RESUMEN
Voltage-gated Na+ channels (Nav ) modulate neuronal excitability, but the roles of the various Nav subtypes in specific neuronal functions such as synaptic transmission are unclear. We investigated expression of the three major brain Nav subtypes (Nav 1.1, Nav 1.2, Nav 1.6) in area CA1 and dentate gyrus of rat hippocampus. Using light and electron microscopy, we found labeling for all three Nav subtypes on dendrites, dendritic spines, and axon terminals, but the proportion of pre- and post-synaptic labeling for each subtype varied within and between subregions of CA1 and dentate gyrus. In the central hilus (CH) of the dentate gyrus, Nav 1.1 immunoreactivity was selectively expressed in presynaptic profiles, while Nav 1.2 and Nav 1.6 were expressed both pre- and post-synaptically. In contrast, in the stratum radiatum (SR) of CA1, Nav 1.1, Nav 1.2, and Nav 1.6 were selectively expressed in postsynaptic profiles. We next compared differences in Nav subtype expression between CH and SR axon terminals and between CH and SR dendrites and spines. Nav 1.1 and Nav 1.2 immunoreactivity was preferentially localized to CH axon terminals compared to SR, and in SR dendrites and spines compared to CH. No differences in Nav 1.6 immunoreactivity were found between axon terminals of CH and SR or between dendrites and spines of CH and SR. All Nav subtypes in both CH and SR were preferentially associated with asymmetric synapses rather than symmetric synapses. These findings indicate selective presynaptic and postsynaptic Nav expression in glutamatergic synapses of CH and SR supporting neurotransmitter release and synaptic plasticity.
Asunto(s)
Hipocampo/citología , Neuronas/fisiología , Densidad Postsináptica/metabolismo , Terminales Presinápticos/metabolismo , Subunidades de Proteína/metabolismo , Canales de Sodio Activados por Voltaje/metabolismo , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/genética , Animales , Células Cultivadas , Embrión de Mamíferos , Células HEK293 , Humanos , Masculino , Canal de Sodio Activado por Voltaje NAV1.1/metabolismo , Canal de Sodio Activado por Voltaje NAV1.1/ultraestructura , Canal de Sodio Activado por Voltaje NAV1.2/metabolismo , Canal de Sodio Activado por Voltaje NAV1.2/ultraestructura , Canal de Sodio Activado por Voltaje NAV1.6/metabolismo , Canal de Sodio Activado por Voltaje NAV1.6/ultraestructura , Plasticidad Neuronal/genética , Neuronas/ultraestructura , Densidad Postsináptica/efectos de los fármacos , Densidad Postsináptica/ultraestructura , Terminales Presinápticos/efectos de los fármacos , Terminales Presinápticos/ultraestructura , Subunidades de Proteína/genética , Ratas , Ratas Sprague-Dawley , Canales de Sodio Activados por Voltaje/genética , Canales de Sodio Activados por Voltaje/ultraestructuraRESUMEN
The axon initial segment (AIS) is the site of initiation of action potentials and influences action potential waveform, firing pattern, and rate. In view of the fundamental aspects of motor function and behavior that depend on the firing of substantia nigra pars compacta (SNc) dopaminergic neurons, we identified and characterized their AIS in the mouse. Immunostaining for tyrosine hydroxylase (TH), sodium channels (Nav ) and ankyrin-G (Ank-G) was used to visualize the AIS of dopaminergic neurons. Reconstructions of sampled AIS of dopaminergic neurons revealed variable lengths (12-60 µm) and diameters (0.2-0.8 µm), and an average of 50% reduction in diameter between their widest and thinnest parts. Ultrastructural analysis revealed submembranous localization of Ank-G at nodes of Ranvier and AIS. Serial ultrathin section analysis and 3D reconstructions revealed that Ank-G colocalized with TH only at the AIS. Few cases of synaptic innervation of the AIS of dopaminergic neurons were observed. mRNA in situ hybridization of brain-specific Nav subunits revealed the expression of Nav 1.2 by most SNc neurons and a small proportion expressing Nav 1.6. The presence of sodium channels, along with the submembranous location of Ank-G is consistent with the role of AIS in action potential generation. Differences in the size of the AIS likely underlie differences in firing pattern, while the tapering diameter of AIS may define a trigger zone for action potentials. Finally, the conspicuous expression of Nav 1.2 by the majority of dopaminergic neurons may explain their high threshold for firing and their low discharge rate.
Asunto(s)
Segmento Inicial del Axón/fisiología , Neuronas Dopaminérgicas/citología , Sustancia Negra/citología , Potenciales de Acción/fisiología , Animales , Ancirinas/metabolismo , Ancirinas/ultraestructura , Segmento Inicial del Axón/ultraestructura , Expresión Génica/fisiología , Imagenología Tridimensional , Masculino , Ratones , Ratones Endogámicos C57BL , Microscopía Electrónica de Transmisión , Microscopía Inmunoelectrónica , Canal de Sodio Activado por Voltaje NAV1.2/genética , Canal de Sodio Activado por Voltaje NAV1.2/metabolismo , Canal de Sodio Activado por Voltaje NAV1.2/ultraestructura , Canal de Sodio Activado por Voltaje NAV1.6/genética , Canal de Sodio Activado por Voltaje NAV1.6/metabolismo , Canal de Sodio Activado por Voltaje NAV1.6/ultraestructura , Neuroimagen , ARN Mensajero/metabolismo , Tirosina 3-Monooxigenasa/metabolismo , Tirosina 3-Monooxigenasa/ultraestructuraRESUMEN
With the ultimate goal of detailed structural analysis of mammalian and particularly human voltage-gated sodium channels (VGSCs), we have investigated the relative stability of human and rat VGSCs and compared them with electric eel VGSC. We found that NaV1.3 from rat was the most stable after detergent solubilisation. The order of stability was rNaV1.3>hNaV1.2>hNaV1.1>hNaV1.6>hNaV1.3>hNaV1.4. However, a comparison with the VGSC from Electrophorus electricus, which is most similar to NaV1.4, shows that the eel VGSC is considerably more stable in detergent than the human VGSCs examined. We conclude that current methods of structural analysis, such as single particle electron cryomicroscopy (cryoEM), may be most usefully targeted to eel VGSC or rNaV1.3, but that structural analysis on the full spectrum of VGSCs, by methods that require greater stability such as crystallisation and X-ray crystallography, will require further stabilisation of the channel.