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.

Chronic activity wheel running reduces the severity of kainic acid-induced seizures in the rat: possible role of galanin.

Studies in both humans and rodents suggest that exercise can be neuroprotective, but the mechanisms by which this occurs are still poorly understood. Three weeks of voluntary, physical activity in rats upregulates prepro-galanin messenger RNA levels in the locus coeruleus. Galanin is a neuropeptide extensively coexisting with norepinephrine that decreases neuronal hyperexcitability both in vivo and in vitro. Thus, exercise may diminish neural hyperexcitability through a galaninergic mechanism. The current experiments tested whether voluntary activity wheel running would protect against kainic acid-evoked seizures and whether galaninergic signaling is a necessary factor in this protection. In experiment 1, rats were given access to running wheels or remained sedentary for three weeks. After this period, rats received an intraperitoneal (i.p.) injection of 0, 7, 10 or 14 mg/kg kainic acid. Exercise decreased the severity of or eliminated seizure behaviors and hippocampal c-fos expression induced by kainic acid. In experiment 2, exercising or sedentary rats were injected intracerebroventricularly (i.c.v.) with 0.2 or 0.4 microg of kainic acid following either an injection of M-40 (a galanin receptor antagonist) or saline. Exercise decreased kainic acid-induced seizures at the 0.2 microg dose, and M-40 (6 nmol) decreased this effect. In contrast, there were no detectable differences between exercising and sedentary rats in behavior at the 0.4 microg dose. The results suggest that the protective effects of exercise against seizures are at least partially mediated by regulation of neural excitability through a process involving galanin.

Effect of semen extender and density gradient centrifugation on the motility and fertility of turkey spermatozoa.

In the absence of commercially viable methods for cryopreserving turkey spermatozoa, new processing methods are required to extend the functional life of stored turkey spermatozoa for artificial insemination. The present study evaluates the efficacy of a new extender (Turkey Semen Extend) and investigates the use of density gradient centrifugation in processing turkey spermatozoa for artificial insemination. The new extender is compared with two commercially available turkey semen extenders, Beltsville Poultry Semen Extender and Ovodyl. Turkey spermatozoa in Turkey Semen Extend were still motile 20 h after collection, representing a considerable improvement over the other semen extenders (40%, 0% and 8% for Turkey Semen Extend, Beltsville Poultry Semen Extender and Ovodyl, respectively). A field trial on a commercial turkey farm showed improved fertilization rates following insemination of turkey hens with semen extended in Turkey Semen Extend (89.7%) compared with Beltsville Poultry Semen Extender (86.9%). This difference is statistically significant (p < 0.05). Processing on a density gradient, optimized for turkey spermatozoa, also increased sperm survival (50% gradient-prepared spermatozoa still motile after 18 h compared with <10% non-processed spermatozoa). Preliminary studies indicate that gradient preparation of spermatozoa may aid survival during cryopreservation.

Reduced senescence and retained nuclear DNA integrity in human spermatozoa prepared by density gradient centrifugation.

PURPOSE: To investigate whether removal of extraneous cells and immotile spermatozoa from a sperm preparation by density gradient centrifugation could help to maintain normal spermatozoa in a viable state and retain their deoxyribonucleic acid integrity. METHODS: Sperm motility was assessed on a daily basis in aliquots of neat semen, extended semen, and spermatozoa prepared on a PureSperm density gradient. At the same time, aliquots of each sperm sample were preserved for TUNEL assay and nick translation. RESULTS: Spermatozoa prepared using density gradient centrifugation survived three times as long as spermatozoa in neat semen or in extended semen. Both deoxyribonucleic acid integrity and sperm motility were retained in the gradient preparations. CONCLUSIONS: Preparing spermatozoa by density gradient centrifugation is advantageous in prolonging sperm survival and maintaining deoxyribonucleic acid integrity, presumably by removing sources of reactive oxygen species. Stored spermatozoa could be used for a second attempt at fertilization if oocyte immaturity was suspected.

An alternative to PVP for slowing sperm prior to ICSI.

BACKGROUND: There is a growing awareness of potential problems in exposing sperm to polyvinylpyrrolidone (PVP) to slow their motility, a procedure commonly used prior to ICSI. The study presented here evaluates an alternative product for slowing sperm motility, which contains hyaluronate, a substance found naturally in the reproductive tract. METHODS: Computerized sperm motility analysis was used to compare the motilities of sperm exposed to either a PVP-containing product (ICSI-100), or a hyaluronate-containing product (SpermCatch), or control sperm resuspended in a sperm maintenance medium. A subjective assessment was made of the ease with which sperm could be isolated and be drawn into, and expelled from, an injection pipette after having their tails nicked. Sperm exposed to either ICSI-100 or SpermCatch were used for ICSI. Fertilization rate, zygote development, grading, and outcome of transfer were recorded for the two treatment groups. RESULTS: The hyaluronate-containing product slowed sperm motility sufficiently for the sperm to be captured in an injection pipette, was easy to draw into and expel from the pipette, prevented sperm sticking to plastic or glassware, and did not affect post-injection zygote development. Clinical pregnancy rates were similar for the two groups. CONCLUSIONS: This product represents an alternative to PVP for slowing sperm motility prior to ICSI.

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.

Olfactory bulbectomy increases met-enkephalin- and neuropeptide-Y-like immunoreactivity in rat limbic structures.

Bilateral olfactory bulbectomy (OBX) in rats produces a well-characterized syndrome of behavioral, physiological, and neurochemical changes identical to those seen in depression. Previous experiments using in situ hybridization histochemistry have demonstrated that OBX increases prepro-neuropeptide-Y (NPY) and prepro-enkephalin (ENK) mRNA levels in limbic structures. The present experiments determined whether increases in peptide immunoreactivity occur in conjunction with increases in mRNA levels following OBX. In situ hybridization analyses in olfactory bulbectomized and sham-operated rats revealed increased prepro-ENK mRNA in the piriform cortex (PIR) and olfactory tubercles (OTs) of bulbectomized rats. Prepro-NPY mRNA levels were significantly increased in the PIR of bulbectomized rats as comapred to controls. Radioimmunoassays (RIAs) revealed significant elevations in ENK-like immunoreactivity in the OTs following OBX. NPY-like immunoreactivity was significantly elevated in the PIR following OBX. These data reveal that OBX-induced increases in ENK-like immunoreactivity occur concomitantly with increases in prepro-ENK mRNA, and NPY-like immunoreactivity occur concomitantly with increases in prepro-NPY mRNA.

Experimentally induced attenuation of neuropeptide-Y gene expression in transgenic mice increases mortality rate following seizures.

Previous experiments have reported increased seizure susceptibility in transgenic mice lacking normal neuropeptide-Y (NPY) gene expression (i.e. NPY 'knock-out' mice). A critical issue inherent in such experiments concerns the confounding of developmental influences of NPY and its neurotransmitter functions in the mature organism. The present experiments directly addressed this issue by studying seizure susceptibility in transgenic mice possessing an inducible antisense transcript that can be experimentally manipulated to attenuate NPY synthesis. NPY-deficient and control mice were injected with kainic acid (40 mg/kg, i.p.) and several seizure-related behaviors were measured. Consistent with previously reported effects in NPY knock-out mice, significantly more NPY-deficient mice died within 24 h than control mice. In situ hybridization analyses confirmed a decrease in prepro-NPY gene expression in transgenic mice. The experiments support the hypothesis that the control of neural excitability is a prominent function of NPY.

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.

Role of aversively motivated behavior in the olfactory bulbectomy syndrome.

The aim of the present studies was to determine the extent to which changes in defensive behaviors could account for some of the behavioral effects of bilateral olfactory bulbectomy (OBX) in rats. Four tests of aversively-motivated behavior were conducted in bulbectomized and sham-operated rats: activity in a dimly lit or brightly lit open field, passive avoidance, foot shock-induced freezing, and defensive withdrawal. OBX reduced the duration of immobility in the open field. Bulbectomized rats exhibited less freezing in response to foot shock than sham-operated rats. In the defensive-withdrawal test, bulbectomized rats made more transitions into and spent less time inside the covered enclosure than sham-operated rats. The experiments thus reveal two novel paradigms for assessing the behavioral effects of OBX. The results also suggest that deficits in aversively-motivated behavior, specifically defensive freezing, may comprehensively explain the putative "hyperactivity" and "passive-avoidance learning deficits" widely associated with the OBX behavioral syndrome.

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.

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.

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.

The antibody-neutralisation of PDGF, CSF-1, TGFb2,3, EGF and EGF-receptor in utero in pre-implantation mice.

The aim of this study was to determine whether specific growth factors, those shown to be involved using PCR and immunohistochemistry, are necessary for the in-vivo mechanisms of normal implantation in mice. The abdomen of pregnant female mice were opened surgically on day 4 to expose the uterine horns, which were microinjected with specific neutralising antibodies against PDGF, CSF-1, TGFb2,3, EGF and EGF-receptor. At autopsy on day 12, the numbers, positions and sizes of all implantation and resorption sites were recorded. Sham-operation controls were utilised to evaluate the implantation model. Normal female mice exhibited a mean of 6.24 implantation sites per uterine horn. Sham-operated mice exhibited a 30.8% reduction in implantation compared with normals, and saline-injected mice exhibited a 45.7% reduction. Antibody-injected horns were compared with horns injected with saline and horns injected with heat deactivated antibody. All neutralising antibodies tested resulted in significant reductions in the implantation rate and the size of the implantation site. These experiments confirm, in vivo, participation of the specific growth factors tested in the mechanisms of murine implantation, as alluded to previously by evidence from PCR in vitro stimulatory and immunohistochemical work.

Pain responses, anxiety and aggression in mice deficient in pre-proenkephalin.

Enkephalins are endogenous opioid peptides that are derived from a pre-proenkephalin precursor protein. They are thought to be vital in regulating many physiological functions, including pain perception and analgesia, responses to stress, aggression and dominance. Here we have used a genetic approach to study the role of the mammalian opioid system. We disrupted the pre-proenkephalin gene using homologous recombination in embryonic stem cells to generate enkephalin-deficient mice. Mutant enk-/- animals are healthy, fertile, and care for their offspring, but display significant behavioural abnormalities. Mice with the enk-/- genotype are more anxious and males display increased offensive aggressiveness. Mutant animals show marked differences from controls in supraspinal, but not in spinal, responses to painful stimuli. Unexpectedly, enk-/- mice exhibit normal stress-induced analgesia. Our results show that enkephalins modulate responses to painful stimuli. Thus, genetic factors may contribute significantly to the experience of pain.

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.

Environmentally induced changes in peripheral benzodiazepine receptors are stressor and tissue specific.

The stress-induced changes in peripheral benzodiazepine receptors (PBR) can be observed in a number of different tissues, depending upon the nature and chronicity of the aversive experience. In addition, virtually all stress procedures that cause rapid changes in PBR simultaneously increase the physical activity or metabolic rate of the subjects. The present study analyzed the contributions of rapid alterations in activity or metabolic rate with and without aversive stimulation and their subsequent impact on PBR. Mechanically induced increases in activity by forced running stress results in a significant reduction in [3H]Ro 5-4864 binding to PBR in olfactory bulb, opposite to the PBR changes in this tissue following forced cold-water swim stress. Pharmacological induction of increased locomotor activity as well as metabolic rate by d-amphetamine causes a significant increase in cardiac PBR binding, again, opposite to the response typically observed following inescapable shock stress. Finally, administration of the anxiogenic beta-carboline, FG-7142, causes increases in both hippocampus and adrenal gland PBR binding reminiscent of acute noise stress exposure. These experiments demonstrate that increased locomotor activity or metabolic rate alone is not a necessary and sufficient condition for previous stress-induced changes in PBR. Conversely, increased metabolic rate coupled with an aversive stimulus appears to be an important factor for inducing stress-like changes in PBR. This data, coupled with previous reports, suggests that rapid alterations in these sites are stressor and tissue dependent. Finally, we propose that the PBR may be involved in many aspects of the stress response including: a) a blowarning system in adrenal gland, b) participation in stress-induced hypertension via renal PBR, and c) a modulator of stress-induced immunosuppression and subsequent recovery of function or recuperation by actions on immune cells.

Chronic social stress increases levels of preprogalanin mRNA in the rat locus coeruleus.

Galanin is a 29 amino acid neuropeptide that coexists with norepinephrine in approximately 80% of locus coeruleus (LC) neurons in the rat. The effects of chronic, naturalistic stress on preprogalanin mRNA in the LC was studied. The visible burrow system (VBS) served as the stress paradigm. Long-Evans rats (three males and two females) were housed together in the VBS for 13 days. The males established dominance hierarchies during this period. On day 14, preprogalanin mRNA in the LC was significantly elevated in subordinate rats compared to dominant and control rats, as measured by quantitative in situ hybridization. Levels of mRNA were positively correlated with the number of wounds by day 7 and day 14 and negatively correlated with body weight gain by day 14. These results suggest that the neuropeptide galanin may be overexpressed during chronic stress in rats.

Lack of effect of chronic morphine treatment and naloxone-precipitated withdrawal on tyrosine hydroxylase, galanin, and neuropeptide Y mRNA levels in the rat locus coeruleus.

Morphine dependence was experimentally induced in rats by daily injection of increasing doses of morphine for seven days. Withdrawal was precipitated in half of the morphine-dependent rats by a single injection of naloxone on day 8. Behavioral signs of withdrawal were evident in the morphine/naloxone group. Gene expression in locus coeruleus (LC) neurons was investigated using quantitative in situ hybridization analysis. Messenger RNA (mRNA) levels for tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis, and for precursors to galanin (GAL) and neuropeptide Y (NPY), peptides that coexist with norepinephrine in LC neurons, were not altered by chronic morphine treatment or naloxone-precipitated withdrawal. In contrast, mRNA levels for c-fos were dramatically elevated in the LC following naloxone-precipitated withdrawal. Chronic morphine treatment caused a small decrease in levels of mRNA encoding the precursor to corticotropin-releasing factor (CRF) in Barrington's nucleus. Although long-term adaptations of LC neurons have previously been implicated in the development of morphine tolerance, dependence, and withdrawal, alterations in the levels of TH, GAL, or NPY mRNA in the LC apparently do not underlie this process.