Journal of Comparative and Physiological Psychology 1975, Vol. 89, No. 9, 1053-1080
Cholinergic Modulation of Tonic Immobility in the Rabbit (Oryctolagus cuniculus) Daniel C. Hatton University of Florida
Michael L. Woodruff Middlebunj College Merle E. Meyer University of Florida
The present study was performed to determine the effects of the anticholinergic agent scopolamine and the cholinergic agent physostigmine on tonic immobility in rabbits. Recordings of the electroencephalographic (EEG) activity from cortex and hippocampus were also made before, during, and after each test session. Scopolamine significantly prolonged the response and produced large amplitude slow wave activity in the EEG of both cortex and hippocampus. Physostigmine significantly shortened the duration of immobility and increased rhythmic slow activity in the frequency range of 5.5-9.1 Hz in the hippocampus while producing a desynchronized cortical rhythm. It is suggested that the cortex and hippocampus play a role in modulating tonic immobility duration by inhibiting the brain-stem structures thought to control this response.
Rabbits generally react to sudden inverIn addition, the electrical activity of the sion and restraint with an inhibition of cortex and hippocampus was monitored movement that may last as long as 30 min before, during, and after induction of TI. or more. This response has been called ani- This was done for two reasons. First, physomal hypnosis but is probably better de- stigmine and scopolamine produce distincscribed by the term "tonic immobility" tive changes in the electroencephalogram (TI; Klemm, 1971b). It is characterized by (EEG) obtained from either neocortex or a reduction in reactivity to external stimu- hippocampus. Scopolamine abolishes the lation, loss of the righting reflex, and depres- 3.5-7.5 Hz activity (theta), which is characsion of flexor and extensor polysynaptic re- teristic of the hippocampus of the rabbit flexes. In a recent report Woodruff, Hatton, (Stumpf, 1965), and induces high-voltage, and Meyer (1975) observed that hippo- irregular slow waves with a mixture of some campal lesions in rabbits significantly pro- lower voltage fast activity in both hippolonged the duration of tonic immobility. campus and neocortex. Physostigmine, on From the data it could be predicted that the the other hand, induces a desynchronization anticholinergic agent scopolamine given to of the neocortical rhythm, while potentiating the rabbit would prolong TI, because, as the theta, or rhythmical slow activity noted by Carlton (1969) and Suits and Isaac- (RSA), of the hippocampus. The EEG reson (1969), scopolamine produces many of cordings were used in the present study as a the same behavioral effects that hippocampal measure of the effectiveness of the drug inlesions produce. It might also be predicted jections, which was separate from the bethat physostigmine, a drug that facilitates havioral measure. Second, Harper (1971) cholinergic activity through its action on reported that low-frequency RSA appeared acetylcholinesterase, would shorten this re- continuously in the hippocampus of the sponse. One purpose of the present study was rabbit during the early stages of TI. This to test the effects of scopolamine and physo- rhythm was replaced by large amplitude, irregular slow activity (LIA) if the response stigmine on TI in the rabbit. continued for a sufficiently long period of Requests for reprints should be sent to Merle E. Meyer, Department of Psychology, University time. Recordings were made of hippocampal electrical activity in undrugged rabbits durof Florida, Gainesville, Florida 32611. 1053
1054
D. C. HATTON, M. L. WOODRUFF, AND M. E. MEYER
ing TI in an attempt to replicate this distinction between the hippocampal EEG correlates of short and long TI responses. METHOD Subjects The subjects were 13 adult male and female Dutch Belt rabbits (Oryctolagus cuniculus), weighing between 1,300 and 2,200 g. They were housed in groups of two or three with food and water available ad lib. A 12:12 hr light/dark cycle was in effect for the colony room.
Preoperative Behavioral Testing Each rabbit was subjected to an immobilization test session prior to implantation of recording electrodes. The rabbit was transported from the colony room to the test room in a large box. The rabbit was taken from the transport box and placed in an upright position in a wooden Vshaped trough as previously described (Woodruff et al., 1975). Immobility was induced 15 sec later by rapidly inverting the rabbit and forcibly restraining it in the inverted position by pressing the thorax with one hand. After 15 sec had elapsed, the hand was slowly withdrawn and a clock started to time the duration of the response. If the rabbit did not become immobile within 15 sec, it was allowed to right itself and remain in the upright position for an additional 15 sec before another trial commenced. Three consecutive induction trials were given in this manner.
Operative Procedure Surgical anesthesia was induced by means of an injection of sodium pentobarbital through the marginal ear vein. No standard dose was used because previous experience indicated that anesthesia could be induced more readily and without loss of animals due to overdose if the corneal reflex and reaction to pinching of the toe were used as indexes of depth of anesthesia. A piece of cord was tied around the upper rib cage to facilitate breathing by reflex excitation of the respiratory centers. The scalp was shaved and the rabbit placed in a stereotaxic instrument. Under clean surgical conditions a midline incision was made in the scalp to expose the dorsolateral skull. Screw holes were drilled over sensorimotor neocortex, and stainless steel screws were epidurally positioned. Leads from these screws were connected to female Amphenol pins which were inserted in an Amphenol strip. An additional screw was implanted over the frontal bone to provide an indifferent recording point. Two additional holes were drilled bilaterally over the region of the dorsal hippocampus, and twisted bipolar electrodes were lowered into this structure. These electrodes were made from .1-mm stainless steel wire insulated
except for .5 mm at the tip and with a tip separation of 1.0 mm. Their resistance in saline was between 40 and 50 kJ2. These electrodes were cemented in place with dental acrylic; their leads, and a lead from the frontal reference screw, were attached to female Amphenol pins which were inserted in the Amphenol strip. Additional acrylic anchored the entire assembly to the rabbit's skull. The scalp was then drawn up around the acrylic and closed with 9.0-mm Clay-Adams wound clips. An injection of 100,000 U of Bicillin was given to combat possible postoperative infection.
Postoperative Behavioral Testing The subjects were allowed 14 days for postoperative recovery. With the same procedure for obtaining TI as was described above, the rabbits were given three consecutive immobility trials to determine the effect of the implant and recording procedures on the response. Fifteen minutes before attempting to induce TI, the rabbit was placed in the trough and connected to four channels of a Grass Model 7 polygraph. The rabbit was allowed 10 min to habituate before recording its EEG. The EEG was taken for 5 min before the first attempt at inducing TI. The EEG recordings were made during all TI inductions and for 5 min following the end of the third TI period.
Drug Testing Each rabbit was tested under the following drug conditions: .25, .1, and .4 mg/kg scopolamine hydrobromide; .015, .04, and .15 mg/kg physostigmine. Seven rabbits received the dosages of scopolamine in a random sequence before receiving a saline control injection followed by physostigmine in a random sequence. For the remaining six the opposite sequence was given. In all cases the saline injection was the fourth injection received. All injections were given intravenously in either the medial or marginal ear vein. The same general procedure as previously described was used to test for immobility in the drug conditions. Upon placement of the rabbit in the trough, the electrodes were connected to the polygraph. After 5 min of recording, the rabbit was given an iv injection of the appropriate drug and an additional 5-min recording was made. Following the three immobility trials, a further 5-min recording was made. Each animal was allowed a minimum of 3 days for recovery following each immobility test session.
Histology Following its final TI test session, each rabbit was sacrificed with an overdose of sodium pentobarbital. It was then intracardially perfused with .9% saline, followed by 10% formalin. The brains were removed, embedded in celloidin, and coronally sectioned at 30 /urn. Every fifth section was retained, slide mounted, and stained with thionin.
CHOLINERGIC SYSTEM AND TONIC IMMOBILITY The sections were examined for electrode place' .„
ment
KESULTS
1055
pocampus were found to have successfully penetrated the dorsal convexity of this structure and were located primarily in the region of the CA-3 field of the pyramidal cells.
Histology
Electrical Recording Before Drug The epidural cortical screws produced no Adrmmstratum observable effect upon the neocortex. The Figure la presents a sample of the neotips of all electrodes directed toward the hip- cortical and hippocampal records from a rab-
MAw*^^ la
RC
3M|jV
RC
RH
/yMl\iA/^W\^ FIGURE 1. Sample electroencephalographic activity from neocortex and hippocampus of a rabbit just prior to immobilization (la) and after 5 min of tonic immobility (Ib). (Abbreviations: LC and RC = left and right sensorimotor cortex; LH and RH = left and right hippocampus. Calibration: a: 100 juV, 1 sec; b: 300 nV, 1 sec.)
1056
D. C. HATTON, M. L. WOODRUFF, AND M. E. MEYER
TABLE 1 MEAN AND RANGE OF FREQUENCY (IN Hz) AND OF AMPLITUDE (IN juV) OF HIPPOCAMPAL AND CORTICAL, ELECTROENCEPHALOGRAM BEFOBE IMMOBILITY, DURING RSA IMMOBILITY, AND DURING LIA IMMOBILITY Before
Electroencephalogram Frequency
During Amplitude
Frequency
RSA
During LIA
Amplitude
Frequency
Amplitude
6.2 5.5-6.8
550-618
7.1 6.2-7.5
549-811
Hippocampus M
Range Cortex M
Range
6.0
240
6.0
242
5.6-6.3
180-287
5.6-6.5
188-281
18.1 16.3-20.1
75 48-91
18.7 16.6-19.8
51-91
68
604 756
Note: Abbreviations: RSA = rhythmical slow activity; LIA = large amplitude, irregular slow activity.
bit sitting upright in the trough just prior to immobilization. Table 1 presents the mean and range of the frequency and amplitude of the neocortical and hippocampal activity as measured with a clear plastic ruler during the 60 sec just prior to inversion of the rabbit. The mean frequency of the hippocampal activity during this period was 6.0 Hz, and the mean amplitude was 240 ftV peak to peak. The waveform, which was nearly sinusoidal, and frequency of this activity were within the limits usually referred to as "theta" or rhythmical slow activity (RSA). The neocortical EEG during this period was characterized by desynchronized activity of about 18 Hz mixed with slower waves in the 6-8 Hz range. The predominant fast activity had a mean amplitude of 75 /u.V, and the slower waves averaged 125 //V. It should be emphasized that the rabbits were not exhibiting gross body movements during this period, though nose and vibrissae were twitching. During the first 175 sec after induction of TI, the hippocampal rhythm continued in the RSA pattern with a mean frequency of 6.0 Hz. The neocortical record did not change either (Table 1). Amplitudes also remained unchanged. For trials in which TI terminated within 175 sec (12 trials in eight rabbits; range 22-175 sec), this pattern of hippocampal and cortical EEG did not change, even up to the point when the rabbits righted themselves. This period of time will be referred to as RSA immobility. The EEG at the time of righting was totally obscured
by movement artifact which continued for 5-15 sec after righting. After the righting movement the rabbits remained sitting in the trough as they had before induction of TI, and the hippocampal and cortical EEG records were indistinguishable from those taken before induction. The initial inspection of the EEG records taken during TI made it apparent that although the EEG activity was unchanged in the first 2 min after induction of TI, alterations did occur when the immobility period extended for about 3 min or more. One rabbit did not immobilize for more than 123 sec in the test session before any drugs were given and never demonstrated an EEG that differed from the EEG it presented before and after TI. The remaining 12 rabbits had 18 TI trials of more than 175 sec. In all 18 rabbits the RSA activity of the hippocampus and the desynchronized rhythm of the cortex were replaced by large amplitude, irregular activity (LIA). As can be seen in Figure Ib and as has been described by Vanderwolf, Bland, and Wishaw (1973), LIA lacks the regularity of RSA and exhibits occasional sharp waves with a duration of between 50-100 msec and amplitude that is often two or more times that of the background. This change occurred from 175 to 201 sec (M = 191 sec) after induction of TI for these 18 trials. This period of TI is referred to as LIA immobility and had a duration of from 22 to 116 sec as measured from the point during TI when the EEG changed. Because the duration of LIA immobility
CHOLINERGIC SYSTEM AND TONIC IMMOBILITY
1057
varied, it seemed of importance to determine Electrical Recording After Drug whether the length of the period influenced Administration the amplitude and frequency of the LIA. To The lowest doses of scopolamine (.025 mg/ this end the frequency and amplitude of kg) and physostigmine (.015 mg/kg) had no three 10-sec periods were measured. The effects on either hippocampal or cortical first 10 sec began with the onset of LIA and EEG, using the recording and measuring was monitored for all TI trials in which LIA techniques of this study. Physostigmine at occurred. The second measurement was the two higher dose levels tended to increase taken from 40 to 50 sec after the beginning both the frequency and amplitude of hippoof LIA in subjects that had remained immo- campal RSA as well as the regularity of the bile for this amount of time (n = 11). The activity. The .04 mg/kg dose of physostigthird measurement was taken for the final mine had the effect of increasing RSA fre10 sec of TI trials that had been included in quency to a mean of 7.2 Hz (Table 2). These the first two measurements and had then measurements were taken as above for the continued for at least 10 sec more (n = 9). 60-sec period just prior to inversion. The amThe results indicated that neither the fre- plitude of RSA did not demonstrate a conquency nor the amplitude showed consistent sistent change. The regularity of the wavechange over time. Therefore, these measure- form appeared to increase. The fast compoments were averaged together. The results nent of the cortical rhythm after .04 mg/kg are presented in Table 1. Mean frequency of physostigmine was unchanged, but the numLIA was within the 5.5-6.8 Hz range for the ber of slower waves diminished. The .15 hippocampus during this period, and the mg/kg dose of physostigmine increased the mean amplitude increased to 604 pV. The mean frequency of hippocampal RSA still neocortical record showed LIA activity of a further to 8.3 Hz (Table 2) as well as increasslightly higher frequency (M — 7.1 Hz) ing the amplitude of the RSA. As can be which was mixed with some faster activity seen in Figure 2a the waveform of the RSA at a lower voltage. The cortical LIA activity also appeared to increase in regularity when had a mean amplitude of 756 /uV. physostigmine was given. The neocortical TABLE 2 MEAN AND RANGE OF FREQUENCY (IN Hz) AND OF AMPLITUDE (IN ^V) OF HIPPOCAMPAL AND CORTICAL ELECTROENCEPHALOGHAPHY UNDER DIFFERENT DOSE LEVELS OF PHYSOSTIGMINE AND SCOPOLAMINE BEFORE INDUCTION OF TONIC IMMOBILITY Encephalography
Frequency
Frequency
Amplitude
Range Cortex
6.1 5.5-6.6
200-248
18.7 15,9-21.2
84 52-97
235
DESYN
M Range
Hippocampus M Range Cortex
M Range
RSA 7.2 6.7-7.8
265 182-317
18.3 16.0-19.8
79 56-93
Scopolamine: .1 mg/kg
RSA 256 178-294
18.8 17.6-20.9
88 61-104
DESYN
307 274-339
18.6 16.2-20.3
75 52-91
.4 mg/kg LIA
LIA 6.3 5.4-6.8
8.3 7.5-9.1
DESYN
DESYN
.025 mg/kg
Amplitude
.15 mg/kg
RSA
RSA
M
Frequency
Physostigmine: .04 mg/kg
.015 mg/kg Hippocampus
Amplitude
7.2 5.1-8.3
334 203-486
4.6 4.2-5.8
308
7.1 5.5-8.1
423 266-574
5.3 4.4-5.9
407 222-591
LIA
LIA
107-412
Note. Abbreviations: RSA = rhythmical slow activity; DESYN = desynchronized; LIA = large amplitude, irregular slo tivity.
1058
D. C. HATTON, M. L. WOODRUFF, AND M. E. MEYER LC
'^M^S^vA^V^AvV'^AVVvrw^
2a
RC
Cholinergic Modulation of Tonic Immobility in the Rabbit (Oryctolagus cuniculus) Daniel C. Hatton University of Florida
Michael L. Woodruff Middlebunj College Merle E. Meyer University of Florida
The present study was performed to determine the effects of the anticholinergic agent scopolamine and the cholinergic agent physostigmine on tonic immobility in rabbits. Recordings of the electroencephalographic (EEG) activity from cortex and hippocampus were also made before, during, and after each test session. Scopolamine significantly prolonged the response and produced large amplitude slow wave activity in the EEG of both cortex and hippocampus. Physostigmine significantly shortened the duration of immobility and increased rhythmic slow activity in the frequency range of 5.5-9.1 Hz in the hippocampus while producing a desynchronized cortical rhythm. It is suggested that the cortex and hippocampus play a role in modulating tonic immobility duration by inhibiting the brain-stem structures thought to control this response.
Rabbits generally react to sudden inverIn addition, the electrical activity of the sion and restraint with an inhibition of cortex and hippocampus was monitored movement that may last as long as 30 min before, during, and after induction of TI. or more. This response has been called ani- This was done for two reasons. First, physomal hypnosis but is probably better de- stigmine and scopolamine produce distincscribed by the term "tonic immobility" tive changes in the electroencephalogram (TI; Klemm, 1971b). It is characterized by (EEG) obtained from either neocortex or a reduction in reactivity to external stimu- hippocampus. Scopolamine abolishes the lation, loss of the righting reflex, and depres- 3.5-7.5 Hz activity (theta), which is characsion of flexor and extensor polysynaptic re- teristic of the hippocampus of the rabbit flexes. In a recent report Woodruff, Hatton, (Stumpf, 1965), and induces high-voltage, and Meyer (1975) observed that hippo- irregular slow waves with a mixture of some campal lesions in rabbits significantly pro- lower voltage fast activity in both hippolonged the duration of tonic immobility. campus and neocortex. Physostigmine, on From the data it could be predicted that the the other hand, induces a desynchronization anticholinergic agent scopolamine given to of the neocortical rhythm, while potentiating the rabbit would prolong TI, because, as the theta, or rhythmical slow activity noted by Carlton (1969) and Suits and Isaac- (RSA), of the hippocampus. The EEG reson (1969), scopolamine produces many of cordings were used in the present study as a the same behavioral effects that hippocampal measure of the effectiveness of the drug inlesions produce. It might also be predicted jections, which was separate from the bethat physostigmine, a drug that facilitates havioral measure. Second, Harper (1971) cholinergic activity through its action on reported that low-frequency RSA appeared acetylcholinesterase, would shorten this re- continuously in the hippocampus of the sponse. One purpose of the present study was rabbit during the early stages of TI. This to test the effects of scopolamine and physo- rhythm was replaced by large amplitude, irregular slow activity (LIA) if the response stigmine on TI in the rabbit. continued for a sufficiently long period of Requests for reprints should be sent to Merle E. Meyer, Department of Psychology, University time. Recordings were made of hippocampal electrical activity in undrugged rabbits durof Florida, Gainesville, Florida 32611. 1053
1054
D. C. HATTON, M. L. WOODRUFF, AND M. E. MEYER
ing TI in an attempt to replicate this distinction between the hippocampal EEG correlates of short and long TI responses. METHOD Subjects The subjects were 13 adult male and female Dutch Belt rabbits (Oryctolagus cuniculus), weighing between 1,300 and 2,200 g. They were housed in groups of two or three with food and water available ad lib. A 12:12 hr light/dark cycle was in effect for the colony room.
Preoperative Behavioral Testing Each rabbit was subjected to an immobilization test session prior to implantation of recording electrodes. The rabbit was transported from the colony room to the test room in a large box. The rabbit was taken from the transport box and placed in an upright position in a wooden Vshaped trough as previously described (Woodruff et al., 1975). Immobility was induced 15 sec later by rapidly inverting the rabbit and forcibly restraining it in the inverted position by pressing the thorax with one hand. After 15 sec had elapsed, the hand was slowly withdrawn and a clock started to time the duration of the response. If the rabbit did not become immobile within 15 sec, it was allowed to right itself and remain in the upright position for an additional 15 sec before another trial commenced. Three consecutive induction trials were given in this manner.
Operative Procedure Surgical anesthesia was induced by means of an injection of sodium pentobarbital through the marginal ear vein. No standard dose was used because previous experience indicated that anesthesia could be induced more readily and without loss of animals due to overdose if the corneal reflex and reaction to pinching of the toe were used as indexes of depth of anesthesia. A piece of cord was tied around the upper rib cage to facilitate breathing by reflex excitation of the respiratory centers. The scalp was shaved and the rabbit placed in a stereotaxic instrument. Under clean surgical conditions a midline incision was made in the scalp to expose the dorsolateral skull. Screw holes were drilled over sensorimotor neocortex, and stainless steel screws were epidurally positioned. Leads from these screws were connected to female Amphenol pins which were inserted in an Amphenol strip. An additional screw was implanted over the frontal bone to provide an indifferent recording point. Two additional holes were drilled bilaterally over the region of the dorsal hippocampus, and twisted bipolar electrodes were lowered into this structure. These electrodes were made from .1-mm stainless steel wire insulated
except for .5 mm at the tip and with a tip separation of 1.0 mm. Their resistance in saline was between 40 and 50 kJ2. These electrodes were cemented in place with dental acrylic; their leads, and a lead from the frontal reference screw, were attached to female Amphenol pins which were inserted in the Amphenol strip. Additional acrylic anchored the entire assembly to the rabbit's skull. The scalp was then drawn up around the acrylic and closed with 9.0-mm Clay-Adams wound clips. An injection of 100,000 U of Bicillin was given to combat possible postoperative infection.
Postoperative Behavioral Testing The subjects were allowed 14 days for postoperative recovery. With the same procedure for obtaining TI as was described above, the rabbits were given three consecutive immobility trials to determine the effect of the implant and recording procedures on the response. Fifteen minutes before attempting to induce TI, the rabbit was placed in the trough and connected to four channels of a Grass Model 7 polygraph. The rabbit was allowed 10 min to habituate before recording its EEG. The EEG was taken for 5 min before the first attempt at inducing TI. The EEG recordings were made during all TI inductions and for 5 min following the end of the third TI period.
Drug Testing Each rabbit was tested under the following drug conditions: .25, .1, and .4 mg/kg scopolamine hydrobromide; .015, .04, and .15 mg/kg physostigmine. Seven rabbits received the dosages of scopolamine in a random sequence before receiving a saline control injection followed by physostigmine in a random sequence. For the remaining six the opposite sequence was given. In all cases the saline injection was the fourth injection received. All injections were given intravenously in either the medial or marginal ear vein. The same general procedure as previously described was used to test for immobility in the drug conditions. Upon placement of the rabbit in the trough, the electrodes were connected to the polygraph. After 5 min of recording, the rabbit was given an iv injection of the appropriate drug and an additional 5-min recording was made. Following the three immobility trials, a further 5-min recording was made. Each animal was allowed a minimum of 3 days for recovery following each immobility test session.
Histology Following its final TI test session, each rabbit was sacrificed with an overdose of sodium pentobarbital. It was then intracardially perfused with .9% saline, followed by 10% formalin. The brains were removed, embedded in celloidin, and coronally sectioned at 30 /urn. Every fifth section was retained, slide mounted, and stained with thionin.
CHOLINERGIC SYSTEM AND TONIC IMMOBILITY The sections were examined for electrode place' .„
ment
KESULTS
1055
pocampus were found to have successfully penetrated the dorsal convexity of this structure and were located primarily in the region of the CA-3 field of the pyramidal cells.
Histology
Electrical Recording Before Drug The epidural cortical screws produced no Adrmmstratum observable effect upon the neocortex. The Figure la presents a sample of the neotips of all electrodes directed toward the hip- cortical and hippocampal records from a rab-
MAw*^^ la
RC
3M|jV
RC
RH
/yMl\iA/^W\^ FIGURE 1. Sample electroencephalographic activity from neocortex and hippocampus of a rabbit just prior to immobilization (la) and after 5 min of tonic immobility (Ib). (Abbreviations: LC and RC = left and right sensorimotor cortex; LH and RH = left and right hippocampus. Calibration: a: 100 juV, 1 sec; b: 300 nV, 1 sec.)
1056
D. C. HATTON, M. L. WOODRUFF, AND M. E. MEYER
TABLE 1 MEAN AND RANGE OF FREQUENCY (IN Hz) AND OF AMPLITUDE (IN juV) OF HIPPOCAMPAL AND CORTICAL, ELECTROENCEPHALOGRAM BEFOBE IMMOBILITY, DURING RSA IMMOBILITY, AND DURING LIA IMMOBILITY Before
Electroencephalogram Frequency
During Amplitude
Frequency
RSA
During LIA
Amplitude
Frequency
Amplitude
6.2 5.5-6.8
550-618
7.1 6.2-7.5
549-811
Hippocampus M
Range Cortex M
Range
6.0
240
6.0
242
5.6-6.3
180-287
5.6-6.5
188-281
18.1 16.3-20.1
75 48-91
18.7 16.6-19.8
51-91
68
604 756
Note: Abbreviations: RSA = rhythmical slow activity; LIA = large amplitude, irregular slow activity.
bit sitting upright in the trough just prior to immobilization. Table 1 presents the mean and range of the frequency and amplitude of the neocortical and hippocampal activity as measured with a clear plastic ruler during the 60 sec just prior to inversion of the rabbit. The mean frequency of the hippocampal activity during this period was 6.0 Hz, and the mean amplitude was 240 ftV peak to peak. The waveform, which was nearly sinusoidal, and frequency of this activity were within the limits usually referred to as "theta" or rhythmical slow activity (RSA). The neocortical EEG during this period was characterized by desynchronized activity of about 18 Hz mixed with slower waves in the 6-8 Hz range. The predominant fast activity had a mean amplitude of 75 /u.V, and the slower waves averaged 125 //V. It should be emphasized that the rabbits were not exhibiting gross body movements during this period, though nose and vibrissae were twitching. During the first 175 sec after induction of TI, the hippocampal rhythm continued in the RSA pattern with a mean frequency of 6.0 Hz. The neocortical record did not change either (Table 1). Amplitudes also remained unchanged. For trials in which TI terminated within 175 sec (12 trials in eight rabbits; range 22-175 sec), this pattern of hippocampal and cortical EEG did not change, even up to the point when the rabbits righted themselves. This period of time will be referred to as RSA immobility. The EEG at the time of righting was totally obscured
by movement artifact which continued for 5-15 sec after righting. After the righting movement the rabbits remained sitting in the trough as they had before induction of TI, and the hippocampal and cortical EEG records were indistinguishable from those taken before induction. The initial inspection of the EEG records taken during TI made it apparent that although the EEG activity was unchanged in the first 2 min after induction of TI, alterations did occur when the immobility period extended for about 3 min or more. One rabbit did not immobilize for more than 123 sec in the test session before any drugs were given and never demonstrated an EEG that differed from the EEG it presented before and after TI. The remaining 12 rabbits had 18 TI trials of more than 175 sec. In all 18 rabbits the RSA activity of the hippocampus and the desynchronized rhythm of the cortex were replaced by large amplitude, irregular activity (LIA). As can be seen in Figure Ib and as has been described by Vanderwolf, Bland, and Wishaw (1973), LIA lacks the regularity of RSA and exhibits occasional sharp waves with a duration of between 50-100 msec and amplitude that is often two or more times that of the background. This change occurred from 175 to 201 sec (M = 191 sec) after induction of TI for these 18 trials. This period of TI is referred to as LIA immobility and had a duration of from 22 to 116 sec as measured from the point during TI when the EEG changed. Because the duration of LIA immobility
CHOLINERGIC SYSTEM AND TONIC IMMOBILITY
1057
varied, it seemed of importance to determine Electrical Recording After Drug whether the length of the period influenced Administration the amplitude and frequency of the LIA. To The lowest doses of scopolamine (.025 mg/ this end the frequency and amplitude of kg) and physostigmine (.015 mg/kg) had no three 10-sec periods were measured. The effects on either hippocampal or cortical first 10 sec began with the onset of LIA and EEG, using the recording and measuring was monitored for all TI trials in which LIA techniques of this study. Physostigmine at occurred. The second measurement was the two higher dose levels tended to increase taken from 40 to 50 sec after the beginning both the frequency and amplitude of hippoof LIA in subjects that had remained immo- campal RSA as well as the regularity of the bile for this amount of time (n = 11). The activity. The .04 mg/kg dose of physostigthird measurement was taken for the final mine had the effect of increasing RSA fre10 sec of TI trials that had been included in quency to a mean of 7.2 Hz (Table 2). These the first two measurements and had then measurements were taken as above for the continued for at least 10 sec more (n = 9). 60-sec period just prior to inversion. The amThe results indicated that neither the fre- plitude of RSA did not demonstrate a conquency nor the amplitude showed consistent sistent change. The regularity of the wavechange over time. Therefore, these measure- form appeared to increase. The fast compoments were averaged together. The results nent of the cortical rhythm after .04 mg/kg are presented in Table 1. Mean frequency of physostigmine was unchanged, but the numLIA was within the 5.5-6.8 Hz range for the ber of slower waves diminished. The .15 hippocampus during this period, and the mg/kg dose of physostigmine increased the mean amplitude increased to 604 pV. The mean frequency of hippocampal RSA still neocortical record showed LIA activity of a further to 8.3 Hz (Table 2) as well as increasslightly higher frequency (M — 7.1 Hz) ing the amplitude of the RSA. As can be which was mixed with some faster activity seen in Figure 2a the waveform of the RSA at a lower voltage. The cortical LIA activity also appeared to increase in regularity when had a mean amplitude of 756 /uV. physostigmine was given. The neocortical TABLE 2 MEAN AND RANGE OF FREQUENCY (IN Hz) AND OF AMPLITUDE (IN ^V) OF HIPPOCAMPAL AND CORTICAL ELECTROENCEPHALOGHAPHY UNDER DIFFERENT DOSE LEVELS OF PHYSOSTIGMINE AND SCOPOLAMINE BEFORE INDUCTION OF TONIC IMMOBILITY Encephalography
Frequency
Frequency
Amplitude
Range Cortex
6.1 5.5-6.6
200-248
18.7 15,9-21.2
84 52-97
235
DESYN
M Range
Hippocampus M Range Cortex
M Range
RSA 7.2 6.7-7.8
265 182-317
18.3 16.0-19.8
79 56-93
Scopolamine: .1 mg/kg
RSA 256 178-294
18.8 17.6-20.9
88 61-104
DESYN
307 274-339
18.6 16.2-20.3
75 52-91
.4 mg/kg LIA
LIA 6.3 5.4-6.8
8.3 7.5-9.1
DESYN
DESYN
.025 mg/kg
Amplitude
.15 mg/kg
RSA
RSA
M
Frequency
Physostigmine: .04 mg/kg
.015 mg/kg Hippocampus
Amplitude
7.2 5.1-8.3
334 203-486
4.6 4.2-5.8
308
7.1 5.5-8.1
423 266-574
5.3 4.4-5.9
407 222-591
LIA
LIA
107-412
Note. Abbreviations: RSA = rhythmical slow activity; DESYN = desynchronized; LIA = large amplitude, irregular slo tivity.
1058
D. C. HATTON, M. L. WOODRUFF, AND M. E. MEYER LC
'^M^S^vA^V^AvV'^AVVvrw^
2a
RC
