Figure 1.
Immunohistochemical localization of torsinA in the cholinergic interneurons of NT and hMT mouse striatum.
Representative confocal images (z-series projections) obtained from coronal slices of NT and hMT mice immunolabelled for ChAT (green, A, D). The macroscopic morphology of ChAT-positive cholinergic interneurons is apparently the same in the two groups at P14. Representative confocal images (single sections) showing the immunostaining of torsinA (red, B and E) in the ChAT-positive cholinergic neurons (merge, C and F) of NT (B–C) and hMT (E–F) mice. The cellular distribution of torsinA is similar between the groups. Scale bars: 10 µm.
Figure 2.
Basic electrophysiological properties of striatal cholinergic interneurons.
Differential interference contrast images showing the morphological properties of striatal cholinergic interneurons in a slice preparation (A) and after enzymatic tissue dissociation (A1). Note the large, polygonal soma. B, B1. Representative traces showing voltage responses to current steps (100 pA, 600 ms) in both depolarizing and hyperpolarizing direction in cholinergic interneurons of hMT recorded at P14 and P90. Note the prominent Ih current evoked by hyperpolarizing step (white arrow) and the robust AHP current following the step ending (black arrow). A single representative action potential is shown at higher sweep speed. C. I-V relationship (10 mV step, 600 ms) recorded in the voltage-clamp mode confirm that no significant difference in membrane properties were detected between hMT and hWT mice.
Table 1.
Intrinsic membrane properties of cholinergic interneurons in NT, hWT and hMT mice.
Figure 3.
Cholinergic interneurons exhibit normal response to M2/M4, GABAB and group I mGlu receptor activation.
Representative traces of cholinergic interneurons recorded in the perforated patch-clamp configuration. Recordings were collected from hMT mice at P14 (left) and P90 (right). A–A1. M2/M4 receptor activation by oxotremorine (300 nM, 3 min) induced a transient firing cessation with a membrane hyperpolarization. No significant differences were measured at both ages. B–B1. Similarly, application of Baclofen, a GABAB receptor agonist (10 µM, 2 min) reversibly blocked firing activity in hMT mice. C–C1. Activation of group I mGlu receptors by 3,5-DHPG (10 µM, 30 sec) induced a prominent membrane depolarization coupled with a transient increase of firing rate.
Figure 4.
Early abnormal responsiveness to D2R stimulation in hMT mice.
A–D. In hMT mice, quinpirole (10 µM, 2 min) caused a paradoxical membrane depolarization coupled to a large increase in the rate of action potentials in cholinergic interneurons recorded from mice at four different developmental ages. A1–D1. Sample traces show, at higher sweep speed, the firing rate measured before and at the peak of the effect of quinpirole application. Note the reduction of mAHP in quinpirole (white and black arrows). Left-shift of the ISI plot (inset) confirm the increased firing frequency induced by quinpirole at all tested ages. E. Graph plots summarize the effect of quinpirole at P10, P14, P20 and P90. The D2R antagonist sulpiride (3 µM) prevented the aberrant response to quinpirole. F. Zoom of action potential recorded in control conditions (black trace) and in the presence of quinpirole (red trace) at P14 and P90. Note the decrease of AP threshold induced by D2R activation. Graphs summarize the changes at both ages. G. Voltage-clamp recordings (holding potential −60 mV) showing that bath-application of quinpirole induces an inward current in hMT mice at P14 and P90. H. Time-course of quinpirole effect on the membrane conductance at P14 and P90. D2R activation induced a significant increase in membrane conductance at both ages.
Figure 5.
Ih and mAHP current modulation by D2R activation.
A. Representative traces of mAHP recorded after a brief depolarizing current pulse (50 pA, 500 ms) in hWT and hMT mice. Note that in hMT mice, quinpirole application (gray trace) significantly reduced mAHP. Summary graph of D2R-mediated effects on mAHP in the three genotypes. B. Hyperpolarizing voltage steps (30 mV, 800 ms) in the presence of TTX (1 µM) were delivered to induce an Ih current. Representative traces recorded in whole-cell configuration before (black) and after (red) quinpirole application in hWT and hMT mice. Graph plot summarizes the effect of D2R activation on the Ih current in the three groups of mice.
Figure 6.
Gi/Go protein involvement in D2R-mediated response.
A. Representative traces of a hMT interneuron recorded in whole-cell current-clamp configuration. In the presence of GDP-β-S in the recording pipette, the response to quinpirole was fully prevented. The graph shows that the instantaneous firing frequency did not change after bath application of quinpirole (10 µM, 2–4 min). B. Data were collected from acutely isolated interneurons of hMT mice. Pipette loading with GDP-β-S was able to block the inhibitory effect of quinpirole onto HVA calcium currents. C. Application of quinpirole (10 µM 2–4 min) produced a robust inhibition of the total HVA calcium currents evoked by voltage-ramp protocols. Time-course of drug application show that the pre-application of N-ethylmaleimide (NEM) fully occluded the inhibitory effect mediated by D2Rs activation. Blue circles represent the HVA currents recorded in control condition. Red circles report the inhibitory effect of quinpirole.
Figure 7.
Calcium ion involvement in D2R-mediated response.
A–A2. Data collected from acutely isolated neurons. A. Graph plot summarizes the inhibitory effect of quinpirole application on HVA calcium current evoked by voltage-ramp protocols. A1. Bar graph shows an increased Cav2.2/N-type fraction of total HVA current in isolated interneurons of hMT mice. A2. Time-course of the peak calcium current before and after drug application. The inhibitory effect produced by quinpirole was occluded by pre-application of ω-conotoxin GVIA. B. Data collected from brain slices. Representative traces of voltage dependent currents evoked by a series of depolarizing steps from an holding potential of −60 mV. Currents were evoked in the presence of TTX (1 µM) in order to eliminate Nav channel-mediated inward currents in the sub- and supra-threshold voltage range. Application of the D2R agonist quinpirole (10 µM, a) clearly reduces the outward current evoked by depolarizing steps. Slices pre-incubation with CdCl2 (300 nM) almost fully abolished the quinpirole-mediated effect (b). Blockade of Cav2.2/N-type calcium channels by ω-conotoxin GVIA (1 µM, c) largely reduced the quinpirole effect on step-evoked current. Graphs report the normalized current recorded for each voltage level. C. Representative traces of cholinergic interneurons recorded in whole-cell current clamp experiments in hMT mice. Intracellular replacement with an high BAPTA concentration (10 mM) fully prevented the abnormal response after quinpirole application. C1. Plot of instantaneous firing frequency confirm that no significant increase of firing rate was caused by D2R activation in the presence of BAPTA.