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Behavioral excitability and the giant-celled pontine reticular formation (nucleus reticularis pontis oralis)
SHORT COMMUNICATIONS
449
Behavioral excitability and the giant-celled pontine reticular formation (nucleus reticularis pontis oralis) Since the experiments of Moruzzi, Magoun, Lindsley, and their associates 13 it has been widely, though not universally (cf. ref. 7) held that the brain stem reticular formation controls levels of behavioral excitability ('arousal'). A specific derivation of this hypothesis is the common pharmacological assumption that the origins of the stimulant properties of amphetamine (and a variety of other drugs) lie within the reticular formation (cf. ref. 3). In view of their widespread acceptance, the experimental bases for these hypotheses are surprisingly meager. The few studies reporting coma after reticular lesions involved massive destruction of extrareticular structures including posterior diencephalon, central gray, and a number of sensory and motor fiber systems 10. On the other hand, several studies have found that discrete lesions of the mesencephalic reticular formation actually increase behavioral activityS,6,s,L To date, behavioral experiments of this type have been performed only on the mesencephalic reticular formation; this is a serious restriction since the origins of many neurophysiological indices of arousal (e.g. EEG desynchrony) have now been localized in areas caudal to this region, specifically in the giant-celled nucleus reticularis pontis oralisL It is possible therefore that the failure to find behavioral depression after discrete reticular lesions is due to a concentration on the wrong subdivision. The present study was prompted by these considerations. In this paper, we report the effects of lesions in the nucleus reticularis pontis oralis on exploratory and amphetamine-induced activity. Thirty-nine naive male albino rats of the Sprague-Dawley strain (pre-operative weight 300-325 g) were used as experimental animals. Prior to the study, several rats were sacrificed and cresyl-violet stained coronal sections made through their brain stems; these sections and the paper of Valverde 15 were used as guides in establishing stereotaxic coordinates for the nucleus reticularis pontis oralis. Animals were operated on following barbiturate (Nembutal) anesthetization and atropine pretreatment; teflon-coated platinum iridium wire of 0.01 in. outside diameter with only the tip of the wire bared was used as the (anodal) electrode with a large rectal cathode. Current was in all cases 1 mA for 30 sec and was generated by a constant current source. All lesions were made bilaterally. Sham controls were treated exactly the same in all details except that the electrode was not lowered into the brain. The animals were given 3-10 days to recover and then were placed in round wiremesh activity cages (diameter 14 in. with a height of 17 in.). Each cage had two photobeams mounted 5 in. off the floor and placed so that their beams bisected each other at the center of the cage. These cages were located inside of wooden boxes with fans and lights attached; the boxes in turn were placed in a semi-isolated experimental room. Activity was measured as the number of photobeam breaks, which were counted and printed every 6 min on a single counter. For each test, the animals were given a 30-min adaptation period after which they were quickly removed from the cages, injected i.p. with 1.0 mg/kg of D-amphetamine sulfate (in a volume of 1 ml/kg of body wt.) and immediately returned to the cages for a 90-min period. Some animals were
Brain Research, 32 (1971) 449--453
450
SHOR 1 ( ' O M M U N I C A 11~)N5
tested twice in this manner with at least 24 h separating tests. Twenty-four animals with lesions and 15 sham-operated rats were tested in this way: 6 additional sham animals were injected with saline alone, to insure that any hyperactivity seen after amphetamine was due to the drug rather than the non-specific effects of handling, injection, etc. We have used these testing situations and procedures extensively in other experiments and have found them to be sensitive to a variety of behavioral stimulants and depressants. The dosage of amphetamine used here is towards the lower end of the dose-response curve for activity 11 obviating problems associated with 'ceiling' effects. Following testing, the animals were given an overdose of barbiturate and perfused with normal saline followed with formalin. Brains were dissected out and serial coronal sections cut at 50 # m prepared. Representative sections through the lesions were then stained with cresyl-violet and mounted. [ ] Fig. 1 is a representative example of the lesions used in this study. Some (N == 9) were clearly asymmetrical or fell outside the reticular formation and are henceforth referred to as 'tegmental lesions' and used as controls. Lesions within the reticular
Fig. 1. Photograph of a brain with representative lesions located inside the nucleus reticularis pontis oralis. Brain Research, 32 (1971) 449-453
S H O R T
451
C O M M U N I C A T I O N S
2,o! 220 ~
".It. st I~---...I 12.----"~
L
2OO 180 160 140 120 IOO 8O
RETICULAR-AMPHETAMINE TEGMENTA L- AMPHETAMINE SHAM-AMPHETAMINE SHAM-SALINE
60 4O
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2O
A
INJECTION
0 I
,
0
PRE- 30 INJECT
,
,
t
,
L
0
I
I
I
'
'
30
'
'
~
*
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POST- 60 INJECT
t
L~
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90
MINUTES
Fig. 2. Photobeam cage activity of rats with pontine reticular lesions and control groups (lesioned and non-lesioned) during habituation to the testing cage and following injection with. 1.0 mg/kg of amphetamine. Also shown are the data for a control group which received an equal volume of saline instead of the amphetamine. formation (N = 15) were all localized rostrocaudally between the area immediately behind the decussation of the superior cerebellar peduncles and the trapezoid bodies. In the transverse plane, the damage was confined to regions medial to the lateral lemniscus and lateral to the raphe group. Most of the individual lesions covered a more limited extent and were centered in the intended area, the rostral pontine reticular formation (Fig. 1). In general, the rats appeared healthy; they did not resist handling, gave no indication that they were impaired with regard to climbing, orienting, and exploratory movements, and maintained weight gain curves comparable to those of the tegmental group. However, a detailed neurological examination (e.g., placing, hopping reactions, etc.) which presumably would have uncovered more subtle deficits was not attempted. Fig. 2 summarizes the activity data for the 4 groups. F r o m an examination of this graph it is evident that the lesions had little effect on activity, either that occurring during adaptation or in response to D-amphetamine. In examining the data for individual animals there was some suggestion that animals with the more caudal lesions (which involved the nucleus reticularis pontis caudalis) were hyperactive but more rats would be needed for a definitive conclusion. In no case, however, was any evidence of behavioral depression detected. There is no suggestion in these results that the giant-celled pontine reticular formation is necessary for the maintenance of normal arousal levels or the production of states of hyperexcitability as indexed by general activity or movement. Combined
Brain Research, 32 (1971) 449-453
452
SHORT COMMUNICA 1IONS;
with results of studies using discrete lesions in various segments of the mesencephalic reticular formation s the present findings point to the more general conclusion that the ponto-mesencephalic reticular system is only secondarily involved in behavioral arousal in rats. They also cast serious doubt on the common assumption that the origins of the hyperactivity produced by amphetamine are to be found in the reticular formation, or at least in its pontine regions. Several reservations, however, need to be established. For one, it is apparent from neurophysiological 1,12,14 and anatomical 4,1~ studies that there are several functionally distinct subdivisions in what is commonly called the reticular system; thus definitive conclusions on the role(s) of the reticular formation in arousal can be obtained only in studies directly comparing the effects of manipulating these. Secondly, current hypotheses about the functions of the reticular formation are based almost exclusively on neurophysiological studies in cats, and the possibility that it is organized differently in other species deserves consideration. That is, the pontine reticular formation may not have special significance for behavioral or neurophysiological indices of arousal in the rat while it does in the cat. Finally, and in contrast to EEG 'arousal', it may be that behavioral arousal is dependent upon the greater part of the reticular formation rather than restricted subdivisions and massive lesions will be required to produce measurable effects. Each of these possibilities is currently under investigation. This research was supported by National Science Foundation Grant GB-16973 and N I H Grant M H 18233-01. Department of Psychobiology, University of California, Irvine, Calif. (U.S.A.)
GARY LYNCH
1 BATINI, C., MORUZZI,O., PALESTRINI,M., ROSSI,G., AND ZANCHETTI, A., Persistent patterns of
wakefulness in the pretrigeminal mid-pontine preparation, Science, t28 (1958) 30-32. 2 BONVALLET, M., AND NEWMAN-TAYLOR, A., Neurophysiological evidence for a differential
organization of the reticular formation, Electroenceph. clin. Neurophysiol., 22 (1967) 54-73. 3 BRADLEY,P. S., Electrophysiologyof psychotomimeticdrugs, Neurosci. Res. Bull., 8 (1970) 55-58. 4 BRODAL,A., The Reticular Formation of the Brain Stem. Anatomical Aspects and Functional Correlations, Oliver and Boyd, Edinburgh, 1957. 5 CAPPS,M. J., ANDSTOCKWELL,C. W., Lesions in the midbrain reticular formation and the startle response in rats, Physiol. Behav., 3 (1968) 661-665. 6 DIRLAM,D. K., The effects of septal, thalamic, and tegmental lesions on general activity in the hooded rat, Canad. J. Psychol., 23 (1969) 303-314. 7 FELDMAN,S., ANDWALLER,H., Dissociation of electrocortical activation and behavioral arousal, Nature (Lond.), 196 (1962) 1320. 8 GLICKMAN,S. E., SRO~ES,R. W., ANDHUNT,J., Brain lesions and locomotor exploration in the albino rat, J. comp. physiol. Psychol., 58 (1964) 93-100. 9 KESNER,R. F., FIEDLER,P., ANDTHOMAS,G. J., Function of the midbrain reticular formation in regulating level of activity and learning in rats, J. comp. physiol. Psychol., 63 (1967) 452~57. 10 LINDSLEY, D. B., SCHREINER, L. H., KNOWLES, W. B., AND MAGOUN, H. W., Behavioral and EEG
changes following chronic brain stem lesions in the cat, Electroenceph. clin. Neurophysiol., 2 (1950) 483-498. Brain Research, 32 (1971) 449-453
SHORT COMMUNICATIONS
453
11 LYNCH,G., BALLANTINE,P., AND CAMPBELL,B., Potentiation of behavioral arousal after cortical damage and subsequent recovery, Exp. Neurol., 23 (1969) 195-206. 12 MAGOUN,H. W., AND RHINES,R., An inhibitory mechanism in the bulbar reticular formation, J. Neurophysiol., 9 (1946) 165. 13 MORUZZt,G., Reticular influences on the EEG, Electroenceph. clin. Neurophysiol., 16 (1964) 2-17. 14 SAUERLAND,E. K., NAKAWAMURA,Y., AND CLEMENTE,C. D., The role of the lower brain stem in cortically induced inhibition of somatic reflex in the cat, Brain Research, 6 (1967) 164-179. 15 VALVERDE,G., Reticular formation of the albino rat's brain stem. Cytoarchitecture and corticofugal connections, J. comp. Neurol., 119 (1962) 25-49. (Accepted June 30th, 1971)
Brain Research, 32 (1971) 449-453
449
Behavioral excitability and the giant-celled pontine reticular formation (nucleus reticularis pontis oralis) Since the experiments of Moruzzi, Magoun, Lindsley, and their associates 13 it has been widely, though not universally (cf. ref. 7) held that the brain stem reticular formation controls levels of behavioral excitability ('arousal'). A specific derivation of this hypothesis is the common pharmacological assumption that the origins of the stimulant properties of amphetamine (and a variety of other drugs) lie within the reticular formation (cf. ref. 3). In view of their widespread acceptance, the experimental bases for these hypotheses are surprisingly meager. The few studies reporting coma after reticular lesions involved massive destruction of extrareticular structures including posterior diencephalon, central gray, and a number of sensory and motor fiber systems 10. On the other hand, several studies have found that discrete lesions of the mesencephalic reticular formation actually increase behavioral activityS,6,s,L To date, behavioral experiments of this type have been performed only on the mesencephalic reticular formation; this is a serious restriction since the origins of many neurophysiological indices of arousal (e.g. EEG desynchrony) have now been localized in areas caudal to this region, specifically in the giant-celled nucleus reticularis pontis oralisL It is possible therefore that the failure to find behavioral depression after discrete reticular lesions is due to a concentration on the wrong subdivision. The present study was prompted by these considerations. In this paper, we report the effects of lesions in the nucleus reticularis pontis oralis on exploratory and amphetamine-induced activity. Thirty-nine naive male albino rats of the Sprague-Dawley strain (pre-operative weight 300-325 g) were used as experimental animals. Prior to the study, several rats were sacrificed and cresyl-violet stained coronal sections made through their brain stems; these sections and the paper of Valverde 15 were used as guides in establishing stereotaxic coordinates for the nucleus reticularis pontis oralis. Animals were operated on following barbiturate (Nembutal) anesthetization and atropine pretreatment; teflon-coated platinum iridium wire of 0.01 in. outside diameter with only the tip of the wire bared was used as the (anodal) electrode with a large rectal cathode. Current was in all cases 1 mA for 30 sec and was generated by a constant current source. All lesions were made bilaterally. Sham controls were treated exactly the same in all details except that the electrode was not lowered into the brain. The animals were given 3-10 days to recover and then were placed in round wiremesh activity cages (diameter 14 in. with a height of 17 in.). Each cage had two photobeams mounted 5 in. off the floor and placed so that their beams bisected each other at the center of the cage. These cages were located inside of wooden boxes with fans and lights attached; the boxes in turn were placed in a semi-isolated experimental room. Activity was measured as the number of photobeam breaks, which were counted and printed every 6 min on a single counter. For each test, the animals were given a 30-min adaptation period after which they were quickly removed from the cages, injected i.p. with 1.0 mg/kg of D-amphetamine sulfate (in a volume of 1 ml/kg of body wt.) and immediately returned to the cages for a 90-min period. Some animals were
Brain Research, 32 (1971) 449--453
450
SHOR 1 ( ' O M M U N I C A 11~)N5
tested twice in this manner with at least 24 h separating tests. Twenty-four animals with lesions and 15 sham-operated rats were tested in this way: 6 additional sham animals were injected with saline alone, to insure that any hyperactivity seen after amphetamine was due to the drug rather than the non-specific effects of handling, injection, etc. We have used these testing situations and procedures extensively in other experiments and have found them to be sensitive to a variety of behavioral stimulants and depressants. The dosage of amphetamine used here is towards the lower end of the dose-response curve for activity 11 obviating problems associated with 'ceiling' effects. Following testing, the animals were given an overdose of barbiturate and perfused with normal saline followed with formalin. Brains were dissected out and serial coronal sections cut at 50 # m prepared. Representative sections through the lesions were then stained with cresyl-violet and mounted. [ ] Fig. 1 is a representative example of the lesions used in this study. Some (N == 9) were clearly asymmetrical or fell outside the reticular formation and are henceforth referred to as 'tegmental lesions' and used as controls. Lesions within the reticular
Fig. 1. Photograph of a brain with representative lesions located inside the nucleus reticularis pontis oralis. Brain Research, 32 (1971) 449-453
S H O R T
451
C O M M U N I C A T I O N S
2,o! 220 ~
".It. st I~---...I 12.----"~
L
2OO 180 160 140 120 IOO 8O
RETICULAR-AMPHETAMINE TEGMENTA L- AMPHETAMINE SHAM-AMPHETAMINE SHAM-SALINE
60 4O
,I.
2O
A
INJECTION
0 I
,
0
PRE- 30 INJECT
,
,
t
,
L
0
I
I
I
'
'
30
'
'
~
*
t,
POST- 60 INJECT
t
L~
L
I
90
MINUTES
Fig. 2. Photobeam cage activity of rats with pontine reticular lesions and control groups (lesioned and non-lesioned) during habituation to the testing cage and following injection with. 1.0 mg/kg of amphetamine. Also shown are the data for a control group which received an equal volume of saline instead of the amphetamine. formation (N = 15) were all localized rostrocaudally between the area immediately behind the decussation of the superior cerebellar peduncles and the trapezoid bodies. In the transverse plane, the damage was confined to regions medial to the lateral lemniscus and lateral to the raphe group. Most of the individual lesions covered a more limited extent and were centered in the intended area, the rostral pontine reticular formation (Fig. 1). In general, the rats appeared healthy; they did not resist handling, gave no indication that they were impaired with regard to climbing, orienting, and exploratory movements, and maintained weight gain curves comparable to those of the tegmental group. However, a detailed neurological examination (e.g., placing, hopping reactions, etc.) which presumably would have uncovered more subtle deficits was not attempted. Fig. 2 summarizes the activity data for the 4 groups. F r o m an examination of this graph it is evident that the lesions had little effect on activity, either that occurring during adaptation or in response to D-amphetamine. In examining the data for individual animals there was some suggestion that animals with the more caudal lesions (which involved the nucleus reticularis pontis caudalis) were hyperactive but more rats would be needed for a definitive conclusion. In no case, however, was any evidence of behavioral depression detected. There is no suggestion in these results that the giant-celled pontine reticular formation is necessary for the maintenance of normal arousal levels or the production of states of hyperexcitability as indexed by general activity or movement. Combined
Brain Research, 32 (1971) 449-453
452
SHORT COMMUNICA 1IONS;
with results of studies using discrete lesions in various segments of the mesencephalic reticular formation s the present findings point to the more general conclusion that the ponto-mesencephalic reticular system is only secondarily involved in behavioral arousal in rats. They also cast serious doubt on the common assumption that the origins of the hyperactivity produced by amphetamine are to be found in the reticular formation, or at least in its pontine regions. Several reservations, however, need to be established. For one, it is apparent from neurophysiological 1,12,14 and anatomical 4,1~ studies that there are several functionally distinct subdivisions in what is commonly called the reticular system; thus definitive conclusions on the role(s) of the reticular formation in arousal can be obtained only in studies directly comparing the effects of manipulating these. Secondly, current hypotheses about the functions of the reticular formation are based almost exclusively on neurophysiological studies in cats, and the possibility that it is organized differently in other species deserves consideration. That is, the pontine reticular formation may not have special significance for behavioral or neurophysiological indices of arousal in the rat while it does in the cat. Finally, and in contrast to EEG 'arousal', it may be that behavioral arousal is dependent upon the greater part of the reticular formation rather than restricted subdivisions and massive lesions will be required to produce measurable effects. Each of these possibilities is currently under investigation. This research was supported by National Science Foundation Grant GB-16973 and N I H Grant M H 18233-01. Department of Psychobiology, University of California, Irvine, Calif. (U.S.A.)
GARY LYNCH
1 BATINI, C., MORUZZI,O., PALESTRINI,M., ROSSI,G., AND ZANCHETTI, A., Persistent patterns of
wakefulness in the pretrigeminal mid-pontine preparation, Science, t28 (1958) 30-32. 2 BONVALLET, M., AND NEWMAN-TAYLOR, A., Neurophysiological evidence for a differential
organization of the reticular formation, Electroenceph. clin. Neurophysiol., 22 (1967) 54-73. 3 BRADLEY,P. S., Electrophysiologyof psychotomimeticdrugs, Neurosci. Res. Bull., 8 (1970) 55-58. 4 BRODAL,A., The Reticular Formation of the Brain Stem. Anatomical Aspects and Functional Correlations, Oliver and Boyd, Edinburgh, 1957. 5 CAPPS,M. J., ANDSTOCKWELL,C. W., Lesions in the midbrain reticular formation and the startle response in rats, Physiol. Behav., 3 (1968) 661-665. 6 DIRLAM,D. K., The effects of septal, thalamic, and tegmental lesions on general activity in the hooded rat, Canad. J. Psychol., 23 (1969) 303-314. 7 FELDMAN,S., ANDWALLER,H., Dissociation of electrocortical activation and behavioral arousal, Nature (Lond.), 196 (1962) 1320. 8 GLICKMAN,S. E., SRO~ES,R. W., ANDHUNT,J., Brain lesions and locomotor exploration in the albino rat, J. comp. physiol. Psychol., 58 (1964) 93-100. 9 KESNER,R. F., FIEDLER,P., ANDTHOMAS,G. J., Function of the midbrain reticular formation in regulating level of activity and learning in rats, J. comp. physiol. Psychol., 63 (1967) 452~57. 10 LINDSLEY, D. B., SCHREINER, L. H., KNOWLES, W. B., AND MAGOUN, H. W., Behavioral and EEG
changes following chronic brain stem lesions in the cat, Electroenceph. clin. Neurophysiol., 2 (1950) 483-498. Brain Research, 32 (1971) 449-453
SHORT COMMUNICATIONS
453
11 LYNCH,G., BALLANTINE,P., AND CAMPBELL,B., Potentiation of behavioral arousal after cortical damage and subsequent recovery, Exp. Neurol., 23 (1969) 195-206. 12 MAGOUN,H. W., AND RHINES,R., An inhibitory mechanism in the bulbar reticular formation, J. Neurophysiol., 9 (1946) 165. 13 MORUZZt,G., Reticular influences on the EEG, Electroenceph. clin. Neurophysiol., 16 (1964) 2-17. 14 SAUERLAND,E. K., NAKAWAMURA,Y., AND CLEMENTE,C. D., The role of the lower brain stem in cortically induced inhibition of somatic reflex in the cat, Brain Research, 6 (1967) 164-179. 15 VALVERDE,G., Reticular formation of the albino rat's brain stem. Cytoarchitecture and corticofugal connections, J. comp. Neurol., 119 (1962) 25-49. (Accepted June 30th, 1971)
Brain Research, 32 (1971) 449-453