Characterization of basal forebrain glutamate neurons suggests a role in control of arousal and avoidance behavior.

The basal forebrain (BF) is involved in arousal, attention, and reward processing but the role of individual BF neuronal subtypes is still being uncovered. Glutamatergic neurons are the least well-understood of the three main BF neurotransmitter phenotypes. Here we analyzed the distribution, size, calcium-binding protein content and projections of the major group of BF glutamatergic neurons expressing the vesicular glutamate transporter subtype 2 (vGluT2) and tested the functional effect of activating them. Mice expressing Cre recombinase under the control of the vGluT2 promoter were crossed with a reporter strain expressing the red fluorescent protein, tdTomato, to generate vGluT2-cre-tdTomato mice. Immunohistochemical staining for choline acetyltransferase and a cross with mice expressing green fluorescent protein selectively in GABAergic neurons confirmed that cholinergic, GABAergic and vGluT2+ neurons represent distinct BF subpopulations. Subsets of BF vGluT2+ neurons expressed the calcium-binding proteins calbindin or calretinin, suggesting that multiple subtypes of BF vGluT2+ neurons exist. Anterograde tracing using adeno-associated viral vectors expressing channelrhodopsin2-enhanced yellow fluorescent fusion proteins revealed major projections of BF vGluT2+ neurons to neighboring BF cholinergic and parvalbumin neurons, as well as to extra-BF areas involved in the control of arousal or aversive/rewarding behavior such as the lateral habenula and ventral tegmental area. Optogenetic activation of BF vGluT2+ neurons elicited a striking avoidance of the area where stimulation was given, whereas stimulation of BF parvalbumin or cholinergic neurons did not. Together with previous optogenetic findings suggesting an arousal-promoting role, our findings suggest that BF vGluT2 neurons play a dual role in promoting wakefulness and avoidance behavior.

Activation of basal forebrain purinergic P2 receptors promotes wakefulness in mice.

The functions of purinergic P2 receptors (P2Rs) for extracellular adenosine triphosphate (ATP) are poorly understood. Here, for the first time, we show that activation of P2Rs in an important arousal region, the basal forebrain (BF), promotes wakefulness, whereas inhibition of P2Rs promotes sleep. Infusion of a non-hydrolysable P2R agonist, ATP-γ-S, into mouse BF increased wakefulness following sleep deprivation. ATP-γ-S depolarized BF cholinergic and cortically-projecting GABAergic neurons in vitro, an effect blocked by antagonists of ionotropic P2Rs (P2XRs) or glutamate receptors. In vivo, ATP-γ-S infusion increased BF glutamate release. Thus, activation of BF P2XRs promotes glutamate release and excitation of wake-active neurons. Conversely, pharmacological antagonism of BF P2XRs decreased spontaneous wakefulness during the dark (active) period. Together with previous findings, our results suggest sleep-wake regulation by BF extracellular ATP involves a balance between excitatory, wakefulness-promoting effects mediated by direct activation of P2XRs and inhibitory, sleep-promoting effects mediated by degradation to adenosine.

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.

Sleep fragmentation reduces hippocampal CA1 pyramidal cell excitability and response to adenosine.

Sleep fragmentation (SF) impairs the restorative/cognitive benefits of sleep via as yet unidentified alterations in neural physiology. Previously, we found that hippocampal synaptic plasticity and spatial learning are impaired in a rat model of SF which utilizes a treadmill to awaken the animals every 2 min, mimicking the frequency of awakenings observed in human sleep apnea patients. Here, we investigated the cellular mechanisms responsible for these effects, using whole-cell patch-clamp recordings. 24h of SF decreased the excitability of hippocampal CA1 pyramidal neurons via decreased input resistance, without alterations in other intrinsic membrane or action potential properties (when compared to cage controls, or to exercise controls that experienced the same total amount of treadmill movement as SF rats). Contrary to our initial prediction, the hyperpolarizing response to bath applied adenosine (30 microM) was reduced in the CA1 neurons of SF treated rats. Our initial prediction was based on the evidence that sleep loss upregulates cortical adenosine A1 receptors; however, the present findings are consistent with a very recent report that hippocampal A1 receptors are not elevated by sleep loss. Thus, increased adenosinergic inhibition is unlikely to be responsible for reduced hippocampal long-term potentiation in SF rats. Instead, the reduced excitability of CA1 pyramidal neurons observed here may contribute to the loss of hippocampal long-term potentiation and hippocampus-dependent cognitive impairments associated with sleep disruption.

Microdialysis elevation of adenosine in the basal forebrain produces vigilance impairments in the rat psychomotor vigilance task.

STUDY OBJECTIVE: The inhibitory neuromodulator adenosine has been proposed as a homeostatic sleep factor that acts potently in the basal forebrain (BF) to increase sleepiness. Here 300 microM of adenosine was dialyzed in the BF of rats, and the effect on vigilance was determined in the rat Psychomotor Vigilance Task (rPVT). DESIGN: Rats experienced all experimental conditions in a repeated-measures, cross-over design. PATIENTS OR PARTICIPANTS: Twelve young adult male Fischer-Norway rats. INTERVENTIONS: Sustained attention performance in the rPVT was evaluated following 2 hours of bilateral microdialysis perfusion of vehicle, adenosine (300 microM), or codialysis of 300 microM of adenosine with the A1 receptor antagonist 8-cyclopentyltheophylline. MEASUREMENTS AND RESULTS: During rPVT performance, response latencies and performance lapses increased significantly after adenosine dialysis when compared with baseline (no dialysis) or vehicle dialysis sessions. The codialysis of 8-cyclopentyltheophylline with adenosine completely blocked the effects produced by adenosine alone, resulting in performance equivalent to that of the vehicle sessions. CONCLUSIONS: Pharmacologic elevation of BF adenosine in rats produced vigilance impairments resembling the effect of sleep deprivation on vigilance performance in both man and rats. This effect of exogenous adenosine was completely blocked by codialysis with an adenosine A1 receptor antagonist. The results are consistent with the hypothesis that sleep loss induces elevations of BF adenosine that, acting via A1 receptors, lead to increased sleepiness and impaired vigilance.