Nanodomain coupling between Ca(2+) channels and sensors of exocytosis at fast mammalian synapses

The physical distance between presynaptic Ca2+ channels and the Ca2+ sensors that trigger exocytosis of neurotransmitter-containing vesicles is a key determinant of the signalling properties of synapses in the nervous system. Recent functional analysis indicates that in some fast central synapses, transmitter release is triggered by a small number of Ca2+ channels that are coupled to Ca2+ sensors at the nanometre scale. Molecular analysis suggests that this tight coupling is generated by protein–protein interactions involving Ca2+ channels, Ca2+ sensors and various other synaptic proteins. Nanodomain coupling has several functional advantages, as it increases the efficacy, speed and energy efficiency of synaptic transmission

Characterization of Sleep in Zebrafish and Insomnia in Hypocretin Receptor Mutants

Sleep is a fundamental biological process conserved across the animal kingdom. The study of how sleep regulatory networks are conserved is needed to better understand sleep across evolution. We present a detailed description of a sleep state in adult zebrafish characterized by reversible periods of immobility, increased arousal threshold, and place preference. Rest deprivation using gentle electrical stimulation is followed by a sleep rebound, indicating homeostatic regulation. In contrast to mammals and similarly to birds, light suppresses sleep in zebrafish, with no evidence for a sleep rebound. We also identify a null mutation in the sole receptor for the wake-promoting neuropeptide hypocretin (orexin) in zebrafish. Fish lacking this receptor demonstrate short and fragmented sleep in the dark, in striking contrast to the excessive sleepiness and cataplexy of narcolepsy in mammals. Consistent with this observation, we find that the hypocretin receptor does not colocalize with known major wake-promoting monoaminergic and cholinergic cell groups in the zebrafish. Instead, it colocalizes with large populations of GABAergic neurons, including a subpopulation of Adra2a-positive GABAergic cells in the anterior hypothalamic area, neurons that could assume a sleep modulatory role. Our study validates the use of zebrafish for the study of sleep and indicates molecular diversity in sleep regulatory networks across vertebrates

Cholinergic filtering in the recurrent excitatory microcircuit of cortical layer 4

Neocortical acetylcholine (ACH) release is known to enhance signal processing by increasing the amplitude and signal-to-noise ratio (SNR) of sensory responses. It is widely accepted that the larger sensory responses are caused by a persistent increase in the excitability of all cortical excitatory neurons. Here, contrary to this concept, we show that ACH persistently inhibits layer 4 (L4) spiny neurons, the main targets of thalamocortical inputs. Using whole-cell recordings in slices of rat primary somatosensory cortex, we demonstrate that this inhibition is specific to L4 and contrasts with the ACH-induced persistent excitation of pyramidal cells in L2/3 and L5. We find that this inhibition is induced by postsynaptic M4-muscarinic ACH receptors and is mediated by the opening of inwardly rectifying potassium (Kir) channels. Pair recordings of L4 spiny neurons show that ACH reduces synaptic release in the L4 recurrent microcircuit. We conclude that ACH has a differential layer-specific effect that results in a filtering of weak sensory inputs in the L4 recurrent excitatory microcircuit and a subsequent amplification of relevant inputs in L2/3 and L5 excitatory microcircuits. This layer-specific effect may contribute to improve cortical SNR

How the “slow” Ca(2+) buffer parvalbumin affects transmitter release in nanodomain coupling regimes at GABAergic synapses

Parvalbumin is thought to act in a manner similar to EGTA, but how a slow Ca2+ buffer affects nanodomain-coupling regimes at GABAergic synapses is unclear. Direct measurements of parvalbumin concentration and paired recordings in rodent hippocampus and cerebellum revealed that parvalbumin affects synaptic dynamics only when expressed at high levels. Modeling suggests that, in high concentrations, parvalbumin may exert BAPTA-like effects, modulating nanodomain coupling via competition with local saturation of endogenous fixed buffers

Cholinergic Signals in Mouse Barrel Cortex during Active Whisker Sensing

Summary: Internal brain states affect sensory perception, cognition, and learning. Many neocortical areas exhibit changes in the pattern and synchrony of neuronal activity during quiet versus active behaviors. Active behaviors are typically associated with desynchronized cortical dynamics. Increased thalamic firing contributes importantly to desynchronize mouse barrel cortex during active whisker sensing. However, a whisking-related cortical state change persists after thalamic inactivation, which is mediated at least in part by acetylcholine, as we show here by using whole-cell recordings, local pharmacology, axonal calcium imaging, and optogenetic stimulation. During whisking, we find prominent cholinergic signals in the barrel cortex, which suppress spontaneous cortical activity. The desynchronized state of barrel cortex during whisking is therefore driven by at least two distinct signals with opposing functions: increased thalamic activity driving glutamatergic excitation of the cortex and increased cholinergic input suppressing spontaneous cortical activity. : Eggermann et al. now find that the desynchronized state of the barrel cortex during active whisker sensing is accompanied by increased cholinergic input, which suppresses slow spontaneous cortical activity in excitatory layer 2/3 barrel cortex neurons

Suprachiasmatic modulation of noradrenaline release in the ventrolateral preoptic nucleus.

As the major brain circadian pacemaker, the suprachiasmatic nucleus (SCN) is known to influence the timing of sleep and waking. We thus investigated here the effect of SCN stimulation on neurons of the ventrolateral preoptic nucleus (VLPO) thought to be involved in promoting sleep. Using an acute in vitro preparation of the rat anterior hypothalamus/preoptic area, we found that whereas single-pulse stimulations of the SCN evoked standard fast ionotropic IPSPs and EPSPs, train stimulations unexpectedly evoked a long-lasting inhibition (LLI). Such LLIs could also be evoked in VLPO neurons by pressure application of NMDA within the SCN, indicating the specific activation of SCN neurons. This LLI was shown to result from the presynaptic facilitation of noradrenaline release, because it was suppressed in presence of yohimbine, a selective antagonist of alpha2-adrenoreceptors. The LLI depended on the opening of a potassium conductance, because it was annulled at E(K) and could be reversed below E(K). These results show that the SCN can provide an LLI of the sleep-promoting VLPO neurons that could play a role in the circadian organization of the sleep-waking cycle

Opposite effects of noradrenaline and acetylcholine upon hypocretin/orexin versus melanin concentrating hormone neurons in rat hypothalamic slices

Hypocretin/orexin (Hcrt/Orx) and melanin concentrating hormone (MCH) are peptides contained in overlapping cell groups of the lateral hypothalamus and commonly involved in regulating sleep-wake states and energy balance, though likely in different ways. To see if these neurons are similarly or differentially modulated by neurotransmitters of the major brainstem arousal systems, the effects of noradrenaline (NA) and carbachol, a cholinergic agonist, were examined on identified Hcrt/Orx and MCH neurons in rat hypothalamic slices. Whereas both agonists depolarized and excited Hcrt/Orx neurons, they both hyperpolarized MCH neurons by direct postsynaptic actions. According to the activity profiles of the noradrenergic locus coeruleus and cholinergic pontomesencephalic neurons across the sleep-waking cycle, the Hcrt/Orx neurons would be excited by NA and acetylcholine (ACh) and thus active during arousal, whereas the MCH neurons would be inhibited by NA and ACh and thus inactive during arousal while disinhibited and possibly active during slow wave sleep. According to the present pharmacological results, Hcrt/Orx neurons may thus stimulate arousal in tandem with other arousal systems, whereas MCH neurons may function in opposition with other arousal systems and thus potentially dampen arousal to promote sleep

Orexins (hypocretins) directly excite tuberomammillary neurons

Wakefulness has recently been shown to depend upon the newly identified orexin (or hypocretin) neuropeptides by the findings that alteration in their precursor protein, their receptors or the neurons that produce them leads to the sleep disorder narcolepsy in both animals and humans. The questions of how and where these brain peptides act to maintain wakefulness remain unresolved. The purpose of the present study was to determine whether the orexins could directly affect hypothalamic histaminergic neurons, which are known to contribute to the state of wakefulness by their diffuse projections through the brain. Using brain slices, we recorded in the ventral tuberomammillary nuclei from neurons identified as histaminergic on the basis of their previously described morphological and electrophysiological characteristics and found that they were depolarized and excited by the orexins through a direct postsynaptic action. We then compared the depolarizing effect of orexin A and B and found that they were equally effective upon these cells. This latter finding suggests that the effect of orexins is mediated by orexin type 2 receptors, which are those lacking in narcoleptic dogs. Our results therefore show that the histaminergic neurons of the tuberomammillary nuclei represent an important target for the orexin system in the maintenance of wakefulness

Selective action of orexin (hypocretin) on nonspecific thalamocortical projection neurons

As is evident from the pathological consequences of its absence in narcolepsy, orexin (hypocretin) appears to be critical for the maintenance of wakefulness. Via diffuse projections through the brain, orexin-containing neurons in the hypothalamus may act on a number of wake-promoting systems. Among these are the intralaminar and midline thalamic nuclei, which project in turn in a widespread manner to the cerebral cortex within the nonspecific thalamocortical projection system. Testing the effect of orexin in rat brain slices, in two nuclei of this system, centromedial (CM) nuclei and rhomboid nuclei, we found that it depolarized and excited all neurons tested through a direct postsynaptic action. An additional analysis of this effect in CM neurons indicates that it results from the decrease of a potassium conductance. By a detailed comparison of the effects of orexin A and B, we established that orexin B was more potent than orexin A, indicating the probable mediation by orexin type 2 receptors. In contrast to its effect on the nonspecific thalamocortical projection neurons, orexin had no effect on the specific sensory relay neurons of the somatic, ventral posterolateral, and visual dorsal lateral geniculate nuclei. Orexin differs in this regard from norepinephrine and acetylcholine, to which neurons in the specific and nonspecific systems are sensitive. Orexin may thus act in the thalamus to promote wakefulness by exciting neurons of the nonspecific thalamocortical projection system, which, through widespread projections to the cerebral cortex, stimulate and maintain cortical activation

Orexins/hypocretins excite basal forebrain cholinergic neurones

The orexins (orexin A and B, also known as hypocretin 1 and 2) are two recently identified neuropeptides (de Lecea et al., 1998; Sakurai et al., 1998) which are importantly implicated in the control of wakefulness (for reviews see Hungs and Mignot, 2001; van den Pol, 2000; Willie et al., 2001 ). Indeed, alteration in these peptides' precursor, their receptors or the hypothalamic neurones that produce them leads to the sleep disorder narcolepsy (Chemelli et al., 1999; Lin et al., 1999; Peyron et al., 2000; Thannickal et al., 2000). The mechanisms by which the orexins modulate wakefulness, however, are still unclear. Their presence in fibres coursing from the hypothalamus (Peyron et al., 1998) up to the preoptic area (POA) and basal forebrain (BF) suggests that they might influence the important sleep and waking neural systems situated there (Jones, 2000). The present study, performed in rat brain slices, demonstrates, however, that the orexins have no effect on the GABA sleep-promoting neurones of the POA, whereas they have a strong and direct excitatory effect on the cholinergic neurones of the contiguous BF. In addition, by comparing the effects of orexin A and B we demonstrate here that orexins' action depends upon orexin type 2 receptors (OX(2)), which are those lacking in narcoleptic dogs (Lin et al., 1999). These results suggest that the orexins excite cholinergic neurones that release acetylcholine in the cerebral cortex and thereby contribute to the cortical activation associated with wakefulness