Figure 5. Y1^Cre Neurons Receive VGAT⁺ and NPY⁺ Synaptic Contacts from NPY::Cre Ins.

(A and B) Examples of GFP-labeled cells from the lumbar spinal cord of a P60 Y1::EGFP; NPY::Cre transgenic mouse. Presynaptic contacts (red) from NPY::Cre INs onto Y1/EGFP⁺ cells were visualized with a Cre-dependent AAV2/1-hSyn-DIO-SypHTom virus. Putative synaptic boutons marked by VGAT (blue; A) and NPY (blue; B) immunoreactivity are indicated by arrowheads.

(C) Examples of synaptic puncta labeled with antibodies against NPY (green) and gephyrin (blue) on a Y1-tdTomato⁺ cell body in the lumbar spinal cord of a P42 Y1^Cre; Ai14^lsl-tdTom mouse (arrowheads).

(D) Schematic illustrating experimental conditions used to assess synaptic connectivity between NPY::Cre INs and Y1^Cre neurons.

(E) ReaChR-mediated activation of NPY::Cre IN generated monosynaptic outward currents in lamina IIi Y1-EGFP⁺ neurons in P14–21 Y1::EGFP; NPY::Cre; Lbx1^FlpO; R26^ds-ReaChR spinal cord slices in the presence of kynurenic acid (KA; 1.5 mM) at a holding potential of —30 mV. These currents were abolished following application of 1 μM strychnine and 60 μM picrotoxin (n = 7 cells from 5 mice). ***p < 0.001. The statistical difference was determined by the two-tailed, paired t test.

(F) Traces recorded from a Y1-EGFP⁺ neuron showing a monosynaptic inhibitory current elicited by NPY::Cre activation (purple trace) and no response following bath application of strychnine and picrotoxin (black trace).

Scale bars: 5 μm.