Brait~ Research, 569 (1992) 210-22(t © 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$03.50

210

BRES 17322

Firing of 'possibly' cholinergic neurons in the rat laterodo tegmental nucleus during sleep and wakefulness

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Yukihiko Kayama, Mamoru Ohta and Eiichi Jodo Department of Physiology, Fukushima Medical College, Fukushima (Japant (Accepted 20 August 1991)

Key words: Cholinergic neuron; Laterodorsal tegmental nucleus; Dorsal raphe nucleus; Single unit; Sleep-waking state; Paradoxical sleep; Rat

To clarify functional roles of mesopontine cholinergic neurons as a component of an activating system, single neuronal activity in the laterodorsal tegmental nucleus (LDT) of undrugged rats. whose head was fixed painlessly, was recorded along with cortical EEG and neck EMG. Activity of some dorsal raphe (DR) neurons was also recorded for comparison. Most of the animals had been sleep-deprived for 24 h. Observation was made only on neurons generating broad spikes, presumed from previous studies to be cholinergic or monoaminergtc, The position of recorded neurons was marked by Pontamine sky blue ejected from the glass pipette microetectrode, and was identified on sections processed for NADPH diaphorase histochemistry which specifically stained cholinerglc neurons. According to their firing rates during wakefulness (AW), slow-wave sleep (SWS) and paradoxical sleep (PS), 46 broad-spike neurons in the LDT were classified into 4 groups: (1) neurons most active during AW and silent during PS (some of these neurons might be serotonergic rather than cholinergic, as all the 9 neurons in the DR); (2) neurons most active during PS and silent during AW; (3) neurons equally more active during AW and PS than SWS; and (4) others mainly characterized by transiently facilitated activity at awakening and/or onset of PS. Neurons of groups 2 and 3 were the major constituents of the LDT. In most neurons change in firing preceded EEG change, except at awakening from PS. These results suggest that: (1) the LDT is composed of cholinergic neurons with heterogenous characteristics in relation to sleep/wakefulness: and (2) some tegmental cholinergic neurons play a pivotal role in induction and maintenance of PS.

INTRODUCTION Since the elaboration of the concept of an 'ascending reticular activating system' by Moruzzi and Magoun 25.

culopontine tegmental nucleus (PPT), i.e. in the reticular formation of the caudal mesencephalon around the superior cerebellar peduncles 2't7'23. These cholinergic neu-

the mesencephalic reticular formation has been consid-

rons send ascending axons diffusely to the various forebrain sites except for most cortical areas 3~'42. That the

ered to be a center for maintaining wakefulness or consciousness. However, it has not been shown which neurons are the real substrate of the system. Many thought that the activating system was associated with acetylcholine as a chemical mediator 11'33. but adequate evidence

ascending cholinergic projection has activating effects on the forebrain has b e e n shown by electrical stimulation of the L D T or PPT in anesthetized animals: it elicits excitation of thalamic relay neurons9't5 along with desynchronization of the cortical E E G 16.

for this idea has not been obtained. O n the other hand, some have proposed that paradoxical sleep (PS), or rapid eye m o v e m e n t ( R E M ) sleep, is initiated by a cholinergic mechanism in the brainstem 1°'13'29'32. It can thus be asked whether a brainstem

To clarify the role of the mesopontine cholinergic system further. El Mansari e t al. 7 and Steriade e t al. 36

cholinergic system is responsible for both activated states of the forebrain, wakefulness and PS. By the mid 1980's immunohistochemical studies for choline acetyltransferase revealed that brainstem cholinergic n e u r o n s gathered tightly in the laterodorsal tegmental nucleus (LDT) in the central gray of the mesencephalon and pons, and were scattered in the pedun-

recently recorded n e u r o n a l activity in the P P T and L D T of the cat during wakefulness and sleep. In the present study we made a similar recording in the L D T of undrugged rats whose head was fixed painlessly, and tried to identify cholinergic versus non-cholinergic n e u r o n s on the basis of spike shape, as done previously with anesthetized rats 14. Characteristics of the possibly cholinergic neurons (see Discussion) in relation to sleep/wakefulness states were compared with those recorded in c a t s 7'36. and also with those of other n e u r o n s projecting diffusely from

Correspondence: Y. Kayama. Department of Physiology, Fukushima Medical College, 1 Hikari-ga-oka. Fukushima 960-I2. Japan. Fax: (81 (245) 48-2571.

211 the brainstem to the forebrain; that is, serotonergic neurons of the dorsal raphe nucleus (DR) and noradrenergic neurons of the locus coeruleus (LC).

trode filled with a 0.5 M sodium acetate solution containing 2% Pontamine sky blue. To avoid penetration of the venous sinus, the electrode was angled posteriorly by 30 ° and lowered through the cerebellum near the primary fissure. Needle electrodes were inserted to the neck muscle for EMG. After all electrodes were connected to amplifiers, the animal was placed in a dimly lit box; they stayed quietly for 3-5 h, repeating sleep and rather short periods of wakefulness. The experiment was discontinued when the animals became slightly restless. Single-unit spikes were amplified with a high-impedance microelectrode amplifier and then a conventional amplifier with low-cut filter of 53 Hz and no high-cut filter. An amplifier with filters of the same values was used for EMG. E E G was amplified through filters of 1-100 Hz; its amplitude was not calibrated exactly, but the largest slow waves shown in the figures were about 0.5 mV. The single-unit activity, E E G and E M G were recorded on magnetic tape with a DAT recorder (Sony PC-108M) and on paper with a thermal array recorder (Nihon-Kohden RTA-1200M) together with outputs of a Schmitt circuit and ratemeter. The latter recorder can follow very high-frequency signals (>10 kHz) so that single-unit spikes could be written directly on paper. To mark positions of neurons recorded from, Pontamine sky blue was ionophoresed from the recording electrode for 5 min with a DC current of 0.01 mA. At most, 3 spots were made in an animal. At the termination of an experiment the animal was deeply anesthetized with urethane (1.5 mg/kg, i.p.), and perfused through the ascending aorta with 50 ml of ice-cold Krebs Ringer solution followed by 250 ml of 4% paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.4). After removal, the brain was rinsed in phosphate buffer containing 10% sucrose for 30 min. The brainstem was then sectioned in the coronal plane at 0.05 mm on a freezing microtome and processed for NADPH-diaphorase histochemistry. In the midbrain and pons only cholinergic neurons of the LDT and PPT were stained darkly blue by this method 4°, while D R was delineated as a mass of cells stained lightly (Fig. 1). The positions of neurons marked could thus be distinguished unequivocally in relation to that of LDT or D R (Fig. 3).

MATERIALS AND M E T H O D S Data were obtained from 40 of 59 male Sprague-Dawley rats (7-12 weeks old), each of which was prepared under pentobarbital anesthesia (initially 40 mg/kg, i.p., supplemented 5-20 mg/kg once or twice) for chronic experimentation. The head was fixed in a stereotaxic instrument by the method of Paxinos and Watson 26. Two sterilized stainless steel bolts (diameter 1.0 mm) with lead wire were screwed to the skull (2 mm lateral to the midline, 2 and 6 mm posterior to the bregma) to touch the dura, either one of which was used to record EEG, with another screw fixed in the sagittal suture between frontal bones as reference electrode. Another 2-4 bolts (diameter 1.5 mm) were fixed to the skull as anchors. Two aluminum tubes, 5 mm in outer diameter and 20 mm long, were placed horizontally at right angles to the sagittal line, 12 mm apart, and were fixed to the skull by embedding them in a mound of dental cement. The mound was made so as to leave a well over the interparietal bone, centered at 0.6 mm lateral to the sagittal line. A small piece of the bone in the well, about 5 mm in diameter, was trephined. Antibiotic ointment (gentamicin) was applied to the dura, and the well filled with sterile bone wax. The animals were treated with antibiotics for 2 days. The aluminum tubes were used to fix the head painlessly in a stereotaxic instrument for chronic experimentation (Narishige, SR8). After recovering from surgery most animals were trained for several days to remain calm while their head was fixed. On 36 animals sleep-deprivation was performed with a method similar to that of Marks and Roffwarg 2~. They were placed in a slowly rotating wheel (diameter 40 cm, width 11 cm, 0.7 rpm) for 24 h until the start of recording. Food and water were available ad libitum. The procedure of sleep-deprivation does not seem to give excessive stress to animals, since no abnormality was observed in their appearance and behavior after release from the rotation, except their readily falling asleep. Even just after the release they could awaken spontaneously. On the recording day, the head was fixed and the bone wax in the well removed. After local anesthesia (2% lidocaine) was applied, the dura was opened under microscopic control. The cerebellar surface was covered with mineral oil to prevent drying. Single unit activity was recorded extracellularly with a glass pipette microelec•

RESULTS

Classification of neurons by spike shape In the present

study using glass microelectrodes

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0.5 rnm Fig. 1. Photomicrographs of coronal sections of caudal mesencephalon (right) and rostral pons (left) processed for N A D P H diaphorase histochemistry. Dotted lines are boundaries of the aqueduct or fourth ventricle, periaqueductal gray and dorsal tegmental nucleus which are observed clearly in color photos. Cholinergic neurons are stained dark, while the D R is delineated lightly (arrowheads). Thick arrows, median line. Thin arrows, spots of Pontamine sky blue deposited heavily (right) or lightly (left).

212 single-unit activity was recorded either as positive (downward in figures) monophasic spikes with a very steep initial downstroke, or as positive-negative biphasic spikes. The results described below were based on observations of only the latter, which were probably generated by neuronal somas but not by axons, as shown by previous workers 4. According to the width of the spikes, neurons were classified into two groups; 'broad-spike' neurons (Fig. 2 A - E ) and 'brief-spike' neurons (Fig. 2F and G. as typical examples of very slender and broader subgroups, respectively). Such classification was possible even with rather small spikes by averaging (Fig, 2 D , D ' ) . The broad spikes were those with a negative component wider than 2.2 ms; all but one (see below) of them had a positive component wider than 0.7 ms (Fig. 2H). In most of them the negative component with a slowly decaying slope was much smaller in amplitude than the positive one, and in some a notch was observed between positive and negative strokes (Fig. 2E). Negative component of the spikes categorized as 'brief' were narrower than 1.9 ms; all of the brief spikes had a positive component narrower than 0.7 ms. It was only one neuron (Fig. 2H, black column} among neurons encountered in or around the L D T that could not be classified with these criteria. (This neuron was not included in the broad class.) The values of these criteria are slightly smaller than those of the previous study in anesthetized animals 14. This may be because neurons firing spikes as large as

those recorded in anesthetized animals were frequently unstable for recording in the awake condition, and soon showed signs of injury, so that most neurons recorded in the present experiments were smaller in spike amplitude than those in the previous study. A n o t h e r possible reason is that in the unanesthetized condition spike shape can be more variable with so far unknown mechanisms: it was sometimes observed that the width of spikes changed reversibly in or without relation to change in the level of activity. Brief-spike neurons were encountered on more occasions than broad-spike neurons, but the number of briefspike neurons in or around the L D T could not be known since their positions were not marked. Spike widths plotted in Fig. 2 (dotted columns) were of those neurons encountered in about the same depths as the LDT. Only a few brief-spike neurons, however, were in the vicinity of broad-spike neurons whose dye-mark was found in the LDT. There were many brief-spike neurons which fired at a rate of 20-80 Hz, while broad-spike neurons rarely discharged faster than 20 Hz except during some transiently activated periods; the majority being slower than 10 Hz. Usually they fired tonically: bursts intermingled sometimes but did not appear repetitively or rhythmically. In the previous study z4 it was concluded that the broad-spike L D T neurons were possibly cholinergic (see Discussion). Therefore the results shown hereafter are all on behaviors of the broad-spike neurons.

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1.4 S W S > A W neurons around the onset of PS (A and C) and around awakening from PS (B and D). Vertical lines show bins (1 s/bin) during which EEG desynchronized (A and C) or EEG theta waves disappeared (B and D). A and B: averages of data obtained at 3 occasions of PS onset and 4 occasions of awakening, respectively, in a neuron Shown in Fig. 5. C: average o f data in 6 neurons in which PS started clearly without repeating short periods of PS and deep SWS. D: that in 6 neurons in which E E G theta Waves were seen clearly.

whether they should belong to AW= PS>SWS group (asterisks in Fig. 3) or to AW>SWS>PS group (closed

( A W > S W S , circles with asterisk in Fig. 3, or P S > S W S , squares with asterisk, respectively), it is not easy to judge

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Fig. 7. Activity of a neuron active during both AW and PS (AW = PS>SWS group). Upper and initial part of lower records are continuous at stars. The record consists of the same combination as that of Fig. 4A. During an interval between two separated Iower records the same PS episode continued for about 70 s. Symbols, the same as those in Figs. 4 and 5.

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Fig. 8. Activity of two neurons (A and B) classified into 'others'. Each record consists of the same combination as that of Fig. 4A (A) or 4B (B). A: a neuron firing tonically throughout all sleep/wakefulness states and facilitated transiently just after awakening or onset of PS. B: a neuron firing intensely only when EEG desynchronized. Activity of this neuron could not be recorded during PS. Symbols, the same as those in Figs. 4 and 5.

circles) or P S > S W S > A W group (closed squares), respectively. With a very rough calculation by p r o p o r t i o n a l allotment in a ratio of two neurons to 5 neurons in the LDT, 15 A W > S W S neurons can be divided into 4 possible A W > S W S > P S and 11 possible A W = P S > S W S neurons. In the same way, 6 P S > S W S neurons can be divided in a ratio of 11 to 5 into 4 possible P S > S W S > A W and two possible A W = P S > S W S neurons. A l t o g e t h e r , in the L D T are 7 neurons (15%) of A W > S W S > P S group (though some of them may be serotonergic), 17 neurons (37%) of P S > S W S > A W group, 18 neurons (39%) of A W - P S > S W S group, and 4 neurons (9%) of others. Thus, although exact estimation of their p r o p o r t i o n is difficult in the present experiments, it seems that the major constituents of the L D T are P S > S W S > A W and A W = P S > S W S groups. DISCUSSION

Identification of 'possibly' cholinergic neurons In the rat L D T cholinergic neurons projecting to forebrain sites gather tightly 2'23'3x'42, but non-cholinergic neurons, some of which also project to the forebrain 34, are intermingling with them. In our previous study on urethane-anesthetized rats, we tried to identify cholinergic neurons electrophysiologically~4. In the L D T two types of neurons, broad- and brief-spike neurons, were encountered, and about 90% of the neurons activated antidromically by stimulation of forebrain sites were of the broad type. Comparing the data with an anatomical

study that about 90% of retrogradely labeled L D T neurons after H R P injection in the thalamus were cholinergic 34, we suggested that the broad-spike L D T neurons may be cholinergic. By using glass pipette but not metal microelectrodes, the same b r o a d spikes were r e c o r d e d in the present experiments. That the b r o a d spikes are generated by cholinergic neurons in the L D T is suggested also by L e o n a r d and Llin~s TM. They r e c o r d e d intracellularly from P P T and L D T neurons in in vitro slice preparations of the mesopontine tegmentum, and classified the neurons into types I, II and III according to their electrophysiological properties. A m o n g them, type II neurons had high-threshold calcium spikes. It was shown that the calcium spike was activated by the ordinary sodium spike and was generated in the falling phase of the latter. Consequently, total width of each spike of type II neurons must be b r o a d e r , though exact values of the spike width were not reported. Following physiological identification, impaled neurons were injected with Lucifer yellow and the tissue was processed histochemically for N A D P H - d i a p h o r a s e . By the presence of both N A D P H - d i a p h o r a s e reaction products and Lucifer yellow in a neuron, L e o n a r d and Llin~s TMconcluded it was clear that type II neurons were cholinergic. The configuration of the b r o a d spikes of L D T neurons was very similar to those of broad-spike D R or L C neurons 14. The latter were d e m o n s t r a t e d to be serotonergic or noradrenergic, respectively ~. Since some anatomical studies have shown that a few serotonergic and norad-

218 renergic neurons are scattered in the periaqueductal gray outside the D R or LC, especially in the circumjacent areas of the L D T ~2'35, the broad-spikes recorded in or around the L D T in the present experiments might be generated by monoaminergic neurons. However, many studies have invariably shown that both serotonergic and noradrenergic neurons have slow, tonic discharges during AW which decrease during SWS and stop almost completely during PS ( A W > S W S > P S group in the present terlTlS) 3'5'19'27'39. This type of behavior was observed in only a very few L D T neurons. Peptidergic neurons may also generate broad spikes as observed in the hypothalamus 22. Though many L D T neurons contain various peptides, they are co-localized in cholinergic neurons 4°. If it is assumed that the ectopic monoaminergic neurons have the same properties as those in the main mass of the D R or LC, the great majority of broad-spike neurons observed in or around the L D T in the present experiments were presumed to be cholinergic. 'Cholinergic neurons' used hereafter will be, in the strict sense, these 'possibly' cholinergic neurons.

Functional roles of the cholinergic neurons Compared with that in cats, recording single neuronal activity during sleep is very difficult in rats, since they, perhaps as a prey species, seldom sleep under the stress of experimental conditions. We had to overcome this situation with sleep-deprivation. This procedure might bias the sampling, for example, in that waking periods were so short that neurons firing only during AW were often difficult to encounter. Even considering this weakness, the following two points can be inferred clearly from the present experiments: (1) the L D T is composed of 'possibly' cholinergic neurons with heterogenous characteristics in relation to sleep and wakefulness; and (2) there were many 'possibly' cholinergic neurons specifically active during PS ( P S > S W S > A W group), in which the increase of firing starts considerably before the onset of PS. These two results are consistent with those obtained in recent studies on PPT and L D T neurons of the cat7'36; we supplemented them by showing that the results were obtained for neurons whose positions and characteristics of action potential were distinguished. A difference is that El Mansari et al. 8 suggested a possible noncholinergic nature of P S > S W S > A W neurons (tonic type II neurons in their term), based upon the fact that neurons of this type were not affected by local application of cholinergic drugs, thus they possibly had no autoreceptors. We suppose that the P S > S W S > A W neurons are also chotinergic, since massive distribution of neurons other than cholinergic ones, which may generate the broad spikes, has not been shown in the LDT. There is

no explanation at present, however, for the discrepancy between the hypotheses of E1 Mansari et al. ~ and ours. In addition to the difference in firing behavior during sleep and wakefulness, another heterogeneity in PPT and L D T neurons was shown in cats: activity of some neurons was modulated with relation to ponto-genicutooccipital (PGO) waves 3°'37. In rats the P G O waves are not observed 38. though a PGO-like activity may be generated 2°. A PGO-related activity was not investigated in the present study. The existence of many LDT neurons of P S > S W S > AW group is especially noteworthy as compared with D R and LC. which are composed of monoaminergic neurons consistently characterized by A W > S W S > P S nature. Except for the subgroup of ~possibly' cholinetgic neurons, no P S > S W S > A W neurons have been identified in the diffuse projection systems. Thus, it can be suggested that at least some of the tegmentat cholinergic neurons have a pivotal role in the induction and maintenance of PS. These cholinerglc neurons may activate: (1) the forebrain structures via ascending projections to the thalamus, hypothalamus and basal forebrain nuclei t5'31"42 that relay into neocortical and limbic systems via thalamocortical projection neurons, basal forebrain cholinergic neurons 6 and/or histaminergic neurons of the hypothalamus: and (2) some brainstem sites where carbachol injection induces PS signs m'13'29"32 via descending projections revealed recently 24'2s'43. Involvement of the tegmental cholinerglc neurons in PS mechanisms is also supported by Webster and Jones 4l who found that neurotoxic destruction of the chotinergic cell area had marked effects upon PS including its elimination. On the other hand. some cholinergic neurons (the AW = PS>SWS group and some of 'others') may be involved in induction and/or maintenance of AW. though Webster and Jones 4~ suggested that a role of the cholinergic neurons in wakefulness itself may be minor, based upon an observation that destruction of cholinergic celt bodies in the brainstem produced no major effects on behavioral wakefulness. How tegmentat cholinergic neurons participate in wakefulness remains to be investigated more clearly. One possible role of the cholinergic neurons during AW is induction of transient global attention, since. according to our preliminary observation, there were many neurons responding phasically to sensory stimuli but the response habituated very strongly by repetition. The hypothesis is not inconsistent with our previous result that a cholinergic facilitatory influence of L D T stimulation on thalamic relay neurons appeared quickly but declined even during on-going stimulation 15. In the present experiments most PS episodes ended by awakening, but change in neuronal activity was not noticeable prior to the awakening indicated by disappear-

219 ance of E E G

t h e t a activity during PS. T h e s a m e p h e -

n o m e n o n was o b s e r v e d by A s t o n - J o n e s and B l o o m 3 in LC neurons (AW>SWS>PS);

discharges a p p e a r e d al-

ways after the a w a k e n i n g f r o m PS, as we o b s e r v e d in a D R n e u r o n . N o study so far has s h o w n p a r t i c i p a t i o n of m a j o r groups or s u b g r o u p s of n e u r o n s with diffuse projection

in

awakening

from

PS.

Mechanisms

to

Acknowledgements. The authors are very grateful to Dr. Robert W. Doty, University of Rochester, for his kind discussion and suggestions in improving the manuscript. This study was presented in abstract form (Soc. Neurosci. Abstr., 16 (1990) 1255). A part of this study was used in a thesis of M.O. This study was supported by a grant from the Ministry of Education, Science and Culture of Japan (No. 01570069).

the

cause(s) of the transition f r o m PS to the a w a k e state r e m a i n s u n k n o w n , and essentially unstudied.

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Firing of 'possibly' cholinergic neurons in the rat laterodorsal tegmental nucleus during sleep and wakefulness.

To clarify functional roles of mesopontine cholinergic neurons as a component of an activating system, single neuronal activity in the laterodorsal te...
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