Changes in oxytocin content in rat brain during morphine withdrawal.

In this study the modification in the oxytocin content in different hypothalamic nuclei during morphine withdrawal was analysed. Male rats were implanted with placebo (naïve) or morphine (tolerant/dependent) pellets for 7 days. On day 7, groups of rats received an acute injection of saline s.c. (control) or naloxone (1 mg/kg s.c.) and were decapitated 30 min later. After administration of naloxone to tolerant rats (withdrawal) an increase in the oxytocin content in the paraventricular nucleus (PVN) and median eminence (ME) was found. No changes were found in the arcuate nucleus (AN) and supraoptic nucleus (SON). Present data demonstrate that administration of naloxone to tolerant rats alters the brain oxytocin system, which suggests that this peptide might contribute to the behavioural, emotional and neuroendocrine response to opioid.

Alterations in corticotropin-releasing factor and vasopressin content in rat brain during morphine withdrawal: correlation with hypothalamic noradrenergic activity and pituitary-adrenal response.

The modification in the activity of noradrenergic neurons projecting to the hypothalamus and the pituitary-adrenal response during morphine withdrawal as well its correlation with alterations in corticotropin-releasing factor (CRF) and vasopressin (AVP) content in different brain areas was analyzed. Male rats were implanted with placebo (naïve) or morphine (tolerant/dependent) pellets for 7 days. On day 8, groups of rats received an acute injection of saline s.c. (control) or naloxone (1 mg/kg s.c.) and were decapitated 30 min later. After administration of naloxone to tolerant rats (withdrawal) we found a striking parallelism between an enhanced activity of hypothalamic noradrenergic neurons and an increased corticosterone secretion; concomitantly, the CRF but not the AVP content in the paraventricular nucleus was decreased, which might reflect an increased release of the peptide. During withdrawal, CRF content also was decreased in the arcuate nucleus, whereas no changes were found in the median eminence, dorsomedial, ventromedial nuclei or in the bed nucleus of the stria terminalis. AVP content levels were not modified in arcuate nucleus, supraoptic or in the suprachiasmatic nuclei. Present data suggest that a hypothalamic noradrenergic hypersecretion may be involved in a selectively increased activity of CRF neurons in the paraventricular nucleus and arcuate nucleus and then in the enhanced release of corticosterone induced by morphine withdrawal. However, we did not find any correlation between opioid withdrawal-induced alterations in the pituitary-adrenal axis and AVP modifications.

Differential regulation of corticotropin-releasing factor and vasopressin in discrete brain regions after morphine administration: correlations with hypothalamic noradrenergic activity and pituitary-adrenal response.

The changes in the content of corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) in discrete brain nuclei during chronic opioids administration have not been well established. We evaluated the effects of acute and chronic morphine administration on the content of CRF and AVP in different hypothalamic and extrahypothalamic (bed nucleus of the stria terminalis, BNST) nuclei in rats. Concomitantly, changes in hypothalamic noradrenaline (NA) turnover [estimated by the 3-methoxy-4-hydroxyphenylethyleneglycol MHPG/NA ratio] and in plasma corticosterone release (as a marker of the activity of the hypothalamus-pituitary-adrenal axis) were determined. Male rats were implanted with placebo (naïve) or morphine (tolerant) pellets for 7 days. On day 8, groups of rats received an acute injection of either saline i.p. or morphine (30 mg/kg i.p.) and were sacrificed 30 min later. Acute morphine injection to naïve rats increased both the release of corticosterone and the hypothalamic NA turnover. CRF and AVP showed no modifications in the paraventricular nucleus (PVN) or in the median eminence (ME). CRF content decreased in the ventromedian nucleus (VMN) and increased in the BNST, but did not change in the arcuate nucleus (AN). AVP was elevated in the supraoptic nucleus (SON) but not changed in the suprachiasmatic nucleus (SCN). In chronic morphine-treated rats, there was a pronounced decrease in the NA turnover and in the release of corticosterone, which indicates that tolerance develops to the acute effects of morphine. Correspondingly, CRF and AVP were enhanced in the PVN and decreased in the ME, when compared with naïve rats injected with morphine. CRF content was decreased in the AN and in the BNST, but increased in the VMN. The AVP content was decreased in the SON, and no modifications were seen in the SCN. The present study shows that, in addition to the modifications in corticosterone secretion and in hypothalamic NA turnover, chronic morphine administration produces a complex response in the CRF and AVP systems. These modifications might contribute to the behavioral, emotional and neuroendocrine alterations produced during opioid tolerance.

Changes in hypothalamic oxytocin levels during morphine tolerance.

The role of hypothalamic oxytocin neurons in the hypothalamus-pituitary-adrenal (HPA) axis adaptation during opioid tolerance has not been explored. In this study the modification of oxytocin levels in different hypothalamic nuclei was determined after acute or chronic morphine exposure. Male rats were implanted with placebo (naïve) or morphine (tolerant) pellets for 7 days. On day 8, groups of rats received an acute injection of either saline i.p. or morphine (30 mg/kg i.p.) and were sacrificed 30 min later. In morphine-tolerant rats, there was a decrease in the oxytocin content in the median eminence (ME) and in the supraoptic nucleus (SO) after acute injection of saline or morphine. No modifications were seen in the paraventricular nucleus (PVN). The present study demonstrates that chronic morphine administration alters the brain oxytocin system, which suggests that this peptide might contribute to the behavioural, emotional and neuroendocrine responses to opioids.

The immunological impairment of vasopressin (AVP) neurons into paraventricular nuclei modifies AVP expression in suprachiasmatic nuclei.

A monoclonal antibody (MAb) to vasopressin (AVP) inhibits the synthesis and the release of AVP when injected near the AVP-producing neurons. In the present experiments, the AVP-MAb was microinjected near the paraventricular (PVN) or the supraoptic (SON) neurons of the rat hypothalamus and the AVP expression was measured in the suprachiasmatic nuclei (SCN). When microinjected near PVN, the AVP-MAb modified the AVP mRNA studied by in situ hybridization, and the AVP immunoreactive content of SCN, whereas it failed to show some effect when injected near SON. This confirms the privileged relationships between AVP-producing neurons in SCN and PVN.

Hypothalamic neuropeptides could mediate the anorectic effects of fenfluramine.

Oxytocin, vasopressin and corticotrophin releasing factor have anorectic properties when injected centrally. We studied the kinetics of these neuropeptides by injecting fenfluramine, a drug which reduces food intake, in Long Evans rats. The drug was injected daily through a double chronic cannula implanted above the paraventricular nucleus of the hypothalamus; the rats had free access to pure macronutrients. The rats lost weight during the treatment. Their total caloric intake decreased mostly because the carbohydrate intake decreased, while the protein intake increased slightly. The synthesis and release of brain oxytocin and vasopressin were increased and the release of corticotrophin releasing factor was stimulated. The neuropeptides could be involved in fenfluramine-triggered mechanisms.

Effect of food deprivation and refeeding on the concentration of vasopressin and oxytocin in discrete hypothalamic sites.

Recent evidence has implicated hypothalamic peptides, such as arginine vasopressin (AVP) and oxytocin (OT) in the control of feeding behavior. In this study, we investigated the impact of food deprivation (48 h) and subsequent refeeding (6 h) on the concentration of AVP and OT in discrete hypothalamic areas, as well as in the neurohypophysis. We also estimated in these rats certain peripheral measures, including hydroelectrolytic parameters, plasma and urine AVP, and plasma corticosterone. The results of this study revealed that food deprivation for 48 h produced little change in OT concentration in the various hypothalamic nuclei studied, including the paraventricular and supraoptic nuclei, with the exception of the median eminence (ME), where a significant decline (-36%; p < 0.05) was detected. This effect was not significantly reversed by 6 h of refeeding. With respect to AVP concentration, food deprivation caused a reliable decline exclusively in the parvocellular subdivision of the paraventricular nucleus (pPVN; -45%; p < 0.01) and in the supraoptic nucleus (SON; -45%; p < 0.01). No change in AVP was detected in the ME or in most other hypothalamic nuclei examined. Refeeding for 6 h actually potentiated the effect of food deprivation, decreasing further from baseline the content of AVP in the pPVN and SON. The only other hypothalamic area to exhibit a change in AVP content was the ventromedial nucleus, where AVP level increased (p < 0.001) after deprivation and declined to normal after 6 h of refeeding. The content of AVP and OT in the neurohypophysis was unaffected by food deprivation and subsequent refeeding.(ABSTRACT TRUNCATED AT 250 WORDS)

Long-term reduction of vasopressin excretion induced by the central injection of an immunoconjugate (antibody to vasopressin linked to ricin A chain).

We have previously demonstrated that vasopressin-producing neurons are the target of monoclonal antibodies to vasopressin microinjected into the brain tissue. At the same time, this central microinjection of vasopressin-monoclonal antibody into the supraoptic nuclei produced hydro-osmotic disorders mimicking the effects of a central diabetes insipidus. In order to investigate the increase in both duration and amplitude of the biological effects seen after the injection of vasopressin-monoclonal antibody, an immunoconjugate was constructed with the vasopressin-monoclonal antibody IgG1k isotype and the cytotoxic part of the ricin molecule, the ricin A chain. The biological parameters, such as diuresis and urine osmolality which are directly regulated by vasopressin, and vasopressin excretion, were measured after the central injection of this immunotoxin/immunoconjugate. The consequences of immunotoxin injection were also studied when immunotoxin was co-injected with monensin (50 nM) which has been shown to decrease the intracellular degradation of immunotoxin, and plasma complement, which has been shown to increase the neuronal uptake of immunotoxin. Single injection of immunotoxin near the hypothalamic supraoptic nuclei significantly increased diuresis and decreased vasopressin excretion. However, these effects were only transient and disappeared 24 h later. Four successive injections of immunotoxin (one per day) with monensin induced a decrease of vasopressin excretion which was still observed after a resting period of four days after the fourth injection. The long-term reduction of vasopressin excretion was induced in rats receiving four successive injections of a mixture consisting of immunotoxin with monensin and plasma complement. In such experiments, the vasopressin content of urine remained low (55% under the baseline value), two weeks after the fourth injection of immunotoxin. At the same time, the diuresis was increased (80% above the baseline value) and urine osmolality lowered (45% under the baseline value). When non-specific IgG replaced specific antibody, vasopressin excretion, diuresis as well as urine osmolality were unchanged. The results of this study demonstrated that the use of a specific immunotoxin results in a local interference with the vasopressinergic neurons and induces a long-term reduction of vasopressin secretion.

Rapid and localized alterations of neuropeptide Y in discrete hypothalamic nuclei with feeding status.

Neuropeptide Y (NPY) is believed to regulate the normal eating behavior and body weight in rats via central mechanisms. We have investigated whether NPY, which stimulates food intake, may in turn be modified by the nutritional state of the animals. Thus the impact of food deprivation (FD) (48 h) and subsequent refeeding on the levels of NPY in discrete hypothalamic areas was examined in this study. The results showed site specific change in only 3 of 7 hypothalamic sites. A 5-fold increment in NPY was reported in the paraventricular nucleus (PVN) and a 10-fold increase was observed in the arcuate nucleus-median eminence (ARC-ME). While subsequent refeeding for 6 h reversed the effect of FD in the ARC-ME, the levels of NPY in the PVN remained high in the refed rats. The perifornical lateral hypothalamus displayed a different pattern, namely, a significant increase in NPY content in refed as compared to satiated and deprived rats. The NPY levels in 4 other hypothalamic sites, namely, the dorsomedian, ventromedian, supraoptic and suprachiasmatic nuclei, and two extrahypothalamic sites, namely caudate nucleus and nucleus accumbens, showed total resistance to any change following deprivation and refeeding. These data emphasize the important and specific role of the paraventricular and arcuate nuclei in NPY's regulation of food intake and provide support for the idea that the variations of hypothalamic NPY after food deprivation reflect a specific physiological response of feeding regulatory system to alterations in the animal nutritional state and body weight.

Comparison of three immunoassays in the screening and characterization of monoclonal antibodies against arginine-vasopressin.

A method for screening monoclonal antibodies (McAbs) to neuropeptides was evaluated using 8-arginine-vasopressin (AVP) as a model. Mice were immunized with AVP-thyroglobulin conjugate and their spleen cells were fused with X 63-Ag8.653 mouse myeloma cells. The resulting hybridoma supernatants were screened for specific antibody production using 3 different assays: solid phase enzyme radioimmunoassay in Terasaki plates (Ter-ELISA), liquid phase radioimmunoassay (LPRIA) and an immunohistochemical technique. From 2 independent fusions, 7 McAbs specific for AVP were obtained. They belonged to the IgG1 subclass and reacted more strongly to the ring part of the nonapeptide. The screening strategy proposed relies upon a crude selection of conjugate-reacting hybridomas, followed by neuropeptide-specific hybridoma identification using both LPRIA (with radioiodinated synthetic peptide) and an immunohistochemical technique (to detect natural neuropeptide). During subcloning steps Ter-ELISA is then chosen, to select for specific clones and to eliminate those reacting with the carrier thyroglobulin.