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.     

Estrogen and progesterone do not activate Fos in AVPV or LHRH neurons in male rats

In rodents, females but not males, in response to escalating levels of estrogen, express a luteinizing hormone (LH) surge that is prompted by a surge in luteinizing hormone-releasing hormone (LHRH). It cannot take place if estrogen-sensitive afferents located in the anteroventral periventricular nucleus (AVPV) are either absent or disabled. Males appear to lack the ability to exhibit an LH surge, but it is unclear what level of the CNS contributes to this dimorphic response. This study was conducted to determine whether estrogen followed by progesterone treatment (E + P) of gonadectomized males evokes Fos activation in LHRH and AVPV neurons as it does in females. The results indicated that, consistent with the males' inability to express an LH surge in response to E + P treatment, LHRH and AVPV neurons in males failed to show increased Fos activation. Examination of neuron nuclear antigen (NeuN, a neuron-specific marker), estrogen receptor (ERalpha) and progesterone receptor (PR) neurons in AVPV neurons indicated that, while essentially all the neurons of the caudal AVPV in males and females are steroid responsive, the male possessed half the number of steroid responsive neurons within the caudal AVPV (where activation of Fos is maximal in females) compared to the female. Together, these data indicate that the male lacks a substantial population of steroid receptive AVPV neurons and is unable to respond to the presence of E and P and activate either AVPV or LHRH neurons.     

Effect of prolonged exposure to low concentrations of formaldehyde on the corticotropin releasing hormone neurons in the hypothalamus and adrenocorticotropic hormone cells in the pituitary gland in female mice

We examine the effect on the hypothalamus-pituitary-adrenal gland (HPA) axis of prolonged exposure to low levels of formaldehyde in female C3H/He mice, using immunocytochemical and RT-PCR methods. Two groups of female mice were exposed to differing concentrations (0, 80, 400, 2000 ppb) of formaldehyde inhalation for 16 h/day, 5 days/week, for 12 weeks. The corticotropin releasing hormone (CRH)-immunoreactive (ir) neurons in the hypothalamus were then examined, together with the adrenocorticotropin hormone (ACTH)-ir cells and ACTH mRNA in the pituitary. One group comprised sham control mice. The other group was made allergic by injection of ovalbumin (OVA) and alum prior to exposure to formaldehyde, since most sick building syndrome (SBS) sufferers are women with allergic disease. These animals were further exposed to aerosolized OVA as a booster four times during the exposure period. Our results showed a dose-dependent increase in the number of CRH-ir neurons in the non-allergy (NAG) group. A similar pattern was found in ACTH-ir cells and ACTH mRNA. The allergy (AG) model group showed an increase in basal levels of all markers of HPA activity. Moreover, the AG mice appeared to respond to the lowest concentration of formaldehyde, and all indices of HPA activity were reduced at the highest concentrations of formaldehyde. These results relate to an important clinical issue and also have implications in the broader area of HPA regulation. We conclude that our experimental system may be a suitable animal model for SBS and/or multiple chemical sensitivity (MCS).     

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.     

Distribution of androgen-binding protein in the rat hypothalamo-neurohypophyseal system, co-localization with oxytocin

Androgen-binding protein (ABP) is known to be expressed in the male and female rat hypothalamus. In the present study, we observed immunocytochemically ABP in neurons of the magnocellular hypothalamic nuclei, in the preoptic region and in the lateral hypothalamus. Dense fiber networks with varicosities, containing ABP immunofluorescence, were visible throughout the hypothalamus, the median eminence and in the posterior pituitary lobe. Double immunostaining revealed a partial coexistence of ABP-and oxytocin immunoreactivity in a portion of the magnocellular perikarya. ABP was isolated by affinity chromatography from hypothalamus homogenates. Western blots resulted in immunoreactive (IR) bands with an approximate molecular weight of 35 and 50 kDa. Mass spectrometry of these preparations confirmed the presence of ABP, which was almost identical to ABP isolated from rat testis. It is likely that ABP, expressed in magnocellular oxytocinergic neurons, is subject to axonal transport and release in the hypothalamo-neurohypophyseal system.     

Tonic angiotensinergic inputs to neurons in the anterior hypothalamic area of rats

We have previously reported that microinjection of angiotensin II into the anterior hypothalamic area (AHA) produces a pressor response in rats and that the angiotensin AT1 receptor antagonist, losartan, similarly injected causes a depressor response in hypertensive rats. In this study, we examined whether endogenous angiotensins are involved in activation of neurons in the AHA. Male Wistar rats were anesthetized and artificially ventilated. Extracellular potentials were recorded from single neurons in the AHA. Pressure-ejected application of angiotensin II and glutamate onto some neurons in the AHA increased their firing rate. The increase of unit firing induced by angiotensin II but not by glutamate was inhibited by losartan. Application of losartan alone inhibited the basal firing rate of angiotensin II-sensitive neurons in a concentration-dependent manner. Application of the angiotensin AT2 receptor antagonist, PD123319, did not affect the increase of unit firing induced by angiotensin II and the basal firing rate of angiotensin II-sensitive neurons. Pressure application of angiotensin I onto angiotensin II-sensitive neurons also increased firing rate and the increase of unit firing by angiotensin I was inhibited by the angiotensin converting enzyme inhibitor, captopril. Captopril alone inhibited the basal firing rate of angitensin II-sensitive neurons. Acetylcholine did not affect unit firing of angiotensin II-sensitive neurons, whereas it increased the firing rate of some angiotensin II-insensitive neurons in the AHA. Increases of blood pressure by intravenous phenylephrine completely inhibited the basal firing rate of angiotensin II-sensitive neurons. These findings suggest that some neurons in the AHA are tonically activated by endogenous angiotensins. It seems likely that newly synthesized angiotensins are used for the angiotensinergic transmission in the AHA.     

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.     

Glutamate agonists activate the hypothalamic-pituitary-adrenal axis through hypothalamic paraventricular nucleus but not through vasopressinerg neurons

The hypothalamic-pituitary-adrenal (HPA) axis plays a crucial role in the stress processes. The nucleus paraventricularis hypothalami (PVN) with corticotropin-releasing hormone (CRH)-containing and arginine vasopressin (AVP)-containing neurons is the main hypothalamic component of the HPA. The glutamate, a well-known excitatory neurotransmitter, can activate the HPA inducing adrenocorticotropin hormone (ACTH) elevation. The aim of our study was to examine the involvement of PVN and especially AVP in glutamate-induced HPA activation using agonists of the N-methyl-d-aspartate (NMDA) and kainate receptors. Two approaches were used: in male Wistar rats the PVN was lesioned, and AVP-deficient homozygous Brattleboro rats were also studied. Blood samples were taken through indwelling cannula and ACTH, and corticosterone (CS) levels were measured by radioimmunoassay. The i.v. administered NMDA (5 mg/kg) or kainate (2.5 mg/kg) elevated the ACTH and CS levels already at 5 min in control (sham-operated Wistar or heterozygous Brattleboro) rats. The PVN lesion had no influence on basal ACTH and CS secretion but blocked the NMDA- or kainate-induced ACTH and CS elevations. The lack of AVP in the Brattleboro animals had no significant influence on the basal or glutamate-agonists-induced ACTH and CS elevations. Our results suggest that NMDA and kainate may activate the HPA axis at central (PVN) level and not at the level of pituitary or adrenal gland and that AVP has minor role in glutamate-HPA axis interaction. The time course of the ACTH secretion was different with NMDA or kainate. If we compared the two curves, the results were not coherent with the general view that NMDA activation requires previous kainate activation. Although it has to be mentioned that the conclusion which can be drawn is limited, the bioavailability of the compounds could be different as well.     

The medial amygdaloid area is involved in activation of angiotensin II-sensitive neurons in the anterior hypothalamic area

We have previously reported that some neurons in the anterior hypothalamic area (AHA) are tonically activated by endogenous angiotensins in rats and that the activities of these AHA angiotensin II-sensitive neurons are enhanced in spontaneously hypertensive rats. It is suggested that there exist neural projections from the medial amygdala to the AHA in rats. In this study, we examined whether neurons in the medial amygdaloid area (MeA) are involved in the 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 glutamate into the MeA caused an increase in the firing rate of AHA angiotensin II-sensitive neurons. The glutamate-induced increase of firing rate was inhibited by pressure application of the AT1 receptor antagonist losartan onto AHA angiotensin II-sensitive neurons. The microinjection of glutamate into the central amygdaloid area also increased the firing rate of AHA angiotensin II-sensitive neurons, but the glutamate-induced increase of firing rate was not affected by pressure application of losartan onto AHA angiotensin II-sensitive neurons. The microinjection of corticotropin-releasing factor (CRF) into the MeA also increased the firing rate of AHA angiotensin II-sensitive neurons, but the CRF-induced increase of firing rate was not inhibited by pressure application of losartan onto AHA angiotensin II-sensitive neurons. Repeated microinjection of glutamate into the MeA caused an increase in the release of angiotensins in the AHA. These findings indicate that neurons in the MeA are involved in the activation of AHA angiotensin II-sensitive neurons. It seems likely that the activation of AHA angiotensin II-sensitive neurons induced by glutamate but not CRF is partly mediated via the release of angiotensins at AHA angiotensin II-sensitive neuron levels.     

Electrophysiological evidences for the contribution of NMDA receptors to the inhibition of clonidine on the RVLM presympathetic neurons

The main objective of this study is to test the hypothesis that N-methyl-D-aspartate (NMDA) receptors within the rostral ventrolateral medulla (RVLM) are involved in the inhibition of clonidine on the RVLM presympathetic neurons. Totally, 22 presympathetic neurons were recorded in anesthetized and paralyzed rats. The majority of these neurons (n=16 of 22) were significantly inhibited by iontophoretic (30 nA) clonidine, the other 6 neurons were insensitive to clonidine. In seven clonidine-sensitive neurons, iontophoretic clonidine (30 nA) antagonized the neuronal excitation of iontophoretic NMDA receptor agonist NMDA (20 nA). In remaining nine clonidine-sensitive neurons, iontophoretic NMDA receptor antagonist MK801 (60 nA) significantly attenuated the neuronal inhibition of iontophoretic (30 nA) clonidine. In conclusion, these results suggest that NMDA receptors contribute to the inhibition of clonidine on the RVLM presympathetic neurons.     

Ionotropic glutamate receptors mediate excitatory drive to caudal medullary expiratory neurons in the rabbit

Most of the neurons of the caudal ventral respiratory group (cVRG) are bulbospinal expiratory neurons that receive their main excitatory drive from more rostral, but not yet defined regions. This study was devoted to investigate the functional role of ionotropic excitatory amino acid (EAA) receptors in the excitatory drive transmission to cVRG expiratory neurons during eupnoeic breathing and some respiratory reflexes including cough induced by mechanical stimulation of the tracheobronchial tree. The experiments were performed on spontaneously breathing rabbits under pentobarbitone anesthesia making use of microinjections (30--50 nl) of EAA receptor antagonists into the cVRG. Phrenic nerve and abdominal muscle activities were recorded. Bilateral microinjections of 50 mM kynurenic acid (KYN), a broad-spectrum EAA antagonist, and 10 mM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a non-NMDA antagonist, or 5 mM 6-nitro-7-sulphamoylbenzo(f)quinoxaline-2,3-dione (NBQX), a more specific non-NMDA antagonist, completely suppressed spontaneous rhythmic abdominal activity and reflex expiratory responses either to tracheal occlusion at end-inspiration (Breuer-Hering inflation reflex) or to expiratory threshold loading (5 cm H(2)O); they also suppressed both the inspiratory and expiratory components of the cough reflex. Spontaneous rhythmic abdominal activity and the reflex respiratory responses were strongly reduced, but not completely abolished by microinjections of 10 mM d(-)-2-amino-5-phosphonopentanoic acid (D-AP5), an NMDA antagonist. The results provide evidence that the excitatory drive to cVRG bulbospinal expiratory neurons during eupnoeic breathing and the investigated respiratory reflexes is mediated by EAA receptors. They also support the view that neurons located in the cVRG are not merely elements of the expiratory output system.     

Responses to GABA(A) receptor activation are altered in NTS neurons isolated from chronic hypoxic rats

The inhibitory amino acid GABA is released within the nucleus of the solitary tract (NTS) during hypoxia and modulates the respiratory response to hypoxia. To determine if responses of NTS neurons to activation of GABA(A) receptors are altered following exposure to chronic hypoxia, GABA(A) receptor-evoked whole cell currents were measured in enzymatically dispersed NTS neurons from normoxic and chronic hypoxic rats. Chronic hypoxic rats were exposed to 10% O(2) for 9-12 days. Membrane capacitance was the same in neurons from normoxic (6.9+/-0.5 pF, n=16) and hypoxic (6.3+/-0.5 pF, n=15) rats. The EC(50) for peak GABA-evoked current density was significantly greater in neurons from hypoxic (21.7+/-2.2 microM) compared to normoxic rats (12.2+/-0.9 microM) (p<0.001). Peak and 5-s adapted GABA currents evoked by 1, 3 and 10 microM were greater in neurons from normoxic compared to hypoxic rats (p<0.05) whereas peak and 5-s adapted responses to 30 and 100 microM GABA were not different comparing normoxic to hypoxic rats. Desensitization of GABA(A)-evoked currents was observed at concentrations greater than 3 microM and, measured as the ratio of the current 5 s after the onset of 100 microM GABA application to the peak GABA current, was the same in neurons from normoxic (0.37+/-0.03) and hypoxic rats (0.33+/-0.04). Reduced sensitivity to GABA(A) receptor-evoked inhibition in chronic hypoxia could influence chemoreceptor afferent integration by NTS neurons.     

Schwann cell: a source of neurotrophic activity on cortical glutamatergic neurons in culture

Glial cells secrete numerous soluble molecules that enhance the development and the survival of different neuronal types cultured in vitro. Schwann cells (SC) play an important role as they are the source of different trophic substances and present a great neurotrophic activity. The aim of this study is to investigate the influence of postnatal SC on embryonic glutamatergic neurons. Co-cultures of SC from sciatic nerve of postnatal rats and neurons from rat embryonic cerebral cortex were successfully established, and cells were immunocytochemically characterized using mono and polyclonal antibodies as different glial and neuronal markers. Furthermore, some neuronal cultures were added with Nerve Growth Factor (NGF) and Insulin-like Growth Factor (IGF) to compare to co-cultures. Our results show that SC promote an increase in the number of glutamatergic cortical neurons; moreover, these neurons present an evidence of dense axonal and dendritic outgrowth even when were fed with conditioned medium obtained from SC cultures. In conclusion, our data suggest that substances produced by SC exert a positive effect on central neuron survival and differentiation as indicated by processes of elongation and that this activity is mediated by soluble factors. Therefore, it is possible to consider the SC as a source of growth factors and might be suitable for the development of a neuroprotective effect in neurodegenerative disorders.     

Bacterial lipopolysaccharide (LPS)-induced type 2 iodothyronine deiodinase (D2) activation in the mediobasal hypothalamus (MBH) is independent of the LPS-induced fall in serum thyroid hormone levels

By administration of bacterial lipopolysaccharide (LPS) to intact and T4-replaced thyroidectomized rats, we demonstrate that in contrast to the cortex and anterior pituitary, there is a persistent increase in type 2 iodothyronine deiodinase (D2) activity in the mediobasal hypothalamus (MBH). We propose that endotoxin-induced D2 activation in the MBH is independent of circulating levels of thyroid hormone and that this mechanism may contribute to central hypothyroidism associated with infection.     

Biosynthesis of gonadotropin releasing hormone (GnRH): present status

Although the mechanism of GnRH synthesis has not yet been elucidated, many experimental data argue in favour of a ribosomal pathway resulting in the biosynthesis of a precursor with a size larger than the decapeptide. The existence of high mol. wt (HMW) immunoreactive forms of GnRH in hypothalamic extracts has been reported but without any evidence that they are precursors of the hypothalamic hormone. Some results obtained with immunocytological methods also suggest that at least a part of immunoreactive GnRH in perikarya might be extended on one or both its terminal ends. In our laboratory, we first investigated the process of GnRH biosynthesis in vivo by infusing tritiated amino acids into the third ventricle of rats. The results, along with the demonstration of the existence of HMW GnRH, support a ribosomal mechanism of GnRH synthesis. Also, preliminary data from our laboratory demonstrate that poly (A+) enriched RNA preparations from rat hypothalami contain compounds capable of hybridizing to a synthetic oligodeoxynucleotide probe which is complementary to a part of the GnRH sequence.     

Involvement of serotoninergic receptors in the anteroventral preoptic region on hypoxia-induced hypothermia

Hypoxia causes a regulated decrease in body temperature (Tb). There is circumstantial evidence that the neurotransmitter serotonin (5-HT) in the anteroventral preoptic region (AVPO) mediates this response. However, which 5-HT receptor(s) is (are) involved in this response has not been assessed. Thus, we investigated the participation of the 5-HT receptors (5-HT1, 5-HT2, and 5-HT7) in the AVPO in hypoxic hypothermia. To this end, Tb of conscious Wistar rats was monitored by biotelemetry before and after intra-AVPO microinjection of methysergide (a 5-HT1 and 5-HT2 receptor antagonist, 0.2 and 2 microg/100 nL), WAY-100635 (a 5-HT(1A) receptor antagonist, 0.3 and 3 microg/100 nL), and SB-269970 (a 5-HT7 receptor antagonist, 0.4 and 4 micro/100 nL), followed by 60 min of hypoxia exposure (7% O2). During the experiments, the mean chamber temperature was 24.6 +/- 0.7 degrees C (mean +/- SE) and the mean room temperature was 23.5 +/- 0.8 degrees C (mean +/- SE). Intra-AVPO microinjection of vehicle or 5-HT antagonists did not change Tb during normoxic conditions. Exposure of rats to 7% of inspired oxygen evoked typical hypoxia-induced hypothermia after vehicle microinjection, which was not affected by both doses of methysergide. However, WAY-100635 and SB-269970 treatment attenuated the drop in Tb in response to hypoxia. The effect was more pronounced with the 5-HT7 antagonist since both doses (0.4 and 4 microg/0.1 microL) were capable of attenuating the hypothermic response. As to the 5-HT(1A) antagonist, the attenuation of hypoxia-induced hypothermia was only observed at the higher dose. Therefore, the present results are consistent with the notion that 5-HT acts on both 5-HT(1A) and 5-HT7 receptors in the AVPO to induce hypothermia, during hypoxia.     

Reticulospinal neurons inactivated by warming of the preoptic area and anterior hypothalamus of rabbits

To identify the premotor neurons for vasoconstrictors of the skin, activities of reticulospinal neurons in the rostroventral medulla, the ear sympathetic nerve (ESNA) and the renal sympathetic nerve (RSNA) were recorded in anesthetized and immobilized Japanese White or New Zealand White rabbits. Two groups of neurons were identified according to their responses to thermal stimulation of the preoptic area and the anterior hypothalamus (POAH) and to electrical stimulation of baroreceptor afferents, the aortic nerve (AN). Neurons (Type I neurons, n = 21) whose activity was inhibited by warm stimulation of the POAH but not inhibited by the AN stimulation were located in sites medial to the rostral ventrolateral medulla (RVLM). The other neurons (Type II neurons, n = 20) whose activity was not inhibited by warm stimulation of the POAH but inhibited by the AN stimulation were located in the RVLM. Because the time course of the inhibitory response of Type I neurons to warm stimulation of the POAH was very similar to that of the inhibitory response of the ESNA and activities of these neurons and the ESNA were not inhibited by the stimulation of the AN, it was suggested the Type I neurons might participate in regulation of activity of the vasoconstrictors of the ear skin. The Type II neurons are considered to be the barosensitive RVLM neurons that regulate systemic arterial pressure by controlling the activity of visceral or muscular sympathetic vasoconstrictors or cardiac sympathetic fibers.     

Characterization of neurons of the nucleus tractus solitarius pars centralis

Esophageal sensory afferent inputs terminate principally in the central subnucleus of the tractus solitarius (cNTS). Neurons of the cNTS comprise two major neurochemical subpopulations. One contains neurons that are nitric oxide synthase (NOS) immunoreactive (-IR) while the other comprises neurons that are tyrosine hydroxylase (TH)-IR. We have shown recently that TH-IR neurons are involved in esophageal-distention induced gastric relaxation. We used whole cell patch clamp techniques in rat brainstem slices combined with immunohistochemical and morphological reconstructions to characterize cNTS neurons. Postrecording reconstruction of cNTS neurons revealed two morphological neuronal subtypes; one group of cells (41 out of 131 neurons, i.e., 31%) had a multipolar soma, while the other group (87 out of 131 neurons, i.e., 66%) had a bipolar soma. Of the 43 cells in which we conducted a neurochemical examination, 15 displayed TH-IR (9 with bipolar morphology, 6 with multipolar morphology) while the remaining 28 neurons did not display TH-IR (18 with bipolar morphology, 10 with multipolar morphology). Even though the range of electrophysiological properties varied significantly, morphological or neurochemical distinctions did not reveal characteristics peculiar to the subgroups. Spontaneous excitatory postsynaptic currents (sEPSC) recorded in cNTS neurons had a frequency of 1.5 +/- 0.15 events s(-1) and an amplitude of 27 +/- 1.2 pA (Vh = -50 mV) and were abolished by pretreatment with 30 muM AP-5 and 10 muM CNQX, indicating the involvement of both NMDA and non-NMDA receptors. Some cNTS neurons also received a GABAergic input that was abolished by perfusion with 30-50 muM bicuculline. In conclusion, our data show that despite the heterogeneity of morphological and neurochemical membrane properties, the electrophysiological characteristics of cNTS neurons are not a distinguishing feature.     

Changes of melanin-concentrating hormone related to LHRH release in the median eminence of rats

The present research was carried out to study the distribution of melanin-concentrating hormone (MCH) fibers in the median eminence of rats and to evaluate if changes in the MCH content of the median eminence could be related to the release of LHRH. Immunocytochemical studies in the median eminence of males and estrous females showed the presence of MCH fibers, mainly in its internal layer. Diestrous and proestrous animals displayed MCH immunoreactivity in both the internal and external layers of the median eminence. Longitudinal sections of the median eminence in proestrous animals showed that MCH-immunoreactive (ir) density is higher at 12 than at 9 h in both layers of the median eminence. MCH was assayed by radioimmunoassay in median eminences of males and in females in all stages of the estrous cycle at 10 h. It was observed that the content of MCH at diestrus-1 and -2 was higher than in estrus and in male rats. In the day of proestrus, MCH and LHRH were assayed at 10, 12, 13, 14, 15 and 17 h. At 12 h, the content of MCH and LHRH showed the maximal values. At 13 h, MCH content showed a decline, while LHRH was still high. At 14 h, the LHRH content started to decrease. The present results suggest that MCH is involved in the regulation of LHRH release in the female rat.     

Tracheal tone and the role of ionotropic glutamate receptors in the nucleus ambiguus

Excitatory amino acid (EAA) transmission in the rostral portion of the ventrolateral medulla (RVLM) appears to be required for reflex airway constriction. The purpose of the present study was to determine whether our prior observations of right sided dominance are also reflected at the level of the RVLM, particularly at the right nucleus ambiguus (NA). Microinjection of glutamate (109 nl, 100 mM) into the right NA of anesthetized dogs produced significant increases in tracheal smooth muscle tone as assessed by pressure changes in a saline filled cuff of the endotracheal tube (Delta15 +/- 2 mm Hg). Increasing inspired CO2 increased tracheal cuff pressure (Delta33 +/- 2 mm Hg) which was abolished by microinjections of the EAA antagonist kynurenic acid (109 nl, 100 mM) or the non-NMDA antagonist 6-nitro-7-sulfamobenzoquinoxaline-2,3-dione (NBQX, 109 nl, 10 mM) into the right NA. These results confirm that EAA receptors in the right NA are capable of modulating tracheal tone and suggest that non-NMDA receptors in the right NA are required for reflex airway constriction produced by systemic hypercapnia. Furthermore, the effects of right-sided NA microinjections in the present study, although not completely definitive, are consistent with previous studies that suggest dominant control of canine tracheal tone by the right vagus.