J.A. Black

Although physiological and pharmacological evidence suggests the presence of multiple tetrodotoxin-resistant (TTX-R) Na channels in neurons of peripheral nervous system ganglia, only one, SNS/PN3, has been identified in these cells to date. We have identified and sequenced a novel Na channel alpha-subunit (NaN), predicted to be TTX-R and voltage-gated, that is expressed preferentially in sensory neurons within dorsal root ganglia (DRG) and trigeminal ganglia. The predicted amino acid sequence of NaN can be aligned with the predicted structure of known Na channel alpha-subunits; all relevant landmark sequences, including positively charged S4 and pore-lining SS1-SS2 segments, and the inactivation tripeptide IFM, are present at predicted positions. However, NaN exhibits only 42-53% similarity to other mammalian Na channels, including SNS/PN3, indicating that it is a novel channel, and suggesting that it may represent a third subfamily of Na channels. NaN transcript levels are reduced significantly 7 days post axotomy in DRG neurons, consistent with previous findings of a reduction in TTX-R Na currents. The preferential expression of NaN in DRG and trigeminal ganglia and the reduction of NaN mRNA levels in DRG after axonal injury suggest that NaN, together with SNS/PN3, may produce TTX-R currents in peripheral sensory neurons and may influence the generation of electrical activity in these cells.

Although it is well established that hyperexcitability and/or increased baseline sensitivity of primary sensory neurons can lead to abnormal burst activity associated with pain, the underlying molecular mechanisms are not fully understood. Early studies demonstrated that, after injury to their axons, neurons can display changes in excitability, suggesting increased sodium channel expression, and, in fact, abnormal sodium channel accumulation has been observed at the tips of injured axons. We have used an ensemble of molecular, electrophysiological, and pharmacological techniques to ask: what types of sodium channels underlie hyperexcitability of primary sensory neurons after injury? Our studies demonstrate that multiple sodium channels, with distinct electrophysiological properties, are encoded by distinct mRNAs within small dorsal root ganglion (DRG) neurons, which include nociceptive cells. Moreover, several DRG neuron-specific sodium channels now have been cloned and sequenced. After injury to the axons of DRG neurons, there is a dramatic change in sodium channel expression in these cells, with down-regulation of some sodium channel genes and up-regulation of another, previously silent sodium channel gene. This plasticity in sodium channel gene expression is accompanied by electrophysiological changes that poise these cells to fire spontaneously or at inappropriate high frequencies. Changes in sodium channel gene expression also are observed in experimental models of inflammatory pain. Thus, sodium channel expression in DRG neurons is dynamic, changing significantly after injury. Sodium channels within primary sensory neurons may play an important role in the pathophysiology of pain.

The expression of sodium channel alpha-, beta 1- and beta 2-subunit mRNAs was examined in adult rat DRG neurons in dissociated culture at 1 day in vitro and within sections of intact ganglia by in situ hybridization and reverse transcription polymerase chain reaction (RT-PCR). The results demonstrate that sodium channel alpha-subunit mRNAs are differentially expressed in small (< 25 microns diam), medium (25-45 microns diam.) and large (> 45 microns diam.) cultured DRG neurons at 1 day in vitro (div). Sodium channel mRNA I is expressed at higher levels in large neurons than small DRG neurons, while sodium channel mRNA II is variably expressed, with most cells lacking or exhibiting low levels of detectable signal of these mRNAs and limited numbers of neurons with moderate expression levels. DRG neurons generally exhibit negligible or low levels of hybridization signal for sodium channel mRNA III. Sodium channel mRNAs Na6 and NaG show similar patterns of expression, with most large and many medium DRG neurons exhibiting high levels of expression. The mRNA for the rat cognate of human sodium channel hNE-Na is detected in virtually every DRG neuron; most cells in all size classes exhibit moderate or high levels of hNE-Na expression. Sodium channel SNS mRNA is expressed in all size classes of DRG neurons, but shows greater expression in small and medium DRG neurons than in large neurons. The mRNA for the rat cognate of mouse sodium channel mNa 2.3 is not detected, or is detected at low levels, in most DRG neurons, regardless of size, although moderate expression is detected in some neurons. Sodium channel beta 1- and beta 2-subunit mRNAs exhibit similar expression patterns; they are detected in most DRG neurons, although the level of expression tends to be greater in large neurons than in small neurons. RT-PCR and in situ hybridization of intact adult DRG showed a similar pattern of expression of sodium channel mRNAs to that observed in DRG neurons in vitro. These results demonstrate that adult DRG neurons express multiple sodium channel mRNAs in vitro and in situ and suggest a molecular basis for the biophysical heterogeneity of sodium currents observed in these cells.

The expression of sodium channel alpha-subunit mRNAs I, II, III, NaG, Na6 and hNE (PN1) was examined in developing (E17-P30) hippocampus, cerebellum, spinal cord and dorsal root ganglia using non-isotopic in situ hybridization cytochemistry. The results showed distinct patterns of expression for each of the sodium channel mRNAs with maturation of the nervous system. In the hippocampus, sodium channel mRNA I was not detected at any developmental time, while mRNA II showed increasing hybridization signal between E17 and P30. Sodium channel mRNA III was more prevalent at late embryonic and early postnatal times, and was barely detectable at P30. The transcript for NaG showed transient expression between P2 and P15, being expressed at low levels at E17 and not being detectable at P30. Sodium channel mRNA Na6 exhibited a high level of expression between E17 and P15 in the hippocampal formation, with an attenuation of the signal by P30. hNE (PN1) mRNA was not detected in the hippocampus at any time examined. In the cerebellum, sodium channel mRNA I was not detected at E17 or P2, but became detectable in Purkinje cells at P15 and continued to show a low level of expression in these cells at P30. mRNA I was not detected at any time examined in granule cells of the cerebellum. Sodium channel mRNA II exhibited increasing expression in the developing cerebellum, and showed increasing signal in Purkinge cells beginning on P2 and granule cells on P15. Sodium channel mRNA III was down-regulated with development in the cerebellum, although mRNA III was readily detected at E17, it was not detected in any layers of the cerebellum by P15. NaG mRNA showed a peak of expression at P2, and was present at low levels at E17 and P15 and not detectable at P30. Na6 mRNA was highly expressed in the E17 cerebellum; this mRNA was present at high levels in Purkinje cells throughout development, although in granule cells the signal was attenuated at P15-P30. Sodium channel hNE (PN1) mRNA was not detected in the cerebellum at any time in development. In the spinal cord, sodium channel mRNA I showed increasing expression beginning at P2 and was highly expressed, particularly in ventral motor neurons, by P30. Sodium channel II mRNA was detected at all stages of development in the spinal cord; in contrast, mRNA III was detected at E17 and P2, but showed very low levels of expression by P30. NaG mRNA exhibited a transient expression in spinal cord at P2, but was not detectable at E17 and P30. Na6 mRNA was detectable at very low levels at E17 and became highly expressed at P2, prior to a reduction of the signal at P15 and P30. hNE (PN1) mRNA was not detected in the spinal cord at any time in development. In the dorsal root ganglia, sodium channel I mRNA hybridization signal was detected in DRG neurons at P2, with slightly increased levels at P15 and P30. Sodium channel II mRNA exhibited a relatively constant, moderate level of expression at all developmental ages. Sodium channel III mRNA was highly expressed in DRG neurons at E17 but was down-regulated with further development so that it was not detectable by P30. NaG mRNA was strongly expressed by some DRG neurons at all stages of development from E17 to P30; in general the level of NaG labelling was greater in larger neurons than in smaller neurons. Na6 mRNA showed increasing expression with development in DRG neurons; at E17, low levels of Na6 mRNA were detected and by P15 to P30 high levels of expression were present in some neurons. hNE (PN1) mRNA was present in DRG neurons at P2, and was up-regulated with further development so that by P30 hNE (PN1) was expressed in all DRG neurons sizes. These results demonstrate that sodium channel alpha-subunit mRNAs I, II, III, NaG, Na6 and hNE (PN1) exhibit distinct spatial and temporal patterns of expression in nervous tissue, and suggest that the expression of the sodium channel alpha-subunits is differentially regulated. (ABSTRACT TRUNCATED)

After transection of their axons within the sciatic nerve, DRG neurons become hyperexcitable. Recent studies have demonstrated the emergence of a rapidly repriming tetrodotoxin (TTX)-sensitive sodium current that may account for this hyperexcitability in axotomized small (<27 microm diam) DRG neurons, but its molecular basis has remained unexplained. It has been shown previously that sciatic nerve transection leads to an upregulation of sodium channel III transcripts, which normally are present at very low levels in DRG neurons, in adult rats. We show here that TTX-sensitive currents in small DRG neurons, after transection of their peripheral axonal projections, reprime more rapidly than those in control neurons throughout a voltage range of -140 to -60 mV, a finding that suggests that these currents are produced by a different sodium channel. After transection of the central axonal projections (dorsal rhizotomy) of these small DRG neurons, in contrast, the repriming kinetics of TTX-sensitive sodium currents remain similar to those of control (uninjured) neurons. We also demonstrate, with two distinct antibodies directed against different regions of the type III sodium channel, that small DRG neurons display increased brain type III immunostaining when studied 7-12 days after transection of their peripheral, but not central, projections. Type III sodium channel immunoreactivity is present within somata and neurites of peripherally axotomized, but not centrally axotomized, neurons studied after <24 h in vitro. Peripherally axotomized DRG neurons in situ also exhibit enhanced type III staining compared with control neurons, including an accumulation of type III sodium channels in the distal portion of the ligated and transected sciatic nerve, but these changes are not seen in centrally axotomized neurons. These observations are consistent with a contribution of type III sodium channels to the rapidly repriming sodium currents observed in peripherally axotomized DRG neurons and suggest that type III channels may at least partially account for the hyperexcitibility of these neurons after injury.