Role of the Lateral Paragigantocellular Nucleus in the Network of Paradoxical (REM) Sleep: An Electrophysiological and Anatomical Study in the Rat

The lateral paragigantocellular nucleus (LPGi) is located in the ventrolateral medulla and is known as a sympathoexcitatory area involved in the control of blood pressure. In recent experiments, we showed that the LPGi contains a large number of neurons activated during PS hypersomnia following a selective deprivation. Among these neurons, more than two-thirds are GABAergic and more than one fourth send efferent fibers to the wake-active locus coeruleus nucleus. To get more insight into the role of the LPGi in PS regulation, we combined an electrophysiological and anatomical approach in the rat, using extracellular recordings in the head-restrained model and injections of tracers followed by the immunohistochemical detection of Fos in control, PS-deprived and PS-recovery animals. With the head-restrained preparation, we showed that the LPGi contains neurons specifically active during PS (PS-On neurons), neurons inactive during PS (PS-Off neurons) and neurons indifferent to the sleep-waking cycle. After injection of CTb in the facial nucleus, the neurons of which are hyperpolarized during PS, the largest population of Fos/CTb neurons visualized in the medulla in the PS-recovery condition was observed in the LPGi. After injection of CTb in the LPGi itself and PS-recovery, the nucleus containing the highest number of Fos/CTb neurons, moreover bilaterally, was the sublaterodorsal nucleus (SLD). The SLD is known as the pontine executive PS area and triggers PS through glutamatergic neurons. We propose that, during PS, the LPGi is strongly excited by the SLD and hyperpolarizes the motoneurons of the facial nucleus in addition to local and locus coeruleus PS-Off neurons, and by this means contributes to PS genesis

Role of the lateral paragigantocellular nucleus in the network of paradoxical sleep in the rat

Le LPGi est la région bulbaire qui contient le plus grand nombre de neurones exprimant Fos pendant l’hypersomnie de SP. 10% de ces neurones Fos dans le LPGi projettent au locus coereleus, une région SP-Off. Récemment, Sapin et al. ont montré que 70% des neurones exprimant Fos pendant l’hypersomnie de SP sont de nature GABAergique. Notre hypothèse est que le LPGi contient des neurones de nature SP-On dont une partie participerait à l’inhibition des noyaux SP-Off. Nous cherchons à vérifier cette hypothèse, d’autre part à identifier les régions afférentes au LPGi actives au cours du SP et enfin, à identifier les projections du LPGi. Nous avons utilisé l’enregistrement extracellulaire des neurones du LPGi chez le rat vigile en contention stéréotaxique. Nous avons couplé cette technique avec le protocole de privation/ rebond de SP par la technique de la piscine. L’analyse du taux de décharge des neurones enregistrés au sein du LPGi montre que celui-ci contient trois types neuronaux différents (SP-On, SP-Off et indifférents). L’analyse des données neuroanatomiques montre que l’afférence majeure du LPGi, active au cours du SP, réside dans le SLD. Le LPGi est donc bien impliqué dans le réseau du SP car il contient des neurones faisant varier leur taux de décharge avec les différents états de vigilance. Le SLD, considéré comme la structure exécutive du SP, de nature glutamatergique, exciterait le LPGi au cours du SP. Ce dernier, de nature GABAergique, inhiberait le noyau moteur facial. Ce travail met pour la première fois en évidence l’activité de la voie SLD-bulbe rachidien ventrolatéral et suggère que le LPGi participe en partie à l’atonie musculaire caractéristique du SPThe LPGi is a ventrolateral medullary area that contains the highest number of Fos-labelled neurons during a paradoxical sleep (PS) hypersomnia. Ten % of these Fos neurons project to the locus coeruleus, a PS-Off area and 70% of these Fos neurons are GABAergic. Our hypothesis is that the LPGi contains PS-On neurons involved in the inhibition of the PS-Off nuclei. Our aim was to check this hypothesis by recording the unit activity of the LPGi neurons, by identifying the afferent areas to the LPGi and determining its projection areas activated during PS. We have used extracellular unit recordings in the unanethetized head-restrained rat model and coupled this method to a selective PS deprivation using the flower-pot method. The analysis of the firing rate of the LPGi neurons showed that there are three different groups, SP-On, SP-Off and indifferent neurons. Moreover, the SLD, a PS-On area, is the main afferent to the LPGi activated during PS rebound. Our conclusion is that the LPGi is involved in the network generating PS. The SLD, considered as the PS executive area through glutamatergic neurons, may excite the LPGi during this state. The LPGi, through its GABAergic neurons, may inhibit the facial motor nucleus. This work provides for the first time evidence for a SLD-ventrolateral medulla pathway and suggests that the LPGi participates in the muscular atonia occurring during P

Rôle du noyau paragigantocellulaire latéral dans le réseau du sommeil paradoxal chez le rat

The LPGi is a ventrolateral medullary area that contains the highest number of Fos-labelled neurons during a paradoxical sleep (PS) hypersomnia. Ten % of these Fos neurons project to the locus coeruleus, a PS-Off area and 70% of these Fos neurons are GABAergic. Our hypothesis is that the LPGi contains PS-On neurons involved in the inhibition of the PS-Off nuclei. Our aim was to check this hypothesis by recording the unit activity of the LPGi neurons, by identifying the afferent areas to the LPGi and determining its projection areas activated during PS. We have used extracellular unit recordings in the unanethetized head-restrained rat model and coupled this method to a selective PS deprivation using the flower-pot method. The analysis of the firing rate of the LPGi neurons showed that there are three different groups, SP-On, SP-Off and indifferent neurons. Moreover, the SLD, a PS-On area, is the main afferent to the LPGi activated during PS rebound. Our conclusion is that the LPGi is involved in the network generating PS. The SLD, considered as the PS executive area through glutamatergic neurons, may excite the LPGi during this state. The LPGi, through its GABAergic neurons, may inhibit the facial motor nucleus. This work provides for the first time evidence for a SLD-ventrolateral medulla pathway and suggests that the LPGi participates in the muscular atonia occurring during PSLe LPGi est la région bulbaire qui contient le plus grand nombre de neurones exprimant Fos pendant l’hypersomnie de SP. 10% de ces neurones Fos dans le LPGi projettent au locus coereleus, une région SP-Off. Récemment, Sapin et al. ont montré que 70% des neurones exprimant Fos pendant l’hypersomnie de SP sont de nature GABAergique. Notre hypothèse est que le LPGi contient des neurones de nature SP-On dont une partie participerait à l’inhibition des noyaux SP-Off. Nous cherchons à vérifier cette hypothèse, d’autre part à identifier les régions afférentes au LPGi actives au cours du SP et enfin, à identifier les projections du LPGi. Nous avons utilisé l’enregistrement extracellulaire des neurones du LPGi chez le rat vigile en contention stéréotaxique. Nous avons couplé cette technique avec le protocole de privation/ rebond de SP par la technique de la piscine. L’analyse du taux de décharge des neurones enregistrés au sein du LPGi montre que celui-ci contient trois types neuronaux différents (SP-On, SP-Off et indifférents). L’analyse des données neuroanatomiques montre que l’afférence majeure du LPGi, active au cours du SP, réside dans le SLD. Le LPGi est donc bien impliqué dans le réseau du SP car il contient des neurones faisant varier leur taux de décharge avec les différents états de vigilance. Le SLD, considéré comme la structure exécutive du SP, de nature glutamatergique, exciterait le LPGi au cours du SP. Ce dernier, de nature GABAergique, inhiberait le noyau moteur facial. Ce travail met pour la première fois en évidence l’activité de la voie SLD-bulbe rachidien ventrolatéral et suggère que le LPGi participe en partie à l’atonie musculaire caractéristique du S

Fos/CTb labeling in the PS recovery condition after injection of CTb in the facial nucleus.

<p>A: CTb injection site. Double-labelled neurons (arrows) in the ipsilateral LPGi (B), in the ipsilateral gigantocellular reticular nucleus, alpha part (GiA)(C) and in the contralateral intermediate reticular nucleus (IRt) (D). Bar  = 500 µm in A, 60 µm in B–D.</p

Electrophysiological characteristics of the three types of neurons recorded in the LPGi.

<p>Data obtained in 21 PS-On, 8 PS-Off and 21 state-indifferent neurons. Figures refer to mean ± S.E.M. N/A, not available, hello P≤0.05, hellohello P≤0.01, hellohellohello P≤0.001 vs Waking;</p>#<p>P≤0.05,</p>##<p>P≤0.01,</p>###<p>P≤0.001 vs SlowWaveSleep;</p>°<p>P≤0.05 vs Waking.</p

Distribution of the single CTb (black dots) and double-labelled Fos/CTb (red dots) neurons, as observed in the hypothalamus and cortex of one PS control, one PS-deprived and one PS-recovery rat after injection of CTb in the LPGi, in the sides ipsilateral (right) and contralateral (left) to the injection site.

<p>Figures refer to the distance from the Bregma. Abbreviations: Au: auditory cortex; CeM: central amygdaloid nucleus; DA: dorsal hypothalamic area; DM: dorsomedial hypothalamic nucleus; Ect: ectorhinal cortex; LH: lateral hypothalamic area; M1: primary motor cortex; M2: secondary motor cortex; PeF: perifornical nucleus; S1: primary somatosensory cortex; S2: secondary somatosensory cortex; ZI: zona incerta.</p

Anterogradely labelled axons in the cranial motor nuclei after injection of PHA-L in the LPGi.

<p>Axons in the hypoglossal (N12) (A, B), facial (N7) (C, D) and trigeminal (N5) (E, F) motor nuclei after injection of PHA-L in the caudal part (A, C, E) and rostral part (B, D, F) of the LPGi. In D note the labelled varicosities (arrows) adjacent to the choline acetyltransferase-immunoreactive motoneurons in the medial part of the nucleus. Abbreviations: 4V, fourth ventricle, int, and med, intermediate and medial parts of the facial nucleus. Bar  = 70 µm in A,B,C,E and F, 30 µm in D.</p

Fos (or TH or ChAT)/CTb labeling after injection of CTb in the LPGi.

<p>A–F and –HJ: PS-recovery condition, G: PS-deprived condition. A: CTb injection site in the LPGi covering Fos nuclei, B: CTb injection site in the LPGi covering TH- neurons of the A1/C1 group, C: CTb injection site in the LPGi, ventrally to the ChAT- neurons of the ambiguus nucleus (NA), D: Contralateral LPGi, E: sublaterodorsal nucleus (SLD) showing many double-labelled Fos-CTb neurons, F: ventrolateral periaqueductal gray (vlPAG) in the PS-recovery condition, G: ventrolateral periaqueductal gray (vlPAG) in the PS-deprivation condition, H: single CTb neurons in the central nucleus of the amygdala (CeM), I: single CTb neurons in the pyramidal layer of the secondary somatosensory cortex (S2), J: lateral hypothalamus (LH). Arrows indicate double-labelled Fos-CTb neurons. Bar  = 500 µm in A-C and 60 µm in D-J.</p

Distribution of the single CTb (black dots) and double-labelled Fos/CTb (red dots) neurons, as observed in the basal forebrain and cortex of one PS control, one PS-deprived and one PS-recovery rat after injection of CTb in the LPGi, in the sides ipsilateral (right) and contralateral (left) to the injection site.

<p>Figures refer to the distance from the Bregma. Abbreviations: BST: bed nucleus of the stria terminalis; GI: insular cortex; LPO: lateral preoptic area; M1: primary motor cortex; M2: secondary motor cortex; MPO: medial preoptic area; S1: primary somatosensory cortex; S2: secondary somatosensory cortex.</p