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The Distribution of Immunoreactivity for
Intracellular Androgen Receptors in the
Cerebral Cortex of Hormonally Intact Adult
Male and Female Rats: Localization in
Pyramidal Neurons Making Corticocortical
Connections
Mar y Kritzer
Gonadal hormones are known to broadly influence cortical information processing. Findings from this study in rats suggest that for
androgens, this influence may include stimulation of underlying
corticocortical connections. First, immunoreactivity for intracellular
androgen receptors, while present in all regions and layers examined, was found to be particularly abundant in sensory and motor
regions, and within these, within their major pyramidal cell layers,
i.e. layers II/III and V/VI. Double labeling immunocytochemical
studies for androgen receptors and for neuron-specific markers then
confirmed that the majority of receptor-bearing cortical cells were
pyramidal neurons. Finally, combined analyses of cortical receptor
immunoreactivity and retrograde labeling produced by tracer injections made in specific subcortical (caudate, nucleus accumbens,
superior colliculus, thalamus) areas yielded only isolated examples
of receptor/tracer overlap. However, injections made within the
cortex itself (sensory, motor, associational areas) retrogradely
labeled cortical cells some 50% or more — especially within injected
hemispheres, were receptor-immunoreactive. Thus, the regional,
laminar, and cellular distributions of immunoreactivity in the rat
cerebrum largely identify pyramidal neurons with connectional
signatures aligning intracellular androgen receptors with the local,
associational, and to a lesser degree, callosal circuits that interlink
territories of the cortical mantle and play key roles in cortical information processing.
(Zhang et al., 2000), and stress-induced rates of dopamine and
noradrenaline turnover in the prefrontal cortices of adult male
rats (Handa et al., 1997) on the other hand, are endpoints that
are stimulated by estrogens but inhibited by androgens.
Although mechanisms are uncertain, one factor that could
help define the spheres of influence and/or divisions of labor
seen in hormone stimulation of the cerebral cortex could be
the specific cortical distributions that intracellular, i.e. nuclear,
estrogen (ER) and androgen receptors (AR) maintain. While
clearly more abundant, however, less is known about the
cortical distribution of intracellular AR as compared with ER
proteins. Available evidence suggests, though, that at least in
rats these two hormone pathways occupy distinct niches
among cerebral cortical circuit structures. Specifically, while
recent light microacopic studies have shown that ER proteins
are present mainly within subsets of the nuclei of nonpyramidal interneurons (Blurton-Jones and Tuszynski, 2002;
Kritzer, 2002), there is some evidence to suggest that androgen
receptors may be preferentially aligned with the pyramidal
neurons of the cortex. In situ hybridization studies, for
example, have shown that cells containing mRNAs for
androgen receptor proteins are particularly concentrated in
cortical layers where pyramidal cells predominate (Simerly et
al., 1990), and immunocytochemical mapping of AR proteins,
although limited to a single representative cortical level,
described the morphology of at least some receptor bearing
cells as pyramidal (Clancy et al., 1992). If indeed intracellular
androgen receptors are predominantly located within the
projection neurons of the cortex then in order to fully define
the anatomical and potential functional contexts of these
target cells, precise information is needed not only about the
regional, laminar, and cellular localization of these receptors
but also about the connections that receptor-bearing neurons
make. Accordingly, the studies presented here used single and
double label immunocytochemistry, respectively, to map and
phenotype intracellular AR-bearing cells in all major cortical
fields of hormonally intact adult male and female rats. A second
series of studies followed that combined receptor immunocytochemistry and retrograde tract tracing to explore the
afferent connections of these receptor-bearing neurons. Some
of this work has been presented in abstract form (Kritzer,
2003).
Keywords: estrogen, gonadal hormones, intracellular receptors, neocortex,
pyramidal neurons, testosterone
Introduction
The cerebral cortices in humans, non-human primates, and
rodents are sensitive to gonadal hormones; the presence of
intracellular androgen and estrogen receptor proteins (e.g.
Clancy et al., 1992; Puy et al., 1995; Finley and Kritzer, 1999;
Blurton-Jones and Tuszynski, 2002; Kritzer, 2002), mRNAs
(e.g. Simerly et al., 1990; Shughrue et al., 1997), and hormone
binding sites (Pfaff and Keiner, 1973; Sar and Stumpf, 1985;
Sholl and Pomerantz, 1986; Sholl and Kim, 1990) within the
cortical mantle are consistent with findings that both testicular
and ovarian hormone-signaling pathways are active in influencing specific endpoints of cortical structure and/or function.
However, in many instances these cortically directed influences are either non-overlapping or diametric. For example, in
rats, estrogen but not androgen stimulation promotes neurite
outgrowth in organotypic cortical cultures (see ToranAllerand, 1991) and modulates the development of cortical
catecholamine activity (Stewart and Rajabi, 1994), while androgens but not estrogens chronically stimulate cortical catecholamine innervation (Kritzer, 2000). The survival and
differentiation of cultured embryonic rat cortical neurons
Cerebral Cortex V 14 N 3 © Oxford University Press 2004; all rights reserved
Department of Neurobiology and Behavior, Stony Brook
University, Stony Brook, New York 11794-5230, USA
Materials and Methods
Tissue
Single and double label immunocytochemistry was carried out in a
total of 10 hormonally intact, adult (200–400 g) Sprague–Dawley rats
(four males, six females); all were subjects in a previous study of
cortical estrogen receptors (Kritzer, 2002). For the females, vaginal
cytology smears were used to verify that animals were either in pro-
Cerebral Cortex March 2004;14:268–280; DOI: 10.1093/cercor/bhg127
estrus (n = 3) or diestrus (n = 3) at the time of euthanasia. An additional 21 animals (17 males, four females) were used in experiments
that combined retrograde tracing and receptor immunocytochemistry; the males were subjects in an unrelated tract-tracing study.
Surgical Procedures: Craniotomy and Intracerebral Injection
Rats were anesthetized with ketamine and xylazine (90 and 10 mg/kg,
respectively, injected i.m.) and placed in a stereotaxic apparatus.
Next, a midline incision was made over the skull, and burr holes were
drilled over sterotaxically determined loci (Paxinos and Watson,
1986). Pulled glass micropipettes filled with the retrograde tracer,
cholera toxin (inactive B-subunit conjugated to colloidal gold, List
Biologicals, Campbell, CA), diluted to 0.025% in sterile water were
then lowered to specific depths, and tracer was ejected into either the
cingulate (n = 3), primary motor (n = 3) or primary somatosensory
cortex (n = 3), the medial or lateral caudate nucleus (n = 4), the
nucleus accumbens (core and shell, n = 2), the superior colliculus
(n = 2) or the ventrolateral/ventral anterior nuclei of the thalamus
(n = 4). At each site, tracer was pressure-injected in roughly 10 nl
increments over a 15–20 min period using visual monitoring of the
meniscus within the calibrated micropipettes to measure the volume
of tracer ejected; total volumes ranged from 50 to 300 nl. After
injecting, pipettes were left in place for 15 min and were then slowly
withdrawn to minimize tracer efflux up the pipette track. A 7–10 day
survival period followed the surgery, after which animals were
transcardially perfused.
Perfusion
Rats were deeply anesthetized with ketamine and xylazine (90 and
10 mg/kg, respectively, injected i.m.). After deep reflexes could no
longer be elicited, animals were transcardially perfused with ∼100 ml
of phosphate-buffered saline (PBS), followed by 500–1000 ml of 2%
paraformaldehyde containing 15% saturated picric acid in 0.1 M PBS,
pH 7.4. Following perfusion, brains were removed and placed in
0.1 M PBS containing 10, 20 and then 30% sucrose for cryoprotection.
After the brains had sunk in the 30% sucrose solution, they were
frozen in powdered dry ice, and serially sectioned on a freezing microtome (40 µm).
Histology
Single Label Androgen Receptor (AR) Immunohistochemistry
To visualize AR-immunoreactivity, sections were rinsed in 0.1 M phosphate buffer (PBS), pH 7.4, incubated in 1% H2O2 in PBS for 45 min,
rinsed again in 0.1M PBS, and were then placed in 1% sodium borohydride in PBS (45 min). Sections were then rinsed thoroughly in
50 mM Tris-buffered saline (TBS), pH 7.4, and placed into a blocking
solution [TBS containing 10% normal swine serum (NSS) for 2 h at
room temperature]. Sections were then incubated in one of two antiAR primary antisera (PG-21 antibody, working dilution1:150, generously provided by Dr G. Greene, Ben May Institute, Chicago, IL; N-20,
working dilution 1:4000, Santa Cruz Biotechnology, Inc., Santa Cruz,
CA) diluted in TBS containing 1% NSS, for 3 days at 4°C. Both antibodies were generated against synthetic peptides corresponding to Nterminal amino acids of the rat androgen receptor, and both have
been proven to be specific for intracellular androgen receptors in this
study and in previous analyses (Greene et al., 1980; Prins et al., 1991;
Kritzer, 1997). Most of the analyses and all of the figures presented in
this study utilized the PG-21 antibody; some single label mapping was
carried out using the N-20 antiserum to supplement the limited
amount to the PG-21 serum available at the time of this study. There
were no obvious differences in the distribution or apparent density of
immunoreactivity when either of the two antibodies was used.
Sections were subsequently rinsed in TBS before being placed in
biotinylated secondary antibodies (2 h, room temperature, working
dilution 1:100, Vector Laboratories, Temecula, CA). Sections were
then rinsed in TBS, and placed in avidin–biotin–complexed horseradish peroxidase (ABC, Vector Laboratories; 2 h, room temperature).
After additional rinses in TBS and subsequent rinses in Tris buffer
(TB), pH 8.0, sections were reacted using 0.07% 3′,3-diaminobenzadine (DAB) and 0.06% nickel ammonium sulfate as the chromagen.
Double Label Immunohistochemistry
For double labeling experiments, antigens were immunolabeled
sequentially beginning with the anti-AR polyclonal antibody. After the
DAB/nickel reaction, sections were rinsed in TBS and replaced in
blocking solution (TBS with 10% NSS, 2 h) prior to being incubated in
one of five primary monoclonal antibodies (1–2 days, 4°C). The monoclonal antibodies used recognized either neuron specific enolase
(NSE; Chemicon International Inc., Temecula, CA, working dilution
1:100), glial fibrillary acidic protein (GFAP; Chemicon International,
working dilution 1:300) an oligodendrocyte-specific moiety [anti-RIP
developed by Dr Susan Hockfield, Yale University, New Haven, CT,
obtained from the Developmental Studies Hybridoma Bank, a facility
maintained by the Department of Biological Sciences, University of
Iowa, Iowa City, IA, under contract NO1-HD-7-3263 from the National
Institute of Child Health and Development (NICHD), working dilution
of 1:3], parvalbumin (PV, Sigma Chemical Corp., St Louis, MO,
working dilution 1:1000) or calbindin (CB, Sigma, working dilution
1:1000). After incubations in biotinylated secondary antibodies and in
ABC solution, sections were rinsed in TBS, then rinsed in TB, pH 7.6,
and finally reacted a second time using DAB as chromagen.
Tracer Visualization
Sections were reacted to visualize the gold-conjugated cholera toxin
using a silver enhancement technique. For this procedure, sections
were first rinsed in 0.1 M PBS, and were then silver enhanced for
90–120 min in a solution composed of equal volumes of the IntenSE
Initiator and Enhancer solutions (Amersham, Arlington Heights, IL).
Sections were then rinsed in 0.1 M PBS, fixed in 2.5% sodium thiosulfate (10 min), washed extensively again in 0.1 M PBS, and were
finally slide mounted, dehydrated and placed under coverslips. Some
sections were counterstained with 1% cresyl violet.
Combined AR-immunocytochemistry and Tracer Visualization
To combine AR-immunocytochemistry and retrograde tracing, prior
to the silver enhancement procedure above, sections were rinsed in
1% H2O2 for 30 min. After the enhancement procedure, sections were
rinsed in TBS, pH 7.4, blocked for 2 h in TBS containing 10% NSS and
then immunoreacted as described for the single procedure (above).
Control Experiments
All immunohistochemical labeling protocols (single, double and in
combination with retrograde tracing) were carried out on representative sections from male and female animals with the omission of
primary or secondary antibodies, or using antibodies against AR that
had been preabsorbed for 5 h with a 10 M excess of the immunizing
AR peptide (AR-21, provided by Dr G. Greene, Ben May Institute;
sc-816 P, Santa Cruz Biotechnology). In the injected animals, receptor
immunoreactivity was also carried out in representative sections that
were not silver enhanced to determine whether this procedure
affected detectability of AR-immunoreactivity.
Data Analysis
Cellular Phenotyping
The sections used to assess the cellular identity of AR-immunoreactive
(AR-IR) cells in the rat cerebrum (double label immunocytochemistry)
were taken at roughly 400 µm intervals from the rostral to caudal
poles of the brains of uninjected animals. Within these sections,
sensory, motor, association, and allocortical regions were examined.
Colocalization between AR-immunoreactivity and immunoreactivity
for NSE, GFAP, RIP, PV or CB was defined as a cell that had a blue/
black DAB/nickel labeled nucleus that lay within the same focal plane
as a surrounding brown, DAB labeled cytoplasm (see Kritzer, 1997;
Creutz and Kritzer, 2002).
Receptor Mapping
For mapping of AR-immunoreactivity, cytoarchitectonic and laminar
boundaries were visualized using nissl counterstaining (1% cresyl
violet) in an adjacent series of the tissue sections, and in some representative immunoreacted sections. Cortical regions and layers were
identified according to Zilles and Wree (1985). Detailed assessments
of labeling distribution were made that included evaluation of the
Cerebral Cortex March 2004, V 14 N 3 269
regional and laminar locations and relative densities of AR-IR nuclei.
For each animal subject, a 1-in-6 series of sections was evaluated.
Connections of AR-IR cells: Qualitative and Quantitative Analysis
Cases of injected animals were selected for analysis based on the positioning of injection sites determined in a 1-in-6 series of silver
enhanced and cresyl violet counterstained sections. Using a Zeiss
Axioskop and a 5× objective, darkfield illumination revealed injection
sites as dense accumulations of silver-enhanced gold particles within
which cellular labeling was completely obscured (see Venkatesan and
Kritzer, 1999); brightfield optics were used to precisely place sites
with respect to cytoarchitecture. The target regions injected were the
caudate nucleus (n = 4), nucleus accumbens (n = 2), thalamus (n = 4),
superior colliculus (n = 2) and the sensory (n = 3), motor (n = 3) and
cingulate (n = 3) cortex.
For each of the 21 selected cases, a separate 1-in-6 series of sections
were silver enhanced and immunoreacted for AR; from these qualitative analyses of the two labels were made, and five sections located at
and anteriorly and posteriorly adjacent to the apparent central foci of
the cortical field or fields of labeling were quantitatively analyzed.
Because the seemingly most densely labeled sections were purposefully selected for assessment, these analyses did not utilize a systematic random sampling approach. However, in including a series of
sections located anterior and posterior to these central starting points,
in all cases the labeling evaluated included central to peripheral
portions of the labeled fields. Neurons within these sections were
identified as retrogradely labeled if the number of gold particles in
their somata exceeded four-times background levels. All such cells
located within the cerebral cortex were then recorded in camera
lucida drawings using darkfield optics and a 20× objective, and were
assigned to cortical regions and layers identified in nissl counterstaining in the same or adjacent sections. Separate drawings were
made of labeling in injected and where applicable, uninjected hemispheres, and superimposed on these were the locations of AR-IR
nuclei visualized using brightfield illumination. All cells with potential
receptor/tracer overlap were further examined using brightfield
optics and a 40× or 63× objective to verify cellular colocalization, i.e.
that immunoreactive nuclei were situated at appropriate planes with
respect to silver grain-containing cytoplasms. For each injection site
evaluated the drawings produced included totals of between 200 and
600 retrogradely labeled cells per animal subject, per hemisphere, per
injection site. From each of these drawings the proportions (percentages) of the total numbers of retrogradely labeled cells identified that
were also AR-immunoreactive were calculated. Because for each injection site evaluated the proportions from the five sections evaluated all
lay within a 5% of one another, cell counts were summed across
sections and the overall proportion of doubly labeled cells was calculated on per hemisphere bases; from these figures, mean proportions
and standard deviations were then computed for labeling produced
by comparably placed injection sites across animal subjects. For
subcortical injection sites, labeling in supragranular and infragranular
layers was considered together; for cortical injections, labeling in
upper versus lower cortical layers was evaluated separately.
Photomicroscopy
All of the images presented were captured using a Zeiss Axiocam
digital camera and a Zeiss Axiplan II light microscope interfaced with
Axiovision Software (Carl Zeiss, Inc., Thornwood, New York). Some
adjustments were made to the brightness and contrast of captured
images. All images of androgen receptor immunoreactivity were
generated using the PG-21 antibody, and all examples shown are from
male animal subjects.
Results
Appearance, Specificity and Patency of
Immunoreactivity
With the exception of the PG-21 antibody, all of the antibodies
used in this study are commercially available, and all gave rise
to expected patterns of labeling. Thus, the AR antibodies both
270 Androgen Receptors in Rat Cerebral Cortex • Kritzer
produced labeling that was localized mainly over cell nuclei in
the cerebral cortex and adjacent allocortical and subcortical
structures in patterns matching previous descriptions of
receptor distribution (e.g. Simerly et al., 1990; Xiao and Jordan,
2002). Immunoreactivity for oligodendrocytes (anti-RIP, Fig.
2B), astrocytes (anti-glial fibrillary acidic protein) and neurons
(anti-neuron-specific enolase, anti-parvalbumin, anti-calbindin)
also labeled subsets of cells that displayed morphological
features and distributions consistent with previous descriptions of identified cortical glial cells and neurons (e.g. see
Debus et al., 1983; Friedman et al., 1989; Marone et al., 1995;
Hof et al., 1999). In addition, no patterned labeling was evident
in control experiments in which the primary or secondary antibodies were omitted from immunolabeling protocols, or in
control studies in which anti-AR antibodies were preabsorbed
with immunizing peptides (see Materials and Methods). Taken
together, these findings support the selectivity of the antibodies used to label intended antigens.
It has been previously noted that the double labeling immunocytochemical method used to coincidently visualize intracellular gonadal hormone receptors and cytoplasmic markers
does not appreciably diminish the immunostaining of either
antigen (Kritzer, 1997; Creutz and Kritzer, 2002). Because the
combination of hormone receptor immunocytochemistry and
silver enhancement of the gold-conjugated retrograde tracer
was used here for the first time, initial studies were conducted
on tissue sections from injected rats to compare patterns of
retrogradely and immunoreacted cells visualized separately on
adjacent sections or co-labeled sequentially in a single section.
These assessments revealed some attenuation of the intensity
of AR-immunoreactivity in the combined procedure. However,
there were no appreciable effects on the density of either ARIR nuclei or of retrogradely labeled cells.
Cortical AR-IR Cells
The subjects used in these studies included hormonally intact
adult male and female rats. Visual comparisons, however,
revealed no obvious qualitative or quantitative differences in
either single or double labeling immunocytochemistry
between these groups. Thus, the descriptions below apply to
males, diestrus females and proestrus females alike. Separate
assessments were also made of labeling in the left and right
hemispheres; as these revealed no obvious hemispheric asymmetries in receptor distribution or density, the data below also
reflect receptor distributions in the left and right cortical hemifields.
Cellular Phenotype
Representative sections of various levels of the cortex that
were immunoreacted for AR proteins revealed labeled profiles
that corresponded mainly to cell nuclei, with staining in the
surrounding cytoplasm usually remaining near background
levels (Fig. 1A–C). Most of these immunoreactive nuclei were
round, although occasionally more oblong shaped profiles
were observed in layer I (Fig. 1A). The nuclei in layer V also
stood out as noticeably larger (10–15 µm in diameter, Fig. 1C)
than the nuclei present in other layers that were usually
between 2–5 µm in diameter (Fig. 1B). Even these smaller
dimensions, however, fell within a range more typical of the
nuclei of cortical neurons rather than glial cells. Thus, it
seemed likely that AR-IR nuclei belonged to cortical neurons.
This was verified in experiments that combined immunoreac-
Figure 1. Representative high power photomicrographs showing the appearance of nuclei immunoreactive for intracellular androgen receptors in primary somatosensory cortex.
Nuclei in layer I are typically small and oblong (A), those in layers II/III are round and small in size (B), and those present in layer V are round and considerably larger (C); the cytoplasm
surrounding these immunoreactive nuclei is not appreciably labeled. (D–F) Low-power photomicrographs demonstrating the laminar distributions and low to modest densities of
immunoreactive nuclei typical for association areas of the cerebral cortex. Extremely sparse and scattered labeling is present in entorhinal (ENT; D) and insular areas (AIP; E). In
cingulate (Cg1; F) and retrosplenial areas (RSG, RSA; G), immunoreactive nuclei are slightly more dense and localized to supragranular layers. Roman numerals in D–G mark the
locations of cortical layers. Other abbreviations: Par2, secondary somatosensory cortex; Pir, piriform cortex; Fr2, premotor cortex. Scale bars: A–C = 25 µm; D–G = 500 µm.
tivity for AR with that for the neuronal marker neuron-specific
enolase (NSE) that showed that virtually all of the cortical
receptor-bearing nuclei identified were surrounded by cytoplasm that was immunoreactive for NSE (Fig. 2A,B). As further
verification, double labeling for immunoreactivity for AR and
for astrocytes (anti-glial fibrillary acidic protein, GFAP, Fig. 2F)
or for oligodendrocytes (anti-RIP) was carried out; these
analyses failed to reveal any examples of cortical AR-IR cells
that were also immunoreactive for these glial markers.
The somatic and dendritic morphology revealed in the NSEimmunoreactivity identified many AR/NSE-IR cells as pyramidal
neurons (Fig. 2A,B). The predominantly pyramidal character of
AR-IR cortical cells was also supported in double labeling
experiments combining AR-immunoreactivity and immunoreactivity for the calcium binding protein calbindin (CB), which
showed that as many as half of the smaller receptor-bearing
neurons in the supragranular layers were small, CB-IR pyramids
(Fig. 2C). Further, in most areas of the cortex virtually none of
the non-pyramidal cells labeled by immunoreactivity for CB
(Fig. 2D) or for the calcium binding protein parvalbumin (PV,
Fig. 2E) were found to be immunoreactive for the intracellular
hormone receptor. There were, however, two exceptions to
this rule. Specifically, in the proisocortical areas of the piriform
and entorhinal cortices, up to three quarters of the AR-IR
cells present were either CB- or PV-IR, non-pyramidal
neurons. In the remainder of the cortical mantle, it was only
the small numbers of AR-IR neurons present in layer I — where
all neurons are presumed GABAergic (Jones and Powell, 1970),
that were also likely to be non-pyramidal cells.
Regional and Laminar Distribution
The distribution of AR-immunoreactivity was next mapped in
relation to cytoarchitectonic divisions of the rat cerebrum
(Zilles and Wree, 1985). Virtually all of the cortical areas and
layers examined contained at least some AR-IR neurons.
However, across the cortex differences in the laminar distributons and/or apparent densities of AR-IR cells coincided with
certain recognized anatomical subdivisions of the cortical
mantle. The most obvious was the broad division of the cortex
into densely versus much more diffusely immunoreactive
zones — a separation that followed the division of the rat cerebrum into its isocortical versus periallocortical/proisocortical
domains. As described below, within each of these two cortical
categories there were also instances of more subtle regional
Cerebral Cortex March 2004, V 14 N 3 271
1D), immunoreactive nuclei were scattered among all major
cellular layers and did not show any obvious preference or
concentration in any particular layer or layers.
Figure 2. Representative high power photomicrographs showing the appearance and
colocalization of immunoreactivity for intracellular androgen receptors (AR) and
neurons specific enolase (NSE; A, B), calbindin (CB; C, D), parvalbumin (PV; E) and glial
fibrillary acidic protein (GFAP; F). Colocalization with NSE and CB and the lack of
colocalization with PV and GFAP indicates that the majority of AR are found in pyramidal
neurons. Scale bar (E) = 25 µm.
differences in receptor distribution that distinguished some of
the individual component areas.
Periallocortex/Proisocortex
The periallocortical/proisocortical fields in rats include the
cingulate and retrosplenial areas of the medial cortical wall,
and the ventrolaterally located insular and perirhinal cortices.
Compared with the rest of the cortex, AR-immunoreactivity in
these regions was noticeably sparse, and was weakest overall
in the insular (Fig. 1E) and perirhinal cortices. In each of these
three areas, the very few AR-IR cells that were present were
scattered across all of the principal cellular layers of these areas
(Fig. 1E). This contrasted the pattern observed in remaining
allocortices, where receptors overall appeared to be somewhat
more prevalent, and more clearly concentrated in the supragranular layers; in cingulate cortex (Cg1, 2, 3) AR-IR nuclei
were most plentiful in layers II and upper III (Fig. 1F), and in
retrosplenial areas (RSG, RSA), immunoreactive nuclei were
concentrated mainly in layer II (Fig. 1G).
The entorhinal and prifirom cortices of the medial temporal
lobe were also evaluated in this study. Like the prefrontal and
retrosplenial areas (above), AR-immunoreactivity in these
regions was sparse. In both pirifirm and entorhinal areas (Fig.
272 Androgen Receptors in Rat Cerebral Cortex • Kritzer
Isocortex
In rats, the isocortices occupy the lateral cortical surface and
include all major primary and secondary sensory and motor
areas. In each of these regions, AR-IR cells were much more
numerous than in the allocortical fields, and were prominent in
both supra- and infragranular cortical layers. These marked
differences in the density of immunoreactivity highlighted the
dorsomedial (Fig. 1F) and ventrolateral (Fig. 1E) boundary
regions where areas of allo- and isocortices abut. The border
separating area Cg1 from the premotor cortex (area Fr2), for
example, coincided with a clear rise in the number and density
of AR-IR neurons in layers II/IIIa, and the emergence of a
second band of strong receptor-immunoreactivity in layers V
and VI (Fig. 1F). This broad bilaminar pattern of labeling
continued from premotor areas without obvious interruption
into the laterally adjacent primary motor fields (areas Fr1, FL,
HL; Fig. 3A). At the borders separating motor and sensory
cortices, however, labeling took on a more conspicuously
stratified pattern (Fig. 3C). Thus, in the primary and secondary
somatosensory (Par1, Par2), auditory (Te1, Te2) and visual
cortices (Oc1, Oc2) alike, AR-immunoreactivity conformed to
a dense stratum of cells in layers II/IIIa, and a second, deeper
and more discrete band of labeling that was dominated by
nuclear immunoreactivity within the large pyramidal neurons
of the lower half of layer V (Fig. 3B). Often seen especially
within the sensory cortices as well was a periodic waxing and
waning in the apparent density of supragranular labeling.
Although varying in crispness from region to region, from
animal to animal, and in some cases, from section to section, in
virtually all subjects it was possible to discern clusters of
denser labeling, usually 500–750 µm across, that alternated
with narrower zones (∼250 µm wide) of reduced AR-immunoreactivity (Fig. 3D,E). There was no obvious tangential organization to the labeling in infragranular layers.
Connections of Cortical AR-IR Neurons
The abundance of AR-IR pyramids in layers II/III, V and to a
lesser extent layer VI suggested that the afferent targets of
these cells could include the neostriatum, thalamus, superior
colliculus and/or cerebral cortex, as each of these regions
receives a major cortical input from cells located in one or
more of these layers (e.g. Donoghue and Parham, 1983;
Olavarria and Van Sluyters, 1983; Isseroff et al., 1984; Miller
and Vogt, 1984; Killackey et al., 1989; Hayama and Ogawa,
1997; Desbois and Villanueva, 2001; Ding et al., 2001). These
predictions were tested by co-mapping and quantifying the
retrograde labeling produced by injections made in either the
thalamus (ventrolateral and ventral anterior nuclei; Fig. 4A),
the superior colliculus (Fig. 4D), the caudate/putamen (all
quadrants; Fig. 4B), the nucleus accumbens (core and shell;
Fig. 4C), or in the somatosensory, primary motor, or cingulate
(Fig. 4E) divisions of the cerebral cortex (see Fig 4), with ARimmunoreactivity (e.g. Fig. 5).
Injections made in each of the subcortical target areas evaluated produced fields of retrogradely labeled pyramidal cells
that conformed to expected patterns and that at least partially
overlapped with fields of AR-immunoreactivity (Figs 6–8).
Thus, large injections in the medial and lateral caudate back-
Figure 3. Representative low-power photomicrographs showing the laminar distribtutions and apparent densities of nuclei immunoreactive for intracellular androgen receptors in
representative regions of isocortex. Labeling in motor areas (Fr1; A, C) corresponds to a bilaminer pattern with nuclei concentrated in layers II/upper III and in V/VI, and separated
by a stratum of reduced label density in lower layer III. In sensory cortices including primary somatosensory cortex (Par1; B–E), nuclei are also dense in layers II/III but are restricted
in infragranular layers to lower layer V. The labeling in supragranular layers of these areas also shows periodic waxing and waning in density (arrowheads, D, E). Roman numerals
(right) mark locations of cortical layers. Asterisks mark sectioning artifacts made to identify left hemispheres. Scale bars: A–C = 1 mm; D, E = 500 µm.
labeled cells in layer V and to a lesser extent, layers III and VI
of ipsilateral cingulate and sensorimotor cortices, respectively,
as well as in corresponding layers of the contralateral hemifields (Fig. 6); injections in the nucleus accumbens most
strongly labeled the infragranular layers of ipsilateral cingulate
and insular cortex (Fig. 7A,B) and yielded much sparser
labeling in these areas contralaterally; injections in the superior
colliculus produced labeling mainly in layers V and VI of the
ipsilateral retrosplenial cortex, and layer V of ipsilateral visual
and adjacent auditory cortices (Fig. 7C,D); and the thalamic
injections retrogradely labeled dense populations of cells in
layers V and VI of ipsilateral and to a minimal extent, contralateral somatosensory and pre- and primary motor regions (Fig.
8). For each of the subcortical sites evaluated, however, cellby-cell examinations carried out across a series of five sections
revealed on average <5% coincidence between retrogradely
labeled cortical cells and those bearing AR-immunoreactivity
(Fig. 9A). Although such doubly labeled cells were readily
identified by their blackened nuclei and surrounding grainfilled cytoplasm (Fig. 5A,B), the thalamic injections tended to
produce the greatest coincidence among labeling which rarely
exceeded more than to two or three double labeled cells
within a given section (Figures 8 and 9A). More typical for the
subcortical sites evaluated were sections from for example,
animals in which the nucleus accumbens or caudate nucleus
had been injected in which the two labels marked non-overlapping subsets of pyramidal neurons (Figs 5C–E, 6, 7 and 9A).
In other sets of animals, injections were made in cingulate
(deep layers only), primary motor, and primary somatosensory
cortex (supragranular and infragranular injections). As
expected, each site produced cortical labeling that involved
specific cytoarchitectonic fields in both cerebral hemispheres,
and specific sets of cortical layers (e.g. Donoghue and Parham,
1983; Olavarria and Van Sluyters, 1983; Isseroff et al., 1984;
Cerebral Cortex March 2004, V 14 N 3 273
Figure 4. Representative low-power photomicrographs showing example injection sites (gold-conjugted cholera toxin, inactive B subunit) in the thalamus (TH; A), caudate nucleus
(Cd; B), nucleus accumbens (Nac; C), superior colliculus (SC; D) and primary motor cortex (Fr1; E). The zones of effective tracer uptake appear as blackened zones within which no
cellular labeling can be discerned. Sections are counterstained (1% cresyl violet) to localize injection sites with respect to cytoarchitecture. Additional abbreviations: HP,
hippocampus; HYP, hypothalamus; AMG, amygdala; SA, septal area; PAG, periaqueductal gray; SN, substantia nigra; Fr2, premotor cortex; Par1, primary somatosensory cortex.
Scale bar (E) = 1 mm.
Miller and Vogt, 1984). When examined for registry with ARimmunoreactivity, considerably higher proportions of these
retrogradely labeled cells were found to be immunoreactive for
AR compared with those labeled by injections made any of the
subcortical target areas investigated (Figs 9–11). Some
apparent differences in the prevalence of AR-immunoreactive
retrogradely labeled cells were also noted, depending on the
cortical region injected and on the cortical region, layer, and/
or hemisphere evaluated (Fig. 9B). Injections made in infragranular cingulate cortex for example, produced retrograde
labeling in the medial prefrontal cortex that was most dense in
layers III and V, and more scattered in layer II (Figs 9B and
11A,B). In the supragranular layers where receptor immunoreactivity was also abundant between 45 and 54% of the retrogradely labeled cells sampled within the injected hemisphere
were also AR-immunoreactive (Fig. 9B). More modest degrees
of coincidence were seen, however, in the deeper layers
where retrogradely labeled cells were plentiful but AR-IR
cells were scarce (Figs 9B and 11A,B). The callosal labeling
produced by cingulate injections also occupied layers II, III and
V, but overall was lighter than the labeling present in the
274 Androgen Receptors in Rat Cerebral Cortex • Kritzer
injected hemisphere, and in both supragranular and infragranular strata showed slightly lower degrees of overlap with
AR-immunoreactivity as well, with roughly 35% of the callosally labeled cells in the supragranular layers and between 6 and
8% of the cells sampled in the infragranular layers being ARimmunopositive (Figs 9B and 11A,B).
Injections in somatosensory and motor areas produced characteristic, laterally widespread labeling; for motor injections,
labeling tended to spread laterally into adjacent sensory territories, and for sensory injections, label spread medially into
motor areas. For both areas, superficially placed injections
produced discrete columns of labeling that were most cell
dense in layers II/III, and narrower and more cell sparse in
layers V/VI. Evaluation of double labeling in representative
sections revealed that in the injected hemispheres, 31–51% of
the cells making up these columns in motor cortex, and
70–82% of these cells in somatosensory fields, whether located
adjacent (Figs 9B and 11C) or several millimeters lateral to the
parent injection site (Fig. 11E), were also AR-immunoreactive.
Callosal labeling, on the other hand, showed lower degrees of
overlap with AR-immunoreactivity in both motor (22–25% of
Figure 5. Representative photomicrographs showing sections sequentially labeled
for intracellular androgen receptor immunoreactivity and retrograde labeling. Dually
labeled cells are identified by black immunoreactive nuclei surrounded by cytoplasm
containing gold particles (A, B). Injections made in subcortical areas including the
superior colliculus and caudate nucleus retrogradely label dense populations of cells
in layer V of visual (C: Oc1, white arrows) and motor areas (D, E: Fr1, arrowheads),
respectively; few to none of these cells contain nuclei that are immunoreactive for
androgen receptors. Panels D and E represent the same section photographed under
darkfield illumination (D) to highlight retrograde labeling and using brightfield optics
(E) to optimize visualization of immunoreactive nuclei; the different colored
arrowheads in these images point to the same sets of cells. Scale bars A–C =
25 µm; D, E = 75 µm.
sampled cells) and somatosensory cortex (21–33% of sampled
cells; see Figs 9B and 11D). The more superficial as well as
more deeply placed injections sites in sensory and motor
cortices also produced strong bands of laterally extensive labeling in layers V and VI (Fig. 11F,G). In injected hemispheres,
about a third of these labeled cells in layers V/VI motor cortex,
and 75–80% of the labeled cells in layer Vb of somatosensory
fields were AR-immunoreactive (Fig. 9B). However, there was
little if any overlap observed between retrograde labeling and
the relatively few AR-IR pyramids present in layers Va and VI of
the somatosensory cortex (Fig. 11F). As for all cortical injections, the patterns of retrograde labeling in these infragranular
strata were largely repeated in the uninjected hemisphere,
albeit with a reduced density of retrograde labeling overall, and
with markedly reduced degrees of overlap between retrograde
labeling and hormone receptor immunoreactivity in both
motor cortex (7–13% of sampled cells) and in somtosensory
fields where overlap was negligible (Figs 9B and11G).
Discussion
Androgens impact an array of cortical structures and functions
both during development and in adulthood (e.g. Leranth et al.,
Figure 6. Schematic representation of camera lucida drawings charting the locations
of cells that are immunoreactive for intracellular androgen receptors (gray dots),
retrogradely labeled by injections made in the lateral caudate nucleus (open triangles)
or both (triangles containing black dots). Labeling from representative sections of
primary motor (Fr1; A, B) and primary somatosensory (Par1; C, D) cortex in injected
(Ispi.; A, C) and uninjected hemispheres (Contra.; B, D) are shown. Although there is
spatial overlap between retrograde labeling and receptor immunoreactivity, very few
cells contain both labels. Scale bar = 100 µm.
2003; Kritzer et al., 2001; Kritzer, 2000; Christiansen and
Knussman, 1987; Stenn et al., 1972). Despite the prevalence of
intracellular nuclear androgen receptors, at least in comparison with other hormone receptors, little is known about where
in the cortex this receptive machinery lies. As these receptors
are likely to contribute at least in part to observed androgen
effects on the cerebrum, it is important to determine where
within the cortical mantle these signaling pathways may be
brought to bear. To address this issue, a comprehensive series
of studies was undertaken to map the distributions of intracellular AR proteins in the cerebral cortices of hormonally
intact adult male and female rats. Using combined immunocytochemical and tract tracing methods, the regional, laminar,
cellular and connectional signatures of AR-immunoreactivity
were identified in all major divisions of the cortical mantle. In
all, three basic patterns of receptor distribution were found;
light, diffuse and concentrated mainly in the supragranular
layers in association areas of the cortex; densely distributed in
layers II/III and V/VI in motor areas; and dense in layers II/III
and Vb in primary and secondary sensory cortical regions. As
discussed below, however, a common theme to each of these
Cerebral Cortex March 2004, V 14 N 3 275
Figure 8. Schematic representation of camera lucida drawings charting the locations
of cells that are immunoreactive for intracellular androgen receptors (gray dots),
retrogradely labeled by injections made in the ventrolateral thalamus (open triangles),
or both (triangles containing black dots). Labeling in representative sections of primary
motor cortex (Fr1) are shown; labeling in layers V (A, B) and VI (C,D) and from the
injected (Ispi.; A, C) and uninjected hemisphere (Contra.; B, D) are shown separately.
Populations of retrogradely and immunoreactive cells especially in the injected
hemispheres are often punctuated by modest numbers of cells containing both labels.
Scale bar = 50 µm.
Figure 7. Schematic representation of camera lucida drawings charting the locations
of cells that are immunoreactive for intracellular androgen receptors (gray dots),
retrogradely labeled by injections made in the nucleus accumbens (A, B) and superior
colliculus (C, D) (open triangles), or both (triangles containing black dots). For the
accumbal injection, labeling from representative sections of anterior insular (AID; A)
and cingulate (Cg1; B) cortex is shown; for the colllicular injection, labeling from
representative sections of primary visual (Oc1) and primary auditory (Te) cortex is
shown. All sections are from the injected hemisphere. Despite prominent spatial
overlap between retrograde labeling and receptor immunoreactive cells, few cells
contain both labels. Scale bar = 100 µm.
patterns was the prominent alignment of AR-immunoreactivity
with cells making corticocortical and to a lesser extent corticothalamic connections — both pivotal among circuit systems
contributing to cortical information processing.
AR in the Cerebrum: Comparisons to Previous Studies
Androgen receptors have been mapped in forebrain areas of
rats including the cerebral cortices using methods of ligand
binding (Sar and Stumpf, 1985), in situ hybridization (Simerly
et al., 1990), and immunocytochemistry (Clancy et al., 1992).
All of these studies share basic conclusions concerning the
cortical distributions of the intracellular AR markers examined.
First, all agree that there is an abundance of androgen receptors in the adult rat cerebral cortex. Further, in studies where
receptors were compared across males and females, no
obvious sex differences in receptor distribution or apparent
density were found (Simerly et al., 1990; Clancy et al., 1992).
Finally, many of the anatomical details of cortical receptor
distribution are consistent. For example, similar to the regional
differences noted in this study, previous in situ hybridization
studies revealed moderate to dense numbers of AR mRNA
276 Androgen Receptors in Rat Cerebral Cortex • Kritzer
containing cells in isocortical areas, and sparser receptor
signals in piriform and entorhinal areas (Simerly et al., 1990).
These studies also reported a bilaminar distribution of receptor
mRNA in layers II/III and V/VI that matches the supragranular
and infragranular concentrations of AR-immunoreactivity
identifed in this study for most lateral cortical regions. The
only previous immunocytochemical investigation of AR in rat
cerebral cortex was limited to a single cortical level and used a
different antibody to label receptor proteins than either of the
two used here; while this study described receptor immunoreactivity only in infragranular layers, the receptor-immunopositive cells within these layers were described as pyramidal
or fusiform in morphology (Clancy et al., 1992), which is
consistent with the appearance AR/NSE-IR neurons identified
in deep cortical strata in this study.
In contrast to studies in rats, studies of AR-immunoreactivity
in the cortex of human and non-human primates have
produced results that are quite different from those obtained in
rodents. For example, in rats there are fewer androgen receptors in prefrontal compared with sensorimotor cortices
(Simerly et al., 1990; present study), whereas in juvenile
monkeys the association areas of the frontal lobe contained
among the highest levels of androgen binding (Clark et al.,
1988). Further, while in rats AR-immunoreactivity tends to be
fairly well localized to particular layers, in primates ARimmunoreactivity is more evenly distributed and is modest to
dense in all major laminae, including layer I (e.g. Clancy et al.,
1992; Finley and Kritzer, 1999). Finally, the double-labeling
strategies used in this study showed that in rats outside of piriform and entorhinal areas, cortical AR-immunoreactivity is
sequestered mainly in pyramidal neurons. In biopsied samples
of human temporal cortex, however, analyses of adjacent
Figure 9. Bar graphs showing mean numbers of cells retrogradely labeled by
injections made in the nucleus accumbens (N Acc), thalamus (Thal), superior
colliculus (S Coll) or caudate nucleus (Cd), or in the cingulated (Cg1), primary motor
(Fr1) or primary somatosensory cortex (Par1) that are also immunoreactive for
intracellular androgen receptors (AR-IR). The error bars in these graphs represent
standard errors (SD). For caudate injections, data from the injected (cCd) and
uninjected (cCd) hemispheres are shown separately. For the cortical injections, data
from layers II/III and V/VI from injected (ipsi) and uninjected (contra) hemispheres are
shown separately. The gray marks superimposed on the bar graphs represent data
points from the individual animal subjects that contributed to the group means
illustrated in the black bars. Negligible overlap was seen between retrograde labeling
and receptor immunoreactivity in the prefrontal and insular cortices following
injections in the nucleus accumbens (A), and in the deep layers of the contralateral
somatosensory cortex following injections of area Par1 (B). Other subcortical
injections produced less than 5% overlap between receptor and retrograde labeling
(A), whereas in superficial layers especially, cortical injections yielded incidences of
overlap between these two that ranged between 30 and 80% (B).
paraffin sections localized AR-IR nuclei to neurons of varied
morphology, as well as to GFAP-immunreactive astrocytes,
microglia identified by the lectin Ricinus communis agglutinin-1, and to histologically identified oligodendrocytes (Puy
et al., 1995). Double-label immunocytochemistry in the
prefrontal cortices of rhesus monkeys also identified as many
as half of all cortical AR-IR profiles as GFAP-immunoreactive
astrocytes (Finley and Kritzer, 1999). That this latter study had
both histochemical procedures and immunochemical markers
in common with the present study adds strength to conclusions that there may be qualitative differences in the regional,
laminar and cellular distributions of androgen receptor-bearing
cells — at least in association cortices, in rats versus primates.
These differences should be borne in mind, especially when
considering how experimental findings in rodents relate to
observed sex differences and/or androgen stimulation of
higher order cortical function and cortical dysfunction in
disease in man (Stenn et al., 1972; Christiansen and Knussman,
1987).
Figure 10. Representative high power photomicrographs showing sections of primary
somatosensory cortex (Par1) through layers II/II (A) and V (B, C) that contain cells
retrogradely labeled by an injection site made in the ispilateral motor cortex (black and
white arrows). Black androgen receptor immunoeactive nuclei are visible in each of
these sections, and cells that are both receptor-immunopostive and retrogradely
labeled are marked by black arrows. In any given field, a majority of cells retrogradely
intrahemispherically labeled by cortical injections are also immunoreactive for the
intracellular androgen receptors. Scale bars = 25 µm.
AR in the Adult Rat Cerebrum: Comparison with ER
Previous light microscopic studies examining the regional,
laminar, and cellular distributions of immunoreactivity for the
classical and beta isoforms of intracellular estrogen receptors
(ERα, ERβ) in the rat cerebrum revealed that these two
receptor subtypes are sequestered in the nuclei of non-overlapping subpopulations of cortical neurons (Blurton-Jones and
Tuszynski, 2002; Kritzer, 2002). Based on findings from the
present study, there seems little possibility of anatomical
overlap between nuclear estrogen receptors and AR-immunoreactivity as in most areas of the cortex their distributions are
complementary. For example, while AR-immunoreactivity is
least dense in areas surrounding the rhinal sulcus, these
insular cortices are precisely where nuclear ERα-immunoreactivity is most enriched (Kritzer, 2002). Further, for cortical
regions and layers where ERα-, ERβ- and AR-IR nuclei may be
spatially overlapping, ER-immunoreactivity is found in the
nuclei of either PV-immunonegative and -immunopositive nonpyramidal interneurons (Blurton-Jones and Tuszynski, 2002;
Kritzer, 2002) while AR-immunoreactivity is usually associated
with the nuclei of pyramidal cells. There may be exceptions to
Cerebral Cortex March 2004, V 14 N 3 277
colocalize ERα and ERβ to individual, identified cholinergic
neurons in rat basal forebrain (Shughrue et al., 2000).
Other than the possibilities sited above, immunoreactivity at
least for nuclear intracellular ER and AR seems to occupy
antomically and functionally distinct sets of circuit elements in
both cerebal and hippocampal cortex (Milner et al., 2001).
These differences in distribution, and in particular the alignment of AR with excitatory projection versus the alignment of
ER with local inhibitory cortical cells, could be substrates for
the separate transcriptional actions of estrogens versus androgens in stimulating the cerebral cortex, perhaps including their
opposing influences on, for example, endpoints of cortical
development (Zhang et al., 2000) and neurotransmitter physiology (Handa et al., 1997).
Figure 11. Schematic representation of camera lucida drawings charting the
locations of cells that are immunoreactive for intracellular androgen receptors (gray
dots), retrogradely labeled by injections made in the cingulate (Cg1; A, B) and primary
motor (Fr1; C–G) cortex (open triangles), or both (triangles containing black dots).
Labeling in sections from injected (Ipsi.; A, C, E, F) and uninjected hemispheres (B, D,
G) are shown; for the motor injection, labeling in layers II/III (C–E) and V/VI (E, G) are
shown separately. For each injection shown, significant proportions of retrogradely
labeled neurons in the injected hemisphere are immunoreactive for the intracellular
androgen receptor; even the laterally extensive labeling produced by the motor cortex
injection located several millimeters millimeters away from the parent injection (Par1;
E) shows a high degree of overlap with receptor immunoreactivity. For all sites as well,
markedly more modest proportions of dually labeled cells are present in the uninjected
hemispheres. Scale bar = 100 µm.
this separation of signaling pathways, however: for example, in
piriform and entorhinal cortices, where ERα, ERβ and AR-IR
nuclei have all been found within CB-IR and PV-IR interneurons. Also, recent electron microscopic studies have identified non-nuclear, presumed membrane ERα in the dendrites
and spines of subsets of pyramidal neurons in the rat hippocampus (Milner et al., 2001; Adams et al., 2002). A similar
scenario in the cerebral cortex could provide substrates for
some intersection between the rapid estrogen-mediated effects
that are presumed to be regulated by these non-nuclear sites
and the transcriptional events mediated by androgen stimulation of its cognate intracellular receptor. Because the antireceptor antibodies used in this study and elsewhere are
typically all rabbit polyclonals, however, verification of dually
receptive cells at least at the light microscopic level will
require a combination of, for example, in situ hybridization
and immunocytochemistry to label multiple receptor subtypes
within single cells. This approach has been used to successfully
278 Androgen Receptors in Rat Cerebral Cortex • Kritzer
AR in the Adult Rat Cerebrum: Connectional and
Functional Implications
The localization of AR-IR within the nuclei of PV-IR or CB-IR
interneurons in the entorhinal and pirifirom cortex gives intracellular androgen receptors in these phylogenetically older
regions functional connotations of local inhibition. The
calcium binding protein signatures of these cells may also be
telling of the synaptic relations maintained by AR-IR inhibitory
neurons, as in other areas of rat cortex PV-immunoreactive
interneurons are either basket or chandelier cells that synapse
on the somata or axon initial segments of target neurons, and
CB-immunoreactive neurons include double bouquet, neurogliaform and Martinotti cells, whose terminals contact the
dendritic domains of postsynaptic cells (Celio, 1990; Kubota et
al., 1994; Gabbott et al., 1997). In areas beyond the medial
temporal lobes, however, AR-IR nuclei are sequestered
perhaps exclusively in pyramidal neurons, thus linking
androgen signaling to excitatory, projection rather than local
inhibitory cortical neurons.
Although the layers where AR-IR pyramids are especially
enriched (layers II/III, V/VI) house cells of origin of multiple
afferent systems, studies combining retrograde tracing and
immunocytochemistry indicated that few to no AR-IR cells
project to the superior colliculus, thalamus, or basal ganglia.
However, injections made within the cortex itself revealed that
substantial proportions of potentially androgen-sensitive pyramids are involved in making corticocortical connections.
Further, there seems to be a relatively stronger relationship
between androgen receptors and associationally projecting
neurons that interlink cortical regions within the same hemisphere. This was especially so for sensory and motor cortices
where it was consistently found that significantly more retrogradely labeled cells in injected hemispheres cells were
receptor-immunoreactive compared with the callosally projecting cells labeled by the same injections. This finding was
surprising in view of the literature identifying sex differences
in and androgen stimulation of the morphometrics and/or axon
density of the corpus callosum in rats (Berrebi et al., 1988;
Fitch et al., 1991; Nunez and Juraska, 1998).
While the richness of colocalization seen between cortically
projecting cells and AR-immunoreactivity was impressive, it is
tempting to speculate that analyses of larger cortical injection
sites, which tend to produce more densely labeled cortical
fields (Kritzer et al., 1992; Kritzer and Goldman-Rakic, 1995)
could uncover even higher degrees of overlap between retrograde labeling and androgen receptors. At the same time,
however, it is clear that not all of the AR-IR cells of the cortex
were accounted for by the projection systems evaluated in this
study. Connectional signatures for the AR-IR neurons scattered
within in layers IV and lower VI, for example, were not identified. These and perhaps other groups of AR-IR neurons could
represent populations of androgen-sensitive cells that project
to as yet unexplored brain areas, e.g. brainstem or spinal cord
(Killackey et al., 1989). Nonetheless, substantial proportions of
AR-IR neurons in the rat cerebral cortex do participate in
making local, long lateral intrahemispheric, and to a lesser
extent interhemispheric cortical connections. Because these
connections have established roles in sculpting cortical receptive field properties (e.g. Gilbert, 1983; see Katz and Callaway,
1992), and in networking the functionally co-activated cortical
regions engaged in sensory, motor, and higher order tasks (see
Goldman-Rakic, 1987), androgen signaling pathways may be
aligned with elements of circuit structure that have critical
involvement in the cortical processing of sensory, motor, and
higher order information at some of its most complex levels.
Notes
Ms Lela Creutz, Ms Aiying Liu and Mr Jonathan Reinstein are thanked
for their excellent technical assistance in all aspects of this project.
This work supported by an RO1 Award from the NINDS (NS41966).
Address correspondence to Mary Kritzer, Department of Neurobiology and Behavior, Stony Brook University, Stony Brook, New York
11794-5230, USA. Email: [email protected].
References
Adams MM, Fink SE, Shah RA, Janssen WGM, Hayashi S, Milner TA,
McEwen BS, Morrison JH (2002) Estrogen and aging affect the
subcellular distribution of estrogen receptor-α in the hippocampus
of female rats. J Neurosci 22: 3608–3614.
Berrebi AS, Fitch RH, Ralphe DL, Denenberg JO, Friedrich VL Jr,
Denenberg VH (1988) Corpus callosum: region-specific effects of
sex, early experience and age. Brain Res 438:216–224.
Blurton-Jones M, Tuszynski MH (2002) Estrogen receptor-beta colocalizes extensively with parvalbumin-labeled inhibitory neurons in the
cortex, amygdala, basal forebrain, and hippocampal formation of
intact and ovariectomized adult rats. J Comp Neurol 452:276–287.
Celio MR (1990) Calbindin-D-28k and parvalbumin in the rat nervous
system. Neuroscience 35:375–475.
Christiansen K, Knussman R (1987) Sex hormones and cognitive functioning in men. Neuropsychobiology 18:27–36.
Clancy AN, Bonsall RW, Michael RP (1992) Immunohistochemical
labeling of androgen receptors in the brain of rat and monkey. Life
Sci 50:409–417.
Clark AS, MacLusky NJ, Goldman-Rakic PS (1988) Androgen binding
and metabolism in the cerebral cortex of the developing rhesus
monkey. Endocrinology 123:932–940.
Creutz LM, Kritzer MF (2002) Estrogen receptor beta immunoreactivity
in the midbrain of adult rats: regional, subregional and cellular localization in the A10, A9 and A8 dopamine cell groups. J Comp Neurol
446:288–300.
Debus E, Weber K, Osborn M (1983) Monoclonal antibodies specific for
glial fibrillary acidic (GFA) protein and for each of the neurofilament
triplet polypeptides. Differentiation 25:193–203.
Desbois C, Villanueva L (2001) The organization of lateral ventromedial
thalamic connections in the rat: a link for the distribution of nociceptive signals to widespread cortical regions. Neuroscience
102:885–898.
Ding DC, Gabbott PL, Totterdell S (2001) Differences in the laminar
origin of projections from the medial prefrontal cortex to the
nucleus accumbens shell and core regions in the rat. Brain Res
917:81–89.
Donoghue JP, Parham C (1983) Afferent connections of the lateral
agranular field of the rat motor cortex. J Comp Neurol 217:390–404.
Finley SK, Kritzer MF (1999) Immunoreactivity for intracellular
androgen receptors in identified subpopulations of neurons, astrocytes and oligodendrocytes in primate prefrontal cortex. J Neurobiol 40:446–457.
Fitch RH, Cowell PE, Schrott LM, Denenberg VH (1991) Corpus
callosum: demasculinization via perinatal anti-androgen. Int J Dev
Neurosci 9:35–38.
Friedman B, Hockfield S, Black JA, Woodruff KA, Waxman SG (1989) In
situ demonstration of mature oligodendrocytes and their processes:
an immunocytochemical study with a new monoclonal antibody,
Rip. Glia 2:380–390.
Gabbott PLA, Dickie GM, Vaid RR, Headlam AJN, Bacon SJ (1997) Local
circuit neurons in the medial prefrontal cortex (areas 25, 32 and
24b) in the rat: morphology and quantitative distribution. J Comp
Neurol 377:465–499.
Gilbert CD (1983) Microcircuitry of the visual cortex. Annu Rev
Neurosci 6:217–247.
Goldman-Rakic PS (1987) Circuit basis of cognitive function in nonhuman primates. In Cognitive neurochemistry (Iversen S, Strauss J,
eds), pp. 91–110. New York: Oxford University Press.
Greene GL, Nolan C, Engler JP, Jensen EV (1980) Monoclonal antibodies
to human estrogen receptor. Proc Natl Acad Sci USA 77:5115–5119.
Handa RJ, Hejna GM, Lorens SA (1997) Androgen inhibits neurotransmitter turnover in the medial prefrontal cortex of the rat following
exposure to a novel environment. Brain Res 751:131–138.
Hayama T, Ogawa H (1997) Regional differences of callosal connections
in the granular zones of the primary somatosensory cortex in rats.
Brain Res 43:341–347.
Hof PR, Glezer II, Conde F, Flagg RA, Rubin MB, Nimchinsky EA, Vogt
Weisenhorn DM (1999) Cellular distribution of the calcium-binding
proteins parvalbumin, calbindin, and calretinin in the neocortex of
mammals: phylogenetic and developmental patterns. J Chem Neuroanat 16:77–116.
Isseroff A, Schwartz ML, Dekker JJ, Goldman-Rakic PS (1984) Columnar
organization of callosal and associational projections from rat frontal
cortex. Brain Res 293:213–223.
Jones EG, Powell TPS (1970) Electron microscopy of the somatic
sensory cortex of the cat. II., The fine structures of layers I–II. Philos
Trans R Soc Lond B Biol Sci 257:13–21.
Katz LC, Callaway EM (1992) Development of local circuits in
mammalian visual cortex. Annu Rev Neurosci 15:31–56.
Killackey HP, Koralek KA, Chiaia NL, Rhodes RW (1989) Laminar and
real differences in the origin of the subcortical projections neurons
of the rat somatosensory cortex. J Comp Neurol 282:428–445.
Kritzer MF (1997) Selective colocalization of immunoreactivity for intracellular gonadal hormone receptors and tyrosine hydroxylase in the
ventral tegmental area, substantia nigra and retrorubral fields in the
rat. J Comp Neurol 379:247–260.
Kritzer MF (2000) The effects of acute and chronic gonadectomy on the
catecholamine innervation of the cerebral cortex in adult male rats:
insensitivity of axons immunoreactive to dopamine-β-hydroxylase to
gonadal steroids, and differential sensitivity of axons immunoreactive for tyrosine hydroxylase to ovarian and testicular hormones. J
Comp Neurol 427:617–633.
Kritzer MF (2002) Regional, laminar, and cellular distribution of
immunoreactivity for ERα and ERβ in the cerebral cortex of
hormonally intact. Adult male and female rats. Cereb Cortex
12:116–128.
Kritzer MF (2003) Androgen receptor immunoreactivity identifies
subsets of corticocortically projecting pyramidal neurons in the
cerebral cortex of adult rats. Horm Behav 44:59.
Kritzer MF, Goldman-Rakic PS (1995) Intrinsic circuit organization of
the major layers and sublayers of the dorsolateral prefrontal cortex
in the rhesus monkey. J Comp Neurol 359:131–143.
Kritzer MF, Cowey A, Somogyi P (1992) Patterns of inter- and intralaminar GABAergic connections distinguish striate (V1) and extrastriate
(V2, V4) visual cortices and their functionally specialized subdivisions in the rhesus monkey. J Neurosci 12:4545–4564.
Kritzer MF, McLaughlin PJ, Smirlis T, Robinson JK (2001) Gonadectomy
impairs T-maze acquisition in adult male rats. Horm Behav
39:167–174.
Cerebral Cortex March 2004, V 14 N 3 279
Kubota Y, Hattori R, Yui Y (1994) Three distinct subpopulations of
GABAergic neurons in rat frontal agranular cortex. Brain Res
639:159–173.
Leranth C, Petnehazy O, MacLusky NJ (2003) Gonadal hormones affect
spine density in the CA1 hippocampal subfield of male rats. J
Neurosci 23:1588–1592.
Marone M, Quinones-Jenab V, Meiners S, Nowakowski RS, Ho SY,
Geller HM (1995) An immortalized mouse neuroepithelial cell line
with neuronal and glial phenotypes. Dev Neurosci 14:311–323.
Miller MW, Vogt BA (1984) Direct connections of rat visual cortex with
sensory, motor, and association cortices. J Comp Neurol
226:184–202.
Milner TA, McEwen BS, Hayashi S, Li CJ, Reagan LP, Alves SE (2001)
Ulrastructural evidence that hippocampal alpha estrogen receptors
are located at extranuclear sites. J Comp Neurol 429:355–371.
Nunez JL, Juraska JM (1998) The size of the splenium of the rat corpus
callosum: influence of hormones, sex ratio, and neonatal cryoanesthesia. Dev Psychobiol 33:295–303.
Olavarria J,Van Sluyters RC (1983) Widespread callosal connections in
infragranular visual cortex of the rat. Brain Res 279:233–237.
Paxinos G, Watson C (1986) The rat brain in stereotaxic coordinates.
New York: Academic Press.
Pfaff D, Keiner M (1973) Atlas of estradiol-concentrating cells in the
central nervous system of the female rat. J Comp Neurol
151:121–158.
Prins GS, Birch L, Greene GL (1991) Androgen receptor localization in
different cell types of the adult rat prostate. Endocrinology
129:3187–3199.
Puy L, MacLusky NJ, Becker L, Karsan N, Trachtenberg J, Brown TJ
(1995) Immunocytochemical detection of androgen receptor in
human temporal cortex: characterization and application of polyclonal androgen receptor antibodies in frozen and paraffinembedded tissues. J Steroid Biochem Mol Biol 55:197–209.
Sar M, Stumpf WE (1985) Distribution of androgen-concentrating
neurons in rat brain. In: Anatomical neuroendocrinology (Stumpf,
WE, Grant LD, eds), pp. 120–133. Basel: Karger.
280 Androgen Receptors in Rat Cerebral Cortex • Kritzer
Sholl SA, Kim KL (1990) Androgen receptors are differentially distributed between right and left cerebral hemispheres of the fetal male
rehsys monkey. Brain Res 516:122–126.
Sholl SA, Pomerantz SM (1986) Androgen receptors in the cerebral
cortex of fetal female rhesus monkeys. Endocrinology
119:1625–1631.
Shughrue PJ, Lane MV, Mercenthaler I (1997) The comparative distribution of estrogen receptor α- and β mRNA in the rat central nervous
system. J Comp Neurol 388:507–525.
Shughrue PJ, Scrimo PJ, Mercenthaler I (2000) Estrogen binding and
estrogen receptor characterization (ERα and ERβ) in the cholinergic
neurons of the rat basal forebrain. Neuroscience 96:41–49.
Simerly RB, Chang C, Muramatsu M, Swanson LW (1990) Distribution of
androgen and estrogen receptor mRNA-containing cells in the rat
brain: an in situ hybridization study. J Comp Neurol 294:76–95.
Stenn PG, Klaiber EL, Vogel W, Broverman DM (1972) Testosterone
effects on photic stimulation of the EEG and mental performances of
humans. Percept Motor Skills 34:371–378.
Stewart J, Rajabi H (1994) Estradiol derived from testosterone in
prenatal life affects the development of catecholamine systems in
the frontal cortex in the male rat. Brain Res 646:157–160.
Toran-Allerand CD (1991) Organotypic culture of the developing cerebral cortex and hypothalamus: relevance to sexual differentiation.
Psychoneuroendocrinology 16:7–24.
Venkatesan C, Kritzer MF (1999) Perinatal gonadectomy affects corticocortical connections in motor but not visual cortex in adult male
rats. J Comp Neurol 415:240–265.
Xiao L, Jordan CL (2002) Sex differences,laterality, and hormonal regulation of androgen receptor immunoreactivity in rat hippocampus.
Horm Behav 42:327–336.
Zhang L, Chang YH, Barker JL, Hu Q, Zhang L, Maric D, Li B-S, Rubinow
DR (2000) Testosterone and estrogen affect neuronal differentiation
but not proliferation in early embryonic cortex of the rat: the
possible roles of androgen and estrogen receptors. Neurosci Lett
281:57–60.
Zilles K, Wree A (1985) Cortex: Areal and laminar structure. In: The rat
nervous system. Vol. 1. Forebrain and midbrain (Paxinos G, ed.), pp.
375–416. Orlando, FL: Academic Press.