Effects of galanin receptor 2 and receptor 3 knockout in mouse models of acute seizures.

There exists solid evidence that endogenous galanin and galanin agonists exert anticonvulsive actions mediated both by galanin 1 receptor (GAL1-R) and galanin 2 receptor (GAL2-R). We have now investigated whether depletion of the recently identified third galanin receptor, GAL3-R, and that of GAL2-R, alters the threshold to the systemically applied γ-aminobutyric acid (GABA) antagonist pentylenetetrazole (PTZ) or to intrahippocampally administered kainic acid (KA). In neither model, GAL3-KO mice differed in their latency to the first seizure, mean seizure duration, total number of seizures, or time spent in seizures compared to wild-type controls. In addition, consistent with previous data, the response to PTZ was not altered in GAL2-KO mice. In contrast, intrahippocampal KA resulted in a significantly increased number of seizures and time spent in seizures in GAL2-KO mice, although the latency to the first seizure and the duration of individual seizures was not altered. These results are consistent with the previous data showing that GAL2-R knockdown does not affect the number of perforant path stimulations required for initiating status epilepticus but significantly increases the seizure severity during the ongoing status. In conclusion, our data support a specific role of GAL2-R but not of GAL3-R in mediating the anticonvulsive actions of endogenous galanin.

A role for galanin N-terminal fragment (1-15) in anxiety- and depression-related behaviors in rats.

BACKGROUND: Galanin (GAL) plays a role in mood regulation. In this study we analyzed the action of the active N-terminal fragment [GAL(1-15)] in anxiety- and depression-related behavioral tests in rats. METHODS: The effect of GAL(1-15) was analyzed in the forced swimming test, tail suspension test, open field test, and light/dark test. The proximity of GAL1 and GAL2 receptors was examined with the proximity ligation assay (PLA). We tested the GAL receptors involved in GAL(1-15) effects with the GAL2 receptor antagonist M871 and with an in vivo model of siRNA GAL2 receptor knockdown or siRNA GAL1 receptor knockdown rats. The effects of GAL(1-15) were also studied in the cell line RN33B. RESULTS: GAL(1-15) induced strong depression-like and anxiogenic-like effects in all the tests. These effects were stronger than the ones induced by GAL. The involvement of the GAL2 receptor was demonstrated with M871 and with the siRNA GAL2 receptor knockdown rats. The PLA indicated the possible existence of GAL1 and GAL2 heteroreceptor complexes in the dorsal hippocampus and especially in the dorsal raphe nucleus. In the siRNA GAL1 receptor knockdown rats the behavioral actions of GAL(1-15) disappeared, and in the siRNA GAL2 receptor knockdown rats the reductions of the behavioral actions of GAL(1-15) was linked to a disappearance of PLA. In the cell line RN33B, GAL(1-15) decreased 5-HT immunoreactivity more strongly than GAL. CONCLUSIONS: Our results indicate that GAL(1-15) exerts strong depression-related and anxiogenic-like effects and may give the basis for the development of drugs targeting GAL1 and GAL2 heteroreceptor complexes in the raphe-limbic system for the treatment of depression and anxiety.

Phenotypic analysis of mice deficient in the type 2 galanin receptor (GALR2).

Galanin is a neuropeptide implicated in the regulation of feeding, reproduction, cognition, nociception, and seizure susceptibility. There are three known galanin receptor (GALR) subtypes (GALR1, GALR2, and GALR3), which bind to galanin with different affinities and have their own unique distributions, signaling mechanisms, and putative functions in the brain and peripheral nervous system. To gain further insight into the possible physiological significance of GALR2, we created mutant mice that were deficient in GALR2 and compared their phenotype to that of wild-type (WT) littermate or age-matched controls, with respect to basic motor and sensory function, feeding behavior, reproduction, mood, learning and memory, and seizure susceptibility. Phenotypic analysis revealed that animals bearing a deletion of GALR2 did not differ significantly from their WT controls in any of the measured variables. We conclude that either GALR2 plays no role in these physiological functions or through redundancy or compensation these mutant animals can adapt to the congenital absence of GALR2. It is also conceivable that GALR2 plays only a subtle role in some of these functions and that the impact of its loss could not be detected by the analytical procedures used here.

Endogenous galanin protects mouse hippocampal neurons against amyloid toxicity in vitro via activation of galanin receptor-2.

Expression of the neuropeptide galanin is known to be upregulated in the brain of patients with Alzheimer's disease (AD). We and others have shown that galanin plays a neuroprotective role in a number of excitotoxic injury paradigms, mediated by activation of the second galanin receptor subtype (GAL2). In the present study, we investigated whether galanin/GAL2 plays a similar protective role against amyloid-ß(Aß) toxicity. Here we report that galanin or the GAL2/3-specific peptide agonist Gal2-11, both equally protect primary dispersed mouse wildtype (WT) neonatal hippocampal neurons from 250 nM Aß1-42 toxicity in a dose dependent manner. The amount of Aß1-42 induced cell death was significantly greater in mice with loss-of-function mutations in galanin (Gal-KO) or GAL2 (GAL2-MUT) compared to strain-matched WT controls. Conversely, cell death was significantly reduced in galanin over-expressing (Gal-OE) transgenic mice compared to strain-matched WT controls. Exogenous galanin or Gal2-11 rescued the deficits in the Gal-KO but not the GAL2-MUT cultures, confirming that the protective effects of endogenous or exogenous galanin are mediated by activation of GAL2. Despite the high levels of endogenous galanin in the Gal-OE cultures, the addition of exogenous 100 nM or 50 nM galanin or 100 nM Gal2-11 further significantly reduced cell death, implying that GAL2-mediated neuroprotection is not at maximum in the Gal-OE mice. These data further support the hypothesis that galanin over-expression in AD is a neuroprotective response and imply that the development of a drug-like GAL2 agonist might reduce the progression of symptoms in patients with AD.

Sensory neuronal phenotype in galanin receptor 2 knockout mice: focus on dorsal root ganglion neurone development and pain behaviour.

Galanin is a 29-amino-acid peptide expressed in dorsal root ganglion (DRG) neurones and spinal dorsal horn neurones. It affects pain threshold and has developmental and trophic effects. Galanin acts at three G-protein-coupled receptors, galanin receptors (GalR1-3), each expressed in the DRGs as suggested by in situ hybridization and/or reverse transcriptase-polymerase chain reaction. The GalR2 knockout (-/-) mice permit studies on the contributions of this receptor subtype to the role of galanin at the spinal level. At 1 week after sciatic nerve transection (axotomy), there were 16-20% fewer neurones in intact and contralateral DRGs of -/- mice as compared with wild-type (WT) mice. In addition, a significant neurone loss (26% reduction) was found in the ipsilateral DRGs of WT mice, whereas no further neurone loss was seen in -/- mice. Expression of several peptides has been examined after axotomy, including galanin, neuropeptide Y and two of its receptors as well as substance P, and no significant differences were found between -/- and WT mice in either ipsi- or contralateral DRGs, respectively. After thermal injury and spinal nerve ligation, onset and duration of hyperalgesia in the injured paw were similar in GalR2-/- and WT animals. Recovery from spinal nerve ligation-caused allodynia had the same kinetics in -/- and WT animals. These data are in line with earlier observations from the peripheral and central nervous system, suggesting that galanin actions mediated by GalR2 subtype are of importance in neurodevelopment and neuroprotection.

Analysis of the contribution of galanin receptors 1 and 2 to the central actions of galanin-like peptide.

Galanin-like peptide (GALP) shares partial sequence identity with galanin and exhibits agonistic activity at two of the galanin receptor subtypes (GALR1 and GALR2) in vitro. The goal of these experiments was to determine whether galanin receptors mediate the effects of central GALP administration on food intake, body weight, and luteinizing hormone (LH) secretion in the mouse. We first evaluated the effects of intracerebroventricular injections of GALP or its vehicle alone in GALR1 knockout mice, GALR2 knockout mice, and their respective wild-type controls. GALP reduced food intake and body weight after 24 h to a similar degree in wild-type, GALR1 knockout, and GALR2 knockout mice. The wild-type, GALR1 knockout, and GALR2 knockout mice also exhibited significant increases in serum levels of LH following the GALP injections. To help delineate the biologically active moiety of the GALP molecule, we injected wild-type mice with shorter fragments of the full-length GALP peptide. Neither GALP((1-21)) (the fragment containing the galanin-homologous sequence) nor GALP((22-60)) (the C-terminal portion of the GALP molecule lacking sequence identity with galanin) had any discernable effect on food intake, body weight or circulating LH. These observations demonstrate that neither GALR1 nor GALR2 are essential for mediating the effects of GALP on feeding, body weight or LH secretion. Furthermore, the galanin-homologous region of the GALP molecule is not sufficient to mimic the effects of full-length GALP. Together, these findings argue against the hypothesis that GALP signals solely through galanin receptors in vivoand suggest the existence of a yet-to-be-identified GALP-specific receptor.