Fig. 6: DRNGAD2 neurons send monosynaptic inhibitory inputs to PVT neurons.
From: A midbrain GABAergic circuit constrains wakefulness in a mouse model of stress
a AAV-EF1α-DIO-EGFP expression in the DRN. Aq, aqueduct. b Representative images showing the distribution of axon fibers of DRNGAD2 neurons in the PVT. D3V, dorsal third ventricle. c Quantification of relative projection density of DRNGAD2 neurons along rostral to caudal axis of the PVT. n = 4 mice, Kruskal-Wallis one way ANOVA on ranks, H = 20.46, P = 0.001. d Schematic of viral injection for retrograde tracing the presynaptic inputs of PVT glutamatergic neurons. e Representative images showing the ___location of start cells in the PVT. Scale bar in the right panel is 20 μm. f Images showing the RV-dsRed-labeled DRN neurons and their co-localization with GAD2. Arrowheads indicate dsRed and GAD2 co-labeled neurons. Scale bar in the right panels is 20 μm. g Quantification the percentage of GAD2-, Tph2-, or TH-positive DRN neurons projecting to PVT glutamatergic neurons. n = 4 mice, Kruskal-Wallis one way ANOVA on ranks following Student-Newman-Keuls multiple comparisons test, H = 9.881, GAD2 vs Tph2: P = 0.021; GAD2 vs TH: P = 0.005. h Schematic diagram of whole-cell recording in the PVT. AAV-DIO-EF1α-ChR2-mCherry was injected into the DRN of GAD2-Cre mice. Recordings were made from PVT neurons with simultaneous optogenetic stimulation of terminals of DRNGAD2 neurons. i Light-evoked action potentials of DRNGAD2 neurons verify the expression efficacy of ChR2-mCherry. j Light-evoked postsynaptic currents in recorded PVT neurons. The dark lines indicate average traces and gray lines indicate responses of individual cells. n = 10 cells. ACSF artificial cerebrospinal fluid, TTX tetrodotoxin, 4-AP 4-Aminopyridine. k Latency (left) and amplitude (right) of light-evoked postsynaptic currents in ASCF conditions. n = 10 cells. l Summary of the amplitude of light-evoked currents normalized to that in ACSF. n = 10 cells, Friedman repeated measures ANOVA on ranks following Student-Newman-Keuls multiple comparisons test, H = 33.515, ACSF vs TTX: P = 5.4 × 10−5; TTX + 4AP vs TTX + 4AP+ bicuculine: P = 5.4 × 10−5; TTX vs TTX + 4AP: P = 0.9. Norm., normalized. m Bicuculine blocks the 10 Hz and 20 Hz light pulses-induced postsynaptic currents. n = 6 cells. *P < 0.05, ***P < 0.001, n.s., not significant. Data (c, g, k, l) are presented as mean ± SEM.
