Muscarinic facilitation of GABA release in substantia gelatinosa of the rat spinal dorsal horn

Abstract

1. Blind patch clamp recordings were made from substantia gelatinosa (SG) neurones in the adult rat spinal cord slice to study the mechanisms of cholinergic modulation of GABAergic inhibition. 2. In the majority of SG neurones tested, carbachol (10 microM) increased the frequency (677 % of control) of spontaneous GABAergic inhibitory postsynaptic currents (IPSCs). A portion of these events appeared to result from the generation of spikes by GABAergic interneurones, since large amplitude IPSCs were eliminated by tetrodotoxin (1 microM). 3. The effect of carbachol on spontaneous IPSCs was mimicked by neostigmine, suggesting that GABAergic interneurones are under tonic regulation by cholinergic systems. 4. The frequency of GABAergic miniature IPSCs in the presence of tetrodotoxin (1 microM) was also increased by carbachol without affecting amplitude distribution, indicating that acetylcholine facilitates quantal release of GABA through presynaptic mechanisms. 5. Neither the M1 receptor agonist McN-A-343 (10-300 microM) nor the M2 receptor agonist, arecaidine (10-100 microM), mimicked the effects of carbachol. All effects of carbachol and neostigmine were antagonized by atropine (1 muM), while pirenzepine (100 nM), methoctramine (1 microM) and hexahydrosiladifenidol hydrochloride, p-fluoro-analog (100 nM) had no effect. 6. Focal stimulation of deep dorsal horn, but not dorsolateral funiculus, evoked a similar increase in IPSC frequency to that evoked by carbachol and neostigmine. The stimulation-induced facilitation of GABAergic transmission lasted for 2-3 min post stimulation, and the effect was antagonized by atropine (100 nM). 7. Our observations suggest that GABAergic interneurones possess muscarinic receptors on both axon terminals and somatodendritic sites, that the activation of these receptors increases the excitability of inhibitory interneurones and enhances GABA release in SG and that the GABAergic inhibitory system is further controlled by cholinergic neurones located in the deep dorsal horn. Those effects may be responsible for the antinociceptive action produced by the intrathecal administration of muscarinic agonists and acetylcholinesterase inhibitors.

Figure 4. Carbachol increases the amplitude of spontaneous IPSCs

Records were obtained from the same neurone as in Fig. 2. In A, the top, middle and bottom show, respectively, the amplitude histograms for IPSCs recorded in control, carbachol (10 μm) and carbachol (10 μm) with TTX (1 μm) bath solutions. Each histogram was constructed from 60 s of continuous recording. Top: in the control bath, the mean IPSC amplitude was 12.0 ± 0.8 pA and the maximum value was 127 pA. Middle: in the presence of carbachol, both the mean and maximum IPSC amplitudes were increased to 52.8 ± 0.8 pA and 233 pA, respectively. Bottom: TTX reduced the mean amplitude and maximum value, respectively, to 10.5 ± 0.2 pA and 43.5 pA. The carbachol-induced large amplitude IPSCs were eliminated by TTX, but small amplitude mIPSCs persisted. The maximum values of IPSC amplitudes are indicated by arrows. B, cumulative histograms of IPSC amplitude before and during carbachol application, and co-application of carbachol and TTX. Carbachol apparently shifted the curve to the right, which was blocked by TTX. C, an illustration of how the amplitude of each IPSC was measured, especially in cases where the baseline current was elevated by the summation of IPSCs.

Figure 1. Carbachol increases the frequency of spontaneous GABAergic IPSCs

Records shown were obtained from two different neurones (A and B). At a holding potential of 0 mV, IPSCs were recorded as upward deflections in the membrane current trace. The bars above each trace indicate the application of carbachol. A, bath application of carbachol (10 μm) evoked repetitive large amplitude IPSCs. In this cell, a prominent summation of IPSCs produced a persistent elevation in the baseline current. B, in a different neurone, relatively small IPSCs began to increase prior to the appearance of large IPSCs. Unlike the cell above, this neurone did not demonstrate a pronounced summation of IPSCs in the presence of carbachol.

Figure 2. The effects of tetrodotoxin on carbachol-induced IPSC frequency

Records were obtained from one neurone. A, carbachol (10 μm) increased IPSC frequency and produced an outward current. Increased IPSC frequency was reduced, but not completely blocked, by TTX (1 μm). The outward current was inhibited by TTX and IPSC peaks were truncated. B, carbachol increased the frequency of spontaneous IPSCs from 5.7 to 48.5 Hz, and the carbachol-induced increase in IPSC frequency was reduced by TTX to 10.1 Hz.

Figure 3. Atropine and bicuculline antagonize the carbachol-induced increase in IPSC frequency

Records shown were obtained from two different neurones (A and B). A, three sets of traces show IPSCs recorded in control, carbachol and carbachol + atropine bath solutions on an expanded time base. Carbachol (10 μm) increased the frequency of IPSCs, and this effect was blocked by the co-application of atropine (1 μm). B, in a different neurone, three sets of traces show IPSCs recorded in control, carbachol and carbachol with bicuculline bath solutions. Carbachol (10 μm) again increased the frequency of IPSCs, and all IPSCs were completely eliminated by the simultaneous application of bicuculline (20 μm).

Figure 5. Carbachol increases the frequency of miniature IPSCs

Records were obtained from one neurone in the presence of TTX (1 μm) and strychnine (2 μm). A, recordings before and during the application of carbachol (10 μm). Carbachol increased the frequency of mIPSCs. B and C, amplitude histograms of mIPSCs before (B) and during (C) carbachol application. Each histogram was constructed from 60 s of continuous recording. Carbachol increased mIPSC frequency from 6.4 to 26.3 Hz without affecting the mean amplitude (control, 14.1 ± 0.5 pA vs. carbachol, 13.6 ± 0.2 pA). D, cumulative histograms of mIPSC amplitude before and during carbachol application. Carbachol had no effect on the distribution of mIPSCs (Kolmogorov-Smirnov test, P= 0.38, 383 and 1574 events analysed for the two curves).

Figure 6. Neostigmine mimics the effect of carbachol on spontaneous IPSCs

Records were obtained from one neurone. A, neostigmine (10 μm) increased the frequency of spontaneous IPSCs from 9.3 to 32.0 Hz and also produced an elevation of baseline current. B, on an expanded time scale, it is possible to clearly observe the change in IPSC frequency as well as the presence of large amplitude IPSCs which were absent in the control recording. C, amplitude histograms before and during neostigmine application. Each histogram was constructed from 60 s of continuous recording. Neostigmine increased the mean IPSC amplitude from 8.0 ± 0.3 to 23.4 ± 0.5 pA. This is in contrast to the increase in maximum IPSC amplitude by carbachol, which increased amplitude from 34 to 118 pA. The maximum values of IPSC amplitudes are indicated by arrows.

Figure 7. Neostigmine, unlike carbachol, fails to increase mIPSC frequency

Records were obtained from two different neurones (A and B). A, in the presence of TTX (0.5 μm) and strychnine (2 μm), neostigmine (10 μm) had no effect on mIPSC frequency. After TTX washout, neostigmine increased the frequency of spontaneous IPSCs from 4.5 to 14.8 Hz. B, in a different neurone, neostigmine again did not increase mIPSC frequency. The frequency of mIPSCs before and during application of neostigmine (10 μm) was 6.2 and 6.3 Hz, respectively. Carbachol (10 μm), however, increased frequency to 16.9 Hz.

Figure 8. Increase of GABAergic IPSCs in response to focal stimulation of deep dorsal horn

Records were obtained from one neurone. A, repetitive focal stimuli (100 μA, 0.4 ms, 20 pulses at 20 Hz) applied to the deep dorsal horn (lamina IV–V) with a monopolar electrode elicited the increase in IPSC frequency. The facilitation of GABAergic transmission started several seconds after the end of the stimuli and lasted for 2–3 min. The facilitation of GABAergic transmission evoked by focal stimulation was reversibly blocked by atropine (100 nm). B, the increase of GABAergic IPSCs are shown with a faster time scale. Note that focal stimulation induced repetitive IPSCs with large amplitude, not seen before stimulation. The effect is similar to that elicited by application of carbachol and neostigmine.