RESUMEN
In this study, the effect of extracellular pH on glutamatergic synaptic transmission was examined in mechanically dissociated rat hippocampal CA3 pyramidal neurons using a whole-cell patch-clamp technique under voltage-clamp conditions. Native synaptic boutons were isolated without using any enzymes, using a so-called "synapse bouton preparation," and preserved for the electrical stimulation of single boutons. Both the frequency and amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) were found to decrease and increase in response to modest acidic (~pH 6.5) and basic (~pH 8.5) solutions, respectively. These changes in sEPSC frequency were not affected by the addition of TTX but completely disappeared by successive addition of Cd2+. However, changes in sEPSC amplitude induced by acidic and basic extracellular solutions were not affected by the addition of neither TTX nor Cd2+. The glutamate-induced whole-cell currents were decreased and increased by acidic and basic solutions, respectively. Acidic pH also decreased the amplitude and increased the failure rate (Rf) and paired-pulse rate (PPR) of glutamatergic electrically evoked excitatory postsynaptic currents (eEPSCs), while a basic pH increased the amplitude and decreased both the Rf and PPR of eEPSCs. The kinetics of the currents were not affected by changes in pH. Acidic and basic solutions decreased and increased voltage-gated Ca2+ but not Na+ channel currents in the dentate gyrus granule cell bodies. Our results indicate that extracellular pH modulates excitatory transmission via both pre- and postsynaptic sites, with the presynaptic modulation correlated to changes in voltage-gated Ca2+ channel currents.NEW & NOTEWORTHY The effects of external pH changes on spontaneous, miniature, and evoked excitatory synaptic transmission in CA3 hippocampal synapses were examined using the isolated nerve bouton preparation, which allowed for the accurate regulation of extracellular pH at the synapses. Acidification generally reduced transmission, partly via effects on presynaptic Ca2+ channel currents, while alkalization generally enhanced transmission. Both pre- and postsynaptic sites contributed to these effects.
Asunto(s)
Región CA3 Hipocampal/fisiología , Potenciales Postsinápticos Excitadores/fisiología , Concentración de Iones de Hidrógeno , Terminales Presinápticos/fisiología , Células Piramidales/fisiología , Animales , Región CA3 Hipocampal/química , Femenino , Ácido Glutámico/metabolismo , Masculino , Técnicas de Placa-Clamp , Terminales Presinápticos/química , Células Piramidales/química , Ratas , Ratas WistarRESUMEN
Spinocerebellar ataxia type 6 (SCA6) is a neurodegenerative disease caused by the expansion of a polyglutamine tract in the Ca(v)2.1 voltage-gated calcium channel. To elucidate how the expanded polyglutamine tract in this plasma membrane protein causes the disease, we created a unique knockin mouse model that modestly overexpressed the mutant transcripts under the control of an endogenous promoter (MPI-118Q). MPI-118Q mice faithfully recapitulated many features of SCA6, including selective Purkinje cell degeneration. Surprisingly, analysis of inclusion formation in the mutant Purkinje cells indicated the lysosomal localization of accumulated mutant Ca(v)2.1 channels in the absence of autophagic response. The lack of cathepsin B, a major lysosomal cysteine proteinase, exacerbated the loss of Purkinje cells and was accompanied by an acceleration of inclusion formation in this model. Thus, the pathogenic mechanism of SCA6 involves the endolysosomal degradation pathway, and unique pathological features of this model further illustrate the pivotal role of protein context in the pathogenesis of polyglutamine diseases.
Asunto(s)
Modelos Animales de Enfermedad , Lisosomas/fisiología , Células de Purkinje/patología , Ataxias Espinocerebelosas/patología , Animales , Autofagia , Ratones , Ratones TransgénicosRESUMEN
Presynaptic elements of axons, in which action potentials (APs) cause release of neurotransmitter, are sites of high densities and complex interactions of proteins. We report that the presence of K(v)3 channels in addition to K(v)1 at glutamatergic mossy fiber boutons (MFBs) in rat hippocampal slices considerably limits the number of fast, voltage-activated potassium channels necessary to achieve basal presynaptic AP repolarization. The â¼ 10-fold higher repolarization efficacy per K(v)3 channel compared with presynaptic K(v)1 results from a higher steady-state availability at rest, a better recruitment by the presynaptic AP as a result of faster activation kinetics, and a larger single-channel conductance. Large-conductance calcium- and voltage-activated potassium channels (BK(Ca)) at MFBs give rise to a fast activating/fast inactivating and a slowly activating/sustained K(+) current component during long depolarizations. However, BK(Ca) contribute to MFB-AP repolarization only after presynaptic K(v)3 have been disabled. The calcium chelators EGTA and BAPTA are equally effective in preventing BK(Ca) activation, suggesting that BK(Ca) are not organized in nanodomain complexes with presynaptic voltage-gated calcium channels. Thus, the functional properties of K(v)3 channels at MFBs are tuned to both promote brevity of presynaptic APs limiting glutamate release and at the same time keep surface protein density of potassium channels low. Presynaptic BK(Ca) channels are restricted to limit additional increases of the AP half-duration in case of K(v)3 hypofunction, because rapid membrane repolarization by K(v)3 combined with distant calcium sources prevent BK(Ca) activation during basal APs.
Asunto(s)
Potenciales de Acción/fisiología , Fibras Musgosas del Hipocampo/fisiología , Canales de Potasio/fisiología , Terminales Presinápticos/fisiología , Canales de Potasio Shaw/fisiología , Potenciales de Acción/efectos de los fármacos , Animales , Región CA3 Hipocampal/efectos de los fármacos , Región CA3 Hipocampal/fisiología , Calcio/metabolismo , Quelantes/farmacología , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Femenino , Técnicas In Vitro , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio , Masculino , Fibras Musgosas del Hipocampo/efectos de los fármacos , Técnicas de Placa-Clamp/métodos , Células Piramidales/fisiología , Ratas , Ratas Wistar , Canales de Potasio de la Superfamilia Shaker/fisiología , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/fisiologíaRESUMEN
Presynaptic glycine receptors (GlyRs) have been implicated in the regulation of glutamatergic synaptic transmission. Here, we characterized presynaptic GlyR-mediated currents by patch-clamp recording from mossy fiber boutons (MFBs) in rat hippocampal slices. In MFBs, focal puff-application of glycine-evoked chloride currents that were blocked by the GlyR antagonist strychnine. Their amplitudes declined substantially during postnatal development, from a mean conductance per MFB of approximately 600 pS in young to approximately 130 pS in adult animals. Single-channel analysis revealed multiple conductance states between approximately 20 and approximately 120 pS, consistent with expression of both homo- and hetero-oligomeric GlyRs. Accordingly, estimated GlyRs densities varied between 8-17 per young, and 1-3 per adult, MFB. Our results demonstrate that functional presynaptic GlyRs are present on hippocampal mossy fiber terminals and suggest a role of these receptors in the regulation of glutamate release during the development of the mossy fiber--CA3 synapse.
Asunto(s)
Fibras Musgosas del Hipocampo/crecimiento & desarrollo , Receptores de Glicina/fisiología , Receptores Presinapticos/fisiología , Animales , Glicina/metabolismo , Glicina/farmacología , Glicina/fisiología , Fibras Musgosas del Hipocampo/efectos de los fármacos , Técnicas de Placa-Clamp , Ratas , Ratas Wistar , Receptores de Glicina/metabolismo , Receptores Presinapticos/metabolismo , Estricnina/farmacologíaRESUMEN
Effects of xenon (Xe) on whole-cell currents induced by glutamate (Glu), its three ionotropic subtypes, and GABA, as well as on the fast synaptic glutamatergic and GABAergic transmissions, were studied in the mechanically dissociated "synapse bouton preparation" of rat spinal sacral dorsal commissural nucleus (SDCN) neurons. This technique evaluates pure single or multi-synapse responses from native functional nerve endings and enables us to quantify how Xe influences pre- and postsynaptic transmissions accurately. Effects of Xe on glutamate (Glu)-, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-, kainate (KA)- and N-methyl-d-aspartate (NMDA)- and GABAA receptor-mediated whole-cell currents were investigated by the conventional whole-cell patch configuration. Excitatory and inhibitory postsynaptic currents (EPSCs and IPSCs) were measured as spontaneous (s) and evoked (e) EPSCs and IPSCs. Evoked synaptic currents were elicited by paired-pulse focal electric stimulation. Xe decreased Glu, AMPA, KA, and NMDA receptor-mediated whole-cell currents but did not change GABAA receptor-mediated whole-cell currents. Xe decreased the frequency and amplitude but did not affect the 1/e decay time of the glutamatergic sEPSCs. Xe decreased the frequency without affecting the amplitude and 1/e decay time of GABAergic sIPSCs. Xe decreased the amplitude and increased the failure rate (Rf) and paired-pulse ratio (PPR) without altering the 1/e decay time of both eEPSC and eIPSC, suggesting that Xe acts on the presynaptic side of the synapse. The presynaptic inhibition was greater in eEPSCs than in eIPSCs. We conclude that Xe decreases glutamatergic and GABAergic spontaneous and evoked transmissions at the presynaptic level. The glutamatergic presynaptic responses are the main target of anesthesia-induced neuronal responses. In contrast, GABAergic responses minimally contribute to Xe anesthesia.
Asunto(s)
Potenciales Postsinápticos Excitadores/efectos de los fármacos , Ácido Glutámico/farmacología , Potenciales Postsinápticos Inhibidores/efectos de los fármacos , Xenón/farmacología , Ácido gamma-Aminobutírico/farmacología , Anestésicos por Inhalación/farmacología , Animales , Neuronas/efectos de los fármacos , Terminales Presinápticos/efectos de los fármacos , Ratas Wistar , Transmisión Sináptica/fisiologíaRESUMEN
Neurons possess multiple types of voltage-dependent calcium channels. These channels are classified as either low-voltage-activated (LVA/T-type) or high-voltage-activated (HVA) consisting of L, N, P, Q and R subtypes. T-type Ca channels can be opened by small depolarization that are under the threshold for action potential generation. These T-type Ca channels may be involved in the sub threshold depolarizations that underlies neuronal bursting activities in neurons. Such burst firing might originate in the postsynaptic membrane of the neuronal dendrites.
RESUMEN
Benzodiazepines act mainly at postsynaptic gamma-aminobutyric acid type A (GABA(A)) receptors. In rat neocortical layer V pyramidal neurons, we found that midazolam (MDZ), a benzodiazepine, increases the frequency of GABAergic miniature inhibitory postsynaptic currents (mIPSCs) via insertion of alpha7 nicotinic acetylcholine receptors (nAChRs) at presynaptic GABAergic boutons. Although nicotine alone had no effect, MDZ plus nicotine dramatically increased mIPSC frequency. Neostigmine, an acetylcholinesterase inhibitor, mimicked the actions of nicotine. MDZ increased the number of alpha-bungarotoxin-bound boutons that were blocked by protein kinase C (PKC) inhibitors, as revealed by confocal imaging of a neuron-synaptic bouton preparation. Thus, MDZ may induce membrane translocation of alpha7 nAChRs on GABAergic boutons via activation of PKC, enabling endogenous acetylcholine to increase GABA release. The above actions seem unique to MDZ because neither other benzodiazepines (diazepam and flunitrazepam) nor zolpidem had this effect. The findings reveal both a novel cholinergic modulatory mechanism affecting GABAergic transmission and a novel action of some general anesthetics.
Asunto(s)
Moduladores del GABA/farmacología , Midazolam/farmacología , Neocórtex/efectos de los fármacos , Neocórtex/metabolismo , Receptores Nicotínicos/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Animales , Benzodiazepinas/farmacología , Fibras Colinérgicas/efectos de los fármacos , Fibras Colinérgicas/metabolismo , Inhibidores de la Colinesterasa/farmacología , Neostigmina/farmacología , Técnicas de Cultivo de Órganos , Técnicas de Placa-Clamp , Terminales Presinápticos/efectos de los fármacos , Terminales Presinápticos/metabolismo , Proteína Quinasa C/metabolismo , Ratas , Ratas Wistar , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/fisiología , Receptor Nicotínico de Acetilcolina alfa 7RESUMEN
The cortical migration process depends on a number of trophic factors and on the activation of different voltage- and ligand-gated channels. We investigated the role of gamma-aminobutyric acid (GABA) type A receptors in the neuronal migration process of the newborn rat parietal cortex in vivo and in vitro. Local in vivo application of the GABA-A antagonist bicuculline methiodide (BMI) or the agonist muscimol via cortical surface Elvax implants induced prominent alterations in the cortical architecture when compared with untreated or sham-operated controls. BMI- and muscimol-treated animals revealed heterotopic cell clusters in the upper layers and a complete loss of the cortical lamination in the region underlying the Elvax implant. Immunocytochemical staining for glial fibrillary acidic protein, N-methyl-D-aspartate receptors, and GABA demonstrated that heterotopia was not provoked by glial proliferation and confirmed the presence of both glutamatergic and GABAergic neurons. In organotypic neocortical slices from embryonic day 18-19 embryos, application of BMI and to a lesser extent also muscimol induced an increase in the migration speed and an accumulation of neurons in the upper cortical layers. Spontaneous intracellular calcium ([Ca2+]i) oscillations in neocortical slices from newborn rats were abolished by BMI (5 and 20 microM) and muscimol (1 and 10 microM), indicating that both compounds interfere with [Ca2+]i signaling required for normal neuronal migration. Electrophysiological recordings from migrating neurons in newborn rat neocortical slices indicate that long-term application of muscimol causes a pronounced reduction (1 microM muscimol) or blockade (10 microM) in the responsiveness of postsynaptic GABA-A receptors due to a pronounced receptor desensitization. Our results indicate that modulation of GABA-A receptors by compounds acting as agonists or antagonists may profoundly influence the neuronal migration process in the developing cerebral cortex.
Asunto(s)
Neocórtex/fisiología , Neuronas/fisiología , Receptores de GABA-A/fisiología , Animales , Animales Recién Nacidos/fisiología , Antimetabolitos , Bicuculina/farmacología , Bromodesoxiuridina , Calcio/metabolismo , Calcio/fisiología , Movimiento Celular , Implantes de Medicamentos , Electrofisiología , Agonistas del GABA/farmacología , Antagonistas del GABA/farmacología , Proteína Ácida Fibrilar de la Glía/metabolismo , Inmunohistoquímica , Técnicas In Vitro , Cinética , Muscimol/farmacología , Neocórtex/citología , Técnicas de Placa-Clamp , Ratas , Ratas Wistar , Receptores de N-Metil-D-Aspartato/metabolismoRESUMEN
Collybistin (Cb) is a brain-specific guanine nucleotide exchange factor that has been implicated in plasma membrane targeting of the postsynaptic scaffolding protein gephyrin found at glycinergic and GABAergic synapses. Here we show that Cb-deficient mice display a region-specific loss of postsynaptic gephyrin and GABA(A) receptor clusters in the hippocampus and the basolateral amygdala. Cb deficiency is accompanied by significant changes in hippocampal synaptic plasticity, due to reduced dendritic GABAergic inhibition. Long-term potentiation is enhanced, and long-term depression reduced, in Cb-deficient hippocampal slices. Consistent with the anatomical and electrophysiological findings, the animals show increased levels of anxiety and impaired spatial learning. Together, our data indicate that Cb is essential for gephyrin-dependent clustering of a specific set of GABA(A) receptors, but not required for glycine receptor postsynaptic localization.
Asunto(s)
Proteínas Portadoras/fisiología , Factores de Intercambio de Guanina Nucleótido/fisiología , Hipocampo/fisiología , Proteínas de la Membrana/fisiología , Plasticidad Neuronal , Receptores de GABA-A/fisiología , Sinapsis/fisiología , Transmisión Sináptica , Amígdala del Cerebelo/fisiología , Animales , Factores de Intercambio de Guanina Nucleótido/genética , Potenciación a Largo Plazo , Depresión Sináptica a Largo Plazo , Aprendizaje por Laberinto , Ratones , Ratones Noqueados , Actividad Motora , Técnicas de Placa-Clamp , Terminales Presinápticos/fisiología , Receptores de Glicina/metabolismo , Factores de Intercambio de Guanina Nucleótido RhoRESUMEN
The mechanisms underlying cyclic AMP modulation of action potential-dependent and -independent (spontaneous) release of glycine from terminals synapsing onto sacral dorsal commissural nucleus neurons of lamina X were studied in spinal cord slices using conventional patch-clamp recordings. 3-Isobutyl-1-methylxanthine (IBMX), a phosphodiesterase inhibitor, and forskolin increased the amplitude of evoked inhibitory postsynaptic currents (eIPSCs) in a sensitive manner to protein kinase A (PKA) inhibition (with KT-5720). Direct activation (with adenosine 3',5'-cyclic-monophosphothioate, Sp-isomer) and inhibition (with adenosine 3',5'-cyclic-monophosphothioate, Rp-isomer) of PKA increased and decreased the eIPSC amplitude, respectively. Paired pulse experiments and direct injection of PKA inhibitor fragment 6-22 amide (PKI(6-22)) into the recording neuron revealed that these effects on eIPSC amplitude occurred presynaptically, indicating that evoked glycine release is regulated by presynaptic cAMP via changes in PKA activity. Increasing cAMP also increased spontaneous release of glycine, causing an increased frequency of miniature IPSCs (mIPSCs). In contrast to the effects on evoked release, this response was not solely mediated via PKA, as it was not occluded by PKA inhibition, and both direct inhibition and direct activation of PKA actually enhanced mIPSC frequency. Direct inhibition of cAMP (with SQ 22536) did, however, reduce mIPSC frequency. These results suggest cAMP modulation of evoked and spontaneous release involves different presynaptic mechanisms and proteins.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Glicina/metabolismo , Terminales Presinápticos/metabolismo , Transducción de Señal/fisiología , Médula Espinal/metabolismo , Animales , Colforsina/farmacología , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Técnicas In Vitro , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Inhibición Neural/efectos de los fármacos , Inhibición Neural/fisiología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/fisiología , Técnicas de Placa-Clamp , Hidrolasas Diéster Fosfóricas/efectos de los fármacos , Terminales Presinápticos/efectos de los fármacos , Ratas , Ratas Wistar , Región Sacrococcígea , Médula Espinal/efectos de los fármacosRESUMEN
The transduction mechanisms underlying presynaptic GABAB receptor-mediated inhibition of transmitter release have been characterized for a variety of synapses in the central nervous system (CNS). These studies have suggested a range of transduction mechanisms, including a role for second messengers such as protein kinases A (PKA) and C (PKC). In the present study, we have examined the intracellular signalling pathways underlying baclofen-induced inhibition of GABA release from terminals synapsing onto rat basalis of Meynert neurons using patch-clamp recordings. Baclofen, a selective GABAB receptor agonist, reversibly decreased both evoked and spontaneous, miniature, GABAergic inhibitory postsynaptic currents (eIPSCs and mIPSCs, respectively). Such baclofen actions were completely abolished by CGP55845A, a selective GABAB receptor antagonist, and by staurosporine, a non-selective PKA and PKC inhibitor. The mIPSC frequency was still decreased by baclofen even in the presence of 4 AP, a K+ channel blocker, and Cd2+, a voltage-dependent calcium channel blocker. Pharmacological activation or inhibition of PKC activity affected basal GABA release and mildly affected the response to baclofen. Inhibition of the cAMP/PKA cascade also affected basal GABA release and, in a subset of neurons, occluded the effects of baclofen, suggesting that the GABAB receptor-mediated inhibitory action on GABA release was mediated via decreases in PKA activity. In addition, PKA inhibition occluded the effects of PKC modulation on both basal GABA release and on the response to baclofen. Our results characterize the transduction pathway of baclofen at these nucleus basalis of Maynert (nBM) synapses and show, for the first time, some cross-talk between the cAMP/PKA and PKC pathways in mammalian presynaptic nerve terminals.
Asunto(s)
Adenina/análogos & derivados , Núcleo Basal de Meynert/fisiología , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Terminales Presinápticos/fisiología , Proteína Quinasa C/fisiología , Receptores de GABA-B/fisiología , Transducción de Señal/fisiología , Sinapsis/fisiología , Adenina/farmacología , Animales , Baclofeno/farmacología , Núcleo Basal de Meynert/citología , Canales de Calcio/fisiología , AMP Cíclico/antagonistas & inhibidores , Conductividad Eléctrica , Inhibidores Enzimáticos/farmacología , Agonistas del GABA/farmacología , Proteínas de Unión al GTP/fisiología , Inhibición Neural/fisiología , Neuronas/fisiología , Canales de Potasio/fisiología , Ratas , Ratas Wistar , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Corelease of glycine and GABA from the single synaptic terminal (synaptic bouton) is well accepted in immature rat spinal cord and brainstem. However, it raises the question of how glycine and GABA are accumulated in the same synaptic vesicles and coreleased. To address this issue, spontaneous miniature inhibitory postsynaptic currents (mIPSCs) and focally evoked IPSCs (eIPSCs) mediated via a single synapse were recorded from synaptic bouton preparations of the rat immature sacral dorsal commissural nucleus (SDCN) neurones by whole-cell patch recording. Focal stimulation of a single synaptic bouton revealed that three different quantal releases occur from a single synaptic bouton: i.e. pure glycine, pure GABA, and mixed. Prolonged treatment with bafilomycin A1, a vacuolar-type H+/ATPase inhibitor, to the SDCN neurone greatly suppressed frequency and amplitude of the mIPSCs. During washing out of bafilomycin A1, complete recovery in the amplitude of glycinergic mIPSCs was observed, while that of GABAergic and mixed mIPSCs was incomplete. These observations indicate that three types of vesicles coexist in single synaptic terminals, and that refilling of glycine into the synaptic vesicle predominantes over GABA after pretreatment with bafilomycin A1 in immature rats. This could be explained by the decrease in the cytosolic concentration of GABA, or by the presence of subtypes of vesicular inhibitory amino acid transporter in the synaptic vesicle membrane.