RESUMEN
Thalamocortical (TC) circuits are essential for sensory information processing. Clinical and preclinical studies of autism spectrum disorders (ASDs) have highlighted abnormal thalamic development and TC circuit dysfunction. However, mechanistic understanding of how TC dysfunction contributes to behavioral abnormalities in ASDs is limited. Here, our study on a Shank3 mouse model of ASD reveals TC neuron hyperexcitability with excessive burst firing and a temporal mismatch relationship with slow cortical rhythms during sleep. These TC electrophysiological alterations and the consequent sensory hypersensitivity and sleep fragmentation in Shank3 mutant mice are causally linked to HCN2 channelopathy. Restoring HCN2 function early in postnatal development via a viral approach or lamotrigine (LTG) ameliorates sensory and sleep problems. A retrospective case series also supports beneficial effects of LTG treatment on sensory behavior in ASD patients. Our study identifies a clinically relevant circuit mechanism and proposes a targeted molecular intervention for ASD-related behavioral impairments.
Asunto(s)
Trastorno del Espectro Autista , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización , Proteínas del Tejido Nervioso , Tálamo , Animales , Tálamo/metabolismo , Tálamo/patología , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/metabolismo , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/genética , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Ratones , Trastorno del Espectro Autista/genética , Trastorno del Espectro Autista/metabolismo , Trastorno del Espectro Autista/fisiopatología , Trastorno del Espectro Autista/patología , Lamotrigina/farmacología , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Canalopatías/genética , Canalopatías/metabolismo , Canalopatías/patología , Humanos , Modelos Animales de Enfermedad , Masculino , Neuronas/metabolismo , Femenino , Ratones Endogámicos C57BL , Mutación/genética , Sueño/fisiología , Sueño/efectos de los fármacos , Sueño/genética , Canales de PotasioRESUMEN
Schizophrenia is a heterogeneous psychiatric disorder with a strong genetic basis, whose etiology and pathophysiology remain poorly understood. Exome sequencing studies have uncovered rare, loss-of-function variants that greatly increase risk of schizophrenia [1], including loss-of-function mutations in GRIN2A (aka GluN2A or NR2A, encoding the NMDA receptor subunit 2A) and AKAP11 (A-Kinase Anchoring Protein 11). AKAP11 and GRIN2A mutations are also associated with bipolar disorder [2], and epilepsy and developmental delay/intellectual disability [1, 3, 4], respectively. Accessible in both humans and rodents, electroencephalogram (EEG) recordings offer a window into brain activity and display abnormal features in schizophrenia patients. Does loss of Grin2a or Akap11 in mice also result in EEG abnormalities? We monitored EEG in heterozygous and homozygous knockout Grin2a and Akap11 mutant mice compared with their wild-type littermates, at 3- and 6-months of age, across the sleep/wake cycle and during auditory stimulation protocols. Grin2a and Akap11 mutants exhibited increased resting gamma power, attenuated auditory steady-state responses (ASSR) at gamma frequencies, and reduced responses to unexpected auditory stimuli during mismatch negativity (MMN) tests. Sleep spindle density was reduced in a gene dose-dependent manner in Akap11 mutants, whereas Grin2a mutants showed increased sleep spindle density. The EEG phenotypes of Grin2a and Akap11 mutant mice show a variety of abnormal features that overlap considerably with human schizophrenia patients, reflecting systems-level changes caused by Grin2a and Akap11 deficiency. These neurophysiologic findings further substantiate Grin2a and Akap11 mutants as genetic models of schizophrenia and identify potential biomarkers for stratification of schizophrenia patients.
Asunto(s)
Proteínas de Anclaje a la Quinasa A , Epilepsia , Receptores de N-Metil-D-Aspartato , Esquizofrenia , Animales , Humanos , Ratones , Proteínas de Anclaje a la Quinasa A/genética , Electroencefalografía/métodos , Mutación , Esquizofrenia/genética , Sueño , Receptores de N-Metil-D-Aspartato/genéticaRESUMEN
Machine learning has been applied in recent years to categorize sleep stages (NREM, REM, and wake) using electroencephalogram (EEG) recordings; however, a well-validated sleep scoring automatic pipeline in rodent research is still not publicly available. Here, we present IntelliSleepScorer, a software package with a graphic user interface to score sleep stages automatically in mice. IntelliSleepScorer uses the light gradient boosting machine (LightGBM) to score sleep stages for each epoch of recordings. We developed LightGBM models using a large cohort of data, which consisted of 5776 h of sleep EEG and electromyogram (EMG) signals across 519 unique recordings from 124 mice. The LightGBM model achieved an overall accuracy of 95.2% and a Cohen's kappa of 0.91, which outperforms the baseline models such as the logistic regression model (accuracy = 93.3%, kappa = 0.88) and the random forest model (accuracy = 94.3%, kappa = 0.89). The overall performance of the LightGBM model as well as the performance across different sleep stages are on par with that of the human experts. Most importantly, we validated the generalizability of the LightGBM models: (1) The LightGBM model performed well on two publicly available, independent datasets (kappa > = 0.80), which have different sampling frequency and epoch lengths; (2) The LightGBM model performed well on data recorded at a lower sampling frequency (kappa = 0.90); (3) The performance of the LightGBM model is not affected by the light/dark cycle; and (4) A modified LightGBM model performed well on data containing only one EEG and one EMG electrode (kappa > = 0.89). Taken together, the LightGBM models offer state-of-the-art performance for automatic sleep stage scoring in mice. Last, we implemented the IntelliSleepScorer software package based on the validated model to provide an out-of-box solution to sleep researchers (available for download at https://sites.broadinstitute.org/pan-lab/resources ).
Asunto(s)
Fases del Sueño , Sueño , Humanos , Ratones , Animales , Polisomnografía , Algoritmos , Electroencefalografía , Programas InformáticosRESUMEN
Neuropsychiatric disorders are often accompanied by cognitive impairments/intellectual disability (ID). It is not clear whether there are converging mechanisms underlying these debilitating impairments. We found that many autism and schizophrenia risk genes are expressed in the anterodorsal subdivision (AD) of anterior thalamic nuclei, which has reciprocal connectivity with learning and memory structures. CRISPR-Cas9 knockdown of multiple risk genes selectively in AD thalamus led to memory deficits. While the AD is necessary for contextual memory encoding, the neighboring anteroventral subdivision (AV) regulates memory specificity. These distinct functions of AD and AV are mediated through their projections to retrosplenial cortex, using differential mechanisms. Furthermore, knockdown of autism and schizophrenia risk genes PTCHD1, YWHAG, or HERC1 from AD led to neuronal hyperexcitability, and normalization of hyperexcitability rescued memory deficits in these models. This study identifies converging cellular to circuit mechanisms underlying cognitive deficits in a subset of neuropsychiatric disease models.
Asunto(s)
Núcleos Talámicos Anteriores/fisiopatología , Disfunción Cognitiva/fisiopatología , Vías Nerviosas/fisiopatología , Núcleos Talámicos/fisiopatología , Animales , Núcleos Talámicos Anteriores/fisiología , Corteza Cerebral/fisiopatología , Cognición/fisiología , Ratones , Vías Nerviosas/fisiología , Núcleos Talámicos/fisiologíaRESUMEN
Effects of violent video games on aggression remain contentious despite decades of empirical investigations. Using eight-wave panel data collected from 662 South Korean adolescents (grades 4, 7, and 10) for a 4-year period (number of observations = 5,296), the current research critically tested competing hypotheses concerning the relations between violent video games and aggression. In so doing, we directly compared the results from dynamic fixed-effects (FE) regression with those from conventional contemporaneous FE regression to observe if different statistical models yield different outcomes. Consistent with the catharsis hypothesis, the dynamic FE model showed that violent (vs. nonviolent) game playing significantly lowered both verbal and physical aggression among heavy players, with no corresponding effect of the game type for light players. By contrast, results from the contemporaneous FE model lent support to the stimulation hypothesis, with violent game playing leading to increased physical aggression as well as verbal aggression among heavy players. Violent game playing did not significantly affect anger and hostility, but overall game time did, although in opposite directions depending on the statistical model. Specifically, the dynamic FE model indicated a significant reduction of the negative emotions as a result of increased game playing, whereas the contemporaneous FE model showed a significant increase in both emotions. Methodological implications and directions for future research are discussed.
Asunto(s)
Conducta del Adolescente/psicología , Agresión/psicología , Catarsis , Modelos Psicológicos , Juegos de Video/psicología , Violencia/psicología , Adolescente , Niño , Emociones , Femenino , Humanos , Estudios Longitudinales , Masculino , Análisis de Regresión , República de CoreaRESUMEN
Waterproof membranes have higher initial strength, faster construction, and better waterproofing than conventional sheet membranes. In addition, their polymer constituents have much higher interfacial adhesion and tensile strength than those of conventional materials. However, despite their advantages, waterproof membranes are not widely used in civil construction. This study evaluates the material properties and interface parameters of a waterproof membrane by considering the results of laboratory experiments and numerical analysis. Since the contact behavior of a membrane at its interface with shotcrete is important for understanding the mechanism of the support it offers known as a shotcrete tunnel lining, modeling should adopt appropriate contact conditions. The numerical analysis identifies the suitability and contact conditions of the waterproof membrane in various conditions.
RESUMEN
The thalamic reticular nucleus (TRN), the major source of thalamic inhibition, regulates thalamocortical interactions that are critical for sensory processing, attention and cognition1-5. TRN dysfunction has been linked to sensory abnormality, attention deficit and sleep disturbance across multiple neurodevelopmental disorders6-9. However, little is known about the organizational principles that underlie its divergent functions. Here we performed an integrative study linking single-cell molecular and electrophysiological features of the mouse TRN to connectivity and systems-level function. We found that cellular heterogeneity in the TRN is characterized by a transcriptomic gradient of two negatively correlated gene-expression profiles, each containing hundreds of genes. Neurons in the extremes of this transcriptomic gradient express mutually exclusive markers, exhibit core or shell-like anatomical structure and have distinct electrophysiological properties. The two TRN subpopulations make differential connections with the functionally distinct first-order and higher-order thalamic nuclei to form molecularly defined TRN-thalamus subnetworks. Selective perturbation of the two subnetworks in vivo revealed their differential role in regulating sleep. In sum, our study provides a comprehensive atlas of TRN neurons at single-cell resolution and links molecularly defined subnetworks to the functional organization of thalamocortical circuits.
Asunto(s)
Redes Reguladoras de Genes , Núcleos Talámicos/citología , Núcleos Talámicos/metabolismo , Animales , Análisis por Conglomerados , Femenino , Perfilación de la Expresión Génica , Hibridación Fluorescente in Situ , Metaloendopeptidasas/metabolismo , Ratones , Vías Nerviosas , Neuronas/metabolismo , Osteopontina/metabolismo , Técnicas de Placa-Clamp , RNA-Seq , Análisis de la Célula Individual , Sueño/genética , Sueño/fisiología , Núcleos Talámicos/fisiología , TranscriptomaRESUMEN
CACNA1I, a schizophrenia risk gene, encodes a subtype of voltage-gated T-type calcium channel CaV3.3. We previously reported that a patient-derived missense de novo mutation (R1346H) of CACNA1I impaired CaV3.3 channel function. Here, we generated CaV3.3-RH knock-in animals, along with mice lacking CaV3.3, to investigate the biological impact of R1346H (RH) variation. We found that RH mutation altered cellular excitability in the thalamic reticular nucleus (TRN), where CaV3.3 is abundantly expressed. Moreover, RH mutation produced marked deficits in sleep spindle occurrence and morphology throughout non-rapid eye movement (NREM) sleep, while CaV3.3 haploinsufficiency gave rise to largely normal spindles. Therefore, mice harboring the RH mutation provide a patient derived genetic model not only to dissect the spindle biology but also to evaluate the effects of pharmacological reagents in normalizing sleep spindle deficits. Importantly, our analyses highlighted the significance of characterizing individual spindles and strengthen the inferences we can make across species over sleep spindles. In conclusion, this study established a translational link between a genetic allele and spindle deficits during NREM observed in schizophrenia patients, representing a key step toward testing the hypothesis that normalizing spindles may be beneficial for schizophrenia patients.
Asunto(s)
Canales de Calcio Tipo T , Esquizofrenia , Animales , Electroencefalografía , Humanos , Ratones , Esquizofrenia/genética , Sueño , Sueño REMRESUMEN
A crucial pathophysiological issue concerning central neuropathic pain is the modification of sensory processing by abnormally increased low-frequency brain rhythms. Here we explore the molecular mechanisms responsible for such abnormal rhythmicity and its relation to neuropathic pain syndrome. Toward this aim, we investigated the behavioral and electrophysiological consequences of trigeminal neuropathic pain following infraorbital nerve ligations in CaV3.1 T-type Ca(2+) channel knockout and wild-type mice. CaV3.1 knockout mice had decreased mechanical hypersensitivity and reduced low-frequency rhythms in the primary somatosensory cortex and related thalamic nuclei than wild-type mice. Lateral inhibition of gamma rhythm in primary somatosensory cortex layer 4, reflecting intact sensory contrast, was present in knockout mice but severely impaired in wild-type mice. Moreover, cross-frequency coupling between low-frequency and gamma rhythms, which may serve in sensory processing, was pronounced in wild-type mice but not in CaV3.1 knockout mice. Our results suggest that the presence of CaV3.1 channels is a key element in the pathophysiology of trigeminal neuropathic pain.
Asunto(s)
Canales de Calcio Tipo T/fisiología , Neuralgia/fisiopatología , Neuralgia del Trigémino/fisiopatología , Animales , Canales de Calcio Tipo T/deficiencia , Canales de Calcio Tipo T/genética , Ritmo Delta/genética , Ritmo Delta/fisiología , Fenómenos Electrofisiológicos , Femenino , Ritmo Gamma/genética , Ritmo Gamma/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuralgia/genética , Corteza Somatosensorial/fisiopatología , Núcleos Talámicos/fisiopatología , Neuralgia del Trigémino/genéticaRESUMEN
In unconscious status (e.g., deep sleep and anesthetic unconsciousness) where cognitive functions are not generated there is still a significant level of brain activity present. Indeed, the electrophysiology of the unconscious brain is characterized by well-defined thalamocortical rhythmicity. Here we address the ionic basis for such thalamocortical rhythms during unconsciousness. In particular, we address the role of CaV3.1 T-type Ca(2+) channels, which are richly expressed in thalamic neurons. Toward this aim, we examined the electrophysiological and behavioral phenotypes of mice lacking CaV3.1 channels (CaV3.1 knockout) during unconsciousness induced by ketamine or ethanol administration. Our findings indicate that CaV3.1 KO mice displayed attenuated low-frequency oscillations in thalamocortical loops, especially in the 1- to 4-Hz delta band, compared with control mice (CaV3.1 WT). Intriguingly, we also found that CaV3.1 KO mice exhibited augmented high-frequency oscillations during unconsciousness. In a behavioral measure of unconsciousness dynamics, CaV3.1 KO mice took longer to fall into the unconscious state than controls. In addition, such unconscious events had a shorter duration than those of control mice. The thalamocortical interaction level between mediodorsal thalamus and frontal cortex in CaV3.1 KO mice was significantly lower, especially for delta band oscillations, compared with that of CaV3.1 WT mice, during unconsciousness. These results suggest that the CaV3.1 channel is required for the generation of a given set of thalamocortical rhythms during unconsciousness. Further, that thalamocortical resonant neuronal activity supported by this channel is important for the control of vigilance states.
Asunto(s)
Canales de Calcio Tipo T/fisiología , Corteza Cerebral/fisiología , Ritmo Circadiano , Tálamo/fisiología , Inconsciencia , Animales , Canales de Calcio Tipo T/genética , RatonesRESUMEN
We have examined the effects of RNS60, a 0.9% saline containing charge-stabilized oxygen nanobubble-based structures. RNS60 is generated by subjecting normal saline to Taylor-Couette-Poiseuille (TCP) flow under elevated oxygen pressure. This study, implemented in Xenopus laevis oocytes, addresses both the electrophysiological membrane properties and parallel biological processes in the cytoplasm. Intracellular recordings from defolliculated X. laevis oocytes were implemented in: (1) air oxygenated standard Ringer's solution, (2) RNS60-based Ringer's solution, (3) RNS10.3 (TCP-modified saline without excess oxygen)-based Ringer's, and (4) ONS60 (saline containing high pressure oxygen without TCP modification)-based Ringer's. RNS60-based Ringer's solution induced membrane hyperpolarization from the resting membrane potential. This effect was prevented by: (1) ouabain (a blocker of the sodium/potassium ATPase), (2) rotenone (a mitochondrial electron transfer chain inhibitor preventing usable ATP synthesis), and (3) oligomycin A (an inhibitor of ATP synthase) indicating that RNS60 effects intracellular ATP levels. Increased intracellular ATP levels following RNS60 treatment were directly demonstrated using luciferin/luciferase photon emission. These results indicate that RNS60 alters intrinsic the electrophysiological properties of the X. laevis oocyte membrane by increasing mitochondrial-based ATP synthesis. Ultrastructural analysis of the oocyte cytoplasm demonstrated increased mitochondrial length in the presence of RNS60-based Ringer's solution. It is concluded that the biological properties of RNS60 relate to its ability to optimize ATP synthesis.
RESUMEN
Superfusion of the squid giant synapse with artificial seawater (ASW) based on isotonic saline containing oxygen nanobubbles (RNS60 ASW) generates an enhancement of synaptic transmission. This was determined by examining the postsynaptic response to single and repetitive presynaptic spike activation, spontaneous transmitter release, and presynaptic voltage clamp studies. In the presence of RNS60 ASW single presynaptic stimulation elicited larger postsynaptic potentials (PSP) and more robust recovery from high frequency stimulation than in control ASW. Analysis of postsynaptic noise revealed an increase in spontaneous transmitter release with modified noise kinetics in RNS60 ASW. Presynaptic voltage clamp demonstrated an increased EPSP, without an increase in presynaptic ICa(++) amplitude during RNS60 ASW superfusion. Synaptic release enhancement reached stable maxima within 5-10 min of RNS60 ASW superfusion and was maintained for the entire recording time, up to 1 h. Electronmicroscopic morphometry indicated a decrease in synaptic vesicle density and the number at active zones with an increase in the number of clathrin-coated vesicles (CCV) and large endosome-like vesicles near junctional sites. Block of mitochondrial ATP synthesis by presynaptic injection of oligomycin reduced spontaneous release and prevented the synaptic noise increase seen in RNS60 ASW. After ATP block the number of vesicles at the active zone and CCV was reduced, with an increase in large vesicles. The possibility that RNS60 ASW acts by increasing mitochondrial ATP synthesis was tested by direct determination of ATP levels in both presynaptic and postsynaptic structures. This was implemented using luciferin/luciferase photon emission, which demonstrated a marked increase in ATP synthesis following RNS60 administration. It is concluded that RNS60 positively modulates synaptic transmission by up-regulating ATP synthesis, thus leading to synaptic transmission enhancement.
RESUMEN
Filamentous tau inclusions are hallmarks of Alzheimer's disease and related neurodegenerative tauopathies, but the molecular mechanisms involved in tau-mediated changes in neuronal function and their possible effects on synaptic transmission are unknown. We have evaluated the effects of human tau protein injected directly into the presynaptic terminal axon of the squid giant synapse, which affords functional, structural, and biochemical analysis of its action on the synaptic release process. Indeed, we have found that at physiological concentration recombinant human tau (h-tau42) becomes phosphorylated, produces a rapid synaptic transmission block, and induces the formation of clusters of aggregated synaptic vesicles in the vicinity of the active zone. Presynaptic voltage clamp recordings demonstrate that h-tau42 does not modify the presynaptic calcium current amplitude or kinetics. Analysis of synaptic noise at the post-synaptic axon following presynaptic h-tau42 microinjection revealed an initial phase of increase spontaneous transmitter release followed by a marked reduction in noise. Finally, systemic administration of T-817MA, a proposed neuro-protective agent, rescued tau-induced synaptic abnormalities. Our results show novel mechanisms of h-tau42 mediated synaptic transmission failure and identify a potential therapeutic agent to treat tau-related neurotoxicity.
RESUMEN
The role of P/Q- and T-type calcium channels in the rhythmic oscillatory behaviour of inferior olive (IO) neurons was investigated in mutant mice. Mice lacking either the CaV2.1 gene of the pore-forming alpha1A subunit for P/Q-type calcium channel, or the CaV3.1 gene of the pore-forming alpha1G subunit for T-type calcium channel were used. In vitro intracellular recording from IO neurons reveals that the amplitude and frequency of sinusoidal subthreshold oscillations (SSTOs) were reduced in the CaV2.1-/- mice. In the CaV3.1-/- mice, IO neurons also showed altered patterns of SSTOs and the probability of SSTO generation was significantly lower (15%, 5 of 34 neurons) than that of wild-type (78%, 31 of 40 neurons) or CaV2.1-/- mice (73%, 22 of 30 neurons). In addition, the low-threshold calcium spike and the sustained endogenous oscillation following rebound potentials were absent in IO neurons from CaV3.1-/- mice. Moreover, the phase-reset dynamics of oscillatory properties of single neurons and neuronal clusters in IO were remarkably altered in both CaV2.1-/- and CaV3.1-/- mice. These results suggest that both alpha1A P/Q- and alpha1G T-type calcium channels are required for the dynamic control of neuronal oscillations in the IO. These findings were supported by results from a mathematical IO neuronal model that incorporated T and P/Q channel kinetics.
Asunto(s)
Canales de Calcio Tipo P/metabolismo , Canales de Calcio Tipo Q/metabolismo , Canales de Calcio Tipo T/metabolismo , Señalización del Calcio , Neuronas/metabolismo , Núcleo Olivar/metabolismo , Animales , Canales de Calcio Tipo N , Canales de Calcio Tipo P/deficiencia , Canales de Calcio Tipo P/genética , Canales de Calcio Tipo Q/deficiencia , Canales de Calcio Tipo Q/genética , Canales de Calcio Tipo T/deficiencia , Canales de Calcio Tipo T/genética , Simulación por Computador , Cinética , Potenciales de la Membrana , Ratones , Ratones Noqueados , Modelos Neurológicos , Núcleo Olivar/citologíaRESUMEN
The rhythmic motor pathway activation by pacemaker neurons or circuits in the brain has been proposed as the mechanism for the timing of motor coordination, and the abnormal potentiation of this mechanism may lead to a pathological tremor. Here, we show that the potentiation of Ca(V)3.1 T-type Ca(2+) channels in the inferior olive contributes to the onset of the tremor in a pharmacological model of essential tremor. After administration of harmaline, 4- to 10-Hz synchronous neuronal activities arose from the IO and then propagated to cerebellar motor circuits in wild-type mice, but those rhythmic activities were absent in mice lacking Ca(V)3.1 gene. Intracellular recordings in brain-stem slices revealed that the Ca(V)3.1-deficient inferior olive neurons lacked the subthreshold oscillation of membrane potentials and failed to trigger 4- to 10-Hz rhythmic burst discharges in the presence of harmaline. In addition, the selective knockdown of Ca(V)3.1 gene in the inferior olive by shRNA efficiently suppressed the harmaline-induced tremor in wild-type mice. A mathematical model constructed based on data obtained from patch-clamping experiments indicated that harmaline could efficiently potentiate Ca(V)3.1 channels by changing voltage-dependent responsiveness in the hyperpolarizing direction. Thus, Ca(V)3.1 is a molecular pacemaker substrate for intrinsic neuronal oscillations of inferior olive neurons, and the potentiation of this mechanism can be considered as a pathological cause of essential tremor.
Asunto(s)
Canales de Calcio Tipo T/metabolismo , Núcleo Olivar/metabolismo , Temblor/metabolismo , Animales , Canales de Calcio Tipo T/deficiencia , Canales de Calcio Tipo T/genética , Línea Celular , Modelos Animales de Enfermedad , Harmalina , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Núcleo Olivar/fisiopatología , Interferencia de ARN , Temblor/inducido químicamente , Temblor/genética , Temblor/fisiopatologíaRESUMEN
Small GTPase Rab is a member of a large family of Ras-related proteins, highly conserved in eukaryotic cells, and thought to regulate specific type(s) and/or specific step(s) in intracellular membrane trafficking. Given our interest in synaptic transmission, we addressed the possibility that Rab27 (a close isoform of Rab3) could be involved in cytosolic synaptic vesicle mobilization. Indeed, preterminal injection of a specific antibody against squid Rab27 (anti-sqRab27 antibody) combined with confocal microscopy demonstrated that Rab27 is present on squid synaptic vesicles. Electrophysiological study of injected synapses showed that the anti-sqRab27 antibody inhibited synaptic release in a stimulation-dependent manner without affecting presynaptic action potentials or inward Ca(2+) current. This result was confirmed in in vitro synaptosomes by using total internal reflection fluorescence microscopy. Thus, synaptosomal Ca(2+)-stimulated release of FM1-43 dye was greatly impaired by intraterminal anti-sqRab27 antibody. Ultrastructural analysis of the injected giant preterminal further showed a reduced number of docked synaptic vesicles and an increase in nondocked vesicular profiles distant from the active zone. These results, taken together, indicate that Rab27 is primarily involved in the maturation of recycled vesicles and/or their transport to the presynaptic active zone in the squid giant synapse.
Asunto(s)
Sinapsis Eléctricas/fisiología , Vesículas Sinápticas/metabolismo , Proteínas de Unión al GTP rab/fisiología , Potenciales de Acción , Animales , Anticuerpos/farmacología , Transporte Biológico , Calcio/metabolismo , Decapodiformes , Electrofisiología , Microscopía , Datos de Secuencia Molecular , Vesículas Sinápticas/química , Proteínas de Unión al GTP rab/análisis , Proteínas de Unión al GTP rab/inmunologíaRESUMEN
To assess the role of alpha(1G) T-type Ca2+ channels in neuropathic pain after L5 spinal nerve ligation, we examined behavioral pain susceptibility in mice lacking CaV3.1 (alpha1G(-/-)), the gene encoding the pore-forming units of these channels. Reduced spontaneous pain responses and an increased threshold for paw withdrawal in response to mechanical stimulation were observed in these mice. The alpha1G(-/-) mice also showed attenuated thermal hyperalgesia in response to both low-(IR30) and high-intensity (IR60) infrared stimulation. Our results reveal the importance of alpha(1G) T-type Ca2+ channels in the development of neuropathic pain, and suggest that selective modulation of alpha1G subtype channels may provide a novel approach to the treatment of allodynia and hyperalgesia.
Asunto(s)
Canales de Calcio Tipo T/deficiencia , Neuralgia/metabolismo , Animales , Canales de Calcio Tipo T/metabolismo , Rayos Infrarrojos , Ratones , Ratones Endogámicos C57BL , Dimensión del Dolor , Tiempo de ReacciónRESUMEN
Low-voltage-activated calcium channels, also known as T-type calcium channels, are widely expressed in various types of neurons. In contrast to high-voltage-activated calcium channels which can be activated by a strong depolarization of membrane potential, T-type channels can be activated by a weak depolarization near the resting membrane potential once deinactivated by hyperpolarization, and therefore can regulate the excitability and electroresponsiveness of neurons under physiological conditions near resting states. Recently, the molecular diversity and functional multiplicity of T-type channels have been demonstrated through molecular genetic studies coupled with physiological and behavioral analysis. Understanding the functional consequences of modulation of each subtype of these channels in vivo could point to the right direction for developing therapeutic tools for relevant diseases.
Asunto(s)
Bloqueadores de los Canales de Calcio/uso terapéutico , Canales de Calcio Tipo T/fisiología , Epilepsia Tipo Ausencia/tratamiento farmacológico , Dolor/tratamiento farmacológico , Animales , Canales de Calcio Tipo T/efectos de los fármacos , Corteza Cerebral/fisiología , Epilepsia Tipo Ausencia/etiología , Humanos , Ratones , Ratones Noqueados , Dolor/etiología , Sueño/fisiología , Médula Espinal/fisiología , Tálamo/fisiologíaRESUMEN
Thalamocortical in vivo and in vitro function was studied in mice lacking P/Q-type calcium channels (Cav2.1), in which N-type calcium channels (Cav2.2) supported central synaptic transmission. Unexpectedly, in vitro patch recordings from thalamic neurons demonstrated no gamma-band subthreshold oscillation, and voltage-sensitive dye imaging demonstrated an absence of cortical gamma-band-dependent columnar activation involving cortical inhibitory interneuron activity. In vivo electroencephalogram recordings showed persistent absence status and a dramatic reduction of gamma-band activity. Pharmacological block of T-type calcium channels (Cav3), although not noticeably affecting normal control animals, left the knockout mice in a coma-like state. Hence, although N-type calcium channels can rescue P/Q-dependent synaptic transmission, P/Q calcium channels are essential in the generation of gamma-band activity and resultant cognitive function.
Asunto(s)
Canales de Calcio Tipo N/genética , Corteza Cerebral/patología , Transmisión Sináptica/fisiología , Tálamo/patología , Animales , Canales de Calcio Tipo N/deficiencia , Canales de Calcio Tipo N/fisiología , Corteza Cerebral/fisiopatología , Cognición/fisiología , Electroencefalografía , Ratones , Ratones Noqueados , Ratones Mutantes , Técnicas de Placa-Clamp , Tálamo/fisiopatologíaRESUMEN
Alterations in thalamic T-type Ca2+ channels are thought to contribute to the pathogenesis of absence seizures. Here, we found that mice with a null mutation for the pore-forming alpha1A subunits of P/Q-type channels (alpha1A-/- mice) were prone to absence seizures characterized by typical spike-and-wave discharges (SWDs) and behavioral arrests. Isolated thalamocortical relay (TC) neurons from these mice showed increased T-type Ca2+ currents in vitro. To examine the role of increased T-currents in alpha1A-/- TC neurons, we cross-bred alpha1A-/- mice with mice harboring a null mutation for the gene encoding alpha1G, a major isotype of T-type Ca2+ channels in TC neurons. alpha1A-/-/alpha1G-/- mice showed a complete loss of T-type Ca2+ currents in TC neurons and displayed no SWDs. Interestingly, alpha1A-/-/alpha1G+/- mice had 75% of the T-type Ca2+ currents in TC neurons observed in alpha1A+/+/alpha1G+/+ mice and showed SWD activity that was quantitatively similar to that in alpha1A-/-/alpha1G+/+ mice. Similar results were obtained using double-mutant mice harboring the alpha1G mutation plus another mutation also used as a model for absence seizures, i.e., lethargic (beta4(lh/lh)), tottering (alpha1A(tg/tg)), or stargazer (gamma2(stg/stg)). The present results reveal that alpha1G T-type Ca2+ channels play a critical role in the genesis of spontaneous absence seizures resulting from hypofunctioning P/Q-type channels, but that the augmentation of thalamic T-type Ca2+ currents is not an essential step in the genesis of absence seizures.