Excitatory Postsynaptic Potentials

AC-coupled recordings were made of the cord dorsum potentials for incoming afferent volleys and the electroneurograms (ENGs: the phrenic nerve; the external intercostal nerve in one cat; when appropriate, see below, the dissected hindlimb nerves as listed above). Intracellular recordings were DC-coupled, but a high gain output channel high pass filtered at 1 Hz was also included. Intracellular recordings were made from antidromically identified motoneurons, using an Axoclamp 2B amplifier (Axon Instruments) in either standard bridge mode, or in discontinuous current clamp (DCC) mode. Microelectrodes (typical impedance 5 MΩ) were filled with 2 M potassium acetate, and contained the local anesthetic derivative QX-314 (50 mm) to block actions potentials, so as to facilitate the study of the size of EPSPs at different membrane potentials. Note that in several of the records illustrated, a few action potentials survived, showing the QX-314 block to be incomplete at those times. DCC mode was used to allow for more accurate measurements of membrane potential despite changes in electrode resistance with injected current. The DCC cycling rate was typically around 3 kHz with optimal capacitance compensation. Most often slow depolarizing and hyperpolarizing ramps of currents were used (triangular current ramps), but some step changes of constant current levels were also employed. During many of the motoneuron recordings we also recorded efferent discharges from the hindlimb nerves via the same electrodes as used for antidromic identification purposes. This was rarely done in the early experiments, where the focus was on the voltage-dependent amplification of synaptic potentials, but once it was realized that a locomotor drive was sometimes present in the recordings, then these electrodes were switched to their recording mode as soon as antidromic identification had been confirmed. The ENG recordings were done with custom built amplifiers and analog filtering (1–10 kHz) and digitized at a rate of 10 kHz. Full wave rectification and additional filtering was done during analysis so that the onset and the offset of ENG bursts in each nerve were identified by visual inspection of ENG levels crossing a baseline defined by no activity periods. These onset and offset points were used during cycle-based averaging of ENG activity. The data were collected and analyzed with a Canadian software-based QNX-system, developed by the Winnipeg Spinal Cord Research Center to run under a real-time Unix personal computer, usually using separate runs of 200 s duration.

Using liquid nitrogen, fresh young leaves were ground to powder, and genomic DNA was extracted using the cetyltrimethylammonium bromide method (Fütterer et al., 1995 ). The forward primer for HMGR, FPS, DBR2, and HD1 was chosen from the EPSP marker gene. Because AaORA was driven by the CYP71AV1 promoter, the forward primer was designed within its sequence, and the reverse primer was selected from the CDS of AaORA.Table 1 shows a list of primers. The PCR reaction was carried out in a 20 μL tube using the LA TaqR Kit (Takara). DNA denaturation was set at 94° C for 3 minutes, followed by 30 cycles of 94° C for 30 seconds 57° C for 30 seconds, and 60 seconds at 72°C, and a final extension of five minutes at 72° C. For product determination, 1.0% agarose gel electrophoresis was performed.

We had previously amplified the AaHMGR (AF142473), AaFPS (AF112881), and AaDBR2 (PWA95605.1) Open Reading Frames (Wang et al., 2011 ; Shen et al., 2018 (link)). PCR products were separated on 1% Agarose gel electrophoresis and purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, USA). Purified PCR products were then cloned into the pJET2.1 vector (Promega), transformed into DH5α high-efficiency competent cells, and grown on LB medium supplemented with Carbenicillin (Cb). Three positive colonies were subjected to colony PCR and submitted for sequence analysis (Sangon sequencing company, Shanghai). For sequence alignment, the DNAMAN software (version 5.6) was utilized. EPSPS was substituted for the hygromycin gene in the XhoI restriction site of pCAMBIA1305. The vector pCAMBIA1305.1-EPSPS-HMGR was created by inserting 35S-HMGR-nos into the HindIII and EcoRI restriction sites of pCAMBIA1305-EPSPS using 5× In-fusion HD Enzyme Primix (Clontech, Dalian, China). The FPS-nos-35S-DBR2 fragment was then inserted into the NcoI and BstEII restriction sites of pCAMBIA1305.1-EPSPS-HMGR (Figure 2A).
The full-length CDS for ORA (JQ797708) was amplified and digested with BamHI and SacI. The full-length ORF was then cloned into the BamHI and SacI sites of the pCAMBIA2300+ vector under the CYP71AV1 promoter to create pCAMBIA2300-proCYP71AV1::AaORA::NOS (Figure 2B).
The ORF of AaHD1 (KU744599), was amplified and cloned into the PHB vector (Figure 2C). After validating the transformation, the positive colonies were transferred into Agrobacterium tumefaciens strain EHA105 and inoculated into A. annua.

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 in^{20 (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 GABA_AR 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 mice^{9 (link)}.