Activity during active sleep of bulbar reticular neurons firing rhythmically during mastication in cats

EXPERIMENTAL

NEUROLOGY

85,

178-186 ( 1984)

Activity during Active Sleep of Bulbar Reticular Neurons Firing Rhythmically during Mastication in Cats Y. NAKAMURA, K. HIRABA, M. TAIRA, Y. SAHARA, S. ENOMOTO, M. KATOH, AND A. IRIKI’ Department of Physiology, Faculty of Dentistry, Tokyo Medical and Dental University, Tokyo 113, Japan Received December 20, 1983; revision received March IS, 1984 Unitary activity was recorded from 17 bulbar reticular neurons, which fired rhythmically during mastication, in unanesthetized, spontaneously respiring cats during sleep and wakefulness. All these neurons showed the highest mean firing rate during food ingestion, and none of them showed any tonic discharge during active sleep. The results are discussedin terms of a functional differentiation of bulbar reticular inhibitory neurons projecting to jaw-closer motoneurons in relation to phasic inhibition during mastication and tonic inhibition during active sleep of jaw-closer motoneurons.

INTRODUCTION Two kinds of central inhibition of jaw-closer motoneurons have been reported in relation to mastication and sleep. One is the rhythmic inhibition of jaw-closer motoneurons during the jaw-opening phase of mastication in the cat and guinea pig (3, 8), which inhibits stretch reflexes of jaw-closing muscles ( 1,6,7, 10, 13) by antagonizing the excitatory effect of muscle spindle afferent fibers from jaw-closing muscles on jaw-closer motoneurons during the jaw-opening phase (5, 14). The other is the tonic inhibition of jaw-closer motoneurons throughout REM sleep in the cat, which is responsible for atonia of jaw-closing muscles during this sleep state (2, 4). Inhibitory premotor neurons directly projecting to jaw-closer motoneurons were shown to be situated in the bulbar reticular formation in cats (12). Corresponding to the two kinds of central inhibition of jaw-closer motoAbbreviations: W-wakefulness, QS-quiet sleep, AS-active sleep. ’ This study was supported by a grant-in-aid for Special Project Research from the Ministry of Education, Science and Culture of Japan. 178 0014-4886/84 $3.00 Copytigbt 8 1984 by Academic Pres. Inc. All rights of reprqduclion in any form -4.

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neurons, two kinds of discharge patterns were reported in these inhibitory premotor neurons. One was a rhythmic burst activity coincident with the jaw-opening phase of mastication (11). The other was a tonic firing pattern, the frequency of which was markedly higher during REM sleep than during either NREM sleep or quiet wakefulness (9). Thus, the bulbar reticular neurons were proposed to be responsible for the rhythmic phasic inhibition during mastication as well as the tonic inhibition during REM sleep of jaw-closer motoneurons in cats (9, 11). The purpose of this study was to find out whether or not the two kinds of inhibition of jaw-closer motoneurons are mediated by the same or separate populations of bulbar reticular neurons. METHODS Three adult cats were used. Under pentobarbital anesthesia (35 mg/kg, i.p.), surgical operations were carried out for recording single-unit activity from brain stem neurons together with polygrams in unanesthetized chronic conditions, following the procedure described in detail elsewhere (4). In brief, screw electrodes for recording the EEG were affixed bilaterally to the skull overlying the frontal, parietal, and occipital regions. An adapter to a head holder was mounted on the skull with acrylic resin to fix the animal’s head stereotaxically without pain or pressure during unitary recording. A hole was drilled rostrally to the lambdoidal ridge on the right side in order to insert tungsten microelectrodes through the cerebellum into the bulbar reticular formation. The hole was sealed with bone wax until the start of recording and between successive recording sessions. Recording was initiated 1 week after surgery. Before each recording session, ketamine hydrochloride ( 15 mg/kg) was administered intramuscularly, and a pair of thin metal wire electrodes (200 pm in diameter, insulated except for 4 mm at the tip) were inserted 6 to 8 mm apart into the posterior neck muscle, the masseter and anterior digastric muscles on the right side for recording EMG, and into the skin just lateral to the external canthus bilaterally for recording the EGG. The head of the animal was fixed stereotaxically in a prone position. A small light bulb was attached with sticky wax to the resin mold, which was built on the symphysis of the mandible in advance during surgery for chronic preparations, and was used as the light source to monitor jaw movement on the sag&al plane during food ingestion by a semiconductor position detector. Unitary recording was initiated about 2 h after injection of ketamine hydrochloride. An electropolished enamel-coated tungsten microelectrode (shaft: 100 pm in diameter, tip resistance: 3 to 10 MQ) was mounted on a

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microdrive and was inserted stereotaxically into the bulbar reticular formation with an inclination of 20” anteroposteriorly to avoid the tentorium cerebelli. During food ingestion, unitary activity was displayed on an oscilloscope together with the EMG of the masticatory muscles and the jaw movement. When the unit rhythmically modulated its firing rate in the same rhythm as the masticatory jaw movement, the unitary activity was stored on a magnetic tape for later analysis together with the EMG of the neck and masticatory muscles, the EEG, and the EOG. As the animal went to sleep, the unitary activity, EMG, EEG, and EOG were continuously recorded on the magnetic tape. Behavioral states of wakefulness (W), NREM sleep (quiet sleep, QS), and REM sleep (active sleep, AS) were judged by the polygram consisting of the neocortical EEG, neck EMG, and EOG. At termination of the last recording session, an electrolytic lesion was made with a tungsten microelectrode at a standard point on the right side (P 9.5, L 1.5, H -7.0) under pentobarbital anesthesia. The point was used as a reference for histological determination of the recording sites. RESULTS Single-unit activity was recorded from a total of 85 neurons in the bulbar reticular formation (P 7.0 to 11.O, L 1.Oto 3.5 coordinates). Seventeen neurons clearly showed rhythmic modulation of firing rate corresponding with the rhythmic jaw movement during ingestion of food (canned fish and milk). Figure 1 illustrates the rhythmic firing of a bulbar reticular neuron coinciding with the rhythmic jaw movement while chewing fish (Fig. 1A) or lapping milk (Fig. 1B). In this neuron, each spike burst coincided with the jawopening phase (i.e., the period from the jaw-closed position to the maximum jaw-open position in each chewing or lapping stroke). Of the 17 neurons, 7 neurons showed bursts or discharge at the highest frequency during the jawopening phase (the opening type), 3 neurons during the jaw-closing phase (i.e., the period from the maximum jaw-open position to the jaw-closed position, the closing type) and 7 neurons during transition from the jawopening to -closing phase (the transitional type) (Table 1). Activity of the 17 neurons was recorded during one or more sleep-waking cycles, consisting of the sequence of quiet W to QS to AS and return to quiet W. Figure 1C and D shows the discharge pattern of the neuron illustrated in Fig. 1A and B during sleep and wakefulness. During food ingestion (period “a”), this neuron fired at the mean firing rate of 33.3/s, which was much higher than that at 16.1/s during quiet W (period “b”). During QS, it fired in burst at irregular intervals at the mean firing rate of 6.5/s (period “c”). At the transition from QS to AS, the burst activity began to decrease about 10 s preceding the loss of the neck EMG activity and a very low level of

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FIG. 1. Firing patterns of a bulbar reticular neuron during food ingestion and a sleep-waking cycle. A and B-rhythmic activity in a bulbar reticular neuron during ingestion of canned fish (A) and milk (B). M: EMG of right masseter muscle. D: EMG of right anterior digastric muscle. JM: jaw position on the sagittal plane in dorsoventral (upper trace) and anteroposterior directions (lower trace); upward deflection corresponds to closing direction (dorsal and anterior displacement of the jaw). U: time of spike firing shown by pulses obtained from the output of an amplitude discriminator. C-E-firing pattern of the same neuron in A and B during sleep and wakefulness. C and D are a continuous polygram. From top to bottom, neocortical EEG, EGG, neck EMG, and firing rate (FIX, i.e., spike numbers in each bin of 250 ms, expressed as the number of spikes per second). Calibration and time base in D also apply to C. E-interspike interval histograms of spike firing shown in C and D during sleep and wakefulness. Each histogram shows spike firing during periods denoted “a,” “b,” “c,” and “d” in C and D, respectively. Abscissae: interspike interval (4 ms/bin); ordinates: frequency of interspike intervals (total numbers are shown in each of a-d). Interspike intervals longer than 200 ms are summed at the extreme right of each histogram. W-MASTICATION: the period of food ingestion, W-QUIET: the period not ingesting food during wakefulness. Interspike interval x f SD: (a) 30.0 + 63.5 ms, (b) 62.0 + 79.0 ms, (c) 154.0 + 419.1 ms, and (d) 178.0 f 500.5 ms.

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TABLE 1 Mean Firing Rates of the 17 Bulbar Reticular Neurons during Sleep and Wakefulness, which Showed Rhythmic Bursts during Jaw Opening (Opening Type), Jaw Closing (Closing Type), or Transition from the Former to the Latter (Transitional Type)” Type

W r; QS < AS W Z QS > AS W > QS = AS W = QS = AS QS > W > AS

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2

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3

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’ Signsof inequality and equality representthe presenceor absenceof a statisticallysignificantdifference (P < 0.05, I test)betweenbehavioral statesof quiet W, QS, and AS, respectively. Numbers in the field indicate the numbers of neurons belonging to the respectivegroups.

firing rate continued throughout AS except for sporadic bursts, which appeared with apparently no correlation with REMs (period ‘d”). The mean firing rate was 5.6/s during AS. Thus, in this neuron the mean firing rate was higher during food ingestion than during quiet W (P < 0.05, t test); it was higher during quiet W than during either QS or AS (P c 0.05); there was no difference in the mean firing rates between QS and AS (P > 0.05). Of the 17 neurons, the mean firing rate in 6 neurons was lower during AS than during either QS or quiet W; in one neuron shown in Fig. 1, it was lower during AS than during quiet W and was not significantly different from that during QS; in 2 neurons there was no difference among quiet W, QS, and AS (Table 1). Figure 1E illustrates the interspike interval histograms of neuronal firing during periods denoted a through d in Fig. 1C and D. The intervals ranged from 4.0 ms to 0.5 s in a and b, from 4.0 ms to 2.5 s in c, and from 4.0 ms to 7.0 s in d. In a through d, the median intervals were 15.4, 39.7, 3 1.2, and 34.2 ms, respectively, and the modes were 10.0, 12.0 to 24.0, 14.0, and 10.0 ms, respectively. In the remaining 8 of 17 neurons, the mean firing rate was higher during AS than during either QS or quiet W (Table 1). Four of seven opening-type neurons belonged to this group. Figure 2 shows an example of these openingtype neurons. During food ingestion (period a), this neuron showed rhythmic burst activity at the mean firing rate of 29.4/s. During QS and quiet W, spontaneous activity was rarely seen (b and c, respectively). The mean firing rate was 1.9 and 3.1 /s during quiet W and QS, respectively. During transition from QS to AS, the discharge rate began to increase slightly about 10 s preceding the loss of the neck EMG. During AS, it showed phasic burst activities, and the highest firing rate attained more than 200/s (d), although the mean firing rate during AS remained as low as 7.2/s. Thus, the mean

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FIG. 2. Firing pattern of a bulbar reticular inhibitory premotor neuron during sleep and wakefulness. A, B, and C are a continuous polygram. D, interspike interval histograms during sleep and wakefulness. A-D am shown in the same manner as in Fig. 1 C-E. Interspike interval a& SD: (a) 34.0 + 167.0 ms, (b) 534.0 ? 365.2 ms, (c) 322.0 + 250.7 ms, and(d) 138.0 + 446.7 ms. E-averaged records of the EMG of the right masseter (top) and anterior digastric muscles (middle) triggered by spontaneous spikes of the bulbar reticular neuron (bottom) illustrated in A-D. The EMG was full-rectified and averaged 500 times by the spike potentials. In the top and middle traces the upward deflection indicates the increase in EMG activity, and in the bottom trace it shows positivity. Arrow indicates the onset of a decrease in the EMG after 1.8 ms from the onset of the spike potential (vertical broken line). Time base applies to all traces.

firing rate in this neuron was sequentially higher in the following order: quiet W, QS, AS, and food ingestion period (P < 0.05). Histograms in Fig. 2D illustrate the distribution of interspike intervals during periods a through d in Fig. 2A-C. The intervals ranged from 4.0 ms to 6.5 s in a, from 4.0 ms to 10.5 s in b, from 4.0 ms to 14.0 s in c, and from 4.0 ms to 20.5 s in d. In a through d, the median intervals were 8.7, 33.0, 17.2, and 18.3 ms, respectively, and the modes were 6.0, 14.0 and 22.0, 10.0, and 6.0 ms, respectively. In all 17 neurons, the mean firing rate was higher during food ingestion than during either quiet W, QS, or AS. None of the 17 neurons, irrespective

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of whether or not their mean firing rate was higher during AS than either QS or quiet W, showed any tonic firing during AS. The higher mean firing rate during AS than either QS or quiet W was due to the high-frequency discharge in burst during AS. Histological examination confirmed that the 17 neurons were recorded from the lateral part of the nucleus reticularis gigantocellularis, the nucleus reticularis magnocellularis, and the medial portion of the nucleus reticularis parvocellularis at the level from the nucleus n. facialis to the middle portion of the nucleus olivaris inferior. There was no separation in loci between the neurons firing at a higher mean firing rate during AS than QS and those firing at a lower firing rate during AS than QS. We tried to find whether or not the seven opening-type neurons make direct inhibitory synaptic connection with jaw-closer motoneurons, using the spike-triggered averaging technique as described elsewhere (1 l), i.e., averaging of the full-rectified EMG of the jaw-closing muscles by the spontaneous spikes of bulbar reticular neurons. Of the seven opening-type neurons, one neuron illustrated in Fig. 2, which showed a higher mean firing rate during AS than during either QS or quiet W, was found to make a direct inhibitory synaptic linkage with masseter motoneurons. As seen in Fig. 2E, a decrease was detected in the EMG activity of the masseter muscle (Fig. 2E top trace) after a latency of 1.8 ms from the onset of the triggering spikes of the bulbar reticular neuron (Fig. 2E, bottom trace) with no change in the digastric EMG (Fig. 2E, middle trace). The latency was within the range of the expected latency of decrease in the masseter EMG when the masseter motoneurons were monosynaptically inhibited by spikes of this neuron (11). DISCUSSION The incidence of the monosynaptic inhibitory connection with jaw-closer motoneurons found in one of seven opening-type neurons ( 14%) is comparable to that in a previous study which showed that four of 22 opening-type neurons (18%) projected directly to jaw-closer motoneurons to make inhibitory synaptic connection with them (11). The remaining opening-type neurons which failed to show inhibitory synaptic linkage with jaw-closer motoneurons may possibly project either to other cranial motoneurons which show rhythmic activities during mastication, e.g., hypoglossal and facial motoneurons, or to the premotor neurons which project to the cranial motoneurons participating in mastication. Thus, in this study, we found that none of the bulbar reticular neurons, that rhythmically modulated the firing rate during mastication in the same rhythm as the masticatory jaw movement, showed any tonic firing during AS, though the mean firing rate was higher during AS than during QS or

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quiet W in nearly half of them, including inhibitory premotor neurons projecting to jaw-closer motoneurons. The results imply that the bulbar reticular inhibitory premotor neurons, which project to jaw-closer motoneurons and rhythmically modulate the firing rate during mastication in the same rhythm as the mastication, would be within the separate populations from the presumed bulbar reticular inhibitory premotor neurons which show a tonic firing at the highest frequency during AS and participate in tonic inhibition of jawcloser motoneurons during that sleep state. Bulbar reticular inhibitory premotor neurons, which show phasic bursts during AS and the higher mean firing rate during that sleep state than during QS or quiet W, may contribute to the tonic inhibition of jaw-closer motoneurons during AS. But they may not be the inhibitory premotor neurons which play so critical a role in the tonic inhibition of jaw-closer motoneurons during AS, if it were not for a mechanism to set the timing of the burst of these neurons successively so as to maintain the amount of converging inhibitory input to jaw-closer motoneurons more or less constant throughout AS. Thus, our study provides evidence for a functional differentiation among inhibitory premotor neurons in the bulbar reticular formation projecting to jaw-closer motoneurons: those which underlie the rhythmic inhibition in the jaw-opening phase during mastication by phasically firing coincidentally with this phase would be within a population different from those which are primarily responsible for the tonic inhibition during AS by tonically discharging throughout that sleep state. REFERENCES 1. APPENTENG, K., M. J. O’DONOVAN, G. SOMJEN, J. A. STEPHENS,AND A. TAYLOR. 1978. The projection of jaw elevator muscle spindle afferents to fifth nerve motoneurones in the cat. J. Physiol. (London) 279: 409-423. 2. CHANDLER, S. H., M. H. CHASE,ANDY. NAKAMURA. 198O.IntraceIIularanaIysi.s ofsynaptic mechanisms controlling trigeminal motoneuron activity during sleep and wakefulness. J. Neurophysiol. 44: 359-37 1. 3. CHANDLER, S. H., AND L. J. GOLDBERG. 1982. Intracellular analysis of synaptic mechanisms controlling spontaneous and cortically induced rhythmical jaw movements in the guinea pig. J. Neurophysiol. 48: 126-138. 4. CHASE, M. H., S. H. CHANDLER, AND Y. NAKAMURA. 1980. Intracellular determination of membrane potential of trigeminal motoneurons during sleep and wakefulness. J. Neurophysiol.

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5. CODY, F. W. J., L. M. HARRISON, AND A. TAYLOR. 1975. Analysis of activity of muscle spindles of the jaw-closing muscles during normal movements in the cat. J. Physiol. (London)

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6. HLJGELIN,A., AND M. RONVALLET. 1957. etude oscillographique d’un &exe monosynaptique ctinien (&Iexe ma&term). J. Physiol. (Paris) 49: 2 IO-2 11. 7. KIDOKORO, Y., K. KUBOTA, S. SHUTO, AND R. SUMINO. 1968. Reflex organization of cat masticatory muscles. J. Neurophysiol. 31: 695-708.

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8. Kuno, Y., S. ENOMOTO, AND Y. NAKAMURA. 1981. Synaptic basis of orbital cortically induced rhythmical masticatory activity of trigeminaf motoneurons in immobilized cats. Bruin Res. 230: 97-l 10. 9. NAKAMURA, Y., S. ENOMOTO, M. KATOH, K. HIRABA, Y. SAHARA, M. TAIRA, AND M. H. CHASE. 1982. Bulbar reticular neurons: possible involvement in tonic inhibition of trigeminal motoneurons during active sleep. Neurosci. Lett. Suppl. 9: S14. 10. NAKAMURA, Y., L. J. GOLDBERG, AND C. D. CLEMENTE. 1967. Nature of suppression of the masseteric monosynaptic reflex induced by stimulation of the orbital gyrus of the cat. Brain Res. 6: 184-198. 1 I. NAKAMURA, Y., K. HIRABA, S. ENOMOTO, AND Y. SAHARA. 1982. Bulbar reticular unit activity during food ingestion in the cat. Bruin Res. 253: 312-316. 12. NOZAKI, S., S. ENOMOTO, AND Y. NAKAMURA. 1983. Identification and input-output prop erties of bulbar reticular neurons involved in the cerebral cortical control of trigeminal motoneurons in cats. Exp. Brain Res. 49: 363-372. 13. SZENT~OTHAI, J. 1948. Anatomical considerations of monosynaptic reflex arcs. J. Neurophysiol. 11: 445-454. 14. TAYLOR, A., AND F. W. J. CODY. 1974. Jaw muscle spindle activity in the cat during normal movements of eating and drinking. Bruin Res. 71: 523-530.