Brains of slightly anesthetized mice (P21–P53; isoflurane) were prepared into ice-cold sucrose-based cutting solution (in mM: 85 sucrose, 60 NaCl, 3.5 KCl, 6 MgCl2, 0.5 CaCl2, 38 NaHCO3, 1.25 NaH2PO4, 10 HEPES, 25 glucose). Coronal slices (250 µm) were cut (Vibroslice 7000smz, Campden Instruments, UK), incubated in artificial cerebrospinal fluid (aCSF; in mM: 120 NaCl, 3.5 KCl, 1 MgCl2, 2 CaCl2, 30 NaHCO3, 1.25 NaH2PO4, 15 glucose) supplemented with 5 mM HEPES, 1 MgCl2 for 30 min at 35 °C and allowed to recover at room temperature for at least 40 min.
MSN were identified as in20 (link). They were recorded in the current clamp configuration with the bridge mode enabled (EPC-10 amplifier, Patch- and Fitmaster software; HEKA, Lambrecht, Germany). The internal solution contained (in mM): 150 K-gluconate, 10 NaCl, 3 Mg-ATP, 0.5 GTP, 10 HEPES and 0.05 EGTA adjusted to pH = 7.3 and 310 mOsm with the liquid junction potential (15 mV) corrected online. Slices were perfused (2–3 ml/min, aCSF, 21–24 °C) in presence of the GABAAR antagonist gabazine (SR-95531, 10 µM, Sigma). All solutions were continuously oxygenated with 95% O2, 5% CO2 gas.
Glutamatergic excitatory afferents where stimulated intrastriatally with aCSF-filled theta-glass electrodes typically ~ 100–150 µm away from the MSN soma (position of stimulation electrode between MSN and corpus callosum). A bipolar voltage pulse (0.1 ms, ± 5 to ± 30 V) at 0.2 Hz induced subthreshold excitatory postsynaptic potentials (EPSPs; 4–10 mV). Following 10–15 min baseline recording synaptic plasticity was induced by a high frequency protocol (four 100 Hz tetani, 3 s long, separated by 30 s; holding potential − 70 mV). Recordings were rejected if the membrane potential was more positive than − 80 mV or the input resistance changed by more than 30%. We verified that no background long-term potentiation was present as APV ((2R)-amino-5-phosphonovaleric acid), a specific blocker of a subtype of glutamate receptors, did not alter the effect in wildtype mice9 (link).
MSN were identified as in20 (link). They were recorded in the current clamp configuration with the bridge mode enabled (EPC-10 amplifier, Patch- and Fitmaster software; HEKA, Lambrecht, Germany). The internal solution contained (in mM): 150 K-gluconate, 10 NaCl, 3 Mg-ATP, 0.5 GTP, 10 HEPES and 0.05 EGTA adjusted to pH = 7.3 and 310 mOsm with the liquid junction potential (15 mV) corrected online. Slices were perfused (2–3 ml/min, aCSF, 21–24 °C) in presence of the GABAAR antagonist gabazine (SR-95531, 10 µM, Sigma). All solutions were continuously oxygenated with 95% O2, 5% CO2 gas.
Glutamatergic excitatory afferents where stimulated intrastriatally with aCSF-filled theta-glass electrodes typically ~ 100–150 µm away from the MSN soma (position of stimulation electrode between MSN and corpus callosum). A bipolar voltage pulse (0.1 ms, ± 5 to ± 30 V) at 0.2 Hz induced subthreshold excitatory postsynaptic potentials (EPSPs; 4–10 mV). Following 10–15 min baseline recording synaptic plasticity was induced by a high frequency protocol (four 100 Hz tetani, 3 s long, separated by 30 s; holding potential − 70 mV). Recordings were rejected if the membrane potential was more positive than − 80 mV or the input resistance changed by more than 30%. We verified that no background long-term potentiation was present as APV ((2R)-amino-5-phosphonovaleric acid), a specific blocker of a subtype of glutamate receptors, did not alter the effect in wildtype mice9 (link).
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