Hypothalamic Cell Groups Research Articles

Reduced Numbers of Corticotropin‐Releasing Hormone Neurons in Narcolepsy Type 1

Narcolepsy type 1 (NT1) is a chronic sleep disorder correlated with loss of hypocretin(orexin). In NT1 post‐mortem brains, we observed 88% reduction in corticotropin‐releasing hormone (CRH)‐positive neurons in the paraventricular nucleus (PVN) and significantly less CRH‐positive fibers in the median eminence, whereas CRH‐neurons in the locus coeruleus and thalamus, and other PVN neuronal populations were spared: that is, vasopressin, oxytocin, tyrosine hydroxylase, and thyrotropin releasing hormone‐expressing neurons. Other hypothalamic cell groups, that is, the suprachiasmatic, ventrolateral preoptic, infundibular, and supraoptic nuclei and nucleus basalis of Meynert, were unaffected. The surprising selective decrease in CRH‐neurons provide novel targets for diagnostics and therapeutic interventions. ANN NEUROL 2022;91:282–288

Peptidergic innervation of the olfactory bulb: a sleep/wake-regulatory route through the nose

Olfactory epithelium receptor neurons in the nasal cavity, which are exposed to the external environment, reach the olfactory bulb (OB), representing a direct port of entry to the brain. Through retrograde axonal transport, pathogens, toxins and misfolded proteins can reach brain cell groups which innervate the OB and result in functional alterations. Indeed, influenza virus nasal instillation was found to target brainstem and hypothalamic cell groups and result in narcoleptic-like sleep/wake changes [1]. These cell groups included the wake-promoting orexin (OX)-containing neurons, and the sleep-promoting melanin-concentrating hormone (MCH)-containing neurons [1]. Orexinergic innervation of the OB has been reported, but OX and MCH neurons innervating the OB have never been visualized. OX immunoreactivity in the mouse olfactory receptor neurons has been ascribed to the olfactory mucosa. Sources of input to the OB have been studied [2] before the discovery of OX in 1998. Orexinergic innervation of the prefrontal cortex is instead well established. Aim of this study was to reveal OX- and MCH-containing neurons projecting to the OB. Unilateral injections of the retrograde fluorescent tracer Fluoro-Gold (FG) confined to the OB of adult mice were combined with immunophenotyping and quantitative analysis of retrogradely labeled neurons. The findings were compared with those obtained after FG injections in the prefrontal cortex. Following FG injections in the OB, labeled neurons were found in the ipsilateral lateral hypothalamus, and included intermingled OX-A- or MCH-immunoreactive cells. About 8% of orexinergic neurons were labeled when the tracer was confined to the OB. This proportion increased (13±2.49 %) in cases in which a faint halo of tracer diffusion to the lateral portion of the prefrontal cortex was observed. Preliminary data indicate retrograde labeling from the OB of almost 15% of MCH-containing neurons. The findings demonstrate that OX and MCH neurons reach the OB directly, thus providing to environmental agents a route to sleep/wake-regulatory nodes via the nasal cavity.

Neuroendocrine Function After Hypothalamic Depletion of Glucocorticoid Receptors in Male and Female Mice.

Glucocorticoids act rapidly at the paraventricular nucleus (PVN) to inhibit stress-excitatory neurons and limit excessive glucocorticoid secretion. The signaling mechanism underlying rapid feedback inhibition remains to be determined. The present study was designed to test the hypothesis that the canonical glucocorticoid receptors (GRs) is required for appropriate hypothalamic-pituitary-adrenal (HPA) axis regulation. Local PVN GR knockdown (KD) was achieved by breeding homozygous floxed GR mice with Sim1-cre recombinase transgenic mice. This genetic approach created mice with a KD of GR primarily confined to hypothalamic cell groups, including the PVN, sparing GR expression in other HPA axis limbic regulatory regions, and the pituitary. There were no differences in circadian nadir and peak corticosterone concentrations between male PVN GR KD mice and male littermate controls. However, reduction of PVN GR increased ACTH and corticosterone responses to acute, but not chronic stress, indicating that PVN GR is critical for limiting neuroendocrine responses to acute stress in males. Loss of PVN GR induced an opposite neuroendocrine phenotype in females, characterized by increased circadian nadir corticosterone levels and suppressed ACTH responses to acute restraint stress, without a concomitant change in corticosterone responses under acute or chronic stress conditions. PVN GR deletion had no effect on depression-like behavior in either sex in the forced swim test. Overall, these findings reveal pronounced sex differences in the PVN GR dependence of acute stress feedback regulation of HPA axis function. In addition, these data further indicate that glucocorticoid control of HPA axis responses after chronic stress operates via a PVN-independent mechanism.

Neuronal expression of SOX2 is enriched in specific hypothalamic cell groups

The transcription factor SOX2 has many established roles in neural development but is generally considered to have limited activity in the adult brain. As part of a study of neuronal phenotypes in the adult rodent hypothalamus, we have now used immunohistochemical analysis to investigate the expression of SOX2 in the adult rat and mouse hypothalamus. Our analysis has revealed that SOX2 protein is extensively expressed in cells of the suprachiasmatic nucleus (SCN). Co-localization with the nuclear marker proteins NeuN and MeCP2 confirmed SOX2 expression in mature neurons of the rat SCN, and the functional integrity of these SOX2+ neurons was also confirmed by demonstrating co-localization with light-induced EGR1 protein. In addition to the SCN, we have also revealed a population of SOX2+/(NeuN+/MeCP2+) neurons in the rat periventricular nucleus (PeN). However, in other hypothalamic nuclei such as the supraoptic nucleus (SON) SOX2+ cells were rare. In extra-hypothalamic areas, SOX2+ cells were also scarce although we have confirmed populations of non-neuronal SOX2+ cells in both the rat sub-ventricular zone (SVZ) and sub-granular zone (SGZ) of the hippocampus. In addition, we have identified an extensive, novel population of non-neuronal SOX2+ cells in the rat subfornical organ (SFO). Our findings provide further evidence of 'immature' phenotypes in rodent SCN neurons and, given the extensive expression of SOX2 across these hypothalamic neurons, may identify a common regulatory factor that maintains this unusual neuronal phenotype. Conservation of SCN SOX2 expression in both rat and mouse indicates a functional requirement for this transcription factor that may be integral to the role of these SCN neurons in mediating daily physiological rhythms.

Intrahypothalamic pituitary adenylate cyclase-activating polypeptide regulates energy balance via site-specific actions on feeding and metabolism

Numerous studies have demonstrated that both the hypothalamic paraventricular nuclei (PVN) and ventromedial nuclei (VMN) regulate energy homeostasis through behavioral and metabolic mechanisms. Receptors for pituitary adenylate cyclase-activating polypeptide (PACAP) are abundantly expressed in these nuclei, suggesting PACAP may be critical for the regulation of feeding behavior and body weight. To characterize the unique behavioral and physiological responses attributed to select hypothalamic cell groups, PACAP was site-specifically injected into the PVN or VMN. Overall food intake was significantly reduced by PACAP at both sites; however, meal pattern analysis revealed that only injections into the PVN produced significant reductions in meal size, duration, and total time spent eating. PACAP-mediated hypophagia in both the PVN and VMN was abolished by PAC1R antagonism, whereas pretreatment with a VPACR antagonist had no effect. PACAP injections into the VMN produced unique changes in metabolic parameters, including significant increases in core body temperature and spontaneous locomotor activity that was PAC1R dependent whereas, PVN injections of PACAP had no effect. Finally, PACAP-containing afferents were identified using the neuronal tracer cholera toxin subunit B (CTB) injected unilaterally into the PVN or VMN. CTB signal from PVN injections was colocalized with PACAP mRNA in the medial anterior bed nucleus of the stria terminalis, VMN, and lateral parabrachial nucleus (LPB), whereas CTB signal from VMN injections was highly colocalized with PACAP mRNA in the medial amygdala and LPB. These brain regions are known to influence energy homeostasis perhaps, in part, through PACAP projections to the PVN and VMN.

Pubertally born neurons and glia are functionally integrated into limbic and hypothalamic circuits of the male Syrian hamster

During puberty, the brain goes through extensive remodeling, involving the addition of new neurons and glia to brain regions beyond the canonical neurogenic regions (i.e., dentate gyrus and olfactory bulb), including limbic and hypothalamic cell groups associated with sex-typical behavior. Whether these pubertally born cells become functionally integrated into neural circuits remains unknown. To address this question, we gave male Syrian hamsters daily injections of the cell birthdate marker bromodeoxyuridine throughout puberty (postnatal day 28-49). Half of the animals were housed in enriched environments with access to a running wheel to determine whether enrichment increased the survival of pubertally born cells compared with the control environment. At 4 wk after the last BrdU injection, animals were allowed to interact with a receptive female and were then killed 1 h later. Triple-label immunofluorescence for BrdU, the mature neuron marker neuronal nuclear antigen, and the astrocytic marker glial fibrillary acidic protein revealed that a proportion of pubertally born cells in the medial preoptic area, arcuate nucleus, and medial amygdala differentiate into either mature neurons or astrocytes. Double-label immunofluorescence for BrdU and the protein Fos revealed that a subset of pubertally born cells in these regions is activated during sociosexual behavior, indicative of their functional incorporation into neural circuits. Enrichment affected the survival and activation of pubertally born cells in a brain region-specific manner. These results demonstrate that pubertally born cells located outside of the traditional neurogenic regions differentiate into neurons and glia and become functionally incorporated into neural circuits that subserve sex-typical behaviors.

The role of the dopamine D2 receptor in descending control of pain induced by motor cortex stimulation in the neuropathic rat

We studied in rats with a spinal nerve ligation-induced neuropathy whether dopamine D2 receptors (D2Rs) play a role in descending control of pain induced by stimulation of the primary motor cortex (M1). Noxious heat-evoked responses were determined in spinal dorsal horn wide-dynamic range (WDR) and nociceptive-specific (NS) neurons, with and without electrical M1 stimulation. A D2R antagonist, raclopride, was administered into the dorsal striatum or spinally in attempts to reverse spinal antinociception induced by M1 stimulation. Moreover, influence of M1 stimulation on the noxious heat-induced limb withdrawal reflex was determined following block of spinal D2Rs with raclopride or a lidocaine-induced block of the hypothalamic A11 cell group, the main source of spinal dopamine. Striatal administration of raclopride enhanced the heat-evoked baseline responses of WDR but not NS neurons and reversed the M1 stimulation-induced suppression of the heat response in WDR neurons. Following spinal administration of raclopride, M1 stimulation failed to suppress the heat response of WDR neurons, whereas the heat response of NS neurons was enhanced by M1-stimulation. After blocking the A11 with lidocaine or spinal D2Rs with raclopride, M1 stimulation failed to suppress the noxious heat-evoked withdrawal reflex. The results indicate that descending pain control induced by stimulation of the M1 cortex in neuropathic animals involves supraspinal (presumably striatal) and, through A11, spinal D2Rs. Supraspinal and spinal D2Rs have partly dissociative effects on spinal dorsal horn WDR and NS neurons, possibly reflecting differential roles and wirings that these sensory neurons have in pain-processing circuitries.

The periaqueductal gray as a critical site to mediate reward seeking during predatory hunting

Previous studies using morphine-treated dams reported a role for the rostral lateral periaqueductal gray (rlPAG) in the behavioral switching between nursing and insect hunting, likely to depend on an enhanced seeking response to the presence of an appetitive rewarding cue (i.e., the roach). To elucidate the neural mechanisms mediating such responses, in the present study, we first observed how the rlPAG influences predatory hunting in male rats. Our behavioral observations indicated that bilateral rlPAG NMDA lesions dramatically interfere with prey hunting, leaving the animal without chasing or attacking the prey, but do not seem to affect the general levels of arousal, locomotor activity and regular feeding. Next, using Phaseolus vulgaris-leucoagglutinin (PHA-L), we have reviewed the rlPAG connection pattern, and pointed out a particularly dense projection to the hypothalamic orexinergic cell group. Double labeled PHA-L and orexin sections showed an extensive overlap between PHA-L labeled fibers and orexin cells, revealing that both the medial/perifornical and lateral hypothalamic orexinergic cell groups receive a substantial innervation from the rlPAG. We have further observed that both the medial/perifornical and lateral hypothalamic orexinergic cell groups up-regulate Fos expression during prey hunting, and that rlPAG lesions blunted this Fos increase only in the lateral hypothalamic, but not in the medial/perifornical, orexinergic group, a finding supposedly associated with the lack of motivational drive to actively pursue the prey. Overall, the present results suggest that the rlPAG should exert a critical influence on reward seeking by activating the lateral hypothalamic orexinergic cell group.

Puberty and Brain Sexual Differentiation: Getting Organized

The adolescent brain undergoes substantial remodeling and is a target of pubertal gonadal steroid hormones. Our work in the Syrian hamster shows that when gonadal hormones are absent during adolescent development, male-typical social behaviors are impaired. In contrast, when present during adolescent development, gonadal hormones program sex-typical adult social behaviors by organizing the adolescent brain. Using rats, we found that during puberty, new cells, including neurons and glia, are added to sexually dimorphic cell groups in the brain. The anteroventral periventricular area (AVPV) is larger in females than in males and is essential for the cyclic production of ovarian hormones. The sexually dimorphic nucleus (SDN) and medial amygdala (ME) are larger in males and functionally linked to male sexual behavior and processing of social sensory stimuli. During puberty, more new cells are added to the male than to the female SDN and ME, whereas more new cells are added to the female than male AVPV. Importantly, prepubertal gonadectomy obliterates these sex differences. Hormone-modulated addition of new cells to hypothalamic and limbic cell groups may be an active mechanism for preserving sexual dimorphisms during remodeling of the adolescent brain or for establishing new functional and behavioral sexual dimorphisms that emerge with adolescent development.

Location of glutamatergic/aspartatergic neurons projecting to the hypothalamic ventromedial nucleus studied by autoradiography of retrogradely transported [3H]d-aspartate

The hypothalamic ventromedial nucleus is a prominent cell group, which is involved in the control of feeding, sexual behavior and cardiovascular function as well as having other functions. The nucleus receives inputs from various forebrain structures and has a dense glutamatergic innervation. The aim of the present investigations was to reveal the location of glutamatergic neurons in the telencephalon and diencephalon projecting to this hypothalamic cell group. [(3)H]d-aspartate retrograde autoradiography was used injecting the tracer into the ventromedial nucleus. We detected radiolabeled neurons in telencephalic structures including the lateral septum, bed nucleus of the stria terminalis and the amygdala, and in various diencephalic regions, such as the medial preoptic area, hypothalamic paraventricular nucleus, periventricular nucleus, anterior hypothalamic area, ventral premamillary nucleus, thalamic paraventricular and parataenial nuclei and in the hypothalamic ventromedial nucleus itself. Our observations are the first data on the location of glutamatergic neurons terminating in the hypothalamic ventromedial nucleus. The findings indicate that glutamatergic innervation of the ventromedial nucleus is very complex.

Restraint stress activates nesfatin-1-immunoreactive brain nuclei in rats

Nesfatin-1 is a newly discovered peptide that was reported to reduce food intake when injected centrally. We recently described its wide distribution in rat brain autonomic nuclei which implies potential recruitment of nesfatin-1 by stress. We investigated whether restraint, a mixed psychological and physical stressor, activates nesfatin-1-immunoreactive (ir) neurons in the rat brain. Male Sprague-Dawley rats were either subjected to 30 min restraint or left undisturbed and 90 min later brains were processed for double immunohistochemical labeling of Fos and nesfatin-1. Restraint induced significant Fos expression in neurons of the supraoptic nucleus (SON), paraventricular nucleus (PVN), locus coeruleus (LC), rostral raphe pallidus (rRPa), nucleus of the solitary tract (NTS), and ventrolateral medulla (VLM). Double Fos/nesfatin-1 labeling revealed that Fos-ir neurons comprised 95% of nesfatin-1-ir cells in the SON, 90% in the VLM, 80% in the LC, 48% in the caudal NTS, 57% in the rRPa, 48% in the anterior parvicellular PVN, 27% in the medial magnocellular PVN, 18% in the lateral magnocellular PVN and 10% in the medial parvicellular PVN. These data demonstrate that nesfatin-1 neurons are part of the hypothalamic and hindbrain neuronal cell groups activated by restraint suggesting a possible role of nesfatin-1 in the response to stress.

Lipopolysaccharide challenge-induced suppression of Fos in hypothalamic orexin neurons: Their potential role in sickness behavior

Orexin neurons in the lateral hypothalamus constitute a critical component in regulation of waking, feeding, and reward-related behaviors. In this study we examined the effects of lipopolysaccharide (LPS) challenge on Fos expression in orexin neurons in rats, to determine changes during sickness in two different behavioral contexts. One cohort of rats was treated with saline or LPS during the daytime, and then tested on an elevated plus maze (EPM) or left in their home cage until sacrifice. Another cohort received LPS or saline shortly before dark onset and was sacrificed 90min into the dark period. The brains were double-stained for Fos and orexin-A immunoreactivity (both cohorts) and for Fos and histidine decarboxylase (dark period cohort). Orexin neurons were strongly activated in context of exploratory behavior (double-labeled for Fos in both medial and lateral portions). LPS challenge prior to maze exposure diminished this activation, most notably among the lateral orexin neurons. In home cage controls, LPS challenge lead to increased Fos expression, most notably in the medial orexin neurons, when compared to saline-injected home cage controls that show little or no Fos during the daytime. In the dark period, Fos expression in both orexin and histaminergic neurons was abundant, which LPS challenge strongly suppressed. These findings are consistent with the hypothesis that the orexin neurons, in conjunction with the histaminergic system, represent a potential target of the neurocircuitry that drives sickness behavior due to peripheral inflammation, likely through functional inhibition of these hypothalamic cell groups.

Descending modulation of neuropathic hypersensitivity by dopamine D2 receptors in or adjacent to the hypothalamic A11 cell group

We determined the role of the dopamine D2 receptor in or adjacent to the dopaminergic A11 cell group in descending modulation of neuropathic hypersensitivity. Moreover, we determined the spinal neurotransmitter receptors mediating the modulatory effect. Neuropathy was produced by spinal nerve ligation in the rat that had a chronic cannula for drug delivery into A11 or a control site in the locus coeruleus, and a catheter for spinal drug delivery. Hypersensitivity was assessed by a withdrawal response to monofilaments. Quinpirole (a dopamine D2/D3 receptor agonist) in A11 attenuated hypersensitivity, without influencing thermal nociception in the uninjured tail. Quinpirole in the locus coeruleus failed to influence hypersensitivity. L-741,626 (a dopamine D2 receptor antagonist), raclopride (a dopamine D2/D3 receptor antagonist) and bicuculline (a GABA(A) receptor antagonist) in A11 reversed the antihypersensitivity effect of quinpirole. Raclopride or bicuculline alone in A11 had no effects, whereas muscimol (a GABA(A) receptor agonist) alone in A11 suppressed hypersensitivity. Spinal administration of atipamezole (an alpha(2)-adrenoceptor antagonist) or marginally also WAY-100635 (a 5-HT(1A) receptor antagonist), but not raclopride or bicuculline, reduced the antihypersensitivity effect induced by quinpirole in A11. Electrical stimulation of A11 produced thermal antinociception following intrathecal administration of saline but not raclopride. The results indicate that activation of the dopamine D2 receptor in A11 may selectively suppress neuropathic hypersensitivity, due to mechanisms that involve GABA(A) receptors in the hypothalamus and descending noradrenergic pathways acting on spinal alpha(2)-adrenoceptors, possibly together with a slight contribution of descending serotoninergic pathways acting on spinal 5-HT(1A) receptors.

Glutamatergic Neurons Projecting to the Hypothalamic Ventromedial Nucleus of the Rat

Event Abstract Back to Event Glutamatergic Neurons Projecting to the Hypothalamic Ventromedial Nucleus of the Rat Agnes Csaki1*, Jozsef Kiss2 and Bela Halasz2 1 Semmelweis University, Department of Human Morphology and Developmental Biology, Hungary 2 Hungarian Academy of Science - Semmelweis University, Hungary The hypothalamic ventromedial nucleus (VMN) being involved in the regulation of various functions, primarily in the control of sexual and feeding behaviours, has rich glutamatergic innervation. The localization of the glutamatergic neurons projecting to this prominent hypothalamic cell group is unknown. The aim of the present investigations was to clarify this question. The transmitter selective [3H]D- aspartate retrograde transport method was used, injecting the tracer into a narrow rostrocaudal extent of the VMN. The radioactive tracer was visualized by autoradiography on vibratome sections which were previously treated for immunocytochemistry. Retrogradely radiolabelled neurons were detected in distinct intra-, and extrahypothalamic areas. Nuclei including both sides of the VMN, hypothalamic paraventicular nucleus, preoptic area, ventral premamillary nucleus, anterior hypothalamic area, bed nucleus of the stria terminalis, medial-, basomedial- and central nuclei of the amygdaloid complex contained neurons undoubtedly radiolabelled by the retrograde transport of radioactive aspartate. In the majority of the examined regions numerous radiolabelled neurons were calretinin or calbindin immunopositive. In the paraventicular nucleus numerous radiolabelled cells were found on both sides of the nucleus with no immunoreactivity for arginin-vasopressin or oxytocin. Our findings indicate that most of the intahypothalamic and several of the telencephalic afferents to the VMN contain glutamatergic/aspartatergic fibres; secondly our observations are in accordance with reports demonstrating the presence of glutamatergic neurons in this cell group; thirdly, they suggest that the glutamatergic/aspartatergic innervation of this prominent structure is very complex. Supported by OTKA T-049455, KJ Conference: 12th Meeting of the Hungarian Neuroscience Society, Budapest, Hungary, 22 Jan - 24 Jan, 2009. Presentation Type: Poster Presentation Topic: Homeostatic regulatory mechanisms Citation: Csaki A, Kiss J and Halasz B (2009). Glutamatergic Neurons Projecting to the Hypothalamic Ventromedial Nucleus of the Rat. Front. Syst. Neurosci. Conference Abstract: 12th Meeting of the Hungarian Neuroscience Society. doi: 10.3389/conf.neuro.01.2009.04.004 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 24 Feb 2009; Published Online: 24 Feb 2009. * Correspondence: Agnes Csaki, Semmelweis University, Department of Human Morphology and Developmental Biology, Budapest, Hungary, csaki@ana2.sote.hu Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Agnes Csaki Jozsef Kiss Bela Halasz Google Agnes Csaki Jozsef Kiss Bela Halasz Google Scholar Agnes Csaki Jozsef Kiss Bela Halasz PubMed Agnes Csaki Jozsef Kiss Bela Halasz Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Regulation of Serine (Ser)-31 and Ser40 Tyrosine Hydroxylase Phosphorylation during Morphine Withdrawal in the Hypothalamic Paraventricular Nucleus and Nucleus Tractus Solitarius-A2 Cell Group: Role of ERK1/2

Our previous studies have shown that naloxone-induced morphine withdrawal increases the hypothalamic-pituitary-adrenocortical (HPA) axis activity, which is dependent on a hyperactivity of noradrenergic pathways [nucleus tractus solitarius (NTS) A(2)] innervating the hypothalamic paraventricular nucleus (PVN). Short-term regulation of catecholamine biosynthesis occurs through phosphorylation of tyrosine hydroxylase (TH), which enhances enzymatic activity. In the present study, the effect of morphine withdrawal on site-specific TH phosphorylation in the PVN and NTS-A(2) was determined by quantitative blot immunolabeling and immunohistochemistry using phosphorylation state-specific antibodies. We show that naloxone-induced morphine withdrawal phosphorylates TH at Serine (Ser)-31 but not Ser40 in PVN and NTS-A(2), which is associated with both an increase in total TH immunoreactivity in NTS-A(2) and an enhanced TH activity in the PVN. In addition, we demonstrated that TH neurons phosphorylated at Ser31 coexpress c-Fos in NTS-A(2). We then tested whether pharmacological inhibition of ERK activation by ERK kinase contributes to morphine withdrawal-induced phosphorylation of TH at Ser31. We show that the ability of morphine withdrawal to stimulate phosphorylation at this seryl residue is reduced by SL327, an inhibitor of ERK(1/2) activation. These results suggest that morphine withdrawal increases noradrenaline turnover in the PVN, at least in part, via ERK(1/2)-dependent phosphorylation of TH at Ser31.

Vesicular glutamate transporter 2 protein and mRNA containing neurons in the hypothalamic suprachiasmatic nucleus of the rat

The hypothalamic suprachiasmatic nucleus is the key structure of the control of circadian rhythms and has a rich glutamatergic innervation. Besides the presence of glutamatergic afferents, several findings also suggest the existence of glutamatergic efferents from the suprachiasmatic nucleus to its target neurons in various prominent hypothalamic cell groups. However, there is no direct neuromorphological evidence for the presence of glutamatergic neurons in the suprachiasmatic nucleus. Therefore, the purpose of the present investigations was to try to clarify this question. Immunocytochemistry was used at the light and electron microscopy level to identify vesicular glutamate transporter type 2 (VGluT2) immunopositive (presumed glutamatergic) neurons in the rat suprachiasmatic nucleus. In addition VGluT2 mRNA expression in neurons of the nucleus was also addressed with radioisotopic in situ hybridization. Both at the light and electron microscopy level we detected VGluT2 positive neurons, which did not contain GABA, vasoactive intestinal polypeptide or vasopressin. Further, we demonstrated the expression of VGluT2 mRNA in a few cells within the suprachiasmatic nucleus; these glutamatergic cells were distinct from somatostatin mRNA expressing neurons. As VGluT2 is a selective marker of glutamatergic neuronal elements, the present observations provide direct neuromorphological evidence for the presence of glutamatergic neurons in the cell group.

Restless legs syndrome

Restless legs syndrome (RLS) involves abnormal limb sensations that diminish with motor activity, worsen at rest, have a circadian peak in expression in the evening and at night, and can severely disrupt sleep. Primary treatment is directed at CNS dopaminergic systems, particularly activation of D(2)-like (D(2), D(3), and D(4)) receptors. Although RLS affects 2% to 15% of the general population, the neural circuitry contributing to RLS remains speculative, and there is currently no accepted animal model to enable detailed mechanistic analyses. Traditional views suggest that RLS arises from supraspinal sources which favor facilitation of the flexor reflex and emergence of the RLS phenotype. The authors forward the hypothesis that RLS reflects a dysfunction of the little-studied dorsoposterior hypothalamic dopaminergic A11 cell group. They assert that, as the sole source of spinal dopamine, reduced drive in this system can lead to spinal network changes wholly consistent with RLS. The authors summarize their recent investigations on spinal cord dopamine dysfunction that rely on lesions centered on A11, and on studies in D(3) receptor knockout (D(3)KO) mice. Excessive locomotor behavior is evident in both sets of animals, and D(3)KO mice exhibit facilitation rather than the expected depression of spinal reflexes in the presence of dopamine as well as a reversal in their circadian expression of the rate-limiting enzyme for dopamine synthesis, tyrosine hydroxylase. Taken together, these findings are consistent with an involvement of spinal dopamine dysfunction in the etiology of RLS, and they argue that the D(3)KO mouse might serve as a relevant animal model to study the underlying mechanisms of RLS.

Expression of Pituitary Adenylate Cyclase Activating Polypeptide Type 1 Receptor (PAC1R) in the Ewe Hypothalamus: Distribution and Colocalization with Tyrosine Hydroxylase‐Immunoreactive Neurones

We have examined the distribution of the pituitary adenylate cyclase activating polypeptide type I receptor (PAC1R) in the ewe hypothalamus by reverse transcription-polymerase chain reaction, in situ hybridization and immunohistochemistry. PAC1R mRNA was highly expressed in the mediobasal hypothalamus of the ewe, particularly in the arcuate nucleus and ventromedial hypothalamus, compared to other hypothalamic regions. Similar results were obtained from immunohistochemistry using a specific PAC1R antibody. Intense immunolabelling was observed in the arcuate nucleus, external zone of the median eminence and ventromedial hypothalamus. Only relatively weak immunolabelling was observed in other hypothalamic regions, including the paraventricular nucleus and supraoptic nucleus. In the ewe, PACAP acts via the arcuate nucleus to suppress prolactin secretion. Therefore we examined whether PAC1R was present on the tuberoinfundibular dopamine (TIDA) neurones in this nucleus. Dual immunofluorescence labelling for PAC1R and tyrosine hydroxylase revealed that 21.2 +/- 1.7% of dopaminergic neurones in the arcuate nucleus (A12 cell group) also stained for PAC1R. By contrast, other hypothalamic dopaminergic cell groups (A11, A13, A14 and A15) exhibited little (< 3%) or no colocalization. Overall, our results indicate that, in the ewe hypothalamus, PAC1R is most concentrated in the arcuate nucleus, where it is localized on a substantial proportion of dopaminergic neurones. These observations, together with previous in vivo studies, suggest that PACAP could act directly on TIDA neurones via PAC1R to increase dopamine release and consequently inhibit prolactin secretion in the sheep.

Lipopolysaccharide has selective actions on sub-populations of catecholaminergic neurons involved in activation of the hypothalamic–pituitary–adrenal axis and inhibition of prolactin secretion

Immune activation results in adaptive neuroendocrine responses, including activation of the hypothalamic-pituitary-adrenal axis, which are dependent on the integrity of medullary catecholaminergic (CA) systems. In contrast, although specific roles of pontine, midbrain, and hypothalamic CA systems in neuroendocrine function have been described, the functional roles of these CA systems in modulating neuroendocrine function during immune responses have not been investigated. We have, therefore, investigated the effects of immune activation on the various CA systems of the central nervous system (CNS) and explored this relationship with changes in plasma corticosterone and plasma prolactin. Male BALB/c mice were injected with lipopolysaccharide (LPS, 500 microg/kg i.p.) and 2 h later cardiac blood was taken and mice were perfused with fixative. Immunostaining procedures were performed using antibodies raised against c-Fos and tyrosine hydroxylase, a marker of CA neurons, and detailed topographical analysis of the CA systems within the CNS was performed. LPS-injected mice had increased concentrations of plasma corticosterone and decreased concentrations of plasma prolactin compared with vehicle-injected controls. LPS-injected mice had increased numbers of c-Fos-positive CA neurons within the medullary (A1, A2, C1, C2), pontine (A6) and midbrain (A10) cell groups when compared with vehicle-injected controls. Among hypothalamic CA cell groups, LPS had differential effects on the numbers of c-Fos-positive CA neurons in topographically organised subdivisions of the arcuate nucleus (A12). Changes in plasma prolactin concentrations correlated with the numbers of c-Fos-positive CA neurons within the area postrema, the medullary CA cell groups, the medial posterior division of the arcuate, and the zona incerta. The present study identifies topographically organised, anatomically distinct CA systems that are likely to modulate some of the neuroendocrine responses to immune activation, and may provide novel targets for the relief of symptoms associated with illness and disease.

Glutamatergic stimulation of the medial amygdala induces steroid dependent c-fos expression within forebrain nuclei responsive to mating stimulation

Neurons within the posterodorsal medial amygdala of female rats are known to process vaginocervical stimulation received during mating through N-methyl-D-aspartate channel activation, conveying information to downstream hypothalamic cell groups that modulate neuroendocrine function. Stimulation of these neurons with an excitatory amino acid cocktail of glutamate, aspartate and glycine initiates 10-12 days of prolactin surge secretion that normally are observed only after the receipt of vaginocervical stimulation. Posterodorsal medial amygdala neurons responsive to vaginocervical stimulation also contain estrogen and progesterone receptors. The present experiment examined which downstream sites involved in prolactin secretion show c-fos expression following glutamate receptor activation within the posterodorsal medial amygdala and whether ovarian steroids influence cellular activation in these areas. Ovariectomized female rats implanted with unilateral cannulas directed at the posterodorsal medial amygdala received injections of estradiol benzoate and progesterone or oil before infusion treatment with either excitatory amino acid or control PBS. An additional group of estradiol benzoate+progesterone-treated females was infused with 1.0 microM glycine alone in PBS. Infusions were administered three times at 30 min intervals. FOS induction 90 min after infusion was determined immunohistochemically on the sides ipsilateral and contralateral to the infusion. Of the examined regions, excitatory amino acid treatment and hormone treatment induced three patterns of c-fos expression: 1) responses to both excitatory amino acid and hormone treatment [posterodorsal medial amygdala, medial preoptic area, ventrolateral ventromedial hypothalamic nucleus, bed nucleus of the stria terminalis]; 2) responses to estradiol benzoate+progesterone treatment only [anteroventral periventricular nucleus and dorsomedial nucleus]; and 3) responses to excitatory amino acid only [arcuate nucleus, suprachiasmatic nucleus, and paraventricular nucleus]. These data identify possible circuits by which vaginocervical stimulation, via activation of posterodorsal medial amygdala glutamate-type receptors, initiates and coordinates a series of events within a larger neuroendocrine circuit important for pregnancy.