The locus coeruleus (LC), a diffusely-projecting noradrenergic nucleus located in the rostral pons, has been recognized as playing a role in arousal, sleep-wake cycles, and chemoreception. Clinically, the LC has been linked with diseases such as depression, anxiety, panic disorder, congenital central hypoventilation, and opiate withdrawal. Nevertheless, previous efforts to specifically alter LC activity with pharmacologic lesioning and genetic ablation have failed to elucidate LC function. Now, Carter et al in a study led by researchers at Stanford University (Nature Neuroscience 13: 1526-1535, 2010) have applied a new and sophisticated tool to the study of the LC: optogenetics. Optogenetics allows one to turn neurons “on” or “off” with light. The temporal and spatial precision of optogenetics is extremely high since only neurons made to express light-sensitive ion channels or pumps will respond to certain wavelengths of light. Halorhodopsin (eNpHR) is a chloride pump sensitive to yellow light which hyperpolarizes or inhibits neurons. Channelrhodopsin-2 (ChR2) is a cation channel sensitive to blue light which depolarizes or stimulates neurons.
The group used an adeno-associated virus to introduce express either eNpHR or ChR2 selectively in the tyrosine hydroxylase expressing neurons of the LC in mice. After confirming robust and selective transgene expression within the LC, the researchers confirmed with the whole-cell patch clamp technique that these transduced LC cells expressed functional light-sensitive ion channels. This in vitro work set the stage for an unprecedented series of experiments that examined the consequences of rapid, light-mediated LC modulation in vivo. Stereotactic surgery was performed in mice to introduce the photosensitive proteins selectively into LC neurons. Next, brain cannulas were stereotactically placed above the LC in these mice in order to facilitate the introduction of fiberoptic cables, and EEG and EMG electrodes were placed to analyze brain wave activity and level of arousal.
Yellow light LC photoinhibition in mice expressing eNpHR within the LC significantly decreased wakefulness, increased NREM sleep, and increased wake-to-NREM transitions in comparison to control mice. Next, mice with an LC expressing ChR2 were exposed to blue light in order to study the effect of LC stimulation. Blue light in these mice led to immediate sleep-to-wake transitions, especially REM sleep-to-wake transitions, within 5 seconds of the onset of stimulation. This wakefulness induced by in vivo LC photostimulation was shown to be dependent, at least in part, on norepinephrine transmission since it could be blocked with clonidine and prazosin.
Next, knowing that physiologic recordings of the LC have revealed both tonic and phasic (short bursts) neuronal firing, the scientists examined the effect of long-term tonic and phasic LC photostimulation. Interestingly, although both tonic and phasic photostimulation of the LC increased total wakefulness, tonic and phasic modes of stimulation have completely opposite effects on locomotor activity in mice. Tonic stimulation caused a significant increased in locomotor activity while phasic stimulation caused a significant decrease in locomotor activity. Finally, this group described a previously unreported, reversible behavioral arrest in mice exposed to LC photostimulation at high frequencies (> 5 Hz). The latency to arrest decreased with increasing frequency of photostimulation and during these arrests, mice were immobile with their eyes open but unresponsive to tail and toe pinches.
Carter and colleagues have made several important contributions to neuroscience with this report. First, they have carefully demonstrated via optogenetics that LC activity is necessary to maintain normal durations of wakefulness and also contributes to sleep-wake transitions and locomotor activity. In addition, the fact that LC photoinhibition did not prevent sleep-to-wake transitions or increase the duration of sleep episodes suggests that other nuclei in the brain also promote sleep-to-wake transitions. Thus, although the LC appears sufficient to wake someone up, there are backup systems in the brain which can compensate for reduced LC activity. This redundancy in the brain arousal system makes sense from an evolutionary standpoint; it would be a precarious situation if an organism's wakefulness was maintained solely by a single foci of neurons in the brain. Moreover, they have shown that tonic and phasic stimulation of the LC had differing effects on locomotion. So it would appear that the LC not only keeps us awake but also encourages us to move or stop moving in response to certain stimuli in the environment.
This group has also demonstrated the potential of optogenetics to be an important tool in neuromodulation. Although current brain stimulation in humans utilizes wires and pulsed electric fields, optogenetics may be applied to human brain modulation in the future. Optogenetics, by only influencing neurons that have been treated with gene therapy, holds the promise of being a more focused form of brain modulation as compared to electrical stimulation, which nonspecifically influences neurons by a diffuse electric field. Also, this paper begs the question of whether LC modulation should be investigated for human diseases. LC stimulation may be able to benefit patients with disorders of impaired arousal such as narcolepsy, sudden infant death syndrome, or the persistent vegetative state. Ultimately, this study has not only expanded the tool chest of the stereotactic surgeon but has also brought to light a new target of brain modulation.
Nestor D. Tomycz
Robert M. Friedlander