Icv Infusion Research Articles

Chronic ICV infusion of morphine and NPFF down-regulate mu opioid receptors in rat brain: A quantitative autoradiographic study

Regulation of the opioid μ receptor by pharmacological manipulation has been extensively investigated using various radioreceptor binding techniques. However, unlike most receptor systems, where chronic agonist administration induces a characteristic down-regulation in receptor density, prolonged morphine treatment has shown conflicting results. Both up- and down-regulation of μ receptors by morphine have been reported (for review see (1)). Our lab recently demonstrated that morphine modulating peptide, NPFF (Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH 2), down-regulates opioid μ receptors (2). The purpose of this study was to compare the effects of chronic ICV infusion of morphine and NPFF on μ opioid binding sites using the in vitro techniques of whole brain homogenate receptor binding and autoradiography.

Effect of tachykinins on the need-free sodium intake of female rats: A continuous intracerebroventricular infusion study

The present study investigated the effect of 24-h continuous ICV infusion of four different tachykinins on the enhanced need-free sodium intake induced by previous repeated sodium depletions in female rats. Female rats were employed because, in response to sodium depletions, they develop a higher need-free sodium intake than male rats. The following tachykinins were used: eledoisin, substance P (SP), [Sar 9,Met(O 2) 11]SP and [Asp 5,6,MePhe 8]SP(5–11), also referred to as NH 2-senktide, all at the same doses of 300 or 600 ng/h × 24 h. Food pellets, water, and 3% NaCl sodium solution were freely available. Eledoisin and NH 2-senktide were more potent than SP in reducing the need-free sodium intake. On the other hand, [Sar 9,Met(O 2) 11]SP had no effect. None of the tachykinins employed completely blocked the intake. Water intake was reduced, but this reduction was apparently a consequence of reduced intake of hypertonic sodium cloride solution, because at the same doses TKs did not inhibit water intake in a single-bottle test. Food intake remained unchanged at either dose used. These findings confirm previous studies in which pulse injection of the same drugs potently inhibited sodium intake. They also demonstrate that tachykinins endowed with high affinity for the NK 3 receptor are the most potent in inhibiting sodium intake. Furthermore, these findings indicate that the tachykinins reduce the need-free sodium intake only during the infusion period, indicating that in these conditions they do not evoke either aversion for salt, or toxic consequences in the follow-up period.

Reducing brain sodium concentration prevents post‐prandial and dehydration‐induced natriuresis in sheep

Renal Na excretion during the 24 h following feeding was studied in sheep. A pronounced natriuresis occurred 3.5-5.5 h after feeding. Na excretion then fell to low levels in animals allowed to drink water, but was significantly elevated above this level in water-deprived sheep for most of the remaining period. Both the post-prandial and dehydration-induced natriuresis were prevented by intracerebroventricular (icv) infusions of low Na concentration 0.3 mol l-1 mannitol at 1 ml h-1, and a water diuresis also occurred. These effects were not caused by icv infusion of artificial cerebrospinal fluid (Na concentration = 150 mmol l-1). As a result, there was a much greater increase in plasma Na concentration and osmolality in the sheep given icv mannitol. Intravenous infusion of vasopressin prevented the water diuresis induced by icv mannitol, but the inhibition of natriuresis was still observed and plasma Na concentration increased by 8 mmol l-1 over 24 h compared with an increase of 3 mmol l-1 in dehydrated sheep infused icv with artificial cerebrospinal fluid. The results show that the ambient Na concentration in the brain plays an important role in the normal homeostatic regulation of Na balance by the kidney in sheep.

Effects of morphine on hypothalamic tyrosine hydroxylase mRNA levels in dopaminergic neurons and on preoptic DOPAC levels measured by microdialysis

Morphine not only suppresses norepinephrine-induced increases in LHRH mRNA levels but, in these same animals, it simultaneously amplifies norepinephrine (NE)-induced LH release. These observations suggest that NE may activate parallel mechanisms which independently increase LHRH mRNA levels and LHRH release and suggest that some of these effects could be mediated indirectly via morphine's action on different components of the hypothalamic dopamine (DA) system. Accordingly, in the present studies we examined the effects of morphine on various components of this dopamine system using as our index of altered DA neuronal activity, the changes which occur in tyrosine hydroxylase (TH) mRNA levels following morphine. As an ancillary index of changes which occur in dopamine neuronal activity, we measured, by microdialysis, the changes which occur in preoptic dihydroxyphenylacetic acid (DOPAC) levels after either subcutaneous injections or following microinfusions of morphine into the zona incerta (ZI). In a final study, we evaluated whether DA when given alone (icv infusion) or prior to icv NE would altered LH release. Single cell levels of TH mRNA in preoptic A15 and periventricular anterior hypothalamic A14 DA neurons were not affected by morphine 1, 5 and 24 h later. In contrast, within 1 h after morphine, TH mRNA levels in ZI A13 neurons were significantly elevated and they remained high at 5 and 24 h compared to controls. Morphine also resulted in a significant rise in TH mRNA levels in tuberoinfundibular DA neurons (TIDA) (A12) within 1 h and these levels remained high to 5 h. Thereafter, by 24 h, message levels had returned to control values. Morphine also resulted in a rapid rise in plasma prolactin (Prl) with peak values occurring at 20 min and then returning to baseline by 90 min. When morphine was given sc it resulted, within 15 min, in a rapid rise in preoptic DOPAC levels and these levels continued to rise such that they were 217% higher than pretreatment values by 105 min. Preoptic 5-hydroxyindoleacetic acid (5-HIAA) levels also increased by 25–75% after sc morphine. The microinfusion of morphine into ZI also resulted in elevated preoptic DOPAC but not 5-HIAA levels within 15 min. The icv infusion of DA alone had no effect on plasma LH whereas, NE (icv) produced a modest but significant increase in plasma LH. When DA was given 15 min prior to the infusion of NE, neither amplification nor inhibition of NE-induced LH release occurred. From these and other studies we conclude that the morphine-induced suppression of TIDA neuronal activity may allow NE to release greater amounts of LHRH from axon terminals in the median eminence. On the other hand, we doubt that the rise in preoptic DA (DOPAC) which occurs after morphine plays a role in augmenting the action of NE on LHRH release since icv DA does not alter the patterns or concentrations of plasma LH which occur after NE alone.

Glutamate, learning and dementia-selection of evidence

Initial suggestions on the involvement of glutamate in memory came from electrophysiological studies on LTP that is blocked by NMDA-antagonists. Then Morris and colleagues (1986) provided the first evidence that icv infusion of the competitive NMDA antagonist 2-amino-5-phosphonovaleric acid (APV) to rats, inhibits both LTP in vivo and spatial learning in a Morris water maze. This was followed by a great amount of evidence confirming the initial finding in various learning tasks. The present paper is devoted to critical review of the literature focusing on the following problems: which glutamate receptors are involved?, in which tests NMDA antagonists inhibit learning?; which types of memory are affected?; which brain structures are involved?; do NMDA receptor antagonists invariably impair learning?; is the effect of NMDA receptors antagonists on learning specific?; does the stimulation of NMDA receptors result in cognitive enhancement?.

Chronic intracerebroventricular infusion of the antiopioid peptide, Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH 2 (NPFF), downregulates mu opioid binding sites in rat brain

Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2 (NPFF), an endogenous mammalian antiopioid peptide, has been shown by other laboratories to attenuate the acute antinociceptive effects of morphine, the development of morphine tolerance, and naloxone-induced withdrawal in morphine-dependent rats. The present study determined the effect of chronic NPFF on mu opioid receptors and mRNA for the endogenous opioids dynorphin and enkephalin. Rats received ICV infusions of either saline or NPFF (5 μg/h) for 13 days via Alzet 2002 osmotic minipumps. Homogenate binding studies, which used whole brain membranes, demonstrated that NPFF decreased the B max of mu binding sites (labeled by [ 3H][ d-Ala 2-MePhe 4,Gly-ol 5]enkephalin) from 262 ± 12 to 192 ± 12 fmolmg protein, and increased the K d from 1.1 to 2.3 nM . Quantitative receptor autoradiography and in situ hybridization experiments were conducted with sections collected at the level of the striatum. The density of mu opioid binding sites labeled by [ 3H][ d-Ala 2-MePhe 4,Gly-ol 5]enkephalin was decreased in all brain areas measured except the corpus callosum, and there was no change in dynorphin mRNA or enkephalin mRNA in the caudate, the nucleus accumbens, or the ventral pallidum. Rats chronically administered ICV morphine sulfate (20 μg/h) for 14 days developed tolerance to morphine and a low degree of dependence, as measured by naloxone-precipitated withdrawal. Chronic administration of NPFF concurrently with morphine sulfate did not significantly alter naloxone-induced withdrawal signs or the development of morphine tolerance. Viewed collectively with previous findings that chronic ICV infusion of anti-NPFF IgG upregulates mu receptors, these data provide additional evidence that the density of CNS mu receptors is tonically regulated by NPFF in the extracellular fluid. The action of NPFF to decrease mu receptors is consistent with an antiopioid role for this peptide; however, the fact that NPFF (administered into the lateral ventricle) did not appreciably alter expression of morphine tolerance and dependence contrasts with previous findings and reinforces the view that this effect is most reliably seen after third ventricle administration.

Role of arteria baroreceptor input on thirst and urinary responses to intracerebroventricular angiotensin II

Intracerebroventricular (icv) infusion of angiotensin II (ANG II) in rats elicits greater water intake under hypotensive, compared with normotensive, conditions. The present experiments used sinoaortic baroreceptor-denervated (SAD) rats and sham-operated rats to examine if the modulatory effects of arterial blood pressure on water intake in response to icv ANG II are mediated by arterial baroreceptors. Mean arterial blood pressure (MAP) was raised or lowered by intravenous (i.v.) infusions of phenylephrine (1 or 10 micrograms.kg-1 x min-1) or minoxidil (25 micrograms.kg-1 x min-1), respectively. The angiotensin-converting enzyme inhibitor captopril (0.33 mg/min) was infused i.v. to prevent the endogenous formation of ANG II during testing. Urinary excretion of water and solutes was measured throughout. Water intake elicited by icv ANG II was inversely related to changes in MAP. Specifically, rats drank more water in response to icv ANG II when MAP was reduced by minoxidil but drank less water when MAP was elevated by phenylephrine. The influence of changing MAP on the icv ANG II-induced drinking responses was not affected by SAD. These results suggest that the modulatory effects of arterial blood pressure on icv ANG II-induced drinking can occur in the absence of sinoaortic baroreceptor input.

Memory facilitation educed by food intake

Acidic fibroblast growth factor (aFGF) in rat CSF increased 1000 times in the 2-h period after food intake, or IP, or ICV glucose infusion. The ICV application of aFGF dose dependently depresses and anti-aFGF antibody facilitates food intake. aFGF is produced in the ependymal cells and released into the CSF in response to increased glucose in the CSF caused by food intake. Released aFGF diffused into the brain parenchyma and was taken up into neurons in the hypothalamus, hippocampus, amygdala, etc. IP injection of glucose 2 h before a task that combined acquisition with passive avoidance significantly increased retention of avoidance by mice tested 24 h later. In a Morris water maze task, IP glucose injection 2 h before a first trial block reduced time to find and climb onto a platform hidden just below the water surface. The glucose facilitation of these affective and spatial memory were abolished by pretreatment with anti-aFGF antibody applied ICV. Continuous ICV infusion of aFGF into rats also significantly increased the reliability of passive avoidance for several days. After food intake, centrally released aFGF reaches the hippocampus and facilitates memory; peripherally released cholecystokinin reaches the endings of the afferent vagal nerves in the portal vein and changes their activity, which modulates hippocampal activity, to lead to memory facilitation. This, however, is blocked by vagotomy below the diaphragm. The results indicate the importance of food intake, not only to maintain homeostasis, but also to prepare a readiness state for memory facilitation.

Stimulation of prolactin secretion in the pig: central effects of relaxin and the antiprogesterone RU 486

Our previous study has shown that oral administration of a potent progesterone antagonist, RU 486, caused a marked elevation of plasma concentrations of both PRL and progesterone in hysterectomized pigs bearing aging corpora lutea. Hysterectomized pigs (hysterectomy performed on day 8; estrus = day 0) were subjected to cranial surgery for chronic placement of a head-mounted stereotaxic apparatus for intracerebroventricular (icv) administration of relaxin (300 U once daily on days 111 and 113; n = 6) and RU 486 (4 mg once daily on days 111, 113, and 115; n = 5) to test whether relaxin and RU 486 exert their actions within the central nervous system and/or pituitary gland to affect PRL and GH secretion. Control pigs (n = 3) received icv injection of vehicle. Intensive blood sampling revealed that icv injection of relaxin on day 111 markedly increased the plasma PRL concentration from 8 to 38 ng/ml within 10 min (P < 0.01). An identical icv injection of relaxin on day 113 caused only a modest increase in PRL, but the overall mean concentration of PRL after relaxin treatment was greater than that before treatment (14 vs. 8 ng/ml; P < 0.05). Intracerebroventricular injection of RU 486 on day 111 greatly elevated plasma PRL. The increase in PRL lasted more than 2 h, with several peak increases of 18-29 ng/ml (P < 0.01). The PRL response to subsequent icv infusion of RU 486 on days 113 and 115 was blunted, but the overall mean concentration of PRL (14 ng/ml) after icv injection of RU 486 remained greater (P < 0.01) than that before treatment (9 ng/ml). In contrast, PRL concentrations in the control group remained unchanged after injection. Plasma concentrations of GH, relaxin, and progesterone were significantly altered in neither hormone- nor vehicle-treated groups during this brief period of sequential blood sampling. This study provides strong evidence that relaxin has a central role in modulating PRL secretion in the pig. In addition, the antagonistic effects on progesterone receptor by RU 486 in the central nervous system and/or pituitary gland caused an abrupt increase in PRL secretion in these hysterectomized gilts.

Stimulation of prolactin secretion in the pig: central effects of relaxin and the antiprogesterone RU 486.

Our previous study has shown that oral administration of a potent progesterone antagonist, RU 486, caused a marked elevation of plasma concentrations of both PRL and progesterone in hysterectomized pigs bearing aging corpora lutea. Hysterectomized pigs (hysterectomy performed on day 8; estrus = day 0) were subjected to cranial surgery for chronic placement of a head-mounted stereotaxic apparatus for intracerebroventricular (icv) administration of relaxin (300 U once daily on days 111 and 113; n = 6) and RU 486 (4 mg once daily on days 111, 113, and 115; n = 5) to test whether relaxin and RU 486 exert their actions within the central nervous system and/or pituitary gland to affect PRL and GH secretion. Control pigs (n = 3) received icv injection of vehicle. Intensive blood sampling revealed that icv injection of relaxin on day 111 markedly increased the plasma PRL concentration from 8 to 38 ng/ml within 10 min (P < 0.01). An identical icv injection of relaxin on day 113 caused only a modest increase in PRL, but the overall mean concentration of PRL after relaxin treatment was greater than that before treatment (14 vs. 8 ng/ml; P < 0.05). Intracerebroventricular injection of RU 486 on day 111 greatly elevated plasma PRL. The increase in PRL lasted more than 2 h, with several peak increases of 18-29 ng/ml (P < 0.01). The PRL response to subsequent icv infusion of RU 486 on days 113 and 115 was blunted, but the overall mean concentration of PRL (14 ng/ml) after icv injection of RU 486 remained greater (P < 0.01) than that before treatment (9 ng/ml). In contrast, PRL concentrations in the control group remained unchanged after injection. Plasma concentrations of GH, relaxin, and progesterone were significantly altered in neither hormone- nor vehicle-treated groups during this brief period of sequential blood sampling. This study provides strong evidence that relaxin has a central role in modulating PRL secretion in the pig. In addition, the antagonistic effects on progesterone receptor by RU 486 in the central nervous system and/or pituitary gland caused an abrupt increase in PRL secretion in these hysterectomized gilts.

Patent Evaluation: Thienopyrazine-2,3-diones as NMDA antagonists

SummaryNovelty: Novel thienopyrazine-2,3-diones are claimed along with their use for the treatment of CNS disorders. These compounds are described as potent, selective antagonists at the glycine binding site on the NMDA receptor complex.Biology: Data are given for five compounds in both in vitro glycine binding and an in vivo convulsion induced by icv infusion of an NMDA model. The most potent compounds in vitro were the 6-chloro-7-propyl derivative (IC50 = 0.18μM, ED50 = 104μg/kg) and the 7-bromo derivative (IC50 = 0.18μM, ED50 = 70 μg/kg) but the most potent compound in vivo is the 7-methyl derivative (IC50 = 1.0 μM, ED50 = 46.4 μg/kg).Chemistry: This series of compounds can be prepared from the methyl 3-aminothiophene-2-carboxylate via a Curtius rearragement on the N-Boc protected carboxylic acid using diphenylphosphoryl azide. Forty compounds are named with the claims, including 6-chloro-7-propylthieno[2,3-b]pyrazine-2,3-(1H,4H)dione.

Differential effects of central and peripheral administration of growth hormone (GH) and insulin-like growth factor on hypothalamic GH- releasing hormone and somatostatin gene expression in GH-deficient dwarf rats

The roles of GH and insulin-like growth factor-I (IGF-I) in the regulation of hypothalamic GH-releasing hormone (GRH) and somatostatin (SRIH) gene expression were investigated in the GH-deficient dwarf (dw) rat, in which endogenous feedback signals are lacking. Adult male and female dw rats were treated with GH or IGF-I by systemic (sc) administration or intracerebroventricular (icv) infusion, and hypothalamic GRH and SRIH mRNA were determined by Northern blotting and densitometric analysis. Systemic sc injection of rGH (75 micrograms every 12 h for 3 days) decreased GRH mRNA levels in both sexes. However, systemic sc injection of human IGF-I (150 micrograms every 12 h for 3 days) did not affect GRH mRNA levels in either sex despite significant stimulation of body weight gain. The use of a continuous sc infusion, which normalized serum IGF-I levels, and prolongation of the treatment period to 7 days also failed to change GRH mRNA levels. SRIH mRNA was unaffected by systemic administration of either GH or IGF-I. Continuous icv infusion of GH (1 microgram/h for 7 days) decreased GRH mRNA levels in both sexes, but did not alter SRIH mRNA levels. Continuous icv infusion of IGF-I (100 ng/h for 7 days) decreased GRH mRNA in both sexes. In contrast, SRIH mRNA levels were increased in both sexes. IGF-I decreased GRH mRNA levels at icv infusion rates of 100 and 300 ng/h and stimulated SRIH mRNA levels at infusion rates of 30 and 100 ng/h. Food intake was unaffected at these infusion rates. Changes in GRH and SRIH mRNA levels in response to systemic or central GH and IGF-I administration were similar in both sexes, except that the decrease in GRH mRNA levels produced by the icv infusion of IGF-I was greater in female than in male rats. The results provide evidence for a direct inhibitory feedback effect of GH in the central nervous system on the regulation of hypothalamic GRH gene expression that is independent of peripheral IGF-I. IGF-I feedback, in contrast, appears to originate with central nervous system-derived IGF-I and exhibits a stimulatory effect on SRIH mRNA as well as an inhibitory effect on GRH mRNA.

Differential effects of central and peripheral administration of growth hormone (GH) and insulin-like growth factor on hypothalamic GH-releasing hormone and somatostatin gene expression in GH-deficient dwarf rats.

The roles of GH and insulin-like growth factor-I (IGF-I) in the regulation of hypothalamic GH-releasing hormone (GRH) and somatostatin (SRIH) gene expression were investigated in the GH-deficient dwarf (dw) rat, in which endogenous feedback signals are lacking. Adult male and female dw rats were treated with GH or IGF-I by systemic (sc) administration or intracerebroventricular (icv) infusion, and hypothalamic GRH and SRIH mRNA were determined by Northern blotting and densitometric analysis. Systemic sc injection of rGH (75 micrograms every 12 h for 3 days) decreased GRH mRNA levels in both sexes. However, systemic sc injection of human IGF-I (150 micrograms every 12 h for 3 days) did not affect GRH mRNA levels in either sex despite significant stimulation of body weight gain. The use of a continuous sc infusion, which normalized serum IGF-I levels, and prolongation of the treatment period to 7 days also failed to change GRH mRNA levels. SRIH mRNA was unaffected by systemic administration of either GH or IGF-I. Continuous icv infusion of GH (1 microgram/h for 7 days) decreased GRH mRNA levels in both sexes, but did not alter SRIH mRNA levels. Continuous icv infusion of IGF-I (100 ng/h for 7 days) decreased GRH mRNA in both sexes. In contrast, SRIH mRNA levels were increased in both sexes. IGF-I decreased GRH mRNA levels at icv infusion rates of 100 and 300 ng/h and stimulated SRIH mRNA levels at infusion rates of 30 and 100 ng/h. Food intake was unaffected at these infusion rates. Changes in GRH and SRIH mRNA levels in response to systemic or central GH and IGF-I administration were similar in both sexes, except that the decrease in GRH mRNA levels produced by the icv infusion of IGF-I was greater in female than in male rats. The results provide evidence for a direct inhibitory feedback effect of GH in the central nervous system on the regulation of hypothalamic GRH gene expression that is independent of peripheral IGF-I. IGF-I feedback, in contrast, appears to originate with central nervous system-derived IGF-I and exhibits a stimulatory effect on SRIH mRNA as well as an inhibitory effect on GRH mRNA.

Intraventricular administration of histidyl-proline-diketopiperazine [Cyclo(His-Pro)] suppresses prolactin secretion and synthesis: a possible role of Cyclo(His-Pro) as dopamine uptake blocker in rat hypothalamus.

Histidyl-proline-diketopiperazine [Cyclo (His-Pro) (CHP)] was discovered to be one of the metabolites of TRH. To understand the specific role of CHP in rat hypothalamic dopamine neurons, we examined the in vivo effects of intraventricular (icv) infusion of CHP on the release and synthesis of PRL in the rat pituitary and the 3,4-dihydroxyphenylacetic acid (DOPAC)/dopamine ratio in the rat hypothalamus. We also examined the in vitro effects of CHP on the release of [3H]dopamine from dispersed tuberoinfundibular dopamine neurons, [3H]dopamine reuptake in hypothalamic membrane fractions, and PRL release from rat pituitary cultured cells. Female rats were treated by icv infusion of 1 microM CHP daily for 1, 3, and 7 days, using Alzet osmotic pumps. After 1 day of treatment, the serum PRL concentration was significantly decreased. Northern blot analysis of the total RNA isolated from the pituitary glands of control animals using 32P-labeled PRL cDNA as a probe indicated the presence of PRL gene transcript, 1.0 kilobase in size, and its amount was decreased by CHP treatment. CHP did not affect [3H]dopamine release from dispersed tuberoinfundibular dopaminergic neurons at any concentration up to 1 microM. CHP did not inhibit PRL release from cultured pituitary cells at low concentrations (1-100 nM), but it stimulated PRL release at high concentrations (1 and 10 microM). We also examined the concentrations of dopamine and DOPAC in the rat hypothalamus when CHP was administered icv for 1 or 7 days. There was a significant decrease in the DOPAC/dopamine ratio after CHP treatment for 1 day. Furthermore, CHP caused dose-dependent inhibition of [3H]dopamine uptake by the rat hypothalamus similar to other dopamine uptake blockers, such as benztropine and GBR12909. These data suggest that icv administration of CHP might decrease both PRL secretion and accumulation of PRL gene transcripts in the pituitary by decreasing the DOPAC/dopamine ratio and inhibiting dopamine reuptake in the rat hypothalamus.

Unsustained dipsogenic response to chronic central infusion of angiotensin-III in spontaneously hypertensive rats

We evaluated the chronic effect of angiotensin-III (AIII) in the promotion of drinking behavior in spontaneously hypertensive (SH) and normotensive Wistar-Kyoto (WKY) rats, using conscious, freely moving, male, adult animals that had been instrumented with an intracerebroventricular (icv) cannula connected to an osmotic mini-pump for 7-day infusion. Chronic icv infusion of AIII (5 or 10 pmol/min) elicited robust, dose-dependent, and Ile7-AIII (100 pmol/min; as specific antagonist)-reversible dipsogenesis in both SH and WKY rats, with higher water intake in the former strain. However, the drinking response in the SHRs exhibited a sharp drop after 3 days of AIII infusion, during which acute AIII (80 pmol, icv) challenges also failed to induce dipsogenesis. Chronic icv infusion of bestatin (150 pmol/min), an aminopeptidase-B inhibitor, did not by itself discernibly affect basal drinking. When combined with AIII (5 or 10 pmol/min), however, bestatin, respectively, suppressed and augmented the dipsogenic response of SH and WKY rats to the heptapeptide. These results suggest that chronic administration of AIII did not produce sustained drinking behavior in SHRs, possibly because of the development of early desensitization of the angiotensin receptors.

Differential induction of c-Fos immunoreactivity in hypothalamus and brain stem nuclei following central and peripheral administration of endotoxin

Lipopolysaccharide (LPS), an endotoxin associated with gram-negative bacteria, is a potent activator of the immune system. We have tested the effects of ICV infusions of LPS (10 ng) or Ringer's solution on the induction of the proto-oncogene protein c-Fos in the brain as well as plasma levels of corticosterone and splenic concentrations of norepinephrine (NE) and VIP. At 3 h post-ICV infusion of LPS, numerous labeled neurons were observed in the paraventricular nucleus (PVN) of the hypothalamus and the nucleus tractus solitarius (A2) region of the brain stem. Also, corticosterone and splenic NE and VIP levels were all elevated post-ICV LPS. Analysis of the time course for the induction of c-Fos protein in the brain following IP injections of LPS indicated that, relative to control injections, increased numbers of c-Fos-positive cells were detected in the PVN 0.5 h following IP injections (100 μg), peaked at 2–3 h postinjection, and then returned to control levels at later intervals. Additional dose-response data for IP LPS indicated a small increase in the number of labeled cells at a dose of 4.0 μg, and the number and staining intensity increased up to a dose of 100 μg- Corticosterone levels followed a similar pattern and were elevated at the 4.0 μg IP dose of LPS and increased to peak levels at 40 μg and higher. In contrast to ICV injections, splenic NE levels were unaltered by IP injections of LPS. An additional difference noted between ICV and IP injections of LPS was the presence of c-Fos-labeled neurons in the supraoptic nucleus, arcuate nucleus, and the ventrolateral (A1) region of the brain stem at IP doses of 40 μg and higher. These results indicate selective and differential effects of central and peripheral LPS on the induction of c-Fos protein in hypothalamic and brain stem nuclei. The concurrent changes in corticosterone and splenic neurotransmitter levels produced by ICV LPS suggest that the endocrine and autonomie nervous systems are primary targets for these activational effects of endotoxin.

Neuropeptide Y administered chronically into the lateral ventricle profoundly inhibits both the gonadotropic and the somatotropic axis in intact adult female rats.

Neuropeptide Y (NPY) is known to be involved in the central regulation of appetite, sexual behavior, and reproductive functions. Whereas central administration of NPY strongly stimulates feeding in satiated animals, diet restriction produces overexpression of NPY in the arcuate and paraventricular nuclei that might reflect behavioral adaptations to shortage of food. Previous studies indicated that central administration of NPY resulted in controversial actions on LH secretion, either stimulatory or inhibitory. In order to analyze the chronic effect on pituitary function of centrally administered NPY, stainless steel cannulae were implanted in the right lateral ventricles of intact 45-day-old Sprague-Dawley female rats. Ten days later, Alzet osmotic minipumps filled with saline or different concentrations of NPY adjusted to deliver 3, 6, 12, or 18 micrograms/day were connected to the intracerebroventricular (icv) cannulae, implanted sc dorsally, and the effects of these treatments evaluated after 7 days. Chronic icv infusion of NPY produced the expected dose-related increase in food intake [25.3 +/- 0.8 g/day (basal) to 47.9 +/- 4.3 g/day (highest NPY dose)] and body wt gain (3.7 +/- 0.4-11.5 +/- 1.4 g/day). Basal insulinemia was highly correlated to the increase in food intake. This orexigenic action of NPY was accompanied by a drastic dose-related decrease in pituitary wt (14.0 +/- 0.5-8.3 +/- 0.3 mg), pituitary concentration of GnRH receptors, a known marker of the activity of the hypothalamo-pituitary gonadal axis (15.2 +/- 1.7-5.2 +/- 0.5 fmol/mg), and ovarian wt (84.0 +/- 4.2-49 +/- 6.7 mg). Ovulation was impaired in NPY-treated animals as seen by daily inspection of vaginal smears. A sharp dose-dependent decrease in plasma levels of insulin-like growth factor I was also observed [934 +/- 64 ng/ml (basal) to 385 +/- 26 ng/ml (highest NPY dose)], probably secondary to a decrease in GH secretion. Whereas these data confirm the central action of NPY to stimulate appetite in satiated animals, they provide the first demonstration that chronic icv administration of NPY unequivocally inhibits gonadotropin secretion and sexual function in intact female rats. These data also confirm that NPY can suppress GH secretion and other anabolic hormones. In conclusion, these results may indicate a physiological role of NPY as an integrator of different adaptive behaviors in periods of unfavorable metabolic conditions such as diet restriction, extending its action to inhibition of sexual functions and anabolic processes.

Unsustained dipsogenic response to chronic central infusion of angiotensin-III in spontaneously hypertensive rats.

We evaluated the chronic effect of angiotensin-III (AIII) in the promotion of drinking behavior in spontaneously hypertensive (SH) and normotensive Wistar-Kyoto (WKY) rats, using conscious, freely moving, male, adult animals that had been instrumented with an intracerebroventricular (icv) cannula connected to an osmotic mini-pump for 7-day infusion. Chronic icv infusion of AIII (5 or 10 pmol/min) elicited robust, dose-dependent, and Ile7-AIII (100 pmol/min; as specific antagonist)-reversible dipsogenesis in both SH and WKY rats, with higher water intake in the former strain. However, the drinking response in the SHRs exhibited a sharp drop after 3 days of AIII infusion, during which acute AIII (80 pmol, icv) challenges also failed to induce dipsogenesis. Chronic icv infusion of bestatin (150 pmol/min), an aminopeptidase-B inhibitor, did not by itself discernibly affect basal drinking. When combined with AIII (5 or 10 pmol/min), however, bestatin, respectively, suppressed and augmented the dipsogenic response of SH and WKY rats to the heptapeptide. These results suggest that chronic administration of AIII did not produce sustained drinking behavior in SHRs, possibly because of the development of early desensitization of the angiotensin receptors.

Differential effects of octreotide on motor responses to nicotine in rats

We tested the hypothesis that brain somatostatin levels modify two motor behaviors evoked by ICV infusions of nicotine. Unrestrained, awake rats were given fixed-concentration infusions of nicotine until the prostration/immobility (PI) syndrome and convulsions were produced. Infusion duration ranged from 0.9 to 1.2 min for the PI syndrome and 2.5 to 4.9 min for the convulsions. Octreotide, a stable somatostatin analog (4.5 μg, ICV), significantly raised the threshold for nicotine convulsions 1.0 and 5.5 h after pretreatment but not at 24 or 48 h. Cysteamine, a somatostatin releaser and depletor (0.35–0.75 mg/rat, ICV), also caused a dose-dependent increase in seizure threshold. Similarities in the responses to octreotide and cysteamine suggest that depression of nicotine convulsions by cysteamine may be mediated by release of endogenous somatostatin. Neither octreotide nor cysteamine altered the threshold for the PI syndrome. These results support the view that one motor behavior evoked by nicotine is subject to control by somatostatin whereas another is not.

Blockade of Glutamatergic Neurotransmission in the Suprachiasmatic Nucleus Prevents Cellular and Behavioural Responses of the Circadian System to Light.

The aim of this study was to test the role of glutamatergic neurotransmission in photic entrainment of the circadian oscillator of the suprachiasmatic nuclei (SCN) in the Syrian hamster. The response of the oscillator to a brief pulse of light was assessed using two independent indices, the phase shift of the free-running activity rhythm, and the photically induced expression of the immediate-early gene c-fos within neurons of the SCN. The behavioural and the cellular responses to light were compared in animals which received intracerebroventricular (icv) infusions into the region of the SCN of either a vehicle solution or a solution of gammad-glutamyl-glycine (DGG), a competitive antagonist at both N-methyl-d-aspartate (NMDA) and non-NMDA types of glutamate receptor. Infusions of vehicle or DGG (200 nmol) were given 10 min before presentation of a 15-min light pulse at either circadian time (CT) 14 or CT20 (onset of activity defined as CT12). As anticipated, animals treated with vehicle and light at CT14 exhibited phase delays in the activity rhythm, whereas animals treated at CT20 exhibited phase advances. Central infusion of DGG prior to a light pulse at CT14 blocked the phase-delaying effect of light, and DGG delivered before a light pulse at CT20 markedly attenuated the phase-advancing effect of light. In a separate group of animals, the expression of the immediate-early gene c-fos was assessed by immunocytochemical staining for its protein product Fos. Exposure of vehicle-infused animals to light at CT14 caused extensive expression of c-fos throughout the retinorecipient region of the SCN. However, when the light pulse was preceded by icv fusion of DGG at a dose which would block the phase-shifting response to light, the total number of neurons immunopositive for Fos was significantly reduced ( approximately 50%) and the expression was confined to a restricted area of the dorsolateral SCN. The precise correlation between the effects of glutamatergic blockade upon both the behavioural and the cellular responses of the circadian system to light demonstrates that effective glutamatergic neurotransmission within or adjacent to the SCN is a necessary component of the mechanism which mediates photic entrainment of the circadian clock. The results further demonstrate a pharmacological and anatomical compartmentalization of the retinorecipient zone of the SCN, consistent with the view that retinal afferents to the ventral region employ glutamate as a transmitter, whereas more dorsal input may be dependent upon non-glutamatergic (DGG-insensitive) pathways.