- Email: [email protected]
Sparing of two types of hippocampal rhythmical slow activity (RSA, theta) in adult rats decorticated neonatally
Brain Research Bulletin, Vol. 26, pp. 425427.
0361-9230191 $3.00 + .OO
0 Pergamon Press plc, 1991. Printed in the U.S.A
RAPID COMMUNICATION
Sparing of Two Types of Hippocampal Rhythmical Slow Activity (RSA, Theta) in Adult Rats Decorticated Neonatally IAN Q. WHISHAW,’
RICHARD
DYCK
AND BRYAN
KOLB
Department of Psychology, University of Lethbridge, Lethbridge, Alberta, Canada, TlK 3M4 Received
4 September
1990
WHISHAW, I. Q., R. DYCK AND B. KOLB. Sparing of two types of hippocampal rhythmical slow activity (RSA, theta) in adult rats decorticated neonatally. BRAIN RES BULL 26(3) 425427, 1991 .-The electroencephalographic (EEG) activity of the hippocampus was examined in adult freely moving rats that had had all of the neocortex, including the cingulate cortex and cingulum, removed at birth. Both the cholinergic and serotonergic forms of rhythmical slow activity (RSA or theta) were present in the dorsal hippocampus and retained their normal relation to behavior. The results show that following decortication, inputs to and connections within the hippocampus apparently retain the ability to produce normal EEG. Since adult decortications abolish serotonergic RSA, the results also suggest that following neonatal injury there is reorganization within remaining serotonergic projections to the hippocampus that spare this form of RSA. Theta rhythm
Hippocampus
Decorticate
rat
Rhythmical
dura. For the neonatally lesioned rats, the tissue covering the trepinated skull was cut and the hippocampus was visualized through a dissecting microscope. The electrodes were lowered so that the longer pole was inserted about 1 mm into the hippocampus and the shorter pole of the electrode rested on the surface of the hippocampus. At the completion of the behavioral experiments, the rats were sacrificed under deep pentobarbital anesthesia, perfused with saline and formalin, and their brains were removed. The location of the recording electrodes were subsequently confirmed in 40-p thick cresyl violet stained sections taken through the hippocampus (7). Electrodes were connected to preamplifiers on a Grass polygraph by a light flexible cable and grounded with shielding connected to a ground screw implanted into the skull overlying the cerebellum. Slow waves were analyzed by inspection, rating on a 4-point scale, and measurement with a plastic ruler. In addition, representative samples of slow-wave activity were stored on FM tape and were digitized so autopower and crosspower spectra could be constructed. All rats were tested in at least four recording sessions during which recordings were made during spontaneous behaviors including walking, rearing, immobility, grooming, eating and drinking, slow-wave and paradoxical sleep. Before one additional session, the rats were given atropine sulfate (50 mg/kg) and after samples of behavior were obtained, they were lightly anes-
removal of the neocortex and midline neonatal or adult rats, the hippocampal formation remains intact, its cross-sectional area remains unchanged, and its connections with the contralateral hippocampus, nucleus reunions, septum, diagonal band of Broca, and Raphe, etc., are retained (1, 2, 6-9). The present study reports that if rats are decorticated neonatally, then the EEG activity of the hippocampus also appears normal. FOLLOWING limbic cortex
complete
in either
METHOD The experiments used 12 male Long-Evans strain rats. Within hours of birth, the rats were anesthetized by cooling on ice. In six rats, the skull overlying the dorsal neocortex was removed and all of their neocortex from the midline, including cingulate and medial frontal cortex, lateral to the rhinal fissure, was removed by aspiration. The rats were then returned to their mothers. When the rats were adult (90 to 120 days) EEG recording electrodes were implanted. The electrodes consisted of bipolar 125pm stainless steel wires coated with Teflon except for the tip and attached to female Amphenol connectors. The rats were anesthetized with sodium pentobarbital (50 mg/kg) for stereotaxic implantation. In the control rats, electrodes were implanted 3 mm posterior to bregma, 2.5 mm lateral to the midline, and 2.5 mm ventral to the ‘Requests for reprints should be addressed
to Dr. I. Q. Whishaw,
slow activity
Department
3M4: Email: [email protected].
425
of Psychology,
University
of L&bridge,
Lethbridge,
Alberta,
TlK
426
WHISHAW,
walk
DYCK AND KOLH
CONTROL -
walk
atlll
walk
-
ATROPINE
walk
----
atIll
ETHER AND ATROPINE
----
ethor and
other and atroplnr
10 20 30 40 1 l ec
FREOUENCY
50
(C/S)
FIG. 2. Polygraph records and power spectra during walking and immobility in one decorticated rat: Control, undrugged; Atropine, following 50 mg/kg atropine sulfate. Ether and atropine, the rat was lightly anesthetized with ether and RSA was elicited with a light tail pinch and the procedure was repeated after atropine sulfate. Power on a logarithmic scale is plotted against frequency (O-100 Hz) on a linear scale. The power spectra shows RSA peaks at 8 Hz during the control and atropine conditions when the rat is walking but not when it is still. A 5-7 Hz RSA peak is shown under ether but not under ether and atropine. FIG. 1. Photomicrographs - _ of the extent of the decortication. The placement of the lower tip of the recording electrodes is shown in both hippo-
campi, just above the granule cells, in the third section (cresyl violet). For descriptions of anatomical peculiarities, i.e., subiculum (right) see (1, 2).
thetized with ether. In another session they were given ether followed by atropine sulfate. RESULTS
Ten of 12 electrode placements in the control rats produced good recordings of RSA (300-1000 (*V). Similar good recordings were obtained from at least one hippocampus in each decorticate rat, and from both hippocampi in two rats. In all eight placements in the decorticate rats, the electrodes were successfully placed with recording tips straddling CA1 (Fig. 1). For the
remaining placements the tips of the electrodes were not in the hippocampus or implanted into a portion of the hippocampus that was damaged. The EEG analysis was done on only those placements that were found on histological inspection to have been in the hippocampus. Electrical activity recorded from appropriately placed electrodes in the decorticate rats could not be distinguished by visual inspection from the EEG recorded from the control rats. For each rat, one hundred l-s samples of EEG were rated for the behaviors of immobility, chewing food, lapping water, and grooming. Large amplitude irregular activity (LIA) was the predominant waveform in these samples and there was no difference in its incidence between the two groups [>80% of samples in each group were LIA, rs(lO)< 1, p>O.O5]. Similar analysis of EEG during walking indicated that RSA was invariably present in both
THETA ACTIVITY IN DECORTICATE
421
RATS
groups [>95% of samples in each group were RSA, rs(lO)O.O5]. The samples of EEG recorded during walking indicated that both groups had RSA of similar frequency [mean frequency of control=7.24 Hz vs. decorticate=7.35 Hz, t(lO)O.O5]. An example of a representative power spectra for one decorticate rat is shown in (Fig. 2). Samples of EEG recorded during sleep indicated that LIA was the major waveform in all rats during quiet sleep. When the rats lost muscle tone and entered REM sleep RSA was present. In both groups of rats, RSA had mean frequencies of between 5 to 7 Hz during nonmovement REM sleep and there was no group difference, t( lo)< 1, p>O.O5). RSA frequency and amplitude increased sharply during periods of vibrissae twitching in REM sleep, giving mean frequencies of RSA of between 7.3 and 8.6 Hz but again there was no group difference, r(lO)O.O5. Between 10 to 30 min following atropine sulfate, the rats were placed on an open table in order to elicit walking. RSA in the best electrode from each rat was rated during at least ten 5-s periods of immobility and walking in each rat. During immobility, RSA was absent (>90% samples) and LIA was recorded. During walking, RSA with a frequency of 7.5 to 8.7 Hz was obtained in more than 80% of all samples (Fig. 2, middle). Under ether anesthesia 4 to 7 Hz RSA occurred spontaneously in the records of all rats and could also be elicited by light tail pinches. When atropine sulfate was administered RSA was no longer recorded (Fig. 2, bottom).
resistant RSA (4-6 Hz) is recorded during the immobility associated with preparation to walk or jump and during nonmovement periods of paradoxical sleep (5). The atropine-resistant RSA is thought to be serotonergic and its pathway is thought to project from the median raphe nucleus, through the lateral hypothalamic and septal regions, to the cingulum and supracallosal striae to enter the hippocampal formation via the entorhinal cortex. Atropine-sensitive RSA is thought to be cholinergic and it is sustained by projections from the medial septum (4,5). After removal of all neocortex, including the cingulate cortex in rats shortly after birth, we found apparently normal EEG in adulthood during spontaneous movements and during sleep. Furthermore, both the atropineresistant and atropine-sensitive forms of RSA were present. Therefore, the main finding of the present study is that all of the forms of hippocampal EEG activity recorded in the normal rat can be obtained in the neonatally decorticated rat. One additional novel feature of the present results is that atropine-resistant RSA can be recorded after complete neocortical removal. When the cingulate cortex or entorhinal cortex is removed in adult rats, this form of EEG is abolished. This has been taken as suggesting that the serotonergic pathway sustaining this EEG courses through these structures. Since this pathway was cut by the neonatal decortications, it must be the case that: 1) serotonergic fibers that normally project through the cingulum bundle take an alternate route to the hippocampus following neonatal lesions or, 2) other serotonergic pathways (3) to the hippocampus are modified to support RSA.
DISCUSSION
The normal rat hippocampus produces two kinds of RSA. Atropine-resistant (and ether sensitive) RSA (6-12 Hz) is recorded during movements, such as walking or rearing or during movements in paradoxical sleep, whereas atropine-sensitive and ether-
ACKNOWLEDGEMENTS
This research was supported by grants from the Natural Sciences and Engineering Council of Canada. The authors thank Stan L.-W. Leung for assistance with the experiments.
REFERENCES 1. Kolb, B.; Sutherland, R. J.; Whishaw, I. Q. Abnormalities in cortical and subcortical morphology after neonatal neocortical lesions in rats. Exp. Neural. 79:223-244; 1983. 2. Kolb, B.; Whishaw, I. Q.; Van Der Kooy. Brain development in the neonatally decorticated rat. Brain Res. 379:315-326; 1986. 3. T&k, I. Raphe nuclei and serotonin containing systems. In: Paxinos, G., ed. The rat nervous system. vol. 2. Sydney: Academic Press; 1985:43-78. 4. Vanderwolf, C. H.; Leung, L.-W. S. Hippocampal rhythmical slow activity: A brief history and the effects of entorhinal lesions and phencyclidine. In: Seifert, W., ed. Neurobiology of the hippocampus.-New York Academic Press; 1983:275-302. 5. Vanderwolf, C. H.; Leung, L.-W. S.; Stewart, D. J. Two afferent pathways mediating hippocampal rhythmical slow activity. In: G. Buzsaki; C. H. Vanderwolf, eds. Electrical activity of the archicortex.
Budapest: Akademiai Kiado; 1985:4766. Whishaw, I. Q. The decorticate rat. In: Kolb, B.; Tees, R. C., eds. The cerebral cortex of the rat. Cambridge, MA: The MIT Press; 1990: 239-268. Whishaw, I. Q.; Dyck, R.; Kolb, B. Two types of rhythmical slow activity (theta) in the hippocampus of neonatally decorticate rats. Sot. Neurosci. Abstr. 9:1197; 1983. Whishaw, I. Q.; Kolb, B. Behavioral and anatomical studies of rats with complete or partial decortication in infancy. In: Finger, S.; Almli, C. R., eds. Early brain damage. vol. 2. New York Academic Press; 1984:117-138. Whishaw, I. Q.; Schallert, T.; Kolb, B. An analysis of feeding and sensorimotor abilities of rats after decortication. J. Comp. Physiol. Psychol. 95:85-103; 1981.
0361-9230191 $3.00 + .OO
0 Pergamon Press plc, 1991. Printed in the U.S.A
RAPID COMMUNICATION
Sparing of Two Types of Hippocampal Rhythmical Slow Activity (RSA, Theta) in Adult Rats Decorticated Neonatally IAN Q. WHISHAW,’
RICHARD
DYCK
AND BRYAN
KOLB
Department of Psychology, University of Lethbridge, Lethbridge, Alberta, Canada, TlK 3M4 Received
4 September
1990
WHISHAW, I. Q., R. DYCK AND B. KOLB. Sparing of two types of hippocampal rhythmical slow activity (RSA, theta) in adult rats decorticated neonatally. BRAIN RES BULL 26(3) 425427, 1991 .-The electroencephalographic (EEG) activity of the hippocampus was examined in adult freely moving rats that had had all of the neocortex, including the cingulate cortex and cingulum, removed at birth. Both the cholinergic and serotonergic forms of rhythmical slow activity (RSA or theta) were present in the dorsal hippocampus and retained their normal relation to behavior. The results show that following decortication, inputs to and connections within the hippocampus apparently retain the ability to produce normal EEG. Since adult decortications abolish serotonergic RSA, the results also suggest that following neonatal injury there is reorganization within remaining serotonergic projections to the hippocampus that spare this form of RSA. Theta rhythm
Hippocampus
Decorticate
rat
Rhythmical
dura. For the neonatally lesioned rats, the tissue covering the trepinated skull was cut and the hippocampus was visualized through a dissecting microscope. The electrodes were lowered so that the longer pole was inserted about 1 mm into the hippocampus and the shorter pole of the electrode rested on the surface of the hippocampus. At the completion of the behavioral experiments, the rats were sacrificed under deep pentobarbital anesthesia, perfused with saline and formalin, and their brains were removed. The location of the recording electrodes were subsequently confirmed in 40-p thick cresyl violet stained sections taken through the hippocampus (7). Electrodes were connected to preamplifiers on a Grass polygraph by a light flexible cable and grounded with shielding connected to a ground screw implanted into the skull overlying the cerebellum. Slow waves were analyzed by inspection, rating on a 4-point scale, and measurement with a plastic ruler. In addition, representative samples of slow-wave activity were stored on FM tape and were digitized so autopower and crosspower spectra could be constructed. All rats were tested in at least four recording sessions during which recordings were made during spontaneous behaviors including walking, rearing, immobility, grooming, eating and drinking, slow-wave and paradoxical sleep. Before one additional session, the rats were given atropine sulfate (50 mg/kg) and after samples of behavior were obtained, they were lightly anes-
removal of the neocortex and midline neonatal or adult rats, the hippocampal formation remains intact, its cross-sectional area remains unchanged, and its connections with the contralateral hippocampus, nucleus reunions, septum, diagonal band of Broca, and Raphe, etc., are retained (1, 2, 6-9). The present study reports that if rats are decorticated neonatally, then the EEG activity of the hippocampus also appears normal. FOLLOWING limbic cortex
complete
in either
METHOD The experiments used 12 male Long-Evans strain rats. Within hours of birth, the rats were anesthetized by cooling on ice. In six rats, the skull overlying the dorsal neocortex was removed and all of their neocortex from the midline, including cingulate and medial frontal cortex, lateral to the rhinal fissure, was removed by aspiration. The rats were then returned to their mothers. When the rats were adult (90 to 120 days) EEG recording electrodes were implanted. The electrodes consisted of bipolar 125pm stainless steel wires coated with Teflon except for the tip and attached to female Amphenol connectors. The rats were anesthetized with sodium pentobarbital (50 mg/kg) for stereotaxic implantation. In the control rats, electrodes were implanted 3 mm posterior to bregma, 2.5 mm lateral to the midline, and 2.5 mm ventral to the ‘Requests for reprints should be addressed
to Dr. I. Q. Whishaw,
slow activity
Department
3M4: Email: [email protected].
425
of Psychology,
University
of L&bridge,
Lethbridge,
Alberta,
TlK
426
WHISHAW,
walk
DYCK AND KOLH
CONTROL -
walk
atlll
walk
-
ATROPINE
walk
----
atIll
ETHER AND ATROPINE
----
ethor and
other and atroplnr
10 20 30 40 1 l ec
FREOUENCY
50
(C/S)
FIG. 2. Polygraph records and power spectra during walking and immobility in one decorticated rat: Control, undrugged; Atropine, following 50 mg/kg atropine sulfate. Ether and atropine, the rat was lightly anesthetized with ether and RSA was elicited with a light tail pinch and the procedure was repeated after atropine sulfate. Power on a logarithmic scale is plotted against frequency (O-100 Hz) on a linear scale. The power spectra shows RSA peaks at 8 Hz during the control and atropine conditions when the rat is walking but not when it is still. A 5-7 Hz RSA peak is shown under ether but not under ether and atropine. FIG. 1. Photomicrographs - _ of the extent of the decortication. The placement of the lower tip of the recording electrodes is shown in both hippo-
campi, just above the granule cells, in the third section (cresyl violet). For descriptions of anatomical peculiarities, i.e., subiculum (right) see (1, 2).
thetized with ether. In another session they were given ether followed by atropine sulfate. RESULTS
Ten of 12 electrode placements in the control rats produced good recordings of RSA (300-1000 (*V). Similar good recordings were obtained from at least one hippocampus in each decorticate rat, and from both hippocampi in two rats. In all eight placements in the decorticate rats, the electrodes were successfully placed with recording tips straddling CA1 (Fig. 1). For the
remaining placements the tips of the electrodes were not in the hippocampus or implanted into a portion of the hippocampus that was damaged. The EEG analysis was done on only those placements that were found on histological inspection to have been in the hippocampus. Electrical activity recorded from appropriately placed electrodes in the decorticate rats could not be distinguished by visual inspection from the EEG recorded from the control rats. For each rat, one hundred l-s samples of EEG were rated for the behaviors of immobility, chewing food, lapping water, and grooming. Large amplitude irregular activity (LIA) was the predominant waveform in these samples and there was no difference in its incidence between the two groups [>80% of samples in each group were LIA, rs(lO)< 1, p>O.O5]. Similar analysis of EEG during walking indicated that RSA was invariably present in both
THETA ACTIVITY IN DECORTICATE
421
RATS
groups [>95% of samples in each group were RSA, rs(lO)
resistant RSA (4-6 Hz) is recorded during the immobility associated with preparation to walk or jump and during nonmovement periods of paradoxical sleep (5). The atropine-resistant RSA is thought to be serotonergic and its pathway is thought to project from the median raphe nucleus, through the lateral hypothalamic and septal regions, to the cingulum and supracallosal striae to enter the hippocampal formation via the entorhinal cortex. Atropine-sensitive RSA is thought to be cholinergic and it is sustained by projections from the medial septum (4,5). After removal of all neocortex, including the cingulate cortex in rats shortly after birth, we found apparently normal EEG in adulthood during spontaneous movements and during sleep. Furthermore, both the atropineresistant and atropine-sensitive forms of RSA were present. Therefore, the main finding of the present study is that all of the forms of hippocampal EEG activity recorded in the normal rat can be obtained in the neonatally decorticated rat. One additional novel feature of the present results is that atropine-resistant RSA can be recorded after complete neocortical removal. When the cingulate cortex or entorhinal cortex is removed in adult rats, this form of EEG is abolished. This has been taken as suggesting that the serotonergic pathway sustaining this EEG courses through these structures. Since this pathway was cut by the neonatal decortications, it must be the case that: 1) serotonergic fibers that normally project through the cingulum bundle take an alternate route to the hippocampus following neonatal lesions or, 2) other serotonergic pathways (3) to the hippocampus are modified to support RSA.
DISCUSSION
The normal rat hippocampus produces two kinds of RSA. Atropine-resistant (and ether sensitive) RSA (6-12 Hz) is recorded during movements, such as walking or rearing or during movements in paradoxical sleep, whereas atropine-sensitive and ether-
ACKNOWLEDGEMENTS
This research was supported by grants from the Natural Sciences and Engineering Council of Canada. The authors thank Stan L.-W. Leung for assistance with the experiments.
REFERENCES 1. Kolb, B.; Sutherland, R. J.; Whishaw, I. Q. Abnormalities in cortical and subcortical morphology after neonatal neocortical lesions in rats. Exp. Neural. 79:223-244; 1983. 2. Kolb, B.; Whishaw, I. Q.; Van Der Kooy. Brain development in the neonatally decorticated rat. Brain Res. 379:315-326; 1986. 3. T&k, I. Raphe nuclei and serotonin containing systems. In: Paxinos, G., ed. The rat nervous system. vol. 2. Sydney: Academic Press; 1985:43-78. 4. Vanderwolf, C. H.; Leung, L.-W. S. Hippocampal rhythmical slow activity: A brief history and the effects of entorhinal lesions and phencyclidine. In: Seifert, W., ed. Neurobiology of the hippocampus.-New York Academic Press; 1983:275-302. 5. Vanderwolf, C. H.; Leung, L.-W. S.; Stewart, D. J. Two afferent pathways mediating hippocampal rhythmical slow activity. In: G. Buzsaki; C. H. Vanderwolf, eds. Electrical activity of the archicortex.
Budapest: Akademiai Kiado; 1985:4766. Whishaw, I. Q. The decorticate rat. In: Kolb, B.; Tees, R. C., eds. The cerebral cortex of the rat. Cambridge, MA: The MIT Press; 1990: 239-268. Whishaw, I. Q.; Dyck, R.; Kolb, B. Two types of rhythmical slow activity (theta) in the hippocampus of neonatally decorticate rats. Sot. Neurosci. Abstr. 9:1197; 1983. Whishaw, I. Q.; Kolb, B. Behavioral and anatomical studies of rats with complete or partial decortication in infancy. In: Finger, S.; Almli, C. R., eds. Early brain damage. vol. 2. New York Academic Press; 1984:117-138. Whishaw, I. Q.; Schallert, T.; Kolb, B. An analysis of feeding and sensorimotor abilities of rats after decortication. J. Comp. Physiol. Psychol. 95:85-103; 1981.