Are There Non-Monoaminergic Paradoxical Sleep-Off Neurons in the Brainstem?

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1 Sleep Research Online 2(3): 57-63, Printed in the USA. All rights reserved X 1999 WebSciences Are There Non-Monoaminergic Paradoxical Sleep-Off Neurons in the Brainstem? Kazuya Sakai 1 and Norio Kanamori 2 1 INSERM U480, Département de Médecine Expérimentale, Université Claude Bernard, Lyon 69373, France and 2 Department of Physiology, Tokushima University School of Dentistry, Tokushima 770, Japan Using extracellular single unit recording in the medulla of freely moving cats, we have found a population of PS-off ("Type II") neurons that are distinct from the classically described monoaminergic PS-off ("Type I") neurons. The presumed nonmonoaminergic Type II PS-off neurons (n=22) showed a relatively high rate of tonic discharge during both quiet waking and slow-wave sleep (10.4±4.1 and 9.3±3.1 spikes/sec, mean ± S.D., respectively) and a marked overall decrease in discharge rate during PS (0.3±0.4 spikes/sec). In contrast to the presumed monoaminergic PS-off neurons (n=62), Type II PS-off neurons showed short-lasting phasic discharges during PS, often in association with rapid eye movement and PGO wave bursts. These Type II neurons were all characterized by a short action potential which was significantly different from that of the monoaminergic PS-off neurons described so far. Five out of 22 neurons were identified antidromically by stimulation of the ventrolateral reticulospinal tract (vlrst) at the caudal medulla, while 2 of the 22 were identified antidromically by stimulation of the peri-locus coeruleus alpha of the mediodorsal pontine tegmentum. Their mean conduction velocity (7.2±1.9 m/sec) was significantly higher than that (0.9±0.3 m/sec) of the presumed monoaminergic PS-off neurons which were identified exclusively by stimulation of the vlrst. In addition, when examined during the sleep-waking cycle, the antidromic responses of Type II PS-off neurons were either completely blocked or reduced, with a prolongation of antidromic latency during PS. Most of these neurons were located in medullary structures containing no, or virtually no, monoaminergic neurons, and none responded by inhibition to systemic administration of serotonergic or adrenergic autoreceptor agonists. These findings indicate the existence, in the medulla, of non-monoaminergic PS-off neurons that would play an important role in PS generation. CURRENT CLAIM: Non-monoaminergic PS-off neurons exist in the brainstem. Single unit recording studies in freely moving cats have demonstrated the existence of two distinct populations of statespecific neurons in the brainstem, one, termed PS-on neurons, showing a tonic discharge just prior to and throughout paradoxical sleep (PS), and the other, PS-off neurons, exhibiting a marked reduction or complete cessation of discharge during PS (see Hobson et al., 1986; Sakai, 1988). It is generally accepted that PS-on neurons are either cholinergic or cholinoceptive, while PS-off neurons are serotonergic, noradrenergic, histaminergic, and possibly adrenergic, but not dopaminergic (Jacobs, 1985; Steriade and McCarley, 1990; Sakai et al., 1990a; Jones, 1991). It has been proposed that PS is generated as a result of tonic excitation of PS-on neurons and inhibition of PS-off neurons (reciprocal interaction hypotheses) (Hobson et al., 1975, 1986; Sakai, 1984, 1988). We have demonstrated recently the existence of two different groups of PS-on neurons in the dorsal pontine tegmentum (Sakai and Koyama, 1996), one of which is characterized by a broad action potential, a slow conduction velocity, and an inhibitory response to iontophoretically applied carbachol (Carb-I PS-on neurons), and another which is characterized by a short action potential, a fast conduction velocity, and an excitatory response to applied carbachol (Carb-E PS-on neurons). Carb-I PS-on neurons are located exclusively in the mediodorsal pontine tegmentum which contains cholinergic neurons, especially in the rostral part of the nucleus peri-locus coeruleus alpha (peri-lcalpha), while Carb-E PS-on neurons are found in both the cholinergic (rostral) and non-cholinergic (caudal) regions of the peri- LCalpha. In light of these findings, we have suggested the cholinergic nature of Carb-I PS-on neurons and noncholinergic nature of Carb-E PS-on neurons. Here we report the existence of presumed non-monoaminergic PS-off neurons in the medulla. Differing from the classically described presumed monoaminergic PS-off neurons, which are characterized by a broad action potential, a slow and regular discharge during quiet waking (QW), a slow conduction velocity and their location in monoaminergic structures, the presumed non-monoaminergic PS-off neurons are characterized by a short action potential, a high rate of spontaneous discharge during QW, a fast conduction velocity, their location in non-monoaminergic structures, and their insensitivity to serotonergic or adrenergic autoreceptor agonists. METHODS Experiments were performed on eight adult cats with chronically implanted electrodes for standard polygraphic recordings. Single units were recorded through chronically Correspondence: Kazuya Sakai, Ph.D., INSERM U480, Université Claude Bernard, Lyon 1, 8 Avenue Rockefeller, Lyon Cedex 08, France, Tel: , Fax: , sakai@univ-lyon1.fr.

2 58 SAKAI AND KANAMORI Figure 1. Averaged spike shape of putative monoaminergic (Type I) and non-monoaminergic (Type II) PS-off neurons showing their respective broad and short action potential. Sixty-four spikes were averaged. implanted flexible Formvar-coated stainless steel wires (32 µm diameter) in the unrestrained, freely moving condition. These microelectrodes were assembled into bundles of 6 wires that could be lowered by means of an attached microdrive assembly. The microwires were inserted through the cerebellum into the medulla at an angle of 72 to the horizontal plane. Recordings of the cortical, hippocampal, and dorsal lateral geniculate EEG rhythms, EOG, and neck EMG were also made using standard techniques. In addition, bipolar stimulation electrodes, consisting of two stainless-steel wires (200 µm diameter, mm apart and bared 0.5 mm at the tip), were implanted stereotaxically into the lateral posterior hypothalamus (A9.0, L3.0, HC -4.0), peri-locus coeruleus alpha (peri-lcalpha) (P2.0, L2.0, HC-3.5), and ventrolateral lateral reticulospinal tract (vlrst) (P15.0, L2.5, HC-10.0). Stimulation was performed with square pulses ( ms, ma, Hz) below the threshold for movements using a WPI 302 stimulator via a stimulus isolation unit. The main criteria for recognizing antidromic responses were a fixed latency, a collision test with spontaneous spikes, and the ability to follow high frequency stimulation (Lipski, 1981). Conduction velocity was estimated from the straight line distance between recording and stimulating sites and the shortest antidromic latency when the antidromic latency was shortened in steps with increasing strength of stimulation. The unit activities were amplified using a conventional amplifier with low and high cut-off filters of 100 Hz and 10 Figure 2. Drawings of frontal sections showing the localization of "Type I" PS-off neurons (A), serotonin (5-HT)-immunoreactive (filled circles) or tyrosine hydroxylase (TH)-immunoreactive neurons (empty circles) (B) and "Type II" PS-off neurons (C). Note that Type I PS-off neurons are located in medullary structures containing mostly serotonergic neurons, whereas Type II PS-off neurons are found in structures containing no, or few, monoaminergic neurons. Abbreviations: 7, facial nucleus; Gc, Mc, Pc, nuclei reticularis gigantocellularis, magnocellularis and parvocellularis, respectively; IO, inferior olivary complex; LR, lateral reticular nucleus; PGCL, nucleus paragigantocellularis lateralis; RM, RPa, ROb, nuclei raphe magnus, pallidus and obscurus, respectively; rs, reticulospinal tract; s, S, solitary tract and nucleus of the solitary tract, respectively.

3 NON-MONOAMINERGIC PS-OFF NEURONS 59 Figure 3. Unit activity for two putative serotonergic (Type I) PS-off neurons (A, B) during different sleep-wake states. Note there is either a complete cessation of discharge during PS (A) or a transient suppression of sustained discharge in association with rapid eye movement (EOG) or PGO wave (LGN) bursts (B). khz, respectively. The action potentials were displayed on a digital memory oscilloscope equipped with a processor for spike waveform averaging, and 64 or 128 action potentials were averaged for each neuron to determine the spike shape. Analysis of unitary activity, such as discharge rate, interspike and post-stimulus time histograms, and auto- and crosscorrelograms, was performed using the CED 1401 data processor and Spike 2 software. The mean firing rates were calculated from 100-sec recordings using 5- or 10-sec bins. Statistical analysis was carried out using Student's t-test or analysis of variance (ANOVA). The drugs used, 5-methoxy- N,N-dimethyltryptamine (5-MeODMT) and clonidine hydrochloride (both from Sigma), were dissolved in physiological saline immediately before use and injected systemically (i.m.) in a volume of ml. 5-MeODMT was used rather than 8-OH-DPAT, a selective 5-HT1A agonist, since our preliminary study showed that 8-OH-DPAT induced marked behavioral agitation in the animals when administered at the same dose and by the same route. At the end of the experiments, the location of the end and several passage points of the track of the microelectrodes were examined histologically using the Prussian blue reaction technique on Nissl-stained sections. The immunohistochemical procedures used for the localization of serotonin (5-HT), tyrosine hydroxylase (TH), phenylethanolamine N- methyltransferase (PNMT) or choline acetyltransferase (ChAT) to identify serotonergic, catecholaminergic, adrenergic and cholinergic neurons have previously been described in detail (Sakai et al., 1990b). RESULTS The results presented here were obtained from 84 PS-off neurons recorded in the rostral medulla. PS-off neurons were defined as those showing the highest rate of tonic spontaneous discharge during waking (W), a reduction of discharge rate Figure 4. Autocorrelograms for Type I and Type II PS-off neurons during quiet waking, showing their regular pattern of spontaneous activity and their respective low and high discharge rates. during slow-wave sleep (SWS), and a further reduction, or even complete cessation, of discharge during paradoxical sleep (PS). Of the 84 neurons, 62 exhibited the characteristics of the classical PS-off neurons that have been described in medullary structures containing serotonergic neurons, such as the caudal raphe nuclei and the nucleus paragigantocellularis lateralis (PGCL) (Heym et al., 1982; Trulson and Trulson, 1982; Sakai et al., 1983; Fornal et al., 1985). In the present study, these will be described as "Type I" PS-off neurons. Type I PS-off neurons were all characterized by a broad action potential with 3 components on positive deflection (Figure 1). The mean value (± S.D.) for the duration of the action potential was 1.71±0.33 msec (range msec) measured from the onset of the first deflection to the peak of the largest positive deflection. These neurons were located in the raphe magnus (RM), pallidus (RPa) or obscurus (ROb), as well as in and around the PGCL (Figure 2A), which mainly contain serotonergic neurons (Figure 2B). As previously described (Heym et al., 1982; Sakai et al., 1983; Fornal et al., 1985), they exhibited a slow and regular discharge pattern (Figures 3 and 4). Their mean firing rate during quiet waking (QW) was 3.7±1.9 (S.D.) spikes/sec. As also previously described (Sakai et al., 1983), two groups of neurons were distinguished on the basis of their firing pattern during PS (Figure 3). The first group was characterized by a complete cessation of discharge during PS (referred to as "complete type" in the previous paper) (Figure 3A), whereas the second was characterized by a maintained slow tonic discharge during PS (referred to as "incomplete type"); the latter group, however, consistently showed a reduction, or cessation, of discharge in conjunction with bursts of rapid eye

4 60 SAKAI AND KANAMORI Figure 5. Effects of systemic administration of 5-methoxy-N,Ndimethyltryptamine (5-MeODMT) on an "incomplete" Type I (A), a "complete" Type I (C1) and two Type II (B, C2) PS-off neurons. Note that the Type I PS-off cells are inhibited by 5-MeODMT, whereas the Type II PS-off cells exhibit no inhibitory response to the serotonergic autoreceptor agonist. Note also that the Type I and Type II PS-off cells were recorded simultaneously in C. Arrows indicate 5-MeODMT injection points. movement (REM) and PGO waves occurring during PS episodes (84% mean percent reduction in firing rate relative to QW) (Figure 3B), compared with a 62% mean percent reduction in firing rate in PS without REM and PGO wave bursts relative to QW. When examined using systemic administration of 5-MeODMT (250 µg/kg, i.m.), all neurons responded to the serotonergic autoreceptor agonist with a significant reduction (n=11) (Figure 5A), or cessation (n=10) (Figure 5C-1), of discharge, as previously reported by Jacobs et al. (1983). Seven out of 62 Type I PS-off neurons were identified antidromically by stimulation of the ventrolateral reticulospinal tract (vlrst) at the caudal medulla. Their mean conduction velocity was 0.9±0.3 (S.D.) m/sec (range: m/sec). None of the 62 responded antidromically to stimuli applied to the peri-locus coeruleus alpha (peri-lcalpha) of the mediodorsal pontine tegmentum or posterior hypothalamus. In contrast to the Type I PS-off neurons described above, 22 of the 88 PS-off neurons, which we will designate as "Type II PS-off neurons", were characterized by a short action potential, a fast conduction velocity, their location outside monoaminergic structures, short phasic discharges with REM and PGO wave bursts, and a lack of inhibitory response to 5- MeODMT or clonidine, an alpha2 adrenergic agonist. As shown in Figure 1, the mean duration of the action potential was 0.60±0.14 msec (range msec), distinct from, and significantly shorter (p<0.001, two-tailed t-test) than, that of Figure 6. Discharge pattern of a Type II PS-off cell during the sleepwaking cycle. Note suppression of spontaneous tonic discharge during paradoxical sleep (PS) and the occurrence of phasic spontaneous activity, often in conjunction with rapid eye movement (EOG) and PGO wave (LGN) bursts. Type I PS-off neurons. Although some neurons were found in the raphe nuclei, the majority were located in structures that contain no, or virtually no, monoaminergic neurons, e.g., the nuclei reticularis magnocellularis (Mc), gigantocellularis (Gc), and parvocellularis (Pc) (Figure 2C). As shown in Figures 4-6, they displayed high rates of tonic discharges as compared to Type I PS-off neurons, e.g., their mean discharge rate during QW was 10.4±4.1 spikes/sec, significantly different (p>0.001, two-tailed t-test) from the 3.7±1.9 spikes/sec seen in Type I PSoff neurons, while those during SWS without PGO waves, PS without REM and PGO wave bursts, and PS with REM and PGO wave bursts were 9.3±3.8, 0.3±0.4, and 4.4±3.9 spikes/sec, respectively. Although Type II PS-off neurons showed significant phasic changes in discharge rates during active waking (AW) in relation to body movements, they displayed a regular discharge pattern during both QW and SWS, as shown in Figure 4. In sharp contrast to Type I PS-off neurons, Type II PS-off neurons exhibited short-lasting phasic firing during PS, often in association with REM and PGO wave bursts (Figure 6); no specific correlation with phasic motor activities, such as eye or pinnae movement, was observed. As illustrated in Figure 5B and 5C-2, Type II PS-off neurons did not respond with reduction of discharge to either 5-MeODMT (250 µg /kg, i.m., n=6) or clonidine (25 µg /kg, i.m., n=1; data not shown). Seven of the 22 Type II PS-off neurons were identified antidromically by stimulation of either the vlrst (n=5) or the peri-lcalpha (n=2). Their mean conduction velocity was

5 NON-MONOAMINERGIC PS-OFF NEURONS Figure 7. Antidromic responses of a Type II PS-off cell following stimulation of the peri-locus coeruleus alpha (Peri-LCalpha) of the mediodorsal pontine tegmentum during waking (A) and sleep (B). Their fixed latency, collision with a spontaneous spike(s), and ability to follow high frequency stimulation are shown, respectively, in A1, A2 and A3. In A4, the cell displays a blockade of the somatodendritic spike (indicated by an arrow) at a high rate of repetitive stimulation. Note also that the cell responds faithfully to each stimulus during both quiet waking and slow-wave sleep (SWS) (B1), whereas during paradoxical sleep (PS), the same stimulus results in complete blockade of antidromic responses of both the initial segment and somatodendritic spikes (B2), a break of IS and SD spikes (B3, indicated by an arrow), or a blockade of the SD spike (B4, indicated by an arrow). 7.3±1.9 m/sec (range m/sec), significantly different (p<0.001, two-tailed t-test) from that of 0.9±0.3 m/sec seen for Type I PS-off neurons. Although it was unsuccessful for Type I PS-off neurons, the antidromic responses of two Type II neurons could be examined in detail during sleep-wake states using the same stimulus intensity. As shown in Figure 7, one cell showed faithful antidromic responses during both QW and SWS, but, during PS displayed a complete blockage of antidromic responses of both initial segment (IS) and somatodendritic (SD) spikes, IS-SD break or a blockage of the SD spike alone (Figure 7B). The other cell exhibited a greater than 10% decrease in antidromic responsiveness during PS to stimulation of the vlrst and displayed prolongation of antidromic latencies from 0.80 msec in SWS to 0.90 msec in PS (data not shown), suggesting inhibitory synaptic volleys impinging on non-monoaminergic PS-off neurons during PS (see Lipski, 1981). DISCUSSION The present findings strongly suggest the existence of nonmonoaminergic PS-off neurons in the medulla, which are characterized by a short action potential, a fast conduction velocity, a high rate of spontaneous discharge during both W and SWS, unresponsiveness to serotonergic or adrenergic autoreceptor agonists, and their location essentially in nonmonoaminergic structures. Until now, all PS-off neurons showing PS-selective discharge cessation have been regarded as monoaminergic, usually serotonergic, noradrenergic or histaminergic, being reported in the raphe nuclei and ventrolateral medulla, containing serotonergic neurons (McGinty et al., 1973; Heym et al., 1982; Sakai et al., 1983; Rasmussen et al., 1984; Fornal et al., 1985), the dorsolateral pontine tegmentum, containing noradrenergic neurons (Hobson et al., 1973; McGinty and Sakai, 1973; Saito et al., 1977; Sakai, 1980; 1991; Aston-Jones and Bloom, 1981; Reiner, 1986), and in the posterior hypothalamus, containing histaminergic neurons (Vanni-Mercier et al., 1984; Sakai et al., 1990a). Features common to these putative monoaminergic neurons are their long duration of action potential and a slow and fairly regular pattern of spontaneous activity during QW (see Jacobs, 1985; Steriade and McCarley, 1990; Sakai et al., 1990; Jacobs and Azmitia, 1992 for review). Unlike these presumed monoaminergic PS-off neurons, the presumed nonmonoaminergic Type II PS-off neurons all displayed a short action potential and a higher rate of spontaneous discharge during QW. Type II PS-off neurons appear to be noncatecholaminergic, since they are not located in medullary regions containing tyrosine hydroxylase (TH)-immunoreactive noradrenergic or adrenergic neurons (Figure 2). In addition, they have higher conduction velocities (7.3±1.9 m/sec) than putative adrenergic C1 neurons (0.9±0.1 m/sec; mean ± S.E.M.) (Haselton and Guyenet, 1989), which is in line with the demonstration that most C1 neurons give rise to unmyelinated axons (Milner et al., 1987). Although the clonidine unresponsiveness found in the one Type II PS-off cell tested is also in favor of the assumption of the non-adrenergic nature of Type II PS-off cells, further pharmacological examination of a large sample of cells is needed for this issue. The activity of both adrenergic and noradrenergic medullary neurons during the sleep-waking cycle is not yet known and will be an important subject for future studies. Type II PS-off cells also appear to be non-serotonergic, since they were mainly located in medullary regions containing no, or few, serotonergic neurons; the present findings on their fast conduction velocity and insensitivity to the systemic administration of 5-MeODMT, a serotonergic autoreceptor agonist, further strengthen this assumption. A basic question therefore arises as to the neurochemical nature and functional roles of these putative non-monoaminergic Type II PS-off neurons. Although the firing pattern of Type II PS-off neurons was variable during AW, they showed a relatively high rate (about 10 Hz) of regular discharge pattern during QW and SWS (Figure 4), during which multiple phasic synaptic inputs should be considerably reduced. The majority of Type II PS-off neurons were found in the rostral and ventrolateral medullary region. In this regard, it is worth mentioning the presence of non-monoaminergic neurons showing a "pacemaker-like" discharge pattern that have been reported in the rat rostral 61

6 62 SAKAI AND KANAMORI ventrolateral medulla, both in vitro (Sun et al., 1988a) and in vivo, particularly in the presence of a glutamate receptor antagonist (Sun et al., 1988b; see also Granata and Kitai, 1992). These neurons have been supposed to be glutamatergic in nature and sympathoexcitatory in function via direct projections to the spinal cord (Sun et al., 1988a,b). Their firing rate (9 spikes/sec in vitro; Sun et al., 1988a) and regular activity, as well as their projection to the spinal cord are reminiscent of the characteristics of descending Type II PS-off neurons, as described in the present study. Further studies are needed to determine whether these neurons are implicated in changes in autonomic events during the sleep-waking cycle. Although once supposed to be glutamatergic, the exact neurochemical identity of putative sympathoexcitatory bulbospinal neurons is not yet known. Histochemical studies of the rostral ventrolateral medulla have demonstrated the presence of many putative neurotransmitters, including biogenic amines, acetylcholine, neuropeptides and both excitatory and inhibitory amino acids, within the cell bodies (see Chalmers and Pilowsky, 1991 for review). Recently, the existence in the rostral ventrolateral medulla of GABAergic pacemaker neurons having a sympathoinhibitory function has been suggested (Hayer et al., 1996). It is especially worth speculating about the GABAergic neurochemical identity of Type II PS-off neurons when we consider their roles in the mechanisms underlying PS generation. In previous reports of studies in freely moving cats, PS-on neurons showing a high rate of tonic discharge just prior to and throughout PS were described in the nuclei reticularis magnocellularis (Mc), parvocellularis (Pc) and paragigantocellularis lateralis (PGCL) (Sakai et al., 1979; Kanamori et al., 1980; Sakai, 1988), areas in which Type II PS-off neurons were recorded in the present study. It seems likely that Type II PS-off neurons exert direct inhibition on medullary PS-on neurons through a local circuit. The present finding on the ascending projection of some Type II PS-off neurons to the peri-lcalpha, which also contains PSon neurons (Sakai, 1980; 1988), allows further speculation on a direct inhibition of pontine PS-on neurons via medullary GABAergic PS-on neurons, an idea suggested by the facts that iontophoretically applied bicuculline, a GABA A receptor antagonist, produces excitation of PS-on neurons and subsequently induces PS (Sakai and Koyama, 1996) and that, conversely, microdialysis application of muscimol ( mm), a potent GABA A receptor agonist, to the peri-lcalpha results in inhibition of PS (Sakai, Onoe and Crochet, unpublished data). Although GABAergic neurons are widely distributed in the brainstem, the existence in the brain of nonmonoaminergic PS-off neurons having a PS-inhibitory function has not been reported until the present study which showed their presence in the medulla. Both pontine and medullary PS-on neurons discharge tonically and selectively just prior to, and throughout, periods of PS, satisfying the selectivity, tonicity, and tonic PS-latency criteria necessary for being PS-generator neurons (Sakai, 1988). There is a mirror image, or exactly inverse relationship in terms of cellular discharge between PS-on and putative monoaminergic PS-off neurons that cease firing selectively during PS, suggesting a mutual "inhibitory" interaction between the two distinct neuronal populations (Sakai, 1984; 1988). In keeping with the mutual inhibitory interaction hypothesis, presumed serotonergic medullary PS-off neurons cease firing during PS, especially during PS episodes marked by the presence of REM and PGO wave bursts (Sakai et al., 1983; and the present study), in which PS-on neurons exhibit an increase in tonic discharge rate (Sakai, 1984; 1988). However, during PS, Type II PS-off neurons exhibited phasic discharges in association with the bursts of REMs and PGO waves accompanying an increase in activity of PS-on neurons, suggesting a possible excitatory action of PS-on neurons on non-monoaminergic PS-off neurons, and thereby supporting, in part, the reciprocal "excitatory-inhibitory" interaction hypothesis between REM-on and REM-off cells, as originally proposed by Hobson and McCarley between cholinergic REMon and monoaminergic REM-off cells (Hobson et al., 1975; 1986). In conclusion, the present study demonstrates the existence of non-monoaminergic PS-off neurons in the medulla and thus opens up the possibility of multiple interactions between PS-on and monoaminergic and non-monoaminergic PS-off neurons. ACKNOWLEDGMENTS This work was supported by INSERM U480. REFERENCES 1. Aston-Jones G, Bloom FE. Activity of norepinephrinecontaining locus coeruleus neurons in behaving rats anticipates fluctuations in the sleep-waking cycle. 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