Ontogenic development of the TTX-sensitive and TTX-insensitive Na+ channels in neurons of the rat dorsal root ganglia

Developmental Brain Research, 65 (1992) 93-100 (~ Elsevier Science Publishers B.V. All rights reserved. 0165-3806/92/$05.00

93

BRESD 51394

Ontogenic development of the TTX-sensitive and TTX-insensitive Na + channels in neurons of the rat dorsal root ganglia Nobukuni

Ogata

and Hideharu

Tatebayashi

Department of Pharmacology, Faculty of Medicine, Kyushu University, Fukuoka (Japan) (Accepted 24 September 1991)

Key words: Sodium channel; Dorsal root ganglion; Tetrodotoxin; Culture; Sensory neuron; Patch-clamp; Neuron size

Developmental changes in the sensitivity of neurons to tetrodotoxin (TIX) were studied in relation to the cell size in rat dorsal root ganglia (DRG). Na + currents were recorded from neurons of various stages of development. Two types of Na + channels were identified on the basis of their sensitivity to Trx. One type was insensitive to a very high concentration (0.1 raM) of TIX, while the other type was blocked by a low concentration (1 nM) of TI'X. These two types of Na + channels were observed throughout the developmental stages examined from day 17 of gestation and adulthood. Thus, both types of Na+ channels are already established at the early stage of neuronal development and appear to be retained throughout the life-span of the DRG neuron. The concentration-response relationships for the block of TIX-sensitive Na + current by TI'X did not appreciably change during development. Although two types of Na* channels had strikingly different kinetic properties, the kinetic properties of each channel type were basically similar throughout development. The TI'X-sensitive Na + channels were mainly concentrated in cells with large cell diameters th.~oughont developmental stages examined. These large cells appear to correspond to the 'large-light' cells. On the contrary, the TrX.insensitive Na + channels were found in smaller diameter cells which may correspond to the 'small-dark' cells. Thus, it is concluded that there are heterogeneous categories of neurons which have Na + channels with different physiological and pharmacological properties. Since Na+ channels play a pivotal role in the action potential generation~,these heterogeneity of DRG neurons appear to be instrumental in integrating the sensory signals. INTRODUCTION Neurons in the mammalian dorsal root ganglia (DRG) have been used extensively for the investigation of neuronal development becausr these cells can be examined at different embryonic and postnatal ages xe. The neurons in the dorsal root ganglia (DRG) are composed of two major categories on the basis of their microscopic observations, the large-light cells and the small-dark cells t2. It has been shown that peripheral C fibers originate from neurons in the size range of the small-dark cell population and that peripheral A a and Ap fibers originate from neurons in the size range of the large-light cell population 9'13 indicating that the two types of cells play different functional roles in sensory integration. However, it is still not clear whether these two cell types are from separate populations or merely examples of the extreme ends of a single distribution. In addition, there has been little attempt to clarify physiological properties of ion channels of the large-light and small-dark cell populations. It has been reported that a part of the action potentials observed in neurons of the mammalian D R G are resistant to tetrodotoxin ( T I X ) 7, a well-known specific

blocker of the voltage-gated Na + channels 17. Subsequently, TI'X-insensitive Na + channel has been identified using a voltage-clamp method in cranial sensory neurons 3'1°'1t. TIX-insensitive Na + channels are found not only in the immature neurons but also in the adult neurons, suggesting that this type of channel may not be a temporary expression during early stage of development but rather may have a functional role in the sensory integration. However, little is known about the expression of different types of Na + channels during neuronal growth and differentiation. In particular, very little information is available about the relationship between cell siz,~ and the neuronal TIX-sensitivity, i.e. whether the small-dark or large-light morphological configurations correspond to the Tl'X-sensitive or "FIX-insensitive categcJries. In this paper, the ontogenic development of Tl'X-sensitivity of Na + channels was investigated in relation to the distribution of neuronal size, using immature developing and adult neurons dissociated from rat DRG. The results show that the size distributions of the Tl'X-sensitive and TIX-insensitive neurons closely correlated with those of the large-light and small-dark morphological configurations.

Correspondence: N. Ogata, Department of Pharmacology, Faculty of Medicine, Kyushu University, Fukuoka 812, Japan.

94

Na+

MATERIALS AND METHODS

Dissociation and culture procedures Procedures for dissociation and culture of neurons in the dorsal root ganglia (DRG) were as described previously2°. The preparations were obtained from fetal (day !7 of gestation), newborn (1-2 days postnatal), and adult (older than 3 months) rats thus allowing developmental studies to be performed. The DRG were dissected out and incubated at 36°C for 30-'~ min in Ca2+- and Mg2+-free saline, containing 0.25% trypsin (Type XI, Sigma). The ganglia were then mechanically dissociated with a fire-polished Pasteur pipette. Dissociated cells were kept in the Krebs solution at a room temperature and used for patch-clamp experiments. Neurons dispersed from the newborn rat were also subjected to culture. The cells were plated on glass cover-slips coated with poly-L-lysine (Sigma) and maintained in a humidified incubator containing 5% COz in air at 35°C in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum (Gi~_~),. (,~ and streptomycin (40 ng/ml). After 1-2 days in culture, cytosine /~-D-arabinofuranoside (Sigma) was added to cultures to suppress the growth of non-neuronal cells. Subsequent medium changes were done at 3-4 day intervals. Cells between 3 and 4 weeks in culture were used.

~i.~'I~.!~. l,Ulml),

Electrical recording The methods for electrical recording used in the present study were similar to those previously described 21. Membrane currents were recorded with the whole-cell patch-clamp techniques. The DC resistance of suction electrodes was 0.5-0.8 MQ. The pipette solution contained (in mM): 120 Cs-glutamate, 10 NaCl, 2.5 MgCl2, 5 glucose, 5 HEPES, and 5 EGTA. The pH of the pipette solution was adjusted to 7.0 with CsOH. The external solution contained (in mM): 100 NaCI, 5 CsCI, 1.8 CaC!2, 1 MgCI2, 5 HEPES, 20 tetraethylammonium-CI, 2 CoCi,, 25 glucose. The pH of the external solution was adjusted to 7.4 with NaOH. All the experiments were performed with an on-line system which has been developed by M. Yoshii and N. Ogata, using a personal computer (PC-286V, Epson, Tokyo, Japan). Membrane currents passing through the pipette were recorded by a current-to-voltage converter designed by M. Yoshii21. Compensation for the series resistance was performed by adding a part of the output voltage of the current recording to the command pulse. Capacitive and leakage currents were subtracted digitally by the P-P/4 procedure2. In addition, the TTX.sensitive component of the Na + current was subtracted with the current remaining after application of 1/~M TTX. The liquid junction potential between internal and external solutions was about 11 mV. The data shown here had compensated for this effect by adjusting the zero-current potential to the liquid junction potential. Only cells showing an adequate voltage and space clamp19 were used. The size of cell soma was measured under a phase-contrast microscope (400x magnification) using a Nikon particle analyzer. The cell diameter was expressed by (the longest dimension + the shortest dimension)12. All experiments were performed at room temperature (20-22°C). Results are expressed as means -- S.E.M., and n represents the number of cells.

the third group of neurons, current was partially blocked by "I'I"X (bimodal cells; C). These 3 types of cells were found t h r o u g h o u t the d e v e l o p m e n t a l stages examined, i.e. day 17 of gestation, 1-2 days postnatal, 3 - 4 weeks of culture of the 1-2 day postnatal D R G , and adulthood. In the bimodal cells, the p r o p o r t i o n of the TrX-sensitive c o m p o n e n t of Na + current to the T r X insensitive c o m p o n e n t varied from cell to cell. The kinetic property of the TTX-sensitive c o m p o n e n t of Na + current in the bimodal cell was identical to that of the Na + current in the Tl"X-sensitive cell (data not shown). Similarly, the kinetic property of the Tl'X-insensitive c o m p o n e n t of the Na + current in the bimodal cell was identical to that of the Na current in the TTX-insensitive cells. As shown in the traces in Fig. 1 where current amplitudes were normalized to facilitate the comparison, the T r X - s e n s i t i v e Na + current had m u c h faster activa-

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TTX-sensitivity of Na + currents Neurons in the DRG could be divided into 3 groups according to their responsiveness to TI'X (Fig. 1). The first group was characteri-ed by Na + current which was totally suppressed by a low concentration of TTX (TI'Xsensitive cells; A). The second group had Na + current which is insensitive to "ITX (TrX-insensitive cells; B). In

Fig. 1. Two types of Na + channels in neurons of the rat dorsal root ganglia (DRG). Na + currents were evoked by 30 ms voltage steps to -10 mV from a holding potential (Vh) of-80 inV. In upper traces in A-C (records 1), currents in the control solution and in the presence of 0.1 ~M TTX were superimposed. Lower traces (records 2) were computed by subtracting the current in the presence of TI'X from the current in the control solution. Cells shown in A-C are TgX-sensitive (A), TFX-insensitive (B), and partially sensitive to TTX (C). In this and subsequent figures, currents were recorded from cultured neurons of the newborn rat DRG and downward deflection represents an inward current.

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Fig. 2. Dependence of TrX-insensitive Na+ current on the external Na+. A family of currents were evoked by a 30 ms step to the various potentials (-80 to +60 mY) from a Vh of -80 mV in the control solution (A), in the presence of 0.1/~M TTX (B), in the presence of T r x plus in the absence of external Na+ (C). In C, total amounts of external NaCl were substituted with equimolar amounts of tetramethylammonium-Ci. B1 and B2 were the same traces with different magnifications, D: current-voltage curves obtained from traces shown in A-C. Peak amplitudes of the Na+ currents were plotted against the test potentials. For further explanation, see text.

tion and inactivation time courses as compared with the TTX-insensitive Na + current. Fig. 2 confirms that the TTX-insensitive current is indeed produced by an influx of Na + ions. A family of inward currents in the control solution (Fig. 2A) was decreased by the addition of 0.1/~M TYX to the medium (Fig. 2B). However, the reversal potential remained unaffected (see the current-voltage curves in D). When the Na + ions in the medium were totally removed in the presence of TTX, the current traces become much smaller and the direction of the current became outward (Fig. 2C). As plotted in Fig. 2D, two types of Na + currents had strikingly different kinetics. The activation levels were about -60 (-57.4 - 1.1, n ffi 7) mV for the TTX-sensitive current and about -40 (-39.6 -- 1.2, n = 7) mV for the TTX-insensitive current. The potential at which Na + conductance was 50% or" the maximal was -39.6 -- 0.7 (n ffi 4) an -15.1 +- 0.7 (n = 5) for the

TrX-sensitive and TYX-insensitive currents, respectively. Peak amplitudes were obtained at -30 (-28.9 -1.0, n = 7) mV and -10 (-5.4 _+ 1.6, n - 7) mV, respectively for the TTX-sensitive and TI'X-insensitive currents (for the TI'X-sensitive current, see the subtracted current-voltage curve indicated by open squares). Thus, it is concluded that the TI'X-insensitive Na + current is in fact generated by an influx of Na ÷ ions. TTX-insensitive Na + current and Ca 2+ current Since in our experiments, Co 2+ (2 mM) was added in the external solution which completely suppresses Ca 2+ currents re'z9, the possibility of an involvement of Ca 2+ currents can be ruled out. This was further confirmed in the experiment shown in Fig. 3. A step depolarization to -50 mV in the medium in which TTX (1 #M) was included but Co 2+ was removed produced an inward current which had a slow activation phase (upper trace of '-50 mV'). This current showed a slight inactivation during a 100 ms depolarization. At step depolarizations to -40 and -30 mV, two additional inward currents were induced, one, a fast inward current having a rapid inactivation, and the other, a slow sustained inward current. When Co 2+ was added to the medium, the slow inward current observed at -50 mV and the sustained inward culTent observed at -40 and -30 mV were all abolished, whereas the fast inward current observed at -40 and -30 mV was not abolished. On the contrary, when Na t ions in the medium were totally removed, all the slow inward currents remained almost unaffected, whereas the fast inward current was abolished. Similar results were obtained from all of the 5 neurons tested. These results suggest that the sharp inward deflection represents the TTX-insensitive Na t current and is distinct from the Ca z+ currents remaining in the Na+-free solution. Concentration-response relationships for T T X TrX-insensitive Na + current was not affected by an extremely high concentration of TYX (100 ~tM) throughout the developmental stages examined. On the contrary, T r x in nanomolar concentrations effectively blocked the TrX.sensitive Na t current (Fig. 4A). The concentration-response relationships for the block of TrX-sensitive Na + current was remarkably similar among neurons of different developmental stages (Fig. 4B). The half-maximal concentration for Na t current block was within a range from 3.3 to 4.1 nM throughout the developmental stages. As shown in Fig. 4B, all the measurements from different developmental stages fit the curve calculated on the basis of 2-to-1 stoichiometry. This stoichiometry is in remarkable contrast with the 1-to-1 stoichiometry reported for other preparations (e.g. rabbit Purkinje fiberss, neuroblastoma cells~). This dif-

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Fig. 3. Contrasting properties of the TI"X-insensitive Na + current and Ca 2+ current. Currents were evoked by a step depolarization to -50 mV (left column), --40 mV (middle column) and -30 mV (right column) from a Vh of -80 mV. All the traces were recorded in the presence of 1/~M T r x . Upper traces: control. Middle traces: in the presence of 2 mM Co 2+. Lower traces: in the absence of external Na +. Total amounts of NaCI in the external solution were replaced with equimolar amounts of tetramethylammonium-Cl. All the traces were recorded from the same neuron. Further explanation, see text.

ference will be discussed elsewhere in detail (manuscript in preparation). Correlation between cell size and TTX-sensitivity The cumulative size distribution of cell soma in different developmental stages were examined rising freshly dissociated neurons (Fig. 5). Regardless of the age studied, the cell size distributions were characterized by two distinct overlapping cell populations. In each developmental stage, the peak height of the percentages for cells with smaller cell diameters was about 2-3 times greater than that for cells with larger cell diameters. The mean cell size was 20.8 - 0.9 for the day 17 fetal, 27.1 ± 0.9

for 1-2 days postnatal, and 32.2 ± 1.0 for the adult (n --300, respectively). Fig. 6 illustrates the relationship between the cell diameter and the TI'X-sensitivity in day 17 of gestation (A) end adult (B). In both developmental stages, the TTX-sensitive cells were distributed preferentially to the larger diameters, while the Tl'X-insensitive cells were distributed to smaller diameters. The bimodal cell groups were distributed throughout the whole diameter range. Thus, there was a close relationship between the cell size and the TTX-sensitivity. The size distribution for cultured newborn DRG neurons are shown in Fig. 7A. Filled columns were compiled

TABLE I

Relationships between the cell size and TTX.sensitivity in DRG neurons of different ontogenic stages Neurons were dissociated from fetal (17 day), adult DRG or cultured from newborn rat DR(}.

TTX.sensitivity of DRG neurons Fetal

Cultured newborn

Adult

TTX-sensitive

n % Diameter ~ m ) *

14 25.5 30,3 4- 0,7

48 !8.9 39.4 4- 0,9

12 26.1 46.2 4- 1.4

TTX-insensitive

n % Diameter ~ m ) *

24 43,6 15.6 4- 0.7

112 44.1 20.5 4- 0;6

16 34.8 25.9 --. 1.3

Bimodal

n % Diameter (~m)*

17 30.9 22.6 4- 1.6

94 37.0 27.7 - 1.2

18 39.1 36.4 4- 2.4

Total number of cells * Mean -+ S.E,M,

55

254

46

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Fig. 6. Correlatio~zbetween cell size and TrX-sensitivity in neurons of the fetal (A) and adult (B) rat DRG. Each symbol represents the cell with different TrX-sensitivity. Circles, TFX-sensitive cells; triangles, TrX-insensitive cells; squares, cells partially sensitive to T I ~ (bimodal). Bars indicate the mean cell diameters of respective cell types.

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Fig. 4. Concentration-response relationships for the inhibition of

Na+ current by Trx. A: currents were evoked by a step depolarization to -10 mV from a Vh of-80 mV in the cultured TI'X. sensitive cell of the newborn rat DRG. Each pair of superimposed current traces was recorded in the presence or absence of Trx. B: concentration-response curves for the TTX-sensitive cells obtained from different stages of development. The data points are fitted by sigmoidal curves calculated by the following equation based on 2-to-1 stoichiometry for interaction of T r x molecules with binding sites for the apparent dissociation constant (Kd) of 4.0 (newborn acute), 3.7 (17 day fetal), 3.4 (adult) and 3.3 (newborn culture). IN, = 1/{l+([TrX]/gd) 2} where INa and [TTX] represent the Na + current amplitude and 'ITX concentration. The vertical lines represent S.E.M. of the data for cultured newborn cells. The S.E.M. for the remaining 3 cell groups was not shown for clarity.

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5

10

15 20 25 30 35 40 45 50

1

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55 60

Cell diameter (lUn) Fig. 5. Distribution histogram of cell size of neurons from D R G of

various developmental stages. The diameter histograms were compiled by counting the number of cells whose size was within bin width (5/~m).

from the size measurement in which all of the viable D R G neurons on the culture disc were counted. Open columns represent the results from cumulative size measurements done at patch-clamp recording. The size distribution for cultured cells was as a whole similar to those for freshly dissociated cells. However, the ratio of the peak of the smaller diameter cells to the peak of the larger diameter cells was 5.0 for cultured cells (filled columns of Fig. 7A), in contrast to 2.2 (an average of 3 stages in Fig. 5) for the freshly dissociated cells. This was probably due to the fact that the survival of smaller neurons during long-term culture was usually much higher than that of larger neurons. Nonetheless, two distinct overlapping cell components were obvious also in cultured cells. The size distribution of neurons having different TrX.sensitivity in cultured cells was basically similar to the size distributions in freshly dissociated cells, although size distributions of smaller and larger cell components overlapped more widely in cultured cells (Fig. 7B). Table I summarizes the relationships between the cell size and TrX-sensitivity in different developmental stages. It should be noted that the percentage of adult cells which are responsive to TFX (~TX-sensitive cells plus bimodal cells; 65.2%) are in good agreement with the report of Yoshida et al. 24. They reported that about 68% of adult mouse D R G neurons showed TI~-sensitive action potentials.

98

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Fig. 7. A: distribution histogram of cell size of cultured DRG neurons. The diameter histograms were compiled by counting the number of cells whose size were within bin width (5/~m). Filled columns represent the percentages of cells found in the same batch. Open columns were compiled from cells in different batches used for patch.clamp experiments. B: correlation between cell size and TTX.sensitivity in cultured DRG neurons. Open columns represent the percentage of cells whose diameters were within the bin width. Since the smaller diameter cells were much more abundant in number than the larger diameter cells in culture, the corrected size distribution (filled columns) was compiled in a given bin as follows. The percentage shown by the open column was divided by the. percentage for the same bin of the total size distribution (filled columns in A), and the obtained value was expressed as a percentage. Bt, TTX-sensitive cells; B2, TrX-insensitive cells; B3, bimodal cells. DISCUSSION Present results show that D R G neurons have two types of Na ÷ channels with different physiological and pharmacological properties. One type of Na ÷ channel was activated at relatively negative membrane potentials and its activation and inactivation kinetics were relatively fast (thus, fast and low-threshold). This type of Na ÷ channel was sensitive to T I X in nM concentrations. The other type was activated at more positive membrane potentials and had a slow channel kinetics (thus, slow and highthreshold). This slow and high-threshold Na ÷ channel was insensitive to a very high concentration (0.1 mM) of TTX. Although it has been reported that trypsin inhibits

the action of TTX on Na ÷ channels in Helix neurons ~4, the sensitivity of neurons to TTX was not affected in our study, since acutely dissociated or cultured newborn D R G neurons had the same TTX-sensitivity. Thus, the insensitiveness of one type of the Na ÷ currents to TTX was not due to enzymatic treatment. The slow TIX-insensitive Na ÷ channel is reminiscent of the Ca 2÷ channel in its insensitiveness to TFX and relatively slow time course of activation and inactivation. Thus, there is a possibility that the TTX-insensitive Na ÷ current observed in the present study is mediated by an influx of ions through Ca 2÷ channels. However, this possibility can be excluded from the following observations. The Ca 2÷ channel had the activation threshold which was apparently more negative than the activation level for the TTX-insensitive Na ÷ current, and the overall time course of the Ca 2÷ current was much longer than the TTX-insensitive Na ÷ current (Fig. 3). Convincing evidence for the notion that the TTX-insensitive Na ÷ current is actually produced by an influx of Na ÷ ions through the Na ÷ channel is presented by the observation that the reversal potential for this ct~rrent was dependent on the external Na ÷ concentration (Fig. 2C,D). Furthermore, the ITX-insensitive Na ÷ current was resistant to 2 ~tM Co 2÷, whereas the Ca 2÷ channel current was totally blocked (Fig. 3). Most of the voltage-gated Na ÷ channels observed in cells from a variety of preparations are blocked by T r x iT. Some ~ypes of cells such as cardiac myocytes are somewhat resistant to T r x 4'6. However, even in these cells, concentrations of 'ITX lower than 1/~M are sufficient to block the Na + channel 4'~. In this respect, the Tl'X-insensitive Na channel observed in the prese~nt study is quite unique in that it was insensitive to a very high concentration (100 /~M) of TTX. The TTXinsensitive Na ÷ channel has also been found in nodose 3'I°'11 and superior cervical 23 ganglia. As far as we know, this type of Na + channel has not been reported in central or other peripheral nervous systems. Therefore, this type of Na + channel appears to be a common expression of neurons in peripheral sensory neurons, suggesting its role in sensory integration. The expression of membrane channels respon~;ible for active ionic currents is an important process of neuronal cytodifferentiation. The expression of the Na ÷ current in rat D R G was unexpectedly very similar throughout the developmental stages examined. Our results demonstrate that the two types of Na + channels with different pharmacological and physiological properties are functioning already at a very early stage of phenotypic maturation of neurons (day 17 of gestation). Similar results have been reported by Matsuda et al. 15 on the basis of microelectrode experiments. The above finding is consistent with

99 the suggestion that the Na ÷ current was already present at the earliest stages of d=velopment at which neurons could be morphologically ~dentified 1. Since these properties are retained in neurons derived from adult animals (more than 6 months), these two types of Na + channels are not the temporary expression oi t,~,c developmental feature. In addition, the proportion of celi~ showing each channel type remained constant with development (Figs. 6 and 7). Thus, a transformation of cells from one type into the other appears to be unlikely, and the two channel types are not temporary expressions of the developmental stages in a homogeneous cell population. The size distribution of the D R G neurons as measured in freshly dissociated living neurons (Fig. 5) is thought to approximate the actual size distribution in vivo, because most of the cells were viable and counted in the freshly dissociated tissues. The pattern of the size distribution was similar throughout the developmental stages (Fig. 6). In addition, the size distribution was closely correlated with that measured according to histological criteria TM. The smaller and larger subpopulations revealed in this study appear to correspond to the small-dark and the large-light morphological popdlations, respectively. Since the TrX-sensitive cells were distributed mainly to neurons with larger cell diameters, while the TIX-insensitive cells were observed in neurons with smaller cell diameters (Figs. 6 and 7), the large-light and the small-dark populations may be mainly composed of the TI'Xsensitive and TI'X-insensitive cells, respectively. The "FIX-sensitive Na + channel has lower activation level (about -60 mY) and faster channel kinetics as compared with the TrX.insensifive Na + channel (Fig. 2D). Therefore, activities of these: two types of Na + channels can be modulated in a different manner by the mem-

brane potential. When the membrane depolarization is small (less than the threshold for the TI'X-insensitive Na + channel o f - 4 0 mV), only the large-light cell population could be excited, because this population has low-threshold Na + channels. The small-dark cell population becomes excited only when the sufficiently strong depolarizing stimuli are applied. It has been shown from measurement of nerve conduction velocities that peripheral unmyelinated C fibers originate from the smail-dark cell population and that peripheral myelinated A a and A~ fibers originate front the large-light cell population 9'13. Assuming that cell body and its processes have the same type of Na + channels, propagation of the action potential could be further slowed down in the small-dark cells, since Na + channels in these cells has a slow activation and inactivation kinetics (Fig. 1). In conclusion, the results presented in this paper indicate that, in rat D R G , two types of Na + channels are already demonstrable at a very early stage of nervous system organogenesis and retained throughout the lifespan. The slow, high-threshold, and TrX-insensitive Na + channels are preferentially distributed on the cell population with smaller cell diameter which might correspond to the morphological small-dark cell population, while the fast, low-threshold, and TFX-sensitive Na + channels are preferentially distributed on the larger cell population which may correspond to the large-light cells. These .:wo types of Na + channels appear to play an important role in the processing of the primary sensory information.

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Acknowledgements. We thank Prof. H. Kuriyama for his advice. This study was supported by the Japanese Ministry of Education (Scientific Resear~:h 63570096).

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