Inhibition of neuronal nicotinic acetylcholine receptors by the abused solvent, toluene

Abstract

1 Toluene is a representative example of a class of industrial solvents that are voluntarily inhaled as drugs of abuse. Previous data from this lab and others has shown that toluene modulates the function of N-methyl-D-aspartate (NMDA), gamma-aminobutyric acid (GABA) and glycine receptors at concentrations that do not affect non-NMDA receptors. 2 We utilized two-electrode voltage-clamp and whole cell patch-clamp techniques to assess the effects of toluene on neuronal nicotinic acetylcholine receptors expressed in oocytes and cultured hippocampal neurons. Toluene (50 micro M to 10 mM) produced a reversible, concentration-dependent inhibition of acetylcholine-induced current in Xenopus oocytes expressing various nicotinic receptor subtypes. The alpha4beta2 and alpha3beta2 subunit combinations were significantly more sensitive to toluene inhibition than the alpha4beta4, alpha3beta4 and alpha7 receptors. 3 Receptors composed of alpha4 and beta2(V253F) subunits showed alpha4beta4-like toluene sensitivity while those containing alpha4 and beta4(F255V) subunits showed alpha4beta2-like sensitivity. 4 In hippocampal neurons, toluene (50 micro M to 10 mM) dose-dependently inhibited ACh-mediated responses with an IC(50) of 1.1 mM. 5 Taken together, these results suggest that nicotinic receptors, like NMDA receptors, show a subunit-dependent sensitivity to toluene and may represent an important site of action for some of the neurobehavioural effects of toluene.

Figure 1

Effects of toluene on α4β2, α3β2, α3β4 and α4β4 nAChRs expressed in Xenopus oocytes. Representative traces are shown for each receptor subtype in response to ACh (1–30 μM) with and without toluene. Each set of the three traces comes from a separate oocyte voltage clamped at −80 mV.

Figure 2

Concentration-response curves for toluene-inhibition of ACh-induced currents in oocytes expressing different nAChR subtypes. Each point represents the per cent inhibition of the steady state response by toluene and is the mean (±s.e.mean) from 5–12 oocytes from at least two separate frogs. The IC₅₀ values for the nAChRs are listed in Table 1.

Figure 3

Inhibitory effects of toluene on the α7 nAChR. (A) Representative traces from an oocyte expressing α7 receptors. The lines above each current represent the length of time that the oocyte was perfused with either 150 μM ACh or 300 μM toluene. At this concentration, ACh-mediated responses were inhibited by 20% in comparison to control. (B) Concentration response curves were generated for the inhibition of the α7 receptor at both the peak and steady state portions of the current. Each point represents the per cent inhibition of the response by toluene and is the mean (±s.e.mean) from 5–8 oocytes from at least two separate frogs. IC₅₀ values for the inhibition at both these portions are listed in Table 1 and do not differ significantly.

Figure 4

Effects of toluene on current-voltage (I-V) relationships of nAChRs expressed in Xenopus oocytes. Toluene inhibition for α4β2 (A), α3β2 (B), and α7 (C) nAChRs was calculated at each voltage (n=4–5 oocytes) and plotted. None of the values were significant with respect to each other (one-way ANOVA).

Figure 5

Per cent inhibition of nAChRs by toluene in the presence of increasing ACh concentrations. Oocytes expressing the indicated nAChR were exposed to 1 mM (α4β2, α3β2, α3β4, α7) or 3 mM (α4β4) toluene at different ACh concentrations. Values represent the per cent inhibition of the corresponding control current by toluene and are the mean (±s.e.mean) from 5–8 oocytes from at least two separate frogs.

Figure 6

Effects of toluene on α4β2(V253F) and α4β4(F255V) nAChRs expressed in Xenopus oocytes. Representative traces are shown for each receptor subtype in response to ACh (1 or 10 μM) with and without 1 mM toluene. Each set of the three traces comes from a separate oocyte voltage-clamped at −80 mV.

Figure 7

Concentration-response curves for toluene-inhibition of ACh-induced currents in oocytes expressing mutant nAChR subtypes. Each point on the curve represents the per cent inhibition of the steady state response by toluene and is the mean (±s.e.mean) from 5–7 oocytes from at least two separate frogs. Concentration-response curves for the corresponding wild type nAChR (α4β2 and α4β4) receptors are displayed as dashed lines and are taken from Figure 2. The IC₅₀ values for the mutant nAChRs are listed in Table 1.

Figure 8

Toluene inhibition of ACh-mediated currents in cultured hippocampal neurons. (A) A representative set of currents generated from one neuron. Traces represent currents induced by ACh (3 mM) in the absence and presence of toluene (3 mM) from a neuron voltage-clamped at −50 mV. The line above each current indicates the specified treatment (3 mM ACh or 3 mM ACh/3 mM toluene). (B) A dose response curve for the toluene-inhibition of ACh-induced currents in hippocampal neurons is shown. Each point represents the per cent inhibition of control currents by toluene and is the mean (±s.e.mean) from 5–9 neurons. The IC₅₀ value of toluene for the ACh response was 1.1 mM (0.65–1.9 mM).