Structural characteristics of endogenous sugar acids and relations to feeding modulation

Structural specificity among short-chain organic acids for effects on feeding behavior, blood glucose and insulin was investigated by infusion of 1 exogenous and 6 endogenous derivatives into the rat third cerebral ventricle. Glyceric acid (GEA) (1.0 mumol), 3,4-dihydroxybutanoic acid gamma-lactone (3,4-DB) and 3,4,5-trihydroxypentanoic acid gamma-lactone (3,4,5-TP) (2.50 mumol) decreased food intake for, at most, 24 h. These acids depressed the size of the first meal after infusion, but did not affect latency to the first meal, eating speed, drinking or ambulation. Infusion of 2,4-dihydroxybutanoic acid gamma-lactone (2,4-DB) (1.25 mumol), 2,4,5-trihydroxypentanoic acid gamma-lactone (2,4,5-TP), and an exogenous compound, 2,4,5,6-tetrahydroxyhexanoic acid gamma-lactone (2,4,5,6-TH) (2.50 mumol), induced transient initial feeding which was not necessarily accompanied by periprandial drinking. Ambulation was concomitantly increased. Of these organic acids, 3,4-DB and 2,4,5-TP were most potent in their effects on feeding. Hyperglycemia was induced by 2.50 mumol 3,4-DB leaving insulin unaffected; 2.50 mumol 2,4,5-TP caused hypoglycemia, with a persistent but not significant rise in insulin. The results suggest that slight structural differences of endogenous organic acids, in particular the positions of hydroxyl groups on the lactone ring of 4-butanolide, may be important in feeding modulation by conveying intrinsically reciprocal signals to neurons involved in feeding and satiety.     

Structural and stereoisomeric specificity of serum-borne sugar acids related to feeding control by rats

Specificity of chemical structures and stereoisomers among serum-borne short-chain organic acids in rats were assessed for their effects on feeding behavior and humoral factors by infusion into the rat third cerebroventricle. Infusion of glyceric acid (1.0 mumol), 3,4-dihydroxybutanoic acid gamma-lactone (3,4-DB) or 3,4,5-trihydroxypentanoic acid gamma-lactone (2.50 mumol) immediately before the dark phase decreased food intake for, at most, 24 h. These acids did not significantly affect drinking or ambulation. Initial feeding, not necessarily accompanied by periprandial drinking, was induced after infusion of 2,4-dihydroxybutanoic acid gamma-lactone, 2,4,5-trihydroxypentanoic acid gamma-lactone (2,4,5-TP) or exogenous 2,4,5,6-tetrahydroxyhexanoic acid gamma-lactone (2.50 mumol) in the light phase. Of these acids, 3,4-DB most potently suppressed and 2,4,5-TP most potently enhanced feeding. Of these, the 2S,4S-isomer and the 3S-isomer were the most potent of 2,4,5-TP and 3,4-DB, respectively. Only the 2S,4S-isomer of 2,4,5-TP induced hypoglycemia with hyperinsulinemia, whereas opposite effects were produced by the 3S-isomer of 3,4-DB. The results indicate that the positions of the hydroxyl groups on 4-butanolide and the S- and S,S-stereoisomers are important in modulating food intake through the hypothalamus.     

Feeding regulation by endogenous sugar acids through hypothalamic chemosensitive neurons

Analysis of blood of fasted rats revealed two endogenous sugar acids, 3,4-dihydroxybutanoic acid (2-deoxytetronic acid; 2-DTA) and 2,4,5-trihydroxypentanoic acid (3-deoxypentonic acid; 3-DPA), that might be related to food intake control. Injection of 2-DTA into the third cerebral ventricle reduced food intake for 24 hr in 72 hr deprived rats and depressed single neurons activity in the lateral hypothalamus (LHA). The same amounts of 3-DPA elicited feeding in a dose-related fashion, and increased LHA single neuron activity with 6 to 8 min latency. Intravenous injection of 3-DPA, but not 2-DTA, was effective. Liposome encapsulation of 2-DTA enhanced its potency after intraperitoneal injection, probably by allowing passage across the blood-brain barrier. Electrophoretic application of 2-DTA significantly and specifically suppressed, and 3-DPA facilitated activity of glucose-sensitive (GS) neurons in the LHA. Neither affected glucose insensitive LHA neurons. Both sugar acids affected glucoreceptor (GR) neuron activity oppositely in the ventromedial hypothalamic nucleus (VMH). Intracellular recordings verified that the effect of 2-DTA on the GS and GR neurons was the same as glucose. Hyperpolarization of GR neurons with a membrane conductance increase was brought about by 3-DPA. The levels of plasma glucose and insulin changed oppositely by 2-DTA and 3-DPA, respectively when these were applied into the third cerebral ventricle. Feeding behavior and LHA and VMH neuron activity changes after injection suggest 2-DTA may be an endogenous satiety substance and 3-DPA a hunger substance, with effects mediated by GS neurons in LHA and GR neurons in VMH. Effects of 3-hydroxybutyric acid were also verified and discussed.     

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.     

Endogenous sugar acid control of hypothalamic neuron activity and gastric acid secretion in rats

The feeding related endogenous sugar acids, 2-deoxytetronic acid, 2-DTA and 3-deoxypentonic acid, 3-DPA, were investigated for their peripheral and central (hypothalamic) control of gastric acid secretion, and effects on activity of lateral hypothalamic (LHA) neurons in rats. Peripheral gastric acid secretion was not affected by either 2-DTA or 3-DPA. Slight gastric acid secretion was elicited by 3-DPA only when it was applied directly into the gastric related site of the LHA. Gastric acid secretion induced by 2-deoxy-D-glucose (2DG) was suppressed by application of 2-DTA in the LHA. 3-DPA had no effect on 2DG induced secretion. Electrophoretic application of 2-DTA significantly inhibited the activity of both gastric and non-gastric type glucose-sensitive neurons in the LHA, and 3-DPA significantly excited both types of glucose-sensitive neurons. The results agree with previous reports that 2-DTA and 3-DPA are endogenous satiety and hunger factors, respectively, and act by modulating hypothalamic control of gastric acid secretion which is mediated through gastric type and non-gastric type glucose-sensitive neurons.     

Central control of gastric acid secretion by extralateralhypothalamic nuclei

Whether secretion of gastric acid (GAS) is in response to peripheral and/or central administration of chemical or electrical stimuli can be differentiated by vagotomy. GAS has been shown to be controlled by specific lateral hypothalamic (LHA) neurons. Application of 2-deoxy-D-glucose (2-DG) or insulin to the LHA by microinjection or iontophoresis has experimentally induced GAS. The paraventricular nucleus (PVN) has now been found to also affect GAS. GAS was produced more copiously and more quickly by rostral PVN lesion than by lesion of the ventromedial (VMH) or dorsomedial (DMH) nucleus, and nearly as much by caudal PVN lesion. Microinjection of 2-DG into the LHA induced GAS more potently in animals with rostral PVN lesions than in those with caudal PVN, VMH or DMH lesions, or in intact animals. Results indicate that the PVN may be an additional central site from which GAS is affected.     

Independent glucose effects on rat hypothalamic neurons: an in vitro study

The effects of changes in glucose concentration were studied on 256 ventromedial (VMH), 212 dorsomedial (DMH) and 59 lateral (LHA) neurons recorded from coronally-oriented rat hypothalamic slices. When glucose concentration of the medium was increased (5.5-20 mM), these neurons exhibited 3 response patterns: excitation, excitation followed by inhibition, and inhibition. Twenty percent of neurons in VMH, 33% in DMH, and 41% in LHA responded to increases in glucose concentration. The majority in VMH and DMH were excited, and in LHA, inhibited. Only minor modifications of these ratios were obtained by isolating VMH from the other areas. In isolated DMH, equal numbers were excited and inhibited. Both glucose-responsive and non-glucose-responsive neurons in VMH and DMH were identified by intracellular horseradish peroxidase staining. The dendritic arborizations of glucose-responsive neurons were richer than in non-glucose-responsive neurons. These results suggest that the different populations of glucose-responsive neurons in the VMH, DMH and LHA might have different functions in the regulation of glucose.     

Hypothalamic control of gastric acid secretion

Study of hypothalamic control of gastric acid scretion (GAS) has revealed GAS-related neurons, their location in the lateral hypothalamic area (LHA), their characteristics, and implications of their relations to feeding and other functions. Some LHA glucose-sensitive neurons are referred to as gastric type because of their effects on gastric oxyntic cells via specific gastric related neurons of the medulla oblongata and the vagus. The 2-deoxy-D-glucose (2-DG), or insulin induced GAS was completely abolished by bilateral subdiaphragmatic vagotomy, or micro-lesions in specific sites of the LHA. These gastric type glucose-sensitive neurons were thus believed to contribute to control of GAS. The paraventricular nucleus (PVN) was also found to affect GAS. GAS-related PVN neurons were observed in the rostral PVN. Electrophoretic application of various chemicals, especially glucose, also affected neurons in the rostral PVN. Electrophoretically applied norepinephrine (NE) increased PVN single neuron activity and suppressed GAS. Results suggest that the rostral PVN may be another site to modulate LHA control of GAS, and NE may be a transmitter or modulator.     

Role of hindbrain adenosine 5′-monophosphate-activated protein kinase (AMPK) in hypothalamic AMPK and metabolic neuropeptide adaptation to recurring insulin-induced hypoglycemia in the male rat

Glucose counter-regulatory dysfunction correlates with impaired activation of the hypothalamic metabolic sensor adenosine 5′-monophosphate-activated protein kinase (AMPK). Hypothalamic AMPK is controlled by hindbrain energy status; we examined here whether hindbrain AMPK regulates hypothalamic AMPK and metabolic neurotransmitter maladaptation to recurring insulin-induced hypoglycemia (RIIH). Brain tissue was harvested after single versus serial insulin (I) dosing for Western blot analysis of AMPK, phospho-AMPK (pAMPK), and relevant biosynthetic enzyme/neuropeptide expression in micro-punch dissected arcuate (ARH), ventromedial (VMH), dorsomedial (DMH) nuclei and lateral hypothalamic area (LHA) tissue. The AMPK inhibitor compound c (Cc) or vehicle was administered to the caudal fourth ventricle ahead of antecedent I injections. RIIH caused site-specific elevation (ARH, VMH, LHA) or reduction (DMH) of total AMPK protein versus acute hypoglycemia; Cc respectively exacerbated or attenuated this response in the ARH and VMH. Hindbrain AMPK correspondingly inhibited or stimulated LHA and DMH pAMPK expression during RIIH. RIIH elicited Cc-reversible augmentation of VMH glutamate decarboxylase profiles, but stimulated (ARH pro-opiomelanocortin; LHA orexin-A) or decreased (VMH nitric oxide synthase) other metabolic neurotransmitters without hindbrain sensor involvement. Results demonstrate acclimated up-regulation of total AMPK protein expression in multiple hypothalamic loci during RIIH, and document hindbrain sensor contribution to amplification of this protein profile in the VMH. Concurrent lack of net change in ARH and VMH tissue pAMPK implies adaptive reductions in local sensor activity, which may/may not reflect positive gain in energy state. It remains unclear if ‘glucose-excited’ VMH GABAergic and/or ARH pro-opiomelanocortin neurons exhibit AMPK habituation to RIIH, and whether diminished sensor activation in these and other mediobasal hypothalamic neurotransmitter populations may contribute to HAAF.     

Effects of endogenous sugar acids on the ventromedial hypothalamic nucleus of the rat

Effects of certain endogenous sugar acids such as 2-deoxytetronic acid (2-DTA) and 3-deoxypentonic acid (3-DPA) were investigated on the neuronal activity of the ventromedial hypothalamic nucleus (VMH) in the rat. Electrophoretically applied 2-DTA significantly and specifically facilitated the activity of glucoreceptor neurons in the VMH, while 3-DPA suppressed their activity. However, non-glucoreceptor neurons were not affected by these sugar acids. These results reinforces the previous suggestion that 2-DTA and 3-DPA also might act as endogenous satiety and hunger substances, respectively.     

Effect of intestinal amino acid infusions on hypothalamic single unit activity in the anesthetized cat

Single unit discharges in the ventromedial hypothalamic nucleus (VMH) and lateral hypothalamic area (LH) were recorded extracellularly in anesthetized cats, while amino acid solutions were perfused through the small intestine via implanted cannulae. Test infusions consisted of 5 amino acid mixtures (arginine, leucine, phenylalanine, tryptophan, alanine: 50 mM each), 5.0% casein hydrolysate; arginine 125 mM (2.2%); leucine 125 mM (1.6%). Sixty-six units were recorded in 30 cats. Of 52 tested in the LH, 19 or 37% were affected: 14 increased and 5 decreased in firing rate in response to amino acid intestinal perfusions with a very short latency consistent with activation of intestinal amino acid receptors. Of 14 neurons tested in VMH, only 3 or 21% were activated by amino acid infusions but with a long latency of several minutes which cannot exclude the involvement of a postabsorptive signal. Results are discussed in terms of intestinal afferent control over hypothalamic neuronal activity related to amino acid induced satiety.     

Functional heterogeneity of the monkey lateral hypothalamus in the control of feeding

Regional differences in the effects of electrical (ES) and chemical stimulation on execution of a bar-press feeding task, and in neuronal activity related to feeding, glucose sensitivity, and odor responsiveness were examined in the lateral hypothalamic area (LHA) of monkeys. In satiated animals, ES of the far lateral and ventral LHA induced bar-press feeding. In hungry animals, ES of the dorsal LHA suppressed the feeding task only during the stimulation period, but prolonged feeding suppression that occurred after ES of the ventromedial LHA. Microinjection of Na-glutamate into LHA sites where ES was effective in suppressing feeding had no effect, but it was effective in the medial hypothalamus. Glucose-sensitive (GS) neurons decreased in activity during bar pressing and/or during the ingestion period. Glucose-insensitive (GIS) neurons showed a cue-related excitation more often than GS neurons. Odor-responding GS and GIS cells were localized in ventromedial and lateral LHA sites, respectively. The present study suggests the regional heterogeneity of the LHA in feeding regulation, depending on both hunger and satiety states.     

Concurrent measurement of serotonin metabolism and single neuron activity changes in the lateral hypothalamus of freely behaving rat.

To further investigate the activity of serotonin neurons in relation to feeding behavior, the metabolic activity of the serotonergic system and single neuron activity changes in the lateral hypothalamic area (LHA) were investigated concurrently in freely behaving rats. The extracellular concentration of 5-hydroxyindoleacetic acid (5-HIAA), a metabolic product of serotonin in the LHA, began to increase concomitantly with the early stage of nocturnal eating. The increased 5-HIAA returned to the basal level within 3 or 4 h. In conjunction with the increase in serotonin metabolism, activity of 12 out of 30 LHA neurons (40%) increased, whereas it decreased in 7 (23%), and in 11 (37%) it showed no change. An intracerebroventricular injection of lisuride suppressed the increased activity in 7 of the 12 neurons, but had no effect on the others. These results suggest that the concurrent increase in serotonin metabolism and neuron activity changes in the LHA may occur in the early portion of the nocturnal eating period, and may be important in controlling feeding behavior.     

Complex attributes of lateral hypothalamic neurons in the regulation of feeding of alert rhesus monkeys

To elucidate the roles of glucose-sensitive (GS) and glucose-insensitive (GIS) cells of the lateral hypothalamic area (LHA), single neuron activity was recorded during 1) microelectrophoretic administration of chemicals, 2) a conditioned bar press feeding task, 3) gustatory, 4) olfactory, and 5) electrical brain stimulation. GS and GIS neurons showed different firing rate changes during phases of the task, and the responses were highly influenced by the palatability of the food and the motivational (hunger or satiety) state of the animal. The two groups of cells also differed in their responsiveness to gustatory and olfactory stimuli: GS neurons were more likely to respond to tastes and odors than GIS cells. Taste- and odor-responsive GS neurons were primarily suppressed by electrophoretically applied noradrenaline and were localized ventromedially within the LHA. The chemosensitive GIS cells, being organized along a dorsolateral axis, were especially excited by dopamine. The two sets of neurons had distinct connections with associative (orbitofrontal, prefrontal) cortical areas. GS and GIS cells, thus, appear to have differential and complex attributes in the control of feeding.     

Phaseolus vulgaris leuco-agglutinin tracing of intrahypothalamic connections of the lateral, ventromedial, dorsomedial and paraventricular hypothalamic nuclei in the rat

Intrahypothalamic connections of the lateral (LHA), ventromedial (VMH), dorsomedial (DMH) and paraventricular (PVN) hypothalamic nuclei were studied with anterograde transport of iontophoretically injected Phaseolus vulgaris leuco-agglutinin and the immunocytochemical detection of labeled structures. The LHA was found to give rise to a minor projection in the VMH, whereas the VMH in reverse maintains few connections with the ventromedial part of the tuberal LHA. Tracer deposits in both the LHA and VMH resulted in anterograde terminal labeling in the DMH. The DMH, in turn, donates a small number of projections to the LHA and VMH. The main projection of the DMH is aimed at the parvocellular paraventricular nucleus. Direct outflow pathways from the VMH to the PVN were not found, but lectin injections in the LHA on the other hand gave rise to terminal labeling in both the parvocellular and magnocellular divisions of the PVN. The PVN in turn was found to give only minor reciprocal projections to the LHA, DMH and VMH. These findings indicate that the main stream of connections in the hypothalamus runs from the LHA and VMH to the DMH, and from the DMH to the PVN. The identified circuitry patterns were discussed with respect to the role of the hypothalamus in the control of homeostasis and metabolic regulation, and more specifically in relation to the modulation of the hormone release from the pancreas and adrenal glands.     

NOS isoenzyme content in brain nuclei as related to food intake in experimental cancer cachexia

Evidence implies that nitric oxide (NO) in the central nervous systems mediates anorexia in tumor-bearing hosts. We have therefore evaluated, by immunohistochemical image analyses, net alterations of nitric oxide synthases (nNOS, eNOS, iNOS) in brain nuclei [paraventricular hypothalamic nucleus (PVN), medial habenular nucleus (MHB), lateral habenular nucleus (LHB), paraventricular thalamic nucleus (PV), lateral hypothalamic area (LHA), ventromedial hypothalamic nucleus (VMH), nucleus of the solitary tract (NTS)] of tumor-bearing mice (TB) with prostanoid-related anorexia. Pair-fed (PF) and freely fed (FF) non-tumor-bearing mice were used as controls. c-fos was analyzed as indicator of neuronal activation. nNOS was significantly increased in VMH and PVN from TB mice, while eNOS was significantly increased in LHB and LHA. iNOS was significantly increased in LHA and PVN nuclei, but decreased in MHB, LHB and VMH from tumor-bearers. However, several of these alterations were similarly observed in brain nuclei from pair-fed controls. Provision of unspecific NOS-antagonists to TB mice increased nNOS, eNOS and iNOS in several brain nuclei (PVN, LHA, VMH), but left tumor-induced anorexia unchanged. c-fos was significantly increased in all brain nuclei in PF mice except for NTS, LHA and PVN compared to controls, while tumor-bearing mice had increased c-fos in LHA and PVN only compared to controls. Our results demonstrate a complex picture of NOS expression in brain areas of relevance for appetite in tumor-bearing hosts, where most changes seemed to be secondary to stress during negative energy balance. By contrast, NOS content in PVN and LHA nuclei remains candidate behind anorexia in tumor disease. However, nitric oxide does not seem to be a primary mediator behind tumor-induced anorexia. NO may rather secondarily support energy intake in conditions with negative energy balance.     

Effects of auricular stimulation on feeding-related hypothalamic neuronal activity in normal and obese rats

It is known that auriculotherapy occasionally affects dramatic body weight reduction for obese patients, although the physiological and anorexigenic functions are not clear. Effects of auricular stimulation on feeding-related lateral (LHA) and ventromedial (VMH) hypothalamic neuronal activity in normal and experimental (hypothalamic and dietary) obese rats were investigated. The LHA and/or VMH neuronal activity were recorded from feeding-related regions in Wistar SPFNAF male and experimental (hypothalamic and dietary) obese rats, anesthetized with urethane-chloralose, under stereotaxic coordination. Recording was through 3 M KCI glass microelectrodes, while stimulating the ipsilateral vagal innervated region of the auricle. This is equivalent to the cavum conchae in the human, and was identified by resistance less than 10–50 kπ The stimulating electrode was a stainless steel ear acupuncture (0.12 x 2.0 mm). The latency of potentials evoked in the LHA by unilateral stimulation of a specific site in the ear was 28.1 ± 3.3 ms (8–92, n = 41). LHA neuronal activity was depressed 45.6% (n = 12, p < 0.01, and VMH activity was excited (60.5%, n = 18, p < 0.01. The auricular acupuncture stimulation clearly modulates feeding-related hypothalamic neuronal activity of experimental (both hypothalamic and dietary) obese rats. These auricle acupuncture stimulation effects were correlated to the degree of obesity. In conclusion, the results suggest that auricular acupuncture stimulation may not reduce appetite, but is more likely concerned with satiation formation and preservation. Thus, auricular acupuncture should be more effective on obese rats than on normal rats.     

Study of the selectivity of the impregnation of neurons by the Golgi method

The selectivity of the impregnation of neurons by the Golgi method was examined by comparing Nissl-stained specimens with Golgi-stained specimens in terms of the somatic orientation, cross-sectional area, and long and short axes of the soma in the ventromedial hypothalamic nucleus (VMH) and lateral hypothalamic area (LHA) of 5-week-old rats. With regard to somatic orientation, we made comparisons directly between the Nissl distribution pattern and the Golgi pattern. The two distribution patterns did not differ significantly in either VMH or LHA (Smirnov test, P greater than 0.05). For somatic cross-sectional area and diameter (long and short), a mean value of every 10% of the total frequency was estimated for each histogram; the histogram was segmented into 10 percentile frequency domains, and a mean value for each domain was estimated. A linear regression was calculated between Golgi mean values and Nissl's at the same place. By use of the linear regression, all of the observed values on Nissl sections for somatic cross-sectional area and diameter were transformed into their corresponding Golgi values. The frequency distribution of these transformed Nissl values was compared with that of the actually observed Golgi values. There was a high degree of similarity between the two distribution patterns in all regions examined for long and short diameters as well as cross-sectional area of the soma (Smirnov test, P greater than 0.05). These findings suggest that the sampling of VMH and LHA neurons in the Golgi method used in the present study is similar to the corresponding sampling in Nissl preparation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regional and subcellular distribution of gold in brain of gold thioglucose obese mouse

By employing neutron activation analysis, endogenous content of gold was estimated quantitatively in discrete brain areas and in subcellular fractions of the hypothalamus of gold thioglucose (GTG) induced obese mice. The highest concentration of gold was obtained in the ventromedial hypothalamus (VMH) reaching approximately 100 ng/mg wet tissue. Significantly higher concentrations were observed in other hypothalamic subareas followed by certain limbic areas and the thalamus, while in the cerebral and the cerebellar cortex the gold concentration was very low. Subcellularly, the hypothalamic gold was principally recovered in the supernatant fraction particularly after a hyposmotic shock treatment of the crude mitochondrial fraction. Contrary to GTG, treatment with gold thiomalate (GTM) did not induce obesity in the mouse, although considerable amount of gold was observed in the VMH, a finding suggesting the existence in the VMH of at least a two step mechanisms for inducing GTG obesity. To identify the satiety neuron transmitter, an analysis of certain enzyme activities involved in the synthesis of known transmitters, such as acetylcholine or γ-aminobutyric acid (GABA), was made in the GTG-obese mice. There were no significant changes in any of the areas functionally related to the VMH.