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. 1998 Apr 1;508(Pt 1):83–93. doi: 10.1111/j.1469-7793.1998.083br.x

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

H Baba *, T Kohno *, M Okamoto *, P A Goldstein *, K Shimoji *, M Yoshimura †
PMCID: PMC2230847  PMID: 9490821

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 μm) 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 μm).

  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 μm) 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 μm) nor the M2 receptor agonist, arecaidine (10–100 μm), mimicked the effects of carbachol. All effects of carbachol and neostigmine were antagonized by atropine (1 μm), while pirenzepine (100 nm), methoctramine (1 μm) 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.

Intrathecal administration of muscarinic agonists or acetylcholinesterase inhibitors produces analgesia in animals (Smith, Yang, Nha & Buccafusco, 1989; Gillberg et al. 1990; Abram & O'Connor, 1995; Bouaziz, Tong & Eisenach, 1995) and clinical investigations have also demonstrated the effectiveness of intrathecally administered acetylcholinesterase inhibitors as analgesics (Hood, Mallak, Eisenach & Tong, 1996). It appears that these drugs exert their analgesic effect through muscarinic receptors since muscarinic, but not nicotinic, agonists are effective (Smith et al. 1989; Gillberg et al. 1990), and the muscarinic antagonist atropine inhibits the analgesia produced by both muscarinic agonists and acetylcholinesterase inhibitors (Zhuo & Gebhart, 1991; Naguib & Yaksh, 1994). The mechanism of this muscarinic effect in the spinal cord, however, is not fully understood. Autoradiographic studies have demonstrated that the highest density of muscarinic receptors in the spinal cord is located in Rexed's lamina II (substantia gelatinosa, SG), both in rats (Yamamura, Wamsley, Deshmukh & Roeske, 1983) and in humans (Scatton, Dubois, Javoy-Agid & Camus, 1984; Villiger & Faull, 1985). In addition, dorsal rhizotomies have been shown to reduce, but not abolish, the level of muscarinic binding in the spinal dorsal horn (Gillberg & Wiksten, 1986; Gillberg & Askmark, 1991). Such observations indicate that muscarinic receptors are located on a subpopulation of spinal interneurones. Furthermore, most Aδ and C fibres carrying nociceptive information preferentially terminate in the superficial dorsal horn, especially in the SG, a region which has been considered as a critical site for modulating nociceptive information and controlling the activity of projection neurones (Kumazawa & Perl, 1978; Yoshimura & Jessell, 1989). It is expected, therefore, that a cholinergic mechanism in SG accounts for the analgesic effect of muscarinic agonists. Direct analysis of responses of SG neurones to these drugs, however, has not been undertaken because of the difficulty of obtaining stable intracellular recordings from small SG neurones in vivo. Conventional intracellular recording requires the use of a high impedance microelectrode because of the small size of SG neurones (5–20 μm in diameter; Brown, 1981), which makes voltage clamp recording and direct measurement of quantal release events difficult; the analysis of small miniature synaptic events is difficult because of the problems of signal-to-noise ratio. Recently, we have developed a technique for whole-cell voltage clamp recordings from SG neurones in thick slices of the adult rat spinal cord which overcomes these problems (Yoshimura & Nishi, 1993). Using blind patch clamp recording from SG neurones in the adult rat spinal cord slice, we have studied the action of muscarinic agonists and acetylcholinesterase inhibitors on GABAergic IPSCs, which are thought to be involved in spinal antinociception (Yoshimura & Nishi, 1995).

METHODS

The methods for obtaining slices of the adult rat spinal cord and for blind patch clamp recordings from SG neurones have been described in detail elsewhere (Yoshimura & Jessell, 1989; Yoshimura & Nishi, 1993). Briefly, a portion of the lumbosacral spinal cord was removed from an adult rat (8–16 weeks, 200–300 g; n= 34) under anaesthesia with urethane (1.5–2.0 g kg−1, i.p.). Rats were killed by cutting the descending aorta. The isolated spinal cord was then placed in pre-oxygenated cold Krebs solution (2–4°C). After removal of the dura mater, all ventral and dorsal roots were cut and then the pia-arachnoid membrane was removed. The spinal cord was placed in a shallow groove formed in an agar block and glued to the bottom of the microslicer stage with cyanoacrylate adhesive and held in place by the agar block. The spinal cord was immersed in cold Krebs solution and a 450–500 μm thick transverse slice was cut on a vibrating microslicer (DTK1500; Dosaka Co. Ltd, Kyoto, Japan). The spinal cord slice was then placed on a nylon mesh in the recording chamber and perfused with Krebs solution (20 ml min−1) saturated with 95 % O2 and 5 % CO2 at 36 ± 1°C. The Krebs solution contained (mm): NaCl, 117; KCl, 3.6; CaCl2, 2.5; MgCl2, 1.2; NaH2PO4, 1.2; NaHCO3, 25; glucose, 11. Unless otherwise indicated, IPSCs were recorded in the presence of dl-2-amino-5-phosphonovaleric acid (APV; 25 μm), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 μm) and strychnine (1–2 μm) so as to block, respectively, N-methyl-d-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and glycine receptors. Blind whole-cell patch clamp recordings were made from neurones located in SG (Yoshimura & Nishi, 1993). The pipette solution contained (mm): caesium sulphate, 110; CaCl2, 0.5; MgCl2, 2; EGTA, 5; Hepes, 5; TEA, 5; MgATP, 5; guanosine-5′-O-(2-thiodiphosphate) (GDP-β-S), 1. The resistance of a typical patch pipette was 5–10 MΩ. GDP-β-S was added to the pipette solution to block postsynaptic effects mediated by cholinergic agonists through G proteins. Focal stimulation was made with a monopolar silver wire electrode (50 μm diameter), insulated except at the tip, positioned in the deep dorsal horn (lamina IV, V) or dorsolateral funiculus to activate fibres of interneurones or descending fibres from the brainstem (Yajiri, Yoshimura, Okamoto, Takahashi & Higashi, 1997). Constant-current pulses (50–100 μA, 0.4 ms) were applied with this electrode. Voltage-clamped neurones were held at a membrane potential of 0 mV (unless otherwise stated). Membrane currents were amplified with an Axopatch 200A amplifier (Axon Instruments) in voltage clamp mode. Signals were filtered at 2 kHz and digitized at 5 kHz. Data were stored in a personal computer using pCLAMP 6 software (Axon Instruments). Frequencies and amplitudes of spontaneous and miniature IPSCs were measured using AxoGraph 3 software (Axon Instruments). The amplitudes of each IPSC were measured from the initial point (not from the baseline) to the peak of the IPSC (Fig. 4C). Numerical data are presented as the means ±s.e.m. Drugs were applied by exchanging the perfusion solution with one containing a known drug concentration, without altering the perfusion rate and temperature. The time required for the drug-containing solution to flow from the three-way stopcock to the recording chamber was about 3 s. Drugs used were carbamylcholine chloride (carbachol; Sigma), (+)-muscarine chloride (RBI), oxotremorine methiodide (RBI), bethanechol chloride (Sigma), arecaidine but-2-ynyl ester tosylate (Tocris Cookson), McN-A-343 (RBI), neostigmine bromide (Sigma), dl-2-amino-5-phosphonovaleric acid (APV; Sigma), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; Tocris Cookson), strychnine (Sigma), bicuculline (Sigma), atropine sulphate monohydrate (WAKO, Osaka, Japan), hexamethonium dichloride (RBI), D-tubocurarine chloride (RBI), pirenzepine dihydrochloride (Sigma), methoctramine tetrahydrochloride (RBI), hexahydrosiladifenidol hydrochloride, p-fluoro-analog (p-F-HHSiD; RBI), tetrodotoxin (TTX; WAKO) and guanosine-5′-O-(2-thiodiphosphate) (GDP-β-S; Sigma).

Figure 4. Carbachol increases the amplitude of spontaneous IPSCs.

Figure 4

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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.

RESULTS

All SG neurones tested exhibited spontaneous EPSCs and IPSCs at holding potentials of −70 mV and more positive than −60 mV, respectively. EPSCs observed at −70 mV were completely blocked by CNQX, suggesting that they were mediated by non-NMDA glutamate receptors. Two distinct types of IPSCs were observed, according to their decay time course. One type of IPSC had a short duration (10–20 ms) and was antagonized by strychnine (1 μm); the other had a relatively long duration (50–100 ms) and was antagonized by bicuculline (10 μm), suggesting that the two types of IPSCs were mediated by glycine and GABAA receptors, respectively (Yoshimura & Nishi, 1993). All of the present data were obtained in the presence of strychnine (1–2 μm) in order to isolate only GABAergic IPSCs. Shortly after establishing the whole-cell patch clamp configuration, bath-applied carbachol elicited either outward or inward currents in the majority of SG neurones at a holding potential of −70 mV. These currents disappeared within a few minutes of carbachol application when the intracellular solution contained Cs+, TEA and GDP-β-S, suggesting that they were mediated by G protein-coupled K+ currents as reported previously (Yajiri et al. 1997).

Carbachol increases the frequency and mean amplitude of spontaneous IPSCs

As shown in Fig. 1, carbachol markedly increased the frequency of spontaneous (as distinct from evoked) GABAergic IPSCs. The baseline frequency of IPSCs was 6.3 ± 0.4 Hz (n= 69; range, 1.6–18.2 Hz). Carbachol (10–30 μm) increased the frequency of IPSCs in sixty-four of seventy-one SG neurones tested (677.4 ± 50.4 % of control for 10 μm carbachol, n= 24). In the majority of SG neurones tested, carbachol induced repetitive IPSCs with large amplitudes, which were not seen in the control state (Figs 1A and 4A). In some SG neurones, however, an increase in the frequency of the small amplitude IPSCs was observed which was then followed by repetitive large amplitude IPSCs (Fig. 1B). The effect of carbachol on IPSC frequency reached a steady state within 2 min and showed no evidence of desensitization over at least 30 min. Following carbachol washout, IPSC frequency slowly returned to baseline (recovery time was usually more than 5 min). Other muscarinic agonists, muscarine (10–20 μm, n= 6), oxotremorine (10 μm, n= 12) and bethanechol (30–100 μm, n= 8), also increased the frequency of spontaneous IPSCs (290–1155 % of control frequency) in a manner similar to that of carbachol. In some SG neurones, the increase in IPSC frequency resulted in a prominent summation of membrane currents that produced a persistent elevation of baseline current. The elevation of baseline current was blocked by TTX to control level (Fig. 2A), leaving small amplitude IPSCs which had a much higher frequency than before application of carbachol (Fig. 2B). Addition of the muscarinic antagonist atropine (1 μm) blocked the increase in frequency of IPSCs induced by carbachol back to control levels (n= 4, Fig. 3A). Facilitated IPSCs were confirmed to be mediated by GABAA receptors as evidenced by their complete elimination in the presence of 20 μm bicuculline (n= 5, Fig. 3B). Hexamethonium (50 μm, n= 12) and D-tubocurarine (10 μm) failed to block the action of carbachol (data not shown), indicating that nicotinic acetylcholine (ACh) receptors did not contribute to the increase in IPSC frequency. In addition to the change in frequency, an IPSC amplitude was also augmented. The mean amplitude of IPSCs before application of carbachol was 13.9 ± 0.8 pA (n= 6; range, 11.5–16.6 pA). In six of six cells, carbachol increased the mean amplitude of IPSCs (280 ± 53 % of control; range, 167–439 %) as well as their frequency. This was especially evident for large amplitude IPSCs, which were not seen in the control state (Fig. 4A; top and middle histograms). Under control conditions, IPSC amplitude tended to cluster between 0 and 20 pA, with a maximum amplitude of 127 pA (indicated by an arrow in Fig. 4A, top histogram). This is in contrast to the amplitude distribution in the presence of carbachol, which exhibited a significant number of IPSCs with an amplitude in the 20–120 pA range with a maximum amplitude of 233 pA (indicated by an arrow in Fig. 4A, middle histogram). These large IPSCs were not due to miniature IPSCs (mIPSCs) reflecting quantal release, but in fact corresponded to synaptic release caused by carbachol-induced firing of GABAergic interneurones. This view is supported by the observation that the large amplitude IPSCs (> 45 pA) were abolished by the addition of TTX (1 μm) to the bathing medium (Figs 2B and 4A, bottom histogram). This observation also indicates that even in the control state, a portion of spontaneous IPSCs (especially those with an amplitude of > 40 pA) result from the spontaneous firing of GABAergic interneurones (Fig. 4A, top and bottom histograms). The change in amplitude distribution was clearly shown in the cumulative histogram (Fig. 4B). In the presence of carbachol, the relative frequency curve was shifted to the right and this shift was markedly inhibited by TTX, indicating that carbachol enhanced GABA release due to the activation of somadendritic spiking. The carbachol-induced increase in IPSC frequency, however, could not be completely blocked by TTX (202 ± 21 % of control; n= 5; Fig. 2B) as the frequency of the relatively small amplitude IPSCs remained elevated even in the presence of TTX, raising the possibility that carbachol also affected quantal release of GABA from presynaptic terminals.

Figure 1. Carbachol increases the frequency of spontaneous GABAergic IPSCs.

Figure 1

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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.

Figure 2

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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.

Figure 3

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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).

Carbachol increases the mIPSC frequency

We next examined, therefore, whether carbachol affected the frequency of mIPSCs. In the presence of TTX (1 μm) and strychnine (2 μm), the frequency of GABAergic mIPSCs was 4.2 ± 0.6 Hz (n= 14; range, 1.5–8.0 Hz). Carbachol (10–30 μm) increased the frequency of mIPSCs in all SG neurones tested (n= 18); in the presence of carbachol (10 μm), mIPSC frequency was 308 ± 49 % of control (n= 10; Fig. 5A). Amplitude histograms were constructed for four cells in which mIPSCs were recorded. In all four cells, the amplitude distribution of mIPSCs was not affected by carbachol, while there was an apparent increase in event frequency (Fig. 5B-D). The carbachol-induced increase in mIPSCs was also reversed by atropine (1 μm; n= 3).

Figure 5. Carbachol increases the frequency of miniature IPSCs.

Figure 5

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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).

Carbachol increases spontaneous IPSCs via non-M1, M2 and M3 muscarinic receptors

To identify the muscarinic receptor subtype involved in carbachol-induced increase in spontaneous IPSC frequency, the effects of the M1 receptor agonist McN-A-343, and the M2 receptor agonist arecaidine on the frequency of spontaneous IPSCs were tested. In all SG neurones tested (n= 8), both McN-A-343 (10–300 μm) and arecaidine (10–100 μm) had no significant effect on the frequency of IPSCs. We next tested the effects of the M1 receptor antagonist pirenzepine, the M2 receptor antagonist methoctramine and the M3 receptor antagonist p-F-HHSiD, on carbachol-induced increase in IPSC frequency. Pirenzepine (at 1 μm; n= 5), but not pirenzepine (at 100 nm; n= 4), attenuated the carbachol-induced increase in IPSC frequency. Methoctramine (1–3 μm; n= 6) failed to block the effect of carbachol. p-F-HHSiD at 1 μm (n= 6) attenuated the effect of carbachol, but p-F-HHSiD at 100 nm (n= 4) had no effect. These observations suggest that the carbachol-induced increases in IPSCs are mediated by non-M1, M2 and M3 muscarinic receptors.

Endogenous ACh increases the frequency of spontaneous, but not miniature, IPSCs

We next tested the effect of neostigmine, an agent that blocks the metabolism of ACh, on both spontaneous and miniature GABAergic IPSCs. Neostigmine (10 μm) increased the frequency and mean amplitude of spontaneous IPSCs in a manner similar to that of carbachol in twelve of fifteen cells tested (501 ± 103 % of control frequency; n= 12; Fig. 6A-C). The addition of TTX (1 μm) after the neostigmine-induced increase in IPSC activity reduced the frequency of IPSCs to less than control levels in all cells tested (62.6 ± 6.5 % of control; n= 6). The large IPSCs induced by neostigmine were also abolished by TTX. The effect of neostigmine was reversed by atropine (1 μm). In contrast to carbachol, the frequency of mIPSCs was not affected by neostigmine (10 μm; 96.8 ± 4.2 % of control) in nine of eleven cells tested (Fig. 7). In the remaining two cells, mIPSCs were slightly increased (131 and 129 % of control frequency, respectively).

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

Figure 6

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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.

Figure 7

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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.

Focal stimulation of deep dorsal horn mimics the effect of carbachol and neostigmine

To clarify the origin of the muscarinic innervation, we examined the effects of focal stimulation within the slice (deep dorsal horn and dorsolateral funiculus). Repetitive focal stimuli (50–100 μA, 0.4 ms at 20 Hz) applied to the deep dorsal horn (lamina IV–V) ipsilateral to the recorded neurone elicited an increase of IPSC frequency in eleven of sixteen SG neurones (Fig. 8A, top). The focal stimulation-induced repetitive IPSCs with large amplitude were similar to those elicited by application of carbachol and neostigmine (Fig. 8B). The facilitation of GABAergic transmission usually started from several seconds after the end of stimuli, and lasted for 2–3 min. Atropine (100 nm) reversibly attenuated the stimulation-induced increase in IPSCs (Fig. 8A, middle). However, the stimulation of the dorsolateral funiculus of the ipsilateral side, which is thought to contain descending fibres, failed to evoke the increase in IPSCs in all SG neurones tested (n= 16). These results suggest that the origin of the muscarinic innervation is in nearby laminae (maybe deep dorsal horn), but not descending fibres from the brainstem.

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

Figure 8

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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.

DISCUSSION

The mechanisms of cholinergic modulation of GABAergic synaptic transmission in the substantia gelatinosa of rat spinal dorsal horn were examined. Both carbachol and neostigmine increased TTX-sensitive GABAergic IPSCs in the majority of SG neurones tested. However, carbachol, but not neostigmine, also increased quantal release of GABA from presynaptic terminals. The facilitatory effect of carbachol on GABAergic transmission was antagonized by atropine, but not by nicotinic antagonists. Focal stimulation of deep dorsal horn, but not dorsolateral funiculus, mimicked the effect of carbachol and neostigmine. Our observations indicate that cholinergic modulation of GABA-mediated transmission results from the activation of muscarinic receptors located at both somatodendritic sites and on the presynaptic terminals of GABAergic interneurones.

Cholinergic excitation of GABAergic neurones

In the majority of SG neurones tested, carbachol increased GABAergic spontaneous IPSCs. The amplitude histogram for IPSCs recorded in the presence of carbachol demonstrated an apparent increase in mean amplitude as well as frequency. A portion of these events appeared to result from the conduction of action potentials from the soma to the presynaptic terminal due to the repetitive firing of GABAergic interneurones within the slice, as evidenced by the fact that carbachol-induced large amplitude IPSCs were blocked by TTX. These large IPSCs suggest the possibility that GABAergic interneurones which synapse onto SG neurones are depolarized by carbachol via muscarinic receptors, a mechanism that has been reported in many brain areas (Madison, Lancaster & Nicoll, 1987; Lacey, Calabresi & North, 1990; Uchimura & North, 1990).

The receptor subtype involved in the carbachol action appears to be non-M1, M2 and M3 muscarinic receptors, since McN-A-343 and arecaidine, at high concentrations, could not mimic the effects of carbachol and neostigmine. In addition, pirenzepine (100 nm), methoctramine (1 μm) and p-F-HHSiD (100 nm) were ineffective (Buckley, Bonner, Buckley & Brann, 1989; Dorje, Wess, Lambrecht, Tacke, Mutschler & Brann, 1991).

The cholinergic activation of GABA release has been reported in many other parts of the CNS (Benardo & Prince, 1982; McCormick & Prince, 1986; Pitler & Alger, 1992), including the trigeminal nucleus (Travagli, 1996). However, the cholinergic excitation of GABAergic neurones shown in these reports was all TTX sensitive, indicating somatodendritic activation via the muscarinic receptors. In this paper, we also demonstrated that quantal release of GABA from presynaptic terminals was facilitated by carbachol which, in conjunction with the discussion above, suggests that GABAergic inhibitory interneurones possess muscarinic receptors on both axon terminals and somatodendritic sites. The carbachol-induced increase in frequency of GABAergic IPSCs was not secondary to changes in the postsynaptic cell. If carbachol produced an increase in the sensitivity of postsynaptic GABAA receptors, it would potentiate a population of subliminal IPSCs to an extent that they would become visible, which would increase the apparent IPSC frequency. Additionally, if carbachol resulted in the recruitment of ‘silent’ inhibitory synapses in a manner analogous to that described for excitatory synapses (Malgaroli & Tsien, 1992; Liao, Hessler & Malinow, 1995), then such recruitment would be expected to increase the apparent mIPSC frequency. Such enhancement is unlikely, however, because muscarinic receptors act via G proteins, and these should have been inhibited by the G protein blocker GDP-β-S present in the intracellular solution. In addition, carbachol did not affect the amplitude distribution of mIPSCs although it did produce an apparent increase in frequency. Thus, carbachol does not alter postsynaptic responsiveness to GABA although it does appear to homogeneously influence presynaptic GABAergic terminals which synapse onto SG neurones.

Although the exact location of GABAergic neurones is unknown, it is possible that they reside within the SG, as GABA has been shown to be present in cell bodies and terminals within the SG (Todd & Sullivan, 1990). Furthermore, autoradiographic mapping indicates that muscarinic receptors in the spinal dorsal horn are concentrated in the SG (Yamamura et al. 1983). Indeed, muscarine depolarized a subset of trigeminal SG neurones (Travagli, 1996) and carbachol produced inward currents in some spinal SG neurones when the intracellular solution did not contain GDP-β-S (H. Baba, unpublished observations). However, these inward currents were antagonized by pirenzepine, suggesting the mediation of M1 receptors. Alternatively, GABA-containing interneurones which synapse onto SG neurones in nearby lamina (possibly lamina III) may be depolarized via non-M1, M2 and M3 receptors.

ACh release

Our data suggest that endogenous ACh is spontaneously released at somatodendritic sites on GABAergic neurones, and that a population of SG neurones is under tonic regulation by the cholinergic system. A similar conclusion for tonic regulation has been proposed by Zhuo & Gebhart (1991) based on the results of their behavioural study on the effects of the intrathecal administration of atropine and physostigmine in the awake rat. Unlike carbachol, however, neostigmine had little effect on mIPSC frequency and the neostigmine-induced increase in IPSC frequency was completely blocked by TTX. These observations suggest that cholinergic axo-axonic synapses onto presynaptic terminals of GABAergic neurones may not exist or be rare. The source of ACh for activation of the presynaptic muscarinic receptors could be spillover from the axosomatic or axodendritic synapses. More likely, the ACh is released from the presynaptic terminal together with GABA, since ACh and GABA are thought to co-localize in at least some dorsal horn neurones (Spike, Todd & Johnston, 1993; Laing, Todd, Heizmann & Schmidt, 1994). However, this possibility is inconsistent with the observation that neostigmine had no significant effect on mIPSCs.

We also demonstrated that the stimulation of the deep dorsal horn, but not the dorsolateral funiculus, had a similar effect to carbachol and neostigmine on IPSC frequency. It is possible that the activation of cholinergic neurones located in the deep dorsal horn by focal stimulation may evoke muscarinic slow EPSPs in GABA-containing interneurones, since the facilitation effect elicited by stimulation lasted for 2–3 min.

Several lines of evidence have indicated the origin of muscarinic innervation in nearby laminae, although there are some reports which suggest cholinergic descending fibres from the brainstem (Jones, Pare & Beaudet, 1986; Zhuo & Gebhart, 1990). Immunocytochemical studies on choline acetyltransferase revealed that intrinsic spinal cholinergic neurones located in the deep dorsal horn, especially in lamina III, send a dense network of fibres to the superficial dorsal horn (Borges & Iversen, 1986; Ribeiro-da-Silva & Cuello, 1990). These studies could not find any descending cholinergic fibres. Our data also suggest that the origin of the muscarinic innervation is in the deep dorsal horn, but not from the brainstem.

Functional consideration

GABA may play an important role in the processing of nociceptive transmission in the SG. GABAA receptor antagonists often produce a burst of EPSPs (in current clamp condition) in response to a single stimulus which previously had evoked a single EPSP (Grudt & Williams, 1994; Yoshimura & Nishi, 1995). These observations suggest that excitatory inputs onto SG neurones are under the control of GABAergic inhibition. Our present study provides evidence that the GABAergic inhibitory system is further controlled by cholinergic inputs acting through a muscarinic receptor. Because GABAergic IPSPs exhibit relatively longer time courses than glycinergic IPSPs and show marked summation (Yoshimura & Nishi, 1995), repetitive spike firing of GABAergic neurones by ACh as shown in the present study may cause a substantial change in IPSP amplitude and duration. Our present study, moreover, provides a possible physiological underpinning for behavioural studies which have demonstrated an antinociceptive effect of spinal muscarinic agonists and acetylcholinesterase inhibitors.

In conclusion, we have provided electrophysiological evidence that the muscarinic receptor-mediated response is exerted via receptors located on GABAergic neurones. The inhibition of SG neurones through the cholinergic activation of GABAergic neurones might be one way in which muscarinic agonists and cholinesterase inhibitors inhibit the transfer of noxious input to other central sites in the pain pathway.

Acknowledgments

We would like to thank Dr C. J. Woolf for critically reading the manuscript. This work was supported by Grant-In-Aid for Scientific Research on Priority Areas (No. 07278214) and Grant-In-Aid for Scientific Research (B) (No. 07457351) by the Ministry of Education, Science, Sports and Culture of Japan and the Human Frontier Science Program (M. Y.).

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