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Effects of testosterone on the synaptology of the medial preoptic nucleus of male Japanese quail
Brain Research Bulletin, Vol. 50, No. 4, pp. 241–249, 1999 Copyright © 1999 Elsevier Science Inc. Printed in the USA. All rights reserved 0361-9230/99/$–see front matter
PII S0361-9230(99)00193-8
Effects of testosterone on the synaptology of the medial preoptic nucleus of male Japanese quail C. Castagna,1 A. Obole,1 C. Viglietti-Panzica,1 J. Balthazart2 and G. C. Panzica1* 1
Department of Anatomy, Pharmacology and Forensic Medicine, University of Torino, Torino, Italy; and Laboratory of Neurochemistry, Research Group in Behavioral Neuroendocrinology, University of Lie`ge, Lie`ge, Belgium
2
[Received 24 March 1999; Revised 6 July 1999; Accepted 19 July 1999] ABSTRACT: The medial preoptic nucleus (POM) of male Japanese quail is a sexually dimorphic testosterone-dependent structure that plays a key role in the activation of male sexual behavior. Both the total volume of the nucleus and the size of the dorsolateral neurons are decreased in gonadectomized males. Immunocytochemical studies have revealed a complex pattern of innervation: immunopositive fibers for several neuropeptides and neurotransmitters have been detected in the POM; some of them (e.g. vasotocin-immunoreactive fibers) are sexually dimorphic and testosterone-dependent. To understand the anatomical bases of these testosterone-dependent neurochemical changes, we performed an ultrastructural study of the POM neuropil in intact sexually mature, gonadectomized, or testosterone-treated gonadectomized males. A complex synaptic organization of the POM neuropil was observed in intact male quail reflecting the heterogeneity of the neurotransmitters and neuropeptides present in this nucleus. Changes in this organization were observed after the endocrine manipulations. The number of axosomatic synapses per cell body decreased after gonadectomy and was restored to the level observed in the intact group after the administration of testosterone. By contrast, no significant change was observed in the density of axodendritic and axospinal synapses after hormonal manipulations which suggests that the total number of synapses in the nucleus should be affected by testosterone (constant density in a changing total volume). The cross-sectional area of synaptic boutons was also decreased by castration and restored to intact level by testosterone. The action of testosterone on the activation of male copulatory behavior in gonadectomized birds is hence paralleled by an extensive rearrangement of neuropil in the POM. © 1999 Elsevier Science Inc.
dictions have been extensively documented by experimental studies carried out mostly during the last 50 years [23,31,40,46,62]. In particular, several studies have documented, both at the light and electron microscopic levels, the dramatic anatomical changes produced by gonadal steroid action. They include changes in the volume of brain nuclei, in the size and staining of neurons, in the innervation pattern, as well as changes in the ultrastructural organization of cell bodies and neuropil [46]. In adult Japanese quail, it has been shown that several morphological characteristics of the medial preoptic nucleus (POM), a sexually dimorphic structure controlling male copulatory behavior [20 –22], change as a function of the levels of circulating testosterone (T). The total volume of the nucleus, the cell size of the dorsolateral population, as well as the presence of neurochemical markers within the neurons are affected [52,53]. In a recent study [48], we investigated the effects of T on the ultrastructural organization of cell bodies in the male quail POM. In castrated birds, the amount and size of organelles were strongly decreased compared to sexually mature males, and a 2-week treatment with T was able to restore an ultrastructural organization that is typical of that found in a control sexually active male. In particular, the organelles involved in protein synthesis (rough endoplasmic reticulum, Golgi complex, secretion vesicles) almost totally disappear in the cell bodies of castrated quail. These changes may reflect the reduction after castration of the concentration of aromatase, the enzyme that catalyzes the conversion of T into estradiol (E2). Aromatase-immunoreactive neurons represent a large fraction of the POM cells and constitute a neuronal marker of the cytoarchitectonic boundaries of the POM [16]. Immunocytochemical studies demonstrated the existence within this region of cell bodies and fibers immunopositive for neuropeptide Y, substance P, NADPH-diaphorase, gonadotropin hormone-releasing hormone (GnRH), vasotocin (VT), and neurotensin. Moreover, immunoreactive fibers for corticotropin-releasing factor, tyrosine hydroxylase, and serotonin were also described [8 –10,45,53]. Recently, we have shown that the VT-immunoreactive (VT-ir) elements of the POM are sexually dimorphic [5], and T-dependent: the density of these fibers is markedly decreased by castration and increased by T administration [63].
KEY WORDS: Gonadal hormones, Sexual behavior, Preoptic area, Synapses, Ultrastructure.
INTRODUCTION The presence of gonadal steroid receptors within the central nervous system (CNS) suggests that steroids could have specific effects on the development and differentiation of the neural circuits, on their maintenance in adulthood, as well as on the control of endocrine and behavioral aspects of reproduction. These pre-
* Address for correspondence: Dr. G. C. Panzica, Department of Anatomy, Pharmacology, and Forensic Medicine, University of Torino, Corso M. D’Azeglio 52, I-10126 Torino, Italy. Fax: ⫹39-011-670-7732; E-mail: [email protected]
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In parallel with these morphological changes, T action in the POM is also essential for the activation of copulatory behavior in adult male quail [23,43]. Electrolytical lesions of the POM abolish male copulatory behavior and T implants within this nucleus, but not in its immediate vicinity, activate behavior in castrated subjects. It is therefore assumed that the anatomical and neurochemical changes (volume, cell size, cell ultrastructure, innervation) seen in this brain region after a treatment with T represent the morphological signature of the mechanisms underlying the behavioral activation [53]. Studies performed in a variety of animal models have demonstrated that in androgen-target regions the ultrastructure of the neuropil shows marked changes as a function of the circulating levels of T [3,26,35,38,49,58]. The immunocytochemical studies of the quail POM described above suggested that in this model, the innervation of the nucleus is also affected by T [52]. Therefore, in the present study we searched whether similar changes in the synaptic organization could be detected in quail after castration and treatment with exogenous T. This study focused on the dorsolateral portion of the POM that has been shown to be very sensitive to T action and probably plays a pivotal role in the control of sexual behavior [12]. MATERIALS AND METHODS Animals This study was performed on the brain of 15 male Japanese quail (Coturnix japonica) that had been previously used to analyze the effects of T on the neuronal ultrastructure in the POM. The in vivo treatment of these birds, fixation and histological procedures have been previously described in detail [48] and will therefore be presented here in a succinct form only. Birds were housed, manipulated, and sacrificed according to the Principle of Laboratory Animal Care (National Institutes of Health), and to the relevant Italian and Belgian laws on the protection of animals. The birds were bought from a local breeder (Lefe`re, Boneffe, Belgium) at the age of 3 weeks and then housed in individual cages on a 16-h light:8-h dark schedule simulating long days, at controlled temperature with water and food available ad libitum. Hormonal Treatments Ten birds were gonadectomized and 5 were sham-operated under anaesthesia (Hypnodil, Janssen Pharmaceutica, Belgium; 15 mg/kg body weight) at the age of 3 weeks using previously described methods [60]. Nine days later, gonadectomized birds were either treated with 40 mm (2 ⫻ 20 mm) Silastic implants filled with T (CX ⫹ T) or left empty (CX). In the third group the sham-operated males (Intact) also received empty implants. The implants were prepared with Silastic tube (Dow Corning tubing, number 602-252; inner diameter: 1.57 mm, outer diameter: 2.41 mm) filled with crystalline T (Sigma T-1500). It has been previously demonstrated that this type of T treatment restores in both males and females plasma levels of the steroid that are similar to those observed in intact sexually active males [14,61]. Approximately 2 weeks later, birds were tested twice for sexual behavior (observed for 5 min in the presence of a sexually mature female) and their body weight and cloacal gland size (a T-dependent structure [59]) were measured as previously described [18,56]. The animals were sacrificed and the brains were collected for ultrastructural observations during the following week when they were 8 weeks old. Fixation and Embedding Procedures The detailed procedure of fixation, dissection, and sampling was described elsewhere [48,50]. Briefly, the birds were transcar-
dially perfused with two different fixatives used in sequence: fixative no. 1 (1% paraformaldehyde ⫹ 1% glutaraldehyde in 0.1M Na-K phosphate buffer, pH 7.2–7.4 containing 0.5% of CaCl2 and MgCl2) for the first 10 min followed by fixative no. 2 (2% paraformaldehyde ⫹ 3% glutaraldehyde in the same buffer). The dissected brains were left into fixative no. 2 for about 2–3 h at 4°C. After washing overnight in sucrose-containing buffer (Na-K phosphate buffer 0.1 M, pH 7.2–7.4 containing 10% sucrose to maintain osmolality), specimens were sectioned in the coronal plane with a vibratome and 200-m-thick sections were collected in the same buffer. The sections were quickly stained with toluidine blue (1% in buffer [27]) and observed with a light microscope in order to select serial levels through the preoptic region along the rostrocaudal axis. The left and right POM were dissected from these sections keeping the ependymal wall as an orientation landmark. The 200-m-thick sections were then postfixed in a solution of 1% osmium tetroxide in 0.1 M phosphate buffer containing 2% dextrose, for 1 h at 4° C in the dark, dehydrated in alcohol, and flat embedded in a mixture of Epon and Araldite (1:1) for 48 h at 60°C. Blocks corresponding to the section located immediately before the anterior commissure were selected for quantitative analysis because this level has been shown to be most closely associated with the activation by steroids of sexual behavior [22] and it was always analyzed in previous light- and electron-microscopic studies [6,7,48,56]. Identical levels of the dorsolateral part of the POM were carefully selected in individual samples based on the observation of 1-m-thick semi-thin sections cut from these blocks and stained with toluidine blue (0.1% in borax, see also our previous study [50]). Ultrathin sections were then cut, contrasted with uranyl acetate and lead citrate, and examined with a Philips 410 transmission electron microscope at 80 kV. Quantitative Analysis Criteria applied for the identification and counting of synapses were the presence of pre- and postsynaptic densities associated with local concentration of synaptic vesicles in the axon terminals. Synapses were subdivided in two main groups: axosomatic and axodendritic. The number of axosomatic synapses per cell was counted in the three experimental groups on micrographs of neurons taken at a magnification of 7,100⫻ and enlarged to a final magnification of 14,200⫻. A total of 10 to 30 neurons per animal were randomly collected [14 ⫾ 5 neurons in Intact, 18 ⫾ 8 neurons in CX, and 16 ⫾ 4 neurons in CX⫹T birds (all means ⫾ standard deviation)]. Neurons were considered only if they could be entirely observed and only if they contained a nucleus. Axodendritic synapses were counted on micrographs covering an area of at least 2,500 2 of neuropil (excluding neurons, glial cells, and blood vessels) per bird (range 2,500 – 4,000 2), at a final magnification of 17,750⫻. They were subdivided in two groups: shaft and spine synapses (see Fig. 2). Axodendritic synapses were further subdivided into 4 main types (A, B, C, and D) based on the presence of clear and dense-core vesicles and on the diameter of these vesicles (see Results for detail, Fig. 1). Results were then standardized and expressed in terms of synaptic density (number of synapses per 1000 m2 of examined area). Morphometric Analysis The micrographs were then digitized by an AppleOne scanner connected to a Macintosh computer (Performa 630). Each synapse was outlined by the operator (A.O.) using the mouse; the area value was automatically calculated by the software NIH-Image
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FIG. 1. Photomicrographs illustrating the synaptic organization of the medial preoptic (POM) neuropile at the ultrastructural level. Synaptic terminals are characterized by different types of vesicles. In type-A synapses (A) presynaptic terminals contain small spherical and pleiomorphic clear vesicles associated with large spherical dense-core vesicles. In type-B synapses (B) presynaptic terminals contain small spherical clear vesicles associated with small spherical dense-core vesicles. Type-C synapses (C) are characterized by presynaptic terminals that contain small spherical clear vesicles while in type-D synapses (D), presynaptic terminals contain small spherical clear vesicles clustered in a regular clump, associated with rare dense-core vesicles. Abbreviation: d, dendrite. Bar ⫽ 1 m.
(version 1.60, Wayne Rasband, National Institutes of Health, Bethesda, MD, USA). Statistical Analysis For each dependent variable, we calculated the mean of all determinations in each bird. Values referring to synaptic densities were used to perform one-way analyses of variance (ANOVA)
comparing the different groups. When appropriate, post-hoc testing was carried out to compare the three groups 2 ⫻ 2 using the Tukey test, that provides an optimal compromise between the risks of type I and type II (program Super ANOVA, Abacus Concepts Inc, Berkeley, CA, USA on MacIntosh computers). Means ⫾ standard errors were systematically used as measures of central tendency and dispersion. Differences were considered significant for p ⬍ 0.05.
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Behavioral and Physical Parameters The behavioral and morphological data concerning the subjects of this experiment have been reported in a previous study [48]. Briefly, the body weight in the three groups of subjects was similar at the beginning and at the end of the experiment (ANOVA; p ⬎ 0.05). Before the experiment, the cloacal gland area of castrated birds was smaller (mean below 50 mm2) than in intact subjects (184 mm2). After T implantation the size of the gland increased very markedly and was restored to values similar to those observed in gonadally intact subjects while it remained significantly smaller in the CX group [F(2,12) ⫽ 70.84, p ⬍ 0.0001]. Intact and CX⫹T birds regularly showed sexual behavior while castrates never did. The frequencies of mount attempts were significantly different [F(2,12) ⫽ 11.62, p ⫽ 0.0016]. These data confirm previously reported results and indicate that the experimental treatments produced the expected endocrine changes [17,19,56]. Synaptology of the Medial Preoptic Nucleus At ultrastructural level the dorsolateral part of the POM exhibited the features of a complex synaptic organization (Figs. 1A–D). The POM neuropil was characterized by numerous nerve endings making synaptic contact with dendrites (axodendritic synapses; Figs. 2A,B) or with neuronal cell bodies (axosomatic synapses; Fig. 3). Axodendritic synapses can be subdivided into two main groups depending on whether the synaptic bouton contacts a portion of the dendritic contour that is not modified (shaft synapses) or contacts a fungiform protuberance of the dendrite (spine synapses). Axosomatic synapses. Axosomatic synapses were frequently observed on neuronal cell bodies in the lateral POM: a mean of 4 to 8 small nerve endings (identified by the presence of synaptic vesicles and presynaptic and postsynaptic densities) per cell body were detected (Fig. 3). Axosomatic synapses were not uniformly distributed around the cell surface, but they were clustered in selected regions of the perikaryon. Synaptic vesicles were small, clear, and pleiomorphic, but in a limited number of cases they were larger and contained a dense core. The active zones of the presynaptic and postsynaptic membrane in axosomatic synapses were identified by the presence of similar amounts of electron-dense material (symmetrical or Gray’s type II synapses; see also [44,57]). Axodendritic synapses. Transversally cut axons and long neuronal processes were frequently observed within the neuropil. Neuronal processes showed mitochondria, tubules, and filaments, as well as, in most cases, rare ribosomes, and were therefore identified as dendrites (Figs. 1A–D) These processes either exhibited a smooth surface bearing synapses (shaft synapses) or displayed short spines, surrounded by synaptic endings (spine synapses; Figs. 2A,B). A variety of axon terminals could be distinguished in the neuropil according to the morphology of their vesicles and synaptic densities. We classified axodendritic synapses in 4 groups on the basis of their morphology and content of vesicles (Figs. 1A–D): ●
● ● ●
Type A, synapses containing small spherical and pleiomorphic clear vesicles associated with large spherical dense-core vesicles; Type B, synapses containing small spherical clear vesicles associated with small spherical dense-core vesicles; Type C, synapses containing spherical clear vesicles; Type D, synapses containing numerous small spherical clear vesicles clustered in a regular clump associated with rare densecore vesicles.
In addition to the vesicular components, the synaptic terminals contained small mitochondria (Figs. 1B,C). The active zone of the presynaptic membrane was identified by the presence of dense projections constituted by a variable amount of electron-dense material. In many cases the postsynaptic membrane also demonstrated a clearly visible postsynaptic density (Figs. 1 A–D). These synapses can therefore be classified as asymmetrical (or Gray’s type I). However, several endings lacked a conspicuous postsynaptic density, and their synaptic cleft was thinner (Fig. 2A). These were identified as symmetrical synapses (or Gray’s type II). Quantitative and Morphometric Analysis The quantitative analysis of the neuropil in the lateral POM revealed a massive rearrangement of the synaptic inputs after endocrine manipulations (Table 1 and Fig. 4). The number of axosomatic synapses was significantly affected by treatments (one-way ANOVA; p ⬍ 0.0004). Tukey tests comparing the three experimental groups 2 ⫻ 2 indicated that castration decreased and T increased in a significant manner the number of axosomatic contacts per cell (Table 1). In contrast, no significant change was observed in the total number of axodendritic contacts per unit area (1000 m2) under the same conditions (Table 1). Similarly the number of shaft or spine contacts per unit area (1000 m2) was not affected by the experimental treatments (Table 1). When terminals were subdivided in specific groups based on the morphological classification described above, a decrease in the number of type A-synapses was observed after gonadectomy, but this decrease was only partly and non-significantly restored by T replacement (p ⫽ 0.0032 in the general ANOVA comparing the three groups, see Table 1 for the detail of Tukey tests comparing the three experimental groups 2 ⫻ 2). No significant variation was detected in the number of the other types of synaptic contacts (types B,C, and D) under the same conditions (Table 1). Morphometric data on the cross-sectional area (size) were subjected to a two-way ANOVA (treatment and type as factors) that indicated the presence of a significant overall effect of treatment on the synaptic endings size [F(2,11) ⫽ 11.045, p ⫽ 0.0023], a highly significant effect of the synaptic type [F(3,33) ⫽ 9.09, p ⫽ 0.0002], and no significant interaction between the two factors [F(6,33) ⫽ 1.339, p ⫽ 0.2681] Subsequent one-way ANOVA analysis of each type of synapse demonstrated that the size of the type A, B and C synapses (Fig. 4) was significantly affected by the experimental (p ⫽ 0.031 for type A, p ⫽ 0.005 for type B, and p ⫽ 0.012 for type C). Type D synapses (Fig. 4) tended to display similar changes in size but these changes were not statistically significant (one-way ANOVA; p ⫽ 0.081). Tukey post-hoc tests comparing the three experimental groups 2 ⫻ 2 confirmed that castration decreased and T increased (i.e., restored to intact level) the size of type A, B, and C synaptic endings, respectively, in a statistically significant manner. DISCUSSION The present study confirms the complexity of the synaptic component in the dorsolateral portion of the male quail POM that was first demonstrated in our previous study [50]. The morphometrical data collected in the control group of sexually mature gonadally intact males characterize more precisely the morphology, the qualitative and the quantitative distribution of synaptic endings in this region crucial for the control of the reproduction in male quail [53]. The vast majority of synapses contact a smooth dendritic surface, whereas only about 15% of total synapses are located on spines. This percentage appears to be higher than that previously observed in the domestic fowl preoptic [44] and supra-
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FIG. 2. (A,B) Photomicrographs showing long dendrites (d), longitudinally sectioned and associated with several synaptic endings. Two types of synapses are detectable: axodendritic or shaft synapses (thin arrows), in which the dendritic contour is not modified, and axospinal or spine synapses (thick arrows), characterized by a bouton contacting fungiform protuberances of the dendrite (spine). A symmetrical synapse can also be detected in A (asterisk). Bar ⫽ 1 m.
chiasmatic areas [47]. No detailed studies based on Golgi impregnation have been published on the quail preoptic neurons, but preliminary results [28] suggest that these neurons bear a larger number of spines than those in the domestic fowl [51]. It is therefore possible that the higher density of spine synapses in the quail compared to the fowl simply reflects the abundance of spines in the former species.
The morphological characterization of synapses utilized in the present study is based on our previous investigations of the anterior hypothalamus of the domestic fowl [47,49,55]. B-type synapses represent the majority of the endings observed in the dorsolateral part of the POM (close to 60% of the total number). Due to the presence of small dense core vesicles, these synapses may be considered to belong to catecholaminergic, serotoninergic, or pep-
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FIG. 3. Photomicrograph illustrating axosomatic synapses (arrowheads) showing similar pre- and postsynaptic densities (symmetrical synapses). Abbreviations: G, Golgi complex; N, neuronal nucleus; RER, rough endoplasmic reticulum. Bar ⫽ 1 m.
tidergic types (see [49]). This interpretation is in good agreement with light microscopic immunocytochemical studies previously performed on the same region: these studies demonstrated a large supply of tyrosine hydroxylase-positive fibers, as well as of peptidergic elements (cells and fibers) in the lateral part of the POM [8 –11,15,25,45]. Synapses showing large dense-core vesicles (type A) represent a scarce population (around the 3% of the total). Due to the presence of large dense core vesicles, these elements are probably peptidergic terminals of neurosecretory type [55,64], and they probably contain some of the neuropeptides that have been demonstrated in this region (e.g., neuropeptide Y, substance P, and VT [5,8 –10,63]). Although these were not evaluated quantitatively, it must also be noted that the number of non-synaptic profiles showing large dense-core vesicles appeared to be much larger than the number of true synapses (i.e., those showing synaptic densities). This finding is in agreement with our recent observations on the relationships between VT-immunopositive fibers and aromatase cells in the POM: only few VT-immunoreactive terminal boutons can be observed in contact with aromatase-immunoreactive cell bodies and along the dendrites of aromatase-immunoreactive fibers
but large number of VT-immunoreactive fibers are present in the POM [13]. Synapses containing small spherical clear vesicles (type C) may contain a large variety of neurotransmitters; their neurochemical identification is therefore impossible on the basis of morphological criteria. Synapses with numerous small spherical clear vesicles clustered in regular clumps associated with rare densecore vesicles (type D) were previously described in the arcuate nucleus of female rats [29], and in the median eminence of the rat and squirrel monkey [32,65]. Our morphometric analysis revealed major changes in the synaptic organization of the dorsolateral region in the medial preoptic nucleus of the male Japanese quail specifically induced by castration and T replacement therapy. Previous studies of estradiol-sensitive systems in the female rat [4,24,29,34,41,42] or of androgen-sensitive systems in the male rat brain [33,35,36,37,38,49,58] also demonstrated a massive rearrangement of the synaptic pattern after steroid manipulation. These changes are confirmed in the present ultrastructural study in the quail. In the present study, the most obvious changes were detected in
TESTOSTERONE EFFECTS ON SYNAPSES IN POM
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ANALYSIS OF SYNAPTIC DENSITY IN THE DORSOLATERAL MEDIAL PREOPTIC NUCLEUS Synaptic Density
p
Intact
Axosomatic synapses (no. of synapses/cell) Axondendritic synapses (no. of synapses/1000 m2) Shaft synapses Spine synapses Synapses of type A
ⴱⴱⴱ
6.20 ⫾ 0.49
n.s.
108.20 ⫾ 7.52
n.s. n.s. ⴱⴱ
92.60 ⫾ 6.24 15.60 ⫾ 2.27 3.00 ⫾ 0.63
Synapses of type B Synapses of type C Synapses of type D
n.s. n.s. n.s.
58.40 ⫾ 6.68 43.80 ⫾ 5.51 1.06 ⫾ 0.76
CX
CX ⫹ T
2.20 ⫾ 0.49 ⴱ 103.75 ⫾ 11.93
6.40 ⫾ 0.75 # 95.60 ⫾ 6.56
84.25 ⫾ 11.20 19.50 ⫾ 1.19 0.33 ⫾ 0.12 ⴱ 54.25 ⫾ 6.47 48.75 ⫾ 6.52 0.43 ⫾ 0.28
80.00 ⫾ 5.15 15.60 ⫾ 2.06 0.82 ⫾ 0.14 ⴱ 46.60 ⫾ 2.38 50.00 ⫾ 6.04 0.22 ⫾ 0.14
Data listed mean ⫾ SE. They were compared by one-way ANOVA whose results are reported in the first column (ⴱⴱp ⬍ 0.01; ⴱⴱⴱp ⬍ 0.001; n.s. ⫽ p ⬎ 0.05). When appropriate ANOVA was followed by Tukey post-hoc tests whose results are indicated under the corresponding means and SE (ⴱp ⬍ 0.05 vs. Intact and #p ⬍ 0.05 vs. CX groups). No statistically significant difference was detected between the Intact and CX ⫹ T groups except for type A synapses.
the axosomatic component, so that a dramatic reduction in the number of synapses per cell body was observed after castration. Previous studies have also shown that castration induces a marked decrease of the neuronal size in the POM [48]. This effect of castration is in agreement with data reporting changes of the synaptic inputs on the motoneurons of the adult spinal cord in male rats [35,39]. Axosomatic synapses observed in the POM are prevalently of the symmetrical type and are therefore generally considered as inhibitory synapses [30]. Therefore, the reduction in the number of axosomatic synapses suggests that inhibitory control could be decreased in the POM of castrated male quail. The quantitative analysis of POM neuropil did not reveal significant differences in the density of axodendritic shaft and
FIG. 4. Bar graphs illustrating the effects of castration associated or not with testosterone (T)-treatment on the cross-sectional area (mean ⫾ SE) of type A, B, C, and D synapses in the medial preoptic nucleus (POM) neuropile of male quail. Data relative to each synapse type were compared by one-way ANOVA followed when appropriate (i.e., when the ANOVA indicated the presence of significant differences) by comparisons of the groups two by two with the Tukey post-hoc test. Results of these post-hoc tests are indicated at the top columns as follows: *p ⬍ 0.05 vs. Intact group; #p ⬍ 0.01 vs. CX group. No statistically significant difference was detected between the Intact and CX⫹T groups.
axodendritic spine synapses between control, castrated and Ttreated quails. However, it is firmly established that in the male quail castration decreases the volume of the POM by about 30% and this effect is reversed by a replacement therapy with exogenous T [54,56]. The apparent stability of synapses density within the neuropil, associated with major changes in the total volume of the structure of interest, may therefore indicate a marked alteration following castration or T treatment in the total number of synapses in the nucleus. This conclusion must, however, be considered with caution since it derives from the comparison between the wellknown decrease in POM volume and the apparent lack of change in density of axodendritic synapses, rather than from a direct count of synapses in POM that is impossible to perform for obvious technical reasons. These data also suggest that castration would apparently affect in the same way all the different morphological types of synapses recognized in the present study, with the possible exception of type-A terminals. Such subtype of synapses was indeed present at a lower density in castrated than in intact sexually mature birds, and this could indicate that they are more affected by castration than the other subtypes (decrease in density superimposed to the decrease in global volume). This interpretation has to be presented cautiously because the replacement therapy with T failed to fully restore type-A synapse density to the level seen in intact birds (a twofold increase of density was nevertheless detected). It must be noted, however, that castrated birds were exposed to exogenous T only for about 2 weeks in the present study and a longer treatment may be necessary to induce a full recovery. This interpretation should be tested in future experiments. Synaptic plasticity was also observed in the present study in relation to another morphometric parameter, i.e., the size of the terminal boutons. Measurements revealed that castration induces a significant drop of the cross-sectional area of the synaptic terminals (a decrease of about 30%), whereas the treatment with T induced a full restoration of the size to the level typical of control birds. These changes are statistically significant for synapses of the A, B, and C types, but they presumably also involved synapses of the D type, although their changes did not reach statistical significance. The functional implications of changes in synaptic size are difficult to ascertain. However, changes in synapse sizes have been frequently associated with maturational events. Newly formed
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adult hippocampal synapses have smaller synaptic components (resembling synapses seen during development) than adult synapses and they increase in size over time with usage, suggesting some sort of correlation between the efficacy of synaptic transmission and the size of synaptic contacts [2]. Previous studies indicated that changes in steroid levels induced by castration or T replacement therapy produce a major reorganization of the male quail dorsolateral portion of the POM. This reorganization was already studied at the light microscopic level [54,56] and at the electron microscopic level, but only in the neuronal compartment. The present ultrastructural data indicate that the castration-associated endocrine changes also produce profound alterations in the number and morphology of synapses. These data confirm and extend light microscopic immunocytochemical data showing that castration or T treatment affect the density of fibers located within the POM that are immunoreactive for neuropeptides, such as VT or neurotensin [1,63]. These immunocytochemical studies indicated the existence of marked decreases in peptidergic innervation following castration but could not demonstrate a true loss of synapses. The disappearance of immunoreactive fibers and punctate structures could only reflect a loss of immunoreactivity induced by a decreased synthesis or increased secretion of the neuropeptide. The present data do not exclude this interpretation but suggest that, in addition, the total number of synapses and their size change as a function of the circulating T levels. Because the POM, and in particular its dorsolateral division, is a crucial station in the pathway controlling male quail copulatory behavior [12,53], it appears likely that the T-dependent synaptic changes identified in the present study represent a morphological signature of the neurochemical events and synaptic reorganization that underlie the activation by steroids of reproductive behavior. Future studies should now be designed to better characterize these synaptic events and their specific connection with behavior.
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ACKNOWLEDGEMENTS
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Work supported by grants from the CNR (97.04308.CT04, 98.03164.CT04) to CVP, from the University of Torino to GCP and CVP, and from the National Institute on Mental Health (MH 50388), the Belgian FRFC (Nbr. 9.4565.96F), the French Community of Belgium (ARC 99/ 04-241) and the University of Lie`ge (Cre´dits spe´ciaux) to J.B.
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PII S0361-9230(99)00193-8
Effects of testosterone on the synaptology of the medial preoptic nucleus of male Japanese quail C. Castagna,1 A. Obole,1 C. Viglietti-Panzica,1 J. Balthazart2 and G. C. Panzica1* 1
Department of Anatomy, Pharmacology and Forensic Medicine, University of Torino, Torino, Italy; and Laboratory of Neurochemistry, Research Group in Behavioral Neuroendocrinology, University of Lie`ge, Lie`ge, Belgium
2
[Received 24 March 1999; Revised 6 July 1999; Accepted 19 July 1999] ABSTRACT: The medial preoptic nucleus (POM) of male Japanese quail is a sexually dimorphic testosterone-dependent structure that plays a key role in the activation of male sexual behavior. Both the total volume of the nucleus and the size of the dorsolateral neurons are decreased in gonadectomized males. Immunocytochemical studies have revealed a complex pattern of innervation: immunopositive fibers for several neuropeptides and neurotransmitters have been detected in the POM; some of them (e.g. vasotocin-immunoreactive fibers) are sexually dimorphic and testosterone-dependent. To understand the anatomical bases of these testosterone-dependent neurochemical changes, we performed an ultrastructural study of the POM neuropil in intact sexually mature, gonadectomized, or testosterone-treated gonadectomized males. A complex synaptic organization of the POM neuropil was observed in intact male quail reflecting the heterogeneity of the neurotransmitters and neuropeptides present in this nucleus. Changes in this organization were observed after the endocrine manipulations. The number of axosomatic synapses per cell body decreased after gonadectomy and was restored to the level observed in the intact group after the administration of testosterone. By contrast, no significant change was observed in the density of axodendritic and axospinal synapses after hormonal manipulations which suggests that the total number of synapses in the nucleus should be affected by testosterone (constant density in a changing total volume). The cross-sectional area of synaptic boutons was also decreased by castration and restored to intact level by testosterone. The action of testosterone on the activation of male copulatory behavior in gonadectomized birds is hence paralleled by an extensive rearrangement of neuropil in the POM. © 1999 Elsevier Science Inc.
dictions have been extensively documented by experimental studies carried out mostly during the last 50 years [23,31,40,46,62]. In particular, several studies have documented, both at the light and electron microscopic levels, the dramatic anatomical changes produced by gonadal steroid action. They include changes in the volume of brain nuclei, in the size and staining of neurons, in the innervation pattern, as well as changes in the ultrastructural organization of cell bodies and neuropil [46]. In adult Japanese quail, it has been shown that several morphological characteristics of the medial preoptic nucleus (POM), a sexually dimorphic structure controlling male copulatory behavior [20 –22], change as a function of the levels of circulating testosterone (T). The total volume of the nucleus, the cell size of the dorsolateral population, as well as the presence of neurochemical markers within the neurons are affected [52,53]. In a recent study [48], we investigated the effects of T on the ultrastructural organization of cell bodies in the male quail POM. In castrated birds, the amount and size of organelles were strongly decreased compared to sexually mature males, and a 2-week treatment with T was able to restore an ultrastructural organization that is typical of that found in a control sexually active male. In particular, the organelles involved in protein synthesis (rough endoplasmic reticulum, Golgi complex, secretion vesicles) almost totally disappear in the cell bodies of castrated quail. These changes may reflect the reduction after castration of the concentration of aromatase, the enzyme that catalyzes the conversion of T into estradiol (E2). Aromatase-immunoreactive neurons represent a large fraction of the POM cells and constitute a neuronal marker of the cytoarchitectonic boundaries of the POM [16]. Immunocytochemical studies demonstrated the existence within this region of cell bodies and fibers immunopositive for neuropeptide Y, substance P, NADPH-diaphorase, gonadotropin hormone-releasing hormone (GnRH), vasotocin (VT), and neurotensin. Moreover, immunoreactive fibers for corticotropin-releasing factor, tyrosine hydroxylase, and serotonin were also described [8 –10,45,53]. Recently, we have shown that the VT-immunoreactive (VT-ir) elements of the POM are sexually dimorphic [5], and T-dependent: the density of these fibers is markedly decreased by castration and increased by T administration [63].
KEY WORDS: Gonadal hormones, Sexual behavior, Preoptic area, Synapses, Ultrastructure.
INTRODUCTION The presence of gonadal steroid receptors within the central nervous system (CNS) suggests that steroids could have specific effects on the development and differentiation of the neural circuits, on their maintenance in adulthood, as well as on the control of endocrine and behavioral aspects of reproduction. These pre-
* Address for correspondence: Dr. G. C. Panzica, Department of Anatomy, Pharmacology, and Forensic Medicine, University of Torino, Corso M. D’Azeglio 52, I-10126 Torino, Italy. Fax: ⫹39-011-670-7732; E-mail: [email protected]
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In parallel with these morphological changes, T action in the POM is also essential for the activation of copulatory behavior in adult male quail [23,43]. Electrolytical lesions of the POM abolish male copulatory behavior and T implants within this nucleus, but not in its immediate vicinity, activate behavior in castrated subjects. It is therefore assumed that the anatomical and neurochemical changes (volume, cell size, cell ultrastructure, innervation) seen in this brain region after a treatment with T represent the morphological signature of the mechanisms underlying the behavioral activation [53]. Studies performed in a variety of animal models have demonstrated that in androgen-target regions the ultrastructure of the neuropil shows marked changes as a function of the circulating levels of T [3,26,35,38,49,58]. The immunocytochemical studies of the quail POM described above suggested that in this model, the innervation of the nucleus is also affected by T [52]. Therefore, in the present study we searched whether similar changes in the synaptic organization could be detected in quail after castration and treatment with exogenous T. This study focused on the dorsolateral portion of the POM that has been shown to be very sensitive to T action and probably plays a pivotal role in the control of sexual behavior [12]. MATERIALS AND METHODS Animals This study was performed on the brain of 15 male Japanese quail (Coturnix japonica) that had been previously used to analyze the effects of T on the neuronal ultrastructure in the POM. The in vivo treatment of these birds, fixation and histological procedures have been previously described in detail [48] and will therefore be presented here in a succinct form only. Birds were housed, manipulated, and sacrificed according to the Principle of Laboratory Animal Care (National Institutes of Health), and to the relevant Italian and Belgian laws on the protection of animals. The birds were bought from a local breeder (Lefe`re, Boneffe, Belgium) at the age of 3 weeks and then housed in individual cages on a 16-h light:8-h dark schedule simulating long days, at controlled temperature with water and food available ad libitum. Hormonal Treatments Ten birds were gonadectomized and 5 were sham-operated under anaesthesia (Hypnodil, Janssen Pharmaceutica, Belgium; 15 mg/kg body weight) at the age of 3 weeks using previously described methods [60]. Nine days later, gonadectomized birds were either treated with 40 mm (2 ⫻ 20 mm) Silastic implants filled with T (CX ⫹ T) or left empty (CX). In the third group the sham-operated males (Intact) also received empty implants. The implants were prepared with Silastic tube (Dow Corning tubing, number 602-252; inner diameter: 1.57 mm, outer diameter: 2.41 mm) filled with crystalline T (Sigma T-1500). It has been previously demonstrated that this type of T treatment restores in both males and females plasma levels of the steroid that are similar to those observed in intact sexually active males [14,61]. Approximately 2 weeks later, birds were tested twice for sexual behavior (observed for 5 min in the presence of a sexually mature female) and their body weight and cloacal gland size (a T-dependent structure [59]) were measured as previously described [18,56]. The animals were sacrificed and the brains were collected for ultrastructural observations during the following week when they were 8 weeks old. Fixation and Embedding Procedures The detailed procedure of fixation, dissection, and sampling was described elsewhere [48,50]. Briefly, the birds were transcar-
dially perfused with two different fixatives used in sequence: fixative no. 1 (1% paraformaldehyde ⫹ 1% glutaraldehyde in 0.1M Na-K phosphate buffer, pH 7.2–7.4 containing 0.5% of CaCl2 and MgCl2) for the first 10 min followed by fixative no. 2 (2% paraformaldehyde ⫹ 3% glutaraldehyde in the same buffer). The dissected brains were left into fixative no. 2 for about 2–3 h at 4°C. After washing overnight in sucrose-containing buffer (Na-K phosphate buffer 0.1 M, pH 7.2–7.4 containing 10% sucrose to maintain osmolality), specimens were sectioned in the coronal plane with a vibratome and 200-m-thick sections were collected in the same buffer. The sections were quickly stained with toluidine blue (1% in buffer [27]) and observed with a light microscope in order to select serial levels through the preoptic region along the rostrocaudal axis. The left and right POM were dissected from these sections keeping the ependymal wall as an orientation landmark. The 200-m-thick sections were then postfixed in a solution of 1% osmium tetroxide in 0.1 M phosphate buffer containing 2% dextrose, for 1 h at 4° C in the dark, dehydrated in alcohol, and flat embedded in a mixture of Epon and Araldite (1:1) for 48 h at 60°C. Blocks corresponding to the section located immediately before the anterior commissure were selected for quantitative analysis because this level has been shown to be most closely associated with the activation by steroids of sexual behavior [22] and it was always analyzed in previous light- and electron-microscopic studies [6,7,48,56]. Identical levels of the dorsolateral part of the POM were carefully selected in individual samples based on the observation of 1-m-thick semi-thin sections cut from these blocks and stained with toluidine blue (0.1% in borax, see also our previous study [50]). Ultrathin sections were then cut, contrasted with uranyl acetate and lead citrate, and examined with a Philips 410 transmission electron microscope at 80 kV. Quantitative Analysis Criteria applied for the identification and counting of synapses were the presence of pre- and postsynaptic densities associated with local concentration of synaptic vesicles in the axon terminals. Synapses were subdivided in two main groups: axosomatic and axodendritic. The number of axosomatic synapses per cell was counted in the three experimental groups on micrographs of neurons taken at a magnification of 7,100⫻ and enlarged to a final magnification of 14,200⫻. A total of 10 to 30 neurons per animal were randomly collected [14 ⫾ 5 neurons in Intact, 18 ⫾ 8 neurons in CX, and 16 ⫾ 4 neurons in CX⫹T birds (all means ⫾ standard deviation)]. Neurons were considered only if they could be entirely observed and only if they contained a nucleus. Axodendritic synapses were counted on micrographs covering an area of at least 2,500 2 of neuropil (excluding neurons, glial cells, and blood vessels) per bird (range 2,500 – 4,000 2), at a final magnification of 17,750⫻. They were subdivided in two groups: shaft and spine synapses (see Fig. 2). Axodendritic synapses were further subdivided into 4 main types (A, B, C, and D) based on the presence of clear and dense-core vesicles and on the diameter of these vesicles (see Results for detail, Fig. 1). Results were then standardized and expressed in terms of synaptic density (number of synapses per 1000 m2 of examined area). Morphometric Analysis The micrographs were then digitized by an AppleOne scanner connected to a Macintosh computer (Performa 630). Each synapse was outlined by the operator (A.O.) using the mouse; the area value was automatically calculated by the software NIH-Image
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FIG. 1. Photomicrographs illustrating the synaptic organization of the medial preoptic (POM) neuropile at the ultrastructural level. Synaptic terminals are characterized by different types of vesicles. In type-A synapses (A) presynaptic terminals contain small spherical and pleiomorphic clear vesicles associated with large spherical dense-core vesicles. In type-B synapses (B) presynaptic terminals contain small spherical clear vesicles associated with small spherical dense-core vesicles. Type-C synapses (C) are characterized by presynaptic terminals that contain small spherical clear vesicles while in type-D synapses (D), presynaptic terminals contain small spherical clear vesicles clustered in a regular clump, associated with rare dense-core vesicles. Abbreviation: d, dendrite. Bar ⫽ 1 m.
(version 1.60, Wayne Rasband, National Institutes of Health, Bethesda, MD, USA). Statistical Analysis For each dependent variable, we calculated the mean of all determinations in each bird. Values referring to synaptic densities were used to perform one-way analyses of variance (ANOVA)
comparing the different groups. When appropriate, post-hoc testing was carried out to compare the three groups 2 ⫻ 2 using the Tukey test, that provides an optimal compromise between the risks of type I and type II (program Super ANOVA, Abacus Concepts Inc, Berkeley, CA, USA on MacIntosh computers). Means ⫾ standard errors were systematically used as measures of central tendency and dispersion. Differences were considered significant for p ⬍ 0.05.
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Behavioral and Physical Parameters The behavioral and morphological data concerning the subjects of this experiment have been reported in a previous study [48]. Briefly, the body weight in the three groups of subjects was similar at the beginning and at the end of the experiment (ANOVA; p ⬎ 0.05). Before the experiment, the cloacal gland area of castrated birds was smaller (mean below 50 mm2) than in intact subjects (184 mm2). After T implantation the size of the gland increased very markedly and was restored to values similar to those observed in gonadally intact subjects while it remained significantly smaller in the CX group [F(2,12) ⫽ 70.84, p ⬍ 0.0001]. Intact and CX⫹T birds regularly showed sexual behavior while castrates never did. The frequencies of mount attempts were significantly different [F(2,12) ⫽ 11.62, p ⫽ 0.0016]. These data confirm previously reported results and indicate that the experimental treatments produced the expected endocrine changes [17,19,56]. Synaptology of the Medial Preoptic Nucleus At ultrastructural level the dorsolateral part of the POM exhibited the features of a complex synaptic organization (Figs. 1A–D). The POM neuropil was characterized by numerous nerve endings making synaptic contact with dendrites (axodendritic synapses; Figs. 2A,B) or with neuronal cell bodies (axosomatic synapses; Fig. 3). Axodendritic synapses can be subdivided into two main groups depending on whether the synaptic bouton contacts a portion of the dendritic contour that is not modified (shaft synapses) or contacts a fungiform protuberance of the dendrite (spine synapses). Axosomatic synapses. Axosomatic synapses were frequently observed on neuronal cell bodies in the lateral POM: a mean of 4 to 8 small nerve endings (identified by the presence of synaptic vesicles and presynaptic and postsynaptic densities) per cell body were detected (Fig. 3). Axosomatic synapses were not uniformly distributed around the cell surface, but they were clustered in selected regions of the perikaryon. Synaptic vesicles were small, clear, and pleiomorphic, but in a limited number of cases they were larger and contained a dense core. The active zones of the presynaptic and postsynaptic membrane in axosomatic synapses were identified by the presence of similar amounts of electron-dense material (symmetrical or Gray’s type II synapses; see also [44,57]). Axodendritic synapses. Transversally cut axons and long neuronal processes were frequently observed within the neuropil. Neuronal processes showed mitochondria, tubules, and filaments, as well as, in most cases, rare ribosomes, and were therefore identified as dendrites (Figs. 1A–D) These processes either exhibited a smooth surface bearing synapses (shaft synapses) or displayed short spines, surrounded by synaptic endings (spine synapses; Figs. 2A,B). A variety of axon terminals could be distinguished in the neuropil according to the morphology of their vesicles and synaptic densities. We classified axodendritic synapses in 4 groups on the basis of their morphology and content of vesicles (Figs. 1A–D): ●
● ● ●
Type A, synapses containing small spherical and pleiomorphic clear vesicles associated with large spherical dense-core vesicles; Type B, synapses containing small spherical clear vesicles associated with small spherical dense-core vesicles; Type C, synapses containing spherical clear vesicles; Type D, synapses containing numerous small spherical clear vesicles clustered in a regular clump associated with rare densecore vesicles.
In addition to the vesicular components, the synaptic terminals contained small mitochondria (Figs. 1B,C). The active zone of the presynaptic membrane was identified by the presence of dense projections constituted by a variable amount of electron-dense material. In many cases the postsynaptic membrane also demonstrated a clearly visible postsynaptic density (Figs. 1 A–D). These synapses can therefore be classified as asymmetrical (or Gray’s type I). However, several endings lacked a conspicuous postsynaptic density, and their synaptic cleft was thinner (Fig. 2A). These were identified as symmetrical synapses (or Gray’s type II). Quantitative and Morphometric Analysis The quantitative analysis of the neuropil in the lateral POM revealed a massive rearrangement of the synaptic inputs after endocrine manipulations (Table 1 and Fig. 4). The number of axosomatic synapses was significantly affected by treatments (one-way ANOVA; p ⬍ 0.0004). Tukey tests comparing the three experimental groups 2 ⫻ 2 indicated that castration decreased and T increased in a significant manner the number of axosomatic contacts per cell (Table 1). In contrast, no significant change was observed in the total number of axodendritic contacts per unit area (1000 m2) under the same conditions (Table 1). Similarly the number of shaft or spine contacts per unit area (1000 m2) was not affected by the experimental treatments (Table 1). When terminals were subdivided in specific groups based on the morphological classification described above, a decrease in the number of type A-synapses was observed after gonadectomy, but this decrease was only partly and non-significantly restored by T replacement (p ⫽ 0.0032 in the general ANOVA comparing the three groups, see Table 1 for the detail of Tukey tests comparing the three experimental groups 2 ⫻ 2). No significant variation was detected in the number of the other types of synaptic contacts (types B,C, and D) under the same conditions (Table 1). Morphometric data on the cross-sectional area (size) were subjected to a two-way ANOVA (treatment and type as factors) that indicated the presence of a significant overall effect of treatment on the synaptic endings size [F(2,11) ⫽ 11.045, p ⫽ 0.0023], a highly significant effect of the synaptic type [F(3,33) ⫽ 9.09, p ⫽ 0.0002], and no significant interaction between the two factors [F(6,33) ⫽ 1.339, p ⫽ 0.2681] Subsequent one-way ANOVA analysis of each type of synapse demonstrated that the size of the type A, B and C synapses (Fig. 4) was significantly affected by the experimental (p ⫽ 0.031 for type A, p ⫽ 0.005 for type B, and p ⫽ 0.012 for type C). Type D synapses (Fig. 4) tended to display similar changes in size but these changes were not statistically significant (one-way ANOVA; p ⫽ 0.081). Tukey post-hoc tests comparing the three experimental groups 2 ⫻ 2 confirmed that castration decreased and T increased (i.e., restored to intact level) the size of type A, B, and C synaptic endings, respectively, in a statistically significant manner. DISCUSSION The present study confirms the complexity of the synaptic component in the dorsolateral portion of the male quail POM that was first demonstrated in our previous study [50]. The morphometrical data collected in the control group of sexually mature gonadally intact males characterize more precisely the morphology, the qualitative and the quantitative distribution of synaptic endings in this region crucial for the control of the reproduction in male quail [53]. The vast majority of synapses contact a smooth dendritic surface, whereas only about 15% of total synapses are located on spines. This percentage appears to be higher than that previously observed in the domestic fowl preoptic [44] and supra-
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245
FIG. 2. (A,B) Photomicrographs showing long dendrites (d), longitudinally sectioned and associated with several synaptic endings. Two types of synapses are detectable: axodendritic or shaft synapses (thin arrows), in which the dendritic contour is not modified, and axospinal or spine synapses (thick arrows), characterized by a bouton contacting fungiform protuberances of the dendrite (spine). A symmetrical synapse can also be detected in A (asterisk). Bar ⫽ 1 m.
chiasmatic areas [47]. No detailed studies based on Golgi impregnation have been published on the quail preoptic neurons, but preliminary results [28] suggest that these neurons bear a larger number of spines than those in the domestic fowl [51]. It is therefore possible that the higher density of spine synapses in the quail compared to the fowl simply reflects the abundance of spines in the former species.
The morphological characterization of synapses utilized in the present study is based on our previous investigations of the anterior hypothalamus of the domestic fowl [47,49,55]. B-type synapses represent the majority of the endings observed in the dorsolateral part of the POM (close to 60% of the total number). Due to the presence of small dense core vesicles, these synapses may be considered to belong to catecholaminergic, serotoninergic, or pep-
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FIG. 3. Photomicrograph illustrating axosomatic synapses (arrowheads) showing similar pre- and postsynaptic densities (symmetrical synapses). Abbreviations: G, Golgi complex; N, neuronal nucleus; RER, rough endoplasmic reticulum. Bar ⫽ 1 m.
tidergic types (see [49]). This interpretation is in good agreement with light microscopic immunocytochemical studies previously performed on the same region: these studies demonstrated a large supply of tyrosine hydroxylase-positive fibers, as well as of peptidergic elements (cells and fibers) in the lateral part of the POM [8 –11,15,25,45]. Synapses showing large dense-core vesicles (type A) represent a scarce population (around the 3% of the total). Due to the presence of large dense core vesicles, these elements are probably peptidergic terminals of neurosecretory type [55,64], and they probably contain some of the neuropeptides that have been demonstrated in this region (e.g., neuropeptide Y, substance P, and VT [5,8 –10,63]). Although these were not evaluated quantitatively, it must also be noted that the number of non-synaptic profiles showing large dense-core vesicles appeared to be much larger than the number of true synapses (i.e., those showing synaptic densities). This finding is in agreement with our recent observations on the relationships between VT-immunopositive fibers and aromatase cells in the POM: only few VT-immunoreactive terminal boutons can be observed in contact with aromatase-immunoreactive cell bodies and along the dendrites of aromatase-immunoreactive fibers
but large number of VT-immunoreactive fibers are present in the POM [13]. Synapses containing small spherical clear vesicles (type C) may contain a large variety of neurotransmitters; their neurochemical identification is therefore impossible on the basis of morphological criteria. Synapses with numerous small spherical clear vesicles clustered in regular clumps associated with rare densecore vesicles (type D) were previously described in the arcuate nucleus of female rats [29], and in the median eminence of the rat and squirrel monkey [32,65]. Our morphometric analysis revealed major changes in the synaptic organization of the dorsolateral region in the medial preoptic nucleus of the male Japanese quail specifically induced by castration and T replacement therapy. Previous studies of estradiol-sensitive systems in the female rat [4,24,29,34,41,42] or of androgen-sensitive systems in the male rat brain [33,35,36,37,38,49,58] also demonstrated a massive rearrangement of the synaptic pattern after steroid manipulation. These changes are confirmed in the present ultrastructural study in the quail. In the present study, the most obvious changes were detected in
TESTOSTERONE EFFECTS ON SYNAPSES IN POM
247 TABLE 1
ANALYSIS OF SYNAPTIC DENSITY IN THE DORSOLATERAL MEDIAL PREOPTIC NUCLEUS Synaptic Density
p
Intact
Axosomatic synapses (no. of synapses/cell) Axondendritic synapses (no. of synapses/1000 m2) Shaft synapses Spine synapses Synapses of type A
ⴱⴱⴱ
6.20 ⫾ 0.49
n.s.
108.20 ⫾ 7.52
n.s. n.s. ⴱⴱ
92.60 ⫾ 6.24 15.60 ⫾ 2.27 3.00 ⫾ 0.63
Synapses of type B Synapses of type C Synapses of type D
n.s. n.s. n.s.
58.40 ⫾ 6.68 43.80 ⫾ 5.51 1.06 ⫾ 0.76
CX
CX ⫹ T
2.20 ⫾ 0.49 ⴱ 103.75 ⫾ 11.93
6.40 ⫾ 0.75 # 95.60 ⫾ 6.56
84.25 ⫾ 11.20 19.50 ⫾ 1.19 0.33 ⫾ 0.12 ⴱ 54.25 ⫾ 6.47 48.75 ⫾ 6.52 0.43 ⫾ 0.28
80.00 ⫾ 5.15 15.60 ⫾ 2.06 0.82 ⫾ 0.14 ⴱ 46.60 ⫾ 2.38 50.00 ⫾ 6.04 0.22 ⫾ 0.14
Data listed mean ⫾ SE. They were compared by one-way ANOVA whose results are reported in the first column (ⴱⴱp ⬍ 0.01; ⴱⴱⴱp ⬍ 0.001; n.s. ⫽ p ⬎ 0.05). When appropriate ANOVA was followed by Tukey post-hoc tests whose results are indicated under the corresponding means and SE (ⴱp ⬍ 0.05 vs. Intact and #p ⬍ 0.05 vs. CX groups). No statistically significant difference was detected between the Intact and CX ⫹ T groups except for type A synapses.
the axosomatic component, so that a dramatic reduction in the number of synapses per cell body was observed after castration. Previous studies have also shown that castration induces a marked decrease of the neuronal size in the POM [48]. This effect of castration is in agreement with data reporting changes of the synaptic inputs on the motoneurons of the adult spinal cord in male rats [35,39]. Axosomatic synapses observed in the POM are prevalently of the symmetrical type and are therefore generally considered as inhibitory synapses [30]. Therefore, the reduction in the number of axosomatic synapses suggests that inhibitory control could be decreased in the POM of castrated male quail. The quantitative analysis of POM neuropil did not reveal significant differences in the density of axodendritic shaft and
FIG. 4. Bar graphs illustrating the effects of castration associated or not with testosterone (T)-treatment on the cross-sectional area (mean ⫾ SE) of type A, B, C, and D synapses in the medial preoptic nucleus (POM) neuropile of male quail. Data relative to each synapse type were compared by one-way ANOVA followed when appropriate (i.e., when the ANOVA indicated the presence of significant differences) by comparisons of the groups two by two with the Tukey post-hoc test. Results of these post-hoc tests are indicated at the top columns as follows: *p ⬍ 0.05 vs. Intact group; #p ⬍ 0.01 vs. CX group. No statistically significant difference was detected between the Intact and CX⫹T groups.
axodendritic spine synapses between control, castrated and Ttreated quails. However, it is firmly established that in the male quail castration decreases the volume of the POM by about 30% and this effect is reversed by a replacement therapy with exogenous T [54,56]. The apparent stability of synapses density within the neuropil, associated with major changes in the total volume of the structure of interest, may therefore indicate a marked alteration following castration or T treatment in the total number of synapses in the nucleus. This conclusion must, however, be considered with caution since it derives from the comparison between the wellknown decrease in POM volume and the apparent lack of change in density of axodendritic synapses, rather than from a direct count of synapses in POM that is impossible to perform for obvious technical reasons. These data also suggest that castration would apparently affect in the same way all the different morphological types of synapses recognized in the present study, with the possible exception of type-A terminals. Such subtype of synapses was indeed present at a lower density in castrated than in intact sexually mature birds, and this could indicate that they are more affected by castration than the other subtypes (decrease in density superimposed to the decrease in global volume). This interpretation has to be presented cautiously because the replacement therapy with T failed to fully restore type-A synapse density to the level seen in intact birds (a twofold increase of density was nevertheless detected). It must be noted, however, that castrated birds were exposed to exogenous T only for about 2 weeks in the present study and a longer treatment may be necessary to induce a full recovery. This interpretation should be tested in future experiments. Synaptic plasticity was also observed in the present study in relation to another morphometric parameter, i.e., the size of the terminal boutons. Measurements revealed that castration induces a significant drop of the cross-sectional area of the synaptic terminals (a decrease of about 30%), whereas the treatment with T induced a full restoration of the size to the level typical of control birds. These changes are statistically significant for synapses of the A, B, and C types, but they presumably also involved synapses of the D type, although their changes did not reach statistical significance. The functional implications of changes in synaptic size are difficult to ascertain. However, changes in synapse sizes have been frequently associated with maturational events. Newly formed
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adult hippocampal synapses have smaller synaptic components (resembling synapses seen during development) than adult synapses and they increase in size over time with usage, suggesting some sort of correlation between the efficacy of synaptic transmission and the size of synaptic contacts [2]. Previous studies indicated that changes in steroid levels induced by castration or T replacement therapy produce a major reorganization of the male quail dorsolateral portion of the POM. This reorganization was already studied at the light microscopic level [54,56] and at the electron microscopic level, but only in the neuronal compartment. The present ultrastructural data indicate that the castration-associated endocrine changes also produce profound alterations in the number and morphology of synapses. These data confirm and extend light microscopic immunocytochemical data showing that castration or T treatment affect the density of fibers located within the POM that are immunoreactive for neuropeptides, such as VT or neurotensin [1,63]. These immunocytochemical studies indicated the existence of marked decreases in peptidergic innervation following castration but could not demonstrate a true loss of synapses. The disappearance of immunoreactive fibers and punctate structures could only reflect a loss of immunoreactivity induced by a decreased synthesis or increased secretion of the neuropeptide. The present data do not exclude this interpretation but suggest that, in addition, the total number of synapses and their size change as a function of the circulating T levels. Because the POM, and in particular its dorsolateral division, is a crucial station in the pathway controlling male quail copulatory behavior [12,53], it appears likely that the T-dependent synaptic changes identified in the present study represent a morphological signature of the neurochemical events and synaptic reorganization that underlie the activation by steroids of reproductive behavior. Future studies should now be designed to better characterize these synaptic events and their specific connection with behavior.
8.
9.
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ACKNOWLEDGEMENTS
18.
Work supported by grants from the CNR (97.04308.CT04, 98.03164.CT04) to CVP, from the University of Torino to GCP and CVP, and from the National Institute on Mental Health (MH 50388), the Belgian FRFC (Nbr. 9.4565.96F), the French Community of Belgium (ARC 99/ 04-241) and the University of Lie`ge (Cre´dits spe´ciaux) to J.B.
19.
20.
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