Selective localization of cardiac SCN5A sodium channels in limbic regions of rat brain

Abstract

Voltage-dependent sodium channels have a critical role in membrane electrogenesis and repetitive firing in excitable cells. Members of this gene superfamily exhibit diffuse, tissue-specific expression in nervous, muscle and cardiac tissues 1 . Tetrodotoxin (TTX) sensitivity and resistance to zinc blockade were thought to distinguish brain from heart sodium channels, but neuronal Na + currents with heart-like properties have been found in brain 2 .Here we report selective expression of the cardiac sodium channel gene SCN5A in neurons within specific limbic pathways. In-situ hybridization and RT-PCR analysis revealed SCN5A mRNA in piriform cortex and subcortical limbic nuclei. SCN5A is the first neuronal sodium channel to show a restricted forebrain expression pattern, and provides a mechanism to regulate excitability and rhythmic firing within these circuits. Limbic networks show striking propensity for epileptogenesis, and seizures are associated with SCN5A mutations linked to human cardiac long-QT syndrome and ventricular fibrillation. Our results suggest that such arrhythmias of heart and brain may be related and implicate SCN5A in some forms of primary inherited epilepsy. Na + currents with TTX resistance have been observed in neurons of the entorhinal cortex 2 , neocortex 3 , hippocampus and striatum 4 , although the genes corresponding to these currents in the CNS are unknown. Northern blots of total brain RNA failed to demonstrate expression of SCN5A mRNA 5 ; however, the more sensitive polymerase chain reaction (PCR) amplified low levels of SCN5A cDNA from rat cortex 6 .We used in-situ hybridization to anatomically locate SCN5A expression more precisely in mammalian brain. Two sets of sense and antisense probes were designed to unique regions of the SCN5A cDNA sequence 7 . The first set (PR1) included two 45-mer oligonucleotides containing sequence in the coding region of rH1 (SCN5A; residues 638-652, bases 1912-1956). The second set (PR2) comprised two 50-mer oligonucleotides for sequence in the 3¢ untranslated region of rH1 (bases 6301-6350). Probes were 3¢-end-labeled using terminal deoxynucleotidyl transferase and [ 35 S]adATP (1300 Ci per mmol) to a specific activity of ~10 9 dpm per mg, purified on a 1-ml column spun at 25 ´g to remove unincorporated radiolabeled nucleotides and hybridized to adult rat brain sections.Autoradiograms using SCN5A antisense probes revealed a sharply demarcated expression pattern in deep layers of the piriform cortex and within subcortical limbic circuitry (Fig. 1). Maximal specific labeling was observed throughout lateral septal nuclei, the bed nucleus of stria terminalis, piriform cortex, amygdala, periamygdaloid cortex and medial hypothalamic nuclei. PR1 and PR2 probes showed identical results. Very little SCN5A transcript was detected in the hippocampal formation. Hybridization in other brain regions was comparable to that detected with the non-specific sense strand and to those in control hybridizations of the labeled antisense oligomer in the presence of excess unla...

Localization of Nav1.5 sodium channel protein in the mouse brain

“…Our study represents the first detailed characterization of Na v 1.5 protein distribution in the mouse brain. Consistent with the distribution of Na v 1.5 transcripts in rat and human brain [2,12], the mouse Na v 1.5 protein was present in the limbic system, olfactory system, thalamus, and hypothalamus. However, our study revealed localization of Na v 1.5 protein in several brain regions that were not described or analyzed in previous studies.…”

“…2d), and weak staining in the olfactory tubercule (data not shown). Meanwhile, in the limbic system where Na v 1.5 mRNA was reported to be strongly expressed [2,12], moderate Na v 1.5 immunoreactivity was detected in the nerve fibers, but no or little Na v 1.5 immunoreactivity was detected in the cell bodies in any of the limbic nuclei (Fig. 2a).…”

“…Na v 1.5 was originally identified as a cardiac sodium channel. Subsequently, it was shown to be expressed in the brain at the mRNA level [2,12], and these findings may explain whyNa v 1.5 mutations are associated with seizures. However, the distribution of Na v 1.5 in the brain at the protein level has not been investigated previously.…”

Gene Duplications and Evolution of Vertebrate Voltage-Gated Sodium Channels

“…SCN5A expression also occurs in central nervous system regions associated with the limbic system (Donahue et al 2000;Hartmann et al 1999). In zebrafish, both scn5Laa and scn5Lab are expressed in adult cardiac tissue as well as in neurons, similar to SCN5A (Fig.…”

“…Both scn5Laa and scn5Lab were also expressed in nervous and cardiac tissues. In mammals, SCN5A is expressed in cardiac muscle and limited regions of the brain (Hartmann et al 1999;Rogart et al 1989), while SCN10A and SCN11A are expressed in peripheral nociceptive sensory neurons (Akopian et al 1996;Dib-Hajj et al 1999a;Sangameswaran et al 1996). However, mammalian expression of SCN5A, SCN10A or SCN11A has not been reported in spinal cord or eye.…”