Liraglutide: short-lived effect on gastric emptying -- long lasting effects on body weight.

AIM: Previous studies with the novel once daily glucagon-like peptide-1 (GLP-1) analogue liraglutide and the GLP-1 receptor agonist exenatide have revealed profound insulinotrophic and antidiabetic effects, but also potent effects on gastric emptying (GE) and long-term and lasting reductions in body weight. In this study, we examined the acute and chronic effects of two different GLP-1 analogues with different pharmacokinetic profiles on GE, food intake and body weight. METHODS: On the basis of a series of dose-finding studies, the doses of exenatide and liraglutide with similar acute anorectic effects were identified. GE was assessed using a standard acetaminophen release assay. After the acute test, rats were dosed bi-daily for 14 days in which period food intake and body weight was monitored. On day 14, the GE rate was reassessed. RESULTS: While both compounds exerted robust acute reductions in GE, the effect was markedly diminished following 14 days of dosing with liraglutide. In contrast, exenatide-treated rats still displayed a profound reduction in GE at the 14-day time-point. Both compounds exerted similar effects on body weight. CONCLUSION: The data suggest that the 'gastric inhibitory' GLP-1 receptors in rats are subject to desensitization/tachyphylaxis but that this effect is dependent on full 24-h exposure as obtained by liraglutide. The body weight-lowering effects of GLP-1 receptor stimulation are not subject to desensitization. These data indicate that regulation of appetite signals in the brain, and not GE, is the main mechanism for liraglutide-induced weight loss.

The proglucagon-derived peptide, glucagon-like peptide-2, is a neurotransmitter involved in the regulation of food intake.

The dorsomedial hypothalamic nucleus harbors leptin sensitive neurons and is intrinsically connected to hypothalamic nuclei involved in feeding behavior. However, it also receives ascending input from the visceroceptive neurons of the brainstem. We have identified a unique glucagon-like-peptide-2 containing neuronal pathway connecting the nucleus of the solitary tract with the dorsomedial hypothalamic nucleus. A glucagon-like-peptide-2 fiber plexus targets neurons expressing its receptor within the dorsomedial hypothalamic nucleus. Pharmacological and behavioral studies confirmed that glucagon-like-peptide-2 signaling is a specific transmitter inhibiting rodent feeding behavior and with potential long-term effects on body weight homeostasis. The glucagon-like-peptide-1 receptor antagonist, Exendin (9-39) is also a functional antagonist of centrally applied glucagon-like-peptide-2.

Identification of cannabinoid type 1 receptor expressing cocaine amphetamine-regulated transcript neurons in the rat hypothalamus and brainstem using in situ hybridization and immunohistochemistry.

Recent data have indicated that the neuropeptide cocaine amphetamine-regulated transcript (CART) may be a downstream mediator of the effect of CB1 receptor antagonist on appetite regulation. In order to identify possible interactions between CART and central CB1R expressing neurons, a detailed mapping of CART and CB1R expression and immunoreactivity in the brain was initiated. Single radioactive in situ hybridizations revealed a predominant overlap between CART and CB1R mRNA in hypothalamic and lower brainstem nuclei. Using double in situ hybridization, co-localization between CART and CB1R mRNA expressing neurons was observed to be most pronounced in the retrochiasmatic and lateral hypothalamic areas, as well as in all parts of the dorsal vagal complex. Further attempts to immunohistochemically characterize the distribution of CB1R were, however, deemed impossible as any of eight commercially available antibodies/antisera gave rise to non-specific staining patterns. Furthermore, the staining pattern obtained was not discriminate between CB1R knockout mice and wild type mice. Collectively, we demonstrate at the messenger level that CB1R expressing perikarya colocalize with CART expressing neurons in hypothalamic and brainstem areas known to be important in appetite control, whereas interactions at the protein level necessitate a demand for cautious interpretations of immunohistochemical results.

Combination of the insulin sensitizer, pioglitazone, and the long-acting GLP-1 human analog, liraglutide, exerts potent synergistic glucose-lowering efficacy in severely diabetic ZDF rats.

OBJECTIVE: Severe insulin resistance and impaired pancreatic beta-cell function are pathophysiological contributors to type 2 diabetes, and ideally, antihyperglycaemic strategies should address both. RESEARCH DESIGN AND METHODS: Therapeutic benefits of combining the long-acting human glucagon-like peptide-1 (GLP-1) analog, liraglutide (0.4 mg/kg/day), with insulin sensitizer, pioglitazone (10 mg/kg/day), were assessed in severely diabetic Zucker diabetic fatty rats for 42 days. Impact on glycaemic control was assessed by glycated haemoglobin (HbA(1C)) at day 28 and by oral glucose tolerance test at day 42. RESULTS: Liraglutide and pioglitazone synergistically improved glycaemic control as reflected by a marked decrease in HbA(1C) (liraglutide + pioglitazone: 4.8 +/- 0.3%; liraglutide: 8.8 +/- 0.6%; pioglitazone: 7.9 +/- 0.4%; vehicle: 9.7 +/- 0.3%) and improved oral glucose tolerance at day 42 (area under the curve; liraglutide + pioglitazone: 4244 +/- 445 mmol/l x min; liraglutide: 7164 +/- 187 mmol/l x min; pioglitazone: 7430 +/- 446 mmol/l x min; vehicle: 8093 +/- 139 mmol/l x min). A 24-h plasma glucose profile at day 38 was significantly decreased only in the liraglutide + pioglitazone group. In addition, 24-h insulin profile was significantly elevated only in the liraglutide + pioglitazone group. Liraglutide significantly decreased food intake alone and in combination with pioglitazone, while pioglitazone alone increased cumulated food intake. As a result, rats on liraglutide alone gained significantly less weight than vehicle-treated rats, whereas rats on pioglitazone alone gained significantly more body weight than vehicle-treated rats. However, combination therapy with liraglutide and pioglitazone caused the largest weight gain, probably reflecting marked improvement of energy balance because of reduction of glucosuria. CONCLUSIONS: Combination therapy with insulinotropic GLP-1 agonist liraglutide and insulin sensitizer, pioglitazone, improves glycaemic control above and beyond what would be expected from additive effects of the two antidiabetic agents.

Chronic 5-HT6 receptor modulation by E-6837 induces hypophagia and sustained weight loss in diet-induced obese rats.

E-6837 is a novel, selective and high-affinity 5-HT(6) receptor ligand (pK(i): 9.13) which in vitro demonstrates partial agonism at a presumably silent rat 5-HT(6) receptor and full agonism at a constitutively active human 5-HT(6) receptor by monitoring the cAMP signaling pathway.The effects of chronic treatment with E-6837 were determined in diet-induced obese (DIO)-rats on changes in body weight, food and water intake, plasma indices of comorbid risk factors, and weight regain on compound withdrawal. The centrally acting antiobesity drug, sibutramine, was used as the reference comparator. Sustained body weight loss and decreased cumulative food intake of DIO-rats was observed with E-6837 (30 mg kg(-1), p.o., twice a day) during the 4-week treatment period. The onset of the E-6837 effect on body weight was slower than that of sibutramine (5 mg kg(-1), p.o.), while its maximal effect was greater, that is -15.7 versus -11.0%.E-6837-induced weight loss was exclusively mediated by a decrease (31.7%) in fat mass, with a concomitant reduction (49.6%) in plasma leptin. Reduced obesity was also reflected in improved glycemic control. Although weight regain occurred after withdrawal from either compound, the body weights after E-6837 (-6.6%) remained lower than after sibutramine (-3.8%) indicating that the greater efficacy of the former did not result in profound rebound hyperphagia/weight gain. These results show that the 5-HT(6) receptor partial agonist, E-6837, is a promising new approach to the management of obesity with the potential to produce greater sustained weight loss than sibutramine.

Neurochemical characterization of hypothalamic cocaine- amphetamine-regulated transcript neurons.

The novel neuropeptide cocaine-amphetamine-regulated transcript (CART) is expressed in several hypothalamic regions and has recently been shown to be involved in the central control of food intake. To characterize the hypothalamic CART neurons and understand the physiological functions they might serve, we undertook an in situ hybridization and immunohistochemical study to examine distribution and neurochemical phenotype of these neurons. In situ hybridization studies showed abundant CART mRNA in the periventricular nucleus (PeV), the paraventricular nucleus of the hypothalamus (PVN), the supraoptic nucleus (SON), the arcuate nucleus (Arc), the zona incerta, and the lateral hypothalamic area. The distribution of CART-immunoreactive neurons as revealed by a monoclonal antibody raised against CART(41-89) displayed complete overlap with CART mRNA. Double immunohistochemistry showed co-existence of CART immunoreactivity (CART-IR) and somatostatin in some neurons of the PeV. In the magnocellular division of the PVN as well as the SON, CART-IR was demonstrated in both oxytocinergic and vasopressinergic perikarya. In the medial parvicellular region of the PVN a few CART-IR neurons co-localized galanin, but none was found to co-localize corticotropin-releasing hormone. In the Arc, almost all pro-opiomelanocortinergic neurons were shown to contain CART, whereas no co-localization of CART with NPY was found. In the lateral hypothalamic area nearly all CART neurons were found to contain melanin-concentrating hormone. The present data support a role for CART in neuroendocrine regulation. Most interestingly, CART is co-stored with neurotransmitters having both positive (melanin-concentrating hormone) as well as a negative (pro-opiomelanocortin) effect on food intake and energy balance.

Glucagon-like peptide 1(7-36) amide's central inhibition of feeding and peripheral inhibition of drinking are abolished by neonatal monosodium glutamate treatment.

In the rat, the glucagon-like peptide 1 (GLP-1)(7-36) amide inhibits neurones in the central nervous system responsible for food and water intake. GLP-1-induced inhibition of food intake may involve the hypothalamic arcuate nucleus, whereas rostral sensory circumventricular organs may be responsible for the inhibitory action of GLP-1 on drinking. To further investigate the role of these blood-brain-barrier-free areas in GLP-1-induced inhibition of ingestive behavior, neonatal Wistar rats were subjected to monosodium glutamate (MSG) treatment, which causes extensive damage to the arcuate nucleus as well as to parts of the sensory circumventricular organs. The inhibitory effect of GLP-1 on feeding induced by food deprivation was completely abolished in MSG-lesioned rats. This effect was not due to either a loss of sensitivity to anorectic agents or a loss of taste aversion because MSG-treated animals displayed normal anorectic responses to central administration of corticotropin-releasing factor and normal aversive responses to peripheral administration of both lithium chloride and D-amphetamine. In non-lesioned rats, neuropeptide Y (NPY)-induced feeding was significantly reduced by concomitant GLP-1 administration. In contrast, GLP-1 had no effect on NPY-induced feeding in MSG-lesioned rats, suggesting that the GLP-1 receptors that mediate inhibition of feeding are localized upstream to the NPY-sensitive neurones inducing feeding behavior. The inhibitory effect of GLP-1 on water intake was tested using an ANG II-elicited drinking paradigm. Central administration of GLP-1 inhibited ANG II drinking in both MSG-treated rats and their nontreated littermates. In contrast, peripheral administration of GLP-1 did not inhibit ANG II-induced drinking behavior in MSG-treated rats. Thus it is evident that centrally acting GLP-1 modulates feeding and drinking behavior via neurones sensitive to MSG lesioning in the arcuate nucleus and circumventricular organs, respectively.

Light-dependent induction of cFos during subjective day and night in PACAP-containing ganglion cells of the retinohypothalamic tract.

Environmental light stimulation via the retinohypothalamic tract (RHT) is necessary for stable entrainment of circadian rhythms generated in the suprachiasmatic nucleus (SCN). In the current report, the authors characterized the functional activity and phenotype of retinal ganglion cells that give rise to the RHT of the rat. Retinal ganglion cells that give rise to the RHT were identified by transsynaptic passage of an attenuated alpha herpesvirus known to have selective affinity for this pathway. Dual labeling immunocytochemistry demonstrated co-localization of viral antigen and pituitary adenylate cyclase activating polypeptide (PACAP) in retinal ganglion cells. This was confirmed using the anterograde tracer cholera toxin subunit B (ChB). In normal and retinally degenerated monosodium glutamate (MSG)-treated rats, ChB co-localized with PACAP in axons of the retinorecipient zone of the SCN. Light-induced Fos-immunoreactivity (Fos-IR) was apparent in all PACAP-containing retinal ganglion cells and a population of non-PACAP-containing retinal ganglion cells at dawn of normal and MSG-treated animals. Within the next 3 h, Fos disappeared in all non-PACAP-immunoreactive cells but persisted in all PACAP-containing retinal ganglion cells until dusk. When animals were exposed to constant light, Fos-IR was sustained only in the PACAP-immunoreactive (PACAP-IR) retinal ganglion cells. Darkness eliminated Fos-IR in all PACAP-IR retinal ganglion cells, demonstrating that the induction of Fos gene expression was light dependent. When animals were maintained in constant darkness and exposed to light pulses at ZT 14, ZT 19, or ZT 6, Fos-IR was induced in PACAP-IR retinal ganglion cells in a pattern similar to that seen at dawn. Collectively, these data indicate that PACAP is present in ganglion cells that give rise to the RHT and suggest a role for this peptide in the light entrainment of the clock.

Central administration of cocaine-amphetamine-regulated transcript activates hypothalamic neuroendocrine neurons in the rat.

We have recently shown that intracerebroventricular (i.c.v.) administration of the hypothalamic neuropeptide cocaine-amphetamine-regulated transcript (CART) inhibits food intake and induces the expression of c-fos in several nuclei involved in the regulation of food intake. A high number of CART-induced c-Fos-positive nuclei in the paraventricular nucleus of the hypothalamus prompted us to examine the effect of i.c.v. recombinant CART-(42-89) on activation of CRH-, oxytocin-, and vasopressin-synthesizing neuroendocrine cells in the paraventricular nucleus (PVN). In addition, plasma levels of glucose were examined after central administration of CART-(42-89). Seventy-six male Wistar rats were fitted with i.c.v. cannulas and singly housed under 12-h light, 12-h dark conditions. One week postsurgery the animals were injected i.c.v. in the morning with 0.5 microg recombinant CART-(42-89) or saline. Trunk blood was collected by decapitation at 0 (baseline), 10, 20, 40, 60, 120, or 240 min. CART caused a strong increase in circulating corticosterone that was significantly different from saline at 20, 40, 60, and 120 min postinjection (P<0.05). Furthermore, CART caused a transient rise in plasma oxytocin levels (P<0.05 at 10 and 20 min postinjection), whereas plasma vasopressin levels were unaffected by i.c.v. CART. Animals injected i.c.v. with CART showed a rise in blood glucose levels 10 min postinjection (P<0.05). To examine whether the stimulatory effect of i.c.v. CART on corticosterone and oxytocin secretion is caused by activation of paraventricular nucleus/supraoptic nucleus (PVN/SON) neuroendocrine neurons, we used c-Fos as a marker of neuronal activity. Animals injected with CART showed a strong increase in c-Fos-immunoreactive nuclei in the PVN. Double immunohistochemistry revealed that a high (89+/-0.4%) number of CRH-immunoreactive neurons in the PVN contained c-Fos after CART i.c.v.. c-Fos expression was also observed in oxytocinergic cells (in both magnocellular and parvicellular PVN neurons as well as in the supraoptic nuclei) 120 min after CART administration, whereas none of the vasopressinergic neurons contained c-Fos. Triple immunofluorescence microscopy revealed that CART-immunoreactive fibers closely apposed c-Fos-positive CRH neurons, suggestive of a direct action of CART on PVN CRH neurons. In summary, i.c.v. CART activates central CRH neurons as well as both magnocellular (presumably neurohypophysial) and parvicellular (presumably descending) oxytocinergic neurons of the PVN. The effect of CART on CRH neurons most likely leads to corticosterone secretion from the adrenal gland, which may contribute to the inhibitory effects of CART on feeding behavior.

Neuronal projections from the mesencephalic raphe nuclear complex to the suprachiasmatic nucleus and the deep pineal gland of the golden hamster (Mesocricetus auratus).

Neuronal projections from the mesencephalic raphe system to the suprachiasmatic nucleus and the pineal complex were mapped in this study of the golden hamster, by use of the anterograde tracer Phaseolus vulgaris-leucoagglutinin and the retrograde tracer cholera toxin subunit B. From the median raphe nucleus, a rostral projection ascended in the ventral part of the mesencephalon to continue in the medial forebrain bundle of the forebrain. Nerve fibres from this bundle innervated the ventral and medial parts of the suprachiasmatic nucleus. At the level of the interpeduncular nucleus of the mesencephalon, fibres of the ventral bundle bent dorsally to reach the epithalamic area and to continue in the forebrain in a periventricular position. Some of these fibres innervated the dorsal tip of the suprachiasmatic nucleus. The dorsal raphe nucleus was the origin of a nerve fibre bundle, located in the periaqueductal gray of the mesencephalon, innervating the deep pineal gland and pineal stalk. Injection of cholera toxin B into the suprachiasmatic nucleus labelled cells in the median raphe. Combination of the retrograde tracing from the suprachiasmatic nucleus and serotonin transmitter immunohistochemistry showed that some of the cholera toxin B-immunoreactive nerve cells also contained serotonin. Thus, this study of the golden hamster shows a serotonergic projection from the median raphe nucleus to the suprachiasmatic nucleus and a projection from the dorsal raphe nucleus to the deep pineal gland supporting physiological indications of an influence of serotonin on the photoreceptive circadian system of the brain.

Topographical organization of the rat suprachiasmatic-paraventricular projection.

The suprachiasmatic nucleus (SCN) is a dominant pacemaker involved in the generation of circadian rhythms in mammals. Surprisingly, the expression of the many rhythms appears to be mediated via a limited efferent projection system of the pacemaker, of which the largest pathway terminates in the subparaventricular area and in the paraventricular nucleus of the hypothalamus. In order to investigate a possible topographical organization of this major outflow pathway of the SCN, microiontophoretic injections of the anterograde tracer Phaseolus vulgaris-leucoagglutinin (PHA-L) or the retrograde tracer cholera toxin subunit B (ChB) were centered in distinct subparts of the SCN (PHA-L) or in the subparaventricular area-paraventricular nucleus (ChB), respectively. PHA-L injections involving the entire SCN revealed not only a major projection to the subparaventricular area, but also one directed towards the medial and dorsal parvicellular subnuclei of the paraventricular nucleus. As opposed to injections involving the entire nucleus, injections of PHA-L centered in the dorsomedial subdivision of the SCN resulted in a relatively larger number of PHA-L-immunoreactive fibers in the parvicellular subdivisions of the PVN, whereas the terminal field in the subparaventricular area was less substantial. A topography of the SCN efferent output system was also revealed by the retrograde tracing with ChB. Injections of ChB in the dorsal part of the paraventricular hypothalamic nucleus, not involving the underlying subparaventricular area, gave rise to a population of retrogradely labeled cells in the dorsomedial part of the SCN. In contrast, ChB injections in the subparaventricular area resulted in labeling of neurons clustered in a more ventrolateral aspect of the SCN. The present data provide evidence for a topography in the major efferent projection system from the SCN, implying that different subparts of the rat SCN, presumably containing partly different potential neurotransmitter substances, may regulate different circadian rhythms.

A direct pretectosuprachiasmatic projection in the rat.

The major afferent projections of the suprachiasmatic nuclei originate in the retina and the intergeniculate leaflet of the lateral geniculate nucleus and are important in the entrainment of endogenous circadian rhythms. A characteristic feature of the suprachiasmatic nucleus and the intergeniculate leaflet of the thalamus is that they are bilaterally innervated from the retina. However, parts of the olivary and posterior pretectal nuclei have been shown to be bilaterally innervated from the retina as well. We therefore aimed to explore whether these two nuclei, in the rat, were anatomically related to the suprachiasmatic nucleus. The anterograde neuronal tract-tracer, Phaseolus vulgaris-leucoagglutinin, was injected iontophoretically into different pretectal nuclei. Pretectal injections centered only in the medial part of the pretectum, i.e. involving the olivary and posterior pretectal nuclei, gave rise to a substantial bilateral innervation of the suprachiasmatic nucleus. From the site of injection, Phaseolus vulgaris-leucoagglutinin-immunoreactive nerve fibers coursed laterally and rostrally into the optic tract, and within the optic tract and chiasm, under the diencephalon to penetrate dorsally into the suprachiasmatic nucleus. Varicose Phaseolus vulgaris-leucoagglutinin-labeled nerve fibers were found exclusively in the ventrolateral part of the suprachiasmatic nucleus, mostly on the ipsilateral side. To determine the precise location of the projecting neurons, the retrograde tracer Cholera toxin, subunit B, was iontophoretically injected into the suprachiasmatic nucleus. The presence of of labeled neurons scattered in both the posterior and olivary pretectal nuclei was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Origin of projections from the midbrain raphe nuclei to the hypothalamic paraventricular nucleus in the rat: a combined retrograde and anterograde tracing study.

A number of neuronal functions governed by the hypothalamic paraventricular nucleus are influenced by serotonin, and it is generally believed that the moderate density of serotonin-immunoreactive fibres and terminals within the paraventricular nucleus originates from the midbrain dorsal and median raphe nuclei. To further evaluate the intricate anatomy of projections from brain stem raphe nuclei of the rat, a combination of retrograde and anterograde tracing experiments were conducted to determine the medullary raphe nuclei projection to the paraventricular nucleus. Rhodamine-labelled latex microspheres, Cholera toxin subunit B and FluoroGold we used as retrograde tracers. Intracerebroventricular injections into the third ventricle of all retrograde tracers labelled a distinct population of neurons in the dorsal raphe situated in the subependymal stratum adjacent to the cerebral aqueduct indicating that these cells take up the tracer from the cerebrospinal fluid. Very few retrogradely labelled neurons were seen in the median raphe after i.c.v. administration of the tracers. Retrograde tracers delivered into the medial part of the paraventricular nucleus labelled no further cells in the midbrain dorsal and median raphe nuclei, whereas a substantial number of retrogradely labelled cells emerged in the pontine raphe magnus. However, when the retrograde tracers were delivered into the lateral part of the paraventricular nucleus, avoiding leakage of the tracer into the ventricle, very few labelled neurons were seen in the dorsal and median raphe, whereas the prominent labelling of raphe magnus neurons persisted. The anatomical organization of nerve fibres terminating in the area of the paraventricular nucleus originating from midbrain raphe nuclei was studied in a series of anterograde tracing experiments using the plant lectin Phaseolus vulgaris leucoagglutinin. Injections delivered into the dorsal raphe or median raphe labelled but a few fibres in the paraventricular nucleus proper. A high number of fine calibered nerve fibres overlying the ependyma adjacent to the paraventricular nucleus was, however, seen after the injections into the subependymal rostral part of the dorsal raphe. Injections delivered into the raphe magnus gave rise to a dense plexus of terminating fibres in the parvicellular parts of the paraventricular nucleus and moderately innervated the posterior magnocellular part of the paraventricular nucleus as well as the magnocellular supraoptic nucleus. Concomitant visualization of serotonin-immunoreactive neurons and retrograde FluoroGold-tracing from the paraventricular nucleus revealed that none of the serotonergic neurons of the raphe magnus projects to this nucleus, while a few of the neurons putatively projecting to the paraventricular nucleus from the median raphe are serotonergic. The current observations suggest that the raphe magnus constitute by far the largest raphe input to the paraventricular nucleus and strongly questions the earlier held view that most raphe fibres innervating the paraventricular nucleus are derived from the midbrain dorsal and median raphe. However, the source of serotonergic innervation of the paraventricular nucleus remains elusive.

The diurnal expression of genes encoding vasopressin and vasoactive intestinal peptide within the rat suprachiasmatic nucleus is influenced by circulating glucocorticoids.

The mammalian suprachiasmatic nucleus (SCN) is the endogenous pacemaker generating the diurnal rhythm of the stress hormones ACTH and glucocorticoid secretion. In the present study, we have employed male rats entrained to a 12:12 h (light:dark) photoperiod to investigate the effects of chronic and acute administration of exogenous glucocorticoids upon the diurnal expression of vasopressin and vasoactive intestinal peptide (VIP) mRNA in the SCN by semiquantitative in situ hybridization histochemistry. Chronic administration of exogenous glucocorticoids significantly enhanced vasopressin mRNA expression only at zeitgeber time (ZT) 5, while the otherwise rhythmic expression of vasopressin mRNA was unaffected at ZT11, ZT17 and ZT23. In contrast, the same treatment abolished the rhythmic expression of VIP mRNA resulting in constantly elevated mRNA levels. In adrenalectomized rats given an overnight supplement of dexamethasone in their drinking water, the expression of both vasopressin and VIP mRNA in the SCN was elevated the following morning at ZT6 when compared to adrenalectomised rats kept on 0.9% saline. These results suggest that glucocorticoids influence the expression of vasopressin during a narrow window of time in the diurnal cycle coinciding with the time where entrainment of the circadian pacemaker with non-photic cues is possible. Constantly elevated levels of glucocorticoids may also interfere with the suprachiasmatic expression of VIP mRNA which is thought to be driven by photic cues.

Effects of leptin on arcuate pro-opiomelanocortin and cocaine-amphetamine-regulated transcript expression are independent of circulating levels of corticosterone.

In the hypothalamic arcuate nucleus, neurones that coexpress cocaine-amphetamine-regulated transcript (CART) and alpha-melanocyte-stimulating hormone [alpha-MSH; pro-opiomelanocortin (POMC) derived] peptides exert catabolic actions and are stimulated by leptin. However, leptin treatment also affects other circulating factors that influence hypothalamic gene expression. Notably, the hypercorticosteronaemia of ob/ob mice is lowered by leptin treatment. To examine the interaction between glucocorticoids and leptin on POMC/CART mRNA expression, an experiment combining leptin and adrenalectomy (ADX) in leptin deficient ob/ob mice was carried out. Obese ob/ob and lean littermate Ob/? mice were ADX or sham-operated. ADX mice received a pellet containing 25% corticosterone subcutaneously. Seven days postoperatively, mice were injected intraperitoneally for 5 days with either recombinant human leptin or vehicle. On the sixth day, the mice were decapitated and the brains removed and trunk blood was collected for corticosterone analysis. Plasma concentrations of corticosterone were elevated in all ob/ob groups compared to Ob/?. For both ob/ob and Ob/? groups, corticosterone concentrations exhibited a decline across groups: vehicle-sham>leptin-sham>ADX-vehicle>ADX-leptin. Leptin inhibited food intake and bodyweight in ob/ob-sham and ob/ob-ADX to a similar extent, whereas no effect of leptin was observed in Ob/? mice. Similarly, leptin caused an identical increase in arcuate POMC and CART mRNA expression in ob/ob-sham and ob/ob-ADX compared to vehicle. The present data support the view that leptin influences arcuate POMC and CART mRNA expression directly, and that the effect is not modulated by corticosterone across a wide range of circulating corticosterone concentrations.

Cocaine- and amphetamine-regulated transcript is present in hypothalamic neuroendocrine neurones and is released to the hypothalamic-pituitary portal circuit.

Cocaine- and amphetamine-regulated transcript (CART) is present in a number of hypothalamic nuclei. Besides actions in circuits regulating feeding behaviour and stress responses, the hypothalamic functions of CART are largely unknown. We report that CART immunoreactivity is present in hypothalamic neuroendocrine neurones. Adult male rats received a systemic injection of the neuronal tracer Fluorogold (FG) 2 days before fixation, and subsequent double- and triple-labelling immunoflourescence analysis demonstrated that neuroendocrine CART-containing neurones were present in the anteroventral periventricular, supraoptic, paraventricular (PVN) and periventricular nuclei of the hypothalamus. In the PVN, CART-positive neuroendocrine neurones were found in all of cytoarchitectonically identified nuclei. In the periventricular nucleus, approximately one-third of somatostatin cells were also CART-immunoreactive. In the medial parvicellular subnucleus of the PVN, CART and FG coexisted with thyrotrophin-releasing hormone, whereas very few of the corticotrophin-releasing hormone containing cells were CART-immunoreactive. In the arcuate nucleus, CART was extensively colocalized with pro-opiomelanocortin in the ventrolateral part, but completely absent from neuroendocrine neurones of the dorsomedial part. To assess the possible role of CART as a hypothalamic-releasing factor, immunoreactive CART was measured in blood samples from the long portal vessels connecting the median eminence with the anterior pituitary gland. Adult male rats were anaesthetized and the infundibular stalk exposed via a transpharyngeal approach. The long portal vessels were transected and blood collected in 30-min periods (one prestimulatory and three poststimulatory periods). Compared to systemic venous plasma samples, baseline concentrations of immunoreactive CART were elevated in portal plasma. Exposure to sodium nitroprusside hypotension triggered a two-fold elevation of portal CART42-89 immunoreactivity throughout the 90-min stimulation period. In contrast, the concentration of portal plasma CART immunoreactivity dropped in the vehicle infused rats. The current study provides further evidence that CART is a neuroendocrine-releasing factor with a possible impact on anterior pituitary function during states of haemodynamic stress.

Gating of retinal inputs through the suprachiasmatic nucleus: role of excitatory neurotransmission.

The mammalian circadian clock, located in the hypothalamic suprachiasmatic nucleus (SCN) is important in the regulation of many circadian rhythms, including regulation of pineal gland metabolism and melatonin secretion. Transsection of the optic nerves, disrupting the retinohypothalamic pathway, lesion of the SCN, or lesion of the hypothalamic paraventricular nucleus (PVN) abolish the regulation of pineal serotonin N-acetyltransferase activity by light. Therefore, the pathways linking the retina and the pineal gland must be channelled from the retina through the SCN and the PVN. Many lines of evidence indicate that the major neurotransmitter in the retinal afferents is glutamate. The first aim was therefore to study the retinal target neurons by localising glutamate receptors in the rodent SCN. Using in situ hybridisation, we detected NMDA-R1 and NMDA-R2C mRNA subunits in the SCN. Using immunocytochemistry, immunoreactivity for the AMPA type receptors GluR1, GluR2,3 and GluR4 was also detected in the SCN. Presentation of a short light pulse during the subjective night [i.e. circadian time (CT) 14 or 19], when light induced phase-shifting of activity-rest cycles can be accomplished, also induces expression of the immediate early-genes c-fos and junB in the rodent SCN. The second aim was to use this cellular correlate of behavioural function to determine the location of potential retinal target neurons in the SCN, and to investigate the hypothesis that glutamatergic neurotransmission mediates the effects of light on the circadian system. Thus, the ability of the NMDA receptor antagonist MK-801 to block light-induced c-fos expression in the SCN was studied. In the rat, this antagonist blocked c-fos mRNA expression in a subpopulation of cells in the ventral SCN at doses of 6, but not 2 mg/kg. In contrast, in the hamster both doses blocked light-induced c-fos expression in the ventral SCN. These data provide support for the hypothesis that glutamate mediates effects of light in the SCN, although it appers that the complexes of NMDA receptor subunits, which are involved in light-induced expression of c-fos after light, are relatively insensitive to MK-801. The diversity, heterogeneous distribution, and complexity of glutamate receptor subunits in the SCN suggest that processing of light pulses in the SCN is mediated by several cell types in the SCN. Via an integration process in the clock, the transmission of photic information takes place to other brain structures.

The arcuate nucleus is pivotal in mediating the anorectic effects of centrally administered leptin.

The adipose tissue hormone leptin, which is secreted to the general circulation and transported into the brain in a facilitated manner, possibly acts via hypothalamic neurones to reduce food intake and increase energy expenditure. To evaluate the involvement of importance of the arcuate nucleus in leptin induced anorexia, groups of rats treated neonatally with monosodium-glutamate (MSG; arcuate lesioned) and littermate controls were injected centrally with 5 microg recombinant leptin or saline daily for three consecutive days. Leptin significantly inhibited food intake and caused weight-loss in non-MSG rats (-14.5+/-3.0 g vs. 10.2+/-4.3 g; mean +/-s.e.m.; leptin vs. vehicle) whereas MSG-treated rats were unresponsive to leptin treatment (5.0+/-2.2 g vs. 0.8+/-3.8 g; leptin vs. vehicle). The present data indicate that an intact arcuate nucleus is necessary for leptins actions on food intake and body weight.

Neurones projecting to the hypothalamus from the brainstem A1 catecholaminergic cell group express glutamate-R2,3 receptor immunoreactivity.

Stimulation of ascending catecholaminergic neurones of the A1 region in ventrolateral medulla by excitatory amino acids mediate neurohypophysial vasopressin secretion triggered by hypovolemic hypotension. Recent cloning of the ionotrophic excitatory amino acid receptors of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-type and subsequent production of receptor recognizing antisera have made immunocytochemical detection of receptor proteins in phenotypically characterized neurones possible. Using single immunocytochemical detection of glutamate GluR1, GluR2,3, GluR4 receptor proteins we have investigated the distribution of GluR-receptor proteins in the caudal ventrolateral medulla. In the neurones of the A1 cell group, only GluR2,3-immunoreactivity was expressed whereas GluR1-immunoreactive neurones were seen in the adjacent reticular formation. Using dual immunocytochemistry in combination with retrograde Fluorogold tracing we determined the extent of co-expression of tyrosine-hydroxylase and glutamate GluR2,3 receptor protein immunoreactivity in neurones of the A1 cell group in the ventrolateral medulla that project to the area of the paraventricular nucleus of the hypothalamus. It was seen that the majority of catecholaminergic A1 neurones of the caudal VLM that project directly to the paraventricular nucleus are also immunoreactive to the Glu R2,3 receptor protein further substantiating that these neurones are directly influenced by the excitatory amino acid glutamate.

Direct projection from the suprachiasmatic nucleus to hypophysiotrophic corticotropin-releasing factor immunoreactive cells in the paraventricular nucleus of the hypothalamus demonstrated by means of Phaseolus vulgaris-leucoagglutinin tract tracing.

The diurnal rhythm of the activity of the hypothalamo-pituitary-adrenal axis is generated by the circadian pacemaker located in the suprachiasmatic nuclei (SCN). However, the neuronal circuit connecting the SCN with the neurosecretory corticotropin-releasing factor (CRF) neurons in the paraventricular nucleus of the hypothalamus is not clear. To investigate the existence of a direct link between the SCN and the CRF neurons in the PVN we combined microiontopheretic injections of the anterograde tracer Phaseolus vulgaris-leucoagglutinin (PHA-L) into the SCN with immunohistochemical detection of CRF in adrenalectomized male rats. The majority of the PHA-L-ir axons originating from the SCN terminated in the subparaventricular area. A minor contingent of fibers continued into the PVN proper, involving the medial and dorsal parvicellular subnuclei of the PVN. All PHA-L injections involving the entire SCN gave rise to PHA-L positive fibers endowed with boutons en passage and terminal boutons contacting CRF positive cell bodies in the PVN. Notably, varicosities on the PHA-L labelled fibers were present in close proximity to cell bodies and proximal dendrites of a subportion of the CRF neurons located in the periphery of the CRF cell cluster. The present study provides the first evidence to suggest a direct connection between the SCN and the CRF producing neurons of the hypothalamo-pituitary-adrenocortical axis in the PVN. Considering the sparse number of PHA-L-ir varicosities in close proximity to the CRF-ir cells, it seems likely that this direct pathway constitutes but a part of a projection system from the SCN, possibly involving multisynaptic pathways, influencing the hypothalamo-pituitary-adrenocortical axis.