RESUMEN
Chronically elevated sympathetic nervous activity underlies many cardiovascular diseases. Elucidating the mechanisms contributing to sympathetic nervous system output may reveal new avenues of treatment. The contribution of the gap junctional protein connexin 36 (Cx36) to the regulation of sympathetic activity and thus blood pressure and heart rate was determined using a mouse with specific genetic deletion of Cx36. Ablation of the Cx36 protein was confirmed in sympathetic preganglionic neurons of Cx36-knockout (KO) mice. Telemetric analysis from conscious Cx36 KO mice revealed higher variance in heart rate and blood pressure during rest and activity compared to wild-type (WT) mice, and smaller responses to chemoreceptor activation when anesthetized. In the working heart-brain stem preparation of the Cx36-KO mouse, respiratory-coupled sympathetic nerve discharge was attenuated and responses to chemoreceptor stimulation and noxious stimulation were blunted compared to WT mice. Using whole cell patch recordings, sympathetic preganglionic neurons in spinal cord slices of Cx36-KO mice displayed lower levels of spikelet activity compared to WT mice, indicating reduced gap junction coupling between neurons. Cx36 deletion therefore disrupts normal regulation of sympathetic outflow with effects on cardiovascular parameters.-Lall, V. K., Bruce, G., Voytenko, L., Drinkhill, M., Wellershaus, K., Willecke, K., Deuchars, J., Deuchars, S. A. Physiologic regulation of heart rate and blood pressure involves connexin 36-containing gap junctions.
Asunto(s)
Presión Sanguínea/fisiología , Conexinas/metabolismo , Uniones Comunicantes/fisiología , Frecuencia Cardíaca/fisiología , Animales , Células Quimiorreceptoras/efectos de los fármacos , Conexinas/genética , Fenómenos Electrofisiológicos , Femenino , Masculino , Ratones , Ratones Noqueados , Cianuro de Sodio/farmacología , Sistema Nervioso Simpático/fisiología , Proteína delta-6 de Union ComunicanteRESUMEN
The level of electrotonic coupling in the inferior olive is extremely high, but its functional role in cerebellar motor control remains elusive. Here, we subjected mice that lack olivary coupling to paradigms that require learning-dependent timing. Cx36-deficient mice showed impaired timing of both locomotion and eye-blink responses that were conditioned to a tone. The latencies of their olivary spike activities in response to the unconditioned stimulus were significantly more variable than those in wild-types. Whole-cell recordings of olivary neurons in vivo showed that these differences in spike timing result at least in part from altered interactions with their subthreshold oscillations. These results, combined with analyses of olivary activities in computer simulations at both the cellular and systems level, suggest that electrotonic coupling among olivary neurons by gap junctions is essential for proper timing of their action potentials and thereby for learning-dependent timing in cerebellar motor control.
Asunto(s)
Cerebelo/fisiología , Uniones Comunicantes/fisiología , Aprendizaje/fisiología , Neuronas/fisiología , Núcleo Olivar/citología , Estimulación Acústica/efectos adversos , Potenciales de Acción/fisiología , Animales , Parpadeo/fisiología , Simulación por Computador , Conexinas/deficiencia , Locomoción/genética , Ratones , Ratones Noqueados , Modelos Neurológicos , Técnicas de Placa-Clamp/métodos , Tiempo de Reacción/fisiología , Factores de Tiempo , Proteína delta-6 de Union ComunicanteRESUMEN
BACKGROUND: The visual system can adjust itself to different visual environments. One of the most well known examples of this is the shift in spatial tuning that occurs in retinal ganglion cells with the change from night to day vision. This shift is thought to be produced by a change in the ganglion cell receptive field surround, mediated by a decrease in the coupling of horizontal cells. METHODOLOGY/PRINCIPAL FINDINGS: To test this hypothesis, we used a transgenic mouse line, a connexin57-deficient line, in which horizontal cell coupling was abolished. Measurements, both at the ganglion cell level and the level of behavioral performance, showed no differences between wild-type retinas and retinas with decoupled horizontal cells from connexin57-deficient mice. CONCLUSION/SIGNIFICANCE: This analysis showed that the coupling and uncoupling of horizontal cells does not play a dominant role in spatial tuning and its adjustability to night and day light conditions. Instead, our data suggest that another mechanism, likely arising in the inner retina, must be responsible.
Asunto(s)
Luz , Células Ganglionares de la Retina/metabolismo , Células Horizontales de la Retina/metabolismo , Percepción Espacial , Campos Visuales/fisiología , Animales , Conducta Animal , Conexinas/fisiología , Dopamina/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células Fotorreceptoras/efectos de la radiación , Células Ganglionares de la Retina/efectos de la radiación , Células Horizontales de la Retina/efectos de la radiaciónRESUMEN
Connexin36 (Cx36) is the main connexin isoform expressed in neurons of the central nervous system (CNS) and in pancreatic beta-cells, i.e. two types of excitable cells that share - in spite of their different origins - a number of common features. Previous studies on Cx36 deficient mice have documented that loss of Cx36 resulted in phenotypic abnormalities in both the CNS and the pancreas which, however, could not be attributed to specific cell types due to the general deletion nature of the animal model used. Attempts to address this limitation using cell type specific deletions generated by the Cre/loxP strategy have so far been complicated by the lack of Cx36 expression from the floxed allele. We have now generated a conditional Cx36 deficient mouse mutant in which the coding region of Cx36 is flanked by loxP sites, followed by a cyan fluorescent protein (CFP) reporter gene. Here we show that Cx36 was still expressed from the floxed allele in neurons and pancreatic beta-cells. In these cells, a 30-60% decrease of this protein, relative to the expression level of the wildtype allele, did not significantly perturb cell coupling. The deletion of Cx36 by ubiquitously and cell type specifically expressed Cre recombinases revealed that CFP functions as a reliable reporter for Cx36 expression in brain neurons and to some extent in retina neurons, but not in pancreas. Loss of Cx36 by Cre-mediated recombination was documented at transcript and protein levels. Cell type specific deletion of Cx36 in the endocrine pancreas revealed major alterations in the basal as well as the glucose-induced insulin secretion, hence specifically attributing to pancreatic Cx36 an important regulatory role in the control of beta-cell function. Cell type specific deletion of Cx36 in the CNS by suitable Cre recombinases should also help to elucidate the functional role of Cx36 in different neuronal subtypes.
Asunto(s)
Conexinas/genética , Conexinas/fisiología , Células Secretoras de Insulina/química , Neuronas/química , Animales , Encéfalo/citología , Conexinas/deficiencia , Regulación de la Expresión Génica , Insulina/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/metabolismo , Integrasas , Ratones , Retina/citología , Distribución Tisular , Proteína delta-6 de Union ComunicanteRESUMEN
Electrical synapses, particularly gap junctions composed of connexin (Cx) 36, have been suggested to synchronize neuronal network oscillations. Recently, we generated Cx30.2-deficient mice which express beta-galactosidase under control of Cx30.2 gene regulatory elements. In the central nervous system beta-galactosidase activity representing Cx30.2 expression was restricted to NeuN-positive cells, thus identifying Cx30.2 as new neuronal connexin. In the hippocampus, co-immunofluorescence analyses revealed beta-galactosidase/Cx30.2 expression in GABAergic inhibitory interneurons such as parvalbumin- and somatostatin-positive basket, axo-axonic, bistratified or oriens lacunosum-moleculare cells. approximately 94% of the Cx30.2 expressing parvalbumin-positive interneurons also expressed Cx36. Performing field potential recordings from hippocampal slices we found no differences in basal excitation and excitation-inhibition balance between Cx30.2+/+ and Cx30.2LacZ/LacZ)mice. Furthermore, frequency and power of gap junction dependent gamma and ripples oscillations were similar in these animals. This suggests that the lack of Cx30.2 in interneurons can be largely compensated by other connexins, most likely Cx36.
Asunto(s)
Sistema Nervioso Central/citología , Conexinas/metabolismo , Regulación del Desarrollo de la Expresión Génica/fisiología , Interneuronas/metabolismo , Potenciales de Acción/genética , Potenciales de Acción/fisiología , Animales , Animales Recién Nacidos , Conexinas/genética , Estimulación Eléctrica/métodos , Embrión de Mamíferos , Técnicas In Vitro , Ratones , Ratones Transgénicos , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Factores de Tiempo , beta-Galactosidasa/metabolismo , Proteína delta-6 de Union ComunicanteRESUMEN
To further characterize the recently described gap junction gene connexin 47 (Cx47), we generated Cx47-null mice by replacing the Cx47 coding DNA with an enhanced green fluorescent protein (EGFP) reporter gene, which was thus placed under control of the endogenous Cx47 promoter. Homozygous mutant mice were fertile and showed no obvious morphological or behavioral abnormalities. Colocalization of EGFP fluorescence and immunofluorescence of cell marker proteins revealed that Cx47 was mainly expressed in oligodendrocytes in highly myelinated CNS tissues and in few calcium-binding protein S100beta subunit-positive cells but not in neurons or peripheral sciatic nerve. This corrects our previous conclusion that Cx47 mRNA is expressed in brain and spinal cord neurons (Teubner et al., 2001). Cx47 protein was detected by Western blot analysis after immunoprecipitation in CNS tissues of wild-type mice but not in heart or Cx47-deficient tissues. Electron microscopic analysis of CNS white matter in Cx47-deficient mice revealed a conspicuous vacuolation of nerve fibers, particularly at the site of the optic nerve where axons are first contacted by oligodendrocytes and myelination starts. Initial analyses of Cx32/Cx47-double-deficient mice showed that these mice developed an action tremor and died on average at 51 d after birth. The central white matter of these double-deficient mice exhibited much more abundant vacuolation in nerve fibers than mice deficient only in Cx47.