Increase In Galanin mRNA Research Articles

Antidepressant and anticonvulsant effects of exercise in a rat model of epilepsy and depression comorbidity

The bidirectional comorbidity between epilepsy and depression is associated with severe challenges for treatment efficacy and safety, often resulting in poor prognosis and outcome for the patient. We showed previously that rats selectively bred for depression-like behaviors (SwLo rats) also have increased limbic seizure susceptibility compared with their depression-resistant counterparts (SwHi rats). In this study, we examined the therapeutic efficacy of voluntary exercise in our animal model of epilepsy and depression comorbidity. We found that chronic wheel running significantly increased both struggling duration in the forced swim test and latency to pilocarpine-induced limbic motor seizure in SwLo rats but not in SwHi rats. The antidepressant and anticonvulsant effects of exercise were associated with an increase in galanin mRNA specifically in the locus coeruleus of SwLo rats. These results demonstrate the beneficial effects of exercise in a rodent model of epilepsy and depression comorbidity and suggest a potential role for galanin.

Regulation of galanin gene expression in human keratinocytes

Recently, we have detected abundant expression of specific immunoreactivity for galanin (GAL), a 29–30 amino acid neuropeptide, in the epidermis and in sweat glands in a non‐neuronal distribution and to a lesser extent in nerve fibre bundles and smooth muscle of blood vessels. In order to uncover a possible function of GAL in human epidermis, we investigated the regulation of GAL mRNA expression in cultured primary human keratinocytes (KCs). First, we determined whether PMA, a protein kinase C activator which is known to be a potent upregulator of GAL gene expression in chromaffin cells, has similar effects in KCs. Stimulation of human cultured primary KC with PMA resulted in a sixfold increase of GAL mRNA. Furthermore, we exposed KCs to a combination of the proinflammatory cytokines IL‐1β and TNF‐α which resulted in a twofold induction of GAL mRNA in comparison with a sixfold induction of ICAM‐1. Most interestingly, capsaicine, which usually leads to a secretion of neuropeptides from nerves endings, did not induce the secretion of GAL of KCs. In contrast, constitutive GAL mRNA expression and peptide secretion were reduced twofold upon treatment of KC with 10 µm capsaicine for 20 min and 24 h, respectively. To our knowledge, this is the first report on a direct regulatory effect by capsaicine on neuropeptide mRNA expression in non‐neuronal cells.

Increase of galanin mRNA in lumbar dorsal root ganglion neurons of adult rats after partial sciatic nerve ligation

Partial sciatic nerve ligation (PSNL) is a widely used model for the study of neuropathic pain. However, there is little information on neuropeptide expression in primary sensory neurons after PSNL. We examined galanin (GAL) mRNA expression in L4 and L5 dorsal root ganglion (DRG) neurons of adult rats after PSNL. We found that 4 and 14 days after PSNL the percentages of GAL mRNA positive neurons were significantly increased in the ipsilateral DRG compared to the contralateral side. Using combined retrograde fluorescent dye tracing and in situ hybridization, we found that 47% of the injured neurons and 10% of the spared neurons were GAL mRNA positive. Since only 2–3% of neurons in the contralateral uninjured DRG were GAL mRNA positive, PSNL induced up-regulation of GAL mRNA in both injured and spared DRG neurons.

NGF and LIF both regulate galanin gene expression in primary DRG cultures.

Both target-derived and injury-induced factors could be involved in the axotomy-induced increases in galanin expression in dorsal root, ganglion (DRG) neurons. Galanin mRNA levels were studied in primary cultures of E13.5 embryos, grown for 14 days in culture, in response to two candidate molecules, nerve growth factor (NGF) and leukemia inhibitory factor (LIF). In these cultures, NGF withdrawal alone resulted in a significant increase in galanin mRNA. Addition of LIF onto NGF-containing cultures did not produce a significant increase, while addition of LIF to NGF-deprived cultures caused an upregulation of galanin mRNA which was significantly stronger than that of NGF withdrawal alone. Thus, NGF withdrawal and LIF increase act together to up-regulate galanin gene transcription in DRG neurons.

Inhibition of Steroid‐Induced Galanin mRNA Expression in GnRH Neurons by Specific NMDA‐Receptor Blockade

Galanin mRNA levels in GnRH neurons increase in association with a steroid-induced LH surge in female rats. Both the steroid-induced LH surge and the concomitant increase of galanin mRNA in GnRH neurons are blocked by non-specific inhibition of central nervous system activity imposed by pentobarbital and specific central alpha-adrenergic receptor blockade. Based on these observations, we hypothesized that galanin gene expression in GnRH neurons is induced whenever GnRH neurons become activated to generate an LH surge. If this were the case, then any neurotransmitter receptor blocking agent that inhibits the LH surge by central mechanisms would likewise block the associated increase in galanin mRNA in GnRH neurons. We tested this hypothesis by examining the effects of an N-methyl-D-aspartate (NMDA) receptor antagonist on the steroid-induced LH surge and on levels of galanin mRNA in GnRH neurons. Three groups of ovariectomized rats were used: Group 1 -treated with estradiol and progesterone (E/P) and sacrificed at the peak of the LH surge; Group 2-treated the same as Group 1 except that dizocilpine (MK801, an NMDA receptor antagonist) was used to block the LH surge; and Group 3-treated the same as Group 1 except they received vehicle instead of E/P. Double-and single-label in situ hybridization followed by computerized image analysis were used to measure levels of galanin mRNA and GnRH mRNA in GnRH neurons [as grains/cell (g/c)]. E/P treatment induced a 3-fold increase in LH levels and a 5-fold increase in the galanin mRNA signal content of GnRH neurons. Treatment with MK801 completely prevented the LH surge in all animals and also blocked the steroid-induced increase in galanin mRNA in GnRH neurons. As assessed by 2 independent GnRH single-labeled assays, neither GnRH message content nor the number of identifiable GnRH neurons differed among the experimental groups. We conclude that the increase in galanin mRNA levels in GnRH neurons is tightly coupled to the occurrence of a steroid-evoked LH surge, and we infer that induction of galanin gene expression in GnRH neurons is induced as a consequence of synaptic activation of GnRH neurons.

Activation-dependent regulation of galanin gene expression in gonadotropin-releasing hormone neurons in the female rat.

In rats, galanin is colocalized in GnRH neurons, and galanin mRNA in GnRH neurons is increased coincidentally with the preovulatory gonadotropin surge. Whether the induction of galanin mRNA in GnRH neurons at proestrus reflects the action of sex steroids is unknown. We tested this hypothesis by challenging ovariectomized rats (n = 7) with estrogen and progesterone (E/P) to induce a LH surge and measuring galanin mRNA in GnRH neurons to determine whether there was an associated induction of galanin message in these cells. We used single and double label in situ hybridization and image analysis to compare among groups the levels of both galanin mRNA and GnRH mRNA in GnRH neurons. We found that steroid-primed animals showed an approximately 400% induction of galanin mRNA signal in GnRH neurons over that in vehicle-treated animals. Second, we hypothesized that steroid-dependent events which induce the expression of galanin mRNA in GnRH neurons depend on transsynaptic input to GnRH neurons. We tested this hypothesis by examining the effect of a pharmacological blockade of the steroid-induced activation of GnRH neurons on levels of galanin mRNA in these cells. We killed groups of ovariectomized adult female rats at the peak of a E/P-primed LH surge (n = 7) and after steroid priming followed by blockade of the LH surge with either the general anesthetic pentobarbital (n = 7) or the specific alpha-adrenergic receptor blocker phenoxybenzamine (n = 7). When we examined signal levels representing galanin mRNA content in GnRH neurons, we observed a 4-fold increase in signal for galanin mRNA in the GnRH neurons of steroid-primed (E/P surge) animals compared with that in oil-treated controls (P < 0.0004). This increase in galanin mRNA was prevented when the LH surge was blocked by treatment with either pentobarbital or phenoxybenzamine (P < 0.03 and P < 0.0001 vs. E/P surge controls, respectively). Cellular levels of GnRH mRNA were not different among control, E/P, and E/P plus pentobarbital groups (P > 0.2). These observations suggest that an increase in galanin mRNA levels in GnRH neurons is tightly coupled to the occurrence of a LH surge. By inference, induction of galanin mRNA in GnRH neurons reflects their activation, possibly via afferent neurons that transduce the steroid signal to GnRH neurons.

Galanin gene expression is increased in the anterior pituitary gland of the human growth hormone-releasing hormone transgenic mouse.

The peptide galanin is synthesized within and secreted from specific cells of the anterior pituitary gland. Previous studies showed that GH-releasing hormone (GHRH) stimulates galanin release from pituitary cells in vitro. In the present study we used human (h) GHRH transgenic mice to examine the effects of high circulating levels of GHRH on pituitary galanin gene expression in vivo. Moreover, the hGHRH transgenic mice develop pituitary tumors and, thus, may be used as a model of estrogen-independent pituitary adenoma formation. We examined male hGHRH transgenic mice and nontransgenic siblings at 2, 4, 6, 8, and 10 months of age. Transgenic mice were identified using the polymerase chain reaction. Body weights and plasma mGH levels were higher in transgenic mice at all ages. Total protein contents in the anterior pituitary glands of transgenic mice were significantly greater at each age. Galanin peptide contents in the anterior pituitary gland of hGHRH mice were normalized for differences in total protein content and were significantly elevated at all ages examined. At 10 months of age, anterior pituitary galanin peptide concentrations were increased 7-fold. Hypothalamic concentrations of galanin peptide were also increased in hGHRH transgenic mice, but were not greater than those in nontransgenic siblings until 4 months of age. In contrast, no significant differences in galanin peptide concentrations of the neurointermediate lobes were evident. Galanin mRNA concentrations in the anterior pituitary of 6-month-old transgenic mice were increased 4-fold. In conclusion, 1) galanin peptide concentrations in the anterior pituitary gland and hypothalamus are increased in hGHRH transgenic mice compared to those in nontransgenic siblings, whereas galanin peptide concentrations in the neurointermediate lobe are not different; 2) pituitary galanin mRNA concentrations are increased 4-fold in 6-month-old transgenic mice; and 3) the development of pituitary hyperplasia is correlated to the increase in galanin mRNA and peptide concentrations.

Adrenalectomy alters discrete galanin mRNA levels in the hypothalamus and mesencephalon of the rat

Using solution and in situ hybridization techniques we have studied the effects of adrenalectomy with or without restitution therapy with corticosterone on galanin mRNA levels in discrete regions of the male rat brain. Galanin peptide levels were also measured using a radioimmunoassay. The solution hybridization showed a two-fold increase in galanin mRNA 7 days, but not 14 days, after adrenalectomy in the preoptic area including the hypothalamic paraventricular nucleus (PVN). No changes were observed in the mediobasal hypothalamus including the arcuate nucleus. In situ hybridization showed that the increase in galanin mRNA was localized to the PVN and that the arcuate nucleus was not affected. The changes observed could be fully counteracted by corticosterone treatment. Radioimmunoassay showed decreased galanin levels in the median eminence 14 days, but not 7 days, after adrenalectomy and increased levels in the anterior pituitary and neurointermediate lobe. The results give evidence for a regional regulation of galanin gene expression and galanin peptide synthesis by adrenocortical steroids.

Expression and sexual dimorphism of galanin messenger ribonucleic acid in growth hormone-releasing hormone neurons of the rat during development

In the rat, the secretion of GH is episodic and sexually dimorphic. The development and regulation of this patterning of GH secretion are governed by the reciprocal influence of the hypothalamic peptide somatostatin and GH-releasing hormone (GHRH). Galanin is a neuropeptide that is colocalized with GHRH in hypothalamic neurons and is thought to be involved in generating the episodic pattern of GH secretion. We hypothesized that galanin mRNA expression in GHRH neurons increases over development in both sexes, and that in the adults, galanin expression in GHRH neurons is greater in males than in females. To test these hypotheses, we used a double label in situ hybridization procedure to detect and measure galanin mRNA expression in GHRH neurons in the rat brain. GHRH mRNA-positive cells were visualized by an alkaline phosphatase color reaction, and galanin mRNA levels were measured by counting autoradiographic grains over individual GHRH mRNA-positive cells. Galanin mRNA coexpression was found in GHRH mRNA-containing cells of the arcuate nucleus, periarcuate area, and ventromedial hypothalamus. In both males and females there was a significant increase in galanin mRNA in GHRH neurons over development. Galanin mRNA levels in GHRH neurons of 10- and 25-day-old rats were higher in females than in males [10-day-old: females, 12 +/- 2; males, 6 +/- 1 grains/cell (P < 0.05); 25-day-old: females, 28 +/- 4; males, 15 +/- 3 grains/cell (P < 0.02)]. In adults (70 days), galanin mRNA levels in GHRH neurons were significantly higher in males than in females (males, 54 +/- 4; females, 32 +/- 3 grains/cell; P < 0.005). In the adult rat, galanin mRNA levels in the individual hypothalamic areas exhibited a significant sexual dimorphism in the arcuate nucleus and periarcuate area, with higher levels in the male, whereas no sexual dimorphism was observed in the ventromedial hypothalamus. To determine whether galanin gene expression is influenced by circulating levels of testosterone, we measured galanin mRNA levels in castrated male rats with and without testosterone replacement. Castration reduced galanin message levels in GHRH neurons (intact, 73 +/- 6; castrate, 57 +/- 4 grains/cell), and although this reduction was not statistically significant (P = approximately 0.07), testosterone replacement significantly increased galanin message content (castrate/sham, 58 +/- 4 grains/cell; castrate plus testosterone replacement, 77 +/- 5 grains/cell; P < 0.02) to intact levels (intact, 73 +/- 6 grains/cell). In summary, galanin message expression in GHRH neurons of both male and female rats increases over development.

Expression and sexual dimorphism of galanin messenger ribonucleic acid in growth hormone-releasing hormone neurons of the rat during development.

In the rat, the secretion of GH is episodic and sexually dimorphic. The development and regulation of this patterning of GH secretion are governed by the reciprocal influence of the hypothalamic peptide somatostatin and GH-releasing hormone (GHRH). Galanin is a neuropeptide that is colocalized with GHRH in hypothalamic neurons and is thought to be involved in generating the episodic pattern of GH secretion. We hypothesized that galanin mRNA expression in GHRH neurons increases over development in both sexes, and that in the adults, galanin expression in GHRH neurons is greater in males than in females. To test these hypotheses, we used a double label in situ hybridization procedure to detect and measure galanin mRNA expression in GHRH neurons in the rat brain. GHRH mRNA-positive cells were visualized by an alkaline phosphatase color reaction, and galanin mRNA levels were measured by counting autoradiographic grains over individual GHRH mRNA-positive cells. Galanin mRNA coexpression was found in GHRH mRNA-containing cells of the arcuate nucleus, periarcuate area, and ventromedial hypothalamus. In both males and females there was a significant increase in galanin mRNA in GHRH neurons over development. Galanin mRNA levels in GHRH neurons of 10- and 25-day-old rats were higher in females than in males [10-day-old: females, 12 +/- 2; males, 6 +/- 1 grains/cell (P < 0.05); 25-day-old: females, 28 +/- 4; males, 15 +/- 3 grains/cell (P < 0.02)]. In adults (70 days), galanin mRNA levels in GHRH neurons were significantly higher in males than in females (males, 54 +/- 4; females, 32 +/- 3 grains/cell; P < 0.005). In the adult rat, galanin mRNA levels in the individual hypothalamic areas exhibited a significant sexual dimorphism in the arcuate nucleus and periarcuate area, with higher levels in the male, whereas no sexual dimorphism was observed in the ventromedial hypothalamus. To determine whether galanin gene expression is influenced by circulating levels of testosterone, we measured galanin mRNA levels in castrated male rats with and without testosterone replacement. Castration reduced galanin message levels in GHRH neurons (intact, 73 +/- 6; castrate, 57 +/- 4 grains/cell), and although this reduction was not statistically significant (P = approximately 0.07), testosterone replacement significantly increased galanin message content (castrate/sham, 58 +/- 4 grains/cell; castrate plus testosterone replacement, 77 +/- 5 grains/cell; P < 0.02) to intact levels (intact, 73 +/- 6 grains/cell). In summary, galanin message expression in GHRH neurons of both male and female rats increases over development.(ABSTRACT TRUNCATED AT 400 WORDS)

Critical effects of aging and nutritional state on hypothalamic neuropeptide Y and galanin gene expression in lean and genetically obese Zucker rats

We have investigated, by Northern blot analysis, the hypothalamic gene expression [messenger RNA (mRNA)] of two appetite stimulating neuropeptides, neuropeptide Y (NPY) and galanin (GAL) in lean (+/+) and genetically obese ( fa/fa) Zucker rats at 11, 24 and 40 weeks of age and their responsiveness to food deprivation. At 11 weeks of age, hypothalamic NPY mRNA levels of fa/fa rats were similar to those observed in lean littermates. However, NPY mRNA levels of fa/fa rats were significantly greater than those of lean rats at 24 (+126%; P < 0.01) and 40 (+65%; P < 0.05) weeks of age. Food deprivation caused a significant increase in NPY mRNA levels in both lean and fa/fa Zucker rats at 11 and 24 weeks of age, but not at 40 weeks old rats. Hypothalamic GAL mRNA showed a different pattern of change. The relative content of GAL mRNA in 11 week old obese rats was significantly lower (−68%; P < 0.05) than that of lean rats, while GAL and mRNA was significantly higher in 40 week old (+57%; P < 0.05) obese rats compared to their lean littermates. At 24 weeks of age, hypothalamic GAL mRNA levels did not differ between lean and obese rats. Food deprivation induced no change in hypothalamic GAL mRNA in lean rats of all 3 ages; however, it caused an increase of GAL mRNA in obese rats at 11 (+60%; P < 0.05) and 24 (+44%; P < 0.05) weeks, but not at 40 weeks. These findings demonstrate that hypothalamic NPY and GAL gene expression differs between lean and obese rats, as a function of age and nutritional state and they also suggest that alterations in NPY and GAL mRNA levels of the obese rat are secondary manifestations of the obese state, rather than the primary cause of obesity.

Regulation of galanin gene expression in gonadotropin-releasing hormone neurons during the estrous cycle of the rat

Galanin is colocalized with GnRH in neurons of the hypothalamus and basal forebrain of female rats, and this neuropeptide may play a role in the generation of the midcycle surge of gonadotropin secretion. We tested the hypothesis that galanin gene expression in GnRH cells increases during proestrus. To accomplish this, we killed groups of adult female rats at 1200 and 1800 h on the day of proestrus as well as at 1800 h on the day of estrus and used double labeling in situ hybridization and image analysis to estimate and compare the levels of galanin mRNA in cells coexpressing GnRH mRNA. GnRH mRNA was detected with an antisense cRNA probe labeled with the hapten digoxigenin, while the galanin cRNA probe was labeled with 35S and detected by autoradiography. There was no significant difference in the total number of GnRH cells identified in each animal in any of the different groups in any experiment. The relative number of silver grains over these cells, reflecting galanin mRNA content in GnRH neurons (identified by their purple color), was counted with a computerized image analysis system. In an initial experiment, we observed a 2-fold (P < 0.03) higher galanin mRNA signal level in the animals killed at 1800 h than in those killed at 1200 h on the day of proestrus. Animals killed at 1800 h on the day of estrus had galanin mRNA signal levels that were not statistically different from those in the proestrous 1800 h group, indicating that the increase in galanin mRNA at proestrus is maintained for at least 24 h. Galanin mRNA levels in GnRH neurons returned to basal levels equivalent to those in the proestrous 1200 h group by 1000 h on diestrous day 1. In conjunction with the studies of galanin gene expression in GnRH neurons, we compared the relative cellular contents of GnRH mRNA among the same groups. Here, we used single labeling isotopic in situ hybridization for GnRH mRNA and computerized image analysis to count the resulting silver grains. We could detect no difference in GnRH mRNA signal levels (proestrus, 1200 h vs. proestrus, 1800 h vs. estrus, 1800 h). In a final experiment, we investigated the possible role of estrogen in the induction of galanin mRNA expression at proestrus by comparing relative galanin mRNA contents in GnRH neurons among groups of ovariectomized, intact (diestrous day 1), and ovariectomized 17 beta-estradiol-replaced female rats.

Regulation of galanin gene expression in gonadotropin-releasing hormone neurons during the estrous cycle of the rat.

Galanin is colocalized with GnRH in neurons of the hypothalamus and basal forebrain of female rats, and this neuropeptide may play a role in the generation of the midcycle surge of gonadotropin secretion. We tested the hypothesis that galanin gene expression in GnRH cells increases during proestrus. To accomplish this, we killed groups of adult female rats at 1200 and 1800 h on the day of proestrus as well as at 1800 h on the day of estrus and used double labeling in situ hybridization and image analysis to estimate and compare the levels of galanin mRNA in cells coexpressing GnRH mRNA. GnRH mRNA was detected with an antisense cRNA probe labeled with the hapten digoxigenin, while the galanin cRNA probe was labeled with 35S and detected by autoradiography. There was no significant difference in the total number of GnRH cells identified in each animal in any of the different groups in any experiment. The relative number of silver grains over these cells, reflecting galanin mRNA content in GnRH neurons (identified by their purple color), was counted with a computerized image analysis system. In an initial experiment, we observed a 2-fold (P < 0.03) higher galanin mRNA signal level in the animals killed at 1800 h than in those killed at 1200 h on the day of proestrus. Animals killed at 1800 h on the day of estrus had galanin mRNA signal levels that were not statistically different from those in the proestrous 1800 h group, indicating that the increase in galanin mRNA at proestrus is maintained for at least 24 h. Galanin mRNA levels in GnRH neurons returned to basal levels equivalent to those in the proestrous 1200 h group by 1000 h on diestrous day 1. In conjunction with the studies of galanin gene expression in GnRH neurons, we compared the relative cellular contents of GnRH mRNA among the same groups. Here, we used single labeling isotopic in situ hybridization for GnRH mRNA and computerized image analysis to count the resulting silver grains. We could detect no difference in GnRH mRNA signal levels (proestrus, 1200 h vs. proestrus, 1800 h vs. estrus, 1800 h). In a final experiment, we investigated the possible role of estrogen in the induction of galanin mRNA expression at proestrus by comparing relative galanin mRNA contents in GnRH neurons among groups of ovariectomized, intact (diestrous day 1), and ovariectomized 17 beta-estradiol-replaced female rats.(ABSTRACT TRUNCATED AT 400 WORDS)

Tyrosine hydroxylase and galanin mRNA levels in locus coeruleus neurons are increased following reserpine administration

The neuropeptide galanin coexists in 80-90% of the norepinephrine-containing neurons in the locus coeruleus. In situ hybridization histochemistry was used to examine the effects of reserpine treatment or swim stress on tyrosine hydroxylase and galanin mRNA concentrations in locus coeruleus neurons. Reserpine administration significantly increased tyrosine hydroxylase and galanin mRNA levels in the locus coeruleus. The reserpine-induced increase in tyrosine hydroxylase mRNA was significantly correlated with the reserpine-induced increase in galanin mRNA. Three consecutive days of swim stress did not significantly alter either tyrosine hydroxylase or galanin mRNA concentrations in the locus coeruleus. These data suggest that both tyrosine hydroxylase and galanin gene expression in locus coeruleus neurons may be regulated by a reserpine-sensitive mechanism.