Effect of dexamethasone implanted in different brain areas on the morphine-induced PRL, GH and ACTH/corticosterone secretion

We studied the effect of dexamethasone (DEX) implantation in male Wistar rats to elucidate the site of action of morphine-induced prolactin (PRL), growth hormone (GH), adrenocorticotropic hormone (ACTH) and corticosterone (B) secretion. DEX or cholesterol was implanted in the close vicinity of the paraventricular (PVN), or the arcuate nuclei (ARN) of the hypothalamus or into the hippocampus. Five days after implantation blood samples were taken 30 min after i.p. morphine by decapitation or through an indwelling cannula 15, 30, 60 min after i.v. injection. DEX implanted near the PVN resulted in a blockade of morphine-induced ACTH and B secretion. In contrast, GH response to morphine was enhanced, while that of PRL was unchanged. DEX implanted near the ARN significantly inhibited the PRL-releasing effect of morphine, but was without any influence on the PRL secretion induced by haloperidol. There was a partial reduction in the B response to morphine, and GH secretion was unchanged. Dorsal hippocampal implants were without any effect on the morphine-induced GH, PRL or B secretion. We suggest that the site of glucocorticoid inhibitory action in the hypothalamus is the PVN for the opiate-induced ACTH/B secretion, and the ARN for the morphine-induced PRL release. The enhanced GH response to morphine observed in DEX-PVN implanted rats might be due to a decreased somatostatin tone.     

c-fos expression in the rat brain following central administration of neuropeptide Y and effects of food consumption

Administration of neuropeptide Y (NPY) intracerebroventricularly (i.c.v.) results in the release of a number of hypothalamic and pituitary hormones and stimulation of feeding and suppression of sexual behavior. In this study, we sought to identify cellular sites of NPY action by evaluating perikaryal Fos-like immunoreactivity (FLI), a marker of cellular activation, in those hypothalamic and extrahypothalamic sites previously implicated in the control of neuroendocrine function and feeding behavior. Additionally, we compared the topography of FLI in these brain sites when food was either available ad libitum or withheld after NPY injection (1 nmol/3 μl, i.c.v.). The results showed that one hour after NPY injection a large number of cells in the parvocellular region of the paraventricular nucleus (PVN) were FLI-positive in the absence of food consumption. However, in association with food intake, a significant number of cells were intensely stained in the magnocellular region of the PVN. An analogous increase in FLI in association with feeding was apparent in the supraotic nucleus (SON), the dorsomedial nucleus and the bed nucleus of the stria terminalis in the hypothalamus. Anong the extrahypothalamic sites, feeding facilitated FLI in a large number of cells located in the lateral subdivision of the central amygdaloid nucleus and the lateral subdivision of the solitary tract. FLI was observed in a moderate number of cells in the hypothalamic arcuate nucleus (ARC) and ventromedial nucleus, and this response was not changed by feeding. Cumulatively, these results show that neurons in a number of discrete hypothalamic and extrahypothalamic sites, previously implicated in the control of neuroendocrine function and feeding behavior, are activated by NPY and further, a divergent pattern of c-fos expression emerged in some of these sites if feeding occurs after NPY injection. Stimulation of FLI in cells of the PVN, SON and ARC by NPY imply the presence of NPY target cells that play a role in the neuroendocrine control of pituitary function. The finding that NPY induced FLI in cells located in the parvocellular subdivision of the PVN even in the absence of feeding, imply that cells involved in initiation of food intake by NPY may reside in this subdivision of the PVN. On the other hand, the appearance of Fos-cells in the magnocellular subdivision of the PVN in response to feeding, suggests neural mechanisms that operate during the post-ingestion period, including those associated with termination of NPY-induced feeding, may impinge upon this subdivision of the PVN.     

Differential effect of ACTH1–24 and α-MSH induced grooming in the paraventricular nucleus of the hypothalamus

Injection of ACTH_1–24 as well as α-MSH in the paraventricular nucleus of the hypothalamus (PVH) induces intense grooming in the rat. While comparing the details of MSH, ACTH and control grooming, we found that the induction of grooming was highly site specific. Even injection of saline in that specific area produced some grooming, possibly due to the release of endogenous substances. To distinguish between effects caused by the peptides and the effects caused by the injection procedure, we compared the behavioural effects of saline and peptide injections in sites with exactly the same location in the PVH, in a post-hoc matched pairs design. Using this design we found that the grooming response induced by saline is of a limited. ACTH_1–24 and α-MSH prolong grooming beyond that period. Interestingly, rats receiving α-MSH continued to groom, while rats receiving ACTH_1–24 changed to scratching. This confirms earlier findings suggesting that grooming and scratching have a differential organization at the level of the PVH. Whether the peptides also have a role in the initiation of the grooming response, or just prolong a response caused by other local factors requires another experimental approach.     

Differential effects of central and peripheral injection of interleukin-1 beta on brain c-fos expression and neuroendocrine functions.

Cytokines such as interleukin-1 beta (IL-1 beta) alter the activity of the hypothalamic-pituitary-adrenal (HPA) and hypothalamic-pituitary-gonadal (HPG) axes in the rat. However, the brain sites at which IL-1 beta exerts these effects have not been well identified. The present study sought to identify some of these sites, using c-fos protein expression as an index of cellular activation. We also attempted to determine possible differences between the effects of peripheral and central injection of IL-1 beta on the activation of specific brain areas. Castrated male rats received intravenous (i.v.) or intracerebroventricular (i.c.v.) injections of IL-1 beta through a jugular catheter or a permanent cannula implanted in the right lateral ventricle, respectively. Blood samples were taken before, as well as 30 and 120 min after i.v. or i.c.v. IL-1 beta infusion in order to measure plasma ACTH and LH levels. Immediately thereafter, the rats were anesthetized with pentobarbital, then perfused. Their brains were removed and postfixed for one hour. Thirty-microns frozen sections were cut and approximately every fourth tissue section was processed for c-fos expression by an avidin-biotin-peroxidase method. Both i.v. (1 microgram) and i.c.v. (100 ng) injection of IL-1 beta significantly increased plasma ACTH levels, but only i.c.v. treatment measurably inhibited LH secretion. I.c.v. infusion of the cytokine markedly augmented c-fos expression in the paraventricular nucleus (PVN) and the arcuate nucleus (ARC) of the hypothalamus. A large amount of CRF cells in the PVN contained labelled c-fos protein (as measured by a double labelling technique), which indicates that CRF perikarya in this hypothalamic region are activated by the central administration of IL-1 beta. In contrast, i.v. injection of IL-1 beta did not significantly alter c-fos expression in the PVN or the ARC of the hypothalamus. These results suggest that the increased HPA axis activity which follows the peripheral IL-1 beta administration, a phenomenon previously shown to depend on endogenous CRF, does not require immediate activation of hypothalamic CRF perikarya. Thus our results indicate that the stimulatory effect of blood-born cytokine may be exerted at the level of nerve terminals in the median eminence. In contrast, i.c.v.-injected IL-1 beta appears to activate the HPA axis through a stimulation of CRF neurons within the parvocellular part of PVN.(ABSTRACT TRUNCATED AT 400 WORDS)     

Immunoreactive interleukin-1β localization in the rat forebrain

To determine whether the cytokine, interleukin-1β, is present in the rat brain as has been reported in human brain, immunocytochemical studies were performed using an antiserum that recognizes recombinant, rat IL-1β. Immunoreaction product was present in the periventricular and medial hypothalamus, mossy fiber distribution in the hippocampus and olfactory tubercle. These studies demonstrate that IL-1β is part of a diffuse intrinsic neural system in the rat central nervous system, associated with regions involved with hypophysiotropic, autonomic, limbic and extrapyramidal functions.     

The effect of adrenalectomy on stress-induced c-fos mRNA expression in the rat brain

Previously, we determined the pattern of stress-induced c-fos mRNA expression throughout the brain in order to gain further insight into the identification of the neural circuits mediating stress-induced regulation of the hypothalamic-pituitary-adrenal axis. In the present study, we determined if rapid effects of increased glucocorticoid levels after stress contribute to changes in c-fos mRNA expression. To this end, stress-induced c-fos expression was characterized in adrenalectomized (ADX) or adrenalectomized and corticosterone replaced (ADX/B) male rats. Animals were sacrificed 30 min post-onset of a 10 min swim stress, and in situ hybridization histochemistry was used to detect c-fos mRNA throughout the brain. The pattern of c-fos induction in the ADX and ADX/B animals was similar to that observed in the sham operated animals. Additionally, densitometric measurements were made to quantify the c-fos response in the paraventricular nucleus of the hypothalamus and the CA1/2 region of the hippocampus. We found that ADX did not alter the magnitude of the c-fos response to stress in these areas, but there was a slight dampening of the response in ADX/B animals. In sum, these results suggest that the pattern of c-fos expression observed 30 min post-stress is independent of stress-induced increases in circulating glucocorticoid concentrations.     

Intracerebroventricular injection of dibutyryl cyclic adenosine 3′,5′-monophosphate increases hypothalamic levels of neuropeptide Y

This investigation examined in vivo the relationship between the nucleotide cAMP and hypothalamic levels of two peptides, neuropeptide Y (NPY) and galanin (GAL), which are known to potentiate feeding behavior. In brain-cannulated rats, third ventricular injections of N⁶,2′-O-dibutyryl cyclic adenosine 3′,5′-monophosphate ((Bu)₂cAMP, 25 μg), compared to saline, caused a significant increase in NPY levels in the arcuate nucleus (ARC) and medial parvocellular portion of the paraventricular nucleus (mPVN), while having no impact in other hypothalamic areas. These site-specific changes in NPY occurred in the absence of any alteration in circulating levels of insulin, corticosterone, aldosterone or glucose, or of changes in hypothalamic levels of GAL. These findings implicate cAMP as having regulatory functions within specific hypothalamic NPY-synthesizing neurons, projecting from the ARC to the mPVN, that are believed to be involved in energy homeostasis.     

Fos expression in the brain induced by peripheral injection of CCK or leptin plus CCK in fasted lean mice

We previously reported a synergistic interaction between leptin and cholecystokinin (CCK) to reduce food intake through CCK-A receptors in lean mice fasted for 24 h. To identify the activated neuronal pathways, we investigated changes in Fos expression in brain nuclei 2 h after single or combined intraperitoneal (i.p.) injections of leptin (120 μg/kg) and sulfated CCK-8 (3.5 μg/kg) in male lean mice (C57BL/6) fasted for 24 h using immunohistochemistry for Fos, the protein product of the early gene, c-fos. Leptin did not increase Fos expression in the brain compared with vehicle-treated mice. CCK increased the numbers of Fos-positive neurons in the nucleus of the solitary tract (NTS)/area postrema (AP), central nucleus of the amygdala (CeA) and, to a smaller extent, in the paraventricular nucleus of the hypothalamus (PVN) (5.2-, 2.3- and 0.3-fold respectively). Injections of leptin–CCK further enhanced Fos expression by 40% in the PVN compared with that induced by CCK alone, but not in the other nuclei. Devazepide (a CCK-A receptor antagonist, 1 mg/kg, i.p.) prevented the increase in Fos expression induced by leptin–CCK in the PVN and by CCK alone in the PVN, CeA and NTS/AP. These results indicate that in fasted mice, i.p. injection of CCK increases Fos expression in specific brain nuclei through CCK-A receptors while leptin alone had no effect. Leptin in conjunction with CCK selectively enhanced Fos expression in the PVN. The PVN may be an important site mediating the synergistic effect of leptin–CCK to regulate food intake.     

Blockade by interleukin-1 receptor antagonist of IL-1β-induced neuronal activity in guinea pig preoptic area slices

Interleukin-1α (IL-1α) and interleukin-1β (IL-1β) are thought to be endogenous pyrogens, i.e., to mediate fever production; warm-sensitive (W) and cold-sensitive (C) neurons in the preoptic area (POA) are presumed to be the ultimate targets of endogenous pyrogens. The recent purification of an IL-1 receptor antagonist (IL-1ra) has provided a means for verifying the presumptive action of IL-1 on these neurons. This study was undertaken, therefore, to investigate whether IL-1ra may block the IL-1α and IL-1β effects on the firing rates (FR) of W and C neurons in guinea pig POA slices. Human recombinant (hr) IL-1β (500 ng/ml) reduced the FR of 26 W neurons and increased those of 3 C neurons recorded; it had no effect on 8 thermally insensitive neurons. hrIL-1α (200–600 ng/ml) did not change the FR of any neuron. IL-1ra (0.01–0.5 mg/ml) had no effect by itself on the FR of all the neurons, but it blocked the hrIL-1β-induced FR changes of 24 of the 26 W and of all 3 C neurons when given before the cytokine. The lowest effective dose was 0.05 mg/ml. These results support the hypothesis, therefore, that POA thermosensitive neurons may be direct targets of IL-1β and that it may be an endogenous pyrogen acting on these units to induce fever production.     

Differential effects of neuropeptide Y and the μ-agonist DAMGO on ‘palatability' vs. ‘energy'

Differential effects of neuropeptide Y (NPY) and μ-opioid DAMGO on ‘palatability' vs. ‘energy'. A variety of studies suggest that NPY is an important manager of energy metabolism. In contrast, the opioid peptides appear to influence the ‘rewarding' aspects of feeding. In the current study, we stimulated feeding by injecting NPY (110 pmol) or the μ-opioid agonist DAMGO (2 nmol) into the paraventricular nucleus of rats. Following injection, rats were given free access to laboratory chow and a 10% sucrose solution. Animals injected with saline derived 10% of their kilocalories from the chow and 90% from the sucrose solution (total kcal/4 h=12.2±1.0). Those rats injected with NPY derived 48% of their energy from chow and 52% from the sucrose solution (total kcal/4 h=24.8±1.7). The DAMGO-injected rats derived only 15% of their kilocalories from chow and the remainder from the sucrose solution (total kcal/4 h=23.0±2.3). Thus, while NPY and DAMGO both stimulated energy intake compared to saline controls (P<0.0001), the effect on intake of a palatable dilute energy solution (0.4 kcal/g) vs. a ‘bland' laboratory chow (3.95 kcal/g) was different. The results of this study reinforce the notion that NPY has a major effect on energy needs, whereas opioids influence the ‘rewarding' characteristics of foods.     

α2-Adrenoceptor-mediated restraint of norepinephrine synthesis, release, and turnover during immobilization in rats

Stress-related release of norepinephrine (NE) in the brain and periphery probably underlies several neuroendocrine and neurocirculatory responses. NE might influence its own synthesis, release, and turnover, by negative feedback regulation via α₂-adrenoceptors. We examined central and peripheral noradrenergic function by measuring concentrations of NE, dihydroxyphenylglycol (DHPG), and dihydroxyphenylacetic acid (DOPAC) in hypothalamic paraventricular nucleus (PVN) microdialysate and arterial plasma simultaneously during immobilization (IMMO) in conscious rats. The α₂-adrenoceptor antagonist yohimbine (YOH) was injected i.p. or perfused locally into the PVN via the microdialysis probe. The i.p. YOH increased plasma NE, epinephrine (EPI), DHPG, dihydroxyphenylalanine, and DOPAC levels by 4.3, 7.3, 2.5, 0.6 and 1.8-fold and PVN microdialysate NE, DHPG, and DOPAC by 1.2, 0.6 and 0.5-fold. The i.p. YOH also enhanced effects of IMMO on plasma and microdialysate NE, DHPG, and DOPAC. YOH delivered via the PVN microdialysis probe did not affect microdialysate or plasma levels of the analytes at baseline and only slightly augmented microdialysate NE responses to IMMO. The results indicate that α₂-adrenoceptors tonically restrain NE synthesis, release, and turnover in sympathetic nerves and limit IMMO-induced peripheral noradrenergic activation. In the PVN, α₂-adrenoceptors do not appear to contribute to these processes tonically and exert relatively little restraint on IMMO-induced local noradrenergic activation.     

Differential effects of interleukin-1β and interleukin-2 on glia and hippocampal neurons in culture

The present study was undertaken to assess the effects of interleukin-1β (IL-1β) and interleukin-2 (IL-2) on glial and neuronal cells in culture. The presence of IL-1β-like and IL-2-like immunoreactivity was detected in media collected from both astroglial and microglial cultures, indicating that both lymphokines can be released from either cell type. However, the levels measured in microglial media were significantly higher than in the astroglial media. Moreover, the content of IL-1β-like immunoreactive material in the media was approximately five- to 10-fold greater than that of IL-2, although exposure of both microglial and astroglial cultures to IL-1β significantly enhanced this measure. A possible role for this glial-derived IL-1β as an astroglial growth factor was substantiated by experiments showing that the lymphokine increased the incorporation of [³H]thymidine into astroglial, but not microglial cultures. In contrast, IL-2 did not significantly alter glial proliferation. In hippocampal neuronal cultures, these lymphokines affected neuronal survival differently. Thus, only the highest concentration (500 ng/ml) of IL-1β tested decreased the long-term (three day), but not the short-term (one day), survival of these neurons, whereas neuronal survival was compromised by IL-2 even after short-term (one day) exposure. In addition, in the long-term (three-day-old) neuronal cultures exposed to IL-2, extensive cellular swelling, vacuolations and neurite retractions were noted, even in cultures exposed to relatively low concentrations (< 10 ng/ml) of the lymphokine. These effects were not apparent with IL-1β or the other lymphokines tested, including IL-3, IL-4 and IL-8. The results suggests that the glial-derived lymphokines IL-1β and IL-2 may have different functions in the CNS. Whereas IL-1β may have an important role in the developing brain as a maintenance and growth-promoting factor, IL-2 may function as an inhibitory factor, and may be of significance only in instances during which it accumulates in sufficiently high concentrations in the vicinity of neurons.     

Specificity of interleukin–1β-induced changes in monoamine concentrations in hypothalamic nuclei: blockade by interleukin-1 receptor antagonist

The purpose of this study was to examine specificity in the effects of interleukin-1β (IL-1β) on monoamines in various areas of the hypothalamus. Adult male rats were injected i.p. with saline or 2.5 or 5.0 μg of IL-1β or were pretreated with 500 μg of IL-1 receptor antagonist (IL-1ra) followed 5 min later by 5 μg of IL-1β. The paraventricular nucleus (PVN), arcuate nucleus (AN), median eminence (ME), and medial preoptic area (MPA) were microdissected and analyzed for neurotransmitter concentrations by high-performance liquid chromatography with electrochemical detection (HPLC-EC). In the PVN, IL treatment produced significant increases in the concentrations of norepinephrine (NE), dopamine (DA), DA metabolite dihydroxyphenylacetic acid (DOPAC), serotonin (5-HT), and its metabolite 5-hydroxyindoleacetic acid (5-HIAA). IL-1 treatment increased the concentrations of NE and DA in the AN but only of NE in the ME, and it was without any effect in the MPA. Pretreatment with IL-1ra completely blocked the IL-1 effects. It is concluded that IL-1 induces highly specific changes in monoamine metabolism in the hypothalamus, and the nature of these changes depends on specific hypothalamic nuclei.     

Role of the α1- and α2-adrenoceptors of the paraventricular nucleus on the water and salt intake, renal excretion, and arterial pressure induced by angiotensin II injection into the medial septal area

In this study we investigated the influence of α-adrenergic antagonists injections into the paraventricular nucleus (PVN) of the hypothalamus on the thirst and salt appetite, diuresis, natriuresis, and pressor effects of angiotensin II (ANG II) stimulation of medial septal area (MSA). ANG II injection into the MSA induced water and sodium intake, diuresis, natriuresis, and pressor responses. The previous injection of prazosin (an α₁-adrenergic antagonist) into the PVN abolished, whereas previous administration of yohimbine (an α₂-adrenergic antagonist) into the PVN increased the water and sodium intake, urinary, natriuretic, and pressor responses induced by ANG II injected into the MSA. Previous injection of a nonselective α-adrenergic antagonist, regitin, into the PVN blocked the urinary excretion, and reduced the water and sodium intake, sodium intake, and pressor responses induced by ANG II injected into the MSA. The present results suggest that α-adrenergic pathways involving the PVN are important for the water and sodium excretion, urine and sodium excretion, and pressor responses, induced by angiotensinergic activation of the MSA.     

Novel effects of estradiol and estrogen receptor α and β on cognitive function

Estrogen influences the development of memory function in humans and rodents and can modulate memory in adults. In these studies we examined the role of the estrogen receptors α (ERα) and β (ERβ) in mediating performance on a hippocampal-dependent, hormone-sensitive task, inhibitory avoidance (IA). Ovariectomized (OVX) estrogen receptor-α-knockout (ERαKO) mice displayed impaired performance on the IA task and OVX heterozygotic (HET) mice exhibited performance that was intermediate between ERαKO and wild-type (WT) mice. Impaired performance by ERαKO mice was rescued by E₂ treatment. The ER antagonist, tamoxifen, did not block enhancement of retention by E₂ suggesting that E₂ mediated modulation of memory is not caused by known genomic receptor mechanisms. In contrast to ERαKO mice, IA performance by OVX estrogen receptor-β-knockout (ERβKO) mice was not compromised. The results indicate an important role for ERα, relative to ERβ, in the establishment of cognitive function and suggest that E₂ modulates memory function via a novel estrogenic mechanism.     

Differential effects of α-, β- and γ-endorphins on dopamine metabolism in the mouse brain

The effects of intracerebral injection of α-, β- and γ-endorphins on the mouse brain dopamine (DA) metabolism were contrasted in relation to the previously identified patterns of behavior. α-Endorphin (20 μg) decreased the content of homovanillic acid (HVA) in the striatum, while γ-endorphin (10 μg) the contents of DA, 3,4-dihydroxyphenylacetic acid and HVA. β-Endorphin (1 and 2 μg) had no effects on the mouse DA metabolism in the brain. The changes in the DA metabolism induced by α- and γ-endorphins were readily reversed by the pretreatment with naloxone (1 mg/kg). Results suggested: (1) the patterns of behavior in mice treated with endorphins are mediated by these differences in DA metabolism; (2) changes in DA metabolism induced by α- and γ-endorphins occur via opiate receptors in the mouse brain.     

Coordinated upregulation of α1- and β11-tubulin mRNAs during collateral axonal sprouting of central peptidergic neurons

An in situ hybridization study was performed to determine the relationship between levels of mRNAs for the axonal growth-associated αl-tubulin and βZII-tubulin isotypes and the process of collateral axonal sprouting by identified central nervous system (CNS) neurons. A unilateral hypothalamic knife-cut was used to hemisect the hypothalarnoneurohypophysial tract, which results in a robust collateral sprouting response by the uninjured neurons of the contralateral supraoptic nucleus (SON) (Watt and Paden: Exp Neurol 111:9-24, 1991). At 10 and 30-35 days after the lesion, cryosections of the SON were obtained and hybridized with ³⁵S-labeled cDNA probeses specific to αl- and β11-tubulin mRNAs. Quantitative evaluation of the resulting autoradiographs revealed that αl-tubulin mRNA levels were significantly increased by 10 days in SON neurons that were undergoing collateral sprouting compared to controls and that this increase was sustained at 30-35 days post-lesion. Less marked increases in hybridization intensity of the βII-tubulin probe were also apparent in sprouting neurons at both 10 and 30-35 days after the lesion, but were statistically significant only at 10 days. The measured increases in intensity of hybridization of αl- and β11-tubulin probes are likely to be conservative estimates of the underlying increase in αl- and β11-tubulin mRNA levels because sprouting SON neurons undergo significant hypertrophy. High levels of both αl- and βII-tubulin mRNAs were also seen in surviving axotomized SON neurons ipsilateral to the hypothalamic lesion. We conclude that the pattern of regulation of αl- and β11-tubulin mRNAs in CNS neurons which are capable of supporting new axonal growth includes three elements: maintenance of significant basal αl- and β11-tubulin mRNA pools in mature neurons, rapid increases in the pool size of the mRNAs following stimulation of collateral sprouting, and sustained elevation of mRNA levels during the period of axonal sprouting. © 1995 Wiley-Liss, Inc.