Cortical and Subcortical Patterns of Response to Afferent Vagal Stimulation

Transcription

1 EXPERIMEXTAL NEUROLOGY 16, (1966) Cortical and Subcortical Patterns of Response to Afferent Vagal Stimulation M. H. CHASE, M. B. STERMAN, AND C. D. CLEMENTE~ Departments of Anatomy and Physiology and the Brain Research Institute, University of California at Los Angeles and the Veterans Administration Hospital, Sepulveda, California Received April 25, 1966 Cortical and subcortical patterns of activity were studied upon electrical stimu!ation of the cut central end of the cervical vagus nerve in acutely prepared adult cats. The effects of different frequencies and voltages of afferent vagal stimulation were studied while the duration of the stimulus pulses was kept constant (0.75 msec). High-frequency (> 7O/sec) low voltage (< 3v) stimulation resulted in the induction of cortical EEG synchronization, while all other combinations of frequency and voltage gave rise to cortical activation. Subcortical structures responded in a manner consistent with the cortical observations. The cerebral cortex and thalamic nuclei responded to synchronogenic vagal afferent stimulus parameters only after an initial latent period, while the hippocampus responded immediately. All responses were bilaterally equivalent and could be induced by either vagus nerve. Severance of both sympathetic trunks and vagi and transection of the neuraxis at the spinomedullary junction indicated that the central responses were neurogenic in origin. These and other studies presently in progress offer convincing evidence that the different cortical responses (synchronization or desynchronization) observed by various investigators from stimulation of the central end of the cervical vagus nerve result from the differential stimulation of specific afferent fiber systems within that nerve. Introduction Zanchetti, Wang and Moruzzi (24) reported that the EEG response to afferent cervical vagal stimulation was one of cortical activation. Grastybn and his collaborators (9) not only confirmed this observation, but also described the occasional appearance of cortical spindles and slow waves during periods of afferent vagal stimulation. Other investigators have since published articles related to this same subject (5, 8, 17)) but no unifying concept has emerged, due to the diversity of their results. In addition to discrepancies regarding the cortical patterns of response, the brain mechanisms underlying the effects observed also remain obscure. Grastyan et 1 This research was supported by a grant from the United States Public Health Service (MH 10083), and the Veteran s Administration. 36

2 VAGAL AFFERENT STIMULATION 37 al. (9) believed that the vagus exerts its effect on the cortex via the reticular activating system of the brain stem, whereas Garnier and Aubert (8) stressed a direct neuronal action on the cerebral vasculature. It has also been postulated that the cortical response induced by central stimulation of the cut cervical vagus may result from humoral mechanisms, since it was found to be abolished when the contralateral nerve was cut (17). Pefialoza-Rojas (19) observed that blockade of the intact cervical vagus nerve by direct current stimulation induced EEG synchronization, and he concluded that the cessation of vagal input was responsible for the cortical slow waves. The present research was undertaken with the objective of clarifying these diverse observations. We were interested, additionally, in correlating changes in subcortical activity with the induced EEG patterns. Materials and Methods Experiments were performed on eighty adult male and female cats weighing between 2.5 and 3.5 kg. Due to the different experimental questions posed, variations from a standard preparation were employed, and these will be described when pertinent. The standard preparation involved a series of surgical procedures all performed under ether anesthesia. The initial steps included cannulation of the trachea and saphenous vein and unilateral separation of the cervical vagus nerve from the carotid artery and cervical sympathetic trunk. The animal was then placed in a stereotaxic instrument, the skull was exposed, and small stainless-steel recording screws were inserted into the calvarium over frontal, parietal and occipital areas of the cerebral cortex. Concentric bipolar electrodes were lowered through trephined holes into predetermined subcortical structures. The dissected vagus nerve was then severed at a low cervical level and its central end was placed in a Harvard peripheral nerve stimulating electrode. At this point xylocaine was liberally applied to all pressure points and wound edges (subsequently repeated at 90-min intervals), and an immobilizing dose of Flaxedil was administered intravenously. This type of preparation was necessary since the experimental objective required a nonanesthetized cerebral cortex in order to observe the effects of vagal stimulation on the EEG. The animal s limbs were comfortably placed and the preparation was maintained by artificial respiration; the experimental observations were made 2 hours after termination of ether anesthesia. This was necessary since the main objective of the experiment was to determine the effects of afferent vagal stimulation on the unanesthetized responsive cerebral cortex. Exposed tissues and the isolated nerve were kept viable by the repeated application of warm mineral oil,

3 38 CHASE, STERMAN, AND CLEMENTE body temperature was maintained constant at 36C, and the animal s limbs were comfortably supported. In order to establish an effective preparation, capable of yielding consistent results, certain points required special attention. When the vagus nerve was isolated from the carotid sheath and sympathetic trunk it was necessary to avoid trauma to it such as would result from stretching, crushing, dehydration or thermal variations. Care was taken to keep the state of CNS within physiological limits and constant attention was paid to ventilation, heart rate, pupillary reactivity and body temperature. Vagal stimulation was accomplished by means of square-wave pulses delivered from a Grass S-4 stimulator via its isolation unit. The electrical activity of cortical and subcortical structures was recorded on a Grass model VI polygraph or on an oscilloscope. At the end of each experiment, the animal was killed with an overdose of Nembutal and the brain was removed and fixed in 10% formalin. It was then sectioned serially and prepared histologically for microscopic verification of electrode placements. Results Electrical stimulation of the cut central end of the cervical vagus nerve was capable of eliciting both synchronized and desynchronized patterns of activity from the cerebral cortex and certain subcortical structures. The nature and degree of the induced EEG patterns were found to be dependent upon the specific parameters of stimulation employed. Cortical Responses to Synchronogenic Stimulation. In an evaluation of various parameters of vagal stimulation, it was observed that stimulation at frequencies greater than 70 pulse/set combined with voltages of 3 or less was capable of inducing EEG synchronization (Fig. 1). The cortical response elicited under these conditions appeared indistinguishable from the EEG patterns typically seen during spontaneous slow-wave sleep in the cat. All of the following characteristics of the induced cortical response were frequently observed in the same preparation. Initial response to stimulation. The EEG spindles and slow waves developed from an activated background with a latency of from 10 to 40 set; they never appeared coincident with the onset of stimulation (Fig. 1). Vagal stimulation at these parameters was without dramatic effect if the EEG was previously synchronized, although an augmentation in the background state of synchronization at times was observed. Occasionally, upon a synchronized EEG background, vagal stimulation resulted in a short period of desynchronization (approximately 10 set) which reverted once again to cortical synchronization. Vagal stimulation, even at these synchronogenic parameters, was on occasion ineffective in inducing spin-

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5 40 CHASE, STERMAN, AND CLEMENTE dies if an intense, prolonged and invariant background of EEG desynchronization preceded the stimulation. Frequently, EEG synchronization was observed on the first vagal stimulation trial, but more often, clear-cut responses appeared after a series of tests. Interspersed with effective trials were periods during which no EEG spindles could be elicited, although vagal stimulation almost always resulted in a decrease in the EEG pattern frequency. Response to continued stimulation. When EEG synchronization resulted from vagal stimulation it usually persisted throughout the entire period of nerve excitation. At times the slow waves were interrupted by brief intervals of desynchronization which lasted approximately 10 sec. Very infrequently the slow waves were permanently replaced by lowvoltage fast activity even though afferent vagal stimulation was continued. Response to cessation of stimulation. At the termination of stimulation, synchronization would either cease immediately (Fig. 1A) or continue uninterrupted for an indefinite period of time (Fig. 1B). When this latter pattern occurred, there was no observable EEG reaction to the cessation of stimulation. Occasionally, termination of stimulation resulted in the appearance of a burst of intense synchronization with waves of high amplitude. Cortical Responses to Desynchronogenic Stimulation. The consistent and immediate EEG response to stimuli of higher voltage (> 3v) delivered at high frequencies (> 70 pulse/set) to the central end of the vagus nerve was cortical desynchronization (Fig. 2B). This resulting EEG activation persisted beyond the period of stimulation and remained unaffected by the cessation of stimulation. Against a synchronized EEG background, low frequency (< 70 pulse/set) stimulation at voltages greater than 0.5 produced dramatic cortical desynchronization (Figs. 2A and 3). This pattern was achieved from 1 to 10 set after the onset of stimulation and persisted for the duration of stimulation. In summary, it was found that high-frequency, low-voltage stimulation of central vagal afferents elicits EEG synchronization. All other combinations of frequency and voltage resulted in the induction of the opposite EEG pattern, namely, desynchronization. Subcortical Responses. Subcortical structures were studied which were known to present different electrical patterns of activity during spontaneous sleep and wakefulness in the normal behaving animal, and a close functional relationship with the systems implicated in sleep and wakefulness cycles. The structures studied included the hippocampus, certain nonspecific thalamic nuclei (centrum medianum, centralis lateralis and medialis dorsalis) and two specific sensory thalamic nuclei (ventralis posterolateralis and lateral geniculate).

6 FIG. 2. Cortical and hippocampal responses to high voltage (> 3~). Excitation of the central end of the vagus, irrespective of the frequency of stimulation, are typically those associated with arousal. These effects appear at the onset of stimulation and upon cessation of stimulation there is a gradual reduction in the degree of cortical activation and hippocampal theta. Unilateral vagal stimulation. 4. c I 25/set 4 V 0.75msec I 200/set 4V 0.75msec

7 FIG. 3. Cortical and subcortical (hippocampus and nucleus centrum medianum) responses to high- and low-frequency excitation of the central end of the vagus nerve. This continuous record demonstrates the differential responses which occur when the voltage is held constant, but at a low level, and only the frequency of nerve stimulation is altered. The immediate activa- tion, at all levels, with low-frequency stimulation is contrasted with the delayed EEG and thalamic synchronization elicited by the increase in frequency. Right central vagal stimulation. R L.P.-OK COR.ynw#q+iw.~~~ R.P.-OCC. COR.4 RT HIPPO. CENT: MED.,25/set I V 75msec ON, 200sec I V.75 msec.

8 VACAL AFFERENT STIMULATION 43 Hippocampus. The predominant pattern of electrical activity recorded from the cat hippocampus during periods of alert behavior is the 4-8 cyclejsec theta rhythm. \Vith the onset of spontaneous sleep, this rhythmic pattern is gradually replaced by a pattern of high-frequency discharge superimposed upon a background of intermittent high-voltage spikes. Central cervical vagal stimulation produced both of these hippocampal patterns, once again dependent on the parameters of stimulation employed. High-frequency, low-voltage pulses delivered to the central end of the cervical vagus nerve led to the immediate disruption (1-2 set) of the ongoing theta rhythm (Figs. 3 and 4). As described above, the cortical response to these parameters of stimulation was synchronization, although it was visualized after 10 to 40 sec. Not only was the latency of the hippocampal response shorter, but it was observed even more consistently than the cortical response. Cortical synchronization and a disrupted theta rhythm are the electrical correlates of the initial stages of behavioral sleep. At times the hippocampal response occurred even without concomitant cortical spindles (Fig. 5). Vagal induced cortical synchronization, on the other hand, was never observed while the hippocampus displayed theta activity. In response to all other parameters of afferent vagal stimulation, the hippocampus showed dramatic theta activity occurring within 1 or 2 set after the onset of stimulation (Figs. 2 and 3). In these instances both the direction of change and the latency were similar to the response observed in the cerebral cortex. Cortical activation and the hippocampal theta rhythm are electrical correlates of behavioral arousal. Thalamus. The thalamic nuclei studied in these experiments responded in an expected manner and consistent with observations made at the cortical level. High-frequency, low-voltage stimulation resulted in the delayed onset of high amplitude slow waves characteristic of the electrical patterns in these structures during spontaneous sleep (Figs. 3, and 4B, C). All other stimulation parameters resulted in desynchronization (Fig. 3). Laterality, Localization and Peripheral Factors. Another series of acute experiments was undertaken in order to evaluate the relative effectiveness of right versus left vagal stimulation and the cerebral localization of the induced responses. Both vagus nerves were comparably effective in their induction of synchronization or desynchronization, but bilateral vagal stimulation did not promote more intense cortical or subcortical responses than unilateral stimulation. Vagal induced EEG synchronization appeared equivalent over both cortices and was similar in wave form to that occurring spontaneously or in response to hyperventilation. By increasing the voltage or lowering the frequency of vagal stimulation, widespread

9 44 CHASE, STERMAN, AND CLEMENTE CL0 ILn

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11 46 CHASE, STERMAN, AND CLEMENTE cortical desynchronization appeared bilaterally in a fashion similar to that observed following alerting or nociceptive stimulation. Other experiments were carried out in order to determine the possible roles of peripheral structures and systemic blood pressure in the etiology of the EEG responses observed. In addition to the recording of blood pressure and heart rate, in these experiments the sympathetic trunks were exposed along with the contralateral vagus and the spinomedullary junction. Variations in blood pressure were not found to be related to the EEG synchronization response to afferent vagal stimulation. Sequential severance of the ipsilateral sympathetic trunk, contralateral sympathetic trunk, contralateral vagus nerve and the neuraxis at the spinomedullary junction did not alter the EEG synchronization response. Time was allowed after the severance of each structure for the heart rate and blood pressure to stabilize before the vagal afferent effect was tested. Discussion Synchronization of EEG and behavioral sleep have both been observed following direct electrical stimulation of the brain (1, 11, 23), intracerebra1 injection of certain chemicals (lo), changes in ambient temperature and barometric pressure (14, 15) and the presentation of adequate stimuli to peripheral receptors (13, 18). Yet, it is common knowledge that EEG desynchronization and arousal can be obtained by these same stimuli if their mode of presentation or the animal s previous conditioning experiences are altered. It appeared to us that the diverse reports published relating EEG changes to afferent vagal stimulation might be explained on this basis, and the results of our experiments confirm this hypothesis. The studies by other investigators most relevant to the experiments reported in this communication are those describing EEG synchronization upon stimulation of various peripheral structures. These studies can be grouped into two categories dependent on the stimulation sites and requirements necessary to induce the EEG effects: (i) Prolonged repetitive stimulation (3, 18, 20-22). Prolonged repetitive stimulation of peripheral nerves or receptors has been found effective in the induction of EEG synchronization. Not only has this been observed through direct stimulation of peripheral nerves, but of special sensory receptors as well. Oswald (18), for example, found that male human volunteers went rapidly to sleep, even though their eyes were glued widely open, when synchronized electric shocks (to the leg), auditory and visual stimuli were simultaneously delivered. He interpreted this response as a consequence to sensory habituation, The EEG synchronization occurring as a manifestation of habituation is generally preceded by desynchronization upon initial presentation of the sensory stimulus. In our experiments

12 VAGAL AFFERENT STIMULATION 47 this did not occur when the proper synchronogenic parameters were delivered to afferent vagal fibers. It was observed that cortical spindles appeared within 10 set after the initial presentation of the vagal stimulus, an observation differentiating this response from the process of habituation. (ii) Sensory stimuli inducing specific synchronogenic patterns. Spindle bursts time-locked to specific sensory stimuli have been observed by several authors (13, 20, 21). These studies and those of Roitbak (22) indicate that, upon low frequency peripheral stimulation, tripped slow waves or spindles are induced following each stimulus. The EEG synchronization from afferent vagal stimulation differs fundamentally from this directed synchronogenic response both in regard to the parameters of stimulation necessary to induce the EEG change and the wave form and characteristics of the synchronization observed. The EEG synchronization resulting from afferent vagal excitation is similar in its electrical characteristics to that occurring during natural sleep. Pompeiano and Swett (2 1) concluded that stimulation of peripheral fiber systems at high frequencies resulted in desynchronization while stimulation of these same fibers at low frequencies resulted in EEG synchronization. In the vagus nerve the opposite was observed. High frequencies were most effective in inducing synchronization, whereas stimulation of the same fiber groups as visualized in a neurogram analysis (6) at lower frequencies and voltages resulted in only a diminution of the synchronization response, and activation certainly did not ensue. With voltages raised to levels exciting vagal fibers with higher thresholds, desynchronization results. The point to be stressed is that by increasing the frequency and holding constant the fiber type stimulated, cutaneous excitation resulted in cortical activation, while vagal stimulation under these conditions resulted in an enhanced EEG synchronization. We have found that one component of afferent vagal fiber groups is capable of inducing a unidirectional effect upon the EEG, namely, the induction of synchronization, whereas other vagal afferents induce EEG activation ( 6). Other experiments on visceral systems implicate the synchronogenic effects of maintained stimulation of visceral afferent fibers. EEG synchronization has been induced by the injection of milk into the stomach (2) while mechanical stimulation of the carotid sinus or electrical stimulation of the sinus depressor nerve results in a well-documented EEG synchronization response (4, 7, 16). It is also well known that visceral stimulation can lead to cortical activation ( 12 ). The potential for the induction of both EEG activation and synchronization resides within fibers carrying impulses from visceral receptors. Inasmuch as the vagus is composed of a wide fiber spectrum, one might look to the differential

13 48 CHASE, STERMAN, AND CLEMENTE excitability and refractory coefficients of the different afferent vagal fibers in an attempt to determine the basis for these differing cortical responses. Recent research analyzing the vagal neurogram with the known anatomical characteristics of cervical vagal fibers confirms this hypothesis (6). References 1. AKERT, K., W. P. KOELLA, and R. HESS, JR Sleep produced by electrical stimulation of the thalamus. Am. J. Physiol. 168: ANOKHIN, P. K The multiple ascending influence of the subcortical centers on the cerebral cortex, pp In Brain and Behavior, M. A. B. Brazier red.]. American Institute of Biological Sciences, Washinlgton, D.C. 3. ARDUINI, A., and T. Hm~o EEG synchronization elicited by light. Arch. Ital. Biol. 98: 27% BONVALLET, M., P. DELL, and G. HIEBEL Tonus sympathique et activite electrique corticale. Electroencephalog. Clin. Neurophysiol. 6: BONVALLET, M., and B. SEC Etude electrophysiologique des afferentes vagales au niveau de leur penetration dans le bulbe. J. Physiol. London 60: CHASE, M. H., Y. NAKAMURA, and M. B. STERMAN Neurographic correlates of cortical synchronization and desynchronization induced by afferent vagal stimulation. Anat. Record 154: GARCIA-MULLIN, R., and E. GARCIA-AUSTT Effects of carotid sinus stimulation on the EEG. Acta Neurol. Latinoam. 3: GARNIER, L., and M. AUBEXT Modifications de l electroencephalogramme du chat consecutives a la stimulation du nerf vague. Compt. Rend. Sot. Biol. 158: GRASTY~N, E., T. HASZNOS, K. LISSAK, L. MOLNAR, and 2. RUZSONYI Activation of the brain stem activating system by vegetative afferents. Acta Physiol. Acad. Sci. Hung. 3: HE~~NDEZ-PENN, R., G. CBAVEZ-IBARRA, P. MORGANE, and C. TIMO-IARIA Limbic choline@ pathways involved in sleep and emotional behavior. Exptl. Neural. 8: HESS, W. R Diencephalon, Autonomic and Extrapyramidal Functions, Grune and Stratton, New York. 12. HUGELIN, A., M. BONVALI.ET, and P. DELL Activation reticulaire et corticale d origine chemoceptive au tours de I hypoxie. Electroencephalog. Clin. Neurophysiol. 11: KAWAMURA, H., and K. YAMAMOTO Specific activation and induced spindle bursts in the auditory system. Japan. J. Physiol. 11: LECHAT, P., and G. STREICHENBERGER. 1964a. Sur le reendormissemment provoque chez le Rat par une variation brusque de temperature ambiente. Corn@. Rend. 968: LECHAT, P., and G. STREICHENBERGER. 1964b. Effets d une agression thermique sur l evolution du sommeil provoque chez le rat. Rev. Agressologie 5: MAZZELLA, H., E. GARCIA-AUSTT, and R. GARCIA-MULLS Carotid sinus and EEG. Electroencephalog. Clin. Neurophysiol. 8: NAKAMURA, Y., and T. HIRAO Spindle inducement and its correlation with hippocampal EEG. Japan. J. Physiol. 14:

14 VAGAL AFFERENT STIMULATION OSWALD, I Falling asleep open-eyed during intense rhythmic stimulation. Bvit. Med. /. 1: PENALOZA-ROJAS, J. H Electroencephalographic synchronization resulting from direct current application to the vagus nerv-e. Exptl..% ewol. 9: POMPEIANO, O., and J. SWETT EEG synchronization produced by peripheral nerve stimulation. Experientiu 17: POMPEIANO, O., and J. SWETT EEC and behavioral manifestations of sleep induced by cutaneous nerve stimulation in normal cats. Arch. Ital. Biol. 100: ROITBAK, A. I Electrical phenomena in the cerebral cortex during the extinction of orientation and conditioned reflexes. Moscow Colloquium. Electroencephalog. Clin. Neurophysiol. Suppl. 13: STERMAN, M. B., and C. D. CLEMENTE Forebrain inhibitory mechanisms: Sleep patterns induced by basal forebrain stimulation in the behaving cat. Exptl. Neuuol. 6: ZANCHETTI, A., S. C. WANG, and G. MORRUZZI The effect of vagal stimulation on the EEG pattern of the cat. Electroencephalog. Clin. Neurophysiol. 4:

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