EXPERMENTAL
64,98- 117 (1979)
NEUROLOGY
Brain Stem Control of Masseteric Reflex Activity during Sleep and Wakefulness: Mesencephalon and Pons NANCY WILLS AND MICHAEL H. CHASE’ Departments
of Physiology
Received
and Anatomy and the Brain Research California, Los Angeles, California 90024 May
11, 1978; revision
received
September
institute,
University
of
5. 1978
Previous studies from our laboratories showed that stimulation of the pontomesencephalic reticular formation resulted in two distinct changes in masseteric reflex excitability which were dependent on the behavioral state of the animal (Chase, M. H., and M. Babb. 1973. Brain Res. 59: 421-426). During wakefulness and quiet sleep, reticular stimulation resulted in an increase in reflex excitability. However, during active sleep, the identical stimulus delivered to the same reticular site led to profound reflex suppression. This phenomenon was termed “response-reversal.” The present study was designed to explore the presence of state-dependent control of motor excitability at rostra1 mesencephalic and pontine levels of the brain stem in chronic freely moving cats during sleep and wakefulness. Conditioning stimulation of mesodiencephalic sites induced only slight reflex facilitation or was without effect during wakefulness and quiet sleep; however, a dramatic suppression of reflex excitability was evoked with the identical stimulus during active sleep. At the level of the pontomesencephalic junction an effective region for “response-reversal” was found to coincide with the nuclei reticularis mesencephali and pontis oralis. The major effect resulting from stimulation of sites surrounding this region occurred exclusively during active sleep and consisted of reflex suppression. During active sleep, from all sites at both levels of the brain stem, only reflex suppression was obtained in conjunction with conditioning stimulation. These findings are discussed in terms of a model of state-dependent regulation of motor activity which accounts for the emergent capability of widespread regions of the brain stem to suppress reflex excitability solely during active sleep. Abbreviations: EEG-electroencephalogram; EOG-eelectrooculogram; EMGelectromyogram. 1 This work was supported by U.S. Public Health Service grant NS-09999. Dr. Wills’ present address is Department of Physiology, School of Medicine, Yale University, New Haven, CN 06510. Address reprint requests to Dr. Michael Chase, Department of Physiology, University of California, Los Angeles, CA 90024. 98 0014-4886/79DlQO98-20$02.00/O Copyright 0 1979 by Academic Press, Inc. AI1 rights of reproduction in any fotm reserved.
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INTRODUCTION Active sleep is characterized by intense brain stem neural activity in the presence of profound motor suppression (23, 26). This relationship is seemingly paradoxical, for many brain stem regions that are most active during this state also induce motor facilitation during wakefulness (20,26). A case in point is the pontomesencephalic reticular formation, where neurons have been found to discharge at higher average rates during active sleep than during wakefulness or quiet sleep (26). This region, which reticular activating system” (21), was encompasses the “ascending thought traditionally to promote wakefulness and facilitate somatomotor activity (1, 14, 17,21). More recently, a contradictory picture has emerged, for areas within the pontomesencephalic reticular formation have been implicated in active sleep and motor suppression (3, 6, 12, 20). How, then, can one resolve the paradox that this neural region plays a key role in the maintenance of active sleep and motor suppression in the face of what appears to be its well-documented function of inducing arousal and motor facilitation? Are there two systems, so closely intertwined, that it falsely appears as if one neuronal population exerts diametrically opposite effects? Or perhaps there is a fundamental change in neuronal circuitry in distant regions during active sleep, so that the output of this reticular locus no longer promotes wakefulness and motor facilitation, but rather induces activity in inhibitory systems which in turn suppress motor behavior and maintain active sleep. Recent evidence from our laboratory indicates that the brain stem control of somatomotor reflex activity may be functionally different during the states of sleep and wakefulness. We showed that a region in the pontomesencephalic reticular tegmentum (in the vicinity of the nucleus pontis oralis) exerts two distinct influences on somatomotor processes which are determined by the behavioral state of the animal (6). During wakefulness and quiet sleep, stimulation of this region leads to facilitation of somatic reflex activity. However, during active sleep, the identical stimulus delivered to the same reticular site results in reflex suppression. This phenomenon, which was termed reticular “response-reversal” (6), may provide a basis for resolving the apparently paradoxical involvement of the reticular tegmentum of the brain stem in the regulation of the behaviorally opposed states of sleep and wakefulness. The experiments reported in this paper were undertaken to assess whether similar or different state-dependent processes can be induced by the excitation of other brain stem regions and to determine the topographic boundaries of the response-reversal phenomenon. We sought to answer the following questions: First, is “response-reversal” restricted to the
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pontomesencephalic reticular tegmentum, or is it a pervasive pattern common to the entire brain stem reticular formation; second, do brain stem regions other than the reticular core exhibit state-dependent motor control and, if they do, what are their patterns of motor modulation and to what extent are those patterns related to reticular pontomesencephalic response-reversal? To answer these questions the brain stem was studied in cross-sectional planes at mesencephalic (mesodiencephalic junction), pontine (pontomesencephalic junction), and medullary (pontomedullary junction) levels. The present report describes the effects of conditioning stimulation of the two rostra1 levels of the brain stem on monosynaptic masseteric reflex excitability during sleep and wakefulness. The pattern of motor control exerted across behavioral states by sites at the level of the pontomedullary junction is presented in the following paper (10). METHODS Surgical Procedures. The present study utilized 10 adult cats. The animals were initially anesthetized (Nembutal, 3.5 mg/kg, i.p.), and permanent electrodes were implanted. A bipolar stimulating electrode was positioned stereotaxically in the mesencephalic nucleus of the Vth nerve to evoke the masseteric reflex. Bipolar electrodes were implanted in the masseter muscle to record the masseteric reflex as the monosynaptically induced electromyographic response following stimulation of the ipsilatera1 mesencephalic Vth nucleus. [Details of the procedure for inducing and recording the masseteric reflex were published previously (7).] Each animal was also implanted with bipolar electrodes for stimulation of ipsilateral brain stem sites (24). The electrodes were constructed of two insulated Nichrome wires mounted on a central shaft (tip: diameter = 0.2 mm, exposure = 0.5 mm, separation = 0.5 mm). Each electrode was attached to a miniature microdrive carrier which permitted vertical movement of the electrode during the experimental procedures described below. In addition, electroencephalographic (EEG), electrooculographic (EOG), and electromyographic (EMG) electrodes were implanted for monitoring sleep and wakefulness (7). Insulated wires from all electrodes were soldered to a Winchester connector which was secured to the skull with acrylic cement. Stimulation and Recording Procedures. After recovery from the surgical procedures (approximately 2 weeks), the cat was placed in an electrically shielded, environmentally controlled chamber. The animal was connected to stimulating and recording equipment by a lightweight rotary cable assembly which permitted the animal to move freely within the chamber. Food and water were available ad libitum.
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Stimuli were generated by a Grass Model S88 stimulator in series with Grass Model 5 stimulus isolation units. Reflex-inducing stimuli consisted of single biphasic pulses delivered to the mesencephalic Vth nucleus (frequency = l/s; pulse duration = 0.75 ms). Stimulus current for reflex induction was 0.2 to 0.6 mA. Stimulation of sites in the mesencephalon and pons was accomplished by the delivery of brief trains of three biphasic pulses (frequency = 400 pulses per second; pulse duration = 0.75 ms; train duration = 5 ms). Current for conditioning brain stem stimulation was 0.2 to 1.5 mA. These stimulus parameters were found, in pilot experiments, to cover the effective range of values for reflex modulation. Stimulus current was calculated by Ohm’s law from the measured voltage drop across a 5-n resistor placed in series with one lead from the stimulus isolation unit. Reflex activity was displayed on an oscilloscope and measured by a peak-amplitude and window discriminator which was interfaced with a digital printer and a signal generator. The latter device provided a saw-tooth input to one channel of a Grass model 7B polygraph. Thus, the amplitude of the reflex was recorded and correlated with the on-line polygraphic record of EEG, EOG, and EMG activity. During experimentation the reflex was induced repeatedly (at a rate of l/s) throughout spontaneously occurring cycles of sleep and wakefulness. During wakefulness, testing was conducted when the animal was alert but not moving. During active sleep, trials in which rapid eye movements occurred were discarded and repeated later in the testing session. In no instance was current used which disrupted the animal’s ongoing state. Experimental Procedures. Because the amplitude of the masseteric reflex decreases as an animal passes from wakefulness to quiet sleep to active sleep (8), the stimulus current for reflex induction was adjusted so that an equivalent control reflex could be obtained during all states. Previous experiments and pilot studies indicated that such increases in current do not influence the results of conditioning-test trials (6, 7). The control reflex used was one whose amplitude during wakefulness was approximately 50% of the maximum amplitude that could be induced by stimulation of the mesencephalic Vth nucleus. Reflex modulation was analyzed by averaging the amplitude of control and experimental reflexes. A 50% or greater increase or decrease from control was defined as facilitation or suppression, respectively. To assess the effects of rostra1 mesencephalic and rostral pontine stimulation upon masseteric reflex excitability, a conditioning-test procedure was utilized wherein a train of stimuli was delivered to brain stem sites at specific latencies prior to the induction of the reflex. A conditioning-test trial consisted of three consecutive control reflexes which
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were followed by three reflexes conditioned by prior stimulation of the anterior mesencephalon or pons at a fixed latency. The mean amplitude of the control reflexes was then compared with that of the conditioned reflexes. This alternating pattern of three “control” reflexes followed by three “conditioned” reflexes was repeated continuously throughout each testing period. At each site the following three paradigms were used to characterize the effects of brain stem stimulation during wakefulness, quiet sleep, and active sleep. (i) A fixed conditioning-test interval of 20 ms was used to assess the basic pattern of reflex response at high (1 .O mA) and low (0.2 mA) values of conditioning current. [The 20-ms latency was chosen because the effect of brain stem stimulation at this conditioning-test interval was found to be the most reliable and effective for reflex modification (see Results).] (ii) The threshold for reflex modulation at each site was defined as the minimum amount of current required to alter reflex amplitude by at least 50% of its control value. The procedure for assessing threshold involved decreasing the conditioning stimulus from 1.5 mA in 0.2-mA increments until no effect was observed. (iii) The time course of response to conditioning stimulation was determined by examining conditioning-test intervals between 5 and 70 ms. For each of the preceding paradigms, three complete conditioning-test trials at each site were obtained during each behavioral state. A total of nine observations at each interval was obtained. After these data were collected during wakefulness, quiet sleep, and active sleep (N = 9 for each state), the electrode used for conditioning stimulation was lowered 1 mm, and the preceding experimental paradigms were repeated. Histology. After the experiment, each animal was dispatched, and its brain was perfused with 0.9% saline followed by 10% formalin. The brain was then removed and stored 1 to 2 weeks in formalin. Subsequently, the tissue was frozen, and sections 60 pm thick were obtained for histological evaluation of the electrode tracks. The location of the electrode for reflex induction was determined. The vertical sites in each track of the conditioning electrode were calculated from the point of lowest descent. RESULTS Mesencephalon: Patterns
of Reflex
Modulation
Mesodiencephalic and Their
Junction Topographic
Distribution.
Figure 1 summarizes the pattern of reflex modulation obtained with a fixed 20-ms conditioning-test interval from a total of 5 1 stimulation sites during wakefulness, quiet sleep, and active sleep. The position of each site is represented on a schematic brain stem cross section on the left side of the
Mesencephalon
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FIG. 1. Summary of state-dependent effects of anterior mesencephalic conditioning stimulation on masseteric reflex excitability during wakefulness (W), quiet sleep (QS), and active sleep (AS). A schematic histologic section representing the mesodiencephalic junction is presented on the left. Two levels of conditioning current are shown (1 .O and 0.2 mA). A through F summarize sites which yielded 50% facilitation (+) or suppression (-) of reflex activity. Note the widespread suppression of reflex excitability in C and the ventral concentration of the suppressor region which emerged at the lower values of conditioning stimulation in F; conditioning-test interval = 20 ms. Abbreviations in this and the following figure: BCC-brachium of the inferior colliculus; CLM-penduncle of the mammilary bodies; CM-centre median; GLD, GLV-dorsal, ventral divisions of the lateral geniculate body; GM-medial geniculate body; HM-medial habenular nucleus; LP-lateral reticular formation; NHL-lateral habenular nucleus; OT-optic tract; thalamic nucleus, posterior division; MRF-mesencephalic P-pyramidal tract; PL-pulvinar nucleus; SGC-stratum griseum centrale; SN-substantia nigra; VPM-ventralis posterior medialis (thalamus).
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FIG. 6. Time course of the effects of stimulation of six different sites at the pontomesencephalic junction during wakefulness, quiet sleep, and active sleep. A-D illustrate patterns of “response-reversal.” E and F portray profound reflex suppression during active sleep in the absence of any predominant response during wakefulness or quiet sleep. Each point on the graphs was obtained by comparing nine control and nine conditioned reflexes. The time course in A was obtained from A 1, L 2.5, H -0.5; in B from A 1.5, L 2.5, H - 1.5; in C from A 1, L 3, H - 1.5; in D from A 1, L 2.5, H -2.5; in E from A 1, L 3.5, H +4; and in F from A 1, L 6.5, H -2.5. Pontomesencephalic conditioning current = 1.0 mA; conditioning-test latency = 20 ms.
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FIG. 3. Summary of the state-dependent effects of mesodiencephalic conditioning stimulation and examples of the two principal time course patterns of response. A-reflex suppression during active sleep in the absence of any striking effect during wakefulness or quiet sleep (obtained from A 6.5, L 4.5, H -3). B-a “response-reversal” pattern (obtained from A 6.5, L 5, H -2.5). Note the peakfacilitatory and suppressor response at 20 ms, and the similarity of the time course for active sleep in A and B. Each point was obtained by comparing nine control with nine conditioned reflexes.
to produce a 50% change from the control amplitude (Figs. 2A, B). However, during active sleep, low-threshold sites were widely distributed and scattered among those of high threshold (Fig. 2C). Consequently, no locus or “hot-spot” of suppression was found during active sleep, although the sites with the lowest thresholds tended to be situated ventrally. Time Course ofResponse. Figure 3 presents the principal time course of response to mesodiencephahc stimulation. The predominant response at this level of the neuraxis, i.e., state-dependent suppression of the masseteric reflex that emerges only during active sleep, is shown in Fig. 3A. Reflex suppression began at 10 to 15 ms, was maximum at 20 ms, and continued until 30 ms. An example of the time course of response from one of the few sites which demonstrated response-reversal is presented in Fig. 3B. Stimulation of this site produced facilitation during wakefulness and quiet sleep, with a peak at a conditioning-test latency of 20 ms.
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The pattern of suppression during active sleep was similar to the active sleep time course in the preceding example (Fig. 3A). Indeed, there was a high degree of similarity between the time-course of response from all sites during active sleep. All curves were comparable in that each exhibited peak suppression at approximately 20 ms. In addition, the onset of suppression was comparable across sites, occurring at 10 ms and preceded occasionally by a brief period of weak facilitation. The duration of suppression lasted 25 to 30 ms. Pons: Pontomesencephalic
Junction
Patterns of Reflex Modulation and Their Topographic Distribution. The 40 stimulation sites examined at this level of the brain stem are presented in Fig. 4. The distribution of these sites is represented on a schematic section of the pontomesencephalic junction shown on the left. The patterns of response at a standard conditioning-test interval of 20 ms are summarized on the right. During wakefulness and quiet sleep, stimulation induced either reflex facilitation or was ineffective. A well defined locus for the facilitatory effect was found when a stimulus current of 1.0 mA was used (Figs. 4A, B). The effective region was in the vicinity of the nuclei pontis oralis and reticularis mesencephali. This region was surrounded by an area which produced no modification of reflex excitability. With lower levels of stimulation (0.2 mA) there was a marked reduction in the number of sites which yielded facilitatory effects during wakefulness or quiet sleep (Figs. 4D, E). During active sleep, stimulation of almost all sites led to reflex suppression at a current of 1 .O mA. The effective region encompassed not only the reticular nuclei but also extended into the central grey, and included the superior colliculus. Although reflex suppression during active sleep was a widespread phenomenon, the effect was not prominent medially. With a lower conditioning current (0.2 mA) the demarcation between medial and lateral regions became more evident. However, even at 0.2 mA, an extensive reflex suppressor region was still noted in the core of the brain stem. Generally, the effect of lower conditioning current was to decrease the magnitude of reflex suppression and to reduce the size of the effective region. During active sleep, high (1.5 mA) values of conditioning current in five sites from two animals led to a decrease in the magnitude of suppression. However, the direction of response remained the same. These sites yielded strong motor reactions and the animal awakened within 2 to 3 s after stimulation. The region from which this response was induced was in the central portion of the nucleus reticularis mesencephali. However, we emphasize that this was an aberrant response, for other sites in this same region yielded a typical pattern of response-reversal which was
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FIG. 4. Summary of state-dependent effects of pontomesencephalic stimulation on masseteric reflex excitability during wakefulness (W), quiet sleep (QS), and active sleep (AS). On the left is a schematic cross section of the pontomesencephalic junction. Stimulation sites are indicated by solid triangles. The reflex response following the application of two levels of conditioning current (1 .O and 0.2 mA) at a conditioning-test latency of 20 ms are presented. A ” +” indicates an increase of 50% or greater in the conditioned (test) reflex amplitude; a ‘* -” indicates a 50% or greater reduction in amplitude. Note that all effective sites for reflex modulation yielded only facilitation during both wakefulness and quiet sleep, and only suppression during active sleep. Abbreviations used in this and the following figure: EC-brachium conjunctivum; BP-brachium pontis; FCT-central tegmental tract; IP-nucleus interpenduncularis; LM-medial lemniscus; MES-nucleus of the mesencephalic tract of V; NPO-nucleus pontis oralis; NRM-nucleus reticularis mesencephali; P-pyramidal tract: SC-superior colliculus; SCG-stratum griseum centrale; TOL-tectoolivary tract; TS-tectospinal tract.
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Response Thresholds. In practically
all cases higher conditioning current was required to induce reflex facilitation during wakefulness and quiet sleep than was necessary for reflex suppression during active sleep. This finding is illustrated in Fig. 5 which shows the relative threshold of the tested sites for the induction of a change in reflex excitability. The integers shown in A through C represent the minimum amount of current which produced reflex facilitation or suppression. There was no clear differentiation of response threshold between wakefulness and quiet sleep; most sites required a relatively high current to induce facilitation during these states (Figs. 5A, B). The few low-threshold sites were clustered in the general vicinity of the reticular nuclei. In contrast, during active sleep, sensitive sites with very low-current thresholds were widely distributed throughout the tectum, central grey, and ventral tegmentum (Fig. 5C).3 Time Course of Response. The most prevalent time course patterns of reflex modulation are shown in Figs. 6 and 7. During wakefulness and quiet sleep, facilitation usually began at a short conditioning-test interval of 5 to 10 ms and reached a peak at 20 or 2.5 ms (Figs. 6A-C). From some sites there was only slight facilitation during wakefulness or quiet sleep (Fig. 6D); however, the overall time course was consistent with that described above. During active sleep, a slight degree of facilitation was usually present between 5 and 10 ms (Fig. 6B-D). Profound reflex suppression consistently occurred between 10 and 30 ms (Fig. 6A-F) with a return to the control value at about 30 to 40 ms. There sometimes followed, however, another period of longer latency, low-level suppression (Fig. 6C), or slight facilitation (Fig. 6D-F). In some cases there was either sustained facilitation or suppression beyond 30 ms as shown in Fig. 6A-C. From a small number of sites another pattern of response was observed which consisted of prominent reflex suppression during active sleep with virtually no effect during wakefulness or quiet sleep (Figs. 6E, F). In these instances the time course of suppression during active sleep paralleled that described above for other sites during this state. In summary, from all sites there was a remarkably consistent suppression of reflex activity at a conditioning-test interval of 10 to 30 ms during active sleep (Fig. 7). Short-latency facilitation and long-latency suppression during active sleep were usually present after conditioning stimulation of most sites, although these responses were of smaller magnitude (Fig. 7). 3 If all effective sites 50.4 mA are considered, then the behavioral states would be ranked as follows: wakefulness, 43%; quiet sleep, 40%; and active sleep, 89%.
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FIG. 5. Conditioning threshold for sites shown in the cross section of the pontomesencephalic junction. Integers indicate the minimum amount of current (in milliamperes) delivered to the indicated sites which was required to increase reflex amplitude by at least 50% of its control during wakefulness (W) or quiet sleep (QS) or induce a decrease in amplitude of at least 50% of control during active sleep (AS). Solid circles indicate sites which were ineffective in modifying reflex amplitude. A standard conditioning-test interval of 20 ms was used.
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DISCUSSION The results of this study reveal dramatic state-dependent control of reflex excitability exerted by the anterior mesencephalon. Stimulation of most sites with a low current resulted in powerful suppression of the test masseteric reflex during active sleep but was relatively ineffective in influencing reflex excitability during wakefulness or quiet sleep. However, structures in this region have not been considered to play a major role in the suppression of somatomotor activity during active sleep, although Hobson (13) reported a slight reduction in active sleep atonia after lesions of the mesencephalon. Thus, the strong involvement of anterior mesencephalic structures or fibers passing through the mesodiencephalic junction in the suppression of reflex activity constitute a new finding concerning the brain stem modulation of motor activity. As emergent reflex suppression during active sleep was both the predominant and low-threshold effect at this level of the neuraxis, we will concentrate in our discussion on an analysis of this response pattern. The emergence of a reflex suppressor influence, that exists only during active sleep (throughout widespread areas of the rostra1 mesencephalon), demonstrates the dynamic and state-dependent nature of somatomotor control. It appears that the entire mesencephalon (either resident nuclear groups and/or fibers of passage) undergoes a functional reorganization during active sleep in which all ineffective or facilitatory stimuli delivered during other states yield reflex suppression during this state. The question then arises as to the system(s) mediating this statedependent pattern of suppression of reflex activity. First, the distribution of the responsive sites do not suggest the exclusive participation of any known neuroanatomical fiber tract (1, 2). Second, although a number of biochemically defined systems course through the rostra1 mesencephalon, the pathways are largely ascending and their distribution is not aligned with the responsive sites we have described (15, 19). Finally, the participation of nuclei with diverse functions (such as the geniculate body and ventralis posterior medialis) in this state-dependent process does not argue for the eventual identification of a single structure mediating this effect. Similarly, widespread diffusely organized reticular-cortical influences were reported by Candia and co-workers, who were unable to localize any brain stem pathway mediating cortical desynchronization during active sleep (3). We hypothesize that reflex suppression which emerges during active sleep from widespread regions may be dependent on circuitry in the caudal brain stem rather than in the pons or mesencephalon (10). Pons: Pontomesencephalic Junction. The data obtained from an analysis Mesencephalon:
Mesodiencephalic
Junction.
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FIG. 6. Time course of the effects of stimulation of six different sites at the pontomesencephalic junction during wakefulness, quiet sleep, and active sleep. A-D illustrate patterns of “response-reversal.” E and F portray profound reflex suppression during active sleep in the absence of any predominant response during wakefulness or quiet sleep. Each point on the graphs was obtained by comparing nine control and nine conditioned reflexes. The time course in A was obtained from A 1, L 2.5, H -0.5; in B from A 1.5, L 2.5, H - 1.5; in C from A 1, L 3, H - 1.5; in D from A 1, L 2.5, H -2.5; in E from A 1, L 3.5, H +4; and in F from A 1, L 6.5, H -2.5. Pontomesencephalic conditioning current = 1.0 mA; conditioning-test latency = 20 ms.
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FIG. 7. The filled circles in the brain stem cross section indicate the sites which yielded reticular conditioned masseteric reflex facilitation during wakefulness and quiet sleep, and suppression during active sleep. The average time course of this pattern of response is shown in A-C. Bars indicate standard errors of the means. Note the potent long-duration facilitatory effect during wakefulness and quiet sleep (A, B) and the biphasic period of suppression during active sleep (C). We occasionally observed sustained suppression during active sleep when the return to baseline of the response at 40 ms was absent. In many animals a brief period of low-amplitude facilitation persisted during active sleep at a conditioning-test latency of 5 to 10 ms. All the sites (unfilled as well as filled circles) shown in this brain stem section induced a pattern of suppression during active sleep similar to that shown in C. This response was observed even when no effect was found during wakefulness or quiet sleep.
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of this region confirm our previous finding that the reflex modulating influence of the pontomesencephalon varies according to the behavioral state of the animal (6, 9). The sites which demonstrate response-reversal were found to be co-extensive with the major reticular nuclei, specifically the nucleus reticularis pontis oralis and reticularis mesencephali. From surrounding regions a new pattern of reflex modulation at this brain stem level was observed. It consisted of reflex suppression, which was present only during active sleep, together with the absence of any effect of conditioning stimulation during wakefulness or quiet sleep. These results extend our finding at the mesodiencephalic level, namely, that widely dispersed regions of the brain stem are capable of influencing reflex activity only during active sleep and that the direction of the reflex response is solely one of motor suppression. Comparison of Mesodiencephalic and Pontomesencephalic Patterns of Rejlex Modulation. An examination of the mesodiencephalic and
pontomesencephalic patterns of response indicate that either there was no effect or that reflex facilitation was the only response evoked during wakefulness and quiet sleep. The responsible region at these brain stem levels is, in general, delineated by the rostra1 and caudal borders of the nucleus reticularis mesencephali and nucleus pontis oralis. However, many more sites led to reflex facilitation at the pontomesencephalic level of the brain stem, where the magnitude of facilitation was greater and could be produced with a lower conditioning current. During active sleep, stimulation of widespread regions at both brain stem levels produced reflex suppression; during this state reflex facilitation was never observed. The time course of suppression was remarkably similar from all sites and was induced with the lowest conditioning current from sites at the pontomesencephalic junction. The response threshold during different states at each brain stem level followed a similar pattern. From most sites less conditioning current was required during active sleep to suppress the masseteric reflex than to facilitate it during wakefulness or quiet sleep. Thus, after reducing the level of conditioning current, the only remaining effect was reflex suppression during active sleep from those brain stem sites that also yielded facilitation during wakefulness or quiet sleep. Moreover, although motor facilitation required higher conditioning current, as noted above, this response pattern was present only during wakefulness or quiet sleep (at conditioning-test intervals greater than 10 ms). The classic pattern of response-reversal, on the basis of amplitude of effect, threshold, and number of effective sites, was more closely associated with excitation of structures at the pontomesencephalic junction than at the more rostra1 brain stem level. These experiments demonstrate that widespread regions at the rostra1
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and caudai borders of the mesencephalon are effective in evoking suppression of reflex activity during active sleep. The lack of a circumscribed effective zone indicates that at these brain stem levels neither a single neuronal pathway nor nuclear group is involved in mediating this state-dependent pattern of motor control. Although state-dependent reflex facilitation during wakefulness and quiet sleep is a more localized and regional phenomenon, it also does not encompass a definable nuclear region or fiber pathway. Putative Neural Circuitry Responsible for State-dependent ReJEex Modulation. The widespread distribution and functional heterogeneity of structures whose excitation induce similar patterns of reflex suppression during active sleep, and the more circumscribed regions yielding reflex facilitation during wakefulness and quiet sleep (which overlap with the active sleep suppressor sites), led us to suggest that the responsible circuitry for these different response patterns may involve a neurophysiologic “gating” mechanism (4, 27). We hypothesize that there is synaptic circuitry functioning as a “gate” which alters the motor effect of brain stem discharge from somatomotor facilitation during wakefulness and quiet sleep to suppression during active sleep. During active sleep, we suggest that the gate is open, so that activity arising in the brain stem (and more than likely other areas as well) is funneled, or allowed access, to the input side of a powerful motor inhibitory system. The responsible inhibitory system may be located in the medullary reticular formation (18). There are fiber projections from pontomesencephalic to medullary reticular regions (11, 16). In turn, neurons in the region of the medullary reticular formation have postsynaptic inhibitory input onto masseter motoneurons (25). We suggest that the excitability of the medullary reticular formation increases during active sleep due to the opening of a neural gate during this state (4, 5). Widespread regions in the pons and mesencephalon (as well as forebrain structures), by virtue of their connection with pontomedullary reticular sites (1,3) which send excitatory projections to the lower brain stem (5), might then induce reflex suppression only during active sleep. Thus, suppression of reflex activity during active sleep could result from the activation of a motor inhibitory system by a “facilitatory” system with widespread afferent input, such as the “pontomesencephalic reticular activating system.” In this manner, activity arising in or coursing through the brain stem would be effective in suppressing reflex excitability during active sleep due to a neural gate that is patent during this state, thus allowing alink to be formed between rostra1 and caudal reticular regions. During wakefulness and quiet sleep we suggest that the gate is “normally closed” and motor facilitation ensues. This concept assumes an indirect route via the aforementioned neural gate for the motor suppressor path. Accordingly, the onset of suppression
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during active sleep would be expected to arise with a slightly longer latency than facilitation during wakefulness or quiet sleep. The time course of response which we observed supports this line of reasoning, for the onset of facilitation during wakefulness and quiet sleep in most cases occurred before the onset of suppression during active sleep. Thus, during active sleep, we would expect that only the very earliest component of motor facilitation would persist, due presumably to the longer activation latency of a suppressor system. Indeed, this is precisely what we found, for the early period of reflex facilitation often did not reverse as the animal changed state (Fig. 3). We recently carried out an intracellular analysis of the phenomenon of response-reversal in order to differentiate the synaptic events and circuitry by which it is mediated (22). We found that during wakefulness and quiet sleep stimulation of “response-reversal” sites induced in trigeminal jaw-closer motoneurons a short-latency (2.5 to 5 ms) depolarizing potential of lo- to 2%ms duration. When the animal passed into active sleep, a hyperpolarizing potential emerges at a latency of approximately 10 ms with a peak at about 20 ms. The early phase of the depolarizing potential, although somewhat diminished in amplitude, appeared to persist during active sleep. Thus the state-dependent change in motoneuron membrane potential mirrored the electromyographically recorded pattern of somatic reflex modulation reported in this paper. In summary, we suggest there is a selective gating mechanism which allows for the excitation of inhibitory neuronal systems by a “facilitatory” system during active sleep and prevents such activation during wakefulness or quiet sleep. At the present time we can offer only hypotheses as to the specific region and site of action of the neuronal mechanisms comprising the putative gate. Our current intracellular experiments are designed to shed light on the underlying synaptic circuitry (22). REFERENCES 1.
A. 1951. The Reticular Formation of the Brain Stem. Anatomical Functional Correlations, The Henderson Trust Lecture. Oliver and 2. BRODAL, A. 1969. Neurological Anatomy in Relation to Clinical Medicine. BRODAL,
Aspects
and
Boyd, London. Oxford Press,
New York. 0.. G. ROSSI, AND T. SEKINO. 1967. Brain stem structures responsible for the electroencephalographic patterns of desynchronized sleep. Science 155: 720-722. 4. CHASE, M. H. 1976. A model of central neural processes controlling motor behavior during active sleep and wakefulness. Pages 99-121 in T. DESIRAJU, Ed., Mechanisms in Transmission of Signals for Conscious Behavior. Elsevier, New York. 5. CHASE, M. H. 1978. State-dependent reversal of a brainstem reflex in Felix domesticus. In Society for Neuroscience Symposia, Vol. 3 pp. 33-65, 1978. 6. CHASE, M. H., AND M. BABB. 1973. Masseteric reflex response to reticular stimulation reverses during active sleep compared with wakefulness or quiet sleep. Brain Res. 59: 421-426. 3. CANDIA,
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7. CHASE, M. H., AND D. J. MCGINTY. 1970. Somatomotor inhibition and excitation by forebrain stimulation during sleep and wakefulness: orbital cortex. Bruin Res. 19: 127- 136. 8. CHASE, M. H., D. J. MCGINTY, AND M. B. STERMAN. 1968. Cyclic variations in the amplitude of a brain stem reflex during sleep and wakefulness. Experientiu 24: 47-48. 9. CHASE, M. H., R. MONOSON, K. WATANABE, AND M. BABB. 1976. Somatic reflex response-reversal of reticular origin. Exp. Neural. 50: 561-568. 10. CHASE, M. H., AND N. WILLS. 1979. Brain stem control of masseteric reflex activity during sleep and wakefulness: Medulla. Exp. Neurol. 64: 118- 131. 11. EDWARDS, S. 1975. Audioradiographic studies of the projections ofthe midbrain reticular formation: Descending projections of the nucleus cuneformis. J. Camp. Neural. 161: 341-358. 12. HENLEY, K., AND A. MORRISON. 1974. A re-evaluation of the effects of lesion of the pontine tegmentum and locus coeruleus on phenomena of paradoxical sleep in the cat. Acta Neurobiol. Exp. 34: 215-232. 13. HOBSON, J. A. 1965. The effect of chronic brain stem lesions on cortical and muscular activity during sleep and waking in the cat. Electroencephalogr. Clin. Neurophysiol. 19: 41-62. 14. HUGELIN, A., AND M. BONVALLET. 1957. Tonus cortical et controle de la facilitation motice d’orgine reticulaire. J. Physiol. (Paris) 49: 1171- 1200. 15. JOUVET, M. 1972. The role of monoamines and acetylcholine-containing neurons in the regulation of the sleep-waking cycle. Ergeb. Physiol. 64: 166-307. 16. KAWAMURA, Y., A. BRODAL, AND G. HODDEVIK. 1974. The projections of the superior colliculus onto the reticular formation of the brainstem: An experimental study in the cat. Exp. Brain Res. 19: l-19. 17. LINDSLEY, D. B., L. H. SCHREINER, E. B. B. KNOWLES, AND H. W. MAGOUN. 1950. Behavioral and EEG changes following chronic brain stem lesions in the cat. Electroencephalogr. Clin. Neurophysiol. 2: 483-498. 18. MAGOUN, H. W., AND R. RHINES. 1946. An inhibitory mechanism in the bulbar reticular formation. J. Neurophysiol. 9: 165- 171. 19. MORGANE, P., AND W. STERN. 1974. Chemical anatomy of brain circuits in relation to sleep and wakefulness. Pages l-131 in E. WEITZMAN, Ed., Advances in Sleep Research. Vol. 1. Spectrum Publishing Company, New York. 20. MORUZZI, G. 1972. The sleep-waking cycle. Ergeb. Physiol. 64: l-165. 21. MORUZZI, G., AND H. W. MAGOUN. 1949. Brain stem reticular formation and activation of the EEG. Electroencephalogr. Clin. Neurophysiol. 1: 455-473. 22. NAKAMURA, Y., L. GOLDBERG, S. CHANDLER, AND M. CHASE. 1978. Intracelhdar analysis of trigeminal motoneuron activity during sleep in the cat. Science 199: 204-207. 23. POMPEIANO, 0. 1967. The neurophysiological mechanisms of the postural and motor events during desynchronized sleep. Res. Publ. Assoc. Res. Nerv. Ment. Dis. 45% 351-423. 24. REINSO-SUAREZ, R. 1961. Topographisher Himatlas der Katze. Herausgegeben von E. Merck A. G., Darmstadt. 25. SAUERLAND, E. K., Y. NAKAMURA, AND C. D. CLEMENTE. 1967. The role of the lower brainstem in cortically induced inhibition of somatic reflexes in the cat. Bruin Res. 6: 164-180. 26. STERIADE, M., AND J. A. HOBSON. 1976. Neuronal activity during the sleep-waking cycle. Prog. Neurobiol. 6: 155-156. 27. WILLS, N. 1977. Brainstem Influences on the Masseteric Reflex during Sleep and Wakefulness. Unpublished doctoral dissertation. University of Virginia.
64,98- 117 (1979)
NEUROLOGY
Brain Stem Control of Masseteric Reflex Activity during Sleep and Wakefulness: Mesencephalon and Pons NANCY WILLS AND MICHAEL H. CHASE’ Departments
of Physiology
Received
and Anatomy and the Brain Research California, Los Angeles, California 90024 May
11, 1978; revision
received
September
institute,
University
of
5. 1978
Previous studies from our laboratories showed that stimulation of the pontomesencephalic reticular formation resulted in two distinct changes in masseteric reflex excitability which were dependent on the behavioral state of the animal (Chase, M. H., and M. Babb. 1973. Brain Res. 59: 421-426). During wakefulness and quiet sleep, reticular stimulation resulted in an increase in reflex excitability. However, during active sleep, the identical stimulus delivered to the same reticular site led to profound reflex suppression. This phenomenon was termed “response-reversal.” The present study was designed to explore the presence of state-dependent control of motor excitability at rostra1 mesencephalic and pontine levels of the brain stem in chronic freely moving cats during sleep and wakefulness. Conditioning stimulation of mesodiencephalic sites induced only slight reflex facilitation or was without effect during wakefulness and quiet sleep; however, a dramatic suppression of reflex excitability was evoked with the identical stimulus during active sleep. At the level of the pontomesencephalic junction an effective region for “response-reversal” was found to coincide with the nuclei reticularis mesencephali and pontis oralis. The major effect resulting from stimulation of sites surrounding this region occurred exclusively during active sleep and consisted of reflex suppression. During active sleep, from all sites at both levels of the brain stem, only reflex suppression was obtained in conjunction with conditioning stimulation. These findings are discussed in terms of a model of state-dependent regulation of motor activity which accounts for the emergent capability of widespread regions of the brain stem to suppress reflex excitability solely during active sleep. Abbreviations: EEG-electroencephalogram; EOG-eelectrooculogram; EMGelectromyogram. 1 This work was supported by U.S. Public Health Service grant NS-09999. Dr. Wills’ present address is Department of Physiology, School of Medicine, Yale University, New Haven, CN 06510. Address reprint requests to Dr. Michael Chase, Department of Physiology, University of California, Los Angeles, CA 90024. 98 0014-4886/79DlQO98-20$02.00/O Copyright 0 1979 by Academic Press, Inc. AI1 rights of reproduction in any fotm reserved.
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INTRODUCTION Active sleep is characterized by intense brain stem neural activity in the presence of profound motor suppression (23, 26). This relationship is seemingly paradoxical, for many brain stem regions that are most active during this state also induce motor facilitation during wakefulness (20,26). A case in point is the pontomesencephalic reticular formation, where neurons have been found to discharge at higher average rates during active sleep than during wakefulness or quiet sleep (26). This region, which reticular activating system” (21), was encompasses the “ascending thought traditionally to promote wakefulness and facilitate somatomotor activity (1, 14, 17,21). More recently, a contradictory picture has emerged, for areas within the pontomesencephalic reticular formation have been implicated in active sleep and motor suppression (3, 6, 12, 20). How, then, can one resolve the paradox that this neural region plays a key role in the maintenance of active sleep and motor suppression in the face of what appears to be its well-documented function of inducing arousal and motor facilitation? Are there two systems, so closely intertwined, that it falsely appears as if one neuronal population exerts diametrically opposite effects? Or perhaps there is a fundamental change in neuronal circuitry in distant regions during active sleep, so that the output of this reticular locus no longer promotes wakefulness and motor facilitation, but rather induces activity in inhibitory systems which in turn suppress motor behavior and maintain active sleep. Recent evidence from our laboratory indicates that the brain stem control of somatomotor reflex activity may be functionally different during the states of sleep and wakefulness. We showed that a region in the pontomesencephalic reticular tegmentum (in the vicinity of the nucleus pontis oralis) exerts two distinct influences on somatomotor processes which are determined by the behavioral state of the animal (6). During wakefulness and quiet sleep, stimulation of this region leads to facilitation of somatic reflex activity. However, during active sleep, the identical stimulus delivered to the same reticular site results in reflex suppression. This phenomenon, which was termed reticular “response-reversal” (6), may provide a basis for resolving the apparently paradoxical involvement of the reticular tegmentum of the brain stem in the regulation of the behaviorally opposed states of sleep and wakefulness. The experiments reported in this paper were undertaken to assess whether similar or different state-dependent processes can be induced by the excitation of other brain stem regions and to determine the topographic boundaries of the response-reversal phenomenon. We sought to answer the following questions: First, is “response-reversal” restricted to the
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pontomesencephalic reticular tegmentum, or is it a pervasive pattern common to the entire brain stem reticular formation; second, do brain stem regions other than the reticular core exhibit state-dependent motor control and, if they do, what are their patterns of motor modulation and to what extent are those patterns related to reticular pontomesencephalic response-reversal? To answer these questions the brain stem was studied in cross-sectional planes at mesencephalic (mesodiencephalic junction), pontine (pontomesencephalic junction), and medullary (pontomedullary junction) levels. The present report describes the effects of conditioning stimulation of the two rostra1 levels of the brain stem on monosynaptic masseteric reflex excitability during sleep and wakefulness. The pattern of motor control exerted across behavioral states by sites at the level of the pontomedullary junction is presented in the following paper (10). METHODS Surgical Procedures. The present study utilized 10 adult cats. The animals were initially anesthetized (Nembutal, 3.5 mg/kg, i.p.), and permanent electrodes were implanted. A bipolar stimulating electrode was positioned stereotaxically in the mesencephalic nucleus of the Vth nerve to evoke the masseteric reflex. Bipolar electrodes were implanted in the masseter muscle to record the masseteric reflex as the monosynaptically induced electromyographic response following stimulation of the ipsilatera1 mesencephalic Vth nucleus. [Details of the procedure for inducing and recording the masseteric reflex were published previously (7).] Each animal was also implanted with bipolar electrodes for stimulation of ipsilateral brain stem sites (24). The electrodes were constructed of two insulated Nichrome wires mounted on a central shaft (tip: diameter = 0.2 mm, exposure = 0.5 mm, separation = 0.5 mm). Each electrode was attached to a miniature microdrive carrier which permitted vertical movement of the electrode during the experimental procedures described below. In addition, electroencephalographic (EEG), electrooculographic (EOG), and electromyographic (EMG) electrodes were implanted for monitoring sleep and wakefulness (7). Insulated wires from all electrodes were soldered to a Winchester connector which was secured to the skull with acrylic cement. Stimulation and Recording Procedures. After recovery from the surgical procedures (approximately 2 weeks), the cat was placed in an electrically shielded, environmentally controlled chamber. The animal was connected to stimulating and recording equipment by a lightweight rotary cable assembly which permitted the animal to move freely within the chamber. Food and water were available ad libitum.
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Stimuli were generated by a Grass Model S88 stimulator in series with Grass Model 5 stimulus isolation units. Reflex-inducing stimuli consisted of single biphasic pulses delivered to the mesencephalic Vth nucleus (frequency = l/s; pulse duration = 0.75 ms). Stimulus current for reflex induction was 0.2 to 0.6 mA. Stimulation of sites in the mesencephalon and pons was accomplished by the delivery of brief trains of three biphasic pulses (frequency = 400 pulses per second; pulse duration = 0.75 ms; train duration = 5 ms). Current for conditioning brain stem stimulation was 0.2 to 1.5 mA. These stimulus parameters were found, in pilot experiments, to cover the effective range of values for reflex modulation. Stimulus current was calculated by Ohm’s law from the measured voltage drop across a 5-n resistor placed in series with one lead from the stimulus isolation unit. Reflex activity was displayed on an oscilloscope and measured by a peak-amplitude and window discriminator which was interfaced with a digital printer and a signal generator. The latter device provided a saw-tooth input to one channel of a Grass model 7B polygraph. Thus, the amplitude of the reflex was recorded and correlated with the on-line polygraphic record of EEG, EOG, and EMG activity. During experimentation the reflex was induced repeatedly (at a rate of l/s) throughout spontaneously occurring cycles of sleep and wakefulness. During wakefulness, testing was conducted when the animal was alert but not moving. During active sleep, trials in which rapid eye movements occurred were discarded and repeated later in the testing session. In no instance was current used which disrupted the animal’s ongoing state. Experimental Procedures. Because the amplitude of the masseteric reflex decreases as an animal passes from wakefulness to quiet sleep to active sleep (8), the stimulus current for reflex induction was adjusted so that an equivalent control reflex could be obtained during all states. Previous experiments and pilot studies indicated that such increases in current do not influence the results of conditioning-test trials (6, 7). The control reflex used was one whose amplitude during wakefulness was approximately 50% of the maximum amplitude that could be induced by stimulation of the mesencephalic Vth nucleus. Reflex modulation was analyzed by averaging the amplitude of control and experimental reflexes. A 50% or greater increase or decrease from control was defined as facilitation or suppression, respectively. To assess the effects of rostra1 mesencephalic and rostral pontine stimulation upon masseteric reflex excitability, a conditioning-test procedure was utilized wherein a train of stimuli was delivered to brain stem sites at specific latencies prior to the induction of the reflex. A conditioning-test trial consisted of three consecutive control reflexes which
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were followed by three reflexes conditioned by prior stimulation of the anterior mesencephalon or pons at a fixed latency. The mean amplitude of the control reflexes was then compared with that of the conditioned reflexes. This alternating pattern of three “control” reflexes followed by three “conditioned” reflexes was repeated continuously throughout each testing period. At each site the following three paradigms were used to characterize the effects of brain stem stimulation during wakefulness, quiet sleep, and active sleep. (i) A fixed conditioning-test interval of 20 ms was used to assess the basic pattern of reflex response at high (1 .O mA) and low (0.2 mA) values of conditioning current. [The 20-ms latency was chosen because the effect of brain stem stimulation at this conditioning-test interval was found to be the most reliable and effective for reflex modification (see Results).] (ii) The threshold for reflex modulation at each site was defined as the minimum amount of current required to alter reflex amplitude by at least 50% of its control value. The procedure for assessing threshold involved decreasing the conditioning stimulus from 1.5 mA in 0.2-mA increments until no effect was observed. (iii) The time course of response to conditioning stimulation was determined by examining conditioning-test intervals between 5 and 70 ms. For each of the preceding paradigms, three complete conditioning-test trials at each site were obtained during each behavioral state. A total of nine observations at each interval was obtained. After these data were collected during wakefulness, quiet sleep, and active sleep (N = 9 for each state), the electrode used for conditioning stimulation was lowered 1 mm, and the preceding experimental paradigms were repeated. Histology. After the experiment, each animal was dispatched, and its brain was perfused with 0.9% saline followed by 10% formalin. The brain was then removed and stored 1 to 2 weeks in formalin. Subsequently, the tissue was frozen, and sections 60 pm thick were obtained for histological evaluation of the electrode tracks. The location of the electrode for reflex induction was determined. The vertical sites in each track of the conditioning electrode were calculated from the point of lowest descent. RESULTS Mesencephalon: Patterns
of Reflex
Modulation
Mesodiencephalic and Their
Junction Topographic
Distribution.
Figure 1 summarizes the pattern of reflex modulation obtained with a fixed 20-ms conditioning-test interval from a total of 5 1 stimulation sites during wakefulness, quiet sleep, and active sleep. The position of each site is represented on a schematic brain stem cross section on the left side of the
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FIG. 3. Summary of the state-dependent effects of mesodiencephalic conditioning stimulation and examples of the two principal time course patterns of response. A-reflex suppression during active sleep in the absence of any striking effect during wakefulness or quiet sleep (obtained from A 6.5, L 4.5, H -3). B-a “response-reversal” pattern (obtained from A 6.5, L 5, H -2.5). Note the peakfacilitatory and suppressor response at 20 ms, and the similarity of the time course for active sleep in A and B. Each point was obtained by comparing nine control with nine conditioned reflexes.
to produce a 50% change from the control amplitude (Figs. 2A, B). However, during active sleep, low-threshold sites were widely distributed and scattered among those of high threshold (Fig. 2C). Consequently, no locus or “hot-spot” of suppression was found during active sleep, although the sites with the lowest thresholds tended to be situated ventrally. Time Course ofResponse. Figure 3 presents the principal time course of response to mesodiencephahc stimulation. The predominant response at this level of the neuraxis, i.e., state-dependent suppression of the masseteric reflex that emerges only during active sleep, is shown in Fig. 3A. Reflex suppression began at 10 to 15 ms, was maximum at 20 ms, and continued until 30 ms. An example of the time course of response from one of the few sites which demonstrated response-reversal is presented in Fig. 3B. Stimulation of this site produced facilitation during wakefulness and quiet sleep, with a peak at a conditioning-test latency of 20 ms.
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The pattern of suppression during active sleep was similar to the active sleep time course in the preceding example (Fig. 3A). Indeed, there was a high degree of similarity between the time-course of response from all sites during active sleep. All curves were comparable in that each exhibited peak suppression at approximately 20 ms. In addition, the onset of suppression was comparable across sites, occurring at 10 ms and preceded occasionally by a brief period of weak facilitation. The duration of suppression lasted 25 to 30 ms. Pons: Pontomesencephalic
Junction
Patterns of Reflex Modulation and Their Topographic Distribution. The 40 stimulation sites examined at this level of the brain stem are presented in Fig. 4. The distribution of these sites is represented on a schematic section of the pontomesencephalic junction shown on the left. The patterns of response at a standard conditioning-test interval of 20 ms are summarized on the right. During wakefulness and quiet sleep, stimulation induced either reflex facilitation or was ineffective. A well defined locus for the facilitatory effect was found when a stimulus current of 1.0 mA was used (Figs. 4A, B). The effective region was in the vicinity of the nuclei pontis oralis and reticularis mesencephali. This region was surrounded by an area which produced no modification of reflex excitability. With lower levels of stimulation (0.2 mA) there was a marked reduction in the number of sites which yielded facilitatory effects during wakefulness or quiet sleep (Figs. 4D, E). During active sleep, stimulation of almost all sites led to reflex suppression at a current of 1 .O mA. The effective region encompassed not only the reticular nuclei but also extended into the central grey, and included the superior colliculus. Although reflex suppression during active sleep was a widespread phenomenon, the effect was not prominent medially. With a lower conditioning current (0.2 mA) the demarcation between medial and lateral regions became more evident. However, even at 0.2 mA, an extensive reflex suppressor region was still noted in the core of the brain stem. Generally, the effect of lower conditioning current was to decrease the magnitude of reflex suppression and to reduce the size of the effective region. During active sleep, high (1.5 mA) values of conditioning current in five sites from two animals led to a decrease in the magnitude of suppression. However, the direction of response remained the same. These sites yielded strong motor reactions and the animal awakened within 2 to 3 s after stimulation. The region from which this response was induced was in the central portion of the nucleus reticularis mesencephali. However, we emphasize that this was an aberrant response, for other sites in this same region yielded a typical pattern of response-reversal which was
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Response Thresholds. In practically
all cases higher conditioning current was required to induce reflex facilitation during wakefulness and quiet sleep than was necessary for reflex suppression during active sleep. This finding is illustrated in Fig. 5 which shows the relative threshold of the tested sites for the induction of a change in reflex excitability. The integers shown in A through C represent the minimum amount of current which produced reflex facilitation or suppression. There was no clear differentiation of response threshold between wakefulness and quiet sleep; most sites required a relatively high current to induce facilitation during these states (Figs. 5A, B). The few low-threshold sites were clustered in the general vicinity of the reticular nuclei. In contrast, during active sleep, sensitive sites with very low-current thresholds were widely distributed throughout the tectum, central grey, and ventral tegmentum (Fig. 5C).3 Time Course of Response. The most prevalent time course patterns of reflex modulation are shown in Figs. 6 and 7. During wakefulness and quiet sleep, facilitation usually began at a short conditioning-test interval of 5 to 10 ms and reached a peak at 20 or 2.5 ms (Figs. 6A-C). From some sites there was only slight facilitation during wakefulness or quiet sleep (Fig. 6D); however, the overall time course was consistent with that described above. During active sleep, a slight degree of facilitation was usually present between 5 and 10 ms (Fig. 6B-D). Profound reflex suppression consistently occurred between 10 and 30 ms (Fig. 6A-F) with a return to the control value at about 30 to 40 ms. There sometimes followed, however, another period of longer latency, low-level suppression (Fig. 6C), or slight facilitation (Fig. 6D-F). In some cases there was either sustained facilitation or suppression beyond 30 ms as shown in Fig. 6A-C. From a small number of sites another pattern of response was observed which consisted of prominent reflex suppression during active sleep with virtually no effect during wakefulness or quiet sleep (Figs. 6E, F). In these instances the time course of suppression during active sleep paralleled that described above for other sites during this state. In summary, from all sites there was a remarkably consistent suppression of reflex activity at a conditioning-test interval of 10 to 30 ms during active sleep (Fig. 7). Short-latency facilitation and long-latency suppression during active sleep were usually present after conditioning stimulation of most sites, although these responses were of smaller magnitude (Fig. 7). 3 If all effective sites 50.4 mA are considered, then the behavioral states would be ranked as follows: wakefulness, 43%; quiet sleep, 40%; and active sleep, 89%.
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FIG. 5. Conditioning threshold for sites shown in the cross section of the pontomesencephalic junction. Integers indicate the minimum amount of current (in milliamperes) delivered to the indicated sites which was required to increase reflex amplitude by at least 50% of its control during wakefulness (W) or quiet sleep (QS) or induce a decrease in amplitude of at least 50% of control during active sleep (AS). Solid circles indicate sites which were ineffective in modifying reflex amplitude. A standard conditioning-test interval of 20 ms was used.
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DISCUSSION The results of this study reveal dramatic state-dependent control of reflex excitability exerted by the anterior mesencephalon. Stimulation of most sites with a low current resulted in powerful suppression of the test masseteric reflex during active sleep but was relatively ineffective in influencing reflex excitability during wakefulness or quiet sleep. However, structures in this region have not been considered to play a major role in the suppression of somatomotor activity during active sleep, although Hobson (13) reported a slight reduction in active sleep atonia after lesions of the mesencephalon. Thus, the strong involvement of anterior mesencephalic structures or fibers passing through the mesodiencephalic junction in the suppression of reflex activity constitute a new finding concerning the brain stem modulation of motor activity. As emergent reflex suppression during active sleep was both the predominant and low-threshold effect at this level of the neuraxis, we will concentrate in our discussion on an analysis of this response pattern. The emergence of a reflex suppressor influence, that exists only during active sleep (throughout widespread areas of the rostra1 mesencephalon), demonstrates the dynamic and state-dependent nature of somatomotor control. It appears that the entire mesencephalon (either resident nuclear groups and/or fibers of passage) undergoes a functional reorganization during active sleep in which all ineffective or facilitatory stimuli delivered during other states yield reflex suppression during this state. The question then arises as to the system(s) mediating this statedependent pattern of suppression of reflex activity. First, the distribution of the responsive sites do not suggest the exclusive participation of any known neuroanatomical fiber tract (1, 2). Second, although a number of biochemically defined systems course through the rostra1 mesencephalon, the pathways are largely ascending and their distribution is not aligned with the responsive sites we have described (15, 19). Finally, the participation of nuclei with diverse functions (such as the geniculate body and ventralis posterior medialis) in this state-dependent process does not argue for the eventual identification of a single structure mediating this effect. Similarly, widespread diffusely organized reticular-cortical influences were reported by Candia and co-workers, who were unable to localize any brain stem pathway mediating cortical desynchronization during active sleep (3). We hypothesize that reflex suppression which emerges during active sleep from widespread regions may be dependent on circuitry in the caudal brain stem rather than in the pons or mesencephalon (10). Pons: Pontomesencephalic Junction. The data obtained from an analysis Mesencephalon:
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FIG. 6. Time course of the effects of stimulation of six different sites at the pontomesencephalic junction during wakefulness, quiet sleep, and active sleep. A-D illustrate patterns of “response-reversal.” E and F portray profound reflex suppression during active sleep in the absence of any predominant response during wakefulness or quiet sleep. Each point on the graphs was obtained by comparing nine control and nine conditioned reflexes. The time course in A was obtained from A 1, L 2.5, H -0.5; in B from A 1.5, L 2.5, H - 1.5; in C from A 1, L 3, H - 1.5; in D from A 1, L 2.5, H -2.5; in E from A 1, L 3.5, H +4; and in F from A 1, L 6.5, H -2.5. Pontomesencephalic conditioning current = 1.0 mA; conditioning-test latency = 20 ms.
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FIG. 7. The filled circles in the brain stem cross section indicate the sites which yielded reticular conditioned masseteric reflex facilitation during wakefulness and quiet sleep, and suppression during active sleep. The average time course of this pattern of response is shown in A-C. Bars indicate standard errors of the means. Note the potent long-duration facilitatory effect during wakefulness and quiet sleep (A, B) and the biphasic period of suppression during active sleep (C). We occasionally observed sustained suppression during active sleep when the return to baseline of the response at 40 ms was absent. In many animals a brief period of low-amplitude facilitation persisted during active sleep at a conditioning-test latency of 5 to 10 ms. All the sites (unfilled as well as filled circles) shown in this brain stem section induced a pattern of suppression during active sleep similar to that shown in C. This response was observed even when no effect was found during wakefulness or quiet sleep.
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of this region confirm our previous finding that the reflex modulating influence of the pontomesencephalon varies according to the behavioral state of the animal (6, 9). The sites which demonstrate response-reversal were found to be co-extensive with the major reticular nuclei, specifically the nucleus reticularis pontis oralis and reticularis mesencephali. From surrounding regions a new pattern of reflex modulation at this brain stem level was observed. It consisted of reflex suppression, which was present only during active sleep, together with the absence of any effect of conditioning stimulation during wakefulness or quiet sleep. These results extend our finding at the mesodiencephalic level, namely, that widely dispersed regions of the brain stem are capable of influencing reflex activity only during active sleep and that the direction of the reflex response is solely one of motor suppression. Comparison of Mesodiencephalic and Pontomesencephalic Patterns of Rejlex Modulation. An examination of the mesodiencephalic and
pontomesencephalic patterns of response indicate that either there was no effect or that reflex facilitation was the only response evoked during wakefulness and quiet sleep. The responsible region at these brain stem levels is, in general, delineated by the rostra1 and caudal borders of the nucleus reticularis mesencephali and nucleus pontis oralis. However, many more sites led to reflex facilitation at the pontomesencephalic level of the brain stem, where the magnitude of facilitation was greater and could be produced with a lower conditioning current. During active sleep, stimulation of widespread regions at both brain stem levels produced reflex suppression; during this state reflex facilitation was never observed. The time course of suppression was remarkably similar from all sites and was induced with the lowest conditioning current from sites at the pontomesencephalic junction. The response threshold during different states at each brain stem level followed a similar pattern. From most sites less conditioning current was required during active sleep to suppress the masseteric reflex than to facilitate it during wakefulness or quiet sleep. Thus, after reducing the level of conditioning current, the only remaining effect was reflex suppression during active sleep from those brain stem sites that also yielded facilitation during wakefulness or quiet sleep. Moreover, although motor facilitation required higher conditioning current, as noted above, this response pattern was present only during wakefulness or quiet sleep (at conditioning-test intervals greater than 10 ms). The classic pattern of response-reversal, on the basis of amplitude of effect, threshold, and number of effective sites, was more closely associated with excitation of structures at the pontomesencephalic junction than at the more rostra1 brain stem level. These experiments demonstrate that widespread regions at the rostra1
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and caudai borders of the mesencephalon are effective in evoking suppression of reflex activity during active sleep. The lack of a circumscribed effective zone indicates that at these brain stem levels neither a single neuronal pathway nor nuclear group is involved in mediating this state-dependent pattern of motor control. Although state-dependent reflex facilitation during wakefulness and quiet sleep is a more localized and regional phenomenon, it also does not encompass a definable nuclear region or fiber pathway. Putative Neural Circuitry Responsible for State-dependent ReJEex Modulation. The widespread distribution and functional heterogeneity of structures whose excitation induce similar patterns of reflex suppression during active sleep, and the more circumscribed regions yielding reflex facilitation during wakefulness and quiet sleep (which overlap with the active sleep suppressor sites), led us to suggest that the responsible circuitry for these different response patterns may involve a neurophysiologic “gating” mechanism (4, 27). We hypothesize that there is synaptic circuitry functioning as a “gate” which alters the motor effect of brain stem discharge from somatomotor facilitation during wakefulness and quiet sleep to suppression during active sleep. During active sleep, we suggest that the gate is open, so that activity arising in the brain stem (and more than likely other areas as well) is funneled, or allowed access, to the input side of a powerful motor inhibitory system. The responsible inhibitory system may be located in the medullary reticular formation (18). There are fiber projections from pontomesencephalic to medullary reticular regions (11, 16). In turn, neurons in the region of the medullary reticular formation have postsynaptic inhibitory input onto masseter motoneurons (25). We suggest that the excitability of the medullary reticular formation increases during active sleep due to the opening of a neural gate during this state (4, 5). Widespread regions in the pons and mesencephalon (as well as forebrain structures), by virtue of their connection with pontomedullary reticular sites (1,3) which send excitatory projections to the lower brain stem (5), might then induce reflex suppression only during active sleep. Thus, suppression of reflex activity during active sleep could result from the activation of a motor inhibitory system by a “facilitatory” system with widespread afferent input, such as the “pontomesencephalic reticular activating system.” In this manner, activity arising in or coursing through the brain stem would be effective in suppressing reflex excitability during active sleep due to a neural gate that is patent during this state, thus allowing alink to be formed between rostra1 and caudal reticular regions. During wakefulness and quiet sleep we suggest that the gate is “normally closed” and motor facilitation ensues. This concept assumes an indirect route via the aforementioned neural gate for the motor suppressor path. Accordingly, the onset of suppression
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during active sleep would be expected to arise with a slightly longer latency than facilitation during wakefulness or quiet sleep. The time course of response which we observed supports this line of reasoning, for the onset of facilitation during wakefulness and quiet sleep in most cases occurred before the onset of suppression during active sleep. Thus, during active sleep, we would expect that only the very earliest component of motor facilitation would persist, due presumably to the longer activation latency of a suppressor system. Indeed, this is precisely what we found, for the early period of reflex facilitation often did not reverse as the animal changed state (Fig. 3). We recently carried out an intracellular analysis of the phenomenon of response-reversal in order to differentiate the synaptic events and circuitry by which it is mediated (22). We found that during wakefulness and quiet sleep stimulation of “response-reversal” sites induced in trigeminal jaw-closer motoneurons a short-latency (2.5 to 5 ms) depolarizing potential of lo- to 2%ms duration. When the animal passed into active sleep, a hyperpolarizing potential emerges at a latency of approximately 10 ms with a peak at about 20 ms. The early phase of the depolarizing potential, although somewhat diminished in amplitude, appeared to persist during active sleep. Thus the state-dependent change in motoneuron membrane potential mirrored the electromyographically recorded pattern of somatic reflex modulation reported in this paper. In summary, we suggest there is a selective gating mechanism which allows for the excitation of inhibitory neuronal systems by a “facilitatory” system during active sleep and prevents such activation during wakefulness or quiet sleep. At the present time we can offer only hypotheses as to the specific region and site of action of the neuronal mechanisms comprising the putative gate. Our current intracellular experiments are designed to shed light on the underlying synaptic circuitry (22). REFERENCES 1.
A. 1951. The Reticular Formation of the Brain Stem. Anatomical Functional Correlations, The Henderson Trust Lecture. Oliver and 2. BRODAL, A. 1969. Neurological Anatomy in Relation to Clinical Medicine. BRODAL,
Aspects
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Boyd, London. Oxford Press,
New York. 0.. G. ROSSI, AND T. SEKINO. 1967. Brain stem structures responsible for the electroencephalographic patterns of desynchronized sleep. Science 155: 720-722. 4. CHASE, M. H. 1976. A model of central neural processes controlling motor behavior during active sleep and wakefulness. Pages 99-121 in T. DESIRAJU, Ed., Mechanisms in Transmission of Signals for Conscious Behavior. Elsevier, New York. 5. CHASE, M. H. 1978. State-dependent reversal of a brainstem reflex in Felix domesticus. In Society for Neuroscience Symposia, Vol. 3 pp. 33-65, 1978. 6. CHASE, M. H., AND M. BABB. 1973. Masseteric reflex response to reticular stimulation reverses during active sleep compared with wakefulness or quiet sleep. Brain Res. 59: 421-426. 3. CANDIA,
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7. CHASE, M. H., AND D. J. MCGINTY. 1970. Somatomotor inhibition and excitation by forebrain stimulation during sleep and wakefulness: orbital cortex. Bruin Res. 19: 127- 136. 8. CHASE, M. H., D. J. MCGINTY, AND M. B. STERMAN. 1968. Cyclic variations in the amplitude of a brain stem reflex during sleep and wakefulness. Experientiu 24: 47-48. 9. CHASE, M. H., R. MONOSON, K. WATANABE, AND M. BABB. 1976. Somatic reflex response-reversal of reticular origin. Exp. Neural. 50: 561-568. 10. CHASE, M. H., AND N. WILLS. 1979. Brain stem control of masseteric reflex activity during sleep and wakefulness: Medulla. Exp. Neurol. 64: 118- 131. 11. EDWARDS, S. 1975. Audioradiographic studies of the projections ofthe midbrain reticular formation: Descending projections of the nucleus cuneformis. J. Camp. Neural. 161: 341-358. 12. HENLEY, K., AND A. MORRISON. 1974. A re-evaluation of the effects of lesion of the pontine tegmentum and locus coeruleus on phenomena of paradoxical sleep in the cat. Acta Neurobiol. Exp. 34: 215-232. 13. HOBSON, J. A. 1965. The effect of chronic brain stem lesions on cortical and muscular activity during sleep and waking in the cat. Electroencephalogr. Clin. Neurophysiol. 19: 41-62. 14. HUGELIN, A., AND M. BONVALLET. 1957. Tonus cortical et controle de la facilitation motice d’orgine reticulaire. J. Physiol. (Paris) 49: 1171- 1200. 15. JOUVET, M. 1972. The role of monoamines and acetylcholine-containing neurons in the regulation of the sleep-waking cycle. Ergeb. Physiol. 64: 166-307. 16. KAWAMURA, Y., A. BRODAL, AND G. HODDEVIK. 1974. The projections of the superior colliculus onto the reticular formation of the brainstem: An experimental study in the cat. Exp. Brain Res. 19: l-19. 17. LINDSLEY, D. B., L. H. SCHREINER, E. B. B. KNOWLES, AND H. W. MAGOUN. 1950. Behavioral and EEG changes following chronic brain stem lesions in the cat. Electroencephalogr. Clin. Neurophysiol. 2: 483-498. 18. MAGOUN, H. W., AND R. RHINES. 1946. An inhibitory mechanism in the bulbar reticular formation. J. Neurophysiol. 9: 165- 171. 19. MORGANE, P., AND W. STERN. 1974. Chemical anatomy of brain circuits in relation to sleep and wakefulness. Pages l-131 in E. WEITZMAN, Ed., Advances in Sleep Research. Vol. 1. Spectrum Publishing Company, New York. 20. MORUZZI, G. 1972. The sleep-waking cycle. Ergeb. Physiol. 64: l-165. 21. MORUZZI, G., AND H. W. MAGOUN. 1949. Brain stem reticular formation and activation of the EEG. Electroencephalogr. Clin. Neurophysiol. 1: 455-473. 22. NAKAMURA, Y., L. GOLDBERG, S. CHANDLER, AND M. CHASE. 1978. Intracelhdar analysis of trigeminal motoneuron activity during sleep in the cat. Science 199: 204-207. 23. POMPEIANO, 0. 1967. The neurophysiological mechanisms of the postural and motor events during desynchronized sleep. Res. Publ. Assoc. Res. Nerv. Ment. Dis. 45% 351-423. 24. REINSO-SUAREZ, R. 1961. Topographisher Himatlas der Katze. Herausgegeben von E. Merck A. G., Darmstadt. 25. SAUERLAND, E. K., Y. NAKAMURA, AND C. D. CLEMENTE. 1967. The role of the lower brainstem in cortically induced inhibition of somatic reflexes in the cat. Bruin Res. 6: 164-180. 26. STERIADE, M., AND J. A. HOBSON. 1976. Neuronal activity during the sleep-waking cycle. Prog. Neurobiol. 6: 155-156. 27. WILLS, N. 1977. Brainstem Influences on the Masseteric Reflex during Sleep and Wakefulness. Unpublished doctoral dissertation. University of Virginia.
