Figure 4: Deleting Ube3a in TH-positive neurons decreases GABA co-release and enhances motivational behaviour.

(a) Average nose pokes for inactive and active ports triggering 30 Hz optical intracranial self-stimulation in a 60 min behavioural session (n=5 and 7, one-way ANOVA *P<0.02). Experimental design was similar to that schematized in Fig. 1a, except that TH^CRE::Ube3a^FLOX/p+ and TH^CRE::Ube3a^m+/p+ mice were examined to selectively delete Ube3a and optically stimulate TH⁺ VTA-to-NAc terminals. (b) Representative fast-scan cyclic voltammograms assessing dopamine release within the ventral striatum of TH^CRE::Ube3a^FLOX/p+ and TH^CRE::Ube3a^m+/p+ mice. Dopamine release was evoked by 30 Hz (5-pulses) optical stimulation. Insets represent background-subtracted electrochemical signal characteristic of oxidized dopamine. (c) Averaged optically evoked dopamine release at a range of frequencies demonstrates that there are no statistical differences between genotypes (n=6 and 7, one-way ANOVA P>0.05). (d) Schematic representing protocol for whole-cell optical IPSC (oIPSC) recordings within the NAc of TH^CRE::Ube3a^FLOX/p+ mice. IPSCs were pharmacologically isolated (see the Methods section). (e) Representative oIPSC traces measured in TH^CRE::Ube3a^FLOX/p+ and TH^CRE::Ube3a^m+/p+ mice evoked by single pulses (20 ms) or 30 Hz trains of stimulation. The pharmacological isolation of GABAergic currents was validated by application of the antagonist SR95531. (f) Average peak amplitude of single oIPSCs demonstrate a significant reduction of GABAergic currents in Ube3a^FLOX/p+ mice (n=13 and 15, **P<0.05). (g) Average decay kinetics of oIPSCs analysed in f (Student’s t-test, P=0.61). Representative traces are shown with normalized amplitude to demonstrate similar current decay kinetics. (h) Average oIPSC charge evoked using a similar 30 Hz optical stimulation paradigm used behaviourally (see a) and tested in vitro (bottom traces of e). TH^CRE::Ube3a^FLOX/p+ mice showed a significant decrease in GABA co-release (n=10 and 14, Student’s t-test **P<0.05). All bars represent the mean±s.e.m.