Regionally Selective Effects of Gonadectomy on Cortical Catecholamine Innervation in Adult Male Rats Are Most Disruptive to Afferents in Prefrontal Cortex

M.F. Kritzer, A. Adler, J. Marotta and T. Smirlis

Department of Neurobiology and Behavior, State University of New York at Stony Brook, Stony Brook, New York, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Changes in gonadal hormones induced early in life produce substantial, seemingly permanent decreases in tyrosine hydroxylase (TH)-immunoreactive axon density in sensory, motor and prefrontal regions in the rat cerebral cortex. Less is known, however, about the responsiveness of cortical catecholamines to hormone stimulation during adulthood. In this study we expanded upon an earlier analysis of the effects of acute (4 day) and chronic (28 day) gonadectomy in adult male rats on TH innervation in right hemifield of the cingulate cortex to include assessment of sensorimotor areas previously examined following perinatal gonadectomy, the left cingulate hemifield, and one additional prefrontal area – the dorsal anterior insular cortex. Qualitative and quantitative analyses of immunoreactivity revealed modest, transient declines in innervation in sensorimotor areas 4 days after gonadectomy, and a return to normal innervation densities by 28 days after surgery. In cingulate and insular cortices, however, strikingly depleted axon densities observed following acute gonadectomy rebounded to significantly higher than normal levels of innervation 3 weeks later. All effects were attenuated in gonadectomized animals supplemented with testosterone. Thus, for cortical catecholamine innervation, as for other endpoints of hormone stimulation, gonadal steroid sensitivity appears to change dramatically with lifestage. In adult male rats, this sensitivity is also marked by a seemingly selective vulnerability of catecholamine innervation in prefrontal areas to changes in the hormone environment induced by gonadectomy.


    Introduction
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Sex differences and/or hormone malleability of sensory, motor and cognitive skills in juvenile monkeys (Goy and Resko, 1972Go; Clark and Goldman-Rakic, 1989Go) [for review see Bachevalier and Hagger (Bachevalier and Hagger, 1991Go)] and human infants (Diamond, 1985Go; Overmann et al., 1996Go) suggest that gonadal hormones provide a potent stimulus for emergent cortical information processing. In human infants in particular, sex differences in the acquisition of motor skills (Butler, 1984Go), as well as perceptual (Tighe and Polison, 1978; Bauer et al., 1986Go), language (Hutt, 1972Go) and mnemonic processing (Diamond, 1985Go) indicate that a spectrum of developing cortical functions are sensitive to gonadal steroid stimulation. The perinatal hormone environment also influences cortical structure in a number of functionally specialized subdivisions. Perinatal gonadectomy in rats, for example, has been shown to significantly affect hemisphere-specific patterns of cortical thickness in five of seven sensory, associational and motor regions examined (Diamond, 1991Go) and influence dendrite structure in cingulate and insular prefrontal cortices (Kolb and Stewart, 1991Go).

Hormone manipulation induced early in postnatal life also appears to have relatively widespread influence on the catecholamine innervation of the cerebral cortex. In male and female rats, perinatal gonadectomy has been shown to affect the tempo of monoamine maturation in cingulate, parietal and occipital cortices (Stewart et al., 1991Go), and in males imposes lasting decreases in tyrosine hydroxylase (TH)-immunoreactive axon density in somatosensory, motor, premotor and cingulate cortices (Kritzer, 1998Go). However, in many neuroendocrine and reproductive brain areas, endpoints of steroid stimulation — including effects on neurotransmitters (Herbison and Dye, 1993Go) — can significantly change and even disappear over the course of the lifespan (Arnold and Gorski, 1984Go). There is some evidence that the hormone sensitivity of cortical catecholamines may also vary with maturity. Specifically, whereas long-term effects of perinatal gonadectomy decreased TH-immunoreactive axon density in areas including the cingulate cortex (Kritzer, 1998Go), a previous study has shown that there is an increase in innervation density in the right hemifield of this region following adult-stage gonadectomy (Adler et al., 1999Go).

Because prior studies in adult animals were limited to the right cingulate cortex (Adler et al., 1999Go), it is unknown whether the catecholamine innervation of sensorimotor areas — which respond vigorously to perinatal gonadectomy (Kritzer, 1998Go) — retain hormone sensitivity in adulthood. Further, it is unknown whether effects of adult-stage gonadectomy are lateralized, as they can be following perinatal gonadectomy (Kritzer, 1998Go). To address these issues, catecholamine innervation was examined in the left and right hemifields of primary somatosensory, primary motor and premotor cortices, and of two prefrontal association areas — the anterior cingulate and dorsal anterior insular cortices — in adult male rats gonadectomized 4 or 28 days prior to being killed. In the cingulate region, axons in the right hemisphere were reevaluated (Adler et al., 1999Go), while those in the left are studied here for the first time. By using immunocytochemistry for TH, axon innervation was qualitatively and quantitatively compared in each of these five areas in gonadectomized animals, gonadectomized subjects supplemented with testosterone proprionate, and in sham-operated controls to explore regional patterns of cortical catecholamine sensitivity to experimentally induced changes in the mature hormonal milieu.


    Materials and Methods
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Animal Subjects

Twenty-five adult male Sprague–Dawley rats (Taconic Farms, Germantown, NY) were used; all animals had served as subjects in a previous study of catecholamine innervation in the right cingulate hemifield (Adler et al., 1999Go). All procedures involving animals were approved by the Institutional Animal Care and Use Committee, SUNY at Stony Brook, and minimized the use of animals and their discomfort. Animals were housed with food and water freely available under a 12 h light/dark cycle. Five animals were sham-operated (CTRL); the remaining 20 rats were gonadectomized and implanted with pellets containing either biodegradable matrix, i.e. placebo (GDX-pl), or testosterone proprionate (GDX-TP). All animals were killed 4 or 28 days after surgery (see below).

Surgical Procedures

All surgical procedures were performed under aseptic conditions and used a mixture of ketamine (0.09 ml/100 g) and xylazine (0.05 ml/100 g) for anesthesia. For gonadectomies and sham operations, the sac of the scrotum and underlying tunica were incised. For gonadectomies, the vas deferens was ligated bilaterally, the testes were removed and slow-release pellets were implanted (see below). For all surgeries, incisions were closed using 6–0 silk sutures.

Depending on survival time, gonadectomized animals received either 21 day slow-release pellets (4 day survival) or 60 day slow-release pellets (28 day survival) that contained either TP in biodegradable matrix, or a placebo containing the biodegradable matrix only (cholesterol, microcrystalline cellulose, {alpha}-lactose, diand tri-calcium phosphate, calcium and magnesium stearate, and stearic acid; Innovative Research of America, Toledo, OH). The TP-containing pellets release ~3–4 ng of TP/ml blood/day, and have been used successfully in previous investigations to maintain circulating levels of testicular hormones in gonadectomized rats (Carmignac et al., 1994; Collins et al., 1992Go).

Euthanasia

Four or 28 days after gonadectomy or sham surgery, rats were deeply anesthetized with an i.m. injection of a mixture of ketamine (0.09 ml/ 100 g) and xylazine (0.05 ml/100 g). After corneal reflexes could no longer be elicited, rats were transcardially perfused with 50–100 ml of 0.1 M phosphate buffer (PB) followed by two paraformaldehyde fixative solutions: 4% paraformaldehyde in 0.1 M PB, pH 6.5 (flow rate 30 ml/min, duration 5 min), and then 4% paraformaldehyde, in 0.1 M borate buffer, pH 9.5 (flow rate 35 ml/min, duration 20 min). These parameters were kept constant to maximize comparable preservation of tissue antigens across animals. After perfusion, the brains were removed, blocked and cryoprotected in 0.1 M PB containing 30% sucrose prior to rapid freezing in powdered dry-ice and storage at –80°C. The medial, ventral and lateral bulbocavernosus muscles were also dissected out and weighed at the time of death; mean muscle weights, whole body weights and percent of whole body weight represented in the dissected muscle mass have been published elsewhere (Adler et al., 1999Go).

Immunocytochemistry

Tissue blocks that included the rostral caudate and septal nuclei were frozen-sectioned at a thickness of 40 µm in the coronal plane; left hemispheres were marked with subcortically placed sectioning artifacts (see Fig. 1Go). A rostrocaudal series of sections from each animal was then immunoreacted using antibodies recognizing the dopamine-synthesizing enzyme TH. Briefly, sections were rinsed in 0.1 M PB, washed in 1% H2O2 (45 min), treated with 1% sodium borohydride in PB (45 min), and then rinsed in 50 mM Tris-buffered saline (TBS), pH 7.4. Sections were then incubated in blocking solution (50 mM TBS containing 10% normal swine serum, NSS) for 2 h, prior to being placed in anti-TH antibody (2–3 days, diluted in TBS containing 1% NSS, 4°C). The primary antibody, obtained from Chemicon International Inc. (Temecula, CA) was used at working dilution of 1:1000. The tissue sections were then rinsed in TBS, incubated in biotinylated secondary antibodies (Vector, Burlingame, CA, 2 h, room temperature, working dilution 1:100), rinsed further in TBS, and then placed in avidin–biotin-complexed horseradish peroxidase (ABC, Vector, 2 h, room temperature). After this final incubation, sections were thoroughly rinsed in Tris buffer, pH 7.6, and reacted using 0.07% 3,3'-diaminobenzidine (DAB) as chromagen. As a control, these immunocytochemical labeling procedures were carried out on representative sections with the omission of primary antiserum or secondary antibodies. The specificity of immunolabeling for TH was further supported in parallels in the patterns of cortical labeling obtained with control animals compared to those documented in previous studies of cortical catecholamine innervation in rats (Levitt and Moore, 1978Go; Morrison et al., 1978Go; Lewis et al., 1979Go; Berger et al., 1985Go; Van Eden et al., 1987Go; Papadapoulos et al., 1989) (see Results).



View larger version (22K):
[in this window]
[in a new window]
 
Figure 1.  Schematic diagram of a representative cross section near the anteroposterior midpoint of the septal nucleus. The locations of area Cg1, Par 1 and AID (Zilles, 1990), and of areas AgM and AgL (Donoghue and Wise, 1982) are shown. For each of these five areas, qualitative analyses were carried out at representative rostrocaudal levels, and quantitative measures were derived from sections matching the one illustrated. Quantitation was based on camera lucida drawings of TH-immunoreactive fibers in layer II/III and layer V. These drawings subtended the entire spans of areas Cg1, AgM, AgL and AID present; the stippled area within area Par1 represents the subportion of this region from which quantitative measures were derived. Abbreviations: art, sectioning artifact; cc, corpus callosum; cd, caudate nucleus; sn, septal nucleus; rf, rhinal fissure; L, left hemisphere; R, right hemisphere.

 
Silver/Gold Intensification

The DAB immunoreaction product was intensified using methods of Kitt et al. (Kitt et al., 1988Go). For this procedure, DAB-reacted, slide-mounted sections were incubated in 1% silver nitrate (pH 7.0, 50 min, 55°C, in the dark), rinsed in running distilled water, placed in 0.2% gold chloride (15 min, room temperature, in the dark), rinsed again in distilled water, and fixed in 5% sodium thiosulfate (10 min, room temperature). The intensified sections were then counterstained with 1% cresyl violet and placed under coverslips. Sections used in control studies (above) were intensified side-by-side with normally immunoreacted slides.

Qualitative Evaluation

Detailed examination of the laminar distribution, orientation, approximate density and the morphology of TH-immunoreactive axons was carried out in representative sections throughout the rostrocaudal extent of the left and right hemifields of the anterior dorsal cingulate cortex (area Cg1), the primary motor cortex (area AgL), primary somatosensory cortex (area Par1), the premotor cortex (area AgM) and the dorsal anterior insular cortex (area AID) (Zilles, 1990Go; Donoghue and Wise, 1982Go) (see Fig. 1Go) in each of the five groups of animals. At least two series of sections (immunoreacted on different days) were qualitatively examined from each animal.

Quantitative Evaluation

Because subtle rostral-to-caudal gradients exist in the density of catecholamine innervation in some of the regions analyzed (Van Eden et al., 1987Go), a single anteroposterior cortical level, transecting the mid-septal nucleus (Fig. 1Go), was selected for quantitative study. Sections at this level contained representations of each of the five cytoarchitectonic fields examined. Tissue sections for these analyses were cut from all animals on the same day and immunoreacted as a group to maximize intersubject consistency in labeling; all slides were coded prior to analysis, and a single observer performed all quantitative analysis for a given cortical region. Quantitative analyses of fiber density and orientation were carried out by first making camera lucida drawings of immunoreactive fibers, visualized under brightfield illumination using a 63x oil immersion objective, in layers II/II and V of the left and right hemifields; section thickness was always measured beforehand (by using roll-focusing from surface-tosurface and the calibrated fine-focus of the microscope, Zeiss Axioskop) to ensure uniformity in section breadth. Individual drawings subtended widths of ~100–300 µm (measured parallel to the pial surface), a height dictated by the thickness of the layer, and a depth covering the full thickness of the section. Three non-overlapping drawings were obtained from each cortical layer: from each area of interest, from each hemisphere, from each animal. Per hemisphere, drawings from areas AID, AgM, Cg1 and AgL subtended essentially the entire cytoarchitectonic representation present (Fig. 1Go); in area Par1, a subregion that occupied an approximate 3 o'clock position within the hemisphere that lay in radial alignment with a local maxima in cell density of layer IV was analyzed. No other attempts were made to preselect drawing locations. All drawings were then digitized, and measures of mean pixel density from skeletinized images (NIH Image 1. 58) provided fiber density estimates (Kritzer and Kohama, 1998Go).

Statistical Analyses

Measures of axon density, and of body weight and bulbocavernosus muscle mass were evaluated using ANOVA, followed by allowed Student– Newman–Keuls post-hoc comparisons (Super ANOVA 1.11). Sample sizes of all data sets compared were equal. Prior to analysis of variance, descriptive statistical analyses were performed on each data set (Stat View 4.5) to evaluate sample distribution and variance.


    Results
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Efficacy of Hormone Treatments

Adult-stage gonadectomy and gonadectomy paired with hormone replacement are established means of experimentally diminishing and maintaining circulating gonadal hormone levels respectively (Collins et al., 1992Go). The efficacy of these experimental treatments in shaping hormone levels in the animals of this study was evinced by group-specific differences in the weights of the androgen-sensitive bulbocavernosus muscles, which is a proven and highly sensitive index of circulating testicular hormones in rats (Wainman and Shipounoff, 1941Go). As anticipated in previous investigations (Collins et al.,. 1992Go), bulbocavernosus muscle mass was ~56% and ~32% of normal in rats gonadectomized and placebo-implanted for 4 and 28 days respectively, and 70–88% of normal in the gonadectomized animals supplemented with TP for the same amounts of time (Adler et al., 1999Go). The reliability of these measures was supported in statistical evaluations. First, an ANOVA identified significant effects of treatment [F(2,21) = 41.76, P < 0.0001] and excluded individual animals as probably sources of variance in the data (P >1.0). The allowed post-hoc comparisons revealed that the reduction in muscle mass in the chronically gonadectomized, placebo-treated group was significant (Student–Newman-Keuls, P < 0.05) (Adler et al., 1999Go).

Specificity of TH Immunoreactivity

Tyrosine hydroxylase immunoreactivity was examined in the left and right cerebral hemifields of two prefrontal regions — the dorsal anterior cingulate (area Cg1) and dorsal anterior insular (area AID) cortices — in the primary somatosensory (area Par1) and primary motor (area AgL) cortices, and in the premotor area (AgM), in hormonally manipulated and control animals. The morphology, distribution and the density of TH-immunoreactive axons has been previously described for these areas in hormonally intact adult rats (Berger et al., 1985Go; Van Eden et al., 1987Go; Papadapoulos et al., 1989). In all regions, the appearance and apparent density of immunolabeling in control animals of this study were highly reminiscent of these descriptions (see below). These parallels with an established literature indicate the specificity of immunolabeling in this study for cortical catecholaminergic fibers, a conclusion also supported by the elimination of any obvious patterned staining upon removal of primary or secondary antibodies from the immunolabeling procedures.

Visual inspection alone revealed clear departures from normal patterns of catecholamine innervation in gonadectomized rats. However, it was also obvious that the effects of acute (4 day) and chronic (28 day) gonadectomy were markedly different from one another. The qualitative and quantitative details of these two outcomes are described separately below.

Tyrosine Hydroxylase Immunoreactivity in Acutely Gonadectomized Rats

The catecholamine innervation of the adult rat cerebrum is characterized by smooth gradients in innervation along its major, e.g. anteroposterior, axes that are interrupted in some cases by more abrupt transitions in axon density or orientation at certain cytoarchitectonic boundaries (Van Eden et al., 1987Go). These features were clearly recognizable in the TH immunoreactivity of sham-operated control animals. For example, extremely dense accumulations of TH-immunoreactive fibers occupied the cingulate and insular cortices, and more moderate levels of innervation were present in adjacent premotor and somatosensory regions. Innervation also displayed expected regionand layer-specific patterns of axon orientation (Berger et al., 1985Go; Febvret et al., 1991Go). Whereas axons in areas Cg1 and AID corresponded mainly to short, randomly arranged processes, fibers in motor, premotor and somatosensory areas were most often longer and more radially oriented (Fig. 2A,FGo). The TH-immunoreactive axons also corresponded to morphological subtypes similar to those that have been previously described (Berger et al., 1985Go; Febvret et al., 1991Go), including small populations of thick, straight, smooth axons, greater numbers of medium caliber, beaded fibers, and extremely fine, sparsely varicose immunoreactive axons (Fig. 2A,FGo).



View larger version (89K):
[in this window]
[in a new window]
 
Figure 2.  Representative brightfield photomicrographs showing the morphology and orientation of axons immunoreactive for TH in layer V of the dorsal anterior insular cortex (AID, A–E) and layer III of the primary somatosensory cortex (area Par1, F–J) in controls (CTRL), rats gonadectomized and placebo-implanted 4 or 28 days prior to being killed (4d GDX-pl, 28d GDX-pl), and rats gonadectomized and supplemented with TP 4 or 28 days prior to being killed (4d GDX-TP, 28d GDX-TP). All sections were immunoreacted for TH and counterstained with 1% cresyl violet; none of the cellular staining visible corresponds to tyrosine-immunopositive somata. In both regions shown, features of axon morphology and orientation characteristic of control animals are also present in gonadectomized rats. Distinctive features such as the short, randomly oriented processes in layer V of AID (A–E), and the long radial fibers in layer III of area Par1 (F–J) are preserved across experimental groups. Decreases in the number of immunoreactive axon segments, however, distinguish both area AID and Par 1 in the 4d GDX-pl animal; for Par1 remaining panels are quantitatively as well as qualitatively similar to the control. In area AID, the 28d GDX-pl panel stands out for a greater than normal complement of fibers; the 4d GDX-TP and 28d GDX-TP panels are similar to controls. Scale bars: (A–E) 100 µm; (F–J) 200 µm.

 
Qualitatively normal features of axon morphology and orientation were also observed in the cortices of rats that had been gonadectomized and placebo-implanted 4 days prior to being killed (4day GDX-pl, see Fig. 2B,GGo). However, in all but the premotor cortex, there were visibly fewer immunoreactive fibers present per unit area. This decrement was severe in areas Cg1 and AID where what should have been extremely dense axon networks corresponded to sparsely distributed sets of fibers (Fig. 3Go). Immunoreactivity in the primary motor and somatosensory areas was also clearly reduced (Fig. 4Go). In layers II/III, for example, there was an obvious reduction in the number of radially oriented axon segments. In the premotor cortex, however, it was difficult to discern a decrease in immunoreactive axon density. Rather, in both premotor hemifields, moderately dense levels of immunoreactivity seemed to be reminiscent of control levels. This was difficult to judge by eye, however, since these fields of relatively dense innervation were flanked by cingulate and primary motor areas where labeling was markedly diminished.



View larger version (93K):
[in this window]
[in a new window]
 
Figure 3.  Representative camera lucida drawings of TH-immunoreactive fibers within the dorsal anterior cingulate cortex (area Cg1) and the dorsal anterior insular cortex (AID) of the left hemisphere of a control (CTRL) animal, an animal gonadectomized and placebo-implanted (GDX-pl) and an animal gonadectomized and supplemented with testosterone proprionate (GDX-TP); all surgeries took place 4 days before the animals were killed. Laminar boundaries are marked by Roman numerals on the left. Comparison across the animal groups represented illustrates the significant loss of axon density in both cingulate and insular cortex of gonadectomized, placebo-implanted animals, and the virtually normal levels of innervation in gonadectomized animals implanted with TP. Across groups, seemingly normal, layer-specific patterns of TH-immunoreactive fiber orientation are preserved. Abbreviation: wm, white matter.

 


View larger version (119K):
[in this window]
[in a new window]
 
Figure 4.  Representative camera lucida drawings of TH-immunoreactive fibers within the premotor cortex (area AgM) and within the primary motor (AgL) and primary somatosensory (Par1) cortices. The left hemispheres of a control (CTRL) animal, an animal gonadectomized and placebo-implanted (GDX-pl) and an animal gonadectomized and supplemented with testosterone proprionate (GDX-TP) are shown; all surgeries took place 4 days before the animals were killed. Laminar boundaries are marked by Roman numerals on the left. Comparison across the animal groups represented illustrates a minimal decline in immunoreactivity in area AgM, and more substantial axon losses in areas AgL and Par1 of gonadectomized, placebo-implanted animals. Seemingly normal levels of innervation are found in all areas of gonadectomized animals that received implants of TP. Intact, layer-specific patterns of TH-immunoreactive fiber orientation are also preserved in all animal groups. Abbreviation: wm, white matter.

 
Quantitative estimates of TH-immunoreactive axon density in representative supragranular (layers II or III) and infragranular (layer V) layers confirmed that there were relatively small effects of acute gonadectomy on catecholamine axons in area AgM. Thus, in this region, axon density measures in 4 day GDX-pl rats were diminished by no more than 20% (between 80 and 100% of normal; see Figs 4 and 5GoGo). Corresponding analyses in prefrontal, primary somatosensory and primary motor cortices also provided additional insights into the clearly greater hormone sensitivity of catecholamine axons in these regions. For example, as anticipated in a previous examination of the right cingulate hemifield (Adler et al., 1999Go), in area Cg1 axon density estimates in this study showed that innervation on both the left and right of the 4 day GDX-pl animals fell to between 50 and 55% of normal in layer V, and dropped even more sharply to ~30% of normal in layers II/III (Fig. 6Go). In area AID, on the other hand, innervation was similarly and dramatically reduced to values of near 30% of normal in both supragranular and infragranular layers (Fig. 7Go). Less hemispherically symmetric and less severe were the responses of catecholamine axons in the primary motor and somatosensory cortices. In motor cortex, innervation in the supragranular and infragranular layers corresponded to 41 and 46% of normal on the right, and to 50 and 58% of normal on the left (Fig. 8Go). Finally, in primary somatosensory cortex, axon density was least affected, dropping in layers II/III to a mean of 69 and 64% of normal on the left and right respectively, and in layer V to 84% of normal in the left hemifield and 75% on the right (Fig. 9Go).



View larger version (18K):
[in this window]
[in a new window]
 
Figure 5.  Scatter plots of mean pixel density measures derived from camera lucida drawings of TH-immunoreactive fibers in layers II/III and V of the left and right premotor (AgM) hemifields of control animals (CTRL), rats gonadectomized and placebo-implanted 4 or 28 days prior to being killed (4D GDX, 28D GDX), and gonadectomized rats implanted with testosterone proprionate 4 or 28 days prior to being killed (4D GDX-TP, 28D GDX-TP). The plots represent raw data points that are inclusive of all measures obtained from each of the five individual animals in a given experimental group. Horizontal bars mark group means. Measures of axon density in control, GDX and GDX-TP groups are essentially overlapping, and none of the differences in group mean values among the groups are statistically significant.

 


View larger version (18K):
[in this window]
[in a new window]
 
Figure 6.  Scatter plots of mean pixel density measures derived from camera lucida drawings of TH-immunoreactive fibers in layers II/III and V of the left and right cingulate (Cg1) hemifields of control animals (CTRL), rats gonadectomized and placebo-implanted 4 or 28 days prior to being killed (4D GDX, 28D GDX), and gonadectomized rats implanted with testosterone proprionate 4 or 28 days prior to being killed (4D GDX-TP, 28D GDX-TP). The plots represent raw data points inclusive of all measures obtained from each of the five individual animals comprising a given experimental group. Horizontal bars mark the numerical group means of pixel density measures. The large, statistically significant decreases in axon density in 4D GDX animals are similar in left and right hemispheres, but are proportionately greater in layer II/III than in layer V. In 28D GDX animals, axon density is closer to yet statistically less than normal in layer II/III, but rises to levels that are significantly greater than normal in layer V. Axon density in gonadectomized animals implanted with TP are statistically normal 4 and 28 days after surgery in both layers II/III and V.

 


View larger version (18K):
[in this window]
[in a new window]
 
Figure 7.  Scatter plots of mean pixel density measures derived from camera lucida drawings of TH-immunoreactive fibers in layers II/III and V of the left and right insular (AID) hemifields of control animals (CTRL), rats gonadectomized and placebo-implanted 4 or 28 days prior to being killed (4D GDX, 28D GDX), and gonadectomized rats implanted with testosterone proprionate 4 or 28 days prior to killed (4D GDX-TP, 28D GDX-TP). The plots represent raw data points inclusive of all measures obtained from each of the five individual animals comprising a given experimental group. Horizontal bars mark the numerical group means of pixel density measures. Large statistically significant decreases in axon density in 4D GDX animals are proportionate in both layers and hemispheres examined. In 28D GDX animals, axon density returns to statistically normal values in layer II/III, but rebounds to levels that are significantly greater than normal in layer V. Axon density in gonadectomized animals implanted with TP are statistically normal 4 and 28 days after surgery in layers II/III and V.

 


View larger version (18K):
[in this window]
[in a new window]
 
Figure 8.  Scatter plots of mean pixel density measures derived from camera lucida drawings of TH-immunoreactive fibers in layers II/III and V of the left and right primary motor (AgL) hemifields of control animals (CTRL), rats gonadectomized and placebo-implanted 4 or 28 days prior to being killed (4D GDX, 28D GDX), and gonadectomized rats implanted with testosterone proprionate 4 or 28 days prior to being killed (4D GDX-TP, 28D GDX-TP). The plots represent raw data points inclusive of all measures obtained from each of the five individual animals comprising a given experimental group. Horizontal bars mark the numerical group means of pixel density measures. Large statistically significant decreases in axon density in 4D GDX animals are proportionate in both layers but differ slightly across the two hemispheres. In 28D GDX animals, axon density returns to statistically normal values in layer II/III and V. Axon density in gonadectomized animals implanted with TP are statistically normal 4 and 28 days after surgery in layers II/III and V.

 


View larger version (18K):
[in this window]
[in a new window]
 
Figure 9.  Scatter plots of mean pixel density measures derived from camera lucida drawings of TH-immunoreactive fibers in layers II/III and V of the left and right primary somatosensory (Par1) hemifields of control animals (CTRL), rats gonadectomized and placebo-implanted 4 or 28 days prior to being killed (4D GDX, 28D GDX), and gonadectomized rats implanted with testosterone proprionate 4 or 28 days prior to being killed (4D GDX-TP, 28D GDX-TP). The plots represent raw data points inclusive of all measures obtained from each of the five individual animals comprising a given experimental group. Horizontal bars mark the numerical group means of pixel density measures. Large statistically significant decreases in axon density in 4D GDX animals are found in layer II/III in both hemispheres. Axon density is also decreased in these animals in layer V, but this trend did not reach statistical significance. In 28D GDX animals, axon density returns to statistically normal values in layer II/III and V. Axon density in gonadectomized animals implanted with TP are statistically normal 4 and 28 days after surgery in layers II/III and V.

 
Statistical evaluations (region-by-region ANOVAs, including animals receiving chronic gonadectomy and hormone replacement, below) of axon density measures identified significant main effects of treatment (gonadectomy) in all but area AgM [AgL: F(4,20) = 57.84, P < 0.0001; AID: F(4,20) = 33.31, P < 0.0001; Cg1: F(4,20) = 28.25, P < 0.001; Par1: F(4,20) = 6.46, P < 0.0017]. In the cingulate and somatosensory regions, significant treatment-by-layer interactions were also revealed [Cg1: F(4,20) = 34.03, P < 0.001; Par1: F(4,20) = 3.19, P < 0.352], consonant with the differences in the degree to which gonadectomy affected TH innervation in the supragranular and infragranular layers (see Fig. 6Go). In no cases, however, were significant treatment-by-hemisphere interactions revealed, and in no cases were individual animals found to be significant sources of variability in the data [P > 1.0]. Post-hoc comparison (Student– Newman–Keuls) revealed that with the exception of layer V in somatosensory cortex, all of the diminished values of axon density in areas Cg1, AID, AgL and Par1 were significantly different from controls at a P < 0.05 level (Figs 6–9GoGoGoGo). In contrast, none of the measures of axon density from area AgM of 4 day GDX-pl rats were significantly different from controls (Fig. 5Go).

Tyrosine Hydroxylase Immunoreactivity in Chronically Gonadectomized Rats

Immunoreactivity in sham-operated control animals was also compared to immunolabeling in animals that had been gonadectomized and placebo-implanted 28 days prior to being killed (28 day GDX-pl). In contrast to the fairly widespread depletion of immunoreactivity in acutely operated animals, labeling in most of the cortical areas of 28 day GDX-pl animals seemed qualitatively and quantitatively normal. In premotor, motor and somatosensory areas, for example, not only were axon morphology and orientation intact, but normal appearing axon densities were also observed (Fig. 10Go). Quantitative estimates of axon density substantiated these observations; in area AgM, axon density ranged from 98% (layer V, left hemisphere) to 113% (layer II/III, left hemisphere) of normal (Fig. 5Go), in area AgL values were between 92% (layer II/III, right hemisphere) and 104% (layer II/III, left hemisphere) of controls (Fig. 8Go), and in area Par1 axon density lay between 89% (layer II/III, right hemisphere) and 123% (layer V, right hemisphere) of values obtained in sham-operated animals (Fig. 9Go). Statistical analyses (ANOVA, followed by Student–Newman–Keuls post–hoc comparison) showed that none of these axon density measures in any of these areas were statistically different from controls at a P < 0.05 level.



View larger version (129K):
[in this window]
[in a new window]
 
Figure 10.  Representative camera lucida drawings of TH-immunoreactive fibers within the premotor cortex (area AgM) and within the primary motor (AgL) and primary somatosensory (Par1) cortices. The left hemispheres of a control (CTRL) animal, an animal gonadectomized and placebo-implanted (GDX-pl) and an animal gonadectomized and supplemented with testosterone proprionate (GDX-TP) are shown; all surgeries took place 28 days before the animals were killed. Laminar boundaries are marked by Roman numerals on the left. Comparison across the animal groups represented shows that in areas AgM, AgL and Par 1, axon orientation and density are similar in all layers for all animal groups. Abbreviation: wm, white matter.

 
In contrast and as anticipated in a previous study of the right cingulate hemifield (Adler et al., 1999Go), axon density in the deep layers of area Cg1 (left and right hemispheres) and in the infragranular layers of area AID was noticeably elevated in chronically gonadectomized rats (Fig. 11Go). Quantitative evaluation showed that these differences were also statistically significant. Thus, in area Cg1 axon density was slightly albeit significantly (P < 0.05) lower than normal in the supragranular layers (~80% of normal on the left and ~58% of normal in the right hemisphere), and was significantly higher than normal (172% and 169% of controls on the left and right respectively ) in layer V (Fig. 6Go). In area AID, on the other hand, innervation was statistically similar to control values in layers II/III (between 98 and 105% in the left and right hemispheres respectively), but was significantly elevated (between 155 and 166% of normal on the left and right respectively) in layer V (Fig. 7Go).



View larger version (106K):
[in this window]
[in a new window]
 
Figure 11.  Representative camera lucida drawings of TH-immunoreactive fibers within the dorsal anterior cingulate cortex (area Cg1) and the dorsal anterior insular cortex (AID) of the left hemisphere of a control (CTRL) animal, an animal gonadectomized and placebo-implanted (GDX-pl) and an animal gonadectomized and supplemented with testosterone proprionate (GDX-TP) are shown; all surgeries took place 28 days before the animals were killed. Laminar boundaries are marked by Roman numerals on the left. Comparison across the animal groups represented illustrates that in both areas there are near normal axon orientations and densities in upper layers, but that the deep layers of GDX animals have higher than normal innervation densities. Abbreviation: wm, white matter.

 
Tyrosine Hydroxylase Immunoreactivity in Gonadectomized Rats with Hormone Replacement

Qualitative examination of immunoreactivity in both acutely and chronically gonadectomized animals supplemented with testosterone proprionate (GDX-TP) revealed a normal appearance (Fig. 2Go) and complement of catecholamine axons in all five areas examined (Figs 3, 4, 10, 11GoGoGoGo). Quantitative analyses showed that in every area, layer and hemisphere evaluated, axon density estimates in GDX-TP rats were within 25% of normal, and that only in layer V of area Cg1 were measures of axon density obtained significantly different from controls at a P < 0.05 level (Figs 5–9GoGoGoGoGo).


    Discussion
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Previous studies have shown that the perinatal hormone environment influences the maturation of cortical catecholamine innervation (Stewart et al., 1991Go; Stewart and Rajabi, 1994Go), and imparts seemingly permanent changes on TH immunoreactivity in sensory, motor and prefrontal areas (Kritzer, 1998Go). Cortical catecholamine innervation also responds to changes in circulating gonadal steroids induced in adulthood (Battaner et al., 1987Go; Adler et al., 1999Go). However, in the right hemifield of the cingulate cortex — the only area and hemisphere where direct comparisons had been previously possible — the effects of gonadectomy performed early versus later in life were qualitatively different from one another. In order to determine whether and to what extent hormone effects in other cortical areas changed with maturation, and to additionally explore the possibility of lateralized effects (Kritzer, 1998Go), a broader investigation of hormone-induced plasticity of catecholamines in the adult cerebrum was undertaken. Specifically, TH-immunoreactive axon density was assessed in the same sensory, motor and prefrontal cortices examined after perinatal gonadectomy (Kritzer, 1998Go) but this time experimental subjects were adult male rats gonadectomized 4 or 28 days prior to being killed. In addition to a reevaluation of the right hemisphere (Adler et al., 1999Go) and new evaluation of the left cingulate hemifield, a second prefrontal region — the dorsal anterior insular cortex — was also bilaterally examined. These analyses revealed that TH immunoreactivity was minimally affected by adult-stage gonadectomy in premotor cortex, that afferents in primary somatosensory and motor cortices were transiently diminished (to between 50 and 70% of normal) 4 days after gonadectomy, and that most sensitive to hormone manipulation were THimmunopositive axons in cingulate and insular prefrontal cortices. In both of these regions, TH axon density rebounded from markedly depressed levels of innervation 4 days after gonadectomy (30–50% of normal) to abnormally high densities (155–170% of normal) in the chronic condition.

That changes in the levels of circulating hormones were principally responsible for the complex outcomes observed is supported in two lines of evidence. First, all of the longand short-term effects on TH immunoreactivity in gonadectomized rats were essentially absent in gonadectomized animals supplemented with TP; in both acutely and chronically manipulated animals, TP replacement maintained both the mass of the androgen-sensitive bulbocavernosus muscles, and statistically normal levels of TH innervation in all but layer V of area Cg1. In addition, experimental methods were used that minimized variability in axon labeling and analysis (Kritzer, 1998Go; Adler et al., 1999Go) (see Materials and Methods). Consistency in parameters of tissue preservation and concurrent immunoprocessing of tissue samples, for example, provided uniformity in axon labeling, while sampling strategies and assessment of section thickness and axon orientation minimized vagaries that could have been introduced in the extraction of quantitative information from the camera lucida drawings (Kritzer and Kohama, 1998Go). These factors combine to support conclusions that catecholamine afferents in the sensorimotor regions examined are either insensitive or only transiently sensitive to changes in the levels of circulating gonadal steroids, whereas the innervation of the two prefrontal cortices assessed was unique for vigorous and sustained responses to changes in the hormonal milieu.

The Effects of Perinatal versus Adult-stage Gonadectomy

A characteristic common to many hormone-sensitive structures in brain and spinal cord is that they are not always nor equally responsive to gonadal steroid stimulation; at an extreme are critical periods of hormone sensitivity — discrete windows of time when and only when a given structure or endpoint is influenced by gonadal steroid exposure (Arnold and Gorski, 1984Go). Although the data do not speak to critical periods of hormone sensitivity in the cerebrum, comparison of the current findings with previously described effects of perinatal gonadectomy (Kritzer, 1998Go) does suggest significant change in the hormone sensitivity of cortical TH axons before and after puberty. For example, perinatal gonadectomy produces lasting, highly lateralized decrements in TH innervation in primary somatosensory and motor cortices (Kritzer, 1998Go), whereas the same manipulation in adult animals only transiently diminished catecholamine afferents in these fields. Even more striking, however, was that while perinatal gonadectomy affects axon density in sensory, motor and association areas similarly (Kritzer, 1998Go), catecholamine axons in the two prefrontal cortices examined were much more sensitive to acute and chronic gonadectomy performed in adulthood than afferents in sensory and motor regions. Four days after surgery, for example, decrements in axon density in cingulate and insular cortices were proportionately nearly twice those observed in somatosensory and motor areas, and by 28 days, it was only in prefrontal regions that aberrant patterns of innervation persisted. In further contradistinction, the enduring elevations in TH immunoreactivity that were observed in the cingulate cortex (Adler et al., 1999Go) provide a striking contrast to the chronic depression of axon density in this region that follows perinatal gonadectomy (Kritzer, 1998Go).

There are also some subtle differences in the degree to which TP replacement attenuates the effects of perinatal versus adult-stage gonadectomy. Specifically, the nearly pan-cortical effectiveness of the slow-release TP pellets implanted in animals gonadectomized as adults in stimulating normal levels of catecholamine innervation contrasts with the more regionally selective ability of daily injections of TP (adjusted weekly for changes in body weight) to sustain innervation in perinatally gonadectomized rats (Kritzer, 1998Go). Although both methods achieve near physiological levels of circulating testicular hormones (Sodersten, 1984Go; Collins et al., 1992Go; Carmignac et al., 1993Go), these differences could be related to methods of hormone supplementation. However, they may also reflect a greater degree of similarity between the square pulses of experimentally introduced steroids and the native hormonal milieu of postpubescent male rat brain. In contrast to the series of conspicuous, stereotyped surges in circulating hormones that mark prepubertal life stages, the adult brain is exposed to more consistent levels of gonadal steroids (Resko et al., 1968Go). Because these relatively unchanging hormone levels are likely to be more closely approximated by the levels of testicular hormones introduced by either daily injections and slow-release pellets, an end result of a more effective means of stimulating normal cortical catecholamine innervation in the adult brain may not be surprising.

Possible Underlying Mechanisms

As developmental mitogens and in their capacity as neurotransmitters, the catecholamines provide a functionally critical, lifelong influence to the cerebral cortex. The present study suggests that testicular hormones may provide regulatory influence over these important afferents in the adult brain. Although not directly examined in this study, some speculation about mechanisms that could underlie this influence may be forthcoming. For example, the fact that changes in catecholamine axons are region-specific suggests that hormone effects on TH-immunoreactive axons are somehow targeted rather than occurring secondarily to non-specific, perhaps diffuse metabolic changes. This in turn brings to mind intracellular estrogen receptors (ER) and androgen receptors (AR), whose cortical distributions show intriguing parallels with patterns of TH-immunoreactive axon responsiveness. Thus, whereas the widespread distribution of classical ERs (ER{alpha}s) and ARs (Shughrue et al., 1990Go; Simerly et al., 1990Go), and the left/right differences in ER{alpha}s in neonatal rat cortex (Sandhu et al., 1986Go) are consonant with the broadly cast and sometimes lateralized effects of perinatal gonadectomy (Kritzer, 1998Go), the relative confinement of AR and ER{alpha} binding (Shughrue et al., 1990Go; Miranda and Toran-Allerand, 1992Go) and/or mRNAs (Simerly et al., 1990Go) to medial and perirhinal cortices in the adult brain parallels the particular vulnerability of TH immunoreactivity in these regions to changes induced by adult-stage gonadectomy. There may also be correspondence with the distribution of the more recently described beta ERs, where in situ hybridization studies in adult rats have shown that mRNAs for this receptor subtype are present throughout the cortex but are particularly abundant in perirhinal cortex (Shughrue et al., 1997Go).

The striking differences in the effects of acute versus chronic gonadectomy on cortical immunoreactivity, however, may also have parallels to the hormone regulation of TH mRNA levels and transcription in ERand AR-containing cells of the hypothalamus. In the anteroventral periventricular nucleus, for example, TH mRNA levels have been shown to be conspicuously elevated 7 days after gonadectomy in both male and female rats, but indistinguishable from controls 10 weeks after surgery (Simerly, 1989Go). Similarly, nuclear run-on assays in the arcuate nucleus have identified acute but not chronic effects of estrogen treatment on the rate of TH mRNA transcription in ovariectomized rats (Blum et al., 1987Go). It is possible that the changes in cortical TH immunoreactivity, which are either transient or change significantly in the acute and chronic condition, may be endproducts of similar hormone receptor-mediated transcriptional regulation of TH mRNA in cortical catecholaminergic cells of origin. Consistent with this possibility are findings that subsets of noradrenergic cells in the locus coeruleus and dopaminergic cells in the substantia nigra, ventral tegmental area, and in the retrorubral fields contain ER and AR (Heritage et al., 1981Go; Kritzer, 1997Go; Shughrue et al., 1997Go). Further, there is a particularly good match between the distribution of ERß, AR and prefrontally projecting midbrain dopamine neurons (Kritzer, 1997Go; Shughrue et al., 1997Go).

While supporting arguments can be mounted for mechanisms involving either cortical or midbrain/brainstem hormone receptors, additional possibilities must also be considered, including transneuronal and even non-genomic routes of hormone influence (see McEwen, 1991). It may also be significant that the behavior of gonadectomized animals in open field testing differs from hormonally intact animals (Adler et al., 1999Go) and that these changes in behavior somehow effect cortical catecholamines. An important requisite to distinguishing among these and other possible mechanisms is to clarify whether the observed effects involve dopaminergic or noradrenergic afferents, or both. This was not possible in the present study because catecholamine axons were assessed using immunoreactivity for TH, a biosynthetic enzyme common to dopaminergic, noradrenergic and adrenergic axons. It has, however, been argued that this marker has some selectivity for dopamine axons in the prefrontal regions of the rat cortex (Lewis et al., 1979Go; Berger et al., 1985Go). Further, previous analyses of homogenates of rat cortex indicate that adult-stage gonadectomy increases dopamine and its major metabolites, but has no significant effect on noradrenalin (Battaner et al., 1987Go). Thus, the results of this study may specifically reflect sensitivity of the dopamine innervation of rat cortex. This question is being pursued in studies of the effects of gonadectomy on cortical dopamine ß-hydroxylase immunoreactivity, an enzyme marker of adrenergic axons.

Whether the axons involved are dopaminergic, noradrenergic or both, there is clear evidence for exquisite, regionally selective hormone stimulation of cortical catecholamine innervation in the adult rat brain. The present data further suggest that with maturation, the initially widespread long-term consequences of perinatal gonadectomy for sensory, motor and prefrontal (cingulate) areas (Kritzer, 1998Go) are replaced by an especial and perhaps selective disruption of catecholamine innervation in the two prefrontal cortices studied. Given the reliance of prefrontal cortical operations on their catecholamine inputs (Brozoski et al., 1979Go; Stam et al., 1989Go; Wilcott and Xuemei, 1990Go), this enduring responsiveness to changes in the hormonal milieu could have relevance for observed relationships between cognitive information processing and circulating hormone levels in adult men and women (Hampson, 1990Go). Further, that one outcome of the hormone manipulation paradigms used was a higher than normal catecholamine innervation in deep layers of these prefrontal cortices may also be of interest in relation to schizophrenia — a disorder in which hypodopaminergia in prefrontal cortex has been implicated in its negative symptoms [e.g. anhedonia, poor planning and motivation (Davis et al., 1991Go; Goldman-Rakic, 1991Go)] which are also those that tend to be most frequent and most resilient to pharmacological treatment in males (Seeman and Lang, 1990).


    Notes
 
This work was supported by a FIRST Award R29NS35422 to M.F.K.

Address correspondence to: Mary Kritzer, Department of Neurobiology and Behavior, SUNY at Stony Brook, Stony Brook, NY 11794-5230, USA. Email: mkritzer{at}neurobio.sunysb.edu.


    References
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Adler AP, Vescovo P, Robinson J, Kritzer MF (1999) Gonadectomy in adulthood increases tyrosine hydroxylase immunoreactivity in the prefrontal cortex and decreases open field activity in male rats. Neuroscience 89:939–954.[ISI][Medline]

Arnold AP, Gorski RA (1984) Gonadal steroid induction of structural sex differences in the central nervous system. Annu Rev Neurosci 7:413–442.[ISI][Medline]

Bachevalier J, Hagger C (1991) Sex differences in the development of learning abilities in primates. Psychoneuroendocrinology 16: 177–188.[ISI][Medline]

Battaner E, del Castillo AR, Guerra M, Mas M (1987) Gonadal influences on spinal cord and brain monoamines in male rats. Brain Res 425:391–394.[ISI][Medline]

Bauer JA, Shimojo S, Gwiazda J, Held R (1986) Sex differences in the development of binocularity in human infants. Invest Ophthamol Vis Sci 27:265.

Berger B, Verney C, Alvarez C, Vigny C, Helle KB (1985) New dopaminergic terminal fields in the motor and retrosplenial cortex in the young and adult rat. Immunocytochemical and catecholamine histochemical analyses. Neuroscience 15:983–998.[ISI][Medline]

Blum M, McEwen BS, Roberts JL (1987) Transcriptional analysis of tyrosine hydroxylase gene expression in the tuberoinfundibular dopaminergic neurons of the rat arcuate nucleus after estrogen treatment. J Biol Chem 262:817–821.[Abstract/Free Full Text]

Brozoski TJ, Brown RM, Rosvold HE, Goldman PS (1979) Cognitive deficit caused by regional depletion of dopamine in prefrontal cortex of rhesus monkey. Science 205:929–932.[ISI][Medline]

Butler S (1984) Sex differences in human cerebral function. Prog Brain Res 61:443–455.[ISI][Medline]

Carmignac DF, Gabrielsson BG, Robinson IC (1993) Growth hormone binding protein in the rat: effects of gonadal steroids. Endocrinology 133:2445–2452.[Abstract]

Clark AS, Goldman-Rakic PS (1989) Gonadal hormones influence the emergence of cortical function in nonhuman primates. Behav Neurosci 103:1287–1295.[ISI][Medline]

Collins WF III, Seymour AW, Klugewicz SW (1992) Differential effect of castration on the somal size of pudenal motoneurons in the adult male rat. Brain Res 577:326–330.[ISI][Medline]

Davis KL, Kahn RS, Ko G, Davidson M (1991) Dopamine in schizophrenia: a review and reconceptualization. Am J Psychiat 148:1474–1486.[Abstract]

Diamond A (1985). Development of the ability to use recall to guide action, as indicated by infants' performance on AB. Child Devel 56:868–883.[ISI][Medline]

Diamond MC (1991) Hormonal effects on the development of cerebral lateralization. Psychoneuroendocrinology 16:121–129.[ISI][Medline]

Donoghue JP, Wise SP (1982) The motor cortex of the rat: cytoarchitecture and microstimulation mapping. J Comp Neurol 212:76–88.[ISI][Medline]

Febvret A, Berger B, Gaspar P, Verney C (1991) Further indication that distinct dopaminergic subsets project to the rat cerebral cortex: lack of colocalization with neurotensin in the superficial dopaminergic fields of the anterior cingulate, motor, retrosplenial and visual cortices. Brain Res 547:37–52.[ISI][Medline]

Goldman-Rakic PS (1991) Prefrontal cortical dysfunction in schizophrenia: the relevance of working memory. In: Psychopathology and the brain (Carroll BJ, Barrett JE, eds), pp. 1–23. New York: Raven Press.

Goy RW, Resko JA (1972) Gonadal hormones and behavioral of normal and pseudohermaphroditic nonhuman female primates. Rec Prog Horm Res 28:707–733.[Medline]

Hampson E (1990) Estrogen-related variations in human spatial and articulatory-motor skills. Psychoneuroendocrinology 15:97–111.[ISI][Medline]

Herbison AE, Dye S (1993) Perinatal and adult factors responsible for the sexually dimorphic calcitonin gene-related peptide-containing cell population in the rat preoptic area. Neuroscience 54:991–999.[ISI][Medline]

Heritage AS, Stumpf WE, Sar M, Grant LD (1981) (3H)-Dihydrotestosterone in catecholamine neurons of rat brain stem: combined localization by autoradiography and formaldehyde-induced fluorescence. J Comp Neurol 200:289–307.[ISI][Medline]

Hutt C (1972) Males and females. Harmondsworth: Penguin.

Kitt CA, Levey AI, Friedmont DP, Walker LC, Koliatsos VE, Raskin LS, Price DL (1988) Immunocytochemical visualization of cholinergic fibers in monkey neocortex: enhanced visualization using silver nitrate. Soc Neurosci Abstr 14:631.

Kolb B, Stewart J (1991) Sex-related differences in dendritic branching of cells in the prefrontal cortex of rats. J Neuroendocrinology 3:95–99.[ISI]

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.[ISI][Medline]

Kritzer MF (1998) Perinatal gonadectomy exerts regionally selective, lateralized effects on the density of axons immunoreactive for tyrosine-hydroxylase in the cerebral cortex of adult male rats. J Neuroscience 24:10735–10748.

Kritzer MF, Kohama SG (1998) Ovarian hormones influence the morphology, distribution, and density of tyrosine hydroxylase immunoreactive axons in the dorsolateral prefrontal cortex of adult rhesus monkeys. J Comp Neurol 395:1–17.[ISI][Medline]

Levitt P, Moore RY (1978) Noradrenalin neuron innervation of the neocortex in the rat. Brain Res 139: 219–231.[ISI][Medline]

Lewis MS, Molliver ME, Morrison JH, Lidow HGW (1979) Complementarity of dopaminergic and noradrenergic innervation in anterior cingulate cortex of the rat. Brain Res 164:328–333.[ISI][Medline]

McEwen BS (1991) Non-genomic and genomic effects of steroids on neural activity. Trends Pharmacol Sci 12:141–147.[ISI][Medline]

Miranda R., Toran-Allerand CD (1992) Developmental expression of estrogen receptor mRNA in the rat cerebral cortex: a nonisotopic in situ hybridization histochemistry study. Cereb Cortex 2:1–15.[Abstract]

Morrison JH, Grzanna R, Molliver ME, Coyle JT (1978) The distribution and orientation of noradrenergic fibers in neocortex of the rat: an immunofluorescence study. J Comp Neurol 181:17–40.[ISI][Medline]

Overmann WH, Bachevalier J, Schumann E, and Ryan P (1996) Cognitive gender differences in very young children parallel biologically based cognitive gender differences in monkeys. Behav Neurosci 110: 673–684.[ISI][Medline]

Papadopoulos GC, Parnavelas JG, Buijs RM (1989) Light and electron microscopic immunocytochemical analysis of the dopamine innervation of the rat visual cortex. J Neurocytology 18:303–310.[ISI][Medline]

Resko JA, Feder HH, Goy RW (1968) Androgen concentration in plasma and testis of developing rats. J Endocrinology 40:485.[ISI][Medline]

Sandhu S, Cook P, Diamond MC (1986) Rat cerebral cortical estrogen receptors: male–female, right–left. Exp Neurol 92:186–196.[ISI][Medline]

Seeman MV, Lang M (1991) The role of estrogens in schizophrenia gender differences. Schizophrenia Bull 16:185–191[ISI]

Shughrue PJ, Lane MV, Mercenthaler I (1997) Comparative distribution of estrogen receptor-{alpha} and ß mRNA in the rat central nervous system. J Comp Neurol 388:507–525.[ISI][Medline]

Shughrue PJ, Stumpf WE, MacLusky NJ, Zielinski JE, Hochberg RB (1990) Developmental changes in estrogen receptors in mouse cerebral cortex between birth and postweaning: studied by autoradiography with 11-beta methoxy-16-alpha [125I] iodoestradiol. Endocrinology 126:1112–1124.[Abstract]

Simerly RB (1989) Hormonal control of the development and regulation of tyrosine hydroxylase expression within a sexually dimorphic population of dopaminergic cells in the hypothalamus. Mol. Brain Res 6:297–310.

Simerly RB, Chang C, Muramatsu M, and 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.[ISI][Medline]

Sodersten P (1984) Sexual differentiation: do males differ from females in behavioral sensitivity to gonadal hormones. Prog Brain Res 61:257–281.[ISI][Medline]

Stam C, deBruin J, vanHaelst A, van der Gugten J, Kalsbeek A (1989) Influence of the mesocortical dopaminergic system on activity, food hoarding, social-agonistic behavior, and spatial delayed alternation in male rats. Behav Neurosci 103:24–35.[ISI][Medline]

Stewart J, Kuhnemann S, Rajabi H (1991) Neonatal exposure to gonadal hormones affects the development of monoamine systems in rat cortex. J Neuroendocrinol 3:85–93.[ISI]

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.

Tighe TJ, Powlison LB (1978) Sex differences in infant habituation research: a survey and some hypotheses. Bull Psychonom Soc 12:337–340.[ISI]

Van Eden CG, Hoorneman EMD, Bujis RM, Matthijssen MAH, Geffard M, Uylings HBM (1987) Immunocytochemical localization of dopamine in the prefrontal cortex of the rat at the light and electron microscopical level. Neuroscience 22:849–862.[ISI][Medline]

Wainman P Shipounoff GC (1941) The effects of castration and testosterone proprionate on the striated perineal musculature in the rat. Endocrinology 29:975–978.

Wilcott R, Xuemei Q (1990) Delayed response, preoperative overtraining, and prefrontal lesions in the rat. Behav Neurosci 104:74–83.[ISI][Medline]

Zilles K (1990) Anatomy of the neocortex: cytoarchitecture and myeloarchitecture. In: The cerebral cortex of the rat (Kolb B, Tees RC, eds), pp. 77–112. Cambridge, MA: MIT Press.