Physiology and Behavior, Vol. 14, pp. 449-456. Brain Research Publications Inc., 1975. Printed in the U.S.A.
Classical Conditioning of Heart Rate in Rats Using Direct Vagal Stimulation as a US ROBERT D. FITZGERALD, 2 GLEN K. MARTIN AND JOHN W. HOFFMAN University o f Oregon Medical School
(Received 22 August 1974)
FITZGERALD, R. D., G. K. MARTIN AND J. W. HOFFMAN. Classical conditioning of heart rate in rats using direct vagal stimulation as a US. PHYSIOL. BEHAV. 14(4) 449-456, 1975. - Classically conditioned heart-rate deceleration was established in rats using a 2-sec stimulation of the intact cervical vagus nerve as the US. Although small in magnitude, changes in the form of the CR over training indicated that there may have been inhibition of delay similar to that obtained using a traditional painful shock US. Independent assessments of motivational potency revealed that the vagal US did not lead to conditioned lever-press suppression in a CER procedure, but did produce reliable escape responding in a shuttle-box preference situation. It was suggested that the mildly aversive motivational effects of vagal stimulation may have stemmed from the activation of a vagally mediated system involved in the regulation of hunger. Heart rate
Classicalconditioning
Vagal stimulation
SEVERAL lines of evidence point to the importance of the motivational potency of the US in producing classically conditioned changes in the cardiovascular system. In two related studies [20,21] it was observed that intracranial stimulation functioned as an effective US for heart-rate (HR) conditioning in rabbits provided the stimulation had a sufficiently strong motivational component as measured independently in self-stimulation and shuttle-box preference situations. Low intensity stimulation that produced a HR UR but relatively weak motivational effects did not lead to conditioning [21]. Teitelbaum, Gantt, and Stone [23] employed a remotely controlled catheterization technique that allowed pain-free intravenous injections of acetylcholine without visible motivational side effects. Although the injections elicited vigorous cardiac URs no evidence of a conditioned response was found. In a second phase of the study a painful shock US was substituted for the drug and a conditioned HR response rapidly developed. Referring to earlier experiments [3] in which intravenous acetylcholine and other peripheral acting drugs supported conditioned cardiac responses, Teitelbaum et al. [23] noted that the presence of the experimenter and the repeated needle punctures from the injections may have created enough emotional disturbance to motivate conditioning. Finally, Bruner [2] was unable to condition HR in cats using a threshold level of shock for leg flexion as a US, even though a HR UR was present. When the intensity of the US was increased conditioned HR changes readily appeared.
Although the results of these studies generally support the view that a motivating US is essential for cardiac conditioning to occur, two investigations [ 11,12 ] in the Russian literature cited by Figar [5] cast some doubt on this r e q u i r e m e n t . According to these reports conditioned cardiac responses in dogs were generated by direct stimulation of the vagus nerve as a US. Kozenko [12] stimulated the peripheral end of the sectioned cervical vagus for up to 10 sec and observed conditioned decreases in blood pressure (BP) within 50 to 100 conditioning trials. Specific quantitative data were not provided but it was noted that the BP CR was transitory, appearing on some trials and not on others. Since the conditioning session began 1 - 2 hr after the stimulating electrodes were implanted, the traumatized neck area was treated with a 5% solution of Novocaine to lessen the residual pain from the surgery. More recently, Andrus, Gantt, Plumlee, and Gross [ 1] sectioned either the left or right vagosympathetic trunk below the larynx in dogs and stimulated the central portion of the nerve for 6 sec as a US. Although several hundred conditioning trials were given some dogs, cardiac CRs were not established. The possibility that vagal stimulation may lack motivational consequences is supported primarily by the failure of anatomical and neurophysiological studies to show conclusively that pain fibers are present in the vagus [14,18 ]. While these findings suggest that vagal stimulation may not be aversive the results of several recent studies [15,16]
~The research was supported by a National Science Foundation Grant GB-28222 and in part by a U.S. Public Health Service Grant 5 S01 RR05412 and a National Institute of General Medical Science Grant 5 T01 GM01495. ~Requests for reprints should be sent to Robert D. Fitzgerald, Department of Medical Psychology, University of Oregon Medical School, Portland, Oregon 97201. 449
450
FITZGERALD, MARTIN AND ttOFFMAN
involving cats and rabbits indicate that vagal afferent sensory fibers mediating the abdominal visera may represent an important segment of a hunger related motivational system controlling consummatory behavior. It is conceivable that the extent to which vagal stimulation serves as an effective US for cardiac conditioning may depend upon the activation of this system. The two objectives of the present series of experiments were to determine whether stimulation of the intact cervical vagus in rats would function as a US for classically conditioned HR and to test the motivational potency of vagal stimulation in lever-press conditioned suppression and shuttle-box preference situations. EXPERIMENT 1
Method Animals. Forty-two female Long-Evans rats ranging 1 9 0 - 2 5 0 g in weight and approximately 9 0 - 1 2 0 days old were used. They were purchased from Simonsen Laboratories and maintained under conditions of continuous illumination with free access to food and water. One week prior to surgery the animals were separated and placed in individual cages. Stimulating electrodes and surgical implantation. The electrodes for stimulating the vagus nerve consisted of two 11.0 -cm lengths of 0.25-mm teflon-coated silver wire. The ends of the wires to be attached to the nerve were shaped to form small hooks and the teflon insulation was scraped from the hooks exposing approximately 3.0 mm of bare wire. Male Amphenol reliatac connector pins were soldered to the opposite ends of the wires. Both wires were inserted through a short section of silastic tubing and a knot was loosely tied toward the end of the tubing for anchoring to the animal's head. Animals were given an injection of atropine sulfate (0.1 mg/kg of body weight) 15 min prior to being anesthesitized with sodium pentobarbital (40 mg/kg of body weight). Additional administrations of small amounts of Penthrane were given, as needed, to maintain proper depth of anesthesia. An incision was made on the ventral aspect of the neck extending approximately 3 cm from the posterior portion of the jaw to the anterior sternum. Following exposure of the right vagus nerve and removal of the carotid sheath covering the nerve the hooked ends of the electrode wires were speared under and around the nerve. The uninsulated sections of the wires were separated by 1 . 0 - 2 . 0 mm and secured and insulated at that distance b y a 1:1 mixture of Dow Coming Medical Elastomer 302 and Medical Fluid 360 with catalyst. The other ends of the wires were tunneled under the skin and pulled through a small incision located midway between the animal's ears. The wires were then anchored in place by suturing the silastic tubing to the surrounding tissues and imbedding the amphenol pins in Dow Coming silicone type A adhesive. All animals were given 4 days to recover from surgery before conditioning was started. Apparatus. The rats were restrained in a U-shaped plastic-dome animal holder manufactured by E & M Instrument Company. Adjustable guillotine-type inserts at both ends of the holder were positioned to hold the rats securely. The holder was located in an Industrial Acoustic sound-isolation chamber equipped with a 10-W house light recessed in the ceiling and a 6-in. speaker mounted on the wall facing the animal. Two rats were trained concurrently,
each in a separate identical chamber with trials alternating between the rats. The electrocardiogram (EKG) was recorded on a Grass polygraph from 20-ga. hypodermic needles located on either side of the rat's thoracic cavity. The number of heart beats occurring in consecutive time intervals within a trial was automatically tabulated by means of an on-line recording system similar to the one described by Fitzgerald, Vardaris, and Teyler [9 ]. Basically, the system consisted of a lever type microswitch mounted on a Plexiglas plate directly above the EKG polygraph pen with the arm of the switch connected to the tip of the pen. The position of the switch was adjusted so that it was actuated by the R wave of the QRS complex. The number of R waves was accumulated on a BCD counter and punched on a high-speed paper-tape punch. The US was a 2-sec train of 1-msec biphasic pulses at a frequency of 10 Hz produced by a Grass S-4 stimulator. The voltage level of the stimulus was adjusted in 0.5-V increments for each animal to elicit vagal blockade of HR. The voltage drop across a fixed resistor in series with the animal was measured with an oscilloscope to determine stimulus current. The mean stimulus current received by the groups was 4.75 ma with a standard deviation of 1.68 ma. The CS was an 8.0-sec k-kHz tone presented at 75-dB sound pressure level (re .0002 dyne/cm2). The CS-US interval was 6 sec with the US overlapping the last 2 sec of the CS. Trials were started automatically by a film-tape programmer with the stimulus events and HR counting intervals occurring within a trial programmed and timed by solid state logic modules. Procedure. The basic design of the experiment was comprised of an experimental group (N = 15) receiving paired presentations of the CS and US and a conditioning control group (N = 15) receiving unpaired presentations with the CS following the US by an interval of 60, 80, or 100 sec (M = 80 sec). Following the completion of the main experiment 12 additional animals were given conditioning training in order to determine whether the original findings could be replicated. Since the conditioning performance level of this additional group and that of the original group were both significantly different from the control group and did not differ significantly from each other, the two experimental groups were combined. All animals were given 30 rain of adaptation in the restraining device prior to conditioning. At the beginning of this period, animals were given two or three vagal stimulations to determine the appropriate voltage level of the US. Once selected the intensity for a given rat was not changed during conditioning. At the end of adaptation, each animal received 20 pretest trials with CS alone at intertrial intervals (ITI) of either 70, 90, or 110 sec (M -- 90 sec), followed by 40 acquisition trials with an ITI of either 270, 290, or 310 sec (M = 290 sec). Immediately following acquisition the animals were released from the restraining device and permitted to explore the inside of the sound isolation chamber. They were then given at least six presentations of the US alone and visual observations were made of the effects of these stimulations on the rat's general behavior. The HR responses to the CS on the acquisition trials were obtained for each rat by subtracting the number of heart beats during the 6 sec immediately preceding the onset of the CS (pre-CS period) from the number during the 6-sec CS-US interval. These difference scores were then converted to a beats-per-minute (bpm) index.
HEART RATE CONDITIONING AND VAGAL STIMULATION
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Experimental Control
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FIG. 2. Mean adjusted heart-rate changes of the experimental and control groups in successive 2-see time periods after CS onset averaged over blocks of 10 trials in acquisition.
Results The mean CS minus pre-CS HR responses of the experimental and control groups during successive four-trial blocks of acquisition are plotted in Fig. 1. The figure reveals that a conditioned decelerative HR response was established in the experimental group. The magnitude of the CR increased relatively rapidly and reached a terminal level of approximately 8 bpm. On the other hand, the HR responses shown by the control groups fluctuated around zero. An analysis of variance comparing the two groups provided a significant experimental vs. control group effect
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FIG. 1. Mean CS minus pre-CS heart-rate changes of the experimental and control groups in successive four-trial blocks of acquisition.
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FIG. 3. Mean adjusted heart-rate changes of the experimental and control groups in successive time periods after vagal stimulation averaged over the forty trials of acquisition. (F = 11.59, dr= 1/40, p
Classical Conditioning of Heart Rate in Rats Using Direct Vagal Stimulation as a US ROBERT D. FITZGERALD, 2 GLEN K. MARTIN AND JOHN W. HOFFMAN University o f Oregon Medical School
(Received 22 August 1974)
FITZGERALD, R. D., G. K. MARTIN AND J. W. HOFFMAN. Classical conditioning of heart rate in rats using direct vagal stimulation as a US. PHYSIOL. BEHAV. 14(4) 449-456, 1975. - Classically conditioned heart-rate deceleration was established in rats using a 2-sec stimulation of the intact cervical vagus nerve as the US. Although small in magnitude, changes in the form of the CR over training indicated that there may have been inhibition of delay similar to that obtained using a traditional painful shock US. Independent assessments of motivational potency revealed that the vagal US did not lead to conditioned lever-press suppression in a CER procedure, but did produce reliable escape responding in a shuttle-box preference situation. It was suggested that the mildly aversive motivational effects of vagal stimulation may have stemmed from the activation of a vagally mediated system involved in the regulation of hunger. Heart rate
Classicalconditioning
Vagal stimulation
SEVERAL lines of evidence point to the importance of the motivational potency of the US in producing classically conditioned changes in the cardiovascular system. In two related studies [20,21] it was observed that intracranial stimulation functioned as an effective US for heart-rate (HR) conditioning in rabbits provided the stimulation had a sufficiently strong motivational component as measured independently in self-stimulation and shuttle-box preference situations. Low intensity stimulation that produced a HR UR but relatively weak motivational effects did not lead to conditioning [21]. Teitelbaum, Gantt, and Stone [23] employed a remotely controlled catheterization technique that allowed pain-free intravenous injections of acetylcholine without visible motivational side effects. Although the injections elicited vigorous cardiac URs no evidence of a conditioned response was found. In a second phase of the study a painful shock US was substituted for the drug and a conditioned HR response rapidly developed. Referring to earlier experiments [3] in which intravenous acetylcholine and other peripheral acting drugs supported conditioned cardiac responses, Teitelbaum et al. [23] noted that the presence of the experimenter and the repeated needle punctures from the injections may have created enough emotional disturbance to motivate conditioning. Finally, Bruner [2] was unable to condition HR in cats using a threshold level of shock for leg flexion as a US, even though a HR UR was present. When the intensity of the US was increased conditioned HR changes readily appeared.
Although the results of these studies generally support the view that a motivating US is essential for cardiac conditioning to occur, two investigations [ 11,12 ] in the Russian literature cited by Figar [5] cast some doubt on this r e q u i r e m e n t . According to these reports conditioned cardiac responses in dogs were generated by direct stimulation of the vagus nerve as a US. Kozenko [12] stimulated the peripheral end of the sectioned cervical vagus for up to 10 sec and observed conditioned decreases in blood pressure (BP) within 50 to 100 conditioning trials. Specific quantitative data were not provided but it was noted that the BP CR was transitory, appearing on some trials and not on others. Since the conditioning session began 1 - 2 hr after the stimulating electrodes were implanted, the traumatized neck area was treated with a 5% solution of Novocaine to lessen the residual pain from the surgery. More recently, Andrus, Gantt, Plumlee, and Gross [ 1] sectioned either the left or right vagosympathetic trunk below the larynx in dogs and stimulated the central portion of the nerve for 6 sec as a US. Although several hundred conditioning trials were given some dogs, cardiac CRs were not established. The possibility that vagal stimulation may lack motivational consequences is supported primarily by the failure of anatomical and neurophysiological studies to show conclusively that pain fibers are present in the vagus [14,18 ]. While these findings suggest that vagal stimulation may not be aversive the results of several recent studies [15,16]
~The research was supported by a National Science Foundation Grant GB-28222 and in part by a U.S. Public Health Service Grant 5 S01 RR05412 and a National Institute of General Medical Science Grant 5 T01 GM01495. ~Requests for reprints should be sent to Robert D. Fitzgerald, Department of Medical Psychology, University of Oregon Medical School, Portland, Oregon 97201. 449
450
FITZGERALD, MARTIN AND ttOFFMAN
involving cats and rabbits indicate that vagal afferent sensory fibers mediating the abdominal visera may represent an important segment of a hunger related motivational system controlling consummatory behavior. It is conceivable that the extent to which vagal stimulation serves as an effective US for cardiac conditioning may depend upon the activation of this system. The two objectives of the present series of experiments were to determine whether stimulation of the intact cervical vagus in rats would function as a US for classically conditioned HR and to test the motivational potency of vagal stimulation in lever-press conditioned suppression and shuttle-box preference situations. EXPERIMENT 1
Method Animals. Forty-two female Long-Evans rats ranging 1 9 0 - 2 5 0 g in weight and approximately 9 0 - 1 2 0 days old were used. They were purchased from Simonsen Laboratories and maintained under conditions of continuous illumination with free access to food and water. One week prior to surgery the animals were separated and placed in individual cages. Stimulating electrodes and surgical implantation. The electrodes for stimulating the vagus nerve consisted of two 11.0 -cm lengths of 0.25-mm teflon-coated silver wire. The ends of the wires to be attached to the nerve were shaped to form small hooks and the teflon insulation was scraped from the hooks exposing approximately 3.0 mm of bare wire. Male Amphenol reliatac connector pins were soldered to the opposite ends of the wires. Both wires were inserted through a short section of silastic tubing and a knot was loosely tied toward the end of the tubing for anchoring to the animal's head. Animals were given an injection of atropine sulfate (0.1 mg/kg of body weight) 15 min prior to being anesthesitized with sodium pentobarbital (40 mg/kg of body weight). Additional administrations of small amounts of Penthrane were given, as needed, to maintain proper depth of anesthesia. An incision was made on the ventral aspect of the neck extending approximately 3 cm from the posterior portion of the jaw to the anterior sternum. Following exposure of the right vagus nerve and removal of the carotid sheath covering the nerve the hooked ends of the electrode wires were speared under and around the nerve. The uninsulated sections of the wires were separated by 1 . 0 - 2 . 0 mm and secured and insulated at that distance b y a 1:1 mixture of Dow Coming Medical Elastomer 302 and Medical Fluid 360 with catalyst. The other ends of the wires were tunneled under the skin and pulled through a small incision located midway between the animal's ears. The wires were then anchored in place by suturing the silastic tubing to the surrounding tissues and imbedding the amphenol pins in Dow Coming silicone type A adhesive. All animals were given 4 days to recover from surgery before conditioning was started. Apparatus. The rats were restrained in a U-shaped plastic-dome animal holder manufactured by E & M Instrument Company. Adjustable guillotine-type inserts at both ends of the holder were positioned to hold the rats securely. The holder was located in an Industrial Acoustic sound-isolation chamber equipped with a 10-W house light recessed in the ceiling and a 6-in. speaker mounted on the wall facing the animal. Two rats were trained concurrently,
each in a separate identical chamber with trials alternating between the rats. The electrocardiogram (EKG) was recorded on a Grass polygraph from 20-ga. hypodermic needles located on either side of the rat's thoracic cavity. The number of heart beats occurring in consecutive time intervals within a trial was automatically tabulated by means of an on-line recording system similar to the one described by Fitzgerald, Vardaris, and Teyler [9 ]. Basically, the system consisted of a lever type microswitch mounted on a Plexiglas plate directly above the EKG polygraph pen with the arm of the switch connected to the tip of the pen. The position of the switch was adjusted so that it was actuated by the R wave of the QRS complex. The number of R waves was accumulated on a BCD counter and punched on a high-speed paper-tape punch. The US was a 2-sec train of 1-msec biphasic pulses at a frequency of 10 Hz produced by a Grass S-4 stimulator. The voltage level of the stimulus was adjusted in 0.5-V increments for each animal to elicit vagal blockade of HR. The voltage drop across a fixed resistor in series with the animal was measured with an oscilloscope to determine stimulus current. The mean stimulus current received by the groups was 4.75 ma with a standard deviation of 1.68 ma. The CS was an 8.0-sec k-kHz tone presented at 75-dB sound pressure level (re .0002 dyne/cm2). The CS-US interval was 6 sec with the US overlapping the last 2 sec of the CS. Trials were started automatically by a film-tape programmer with the stimulus events and HR counting intervals occurring within a trial programmed and timed by solid state logic modules. Procedure. The basic design of the experiment was comprised of an experimental group (N = 15) receiving paired presentations of the CS and US and a conditioning control group (N = 15) receiving unpaired presentations with the CS following the US by an interval of 60, 80, or 100 sec (M = 80 sec). Following the completion of the main experiment 12 additional animals were given conditioning training in order to determine whether the original findings could be replicated. Since the conditioning performance level of this additional group and that of the original group were both significantly different from the control group and did not differ significantly from each other, the two experimental groups were combined. All animals were given 30 rain of adaptation in the restraining device prior to conditioning. At the beginning of this period, animals were given two or three vagal stimulations to determine the appropriate voltage level of the US. Once selected the intensity for a given rat was not changed during conditioning. At the end of adaptation, each animal received 20 pretest trials with CS alone at intertrial intervals (ITI) of either 70, 90, or 110 sec (M -- 90 sec), followed by 40 acquisition trials with an ITI of either 270, 290, or 310 sec (M = 290 sec). Immediately following acquisition the animals were released from the restraining device and permitted to explore the inside of the sound isolation chamber. They were then given at least six presentations of the US alone and visual observations were made of the effects of these stimulations on the rat's general behavior. The HR responses to the CS on the acquisition trials were obtained for each rat by subtracting the number of heart beats during the 6 sec immediately preceding the onset of the CS (pre-CS period) from the number during the 6-sec CS-US interval. These difference scores were then converted to a beats-per-minute (bpm) index.
HEART RATE CONDITIONING AND VAGAL STIMULATION
-12 -
Experimental Control
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FIG. 2. Mean adjusted heart-rate changes of the experimental and control groups in successive 2-see time periods after CS onset averaged over blocks of 10 trials in acquisition.
Results The mean CS minus pre-CS HR responses of the experimental and control groups during successive four-trial blocks of acquisition are plotted in Fig. 1. The figure reveals that a conditioned decelerative HR response was established in the experimental group. The magnitude of the CR increased relatively rapidly and reached a terminal level of approximately 8 bpm. On the other hand, the HR responses shown by the control groups fluctuated around zero. An analysis of variance comparing the two groups provided a significant experimental vs. control group effect
-
~,-40 ~
FIG. 1. Mean CS minus pre-CS heart-rate changes of the experimental and control groups in successive four-trial blocks of acquisition.
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FIG. 3. Mean adjusted heart-rate changes of the experimental and control groups in successive time periods after vagal stimulation averaged over the forty trials of acquisition. (F = 11.59, dr= 1/40, p
