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Biotelemetry: Adjustment of a telemetry system for simultaneous measurements of acute heart rate changes and behavioral events in unrestrained rats
Physiology&Behavior,Vol. 53, pp. 1121-1126, 1993
0031-9384/93 $6.00 + .00 Copyright © 1993PergamonPress Ltd.
Printed in the USA.
Biotelemetry: Adjustment of a Telemetry System for Simultaneous Measurements of Acute Heart Rate Changes and Behavioral Events in Unrestrained Rats MICHAELA
D I A M A N T , *l L E O V A N W O L F S W I N K E L , I -
BAS A L T O R F F E R : ~ A N D D A V I D
DE WIED*
*Rudolf Magnus Institute, Department of Pharmacology, Medical Faculty, University of Utrecht, Utrecht, The Netherlands, tUniversity Hospital for Children and Youth, 'Her Wilhelminakinderziekenhuis," Department of Anaesthesiology, Utrecht, The Netherlands, and qtLaboratory for Physiology, Department of Electrotechnology, Medical Faculty, University of Utrecht, Utrecht, The Netherlands R e c e i v e d 3 A u g u s t 1992 DIAMANT, M., L. VAN WOLFSWINKEL, B. ALTORFFER AND D. DE WIED, Biotelemetry:Adjustment of a telemetry systemfor simultaneous measurementsofacuteheartratechangesand behavioraleventsin unrestrainedrats. PHYSIOL BEHAV 53(6) 1121-1126, 1993.--The radiotelemetry system described in this paper consists of an implantable transmitter and a receiver, connected to a microcomputer. The hardware and software belonging to Mini-Mitters, for the collection and analysis of heart rate (HR), core temperature (CT), and gross activity data, do not possess the flexibility to detect acute changes in HR nor to discriminate among simultaneously occurring different types of behavior. In order to study short-term changes in HR in response to stress or drugs, in relation to behavioral responses, an inexpensive computer interface and a software program (CARDIAQ) were developed to collect data from Mini-Mitters. The interface conveys the QRS signal, which is converted to a TTL pulse train, to the parallel printer adapter (LPTI) of an IBM-compatible computer. Heart rate is determined by measuring single interbeat intervals (IBI). The software controls the sampling schedule and stores the collected data in a format compatible with a commercial spreadsheet package. The program calculates the median IBI per s, mean _+ SD IBI, variance, skewness, and kurtosis of the IBI distribution. In addition, it enables simultaneous recording of behavior by entering data through the keyboard at the occurrence of each event. In this paper, we describe the CARDIAQ program and provide examples of its application together with the implantable transmitters in freely moving rats. Telemetry Autonomic nervous system Conscious rats
Heart rate
Interbeat interval
E N V I R O N M E N T A L events of a stressful nature elicit simultaneous changes in autonomic and behavioral activity (2,4,5,9,10). Various biotelemetry systems for small animals have been developed to record alterations in autonomic and behavioral activity (3,8,11-13). The major asset of the telemetry method is the possibility of recording various parameters at a time in the unrestrained, conscious animal ( 1,3,7,11,14). In previous studies, we have employed a wireless telemetry system (Mini-Mitter Co., Sunriver, OR) to assess heart rate (HR), core temperature (CT), and gross locomotor activity in freely moving
Behavior
Acute stress
rats while performing a behavioral task (5,14). The telemetry system consists of a small implantable transmitter, a receiver connected to a computer with data acquisition controlled by a computer board, and software package (DATAQUEST III). However, we have encountered various limitations of the method. This system has been developed for long-term monitoring of the above-mentioned parameters in small animals, rather than for measuring rapid H R responses. Because the shortest sampling period in the D A T A Q U E S T m e n u is l0 s, H R changes occurring within 10 s after a stimulus is applied
The CARDIAQ program is available from Mini-Mitter Co., Inc., PO BOX 3386, Sunriver, OR 97707; Phone: (503) 593-8639; FAX: (503) 5935604. i Requests for reprintsshould be addressed to Michaela Diamant, M.D., Rudolf Magnus Institute,Department of Pharmacology, Medical Faculty, Universityof Utrecht, Vondellaan 6, 3521 GD Utrecht, The Netherlands.
1121
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DIAMgNI
TABLE 1 COMPARISON OF HR MEASURED BY DAEAQUES'I AND CARDIAQ
li1 AI
after exposure of a rat to a novel stimulus will be demonstrated.
CARDIAQ
t (s)
DATAQUEST HR (bpm)
HR (bpm)
IBI (ms)
~Beats
0-60 180-240 300-360 540-600
329 366.2 352.6 355.1
328 368 351.2 356.5
183.1 163.0 170.5 168.3
328 351 352 356
Mean (0-600)
345.8
345.3
173.9
343.5
METHOD
Mean HR of a rat under resting conditions, simultaneously recorded by DATAQUEST III and CARDIAQ during a 10-min period. DATAQUEST was set to continuously sample HR at a sampling period of 10 s (column 2). Separate beat-to-beat recordings [(interbeat interval (IBI)] were performed by CARDIAQ for 60 s every other minute (column 3 and 5). Heart rate was calculated from the IBI by CARDIAQ (column 3). Representative data collected at t = 0-1, 3-4, 5-6, and 9-10 min are shown. For details see text.
would be undetected. Furthermore, the system collects so-called gross activity counts only, without specification of the actual types of behavior of the animal. In view of this, we have adjusted and extended the data acquisition system, by developing a program C A R D I A Q (Computer-Assisted Recording of Digitized Interbeat intervals Analyzed by Quattro, v 1.1), which enables beat-to-beat recording o f H R by d e t e r m i n a t i o n o f the interbeat interval (IBI) in ms. In addition, C A R D I A Q allows differentiation of various types of behavior. By entering data through precoded keys on the keyboard, various behaviors can be scored, the frequency and duration of which can be calculated off-line. Both the D A T A Q U E S T and the C A R D I A Q methods were c o m p a r e d in their efficacy to m o n i t o r changes in HR. Examples of m e a s u r e m e n t s by both systems of acute beat-to-beat changes in H R and behavioral responses
The CardioTel¢~ Biotelemetry and DA TAQUEST Ill Data Acquisition System~ Both systems have in part been described elsewhere (4,14). A wireless, battery-operated transmitter and a receiver connected to an IBM-compatible computer, which is operated by the DAT A Q U E S T hardware and software package, enable the monitoring and on-line analysis of heart rate (HR), core temperature (CT), and gross activity in laboratory animals. The implantable transmitter ( C a r d i o T e l ~ model CTT85-SA, Mini-Mitter Co., Inc.) consists of a resistor-capacitor (RC) oscillator in which the value of the resistor and consequently the output frequency is temperature dependent. The output from the transmitter consists of a train of 455 kHz bursts with the average frequency of these bursts coding for the telemetered temperature data and the shortterm variations in the frequency of the pulse train being proportional to the EKG signal. The output frequency of the CTT85SA transmitters can be received up to 30 cm away by a simple AM receiver. If the transmitters are used to measure the animal's CT. calibration in a water bath at two different temperatures is required prior to implantation, because each transmitter has a unique characteristic of frequency output vs. temperature. The oscillator, transmitter, and battery are enclosed in a plastic capsule and coated with wax to seal against moisture. The proportions of the package are 3 X 1.5 × 0.8 cm at the largest, flat and irregularly rectangular in shape, weighing 4 g. The battery has a lifetime of about 6 months. Heart potentials are sensed by the bare ends of the two leads extending from the package. After implantation of the package into the right lower quadrant of the abdominal cavity, the electrodes are tunnelled subcutaneously on either side of the thorax and the tips placed in a position that provides sufficient signal (>0.1 millivolt for the standard modeL). The receiver unit, placed under the animal's cage in the case of the model used in our experiments (model CTR85-SA, Mini-
TABLE 2 PRINTOUT OF THE .SUM FILE MADE BY CARDIAQ DATE
TIME
START
AUTO
RAT
TRT
PER.
ZBEAT
IBI
VAR
SKEW
KURT
22-04-91 22-04-9l 22-04-91 22-04-91 22-04-91 22-04-91 :
15:15:11 15:16:11 15:17:11 15:18:11 15:19:11 15:20:11 :
FI FI3 FI3 F13 FI3 Ft3 :
FI3 F13 FI3 FI3 FI3 FI3 :
3 3 3 3 3 3 :
0 0 0 0 0 0 :
60 60 60 60 60 60 :
331 328 330 322 351 354 :
180.73 183.06 181.75 185.74 163.01 169.43 :
6.76 3.80 6.78 8.89 12.03 6.18 :
-0.48 0.01 -0.10 -1.40 0.09 -0.52 :
0.44 -0.71 0.44 2.58 0.26 0.40 :
22-04-91
15:5'0:34
F1
FI 3
3
0
60
354
165.51
15.56
2.05
36.65
22-04-91 22-04-91 22-04-91
16:52:48 17:05:53 17:21:47
F1 F2 F1
F13 FI3 FI3
1 2 3
10 11 12
600 600 600
3710 3784 4900
133.68 150.53 120.44
38.34 21.55 17.77
3.72 4.46 9.62
15.75 31.32 I t 1.83
The upper part of the printout (above marker-line) summarizes Experiment 1; the three lines under the marker-line show data from Experiment 2. Rows 3 and 4 show the function keys pressed to start and restart sampling, respectively. RAT, rat code number; TRT, treatment; PER, sampling period (s); ZBEAT, total amount of beats recorded during sampling period; IB1, interbeat interval (ms); VAR, variation; SKEW, skewness; KURT, kurtosis; F13, programmed automatic restart of sampling; F I 2, manual interruption.
MONITORING OF HR AND BEHAVIOR BY TELEMETRY IN RATS
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connecting the receiver to the consolidation matrix and optically isolated interfaced to the ACK output of the parallel printer port (LPTI). This option was considered best for reasons of expense and compatibility.
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FIG. 1. Changes in gross activity (A) and heart rate (B) during a 10-min exposure to a mild novelty stress in rats 30 min after IP treatment with saline, atropine, or propranolol, reconstructed from data obtained by DATAQUEST.
Hardware Adaptation The digital HR pulse generated by the receiver (upon triggering by the R-wave of the EKG) was branched off the cable
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Mitter Co., Inc.), contains two perpendicularly placed antennas connected to AM receivers. This receiver provides an EKG analog output, a digital composite temperature and EKG output, and a digital activity output. The receiver detects R-waves in the EKG waveform and provides a digital pulse for each heart beat. Digital activity output from the receiver is obtained by continuous monitoring of the received signal strength, which varies as a result of movement-inducedchanges in the orientation between the transmitter and the receiving antenna. A cable with a modular plug at both ends directly conveys the composite EKG and temperature signals as well as the digital activity output to a consolidation matrix (BCM-100) for multiplexing the frequency and activity signals. When the telemetry system is used with the DATAQUEST data acquisition system, the data from the receivers are routed through the consolidation block onto a 26-conductor fiat cable, six receivers to a block. In our laboratory, the DATAQUEST III system, consisting of a printed circuit card (DQ1088, Data Sciences®) and a menu-driven software program for system operation, runs on an IBM XT computer.
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FIG. 2. Changesin gross activity(A), heart rate (B), and interbeat interval (C) over 60 s during period a 10-rain exposure to a mild novelty stress in rats treated with saline, atropine, or pmpranolol, respectively.Gross activity and heart rate were assessed by DATAQUEST, changes in interbeat interval were recorded by CARDIAQ.
1124
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TABLE 3 COMPARISON OF RESULTS BY DATA0[3EST AND CARDIAQ DATAQUEST
CARDIAQ
Rat No.
TRT
HR [_+SD (SEM) var] (bpm)
GLA (counts}
IBI (ms)
HR (bpm)
GR (s)
[OC (s)
REAR (s)
VAR Is)
1 2 3
SAL PROP ATR
447 [_+99.8(12.7) 1268] 398 [_+36.5 (4.7) 1333] 500 [_+25.5(3.3) 650]
67.7 33.1 36.6
133,7 150.5 120.4
448.9 398.6 498.2
187 92 73
120 85 67
238 193 194
55 230 266
Results were obtained during a 10-min exposure (t = 0-10 min) of rats to a novelty stimulus, 30 min followinga single IP treatment of saline (rat 1), propranolol (rat 2), or atropine (rat 3). The values are calculated off-line.TRT, treatment; HR, heart rate; var, variance; GLA, gross locomotor activity; IBI, interbeat interval; SAL, saline; PROP, propranolol; ATR, atropine; counts, counts/time unit; GR, grooming; LOC, locomotion; REAR, rearing; VAR, various other types of behavior. For details see text.
Software
Experimental Protocols
The CARDIAQ program was written in Turbo Pascal (version 5.5). It does not use the already installed DATAQUEST III system. The program was designed for recording one animal at a time, mainly to maximize the number of events capable of being entered. The program determines the intervals between subsequent hardware interrupts from the ACK input of the parallel printer port (LPTI). These interrupts are generated by the Rwave of the rat EKG. The system clock chip, which normally produces an interrupt every 55 ms, has been reprogrammed so as to generate an interrupt every ms. This interrupt increments a memory-variable that is used as an interbeat interval timer. Using another variable, the system interrupt is activated each 55 ms to update the bios variables in the usual way. Thus, the program gives a 1-ms resolution of the R - R interbeat. Every heartbeat interrupt causes the ms counter to be stored in a buffer array, after which the counter is zeroed. To reduce artifacts, threshold values are used for maximum and minimum acceptable IBis. From the buffer array, the median IBI in each s is calculated. Also, the mean (absolute) difference between subsequent IBis in that s is calculated, as this may serve as an index for the beat-to-beat variability. These values, together with the raw data, are written to an output file in ASCII format, which can be handled by a commercial spreadsheet package (e.g., Borland Quattro or Lotus 123). This file, provided with the extension .DAT, also contains the animal number, the treatment code, date and time of sampling, the function key(s) used to start data acquisition, and keys pressed to mark the occurrence of events. Another output file, with the extension .SUM, contains statistical parameters calculated over each sampling interval, which was started by pressing a function key. The following experiments were conducted to validate the data obtained by this new data acquisition program together with the radiotelemetry system.
Rats were handled for 5 consecutive days following surgery for weighing and habituation purposes. On days 5 and 6, baseline HR values were recorded. To this purpose, the rat in its home cage was brought to the experimental room, placed onto a receiver, and allowed to habituate to the new situation for 60 to 90 min. When the animal was at rest, basal HR values were obtained by sampling with the DATAQUEST system, with the sampling period set at 30 s for 15-30 consecutive min.
Animals and Surgery Male Wistar rats of an inbred strain (weighing 200-220 g at the start of the experiments) were used. Under ether anaesthesia and sterile conditions, the transmitters were implanted intraperitoneally, the electrodes were guided subcutaneously on either side of the thorax, and the tips were sutured in place. Following surgery, the animals were housed individually and kept under conditions controlled for temperature (22 + 2°C) and light (lights on from 0600 to 2000 h). The animals were allowed to recover for 5 days.
Experiment 1--Validation ().['the CARDIAQ Data Acquisition Program In order to establish the compatibility of HR data recorded by the CARDIAQ program with HR values assessed by DATAQUEST III, the CARDIAQ software was installed to an IBM computer, different from the one configured with the DATAQUEST III system, and both systems were used simultaneously to monitor the HR of a rat under resting conditions. The sampling time in the DATAQUEST system was set at 10 s, the temperature channel was turned off. Data acquisition started at t = 0 after the animal was adapted to the experimental room and continued for two subsequent periods of 10 and 20 rain, respectively. During the first 10 min, CARDIAQ was set to simultaneously record IBis, starting at t = 0. Thus, for six data points obtained by the DATAQUEST system in 1 min, CARDIAQ provided a number of points equivalent to the number of impulses collected from the receiver and conveyed to the LPT1 during a 60-s recording period (beats per min). During the 20-min period, for the sake of data reduction, CARDIAQ was set to take 60-s samples every 5 rain. Mean HR values were calculated and compared over the various time periods.
Experiment 2--Detection of Acute Changes in HR Associated With Behavior Behavioral and cardiac responses to a mild stress after autonomic blockade were measured simultaneously by the DATAQUEST and CARDIAQ system. A t0-min exposure to a novel cage was used as a mild stressor. At 30 rain (t = -30) before the exposure to the novelty stimulus, rats were treated intraperitoneally (IP) with saline (SAL), atropine sulphate (ATR; 2 mg/kg b.wt.), or propranolol HCI (PROP; 2.5 mg/kg b.wt.). The injection volume was 0.5 ml. After the injections, the animals were returned to their home cages. At t = 0, one rat was placed into a novel cage and both computers were switched to the data acquisition mode. A receiver, which was connected to
MONITORING OF HR AND BEHAVIOR BY TELEMETRY IN RATS
both computers, was placed under the novel cage. The DATAQUEST system was set to sample HR and gross activity at 10-s sampling periods. Options from the CARDIAQ menu were defined to sample heartbeats for 600 s, while the observer rated the animal's behavior by pressing precoded keys at the onset of one of four predefined behavioral responses. Thus, G(rooming), R(earing), L(ocomotion), and V(arious) behaviors were scored.
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Experiment 1 Table 1 shows summarized baseline HR values of a rat computed from data collected simultaneously by the DATAQUEST and CARDIAQ data acquisition systems. As the rat was under resting conditions (sleep, with mean gross activity 0), no significant variation in HR was observed during the 10-min recording period (evaluated from previous DATAQUEST recordings in sleeping rats for 30-90 min, data not shown). On these grounds, a discontinuous HR recording by CARDIAQ was justified. The mean HR over the 10-min period was determined at 345.8 bpm by DATAQUEST by averaging 60 data points, and at 345.7 bpm by CARDIAQ, calculated from mean IBI (Table 1). Additional comparisons for HR recorded by both systems were made (Table 1). The maximum difference between DATAQUEST-measured and CARDIAQ-computed HR over a 1-min time period was less than 5% (Table 1). Mean HR over the 20rain period was 340.5 for DATAQUEST (based on 119 data points) and 341.2 bpm for CARDIAQ (obtained from four 60s recordings, starting at, respectively, t = 0, 5, 10, 15, and calculated from mean IBI). Table 2 shows an adaptation of a typical print-out of a CARDIAQ.SUM file. The upper part of Table 2 contains data from the 10-min recording period, described above and elaborated in Table 1. The discrepancy between HR data in the Y~beats column and the corresponding HR values, calculated from the mean IBI, may result from artifacts at the level of the transmitter-receiver interaction, rather than being produced during system-scheduled data collection from the receiver. Due to this dropped-beat phenomenon, which is occasionally observed in both recording systems whenever rats engage in too vigorous somatomotor activity, the first value shows a lower HR than that calculated from mean IBI (Table 2). These results indicate that the CARDIAQ program reliably collects and analyzes HR data obtained by a wireless radiotelemetry system.
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Figure 1A and B show the respective time courses of gross activity and HR of three differently treated rats during 10 min of exposure to the novel cage, recorded by the DATAQUEST system. In Fig. 2A and B, DATAQUEST-collected gross activity and HR over the first 60-s period of the novel-cage test are depicted; Fig. 2C shows the corresponding IBI measurement by CARDIAQ. The IBI data points represent mean IBI per s. From the IBI recording, initial slowing and subsequent acceleration of HR in the SAL-treated (429 and 469 bpm, respectively) and in the ATR-treated (444 and 480 bpm) rat are observed (Fig. 2C). This short-lasting effect is not detected by DATAQUEST (Fig. 2B). In Table 3, an off-line analysis of HR and gross locomotor activity by DATAQUEST III, and of HR and behavioral responses recorded and calculated by CARDIAQ and a spread-sheet program, are compared. Mean HRs over the 10-min period were different for the animals, and in accordance with their respective treatments. Thus, the PROP-treated rat
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FIG. 3. Time course of changes in interbeat interval related to behavioral responses over a 60-s period during exposure to a novel cage in rats 30 min after IP treatment with saline (upper), propranolol (middle), and atropine (lower), reconstructed from data measured by CARDIAQ. LOC, locomotor activity; REAR, rearing; VAR, various.
showed the lowest and the ATR-administered rat the highest HR. Overall HR, calculated by DATAQUEST from 59 points and by CARDIAQ from mean IBI, differed <0.5%. Interestingly, the variance in HR during the 10-rain test for the PROP-treated rats was even higher than in controls, when computed by DATAQUEST, whereas from the calculation by CARDIAQ, the/3sympatholytic effect is reflected in a lower variance of the IBI [Table 2; (6)]. The marked reduction of HR variance due to ATR treatment, however, was detected by both recording methods [(6); Tables 2, 3].
1126
DIAMANi
D A T A Q U E S T gives a global impression of the rats' overall somatomotor activity in the novel environment by the recording of gross locomotor activity counts. Inhibition of either branch of the ANS resulted in a decrease in gross activity, respective to vehicle treatment, which was found most marked in the PROPtreated rat (Table 3). However, specification of behavioral activity by C A R D I A Q revealed that rats given A T R showed less grooming and locomotor activity than rats given PROP. This discrepancy may be explained by the mode D A T A Q U E S T obtains the activity data. As briefly indicated in the Method section, a critical change in the orientation of the (implanted) transmitter over the receiver, due to the rat's movement, results in change of the signal strength as detected by the receiver card, and hence a pulse or count of activity is recorded. This pulse of activity is recorded and increments the counter associated with a specific channel. Each input is compared with the state recorded on the previous sample, and, as a consequence, if the animal has not moved from its previous position, activity will not be incremented. Therefore, grooming behavior and other behaviors that do not cause a critical change in the orientation of the transmitter over the receiver will remain undetected as an activity pulse, whereas they can be recorded as coded event by C A R D I A Q . Figure 3 shows the relation among IBI and behaviors in the three differently treated rats during 60 s in the novel cage. DISCUSSION This report describes a software/hardware adaptation of a commercially available acquisition system for telemetered H R and gross activity data in freely moving rats. The newly developed system (CARDIAQ) determines H R by beat-to-beat measurements and therefore enables the detection of acute changes in cardiac activity. In addition, the method allows differentiation
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for various types of behavior exhibited by an animal m response to a stimulus, by simply entering data through the keyboard. The experiments described in the paper merely serve to illustrate the use of this HR recording telemetry system m behavioral research. The results of the first experiment demonstrate that beat-to-beat measurements from Mini-Mitters by the C A R D I A Q program is fully compatible with the H R data acquired and analyzed by the D A T A Q U E S T III system. In the second experiment, we have demonstrated the use of the C A R D I A Q program in detecting acute and short-lived changes in autonomic responses related to differential behavioral patterns. The C A R D I A Q program has several advantages. First, no expensive additional computer cards are necessary to record and store H R data on a beat-to-beat base. Second, the use of the LPTI port as a gate for the incoming heart beats allows the use of any other IBM-compatible computer, without requiring further hardware adaptation. Third, specific behavior-related changes in cardiac activity can be assessed and vice versa. Fourth, the possibility of data entry by the user makes this program suitable for use in an extensive variety of experimental protocols. As an asset over the D A T A Q U E S T system, C A R D I A Q enables the evaluation of H R concomitants of various behaviors and as a result, provides a more differentiated picture of drug- or stimulus-specific autonomic and behavioral interrelationships. In sum, the Mini-Mitters, used in combination with DAT A Q U E S T system hardware and software, provide a suitable tool for long-term monitoring of H R in freely moving animals. The limitations of this system to detect small changes in cardiac action, related to behavioral patterns, have been described in this paper. Therefore, the C A R D I A Q program, sampling HR on a beat-to-beat base and allowing simultaneous recording of various behaviors, may further extend the possibilities of the biotelemetry method.
REFERENCES 1. Barringer, D. L.; Bufiag, R. D. Differential anaesthetic depression of chronotropic baroreflexes in rats. J. Cardiovasc. Pharmacol. 15: 10-15; 1990. 2. Bohus, B. Telemetered heart rate responses of the rat during free and learned behavior. In: Biotelemetry, vol. 1. Basel: Karger; 1974: 193-201, 3. Bohus, B. Behavioral and methodological factors influencing heart rate of freely moving rats. In: Biotelemetry, vol. 2. Basel: Karger; 1974:185-187. 4. Diamant, M.; De Wied, D. Autonomic and behavioral effects of centrally administered corticotropin-releasing factor in rats. Endocrinology 129:446-454; 1991. 5. Diamant, M.; Croiset, G,; De Zwart, N,; De Wied, D. Shock-prod burying behavior in rats: Autonomic and behavioral responses. Physiol. Behav. 50:23-32; 1991. 6. Ferrari, A.; Daffonchio, U. A.; Albergati, F.; Mancia, G. Inverse relationship between heart rate and blood pressure variabilities in rats. Hypertension 10:533-537; 1987. 7. Fluckiger, J-P.; Sonnay, M.; Boillat, N.; Atkinson, J. Attenuation of the baroreceptor reflex by general anesthetic agents in the normotensive rat. Eur. J. Pharmacol. 109:105-109; 1985.
8. Gallaher, E. J.; Egner, D. A.; Swen, J. W, Automated remote temperature measurement in small animals using a telemetry/ microcomputer interface. Comput. Biol. Med. 15:103-110; 1985. 9. Meinrath, M.; D'Amato, M. R. Interrelationships among heart rate, activity, and body temperature in the rat. Physiol. Behav. 22:491498; 1979. 10. Obrist, P. A. Cardiovascular psychobiology: A perspective. New York: Plenum Publishing; 1981. 11. Schlatter, J.; Elsner, J.; Zbinden, G. Correlation of telemetered heart rate and locomotor behavior in cyclazocine treated rats. Neurobehav. Toxicol. Teratol. 5:413-419; 1983. 12. Strhr, W. Long-term heart-rate telemetry in small mammals: A comprehensive approach as a prerequisite for valid results. Physiol. Behav. 43:567-576; 1988. 13. Sugiura, T.: Kimura, M.; Hasepawa, T.; Yoshimura, K.; Harada, Y. Telemetry system for heart-rate and blood temperature using a microcomputer. J. Med. Eng. Technol. 9:5-9; 1985. 14. Wan, R.; Diamant, M.; De Jong, W.; De Wied, D. Changes in heart rate and body temperature during passive avoidance behavior in rats. Physiol. Behav. 47:493-499; 1990.
0031-9384/93 $6.00 + .00 Copyright © 1993PergamonPress Ltd.
Printed in the USA.
Biotelemetry: Adjustment of a Telemetry System for Simultaneous Measurements of Acute Heart Rate Changes and Behavioral Events in Unrestrained Rats MICHAELA
D I A M A N T , *l L E O V A N W O L F S W I N K E L , I -
BAS A L T O R F F E R : ~ A N D D A V I D
DE WIED*
*Rudolf Magnus Institute, Department of Pharmacology, Medical Faculty, University of Utrecht, Utrecht, The Netherlands, tUniversity Hospital for Children and Youth, 'Her Wilhelminakinderziekenhuis," Department of Anaesthesiology, Utrecht, The Netherlands, and qtLaboratory for Physiology, Department of Electrotechnology, Medical Faculty, University of Utrecht, Utrecht, The Netherlands R e c e i v e d 3 A u g u s t 1992 DIAMANT, M., L. VAN WOLFSWINKEL, B. ALTORFFER AND D. DE WIED, Biotelemetry:Adjustment of a telemetry systemfor simultaneous measurementsofacuteheartratechangesand behavioraleventsin unrestrainedrats. PHYSIOL BEHAV 53(6) 1121-1126, 1993.--The radiotelemetry system described in this paper consists of an implantable transmitter and a receiver, connected to a microcomputer. The hardware and software belonging to Mini-Mitters, for the collection and analysis of heart rate (HR), core temperature (CT), and gross activity data, do not possess the flexibility to detect acute changes in HR nor to discriminate among simultaneously occurring different types of behavior. In order to study short-term changes in HR in response to stress or drugs, in relation to behavioral responses, an inexpensive computer interface and a software program (CARDIAQ) were developed to collect data from Mini-Mitters. The interface conveys the QRS signal, which is converted to a TTL pulse train, to the parallel printer adapter (LPTI) of an IBM-compatible computer. Heart rate is determined by measuring single interbeat intervals (IBI). The software controls the sampling schedule and stores the collected data in a format compatible with a commercial spreadsheet package. The program calculates the median IBI per s, mean _+ SD IBI, variance, skewness, and kurtosis of the IBI distribution. In addition, it enables simultaneous recording of behavior by entering data through the keyboard at the occurrence of each event. In this paper, we describe the CARDIAQ program and provide examples of its application together with the implantable transmitters in freely moving rats. Telemetry Autonomic nervous system Conscious rats
Heart rate
Interbeat interval
E N V I R O N M E N T A L events of a stressful nature elicit simultaneous changes in autonomic and behavioral activity (2,4,5,9,10). Various biotelemetry systems for small animals have been developed to record alterations in autonomic and behavioral activity (3,8,11-13). The major asset of the telemetry method is the possibility of recording various parameters at a time in the unrestrained, conscious animal ( 1,3,7,11,14). In previous studies, we have employed a wireless telemetry system (Mini-Mitter Co., Sunriver, OR) to assess heart rate (HR), core temperature (CT), and gross locomotor activity in freely moving
Behavior
Acute stress
rats while performing a behavioral task (5,14). The telemetry system consists of a small implantable transmitter, a receiver connected to a computer with data acquisition controlled by a computer board, and software package (DATAQUEST III). However, we have encountered various limitations of the method. This system has been developed for long-term monitoring of the above-mentioned parameters in small animals, rather than for measuring rapid H R responses. Because the shortest sampling period in the D A T A Q U E S T m e n u is l0 s, H R changes occurring within 10 s after a stimulus is applied
The CARDIAQ program is available from Mini-Mitter Co., Inc., PO BOX 3386, Sunriver, OR 97707; Phone: (503) 593-8639; FAX: (503) 5935604. i Requests for reprintsshould be addressed to Michaela Diamant, M.D., Rudolf Magnus Institute,Department of Pharmacology, Medical Faculty, Universityof Utrecht, Vondellaan 6, 3521 GD Utrecht, The Netherlands.
1121
1122
DIAMgNI
TABLE 1 COMPARISON OF HR MEASURED BY DAEAQUES'I AND CARDIAQ
li1 AI
after exposure of a rat to a novel stimulus will be demonstrated.
CARDIAQ
t (s)
DATAQUEST HR (bpm)
HR (bpm)
IBI (ms)
~Beats
0-60 180-240 300-360 540-600
329 366.2 352.6 355.1
328 368 351.2 356.5
183.1 163.0 170.5 168.3
328 351 352 356
Mean (0-600)
345.8
345.3
173.9
343.5
METHOD
Mean HR of a rat under resting conditions, simultaneously recorded by DATAQUEST III and CARDIAQ during a 10-min period. DATAQUEST was set to continuously sample HR at a sampling period of 10 s (column 2). Separate beat-to-beat recordings [(interbeat interval (IBI)] were performed by CARDIAQ for 60 s every other minute (column 3 and 5). Heart rate was calculated from the IBI by CARDIAQ (column 3). Representative data collected at t = 0-1, 3-4, 5-6, and 9-10 min are shown. For details see text.
would be undetected. Furthermore, the system collects so-called gross activity counts only, without specification of the actual types of behavior of the animal. In view of this, we have adjusted and extended the data acquisition system, by developing a program C A R D I A Q (Computer-Assisted Recording of Digitized Interbeat intervals Analyzed by Quattro, v 1.1), which enables beat-to-beat recording o f H R by d e t e r m i n a t i o n o f the interbeat interval (IBI) in ms. In addition, C A R D I A Q allows differentiation of various types of behavior. By entering data through precoded keys on the keyboard, various behaviors can be scored, the frequency and duration of which can be calculated off-line. Both the D A T A Q U E S T and the C A R D I A Q methods were c o m p a r e d in their efficacy to m o n i t o r changes in HR. Examples of m e a s u r e m e n t s by both systems of acute beat-to-beat changes in H R and behavioral responses
The CardioTel¢~ Biotelemetry and DA TAQUEST Ill Data Acquisition System~ Both systems have in part been described elsewhere (4,14). A wireless, battery-operated transmitter and a receiver connected to an IBM-compatible computer, which is operated by the DAT A Q U E S T hardware and software package, enable the monitoring and on-line analysis of heart rate (HR), core temperature (CT), and gross activity in laboratory animals. The implantable transmitter ( C a r d i o T e l ~ model CTT85-SA, Mini-Mitter Co., Inc.) consists of a resistor-capacitor (RC) oscillator in which the value of the resistor and consequently the output frequency is temperature dependent. The output from the transmitter consists of a train of 455 kHz bursts with the average frequency of these bursts coding for the telemetered temperature data and the shortterm variations in the frequency of the pulse train being proportional to the EKG signal. The output frequency of the CTT85SA transmitters can be received up to 30 cm away by a simple AM receiver. If the transmitters are used to measure the animal's CT. calibration in a water bath at two different temperatures is required prior to implantation, because each transmitter has a unique characteristic of frequency output vs. temperature. The oscillator, transmitter, and battery are enclosed in a plastic capsule and coated with wax to seal against moisture. The proportions of the package are 3 X 1.5 × 0.8 cm at the largest, flat and irregularly rectangular in shape, weighing 4 g. The battery has a lifetime of about 6 months. Heart potentials are sensed by the bare ends of the two leads extending from the package. After implantation of the package into the right lower quadrant of the abdominal cavity, the electrodes are tunnelled subcutaneously on either side of the thorax and the tips placed in a position that provides sufficient signal (>0.1 millivolt for the standard modeL). The receiver unit, placed under the animal's cage in the case of the model used in our experiments (model CTR85-SA, Mini-
TABLE 2 PRINTOUT OF THE .SUM FILE MADE BY CARDIAQ DATE
TIME
START
AUTO
RAT
TRT
PER.
ZBEAT
IBI
VAR
SKEW
KURT
22-04-91 22-04-9l 22-04-91 22-04-91 22-04-91 22-04-91 :
15:15:11 15:16:11 15:17:11 15:18:11 15:19:11 15:20:11 :
FI FI3 FI3 F13 FI3 Ft3 :
FI3 F13 FI3 FI3 FI3 FI3 :
3 3 3 3 3 3 :
0 0 0 0 0 0 :
60 60 60 60 60 60 :
331 328 330 322 351 354 :
180.73 183.06 181.75 185.74 163.01 169.43 :
6.76 3.80 6.78 8.89 12.03 6.18 :
-0.48 0.01 -0.10 -1.40 0.09 -0.52 :
0.44 -0.71 0.44 2.58 0.26 0.40 :
22-04-91
15:5'0:34
F1
FI 3
3
0
60
354
165.51
15.56
2.05
36.65
22-04-91 22-04-91 22-04-91
16:52:48 17:05:53 17:21:47
F1 F2 F1
F13 FI3 FI3
1 2 3
10 11 12
600 600 600
3710 3784 4900
133.68 150.53 120.44
38.34 21.55 17.77
3.72 4.46 9.62
15.75 31.32 I t 1.83
The upper part of the printout (above marker-line) summarizes Experiment 1; the three lines under the marker-line show data from Experiment 2. Rows 3 and 4 show the function keys pressed to start and restart sampling, respectively. RAT, rat code number; TRT, treatment; PER, sampling period (s); ZBEAT, total amount of beats recorded during sampling period; IB1, interbeat interval (ms); VAR, variation; SKEW, skewness; KURT, kurtosis; F13, programmed automatic restart of sampling; F I 2, manual interruption.
MONITORING OF HR AND BEHAVIOR BY TELEMETRY IN RATS
1123
connecting the receiver to the consolidation matrix and optically isolated interfaced to the ACK output of the parallel printer port (LPTI). This option was considered best for reasons of expense and compatibility.
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FIG. 1. Changes in gross activity (A) and heart rate (B) during a 10-min exposure to a mild novelty stress in rats 30 min after IP treatment with saline, atropine, or propranolol, reconstructed from data obtained by DATAQUEST.
Hardware Adaptation The digital HR pulse generated by the receiver (upon triggering by the R-wave of the EKG) was branched off the cable
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Mitter Co., Inc.), contains two perpendicularly placed antennas connected to AM receivers. This receiver provides an EKG analog output, a digital composite temperature and EKG output, and a digital activity output. The receiver detects R-waves in the EKG waveform and provides a digital pulse for each heart beat. Digital activity output from the receiver is obtained by continuous monitoring of the received signal strength, which varies as a result of movement-inducedchanges in the orientation between the transmitter and the receiving antenna. A cable with a modular plug at both ends directly conveys the composite EKG and temperature signals as well as the digital activity output to a consolidation matrix (BCM-100) for multiplexing the frequency and activity signals. When the telemetry system is used with the DATAQUEST data acquisition system, the data from the receivers are routed through the consolidation block onto a 26-conductor fiat cable, six receivers to a block. In our laboratory, the DATAQUEST III system, consisting of a printed circuit card (DQ1088, Data Sciences®) and a menu-driven software program for system operation, runs on an IBM XT computer.
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FIG. 2. Changesin gross activity(A), heart rate (B), and interbeat interval (C) over 60 s during period a 10-rain exposure to a mild novelty stress in rats treated with saline, atropine, or pmpranolol, respectively.Gross activity and heart rate were assessed by DATAQUEST, changes in interbeat interval were recorded by CARDIAQ.
1124
I)IAMANI 1!1- Al.
TABLE 3 COMPARISON OF RESULTS BY DATA0[3EST AND CARDIAQ DATAQUEST
CARDIAQ
Rat No.
TRT
HR [_+SD (SEM) var] (bpm)
GLA (counts}
IBI (ms)
HR (bpm)
GR (s)
[OC (s)
REAR (s)
VAR Is)
1 2 3
SAL PROP ATR
447 [_+99.8(12.7) 1268] 398 [_+36.5 (4.7) 1333] 500 [_+25.5(3.3) 650]
67.7 33.1 36.6
133,7 150.5 120.4
448.9 398.6 498.2
187 92 73
120 85 67
238 193 194
55 230 266
Results were obtained during a 10-min exposure (t = 0-10 min) of rats to a novelty stimulus, 30 min followinga single IP treatment of saline (rat 1), propranolol (rat 2), or atropine (rat 3). The values are calculated off-line.TRT, treatment; HR, heart rate; var, variance; GLA, gross locomotor activity; IBI, interbeat interval; SAL, saline; PROP, propranolol; ATR, atropine; counts, counts/time unit; GR, grooming; LOC, locomotion; REAR, rearing; VAR, various other types of behavior. For details see text.
Software
Experimental Protocols
The CARDIAQ program was written in Turbo Pascal (version 5.5). It does not use the already installed DATAQUEST III system. The program was designed for recording one animal at a time, mainly to maximize the number of events capable of being entered. The program determines the intervals between subsequent hardware interrupts from the ACK input of the parallel printer port (LPTI). These interrupts are generated by the Rwave of the rat EKG. The system clock chip, which normally produces an interrupt every 55 ms, has been reprogrammed so as to generate an interrupt every ms. This interrupt increments a memory-variable that is used as an interbeat interval timer. Using another variable, the system interrupt is activated each 55 ms to update the bios variables in the usual way. Thus, the program gives a 1-ms resolution of the R - R interbeat. Every heartbeat interrupt causes the ms counter to be stored in a buffer array, after which the counter is zeroed. To reduce artifacts, threshold values are used for maximum and minimum acceptable IBis. From the buffer array, the median IBI in each s is calculated. Also, the mean (absolute) difference between subsequent IBis in that s is calculated, as this may serve as an index for the beat-to-beat variability. These values, together with the raw data, are written to an output file in ASCII format, which can be handled by a commercial spreadsheet package (e.g., Borland Quattro or Lotus 123). This file, provided with the extension .DAT, also contains the animal number, the treatment code, date and time of sampling, the function key(s) used to start data acquisition, and keys pressed to mark the occurrence of events. Another output file, with the extension .SUM, contains statistical parameters calculated over each sampling interval, which was started by pressing a function key. The following experiments were conducted to validate the data obtained by this new data acquisition program together with the radiotelemetry system.
Rats were handled for 5 consecutive days following surgery for weighing and habituation purposes. On days 5 and 6, baseline HR values were recorded. To this purpose, the rat in its home cage was brought to the experimental room, placed onto a receiver, and allowed to habituate to the new situation for 60 to 90 min. When the animal was at rest, basal HR values were obtained by sampling with the DATAQUEST system, with the sampling period set at 30 s for 15-30 consecutive min.
Animals and Surgery Male Wistar rats of an inbred strain (weighing 200-220 g at the start of the experiments) were used. Under ether anaesthesia and sterile conditions, the transmitters were implanted intraperitoneally, the electrodes were guided subcutaneously on either side of the thorax, and the tips were sutured in place. Following surgery, the animals were housed individually and kept under conditions controlled for temperature (22 + 2°C) and light (lights on from 0600 to 2000 h). The animals were allowed to recover for 5 days.
Experiment 1--Validation ().['the CARDIAQ Data Acquisition Program In order to establish the compatibility of HR data recorded by the CARDIAQ program with HR values assessed by DATAQUEST III, the CARDIAQ software was installed to an IBM computer, different from the one configured with the DATAQUEST III system, and both systems were used simultaneously to monitor the HR of a rat under resting conditions. The sampling time in the DATAQUEST system was set at 10 s, the temperature channel was turned off. Data acquisition started at t = 0 after the animal was adapted to the experimental room and continued for two subsequent periods of 10 and 20 rain, respectively. During the first 10 min, CARDIAQ was set to simultaneously record IBis, starting at t = 0. Thus, for six data points obtained by the DATAQUEST system in 1 min, CARDIAQ provided a number of points equivalent to the number of impulses collected from the receiver and conveyed to the LPT1 during a 60-s recording period (beats per min). During the 20-min period, for the sake of data reduction, CARDIAQ was set to take 60-s samples every 5 rain. Mean HR values were calculated and compared over the various time periods.
Experiment 2--Detection of Acute Changes in HR Associated With Behavior Behavioral and cardiac responses to a mild stress after autonomic blockade were measured simultaneously by the DATAQUEST and CARDIAQ system. A t0-min exposure to a novel cage was used as a mild stressor. At 30 rain (t = -30) before the exposure to the novelty stimulus, rats were treated intraperitoneally (IP) with saline (SAL), atropine sulphate (ATR; 2 mg/kg b.wt.), or propranolol HCI (PROP; 2.5 mg/kg b.wt.). The injection volume was 0.5 ml. After the injections, the animals were returned to their home cages. At t = 0, one rat was placed into a novel cage and both computers were switched to the data acquisition mode. A receiver, which was connected to
MONITORING OF HR AND BEHAVIOR BY TELEMETRY IN RATS
both computers, was placed under the novel cage. The DATAQUEST system was set to sample HR and gross activity at 10-s sampling periods. Options from the CARDIAQ menu were defined to sample heartbeats for 600 s, while the observer rated the animal's behavior by pressing precoded keys at the onset of one of four predefined behavioral responses. Thus, G(rooming), R(earing), L(ocomotion), and V(arious) behaviors were scored.
1125
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RESULTS
Experiment 1 Table 1 shows summarized baseline HR values of a rat computed from data collected simultaneously by the DATAQUEST and CARDIAQ data acquisition systems. As the rat was under resting conditions (sleep, with mean gross activity 0), no significant variation in HR was observed during the 10-min recording period (evaluated from previous DATAQUEST recordings in sleeping rats for 30-90 min, data not shown). On these grounds, a discontinuous HR recording by CARDIAQ was justified. The mean HR over the 10-min period was determined at 345.8 bpm by DATAQUEST by averaging 60 data points, and at 345.7 bpm by CARDIAQ, calculated from mean IBI (Table 1). Additional comparisons for HR recorded by both systems were made (Table 1). The maximum difference between DATAQUEST-measured and CARDIAQ-computed HR over a 1-min time period was less than 5% (Table 1). Mean HR over the 20rain period was 340.5 for DATAQUEST (based on 119 data points) and 341.2 bpm for CARDIAQ (obtained from four 60s recordings, starting at, respectively, t = 0, 5, 10, 15, and calculated from mean IBI). Table 2 shows an adaptation of a typical print-out of a CARDIAQ.SUM file. The upper part of Table 2 contains data from the 10-min recording period, described above and elaborated in Table 1. The discrepancy between HR data in the Y~beats column and the corresponding HR values, calculated from the mean IBI, may result from artifacts at the level of the transmitter-receiver interaction, rather than being produced during system-scheduled data collection from the receiver. Due to this dropped-beat phenomenon, which is occasionally observed in both recording systems whenever rats engage in too vigorous somatomotor activity, the first value shows a lower HR than that calculated from mean IBI (Table 2). These results indicate that the CARDIAQ program reliably collects and analyzes HR data obtained by a wireless radiotelemetry system.
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Figure 1A and B show the respective time courses of gross activity and HR of three differently treated rats during 10 min of exposure to the novel cage, recorded by the DATAQUEST system. In Fig. 2A and B, DATAQUEST-collected gross activity and HR over the first 60-s period of the novel-cage test are depicted; Fig. 2C shows the corresponding IBI measurement by CARDIAQ. The IBI data points represent mean IBI per s. From the IBI recording, initial slowing and subsequent acceleration of HR in the SAL-treated (429 and 469 bpm, respectively) and in the ATR-treated (444 and 480 bpm) rat are observed (Fig. 2C). This short-lasting effect is not detected by DATAQUEST (Fig. 2B). In Table 3, an off-line analysis of HR and gross locomotor activity by DATAQUEST III, and of HR and behavioral responses recorded and calculated by CARDIAQ and a spread-sheet program, are compared. Mean HRs over the 10-min period were different for the animals, and in accordance with their respective treatments. Thus, the PROP-treated rat
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FIG. 3. Time course of changes in interbeat interval related to behavioral responses over a 60-s period during exposure to a novel cage in rats 30 min after IP treatment with saline (upper), propranolol (middle), and atropine (lower), reconstructed from data measured by CARDIAQ. LOC, locomotor activity; REAR, rearing; VAR, various.
showed the lowest and the ATR-administered rat the highest HR. Overall HR, calculated by DATAQUEST from 59 points and by CARDIAQ from mean IBI, differed <0.5%. Interestingly, the variance in HR during the 10-rain test for the PROP-treated rats was even higher than in controls, when computed by DATAQUEST, whereas from the calculation by CARDIAQ, the/3sympatholytic effect is reflected in a lower variance of the IBI [Table 2; (6)]. The marked reduction of HR variance due to ATR treatment, however, was detected by both recording methods [(6); Tables 2, 3].
1126
DIAMANi
D A T A Q U E S T gives a global impression of the rats' overall somatomotor activity in the novel environment by the recording of gross locomotor activity counts. Inhibition of either branch of the ANS resulted in a decrease in gross activity, respective to vehicle treatment, which was found most marked in the PROPtreated rat (Table 3). However, specification of behavioral activity by C A R D I A Q revealed that rats given A T R showed less grooming and locomotor activity than rats given PROP. This discrepancy may be explained by the mode D A T A Q U E S T obtains the activity data. As briefly indicated in the Method section, a critical change in the orientation of the (implanted) transmitter over the receiver, due to the rat's movement, results in change of the signal strength as detected by the receiver card, and hence a pulse or count of activity is recorded. This pulse of activity is recorded and increments the counter associated with a specific channel. Each input is compared with the state recorded on the previous sample, and, as a consequence, if the animal has not moved from its previous position, activity will not be incremented. Therefore, grooming behavior and other behaviors that do not cause a critical change in the orientation of the transmitter over the receiver will remain undetected as an activity pulse, whereas they can be recorded as coded event by C A R D I A Q . Figure 3 shows the relation among IBI and behaviors in the three differently treated rats during 60 s in the novel cage. DISCUSSION This report describes a software/hardware adaptation of a commercially available acquisition system for telemetered H R and gross activity data in freely moving rats. The newly developed system (CARDIAQ) determines H R by beat-to-beat measurements and therefore enables the detection of acute changes in cardiac activity. In addition, the method allows differentiation
t:i1 \ I .
for various types of behavior exhibited by an animal m response to a stimulus, by simply entering data through the keyboard. The experiments described in the paper merely serve to illustrate the use of this HR recording telemetry system m behavioral research. The results of the first experiment demonstrate that beat-to-beat measurements from Mini-Mitters by the C A R D I A Q program is fully compatible with the H R data acquired and analyzed by the D A T A Q U E S T III system. In the second experiment, we have demonstrated the use of the C A R D I A Q program in detecting acute and short-lived changes in autonomic responses related to differential behavioral patterns. The C A R D I A Q program has several advantages. First, no expensive additional computer cards are necessary to record and store H R data on a beat-to-beat base. Second, the use of the LPTI port as a gate for the incoming heart beats allows the use of any other IBM-compatible computer, without requiring further hardware adaptation. Third, specific behavior-related changes in cardiac activity can be assessed and vice versa. Fourth, the possibility of data entry by the user makes this program suitable for use in an extensive variety of experimental protocols. As an asset over the D A T A Q U E S T system, C A R D I A Q enables the evaluation of H R concomitants of various behaviors and as a result, provides a more differentiated picture of drug- or stimulus-specific autonomic and behavioral interrelationships. In sum, the Mini-Mitters, used in combination with DAT A Q U E S T system hardware and software, provide a suitable tool for long-term monitoring of H R in freely moving animals. The limitations of this system to detect small changes in cardiac action, related to behavioral patterns, have been described in this paper. Therefore, the C A R D I A Q program, sampling HR on a beat-to-beat base and allowing simultaneous recording of various behaviors, may further extend the possibilities of the biotelemetry method.
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