Generation of rhythmic patterns of activity by ventral interneurones in rat organotypic spinal slice culture

Abstract

1. In the presence of certain excitatory substances the rat isolated spinal cord generates rhythmic oscillations believed to be an in-built locomotor programme (fictive locomotion). However, it is unknown whether a long-term culture of the same tissue can express rhythmic activity. Such a simplified model system would provide useful data on the minimal circuitry involved and the cellular mechanisms mediating this phenomenon. For this purpose we performed patch clamp recording (under whole-cell voltage or current clamp conditions) from visually identified ventral horn interneurones of an organotypic slice culture of the rat spinal cord. 2. Ventral horn interneurones expressed rhythmic bursting when the extracellular [K+] was raised from 4 to 6-7 mM. Under voltage clamp this activity consisted of composite synaptic currents grouped into bursts lasting 0.9 +/- 0.5 s (2.8 +/- 1.5 s period) and was generated at network level as it was blocked by tetrodotoxin or low-Ca2+-high-Mg2+ solution and its periodicity was unchanged at different potential levels. 3. In current clamp mode bursting was usually observed as episodes comprising early depolarizing potentials followed by hyperpolarizing events with tight temporal patterning. Bursting was fully suppressed by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and reduced in amplitude and duration by N-methyl-D-aspartate (NMDA) receptor antagonism without change in periodicity. Extracellular field recording showed bursting activity over a wide area of the ventral horn. 4. Regular, rhythmic activity similar to that induced by K+ also appeared spontaneously in Mg2+-free solution. The much slower rhythmic pattern induced by strychnine and bicuculline was also accelerated by high-K+ solution. 5. The fast and regular rhythmic activity of interneurones in the spinal organotypic culture is a novel observation which suggests that the oversimplified circuit present in this culture is a useful model for investigating spinal rhythmic activity.

Figure 6. Rhythmic activity induced by high-K⁺ solution under current clamp recording

A, rhythmic episodes evoked by 7 mM K⁺ recorded at three different levels of resting membrane potential (indicated by each trace) from the same cell patch clamped with an intracellular solution containing 4 mM Cl⁻ and 0.5 mM QX-314 (see Methods). At a resting membrane potential of -42 mV, depolarizing potentials (▾) preceded hyperpolarizing components (▵). At -66 mV, only large depolarizing events were manifested while at -12 mV events consisted mainly of large hyperpolarizing potentials. B, EPSP cycle period versus IPSP cycle period during rhythmic bursting evoked by K⁺ at a resting membrane potential of -42 mV. Data are from the cell shown in A. C, representative current clamp trace of spike activity recorded from a ventral interneurone (at -56 mV) following intracellular injection of a 500 ms depolarizing current pulse. In this case, QX-314 was omitted from the intracellular solution. Note that spike activity was maintained throughout the train without apparent accommodation.

Figure 1. Choline acetyltransferase (ChAT) immunocytochemical or biocytin-stained cells in an organotypic spinal cord slice

A, localization of ChAT-positive cells (arrow) in a 14 DIV spinal cord culture. The central fissura (asterisk) is a topographical marker of the ventral area. Note the ventral, bilateral distribution of positive cells. Calibration, 200 μm (× 5 objective). B, ventral motoneurones visualized by ChAT immunostaining. Same slice as in A (arrow). Note the large somata and the multipolar dendrites of these cells. The central fissura (asterisk) is outlined with a dashed line. Calibration (see bar in A), 75 μm (× 20 DIC (differential interference contrast) objective). C, double staining in a different spinal cord culture (14 DIV). Note the ChAT-positive neurone (arrow) and the ChAT-negative neurone (arrowhead). Calibration (see bar in A), 50 μm (× 20 objective). D, ChAT-positive neurone; note the large and triangle-shaped soma. Calibration (see bar in A), 25 μm (× 40 objective). E, ChAT-negative neurone; note the small dimension of the cell soma. The long time taken for the cell to be filled with biocytin from the patch pipette (the position is indicated by the arrowhead; see Methods) resulted in the soma being stained dark brown. Calibration (see bar in A), 25 μm (× 40 objective).

Figure 2. Increased extracellular [K⁺] induces patterned activity in ventral interneurone

A: left, spontaneous synaptic activity in control solution. Middle, increased extracellular K⁺ (7 mM; arrow indicates the start of application) induced a slow inward current which reached a peak value of -65 pA 40 s later (note smaller gain). This response was accompanied by a large increase in spontaneous synaptic activity and a gradual decline of the inward current towards baseline during continuous K⁺ superfusion. In the presence of high K⁺, spontaneous synaptic activity turned into a patterned activity within 2 min from the start of K⁺ application (expanded trace in B, middle), consisting of rapid bursts of inward current. Right, this pattern persisted with a regular although slower rhythm at 5 min of K⁺ superfusion. B, left trace depicts an average of 20 events taken at 5 min of K⁺-induced bursting (same cell as in A). Right trace, in the presence of K⁺ spontaneous bursting was suppressed by CNQX (same cell as in A). C, graphs showing burst cycle period (left) and duration (right) plotted against three different concentrations of extracellular K⁺. Cycle period and burst duration values were collected at 2 min (▪) or 5 min (▴) of the K⁺-induced patterned activity. Note the significant (**P < 0.001) shortening in cycle period and burst duration with 8 mM K⁺. Data were pooled from a population of ventral interneurones.

Figure 3. K⁺-induced activity is present over a wide area of the organotypic spinal cord culture

A, simultaneous field (a) and whole-cell recordings (b). Perfusion of K⁺ (arrowhead) induced an inward current in the recorded interneurone (b) with enhanced spontaneous synaptic activity, shown on a slow time base. In the presence of K⁺ this activity developed into a regular pattern, which could be detected with both electrodes although with opposite polarity, as more clearly shown by the faster records on the right. Note the similarity in cycle period and burst duration between the two recordings. The right-hand time calibration in b also applies to a. B: left, schematic drawing of the position of the field (Extra) and patch (WCR) electrodes within the organotypic slice. Note that the two electrodes were placed in the two contralateral ventral horns. In the presence of high K⁺, spontaneous rhythmic activity stabilized at 5 min in both ventral horns as shown by the two tracings (field (a) and patch (b) recordings). Note that the extracellular recorded events occurred with a delay from the patch recorded ones. The current calibration bar in B also applies to A.

Figure 4. Effect of the NMDA antagonist CPP on the K⁺-induced pattern

The NMDA receptor antagonist CPP was superfused after the K⁺-induced rhythm had stabilized. A: left, histograms of cycle period and burst duration recorded after 15 min application of CPP (10 μm) in 5 interneurones, expressed as a percentage of the rhythm observed in high-K⁺ solution. A significant reduction (**P < 0.001) in burst duration was observed. Right, averaged traces of 5 consecutive burst events in the absence or in the presence (arrow) of CPP are superimposed. Note the reduction in amplitude and duration brought about by CPP. B: left, example of 7 mM K⁺-induced rhythmic activity. Right, the burst duration and amplitude were reduced by CPP. Different cell from A (right).

Figure 5. Effect of different holding potentials on K⁺-induced rhythmic activity

A, bursting currents were inward at a V_h of -66 mV (top), decreased in amplitude at -36 mV (middle) and were outward at a V_h of +16 mV (bottom). Traces were digitized at 5 kHz. The current calibration for the middle trace also applies to the top trace. B, data shown are from 6 interneurones for which cycle period (top) and burst duration (bottom) are plotted against V_h. Values are expressed as a percentage of cycle period and burst duration recorded in each neurone at a V_h of -56 mV. Note that these values were not affected by changes in V_h. The inset shows the I-V curve obtained by plotting burst amplitude against V_h. Note the non-linearity in the negative potential range and that the calculated reversal potential was -18 mV.

Figure 7. High-K⁺ effects on bursting induced by coapplication of strychnine and bicuculline

A: left, strychnine and bicuculline induced sustained rhythmic bursting activity of a ventral horn interneurone. Right, an increased K⁺ concentration in the presence of strychnine and bicuculline shortened both cycle period and duration of bursting. B: left, expanded record of an individual event elicited by strychnine and bicuculline (segment of trace shown at higher gain and speed is indicated by dashed lines). Middle, plot of cycle period values observed in strychnine plus bicuculline solution (S + B) and after the subsequent increase in extracellular K⁺ to 8 mM, which significantly (**P < 0.0001; n = 5 cells) reduced them. Right, plot of burst duration values calculated for the same events under the same experimental conditions in which 8 mM K⁺ also decreased burst duration (**P < 0.0001). Data in 8 mM K⁺ were normalized with respect to those obtained in strychnine plus bicuculline solution. C: left, burst amplitude in the presence of strychnine and bicuculline plotted against V_h. I-V relations before (•) and after (▾) high-K⁺ application are shown (same cell as in A; note that burst amplitude in the presence of K⁺ was reduced and that the two curves displayed the same reversal potential). Right, burst amplitude versus V_h in the presence of strychnine and bicuculline before (□) and after (▵) 10 μm CPP superfusion (different cell from that in A and C). Note that the plot became linear after application of CPP.

Figure 8. Induction of rhythmic activity by high K⁺ is impaired when the bath concentration of Mg²⁺ and Ca²⁺ is changed

A, spontaneous synaptic activity under control conditions (left) developed into a patterned activity in the presence of high K⁺ (5 min; right). B, in the same cell spontaneous synaptic activity was greatly reduced in the presence of external solution with a 5/1 ratio of [Mg²⁺]/[Ca²⁺] (left; see Methods). Under these conditions addition of 6 mM K⁺ failed to induce rhythmic bursting (right). As these traces were not DC mounted, the steady inward current elicited by 6 mM K⁺ is not shown. C, synaptic currents evoked by DRG stimulation (see Methods) under control conditions (top; same cell as in A and B) disappeared in the presence of the 5/1 external solution (middle) and recovered after 10 min washout in control solution (bottom). Each panel is an average of 5 consecutive evoked responses.

Figure 9. Sensitivity of Mg²⁺-free-induced activity to membrane potential changes or glutamate receptor blockers

A: left, in the presence of Mg²⁺-free solution rhythmic bursting developed spontaneously after 5-10 min. Right, bursting currents were inward at a V_h of -66 mV, and outward at -10 mV. B, I-V relation of burst amplitude against V_h (same cell as in A). Note that the reversal potential was -20 mV. C, rhythmic bursting induced by superfusion with Mg²⁺-free solution (left) was fully blocked by CPP application (top right) but persisted in the presence of CNQX (bottom right), although with reduced amplitude and duration. In this cell, cycle period was not affected by CNQX. Different cell from A.