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Intracellular recording in trigeminal motoneurons of the anesthetized guinea pig during rhythmic jaw movements
EXPERIMENTAL
NEUROLOGY
58, loi?-
(1978)
Intracellular Recording in Trigeminal Motoneurons Anesthetized Guinea Pig during Rhythmic Jaw Movements LOUIS J.
GOLDBERG
AND
MICHAEL
TAL
of the
l
Departments of Oral Biology and Anatomy a.4 Dental Research Institute, Schools of Dentistry and Medicine, University of C3alifornia, Los Angeles, California 90024 Received
July
28, 1977
Intracellular recordings were obtained from guinea pig trigeminal motoneurons during rhythmic jaw movements. The cells were identified by stimulation of the trigeminal mesencephalic nucleus which evokes excitatory postsynaptic potentials and spikes in jaw-closer motoneurons. The anesthetized guinea pig in the stereotaxic apparatus demonstrated spontaneous rhythmic jaw movements which were characterized by hyperpolarization of jaw-closer motoneurons, occurring concurrently with digastric muscle excitation during the jaw-opening phase of the cycle. The anesthetized guinea pig could also be induced to rhythmically clench and release a stick placed between the molar teeth. During this behavior intracellular recordings in jawcloser motoneurons revealed a rapid depolarization leading to bursts of action potentials following the hyperpolarization which occurred during the jawopening phase of the cycle. The results demonstrate the feasibility of intracellular recording in guinea pig trigeminal motoneurons during rhythmic jaw movements. It was also shown that during the opening phase of the rhythmic jaw movement cycle there is a pronounced hyperpolarization present in the membrane potential of jaw-closer motoneurons. Resolution of the problem of central vs. peripheral origin of this hyperpolarization is significant for our understanding of the motor control of the jaw.
INTRODUCTION Intracellular obtained in
recordings anesthetized
in trigeminal motoneurons have previously been or decerebrate cats immobilized with neuro-
EPSP-excitatory postsynaptic potential ; Abbreviations : EMG-electromyogram; im-intramuscular ; iv-intravenous. 1 This research was supported in whole by the National Institute of Dental Research, National Institutes of Health, Research Grant ROl DE 4166. 102
0014-4886/78/0581-0102$02.00/0 Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
GU1NEA
PIG
TKIC~MINAL
MOTONEUROKS
103
muscular blocking agents (5, S, 11, 14). Those studies were invaluable in elucidating the synaptic mechanisms underlying the effects of peripheral and central inputs on the activity of jaw-opener and jaw-closer motoneurons. However, the attempt to understand how the central nervous system controls jaw movements in complex behaviors such as chewing would be greatly assisted by a preparation in which intracellular recordings could be obtained in trigeminal motoneurons during the actual performance of such behaviors. This requires a preparation in which the animal is essentially intact and, obviously, not immobilized. In the present paper, we describe such a preparation and the results of recordings from trigeminal jaw-closer motoneurons during two types of rhythmic jaw movements in the anesthetized guinea pig. METHODS The experiments were done on 11 albino guinea pigs (English Short Hair) weighing 400 to 850 g. Atropine sulfate (0.1 mg/kg, intraperitoneal) and Thorazine [chlorpromazine hydrochloride ; 12.5 mg, intramuscular (im)] were given 30 min prior to administering the anesthetic agent. The animals were anesthetized with ketamine HCl (100 mg/kg, im), with maintenance doses of 10 mg/kg intravenous (iv) when necessary. Cortisone acetate (2.8 mg/kg, im) was given at the time of the initial dose of anesthesia. A heating pad was used to assist in the maintenance of body temperature. A midline incision was made from the mental symphysis at the inferior surface of the mandible to the sternum. A tracheotomy was carried out (however, artificial ventilation was not needed during these experiments) and a cannula was placed in the right external jugular vein. A bipolar electromyographic (EMG) hook electrode (Teflon-insulated, 76pm-diameter stainless-steel wires ; interpolar distance 5 mm) was inserted into the anterior belly of the left digastric muscle with a 22-gauge hypodermic needle. The incision was then closed and the animal positioned in a stereotaxic apparatus. A bipolar needle stimulating electrode was inserted into the left side of the tongue (interpolar distance 2 mm). A midline incision over the skull was made and a small (approximately 4 mm in diameter) circular piece of bone was removed on the left side at the region of the interaural line. The dura was cut and a concentric bipolar stainlesssteel electrode (inner core 0.2 mm, sheath 0.5 mm, interpolar distance 0.5 mm) was inserted for stimulation of the left trigeminal mesencephalic nucleus (Fig. 1A) at a position 1.5 mm lateral to the midline and 0.5 mm anterior to the interaural line. Beveled glass micropipets (5 to 10 Ma) filled with 2 M potassium citrate were inserted with a microdrive into the left trigeminal motor nucleus which is situated approximately 3 mm lateral from the midline and 1 mm posterior to the interaural line. Recordings
104
GOLDBERG
AND
TAL
made from the EMG and glass microelectrodes tape for later data analysis.
were recorded on magnetic
RESULTS Identification of jaw-closer motoneurons was accomplished in the following manner. Electrical shocks delivered through the electrode placed near the mesencephalic nucleus (Fig. 1) evoked a large field potential in the jaw-closer motoneuron pool of the trigeminal motor nucleus (Fig. 1B). Intracellular recordings from trigeminal jaw-closer motoneurons were obtained in the area of the field potential (Figs. lC, D). It was found that the most effective stimulus paradigm was three brief shocks (10 to 50 ps in duration) at 750 pulses per second (Fig. 1B). Stimulation of the mes-
esencephalic
Jaw Mot’
nucleus
Iv
of P
2mV
lmsec
FIG. 1. A-Schematic representation of the stimulating and recording sites. B-Field potential recorded in the motoneuron pool of the jaw-closing muscles following three shocks delivered to the mesencephalic nucleus of V. C and D-Intracellular recordings obtained from a jaw-closer motoneuron. Each trace in B, C, and D is the photographic superimposition of 10 oscilloscope sweeps.
CIJJNEA
PIG
TRTCERIINAL
MOTONEURONS
10.5
encephalic nucleus has been shown to evoke monosynaptic excitatory postsynaptic potentials (EPSPs) and spikes in the motoneurons of the jawclosing temporal and masseter muscles (8, 11). The effectiveness of the multiple shocks is due to the addition of EPSPs which result in bringing increased numbers of motoneurons in the motor pool to threshold for spike initiation. This phenomenon is clearly seen in Figs. lC, D; in Fig. 1C a single shock to the mesencephalic nucleus evokes an EPSP in the motoneuron whereas two shocks, in this case, sum sufficiently to induce an action potential (Fig. 1D). The multiple-shock technique is also important in that it permits the shocks delivered in the mesencephalic nucleus to be of short duration and low intensity, thereby minimizing the size of the stimulus field. The latency from the last shock in the burst delivered to the mesencephalic nucleus to the start of the large negative deflection in the field potential (Fig. lB, arrow), and to the initiation of spikes in the closer motoneurons (Fig. lD), was approximately 0.5 ms. Similar latencies (approximately 0.6 ms) were reported in the cat (8, 11). This technique of identifying jaw-closer motoneurons is similar to that used in a recent study involving intracellular recordings from trigeminal motoneurons in the fully conscious, normally respiring, unanesthetized cat during naturally occurring sleep ( 12). When the anesthetized guinea pig is the stereotaxic apparatus there is a separation of approximately 1 mm between the molar teeth and 3 mm between the incisal edges of the incisor teeth. From this static position we have observed that all animals spontaneously begin to produce rhythmic jaw movements. This activity occurs in the absence of movement of any part of the body. These rhythmic jaw movements consist of a slight opening of the jaw (approximately 4 mm) followed by a return to the original teethapart position. Intracellular recordings from a jaw-closer motoneuron obtained during spontaneous rhythmic jaw movements are shown in Fig. 2A in which an 8-s consecutive sample obtained during this behavior is illustrated. The upper trace is the intracellular record, and the lower trace the digastric muscle EMG. Electromyographic activity in the digastric muscle is associated with the opening phase of the cycle; the rhythmic movements had a frequency of 2 to 3/s and are approximately 2/s in Fig. 2A. We consistently observed that the digastric muscle EMG activity is accompanied by membrane hyperpolarization in jaw-closer motoneurons (Figs. 2A, C). Furthermore, a dramatic increase in synaptic activity is seen during this period of hyperpolarization (Figs. 2A, C). The appearance of rhythmic jaw movements is, to some extent, a function of depth of anesthesia because this behavior could be stopped in all cases by iv injection of ketamine (10 mg/ kg)Stimulation of the tongue evoked a hyperpolarizing potential after a
106
GOLDBERG
AND
TAL
latency of 3.2 ms (Fig. 2B) in the same closer motoneuron shown in Figs. 2A, C, during a period in which no spontaneous jaw movements were present; the same stimulus evoked a large EMG response in the digastric A
FIG. 2. Intracellular recording in jaw-closer motoneurons during rhythmic jaw movements. A-Raster display, obtained during spontaneous rhythmic jaw movements, of the simultaneously recorded membrane potential of a jaw-closer motoneuron (upper trace) and digastric muscle EMG (lower trace). The bar in A shows that part of the record of the second sweep which is illustrated at higher gain and faster sweep speed in C. B-Response to electrical stimulation of the tongue in ‘the same motoneuron and digastric muscle shown in A and C (each trace is the photographic superimposition of 10 sweeps). D-Same as A, recorded from another animal during chewing on a stick placed between the molar teeth. Approximately the first half of sweep 2 in D is shown in E, at a higher gain and faster sweep speed. All calibration bars for the intracellular recordings are 5 mV except for D, which is 50 mV. All calibration bars for the digastric EMG recordings are 500 @V except for E, which is 200 pV. The raster displays in A and D illustrate continuous recordings during rhythmic jaw movements; however, a small part of the record which occurs between the end of one sweep and the beginning of the next is not shown.
GUINEA
PIG
TRIGEMINAL
MOTONEURONS
107
muscle beginning after a latency of 8 ms. This response represents the jawopening reflex (excitation of jaw-opener and inhibition of jaw-closer motoneurons) which was previously described in the cat after stimulation of both lingual (5) and inferior dental nerves (8). Stimulation of these nerves in the cat has been shown to evoke disynaptic inhibitory postsynaptic potentials (2.5~ms latency) in jaw-closing masseter and temporal motoneurons (5, 8) and EPSPs and spikes in digastric motoneurons [ 3- to 4-ms latency (8) 1. The latencies reported here for the guinea pig are longer than those in the cat due to the fact that in the present case we are stimulating in the tongue rather than in the nerve trunk and are recording from the muscle rather than from the motoneurons or the digastric nerve. Note that during the hyperpolarization evoked by tongue stimulation there are no membrane fluctuations associated with digastric muscle EMG activity (Fig. 2B), in contrast to the large fluctuations present during the hyperpolarization which occurs in spontaneous rhythmic jaw movements (Figs. ZA, C). A second type of rhythmic jaw movement was observed in the anesthetized guinea pig which more resembled chewing than did the previously described behavior. When a wooden stick (2 mm in diameter) was placed between the molar teeth on one side, cyclic chewing movements with alternating clenching and releasing of the stick could be evoked. (Because the occlusal surfaces of the molar teeth were approximately 1 mm apart, the stick initially acted to force the jaws further apart.) This behavior could be evoked in guinea pigs which were not producing any jaw movements prior to the introduction of the stick. The chewing behavior stopped if the stick was removed from the mouth. In Fig. 2D, a 16-s consecutive sample of this behavior is shown. The upper trace is the intracellular record (obtained from the same jaw-closer motoneuron shown in Figs. lC, D), and the lower trace is the simuitaneous record of the digastric muscle EMG. In contrast to the record obtained during spontaneous rhythmic jaw movements (Fig, 2A), rapid depolarization of the motoneuron leading to spiking activity can be observed after the hyperpolarization accompanying digastric muscle EMG activity (Figs. 2D, E). During those bursts of spikes in the jaw-closer motoneuron the stick was observed to be firmly gripped by the molar teeth. During the periods of digastric muscle activity the stick was released as the jaw opened. Note the synaptic activity which can be observed during the hyperpolarization which occurs prior to the spike bursts (Fig. 2E). This synaptic activity occurs during the period in which the digastric muscle is active both in spontaneous rhythmic jaw movements (Figs, 2A, C) and in stick chewing (Figs. 2D, E) . DISCUSSION These experiments demonstrate that the guinea pig is an excellent animal model for the study of the central nervous system control of rhythmic jaw
108
GOLDBERG
AND
TAL
movements. Under anesthesia in a stereotaxic apparatus the guinea pig ‘exhibits spontaneous rhythmic jaw movements as well as rhythmic chewing movements during which there is alternating clenching and releasing of an object placed between the molar teeth. This proclivity of the guinea pig to exhibit rhythmic jaw movements under anesthesia is not surprising in the light of studies which have shown that the molar teeth of guinea pig fetuses exhibit occlusal wear 10 days prior to birth, indicating that significant grinding of the teeth occurs in utero (1). The central nervous system components responsible for chewing patterns are obviously well developed in this animal, We also demonstrated a method for the identification of jawcloser motoneurons in the intact guinea pig as well as establishing the feasibility of intracellular recording in trigeminal motoneurons during the above-described rhythmic jaw movements. Action potentials were not observed in jaw-closer motoneurons during spontaneous rhythmic jaw movements. However, a striking finding was the pronounced hyperpolarization and increase in synaptic activity that accompanied the digastric muscle activity associated with the jaw-opening phase of the cycle (Figs. ZA, C-E). It is well known from the EMG studies that jaw-closer motoneurons are not active in the jaw-opening phase of the chewing cycle (2, 10, 16). At present there is no information indicating whether this silence is a function of a simple absence of excitation or whether there are active inhibitory processes at work. The results of intracellular recordings in the jaw-closer motoneurons of the guinea pig provide direct evidence for the presence of an active inhibitory process. There is previous evidence indicating that the observed hyperpolarization in closer motoneurons during rhythmic jaw movement could be induced either by peripheral or central mechanisms. In the spontaneous rhythmic jaw movements, the hyperpolarization in the closer motoneurons appears to closely correspond to activity in the digastric muscle (Figs. 2A, C) . Recent studies indicated the presence of proprioceptors in the anterior belly of the digastric muscle in cats (15) and humans (7). On the other hand, it was reported that stimulation of sites in the medial bulbar reticular formation produces monosynaptic inhibition of jaw-closer motoneurons and reciprocal excitation of opener motoneurons (14). If this reticular formation site is involved in the central generation of rhythmic jaw movements, then one could speculate that the spontaneous rhythmic jaw movements in the guinea pig are produced by a central pattern generator that would simultaneously excite digastric motoneurons and inhibit closer motoneurons and thereby produce the pattern observed in Figs. 2A, C. Whether the origin of the hyperpolarization is central, peripheral, or a combination of both, its function may be to prevent the monosynaptic excitation of jaw-closer motoneurons by spindle afferents activated by stretch
GUINEA
PIG
TRIGEMINAL
MOTONEURONS
109
of the jaw-closing muscles which accompanies jaw opening. It has been shown in the cat (2) and monkey (4) that stretch of the jaw-closing muscles, which occurs during the rapid jaw-opening phase of chewing, induces vigorous activity in spindle afferents from jaw-closing muscles. Such activation could result in inappropriate excitation of jaw-closing muscles and thereby effectively splint the jaw and restrict opening. Presynaptic inhibition of this reflex in the cat by depolarization of the central terminals of spindle afferents was recently proposed as one mechanism for the prevention of unwanted excitation of jaw-closing muscles during the opening phase of the chewing cycle (6). The present study provides evidence suggesting that postsynaptic inhibition of jaw-closer motoneurons also occurs during jaw opening, indicating that both presynaptic and postsynaptic inhibition plays a role in assuring the inactivity of jaw-closing muscles when the jaw is being opened. As previously stated, action potentials were not observed in jaw-closer motoneurons during the spontaneous rhythmic jaw movements and preliminary EMG recordings in both the masseter and temporal muscle indicate that they are not active during this behavior. At the present time, we do not have evidence indicating the mechanism responsible for bringing the jaw back to the initial rest position after the opening phase of the cycle is completed. Several possibilities exist: There may be activity that we have not observed in the masseter and temporal muscles, the medial pterygoid muscle may be involved, or the return might be a function of the passive elastic component of the closing muscles. It is clear, however, that in the chewing movements which can be induced when the stick is placed between the molar teeth, both the temporal and masseter muscles are active and bursts of spikes can be observed in closer motoneurons (Figs. 2D, E). The bursting activity in the closer motoneurons which we observed appears to be present only when the guinea pigs are clenching an object placed between the teeth. In the spontaneous rhythmic jaw movements, no tooth contact occurs and no jaw-closer motoneuron activation has so far been observed. Studies in the cat (13) and rabbit (3, 9) demonstrated the presence of a central pattern generator which produces rhythmic alternating activity in jaw-opener and jaw-closer motoneurons. Similar alternating activity was observed in the anesthetized guinea pig during chewing behavior. We also observed spontaneous rhythmic jaw movements accompanied by rhythmic digastric muscle activity and concurrent closer motoneuron hyperpolarization. Experiments are currently underway to determine the origin of the observed hyperpolarization and to examine the effects of interaction between the central pattern generator and feedback from both jaw muscle
110 proprioceptors motoneurons.
GOLDBERG
and intraoral
AND
TAL
mucosal receptors
on guinea pig jaw muscle
REFERENCES 1. AINAMO, J. 1971. Prenatal occlusal wear in guinea pig molars. Stand. J. Dent. RES. 79 : 69-71. 2. 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. (Lo&on) 253 : 565-582. 3. DELLOW, P. G., AND J. P. LUND. 1971. Evidence for central timing of rhythmical mastication. J. Physiol. (London) 215 : 1-13. 4. GOODWIN, G. M., AND E. S. LUSCHEI. 1975. Discharge of spindle afferents from jaw-closing muscles during chewing in alert monkeys. J. Neurophysiol. 38 : 560571. 5. GOLDBERG, L. J., AND Y. NAKAMURA. 1968. Lingually induced inhibition of masseteric motoneurons. Erperientia 24 : 371373. 6. GOLDBERG, L. J., AND Y. NAKAMURA. 1977. Production of primary afferent depolarization in Group Ia fibers from the masseter muscle. by stimulation of trigeminal cutaneous afferents. Brain Res. (in press). 7. HELLSING, G. 1977. A tonic vibration reflex evoked in the jaw opening muscles in man. 1977. Arch. Oral Biol. 22 : 175-180. 8. KIDOKORO, Y., K. KUBOTA, S. SHUTO, AND R. SUMINO. 1968. Reflex organization of cat masticatory muscles. J. Newophysiol. 31: 695-708. 9. LUND, J. P., AND P. G. DELLOW. 1971. The influence of interactive stimuli on rhythmical masticatory movements in rabbits. Arch. Oral Biol. 16: 215-223. 10. LUSCHEI, E. S., AND G. M. GOODWIN. 1974. Patterns of mandibular movement and jaw muscle activity during mastication in the monkey. J. Neurophysiol. 37: 954966. 11. 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. 12. NAKAMURA, Y., L. J. GOLDBERG, S. H. CHANDLER, AND M. H. CHASE. 1977. Intracellular analysis of trigeminal motoneuron activity during sleep in the cat. Science (in press). 13. NAKAMURA, Y., Y. KUBO, S. NOZAKI, AND M. TAKATORI. 1976. Cortically induced masticatory rhythm and its modification by tonic peripheral inputs in immobilized cats. Bull. Tokyo Med. Dent. Univ. 23 : 101-107. 14. NAKAMURA, Y., M. TAKATORI, S. NOZAKI, AND M. KIKUCHI. 1975. Monosynaptic reciprocal control of trigeminal motoneurons from the medial bulbar reticular formation. Brain. Res. 89 : 144-148. 15. SAUERLAND, E. K., AND H. THIELE. 1970. Presynaptic depolarization of lingual and glossopharyngeal nerve afferents induced by stimulation of trigeminal proprioceptive fibers. Exj. Neural. 28 : 344-355. 16. TAYLOR, A., AND F. W. J. CODY. 1974. Jaw muscle spindle activity in the cat during normal movements of eating and drinking. Brain Res. 71: 523-530.
NEUROLOGY
58, loi?-
(1978)
Intracellular Recording in Trigeminal Motoneurons Anesthetized Guinea Pig during Rhythmic Jaw Movements LOUIS J.
GOLDBERG
AND
MICHAEL
TAL
of the
l
Departments of Oral Biology and Anatomy a.4 Dental Research Institute, Schools of Dentistry and Medicine, University of C3alifornia, Los Angeles, California 90024 Received
July
28, 1977
Intracellular recordings were obtained from guinea pig trigeminal motoneurons during rhythmic jaw movements. The cells were identified by stimulation of the trigeminal mesencephalic nucleus which evokes excitatory postsynaptic potentials and spikes in jaw-closer motoneurons. The anesthetized guinea pig in the stereotaxic apparatus demonstrated spontaneous rhythmic jaw movements which were characterized by hyperpolarization of jaw-closer motoneurons, occurring concurrently with digastric muscle excitation during the jaw-opening phase of the cycle. The anesthetized guinea pig could also be induced to rhythmically clench and release a stick placed between the molar teeth. During this behavior intracellular recordings in jawcloser motoneurons revealed a rapid depolarization leading to bursts of action potentials following the hyperpolarization which occurred during the jawopening phase of the cycle. The results demonstrate the feasibility of intracellular recording in guinea pig trigeminal motoneurons during rhythmic jaw movements. It was also shown that during the opening phase of the rhythmic jaw movement cycle there is a pronounced hyperpolarization present in the membrane potential of jaw-closer motoneurons. Resolution of the problem of central vs. peripheral origin of this hyperpolarization is significant for our understanding of the motor control of the jaw.
INTRODUCTION Intracellular obtained in
recordings anesthetized
in trigeminal motoneurons have previously been or decerebrate cats immobilized with neuro-
EPSP-excitatory postsynaptic potential ; Abbreviations : EMG-electromyogram; im-intramuscular ; iv-intravenous. 1 This research was supported in whole by the National Institute of Dental Research, National Institutes of Health, Research Grant ROl DE 4166. 102
0014-4886/78/0581-0102$02.00/0 Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
GU1NEA
PIG
TKIC~MINAL
MOTONEUROKS
103
muscular blocking agents (5, S, 11, 14). Those studies were invaluable in elucidating the synaptic mechanisms underlying the effects of peripheral and central inputs on the activity of jaw-opener and jaw-closer motoneurons. However, the attempt to understand how the central nervous system controls jaw movements in complex behaviors such as chewing would be greatly assisted by a preparation in which intracellular recordings could be obtained in trigeminal motoneurons during the actual performance of such behaviors. This requires a preparation in which the animal is essentially intact and, obviously, not immobilized. In the present paper, we describe such a preparation and the results of recordings from trigeminal jaw-closer motoneurons during two types of rhythmic jaw movements in the anesthetized guinea pig. METHODS The experiments were done on 11 albino guinea pigs (English Short Hair) weighing 400 to 850 g. Atropine sulfate (0.1 mg/kg, intraperitoneal) and Thorazine [chlorpromazine hydrochloride ; 12.5 mg, intramuscular (im)] were given 30 min prior to administering the anesthetic agent. The animals were anesthetized with ketamine HCl (100 mg/kg, im), with maintenance doses of 10 mg/kg intravenous (iv) when necessary. Cortisone acetate (2.8 mg/kg, im) was given at the time of the initial dose of anesthesia. A heating pad was used to assist in the maintenance of body temperature. A midline incision was made from the mental symphysis at the inferior surface of the mandible to the sternum. A tracheotomy was carried out (however, artificial ventilation was not needed during these experiments) and a cannula was placed in the right external jugular vein. A bipolar electromyographic (EMG) hook electrode (Teflon-insulated, 76pm-diameter stainless-steel wires ; interpolar distance 5 mm) was inserted into the anterior belly of the left digastric muscle with a 22-gauge hypodermic needle. The incision was then closed and the animal positioned in a stereotaxic apparatus. A bipolar needle stimulating electrode was inserted into the left side of the tongue (interpolar distance 2 mm). A midline incision over the skull was made and a small (approximately 4 mm in diameter) circular piece of bone was removed on the left side at the region of the interaural line. The dura was cut and a concentric bipolar stainlesssteel electrode (inner core 0.2 mm, sheath 0.5 mm, interpolar distance 0.5 mm) was inserted for stimulation of the left trigeminal mesencephalic nucleus (Fig. 1A) at a position 1.5 mm lateral to the midline and 0.5 mm anterior to the interaural line. Beveled glass micropipets (5 to 10 Ma) filled with 2 M potassium citrate were inserted with a microdrive into the left trigeminal motor nucleus which is situated approximately 3 mm lateral from the midline and 1 mm posterior to the interaural line. Recordings
104
GOLDBERG
AND
TAL
made from the EMG and glass microelectrodes tape for later data analysis.
were recorded on magnetic
RESULTS Identification of jaw-closer motoneurons was accomplished in the following manner. Electrical shocks delivered through the electrode placed near the mesencephalic nucleus (Fig. 1) evoked a large field potential in the jaw-closer motoneuron pool of the trigeminal motor nucleus (Fig. 1B). Intracellular recordings from trigeminal jaw-closer motoneurons were obtained in the area of the field potential (Figs. lC, D). It was found that the most effective stimulus paradigm was three brief shocks (10 to 50 ps in duration) at 750 pulses per second (Fig. 1B). Stimulation of the mes-
esencephalic
Jaw Mot’
nucleus
Iv
of P
2mV
lmsec
FIG. 1. A-Schematic representation of the stimulating and recording sites. B-Field potential recorded in the motoneuron pool of the jaw-closing muscles following three shocks delivered to the mesencephalic nucleus of V. C and D-Intracellular recordings obtained from a jaw-closer motoneuron. Each trace in B, C, and D is the photographic superimposition of 10 oscilloscope sweeps.
CIJJNEA
PIG
TRTCERIINAL
MOTONEURONS
10.5
encephalic nucleus has been shown to evoke monosynaptic excitatory postsynaptic potentials (EPSPs) and spikes in the motoneurons of the jawclosing temporal and masseter muscles (8, 11). The effectiveness of the multiple shocks is due to the addition of EPSPs which result in bringing increased numbers of motoneurons in the motor pool to threshold for spike initiation. This phenomenon is clearly seen in Figs. lC, D; in Fig. 1C a single shock to the mesencephalic nucleus evokes an EPSP in the motoneuron whereas two shocks, in this case, sum sufficiently to induce an action potential (Fig. 1D). The multiple-shock technique is also important in that it permits the shocks delivered in the mesencephalic nucleus to be of short duration and low intensity, thereby minimizing the size of the stimulus field. The latency from the last shock in the burst delivered to the mesencephalic nucleus to the start of the large negative deflection in the field potential (Fig. lB, arrow), and to the initiation of spikes in the closer motoneurons (Fig. lD), was approximately 0.5 ms. Similar latencies (approximately 0.6 ms) were reported in the cat (8, 11). This technique of identifying jaw-closer motoneurons is similar to that used in a recent study involving intracellular recordings from trigeminal motoneurons in the fully conscious, normally respiring, unanesthetized cat during naturally occurring sleep ( 12). When the anesthetized guinea pig is the stereotaxic apparatus there is a separation of approximately 1 mm between the molar teeth and 3 mm between the incisal edges of the incisor teeth. From this static position we have observed that all animals spontaneously begin to produce rhythmic jaw movements. This activity occurs in the absence of movement of any part of the body. These rhythmic jaw movements consist of a slight opening of the jaw (approximately 4 mm) followed by a return to the original teethapart position. Intracellular recordings from a jaw-closer motoneuron obtained during spontaneous rhythmic jaw movements are shown in Fig. 2A in which an 8-s consecutive sample obtained during this behavior is illustrated. The upper trace is the intracellular record, and the lower trace the digastric muscle EMG. Electromyographic activity in the digastric muscle is associated with the opening phase of the cycle; the rhythmic movements had a frequency of 2 to 3/s and are approximately 2/s in Fig. 2A. We consistently observed that the digastric muscle EMG activity is accompanied by membrane hyperpolarization in jaw-closer motoneurons (Figs. 2A, C). Furthermore, a dramatic increase in synaptic activity is seen during this period of hyperpolarization (Figs. 2A, C). The appearance of rhythmic jaw movements is, to some extent, a function of depth of anesthesia because this behavior could be stopped in all cases by iv injection of ketamine (10 mg/ kg)Stimulation of the tongue evoked a hyperpolarizing potential after a
106
GOLDBERG
AND
TAL
latency of 3.2 ms (Fig. 2B) in the same closer motoneuron shown in Figs. 2A, C, during a period in which no spontaneous jaw movements were present; the same stimulus evoked a large EMG response in the digastric A
FIG. 2. Intracellular recording in jaw-closer motoneurons during rhythmic jaw movements. A-Raster display, obtained during spontaneous rhythmic jaw movements, of the simultaneously recorded membrane potential of a jaw-closer motoneuron (upper trace) and digastric muscle EMG (lower trace). The bar in A shows that part of the record of the second sweep which is illustrated at higher gain and faster sweep speed in C. B-Response to electrical stimulation of the tongue in ‘the same motoneuron and digastric muscle shown in A and C (each trace is the photographic superimposition of 10 sweeps). D-Same as A, recorded from another animal during chewing on a stick placed between the molar teeth. Approximately the first half of sweep 2 in D is shown in E, at a higher gain and faster sweep speed. All calibration bars for the intracellular recordings are 5 mV except for D, which is 50 mV. All calibration bars for the digastric EMG recordings are 500 @V except for E, which is 200 pV. The raster displays in A and D illustrate continuous recordings during rhythmic jaw movements; however, a small part of the record which occurs between the end of one sweep and the beginning of the next is not shown.
GUINEA
PIG
TRIGEMINAL
MOTONEURONS
107
muscle beginning after a latency of 8 ms. This response represents the jawopening reflex (excitation of jaw-opener and inhibition of jaw-closer motoneurons) which was previously described in the cat after stimulation of both lingual (5) and inferior dental nerves (8). Stimulation of these nerves in the cat has been shown to evoke disynaptic inhibitory postsynaptic potentials (2.5~ms latency) in jaw-closing masseter and temporal motoneurons (5, 8) and EPSPs and spikes in digastric motoneurons [ 3- to 4-ms latency (8) 1. The latencies reported here for the guinea pig are longer than those in the cat due to the fact that in the present case we are stimulating in the tongue rather than in the nerve trunk and are recording from the muscle rather than from the motoneurons or the digastric nerve. Note that during the hyperpolarization evoked by tongue stimulation there are no membrane fluctuations associated with digastric muscle EMG activity (Fig. 2B), in contrast to the large fluctuations present during the hyperpolarization which occurs in spontaneous rhythmic jaw movements (Figs. ZA, C). A second type of rhythmic jaw movement was observed in the anesthetized guinea pig which more resembled chewing than did the previously described behavior. When a wooden stick (2 mm in diameter) was placed between the molar teeth on one side, cyclic chewing movements with alternating clenching and releasing of the stick could be evoked. (Because the occlusal surfaces of the molar teeth were approximately 1 mm apart, the stick initially acted to force the jaws further apart.) This behavior could be evoked in guinea pigs which were not producing any jaw movements prior to the introduction of the stick. The chewing behavior stopped if the stick was removed from the mouth. In Fig. 2D, a 16-s consecutive sample of this behavior is shown. The upper trace is the intracellular record (obtained from the same jaw-closer motoneuron shown in Figs. lC, D), and the lower trace is the simuitaneous record of the digastric muscle EMG. In contrast to the record obtained during spontaneous rhythmic jaw movements (Fig, 2A), rapid depolarization of the motoneuron leading to spiking activity can be observed after the hyperpolarization accompanying digastric muscle EMG activity (Figs. 2D, E). During those bursts of spikes in the jaw-closer motoneuron the stick was observed to be firmly gripped by the molar teeth. During the periods of digastric muscle activity the stick was released as the jaw opened. Note the synaptic activity which can be observed during the hyperpolarization which occurs prior to the spike bursts (Fig. 2E). This synaptic activity occurs during the period in which the digastric muscle is active both in spontaneous rhythmic jaw movements (Figs, 2A, C) and in stick chewing (Figs. 2D, E) . DISCUSSION These experiments demonstrate that the guinea pig is an excellent animal model for the study of the central nervous system control of rhythmic jaw
108
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movements. Under anesthesia in a stereotaxic apparatus the guinea pig ‘exhibits spontaneous rhythmic jaw movements as well as rhythmic chewing movements during which there is alternating clenching and releasing of an object placed between the molar teeth. This proclivity of the guinea pig to exhibit rhythmic jaw movements under anesthesia is not surprising in the light of studies which have shown that the molar teeth of guinea pig fetuses exhibit occlusal wear 10 days prior to birth, indicating that significant grinding of the teeth occurs in utero (1). The central nervous system components responsible for chewing patterns are obviously well developed in this animal, We also demonstrated a method for the identification of jawcloser motoneurons in the intact guinea pig as well as establishing the feasibility of intracellular recording in trigeminal motoneurons during the above-described rhythmic jaw movements. Action potentials were not observed in jaw-closer motoneurons during spontaneous rhythmic jaw movements. However, a striking finding was the pronounced hyperpolarization and increase in synaptic activity that accompanied the digastric muscle activity associated with the jaw-opening phase of the cycle (Figs. ZA, C-E). It is well known from the EMG studies that jaw-closer motoneurons are not active in the jaw-opening phase of the chewing cycle (2, 10, 16). At present there is no information indicating whether this silence is a function of a simple absence of excitation or whether there are active inhibitory processes at work. The results of intracellular recordings in the jaw-closer motoneurons of the guinea pig provide direct evidence for the presence of an active inhibitory process. There is previous evidence indicating that the observed hyperpolarization in closer motoneurons during rhythmic jaw movement could be induced either by peripheral or central mechanisms. In the spontaneous rhythmic jaw movements, the hyperpolarization in the closer motoneurons appears to closely correspond to activity in the digastric muscle (Figs. 2A, C) . Recent studies indicated the presence of proprioceptors in the anterior belly of the digastric muscle in cats (15) and humans (7). On the other hand, it was reported that stimulation of sites in the medial bulbar reticular formation produces monosynaptic inhibition of jaw-closer motoneurons and reciprocal excitation of opener motoneurons (14). If this reticular formation site is involved in the central generation of rhythmic jaw movements, then one could speculate that the spontaneous rhythmic jaw movements in the guinea pig are produced by a central pattern generator that would simultaneously excite digastric motoneurons and inhibit closer motoneurons and thereby produce the pattern observed in Figs. 2A, C. Whether the origin of the hyperpolarization is central, peripheral, or a combination of both, its function may be to prevent the monosynaptic excitation of jaw-closer motoneurons by spindle afferents activated by stretch
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of the jaw-closing muscles which accompanies jaw opening. It has been shown in the cat (2) and monkey (4) that stretch of the jaw-closing muscles, which occurs during the rapid jaw-opening phase of chewing, induces vigorous activity in spindle afferents from jaw-closing muscles. Such activation could result in inappropriate excitation of jaw-closing muscles and thereby effectively splint the jaw and restrict opening. Presynaptic inhibition of this reflex in the cat by depolarization of the central terminals of spindle afferents was recently proposed as one mechanism for the prevention of unwanted excitation of jaw-closing muscles during the opening phase of the chewing cycle (6). The present study provides evidence suggesting that postsynaptic inhibition of jaw-closer motoneurons also occurs during jaw opening, indicating that both presynaptic and postsynaptic inhibition plays a role in assuring the inactivity of jaw-closing muscles when the jaw is being opened. As previously stated, action potentials were not observed in jaw-closer motoneurons during the spontaneous rhythmic jaw movements and preliminary EMG recordings in both the masseter and temporal muscle indicate that they are not active during this behavior. At the present time, we do not have evidence indicating the mechanism responsible for bringing the jaw back to the initial rest position after the opening phase of the cycle is completed. Several possibilities exist: There may be activity that we have not observed in the masseter and temporal muscles, the medial pterygoid muscle may be involved, or the return might be a function of the passive elastic component of the closing muscles. It is clear, however, that in the chewing movements which can be induced when the stick is placed between the molar teeth, both the temporal and masseter muscles are active and bursts of spikes can be observed in closer motoneurons (Figs. 2D, E). The bursting activity in the closer motoneurons which we observed appears to be present only when the guinea pigs are clenching an object placed between the teeth. In the spontaneous rhythmic jaw movements, no tooth contact occurs and no jaw-closer motoneuron activation has so far been observed. Studies in the cat (13) and rabbit (3, 9) demonstrated the presence of a central pattern generator which produces rhythmic alternating activity in jaw-opener and jaw-closer motoneurons. Similar alternating activity was observed in the anesthetized guinea pig during chewing behavior. We also observed spontaneous rhythmic jaw movements accompanied by rhythmic digastric muscle activity and concurrent closer motoneuron hyperpolarization. Experiments are currently underway to determine the origin of the observed hyperpolarization and to examine the effects of interaction between the central pattern generator and feedback from both jaw muscle
110 proprioceptors motoneurons.
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and intraoral
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mucosal receptors
on guinea pig jaw muscle
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