The GABA_AR reversal potential is near −60 mV in both young and adult SPNs.

(A) Clomeleon-expressing iSPNs allowed visual identification of dendrites in gramicidin perforated-patch recording conditions where cells cannot be loaded with dyes via internal-pipette solution (920 nm laser maximum projection image, scale bar = 40 μm). When low (<100 MΩ) access-resistance was achieved in voltage-clamp mode, RuBi-GABA (10 μM) was uncaged with a 473 nm laser spot (approximately 1 μm diameter, 1 ms) in the presence of the synaptic blockers: TTX (1 μM), AP5 (50 μM), NBQX (5 μM), CGP-55845 (1 μM). The laser was targeted to the somatic region or to distal dendrites (blue spots, projection image). (B) Representative voltage traces showing GABA responses, recorded in serial, from the soma (top traces, scale bars = 20 pA/2 s) or the dendrite (lower traces, scale bars = 10 pA/2 s) as the membrane was manually stepped from −70 mV to −50 mV. (C) Plot of the current/voltage relationship between somatic and dendritic activation. The data, represented by medians with interquartile ranges, did not differ significantly between the soma and dendritic compartments (n = 5 each; soma, dendrite; slope = 0.96, 0.71; x-intercept = −55.9, −59.6 mV; R² = 0.77, 0.85, respectively). Current measurements were rounded to the nearest 0.5 pA. (D) Current-clamp experiments in gramicidin perforated-patch mode were performed to examine age-dependent shifts in reversal potential. Here, Adora2a-eGFP positive iSPNs could be visually identified and patched. When low (<100 MΩ) access-resistance was achieved in current-clamp mode, the resting membrane potential along with series of hyperpolarizing and depolarizing steps were used to examine cell health (traces, scale bars = 20 mV/200 ms). (E) RuBi-GABA (10 μM) was uncaged over the full-field (3 ms duration, 60× lens) with a 473 nm LED in the presence of the synaptic blockers: AP5 (50 μM), NBQX (5 μM), CGP-55845 (1 μM). Representative current traces showing GABA responses as the membrane was manually stepped from −80 mV to −50 mV, scale bars = 5 mV/200 ms. (F) Plot of the change in PSP amplitude at P30, P90, and P270. The data, represented by medians with interquartile ranges, did not differ significantly between the 3 ages tested (n = 5 each P30, P90, P270; slope = 0.75, 0.70, 0.75; x-intercept = −61.6, −61.5, −61.5 mV; R² = 0.93, 0.91, 0.94, respectively). Values were calculated to the nearest 0.5 mV for the ΔV/V measurements. The data shows that the reversal for GABA-induced current is a full 20 mV+ above the resting membrane potential for SPNs, typically, −80 to −85 mV. (G) The addition of bumetanide (NKCC1 blocker, 10 μM) did not change the GABA_AR reversal potential significantly (n = 5, p = 0.4076). (H) Perforated patch recordings were obtained from Adora2a-eGFP iSPNs and then the reversal potential of GABA_ARs determined before and after inhibition of CA with acetazolamide (aceta, 10 μM). (I) Representative traces recorded from a visually identified iSPN from an Adora2a-eGFP mouse in gramicidin perforated patch in current-clamp mode in the synaptic blockers: AP5 (50 μM), NBQX (5 μM), CGP-55845 (1 μM), MPEP (1 μM), and CPCCOEt (50 μM). RuBi-GABA (15 μM) was uncaged using a single LED pulse (470 nm, 25 ms). The pulse was applied at an interval of 30 s while manually stepping the cell to different potentials from −80 to −50 mV, scale bars = 10 mV/100 ms. (J) Summary data shows that application of acetazolamide shifted the reversal of the GABA-induced current to more negative potentials (p = 0.03125, n = 6). Membrane potentials were adjusted to correct for the estimated liquid junction potential and then binned into 5 mV increments (-70, -65, -60, -55 and -50 mV). The data underlying the graphs shown in the figure can be found in dx.doi.org/10.5281/zenodo.10386854. CA, carbonic anhydrase; PSP, postsynaptic potential; SPN, spiny projection neuron.