The Involvement Of Galanin In Neuronal Development

Galanin is a neuropeptide that has a widespread distribution throughout the mammalian brain.The actions of galanin mediated by several galanin receptor subtypes are involved in widespread physiological and pathophysiological responses.Previous studies have investigated the galanin and galanin receptor system in the nervous system of the rat by using in situ hybridization and receptor binding assay.Galanin and GalRs were found in the neurogenetic regions inclding subventricular zone(SVZ),dentate gyrus(DG)and rostral migratory stream(RMS)of adult and postnatal rat.In addition to its function as a modulator of fast transmission in adult brain,more and more evidence suggests that galanin also acts as atrophic factor during neurogenesis and neural injury and repair. Recent studies published have described the distribution of galanin mRNA and immunoreactive cell bodies and nerve fibers in mouse brain.But there is still limited comparable information on galanin receptors in the brain of normal mice.In this study,first we examined GalRs distribution by autoradiographic localization of [¹²⁵I]-galanin binding sites in brain of the C57BL/6J mice.[¹²⁵I]-galanin binding sites had been detected throughout the brain,including moderate-high relative densities in the basal ganglia,limbic regions,centro-lateral/medial thalamic nuclei,preoptic/lateral hypothalamus,midbrain,pons/medulla oblongata and cerebellar cortex.[¹²⁵I]-Galanin binding was absent or present in low levels in olfactory bulb,neocortex,dorsal hippocampus.This evaluation of galanin receptor site distribution in mouse brain has revealed similarities and some differences with the equivalent system in rat and provides a valuable reference for future comparative studies of central galanin transmission. Immunohistochemistry assay was also employed on the presence and distribution of galanin-like-immunoreactivity(-LI)in the brain.The distribution of galanin-LI in the mouse is consistent with the distribution of galanin mRNA published.These anatomical studies have demonstrated the presence of galanin and GalRs in the SVZ,RMS and DG which is in good agreement with the data from rat.Then,in situ hybridization was used to elucidate the expression of galanin,GalR1 and GalR2 in these regions of a serial of postnatal mice brain.We found that the expressions of galanin,GalR1 and GalR2 are detectable at DG and RMS on dayl or day4 and go stronger gradually.Expression signals in the SVZ could be detected from day 10.These findings suggest galanin may play an important role in the regulation of neuro(glio)genesis under normal and/or pathophysiological conditions in the mature murine brain.Recently,galanin expression was detected in mouse embryonic stem cells and suggested to have a regulatory role in embryonic stem cell biology.To our knowledge however, there is still little or no documented evidence of the involvement of galanin and galanin receptors in neural stem cell(NSC)or progenitor development and differentiation.Here, we established the SVZ-derived NSCs primary culture from wild-type and Gal KO mice. It is notable that differentiated GalKO NSCs produce significantly shorter neurites compared with wild-type culture after 3 days differentiation.The presence of galanin and 3 Gal receptor subtypes' expression were identified by RT-PCR in both cultures except absence of galanin in GalKO culture.Then,galanin,the GalR2/3 agonist,GAL(2-11), and the galanin receptor antagonist,M35 were used to investigate the effect of galanin on the neurite outgrowth in differentiated NSCs.Incubation of galanin and GAL(2-11)with these mouse NSCs,grown under differentiation conditions,significantly increased the percentage of cells producing neurites after 4h differentiation and the length of neurites outgrowth ofβⅢ-tubulin-positive cells.This effect could be reduced by treatment with the non-selective galanin receptor antagonist,M35. As last part in this research,the mechanism of galanin's effect on NSCs differentiation was studied.From the data on PC12 cell,Galanin is known to alter the activity of extracellular signal-regulated kinase(ERK)via GalR2 activation.To determine which GalR involved in this process,GalR1KO NSCs was set up as well.Treatment of mouse NSCs with an inhibitor of ERK or protein kinase C blocked the galanin-induced increase in neurite outgrowth and the increase in ERK phosphorylation levels in cultures from wild-type and GalR1-KO mice.In contrast,the addition of an inhibitor of protein kinase A had no effect on neuritogenesis or ERK phosphorylation.We also examined the galanin receptors' mRNA expression after galanin,Gal(2-11)and M35 treatment in wild-type and GalR1-KO NSCs cultures.Levels of GalR2 mRNA in the differentiating NSCs were higher than those of GalR1 and GalR3,and incubation in galanin or GAL(2-11) up-regulated GalR2 mRNA expression,relative to control.These results suggest that galanin promotes neurite extension in differentiating mouse NSCs,a process that is mediated by activation of GalR2 and downstream PKC-ERK signaling. Conclusion:1.Data from[¹²⁵I]-galanin receptor binding,immunohistochemistry and in situ hybridiztion assay reveals the presence of galanin and GalRs(peptide or mRNA)in the SVZ,DG and RMS regions of postnatal and adult mice.2.Galanin and GalRs mRNA expression were detectable in SVZ-derived NSCs. Exogenous galanin and Gal(2-11)have stimulative effect on the neurites outgrowth of differentiating NSCs.3.Results from pharmacological experiment suggest that galanin's effect on the neurite extension in NSCs is mainly mediated by activation of GalR2 and downstream PKC-ERK signaling.