Distinct lateral hypothalamic CaMKIIα neuronal populations regulate wakefulness and locomotor activity.

For nearly a century, evidence has accumulated indicating that the lateral hypothalamus (LH) contains neurons essential to sustain wakefulness. While lesion or inactivation of LH neurons produces a profound increase in sleep, stimulation of inhibitory LH neurons promotes wakefulness. To date, the primary wake-promoting cells that have been identified in the LH are the hypocretin/orexin (Hcrt) neurons, yet these neurons have little impact on total sleep or wake duration across the 24-h period. Recently, we and others have identified other LH populations that increase wakefulness. In the present study, we conducted microendoscopic calcium imaging in the LH concomitant with EEG and locomotor activity (LMA) recordings and found that a subset of LH neurons that express Ca2+/calmodulin-dependent protein kinase IIα (CaMKIIα) are preferentially active during wakefulness. Chemogenetic activation of these neurons induced sustained wakefulness and greatly increased LMA even in the absence of Hcrt signaling. Few LH CaMKIIα-expressing neurons are hypocretinergic or histaminergic while a small but significant proportion are GABAergic. Ablation of LH inhibitory neurons followed by activation of the remaining LH CaMKIIα neurons induced similar levels of wakefulness but blunted the LMA increase. Ablated animals showed no significant changes in sleep architecture but both spontaneous LMA and high theta (8 to 10 Hz) power during wakefulness were reduced. Together, these findings indicate the existence of two subpopulations of LH CaMKIIα neurons: an inhibitory population that promotes locomotion without affecting sleep architecture and an excitatory population that promotes prolonged wakefulness even in the absence of Hcrt signaling.

Optogenetic activation of striatal cholinergic interneurons regulates L-dopa-induced dyskinesias.

L-dopa-induced dyskinesias (LIDs) are a serious complication of L-dopa therapy for Parkinson's disease. Emerging evidence indicates that the nicotinic cholinergic system plays a role in LIDs, although the pathways and mechanisms are poorly understood. Here we used optogenetics to investigate the role of striatal cholinergic interneurons in LIDs. Mice expressing cre-recombinase under the control of the choline acetyltransferase promoter (ChAT-Cre) were lesioned by unilateral injection of 6-hydroxydopamine. AAV5-ChR2-eYFP or AAV5-control-eYFP was injected into the dorsolateral striatum, and optical fibers implanted. After stable virus expression, mice were treated with L-dopa. They were then subjected to various stimulation protocols for 2h and LIDs rated. Continuous stimulation with a short duration optical pulse (1-5ms) enhanced LIDs. This effect was blocked by the general muscarinic acetylcholine receptor (mAChR) antagonist atropine indicating it was mAChR-mediated. By contrast, continuous stimulation with a longer duration optical pulse (20ms to 1s) reduced LIDs to a similar extent as nicotine treatment (~50%). The general nicotinic acetylcholine receptor (nAChR) antagonist mecamylamine blocked the decline in LIDs with longer optical pulses showing it was nAChR-mediated. None of the stimulation regimens altered LIDs in control-eYFP mice. Lesion-induced motor impairment was not affected by optical stimulation indicating that cholinergic transmission selectively regulates LIDs. Longer pulse stimulation increased the number of c-Fos expressing ChAT neurons, suggesting that changes in this immediate early gene may be involved. These results demonstrate that striatal cholinergic interneurons play a critical role in LIDs and support the idea that nicotine treatment reduces LIDs via nAChR desensitization.

A role for cortical nNOS/NK1 neurons in coupling homeostatic sleep drive to EEG slow wave activity.

Although the neural circuitry underlying homeostatic sleep regulation is little understood, cortical neurons immunoreactive for neuronal nitric oxide synthase (nNOS) and the neurokinin-1 receptor (NK1) have been proposed to be involved in this physiological process. By systematically manipulating the durations of sleep deprivation and subsequent recovery sleep, we show that activation of cortical nNOS/NK1 neurons is directly related to non-rapid eye movement (NREM) sleep time, NREM bout duration, and EEG δ power during NREM sleep, an index of preexisting homeostatic sleep drive. Conversely, nNOS knockout mice show reduced NREM sleep time, shorter NREM bouts, and decreased power in the low δ range during NREM sleep, despite constitutively elevated sleep drive. Cortical NK1 neurons are still activated in response to sleep deprivation in these mice but, in the absence of nNOS, they are unable to up-regulate NREM δ power appropriately. These findings support the hypothesis that cortical nNOS/NK1 neurons translate homeostatic sleep drive into up-regulation of NREM δ power through an NO-dependent mechanism.

Neocortical long-range inhibition promotes cortical synchrony and sleep.

Behavioral states such as sleep and wake are highly correlated with specific patterns of rhythmic activity in the cortex. During low arousal states such as slow wave sleep, the cortex is synchronized and dominated by low frequency rhythms coordinated across multiple regions. Although recent evidence suggests that GABAergic inhibitory neurons are key players in cortical state modulation, the in vivo circuit mechanisms coordinating synchronized activity among local and distant neocortical networks are not well understood. Here, we show that somatostatin and chondrolectin co-expressing cells (Sst-Chodl cells), a sparse and unique class of neocortical inhibitory neurons, are selectively active during low arousal states and are largely silent during periods of high arousal. In contrast to other neocortical inhibitory neurons, we show these neurons have long-range axons that project across neocortical areas. Activation of Sst-Chodl cells is sufficient to promote synchronized cortical states characteristic of low arousal, with increased spike co-firing and low frequency brain rhythms, and to alter behavioral states by promoting sleep. Contrary to the prevailing belief that sleep is exclusively driven by subcortical mechanisms, our findings reveal that these long-range inhibitory neurons not only track changes in behavioral state but are sufficient to induce both sleep-like cortical states and sleep behavior, establishing a crucial circuit component in regulating behavioral states.

Shift in the balance between excitation and inhibition during sensory adaptation of S1 neurons.

Sustained stimulation of sensory organs results in adaptation of the neuronal response along the sensory pathway. Whether or not cortical adaptation affects equally excitation and inhibition is poorly understood. We examined this question using patch recordings of neurons in the barrel cortex of anesthetized rats while repetitively stimulating the principal whisker. We found that inhibition adapts more than excitation, causing the balance between them to shift toward excitation. A comparison of the latency of thalamic firing and evoked excitation and inhibition in the cortex strongly suggests that adaptation of inhibition results mostly from depression of inhibitory synapses rather than adaptation in the firing of inhibitory cells. The differential adaptation of the evoked conductances that shifts the balance toward excitation may act as a gain mechanism which enhances the subthreshold response during sustained stimulation, despite a large reduction in excitation.

Parallel Arousal Pathways in the Lateral Hypothalamus.

Until recently, hypocretin (Hcrt) neurons were the only known wake-promoting neuronal population in the lateral hypothalamus (LH), but subpopulations of inhibitory neurons in this area and glutamatergic neurons in the nearby supramammillary nucleus (SuM) have recently been found that also promote wakefulness. We performed chemogenetic excitation of LH neurons in mice and observed increased wakefulness that lasted more than 4 h without unusual behavior or EEG anomalies. The increased wakefulness was similar in the presence or absence of the dual orexin receptor blocker almorexant (ALM). Analysis of hM3Dq transfection and c-FOS expression in LH inhibitory neurons and in the SuM failed to confirm that the increased wakefulness was due to these wake-promoting populations, although this possibility cannot be completely excluded. To evaluate the relationship to the Hcrt system, we repeated the study in Orexin-tTA mice in the presence or absence of dietary doxycycline (DOX), which enabled us to manipulate the percentage of Hcrt neurons that expressed hM3Dq. In DOX-fed mice, 18% of Hcrt neurons as well as many other LH neurons expressed hM3Dq; these mice showed a profound increase in wake after hM3Dq activation even in the presence of ALM. In mice switched to normal chow, 62% of Hcrt neurons expressed hM3Dq along with other LH cells; chemogenetic activation produced even more sustained arousal which could be reduced to previous levels by ALM treatment. Together, these results indicate an LH neuron population that promotes wakefulness through an Hcrt-independent pathway that can act synergistically with the Hcrt system to prolong arousal.

Cross-whisker adaptation of neurons in the rat barrel cortex.

Neurons in the barrel cortex and the thalamus respond preferentially to stimulation of one whisker (the principal whisker) and weakly to several adjacent whiskers. Cortical neurons, unlike thalamic cells, gradually adapt to repeated whisker stimulations. Whether cortical adaptation is specific to the stimulated whisker is not known. The aim of this intracellular study was to determine whether the response of a cortical cell to stimulation of an adjacent whisker would be affected by previous adaptation induced by stimulation of the principal whisker and vice versa. Using a high-frequency stimulation that causes substantial adaptation in the cortex and much less adaptation in the thalamus, we show that cortical adaptation evoked by a train of stimuli applied to one whisker does not affect the synaptic response to subsequent stimulation of a neighboring whisker. Our data indicate that intrinsic mechanisms are not involved in cortical adaptation. Thalamic recordings obtained under the same conditions demonstrated that an adjacent whisker response was not generated in the thalamus, indicating that the observed whisker-specific adaptation results from diverging thalamic inputs or from cortical integration.

Extracting fuzzy rules from polysomnographic recordings for infant sleep classification.

A neuro-fuzzy classifier (NFC) of sleep-wake states and stages has been developed for healthy infants of ages 6 mo and onward. The NFC takes five input patterns previously identified on 20-s epochs from polysomnographic recordings and assigns them to one out of five possible classes: Wakefulness, REM-Sleep, Non-REM Sleep Stage 1, Stage 2, and Stage 3-4. The definite criterion for a sleep state or stage to be established is duration of at least 1 min. The data set consisted of a total of 14 continuous recordings of naturally occurring naps (average duration: 143 +/- 39 min), corresponding to a total of 6021 epochs. They were divided in a training, a validation and a test set with 7, 2, and 5 recordings, respectively. During supervised training, the system determined the fuzzy concepts associated to the inputs and the rules required for performing the classification, extracting knowledge from the training set, and pruning nonrelevant rules. Results on an independent test set achieved 83.9 +/- 0.4% of expert agreement. The fuzzy rules obtained from the training examples without a priori information showed a high level of coincidence with the crisp rules stated by the experts, which are based on internationally accepted criteria. These results show that the NFC can be a valuable tool for implementing an automated sleep-wake classification system.

Simultaneous Bayesian Estimation of Excitatory and Inhibitory Synaptic Conductances by Exploiting Multiple Recorded Trials.

Advanced statistical methods have enabled trial-by-trial inference of the underlying excitatory and inhibitory synaptic conductances (SCs) of membrane-potential recordings. Simultaneous inference of both excitatory and inhibitory SCs sheds light on the neural circuits underlying the neural activity and advances our understanding of neural information processing. Conventional Bayesian methods can infer excitatory and inhibitory SCs based on a single trial of observed membrane potential. However, if multiple recorded trials are available, this typically leads to suboptimal estimation because they neglect common statistics (of synaptic inputs (SIs)) across trials. Here, we establish a new expectation maximization (EM) algorithm that improves such single-trial Bayesian methods by exploiting multiple recorded trials to extract common SI statistics across the trials. In this paper, the proposed EM algorithm is embedded in parallel Kalman filters or particle filters for multiple recorded trials to integrate their outputs to iteratively update the common SI statistics. These statistics are then used to infer the excitatory and inhibitory SCs of individual trials. We demonstrate the superior performance of multiple-trial Kalman filtering (MtKF) and particle filtering (MtPF) relative to that of the corresponding single-trial methods. While relative estimation error of excitatory and inhibitory SCs is known to depend on the level of current injection into a cell, our numerical simulations using MtKF show that both excitatory and inhibitory SCs are reliably inferred using an optimal level of current injection. Finally, we validate the robustness and applicability of our technique through simulation studies, and we apply MtKF to in vivo data recorded from the rat barrel cortex.

Alterations in High-Frequency Neuronal Oscillations in a Cynomolgus Macaque Test of Sustained Attention Following NMDA Receptor Antagonism.

A growing body of evidence indicates that neuronal oscillations in the gamma frequency range (30-80 Hz) are disturbed in schizophrenic patients during cognitive processes and may represent an endophenotype of the disease. N-methyl-D-aspartate (NMDA) receptor antagonists have been used experimentally to induce schizophrenia-like symptoms including cognitive deficits in animals and humans. Here we characterized neuronal oscillations and event-related potentials (ERPs) in Cynomolgus macaques fully trained to perform a continuous performance test (CPT) in the presence and absence of the NMDA antagonist phencyclidine (PCP). Macaques (n=8) were trained to touch 'target' stimuli and ignore 'distractor' stimuli presented randomly on a touchscreen. Subsequently, all subjects were implanted with epidural EEG electrodes over frontal (FC) and parietal cortices (PC) and later tested under vehicle (saline, i.m.) or acute PCP (0.1-0.3 mg/kg, i.m.) conditions. Compared with vehicle treatment, PCP produced a significant dose-dependent decrease in CPT performance accuracy and increased reaction times. Furthermore, PCP elevated the amplitudes of 'low' (30-50 Hz) and 'high' (51-80 Hz) gamma oscillations in FC and PC around target presentations for all correct responses. The CPT accuracy was inversely correlated with the gamma band amplitude in the presence of PCP. Additionally, PCP delayed the N100 peak latency in FC, and prolonged and suppressed the cognitively relevant P300 component of mean ERPs in FC and PC, respectively. The NMDA receptor antagonist-induced alteration in neuronal oscillations and ERPs may contribute to the observed cognitive deficits in macaques, and enhance our understanding of EEG recordings as a translatable biomarker.

Cortical nNOS neurons co-express the NK1 receptor and are depolarized by Substance P in multiple mammalian species.

We have previously demonstrated that Type I neuronal nitric oxide synthase (nNOS)-expressing neurons are sleep-active in the cortex of mice, rats, and hamsters. These neurons are known to be GABAergic, to express Neuropeptide Y (NPY) and, in rats, to co-express the Substance P (SP) receptor NK1, suggesting a possible role for SP in sleep/wake regulation. To evaluate the degree of co-expression of nNOS and NK1 in the cortex among mammals, we used double immunofluorescence for nNOS and NK1 and determined the anatomical distribution in mouse, rat, and squirrel monkey cortex. Type I nNOS neurons co-expressed NK1 in all three species although the anatomical distribution within the cortex was species-specific. We then performed in vitro patch clamp recordings in cortical neurons in mouse and rat slices using the SP conjugate tetramethylrhodamine-SP (TMR-SP) to identify NK1-expressing cells and evaluated the effects of SP on these neurons. Bath application of SP (0.03-1 µM) resulted in a sustained increase in firing rate of these neurons; depolarization persisted in the presence of tetrodotoxin. These results suggest a conserved role for SP in the regulation of cortical sleep-active neurons in mammals.