RESUMEN
OBJECTIVE: KCNA1 mutations are associated with a rare neurological movement disorder known as episodic ataxia type 1 (EA1), and epilepsy is a common comorbidity. Current medications provide only partial relief for ataxia and/or seizures, making new drugs needed. Here, we characterized zebrafish kcna1a-/- as a model of EA1 with epilepsy and compared the efficacy of the first-line therapy carbamazepine in kcna1a-/- zebrafish to Kcna1-/- rodents. METHODS: CRISPR/Cas9 mutagenesis was used to introduce a mutation in the sixth transmembrane segment of the zebrafish Kcna1 protein. Behavioral and electrophysiological assays were performed on kcna1a-/- larvae to assess ataxia- and epilepsy-related phenotypes. Real-time quantitative polymerase chain reaction (qPCR) was conducted to measure mRNA levels of brain hyperexcitability markers in kcna1a-/- larvae, followed by bioenergetics profiling to evaluate metabolic function. Drug efficacies were tested using behavioral and electrophysiological assessments, as well as seizure frequency in kcna1a-/- zebrafish and Kcna1-/- mice, respectively. RESULTS: Zebrafish kcna1a-/- larvae showed uncoordinated movements and locomotor deficits, along with scoliosis and increased mortality. The mutants also exhibited impaired startle responses when exposed to light-dark flashes and acoustic stimulation as well as hyperexcitability as measured by extracellular field recordings and upregulated fosab transcripts. Neural vglut2a and gad1b transcript levels were disrupted in kcna1a-/- larvae, indicative of a neuronal excitatory/inhibitory imbalance, as well as a significant reduction in cellular respiration in kcna1a-/- , consistent with dysregulation of neurometabolism. Notably, carbamazepine suppressed the impaired startle response and brain hyperexcitability in kcna1a-/- zebrafish but had no effect on the seizure frequency in Kcna1-/- mice, suggesting that this EA1 zebrafish model might better translate to humans than rodents. SIGNIFICANCE: We conclude that zebrafish kcna1a-/- show ataxia and epilepsy-related phenotypes and are responsive to carbamazepine treatment, consistent with EA1 patients. These findings suggest that kcna1-/- zebrafish are a useful model for drug screening as well as studying the underlying disease biology.
Asunto(s)
Epilepsia , Pez Cebra , Humanos , Ratones , Animales , Ataxia/tratamiento farmacológico , Ataxia/genética , Ataxia/complicaciones , Convulsiones/complicaciones , Carbamazepina/farmacología , Carbamazepina/uso terapéutico , Canal de Potasio Kv.1.1/genéticaRESUMEN
BACKGROUND: Cannabinoid hyperemesis syndrome (CHS) is a poorly understood vomiting disorder associated with chronic cannabis use. AIMS: To characterise patients experiencing CHS in North America and to obtain a population-based estimate of CHS treatment prevalence in Canada before and during the Covid-19 pandemic METHODS: Internet survey of 157 CHS sufferers in Canada and the United States. Administrative health databases for the province of Alberta (population 5 million) were accessed to measure emergency department (ED) visits for vomiting, with a concurrent diagnostic code for cannabis use. Three time periods of 1 year were assessed: prior to recreational cannabis legalisation (2017-2018), after recreational legalisation (2018-2019) and during the first year of the Covid-19 pandemic (2020-2021). RESULTS: Problematic cannabis use (defined as a CUDIT-R score ≥8) was universal among the survey cohort, and 59% and 68% screening for moderate or worse anxiety or depression, respectively. The overall treatment prevalence of CHS across all ages increased from 15 ED visits per 100,000 population (95% CI, 14-17) prior to legalisation, to 21 (95% CI, 20-23) after legalisation, to 32 (95% CI, 31-35) during the beginning of the Covid-19 pandemic (p < 0.001). Treatment prevalence among chronic cannabis users was as high as 6 per 1000 in the 16-24 age group. CONCLUSION: Survey data suggest patients with CHS almost universally suffer from a cannabis use disorder, which has significant treatment implications. Treatment prevalence in the ED has increased substantially over a very short time period, with the highest rates seen during the Covid-19 pandemic.
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COVID-19 , Cannabinoides , Humanos , Cannabinoides/efectos adversos , Prevalencia , COVID-19/epidemiología , Pandemias , Vómitos/inducido químicamente , Vómitos/epidemiología , Síndrome , América del NorteRESUMEN
The physiological functions controlled by T-type channels are intrinsically dependent on their gating properties, and alteration of T-type channel activity is linked to several human disorders. Therefore, it is essential to develop a clear understanding of the structural determinants responsible for the unique gating features of T-type channels. Here, we have investigated the specific role of the carboxy terminal region by creating a series a deletion constructs expressed in tsA-201 cells and analyzing them by patch clamp electrophysiology. Our data reveal that the proximal region of the carboxy terminus contains a structural determinant essential for shaping several gating aspects of Cav3.3 channels, including voltage-dependence of activation and inactivation, inactivation kinetics, and coupling between the voltage sensing and the pore opening of the channel. Altogether, our data are consistent with a model in which the carboxy terminus stabilizes the channel in a closed state.
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Canales de Calcio Tipo T/metabolismo , Activación del Canal Iónico , Secuencia de Aminoácidos , Células HEK293 , Humanos , Cinética , Relación Estructura-ActividadRESUMEN
Despite the development of newer anti-seizure medications over the past 50 years, 30-40% of patients with epilepsy remain refractory to treatment. One explanation for this lack of progress is that the current screening process is largely biased towards transmembrane channels and receptors, and ignores intracellular proteins and enzymes that might serve as efficacious molecular targets. Here, we report the development of a novel drug screening platform that harnesses the power of zebrafish genetics and combines it with in vivo bioenergetics screening assays to uncover therapeutic agents that improve mitochondrial health in diseased animals. By screening commercially available chemical libraries of approved drugs, for which the molecular targets and pathways are well characterized, we were able to reverse-identify the proteins targeted by efficacious compounds and confirm the physiological roles that they play by utilizing other pharmacological ligands. Indeed, using an 870-compound screen in kcna1-morpholino epileptic zebrafish larvae, we uncovered vorinostat (Zolinza™; suberanilohydroxamic acid, SAHA) as a potent anti-seizure agent. We further demonstrated that vorinostat decreased average daily seizures by â¼60% in epileptic Kcna1-null mice using video-EEG recordings. Given that vorinostat is a broad histone deacetylase (HDAC) inhibitor, we then delineated a specific subset of HDACs, namely HDACs 1 and 3, as potential drug targets for future screening. In summary, we have developed a novel phenotypic, metabolism-based experimental therapeutics platform that can be used to identify new molecular targets for future drug discovery in epilepsy.
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Histona Desacetilasas/metabolismo , Convulsiones/metabolismo , Convulsiones/terapia , Animales , Animales Modificados Genéticamente , Anticonvulsivantes/uso terapéutico , Modelos Animales de Enfermedad , Sistemas de Liberación de Medicamentos , Evaluación Preclínica de Medicamentos , Electrochoque/efectos adversos , Embrión no Mamífero , Metabolismo Energético/efectos de los fármacos , Metabolismo Energético/genética , Inhibidores de Histona Desacetilasas/uso terapéutico , Canal de Potasio Kv.1.1/genética , Canal de Potasio Kv.1.1/metabolismo , Ratones , Morfolinos , Pentilenotetrazol/toxicidad , Desempeño Psicomotor/fisiología , Convulsiones/etiología , Convulsiones/genética , Vorinostat/uso terapéutico , Pez CebraRESUMEN
Night blindness can result from impaired photoreceptor function and a subset of cases have been linked to dysfunction of Cav1.4 calcium channels and in turn compromised synaptic transmission. Here, we show that active zone proteins RIM1/2 are important regulators of Cav1.4 channel function in mouse rod photoreceptors and thus synaptic activity. The conditional double knock-out (cdko) of RIM1 and RIM2 from rods starting a few weeks after birth did not change Cav1.4 protein expression at rod ribbon synapses nor was the morphology of the ribbon altered. Heterologous overexpression of RIM2 with Cav1.4 had no significant influence on current density when examined with BaCl2 as the charge carrier. Nonetheless, whole-cell voltage-clamp recordings from cdko rods revealed a profound reduction in Ca(2+) currents. Concomitantly, we observed a 4-fold reduction in spontaneous miniature release events from the cdko rod terminals and an almost complete absence of evoked responses when monitoring changes in membrane incorporation after strong step depolarizations. Under control conditions, 49 and 83 vesicles were released with 0.2 and 1 s depolarizations, respectively, which is close to the maximal number of vesicles estimated to be docked at the base of the ribbon active zone, but without RIM1/2, only a few vesicles were stimulated for release after a 1 s stimulation. In conclusion, our study shows that RIM1/2 potently enhance the influx of Ca(2+) into rod terminals through Cav1.4 channels, which is vitally important for the release of vesicles from the rod ribbon. Significance statement: Active zone scaffolding proteins are thought to bring multiple components involved in Ca(2+)-dependent exocytosis into functional interactions. We show that removal of scaffolding proteins RIM1/2 from rod photoreceptor ribbon synapses causes a dramatic loss of Ca(2+) influx through Cav1.4 channels and a correlated reduction in evoked release, yet the channels remain localized to synaptic ribbons in a normal fashion. Our findings strongly argue that RIM1/2 facilitate Ca(2+) entry and in turn Ca(2+) evoked release by modulating Cav1.4 channel openings; however, RIM1/2 are not needed for the retention of Cav1.4 at the synapse. In summary, a key function of RIM1/2 at rod ribbons is to enhance Cav1.4 channel activity, possibly through direct or indirect modulation of the channel.
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Fenómenos Biofísicos/genética , Canales de Calcio/metabolismo , Calcio/metabolismo , Proteínas de Unión al GTP/metabolismo , Regulación de la Expresión Génica/genética , Células Fotorreceptoras Retinianas Bastones/fisiología , Proteínas de Unión al GTP rab3/metabolismo , Animales , Ácido Aspártico/farmacología , Compuestos de Bario/farmacología , Fenómenos Biofísicos/efectos de los fármacos , Canales de Calcio/genética , Canales de Calcio Tipo L , Cloruros/farmacología , Fármacos actuantes sobre Aminoácidos Excitadores/farmacología , Proteínas de Unión al GTP/genética , Regulación de la Expresión Génica/efectos de los fármacos , Células HEK293 , Humanos , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Terminales Presinápticos/metabolismo , Terminales Presinápticos/ultraestructura , Retina/citología , Células Fotorreceptoras Retinianas Bastones/efectos de los fármacos , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/genética , Vesículas Sinápticas/efectos de los fármacos , Vesículas Sinápticas/metabolismo , Vesículas Sinápticas/ultraestructura , Proteínas de Unión al GTP rab3/genéticaRESUMEN
The L-type voltage-gated calcium channel Cav1.2 and the calcium-activated CaM kinase cascade both regulate excitation transcription coupling in the brain. CaM kinase is known to associate with the C terminus of Cav1.2 in a region called the PreIQ-IQ domain, which also binds multiple calmodulin molecules. Here we identify and characterize a second CaMKII binding site in the N terminus of Cav1.2 that is formed by a stretch of four amino residues (cysteine-isoleucine-serine-isoleucine) and which regulates channel expression and function. By using live cell imaging of tsA-201 cells we show that GFP fusion constructs of the CaMKII binding region, termed N2B-II co-localize with mCherry-CaMKII. Mutating CISI to AAAA ablates binding to and colocalization with CaMKII. Cav1.2-AAAA channels show reduced cell surface expression in tsA-201 cells, but interestingly, display an increase in channel function that offsets the trafficking deficit. Altogether our data reveal that the proximal N terminus of Cav1.2 contains a CaMKII binding region which contributes to channel surface expression and function.
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Canales de Calcio Tipo L/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Potenciales de Acción , Secuencia de Aminoácidos , Animales , Sitios de Unión , Canales de Calcio Tipo L/química , Membrana Celular/metabolismo , Células HEK293 , Humanos , Datos de Secuencia Molecular , Unión Proteica , Transporte de Proteínas , RatasRESUMEN
Calcium-activated potassium channels of the KCa1.1 class are known to regulate repolarization of action potential discharge through a molecular association with high voltage-activated calcium channels. The current study examined the potential for low voltage-activated Cav3 (T-type) calcium channels to interact with KCa1.1 when expressed in tsA-201 cells and in rat medial vestibular neurons (MVN) in vitro. Expression of the channel α-subunits alone in tsA-201 cells was sufficient to enable Cav3 activation of KCa1.1 current. Cav3 calcium influx induced a 50 mV negative shift in KCa1.1 voltage for activation, an interaction that was blocked by Cav3 or KCa1.1 channel blockers, or high internal EGTA. Cav3 and KCa1.1 channels coimmunoprecipitated from lysates of either tsA-201 cells or rat brain, with Cav3 channels associating with the transmembrane S0 segment of the KCa1.1 N-terminus. KCa1.1 channel activation was closely aligned with Cav3 calcium conductance in that KCa1.1 current shared the same low voltage dependence of Cav3 activation, and was blocked by voltage-dependent inactivation of Cav3 channels or by coexpressing a non calcium-conducting Cav3 channel pore mutant. The Cav3-KCa1.1 interaction was found to function highly effectively in a subset of MVN neurons by activating near -50 mV to contribute to spike repolarization and gain of firing. Modelling data indicate that multiple neighboring Cav3-KCa1.1 complexes must act cooperatively to raise calcium to sufficiently high levels to permit KCa1.1 activation. Together the results identify a novel Cav3-KCa1.1 signaling complex where Cav3-mediated calcium entry enables KCa1.1 activation over a wide range of membrane potentials according to the unique voltage profile of Cav3 calcium channels, greatly extending the roles for KCa1.1 potassium channels in controlling membrane excitability.
Asunto(s)
Calcio/metabolismo , Caveolina 3/metabolismo , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/metabolismo , Neuronas/metabolismo , Subunidades de Proteína/metabolismo , Transducción de Señal/fisiología , Núcleos Vestibulares/metabolismo , Potenciales de Acción/fisiología , Secuencia de Aminoácidos , Animales , Bloqueadores de los Canales de Calcio/farmacología , Caveolina 3/genética , Línea Celular Transformada , Expresión Génica , Humanos , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/genética , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Neuronas/citología , Neuronas/efectos de los fármacos , Técnicas de Placa-Clamp , Estructura Terciaria de Proteína , Subunidades de Proteína/genética , Ratas , Núcleos Vestibulares/citología , Núcleos Vestibulares/efectos de los fármacosRESUMEN
Encoding sensory input requires the expression of postsynaptic ion channels to transform key features of afferent input to an appropriate pattern of spike output. Although Ca(2+)-activated K(+) channels are known to control spike frequency in central neurons, Ca(2+)-activated K(+) channels of intermediate conductance (KCa3.1) are believed to be restricted to peripheral neurons. We now report that cerebellar Purkinje cells express KCa3.1 channels, as evidenced through single-cell RT-PCR, immunocytochemistry, pharmacology, and single-channel recordings. Furthermore, KCa3.1 channels coimmunoprecipitate and interact with low voltage-activated Cav3.2 Ca(2+) channels at the nanodomain level to support a previously undescribed transient voltage- and Ca(2+)-dependent current. As a result, subthreshold parallel fiber excitatory postsynaptic potentials (EPSPs) activate Cav3 Ca(2+) influx to trigger a KCa3.1-mediated regulation of the EPSP and subsequent after-hyperpolarization. The Cav3-KCa3.1 complex provides powerful control over temporal summation of EPSPs, effectively suppressing low frequencies of parallel fiber input. KCa3.1 channels thus contribute to a high-pass filter that allows Purkinje cells to respond preferentially to high-frequency parallel fiber bursts characteristic of sensory input.
Asunto(s)
Canales de Potasio de Conductancia Intermedia Activados por el Calcio/fisiología , Células de Purkinje/fisiología , Animales , Femenino , Inmunohistoquímica , Embarazo , Células de Purkinje/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
A-type potassium current generated by the K(V)4 family of channels is an important factor regulating the frequency, latency and dendritic backpropagation of spike discharge. The K(V)4.2 complex of K(V)4.2-KChIP3-DPP10c was recently shown to form a novel signaling complex through its association with T-type Ca(V)3.2 or Ca(V)3.3 calcium channel isoforms. Ca(V)3-mediated calcium entry was shown to selectively right-shift the inactivation voltage of K(V)4.2 into the physiological range to modulate cerebellar stellate cell latency and gain. We now show that Ca(V)3.1 calcium channels can also associate with the K(V)4.2 complex to effect similar regulation of K(V)4.2 inactivation voltage. By comparison, no calcium-dependent shift in K(V)4.2 inactivation properties was elicited by any of Ca(V)1.4, Ca(V)2.1 or Ca(V)2.3 calcium channels coexpressed with the K(V)4.2 complex, emphasizing the important role for low voltage-activated Ca(V)3 channels in this signaling complex.
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Canales de Calcio Tipo T/fisiología , Canales de Potasio Shal/metabolismo , Potenciales de Acción , Animales , Calcio/metabolismo , Calcio/farmacología , Línea Celular , Electrofisiología , Técnicas de Placa-Clamp , Ratas , TransfecciónRESUMEN
Kv4 low voltage-activated A-type potassium channels are widely expressed in excitable cells, where they control action potential firing, dendritic activity and synaptic integration. Kv4 channels exist as a complex that includes K(+) channel-interacting proteins (KChIPs), which contain calcium-binding domains and therefore have the potential to confer calcium dependence on the Kv4 channel. We found that T-type calcium channels and Kv4 channels form a signaling complex in rat that efficiently couples calcium influx to KChIP3 to modulate Kv4 function. This interaction was critical for allowing Kv4 channels to function in the subthreshold membrane potential range to regulate neuronal firing properties. The widespread expression of these channels and accessory proteins indicates that the Cav3-Kv4 signaling complex is important for the function of a wide range of electrically excitable cells.
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Canales de Calcio Tipo T/metabolismo , Neuronas/fisiología , Canales de Potasio Shal/metabolismo , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Animales , Encéfalo/efectos de los fármacos , Encéfalo/fisiología , Calcio/metabolismo , Canales de Calcio Tipo T/genética , Línea Celular , Espacio Extracelular/metabolismo , Humanos , Técnicas In Vitro , Cinética , Proteínas de Interacción con los Canales Kv/metabolismo , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Neuronas/efectos de los fármacos , Ratas , Ratas Sprague-DawleyRESUMEN
OBJECTIVE: The deleterious effects of glutamate excitotoxicity are well described for central nervous system gray matter. Although overactivation of glutamate receptors also contributes to axonal injury, the mechanisms are poorly understood. Our goal was to elucidate the mechanisms of kainate receptor-dependent axonal Ca(2+) deregulation. METHODS: Dorsal column axons were loaded with a Ca(2+) indicator and imaged in vitro using confocal laser-scanning microscopy. RESULTS: Activation of glutamate receptor 6 (GluR6) kainate receptors promoted a substantial increase in axonal [Ca(2+)]. This Ca(2+) accumulation was due not only to influx from the extracellular space, but a significant component originated from ryanodine-dependent intracellular stores, which, in turn, depended on activation of L-type Ca(2+) channels: ryanodine, nimodipine, or nifedipine blocked the agonist-induced Ca(2+) increase. Also, GluR6 stimulation induced intraaxonal production of nitric oxide (NO), which greatly enhanced the Ca(2+) response: quenching of NO with intraaxonal (but not extracellular) scavengers, or inhibition of neuronal NO synthase with intraaxonal Nomega-nitro-L-arginine methyl ester, blocked the Ca(2+) increase. Loading axons with a peptide that mimics the C-terminal PDZ binding sequence of GluR6, thus interfering with the coupling of GluR6 to downstream effectors, greatly reduced the agonist-induced axonal Ca(2+) increase. Immunohistochemistry showed GluR6/7 clusters on the axolemma colocalized with neuronal NO synthase and Ca(v)1.2. INTERPRETATION: Myelinated spinal axons express functional GluR6-containing kainate receptors, forming part of novel signaling complexes reminiscent of postsynaptic membranes of glutamatergic synapses. The ability of such axonal "nanocomplexes" to release toxic amounts of Ca(2+) may represent a key mechanism of axonal degeneration in disorders such as multiple sclerosis where abnormal accumulation of glutamate and NO are known to occur.
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Axones/metabolismo , Fibras Nerviosas Mielínicas/metabolismo , Receptores de Ácido Kaínico/fisiología , Raíces Nerviosas Espinales/citología , Animales , Axones/efectos de los fármacos , Calcio/metabolismo , Canales de Calcio Tipo L/fisiología , Cisteína/metabolismo , Ácido Egtácico/análogos & derivados , Antagonistas de Aminoácidos Excitadores/farmacología , Ácido Glutámico/farmacología , Hidroxocobalamina/farmacología , Masculino , Microscopía Confocal/métodos , Mioglobina/farmacología , Fibras Nerviosas Mielínicas/efectos de los fármacos , Óxido Nítrico/metabolismo , Óxido Nítrico/farmacología , Dominios PDZ/fisiología , Péptidos/metabolismo , Multimerización de Proteína/fisiología , Ratas , Ratas Long-Evans , Receptores de Ácido Kaínico/química , Rianodina/farmacología , Traumatismos de la Médula Espinal/metabolismo , Receptor de Ácido Kaínico GluK2RESUMEN
We have generated a syntaxin 1A knockout mouse by deletion of exons 3 through 6 and a concomitant insertion of a stop codon in exon 2. Heterozygous knockout animals were viable with no apparent phenotype. In contrast, the vast majority of homozygous animals died in utero, with embryos examined at day E15 showing a drastic reduction in body size and development when compared to WT and heterozygous littermates. Surprisingly, out of a total of 204 offspring from heterozygous breeding pairs only four homozygous animals were born alive and viable. These animals exhibited reduced body weight, but showed only mild behavioral deficiencies. Taken together, our data indicate that syntaxin 1A is an important regulator of normal in utero development, but may not be essential for normal brain function later in life.
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Desarrollo Embrionario , Muerte Fetal , Sintaxina 1/fisiología , Útero/fisiología , Animales , Codón de Terminación/genética , Desarrollo Embrionario/genética , Exones/genética , Femenino , Muerte Fetal/genética , Peso Fetal/genética , Humanos , Ratones , Ratones Noqueados , Ratas , Eliminación de Secuencia , Sintaxina 1/genéticaRESUMEN
The Cacna1f(nob2) mouse is reported to be a naturally occurring null mutation for the Ca(v)1.4 calcium channel gene and the phenotype of this mouse is not identical to that of the targeted gene knockout model. We found two mRNA species in the Cacna1f(nob2) mouse: approximately 90% of the mRNA represents a transcript with an in-frame stop codon within exon 2 of CACNA1F, while approximately 10% of the mRNA represents a transcript in which alternative splicing within the ETn element has removed the stop codon. This latter mRNA codes for full length Ca(v)1.4 protein, detectable by Western blot analysis that is predicted to differ from wild type Ca(v)1.4 protein in a region of approximately 22 amino acids in the N-terminal portion of the protein. Electrophysiological analysis with either mouse Ca(v)1.4(wt) or Ca(v)1.4(nob2) cDNA revealed that the alternatively spliced protein does not differ from wild type with respect to activation and inactivation characteristics; however, while the wild type N-terminus interacted with filamin proteins in a biochemical pull-down experiment, the alternatively spliced N-terminus did not. The Cacna1f(nob2) mouse electroretinogram displayed reduced b-wave and oscillatory potential amplitudes, and the retina was morphologically disorganized, with substantial reduction in thickness of the outer plexiform layer and sprouting of bipolar cell dendrites ectopically into the outer nuclear layer. Nevertheless, the spatial contrast sensitivity (optokinetic response) of Cacna1f(nob2) mice was generally similar to that of wild type mice. These results suggest the Cacna1f(nob2) mouse is not a CACNA1F knockout model. Rather, alternative splicing within the ETn element can lead to full-length Ca(v)1.4 protein, albeit at reduced levels, and the functional Ca(v)1.4 mutant may be incapable of interacting with cytoskeletal filamin proteins. These changes, do not alter the ability of the Cacna1f(nob2) mouse to detect and follow moving sine-wave gratings compared to their wild type counterparts.
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Empalme Alternativo , Canales de Calcio Tipo L/genética , Canales de Calcio/genética , Exones , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Canales de Calcio/metabolismo , Canales de Calcio Tipo L/metabolismo , Ratones , Datos de Secuencia Molecular , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
T-type calcium channels are thought to transform neuronal output to a burst mode by generating low voltage-activated (LVA) calcium currents and rebound burst discharge. In this study we assess the expression pattern of the three different T-type channel isoforms (Ca(v)3.1, Ca(v)3.2, and Ca(v)3.3) in cerebellar neurons and focus on their potential role in generating LVA spikes and rebound discharge in deep cerebellar nuclear (DCN) neurons. We detected expression of one or more Ca(v)3 channel isoforms in a wide range of cerebellar neurons and selective expression of different isoforms in DCN cells. We further identify two classes of large-diameter DCN neurons that exhibit either a strong or weak capability for rebound discharge, despite the ability to generate LVA spikes when calcium currents are pharmacologically isolated. By correlating the Ca(v)3 channel expression pattern with the electrophysiological profile of identified DCN cells, we show that Ca(v)3.1 channels are expressed in isolation in DCN-burst cells, whereas Ca(v)3.3 is expressed in DCN-weak burst cells. Ca(v)3.1-expressing DCN cells correspond to excitatory or GABAergic neurons, whereas Ca(v)3.3-expressing cells are non-GABAergic. The Ca(v)3 class of LVA calcium channels is thus expressed in specific combinations in a wide range of cerebellar neurons but contributes to rebound burst discharge in only a select number of cell classes.