Nanofil needle

Stereotaxic injections were performed in heterozygous DAT-cre⁺ and wild-type (WT) mice of either sex at 8 to 11 weeks of age^{96 (link)}. An Adeno-Associated Virus (AAV) containing an inverted sequence of EGFP (AAV1 pCAG-FLEX-EGFP-WPRE, University of Pensylvania)^{22 (link)} or mNeongreen (AAV1 pCAG-FLEX-mNeongreen-WPRE)^{97 (link)} coding gene flanked by loxP-sites was injected into DAT-cre⁺ mice (Fig. 1 Panel 1). Saline injected littermates were used as autofluorescence controls. The stereotaxic injections were performed in Isoflurane-anesthetized mice using a 10 μl NanoFil syringe and a 35 G beveled NanoFil needle (World Precision Instruments). Injection coordinates for the Substantia Nigra pars compacta (SNc) were anterior/posterior (A/P)− 3.6 mm, lateral (L)± 1.3 mm, dorsal/ventral (D/V)− 4.2 mm. Injection coordinates for the Ventral Tegmental (VTA) were with a 12° angle A/P− 2.9 mm, L± 1.6 mm; D/V− 4.6 mm. A/P and L coordinates are given with respect to the bregma, whereas D/V coordinates are given with respect to the brain surface (Fig. 1 Panel 1). The animals were euthanized after 28–35 days at the maximal viral EGFP/mNeongreen expression. For each fluorescence-activated synaptosome sorting (FASS) experiment, three to six DAT-cre⁺ mice and one WT mouse were used independently of their sex.

The adeno-associated virus (AAV) rAV-hSyn-mCherry-5’miR-30a-shRNA-3’miR-30a-WPREs (Shumi Technology, Wuhan, China) was used for TLR3 neural-specific knockdown. The transfection reagent (Engreen, Beijing, China) was used to dilute the primary solution of AAV-TLR3 (AAV⁺) and AAV-control (AAV⁻). A stereotaxic apparatus (RWD Life Science, Shenzhen, China) was used for the injection of the hippocampus. AAV solution (0.2 µl) was slowly injected into the hippocampus by a NanoFil needle, a NanoFil syringe and a MicroSyringe Pump Controller (World Precision Instruments, Sarasota, FL, USA). The coordinates are antero-posterior = −2.00 mm, medio-lateral = ±1.8 mm and dorso-ventral= −1.7 mm. Bilateral intrahippocampal injection of AAV-TLR3 was the same as AAV⁻ control.

Twelve- to 14-week-old male mice were used for the chronic monitoring of the EEG/EMG signals. The mice were anesthetized with 0.2% tribromoethanol (20 ml/kg, intraperitoneal injection; Sigma-Aldrich, St. Louis, MO, USA) and placed on a stereotaxic frame (David Kopf Instruments, Tujunga, CA, USA). Cre-inducible vectors containing adeno-associated virus (AAV9.hsyn.HI.eGFP-Cre.WPRE.SV40) were bilaterally injected into the ventrobasal complex region of the thalamus (anteroposterior, −1.82 mm; lateral, ±1.75 mm; ventral, −3.5 and −3.0 mm, 1 μℓ in each site) using a NanoFil needle (NF33BL; World Precision Instruments, Sarasota, FL, USA) with a Hamilton syringe and pump (SP100IZ; World Precision Instruments, USA). AAV9.hsyn.eGFP.WPRE.bGH was used as control. For the EEG recordings, epidural electrodes with a 6-pin surface mount connector with EMG leads (8231-SM; Pinnacle Technology, USA) were implanted in the frontal and parietal lobes. For the EMG signal recordings, EMG leads were inserted into the nuchal musculature and a grounding electrode was implanted in the occipital region of the skull.