Hypothalamic regulation of histidyl-proline diketopiperazine binding sites in the rat liver

The effects of hypothalamic hormones and electrolytic lesioning of the ventromedial hypothalamic nuclei (VMH) on histidyl-proline diketopiperazine (cyclo(His-Pro] binding in the rat liver were studied. VMH-lesioning markedly decreased cyclo(His-Pro) binding in the liver. Scatchard analysis revealed that the loss of cyclo(His-Pro) binding induced by VMH lesioning was due to a decrease in the number and affinity of binding sites. Somatostatin (SS) administration decreased cyclo(His-Pro) binding. The SS-induced changes in cyclo(His-Pro) binding were due to changes in the binding affinity. On the other hand, the administration of TRH or LH-RH did not affect cyclo(His-Pro) binding in the liver, although cyclo(His-Pro) has been proposed to be a metabolite of TRH. These findings suggest that the hypothalamus may regulate the cyclo(His-Pro) binding sites in the liver probably by controlling pancreatic SS secretion, since a VMH-lesion is reported to cause hypersecretion of pancreatic SS.     

Neuropeptide-dopamine interactions. I. Dopaminergic mechanisms in cyclo(His-Pro)-mediated hypothermia in rats

Administration of cyclo(His-Pro) to rats produces a dose-dependent hypothermia that is attenuated by dopaminergic antagonists. Chronic treatment with cyclo(His-Pro) potentiates hypothermia induced by apomorphine. These results suggest that cyclo(His-Pro) acts via a dopaminergic mechanism to modulate body temperature.     

Cyclo(His-Pro) and the development of tolerance to the hypothermic effect of ethanol

Acute intraperitoneal administration of ethanol to rats causes a dose-dependent transient hypothermia. On repeated exposure, however, rats develop tolerance to hypothermic effects of ethanol. Cyclo(His-Pro), an endogenous brain peptide, modifies both acute and chronic themomodulatory effects of alcohol. For example, a) acute pretreatment of rats with increasing amounts of cyclo(His-Pro) produces a progressive decrease in ethanol hypothermia, and b) chronic cyclo(His-Pro) administration augments the development of tolerance to hypothermic effects of alcohol. While the mechanism of cyclo(His-Pro) action is not clear, these data are interpreted to suggest that this peptide may play important roles in ethanol intoxication, preference, tolerance, and/or addiction.     

The distribution of Histidyl-Proline Diketopiperazine [cyclo(His-Pro)] in discrete rat hypothalamic nuclei

The distribution of Histidyl-Proline Diketopiperazine [cyclo(His-Pro)], a metabolite of thyrotropin-releasing hormone (TRH), was determined by specific RIA in Palkovits micropunch pools derived from discrete hypothalamic nuclei. Highest concentrations of cyclo(His-Pro) were identified in the anterior nucleus (3.5 ng/mg protein) and the paraventricular nucleus (2.95 ng/mg protein), while lower concentrations of cyclo(His-Pro) were seen in the other 6 nuclei. In contrast, TRH concentrations were highest in the ventromedial nucleus pars medialis (3.2 ng/mg protein) and arcuate nucleus (2.7 ng/mg protein). This qualitatively different distribution of cyclo(His-Pro) from that of TRH suggests that not all of cyclo(His-Pro) is derived exclusively from TRH.     

Influence of midbrain stimulation on the excitability of neurons in the medial hypothalamus of the rat

Extracellular action potentials were recorded from 1098 neurons in the medial hypothalamus of pentobarbital anesthetized male rats. Their excitability was analyzed after single 1 Hz stimulation of the midbrain periaqueductal gray (PAG) or adjacent reticular formation. Cells were also examined for their response to median eminence (ME), amygdala, lateral septum (LS) or anterior hypothalamic/preoptic area (AHA/POA) stimulation. Antidromic invasion from midbrain stimulation was recorded from 110 neurons. Eight of these neurons showed features of axon branching and displayed antidromic invasion from both midbrain and amygdala (2 cells) or AHA/POA (6 cells). Many neurons with midbrain projections displayed orthodromic responses to stimulation in the amygdala, but few responded to AHA/POA or LS stimulation. Midbrain stimulation evoked orthodromic responses from 99 medial hypothalamic neurons. Many of these cells also displayed orthodromic responses to amygdala or AHA/POA stimulation, whereas a small number were activated antidromically by stimulation in these sites. None of 42 neurons activated antidromically from median eminence stimulation were responsive to midbrain stimulation. These results provide electrophysiological evidence of reciprocal connections between medial hypothalamic and medial midbrain areas, and indicate that medial hypothalamic neurons with midbrain connections are subject to influences from other extrahypothalamic areas.     

Neuropeptide-dopamine interactions. II. Cyclo (His-Pro) augmentation of amphetamine- but not apomorphine-induced stereotypic behavior

Histidyl-proline diketopiperazine (cyclo [His-Pro]) not only exists in the basal ganglia of rodents, monkeys, and humans, but also exhibits a variety of biologic activities, some of which appear to be mediated via dopaminergic mechanisms. We investigated the potential modulation by cyclo (His-Pro) of amphetamine- and apomorphine-induced stereotypic behavior, a behavior that is associated with the activation of postsynaptic dopamine receptor. Administration of amphetamine to rats resulted in a dose-dependent increase in stereotypic behavior that was further augmented if animals were pretreated with cyclo (His-Pro). Although apomorphine also led to a dose-related progression in the stereotypic behavior, the apomorphine effects were not modified by cyclo (His-Pro) pretreatment. We conclude that cyclo (His-Pro) either acts indirectly at the presynaptic dopamine site or modulates other neurotransmitters to potentiate actions of amphetamine.     

Long-term reorganization of afferents to the mediobasal hypothalamus following retrochiasmatic knife cuts. A study using horseradish peroxidase

Retrochiasmatic knife cuts produce a series of dynamic changes across time in the luteinizing hormone-releasing hormone and catecholamine content of the mediobasal hypothalamus (MBH). We have examined the sources of afferents projecting to the mediobasal hypothalamus of female rats at 7, 60 and 90 days following retrochiasmatic frontal cut (FC) surgery using the intra-axonal retrograde transport of horseradish peroxidase (HRP) in order to better define the functional plasticity demonstrated by the MBH at these time periods after damage. Age-matched, previously unoperated, female rats served as controls. Small HRP injections placed in the ventromedial nucleus (VMN) in control animals labelled neurons within the VMN, dorsomedial nucleus (DMN) and lateral hypothalamic area (LHA). Larger injections also labelled neurons in the anterior hypothalamic area (AHA), preoptic area (POA), septal nuclei, periventricular nuclei (PVN), supraoptic nucleus (SON), zona incerta (ZI) and suprachiasmatic nucleus (Schn). Seven days after surgery, no labelled neurons could be detected rostral to the knife cut when the injection site was confined to the boundary of the glial scar. At 60 days, labelled soma were observed in LPOA, POA, AHA, PVN, SON and ZI. At 90 days only the SON contained labelled neurons rostral to the knife cut. These results suggest a dynamically changing pattern of innervation to the MBH following damage.     

Antidromic identification of neurons in the ventrolateral part of the medulla oblongata with ascending projections to the preoptic and anterior hypothalamic area (POA/AHA)

Seventy neurons in the ventrolateral medulla oblongata were antidromically activated by electrical stimulation of the preoptic and anterior hypothalamic area (POA/AHA) in female rats under urethane anesthesia. These identified cells were located within and adjacent to the nucleus reticularis lateralis and could be readily distinguished into at least two types of neurons, designated as ‘fast’ and ‘slow’ cells, on the basis of their waveform and conduction velocity.     

Mapping of hypothalamic sites involved in the effects of NPY on body temperature and food intake

The objective of the present study was to identify hypothalamic sites that might be implicated in the effects of neuropeptide Y (NPY) on both body temperature and food intake. For this purpose, the effects of direct microinjections of NPY in several doses (0.156–20 μg) into discrete hypothalamic nuclei on body temperature were examined in rats. To examine specificity of effects, food consumption of animals following injections was also measured. Results indicate that the influence of NPY on body temperature varies with the hypothalamic region where the peptide is administered. NPY had no effect on temperature after administration into the ventromedial (VMH) and the perifornical hypothalamus (PeF). However, a significant hypothermia was seen following administration into the preoptic (POA) and arcuate nucleus (Arc), and hyperthermia was seen after injection into the paraventricular nucleus (PVN). Finally, a biphasic effect was observed after injection into the lateral hypothalamus (LH): hyperthermia with relatively small doses and hypothermia with higher doses. Similar effects were obtained when administred into the third ventricle (3V) but in an inverted dose-related fashion: hypothermia at low and hyperthermia at higher doses. For feeding, NPY consistently increased food intake in all regions examined, with the strongest effect obtained after administration into the PeF. The present results clearly dissociate the effects of NPY on food intake and body temperature, and demonstrate that these effects are related to specific hypothalamic nuclei.     

Lipopolysaccharide-induced hypotension is mediated by a neural pathway involving the vagus nerve, the nucleus tractus solitarius and alpha-adrenergic receptors in the preoptic anterior hypothalamic area

We recently reported that the preoptic anterior hypothalamic area (POA) mediates the hypotensive response evoked by lipopolysaccharide (LPS). In this study, we investigated how the inflammatory signal induced by LPS reaches the POA. Subdiaphragmatic vagotomy and abdominal perivagal lidocaine administration, or lidocaine injection into the nucleus tractus solitarius (NTS) prevented LPS hypotension. Microinjection of the alpha-adrenergic receptor antagonist phentolamine into the POA, blocked initiation of the hypotensive response and prevented the late decompensatory phase. These data suggest that LPS hypotension is mediated by the vagus nerve which conveys the signal to the NTS and, in turn, stimulates norepinephrine release within the POA.     

5-HT receptors and hyper- or hypothermia: Elucidation by catecholamine antagonists injected into the cat hypothalamus

The receptor mechanisms in the hypothalamus and preoptic area which mediate thermoregulatory changes produced by serotonin (5-HT) were examined in the unrestrained cat. Stainless steel guide tubes were implanted just above the rostral diencephalon or preoptic area in each of twelve cats. 5-HT in a dose of 1.5 to 10.5 μg was micro-injected in a volume of 0.75 to 1.5 μl into a total of 256 test sites in the hypothalamus. An individual site at which 5-HT evoked a rise or a fall in core temperature of 0.5°C within 30 min was considered to be reactive to the indoleamine. Within such a region of maximum sensitivity, i.e., the cat's anterior hypothalamic, preoptic area (AH/POA), either norepinephrine (NE) or dopamine (DA) micro-injected in a dose of 2.33 to 14.0 μg in similar volumes evoked only a dose-dependent decline in the body temperature of the cat. 5-HT was found to cause either hyper- or hypothermia. The direction of temperature change caused by 5-HT was dependent principally upon the neuroanatomical locus of injection. Each of the 5-HT reactive sites was characterized pharmacologically by their pre-treatment with phentolamine (PHT), d-butaclamol (BUTAC) or methysergide (METHY) in doses of 1.0 to 10.0 μg. At the most rostral sites in POA, the catecholamine receptor antagonists selectively delayed the onset of the 5-HT hypothermia and limited the magnitude of the response. Within sites located in the AH in which 5-HT induced only a rise in body temperature, prior treatment of the site with the catecholamine receptor antagonists PHT or BUTAC failed to modify the response. These results indicate that 5-HT in producing hypothermia not only may saturate the 5-HT receptor sites but also may be taken up by catecholamine receptors which mediate the diencephalic heat loss pathway.     

Distribution of histidyl-proline diketopiperazine [cyclo (His-Pro)] and thyrotropin-releasing hormone (TRH) in the primate central nervous system

The concentrations of cyclo (His-Pro) and its precursor, thyrotropin-releasing hormone (TRH) were measured in 47 different loci of monkey brain using specific radioimmunoassays. Cyclo (His-Pro) concentrations were higher than those of TRH in all loci excepting the hypothalamus, where TRH concentration was found to be the highest of all the loci and twice those of cyclo (His-Pro). The high levels of cyclo (His-Pro) were seen within the cerebellar system (inferior olivary nucleus>nucleus interpositus>fastigial nucleus>posterior vermis). The great variations in TRH-cyclo (His-Pro) ratios among different loci suggest that other factors in addition to TRH concentration must play roles in determining the unique distribution pattern of cyclo (His-Pro) in the primate brain.     

Failure of cyclo (His-Pro) to exhibit natriuretic activity

Administration of exogenous cyclo (His-Pro) to dogs has been reported to elicit natriuresis. In contrast, our data fail to show any natriuretic activity of cyclo (His-Pro) in dogs or rats. The possible reasons underlying this discrepancy are discussed.     

Thyrotropin-releasing hormone: its distribution and metabolism during development in bullfrog (Rana catesbeiana)

The development changes in the metabolism of thyrotropin-releasing hormone (TRH), cyclo (His-Pro) formation from TRH, and the levels of endogenous TRH in frog brain and skin were determined. The results indicated that TRH concentrations were considerably higher in brain than in skin, and in both of these structures TRH content increased significantly following metamorphosis to adulthood. This increase in TRH concentration is probably a reflection of a marked decrease in TRH-metabolism in adult frogs compared to tadpoles. However, the formation of cyclo (His-Pro) from TRH increased during the developmental period reaching to a maximum in adulthood. The possible role of cyclo (His-Pro) in the amphibian developmental process is discussed in relation to our recent observation showing cyclo (His-Pro) inhibition of prolactin secretion.     

Neuroanatomical mapping of hypothalamic regions mediating verapamil hyper- and hypothermia in the cat

Guide cannulae for microinjection and push-pull perfusion in the unrestrained cat were implanted bilaterally in the anterior hypothalamic, preoptic area (AH/POA) and posterior hypothalamus (PH). Postoperatively, the region was first identified in AH/POA which was reactive to norepinephrine or in PH to excess Ca++ ions; in both cases a hypothermic response was produced. Then either an artificial CSF control vehicle or the Ca++ ion channel blocking agent, verapamil, was perfused for 30 min by means of push-pull cannulae at a rate of 25.0 microliters/min. Verapamil 0.4, 2.0 and 4.0 micrograms/microliter) induced a concentration-dependent hypothermia when perfused within AH/POA sites but hyperthermia when perfused in the caudal hypothalamus. An anatomical analysis of the sites of perfusion revealed that verapamil's thermolytic effect was localized within the classical thermosensitive region of the cat's diencephalon, a region ventral to the anterior commissure and dorsal to the optic chiasm. On the other hand, the loci in which verapamil evoked thermogenesis were localized to a region dorsal to the mammillary bodies and caudal to the descending columns of the fornix. It is suggested that verapamil interferes with Ca++ ion channels in the PH to shift the cat's "set-point" temperature. Conversely, however, verapamil apparently could act on catecholaminergic terminals in AH/POA to enhance the presynaptic release of norepinephrine which, in turn, stimulates the heat loss pathway to yield hypothermia.     

Prostanoids in the preoptic hypothalamus mediate systemic lipopolysaccharide-induced hyperalgesia in rats

The systemic administration of lipopolysaccharide (LPS), an experimental model of systemic bacterial infection is known to modulate nociception. It increases the prostaglandin E₂ (PGE₂) levels in the preoptic area of the hypothalamus (POA) and the microinjection of PGE₂ into the POA and the neighboring basal forebrain induces hyperalgesia. We, therefore, hypothesized that the PGE₂ synthesized in these regions mediates intravenous (i.v.) LPS-induced hyperalgesia. To test this hypothesis, we microinjected cyclooxygenase (COX) inhibitors into several sites in the rat hypothalamus and observed their effects on the LPS (0.1–100 μg/kg, i.v.)-induced changes in nociceptive behavior as assessed by a plantar test. LPS (10 and 100 μg/kg, i.v.) reduced the paw-withdrawal latency at 90 min and 45–60 min after injection, respectively, both thus indicating a hyperalgesic effect. This hyperalgesia was observed only in the period before the development of fever which started 120–135 min after the LPS injection. The LPS (100 μg/kg, i.v.)-induced hyperalgesia was completely abolished by pretreatment with the microinjection of diclofenac (an inhibitor of COX-1 and 2) at 1.0 ng into the bilateral POA. Furthermore, it was also blocked by the microinjection of NS-398 (a selective COX-2 inhibitor) at 1.0 ng into the bilateral POA and the horizontal limb of the diagonal band of Broca (DBB), but not the lateral hypothalamic area, the paraventricular hypothalamic nucleus, and the ventromedial hypothalamic nucleus. These findings suggest that LPS (i.v.)-induced hyperalgesia is mediated predominantly through a COX-2 induced prostanoids in the POA and the DBB in rats.     

Direct pyrogenic input from prostaglandin EP3 receptor-expressing preoptic neurons to the dorsomedial hypothalamus

Fever is induced by a neuronal mechanism in the brain. Prostaglandin (PG) E2 acts as a pyrogenic mediator in the preoptic area (POA) probably through the EP3 subtype of PGE receptor expressed on GABAergic neurons, and this PGE2 action triggers neuronal pathways for sympathetic thermogenesis in peripheral effector organs including brown adipose tissue (BAT). To explore pyrogenic efferent pathways from the POA, we determined projection targets of EP3 receptor-expressing POA neurons with a special focus on rat hypothalamic regions including the dorsomedial hypothalamic nucleus (DMH), which is known as a center for autonomic responses to stress. Among injections of cholera toxin b-subunit (CTb), a retrograde tracer, into hypothalamic regions at the rostrocaudal level of the DMH, injections into the DMH, lateral hypothalamic area (LH) and dorsal hypothalamic area (DH) resulted in EP3 receptor immunolabelling in substantial populations of CTb-labeled neurons in the POA. Bilateral microinjections of muscimol, a GABA(A) receptor agonist, into the DMH and a ventral region of the DH, but not those into the LH, inhibited thermogenic (BAT sympathetic nerve activity, BAT temperature, core body temperature and expired CO2) and cardiovascular (arterial pressure and heart rate) responses to an intra-POA PGE2 microinjection. Further immunohistochemical observations revealed a close association of POA-derived GABAergic axon swellings with DMH neurons projecting to the medullary raphe regions where sympathetic premotor neurons for febrile and thermoregulatory responses are localized. These results suggest that a direct projection of EP3 receptor-expressing POA neurons to the DMH/DH region mediates febrile responses via a GABAergic mechanism.     

Organization of central hyperthermic mechanisms in helium-cold hypothermic hamsters

We have investigated the ability of three hyperthermic stimuli (PGE2, 5-HT and ACh) to elicit hyperthermia in the Helium-Cold (He-Cold) hypothermic hamster. Hamsters in these conditions are poikilothermic and will passively follow room temperature in a regulated cold room. Animals were injected centrally at AH/POA sites via an indwelling guide tube at body temperatures maintained between 9-12 degrees C. Active sites in the AH/POA were determined prior to the experiment by PGE2 injection. PGE2 injection at an effective AH/POA site immediately reversed the anesthetic induced hypothermia in warm air. Hamsters were induced into hypothermia by the He-Cold induction method and body temperatures were maintained in a 9 degrees C cold room. Colonic temperatures were monitored at 5 minute intervals by a YSI thermistor probe and telethermometer. Central injections of 5-HT (2 micrograms/microliter) and ACh (50 micrograms/microliter) at effective AH/POA sites evoked significant increases in colonic temperature in He-Cold hamsters. PGE2 injections were not different from saline control injections and did not elicit pronounced temperature changes in these animals. Specific blockade of the 5-HT and ACh temperature increases was demonstrated with specific antagonist injections. The results suggest that the central organization of heat-gain mechanisms in the AH/POA is the same as normothermic animals even at temperatures well below those previously investigated.     

Bombesin produces hypothermia in rats pretreated with 2-deoxy-D-glucose

Previous research has shown that microinfusion of bombesin into the preoptic area (POA) decreases core body temperature in rats that are food-deprived or made hypoglycemic with insulin. The present study employed 2-deoxy-D-glucose, a competitive inhibitor of glycolysis, to further investigate the importance of a reduction in glucose utilization in the production of bombesin-induced hypothermia. Rats (n = 7) were pretreated with 2-DG (0, 25, 50, 100, 200 mg/kg; IP) followed by bombesin (100 ng/1.0 microliters) microinfusions into the POA. The highest dose of 2-DG (200 mg) was also tested in the absence of bombesin as a control. Pretreatment with 2-DG resulted in a dose-related reduction in Tb following bombesin. Injections of 2-DG alone did not significantly alter Tb. The results provide additional evidence that the production of bombesin-induced hypothermia in fasted rats is linked to a reduction in glucose utilization.     

ACh and 5-HT stimulated thermogenesis at different core temperatures in the He-Cold hypothermic hamster

Hamsters in deep experimentally induced hypothermia, at body temperatures between 7 degrees C and 11.5 degrees C, were microinjected with 5-HT and ACh at brain sites in the anterior-preoptic area of the hypothalamus (AH/POA). ACh or 5-HT was injected into an AH/POA site at different starting core temperatures in different groups of hypothermic hamsters. Colonic temperatures (Tc) were maintained, following He-Cold induction, in a temperature controlled environmental chamber and measured with a YSI thermister probe and YSI telethermometer. Injections of either 5-HT or ACh at Tc's between 7.0 degrees C and 9.0 degrees C elicited only modest increases in Tc i.e., 0.3 degrees C--0.6 degrees C, respectively. As Tc increased, however, to ranges between 9.1 degrees C--10.0 degrees C and in different animals to greater than 10 degrees C both ACh and 5-HT at the same sites elicited significant increases in Tc, 1.5 degrees C for 5-HT and 2.2 degrees C for ACh compared to saline injections. These data suggest that at the lowest Tc's we are observing a "cold block" of temperature sensitive sites in the AH/POA. Increasing the starting Tc beyond 9.0 degrees C however, evokes significant increases in heat-gain following AH/POA injection of either ACh or 5-HT. These data are consistent with Myers' observations concerning the organization of heat-gain mechanisms at AH/POA sites. In addition, they suggest that both the afferent limb of the heat-gain circuit (5-HT) and the efferent limb of the circuit (ACh) are functionally impaired when Tc is close to the physiological limit in the He-Cold hypothermic hamster.