RESUMEN
Temporal lobe epilepsy (TLE) is the most common type of epilepsy in humans. Cognitive impairment and memory consolidation problems are common among TLE patients. To understand the changes in the cellular process of memory in TLE, we studied the long-term depression (LTD) in Schaffer-collateral (Sc) CA1 synapses in an epilepsy model. Long-term potentiation (LTP) was investigated in patient samples and animal models by several groups, but LTD was not studied with the same interest in epilepsy research. Here we induced epileptiform activity in rat hippocampal slices using magnesium-free high-potassium (7.5 mM K +) artificial cerebrospinal fluid (HK-ACSF) and characterized the LTD in Sc-CA1 synapses. We found that epileptiform activity abolished/impaired LTD and depotentiation in the Sc-CA1 synapses. In control slices, application of NMDA (30 µM for 3 min) induced chemical LTD (c-LTD) in Sc-CA1 synapses, whereas epileptiform activity induced slices showed slow onset potentiation. Induction of LTD using 1 Hz, 900 pulses yielded a similar outcome as c-LTD. Both forms of LTD were NMDA receptor dependent. In addition, we found that the polarity changes in the synaptic plasticity in epileptiform-induced slices were blocked by GluN2B antagonists ifenprodil and Ro 25-6981. Our data suggest that epileptiform-induced metaplasticity inhibits LTD in Sc-CA1 synapses. We provide new insight into the cellular mechanism of memory formation during epilepsy.
Asunto(s)
Epilepsia , Receptores de N-Metil-D-Aspartato , Humanos , Ratas , Animales , Sinapsis , Potenciación a Largo Plazo , Hipocampo/metabolismo , Plasticidad NeuronalRESUMEN
Interneurons of the spinal dorsal horn are central to somatosensory and nociceptive processing. A mechanistic understanding of their function depends on profound knowledge of their intrinsic properties and their integration into dorsal horn circuits. Here, we have used BAC transgenic mice expressing enhanced green fluorescent protein (eGFP) under the control of the vesicular glutamate transporter (vGluT2) gene (vGluT2::eGFP mice) to perform a detailed electrophysiological and morphological characterisation of excitatory dorsal horn neurons, and to compare their properties to those of GABAergic (Gad67::eGFP tagged) and glycinergic (GlyT2::eGFP tagged) neurons. vGluT2::eGFP was detected in about one-third of all excitatory dorsal horn neurons and, as demonstrated by the co-expression of vGluT2::eGFP with different markers of subtypes of glutamatergic neurons, probably labelled a representative fraction of these neurons. Three types of dendritic tree morphologies (vertical, central, and radial), but no islet cell-type morphology, were identified in vGluT2::eGFP neurons. vGluT2::eGFP neurons had more depolarised action potential thresholds and longer action potential durations than inhibitory neurons, while no significant differences were found for the resting membrane potential, input resistance, cell capacitance and after-hyperpolarisation. Delayed firing and single action potential firing were the single most prevalent firing patterns in vGluT2::eGFP neurons of the superficial and deep dorsal horn, respectively. By contrast, tonic firing prevailed in inhibitory interneurons of the dorsal horn. Capsaicin-induced synaptic inputs were detected in about half of the excitatory and inhibitory neurons, and occurred more frequently in superficial than in deep dorsal horn neurons. Primary afferent-evoked (polysynaptic) inhibitory inputs were found in the majority of glutamatergic and glycinergic neurons, but only in less than half of the GABAergic population. Excitatory dorsal horn neurons thus differ from their inhibitory counterparts in several biophysical properties and possibly also in their integration into the local neuronal circuitry.
Asunto(s)
Potenciales de Acción , Células del Asta Posterior/fisiología , Potenciales Sinápticos , Animales , Neuronas GABAérgicas/citología , Neuronas GABAérgicas/fisiología , Interneuronas/citología , Interneuronas/fisiología , Ratones , Células del Asta Posterior/citología , Sinapsis/fisiologíaRESUMEN
Tissue-specific implications of SARS-CoV-2-encoded accessory proteins are not fully understood. SARS-CoV-2 infection can severely affect three major organs-the heart, lungs, and brain. We analyzed SARS-CoV-2 ORF3a interacting host proteins in these three major organs. Furthermore, we identified common and unique interacting host proteins and their targeting miRNAs (lung and brain) and delineated associated biological processes by reanalyzing RNA-seq data from the brain (COVID-19-infected/uninfected choroid plexus organoid study), lung tissue from COVID-19 patients/healthy subjects, and cardiomyocyte cells-based transcriptomics analyses. Our in silico studies showed ORF3a interacting proteins could vary depending upon tissues. The number of unique ORF3a interacting proteins in the brain, lungs, and heart were 10, 7, and 1, respectively. Though common pathways influenced by SARS-CoV-2 infection were more, unique 21 brain and 7 heart pathways were found. One unique pathway for the heart was negative regulation of calcium ion transport. Reported observations of COVID-19 patients with a history of hypertension taking calcium channel blockers (CCBs) or dihydropyridine CCBs had an elevated rate of intubation or increased rate of intubation/death, respectively. Also, the likelihood of hospitalization of chronic CCB users with COVID-19 was greater in comparison to long-term angiotensin-converting enzyme inhibitors/angiotensin receptor blockers users. Further studies are necessary to confirm this. miRNA analysis of ORF3a interacting proteins in the brain and lungs revealed 3 of 37 brain miRNAs and 1 of 25 lung miRNAs with high degree and betweenness indicating their significance as hubs in the interaction network. Our study could help in identifying potential tissue-specific COVID-19 drug/drug repurposing targets.
RESUMEN
Spinal dorsal horn GABA(A) receptors are found both postsynaptically on central neurons and presynaptically on axons and/or terminals of primary sensory neurons, where they mediate primary afferent depolarization (PAD) and presynaptic inhibition. Both phenomena have been studied extensively on a cellular level, but their role in sensory processing in vivo has remained elusive, due to inherent difficulties to selectively interfere with presynaptic receptors. Here, we address the contribution of a major subpopulation of GABA(A) receptors (those containing the α2 subunit) to spinal pain control in mice lacking α2-GABA(A) receptors specifically in primary nociceptors (sns-α2(-/-) mice). sns-α2(-/-) mice exhibited GABA(A) receptor currents and dorsal root potentials of normal amplitude in vitro, and normal response thresholds to thermal and mechanical stimulation in vivo, and developed normal inflammatory and neuropathic pain sensitization. However, the positive allosteric GABA(A) receptor modulator diazepam (DZP) had almost completely lost its potentiating effect on PAD and presynaptic inhibition in vitro and a major part of its spinal antihyperalgesic action against inflammatory hyperalgesia in vivo. Our results thus show that part of the antihyperalgesic action of spinally applied DZP occurs through facilitated activation of GABA(A) receptors residing on primary nociceptors.
Asunto(s)
Hiperalgesia/fisiopatología , Neuralgia/fisiopatología , Neuronas Aferentes/fisiología , Receptores de GABA-A/fisiología , Receptores Presinapticos/fisiología , Raíces Nerviosas Espinales/fisiología , Animales , Diazepam/administración & dosificación , Diazepam/farmacología , Regulación de la Expresión Génica/genética , Regulación de la Expresión Génica/fisiología , Hiperalgesia/tratamiento farmacológico , Inyecciones Espinales , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/genética , Potenciales de la Membrana/fisiología , Ratones , Ratones Noqueados , Ratones Transgénicos , Neuralgia/tratamiento farmacológico , Neuronas Aferentes/efectos de los fármacos , Neuronas Aferentes/metabolismo , Nociceptores/efectos de los fármacos , Nociceptores/fisiología , Técnicas de Placa-Clamp , Receptores de GABA-A/biosíntesis , Receptores de GABA-A/genética , Receptores Presinapticos/efectos de los fármacos , Raíces Nerviosas Espinales/efectos de los fármacosRESUMEN
Neuroplastic changes at the spinal synapses between primary nociceptors and second order dorsal horn neurons play key roles in pain and analgesia. NMDA receptor-dependent forms of long-term plasticity have been studied extensively at these synapses, but little is known about possible contributions of the endocannabinoid system. Here, we addressed the role of cannabinoid (CB)1 receptors in activity-dependent plasticity at these synapses. We report that conditional low-frequency stimulation of high-threshold primary sensory nerve fibres paired with depolarisation of the postsynaptic neuron evoked robust long-term depression (LTD)of excitatory synaptic transmission by about 40% in the vast majority (90%) of recordings made in wild-type mice. When recordings were made from global or nociceptor-specific CB(1) receptor-deficient mice (CB(1) (−/− ) mice and sns-CB(1)(−/−) mice), the portion of neurons exhibiting LTD was strongly reduced to about 25%. Accordingly, LTD was prevented to a similar extent by the CB1 receptor antagonist AM251 and mimicked by pharmacological activation of CB1 receptors. In a subset of neurons with EPSCs of particularly high stimulation thresholds, we furthermore found that the absence of CB(1) receptors in CB(1)(−/−) and sns-CB(1)(−/−) mice converted the response to the paired conditioning stimulation protocol from LTD to long-term potentiation (LTP). Our results identify CB1 receptor-dependent LTD as a form of synaptic plasticity previously unknown in spinal nociceptors. They furthermore suggest that prevention of LTP may be a second hither to unknown function of CB1 receptors in primary nociceptors. Both findings may have important implications for our understanding of endogenous pain control mechanisms and of analgesia evoked by cannabinoid receptor agonists.
Asunto(s)
Plasticidad Neuronal/fisiología , Receptor Cannabinoide CB1/fisiología , Médula Espinal/fisiología , Animales , Endocannabinoides/fisiología , Potenciales Postsinápticos Excitadores , Femenino , Técnicas In Vitro , Masculino , Ratones , Ratones Transgénicos , Nociceptores/fisiología , Sinapsis/fisiologíaRESUMEN
NMDA receptors (NMDARs) play a key role in synaptic plasticity and excitotoxicity. Subtype-specific role of NMDAR in neural disorders is an emerging area. Recent studies have revealed that mutations in NMDARs are a cause for epilepsy. Hippocampus is a known focal point for epilepsy. In hippocampus, expression of the NMDAR subtypes GluN1/GluN2A and GluN1/GluN2B is temporally regulated. However, the pharmacological significance of these subtypes is not well understood in epileptic context/models. To investigate this, epilepsy was induced in hippocampal slices by the application of artificial cerebrospinal fluid that contained high potassium but no magnesium. Epileptiform events (EFEs) were recorded from the CA1 and DG areas of hippocampus with or without subtype-specific antagonists. Irrespective of the age group, CA1 and DG showed epileptiform activity. The NMDAR antagonist AP5 was found to reduce the number of EFEs significantly. However, the application of subtype-specific antagonists (TCN 201 for GluN1/GluN2A and Ro 25-69811 for GluN1/GluN2B) revealed that EFEs had area-specific and temporal components. In slices from neonates, EFEs in CA1 were effectively reduced by Ro 25-69811, but were largely insensitive to TCN 201. In contrast, EFEs in DG were equally sensitive to both of the subtype-specific antagonists. However, the differential sensitivity for the antagonists observed in neonates was absent in later developmental stages. The study provides a functional insight into the NMDAR subtype-dependent contribution of EFEs in hippocampus of young rats, which may have implications in treating childhood epilepsy and avoiding unnecessary side effects of broad spectrum antagonists.
Asunto(s)
Región CA1 Hipocampal/fisiopatología , Giro Dentado/fisiopatología , Epilepsia/fisiopatología , Antagonistas de Aminoácidos Excitadores/farmacología , Fenoles/farmacología , Piperidinas/farmacología , Receptores de N-Metil-D-Aspartato/fisiología , Sulfonamidas/farmacología , 2-Amino-5-fosfonovalerato/farmacología , Factores de Edad , Animales , Animales Recién Nacidos , Región CA1 Hipocampal/efectos de los fármacos , Región CA1 Hipocampal/embriología , Región CA1 Hipocampal/crecimiento & desarrollo , Giro Dentado/efectos de los fármacos , Giro Dentado/embriología , Giro Dentado/crecimiento & desarrollo , Epilepsia/inducido químicamente , Femenino , Masculino , Especificidad de Órganos , Ratas , Ratas Wistar , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidoresRESUMEN
In eukaryotes, the cellular functions are segregated to membrane-bound organelles. This inherently requires sorting of metabolites to membrane-limited locations. Sorting the metabolites from ribosomes to various organelles along the intracellular trafficking pathways involves several integral cellular processes, including an energy-dependent step, in which the sorting of metabolites between organelles is catalyzed by membrane-anchoring protein Rab-GTPases (Rab). They contribute to relaying the switching of the secretory proteins between hydrophobic and hydrophilic environments. The intracellular trafficking routes include exocytic and endocytic pathways. In these pathways, numerous Rab-GTPases are participating in discrete shuttling of cargoes. Long-distance trafficking of cargoes is essential for neuronal functions, and Rabs are critical for these functions, including the transport of membranes and essential proteins for the development of axons and neurites. Rabs are also the key players in exocytosis of neurotransmitters and recycling of neurotransmitter receptors. Thus, Rabs are critical for maintaining neuronal communication, as well as for normal cellular physiology. Therefore, cellular defects of Rab components involved in neural functions, which severely affect normal brain functions, can produce neurological complications, including several neurodegenerative diseases. In this review, we provide a comprehensive overview of the current understanding of the molecular signaling pathways of Rab proteins and the impact of their defects on different neurodegenerative diseases. The insights gathered into the dynamics of Rabs that are described in this review provide new avenues for developing effective treatments for neurodegenerative diseases-associated with Rab defects.
Asunto(s)
Proteínas del Tejido Nervioso/fisiología , Enfermedades Neurodegenerativas/enzimología , Neuronas/metabolismo , Proteínas de Unión al GTP rab/fisiología , Compartimento Celular , Cilios/enzimología , Endocitosis , Exocitosis , Glicosilfosfatidilinositoles/fisiología , Homeostasis , Humanos , Modelos Moleculares , Proteínas del Tejido Nervioso/química , Enfermedades del Sistema Nervioso/enzimología , Neuritas/fisiología , Enfermedades Neurodegenerativas/fisiopatología , Neuronas/ultraestructura , Conformación Proteica , Prenilación de Proteína , Sistemas de Translocación de Proteínas/fisiología , Transporte de Proteínas , Vesículas Sinápticas/metabolismo , Proteínas de Unión al GTP rab/químicaRESUMEN
NMDA receptor (NMDAR) 2A (NR2A)- and NR2B-type NMDARs coexist in synapses of CA1 pyramidal cells. Recent studies using pharmacological blockade of NMDAR subtypes proposed that the NR2A type is responsible for inducing long-term potentiation (LTP), whereas the NR2B type induces long-term depression (LTD). This contrasts with the finding in genetically modified mice that NR2B-type NMDARs induce LTP when NR2A signaling is absent or impaired, although compensatory mechanisms might have contributed to this result. We therefore assessed the contribution of the two NMDAR subtypes to LTP in mouse hippocampal slices by different induction protocols and in the presence of NMDAR antagonists, including the NR2A-type blocker NVP-AAM077, for which an optimal concentration for subtype selectivity was determined on recombinant and native NMDARs. Partial blockade of NMDA EPSCs by 40%, either by preferentially antagonizing NR2A- or NR2B-type NMDARs or by the nonselective antagonist D-AP-5, did not impair LTP, demonstrating that hippocampal LTP induction can be generated by either NMDAR subtype.
Asunto(s)
Hipocampo/fisiología , Potenciación a Largo Plazo/fisiología , Receptores de N-Metil-D-Aspartato/fisiología , Animales , Línea Celular , Estimulación Eléctrica , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Hipocampo/citología , Humanos , Riñón/citología , Ratones , Técnicas de Cultivo de Órganos , Piperidinas/farmacología , Células Piramidales/fisiología , Quinoxalinas/farmacología , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Receptores de N-Metil-D-Aspartato/genética , Proteínas Recombinantes/genética , TransfecciónRESUMEN
Excitatory neurotransmission mediated by N-methyl-d-aspartate receptors (NMDARs) is fundamental to learning and memory and, when impaired, causes certain neurological disorders. NMDARs are heterotetrameric complexes composed of two GluN1 [NR1] and two GluN2(A-D) [NR2(A-D)] subunits. The GluN2 subunit is responsible for subunit-specific channel activity and gating kinetics including activation (rise time), peak open probability (peak Po) and deactivation (decay time). The peak Po of recombinant NMDARs was recently described to be controlled by the extracellular GluN2 N-terminal domain (NTD). The cytoplasmic GluN2 C-terminal domain (CTD) could also be involved, because the Po of synaptic NMDARs is reduced in mice expressing C-terminally truncated GluN2 subunits. Here, we examined the role of the GluN2 cytoplasmic tail for NMDAR channel activity and gating in HEK-293 cells. C-terminal truncation of GluN2A, GluN2B or GluN2C did not change the subunit-specific rise time but accelerated the decay time of glutamate-activated currents. Furthermore, the peak Po was reduced by about 50% for GluN2A and GluN2B but not for GluN2C. These results indicated that the CTD of GluN2 has a modulating role in NMDAR gating even in the absence of interacting synaptic proteins. Reduction of peak Po and deactivation kinetics following GluN2 C-terminal truncation were reversed by re-introducing a CTD from a different GluN2 subunit. Thus, the CTDs of GluN2 subunits behave as constitutive structural elements required for normal functioning of NMDARs but are not involved in determining the subunit-specific gating properties of NMDARs.
Asunto(s)
Ácido Glutámico/metabolismo , Subunidades de Proteína/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Transducción de Señal/fisiología , Células HEK293 , Humanos , Transmisión Sináptica/fisiologíaRESUMEN
The genetic heterogeneity of autism poses a major challenge for identifying mechanism-based treatments. A number of rare mutations are associated with autism, and it is unclear whether these result in common neuronal alterations. Monogenic syndromes, such as fragile X, include autism as one of their multifaceted symptoms and have revealed specific defects in synaptic plasticity. We discovered an unexpected convergence of synaptic pathophysiology in a nonsyndromic form of autism with those in fragile X syndrome. Neuroligin-3 knockout mice (a model for nonsyndromic autism) exhibited disrupted heterosynaptic competition and perturbed metabotropic glutamate receptor-dependent synaptic plasticity, a hallmark of fragile X. These phenotypes could be rescued by reexpression of neuroligin-3 in juvenile mice, highlighting the possibility of reverting neuronal circuit alterations in autism after the completion of development.