ABSTRACT
The characterization of neuronal properties is a necessary first step toward understanding how the ventrolateral preoptic nucleus (VLPO) neuronal network regulates slow-wave sleep (SWS). Indeed, the electrophysiological heterogeneity of VLPO neurons suggests the existence of subtypes that could differently contribute in SWS induction and maintenance. The aim of the present study was to define cell classes in the VLPO using an unsupervised clustering classification method. Electrophysiological features extracted from 289 neurons recorded in whole-cell patch-clamp allowed the identification of three main classes of VLPO neurons subdivided into five distinct subpopulations (cluster 1, 2a, 2b, 3a and 3b). The high occurrence of a low-threshold calcium spike (LTS) was one of the most distinctive features of cluster 1 and 3. Since sleep-promoting neurons are generally identified by their ability to generate an LTS and by their inhibitory response to noradrenaline (NA), 189 neurons from our dataset were also tested for this neurotransmitter. Neurons from cluster 3 were the most frequently inhibited by NA. Biocytin labeling and Neurolucida reconstructions of 112 neurons furthermore revealed a small dendritic arbor of cluster 3b neurons compared, in particular, to cluster 2b neurons. Altogether, we performed an exhaustive characterization of VLPO neuronal subtypes that is a crucial step toward a better understanding of the neuronal network within the VLPO and thereby sleep physiology.
Subject(s)
Action Potentials/physiology , Nerve Net/physiology , Neurons/physiology , Preoptic Area/cytology , Synaptic Potentials/physiology , Action Potentials/drug effects , Animals , Animals, Newborn , Biophysics , Cluster Analysis , Electric Stimulation , In Vitro Techniques , Male , Mice , Mice, Inbred C57BL , Models, Neurological , Neurons/classification , Neurons/drug effects , Norepinephrine/pharmacology , Patch-Clamp Techniques , Serotonin/pharmacology , Statistics, Nonparametric , Synaptic Potentials/drug effectsABSTRACT
The role of serotonin (5-HT) in sleep-wake regulation has been a subject of intense debate and remains incompletely understood. In the ventrolateral preoptic nucleus (VLPO), the main structure that triggers non-rapid eye movement (NREM) sleep, putative sleep-promoting (PSP) neurons were shown ex vivo to be either inhibited (Type-1) or excited (Type-2) by 5-HT application. To determine the complex action of this neurotransmitter on PSP neurons, we recorded spontaneous and miniature excitatory and inhibitory postsynaptic currents (sEPSCs, sIPSCs, mEPSCs and mIPSCs) in response to bath application of 5-HT. We established in mouse acute VLPO slices that 5-HT reduces spontaneous and miniature EPSC and IPSC frequencies to Type-1 neurons, whereas 5-HT selectively increases sIPSC and mIPSC frequencies to Type-2 VLPO neurons. We further determined that Type-1 neurons display a lower action potential threshold and a smaller soma size than Type-2 neurons. Finally, single-cell RT-PCR designed to identify the 13 serotonergic receptor subtypes revealed the specific mRNA expression of the 5-HT1A,B,D,F receptors by Type-1 neurons. Furthermore, the 5-HT2A-C,4,7 receptors were found to be equivalently expressed by both neuronal types. Altogether, our results establish that the excitatory and inhibitory inputs to Type-1 and Type-2 VLPO PSP neurons are differentially regulated by 5-HT. Electrophysiological, morphological and molecular differences were also identified between these two neuronal types. Our results provide new insights regarding the orchestration of sleep regulation by 5-HT release, and strongly suggest that Type-2 neurons could play a permissive role, whereas Type-1 neurons could have an executive role in sleep induction and maintenance.
