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Neural Substrates of Awakening Probed With Optogenetic Control of Hypocretin Neurons


Neural Substrates of Awakening Probed With Optogenetic Control of Hypocretin Neurons

Antoine R Adamantidis et al. Nature.


The neural underpinnings of sleep involve interactions between sleep-promoting areas such as the anterior hypothalamus, and arousal systems located in the posterior hypothalamus, the basal forebrain and the brainstem. Hypocretin (Hcrt, also known as orexin)-producing neurons in the lateral hypothalamus are important for arousal stability, and loss of Hcrt function has been linked to narcolepsy. However, it is unknown whether electrical activity arising from Hcrt neurons is sufficient to drive awakening from sleep states or is simply correlated with it. Here we directly probed the impact of Hcrt neuron activity on sleep state transitions with in vivo neural photostimulation, genetically targeting channelrhodopsin-2 to Hcrt cells and using an optical fibre to deliver light deep in the brain, directly into the lateral hypothalamus, of freely moving mice. We found that direct, selective, optogenetic photostimulation of Hcrt neurons increased the probability of transition to wakefulness from either slow wave sleep or rapid eye movement sleep. Notably, photostimulation using 5-30 Hz light pulse trains reduced latency to wakefulness, whereas 1 Hz trains did not. This study establishes a causal relationship between frequency-dependent activity of a genetically defined neural cell type and a specific mammalian behaviour central to clinical conditions and neurobehavioural physiology.


Figure 1 |
Figure 1 |. Genetically targeted cell-type-specific optical control of Hcrt neurons using ChR2.
a, Schematic diagram showing lentiviral vectors carrying the genes for ChR2-mCherry or mCherry driven by the 3.1-kb mouse Hcrt promoter,. The lentiviral backbone is derived from pLenti-CaMKIIα-ChR2-mCherry. LTR, long terminal repeats; RRE, Rev responsive element; WPRE, woodchuck post-transcriptional regulatory element. b, Images of Hcrt neurons (green, Hcrt::EGFP) co-expressing ChR2–mCherry protein (red) in the adult mouse lateral hypothalamus (scale bar, 20 μm). Hcrt and mCherry expression was detected by double-fluorescent immunohistochemistry (see Methods). c, Lentiviral Hcrt::ChR2-mCherry expression is highly specific to the Hcrt::EGFP neurons. d, Hcrt::ChR2-mCherry neurons in the lateral hypothalamus can be electrically controlled with light. Voltage-clamp recording of a neuron expressing ChR2–mCherry in acute lateral hypothalamus slice shows inward photocurrent evoked by illumination with blue light. e, Neurons expressing Hcrt::ChR2-mCherry in acute lateral hypothalamus brain slices under current-clamp conditions fire action potentials on illumination with 1 s of continuous blue light. Two sweeps are superimposed. f, Blue-light pulse trains (15 ms per pulse, 20 Hz) evoked reliable firing of action potential trains. Two consecutive sweeps are superimposed, showing temporal precision of evoked action potential trains even in the presence of basal spontaneous activity (spontaneous activity in Hcrt neurons in vitro is expected, as previously reported). Fifteen-millisecond light pulses are indicated by blue bars. g, Light-evoked spike trains are reliable over a range of frequencies. The percentage of action potentials evoked by 20 light pulses at the indicated frequency (15–50 Hz) is shown (n = 6). h, Comparison between the resting membrane potential of ChR2–mCherry/EGFP double-positive (n = 7) and EGFP-only (n = 5) neurons; ChR2–mCherry expression does not significantly alter basal electrical properties of the Hcrt neurons. Error bars, s.e.m.
Figure 2 |
Figure 2 |. Integrated in vivo optical and physiological system for control of the lateral hypothalamus in the setting of behavior alanalysis.
a, Schematic of the behavioural set-up used for in vivo deep-brain photostimulation in mice. Magnification (inset) shows the EEG/EMG connector used for sleep recording and the cannula guide used for lateral hypothalamus light delivery through an optical fibre. b, Schematic of experimental set-up, showing relationship between the optical fibre, brain tissue and attenuating light. r, optical fibre radius; z, tissue depth from fibre end; qdiv, the half-angle of divergence. LH, lateral hypothalamus. c, Normalized light intensity(mW mm−2) as a function of lateral hypothalamus tissue depth z. Values were experimentally determined as previously described by measuring the light intensity after transmission through a given tissue thickness and dividing by the intensity of the light emanating from the optical fibre tip. Tissue of different thickness was prepared in the form of acute brain slices from adult C57BL/6 mice. Error bars indicate one standard deviation from the mean. Sample size: 0.2 mm, n = 8; 0.4mm, n = 6; 1mm, n = 2. Fits were produced using the Kubelka–Munk model of light transmission through diffuse scattering media as previously described. We estimate that by placing the tip of the optical fibre at the upper limit of the lateral hypothalamus, at least 1 mW mm−2 of light, which is sufficient to activate ChR2, reaches the entire Hcrt field (0.75 mm (medial-lateral) × 0.75mm (dorso-ventral) × 1mm (rostro-caudal)).
Figure 3 |
Figure 3 |. In vivo photostimulation of Hcrt neurons drives sleep-to-wake transitions.
a, Representative EEG/EMG recordings showing awakenings after a single bout of photostimulation (15ms, 20Hz, 10s) in Hcrt::ChR2-mCherry and Hcrt::mCherry (control) animals during SWS (upper traces) and REM sleep (lower traces). Light stimulations are represented by horizontal bluebars. Awakening events are indicated by vertical black arrows according to the described criteria (see Methods). Panels (right) show representative relative cortical EEG power spectra corresponding to the SWS and REM sleep-to-wake transitions highlighted with boxes on the EEG traces (left). b, c, Latencies of wake transitions during SWS (b) and REM sleep (c) of Hcrt::ChR2-mCherry transduced animals (n = 7) and their controls (n = 6) after a single photostimulation bout at different frequencies (15-ms light pulses, at 1–30 Hz, during 10 s; ON, continuous light illumination of 10 s). Data analysis is based on an average of 15 and 5 stimulations per frequency and per mouse during SWS and REM sleep, respectively. Paired comparison between control conditions for SWS and REM sleep-to-wake transitions did not reveal any significant differences (P>0.05, two-tailed Student’s t-test). Latencies are represented as mean ± s.e.m. Asterisk, P<0.05; double asterisk, P<0.001; triple asterisk, P<0.0001 using a two-tailed Student’s t-test between mCherry control (red) and ChR2 animals (blue) for each frequency. d, Cumulative probability distribution of latencies from SWS to wakefulness after light stimulation (mCherry control, red curve; ChR2, blue curve). e, Cumulative probability distribution of latencies from REM sleep to wakefulness after light stimulation (mCherry control, red curve; ChR2, blue curve).
Figure 4 |
Figure 4 |. Behavioural transitions induced by photostimulation are mediated by Hcrt.
a, b, Effect of the Hcrt receptor 1 antagonist SB334867 (ref. 24) on latencies of light-induced wake events during SWS (a) and REM sleep (b) in Hcrt::ChR2-mCherry transduced animals (n = 3) and their controls (n = 3) after single bouts of 20 Hz photostimulation (15 ms, 10 s). Data analysis is based on an average of ten and three stimulations per frequency and per mouse for SWS and REM sleep, respectively. At the doses tested here, SB334867 had no effect on the latency of SWS and REM sleep-to-wake transitions after photostimulation in Hcrt::mCherry control animals. Latencies are represented as mean ± s.e.m. Asterisk, P<0.05 using a paired Student’s t-test between saline and drug conditions. c, Latencies of SWS sleep-to-wake transitions of Hcrt knockout (KO) animals compared with our control data; three independent mice were transduced with Hcrt::ChR2-mCherry lentiviruses and received a single photostimulation bout spanning the relevant frequencies (1–30 Hz, 15-ms light pulses, during 10 s). Hcrt knockout data analysis is based on an average of 20 stimulations at different frequencies (1–30 Hz) per mouse during SWS. Latencies are represented as pooled mean ± s.e.m. values. Triple asterisk, P<0.0001, indicates the significant difference between control mCherry and ChR2 animals using a two-tailed Student’s t-test. Asterisk, P<0.05, indicates significant difference using a two-tailed Student’s t-test between ChR2 and Hcrt knockout ChR2 animals. No significant differences in latencies to wakefulness were found between control mCherry and Hcrt knockout ChR2 animals.

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