510
Electroencephalography and clinical Neurophysiology , 79 ( 1991) 510-512 © 1991 Elsevier Scientific Publishers Ireland, Ltd. 0013-4649/91/$03.50
EEG 91068
Short communication
Ambulatory EEG cassette recorders for prolonged electroencephalographic monitoring in animals E . H . B e r t r a m * a n d E . W . L o t h m a n ** Department of Neurology, UniL,ersity of Virginia Health Sciences Center, Charlottesville, VA 22908 (U.S.A.) (Accepted for publication: 27 August 1991)
Summary We describe the use of ambulatory cassette EEG recorders for monitoring in vivo neurophysiologic signals from multiple animals over prolonged periods of time. This technique centers around a simple interface device that attenuates the intracerebral signals to the input range of the recording device and around a common indifferent input for all animals. The resulting analog recordings have the advantage of good signal resolution and rapid review of 24 h of recorded data. Key words: Ambulatory EEG; Epilepsy; Seizure models; Chronic neurophysiology
Recent studies have examined the electroencephalogram of unanesthetized animals over prolonged periods (hours to days) for spontaneous interictal and ictal events in cats and rats previously exposed to an epileptogenic procedure (Cavalheiro et al. 1982; Tanaka et al. 1985; Griffith et al. 1987; Hawkins and Mellanby 1987; Shouse et al. 1990). These studies have used standard paper tracings of the electroencephalogram to archive the physiologic signals for analysis at a later time. There are 2 common techniques for recording these signals: (1) relatively slow paper speeds that compress the recorded signals or (2) faster paper speeds that maintain more details of the signal. The former has the advantage of using less paper thereby speeding review of the information, but this method has the disadvantage of losing considerable signal resolution, making it more difficult to differentiate between true physiologic signals and artifact. The latter method has not only the greater cost of increased paper use but also of additional review time. Because neither of these techniques is without significant difficulties, we were interested in developing a new means of recording rat electroencephalograms which could be reviewed rapidly with good preservation of signal detail. A method has been developed in the clinical setting that fulfills both requirements: the ambulatory electroencephalogram cassette recorder (Ives 1989). This device can record up to 24 h of physiologic signal in an analog form that can be reviewed rapidly (24 h in as little as 24 min). Signal morphology is well preserved, and the data can be transcribed to paper through a standard chart recorder. Paper speeds of the chart recorder can be varied to emphasize specific aspects of the signal. The currently available recorders have 2 major technical limitations that must be overcome before this system can be employed routinely in the laboratory. First, the maximum input voltage is 800 p.V peak-to-peak, while the input signal from intracerebral electrodes in rats is frequently greater than 1 inV. Second, the recorder
Correspondence to: Edward H. Bertram, M.D., Department of Neurology, Box 394, University of Virginia Health Sciences Center, Charlottesville, VA 22908 (U.S.A.). Tel.: (804) 924-5233; FAX: (804) 982-1726. This work was supported in part by NIH-NINDS Grants NS-01324 *, NS-28073 * and NS-21617 **
has a single ground input, which makes simultaneous recording from multiple animals difficult. In this report we describe an interface device that overcomes these technical problems and allows for prolonged paperless EEG recording in rats.
Methods
Rats were prepared by standard laboratory methods (Lothman et al. 1990). Briefly, under ketamine-xylazine anesthesia bipolar stainless steel electrodes were placed in the posterior ventral hippocampus and an indifferent electrode was placed in the frontal sinus. After a 7 day recovery period, the rats were stimulated "continuously" through the hippocampal electrode (50 Hz, 1 msec biphasic square waves 400 /xA peak-to-peak in 10 sec trains delivered every 11 sec for 90 rain). This procedure resulted in a prolonged period of self-sustaining limbic status epilepticus that lasted up to 24 h. One month after this stimulation the rats underwent a recording protocol to determine the occurrence and frequency of spontaneous limbic seizures, a phenomenon which we have recently detailed (Lothman et al. 1990). The rat EEGs were recorded using an Oxford Medilog 8-channel recorder, with as many as 7 rats recorded simultaneously, 1 rat to each channel. A voltage attenuator was used to bring the voltages (frequently many millivolts) of the physiologic signals into the recorder's range. To accomplish this voltage reduction, the 2 hippocampal input electrodes were linked to poles 1 and 3 of a 1 MI2 trimmer potentiometer. Hippocampal lead 1 was subsequently joined to input 1 of one of EEG channels in the cassette recorder indirectly via pole 2 of the potentiometer. Hippocampal lead 2 was directly linked to input 2 of the cassette recorder through pole 3 of the potentiometer (Fig. 1). Eight such input modules were placed in a single acrylic box to allow for separate inputs to each EEG channel. The indifferent electrodes of all rats were linked together and joined to the ground input of the recorder. This latter step was necessary to allow the simultaneous recordings from multiple animals. The rats were linked through the attenuator box to the cassette recorder. This combination formed the basic recording unit. Before a recording session began, physiologic signals were verified and the voltage attenuator adjusted to keep signal amplitude within the
511
C A S S E T T E EEG F O R A N I M A L M O N I T O R I N G range of the amplifiers. For this reason an output signal from the cassette recorder was sent to an E E G machine via a proprietary interface box from the Oxford company. Using continuous paper writeout of the signal for visual feedback the cassette recorder channels were adjusted individually by turning the potentiometers in the attenuator box. The adjustments were made to allow for good visualization of the E E G from each rat while remaining within the voltage limits of the amplifiers. Once the adjustments were made calibration signals could be sent through the attenuator to document true voltage amplitudes for later analysis. The recording sessions could then continue for periods up to 24 h. With the interface between the cassette recorder and the polygraph the system could be checked intermittently for signal quality without interrupting the ongoing recording. At the end of each session, the cassette tape was removcd and, if anothcr recording period was desired, a new tape replaced. This action interrupted recording for less than 1 rain. T h e quality and amplitude of the signal was rechecked using the adjustment module, and the recording was continued as before. The data from the previous day were then visually reviewed off-line at a proprietary review station which allowed the review of the previous 24 h in as little as 24 rain, although an average review of a day's tape lasted 40 rain. Segments of data that were of interest could be transcribed to paper with an E E G machine that was connected to the reading station.
CASSETTE CHANNEL 1
CASSETTE CHANNEL 2
Inputs
I
Inputs
2
I
2 -
.?
1
/~11rna .-- leads
Incllfferent
I
l
RAT
I
I
RAT 2 ]
Fig. 1. Schematic diagram for 2 channels of an 8-channel voltage attenuator box. The 2 electrode leads from one animal were connected to the 2 ends of a 1 M,Q trimmer potentiometer. O n e of these ends was, in turn, connected to one of the two inputs of one channel of the cassette recorder; the other animal lead was connected to second input indirectly through the wiper pole (2) of the potentiometer. A key feature of this device is the linked indifferent input which connects the indifferent electrodes from all the rats to a common ground input in the recorder.
I !
i:
Fig. 2. Sample of E E G writeout of 7 rats recorded simultaneously with one rat (fifth line) experiencing an electrographic seizure. There was no spillover of activity among the other channels. Time line equals 2 sec. Vertical calibration before each channel equals 1 inV. Results and discussion
This technique produced consistently good quality tracings. Interictal spikes and spontaneous seizures were well defined, and there was no discernible interference ("cross-talk') among the channels coming from the different rats (Fig. 2). On some occasions non-physiologic artefact appeared simultaneously in a n u m b e r of channels, but this finding never posed a problem for record interpretation. The use of the ambulatory cassette recorder clearly allowed for rapid review of lengthy recordings while preserving the morphology of the signals to help differentiate between true physiologic activity and artefact. The only major drawback of this system is the frequency response which is 1-40 Hz, which clearly limits the ability to record high frequency signals. This limitation, however, caused no problems for the studies of concern to us. The input impedance into the cassette recorder is 1 M,Q and the common mode rejection is 100 dB (Oxford technical manual specifications). The intereleetrode resistance, when measured between animal leads 1 and 2 (or potentiometer poles 1 and 3, Fig. 1), averaged 75 k,(L When measured between inputs 1 and 2 at the cassette recorder (or poles 2 and 3 of the potentiometer) the resistance varied with the position of the slider (pole 2). When it was positioned at pole 1 the resistance was 75 k~2, but the resistance increased to a peak of 250-300 k J2 when the slider was positioned midway between poles 1 and 3, and then it fell rapidly to 0 as the slider approached pole 3. The recordings were generally made with the resistances less than 75 k$2 and greater than 1 ks2, that is with the slider approaching but not at pole 3. The key element to the success of this method is the voltage a t t e n u a t o r / c o m m o n ground box which allowed for the simultaneous recording of multiple animals. The attenuator reduced the signal amplitude without altering wave morphology. By the use of this interface device, we have been able to bring a useful clinical tool into the basic research lab. As a result the recording and interpretation of prolonged in vivo neurophysiologic recordings have been greatly simplified. The authors wish to thank D e a n n a Kirby, John Williamson and John Cornett for technical advice and assistance and Barbara Sisk for manuscript polish.
References
Cavalheiro, E.A., Riche, D.A. and Le Gal La Salle, G. Long-term effects of intrahippocampal kainie acid injection in rats: a method
512 for inducing spontaneous recurrent seizures. Electroenceph. clin. Neurophysiol., 1982, 53: 581-589. Griffith, N., Engel, Jr., J. and Bandler, R. Ictal and enduring interictal disturbances in emotional behaviour in an animal model of temporal lobe epilepsy. Brain Res., 1987, 400: 360-364. Hawkins, C.A. and Mellanby, J.H. Limbic epilepsy induced by tetanus toxin: a longitudinal electroencephalographic study. Epilepsia, 1987, 28: 431-444. Ives, J.R. Evolution of ambulatory cassette EEG. In: J.S. Ebersole (Ed.), Ambulatory EEG Monitoring. Raven Press, New York, 1989: 1-12.
E.H. BERTRAM, E.W. LOTHMAN Lothman, E.W., Bertram, E.H., Kapur, J. and Stringer, J.L. Recurrent spontaneous bippocampal seizures in the rat as a chronic sequela to limbic status epilepticus. Epilepsy Res., 1990, 6: 110118. Shouse, M.N., Langer, J.V. and Dittes, P.R. Spontaneous sleep epilepsy in amygdala-kindled kittens: a preliminary report. Brain Res., 1990, 535: 163-168. Tanaka, T., Kaijima, M., Yonemasu, Y. and Cepeda, C. Spontaneous secondarily generalized seizures induced by a single microinjection of kainic acid into unilateral amygdala in cats. Electroenceph. clin. Neurophysiol., 1985, 61: 422-429.
Electroencephalography and clinical Neurophysiology , 79 ( 1991) 510-512 © 1991 Elsevier Scientific Publishers Ireland, Ltd. 0013-4649/91/$03.50
EEG 91068
Short communication
Ambulatory EEG cassette recorders for prolonged electroencephalographic monitoring in animals E . H . B e r t r a m * a n d E . W . L o t h m a n ** Department of Neurology, UniL,ersity of Virginia Health Sciences Center, Charlottesville, VA 22908 (U.S.A.) (Accepted for publication: 27 August 1991)
Summary We describe the use of ambulatory cassette EEG recorders for monitoring in vivo neurophysiologic signals from multiple animals over prolonged periods of time. This technique centers around a simple interface device that attenuates the intracerebral signals to the input range of the recording device and around a common indifferent input for all animals. The resulting analog recordings have the advantage of good signal resolution and rapid review of 24 h of recorded data. Key words: Ambulatory EEG; Epilepsy; Seizure models; Chronic neurophysiology
Recent studies have examined the electroencephalogram of unanesthetized animals over prolonged periods (hours to days) for spontaneous interictal and ictal events in cats and rats previously exposed to an epileptogenic procedure (Cavalheiro et al. 1982; Tanaka et al. 1985; Griffith et al. 1987; Hawkins and Mellanby 1987; Shouse et al. 1990). These studies have used standard paper tracings of the electroencephalogram to archive the physiologic signals for analysis at a later time. There are 2 common techniques for recording these signals: (1) relatively slow paper speeds that compress the recorded signals or (2) faster paper speeds that maintain more details of the signal. The former has the advantage of using less paper thereby speeding review of the information, but this method has the disadvantage of losing considerable signal resolution, making it more difficult to differentiate between true physiologic signals and artifact. The latter method has not only the greater cost of increased paper use but also of additional review time. Because neither of these techniques is without significant difficulties, we were interested in developing a new means of recording rat electroencephalograms which could be reviewed rapidly with good preservation of signal detail. A method has been developed in the clinical setting that fulfills both requirements: the ambulatory electroencephalogram cassette recorder (Ives 1989). This device can record up to 24 h of physiologic signal in an analog form that can be reviewed rapidly (24 h in as little as 24 min). Signal morphology is well preserved, and the data can be transcribed to paper through a standard chart recorder. Paper speeds of the chart recorder can be varied to emphasize specific aspects of the signal. The currently available recorders have 2 major technical limitations that must be overcome before this system can be employed routinely in the laboratory. First, the maximum input voltage is 800 p.V peak-to-peak, while the input signal from intracerebral electrodes in rats is frequently greater than 1 inV. Second, the recorder
Correspondence to: Edward H. Bertram, M.D., Department of Neurology, Box 394, University of Virginia Health Sciences Center, Charlottesville, VA 22908 (U.S.A.). Tel.: (804) 924-5233; FAX: (804) 982-1726. This work was supported in part by NIH-NINDS Grants NS-01324 *, NS-28073 * and NS-21617 **
has a single ground input, which makes simultaneous recording from multiple animals difficult. In this report we describe an interface device that overcomes these technical problems and allows for prolonged paperless EEG recording in rats.
Methods
Rats were prepared by standard laboratory methods (Lothman et al. 1990). Briefly, under ketamine-xylazine anesthesia bipolar stainless steel electrodes were placed in the posterior ventral hippocampus and an indifferent electrode was placed in the frontal sinus. After a 7 day recovery period, the rats were stimulated "continuously" through the hippocampal electrode (50 Hz, 1 msec biphasic square waves 400 /xA peak-to-peak in 10 sec trains delivered every 11 sec for 90 rain). This procedure resulted in a prolonged period of self-sustaining limbic status epilepticus that lasted up to 24 h. One month after this stimulation the rats underwent a recording protocol to determine the occurrence and frequency of spontaneous limbic seizures, a phenomenon which we have recently detailed (Lothman et al. 1990). The rat EEGs were recorded using an Oxford Medilog 8-channel recorder, with as many as 7 rats recorded simultaneously, 1 rat to each channel. A voltage attenuator was used to bring the voltages (frequently many millivolts) of the physiologic signals into the recorder's range. To accomplish this voltage reduction, the 2 hippocampal input electrodes were linked to poles 1 and 3 of a 1 MI2 trimmer potentiometer. Hippocampal lead 1 was subsequently joined to input 1 of one of EEG channels in the cassette recorder indirectly via pole 2 of the potentiometer. Hippocampal lead 2 was directly linked to input 2 of the cassette recorder through pole 3 of the potentiometer (Fig. 1). Eight such input modules were placed in a single acrylic box to allow for separate inputs to each EEG channel. The indifferent electrodes of all rats were linked together and joined to the ground input of the recorder. This latter step was necessary to allow the simultaneous recordings from multiple animals. The rats were linked through the attenuator box to the cassette recorder. This combination formed the basic recording unit. Before a recording session began, physiologic signals were verified and the voltage attenuator adjusted to keep signal amplitude within the
511
C A S S E T T E EEG F O R A N I M A L M O N I T O R I N G range of the amplifiers. For this reason an output signal from the cassette recorder was sent to an E E G machine via a proprietary interface box from the Oxford company. Using continuous paper writeout of the signal for visual feedback the cassette recorder channels were adjusted individually by turning the potentiometers in the attenuator box. The adjustments were made to allow for good visualization of the E E G from each rat while remaining within the voltage limits of the amplifiers. Once the adjustments were made calibration signals could be sent through the attenuator to document true voltage amplitudes for later analysis. The recording sessions could then continue for periods up to 24 h. With the interface between the cassette recorder and the polygraph the system could be checked intermittently for signal quality without interrupting the ongoing recording. At the end of each session, the cassette tape was removcd and, if anothcr recording period was desired, a new tape replaced. This action interrupted recording for less than 1 rain. T h e quality and amplitude of the signal was rechecked using the adjustment module, and the recording was continued as before. The data from the previous day were then visually reviewed off-line at a proprietary review station which allowed the review of the previous 24 h in as little as 24 rain, although an average review of a day's tape lasted 40 rain. Segments of data that were of interest could be transcribed to paper with an E E G machine that was connected to the reading station.
CASSETTE CHANNEL 1
CASSETTE CHANNEL 2
Inputs
I
Inputs
2
I
2 -
.?
1
/~11rna .-- leads
Incllfferent
I
l
RAT
I
I
RAT 2 ]
Fig. 1. Schematic diagram for 2 channels of an 8-channel voltage attenuator box. The 2 electrode leads from one animal were connected to the 2 ends of a 1 M,Q trimmer potentiometer. O n e of these ends was, in turn, connected to one of the two inputs of one channel of the cassette recorder; the other animal lead was connected to second input indirectly through the wiper pole (2) of the potentiometer. A key feature of this device is the linked indifferent input which connects the indifferent electrodes from all the rats to a common ground input in the recorder.
I !
i:
Fig. 2. Sample of E E G writeout of 7 rats recorded simultaneously with one rat (fifth line) experiencing an electrographic seizure. There was no spillover of activity among the other channels. Time line equals 2 sec. Vertical calibration before each channel equals 1 inV. Results and discussion
This technique produced consistently good quality tracings. Interictal spikes and spontaneous seizures were well defined, and there was no discernible interference ("cross-talk') among the channels coming from the different rats (Fig. 2). On some occasions non-physiologic artefact appeared simultaneously in a n u m b e r of channels, but this finding never posed a problem for record interpretation. The use of the ambulatory cassette recorder clearly allowed for rapid review of lengthy recordings while preserving the morphology of the signals to help differentiate between true physiologic activity and artefact. The only major drawback of this system is the frequency response which is 1-40 Hz, which clearly limits the ability to record high frequency signals. This limitation, however, caused no problems for the studies of concern to us. The input impedance into the cassette recorder is 1 M,Q and the common mode rejection is 100 dB (Oxford technical manual specifications). The intereleetrode resistance, when measured between animal leads 1 and 2 (or potentiometer poles 1 and 3, Fig. 1), averaged 75 k,(L When measured between inputs 1 and 2 at the cassette recorder (or poles 2 and 3 of the potentiometer) the resistance varied with the position of the slider (pole 2). When it was positioned at pole 1 the resistance was 75 k~2, but the resistance increased to a peak of 250-300 k J2 when the slider was positioned midway between poles 1 and 3, and then it fell rapidly to 0 as the slider approached pole 3. The recordings were generally made with the resistances less than 75 k$2 and greater than 1 ks2, that is with the slider approaching but not at pole 3. The key element to the success of this method is the voltage a t t e n u a t o r / c o m m o n ground box which allowed for the simultaneous recording of multiple animals. The attenuator reduced the signal amplitude without altering wave morphology. By the use of this interface device, we have been able to bring a useful clinical tool into the basic research lab. As a result the recording and interpretation of prolonged in vivo neurophysiologic recordings have been greatly simplified. The authors wish to thank D e a n n a Kirby, John Williamson and John Cornett for technical advice and assistance and Barbara Sisk for manuscript polish.
References
Cavalheiro, E.A., Riche, D.A. and Le Gal La Salle, G. Long-term effects of intrahippocampal kainie acid injection in rats: a method
512 for inducing spontaneous recurrent seizures. Electroenceph. clin. Neurophysiol., 1982, 53: 581-589. Griffith, N., Engel, Jr., J. and Bandler, R. Ictal and enduring interictal disturbances in emotional behaviour in an animal model of temporal lobe epilepsy. Brain Res., 1987, 400: 360-364. Hawkins, C.A. and Mellanby, J.H. Limbic epilepsy induced by tetanus toxin: a longitudinal electroencephalographic study. Epilepsia, 1987, 28: 431-444. Ives, J.R. Evolution of ambulatory cassette EEG. In: J.S. Ebersole (Ed.), Ambulatory EEG Monitoring. Raven Press, New York, 1989: 1-12.
E.H. BERTRAM, E.W. LOTHMAN Lothman, E.W., Bertram, E.H., Kapur, J. and Stringer, J.L. Recurrent spontaneous bippocampal seizures in the rat as a chronic sequela to limbic status epilepticus. Epilepsy Res., 1990, 6: 110118. Shouse, M.N., Langer, J.V. and Dittes, P.R. Spontaneous sleep epilepsy in amygdala-kindled kittens: a preliminary report. Brain Res., 1990, 535: 163-168. Tanaka, T., Kaijima, M., Yonemasu, Y. and Cepeda, C. Spontaneous secondarily generalized seizures induced by a single microinjection of kainic acid into unilateral amygdala in cats. Electroenceph. clin. Neurophysiol., 1985, 61: 422-429.
