Figure 5.
Optogenetic activation of VPVglut2-LH pathway increases wakefulness
(A) Schematic showing in vivo optogenetic stimulation of VPVglut2-LH projections in Vglut2-Cre mice. AAV-DIO-hSyn-ChR2-mCherry construct was bilaterally expressed in the VP, and two optical fibers that were used for LH light delivery were implanted.
(B) Representative images showing that ChR2-mCherry-positive cell bodies in the VP (b1), and the tip of fiber optic above the LH (b2). Dense axonal terminals of VPVglut2 neurons were found in the LH (b3). Scale bars = 100 μm.
(C) EEG/EMG traces and EEG heatmap showed that optogenetic activation of VPVglut2-LH pathway induced wakefulness in ChR2 mice, but not mCherry control mice.
(D) Latencies of transitions from NREM sleep to wakefulness after optogenetic stimulation at different frequencies. ∗∗p < 0.01, unpaired t-test.
(E) Sleep stage after blue-light stimulation. For each stimulation trial, EEG was analyzed for 6 min with a 2-min baseline, and the light-on time was 20 s at 20 Hz. The percentages of NREM sleep, REM sleep, and wakefulness were analyzed during the short-stimulation experiment.
(F) One-hour optogenetic stimulation of VPVglut2-LH pathway increased the amount of wakefulness but decreased NREM sleep and REM sleep during the inactive period. n = 5 mice, ∗∗p < 0.01 using repeated-measures ANOVA, followed by paired t-test.
(G) Diagram for in vitro optogenetic stimulation of VPVglut2-LH pathway.
(H) Proportions of connected and unconnected GFP+ and GFP- neurons (n = 28 GFP+ neurons, n = 19 GFP- neurons, from 8 mice. Chi-square test; p = 0.1506).
(I and J) Latency (I) and amplitude (J) of light-evoked EPSCs in LH GFP+ and GFP- neurons. (K) Typical example of a biocytin-labeled neuron (blue) that was GFP positive and responsive to light stimulation. Scale bar = 20 μm. (L) Electrophysiological properties of a GFP+ cell in the LH (left). Light-evoked postsynaptic currents were EPSCs, which were blocked by AMPA and NMDA receptor antagonists, NBQX and D-APV (right).
(M) Typical example of a biocytin-labeled neuron (blue) that was GFP negative and responsive to light stimulation. Scale bar = 20 μm.
(N) Electrophysiological properties of a GFP negative cell in the LH (left). Light-evoked postsynaptic currents were EPSCs (right).
(O) Optogenetic activation of VPVglut2-LH projections did not alter locomotion and time spent in the center area in the open field test. n = 8 mice per group, p > 0.05, unpaired t-test.
(P and Q) Optogenetic activation of VPVglut2-LH projections did not alter immobility time in the forced swimming (P) and TS (Q) tests. n = 8 mice per group, p > 0.05, unpaired t-test.