Original Article

Introduction

n living organisms, from fungi to humans, many structural and functional properties are determined by sexual and nonsexual steroid hormones, which are effective at almost all levels of biological organization.¹ The distribution and concentration of sex steroids in the various central nervous system (CNS) regions depend not only on the serum level, but also on the local synthesis of the steroids.² However, it is now known that levels of circulating sex steroid hormones, during development and in adulthood, play a critical role in determining physiology and behavior in adulthood.^{3, 4} The neural mechanisms

underlying hormonal effects on brain function and morphology have been the subject of numerous studies. Traditionally, steroid hormonal effects have been classified into 2 categories; organizational or genomic and activational or nongenomic.⁵ Whereas, organizational effects have been defined as permanent structural changes induced by hormones during development; activational effects have been defined as the turning on or on/off of previously established neuronal circuits in adulthood.^{1, 5} Since the morphologic characteristics of neurons have been shown to influence the functional properties of the neurons,^{6 – 8} it is likely that these hormone-induced structural changes contribute significantly to the activation of neural circuits necessary for certain behaviors.⁹ Recent findings suggest that manipulation of sex steroid hormone levels may induce dramatic macroscopic and microscopic structural changes in certain regions of the central nervous system, such as neurons of adult avian song system,^{10, 11} corpus callosum and anterior commissure,^12,
13 bulbocavernosus spinal nucleus,^{14, 15} spinal motorneurons,¹⁶ rat Purkinje cell,¹⁷ sexual dimorphic nucleus of preoptic area of hypothalamus (SDN-POA) of hypothalamus,¹⁸ hippocampal pyramidal cells,¹⁹ bed nucleus of human stria terminalis,²⁰ nigrostriatal dopamine neurons,²¹ rat arcuate nucleus,^{22 – 24} human median raphe nucleus,²⁵ and substantia nigra.²⁶ Dorsal raphe nucleus (DRN) is the largest of all raphe nuclei in rat brainstem, and a part of serotonergic system.²⁷ The serotonergic nuclei and their neurotransmitters are involved in a number of important physiological and pathological functions such as movement, sleep-wake cycle, memory, and sexual behavior.^{28, 29} Neurons of adult DRN appear to be particularly sensitive to female gonadal steroids as alterations in level of female sex hormones dramatically change the rate of serotonin turnover and synthesis, and thereby, the serotonergic physiology.^{30, 31} It is not known, however, if manipulations in ovarian steroid level also influence the structure of adult dorsal raphe neurons. We examined the effect of manipulation in ovarian steroids via ovariectomy, on the density of dendritic spines from primary dendrites of dorsal raphe neurons to determine whether the morphological characteristics of dorsal raphe neurons are also affected by changes in the circulating levels of female sex hormones.

Figure 1. Dorsal raphe nucleus (A, scale bar 50 μm), neurons of dorsal raphe nucleus (B, scale bar 30 μm, C and D, scale bar 10 μm).

Figure 2. Camera lucida drawing of representative polygonal dorsal raphe neurons before (A, B) and after (A’, B’) ovariectomy. The regions located between arrowheads demonstrate dendritic segments appropriate for analysis (see text).

Materials and Methods

Twenty-one virgin, female Sprague-Dawley (Hesarak Institute, Karaj) rats (200 – 250 g, 90 days old) were used for this study. The animals were maintained in a standardized environment with respect to both photoperiod cycle (12 hr light, light at 6 am) and temperature (22ºC). Food and water were provided ad libitum throughout the experiments. The animals were sham and intact control groups (n = 7) and were put into ovariectomies (OVX). For the OVX group, under sterile conditions, the animals were anesthetized by an intraperitoneal (IP) injection of sodium pentobarbital (40 mg/kg). Bilateral ovariectomy was done through a ventral incision of shaved abdominal skin. The animals were sacrificed three weeks later.³² Sham animals were put through the same surgical procedures but ovariectomy. These rats were perfused and the brainstems were processed using the version of single-section Golgi-impregnation method as follows.³³ All of the rats were deeply anesthetized with IP injection of sodium pentobarbital (50 mg/kg) and transcardially perfused with 300 mL of 1% paraformaldehyde and 1% glutaraldehyde in 0.1 M phosphate-buffered saline (PBS; pH 7.6) followed by 450 mL of second fixative containing 6% potassium dichromate, 6% chloral hydrate, and 10% formaldehyde in 0.1 M phosphate-buffered saline (PBS; pH 7.6). Brainstems were removed; 3 mm thick blocks containing DRN were prepared and postfixed in the second fixative for 24 hr. Then, the blocks were put in the solution of 3.5% potassium dichromate in distilled water for 48 hr, followed by storage in 1.5% silver nitrate for 72 hr in a dark room. Dehydration and paraffin embedding were done, and by using a rotatory microtome coronal sections, 100 µm-thick were cut, mounted, and coversliped.

Figure 3. Number of primary dendritic spines. Values present mean ± SEM. *Significant difference between ovariectomized (OVX) + control (Cont) (p < 0.05).

Figure 4. Number of secondary dendritic spines. Values present mean ± SEM. *Significant difference between OVX + Cont (p < 0.05).

Slides containing Golgi-impregnated brain- stems were coded prior to quantitative analysis. The code was not broken before the completion of the analysis. Camera lucida drawings were obtained from selected dendritic segments that remained within the plane of focus. All spines of the selected dendritic segments were counted, and spine density values were expressed as the number of spines/20 µm of the primary and secondary dendrites. The length of the selected segments was determined through the image analysis system. For each animal, a total of 30 dendritic branches (15 for each primary and secondary dendrites) were analyzed. Using SPSS, variable means were determined and the data were subjected to 1-way ANOVA.

Results

Light microscopic examination of Golgi-impregnated tissue revealed reliable and consistent staining throughout the DRN of all brainstems. In particular, three neuronal populations of DRN including polygonal, fusiform, and round neurons were always well represented and easily identifiable in all parts of DRN of the Golgi-stained sections (Figure 1). Camera lucida tracing of the selected portions of dendrites of OVX rats revealed significant changes in the number of dendritic spines (Figure 2). Quantitative analysis of the spine density of dorsal raphe neurons clearly revealed a significant decrease in the number of these spines. Three weeks after surgery, OVX rats showed significantly fewer dendritic spines than intact and sham female rats did (p < 0.01; Figures 3 and 4). In addition to changes in the number of spines per selected length of dendrite, the shape of remaining spines appeared to be different between OVX and intact rats. Dendritic spines of OVX rats showed fewer and less pronounced heads compared to intact females (Figures 5 and 6). Observation of cell body area did not differ significantly between OVX and intact animals.

Discussion

Results of the present study show that female steroid hormones play a critical role in maintaining the number and shape of dendritic spines of the adult dorsal raphe neurons and also exert very rapid effects after gonadal steroid deprivation. Short-term sex steroid deprivation by gonadectomy resulted in significant decrease of dendritic spine density compared to that of intact females.

Various functions have been ascribed to denderitic spines. Dendritic spines are thought to represent postsynaptic sites for synaptic transmission. Formation of synapses, including the presynaptic and postsynaptic elements during development, is thought to depend upon both genetic and environmental influences. Initial establishment of synaptic connection occurs independent of environmental factors. However, research over the past decade has provided evidence for extensive steroid-dependent plasticity in the adult brain. Neuroactive steroid compounds apply to those steroids that have biological effects or activities in nervous system which may originate from various sources: a) the endocrine system mainly adrenal and sexual glands^{34, 35}; b) the nervous system itself, where steroids can be synthesized de novo^{36, 37, 21}; and c) exogenous from various environmental sources.³⁸ Original studies demonstrating different effects of steroids on the brain were carried out in rodents, but more recently these studies have been extended to primates, including human.^{8, 21, 39} Sex steroids regulate neural function by influencing neurons via steroid receptors, which have been defined in many parts of the brain, whether involving reproductive behavior or not.^{21, 40} Neurons responsive to gonadal steroids may have at least three types of steroids receptors acting through different mechanisms: 1) the well-known intracellular steroid receptors responsible for steroidal genomic action. ^{41
– 43} Once activated will act as transcription factors and may trigger gene expression, and are typically responsible for late and long-lasting neuronal response^{5, 44, 45};2) effects mediated by membrane receptors for nongenomic actions; and 3) the recently described membrane steroidal receptors^{46, 47}which may be coupled directly to membrane ion channels or second messengers system that elicit rapid and transitory changes on neural structure.^{48 – 50} Steroidal effects on the serotonergic nuclei, appears to be linked to estrogen- and progesteron-sensitive neurons in the midbrain raphe nuclei.

Figure 5. Photomicrograph montage dendritic spines from dorsal raphe neurons of ovariectomized (OVX) animals (A) and control (B). Observe the decrease of dendritic spine in A compared to B; arrows indicate dendritic spines in B. Scale bar, 10 μm (applies to both A and B).

Figure 6. Camera Lucida drawings of the selected dendritic spines partially depicted in Figure 5. Observe the decrease in dendritic spines with pronounced heads in B compared to A. Scale bar, 10 μm (applies to both A and B).

From the neuroplastical point of view, it is generally accepted that the dendritic spines are the most plastic site in the neuron, so that any changes in their number and shape via possibly above-mentioned mechanisms, during development or in response to other pathological factors, have been positively correlated with changes in synaptic plasticity. It is thereby reasonable that the spine changes observed in this report reflected changes in synapses and thus changes in behaviors. Clinically, it is acceptable that the neuronal and morphological changes induced by physiological and pathological sex steroidal deprivation would affect a number of processes, such as psychotic and cognitive functions.

In summary, cross talk between ovarian hormones and CNS play important roles in regulating behaviors by regulating neural structure and function. The authors conclude that sex hormones drive structural plasticity in dendrites, which may underlie reorganization of neuronal circuits during plasticity.

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	Original Article
	Original Article