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The relationship between type 2 theta and behavior
Physiology & Behavior, Vol. 33, pp. 621-626. Copyright 0 Pergnrnon Press Ltd., 1984. Printed in the U.S.A.
0031-9384/&i $3.00 + .oo
The Relationship Between TYPe 2 Theta and Behavior’ R. S. SAINSBURY
Department
of Psychology,
AND C. P. MONTOYA
University of Calgary, Calgary, Alberta, Canada Received
27 October
1983
SAINSBURY, R. S. AND C. P. MONTOYA. The relationships between type 2 theta and behoviur. PHYSIOL BEHAV 33(4) 621-626, 1984.-Although there is extensive literature on the correlation between Type 1 theta activity in the hippocampus and behavior, little is known about the behavioral correlates of “Type 2” (immobility) theta. In the present study, guinea pigs were exposed to three high “arousal” situations. These situations produced reliable trains of “Type 2” theta. Atropine sulfate abolished ‘arousal” and Type 2 theta. In the second study, we determined whether the Type 2 theta response was produced by specific stimuli or by a generalized sensitization to incoming sensory stimuli created by the testing situation. In this study, a stimulus which had not previously elicited Type 2 theta was presented a second time after a presentation of a stimulus which did elicit Type 2 theta. The previously neutral stimulus now elicited Type 2 theta. These results were interpreted within an “arousaI” theory framework.
Theta
Type 2
Atropine
Arousal
tones, hand claps, hand waves, stroking of the fur). The rat rarely produces Type 2 theta when immobility occurs during spontaneous behavior. However, short trains of Type 2 theta have been observed in the rat during conditioning studies 1231. The existence of a correlation between Type 1 or Type 2 theta and behavior in the cat is more controversial Several investigators [4, 6, 121 have found theta occurring both during movement and during immobility. Preliminary work in our laboratory demonstrated a lack of correlation between motor movements and theta rhythms indicating that cats may only generate Type 2 theta. Systematic attempts to correlate Type 2 theta with behavior are lacking. Although some investigators have suggested that Type 2 theta occurs during sensory processing in the rabbit [2, 3, 41, it is difficult to specify the conditions necessary for its elicitation as the rabbit seems to produce Type 2 theta in response to a wide variety of sensory stimuli. Since the rat rarely produces Type 2 theta in the restricted environmental setting and cat theta remains in dispute, we have chosen the guinea pig as a model to systematically investigate the relationship between behavior and Type 2 theta. The guinea pig shows an excellent motor correlation with Type 1 theta [ 161 and they also show some small trains of spontaneous Type 2 theta. In an attempt to reliably produce Type 2 theta, we presented a number of stimuli, including tones and tape recordings of owl calls as well as the physical presence of female conspecifics and live snakes. In the second study we manipulated the presentation of the stimuli in order to understand the nature of the behavioral correlation.
A number of earlier papers [13, 15, 17, 201 have demonstrated a correlation between hippocampal theta activity and what has been termed “voluntary” motor activities in the rat, gerbil and guinea pig. Several studies reported observing theta activity coincident with immobility in rabbits and cats [ 11, 12, 13, 261. Subsequent behavioral and pharmacological manipulations demonstrated two types of theta activity in both the rat and the rabbit [14,21,22, 231. One type (labelled Type 1) has a higher overall frequency range (6-12 Hz), occurs during voluntary movement (e.g., walking, running, rearing), and is resistant to atropine, but sensitive to anesthetic agents such as urethane, ethyl ether and alcohol. The second type (labelled Type 2 theta) has a lower frequency range (4-9 Hz), occurs during immobility, and is resistant to most anesthetics but sensitive to atropine. These data, together with the finding that eserine elicits Type 2 theta in both rats and rabbits [21] suggested that Type 2 theta is mediated by cholinergic inputs to the hippocampal formation. Electrophysiological studies have shown that theta is generated in the stratum oriens layer of the CA1 pyramidal region of the hippocampus proper and the stratum moleculare layer of the dentate gyrus of the rat [6,27], rabbit [28] and cat [I]. Both types of theta can be recorded from these two generators, and suggests that theta activity results from cholinergic and noncholinergic inputs to each generator. While it appears that rats and rabbits exhibit both types of theta, there are obvious species differences with respect to the production of Type 2 theta during spontaneouslyoccurring behavor [ 151. The rabbit can produce long trains of Type 2 theta during immobility, both spontaneously and in response
to various types of sensory
‘This research
was supported
stimulation
by a grant (A0361)
(whistles,
from the Natural
Sciences and Engineering Research Council of Canada.
621
SAINSBURY
622
EXPERIMENT
1
METHOD
Subjects
Forty mixed strain male guinea pigs ranging in weight from 3OW300 grams, were used as subjects in this experiment. Twenty mixed strain female guinea pigs were used as stimuli during the experiment. Testing occurred during daylight hours. Surgery Subjects were anesthetized with sodium pentobarbital(25 mg/kg IP) and electrodes were stereotaxically implanted in each hippocampus. Movable electrolytically sharpened kynar insulated tungsten microelectrodes (1 tip; 5-10 megohm at 100 Hz) were used for recording. The ground electrode was a male subminiature Winchester connector soldered to a jeweler’s screw placed in the skull. A detailed description of the implantation and recording technique has been reported elsewhere [17]. The data presented here were all recorded from the dentate gyrus of the hippocampus. Signals from the brain were passed through a 9 pin electromechanical commutator and fed into a Grass Model 7 polygraph. The polygraph amplifiers were set at 1 and 35 Hz for the half amplitude low and high filter settings, respectively. In addition to chart recording, the signal was taken from the 56 output, frequency modulated, and stored on the audio channels of a BASF L750 chrome video cassette. Also stored at the same time and on the same tape was a split screen image of both the animal and the concomitant EEG tracing. A Sony SLO 2300 Beta Max recorder was used for recording the information. After the data was collected, portions of the taped recording were fed into a Cromemco System 3 computer. In order to maintain sample accuracy the EEG signal was sampled at a rate of 5120 samples/set. Slow motion playback of the video recorder permitted exact correlations between EEG recordings and animal behavior. Samples totalling at least 2 min of Type 1 and Type 2 theta were subjected to a Fast Fourier Transform which was then plotted. No apodizing or smoothing technique was used on the data. In addition, a frequency analysis was made of the first and second set of three waves in a theta segment. Behavioral
Testing
All testing was carried out in one of two boxes placed inside a 1.2~ 1.28~ 2.25 m sound-resistant room. Testing during sexual and snake presentation behavior was carried out in an open-topped 60x 105 x 26 cm copper-shielded plywood box. The remainder of the testing (i.e., auditory stimulation) was carried out in a shielded box (57.5x35x41.25 cm) containing a 6 in. 8 ohm speaker. Sounds were presented by a Sony TC-399 reel-to-reel tape recorder. Test stimuli. Each of the subjects received three presentations of the following stimuli in separate test sessions. In addition, subjects were given one session with each of the same stimuli following a 50 mg/kg injection of atropine sulfate (N=5). Subjects were also given a separate session with atropine methyl nitrate (N=5, 50 mg/kg) as a control for the peripheral vs. central effects of atropine. During all sessions motor behaviors were recorded including walking, running, and head movements. In addition motionless behavior was also continuously recorded.
AND MONTOYA
Snake stimulus. Each subject was placed in the recording cage with one or more 1.3 m garter snakes (Thamnophis elegans). Extreme care was taken not to allow the subject to physically injure the snake. Intervention was required on only two occasions. Owl stimulus. Subjects were presented with the sound of a great homed owl (10 set) embedded in a 5 min tape containing hawk, kestrel and owl sounds. The most effective sound was that of the great homed owl. Sounds were taken from a commercially available record [lo] and signal strength was maintained between 75 and 80 dB. Sexual stimulus. Subjects were placed with two or more female guinea pigs. The presence of female partners will always elicit sexual behavior from male guinea pigs. A complete description of the mating pattern has been reported elsewhere [ 161. RESULTS AND DISCUSSION
Behavioral Responses
to Test Stimuli
Snake stimulus. The responses of guinea pigs to a garter snake fell into three general categories. Approximately 90 percent of the animals showed extremely fast running movements to all areas of the cage, followed by rump swaggering [ 161 and piloerection. The last two components invariably occurred while the guinea pig was facing the snake. Brief (l-3 set) periods of immobility occurred during all of these components. In the second general category (5% of subjects) the above components were present but were followed by rapid approach and withdrawal movements towards the snake. During the approach or attack segment the guinea pig attempted to bite the snake. The third category (5% of subjects) of response could best be described as escape responding. Here the guinea pigs showed fast running around the cage but avoided any contact with the snake. In addition these subjects spent considerable time facing a comer away from the snake. If the snake approached, the guinea pig fled. The response of the snake was very much determined by the response of the guinea pig. If the guinea pig was aggressive the snake would attempt to flee and often strike at the guinea pig. If, on the other hand, the guinea pig attempted to escape, the snake would slowly pursue the guinea pig. However, in this instance aggression was not seen from the snake. Owl stimulus. Initially the owl sound produced freezing in all animals. However after approximately 15-20 presentations within a session movements would occur. These usually involved walking and small head movements. Responses to the other hawk and kestrel sounds produced much less immobility, and indeed during some segments, chewing and grooming, as well as slow walking would occur. Sexual behavior. A complete description of the mating pattern of guinea pigs has been reported elsewhere [ 161. Spontaneous Type 2 theta rarely occurred during our experiments. Extraneous room noises normally failed to produce Type 2 theta. Type 2 theta could, however, be reliably produced during the three test situations. Figure 1 shows examples of both Type 1 and Type 2 theta taken during the various test stimuli. In this particular example of the snake stimulus, the guinea pig was motionless while staring at a coiled snake. The immobility period was both preceded and followed by a body movement. In the owl sound segment the animal remained motionless both prior to and after the owl sound. Periods of immobility during sexual stimulation resemble the
TYPE 2 THETA
623
AND BEHAVIOR
TYPE 1 THETFI
TYPE
NORMAL CONOITION
I THETR:
TYPE 2 THETFl A tsndd
FREQUENCY TYPE
II
0
THETA:
IN HZ
NORMAL CONDITION
m 0
FREQUENCY
FIG. 1. Examples of Type 1 and Type 2 theta taken from a single guinea pig. The second row illustrates large irregular waves (LIA) which normally occur during immobility. In contrast the three Type 2 examples show good theta during immobility.
Type 2 theta seen in the snake section of Fig. 1, i.e., sandwiched between body movements. Type 2 theta showed no signs of habituation either within or between sessions where sexual stimulation or snakes were used. Habituation occurred after approximately 20 presentations of the owl sound within a session but would reappear between sessions. A frequency histogram of Type 1 and Type 2 theta may be found in Fig. 2. The histograms depicted in Fig. 2 are each composed to a total of two min of randomly sampled theta segments taken from all subjects used in the experiment. From Fig. 2 one can see that the peak frequency of 2 min of sampled Type 1 theta was 8 cps while Type 2 theta had a peak frequency of 7 cps. A detailed analysis of the first and second set of three waves in a theta sequence revealed a_higher mean frequency for the first three waves (Type 1; X=8.65 N=60; Type 2; X=7.65 N=60) than for the second three waves (Type 1; X=7.87 N=60, Type 2; X=6.63 N=60). Although the overall mean indicated that the first three waves were of a higher frequency, this was not the case in 20% of the samples. Thus the results of the first phase of this study indicate that we have established good stimulus control over Type 2 theta in the guinea pig. As atropine sulfate has proven effective in eliminating Type 2 theta, the experiment was repeated using atropine sulfate as an experimental condition and atropine methyl nitrate as a control condition. Atropine sulfate abolished Type 2 theta during our stimulus conditions. An example of the effect may be seen in Fig. 3. The owl produced no theta
IN
HZ
FIG. 2. A Fast Fourier Transform analysis of a random 2 min sample of Type 1 theta (top) and Type 2 theta (bottom graph). The mean frequencies of Type 1 and Type 2 were 8.01 Hz and 7.13 Hz respectively.
ATS04
RTN03
FrCXZe sou-ld
owl
0.5mV
FIG. 3. The effects of atropine sulfate and atropine methyl nitrate on hippocampal EEG activity during the presentation of the owl sound stimulus. As one can see there is no evidence of theta rhythm when atropine sulfate is given.
activity in the guinea pig when the animal was treated with atropine sulfate while the response under atropine methyl nitrate appeared normal. Motor movements did, however, continue to produce Type 1 theta. While the elimination of Type 2 theta on the surface appears to be due to a strong drug effect, the site of action is not clear. While Type 2 theta was effectively abolished with atropine sulfate, the behavior of the animal was also drastically altered. This effect occurred in all animals and was most dramatically illustrated by
624
SAINSBURY
TYPE I THETA:
RTROPINE METHYL NITRATE CONOITION
g 2i E g E $
FREQUPKYIN HZ TYPE I THETA:
Type 2 theta under normal (X=7.13) or atropine methyl nitrate (X=7.10) conditions (diff=0.033, SSR=0.389, ~~0.05). Type 2 theta was however significantly slower than Type 1 theta regardless of condition (pcO.01 in all cases). Finally, we found that Type 1 theta under the atropine sulfate (x=8.42) condition had a significantly higher frequency than all other groups. (p
ATROPINE SULFATE CONDITION
1
E Z c( 6 g c $ FREOUENCY IN HZ FIG. 4. A Fast Fourier Transform analysis of a random sample of Type 1 theta under the ATNO condition (top) and Type 1 theta under the ATSO, condition (lower graph). Note the increase in frequency under ATSO, and the relative increase in amplitude of background frequencies.
the snake stimulus. Without exception animals treated with atropine sulfate failed to make any physical response to the snake. They remained motionless while the snake moved over or under them. This is in distinct contrast to the animated responses given in either the normal or atropine methyl nitrate condition. Similar results were found with our other stimuli. During the owl stimulus presentation the animal simply remained motionless. During sexual behavior, the animals moved only when pushed or sexually approached by other males. However, they showed no sexual behavior, and if not touched by the other animals, remained motionless. A frequency histogram of Type 1 theta under the atropine sulfate and the atropine methyl nitrate conditions may be found in Fig. 4. The data presented here are 2 min random samples taken from all subjects in each condition.As the histogram indicates Type 1 theta under the atropine sulfate condition is less consistent in frequency than theta seen when atropine methyl nitrate is used. An analysis of variance was performed on the mean frequencies seen during Type 2 theta (both under normal conditions and under atropine methyl nitrate) and Type 1 theta (under normal conditions and with atropine sulfate and atropine methyl nitrate). As the F value was significant, F(295)=24.5, ~~0.01, we performed a Duncan’s New Multiple Range Test. We found no difference between Type 1 theta under normal conditions (X=8.01) or when atropine methyl nitrate (X=7.45) was used (ditT=0.06, SSR=0.4173, p>O.O5). There was also no difference in the frequency of
AND MONTOYA
EXPERIMENT
2
In searching for stimuli which would produce Type 2 theta in our pilot research, we used many stimuli which had no apparent effect on the hippocampal EEG. These stimuli included pure tones, white noise, flashing lights and buzzers. In order to test the two hypotheses, animals were pretested with a stimulus which produced no EEG effect (neutral stim.) then tested with the owl stimulus and then tested again with the neutral stimulus. If the owl stimulus produced sensitization to incoming stimuli, then one would expect the animals to produce theta during the second presentation of the neutral stimulus. If, on the other hand, Type 2 theta production is linked directly to the types of stimuli, then no theta
should
be seen during
the second
presentation.
METHOD AND RESULTS
Ten male guinea pigs were used as subjects. techniques were as described in Experiment 1.
Recording
Procedure
Subjects were given daily presentations of the following sequence. They were placed in the recording box for 5 min then presented with one 1200 Hz tone for 8 set followed 30 set later by an 8 min prerecorded tape of owls, hawks and kestrels. Finally they were presented with an 8 set presentation of the 1200 Hz tone after a 30 set delay. All sounds were kept between 75-80 dB. An example of the response to the tone may be found in Fig. 5. All animals produced Type 2 theta in response to the owl tape and, in addition, produced Type 2 theta in response to the second presentation of the tone. We have reproduced this effect using numerous stimuli (e.g., lights, and other auditory stimuli). Thus, the results indicated that the owl stimulus sensitizes the animal to respond to a previously neutral stimulus with Type 2 theta. GENERAL
DISCUSSION
The results of the first experiment indicate that Type 2 theta is produced in situations which increase the animals’ “arousal.” Since at least two separate motivational states were used (i.e., fear and sex), the response is not motivation specific. Experiment 2 indicates that the production of Type 2 theta in the presence of a stimulus is dependent on the “arousal” level of the animal. Incidental evidence from individual differences seen between animals also supports this view. In the presence of both the snake and the female guinea pigs, subjects displaying the most agitation also
TYPE 2 THETA
625
AND BEHAVIOR
is not directly related to arousal but rather to sensory stimuli which occur during a high arousal state. The lower frequency of Type 2 theta as compared with Type 1 theta is consistent with data from both the rat and the rabbit [15]. The relationship between the frequency of the first three waves and the second three waves is of considerable interest. From Experiment 1 it appears that the onset frequency of a train of theta is of a higher frequency than the subsequent theta. While this result has previously been reported for Type 1 theta [5], it has not been noted for Type 2 theta. This finding suggests that a more detailed correlation should be made between onset frequency of Type 2 theta and the type of movement that does, on occasion, occur. In our analysis we found considerable overlap in the onset frequencies of Type 1 and Type 2 theta. This result suggests that overall analysis of a complete train of theta may be somewhat misleading. While the present experiments do not directly assess the relationship between Type 1 and Type 2 theta it does suggest that the behavioral correlates are different. There are three ways of looking at the relationship between the two types of theta. First, a number of investigators have suggested that the functional and pharmacological relationships between Type 1 and Type 2 theta are quite distinct [14, 15, 21, 221. The second conceptualization is to regard Type 2 theta as a precursor of Type 1 theta; that is, Type 2 theta occurs during the analysis of incoming sensory information and may concomitantly be related to the setting up of a subsequent motor program (e.g., an escape route) [3,4]. Unfortunately it is difficult to test this hypothesis because Type 2 theta is not always followed by a motor movement. However, it should be possible on those occasions when it does, to correlate the frequency of the Type 2 theta and the succeeding motor movements. If a relationship does exist this would suggest a motor program connection between Type 1 and Type 2 theta. The third possibility is that both Type 1 and Type 2 theta are correlated with sensory motor processing. Type 2 theta appears to occur during sensory processing and may or may not culminate in a motor movement. Type 1 theta may also be indicative of this kind of sensory processing but occurs while the organism is producing “voluntary” movements. By definition, voluntary movement requires an interaction with the environment. Thus, while the correlation is present with the movement, it would also be present with sensory processing. The differentiation of these three hypotheses remains to be tested. correlation
Pi-e Owl Stlm.
Post
owl stin. B.SnV I
l
TONE WHILE STILL
FIG. 5. The hippocampal EEG response to a 1200 Hz tone. In the top row little or no theta is present when a tone is presented before the presentation of the owl stimulus. The second row shows the response to the tone 30 set after the presentation of the owl sound stimulus. Type 2 theta is clearly evident.
tended to produce the most Type 2 theta during immobility. Thus, it appears that high arousal situations sensitize the animal to produce Type 2 theta in response to incoming sensory information. Data collected while the animal was under the influence of atropine sulfate are entirely consistent with that seen with the rabbit [14,21]. Earlier studies [18,19] have also found a cessation of sexual behavior following much lower doses of atropine than those used in the present study. In these earlier studies however, general motor activity was not reduced The large doses of atropine used in this study are levels that are necessary to abolish Type 2 theta [14,21] in most of the species tested. In the present study, animals given atropine sulfate showed very little motor activity, and showed essentially no physical response to the test stimuli. Animals no longer engaged in sexual behavior and became impassive to the snake even when the snake was under the subject’s body or moving over the subject’s head. These results indicate that a very low “arousal” level was in effect, and brings into question the exact nature of the atropine sulfate effect. It is now unclear whether atropine sulfate affects hippocampal production of theta per se or affects some other neural substrate which in turn produces a very low arousal level thus blocking a Type 2 theta response to incoming stimuli. The increase in frequency of Type 1 theta which accompanied the use of atropine sulfate suggests that this drug is not totally selective to the Type 2 system. The notion of “arousal” being linked to the production of theta is not new [9,12]. The difference noted here is that the
ACKNOWLEDGEMENT
Our thanks to B. H. Bland for his comments on the manuscript.
REFERENCES Bland, B. H., R. S. Sainsbury and B. Creery. Anatomical correlates of rhythmical slow wave activity (theta) in the hippocampal formation of the cat. Brain Res 161: 199-209, 1979. Bland, B. H., R. S. Sainsbury, M. Seto, B. R. Sinclair and I. Q. Whishaw. The use of sodium pentobarbital for the study of immobility-related (Type 2) hippocampal theta. Physiol Eehav 27: 363-368, 1981. Bland, B. H., M. Seto and C. Rowntree. The relation of multiple theta cell discharge rates to slow wave theta frequency. Physiol Behav
31:
111-I 17, 1983.
4. Bland, B. H., M. Seto, B. Sinclair and S. Fraser. The pharmacology of hippocampal theta cells: Evidence that the sensory process& correlate is-cholinergic. Brain Res, in press. 5. Bland. B. H. and C. H. Vanderwolf. Diencephalic and hippo&pal mechanisms of motor activity in the rat: Effects of posterior hypothalamic stimulation on behavior and hippocampal slow wave activity. Brain Res 43: 67-88, 1972. 6. Bland, B. H. and I. Q. Whishaw. Generators and topography of hippocampal theta (RSA) in the anaesthetized and freely moving rat. Brain Res 1lS: 259-280, 1976.
SAINSBURY
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7. Grastyan, E., Cl. Karmos, L. Vereczkey and L. Kelleyni. The hippocampal electrical correlates of the homeostatic regulation of motivation. Electroencephalogr Clin Neurophysiol21: 34-53, 1966. 8. Grastyan, E., K. Lissak, I. Madarasz and H. Donhoffer. Hippocampal electrical activity during the development of conditioned reflexes, Electroencephalogr Clin Neurophysiol 11: 4w30, 1959. 9. Green, J. D. and A. Arduini. Hippocampal electrical activity in arousal J Neurophysiol 17: 532-557, 1954. 10. Chum, W. W. H and P. P. Kellog (eds.) Peterson’s A Field Guide to Western Bird Songs. Boston, MA: Houghton Mifflin Company, 1975. 11. Harper, R. M. Frequency changes in hippocampal electrical activity during movement and tonic immobility. Physiol Behav 7:
18. Singer, J. The effects of atropine upon the female and male sexual behavior of female rats. Physiol Behav 3: 377-378, 1968. 19. Soulairac, A. and M. Soulairac. Action de L’Amphet-Amine, de L’Adrenaline et de L’Atropine sur le comportement: Sexuel du Rat Male. J Physiol (Paris) 49: 381-385, 1957. 20. Vanderwolf, C. H. Hippocampal electrical activity and volunClin Neurophystary movement in the rat. Electroencephalogr iol 26: 407-418, 1969. 21. Vanderwolf, C. H. Neocortical and hippocampal activation in relation to behavior: Effects of atropine, eserine, phenothiazines and amphetamine. J Comp Physiol Psycho1 88: 300-332, 1975. 22. Vanderwolf,
55-58, 1971. 12. Kemp, I. R. and B. R. Kaada. The relation of hippocampal theta
activity to arousal, attentive behaviour and somato-motor movements in unrestrained cats. Brain Res 95: 323-342, 1975. 13. Klemm, W. R. EEG and multiple-unit activity in limbic and motor systems during movement and immobility. Physiol Behav
23.
7: 337-343, 1971. 14. Kramis, R., C. H. Vanderwolf
24.
and B. H. Bland. Two types of hippocampal rhythmical slow activity (RSA) in both the rabbit and the rat: Relations to behavior and effects of atropine, diethyl ether, urethane, and pentobarbital. Exp New-01 49:
25.
58-85, 1975. 15. Robinson, T. E. Hippocampal
rhythmical slow activity (RSA; theta): A critical analysis of selected studies and discussion of possible species-differences. Brain Res Rev 2: 69-101, 1980. 16. Sainsbury, R. S. Hippocampal activity during natural behavior in the guinea pig. Physiol Behav 5: 317-324, 1970. 17. Sainsbury, R. S., C. P. Montoya, I. Westra and T. Mani. An inexpensive, direct-line, non-rotating, chronically implanted microdrive for small rodents. Physiol Behav 31: 729-731, 1983.
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26.
C. H., R. Kramis, L. A. Gillespie and B. H. Bland. Hippocampal rhythmical slow activity and neocortical lowvoltage fast activity. Relations to behavior. In: The Hippocampus, ~012, edited by R. L. Isaacson and K. H. Pribram. New York: Plenum Press, 1975, pp. 101-128. Whishaw, I. Q. Hippocampal electroencephalographic activity in the Mongolian gerbil during natural behaviors and wheel running and in the rat during wheel running and conditioned immobility. Can J Psycho1 26: 219-239, 1972. Whishaw, I. Q. A simple behavioral paradigm for the study of type I hippocampal rhythmical slow activity (RSA) frequency shifts. Physiol Behuv 29: 751-753, 1982. Whishaw, I. Q., B. H. Bland, T. E. Robinson and C. H. Vanderwolf. Two types of rhythmical slow activity (RSA) in hippocampus and dentate gyrus during neuromuscular blockage. Brain Res Bull 1: 573-581, 1976. Winson, J. Interspecies differences in the occurrence of theta.
Behuv Biol 7: 479-487, 1972. 27. Winson, J. Patterns of hippocampal theta rhythm in the freely Clin Neurophysiol36: 291-301, moving rat. Electroencephulogr 1974. 28. Wihson, J. Hippocampal theta rhythm: II. Depth profiles in the freely moving rabbit. Bruin Res 103: 71-79, 1976.
0031-9384/&i $3.00 + .oo
The Relationship Between TYPe 2 Theta and Behavior’ R. S. SAINSBURY
Department
of Psychology,
AND C. P. MONTOYA
University of Calgary, Calgary, Alberta, Canada Received
27 October
1983
SAINSBURY, R. S. AND C. P. MONTOYA. The relationships between type 2 theta and behoviur. PHYSIOL BEHAV 33(4) 621-626, 1984.-Although there is extensive literature on the correlation between Type 1 theta activity in the hippocampus and behavior, little is known about the behavioral correlates of “Type 2” (immobility) theta. In the present study, guinea pigs were exposed to three high “arousal” situations. These situations produced reliable trains of “Type 2” theta. Atropine sulfate abolished ‘arousal” and Type 2 theta. In the second study, we determined whether the Type 2 theta response was produced by specific stimuli or by a generalized sensitization to incoming sensory stimuli created by the testing situation. In this study, a stimulus which had not previously elicited Type 2 theta was presented a second time after a presentation of a stimulus which did elicit Type 2 theta. The previously neutral stimulus now elicited Type 2 theta. These results were interpreted within an “arousaI” theory framework.
Theta
Type 2
Atropine
Arousal
tones, hand claps, hand waves, stroking of the fur). The rat rarely produces Type 2 theta when immobility occurs during spontaneous behavior. However, short trains of Type 2 theta have been observed in the rat during conditioning studies 1231. The existence of a correlation between Type 1 or Type 2 theta and behavior in the cat is more controversial Several investigators [4, 6, 121 have found theta occurring both during movement and during immobility. Preliminary work in our laboratory demonstrated a lack of correlation between motor movements and theta rhythms indicating that cats may only generate Type 2 theta. Systematic attempts to correlate Type 2 theta with behavior are lacking. Although some investigators have suggested that Type 2 theta occurs during sensory processing in the rabbit [2, 3, 41, it is difficult to specify the conditions necessary for its elicitation as the rabbit seems to produce Type 2 theta in response to a wide variety of sensory stimuli. Since the rat rarely produces Type 2 theta in the restricted environmental setting and cat theta remains in dispute, we have chosen the guinea pig as a model to systematically investigate the relationship between behavior and Type 2 theta. The guinea pig shows an excellent motor correlation with Type 1 theta [ 161 and they also show some small trains of spontaneous Type 2 theta. In an attempt to reliably produce Type 2 theta, we presented a number of stimuli, including tones and tape recordings of owl calls as well as the physical presence of female conspecifics and live snakes. In the second study we manipulated the presentation of the stimuli in order to understand the nature of the behavioral correlation.
A number of earlier papers [13, 15, 17, 201 have demonstrated a correlation between hippocampal theta activity and what has been termed “voluntary” motor activities in the rat, gerbil and guinea pig. Several studies reported observing theta activity coincident with immobility in rabbits and cats [ 11, 12, 13, 261. Subsequent behavioral and pharmacological manipulations demonstrated two types of theta activity in both the rat and the rabbit [14,21,22, 231. One type (labelled Type 1) has a higher overall frequency range (6-12 Hz), occurs during voluntary movement (e.g., walking, running, rearing), and is resistant to atropine, but sensitive to anesthetic agents such as urethane, ethyl ether and alcohol. The second type (labelled Type 2 theta) has a lower frequency range (4-9 Hz), occurs during immobility, and is resistant to most anesthetics but sensitive to atropine. These data, together with the finding that eserine elicits Type 2 theta in both rats and rabbits [21] suggested that Type 2 theta is mediated by cholinergic inputs to the hippocampal formation. Electrophysiological studies have shown that theta is generated in the stratum oriens layer of the CA1 pyramidal region of the hippocampus proper and the stratum moleculare layer of the dentate gyrus of the rat [6,27], rabbit [28] and cat [I]. Both types of theta can be recorded from these two generators, and suggests that theta activity results from cholinergic and noncholinergic inputs to each generator. While it appears that rats and rabbits exhibit both types of theta, there are obvious species differences with respect to the production of Type 2 theta during spontaneouslyoccurring behavor [ 151. The rabbit can produce long trains of Type 2 theta during immobility, both spontaneously and in response
to various types of sensory
‘This research
was supported
stimulation
by a grant (A0361)
(whistles,
from the Natural
Sciences and Engineering Research Council of Canada.
621
SAINSBURY
622
EXPERIMENT
1
METHOD
Subjects
Forty mixed strain male guinea pigs ranging in weight from 3OW300 grams, were used as subjects in this experiment. Twenty mixed strain female guinea pigs were used as stimuli during the experiment. Testing occurred during daylight hours. Surgery Subjects were anesthetized with sodium pentobarbital(25 mg/kg IP) and electrodes were stereotaxically implanted in each hippocampus. Movable electrolytically sharpened kynar insulated tungsten microelectrodes (1 tip; 5-10 megohm at 100 Hz) were used for recording. The ground electrode was a male subminiature Winchester connector soldered to a jeweler’s screw placed in the skull. A detailed description of the implantation and recording technique has been reported elsewhere [17]. The data presented here were all recorded from the dentate gyrus of the hippocampus. Signals from the brain were passed through a 9 pin electromechanical commutator and fed into a Grass Model 7 polygraph. The polygraph amplifiers were set at 1 and 35 Hz for the half amplitude low and high filter settings, respectively. In addition to chart recording, the signal was taken from the 56 output, frequency modulated, and stored on the audio channels of a BASF L750 chrome video cassette. Also stored at the same time and on the same tape was a split screen image of both the animal and the concomitant EEG tracing. A Sony SLO 2300 Beta Max recorder was used for recording the information. After the data was collected, portions of the taped recording were fed into a Cromemco System 3 computer. In order to maintain sample accuracy the EEG signal was sampled at a rate of 5120 samples/set. Slow motion playback of the video recorder permitted exact correlations between EEG recordings and animal behavior. Samples totalling at least 2 min of Type 1 and Type 2 theta were subjected to a Fast Fourier Transform which was then plotted. No apodizing or smoothing technique was used on the data. In addition, a frequency analysis was made of the first and second set of three waves in a theta segment. Behavioral
Testing
All testing was carried out in one of two boxes placed inside a 1.2~ 1.28~ 2.25 m sound-resistant room. Testing during sexual and snake presentation behavior was carried out in an open-topped 60x 105 x 26 cm copper-shielded plywood box. The remainder of the testing (i.e., auditory stimulation) was carried out in a shielded box (57.5x35x41.25 cm) containing a 6 in. 8 ohm speaker. Sounds were presented by a Sony TC-399 reel-to-reel tape recorder. Test stimuli. Each of the subjects received three presentations of the following stimuli in separate test sessions. In addition, subjects were given one session with each of the same stimuli following a 50 mg/kg injection of atropine sulfate (N=5). Subjects were also given a separate session with atropine methyl nitrate (N=5, 50 mg/kg) as a control for the peripheral vs. central effects of atropine. During all sessions motor behaviors were recorded including walking, running, and head movements. In addition motionless behavior was also continuously recorded.
AND MONTOYA
Snake stimulus. Each subject was placed in the recording cage with one or more 1.3 m garter snakes (Thamnophis elegans). Extreme care was taken not to allow the subject to physically injure the snake. Intervention was required on only two occasions. Owl stimulus. Subjects were presented with the sound of a great homed owl (10 set) embedded in a 5 min tape containing hawk, kestrel and owl sounds. The most effective sound was that of the great homed owl. Sounds were taken from a commercially available record [lo] and signal strength was maintained between 75 and 80 dB. Sexual stimulus. Subjects were placed with two or more female guinea pigs. The presence of female partners will always elicit sexual behavior from male guinea pigs. A complete description of the mating pattern has been reported elsewhere [ 161. RESULTS AND DISCUSSION
Behavioral Responses
to Test Stimuli
Snake stimulus. The responses of guinea pigs to a garter snake fell into three general categories. Approximately 90 percent of the animals showed extremely fast running movements to all areas of the cage, followed by rump swaggering [ 161 and piloerection. The last two components invariably occurred while the guinea pig was facing the snake. Brief (l-3 set) periods of immobility occurred during all of these components. In the second general category (5% of subjects) the above components were present but were followed by rapid approach and withdrawal movements towards the snake. During the approach or attack segment the guinea pig attempted to bite the snake. The third category (5% of subjects) of response could best be described as escape responding. Here the guinea pigs showed fast running around the cage but avoided any contact with the snake. In addition these subjects spent considerable time facing a comer away from the snake. If the snake approached, the guinea pig fled. The response of the snake was very much determined by the response of the guinea pig. If the guinea pig was aggressive the snake would attempt to flee and often strike at the guinea pig. If, on the other hand, the guinea pig attempted to escape, the snake would slowly pursue the guinea pig. However, in this instance aggression was not seen from the snake. Owl stimulus. Initially the owl sound produced freezing in all animals. However after approximately 15-20 presentations within a session movements would occur. These usually involved walking and small head movements. Responses to the other hawk and kestrel sounds produced much less immobility, and indeed during some segments, chewing and grooming, as well as slow walking would occur. Sexual behavior. A complete description of the mating pattern of guinea pigs has been reported elsewhere [ 161. Spontaneous Type 2 theta rarely occurred during our experiments. Extraneous room noises normally failed to produce Type 2 theta. Type 2 theta could, however, be reliably produced during the three test situations. Figure 1 shows examples of both Type 1 and Type 2 theta taken during the various test stimuli. In this particular example of the snake stimulus, the guinea pig was motionless while staring at a coiled snake. The immobility period was both preceded and followed by a body movement. In the owl sound segment the animal remained motionless both prior to and after the owl sound. Periods of immobility during sexual stimulation resemble the
TYPE 2 THETA
623
AND BEHAVIOR
TYPE 1 THETFI
TYPE
NORMAL CONOITION
I THETR:
TYPE 2 THETFl A tsndd
FREQUENCY TYPE
II
0
THETA:
IN HZ
NORMAL CONDITION
m 0
FREQUENCY
FIG. 1. Examples of Type 1 and Type 2 theta taken from a single guinea pig. The second row illustrates large irregular waves (LIA) which normally occur during immobility. In contrast the three Type 2 examples show good theta during immobility.
Type 2 theta seen in the snake section of Fig. 1, i.e., sandwiched between body movements. Type 2 theta showed no signs of habituation either within or between sessions where sexual stimulation or snakes were used. Habituation occurred after approximately 20 presentations of the owl sound within a session but would reappear between sessions. A frequency histogram of Type 1 and Type 2 theta may be found in Fig. 2. The histograms depicted in Fig. 2 are each composed to a total of two min of randomly sampled theta segments taken from all subjects used in the experiment. From Fig. 2 one can see that the peak frequency of 2 min of sampled Type 1 theta was 8 cps while Type 2 theta had a peak frequency of 7 cps. A detailed analysis of the first and second set of three waves in a theta sequence revealed a_higher mean frequency for the first three waves (Type 1; X=8.65 N=60; Type 2; X=7.65 N=60) than for the second three waves (Type 1; X=7.87 N=60, Type 2; X=6.63 N=60). Although the overall mean indicated that the first three waves were of a higher frequency, this was not the case in 20% of the samples. Thus the results of the first phase of this study indicate that we have established good stimulus control over Type 2 theta in the guinea pig. As atropine sulfate has proven effective in eliminating Type 2 theta, the experiment was repeated using atropine sulfate as an experimental condition and atropine methyl nitrate as a control condition. Atropine sulfate abolished Type 2 theta during our stimulus conditions. An example of the effect may be seen in Fig. 3. The owl produced no theta
IN
HZ
FIG. 2. A Fast Fourier Transform analysis of a random 2 min sample of Type 1 theta (top) and Type 2 theta (bottom graph). The mean frequencies of Type 1 and Type 2 were 8.01 Hz and 7.13 Hz respectively.
ATS04
RTN03
FrCXZe sou-ld
owl
0.5mV
FIG. 3. The effects of atropine sulfate and atropine methyl nitrate on hippocampal EEG activity during the presentation of the owl sound stimulus. As one can see there is no evidence of theta rhythm when atropine sulfate is given.
activity in the guinea pig when the animal was treated with atropine sulfate while the response under atropine methyl nitrate appeared normal. Motor movements did, however, continue to produce Type 1 theta. While the elimination of Type 2 theta on the surface appears to be due to a strong drug effect, the site of action is not clear. While Type 2 theta was effectively abolished with atropine sulfate, the behavior of the animal was also drastically altered. This effect occurred in all animals and was most dramatically illustrated by
624
SAINSBURY
TYPE I THETA:
RTROPINE METHYL NITRATE CONOITION
g 2i E g E $
FREQUPKYIN HZ TYPE I THETA:
Type 2 theta under normal (X=7.13) or atropine methyl nitrate (X=7.10) conditions (diff=0.033, SSR=0.389, ~~0.05). Type 2 theta was however significantly slower than Type 1 theta regardless of condition (pcO.01 in all cases). Finally, we found that Type 1 theta under the atropine sulfate (x=8.42) condition had a significantly higher frequency than all other groups. (p
ATROPINE SULFATE CONDITION
1
E Z c( 6 g c $ FREOUENCY IN HZ FIG. 4. A Fast Fourier Transform analysis of a random sample of Type 1 theta under the ATNO condition (top) and Type 1 theta under the ATSO, condition (lower graph). Note the increase in frequency under ATSO, and the relative increase in amplitude of background frequencies.
the snake stimulus. Without exception animals treated with atropine sulfate failed to make any physical response to the snake. They remained motionless while the snake moved over or under them. This is in distinct contrast to the animated responses given in either the normal or atropine methyl nitrate condition. Similar results were found with our other stimuli. During the owl stimulus presentation the animal simply remained motionless. During sexual behavior, the animals moved only when pushed or sexually approached by other males. However, they showed no sexual behavior, and if not touched by the other animals, remained motionless. A frequency histogram of Type 1 theta under the atropine sulfate and the atropine methyl nitrate conditions may be found in Fig. 4. The data presented here are 2 min random samples taken from all subjects in each condition.As the histogram indicates Type 1 theta under the atropine sulfate condition is less consistent in frequency than theta seen when atropine methyl nitrate is used. An analysis of variance was performed on the mean frequencies seen during Type 2 theta (both under normal conditions and under atropine methyl nitrate) and Type 1 theta (under normal conditions and with atropine sulfate and atropine methyl nitrate). As the F value was significant, F(295)=24.5, ~~0.01, we performed a Duncan’s New Multiple Range Test. We found no difference between Type 1 theta under normal conditions (X=8.01) or when atropine methyl nitrate (X=7.45) was used (ditT=0.06, SSR=0.4173, p>O.O5). There was also no difference in the frequency of
AND MONTOYA
EXPERIMENT
2
In searching for stimuli which would produce Type 2 theta in our pilot research, we used many stimuli which had no apparent effect on the hippocampal EEG. These stimuli included pure tones, white noise, flashing lights and buzzers. In order to test the two hypotheses, animals were pretested with a stimulus which produced no EEG effect (neutral stim.) then tested with the owl stimulus and then tested again with the neutral stimulus. If the owl stimulus produced sensitization to incoming stimuli, then one would expect the animals to produce theta during the second presentation of the neutral stimulus. If, on the other hand, Type 2 theta production is linked directly to the types of stimuli, then no theta
should
be seen during
the second
presentation.
METHOD AND RESULTS
Ten male guinea pigs were used as subjects. techniques were as described in Experiment 1.
Recording
Procedure
Subjects were given daily presentations of the following sequence. They were placed in the recording box for 5 min then presented with one 1200 Hz tone for 8 set followed 30 set later by an 8 min prerecorded tape of owls, hawks and kestrels. Finally they were presented with an 8 set presentation of the 1200 Hz tone after a 30 set delay. All sounds were kept between 75-80 dB. An example of the response to the tone may be found in Fig. 5. All animals produced Type 2 theta in response to the owl tape and, in addition, produced Type 2 theta in response to the second presentation of the tone. We have reproduced this effect using numerous stimuli (e.g., lights, and other auditory stimuli). Thus, the results indicated that the owl stimulus sensitizes the animal to respond to a previously neutral stimulus with Type 2 theta. GENERAL
DISCUSSION
The results of the first experiment indicate that Type 2 theta is produced in situations which increase the animals’ “arousal.” Since at least two separate motivational states were used (i.e., fear and sex), the response is not motivation specific. Experiment 2 indicates that the production of Type 2 theta in the presence of a stimulus is dependent on the “arousal” level of the animal. Incidental evidence from individual differences seen between animals also supports this view. In the presence of both the snake and the female guinea pigs, subjects displaying the most agitation also
TYPE 2 THETA
625
AND BEHAVIOR
is not directly related to arousal but rather to sensory stimuli which occur during a high arousal state. The lower frequency of Type 2 theta as compared with Type 1 theta is consistent with data from both the rat and the rabbit [15]. The relationship between the frequency of the first three waves and the second three waves is of considerable interest. From Experiment 1 it appears that the onset frequency of a train of theta is of a higher frequency than the subsequent theta. While this result has previously been reported for Type 1 theta [5], it has not been noted for Type 2 theta. This finding suggests that a more detailed correlation should be made between onset frequency of Type 2 theta and the type of movement that does, on occasion, occur. In our analysis we found considerable overlap in the onset frequencies of Type 1 and Type 2 theta. This result suggests that overall analysis of a complete train of theta may be somewhat misleading. While the present experiments do not directly assess the relationship between Type 1 and Type 2 theta it does suggest that the behavioral correlates are different. There are three ways of looking at the relationship between the two types of theta. First, a number of investigators have suggested that the functional and pharmacological relationships between Type 1 and Type 2 theta are quite distinct [14, 15, 21, 221. The second conceptualization is to regard Type 2 theta as a precursor of Type 1 theta; that is, Type 2 theta occurs during the analysis of incoming sensory information and may concomitantly be related to the setting up of a subsequent motor program (e.g., an escape route) [3,4]. Unfortunately it is difficult to test this hypothesis because Type 2 theta is not always followed by a motor movement. However, it should be possible on those occasions when it does, to correlate the frequency of the Type 2 theta and the succeeding motor movements. If a relationship does exist this would suggest a motor program connection between Type 1 and Type 2 theta. The third possibility is that both Type 1 and Type 2 theta are correlated with sensory motor processing. Type 2 theta appears to occur during sensory processing and may or may not culminate in a motor movement. Type 1 theta may also be indicative of this kind of sensory processing but occurs while the organism is producing “voluntary” movements. By definition, voluntary movement requires an interaction with the environment. Thus, while the correlation is present with the movement, it would also be present with sensory processing. The differentiation of these three hypotheses remains to be tested. correlation
Pi-e Owl Stlm.
Post
owl stin. B.SnV I
l
TONE WHILE STILL
FIG. 5. The hippocampal EEG response to a 1200 Hz tone. In the top row little or no theta is present when a tone is presented before the presentation of the owl stimulus. The second row shows the response to the tone 30 set after the presentation of the owl sound stimulus. Type 2 theta is clearly evident.
tended to produce the most Type 2 theta during immobility. Thus, it appears that high arousal situations sensitize the animal to produce Type 2 theta in response to incoming sensory information. Data collected while the animal was under the influence of atropine sulfate are entirely consistent with that seen with the rabbit [14,21]. Earlier studies [18,19] have also found a cessation of sexual behavior following much lower doses of atropine than those used in the present study. In these earlier studies however, general motor activity was not reduced The large doses of atropine used in this study are levels that are necessary to abolish Type 2 theta [14,21] in most of the species tested. In the present study, animals given atropine sulfate showed very little motor activity, and showed essentially no physical response to the test stimuli. Animals no longer engaged in sexual behavior and became impassive to the snake even when the snake was under the subject’s body or moving over the subject’s head. These results indicate that a very low “arousal” level was in effect, and brings into question the exact nature of the atropine sulfate effect. It is now unclear whether atropine sulfate affects hippocampal production of theta per se or affects some other neural substrate which in turn produces a very low arousal level thus blocking a Type 2 theta response to incoming stimuli. The increase in frequency of Type 1 theta which accompanied the use of atropine sulfate suggests that this drug is not totally selective to the Type 2 system. The notion of “arousal” being linked to the production of theta is not new [9,12]. The difference noted here is that the
ACKNOWLEDGEMENT
Our thanks to B. H. Bland for his comments on the manuscript.
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