Galanin receptor-1 knockout mice exhibit spontaneous epilepsy, abnormal EEGs and altered inhibition in the hippocampus.

Galanin is a widely-distributed neuropeptide that acts as an endogenous anticonvulsant. We have recently generated a galanin receptor type 1 knockout mouse (Galr1(-/-)) that develops spontaneous seizures. Our aim here was to characterize the seizures by making electroencephalogram (EEG) recordings from this animal, and also to elucidate the cellular basis of its epileptic phenotype by studying the neurophysiology of CA1 pyramidal neurons in acute hippocampal slices. EEGs showed that major seizures had a partial onset with secondary generalization, and that paroxysms of spike-and-slow waves occurred and were associated with hypoactivity. The interictal EEG was also abnormal, with a marked excess of spike-and-slow waves. Slice experiments showed that resting potential, input resistance, intrinsic excitability, paired-pulse facilitation of excitatory and inhibitory postsynaptic currents (EPSCs and IPSCs), stimulus--response plots for EPSCs, and several properties of spontaneous miniature EPSCs and IPSCs were all unchanged in the mutant mouse compared with wildtype. However, the frequency of miniature IPSCs was significantly reduced in the mutants. These results suggest that impaired synaptic inhibition in the hippocampus may contribute to the local onset of seizures in the Galr1(-/-) mouse.

The second galanin receptor GalR2 plays a key role in neurite outgrowth from adult sensory neurons.

Expression of the neuropeptide galanin is markedly upregulated within the adult dorsal root ganglion (DRG) after peripheral nerve injury. We demonstrated previously that the rate of peripheral nerve regeneration is reduced in galanin knock-out mice, with similar deficits observed in neurite outgrowth from cultured mutant DRG neurons. Here, we show that the addition of galanin peptide significantly enhanced neurite outgrowth from wild-type sensory neurons and fully rescued the observed deficits in mutant cultures. Furthermore, neurite outgrowth in wild-type cultures was reduced to levels observed in the mutants by the addition of the galanin antagonist M35 [galanin(1-13)bradykinin(2-9)]. Study of the first galanin receptor (GalR1) knock-out animals demonstrated no differences in neurite outgrowth compared with wild-type animals. Similarly, use of a GalR1-specific antagonist had no effect on neuritogenesis. In contrast, use of a GalR2-specific agonist had equipotent effects on neuritogenesis to galanin peptide, and inhibition of PKC reduced neurite outgrowth from wild-type sensory neurons to that observed in galanin knock-out cultures. These results demonstrate that adult sensory neurons are dependent, in part, on galanin for neurite extension and that this crucial physiological process is mediated by activation of the GalR2 receptor in a PKC-dependent manner.

Galanin GAL-R1 receptor null mutant mice display increased anxiety-like behavior specific to the elevated plus-maze.

The neuropeptide galanin coexists with norepinephrine and serotonin in neural systems mediating emotion. Previous findings suggested that galanin modulates anxiety-related behaviors in rodents. Three galanin receptor subtypes have been cloned; however, understanding their functions has been limited by the lack of galanin receptor subtype-selective ligands. To study the role of the galanin GAL-R1 receptor subtype in mediating anxiety-related behavior, we generated mice with a null mutation in the Galr1 gene. GAL-R1 -/- are viable and show no abnormalities in health, neurological reflexes, motoric functions, or sensory abilities. On a battery of tests for anxiety-like behavior, GAL-R1 -/- showed increased anxiety-like behavior on the elevated plus-maze test. Anxiety-related behaviors on the light/dark exploration, emergence, and open field tests were normal in GAL-R1 -/-. This test-specific anxiety-like phenotype was confirmed in a second, independent cohort of GAL-R1 null mutant mice and +/+ controls. Principal components factor analysis of behavioral scores from 279 mice suggested that anxiety-like behavior on the elevated plus-maze was qualitatively distinct from behavior on other tests in the battery. In addition, exposure to the elevated plus-maze produced a significantly greater neuroendocrine response than exposure to the light/dark exploration test, as analyzed in normal C57BL/6J mice. These behavioral findings in the first galanin receptor null mutant mouse are consistent with the hypothesis that galanin exerts anxiolytic actions via the GAL-R1 receptor under conditions of relatively high stress.

Galanin and neuropeptide Y reduce cholinergic transmission in the heart of the anaesthetised mouse.

(1) This study investigated the effects of galanin (GAL) on inhibition of cholinergic (vagal) activity in the mouse heart using control galanin knockout (GAL-KO) and GAL-1R receptor knockout (GAL-1R-KO) mice. (2) In pentobarbitone anaesthetised mice, supramaximal stimulation every 30 s of the vagus nerve innervating the heart, increased pulse interval (PI) by approximately 50 ms or decreased heart rate by approximately 100 beats min-1. This response was attenuated by intravenous administration of GAL (dose ranged from 0.8 to 13 nmol kg-1) in a dose-dependent manner. (3) In GAL-KO mice, the magnitude of inhibition of the increase in PI (DeltaPI) following a bolus dose of GAL was not different from the DeltaPI in control mice, and neuropeptide Y (NPY), previously shown to attenuate vagal inhibitory activity in mice, evoked a comparative inhibition of DeltaPI in GAL-KO mice. (4) In GAL-1R-KO mice, an intravenous, bolus injection of GAL had no inhibitory effect on vagal activity. (5) In control mice, stimulation of the sympathetic nerve at 25 V, 10 Hz for 2 min in the presence of propranolol evoked a long-lasting attenuation of DeltaPI. The inhibitory effect on DeltaPI was reduced in the presence of the NPY Y2 antagonist, BIIE0246. (6) In GAL-1R-KO mice, stimulation of the sympathetic nerve in the presence of propranolol evoked an attenuation of DeltaPI not significantly different from the response in control mice in the presence of BIIE0246. Following administration of BIIE0246 in GAL-1R-KO mice, the inhibition of DeltaPI that followed stimulation of the sympathetic nerve was abolished. (7) These findings support the view that the nerve terminals of parasympathetic neurons in the mouse heart possess both GAL-1R and NPY Y2 receptors which, when activated, reduce acetylcholine release.

Flexor reflex excitability in mice lacking galanin receptor galanin-R1.

This study was conducted to examine the excitability of the nociceptive flexor reflex and its sensitization by repetitive stimulation of C-fibers in anesthetized mice that lack the galanin-R1 receptor. Repetitive stimulation of C-fibers induced a gradual increase in reflex magnitude during the stimulation (wind-up), and a subsequent increase in spinal reflex excitability (central sensitization). This occurred in GAL-R1 -/-, GAL-R1 +/-, and +/+ wild-type controls, with no significant differences observed between genotypes. Intrathecal administration of galanin markedly blocked the sensitization following the repetitive stimulation in all three groups. No differences between wild-type or galanin-R1 receptor knockout mice were seen. These results confirm previous studies in rats, showing that intrathecal galanin reduces the central sensitization following wind-up. The present data indicate that this effect is probably mediated by receptors other than GAL-R1.

Galanin negatively modulates opiate withdrawal via galanin receptor 1.

RATIONALE: The neuropeptide galanin has been shown to modulate opiate dependence and withdrawal. These effects could be mediated via activation of one or more of the three distinct G protein-coupled receptors, namely galanin receptors 1 (GalR1), 2 (GalR2), and 3 (GalR3). OBJECTIVES: In this study, we used several transgenic mouse lines to further define the mechanisms underlying the role played by galanin and its receptors in the modulation of morphine dependence. First, transgenic mice expressing ß-galactosidase under the control of the galanin promoter were used to assess the regulation of galanin expression in response to chronic morphine administration and withdrawal. Next, the behavioral responses to chronic morphine administration and withdrawal were tested in mice that over-express galanin, lack the GalR1 gene, or lack the GalR2 gene. METHODS: Transgenic and matched wild-type mice were given increasing doses of morphine followed by precipitation of withdrawal by naloxone and behavioral responses to withdrawal were assessed. RESULTS: Both morphine administration and withdrawal increased galanin gene transcription in the locus coeruleus (LC). Increasing galanin levels in the brain reduced signs of opiate withdrawal. Mice lacking GalR1 undergo more severe opiate withdrawal, whereas mice lacking GalR2 show no significant difference in withdrawal signs, compare with matched wild-type controls. CONCLUSIONS: Opiate administration and withdrawal increase galanin expression in the LC. Galanin opposes the actions of morphine which leads to opiate dependence and withdrawal, an effect that is mediated via GalR1.

Presence of transient helical segments in the galanin-like peptide evident from (1)H NMR, circular dichroism, and prediction studies.

Galanin and its newly discovered relative galanin-like peptide (GALP) are neuropeptides that are implicated in the neuroendocrine regulation of body weight and reproduction. GALP encompasses within its sequence the first 13 residues of galanin, known to be crucial to binding and activation of galanin receptor (GalR) subtypes. Using 2D-NMR and circular dichroism spectroscopy we demonstrated that GALP does not adopt a preferred conformation in pure water alone. However, it shows characteristics of transient turn-like structures in two distinct regions of its sequence, 11-23 and 41-49. These transient ordered structures, nascent helices, probably form stable helical structures upon addition of the helix-inducing solvent, trifluoroethanol, as determined by circular dichroism studies. Secondary structure prediction methods also predict the presence of two helical regions in the sequence of GALP overlapping reasonably with those regions identified as nascent helical structures by experimental methods.

The neuropeptide galanin augments lobuloalveolar development.

Mammary lobuloalveolar development during pregnancy is controlled by ovarian sex steroids and pituitary prolactin release. In organ culture these hormones are incapable of reproducing the density and size of lobuloalveoli seen in mice, suggesting the existence of other undiscovered factors. We showed previously that galanin knockout mice fail to lactate sufficiently for pup survival following their first pregnancy. Here we demonstrate that prolactin treatment of galanin knockout mice allows pup survival but does not completely rescue lobuloalveolar development or reduced milk protein expression. When galanin was used in combination with prolactin in mammary organ culture, larger and more numerous lobules were produced than with prolactin alone. Galanin alone produced sustained activation of STAT5a and the induction of milk protein expression but did not induce lobulogenesis. Examination of the transcriptional interaction between galanin and prolactin using oligonucleotide microarrays demonstrated synergistic and antagonistic modes of interaction between these hormones. These data establish a new role for galanin as a hormone augmenting mammary development during pregnancy in concert with prolactin.

Altered hippocampal expression of neuropeptides in seizure-prone GALR1 knockout mice.

PURPOSE: Mice carrying a deletion of the GALR1 galanin receptor have recently showed spontaneous seizure phenotype with 25% penetrance. To better understand the role of neuropeptides, which are known to undergo complex plasticity changes with development of epileptic seizures, we characterized their expression in the hippocampal formation in GALR1- knockout (-KO) mice with or without seizures and in wild-type (WT) mice. METHODS: Immunohistochemistry and in situ hybridization were used to study expression of galanin, neuropeptide Y (NPY), substance P, enkephalin, dynorphin, and cholecystokinin (CCK). RESULTS: In GALR1-KO mice that had been displaying seizures, a strong upregulation of galanin immunoreactivity (ir) and messenger RNA (mRNA) was found in the polymorph layer of the dentate gyrus; galanin-ir also appeared in a dense fiber network in the supragranular layer. A strong upregulation of enkephalin was found in the granule cells/mossy fibers, whereas dynorphin mRNA levels were modestly decreased. NPY was strongly expressed in the granule cells/mossy fibers, and an increase of NPY mRNA levels in the polymorph cells was paralleled by an increase of NPY-ir in the molecular layer. An upregulation of substance P-ir was confined to the fibers in the granule and molecular layers, whereas substance P mRNA was increased in the cells of the polymorph layer. Both CCK-ir and mRNA were strongly downregulated in the granule cell/mossy fiber system, but CCK-ir appeared increased in the supragranular and molecular layers. No changes in neuropeptide-ir were found in GALR1-KO mice not displaying seizures. CONCLUSIONS: Complex changes in neuropeptide expression in some principal hippocampal neurons and interneurons appear as a characteristic feature of the spontaneous-seizure phenotype in GALR1-KO mice. However, to what extent causal relations exist between this "epilepsia peptidergic profile" and development of seizures requires further clarification.

Elevated expression of galanin receptors in childhood neuroblastic tumors.

The neuropeptide galanin (GAL) has been shown to be present in certain brain tumors. In order to learn more about GAL and its receptors in human tumors of the peripheral nervous system, we investigated the expression of the GAL peptide and the GAL receptors in tumor tissue from childhood neuroblastic tumors. GAL peptide concentrations up to 674 +/- 166 fmol/mg of tissue were detected by radioimmunoassay, but no significant correlation with standard tumor markers or the prognosis of the 14 patients investigated was observed. Ligand binding experiments showed different levels of GAL binding in all 28 primary neuroblastomas and 7 ganglioneuromas investigated. All three human GAL receptor subtypes cloned to date could be detected, with the GALR1 receptor subtype being expressed most prominently. GAL binding did not significantly correlate with genetic markers such as unfavorable DNA ploidy, amplification of the oncogene MYCN and allelic loss of chromosome 1p. However, low galanin binding was significantly correlated with survival (p = 0.021) in this limited analysis of neuroblastic tumor samples. These results raise the possibility that the expression of GAL binding sites may play a role in neuroblastic tumor biology and behavior.

Learning and memory performance in mice lacking the GAL-R1 subtype of galanin receptor.

The neuropeptide galanin induces performance deficits in a wide range of cognitive tasks in rodents. Three G-protein-coupled galanin receptor subtypes, designated GAL-R1, GAL-R2 and GAL-R3, have been cloned. The present study examined the role of GAL-R1 in cognition by testing mice with a null mutation in Galr1 on several different types of learning and memory tasks. Assessments of general health, neurological reflexes, sensory abilities and motor functions were conducted as control measures. Mutant mice were unimpaired in social transmission of food preference and the Morris water maze. In tests of fear conditioning, mutant mice were unimpaired in a delay version of cued fear conditioning. However, mice homozygous for the null mutation were impaired in a trace version of cued fear conditioning. Mutant mice were unimpaired in contextual fear conditioning, whether training was by the delay or trace protocol. General health, neurological reflexes, sensory abilities and motor functions did not differ across genotypes, indicating that the trace fear conditioning deficit was not an artifact of procedural disabilities. The findings of normal performance on several cognitive tasks and a selective deficit in trace cued fear conditioning in homozygous GAL-R1 mutant mice are discussed in terms of hypothesized roles of the GAL-R1 subtype. The generally normal phenotype of GAL-R1 null mutants supports the use of this line for identification of the receptor subtypes that mediate the cognitive deficits produced by exogenous galanin.