Abused inhalants enhance GABA-mediated synaptic inhibition

Abstract

Abused inhalants are widely used, especially among school-age children and teenagers, and are 'gateway' drugs leading to the abuse of alcohol and other addictive substances. In spite of this widespread use, little is known about the effects produced by inhalants on the central nervous system. The similarity in behavioral effects produced by inhalants and inhaled anesthetics, together with their common chemical features, prompted this study of inhalant actions on a well-characterized anesthetic target, GABA synapses. Whole-cell patch clamp recordings were conducted on CA1 pyramidal neurons in rat hippocampal brain slices to measure effects on resting membrane properties, action potential discharge, and GABA-mediated inhibitory responses. Toluene, 1,1,1-trichloroethane, and trichloroethylene depressed CA1 excitability in a concentration-dependent and reversible manner. This depression appeared to involve enhanced GABA-mediated inhibition, evident in its reversal by a GABA receptor antagonist. Consistent with this, the abused inhalants increased inhibitory postsynaptic potentials produced using minimal stimulation of stratum radiatum inputs to CA1 neurons, in the presence of CNQX and APV to block excitatory synaptic responses and GGP to block GABA(B) responses. The enhanced inhibition appeared to come about by a presynaptic action on GABA nerve terminals, because spontaneous inhibitory postsynaptic current (IPSC) frequency was increased with no change in the amplitude of postsynaptic currents, both in the presence and absence of tetrodotoxin used to block interneuron action potentials and cadmium used to block calcium influx into nerve terminals. The toluene-induced increase in mIPSC frequency was blocked by dantrolene or ryanodine, indicating that the abused inhalant acted to increase the release of calcium from intracellular nerve terminal stores. This presynaptic action produced by abused inhalants is shared by inhaled anesthetics and would contribute to the altered behavioral effects produced by both classes of drugs, and could be especially important in the context of a disruption of learning and memory by abused inhalants.

Figure 1

Toluene depressed excitability of CA1 pyramidal neurons, but produced very little effect on resting membrane responses. A, Direct current evoked discharge frequency was slowed by toluene (950 μM; top recordings), but there was no apparent change in spike threshold, action potential rise time, amplitude or decay time (bottom recordings). B, Group data based on measures from 10 pyramidal neurons shows a small, but significant (ANOVA p < 0.05), depression of discharge produced by toluene. This depression was reversed by a GABA antagonist, gabazine, indicating a possible involvement of enhanced inhibition in the effect produced by toluene (top graph). Neither toluene nor gabazine had any apparent effect on resting membrane potential but membrane resistance followed the same trends as spike discharge, however only the gabazine effect was significant (compared to toluene; p < 0.1; bottom graphs; p < 0.05 for normalized data, not shown).

Figure 2

Toluene enhanced GABA-mediated inhibition measured using monosynaptically-evoked IPSPs recorded from CA1 pyramidal neurons (top recordings) and this effect recovered following washout of the inhalant. IPSP response amplitude was measured from pre-stimulus baseline to peak negativity, as indicated by the arrows. An increased IPSP amplitude was clearly evident in the overlay of control and toluene recordings (left, middle). The dashed lines in the overlay plot are fits to a single exponential, used to measure IPSP decay times (see results). Neither the rise time nor decay time of IPSPs was altered by toluene, as shown in the expanded overlay of recordings (left, bottom) in which the control response was scaled to the same peak amplitude as the toluene IPSP. The toluene effect on GABA-mediated IPSPs was concentration-dependent and was also evident for other abused inhalants (trichloroethane – TCE and trichloroethylene – TCY). Each point in the concentration-effect graph (lower right) represents the mean ± SD for at least five measures. The dashed lines are fits to the Hill equation using a least squares approach. All of the abused inhalants appeared to be equally efficacious, but TCY was approximately three times more potent than either toluene or TCE.

Figure 3

Toluene enhanced GABA inhibition by a presynaptic mechanism, evident in spontaneous IPSC recordings from CA1 pyramidal neurons. Recordings on top show 1.0 second long consecutive traces in control or after exposure to 950 μM) toluene. Unlike inhaled anesthetics, toluene did not appear to increase the decay time constants of these inhibitory currents, even for larger amplitude IPSCs (lower left). Toluene produced a marked increase in the frequency of spontaneous IPSCs (ANOVA p < 0.01), with no significant change in current rise time, amplitude, decay time or duration (lower right grouped data from 10 experiments).

Figure 4

Toluene appeared to act directly at GABA nerve terminals, since an increase in miniature IPSC frequency was evident in the presence of tetrodotoxin used to block action potentials in the inhibitory interneurons. A, Consecutive five second long recordings of miniature IPSCs for control and in the presence of toluene. B, Rate meter plot showing the time course of toluene-induced increase in the frequency of IPSCs and recovery following washout of the abused inhalant. The GABA receptor antagonist, gabazine was applied at the end of this experiment to demonstrate that these synaptic currents were all GABA-dependent. C, Summary data from ten experiments showing that toluene produced a significant increase in IPSC frequency (mean ± SD, * - p < 0.05; ** - p < 0.001).

Figure 5

Toluene appeared to selectively enhance the frequency of GABA_{A slow} synaptic currents. In some experiments (3 of 10), two kinds of miniature IPSC kinetics were evident: fast and slow (recording on top). In these experiments, toluene selectively enhanced the proportion of slow IPSCs, evident in the graphs of rise time and decay shown below. GABA_{A fast} IPSCs typically exhibit rise times less than 2.0 ms and decay times less than 20 ms. GABA_{A slow} IPSCs, in contrast, exhibit rise times of 3.0 ms and decay times over 20 ms (often 30 to 60 ms). Toluene skewed the distribution of IPSC rise and decay times in favor of GABA_{A slow} IPSCs (bottom graphs), indicating a selective effect on nerve terminals that give rise to these slower synaptic currents. For kinetic analysis, all mIPSCs in the 3 neurons during 30 s recordings in control (356 events) and drug conditions (428 events) were used. For rise times, 0.2 ms bins were used and for decay times, 1.0 ms bins were used.

Figure 6

Toluene increased IPSC frequency by releasing calcium from intracellular stores, since the effect persisted in the presence of Cd⁺⁺, used to block calcium entry through nerve terminal membrane channels, but was blocked when calcium stores were depleted, or when ryanodyne receptor/channels were blocked. Recordings on the top show mIPSCs in the presence of CNQX and APV used to block glutamate synaptic currents, TTX used to block presynaptic action potentials, and dantrolene (DAN) used to deplete intracellular calcium stores. Toluene no longer produced an increase in mIPSC frequency when calcium stores were depleted. The bar graph on the bottom compares the effect produced by toluene on mIPSC frequency in control conditions, the lack of effect produced by Cd⁺⁺ block, and lack of effect seen with thapsigargin (THAP), used to block calcium release from IP₃ sensitive stores. Both dantrolene and ryanodine (RYAN) blocked the toluene-induced increase in mIPSC frequency indicating a selective effect on caffeine sensitive calcium stores. Each bar represents the mean ± SD for at least 5 determinations of the toluene-induced effect from separate experiments. Statistical comparisons were done using ANOVA, with Cd, THAP,DAN and RYAN data compared to the control (toluene-induced) response.