Excitation of medium spiny neurons by 'inhibitory' ultrapotent chemogenetics via shifts in chloride reversal potential

Abstract

Ultrapotent chemogenetics, including the chloride-permeable inhibitory PSAM⁴-GlyR receptor, were recently proposed as a powerful strategy to selectively control neuronal activity in awake, behaving animals. We aimed to validate the inhibitory function of PSAM⁴-GlyR in dopamine D1 receptor-expressing medium spiny neurons (D1-MSNs) in the ventral striatum. Activation of PSAM⁴-GlyR with the uPSEM⁷⁹² ligand enhanced rather than suppressed the activity of D1-MSNs in vivo as indicated by increased c-fos expression in D1-MSNs and in vitro as indicated by cell-attached recordings from D1-MSNs in mouse brain slices. Whole-cell recordings showed that activation of PSAM⁴-GlyR depolarized D1-MSNs, attenuated GABAergic inhibition, and shifted the reversal potential of PSAM⁴-GlyR current to more depolarized potentials, perpetuating the depolarizing effect of receptor activation. These data show that 'inhibitory' PSAM⁴-GlyR chemogenetics may activate certain cell types and highlight the pitfalls of utilizing chloride conductances to inhibit neurons.

Keywords: D1 medium spiny neuron; PSAM; chemogenetics; chloride permeability; mouse; neuroscience; uPSEM.

Figure 1.. In vivo activation of PSAM⁴-GlyR enhances c-fos expression in D1-MSNs.

(A) Left, cartoon of viral injection site in the ventral striatum. Right, representative confocal images of Cre-dependent PSAM⁴-GlyR expression (eGFP) in Cre-expressing D1-MSNs (tdTomato). (B) Experimental design for measuring c-fos expression after activation of PSAM⁴-GlyR. (C) Representative maximum intensity Z-stack confocal images of c-fos immunostaining (red) in D1-MSNs with or without activation of PSAM⁴-GlyR, after saline vs. fentanyl injection (0.2 mg/kg, i.p.). PSAM⁴-GlyR* refers to PSAM⁴-GlyR activated with uPSEM⁷⁹² (3 mg/kg, i.p.). The ‘Control’ group includes pooled data from mice with (control virus + uPSEM⁷⁹² 1^st i.p. injection) and (PSAM⁴-GlyR virus + saline 1^st i.p. injection). Scale bars: 50 µm. (D) Activation of PSAM⁴-GlyR with uPSEM⁷⁹² increased c-fos expression in PSAM⁴-GlyR expressing D1-MSNs compared to control (two-way ANOVA with Sidak’s multiple comparisons test). Data are presented as % colocalization of c-fos and eGFP/eYFP⁺ in transduced D1-MSNs. (E) Activation of PSAM⁴-GlyR did not increase c-fos expression in non-transduced cells (calculated as the number of c-fos expressing non-transduced cells/total number of non-transduced cells). Data represent mean ± SEM. * indicates statistical significance, ns denotes not significant.

Figure 2.. Activation of PSAM⁴-GlyR in D1-MSNs produces an inward current.

(A) Representative traces of whole-cell voltage-clamp recordings (V_hold −88 mV) demonstrate no effect of uPSEM⁷⁹² (10 or 50 nM) in control neurons. In PSAM⁴-GlyR⁺ neurons, 10 or 50 nM uPSEM⁷⁹² produced an inward current. Dashed line is baseline whole-cell current for ease of visualization. (B) Plot of the magnitude of the inward current produced by 10, 50, or 100 nM uPSEM⁷⁹² in PSAM⁴-GlyR⁺ neurons. Line and error bars represent means ± SEM. (C) To measure membrane resistance (R_m), a 10 mV voltage step (−88 to −78 mV) was made in aCSF and during the uPSEM⁷⁹²-induced inward current, and the instantaneous change in current following the capacitive transient was measured (ΔI). Representative traces are shown below the voltage step command. Dashed line is 0 pA. (D) In PSAM⁴-GlyR⁺ neurons, 50 nM uPSEM⁷⁹² significantly decreased R_m (paired t-test, p<0.0001) while 10 nM showed a trend toward lower R_m (paired t-test, p=0.08). * indicates statistical significance, ns denotes not significant.

Figure 3.. Activation of PSAM⁴-GlyR depolarizes D1-MSNs and does not inhibit firing.

(A) In PSAM⁴-GlyR⁺ neurons, activation of PSAM⁴-GlyR with uPSEM⁷⁹² (10 and 50 nM) significantly depolarized the membrane potential (V_m) compared to control (non-PSAM⁴-GlyR) neurons, measured in current-clamp (two-way ANOVA with Sidak’s multiple comparisons test). (B) Representative traces of current-clamp recordings from a control (top) or PSAM⁴-GlyR⁺ neuron (bottom). Action potential firing was evoked by somatic current injection (250 pA, 2 s) in aCSF (left) and 50 nM uPSEM⁷⁹² (right). Dashed line is −80 mV. (C) Distribution of firing response of PSAM⁴-GlyR⁺ neurons after 10 or 50 nM uPSEM⁷⁹² application. In 10 nM uPSEM⁷⁹², 14/14 neurons continued to fire with current injection. In 50 nM, 9/16 neurons fired with current injection (induced firing), 1/16 fired spontaneously without a current injection (spont. firing), and 6/16 went into depolarization block (DP block). (D) Representative traces of current-clamp recordings from a PSAM⁴-GlyR⁺ neuron that went into depolarization block with uPSEM⁷⁹². Response to somatic current injection (250 pA, 2 s) in aCSF (gray) and after uPSEM⁷⁹² application (black). The membrane potential in uPSEM⁷⁹² was more depolarized than the potential where the neuron entered depolarization block in aCSF. Dashed line is −80 mV. (E) Plot of change in the minimum current needed to induce firing (approximate rheobase: ~rb) induced by uPSEM⁷⁹² (10 and 50 nM) in control (gray) and PSAM⁴-GlyR⁺ (black) in cells that continued to fire (10/16). No significant change of ~rb was observed. (F) Plot of number of action potentials (APs) fired versus injected current in control neurons in aCSF (open) and in uPSEM⁷⁹² (closed). No significant difference was observed. (G) Plot of AP firing frequency versus injected current in control neurons in aCSF (open) and in uPSEM⁷⁹² (closed). No significant difference was observed. (H) Plot of number of APs fired versus injected current in PSAM⁴-GlyR⁺ neurons in aCSF (open) and in uPSEM⁷⁹² (closed). No significant difference was observed. (I) Plot of AP firing frequency versus injected current in PSAM⁴-GlyR⁺ neurons in aCSF (open) and in uPSEM⁷⁹² (closed). No significant difference was observed. Line and error bars represent means ± SEM, * indicates statistical significance, ns denotes not significant.

Figure 4.. In cell-attached configuration, activation of PSAM⁴-GlyR triggers firing of D1-MSNs when the membrane is depolarized to subthreshold potentials.

(A) Representative traces of cell-attached recordings from a control (gray) or PSAM⁴-GlyR⁺ neuron in standard aCSF (4.5 mM [K⁺]). Action currents were evoked following uPSEM⁷⁹² by applying a high external concentration of potassium (high K⁺, ~40 mM). The insets below the high K⁺ traces represent an expanded timescale at the beginning of firing; Inset scale bar = 200 ms. (B) In PSAM⁴-GlyR⁺ neurons, activation of PSAM⁴-GlyR with uPSEM⁷⁹² had no effect on the frequency of action currents in response to high K⁺. (C) Representative traces of cell-attached recordings from a control (gray) or PSAM⁴-GlyR⁺ neuron in aCSF containing 10–13 mM [K⁺]. In PSAM⁴-GlyR⁺ neurons, activation of PSAM⁴-GlyR with uPSEM⁷⁹² produced firing. There was no further firing with application of high K⁺. uPSEM⁷⁹² had no effect on firing in control neurons. (D) Plot of the frequency of action currents in control and PSAM⁴-GlyR⁺ neurons in aCSF, uPSEM⁷⁹² and high K⁺ (mixed effect two-way ANOVA with Sidak’s multiple comparisons test). Line and error bars represent means ± SEM. * indicates statistical significance, ns denotes not significant.

Figure 5.. Current–voltage relationship of PSAM⁴-GlyR current shows rightward shift in reversal potential and transient or small outward currents at depolarized potentials.

(A) Current was recorded in response to voltage steps (1 s, −118 to −28 mV, 10 mV) from V_hold −88 mV in aCSF and after application of uPSEM⁷⁹² (50 or 100 nM). uPSEM⁷⁹²-induced current was isolated by subtraction. Representative traces of uPSEM⁷⁹²-induced current during voltage steps in a control (left) and a PSAM⁴-GlyR⁺ neuron (right). Inset numbers by trace indicate transient (Roth, 2016), steady-state (Saloman et al., 2016), and tail (Gomez et al., 2017) currents. (B) Expanded timescale of transient currents (inset ‘1’ in A) in a control and a PSAM⁴-GlyR⁺ neuron. (C) Plot of current–voltage relationship of the transient current (I_transient) in control (n = 11) vs. PSAM⁴-GlyR⁺ neurons (n = 21). The reversal potential of transient current in PSAM⁴-GlyR⁺ neurons was −61 mV. (D) Plot of current–voltage relationship of the transient (inset ‘1’ in A, same as in C) vs. steady-state uPSEM⁷⁹²-induced (inset ‘2’ in A) currents in PSAM⁴-GlyR⁺ neurons. At steady state, the reversal potential of uPSEM⁷⁹²-induced current shifted to −44 mV. (E) Plot of the pairwise shift of reversal potential (E_rev) between transient (I_tran) and steady-state currents (I_ss). (F) Expanded timescale of tail currents (inset ‘3’ in A) in a control and a PSAM⁴-GlyR⁺ neuron. (G) Plot of the amplitude of tail currents measured at V_hold −88 mV (I_tail) versus holding potential of the preceding voltage step in control and PSAM⁴-GlyR⁺ neurons. uPSEM⁷⁹²-induced tail current at −88 mV demonstrated robust augmentation with prior depolarization and reduction with prior hyperpolarization. (H) The magnitude of the rightward shift in reversal potential between transient and steady-state current was positively correlated with the PSAM⁴-GlyR conductance (G in nS). Line and error bars represent means ± SEM, * indicates statistical significance.

Figure 6.. PSAM⁴-GlyR activation in D1-MSNs reduces GABAergic synaptic inhibition.

(A) Representative traces of GABA-A receptor-mediated synaptic currents in a PSAM⁴-GlyR⁺ neuron in aCSF, evoked during voltage steps from V_hold −88 mV (voltage steps: 1 s, −118 to −28 mV, 10 mV; electrical stimulation at 850 ms into voltage steps). Panel on the right shows the traces in expanded timescale corresponding to the gray rectangle on the left. (B) Representative traces of GABA-A receptor-mediated currents evoked in the same neuron from V_hold −28 mV (outward) and −118 mV (inward) in aCSF (gray) and uPSEM⁷⁹² (black). (C) Plot of the current–voltage relationship of the GABA-A receptor synaptic currents in aCSF and uPSEM⁷⁹² in neurons where the reversal potential of PSAM⁴-GlyR current shifted by >5 mV (n = 5, average shift of 17.0 mV). The reversal potential of GABA-A receptor-mediated current was −60.9 mV in aCSF and −51.6 mV in uPSEM⁷⁹². Inset (top left) shows values of GABA-A receptor synaptic currents in uPSEM⁷⁹² near the reversal potential in an expanded scale. (D) Plot of the current–voltage relationship of the GABA-A receptor-mediated synaptic current in aCSF and uPSEM⁷⁹² in neurons where the reversal potential of PSAM⁴-GlyR current did not shift (n = 3, average shift of 2.1 mV). The reversal potential of GABA-A receptor-mediated synaptic currents was similar in aCSF (−64.7 mV) and uPSEM⁷⁹² (−64.9 mV). Line and error bars represent means ± SEM, * indicates statistical significance.