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Frequency analysis of rat hippocampal electrical activity
Physiology and Behavior, Vol. 14, pp. 391-394. Brain Research Publications Inc., 1975. Printed in the U.S.A.
BRIEF COMMUNICATION Frequency Analysis of Rat Hippocampal Electrical Activity A. M. L. COENEN l
Department of Comparative and Physiological Psychology University of Ni/megen, Erasmuslaan 16, Ni/megen, The Netherlands
(Received 4 June 1974) COENEN, A.M.L. Frequency analysis of rat hippocampal electrical activity. PHYSIOL. BEHAV. 14(3) 391-394, 1975. - Hippocampal electrical activity was recorded in free-moving rats and behavioral observations carried out simultaneously. The hippocampal EEG was analysed by an EEG spectrograph giving the amplitudes of 20 frequencies in the 2-30 Hz band in the form of spectrograms. Twenty spectrograms per sec were generated, often showing considerable fluctuations even during any one behavioral pattern. Therefore mean spectrograms of several behavioral patterns were constructed by averaging 256 spectrograms, which corresponds to an analysis time of 12.8 sec per behavioral pattern. This was also performed, in modified form, for the hippocampal EEG accompanying sleep. Spectrograms could be classified into three groups. The first group includes the spectrograms, composed of a theta EEG, made during patterns of voluntary behavior (walking, rearing, sniffing objects) and paradoxical sleep. The spectrograms accompanying patterns of automatic behavior (grooming head and body, eating, sniffing air) belong to the second group whereas those made during immobile behavior (sitting, lying, slow wave sleep) form the third group. In using this method the results of Vanderwolf and Whishaw were generally confirmed in that there exists a strong temporal relationship between hippocampal electrical activity and behavior. Hippocampal EEG
Frequency analysis
Averagingprocedure
A common procedure employed to obtain a quantitative analysis of a fluctuating signal is to determine its mean characteristics with their variances by means of an averaging technique. It is impossible, however, to apply such a technique directly to a spontaneously running EEG. In this paper a method is described in which the use of an EEG spectrograph, performing a frequency analysis and thereby generating series of discrete spectrograms, offers an opportunity to introduce an averaging procedure. This method is applied to the investigation into the relationship between behavior and electrical activity of the hippocampus. Basically two main patterns can be distinguished in this activity: rhythmical slow activity or theta rhythm and large amplitude irregular activity. Vanderwolf [3] and Whishaw and Vanderwolf [4] have shown that a number of behavioral patterns such as running, walking, rearing and swimming are accompanied by theta rhythm, whereas others such as grooming, licking, biting and immobile behavior are accompanied by large amplitude irregular activity. In the light of these data Vanderwolf [3 ] has postulated a theory suggesting that theta rhythm accompanies patterns of voluntary behavior whereas large amplitude irregular activity accompanies patterns of automatic and immobile behavior.
METHOD A tripolar electrode set (Plastic Products Company, MS 333) was chronically implanted in 15 well-handled adult male Wistar rats of about 3 0 0 - 4 0 0 g. The three electrodes were placed in the same coronal plane. Two electrodes, with vertical and lateral tip distances of 0.5 to 1 mm, were aimed at the left hippocampus and the third served as an earth electrode. Hippocampal coordinates, with skull surface horizontal, were 4.0 mm posterior to bregma, 3.0 mm lateral to the midline and 3.0 mm ventral to the skull surface. After allowing at least two weeks of recovery the experiments were started. The rat was connected to the equipment via an electrode cord and placed in a Plexiglas observation box (50 × 30 x 40 cm), with a layer of sawdust and some food pellets. Experimental sessions always started at the beginning of the active (dark) period of the d a y - n i g h t cycle and were carried out in red light for which the albino rat is particularly insensitive. In this way an observation period of about 1 hr proved sufficient for all types of investigated behavior. Behavioral patterns were manually coded on the channels of a tape recorder (Analog-7, Philips). One channel was used to record the electroencephalographic (EEG) activity,
The author is indebted to Prof. J. Vossen for supporting this investigation and to H. Bexkens and J. Arenis for assistance. 391
392
('OENEN
having a bandwidth of 1 - 7 0 Hz. The EEG and the behavioral codes could also be recorded on a polygraph (Minograf-800, Elema-Schbnander). The EEG was analysed by visual inspection of the polygraph recordings as well as more quantitatively by using an EEG spectrograph [1]. The spectrograph consisted of 20 parallel bandpass filters with a maximum response at respectively 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 24 and 28 Hz. (Fig. 1). The outputs of these filters were displayed as vertical lines on the screen of an oscilloscope. Consequently 20 vertical lines are visible; the horizontal position of the line indicating frequency, and the height of the line indicating amplitude. The bank of filters are scanned every 50 msec thus generating 20 spectrograms per sec. In this way an immediate visual display of the EEG characteristics is obtained. For further details of this technique see the paper of Bekkering et al. [ 1 ].
0.5
0,
FIG. 1. Amplitude and frequency characteristics of the filters in the EEG spectrograph. Spectrograms were averaged by a signal averager (Hewlett Packard 5480). The averager was triggered only during the presence of a specific behavioral code. In this way the mean frequency spectrograms of several behavioral patterns were obtained. Generally 256 spectrograms were averaged, corresponding to 12.8 sec of EEG analysis of any type of behvior. Significant changes after 256 sweeps were not obtained which indicates that this time period was sufficient. Spectrograms could also be reproduced on paper using an X/Y plotter. RESULTS AND DISCUSSION The behavioral patterns taken into account were those commonly occuring in a small observation box. The following patterns were distinguished: walking, rearing, sniffing in two forms, grooming also in two forms, eating, sitting and lying. Some comments must be made about these patterns of behavior. Walking was rather slow, occuring frequently but lasting only for short periods. This was also the case with rearing, by which is meant standing upright against a wall of the cage. Rearing is mostly accompanied by sniffing. The behavior we termed sniffing indicates sniffing only when the rat is standing still on four legs. Two types of sniffing are distinguished: sniffing objects such as pieces of sawdust, food pellets or faeces, and sniffing in the air. Both types of sniffing are usually accompanied by small head movements. Eating includes biting and gnawing on food pellets and is often accompanied by manipulating movements. Grooming is also distinguished by two distinct patterns: grooming of the head, whereby the rat sits on the hind legs washing the head and grooming of the body where
licking and biting of the fur is sometimes accompanied by rather gross movements. Sitting and lying are never accompanied by sniffing and other movements and did not include drowsy or sleepy states. A l m o s t i d e n t i c a l hippocampal EEG results were obtained from 12 out of 15 rats. Histological verification of the position of the electrode tips showed that in these rats the electrodes were implanted in the dorsal hippocampus close to the pyramidal cell layer, whereas the other rats had variable electrode placings. Mean spectrograms can be divided into three groups. The first group includes the spectrograms composed of a theta EEG with the main peak at about 8 Hz and smaller but clear one at 1 6 - 2 0 Hz. The spectrograms of walking, rearin_g and sniffing objects which are all types of voluntary behavior belong to this group. In the sequence given there is a small decrease in frequency of the main peak, a large decrease in amplitude of the 1 6 - 2 0 Hz peak and an increase of the standard deviations of most frequencies corresponding to a decrease in the regularity of the theta rhythm (Fig. 2). The EEG accompanying sniffing in the air, eating and grooming of the body and head display ahnost identical characteristics: a large amplitude irregular activity but still interspersed by theta waves. The spectrograms of these behavioral patterns, forming the second group, are characterized by a small, variable and relatively broad peak at about 7 Hz and an increased amount of 2 - 5 Hz activity (Fig. 2). The nature of the behavioral patterns included in this group strongly suggest a correspondence with what Vanderwolf [3]has termed automatic movements. It is remarkable that despite the gradual transition between the two groups, sniffing objects falls into the first group whereas sniffing in the air belongs to the second. This may indicate a more automatic or stereotyped behavior than is the case with sniffing objects. To the third group belong the spectrograms of sitting and lying which are both types of immobile behavior. This group is characterized by rather flat spectrograms composed of large amplitude irregular activity. The irregular character is clearly expressed in the increased variability of the frequencies in the spectrograms (Fig. 2). We were also interested in the EEG spectrograms of the different sleep phases of the hippocampal EEG. The method used for free moving rats, however, cannot be employed since it is difficult to distinguish these different phases by simple observation of the sleeping rat. At first, therefore, the hippocampal sleep EEG was classified by visual inspection into slow wave sleep and paradoxical sleep and mean spectrograms were separately constructed for these phases (Fig. 2). The mean spectrogram of slow wave sleep corresponds closely with those of immobile behavior and also displays a high variability. In the mean spectrogram of paradoxical sleep a peak at 8 Hz is visible and again a smaller one at 1 6 - 2 0 Hz. The spectrogram resembles those of voluntary behavior with a slightly increased variability in both frequency and amplitude which presumably can be explained through the fact that the presence or absence of movements was neglected during this type of sleep. The 1 6 - 2 0 peak is produced during various types of voluntary behavior as well as during paradoxical sleep. This is in contrast with Harper's work [2] on the rabbit in which the peak occurred during paradoxical sleep only. Presumably this peak is a second harmonic of the theta rhythm.
HIPPOCAMPAL ELECTRICAL ACTIVITY
WALKING
SNIFFINGOBJECT
SNIFFINGAIR
LYING ?
393
REhR~NG
PARADOXICALSLEEP
GROOMINGHEAD
SLOWWA~,~SLEEP
2~3 4 56 7 B 9 101112111415161~ ?4L~
FIG. 2. Examples of mean spectrograms (N = 256) of the hippocampal EEG of a representative rat. Each block of a spectrogram represents one frequency, the height indicates the amplitude of that frequency in arbitrary units. Standard deviations are indicated.
During this investigation into sleep a number of cases were noted in which a clear small amplitude irregular activity occurred following just after paradoxical sleep. The duration of this phase was some seconds and the main frequency about 4 0 - 5 0 Hz. At the m o m e n t the exact relationship between behavior and this EEG activity is still unclear and is under further investigation.
The method described in this paper allows accurate quantification of the hippocampal EEG and would be suitable for similar investigations. Implementation of the technique requires, however, that the EEG obtained during any given behavioral pattern does not significantly change but rather fluctuates around a distinct pattern only. This is the case in the hippocampal EEG, excepting the first few
394
('Ot:N F.N
h u n d r e d s of milliseconds of a v o l u n t a r y b e h a v i o r d u r i n g w h i c h m o v e m e n t i n i t i a t i o n is a c c o m p a n i e d b y a higher t h e t a f r e q u e n c y t h a n is t h e case d u r i n g s t e a d y m o v e m e n t [ 4 ] . In particular w h e n m a n y b r i e f periods are averaged (e.g., walking) this m a y lead to an increase of the peak
frequency. initiations time being differences
However, for the most part these m o v e m e n t will be cancelled o u t b y the o b s e r v e r ' s r e a c t i o n of the same o r d e r o f m a g n i t u d e so t h a t actual will be very small.
REFERENCES 1. Bekkering, D. H., A. Kamp and W. Storm van Leeuwen. The EEG - spectrograph. Electroenceph. clin. Neurophysiol. 10: 5 5 5 - 5 5 9 , 1958. 2. Harper, R.M. Frequency changes in hippocampat electrical activity during movement and tonic immobility. Physiol. Behav. 7: 55--58, 1971.
3. Vanderwolf, C. H. Hippocampal electrical activity and voluntary movement in the rat. Electroenceph. din. Neurophysiol. 26: 4 0 7 - 4 1 8 , 1969. 4. Whishaw, I.Q. and C.H. Vanderwolf. Hippocampal EEG and behavior: Change in amplitude and frequency of RSA (theta rhythm) associated with spontaneous and learned movement patterns in rats and cats. Behav. Biol. 8:461 484, 1973.
BRIEF COMMUNICATION Frequency Analysis of Rat Hippocampal Electrical Activity A. M. L. COENEN l
Department of Comparative and Physiological Psychology University of Ni/megen, Erasmuslaan 16, Ni/megen, The Netherlands
(Received 4 June 1974) COENEN, A.M.L. Frequency analysis of rat hippocampal electrical activity. PHYSIOL. BEHAV. 14(3) 391-394, 1975. - Hippocampal electrical activity was recorded in free-moving rats and behavioral observations carried out simultaneously. The hippocampal EEG was analysed by an EEG spectrograph giving the amplitudes of 20 frequencies in the 2-30 Hz band in the form of spectrograms. Twenty spectrograms per sec were generated, often showing considerable fluctuations even during any one behavioral pattern. Therefore mean spectrograms of several behavioral patterns were constructed by averaging 256 spectrograms, which corresponds to an analysis time of 12.8 sec per behavioral pattern. This was also performed, in modified form, for the hippocampal EEG accompanying sleep. Spectrograms could be classified into three groups. The first group includes the spectrograms, composed of a theta EEG, made during patterns of voluntary behavior (walking, rearing, sniffing objects) and paradoxical sleep. The spectrograms accompanying patterns of automatic behavior (grooming head and body, eating, sniffing air) belong to the second group whereas those made during immobile behavior (sitting, lying, slow wave sleep) form the third group. In using this method the results of Vanderwolf and Whishaw were generally confirmed in that there exists a strong temporal relationship between hippocampal electrical activity and behavior. Hippocampal EEG
Frequency analysis
Averagingprocedure
A common procedure employed to obtain a quantitative analysis of a fluctuating signal is to determine its mean characteristics with their variances by means of an averaging technique. It is impossible, however, to apply such a technique directly to a spontaneously running EEG. In this paper a method is described in which the use of an EEG spectrograph, performing a frequency analysis and thereby generating series of discrete spectrograms, offers an opportunity to introduce an averaging procedure. This method is applied to the investigation into the relationship between behavior and electrical activity of the hippocampus. Basically two main patterns can be distinguished in this activity: rhythmical slow activity or theta rhythm and large amplitude irregular activity. Vanderwolf [3] and Whishaw and Vanderwolf [4] have shown that a number of behavioral patterns such as running, walking, rearing and swimming are accompanied by theta rhythm, whereas others such as grooming, licking, biting and immobile behavior are accompanied by large amplitude irregular activity. In the light of these data Vanderwolf [3 ] has postulated a theory suggesting that theta rhythm accompanies patterns of voluntary behavior whereas large amplitude irregular activity accompanies patterns of automatic and immobile behavior.
METHOD A tripolar electrode set (Plastic Products Company, MS 333) was chronically implanted in 15 well-handled adult male Wistar rats of about 3 0 0 - 4 0 0 g. The three electrodes were placed in the same coronal plane. Two electrodes, with vertical and lateral tip distances of 0.5 to 1 mm, were aimed at the left hippocampus and the third served as an earth electrode. Hippocampal coordinates, with skull surface horizontal, were 4.0 mm posterior to bregma, 3.0 mm lateral to the midline and 3.0 mm ventral to the skull surface. After allowing at least two weeks of recovery the experiments were started. The rat was connected to the equipment via an electrode cord and placed in a Plexiglas observation box (50 × 30 x 40 cm), with a layer of sawdust and some food pellets. Experimental sessions always started at the beginning of the active (dark) period of the d a y - n i g h t cycle and were carried out in red light for which the albino rat is particularly insensitive. In this way an observation period of about 1 hr proved sufficient for all types of investigated behavior. Behavioral patterns were manually coded on the channels of a tape recorder (Analog-7, Philips). One channel was used to record the electroencephalographic (EEG) activity,
The author is indebted to Prof. J. Vossen for supporting this investigation and to H. Bexkens and J. Arenis for assistance. 391
392
('OENEN
having a bandwidth of 1 - 7 0 Hz. The EEG and the behavioral codes could also be recorded on a polygraph (Minograf-800, Elema-Schbnander). The EEG was analysed by visual inspection of the polygraph recordings as well as more quantitatively by using an EEG spectrograph [1]. The spectrograph consisted of 20 parallel bandpass filters with a maximum response at respectively 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 24 and 28 Hz. (Fig. 1). The outputs of these filters were displayed as vertical lines on the screen of an oscilloscope. Consequently 20 vertical lines are visible; the horizontal position of the line indicating frequency, and the height of the line indicating amplitude. The bank of filters are scanned every 50 msec thus generating 20 spectrograms per sec. In this way an immediate visual display of the EEG characteristics is obtained. For further details of this technique see the paper of Bekkering et al. [ 1 ].
0.5
0,
FIG. 1. Amplitude and frequency characteristics of the filters in the EEG spectrograph. Spectrograms were averaged by a signal averager (Hewlett Packard 5480). The averager was triggered only during the presence of a specific behavioral code. In this way the mean frequency spectrograms of several behavioral patterns were obtained. Generally 256 spectrograms were averaged, corresponding to 12.8 sec of EEG analysis of any type of behvior. Significant changes after 256 sweeps were not obtained which indicates that this time period was sufficient. Spectrograms could also be reproduced on paper using an X/Y plotter. RESULTS AND DISCUSSION The behavioral patterns taken into account were those commonly occuring in a small observation box. The following patterns were distinguished: walking, rearing, sniffing in two forms, grooming also in two forms, eating, sitting and lying. Some comments must be made about these patterns of behavior. Walking was rather slow, occuring frequently but lasting only for short periods. This was also the case with rearing, by which is meant standing upright against a wall of the cage. Rearing is mostly accompanied by sniffing. The behavior we termed sniffing indicates sniffing only when the rat is standing still on four legs. Two types of sniffing are distinguished: sniffing objects such as pieces of sawdust, food pellets or faeces, and sniffing in the air. Both types of sniffing are usually accompanied by small head movements. Eating includes biting and gnawing on food pellets and is often accompanied by manipulating movements. Grooming is also distinguished by two distinct patterns: grooming of the head, whereby the rat sits on the hind legs washing the head and grooming of the body where
licking and biting of the fur is sometimes accompanied by rather gross movements. Sitting and lying are never accompanied by sniffing and other movements and did not include drowsy or sleepy states. A l m o s t i d e n t i c a l hippocampal EEG results were obtained from 12 out of 15 rats. Histological verification of the position of the electrode tips showed that in these rats the electrodes were implanted in the dorsal hippocampus close to the pyramidal cell layer, whereas the other rats had variable electrode placings. Mean spectrograms can be divided into three groups. The first group includes the spectrograms composed of a theta EEG with the main peak at about 8 Hz and smaller but clear one at 1 6 - 2 0 Hz. The spectrograms of walking, rearin_g and sniffing objects which are all types of voluntary behavior belong to this group. In the sequence given there is a small decrease in frequency of the main peak, a large decrease in amplitude of the 1 6 - 2 0 Hz peak and an increase of the standard deviations of most frequencies corresponding to a decrease in the regularity of the theta rhythm (Fig. 2). The EEG accompanying sniffing in the air, eating and grooming of the body and head display ahnost identical characteristics: a large amplitude irregular activity but still interspersed by theta waves. The spectrograms of these behavioral patterns, forming the second group, are characterized by a small, variable and relatively broad peak at about 7 Hz and an increased amount of 2 - 5 Hz activity (Fig. 2). The nature of the behavioral patterns included in this group strongly suggest a correspondence with what Vanderwolf [3]has termed automatic movements. It is remarkable that despite the gradual transition between the two groups, sniffing objects falls into the first group whereas sniffing in the air belongs to the second. This may indicate a more automatic or stereotyped behavior than is the case with sniffing objects. To the third group belong the spectrograms of sitting and lying which are both types of immobile behavior. This group is characterized by rather flat spectrograms composed of large amplitude irregular activity. The irregular character is clearly expressed in the increased variability of the frequencies in the spectrograms (Fig. 2). We were also interested in the EEG spectrograms of the different sleep phases of the hippocampal EEG. The method used for free moving rats, however, cannot be employed since it is difficult to distinguish these different phases by simple observation of the sleeping rat. At first, therefore, the hippocampal sleep EEG was classified by visual inspection into slow wave sleep and paradoxical sleep and mean spectrograms were separately constructed for these phases (Fig. 2). The mean spectrogram of slow wave sleep corresponds closely with those of immobile behavior and also displays a high variability. In the mean spectrogram of paradoxical sleep a peak at 8 Hz is visible and again a smaller one at 1 6 - 2 0 Hz. The spectrogram resembles those of voluntary behavior with a slightly increased variability in both frequency and amplitude which presumably can be explained through the fact that the presence or absence of movements was neglected during this type of sleep. The 1 6 - 2 0 peak is produced during various types of voluntary behavior as well as during paradoxical sleep. This is in contrast with Harper's work [2] on the rabbit in which the peak occurred during paradoxical sleep only. Presumably this peak is a second harmonic of the theta rhythm.
HIPPOCAMPAL ELECTRICAL ACTIVITY
WALKING
SNIFFINGOBJECT
SNIFFINGAIR
LYING ?
393
REhR~NG
PARADOXICALSLEEP
GROOMINGHEAD
SLOWWA~,~SLEEP
2~3 4 56 7 B 9 101112111415161~ ?4L~
FIG. 2. Examples of mean spectrograms (N = 256) of the hippocampal EEG of a representative rat. Each block of a spectrogram represents one frequency, the height indicates the amplitude of that frequency in arbitrary units. Standard deviations are indicated.
During this investigation into sleep a number of cases were noted in which a clear small amplitude irregular activity occurred following just after paradoxical sleep. The duration of this phase was some seconds and the main frequency about 4 0 - 5 0 Hz. At the m o m e n t the exact relationship between behavior and this EEG activity is still unclear and is under further investigation.
The method described in this paper allows accurate quantification of the hippocampal EEG and would be suitable for similar investigations. Implementation of the technique requires, however, that the EEG obtained during any given behavioral pattern does not significantly change but rather fluctuates around a distinct pattern only. This is the case in the hippocampal EEG, excepting the first few
394
('Ot:N F.N
h u n d r e d s of milliseconds of a v o l u n t a r y b e h a v i o r d u r i n g w h i c h m o v e m e n t i n i t i a t i o n is a c c o m p a n i e d b y a higher t h e t a f r e q u e n c y t h a n is t h e case d u r i n g s t e a d y m o v e m e n t [ 4 ] . In particular w h e n m a n y b r i e f periods are averaged (e.g., walking) this m a y lead to an increase of the peak
frequency. initiations time being differences
However, for the most part these m o v e m e n t will be cancelled o u t b y the o b s e r v e r ' s r e a c t i o n of the same o r d e r o f m a g n i t u d e so t h a t actual will be very small.
REFERENCES 1. Bekkering, D. H., A. Kamp and W. Storm van Leeuwen. The EEG - spectrograph. Electroenceph. clin. Neurophysiol. 10: 5 5 5 - 5 5 9 , 1958. 2. Harper, R.M. Frequency changes in hippocampat electrical activity during movement and tonic immobility. Physiol. Behav. 7: 55--58, 1971.
3. Vanderwolf, C. H. Hippocampal electrical activity and voluntary movement in the rat. Electroenceph. din. Neurophysiol. 26: 4 0 7 - 4 1 8 , 1969. 4. Whishaw, I.Q. and C.H. Vanderwolf. Hippocampal EEG and behavior: Change in amplitude and frequency of RSA (theta rhythm) associated with spontaneous and learned movement patterns in rats and cats. Behav. Biol. 8:461 484, 1973.