- Email: [email protected]
Hyperpolarizing membrane responses induced in lumbar motoneurons by stimulation of the nucleus reticularis pontis oralis during active sleep
Brain Research, 248 (1982) 267-273 Elsevier Biomedical Press
267
Hyperpolarizing Membrane Responses Induced in Lumbar Motoneurons by Stimulation of the Nucleus Reticularis Pontis Oralis During Active Sleep SIMON J. FUNG, PETER A. BOXER, FRANCISCO R. MORALES and MICHAEL H. CHASE* Brain Research Institute and Departments of Physiology and Anatomy, University of California, Los Angeles, CA 90024 (U.S.A.)
(Accepted March llth, 1982) Key words: intracellular recording - - lumbar motoneurons - - motoneurons - - reticular formation - nucleus reticularis pontis oralis - - sleep - - active sleep
Intracellular recordings were obtained from lumbar motoneurons in unanesthetized, undrugged, normally respiring cats during the states of wakefulness, quiet sleep and active sleep. The objective was to examine the state-dependent control of spinal cord motoneurons exerted by the pontomesencephalic reticular formation. Accordingly, electrical stimulation was applied to the nucleus reticularis pontis oralis while the membrane potential of lumbar motoneurons was recorded. Short latency depolarizing and/or hyperpolarizing potentials were observed throughout sleep and wakefulness; no state-dependent pattern was found in the direction of polarization or amplitude for these early potentials. However, a long latency hyperpolarizing potential emerged exclusively during active sleep; it was characterized by a peak latency of 45 ± 1 (S.E.M.) ms, a duration of 40 4- 2 ms, and an amplitude of 3 0.5 mV. This active sleep-selective potential was capable of inhibiting spontaneous motoneuron activity. These and previously obtained data support the notion that excitation of the nucleus reticularis pontis oralis results in somatomotor inhibition at the level of the spinal cord and brainstem selectively during the state of active sleep. INTRODUCTION T h e b r a i n s t e m reticular f o r m a t i o n has been implic a t e d in a v a r i e t y o f b e h a v i o r a l processes, including m o t o r c o n t r o l a n d the r e g u l a t i o n o f sleep a n d w a k e fulness 2s. I n the context o f m o t o r c o n t r o l m e c h a nisms, i n d i v i d u a l reticular areas exhibit f u n c t i o n a l specificity22. In p a r t i c u l a r , the nucleus reticularis p o n t i s oralis ( N P O ) has b e e n shown, b y lesion 2 a n d p h a r m a c o l o g i c a l 29,3z studies, to p l a y a n i m p o r t a n t role in the b e h a v i o r a l ( m o t o r ) expression o f active sleep (AS). S p o n t a n e o u s discharge o f N P O cells occurs in c o n j u n c t i o n with a decrease in muscle t o n e 14. F u r t h e r m o r e , n e u r o n a l d e s t r u c t i o n within a n d in the vicinity o f the N P O diminishes o r abolishes the a t o n i a o f A S 13. R e c e n t studies have f u r t h e r d e m o n s t r a t e d t h a t N P O s t i m u l a t i o n p r o d u c e s facilitation
o f the masseteric reflex a n d a d e p o l a r i z i n g p o t e n t i a l in t r i g e m i n a l j a w - c l o s e r m o t o n e u r o n s d u r i n g wakefulness (W) a n d quiet sleep (QS), while the identical stimulus d u r i n g A S induces reflex i n h i b i t i o n a n d a c o r r e s p o n d i n g h y p e r p o l a r i z i n g potential4,L T h e current s t u d y was designed to examine the effects o f N P O s t i m u l a t i o n on the m e m b r a n e p o t e n t i a l o f ~m o t o n e u r o n s at the level o f the spinal c o r d d u r i n g sleep a n d wakefulness. P r e l i m i n a r y d a t a have been r e p o r t e d elsewhere6,10. METHODS E x p e r i m e n t s were p e r f o r m e d on 3 unanesthetized, u n d r u g g e d , n o r m a l l y respiring cats d u r i n g W , QS a n d AS. In o r d e r to c a r r y o u t these studies, which r e q u i r e d s i m u l t a n e o u s e x p l o r a t i o n s within the
* To whom correspondence should be addressed at: Department of Physiology, Center for the Health Sciences, University of California, Los Angeles, CA 90024, U.S.A. 0006-8993/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press
268 brainstem (site of stimulation) and spinal cord (site of intracellular recording), two separate, interdependent surgical procedures were undertaken while the animals were anesthetized with sodium pentobarbital (40 mg/kg, i.p.). The procedures for preparing the head region in the chronic cat are described in detail in Chase et al. 7. Specific procedures for intracellular recording from spinal cord motoneurons in the cat are presented in Morales et al. ~1. Consequently, in this report, only an overview of the key elements of the preceding methodologies are presented. Chronic recording screw electrodes were placed in the calvarium to monitor E E G activity. Bipolar strut electrodes were implanted stereotaxically (A6, L10.5, H3) to record pontogeniculate-occipital (PGO) waves. These electrodes were connected to a female Winchester plug which was bonded to the calvarium with acrylic resin. A head-restraining device was implanted for fixing the head of the cat in a standard 3-dimensional stereotaxic position without pain or discomfort. The area of the calvarium overlying the region for electrical stimulation was left uncovered for later trephination before the initial experimental session. Next, the spinal cord was prepared for intracellular recording in the chronic preparation. Hard plastic clamps were mounted around L3, L4 and L6 vertebrae. Stainless steel screws were put in the articular processes and transverse steel bars were placed next to the vertebral clamps. Mechanical immobilization of the vertebral column was achieved by binding the screws, clamps and bars together with acrylic resin; a circle (4 mm in diameter) in the lamina of L5 was left free of acrylic resin in order to permit microelectrode exploration of the underlying spinal cord. All wound margins were sutured and postoperative care was administered to avoid infection. Prior to the initial experimental session each animal was anesthetized with 4 ~ halothane. Chronic stimulating cuff electrodes were placed around the tibial and/or sciatic nerves. A small hole was drilled in the calvarium for brain stimulation and another in the lamina of L5 for microelectrode penetration; both holes were ipsilateral to the nerve electrode. The dura mater was cut to expose the dorsal root entry zone of L7.
Data were collected for 3 consecutive days on each animal over a period of 6-8 weeks. During each session, the animal was placed in the stereotaxic apparatus so that its head and spinal cord were returned to the original orientation assumed during the preceding surgical procedures. Pin electrodes were placed in the skin at the lateral margins of each eye and in the nuchal musculature to record EOG and E M G activity, respectively. A male connector was attached to the female plug to monitor EEG and PGO activity. A bipolar strut electrode (0.5 mm tip exposure, 1 mm interpolar distance) was placed stereotaxically (A1 to P1, L2 to 3, H - - 2 to --4) to stimulate the region of the NPO (2-4 pulses, 0.5 ms, 800 cps, 0.8-6 V). Current intensities were determined by recording the voltage drop across a 100 kf~ resistor placed in series with the stimulation circuitry. Stimulus strength ranged from 7 to 70 uA. Glass micropipettes filled with 2 M potassium citrate (6-15 Mf~ resistance) were employed to record intracellularly from motoneurons. A reference Ag-AgC1 electrode was placed subcutaneously lateral to the vertebral column. Motoneurons were identified by antidromic activation via the nerve stimulating electrode. At the conclusion of the experimental sessions, the animal was anesthetized with sodium pentobarbital and reference points in the brainstem were made at APO, L3, H - - 3 and at APO, L3, H - - 6 by delivering anodal current of 100 #A, which was passed through a stainless steel microelectrode for 30 s. The animal was sacrificed with an overdose of sodium pentobarbital and perfused with 1 0 ~ formalin and 2 ~ potassium ferrocyanide (Prussian blue reaction). Frozen transverse sections (50 #m in thickness) were cut and stained with thionin. Stimulation sites in the NPO were reconstructed according to their stereotaxic coordinates, based upon the location of the two reference points. RESULTS Data were collected following stimulation of 12 sites which were histologically verified to be located within the core of the NPO, according to Snider and Niemer a°. Based upon a maximal intensity of 70 #A that was employed, the zone of excitation can be considered to be limited. This conclusion is further
269
A
WAKEFULNESS INDIVIDUAL
AVERAGED POTENTIALS
N: 8
POTENTIALS
2 '&,,u,..,~
EOG * ' * " - - - - " - ~ ~ E M G ....... . - . . . . . . . . . . . . . . . . . . . . . . ~ll
B
QUIETSLEEP .1i J..,,khJ . J . . . |
_J..,.J,._L.,,[I.,J, ~,,IL
I
,.,, , , . r , T ,
~ /-L/
,.
C
z
~
3 'k,+.~-,,.
ACTIVESLEEP
,
tlo0~v 2 sec
4 ?v~, 20 msec
I ZmV
20 msec
Fig. 1. Nucleus reticularis pontis oralis (NPO)-induced motoneuron responses throughout the sleep-waking cycle. Left column: polygraphic recordings of EEG, EOG and EMG activity during wakefulness (A), quiet sleep (B), and active sleep (C). Middle column: averaged traces of eight consecutive samples of NPO-induced membrane responses. Note the presence of active sleepspecific hyperpolarizing potentials in C1-4. Right column: individual samples selected from the eight employed in generating the averaged potential. Sciatic motoneuron: antidromic spike potential = 69 mV, NPO stimulation: 3 pulses, 0.5 ms, 800 cps. substantiated by the observation that N P O stimulation did not induce muscle contractions of the neck musculature. Hence the stimulus did not spread to neighboring structures, such as the tectospinal tract, which lies immediately adjacent to the NPO. We cannot, however, exclude the possible coexcitation of descending fiber tracts coursing through the NPO. Measurements of intracellular potentials were performed in 24 motoneurons; the mean antidromic spike amplitude was 69.6 zk 11.2 mV (range = 55-98 mV). For data analysis, 8-32 consecutive NPO-induced potentials were routinely averaged. Data during AS were not analyzed during those
periods of prominent subthreshold membrane activities which occur in conjunction with rapid eye movement episodesL The general characteristics of the motoneuronal response to N P O stimulation during W, QS and AS are illustrated in Fig. 1. During these three states, reticular stimulation induced an early synaptic response at latencies of 2-15 ms; in some motoneurons they were depolarizing, and in others hyperpolarizing (Figs. 1-3). The amplitude of this potential was relatively constant across states or varied in a nonsystematic fashion. The initial potential induced by N P O stimulation was therefore nonstate-dependent; consequently, no specific analysis
270 of its characteristics was performed. At a latency greater than that of the early potential, no distinct additional evoked activity was registered during W or QS (Figs. 1A, B). In contrast, at the intensity of NPO stimulation that was employed, a large amplitude hyperpolarizing potential emerged during AS in 6 of 8 motoneurons showing state transitions (Fig. 1C). This potential arose during the transition period from QS to AS in conjunction with the development of PGO waves and reduction in muscle tone. It was present for the duration of AS and disappeared abruptly when the animal awoke. In 7 of 8 other motoneurons examined only during AS, a similar hyperpolarizing response was obtained. The remaining 8 motoneurons tested in QS alone did not exhibit a late hyperpolarizing potential; only the early response was present, Averages from 8-32 consecutive trials were used to analyze the NPO-induced hyperpolarizing potential of AS according to the schema shown in Fig. 2A. A determination of its latency and duration could not be obtained in all cells since the initial short latency potential sometimes obscured the onset of the succeeding AS-specific hyperpolarizing potential; in addition, its return to baseline was not
_~,,~_f~.~_~i,~y__T_~Z~-~_ _ DURATION ~ L A T E N C Y -----~ - . . . . . . . ~ ...........
• ~
. -T2
7~-=~'---~-~-
A. TRANSITION (QUIET SLEEP---> ACTIVE SLEEP)
25 mV
0.5 sec
B. QUIET SLEEP
C. ACTIVE SLEEP
I
' 5mV
I 20 msec
Fig. 3. Single traces of postsynaptic responses following nucleus reticularis pontis oralis (NPO) stimulation at the transition from quiet sleep to active sleep (A1 and A2) and during quiet sleep (B) and active sleep (C). The active sleepspecific hyperpolarizing response inhibited spontaneous motoneuron discharge as indicated in A1 and A2. The NPOinduced synaptic response is portrayed in the bottom portion of A at a faster sweep speed. Tibial motoneuron : antidromic spike potential = 73 mV; NPO stimulation: 3 pulses, 0.5 ms, 800 cps.
PEAK
AMPLITUDE
/
/ 20 msec
• LATENCY TO P
LATENCY
E
A
K
LATENCYTO PEAK (msec)
~
PEAK AMPLITUDE (mV)
DURATION HALF'WIDTH (msec)
(msec)
2ZO
44.9
5.I
40.1
24.1
S.E.M.
±1.7
±1.2
±0.5
±2.1
±1.5
RANGE
t9.3 - 3 8 . 3
58.0-53.0
29.7-46.6
14Z-36.5
(msec)
1.0 - 6 7
Fig. 2. Averaged record (A) of 8 consecutive nucleus reticularis pontis oralis (NPO)-induced membrane responses during active sleep. A quantitative determination of the active sleepspecific NPO-induced hyperpolarizing potential according to the diagram in A is presented in B. Details in text. In A, tibial motoneuron: antidromic spike potential = 79 mV; NPO stimulation: 4 pulses, 0.5 ms, 800 cps.
always dearly defined. For those cells that could be subjected to analysis, a baseline was first established prior to the artifacts of the stimulating pulse train. The peak amplitude of the hyperpolarizing potential was 3.1 4- 0.5 mV (X :k S.E.M.) (n = 11) (Fig. 2B). Its latency from the initiation of the reticular stimuli was 27.0 4- 1.7 ms (n = 10) (Fig. 2B). The latency to peak was 44.9 4- 1.2 ms (n = 11) (Fig. 2B) and its duration was 40.1 4- 2.1 ms (n = 7) (Fig. 2B). The half-width (the duration at half of the peak amplitude) was found to be 24. l 4- 1.5 ms (n = 10) (Fig. 2B). To summarize, the NPO-induced AS-specific response in lumbar motoneurons was typified by a 3 mV hyperpolarizing potential with a long latency to peak (45 ms) and a slow time course (half-width = 24 ms). The relation of the hyperpolarizing potential to spontaneous motoneuron activity is shown in Fig. 3.
271 ACTIVE
A.
EMG
-'" .
.
.
SLEEP
.
I
1
o. i n t r a c e l l u l a r potential
B.
| ..L. L ~ . ~ . . . If T "~" ~ - "
'. . . . . . . . . .
,k "
I
1 ~O0,~V
2 s--ec
]
b. ~ % ~ 2mV 2 0 msec
Fig. 4. Abolition of the hyperpolarizing potential concomitant with the occurrence of muscle activity and rapid eye movements during active sleep. Intracellular potential traces in a and b were averaged signals from four consecutive samples of nucleus reticularis pontis oralis (NPO)-induced responses during the periods marked beneath EMG traces in A and B. Stimuli to the NPO produced a hyperpolarizing potential of approximately 2 mV when atonia was present during active sleep (a). The active sleep-specific hyperpolarizing response disappeared (b) in conjunction with the occurfence of a brief episode of neck muscle activity (B), only to reappear once again at the end of the episode. Sciatic motoneuron: antidromic spike potential = 76 mV; NPO stimulation: 3 pulses, 0.5 ms, 800 cps.
In the example which is presented (Fig. 3A), during a postural readjustment just prior to AS, the motoneuron discharged spontaneously for a finite period (it ceased discharge once the AS state had been fully established). The NPO-induced hyperpolarizing potential arose during the transition from QS to AS while the motoneuron was still discharging (Fig. 3A). Based on the pattern of rhythmic activity just prior to NPO stimulation, it was evident that spontaneous action potentials were completely suppressed in the time slot corresponding to the period of the long latency hyperpolarizing potential (Fig. 3AI, 3A2). Muscle tone during AS is not always abolished; there are brief periods of partial resumption of EMG activity and/or muscle twitches. During these episodes of EMG activity, the NPO-induced synaptic potential was no longer present (Fig. 4B). The hyperpolarizing potential returned only after EMG activity had once again disappeared (Fig. 4A). DISCUSSION
The principal finding of the present study is that stimulation of the NPO induces a long latency hyperpolarizing potential in lumbar motoneurons exclusively during AS. This observation could not
have been made by previous investigators, since all other comparable experiments have been performed on acutely anesthetized or decerebrate animals. In these acute studies, however, a short latency potential was found whose characteristics were comparable to those which we observed; therefore, this potential will be discussed prior to an examination of the state-selective long latency potential. In acute cats, descending reticulofugal impulses originating from the medial pontomeduUary reticular formationa,15,28,26,27 and the medial longitudinal fasciculus of the medulla11,12,a4 impinge on lumbar motoneurons at a relatively short latency ranging from 2.5 (ref. 23) to 15 ms (ref. 26).Membrane responses to these descending volleys include both excitatory and inhibitory postsynaptic potentials. The early synaptic response which we observed exhibited a comparable latency and configuration and thus we can corroborate the intracellular findings which have revealed reticulospinal projections to lumbar motoneurons in acute cats. An anatomical basis for these electrophysiological findings encompasses a reticulospinal link between the NPO and the lumbar cord, which has been demonstrated in the cat by retrograde chromatolysis a2 and fiber degeneration 24, as well as by retrograde horseradish peroxidase transport 1. The early potential did not vary in configuration and exhibited only minor fluctuations in amplitude as a function of the animal's state; across neurons, no consistent pattern was observed for either hyperpolarizing or depolarizing activity. The presence of this early potential throughout the sleep-waking continuum indicates that a high safety factor is involved in its transmission and that it is not involved in sleep-specific functions. In contrast to the non-state-selective presence of the early potential, the succeeding potential was always hyperpolarizing and appeared exclusively during AS. Previous studies have demonstrated a state-dependent effect of NPO stimulation on the masseteric reflex and trigeminal motoneurons in the chronic cat (for review, see ref. 5). During AS, stimulation led to masseteric reflex suppression at conditioning-test intervals of 15-25 ms; at a similar peak latency a hyperpolarizing potential was observed in masseter motoneurons when intracellular recordings were obtained 4.
272 A comparison of the results of the brainstem and spinal cord experiments indicates that a hyperpolarizing potential arises in both lumbar (Fig. 2A) and trigeminal motoneurons exclusively during AS following excitation of the NPO 4. This hyperpolarizing potential of NPO origin inhibits spontaneous discharge in spinal motoneurons (Fig. 3) and synaptically induced spike potentials in trigeminal motoneurons 4. These data support the notion that stimulation of the NPO results in state-dependent somatomotor inhibition within the spinal cord as well as at the level of the brainstem. At both neuraxial levels the potential has a slow rise time, slow decay and prolonged half-width, which may be attributed to the temporal dispersion of impulses impinging onto the motoneuron membrane. It is unlikely that an asynchronous arrival of spike potentials could be explained solely by the heterogeneous conduction velocities of reticulospinal fibersaS; rather, the average duration of the hyperpolarizing potential (circa 40 ms) is suggestive of sustained inhibitory synaptic bombardment, probably via neuronal populations recruited by the NPO. Our present observations concerning the AS-specific hyperpolarizing potential favor the interpretation that the NPO stimulation promotes an inhibitory postsynaptic response in spinal motoneurons indirectly via an inhibitory relay. A possible relay site for the NPO action during AS is the medullary inhibitory area described by Magoun and Rhines 19, stimulation of which produces motor inhibition and diminished muscle tone in acute experiments. In decerebrate cats, several studies report inhibitory postsynaptic potentials in both flexor and extensor hindlimb motoneurons followREFERENCES 1 Basbaum, A. L. and Fields, H. L., The origin of descending pathways in the dorsolateral funiculus of the spinal cord of the cat and rat: further studies on the anatomy of pain modulation, J. comp. neuroL, 187 (1979) 513-532. 2 Carli, G. and Zanchetti, A., A study of pontine lesions suppressing deep sleep in the cat, Arch. itaL BioL, 103 (1965) 751-788. 3 Chan, S. H. H. and Barnes, C. D., Postsynaptic effects evoked from brain stem reticular formation in lumbar cord and their temporal correlation with a presynaptic mechanism, Arch. ital. BioL, 112 (1974) 81-97. 4 Chandler, S. H., Nakamura, Y. and Chase, M. H., Intra-
ing electrical stimulation of this area 15,17,1s. Further evidence of a medullary relay derives from the recent finding of tonic membrane depolarization of gigantocellular reticular neurons that occurs selectively during AS s and an AS-specific increase in the unit activity of bulbar magnocellular reticular neurons 16. In addition, there are both anatomical 25 and physiological z° links between the medullary and pontomesencephalic reticular regions. Activation of the medullary area would then generate suppression of motor activity by activation of a population of inhibitory interneurons. What might we conclude from our finding that hyperpolarizing potentials in motoneurons are induced by NPO stimulation specifically during AS? A parsimonious explanation would be that this area, situated within the core of the reticular formation, would be subjected to excitatory input during AS, as it is throughout the waking state. We propose, in order to maintain the integrity of the AS state by preventing the motor expression of this activity, that it is converted to an inhibitory drive by intrareticular processes. To summarize, there is a striking AS-selective action on motoneurons resulting from stimulation of the NPO. Thus, it is reasonable to expect that the locus of the NPO participates in or is responsible for certain of the processes underlying somatomotor atonia during AS. ACKNOWLEDGEMENTS This work was supported by grants from the National Science Foundation (BNS 790-12897) and the U.S. Public Health Service (NS-09999). cellular analysis of synaptic potentials induced in trigeminal jaw-closer motoneurons by pontomesencephalicreticular stimulation during sleep and wakefulness, J. Neurophysiol., 44 (1980) 372-382. Chase, M. H., The motor functions of the reticular formation are multifaceted and state-determined. In J. A. Hobson, and M. A. B. Brazier (Eds.), The Reticular Formation Revisited, Raven Press, New York, 1980, pp. 449-472. Chase, M. H., Boxer, P. A., Fung, S. J. and Morales, F. R., Pontomesencephalic reticular inhibition of lumbar motoneurons during active sleep. In M. H. Chase, D. F. Kripke and P. L. Walter (Eds.), Sleep Research, Vol. 10, 1981, p. 25.
273 7 Chase, M. H., Chandler, S. H. and Nakamura, Y., Intracellular determination of membrane potential of trigemihal motoneurons during sleep and wakefulness, J. Neurophysiol., 44 (1980) 349-358. 8 Chase, M. H., Enomoto, S., Murakami, T., Nakamura, Y. and Taira, M., Intracellular potential of medullary reticular neurons during sleep and wakefulness, Exp. Neurol., 71 (1981) 226-233. 9 Chase, M. H. and Morales, F. R., Subthreshold membrane activity in spinal cord motoneurons during active sleep, Neurosci. Abstr., 5 (1979) 365. 10 Fung, S. J., Boxer, P. A., Morales, F. R. and Chase, M. H., Pontomesencephalic induced hyperpolarizing potentials in lumbar motoneurons during active sleep, Neurosci. ,4bstr., 7 (1981) 361. 11 Grillner, S., Hongo, T. and Lund, S., Reciplocal effects between two descending bulbospinal systems with monosynaptic connections to spinal motoneurones, Brain Research, 10 (1968) 477-480. 12 Grillner, S. and Lund, S., The origin of descending pathway with monosynaptic action on flexor motoneurones, ,4cta physiol, scand., 74 (1968) 274-284. 13 Henley, K. and Morrison, A. D., A re-evaluation of the effects of lesions of the pontine tegmentum and locus coeruleus on phenomena of paradoxical sleep in the cat, Acta neurobiol, exp., 34 (1974) 215-232. 14 Hoshino, K. and Pompeiano, O., Selective discharge of pontine neurons during the postural atonia produced by an anticholinesterase in the decerebrate cat, Arch. ital. Biol., 114 (1976) 244-277. 15 Jankowska, E., Lund, S., Lundberg, A. and Pompeiano, O., Inhibitory effects evoked through ventral reticulospinal pathways, Arch. ital. Biol., 106 (1968) 124-140. 16 Kanamori, N., Sakai, K. and Jouvet, M., Neuronal activity specific to paradoxical sleep in the ventromedial medullary reticular formation of unrestrained cats, Brain Research, 189 (1980) 251-255. 17 Llinas, R. and Terzuolo, C. A., Mechanisms of supraspinal actions upon spinal cord activities. Reticular inhibitory mechanisms on alpha-extensor motoneurons, J. Neurophysiol., 27 (1964) 579-591. 18 Llinas, R. and Terzuolo, C. A., Mechanisms of supraspinal actions upon spinal cord activities. Reticular inhibitory mechanisms upon flexor motoneurons, J. Neurophysiol., 28 (1965) 413-422. 19 Magoun, H. W. and Rhines, R., An inhibitory mechanism in the bulbar reticular formation, J. Neurophysiol., 9 (1946) 165-171. 20 Mancia, M., Mariotti, M. and Speafico, R., Caudo-rostral brain stem reciprocal influences in the cat, Brain Research, 80 (1974) 41-51.
21 Morales, F. R., Schadt, J. C. and Chase, M. H., Intracellular recording from spinal cord motoneurons in the chronic cat, Physiol. Behav., 27 (1981) 355-362. 22 Peterson, B. W., Reticulospinal projections to spinal motor nuclei, Ann. Rev. Physiol., 41 (1979) 127-140. 23 Peterson, B. W., Pitts, N. G. and Fukushima, K., Reticulospinal connections with limb and axial motoneurons, Exp. Brain Res., 36 (1979) 1-20. 24 Petras, J. M., Cortical, tectal and tegmental fiber connections in the spinal cord of the cat, Brain Research, 6 (1967) 275-324. 25 Sakai, K., Sastre, J. P., Salvert, D., Touret, M., Tohyama, M. and Jouvet, M., Tegmentoreticular projections with special reference to the muscular atonia during paradoxical sleep in the cat: an HRP study, Brain Res., 176 (1979) 233-254. 26 Sasaki, K., Tanaka, T. and Mori, K., Effects of stimulation of pontine and bulbar reticular formation upon spinal motoneurons of the cat, Jap. J. Physiol., 12 (1962) 45-62. 27 Shapovalov, A. I., Posttetanic potentiation of monosynaptic and disynaptic actions from supraspinal structures on lumbar motoneurons, J. Neurophysiol., 32 (1969) 948-959. 28 Seigel, J. M., Behavioral functions of the reticular formation, Brain Res. Rev., 1 (1979) 69-105. 29 Silberman, E. K., Vivaldi, E., Garfield, J., McCarley, R.W. and Hobson, J. A., Carbachol triggering of desynchronized sleep phenomena: enhancement via small volume infusions, Brain Research, 191 (1980) 215-224. 30 Snider, R. S. and Niemer, W. T., .4 Stereotaxic Atlas of the Cat Brain, University of Chicago Press, Chicago 1961. 31 Steriade, M., and Hobson, J. A., Neuronal activity during the sleep-waking cycle, Progr. Neurobiol., 6 (1976) 155-376. 32 Torvik, A. and Brodal, A., The origin of reticulospinal fibers in the cat: an experimental study, Anat. Rec., 128 (1957) 113-135. 33 Van Dongen, P. A. M., Broekkamp, C. L. E. and Cools, A. K., Atonia after carbachol microinjection near the locus coeruleus in cats, Pharrnacol. Biochem. Behav., 8 (1978) 527-532. 34 Wilson, V. J. and Yoshida, M., Comparison of effects of stimulation of Deiters' nucleus and medial longitudinal fasciculus on neck, forelimb, and hindlimb motoneurons, J. Neurophysiol., 32 (1969) 743-758. 35 Wolstencroft, J. H., Reticulospinal neurones, J. Physiol. (Lond.), 174 (1964) 91-108.
267
Hyperpolarizing Membrane Responses Induced in Lumbar Motoneurons by Stimulation of the Nucleus Reticularis Pontis Oralis During Active Sleep SIMON J. FUNG, PETER A. BOXER, FRANCISCO R. MORALES and MICHAEL H. CHASE* Brain Research Institute and Departments of Physiology and Anatomy, University of California, Los Angeles, CA 90024 (U.S.A.)
(Accepted March llth, 1982) Key words: intracellular recording - - lumbar motoneurons - - motoneurons - - reticular formation - nucleus reticularis pontis oralis - - sleep - - active sleep
Intracellular recordings were obtained from lumbar motoneurons in unanesthetized, undrugged, normally respiring cats during the states of wakefulness, quiet sleep and active sleep. The objective was to examine the state-dependent control of spinal cord motoneurons exerted by the pontomesencephalic reticular formation. Accordingly, electrical stimulation was applied to the nucleus reticularis pontis oralis while the membrane potential of lumbar motoneurons was recorded. Short latency depolarizing and/or hyperpolarizing potentials were observed throughout sleep and wakefulness; no state-dependent pattern was found in the direction of polarization or amplitude for these early potentials. However, a long latency hyperpolarizing potential emerged exclusively during active sleep; it was characterized by a peak latency of 45 ± 1 (S.E.M.) ms, a duration of 40 4- 2 ms, and an amplitude of 3 0.5 mV. This active sleep-selective potential was capable of inhibiting spontaneous motoneuron activity. These and previously obtained data support the notion that excitation of the nucleus reticularis pontis oralis results in somatomotor inhibition at the level of the spinal cord and brainstem selectively during the state of active sleep. INTRODUCTION T h e b r a i n s t e m reticular f o r m a t i o n has been implic a t e d in a v a r i e t y o f b e h a v i o r a l processes, including m o t o r c o n t r o l a n d the r e g u l a t i o n o f sleep a n d w a k e fulness 2s. I n the context o f m o t o r c o n t r o l m e c h a nisms, i n d i v i d u a l reticular areas exhibit f u n c t i o n a l specificity22. In p a r t i c u l a r , the nucleus reticularis p o n t i s oralis ( N P O ) has b e e n shown, b y lesion 2 a n d p h a r m a c o l o g i c a l 29,3z studies, to p l a y a n i m p o r t a n t role in the b e h a v i o r a l ( m o t o r ) expression o f active sleep (AS). S p o n t a n e o u s discharge o f N P O cells occurs in c o n j u n c t i o n with a decrease in muscle t o n e 14. F u r t h e r m o r e , n e u r o n a l d e s t r u c t i o n within a n d in the vicinity o f the N P O diminishes o r abolishes the a t o n i a o f A S 13. R e c e n t studies have f u r t h e r d e m o n s t r a t e d t h a t N P O s t i m u l a t i o n p r o d u c e s facilitation
o f the masseteric reflex a n d a d e p o l a r i z i n g p o t e n t i a l in t r i g e m i n a l j a w - c l o s e r m o t o n e u r o n s d u r i n g wakefulness (W) a n d quiet sleep (QS), while the identical stimulus d u r i n g A S induces reflex i n h i b i t i o n a n d a c o r r e s p o n d i n g h y p e r p o l a r i z i n g potential4,L T h e current s t u d y was designed to examine the effects o f N P O s t i m u l a t i o n on the m e m b r a n e p o t e n t i a l o f ~m o t o n e u r o n s at the level o f the spinal c o r d d u r i n g sleep a n d wakefulness. P r e l i m i n a r y d a t a have been r e p o r t e d elsewhere6,10. METHODS E x p e r i m e n t s were p e r f o r m e d on 3 unanesthetized, u n d r u g g e d , n o r m a l l y respiring cats d u r i n g W , QS a n d AS. In o r d e r to c a r r y o u t these studies, which r e q u i r e d s i m u l t a n e o u s e x p l o r a t i o n s within the
* To whom correspondence should be addressed at: Department of Physiology, Center for the Health Sciences, University of California, Los Angeles, CA 90024, U.S.A. 0006-8993/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press
268 brainstem (site of stimulation) and spinal cord (site of intracellular recording), two separate, interdependent surgical procedures were undertaken while the animals were anesthetized with sodium pentobarbital (40 mg/kg, i.p.). The procedures for preparing the head region in the chronic cat are described in detail in Chase et al. 7. Specific procedures for intracellular recording from spinal cord motoneurons in the cat are presented in Morales et al. ~1. Consequently, in this report, only an overview of the key elements of the preceding methodologies are presented. Chronic recording screw electrodes were placed in the calvarium to monitor E E G activity. Bipolar strut electrodes were implanted stereotaxically (A6, L10.5, H3) to record pontogeniculate-occipital (PGO) waves. These electrodes were connected to a female Winchester plug which was bonded to the calvarium with acrylic resin. A head-restraining device was implanted for fixing the head of the cat in a standard 3-dimensional stereotaxic position without pain or discomfort. The area of the calvarium overlying the region for electrical stimulation was left uncovered for later trephination before the initial experimental session. Next, the spinal cord was prepared for intracellular recording in the chronic preparation. Hard plastic clamps were mounted around L3, L4 and L6 vertebrae. Stainless steel screws were put in the articular processes and transverse steel bars were placed next to the vertebral clamps. Mechanical immobilization of the vertebral column was achieved by binding the screws, clamps and bars together with acrylic resin; a circle (4 mm in diameter) in the lamina of L5 was left free of acrylic resin in order to permit microelectrode exploration of the underlying spinal cord. All wound margins were sutured and postoperative care was administered to avoid infection. Prior to the initial experimental session each animal was anesthetized with 4 ~ halothane. Chronic stimulating cuff electrodes were placed around the tibial and/or sciatic nerves. A small hole was drilled in the calvarium for brain stimulation and another in the lamina of L5 for microelectrode penetration; both holes were ipsilateral to the nerve electrode. The dura mater was cut to expose the dorsal root entry zone of L7.
Data were collected for 3 consecutive days on each animal over a period of 6-8 weeks. During each session, the animal was placed in the stereotaxic apparatus so that its head and spinal cord were returned to the original orientation assumed during the preceding surgical procedures. Pin electrodes were placed in the skin at the lateral margins of each eye and in the nuchal musculature to record EOG and E M G activity, respectively. A male connector was attached to the female plug to monitor EEG and PGO activity. A bipolar strut electrode (0.5 mm tip exposure, 1 mm interpolar distance) was placed stereotaxically (A1 to P1, L2 to 3, H - - 2 to --4) to stimulate the region of the NPO (2-4 pulses, 0.5 ms, 800 cps, 0.8-6 V). Current intensities were determined by recording the voltage drop across a 100 kf~ resistor placed in series with the stimulation circuitry. Stimulus strength ranged from 7 to 70 uA. Glass micropipettes filled with 2 M potassium citrate (6-15 Mf~ resistance) were employed to record intracellularly from motoneurons. A reference Ag-AgC1 electrode was placed subcutaneously lateral to the vertebral column. Motoneurons were identified by antidromic activation via the nerve stimulating electrode. At the conclusion of the experimental sessions, the animal was anesthetized with sodium pentobarbital and reference points in the brainstem were made at APO, L3, H - - 3 and at APO, L3, H - - 6 by delivering anodal current of 100 #A, which was passed through a stainless steel microelectrode for 30 s. The animal was sacrificed with an overdose of sodium pentobarbital and perfused with 1 0 ~ formalin and 2 ~ potassium ferrocyanide (Prussian blue reaction). Frozen transverse sections (50 #m in thickness) were cut and stained with thionin. Stimulation sites in the NPO were reconstructed according to their stereotaxic coordinates, based upon the location of the two reference points. RESULTS Data were collected following stimulation of 12 sites which were histologically verified to be located within the core of the NPO, according to Snider and Niemer a°. Based upon a maximal intensity of 70 #A that was employed, the zone of excitation can be considered to be limited. This conclusion is further
269
A
WAKEFULNESS INDIVIDUAL
AVERAGED POTENTIALS
N: 8
POTENTIALS
2 '&,,u,..,~
EOG * ' * " - - - - " - ~ ~ E M G ....... . - . . . . . . . . . . . . . . . . . . . . . . ~ll
B
QUIETSLEEP .1i J..,,khJ . J . . . |
_J..,.J,._L.,,[I.,J, ~,,IL
I
,.,, , , . r , T ,
~ /-L/
,.
C
z
~
3 'k,+.~-,,.
ACTIVESLEEP
,
tlo0~v 2 sec
4 ?v~, 20 msec
I ZmV
20 msec
Fig. 1. Nucleus reticularis pontis oralis (NPO)-induced motoneuron responses throughout the sleep-waking cycle. Left column: polygraphic recordings of EEG, EOG and EMG activity during wakefulness (A), quiet sleep (B), and active sleep (C). Middle column: averaged traces of eight consecutive samples of NPO-induced membrane responses. Note the presence of active sleepspecific hyperpolarizing potentials in C1-4. Right column: individual samples selected from the eight employed in generating the averaged potential. Sciatic motoneuron: antidromic spike potential = 69 mV, NPO stimulation: 3 pulses, 0.5 ms, 800 cps. substantiated by the observation that N P O stimulation did not induce muscle contractions of the neck musculature. Hence the stimulus did not spread to neighboring structures, such as the tectospinal tract, which lies immediately adjacent to the NPO. We cannot, however, exclude the possible coexcitation of descending fiber tracts coursing through the NPO. Measurements of intracellular potentials were performed in 24 motoneurons; the mean antidromic spike amplitude was 69.6 zk 11.2 mV (range = 55-98 mV). For data analysis, 8-32 consecutive NPO-induced potentials were routinely averaged. Data during AS were not analyzed during those
periods of prominent subthreshold membrane activities which occur in conjunction with rapid eye movement episodesL The general characteristics of the motoneuronal response to N P O stimulation during W, QS and AS are illustrated in Fig. 1. During these three states, reticular stimulation induced an early synaptic response at latencies of 2-15 ms; in some motoneurons they were depolarizing, and in others hyperpolarizing (Figs. 1-3). The amplitude of this potential was relatively constant across states or varied in a nonsystematic fashion. The initial potential induced by N P O stimulation was therefore nonstate-dependent; consequently, no specific analysis
270 of its characteristics was performed. At a latency greater than that of the early potential, no distinct additional evoked activity was registered during W or QS (Figs. 1A, B). In contrast, at the intensity of NPO stimulation that was employed, a large amplitude hyperpolarizing potential emerged during AS in 6 of 8 motoneurons showing state transitions (Fig. 1C). This potential arose during the transition period from QS to AS in conjunction with the development of PGO waves and reduction in muscle tone. It was present for the duration of AS and disappeared abruptly when the animal awoke. In 7 of 8 other motoneurons examined only during AS, a similar hyperpolarizing response was obtained. The remaining 8 motoneurons tested in QS alone did not exhibit a late hyperpolarizing potential; only the early response was present, Averages from 8-32 consecutive trials were used to analyze the NPO-induced hyperpolarizing potential of AS according to the schema shown in Fig. 2A. A determination of its latency and duration could not be obtained in all cells since the initial short latency potential sometimes obscured the onset of the succeeding AS-specific hyperpolarizing potential; in addition, its return to baseline was not
_~,,~_f~.~_~i,~y__T_~Z~-~_ _ DURATION ~ L A T E N C Y -----~ - . . . . . . . ~ ...........
• ~
. -T2
7~-=~'---~-~-
A. TRANSITION (QUIET SLEEP---> ACTIVE SLEEP)
25 mV
0.5 sec
B. QUIET SLEEP
C. ACTIVE SLEEP
I
' 5mV
I 20 msec
Fig. 3. Single traces of postsynaptic responses following nucleus reticularis pontis oralis (NPO) stimulation at the transition from quiet sleep to active sleep (A1 and A2) and during quiet sleep (B) and active sleep (C). The active sleepspecific hyperpolarizing response inhibited spontaneous motoneuron discharge as indicated in A1 and A2. The NPOinduced synaptic response is portrayed in the bottom portion of A at a faster sweep speed. Tibial motoneuron : antidromic spike potential = 73 mV; NPO stimulation: 3 pulses, 0.5 ms, 800 cps.
PEAK
AMPLITUDE
/
/ 20 msec
• LATENCY TO P
LATENCY
E
A
K
LATENCYTO PEAK (msec)
~
PEAK AMPLITUDE (mV)
DURATION HALF'WIDTH (msec)
(msec)
2ZO
44.9
5.I
40.1
24.1
S.E.M.
±1.7
±1.2
±0.5
±2.1
±1.5
RANGE
t9.3 - 3 8 . 3
58.0-53.0
29.7-46.6
14Z-36.5
(msec)
1.0 - 6 7
Fig. 2. Averaged record (A) of 8 consecutive nucleus reticularis pontis oralis (NPO)-induced membrane responses during active sleep. A quantitative determination of the active sleepspecific NPO-induced hyperpolarizing potential according to the diagram in A is presented in B. Details in text. In A, tibial motoneuron: antidromic spike potential = 79 mV; NPO stimulation: 4 pulses, 0.5 ms, 800 cps.
always dearly defined. For those cells that could be subjected to analysis, a baseline was first established prior to the artifacts of the stimulating pulse train. The peak amplitude of the hyperpolarizing potential was 3.1 4- 0.5 mV (X :k S.E.M.) (n = 11) (Fig. 2B). Its latency from the initiation of the reticular stimuli was 27.0 4- 1.7 ms (n = 10) (Fig. 2B). The latency to peak was 44.9 4- 1.2 ms (n = 11) (Fig. 2B) and its duration was 40.1 4- 2.1 ms (n = 7) (Fig. 2B). The half-width (the duration at half of the peak amplitude) was found to be 24. l 4- 1.5 ms (n = 10) (Fig. 2B). To summarize, the NPO-induced AS-specific response in lumbar motoneurons was typified by a 3 mV hyperpolarizing potential with a long latency to peak (45 ms) and a slow time course (half-width = 24 ms). The relation of the hyperpolarizing potential to spontaneous motoneuron activity is shown in Fig. 3.
271 ACTIVE
A.
EMG
-'" .
.
.
SLEEP
.
I
1
o. i n t r a c e l l u l a r potential
B.
| ..L. L ~ . ~ . . . If T "~" ~ - "
'. . . . . . . . . .
,k "
I
1 ~O0,~V
2 s--ec
]
b. ~ % ~ 2mV 2 0 msec
Fig. 4. Abolition of the hyperpolarizing potential concomitant with the occurrence of muscle activity and rapid eye movements during active sleep. Intracellular potential traces in a and b were averaged signals from four consecutive samples of nucleus reticularis pontis oralis (NPO)-induced responses during the periods marked beneath EMG traces in A and B. Stimuli to the NPO produced a hyperpolarizing potential of approximately 2 mV when atonia was present during active sleep (a). The active sleep-specific hyperpolarizing response disappeared (b) in conjunction with the occurfence of a brief episode of neck muscle activity (B), only to reappear once again at the end of the episode. Sciatic motoneuron: antidromic spike potential = 76 mV; NPO stimulation: 3 pulses, 0.5 ms, 800 cps.
In the example which is presented (Fig. 3A), during a postural readjustment just prior to AS, the motoneuron discharged spontaneously for a finite period (it ceased discharge once the AS state had been fully established). The NPO-induced hyperpolarizing potential arose during the transition from QS to AS while the motoneuron was still discharging (Fig. 3A). Based on the pattern of rhythmic activity just prior to NPO stimulation, it was evident that spontaneous action potentials were completely suppressed in the time slot corresponding to the period of the long latency hyperpolarizing potential (Fig. 3AI, 3A2). Muscle tone during AS is not always abolished; there are brief periods of partial resumption of EMG activity and/or muscle twitches. During these episodes of EMG activity, the NPO-induced synaptic potential was no longer present (Fig. 4B). The hyperpolarizing potential returned only after EMG activity had once again disappeared (Fig. 4A). DISCUSSION
The principal finding of the present study is that stimulation of the NPO induces a long latency hyperpolarizing potential in lumbar motoneurons exclusively during AS. This observation could not
have been made by previous investigators, since all other comparable experiments have been performed on acutely anesthetized or decerebrate animals. In these acute studies, however, a short latency potential was found whose characteristics were comparable to those which we observed; therefore, this potential will be discussed prior to an examination of the state-selective long latency potential. In acute cats, descending reticulofugal impulses originating from the medial pontomeduUary reticular formationa,15,28,26,27 and the medial longitudinal fasciculus of the medulla11,12,a4 impinge on lumbar motoneurons at a relatively short latency ranging from 2.5 (ref. 23) to 15 ms (ref. 26).Membrane responses to these descending volleys include both excitatory and inhibitory postsynaptic potentials. The early synaptic response which we observed exhibited a comparable latency and configuration and thus we can corroborate the intracellular findings which have revealed reticulospinal projections to lumbar motoneurons in acute cats. An anatomical basis for these electrophysiological findings encompasses a reticulospinal link between the NPO and the lumbar cord, which has been demonstrated in the cat by retrograde chromatolysis a2 and fiber degeneration 24, as well as by retrograde horseradish peroxidase transport 1. The early potential did not vary in configuration and exhibited only minor fluctuations in amplitude as a function of the animal's state; across neurons, no consistent pattern was observed for either hyperpolarizing or depolarizing activity. The presence of this early potential throughout the sleep-waking continuum indicates that a high safety factor is involved in its transmission and that it is not involved in sleep-specific functions. In contrast to the non-state-selective presence of the early potential, the succeeding potential was always hyperpolarizing and appeared exclusively during AS. Previous studies have demonstrated a state-dependent effect of NPO stimulation on the masseteric reflex and trigeminal motoneurons in the chronic cat (for review, see ref. 5). During AS, stimulation led to masseteric reflex suppression at conditioning-test intervals of 15-25 ms; at a similar peak latency a hyperpolarizing potential was observed in masseter motoneurons when intracellular recordings were obtained 4.
272 A comparison of the results of the brainstem and spinal cord experiments indicates that a hyperpolarizing potential arises in both lumbar (Fig. 2A) and trigeminal motoneurons exclusively during AS following excitation of the NPO 4. This hyperpolarizing potential of NPO origin inhibits spontaneous discharge in spinal motoneurons (Fig. 3) and synaptically induced spike potentials in trigeminal motoneurons 4. These data support the notion that stimulation of the NPO results in state-dependent somatomotor inhibition within the spinal cord as well as at the level of the brainstem. At both neuraxial levels the potential has a slow rise time, slow decay and prolonged half-width, which may be attributed to the temporal dispersion of impulses impinging onto the motoneuron membrane. It is unlikely that an asynchronous arrival of spike potentials could be explained solely by the heterogeneous conduction velocities of reticulospinal fibersaS; rather, the average duration of the hyperpolarizing potential (circa 40 ms) is suggestive of sustained inhibitory synaptic bombardment, probably via neuronal populations recruited by the NPO. Our present observations concerning the AS-specific hyperpolarizing potential favor the interpretation that the NPO stimulation promotes an inhibitory postsynaptic response in spinal motoneurons indirectly via an inhibitory relay. A possible relay site for the NPO action during AS is the medullary inhibitory area described by Magoun and Rhines 19, stimulation of which produces motor inhibition and diminished muscle tone in acute experiments. In decerebrate cats, several studies report inhibitory postsynaptic potentials in both flexor and extensor hindlimb motoneurons followREFERENCES 1 Basbaum, A. L. and Fields, H. L., The origin of descending pathways in the dorsolateral funiculus of the spinal cord of the cat and rat: further studies on the anatomy of pain modulation, J. comp. neuroL, 187 (1979) 513-532. 2 Carli, G. and Zanchetti, A., A study of pontine lesions suppressing deep sleep in the cat, Arch. itaL BioL, 103 (1965) 751-788. 3 Chan, S. H. H. and Barnes, C. D., Postsynaptic effects evoked from brain stem reticular formation in lumbar cord and their temporal correlation with a presynaptic mechanism, Arch. ital. BioL, 112 (1974) 81-97. 4 Chandler, S. H., Nakamura, Y. and Chase, M. H., Intra-
ing electrical stimulation of this area 15,17,1s. Further evidence of a medullary relay derives from the recent finding of tonic membrane depolarization of gigantocellular reticular neurons that occurs selectively during AS s and an AS-specific increase in the unit activity of bulbar magnocellular reticular neurons 16. In addition, there are both anatomical 25 and physiological z° links between the medullary and pontomesencephalic reticular regions. Activation of the medullary area would then generate suppression of motor activity by activation of a population of inhibitory interneurons. What might we conclude from our finding that hyperpolarizing potentials in motoneurons are induced by NPO stimulation specifically during AS? A parsimonious explanation would be that this area, situated within the core of the reticular formation, would be subjected to excitatory input during AS, as it is throughout the waking state. We propose, in order to maintain the integrity of the AS state by preventing the motor expression of this activity, that it is converted to an inhibitory drive by intrareticular processes. To summarize, there is a striking AS-selective action on motoneurons resulting from stimulation of the NPO. Thus, it is reasonable to expect that the locus of the NPO participates in or is responsible for certain of the processes underlying somatomotor atonia during AS. ACKNOWLEDGEMENTS This work was supported by grants from the National Science Foundation (BNS 790-12897) and the U.S. Public Health Service (NS-09999). cellular analysis of synaptic potentials induced in trigeminal jaw-closer motoneurons by pontomesencephalicreticular stimulation during sleep and wakefulness, J. Neurophysiol., 44 (1980) 372-382. Chase, M. H., The motor functions of the reticular formation are multifaceted and state-determined. In J. A. Hobson, and M. A. B. Brazier (Eds.), The Reticular Formation Revisited, Raven Press, New York, 1980, pp. 449-472. Chase, M. H., Boxer, P. A., Fung, S. J. and Morales, F. R., Pontomesencephalic reticular inhibition of lumbar motoneurons during active sleep. In M. H. Chase, D. F. Kripke and P. L. Walter (Eds.), Sleep Research, Vol. 10, 1981, p. 25.
273 7 Chase, M. H., Chandler, S. H. and Nakamura, Y., Intracellular determination of membrane potential of trigemihal motoneurons during sleep and wakefulness, J. Neurophysiol., 44 (1980) 349-358. 8 Chase, M. H., Enomoto, S., Murakami, T., Nakamura, Y. and Taira, M., Intracellular potential of medullary reticular neurons during sleep and wakefulness, Exp. Neurol., 71 (1981) 226-233. 9 Chase, M. H. and Morales, F. R., Subthreshold membrane activity in spinal cord motoneurons during active sleep, Neurosci. Abstr., 5 (1979) 365. 10 Fung, S. J., Boxer, P. A., Morales, F. R. and Chase, M. H., Pontomesencephalic induced hyperpolarizing potentials in lumbar motoneurons during active sleep, Neurosci. ,4bstr., 7 (1981) 361. 11 Grillner, S., Hongo, T. and Lund, S., Reciplocal effects between two descending bulbospinal systems with monosynaptic connections to spinal motoneurones, Brain Research, 10 (1968) 477-480. 12 Grillner, S. and Lund, S., The origin of descending pathway with monosynaptic action on flexor motoneurones, ,4cta physiol, scand., 74 (1968) 274-284. 13 Henley, K. and Morrison, A. D., A re-evaluation of the effects of lesions of the pontine tegmentum and locus coeruleus on phenomena of paradoxical sleep in the cat, Acta neurobiol, exp., 34 (1974) 215-232. 14 Hoshino, K. and Pompeiano, O., Selective discharge of pontine neurons during the postural atonia produced by an anticholinesterase in the decerebrate cat, Arch. ital. Biol., 114 (1976) 244-277. 15 Jankowska, E., Lund, S., Lundberg, A. and Pompeiano, O., Inhibitory effects evoked through ventral reticulospinal pathways, Arch. ital. Biol., 106 (1968) 124-140. 16 Kanamori, N., Sakai, K. and Jouvet, M., Neuronal activity specific to paradoxical sleep in the ventromedial medullary reticular formation of unrestrained cats, Brain Research, 189 (1980) 251-255. 17 Llinas, R. and Terzuolo, C. A., Mechanisms of supraspinal actions upon spinal cord activities. Reticular inhibitory mechanisms on alpha-extensor motoneurons, J. Neurophysiol., 27 (1964) 579-591. 18 Llinas, R. and Terzuolo, C. A., Mechanisms of supraspinal actions upon spinal cord activities. Reticular inhibitory mechanisms upon flexor motoneurons, J. Neurophysiol., 28 (1965) 413-422. 19 Magoun, H. W. and Rhines, R., An inhibitory mechanism in the bulbar reticular formation, J. Neurophysiol., 9 (1946) 165-171. 20 Mancia, M., Mariotti, M. and Speafico, R., Caudo-rostral brain stem reciprocal influences in the cat, Brain Research, 80 (1974) 41-51.
21 Morales, F. R., Schadt, J. C. and Chase, M. H., Intracellular recording from spinal cord motoneurons in the chronic cat, Physiol. Behav., 27 (1981) 355-362. 22 Peterson, B. W., Reticulospinal projections to spinal motor nuclei, Ann. Rev. Physiol., 41 (1979) 127-140. 23 Peterson, B. W., Pitts, N. G. and Fukushima, K., Reticulospinal connections with limb and axial motoneurons, Exp. Brain Res., 36 (1979) 1-20. 24 Petras, J. M., Cortical, tectal and tegmental fiber connections in the spinal cord of the cat, Brain Research, 6 (1967) 275-324. 25 Sakai, K., Sastre, J. P., Salvert, D., Touret, M., Tohyama, M. and Jouvet, M., Tegmentoreticular projections with special reference to the muscular atonia during paradoxical sleep in the cat: an HRP study, Brain Res., 176 (1979) 233-254. 26 Sasaki, K., Tanaka, T. and Mori, K., Effects of stimulation of pontine and bulbar reticular formation upon spinal motoneurons of the cat, Jap. J. Physiol., 12 (1962) 45-62. 27 Shapovalov, A. I., Posttetanic potentiation of monosynaptic and disynaptic actions from supraspinal structures on lumbar motoneurons, J. Neurophysiol., 32 (1969) 948-959. 28 Seigel, J. M., Behavioral functions of the reticular formation, Brain Res. Rev., 1 (1979) 69-105. 29 Silberman, E. K., Vivaldi, E., Garfield, J., McCarley, R.W. and Hobson, J. A., Carbachol triggering of desynchronized sleep phenomena: enhancement via small volume infusions, Brain Research, 191 (1980) 215-224. 30 Snider, R. S. and Niemer, W. T., .4 Stereotaxic Atlas of the Cat Brain, University of Chicago Press, Chicago 1961. 31 Steriade, M., and Hobson, J. A., Neuronal activity during the sleep-waking cycle, Progr. Neurobiol., 6 (1976) 155-376. 32 Torvik, A. and Brodal, A., The origin of reticulospinal fibers in the cat: an experimental study, Anat. Rec., 128 (1957) 113-135. 33 Van Dongen, P. A. M., Broekkamp, C. L. E. and Cools, A. K., Atonia after carbachol microinjection near the locus coeruleus in cats, Pharrnacol. Biochem. Behav., 8 (1978) 527-532. 34 Wilson, V. J. and Yoshida, M., Comparison of effects of stimulation of Deiters' nucleus and medial longitudinal fasciculus on neck, forelimb, and hindlimb motoneurons, J. Neurophysiol., 32 (1969) 743-758. 35 Wolstencroft, J. H., Reticulospinal neurones, J. Physiol. (Lond.), 174 (1964) 91-108.