Helena Pinos

The development of sex differences in the locus coeruleus of the rat

Development of sex differences in the locus coeruleus (LC) is investigated. The LC is a sexually dimorphic structure in which the female manifests a larger volume and greater number of neurons than do males. Male and female Wistar rats were sacrificed on prenatal days (E) 16 and 20 and postnatally (P) on days 1, 3, 7, 15, 35, 45, 60, and 90. Male and female rats show a continuous increase in the number of neurons after birth that stops in the males by P45 and in females by P60. These findings point out the existence of different patterns of development in male and female rats and may suggest that sex differences could be established because of the existence of a differential period of neurogenesis in both sexes in the postpubertal period.

The role of the androgen receptor in CNS masculinization

The medial posterior region of the bed nucleus of the stria terminalis (BSTMP) and the locus coeruleus (LC) show opposite patterns of sexual dimorphism. The BSTMP in males is greater in volume and number of neurons than in females (male > female) while in the LC, the opposite is true (female > male). To investigate the possible role of the androgen receptor (AR) in the masculinization of these two structures, males with the testicular feminization mutation (Tfm) were compared to their control littermate males. No differences were seen in the number of neurons of the BSTMP between Tfm and their control littermate males, while in the LC, Tfm males have a greater number of neurons than their control littermate males. These results show that the AR is involved in the control of neuron number in the LC but not in the BSTMP. Results based on the LC suggest that when females have a larger brain area than males, masculinization in males may be achieved through the AR, with androgens perhaps decreasing cell survival.

Morphometrical and neurochemical changes in the anteroventral subdivision of the rat medial amygdala during estrous cycle

The anteroventral subdivision of the medial amygdala (MeAV) is one of the vomeronasal structures involved in the control of hormonally dependent behaviors such as sexual and agonistic behaviors in rats. The present study investigates some anatomical and neurochemical parameters of this nucleus (volume, number of neurons, number of glial elements, and of NADPH-diaphorase-positive neurons) in females in two estrous cycle phases (diestrous and estrous) and in males. We also investigate the possible existence of adult neurogenesis in this nucleus in the females. Results showed that volume and estimated number of Nissl-stained neurons in the MeAV vary with the estrous cycle phase: estrous females have greater values than diestrous females. As a consequence of these variations, there is a transient sex difference between males and diestrous females. Two subpopulations of NADPH-diaphorase-positive neurons were detected: intensely stained and medium stained. The intensely stained neurons were more numerous in the estrous than the diestrous females. Neither BrdU nor GFAP inmunostaining revealed significant differences between the two groups, suggesting that adult cell generation, i.e., increases in the number of glial elements, has no significant role in the changes detected in the number of Nissl-stained sections. In conclusion, the MeAV shows functional diergism, due to plastic changes in the female rat brain probably linked to the increase of estradiol during estrous. Finally, these changes are probably functionally related to changes in the behaviors that are controlled through this nucleus.

Sexual dimorphism in the vomeronasal system of the rabbit.

Studies have shown that the vomeronasal system (VNS), an olfactory neural network that participates in the control of reproductive physiology and behavior, is sexually dimorphic in the rat. These works have also shown two main characteristics of brain sexual dimorphism: (a) dimorphism appears in neural networks related to reproduction and (b) it can present two morphological patterns: one in which males present greater morphological measures than females (male > female) and another in which the opposite is true (female > male). The present work extends the hypothesis to the rabbit, as a representative species of Lagomorpha. In addition, the locus coeruleus (LC), which is known to send rich noradrenergic projections to VNS structures, was also studied. Sex differences were found in: (a) the number of mitral, and dark and light granule cells (female > male) of the accessory olfactory bulb (AOB); (b) the medial amygdala (Me) and its dorsal (Med) and ventral (Mev) subdivisions, males showing greater values than females in volume and number of neurons, while in the posteromedial cortical amygdala (PMCo or C(3)), females show greater density of neurons than males and (c) the posteromedial division of the bed nucleus of the stria terminalis (BSTMP) in which males have more neurons than females. No sex differences were seen in the bed nucleus of the accessory olfactory tract (BAOT) and the LC. These results evidence that, as it was observed in rodents, sex differences are also seen in the VNS of Lagomorpha and that these sex differences present the two morphological patterns seen in Rodentia. Differences between orders are discussed with respect to the species-specific physiological and behavioral peculiarities.

The expression of brain sexual dimorphism in artificial selection of rat strains

Central nervous system sex differences have two morphological patterns. In one pattern, males show larger measurements (volume, number of neurons) than females (male > female; m > f) and, in the other, the opposite is true (female > male; f > m). The bed nucleus of the stria terminalis (BST) is a unique model for the study of sex differences because it has dimorphic and isomorphic subdivisions, with the former showing the two sexually differentiated morphological patterns. Meanwhile, other CNS structures, like the locus coeruleus (LC), present the f > m pattern. The philogenetic maintenance of the two patterns of sexual differentiation can help to disentangle the functional meaning of sex differences. Laboratory rat strains, whether albino or pigmented, descend from the Wistar strain through artificial selection. The present work compares the BST and LC of Wistar and Long-Evans rats. The medial posterior subdivision of the BST (BSTMP) is sexually dimorphic (m > f pattern) in the original (Wistar) and derived (Long-Evans) strains, while the lateral anterior and medial anterior subdivisions of the BST and the LC only present sex differences (f > m pattern) in the ancestor Wistar strain. Isomorphic BST regions are the same in both strains. The fact that the BSTMP, which is implicated in male copulatory behavior, is sexually dimorphic in both strains, as well as in other species, including humans, indicates the relevance of this structure in male sexual behavior in mammals.

NADPH-diaphorase activity increases during estrous phase in the bed nucleus of the accessory olfactory tract in the female rat.

We investigated the presence of nitric oxide in the bed nucleus of the accessory olfactory tract (BAOT) in males, diestrous females and estrous females using NADPH-diaphorase. Our results demonstrate a significant increase in the density of the medium-stained cells in the estrous female rats suggesting that during estrous a specific subpopulation of nitrinergic cells are activated in the BAOT. This might be related to the physiological and behavioral changes that occurs in estrous.

Effects of undernourishment on the hypothalamic orexinergic system

The present study examined the effects of a severely restricted diet during the pre- and postnatal periods with later nutritional rehabilitation on orexin hypothalamic neurons in male and female Wistar rats. Immunocytochemistry was used to reveal orexin-immunoreactive (orexin-ir) cells in the ventromedial hypothalamus (VMH), dorsomedial hypothalamus (DMH), lateral hypothalamic area (LH) and the perifornical nucleus (PF). Dietary restriction decreased the number of orexin-ir cells in the LH, whereas DMH or PF orexin-ir populations were not affected in either male or female rats. Nutritional rehabilitation resulted in a differential recovery that depended on the period during which rehabilitation occurred and on the sex of the animal. In summary, our study suggests that the hypothalamic nuclei implicated in eating behavior present a differential vulnerability to adverse environmental conditions during development. Specifically, among the studied nuclei only the LH orexin-ir cells were sensitive to severe food deprivation during development in male and female rats. These results suggest that starvation interferes with developmental events that occur during CNS sexual differentiation.

The effects of partial and complete masculinization on the sexual differentiation of nuclei that control lordotic behavior in the male rat

Male rats, under certain experimental conditions, may show lordosis, the typical expression of female sexual receptivity. This work studies the sexual morphological pattern of facilitatory and inhibitory structures that control lordosis. Three groups of males were neonatally subjected to a gradient of androgen exposure (castrated plus injected oil (GxM+oil); castrated plus androstenedione treated (GxM+AND); and sham operated [CM]); a group of control females (CF) was also added. Lordotic response after these different hormonal and neonatal surgical treatments, as well as the volume or number of neurons in facilitatory (ventromedial nucleus of the hypothalamus [VMN]) and inhibitory (the intermediate region of the lateral septum [LSi] and accessory olfactory bulb [AOB]) nuclei involved in lordosis was studied in adults. The inhibition of lordosis in the males seems to be associated to the neonatal presence of testosterone and the consequent masculinization of the VMN, VMNvl, LSi and AOB. It is suggested that one of the functions of the sex differences consistently seen in these structures might be to inhibit the lordosis response in the male.

Early undernutrition decreases the number of neurons in the locus coeruleus of rats

Abstract The effects of perinatal undernutrition on the number of neurons and apoptotic cells of the locus coeruleus (LC) of female and male rats at postpartum days 7, 12, 20, 30 and 60 were studied. Undernutrition reduces the number of neurons in both sexes without affecting cell death, as indicated by the ratio of apoptotic cells to neurons. The data suggest that in the undernourished groups lower rates of neurogenesis and proliferation (neurogenetic/proliferation rates) might avoid these animals achieving the number of LC neurons as in the control subjects. Although food restriction in both sexes apparently provokes the loss of cells, the effect does not appear to be equal in females and males, as shown by post weaning food rehabilitation. The results suggest that severe food deprivation may interfere with the ontogenetic processes underlying neuronal differentiation of the LC. Morphological damage in the LC due to undernutrition might alter the physiology of sexual and/or feeding behaviours in which this structure is implicated.

Role of oestrogen receptors on the modulation of NADPH-diaphorase-positive cell number in supraoptic and paraventricular nuclei of ovariectomised female rats.

Modulation of the nitric oxide producing system (demonstrated via the NADPH‐diaphorase histochemical reaction) by oestradiol has been established in several structures of the rat brain. The present study aimed to explore the possible regulation of NADPH‐diaphorase activity by oestradiol in neurones of the supraoptic (SON) and paraventricular (PVN) nuclei and the role of oestrogen receptors (ERα and ERβ) in this regulation. Adult ovariectomised rats were divided into six groups and injected either with vehicle or a single dose of oestradiol, a selective ERα agonist‐PPT [4,4′,4″‐(4‐propyl‐[1H]‐pyrazole‐1,3,5‐triyl)trisphenol], a selective ERβ agonist‐DPN [2,3‐bis(4‐hydroxyphenyl)‐propionitrile], a selective ERα antagonist‐MPP [1,3‐bis(4‐hydroxyphenyl)‐4‐methyl‐5‐[4‐(2‐piperidinylethoxy)phenol]‐1H‐pyrazole dihydrochloride] or a selective ERβ antagonist‐PHTPP (4‐[2‐phenyl‐5,7‐bis(trifluoromethyl)pyrazolo[1,5‐a]pyrimidin‐3‐yl]phenol). The number of NADPH‐diaphorase positive elements in the SON and the PVN was modulated by both ERs but, depending on the nucleus, ERα and ERβ ligands induced different effects. These results suggest that the regulation of nitrergic system by ERs may play a role in the control of oestrogen‐dependent physiological mechanisms regulated by the SON and the PVN.