Hypocretin (orexin) enhances neuron activity and cell synchrony in developing mouse GFP‐expressing locus coeruleus

Abstract

The noradrenergic neurons of the locus coeruleus (LC) play an important role in modulating arousal and selective attention. A similar function has been attributed to the hypocretin neurons of the hypothalamus which maintain a strong synaptic projection to the LC. As the LC can be difficult to detect in the embryonic and neonatal mouse brain, we used a new transgenic mouse with strong GFP expression in the LC under the regulation of a mouse prion promoter. GFP colocalized with immunoreactive tyrosine hydroxylase in sections and dispersed cultures of the LC, allowing visualization and whole cell or single‐unit recording from the LC in early stages of cellular development. GFP expression in the LC had no apparent effect on cellular physiology, including resting membrane potential, input resistance, spike threshold, depolarization‐induced spike frequency increase, current‐voltage relations, or hypocretin responses. In slices of the mature mouse and rat LC, hypocretin‐1 and −2 increased spike frequency, with hypocretin‐1 being an order of magnitude more potent. In the postnatal day (P) 0‐2 developing mouse slice during a developmental period when spikes could be elicited in some cells, other developing LC neurons showed rhythmic, subthreshold oscillations (≈1 Hz) in membrane potential (2.9‐7.4 mV amplitude); others were arrhythmic. Hypocretin‐1 depolarized the membrane potential, resulting in the appearance of spikes in developing LC cells that showed no spikes under control conditions. In the presence of TTX and glutamate receptor antagonists, hypocretin‐1‐mediated inward currents were blocked by substitution of choline‐Cl for NaCl, suggesting an excitatory mechanism based on an inward cation current. Hypocretin‐1 initiated strong regular membrane voltage oscillations in arrhythmic immature neurons. Hypocretin increased the temporal synchrony of action potentials studied with dual‐cell recording in P1‐P5 mouse LC slices, consistent with the view that synchrony of LC output, associated with improved cognitive performance, may be increased by hypocretin. Together these data suggest that the hypothalamus, via hypocretin projections, may therefore be in a position to enhance arousal and modulate plasticity in higher brain centres through the developing LC.

Mechanism for Hypocretin-mediated sleep-to-wake transitions

“…5). These results build upon previous in vivo electrophysiological experiments demonstrating that Hcrts depolarize LC neurons (12,18,31) and increase LC discharge rates (12,17,(19)(20)(21). We conclude that the LC gates Hcrt-mediated sleep-to-wake transitions during NREM sleep and serves as a necessary downstream effector for Hcrt function in promoting arousal.…”

“…To gain insight into the effects of Hcrt stimulation on LC membrane potential, we built a minimal realistic model of Hcrt-LC circuitry, using previous biophysical studies (18,29,31). Our model focuses on the effect of Hcrt activity on downstream LC neurons, showing that Hcrt stimulation slowly (over several seconds) increases the membrane potential of LC neurons from baseline (<1 Hz) to tonic discharge (1-3 Hz) (Fig.…”

“…Indeed, the LC receives the densest afferent projections from Hcrt neurons (14,17). Application of Hcrts into the LC region depolarizes LC neurons (12,18) and increases LC discharge rates (12,17,(19)(20)(21). Interestingly, RNAi-mediated knockdown of Hcrt receptor signaling in the LC region increases REM sleep (22).…”

Identification of connexin36 in gap junctions between neurons in rodent locus coeruleus

“…For FRIL, P7-P21 and adult Sprague-Dawley rats were obtained from Laboratory Animal Services at Colorado State University. In addition, mice whose LC neurons express Enhanced Green Fluorescent Protein (EGFP) (van den Pol et al, 2002) were obtained from a colony established courtesy of Anthony N. van den Pol, Yale University School of Medicine, New Haven, CT.…”

Overview of orexin/hypocretin system

“…After this epoch-making discovery, a whole series of studies confirmed that the orexin system plays a central role in the regulation of sleep/wakefulness. Neurons expressing orexin (orexin neurons) activate nuclei considered wake-promoting, including LC noradrenergic neurons (Horvath et al, 1999;van den Pol et al, 2002), raphe serotonergic neurons Liu et al, 2002), TMN histaminergic neurons Yamanaka et al, 2002), and PPT/LDT and basal forebrain cholinergic neurons Eriksson et al, 2001). Central administration of orexin-A in rodents reduces REM and non-REM sleep, and increases wakefulness time (Hagan et al, 1999).…”