Ovarian Steroid Regulation of Estrogen and Progesterone Receptor Messenger Ribonucleic Acid in the Anteroventral Periventricular Nucleus of the Rat

The anteroventral periventricular nucleus of the preoptic region (AVPV) represents a key site for hormonal feedback on gonadotropin secretion. It plays a critical role in the neural control of luteinizing hormone secretion and contains high densities of neurons that express receptors for estrogen and progesterone. In this study in situ hybridization was used to examine the expression of mRNAs encoding the estrogen (ER) and progesterone (PR) receptors in the AVPV during the estrous cycle. ER gene expression fluctuated during the cycle with the lowest levels of ER mRNA observed in animals killed on the afternoon of proestrus, and the highest levels present in animals killed during metestrus. This apparent inverse relationship between circulating levels of estradiol (E2) and ER mRNA levels in AVPV neurons was supported by the observation that treatment of ovariectomized rats with E2 suppressed expression of ER mRNA in the AVPV. The influence of progesterone (P4) on ER expression was less pronounced, but a significant increase in ER mRNA in the AVPV was detected 3 h after treatment with P4. In contrast, PR mRNA levels were highest in the AVPV during diestrus and lowest on the morning of proestrus suggesting that PR expression in the AVPV is regulated in a complex manner that may reflect the combined regulatory effects of E2 and P4. E2 treatment caused a dramatic induction of PR mRNA in the AVPV, but P4 did not affect PR mRNA expression acutely, although PR mRNA appears to be attenuated in the AVPV 27 h after P4 treatment. These findings suggest that ovarian steroid hormones regulate ER and PR gene expression in the AVPV during the estrous cycle, which may represent molecular events that contribute to cyclic changes in the responsiveness of AVPV neurons to steroid hormones.     

Sexually Dimorphic Expression of Androgen Receptor Immunoreactivity by Somatostatin Neurones in Rat Hypothalamic Periventricular Nucleus and Bed Nucleus of the Stria Terminalis

Gonadal steroids exert important regulatory actions on the hypothalamic neurones regulating growth hormone secretion and are believed to play a role in generating its sexually dimorphic pattern of secretion. Recent evidence indicates that estrogen actions on one of these neural populations, the periventricular somatostatin (SOM) neurones, are likely to be indirect as they do not possess nuclear estrogen receptors in either sex although androgen receptors (ARs) have been reported within these cells in male rats. The present study has used double-labelling immunocytochemistry procedures to examine whether sex differences exist in AR expression by SOM neurones located in the periventricular nucleus and bed nucleus of the stria terminalis (BNST). Within the hypothalamus, SOM-immunoreactive neurones were found concentrated in the periventricular nucleus while both anterior and posterior divisions of the BNST contained scattered populations of SOM cells. Cells immunoreactive for the AR were detected in all of these areas. Although the intensity of AR cell nuclei staining was equivalent in males and females in regions such as the lateral septum, the intensity of AR staining in many individual cells of the periventricular nucleus and posterior BNST of the female was reduced when compared with the male. Double-labelling experiments revealed that approximately 40% of periventricular SOM neurones expressed AR immunoreactivity in the male compared with significantly (P<0.01) fewer cells in the female (～7%). In the BNST, double-labelled cells were only detected within the principle encapsulated, interfascicular and transverse nuclei of its posterior division. Approximately 60% of SOM cells in these nuclei expressed AR immunoreactivity in the male while significantly (P<0.01) fewer did so in the female (～25%). These results indicate that substantial sex differences exist in AR expression by SOM neurones in both the periventricular nucleus and BNST. Such differences in AR expression by periventricular SOM cells may contribute to their sexually dimorphic nature and, consequently, sex differences in growth hormone secretion.     

Induction of Substance P-immunoreactivity by Estrogen in Neurons Containing Estrogen Receptors in the Anterovental Periventricular Nucleus of Female but not Male Rats

Effects of gonadal steroids on numbers of neurons containing estrogen receptor (ER) and/or substance P (SP) were examined in the anteroventral periventricular nucleus (AVPV) of female and male rats by double-labeling immunohistochemistry employing antibodies specific for ER and SP. Animals were gonadectomized and received subcutaneously either oil alone (Control group), sequential injections of estradiol benzoate and oil (EB + Oil group), or those of EB and progesterone (EB + P group). In the female control rat, a large population of ER-immunoreactive (IR) cells were found clustered throughout the AVPV. They were counted more than 2,000 in total of 4 sections in this nucleus. On the contrary, SP-IR neurons were scarcely observed in the same area of this group. Administration of estrogen to female animals decreased the total number of ER-IR cells to 67% of the control group. In contrast to the supressive effect of estrogen to its own receptor, it induced SP-IR neurons in the AVPV of the female. Approximately 50–80 SP-IR neurons were counted in the 4 sections, and 59% of these neurons expressed ER-IR material in their nuclei. In the female EB + P group, the number of ER-IR neurons also decreased to 79% of the control group. Although the number of SP-IR neurons in this group decreased to 32% of that in the EB + Oil group, a ratio of coexistence of ER-IR material in these neurons increased to 75%. The male control group contained a smaller population of ER-IR cells relative to the female control (1497 vs 2143). SP-IR neurons were rarely observed as were in the female control. Administration of estrogen to the male also decreased the number of ER-IR cells in a manner similar to that in the female. However, unlike the female, the steroid failed to induce the SP-IR neurons in the male. These results demonstrate sexual dimorphism in the AVPV not only in the number of ER-IR neurons but also in the responsiveness of SP neurons to estrogen. They further provide anatomical evidence that a subset of SP neurons are regulated by estradiol in estrogen sensitive neurons in the female rat. The data also suggest that this peptide is involved in mechanisms of luteinizing hormone surge by mediating actions of gonadal steroids in the AVPV.     

Neurotensin-lmmunoreactive Neurons in the Rat Medial Preoptic Area are Oestrogen-Receptive

The identity of neurons influenced by oestrogen is critical for the understanding of ovarian steroid actions in the brain. The medial preoptic area (MPOA) contains one of the largest oestrogen-receptive cell populations in the rat brain and participates in several oestrogen-dependent functions, including the regulation of luteinizing hormone (LH) secretion and sexual behaviour. Using double immunostaining procedures with antibodies specific for the oestrogen receptor and neurotensin, a neuropeptide implicated in the regulation of LH secretion within this area, we found that approximately half of the neurotensin-immunoreactive neurons in the MPOA also displayed immunoreactivity for the oestrogen receptor. We estimate that oestrogen-receptive neurotensin neurons represent 5% to 12% of all oestrogen receptor-positive cells in the MPOA. Our results provide morphological evidence that neurotensin mediates oestrogen-dependent mechanisms within the brain and suggest that oestrogen may act through preoptic neurotensin neurons to aid in the generation of the LH surge.     

Testosterone or Oestradiol Implants in the Medial Preoptic Area Induce Mating in Noncopulating Male Rats

Noncopulating (NC) male rats are those males that do not mount, intromit or ejaculate when repeatedly tested with receptive females. The lack of sexual behaviour in these males is not associated with alterations in testosterone or oestradiol (E₂) plasma concentrations. Instead, it has been shown that androgen receptors are higher and oestrogen receptors are lower in the medial preoptic area (MPOA) of NC male rats than those observed in copulating (C) male rats. We have also observed reduced aromatase activity in the MPOA (but not in other brain regions) of NC male rats. The aim of the present study was to determine whether testosterone or E₂ implants in the MPOA of NC male rats could induce sexual behaviour. Accordingly, in Experiment 1, we evaluated the long‐term effects of testosterone or E₂ implants in the MPOA, the ventromedial nucleus of the hypothalamus or the medial amygdala with respect to inducing sexual behaviour in castrated C male rats. Male rats were bilaterally implanted with a guide cannula, either empty or containing testosterone or E₂. Starting 1 week later, all male rats were mated once weekly for 5 months. As described previously, the site where hormone implants most consistently induced sexual behaviour in castrated C male rats was the MPOA. Experiment 1 extended these findings showing that the males continued mating even 5 months after the implant. In the second experiment, NC males were implanted in the MPOA with a guide cannula empty or filled with testosterone or E_2. One week after the testosterone or E₂ implant, the percentage of males that mounted and intromitted started to increase and, 5 weeks after the implant, 50% of the subjects displayed mounts and intromissions. All NC males implanted with testosterone ejaculated consistently from week 11 after the implant until the end of testing (5 months), whereas all subjects implanted with E₂ ejaculated from week 16 after the implant until the end of testing. These results support the hypothesis that, in the MPOA of NC male rats, there is a hormonal alteration associated with the lack of sexual behaviour.     

Estrogenic Induction of NADPH- Diaphorase Activity in the Preoptic Neurons Containing Estrogen Receptor Immunoreactivity in the Female Rat

Nitric oxide and estrogen have been shown to play a critical role in the control of female reproductive function. In order to determine an anatomical relationship between nitric oxide generating neurons and estrogen target neurons, NADPH-diaphorase histochemistry was combined with estrogen receptor immunohistochemistry in the female medial preoptic area. While only a few weakly stained neurons for NADPH-diaphorase were found in ovariectomized control rats, a drastic increase in NADPH-diaphorase activity was observed in the medial preoptic nucleus of estradiol-treated ovariectomized animals. The total number of NADPH-diaphorase neurons in the estradiol-treated group increased three-fold relative to controls, and more than 80% of those neurons contained estrogen receptor-immunoreactivity in their nuclei. Since neuronal NADPH-diaphorase is nitric oxide synthase, the present result suggests that nitric oxide synthase activity can be positively regulated by estradiol in neurons containing estrogen receptor in the female medial preoptic nucleus.     

Preoptic formation of 17β-oestradiol is influenced by behavioural stimuli in the dove

In vitro study of testosterone (T) metabolism shows that formation of 17β-oestradiol (E₂) in the preoptic area is higher in male doves exposed to behavioural stimuli from interacting pairs than in visually isolated males or males that have interacted with females. Formation of 5-dihydrotestosterone, 5β-dihydrotestosterone and 5β-androstane-3,17β-diol was similar in all groups, indicating that exposure to stimuli from interacting pairs influences aromatization of T and not other pathways of androgen metabolism. The results suggests that sexual stimuli, which do not involve tactile cotact between male and female, affect local E₂ formation in the brain.     

Mating-lnduced FOS-Like Immunoreactivity in the Rat Forebrain: A Sex Comparison and a Dimorphic Effect of Pelvic Nerve Transection

Previous research has shown that mating induces the expression of the immediate-early gene, c-fos, as detected by the increased presence of nuclear FOS-like immunoreactivity (FOS-IR), in specific forebrain regions of both male and female rats. In the male both olfactory/vomeronasal (O/V) and genital/somatosensory (G/S) inputs appear to contribute to the neural FOS response to mating whereas in the female G/S input carried by the pelvic nerves appears to mediate the forebrain FOS response. To date, however, no direct sex comparison of the mating-induced forebrain FOS response has been made in rats maintained under the same steroidal conditions nor has the contribution of afferent sensory input from the pelvic nerves been assessed in males. We first compared the level of FOS-IR in brain regions of mated and unpaired gonadectomized male and female rats given 5μg/kg estradiol benzoate (EB) for 7 days and 500 μg progesterone (P) 4 h prior to testing. One h after experiencing 1 ejaculation, both sexes showed increased FOS-IR in the medial preoptic area (MPOA), bed nucleus of the stria terminalis (BNST), medial amygdala, the ventro-lateral portion of the ventromedial nucleus of the hypothalamus (VMN), and the midbrain central tegmental field (CTF). This increase was significantly greater in the MPOA and medial amygdala of mated females than of males. Bilateral transection of the pelvic nerves significantly attenuated the increase in FOS-IR after mating in the CTF of male rats and in the MPOA, BNST, VMN, medial amygdala and CTF of females. Thus, following mating there is no sex difference in the brain regions which express c-fos, but there is a dimorphism in the contribution of afferent information conveyed by the pelvic nerves to the mating-induced FOS response. The neural FOS response of the female to mating is heavily dependent upon the G/S afferent inputs carried by the pelvic nerves whereas the male's neural c-fos response may depend on O/V input plus G/S input conveyed via other afferent pathways such as the pudendal nerves.     

Infusions of Lidocaine into the Amygdala, but not the Preoptic Area, Block Pseudopregnancy in the Rat

In the rat, vaginocervical stimulation (VCS) received during mating is required for the subsequent expression of 10–12 days of twice-daily prolactin surges that are necessary for pregnancy or pseudopregnancy (PSP). This temporal separation of sensory stimulus and neuroendocrine response suggests that a mnemonic of the vaginocervical stimulation is created in the brain that triggers and sustains the daily prolactin surges. We investigated the possible involvement of the medial preoptic area (mPOA) and the medial amygdala (mAMYG) as potential neural sites involved in the processing of this neuroendocrine arc. Cycling female rats were bilaterally implanted with intracerebral cannulae in either the mPOA or mAMYG. On proestrus, females were manually palpated to confirm sexual receptivity and then received bilateral infusions of either the local anesthetic lidocaine, the Ca⁺⁺ channel blocker, verapamil, or phosphate-buffered saline (PBS) into either brain site before or both before and after receipt of 15 intromissions from an experienced male. Unmated control females received comparable infusions of lidocaine or verapamil, and were placed in the empty test arena for 10 min. Infusions consisted of either a single bilateral infusion 15 min before mating (Expt. 1), bilateral infusions both 15 min before and after mating (Expt. 2) or eight bilateral infusions separated by 30 min intervals spanning a period beginning 45 min before and ending 2 h 45 min after mating (Expt. 3). None of the lidocaine infusions into the mPOA prevented the establishment of PSP, and neither verapamil infusions into the mAMYG nor the shorter-term neural block (i.e. single or double lidocaine infusions) of the mAMYG prevented mating-induced PSP. However, the longer-term neural block (i.e. multiple lidocaine infusions) of the mAMYG significantly reduced the incidence of PSP. These data support previous findings that the mAMYG receives sensory input from VCS, and suggest that the mAMYG is a site at which a mnemonic of VCS is established.     

Pre- and postnatal influence of testosterone propionate and diethylstilbestrol on differentiation of the sexually dimorphic nucleus of the preoptic area in male and female rats

The volume of the sexually dimorphic nucleus in the preoptic area (SDN-POA) of the rat brain is several fold larger in males than in females. When female rats were treated pre- and postnatally with testosterone propionate (TP) or with diethylstilbestrol (DES) they became anovulatory and their SDN-POA developed equivalent in size to that of normal males. Identical treatment of male rats resulted in deficient testicular development, but had no influence on SDN-POA volume. The results indicate that the gross morphological sex difference in SDN-POA volume can exclusively be controlled by the hormonal environment during the critical period of sexual brain differentiation, and that non-steroidal estrogens are just as effective as convertible androgens in stimulating SDN-POA differentiation.     

The Luteinising Hormone Surge‐Generating System is Functional in Male Goats as in Females: Involvement of Kisspeptin Neurones in the Medial Preoptic Area

A luteinising hormone (LH) surge is fundamental to the induction of ovulation in mammalian females. The administration of a preovulatory level of oestrogen evokes an LH surge in ovariectomised females, whereas the response to oestrogen in castrated males differs among species; namely, the LH surge‐generating system is sexually differentiated in some species (e.g. rodents and sheep) but not in others (e.g. primates). In the present study, we aimed to determine whether there is a functional LH surge‐generating system in male goats, and whether hypothalamic kisspeptin neurones in male goats are involved in the regulation of surge‐like LH secretion. By i.v. infusion of oestradiol (E₂; 6 μg/h) for 16 h, a surge‐like LH increase occurred in both castrated male and ovariectomised female goats, although the mean peak LH concentration was lower and the mean peak of the LH surge was later in males compared to females. Dual staining with KISS1 in situ hybridisation and c‐Fos immunohistochemistry revealed that E₂ treatment significantly increased c‐Fos expression in the medial preoptic area (mPOA) KISS1 cells in castrated males, as well as ovariectomised females. By contrast, dual‐labelled cells were scarcely detected in the arcuate nucleus (ARC) after E₂ treatment in both sexes. These data suggest that kisspeptin neurones in the mPOA, but not those in the ARC, are involved in the induction of surge‐like LH secretion in both male and female goats. In summary, our data show that the mechanism that initiates the LH surge in response to oestrogen, the mPOA kisspeptin neurones, is functional in male goats. Thus, sexual differentiation of the LH surge‐generating system would not be applicable to goats.     

Sexual dimorphism in the preoptic/anterior hypothalamic area of ferrets: Effects of adult exposure to sex steroids

The organization of neuronal cell bodies in the caudal preoptic area (POA) and rostral anterior hypothalamic area (AH) was studied in Nissl-stained brain sections from adult male and female ferrets. Computer-assisted image-analysis procedures were developed to help estimate the areas of cellular density and the sizes of individual perikarya. At the junction of the POA and AH, a bilateral dorsal-medial group of neurons was apparent only in male ferrets (dorsal nucleus). At the same coronal level, a ventral-medial group of neurons was apparent bilaterally in both males and females (ventral nucleus). The mean somal area of cells in the dorsal nucleus of males was significantly greater than the mean somal area of cells in the corresponding dorsal region of females or in the ventral nucleus of both sexes. The dorsal nucleus was clearly discernible in adult males regardless of their hormonal status, although cells in the dorsal nucleus were larger in intact breeding males or gonadectomized males given testosterone, estradiol or dihydrotestosterone than in gonadectomized males given no gonadal hormones or given progesterone. Neither the grouping of large cells nor the steroid-induced increase in cell size, characteristic of the male dorsal nucleus, was seen in the comparable dorsal region of females. The sex difference in cellular organization observed in the ferret at the junction of the POA and AH is the first difference of this type to be seen in the POA/AH of a non-rodent mammalian species. Its identification will, hopefully, aid in the analysis of the neural mechanisms that control various sex-specific behaviors in this species.     

Effects of prenatal stress on differentiation of the sexually dimorphic nucleus of the preoptic area (SDN-POA) of the rat brain

The present study was designed to determine the effects of prenatal malnutrition or environmental stress on the development of the sexually dimorphic nucleus of the preoptic area (SDN-POA). Pregnant rats were divided into a control group and two treatment groups (immobilization-illumination-heat or environmental stress, and nutritional stress). The two forms of stress were administered during the third trimester of gestation (days 14-20). Male and female offspring were sacrificed at birth, 20, and 60 days postnatally. The cross-sectional area of the SDN-POA was identified under light microscopy and was measured. The data confirm previous studies by showing a significant sex difference in the SDN-POA between control male and female rats. Prenatally stressed males sacrificed 20 and 60 days after birth showed SDN-POA areas 50% smaller than the nuclear areas of control males. The size of the SDN-POA of female offspring, however, was not significantly altered by prenatal treatments.     

The effect of neonatal testosterone on specific male and female patterns of phosphorylated cytosolic proteins in the rat preoptic-hypothalamus, cortex and amygdala

The original evidence that spared cutaneous nerves will sprout following partial denervation of skin was obtained in adult rabbits, in which nociceptive function returned to the deprived areas. Recently we reported that in the adult rat intact touch-sensitive (low-threshold mechanosensory) nerves fail to establish new functional endings in adjacent denervated skin. We have now investigated low-threshold mechanosensory nerves in the skin of adult rabbits after partially denervating the ear or hind limb; the sprouting of new functional endings into neighboring skin would be revealed by an expansion of the low-threshold receptive fields of cutaneous nerves (the areas of skin from which impulses could be evoked by stroking with a bristle). The results show that intact low-threshold mechanosensory nerves do not establish functional endings in adjacent denervated skin in the adult rabbit. These findings, together with those now reported for the cat as well as the rat, support the conclusion that possibly in all adult mammals intact low- and high-threshold nerves differ in their ability to respond to the presence of adjacent denervated skin by sprouting.     

Age- and Hormone-Regulation of N-Methyl-d-Aspartate Receptor Subunit NR2b in the Anteroventral Periventricular Nucleus of the Female Rat: Implications for Reproductive Senescence

Glutamate, acting through its N‐methyl‐d ‐aspartate (NMDA) and non‐NMDA receptors in the hypothalamus, regulates reproductive neuroendocrine functions via direct and indirect actions upon gonadotrophin‐releasing hormone (GnRH) neurones. Previous studies indicate that the NMDA receptor subunit NR2b undergoes changes in protein and gene expression in the hypothalamus in general, and on GnRH neurones in particular, during reproductive ageing. In the present study, we examined whether the NR2b‐expressing cell population, both alone and in association with the NR1 subunit (i.e. the latter subunit is necessary for a functional NMDA receptor), is altered as a function of age and/or steroid hormone treatment. Studies focused on the anteroventral periventricular (AVPV) nucleus of the hypothalamus, a region critically involved in the control of reproduction. Young (3–5 months), middle‐aged (9–12 months), and aged (approximately 22 months) female rats were ovariectomised and, 1 month later, they were treated sequentially with oestradiol plus progesterone, oestradiol plus vehicle, or vehicle plus vehicle, then perfused. Quantitative stereologic analysis of NR2b‐immunoreactive cell numbers in the AVPV showed an age‐associated decrease in the density of NR2b‐immunoreactive cells, but no effect of hormone treatment. In a second study, immunofluorescent double labelling of NR2b and NR1 was analysed by confocal microscopy of fraction volume, a semi‐quantitative measure of fluorescence intensity. No effect of ageing was detected for immunofluorescent NR1 or NR2b alone, whereas the NR2b fraction volume increased in the oestradiol plus vehicle group. With ageing, the fraction volume of the NR2b/NR1‐colocalised subunits increased. Together with the stereology results, this suggests that, although fewer cells express the NR2b subunit in the ageing AVPV, a greater percentage of these subunits are co‐expressed with NR1. Our results suggest that the subunit composition of NMDA receptors in the AVPV undergo both age‐ and hormonal‐regulation, which may be related to previous observations of changes in functional responses of reproductive neuroendocrine systems to NMDA receptor modulators with ageing.     

Effects of Paced Mating on c-fos Gene Expression in the Female Rat Brain

When estrous female rats control or pace (P) their sexual contacts with males, several neuroendocrine and behavioral responses to mating occur that are not observed or are greatly attenuated after nonpaced mating. The present study examined whether the distribution and amount of FOS immunoreactivity (FOS-IR) induced in brain by mating would be altered in females receiving paced rather than nonpaced mating stimulation. In the first experiment, females received 5 or 15 intromissions during paced mating tests (5P and 15P), 5 or 15 intromissions during nonpaced mating tests (5NP and 15NP), 15 mounts-without-intromission (MO) or remained in their homecages (HC). Selective increases 1 h after paced mating stimulation were observed in the posterodorsal medial amygdala (MePD), where significantly more FOS-IR cells were present in the 5P and 15P groups than in the respective NP groups. The 5P, 5NP and 15NP had significantly more FOS-IR than the HC, MO, and 5NP groups, and the 5P group had levels of FOS-IR which were equivalent to that seen in the 15NP group. In the posteromedial portion of the bed nucleus of the stria terminalis (BNSTpm) and the ventrolateral portion of the ventromedial nucleus of the hypothalamus (VMHvl), paced mating induced significantly greater numbers of FOS-IR cells than did either MO or HC treatments; increases induced by nonpaced mating were not statistically greater than HC controls. No differences between groups were seen in the medial preoptic area (mPOA). In the second experiment, experimentally lengthening the interintromission interval (III) as well as increasing the intromission duration to mimic the characteristics of paced mating, resulted in significant increases in FOS-IR in the MePD but not in the other three brain regions. These results demonstrate that paced mating is more effective in inducing c-fos expression than nonpaced mating, and that the MePD is particularly sensitive to differing characteristics of the mating stimuli received.     

Interaction between Dopamine‐ and Isotocin‐Containing Neurones in the Preoptic Area of the Catfish,Clarias batrachus: Role in the Regulation of Luteinising Hormone Cells

Apart from gonadotrophin‐releasing hormone (GnRH) and dopamine (DA), oxytocin has emerged as an important endogenous agent that regulates reproduction. Although the interaction between these factors has been extensively studied in mammals, parallel information in teleosts is much limited. We studied the organisation of tyrosine hydroxylase (TH; a marker for dopamine) and isotocin neurones in the preoptic area (POA) and hypothalamus of the catfish, Clarias batrachus and its implication in the regulation of luteinising hormone (LH) cells in the pituitary. Nucleus preopticus periventricularis (NPP), a major dopaminergic centre in the brain, consists of anterior (NPPa) and posterior (NPPp) subdivisions. Using retrograde neuronal tracing, we found that majority of the DA neurones in NPPa, but none from NPPp, project to the pituitary. The nucleus preopticus (NPO) of C. batrachus contains a conspicuous assemblage of large isotocin‐positive neurones. It consists of a paraventricular subdivision (NPOpv) located on either side of the third ventricle and lies roughly sandwiched between the dopaminergic neurones of NPPa and NPPp. An additional subset of isotocin neurones was located above the optic chiasm in the supraoptic subdivision of the NPO (NPOso). Isotocin‐containing neurones in both the subdivisions of NPO were densely innervated by DA fibres. Superfusion of the POA‐containing brain slices with DA D₁‐like receptor agonist (SKF‐38393) resulted in significant increase in isotocin immunoreactivity in the NPOpv neurones; NPOso neurones did not respond. However, treatment with DA D₂‐like receptor agonist (quinpirole) reduced isotocin immunoreactivity in the NPOso, but not in the NPOpv. Thus, DA appears to differentially regulate the components of isotocinergic system. Isotocin fibres extend to the pituitary and terminate on LH cells and the superfused pituitary slices treated with isotocin caused significant reduction in LHβ‐immunoreactivity. An elaborate interplay between the DA and isotocin systems appears to be an important component of the LH regulatory system.     

Neurons in the Paraventricular Nucleus of the Hypothalamus that Project to the Sexually Dimorphic Lower Lumbar Spinal Cord Concentrate 3H-Estradiol in the Male Rat

The location and distribution of estradiol-concentrating neurons in the hypothalamus afferent to segments of lumbar spinal cord that contain the sexually dimorphic spinal nucleus of the bulbocavernosus (SNB) were determined by combining retrograde fluorescent tract tracing with steroid hormone autoradiography. Injections of Fluorogold were made into segments of L5-L6 of the spinal cord of adult male rats and 12 days later animals were castrated. One week following castration, males received injections of [³H]estradiol and were perfused. Their brains were then processed for steroid hormone autoradiography. Following exposure times of 11 to 12 months, autoradiograms were developed and the hypothalamus was analyzed for neurons that concentrate estradiol and project to the spinal cord. Numerous neurons in the hypothalamus projected to the spinal cord, specifically neurons in the paraventricular nucleus (PVN), the lateral hypothalamus and the dorsal area of the hypothalamus. Although many subnuclei of PVN, as well as lateral hypothalamus, contained Fluorogold labelled neurons and estradiol concentrating neurons, the majority of double labeled cells were found in the lateral parvocellular (lp) subnucleus of PVN. Approximately 30% of the neurons in the lp subnucleus that projected to spinal cord also concentrated estradiol. Up to one half of the estradiol-concentrating neurons in Ip sent axons to the lower lumbar spinal cord. These results suggest that some of the effects of gonadal steroid hormones on SNB development, plasticity and function may in fact, be indirect, via steroid-sensitive afferents.     

Tyrosine Hydroxylase in the Olfactory System,Forebrain and Pituitary of the Indian Major Carp,Cirrhinus cirrhosus: Organisation and Interaction with Neuropeptide Y in the Preoptic Area

Dopamine (DA) inhibits, whereas gonadotrophin‐releasing hormone (GnRH) stimulates, luteinisiing (LH) cells in the pituitary of some but not all teleosts. A reduction in the hypophysiotropic dopaminergic tone is necessary for the stimulatory effect of GnRH on LH cells. Neuropeptide Y (NPY) has emerged as one of the potent, endogenous agent that modulates LH secretion directly or indirectly via GnRH. Involvement of NPY in the regulation of hypophysiotropic DA neurones, however, is not known, but there is good evidence suggesting an interaction in the mammalian hypothalamus. DA neurones, identified by tyrosine hydroxylase (TH)‐immunoreactivity, were observed widely throughout the brain of the Indian major carp, Cirrhinus cirrhosus. The granule cells and ganglion cells of terminal nerve in the olfactory bulb, and cells in ventral telencephalon and preoptic area (POA) showed conspicuous TH immunoreactivity. In the POA, the nucleus preopticus periventricularis (NPP), divisible into anterior (NPPa) and posterior (NPPp) components, showed prominent TH‐immunoreactivity. The majority of TH neurones in NPPa showed axonal extensions to the pituitary and were closely associated with LH cells. The NPPa also appeared to be the site for intense interaction between NPY and DA because it contains a rich network of NPY fibres and few immunoreactive cells. Approximately 89.7 ± 1.5% TH neurones in NPPa were contacted by NPY fibres. Superfused POA slices treated with a NPY Y₂‐receptor agonist, NPY 13‐36 resulted in a significant (P < 0.001) reduction in TH‐immunoreactivity in NPPa. TH neurones in NPPa did not respond to NPY Y₁‐receptor agonist, [Leu³¹, Pro³⁴] Neuropeptide Y treatment. We suggest that, by inhibiting DAergic neurones in NPPa via Y₂‐receptors, NPY may contribute to the up‐regulation of the GnRH–LH cells axis. The microcircuitry of DA and NPY and their interaction in NPPa might be a crucial component in the central regulation of LH secretion in the teleosts.     

Ontogeny of opiate receptors in the rat medial preoptic area: Critical periods in regional development

Opiate receptor labeling was examined throughout the early postnatal period using autoradiography to localize and quantify [³H]naloxone binding to μ-type opiate receptors in the medial preoptic area (MPOA). This region begins to exhibit sexual dimorphism of volume and dendritic growth shortly after birth. A distinct concentration of opiate receptor labeling appears on postnatal day 3 in females: this labeling is directly associated with the sexually dimorphic nucleus of the preoptic area (SDN-POA). SDN-POA labeling becomes denser through postnatal day 10 in females and the densely labeled area increases in size to encompass and surround the SDN-POA. These changes in opiate receptor labeling occur only in females, since males show relatively uniform labeling across the region throughout the early postnatal period.The critical time of formation of dense MPOA opiate receptor labeling may be related to endogenous MPOA opioid function and to the critical period of dendritic growth of SDN-POA neurons. The timing of these critical periods and their focus in the SDN-POA are coincident. The possible role of MPOA opiate receptors in modulating growth of MPOA neurons is discussed.