Fig 1.
AAV FLEX-switch delivery system promotes selective Cre and hM3D(Gq) expression in primary cortical neurons.
The current experiment utilized 2 viral vectors shown at the top of panel B. At the C-terminal of the AAV-CMV-GFP/Cre (serotype 9) virus, Cre was fused with green fluorescent protein (GFP) that can be observed in infected cells using a confocal microscope. The AAV-hSyn-DIO-hM3D(Gq)-mCherry virus contains a double inverted coding sequence promoted by the neuron-specific promoter, hSyn, and the mCherry red fluorescent marker that can similarly be observed in infected cells using a confocal microscope. A) A representative confocal image of GFP/Cre and hM3D(Gq)-mCherry expression in a primary cortical neuron is shown. Here, the GFP green fluorescence indicates the expression of Cre and is localized in the nuclei of cells. mCherry red fluorescence indicates the expression of hM3D(Gq) receptors after the vector was reoriented by GFP/Cre recombinase in vitro. 3-D reconstruction based on Z-stack scanning verified that mCherry fluorescence exhibited a membrane-specific distribution. B) When injected into the AcbSh of Fisher 344 rats, GFP/Cre is retrogradely transported along the axons of VTA neurons to the soma where it reorients hM3D(Gq)-mCherry as represented in the drawing.
Fig 2.
Locomotor response to CNO under normal conditions.
A) Four animals that had received viral infusions and nine that received sham surgeries were exposed to various doses of CNO (0, 0.15, 0.3, 0.6, or 1.0 mg/kg) across 5 days under normal/no novelty present conditions. The mean number of total ambulations (± SEM) did not significantly change as a function of CNO dose, indicating that the administered doses of CNO did not elicit unforeseen locomotor effects. B) 12 animals (2 sham and 10 DREADDs) were administered 1 ml/kg 0.9% saline prior to a 60 min normal/no novelty present testing session and a novelty present testing session. The absence and presence of white background noise is indicated by the white and grey scale backgrounds, respectively. Only activity recorded after the 30-min time point (when white noise was temporarily turned off during the novelty present condition) was analyzed and is presented here. The mean number of total ambulations generally decreased over 30–60 min. However, in the presence of novelty, animals increased activity and sustained high activity for approximately 15–20 min. C) Twenty-two animals (10 DREADDs and 12 sham) were administered 1 mg/kg CNO prior to four novelty present testing sessions. The mean number of total ambulations increased after white noise was removed from the environment but gradually decreased once the novelty stimulus was removed. Animals that received viral infusions rather than sham surgeries increased activity in response to novelty to a greater degree and sustained high levels of activity for a longer period of time, suggesting that stimulation of hM3D(Gq) receptors on neurons of the VTA-AcbSh circuitry prolongs animals’ locomotor response to novelty. D) Following the conclusion of locomotor testing, all animals were administered 1 mg/kg saline or 1 mg/kg CNO prior to gap-PPI testing sessions to ensure that no animals exhibited abnormal auditory processing. Mean peak ASR amplitude (±SEM) across 2 days is displayed, with maximal inhibition observed at the 50 msec ISI. Neither viral infusions nor CNO administration significantly disrupted inhibition of the ASR, as indicated by statistically similar ASR amplitude curves.
Fig 3.
Histological identification of DREADDs expression.
Virus-infused cortical tissues were sectioned into 50μm thick coronal slices to determine which neuronal VTA subpopulations were infected by the AAV-hSyn-DIO-hM3D(Gq)-mCherry virus and stimulated by CNO. hM3D(Gq) receptors (as indicated by mCherry expression) were predominantly expressed in neurons of the pVTA. Blue DAPI (4’,6-diamidino-2-phenylindole) staining suggest that hM3D(Gq)-mCherry expression was limited to a subset of pVTA neurons that communicate with the AcbSh.