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Neuronal activities in ventrobasal complex of thalamus and in trigeminal main sensory nucleus during EEG desynchronization in anesthetized rats
Brain Rest,arch, 379 (1986) 9t!--97 Elsevier
90
BRE 11879
Neuronal Activities in Ventrobasal Complex of Thalamus and in Trigeminal Main Sensory Nucleus During EEG Desynchronization in Anesthetized Rats KAZUYUKI KANOSUE, TERUO NAKAYAMA, PAUL D. ANDREW, ZAIWEN SHEN and MAKOTO SATO
Department of Physiology, Osaka University Medical School, OSaka (Japan) (Accepted December 10th, !985)
Key words: thalamus - - trigeminal main sensory nucleus - - somatosensory neuron - - electroencephalogram (EEG)
Activities of somatosensory relay neurons responding to orofacial mechanical stimulation were examined in the ventrobasal complex of the thalamus (VB) and in the trigeminal main sensory nucleus (MSN) during EEG desynchronization in Urethane-anesthetized rats. EEG desynchronization was induced by scrotal warming in a temperature range of 35-40 °C. Responses of most VB neurons to receptive-field stimulation were augmented during EEG desynchr0nization, when compared to responses during synchronization. Spontaneous activity of VB neurons also increased with EEG desynchronization. Responses of MSN neurons to reCeptive-fieidstimulation did not change appreciably when the EEG pattern was altered. If a VB neuron was induced by i0ntophoretic application of glutamate to fire at the same rate as seen during EEG desynchronization, a similar increased response to receptive-field stimuli was also observed. The augmented response of the VB neuron during desynchronization may thus have resulted from increased excitability of the neuron itself.
INTRODUCTION
ulate the same receptive field repeatedly for a sufficiently long time. We investigated the n e u r o n a l acti-
Studies on how sleep or wakefulness influence afferent transmission through the somatosensory pathways have for the most part involved responses to
vines during E E G synchromzation-and desynchronization. In previous studies, we found that scrotal
electrical stimulation of peripheral or central nervous structures t,2,9'tl,19. Results thus far obtained indicate that afferent transmission at the level of the ventrobasal complex of the thalamus (VB) may be facilitated during arousal and R E M sleep, as compared to slow-wave sleep 1,9'24. Many details on the nature of such facilitation, however, remain to be clarified. Not yet k n o w n , for example, is the extent to which sleep or wakefulness affects the usual responses of individual VB n e u r o n s , particularly when those responses are to natural stimuli. In the present experiments, we examined this problem by recording unit discharges of VB n e u r o n s in anesthetized rats. We chose this preparation because in unanesthetized animals it is difficult to stim-
warming in the temperature range of 32-40 °C produced E E G desynchronization in urethane-anesthetized rats 15"16. Thus. either synchronized o r desynchronized patterns in the E E G could be arbitrarily obtained by manipulating scrotal temperature. We adopted this m e t h o d to control the E E G pattern. In addition to responses of VB neurons, we examined responses of individual n e u r o n s in the trigeminal main sensory nucleus (MSN) as well. Electrophysiological and anatomical studies in cats have indicated that this first somatosensory relay station receives inputs from the cerebral cortex ~67-stT"2°'25'27. thalamus 7 and brainstem 3. This suggests that modulation of afferent transmission m the MSN may occur with changes in E E G pattern. Studies using evoked potentials in the trigeminal system have not yet pro-
Correspondence: T. Nakayama, Department of Physiology. Osaka University Medical School. Nakanoshima 4-3-57. Kita-ku, Osaka 530, Japan. 0006-8993/86/$03.50© 1986 Elsevier Science Publishers B .V. t Biomedical Division J
91 vided a conclusive answer to this question.
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MATERIALS AND METHODS Twenty-two male Wistar rats weighing between 300 and 400 g were anesthetized with 1.2-1.5 g/kg urethane injected intraperitoneally. The rats were mounted stereotaxically with the heads fixed according to the coordinate system of Paxinos and Watson 22. Rectal t e m p e r a t u r e was maintained at 36-38 °C by a water-perfused heating pad. F o r thermal stimulation, the scrotal skin was clipped and placed lightly against a rectangular t h e r m o d e (Peltier Device, 30 x 30 ram) and then covered with a thin film of liquid paraffin to ensure full contact. Scrotal t e m p e r a t u r e was measured with a thermocouple cem e n t e d to the surface of the t h e r m o d e in contact with the skin. To stimulate the medial lemniscus, a bipolar electrode consisting of two insulated wires (0.2 m m diameter) with bared tips 0.5 mm apart was inserted stereotaxically 1.5 mm anterior to the interaural line, 0.8 mm left the surface was placed stimulation
of the midline, and about 9.5 mm beneath of the skull. The stimulating electrode where e v o k e d responses to mechanical of vibrissae were found to be maximum.
Stimulus pulses from this electrode were 0.05 ms in duration with intensities less than 1 m A . Neuronal activities were recorded extracellularly from the VB and from the MSN (Fig. 1). To record from the VB, the electrode was first placed on the surface of the cerebral cortex 3 . 0 - 4 . 5 mm posterior to the bregma and 2 . 5 - 3 . 5 mm left of the midline, and then advanced vertically 5 . 5 - 6 . 5 mm into the brain. VB relay neurons were identified as those responding to single shocks of the medial lemniscus bv single spikes at latencies of 1.0-2.5 ms, typically followed by a series of grouped discharges. To record MSN neurons, the electrode was inserted into a point 4 . 5 - 5 . 0 mm posterior to the lambda and 3.0 mm right of the midline, directed 30 ° anteriorly from vertical orientation and advanced 6 . 0 - 7 . 5 mm deep from the surface of the cerebellum. MSN relay neurons were identified by their antidromic responses to electrical stimulation of the contralateral medial lemniscus. A response was considered antidromic if it had a constant latency, if it could consistently a p p e a r even when stimuli were de-
~-'~Glutamate
VB(.) ML
Fig. 1. Experimental arrangement for observing changes in neuronal activities in the VB and in the trigeminal MSN during EEG desynchronization induced by scrotal warming ML, medial lemniscus: Cx, cerebral cortex.
livered beyond 100 Hz, and if it could disappear by collision with a spontaneous o r t h o d r o m i c impulse. The recording electrode was a glass microelectrode filled with a solution of Pontamine sky blue in 0.5 M sodium acetate. The dye was deposited bv iontophoresis to mark the tip position for histological examination. The resistance of the electrode was 8 - 1 5 M Q for VB recordings and 2 0 - 3 0 Mr2 for MSN recordings. Neuronal impulses were counted bv a custom-made rate meter and displayed on a chart recorder along with scrotal t e m p e r a t u r e and E E G activity. A stainless steel screw electrode was placed in the skull over the occipital cortex for m o n o p o l a r E E G recordings. A n o t h e r screw in the skull, over the frontal sinus, served as an indifferent electrode. In the VB experiments, another micropipette, filled with monosodium-L-glutamate (pH 8.0), was attached to the recording electrode for iontophoretic application of glutamate. This pipette had a resistance of 4 0 - 7 0 M ~ . The iontophoretic current was in a range of 20-200 nA. If the VB or MSN neuron responded to mechanical stimulation of a vibrissa, a slender but stiff metal rod was set up to move the vibrissa. If the neuron reacted more readily to skin press or touch, a probe was placed so that its blunt end could activate the neuron by abutting against the skin surface. In either case an electric m o t o r o p e r a t e d the rod or probe to stimulate the receptive field periodically (once every several
92 means for arbitrarily controlling the general pattern of the EEG. Following each experiment the rat was perfused with saline solution followed b3 1()% formalin. Frozen sections of the brain were prepared in 4(1 ~m thick slices and stained with cresyl violet to verify the position of the electrode tip.
seconds). The electric signal running the electric motor and the electrical activity of the neuron were sent on-line to a signal processor (San-ei 7T07A) for immediate construction of a peristimulus time histogram (PSTH) by adding together 5-10 successive responses. Scrotal temperature was raised or lowered to control the E E G pattern. Under the present anesthetic condition and at a scrotal temperature of 30 °C, the E E G exhibited a maintained synchronized pattern in some animals and spontaneous fluctuation between synchronized and desynchronized patterns in other animals. In rats showing maintained synchronization at a scrotal temperature of 30 °C, scrotal warming to about 40 °C produced E E G desynchronization that lasted as long as the higher temperature was maintained, but which immediately gave way to a synchronized pattern after the temperature was lowered to the control level (Fig. 2). In rats showing transient spontaneous desynchronization, scrotal warming applied during the desynchronization produced no obvious change in E E G , but a fall of scrotal temperature was typically accompanied by a switch to an E E G synchronization that would last longer than the synchronization seen during spontaneous fluctuation (Fig. 5). Scrotal warming thus provided a convenient
(°C)
RESULTS
E E G desynchronization and VB relay neurons We recorded 40 VB relay neurons that had clearly demonstrable receptive fields in the contralateral area innervated by the trigeminat nerve. Hereinafter, spontaneous discharge refers to a discharge observed when no receptive-field stimulus is being applied or to a discharge observed between two stimuli at a point when the effects of the preceding stimulus have become negligible. While the E E G was displaying a synchronized pattern, the spontaneous discharge rates of the VB neurons were relatively stow, less than 1 Hz in most neurons, and 14 neurons showed no activity at all. Mean spontaneous firing rate of the 40 neurons was 0.78 _+ 1:30 (S.D,) Hz during E E G synchronization. When the E E G pattern became desynchronized, the spontaneous discharge
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Fig. 2. Response of a VB neuron to dorsal displacement of vibrissa B2. From top to bottom: scrotal temperature (Tscr), cortical E E G , firing rate (rate-meter recording) of the neuron and marks indicating receptive-field stimulation. Peristimulus Ume histograms (PSTHs) are cumulative composites of response profiles to 10 consecutive stimuli under desynchronized (left) and synchronized (right) conditions of EEG. See text for explanation of the hatched areas in the PSTHs.
93 increased in most of these neurons, but 7 neurons silent during synchronized E E G remained so during desynchronization as well. The mean spontaneous discharge rate of the 40 neurons during desynchronization was 5.02 + 6.16 Hz, significantly higher than during synchronization (t-test, P < 0.01). Fig. 2, which shows a typical response pattern of the VB relay neurons, illustrates clearly our usual finding that the neuronal response to the receptivefield stimuli was enhanced during E E G desynchronization. When scrotal temperature was low and the E E G exhibited a synchronized pattern, this neuron showed no spontaneous discharge. When the scrotum was warmed and its temperature reached about 40 °C, the E E G suddenly changed to a desynchronized pattern. At about the same time as the E E G change, the neuron began to discharge spontaneously. This increased activity was not a specific response to scrotal temperature change but resulted rather as part of a generalized effect accompanying E E G change >. Responses to receptive-field (vibrissa B2 (ref. 29)) stimuli were augmented in parallel with the increase in spontaneous discharge. During E E G desynchronization (the left PSTH in Fig. 2), this neuron increased activity in response to the receptive-field stimuli, being transiently inhibited immediately following the stimulus period with a subsequent rebound discharge. In response to a lowering of scrotal temperature, the E E G and neuronal activities returned to their original levels. The neuron then produced only an initial phasic response to receptivefield stimuli (the right PSTH in Fig. 2). We used the PSTH to obtain quantitative information on relative sensitivity of the neuron under different E E G conditions. The mean firing rate prior to onset of the stimulus calculated from the PSTH recording was subtracted from the area under the histogram during the stimulus phase. The remainder, illustrated by the hatched area of each PSTH in Fig. 2, provided a quantitative indication of the magnitude of the response for a given neuron under a given set of conditions. We then calculated the ratio of response magnitude during E E G desynchronization (Rdesynch) tO that during synchronization (esynch). This ratio Rdesynch/Rsynch has a value of 12.1 for the neuron shown in Fig. 2. For the 40 VB relay neurons recorded in this study, Rdesynch/Rsynch had a mean value of 3.6 + 2.3, significantly greater than a ratio of one
(P < 0.01). Thus the response magnitudes of the neurons to receptive-field stimulation were, in general, greater during E E G desynchronization than during E E G synchronization. We encountered, in fact, only two instances in which idesynch/Rsynch was less than one, 0.98 and 0.92.
Glutamate iontophoresis and VB relay neurons During E E G desynchronization, not only was the responsiveness of VB relay neurons to receptivefield stimuli augmented, but also the level of spontaneous activity was raised. This latter effect suggested that the soma of the neurons might be undergoing some degree of membrane excitation during E E G desynchronization. To see if membrane excitation was a cause of the increased responsiveness to receptive-field stimuli during desynchronization, glutamate was iontophoretically applied to 24 of the 40 VB neurons during E E G synchronization and responsiveness to receptive-field stimuli was examined. Such application of glutamate generally increased the firing rate, so for each neuron we adjusted the intensity of the iontophoretic current to match the firing rate to the level seen during desynchronization. In some neurons, however, a small increase in current dramatically transformed the spontaneous firing rate of the neuron from practically zero to beyond 20 Hz. In still other instances a fixed amount of current would produce varying firing rates in the same neuron. We thus frequently noted qualitative differences for increasing the firing rate of a given neuron between glutamate induction and induction by E E G desynchronization. Quantitatively, however, the mean spontaneous firing rate of the 24 neurons during glutamate iontophoresis was 5.03 + 4.86 Hz, not significantly different from the mean of 3.32 _+ 5.25 Hz for the same group of 24 neurons recorded during E E G desynchronization produced by scrotal warming. Fig. 3 shows the typical effect of glutamate application on a VB neuron. This neuron had hardly any spontaneous activity during E E G synchronization, but displayed a phasic burst as the cheek was pressed (Fig. 3, left). During desynchronization this neuron spontaneously fired at 1.5 Hz and when the receptive field was stimulated tonic activity appeared (Fig. 3, middle). Rdesynch/Rsynch was 10.57. Glutamate iontophoresis (50 nA) elicited spontaneous firing at a
94
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10s Fig. 3. PSTHs of a VB neuron during EEG synchronization (left), desynchronization induced by scrotal warming (middle), and local iontophoresis of glutamate (right). From top to bottom: PSTH. receptive-field (r.f. I stimulation and cortical EEG. This neuron responded to mechanical pressure of the cheek.
mean rate of 6.6 Hz. Response to the pressing during the glutamate application (Rglut) was augmented (Fig. 3, right). Rgtut/Rsynchwas 7.72. In some neurons. however, glutamate application did not differentially augment the response to receptive-field stimuli so strongly as happened during E E G desynchronization. In Fig. 4, Rglut/Rsync h w a s plotted against Roe_ synch/Rsynch for all the neurons analyzed except two. for which these values could not be calculated bec a u s e Rsynch was almost zero. Neither the intercept nor the slope of the regression line was significantly different from those of the line Rglut/Rsyne h = Rdesynch/Rsynch. Thus, glutamate iontophoresis at an appropriate dosage generally augmented the neuronal response to an extent similar to that seen during E E G desynchronization.
E E G desynchronization and M S N relay neurons We recorded 24 MSN neurons that were antidromically activated by stimulation of the contralateral medial lemniscus. All of them had receptive fields in the ipsilateral orofacial region and responded to innocuous mechanical stimulation of the receptive field. Latencies of the antidromic response ranged from 0.5 to 1.5 ms (0.81 -,- 0.22 ms/. Of the 24 neurons, 20 displayed no spontaneous discharge at all.
regardless of the E E G patterns. Fwo others discharged at a few Hz spontaneously during E E G synchronization and of those one increased its firing rate when the E E G became desynchronized. The remainmg two neurons exhibited no activity during E E G synchronization, but one of them discharged at 6 Hz and the other at less than 1 H z during desynchronization. Under the present experimental conditions, responses to receptive-field stimuli underwent no appreciable changes as the E E G shifted between synchronized and desynchronized stages. Fig. 5 shows a typical example of such a neuronal response to receptive-field stimuli during both E E G synchronization and desynchronization. When this neuron was recorded, the E E G was spontaneously fluctuating between synchronization and desynchronization. Scrotal warming maintained the desynchronized pattern and subsequent return of scrotal temperature to 32 °C led to a relatively prolonged state of synchronization. Both the rate-meter recording and the PSTHs revealed a fairly constant response of the neuron to mechanical stimulation in its receptive field, regardless of the E E G state. Rdesyneh/Rsvncl~was 1.02 in the example illustrated in Fig. 4. For 22 neurons recorded, this value remained in a narrow range from 0.65 to 1.18with a mean value of 0.97 + 0.12.
95
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DISCUSSION
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R desynch / Rsynch Fig. 4. Neuron by neuron comparison for 22 VB neurons of response magnitude seen during local glutamate application (Rglut, ordinate) to response magnitude recorded during E E G desynchronization induced by scrotal warming (Racsynch, abscissa). Both response magnitudes are expressed relative to the response magnitude seen during E E G synchronization (Rsynch). The regression line is not significantly different from RglJRsynch = Rdesynch/Rsynch (broken line).
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Fig. 5. Response of a MSN neuron to mechanical pressure on the cheek. A: antidromic discharge of the neuron in response to a single shock of the contralateral medial lemniscus. The stimulus was applied 0.5 ms after spontaneous discharge. B: note that no response was elicited when the stimulus was applied 0.4 ms after a spontaneous discharge. In both A and B, an arrow indicates the moment of stimulation. C: from top to bottom: scrotal temperature (Tscr), cortical E E G , firing rate (ratemeter recording) of the neuron and marks indicating receptivefield stimulation. PSTHs during E E G desynchronization and synchronization are shown at the bottom. Each PSTH represents the sum of 5 responses. Rdesynch/Rsynch = 1.02.
In VB relay neurons, the responses to receptive field stimuli were on the average 3.6x greater during E E G desynchronization than during synchronization (Fig. 2). Augmentation of responsiveness was observed in almost all the VB neurons. We had obtained similar augmented responses during E E G desynchronization in previous work 16, but our observations were limited to fewer neurons and were only qualitative in nature. In unanesthetized cats as well, stimulating the medial lemniscus has been shown to elicit greater evoked potentials recorded from the VB, or from somesthetic radiations, during arousal and REM sleep than during slow-wave sleep 1'9. Responsiveness of MSN neurons to receptive-field stimuli, on the other hand, changed little, if at all, with change in E E G activity. MSN neuronal responses during E E G desynchronization were similar in size to response during synchronization (Fig. 5). This suggests that the alteration of responses with E E G changes observed in the VB neurons takes place at the VB itself. Two possible factors could then be considered that might bring about the augmentation of responses during E E G desynchronization: (a) increased excitability of VB neurons and (b) increased input by some presynaptic mechanism at the synapses between lemniscal fibers and the VB neurons. Steriade has shown by evoked potentials in the cat that neurons of the ventroposterolateral nucleus, which mediates mainly epicritic sensation from the spinal cord, undergo facilitation only of a postsynaptic nature in response to stimulation of the reticular formation 24. In the present study, spontaneous activity in the VB neurons activated by natural stimulation of the trigeminal area increased during E E G desynchronization, suggesting that the soma was undergoing some degree of membrane excitation. When enough glutamate was iontophoretically applied to a VB neuron to elicit the same degree of spontaneous discharge as seen during desynchronization, the increase in responsiveness to receptive-field stimuli in most cases was likewise similar to that seen during desynchronization (Figs. 3 and 4). This favors the idea that the augmented response during desynchronization may have resulted primarily from heightened excitability of the VB neuron itself.
96 Scrotal warming is known to increase the level of spontaneous activity in a large proportion of neurons in the V B 10"12'14'15"27. These neurons have received considerable analytic attention as being involved in a specific ascending pathway of thermal information. but we have recently shown that increases in activities of VB neurons associated with scrotal warming are non-specific p h e n o m e n a accompanying E E G desynchronization ~6. Thus. past experimental findings of increased spontaneous activity of VB neurons obtained by scrotal warming may merit reinterpretation. Which inputs to the VB influence spontaneous activity of VB neurons? Disrupting the nucleus raphe magnus 28. nucleus raphe dorsalis 1°. or nucleus raphe centralis 1° prevents scrotal warming from elevating spontaneous activities of VB neurons, but the effect on E E G activity has yet to be investigated. Scrotal warming also raises tonic firing rates of neurons in the locus coeruleus (unpublished observations), electrical stimulation of which augments responsiveness of relay neurons in the lateral geniculate body to optic nerve stimulation TM. but no corresponding experiment has been performed on VB neurons. The thalamic reticular nucleus may attenuate the activities of VB relay neurons 2~. Cooling the somatosensory cortex suppresses the facilitating effect of scrotal warming on spontaneous activity of thalamic neurons (including VB neurons) ~4, but most somatosensory cortical neurons are themselves inhibited during scrotal warming t3 Thus. to understand elevation of spontaneous activity and augmented responsiveness to receptivefield stimuli of VB neurons during E E G desynchronization, an overall picture is required that includes effects from brainstem, thalamic and cortical levels. Anatomical and electrophysiological studies in the cat have indicated that the MSN received inputs from
REFERENCES 1 Allison. T.. Cortical and subcortical evoked responses to central stimuli during wakefulness and sleep, Electroenceph. Clin. Neurophysiol.. 18 (1965) 131-139. 2 Allison. T. and Golf. G.D.. Potentials evoked in somatosensory cortex to thalamocortical radiation stimulation during waking, sleep and arousal from sleep, Arch. Ital. Biol. 106 (1968) 41-60. 3 Baldissera, F.. Broggi. G. and Mancia. M.. Depolarization
the
cerebral
c o r t e x 567"8"17"20"24"27
thalamus:
and
brainstem 3. suggesting possibilities for modification of somatosensory input there. A spontaneous increase in excitability of cat trigeminal afferents, suggesting presynaptic inhibition of these fibers, has in fact been reported during R E M sleep and at the moment of arousal 4. but in the present study E E G changes exerted no effect at all on MSN neurons. The MSN in the rat thus appears to function as a stable and reliable relay station. In the present experiment, E E G changes were associated with modulation of somatosensory responses at a level no lower than the thalamus. An analogous p h e n o m e n o n can be found in an auditory study on isolated cat encephalon preparations, in which arousal due to stimulation of the reticular formation failed to have any effect on responses. evoked by clicking sounds, in the inferior colliculus. but did augment responses in the medial geniculate body 26. Furthermore, evidence abounds both from evoked-potential and single-unit analyses that transmission of visual information at the lateral geniculate body is facilitated during wakefulness and R E M sleep but reduced during slow-wave sleep I see Singer 23 for references). These facts combine to suggest that modulating sensory transmtssion according to state of arousal tends to be concentrated at the thalamic level.
ACKNOWLEDGEMENTS The authors are grateful to Drs. Y Kayama. S. Nakamura and A. Shosaku for their valuable comments. This study was supported in part by a Grant-inAid for Scientific Research from the Ministry of Education. Science and Culture of Japan (Grants 59440026 and 60770144 ).
of trigeminal afferents induced by stimulation of brain-stem and peripheral nerves, Exp. Brain Res,, 4 (1967) 1-17. 4 Baldissera, F., Broggi. G. and Mancia, M., Presynaptic inhibition of trigeminal afferent fibres during the rapid eye movements of desynchronized sleep, Experientia, 22 (I966) 754-755. 5 Brodal. A.. Szabo, T. and Torvik, A., Corticofugal fibers to sensory trigeminal nuclei and nucleas of solitary tract. An experimental study in the cat. J. Comp. Neurol.. 106 11956) 527-555.
97 6 Darian-Smith, 1. and Yokota, T., Cortically evoked depolarization of trigeminal cutaneous afferent fibers in the cat, J. Neurophysiol., 29 (1966) 170-184. 7 Dubner, R., Interaction of peripheral and central input in the main sensory trigeminal nucleus of the cat, Exp. Neurol., 17 (1967) 186-202. 8 Dubner, R. and Sessle, B.J., Presynaptic excitability changes of primary afferent and corticofugal fibers projecting to trigeminal brain stem nuclei, Exp. Neurol., 30 (1971) 223-238. 9 Favale, E., Loeb, C., Manfredi, M. and Sacco, G., Somatic afferent transmission and cortical responsiveness during natural sleep and arousal in the cat, Electroenceph. Clin. Neurophysiol., 18 (1965) 354-368. 10 Gottschlich, K.-W. and Werner, J., Effects of medial midbrain lesions on thermoresponsive neurons in the thalamus of the rat, Exp. Brain Res., 57 (1985) 355-361. 11 Hagbarth, K.-E. and Kerr, D.I.B., Central influences of spinal afferent conduction, J. Neurophysiol., 17 (1954) 295-307. 12 Hellon, R.F. and Misra, N.K., Neurons in the ventrobasal complex of the rat thalamus responding to scrotal skin temperature changes, J. Physiol. (London), 232 (1973) 389-399. 13 Hellon, R.F., Misra, N.K. and Provins, K.A., Neurons in the somatosensory cortex of the rat responding to scrotal skin temperature changes, J. Physiol. (London), 232 (1973) 401-411. 14 Hellon, R.F. and Taylor, D.C.M., An analysis of a thermal afferent pathway in the rat, J. Physiol. (London), 326 (1982) 319-328. 15 Kanosue, K., Nakayama, T., Ishikawa, Y. and Hosono, T., Threshold temperatures of diencephalic neurons responding to scrotal warming, Pliigers Arch., 400 (1984) 418-423. 16 Kanosue, K., Nakayama, T., Ishikawa, Y., Hosono, T., Kaminaga, T. and Shosaku, A., Responses of thalamic and hypothalamic neurons to scrotal warming in rats: nonspecific responses?, Brain Research, 328 (1985) 207-213. 17 Kawana, E., Projections of the anterior ectosylvian gyrus to the thalamus, the dorsal column nuclei, the trigeminal
nuclei and the spinal cord in cats, Brain Research, 14 (1969) 117-136. 18 Kayama, Y., Negi, T., Sugitani, M. and Iwama, K., Effects of locus coeruleus stimulation on neuronal activities of dorsal lateral geniculate nucleus and perigeniculate reticular nucleus of the rat, Neuroscience, 7 (1982) 655-666. 19 King, E.E., Naquet, R. and Magoun, H.W., Alterations in somatic afferent transmission through the thalamus by central mechanisms and barbiturates, J. Pharmacol. Exp. Ther., 119 (1957) 48-63. 20 Kuypers, H.G.J.M., An anatomical analysis of cortico-bulbar connexions to the pons and lower brain stem in the cat, J. Anat. (London), 92 (1958) 198-218. 21 Mushiake, S., Shosaku, A. and Kayama, Y., Inhibition of thalamic ventrobasal complex neurons by glutamate infusion into the thalamic reticular nucleus in rats, J. Neurosci. Res., 12 (1984) 93-100. 22 Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, Academic Press, Sydney, 1982. 23 Singer, W., Control of thalamic transmission by corticofugal and ascending reticular pathways in the visual system, Physiol. Rev., 57 (1977) 386-420. 24 Steriade, M., Ascending control of thalamic and cortical re, sponsiveness, Int. Rev. Neurobiol., 12 (1970) 87-144. 25 Stewart, D.H., Jr., Scibetta, C.J. and King, R.B., Presynaptic inhibition in the trigeminal relay nuclei, J. Neurophysiol., 30 (1967) 135-153. 26 Symmes, D. and Anderson, K.V., Reticular modulation of higher auditory centers in monkey, Exp. Neurol., 18 (1967) 161-176. 27 Tashiro, T., Distribution of cortical cells projecting to the main sensory trigeminal nucleus in the cat: a study with the horseradish peroxidase technique, Exp. Neurol., 78 (1982) 561-573. 28 Taylor, D.C.M., The effects of nucleus raphe magnus lesions on an ascending thermal pathway in the rat, J. Physiol. (London), 326 (1982) 309-318. 29 Van der Loos, H. and Woolsey, T.A., Somatosensory cortex: structural alterations following early injury to sense organs, Science. 179 (1973) 395-398.
90
BRE 11879
Neuronal Activities in Ventrobasal Complex of Thalamus and in Trigeminal Main Sensory Nucleus During EEG Desynchronization in Anesthetized Rats KAZUYUKI KANOSUE, TERUO NAKAYAMA, PAUL D. ANDREW, ZAIWEN SHEN and MAKOTO SATO
Department of Physiology, Osaka University Medical School, OSaka (Japan) (Accepted December 10th, !985)
Key words: thalamus - - trigeminal main sensory nucleus - - somatosensory neuron - - electroencephalogram (EEG)
Activities of somatosensory relay neurons responding to orofacial mechanical stimulation were examined in the ventrobasal complex of the thalamus (VB) and in the trigeminal main sensory nucleus (MSN) during EEG desynchronization in Urethane-anesthetized rats. EEG desynchronization was induced by scrotal warming in a temperature range of 35-40 °C. Responses of most VB neurons to receptive-field stimulation were augmented during EEG desynchr0nization, when compared to responses during synchronization. Spontaneous activity of VB neurons also increased with EEG desynchronization. Responses of MSN neurons to reCeptive-fieidstimulation did not change appreciably when the EEG pattern was altered. If a VB neuron was induced by i0ntophoretic application of glutamate to fire at the same rate as seen during EEG desynchronization, a similar increased response to receptive-field stimuli was also observed. The augmented response of the VB neuron during desynchronization may thus have resulted from increased excitability of the neuron itself.
INTRODUCTION
ulate the same receptive field repeatedly for a sufficiently long time. We investigated the n e u r o n a l acti-
Studies on how sleep or wakefulness influence afferent transmission through the somatosensory pathways have for the most part involved responses to
vines during E E G synchromzation-and desynchronization. In previous studies, we found that scrotal
electrical stimulation of peripheral or central nervous structures t,2,9'tl,19. Results thus far obtained indicate that afferent transmission at the level of the ventrobasal complex of the thalamus (VB) may be facilitated during arousal and R E M sleep, as compared to slow-wave sleep 1,9'24. Many details on the nature of such facilitation, however, remain to be clarified. Not yet k n o w n , for example, is the extent to which sleep or wakefulness affects the usual responses of individual VB n e u r o n s , particularly when those responses are to natural stimuli. In the present experiments, we examined this problem by recording unit discharges of VB n e u r o n s in anesthetized rats. We chose this preparation because in unanesthetized animals it is difficult to stim-
warming in the temperature range of 32-40 °C produced E E G desynchronization in urethane-anesthetized rats 15"16. Thus. either synchronized o r desynchronized patterns in the E E G could be arbitrarily obtained by manipulating scrotal temperature. We adopted this m e t h o d to control the E E G pattern. In addition to responses of VB neurons, we examined responses of individual n e u r o n s in the trigeminal main sensory nucleus (MSN) as well. Electrophysiological and anatomical studies in cats have indicated that this first somatosensory relay station receives inputs from the cerebral cortex ~67-stT"2°'25'27. thalamus 7 and brainstem 3. This suggests that modulation of afferent transmission m the MSN may occur with changes in E E G pattern. Studies using evoked potentials in the trigeminal system have not yet pro-
Correspondence: T. Nakayama, Department of Physiology. Osaka University Medical School. Nakanoshima 4-3-57. Kita-ku, Osaka 530, Japan. 0006-8993/86/$03.50© 1986 Elsevier Science Publishers B .V. t Biomedical Division J
91 vided a conclusive answer to this question.
j
r
MATERIALS AND METHODS Twenty-two male Wistar rats weighing between 300 and 400 g were anesthetized with 1.2-1.5 g/kg urethane injected intraperitoneally. The rats were mounted stereotaxically with the heads fixed according to the coordinate system of Paxinos and Watson 22. Rectal t e m p e r a t u r e was maintained at 36-38 °C by a water-perfused heating pad. F o r thermal stimulation, the scrotal skin was clipped and placed lightly against a rectangular t h e r m o d e (Peltier Device, 30 x 30 ram) and then covered with a thin film of liquid paraffin to ensure full contact. Scrotal t e m p e r a t u r e was measured with a thermocouple cem e n t e d to the surface of the t h e r m o d e in contact with the skin. To stimulate the medial lemniscus, a bipolar electrode consisting of two insulated wires (0.2 m m diameter) with bared tips 0.5 mm apart was inserted stereotaxically 1.5 mm anterior to the interaural line, 0.8 mm left the surface was placed stimulation
of the midline, and about 9.5 mm beneath of the skull. The stimulating electrode where e v o k e d responses to mechanical of vibrissae were found to be maximum.
Stimulus pulses from this electrode were 0.05 ms in duration with intensities less than 1 m A . Neuronal activities were recorded extracellularly from the VB and from the MSN (Fig. 1). To record from the VB, the electrode was first placed on the surface of the cerebral cortex 3 . 0 - 4 . 5 mm posterior to the bregma and 2 . 5 - 3 . 5 mm left of the midline, and then advanced vertically 5 . 5 - 6 . 5 mm into the brain. VB relay neurons were identified as those responding to single shocks of the medial lemniscus bv single spikes at latencies of 1.0-2.5 ms, typically followed by a series of grouped discharges. To record MSN neurons, the electrode was inserted into a point 4 . 5 - 5 . 0 mm posterior to the lambda and 3.0 mm right of the midline, directed 30 ° anteriorly from vertical orientation and advanced 6 . 0 - 7 . 5 mm deep from the surface of the cerebellum. MSN relay neurons were identified by their antidromic responses to electrical stimulation of the contralateral medial lemniscus. A response was considered antidromic if it had a constant latency, if it could consistently a p p e a r even when stimuli were de-
~-'~Glutamate
VB(.) ML
Fig. 1. Experimental arrangement for observing changes in neuronal activities in the VB and in the trigeminal MSN during EEG desynchronization induced by scrotal warming ML, medial lemniscus: Cx, cerebral cortex.
livered beyond 100 Hz, and if it could disappear by collision with a spontaneous o r t h o d r o m i c impulse. The recording electrode was a glass microelectrode filled with a solution of Pontamine sky blue in 0.5 M sodium acetate. The dye was deposited bv iontophoresis to mark the tip position for histological examination. The resistance of the electrode was 8 - 1 5 M Q for VB recordings and 2 0 - 3 0 Mr2 for MSN recordings. Neuronal impulses were counted bv a custom-made rate meter and displayed on a chart recorder along with scrotal t e m p e r a t u r e and E E G activity. A stainless steel screw electrode was placed in the skull over the occipital cortex for m o n o p o l a r E E G recordings. A n o t h e r screw in the skull, over the frontal sinus, served as an indifferent electrode. In the VB experiments, another micropipette, filled with monosodium-L-glutamate (pH 8.0), was attached to the recording electrode for iontophoretic application of glutamate. This pipette had a resistance of 4 0 - 7 0 M ~ . The iontophoretic current was in a range of 20-200 nA. If the VB or MSN neuron responded to mechanical stimulation of a vibrissa, a slender but stiff metal rod was set up to move the vibrissa. If the neuron reacted more readily to skin press or touch, a probe was placed so that its blunt end could activate the neuron by abutting against the skin surface. In either case an electric m o t o r o p e r a t e d the rod or probe to stimulate the receptive field periodically (once every several
92 means for arbitrarily controlling the general pattern of the EEG. Following each experiment the rat was perfused with saline solution followed b3 1()% formalin. Frozen sections of the brain were prepared in 4(1 ~m thick slices and stained with cresyl violet to verify the position of the electrode tip.
seconds). The electric signal running the electric motor and the electrical activity of the neuron were sent on-line to a signal processor (San-ei 7T07A) for immediate construction of a peristimulus time histogram (PSTH) by adding together 5-10 successive responses. Scrotal temperature was raised or lowered to control the E E G pattern. Under the present anesthetic condition and at a scrotal temperature of 30 °C, the E E G exhibited a maintained synchronized pattern in some animals and spontaneous fluctuation between synchronized and desynchronized patterns in other animals. In rats showing maintained synchronization at a scrotal temperature of 30 °C, scrotal warming to about 40 °C produced E E G desynchronization that lasted as long as the higher temperature was maintained, but which immediately gave way to a synchronized pattern after the temperature was lowered to the control level (Fig. 2). In rats showing transient spontaneous desynchronization, scrotal warming applied during the desynchronization produced no obvious change in E E G , but a fall of scrotal temperature was typically accompanied by a switch to an E E G synchronization that would last longer than the synchronization seen during spontaneous fluctuation (Fig. 5). Scrotal warming thus provided a convenient
(°C)
RESULTS
E E G desynchronization and VB relay neurons We recorded 40 VB relay neurons that had clearly demonstrable receptive fields in the contralateral area innervated by the trigeminat nerve. Hereinafter, spontaneous discharge refers to a discharge observed when no receptive-field stimulus is being applied or to a discharge observed between two stimuli at a point when the effects of the preceding stimulus have become negligible. While the E E G was displaying a synchronized pattern, the spontaneous discharge rates of the VB neurons were relatively stow, less than 1 Hz in most neurons, and 14 neurons showed no activity at all. Mean spontaneous firing rate of the 40 neurons was 0.78 _+ 1:30 (S.D,) Hz during E E G synchronization. When the E E G pattern became desynchronized, the spontaneous discharge
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Fig. 2. Response of a VB neuron to dorsal displacement of vibrissa B2. From top to bottom: scrotal temperature (Tscr), cortical E E G , firing rate (rate-meter recording) of the neuron and marks indicating receptive-field stimulation. Peristimulus Ume histograms (PSTHs) are cumulative composites of response profiles to 10 consecutive stimuli under desynchronized (left) and synchronized (right) conditions of EEG. See text for explanation of the hatched areas in the PSTHs.
93 increased in most of these neurons, but 7 neurons silent during synchronized E E G remained so during desynchronization as well. The mean spontaneous discharge rate of the 40 neurons during desynchronization was 5.02 + 6.16 Hz, significantly higher than during synchronization (t-test, P < 0.01). Fig. 2, which shows a typical response pattern of the VB relay neurons, illustrates clearly our usual finding that the neuronal response to the receptivefield stimuli was enhanced during E E G desynchronization. When scrotal temperature was low and the E E G exhibited a synchronized pattern, this neuron showed no spontaneous discharge. When the scrotum was warmed and its temperature reached about 40 °C, the E E G suddenly changed to a desynchronized pattern. At about the same time as the E E G change, the neuron began to discharge spontaneously. This increased activity was not a specific response to scrotal temperature change but resulted rather as part of a generalized effect accompanying E E G change >. Responses to receptive-field (vibrissa B2 (ref. 29)) stimuli were augmented in parallel with the increase in spontaneous discharge. During E E G desynchronization (the left PSTH in Fig. 2), this neuron increased activity in response to the receptive-field stimuli, being transiently inhibited immediately following the stimulus period with a subsequent rebound discharge. In response to a lowering of scrotal temperature, the E E G and neuronal activities returned to their original levels. The neuron then produced only an initial phasic response to receptivefield stimuli (the right PSTH in Fig. 2). We used the PSTH to obtain quantitative information on relative sensitivity of the neuron under different E E G conditions. The mean firing rate prior to onset of the stimulus calculated from the PSTH recording was subtracted from the area under the histogram during the stimulus phase. The remainder, illustrated by the hatched area of each PSTH in Fig. 2, provided a quantitative indication of the magnitude of the response for a given neuron under a given set of conditions. We then calculated the ratio of response magnitude during E E G desynchronization (Rdesynch) tO that during synchronization (esynch). This ratio Rdesynch/Rsynch has a value of 12.1 for the neuron shown in Fig. 2. For the 40 VB relay neurons recorded in this study, Rdesynch/Rsynch had a mean value of 3.6 + 2.3, significantly greater than a ratio of one
(P < 0.01). Thus the response magnitudes of the neurons to receptive-field stimulation were, in general, greater during E E G desynchronization than during E E G synchronization. We encountered, in fact, only two instances in which idesynch/Rsynch was less than one, 0.98 and 0.92.
Glutamate iontophoresis and VB relay neurons During E E G desynchronization, not only was the responsiveness of VB relay neurons to receptivefield stimuli augmented, but also the level of spontaneous activity was raised. This latter effect suggested that the soma of the neurons might be undergoing some degree of membrane excitation during E E G desynchronization. To see if membrane excitation was a cause of the increased responsiveness to receptive-field stimuli during desynchronization, glutamate was iontophoretically applied to 24 of the 40 VB neurons during E E G synchronization and responsiveness to receptive-field stimuli was examined. Such application of glutamate generally increased the firing rate, so for each neuron we adjusted the intensity of the iontophoretic current to match the firing rate to the level seen during desynchronization. In some neurons, however, a small increase in current dramatically transformed the spontaneous firing rate of the neuron from practically zero to beyond 20 Hz. In still other instances a fixed amount of current would produce varying firing rates in the same neuron. We thus frequently noted qualitative differences for increasing the firing rate of a given neuron between glutamate induction and induction by E E G desynchronization. Quantitatively, however, the mean spontaneous firing rate of the 24 neurons during glutamate iontophoresis was 5.03 + 4.86 Hz, not significantly different from the mean of 3.32 _+ 5.25 Hz for the same group of 24 neurons recorded during E E G desynchronization produced by scrotal warming. Fig. 3 shows the typical effect of glutamate application on a VB neuron. This neuron had hardly any spontaneous activity during E E G synchronization, but displayed a phasic burst as the cheek was pressed (Fig. 3, left). During desynchronization this neuron spontaneously fired at 1.5 Hz and when the receptive field was stimulated tonic activity appeared (Fig. 3, middle). Rdesynch/Rsynch was 10.57. Glutamate iontophoresis (50 nA) elicited spontaneous firing at a
94
Glutamate
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50 nA
1 300 m s
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10s Fig. 3. PSTHs of a VB neuron during EEG synchronization (left), desynchronization induced by scrotal warming (middle), and local iontophoresis of glutamate (right). From top to bottom: PSTH. receptive-field (r.f. I stimulation and cortical EEG. This neuron responded to mechanical pressure of the cheek.
mean rate of 6.6 Hz. Response to the pressing during the glutamate application (Rglut) was augmented (Fig. 3, right). Rgtut/Rsynchwas 7.72. In some neurons. however, glutamate application did not differentially augment the response to receptive-field stimuli so strongly as happened during E E G desynchronization. In Fig. 4, Rglut/Rsync h w a s plotted against Roe_ synch/Rsynch for all the neurons analyzed except two. for which these values could not be calculated bec a u s e Rsynch was almost zero. Neither the intercept nor the slope of the regression line was significantly different from those of the line Rglut/Rsyne h = Rdesynch/Rsynch. Thus, glutamate iontophoresis at an appropriate dosage generally augmented the neuronal response to an extent similar to that seen during E E G desynchronization.
E E G desynchronization and M S N relay neurons We recorded 24 MSN neurons that were antidromically activated by stimulation of the contralateral medial lemniscus. All of them had receptive fields in the ipsilateral orofacial region and responded to innocuous mechanical stimulation of the receptive field. Latencies of the antidromic response ranged from 0.5 to 1.5 ms (0.81 -,- 0.22 ms/. Of the 24 neurons, 20 displayed no spontaneous discharge at all.
regardless of the E E G patterns. Fwo others discharged at a few Hz spontaneously during E E G synchronization and of those one increased its firing rate when the E E G became desynchronized. The remainmg two neurons exhibited no activity during E E G synchronization, but one of them discharged at 6 Hz and the other at less than 1 H z during desynchronization. Under the present experimental conditions, responses to receptive-field stimuli underwent no appreciable changes as the E E G shifted between synchronized and desynchronized stages. Fig. 5 shows a typical example of such a neuronal response to receptive-field stimuli during both E E G synchronization and desynchronization. When this neuron was recorded, the E E G was spontaneously fluctuating between synchronization and desynchronization. Scrotal warming maintained the desynchronized pattern and subsequent return of scrotal temperature to 32 °C led to a relatively prolonged state of synchronization. Both the rate-meter recording and the PSTHs revealed a fairly constant response of the neuron to mechanical stimulation in its receptive field, regardless of the E E G state. Rdesyneh/Rsvncl~was 1.02 in the example illustrated in Fig. 4. For 22 neurons recorded, this value remained in a narrow range from 0.65 to 1.18with a mean value of 0.97 + 0.12.
95
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DISCUSSION
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R desynch / Rsynch Fig. 4. Neuron by neuron comparison for 22 VB neurons of response magnitude seen during local glutamate application (Rglut, ordinate) to response magnitude recorded during E E G desynchronization induced by scrotal warming (Racsynch, abscissa). Both response magnitudes are expressed relative to the response magnitude seen during E E G synchronization (Rsynch). The regression line is not significantly different from RglJRsynch = Rdesynch/Rsynch (broken line).
1ms
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Fig. 5. Response of a MSN neuron to mechanical pressure on the cheek. A: antidromic discharge of the neuron in response to a single shock of the contralateral medial lemniscus. The stimulus was applied 0.5 ms after spontaneous discharge. B: note that no response was elicited when the stimulus was applied 0.4 ms after a spontaneous discharge. In both A and B, an arrow indicates the moment of stimulation. C: from top to bottom: scrotal temperature (Tscr), cortical E E G , firing rate (ratemeter recording) of the neuron and marks indicating receptivefield stimulation. PSTHs during E E G desynchronization and synchronization are shown at the bottom. Each PSTH represents the sum of 5 responses. Rdesynch/Rsynch = 1.02.
In VB relay neurons, the responses to receptive field stimuli were on the average 3.6x greater during E E G desynchronization than during synchronization (Fig. 2). Augmentation of responsiveness was observed in almost all the VB neurons. We had obtained similar augmented responses during E E G desynchronization in previous work 16, but our observations were limited to fewer neurons and were only qualitative in nature. In unanesthetized cats as well, stimulating the medial lemniscus has been shown to elicit greater evoked potentials recorded from the VB, or from somesthetic radiations, during arousal and REM sleep than during slow-wave sleep 1'9. Responsiveness of MSN neurons to receptive-field stimuli, on the other hand, changed little, if at all, with change in E E G activity. MSN neuronal responses during E E G desynchronization were similar in size to response during synchronization (Fig. 5). This suggests that the alteration of responses with E E G changes observed in the VB neurons takes place at the VB itself. Two possible factors could then be considered that might bring about the augmentation of responses during E E G desynchronization: (a) increased excitability of VB neurons and (b) increased input by some presynaptic mechanism at the synapses between lemniscal fibers and the VB neurons. Steriade has shown by evoked potentials in the cat that neurons of the ventroposterolateral nucleus, which mediates mainly epicritic sensation from the spinal cord, undergo facilitation only of a postsynaptic nature in response to stimulation of the reticular formation 24. In the present study, spontaneous activity in the VB neurons activated by natural stimulation of the trigeminal area increased during E E G desynchronization, suggesting that the soma was undergoing some degree of membrane excitation. When enough glutamate was iontophoretically applied to a VB neuron to elicit the same degree of spontaneous discharge as seen during desynchronization, the increase in responsiveness to receptive-field stimuli in most cases was likewise similar to that seen during desynchronization (Figs. 3 and 4). This favors the idea that the augmented response during desynchronization may have resulted primarily from heightened excitability of the VB neuron itself.
96 Scrotal warming is known to increase the level of spontaneous activity in a large proportion of neurons in the V B 10"12'14'15"27. These neurons have received considerable analytic attention as being involved in a specific ascending pathway of thermal information. but we have recently shown that increases in activities of VB neurons associated with scrotal warming are non-specific p h e n o m e n a accompanying E E G desynchronization ~6. Thus. past experimental findings of increased spontaneous activity of VB neurons obtained by scrotal warming may merit reinterpretation. Which inputs to the VB influence spontaneous activity of VB neurons? Disrupting the nucleus raphe magnus 28. nucleus raphe dorsalis 1°. or nucleus raphe centralis 1° prevents scrotal warming from elevating spontaneous activities of VB neurons, but the effect on E E G activity has yet to be investigated. Scrotal warming also raises tonic firing rates of neurons in the locus coeruleus (unpublished observations), electrical stimulation of which augments responsiveness of relay neurons in the lateral geniculate body to optic nerve stimulation TM. but no corresponding experiment has been performed on VB neurons. The thalamic reticular nucleus may attenuate the activities of VB relay neurons 2~. Cooling the somatosensory cortex suppresses the facilitating effect of scrotal warming on spontaneous activity of thalamic neurons (including VB neurons) ~4, but most somatosensory cortical neurons are themselves inhibited during scrotal warming t3 Thus. to understand elevation of spontaneous activity and augmented responsiveness to receptivefield stimuli of VB neurons during E E G desynchronization, an overall picture is required that includes effects from brainstem, thalamic and cortical levels. Anatomical and electrophysiological studies in the cat have indicated that the MSN received inputs from
REFERENCES 1 Allison. T.. Cortical and subcortical evoked responses to central stimuli during wakefulness and sleep, Electroenceph. Clin. Neurophysiol.. 18 (1965) 131-139. 2 Allison. T. and Golf. G.D.. Potentials evoked in somatosensory cortex to thalamocortical radiation stimulation during waking, sleep and arousal from sleep, Arch. Ital. Biol. 106 (1968) 41-60. 3 Baldissera, F.. Broggi. G. and Mancia. M.. Depolarization
the
cerebral
c o r t e x 567"8"17"20"24"27
thalamus:
and
brainstem 3. suggesting possibilities for modification of somatosensory input there. A spontaneous increase in excitability of cat trigeminal afferents, suggesting presynaptic inhibition of these fibers, has in fact been reported during R E M sleep and at the moment of arousal 4. but in the present study E E G changes exerted no effect at all on MSN neurons. The MSN in the rat thus appears to function as a stable and reliable relay station. In the present experiment, E E G changes were associated with modulation of somatosensory responses at a level no lower than the thalamus. An analogous p h e n o m e n o n can be found in an auditory study on isolated cat encephalon preparations, in which arousal due to stimulation of the reticular formation failed to have any effect on responses. evoked by clicking sounds, in the inferior colliculus. but did augment responses in the medial geniculate body 26. Furthermore, evidence abounds both from evoked-potential and single-unit analyses that transmission of visual information at the lateral geniculate body is facilitated during wakefulness and R E M sleep but reduced during slow-wave sleep I see Singer 23 for references). These facts combine to suggest that modulating sensory transmtssion according to state of arousal tends to be concentrated at the thalamic level.
ACKNOWLEDGEMENTS The authors are grateful to Drs. Y Kayama. S. Nakamura and A. Shosaku for their valuable comments. This study was supported in part by a Grant-inAid for Scientific Research from the Ministry of Education. Science and Culture of Japan (Grants 59440026 and 60770144 ).
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