Abused Inhalants Produce Little effect on Postsynaptic Excitability
Even at high concentrations, toluene produced only a small depression of CA1 neuron excitability (), measured as an ability to generate action potentials in response to depolarizing current injection. There was no effect apparent on action potential peak amplitude, rise time, half-width, or decay phase. At a concentration of 1 mM, toluene depressed action potential discharge from 7 spikes in control to 6.2 ± 0.7 (mean ± SD) spikes in the presence of the inhalant (). Although this depression was statistically significant (p < 0.05; n = 10 experiments, each from different slices, from 8 rats), only a minor degree of inhibition was evident. The depression of spike discharge was not accompanied by a change in the resting membrane potential of CA1 neurons, although a small, statistically insignificant, decrease in membrane resistance was observed.
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 (more ...)
To determine whether the depression of action potential discharge was due to an inhalant-induced enhancement of synaptic inhibition, the ability of a GABA receptor antagonist to reverse the depression was studied. The GABA receptor antagonist, gabazine (SR95531) at a saturating concentration (10 μM) completely reversed the inhalant-induced depression of discharge (8.1 ± 0.6 spikes), indicating that an increase in GABA-mediated inhibition could account for this depression. Gabazine also reversed the small change in membrane resistance that was observed, but the difference was only significant at the p < 0.05 level when gabzine data were compared to the normalized resistance in the presence of toluene (). Gabazine alone had no effect on action potential amplitude, rise time, duration, or on resting membrane potential.
Abused Inhalants Enhanced GABA-Mediated Synaptic Inhibition
To study the effects of abused inhalants on GABA-mediated inhibition, isolated, monosynaptic inhibitory postsynaptic potentials (IPSPs) were evoked with stimulating electrodes placed in stratum radiatum. Glutamate and GABA-B-mediated synaptic responses were blocked using receptor antagonists – CNQX (18 μM and APV 100 μM) and CGP (1.0 μM), respectively. Stimulus location, within stratum radiatum, as well as intensities and polarity were adjusted to produce minimal, stable response amplitudes; typically about 1.5 times threshold for the smallest amplitude responses seen. Using this approach, IPSPs exhibited amplitudes from 5 to 15 mV and failure rates of approximately 20 %. The abused inhalants increased IPSP amplitudes, but did not appear to alter the rise time, decay time, duration or failure rate of these synaptic responses ().
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 (more ...)
Toluene (TOL), trichloroethane (TCE) and trichloroethylene (TCY) appeared to be equally efficacious at increasing GABA-mediated inhibition, enhancing IPSP amplitudes to 129 ± 2.3, 128 ± 1.6 and 127 ± 0.9 % of control responses, respectively. The inhalants differed considerably, however, in potencies with a rank order of TCY > TOL > TCE over a two fold range of concentrations. The concentration producing a half maximal effect (EC50) was determined from Hill equations, fitting the data with a least squares approach. The TCY EC50 was 360 μM, and EC50s for TOL and TCE were 628 μM and 895 μM, respectively. Hill coefficients also varied considerably: 1.2 for TCE, 2.7 for TOL and 4.7 for TCY.
Abused Inhalants Enhanced GABA-Mediated Synaptic Inhibition by a Presynaptic Action
To determine whether the enhanced GABA-mediated inhibition resulted from pre- or post-synaptic mechanisms of action, spontaneous inhibitory postsynaptic currents (IPSCs) were recorded from voltage clamped CA1 neurons (). In control conditions, IPSC amplitudes ranged from 2 pA, just detectable above noise, to 650 pA with an average amplitude of 75.8 ± 14.2 pA seen across 23,720 events recorded from 10 CA1 neurons. Frequency varied considerably, even within each cell, but averaged 4.3 ± 1.7 Hz. IPSC rise time (1.58 ± 0.21 ms), decay time (16.9 ± 5.2 ms) and duration (24.6 ± 7.8 ms) measurements were quite consistent across cells. These IPSCs were completely blocked with 20 μM gabazine (not shown), indicating that they were GABA-mediated synaptic currents.
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 (more ...)
All three abused inhalants significantly increased IPSC frequency: TOL to 134 ± 5.6 % of control, TCE to 130 ± 6.3 % and TCY to 123 ± 7.1 % of control, for concentrations of 940 μM TOL, and 800 μM of both TCE and TCY (). These frequency increases were not accompanied by any measurable effect on holding currents needed to maintain the resting membrane potential at the original control values for the CA1 neurons. No significant effects on IPSC amplitude, rise time, decay time or duration were observed in the presence of any abused inhalant. Thus, the abused inhalant-induced increase in inhibition appears to come about by a presynaptic action to increase the release of GABA from inhibitory nerve terminals.
Abused Inhalants Act Directly on GABA Nerve Terminals
The increased IPSC frequency produced by the inhalants could have come about by increased discharge of action potentials in inhibitory interneurons, or by a direct effect on interneuron nerve terminals. To test whether either or both of these effects contribute to the increased IPSC frequency, miniature IPSCs (mIPSCs) were recorded in the presence of TTX (1.0 μM) to block sodium channels, and, hence, abolish action potential discharge of inhibitory neurons. If the inhalant-induced increase in mIPSC frequency still occurred, then a direct action on GABA nerve terminals would be implicated.
In control conditions, mIPSC amplitudes were much smaller than IPSCs, averaging 27.4 ± 5.3 pA. Rise times and decay times were essentially the same as seen for control conditions in the absence of TTX, however, mIPSC frequency was reduced to 3.25 ± 1.89 Hz across the 9,189 events recorded from 10 CA1 neurons studied. In the neuron shown in , toluene (940 μM) produced a near doubling in mIPSC frequency (from 5.5 to 8.3 Hz), that reached steady state within 10 min and recovered following removal of toluene within 20 min. All of these synaptic currents were blocked by 20 μM gabazine applied at 50 min. Grouped data for all 10 experiments are shown in and the increased frequency of mIPSCs was statistically significant (p < 0.01), as was the gabazine block (p < 0.001), both compared to control frequencies measured before application of drug.
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 (more ...)
For the majority of experiments (7 of 10), neither mIPSC amplitude, rise time, decay time nor duration were altered by the inhalant. In the remaining three experiments, an apparent toluene-induced increase in rise time (from 1.78 ± 0.63 to 2.55 ± 0.98 ms), decay time (from 17.11 ± 11.23 to 29.87 ± 28.65 ms) and duration was seen in the initial analysis. On closer inspection, it was evident that these cells had prominent GABAA Slow mIPSCs mixed in amongst the typical GABAA Fast responses seen during control recordings (). Toluene appeared to have a selective effect to enhance the frequency of GABAA Slow mIPSCs (to 120 % of control) in these cells and this produced an apparent increase in decay time and duration, since more slow events were contributing to the analysis in the presence of inhalant.
Figure 5 Toluene appeared to selectively enhance the frequency of GABAA 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 (more ...)
Previous studies have shown that volatile anesthetics increase mIPSC frequency by acting on intracellular calcium stores in GABA nerve terminals.(Doze and MacIver, 1998
; Yamashita et al, 2001
) To determine whether a similar mechanism contributed to the toluene-induced increase in mIPSC frequency, the effects of blocking calcium influx into nerve terminals was studied. The toluene-induced increase in mIPSC frequency persisted in the presence of 500 μM Cd++
, used to block calcium entry into nerve terminals, thus, toluene appeared to share an action on intracellular stores with volatile anesthetics. Nerve terminals appear to have at least two intracellular calcium storage compartments: a thapsigargin-sensitive store that is associated with IP3
receptor-mediated regulation, and a caffeine-sensitive store that is associated with ryanodine receptor-mediated regulation. Neither thapsigargin, ryanodine nor dantrolene, when applied alone, appeared to alter mIPSC frequency, except for a transient increase (2 to 5 min) following dantrolene exposure. Pretreatment of preparations with thapsigargin did not alter the toluene-induced increase in mIPSC frequency (), however, pretreatment with either dantrolene (a calcium store depletion agent) or with ryanodine (a caffeine store calcium channel blocker) blocked the effect produced by toluene.
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, (more ...)