Opioid-sensitive GABA inputs from rostromedial tegmental nucleus synapse onto midbrain dopamine neurons

Abstract

Opioids increase dopamine release in the brain through inhibition of GABA-A IPSCs onto dopamine cells. Immunolabeling indicates that GABA neurons in the rostromedial tegmental nucleus (RMTg), also known as the tail of the ventral tegmental area, send a dense projection to midbrain dopamine neurons stain for μ-opioid receptors. There is however, little functional evidence that these neurons play a role in the opioid-dependent increase in dopamine neuron activity. The present study used retrograde tracers injected into the ventral tegmental area and substantia nigra (VTA/SN) to identify RMTg neurons that project to the VTA/SN. Whole-cell current-clamp and cell-attached recordings from labeled RMTg neurons were performed in sagittal slices from rat. The rhythmic spontaneous firing rate of RMTg neurons was decreased and the membrane potential was hyperpolarized in response to application of μ-opioid agonist DAMGO. Agonists that act at κ- and δ-opioid receptors (U69593 and DPDPE) failed to hyperpolarize RMTg neurons. Whole-cell recordings made in dopamine neurons revealed rhythmic, large amplitude spontaneous IPSCs that had a similar frequency, pattern and opioid sensitivity to the firing of RMTg neurons. In addition, electrical and channelrhodopsin-2 stimulation within the RMTg evoked GABA-A IPSCs in dopamine neurons that were inhibited by μ-opioid agonists DAMGO, but not κ- and δ-opioid agonists. Thus, this study demonstrates functional connection from the RMTg to the VTA/SN mediated by a dense, opioid-sensitive GABA innervation, and that the RMTg is a key structure in the μ-opioid receptor-dependent regulation of dopamine neurons.

Figure 1.

Hyperpolarization of identified RMTg neurons mediated by μ-opioid agonists. A, Confocal mosaic image of a sagittal slice containing VTA/SN and RMTg. RMTg neurons were identified with elevated cFOS (red) activity 2 h after an injection of amphetamine. The retrograde tracer, cholera toxin subunit B (green), was injected into VTA. cFOS-positive neurons frequently colocalized with retrograde tracer (inset). ml, medial lemniscus; 3n, oculomotor nerve. B, Whole-cell current-clamp recording from retrograde tracer-positive neuron. ME (10 μm) caused hyperpolarization and decreased spontaneous firing. C, Retrogradely labeled neurons in the RMTg were hyperpolarized through an activation of μ-opioid receptors DAMGO. Application of ME and DAMGO but not κ- or δ-opioid agonists (U69593 or DPDPE) significantly induced a hyperpolarization of the membrane potential from the baseline potentials. Number of cells indicated in or above the bars. Error bars indicate SEM. *p < 0.05, ***p < 0.001.

Figure 2.

The firing rate of retrogradely labeled RMTg neurons and rate of spontaneous GABA-A IPSCs were inhibited by μ-opioid agonists. A, Cell-attached recording from a RMTg neuron. Application of μ-opioid agonist DAMGO (1 μm) gradually inhibited the firing of the neuron. This inhibition was reversed by application of opioid antagonist naloxone (1 μm). B, Summary plot of the firing rate of RMTg neurons in control, during application of ME (•; 1 μm) or DAMGO (○; 1 μm), and washout ME or superfusion of naloxone after DAMGO (n = 12). Application of ME or DAMGO significantly reduced frequency of spontaneous firing from control and washout. Bar graphs represent the average firing frequency of shown data. C, Whole-cell voltage-clamp recording from a VTA dopamine neuron. Rhythmic spontaneous GABA-A IPSCs were examined. The μ-opioid agonist DAMGO (1 μm) decreased large amplitude sIPSCs, but not miniature IPSCs and the inhibition was reversed by naloxone (1 μm). D, Summary plot of the frequency of the large amplitude sIPSCs in control, during ME (•; 1 μm) or DAMGO (○; 1 μm), and washout of ME or superfusion of naloxone after DAMGO. Application of ME or DAMGO significantly reduced frequency of sIPSC from control and washout. Bar graphs represent the average sIPSC frequency of shown values. In B and D, lines connecting circles indicate recordings made from the same cell. Error bars indicate SEM. ***p < 0.001.

Figure 3.

Electrical stimulation in the RMTg evoked μ-opioid-sensitive IPSCs in dopamine neurons. A, Superimposed traces of evoked IPSCs in a dopamine neuron (V_m = −60 mV, average of 10). Application of DAMGO, but not U69593 or DPDPE, decreased the amplitude of eIPSCs. B, Representative image indicating the location of an electrical stimulation and site of recording in the sagittal plane. C, Summary graph of eIPSC amplitude (% of control) during μ-opioid agonist (DAMGO), reversal by opioid antagonist (Naloxone), and during κ- or δ-opioid agonists (U69563 or DPDPE). DAMGO significantly reduced eIPSC amplitude compared with 100% control. Number of cells indicated in bars. Error bars indicate SEM. ***p < 0.001.

Figure 4.

DAMGO inhibited ChR2-evoked IPSCs on dopamine neurons. A, Superimposed traces of fIPSCs (0.3 ms; 470 nm) in a dopamine neuron (V_m = −60 mV, average of 10). DAMGO decreased the amplitude of fIPSCs in a concentration-dependent manner. The GABA-A receptor antagonist picrotoxin (100 μm) blocked fIPSCs. B, Confocal image stained for tyrosine hydroxylase (TH; red) and ChR2 conjugated with Venus (green) in VTA. ChR2-expressing axons were densely innervated in the VTA/SN 7 d after AAV viral expression of ChR2 in the RMTg. C, Concentration-response curve of the inhibition of fIPSC by DAMGO. Error bars indicate SEM.

Figure 5.

Activation of ChR2 in the RMTg-evoked IPSCs in dopamine neurons that were inhibited by μ-opioid agonists. A, Superimposed traces of IPSCs in a dopamine neuron evoked by laser flashes (5 ms; 473 nm, average of 5) in the RMTg. DAMGO (1 μm) and picrotoxin (100 μm) inhibited amplitude of IPSCs. Notice the latency between laser stimulation and the onset of the IPSC (5.4 ± 0.5 ms). B, Wide-field mosaic confocal image stained for tyrosine hydroxylase (TH; red) and ChR2 conjugated with Venus (green) in sagittal plane. The star indicates the location of laser stimulation in RMTg. C, Summary graph of flash-evoked IPSC amplitude. ME (1 μm) and DAMGO (1 μm) significantly decreased IPSC amplitude compared with 100% control. The inhibitions were reversed by washout of ME or by naloxone (1 μm) after DAMGO that were not significantly different from 100% control. Number of cells indicated in the bars. Error bars indicate SEM. ***p < 0.001.