Localisation of 11ß-Hydroxysteroid Dehydrogenase Type 2 in Mineralocorticoid Receptor Expressing Magnocellular Neurosecretory Neurones of the Rat Supraoptic and Paraventricular Nuclei.

An accumulating body of evidence suggests that the activity of the mineralocorticoid, aldosterone, in the brain via the mineralocorticoid receptor (MR) plays an important role in the regulation of blood pressure. MR was recently found in vasopressin and oxytocin synthesising magnocellular neurosecretory cells (MNCs) in both the paraventricular (PVN) and supraoptic (SON) nuclei in the hypothalamus. Considering the physiological effects of these hormones, MR in these neurones may be an important site mediating the action of aldosterone in blood pressure regulation within the brain. However, aldosterone activation of MR in the hypothalamus remains controversial as a result of the high binding affinity of glucocorticoids to MR at substantially higher concentrations compared to aldosterone. In aldosterone-sensitive epithelia, the enzyme 11ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD2) prevents glucocorticoids from binding to MR by converting glucocorticoids into inactive metabolites. The present study aimed to determine whether 11ß-HSD2, which increases aldosterone selectivity, is expressed in MNCs. Specific 11ß-HSD2 immunoreactivity was found in the cytoplasm of the MNCs in both the SON and PVN. In addition, double-fluorescence confocal microscopy demonstrated that MR-immunoreactivity and 11ß-HSD2-in situ hybridised products are colocalised in MNCs. Lastly, single-cell reverse transcriptase-polymerase chain reaction detected MR and 11ß-HSD2 mRNAs from cDNA libraries derived from single identified MNCs. These findings strongly suggest that MNCs in the SON and PVN are aldosterone-sensitive neurones.

Transient receptor potential channel m4 and m5 in magnocellular cells in rat supraoptic and paraventricular nuclei.

The neurohypophysial hormones, vasopressin (VP) and oxytocin (OT), are synthesised by magnocellular cells in the supraoptic nucleus (SON) and the paraventricular nucleus (PVN) of the hypothalamus. The release of VP into the general circulation from the neurohypophysis increases during hyperosmolality, hypotension and hypovolaemia. VP neurones increase hormone release by increasing their firing rate as a result of adopting a phasic bursting. Depolarising after potentials (DAPs) following a series of action potentials are considered to be involved in the generation of the phasic bursts by summating to plateau potentials. We recently discovered a fast DAP (fDAP) in addition to the slower DAP characterised previously. Almost all VP neurones expressed the fDAP, whereas only 16% of OT neurones had this property, which implicates the involvement of fDAP in the generation of the firing patterns in VP neurones. Our findings obtained from electrophysiological experiments suggested that the ionic current underlying the fDAP is mediated by those of two closely-related Ca(2+) -activated cation channels: the melastatin-related subfamily of transient receptor potential channels, TRPM4 and TRPM5. In the present study, double/triple immunofluorescence microscopy and reverse transcriptase-polymerase chain reaction techniques were employed to evaluate whether TRPM4 and TRPM5 are specifically located in VP neurones. Using specific antibodies against these channels, TRPM5 immunoreactivity was found almost exclusively in VP neurones, but not in OT neurones in both the SON and PVN. The most prominent TRPM5 immunoreactivity was in the dendrites of VP neurones. By contrast, most TRPM4 immunoreactivity occurred in cell bodies of both VP and OT neurones. TRPM4 and TRPM5 mRNA were both found in a cDNA library derived from SON punches. These results indictate the possible involvement of TRPM5 in the generation of the fDAP, and these channels may play an important role in determining the distinct firing properties of VP neurones in the SON.

Performance, properties and plasticity of identified oxytocin and vasopressin neurones in vitro.

The neurohypophysial hormones oxytocin (OT) and vasopressin (VP) originate from hypothalamic neurosecretory cells in the paraventricular and supraoptic (SON) nuclei. The firing rate and pattern of action potentials arising from these neurones determine the timing and quantity of peripheral hormone release. We have used immunochemical identification of biocytin-filled SON neurones in hypothalamic slices in vitro to uncover differences between OT and VP neurones in membrane and synaptic properties, firing patterns, and plasticity during pregnancy and lactation. In this review, we summarise some recent findings from this approach: (i) VP neuronal excitability is influenced by slow (sDAP) and fast (fDAP) depolarising afterpotentials that underlie phasic bursting activity. The fDAP may relate to a transient receptor potential (TRP) channel, type melastatin (TRPM4 and/or TRPM5), both of which are immunochemically localised more to VP neurones, and especially, to their dendrites. Both TRPM4 and TRPM5 mRNAs are found in the SON, but single cell reverse transcriptase-polymerisation suggests that TRPM4 might be the more prominent channel. Phasic bursting in VP neurones is little influenced by spontaneous synaptic activity in slices, being shaped largely by intrinsic currents. (ii) The firing pattern of OT neurones ranges from irregular to continuous, with the coefficient of variation determined by randomly distributed, spontaneous GABAergic, inhibitory synaptic currents (sIPSCs). These sIPSCs are four- to five-fold more frequent in OT versus VP neurones, and much more frequent than spontaneous excitatory synaptic currents. (iii) Both cell types express Ca(2+)-dependent afterhyperpolarisations (AHPs), including an apamin-sensitive, medium duration AHP and a slower, apamin-insensitive AHP (sAHP). In OT neurones, both AHPs are enhanced during pregnancy and lactation. During pregnancy, the plasticity of the sAHP is blocked by antagonism of central OT receptors. AHP enhancement is mimicked by exposing slices from day 19 pregnant rats to OT and oestradiol, suggesting that central OT and sex steroids programme this plasticity during pregnancy by direct hypothalamic actions. In conclusion, the differences in VP and OT neuronal function are underlain by differences in both membrane and synaptic properties, and differentially modulated by reproductive state.

Central blockade of oxytocin receptors during mid-late gestation reduces amplitude of slow afterhyperpolarization in supraoptic oxytocin neurons.

The neurohypophysial hormone oxytocin (OT), synthesized in magnocellular paraventricular (PVN) and supraoptic (SON) nuclei, is well known for its effects in lactation. Our previous studies showed that central OT receptor (OTR) binding is increased during gestation and that blockade of central OTRs, specifically during mid-late gestation, causes a delay in OT release during suckling and reduces weight gain in pups, suggesting decreased milk delivery. In the present study, we tested whether central OTR blockade during late gestation disrupts the gestation-related plasticity in intrinsic membrane properties. Whole cell current-clamp recordings were performed in OT neurons from pregnant rats (19-22 days in gestation) that were infused with an OTR antagonist (OTA) or artificial cerebrospinal fluid (aCSF) and from virgin rats infused with aCSF into the third ventricle via an osmotic minipump beginning on days 12-14 of gestation. The amplitudes of both Ca(2+)-dependent afterhyperpolarizations (AHPs), an apamin-sensitive medium AHP (mAHP) and an apamin-insensitive slow AHP (sAHP), were significantly increased during late gestation in control pregnant animals. However, the amplitude of the sAHP from pregnant rats treated with the OTA was significantly smaller than that of pregnant control rats and similar to that of virgins. These results indicate that the diminished efficiency in lactation due to OTR blockade may be partly a result of an altered sAHP that would shape OT bursting. These findings suggest that central actions of OT during late gestation are necessary for programming the plasticity of at least some of the intrinsic membrane properties in OT neurons during lactation.

Changes in the active membrane properties of rat supraoptic neurones during pregnancy and lactation.

To better understand the plasticity of intrinsic membrane properties of supraoptic magnocellular neuroendocrine cells associated with reproductive function, intracellular recordings were performed in oxytocin (OT) and vasopressin (VP) neurones from virgin, late pregnant (E19-22), and lactating (8-12 days of lactation) rats in vitro, using hypothalamic explants. OT neurones from virgin rats displayed a narrower spike width than neurones from pregnant and lactating rats, characterized by faster rise and decay times. Spike width changes in VP neurones were not as prominent as those observed in OT neurones. In OT neurones, the amplitude and the decay of the afterhyperpolarization following spike trains was significantly larger and faster, respectively, in pregnant and lactating rats compared to virgin rats. These properties did not change during pregnancy and lactation in VP neurones. The incidence of the depolarizing afterpotential following spikes significantly increased from approximately 20% in virgin rats to 40-50% during pregnancy and lactation in OT neurones, but was stable (80-90%) across states in VP neurones. Repetitive firing properties (frequency adaptation, the first interspike interval frequency and frequency-current (F-I) relationship) were altered during pregnancy and lactation in OT neurones, but not VP neurones. The increased incidence of depolarizing afterpotentials in OT neurones enhances excitability, while the increased afterhyperpolarization results in suppression of firing rate. Thus, the changes may favour the short bursting activity seen in OT neurones during lactation. These results confirmed reproductive state-dependent changes in intrinsic membrane properties of OT neurones during lactation, and suggest these changes are in place during late pregnancy. This argues that the plasticity in the electrical properties in OT neurones associated with lactation is not instigated by suckling.

Double immunocytochemistry of vasoactive intestinal peptide and cGnRH-I in male quail: photoperiodic effects.

Reproduction in Japanese quail is primarily regulated by photoperiod. Vasoactive intestinal peptide (VIP) has been suggested as a transducer of environmental information, especially photoperiodic cues, to the hypothalamo-pituitary-gonadal axis. To investigate the possible interaction of VIP and the reproductive (gonadotropin-releasing hormone, GnRH) system, double-immunocytochemical staining for VIP and cGnRH-I was conducted in sexually mature male quail held under a long-day photoperiod (16L:8D; LD) and in sexually quiescent males held under a short-day photoperiod (8L:16D; SD). VIP-immunoreactive (ir) cells were found primarily in three locations: lateral septal organ (LSO) in nucleus accumbens (Ac), ventral hypothalamus, and infundibular area. VIP-ir cells in LSO displayed characteristics typical of cerebrospinal fluid (CSF)-contacting cells, and co-existed with cGnRH-I-ir cells and beaded fibers. In contrast, VIP-ir cells in the infundibular area did not co-exist with cGnRH-I-ir structures. The number of visible VIP-ir cells in the infundibular area of SD males was significantly lower than that of LD males, while the number of visible VIP-ir cells in Ac/LSO was not altered by photoperiod. A cluster of cGnRH-I-ir cells in the caudalmost septal area was heavily innervated by VIP-ir fibers, which appeared to contact cGnRH-I-ir cells directly at this location. Both VIP- and cGnRH-I-ir fibers heavily innervated the external layer of the median eminence (ME). These data suggest that Ac/LSO, the caudalmost septal area, and ME are possible sites of interaction between the VIP and the GnRH systems.

Changes in immunoreactivity to anti-cGnRH-I and -II are associated with photostimulated sexual status in male quail.

In sexually active males exposed to long-day (LD) photoperiod, perikarya in the olfactory bulb, lobus parolfactorius, n. accumbens, and preoptic region were immunoreactive (ir) to an antiserum against gonadotropin-releasing hormone (anti-cGnRH-I), and a cluster of ir-perikarya was found in the caudal-most septal area. Ir-perikarya in these brain areas of sexually inactive short-day (SD) males were located within more discrete areas than those in LD brain, which were more scattered in appearance. Absolute cell numbers were similar between LD and SD brains. Ir-fibers in LD brains were mostly in the external median eminence, along the lateral ventricle to septum (especially in and about the n. accumbens), in the septal-preoptic area, along the third ventricle, and at the n. commissure palli. There were fewer ir-fibers in SD brain. Many small dark ring-like ir-structures were found in the hyperstriatum, hippocampus, and n. taeniae. Interpreted as being ir-terminals on non-ir perikarya, these were not observed in SD males. cGnRH-II ir-perikarya were observed in only two areas regardless of reproductive status: (1) ventral to the substantia grisea centralis and caudal to the oculomotor complex, and (2) scattered in and about the lateral hypothalamus. Ir-fibers occurred in the habenular area, hyperstriatum, hippocampus, parahippocampal area, cortex piriformis, and n. taeniae. cGnRH-II ir-fibers occurred in the external median eminence but were less intensely stained than cGnRH-I ir-fibers. These fibers in SD males were similar except in the diencephalon, where scattered swellings were observed. Thus, the appearance and distribution of anti-cGnRH-I and -II ir-structures change with the sexual status of male quail, but changes in immunoreactivity to anti-cGnRH-I appear to be more widespread.

Neural sites and pathways regulating food intake in birds: a comparative analysis to mammalian systems.

The paper reviews hypotheses explaining the regulation of food intake in mammals that have addressed specific anatomical structures in the brain. An hypothesis, poikilostasis, is introduced to describe multiple, homeostatic states whereby the regulation of metabolism and feeding occur in birds. Examples are given for both wild and domestic avian species, illustrating dynamic shifts in homeostasis responsible for the changes in body weights that are seen during the course of an annual cycle or by a particular strain of bird. The following neural structures are reviewed as each has been shown to affect food intake in birds or in mammals: ventromedial hypothalamic nucleus (n.), lateral hypothalamic area, paraventricular hypothalamic n., n. tractus solitarius and area postrema, amygdala, parabrachial n., arcuate n. and bed n. of the stria terminalis. Two neural pathways are described which have been proposed to regulate feeding. The trigeminal sensorimotor pathway is the most complete neural pathway characterized for this behavior and encompasses the mechanics of pecking, grasping and mandibulating food particles from the tip of the bill to the back of the buccal cavity. A second pathway, the visceral forebrain system (VFS), affects feeding by regulating metabolism and the balance of the autonomic nervous system. Wild, migratory birds are shown to exhibit marked changes in body weight which are hypothesized to occur due to shifts in balance between the sympathetic and parasympathetic nervous systems. Domestic avian species, selected for a rapid growth rate, are shown to display a dominance of the parasympathetic nervous system. The VFS is the neural system proposed to effect poikilostasis by altering the steady state of the autonomic nervous system in aves and perhaps is applicable to other classes of vertebrates as well.

Development of metabolic response in male quail brain during sexual maturation.

Seasonal reproductive activities of Japanese quail Coturnix japonica are induced most obviously by stimulatory effects of long-day photoperiod. This study addressed the metabolic response, as measured by 2-deoxyglucose (2-DG), in brain of male quail during sexual maturation. At 7 weeks of age, reproductively quiescent quail exposed to a short photoperiod of 6L:18D, received 2-DG on day 0 and +3, +6, +9, +12, +15 and +18 days after onset of 16L:8D. Brains were processed for autoradiography; serum testosterone was measured to indicate reproductive response to photoperiod. Circulating testosterone remained low until day 9, then rose sharply, reaching maximum levels at day 18. Heavily labeled nuclei were identified in some discrete neural pathways: both tectofugal and thalamofugal visual pathways, ascending auditory pathway, efferent vocalization pathway, and limbic structures. Metabolic activity of the terminal nucleus (ectostriatum) of the tectofugal pathway increased significantly by day 18, but in the terminal nuclei (the Wulst) of the thalamofugal visual pathway activity did not change significantly. Energy metabolism of some nuclei of the auditory pathway rose significantly by day 3, although in the vocal pathway it did not show augmentation until days 15-18. The metabolic activity of limbic structures also increased. These results suggest that, in Japanese quail, sensory nuclei and some of their integrative areas become sensitive to environmental cues in response to long-day photoperiod. It is possible that the external environmental cues that affect the reproductive activities of quail act through sensory systems.