James T. McKenna

Control of Sleep and Wakefulness

This review summarizes the brain mechanisms controlling sleep and wakefulness. Wakefulness promoting systems cause low-voltage, fast activity in the electroencephalogram (EEG). Multiple interacting neurotransmitter systems in the brain stem, hypothalamus, and basal forebrain converge onto common effector systems in the thalamus and cortex. Sleep results from the inhibition of wake-promoting systems by homeostatic sleep factors such as adenosine and nitric oxide and GABAergic neurons in the preoptic area of the hypothalamus, resulting in large-amplitude, slow EEG oscillations. Local, activity-dependent factors modulate the amplitude and frequency of cortical slow oscillations. Non-rapid-eye-movement (NREM) sleep results in conservation of brain energy and facilitates memory consolidation through the modulation of synaptic weights. Rapid-eye-movement (REM) sleep results from the interaction of brain stem cholinergic, aminergic, and GABAergic neurons which control the activity of glutamatergic reticular formation neurons leading to REM sleep phenomena such as muscle atonia, REMs, dreaming, and cortical activation. Strong activation of limbic regions during REM sleep suggests a role in regulation of emotion. Genetic studies suggest that brain mechanisms controlling waking and NREM sleep are strongly conserved throughout evolution, underscoring their enormous importance for brain function. Sleep disruption interferes with the normal restorative functions of NREM and REM sleep, resulting in disruptions of breathing and cardiovascular function, changes in emotional reactivity, and cognitive impairments in attention, memory, and decision making.

Hippocampal synaptic plasticity and spatial learning are impaired in a rat model of sleep fragmentation

Sleep fragmentation, a symptom in many clinical disorders, leads to cognitive impairments. To investigate the mechanisms by which sleep fragmentation results in memory impairments, rats were awakened once every 2 min via 30 s of slow movement on an automated treadmill. Within 1 h of this sleep interruption (SI) schedule, rats began to sleep in the 90‐s periods without treadmill movement. Total non‐rapid eye movement sleep (NREM) sleep time did not change over the 24 h of SI, although there was a significant decline in rapid eye movement sleep (REM) sleep and a corresponding increase in time spent awake. In the SI group, the mean duration of sleep episodes decreased and delta activity during periods of wake increased. Control rats either lived in the treadmill without movement (cage controls, CC), or had 10 ‐min periods of movement followed by 30 min of non‐movement allowing deep/continuous sleep (exercise controls, EC). EC did not differ from baseline in the total time spent in each vigilance state. Hippocampal long‐term potentiation (LTP), a long‐lasting change in synaptic efficacy thought to underlie declarative memory formation, was absent in rats exposed to 24 and 72 h SI. In contrast, LTP was normal in EC rats. However, long‐term depression and paired‐pulse facilitation were unaltered by 24 h SI. Twenty‐four hour SI also impaired acquisition of spatial learning in the hippocampus‐dependent water maze test. Twenty‐four hour SI elevated plasma corticosterone (CORT) to levels previously shown to enhance LTP (125 ng/mL). The results suggest that sleep fragmentation negatively impacts spatial learning. Loss of N‐methyl‐d‐aspartate (NMDA) receptor‐dependent LTP in the hippocampal CA1 region may be one mechanism involved in this deficit.

Afferent projections to nucleus reuniens of the thalamus

The nucleus reuniens (RE) is the largest of the midline nuclei of the thalamus and the major source of thalamic afferents to the hippocampus and parahippocampal structures. Nucleus reuniens has recently been shown to exert powerful excitatory actions on CA1 of the hippocampus. Few reports on any species have examined afferent projections to nucleus reuniens. By using the retrograde anatomical tracer Fluorogold, we examined patterns of afferent projections to RE in the rat. We showed that RE receives a diverse and widely distributed set of afferents projections. The main sources of input to nucleus reuniens were from the orbitomedial, insular, ectorhinal, perirhinal, and retrosplenial cortices; CA1/subiculum of hippocampus; claustrum, tania tecta, lateral septum, substantia innominata, and medial and lateral preoptic nuclei of the basal forebrain; medial nucleus of amygdala; paraventricular and lateral geniculate nuclei of the thalamus; zona incerta; anterior, ventromedial, lateral, posterior, supramammillary, and dorsal premammillary nuclei of the hypothalamus; and ventral tegmental area, periaqueductal gray, medial and posterior pretectal nuclei, superior colliculus, precommissural/commissural nuclei, nucleus of the posterior commissure, parabrachial nucleus, laterodorsal and pedunculopontine tegmental nuclei, nucleus incertus, and dorsal and median raphe nuclei of the brainstem. The present findings of widespread projections to RE, mainly from limbic/limbic‐associated structures, suggest that nucleus reuniens represents a critical relay in the transfer of limbic information (emotional/cognitive) from RE to its major targets, namely, to the hippocampus and orbitomedial prefrontal cortex. RE appears to be a major link in the two‐way exchange of information between the hippocampus and the medial prefrontal cortex. J. Comp. Neurol. 480:115–142, 2004.

Cortically projecting basal forebrain parvalbumin neurons regulate cortical gamma band oscillations

Significance When we are awake, purposeful thinking and behavior require the synchronization of brain cells involved in different aspects of the same task. Cerebral cortex electrical oscillations in the gamma (30–80 Hz) range are particularly important in such synchronization. In this report we identify a particular subcortical cell type which has increased activity during waking and is involved in activating the cerebral cortex and generating gamma oscillations, enabling active cortical processing. Abnormalities of the brain mechanisms controlling gamma oscillations are involved in the disordered thinking typical of neuropsychiatric disorders such as schizophrenia. Thus, these findings may pave the way for targeted therapies to treat schizophrenia and other disorders involving abnormal cortical gamma oscillations. Cortical gamma band oscillations (GBO, 30–80 Hz, typically ∼40 Hz) are involved in higher cognitive functions such as feature binding, attention, and working memory. GBO abnormalities are a feature of several neuropsychiatric disorders associated with dysfunction of cortical fast-spiking interneurons containing the calcium-binding protein parvalbumin (PV). GBO vary according to the state of arousal, are modulated by attention, and are correlated with conscious awareness. However, the subcortical cell types underlying the state-dependent control of GBO are not well understood. Here we tested the role of one cell type in the wakefulness-promoting basal forebrain (BF) region, cortically projecting GABAergic neurons containing PV, whose virally transduced fibers we found apposed cortical PV interneurons involved in generating GBO. Optogenetic stimulation of BF PV neurons in mice preferentially increased cortical GBO power by entraining a cortical oscillator with a resonant frequency of ∼40 Hz, as revealed by analysis of both rhythmic and nonrhythmic BF PV stimulation. Selective saporin lesions of BF cholinergic neurons did not alter the enhancement of cortical GBO power induced by BF PV stimulation. Importantly, bilateral optogenetic inhibition of BF PV neurons decreased the power of the 40-Hz auditory steady-state response, a read-out of the ability of the cortex to generate GBO used in clinical studies. Our results are surprising and novel in indicating that this presumptively inhibitory BF PV input controls cortical GBO, likely by synchronizing the activity of cortical PV interneurons. BF PV neurons may represent a previously unidentified therapeutic target to treat disorders involving abnormal GBO, such as schizophrenia.

Sleep fragmentation elevates behavioral, electrographic and neurochemical measures of sleepiness

Sleep fragmentation, a feature of sleep apnea as well as other sleep and medical/psychiatric disorders, is thought to lead to excessive daytime sleepiness. A rodent model of sleep fragmentation was developed (termed sleep interruption, SI), where rats were awakened every 2 min by the movement of an automated treadmill for either 6 or 24 h of exposure. The sleep pattern of rats exposed to 24 h of SI resembled sleep of the apneic patient in the following ways: sleep was fragmented (up to 30 awakening/h), total rapid eye movement (REM) sleep time was greatly reduced, non-rapid eye movement (NREM) sleep episode duration was reduced (from 2 min, 5 s baseline to 58 s during SI), whereas the total amount of NREM sleep time per 24 h approached basal levels. Both 6 and 24 h of SI made rats more sleepy, as indicated by a reduced latency to fall asleep upon SI termination. Electrographic measures in the recovery sleep period following either 6 or 24 h of SI also indicated an elevation of homeostatic sleep drive; specifically, the average NREM episode duration increased (e.g. for 24 h SI, from 2 min, 5 s baseline to 3 min, 19 s following SI), as did the NREM delta power during recovery sleep. Basal forebrain (BF) levels of extracellular adenosine (AD) were also measured with microdialysis sample collection and high performance liquid chromatography detection, as previous work suggests that increasing concentrations of BF AD are related to sleepiness. BF AD levels were significantly elevated during SI, peaking at 220% of baseline during 30 h of SI exposure. These combined findings imply an elevation of the homeostatic sleep drive following either 6 or 24 h of SI, and BF AD levels appear to correlate more with sleepiness than with the cumulative amount of prior wakefulness, since total NREM sleep time declined only slightly. SI may be partially responsible for the symptom of daytime sleepiness observed in a number of clinical disorders, and this may be mediated by mechanisms involving BF AD.

Characterization of GABAergic neurons in rapid-eye-movement sleep controlling regions of the brainstem reticular formation in GAD67–green fluorescent protein knock-in mice

Recent experiments suggest that brainstem GABAergic neurons may control rapid‐eye‐movement (REM) sleep. However, understanding their pharmacology/physiology has been hindered by difficulty in identification. Here we report that mice expressing green fluorescent protein (GFP) under the control of the GAD67 promoter (GAD67‐GFP knock‐in mice) exhibit numerous GFP‐positive neurons in the central gray and reticular formation, allowing on‐line identification in vitro. Small (10–15 µm) or medium‐sized (15–25 µm) GFP‐positive perikarya surrounded larger serotonergic, noradrenergic, cholinergic and reticular neurons, and > 96% of neurons were double‐labeled for GFP and GABA, confirming that GFP‐positive neurons are GABAergic. Whole‐cell recordings in brainstem regions important for promoting REM sleep [subcoeruleus (SubC) or pontine nucleus oralis (PnO) regions] revealed that GFP‐positive neurons were spontaneously active at 3–12 Hz, fired tonically, and possessed a medium‐sized depolarizing sag during hyperpolarizing steps. Many neurons also exhibited a small, low‐threshold calcium spike. GFP‐positive neurons were tested with pharmacological agents known to promote (carbachol) or inhibit (orexin A) REM sleep. SubC GFP‐positive neurons were excited by the cholinergic agonist carbachol, whereas those in the PnO were either inhibited or excited. GFP‐positive neurons in both areas were excited by orexins/hypocretins. These data are congruent with the hypothesis that carbachol‐inhibited GABAergic PnO neurons project to, and inhibit, REM‐on SubC reticular neurons during waking, whereas carbachol‐excited SubC and PnO GABAergic neurons are involved in silencing locus coeruleus and dorsal raphe aminergic neurons during REM sleep. Orexinergic suppression of REM during waking is probably mediated in part via excitation of acetylcholine‐inhibited GABAergic neurons.

Collateral projections from the supramammillary nucleus to the medial septum and hippocampus

Previous reports have shown that the supramammillary nucleus projects to the medial septum and to the hippocampus, and specifically to the dentate gyrus and the CA2/CA3a region of the hippocampus. The aim of the present study was to examine collateral projections from the supramammillary nucleus to the septum and hippocampus. The fluorescent retrograde tracers, Fluororuby and Fluorogold, were injected into regions of the septum and hippocampus, respectively, and the supramammillary nucleus was examined for the presence of single‐ and double‐labeled neurons. The main findings were: 1) pronounced numbers of single‐labeled cells (about 40–60/section) were present in the supramammillary nucleus following retrograde tracer injections in either the septum or hippocampus; 2) single and double retrogradely labeled neurons were intermingled within the supramammillary nucleus and mainly localized to the lateral two‐thirds of the supramammillary nucleus; 3) approximately 5–10% of supramammillary cells were double‐labeled, ipsilaterally, and 2–4%, contralaterally, with injections in medial or lateral parts of the medial septum and the dentate gyrus of the hippocampus; and 4) approximately 3–5% of supramammillary cells were double‐labeled, ipsilaterally, and 1–2%, contralaterally, with injections in the medial septum and CA2/CA3a of the dorsal hippocampus. Cells of the supramammillary nucleus have been shown to fire rhythmically in bursts synchronous with the hippocampal theta rhythm and have been implicated in the generation of the theta rhythm. The supramammillary cells that we identified with collateral projections to the septum and hippocampus may be directly involved in generation of the theta rhythm. Synapse 38:281–293, 2000.

Differential effect of orexins (hypocretins) on serotonin release in the dorsal and median raphe nuclei of freely behaving rats

Orexin (hypocretin)-containing neurons in the perifornical hypothalamus project to widespread regions of the brain, including the dorsal and median raphe nuclei [Peyron C, Tighe DK, van den Pol AN, de Lecea L, Heller HC, Sutcliffe JG, Kilduff TS (1998) Neurons containing hypocretin (orexin) project to multiple neuronal systems. J Neurosci 18:9996-10015; Wang QP, Koyama Y, Guan JL, Takahashi K, Kayama Y, Shioda S (2005) The orexinergic synaptic innervation of serotonin- and orexin 1-receptor-containing neurons in the dorsal raphe nucleus. Regul Pept 126:35-42]. Orexin-A or orexin-B was infused by reverse microdialysis into the dorsal raphe nucleus or median raphe nucleus of freely behaving rats, and extracellular serotonin was simultaneously collected by microdialysis and analyzed by high-performance liquid chromatography. We have found that orexin-A produced a dose-dependent increase of serotonin in the dorsal raphe nucleus, but not in the median raphe nucleus. However, orexin-B elicited a small but significant effect in both the dorsal raphe nucleus and median raphe nucleus. Orexins may have regionally selective effects on serotonin release in the CNS, implying a unique interaction between orexins and serotonin in the regulation of activities including sleep-wakefulness.

Collateral projections from the median raphe nucleus to the medial septum and hippocampus

It has previously been shown that the median raphe nucleus (MR) is a source of pronounced projections to the septum and hippocampus. The present study examined collateral projections from MR to the medial septum (MS) and to various regions of the hippocampus. The fluorescent retrograde tracers, Fluororuby and Fluorogold, were injected into the septum and hippocampus, respectively, and the median raphe nucleus was examined for the presence of single- and double-labeled neurons. The dorsal raphe nucleus (DR) was also examined for the presence of single- and double-labeled cells and comparisons were made with the MR. The main findings were: (1) pronounced numbers of retrogradely labeled cells (approximately 50 cells/section) were present in MR with injections in the MS or in various regions of the hippocampus; (2) approximately 8-12% of MR cells were double-labeled following paired injections in the MS-CA1, MS-CA3, and MS-dentate gyrus of the dorsal hippocampus, the lateral MS-dentate gyrus, and the MS-ventral hippocampus; (3) single- and double-labeled cells were intermingled throughout MR and present in greater numbers in the rostral than caudal MR; and (4) significantly more single- and double-labeled cells were present in MR than in DR with all combinations of injections. These findings demonstrate that MR projects strongly to the MS and hippocampus, and that a significant population of MR neurons (8-12%) sends collateral projections to both sites. It is well established that the MR nucleus serves a direct role in the desynchronization of the electroencephalographic (EEG) activity of the hippocampus-or the blockade of the hippocampal theta rhythm. The MR neurons that we have identified with collateral projections to the septum and hippocampus may be critically involved in the modulation/control of the hippocampal EEG. A role for the MR in memory associated functions of the hippocampus is discussed.

Complex receptor mediation of acute ketamine application on in vitro gamma oscillations in mouse prefrontal cortex: modeling gamma band oscillation abnormalities in schizophrenia.

Schizophrenia (Sz), along with other neuropsychiatric disorders, is associated clinically with abnormalities in neocortical gamma frequency (30-80 Hz) oscillations. In Sz patients, these abnormalities include both increased and decreased gamma activity, and show a strong association with Sz symptoms. For several decades, administration of sub-anesthetic levels of ketamine has provided the most comprehensive experimental model of Sz-symptoms. While acute application of ketamine precipitates a psychotic-like state in a number of animal models, as well as humans, the underlying mechanisms behind this effect, including alteration of neuronal network properties, are incompletely understood, making an in vitro level analysis particularly important. Previous in vitro studies have had difficulty inducing gamma oscillations in neocortical slices maintained in submerged-type recording chambers necessary for visually guided whole-cell recordings from identified neurons. Consequently, here, we validated a modified method to evoke gamma oscillations using brief, focal application of the glutamate receptor agonist kainate (KA), in slices prepared from mice expressing green fluorescent protein in GABAergic interneurons (GAD67-GFP knock-in mice). Using this method, gamma oscillations dependent on activation of AMPA and GABA(A) receptors were reliably elicited in slices containing mouse prelimbic cortex, the rodent analogue of the human dorsolateral prefrontal cortex. Examining the effects of ketamine on this model, we found that bath application of ketamine significantly potentiated KA-elicited gamma power, an effect mimicked by selective NMDAR antagonists including a selective antagonist of NMDARs containing the NR2B subunit. Importantly, ketamine, unlike more specific NMDAR antagonists, also reduced the peak frequency of KA-elicited oscillatory activity. Our findings indicate that this effect is mediated not through NMDAR, but through slowing the decay kinetics of GABA(A) receptor-mediated inhibitory postsynaptic currents in identified GABAergic interneurons. These in vitro findings may help explain the complexities of gamma findings in clinical studies of Sz and prove useful in developing new therapeutic strategies.