(A) AAV9-hSyn-chronos-flex-eGFP was stereotaxically injected into the striatum of two-month-old ChAT-Cre X D1tdTomato mice (Stereotaxic coordinate injection: ML = −1.2, AP = −0.7, DV = −3.4). The coronal confocal slice image shows the expression of Chronos (green cells) in a ChAT-cre neuron (cholinergic interneurons) along with dSPNs expressing tdTomato (red cells, scale bar = 40 μm). The tissue was dissected and recorded from 21 days postinjection. (B) The mean (± SEM) of ChI-evoked EPSP responses recorded from visually identified SPNs in gramicidin perforated patch in current-clamp mode in the presence of synaptic blockers: NBQX (5 μM), AP5 (50 μM), CGP-55845 (1 μM), MPEP (1 μM), and CPCCOEt (50 μM). The LED pulse (470 nm, 5 ms) was applied at an interval of 60 s. The traces recorded before and after the addition of gabazine (10 μM). Scale bars = 1 mV/100 ms. (C) Box plots of data from dSPNs (n = 8) and iSPNs (n = 6). (D) NPY-Cre X D1tdTomato mice were injected as described in (A). Confocal image showing NPY-Cre neurons expressing Chronos (green) and dSPNs expressing tdTomato (red, scale bar = 40 μm). (E) Mean (+ SEM) of NPY-Cre-evoked EPSP responses recorded from visually identified dSPNs in gramicidin perforated patch in current-clamp mode in the presence of blockers as described in (B) before and after the addition of Gabazine (10 μM). Traces from dSPN recorded in NPY (n = 4). Scale bars = 1 mV/100 ms. (F) Summary data for dSPNs (n = 4) and for iSPNs (n = 4). The data underlying the graphs shown in the figure can be found in dx.doi.org/10.5281/zenodo.10386854. ChAT, choline acetyltransferase; ChI, cholinergic interneuron; PSP, postsynaptic potential; SPN, spiny projection neuron.

https://doi.org/10.1371/journal.pbio.3002752.g001

(A) Maximum projection image of a visually identified dSPN from a D1R-tdTomato x ChAT-cre mouse with a high magnification image of a distal dendrite where 720 nm 2PLSM spot uncaging of DNI-Glu (2PLU, 5 mM) was conducted (red dots). Tomato+ dSPNs were patched in whole-cell mode and the cells were loaded with Alexa 568 for clear identification of dendrites and spines. Scale bars = 40 μm cell, 5 μm dendrite. (B) Scheme for interrogating endogenous GABA release from NGFIs onto SPNs via optogenetic stimulation of ChAT-cre mice expressing Chronos. (C, D) Throughout the dendrites, glutamate uPSPs in dSPNs and iSPNs can be evoked by uncaging DNI-Glu (5 mM, 1 × 15 spines, 1 ms pulses at 500 Hz, red traces, 720 nm laser) while stimulating GABA release from NGFIs with the blue laser (1 × 3 ms pulse, blue traces, 473 nm, within approximately 20 μm of the dendrite). From the quiescent down-state, GABA_AR activation is depolarizing and pushes SPNs toward enhanced dendritic integration in both dSPN and iSPN dendrites (Glu-2PLU + GABA_A opto = black trace, scale bars = 5 mV/200 ms). (E) Summary data showing the enhancement in amplitude and duration of the plateaus at ½ the maximum amplitude (1/2max) in iSPNs and dSPNs combined (n = 11 total: 3 iSPNs + 8 dSPNS; p < 0.001 for both amplitude and 1/2max duration, respectively). (F) Scatter plot of duration at ½ maximum amplitude vs. amplitude for clustered glutamate alone (red) and following GABA_AR activation (black). Median effects (open circles) and the median absolute difference as capped lines are also illustrated. All experiments are conducted in the appropriate cocktail of synaptic blockers: CGP-55845 (1 μM), MPEP (1 μM), and CPCCOEt (50 μM). The data underlying the graphs shown in the figure can be found in dx.doi.org/10.5281/zenodo.10386854. 2PLSM, two-photon laser scanning microscopy; ChAT, choline acetyltransferase; ChI, cholinergic interneuron; SPN, spiny projection neuron.

https://doi.org/10.1371/journal.pbio.3002752.g002