Galanin Receptor 1 Deletion Exacerbates Hippocampal Neuronal Loss after Systemic Kainate Administration in Mice

Galanin is a neuropeptide with a wide distribution in the central and peripheral nervous systems and whose physiological effects are mediated through three G protein-coupled receptor subtypes, GalR1, GalR2, and GalR3. Several lines of evidence indicate that galanin, as well as activation of the GalR1 receptor, is a potent and effective modulator of neuronal excitability in the hippocampus.In order to test more formally the potential influence of GalR1 on seizure-induced excitotoxic cell death, we conducted functional complementation tests in which transgenic mice that exhibit decreased expression of the GalR1 candidate mRNA underwent kainate-induced status epilepticus to determine if the quantitative trait of susceptibility to seizure-induced cell death is determined by the activity of GalR1. In the present study, we report that reduction of GalR1 mRNA via null mutation or injection of the GalR1 antagonist, galantide, prior to kainate-induced status epilepticus induces hippocampal damage in a mouse strain known to be highly resistant to kainate-induced neuronal injury. Wild-type and GalR1 knockout mice were subjected to systemic kainate administration. Seven days later, Nissl and NeuN immune- staining demonstrated that hippocampal cell death was significantly increased in GalR1 knockout strains and in animals injected with the GalR1 antagonist. Compared to GalR1-expressing mice, GalR1-deficient mice had significantly larger hippocampal lesions after status epilepticus.Our results suggest that a reduction of GalR1 expression in the C57BL/6J mouse strain renders them susceptible to excitotoxic injury following systemic kainate administration. From these results, GalR1 protein emerges as a new molecular target that may have a potential therapeutic value in modulating seizure-induced cell death

Quantification of kainate (KA)-induced damage in hippocampal subfields in vehicle-injected C57BL/6 (resistant) and GalR1 antagonist treated C57BL/6 mice.

<p>Quantitative analysis of neuron density in the dentate hilus, areas CA3 and CA1, and dentate gyrus of C57BL/6 following intrahippocampal administration of 1 µM galantide. Viable surviving neuronal profiles were estimated by cresyl violet staining and counts were performed in each subfield for the most ventral hippocampal sections and averaged into single values for each animal. Bars denote the percentage of surviving neuronal profiles (as compared with saline-injected control mice) in each hippocampal region. Comparison of neuronal profile counts between treatments revealed statistically significant differences when comparing vehicle + KA versus GalR1 antagonist + KA. Data represent the mean ± SEM of eight to 10 mice/treatment. *P<0.05 as compared with intact mice of the C57BL/6 strain.</p

Kainate-induced cell death is substantially increased in GalR1^−/− mice.

<p>Quantitative analysis of neuronal density in four hippocampal subfields following kainate-induced status epilepticus to young adult mice. Viable surviving neurons were estimated by cresyl violet staining. Bars denote the percentage of surviving neurons (as compared with saline-injected control mice) in each hippocampal region. Differences in the extent of cell loss in three hippocampal subfields were observed 7 days following kainate administration between the GalR1^−/− and GalR1^+/+ mice. Data represent the mean ± SEM of at least 6 mice/condition. CA1 and CA3, hippocampal subfields; Hilus, dentate hilus; DG, dentate gyrus. *P<0.05.</p

C57BL/6 mice pre-treated with the GalR1 antagonist, galantide, show increased susceptibility to the neurotoxic effects of kainate.

<p>Corresponding low-power and high-power photomicrographs of NeuN- stained horizontal sections of the hippocampus showing differential cell loss 7 days after kainate-induced SE (vehicle), and in a representative mouse pre-treated with the GalR1 antagonist, galantide. Note that while hippocampal cell death is essentially non-existent following kainate-induced status epilepticus (Vehicle) to excitotoxin cell death-resistant mice (C57BL/6), a massive loss of neurons, as evidenced by loss of immunostaining in the hilar, CA3, and CA1 fields of the hippocampus, was induced by pre-treatment with galantide prior to KA-induced SE. CA1 and CA3 denote the hippocampal subfields; H, dentate hilus. Scale bars: top panels, 750 µm; bottom panels, 100 µm.</p

GalR1 deficient mice show increased susceptibility to seizure-induced cell death.

<p>Corresponding low-power and high-power photomicrographs of NeuN-immunofluorescent stained horizontal sections of the hippocampus depicting surviving cells throughout the hippocampus 7 days following systemic KA administration to GalR1^−/− and GalR1^+/+ mice. Hippocampal sections from GalR1^+/+ and GalR1^−/− brains were stained with NeuN immunofluorescence to determine the amount of cellular damage. Note the massive loss of neurons, as evidenced by loss of immunostaining, in the hilar, CA3 and CA1 fields of the hippocampus, seven days after a systemic injection of KA inGalR1^−/− mice. In contrast, hippocampal cell death was essentially non-existent throughout all hippocampal subfields in GalR1^+/+ mice. CA1 and CA3 denote the hippocampal subfields; H, dentate hilus. Scale bars = 750 µm (top panels); 100 µm (bottom panels).</p

Histograms of seizure parameters in GalR1 mutant mice following kainic acid-induced status epilepticus.

<p>(A) Data represent latency to severe stage 4/5 seizures in minutes (mean ± SEM) for GalR1 ^+/+ and GalR1 ^−/− mice. No significant differences between groups were observed. (B) Duration of severe stage 4/5 seizures in minutes for GalR1 ^+/+ and GalR1 ^−/− mice was not significantly different among mice. Data represent the mean ± SEM of at least 6 mice per group. *P<0.05.</p

Characterization of mice lacking GalR1.

<p>Real-time PCR quantification of the levels of GalR1, GalR2, and galanin produced in the hippocampus of wildtype (GalR1^+/+) and GalR1 mutants (GalR1^−/−). Expression levels were standardized relative to GAPDH transcript levels using the standard curve method. Values are provided as mean ± SEM from 6–9 mice per strain analyzed in triplicate. Expression studies demonstrated that the hippocampus of knockout animals had a minimal expression of GalR1 when compared to wild-type littermates. In contrast, differences in the expression of GalR2 or galanin between GalR1^−/− mice and their wildtype littermates were not observed in the hippocampus. *P<0.05.</p