Fig. 8. Mutant Nb-Ft-TRPV1^Cl− inhibits neuronal activity of subthalamic nucleus projection neurons and rescues motor impairment in parkinsonian mice.

A. Schema of inhibition system with mutant Nb-Ft-TRPV1^Cl− membrane channel. B. Normalized change in MQAE fluorescence Intensity (ΔF/F0) of Neuro2A cells expressing mCherry, TRPV1^Ca2+ or Nb-Ft-TRPV1^Cl− with low AMF application in vitro. C. AUC analysis of chloride imaging for Nb-Ft-TRPV1^Cl− channel. D. Protocol and schema for generation of parkinsonian PitX-2-Cre mice with unilateral injection of 6 hydroxydopamine (6-OHDA) in the medial forebrain bundle (MFB), followed by randomization based contralateral rotations induced by i.p. administration of apomorphine (0.25mg/kg) and ipsilateral intracranial injection of the double-floxed Cre-dependent AAV vector expressing the mutant Nb-Ft-TRPV1^Cl− under the control of the JET promoter in the subthalamic nucleus (STN). Representative immunostaining of the selective expression of Nb-Ft-TRPV1^Cl− (red) in STN neurons expressing Cre recombinase (Green). E. Percentage of change from pre-DMF treatment in contralateral rotations induced with apomorphine (0.25mg/kg, i.p.) in parkinsonian PitX-2-Cre mice expressing the Nb-Ft-TRPV1^Cl− or the mCherry control viruses in the STN during DMF, and post-DMF. F. Immunostaining for c-fos (cyan) and HA or mCherry (red) of STN of PiX-2-Cre mice euthanized 1 hour following 15 minutes of DMF exposure with apomorphine induced rotations (0.25mg/kg).G. Number of transduced neurons per area in STN between mCherry and Nb-Ft-TRPV1^Ca2+ groups. H. Percentage (%) of c-fos positive (+) neurons over total number of STN neurons expressing HA or RFP (n=4 mice per group/3 sections per mice). Error bars show SEM. * represents p value < 0.05, ** represents p value < 0.01 with two-tailed, unpaired t-test with Welch’s correction. Anti-HA staining to detect Nb-Ft-TRVP1^Ca2+ expression, anti-RFP staining to detect mCherry expression.