Mice selectively bred for high- or low-alcohol-induced locomotion exhibit differences in dopamine neuron function

Abstract

Elevated sensitivity to the euphoric or stimulant effects of ethanol is associated with higher levels of alcohol use in some human populations. Midbrain dopamine neurons are thought to be important mediators of both ethanol reward and locomotor stimulation. Patch-clamp recordings were used to examine the electrical properties of dopamine neurons in a genetic model of heightened (FAST) and reduced (SLOW) sensitivity to the locomotor-activating effects of ethanol. Pacemaker firing of dopamine neurons was faster in FAST than SLOW mice, as was the current density through I(H) channels. Acute administration of ethanol accelerated the firing of dopamine neurons to a greater extent in recordings from FAST than SLOW mice. Dopamine neurons from FAST mice also exhibited reduced GABA(A) receptor-mediated synaptic input, compared with SLOW mice. The results suggest that dopamine neuron I(H) channels, firing rate, and GABAergic input may play a role in sensitivity to the locomotor activation observed at early time points after ethanol administration and could underlie differences in sensitivity to alcohol relevant to risk for alcohol abuse.

Fig. 1.

Dopamine neurons from FAST mice exhibit a more rapid spontaneous firing rate. Midbrain dopamine neurons exhibit a tonic, regular firing pattern when observed in vitro in brain slice preparations. Examination of firing rate using the loose patch cell-attached configuration revealed that the dopamine cell basal firing rate is significantly faster in slices from FAST compared with SLOW mice [A and B, t₍₁₁₃₎ = 4.35, P < 0.0001, n = 55–60 cells from 17–18 mice per group]. Sample recordings in slices taken from a FAST and a SLOW mouse illustrate the difference in pacemaker firing rate (C). The data presented are predominantly from an independent experiment but do include neurons also presented in Figs. 2C and 3A.

Fig. 2.

Dopamine neurons from FAST mice exhibit a higher I_H current density. The nonselective cation conductance I_H was examined in voltage clamp with a hyperpolarization from -60 to -110 mV, measured as the time-dependent component illustrated by the dotted lines (A). Measured as a function of cell capacitance, dopamine neurons from FAST mice exhibited a higher I_H density compared with those from SLOW mice [B, t₍₆₇₎ = 2.37, P = 0.02, n = 33–36 cells from six mice per group], which could contribute to their faster firing rate. When I_H channels were blocked with ZD 7288 (30 μM), spontaneous firing was more substantially slowed in cells from FAST mice [C, t₍₂₂₎ = 2.53, P = 0.019, n = 12 cells from four to six mice per group].

Fig. 3.

Ethanol increases the dopamine neuron firing rate to a greater extent in recordings from FAST mice. Ethanol (50–80 mM) produced a subtle increase in pacemaker firing of dopamine neurons (A). This increase was significantly larger in recordings from FAST mice [F_(1,38) = 6.84, P = 0.013 for main effect of line, n = 9–11 cells from four mice per group]. The time course of the effect of ethanol (80 mM) is illustrated in B (P = 0.035, Tukey's post hoc test for 80 mM ethanol applications between recordings from FAST versus SLOW mice).

Fig. 4.

GIRK channel-mediated inhibitory transmission does not differ between FAST and SLOW mice. Experiments using whole-cell patch clamp of dopamine neurons revealed no difference in GIRK channel-mediated currents in slices obtained from FAST and SLOW mice. GIRK conductance was examined through activation of metabotropic GABA_B and dopamine D2 receptors, both through application of exogenous agonists (30 μM baclofen and dopamine applied through iontophoresis) and by evoking IPSCs in the presence of synaptic blockers (n = 9–14 cells from three to six mice per group; baclofen, P = 0.095; GABA_B IPSC, P = 0.59; dopamine, P = 0.87; D2 IPSC, P = 0.92).

Fig. 5.

GABA_A receptor input to dopamine neurons is larger in recordings from SLOW mice. Endogenous GABA release was evoked with a bipolar-stimulating electrode placed caudal to the dopamine neuron being recorded. A preliminary investigation found that the amplitude of the inhibitory synaptic current was significantly larger in slices obtained from SLOW compared with FAST mice (P = 0.011, n = 30–33 cells from six to seven mice in each group, data not shown). Full stimulus-response curves were subsequently constructed with varying stimulus intensities (A), again demonstrating significantly larger IPSCs in recordings made from SLOW mice [F_(1,18) = 36.7, P = 0.038 for main effect of line; F_(6,108) = 4.71, P = 0.0003 for line-stimulus interaction, n = 9–11 cells from three mice per group]. GABA_A receptor-mediated currents were also larger in recordings from SLOW mice (B) when activated with iontophoresis of exogenous GABA [1 M, t₍₂₀₎ = 2.44, P = 0.024, n = 11 cells from three mice per group]. There was no difference in the paired-pulse ratio of evoked GABA_A receptor currents in brain slices taken from FAST versus SLOW mice (C). Care was taken while conducting the experiments for data shown in A to C to use identical slice configuration, stimulator depth and position, and iontophoretic pipette position relative to each neuron being recorded. Next, GABA release was presynaptically inhibited by activation of μ opioid receptors with the agonist [Met]⁵ enkephalin (10 μM). The inhibition produced by enkephalin was significantly larger in recordings from SLOW mice [D and E, main effect of line, F_(1,20) = 4.76; P = 0.041, n = 10–12 cells from three mice per group].

Fig. 6.

Spontaneous GABA_A IPSCs are larger and more frequent in SLOW mice. In the presence of pharmacological blockers of glycine, N-methyl-d-aspartate, AMPA, and GABA_B receptors, spontaneous and miniature GABA_A IPSCs were recorded in dopamine neurons of slices from FAST and SLOW mice. Tonic GABAergic input was more pronounced in cells from SLOW mice (A, 30 1-s sweeps overlaid). SLOW mice exhibited a greater frequency of sIPSCs (B), and this effect was blocked in the presence of tetrodotoxin (300 nM, mIPSCs). sIPSCs from SLOW mice also exhibited a larger amplitude (C) (n = 15–20 cells from three to four mice per group, Tukey's post hoc test, P = 0.02 for differences in frequency between SLOW sIPSCs and the other groups, P < 0.0001 for differences in amplitude).

Fig. 7.

Ethanol effects on GABAergic input to dopamine neurons do not exhibit a line difference. GABA_A receptor-mediated IPSCs were evoked in midbrain dopamine neurons. Bath perfusion of ethanol (80 mM) increased the area under the curve (A) of the IPSCs. However, there was no difference between the FAST and SLOW lines [n = 14 cells from six to seven mice per group, main effect of line, F_(1,25) = 0.0025, P = 0.96]. Bath perfusion of ethanol had no obvious effects on the frequency (B) or amplitude (data not shown) of sIPSCs recorded from FAST and SLOW mice (n = 7 cells from three mice per group).