Tuning arousal with optogenetic modulation of locus coeruleus neurons

Abstract

Neural activity in the noradrenergic locus coeruleus correlates with periods of wakefulness and arousal. However, it is unclear whether tonic or phasic activity in these neurons is necessary or sufficient to induce transitions between behavioral states and to promote long-term arousal. Using optogenetic tools in mice, we found that there is a frequency-dependent, causal relationship among locus coeruleus firing, cortical activity, sleep-to-wake transitions and general locomotor arousal. We also found that sustained, high-frequency stimulation of the locus coeruleus at frequencies of 5 Hz and above caused reversible behavioral arrests. These results suggest that the locus coeruleus is finely tuned to regulate organismal arousal and that bursts of noradrenergic overexcitation cause behavioral attacks that resemble those seen in people with neuropsychiatric disorders.

Figure 1

Specific and efficient functional expression of optogenetic transgenes in locus coeruleus neurons. (a) Representative photomicrographs depicting tyrosine hydroxylase (TH) immunoreactivity (left column, red), viral eYFP expression (center column, green), and merged images (right column) from an animal unilaterally injected with EF1α::eYFP rAAV virus into the left locus coeruleus region. Top row shows global expression in a coronal section counterstained with DAPI (scale bar, 100μm); middle row shows expression within the full locus coeruleus (scale bar, 25 μm); bottom row shows individual neurons (scale bar, 5 μm). (b) Quantification of co-expression of eYFP and TH immunofluorescence from EF1α::eYFP transduced mice (n=4) in 30 μm brain sections from the rostral-to-caudal ends of the locus coeruleus (anteroposterior, –5.20 to –5.80). Cell counts are represented as mean +/– s.d. Inset represents the statistics of the total co-expression (c) Voltage clamp recording of a neuron expressing eNpHR-eYFP in brainstem slice showing outward current in response to yellow light. (d) Voltage clamp recording of a neuron expressing ChR2-eYFP in brainstem slice showing inward current in response to blue light. (e) Action potential trains recorded under current clamp conditions from a neuron expressing eNpHR-eYFP in brainstem slice for 5 s (top) or 1 min (bottom). (f) Blue-light pulse trains (10 ms per pulse) evoked action potential trains in neurons expressing ChR2-eYFP at various frequencies. (g) Efficiency of action potential trains evoked by blue light pulses in ChR2-eYFP expressing neurons. Data represent mean probability +/– s.e.m. from n=6 neurons.

Figure 2

Photoinhibition of locus coeruleus neurons causes a reduction in the duration of wakefulness. (a) The percentage of time spent in wake, NREM, and REM sleep during 1 h photoinhibition in the active (dark) period. Data represent the mean +/– s.e.m. of 6 separate 1 h sessions, n=6 animals throughout. *P<0.05, two-tailed Student's t-test between transduced animals. (b) The duration of individual wake, NREM, and REM episodes during 1 h photoinhibition during the active period. *P<0.05, two-tailed Student's t-test between transduced animals. (c) The percentage of sleep state transitions relative to baseline levels during 1 h photoinhibition during the active period. **P<0.001, two-tailed Student's t-test between transduced animals. (d) The duration of individual wake episodes in baseline versus photoinhibition conditions (20 episodes per mouse, n=6 mice). *P<0.05, two-way ANOVA between stimulation condition and viral transduction followed by Tukey posthoc test. (e) Relative EEG power of wakefulness between 80-120 s after wake-onset in baseline (top) and photoinhibition (bottom) conditions. Data represent the mean +/– s.e.m. relative power of 0.5 Hz binned frequencies (20 episodes per mouse, n=6 mice).

Figure 3

Photostimulation of locus coeruleus neurons causes immediate sleep-to-wake transitions. (a,d) Representative traces of EEG/EMG recordings showing an immediate (a) NREM or (d) REM sleep-to-wake transition following acute photostimulation (10 ms pulses at 5 Hz for 5 s) of locus coeruleus neurons during the inactive period in a mouse transduced with ChR2-eYFP (bottom) but no awakening in a mouse transduced with eYFP alone (top). Arrow indicates onset of sleep-to-wake transition. (b,e) Cortical EEG traces from ChR2-eYFP mice 5 s prior to the onset of stimulation (black) and 5 s during stimulation (grey). Quantification is based on the average of (b) 15 or (e) 8 stimulations per mouse, n=6 mice. (c,f) Heat maps showing the effects of photostimulation on (c) NREM or (f) REM sleep-to-wake transitions in eYFP (n=6) or ChR2-eYFP (n=6) transduced animals. Each square represents the mean probability of a sleep-to-wake transition within 10 s of the onset of stimulation. Data analysis is based on the average of (c) 15 or (f) 8 stimulations per condition per mouse.

Figure 4

Long-term tonic versus phasic stimulation of the locus coeruleus causes differential promotion of arousal. Tonic (consistent 10 ms pulses at 3 Hz) and phasic (10 ms pulses at 10 Hz for 500 ms occurring every 20 s) stimulation protocols are consistent throughout. (a,b) The effect of (a) tonic or (b) phasic photostimulation for 1 h on sleep architecture in ChR2-eYFP (n=5) or eYFP (n=5) transduced mice. Data represent the mean +/- s.e.m. percentage time over four trials spent in wake, NREM sleep, or REM sleep in baseline conditions or during photostimulation. **P<0.001, ***P<0.0001, two-way ANOVA between stimulation condition and viral transduction, followed by Tukey posthoc test. (c,d) Sleep recordings in the hour following (c) tonic or (d) phasic photostimulation. *P<0.05, ** P<0.001, Student's t-test. (e,f) Representative trace of locomotor activity in an eYFP and ChR2-eYFP transduced mouse during (e) tonic or (f) phasic photostimulation for 1 h during a 10 min wake period. Quantification at right shows the mean +/– s.e.m. distance traveled by eYFP (n=5) or ChR2-eYFP (n=5) transduced animals over the 1 h of photostimulation after 5 sessions of stimulation per mouse. *P<0.05, **P<0.001, two-way ANOVA between stimulation condition and viral transduction, followed by Tukey posthoc test. (g,h) The effect of (g) tonic or (h) phasic photostimulation for 5 h on sleep architecture in ChR2-eYFP (n=5) or eYFP (n=5) transduced mice. *P<0.05, two-way ANOVA between stimulation condition and viral transduction, followed by Tukey posthoc test.

Figure 5

High-frequency photostimulation of the locus coeruleus causes reversible behavioral arrests. (a) Sequence of events in a behavioral arrest. (b) Probability of behavioral arrests depends on photostimulation frequency. Data represent mean +/– s.e.m. of ChR2-eYFP transduced animals (n=4 animals, 10 trials per frequency per mouse). (c) The duration of latencies to arrest (time from light onset until behavioral arrest) and durations of arrest (time between the onset and offset of behavioral arrest) in ChR2-eYFP stimulated animals. Data represent the mean +/– s.e.m. of 20 trials per animal, n=8 animals. *P<0.05, **P<0.001 between frequencies, ANOVA followed by Tukey posthoc test. (d) Representative EEG/EMG trace of a behavioral arrest following 10 Hz photostimulation. Arrows represent the onset and offset of immobility. (e) Relative EEG power of the first 10 s of behavioral arrests across multiple animals. Data represent the mean +/– s.e.m. relative power of 0.5 Hz binned frequencies (20 episodes per mouse, n=6 mice). (f) Measurement of extracellular norepinephrine content in prefrontal cortex during 10 Hz stimulation. Data represent the mean +/– s.e.m. of 3 trials per animal, n=4 animals. **P<0.001, two-way ANOVA between timepoint and virally-transduced animal followed by Bonferroni post-hoc test. (g) The duration of latencies to arrest and durations of arrest in ChR2-eYFP stimulated animals upon administration of the norepinephrine reuptake inhibitors atomoxetine or reboxetine. Data represent the mean +/– s.e.m. of 10 trials per animal, n=4 animals. Increased darkness of bars represents increasing pharmacological dose. *P<0.05, Student's t-test between saline and drug-injected animals.