Short- and long-term enhancement of excitatory transmission in the spinal cord dorsal horn by nicotinic acetylcholine receptors

Abstract

Spinal administration of nicotinic agonists can produce both hyperalgesic and analgesic effects in vivo. The cellular mechanisms underlying these behavioral phenomena are not understood. As a possible explanation for nicotinic hyperalgesia, we tested whether nicotinic acetylcholine receptors (nAChRs) could enhance excitatory transmission onto spinal cord dorsal horn neurons. Whole-cell patch-clamp recordings were performed in neonatal rat spinal cord slices. Activation of nAChRs enhanced glutamatergic synaptic transmission in 59% of dorsal horn neurons tested, and this effect was blocked by methyllycaconitine (10 nM), suggesting a key role for alpha7 nAChRs. Inhibition of acetylcholinesterase with methamidophos also enhanced transmission, demonstrating a similar effect of endogenous acetylcholine. nAChR activation also enhanced transmission by dorsal root entry zone stimulation, suggesting that alpha7 nAChRs on the central terminals of DRG afferents mediate this effect. Paired pre- and postsynaptic stimulation induced long-term potentiation of excitatory inputs to some of the dorsal horn neurons. Long-term potentiation induction was much more prevalent when nicotine was applied during stimulation. This effect also depended on both alpha7 nAChRs and N-methyl-d-aspartate glutamate receptors. Our findings demonstrate that alpha7 nAChRs can contribute to both short- and long-term enhancement of glutamatergic synaptic transmission in the spinal cord dorsal horn and provide a possible mechanism for nicotinic hyperalgesia.

Fig. 1.

Nicotine enhances glutamatergic transmission in the SCDH. (A) Voltage-clamp recording from a SCDH neuron from control and with 1 μM nicotine (Vh = -70 mV). (Bar = 20 pA, 250 ms.) (B) Frequency histogram showing an increase in glutamatergic mEPSCs with 1 μM nicotine. (C) The average normalized frequency data from all neurons (n = 27; *, P < 0.05) compared with control without nicotine (n = 10). (D) Cumulative distributions illustrate no difference in average mEPSC amplitudes during baseline and nicotine treatment (P > 0.05). (E) The locations of recorded neurons presented as nicotine responders (n = 16, filled circles) or nonresponders (n = 11, open circles). (Bar = 250 μm.)

Fig. 2.

Enhancement of synaptic transmission is due to α7 nAChR activation. (A) Raw current traces during baseline and with 1 μM nicotine + 10 nM MLA in the external solutions. (Bar = 20 pA, 250 ms.) (B) mEPSC histogram showing the lack of response to 1 μM nicotine with 10 nM MLA. (C) Summary histogram of the magnitude of nicotine-induced changes in mEPSC frequency in control (177.9 ± 28.8%; n = 27) and with 10 nM MLA (101.2 ± 8.5%; n = 23; *, P < 0.05) or 1 μM mecamylamine (135.9 ± 8.5%; n = 20; P > 0.05). (D) Response prevalence under these three conditions.

Fig. 3.

Enhancement of mEPSC frequency by endogenous ACh. (A) Raw current traces during baseline and with 200 μM methamidophos. (Bar = 20 pA, 250 ms.) (B) mEPSC histogram during control and 200 μM methamidophos exposure. (C) Average change in mEPSC frequency after methamidophos (185.9 ± 3.3%; n = 5) and with 10 nM MLA (100.4 ± 3.2%; n = 6; *, P < 0.05). All data were averaged for responders and nonresponders.

Fig. 4.

Expression of nAChRs on acutely isolated dorsal horn neurons. (A) ACh or choline induced a rapid inward current in an acutely isolated dorsal horn neuron that was insensitive to epibatidine. These α7 currents could be inhibited by MLA (Inset; n = 4) and were seen in 24% of recordings (n = 46). [Bar = 400 pA, 500 ms (Left); 400 pA, 100 ms (Inset).] (B) In a different neuron, ACh or epibatidine induced a current with slower inactivation kinetics that was insensitive to choline. This non-α7 current was see in 20% of recordings (n = 46) and could be inhibited by dihydro-β-erythroidine (Inset; n = 4; Bar = 400 pA, 400 ms) but not MLA (not shown; n = 2).

Fig. 5.

α7 nAChR expression on the central terminals of DRG afferents. (A) Evoked synaptic current amplitudes from one neuron before and after nicotine treatment. (Inset) Currents are individual traces. (Bar = 100 pA, 4 ms.) (B) Averages of evoked current amplitudes and sEPSC frequency for responders (n = 8; open circle) and nonresponders (n = 10; filled circle) (* and **, P < 0.05).

Fig. 6.

LTP and LTD induction in the dorsal horn. The protocol used to induce long-term changes in eEPSC amplitudes consisted of 200 DREZ stimuli at 1Hz (0.5 ms) during simultaneous postsynaptic depolarizations to +10 mV (100 ms). Shown are examples of potentiation and depression induced by this protocol in different neurons.

Fig. 7.

Nicotine enhances LTP induction in the dorsal horn. (A) Average normalized eEPSC amplitudes before and after the stimulation protocol (n = 11). Bar graphs (A–D Right) illustrate the prevalence of LTP, LTD, and no change under each condition. (B) Average normalized eEPSC amplitudes with 1 μM nicotine applied 2 min before and during the simulation paradigm (n = 13). (C) Average normalized eEPSC amplitudes with 1 μM nicotine in the presence of 10 nM MLA (n = 16). (D) Average normalized eEPSC amplitudes with 1 μM nicotine in the presence of 50 μM dl--2-amino-5 phosphonovaleric acid (n = 9).