Differential Responses of Brain Stem Neurons during Spontaneous and Stimulation-Induced Desynchronization of the Cortical EEG in Freely Moving Cats

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1 Sleep Research Online 1(4): , Printed in the USA. All rights reserved X 1998 WebSciences Differential Responses of Brain Stem Neurons during Spontaneous and Stimulation-Induced Desynchronization of the Cortical EEG in Freely Moving Cats Birendra N. Mallick 1, Stephen Thankachan 1 and Fakhrul Islam School of Life Sciences, Jawaharlal Nehru University, New Delhi , India and Department of Medical Elementology and Toxicology, Jamia Hamdard, Hamdard University, New Delhi , India The EEG is desynchronized during wakefulness and REM sleep. There are awake and REM sleep-related neurons in the brain stem. This study was carried out to investigate if the same neuron in the brain stem reticular formation may be responsible for EEG desynchronization during wakefulness and REM sleep. Single neuronal activity was recorded in chronically prepared freely moving normal cats and their activities were correlated with EEG desynchronization during spontaneous wakefulness, REM sleep, and during wakefulness induced by stimulation of the brain stem reticular formation. A majority of the neurons showed an increased firing associated with spontaneous EEG desynchronization during wakefulness and REM sleep, however, about 55% of them showed a similar behavior during stimulation-induced desynchronization. It was found that responses of a majority of the neurons during stimulation-induced desynchronization were similar to that of their firing rate during EEG desynchronization associated with spontaneous wakefulness irrespective of their behavior during REM sleep; the REM-ON neurons were not affected by the stimulation-induced desynchronization. A majority of the neurons which showed an increased firing during spontaneous and stimulation-induced EEG desynchronization received an excitatory input from the brain stem reticular formation. The results of this study suggest that although some neurons may be common, there is a strong possibility that the same neuron in the brain stem reticular formation is not involved in EEG desynchronization during wakefulness and REM sleep. CURRENT CLAIM: Separate groups of neurons are possibly involved in EEG desynchronization during wakefulness and REM sleep. The role of brain stem in EEG desynchronization is well established. The EEG is synchronized in cerveau isolé preparation (brain stem transected at mid-collicular level) while the synchronization alternated with desynchronization in the encéphale isolé (transection was made at the junction of brain stem and the spinal cord C1/C2 level) preparation (Moruzzi, 1972). This was further confirmed by lesion and stimulation studies. The rostral portion of the brain stem was reported to be responsible for wakefulness and EEG desynchronization (Batini et al., 1959; Kasamatsu, 1970). Subsequently, it has been shown in anesthetized as well as in freely moving animals that although neurons related to wakefulness and EEG desynchronization are scattered in the brain stem reticular formation, they are predominantly located in the rostral brain stem (Huttenlocher, 1961; Manohar et al., 1972). The brain stem area which induces EEG desynchronization has been found to affect neurons present in other sleep-waking areas in the brain (Mallick et al., 1986) and vice versa (Fenske et al., 1975; Szymusiak and McGinty, 1989). Recently it has been suggested that brain stem neurons induce EEG desynchronization by acting through the thalamic neurons (Paré et al., 1988; Steriade et al., 1990, 1996). In addition to wakefulness, the EEG is desynchronized during rapid eye movement (REM) sleep as well, and the neurons related to REM sleep are also located in the brain stem reticular formation (Jones, 1991). The aminergic neurons are REM-OFF type (Chu and Bloom, 1974; McGinty and Harper, 1976; Jacobs, 1986), while REM-ON neurons may be cholinergic or non-cholinergic (El Mansari et al., 1989; Kayama et al., 1992; Sakai and Koyama, 1996). Although the mechanism of generation of REM sleep is not yet clear, neurons in the dorsolateral pontine region ventral to the locus coeruleus are attributed to muscle atonia (Sakai, 1985; Lai and Siegel, 1988) and those in the parabrachial area to PGO waves (Datta, 1995) during REM sleep. However, it is not known with certainty which neurons are responsible for EEG desynchronization during REM sleep. Since wakefulness and REM sleep are two distinct behaviors and EEG desynchronization is one of the signs (apparently) common to them, at least two possibilities could exist. One, that the same neurons in the brain stem may be involved in EEG desychronization which switch from one neuronal circuitry responsible for behavioral wakefulness to circuitry responsible for REM sleep or vice versa. Two, that there may be two separate groups of neurons forming separate circuitry responsible for EEG desynchronization during wakefulness and REM sleep behaviors. It was hypothesized that if there were two separate groups of neurons for EEG Correspondence: Birendra N. Mallick, Ph.D., School of Life Sciences, Jawaharlal Nehru University, New Delhi, , India, Tel: x 2522, Fax: , bnm@jnuniv.ernet.in.

2 133 MALLICK ET AL. desynchronization during wakefulness and REM sleep, then neurons related to the former only should respond similarly to brain stem reticular formation stimulation-induced wakefulness and EEG desynchronization. Therefore, the behavior of the same awake and REM sleep related neurons from the reticular formation in freely moving cats was correlated with EEG desynchronization during spontaneous wakefulness as well as REM sleep and also during brain stem stimulation-induced EEG desynchronization. Nevertheless, considering the complexity and redundancy of the nervous system, the other complex possibility, that although there may be a predominance of separate groups of neurons, it cannot be ruled out that a small portion of the neurons may be involved in EEG desynchronization during both the behavioral states. METHODS Experiments were performed on 9 adult cats (5 male and 4 female), weighing between 2.5 and 3.4 kg. Under surgical anesthesia (sodium pentobarbitone, 35 mg/kg, i.v.) cats were prepared for chronic sleep-wakefulness (S-W) recording (Mallick et al., 1989). One pair of small stainless steel screw electrodes was fixed in the skull above the sensorimotor cortex to record bipolar EEG and another pair over the orbital bone of the eye to record electrooculogram (EOG). Two flexible insulated (except a small portion at the end) wires were attached bilaterally to the dorsal neck muscles to record bipolar electromyogram (EMG). Stainless steel insulated (except at the tip) tripolar electrodes were bilaterally implanted at stereotaxic coordinates (Berman, 1968) of A6, L10, H2 to record pontogeniculooccipital (PGO) waves. One stainless steel tripolar stimulating electrode was stereotaxically implanted in the midbrain reticular formation (MRF) at the coordinates of A2, L3, H-1 (Mallick et al., 1986). The microdrives consisted of two thin-walled stainless steel 19G outer barrels (20 mm length) parallely placed 2 mm apart. Two 22G thin-walled stainless steel inner barrels (40 mm length) were passed through the outer barrel and arranged on a screw-nut assembly for smooth and easy vertical movement (McGinty and Harper, 1976). A pair of microdrives was stereotaxically introduced into the brain bilaterally at an angle of 30 o through drill holes made in the skull so as to reach coordinates of AP0-P3, L The microdrives were introduced so that the tips of the inner cannulae reached 6.0 mm above the target H-1. The outer cannulae of the microdrives were cemented on the skull with dental acrylic so that the inner cannulae could move freely dorsoventrally. Four microelectrode bundles each consisting of nine 32 µm Formvar-coated stainless steel wires (California Fine Wire Inc., U.S.A.) soldered to a 9-pin connector were lowered through each of the inner guide cannulae so as to extend 5.0 mm beyond the inner cannulae of the microdrives. Thus, the tips of the microelectrodes were positioned 1.0 mm above the target. The microelectrodes were glued to the inner cannulae so that when the inner cannulae were advanced manually by means of the attached screw, the microelectrodes also advanced through the brain. The microdrives could be moved dorsoventrally by a total length of about 4 mm. Thus, with this arrangement in this study single neuronal activities were recorded within the coordinates of AP0-3, L , H-1 to -4. About 2 weeks were allowed for the cats to recover from surgical trauma. During this recovery period the cats were habituated to the recording chamber. After recovery, electrophysiological signals viz., EEG, EOG, EMG and PGO were recorded simultaneously on a Grass polygraph from unrestrained, freely moving cats. Once a single unit activity (S:N > 3:1) was encountered, it was amplified (A-M Systems Inc., U.S.A.) and fed to an adjustable window discriminator (Fredrick Haer Brunnswick, ME). The raw signal from the window discriminator was monitored on an oscilloscope (TDS 420 Tektronix Inc., U.S.A.). The window-triggered output from the window discriminator was fed into a pulse counter and the output was recorded simultaneously on one of the channels of the polygraph along with other electrophysiological signals in other channels. The signals were also recorded and processed in a computer connected to a CED 1401plus intelligent interface. At least two spontaneous S-W-REM sleep cycles were recorded and then the MRF was stimulated (100 Hz, 300 µs, µa) for 8-12 sec at the background of quiet wakefulness and the effects on sleepwakefulness as well as on the unit activity were recorded simultaneously as long as the effects lasted. Thereafter, in order to study if the neurons received any direct projection from the MRF stimulation site, the response of the neurons to 1 Hz (300 µs, µa) stimulation of MRF was also studied by overlapping stimulus-bound responses on the oscilloscope. Such 1 Hz stimulation of the MRF did not produce wakefulness, unlike the effects on high frequency stimulation. Thus, the effect on the neurons by 1 Hz stimulation was independent of changes in EEG desynchronization and associated behavior. After completion of the recording sessions the animals were deeply anesthetized with sodium pentobarbitone (45 mg/kg) and a small electrolytic lesion ( µa for 10 sec) was made at the tip of the successful unit recording site. The cat was then euthanized by intracardial perfusion with 0.5 L of saline followed by 1 L of 4% paraformaldehyde and 2% potassium ferrocyanide and preserved for histological identification of the lesioned site and reconstruction of the recording sites. The brain stem was trimmed and preserved in 30% sucrose before making 40 µm coronal sections which were processed for staining with cresyl violet or hematoxylin and eosin. Some of the sections were immunostained for tyrosine hydoxylase (TH) by using standard methods (Reiner and Vincent, 1987). The former two stained sections showed recording sites and the latter showed if the REM-OFF neurons were recorded from the TH-positive sites. For immunostaining, the sections were processed using primary antibody (rabbit anti-th polyclonal, 1:1000, Eugene Tech) and incubated in secondary antibody (biotinylated goat anti-rabbit immunoglobulin, 1:400, Vector Lab). This was followed by treatment with avidin-biotin complex (ABC complex, 1:500,

3 UNIT FIRING DURING WAKE AND REM DESYNCHRONIZATION 134 Figure 1. Representative histological section (inset) and schematic diagram through cat brain showing the types of neurons recorded from different sites (A). The recording wire track (arrow) can be seen in the histological section (inset). Schematic diagram of cannulae and recording wire in the brain are shown in (B). A TH immuno-stained section showing TH-positive neurons (arrows) are shown in the inset. Abbreviations of anatomical terms: 7G, genu of the facial nerve; AQ, aqueduct; BC, brachium conjunctivum; BP, brachium pontis; CS, central superior nucleus; DRM, dorsal nucleus of the raphe, medial division; FTC, central tegmental field; FTG, gigantocellular tegmental field; FTM, magnocellular tegmental field; FTP, paralemniscal tegmental field; IC, inferior olive; LC, locus coerulus; LDT, laterodorsal tegmentum; PAG, periaqueductal gray; PH, praepositus hypoglossi; PG, pontine gray; PPT, pedunculopontine tegmentum; TRC, tegmental reticular nucleus; TB; trapezoid body; V4, fourth ventricle.

4 135 MALLICK ET AL. INCREASE Table 1 Brain Stem Neuronal Activity Relationship to AW REM Sleep (84) (84) NO CHANGE DECREASE INCREASE NO CHANGE DECREASE Table 1. The number of brain stem neurons showing significant increase, decrease or no change in their spontaneous firing rates during active wakefulness (AW) (left panel) and rapid eye movement (REM) sleep (right panel) compared to respective spontaneous quiet wake firing rates are shown in this table. Total number of brain stem neurons studied are shown in parentheses. Table 2 Classification of Neurons Spontaneous Spontaneous AW REM Sleep Increase No Change Decrease Increase No Change Decrease Table 2. The neuronal firing rates during spontaneous active wakefulness (AW) and REM sleep states, when the EEG remained desynchronized were statistically compared to respective quiet wakefulness firing rates. Firing rates of some of the neurons changed in the same direction while some others, in the opposite direction, during those two states. Accordingly, the neurons could be classified into seven groups and number of neurons in each of those groups are shown in this table. Vector Lab) and the brown reaction product was visualized by incubating in DAB (Vector Lab) and H The sections were mounted on subbed slides, dehydrated and prepared for microscopic examination. Reconstruction of sections showing unit recording sites and one histological TH-stained section showing microwire track passing through TH-positive neurons are shown in Figure 1. The sleep-wakefulness-rem sleep stages were classified as reported earlier (Ursin and Sterman, 1981; Mallick et al., 1989). In brief, active awake (AW), was characterized by EEG desynchronization accompanied by eye movement, increased EMG tone and/or phasic motor activities; quiet awake (QW) by EEG desynchronization with occasional (<20% time) synchronization and spindling, reduced eye movements and reduced EMG tone (absence of phasic motor activity). Sleep stage was defined by EEG synchronization and spindles, no eye movements, reduced EMG tone and absence of phasic motor activity. REM sleep had characteristic signs of EEG desynchronization, rapid eye movement accompanied by muscle atonia and presence of PGO waves. Mean neuronal discharge rate during each of the sleepwakefulness-rem sleep states was calculated by taking the average of five 60-second epochs of respective states. Mean neuronal activity per second during different states was compared statistically by applying analysis of variance (ANOVA) coupled with Newman-Keul's test taking QW as the basal level to establish the relationship of spontaneous neuronal firing rate to different states. To study the effect of MRF stimulation-induced EEG desynchronization on the unit activity, the firing rate of neuronal activity during poststimulation EEG desynchronization was also compared with that of the rate of firing of the unit activity during prestimulation QW period. The firing rate of the neurons for at least three 30 sec episodes each during pre-stimulation QW and post-stimulation desynchronization were calculated and statistically tested by applying ANOVA and Newman Keul's test. As a rule, firing rate during the period of MRF stimulation was not taken because occasionally the stimulation artifact falling within the window levels could not be avoided. RESULTS Eighty-four well isolated single units were recorded between caudal midbrain to rostral pontine tegmental region of the brain stem from chronically prepared freely moving normally behaving cats. The neuronal activities (spikes per second) during each of the S-W-REM sleep states were compared statistically against QW to establish the relationship of individual neuronal firing rates to different states. It was found that 61 increased, 3 decreased and 20 remained unaffected in relation to AW only; while 70 neurons increased, 7 decreased and 7 remained unaffected in relation to REM sleep only (Table 1). However, the neurons could be classified into 7 groups (Table 2) considering their firing rate both during AW and REM sleep when the EEG during both the behavioral states was desynchronized. The mean firing rates for each of the neurons, in all the 7 groups so classified, during different states are shown in Figure 2. About 62% (52 out of the 84) of the recorded neurons showed significantly increased firing rates (Fig. 2A and B) during AW (p<0.01, F 1,102 =44.81) and during REM sleep (p <0.01, F 1,102 =40.67). The mean firing rates of this group of neurons during AW and REM sleep were 21.1 ± 2.1 spikes/sec and 21.6 ± 2.3 spikes/sec, respectively, while during QW the firing rate was 5.6 ± 1.0 spikes/sec. The second largest group of neurons (15 out of 84) showed a significantly (p<0.01, F 1,28 =8.1) increased firing rate during REM sleep without being significantly affected during AW and sleep states (Figure 2E). The mean firing rates of this group of neurons were 18.4 ± 4.4 spikes/sec during REM sleep, 5.0 ± 1.8 spikes/sec during QW and 5.9 ± 2.0 spikes/sec during AW. There were 4 neurons whose firing rate significantly (p<0.01, F 1,6 =1.33) increased during AW but did not change significantly during REM sleep (Figure 2D). The mean firing rate of this group of neurons during AW, QW and REM sleep was 9.5 ± 3.5 spikes/sec, 4.8

5 UNIT FIRING DURING WAKE AND REM DESYNCHRONIZATION 136 Figure 2. The mean firing rates of all the recorded neurons during spontaneous S-W states as well as during MRF stimulation-induced desynchronization of the EEG are shown in this figure. The neurons could be classified into seven groups (details mentioned in the text). ± 2.1 spikes/sec and 5.6 ± 2.2 spikes/sec, respectively. There were 7 neurons whose firing rates were significantly lower during REM sleep (Figure 2C). Out of these, for 5 neurons the firing rate was higher (p<0.01, F 1,8 =4) during AW and significantly lower (p<0.01, F 1,8 =1.9) during REM sleep compared to QW and their mean firing rates during AW, QW and REM sleep were 14.4 ± 4.4 spikes/sec, 4.7 ± 1.3 spikes/sec and 1.76 ± 1.02 spikes/sec, respectively. The other two neurons decreased their firing rate exclusively during REM sleep (Figure 3) where the mean rate of firing was 1.6 ± 0.6 spikes/sec, 1.85 ± 0.7 spikes/sec and 0.6 ± 0.02 spikes/sec during AW, QW and REM sleep, respectively. These were possibly norepinephrinergic REM-OFF neurons, since their spike shape and duration followed the criteria reported earlier for such neurons (Koyama et al., 1994) and they were recorded from TH-positive sites (when confirmed histologically). For 4 neurons the firing rate during REM sleep was significantly higher (p<0.01, F 1,4 =1.76) while during AW the rate was significantly lower (p<0.01, F 1,4 =14.5) (Fig. 2F). The mean firing rates of this group of neurons during REM sleep, QW and AW were 3.4 ± 1.9 spikes/sec, 0.9 ± 0.15 spikes/sec and 0.2 ± 0.1 spikes/sec, respectively. These neurons may possibly be cholinergic or non-cholinergic REM-ON neurons because the shape and duration of spikes from these neurons followed those of the putative cholinergic neurons as reported by others (El Mansari et al., 1989; Kayama et al., 1992; Sakai and Koyama, 1996). There were three neurons whose firing rates remained unaffected during AW and REM sleep as compared to QW when the mean firing rates were 8.7 ± 1.8, 10.2 ± 8.3 and 9.8 ± 8.0 spikes/sec, respectively. Of these three neurons, one showed increased firing (p<0.01, F 1,2 =30.5), the second decreased firing (p<0.01, F 1,6 =1.49) and the third remained unaffected during sleep compared to QW, when the firing rates were 0.24 ± 0.16, 3.58 ± 0.58 and ± 2.5 spikes/sec, respectively (Figure 2G). Behavior of neurons (classified above) to high frequency (100 Hz) MRF stimulation-induced desynchronization of the EEG 100 Hz stimulation of the MRF induced long-lasting EEG desynchronization and behavioral arousal during and after cessation of the stimulation. The stimulation strength was

6 137 MALLICK ET AL. adjusted such that although the animal was awake there was no significant active movement other than raising head and being attentive. There was no sign of discomfort, fright or any other unusual behavioral response. Effect on group of neurons classified on the basis of their relationship to spontaneous AW only As shown in Table 3, out of the 61 neurons which showed increased firing during spontaneous wakefulness, 35 (57%) showed increased activity during MRF stimulation-induced wakefulness while the other 26 remained unaffected. Two out of the 3 neurons which were inhibited during spontaneous AW were inhibited on MRF stimulation-induced desynchronization while the other remained unaffected and 19 out of 20 neurons which remained unaffected during spontaneous AW remained unaffected on MRF stimulation-induced desynchronization and wakefulness. Effect on group of neurons classified on the basis of their relationship to spontaneous REM sleep only Out of the 70 neurons whose firing rate increased during spontaneous REM sleep, 38 (54%) remained unaffected, 29 (41%) increased while 3 decreased their firing during stimulation-induced desynchronization. Five of the 7 neurons which were inhibited during spontaneous REM sleep showed increased firing during stimulation-induced desynchronization, while 6 of the 7 neurons which remained unaffected during spontaneous REM sleep showed a similar behavior during stimulation-induced desynchronization (Table 4). Effects of MRF stimulation-induced EEG desynchronization on groups of neurons classified based on their firing rate during both spontaneous AW and REM sleep The effect of MRF stimulation-induced EEG desynchronization was studied on 84 neurons and each of their firing rates have been shown in Figure 2. There were 61 neurons which showed increased firing rates during Table 3 Comparative Rates of Neuronal Discharge-Wakefulness 100 Hz Relation to spontaneous AW MRF Increase No Change Decrease Stimulation (61) (20) (3) Increase No Change Decrease Table 3. The firing rates of the brain stem neurons during spontaneous active wakefulness (AW) were statistically compared to respective quiet wakefulness state and the number of neurons which showed significantly increased, decreased or no change in their firing rates are shown in parentheses. The responses (increase, decrease or no change in their firing rates) of number of neurons, in each of those groups, during EEG desynchronization induced by 100Hz stimulation of the midbrain reticular formation (MRF) are shown in this table. Figure 3. Simultaneous recording of EEG, EOG, EMG and spontaneous activity from one REM-OFF neuron during active awake, sleep and REM sleep are shown in this figure. The spontaneous unit activity in the upper trace is shown as rate histogram (SPIKE 2). Table 4 REM Sleep 100 Hz Relation to spontaneous REM Sleep MRF Increase No Change Decrease Stimulation (70) (7) (7) Increase No Change Decrease Table 4. The firing rates of the brain stem neurons during spontaneous rapid eye movement (REM) sleep were statistically compared to that of respective quiet wakefulness state and the number of neurons which showed significantly increased, decreased and no change in their firing rates are shown in parentheses. The responses (increase, decrease or no change in their firing rates) of number neurons, in each of those groups, during EEG desynchronization induced by 100Hz stimulation of the midbrain reticular formation (MRF) are shown in this table.

7 UNIT FIRING DURING WAKE AND REM DESYNCHRONIZATION 138 Figure 4. Simultaneous recording of EEG, EOG, EMG, PGO and one neuronal activity during spontaneous S-W states and after MRF stimulation are shown in this figure. This is an example of a neuron whose firing rate increased during AW and REM sleep. The neuronal activity increased with high frequency stimulation of the MRF which induced EEG desynchronization. The unit activity is 1:5 processed output from the pulse counter. Figure 5. A REM-OFF neuronal activity is shown in this figure. The S-W states are identified by corresponding changes in EEG, EOG and EMG. The unit activity is 1:5 processed output from the pulse counter. The neuronal activity increased firing during waking, reduced during sleep, ceased during REM sleep. The same neuron increased its firing during MRF stimulation-induced desynchronization of the EEG. The stimulationinduced behavior was similar to that of wakefulness and not that of REM sleep while the EEG was desynchronized during both instances. Table 5. The numbers in parentheses show the brain stem neurons showing statistically significant increase, decrease or no change in their firing rates considering both AW and REM sleep (when the EEG remained desynchronized). The responses of those neurons to MRF stimulation induced EEG desynchronization are shown in this table. Abbreviations: S-W-REM sleep: Sleep-wakefulness-rapid eye movement sleep; AW: Active awake; REMS: Rapid eye movement sleep; Stim: Stimulation; MRF: Midbrain Reticular Formation. Meaning of arrows is same as in Fig 2.

8 139 MALLICK ET AL. Figure 6. A representative neuronal behavior along with simultaneous changes in S-W cycle are shown in the upper four traces in each set. The last trace in each set shows the neuronal firing rate during and following MRF stimulation-induced EEG desynchronization. The unit activity is 1:5 processed output from the pulse counter. The firing rate increased during REM sleep. The neuron remained unaffected during stimulation-induced EEG desynchronization in line with that during spontaneous wakefulness. Figure 7. This figure shows simultaneous recording of EEG, EOG, EMG, PGO waves and pulse counter output (1:5) corresponding to neuronal activity during spontaneous behavior as well as during MRF stimulation-induced EEG desynchronization. This is a representative neuron whose firing rate increased during REM sleep only and was not active during wakefulness (REM-ON neuron). The firing rate of this neuron remained unaffected during stimulation-induced desynchronization in line with spontaneous waking behavior. Table 6. The brain stem neurons were classified based on changes in their firing rates corresponding to EEG desynchronization during spontaneous AW, REM sleep and induced by 100 Hz stimulation of the MRF. The numbers in parentheses show the number of neurons in each group so classified. The number of neurons, from each of such classified groups, which showed excitation, inhibition or no change to 1Hz MRF stimulation (when the EEG was not desynchronized) are shown in this table. Abbreviations and signs are same as in Table 5.

9 UNIT FIRING DURING WAKE AND REM DESYNCHRONIZATION 140 Table 7 Comparative Effect of 100 Hz and 1 Hz MRF Stimulation 1 Hz 100 Hz Stimulation Stimulation Increase No Change Decrease (23) (14) (-) Excitation No Change Inhibition Table 7. A comparative response of the same brain stem neuron to 100Hz and 1Hz stimulation of the MRF are shown here. The former induced desynchronization of the EEG while the latter did not. The number of neurons which showed an increase, a decrease or no change in their firing rates during 100 Hz stimulation of the MRF are shown in the parentheses. Number of neurons, out of those classified neurons, which were excited, inhibited or remained unaffected (no change) by 1Hz stimulation are shown in this table. Abbreviations as in Fig. 5. Figure 8. Simultaneous recording of one REM-OFF and one REM- ON neuron during spontaneous S-W cycle and MRF stimulationinduced EEG desynchronization is shown in this figure. The window discriminator output (1:5) corresponding to the neuronal firing along with simultaneous changes in the EEG, EOG, EMG and PGO waves were recorded on different channels of a polygraph. The REM-OFF neuron showed increased firing rate, while the REM-ON neuron did not show any firing during spontaneous wakefulness when the EEG was desynchronized. These neurons behaved similarly during MRF stimulation-induced desynchronization of EEG also. spontaneous EEG desynchronization irrespective of their behavior during spontaneous REM sleep (when EEG was also desynchronized). A total of 37 of these 61 neurons showed a significantly increased (p<0.01) firing rate during EEG desychronization induced by MRF stimulation (Table 5). Twenty-nine out of 52 neurons (55%), which showed increased firing rates during spontaneous AW and REM sleep, showed increased firing rates during MRF stimulation-induced desynchronization (Figure 4). All the neurons showing increased firing during AW and reduced/ceased firing during REM sleep (the REM-OFF neurons) showed a significant increase in their firing rate during MRF stimulation-induced wakefulness and EEG desynchronization (Figure 5). Out of the 4 neurons which showed increased firing during spontaneous AW without being affected during REM sleep, 3 increased while one decreased firing during MRF stimulation-induced desynchronization (Figure 2D). However, out of the three which increased, only one was statistically significant while the other two remained insignificant, possibly because of high standard deviation. There were 17 neurons whose firing rates were affected during spontaneous REM sleep but remained unaffected during spontaneous AW (EEG was desynchronized during both the behaviors). Fifteen of them showed increased, while two showed decreased, firing rates during REM sleep. All except one of these neurons remained unaffected during MRF stimulation-induced EEG desynchronization (Figure 6). Out of three classical REM-ON neurons, where the firing rate decreased during spontaneous AW and increased during spontaneous REM sleep, none showed any firing during or following stimulation-induced desychronization (Figure 7). Two neurons, one REM-ON and the other REM-OFF, were recorded simultaneously. The former increased while the latter decreased firing during spontaneous REM sleep. The firing rate of the REM-OFF neuron increased while the REM-ON neuron was not affected by MRF stimulation-induced desynchronization (Figure 8). The interspike interval histograms (generated by SPIKE2, CED) of these two neurons are shown in Figure 9. All the three neurons which remained unaffected during spontaneous AW and REM sleep remained unaffected during MRF stimulation-induced desynchronization of the EEG (Figure 2G). Effect of 1 Hz stimulation of MRF on the brain stem neurons Stimulation of MRF with 1 Hz did not affect EEG synchronization or desynchronization. Such stimulation showed that MRF may influence neuronal activity irrespective of induced alteration in the EEG. The effect was studied on 37 neurons (Table 6). Out of the 32 neurons which increased firing during spontaneous AW, irrespective of their behavior during REM sleep, 17 were excited, 14 remained unaffected while only one was inhibited by 1 Hz MRF stimulation. A majority

10 141 MALLICK ET AL. A. ACTIVE AWAKE REM SLEEP B. Time second Time second PRE-STIMULATION POST-STIMULATION Time second Time second ACTIVE AWAKE REM SLEEP Time second Time second PRE-STIMULATION POST-STIMULATION Time second Time second Figure 9. Interspike interval histograms (ISIHs) of one each REM-OFF (A) and REM-ON (B) neurons (same neuron as in Figure 8) before and after MRF stimulation-induced desynchronization of the EEG are shown here. It can be seen that the REM-OFF neuron which normally showed increased firing during spontaneous wakefulness also showed increased firing when EEG desynchronization was induced by high frequency stimulation of the MRF. Similarly, the REM-ON neuron which increased firing during rapid eye movement sleep only but did not fire during spontaneous wakefulness, did not show any activity during MRF stimulation-induced EEG desynchronization. Bin width for histograms:10 ms.

11 UNIT FIRING DURING WAKE AND REM DESYNCHRONIZATION 142 A. Stim B. Stim Figure 10. Ten stimulus-bound overlapped responses of 2 brain stem neurons to 1 Hz stimulation of MRF are shown in this figure. The neuron in trace A was excited. The neuron showed increased firing during spontaneous wakefulness and also during REM sleep as well as MRF stimulation-induced EEG desynchronization. Neuron in trace B showed an inhibitory response. This neuron decreased its firing during spontaneous wakefulness and MRF stimulation-induced EEG desynchronization while showing increased firing during spontaneous REM sleep. (17 out of 23) of the neurons which increased firing by high frequency stimulation of the MRF were excited by 1 Hz stimulation (Table 7) with a latency of 5-10 msec (Figure 10A). Two of the 4 neurons which decreased firing during spontaneous AW, increased during spontaneous REM sleep and were not affected by 100 Hz stimulation, decreased (Figure 10B) while the other two remained unaffected by 1 Hz MRF stimulation. Classification of neurons into tonic and phasic types and their responses to MRF stimulation Depending on spontaneous firing pattern, the recorded neurons could be grouped into tonic (N=63) and phasic (N=21) types as reported earlier (El Mansari et al., 1989 and Steriade et al., 1990) (Figure 11). A majority (N=48) of the tonic-type neurons showed a tonic increase in discharge rate during spontaneous AW (p<0.01, F 1,94 =26.97) and during REM sleep (p<0.01, F 1,94 =23.43) (these neurons would fall within the group shown in Fig 2A and B), a characteristic of tonic-type I and 12 neurons showed tonic increase (p<0.01, F 1,22 =1.45) of firing from QW to sleep and highest during REM sleep (neurons within the group shown in Fig 2E and F), a characteristic of tonic-type II. Out of the 21 phasic-type neurons, 13 showed burst firing during AW and REM sleep, while 8 showed burst firing primarily during REM sleep. On high frequency MRF stimulation-induced wakefulness and EEG desynchronization, all the 24 affected neurons (out of 48 tonic-type I) and 9 out of 13 phasic neurons (burst firing during AW and REM sleep) showed an increased firing. On the other hand, all except 3 phasic-type neurons (10 out of 12 tonic-type II and 7 out of 8 phasic type), remained unaffected by high frequency MRF stimulation-induced wakefulness and EEG desynchronization; the 3 affected neurons showed decreased firing rate. On 1 Hz MRF stimulation, when EEG was not desynchronized, 14 out of 25 tonic-type I neurons and 4 out of 7 phasic-type neurons (burst firing during AW and REM sleep) were excited. The effect of 1 Hz stimulation was studied on 2 tonic-type II and 3 phasic-type neurons that showed firing only during REM sleep. The former were inhibited while the latter showed no change. DISCUSSION In this study EEG, EOG, EMG, PGO and single neuronal activity were recorded from brain stem reticular formation in freely moving normally behaving chronically prepared cats. Based on their spontaneous firing rate during wakefulness and REM sleep, when the EEG remained desynchronized, the neurons were classified into seven groups. Although in earlier reports responses of neurons have been characterized and

12 143 MALLICK ET AL. Figure 11. Neurons were grouped into tonic I, tonic II and phasic types depending on their firing pattern during different S-W states. Mean firing rate (± S.E.M) of these groups of neurons during S-W-REM sleep and also during MRF stimulation-induced (STIM) wakefulness accompanied by desynchronization are shown in this figure. * p<0.05 and ** p<0.01 correlated with spontaneous changes in sleep, wakefulness and REM sleep, or specific correlates of REM sleep, their behaviors during spontaneous as well as induced changes in those behaviors or correlates were not studied. In order to study neuronal specificity to a correlate of REM sleep, it is desirable to relate neuronal activity with spontaneous as well as induced changes with the correlate under study. It assumes significance for correlating neuronal activity with EEG desynchronization which is present both during wakefulness as well as REM sleep. The advantage of correlating unit activity with spontaneous and induced changes to a sign is that the latter is induced in a relatively controlled condition. Since this study attempted to identify neuronal specificity to EEG desynchronization during wakefulness and REM sleep, the firing rate of the neurons during quiet wakefulness was taken as baseline because there was absence of phasic muscle activity during that phase although the EEG remained desynchronized. The specificity of the neurons to spontaneous EEG desynchronization during wakefulness was tested by studying

13 UNIT FIRING DURING WAKE AND REM DESYNCHRONIZATION and correlating their behavior during MRF stimulationinduced desynchronization of the EEG. Since the aim of this study was to investigate if the same neuron in the brain stem reticular formation may be responsible for desynchronization of the EEG during both wakefulness and REM sleep, the neuronal activities were recorded from structures lying between caudal midbrain and rostral pons because awake (Steriade and McCarley, 1990) and REM sleep-related (Siegel et al., 1984; Sakai, 1985; Mallick et al., 1989, 1994) neurons are reported to be present in these regions. The absolute firing rate of the neurons during different states varied significantly and the neuronal spike shapes were mono-, bi- or tri-phasic. The sites from where REM-OFF neurons were recorded showed TH immunopositivity (Figure 1). The REM-ON and REM-OFF neurons, as classified in this study, showed spike shape and duration as reported earlier (El Mansari et al., 1989; Kayama et al., 1992; Koyama et al., 1994; Sakai and Koyama, 1996). It was found that a majority of the recorded neurons in this area altered their firing rates whenever the EEG was desynchronized, irrespective of whether the animal was in spontaneous awake or REM sleep state. Almost 95% of the neurons showed an increased firing rate correlated to EEG desynchronization during both wakefulness and REM sleep and 61% of the neurons were common and showed an increased firing rate during both the phases. Since this area of the brain stem is attributed to EEG desynchronization (Moruzzi, 1972), it is reasonable that a majority of the neurons in this area increased firing associated with EEG desynchronization. However, their differential relationship, if any, to the two distinct behaviors wakefulness and REM sleep, when EEG remains desynchronized, was not known. The observation that a majority of the neurons increased firing during EEG desynchronization irrespective of wakefulness or REM sleep behaviors tempts one to suggest that the same neuron in the brain stem could be responsible for EEG desynchronization during both the behavioral states. However, it may be an oversimplification and will be discussed later. Earlier neurons recorded from brain stem reticular formation were classified as tonic-type I, tonic-type II and phasic-type (El Mansari et al., 1989; Steriade et al., 1990). In this study, the number of neurons in those categories (when classified using their criteria) were comparable (Figure 11) with minor differences which could be due to differences in recording area and the techniques used. It may be noted that there were more tonic-type neurons than phasic-type. The former could be related to EEG desynchronization (a tonic phenomenon) and the latter to movements, etc. (a phasic phenomenon). There were some neurons which showed opposite response in firing rate during REM sleep and wakefulness. These neurons may play a significant role in modulating behavior associated to REM sleep and wakefulness, respectively. For example, a varying degree of muscle tone is present during wakefulness, while it is absent in REM sleep. Since the objective of this study was to investigate if the same neuron may be responsible for EEG desynchronization during both the 144 behaviors (REM sleep and wakefulness), it was necessary to study the behavior of the same neuron throughout during spontaneous and stimulation-induced EEG desynchronization. Hence, after classifying them on the basis of their responses to spontaneous behavior, the neurons were studied if they maintained similar behavior during MRF stimulation-induced EEG desynchronization. In addition, since the desynchronization and effect on the units was induced by MRF stimulation, the responsiveness of the neurons to inputs from the MRF without changes in the EEG was also studied. This was achieved by overlapping 1 Hz stimulation bound responses of the units on the oscilloscope. This showed if the recorded neurons received input from the area of stimulation which functionally induced EEG desynchronization. Relationship of neuronal activity to spontaneous and induced EEG desynchronization and its physiological significance Since EEG desynchronization is a common correlate during wakefulness and REM sleep, it is possible that at least some of the neurons could be common for induction of EEG desychronization during both the behaviors. However, it was found that all except one of the neurons which remained unaffected during spontaneous EEG desynchronization accompanied by wakefulness, irrespective of their behavior during REM sleep, remained unaffected during high frequency MRF stimulation-induced EEG desynchronization (Table 5). Similarly, all except one of the REM-specific neurons (REM- ON and REM-OFF) were affected in a manner similar to that of their behavior during spontaneous wakefulness when EEG desynchronization was induced by high frequency stimulation of the MRF. The REM-ON neurons were inhibited while the REM-OFF neurons were either excited or not affected by high frequency MRF stimulation-induced desynchronization of the EEG. Thus, these groups of neurons during stimulationinduced desynchronization of the EEG behaved similarly to their behavior during spontaneous EEG desynchronization accompanied by wakefulness and not as during spontaneous REM sleep. Therefore, it is reasonable that the neurons not related to EEG desynchronization during wakefulness were unaffected; while the so called REM-OFF neurons excited (Figure 10A) and the REM-ON neurons remained inhibited (Figure 10B) on 1 Hz stimulation of the MRF. However, although a majority of the AW-related neurons were affected by 1 Hz MRF stimulation, some were not affected even though they were affected during high frequency stimulation-induced EEG desynchronization. The former could be a cause for EEG desynchronization and the latter an effect of wakefulness and desynchronization of the EEG. The latter neurons may also be related to muscle activity and movement associated to wakefulness. The influence of MRF on the REM sleep-related neurons suggest that there is possibly an interaction between the neurons related to REM sleep and the wakefulnessgenerating area in the brain stem. The excitation and inhibition observed in this study may be a direct excitatory or a disinhibitory effect or an inhibitory or a disfacilitatory effect,

14 145 MALLICK ET AL. respectively, which cannot be confirmed from this study. However, either or both the possibilities may be supported by the presence of excitatory and inhibitory neurons and inputs to this area (Sakai, 1985). One of the limitations of this study is that the neuronal responses to specific muscle movement have been compromised. We are aware of the fact that most of the brain stem neuronal activity could be correlated with some muscle movement (Siegel, 1979; Siegel and Tomaszewki, 1983) during wakefulness-associated desynchronization. In that case it is very difficult to understand and interpret the cause and effect relationship if a neuronal activity is related to movement or wakefulness or EEG desynchronization. Since these phenomena are associated and are correlates of wakefulness they are difficult to isolate behaviorally in in vivo studies. This is an inherent deficiency of behavioral studies, and may be taken care of if the response of the same neuron can be studied after induction of REM sleep in addition to the studies being reported here (preliminary data support this view [Thankachan et al., 1997]). Nevertheless, this limitation has been minimized in this study by recording the neuronal activity during comparable physical conditions, as far as possible, during spontaneous and MRF stimulation-induced wakefulness. Although a good proportion of brain stem neurons showed increased firing rates when the EEG remained desynchronized during spontaneous wakefulness as well as REM sleep, only a little over half of them showed a similar behavior during MRF stimulation-induced desynchronization (Table 5). This is probably because it is unlikely that all the neurons would be involved in desynchronization of the EEG during those behaviors. The neurons which were related to wakefulness and showed a similar behavior during MRF stimulation-induced desynchronization may be responsible for EEG desynchronization, while those which did not behave similarly to MRF stimulation-induced desynchronization of the EEG may be involved in other physiological changes during wakefulness. The neurons which increased firing during spontaneous as well as MRF stimulation-induced EEG desynchronization are likely to be involved in desynchronization of the EEG during wakefulness. The neurons which increased firing during REM sleep only, but were not affected by MRF stimulation, may be involved in EEG desynchronization during REM sleep. This may be supported by the fact that 1 Hz stimulation of MRF significantly increased the firing rate of 50% of the tonic-type and 80% of the phasic-type of neurons. The important fact to be noted is that most of the REM sleep-related neurons (either tonic or phasic category) remained unaffected by such stimulation (Figure 11). None of the studied neurons was antidromically activated by MRF stimulation. This suggests that the recorded neurons neither projected to or passed through MRF, the area of stimulation. Nevertheless, since a majority of wake-active neurons was activated by 1 Hz stimulation of MRF at a latency of 5-10 msec, it is likely that the MRF has a multisynaptic and/or unmyelinated projection to the recorded neurons. Interestingly, among the neurons affected by 1 Hz MRF stimulation, a majority of the neurons showing increased firing during AW as well as during MRF stimulation-induced desynchronization, irrespective of their behavior during REM sleep, received excitatory inputs (Table 6). These MRF inputs to wake active neurons might provide a bias to the reticular neurons for inducing EEG desynchronization during wakefulness. Thus, it is reasonable that REM-OFF neurons were not inhibited (excited), while the REM-ON neurons were either inhibited or remained unaffected by 1 Hz stimulation of the MRF (Table 6). This view may be supported by previous reports (El Mansari et al., 1989; Steriade et al., 1990) that some of the tonic as well as phasic neurons project to the thalamus and might be responsible for EEG desynchronization, possibly during wakefulness. Although thalamic projection of the recorded neurons has not been investigated in this study, it may be said that since the proportion of tonic and phasic neurons are comparable to earlier studies, the tonic neurons in this study are likely to project through the thalamus for modulation of the EEG as hypothesized in those studies. It may also be argued that those neurons which increased firing during REM sleep but were not affected by MRF stimulation-induced desynchronization of the EEG may be related to muscle atonia during REM sleep. Some of these neurons may be responsible for inducing atonia and others for inducing desynchronization of the EEG during REM sleep. The latter view may be supported by earlier reports that depending on the anatomical location of the neurons, some may be involved in atonia and others in EEG desynchronization during REM sleep (Sakai, 1980, 1985). These neurons may be cholinergic as well as non-cholinergic (Sakai and Koyama, 1996). Also, it has been shown that neurons having non-nmda receptors are related to atonia while those with NMDA receptors are not (Lai and Siegel, 1991). Therefore, it may be suggested that the latter neurons may be involved in EEG desynchronization during REM sleep. The neurons which increased firing during wakefulness as well as REM sleep may be involved in common behavior during wakefulness and REM sleep, e.g., eye movement. Since it has been found in this study that most of the neurons during MRF stimulation-induced desynchronization behaved in a manner similar to that of spontaneous desynchronization associated to wakefulness irrespective of their behavior during REM sleep, it suggests that separate groups of neurons are probably involved in EEG desynchronization during wakefulness and REM sleep. However, the observation that neurons showing increased firing during REM sleep and AW and also receiving inputs from MRF may suggest that at least some neurons may be common for EEG desynchronization during REM sleep and wakefulness. This is understandable in view of the complexity of brain function and its redundancy hypothesis. Thus, although the possibility of separate groups of neurons in the brain stem for EEG desynchronization during wakefulness and REM sleep may be suggested, it needs to be confirmed. The effect of MRF stimulation has been investigated in this study; however, the role of induced REM