Blockade of the cerebral aqueduct in rats provides evidence of antagonistic leptin responses in the forebrain and hindbrain.

Previously, we reported that low-dose leptin infusions into the fourth ventricle produced a small but significant increase in body fat. These data contrast with reports that injections of higher doses of leptin into the fourth ventricle inhibit food intake and weight gain. In this study, we tested whether exogenous leptin in the fourth ventricle opposed or contributed to weight loss caused by third ventricle leptin infusion by blocking diffusion of CSF from the third to the fourth ventricle. Male Sprague-Dawley rats received third ventricle infusions of PBS or 0.3 µg leptin/24 h from miniosmotic pumps. After 4 days, rats received a 3-µl cerebral aqueduct injection of saline or of thermogelling nanoparticles (hydrogel) that solidified at body temperature. Third ventricle leptin infusion inhibited food intake and caused weight loss. Blocking the aqueduct exaggerated the effect of leptin on food intake and weight loss but had no effect on the weight of PBS-infused rats. Leptin reduced both body fat and lean body mass but did not change energy expenditure. Blocking the aqueduct decreased expenditure of rats infused with PBS or leptin. Infusion of leptin into the third ventricle increased phosphorylated STAT3 in the VMHDM of the hypothalamus and the medial NTS in the hindbrain. Blocking the aqueduct did not change hypothalamic p-STAT3 but decreased p-STAT3 in the medial NTS. These results support previous observations that low-level activation of hindbrain leptin receptors has the potential to blunt the catabolic effects of leptin in the third ventricle.

Central administration of metformin into the third ventricle of C57BL/6 mice decreases meal size and number and activates hypothalamic S6 kinase.

Administration of metformin is known to reduce both body weight and food intake. Although the hypothalamus is recognized as a critical regulator of energy balance and body weight, there is currently no evidence for an effect of metformin in the hypothalamus. Therefore, we sought to determine the central action of metformin on energy balance and body weight, as well as its potential involvement with key hypothalamic energy sensors, including adenosine monophosphate-activated protein kinase (AMPK) and S6 kinase (S6K). We used meal pattern analysis and a conditioned taste aversion (CTA) test and measured energy expenditure in C56BL/6 mice administered metformin (0, 7.5, 15, or 30 µg) into the third ventricle (I3V). Furthermore, we I3V-administered either control or metformin (30 µg) and compared the phosphorylation of AMPK and S6K in the mouse mediobasal hypothalamus. Compared with the control, I3V administration of metformin decreased body weight and food intake in a dose-dependent manner and did not result in CTA. Furthermore, the reduction in food intake induced by I3V administration of metformin was accomplished by decreases in both nocturnal meal size and number. Compared with the control, I3V administration of metformin significantly increased phosphorylation of S6K at Thr(389) and AMPK at Ser(485/491) in the mediobasal hypothalamus, while AMPK phosphorylation at Thr(172) was not significantly altered. Moreover, I3V rapamycin pretreatment restored the metformin-induced anorexia and weight loss. These results suggest that the reduction in food intake induced by the central administration of metformin in the mice may be mediated by activation of S6K pathway.

Photoperiodic expression of two RALDH enzymes and the regulation of cell proliferation by retinoic acid in the rat hypothalamus.

Retinoic acid (RA) has been found to regulate hypothalamic function, but precisely where it acts is unknown. This study shows expression of retinaldehyde dehydrogenase (RALDH) enzymes in tanycytes that line the third ventricle in an area overlapping with the site of hypothalamic neural stem cells. The influence of RA was examined on the proliferation of progenitors lining the third ventricle using organotypic slice cultures. As has been shown in other regions of neurogenesis, RA was found to inhibit proliferation. Investigations of the dynamics of RALDH1 expression in the rat hypothalamus have shown that this enzyme is in tanycytes under photoperiodic control with highest levels during long versus short days. In parallel to this shift in RA synthesis, cell proliferation in the third ventricle was found to be lowest during long days when RA was highest, implying that RALDH1 synthesized RA may regulate neural stem cell proliferation. A second RA synthesizing enzyme, RALDH2 was also present in tanycytes lining the third ventricle. In contrast to RALDH1, RALDH2 showed little change with photoperiodicity, but surprisingly the protein was present in the apparent absence of mRNA transcript and it is hypothesized that the endocytic tanycytes may take this enzyme up from the cerebrospinal fluid (CSF).

Changes in central sodium and not osmolarity or lactate induce panic-like responses in a model of panic disorder.

Panic disorder is a severe anxiety disorder characterized by recurrent panic attacks that can be consistently provoked with intravenous (i.v.) infusions of hypertonic (0.5 M) sodium lactate (NaLac), yet the mechanism/CNS site by which this stimulus triggers panic attacks is unclear. Chronic inhibition of GABAergic synthesis in the dorsomedial hypothalamus/perifornical region (DMH/PeF) of rats induces a vulnerability to panic-like responses after i.v. infusion of 0.5 M NaLac, providing an animal model of panic disorder. Using this panic model, we previously showed that inhibiting the anterior third ventricle region (A3Vr; containing the organum vasculosum lamina terminalis, the median preoptic nucleus, and anteroventral periventricular nucleus) attenuates cardiorespiratory and behavioral responses elicited by i.v. infusions of NaLac. In this study, we show that i.v. infusions of 0.5 M NaLac or sodium chloride, but not iso-osmolar D-mannitol, increased 'anxiety' (decreased social interaction) behaviors, heart rate, and blood pressure responses. Using whole-cell patch-clamp preparations, we also show that bath applications of NaLac (positive control), but not lactic acid (lactate stimulus) or D-mannitol (osmolar stimulus), increases the firing rates of neurons in the A3Vr, which are retrogradely labeled from the DMH/PeF and which are most likely glutamatergic based on a separate study using retrograde tracing from the DMH/PeF in combination with in situ hybridization for vesicular glutamate transporter 2. These data show that hypertonic sodium, but not hyper-osmolarity or changes in lactate, is the key stimulus that provokes panic attacks in panic disorder, and is consistent with human studies.

Involvement of anteroventral third ventricular AMPA/kainate receptors in both hyperosmotic and hypovolemic AVP secretion in conscious rats.

The area of the brain called the anteroventral third ventricular region (AV3V) includes three different subtypes of glutamate receptor, as well as neural circuits controlling fluid balance and cardiovascular and neuroendocrine functions. Although our previous data indicate the ability of AV3V N-methyl-d-aspartate (NMDA) and metabotropic receptors to provoke vasopressin (AVP)-releasing, pressor and hyperglycemic responses, the roles of non-NMDA receptors selective for alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid and kainate have not been elucidated to date. To address this question, the effects of intracerebral infusion with FWD or NBQX (specific agonist and antagonist for non-NMDA receptors, respectively) on plasma AVP, glucose, osmolality, electrolytes and cardiovascular parameters were examined in conscious rats in the absence or presence of an osmotic or volemic stimulus. When applied topically to AV3V structures such as the median preoptic nucleus, FWD augmented plasma AVP, osmolality, glucose and arterial pressure in a dose-associated fashion. All responses of the variables were abolished by pre-administering NBQX, which exerted no conspicuous effect on any variable except arterial pressure. It was revealed that NBQX administration in AV3V structures such as the median preoptic nucleus and the periventricular nucleus inhibited the rise of plasma AVP in response to intravenous infusion with hypertonic saline or removal of systemic blood through the femoral artery. Elevation of plasma osmolality and sodium evoked by osmotic load, and elevation of plasma osmolality, glucose and angiotensin II and decrease of arterial pressure caused by bleeding, were not significantly affected by NBQX treatment. These results suggest that AV3V non-NMDA receptors, as well as NMDA receptors, may elicit AVP-releasing, pressor and hyperglycemic actions when stimulated in the basal state, and may facilitate AVP secretion under both hyperosmotic and hypovolemic conditions, without contributing to cardiovascular, blood glucose or other responses.

Anteroventral third ventricular N-methyl-D-aspartate receptors, but not metabotropic glutamate receptors are involved in hemorrhagic AVP secretion.

This study aimed to investigate the roles of glutamate (Glu) receptors in the anteroventral third ventricular region (AV3V), a pivotal area for water, cardiovascular and neuroendocrine regulations, in causing vasopressin (AVP) secretion and other phenomena in response to bleeding. The effects of intracerebral infusions of MK-801 [a N-methyl-D-aspartate (NMDA) receptor antagonist] or a metabotropic Glu receptor antagonist (MCPG) on plasma levels of AVP, electrolytes, osmolality and glucose, heart rate and arterial pressure following AV3V administration with NMDA or bleeding stimuli were analyzed in conscious rats. NMDA provoked prominent rises of plasma AVP, osmolality, glucose and arterial pressure, without changing plasma electrolytes or heart rate significantly. All the effects of NMDA were blocked by pre-administration of MK-801 into the same loci. Removal through a femoral arterial line of 10 ml blood per kg body weight did not affect arterial pressure or other variables significantly, although plasma AVP and angiotensin II (ANG II) tended to increase. When bleeding was repeated after 10 min (B2), arterial pressure dropped promptly, and plasma AVP, ANG II, osmolality and glucose augmented remarkably. MK-801 applied 35 min preceding B2, to loci such as the median preoptic nucleus, periventricular nucleus and medial preoptic area inhibited the response of plasma AVP significantly, without exerting any effects on other variables. When MK-801 was administered intracerebroventricularly, or when MCPG was infused into the AV3V, significant alterations did not occur in B2-evoked responses of plasma AVP nor in those of the other variables. In rats given sham bleeding after AV3V infusions of MK-801 or MCPG or intracerebroventricular applications of MK-801, all monitored variables roughly remained at stable levels throughout the experiments. We conclude that NMDA receptors in AV3V, but not metabotropic Glu receptors, may facilitate AVP secretion in hypotensive hypovolemia.

Hypothalamic neuronal histamine in genetically obese animals: its implication of leptin action in the brain.

Leptin regulates feeding behavior and energy metabolism by affecting hypothalamic neuromodulators. The present study was designed to examine hypothalamic neuronal histamine, a recently identified mediator of leptin signaling in the brain, in genetic obese animals. Concentrations of hypothalamic histamine and tele-methylhistamine (t-MH), a major histamine metabolite, were significantly lower in obese (ob/ob) and diabetic (db/db) mice, and Zucker fatty (fa/fa) rats, leptin-deficient and leptin-receptor defective animals, respectively, relative to lean littermates (P < 0.05 for each). A bolus infusion of leptin (1.0 microg) into the lateral ventricle (ilvt) significantly elevated the turnover rate of hypothalamic neuronal histamine, as assessed by pargyline-induced accumulation of t-MH, in ob/ob mice compared with phosphate-buffered saline (PBS) infusions (P < 0.05). However, this same treatment did not affect hypothalamic histamine turnover in db/db mice. In agouti yellow (A(y)/a) mice, animals defective in pro-opiomelanocortin (POMC) signaling, normal levels of histamine, and t-MH were seen in the hypothalamus at 4 weeks of age when obesity had not yet developed. These amine levels in A(y)/a mice showed no change until 16 weeks of age, although the mice were remarkably obese by this time. Infusions of corticotropin releasing hormone (CRH), one of neuropeptide related to leptin signaling, into the third ventricle (i3vt) increased histamine turnover in the hypothalamus of Wistar King A rats (P < 0.05 versus PBS infusion). Infusion of neuropeptide Y (NPY) or alpha-melanocyte stimulating hormone (MSH), a POMC-derived peptide failed to increase histamine turnover. These results indicate that lowered activity of hypothalamic neuronal histamine in ob/ob and db/db mice, and fa/fa rats may be due to insufficiency of leptin action in the brains of these animals. These results also suggest that disruption of POMC signaling in A(y)/a mice may not impact on neuronal histamine. Moreover, CRH but neither POMC-derived peptide nor NPY may act as a signal to neuronal histamine downstream of the leptin signaling pathway.

Contribution of N-methyl-d-aspartate receptors in the anteroventral third ventricular region to vasopressin secretion, but not to cardiovascular responses provoked by hyperosmolality and prostaglandin E2 in conscious rats.

The aim of this study is to pursue roles of N-methyl-d-aspartate (NMDA) receptors in the anteroventral third ventricular region (AV3V; a pivotal area for autonomic functions) in controlling vasopressin (AVP) release and cardiovascular system. In conscious rats, we examined effects of AV3V infusion of MK-801 (a selective antagonist for NMDA receptor) on plasma AVP, osmolality, electrolytes, arterial pressure and heart rate, in the absence or presence of NMDA, hyperosmotic or prostaglandin (PG) E2 stimulus. The AV3V infusion of NMDA caused significant increases in plasma AVP, osmolality and sodium, hematocrit, arterial pressure and heart rate after 5 or 15min. When NMDA was administered into the cerebral ventricle, relatively smaller elevations were observed only in plasma AVP and arterial pressure. The effects of AV3V infusion of NMDA were nearly completely prevented by MK-801 applied to the same region before 15min. The application of MK-801 was also potent to block rises of plasma AVP elicited by AV3V injection of PGE2 or i.v. infusion of hypertonic saline. However, it inhibited neither increases of arterial pressure and heart rate due to the PGE2 treatment nor those of arterial pressure, plasma osmolality and sodium in response to the osmotic load. Histological analysis on the AV3V infusion sites of NMDA, MK-801 and PGE2 indicated that they had been located in the structures such as the median and medial preoptic nuclei, periventricular nucleus and medial preoptic area. These results suggest that stimulation of AV3V NMDA receptors in the basal state may facilitate AVP secretion and cause pressor and tachycardiac actions, and that these receptors may be involved in both the hyperosmolality- and PGE2-induced hormone release, but not in the cardiovascular responses to these stimuli.

Functional relationship between subfornical organ cholinergic stimulation and cellular activation in the hypothalamus and AV3V region.

The subfornical organ (SFO) has been suggested to be important for water intake and secretion of vasopressin (AVP). However, the role of the SFO cholinergic mechanism in the control of body fluid regulation is not clear. This study determined the effects of local cholinergic stimulation in the SFO produced by administration of physostigmine on drinking and cellular excitation in the anterior third ventricle (AV3V) region and in the supraoptic and paraventricular nuclei (SON and PVN). The results showed that injection of physostigmine into the SFO induced water intake and c-fos expression in the AV3V area as well as in the AVP containing neurons in the hypothalamus. Pretreatment of the SFO with mecamylamine, a nicotinic receptor antagonist, had no effect on physostigmine induced behavioral and c-fos responses. The muscarinic receptor blocker atropine, however, abolished both drinking and cellular activation after injection of physostigmine into the SFO. Immunostaining experiments demonstrated positive acetyltransferase (ChAT) in the SFO. Intensive ChAT immunoreactivity was located in the cholinergic fibers in the SFO. Together, the results indicate that SFO cholinergic mechanisms are important in co-operation with the AV3V and hypothalamic neurons in the control of thirst and AVP-mediated body fluid homeostasis.

The role of the hypothalamic melanocortin system in behavioral appetitive processes.

Much evidence suggests that the hypothalamic melanocortin (MC) system plays an important role in the control of food intake. However, investigations of the potential behavioral mechanisms have been limited to measures of aversion. The purpose of the present experiment was to assess whether other behavioral consequences of administration of MC peptides were similar to those produced by 0- or 24-h food deprivation, respectively. Rats were first trained while food deprived that a tone predicted the delivery of peanut oil. They then received exposure to oil under food deprivation, satiation, intra-third-cerebroventricular (i3vt) infusion of MTII (a potent MC agonist) or SHU-9119 (a potent MC antagonist). All rats were then tested during extinction for levels of responding to the tone under food satiation. Previous results demonstrated that sated exposure reduces subsequent test responding to the tone. During the present extinction test, rats that received sated exposure exhibited reduced responding to the tone, relative to rats that received deprived exposure. Unlike satiation, rats that received exposure after MTII exhibited continued high levels of responding to the tone. Further, rats that received SHU-9119 exhibited a small reduction in responding. These data suggest that MTII and SHU-9119 do not influence intake via the same mechanisms as hunger and food satiation, respectively.

Estradiol and progesterone regulate the expression of insulin-like growth factor-I receptor and insulin-like growth factor binding protein-2 in the hypothalamus of adult female rats.

Gonadal hormones interact with insulin-like growthfactor-I (IGF-I) to regulate synaptic plasticity during the estrous cycle in the rat mediobasal hypothalamus. It has been proposed that tanycytes, specialized glial cells lining the ventral region of the third ventricle, may regulate the availability of IGF-I to hypothalamic neurons. IGF-I levels in tanycytes fluctuate during the estrous cycle. Furthermore, estrogen administration to ovariectomized rats increases IGF-I levels in tanycytes, while progesterone, injected simultaneously with estrogen, blocks the estrogen-induced increase of IGF-I levels in tanycytes. To test whether hormonal regulation of IGF-I receptor (IGF-IR) and IGF binding protein-2 (IGFBP-2) may be involved in the accumulation of IGF-I in tanycytes, we assessed the effect of ovarian hormones on the levels of these molecules in the mediobasal hypothalamus of adult female rats. Ovariectomized animals were treated with either oil, estrogen, progesterone, or estrogen and progesterone simultaneously and then killed 6 or 24 h later. Some neurons, some astrocytes, and many tanycytes in the mediobasal hypothalamus were found by confocal microscopy to be immunoreactive for IGF-IR. IGFBP-2 immunoreactivity was restricted almost exclusively to tanycytes and ependymal cells and was colocalized with IGF-IR immunoreactivity in tanycytes. By electron microscope immunocytochemistry using colloidal gold labeling, IGF-IR and IGFBP-2 immunoreactivities were observed in the microvilli of tanycytes in the lumen of the third ventricle. IGF-IR and IGFBP-2 immunoreactive levels on the apical surface of tanycytes were significantly decreased by the administration of progesterone, either alone or in the presence of estradiol. IGF-IR levels in the mediobasal hypothalamus, measured by Western blotting, were not significantly affected by the separate administration of estradiol or progesterone to ovariectomized rats. However, the simultaneous administration of both hormones resulted in a marked decrease in IGF-IR protein levels. Estradiol administration to ovariectomized rats increased IGFBP-2 immunoreactive levels in the hypothalamus. While progesterone did not significantly affect IGFBP-2 expression, the simultaneous injection of estradiol and progesterone resulted in a marked decrease in IGFBP-2 protein levels. The effect of estradiol on IGFBP-2 was observed both in protein and mRNA levels, suggesting a transcriptional regulation. However, the simultaneous administration of progesterone and estradiol had different effects on IGF-IR protein and IGF-IR mRNA levels, as well as on IGFBP-2 protein and IGFBP-2 mRNA levels, suggesting a postranscriptional action. These findings indicate that estradiol and progesterone regulate the expression of IGF-IR and IGFBP-2 in the mediobasal hypothalamus of adult female rats. Regulation of the hypothalamic IGF-I system by ovarian hormones may be physiologically relevant for neuroendocrine regulation and for synaptic plasticity during the estrous cycle. These results do not support the hypothesis that estrogen-induced accumulation of IGF-I by tanycytes is mediated by the hormonal regulation of IGF-IR. However, estrogen-induced up-regulation of IGFBP-2 and progesterone-induced down-regulation of IGF-IR and IGFBP-2 levels in the apical plasma membrane of tanycytes may be involved in the fluctuation of IGF-I levels in the mediobasal hypothalamus during the estrous cycle.

Reliable method for cannulation of the third ventricle of the rat.

A method for chronic or acute cannulation of the third ventricle of the rat is described. This technique, in which successful cannulation is verified by the aspiration of a minute quantity of cerebrospinal fluid, is simple, reliable and produces minimal tissue distortion. The results of an experiment employing the technique and which compared the effects of direct intrahypothalamic and intraventricular administration of phentolamine on stimulus-bound feeding are briefly described.