas reinhardtii
(green algae) and Natronomonas pharaonis (halobacteria),
respectively. Light of an appropriate wavelength causes a
conformational change in the opsin, which opens ChR2 or
NpHR. For ChR2, blue light (⬃470 nm) opens a cation channel
allowing Na⫹ to enter the cell, thus depolarizing it (Nagel et al.
2003). For NpHR, yellow light (⬃590 nm) activates a pump
that transports Cl⫺ into cells and hyperpolarizes transduced
neurons (Zhang et al. 2007). Today, many other variants and
new opsins exist, such as C1V1 [a channelrhodopsin-1
(ChR1)-red-shifted channelrhodopsin-1 (VChR1) chimera]
and archaerhodopsins (Arch and ArchT), with different activation wavelengths and times (Fig. 3A). For comparisons of
activation wavelengths and kinetic properties of different
opsins see Yizhar et al. (2011), Mattis et al. (2012), Tye and
Deisseroth (2012), and Klapoetke et al. (2014). Therefore,
using opsins with distinguishable peak activation profiles enables neuronal populations in one area of the brain to be
selectively excited and/or inhibited (Han and Boyden 2007).
The continuous development of new opsin variants with enhanced or altered properties will increase the use of optogenetics in the future.
Optogenetic stimulation has moved beyond activating or
inhibiting neuronal activity so that it can be used to control
intracellular signaling, DNA binding, and epigenetics (see
Chow and Boyden 2013). More recently, with rhodopsin-G
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VIRAL VECTOR USE IN BASAL GANGLIA
The existence of rapid cerebello-striatal communication,
independent of slower cortical loops, has been proven with
optogenetics. Dorsolateral striatum activity is modulated by
cerebellar projection neurons via a disynaptic pathway through
the centrolateral nucleus (CL) of the thalamus (Chen et al.
2014). Cerebellum-induced responses in the striatum after
electrical or optogenetic stimulation (ChR2) of neurons in the
cerebellar dentate nucleus were prevented by inactivation of
CL by using local injection of tetrodotoxin to block voltagegated sodium channels or by optogenetically silencing CL
neurons expressing ArchT, a light-sensitive (566 nm) extrusion
proton transporter (Chen et al. 2014). This cerebello-striatal
pathway may play an important role coordinating motor outputs in real time.
The functions of SNc- or VTA-derived dopaminergic afferents in the striatum have been separated and characterized with
optogenetics. By combining stimulation of ChR2 expressed in
spatially restricted SNc neurons with fast-scan cyclic voltammetry (FSCV) in the striatum, optical stimulation restricted
dopamine release to the dorsal region (Bass et al. 2010). This
technical advancement is of special interest for understanding
the neural microcircuitry underlying dopamine transmission
since it is nearly impossible to electrically stimulate SNc alone
without also recruiting VTA neurons in adjacent brain regions
or fibers of passage (Bass et al. 2010). Furthermore, Cretransgenic mouse lines (DAT-Cre and VGAT-Cre) combined
with viral vector-based optogenetics (ChR2 activation) enabled
cell type-specific identification of VTA neurons in a reward
paradigm and verified that dopamine neurons and their
GABAergic neighbors in the VTA have distinct physiological
responses to reward (Cohen et al. 2012), which was previously
hypothesized but could not be definitively shown without these
techniques.
Direct optogenetic manipulation of MSN subpopulations has
been used to confirm their roles underlying behavior. Albin et
al. (1989) hypothesized that subpopulations of dorsal striatal
MSNs express D1- or D2-like dopamine receptors and are part
of the promovement, direct or antimovement, indirect pathway
(Fig. 1), respectively, but this could not be explicitly tested
without using viral vectors and optogenetics. Selective optogenetic stimulation of either D1- or D2-like-expressing MSN
populations confirmed that the pathways differentially influence BG output nuclei and movements (Freeze et al. 2013;
Kravitz et al. 2010, 2013). Specifically, the use of D1- and
D2-Cre-transgenic mice in combination with stimulation of
virally introduced ChR2 revealed that the D2-like-expressing
neurons excited SNr neurons and suppressed movements to the
point of inducing parkinsonism, whereas stimulation of D1like neurons inhibited SNr activity and promoted movements
in behaving mice (Freeze et al. 2013; Kravitz et al. 2010). In
the limbic BG pathway, behavior flexibility can be increased
by inhibiting NAc shell MSNs during reward or error feedback
intervals (Aquili et al. 2014). The precise timing of the bilateral
optogenetic inhibition (via eNpHR activation) revealed critical
time periods when NAc shell neurons integrate reward or error
feedback history and use this integrated history to make subsequent decisions (Aquili et al. 2014).
The study of corelease of neurotransmitters has been revolutionized by combining cell type-specific viral constructs,
Cre-transgenic animals, and optogenetics. The role of corelease
of glutamate from some midbrain dopamine neurons has been
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derstand the consequences of altering gene expression in normal and diseased brains. None of these constructs has been
used within BG nuclei.
BG function in health. The BG can be divided into parallel
networks (see Fig. 1), and we focus on motor and limbic
pathways. Optogenetic stimulation is frequently applied in
rodents to modulate neuronal activity in brain structures, including the BG, and alter behavior (e.g., Gradinaru et al. 2009;
Seeger-Armbruster et al. 2015; Tsai et al. 2009), but most
studies in nonhuman primates have been limited to the cerebral
cortex (e.g., Dai et al. 2015; Han et al. 2011), with only one
targeting subcortical nuclei (Galvan et al. 2012). In this section
we summarize how viral vectors and optogenetic experiments
have significantly increased the understanding of the BG networks by dissecting the function of subpathways and subpopulations within or between BG nuclei or between BG nuclei and
other structures that project to or receive information from the
BG in health.
Like electrical stimulation, optogenetic stimulation-induced
neuronal responses are often difficult to interpret and understand because of their complexity. However, compared with
electrical stimulation, selective activation of one cell phenotype and verification of the maximum brain area stimulated
(determined by examining the location of transduced cells
postmortem) aid interpretation of neuronal responses. Striatal,
GPe, and VL thalamus responses following light stimulation of
dendrites, soma, axons, or terminals of virally transduced GPe
or dorsal striatal neurons varied depending on postmortem
ChR2 expression (Galvan et al. 2012). More neurons responded in the striatum and VL (⬃32% of neurons responded)
than GPe (3% responses). Responses included direct and indirect ChR2-induced effects, with indirect effects most likely
mediated through local inhibitory network activation (Galvan
et al. 2012). Together, these data highlighted a difference in the
type and magnitude of responses depending on the BG nucleus.
This study shows that while optogenetic stimulation is a
powerful tool that enables anatomy and function to be dissected, responses are complex and the viral construct, injection
parameters, and light stimulation conditions need to be optimized for each structure.
Optogenetic experiments have shown that the medial prefronto-striosomal circuit selectively affects decision-making in
a cost-benefit task under approach-avoidance conflict conditions known to evoke anxiety in humans. The function of
inputs from the PFC has been thought to be important in
addiction behavior and associated plasticity, mostly through
NAc afferents (see Yager et al. 2015). In contrast to this, a
recent study showed the involvement of prefrontal afferents on
striosomes in the dorsal striatum in decision-making (Friedman
et al. 2015). Striosomes are too small to be identified in human
fMRI, but abnormalities in postmortem tissue are reported in
patients with mood changes and motor deficits (Crittenden and
Graybiel 2011). Modulation of prefrontal-prelimbic cortex
(PFC-PL) afferents innervating dorsal striatum striosomes with
enhanced NpHR (eNpHR) or C1V1 (excited by 532-nm light)
(Friedman et al. 2015) underlies balanced decision-making
disturbances. In particular, eNpHR-mediated intrastriatal inactivation of PFC-PL afferents increased the choice for the
high-cost option, while C1V1-mediated excitation changed the
behavior toward the low-cost and low-benefit option.
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The short-latency cerebellar input to dorsolateral striatum
(Chen et al. 2014) rapidly modulates symptom onset in dystonia-Parkinsonism. Bilateral optogenetic silencing of CL thalamic neurons, functionally severing the link between the
cerebellum and the striatum, abated cerebellum-induced dystonia shortly after light activation was started and dystonia
returned soon after stimulation was terminated. Thus, under
pathological conditions, the cerebello-striatal pathway may
transfer abnormal cerebellar activity to the BG, contributing to
movement disorders such as dystonia (Chen et al. 2014).
Optogenetic experiments have also increased knowledge of
changes in striatal MSNs (Cepeda et al. 2013) and cholinergic
function (Holley et al. 2015) in the R6/2 transgenic mouse
model of HD with a severe, rapidly progressing phenotype.
The cholinergic deficit in HD was previously unresolved, since
large cholinergic interneurons in the striatum are spared in the
disease. In slice preparations, activation of ChR2-expressing
PV interneurons strongly inhibited MSNs in HD model mice
but not in control mice (Cepeda et al. 2013), indicating that PV
interneurons are a possible source of increased GABA synaptic
activity on MSNs. Furthermore, optogenetic stimulation of
somatostatin-expressing interneurons inhibited striatal cholinergic interneurons in the R6/2 mouse model (Holley et al.
2015); thus the cholinergic deficit arises from a change in
activity and not degeneration of these neurons.
The BG, particularly limbic structures, are involved in the
pathology of OCD, and ablation or stimulation can decrease
symptoms in patients (Ahmari and Dougherty 2015). Two
recent studies have investigated the impact of optogenetically
manipulating orbitofrontal cortico (OFC)-striatal projections
on OCD pathology and treatment. Burguiere et al. (2013)
selectively stimulated ChR2-expressing lateral OFC-striatal
neurons and their striatal terminals in SAPAP3 (corticostriatal
postsynaptic density protein)-knockout mice, which display
anxiety and the OCD-like symptom of perseverative grooming.
Stimulation reinstated normal responses, i.e., inhibited conditioned grooming (Burguiere et al. 2013). Another study (Ahmari et al. 2013), published in the same journal issue, activated
OFC-ventromedial striatal neurons in normal mice to mimic
hyperactivity in the corticostriatal-thalamocortical circuit, previously observed in OCD patients (Ahmari and Dougherty
2015). While acute stimulation of ChR2-expressing neurons
did not generate repetitive behaviors, brief repeated activation
over several days resulted in a progressive increase in grooming behavior that correlated with an increase in stimulationevoked firing rate of neurons in the ventromedial striatum
(Ahmari et al. 2013).
The specific timing of dopamine release and NAc activity
could partly underlie conditioned place preference (CPP) behavior. Changes in limbic BG reward system and synaptic
modifications in substance abuse disorders have been investigated with viral vector-mediated optogenetic stimulation in
animal models (for review see Britt and Bonci 2013; Stuber et
al. 2012). Stimulation of ChR2-expressing mesoaccumbens
dopamine neurons resulted in CPP in drug-naive behaving
mice. Phasic, but not tonic, optogenetic stimulation of dopamine somata in VTA induced drug addiction behavior as well
as transient dopamine release measured by FSCV (Tsai et al.
2009). Additional neuronal targets for optogenetic induction of
CPP in mice, without additional drug application, are MSNs in
the NAc (Airan et al. 2009). Activation of an opsin-adreno-
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resolved by combining optogenetic stimulation of ChR2-expressing midbrain dopamine neurons with FSCV or electrophysiological recordings (Stuber et al. 2010; Zhang et al.
2015). In particular, blue light stimulation of transduced dopamine neuron terminals in the NAc shell or dorsal striatum
resulted in dopamine release in both structures but only caused
glutamate-mediated excitatory postsynaptic currents (EPSCs)
in NAc MSNs (Stuber et al. 2010; Zhang et al. 2015). The
additional use of vesicular glutamate transporter 2 (vGlut2)
conditional knockout or Cre-transgenic mice showed that this
glutamate release from dopamine neurons is vGlut2 mediated
(Stuber et al. 2010) and that dopamine and glutamate can be
released from the same mesoaccumbens axon, but not at the
same site or from the same synaptic vesicle (Zhang et al. 2015).
Further to glutamate, midbrain dopamine neurons also corelease GABA from their axons (Tritsch et al. 2012, 2014).
Activation of ChR2-expressing nigrostriatal or mesolimbic
dopaminergic terminals in the dorsal or ventral striatum
showed that GABA corelease rapidly inhibited MSN activity,
which relied on GABA uptake across the plasma membrane
rather than de novo synthesis and the activity of vesicular
monoamine transporter 2 (not vesicular GABA transporter)
(Tritsch et al. 2012, 2014).
Optogenetic stimulation experiments will continue to increase our knowledge of BG connections with other brain
regions as well as interactions of neuronal subpopulations
within healthy BG because specific cell bodies or their projections can be targeted. Given the extensive BG interconnections,
optogenetics allows the function of one projection over another
to be differentiated. Extended knowledge of BG during health
is necessary to understand changes in pathophysiological
conditions.
BG pathophysiology. Changes in BG activity underlying
neurological and psychiatric conditions have been investigated
by optogenetic stimulation in animal models of disease. These
studies have provided new insights into the mechanism of
action of electrical DBS currently used in patients or have
identified potential new treatment strategies.
Animal models for PD have investigated the underlying
mechanisms or new options for DBS. Kravitz et al. (2010)
provided the first evidence that direct-pathway MSN stimulation can ameliorate motor deficits in parkinsonian mice. In
particular, bilateral activation of ChR2-expressing D1-like
MSNs (direct pathway) completely rescued deficits in freezing,
bradykinesia, and deficits initiating locomotion in bilaterally
6-OHDA-lesioned mice, while stimulation of D2-like MSNs
(indirect pathway) induced a parkinsonian state in normal mice
(Kravitz et al. 2010). Optogenetic experiments showed that
inactivation of STN neurons, the hypothesized target effect of
electrical STN DBS in PD patients, may not be the mechanism
responsible for improving motor control (Gradinaru et al.
2009). Neither direct inhibition (via eNpHR stimulation) nor
activation (via ChR2 stimulation) of STN neurons improved
motor symptoms in unilateral 6-OHDA-lesioned rats. On the
other hand, selective stimulation of afferent axons originating
in the motor cortex that project to the STN showed a therapeutic behavioral effect (Gradinaru et al. 2009). These studies
encourage review of current DBS mechanisms and strategies
and provide additional target sites for stimulation therapies.
For an overview on optogenetic insights for PD see Vazey and
Aston-Jones (2013) and Rossi et al. (2015).
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VIRAL VECTOR USE IN BASAL GANGLIA
electrical low-frequency DBS when combined with injection of
a D1-like antagonist. These studies show that knowledge
gained by optogenetic experiments can direct electrical DBS
regimens that could be rapidly translated into the clinic to treat
addictive behavior.
Optogenetic stimulation experiments have revealed new
firing patterns and neural circuit-specific mechanisms of depression that could improve DBS treatment in patients. The
limbic-reward BG pathway is also involved in depression and
can be targeted with electrical DBS to treat patients (see
Williams and Okun 2013) and optogenetic stimulation in
animal models (see Lobo et al. 2012). Phasic, but not tonic,
stimulation of ChR2-expressing dopaminergic VTA-NAc neurons mediates susceptibility to subthreshold social-defeat stress
in freely behaving mice, measured by social avoidance and
decreased sucrose preference, while VTA-medial PFC stimulation was not effective (Chaudhury et al. 2013). Photoinhibition (via NpHR stimulation) of VTA mesoaccumbens neurons
reversed “depression-like” symptoms in previously susceptible
mice, whereas inhibition of VTA-medial PFC neurons promoted susceptibility (Chaudhury et al. 2013).
The use of optogenetics in animal models of BG pathologies
has immensely increased our understanding of neuronal subpopulations (e.g., D1- and D2-expressing MSNs in PD or
addictive behavior) or projection pathways (e.g., VTA-NAc vs.
VTA-mPFC in depression-related stress). Furthermore, this
technology raises questions about previously assumed mechanisms in pathology and treatment that may need to be reassessed. Overall, this knowledge has the potential to improve
treatment options for BG-related disorders, either promptly by
applying new findings to existing treatment options or by
developing new treatment strategies in the long term.
Chemogenetics
Chemogenetic stimulation, a technique that uses small-molecule-mediated activation of engineered proteins, is another
major technology that has increased our understanding of
neuronal circuits in health and disease (see Sternson and Roth
2014). The chemogenetic concept uses a viral vector to introduce a nonnative protein, such as an engineered G proteincoupled receptor that is only activated by a specific nonnative
chemical, into specific brain regions. Similar to optogenetic
stimulation, nonnative protein expression can be restricted to
specific neuronal phenotypes. Following this, systemic administration of a drug that crosses the blood-brain barrier activates
this nonnative chemoreceptor and associated changes in neuronal activity, chemistry, and behavior can be quantified. In
contrast to optogenetic stimulation, the latency (minutes) for
activating or inhibiting nonnative proteins is slow, and once the
chemical has been administered the effects last for hours.
Therefore, chemogenetic stimulation is ideal for investigating
long-term changes within a circuit, chronic rather than acute
effects, or for mimicking physiological parameters that occur
over longer time frames (hours-days), e.g., circadian rhythms.
Finally, chemogenetics has the advantage of selectively activating cellular signaling systems without the need to introduce
a fiber-optic probe into the brain.
The idea to activate receptors by nonnative ligands goes
back to the 1990s (e.g., Coward et al. 1998; Strader et al. 1991)
and led to the generation of several “receptors activated solely
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ceptor chimera, Opto-␣1, in NAc MSNs increased NAc neuronal activity. More interestingly, this stimulation induced CPP
behavior in drug-free mice (Airan et al. 2009). Thus optogenetic stimulation studies have refined the timing of dopamine
release in reward pathways.
Optogenetic stimulation and CPP tasks have also been used
to dissect the structures and pathways involved in drug-induced
CPP. One of the drugs often used to induce CPP in rodents is
the dopamine reuptake blocker cocaine. Similar to the dorsal
striatum, MSNs in the NAc selectively express D1- or D2-like
receptors, which have been selectively targeted in Cre-transgenic mice (Lobo et al. 2010). Combined cocaine administration and stimulation of D1-like MSNs increased CPP, while
cocaine combined with D2-like receptor stimulation attenuated
it. In a control experiment, a HSV-ChR2 vector was injected
into the NAc of wild-type animals to globally activate NAc
neuron activity during cocaine ⫹ blue light stimulation, which
enhanced CPP (Lobo et al. 2010). In light of their results, the
authors concluded that there might be an imbalance in these
two NAc MSN subtypes in the addicted brain, highlighting that
correcting this imbalance could be a treatment strategy. Besides MSNs, the NAc also contains interneurons. Witten et al.
(2010) expressed eNpHR or ChR2 in NAc cholinergic interneurons in ChAT-Cre mice. In vivo NAc light stimulation
revealed that activation of ChAT interneurons inhibited the
majority of MSNs, while inhibition of ChAT interneurons
excited most MSNs. Furthermore, photoinhibition (via eNpHR
stimulation) of ChAT interneurons resulted in reduced cocaine
CPP, illustrating that cholinergic neuron activity has a role
facilitating cocaine conditioning in freely moving animals
(Witten et al. 2010). Recently, selective optogenetic VTA
dopamine neuron self-stimulation in DAT-Cre mice induced
and progressed cocaine addiction and was associated with
increased NAc MSN synaptic plasticity (Pascoli et al. 2015).
In addition to generating or modulating animal models for
addiction behavior, virus-mediated optogenetics has been used
to investigate potential reward system-based treatment options.
Optogenetic inhibition of prelimbic cortico-accumbens neurons can prevent reinstatement of cocaine seeking behavior
(Stefanik et al. 2013). In particular, photoinhibition (via eNpHR or ArchT) of prelimbic cortical neurons or their afferents
in the NAc core prevented reinstatement of cocaine seeking
behavior. This study strengthens knowledge obtained from
pharmacological blockers about the role of prelimbic cortex
and NAc core in cocaine reinstatement behavior (Kalivas et al.
2005). Optogenetic activation of medial PFC afferents in NAc
shell induced long-term potentiation in D1-expressing MSNs,
showing that synaptic plasticity is required for locomotor
sensitization to cocaine (Pascoli et al. 2012). Specifically,
bilateral low-frequency stimulation of ChR2-transduced medial PFC afferents in the NAc shell depotentiated EPSC in
D1-expressing MSNs, abolished locomotor sensitization to
cocaine, and reset behavioral sensitization induced by chronic
cocaine injections. More recently, optogenetic experiments
have revealed that synaptic plasticity of PFC and hippocampal
inputs onto NAc D1 MSNs are associated with cocaine seeking
behavior, with hippocampus conveying information on intensity of cocaine seeking and PFC differentiating outcomes
related to cocaine and noncocaine actions (Pascoli et al. 2014).
Creed et al. (2015) confirmed that low-frequency optogenetic
stimulation prevents cocaine addiction and can be mimicked by
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ferent DREADDs in the same animals can successfully facilitate multiplexed chemogenetic control of behavior.
Parallel to the ongoing development of DREADDs, different
classes of chemogenetic LGIC tools for neuron phenotypespecific perturbation in mammalian brains have been developed (for overview see Sternson and Roth 2014). In the single
BG study, administration of the antiparasitic drug ivermectin
selectively silenced virally transduced BG neurons expressing
a mutated glutamate-gated chloride (GluCl) channel from Caenorhabditis elegans (roundworm) (Lerchner et al. 2007;
Slimko et al. 2002); therefore, LGICs are not discussed below.
Motor and limbic BG pathways (Fig. 1) have been targeted
with chemogenetic experiments, with the majority of studies
performed in mice or rats. Below we discuss the impact
DREADDs have had on increasing knowledge of BG function.
Applications in health. Distinct functional properties of
direct and indirect striatal MSNs have been explored with
DREADDs. Chemogenetic perturbations of MSNs or cortical
inputs to MSNs during striatal excitatory synaptogenesis
(mouse postnatal days 8 –14) disrupted neuronal plasticity and
MSN morphology (Kozorovitskiy et al. 2012). Extensive bilateral inhibition by CNO activation of hM4Di in direct or
indirect MSNs in the dorsolateral striatum resulted in opposing
changes: inhibition of direct (D1-like) MSNs decreased miniature EPSC frequency and spine density, whereas these measures increased for indirect (D2-like) MSNs. In contrast, CNOmediated inhibition of hM4Di-expressing corticostriatal neurons during excitatory synaptogenesis decreased miniature
EPSC frequency and spine density for both direct- and indirectpathway MSNs. For indirect-pathway MSNs, hM4Di activation effects persisted into early adulthood (Kozorovitskiy et al.
2012); however, the effect on direct-pathway MSNs was not
reported. Activity in direct- and indirect-pathway MSNs and
cortical inputs is important for determining formation and
maintenance of corticostriatal synapses and thus has important
implications for overall BG function.
Chemogenetics have been used to explore the role of specific
striatal MSNs and VTA-SN neuron subpopulations in the
control of movement. Direct-pathway MSNs were either inhibited (hM4Di) or activated (rM3Ds) by CNO administration
during discrete periods of training. Inhibition impaired performance, whereas activation significantly enhanced it and altered
behavior by causing a high-reward preference (Ferguson et al.
2013). Recently, Gi-DREADD KORD was combined with
hM3Dq in GABAergic VTA-SN neurons to allow bidirectional
control of behavior in the same animals. While SalB silenced
these neurons and enhanced locomotion, CNO activated them
and decreased locomotion (Vardy et al. 2015).
In addition to DREADD-induced changes in behavior or
neuronal recordings, it can be combined with in vivo imaging.
DREAMM (DREADD-assisted metabolic mapping) generates
whole-brain metabolic maps of cell-specific functional circuits
(Anderson et al. 2013; Michaelides et al. 2013). hM4Dimediated inhibition of MSNs in the NAc shell combined with
[18F]fluorodeoxyglucose (FDG) ␮PET imaging revealed concurrent engagement of distinct corticolimbic networks: inhibition of direct MSNs significantly increased FDG uptake in
posterior VP and VTA-SN, and inhibition of indirect MSNs
decreased FDG uptake in rostral VP (Michaelides et al. 2013).
The same DREAMM technique was employed by Anderson et
al. (2013) in rat models of opiate addiction and depression,
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by synthetic ligands” (RASSLs) (for review see Conklin et al.
2008). However, early RASSLs were not widely used or
adapted in neuroscience because of restricted selectivity of
either the engineered receptors or the nonnative ligands (for
review see Sternson and Roth 2014). A solution for these
problems arose with the development of a platform termed
“designer receptors exclusively activated by designer drugs”
(DREADDs), where intracellular signaling is modulated via
the selective activation of genetically engineered G proteincoupled receptors by pharmacologically inert, small druglike
molecules (Armbruster et al. 2007). In the last decade,
DREADDs have tremendously increased our understanding of
neuronal circuits and the number of publications using DREADDs has increased exponentially (English and Roth 2015).
Chemogenetics can also be used for cell type-specific control
of ion conductance by targeting genetically engineered ligandgated ion channels (LGICs), which enables mimicking of
ionotropic, rather than slower G protein-coupled, modulation
of neuronal activity (see Sternson and Roth 2014).
Background. Similar to optogenetics, virus-mediated gene
transduction is the most common approach for introducing
chemogenetic constructs into neurons. AAVs, HSVs, and lentiviruses are viral vectors routinely used to introduce
DREADDs, with canine adenovirus providing a recent additional option (for overview see Urban and Roth 2015; Zhu and
Roth 2015). Transgenic animals have also been produced
(Alexander et al. 2009; Farrell et al. 2013; Guettier et al. 2009).
The original DREADDs (Armbruster et al. 2007) were engineered from human muscarinic acetylcholine receptors and are
activated by clozapine N-oxide (CNO), an inactive clozapine
metabolite. Three engineered muscarinic DREADDs (see Fig.
3B) that have been frequently used in neuroscience share two
point mutations that make them responsive to CNO and unresponsive to acetylcholine (Farrell and Roth 2013; Urban and
Roth 2015; Wess et al. 2013; Zhu and Roth 2015): hM4Di
(human M4 muscarinic DREADD receptor) is coupled to G␣i
signaling and silences neuronal activity (Armbruster et al.
2007), hM3Dq is coupled to G␣q signaling and increases
neuronal activity (Alexander et al. 2009), and rM3Ds (chimeric
rat M3 muscarinic DREADD receptor with intracellular loops
from the turkey ␤1-adenoceptor) is coupled to G␣s signaling
and modulates neuronal activity (Guettier et al. 2009). The
chimeric rM3Ds (Guettier et al. 2009) expanded the DREADD
toolbox to include Gs-coupled receptors, which no native
muscarinic receptors couple to.
Most DREADDs are activated by CNO, an inert pharmacological agent with known high bioavailability and blood-brain
barrier permeability in humans and mice (Rogan and Roth
2011). CNO undergoes extensive back-metabolism to the pharmacologically active clozapine in humans (Chang et al. 1998),
which might limit rapid translation of this approach to humans
and may also cause confounding effects in animal experiments.
Use of CNO as the common inert ligand limits the effectiveness of these DREADDs for multiplexed and bidirectional
chemogenetic control within the brain. To overcome this,
Vardy et al. (2015) recently developed a new kappa-opioid
receptor DREADD (KORD) coupled to Gi (see Fig. 3B), which
is activated by salvinorin B (SalB), an inactive, druglike
metabolite of the KOR-selective agonist salvinorin A. Vardy et
al. (2015) demonstrated proof of concept that combining dif-
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VIRAL VECTOR USE IN BASAL GANGLIA
(2011) demonstrated that acute drug effects can be determined
by using behavioral adaptations associated with repeated drug
exposure. Inhibition (via hM4Di ⫹ CNO injection) of dorsal
striatum direct- or indirect-pathway MSNs had no effect on
acute amphetamine-induced hyperlocomotion in rats but
caused drug addiction with locomotor sensitization after repeated amphetamine exposure. Transient indirect-pathway inhibition enhanced the development and persistence of sensitization, while inhibition of direct-pathway MSNs impaired
sensitization persistence (Ferguson et al. 2011). These studies
indicate that different neuronal populations in the BG are
involved in specific aspects of amphetamine-induced locomotor activity. Chemogenetics can target subpathways to treat
drug addiction, and findings may help direct future drug
development (Ena et al. 2011).
Another animal model of drug addiction is based on the fact
that cocaine administration increases the amount of ⌬FosB in
NAc neurons of mice (Kelz et al. 1999). In transgenic mice
with increased ⌬FosB expression, recombinant HSV-GluR2
injection into the NAc caused GluR2 overexpression and
enhanced rewarding effects of cocaine. In contrast, overexpression of GluR2(Q) caused drug aversion, highlighting a cocaine
drug addiction mechanism that could be manipulated to treat
this disease (Kelz et al. 1999). VTA and SN dopamine neuron
activity appears to underlie this cocaine-induced behavior.
Inhibition of VTA and SN midbrain neurons expressing KORD
or hM4Di (via SalB or CNO application, respectively) decreased spontaneous and acute cocaine-induced locomotor activity in rats and also inhibited midbrain putative dopamine
neuron activity in brain slices (Marchant et al. 2016).
DREADDs have been used to investigate limbic BG nuclei
cell populations involved in reinstatement of drug seeking, a
model of addiction relapse. Distinct neuronal projections from
VP to VTA underlie specific aspects of reinstatement of cocaine seeking (Mahler et al. 2014). Transient inhibition of
hM4Di-expressing rostral VP neurons blocked reinstatement of
drug-associated, cue-induced cocaine seeking behavior, while
caudal VP neuron inhibition blocked cocaine-primed reinstatement. Chemogenetic manipulation of NAc, inspired by the
finding that NAc lesions reduce relapse rates in alcoholdependent patients (Wu et al. 2010), altered ethanol consumption and self-administration, while sucrose consumption was
unaffected (Bull et al. 2014; Cassataro et al. 2014). Transient
inhibition (via hM4Di) of NAc core and shell neuronal populations reduced ethanol consumption, whereas hM3Dq-mediated transient activation had no effect, consistent with electrolytic lesions of NAc core (Cassataro et al. 2014). NAc core
astrocyte-specific DREADDs (via hM3Dq) can modulate ethanol-related drug-seeking behavior (Bull et al. 2014); activation facilitated intracranial self-stimulation and reduced the
motivation for ethanol self-administration after 3 wk of abstinence. Scofield et al. (2015) recently demonstrated that transient CNO activation of hM3Dq-expressing NAc core astrocytes increased NAc core extracellular glutamate levels in vivo
and inhibited cue-induced reinstatement of cocaine seeking,
probably by inhibiting cue-induced synaptic glutamate spillover. Neuronal subpopulations in VP as well as neuronal and
astrocyte populations in NAc are involved in drug-seeking
behavior.
Virus-mediated chemogenetic experiments have increased
our understanding of physiological interactions within the BG
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showing that hM4Di-mediated inhibition of prodynorphinexpressing neurons in periamygdaloid cortex increased metabolic activity in the extended amygdala, a key structure of the
extrahypothalamic brain stress system. DREADDs have also
been used to image the location and intensity of lentivirusmediated hM4Di expression in the putamen of nonhuman
primates with in vivo PET (Nagai et al. 2014) and matched
with postmortem immunohistochemical analysis of hM4Di
expression. These researchers have shown in other monkeys
that CNO administration to AAV-mediated hM4Di expression
in the ventral striatum altered performance in a reward-size
task, similar to effects seen with muscimol inactivation (Nagai
et al. 2014). Thus DREADDs can produce behavioral changes
in higher-order species. These multidisciplinary studies show
that DREADDs and PET imaging can enhance our understanding of the neuronal mechanisms underlying changes in
behavior.
Applications for neurological and psychiatric disorders.
One strategy to treat neurological disorders is to directly target
neuronal populations affected in the condition. DREADD activation of cholinergic pedunculopontine tegmental nucleus
(PPTg) neurons using hM3Dq increased PPTg spiking activity
and improved motor deficits in PD model rats (Pienaar et al.
2015); thus selective PPTg activation might be a potential
target to treat PD. Another strategy has been to express a
DREADD in transplanted neurons, enabling subsequent control by systemic CNO injections (Dell’Anno et al. 2014). In
particular, unilateral 6-OHDA-lesioned rats were transplanted
with hM3Dq-expressing induced dopaminergic (iDA) neurons,
generated from skin fibroblasts, in the lesioned striatum. The
grafted neurons were functionally integrated, and CNO-activated iDA neurons improved motor deficits, similar to primary
dopamine neurons grafted in lesioned rats (Dell’Anno et al.
2014). Two recent reviews (Sharma and Pienaar 2014; Vazey
and Aston-Jones 2013) discuss the current state and possibilities for future applications of DREADDs in the field of PD
research.
Another strategy to treat disorders is to find neuroprotective
treatments. Chemogenetics have elucidated neuroprotective
effects of the cannabinoid 1 (CB1) receptor in the dorsolateral
striatum in health (Chiarlone et al. 2014). CNO activation of
hM3Dq-expressing corticostriatal neurons enhanced glutamatergic transmission and excitotoxicity, which was associated
with reduced DARPP-32 immunoreactivity and impaired RotaRod performance. CNO effects were abrogated by the selective NMDA receptor antagonist MK-801 or tetrahydrocannabinol (THC), a CB receptor agonist, indicating that striatal CB1
cannabinoid receptors are neuroprotective. This physiological
study has important implications for HD and other neurological
disorders (Chiarlone et al. 2014).
Chemogenetic approaches are increasing knowledge of the
limbic BG pathways involved in substance abuse disorders (for
review see Ferguson and Neumaier 2015; Yager et al. 2015).
HSV vectors with promoters for enkephalin and dynorphin that
express the DREADD hM4Di have been used to examine the
role of striatopalldial or striatonigral pathways in amphetamine-induced locomotor sensitization (Ferguson et al. 2011).
Stimulation of hM4Di by CNO hyperpolarized and silenced
transduced striatopalldial or striatonigral neurons, respectively,
and was associated with increased and decreased amphetamine-induced sensitization. Furthermore, Ferguson et al.
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and BG connections with other brain regions. Furthermore,
chemogenetics has revealed that distinct cell populations (e.g.,
NAc neurons and astrocytes) are involved in addiction-related
behavior and has provided new perspectives for potential PD
treatments (e.g., long activation periods of PPTg neurons or
transplanted neurons). Further development of chemogenetic
technology has the potential to improve pharmacological therapies of BG-related disorders.
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Creating and Treating Animal Models of BG Disorders
Background. Viral vector technology has been routinely
used to alter cellular and genetic functions by increasing or
decreasing protein expression or delivering genes for therapeutic benefit. However, more recently viral vectors have been
used to create genetically modified animals, create disease
models, or alter transcription to study epigenetic changes.
Cre-lox transgenic rodents have been used to develop
“brainbow” labeling (Cai et al. 2013), which has been used to
elucidate the roles of striatopallidal and striatonigral pathways
in health and disorders like PD (Ena et al. 2011; Kravitz et al.
2010). The technology has been combined with bacterial artificial chromosome (BAC) Cre-recombinase driver lines and
AAVs to target expression in specific neuron or glial populations (Gerfen et al. 2013). Cerebral cortex and BG BAC
Cre-driver lines have been characterized (Gerfen et al. 2013)
and will be useful for exploring neuronal circuits.
Viral vectors have been used to insert or delete genes for
gene therapy options. Zinc finger nucleases, transcription activator-like effector nucleases (TALENs) (Joung and Sander
2013), and CRISPR-Cas can be used to edit the genome;
CRISPR/Cas technology is superseding zinc finger nucleases
(Ledford 2015). CRISPR is an acronym for clustered, regularly
interspaced, short palindromic repeats; it identifies the gene to
be edited. Cas stands for CRISPR-associated protein and is a
RNA-guided DNA endonuclease enzyme that cleaves the host
DNA to add or delete the target gene. CRISPR-Cas technology
is still in its infancy and has not been utilized in the BG but
could be used in the future to create animal models of disease
and compile genetic libraries that will aid genetic screening
and eventually therapeutic applications (Sander and Joung
2014).
Animal models. Animal models of BG dysfunction have
been created with viral vectors. In some, primarily early-onset
cases, PD has a genetic component. Based on this, viral vectors
(lentivirus, AAV, HSV, and adenovirus) have been used to
create animal models of these genetic forms (Löw and Aebischer 2012). These viral vector models have been used in rats,
mice, and marmosets and are based on dominant mutations of
alpha-synuclein (in Lewy body aggregates) and leucine-rich
repeat kinase-2 (LRRK-2) genes linked to PD, which cause
overexpression and aggregation of these proteins in the BG and
degeneration of dopaminergic neurons. Recessive mutations of
genes associated with early-onset PD have also been created by
overexpressing parkin substrates, which causes toxic accumulation of parkin and eventually dopaminergic neuron loss in the
substantia nigra (see Löw and Aebischer 2012). Parkin substrates that have been used are Pael-R (Parkin-associated endothelin receptor-like receptor), CDCrel (protein at presynaptic
terminals), p38/JTV (a substrate aminoacyl-tRNA synthetase
cofactor), and synphilin-1 (a neuronal cytoplasmic protein).
AAVs have delivered small interfering RNA to the BG to
decrease TH gene expression, a dopamine biosynthesis enzyme, which resulted in PD-like behavioral deficits, illustrating
that small interfering RNA could be used to create other animal
models or gene therapy treatments (Hommel et al. 2003).
Optogenetic activation in D1- or D2-Cre mice has also resulted
in a behavioral animal model of PD. Activation of ChR2expressing D2-like MSNs in D2-Cre transgenic mice induced
parkinsonism, but activation of the direct pathway in D1-Cre
mice increased movements and decreased freezing and bradykinesia (Kravitz et al. 2010). This model could be used to study
the relationships in changes in BG circuits and motor function
(Kravitz et al. 2010).
To date, the only viral vector-derived animal model for HD
involves lentivirus-mediated expression of the mutant human
huntingtin gene that has 82 CAG repeats, causing formation of
inclusions in striatal neurons and loss of striatal MSNs in
rodents (Ramaswamy et al. 2007), which is similar to the
pathology that occurs in the BG of humans. Currently, these
researchers are developing a nonhuman primate model of HD
(Ramaswamy et al. 2007). Finally, circuitry has been selectively lesioned, using viral vectors to examine how a pathway
normally functions and to consider how it might contribute to
BG disorders. Takada et al. (2013) developed a new vector,
NeuRet, based on HIV with a hybrid of rabies and VSVg
glycoproteins, to deliver immunotoxin to ablate the hyperdirect
pathway neurons in primates, showing that the motor cortex
excites GPi neurons through this pathway, which was later
confirmed with pharmacophysiological and electrical stimulation methods.
Treating BG disorders. To best answer any research question or develop gene therapy strategies using viral vectors, the
optimal vector needs to be chosen. The advantages of one
vector over others is described in Viral Vector Use in Neuroscience; however, most studies that use vectors rarely outline
the rationale for their choice. Choosing the best vector is a
critical step for progressing gene therapy technologies into
clinical trials.
The longevity and specificity of vectors in BG nuclei have
been examined in rodents, pigs, and primates. Lentiviral vectors have been compared to a Moloney murine virus in rodent
striatum and were shown to provide sustained expression for at
least 6 mo and transduced ⬎88% of striatal neurons. A major
advantage of lentiviral vectors is that they can transduce
nondividing cells (Blömer et al. 1997). However, AAVs are
more commonly used for gene therapy in the BG. Recombinant
AAV5 has been preferred over AAV1 because AAV5 also
transduced nonneuronal cells in rat and pig striatum (Kornum
et al. 2010). Studies comparing viral vectors in monkey brains,
because they more closely resemble the human brain, found
that AAV2 injected into the putamen, thalamus, and corona
radiata was easier to visualize by fMRI than AAV1 (Fiandaca
et al. 2009; Fiandaca and Federoff 2014) and thus may translate effectively to patient applications in the clinic. To this end,
AAV2 serotypes have been compared in primates to determine
transduction efficiency. Injection of AAV2 serotypes 1, 2, 5,
and 8 into the caudate putamen of rhesus monkeys revealed
that more cells were transduced with AAV2/8 than the others;
however, this was not specific to neurons (Sanchez et al. 2011).
Several technologies introduced above provide important
potential mechanisms for gene therapy. For example, CRIPSR/
Cas technology could eventually be used for gene therapy
Review
VIRAL VECTOR USE IN BASAL GANGLIA
normal mice, which caused depression-like behaviors (Gelfand
and Kaplitt 2013). Optogenetic stimulation in the pII knockout
produced antidepressant effects. Similar targets have been
identified for treating drug addiction. pII-knockout mice have
increased CPP after cocaine administration, which can be
reversed after AAV pII gene therapy (Kaplitt et al. 2010). Mice
with knockdown of the BDNF receptor TrkB in D1-expressing
MSNs also had increased CPP in response to cocaine administration, while TrKB in D2 MSNs or optogenetic stimulation
of D2 MSNs in the NAc (Lobo et al. 2010) had the opposite
effect. Schizophrenia can have a genetic basis, but further
studies are needed to understand the complexity of schizophrenia symptoms. Genetic deletion of Dgcr8, which is a microRNA processor required for proper dendrite development,
affects working memory (Stark et al. 2008), whereas knocking
out the DISC 1 gene (disrupted in schizophrenia 1) affects
progenitor proliferation in the dentate gyrus (Mao et al. 2009).
A deficiency in the postsynaptic scaffolding protein SAPAP3
(SAP90/PSD95-associated protein 3) may underlie OCD
symptoms because SAPAP3-knockout mice produce OCD
behaviors, and OCD behaviors are reversed with lentiviral
vector delivery of SAPAP3 to the dorsolateral striatum or
administration of serotonin uptake inhibitors (Welch et al.
2007). Another therapeutic target for OCD is the transmembrane protein Slitrk5 (SLIT and NTRK-like protein 5), because
knockout mice express OCD-like behaviors (Gelfand and Kaplitt 2013; Shmelkov et al. 2010). Both SAPAP3 and Slitrk5
could be used as gene therapy targets in OCD. Thus these
molecular BG targets in preclinical studies of psychiatric
disorders could eventually lead to treatment options in patients.
Some preclinical gene therapy experiments have been
moved into patient clinical trials. These human trials were
monitored with PET scanning or ELISA assays and clinical
tests. For PD, clinical trials have reached open label, phase
I–II. Gene therapy delivery of GAD, neurturin, or ProSavin
(AADC, TH, and cyclohydrolase 1) via an AAV or lentiviral
vector to the STN or striatum saw some improvements in
movement deficits in PD patients (Feigin et al. 2007; Kaplitt et
al. 2007; Olanow et al. 2015; Palfi et al. 2014). Although gene
therapy did not always significantly improve clinical measures
(Kaplitt et al. 2007; Olanow et al. 2015), patients did not suffer
any adverse effects from the treatment, indicating that gene
therapy may become an effective treatment for BG disorders.
In the future, gene therapy may become a conventional treatment for many neurological disorders.
Future Directions
Viral vector use in BG research is expected to continue to
increase in the next decade as more groups employ vectors for
optogenetic and chemogenetic stimulation, to trace pathways
and create or treat animal models of BG disorders. The specificity of viral vectors to target a neuronal or glial population
could be used to further delineate BG circuits, connectomes,
and microcircuits. Imaging can be done in vitro or in vivo from
the ultrastructural level through to large blocks of brain, and
continued developments in microscopy and bioimaging (fMRI,
PET) will improve the resolution of images at all levels.
Although injection of viral vectors combined with bioimaging
may not become a routine clinical scan, it is possible that these
technologies could be combined in rare conditions in patients.
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treatments in the BG. First, effective delivery needs to be
achieved, aberrant mutations in the genome avoided, and
ethical issues around being able to change the human genome
addressed (Ledford 2015). Currently, gene therapy is the most
advanced viral vector technology for treatment applications in
humans (Denyer and Douglas 2012; Lowenstein et al. 2014).
Viral vectors have also been used to treat animal models of
BG dysfunction as a preclinical step toward gene therapy
clinical trials. To date, most of the studies focus on PD or HD.
To determine which gene therapy tool may be most efficacious
for PD, viral vector delivery is sometimes combined with
imaging techniques. Glial cell-derived neurotrophic factor
(GDNF) has been delivered in vivo to the striatum with AAV2
vectors in 6-OHDA-lesioned PD model rats and monkeys. In
rodents, the striatum was the most effective site of GDNF
delivery because it reached all affected BG areas compared
with delivery in the substantia nigra (Ciesielska et al. 2011). In
monkeys, low levels of GDNF delivery protected neurons, did
not interrupt dopamine synthesis, and provided behavioral
attenuation of symptoms (Eslamboli et al. 2005). Other approaches include increasing dopamine-synthesis enzymes like
aromatic-amino acid decarboxylase (AADC), GTP cyclohydrolase 1 (GCH), and TH in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) parkinsonian monkeys. Delivery of
these dopaminergic enzymes increased the number of dopamine-positive neurons in the putamen, increased dopamine
levels in the brain, and restored motor function (Muramatsu et
al. 2002).
Gene therapy has also been investigated in animal models of
HD. Neurotropic factors are being used to investigate neuronal
survival in quinolinic acid (QA) or transgenic animal models of
HD. Viral delivery of brain-derived neurotrophic factor
(BDNF) reduced striatal cell loss in QA HD model rats (Henry
et al. 2007; Southwell and Patterson 2011). A member of the
GDNF family, neuretin, similarly rescued striatal MSNs in
models of HD when delivered via viral vector (Southwell and
Patterson 2011). Another use of viral vectors in the treatment
of HD could be the delivery of growth factors like BDNF to
neural stem cells in the subventricular zone, which in turn
proliferate into MSNs and migrate to the striatum (Henry et al.
2007; Southwell and Patterson 2011). Similarly, AAV1/2 delivery of BDNF or GDNF enhanced striatal and dopamine
neuron survival in vitro (Kells et al. 2007). Finally, calmodulin
delivery via AAV has improved motor function, reduced the
number of inclusions and cell loss, and increased body weight,
possibly by improving HD-associated deficits in calcium signaling (Southwell and Patterson 2011).
Although less studied than PD and HD, gene therapy targets
have also been identified in models of psychiatric disorders.
Anatomical and molecular BG targets involving serotonin and
BDNF have been identified (Gelfand and Kaplitt 2013) in
depression, schizophrenia, and OCD, possibly because these
disorders have overlapping symptoms. BDNF levels are decreased in postmortem tissue from depression patients (Deisseroth 2011), and serum response factor, which is an activator
of early genes, is also decreased in NAc in postmortem tissue
(Ramanan et al. 2005). The serotonin receptor 5-HT1b is
thought to be involved in depression. It interacts with a cytoplasmic protein, pII, which causes depression-like symptoms in
knockout mice. AAV-mediated delivery of small interference
RNA has been used to block pII production in the NAc in
2139
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VIRAL VECTOR USE IN BASAL GANGLIA
new treatment strategies. An obvious step in the future will be
the use of optogenetic and chemogenetic stimulation in patients. Development of other viral vector-based treatment options for human BG disorders is initially limited by imagining
new ways to combine these tools and then applying them in
preclinical studies.
GRANTS
This work was supported by grants from the Neurological Foundation of
New Zealand (to L. C. Parr-Brownlie and S. M. Hughes), Health Research
Council of New Zealand (to L. C. Parr-Brownlie and S. M. Hughes), Royal
Society of New Zealand Marsden Fund (to S. M. Hughes), and Brain Research
New Zealand, Centre of Research Excellence (to L. C. Parr-Brownlie and
S. M. Hughes).
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
Author contributions: R.J.S., S.S.-A., S.M.H., and L.C.P.-B. prepared
figures; R.J.S., S.S.-A., S.M.H., and L.C.P.-B. drafted manuscript; R.J.S.,
S.S.-A., S.M.H., and L.C.P.-B. edited and revised manuscript; R.J.S., S.S.-A.,
S.M.H., and L.C.P.-B. approved final version of manuscript.
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Nevertheless, the anatomy and physiology of inputs to and
from the BG will continue to be explored in preclinical studies
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the deficits that occur with BG dysfunction. Such basic science
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drug addiction.
While viral vector technologies have been used in the BG,
additional information would be gained by using multidisciplinary approaches. For example, viral vectors could be combined with tissue clearing techniques, optogenetic or chemogenetic stimulation, tissue processing, and imaging to examine
anatomical and physiological changes at gross and ultrastructural levels in the same tissue (Pastrana 2010; Watanabe et al.
2014). Such approaches would address unanswered questions
such as, “Does altered BG activity in PD change synaptic
structure (number of synapses or dendritic spines) or function
(plasticity mechanisms) downstream from the striatum?” These
new technologies will also permit previously unimagined experiments such as investigating the relationship between
changes in gene expression and behavior underlying drug
addiction by using TALEs and LITEs (Konermann et al. 2013).
An initial challenge for BG researchers is to extend their
understanding and use of these multidisciplinary viral vector
approaches.
Although gene therapy is in its infancy, it has been trialed in
PD patients. Importantly, phase I–II trials have reported that
gene therapy applied to the brain is safe, with few adverse
effects and no reports of tumor development (Feigin et al.
2007; Kaplitt et al. 2007; Olanow et al. 2015; Palfi et al. 2014).
Currently, gene therapy is not more efficient than other treatments, but these studies may be biased because they are usually
offered to patients at later stages of the disease for whom
conventional treatments have side effects. Now that gene
therapy has been shown to be safe, it will be important to trial
future strategies on patients earlier in the disease process.
Controversial future gene therapy options include the use of
CRISPR/Cas technology to edit the human genome to prevent
a BG disorder or reduce its severity, perhaps by removing
many of the CAG repeats in the huntingtin gene of HD
patients. At this stage, CRISPR/Cas gene editing would need to
be performed in preclinical studies aimed at reducing BG
dysfunction and shown to be highly efficacious. Then, the
ethics of CRISPR/Cas use in patients (Ledford 2015) would
need to be debated before the first clinical study could be
conducted.
Preclinical studies continue to explore potential new treatment options for BG conditions. Optogenetic stimulation in the
BG has improved understanding of function (Kravitz et al.
2010) and has continued to tease out the mechanisms of DBS
(Gradinaru et al. 2009). More recently, recordings of VA motor
thalamus showed impaired movement-related modulations in
firing rate and low-threshold calcium spike bursts in PD model
rats (Bosch-Bouju et al. 2014). On the basis of this finding, we
optogenetically stimulated VA motor thalamus with complex
patterns (neuronal activity previously recorded from the VA of
controls) and found that reaching performance improved in PD
model rats (Seeger-Armbruster et al. 2015). Thus application
of findings from basic science can successfully identify novel
treatment approaches for BG disorders, and future chemogenetic studies will provide additional opportunities for creating
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