Effects of gonadal hormones on the morphology of neurons from the medial amygdaloid nucleus of rats

Brain Research Bulletin, Vol. 48, No. 2, pp. 173–183, 1999 Copyright © 1999 Elsevier Science Inc. Printed in the USA. All rights reserved 0361-9230/99/$–see front matter

PII S0361-9230(98)00160-9

Effects of gonadal hormones on the morphology of neurons from the medial amygdaloid nucleus of rats A. A. Rasia-Filho,1* R. G. Londero2 and M. Achaval2 Departments of 1Physiology and 2Morphological Sciences, Universidade Federal do Rio Grande do Sul, Instituto de Cieˆncias Ba´sicas da Sau´de, Porto Alegre, Brazil [Received 15 June 1998; Revised 26 October 1998; Accepted 26 October 1998] ABSTRACT: The medial amygdala (MeA) has receptors for gonadal hormones and is a sexually dimorphic area in rats. The aims of the present work were (1) to look at sex differences and the effect of gonadal hormone withdrawal in males castrated as offspring or at adulthood on neuronal soma area in the anterior and posterior MeA and (2) to study the dendritic branching and the density of dendritic spines in neurons from the MeA of intact males and females. Animals were adult rats, for which the single-section Golgi method was used. Stellate and bitufted cells were found in the MeA. Comparing data among groups, no significant difference in cell body area was found. Dendrites divide sparingly and have very different lengths, and a statistical difference (p < 0.001, males higher than females) in the spine density in the anterior MeA, but not in the posterior MeA, was found. These results suggest that castration does not alter the somal area in males submitted to gonadectomy during the early postnatal period or at adulthood. In addition, the already described sex difference in this nucleus may be more related to the neuropil than the neuronal somal area, which may be relevant for the function of the MeA. © 1999 Elsevier Science Inc.

ences in neural, endocrine and behavioral responses and may also add to the identification of the relationships between morphology and function in specific areas [13,17,42,49,60]. The amygdala is part of the limbic system and is composed of several interconnected nuclei located in the temporal lobe subcortical region [1,63,65]. As suspected by the several afferent and efferent neural pathways of the amygdala, it does not have an elementary functional unity but rather shows an integrative and modulatory role in behavioral, vegetative and endocrine activities relevant for the relationship of the animal with its environment [8,9,12,23,36 –38,65,86]. In rats, the medial amygdala (MeA), which is part of the neural tissue called the extended amygdala [1] (but see [9] and [87]), is involved with the modulation of male [71,86,94] and female reproductive behavior [45]. It was recently suggested that the posterodorsal MeA (MePD) might influence hypothalamic neuroendocrine regulation whereas the anterodorsal MeA (MeAD), the anteroventral MeA (MeAV), and the posteroventral MeA (MePV) may be primarily related with the hypothalamic modulation of reproductive and defensive behaviors [9]. Due to its connections, the MeA may also contribute to associative learning and the modulation of memory and behavior for which emotion is involved [9,12]. Receptors for gonadal hormones are found in the amygdala of rats [39,40,62,74,79,81,84] in a quantity that sometimes resembles that found in the hypothalamus. This finding led to the supposition that the activity of some cells in this nucleus would be affected by the actions of sex steroids [14,82,83]. Males have a greater MeA than female rats [29,54] (but see [21]), and males have more shaft and spine synapses in the MeA than females [59]. Testosterone secreted during the perinatal period is needed for the development of sex differences [57]. Also, androgen secretion at adulthood is necessary for partial maintenance of structural integrity of the posterior MeA in adult male rats because castration reduces its area [44]. Other sets of experiments indicate possible functions for gonadal hormone receptors in the MeA. For example, although androgen receptor blockade in the MeA appears not to inhibit male rat sexual behavior [50], implantation of estradiol into the corticomedial amygdala may increase it in adult castrated male rats [71] and hamsters [92]. In addition, males and females differ in the concentration of cholecystokinin [52,83] and substance P [43,52] in the amygdala.

KEY WORDS: Sex differences, Castration, Sex steroids, Neuron, Golgi impregnation, Amygdala.

INTRODUCTION Traditionally, it has been described that gonadal hormones act in the central nervous system (CNS) during an early “organizational” period to promote the development of long-lasting changes in the structure and neurochemistry of sexually dimorphic areas and circuits, and during an “activational” period, at adulthood, these steroids stimulate the already developed circuits [33]. For example, rats castrated between 1 and 5, but not after 10 or more days of age, show an impaired male sexual behavior when adults [71,85] and, if injected with estrogen and progesterone, display a female-like lordotic behavior when mounted by normal males [22]. Nevertheless, the complete separation of “activational” and “organizational” periods as fully independent events is an over-simplification [2,46,49]. In fact, the sensitivity of some brain circuits to sex steroids [28,51,55] and the possibility of occurrence of plastic changes in the CNS can remain until puberty or adulthood in rats [18,24,44,75,95,96]. Studies of gonadal hormone effects in the CNS may provide some insights on the ontogeny of sex differ-

* Address for correspondence: Alberto A. Rasia-Filho, Universidade Federal do Rio Grande do Sul, Inst. Cieˆncias Ba´sicas da Sau´de, Depto. Fisiologia, R. Sarmento Leite 500, Porto Alegre, RS 90050-170, Brazil. E-mail: [email protected]

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Interestingly, the vomeronasal organ displays some morphological characteristics that are larger in males than in female rats [26]. Sex differences are found in the posterior aspect of the MeA, and structural and biochemical sexual dimorphisms are observed in the vomeronasal pathway, which reach the MeA and may be forming a sexual dimorphic circuit along with interconnected structures [26,94]. The distribution of sex steroid receptors and the amygdaloid connections are suggestive of a gonadal steroid-responsive subcircuit that may integrate chemosensory information and hormonal signals with the intrinsic production of chemical transmitters in the amygdala [3,93,94]. A neuron can perform specific roles according to its synaptic connection, its geometric design, its intrinsic membrane properties and electrical activity. Gonadal hormones may have marked effects on cellular structure. For example, testosterone and estrogen induce the proliferation of neurites in the newborn mouse hypothalamus in vitro [88], and estrogen promotes the development of dendritic branches in MeA neurons in vitro [41]. In the spinal nucleus of the bulbocavernosus muscle, another sexually dimorphic area in rats, castration significantly reduced neuronal soma size [75]. In the ventromedial nucleus of the hypothalamus (VMN), estradiol treatment significantly increased dendritic and soma spine density in peripubertal rats [78] and promoted an increase in the dendritic spine density and in the number of axodendritic synapses in the VMN of adult ovariectomized rats [18,19]. When sex differences or effects of gonadal hormone manipulation are detected in these morphological parameters, they raise the possibility to understand the studied area as having an inherently plastic response to these hormones and/or to be affected by an afferent population of hormone-sensitive neurons [25]. The Golgi method, even giving partial results, is a useful tool for the detection of the neuronal morphology [18,24,48,67,76,89,96]. The objectives of the present study were fourfold. The first goal was to look at sex differences on the somal area of Golgi-impregnated neurons from the anterior MeA and the posterior MeA of rats and to study the effect of gonadal hormone withdrawal in the neurons from the anterior MeA and the posterior MeA of males castrated at different ages. It was questioned whether castration during the early postnatal critical period for sexual differentiation of the CNS or at adulthood, after a relatively short period (8 days) or after a long period following castration (90 days), could alter neuronal soma size in the MeA. At 90 days postcastration, it is expected that male copulatory behavior in most orchidectomized rats has a significant reduction compared to intact animals [11,71]. Thus, it was expected that at this time the effect of sex steroid withdrawal could be markedly seen. The second goal was to study the dendritic branches of neurons from the anterior and posterior aspect of the MeA of intact adult male and female rats. The third goal was to determine the number of dendritic spines per micrometer of dendritic length in neurons from the anterior and posterior aspect of the MeA in intact adult male and female rats. Finally, the fourth goal was to provide a brief discussion about the cellular types that compose the MeA of rats. Some of these data were presented previously in an abstract [69], but the present findings improve and complement the older information. MATERIALS AND METHODS Animals The animals were 63 Wistar rats housed in groups with free access to food and water. Temperature was maintained around 22°C in a 12-h light/dark cycle (lights off at 1700h).

Surgical Procedure For the study of MeA cell body area, rats were divided into the following groups: intact adult male rats (n 5 10), intact adult female rats (n 5 8), males sham operated up to 6 h after birth and sacrificed as adults (n 5 6), males castrated up to 6 h after birth and sacrificed as adults (n 5 8), males sham operated as adults and studied 8 days after surgery (n 5 7), males castrated as adults and studied 8 days after gonadectomy (n 5 6), males sham operated as adults and studied 90 days after surgery (n 5 9), and males castrated as adults and studied 90 days after gonadectomy (n 5 9). For the study of MeA dendritic branches and spine density, only intact adult male rats (n 5 7) and intact adult female rats (n 5 6) were used. Intact adult male rats were 3–7 months of age. Vaginal smears were taken from intact female rats, 3–7 months old. To avoid possible gonadal hormone-mediated variation in the morphological parameters studied according to the phase of the estrous cycle [18,95], females were invariably sacrificed in the afternoon of diestrus. The newborn males were orchidectomized under ether anesthesia via a bilateral incision in the abdominal wall to allow intraperitoneal testes removal. All the newborns returned to their respective mothers after the surgical procedure and stayed with them until the 21st day after birth. Male offspring castrated or sham operated were sacrificed when they were 3–7 months of age. Adult males were castrated via a midline scrotal incision under ether anesthesia. One group of adult castrated males was sacrificed 8 days after gonadectomy and the other at 90 days following gonadectomy. All sham operated animals were submitted to the same anesthetic and surgical procedure as the castrated males but their testes were not removed. These animals were sacrificed 8 days and 90 days following surgery. Histological Procedure The single-section Golgi method was used in this study, as described in Gabbott and Somogy [20] and Woolley and McEwen [96]. All rats were anesthetized with Tionembutalt (sodium thiopental) and transcardially perfused with 4% paraformaldehyde and 1.5% picric acid in 0.1 M phosphate buffer (pH 5 7.4). Following perfusion, brains were postfixed in the same fixative solution for no more than 24 h. The brain was sectioned using a vibratome (Oxford), and the coronal sections (100 mm) were received in a bath of 3% potassium dichromate in distilled water. The sections were incubated in the same solution for 24 h. Afterwards, sections were washed in distilled water, mounted on glass coverslips and impregnated in silver nitrate (1.5%) in the dark for at least 48 h. The coverslips were removed, and the tissue sections were rinsed in distilled water. Unavoidable crystals were removed with the aid of a soft paintbrush. Following this, sections were dehydrated, cleared, mounted on slides and covered with non-acidic synthetic balsam and coverslips. Data Acquisition In the present study, the location of the MeA was based on the descriptions of Alheid et al. [1], taking the direct apposition of the MeA to the ventrolateral side of the optic tract as a reference for its location. For precise localization of the MeA, sections containing this nucleus and its subdivisions were projected onto the schematic drawings of coronal sections of the rat brain obtained from the atlas of Paxinos and Watson [63]. It was tentatively assumed that the general location of the MeA was approximately the same in all studied groups [1]. Nevertheless, we took the precaution of avoiding to study neurons that were in the boundaries of this nucleus, as will be described below.

GONADAL HORMONES AND MeA NEURONS The MeA can be divided in some subnuclei: the MeAD, the MeAV, the MePD and the MePV [63]. Drawings of the cell bodies were sorted according to their location in the MeA, that is, in the anterior (MeAD 1 MeAV, corresponding to plates 26 –29, 1.80 to 2.30 mm posterior to the bregma [63]) or posterior aspect (MePD 1 MePV, corresponding to plates 32–33, 3.14 to 3.30 mm posterior to the bregma [63]). Both left and right sides of each brain were used. Although it is assumed that a certain degree of tissue distortion occurs during its processing, brain sections with approximately the same size were used for all groups whereas those sections that appeared abnormally narrow after processing were not accepted for further study. No correction formula was used in the present study. Because there was a considerable variability in the number of cells in each brain that were satisfactorily stained by the Golgi method, the number of selected neurons in each brain varied between animals. This same procedure was adopted by Gomez and Newman [24] in their Golgi study of MeA neurons in Syrian hamsters. To be selected for further analysis, the neuronal cell body had to possess the following characteristics: (a) be undoubtedly located in the anterior or posterior aspect of the MeA (i.e., cell bodies must be within the boundaries of the nucleus and relatively away from its limits in the anteroposterior, dorsoventral or mediallateral planes), (b) have well-defined borders, (c) be near the middle third of the section, and (d) be relatively isolated from neighboring impregnated cell bodies. Although the number of impregnated neurons was variable from section to section, as a characteristic of the Golgi method, usually a small number of cells in the MeA were completely impregnated in each brain. In some cases, cell bodies from neurons with poor impregnated dendritic branches were also selected for further study. Thus, all cell bodies that could be included in the aforementioned criteria were drawn using a camera lucida (4503, Nikon, Tokyo, Japan). The mean number and SD of cell bodies obtained for each rat was 10.54 6 6.32. All drawings were digitized using a scanner (HP ScanJet II CX, Palo Alto, CA, USA), and the area of the cell bodies was measured using an image analysis system (SigmaScan, San Rafael, CA, USA). Other camera lucida drawings were made at 4503 and at 1,2503 (oil immersion objective) to measure dendritic length and branchpoints from neurons of intact adult males and females. To be selected for further analysis, dendrites had to fulfill the same requirements as described above for the selection of neuronal cell body area. Moreover, dendrites that appeared to be consistently impregnated and that appeared to have a partial decrease in their diameter when they extend through the brain section or when giving rise to successive generations of branches were selected. In some few dendrites, the end of the branch was assumed to occur when the tapered dendritic branch had a terminal cluster of spines. In addition, we tried to avoid as much as possible to include neurons with short cut-off branches or “tangled” dendrites that could not be individualized from adjacent neurons because they could lead to erroneous interpretations of their lengths. The dendritic branches were denoted by their centrifugal order (Fig. 1). The length of primary dendritic branches was measured, after being digitized, with the aid of an image analysis system as described above. Spines were directly counted only from drawings made at 1,2503. Spine density was then expressed as the average number of spines per micrometer of total dendritic length, as was done by Frankfurt et al. [18]. That is, irrespective of the level of the dendritic branch (primary, secondary and so on), the number of spines was counted and divided by the total length of the dendrite that fulfilled the including criteria.

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FIG. 1. Photomicrographs of representative Golgi-impregnated neuronal cell bodies from the medial nucleus of the amygdala of rats. Multipolar neurons were classified as “B”, a typical bitufted cell, and “S”, an example of a stellate one. Numbers represent the orders of dendritic branches: “1” means a primary dendritic branch, “2” a secondary branch and “3” a tertiary branch. Scale bar corresponds to 50 mm.

Statistical Analysis Because the groups of rats were collected at random as treatment finished and because it might have occurred subtle, although unpredictable, variations in fixation and osmolality across experiments, cell body area data were submitted to a one-way nested Analysis of Variance (ANOVA) for each type of neuron in the anterior or posterior aspect of the MeA, including all groups together. Therefore, for each cell category, a separate nested analysis was done, taking into account that there was an unequal number of rats and cells in each group. Previously, results were submitted to a square root transformation to obtain a normal distribution and to meet the requirements for the ANOVA. Spine density did not show a normal distribution and was submitted to a nonparametric Mann-Whitney test to compare data from male and female rats. In both cases, statistical significance was taken as p , 0.05. RESULTS Cellular Types According to the cell body shape and the number of primary dendritic branches, two different kinds of neurons were basically

176 found either in the anterior or in the posterior aspect of the MeA: stellate and bitufted cells (Fig. 1). Both kinds of neurons are multipolar ones. As described by other authors [1,48], MeA neurons are almost entirely small- and medium-sized. In the present study, cell body diameter was (mean 6 SD) 12.08 6 1.88 mm in intact adult males (n 5 10 rats, 256 cells). Stellate cells have three or more primary dendrites that radiate in all directions, having the cell body shape determined by the number and relative width of the primary dendrites (Fig. 1). According to the description of Ferna´ndez et al. [16], bitufted cells are characterized by two practically symmetric dendritic shafts that arise from the soma of the cell. The shape of the cell body can be either fusiform or ovoid (Fig. 1). McDonald [47] used “bipolar” to refer to neurons in the lateral and basolateral nucleus of the amygdala that are “characterized by two primary dendrites that arise from opposite poles of a fusiform cell body” (p. 300). The term bipolar is used in other studies [1,24]. According to Ramo´n y Cajal [68], it is preferable to use the term bipolar cells to those whose “cell bodies are almost always fusiform and give rise to one process from either end. One process can be thought of as central and the other as peripheral ... a cellulipetal process, or dendrite ... and a cellulifugal process, or axon” (pp. 51–52). Likewise, Jones and Cowan [32] describe bipolar cells as “relatively symmetrical, with the axon (or central process) and a single dendritic (or peripheral) process arising from opposite poles of the ovoid or elongated soma” (p. 295). According to these latter authors, we decided to classify the neurons in the MeA as stellate or bitufted (with ovoid or fusiform cell body) cells. Somal Area A total of 2,192 cell bodies was obtained from the studied groups as follows: intact males (n 5 289 cells), females (n 5 230 cells), males that were sham operated (n 5 236 cells) or castrated up to 6 h after birth (n 5 251 cells), males that were sham operated (n 5 384 cells) or castrated as adults and studied 8 days after gonadectomy (n 5 309 cells), males that were sham operated (n 5 239 cells) or castrated as adults and studied 90 days after gonadectomy (n 5 254 cells). Untransformed data are shown in Fig. 2 and Fig. 3, which show the mean (6 SEM) of the cell body area of bitufted and stellate neurons in the anterior and in the posterior MeA of each experimental group. Statistical analysis showed no significant difference among groups for any cell or location in the MeA studied, although a marked difference was found due to the variability of the rats within the groups. That is, for bitufted neurons in the anterior MeA, Fgroups(7,46,53) 5 1.71, p 5 0.12; and, Frats(42,466) 5 2.87, p 5 0.0001. For stellate neurons in the anterior MeA, Fgroups(7,46,75) 5 0.96, p 5 0.46; and, Frats(40,487) 5 2.81, p 5 0.0001. Although not significant, a trend to show statistical difference among groups was observed in bitufted neurons in the posterior MeA where, Fgroups(7,56,03) 5 1.87, p 5 0.09; and, like the other cells, Frats(48,569) 5 2.87, p 5 0.0001. For stellate neurons in the posterior MeA, Fgroups(7,53,74) 5 1.72, p 5 0.12; and, Frats(46,462) 5 2.21, p 5 0.0001. Dendritic Branches In the description of the dendritic length and branching pattern, the methodology was based on the work of Bannister and Larkman [4]. Sixty-four dendritic branches, one of them per neuron, were presently studied. They were obtained as follows: in males, 14 were from the anterior aspect of the MeA and 17 were from the posterior aspect (n 5 7 rats for each aspect of the MeA, mean 6 SD 5 2.00 6 1.15 and 2.42 6 1.90 neurons obtained per rat in the anterior and posterior MeA, respectively); in females, 19 were from the anterior aspect of the MeA and 14 were from the posterior

RASIA-FILHO, LONDERO AND ACHAVAL MeA (n 5 5 rats for the anterior MeA and n 5 6 rats for the posterior MeA, mean 6 SD 5 3.80 6 2.38 and 2.33 6 0.81 neurons per rat, respectively). The dendritic trees of the Golgiimpregnated neurons in the MeA typically branch sparingly and have few branchpoints (Fig. 1), which is similar to previous descriptions [1,48]. In addition, the lengths of dendrites from the anterior and posterior MeA are very different, extending over a wide range of path lengths from the soma. For example, primary branches in males varied from approximately 4.29 to 321.37 mm in the anterior MeA and from 5.30 to 362.93 mm in the posterior MeA (mean 6 SD 5 80.76 6 111.69 mm and 66.19 6 101.62 mm with a coefficient of variation of 138.29% and 153.52%, respectively). In females, primary branch length varied from approximately 5.30 to 288.20 mm in the anterior MeA and from 9.30 to 393.65 mm in the posterior MeA (mean 6 SD 5 45.34 6 62.93 mm and 81.93 6 93.53 mm with a coefficient of variation of 138.79% and 114.15%, respectively). The length of secondary and tertiary dendritic branches was not measured in the present study. Also, it is important to emphasize that these values do not correspond to all dendritic branches from a selected neuron but rather represent the length of the branch that fulfilled the criteria for further study. Due to the great variability in primary dendritic length, we precautionarily decided to develop no statistical test or to reach any conclusion about length of primary dendrites or total dendritic length with the number of dendrites presented here. Spine Density Although spines could also be found in the neuronal cell body in some cells, spines were studied only in the dendrites. The dendritic spines in both aspects of the MeA are pleomorphic and show a morphology that allowed for classification into distinct categories: (1) with thin stalk and a small bulb in its round tip, (2) “mushroom”-like shape with a large ending bulb, (3) stubby protrusions, and (4) with a stalk that apparently gives rise to two or more branched heads [64,66]. Some examples can be observed in Fig. 4. The dendritic spine density was calculated in 50 out of the 64 dendrite branches described above from intact adult males and females. Approximately 14,000 spines were drawn for further study. In males, spines were obtained in 11 dendrites from the anterior aspect of the MeA and another 11 from its posterior aspect (n 5 7 and 5 rats, mean 6 SD 5 1.57 6 0.53 and 2.20 6 1.09 cells per rat, respectively). In females, spines were obtained in 16 dendrites from the anterior aspect of the MeA and another 12 from the posterior MeA (n 5 5 and 6 rats, mean 6 SD 5 3.20 6 2.68 and 2.00 6 0.89 cells per rat, respectively). In the anterior MeA, males have (mean 6 SEM) 1.9 6 0.1 and females have 0.6 6 0.1 spines/mm of dendrite. Median and interquartile range are 2.0 (1.2/2.5) and 0.4 (0.3/0.7), respectively. In the posterior MeA, males have 1.0 6 0.1 and females have 0.8 6 0.1 spines/mm of dendrite. Median and interquartile range are 0.9 (0.7/1.2) and 0.7 (0.6/1.1), respectively. No correction formula was used to adapt spine density to dendritic segment diameter. Data were submitted to a Mann-Whitney test, which showed a statistical difference (p , 0.001, males higher than females) in the spine density in the anterior MeA but not in the posterior MeA (p 5 0.355). DISCUSSION The present results on the cell body area of the neurons that compose the MeA showed that no statistical difference was found in the neuronal somal area of intact males and females and in males castrated during the early postnatal period (up to 6 h after birth) or at adulthood, either after a relatively short period (8 days)

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FIG. 2. Values of untransformed data (mean 6 SEM) of the somal area (mm2) of bitufted and stellate neurons in the anterior medial amygdala from intact adult males (MALE) and females (FEMALE), from adult rats submitted to sham operation up to 6 h after birth (SGDX 6h) and adult males castrated as neonates (GDX 6h), from males sham operated as adults and studied 8 days after surgery (SGDX 8d) and adult castrated males sacrificed at the same time (GDX 8d), from males sham operated as adults and studied 90 days after surgery (SGDX 90d) and adult castrated males sacrificed at the same time (GDX 90d). The numbers in parentheses correspond to the number of studied cells. No statistical difference among groups was found.

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FIG. 3. Values of untransformed data (mean 6 SEM) of the somal area (mm2) of bitufted and stellate neurons in the posterior medial amygdala from intact adult males (MALE) and females (FEMALE), from adult rats submitted to sham operation up to 6 h after birth (SGDX 6h) and adult males castrated as neonates (GDX 6h), from males sham operated as adults and studied 8 days after surgery (SGDX 8d) and adult castrated males sacrificed at the same time (GDX 8d), from males sham operated as adults and studied 90 days after surgery (SGDX 90d) and adult castrated males sacrificed at the same time (GDX 90d). The numbers in parenthesis correspond to the number of studied cells. No statistical difference among groups was found.

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FIG. 4. Photomicrographs of representative Golgi-impregnated dendritic branches from the anterior medial nucleus of the amygdala of intact male (M) and female (F) rats. The arrows point to spines. Note the difference in the density of spines in M and F. In both, scale bar corresponds to 5 mm.

or after a long period following gonadectomy (90 days). Our data regarding MeA neuronal morphology are in accordance with the general description provided by McDonald [48], in which it is stated that “primary dendrites give rise to only a few branches which extend for fairly long distances within the nucleus. The dendritic spine density varies among neurons from moderate to fairly sparse” (p. 82). Nevertheless, it appears that males have a higher dendritic spine density than females in the anterior MeA. These data are in agreement and expand previous work [24] that did not find significant differences in the somal area of neurons from the anterior MeA of intact male hamsters and adult castrated ones. In Fig. 2, it is interesting to note that, respectively, the somal area of bitufted and stellate cells from the anterior MeA is approximately 15% and 16% smaller in adult castrated males sacrificed 90 days following gonadectomy than in the corresponding sham-operated control group. However, the statistical analysis showed no difference among the studied groups in any part of the MeA of rats. On the other hand, in the work of Gomez and Newman [24], castration of adult male hamsters decreased the neuronal cell body area in the posterior MeA. For the cell body area experiment, it could also be argued as to whether the statistical procedure used in this work could detect significant differences, considering the sample size obtained for each cell type and region of the MeA in each experimental group [35]. Considering that the somal area of the MeA neurons is approximately 10% greater in males than in females in anterior

MeA, their means and standard deviations (given a statistical significance with a power of 95% and a significance level set at the 5% level), a minimum of 162 cells per region of the MeA per type of neuron and per group would be necessary to prove statistical significance. Therefore, because of the relatively small difference in the somal area from the neurons studied, a larger sample would be needed to prove the effect of sex steroids in the MeA neurons. It is noteworthy that a similar conclusion about feasibility of experimental procedures to measure neuronal components was obtained by Sasaki and Arnold [75] that found no marked effect of castration and androgen replacement in the dendritic arbor of neurons from the spinal nucleus of the bulbocavernosus in adult male rats. From thionin-stained sections, Hines et al. [29] (but see [21]) described that the volume of the posterodorsal region of the MeA is greater in male than in female rats. Interestingly, the portion of the MeA that highly concentrates androgen is confined within approximately 500 mm in the rostral-caudal plane centered in the middle to posterior part of the MeA [79]. Estradiol receptors a and b [39,62,81,84] and aromatase [80], the enzyme that converts testosterone into estradiol, are also found in the amygdala of rats. Using in situ hybridization, aromatase mRNA is found in the anterior and in the posterior area of the MeA [91]. Aromatase activity in the MePD is high during the neonatal critical period in rats [80] and is significantly higher in the MeA of males as

180 compared with females [73], although no sex difference in the aromatase mRNA was found in the MeA of rats [91]. Our findings suggest that anterior MeA should also be considered as a sexual dimorphic region. Contrary to our expectations, however, no sex differences in the parameters studied here were observed in the posterior MeA. It must be clear that the present results are not denying the existence of sex differences in the rat posterior MeA. It is possible that sex differences and the effects of castration in the somal area may be occurring in a region more caudal in the posterior MeA than the place we selected for our analysis (plates 32–33 from the atlas of Paxinos and Watson). Thus, the hormonal effect evaluated in the posterior MeA refers to these studied plates, which also are within an area sensitive to gonadal hormones. It is possible that differences in apparent soma shape can be affected, to a certain degree, by artifacts of section plane [61]. In addition, because the Golgi method does not sort out those neurons that concentrate from those that do not concentrate sex steroids, it is also possible that a high degree of intra-animal and inter-animal variability can occur due to this unavoidable mixing [24]. Not all cells in a determined area possess receptors for gonadal hormones [88], and those neurons that are sensitive to these steroids are not reliably identifiable by the Golgi method. Nabekura et al. [56] showed that there may be some similar morphological characteristics for some estradiol-responding and nonresponding neurons in MeA slices of rats. These three preceding reasons could account for the statistically significant interanimal variability revealed by the nested ANOVA. However, it appears that the data on the somal area have come from the same subset of neurons because their variation in the anterior and posterior MeA is low, even considering the untransformed results [18]. Finally, it is possible that sex differences in the posterior MeA occur in some quantitative parameters of dendrites that were not evaluated here. Neurogenesis in the MeA occurs approximately until the 16th day of gestation in rats [6]. Nevertheless, the maturation of the MeA continues throughout the neonatal period. Mizukami et al. [54] showed that, although the MeA nuclear size increases between postnatal days (P) 1 and 5 in both sexes, in females the volume attains its adult value at P5, whereas in males it increases more gradually and attains its adult value at P11. This sex difference does not appear to be dependent on body or brain weight and became clearly evident at P21 [54]. These data would suggest that a postnatal action of gonadal hormones is relevant for the development of the MeA of rats. As cited by Kerchner et al. [34], sex difference in the area of the MePD was eliminated by castrating newborn males. Therefore, castration done up to 6 h after delivery could disrupt a sex steroid-dependent normal development of the MeA of rats if the cell body area was sensitive to hormonal manipulation at this period. Because neuronal somal area in both parts of the MeA appears not to be affected by neonatal castration, it is possible that cell body area is not affected by neonatal gonadal hormone withdrawal or, alternatively, that a partial brain masculinization has occurred due to an increase in serum testosterone from the first hours of postnatal life until the moment of castration [70,72]. Additionally, in adult male rats, Malsbury and McKay [44] showed that gonadectomy was able to reduce the size of the MePD and MePV by approximately 27% and 26%, respectively. In adult male hamsters, gonadal hormone withdrawal decreased mean highest dendritic branch and the percentage of neurons with tertiary branch segments in the posterior, but not the anterior aspect of the MeA [24]. Based on the present data, castration also does not appear to alter somal area at adulthood, either after a relatively short period (8 days) or after a long period following castration (90 days). Thus, it is possible that other morphological differences in

RASIA-FILHO, LONDERO AND ACHAVAL the MeA could contribute in a more relevant way to sex difference and the effect of gonadal hormones in the volume of the MeA [44]. Differences in the neuropil (composed of axons, dendrites, their synaptic contacts and glial cells) may account for MeA nuclear volume differences more than the somal area of the neurons that compose it. At least in the guinea pig hypothalamus, estrogen receptors are not only found in the neuronal nucleus and perikarya but also in dendrites and axon terminals [7]. However, some limitations on the analysis of dendritic branching occurred for the MeA. They are basically the following: (1) because the amygdala is a heterogeneous structure, the different subnuclei within the amygdala occupy distinct locations in it and change their size from section to section, which restricts the number of available slices to be studied; (2) the number of sections in which a specific subdivision of a subnuclei can be reliably found is even fewer; (3) the reaction in the Golgi method impregnates cells according to unknown and uncontrollable factors that, therefore, lead to an unpredictable number and pattern of impregnated neurons; (4) in accordance with the work of Gomez and Newman [24], it empirically appears that only a small number of neurons in the MeA are normally impregnated in each brain; (5) neuronal impregnation appears to vary with the age of the animal (i.e., we used adult rats in the present study but the best results are obtained in young animals, at least for the Cox modification of the Golgi method) [67,89]; and (6) for an impregnated neuron to be considered “acceptable”, it must have some specific characteristics such as quality of impregnation, relative isolation from other neighboring cells and be within the thickness of the section that limits the number of cells or their dendritic branches available for further study. Even with all of the efforts to obtain suitable neurons for further study, sectioning tissue at 100 mm may result in cutting dendritic branches. It was suggested that horseradish peroxidase (HRP) injection would give a better estimation of dendritic length than Golgi studies [4], but injection techniques may not sample neurons randomly as obtained with Golgi stains [30]. The degree of tissue distortion after histological procedure can also modify the actual morphometric value of the neuron. In fact, as also described by Jacobs et al. [30], the present results are probably relative rather than absolute values of dendritic length. The Golgi method may lead to an underestimation of the actual spine population because only visible spines are counted. Spines that project from above or below darkened impregnated dendrites may not be seen [30,64]. The shape and size of the spine can also affect the quantification of spines if they are near the resolving power of optical microscopy [5]. But some of these difficulties are also inherent to other currently applied methods for quantitative measurement of neurons, and definitive conclusion about spine morphology should be obtained with electron microscopy. Dendrites are considered to be the main receptive site of the neuron and are formed in different times during development [90]. Cellular local responses can be affected by the degree of dendritic branching and tapering [66]. The density and the functional properties of dendritic ionic channels can affect the membrane potential that will be generated and propagated, altering the local input resistance and time constant and influencing the time window for summation of excitatory and inhibitory postsynaptic potentials [31,53]. The pattern of spacing and the shape of dendritic spines can alter the neuronal excitability [66]. Spines appear to be relevant for induction and endurance of long-term potentiation [10, 27], amplifying the membrane potential, and associating postsynaptic potentials among neighboring spines to coactivate them [27]. Spines also could serve to establish a biochemical compartmentation and to store calcium intracellularly, which might prevent the increase in calcium concentration to a pathological level during

GONADAL HORMONES AND MeA NEURONS normal synaptic transmission [27,77]. Thus, it is possible that processing demands placed on dendritic systems may influence the dendritic morphology and the density of spines in these dendrites [30]. Specifically in the MeA, a sex difference was found in the synaptic pattern and in the number of synapses [57–59]. That is, the occurrence of shaft synapses terminating on dendritic shafts and spine synapses made on dendritic spines showed a significant decrease in the MeA following neonatal orchidectomy of male rats [57]. Interestingly, neurogenesis in the ventral and anterior aspect of the MeA occurs significantly earlier than in the posterodorsal region, which appears to be related to the arriving of olfactory inputs [6]. In addition, the neural connectivity between the MeA and the preoptic area in the female rat is different from that in the males and is markedly influenced by the neonatal steroidal environment [15]. These findings can add to the study of the ontogenetic differences in the establishment of synaptic contacts in the amygdala and their morphological and functional consequences. In conclusion, expanding previous results [24,29,44,54,57–59, 69], the present data suggest that the already described sex difference in the MeA may have a greater effect on the neuropil than on the neuronal somal area and that castration appears not to alter the somal area in males submitted to gonadectomy during the early postnatal period or at adulthood. Bitufted and stellate neurons in both aspects of the MeA appear to have dendrites that show a high variation in their lengths, and, interestingly, gonadal hormones may be altering dendritic spine density and perhaps the ongoing synaptic activity processed by each neuron. The present findings may also help to evaluate sex differences in the pattern of connectivity that occurs in the MeA of rats because it was already described that males have more shaft and spine synapses than females [59]. The present results may also help to understand how gonadal hormones modulate neural activity involved with memory, learning, emotion and the behavioral manifestation observed in normal and pathological conditions for which the MeA is involved. ACKNOWLEDGEMENTS

This study was supported by grants from the Brazilian agencies CAPES, CNPq and FINEP. The authors are very thankful to Drs. Bruce S. McEwen and Ana M. Magarinos (The Rockefeller University, New York, NY, USA) for their kind help and valuable advice used during the execution of the experiments presented here. We are also indebted to Drs. Sı´dia Jacques and Joa˜o Riboldi (UFRGS, Porto Alegre, RS, Brazil) for their help in the statistical analysis and to Dr. Felipe L. Schneider (UFRGS) for the use of the vibratome.

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