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Applicable species: newborn mice P0-P4, adult mice/rats, NHP[12-33,12-39]. Dose range: neonatal P0-P2 1-2×10^10 GC/hemisphere (total 2-4×10^10/mouse); adult mice 1×10^11 vg/side; NHP 4×10^13 vg (bilateral, 2×10^13/ventricular×1 mL) [12-33]. Injection volume: neonates 1-2 μL/side (P0 recommendation ≤1 μL/side); adults 5-10 μL/side; NHP 1 mL/ventricular [12-33]. Needles: newborn 10 μL Hamilton with 32 G needle; adult Kopf stereotaxic + Hamilton 33 G bevel needle; NHP CT/fluoroscopic guided lumbar puncture needle/ventricular catheter [12-33]. Speed: newborn ≤30 nL/s (no exceeding 15 nL/s); adults 1 μL/min[12-33]. Anesthesia: neonatal hypothermic anesthesia on ice for 60 s; adult isoflurane + stereotaxic frame; NHP isoflurane [12-33]. Key points of operation: Percutaneous translucent positioning of neonates (lambda suture to 2/5 of the eye, opening 0.25 mm and inserting needle 2 mm); drilling holes in adults, selecting the dural needle and retaining the needle for 2 minutes; P0 injection of neonates has the widest neuron transduction, and the proportion of astrocytes increases after P3 [12-33]. Coverage: neonates extensively cover the cortex, hippocampus, striatum, brainstem, cerebellum, spinal cord lumbar segment > thoracic segment > cervical segment; in adults only the ependyma + choroid plexus + adjacent brain parenchyma; peak expression is 2-4 weeks [12-33]. Safety: Neonatal injection is too fast (>15 nL/s) or excessive (>2 μL/side) → increased intracranial pressure, cerebral hemorrhage, and 1-week mortality [12-33].