The GABAergic Gudden's dorsal tegmental nucleus: A new relay for serotonergic regulation of sleep-wake behavior in the mouse.

Serotonin (5-HT) neurons are involved in wake promotion and exert a strong inhibitory influence on rapid eye movement (REM) sleep. Such effects have been ascribed, at least in part to the action of 5-HT at post-synaptic 5-HT1A receptors (5-HT1AR) in the brainstem, a major wake/REM sleep regulatory center. However, the neuroanatomical substrate through which 5-HT1AR influence sleep remains elusive. We therefore investigated whether a brainstem structure containing a high density of 5-HT1AR mRNA, the GABAergic Gudden's dorsal tegmental nucleus (DTg), may contribute to 5-HT-mediated regulatory mechanisms of sleep-wake stages. We first found that bilateral lesions of the DTg promote wake at the expense of sleep. In addition, using local microinjections into the DTg in freely moving mice, we showed that local activation of 5-HT1AR by the prototypical agonist 8-OH-DPAT enhances wake and reduces deeply REM sleep duration. The specific involvement of 5-HT1AR in the latter effects was further demonstrated by ex vivo extracellular recordings showing that the selective 5-HT1AR antagonist WAY 100635 prevented DTg neuron inhibition by 8-OH-DPAT. We next found that GABAergic neurons of the ventral DTg exclusively targets glutamatergic neurons of the lateral mammillary nucleus (LM) in the posterior hypothalamus by means of anterograde and retrograde tracing techniques using cre driver mouse lines and a modified rabies virus. Altogether, our findings strongly support the idea that 5-HT-driven enhancement of wake results from 5-HT1AR-mediated inhibition of DTg GABAergic neurons that would in turn disinhibit glutamatergic neurons in the mammillary bodies. We therefore propose a Raphe→DTg→LM pathway as a novel regulatory circuit underlying 5-HT modulation of arousal.

Single intranasal administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in C57BL/6 mice models early preclinical phase of Parkinson's disease.

Many studies have shown that deficits in olfactory and cognitive functions precede the classical motor symptoms seen in Parkinson's disease (PD) and that olfactory testing may contribute to the early diagnosis of this disorder. Although the primary cause of PD is still unknown, epidemiological studies have revealed that its incidence is increased in consequence of exposure to certain environmental toxins. In this study, most of the impairments presented by C57BL/6 mice infused with a single intranasal (i.n.) administration of the proneurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (1 mg/nostril) were similar to those observed during the early phase of PD, when a moderate loss of nigral dopamine neurons results in olfactory and memory deficits with no major motor impairments. Such infusion decreased the levels of the enzyme tyrosine hydroxylase in the olfactory bulb, striatum, and substantia nigra by means of apoptotic mechanisms, reducing dopamine concentration in different brain structures such as olfactory bulb, striatum, and prefrontal cortex, but not in the hippocampus. These findings reinforce the notion that the olfactory system represents a particularly sensitive route for the transport of neurotoxins into the central nervous system that may be related to the etiology of PD. These results also provide new insights in experimental models of PD, indicating that the i.n. administration of MPTP represents a valuable mouse model for the study of the early stages of PD and for testing new therapeutic strategies to restore sensorial and cognitive processes in PD.

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.

[Contribution of animal experimentation to pharmacology]. / Apport de l'animal a la recherche pharmacologique.

Animal experimentation is of considerable importance in pharmacology and cannot yet be avoided when studying complex, highly integrated physiological functions. The use of animals has been drastically reduced in the classical phases of pharmacological research, for example when comparing several compounds belonging to the same pharmacological class. However, animal experiments remain crucial for generating and validating new therapeutic concepts. Three examples of such research, conducted in strict ethical conditions, will be used to illustrate the different ways in which animal experimentation has contributed to human therapeutics.

Interactions between TrkB signaling and serotonin excess in the developing murine somatosensory cortex: a role in tangential and radial organization of thalamocortical axons.

Mice lacking monoamine oxidase A (MAOA) display high levels of brain serotonin during the first postnatal week, causing an exuberant outgrowth of thalamocortical axons (TCAs) in layer IV of the somatosensory cortex (S1). We asked whether this exuberance is attributable to abnormal TrkB signaling, because modulation of TrkB signaling during a critical period dramatically influences the segregation and the morphology of TCAs in layer IV of the visual cortex. Using in situ hybridization and ELISA immunoassays, we showed that the levels of trkB mRNA and BDNF and neurotrophin-4 (NT-4) proteins are normal in the thalamus and the cortex of mice lacking MAOA during barrel field formation. Because the release of BDNF and NT-4 could be abnormal in MAOA knock-out (KO) mice, we tested whether abnormal TrkB signaling is required for TCA exuberance in MAOA-KO mice by generating mice lacking both trkB and MAOA. Surprisingly, these mice exhibited more severe phenotypes than those found in MAOA-KO mice: a widespread tangential expansion of TCAs in layer IV of the cortex, resulting in a fusion of all sensory representations and a radial expansion of TCAs in layers II-III of the cortex. Careful examination of mice lacking trkB alone revealed subtle alterations of TCAs, with abnormal invasion of layer III. This study reveals the following: (1) expression of trkB, BDNF, and NT-4 are not modulated by an excess of serotonin during barrel formation, (2) TrkB signaling limits branching of TCAs in inappropriate supragranular cortical layers, and (3) serotonin and TrkB signaling act together to cluster thalamocortical axons in layer IV.

Adenosine receptor-mediated control of in vitro release of pain-related neuropeptides from the rat spinal cord.

Although it is well established that adenosine exerts antinociceptive effects at the spinal level in various species including human, the mechanisms responsible for such effects are still a matter of debate. We presently investigated whether adenosine-induced antinociception might possibly be related to an inhibitory influence of this neuromodulator on the spinal release of neuropeptides implicated in the transfer and/or control of nociceptive signals. For this purpose, the K(+)-evoked overflow of substance P-, calcitonin gene-related peptide (CGRP)- and cholecystokinin-like materials was measured from slices of the dorsal half of the rat lumbar enlargement superfused with an artificial cerebrospinal fluid supplemented with increasing concentrations of various adenosine receptor ligands. The data showed that stimulation of adenosine A(1) and (possibly) A(3) receptors, but not A(2A) receptors, exerted an inhibitory influence on the spinal release of CGRP-like material. In contrast, none of the adenosine A(1), A(2A) and A(3) receptor agonists tested within relevant ranges of concentrations significantly affected the release of substance P- and cholecystokinin-like materials. These results support the idea that adenosine-induced antinociception at the spinal level might possibly be caused, at least partly, by the stimulation of inhibitory adenosine A(1) receptors located presynaptically on primary afferent fibres containing CGRP but not substance P.