Optogenetic interrogation of neural circuits: technology for probing mammalian brain structures

Elucidation of the neural substrates underlying complex animal behaviors depends on precise activity control tools, as well as compatible readout methods. Recent developments in optogenetics have addressed this need, opening up new possibilities for systems neuroscience. Interrogation of even deep neural circuits can be conducted by directly probing the necessity and sufficiency of defined circuit elements with millisecond-scale, cell type-specific optical perturbations, coupled with suitable readouts such as electrophysiology, optical circuit dynamics measures and freely moving behavior in mammals. Here we collect in detail our strategies for delivering microbial opsin genes to deep mammalian brain structures in vivo, along with protocols for integrating the resulting optical control with compatible readouts (electrophysiological, optical and behavioral). The procedures described here, from initial virus preparation to systems-level functional readout, can be completed within 4–5 weeks. Together, these methods may help in providing circuit-level insight into the dynamics underlying complex mammalian behaviors in health and disease

The evolutionarily conserved miRNA-137 targets the neuropeptide hypocretin/orexin and modulates the wake to sleep ratio.

SignificanceThe hypocretin (Hcrt, also known as orexin) neuropeptides regulate sleep and wake stability, and disturbances of Hcrt can lead to sleep disorders. MicroRNAs (miRNAs) are short noncoding RNAs that fine-tune protein expression levels, and miRNA-based therapeutics are emerging. We report a functional interaction between miRNA (miR-137) and Hcrt. We demonstrate that intracellular miR-137 levels in Hcrt neurons regulate Hcrt expression with downstream effects on wakefulness. Specifically, lowering of miR-137 levels increased wakefulness in mice. We further show that the miR-137:Hcrt interaction is conserved across mice and humans, that miR-137 also regulates sleep-wake balance in zebrafish, and that the MIR137 locus is genetically associated with sleep duration in humans. Together, our findings reveal an evolutionarily conserved sleep-wake regulatory role of miR-137

How REM sleep shapes hypothalamic computations for feeding behavior.

The electrical activity of diverse brain cells is modulated across states of vigilance, namely wakefulness, non-rapid eye movement (NREM) sleep, and rapid eye movement (REM) sleep. Enhanced activity of neuronal circuits during NREM sleep impacts on subsequent awake behaviors, yet the significance of their activation, or lack thereof, during REM sleep remains unclear. This review focuses on feeding-promoting cells in the lateral hypothalamus (LH) that express the vesicular GABA and glycine transporter (vgat) as a model to further understand the impact of REM sleep on neural encoding of goal-directed behavior. It emphasizes both spatial and temporal aspects of hypothalamic cell dynamics across awake behaviors and REM sleep, and discusses a role for REM sleep in brain plasticity underlying energy homeostasis and behavioral optimization

Sleep and the hypothalamus.

Neural substrates of wakefulness, rapid eye movement sleep (REMS), and non-REMS (NREMS) in the mammalian hypothalamus overlap both anatomically and functionally with cellular networks that support physiological and behavioral homeostasis. Here, we review the roles of sleep neurons of the hypothalamus in the homeostatic control of thermoregulation or goal-oriented behaviors during wakefulness. We address how hypothalamic circuits involved in opposing behaviors such as core body temperature and sleep compute conflicting information and provide a coherent vigilance state. Finally, we highlight some of the key unresolved questions and challenges, and the promise of a more granular view of the cellular and molecular diversity underlying the integrative role of the hypothalamus in physiological and behavioral homeostasis

Sleep-wake control and the thalamus

Sleep is an essential component of animal behavior, controlled by both circadian and homeostatic processes. Typical brain oscillations for sleep and wake states are distinctive and reflect recurrent activity amongst neural circuits spanning localized to global brain regions. Since the original discovery of hypothalamic centers controlling both sleep and wakefulness, current views now implicate networks of neuronal and non-neuronal cells distributed brain-wide. Yet the mechanisms of sleep-wake control remain unclear. In light of recent studies, here we review experimental evidence from lesional, correlational, pharmacological and genetics studies, which support a role for the thalamus in several aspects of sleep-wake states. How these thalamo-cortical network mechanisms contribute to other executive functions such as memory consolidation and cognition, remains an open question with direct implications for neuro-psychiatric diseases and stands as a future challenge for basic science and healthcare research

Oscillating circuitries in the sleeping brain

Brain activity during sleep is characterized by circuit-specific oscillations, including slow waves, spindles and theta waves, which are nested in thalamocortical or hippocampal networks. A major challenge is to determine the relationships between these oscillatory activities and the identified networks of sleep-promoting and wake-promoting neurons distributed throughout the brain. Improved understanding of the neurobiological mechanisms that orchestrate sleep-related oscillatory activities, both in time and space, is expected to generate further insight into the delineation of sleep states and their functions

A role for spindles in the onset of rapid eye movement sleep

Sleep spindle generation classically relies on an interplay between the thalamic reticular nucleus (TRN), thalamo-cortical (TC) relay cells and cortico-thalamic (CT) feedback during non-rapid eye movement (NREM) sleep. Spindles are hypothesized to stabilize sleep, gate sensory processing and consolidate memory. However, the contribution of non-sensory thalamic nuclei in spindle generation and the role of spindles in sleep-state regulation remain unclear. Using multisite thalamic and cortical LFP/unit recordings in freely behaving mice, we show that spike-field coupling within centromedial and anterodorsal (AD) thalamic nuclei is as strong as for TRN during detected spindles. We found that spindle rate significantly increases before the onset of rapid eye movement (REM) sleep, but not wakefulness. The latter observation is consistent with our finding that enhancing spontaneous activity of TRN cells or TRN-AD projections using optogenetics increase spindle rate and transitions to REM sleep. Together, our results extend the classical TRN-TC-CT spindle pathway to include non-sensory thalamic nuclei and implicate spindles in the onset of REM sleep

Die normale Schlafphysiologie

Kernaussagen Dieses Kapitel gibt eine Übersicht zur normalen Schlafphysiologie, Erfassung von Schlaf in einem Schlaflabor und Aussagekraft elektrophysiologischer Befunde über eine normale Schlafstruktur. Das Gehirn als übergeordnete Schaltzentrale koordiniert die Wach- und Schlafmechanismen und reguliert zahlreiche biologische Funktionen sowohl der zentralen als auch peripheren Organe, die mit dem Schlaf gekoppelt sind. Durch Schlafentzug sowie Schlafstörungen ergeben sich zahlreiche pathologische Konsequenzen. Anhand des Zwei-Prozess-Modells der Schlafregulation wird gezeigt, wie Schlafdauer und -intensität homöostatisch reguliert werden. Wichtige Theorien der Schlaffunktion werden diskutiert

REM sleep stabilizes hypothalamic representation of feeding behavior

During rapid eye movement (REM) sleep, behavioral unresponsiveness contrasts strongly with intense brain-wide neural network dynamics. Yet, the physiological functions of this cellular activation remain unclear. Using in vivo calcium imaging in freely behaving mice, we found that inhibitory neurons in the lateral hypothalamus (LH$^{vgat}$) show unique activity patterns during feeding that are reactivated during REM, but not non-REM, sleep. REM sleep-specific optogenetic silencing of LH$^{vgat}$ cells induced a reorganization of these activity patterns during subsequent feeding behaviors accompanied by decreased food intake. Our findings provide evidence for a role for REM sleep in the maintenance of cellular representations of feeding behavior

Slow waves promote sleep-dependent plasticity and functional recovery after stroke.

Functional recovery after stroke is associated with a remapping of neural circuits. This reorganization is often associated with low frequency high amplitude oscillations in the peri-infarct zone in both rodents and humans. These oscillations are reminiscent of sleep slow waves (SW) and suggestive of a role for sleep in brain plasticity that occur during stroke recovery, however, direct evidence is missing. Using a stroke model in male mice, we showed that stroke was followed by a transient increase in NREM sleep accompanied by reduced amplitude and slope of ipsilateral NREM sleep SW. We next used 5 ms optical activation of Channelrhodopsin 2-expressing pyramidal neurons, or 200 ms silencing of Archeorhodopsin T-expressing pyramidal neurons, to generate local cortical UP, or DOWN, states, respectively, both sharing similarities with spontaneous NREM SW in freely-moving mice. Importantly, we found that single optogenetically-evoked SW (SWopto) in the peri-infarct zone, randomly distributed during sleep, significantly improved fine motor movements of the limb corresponding to the sensorimotor stroke lesion site, as compared to spontaneous recovery and control conditions, while motor strength remained unchanged. In contrast, SWopto during wakefulness had no effect. Furthermore, chronic SWopto during sleep were associated with local axonal sprouting as revealed by the increase of anatomical pre- and post-synaptic markers in the peri-infarct zone and corresponding contra-lesional areas to cortical circuit reorganization during stroke recovery. These results support a role for sleep SW in cortical circuit plasticity and sensorimotor recovery after stroke and provide a clinically-relevant framework for rehabilitation strategies using neuromodulation during sleep.SIGNIFICANCE STATEMENTBrain stroke is one of the leading causes of death and major disabilities in elderly worldwide. A better understanding of the pathophysiological mechanisms underlying spontaneous brain plasticity after stroke, together with an optimization of rehabilitative strategies, are essential to improve stroke treatments. Here, we investigate the role of optogenetically-induced sleep slow waves in an animal model of ischemic stroke and identify sleep as a window for post-stroke intervention that promotes neuroplasticity and facilitates sensorimotor recovery