Nicotine persistently activates ventral tegmental area dopaminergic neurons via nicotinic acetylcholine receptors containing α4 and α6 subunits

Abstract

Nicotine is reinforcing because it activates dopaminergic (DAergic) neurons within the ventral tegmental area (VTA) of the brain's mesocorticolimbic reward circuitry. This increase in activity can occur for a period of several minutes up to an hour and is thought to be a critical component of nicotine dependence. However, nicotine concentrations that are routinely self-administered by smokers are predicted to desensitize high-affinity α4β2 neuronal nicotinic acetylcholine receptors (nAChRs) in seconds. Thus, how physiologically relevant nicotine concentrations persistently activate VTA DAergic neurons is unknown. Here we show that nicotine can directly and robustly increase the firing frequency of VTA DAergic neurons for several minutes. In mouse midbrain slices, 300 nM nicotine elicited a persistent inward current in VTA DAergic neurons that was blocked by α-conotoxin MII[H9A;L15A], a selective antagonist of nAChRs containing the α6 subunit. α-conotoxin MII[H9A;L15A] also significantly reduced the long-lasting increase in DAergic neuronal activity produced by low concentrations of nicotine. In addition, nicotine failed to significantly activate VTA DAergic neurons in mice that did not express either α4 or α6 nAChR subunits. Conversely, selective activation of nAChRs containing the α4 subunit in knock-in mice expressing a hypersensitive version of these receptors yielded a biphasic response to nicotine consisting of an acute desensitizing increase in firing frequency followed by a sustained increase that lasted several minutes and was sensitive to α-conotoxin MII[H9A;L15A]. These data indicate that nicotine persistently activates VTA DAergic neurons via nAChRs containing α4 and α6 subunits.

Fig. 1.

Characterization of VTA DAergic neurons. A, representative cell-attached recording from a putative DAergic neuron. DAergic neurons had characteristic low baseline firing frequencies (< 8 Hz) and B, expressed the hyperpolarizing activated cation current, I_H. Currents were elicited by 20 -mV hyperpolarizing steps from a holding potential of −60 mV to −120 mV. C, at the end of each recording, the content of each neuron was aspirated into the patch pipette, and TH expression was verified by single-cell reverse transcription-polymerase chain reaction (RT-PCR). A representative DNA agarose gel is shown illustrating a typical result for a DAergic neuron. Only neurons that clearly expressed TH (arrow) and not GAD 65/67 were included in the analysis.

Fig. 2.

Nicotine activation of VTA DAergic neurons. A, representative events frequency histogram from a VTA DAergic neuron before, during, and after bath application of 1 μM nicotine (black bar). Current clamp (I = 0) responses were recorded from visually identified VTA DAergic neurons in C57BL/6J mesocortical coronal slices. Note that nicotine increases DAergic neuron firing frequency for several minutes during and after initial exposure. B, representative current recordings taken from a) baseline, b) 5-min after nicotine exposure, and c) 12-min after nicotine exposure. C, change in average DAergic neuron action potential frequency in response to nicotine (gray bar, 5 min application) and after several minute wash with ACSF (white bar) compared with baseline (dotted line). **, p < 0.01.

Fig. 3.

Direct and persistent activation of VTA DAergic neurons by nicotine. A, representative action potential firing frequency histogram from a VTA DAergic neuron before, during, and after 30-min bath application of 300 nM nicotine. Action potentials were recorded in cell-attached mode. B, change in average firing frequency compared with baseline (1 min before nicotine application, dotted line) 10 and 30 min after nicotine application and after 20-min washout. *, p < 0.05, one-way ANOVA, Tukey post hoc, n = 6. C, representative whole-cell response to 300 nM nicotine in a VTA DAergic neuron. The neuron was voltage-clamped at −60 mV and 300 nM nicotine was bath applied for 10 min (black bar). The dotted line represents baseline current extrapolated from the recording before nicotine application. Responses were recorded in the presence of a cocktail of inhibitors. D, representative whole-cell response to 300 nM nicotine (black bar) in the presence and absence of 100 nM α-conotoxin MII[H9A;L15A] (red bar). E, average current amplitude minus baseline during 1.5-, 5-, and 9-min exposure to 300 nM nicotine. n = 8 neurons. F, average current amplitude minus baseline after a 5-min exposure to 300 nM nicotine (white bar) and after an additional 5 min exposure of nicotine + 100 nM α-conotoxin MII[H9A;L15A]. **, p < 0.01, compared with nicotine-induced current, n = 5 neurons/treatment.

Fig. 4.

The contribution of α4 and α6 subunits to nicotine-induced increases in DAergic neuron firing frequency. A, left, representative action potential firing frequency histogram from a VTA DAergic neuron in a WT midbrain slice. Nicotine (300 nM) was applied over the times indicated by the black bar. After 5 min of nicotine application, 100 nM α-contoxin MII[H9A;L15A] was applied concomitantly with nicotine. Right, change in average firing frequency compared with baseline (dotted line) 5 min after nicotine exposure and after an additional 5-min exposure to both nicotine and α-contoxin MII[H9A;L15A]. *, p < 0.05, compared with nicotine, n = 6. B, left, representative action potential firing frequency histogram from a VTA DAergic neuron in an α4 KO midbrain slice. Right, average firing frequency fold change compared with baseline (dotted line) 5 min after nicotine exposure and after an additional 5-min wash period. C, left, representative action potential firing frequency histogram from a VTA DAergic neuron in an α6 KO midbrain slice. Right, average firing frequency fold change compared with baseline (dotted line) 5 min after nicotine exposure and after an additional 5-min wash period. n = 6 neurons/genotype.

Fig. 5.

Selective activation of α4* nAChRs reveals two receptor subtypes mediating nicotine-induced activation of VTA DAergic neurons. A, representative action potential firing frequency histogram from a VTA DAergic neuron in a L9′A midbrain slice. Nicotine (50 nM) was applied over the times indicated by the black bar. After 5 min of nicotine application, 100 nM α-contoxin MII[H9A;L15A] was applied concomitantly with nicotine (red bar). Representative cell-attached recordings corresponding to individual time points before, during, and after drug treatment are illustrated below the graph. B, representative action potential firing frequency histogram from a VTA DAergic neuron in a L9′A midbrain slice. Nicotine was applied in the presence of 100 nM α-contoxin MII[H9A;L15A] and 100 nM DHβE. Cell-attached recordings are shown below the histogram as in A. C, representative action potential firing frequency histogram from a VTA DAergic neuron from a WT animal. Nicotine (50 nM) was applied to the slice as in A. Cell-attached recordings are shown below the histogram as in A. D, average firing frequency fold change compared with baseline (dotted line) in response to 50 nM nicotine in DAergic neurons from WT and L9′A slices in experiments depicted in A to C. The early phase of the nicotine response (E) is defined as the peak fold change produced by nicotine compared with baseline within the first 2 to 3 min of application, whereas the late phase of the nicotine response (L) is defined as the fold change produced by nicotine compared with baseline after 5-min application of drug.