Effects of leptin and orexin-A on food intake and feeding related hypothalamic neurons.

The lateral hypothalamic area (LHA) and the ventromedial hypothalamic nucleus (VMH) have historically been implicated in ingestive behavior, energy balance and body mass regulation. The LHA is more closely associated with the initiation of eating; whereas the VMH mediates the cessation of eating. The parvocellular part of the paraventricular nucleus (pPVN) is also included in the suppressing mechanism. Recently, two hypothalamic peptides, orexin-A and orexin-B, localized in the posterior and lateral hypothalamic perifornical region were discovered in the rat brain and they increase food intake. Leptin, a protein encoded by an obesity gene, expressed in adipose tissue and released into the blood also affects food intake. Orexin and leptin receptors have been localized in the LHA, pPVN, and VMH. The purpose of this study was to measure food intake in the rat in response to leptin and orexin-A; and to determine their electrophysiological effects on feeding related hypothalamic neurons. Results clearly show that leptin suppresses food intake whereas orexin-A increases food intake. These differences are associated with leptin and orexin-A modulatory effects on LHA, pPVN, and VMH glucose responding neurons. In the LHA, leptin inhibits a larger proportion of both glucose-sensitive neurons (GSNs) and non-GSNs. In the pPVN, leptin increases more GSNs in comparison to non-GSNs. Whereas in the VMH, leptin increases the activity of glucoreceptor neurons (GRNs) in comparison to non-GRNs. Orexin-A had opposite effects: increases activity of GSNs more than the non-GSNs in the LHA and significantly suppresses GRNs in the VMH. In the pPVN, orexin-A had no observable effects on neurons that have a low density of orexin 2 receptors. Results are discussed in terms of hypothalamic neural circuits that are sensitive to endogenous food intake inducing and reducing substances.

Leptin effects on feeding-related hypothalamic and peripheral neuronal activities in normal and obese rats.

We investigated the effects of leptin on central and/or peripheral feeding-related neuronal networks in Wistar male rats either normal (350-450 g) or Zucker obese (500-800 g). Low doses (1-10 pg) of leptin inhibited glucose-sensitive vagal hepatic afferent discharges and facilitated sympathetic efferent discharges to brown and white adipose tissue. Most (40-75%) neurons in the arcuate nucleus were significantly inhibited by superperfusion with leptin (0.1 nM-10 pM) under in vitro conditions. In anesthetized animals, leptin was applied electrophoretically to single hypothalamic neurons. Both glucose-sensitive neurons (GSNs) and non-GSNs in the feeding center (LHA) were significantly inhibited. Most glucoreceptor neurons in the satiety center (VMH) were significantly excited. Their depolarization was confirmed by activation of Na+ and K+ channels by 10(-11) M leptin using the perforate blind patch-clamp method. Although leptin excited GSNs in the parvocellular part of the paraventricular nucleus, the effects of leptin on such neuronal activity were slight or absent in Zucker obese rats. These results suggest that the feeding-suppression effects of leptin are mediated by its effects on signal transduction through both the central and the peripheral nervous systems.

Increased expression of hippocampal cholinergic neurostimulating peptide-related components and their messenger RNAs in the hippocampus of aged senescence-accelerated mice.

Hippocampal cholinergic neurostimulating peptide stimulates cholinergic phenotype development by inducing choline acetyltransferase in the rat medial septal nucleus in vitro. Adult senescence-accelerated-prone mice/8, a substrain of the senescence-accelerated-prone mouse, show a remarkable age-accelerated deterioration in learning and memory. We cloned mouse hippocampal cholinergic neurostimulating peptide precursor protein complementary DNA. The deduced amino acid sequence showed that the neurostimulating peptide itself is the same as that found in the rat. In situ hybridization revealed that the highest expression of the precursor protein messenger RNA was in hippocampal pyramidal neurons. Compared with a strain of senescence-accelerated-resistant mouse (control mouse), adult senescence-accelerated-prone mice/8 showed increased expression of both the precursor messenger RNA and the neurostimulating peptide-related immunodeposits in the hippocampal CA1 field. The deposits were intensely and diffusely precipitated in neuropils throughout the strata oriens and radiatum in senescence-accelerated-prone mice/8, but not in control mice. The neurostimulating peptide content in the hippocampus was higher in senescence-accelerated-prone mice/8 than in control mice, while its precursor protein itself was not different between the two strains. Furthermore, our previous and present data show that the medial septal and hippocampal choline acetyltransferase activity was significantly lower in senescence-accelerated-prone mice/8 than in control mice. The data suggest that, in hippocampal neurons in adult senescence-accelerated-prone mice/8, the production of hippocampal cholinergic neurostimulating peptide precursor protein in neuronal somata, which is associated with an increased expression of its messenger RNA in the CA1 field, occurs as a consequence of low activity in their presynaptic cholinergic neurons. This is followed by accelerated processing to generate bioactive peptide and transport to its functional fields. However, certain mechanisms reduce the release of the peptide and lead to its accumulation in the neuropil. These disturbances of the septohippocampal cholinergic system might be the biochemical mechanism underlying the characteristic deterioration of senescence-accelerated-prone mice/8.

Acidic fibroblast growth factor activates hypothalamic-pituitary-adrenocortical axis in rats.

Effects of acidic fibroblast growth factor (aFGF), an endogenous satiety substance, on the hypothalamic-pituitary-adrenocortical axis were examined under pentobarbital sodium anesthesia in rats. A guide cannula was inserted into the cerebral third ventricle and a vascular indwelling catheter was inserted into the right atrium from the jugular vein 2 wk and 3 days, respectively, before the experiment. A marked dose-dependent increase in plasma corticosterone was detected from 20 min to 2 h after intracerebroventricular administration of aFGF (1-10 ng). Significant increases in plasma adrenocorticotropic hormone (ACTH) were observed from 5 to 150 min after the intracerebroventricular administration of 10 ng aFGF. Significant dose-dependent increases in plasma corticosterone were also observed after intravenous injections of aFGF (1, 10, and 100 ng), together with increases in the plasma ACTH level. Pretreatment with antibody to corticotropin-releasing factor via the intracerebroventricular route abolished the increases in corticosterone induced by intracerebroventricularly administered aFGF, but not those induced by intravenous injection of aFGF. In adrenal glands perfused in situ with artificial medium, the corticosterone secretion rate increased slightly in response to 10(-9) M aFGF. These findings suggest that intracerebroventricular administration of aFGF activates the hypothalamic-pituitary-adrenal axis via corticotropin-releasing factor release in the brain, whereas peripheral administration of aFGF activates adrenocortical secretion mainly via a direct action on ACTH release.

Fibroblast growth factor receptor-1 in the lateral hypothalamic area regulates food intake.

Previous studies have shown that acidic and basic fibroblast growth fa ctor (aFGF and bFGF) and certain fragments of the aFGF N-terminal suppress food intake in rats due to their inhibitory actions on the glucose-sensitive neurons in the lateral hypothalamic area (LHA). The present study was planned to determine the role of FGF receptor-1 (FGFR-1), which was found in the LHA neurons of rats, on feeding regulation. The structure-activity relationship of aFGF fragments in feeding suppression was also investigated. An injection of anti-FGFR-1 antibody (250 and 350 ng) into the bilateral LHA significantly increased food intake. Synthesized aFGF fragments were infused into the III ventricle to elucidate the structure-activity relationship on the inhibition of feeding. Although aFGF-(1-29) did not affect food intake, [Ser16]aFGF-(1-29) (400 ng) and [Glu16]aFGF-(1-29) (400 NG), in which the cysteine residue at position 16 of aFGF(1-29) was replaced with structurally similar serine and glutamic acid, were observed to significantly inhibit food intake. These findings suggest that endogenous FGFR-1 in the LHA plays an important role in FGF-induced feeding suppression, while, in addition, the dissolving disulfide bond formation in aFGF fragments enhances their inhibitory effects on feeding.

Cerebellar afferents to neuroendocrine cells: implications for adaptive responses to simulated weightlessness.

The hypothalamo-neurohypophyseal system as well as the autonomic nervous system is involved in homeostatic responses associated with changes in head position and orthostatic reflex. The responses induced by body tilt on earth are thought to be attributed to changes in inputs from baroreceptors, vestibular organs and proprioreceptors that are normally required for postural control. The information from these organs is sent to the hypothalamus which thereby influences both neuroendocrine and autonomic systems as well as various kinds of emotional behavior. Our findings showing the fastigial input to the hypothalamus suggested that the FN plays a significant role in these homeostatic responses through its connections with the brain stem and the hypothalamus. Figure 4 shows the input-output organization among the hypothalamus, cerebellum and brain stem, described in detail in sections III to V. This hypothesis may help to account for the autonomic and endocrine disorders often observed in weightlessness.

Electrophysiological study of neurotropin-induced responses in guinea pig hypothalamic neurons.

To investigate the direct actions of neurotropin (NSP, a nonproteinaceous extract from inflamed skin of rabbits which is in therapeutic use), intracellular recordings were made from neurons of the ventromedial hypothalamic nucleus (VMH) and lateral hypothalamic area (LHA) in slices of guinea pig brain. In the VMH, NSP, applied by perfusion (0.1-3.0 NU/ml), caused dose-dependent depolarization in 29 of 48 neurons (60%) tested. No change in membrane resistance was observed during the depolarization, which hypothesized that the NSP-induced depolarization might be mediated through the inactivation of the Na-K pump. The NSP-induced depolarization persisted even after the elimination of synaptic activity by perfusion with Ca(2+)-free and high Mg2+ Ringer solution. NSP hyperpolarized the cell membrane of three neurons (6%) while two neurons (4%) showed biphasic responses; transient depolarization followed by long-lasting hyperpolarization. Membrane potential of the remaining 14 neurons was not changed by application of NSP. Of 14 LHA neurons tested for NSP effects, eight (57%) were depolarized, three (21%) were hyperpolarized, and one showed a biphasic response. The present results suggest that NSP significantly modulates hypothalamic neuron activity, and the central modulation of autonomic functions by NSP might be mediated through hypothalamic neurons.

In vivo measurement of hypothalamic serotonin release by intracerebral microdialysis: significant enhancement by immobilization stress in rats.

Intracerebral microdialysis was used to measure extracellular serotonin and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) in the hypothalamus of unanesthetized rats. Increase in the concentration of K+ in the perfusing Ringer solution (70 mM) produced a sharp increase in serotonin release, which was significantly attenuated by omitting Ca2+ from the perfusion medium. Intraperitoneal injection of 5-hydroxytryptophan, a precursor of serotonin, or local perfusion of pargyline, a monoamine oxidase inhibitor, elevated the hypothalamic serotonin. Releasers or uptake inhibitors of serotonin, such as fenfluramine, cocaine, mazindol, or imipramine, when added to the perfusion medium, significantly increased serotonin level, whereas 5-HIAA was unaffected by these substances. Immobilization-stress caused an immediate increase in both the extracellular serotonin and 5-HIAA in the hypothalamus, suggesting that the hypothalamic serotonergic system is activated during immobilization stress. The present study indicates that the brain microdialysis is useful for analysis of local changes in serotonin concentration which directly reflect neuronal transmission.

Feeding suppression elicited by electrical and chemical stimulations of monkey hypothalamus.

The effects of electrical (ES) and chemical stimulations of the hypothalamus were investigated in monkeys during bar-press feeding. ES elicited both prolonged and nonprolonged types of suppression of bar-press feeding in hungry animals. Prolonged type suppression persisted for greater than 1 min beyond one or more post-ES trials and was found after ES of the dorsomedial hypothalamus (DMH), the ventromedial hypothalamus (VMH), and the ventromedial part of the lateral hypothalamic area (LHA). Non-prolonged type suppression was observed only during ES at some sites of both in hypothalamic and extrahypothalamic areas. A microinjection of glutamate into the VMH and the DMH, but not into the LHA, was able to reproduce the ES-induced prolonged type suppression. In contrast, the ES of the LHA, but not the VMH and DMH, in a satiated state provoked feeding. The results, together with the previous findings, suggest that the neuronal inhibitory mechanism of feeding exists in the VMH and DMH, while both the neuronal facilitatory and axonal inhibitory mechanisms in the LHA are involved in the feeding regulation, and these mechanisms of the LHA are affected by the hunger/satiety state.

A new brain glucosensor and its physiological significance.

The concentration of fibroblast growth factor (FGF), which is found in cerebrospinal fluid (CSF), markedly increases after the start of feeding. Food intake was dose-dependently suppressed by picomole doses of FGF and facilitated by anti-FGF antibody. This suppression was caused by activation of protein kinase C in glucose-sensitive neurons in the lateral hypothalamus. In situ hybridization by use of cDNA showed that acidic (a)FGF was produced in ependymal cells. The ependymal cells released aFGF by responding to glucose increase in CSF after feeding. Released aFGF diffused into the brain parenchyma and was taken by neurons. Passive avoidance was significantly more reliable after aFGF infusion into CSF. Clamping cerebral arteries in the gerbil induced ischemia, which damaged neurons in the CA1 layer of the hippocampus. Pretreatment with aFGF prevented this damage. Thus, aFGF is not only the most potent substance yet found for the suppression of feeding, but it is also extremely effective as a neurotrophic and memory facilitating substance.

Effects of fibroblast growth factors and platelet-derived growth factor on food intake in rats.

In the present study, the relations between acidic and basic fibroblast growth factors (aFGF and bFGF, respectively), platelet-derived growth factor (PDGF), and food intake were studied. When aFGF-, bFGF-, and PDGF-like activity in cerebrospinal fluid (CSF) was examined by bioassay, the activity of those factors significantly increased in postfeeding CSF, compared to prefeeding CSF. Injections of aFGF, bFGF, aFGF (synthetic amino-terminal peptide of aFGF), and PDGF into the third cerebral ventricle decreased food intake, and injections of anti-aFGF, anti-bFGF, and anti-aFGF antibodies into the lateral hypothalamus (LHA) increased food intake. The activity of LHA glucose-sensitive neurons was inhibited by electrophoretic application of aFGF. These results suggest that aFGF, bFGF and PDGF have in vivo physiological roles in the central nervous system, distinct from those as mitogens.

Electrical stimulation of male monkey's midbrain elicits components of sexual behavior.

We electrically stimulated the midbrain of male rhesus monkeys seated in a restraint chair facing the female partners and examined whether sexual behavior could be induced. When the midbrain was stimulated (0.2 ms, 50-500 microA and 50 Hz for 2.5 s), the male monkey touched and held the waist of his partner (latency; 0.9 +/- 0.4 s, mean +/- SD, n = 225), and then mounted her when she responded with presenting her hip toward him. However, this mounting, unlike when the hypothalamus was stimulated, did not lead to thrusting or ejaculation even if the stimulation continued. The sites in the midbrain where the stimulation elicited touching and mounting were the ventral tegmental area, the substantia nigra, the nucleus reticularis mesencephali and the nucleus reticularis pontis oralis et caudalis. Touching and mounting were not elicited when the partner was put away from the male or replaced by submissive male monkeys or humans. The findings suggest that the stimulation-evoked touching and mounting are components of copulatory behavior and that the midbrain structures may be involved in the sexual behavior of male monkeys.

17 beta-estradiol depolarization of hypothalamic neurons is mediated by cyclic AMP.

The process by which 17 beta-estradiol rapidly modulates the excitability of neurons in the ventromedial hypothalamus, a facilitation center of female sexual behavior and satiety center of feeding behavior, through mediation by cyclic nucleotides, was investigated by intracellular recording from the guinea pig brain slice preparations. Two types of short-term responses were produced by depolarization with decreased K+ conductance and hyperpolarization with increased K+ conductance. These two responses were enhanced by the phosphodiesterase inhibitor, isobutylmethylxanthine. However, the specific adenylate cyclase activator, forskolin, enhanced only the depolarization. The analogue of cyclic adenosine 3',5'-monophosphate (cAMP), 8-bromo-cAMP, induced only depolarization, the ionic mechanism of which was similar to that of 17 beta-estradiol. In addition, the possibility of non-specific effects of cyclic nucleotides was precluded by an experiment using an analogue of cyclic guanosine 3',5'-monophosphate (cGMP), 8-bromo-cGMP, which hyperpolarized neurons. Thus, the present study strongly suggests that the production of depolarizing responses of neurons in the hypothalamus produced by estradiol is specifically mediated through cAMP.

Central control of sexual behavior.

Neuronal activity changes in the medial preoptic area (MPOA) of the male monkey were related to the commencement of sexual behavior, penile erection and the refractory period following ejaculation. Similarly, changes in the female MPOA were related to the commencement of sexual behavior and presentation. Increased neuronal activity in the dorsomedial hypothalamic nucleus (DMH) in the male monkey and in the ventromedial hypothalamic nucleus (VMH) in the female monkey was synchronized to each mating act. Stimulation study and neuronal activity recordings in the MPOA, DMH and VMH suggest involvement of MPOA neurons in sexual arousal, and of male DMH and female VMH neurons in the copulatory act. Stimulation experiment on the various parts in the hypothalamus of the female monkey also supports the conclusion.

Proceptive presenting elicited by electrical stimulation of the female monkey hypothalamus.

Proceptive presenting by female macaque monkeys was evoked by electrical stimulation of the ventromedial hypothalamic nucleus and the medial preoptic area, under conditions of partial restraint while sitting in a primate chair. This behavior could be elicited only when a male monkey was in close proximity and not when he was removed or was replaced with a female monkey or the human experimenter. This seems to be the first report on the effects of electrical brain stimulation on proceptivity in the female monkey.

Effects of behaviorally rewarding hypothalamic electrical stimulation on intracellularly recorded neuronal activity in the motor cortex of awake monkeys.

Effects of hypothalamic stimulation (HS) were studied in intracellular recordings obtained from 125 neurons of the motor cortex (MC). HS that was effective in reinforcing bar-press behavior, i.e. satisfactory for intracranial self-stimulation (ICSS), evoked short-latency (less than 3 ms) activation of these cortical neurons more frequently (42% of cells tested) than did HS that was ineffective in reinforcing bar-press behavior (7% of cells tested). Longer latency activation (greater than 3 ms) and inhibition (of variable onset) also occurred, but their incidence was not significantly different when HS was effective or ineffective in producing ICSS. Effects of HS that was effective in producing ICSS were also examined in 23 cells in which the spikes were followed by afterhyperpolarization (AHP) of 1.4-10 mV amplitude and 1.7-54 ms duration. The amplitudes of AHPs of greater than 8 ms duration were reduced after presentations of HSs that were effective as a reinforcer for ICSS. These results suggest that: (1) MC neurons receive reward-related hypothalamic information through pathways sufficiently direct to produce short-latency activation; and (2) a modulation of spike afterhyperpolarization can be observed in conjunction with reception of this information.

Effects of hypothalamic stimulation on activity of dorsomedial medulla neurons that respond to subdiaphragmatic vagal stimulation.

1. Effects of hypothalamic stimulation on activity of dorsomedial medulla neurons that responded to subdiaphragmatic vagal stimulation were investigated in urethan-anesthetized rats. 2. Extracellular recordings were made from 231 neurons in the nucleus of the tractus solitarius (NTS) that fired repetitively in response to single-pulse subdiaphragmatic vagal stimulation and from 320 neurons in the dorsal motor nucleus of the vagal nerve (DMV) that responded antidromically to subdiaphragmatic vagal stimulation. The mean latencies of responses to subdiaphragmatic vagal stimulation were 90.3 +/- 17.1 ms (mean +/- SD) for NTS neurons, and 90.8 +/- 11.2 ms for DMV neurons. This indicated that both afferent and efferent subdiaphragmatic vagal fibers were thin and unmyelinated and had a conduction velocity of approximately 1 m/s. 3. In extracellular recordings from 320 DMV neurons, marked inhibition preceded the antidromic response and subdiaphragmatic vagal stimulation evoked orthodromic spikes in only a few neurons. 4. Intracellular recordings from 66 DMV neurons revealed inhibitory postsynaptic potentials (IPSPs) before the antidromic responses. These IPSPs suppressed spontaneous firing and prevented excitatory postsynaptic potentials (EPSPs) from generating action potentials. 5. Stimulation in all hypothalamic loci studied, the ventromedial hypothalamic nucleus (VMH), the lateral hypothalamic area (LHA), and the paraventricular nucleus (PVN), induced responses with similar characteristics of excitation alone or excitation followed by inhibition in most NTS and DMV neurons. 6. No reciprocal effect of VMH and LHA stimulation was observed on NTS and DMV neurons. 7. Intracellular recordings from DMV neurons revealed monosynaptic EPSPs in response to stimulation of the VMH, the LHA, and the PVN. 8. PVN stimulation evoked significantly more responses in NTS and DMV neurons than VMH stimulation and more responses in DMV neurons than LHA stimulation. This suggests a difference in the number of connections between each hypothalamic site and the dorsomedial medulla. 9. The same dorsomedial medulla neurons were tested with VMH and LHA stimulation. The respective mean latencies of the antidromic and the orthodromic NTS neuron responses were 37.3 +/- 3.2 and 39.6 +/- 12.9 ms for VMH stimulation and 29.8 +/- 5.3 and 31.8 +/- 8.7 ms for LHA stimulation. The mean latencies of the orthodromic DMV neuron responses were 39.4 +/- 8.3 ms for VMH stimulation and 31.1 +/- 5.2 ms for LHA stimulation. The estimated conduction velocity from the VMH to the dorsomedial medulla was approximately 0.25 m/s and from the LHA it was approximately 0.33 m/s, which was significantly faster.(ABSTRACT TRUNCATED AT 400 WORDS)