Does Dynorphin Play a Role in the Onset of Puberty in Female Sheep?

Puberty onset involves increased gonadotrophin‐release (GnRH) release as a result of decreased sensitivity to oestrogen (E₂)‐negative feedback. Because GnRH neurones lack E₂ receptor α, this pathway must contain interneurones. One likely candidate is KNDy neurones (kisspeptin, neurokinin B, dynorphin). The overarching hypothesis of the present study was that the prepubertal hiatus in luteinising hormone (LH) release involves reduced kisspeptin and/or heightened dynorphin input. We first tested the specific hypothesis that E₂ would reduce kisspeptin‐immunopositive cell numbers and increase dynorphin‐immunopositive cell numbers. We found that kisspeptin cell numbers were higher in ovariectomised (OVX) lambs than OVX lambs treated with E₂ (OVX+ E₂) or those left ovary‐intact. Very few arcuate dynorphin cells were identified in any group. Next, we hypothesised that central blockade of κ‐opioid receptor (KOR) would increase LH secretion at a prepubertal (6 months) but not postpubertal (10 months) age. Luteinising hormone pulse frequency and mean LH increased during infusion of a KOR antagonist, norbinaltorphimine, in OVX + E₂ lambs at the prepubertal age but not in the same lambs at the postpubertal age. We next hypothesised that E₂ would increase KOR expression in GnRH neurones or alter synaptic input to KNDy neurones in prepubertal ewes. Oestrogen treatment decreased the percentage of GnRH neurones coexpressing KOR (approximately 68%) compared to OVX alone (approximately 78%). No significant differences in synaptic contacts per cell between OVX and OVX + E₂ groups were observed. Although these initial data are consistent with dynorphin inhibiting pulsatile LH release prepubertally, additional work will be necessary to define the source and mechanisms of this inhibition.     

Differential distribution of tight junction proteins suggests a role for tanycytes in blood‐hypothalamus barrier regulation in the adult mouse brain

The median eminence is one of the seven so‐called circumventricular organs. It is located in the basal hypothalamus, ventral to the third ventricle and adjacent to the arcuate nucleus. This structure characteristically contains a rich capillary plexus and features a fenestrated endothelium, making it a direct target of blood‐borne molecules. The median eminence also contains highly specialized ependymal cells called tanycytes, which line the floor of the third ventricle. It has been hypothesized that one of the functions of these cells is to create a barrier that prevents substances in the portal capillary spaces from entering the brain. In this paper, we utilize immunohistochemistry to study the expression of tight junction proteins in the cells that compose the median eminence in adult mice. Our results indicate that tanycytes of the median eminence express occludin, ZO‐1, and claudin 1 and 5, but not claudin 3. Remarkably, these molecules are organized as a continuous belt around the cell bodies of the tanycytes that line the ventral part of the third ventricle. In contrast, the tanycytes at the periphery of the arcuate nucleus do not express claudin 1 and instead exhibit a disorganized expression pattern of occludin, ZO‐1, and claudin 5. Consistent with these observations, permeability studies using peripheral or central injections of Evans blue dye show that only the tanycytes of the median eminence are joined at their apices by functional tight junctions, whereas tanycytes located at the level of the arcuate nucleus form a permeable layer. In conclusion, this study reveals a unique expression pattern of tight junction proteins in hypothalamic tanycytes, which yields new insights into their barrier properties. J. Comp. Neurol. 518:943–962, 2010. © 2009 Wiley‐Liss, Inc.     

Monosynaptic Pathway Between the Arcuate Nucleus Expressing Glial Type II Iodothyronine 5'-Deiodinase mRNA and the Median Eminence-Projective TRH Cells of the Rat Paraventricular Nucleus

Recent evidence suggests that the thyroid regulation of thyrotropin-releasing hormone (TRH)-containing neurons in the paraventricular nucleus of the hypothalamus involves the activation of other hypothalamic neural circuits. For example, the arcuate nucleus and not the paraventricular nucleus contains the highest enzyme activity of 5′-deiodinase type II, an enzyme that is pivotal for the local synthesis of T3. This experiment was undertaken to demonstrate whether a monosynaptic pathway exists between the arcuate nucleus and those TRH cells of the paraventricular nucleus that are neuroendocrine, i.e. project to the external layer of the median eminence. A specific cRNA probe derived from the coding region of deiodinase type II was used for the in situ hybridization histochemistry which was combined with immunocytochemistry for a specific marker of glial cells, glial fibrillary acidic protein (GFAP). The hybridization signals were present within the hypothalamus in the arcuate nucleus–median eminence region and in the periventricular area. The periventricular labeling was localized to the ependymal layer of the third ventricle and no hybridization product was detected in the paraventricular nucleus and other hypothalamic nuclei adjacent to the third ventricle. Within the median eminence, numerous cells containing the hybridization product were located in the internal layer adjacent to the floor of the third ventricle and in the external layer adjacent to the surface of the brain. In the dorso- and ventromedial regions of the arcuate nucleus, deiodinase type II mRNA-containing cells were also detected. Numerous type II deiodinase mRNA-containing cells in the median eminence and arcuate nucleus were also found to be immunopositive for GFAP. The abundance of arcuate cells expressing the hybridization product was lower than those in the periventricular region or in the median eminence. The anterograde tracer, Phaseolus vulgaris leucoagglutinin, was injected into the medial parts of the arcuate nucleus where the in situ hybridization experiment detected deiodinase type II mRNA. Simultaneously with the anterograde tracing, the retrograde tracer, Fluoro-Gold, was injected into either the median eminence or the general circulation. Light and electron microscopic double and triple immunolabeling experiments on vibratome sections of colchicine-pretreated animals revealed that arcuate fibers innervate TRH cells within the parvicellular region of the paraventricular nucleus. Populations of these TRH cells receiving afferents from the arcuate nucleus were also retrogradely labelled from either the median eminence or the general circulation indicating their direct role in the regulation of thyrotropin secretion from the anterior pituitary. The majority of arcuate nucleus efferents on TRH cells were found to establish symmetrical synaptic connections. The present results provided direct evidence of a monosynaptic pathway between the hypothalamic site of local thyroid hormone production, the arcuate nucleus, and neuroendocrine TRH cells in the paraventricular nucleus. This signalling modality may play an important role in thyroid feedback on TRH cells. Since the arcuate nucleus is involved in the regulation of central mechanisms controlling diverse homeostatic functions, including reproduction and feeding, the pathway described in this study may also carry integrated signals related to reproduction and ingestion to TRH-producing cells.     

Distributions of periventricular projections of the paraventricular nucleus to the median eminence and arcuate nucleus

Neuronal projections from the periventricular subnucleus of the hypothalamic paraventricular nucleus to the median eminence and the arcuate nucleus were investigated in the rat by the anterograde tract-tracer, Phaseolus vulgaris leucoagglutinin. The vast majority of labeled fibers coursed ventrally along the third ventricle and distributed in the external layer of the median eminence bilaterally, with ipsilateral predominance moving caudalwards. Periventricular fibers also terminated in the arcuate nucleus, but this innervation was exclusively ipsilateral.     

Hypothalamic Effects of Tamoxifen on Oestrogen Regulation of Luteinising Hormone and Prolactin Secretion in Female Rats

Oestradiol (E₂) acts in the hypothalamus to regulate luteinising hormone (LH) and prolactin (PRL) secretion. Tamoxifen (TX) has been extensively used as a selective oestrogen receptor modulator, although its neuroendocrine effects remain poorly understood. In the present study, we investigated the hypothalamic effects of TX in rats under low or high circulating E₂ levels. Ovariectomised (OVX) rats treated with oil, E₂ or TX, or E₂ plus TX, were evaluated for hormonal secretion and immunohistochemical analyses in hypothalamic areas. Both E₂ and TX reduced LH levels, whereas TX blocked the E₂‐induced surges of LH and PRL. TX prevented the E₂‐induced expression of progesterone receptor (PR) in the anteroventral periventricular nucleus (AVPV) and arcuate nucleus (ARC), although it did not alter PR expression in OVX rats. TX blocked the E₂ induction of c‐Fos in AVPV neurones, consistent with the suppression of LH surge. However, TX failed to prevent E₂ inhibition of kisspeptin expression in the ARC. In association with the blockade of PRL surge, TX increased the phosphorylation of tyrosine hydroxylase (TH) in the median eminence of OVX, E₂‐treated rats. TX also precluded the E₂‐induced increase in TH expression in the ARC. In all immunohistochemical analyses, TX treatment in OVX rats caused no measurable effect on the hypothalamus. Thus, TX is able to prevent the positive‐ but not negative‐feedback effect of E₂ on the hypothalamus. TX also blocks the effects of E₂ on tuberoinfundibular dopaminergic neurones and PRL secretion. These findings further characterise the anti‐oestrogenic actions of TX in the hypothalamus and provide new information on the oestrogenic regulation of LH and PRL.     

Issue Information

January 2016 Issue Highlights:

• Epigenetic changes after neonatal fasting stress protect from future fasting stress
• Estradiol exposure early in life programs activity rhythms in mice
• Tamoxifen suppression of oestrogen effects on the hypothalamus
• 11β‐HSD1 deficiency and ageing alters Npas4 and Arc mRNA
• Sex related differences in rat choroid plexus and cerebrospinal fluid

Short title: Effect of tamoxifen (TX) on the expression of progesterone receptor (PR) in the arcuate nucleus (ARC). Long title: Effect of tamoxifen (TX) on the expression of progesterone receptor (PR) in the arcuate nucleus (ARC). Ovariectomised (OVX) rats were treated with oil (OVX; n = 6), oestradiol (OVX+E2; n = 6), TX (OVX+TX; n = 5) or oestradiol plus TX (OVX+E2TX; n = 5) daily for three days. Blood samples were withdrawn hourly between 13:00 and 18:00 h and rats were perfused at 18:00 h. (A) Schematic diagram representing the area analysed for PR expression in the ARC (black squares). (B–E) Representative photomicrographs of PR expression in the ARC of OVX (B), OVX+TX (C), OVX+E2 (D), and OVX+E2TX (E) rats. (F) Mean ± SEM number of PR‐immunoreactive (ir) neurones/section. **P < 0.01 OVX+E2 versus OVX+E2TX; ###P < 0.001 compared to OVX. 3V, third ventricle. ME, median eminence. Scale bar, 100 µm.     

Immunohistochemical Distribution of Somatostatin and Somatostatin Receptor Subtypes (SSTR1–5) in Hypothalamus of ApoD Knockout Mice Brain

In the present study, the expression of somatostatin (SST) and somatostatin receptor subtypes (SSTR1-5) was determined in the hypothalamus of wild-type (wt) and apolipoprotein D knockout (ApoD(-/-)) mice brain. SST-like immunoreactivity, while comparable in most regions of hypothalamus, diminished significantly in arcuate nucleus of ApoD(-/-) mice. SSTR1 strongly localized in all major hypothalamic nuclei as well as in the median eminence and ependyma of the third ventricle of wt mice brain. SSTR1-like immunoreactivity increases in hypothalamus except in paraventricular nucleus of ApoD(-/-) mice. SSTR2 was well expressed in most of the hypothalamic regions whereas it decreases significantly in ventromedial and arcuate nucleus of ApoD(-/-) mice. SSTR3 and SSTR4-like immunoreactivity increases in ApoD(-/-) mice in all major nuclei of hypothalamus, median eminence, and ependymal cells of third ventricle. SSTR5 is well expressed in ventromedial and arcuate nucleus whereas weakly expressed in paraventricular nucleus. In comparison to wt, ApoD(-/-) mice exhibit increased SSTR5-like immunoreactivity in paraventricular nuclei and decreased receptor expression in ventromedial hypothalamus and arcuate nucleus. In conclusion, the changes in hypothalamus of ApoD(-/-) mice may indicate potential role of ApoD in regulation of endocrine functions of somatostatin in a receptor-dependent manner.     

Enhancement of the luteinising hormone surge by male olfactory signals is associated with anteroventral periventricular Kiss1 cell activation in female rats

Olfactory stimuli play an important role in regulating reproductive functions in mammals. The present study investigated the effect of olfactory signals derived from male rats on kisspeptin neuronal activity and luteinising hormone (LH) secretion in female rats. Wistar‐Imamichi strain female rats were ovariectomised (OVX) and implanted with preovulatory levels of 17β‐oestradiol (E₂). OVX+E₂ rats were killed 1 hour after exposure to either: clean bedding, female‐soiled bedding or male‐soiled bedding. Dual staining for Kiss1 mRNA in situ hybridisation and c‐Fos immunohistochemistry revealed that the numbers of Kiss1‐expressing cells and c‐Fos‐immunopositive Kiss1‐expressing cells in the anteroventral periventricular nucleus (AVPV) were significantly higher in OVX+E₂ rats exposed to male‐soiled bedding than those of the other groups. No significant difference was found with respect to the number of c‐Fos‐immunopositive Kiss1‐expressing cells in the arcuate nucleus and c‐Fos‐immunopositive Gnrh1‐expressing cells between the groups. The number of c‐Fos‐immunopositive cells was also significantly higher in the limbic system consisting of several nuclei, such as the bed nucleus of the stria terminalis, the cortical amygdala and the medial amygdala, in OVX+E₂ rats exposed to male‐soiled bedding than the other groups. OVX+E₂ rats exposed to male‐soiled bedding showed apparent LH surges, and the peak of the LH surge and area under the curve of LH concentrations in the OVX+E₂ group were significantly higher than those of the other two groups. These results suggest that olfactory signals derived from male rats activate AVPV kisspeptin neurones, likely via the limbic system, resulting in enhancement of the peak of the LH surge in female rats. Taken together, the results of the present study suggests that AVPV kisspeptin neurones are a target of olfactory signals to modulate LH release in female rats.     

Evidence of involvement of neurone‐glia/neurone‐neurone communications via gap junctions in synchronised activity of KNDy neurones

Pulsatile secretion of gonadotrophin‐releasing hormone (GnRH)/luteinising hormone is indispensable for the onset of puberty and reproductive activities at adulthood in mammalian species. A cohort of neurones expressing three neuropeptides, namely kisspeptin, encoded by the Kiss1 gene, neurokinin B (NKB) and dynorphin A, localised in the hypothalamic arcuate nucleus (ARC), so‐called KNDy neurones, comprises a putative intrinsic source of the GnRH pulse generator. Synchronous activity among KNDy neurones is considered to be required for pulsatile GnRH secretion. It has been reported that gap junctions play a key role in synchronising electrical activity in the central nervous system. Thus, we hypothesised that gap junctions are involved in the synchronised activities of KNDy neurones, which is induced by NKB‐NK3R signalling. We determined the role of NKB‐NK3R signalling in Ca²⁺ oscillation (an indicator of neuronal activities) of KNDy neurones and its synchronisation mechanism among KNDy neurones. Senktide, a selective agonist for NK3R, increased the frequency of Ca²⁺ oscillations in cultured Kiss1‐GFP cells collected from the mediobasal hypothalamus of the foetal Kiss1‐green fluorescent protein (GFP) mice. The senktide‐induced Ca²⁺ oscillations were synchronised in the Kiss1‐GFP and neighbouring glial cells. Confocal microscopy analysis of these cells, which have shown synchronised Ca²⁺ oscillations, revealed close contacts between Kiss1‐GFP cells, as well as between Kiss1‐GFP cells and glial cells. Dye coupling experiments suggest cell‐to‐cell communication through gap junctions between Kiss1‐GFP cells and neighbouring glial cells. Connexin‐26 and ‐37 mRNA were found in isolated ARC Kiss1 cells taken from adult female Kiss1‐GFP transgenic mice. Furthermore, 18β‐glycyrrhetinic acids and mefloquine, which are gap junction inhibitors, attenuated senktide‐induced Ca²⁺ oscillations in Kiss1‐GFP cells. Taken together, these results suggest that NKB‐NK3R signalling enhances synchronised activities among neighbouring KNDy neurones, and that both neurone‐neurone and neurone‐glia communications via gap junctions possibly contribute to synchronised activities among KNDy neurones.     

Inhibitory Actions of Acute Estradiol Treatment on the Activity and Quantity of Tyrosine Hydroxylase in the Median Eminence of Ovariectomized Rats

The effects of acute estradiol (E₂) treatment on both the activity of tyrosine hydroxylase (TH) in the median eminence and the serum level of prolactin (PRL) were investigated. Twelve-day-ovariectomized rats were injected with 17β-E₂ (25μg sc) at 1100 h and sacrificed hourly from 1200 to 2300 h. TH activity was quantified by measuring the amount of exogenous tyrosine converted to L-DOPA in vitro by aliquots of median eminence homogenates. Serum PRL levels were evaluated by radioimmunoassay. A biphasic response of TH activity to treatment was observed: an immediate decrease occurred—preceding and accompanying a rise in serum PRL—followed by an increase beyond control levels 2 h after the maximal release of PRL. The increase in TH activity could be prevented by the pretreatment of rats with a specific rat PRL antiserum, suggesting it was not due to E₂ per se but rather mediated by the E₂-induced PRL elevation. To pin-point the process underlying the E₂-induced decrease in TH activity, we evaluated the kinetic parameters of TH in the median eminence as well as its quantity (by Western blot analysis) in the median eminence and arcuate nucleus. Finally, we used a sensitive dot-blot assay to quantify specific TH messenger ribonucleic acid in the arcuate nucleus. The decrease in TH activity after E₂ treatment paralleled an immediate decrease in the affinity of TH for its pterin cofactor (6-MPH4), while V_max remained unchanged. A decrease in the amount of TH protein in the arcuate nucleus and median eminence as well as in the TH messenger ribonucleic acid level in the arcuate nucleus was also observed, but the latency of these effects precluded a major involvement in the immediate decline of TH activity. Therefore, when observed separately from those of PRL, E₂ effects on TH in tuberoinfundibular dopaminergic neurons are clearly inhibitory consisting of a ‘deactivation’ of the enzyme together with a reduction of its synthesis.     

Ovarian Regulation of Kisspeptin Neurones in the Arcuate Nucleus of the Rhesus Monkey (Macaca mulatta)

Tonic gonadotrophin secretion throughout the menstrual cycle is regulated by the negative‐feedback actions of ovarian oestradiol (E₂) and progesterone. Although kisspeptin neurones in the arcuate nucleus (ARC) of the hypothalamus appear to play a major role in mediating these feedback actions of the steroids in nonprimate species, this issue has been less well studied in the monkey. In the present study, we used immunohistochemistry and in situ hybridisation to examine kisspeptin and KISS1 expression, respectively, in the mediobasal hypothalamus (MBH) of adult ovariectomised (OVX) rhesus monkeys. We also examined kisspeptin expression in the MBH of ovarian intact females, and the effect of E₂, progesterone and E₂ + progesterone replacement on KISS1 expression in OVX animals. Kisspeptin or KISS1 expressing neurones and pronounced kisspeptin fibres were readily identified throughout the ARC of ovariectomised monkeys but, on the other hand, in intact animals, kisspeptin cell bodies were small in size and number and only fine fibres were observed. Replacement of OVX monkeys with physiological levels of E₂, either alone or with luteal phase levels of progesterone, abolished KISS1 expression in the ARC. Interestingly, progesterone replacement alone for 14 days also resulted in a significant down‐regulation of KISS1 expression. These findings support the view that, in primates, as in rodents and sheep, kisspeptin signalling in ARC neurones appears to play an important role in mediating the negative‐feedback action of E₂ on gonadotrophin secretion, and also indicate the need to study further their regulation by progesterone.     

Kisspeptin Regulates Tuberoinfundibular Dopaminergic Neurones and Prolactin Secretion in an Oestradiol‐Dependent Manner in Male and Female Rats

Prolactin (PRL) secretion is inhibited by hypothalamic dopamine. Kisspeptin controls luteinising hormone (LH) secretion and is also involved in PRL regulation. We further investigated the effect of kisspeptin‐10 (Kp‐10) on the activity of tuberoinfundibular dopaminergic (TIDA) neurones and the role of oestradiol (E₂) in this mechanism. Female and male rats were injected with i.c.v. Kp‐10 and evaluated for PRL release and the activity of dopamine terminals in the median eminence (ME) and neurointermediate lobe of the pituitary (NIL). Kp‐10 at the doses of 0.6 and 3 nmol increased plasma PRL and decreased 4‐dihydroxyphenylacetic acid (DOPAC) levels in the ME and NIL of ovariectomised (OVX), E₂‐treated rats but had no effect in OVX. In gonad‐intact males, 3 nmol Kp‐10 increased PRL secretion and decreased DOPAC levels in the ME but not in the NIL. Castrated males treated with either testosterone or E₂ also displayed increased PRL secretion and reduced ME DOPAC in response to Kp‐10, whereas castrated rats receiving oil or dihydrotestosterone were unresponsive. By contrast, the LH response to Kp‐10 was not E₂‐dependent in either females or males. Additionally, immunohistochemical double‐labelling demonstrated that TIDA neurones of male rats contain oestrogen receptor (ER)‐α, with a higher proportion of neurones expressing ERα than in dioestrous females. The dopaminergic neurones of periventricular hypothalamic nucleus displayed much lower ERα expression. Thus, TIDA neurones express ERα in male and female rats, and kisspeptin increases PRL secretion through inhibition of TIDA neurones in an E₂‐dependent manner in both sexes. These findings provide new evidence about the role of kisspeptin in the regulation of dopamine and PRL.     

Neuronal morphology and neuropil structure in the stomatogastric ganglion of the lobster, Homarus americanus

The hypothalamus is a major site of somatostatin (SST) production and action. SST is synthesized in several hypothalamic nuclei and involved in a variety of functions. Using SST receptor (SSTR)-specific antibodies, we localized SSTR subtypes in the rat hypothalamus. In addition, we also demonstrated SSTRs colocalization with SST, NADPH-diaphorase (NADPH-d), and tyrosine hydroxylase (TH). SSTR1 is strongly localized in neurons in all major hypothalamic nuclei as well as in nerve fibers in the zona externa of the median eminence and the ependyma of the third ventricle. SSTR2 is also well expressed in most regions but with a relatively lower abundance in comparison to SSTR1. In contrast, SSTR3 is localized primarily in the paraventricular nucleus, dorsomedial hypothalamic nucleus, arcuate nucleus, and median eminence. SSTR4-like immunoreactivity is mainly confined to the arcuate nucleus, ventromedial hypothalamic nucleus, median eminence, and ependymal cells of third ventricle, with the rare SSTR4-positive neuron in the paraventricular nucleus. SSTR5 is the least expressed subtype occurring only in few cells in the inner layer of the median eminence. Overall, SSTR1 is the predominant subtype, followed by SSTR2, 4, 3, and 5. Combined immunofluorescence, immunocytochemistry, and histochemistry were used to demonstrate SSTRs colocalization with SST, TH, and NADPH-d. SSTRs colocalization with SST, TH, and NADPH-d displays in a region and receptor specificity. Colocalization of SST and NADPH-d with SSTRs in hypothalamic regions was similar, suggesting that SST and NADPH-d producing cells are same. In contrast, TH was selectively coexpressed with SSTRs in the hypothalamus in a receptor-specific manner. Taken together, these data suggest that SSTRs may interact with NADPH-d and TH to exert a physiological role in concert within the hypothalamus.     

Vasopressin Anti-Idiotypic Antibody Staining in the Rat Brain: Colocalization with [35S][pGlu4, Cyt6]AVP(4–9) Binding Sites

Vasopressin and its fragment peptides such as [pGlu⁴, Cyt⁶]AVP_(4–9) (AVP_(4–9)) represent putative neuromodulators within central nervous homeostatic, memory and behavioural circuits. To localize their central receptor systems, the previously characterized monoclonal anti-idiotypic antibody mAb 237 was employed in immunocytological investigations of rat brain tissue sections. This antibody was raised to the monoclonal idiotypic anti-AVP antibody mAb 113 which preferentially binds to the acyclic C-terminal portion of the AVP molecule and is therefore also capable of binding the naturally occurring AVP_(4–9) fragment. Immunoreactive magnocellular neurones were detected in the AVP-synthesizing supraoptic but not paraventricular nuclei. Dense staining was observed within circumventricular organs lacking a blood-brain barrier (BBB). These structures include the subfornical organ, the organum vasculosum laminae terminalis, the internal layer of the median eminence, the body of the pineal gland, the choroid plexus and the area postrema, where immunoreactivity was found on capillaries, neurones and fibres. Further staining was found in the nucleus of the solitari tract and the arcuate nucleus, endowed with a leaky BBB. Distinct cell patches in the ependymal lining of the third ventricle as well as dendritic processes of juxtaependymal neurones were labelled by the anti-idiotypic antibody mAb 237. The observed staining pattern did not parallel that obtained in autoradiographic studies performed using either radiolabelled AVP or a V₁-receptor antagonist, but that found with the [³⁵S]-labelled AVP_(4–9) fragment. Using [³⁵S]-labelled AVP_(4–9) fragment, specific high density binding sites could be localized autoradiographically in structures within and outside the BBB, in complete agreement with the anti-idiotypic immunoreactivity. Since the anti-idiotypic methodology is based on transfer of complementary structures, and the epitope recognized by the corresponding idiotypic antibody resembles the sequence of AVP_(4–9), the anti-idiotypic antibodies might recognize the AVP_(4–9) receptor with high affinity.     

Light and electron microscopic immunocytochemistry of β-endorphin/β-LPH-like immunoreactive neurons in the arcuate nucleus and surrounding areas of the rat hypothalamus

β-Endorphin/β-LPH-like immunoreactive neurons in the hypothalamic arcuate nucleus and its surrounding areas were visualized by light and electron microscopic immunocytochemistry. Immunoreactive processes were found in the vicinity of the pia mater, in the lateral part of the external layer of the median eminence and near the lateral wall of the third ventricle. Neuronal perikarya contained immunoreactive dense granules as well as developed cell organellae. They received neuronal inputs from other neurons through axoplasmic and axodendritic synapses. Immunoreactive neuronal processes containing dense granules and mitochondria were found as preterminal elements on non-immunoreactive neuronal soma and dendrites. Immunoreactive processes also make intimate contact with capillaries in the arcuate nucleus near the median eminence.     

Evidence for Changes in Numbers of Synaptic Inputs onto KNDy and GnRH Neurones during the Preovulatory LH Surge in the Ewe

Kisspeptin neurones located in the arcuate nucleus (ARC) and preoptic area (POA) are critical mediators of gonadal steroid feedback onto gonadotrophin‐releasing hormone (GnRH) neurones. ARC kisspeptin cells that co‐localise neurokinin B (NKB) and dynorphin (Dyn), are collectively referred to as KNDy (Kisspeptin/NKB/Dyn) neurones, and have been shown in mice to also co‐express the vesicular glutamate transporter, vGlut2, an established glutamatergic marker. The ARC in rodents has long been known as a site of hormone‐induced neuroplasticity, and changes in synaptic inputs to ARC neurones in rodents occur over the oestrous cycle. Based on this evidence, the the present study aimed to examine possible changes across the ovine oestrous cycle in synaptic inputs onto kisspeptin cells in the ARC (KNDy) and POA, and inputs onto GnRH neurones. Gonadal‐intact breeding season ewes were perfused using 4% paraformaldehyde during either the luteal or follicular phase of the oestrous cycle, with the latter group killed at the time of the luteinising hormone (LH) surge. Hypothalamic sections were processed for triple‐label immunodetection of kisspeptin/vGlut2/synaptophysin or kisspeptin/vGlut2/GnRH. The total numbers of synaptophysin‐ and vGlut2‐positive inputs to ARC KNDy neurones were significantly increased at the time of the LH surge compared to the luteal phase; because these did not contain kisspeptin, they do not arise from KNDy neurones. By contrast to the ARC, the total number of synaptophysin‐positive inputs onto POA kisspeptin neurones did not differ between luteal phase and surge animals. The total number of kisspeptin and vGlut2 inputs onto GnRH neurones in the mediobasal hypothalamus (MBH) was also increased during the LH surge, and could be attributed to an increase in the number of KNDy (double‐labelled kisspeptin + vGlut2) inputs. Taken together, these results provide novel evidence of synaptic plasticity at the level of inputs onto KNDy and GnRH neurones during the ovine oestrous cycle. Such changes may contribute to the generation of the preovulatory GnRH/LH surge.     

Neural Systems Mediating Seasonal Breeding in the Ewe

Seasonal reproduction in ewes is caused by a dramatic increase in response to oestradiol (E₂) negative feedback during the nonbreeding (anoestrous) season. Considerable evidence supports the hypothesis that A15 dopaminergic neurones in the retrochiasmatic area (RCh) play a key role in these seasonal changes. These A15 neurones are stimulated by E₂ and inhibit gonadotrophin‐releasing hormone (GnRH) secretion in anoestrus, but not the breeding season. Because A15 neurones do not contain oestrogen receptors‐α (ERα), it is likely that E₂‐responsive afferents stimulate their activity when circulating E₂ levels increase during anoestrus. Retrograde tract tracing studies identified a limited set of ERα‐containing afferents primarily found in four areas [ventromedial preoptic area, RCh, ventromedial and arcuate (ARC) nuclei]. Pharmacological and anatomical data are consistent with GABA‐ and glutamate‐containing afferents controlling A15 activity in anoestrus, with E₂ inhibiting GABA and stimulating glutamate release at this time of year. Tract tracing demonstrated that A15 efferents project posteriorly to the median eminence and the ARC, suggesting possible direct actions on GnRH terminals or indirect actions via kisspeptin neurones in the ARC to inhibit GnRH in anoestrus. Identification of this neural circuitry sets the stage for the development of specific hypotheses for morphological or transmitter/receptor expression changes that would account for seasonal breeding in ewes.     

Distribution of neuropeptide Y-like immunoreactivity in the hypothalamus of the adult golden hamster

The distribution of neuropeptide Y (NPY)-like immunoreactivity within the hypothalamus of the adult golden hamster was investigated with conventional immunohistochemical techniques. Neuropeptide Y immunoreactive cell bodies were found in greatest numbers in the arcuate nucleus while a few stained perikarya were seen in the internal and subependymal zones of the median eminence. Isolated perikarya were observed in the anterior commissure and supracommissural portion of the interstitial nucleus of the stria terminalis. Immunoreactive axons were located throughout the hypothalamus with the highest concentrations in the subependymal and internal zones of the median eminence, the interstitial nucleus of the stria terminalis, the medial preoptic area, and in the following nuclei: periventricular, suprachiasmatic, paraventricular, perifornical, median preoptic, and arcuate. Moderate to dense plexuses of immunoreactive fibers were observed in the anterior, lateral, and posterior hypothalamic areas and in the infundibular stalk. The supraoptic nucleus and lateral preoptic area displayed a small number of labeled axons whereas the ventromedial nucleus contained only a few fibers. NPY immunoreactive fibers were present in the optic tract and in the dorsomedial aspect of the optic chiasm. Labeled fibers penetrated the ependymal lining of the third ventricle throughout the ventral aspect of the periventricular zone. Additional fibers were observed in the pia lining the ventral aspect of the hypothalamus. This systematic analysis of hypothalamic NPY immunoreactivity in the adult golden hamster suggests that a portion of the labeled fibers display a distribution that is similar to previously described noradrenergic fibers in the hypothalamus.     

Ultrastructure of gonadotropin-releasing hormone neuronal structures derived from normal fetal preoptic area and transplanted into hypogonadal mutant (hpg) mice

The hpg mutant mouse lacks the neurohormone gonadotropin-releasing hormone (GnRH) and hence has a reproductive deficit. This deficit can be corrected by placement of normal fetal preoptic area into the third ventricle (see Krieger et al., 1985). We have now used ultrastructural immunocytochemistry to investigate the morphology of GnRH neurons in such intraventricular grafts, the routes that their axons take as they exit into the host, and the neurosecretory terminations that they make in the host median eminence. The GnRH cells in the transplant were similar in morphology to that reported for such cells in the preoptic area of other rodents. There was a large central nucleus, frequently indented and containing 1 or 2 nucleoli. The thin rim of cytoplasm was filled with rough endoplasmic reticulum, Golgi stacks, and mitochondria. Both dendritic and axonal profiles were identified, and a modest synaptic input to the former was found. Between the host and the implant a complex multilayered ependymal zone developed, and it was through this region that GnRH axons exited into the host arcuate nucleus and median eminence, usually surrounded by ependymal or glial elements. Within the median eminence, GnRH terminals were in close association with fenestrated blood vessels forming a normal neurosecretory terminus.