Maternal thyroid hormone is required for parvalbumin neurone development in the anterior hypothalamic area.

Thyroid hormone (TH) is crucial for brain development and function. This becomes most evident in untreated congenital hypothyroidism, leading to irreversible mental retardation. Likewise, maternal hypothyroxinaemia, a lack of TH during pregnancy, is associated with neurological dysfunction in the offspring, such as autism and reduced intellectual capacity. In the brain, TH acts mainly through TH receptor α1 (TRα1). Consequently, mice heterozygous for a dominant-negative mutation in TRα1 display profound neuroanatomical abnormalities including deranged development of parvalbumin neurones. However, the exact timing and orchestration of TH signalling during parvalbumin neurone development remains elusive. In the present study, we dissect the development of parvalbumin neurones in the anterior hypothalamic area (AHA) in male mice using different mouse models with impaired pre- and postnatal TH signalling in combination with bromodeoxyuridine birth dating and immunohistochemistry. Our data reveal that hypothalamic parvalbumin neurones are born at embryonic day 12 and are first detected in the AHA at postnatal day 8, reaching their full population number at P13. Interestingly, they do not require TH postnatally because their development is not impaired in mice with impaired TH signalling after birth. By contrast, however, these neurones crucially depend on TH through TRα1 signalling in the second half of pregnancy, when the hormone is almost exclusively provided by the mother. For the first time, our findings directly link a maternal hormone to a neuroanatomical substrate in the foetal brain, and underline the importance of proper TH signalling during pregnancy for offspring mental health. Given the role of hypothalamic parvalbumin neurones in the central control of blood pressure, the present study advocates the inclusion of cardiovascular parameters in the current discussion on possible TH substitution in maternal hypothyroxinaemia.

A sexually dimorphic hypothalamic circuit controls maternal care and oxytocin secretion.

It is commonly assumed, but has rarely been demonstrated, that sex differences in behaviour arise from sexual dimorphism in the underlying neural circuits. Parental care is a complex stereotypic behaviour towards offspring that is shared by numerous species. Mice display profound sex differences in offspring-directed behaviours. At their first encounter, virgin females behave maternally towards alien pups while males will usually ignore the pups or attack them. Here we show that tyrosine hydroxylase (TH)-expressing neurons in the anteroventral periventricular nucleus (AVPV) of the mouse hypothalamus are more numerous in mothers than in virgin females and males, and govern parental behaviours in a sex-specific manner. In females, ablating the AVPV TH(+) neurons impairs maternal behaviour whereas optogenetic stimulation or increased TH expression in these cells enhance maternal care. In males, however, this same neuronal cluster has no effect on parental care but rather suppresses inter-male aggression. Furthermore, optogenetic activation or increased TH expression in the AVPV TH(+) neurons of female mice increases circulating oxytocin, whereas their ablation reduces oxytocin levels. Finally, we show that AVPV TH(+) neurons relay a monosynaptic input to oxytocin-expressing neurons in the paraventricular nucleus. Our findings uncover a previously unknown role for this neuronal population in the control of maternal care and oxytocin secretion, and provide evidence for a causal relationship between sexual dimorphism in the adult brain and sex differences in parental behaviour.

A newly identified mouse hypothalamic area having bidirectional neural connections with the lateral septum: the perifornical area of the anterior hypothalamus rich in chondroitin sulfate proteoglycans.

While previous studies and brain atlases divide the hypothalamus into many nuclei and areas, uncharacterised regions remain. Here, we report a new region in the mouse anterior hypothalamus (AH), a triangular-shaped perifornical area of the anterior hypothalamus (PeFAH) between the paraventricular hypothalamic nucleus and fornix, that abundantly expresses chondroitin sulfate proteoglycans (CSPGs). The PeFAH strongly stained with markers for chondroitin sulfate/CSPGs such as Wisteria floribunda agglutinin and antibodies against aggrecan and chondroitin 6 sulfate. Nissl-stained sections of the PeFAH clearly distinguished it as a region of comparatively low density compared to neighboring regions, the paraventricular nucleus and central division of the anterior hypothalamic area. Immunohistochemical and DNA microarray analyses suggested that PeFAH contains sparsely distributed calretinin-positive neurons and a compact cluster of enkephalinergic neurons. Neuronal tract tracing revealed that both enkephalin- and calretinin-positive neurons project to the lateral septum (LS), while the PeFAH receives input from calbindin-positive LS neurons. These results suggest bidirectional connections between the PeFAH and LS. Considering neuronal subtype and projection, part of PeFAH that includes a cluster of enkephalinergic neurons is similar to the rat perifornical nucleus and guinea pig magnocellular dorsal nucleus. Finally, we examined c-Fos expression after several types of stimuli and found that PeFAH neuronal activity was increased by psychological but not homeostatic stressors. These findings suggest that the PeFAH is a source of enkephalin peptides in the LS and indicate that bidirectional neural connections between these regions may participate in controlling responses to psychological stressors.

GnRH neuron firing and response to GABA in vitro depend on acute brain slice thickness and orientation.

The GnRH neurons exhibit long dendrites and project to the median eminence. The aim of the present study was to generate an acute brain slice preparation that enabled recordings to be undertaken from GnRH neurons maintaining the full extent of their dendrites or axons. A thick, horizontal brain slice was developed, in which it was possible to record from the horizontally oriented GnRH neurons located in the anterior hypothalamic area (AHA). In vivo studies showed that the majority of AHA GnRH neurons projected outside the blood-brain barrier and expressed c-Fos at the time of the GnRH surge. On-cell recordings compared AHA GnRH neurons in the horizontal slice (AHAh) with AHA and preoptic area (POA) GnRH neurons in coronal slices [POA coronal (POAc) and AHA coronal (AHAc), respectively]. AHAh GnRH neurons exhibited tighter burst firing compared with other slice orientations. Although α-Amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) excited GnRH neurons in all preparations, γ-aminobutyric acid (GABA) was excitatory in AHAc and POAc but inhibitory in AHAh slices. GABA(A) receptor postsynaptic currents were the same in AHAh and AHAc slices. Intriguingly, direct activation of GABA(A) or GABA(B) receptors respectively stimulated and inhibited GnRH neurons regardless of slice orientation. Subsequent experiments indicated that net GABA effects were determined by differences in the ratio of GABA(A) and GABA(B) receptor-mediated effects in "long" and "short" dendrites of GnRH neurons in the different slice orientations. These studies document a new brain slice preparation for recording from GnRH neurons with their extensive dendrites/axons and highlight the importance of GnRH neuron orientation relative to the angle of brain slicing in studying these neurons in vitro.

The retinohypothalamic tract: comparison of axonal projection patterns from four major targets.

The retinohypothalamic tract is one component of the optic nerve that transmits information about environmental luminance levels through medial and lateral branches to four major terminal fields in the hypothalamus. The spatial distribution and organization of axonal projections from each of these four terminal fields were analyzed and compared systematically with the anterograde pathway tracer PHAL in rats where the terminal fields had been labeled with intravitreal injections of a different anterograde pathway tracer, CTb. First, the well-known projections of two medial retinohypothalamic tract targets (the ventrolateral suprachiasmatic nucleus and perisuprachiasmatic region) were confirmed and extended. They share qualitatively similar projections to a well-known set of brain regions thought to control circadian rhythms. Second, the projections of a third medial tract target, the ventromedial part of the anterior hypothalamic nucleus, were analyzed for the first time and shown to resemble qualitatively those from the suprachiasmatic nucleus and perisuprachiasmatic region. And third, projections from the major lateral retinohypothalamic tract target were analyzed for the first time and shown to be quite different from those associated with medial tract targets. This target is a distinct core part of the ventral zone of the anterior group of the lateral hypothalamic area that lies just dorsal to the caudal two-thirds of the supraoptic nucleus. Its axonal projections are to neural networks that control a range of specific goal-oriented behaviors (especially drinking, reproductive, and defensive) along with adaptively appropriate and complementary visceral responses and adjustments to behavioral state.

17beta-Estradiol-induced remodeling of GABAergic axo-somatic synapses on estrogen receptor expressing neurons in the anteroventral periventricular nucleus of adult female rats.

Experimental data demonstrate that the nervous system is widely influenced by sex hormones and the brain is continuously shaped by the changing hormone milieu throughout the whole life. Earlier we demonstrated that on the effect of estradiol there is a cyclic synaptic remodeling, i.e. a transient decrease in the number of GABAergic axo-somatic synapses in the arcuate nucleus. By using preembedding estrogen receptor and postembedding GABA immunostaining, in the present paper we studied the specificity of this effect and we found that in the anteroventral periventricular nucleus (AvPv) of adult female rats 17beta-estradiol treatment does not affect all synapses and neurons. In contrast to the arcuate nucleus, hormonal treatment induces a significant increase of inhibitory axo-somatic synapses in the AvPv and we found selectivity at the level of the postsynaptic neurons, as well. We analyzed the hormone-induced synaptic remodeling in estrogen receptor alpha and beta immunoreactive and non-labeled cells and the change in synapse number was observed only in neurons which express estrogen beta receptor.

Calcitonin gene-related peptide alters the firing rates of hypothalamic temperature sensitive and insensitive neurons.

BACKGROUND: Transient hyperthermic shifts in body temperature have been linked to the endogenous hormone calcitonin gene-related peptide (CGRP), which can increase sympathetic activation and metabolic heat production. Recent studies have demonstrated that these centrally mediated responses may result from CGRP dependent changes in the activity of thermoregulatory neurons in the preoptic and anterior regions of the hypothalamus (POAH). RESULTS: Using a tissue slice preparation, we recorded the single-unit activity of POAH neurons from the adult male rat, in response to temperature and CGRP (10 muM). Based on the slope of firing rate as a function of temperature, neurons were classified as either warm sensitive or temperature insensitive. All warm sensitive neurons responded to CGRP with a significant decrease in firing rate. While CGRP did not alter the firing rates of some temperature insensitive neurons, responsive neurons showed an increase in firing rate. CONCLUSION: With respect to current models of thermoregulatory control, these CGRP dependent changes in firing rate would result in hyperthermia. This suggests that both warm sensitive and temperature insensitive neurons in the POAH may play a role in producing this hyperthermic shift in temperature.

The effects of Cirazoline, an alpha-1 adrenoreceptor agonist, on the firing rates of thermally classified anterior hypothalamic neurons in rat brain slices.

Peripheral exposure to LPS induces a biphasic fever thought to be initiated via vagal afferents to the preoptic area of the anterior hypothalamus (POAH), an important thermoregulatory control center in the brain. Previous studies have shown that norepinephrine synaptically mediates this Prostaglandin E(2) (PGE(2))-dependent change in temperature through the selective activation of alpha-2 adrenoreceptors (AR). However, there is clear evidence that alpha-1 AR activation of thermoregulatory hypothalamic neurons will result in a rapid hyperthermia that is not dependent on PGE(2). This direct action of norepinephrine in the POAH was tested in the present study by recording the single-unit activity of POAH neurons in a tissue slice preparation from the adult male rat, in response to temperature and the selective alpha-1 AR agonist Cirazoline (1-100 microM). Neurons were classified as either warm sensitive or temperature insensitive. Warm sensitive neurons responded to Cirazoline with a decrease in firing rate, while temperature insensitive neurons showed a firing rate increase. These responses are similar to those reported for PGE(2) and suggest that both warm sensitive and temperature insensitive neurons in the POAH are important in mediating this alpha-1 AR-dependent hyperthermic shift in body temperature.

Physiologic regulation of a tetrodotoxin-sensitive sodium influx that mediates a slow afterdepolarization potential in gonadotropin-releasing hormone neurons: possible implications for the central regulation of fertility.

The brain controls fertility through release of gonadotropin-releasing hormone (GnRH), but the mechanisms underlying action potential patterning and GnRH release are not understood. We investigated whether GnRH neurons exhibit afterdepolarizing potentials (ADPs) and whether these are modified by reproductive state. Whole-cell current-clamp recordings of GnRH neurons in brain slices from ovariectomized mice revealed a slow ADP (sADP) after action potentials generated by brief current injection. Generating two or four spikes enhanced sADP amplitude and duration. sADP amplitude was not affected by blocking selected neurotransmitter/neuromodulator receptors, delayed-rectifier potassium channels, calcium-dependent cation channels, or hyperpolarization-activated cation channels but was halved by the calcium channel blocker cadmium and abolished by tetrodotoxin. Cadmium also reduced peak latency. Intrinsic mechanisms underlying the sADP were investigated using voltage-clamp protocols simulating action potential waveforms. A single action potential produced an inward current, which increased after double and quadruple stimulation. Cadmium did not affect current amplitude but reduced peak latency. Pretreatment with blockers of calcium-activated potassium currents (I(KCa)) reproduced this shift and blocked subsequent cadmium-induced changes, suggesting cadmium changes latency indirectly by blocking I(KCa). Tetrodotoxin abolished the inward current, suggesting that it is carried by sodium. In contrast, I(KCa) blockers increased the inward current, indicating that I(KCa) may oppose generation of the sADP. Strong sADPs were suprathreshold, generating repetitive spontaneous firing. I(ADP), sADP, and excitability were enhanced by in vivo estradiol, which triggers a preovulatory surge of GnRH release. Physiological feedback modification of this inward current and resulting sADP may modulate action potential firing and subsequent GnRH release.

IL-1beta induces a MyD88-dependent and ceramide-mediated activation of Src in anterior hypothalamic neurons.

The proinflammatory cytokine interleukin 1beta (IL-1beta), acting at IL-1R1 receptors, affects neuronal signaling under both physiological and pathophysiological conditions. The molecular mechanism of the rapid synaptic actions of IL-1beta in neurons is not known. We show here that within minutes of IL-1beta exposure, the firing rate of anterior hypothalamic (AH) neurons in culture was inhibited. This effect was prevented by pre-exposure of the cells to the Src family inhibitor, PP2, suggesting the involvement of Src in the hyperpolarizing effects of IL-1beta. The IL-1beta stimulation of neurons induced a rapid increase in the phosphorylation of the tyrosine kinase Src and kinase suppressor of Ras (ceramide activated protein kinase (CAPK)/KSR) in neurons grown on glia from IL-1RI(-/-) mice. These effects of IL-1beta were dependent on the association of the cytosolic adaptor protein, MyD88, to the IL-1 receptor, and on the activation of the neutral sphingomyelinase, leading to production of ceramide. A cell-permeable analog of ceramide mimicked the effects of IL-1beta on the cultured AH neurons. These results suggest that ceramide may be the second messenger of the fast IL-1beta actions in AH neurons, and that this IL-1beta/ceramide pathway may underlie the fast non-transcription-dependent, electrophysiological effects of IL-1beta observed in AH neurons in vivo.

Evidence suggesting that angiotensins released not via synaptic inputs are involved in the basal activity of anterior hypothalamic neurons in rats.

Previously, we have demonstrated that angiotensin II-sensitive neurons exist in the anterior hypothalamic area (AHA) and that these neurons are tonically activated by endogenous angiotensins in rats. Chemical stimulation of the lateral septal area (LSV) and medial amygdaloid nucleus (MeA), and intracerebroventricular injection of hypertonic saline, activated AHA angiotensin II-sensitive neurons. To investigate mechanisms of the basal activity of AHA angiotensin II-sensitive neurons, we examined the effect of N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W7), a calmodulin inhibitor, applied onto AHA neurons on the basal activity and the stimulus-evoked activation of these neurons. Male Wistar rats were anesthetized and artificially ventilated. Extracellular potentials were recorded from single neurons in the AHA. Microinjections of carbachol into the LSV and corticotropin-releasing factor into the MeA, and intracerebroventricular injection of hypertonic saline, activated AHA angiotensin II-sensitive neurons. These three kinds of injection-induced activations of AHA neurons were abolished by pressure application of W7 onto the same neurons, while the calmodulin inhibitor did not affect the increase in firing of AHA neurons induced by pressure application of angiotensin II onto the same neurons. The pressure application of W7 did not affect the basal activity of AHA angiotensin II-sensitive neurons, whereas the angiotensin AT1 receptor antagonist losartan similarly applied inhibited it. These findings suggest that the basal activity of AHA angiotensin II-sensitive neurons is mediated by angiotensins released not via synaptic inputs.

Enhanced central hypertonic saline-induced activation of angiotensin II-sensitive neurons in the anterior hypothalamic area of spontaneously hypertensive and Dahl S rats.

High dietary salt intake activates the brain renin-angiotensin system in spontaneously hypertensive rats (SHR) and Dahl S rats, resulting in sympathetic hyperactivity and hypertension. Increases of sodium concentration in cerebrospinal fluid (CSF) and/or enhanced responses to CSF sodium are considered to be involved in the high dietary salt-induced activation of central nervous system pathways in those rats. Previously we have demonstrated that intracerebroventricular injection of hypertonic saline increases the neural activity of angiotensin II-sensitive neurons trans-synaptically via endogenous angiotensins in the anterior hypothalamic area (AHA) of rats. In the present study, we examined whether the AHA angiotensin II-sensitive neuron response to hypertonic saline would differ in SHR and Dahl S rats from those of their controls. Male 15- to 16-week-old SHR and age-matched Wistar Kyoto rats (WKY), Dahl S rats and Dahl R rats and Wistar rats were anesthetized and artificially ventilated. Extracellular potentials were recorded from single neurons in the AHA. Intracerebroventricular injection of hypertonic saline increased the firing rate of AHA angiotensin II-sensitive neurons. The threshold sodium concentration for the central sodium-induced increase of neural firing was lower in SHR than those of WKY, Dahl S rats, Dahl R rats and Wistar rats. The increase in neural firing induced by hypertonic saline (250 mM) was greater in SHR than those of other four kinds of rats. Similarly, the threshold sodium concentration was lower in Dahl S rats than those of WKY, Dahl R rats and Wistar rats and the increase in neural firing induced by hypertonic saline (250 mM) was greater in Dahl S rats than those of WKY, Dahl R rats and Wistar rats. In SHR, intracerebroventricular injection of the amiloride-sensitive sodium channel blocker benzamil abolished the hypertonic saline (250 mM)-induced increase in neural firing, but the sodium channel blocker itself did not affect the basal firing of these neurons. These findings indicate that central sodium-induced activation of AHA angiotensin II-sensitive neurons is enhanced in SHR and Dahl S rats.

Comparative study of the sources of neuronal projections to the site of gonadotrophin-releasing hormone perikarya and to the anteroventral periventricular nucleus in female rats.

The rat ovulatory cycle is dependent on the preoptic region encompassing the gonadotrophin-releasing hormone (GnRH) perikarya and the anteroventral periventricular nucleus (AVPV). Retrograde tract tracing was used to identify and compare the sources of inputs to these sites in female rats. Within the telencephalon and diencephalon, the incidence of retrograde labelling from both sites was moderate to abundant in the ventral lateral septum, posteromedial bed nucleus of the stria terminalis, amygdalohippocampal area and the periventricular, medial preoptic, anterodorsal preoptic, dorsomedial suprachiasmatic, arcuate, and posterior ventrolateral ventromedial hypothalamic nuclei. In these regions, the incidence of retrograde labelling was either greater from the AVPV than from the GnRH perikarya site or similar from both sites. In the medial amygdaloid, parastrial, striohypothalamic, and ventral premammillary nuclei, the retrograde labelling from the AVPV greatly exceeded the sparse incidence from the GnRH perikarya site. In contrast, retrograde labelling from the GnRH perikarya site predominated in the median preoptic, lateroanterior and dorsomedial hypothalamic nuclei, subparaventricular zone, and retrochiasmatic area; it was abundant in the AVPV. Caudal to the diencephalon, retrograde labelling from either site was sparse, except in the lateral parabrachial nucleus, which displayed a particularly high incidence from the GnRH perikarya site. Other mesencephalic regions labelled from either site included the periaqueductal gray and dorsal and median raphe nuclei. The most caudal labelling was found in the ventrolateral medulla and region of the solitary tract nucleus; this was almost exclusively from the GnRH perikarya site. These findings further elucidate the neuroanatomical connections underlying the control of the ovulatory cycle.

Posterior hypothalamus cholinergic stimulation-induced activation of anterior hypothalamic area neurons is enhanced in spontaneously hypertensive rats.

We have previously demonstrated that some neurons in the anterior hypothalamic area (AHA) are tonically activated by endogenous angiotensins in rats and that activities of these AHA neurons are enhanced in spontaneously hypertensive rats (SHR). In addition, we have demonstrated that cholinergic mechanisms in the posterior hypothalamic nucleus (PHN) are involved in the activation of AHA angiotensin-II-sensitive neurons. It has been suggested that cholinergic function in the posterior hypothalamus is enhanced in SHR and that this hyperactivity plays a role in hypertension in SHR. In the present study, we examined whether the PHN cholinergic stimulation-induced activation of AHA angiotensin-II-sensitive neurons is altered in SHR. Male 15- to 16-week-old SHR and age-matched Wistar Kyoto rats (WKY) were anesthetized and artificially ventilated. Extracellular potentials were recorded from single neurons in the AHA. Microinjection of the cholinoceptor agonist carbachol, the cholinesterase inhibitor physostigmine and the excitatory amino acid glutamate into the PHN caused increases in firing rate of AHA angiotensin-II-sensitive neurons in anesthetized WKY and SHR. The increase in firing rate of AHA neurons induced by these drugs was enhanced in SHR as compared to WKY. The enhancement of the physostigmine-induced activation of AHA neurons in SHR was similar to that of the carbachol-induced activation of AHA neurons in SHR. The enhancement of the glutamate-induced activation of AHA neurons in SHR was similar to that of the carbachol-induced activation of AHA neurons in SHR. Microinjection of scopolamine, a cholinoceptor antagonist, into the PHN caused a small but significant decrease of firing rate of AHA angiotensin-II-sensitive neurons in SHR but not in WKY. These findings indicate that the PHN cholinergic stimulation-induced activation of AHA angiotensin-II-sensitive neurons is enhanced in SHR and that PHN cholinergic mechanisms are involved in tonic activation of angiotensin-II-sensitive neurons in the AHA of SHR. It appears that the enhancement of the PHN cholinergic stimulation-induced activation of AHA neurons in SHR results mainly from the enhanced neural reactivity to angiotensins in AHA neurons of SHR.

Cholinergic stimulation in the posterior hypothalamic nucleus activates angiotensin II-sensitive neurons in the anterior hypothalamic area of rats.

We have previously reported that some neurons in the anterior hypothalamic area (AHA) are tonically activated by endogenous angiotensins in rats and that activities of these AHA angiotensin II-sensitive neurons are enhanced in spontaneously hypertensive rats (SHR). Acetylcholine in the posterior hypothalamic nucleus (PHN) has been implicated in hypertension in SHR. It is suggested that there exist neuronal projections from the PHN to the AHA in rats. In this study, we examined whether cholinergic stimulation in the PHN activates AHA angiotensin II-sensitive neurons. Male Wistar rats were anesthetized and artificially ventilated. Extracellular potentials were recorded from single neurons in the AHA. Microinjection of carbachol, physostigmine and glutamate into the PHN caused an increase in firing rate of AHA angiotensin II-sensitive neurons in anesthetized rats. The carbachol-induced increase of firing rate was inhibited by pressure application of the AT1 receptor antagonist losartan onto AHA angiotensin II-sensitive neurons. The glutamate-induced increase of firing rate was also inhibited by the pressure application of losartan. PHN microinjections of carbachol and glutamate did not affect blood pressure in these anesthetized rats. In conscious rats, PHN microinjection of carbachol produced an increase of blood pressure and the carbachol-induced pressor response was inhibited by bilateral microinjections of losartan into the AHA. These findings indicate that cholinergic stimulation in the PHN activates AHA angiotensin II-sensitive neurons. It seems likely that the activation of AHA angiotensin II-sensitive neurons induced by PHN cholinergic stimulation is partly mediated via release of angiotensins at AHA angiotensin II-sensitive neuron levels.

Cholinergic systems in the posterior hypothalamic nucleus are involved in blood pressure decrease-induced excitation of anterior hypothalamic area neurons in rats.

Previously, we have demonstrated that decreases in blood pressure induced by intravenous nitroprusside increase the firing rate of angiotensin II-sensitive neurons in the anterior hypothalamic area (AHA) of rats and that this increase of neural firing rate is blocked by the pressure application of losartan onto the same neurons. It has been suggested that acetylcholine in the posterior hypothalamic nucleus (PHN) serves as a neurotransmitter in a pathway which can modulate baroreceptor reflexes. In the present study, we examined whether acetylcholine in the PHN is involved in the nitroprusside-induced increase of the firing of angiotensin II-sensitive neurons in the AHA of rats. Male Wistar rats were anesthetized and artificially ventilated. Extracellular potentials were recorded from single neurons in the AHA. Decreases in blood pressure induced by intravenous nitroprusside (100 ug/kg) increased the firing rate of AHA angiotensin II-sensitive neurons. The nitroprusside-induced increase of firing rate of AHA neurons was inhibited by PHN microinjection of the cholinoceptor antagonist scopolamine and potentiated by PHN microinjection of the cholinesterase inhibitor physostigmine. Microinjections of carbachol and glutamate into the PHN caused increases of firing rate of AHA neurons. The carbachol-induced but not glutamate-induced increase of unit firing was abolished by the pre-microinjection of scopolamine into the same sites of the PHN. These findings suggest that the nitroprusside-induced increase of firing of AHA neurons is mediated via acetylcholine at the level of the PHN.

Cholinergic stimulation in the lateral septal area activates anterior hypothalamic area neurons via excitatory amino acid receptors in rats.

We have previously reported that some neurons in the anterior hypothalamic area (AHA) are tonically activated by endogenous angiotensins in rats and that activities of these AHA angiotensin II-sensitive neurons are enhanced in spontaneously hypertensive rats. It is suggested that there exist neuronal projections from the lateral septal area (LSV) to the AHA in rats. In this study, we examined whether neurons in the LSV are involved in activation of AHA angiotensin II-sensitive neurons. Male Wistar rats were anesthetized and artificially ventilated. Extracellular potentials were recorded from single neurons in the AHA. Microinjection of carbachol into the LSV caused an increase in firing rate of AHA angiotensin II-sensitive neurons. The carbachol-induced increase of firing rate of AHA angiotensin II-sensitive neurons was inhibited by pressure application of the excitatory amino acid receptor antagonist kynurenate but not by the AT1 receptor antagonist losartan onto the same neurons. Microinjection of carbachol into the LSV also increased the firing rate of AHA ACh-sensitive neurons, and the carbachol-induced increase of firing rate of ACh-sensitive neurons was again abolished by pressure application of kynurenate but not by the muscarinic receptor antagonist scopolamine onto the same neurons. Microinjection of the muscarinic receptor antagonist 4-DAMP into the LSV did not affect the firing rate of AHA angiotensin II-sensitive neurons. These findings indicate that neurons in the LSV are involved in activation of AHA angiotensin II-sensitive neurons. It seems likely that the carbachol-induced activation of AHA angiotensin II-sensitive neurons is mainly mediated via excitatory amino acid receptors at AHA neurons.

Sex differences in the location of immunochemically defined cell populations in the mouse preoptic area/anterior hypothalamus.

The preoptic area/anterior hypothalamus (POA/AH) is sexually dimorphic in many vertebrates. We have defined specific cell populations within the POA/AH using immunocytochemical markers for estrogen receptor beta (ERbeta) and the R1 subunit of the GABA(B) receptor (GABA(B)R1). Our previous finding of sex differences in cell migration in this region in embryonic day 15 mice led us to examine sex differences in the location or size of chemically identified cell groups. At embryonic day 17 (E17), cells containing immunoreactive (ir) ERbeta in females were located more dorsal and lateral than those in males. In contrast to this positional sex difference seen at E17, ERbeta expression at P0 and adulthood showed a sex difference in cell number and area of immunoreactivity with a higher expression of ERbeta in males than females. Furthermore, in animals that were genetically deprived of gonadal and adrenal hormones by virtue of a disrupted gene coding for steroidogenic factor 1, cells containing ir ERbeta followed a female phenotype for location at E17 and a female phenotype for number of ir cells at P0 regardless of genetic sex, suggesting that circulating hormones may be influencing cell position in the POA/AH. A second phenotypically identified cell group containing ir GABA(B)R1 also had a sex difference in cell positions at E17. Females expressed GABA(B)R1 in cells with a more dorsal position than in males. These results provide support for the suggestion that sex differences in cellular organization in the developing hypothalamus arise from sex differences in cell migration.

Melanin concentrating hormone (MCH): a novel neural pathway for regulation of GnRH neurons.

The link between the state of energy balance and reproductive function is well known. Thus, signals denoting negative energy balance and the accompanying hyperphagic drive are likely to be factors in the suppression of gonadotropin releasing hormone (GnRH) activity. We have previously found that appetite-regulating systems, such as neuropeptide Y (NPY) in the arcuate nucleus (ARH) and orexin in the lateral hypothalamic area (LHA), send fiber projections that come in close apposition with GnRH neurons. Furthermore, the appropriate receptors, NPY Y5 and OR-1, respectively, are coexpressed on GnRH neurons, providing neuroanatomical evidence for a direct link between the NPY and orexin systems and GnRH neurons. Therefore, these orexigenic neuropeptide systems are potential candidates that convey information about energy balance to GnRH neurons. The current studies focused on melanin concentrating hormone (MCH), another orexigenic neuropeptide system located in the LHA that is sensitive to energy balance. The results showed that MCH fiber projections came in close apposition with approximately 85-90% of GnRH cell bodies throughout the preoptic area and anterior hypothalamic area in the rat. In addition, the MCH receptor (MCHR1) was coexpressed on about 50-55% of GnRH neurons. These findings present evidence for a possible direct neuroanatomical pathway by which MCH may play a role in the regulation of GnRH neuronal function. Thus, MCH is another potential signal that may serve to integrate energy balance and reproductive function.

A sexually dimorphic hypothalamic nucleus in a macaque species with frequent female-female mounting and same-sex sexual partner preference.

In some captive and free-ranging populations, unmanipulated female Japanese macaques (Macaca fuscata) routinely court, mount (with pelvic thrusting), compete for, and even prefer, on occasion, certain female sexual partners over certain males. The goal of this study was to determine if the cytoarchitecture of the dorsocentral portion of the anterior hypothalamic nucleus (AHdc), was male-typical in female Japanese macaques drawn from one such population. The AHdc is located in the medial preoptic anterior hypothalamus (MPO-AH), a region of the brain that is known to regulate sexual behaviour in primates. Despite their potential for male-typical sexual behaviour and sexual partner preference, our female subjects did not possess male-typical AHdc. The AHdc was significantly larger in males than it was in females, a difference that could be attributed to the significantly larger number of neurons in the male AHdc compared to that of the females. The AHdc of female Japanese macaques were no more male-typical in size than those of female rhesus macaques, a closely related sister species in which females rarely exhibit male-typical sexual behaviour. Although the AHdc may be involved in the regulation of sexual behaviour, this study indicates that a male-typical AHdc is not a prerequisite for the expression of male-typical sexual behaviour and sexual partner preference in Japanese macaques. This study is the first to examine the relationship between sex-atypical sexual activity and the cytoarchitecture of a hypothalamic nucleus in hormonally unmanipulated females.