Véronique Fabre

Contribution of 5-HT2 Receptor Subtypes to Sleep–Wakefulness and Respiratory Control, and Functional Adaptations in Knock-Out Mice Lacking 5-HT2A Receptors

Serotonin (5-hydroxytryptamine; 5-HT) plays key roles in sleep–wakefulness regulation. Evidence indicates that 5-HT2 receptors are involved mainly in non-rapid eye movement sleep (NREMS) regulation and respiratory control. Here, we investigated the relative contribution of 5-HT2A, 5-HT2B, and 5-HT2C receptor subtypes to NREMS and breathing during sleep, using 5-HT2 subtype-selective ligands in wild-type (5-HT2A+/+) and knock-out (5-HT2A–/–) mice that do not express 5-HT2A receptors. Acute blockade of 5-HT2A receptors induced an increase in NREMS in 5-HT2A+/+ mice, but not 5-HT2A–/– mutants, which spontaneously expressed less NREMS than wild-type animals. In 5-HT2A+/+ mice, 5-HT2B receptor blockade produced a reduction of NREMS, whereas receptor activation induced an increase in this sleep stage. These effects were less pronounced in 5-HT2A–/– mice, indicating a lower sensitivity of 5-HT2B receptors in mutants, with no change in 5-HT2B mRNA. Blockade of 5-HT2C receptors had no effect on NREMS in both strains. In addition, an increase in EEG power density after sleep deprivation was observed in 5-HT2A+/+ mice but not in 5-HT2A–/– mice. Whole-body plethysmographic recordings indicated that 5-HT2A receptor blockade in 5-HT2A+/+ mice reduced NREMS apneas and bradypneas that occurred after sighs. In contrast, in 5-HT2A–/– mutants, NREMS apneas were not modified, and bradypnea after sighs were more pronounced. Our results demonstrate that 5-HT exerts a 5-HT2B-mediated facilitation of NREMS, and an influence respectively inhibitory on NREMS and facilitatory on sleep apnea generation, via 5-HT2A receptors. Moreover, 5-HT2A gene knock-out leads to functional compensations yielding adaptive changes opposite to those caused by pharmacological blockade of 5-HT2A receptors in 5-HT2A+/+ mice.

Early Life Blockade of 5-Hydroxytryptamine 1A Receptors Normalizes Sleep and Depression-Like Behavior in Adult Knock-Out Mice Lacking the Serotonin Transporter

In serotonin transporter knock-out (5-HTT−/−) mice, extracellular serotonin (5-HT) levels are markedly elevated in the brain, and rapid eye movement sleep (REMS) is enhanced compared with wild-type mice. We hypothesized that such sleep impairment at adulthood results from excessive serotonergic tone during early life. Thus, we assessed whether neonatal treatment with drugs capable of limiting the impact of 5-HT on the brain could normalize sleep patterns in 5-HTT−/− mutants. We found that treatments initiated at postnatal day 5 and continued for 2 weeks with the 5-HT synthesis inhibitor para-chlorophenylalanine, or for 4 weeks with the 5-HT1A receptor (5-HT1AR) antagonist N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl) cyclohexane carboxamide (WAY 100635), induced total or partial recovery of REMS, respectively, in 5-HTT−/− mutants. Early life treatment with WAY 100635 also reversed the depression-like behavior otherwise observed in these mutants. Possible adaptive changes in 5-HT1AR after neonatal treatment with WAY 100635 were investigated by measuring 5-HT1A binding sites and 5-HT1A mRNA in various REMS- and/or depression-related brain areas, as well as 5-HT1AR-mediated hypothermia and inhibition of neuronal firing in the dorsal raphe nucleus. None of these characteristics were modified in parallel with REMS recovery, suggesting that 5-HT1ARs involved in wild-type phenotype rescue in 5-HTT−/− mutants are located in other brain areas or in 5-HT1AR-unrelated circuits where they could be transiently expressed during development. The reversal of sleep alterations and depression-like behavior after early life blockade of 5-HT1AR in 5-HTT−/− mutants might open new perspectives regarding preventive care of sleep and mood disorders resulting from serotonin transporter impairments during development.

Interaction of the hypocretins with neurotransmitters in the nucleus accumbens

The hypocretins (hcrt1 and hcrt2), also known as orexins, are two neuropeptides derived from the same precursor, expressed in a few thousand cells in the lateral hypothalamus. Hypocretin-containing cells project throughout the brain, including ascending projections to the olfactory bulb and cerebral cortex, through the medial septum and the nucleus accumbens. Here, we have studied the interactions of the hypocretins with different neurotransmitters by patch clamp recording of acutely dissociated cells from the nucleus accumbens. Application of hcrt1 or hcrt2 decreased postsynaptic NMDA currents, enhanced GABA currents but did not affect glycine-activated conductances. Our results strongly suggest that the hypocretin peptides may be inhibitory peptides, probably via binding hcrt receptor 2.

Hypocretins/orexins as integrators of physiological information: lessons from mutant animals

The hypocretins/orexins (hcrts) are two recently described neuropeptides derived from the same precursor and expressed in a few thousand neurons in the perifornical area of the lateral hypothalamus, which project throughout the brain. The hypocretins bind to two G-protein coupled receptors with different selective affinities. Positional cloning of the gene responsible for a canine model of narcolepsy revealed that this disease is caused by mutations in hypocretin receptor type 2. Parallel studies with hypocretin/orexin knockout mice showed behavioral arrests reminiscent of narcolepsy-like attacks. Narcoleptic patients have decreased hypocretin-containing neurons suggesting that narcolepsy in humans is caused by selective neurodegeneration of hypocretinergic neurons. Additional functions for the hypocretins on regulation of energy balance neuroendocrine release and sympathetic outflow have been described. Here we review studies in humans and mutant animals that have provided clues about the functions of the hypocretinergic system, which appear to involve the coherent regulation of networks that dictate the states of arousal.

Cortistatin overexpression in transgenic mice produces deficits in synaptic plasticity and learning

Cortistatin-14 (CST) is a neuropeptide expressed in cortical and hippocampal interneurons that shares 11 of 14 residues with somatostatin. In contrast to somatostatin, infusion of CST decreases locomotor activity and selectively enhances slow wave sleep. Here, we show that transgenic mice that overexpress cortistatin under the control of neuron-specific enolase promoter do not express long-term potentiation in the dentate gyrus. This blockade of dentate LTP correlates with profound impairment of hippocampal-dependent spatial learning. Exogenously applied CST to slices of wild-type mice also blocked induction of LTP in the dentate gyrus. Our findings implicate cortistatin in the modulation of synaptic plasticity and cognitive function. Thus, increases in hippocampal cortistatin expression during aging could have an impact on age-related cognitive deficits.

Altered Sleep Homeostasis after Restraint Stress in 5-HTT Knock-Out Male Mice: A Role for Hypocretins

Restraint stress produces changes in the sleep pattern that are mainly characterized by a delayed increase in rapid eye movement sleep (REMS) amounts. Because the serotonin (5-HT) and the hypocretin (hcrt) systems that regulate REMS are interconnected, we used mutant mice deficient in the 5-HT transporter (5-HTT−/−) to examine the role of 5-HT and hcrt neurotransmissions in the sleep response to stress. In contrast to wild-type mice, restraint stress did not induce a delayed increase in REMS amounts in 5-HTT−/− mice, indicating impaired sleep homeostasis in mutants. However, pharmacological blockade of the hcrt type 1 receptor (hcrt-R1) before restraint stress restored the REMS increase in 5-HTT−/− mice. In line with this finding, 5-HTT−/− mutants displayed after restraint stress higher long-lasting activation of hypothalamic preprohcrt neurons than wild-type mice and elevated levels of the hcrt-1 peptide and the hcrt-R1 mRNA in the anterior raphe area. Thus, hypocretinergic neurotransmission was enhanced by stress in 5-HTT−/− mice. Furthermore, in 5-HTT−/− but not wild-type mice, hypothalamic levels of the 5-HT metabolite 5-hydroxyindole acetic acid significantly increased after restraint stress, indicating a marked enhancement of serotonergic neurotransmission in mutants. Altogether, our data show that increased serotonergic -and in turn hypocretinergic- neurotransmissions exert an inhibitory influence on stress-induced delayed REMS. We propose that the direct interactions between hcrt neurons in the hypothalamus and 5-HT neurons in the anterior raphe nuclei account, at least in part, for the adaptive sleep–wakefulness regulations triggered by acute stress.

Overexpression of the human β-amyloid precursor protein downregulates cortistatin mRNA in PDAPP mice

We measured preprocortistatin mRNA expression in young and aged transgenic (Tg) mice overexpressing the human beta-amyloid precursor protein (hbetaAPP) under the platelet-derived growth factor-beta promoter. Our findings suggest that the significant increase in hippocampal cortistatin mRNA expression during normal aging is significantly attenuated in Tg mice at an age known to exhibit beta-amyloid protein (Abeta) deposition. These deficits in cortistatin expression may play a role in the deficits in hippocampal-dependent spatial learning and sleep/wake states previously demonstrated in aged Tg mice.

Cortistatin radioligand binding in wild-type and somatostatin receptor-deficient mouse brain

Cortistatin-14 (CST-14) is a recently discovered member of the somatostatin family of neuropeptides. It shares 11 of its 14 amino acids with somatostatin-14 (SRIF-14). In the present study, binding sites for cortistatin-14 in the mouse brain were examined and compared to those for somatostatin using iodinated cortistatin-14 and iodinated somatostatin-14. By in vitro receptor autoradiography, high densities of cortistatin-14 and somatostatin-14 specific binding sites were detected in the cortex, hippocampal formation, basolateral amygdala and medial habenula. Unlabeled 100 nM cortistatin-14 inhibited iodinated somatostatin-14 binding in the hippocampus, but not in the cortex or amygdaloid nuclei. In somatostatin receptor subtype-2 knock-out (KO) mice, autoradiographic iodinated somatostatin-14 binding was observed in the hippocampus and habenula but was removed in the cortex and amygdaloid nuclei, specific iodinated cortistatin-14 binding sites were found in the hippocampus, habenula and throughout the cortex. We conclude that the somatostatin receptor subtype-2 is responsible for somatostatin binding in cortical and amygdaloid regions and that cortistatin predominantly interacts with the same receptors as somatostatin.

Cortistatin promotes and negatively correlates with slow-wave sleep

Sleep need is characterized by the level of slow‐wave activity (SWA) and increases with time spent awake. The molecular nature of this sleep homeostatic process is practically unknown. Here, we show that intracerebroventricular administration of the neuropeptide, cortistatin (CST‐14), enhances EEG synchronization by selectively promoting deep slow‐wave sleep (SWS) during both the light and dark period in rats. CST‐14 also increases the level of slow‐wave activity (SWA) within deep SWS during the first two hours following CST‐14 administration. Steady‐state levels of preprocortistatin mRNA oscillate during the light : dark cycle and are four‐fold higher upon total 24‐h sleep deprivation, returning progressively to normal levels after eight hours of sleep recovery. Preprocortistatin mRNA is expressed upon sleep deprivation in a particular subset of cortical interneurons that colocalize with c‐fos. In contrast, the number of CST‐positive cells coexpressing pERK1/2 decreases under sleep deprivation. The capacity of CST‐14 to increase SWA, together with preprocortistatins inverse correlation with time spent in SWS, suggests a potential role in sleep homeostatic processes.

Cortistatin- A Novel Member of the Somatostatin Gene Family

Cortistatin (CST) is a recently discovered neuropeptide from the somatostatin (SST) gene family named after its predominantly cortical expression and ability to depress cortical activity [1]. CST shows many remarkable structural and functional similarities to its related neuropeptide SST. However, the many physiological differences between CST and SST are just as remarkable as the similarities. CST-14 shares 11 of its 14 amino acids with SST-14, including the FWKT tetramer thought to be responsible for SSTs receptor interactions and the pair of cysteine residues that likely render the peptides cyclic [2]. Yet the nucleotide sequences and chromosomal localizations of these genes clearly indicate they are products of separate genes and CSTs activity in the brain is widely distinct from that of SST [3].