The abused inhalant toluene differentially modulates excitatory and inhibitory synaptic transmission in deep-layer neurons of the medial prefrontal cortex

Abstract

Volatile organic solvents such as toluene are voluntarily inhaled for their intoxicating effects. Solvent use is especially prevalent among adolescents, and is associated with deficits in a wide range of cognitive tasks including attention, behavioral control, and risk assessment. Despite these findings, little is known about the effects of toluene on brain areas mediating these behaviors. In this study, whole-cell patch-clamp recordings were used to determine the effect toluene on neurons within the medial PFC, a region critically involved in cognitive function. Toluene had no effect on measures of intrinsic excitability, but enhanced stimulus-evoked γ-amino butyric acid A-mediated inhibitory postsynaptic currents (IPSCs). In the presence of tetrodotoxin (TTX) to block action potentials, toluene increased the frequency and amplitude of miniature IPSCs. In contrast, toluene induced a delayed but persistent decrease in evoked or spontaneous AMPA-mediated excitatory postsynaptic currents (EPSCs). This effect was prevented by an intracellular calcium chelator or by the ryanodine receptor and SERCA inhibitors, dantrolene or thapsigargin, respectively, suggesting that toluene may mobilize intracellular calcium pools. The toluene-induced reduction in AMPA EPSCs was also prevented by a cannabinoid receptor (CB1R) antagonist, and was occluded by the CB1 agonist WIN 55,212-2 that itself induced a profound decrease in AMPA-mediated EPSCs. Toluene had no effect on the frequency or amplitude of miniature EPSCs recorded in the presence of TTX. Finally, toluene dose-dependently inhibited N-methyl-D-aspartate (NMDA)-mediated EPSCs and the magnitude and reversibility of this effect was CB1R sensitive indicating both direct and indirect actions of toluene on NMDA-mediated responses. Together, these results suggest that the effect of toluene on cognitive behaviors may result from its action on inhibitory and excitatory synaptic transmission of PFC neurons.

Figure 1

Toluene has no effects on measures of intrinsic excitability of PFC pyramidal neurons. (a) Representative example of action potentials evoked by direct injection of current into a patch-clamped PFC pyramidal neuron. (b) Traces from a control and toluene exposed neurons are superimposed to show lack of effect of 3 mM toluene on AP parameters. (c) Summary of effects of 3 mM toluene on resting membrane potential, action potential threshold, membrane resistance, number of spikes, spike rise time, or half width, as compared with baseline values. Data are mean (±SEM) from six neurons. The color reproduction of this figure available on the HTML full text version of the paper.

Figure 2

Toluene dose dependently enhances stimulus-evoked GABA-mediated IPSCs in PFC pyramidal neurons. (a) Representative example of stimulus-evoked GABA-mediated IPSCs before, during, and after exposure to 3 mM toluene. (b) Time course of toluene action on amplitude of stimulus-evoked IPSCs. Data are expressed as a percent of the averaged pre-toluene response. (c) Summary of effects of toluene on IPSC peak amplitude. Data are expressed as a percentage of average baseline (pre-toluene exposure) amplitude. ^*Value significantly different from control (repeated measures ANOVA, post hoc Dunnett's multiple comparison test p<0.05). Data are mean±SEM. The color reproduction of this figure available on the HTML full text version of the paper.

Figure 3

Toluene enhances both the frequency and amplitude of mIPSCs in PFC pyramidal neurons. (a) Representative example of GABA-mediated mIPSCs from a mPFC neuron before, during, and after washout of a 3 mM toluene solution. Summary plots below show the cumulative probability for the inter-event interval (b) and amplitude (c) of the same example recording. (d and e) Summary of effects of toluene on miniature IPSC frequency and amplitude. ^*Value significantly different from baseline (p<0.05; repeated measures ANOVA, post hoc Dunnett's multiple comparison test). Data are mean±SEM. The color reproduction of this figure available on the HTML full text version of the paper.

Figure 4

Toluene inhibition of AMPA EPSCs requires postsynaptic Ca²⁺ and the CB1 receptor. (a) Traces are representative examples of AMPA-mediated EPSCs recorded under baseline conditions (left) and following exposure to toluene (right). Normal aCSF and internal solution, BAPTA in the internal solution, AM-281 in the aCSF, and MPEP in the aCSF. (b) Time-course of AMPA-mediated EPSCs during toluene exposure. Data are expressed as a percent of the averaged pre-toluene response under each condition. ^***Values significantly different when recording with intracellular BAPTA or extracellular AM-281, compared with normal aCSF and internal solution. (p<0.001, two-way ANOVA) (c) Summary of effects of toluene on AMPA-mediated currents under various experimental conditions. ^**Values significantly different from baseline amplitudes. (p<0.01, repeated measures ANOVA, post hoc Dunnett's multiple comparison test). Data are mean±SEM. The color reproduction of this figure available on the HTML full text version of the paper.

Figure 5

Toluene-induced inhibition of AMPA EPSCs is blocked by either the ryanodine receptor dantrolene or sarco/endoplasmic reticulum Ca²⁺/ATPase inhibitor thapsigargin. (a) Time-course of AMPA-mediated EPSCs before, during, and after 3 mM toluene exposure. Data are expressed as a percent of averaged baseline response. (b) Summary of effects of toluene on AMPA-mediated currents under control conditions or in the presence of either dantrolene or thapsigargin. ^**Values significantly different from baseline amplitudes. (p<0.01, repeated measures ANOVA, post hoc Dunnett's multiple comparison test). Data are mean±SEM. The color reproduction of this figure available on the HTML full text version of the paper.

Figure 6

The CB1 agonist WIN 55,212-2 occludes toluene inhibition of AMPA EPSCs. (a) Time-course of AMPA-mediated EPSCs before, during, and after application of WIN 55,212-2. Toluene (3 mM) was present during the final 8 min of WIN 55,212-2 application. Data are expressed as a percent of averaged baseline response. (b) Summary of effects of WIN and toluene on AMPA-mediated currents. Data are expressed as peak AMPA EPSC amplitude (pA). Asterisks are the values that are significantly different from baseline amplitudes (^*p<0.05, ^**p<0.01, repeated measures ANOVA, post hoc Bonferroni multiple comparisons test). Data are mean±SEM.

Figure 7

Toluene affects spontaneous but not miniature EPSCs. (a) Representative examples of mEPSCs recorded before, during, or following exposure to 3 mM toluene. Panels below are from the same recording and show a cumulative probability chart of the inter-event interval (IEI, b) and amplitude (c) of mEPSCs. Summary of effects of 3 mM toluene on AMPA mEPSC frequency (d) and amplitude (e). Data are from six neurons. (f) Representative example of spontaneous AMPA-mediated EPSCs. Panels below show cumulative probability charts of IEI (g) and amplitude (h) from the same recording. Summary figure showing the effect of toluene on frequency (i) and amplitude (j). ^*Value significantly different from baseline (p<0.05, paired t-test). Data are mean±SEM. The color reproduction of this figure available on the HTML full text version of the paper.

Figure 8

Toluene dose dependently inhibits NMDA-mediated EPSCs. (a) Traces are representative examples of NMDA-mediated EPSCs recorded before, during, and after washout of a 3 mM toluene solution. (b) Time-course of NMDA-mediated current amplitudes. Data represent the percent of the pre-toluene baseline value. (c) Summary graph of the effects of toluene on NMDA-mediated currents. Data are expressed as the percent of averaged baseline response. Asterisks are the value that are significantly different from baseline (^*p<0.05, ^**p<0.01; repeated measures ANOVA, Dunnett's post hoc comparison test). Data are mean±SEM. The color reproduction of this figure available on the HTML full text version of the paper.

Figure 9

Effects of the CB1 receptor antagonist AM-281 on toluene-induced inhibition of NMDA-mediated EPSCs. (a) Traces show representative examples of stimulus evoked NMDA EPSCs currents before, during, and after washout of a 3 mM toluene solution. All recordings were carried out in the presence of the CB1 antagonist AM-281 (0.75 μM). (b) Time-course of 3 mM toluene's inhibition of NMDA-mediated currents recorded with normal or AM-281 containing aCSF. Data are expressed as a percent of the averaged baseline response (c) Summary of effects of toluene on NMDA-mediated currents in the presence or absence of 0.75 μM AM-281. Data are expressed as a percent of the averaged baseline response. Asterisks are the values that are significantly different from baseline amplitude (^*p<0.05, ^**p<0.01; repeated measures ANOVA, Dunnett's multiple comparison test). Data are mean±SEM. The color reproduction of this figure available on the HTML full text version of the paper.