Functional distribution of three types of Na channel on soma and processes of dorsal horn neurones of rat spinal cord

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1 Keywords: Sodium channel, Dorsal horn, Spinal cord 6668 Journal of Physiology (1997), 503.2, pp Functional distribution of three types of Na channel on soma and processes of dorsal horn neurones of rat spinal cord Boris V. Safronov, Matthias Wolff and Werner Vogel Physiologisches Institut, Justus-Liebig-Universit at Giessen, Aulweg 129, Giessen, Germany 1. Voltage-gated Na channels and their distribution were studied by the patch-clamp technique in intact dorsal horn neurones in slices of newborn rat spinal cord and in neurones isolated from the slice by slow withdrawal of the recording pipette. This new method of neurone isolation was further used to study the roles of soma and axon in generation of action potentials. 2. Tetrodotoxin (TTX)-sensitive Na currents in intact neurones consisted of three components. A fast component with an inactivation time constant (ôf) of ms formed the major part (80 90 %) of the total Na current. The remaining parts consisted of a slowly inactivating component (ôs of 5 20 ms) and a steady-state component. 3. Single fast and slow inactivating Na channels with conductances of 11 6 and 15 5 ps, respectively, were identified in the soma of intact neurones in the slice. Steady-state Na channels were not found in the soma, suggesting an axonal or dendritic localization of these channels. 4. In the whole-cell recording mode, the entire soma of a dorsal horn neurone could be isolated from the slice by slow withdrawal of the recording pipette, leaving all or nearly all of its processes in the slice. The isolated structure was classified as: (1) soma if it lost all of its processes, (2) soma+axon complex if it preserved one process and at least 85 % of its original peak Na current or (3) soma+dendrite complex if it preserved one process but the remaining Na current did not exceed those observed in the isolated somata. 5. The spatial distribution of Na channels in the neurone was studied by comparing Na currents recorded before and after isolation. The isolated soma contained 13 8 ± 1 3 % of inactivating Na current but no steady-state Na current. Soma+axon complexes contained 93 6 ± 1 4 % of inactivating and 46 % of steady-state Na current. 6. In current-clamp experiments, the intact neurones and isolated soma+axon complexes responded with all-or-nothing action potentials to current injections. In contrast, isolated somata showed only passive or local responses and were unable to generate action potentials. 7. It is concluded that dorsal horn neurones of the spinal cord possess three types of TTXsensitive voltage-gated Na channels. The method of entire soma isolation described here shows that the majority of inactivating Na channels are localized in the axon hillock and only a small proportion (ca 1Ï7) are distributed in the soma. Steady-state Na channels are most probably expressed in the axonal and dendritic membranes. The soma itself is not able to generate action potentials. The axon or its initial segment plays a crucial role in the generation of action potentials. Dorsal horn neurones of the spinal cord play a key role in processing the sensory information received from the primary afferent fibres. Several types of dorsal horn neurone have been classified according to their receptive field, the kind of stimulation that leads to neurone excitation and their pattern of firing behaviour (Woolf & Fitzgerald, 1983; Lopez-Garcia & King, 1994). Dorsal horn neurones display a broad range of firing patterns, from tonic firing in response to a sustained depolarization to strongly phasic firing resulting in only one action potential at the beginning of the pulse (Lopez-Garcia & King, 1994). Such complexity in firing behaviour is thought to be determined by the cellspecific expression of ion channels underlying the major membrane conductances. It has been shown that dorsal horn

2 372 B. V. Safronov, M. Wolff and W. Vogel J. Physiol neurones possess inward Na as well as inward Ca and outward K currents (Huang, 1987), which play an important role in the generation of action potentials (Murase & Randic, 1983). Recent molecular biology investigations have undoubtedly indicated the expression of several types of Na channel á_subunit mrna in the dorsal horn of the spinal cord (Westenbroek, Merrick & Catterall, 1989; Black, Yokoyama, Higashida, Ransom & Waxman, 1994). Differences in the developmental regulation of Na channel mrna expression in the spinal cord (Gordon et al. 1987; Beckh, Noda, L ubbert & Numa, 1989) and a variable degree of â1-subunit expression amongst different cell types (Oh, Sashihara & Waxman, 1994) could provide the basis for an additional diversity of Na channels in dorsal horn neurones. Unfortunately, a comprehensive electrophysiological description of voltageactivated Na channels in dorsal horn neurones is lacking at present and the physiological relevance of the molecular biology experiments remains to be proven by direct measurements of Na currents in intact neurones. In the present work we studied Na channels in laminae I III dorsal horn neurones identified in slices of newborn rat spinal cord. It is shown that dorsal horn neurones possess three different types of voltage-gated Na channels: two types of inactivating and one type of non-inactivating steady-state channel. By comparing Na currents recorded from the neurones within the slice with those recorded from isolated somata it was found that the soma of the neurone contained only one-seventh of the inactivating Na channels. The rest of the inactivating Na channels are located on the axon. The steady-state Na channels are mostly distributed over the axonal and dendritic, but not the somatic, membranes. Functionally, it is shown that the soma itself cannot generate action potentials; in spinal dorsal horn neurones, an axon is needed for action potential generation. METHODS Preparation Experiments were performed by means of the patch-clamp technique (Hamill, Marty, Neher, Sakmann & Sigworth, 1981) on 200 ìm thin slices (Edwards, Konnerth, Sakmann & Takahashi, 1989) prepared from the lumbar enlargement (L3 6) of the spinal cord of 2- to 9-day-old rats. Rats were rapidly decapitated and the spinal cords carefully cut out. The slices were prepared and kept according to a description given by Takahashi (1990). The study was performed on 8 12 ìm dorsal horn neurones identified in laminae I III of the spinal cord (Fig. 1A). No cleaning procedure was necessary in the present study, since tens of neurones on the surface of each slice were free from connective tissue and directly accessible for the patch pipette. During experiments the cells were viewed with Nomarski optics. The photographs in Fig. 7B and C were made with a thermal video printer using infrared video microscopy (Stuart, Dodt & Sakmann, 1993). Neurones shown in Fig. 7D and E under fluorescent optics were filled with 0 3 % Lucifer Yellow (dilithium salt; Sigma) dissolved in high-cs solution. Solutions Ringer solution for preparing and maintaining the slices contained (mò): NaCl, 115; KCl, 5 6; CaClµ, 2; MgClµ, 1; glucose, 11; NaHµPOÚ, 1; NaHCO, 25 (ph 7 4 when bubbled with a 95 % Oµ 5 % COµ mixture). During all experiments the slices were perfused with low-ca, high-mg Ringer solution, in order to reduce synaptic activity in neurones. This solution was obtained from the first one by setting the concentrations of Ca and Mg at 0 1 and 5 mò, respectively, and is hence referred to as Ringer solution. External tetraethylammonium (TEA)-containing solution (Ringer-TEA) was made up of (mò): NaCl, 95; KCl, 5 6; CaClµ, 0 1; MgClµ, 5; glucose, 11; NaHµPOÚ, 1; NaHCO, 25; and TEA-Cl, 20 (ph 7 4 by bubbling with Oµ COµ as above). External TEAcontaining solution used for pipette filling in experiments with inside-out patches (Ringer-TEAï) contained (mò): NaCl, 115 8; KCl, 5 6; CaClµ, 0 1; MgClµ, 5; glucose, 11; TEA-Cl, 36; and Hepes, 10 (ph 7 4 adjusted with NaOH to give a final concentration of 5 2 mò). Na -free choline-cl solution contained (mò): choline chloride, 141; KCl, 0 6; CaClµ, 0 1; MgClµ, 5; glucose, 11; and Hepes, 10 (ph adjusted to 7 4 with KOH to give a final concentration of 5 mò). Tetrodotoxin (TTX) was added directly to external solutions. Standard internal solution (high-k ) contained (mò): NaCl, 5; KCl, 144 4; MgClµ, 1; EGTA, 3; and Hepes, 10 (ph adjusted to 7 3 with KOH to give a final concentration of 10 6 mò). Successful isolation ofaneuronefromaslicewasachievedwhenthetotalamountof internal Na was increased to 15 mò. The composition of this internal solution (high-k o) was (mò): NaCl, 5; KCl, 144 4; MgClµ, 1; EGTA, 3; and Hepes, 10 (ph adjusted to 7 3 with NaOH giving a final concentration of 10 mò). Internal solution for studying Na channels (high-cs ) contained (mò): NaCl, 5 8; CsCl, 134; MgClµ, 1; EGTA, 3; and Hepes, 10 (ph adjusted to 7 3 with NaOH to give a final concentration of 9 2 mò). Internal solution with 50 mò CsF contained (mò): NaCl, 11 5; CsCl, 87; CsF, 50; MgClµ, 1; and Hepes, 10 (ph adjusted to 7 3 with 3 5 mò NaOH). Current recordings The patch pipettes were pulled in two stages from borosilicate glass tubes (GC 150, Clark Electromedical Instruments, Pangbourne, UK). The pipettes used for single-channel recordings were coated with Sylgard 184 (Dow Corning) and had a resistance of 8 22 MÙ. The pipettes for whole-cell recordings had a resistance of 2 7 MÙ. All pipettes were fire polished immediately before the experiments. The patch-clamp amplifier was a List EPC_7 (Darmstadt, Germany) in all voltage- and current-clamp experiments. The effective corner frequency of the low-pass filter, unless otherwise stated, was 3 khz. The frequency of digitization was 5 10 khz (except the recording in Fig. 8A). Data were stored and analysed using commercially available software (pclamp, Axon Instruments). Transients and leakage currents were digitally subtracted using records with either negative or positive pulses which activated no channels. Offset potentials were nulled directly before formation of a seal. In fifteen experiments we tried to compensate the error produced by the resistance in series (up to 65 % compensation). However, smooth activation characteristics of Na currents could still not be obtained for neurones in the slices due to incomplete space clamp of remote membrane regions. Therefore no series resistance compensation was employed further in the present study. In the majority of isolated somata, Na currents had smaller amplitudes of pa and voltage error due to series resistance became, in most cases, smaller than 5 mv. The inside-out patches were studied in an additional small chamber as described elsewhere (Safronov & Vogel, 1995) in order to avoid

3 J. Physiol Na channels in dorsal horn neurones 373 the destruction of the whole slice during its superfusion with internal solutions. The present study is based on recordings from 268 intact neurones in the slice, 119 isolated somata, soma+axon and soma+dendrite structures (see Results) and from twenty-nine excised membrane patches. All experiments were carried out at a room temperature of C. Data are given as the means ± s.e.m. RESULTS Identification of dorsal horn neurones Several criteria were used in the present study for distinguishing between dorsal horn neurones and glial cells. It has been shown that some types of glial cells in the grey matter of rat spinal cord slices possess voltage-gated Na channels (Chv atal, Pastor, Mauch, Sykov a & Kettenmann, 1995). However, the amplitude of Na currents was at least oneorderofmagnitudesmallerthanthatofk currents and no glial cells in the spinal cord slice were able to generate action potentials. Unfortunately, the major part of the present experiments was performed using pipettes filled with high-cs solution and no action potentials could be recorded. Therefore, in these experiments dorsal horn neurones were separated from glial cells on the basis of the amplitudes of their peak Na currents. A total of sixty-three neurones and glial cells were first studied in Ringer solution using pipettes filled with standard high-ki solution. Under voltage-clamp conditions, the maximum Na current activated by depolarizing voltage steps from a holding level of 70 mv was determined. This was usually observed at 40 or 30 mv, less frequently at 20 mv. Thereafter, the cell s ability to generate action potentials was tested in current-clamp mode. Figure 1B shows typical recordings obtained from neurones and glial cells. Cells which responded to depolarizing voltage pulses with large na Na currents under voltage-clamp conditions (upper trace) and generated short action potentials in current-clamp mode (lower trace) were considered as Figure 1. Identification of dorsal horn neurones A, slice from the lumbar enlargement of the spinal cord of a 5-day-old rat. The neurones studied were from the dorsal horn region above the dashed line. It corresponded to laminae I III of the grey matter of the spinal cord. B, neurone and 2 types of glial cells under voltage- (upper traces) and current-clamp (lower traces) conditions. In voltage-clamp experiments, Na currents were activated by voltage steps from 70 to 40 or 30 mv. In current-clamp experiments, 60 ms current pulses of different strength were injected into the cells in order to test their ability to generate action potentials. C, distribution of maximum amplitudes of Na currents (INa) recorded in 63 spinal cord cells. Here and in D, the cells which generated action potentials are shown by open bars and those which generated no action potentials by filled bars. D, distribution of Na to leakage current ratios. Data from 63 cells. The vertical dashed lines in C and D indicate the borders of the regions used for selection of neurones as described in the text.

4 374 B. V. Safronov, M. Wolff and W. Vogel J. Physiol neurones. In contrast, cells which showed either none or only very small na Na currents (upper traces) and could not generate action potentials (lower traces) were assumed to be glial cells. One of the reasons why glial cells with Na channels were not able to generate action potentials wasthepresenceofrelativelylargeleakagecurrentsseenin our experiments. Therefore, the ratio of Na currents to leakage currents could be used as a further criterion for the separation of neurones from glial cells. The distribution of maximum Na currents is given in Fig. 1C. The cells which generated no action potentials were considered as glial cells and are shown by filled bars. It could be seen that the cells with peak Na current exceeding 0 4 na were most probably neurones. The ratio of Na currents to leakage currents was defined as the ratio between the maximum Na current activated from a holding potential of 70 mv and the leakage current produced by a hyperpolarizing 40 mv voltage step. This ratiowasbetween0and4forglialcellsandbetween10and 600 for neurones (Fig. 1D). From our data, the following criteria for neurone identification in voltage-clamp experiments were used. Cells with Na currents exceeding 1 na were considered as neurones. Cells with Na current amplitudes between 0 6 and 1 0 na (indicated by the dashed lines in Fig. 1C) were considered as neurones if the ratio of Na current to leakage current exceeded 15 (dashed line in Fig. 1D). All cells with Na current amplitudes less than 0 6 na were discarded regardless of their Na current to leakage current ratio. The neurone identification was often confirmed by spontaneous synaptic currents observed during experiments. No further identification of different types of spinal dorsal horn neurones was undertaken in the present work. The neurones investigated had a high input resistance of 3 7 ± 0 5 GÙ (104 neurones). The neurones investigated with standard high-k o solution showed resting potentials between 50 and 85 mv under the present experimental conditions. Na currents in dorsal horn neurones of rat Voltage-activated Na currents were mainly recorded in external Ringer-TEA solution using pipettes filled with high-cs solution for the suppression of voltage-activated K currents. A typical whole-cell Na current activated by a voltagestepto 30mVinadorsalhornneuroneinthe spinal cord slice is shown in Fig. 2. Three components of whole-cell Na current could be identified on the basis of their inactivation kinetics. A fast component inactivated with a time constant of ms (ôf) and made up % of the total Na current. A slow component inactivated with a time constant (ôs) of 5 20 ms and produced 5 20 % of the total Na current. The third component was a steady-state one, which showed no inactivation and an amplitude of % of the total Na current (mean, 2 1 ± 0 2 %, 60 neurones). Both the slow and the steady-state components began to activate at 60 to 50 mv. The fast component recorded from neurones in the slice activated abruptly at 50 to 40 mv, indicating insufficient space clamp of the neuronal membrane under these experimental conditions (see also Fig. 10A). For the same reason, no reversal potential could be measured for the peak Na currents, since they remained inwardly directed even at nominative potentials as positive as +80 mv (the calculated equilibrium potential for Na (ENa) was +53 mv). In contrast, the steady-state current changed its polarity at potentials of +20 to +40 mv, presumably due to activation at these potentials of non-specific outward Cs current through delayed-rectifier K channels which could not be completely blocked by 20 mò external TEA. There are several indications that none of these components resulted from activation of voltage-gated Ca channels. All three were recorded in external Ringer-TEA solution containing 0 1 mò Ca and 5 mò Mg. They were also recorded when the internal solution contained 50 mò F (17 neurones), known to produce an irreversible block of Ca channels. All three components disappeared in external Na -free choline-cl solution(5neurones)andwereblockedby1ìò external TTX (15 neurones). Kinetics of Na current blockade by TTX In the following experiments the time course of Na current reduction during perfusion of the slice with TTX was studied (Fig. 3). The time elapsed from the start of TTX perfusion is indicated above the corresponding traces. TTX first suppressed the fast component of Na current completely and only several minutes later the block of the slow and Figure 2. Three components of voltage-gated Na currents recorded from a dorsal horn neurone in a spinal cord slice Sodium current was activated by a voltage step to 30 mv following a 50 ms prepulse to 120 mv. Holding potential was 80 mv. The time course of current inactivation was fitted with two exponentials (superimposed dashed line): a fast (ôf) of 0 9 ms (83 6 %) and a slow (ôs) of 8 3 ms (12 9 %). The steady-state current (Iss) made up 3 5 % of the total Na current. Temperature was 23 C.

5 J. Physiol Na channels in dorsal horn neurones 375 steady-state components developed. The effect could be seen more clearly when the current traces were superimposed (Fig. 3, middle). The same time course of Na current block by TTX was observed in ten out of ten dorsal horn neurones. Such an effect could be explained by assuming that the connective tissue in the spinal cord slice impedes the diffusion of the blocker molecules and, therefore, different regions of the neuronal membrane experience different gradients of TTX concentration. It was concluded that the channels underlying the fast and the slow components of whole-cell Na current differ from one another either in their sensitivity to TTX or in their spatial distribution across the neuronal membrane. Single-channel experiments Because of the problems arising from insufficient space clamp, no further investigations of Na currents in neurones in the spinal cord slice were performed. Outside-out membrane patches obtained from the soma of dorsal horn neurones with 9 18 MÙ pipettes contained between two and fifteen Na channels of both fast and slow types. The proportion of fast to slow components in averaged currents (approximately 5 10 : 1) and their inactivation kinetics (ôf of ms and ôs of 5 15 ms) were very similar to those described for whole-cell currents. In contrast to Na currents recorded from neurones in slices, a steady-state component was never seen in recordings from ten outside-out patches. Patches containing only a few Na channels could seldom be obtained in the outside-out recording mode. Therefore, the study of the single-channel properties was mostly performed by using inside-out patches. The pipettes filled with Ringer- TEAï solution had a resistance of 8 22 MÙ. The internal surface of the patch was exposed to high-cs bath solution. Approximately every second inside-out patch contained active Na channels, indicating their relatively low density in the somatic membrane of dorsal horn neurones. Twelve of nineteen inside-out patches with active Na channels contained only the fast-type channels. Figure 4A shows single-channel recordings and an averaged current (lowermost trace) from one such inside-out patch. The channels opened quickly and inactivated completely within 3 4 ms. The time of the last channel closing seen in this patch is indicated by the asterisk above the averaged current. The inactivation kineticsoftheaveragedfastcurrentcouldbesatisfactorily fitted by a monoexponential with a ôf of 1 2 ms. The mean valueofôfat 20 mv was 1 1 ± 0 1 ms (12 patches). Seven of nineteen inside-out patches contained a mixture of oneslowandbetweenoneandfourfastna channels. Figure 4B shows openings of fast (first and second trace) and slow channels (third and fourth trace) recorded from the same patch. The slow channels inactivated within ms. The time of the last channel closing is shown by the asterisk above the averaged current (lowermost trace). Two exponentials, fast and slow, were needed for fitting the inactivation kinetics of the averaged current, revealing a ôf of 1 5 ms (62 %) and a ôs of 11 9 ms (38 %). The mean values of ôf and ôs obtained for these seven patches at 20 mv were 1 1 ± 0 1 ms and 7 2 ± 1 1 ms, respectively. In further experiments the kinetics of the fast Na channel inactivation were studied at different potentials. The averaged currents from one inside-out patch containing four fast Na channels are shown at 40, 20 and 0 mv (Fig. 5). The kinetics of channel inactivation depended on voltage but remained monoexponential at all potentials investigated (4 patches). Figure 3. Kinetics of Na current blockade by TTX Sodium current activated by a voltage step to 30 mv after a 50 ms prepulse to 120 mv during slice perfusion with 1 ìò TTX. The time elapsed from the start of TTX perfusion is indicated above the corresponding traces. Holding potential was 80 mv. The same traces are superimposed in the middle of the figure on a different scale.

6 376 B. V. Safronov, M. Wolff and W. Vogel J. Physiol It can be seen from the recordings shown in Fig. 4B that the currents through slow Na channels were slightly larger than those through fast channels. In order to verify this observation, we constructed point-amplitude histograms for both channel currents recorded at a potential of 20 mv (Fig. 6). Twenty-eight fast channel openings were selected from eight inside-out patches containing only fast Na channels. Ten slow channel openings were selected from three inside-out patches which contained a mixture of both channels. In such patches the averaged currents were first constructed and the ôf and ôs values determined by biexponential fitting of the inactivation kinetics. Each single episode was then analysed. Parts of long openings 3 ² ôf ms after the moment when the averaged Na current reached its peak were taken for the histogram. The fitting of the pointamplitude histograms with two Gaussian functions gave the values of 0 85 pa (variance, ä = 0 20 pa) for the fast channel and 1 13 pa (ä = 0 25 pa) for the slow channel at a potential of 20 mv. Assuming an ENa value of +53 mv, the corresponding conductances were calculated to be 11 6 ps for the fast channel and 15 5 ps for the slow channel. The channels responsible for the steady-state component of the whole-cell Na current were not observed in inside-out patches originating from the soma membrane. On the basis of whole-cell and single-channel experiments it was possible to conclude that the TTX-sensitive Na conductance in spinal dorsal horn neurones consisted of three components. The channels underlying inactivating fast and slow current components could be identified in patches originating from the soma membrane. Insufficient space clamp during whole-cell recording in the spinal cord slice and a low frequency of Na channel appearance in inside-out patches from the soma membrane could indicate that the majority of Na channels are located on the axonal or dendritic membrane. The axons and dendrites of dorsal horn neurones with diameters smaller than 0 5 ìm could barely be resolved even with infrared optics and, therefore, axonal or dendritic localization of Na channels could not be directly shown by single-channel recording. In order to solve this problem we developed a method for investigating the functional distribution of channels in small (ca 10 ìm) cells with fine processes. Themethodofentiresomaisolation In the whole-cell recording mode, entire somata of dorsal horn neurones could easily be isolated from the slice by slow withdrawal of the recording pipette, leaving all or nearly all of their processes in the slice (Fig. 7). The channel distribution could then be studied by comparing the macroscopic Na currents recorded in neurones before and after their isolation. When recording from the neurone in the slice was completed, a slight suction was applied to the recording pipette and it wasgentlywithdrawnuntiltheconnectionbetweenthe Figure 4. Single fast and slow Na channels in dorsal horn neurones Sodium channels in inside-out patches were activated by repetitive depolarizing voltage steps from 80 to 20 mv. A, recordings from an inside-out membrane patch containing only 1 fast Na channel. The lowermost trace is the average of 16 recordings similar to those shown above. * Time of the last channel closing. Inactivation kinetics of the averaged current were fitted with a monoexponential (ôf = 1 2 ms, superimposed dashed line). B, recordings from another inside-out patch which contained one fast (top 2 traces) and one slow channel (next 2 traces). The lowermost trace is the average of 23 recordings. * Time of the last channel closing. The time course of the current inactivation was fitted with two (fast and slow) exponentials: ôf was 1 5 ms (62 %) and ôswas 11 9 ms (38 %; superimposed dashed line).

7 J. Physiol Na channels in dorsal horn neurones 377 Figure 5. Kinetics of the fast Na channel inactivation at different membrane potentials Averaged Na currents at potentials of 40, 20 and 0 mv. Each trace is a mean of recordings from 1 inside-out patch containing fast Na channels only. The channels were activated by voltage steps from a holding potential of 80 mv. The channel inactivation kinetics were fitted with monoexponentials (dashed line). The time constants are indicated near the corresponding traces. soma and the slice was broken (Fig. 7A). The suction applied was similar to that needed to break the membrane during formation of whole-cell mode. The suction was immediately released after the isolation was completed. The isolated structure was classified as a soma if adjacent processes could not be seen either during the experiment or after, when the recording pipette was turned over. The isolated structure was classified as a soma+axon if it contained one ìm process and preserved more than 85 % of the original Na current recorded in the slice before isolation. The structure was considered as a soma+dendrite if one adjacent process was observed but the amplitude of Na current was in the range typical for isolated somata. A total of ninety-four isolations could be classified into seventysix somata, twelve soma+axon and six soma+dendrite complexes. Stable recordings lasting for 1 h and more could be obtained from each of the three isolated structures. In general, the probability of obtaining a certain configuration depended on the age of the animal, the time elapsed since the slice preparation, the duration of the whole-cell recording from the neurone in the slice before isolation, and the type of pipette solution. Theisolationofthe soma fromadorsalhornneuroneina spinal cord slice is shown under infrared optics (Fig. 7B) and fluorescent optics when the neurone was filled with Lucifer Yellow dye (Fig. 7D). The dendritic tree remaining in the slice after soma isolation can be seen in Fig. 7D (middle). Isolated soma+axon complexes obtained from other neurones are shown in Fig. 7C and E. Changes in leakage current and membrane resting potential monitored during isolation of the cell in Ringer solution are shown in Fig. 8. The leakage currents were studied in voltage-clamp mode using pipettes filled with high-cs or high-k o solutions. Leakage current was monitored every second by 100 ms voltage steps from 80 to 120 mv (Fig. 8A). A successful isolation was always accompanied by a considerable reduction in the leakage current, probably Figure 6. Point-amplitude histograms for the fast and slow single Na channel currents at 20 mv A, 28 single-channel openings were selected from 8 inside-out membrane patches containing only fast Na channels. No opening was shorter than 0 3 ms and longer than 1 5 ms. The peaks were fitted with two Gaussian functions, giving a value of 0 85 pa (variance, ä = 0 20 pa) for the amplitude of the fast Na channel at 20 mv. B, 10 openings of slow Na channels were selected from 3 inside-out patches containing a mixture of fast and slow channels. The procedure for the selection of the slow channel openings is given in the text. Fitting of the histogram peaks with two Gaussian functions gave an amplitude for the single slow Na channel current 1 13 pa (ä = 0 25 pa).

8 378 B. V. Safronov, M. Wolff and W. Vogel J. Physiol Figure 7. Method of isolation of somata and soma+axon complexes A, scheme of isolation of soma and soma+axon complex from a dorsal horn neurone in a spinal cord slice. The small arrow indicates the slight suction applied to the pipette during its withdrawal from the slice. B, intact dorsal horn neurones in the spinal cord slice (left), one of these neurones in the slice during wholecell recording (middle) and its isolated soma on the tip of the recording pipette (right). C, isolated soma+axon complex. The process is marked by arrowheads. D, isolationofdorsalhornneuronestained with Lucifer Yellow: neurone in the slice during whole-cell recording (left); dendritic tree remaining in the slice after the soma was isolated (middle, on another focus level); and isolated soma on the tip of the recording pipette (right). All photographs are from the same neurone. E, isolated soma+axon complex.

9 J. Physiol Na channels in dorsal horn neurones 379 due to the loss of the greater part of membrane area. (Compare the area of the isolated soma with that of the processes remaining in the slice in Fig. 7D.) In ninety-three neurones the leakage current was reduced to 50 ± 3 % after isolation. The loss of membrane area led to an essential reduction in electrical noise as well as to an acceleration in the transients, which could no longer be resolved under the recording conditions of Fig. 8A. The change of resting potential during isolation was studied in current-clamp mode using pipettes filled with high-k o pipette solution. Neurones with relatively low resting potentials of 50 to 60 mv hyperpolarized during isolation (Fig. 8B, upper trace), probably due to a decrease in non-specific leakage conductance. Neurones with resting potentials arround 70 mv did not show considerable changes in membrane potential during isolation (Fig. 8B, lower trace). Distribution of Na channels in soma, axon and dendrites The following experiments were performed in external Ringer-TEA solution using pipettes filled with high-cs solution. Na currents recorded from neurones before and after isolation were compared. A typical isolated soma lost the majority of inactivating and all steady-state Na channels (Fig. 9A). The mean inactivating current measured in the soma was 13 8 ± 1 3 % (52 neurones) of that recorded from the neurones in the slice before isolation. The steady-state currents disappeared completely in forty-six of fifty-two somata. Openings of non-inactivating Na channels could not be revealed even when the soma currents were recorded at the high amplification normally used in single-channel experiments. In the remaining six somata the mean steady-state current was 5 8 ± 1 1 pa and openings of the steady-state channels could be seen. It should be noted that these somata preserved relatively large peak Na currents of 570 ± 72 pa. Therefore, it could not be excluded that these six somata may have contained parts of the axon hillock. Isolated soma+axon complexes preserved almost the whole inactivating current (93 6 ± 1 4 %, 6 neurones) and a large part of the steady-state current (Fig. 9B). Unfortunately, it was not possible to estimate the proportion of steady-state current remaining in a soma+axon complex by direct comparison from the same cell. Adequate measurements of the steady-state currents in the neurone within the slice could be achieved no earlier than 3 5 min after formation of the whole-cell recording mode, when the original intracellular K in the small-diameter processes had been Figure 8. Time course of the soma isolation A, change in leakage current monitored during soma isolation in voltage-clamp mode. Voltage pulses (100 ms duration) from 80 to 120 mv were applied once per second. In this neurone the input membrane resistance was increased during isolation from 2 5 to 25 GÙ. The filter frequency was 1 khz. Data were sampled at 10 ms intervals. B, changes in membrane resting potential during soma isolation shown for a neurone with a relatively low initial resting potential of 55 mv (measured in the slice) and for another neurone with a more negative resting potential of about 70 mv.

10 380 B. V. Safronov, M. Wolff and W. Vogel J. Physiol replaced by Cs and outward K currents no longer masked the steady-state Na current. The isolation of such neurones nearly always resulted in formation of a soma configuration. The probability of getting a soma+axon complex was much higher if isolation was completed not later than min after breaking through the membrane. We therefore compared the mean amplitude of the steady-state current measured in six isolated soma+axon complexes with that measured in twenty other intact neurones in the slice. The six isolated soma+axon complexes with a mean peak Na current of 1 6 ± 0 4 na had a mean steady-state current of 23 2 ± 6 5 pa. The reference group of twenty intact neurones, selected for a similar mean peak Na current (1 6 ± 0 1 na), showed a mean steady-state current of 50 2 ± 5 4 pa. Thus, isolated soma+axon complexes preserved 46 % of the steady-state Na currents. It could be supposed that the rest of the steady-state Na channels are located on dendrites. Indeed, small remaining steady-state components (5 25 pa) were seen in two soma+dendrite complexes. Larger currents were not obtained, since only a small part of the dendritic tree could be isolated (see Fig. 7D). Macroscopic Na currents in isolated somata or soma+axon complexes activated and inactivated faster than those recorded from neurones in the slice (Fig. 9). The activation of Na currents in the soma took place over a broader voltage range, indicating improved space clamp conditions. The reversal potential for Na currents became equal to the equilibrium potential for Na ions (ENa) (Fig. 10A).However, decay kinetics of Na current remained biexponential (Fig. 10B) and the ratio between the amplitudes of fast and slow components was similar to that observed for neurones in the slices. In five isolated somata we tested the sensitivity of the fast and slow inactivating components of Na current to TTX. The range of TTX concentrations tested was nò. In isolated structures, 100 nò TTX blocked both components completely. At 3, 10 and 30 nò TTX, a progressive block of both components was seen. The fast component was only slightly more sensitive to TTX, but at no concentration was a selective block of only one component observed. Therefore, thedifferentblockofthefastandslowcomponentsofna current during slice perfusion with 1 ìò TTX (Fig. 3) most probably resulted from a different spatial distribution of fast and slow Na channels across the neuronal membrane. Functional role of soma and axon in action potential generation The role of the soma and axon in the generation of action potential was studied under current-clamp conditions. Dorsalhornneuronesbeforeandafterisolationwerekeptat a potential of 80 or 70 mv by injection of steady-state currents through the recording pipette. Action potentials were evoked by 10 ms depolarizing current pulses of increasing strength. A typical neurone in the slice generated an all-or-nothing action potential in response to a current injection (Fig. 11). Smaller currents were needed for action potential activation in isolated soma+axon complexes (Fig. 11A) probably due to an increase in input resistance Figure 9. Comparison of Na currents recorded from neurones in the slice with those recorded from an isolated soma and from a soma+axon complex A, Na currents activated by depolarizing steps to 30 mv following 50 ms prepulse to 120 mv in a neurone in a spinal cord slice (left) and in its isolated soma (middle). B, Na currents activated by depolarization to 20 mv following 50 ms prepulse to 120 mv in a neurone in slice (left) and in an isolated soma+axon complex (middle). In both A and B, the currents recorded before and after isolation are shown superimposed on the right. The steady-state currents in the insets are given at 10 times higher current magnification. Holding potential was 80 mv.

11 J. Physiol Na channels in dorsal horn neurones 381 during isolation. Injection of larger currents produced almost no changes in the shape of the action potential, in agreement with the all-or-nothing principle of action potential generation. In general, the shape of the action potentials recorded from neurones in slices and from soma+axon complexes were almost identical (Fig. 11A, superimposed traces). Another type of membrane response to current stimulation was observed for isolated somata (Fig. 11B). The cell shown preserved about 10 % of peak Na current after isolation and its input resistance was increased by a factor of 4 2. Much smaller current pulses depolarized the membrane to levels beyond the threshold of the action potential seen in this neurone before isolation (indicated by arrows). Further increases in the amplitude of injected current produced even stronger depolarization but all-or-nothing action potentials were no longer observed. This phenomenon became more evidentwhentheactionpotentialoftheintactneuronein the slice was superimposed with the membrane responses of the isolated soma (Fig. 11B, right). In the following experiments the contribution of voltagegated Na and passive membrane conductances to the soma responses during current stimulation was studied. The passive membrane responses were obtained by injecting hyperpolarizing 10 pa currents both in neurones in slices and in isolated soma (Fig. 11C). The active membrane responses were evoked by injection of depolarizing currents of different strength. The passive responses multiplied by the corresponding factors are shown as dashed lines. It can be seen that the response of the intact neurone was passive until the threshold level was reached and the activating Na conductance induced a steep membrane depolarization, i.e. the action potential (Fig. 11C, left). In contrast, the soma responses remained passive and only a small deflection of membrane potential in the depolarizing direction could indicate the activation of a small Na conductance (Fig. 11C, middle). The soma response to stronger current injection remained passive until the membrane potential deflected in the hyperpolarizing direction (indicated by the asterisk), presumably due to an activation of K conductance (Fig. 11C, right). Isolated somata with a higher percentage of remaining Na channels showed larger local Na responses but they were still not able to generate an all-or-nothing action potential. Another reason why the soma could not generate action potentials was the presence of a large proportion of voltagegated K channels (30 50 %) remaining after soma isolation. Detailed investigation of K channel distribution was beyond the scope of the present study. DISCUSSION We studied voltage-gated Na channels in dorsal horn neurones visually identified in laminae I III of the rat spinal cord. Lamina I III neurones are known to receive most of their information from myelinated Aä and unmyelinated C fibres of primary afferents (Light & Perl, 1979; Ralston & Ralston, 1979; Woolf & Fitzgerald, 1983) which convey information about pain and thermoreception. Neurones were separated from glial cells on the basis of the Figure 10. Kinetics of Na currents in isolated soma A, current voltage relationships for peak Na currents recorded from 1 neurone before (0) and after isolation (1). The isolated structure was classified as a soma. The currents recorded from the soma have been multiplied by a factor of 5. Data points for the neurone in the slice have been connected by straight lines. The points obtained for isolated soma were fitted with the expression: g0ï(1 + exp((e50 E)Ïk))(E ENa), where the maximum conductance, g0, was 3 1 ns, the potential of half-maximum channel activation, E50, was 37 6mV, the steepness factor, k, was 7 0 mv and the equilibrium potential for Na ions, ENa, was +53 mv. B, Na current activated at 40 mv (after 50 ms prepulse to 120 mv) in isolated soma. Inactivation kinetics were fitted with two exponentials with a ôf of 1 8 ms (81 %) and a ôs of 15 0 ms (19 %).

12 382 B. V. Safronov, M. Wolff and W. Vogel J. Physiol amplitudes of their maximum Na currents and the ratio of Na current to leakage current. Three types of Na channel Voltage-activated Na currents in dorsal horn neurones consisted of fast, slow and steady-state components. All of these were also seen in isolated structures and both fast and slow components were additionally observed in excised patches, indicating that the three components did not result from insufficient voltage clamp of remote membrane regions. Our experiments with TTX have further shown that Na channels underlying fast and slow components most probably differ in their spatial distribution over the neuronal membrane. Therefore, it seems to be unlikely that the biexponential inactivation kinetics of the macroscopic currents was produced by changes in the gating mode of one type of channel. Further evidence for the existence of two types of inactivating channels was provided in experiments with Figure 11. Current-clamp recordings from an isolated soma+axon complex and an isolated soma A, recordings from a neurone which, when isolated, formed a soma+axon complex. The resting potential was kept at 80 mv during the whole experiment by the injection of a steady-state holding current through the recording pipette. Action potentials were evoked by short 10 ms current pulses of different strength (indicated near the corresponding traces). The action potentials recorded from the neurone in the slice and in the soma+axon complex (left trace) are superimposed on the right. B, membrane responses to depolarization recorded in an intact neurone (left) and in its isolated soma (middle). The membrane potential was kept at 70 mv throughout the experiment by the injection of steady-state current. Recordings from the intact neurone and its soma are superimposed on the right. The threshold of the action potential of the neurone in the slice is indicated by arrowheads in all traces. C, responses of the isolated soma to current injections. Short 10 ms current pulses were applied to the neurone and its soma, first in the hyperpolarizing direction ( 10 pa, passive response) and then in the depolarizing direction (the current amplitude is indicated by the pulse protocol). The passive responses multiplied by the corresponding factors are shown by dashed lines on each trace. These were then smoothed using the method of moving window least-squares cubic smoothing (Savitzky & Golay, 1964). Ten points were taken within the smoothing window. The passes were repeated 5 times. * Point where the membrane potential deflects in the hyperpolarizing direction. Same neurone as in B.

13 J. Physiol Na channels in dorsal horn neurones 383 inside-out patches. In the majority of patches, only fast inactivating Na channels were seen and the kinetics of channel inactivation remained monoexponential at all potentials investigated. In a few inside-out patches with one fast and one slow channel, the inactivation kinetics were biexponentialandthecontributionoftheslowcomponentwas about 40 %. Furthermore, the single-channel conductance of the slow channel was 25% larger than that of the fast channel. The fast Na channel underlying the major part of Na conductance in dorsal horn neurones had very similar properties to a Na channel type described in the soma of spinal motoneurones (Safronov & Vogel, 1995). The steady-state component made up % of the total Na conductance. The amplitude of the steady-state current was pa and it could therefore produce a strong depolarizationindorsalhornneuroneswithalargeinput resistance of several gigaohms. We investigated the possibility that the steady-state component could be a window current resulting from an overlap of steady-state inactivation and activation characteristics of transient Na currents (Atwell, Cohen, Eisner, Ohba & Ojeda, 1979). Our calculations showed that the maximum amplitude of the window current would be 0 5 % of the peak Na current at potentials of 50 to 40 mv and almost no window current would be seen at potentials positive to 10mV. In contrast, the steady-state currents measured in the present experiments were much larger, reached their maximum at between 30 and 0 mv becoming negligible at potentials of between +30 and +40 mv because of a decrease in the driving force for Na ions and the activation of non-specific outward currents through delayed-rectifier K channels. Differential block of the fast and steady-state currents during slice perfusion with TTX is a further indication that the steady-state current is not a window current and could notresultfromincompleteinactivationofthefastchannels reported for pyramidal neurones from rat sensorimotor cortex (Alzheimer, Schwindt & Crill, 1993). Furthermore, the steady-state current was considerably reduced in soma+axon complexes, whereas the fast and slow components remained unchanged. The steady-state current disappeared completely in the majority of isolated somata although the inactivating components were only reduced by a factor between 3 and 50. The steady-state current was seen in some soma+dendrite complexes in which the inactivating components became very small. The existence of similar fast, slow and persistent Na currents has been reported for several types of central neurones (Huguenard, Hamill & Prince, 1988; French, Sah, Buckett & Gage, 1990; for review see Crill, 1996). In rat neocortical neurones fast and slow components of Na current were shown to differ not only in their electrophysiological properties but also according to their development with age (Huguenard et al. 1988). Further evidence for Na channel diversity comes from molecular biology studies which showed the expression of several types of Na channel á-subunit mrna in the dorsal horn of the spinal cord (Westenbroek et al. 1989; Black et al. 1994) and revealed a different degree of â1-subunit expression between different cell types within the spinal cord (Oh et al. 1994). A direct comparison between wildtype and cloned Na channels is unfortunately not possible at the moment, but it could be of importance for understanding the functional expression of Na channels in dorsal horn neurones. The present results allow us to suggest that Na conductance in spinal dorsal horn neurones is based on three different types of Na channels. However, direct single-channel recordings from the steady-state Na channel still remain to be made. Method of soma and soma+axon isolation In order to study the channel distribution over the neuronal membrane, we developed a method which allowed the direct comparison of currents recorded from the neurone in the slice with those from isolated somata and soma+axon complexes. The method could be applied to small 8 12 ìm neurones with very fine processes which could not be subjected to direct patch-clamp investigation. In comparison with the well-known outside-out (Hamill et al. 1981) and nucleated patch techniques (Sather, Dieudonn e, MacDonald & Ascher, 1992) the present method allows the isolation of the entire neuronalsomaortheentiresomawithanattachedaxonor dendrite. The good physiological state of the isolated somata or soma+axon complexes was confirmed by a decrease in membrane leakage conductance, by stable or even improved membrane resting potentials and by the ability of soma+axon complexes to generate action potentials. Improved space clamp conditions of isolated soma in comparisonwiththeneuroneintheslicemakethepresent method useful also for studying the kinetics and activation characteristics of fast transient Na as well as K currents. Furthermore, somata and soma+axon complexes isolated from surrounding connective tissue could be successfully used in pharmacological studies of different types of identified neurones. Distribution of Na channels between soma, axon and dendrites The present results show that the soma of dorsal horn neurones contained only a small proportion (ca 1Ï7) of inactivating Na channels and almost no steady-state Na channels. The axon contained the rest of the inactivating and only about 50 % of the total steady-state Na channels. Another 50 % of the steady-state Na channels are probably distributed amongst the dendrites of the dorsal horn neurones. The density of inactivating Na channels in the soma membrane could be estimated in two different ways. The mean peak Na current measured in isolated soma was 306 ± 27 pa (52 somata ) at 30 to 10 mv. This gives a mean channel density of 1 0 per ìmâ under the assumption thatthemeansinglena channelcurrentis1paandthe