Enhancement of neurogenesis and cognition through intranasal co-delivery of galanin receptor 2 (GALR2) and neuropeptide Y receptor 1 (NPY1R) agonists: a potential pharmacological strategy for cognitive dysfunctions.

BACKGROUND: Spatial memory deficits and reduced neuronal survival contribute to cognitive decline seen in the aging process. Current treatments are limited, emphasizing the need for innovative therapeutic strategies. This research explored the combined effects of intranasally co-administered galanin receptor 2 (GALR2) and neuropeptide Y1 receptor (NPY1R) agonists, recognized for their neural benefits, on spatial memory, neuronal survival, and differentiation in adult rats. After intranasal co-delivery of the GALR2 agonist M1145 and a NPY1R agonist to adult rats, spatial memory was tested with the object-in-place task 3 weeks later. We examined neuronal survival and differentiation by assessing BrdU-IR profiles and doublecortin (DCX) labeled cells, respectively. We also used the GALR2 antagonist M871 to confirm GALR2's crucial role in promoting cell growth. RESULTS: Co-administration improved spatial memory and increased the survival rate of mature neurons. The positive effect of GALR2 in cell proliferation was confirmed by the nullifying effects of its antagonist. The treatment boosted DCX-labeled newborn neurons and altered dendritic morphology, increasing cells with mature dendrites. CONCLUSIONS: Our results show that intranasal co-delivery of GALR2 and NPY1R agonists improves spatial memory, boosts neuronal survival, and influences neuronal differentiation in adult rats. The significant role of GALR2 is emphasized, suggesting new potential therapeutic strategies for cognitive decline.

The antidepressant-like effect of galanin in the dorsal raphe nucleus of rats involves GAL2 receptors.

Galanin is a neuropeptide distributed in human and rat brain regions that are involved with emotional regulation, such as the dorsal raphe nucleus (DRN). Galanin effects in the DRN are mediated by GAL1 and GAL2 receptors. Intracerebral infusion of a GAL2 (AR-M1896) or a GAL1 (M617) agonist induced either antidepressant or depressive-like effect, respectively, in rats exposed to the forced swimming test (FST). However, it is not clear if GAL1 and/or GAL2 receptors present in the DRN would be involved in such effects. Therefore, we investigated the effects induced by intra-DRN infusion of galanin (0.3 nmol), AR-M1896 (1 nmol, GAL2 agonist), or M617 (GAL1 agonist) in rats exposed to the FST. Galanin and AR-M1896 intra-DRN administration induced antidepressant-like effect in the FST. However, M617 did not induce any change in the FST. Neither M617 nor AR-M1896 changed the locomotor activity of rats in the open field test. Intra-DRN pre-treatment with M871 (1 nmol), a selective GAL2 antagonist, counteracted the antidepressant-like effect induced by galanin. These results suggest that galanin signaling through GAL2 receptors in the DRN produces triggers antidepressant-like effect.

The role of galanin system in modulating depression, anxiety, and addiction-like behaviors after chronic restraint stress.

There is high comorbidity between stress-related psychiatric disorders and addiction, suggesting they may share one or more common neurobiological mechanisms. Because of its role in both depressive and addictive behaviors, the galanin system is a strong candidate for such a mechanism. In this study, we tested if galanin and its receptors are involved in stress-associated behaviors and drug addiction. Mice were exposed to 21 days of chronic restraint stress (CRS); subsequently, mRNA levels of galanin, galanin receptors (GalRs), the rate-limiting enzymes for the synthesis of monoamines, and monoamine autoreceptors were measured in the nucleus accumbens by a quantitative real-time polymerase chain reaction. Moreover, we tested the effects of this stress on morphine-induced addictive behaviors. We found that CRS induced anxiety and depression-like behaviors, impaired the formation and facilitated the extinction process in morphine-induced conditioned place preference (CPP), and also blocked morphine-induced behavioral sensitization. These behavioral results were accompanied by a CRS-dependent increase in the mRNA expression of galanin, GalR1, tyrosine hydroxylase (TH), tryptophan hydroxylase 2, and 5-HT1B receptor. Interestingly, treatment with a commonly used antidepressant, fluoxetine, normalized the CRS-induced behavioral changes based on reversing the higher expression of galanin and TH while increasing the expression of GalR2 and α2A-adrenceptor. These results indicate that activating the galanin system, with corresponding changes to noradrenergic systems, following chronic stress may modulate stress-associated behaviors and opiate addiction. Our findings suggest that galanin and GalRs are worthy of further exploration as potential therapeutic targets to treat stress-related disorders and drug addiction.

Membrane cholesterol in the function and organization of G-protein coupled receptors.

Cholesterol is an essential component of higher eukaryotic membranes and plays a crucial role in membrane organization, dynamics and function. The G-protein coupled receptors (GPCRs) are the largest class of molecules involved in signal transduction across membranes, and represent major targets in the development of novel drug candidates in all clinical areas. Membrane cholesterol has been reported to have a modulatory role in the function of a number of GPCRs. Two possible mechanisms have been previously suggested by which membrane cholesterol could influence the structure and function of GPCRs (i) through a direct/specific interaction with GPCRs, or (ii) through an indirect way by altering membrane physical properties in which the receptor is embedded, or due to a combination of both. Recently reported crystal structures of GPCRs have shown structural evidence of cholesterol binding sites. Against this backdrop, we recently proposed a novel mechanism by which membrane cholesterol could affect structure and function of GPCRs. According to our hypothesis, cholesterol binding sites in GPCRs could represent 'nonannular' binding sites. Interestingly, previous work from our laboratory has demonstrated that membrane cholesterol is required for the function of the serotonin(1A) receptor (a representative GPCR), which could be due to specific interaction of the receptor with cholesterol. Based on these results, we envisage that there could be specific/nonannular cholesterol binding site(s) in the serotonin(1A) receptor. We have analyzed putative cholesterol binding sites from protein databases in the serotonin(1A) receptor. Our analysis shows that cholesterol binding sites are inherent characteristic features of serotonin(1A) receptors and are conserved through natural evolution. Progress in deciphering molecular details of the GPCR-cholesterol interaction in the membrane would lead to better insight into our overall understanding of GPCR function in health and disease, thereby enhancing our ability to design better therapeutic strategies to combat diseases related to malfunctioning of GPCRs.

Differential role of galanin receptors in the regulation of depression-like behavior and monoamine/stress-related genes at the cell body level.

The present study on rat examined the role of galanin receptor subtypes in regulation of depression-like behavior as well as potential molecular mechanisms involved in the locus coeruleus (LC) and dorsal raphe (DR). The effect of intracerebroventricular (i.c.v.) infusion of galanin or galanin receptor GalR1- and GalR2-selective ligands was studied in the forced swim test, followed by quantitative in situ hybridization studies. Naive control, non-treated (swim control), saline- and fluoxetine-treated rats were used as controls in the behavioral and in situ hybridization studies. Subchronic treatment with fluoxetine reduced immobility and climbing time. Intracerebroventricular infusion of galanin, the GalR1 agonist M617 or the GalR2 antagonist M871 increased, while the GalR2(R3) agonist AR-M1896 decreased, immobility time compared to the aCSF-treated animals. Galanin also decreased the time of climbing. Galanin mRNA levels were upregulated by the combination of injection+swim stress in the saline- and the fluoxetine-treated groups in the LC, but not in the DR. Also tyrosine hydroxylase levels in the LC were increased following injection+swim stress in the saline- and fluoxetine-treated rats. Tryptophan hydroxylase 2 and serotonin transporter mRNAs were not significantly affected by any treatment. 5-HT(1A) mRNA levels were downregulated following i.c.v. galanin, M617 or AR-M1896 infusion. These results indicate a differential role of galanin receptor subtypes in depression-like behavior in rodents: GalR1 subtype may mediate 'prodepressive' and GalR2 'antidepressant' effects of galanin. Galanin has a role in behavioral adaptation to stressful events involving changes of molecules important for noradrenaline and/or serotonin transmission.

Selective stimulation of GalR1 and GalR2 in rat substantia gelatinosa reveals a cellular basis for the anti- and pro-nociceptive actions of galanin.

Galanin modulates spinal nociceptive processing by interacting with two receptors, GalR1 and GalR2. The underlying neurophysiological mechanisms were examined by whole-cell recording from identified neurons in the substantia gelatinosa of young adult rats. GalR1 was activated with a 'cocktail' containing the GalR1/2 agonist, AR-M 961 (0.5 microM), in the presence of the GalR2 antagonist, M871 (1.0-2.5 microM). GalR2 was activated with the selective agonist, AR-M 1896 (0.5-1.0 microM). Application of the 'GalR1 agonist cocktail' often activated an inwardly-rectifying conductance in delay firing (excitatory) and tonically firing (inhibitory) neurons. This conductance was not activated by AR-M 1896 which instead decreased or increased an outwardly-rectifying conductance at voltages positive to -70 mV. Despite this variability in its actions on current-voltage relationships, AR-M 1896 very consistently decreased membrane excitability, as measured by cumulative action potential latency in response to a depolarizing current ramp. This strong GalR2-mediated effect was seen in neurons where membrane conductance was decreased, and where membrane excitability might be predicted to increase. GalR2 was also located presynaptically, as AR-M 1896 increased the interevent interval of spontaneous EPSCs in both delay and tonic cells. By contrast, the 'GalR1 agonist cocktail' had little effect on spontaneous EPSCs, suggesting that presynaptic terminals do not express GalR1. These diverse actions of GalR1 and GalR2 activation on both inhibitory and excitatory neurons are discussed in relation to the known spinal antinociceptive and pro-nociceptive actions of galanin, to the possible association of GalR1 with the inhibitory G-protein, G(i/o) and to report that GalR2 activation suppresses Ca2+ channel currents.

The effects of galanin-like peptide on energy balance, body temperature and brain activity in the mouse and rat are independent of the GALR2/3 receptor.

Galanin-like peptide (GALP) is a neuropeptide that is thought to act on the galanin receptors GALR1, GALR2 and GALR3. In rats, i.c.v. injection of GALP has dichotomous actions on energy balance, stimulating feeding over the first hour, but reducing food intake and body weight at 24 h, as well as causing an increase in core body temperature. In mice, GALP only induces an anorexic action, and its effects on core body temperature are unknown. One aim of the present study was to determine the effects of GALP on core body temperature in mice. Intracerebroventricular injection of GALP into conscious mice had no effect on feeding over 1 h, but caused a significant reduction in food intake and body weight at 24 h. It also caused an immediate drop in core body temperature, which was followed by an increase in body temperature. To understand these different effects of GALP on energy balance in mice compared to rats, and to determine the involvement of GALR2 and GALR3, immunohistochemistry was performed to localise c-Fos, a marker of cell activation. Intracerebroventricular injection of GALP induced c-Fos expression in the parenchyma surrounding the ventricles, the ventricular ependymal cells and the meninges in mice and rats. GALP also induced c-Fos expression in the supraoptic nucleus, dorsomedial hypothalamic nucleus, lateral hypothalamus and nucleus tractus solitarius in rats but not in mice. Central administration of a GALR2/3 agonist in rats did not induce c-Fos in any of the brain regions that expressed this protein after GALP injection, and had no effect on food intake, body weight and body temperature in rats or mice. These data suggest that GALP induces differential effects on energy balance and brain activity in mice compared to rats, which are unlikely to be due to activation of the GALR2 or GALR3 receptor.

Galanin receptor subtype 2 (GalR2) null mutant mice display an anxiogenic-like phenotype specific to the elevated plus-maze.

The neuropeptide galanin has been implicated in anxiety-related behaviors, cognition, analgesia, and feeding in rodents. Neuromodulatory actions of galanin are mediated by three G-protein coupled receptors, GalR1, GalR2, and GalR3. The present study investigates the role of the GalR2 receptor by evaluating behavioral phenotypes of mice with a targeted mutation in the GalR2 gene. A three-tiered behavioral phenotyping approach first examined control measures of general health, body weight, neurological reflexes, sensory abilities and motor function. Mice were then assessed on several tests for cognitive and anxiety-like behaviors. GalR2 null mutants and heterozygotes were not significantly different from wildtype littermates on two cognitive tests previously shown to be sensitive to galanin manipulation: acquisition of the Morris water maze spatial task, and trace cued and contextual fear conditioning, an emotional learning and memory task. Two independent cohorts of GalR2 null mutant mice demonstrated an anxiogenic-like phenotype in the elevated plus-maze. No genotype differences were detected on several other measures of anxiety-like behavior. The discovery of an anxiogenic phenotype specific to the elevated plus-maze, similar to findings in GalR1 null mutants, highlights the potential therapeutic efficacy of targeting GalR1 and GalR2 receptors in treating anxiety disorders.

Mice deficient for galanin receptor 2 have decreased neurite outgrowth from adult sensory neurons and impaired pain-like behaviour.

Expression of the neuropeptide galanin is markedly up-regulated within the adult dorsal root ganglia (DRG) following peripheral nerve injury. We have previously demonstrated that galanin knockout (Gal-KO) mice have a developmental loss of a subset of DRG neurons. Galanin also plays a trophic role in the adult animal, and the rate of peripheral nerve regeneration and neurite outgrowth is reduced in adult Gal-KO mice. Here we describe the characterization of mice with an absence of GalR2 gene transcription (GalR2-MUT) and demonstrate that they have a 15% decrease in the number of calcitonin gene-related peptide (CGRP) expressing neuronal profiles in the adult DRG, associated with marked deficits in neuropathic and inflammatory pain behaviours. Adult GalR2-MUT animals also have a one third reduction in neurite outgrowth from cultured DRG neurons that cannot be rescued by either galanin or a high-affinity GalR2/3 agonist. Galanin activates extracellular signal-regulated kinase (ERK) and Akt in adult wild-type (WT) mouse DRG. Intact adult DRG from GalR2-MUT animals have lower levels of pERK and higher levels of pAkt than are found in WT controls. These data suggest that a lack of GalR2 activation in Gal-KO and GalR2-MUT animals is responsible for the observed developmental deficits in the DRG, and the decrease in neurite outgrowth in the adult.

[Galanin increases the survival rate of hippocampal cells injured by H2O2 in vitro].

The method of primary hippocampal nerve cell culture was used to study the injury effect of H2O2 and the protective effect of galanin (GAL) and GAL receptor agonists. Result demonstrated that H2O2 has obvious dose relative toxicity to hippocampal cells in vitro. GAL and GAL's nonselective agonist GAL1-11, GalR2's selective agonist GAL2-11 can increase the survival rate of hippocampal cells suffered form H2O2. All the protective effects can be blocked by nonselective antagonist M35. The result indicates that GAL can protect hippocampal cells from oxidative injury in vitro, which is most probably mediated by GalR2.

Regulation of kindling epileptogenesis by hippocampal galanin type 1 and type 2 receptors: The effects of subtype-selective agonists and the role of G-protein-mediated signaling.

The search for antiepileptic drugs that are capable of blocking the progression of epilepsy (epileptogenesis) is an important problem of translational epilepsy research. The neuropeptide galanin effectively suppresses acute seizures. We examined the ability of hippocampal galanin receptor type 1 (GalR1) and type 2 (GalR2) to inhibit kindling epileptogenesis and studied signaling cascades that mediate their effects. Wistar rats received 24-h-long intrahippocampal infusion of a GalR1/2 agonist galanin(1-29), GalR1 agonist M617 [galanin(1-13)-Gln14-bradykinin(2-9)-amide], or GalR2 agonist galanin(2-11). The peptides were administered alone or combined with an inhibitor of Gi protein pertussis toxin (PTX), Gi-protein activated K+ channels (GIRK) inhibitor tertiapin Q (TPQ), G(q/11) protein inhibitor [D-Arg1,D-Trp(5,7,9),Leu11]-substance P (dSP), or an inhibitor of intracellular Ca2+ release dantrolene. Sixteen hours into drug delivery, the animals were subjected to rapid kindling-60 electrical trains administered to ventral hippocampus every 5 min. M617 delayed epileptogenesis, whereas galanin(1-29) and galanin(2-11) completely prevented the occurrence of full kindled seizures. TPQ abolished anticonvulsant effect of M617 but not of galanin(2-11). PTX blocked anticonvulsant effects of M617 and inversed the action of galanin(1-29) and galanin(2-11) to proconvulsant. dSP and dantrolene did not modify seizure suppression through GalR1 and GalR2, but eliminated the proconvulsant effect of PTX + galanin(1-29) and PTX + galanin(2-11) combinations. We conclude that hippocampal GalR1 exert their disease-modifying effect through the Gi-GIRK pathway. GalR2 is antiepileptogenic through the Gi mechanism independent of GIRK. A secondary proconvulsant pathway coupled to GalR2 involves G(q/11) and intracellular Ca2+. The data are important for understanding endogenous mechanisms regulating epileptogenesis and for the development of novel antiepileptogenic drugs.

Regulation of limbic status epilepticus by hippocampal galanin type 1 and type 2 receptors.

It has been well established that galanin is a potent endogenous anticonvulsant peptide. However, the role of galanin receptor subtypes in mediating anticonvulsant effects of the peptide is poorly understood. Using pharmacological, transgenic and antisense approaches, we examined the involvement of galanin receptors GalR1 and GalR2 in regulating seizures and associated neuronal degenerative changes. In the rat model of status epilepticus (SE) induced by electrical stimulation of perforant path, in vivo uncoupling of G protein coupled receptors (GPCR) through intrahippocampal administration of pertussis toxin (PTX) facilitated the initiation of SE, and increased the severity of the established SE. Injection of a non-selective GalR1/GalR2 agonist galanin (1-29) and a preferential GalR2 agonist galanin (2-11) into the hippocampus of PTX-pretreated rats revealed that while during early phase of SE galanin inhibited seizures predominantly through GalR1, GalR2 mediated anticonvulsant effects of the peptide during advanced stage of SE. GalR1 knockout mice showed increased severity of both pilocarpine- and perforant path stimulation -induced SE, compared to wild type (WT) littermates. In GalR1 knockout animals SE led to more severe and wider-spread hippocampal injury, than in WT. Focal downregulation of GalR2, which had been achieved in rats by intrahippocampal infusion of anti-GalR2 peptide nucleic acid (PNA) antisense, significantly increased the severity of perforant path stimulation- induced SE. Downregulation of GalR2 led to mild injury to hilar interneurons and potentiated seizure-induced hippocampal damage. In conclusion, both GalR1 and GalR2 mediate anticonvulsant effects of galanin. GalR1 and GalR2 exhibit differential effects on the initiation and the maintenance phases of SE. Activation of both galanin receptor subtypes exerts neuroprotective effects under conditions of excitotoxic injury.

Role of galanin receptor 1 and galanin receptor 2 activation in synaptic plasticity associated with 3',5'-cyclic AMP response element-binding protein phosphorylation in the dentate gyrus: studies with a galanin receptor 2 agonist and galanin receptor 1 knockout mice.

The neuropeptide galanin was shown to impair cognitive performance and reduce hippocampal CA1 long-term potentiation (LTP) in rodents. However, the contribution of the two main galanin receptors; GalR1 and GalR2, present in the hippocampus to these effects is not known. In the present study, we determined the protein expression levels of GalR1 and GalR2 in the mouse dentate gyrus (DG) and used galanin (2-11), a recently introduced GalR2 agonist, and GalR1 knockout mice to examine the contribution of GalR1 and GalR2 to the modulation of LTP and 3',5'-cyclic AMP response element-binding protein (CREB)-dependent signaling cascades. In the DG, 57+/-5% of the galanin binding sites were GalR2, and the remaining population corresponded to GalR1. In hippocampal slices, galanin (2-11) fully blocked the induction of DG LTP, whereas galanin (1-29), a high affinity agonist for both GalR1 and GalR2, strongly but not fully attenuated the late phase of LTP by 80+/-1.5%. Application of galanin (1-29) or galanin (2-11) after LTP induction caused a transient reduction in the maintenance phase of LTP, with the larger effect displayed by superfusion of galanin (2-11). The induction and maintenance of DG LTP was not altered in the GalR1 knockout mice. Superfusion of galanin (1-29) or galanin (2-11) blocked the LTP induction to the same degree indicating a role for GalR2 in the induction phase of DG LTP. Furthermore, we analyzed the effects of GalR1 and/or GalR2 activation on DG LTP-induced CREB phosphorylation, associated with the late transcriptional effects of LTP. In the lateral part of the granule cell layer, high-frequency trains stimulation caused a significant increase in the level of CREB phosphorylation, which was significantly reduced by application of either galanin (1-29) or galanin (2-11), indicating that both GalR1 and/or GalR2 can mediate some of their effects on LTP through inhibition of CREB-related signaling cascades.

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.

Galanin type 2 receptors regulate neuronal survival, susceptibility to seizures and seizure-induced neurogenesis in the dentate gyrus.

The neuropeptide galanin has been implicated in inhibiting seizures and protecting hippocampal neurons from excitotoxic injury. In the hippocampus galanin acts through two receptor subtypes, GalR1, expressed in CA1, and GalR2, abundant in dentate gyrus. We developed an approach to induce and to study selective semichronic knockdown of GalR2 in the rat hippocampus. A 50% reduction of GalR2 binding was achieved by continuous infusion of complementary peptide nucleic acid antisense oligonucleotide into the dentate gyrus. This resulted in an increase in the severity of self-sustaining status epilepticus induced by electrical stimulation of the perforant path, induced mild neuronal injury in the dentate hilus, augmented seizure-induced hilar injury and inhibited seizure-induced neurogenesis in the subgranular zone of the dentate gyrus. Our data suggest that in the dentate gyrus, galanin, acting through GalR2, inhibits seizures, promotes viability of hilar interneurons and stimulates seizure-induced neurogenesis. These results are important for understanding the role of galanin and galanin receptor subtypes in the hippocampus both under normal conditions and in excitotoxic injury.

Galanin receptor subtype GalR2 mediates apoptosis in SH-SY5Y neuroblastoma cells.

Recently we have shown that galanin binding significantly correlates with survival in neuroblastoma patients, indicating a possible modulatory role of galanin receptors in neuroblastic tumor biology. However, the molecular mechanisms beyond this correlation have not been elucidated. Here, the cellular effects on activation of specific galanin receptor subtypes in human SH-SY5Y neuroblastoma cells were analyzed using a tetracycline-controlled expression system. Pharmacological studies confirmed the inducible expression of high affinity binding sites for galanin in SH-SY5Y cells transfected with the galanin receptors GalR1 (SY5Y/GalR1) and GalR2 (SY5Y/GalR2). Microphysiometry revealed that both receptor subtypes were able to mediate an intracellular signal upon galanin application. Interestingly, induction of receptor expression and treatment with 100 nm galanin resulted in a dramatic decrease in cell viability in SY5Y/GalR2 cells (93 +/- 3%) compared with a less pronounced effect in SY5Y/GalR1 cells (19 +/- 10%). The antiproliferative potency of galanin was 100-fold higher in SY5Y/GalR2 (50% effective concentration, 1.1 nm) than in SY5Y/GalR1 cells (50% effective concentration, 190 nm). Furthermore, activation of receptor expression and exposure to galanin resulted in apparent morphological changes indicative of apoptosis in SY5Y/GalR2 cells only. Induction of cell death by the apoptotic process was confirmed by poly-(ADP-ribose)-polymerase cleavage, caspase-3 activation, and the typical laddering of DNA. This study indicates that a high level of GalR2 expression is able to inhibit cell proliferation and induce apoptosis in neuroblastoma cells and therefore identifies GalR2 as a possible target for pharmacological intervention in neuroblastoma.