Pituitary adenylate cyclase-activating polypeptide directly modulates the activity of proopiomelanocortin neurons in the rat arcuate nucleus

Pituitary adenylate cyclase-activating polypeptide (PACAP) and the proopiomelanocortin (POMC)-derived peptide alpha-melanocyte-stimulating hormone (alpha-MSH) both regulate multiple neuroendocrine functions and feeding behavior. Two subtypes of PACAP receptor mRNAs, pituitary adenylate cyclase-activating polypeptide-specific receptor (PAC1-R) and pituitary adenylate cyclase-activating polypeptide/vasoactive intestinal polypeptide mutual receptor (VPAC2-R), are actively expressed in the arcuate nucleus of the hypothalamus, where POMC cell bodies are located. This observation led us to investigate the possible regulatory action of PACAP on rat POMC neurons. Double-labeling in situ hybridization histochemistry revealed that approximately 50% of POMC-producing neurons express PAC1-R and/or VPAC2-R mRNAs. The proportion of POMC neurons that also contain PAC1-R mRNA was homogeneous along the rostro-caudal axis of the arcuate nucleus while POMC-positive cell bodies expressing the VPAC2-R subtype were more abundant in the rostral region. Incubation of mediobasal hypothalamic explants with PACAP (10(-7) M; 30 min) increased POMC mRNA expression, and this effect was blocked by PACAP6-38 (10(-6) M). In contrast, incubation with vasoactive intestinal polypeptide (10(-7) M) did not affect POMC mRNA level. Incubation of hypothalamic fragments with PACAP (10(-7) M) caused a significant increase in alpha-MSH content in the tissue and in the incubation medium. Altogether, the present results reveal that exogenous PACAP, acting probably through PAC1-R, regulates the activity of POMC neurons in the rat hypothalamus. These data suggest that the effects of PACAP on the gonadotropin-releasing hormone neuroendocrine axis and the regulation of feeding behavior may be mediated, at least in part, through modulation of POMC neurons.     

Biphasic modulation by galanin of excitatory synaptic transmission in substantia gelatinosa neurons of adult rat spinal cord slices

Although intrathecally administrated galanin modulates nociceptive transmission in a biphasic manner, this has not been fully examined previously. In the present study, the action of galanin on synaptic transmission in the substantia gelatinosa (SG) neurons of adult rat spinal cord slices was examined, using the whole cell patch-clamp technique. Galanin concentration-dependently increased the frequency of spontaneous excitatory postsynaptic current (EPSC; EC(50) = 2.0 nM) without changing the amplitude, indicating a presynaptic effect. This effect was reduced in a Ca(2+)-free, or voltage-gated Ca(2+) channel blocker La(3+)-containing Krebs solution and was produced by a galanin type-2/3 receptor (GalR2/R3) agonist, galanin 2-11, but not by a galanin type-1 receptor (GalR1) agonist, M617. Galanin also concentration-dependently produced an outward current at -70 mV (EC(50) = 44 nM), although this appeared to be contaminated by a small inward current. This outward current was mimicked by M617, but not by galanin 2-11. Moreover, galanin reduced monosynaptic Aδ-fiber- and C-fiber-evoked EPSC amplitude; the former reduction was larger than the latter. A similar action was produced by galanin 2-11, but not by M617. Spontaneous and focally evoked inhibitory (GABAergic and glycinergic) transmission was unaffected by galanin. These findings indicate that galanin at lower concentrations enhances the spontaneous release of l-glutamate from nerve terminals by Ca(2+) entry from the external solution following GalR2/R3 activation, whereas galanin at higher concentrations also produces a membrane hyperpolarization by activating GalR1. Moreover, galanin reduces l-glutamate release onto SG neurons from primary afferent fibers by activating GalR2/R3. These effects could partially contribute to the behavioral effect of galanin.     

Association of interleukin-1 beta polymorphisms with idiopathic Parkinson's disease

We have isolated a novel hypothalamic peptide, Galanin-like peptide (GALP), as a ligand for galanin receptor subtype GalR2. To investigate the physiological role of GALP, we examined the effect of the intracerebroventricular administration of GALP and found that GALP induced food intakes. GALP had ten-fold the orexigenic activity of galanin. We also observed the anxiogenic-like behavior after the administration of 1 nmol GALP. These results suggest that GALP is a novel orexigenic and anxiogenic peptide.     

Restricted distribution of galanin receptor 3 (GalR3) mRNA in the adult rat central nervous system

Recent molecular cloning studies have established the existence of a third rat galanin receptor subtype, GalR3, however its precise distribution in the mammalian central nervous system (CNS) is not well established. In the present study, we examined the regional and cellular distribution of GalR3 mRNA in the CNS of the rat by in situ hybridization. Our findings indicate that GALR3 mRNA expression in the rat brain is discrete and highly restricted, concentrated mainly in the preoptic/hypothalamic area. Within the hypothalamus, GalR3 expression was confined to the paraventricular, ventromedial and dorsomedial hypothalamic nuclei. In addition to these hypothalamic nuclei, GalR3 mRNA-expressing cells were observed in the medial septum/diagonal band of Broca complex, the bed nucleus of the stria terminalis, the medial amygdaloid nucleus, the periaqueductal gray, the lateral parabrachial nucleus, the dorsal raphe nucleus, the locus coeruleus, the medial medullary reticular formation and in one of the circumventricular organs, the subfornical organ. In the spinal cord, a faint but specific ISH signal was observed over the laminae I–II with a few moderately labeled cells distributed in laminae V and X. The neuroanatomical distribution of GalR3 suggests it might be involved in mediating documented effects of galanin on food intake, fluid homeostasis, cardiovascular function and nociception.     

Bidirectional regulation of stress responses by galanin in mice: Involvement of galanin receptor subtype 1

The neuropeptide galanin has been shown to play a role in psychiatric disorders as well as in other biological processes including regulation of pain threshold through interactions with three G-protein coupled receptors, galanin receptor subtypes 1–3 (GalR1−3). While most of the pharmacological studies on galanin in stress-related disorders have been done with rats, the continuous development of genetically engineered mice involving galanin or its receptor subtype(s) validates the importance of mouse pharmacological studies. The present study on mice examined the homeostatic, endocrinological and neuroanatomical effects of the galanin, injected intracerebroventricularly (i.c.v.), in regulation of stress responses after restraint stress. Furthermore, the roles of GalR1 on these effects were studied using GalR1 knockout (KO) mice. The core body temperature and the locomotor activity were monitored with radio telemetry devices. Galanin (i.c.v.) decreased locomotor activity and exerted a bidirectional effect on the restraint stress–induced hyperthermia; a high dose of galanin significantly attenuated the stress-induced hyperthermic response, while a low dose of galanin moderately enhanced this response. The bidirectional effect of galanin was correlated with changes in stress hormone levels (adrenocorticotropic hormone and corticosterone). To neuroanatomically localize the effects of galanin on stress response, cFos immunoreactivity was assessed in galanin receptor rich areas; paraventricular nucleus (PVN) of the hypothalamus and the locus coeruleus (LC), respectively. A high dose of galanin significantly induced cFos activity in the LC but not in the PVN. In GalR1KO mice, a high dose of galanin failed to induce any of the above effects, suggesting the pivotal role of GalR1 in decreased locomotor activity and stress-resistant effects caused by galanin i.c.v. injection studied here.     

Discharge of gonadotrophin-releasing hormone from the mediobasal part of the hypothalamus: Effect of stimulation frequency and gonadal steroids

Summary The release of gonadotrophin-releasing hormone (Gn-RH), in response to electrical stimulation of the mediobasal part of the hypothalamus incubated in vitro, was studied in both male and female rats. In male rats significant release of Gn-RH occurred during the 10-min experimental period only when the incubated tissue was stimulated at frequencies of 10 Hz or greater. There was no release when stimulated at 5 Hz. There was also no release of hormone when the mediobasal hypothalami were incubated in a Ca²⁺ free medium. The amount of Gn-RH released during a 10-min incubation period increased progressively as the frequency of stimulation was raised from 10–100 Hz. During short (4-min) incubation periods the effectiveness of each stimulus pulse for Gn-RH release also increased with the frequency of stimulation. However, when stimulated for 10 min there was no increase in hormone released per stimulus pulse when frequency of stimulation was raised above 10 Hz. The amount of Gn-RH released in response to stimulation at 50 Hz was greater in male rats than in females. For the females, there was no significant difference between the amounts of Gn-RH released at dioestrus and pro-oestrus. In both male and female rats gonadectomised 4 weeks prior to hypothalamic incubation, the response to electrical stimulation at 50 Hz was reduced when compared with intact controls. Indeed, for the females there was no longer a statistically significant increase in the amount of Gn-RH in the incubation medium after 50 Hz stimulation. Ovariectomised female rats, injected twice daily for 3.5 days with 20 g of oestradiol benzoate released Gn-RH in response to 50 Hz stimulation in the same amounts as intact control animals. By contrast, there was no recovery of Gn-RH release to normal levels in castrated male rats similarly treated with 1.25 mg testosterone propionate.     

Sex differences in the cannabinoid modulation of an A-type K+ current in neurons of the mammalian hypothalamus

Cannabinoids regulate biological processes governed by the hypothalamus including, but not limited to, energy homeostasis and reproduction. The present study sought to determine whether cannabinoids modulate A-type K(+) currents (I(A)) in neurons of the hypothalamic arcuate nucleus (ARC). Whole cell patch-clamp recordings were performed in slices through the ARC prepared from castrated female and male guinea pigs. Forty percent of guinea pig ARC neurons exhibited a transient outward current that was antagonized by high (mM) concentrations of 4-aminopyridine and (100 nM) rHeteropodatoxin-2. Five of these neurons also were immunopositive for both beta-endorphin and the Kv4.2 channel subunit. Bath application of the CB1 receptor agonists WIN 55,212-2 (1 microM) or ACEA (1 microM) selectively induced a rightward shift in the inactivation curve for the I(A), significantly increasing the half-maximal voltage without affecting the peak current magnitude, in neurons from female but not male animals. The CB1 receptor antagonist AM251 (1 microM) reversed this action. Collectively, these data reveal that guinea pig ARC neurons, including proopiomelanocortin neurons, express a prominent I(A) that is positively modulated by cannabinoids in a sex-specific way by altering the voltage dependence of its inactivation. The resultant inhibitory effect on this neuronal population may shed some insight into the mechanism(s) by which cannabinoids influence hypothalamic function.     

The role of galanin receptors in anticonvulsant effects of low-frequency stimulation in perforant path-kindled rats

Low-frequency stimulation (LFS) has antiepileptogenic effects on kindled seizures. In the present study, the role of galanin receptors in the inhibitory effect of LFS on perforant path kindling acquisition was investigated in rats. Animals were kindled by perforant path stimulation in a rapid kindling manner (six stimulations per day). LFS (0.1 ms pulses at 1 Hz, 600 pulses, and 80-150 microA) was applied immediately after termination of each kindling stimulation. M35 (0.5 and 1.0 nM per site), a nonselective galanin receptor antagonist and M871 (1.0 microM per site), a selective galanin receptor type 2 (GalR2) antagonist, were daily microinjected into the dentate gyrus before starting the stimulation protocol. The expression of GalR2 in the dentate gyrus was also investigated using semi-quantitative RT-PCR. Application of LFS significantly retarded the kindling acquisition and delayed the expression of different kindled seizure stages. In addition, LFS significantly reduced the increment of daily afterdischarge duration during kindling development. Intra-dentate gyrus microinjection of both M35 and M871 significantly prevented the inhibitory effects of LFS on kindling parameters. During the focal kindled seizure stages (1-3) M871 had no significant effect. However, during generalized seizure stages (4 and 5), M871 had the same effect as M35. Semi-quantitative RT-PCR also showed that after kindling acquisition, the GalR2 mRNA level decreased in the dentate gyrus but application of LFS prevented this decrease. Obtained results show that activation of galanin receptors by endogenous galanin has a role in mediating the inhibitory effect of LFS on perforant path-kindled seizures. This role is exerted through GalR1 during focal- and through GalR2 during generalized-kindled seizures.     

Feedback inhibition of opioid peptide release in the hypothalamus of the rat

Corticotropin-releasing factor initially stimulates the release of beta-endorphin and dynorphin from rat hypothalamic slices in vitro; with time, in the continued presence of corticotropin-releasing factor, the release of both these peptides declines. The studies described here were undertaken to test whether this decline could be due to the operation of inhibitory feedback mechanisms associated with the function of the opioidergic neurons. When the opioid receptor antagonist naloxone was added to the superfusion medium in the presence of corticotropin-releasing factor, the time-related decrease in opioid release was not observed. Potassium ions also caused an increase, followed by a decrease, in opioid peptide release, and naloxone also prevented the latter from occurring. In addition, naloxone on its own, produced a Ca2+-dependent increase in the non-stimulated release of beta-endorphin and dynorphin, and this action was resistant to tetrodotoxin. These findings suggest that opioid receptors mediate inhibitory feedback effects upon the secretory activity of beta-endorphin and dynorphin neurons in the hypothalamus.     

Galanin stimulates the release of vasoactive intestinal polypeptide from perifused hypothalamic fragments in vitro and from periventricular structures into the cerebrospinal fluid in vivo in the rat

Intracerebroventricular injection of galanin (2 micrograms/rat) raised plasma prolactin (PRL) levels in the rat, which was accompanied by an increase in immunoreactive vasoactive intestinal polypeptide (VIP) in the cerebrospinal fluid (CSF). Immunoreactive VIP release from superfused rat hypothalamic fragments in vitro was dose-relatedly stimulated by galanin (10(-7) and 10(-8) M). PRL release from superfused rat anterior pituitary cells was stimulated by TRH (10(-8) M) but not affected by galanin (10(-7) to 10(-5) M). These findings indicate that central galanin has a stimulating role in the release of hypothalamic VIP, which results in pituitary PRL secretion in the rat.     

Central-type benzodiazepines inhibit release of alpha-melanocyte-stimulating hormone from the rat hypothalamus.

In a previous work, we have shown that GABA inhibits the release of alpha-melanocyte-stimulating hormone (alpha-melanotropin) from hypothalamic neurons through activation of GABAA receptors [Delbende et al. (1989) Brain Res. 497, 86-93]. Since GABA-gated channel activity can be allosterically modulated by a variety of compounds including benzodiazepines, we have investigated the effect of benzodiazepines in the control of alpha-melanotropin release by the rat basal hypothalamus. This study was conducted in vitro using perifused rat hypothalamic slices and the amount of alpha-melanotropin release was monitored with a sensitive and highly specific radioimmunoassay. Infusion of clonazepam (50 microM), a selective agonist for central-type benzodiazepine binding sites, induced an inhibition of KCl (50 mM)-evoked alpha-melanotropin release. The inhibitory effect of clonazepam was rapid and reversible. Administration of Ro 15-1788 (100 microM), a specific antagonist for central-type benzodiazepine receptors or SR 95531, a GABAA receptor antagonist, completely reversed the inhibitory effect of clonazepam. In addition, Ro 15-1788 and SR 95531 both enhanced the amplitude of the response observed during prolonged KCl infusion on alpha-melanotropin neurons, suggesting the existence of a tonic inhibitory effect of endogenous GABA and/or benzodiazepines in the release of alpha-melanotropin by hypothalamic neurons. To investigate further the effect of benzodiazepines in the regulation of alpha-melanotropin neurons, rats were treated in vivo with clonazepam (5 mg/kg) or the non-selective benzodiazepine receptor agonist diazepam (3 mg/kg). Both compounds caused a significant increase in the content of alpha-melanotropin and beta-endorphin in the rat hypothalamus within 3 h.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antinociceptive effects of galanin in the nucleus accumbens of rats

It has been demonstrated that galanin plays important roles in the modulation of nociceptive information in rats. The present study is performed to investigate the regulating role of galanin in nociception in the nucleus accumbens (NAc) of rats. Intra-NAc administration of galanin induces dose-dependent increases in the hindpaw withdrawal latency (HWL) to noxious thermal and mechanical stimulation in rats. Furthermore, the galanin-induced antinociceptive effects are blocked by following intra-NAc injection of the galanin receptor antagonist galantide. The results demonstrate that galanin induces antinociceptive effects in the NAc of rats, and galanin receptors are involved in the galanin-induced antinociception effects.     

Activation of hypothalamic arcuate but not paraventricular neurons following carotid body chemoreceptor stimulation in the rat

The effect of carotid body chemoreceptor stimulation on 292 neurons in midline hypothalamic nuclei has been examined electrophysiologically in ethyl carbamate/sodium pentobarbitone anaesthetized rats. Experiments demonstrated that specific stimulation of carotid body chemoreceptors activates a small group (16) of neurons in the mediobasal hypothalamic arcuate nucleus, but has no effect on neurons (157) in the hypothalamic paraventricular nucleus or the anterior hypothalamus. Of 16 arcuate neurons activated by the stimulus, six projected to the median eminence and three projected to the dorsal medulla, as defined by antidromic invasion. Three of the neurons activated from the carotid body also showed a resting discharge that was linked with ventilation rate, suggesting that the arcuate nucleus may have some involvement with respiratory processing. The activation of neurons projecting to the median eminence implies that the release of adenohypophyseal hormones may also be influenced by carotid body chemoreceptors.     

Distribution of galanin messenger RNA-expressing cells in murine brain and their regulation by leptin in regions of the hypothalamus

Galanin is widely distributed throughout the mammalian brain and has been implicated in the regulation of food intake, metabolism and reproduction-functions that are also thought to be under the control of leptin. To investigate the possible role of galanin in mediating the physiological effects of leptin in the mouse, we had three experimental objectives: first, to map the distribution of galanin messenger RNA-expressing cells in the brain of the mouse; second, to assess the effects of leptin on galanin gene expression in areas of the brain thought to be involved in the regulation of body weight and reproduction; and third, to determine whether galanin neurons in these regions express leptin receptor messenger RNA. We found the pattern of galanin messenger RNA expression in the mouse brain to be similar, but not identical, to that in the rat. Leptin treatment (2microg/g for six days) significantly reduced cellular levels of galanin messenger RNA in the hypothalamic periventricular nucleus of leptin-deficient obese (ob/ob) mice (P<0.01) by approximately 30%; however, leptin did not appear to influence the expression of galanin in the arcuate or dorsomedial nucleus of the hypothalamus. Galanin-producing neurons in the arcuate, dorsomedial and periventricular nuclei did not appear to express leptin receptor messenger RNA (P>0.05). These results demonstrate that galanin distribution patterns in the mouse brain are comparable to other species and, yet, possess unique features. In addition, galanin-expressing neurons in the hypothalamic periventricular nucleus are targets for regulation by leptin; however, the effect of leptin on galanin gene expression is likely to be mediated indirectly, perhaps through either proopiomelanocortin- or neuropeptide Y-expressing cells in the hypothalamus.     

GABAergic and catecholaminergic innervation of mediobasal hypothalamic beta-endorphin cells projecting to the medial preoptic area.

In the absence of cellular estrogen receptors or proven direct estrogen action in the rat, it is assumed that estrogen indirectly regulates the secretory activity of the preoptic area luteinizing hormone-releasing hormone-producing cells. We have previously shown that pro-opiomelanocortin neurons in the arcuate nucleus of the rat send axons rostrally to connect with luteinizing hormone-releasing hormone neurons of the preoptic area. An experiment combining retrograde tracing and double-immunostaining was used to test the hypothesis that rat GABAergic and/or catecholaminergic neurons can influence luteinizing hormone-releasing hormone-producing cells via mediobasal hypothalamic beta-endorphin neurons. The retrograde tracer horseradish peroxidase was injected into the medial preoptic area; two days later, arcuate nucleus Vibratome sections were double-immunostained for beta-endorphin and glutamate decarboxylase or tyrosine hydroxylase. Light and electron microscopic analysis of these triple-labeled sections demonstrated that a population of beta-endorphin-immunoreactive neurons concentrated in the ventromedial arcuate nucleus contain retrogradely transported horseradish peroxidase granules and form synaptic contacts with glutamate decarboxylase- and tyrosine hydroxylase-immunoreactive axon terminals. The present data suggest that arcuate nucleus GABA and catecholamine fibers may influence luteinizing hormone-releasing hormone-containing neurons via projective pro-opiomelanocortin cells.     

Effects of neurotransmitters on the release of corticotropin releasing hormone (CRH) by rat hypothalamic tissue in vitro

Summary Effects of neurotransmitters on in vitro release of CRH from rat hypothalamic tissue were investigated. Three whole hypothalami or three mediobasal hypothalami from male rats, adrenalectomized 7 days before, were incubated for 10 min in a medium similar to cerebrospinal fluid at 37 ° C under 95% O₂, 5% CO₂. CRH activity was assayed by radioimmunological measurement of ACTH released by isolated pituitary cells from adrenalectomized rats.Norepinephrine (NE) at a concentration of 0.02×10^–6 M to 2×10^–6 M stimulated dose-related CRH release. At larger concentrations the response decreased again (bell-shaped dose-response relationship). The action of norepinephrine was completely blocked by coincubation with phentolamine. Similar results were obtained whether whole hypothalami or mediobasal hypothalami were incubated with norepinephrine. This suggests that the site of action of NE was within the mediobasal hypothalamus. The possibility that the observed effects were due to the CRH-like activity of vasopressin released from the hypothalamic tissue was excluded by simultaneous measurements of CRH activity and arginine-vasopressin concentrations in the medium. Dopamine at very large concentrations (17.6×10^–6 M) also stimulated CRH release. Acetylcholine, serotonin, histamine and GABA did not influence the basal CRH release at any of the concentrations tested.These results suggest that in the rat, norepinephrine may have a physiological role as stimulator of CRH-release at the level of the mediobasal hypothalamus. However, in view of the conflicting results obtained with similar and other methods, no definite conclusions should yet be drawn.This work was supported by Deutsche Forschungsgemeinschaft. Bonn-Bad Godesberg. SFB 87/B2. K.H. V. is a recipient of a Heisenberg Stipendium