Targeted genetic manipulations of neuronal subtypes using promoter-specific combinatorial AAVs in wild-type animals

Techniques to genetically manipulate the activity of defined neuronal subpopulations have been useful in elucidating function, however applicability to translational research beyond transgenic mice is limited. Subtype targeted transgene expression can be achieved using specific promoters, but often currently available promoters are either too large to package into many vectors, in particular adeno-associated virus (AAV), or do not drive expression at levels sufficient to alter behavior. To permit neuron subtype specific gene expression in wildtype animals, we developed a combinatorial AAV targeting system that drives, in combination, subtype specific Cre-recombinase expression with a strong but non-specific Cre-conditional transgene. Using this system we demonstrate that the tyrosine hydroxylase promoter (TH-Cre-AAV) restricted expression of channelrhodopsin-2 (EF1α-DIO-ChR2-EYFP-AAV) to the rat ventral tegmental area (VTA), or an activating DREADD (hSyn-DIO-hM3Dq-mCherry-AAV) to the rat locus coeruleus (LC). High expression levels were achieved in both regions. Immunohistochemistry (IHC) showed the majority of ChR2+ neurons (>93%) colocalized with TH in the VTA, and optical stimulation evoked striatal dopamine release. Activation of TH neurons in the LC produced sustained EEG and behavioral arousal. TH-specific hM3Dq expression in the LC was further compared with: (1) a Cre construct driven by a strong but non-specific promoter (non-targeting); and (2) a retrogradely-transported WGA-Cre delivery mechanism (targeting a specific projection). IHC revealed that the area of c-fos activation after CNO treatment in the LC and peri-LC neurons appeared proportional to the resulting increase in wakefulness (non-targeted > targeted > ACC to LC projection restricted). Our dual AAV targeting system effectively overcomes the large size and weak activity barrier prevalent with many subtype specific promoters by functionally separating subtype specificity from promoter strength

Cross-hemispheric dopamine projections have functional significance

Decades of research have described dopamine’s importance in reward-seeking behavior and motor control. Although numerous investigations have focused on dopamine’s mechanisms in modulating behavior, the long-standing belief that dopamine neurons project solely unilaterally has limited the exploration of interhemispheric dopamine signaling. Here we resolve disparate descriptions of unilateral vs. bilateral projections by reporting that dopamine neurons can release dopamine in the contralateral hemisphere. Using voltammetry in awake and anesthetized rats, we reveal an unprecedented synchrony of dopamine fluctuations between hemispheres. Via stimulation with amphetamine, we demonstrate functional cross-hemispheric projections in a hemiparkinsonian model. This previously undescribed capacity for interhemispheric dopamine signaling can precipitate new areas of inquiry. Future work may exploit properties of bilateral dopamine release to improve treatments for Parkinson’s disease, including deep brain stimulation

Aversive stimulus differentially triggers subsecond dopamine release in reward regions

Aversive stimuli have a powerful impact on behavior and are considered to be the opposite valence of pleasure. Recent studies have determined some populations of VTA dopaminergic neurons are activated by several types of aversive stimuli while other distinct populations are either inhibited or unresponsive. However, it is not clear where these aversion responsive neurons project, and whether alterations in their activity translate into dopamine release in the terminal field. Here we show unequivocally that the neurochemical and anatomical substrates responsible for the perception and processing of pleasurable stimuli within the striatum are also activated by tail pinch, a classical painful and aversive stimulus. Dopamine release is triggered in the dorsal striatum and nucleus accumbens (NAc) core by tail pinch and is time locked to the duration of the stimulus, indicating that the dorsal striatum and NAc core are neural substrates, which are involved in the perception of aversive stimuli. However, dopamine is released in the NAc shell only when tail pinch is removed, indicating that the alleviation of aversive condition could be perceived as a rewarding event

Short-Term Consequences of Single Social Defeat on Accumbal Dopamine and Behaviors in Rats

The present study aimed to explore the consequences of a single exposure to a social defeat on dopamine release in the rat nucleus accumbens measured with a fast-scan cyclic voltammetry. We found that 24 h after a social defeat, accumbal dopamine responses, evoked by a high frequency electrical stimulation of the ventral tegmental area, were more profound in socially defeated rats in comparison with non-defeated control animals. The enhanced dopamine release was associated with the prolonged immobility time in the forced swim test. The use of the dopamine depletion protocol revealed no alteration in the reduction and recovery of the amplitude of dopamine release following social defeat stress. However, administration of dopamine D2 receptor antagonist, raclopride (2 mg/kg, i.p.), resulted in significant increase of the electrically evoked dopamine release in both groups of animals, nevertheless exhibiting less manifested effect in the defeated rats comparing to control animals. Taken together, our data demonstrated profound alterations in the dopamine transmission in the association with depressive-like behavior following a single exposure to stressful environment. These voltammetric findings pointed to a promising path for the identification of neurobiological mechanisms underlying stress-promoted behavioral abnormalities

Recombinant Adeno-Associated Virus-mediated rescue of function in a mouse model of Dopamine Transporter Deficiency Syndrome

Dopamine Transporter Deficiency Syndrome (DTDS) is a rare autosomal recessive disorder caused by loss-of-function mutations in dopamine transporter (DAT) gene, leading to severe neurological disabilities in children and adults. DAT-Knockout (DAT-KO) mouse is currently the best animal model for this syndrome, displaying functional hyperdopaminergia and neurodegenerative phenotype leading to premature death in ~36% of the population. We used DAT-KO mouse as model for DTDS to explore the potential utility of a novel combinatorial adeno-associated viral (AAV) gene therapy by expressing DAT selectively in DA neurons and terminals, resulting in the rescue of aberrant striatal DA dynamics, reversal of characteristic phenotypic and behavioral abnormalities, and prevention of premature death. These data indicate the efficacy of a new combinatorial gene therapy aimed at rescuing DA function and related phenotype in a mouse model that best approximates DAT deficiency found in DTDS