Drinking patterns in genetic low-alcohol-drinking (LAD) rats after systemic cyanamide and cerebral injections of THP or 6-OHDA.

A key question related to the role of acetaldehyde and aldehyde adducts in alcoholism concerns their relationship to the genetic mechanisms underlying drinking. Experimentally, the low-alcohol-drinking (LAD) rat represents a standard rodent model having a strong aversion to alcohol. In these experiments, preferences for water vs. alcohol, offered in concentrations from 3% to 30%, were determined over 10 days in adult LAD rats (N = 6 per group). Then a saline vehicle or either 10 or 20 mg/kg of the aldehyde dehydrogenase (AIDH) inhibitor, cyanamide, was injected s.c. twice daily for 3 days. Secondly, either 0.5 or 1.0 microg of tetrahydropapaveroline (THP) was infused i.c.v. twice daily for 3 days in LAD rats (N = 8) and, as a genetic control, THP also was infused identically in Sprague-Dawley (SD) rats (N = 8). The results showed that the lower and higher doses of cyanamide augmented alcohol intakes in 33% and 50% of the LAD rats, respectively, with the patterns of drinking resembling that of genetic high-alcohol-drinking HAD or P rats. Although i.c.v. infusions of THP had little effect on alcohol preference of LAD rats, alcohol drinking was enhanced significantly in the SD rats. In a supplementary study, 200 microg of 6-hydroxydopamine (6-OHDA) also was infused i.c.v. in LAD rats (N = 7) on two consecutive days; no change occurred in the characteristic aversion to alcohol. These findings suggest that in certain individuals, a perturbation in the synthesis of AIDH can modify the genetically based aversion to alcohol, thus precipitating the liability for alcoholism. In that neither THP nor 6-OHDA lesioning exerted any effect on the genetic nondrinking LAD animal suggests that an unknown endogenous factor in the brain must underlie the cyanamide-induced shift to alcohol preference. We conclude that the genetic elements that normally prevent the progression to addictive drinking in most individuals appear to be invariant and irreversible.

Antibodies to macrophage inflammatory protein-1beta in preoptic area of rats fail to suppress PGE2 hyperthermia.

This study determined whether macrophage inflammatory protein-1beta (MIP-1beta) plays a role in the hyperthermia caused by prostaglandin E2 (PGE2) given intracerebroventricularly (i.c.v.) in the rat. In these experiments, anti-murine MIP-1beta antibody (anti-MIP-1beta) was micro-injected in the anterior hypothalamic, preoptic area (AH/POA) just before i.c.v. PGE2. The results showed that anti-MIP-1beta failed to alter the PGE2 hyperthermia. However, immunocytochemical studies revealed MIP-1beta immunoreactivity detectable in both the organum vasculosum laminae terminalis (OVLT) and AH/POA in the febrile rat. These data thus demonstrate that MIP-1beta is sequestered in diencephalic structures underlying thermoregulation even though it is not involved in PGE2 hyperthermia. This dissociation supports the viewpoint that at least two distinct systems exist in the brain which underlie a febrile response: MIP-1beta underlies one component whereas PGE2 comprises the other.

Hypothalamic interaction between macrophage inflammatory protein-1 alpha (MIP-1 alpha) and MIP-1 beta in rats: a new level for fever control?

1. The microinjection of macrophage inflammatory protein-1 (MIP-1 alpha; 5.0 and 25 pg) into the anterior hypothalamic, preoptic area (AHPOA) induced a slow onset; monophasic fever in rats that persisted for a long period. Microinjection of 25 pg MIP-1 beta into the AHPOA induced a fever of rapid onset, whereas 5.0 pg MIP-1 beta did not alter body temperature (Tb) significantly. When either MIP-1 alpha or MIP-1 beta was heated to 70 degrees C for 30 min prior to their injection, no pyrexic response was produced. 2. The concurrent microinjection of 25 pg MIP-1 alpha and 25 pg MIP-1 beta into the AHPOA attenuated the effects on Tb of either cytokine alone. However, pretreatment with either 5.0 pg MIP-1 beta or 5.0 pg MIP-1 alpha suppressed the febrile response induced by 25 pg MIP-1 alpha or 25 pg MIP-1 beta, given at the same site, respectively. 3. The present experiments show that MIP-1 alpha and MIP-1 beta are active individually and possess distinct differences in their evocation of a febrile response. Further, our results suggest a functional antagonism between MIP-1 alpha and MIP-1 beta that could represent a new level in the development of fever.

Fever induced in rats by intrahypothalamic macrophage inflammatory protein (MIP)-1 beta: role of protein synthesis.

The effect of macrophage inflammatory protein-1 beta (MIP-1 beta) on body temperature, following its injection into the anterior hypothalamic pre-optic area (AH/POA), was examined by a radiotelemetry system in the freely moving rat. The purpose of this study was to examine the action of an inhibitor of protein synthesis, anisomycin, on the pyrexia which follows intrahypothalamic injection of MIP-1 beta. The micro-injection of 10 to 20 pg MIP-1 beta into the AH/POA induced a dose-dependent monophasic increase in body temperature, whereas a higher dose of 25 pg of the cytokine caused a biphasic febrile response. When MIP-1 beta was heated at 70 degrees C for 30 min prior to its administration, the pyrogenic response was abolished. Pretreatment of the micro-injection site in the AH/POA with 10 micrograms anisomycin did not alter the febrile response to 25 pg MIP-1 beta given at the same site in the AH/POA. When 10 mg/kg anisomycin was administered subcutaneously, the febrile response to 25 pg MIP-1 beta injected in the AH/POA was significantly suppressed. The present results suggest that fever caused by MIP-1 beta within the cells of the AH/POA may not require the synthesis of a new protein factor; however, the de novo synthesis of a protein outside of the AH/POA presumably plays a functional role, at least in part, in the intense fever produced by this cytokine in the hypothalamus.

Anorexic action of a new potential neuropeptide Y antagonist [D-Tyr27,36, D-Thr32]-NPY (27-36) infused into the hypothalamus of the rat.

Neuropeptide Y (NPY) produces a vigorous feeding response in several species when it is injected into hypothalamic structures involved in eating behavior. The purpose of this study was to determine whether a unique carboxy terminal fragment of NPY would alter the pattern of eating induced in the rat either by NPY injected into the hypothalamus or by a 24-h period of food deprivation. In this case, two L-tyrosine residues and one L-threonine residue of the NPY27-36 fragment were transformed to their D-conformation to produce [D-Tyr27,36,D-Thr32]-NPY (27-36), i.e., D-NPY27-36. Guide cannulae for microinjection were implanted stereotaxically just dorsal to the paraventricular nucleus (PVN) or ventromedial hypothalamus (VMH) of 24 adult male Sprague-Dawley rats. Following postoperative recovery, a microinjection of artificial CSF or 1.1 microgram or 3.3 micrograms of a peptide was made directly into the PVN or VMH as follows: native NPY; D-NPY27-36; or [L-Tyr27,36, L-Thr32]-NPY (27-36), i.e., L-NPY27-36. Food intakes were measured at intervals of 0.25, 0.5, 1.1, 2.0, 4.0, and 24 h. When D-NPY27-36 was microinjected at NPY reactive sites in the PVN or VMH of the rat 15 min before a similar microinjection of NPY, the intense eating response induced by the peptide was reduced significantly. Not only was the effect dose dependent, but D-NPY27-36 also augmented the latency to feed. A mixture of the two doses of NPY and D-NPY27-36 injected at the same hypothalamic loci did not attenuate the intake of food but tended to enhance the feeding response in the rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Prostaglandins and hypothalamic neurotransmitter receptors involved in hyperthermia: a critical evaluation.

The role of a prostaglandin of the E series (PGE) in the hypothalamic mechanisms underlying a fever continues to be controversial. This paper reviews the historical literature and current findings on the central action of the PGEs on body temperature (Tb). New experiments were undertaken to examine the local effect of muscarinic, nicotinic, serotonergic, alpha-adrenergic, or beta-adrenergic receptor antagonists at hypothalamic sites where PGE1 caused a rise in Tb of the primate. Guide tubes for microinjection were implanted stereotaxically above sites in and around the anterior hypothalamic, preoptic area (AH/POA) of male Macaque monkeys. Following postoperative recovery, 30-100 ng of PGE1 was micro-injected unilaterally in a volume of 1.0-1.5 microliter at sites in the AH/POA to evoke a rise in Tb, and once identified, pretreated with a receptor antagonist. PGE1 hyperthermia was significantly reduced by microinjections of the muscarinic and nicotinic antagonists, atropine, or mecamylamine, at PGE1 reactive sites in the AH/POA. The serotonergic antagonist, methysergide, injected at PGE1 sensitive sites in the ventromedial hypothalamus also attenuated the rise in Tb. However, the 5-HT reuptake blocker, fluoxetine, and the beta-adrenergic receptor antagonist, propranolol, injected in the AH/POA failed to alter the PGE1 hyperthermia. In contrast, the alpha-adrenergic antagonist, phentolamine, potentiated the increase in Tb at all PGE1 reactive sites in the hypothalamus. An updated model is presented to explain how the concurrent actions of aminergic neurotransmitters acting on their respective receptors in the hypothalamus can interact with a PGE to elicit hyperthermia. Finally, an evaluation of the current literature including recent findings on macrophage inflammatory protein (MIP-1) supports the conclusion that a PGE in the brain is neither an obligatory nor essential factor for the expression of a pyrogen fever.

Norepinephrine, dopamine, and 5-HT release from perfused hypothalamus of the rat during feeding induced by neuropeptide Y.

In the unrestrained rat, the hyperphagic-like ingestion of food evoked by the sustained elevation of neuropeptide-Y (NPY) in the hypothalamus was correlated with the release and turnover of monoaminergic transmitters in this structure. A single guide tube was implanted stereotaxically in the perifornical region of the hypothalamus for localized push-pull perfusion of an artificial CSF vehicle or NPY1-36 in a concentration of 10, 50, or 100 ng/1.0 microliters. After the rat was fully satiated, a site reactive to NPY was perfused repeatedly at a rate of 20 microliters/min for 6.0 min with an interval of 6.0-12 min elapsing between each perfusion. Samples of perfusate were analyzed by HPLC with coulometric detection for DA, HVA, DOPAC, NE, MHPG, 5-HT, and 5-HIAA. Although control perfusions were without effect on feeding or monoamine activity, NPY evoked mean cumulative intakes of food of 14 +/- 2.4, 25.6 +/- 3.0 and 26.5 +/- 3.2 g in response to 10, 50, or 100 ng/microliter concentrations of NPY, respectively, over the 4.0-5.0 hr test interval. HPLC analyses showed that during feeding the release of both NE and DA was enhanced significantly. The turnover of both catecholamines likewise increased significantly as reflected by the elevated levels of MHPG, DOPAC and HVA. However, neither the basal efflux of 5-HT nor its turnover, as reflected by the output of 5-HIAA, was affected during feeding induced by NPY perfused in the hypothalamus. These results suggest that a sustained elevation of NPY in the hypothalamus causes a perturbation in the basal activity of NE and DA which are both implicated in the neuronal mechanism regulating normal eating behavior. Thus, these catecholamine neurotransmitters are envisaged to comprise an intermediary step in the functional role played by NPY in the hypothalamus in integrating the control of energy metabolism and caloric intake.

Action of cyclosporine on fever induced in rats by intrahypothalamic injection of macrophage inflammatory protein-1 (MIP-1).

In order to examine the central mechanism of pyrexic action of macrophage inflammatory protein-1 (MIP-1), guide cannulae for injections were implanted stereotaxically just above the anterior hypothalamic, pre-optic area of the rat. Following post-operative recovery, the body temperature (Tb) of each rat was monitored by a colonic thermistor probe over a test interval of 4 hr. Injected in a 0.5 microliter volume into the anterior hypothalamic pre-optic area, MIP-1, in a dose of 5.6 or 28 pg, evoked an intense fever with a latency of 15-30 min. Pretreatment of the anterior hypothalamic pre-optic area with 1.0 microgram cyclosporine A (CsA), delivered in a volume of 0.5 microliter, delayed the onset of the fever induced by 5.6 pg MIP-1, injected at the same site. Similar injections of CsA also attenuated significantly the magnitude of the fever, following either the 5.6 or 28 pg dose of MIP-1. As a systemic control, 15 mg/kg CsA was administered intraperitoneally, 2.0 hr before the injection of MIP-1 in the anterior hypothalamic pre-optic area. By this route, CsA also delayed the rise in temperature but the fever induced by 5.6 pg MIP-1 reached the same magnitude as that after MIP-1 alone. Conversely, intraperitoneal administration of CsA did not antagonize the pyrexic response evoked by 28 pg MIP-1, injected into the anterior hypothalamic pre-optic area, but rather enhanced the fever.(ABSTRACT TRUNCATED AT 250 WORDS)

Fever induced by macrophage inflammatory protein-1 (MIP-1) in rats: hypothalamic sites of action.

The purpose of this study was to clarify the central site of action as well as functional characteristics of the febrile response of the cytokine, macrophage inflammatory protein-1 (MIP-1). Guide cannulae for microinjection were implanted stereotaxically in the rat just above the pyrogen and thermosensitive area of the anterior hypothalamic, preoptic area (AH/POA). Following postoperative recovery, the body temperature of each rat (Tbo) was monitored during an experiment by a colonic thermistor probe at 0.5-1.0-h intervals. When MIP-1 was microinjected in a 0.5-microliter volume into the AH/POA in one of eight concentrations ranging from 0.0028 nanograms (ng) to 9.0 ng, an intense monophasic or biphasic fever was evoked. The MIP-1-induced increase in the Tbo of the rat was characterized by its short latency of 15 to 30 min and an inverse dose-response curve. Measures of mean latency and maximal rise in Tbo following MIP-1 confirmed the potency of this dose. Although the dose of 0.028 ng produced a fever of over 2.0 degrees C with a latency of only 15 min or less, the hyperthermic response became less intense as the dose of MIP-1 was increased. An anatomical mapping of sites of microinjection which reacted to MIP-1 in mediating fever revealed that the medial portion of the POA of the rat just rostral to the border of the AH was the region of maximum sensitivity to the cytokine.(ABSTRACT TRUNCATED AT 250 WORDS)

Hypothalamic indomethacin fails to block fever induced in rats by central macrophage inflammatory protein-1 (MIP-1).

This investigation examined the extent to which the activity of a prostaglandin (PG) in the anterior hypothalamic, preoptic area (AH/POA) of the rat plays a role in the intense fever induced by macrophage inflammatory protein-1 (MIP-1) applied directly to this anatomical region. For the microinjection of both a PG synthesis inhibitor, indomethacin, and MIP-1 into sites within the AH/POA, guide cannulae were implanted chronically just above this pyrogen-reactive region. Postoperatively, the body temperature (Tb) of each rat was monitored in the unrestrained condition by means of a colonic thermistor probe. MIP-1 microinjected into the AH/POA in a 0.5-microliter volume evoked a biphasic fever when given in a dose of 5.6 picograms (pg) and a monophasic fever in a dose of 28 pg. The latency of the febrile response was ordinarily 15 min with an asymptote of 1.5 degrees C reached ordinarily within 2.0-2.5 h. When the cytokine-reactive site in the AH/POA was pretreated with indomethacin microinjected in an efficacious dose of 0.5 microgram, the MIP-1 fever evoked by 5.6 pg was not inhibited. Further, pretreatment of AH/POA sites with indomethacin prior to the higher 28-pg dose of MIP-1 delayed the febrile response but did not block it. As a systemic control, indomethacin also was administered intraperitoneally in a dose of 5.0 mg/kg, again 15 min prior to the microinjection of MIP-1 into the AH/POA. In this case, indomethacin only partially attenuated but did not block the fever evoked by either dose of MIP-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Macrophage inflammatory protein-1: unique action on the hypothalamus to evoke fever.

Macrophage inflammatory protein (MIP-1) administered systemically causes a fever not blocked by a prostaglandin (PGE) synthesis inhibitor. The purpose of this study was to examine the central mechanism of pyrexic action of this cytokine in the unrestrained rat. After guide cannulae for microinjection were implanted stereotaxically just above the anterior hypothalamic preoptic area (AH/POA), the body temperature of each rat was monitored by a colonic thermistor probe. Saline control vehicle or MIP-1 was microinjected into the AH/POA in one of eight concentrations ranging from 0.0028-9.0 ng per 0.5 mu 1 volume. MIP-1 induced a biphasic or monophasic fever of short latency characterized by an inverse dose-response curve. The potency of MIP-1 was in the femtomolar (10(-15)) range with the lowest dose of 0.028 ng producing a fever of over 2.0 degrees C with a latency of 15 min or less. To determine whether a PGE mediates MIP-1 fever, indomethacin was administered either intraperitoneally in a dose of 5.0 mg/kg or directly into the MIP-1 injection site in a dose of 0.5 microgram/0.5 mu 1, both injected 15 min before MIP-1. Pretreatment of the injection site in the AH/POA with indomethacin failed to prevent the febrile response evoked by MIP-1 injected at the same locus. Further, the dose of systemic indomethacin, which blocks PGE-induced fever in the rat, attenuated only partially the MIP-1 fever. The results demonstrate that MIP-1 is the most potent endopyrogen discovered thus far, and that its action is directly in the region of the hypothalamus which contains both thermosensitive and pyrogen-sensitive neurons. The local action of MIP-1 on cells of the AH/POA in evoking fever is unaffected by the PGE inhibitor which indicates, therefore, that a cellular mechanism operates in the hypothalamus to evoke fever independently of the central synthesis of a PGE.

Neurotensin perfused in hypothalamus of sated or fasted rat: HPLC analysis of release of DA, NE and 5-HT and their metabolites.

This investigation was undertaken in the unrestrained rat to determine the localized effect of neurotensin (NT) on the profile of release and turnover of norepinephrine (NE), dopamine (DA) and serotonin (5-HT) within the hypothalamus. Following stereotaxic implantation of a permanent guide tube, artificial CSF was perfused in the hypothalamus of the freely moving animal by means of push-pull cannulae at a rate of 20 microliters/min and for an interval of 5.0 min. After three 5.0 min control samples were collected, NT in a concentration of 0.1 micrograms/microliter was perfused followed by additional CSF controls. Assay by HPLC-EC of each perfusate showed that when the rat was sated, NT evoked a significant increase in the release of DA and DOPAC from the hypothalamus as well as augmented NE turnover, as reflected by a significant efflux in MHPG. However, when the rat was fasted for 22 hr, the perfusion of NT reduced DA and DOPAC concentrations in the diencephalic perfusate significantly as well as levels of both MHPG and VMA. Under both sated and fasted conditions, NT failed to produce notable changes in the release of 5-HT or its metabolism to 5-HIAA. These findings thus reveal a functional interaction between NT and both of the catecholamine neurotransmitters within hypothalamic neurons, which is clearly dependent upon the nutritional status of the animal.

Rostral hypothalamus: a new neuroanatomical site of neurochemically-induced emesis in the cat.

The localized effect of noradrenergic agonists administered directly in the anterior hypothalamic preoptic area (AH/POA) in inducing emesis in the cat was investigated. Of the noradrenergic agonists tested, which included norepinephrine, clonidine, phenylephrine and methoxamine, only clonidine in doses of 5.0-50.0 micrograms was found to evoke emesis consistently when micro-injected in a volume of 1.0 microliter into AH/POA of the unrestrained cat. The emetic response to clonidine was short-lasting, generally dose-dependent in terms of latency and frequency, and occurred in bouts of one to three episodes. The sequence of the vomiting response, beginning with licking and retching, functionally resembled a normal pattern of an emetic response. The clonidine-induced emesis was not antagonized by the following antagonists micro-injected in AH/POA just prior to clonidine: alpha-adrenergic blocking agents, yohimbine, RX 781094 and phentolamine; the antimuscarinic drug, atropine; the serotonin antagonist, methysergide; the opioid antagonist, naloxone; and the dopamine antagonist, chlorpromazine. Therefore, it would appear that clonidine-induced emesis is not mediated by alpha noradrenergic, serotonergic, dopaminergic, muscarinic and opiate receptor systems within the AH/POA of the cat. Finally, the obtained results show that apart from the area postrema and a circumscribed zone of the brain-stem reticular formation, the hypothalamus is now implicated as a neuroanatomical site in the central nervous system mechanism underlying neurochemically-induced emesis.

Ethanol challenge alters amino acid neurotransmitter release from frontal cortex of the aged rat.

In two groups of male rats having an average age of either 90 days or two years, guide cannulae for bilateral push-pull perfusion were implanted stereotaxically to rest upon the superficial frontal cerebral cortex. On post-operative recovery, either 1.5 or 3.0 g/kg 20% ethanol (V/V) was given by intragastric gavage to each unrestrained rat. Sequential samples of venous blood were obtained from the tail and analyzed for alcohol levels by gas chromatography. A set of push-pull perfusions of the cortical sites was carried out with an artificial CSF before gavage and at 25, 50 and 150 min after the administration of ethanol. An individual perfusion was continued for 5.0 min at a rate of 25 microliters/min. Using high performance liquid chromatography with electrochemical detection (HPLC-EC) each sample of perfusate was then assayed for its content of glutamate (Glu), aspartate (Asp), glutamine (Gln), glycine (Gly), taurine (Tau) and GABA with homoserine used as the internal standard. The results showed that the 3.0 g/kg dose of ethanol resulted in a higher level of blood ethanol in the older animals, which persisted over the 150 min time interval. Further, the 1.5 g/kg dose of ethanol administered to the older rats reduced the cortical activity of Glu and Gln relative to the younger animals. In addition, the 3.0 g/kg dose augmented the cortical efflux of Tau in the aged rats. Neither dose of ethanol affected the efflux of Asp or Gly from the perfused frontal cortex of either the young or old group, nor was the release of GABA detectable under either the control condition or following treatment with ethanol.(ABSTRACT TRUNCATED AT 250 WORDS)

Neurotensin releases norepinephrine differentially from perfused hypothalamus of sated and fasted rat.

The central injection of neurotensin (NT) has been reported to attenuate the intake of food in the fasted animal. To determine whether endogenous norepinephrine (NE) is involved in the satiating effect of NT, the in vivo activity of NE in circumscribed sites in the hypothalamus of the unanesthetized rat was examined. Bilateral guide tubes for push-pull perfusion were implanted stereotaxically to rest permanently above one of several intended sites of perfusion, which included the paraventricular nucleus (PVN), ventromedial nucleus (VMN), and the lateral hypothalamic (LH) area. After endogenous stores of NE at a specific hypothalamic locus were radiolabeled by microinjection of 0.02-0.5 mu Ci of [3H]NE, an artificial cerebrospinal fluid was perfused at the site at a rate of 20 microliter/min over successive intervals of 5.0 min. When 0.05 or 0.1 micrograms/microliter NT (0.03-0.6 mM) was added to the perfusate, the peptide served either to enhance or reduce the local release of NE at 50% of the sites of perfusion. In these experiments, the circumscribed effect of NT on the characteristics of catecholamine efflux depended entirely on the state of hunger or satiety of the rat. That is, when NT was perfused in the fully satiated rat, NE release was augmented within the PVN or VMN; conversely, NE release was inhibited in the LH. In the animal fasted for 18-22 h, NT exerted an opposite effect on the activity of NE within the same anatomical loci in that the efflux of NE was enhanced in the LH but attenuated or unaffected in the PVN or VMN.(ABSTRACT TRUNCATED AT 250 WORDS)

Rate of in vivo verapamil exchange within the hypothalamus of the cat as examined by push-pull perfusion.

To investigate the characteristics of the uptake within hypothalamic tissue of the Ca2+-channel blocker, verapamil, push-pull canulae were implanted bilaterally above the anterior hypothalamic-preoptic area (AH/POA) and posterior hypothalamus (PH) of the cat. The functional reactivity of these two anatomical regions was verified in the unrestrained cat, prior to a push-pull perfusion, by a micro-injection of either 5-7 micrograms norepinephrine (NE) into AH/POA, or by perfusion of 50 mM Ca2+ within the PH, both of which induce a transient decline in the cat's core temperature. Verapamil was perfused in a concentration of 0.4, 2.0 or 4.0 micrograms/microliter for successive 10 and 20 min intervals within these NE and Ca2+-sensitive sites. A quantitative analysis of verapamil in each sample of perfusate was performed double-blind by HPLC-spectrophotometric detection. The results showed that the percent recovery of verapamil after the 10 min interval was always less than that after the next 20 min period of perfusion. These recovery values were independent of the site of perfusion and the concentration of verapamil. However, the mean uptake of verapamil into tissue after 10 min was significantly greater than that after the 20 min period for all concentrations tested. These results demonstrate that the hypothalamus has a time-dependent characteristic to incorporate a Ca2+-channel blocker into the parenchyma. Once the point of tissue saturation is reached, a steady-state level of verapamil uptake is established.

Rapid method for micro-analysis of endogenous amino acid neurotransmitters in brain perfusates in the rat by isocratic HPLC-EC.

A simplified method is described for the isocratic analysis of endogenous amino acid neurotransmitters contained in brain perfusates by high performance liquid chromatography (HPLC) with electrochemical detection (EC). Pre-column o-phthalaldehyde (OPA) tert-butylthiol derivatives of the amino acids were injected into a C18 3 microns column. After linear concentration curves for standard solutions were obtained, the content of 6 amino acid neurotransmitters was analyzed in push-pull perfusates obtained from the hypothalamus and cerebral cortex of the unrestrained rat. Each analysis which included the simultaneous quantification of aspartic acid, glutamic acid, glutamine, glycine, taurine and gamma-aminobutyric acid (GABA), was completed in less than 15 min. The sensitivity of the assay ranged from 1.0 to 5.0 pmol of each amino acid contained within a 20 microliters aliquot of each perfusion sample.

CCK and other peptides modulate hypothalamic norepinephrine release in the rat: dependence on hunger or satiety.

The purpose of this investigation was to determine the functional relationship between putative satiety peptides and endogenous norepinephrine (NE) activity in the hypothalamus. Permanent guide cannulae for push-pull perfusion were implanted stereotaxically in Sprague-Dawley rats so as to rest above the medial or lateral hypothalamus (LH). Post-operatively, the animals were either satiated with food and water, both available ad lib, or fasted for 18-22 hr prior to an experiment. To perfuse a site in the LH, paraventricular (PVN) or ventromedial nucleus (VMN), a concentric 29-23 ga push-pull cannula system was lowered to a pre-determined site, in most cases after catecholamine stores had been pre-labeled with [3H]-NE. During control tests, an artificial CSF was perfused at a rate of 20-25 microliter/min for 5-8 min with a 5 min interval between each sample. The addition of cholecystokinin (CCK) in a concentration of 2.0-6.0 ng/microliter to the CSF perfused in PVN or VMN of the satiated rat enhanced the efflux of NE; however, in the fasted animal CCK often suppressed the catecholamine's release. Perfused in the LH, CCK exerted opposite effects, typically augmenting NE output when the rat was fasted but not affecting the amine's activity during the sated condition. Proglumide (1.2 micrograms/microliter) attenuated CCK's effect in releasing NE when the antagonist was perfused in the PVN of the satiated rat. Similar experiments in which neurotensin (NT) was perfused in the LH, PVN and VMN revealed virtually the same inverse effects on NE release in the fasted and satiated rat, which again were anatomically specific. Finally, insulin and 2-deoxy-D-glucose (2-DG) exerted similar state-dependent effects on the release of NE within LH and PVN. Overall, the results suggest that CCK or other neuroactive peptide could serve as a "neuromodulator" of the pre-synaptic release of NE within classical hypothalamic structures which are thought to underlie both hunger and satiety. The state-dependent nature of the peptides' activity on the noradrenergic feeding mechanism implies that these substances constitute a pivotal portion of the profile of factors which impinge functionally upon the hypothalamic neurons responsible for the feeding response and its cessation.

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.

Release of norepinephrine from the rat's hypothalamus perfused with alcohol: relation to body temperature.

The activity of norepinephrine (NE) within the thermosensitive region of the anterior hypothalamic, pre-optic area (AH/POA) of the rat was examined in relation to changes in core temperature produced by ethyl alcohol. Following stereotaxic implantation of push-pull guide tubes, a specific site in the AH/POA, reactive or non-reactive to NE, was labeled with 1.0 microliter of [3H]-NE. Alcohol in a concentration of 2.75% or 5.5% was then perfused locally at the same site by push-pull cannulae or administered peripherally in a dose of 2.0 g/kg. In control experiments, artificial CSF was perfused alone. The perfusion of alcohol enhanced or delayed the release of [3H]-NE in AH/POA or failed to alter the efflux of the catecholamine, with the specific response dependent principally on the: (1) anatomical site of hypothalamic perfusion, (2) concentration of alcohol, and (3) interval of perfusion itself. During the perfusion of alcohol within a very circumscribed region in the AH/POA, vasodilatation, as reflected by an increase in skin temperature, and a hypothermia of short latency, occurred. The change in core temperature was usually accompanied by a delay in the efflux of [3H]-NE. After the peripheral administration of 2.0 g/kg alcohol, an alteration in NE efflux from the AH/POA was also induced during the course of a hypothermic response accompanied by vasodilatation. These results suggest that alcohol exerts a direct central effect on nerve cells comprising the thermoregulatory mechanism located within the hypothalamus. Further, the well-known thermolytic effect of alcohol could be mediated in part by noradrenergic synapses within AH/POA, by means of their phasic release of NE.