Galanin mediates features of neural and behavioral stress resilience afforded by exercise.

Exercise promotes resilience to stress and increases galanin in the locus coeruleus (LC), but the question of whether changes in galanin signaling mediate the stress-buffering effects of exercise has never been addressed. To test the contributions of galanin to stress resilience, male Sprague Dawley rats received intracerebroventricular (ICV) cannulation for drug delivery and frontocortical cannulation for microdialysis, and were housed with or without a running wheel for 21d. Rats were acutely injected with vehicle or the galanin receptor antagonist M40 and exposed to a single session of either footshock or no stress. Other groups received galanin, the galanin receptor antagonist M40, or vehicle chronically for 21d prior to the stress session. Microdialysis sampling occurred during stress exposure and anxiety-related behavior was measured on the following day in the elevated plus maze. Dendritic spines were visualized by Golgi impregnation in medial prefrontal cortex (mPFC) pyramidal neurons and quantified. Exercise increased galanin levels in the LC. Under non-stressed conditions, anxiety-related behavior and dopamine levels were comparable between exercised and sedentary rats. In contrast, exposure to stress reduced open arm exploration in sedentary rats but not in exercise rats or those treated chronically with ICV galanin, indicating improved resilience. Both exercise and chronic, ICV galanin prevented the increased dopamine overflow and loss of dendritic spines observed after stress in sedentary rats. Chronic, but not acute M40 administration blocked the resilience-promoting effects of exercise. The results indicate that increased galanin levels promote features of resilience at both behavioral and neural levels.

Central and peripheral benzodiazepine receptors: involvement in an organism's response to physical and psychological stress.

The present review discusses the current knowledge of the molecular pharmacology and neuroanatomical and subcellular localization of both the central benzodiazepine/GABA-chloride ionophore receptor complex and the peripheral benzodiazepine receptor. It then reviews all of the literature to date on how these two receptor sites are modulated by environmental stress. The possible role of these sites in learning and memory is also discussed. Finally, a theoretical model is presented which examines the differential, and perhaps complementary, alterations of these two sites in an organism's response to stress.

The role of cyclic adenosine 3',5'-monophosphate in the ovulatory process of the in vitro perfused rabbit ovary.

LH alters ovarian steroidogenesis via adenylate cyclase (AC) activation and cAMP production. Although LH also initiates ovarian follicle rupture, evidence is lacking for involvement of cAMP in this process. This work explores the involvement of cAMP in the ovulation of in vitro perfused rabbit ovaries by comparing LH stimulation of ovaries with that of LH plus 3-isobutyl-1-methyl-xanthine (IBMX), (an inhibitor of phosphodiesterase) and of forskolin (a nonreceptor-specific activator of AC). Venous perfusates were analyzed for cAMP, progesterone, 17 beta-estradiol, and testosterone, ovaries were analyzed for cAMP, and ovulations were noted. LH, LH plus IBMX, and forskolin all increased tissue cAMP levels significantly after 0.5 h, the perfusate levels increasing rapidly thereafter reaching plateau levels, while tissue levels returned to control levels after 2.4 h. LH plus IBMX and forskolin significantly increased cAMP release over LH controls, LH plus IBMX increasing and forskolin decreasing the number of ovulations. Forskolin significantly increased progesterone release over LH controls and, although no other significant steroid differences were seen, strong tendencies existed. Although forskolin could induce ovulations and could induce significantly higher release of cAMP than LH, it resulted in a lower ovulation rate than receptor-specific LH. LH plus IBMX also induced significantly higher cAMP release than LH, as did forskolin, and resulted in a higher ovulation rate than both LH and forskolin. These findings suggest, not only that cAMP production alone is sufficient for ovulation, but also that the receptor specificity of the cAMP production is important for the number of ovulations. Since tissue levels of cAMP peak several hours before ovulation, the cAMP is probably inducing a metabolic pathway leading to ovulation.

Embryonic stem cells express growth hormone receptors: regulation by retinoic acid.

Embryonic and fetal growth are generally considered to be independent of pituitary GH. However, it has been demonstrated recently that 18-day-old rat embryos and rat fetuses express GH receptors, suggesting that GH could play a role in early development. The aim of the present investigation was to determine whether preimplantation embryos also express GH receptors. Germ line competent mouse embryonic stem (ES) cells and cultured mouse preimplantation embryos were examined with Northern blot analysis, RNAse-protection solution-hybridization assays, reverse transcription/polymerase chain reaction assays and immunohistochemistry for the detection of GH receptors. Northern blot analysis of ES cells using a probe corresponding to the extracellular domain of the GH receptor demonstrated the presence of two transcripts (1.2 and 4.5 kilobases). The RNAse-protection solution-hybridization assay revealed that ES cells express approximately one sixth of the GH-receptor messenger RNA (mRNA) levels expressed in liver from pregnant mice. Treatment of cultured ES cells with retinoic acid (100 nM) for 6 days increased GH-receptor mRNA levels (P < 0.01). GH-receptor mRNA was further identified in ES cells, preimplantation embryos, muscle, liver, and placenta by a reverse transcription/polymerase chain reaction assay. In humans it has previously been shown that exon 3 of the GH-receptor is deleted in the placenta. However, none of the studied mouse tissues had a deletion of the GH-receptor mRNA corresponding to exon 3 of the human GH receptor. GH-receptor immunoreactivity was identified on the cultured ES-cells by immunohistochemistry. In conclusion, we have in the present study shown that germline competent ES cells and preimplantation mouse embryos express the GH receptor transcript and that this transcription is increased by retinoic acid in ES cells. Furthermore, the presence of GH-receptor immunoreactivity on the ES cells indicates that the GH-receptor transcript is translated.

Sexual dimorphism of stress-induced changes in renal peripheral benzodiazepine receptors in rat.

Adult male and female rats were exposed to either inescapable shock or no treatment. In vitro [3H]Ro 5-4864 (4'-chlorodiazepam) binding (1 nM) to peripheral benzodiazepine receptors (PRB) in both CNS and peripheral tissues indicated no gender differences in olfactory bulb, heart, lung or adrenal gland but a significant effect was observed in renal tissue. Female rats showed an attenuated stress-induced reduction (23%) in PBR in comparison to males (55%). This difference was shown to be an alteration of Bmax and not kD by Scatchard analysis. These data are the first demonstration of a sexual dimorphism in environmentally-induced alterations in PBR.

Effects of olfactory bulbectomy on neuropeptide gene expression in the rat olfactory/limbic system.

Bilateral olfactory bulbectomy in the rat produces a well-characterized syndrome that is independent of anosmia. This syndrome is reversed by chronic antidepressant administration, which provides the basis for the olfactory bulbectomy model of depression. The present experiments focused on neuropeptide plasticity in central olfactory/limbic structures following olfactory bulbectomy in rats. Male Sprague-Dawley rats received bilateral surgical ablation of the olfactory bulbs, sham surgery, or no surgery and were killed either three, seven, 14 or 28 days later. Relative levels of messenger RNA encoding neuropeptide Y, somatostatin, thyrotropin-releasing hormone, and corticotropin-releasing factor precursors in the forebrain were measured by quantitative in situ hybridization histochemistry using oligonucleotide probes. Prepro-neuropeptide Y messenger RNA levels in the piriform cortex and dentate gyrus were significantly elevated in bulbectomized rats 14 and 28 days after surgery compared to sham-operated and surgically naive rats. Prepro-somatostatin messenger RNA levels in the piriform cortex were marginally increased in bulbectomized rats at these time-points. Thyrotropin-releasing hormone and corticotropin-releasing factor precursor messenger RNA levels were not altered in the brain regions studied. The results indicate that olfactory bulbectomy causes long-term increases in the expression of the neuropeptide Y gene. These findings suggest that neuropeptide Y plasticity in the olfactory/limbic system may contribute to the olfactory bulbectomy syndrome in rats, and they provide further evidence of a role for neuropeptide Y in the pathophysiology of depression.

Stress-induced regulation of the renal peripheral benzodiazepine receptor: possible role of the renin-angiotensin system.

The etiology of the decrease in renal peripheral benzodiazepine receptor (PBR) binding caused by stress was studied in rats. Prior investigations suggest that the response of the renal PBR to stress occurs independently of the hypothalamo-pituitary-adrenal (HPA) axis and sympathetic nervous system. The present experiments tested the hypothesis that the renin-angiotensin system is involved in regulating the PBR. Eighty min of brief, intermittent tailshocks caused increases in plasma renin activity and decreases in renal PBR binding. The stress-induced decrease in renal PBR binding was reversed by pretreatment with captopril. Acute administration of angiotensin II (ANG II) alone caused reductions in PBR binding in kidney, heart, and cerebral cortex. These data suggest that ANG II may be an endogenous factor responsible for regulating the PBR in several tissues during stress.

Olfactory bulbectomy increases prepro-galanin mRNA levels in the rat locus coeruleus.

The effects of olfactory bulbectomy (OBX) on galanin (GAL) gene expression in the locus coeruleus (LC) were examined with quantitative in situ hybridization histochemistry. OBX increased prepro-GAL levels 3 and 14 days after surgery, as compared to sham-operated controls. Levels of mRNA encoding prepro-neuropeptide Y (NPY) were unchanged, and levels of mRNA encoding tyrosine hydroxylase (TH) were elevated in the LC only on day 3. The results indicate that GAL gene expression in the LC increases after lesioning a terminal field.

Prostaglandin-E2 released by pre-implantation human conceptuses.

This work investigates the production of prostaglandin E2 (PGE2), a well known modulator for the suppression of immune cells, by pre-implantation human conceptuses from the 4-cell stage to the hatched blastocyst and by cumulus oophorus cells, these being obtained from an IVF/ET program. Cumulus cell complexes cultured for 48 h produced considerable amounts of PGE2, necessitating complete removal of their influence on the conceptus cultures. All stages of the human conceptus studied produced PGE2 during 48-h cultures, the greatest amounts from late blastocysts, and from those cultured in media containing bovine serum albumin rather than human donor serum. Indomethacin-treated control blastocysts confirmed the synthesis and release from the human conceptuses. The production of PGF2a from human blastocysts could not be demonstrated.

Brain noradrenergic responses to footshock after chronic activity-wheel running.

Effects of physical activity on brain noradrenergic response to footshock were examined. Male Fischer 344 rats were randomly assigned to shoebox cages with (AW) or without (SED) 24-hr access to an activity wheel for 4-5 weeks. Extracellular levels of norepinephrine (NE) and 3,4-dihydroxyphenyl-acetic acid (DOPAC) in the brain frontal cortex were measured in 20-min samples of microdialysate taken during a 2-hr baseline, 40 min of scrambled footshock, and a 1-hr recovery. Levels of messenger RNA (mRNA) for tyrosine hydroxylase (TH), c-fos, and prepro-galanin in the locus coeruleus were measured by in situ hybridization histochemistry with autoradiographic analysis. NE levels were the same for SED and AW rats at baseline but were elevated in SED compared with AW during and after footshock. Levels of mRNA for TH and c-fos were elevated after footshock but did not differ between SED and AW. Our findings suggest that wheel running blunts NE release in the brain frontal cortex in response to footshock but does not influence expression of the gene that encodes TH in the locus coeruleus.

Differential effects of anxiogenic central and peripheral benzodiazepine receptor ligands in tests of learning and memory.

Previous research has demonstrated that low doses of anxiogenic central benzodiazepine receptor (CBR) ligands, the beta-carbolines, improve performance in various learning and memory tests in animals if administered prior to training. The present experiments compared the effect of a beta-carboline (FG 7142) with that of a pharmacologically distinct anxiogenic compound, a peripheral benzodiazepine receptor (PBR) ligand, 4'-chlorodiazepam (Ro5-4864), in two tests of learning and memory in rats. As expected, FG 7142 significantly improved performance in a passive avoidance test. Ro5-4864 was without effect. In a shuttlebox escape test, Ro5-4864 significantly impaired performance while FG 7142 had no effect. The effect of Ro5-4864 was antagonized by the specific peripheral benzodiazepine receptor antagonist, PK 11195. These results indicate that the differential impact of CBR and PBR anxiogenic ligands on performance in aversively-motivated learning tests may be a reflection of their distinct pharmacologies.

Studies on the mechanism of ovulation using the model of the isolated ovary.

Using the isolated perfused rabbit and rat ovaries as experimental models, we have studied various biochemical aspects of the ovulatory process. In rabbits, ovulations were induced by injecting hCG prior to the perfusion or by adding LH directly to the medium. In PMSG-treated rats, ovulations were induced by adding LH to the perfusion system. Steroids and other metabolites were analyzed in the perfusate and in follicular fluid. Steroid levels in follicular fluid were high early in the preovulatory development, but declined to very low levels 4 hours after LH stimulation. Levels of prostaglandins E and F rose as ovulation approached. In both perfusion models, indomethacin blocked ovulation without affecting steroid release or oocyte maturation. In the rabbit, PGF2 alpha reversed the indomethacin-induced inhibition and was able to induce follicular rupture by itself. Manipulations of the follicular fluid content of progesterone and estradiol to supraphysiological levels did not affect follicular rupture or oocyte maturation in the rabbit model. When the initial increase in LH-induced steroidogenesis was blocked by a 3 beta-ol-dehydrogenase inhibitor, ovulation was not affected. In rats, inhibition of estradiol production by an aromatase blocker did not affect the ovulatory process. When the endogenous formation of cyclic AMP is increased by pretreatment with a phosphodiesterase inhibitor, the LH-induced ovulation frequency increases in rabbits. Furthermore, forskolin, which increased the adenylate cyclase activity, stimulated steroidogenesis and induced follicular rupture. Recent experiments in the rat indicate that cyclic AMP acts on the ovulatory process via an effect on prostaglandin synthesis.

Intraventricular administration of galanin does not affect behaviors associated with locus coeruleus activation in rats.

The 29 amino acid peptide galanin (GAL) coexists with norepinephrine in rat locus coeruleus (LC) neurons to a remarkably high degree. The effects of central administration of GAL were examined in three behavioral paradigms that putatively involve increases in the activity of LC neurons. GAL did not affect behavioral signs associated with naloxone-precipitated withdrawal in rats treated chronically with morphine, a condition in which the firing rate of LC neurons is dramatically increased, although the behavioral signs of withdrawal were abolished by clonidine. Foot shock induced freezing behavior was similarly unaffected by either dose of GAL but was significantly diminished by clonidine and the corticotropin-releasing factor (CRF) antagonist alpha-helical CRF. GAL did not influence the decrease in exploratory activity in a novel open field induced by idazoxan. The behavioral activity of the peptide and route of administration were confirmed in a feeding paradigm. Doses of GAL that were inactive in the three paradigms were active in stimulating intake of a palatable food to a similar degree as clonidine-stimulated intake. These results suggest that intraventricularly administered GAL may not influence behaviors thought to be mediated by activation of neurons in the LC.

Olfactory bulbectomy increases prepro-enkephalin mRNA levels in the ventral striatum in rats.

The effects of bilateral olfactory bulbectomy (OBX) on prepro-enkephalin, thyrotropin-releasing hormone, and D-2 receptor mRNA levels in the ventral striatum were examined by in situ hybridization histochemistry. Pre- pro-enkephalin mRNA levels were significantly increased in the olfactory tubercle (OT), but not in the nucleus accumbens, 14 days following bilateral OBX. Levels of D-2 receptor mRNA were also increased in the OT, though to a lesser degree. Prepro-thyrotropin-releasing hormone mRNA was unaffected by OBX. A separate experiment revealed no effect of OBX on enkephalin gene expression 7 days following surgery but a comparable elevation in pre- pro-enkephalin mRNA 14 and 28 days post-surgery. The findings are consistent with previously-reported effects of dopamine lesions on striatal gene expression, suggesting that the observed effects may be mediated by deafferentation-induced alterations in dopaminergic transmission in the OT. Altered dopaminergic function in the OT may be particularly relevant to the 'anhedonia' that has been associated with the olfactory bulbectomized rat model of depression.

Amygdaloid central nucleus lesions and cholinergic blockade attenuate the response of the renal peripheral benzodiazepine receptor to stress.

Previous research has demonstrated that the density of peripheral benzodiazepine receptors (PBR) in rat kidney rapidly drops following exposure to 80 min of stress. The present experiments examined the contribution of the central and autonomic nervous systems in mediating this effect. Ibotenic acid lesions of the amygdaloid central nucleus (ACe), but not the lateral and basolateral amygdala, diminished the magnitude of the reduction in renal PBR binding caused by stress. Pretreating rats with methyl-scopolamine also inhibited the response of the PBR to stress. Adrenergic blockade with nadolol was ineffective. In order to test whether the PBR was under direct or indirect neural control during stress, unilateral renal denervation was performed. The stress-induced reduction in PBR binding persisted in denervated kidneys revealing that any neural control over the PBR that might exist must be indirect. Together the results suggest that the CNS may be involved in regulating the PBR during stress through the activation of intermediate, possibly hormonal, factors. The involvement of the central nervous system in the modulation of the PBR indicates the relevance of the PBR to physiological adaptations to stress.

Pentobarbital blocks the stress-induced decrease in [3H]Ro 5-4864 binding in rat kidney.

Exposure to environmental stress causes changes in the binding of [3H]Ro 5-4864 to peripheral benzodiazepine receptors (PBRs). The influence of the central nervous system (CNS) in these stress-induced modifications is unclear. The present study examined whether pretreatment with a dose-response regimen of sodium pentobarbital would impact the stress-induced reduction in renal PBR. Administration of either a sedative/ataxic (20 mg/kg) or hypnotic (60 mg/kg) dose of pentobarbital prior to stress blocks the stress-induced decrease of [3H]Ro 5-4864 binding to renal PBR in rat. These findings suggest that higher-order, supraspinal mechanisms play a critical role in marshalling the renal PBR changes in response to stress.

Angiotensin II rapidly modulates the renal peripheral benzodiazepine receptor.

The effects of acute exposure to angiotensin II (AII) on the renal peripheral benzodiazepine receptor were studied in rats. As little as 37.5 micrograms of AII injected s.c. over an 80 min period caused immediate reductions in [3H]Ro5-4864 binding. Scatchard analysis revealed that the reduction in [3H]Ro5-4864 binding induced by AII was due to a drop in receptor density or Bmax. The influence of AII on the peripheral benzodiazepine receptor is similar to that of stress.

Neuropeptide-Y exerts antidepressant-like effects in the forced swim test in rats.

The effects of neuropeptide-Y were examined in the forced swim model of depression in rats. Following a 15-min preswim, four groups of rats were given three intracerebroventricular (i.c.v.) injections of neuropeptide-Y (0.5, 5, or 10 microg) or saline over a 24-h period. Several behaviors were subsequently measured during a 5-min forced swim. Neuropeptide-Y treatment dose dependently increased swimming and decreased immobility. The pattern of results is consistent with that produced by serotonergic antidepressant drugs in this model.

Platelet-derived growth factor is detected in human blastocyst culture medium but not in human follicular fluid--a preliminary report.

The content of PDGF in human blastocyst culture medium (n = 8), serum (n = 12), and FF (n = 17) from natural IVF cycles was determined by an RIA specific for PDGF B-chain. The blastocysts were cultured under serum-free conditions throughout development. The findings show that PDGF B-chain is released into the culture medium of human blastocysts and that serum is positive, whereas FF is negative for PDGF.

Prepro-galanin messenger RNA levels are increased in rat locus coeruleus after treadmill exercise training.

The effects of treadmill exercise training on prepro-galanin (GAL) and tyrosine hydroxylase (TH) gene expression in the locus coeruleus (LC) were examined. Male Fischer-344 rats (n=9) were assigned to 6 weeks of treadmill running. An additional group of animals comprised the sedentary home cage control group (n=9). Levels of GAL and TH messenger RNA (mRNA) in the LC were measured using in situ hybridization histochemistry with autoradiography. Levels of GAL mRNA were higher in treadmill trained animals compared to sedentary animals, but there was no effect of treadmill running on TH mRNA. These results suggest that gene expression for galanin is responsive to repeated exercise stress and may have a neuromodulatory role in LC-noradrenergic adaptation to treadmill exercise training.