Mefloquine effects on ventral tegmental area dopamine and GABA neuron inhibition: a physiologic role for connexin-36 GAP junctions

Abstract

Connexin-36 (Cx36) gap junctions (GJs) appear to be involved in the synchronization of GABA interneurons in many brain areas. We have previously identified a population of Cx36-connected ventral tegmental area (VTA) GABA neurons that may regulate mesolimbic dopamine (DA) neurotransmission, a system implicated in reward from both natural behaviors and drugs of abuse. The aim of this study was to determine the effect mefloquine (MFQ) has on midbrain DA and GABA neuron inhibition, and the role Cx36 GJs play in regulating midbrain VTA DA neuron activity in mice. In brain slices from adolescent wild-type (WT) mice the Cx36-selective GJ blocker mefloquine (MFQ, 25 μM) increased VTA DA neuron sIPSC frequency sixfold, and mIPSC frequency threefold. However, in Cx36 KO mice, MFQ only increased sIPSC and mIPSC frequency threefold. The nonselective GJ blocker carbenoxolone (CBX, 100 μM) increased DA neuron sIPSC frequency twofold in WT mice, did not affect Cx36 KO mouse sIPSCs, and did not affect mIPSCs in WT or Cx36 KO mice. Interestingly, MFQ had no effect on VTA GABA neuron sIPSC frequency. We also examined MFQ effects on VTA DA neuron firing rate and current-evoked spiking in WT and Cx36 KO mice, and found that MFQ decreased WT DA neuron firing rate and current-evoked spiking, but did not alter these measures in Cx36 KO mice. Taken together these findings suggest that blocking Cx36 GJs increases VTA DA neuron inhibition, and that GJs play in key role in regulating inhibition of VTA DA neurons. Synapse, 2011. © 2011 Wiley-Liss, Inc.

Fig. 1

Time course of mefloquine effects on VTA dopamine neuron sIPSC frequency. VTA DA neuron sIPSCs were recorded at 15-min intervals in wildtype (WT, open circle) and Cx36 KO (filled squares) mice with bath-applied MFQ (25 μM), D-APV (50 μM), and CNQX (30 μM). MFQ increased sIPSC frequency similar in both WT and Cx36 KO mice until the 60+ min time point when WT sIPSCs were 175% of KO mice sIPSCs, P = 0.0001. WT data: 7.3 ± 1.7 Hz (t = 0); 8.4 ± 0.9 Hz (15 min); 18.9 ± 3.5 Hz (30 min); 19.2 ± 1.4 Hz (45 min); 59.8 ± 4.3 Hz (60 min). Cx36 KO data: 7.3 ± 1.0 Hz (t = 0) to: 7.5 ± 0.9 Hz (15 min); 13.7 ± 1.6 Hz (30 min); 21.9 ± 3.7 Hz (45 min); 32.2 ± 6.5 Hz (60 min).

Fig. 2

Mefloquine effects on VTA dopamine neuron sIPSC frequency, amplitude, and interevent interval in WT and Cx36 KO mice. This figure compares WT (A–C) and KO (D–F) VTA DA neuron sIPSC data after being presoaked with either MFQ (25 μM), CBX (100 μM), ODS (25 μM), or MFQ (25 μM) 1 MEC (10 μM) for ≥60 min. In WT mice (C), sIPSCs were increased similarly by MFQ (533%) and MFQ+MEC (488%), CBX alone increased sIPSC frequency 211%, but ODS alone did not mimic MFQ. MFQ and MFQ+MEC shifted the interevent interval curve to the left (B), while CBX alone shifted the curve slightly to the left. In Cx36 KO mice, only MFQ increased sIPSC frequency (F) and shifted the interevent interval curve to the left (E). MFQ's increase of Cx36 KO mouse sIPSC frequency was about half of its increase of WT sIPSC frequency.

Fig. 3

Mefloquine effects on VTA dopamine neuron mIPSC frequency, amplitude, and interevent interval in WT and Cx36 KO mice: This figure compares WT (A–C) and KO (D–F) VTA DA neuron mIPSC data after being presoaked with MFQ (25 μM) or CBX (100 μM) for ≥60 min. In WT mice (C), mIPSCs were increased (289%) by MFQ but not at all by CBX (100 μM). In WT mice, MFQ but not CBX shifted the interevent interval curve to the left (B). In Cx36 KO mice, MFQ (25 μM) increased mIPSC frequency 358% (F) and shifted the interevent interval plot to the left (E).

Fig. 4

Mefloquine does not affect VTA GABA neuron sIPSC frequency. This figure diagrams presoaked MFQ (25 μM) effects on WT VTA GABA neuron sIPSCs. MFQ did not increase VTA GABA neurons sIPSC frequency (A, C) or shift the interevent distribution plot (B).

Fig. 5

MFQ reduces VTA DA neuron firing rate and current-evoked spiking. This figure compares WT (A–D) and KO (E–H) VTA DA neuron spontaneous firing rate and current-evoked spikes after being presoaked with MFQ (25 μM). (A, E) Raw traces of WT and KO mouse VTA DA neuron spontaneous firing rate voltage spikes. (B, F) These panels show the summary of all spontaneous firing rate experiments in WT and KO mice in which MFQ significantly decreased firing rate in WT mice but not it Cx36 KO mice. Note that Cx36 KO mouse baseline firing rate is lower than WT mouse baseline firing rate. Panels C and G show raw traces of current-evoked spiking in WT and KO mice in which MFQ reduced current-evoked spiking at all currents greater than 125pA (D), but had no effect on already reduced Cx36 KO mouse spiking (H). Note that Cx36 KO mouse control and MFQ-treated current-evoked spiking are similar to WT MFQ-treated current-evoked spiking.