RESUMEN
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.
Asunto(s)
Ansiedad/inducido químicamente , Depresión/inducido químicamente , Galanina/química , Galanina/toxicidad , Animales , Línea Celular Transformada , Adaptación a la Oscuridad/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Conducta Exploratoria/efectos de los fármacos , Galanina/antagonistas & inhibidores , Regulación de la Expresión Génica/efectos de los fármacos , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Masculino , Fragmentos de Péptidos/toxicidad , Núcleos del Rafe/efectos de los fármacos , Núcleos del Rafe/metabolismo , Ratas , Ratas Sprague-Dawley , Ratas Transgénicas , Receptor de Galanina Tipo 1/deficiencia , Receptor de Galanina Tipo 1/genética , Receptor de Galanina Tipo 2/deficiencia , Receptor de Galanina Tipo 2/genética , Serotonina/metabolismo , Factores de TiempoRESUMEN
It is established that the gut peptide galanin reduces neuronal excitability via galanin receptor subtypes GALR1 and GALR3 and increases excitability via subtype GALR2. We have previously shown that galanin potently reduces mechanosensitivity in the majority of gastro-oesophageal vagal afferents, and potentiates sensitivity in a minority. These actions may have implications for therapeutic inhibition of gut afferent signalling. Here we investigated which galanin receptors are likely to mediate these effects. We performed quantitative RT-PCR on RNA from vagal (nodose) sensory ganglia, which indicated that all three GALR subtypes were expressed at similar levels. The responses of mouse gastro-oesophageal vagal afferents to graded mechanical stimuli were investigated before and during application of galanin receptor ligands to their peripheral endings. Two types of vagal afferents were tested: tension receptors, which respond to circumferential tension, and mucosal receptors which respond only to mucosal stroking. Galanin induced potent inhibition of mechanosensitivity in both types of afferents. This effect was totally lost in mice with targeted deletion of Galr1. The GALR1/2 agonist AR-M961 caused inhibition of mechanosensitivity in Galr1+/+ mice, but this was reversed to potentiation in Galr1-/- mice, indicating a minor role for GALR2 in potentiation of vagal afferents. We observed no functional evidence of GALR3 involvement, despite its expression in nodose ganglia. The current study highlights the complex actions of galanin at different receptor subtypes exhibiting parallels with the function of galanin in other systems.
Asunto(s)
Esófago/inervación , Galanina/metabolismo , Mecanotransducción Celular , Neuronas Aferentes/metabolismo , Receptor de Galanina Tipo 1/metabolismo , Receptor de Galanina Tipo 2/metabolismo , Estómago/inervación , Nervio Vago/metabolismo , Animales , Galanina/farmacología , Indoles/farmacología , Mecanotransducción Celular/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas Aferentes/efectos de los fármacos , Ganglio Nudoso/metabolismo , Fragmentos de Péptidos/farmacología , ARN Mensajero/metabolismo , Receptor de Galanina Tipo 1/agonistas , Receptor de Galanina Tipo 1/deficiencia , Receptor de Galanina Tipo 1/genética , Receptor de Galanina Tipo 2/agonistas , Receptor de Galanina Tipo 2/genética , Receptor de Galanina Tipo 3/antagonistas & inhibidores , Receptor de Galanina Tipo 3/metabolismo , Estrés Mecánico , Nervio Vago/citología , Nervio Vago/efectos de los fármacosRESUMEN
The neuropeptide galanin suppresses seizure activity in the hippocampus by inhibiting glutamatergic neurotransmission. Galanin may also modulate limbic seizures through interaction with other neurotransmitters in neuronal populations that project to the hippocampus. We examined the role of galanin receptors types 1 and 2 in the dorsal raphe (DR) in the regulation of serotonergic transmission and limbic seizures. Infusion of a mixed agonist of galanin receptors types 1 and 2 [galanin (1-29)] into the DR augmented the severity of limbic seizures in both rats and wild-type mice and concurrently reduced serotonin concentration in the DR and hippocampus as measured by immunofluorescence or HPLC. In contrast, injection of the galanin receptor type 2 agonist galanin (2-11) mitigated the severity of seizures in both species and increased serotonin concentration in both areas. Injection of both galanin fragments into the DR of galanin receptor type 1 knockout mice exerted anticonvulsant effects. Both the proconvulsant activity of galanin (1-29) and seizure suppression by galanin (2-11) were abolished in serotonin-depleted animals. Our data indicate that, in the DR, galanin receptors types 1 and 2 modulate serotonergic transmission in a negative and a positive fashion, respectively, and that these effects translate into either facilitation or inhibition of limbic seizures.
Asunto(s)
Núcleos del Rafe/metabolismo , Receptor de Galanina Tipo 1/metabolismo , Receptor de Galanina Tipo 2/metabolismo , Convulsiones/metabolismo , Serotonina/metabolismo , Animales , Recuento de Células/métodos , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Estimulación Eléctrica/efectos adversos , Electroencefalografía/métodos , Galanina/farmacología , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Hipocampo/efectos de la radiación , Inmunohistoquímica/métodos , Masculino , Ratones , Ratones Noqueados , Norepinefrina/metabolismo , Fragmentos de Péptidos/farmacología , Núcleos del Rafe/efectos de los fármacos , Ratas , Ratas Wistar , Receptor de Galanina Tipo 1/agonistas , Receptor de Galanina Tipo 1/deficiencia , Receptor de Galanina Tipo 2/agonistas , Convulsiones/etiología , Serotoninérgicos/farmacología , p-Cloroanfetamina/farmacologíaRESUMEN
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.
Asunto(s)
Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Giro Dentado/metabolismo , Potenciación a Largo Plazo/fisiología , Receptor de Galanina Tipo 1/deficiencia , Receptor de Galanina Tipo 1/fisiología , Receptor de Galanina Tipo 2/fisiología , Animales , Estimulación Eléctrica/métodos , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Potenciales Postsinápticos Excitadores/efectos de la radiación , Técnica del Anticuerpo Fluorescente/métodos , Galanina/química , Galanina/farmacocinética , Galanina/farmacología , Técnicas In Vitro , Isótopos de Yodo/farmacocinética , Potenciación a Largo Plazo/efectos de los fármacos , Potenciación a Largo Plazo/efectos de la radiación , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fragmentos de Péptidos/farmacología , Fosforilación , Unión Proteica , Receptor de Galanina Tipo 2/agonistas , Factores de TiempoRESUMEN
Widespread production of knockout and transgenic mice has led to an increased use of mice as animal models for studies of normal- and patho-physiology. Hence, the precise mapping of central transmitter/peptide systems in the mouse has become essential for the interpretation of functional studies and for the correct correlation with findings obtained in the rat, primates and/or human. In this regard, the current study reports the autoradiographic localization of [(125)I]-galanin (GAL) binding sites in brain of the common C57BL/6J and 129OlaHsd mouse strains, as well as in GAL and galanin receptor-1 (GalR1) knockout (KO) mice. In C57BL/6J and 129OlaHsd mice, [(125)I]-GAL binding sites were detected throughout the brain, including moderate-high relative densities in the basal ganglia (caudate putamen, nucleus [n.] accumbens, olfactory tubercle, substantia nigra), limbic regions (septum, bed n. stria terminalis, ventral hippocampus, amygdala), cingulate, retrosplenial, entorhinal cortex, centro-lateral/medial thalamic n., preoptic/lateral hypothalamus, midbrain (superior colliculus, periaqueductal gray), pons/medulla oblongata (parabrachial, pontine reticular and solitary tract n.) and cerebellar cortex. [(125)I]-GAL binding levels were low or absent in main olfactory bulb, neocortex, ventrolateral/geniculate thalamic n., dorsal hippocampus, inferior colliculus and cranial motor n. In simultaneous determinations, relative [(125)I]-GAL binding site densities in brain were generally lower in C57BL/6J than in 129OlaHsd mice, while the density and distribution of central binding in the GAL-KO mouse was essentially identical to that in its background-129OlaHsd strain. In contrast, no specific [(125)I]-GAL binding was detected in any region of GalR1-KO mouse brain, revealing that under the experimental conditions used, the peptide ligand binding is predominantly (exclusively) to the GalR1 subtype. This evaluation of GAL 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 GAL transmission.
Asunto(s)
Encéfalo/metabolismo , Galanina/genética , Galanina/metabolismo , Receptor de Galanina Tipo 1/genética , Receptor de Galanina Tipo 1/metabolismo , Animales , Femenino , Galanina/deficiencia , Radioisótopos de Yodo/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Unión Proteica/genética , Receptor de Galanina Tipo 1/deficiencia , Especificidad de la EspecieRESUMEN
The neuropeptide galanin exhibits anticonvulsant effects in experimental epilepsy. Two galanin receptor subtypes, GalR1 and GalR2, are present in the brain. We examined the role of GalR1 in seizures by studying the susceptibility of GalR1 knockout (KO) mice to status epilepticus (SE) and accompanying neuronal injury. SE was induced in GalR1 KO and wild type (WT) mice by Li-pilocarpine, 60 min electrical perforant path stimulation (PPS), or systemic kainic acid (KA). Seizures were analyzed using Harmonie software. Cell injury was examined by FluoroJade B- and terminal deoxynucleotidyl transferase-mediated uridine triphosphate nick end labeling; neurogenesis was studied using bromodeoxyuridine labeling. Compared with WT littermates, GalR1 KO showed more severe seizures, more profound injury to the CA1 pyramidal cell layer, as well as injury to hilar interneurons and dentate granule cells upon Li-pilocarpine administration. PPS led to more severe seizures in KO, as compared with WT mice. No difference in the extent of neuronal degeneration was observed between the mice of two genotypes in CA1 pyramidal cell layer; however, in contrast to WT, GalR1 KO developed mild injury to hilar interneurons on the side of PPS. KA-induced seizures did not differ between GalR1 KO and WT animals, and led to no injury to the hippocampus in either of experimental group. No differences were found between KO and WT mice in both basal and seizure-induced neuronal progenitor proliferation in all seizure types. Li-pilocarpine led to more extensive glia proliferation in GalR1 KO than in WT, and in both mouse types in two other SE models. In conclusion, GalR1 mediate galanin protection from seizures and seizure-induced hippocampal injury in Li-pilocarpine and PPS models of limbic SE, but not under conditions of KA-induced seizures. The results justify the development and use of GalR1 agonists in the treatment of certain forms of epilepsy.
Asunto(s)
Hipocampo/patología , Hipocampo/fisiopatología , Receptor de Galanina Tipo 1/deficiencia , Estado Epiléptico/patología , Estado Epiléptico/fisiopatología , Animales , Giro Dentado/fisiopatología , Combinación de Medicamentos , Estimulación Eléctrica , Ácido Kaínico , Cloruro de Litio , Ratones , Ratones Noqueados , Neuronas/patología , Vía Perforante , Pilocarpina , Estado Epiléptico/inducido químicamente , Estado Epiléptico/etiología , Células Madre/patologíaRESUMEN
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.
Asunto(s)
Ingestión de Alimentos/fisiología , Péptido Similar a Galanina/fisiología , Hormona Luteinizante/sangre , Receptor de Galanina Tipo 1/metabolismo , Receptor de Galanina Tipo 2/metabolismo , Análisis de Varianza , Animales , Núcleo Arqueado del Hipotálamo/metabolismo , Peso Corporal/fisiología , Péptido Similar a Galanina/administración & dosificación , Inyecciones Intraventriculares , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fragmentos de Péptidos , Receptor de Galanina Tipo 1/deficiencia , Receptor de Galanina Tipo 2/deficienciaRESUMEN
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.
Asunto(s)
Memoria/fisiología , Receptor de Galanina Tipo 1/deficiencia , Animales , Condicionamiento Psicológico/fisiología , Femenino , Aprendizaje/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Desempeño Psicomotor/fisiología , Receptor de Galanina Tipo 1/genéticaRESUMEN
(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.