Pre- and postsynaptic volatile anaesthetic actions on glycinergic transmission to spinal cord motor neurons

Abstract

1. A common anaesthetic endpoint, prevention of withdrawal from a noxious stimulus, is determined primarily in spinal cord, where glycine is an important inhibitory transmitter. To define pre- and postsynaptic anaesthetic actions at glycinergic synapses, the effects of volatile anaesthetic agents on spontaneous and evoked glycinergic currents in spinal cord motor neurons from 6 - 14-day old rats was investigated. 2. The volatile anaesthetic agents enflurane, isoflurane and halothane significantly increased the frequency of glycinergic mIPSCs, enflurane to 190.4% of control+/-22.0 (mean+/-s.e.m., n=7, P<0.01), isoflurane to 199.0%+/-28.8 (n=7, P<0.05) and halothane to 198.2%+/-19.5 (n=7, P<0.01). However without TTX, isoflurane and halothane had no significant effect and enflurane decreased sIPSC frequency to 42.5% of control+/-12.4 (n=6, P<0.01). All the anaesthetics prolonged the decay time constant (tau) of both spontaneous and glycine-evoked currents without increasing amplitude. With TTX total charge transfer was increased; without TTX charge transfer was unchanged (isoflurane and halothane) or decreased (enflurane). 3. Enflurane-induced mIPSC frequency increases were not significantly affected by Cd(2+) (50 microM), thapsigargin (1 - 5 microM), or KB-R7943 (5 microM). KB-R7943 and thapsigargin together abolished the enflurane-induced increase in mIPSC frequency. 4. There are opposing facilitatory and inhibitory actions of volatile anaesthetics on glycine release dependent on calcium homeostatic mechanisms and sodium channels respectively. Under normal conditions (no TTX) the absolute amount of glycinergic inhibition does not increase. The contribution of glycinergic inhibition to anaesthesia may depend on its duration rather than its absolute magnitude.

Figure 1

In spinal cord motor neurons sIPSCs are predominantly glycinergic. Excitatory glutamate receptors were blocked by AP-5 and CNQX. Holding potentials were −60 mV for this and all other experiments. The currents were inward (downward direction) due to the high Cl⁻ concentration in the patch pipette. (A) The GABA_A receptor antagonist bicuculline (5 μM) had little effect on sIPSC frequency or amplitudes. The remaining currents were completely blocked by the glycine antagonist strychnine (400 nM). (B) In another cell, the sequence of application of inhibitory receptor antagonists was reversed. Application of strychnine 400 nM removed the majority of events, leaving a population of sIPSCs that were smaller and had slower kinetics. These events were blocked by bicuculline 5 μM. (C) Cumulative histograms of interval and amplitude in the presence of bicuculline (left two graphs in C) and in the presence of strychnine (right two graphs in C). Bicuculline was not associated with change in frequency or amplitude distribution, whereas strychnine produced a rightward shift in interevent interval (decrease in frequency) and a leftward shift in amplitude distribution.

Figure 2

Histograms comparing glycinergic mIPSC and sIPSC frequency and amplitude. TTX reduced both frequency and amplitude of mIPSCs compared to sIPSCs. The results suggest that the majority of sIPSCs are TTX-sensitive Na⁺ channel-dependent events, and that their amplitudes are larger than those of sodium channel-independent events. n=18 – 21, ^**, significantly different from mIPSCs, P<0.01.

Figure 3

Volatile anaesthetics increased the frequency of miniature glycinergic IPSCs in the presence of TTX. A – C, mIPSCs in control conditions and during exposure to each of the three anaesthetics. Graphs to the right of each set of traces show the time course of the frequency changes during and after anaesthetic exposure in the same neurons. Bin width=10 s.

Figure 4

(A) Histograms showing actions of each anaesthetic on mIPSC frequency, amplitude and decay time constant. n=7 for each anaesthetic, error bars are s.e.m. ^*, ^** significantly different from control at P×0.05 and P<0.01, respectively. (B) Illustrations of the prolongation induced by anaesthetic on mIPSCs. Each trace is the mean IPSC in control and anaesthetic periods from a single cell. Scaled overlay shows anaesthetic current amplitude scaled to match control; the slower-decaying current is in the presence of the anaesthetic. The number of mIPSCs in each mean for enflurane is 166, halothane 133, and isoflurane 299.

Figure 5

Examples of enflurane actions on mIPSC frequency in the presence of agents which block calcium influx and release from intraterminal stores. (A) Cadmium (50 μM), which blocks both entry through calcium channels and extrusion via the Na-Ca transporter; (B) Thapsigargin (5 μM), which blocks release from endoplasmic reticulum; (C) KB-R7943 (5 μM), which blocks influx via the Na-Ca transporter, and (D) the combination of Thapsigargin (1 μM) and KB-R7943 (5 μM). Only the combination prevented the enflurane-induced increase in mIPSC frequency.

Figure 6

Quantitative effects of agents which block Ca influx and release on mIPSC frequency. Numbers over each bar are numbers of cells. ^**, ^*, significantly different from control, P<0.01 and P<0.05 respectively; #, significantly different from enflurane alone, P<0.05.

Figure 7

In some neurons volatile anaesthetic decreased the frequency of glycinergic sIPSCs in the absence of TTX; over the entire sample the decrease was significant only for enflurane. (A – C) Sample records of sIPSCs before and during exposure to each of the three anaesthetic agents. To the right of each set of traces are the time courses of sIPSC frequency from the same neuron during and after anaesthetic exposure.

Figure 8

(A,B) Two examples of enflurane actions on glycine-evoked currents. Glycine pulses of 200 ms were given at the time indicated by the arrows. Sharp deflections are brief hyperpolarizing voltage steps of 10 mV to monitor input resistance. In the example shown in (A) enflurane did not appreciably affect amplitude but prolonged decay. Overlay shows control and enflurane-modified currents scaled to the same amplitude. In the cell shown in (B), enflurane depressed the amplitude of glycine-evoked currents but still prolonged the decay time constant. (C) Histogram summarizing enflurane effects on glycine-evoked currents in motor neurons (n=15). Error bars are s.e.m. Enfluorane significantly prolonged the decay time of the currents (P<0.05, compare to control), but did not significantly alter amplitude or area. (D) Ejection duration was reduced to test the possibility that prolonged glycine exposure altered enflurane actions by saturating receptors or promoting desensitization. Glycine currents were evoked by a graded series of increasing durations of 1 mM glycine application. As was the case with the long duration applications, enflurane (0.6 mM) prolonged the currents evoked by brief glycine applications but did not increase amplitudes in any of the five cells tested. Insets under the shortest duration (2.5 ms) show the small currents at enlarged amplification. (E) The concentration of glycine in the pipette was reduced to 100 μM. Inset shows the currents are prolonged but not increased in amplitude. Graph shows stable amplitude over the course of enflurane application and an increase in area.