Tuning arousal with optogenetic modulation of locus coeruleus neurons.

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.

INTRODUCTION

The locus coeruleus is a noradrenergic brainstem structure that is thought to play a major role in promoting arousal[1-5]. Locus coeruleus neurons fire tonically from 1–3 Hz during awake states, decrease firing during NREM sleep, and are virtually silent during REM sleep[6-8]. The locus coeruleus also fires phasically in short bursts of 8–10 Hz during the presentation of salient stimuli which prolong wake states[7,9]. Importantly, alterations in discharge rate precede changes in sleep-to-wake transitions[6,8,9]. However, whether this activity is causal or submissive remains unresolved, and the specific contributions of tonic versus phasic activity in modulating arousal states is unknown.

Experimentally determining a causal role for the locus coeruleus in promoting and maintaining arousal has remained elusive using traditional pharmacological and electrical techniques due to its small size, unique morphology, and proximity to neighboring brain structures[1,2]. Physical lesions of the locus coeruleus do not elicit consistent changes in cortical electroencephalography (EEG) or behavioral indices of arousal[10-12]. Genetic ablation of dopamine beta-hydroxylase, an enzyme necessary for norepinephrine synthesis, also does not disrupt sleep/wake states[13]. However, central injections of pharmacological antagonists for noradrenergic receptors[14] or agonists for inhibitory autoreceptors[15] cause substantial sedative effects. Alternatively, central administration of norepinephrine directly into the ventricles or forebrain promotes wakefulness[16,17]. Stimulation of locus coeruleus neurons using local microinjections of the cholinergic agonist bethanechol produces rapid activation of forebrain EEG in halothane-anesthetized rats[18]. Taken together, these results imply a role for the locus coeruleus in promoting arousal, but clearly new tools are necessary to selectively manipulate locus coeruleus discharge activity in freely moving, behaving animals at timescales relevant to natural sleep/wake events.

The recent development of optogenetic tools[19,20] provides a valuable opportunity to inhibit or stimulate activity in genetically-targeted neural populations with high spatial and temporal precision[21,22]. Therefore, to determine a conclusive, causal role for the locus coeruleus-norepinephrine system in promoting and maintaining wakefulness, we studied the effects of inhibiting locus coeruleus neurons with halorhodopsin (eNpHR)[23,24], a yellow-light sensitive chloride pump, or stimulating locus coeruleus neurons with channelrhodopsin-2 (ChR2)[25], a blue-light sensitive cation channel. We found that the locus coeruleus was necessary for maintenance of wake episodes but inhibition did not increase the duration of sleep episodes. Stimulation caused immediate sleep-to-wake transitions, extending the duration of wakefulness in a manner consistent with sleep deprivation. The probability of wakefulness during stimulation was finely tuned to both light pulse frequency and the duration of stimulation, indicating that the sleep/wake state of an animal is highly sensitive to activity in the locus coeruleus at a scale of single action potentials. Surprisingly, we also found that sustained (10–15 s) high frequency (>5 Hz) stimulation caused reversible behavioral arrests previously unknown to occur with locus coeruleus stimulation, suggesting a potential mechanism for the behavioral arrests present in some neuropsychiatric disorders.

RESULTS

Genetic targeting of locus coeruleus neurons

We genetically targeted locus coeruleus neurons by stereotaxically injecting a Cre-recombinase-dependent adeno-associated virus (rAAV)[26,27] into knockin mice selectively expressing Cre in tyrosine hydroxylase neurons[28] (). We validated the specificity and efficiency of transgene expression by unilaterally injecting virus into the locus coeruleus region[29] and comparing viral eYFP expression with tyrosine hydroxylase immunofluorescence. eYFP fluorescence was detected throughout the entire locus coeruleus but not in neighboring noradrenergic or dopaminergic regions ( and ). Out of 3463 tyrosine hydroxylase expressing neurons (n=4 mice), 98.1 +/– 1.9% co-expressed eYFP (). Conversely, 97.9 +/– 2.9% of eYFP cells co-expressed tyrosine hydroxylase (), demonstrating the specificity of viral targeting of the locus coeruleus.

To test the functional expression of optogenetic transgenes in locus coeruleus neurons, we recorded from eYFP-positive neurons in acute brainstem slices using the whole-cell patch clamp technique. In voltage-clamp mode, we found that eNpHR-transduced neurons exhibited rapid outward photocurrents upon illumination with yellow light (), with a peak level of 435.5 +/– 44.2 pA and a steady-state level of 302.2 +/– 65.8 pA (mean +/– s.e.m.; n=5 cells). Alternatively, blue light photostimulation of ChR2-transduced neurons caused an inward steady state current of 321 +/– 50 pA (mean +/– s.e.m.; n=6 cells) (). In current-clamp experiments, eNpHR-transduced neurons exhibited yellow light-evoked hyperpolarization, completely blocking endogenous spontaneous action potentials (). In locus coeruleus neurons transduced with ChR2, 10 ms blue-light pulses caused action potentials from 1–30 Hz (). The efficiency of ChR2-mediated spikes decreased with increasing frequency, with 100% efficiency at 20 Hz and below (). Taken together, these in vitro results demonstrate that rAAV-mediated expression of eNpHR or ChR2 is sufficient to inhibit or stimulate action potentials in locus coeruleus neurons, respectively.

To modulate locus coeruleus neural activity in vivo, we bilaterally injected rAAV into each locus coeruleus region (), and implanted a bilateral cannula for subsequent light delivery. Electroencephalographic (EEG) and electromyographic (EMG) electrodes were placed in the skull and neck musculature, respectively, for sleep/wake analysis (). Initial sleep recordings demonstrated that there was no difference in baseline sleep architecture between non-virally and virally transduced animals (n=4 animals per condition) ().

To ensure the correct placement of cannulae and fiber optic cables, we applied long-term photostimulation (10 ms pulses at 3 Hz for 1 h) to the left locus coeruleus in ChR2-eYFP transduced mice or eYFP control mice (n=4 animals per condition) during the inactive (light) period and detected the presence of c-Fos, an indirect marker of neural activity (). We found a significant increase in the number of tyrosine hydroxylase-positive cells that also expressed c-Fos between eYFP animals (17.2 +/–7.2%) and ChR2-eYFP animals (64.7 +/– 13.6%). Furthermore, c-Fos expression was found throughout the entire anteroposterior length of the locus coeruleus (), demonstrating the accuracy of our in vivo light delivery paradigm in stimulating the global population of locus coeruleus neurons.

To validate eNpHR-mediated inhibition of the locus coeruleus in vivo, we tested the effect of photoinhibition on norepinephrine content in prefrontal cortex, a region that only receives noradrenergic projections from the locus coeruleus. We delivered constant yellow light to the locus coeruleus in freely moving animals transduced with eNpHR-eYFP or eYFP (n=4 mice per condition) during the active (dark) period for 10 min while simultaneously using microdialysis to sample extracellular fluid in the prefrontal cortex. We found that 10 min of continuous photoinhibition significantly decreased cortical norepinephrine concentration in eNpHR-eYFP but not eYFP control animals (), demonstrating the utility of eNpHR-mediated photoinhibition in vivo.

Inhibition of the locus coeruleus reduces wakefulness

To determine the necessity of locus coeruleus neural activity in promoting wakefulness, we first tested the effect of long-term photoinhibition of the locus coeruleus (constant yellow light for 1 h) on sleep-wake architecture during both the inactive and active periods. We found no significant difference between eNpHR-eYFP and eYFP control animals (n=6 animals per condition) in the total duration of wake/sleep states during the inactive period (). However, we found a significant decrease in the amount of wakefulness and significant increase in the amount of NREM sleep during the active period (). During the active period, duration of individual wake episodes significantly decreased, but there was no significant change in the duration of NREM or REM episodes (). We also found a significant increase in the number of wake-to-NREM transitions relative to baseline (no photoinhibition), but not an increase in any other sleep-state transitions (), suggesting that the locus coeruleus is necessary for maintaining the normal duration of wake episodes.

To study the effects of locus coeruleus photoinhibition specifically during wakefulness, we restricted photoinhibition only to epochs when the animal was awake based on real-time, online EEG analysis. We found a significant reduction in wake episode duration in eNpHR-eYFP animals (n=6 animals) between baseline (no inhibition) and photoinhibition conditions (), but no significant reduction of wake duration in photoinhibited eYFP control animals. Upon examination of the power spectrum of the cortical EEG towards the end of wake episodes (80–120 s from the onset of wakefulness in wake episodes lasting at least 120 s), we found no difference in the relative slow wave activity (SWA, 0.5–4 Hz), a hallmark of sleep pressure, between eNpHR-eYFP and control animals in baseline (no inhibition) conditions (, top). However, during photoinhibition, there was a statistically significant increase in SWA between eYFP and eNpHR-eYFP mice (P<0.05, Student's t-test between transduced mice), demonstrating an increase in the propensity of eNpHR-eYFP animals to enter NREM sleep (, bottom). Taken together, these results suggest that the locus coeruleus is necessary for maintaining normal durations of wakefulness during the active period.

Stimulation of the locus coeruleus causes wake transitions

We next investigated a causal role for locus coeruleus neurons in promoting wakefulness by testing the effect of photostimulation on sleep-to-wake transitions during the inactive period. We initially tested the effect of 5 Hz blue light stimulation for 5 s (10 ms pulses) during NREM sleep, always beginning stimulation trials 15 s after the onset of NREM sleep. Stimulation at these parameters reliably produced immediate sleep-to-wake transitions within 5 s of the onset of stimulation in ChR2-eYFP animals but not eYFP control animals (n=6 animals per condition) ( and ). In ChR2-eYFP animals, photostimulation produced immediate changes in the cortical EEG with a significant decrease in SWA (P<0.001 using a two-tailed Student's t-test between pre-stimulation and stimulation conditions) (). To precisely determine the effects of various photostimulation frequencies and durations on sleep-to-wake transitions, we stimulated eYFP and ChR2-eYFP animals at frequencies of 1–10 Hz and durations of 1–10 s (all light pulses 10 ms in duration), measuring the probability of a NREM-to-wake transition within 10 s of the onset of stimulation. We found that the probability of a NREM-to-wake transition increased with stimulation frequency and duration in ChR2-eYFP animals but not control animals ().

We then repeated the experiments above but studied the effect of photostimulation on REM sleep-to-wake transitions. Photostimulation at 5 Hz for 5 s (10 ms pulses) in ChR2-eYFP animals also caused immediate REM sleep-to-wake transitions (), with immediate changes in the cortical EEG and a significant decrease in the theta activity (4–9 Hz) that characterizes REM sleep (P<0.001 using a two-tailed Student's t-test between pre-stimulation and stimulation conditions) (). As in NREM sleep, increasing the frequency and duration of stimulation increased the probability of sleep-to-wake transitions ().

The relationship between the duration and frequency of stimulation necessary to cause a 100% probability of sleep-to-wake transitions was linear and inversely proportional during both NREM ( and ) and REM sleep ( and ). We also explored the number of photostimulation pulses required for a sleep-to-wake transition. As the frequency of stimulation increased, the number of pulses required for a 100% probability of awakening decreased for both NREM () and REM (). Interestingly, as the duration of stimulation increased, the number of pulses required for a 100% probability of awakening increased for both NREM () and REM sleep ().

To determine if locus coeruleus-mediated sleep-to-wake transitions depend on norepinephrine release, we repeated photostimulation experiments (10 ms pulses at 5 Hz for 5 s) in ChR2-eYFP animals (n=4) 30 min after administration of the adrenergic α2 receptor agonist clonidine or α1 receptor antagonist prazosin, compounds that both result in suppressing NE transmission. In saline-injected ChR2 animals, photostimulation always caused sleep-to-wake transitions (probability=1.00). We found that administration of both clonidine and prazosin reduced the probability of NREM and REM sleep-to-wake transitions in a dose-dependent manner (). These results demonstrate that locus coeruleus-mediated sleep-to-wake transitions depend, at least in part, on normal NE transmission.

Long-term stimulation of locus coeruleus neurons

To determine the effects of long-term stimulation on arousal, we photostimulated locus coeruleus neurons for one hour during the inactive period and measured changes in sleep/wake duration and general locomotor activity. Previous physiological recordings of the locus coeruleus demonstrate that neurons fire tonically from 1–3 Hz during wakefulness[6-8], and also phasically in short bursts (500 ms) of 8-10 Hz during the presentation of salient stimuli which prolong wake states[7,9]. Therefore, we decided to study the effects of long-term stimulation using both tonic and phasic stimulation paradigms to see if there were differential effects on arousal. Tonic stimulation consisted of constant 10 ms light pulses at 3 Hz while phasic stimulation consisted of 10 ms pulses at 10 Hz for 500 ms (5 light pulses) every 20 s.

We found that both tonic and phasic stimulation paradigms increased the total amount of wakefulness and decreased the total amount of NREM sleep in ChR2-eYFP compared with eYFP animals (n=5 animals per condition) (). The reduction in sleep caused by photostimulation of locus coeruleus neurons at tonic and phasic frequencies can be considered a model of sleep deprivation, as the following hour (without photostimulation) showed hallmarks of sleep pressure including a rebound of NREM sleep and a significant increase in slow wave activity (SWA) in the cortical EEG (P<0.05, Student's t-test between baseline and 1 h post-stimulation conditions) (). Interestingly, we found that tonic versus phasic stimulation of locus coeruleus neurons caused differential effects on locomotor activity: tonic stimulation caused a significant increase in locomotor activity over the hour of stimulation () while phasic stimulation caused a significant decrease (). Finally, we wondered if tonic or phasic stimulation would increase the total duration of wakefulness over longer time periods of 5 hours. We found that, unlike 1 h of stimulation, tonic stimulation at 3 Hz for 5 h did not result in a significant difference in the total duration of wakefulness or NREM sleep states between ChR2-eYFP compared with eYFP animals (n=5 animals per condition) (). Alternatively, phasic stimulation for 5 h resulted in a significant increase in the total amount of wakefulness and decease in the total amount of NREM sleep ().

Taken together, these results indicate that long-term, tonic stimulation of locus coeruleus neurons over 1 h causes an increase in wakefulness and general locomotor activity, but the effects of stimulation decline over a 5 h period. Long-term, phasic stimulation of locus coeruleus neurons causes an increase in wakefulness over both 1 and 5 h periods but with a decrease in locomotor activity.

High-frequency stimulation of locus coeruleus neurons

Surprisingly, we found that sustained photostimulation of the locus coeruleus at frequencies 5 Hz and above caused reversible behavioral arrests ( and ). At the onset of stimulation, mice began a 5–20 s period of increased locomotor behavior leading to eventual immobility. After the laser diode was switched off, mice remained immobile with their eyes open for 15–20 s and then fully recovered movement. During the behavioral arrest, mice were unresponsive to tail and toe pinch. Only stimulation frequencies of 5 Hz and above produced behavioral arrests, with a 100% probability at 15 Hz and above (). These arrests were caused by both unilateral and bilateral photostimulation; unilateral stimulation did not produce any obvious unilateral motor effects such as turning or direction-bias. Increasing stimulation frequencies caused decreases in the latency to arrest (time from light onset to immobility), however, the stimulation frequency that caused the arrest had no effect on the duration of arrest (time from light offset to recovery of movement) ().

We hypothesized that high-frequency stimulation of the locus coeruleus caused behavioral arrests due to seizure or a severe cardiovascular response. However, we detected no signs of spike discharge in the cortical EEG characteristic of seizure activity (). Instead, theta rhythm activity was prominent in the EEG across multiple animals (n=6 mice) throughout most of the behavioral arrest episodes (). We also found no difference in heart rate or systolic blood pressure between baseline conditions and during behavioral arrests (n=4 mice) ().

We also investigated the effect of high-frequency stimulation on NE release. Using in vivo microdialysis to sample extracellular fluid from the prefrontal cortex, we found that extracellular NE concentration substantially decreased during 10 min of continuous stimulation at 10 Hz in ChR2-eYFP compared with eYFP animals (n=4 animals per condition) (). However, there was no statistical difference between the percentage decrease in cortical NE elicited by 10 Hz stimulation in ChR2-eYFP animals (n=4 animals), which caused behavioral arrests, and constant photoinhibition in eNpHR-eYFP animals (n=4 animals; ), which did not cause behavioral arrests (P>0.05, two-way ANOVA between stimulation condition and transgene expression). To determine the effects of increasing extracellular NE concentration on behavioral arrests, we stimulated ChR2-eYFP animals (n=4) 30 minutes after administration of the NE reuptake inhibitors atomoxitine or reboxitine. We found a dose dependent increase in the latency to arrest and decrease in the duration of arrest (). Taken together, these results demonstrate that sustained, high frequency stimulation of locus coeruleus neurons causes a depletion of NE stores and that increasing NE concentration using reuptake inhibitors attenuates the arrests.

DISCUSSION

Tuning arousal with modulation of locus coeruleus neurons

This study demonstrates that neural activity in the locus coeruleus is both necessary for maintaining normal durations of wakefulness as well as sufficient to promote immediate sleep-to-wake transitions, long-term wakefulness, and an increase in locomotor arousal. Taken together, these results suggest that the locus coeruleus is finely-tuned to influence wakefulness, with even subtle differences in stimulation frequency and duration causing different effects on arousal and sleep-to-wake transitions (). Anatomically, the locus coeruleus receives many afferent projections[2,30] and is well-positioned to receive input from hypothalamic and brainstem circuits that sense salient environmental and homeostatic stimuli[31-33], as well as information from higher-cognitive circuits in the prefrontal cortex[34]. In turn, the locus coeruleus projects extensively to virtually all brain regions with the exception of the striatum[2]. Therefore, the anatomical connections of the locus coeruleus, combined with our in vivo behavioral results, suggest that the locus coeruleus integrates information from lower and higher neural circuits and is finely-tuned to affect downstream nuclei and modulate behavior.

Although our in vitro recordings from locus coeruleus neurons in acute brainstem slices demonstrate the efficiency of eNpHR-mediated photoinhibition and the reliability of 10 ms blue-light pulses in causing single spikes, we cannot be certain that these tools modulate neural activity as precisely in vivo. We found that inhibition of the locus coeruleus using yellow light significantly decreased cortical norepinephrine concentration () and that stimulation using blue light significantly increased expression of the immediate early gene c-Fos throughout the entire anteroposterior length of the locus coeruleus (). However, we cannot be certain of the efficiency of these tools in inhibiting or stimulating single action potentials in awake, behaving animals. At the same time, our results clearly show differential effects that vary linearly based on the frequency and duration of stimulation (). Therefore, we conclude that the locus coeruleus is finely-tuned to influence behavior depending on different stimulation parameters.

Previous studies in which the locus coeruleus was pharmacologically lesioned[10-12] or NE transmission was genetically ablated[13] found no significant differences in durations of sleep and wakefulness. In contrast, we found a significant reduction in wake episode duration during 1 h of eNpHR-mediated inhibition of the locus coeruleus during the active period (). A major advantage of eNpHR to traditional pharmacological or genetic loss-of-function methods is the ability to silence neural activity at specifically defined temporal windows with minimal disturbance to the animal. Perhaps permanently ablating locus coeruleus neurons or norepinephrine synthesis causes the brain to adapt using other wake-promoting systems[4], as has been reported for other brain circuits, such as those involved in food intake[35,36]. Furthermore, our rAAV-mediated genetic targeting of the locus coeruleus was able to bilaterally transduce >98% of locus coeruleus neurons, a higher percentage than previous studies using radiofrequency pulses[10], saporin[12], or DSP-4[37]. Therefore, our loss-of-function approach achieved not only acute temporal specificity but also efficient genetic targeting of the locus coeruleus, which may explain our novel results.

eNpHR-mediated inhibition of the locus coeruleus caused a decrease in the duration of wakefulness and an increase in slow-wave activity in the EEG power spectra at the end of wake episodes (). However, inhibition did not prevent sleep-to-wake transitions, nor increase the duration of sleep episodes. In contrast to inhibition, we found that stimulation of the locus coeruleus caused immediate sleep-to-wake transitions (). Therefore, we conclude that the locus coeruleus is sufficient to promote sleep-to-wake transitions, but other nuclei must contribute to sleep-to-wake transitions in a way that is statistically redundant with locus coeruleus activity. Other known arousal-promoting nuclei include the histaminergic tuberomammilary nucleus (TMN)[38], the serotoninergic dorsal raphe nuclei[39], the cholinergic pedunculopontine nucleus and lateral tegmental nucleus[40,41], as well as multiple cell-types in the basal forebrain[42]. Perhaps the locus coeruleus plays a more prominent, necessary role in promoting wakefulness when the animal is already awake, but is sufficient to promote arousal in a way that is redundant with other arousal systems when the animal is asleep.

Importantly, we show differential effects on arousal using naturally occurring tonic versus phasic stimulation paradigms (). Long-term tonic stimulation over 1 h caused an increase in the duration of wakefulness, as well as an increase in locomotor activity; however, this paradigm was not sufficient to increase the duration of wakefulness over 5 h of stimulation. In contrast, long-term phasic stimulation caused an increase in the duration of wakefulness over 1 h and 5 h, but a decrease in locomotor activity. Previous studies in which locus coeruleus activity was recorded in rats and primates demonstrate that tonic locus coeruleus activity is correlated with wakefulness, firing at 3 Hz during active wakefulness, 1-2 Hz during quiet wakefulness, <1 Hz during NREM sleep, and not firing during REM sleep[6-8]. Phasic locus coeruleus activity correlates with salient environmental stimuli[7,9] and has been proposed to underly mechanisms of attention and behavioral/cognitive adaptation to changing environmental circumstances[1,2,5,43-45]. Therefore, perhaps tonic stimulation at low frequencies (3 Hz) specifically causes an increase in arousal, while phasic frequencies may also cause changes in cortical networks and synaptic plasticity that alters cognition or attention, resulting in loss of exploratory/locomotor behavior. Furthermore, presenting phasic stimulation without environmental stimuli that cause endogenous phasic activation of the locus coeruleus may confound cortical networks and result in a lack of locomotion.

Tonic versus phasic frequencies are also thought to differentially affect different categories of noradrenergic receptors[1,2,44]: norepinephrine has the highest affinity for the α2-receptors, which are found on both pre- and post-synaptic terminals and cause hyperpolarizing effects. Norepinephrine has lower affinities for the α1- and β-receptors, which are primarily post-synaptic. Therefore, it is thought that low, tonic release of norepinephrine may preferentially activate α2-receptors, while high-frequency, phasic release of NE may preferentially activate α1- and β-receptors. Perhaps tonic stimulation of locus coeruleus neurons promotes wakefulness during 1 h, but is insufficient to increase arousal at longer time periods due to steady activation of α2-receptors, leading to self-inhibition of the locus coeruleus and inhibition of downstream targets. Alternatively, phasic stimulation of locus coeruleus neurons may cause long-term activation of α1- and β-receptors, allowing animals to consistently maintain wakefulness over longer time periods.

The locus coeruleus can cause behavioral arrests

A surprising and novel finding of our study is that sustained high frequency (>5 Hz) stimulation of the locus coeruleus causes reversible behavioral arrests (). We hypothesized that these arrests were caused by seizure or a severe cardiovascular response, but found no signs of spike discharge in the cortical EEG characteristic of seizure activity or statistically significant differences in heart rate or blood pressure ( and ).

Interestingly, using microdialysis to sample extracellular fluid from the cortex, we found that high-frequency stimulation of the locus coeruleus caused a decrease in cortical norepinephrine concentration (), suggesting that high-frequency, non-physiological levels of stimulation depleted norepinephrine from locus coeruleus terminals. Unfortunately, microdialysis has a temporal resolution of several minutes, preventing detection of norepinephrine discharge rates over the 10-15 s of high frequency stimulation necessary to cause behavioral arrests. Voltammetry techniques have better temporal resolutions but are not good at distinguishing between norepinephrine and other catecholamines. We hypothesize that high-frequency stimulation of the locus coeruleus causes first a rapid increase of extracellular norepinephrine concentration over several seconds, followed by the decrease observed by our microdialysis data. The decrease in norepinephrine content could be due to a breakdown of cortical norepinephrine in the synapse and the inability of locus coeruleus terminals to replenish norepinephrine while still firing at >5 Hz stimulation. At the offset of blue light stimulation, the locus coeruleus terminals could replenish norepinephrine stores, allowing the animal to recover. Indeed, increasing extracellular norepinephrine by preventing reuptake attenuated the arrests ().

However, behavioral arrests cannot be caused solely by a decrease in brain norepinephrine content, as eNpHR-mediated inhibition produced statistically similar decreases in cortical norepinephrine concentrations without producing arrests (). Futhermore, previous studies in which locus coeruleus activity was ablated did not produce behavioral arrests[10-12]. The inability of photoinhibition to cause arrests may be due to the synergistic effects of other neuromodulatory systems. For example, it has been shown that serotonin cell activity (and presumably serotonin release) is greatly decreased in cataplexy[46] and REM sleep[47]. Thus, inhibiting the locus coeruleus during tonic activity of raphe neurons may allow muscle tone to be maintained by serotonin. It has also been shown with microdialysis that GABAergic and glycinergic release is increased during muscle tone suppression[48], causing active inhibition in motoneurons. Excessive firing of norepinephrine neurons may trigger the activity of post-synaptic inhibitory neurons in the brainstem eliciting a behavioral arrest.

Behavioral arrests may be caused by non-endogenous norepinephrine release onto noradrenergic receptors. As discussed above, α1- and β-receptors may be preferentially activated by phasic versus tonic frequencies. Therefore, the high-frequency stimulation that elicits locus coeruleus-mediated behavioral arrests may cause dramatic, non-endogenous activation of α1 and β-receptors, followed by a relative loss of extracellular norepinephrine as indicated by our microdialysis results (). This rapid release followed by depletion is an unexplored phenomenon on post-synaptic terminals, and future in vitro research using slice preparations is necessary to understand the consequences of a rapid efflux of norepinephrine release at postsynaptic sites.

The locus coeruleus-mediated behavioral arrests described here may cause the motor arrests associated with symptoms of some neuropsychiatric disorders with no known etiology. Interestingly, locus coeruleus-mediated behavioral arrests are not inconsistent with a mouse model of cataplexy as recently defined by the International Working Group on Rodent Models of Narcolepsy[49]. This condition is generally defined by an abrupt episode of nuchal atonia lasting at least 10 seconds with EEG characteristic of theta activity and at least 40 seconds of wakefulness preceding the episode. Our characterization of locus coeruleus-mediated behavioral arrests fit these criteria (). However, locus coeruleus-mediated arrests are likely to be analogous rather than homologous to murine cataplexy, as the locus coeruleus has been previously shown to be silent preceding cataplectic attacks[50] rather than exhibiting the high frequencies reported here. Nevertheless, based on our results, the consensus definition of cataplexy may need to be revised, and it will be important for future studies to consider the role of the locus coeruleus in mediating the symptoms of other neuropsychiatric diseases.

SUPPLEMENTARY FIGURE LEGENDS

Supplementary Figure 1. Genetic strategy to target locus coeruleus neurons. (a) We used a double-floxed inverted open reading frame (DIO) construct within a recombinant adeno-associated viral vector (rAAV) to deliver optogenetic transgenes to locus coeruleus neurons. Our optogenetic transgenes (Opsin-eYFP) included eNpHR-eYFP, ChR2-eYFP, or eYFP alone. In the absence of Cre recombinase, the coding sequences for these transgenes reside in the opposite orientation. However, upon Cre-lox mediated recombination, the transgenes flip into the correct orientation so that efficient mRNA and protein expression can occur. (b) High-titer rAAV was stereotaxically injected into the brainstem of TH::IRES-Cre knockin mice in which Cre recombinase is specifically expressed in cells that express tyrosine hydroxylase. Thus, although the rAAV infects multiple neuronal subtypes in the brain, only tyrosine hydroxylase-positive cells in the local site of injection express transgenes in the correct orientation. (c) rAAV was injected just lateral to the locus coeruleus (anteroposterior, –5.45 mm; mediolateral, +/–1.28 mm; dorsoventral, 3.65 mm). Experiments began two weeks after injection to allow for efficient gene expression.

Supplementary Figure 2. Transgene expression in brainstem noradrenergic and dopaminergic nuclei. (a) Sagittal profile of the mouse brain depicting the locations of brainstem noradrenergic and dopaminergic nuclei. (b) Co-expression of tyrosine hydroxylase immunoreactivity (red), and viral eYFP expression (green) in brainstem noradrenergic and dopaminergic nuclei labeled in (a).

Supplementary Figure 3. Surgical implantation of bilateral cannulae and EEG/EMG electrodes for in vivo light delivery and recordings. A bilateral cannula was placed above the locus coeruleus (anteroposterior, –5.45mm; mediolateral, +/–1.0 mm; dorsoventral, 3.0 mm) for in vivo light delivery. An EEG/EMG implant was placed anterior to the cannula. EEG electrodes were placed on the skull above the frontal and parietal lobes, and EMG electrodes were placed within the neck musculature.

Supplementary Figure 4. Baseline sleep architecture of sham and virally-transduced animals. In both the light (inactive) and dark (active) periods, we found no significant difference in the percentage of wakefulness, NREM, or REM sleep between animals injected with saline or transduced with EF1α::eYFP, EF1α::eNpHR-eYFP, or EF1α::ChR2-eYFP (P>0.05, two-way ANOVA, n=4 animals).

Supplementary Figure 5. In vivo photostimulation of locus coeruleus neurons increases c-Fos immunoreactivity in the locus coeruleus. (a) Representative images of the locus coeruleus co-stained for tyrosine hydroxylase (light brown) and c-Fos (black). Photographs depict histological samples from eYFP transduced mice (top row) and ChR2-eYFP transduced mice (bottom row) and represent the side of the brain that received unilateral stimulation (ipsilateral, left column) or the side of the brain that was not stimulated (contralateral, right column). (b) Quantification of the percentage of neurons showing tyrosine hydroxylase immunoreactivity that also express c-Fos. Data represent the mean +/– s.d. from eYFP (n=4) and ChR2-eYFP (n=4) animals. **P<0.001, two-way ANOVA followed by Student's t-test. (c) Quantification of the number of neurons showing tyrosine hydroxylase immunoreactivity that also express c-Fos from adjacent 30 μm brain sections. Data represent the mean +/– s.d. number of neurons that express each label in the ipsilateral locus coeruleus in ChR2-eYFP animals (n=4).

Supplementary Figure 6. In vivo photoinhibition of locus coeruleus neurons causes a decrease in norepinephrine content in prefrontal cortex. Data represent the mean +/– s.e.m. of 4 trials per animal, n=4 animals. **P<0.001, two-way ANOVA between timepoint and virally-transduced animal followed by Bonferroni post-hoc test.

Supplementary Figure 7. The percentage of time spent in wake, NREM, and REM sleep during 1 h photoinhibition in the inactive (light) period. Data represent the mean +/– s.e.m. of 6 separate 1 h sessions, n=6 animals. *P>0.05, two-tailed Student's t-test between transduced animals.

Supplementary Figure 8. Correlation between stimulation parameters and the probability of awakening from NREM (a,c,e) and REM (b,d,f) sleep. Each dot represents the lowest mean value across 6 stimulated ChR2-eYFP animals for which animals exhibited sleep-to-wake transitions in 100% of trials. (a,b) The relationship between the stimulation frequency (1–10 Hz) and duration (1–10 s) that caused a 100% probability of awakening. (c,d) The relationship between the number of pulses necessary to elicit a 100% probability of awakening and the stimulation frequency. (e,f) The relationship between the number of pulses necessary to elicit a 100% probability of awakening and the stimulation duration.

Supplementary Figure 9. The probability of a sleep-to-wake transition from NREM (left) or REM sleep (right) in the 10 s following the onset of photostimulation (10 ms pulses at 5 Hz for 5 s) in ChR2-eYFP transduced animals (n=4) following administration of an α2 receptor agonist (clonidine) or α1 receptor antagonist (prazosin). Data represent the mean +/– s.e.m after 12 trials per condition per mouse. Increased darkness of bars represents increased pharmacological dose. *P<0.05, **P<0.001 two way ANOVA between saline and pharmacological conditions followed by Bonferroni posthoc test.

Supplementary Figure 10. Heart rate and systolic blood pressure before and during behavioral arrests. P>0.05, Student's t-test between epochs, 6 trials per animal in n=6 animals.

Supplementary Figure 11. Summary of the effects of manipulation of locus coeruleus activity on wakefulness. The locus coeruleus can be thought of as a tuning dial, with different frequencies causing different effects on behavior. Red arrows represent the specific frequencies tested in this study. (a) Photoinhibition with yellow light decreases the duration of wake-episodes. (b) Photostimulation with 10 ms pulses of blue light linearly increases the probability of a sleep-to-wake transition. (c) Long-term tonic stimulation with 10 ms pulses at 3 Hz increases wakefulness and locomotor activity over an hour, but the effects are lost over 5 h stimulation. (d) Phasic stimulation with 10 ms pulses at 10 Hz (lasting 500 ms every 20 s) increases wakefulness over both 1–5 hours, while locomotor activity decreases. (e) Phasic stimulation with 10 ms pulses at 5 Hz and above causes reversible behavioral arrests when applied for greater than 5–15 s.

Supplementary Movie 1. Representative sleep-to-wake transition following photostimulation of locus coeruleus neurons during NREM sleep. Photostimulation condition was 10 ms pulses at 5 Hz for 5 s.

Supplementary Movie 2. Representative behavioral arrest following sustained, high-frequency photostimulation of locus coeruleus neurons with 10 ms pulses at 10 Hz.