Ectopic galanin expression and normal galanin receptor 2 and galanin receptor 3 mRNA levels in the forebrain of galanin transgenic mice.

The functional interactions of the neuropeptide galanin (GAL) occur through its binding to three G protein-coupled receptor subtypes: galanin receptor (GALR) 1, GALR2 and GALR3. Previously, we demonstrated that GALR1 mRNA expression was increased in the CA1 region of the hippocampus and discrete hypothalamic nuclei in galanin transgenic (GAL-tg) mice. This observation suggested a compensatory adjustment in cognate receptors in the face of chronic GAL exposure. To evaluate the molecular alterations to GALR2 and GALR3 in the forebrain of GAL overexpressing mice, we performed complementary quantitative, real-time PCR (qPCR), in situ hybridization, and immunohistochemistry in select forebrain regions of GAL-tg mice to characterize the neuronal distribution and magnitude of GAL mRNA and peptide expression and the consequences of genetically manipulating the neuropeptide GAL on the expression of GALR2 and GALR3 receptors. We found that GAL-tg mice displayed dramatic increases in GAL mRNA and peptide in the frontal cortex, posterior cortex, hippocampus, septal diagonal band complex, amygdala, piriform cortex, and olfactory bulb. Moreover, there was evidence for ectopic neuronal GAL expression in forebrain limbic regions that mediate cognitive and affective behaviors, including the piriform and entorhinal cortex and amygdala. Interestingly, regional qPCR analysis failed to reveal any changes in GALR2 or GALR3 expression in the GAL-tg mice, suggesting that, contrary to GALR1, these receptor genes are not under ligand-mediated regulatory control. The GAL-tg mouse model may provide a useful tool for the investigation of GAL ligand-receptor relationships and their role in normal cognitive and affective functions as well as in the onset of neurological disease.

Obesity and endocrine dysfunction in mice with deletions of both neuropeptide Y and galanin.

Neuropeptide Y (NPY) and galanin have both been implicated in the regulation of body weight, yet mice bearing deletions of either of these molecules have unremarkable metabolic phenotypes. To investigate whether galanin and NPY might compensate for one another, we produced mutants lacking both neuropeptides (GAL(-/-)/NPY(-/-)). We found that male GAL(-/-)/NPY(-/-) mice ate significantly more and were much heavier (30%) than wild-type (WT) controls. GAL(-/-)/NPY(-/-) mice responded to a high-fat diet by gaining more weight than WT mice gain, and they were unable to regulate their weight normally after a change in diet. GAL(-/-)/NPY(-/-) mice had elevated levels of leptin, insulin, and glucose, and they lost more weight than WT mice during chronic leptin treatment. Galanin mRNA was increased in the hypothalamus of NPY(-/-) mice, providing evidence of compensatory regulation in single mutants. The disruption of energy balance observed in GAL(-/-)/NPY(-/-) double knockouts is not found in the phenotype of single knockouts of either molecule. The unexpected obesity phenotype may result from the dysregulation of the leptin and insulin systems that normally keep body weight within the homeostatic range.

Distribution and regulation of galanin receptor 1 messenger RNA in the forebrain of wild type and galanin-transgenic mice.

To learn more about molecular alterations in the brain that occur as a consequence of either the chronic excess or absence of peptide neurotransmitters, we examined the impact of genetically manipulating the neuropeptide galanin on the expression of one of its cognate receptors, galanin receptor 1. First, we examined the distribution of galanin receptor 1 messenger RNA in the mouse forebrain, and found it to be abundantly expressed in many brain regions, including in numerous hypothalamic and other forebrain regions associated with neuroendocrine function. The distribution of galanin receptor 1 messenger RNA in the mouse was similar to previous reports in the rat, with additional expression noted in the caudate putamen and in several midbrain regions. Next, using quantitative in situ hybridization, we measured cellular levels of galanin receptor 1 messenger RNA in the brains of mice that either overexpress galanin (galanin transgenic) or lack a functional galanin gene (galanin knockout). We report that relative to wild-type controls, the expression of galanin receptor 1 messenger RNA was increased in discrete areas of the brain in galanin-transgenic mice, but that depletion of galanin/noradrenergic innervation to the hypothalamus with the neurotoxin 6-hydroxydopamine did not alter levels of galanin receptor 1 messenger RNA. We also report that levels of galanin receptor 1 messenger RNA were not different between galanin-knockout and wild-type mice. These results suggest that compensatory adjustments in the expression of cognate receptors represent one mechanism by which the developing nervous system attempts to maintain homeostasis in response to overexpression of a peptidergic transmitter. However, the lack of significant changes in galanin receptor 1 messenger RNA in galanin-knockout mice suggests that developmentally programmed levels of receptor expression are maintained even in the complete absence of ligand.

Genetic comparison of seizure control by norepinephrine and neuropeptide Y.

Epilepsy is a disease of neuronal hyperexcitability, and pharmacological and genetic studies have identified norepinephrine (NE) and neuropeptide Y (NPY) as important endogenous regulators of neuronal excitability. Both transmitters signal through G-protein-coupled receptors, are expressed either together or separately, and are abundant in brain regions implicated in seizure generation. NPY knock-out (NPY KO) and dopamine beta-hydroxylase knock-out (DBH KO) mice that lack NE are susceptible to seizures, and agonists of NE and NPY receptors protect against seizures. To examine the relative contributions of NE and NPY to neuronal excitability, we tested Dbh;Npy double knock-out (DKO) mice for seizure sensitivity. In general, DBH KO mice were much more seizure-sensitive than NPY KO mice and had normal NPY expression, demonstrating that an NPY deficiency did not contribute to the DBH KO seizure phenotype. DKO mice were only slightly more sensitive than DBH KO mice to seizures induced by kainic acid, pentylenetetrazole, or flurothyl, although DKO mice were uniquely prone to handling-induced seizures. NPY contributed to the seizure phenotype of DKO mice at high doses of convulsant agents and advanced stages of seizures. These data suggest that NE is a more potent endogenous anticonvulsant than NPY, and that NPY has the greatest contribution under conditions of extreme neuronal excitability.

Galanin transgenic mice display cognitive and neurochemical deficits characteristic of Alzheimer's disease.

Galanin is a neuropeptide with multiple inhibitory actions on neurotransmission and memory. In Alzheimer's disease (AD), increased galanin-containing fibers hyperinnervate cholinergic neurons within the basal forebrain in association with a decline in cognition. We generated transgenic mice (GAL-tg) that overexpress galanin under the control of the dopamine beta-hydroxylase promoter to study the neurochemical and behavioral sequelae of a mouse model of galanin overexpression in AD. Overexpression of galanin was associated with a reduction in the number of identifiable neurons producing acetylcholine in the horizontal limb of the diagonal band. Behavioral phenotyping indicated that GAL-tgs displayed normal general health and sensory and motor abilities; however, GAL-tg mice showed selective performance deficits on the Morris spatial navigational task and the social transmission of food preference olfactory memory test. These results suggest that elevated expression of galanin contributes to the neurochemical and cognitive impairments characteristic of AD.

Distribution of galanin messenger RNA-expressing cells in murine brain and their regulation by leptin in regions of the hypothalamus.

Galanin is widely distributed throughout the mammalian brain and has been implicated in the regulation of food intake, metabolism and reproduction-functions that are also thought to be under the control of leptin. To investigate the possible role of galanin in mediating the physiological effects of leptin in the mouse, we had three experimental objectives: first, to map the distribution of galanin messenger RNA-expressing cells in the brain of the mouse; second, to assess the effects of leptin on galanin gene expression in areas of the brain thought to be involved in the regulation of body weight and reproduction; and third, to determine whether galanin neurons in these regions express leptin receptor messenger RNA. We found the pattern of galanin messenger RNA expression in the mouse brain to be similar, but not identical, to that in the rat. Leptin treatment (2microg/g for six days) significantly reduced cellular levels of galanin messenger RNA in the hypothalamic periventricular nucleus of leptin-deficient obese (ob/ob) mice (P<0.01) by approximately 30%; however, leptin did not appear to influence the expression of galanin in the arcuate or dorsomedial nucleus of the hypothalamus. Galanin-producing neurons in the arcuate, dorsomedial and periventricular nuclei did not appear to express leptin receptor messenger RNA (P>0.05). These results demonstrate that galanin distribution patterns in the mouse brain are comparable to other species and, yet, possess unique features. In addition, galanin-expressing neurons in the hypothalamic periventricular nucleus are targets for regulation by leptin; however, the effect of leptin on galanin gene expression is likely to be mediated indirectly, perhaps through either proopiomelanocortin- or neuropeptide Y-expressing cells in the hypothalamus.

Modulation of hippocampal excitability and seizures by galanin.

Previous studies have shown that the expression of the neuropeptide galanin in the hippocampus is altered by seizures and that exogenous administration of galanin into the hippocampus attenuates seizure severity. To address the role of endogenous galanin in modulation of hippocampal excitability and its possible role in seizure mechanisms, we studied two types of transgenic mice: mice with a targeted disruption of the galanin gene (GalKO) and mice that overexpress the galanin gene under a dopamine-beta-hydroxylase promoter (GalOE). GalKO mice showed increased propensity to develop status epilepticus after perforant path stimulation or systemic kainic acid, as well as greater severity of pentylenetetrazol-induced convulsions. By contrast, GalOE mice had increased resistance to seizure induction in all three models. Physiological tests of hippocampal excitability revealed enhanced perforant path-dentate gyrus long-term potentiation (LTP) in GalKO and reduced LTP in GalOE. GalKO showed increased duration of afterdischarge (AD) evoked from the dentate gyrus by perforant path simulation, whereas GalOE had increased threshold for AD induction. Depolarization-induced glutamate release from hippocampal slices was greater in GalKO and lower in GalOE, suggesting that alterations of physiological and seizure responses in galanin transgenic animals may be mediated through modulation of glutamate release. Our data provide further evidence that hippocampal galanin acts as an endogenous anticonvulsant and suggest that genetically induced changes in galanin expression modulate both hippocampal excitability and predisposition to epileptic seizures.

Differential role of melanocortins in mediating leptin's central effects on feeding and reproduction.

Leptin serves as a humoral link coupling the status of energy reserves to the functional activity of the reproductive system. Leptin is thought to act through melanocortinergic pathways in the brain to regulate ingestive behaviors; however, whether melanocortins mediate leptin's actions on the neuroendocrine-reproductive axis is unknown. We tested this hypothesis first by determining whether the effects of leptin on feeding behavior and reproduction in the ob/ob mouse could be blocked by the melanocortin receptor (MC-R) antagonist SHU9119 and second, by examining the effects of the MC-R agonist MTII on feeding and the endocrine-reproductive system. Administered by intracerebroventricular injections, leptin inhibited food intake, raised plasma gonadotropin levels, and increased seminal vesicle weights compared with controls; SHU9119 (intracerebroventricularly) attenuated leptin's effects on food intake and body weight but did not alter leptin's stimulatory effect on the reproductive axis. MTII (intracerebroventricularly and intraperitoneally) decreased food intake and increased body temperature compared with controls but had no effect on the reproductive-endocrine axis. These results suggest that although leptin acts centrally through melanocortinergic pathways to inhibit ingestive behaviors and stimulate metabolism, leptin's activational effect on the reproductive axis is likely to be mediated by other, unknown neuroendocrine circuits.

Galanin: analysis of its coexpression in gonadotropin-releasing hormone and growth hormone-releasing hormone neurons.

Galanin is coexpressed in a subset of gonadotropin-releasing hormone (GnRH) and growth hormone-releasing hormone (GHRH) neurons in the brain and has an important role in the neuroendocrine regulation of gonadotropin and growth hormone secretion. Our overall goal has been to understand the functional significance of galanin as a cotransmitter with GnRH and GHRH in the regulation of these important physiologic processes. To this end, we studied the regulation of galanin's expression in GnRH and GHRH neurons under a variety of physiologic and experimental conditions. Using double-label in situ hybridization and computerized image analysis, we observed that in GnRH neurons, galanin's expression is increased over the course of development in both sexes. Galanin achieves a higher basal expression in GnRH neurons in females, and it is sexually differentiated in the adult as a result of the differential exposure to testosterone during the neonatal critical period. Galanin is induced in GnRH neurons coincident with and subsequent to the proestrous luteinizing hormone surge (reflecting the combined action of estradiol and progesterone) acting indirectly on GnRH neurons through a synaptic relay. Galanin's expression in GnRH neurons is inhibited during lactation, when the neuroendocrine reproductive axis is relatively quiescent. In GHRH neurons, the expression of galanin is also induced over the course of development in both sexes. Galanin's expression in GHRH neurons in the adult is sexually differentiated, but in this case, its expression is higher in males than females, reflecting the stimulatory effect of testosterone on galanin in the male. Galanin's expression in GHRH neurons is induced by growth hormone (GH), whereas the absence of GH leads to a reduction of galanin mRNA in these same cells. On the basis of these observations, we conclude that galanin is an important target for regulation by many hormones, and we postulate that as a cotransmitter, galanin acts presynaptically to modulate the secretion of GnRH and GHRH, possibly by altering their pulsatile release patterns, which in turn influences the release of the gonadotropins and GH from the pituitary.