The NeuroD6 Subtype of VTA Neurons Contributes to Psychostimulant Sensitization and Behavioral Reinforcement

Abstract

Reward-related behavior is complex and its dysfunction correlated with neuropsychiatric illness. Dopamine (DA) neurons of the ventral tegmental area (VTA) have long been associated with different aspects of reward function, but it remains to be disentangled how distinct VTA DA neurons contribute to the full range of behaviors ascribed to the VTA. Here, a recently identified subtype of VTA neurons molecularly defined by NeuroD6 (NEX1M) was addressed. Among all VTA DA neurons, less than 15% were identified as positive for NeuroD6. In addition to dopaminergic markers, sparse NeuroD6 neurons expressed the vesicular glutamate transporter 2 (Vglut2) gene. To achieve manipulation of NeuroD6 VTA neurons, NeuroD6(NEX)-Cre-driven mouse genetics and optogenetics were implemented. First, expression of vesicular monoamine transporter 2 (VMAT2) was ablated to disrupt dopaminergic function in NeuroD6 VTA neurons. Comparing Vmat2 lox/lox;NEX-Cre conditional knockout (cKO) mice with littermate controls, it was evident that baseline locomotion, preference for sugar and ethanol, and place preference upon amphetamine-induced and cocaine-induced conditioning were similar between genotypes. However, locomotion upon repeated psychostimulant administration was significantly elevated above control levels in cKO mice. Second, optogenetic activation of NEX-Cre VTA neurons was shown to induce DA release and glutamatergic postsynaptic currents within the nucleus accumbens. Third, optogenetic stimulation of NEX-Cre VTA neurons in vivo induced significant place preference behavior, while stimulation of VTA neurons defined by Calretinin failed to cause a similar response. The results show that NeuroD6 VTA neurons exert distinct regulation over specific aspects of reward-related behavior, findings that contribute to the current understanding of VTA neurocircuitry.

Neurochemical signaling of reward and aversion by ventral tegmental area glutamate neurons

“…Our iGluSnFR recordings mirror these results, identifying one population of glutamate inputs that increases in response to both rewarding and aversive stimuli, and another population of glutamate inputs that decreases in response to reward but increases in response to aversive stimuli. Together, we hypothesize that distinct glutamatergic inputs to VTA VGluT2 neurons drive their signaling of and participation in differently valenced behaviors that have been observed following their optogenetic activation [17][18][19][20] .…”

“…This heterogeneity in firing likely contributes to the functional diversity of these neurons in motivated behavior. Optogenetic stimulation of VTA VGluT2 neurons can result in either place preference or place aversion [17][18][19]20 . Stimulation parameters and projection targets both influence when mice exhibit reward or aversion-based behavior following VTA VGluT2 neuron stimulation, which indicates that the role of these neurons in motivation is complex.…”

Generation of a DAT-Flp mouse line for intersectional genetic targeting of dopamine neuron subpopulations

“…Since both the cell bodies and projection sites of these two functionally distinct subpopulations are in close spatial proximity, it has been a challenge to selectively target them for further study. The restricted projections of Neurod6-expressing DA neurons, as previously defined by retrograde labeling and fluorescent in situ hybridization (FISH), make Neurod6 a compelling marker gene to label MSh-projecting DA neurons (12,38).…”

“…Previous studies have revealed that DA neurons are composed of genetically distinct subclasses (12)(13)(14)(15)37). One of these subpopulations, marked by the expression of the transcription factor Neurod6, is found in the ventromedial VTA and projects to the NAc medial shell (NAc MSh) (12,(37)(38)(39). This subpopulation is of interest because it is selectively spared in a 6-OHDA model of Parkinson's disease (12).…”

“…At the behavioral level, there is strong evidence that MSh-projecting DA neurons in the VTA are activated by unexpected aversive events (8). However, it has also been reported that high power optogenetic activation of NEX-Cre-expressing neurons in the midbrain can lead to realtime place preference, signifying a rewarding outcome (38). Moreover, it has been shown that while increased DA release in the NAc core may signal reward, this is not necessarily correlated with cell body firing in the VTA (54).…”