Evidence for bursting pacemaker neurones in cultured spinal cord cells

Neuroscience Vol. 16, No. 4, pp. 753-761, 1985 Printed in Great Britain

0306-4522/85 $3.00 + 0.00 Pergamon Press Ltd IBRO

EVIDENCE FOR BURSTING PACEMAKER NEURONES IN CULTURED SPINAL CORD CELLS P. LEGENDRE, J. S. MCKENZIE,*

B. Duwuv

and J. D. VINCENT Unit& de Neurobiologie des Comportements, I.N.S.E.R.M. U.176, Domaine de Carreire, Rue Camille Saint-Sa&ns, 33077 Bordeaux Ctdex, France

Abstract-Intracellular recordings were made from dissociated mouse spinal cord cells in primary culture. One type of spinal cord neurone, with a large cell body (4CL50pm), 3-5 short neurites, and a mean resting potential of -65 mV, was found to fire rhythmic bursts of action potentials with a phase duration of approximately Is when the membrane potential was depolarized to -55 mV. These bursts did not arise

from spontaneous synaptic input, but appeared to result from endogenous ionic conductance properties of the membrane resembling those observed in molluscan bursting pacemaker neurones. Ionic conductances underlying this bursting activity were studied pharmacologically by local application of ionic conductance blockers. Pacemaker potentials depended on Na+ conductance, since tetrodotoxin and Na-free medium were the most potent agents for blocking spontaneous rhythmic activity. However, a Ca’+ conductance was involved in the depolarizing phase of membrane potential oscillations, since Ba2+ application increased oscillation amplitude. Action potentials observed during the bursts were Na+- and Cal+-dependent. They did not differ significantly from those observed in other spinal cord neurones in culture. Application of tetraethylammonium, CoCI,, BaCl, and 4-aminopyridine revealed at least three different potassium conductances which controlled this bursting pacemaker activity. A delayed potassium conductance controlled spike duration, a Ca-dependent potassium conductance controlled the duration of the burst and underlay the hyperpolarizing phase terminating the burst, and finally, a transient potassium conductance appeared to be involved in the control of phase duration. The demonstration that spinal cord neurones growing in monolayer culture display typical bursting pacemaker activity raises the possibility that bursting pacemaker neurones in the mammalian spinal cord may be involved in a phasic pattern generator that could control such activities as walking and the respiratory rhythm.

Rhythmic activity of spinal cord neurones occurs in mammals and other vertebrates, even in the absence of rhythmic input, and is possibly related to the generation of local programmes for activating motoneurones underlying locomotion and respiratory movements.27~28~62~63 Extracellular recordings of rhythmic bursting activity in the spinal cord are not conclusive as to the membrane mechanism underlying this pattern of electrical behaviour. Various hypotheses have been proposed according to which the oscillations could be due either to a network of interneuronal connections (the half-center mode141.6s and the loop model”‘) or to coupled endogenous pacemaker neuronesSo (for review see Grillner*‘). Endogenous bursting pacemakers have been described previously in invertebrate neurones from Invertebrate neurones are various species. 2’~23~38~58~64 more accessible for intracellular recording and have enabled, using voltage clamp and current clamp techniques, the investigation of the mechanisms underlying this electrical activity which is now well documented.5~6~7~25~29~37 In mammals, bursting rhythAbbreviations: SRB, spontaneous rhythmic bursting; TEA, tetraethylammonium; TTX, tetrodotoxin. *Present address: University of Melbourne, Department of Physiology, Parkville, Victoria 3052, Australia. 753

mic activity has only recently been analyzed, using in vitro intracellular recording techniques in cerebellum40 in hypothalamic explants** and in slices of thalamus.‘7.35 Observation of rhythmic bursting activity in cultured mammalian spinal cord neurones offer the possibility, as in invertebrates, of distinguishing endogenous pacemaker mechanisms from synaptically driven rhythmic activity and of studying them under controlled conditions. In the present paper, we report on the occurrence of spontaneous bursting pacemaker activity recorded intracellularly, from neurones with a particular morphology, in dissociated mouse spinal cords cultures. MATERIALS AND

METHODS

The techniques for preparing dissociated cell cultures of mouse spinal cord were similar to those previously described.49,57Briefly, foetuses were removed from IOPS/ OF1 mice on the thirteenth day of gestation. Dissociation was performed mechanically, and the cell suspension was dispersed into sterile 35 mm plastic dishes (lux) at approximately IO6 cells per dish. Cultures were incubated at 37°C. in an atmosphere of 95% air and 5% CO,. The culture medium was changed twice per week. The mkdium used for the first week of incubation contained Minimal Essential Medium (Dulbecco) with 10% heat-inactivated horse serum, (Sigma) (IBF) 10% fetal calf serum, (IBF) 0.6% glucose, uridine (Sigma) 30pgml-’ and 5-fluoro-2-

P. LCGENURE et d.

754

deoxyuridine (Sigma) 15 pgml ’ to prevent division ol non-neuronal cells. After 6 days, this medium was changed to one containing Minimal Essential Medium supplemented with 10% inactivated horse serum and 0.6% glucose. On three occasions, foetal spinal cords were divided into three approximately equal parts along their craniocaudal extent. and separate cultures were prepared from each part. Intracellular recordings were made under direct visual control from cells in culture dishes placed on the stage of an inverted phase-contrast microscope, at 200 x magnification. The preparation was perfused continuously by gravity feed with a balanced salt solution consisting of HANK’s medium supplemented by 0.6% glucose, CaCI, (2 mM final concentration) (Gibco) and MgClz (5 mM final concentration), buffered at pH 7.3 with IO mM HEPES, (Sigma) and maintained at constant temperature (36 ‘C) by controlled air flux. Osmolarity was adjusted to 330mosM. The recording pipettes were filled with IM CH,COOK (SCrlOO MR resistance) or with 3M KCI (4660 MR resistance). Recording was performed using a conventional bridge circuit that allowed simultaneous voltage recording and current stimulation through the same electrode (DAGAN SSOO). Membrane potential was recorded with a rectilinear pen recorder (Gould 2400) and photographed from a storage oscilloscope (Tektronix 5 113). Tetrodotoxin (TTX, IO- 6 M Sigma), tetraethylammonium chloride (TEA, 10mM Sigma), 4-aminopyridine (10 mM Merck), CoCl, (10 mM), CdC1, (IO mM), BaCl, (6mM) and Na+-free solution (Tris HCI 100 mM subs&ted) were applied locally through multi-barelled micropipettes, to alter specific ionic currents. Ejection was controlled by gas pressure using a micrOpump (Medical Instrument BH2). The pressure of ejection was recorded on the pen recorder. All applied chemicals were dissolved in recording medium. In some experiments, normal medium was applied from one barrel to test for ejection artefacts. For marking cells with horseradish peroxidase, the microelectrode was withdrawn. and another one, filled with a 4% solution of horseradish peroxidase (Sigma Type VI) in 50mM Tris HCI at OH 8.6 suoolemented with 0.2M KC], .. was inserted into the same cell. The horseradish peroxidase was passed iontophoretically into the cell using 0.5 s positive pulses of 5-IOnA at I Hz. for 5m10min.4h RESULTS

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General ~~lectroph~siologic~~l properties

After intracellular penetration of the recording electrode, correctly impaled SRB neurones had a resting potential (Vm) of about -65 mV (mean = -65 mV i 6.4 SD; 1~= 26). and the intracellular recording remained stable for I h or more. Synaptic events were rarely observed in these cells although the same dish prominent synaptic activity could be recorded in other spinal cord neurones. At the level of resting membrane potential the cells remained generally silent but a burst of action potentials could be evoked by passing a short pulse of depolarizing current of high intensity (Fig. 2A,B). The cells revealed their capacity for rhythmic bursting only when subjected to depolarizing current (Fig. 2C). The threshold level for rhythmic bursting activity was about -55 mV (mean = -55.8 mV k 5.4 SD; n = 26) and at this membrane potential value the bursts occurred rhythmically at l-3 Hz. During spontaneous rhythmic activity, bursts were terminated by a fast repolarization (after potential), followed by a progressive depolarization of the membrane potential preceding the onset of a newburst (Fig. 2D). At threshold level the bursts had a duration varying from IO to 120 ms (mean = 74.4 ms f 38.6 SD; n = 26) and membrane oscillation underlying spike activity had an amplitude varying from 8 to 29 mV (mean = 14.3 mV f 6.2 SD: n = 26). The action potentials during a burst had a duration ol 1 ms and an absolute peak amplitude of about 10 mV (9.1 mV + 2.5 SD; n = 26). The number of spikes per burst was a fixed characteristic of a particular cell at the threshold level and amounted to 2-14 depending on the cell (mean = 5.5 & 3.1 SD; IZ= 26). Bursting rhythmic activity depended upon the membrane potential value. As shown in Fig. 3. increasing the depolarization gave rise to a decrease in duration and an increase in frequenty of the bursts. The rhythmicity of the burst discharge could be modulated by passing hyperpolarizing pulses of current since a pulse applied between two bursts delayed the onset of the next burst.

Spontaneous rhythmic bursting activity (SRB) was recorded from large multipolar neurones in 24-43 day-old cultures (26 in 24 dishes). After intracellular recording, horseradish peroxidase was intracellularly injected to reveal the neurite processes of the cells. These SRB neurones exhibited a constant and characteristic morphology: they had round or ovoid cell bodies of large diameter (40-50 pm) from which arose 3-5 short beaded neurites with sparse branching (Fig. 1). Other categories of neurones have been previously described by different workers.46.48.4y.s7 Passive electrical membrane properties Briefly, two morphologically and electrophysioInput resistance. The input resistance of bursting logically distinct elements were distinguished: (i) neurones was determined using 100 ms current pulses dorsal root ganglion cells characterized by circular of various amplitudes and both polarities (Fig. 4A). somata of various sizes and one or two thin and long The amplitude of the voltage response was measured process; (ii) spinal cord neurones identified by their before the onset of the burst and after the capacivariably shaped somata, less prominent nuclei and tative response. In the hyperpolarizing direction. multiple large processes. However, to our knowledge, the voltage
Fig. I. Light photomicrograph (Nomarski Optics) of a recorded bursting spinal cord neuron (42 days in culture) after intracellular injection of horseradish peroxidase (50 pm bar).

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Fig. 2. Rhythmic bursting activity (A) and (B). Two examples of bursts of different duration recorded in different cells. These bursts were triggered by passing a short pulse of depolarizing current of high intensity (A) resting potential (Vm) = -66 mV; (B) Vm = -60 mV). (C) A long subthreshold depolarizing pulse of current evoked bursting rhythmic activity (Vm = -68 mV). (D) The input resistance fluctuation &il nl : rhythmic activity was measured by passing short pulses of hyperpolarizing current (2 nA, 5 ms, / 20 H.z,). Note that apparent input resistance increased during the depolarizing ramp before the onset of a burst (maximum increase 33%) (holding potential (Vh) = -5OmV).

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Fig. 3. (A) Pen recordings of rhythmic bursting activity evoked by passing depolarizing current of increasing intensities. (B) Relationship between the frequency (a), the burst duration (m) and the intensity of the depolarizing holding current. The burst frequency increased with increasing depolarizing current while burst duration decreased: graph in (B) was derived from the data shown in (A).

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Fig. 4. Passive electrical properties of bursting neurones. (A) V-Z curve. Membrane potential responses were evoked by passing either hyperolarizing or depolarizing current pulses of varying intensities (inset). Note the linear relationship between current intensity and voltage response in the hyperpolarizing direction and the anomalous rectification in the depolarizing direction (membrane potential response value was measured 40ms after the beginning of the response, just before the onset of the burst: arrowhead). (B) The membrane time-constant was determined by plotting the time course of the membrane potential response to a constant hy~r~la~ng ~tang~ar pulse of current (inset). The vertical axis is the percent log change of the membrane potential with respect to time (abscissa). Possible electrode artefacts were ruled out by observing the response evoked by a pulse of current before and after cell penetration. The membrane time constant (3. I8 ms) was calculated as r = -S -’ where S is the slope of the regression line.

and 110 MfZ (mean = 70.4 h4Q 2 33.9 SD; n = 26). No relationship was found between the value of input resistance and the characteristics of bursting activity of these cells. In the depolarizing direction, the voltage-current relationship became non-linear (Fig. 4A). Changes in apparent input resistance during spontaneous rhythmic bursting activity were visualized by applying pulses of hy~rpolarizing current of constant intensity (2 ms, 2 nA). As seen in Fig. 2D the resistance increased progressively, between two successive bursts. This might represent the anomalous rectification classically described for pacemaker neurones,5.13.21.23

Tome-co~tant. The membrane time-constant of bursting neurones was determined by analysing the time course of the membrane voltage response evoked by a constant hyperpolarizing current pulses4 (Fig. 4B). The natural log of the voltage transient response was expressed as the percent of maximal response measured at the stable phase of the membrane response and was determined at every millisecond after the onset of the current pulse. These values were

plotted against time and the membrane time constant (5) calculated as 7 = -S- ’ where S represent the slope of the regression line. In six bursting neurones tested, the time-constant ranged between 2.5 and 5.5 ms (mean = 2.9 + I .09 ms SD). For the six cells tested, the semilogarithmic plot of the rate of change of membrane potential as a function of time was linear. This suggest that the neurites do not con-

tribute to the passive electrical response of the soma of SRB neurones.“s.s4 Pharmacological study of membrane underlying bursting rhythmic activity

conductances

EjFects of Na+ conductance blockers. Application of the Na+ current blocker, tetrodotoxin (TTX, 10m6M in the delivery pipette) or of Na+-free medium, from a delivery pipette near the cell body, completely suppressed the rhythmic bursting electrical activity (Fig SA,B). Recovery occurred at 3&60 s after the end of TTX application (n = 4) and at least 5 s after the end of Na-free medium application (n = 2). Nevertheless, in the presence of TTX, single spikes could be evoked by a pulse of depolarizing current, but not repetitive activity (Fig. 5C). The threshold for these spikes was higher than the threshold for burst generation before TTX application and after recovery. Application of Nat-free medium had the same effect as TTX in producing a slight hyperpolarization. Subsequently imposed depolarization (20 mV or more) failed to restore bursting activity until at least 5 s after terminating the Na +-free medium application. The beginning of recovery from Na+-free medium was characterized at first by isolated bursts and single action potentials developed with irregular membrane potential oscillations; this was followed by the occurrence of normal rhythmic bursts (Fig. 5B). On repeated brief application of Na” -free medium, the initial effect was each time to increase phase and

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Fig. 5. (A) Effect of local application of tetrodotoxin (TTX, 10e6M in the delivery pipette) (Vh = -50 mV). (B) Effect of Na+-free medium on spontaneous rhythmic activity. Both TTX and Na-free medium blocked spontaneous electrical activity (Vh = -48 mV). (C) During TTX application a single spike could still be evoked by a pulse of depolarizing current (Vm = - 76 mV).

burst duration. This was followed by a complete block of electrical activity, and recovery occurred in the reversed order (not shown). Agents modifying calcium conductance. Application of low Ca2+ solution (1 p M) from a delivery pipette near the cell produced a slow depolarization of the membrane potential and modified the firing pattern of the rhythmic bursting activity, by increasing burst duration and firing frequency and by reducing the duration and amplitude of the hyperpolarization

following the bursts (Fig. 6A). In the presence of low Ca2+ concentration the threshold of spikes was reduced, and the pulses of depolarizing current which normally evoked a burst induced a train of action potentials characterized by a progressive accomodation of discharge frequency (Fig. 6B). Application of Co2+ (10 mM in the delivery pipette) blocked the spontaneously electrical activity (Fig. 7A). Recovery occurred progressively. At first, single action potentials appeared. This was followed

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Fig. 6. Effect of low Ca’+ concentration (I PM) on spontaneous bursting activity. (A) During low Ca’+ medium application, burst duration and frequency increased. The hyperpolarizing phase between bursts was partially suppressed during such application (Vh = -50 mV). (B) Bursts evoked by a depolarizing pulse before (I), during (2) and after (3) application of low Ca’+ medium (Vm = -64mV).

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Fig. 7. (A) Effect of Co2+ application (10 mM in the delivery pipette) on rhythmic bursting activity (a). Co*+ blocked the spontaneous bursting rhythmic activity. Continuous firing of action potentials (b) was then observed before the complete recovery of rhythmic bursting activity (c) (Vh = -42 mV). (B) Co2+ application suppressed the burst evoked by a pulse of current, but a single action potential could still be evoked (Vh = - 80 mV).

by intense acceleration of the firing rate (Fig. 7A). Subsequently the action potentials became grouped into bursts with the reappearance of membrane potential oscillation. Only single action potentials could be triggered by a depolarizing pulse of current in the presence of calcium conductance blockers (Fig. 7B). This result was similar to that observed with TTX, but the threshold of this spike was higher than the threshold of the spike triggered under TTX. Also it had a shorter duration than the TTX-resistant action potential.

Application of high Ca’+ solution (10mM in the delivery pipette) near the soma of the cell produced temporary hyperpolarization with blockage of rhythmic activity. However, the rhythmic activity remained blocked during spontaneous return to previous membrane potential (Fig. 8A). On return to spontaneous bursting rhythmic activity with normal Ca*+ concentration, a pronounced hyperpolarization followed each burst with increased phase duration (Fig. 8A). Application of a depolarizing pulse of current during high Ca*+ could evoke a burst of

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Fig. 8. (A) Effect of IOmM calcium application from a delivery pipette on rhythmic bursting activity. IOmM calcium solution blocked reversibly the spontaneous rhythmic activity and transiently hyperpolarized the membrane. Note that the first bursts, observed at the beginning of spontaneous rhythmic bursting recovery, are followed by pronounced hyperpolarizing @t-potentials (Vh = -48 mV). (B) During 10 mM calcium application a depolarizing pulse evoked a burst of shorter duration than observed in normal calcium concentration (Vh = -72 mV).

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Bursting pacemaker neurones in cultured spinal cord

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Fig. 9. (A) Effect of Ba*+ application (6 mM in the delivery pipette) on spontaneous bursting activity (intensity of pressure ejection is noted in bracket). Ba*+ induced a transitory block of the electrical activity and then increased burst amplitude (Vh = -46 mV). (B) The application of Ba*+ plus TTX blocked reversibly the rhythmic spontaneous activity (Vh = -44 mV). (C) In the presence of Ba*+, the application of Co*+ was less effective than of TTX in blocking rhythmic electrical activity (Vh = -44 mV).

potentials (Fig. 8B). This burst had a shorter duration than those evoked with the normal Ca*+ concentration and the membrane depolarization underlying spike firing was of greater amplitude (Fig. 8B). Application of Ba *+ during spontaneous bursting activity increased bursting amplitude after an initial hyperpolarization and a transitory inhibition of the spontaneous electrical activity (Fig. 9A). Application of TTX or Co*+ during Ba*+ application interrupted the spontaneous electrical activity (Fig. 9B,C). TTX had a more pronounced effect than Co*+ on bursting rhythmic activity in the presence of Ba*+. TTX immediately blocked the repetitive electrical activity and slowly depolarized the membrane potential (Fig. 9B). Recovery occurred within 20-30 s after the end of TTX application. Before complete recovery, small membrane potential oscillations appeared and occasionally evoked large Ba*+ spikes followed by a pronounced hyperpolarizing after potential (Fig. 9B). Co*+ application briefly interrupted spontaneous Ba2+ spikes and hyperpolarized the membrane potential by about 2-4mV (Fig. 9C). The recovery of electrical activity occurred immediately after the end of Co*+ application and was characterized by a slow depolarization of the membrane potential which evoked a rebound of high frequency discharge Ba*+ spikes, with increasing amplitude. When a burst was triggered by a depolarizing current, Ba*+ decreased its threshold and increased the amplitude of the membrane potential depolarization wave underlying the spiking activity (Fig. IOA). In some cases, small oscillations occurred in the action

ascending phase of these Ba*+ spikes. Simultaneous application of TTX and Ba*+ had a less pronounced effect than application of Co*+ and Ba*+ on bursts evoked by depolarizing current. TTX application slightly increased the threshold of the Ba*+ spike, slightly reduced its duration, and partially suppressed the small oscillations occurring in the ascending phase, but did not alter its amplitude (Fig. IOB). On the other hand, Co2+ application modified the evoked Ba*+ spike into a single action potential of short duration (Fig. 1OC). This action potential was suppressed by simultaneous application of Ba*+, Co*+ and TTX (Fig. 10D). Effects of potassium conductance blockers. Application of TEA from a delivery pipette near the soma of a recorded neurone (n = 3) resulted in a progressive increase of burst amplitude and a decrease of burst frequency (Fig. 11A). Both amplitude and duration of the action potentials were augmented. In some cases only a single large spike occupying the duration of the burst was observed. The burst was then followed by a pronounced hyperpolarization. After the end of TEA application recovery occurred slowly over IO-20 s. The burst amplitude progressively decreased to reach the pre-TEA value, while at the same time burst frequency increased and the hyperpolarization following the burst returned to its initial value. During TEA application, simultaneous application of TTX or CoCI, from a delivery pipette blocked the spontaneous electrical activity (Fig. 1lB,C). Recovery after TTX ejection was delayed compared to that observed after Co2+ application. However, a spike could still be evoked in the presence of TEA

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Fig. 10. (A) Effect of Ba*+ application on evoked bursts. The threshold level for the burst was reduced by Ba2+ and the amplitude of the oscillation underlying the burst of action potentials was increased (Vh = -74 mV). (B) This Ba2+ spike was not blocked by TTX application but its duration was slightly reduced and its threshold was slightly increased (Vh = -74 mV). (C) Co2+ had a more pronounced effect than TTX on the evoked burst in the presence of Ba2+; in the presence of Co’+ only one short action potential with a higher threshold was evoked by a pulse of depolarizing current (Vh = -74 mV). (D) This action potential evoked in presence of Ba2+ plus Co’+ was completely suppressed by TTX application (Vh = -74 mV).

or of TEA and TTX (Fig. 12). Under these conditions, the duration of the evoked spike was smaller than during TEA alone. The TTXresistant spike was followed by a pronounced hyperpolarization (Fig. 12). Application of Ba2+ during TEA ejection increased the discharge frequency of TEA spikes and depolarized the membrane potential before the onset of a long plateau of depolarization (Fig. 13). These plateaux were briefly interrupted by fast and irregular high-amplitude membrane potential oscillations. The stable depolarized phase of these plateaux was characterized by fast oscillations of low amplitude. After the end of Ba2+ application, the membrane holding potential returned to its previous value. This was followed by bursts which occurred irregularly before the complete recovery of the electrical activity normally observed under TEA. Application of 4-aminopyridine from a delivery pipette transformed rhythmic bursting activity into a continuous discharge of action potentials of longer duration (Fig. 14). Occasionally, the continuous and CoCI,,

firing led to a burst followed by a long hyperpolarizing phase, then by a brief rebound of high frequency discharge followed again by a continuous discharge. Recovery occurred progressively, the spikes tending to fire in short bursts with a high frequency, then in bursts of longer but irregular duration. Finally, the activity returned to its regular pattern of firing. DISCUSSION

Dissociated cell cultures obtained from mouse spinal cord have been extensively studied.“~46~48~49~55~s”~s7 They contain neurones of varying morphology of which electrophysiological properties and synaptic responses have been well documented with intracellular recordings.“.‘4.4s~4h~M.J7 In this paper, we describe a unique type of large neurone displaying a rhythmic pattern of bursting activity. The fact that neurones have never been these “pacemaker” described before, could be explained by their rarity (one or two neurones per dish). However, they can

163

Bursting pacemaker neurones in cultured spinal cord

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Fig. 11. Effect of TEA (10 mM in the delivery pipette) on spontaneous bursting activity. (A) TEA increased spike amplitude and duration. A pronounced afterpotential was then observed (Vh = -48 mV). (B) Simultaneous application of TEA and TTX blocked reversibly the spontaneous electrical activity (record interruption = 10 s). (C) In the presence of TEA, the application of Co2+ was less effective in blocking the spontaneous electrical activity than TTX application.

be easily distinguished from other neurones in the culture by their size (4&5Opm) and by their morphology, consisting of a large ovoid cell body with 3-5 short beaded neurites. Different arguments support the hypothesis that an endogenous pacemaker membrane potential underlies the rhythmic bursting activity of these large neurones. The cells which were silent at the

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membrane potential of -65 mV revealed their rhythmic activity when the holding potential was set by a depolarizing current above a threshold level (-55 mV). The frequency of bursts increased and their duration decreased with increasing depolarization of the holding potentia1.2’~23~38 The apparent input resistance increased with spontaneous ramp depolarization during the intervals between bursts.‘.*‘.”

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Fig. 13. Long depolarizing plateaux were evoked by both TEA and Ba2+. Small, high-frequency oscillations were superimposed on the depolarizing phase of the plateau (Vh = -42 mV).

Fig. 14. Effect of 4-aminopyridine (4AP) application (10 mM in the delivery pipette) on bursting electrical activity. The action potential duration was increased by 4-aminopyridine and the pattern of spontaneous electrical activity was modified from bursting to continuous firing. Recovery of the bursting rhythmic pattern occurred progressively and was characterized by an initial high-frequency discharge of small bursts followed by irregular burst discharge (Vh = -46 mV).

Finally, phase shifts could be induced by the application of hyperpolarizing pulses of current during spontaneous rhythmic bursting activity.8.5’.s2~5’.59 The endogenous bursters recorded from cultured mouse spinal cord have different characteristics from those identified in a number of other cells. The bursts are of shorter duration than those observed in molluscan nervous systems, while their frequency is IO-50 times higher than that of molluscan bursters.2’.23.38,64The values are close to those observed in some crustacean neurones” and in hypothalamic explants from rats.** Some passive membrane properties of the SRB neurones that we have recorded from the cultured spinal cord differ significantly from other spinal neurones’9,49.57and from nigral dopaminergic neurones2h recorded in vitro. The anomalous rectification observed in the V-I curve can be considered as characteristic of a pacemaker neurone.“.“.“,*’ The time constant for SRB neurones was estimated to be about 3 ms, a value lower than that observed in other cultured spinal cord cells (5 ms),” and in cells recorded in oico from spinal cord (5.3 ms)47 or from substantia nigra (12.5 ms).‘” However, this value is similar to that observed in cultures of dorsal root ganglion cells (3 ms).5’ The observation that the semilog plot of the rate of change of membrane potential as a function of time was linear suggests that the processes do not contribute to the passive electrical response of the soma of the SRB neurones.54 This can be explained by the very small surface area of their dendritic tree in comparison with the surface area of their cell body. In comparison, other large neurones in the culture

have a smaller cell body but a more extensive dendritic tree.j’ In order to determine which ionic conductances underlie the electrophysiological properties of SRB neurones, we have used different pharmacotogical agents known for their effects on different ionic channels. Single action potentials observed in SRB neurones do not seem to differ from those observed in other spinal cord neurones in culture.33.34,57Both Na+ and Ca*+ conductances participate in depolarizing inward current since TTX alone is not able to block action potentials which are suppressed by further addition of Co’+. Furthermore, TEA which blocks repolarizing K+ conductance increases the duration of the action potential. These TEA large spikes are not blocked by TTX but are considerably reduced by Co.*’ A Na+ conductance seems to be the major inward current carrier in the depolarizing phase of the pacemaker oscillation, since TTX and Na-free medium block the spontaneous rhythmic activity. This is in agreement with the observations of other workers on the beating or bursting pacemaker cells from invertebrates.5,25.36The role of Ca’+ in bursting activity appears more controversial.‘.h.‘1.‘5.7h.5yIt cannot be excluded that Ca’+ participates in the depolarizing phase, since Ba’+ which increases divalent inward current’ increases the amplitude of the oscillations, after a temporary block of spontaneous activity related to the effect of divalent cation on excitable properties of the membrane.‘~4~“.‘4~“1 Conversely Co2 +. after a temporary block of electrical activity, induces continous firing of action potentials. However, experiments performed on invertebrate neurones

Bursting pacemaker neurones in cultured spinal cord

suggest that Ca’+ is not involved in bursting pacemaker oscillation as a current carrier, but as a modulator of rhythmic activity.‘.6.7.25 Some of our results seem to confirm this hypothesis: high Ca2+ medium suppresses the spontaneous bursting activity while low Ca”+ increases the frequency and the duration of bursts. Other data indicate that a Ca*+-dependent conductance sustains the repolarization phase of the membrane potential after a burst: high Ca’+ medium reduces burst duration and increases both the amplitude of the burst after-hyperpolarization and the phase duration. This conductance, which is modified by the inward Ca*+ current, can be revealed by simultaneous application of Ba*+ and TEA. Such applications evoke long plateau depolarizations which probably are due to the blockage of calciumactivated KC current by Ba”‘.” However, BaZC, like Sr’+, can also affect calcium conductance inactivation.‘8 Thus, the interburst hyperpolarizations observed in SRB neurones may also represent an inactivation of calcium conductance induced by the intracellular accumulation of Ca*+, as recently proposed for Aplysia bursting neurones.39 Our results also suggest that another KC conductance might play a role in controlling the activity of the bursting pacemaker in spinal cord neurones. cl-Aminopyridine modifies dramatically the pattern of rhythmic bursting pacemaker activity. This potassium conductance blocker is generally described as blocking a transient potassium conductance’ involved in the control of phase duration in pacemaker neurones.7.‘h.254-Aminopyridine application increases the spike duration. It is unlikely that this action affects the membrane oscillations, since they are not modified by TEA. It is tempting to speculate that the irregular bursting rhythmic activity observed during 4-aminopyridine is due to partial block of a K+ conductance which resembles the transient outward current already described in invertebrate pacemaker neurones.‘,7.‘6.“4.2S

The demonstration that cultured spinal cord neurones display endogenous bursting pacemaker activity raises the question of pacemaker neurones playing a role as phasic pattern generators at the spinal cord Ievel. It is highly unlikely that the bursting pacemaker activity recorded in vitro is an artefact due to the culture conditions, since it is highly reproducible and is specific to a distinct type of cell. Moreover, numerous studies have demonstrated that

765

phasic generators can persist at the spinal cord level after complete central deafferentation, especially for walking and respiratory behavior.‘“~“~27~28~32~62 However, chemical excitation is necessary to observe this endogenous rhythmic activity. This implies a tonic inhibition of the bursting generator. This latter possibility has been confirmed by Viala and Buser*’ who proposed that the phasic generators are silent in the absence of central afferent activation. This observation seems to be in agreement with our results, which demonstrate that bursting pacemaker neurones in spinal cord culture are silent at resting potential levefs and need a holding depolarizing current for firing in serum free medium (recording medium) and in the absence of specific central inputs. Furthermore, the frequency observed in our cultures (l-2 Hz) is of the same order of magnitude as observed in v~vo.~~~~~ In our experiments, we cultured different segments of the foetal spinal cord to determine at which segmental level of the spinal cord the bursting pacemaker is localized. However, we found cells displaying a typical bursting pacemaker activity in all our cultured segments. These results are consistent with the results obtained in vivo which demonstrate the existence of pattern generators localized at different levels of the spinal cord.h3 These pattern generators are involved in the control of fore- and hindlimb behavior, and in the upper segments they also play a role in rhythmic respiratory activity.62 It will be intersting in the future to find the cell type that displays bursting activity in viva and to compare it with the cells studied in vitro. In vim, it had been proposed that bursting generators were localized in and around lamina VII of Rexed,” where numerous cells are phasically active. In our cultures, the cells that displayed bursting pacemaker activity differ in their morphology from other large multipolar neurones in vitro.” They also differ from motoneurones recorded intracellularly in vivo’2.42.47by their morphology and some of their passive and active electrical properties. Our cultured neurones thus appear to represent a particular population of spinal cord neurones. We are now trying to identify them by their secreted products and by their sensitivity to specific neurotransmitters such as catecholamines and serotonin, which could be involved in the control of spinal bursting-pattern generator activity.6i ~ckno~,~ed~emenrs-Weare grateful to Roger Miguelez for his excellent technical assistance. We thank Isabelle Lefranc for typing the manuscript, This work was supported by grants from the C.N.R.S. (UA 647) and Universiti de Bordeaux II.

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