RESUMEN
Functional magnetic resonance imaging (fMRI) of learned behaviour in 'awake rodents' provides the opportunity for translational preclinical studies into the influence of pharmacological and genetic manipulations on brain function. fMRI has recently been employed to investigate learned behaviour in awake rats. Here, this methodology is translated to mice, so that future fMRI studies may exploit the vast number of genetically modified mouse lines that are available. One group of mice was conditioned to associate a flashing light (conditioned stimulus, CS) with foot shock (PG; paired group), and another group of mice received foot shock and flashing light explicitly unpaired (UG; unpaired group). The blood oxygen level-dependent signal (proxy for neuronal activation) in response to the CS was measured 24 h later in awake mice from the PG and UG using fMRI. The amygdala, implicated in fear processing, was activated to a greater degree in the PG than in the UG in response to the CS. Additionally, the nucleus accumbens was activated in the UG in response to the CS. Because the CS signalled an absence of foot shock in the UG, it is possible that this region is involved in processing the safety aspect of the CS. To conclude, the first use of fMRI to visualise brain activation in awake mice that are completing a learned emotional task is reported. This work paves the way for future preclinical fMRI studies to investigate genetic and environmental influences on brain function in transgenic mouse models of disease and aging.
Asunto(s)
Aprendizaje por Asociación/fisiología , Encéfalo/fisiología , Condicionamiento Psicológico/fisiología , Miedo/fisiología , Imagen por Resonancia Magnética/métodos , Animales , Mapeo Encefálico , Circulación Cerebrovascular/fisiología , Electrochoque , Estudios de Factibilidad , Pie , Masculino , Ratones Endogámicos C57BL , Movimiento (Física) , Vías Nerviosas/fisiología , Oxígeno/sangre , Estimulación Luminosa , Procesamiento de Señales Asistido por Computador , Percepción Visual/fisiología , VigiliaRESUMEN
Transcriptional codes initiated during brain development are ultimately realized in adulthood as distinct cell types performing specialized roles in behavior. Focusing on the mouse external globus pallidus (GPe), we demonstrate that the potential contributions of two GABAergic GPe cell types to voluntary action are fated from early life to be distinct. Prototypic GPe neurons derive from the medial ganglionic eminence of the embryonic subpallium and express the transcription factor Nkx2-1. These neurons fire at high rates during alert rest, and encode movements through heterogeneous firing rate changes, with many neurons decreasing their activity. In contrast, arkypallidal GPe neurons originate from lateral/caudal ganglionic eminences, express the transcription factor FoxP2, fire at low rates during rest, and encode movements with robust increases in firing. We conclude that developmental diversity positions prototypic and arkypallidal neurons to fulfil distinct roles in behavior via their disparate regulation of GABA release onto different basal ganglia targets.
Asunto(s)
Factores de Transcripción Forkhead/metabolismo , Globo Pálido/citología , Globo Pálido/crecimiento & desarrollo , Movimiento/fisiología , Neuronas/clasificación , Neuronas/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Potenciales de Acción/fisiología , Animales , Linaje de la Célula/fisiología , Encefalinas/metabolismo , Globo Pálido/embriología , Ratones , Precursores de Proteínas/metabolismo , Curva ROC , Factor Nuclear Tiroideo 1 , Ácido gamma-Aminobutírico/metabolismoRESUMEN
The N-methyl-D-aspartate (NMDA) receptor plays an essential role in excitatory transmission, synaptic integration, and learning and memory. In the classical view, postsynaptic NMDA receptors act as canonical coincidence detectors providing a 'molecular switch' for the induction of various forms of short- and long-term synaptic plasticity. Over the past twenty years there has been accumulating evidence to suggest that NMDA receptors are also expressed presynaptically and are involved in the regulation of synaptic transmission and specific forms of activity-dependent plasticity in developing neural circuits. However, the existence of presynaptic NMDA receptors remains a contentious issue. In this review, I will discuss the criteria required for identifying functional presynaptic receptors, novel methods for probing NMDA receptor function, and recent evidence to suggest that NMDA receptors are expressed at presynaptic sites in a target-specific manner.
Asunto(s)
Dendritas/metabolismo , Neuronas/citología , Terminales Presinápticos/metabolismo , Receptores de N-Metil-D-Aspartato/fisiología , AnimalesRESUMEN
Inferior olive neurons regulate plasticity and timing in the cerebellar cortex via the climbing fiber pathway, but direct characterization of the output of this nucleus has remained elusive. We show that single somatic action potentials in olivary neurons are translated into a burst of axonal spikes. The number of spikes in the burst depends on the phase of subthreshold oscillations and, therefore, encodes the state of the olivary network. These bursts can be successfully transmitted to the cerebellar cortex in vivo, having a significant impact on Purkinje cells. They enhance dendritic spikes, modulate the complex spike pattern, and promote short-term and long-term plasticity at parallel fiber synapses in a manner dependent on the number of spikes in the burst. Our results challenge the view that the climbing fiber conveys an all-or-none signal to the cerebellar cortex and help to link learning and timing theories of olivocerebellar function.
Asunto(s)
Potenciales de Acción/fisiología , Relojes Biológicos/fisiología , Vías Nerviosas/fisiología , Núcleo Olivar/fisiología , Células de Purkinje/fisiología , Animales , Axones/fisiología , Comunicación Celular/fisiología , Vías Nerviosas/citología , Plasticidad Neuronal/fisiología , Núcleo Olivar/citología , Periodicidad , Ratas , Ratas Sprague-Dawley , Umbral Sensorial/fisiología , Transducción de Señal/fisiologíaRESUMEN
In the cerebellum, the process of retrograde signaling via presynaptic receptors is important for the induction of short- and long-term changes in inhibitory synaptic transmission at interneuron-Purkinje cell (PC) synapses. Endocannabinoids, by activating presynaptic CB1 receptors, mediate a short-term decrease in inhibitory synaptic efficacy, whereas glutamate, acting on presynaptic NMDA receptors, induces a longer-latency sustained increase in GABA release. We now demonstrate that either low-frequency climbing fiber stimulation or direct somatic depolarization of Purkinje cells results in SNARE-dependent vesicular release of glutamate from the soma and dendrites of PCs. The activity-dependent release of glutamate caused the activation of postsynaptic metabotropic glutamate receptor 1 (mGluR1) on PC somatodendritic membranes, resulting in the cooperative release of endocannabinoids and an mGluR1-mediated slow membrane conductance. The activity of excitatory amino acid transporters regulated the spatial spread of glutamate and thus the extent of PC mGluR1 activation. We propose that activity-dependent somatodendritic glutamate release and autocrine activation of mGluR1 on PCs provides a powerful homeostatic mechanism to dynamically regulate inhibitory synaptic transmission in the cerebellar cortex.
Asunto(s)
Comunicación Autocrina/fisiología , Dendritas/metabolismo , Ácido Glutámico/metabolismo , Inhibición Neural/fisiología , Células de Purkinje/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Vías Aferentes/fisiología , Animales , Animales Recién Nacidos , Comunicación Autocrina/efectos de los fármacos , Moduladores de Receptores de Cannabinoides/metabolismo , Cerebelo/citología , Cerebelo/efectos de los fármacos , Cerebelo/fisiología , Dendritas/efectos de los fármacos , Estimulación Eléctrica , Homeostasis/efectos de los fármacos , Homeostasis/fisiología , Inhibición Neural/efectos de los fármacos , Técnicas de Cultivo de Órganos , Células de Purkinje/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Receptores de Glutamato Metabotrópico/efectos de los fármacos , Proteínas SNARE/metabolismo , Membranas Sinápticas/metabolismo , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/fisiología , Vesículas Sinápticas/metabolismoRESUMEN
Ionotropic gamma-amino butyric acid (GABA) receptors composed of heterogeneous molecular subunits are major mediators of inhibitory responses in the adult CNS. Here, we describe a novel ionotropic GABA receptor in mouse cerebellar Purkinje cells (PCs) using agents reported to have increased affinity for rho subunit-containing GABA(C) over other GABA receptors. Exogenous application of the GABA(C)-preferring agonist cis-4-aminocrotonic acid (CACA) evoked whole-cell currents in PCs, whilst equimolar concentrations of GABA evoked larger currents. CACA-evoked currents had a greater sensitivity to the selective GABA(C) antagonist (1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA) than GABA-evoked currents. Focal application of agonists produced a differential response profile; CACA-evoked currents displayed a much more pronounced attenuation with increasing distance from the PC soma, displayed a slower time-to-peak and exhibited less desensitization than GABA-evoked currents. However, CACA-evoked currents were also completely blocked by bicuculline, a selective agent for GABA(A) receptors. Thus, we describe a population of ionotropic GABA receptors with a mixed GABA(A)/GABA(C) pharmacology. TPMPA reduced inhibitory synaptic transmission at interneurone-Purkinje cell (IN-PC) synapses, causing clear reductions in miniature inhibitory postsynaptic current (mIPSC) amplitude and frequency. Combined application of NO-711 (a selective GABA transporter subtype 1 (GAT-1) antagonist) and SNAP-5114 (a GAT-(2)/3/4 antagonist) induced a tonic GABA conductance in PCs; however, TPMPA had no effect on this current. Immunohistochemical studies suggest that rho subunits are expressed predominantly in PC soma and proximal dendritic compartments with a lower level of expression in more distal dendrites; this selective immunoreactivity contrasted with a more uniform distribution of GABA(A) alpha1 subunits in PCs. Finally, co-immunoprecipitation studies suggest that rho subunits can form complexes with GABA(A) receptor alpha1 subunits in the cerebellar cortex. Overall, these data suggest that rho subunits contribute to functional ionotropic receptors that mediate a component of phasic inhibitory GABAergic transmission at IN-PC synapses in the cerebellum.
Asunto(s)
Inhibición Neural/fisiología , Células de Purkinje/metabolismo , Receptores de GABA/metabolismo , Transmisión Sináptica/fisiología , Ácido gamma-Aminobutírico/metabolismo , Animales , Células Cultivadas , Cerebelo/metabolismo , Medicina Basada en la Evidencia , Masculino , Ratones , Subunidades de Proteína/metabolismoRESUMEN
Hyperekplexia is a human neurological disorder characterized by an excessive startle response and is typically caused by missense and nonsense mutations in the gene encoding the inhibitory glycine receptor (GlyR) alpha1 subunit (GLRA1). Genetic heterogeneity has been confirmed in rare sporadic cases, with mutations affecting other postsynaptic glycinergic proteins including the GlyR beta subunit (GLRB), gephyrin (GPHN) and RhoGEF collybistin (ARHGEF9). However, many individuals diagnosed with sporadic hyperekplexia do not carry mutations in these genes. Here we show that missense, nonsense and frameshift mutations in SLC6A5 (ref. 8), encoding the presynaptic glycine transporter 2 (GlyT2), also cause hyperekplexia. Individuals with mutations in SLC6A5 present with hypertonia, an exaggerated startle response to tactile or acoustic stimuli, and life-threatening neonatal apnea episodes. SLC6A5 mutations result in defective subcellular GlyT2 localization, decreased glycine uptake or both, with selected mutations affecting predicted glycine and Na+ binding sites.
Asunto(s)
Proteínas de Transporte de Glicina en la Membrana Plasmática/genética , Mutación , Reflejo de Sobresalto/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Línea Celular , Femenino , Proteínas de Transporte de Glicina en la Membrana Plasmática/química , Proteínas de Transporte de Glicina en la Membrana Plasmática/fisiología , Humanos , Técnicas In Vitro , Recién Nacido , Masculino , Modelos Moleculares , Datos de Secuencia Molecular , Oocitos/metabolismo , Terminales Presinápticos/fisiología , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Reflejo de Sobresalto/fisiología , Transfección , Xenopus laevisRESUMEN
Glycine receptors (GlyRs) and specific subtypes of GABA(A) receptors are clustered at synapses by the multidomain protein gephyrin, which in turn is translocated to the cell membrane by the GDP-GTP exchange factor collybistin. We report the characterization of several new variants of collybistin, which are created by alternative splicing of exons encoding an N-terminal src homology 3 (SH3) domain and three alternate C termini (CB1, CB2, and CB3). The presence of the SH3 domain negatively regulates the ability of collybistin to translocate gephyrin to submembrane microaggregates in transfected mammalian cells. Because the majority of native collybistin isoforms appear to harbor the SH3 domain, this suggests that collybistin activity may be regulated by protein-protein interactions at the SH3 domain. We localized the binding sites for collybistin and the GlyR beta subunit to the C-terminal MoeA homology domain of gephyrin and show that multimerization of this domain is required for collybistin-gephyrin and GlyR-gephyrin interactions. We also demonstrate that gephyrin clustering in recombinant systems and cultured neurons requires both collybistin-gephyrin interactions and an intact collybistin pleckstrin homology domain. The vital importance of collybistin for inhibitory synaptogenesis is underlined by the discovery of a mutation (G55A) in exon 2 of the human collybistin gene (ARHGEF9) in a patient with clinical symptoms of both hyperekplexia and epilepsy. The clinical manifestation of this collybistin missense mutation may result, at least in part, from mislocalization of gephyrin and a major GABA(A) receptor subtype.
Asunto(s)
Proteínas Portadoras/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas de la Membrana/metabolismo , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Encéfalo/citología , Proteínas Portadoras/genética , Células Cultivadas , Epilepsia/complicaciones , Epilepsia/genética , Exones , Femenino , Factores de Intercambio de Guanina Nucleótido/genética , Humanos , Masculino , Proteínas de la Membrana/genética , Ratones , Datos de Secuencia Molecular , Mutación , Neuronas/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estructura Terciaria de Proteína , Transporte de Proteínas , Ratas , Receptores de Glicina/metabolismo , Reflejo de Sobresalto , Factores de Intercambio de Guanina Nucleótido RhoRESUMEN
Synaptic inhibition is a vital component in the control of cell excitability within the brain. Here we report a newly identified form of inhibitory synaptic plasticity, termed depolarization-induced potentiation of inhibition, in rodents. This mechanism strongly potentiated synaptic transmission from interneurons to Purkinje cells after the termination of depolarization-induced suppression of inhibition. It was triggered by an elevation of Ca(2+) in Purkinje cells and the subsequent retrograde activation of presynaptic NMDA receptors. These glutamate receptors promoted the spontaneous release of Ca(2+) from presynaptic ryanodine-sensitive Ca(2+) stores. Thus, NMDA receptor-mediated facilitation of transmission at this synapse provides a regulatory mechanism that can dynamically alter the synaptic efficacy at inhibitory synapses.
Asunto(s)
Cerebelo/citología , Ácido Egtácico/análogos & derivados , Interneuronas/metabolismo , Células de Purkinje/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapsis/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Animales , Animales Recién Nacidos , Cadmio/farmacología , Calcio/metabolismo , Quelantes/farmacología , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Ácido Egtácico/farmacología , Estimulación Eléctrica , Potenciales Evocados/efectos de los fármacos , Potenciales Evocados/efectos de la radiación , Antagonistas de Aminoácidos Excitadores/farmacología , Glutamato Descarboxilasa/metabolismo , Inmunohistoquímica/métodos , Técnicas In Vitro , Isoenzimas/metabolismo , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/efectos de la radiación , N-Metilaspartato/farmacología , Inhibición Neural/fisiología , Plasticidad Neuronal/efectos de los fármacos , Plasticidad Neuronal/fisiología , Plasticidad Neuronal/efectos de la radiación , Técnicas de Placa-Clamp/métodos , Piperidinas/farmacología , Terminales Presinápticos/metabolismo , Pirazoles/farmacología , Ratas , Ratas Sprague-Dawley , Rimonabant , Rianodina/farmacología , Bloqueadores de los Canales de Sodio/farmacología , Transmisión Sináptica/fisiología , Sinaptofisina/metabolismo , Tetrodotoxina/farmacología , Factores de TiempoRESUMEN
Gephyrin (GPHN) is an organizational protein that clusters and localizes the inhibitory glycine (GlyR) and GABAA receptors to the microtubular matrix of the neuronal postsynaptic membrane. Mice deficient in gephyrin develop a hereditary molybdenum cofactor deficiency and a neurological phenotype that mimics startle disease (hyperekplexia). This neuromotor disorder is associated with mutations in the GlyR alpha1 and beta subunit genes (GLRA1 and GLRB). Further genetic heterogeneity is suspected, and we hypothesized that patients lacking mutations in GLRA1 and GLRB might have mutations in the gephyrin gene (GPHN). In addition, we adopted a yeast two-hybrid screen, using the GlyR beta subunit intracellular loop as bait, in an attempt to identify further GlyR-interacting proteins implicated in hyperekplexia. Gephyrin cDNAs were isolated, and subsequent RT-PCR analysis from human tissues demonstrated the presence of five alternatively spliced GPHN exons concentrated in the central linker region of the gene. This region generated 11 distinct GPHN transcript isoforms, with 10 being specific to neuronal tissue. Mutation analysis of GPHN exons in hyperekplexia patients revealed a missense mutation (A28T) in one patient causing an amino acid substitution (N10Y). Functional testing demonstrated that GPHNN10Y does not disrupt GlyR-gephyrin interactions or collybistininduced cell-surface clustering. We provide evidence that GlyR-gephyrin binding is dependent on the presence of an intact C-terminal MoeA homology domain. Therefore, the N10Y mutation and alternative splicing of GPHN transcripts do not affect interactions with GlyRs but may affect other interactions with the cytoskeleton or gephyrin accessory proteins.
Asunto(s)
Proteínas Portadoras/genética , Proteínas de la Membrana/genética , Enfermedad de la Neurona Motora/genética , Receptores de Glicina/metabolismo , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Sitios de Unión/genética , Proteínas Portadoras/metabolismo , Exones/genética , Variación Genética , Humanos , Proteínas de la Membrana/metabolismo , Ratones , Datos de Secuencia Molecular , Enfermedad de la Neurona Motora/metabolismo , Mutación , Unión Proteica , Isoformas de Proteínas/genética , Receptores de Glicina/genética , Alineación de SecuenciaRESUMEN
1. Whole-cell currents were recorded from Xenopus laevis oocytes and human embryonic kidney cells expressing GABA(A) receptor beta3 subunit homomers to search for additional residues affecting Zn(2+) inhibition. These residues would complement the previously identified histidine (H267), present just within the external portal of the ion channel, which modulates Zn(2+) inhibition. 2. Zinc inhibited the pentobarbitone-gated current on beta3(H267A) homomers at pH 7.4, but this effect was abolished at pH 5.4. The Zn(2+)-sensitive spontaneous beta3 subunit-mediated conductance was also insensitive to block by Zn(2+) at pH 5.4. 3. Changing external pH enabled the titration of the Zn(2+) sensitive binding site or signal transduction domain. The pK(a) was estimated at 6.8 +/- 0.03 implying the involvement of histidine residues. 4. External histidine residues in the beta3 receptor subunit were substituted with alanine, in addition to the background mutation, H267A, to assess their sensitivity to Zn(2+) inhibition. The Zn(2+) IC(50) was unaffected by either the H119A or H191A mutations. 5. The remaining histidine, H107, the only other candidate likely to participate in Zn(2+) inhibition, was substituted with various residues. Most mutants were expressed at the cell surface but they disrupted functional expression of beta3 homomers. However, H107G was functional and demonstrated a marked reduction in sensitivity to Zn(2+). 6. GABA(A) receptor beta3 subunits form functional ion channels that can be inhibited by Zn(2+). Two histidine residues are largely responsible for this effect, H267 in the pore lining region and H107 residing in the extracellular N-terminal domain.