Methoxamine is an effective unconditioned stimulus for cardiovascular conditioning

Phystology&Behavior, Vol. 43, pp. 177-185 Copyright©PergamonPress plc, 1988 Pnntedin the U.S.A

0031-9384/88$3 00 + .00

Methoxamine is an Effective Unconditioned Stimulus for Cardiovascular Conditioning C H A R L E S M. G I B B S , B A R B A R A A. S P E N C E R , K U E N - T E N G K A O A N D D. A. P O W E L L

Neuroscience Laboratory, WJB Dorn Veterans' Hospital, Columbia, SC 29201 and Department o f Psychology, University o f South Carolina, Columbia, SC 29208 R e c e i v e d 24 July 1987 GIBBS, C. M., B. A. SPENCER, K.-T. KAO AND D. A POWELL. Methoxamine is an effecttve uncondtttonedstimulus for cardiovascular condztiomng. PHYSIOL BEHAV 43(2) 177-185, 1988.--New Zealand albino rabbits received classical conditioning training in which a 35-sec tone conditioned stimulus was paired with a bolus injection of methoxamine hydrochloride (Vasoxyl), an al-adrenerglc agonist. Heart rate (HR) and blood pressure (BP) responses were recorded. Methoxamine produced a precipitous nse in BP and bradycardia as an uncondmoned response (UR); pairings of tone and methoxamine over a 5-day period resulted m a gradually appearing tachycardiac conditioned response (CR) which occurred shortly following tone onset. On the other hand, the BP CR was a pressor response. Accordingly, the HR CR was Opposite in direction and, thus, apparently compensatory to the UR, whereas the BP CR was similar in direction to the UR. Neither of these cardiovascular changes were observed in control animals receiving either unpaired presentations of tone and methoxamine or tones paired with physiological saline. Most animals receiving either paired or unpaired infusions of methoxamine also showed consistent elevations in basehne HR as training progressed, relative to their respective day 1 levels, thus suggesting the development of compensatory HR CRs to the contextual cues associated with training Pavlovian conditioning Heart rate Stimulus properties of drugs

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A variety of cardiovascular responses are subject to Pavlovian conditioning procedures [6]. The parameters which control this kind of autonomic learning are known to differ from those that control the development of somatomotor CRs, such as the conditioned leg flexion and eyelid responses [27,29]. In this regard, somatomotor CRs have been shown to be highly specific to the particular features of the unconditioned stimuli (USs) employed (e.g., their locus of application); whereas conditioned autonomic changes, which invariably develop during somatomotor conditioning, have typically been described as "nonspecific" to the characteristics of their respective USs (e.g., [28,33]). This dichotomy is not surprising, since most previous studies of conditioned autonomic changes have employed aversive USs that directly elicit somatomotor defense responses but only indirectly influence autonomic variables. There is a reason to believe, however, that some conditioned autonomic changes may be routinely established which are, in fact, "specific," since their development resuits from USs which directly affect visceral response systems. For example, taste aversion learning apparently involves visceral CRs established by one or more pairings of drug-induced illness with a gustatory stimulus [9]. Similarly, situation-specific drug tolerance [32] may involve an association between external, contextual stimuli and pharmacological changes that are specifically induced by drug

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administration. An understanding of the parametric features of this kind of conditioning would certainly have implications for understanding the mechanisms underlying learned tolerance and addiction [32]. Nevertheless, although animal models of conditioned taste aversion (e.g., [9]), as well as situation-specific tolerance (e.g., [23,31]), have existed for some time, no such model has been developed in which the traditional methodolgy involving repeated pairings of a discrete conditioned stimulus (CS) and a US is employed. And yet, the use of a discrete CS would clearly facilitate the parametric evaluation of these forms of visceral learning. The present experiments represent an initial attempt to develop such a model. To this end, the al-adrenergic agonist, methoxamine hydrochloride, was employed as a US and paired with a 75-dB, 1216-Hz tone. This tonal CS has been used in a number of previous investigations of learned cardiovascular adjustments associated with classical eyelid conditioning [4, 12, 26, 27]. In the present instance, however, a much longer CS-US interval was employed, since the latency for drug-induced cardiovascular changes is appreciably longer than for changes produced by a peripheral US such as paraorbital electric shock. The unconditioned response (UR) produced by methoxamine consists of a pressor response and bradycardia, the latter reflecting baroreceptor activation of vagal tone [2]. We initially hypothesized that the CRs in the present studies would consist of tachycardia

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and depressor responses. This hypothesis stemmed from the idea that autonomic CRs are often observed to be compensatory, that is, opposite in sign, to their respective USs as elicited by either exteroceptive or pharmacological USs (see reviews in [8] and [30]). As described below, however, this hypothesis was only partially confirmed. EXPERIMENT 1

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Animals The animals were 10 male and 10 female experimentally naive New Zealand albino rabbits (Oryctolagus cuniculus), approximately 6 months of age and weighing 2.7-3.5 kg upon arrival in the laboratory. Animals were individually housed within a climate-controlled, AAALAC-accredited animal colony and maintained on ad lib food and water. All training sessions were conducted during the daylight portion of a 0700-1900 hours light-dark cycle.

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Apparatus During training, each animal was positioned in a Plexiglas rabbit restrainer [14] which was placed within a ventilated, sound-attenuating experimental enclosure (Industrial Acoustics Inc.). Behavioral responses were recorded on a Grass Model 7D polygraph equipped with appropriate preamplifiers. Experimental events were controlled by a Heath LSI-11 microcomputer, which also measured and time-documented behavioral data from the polygraph amplifiers. The auditory CS was a 35-sec, 1216-Hz, 75 dB re 20 ~ N / m 2 square-wave tone generated by a sohd state audio oscillator and delivered through a Quam speaker located approximately 30 cm above the animal's head. The drug US was a 100/zg/kg body wt. dose of methoxamine hydrochloride delivered through a 26-g hypodermic needle inserted into the marginal ear vein. This needle was coupled to a 5-ml syringe driven by a remotely controlled infusion pump (Harvard Apparatus Model 975) located outside the experimental chamber. The concentration of the drug in heparinized saline was adjusted for each animal so that the entire dose could be administered in a 0.095-ml, 1-sec bolus. These drug parameters were established in a pilot study in which HR and BP changes were recorded in three rabbits in response to several different doses of methoxamine. The 100 /~g/kg dose was selected as the US based on the observation that it resulted in a profound pressor response (<30 mmHg) and bradycardia (<50 beats/min) in all three animals; both HR and BP returned to baseline within 15 min following injection.

Response Measurement HR was measured in beats/min (bpm) on each trial during successive baseline, tone, and posttone periods of 5, 35, and 25 sec duration, respectively. The HR electrodes were stainless steel safety pins inserted subcutaneously over the right shoulder and left flank, which were connected to a Grass Model 7P4F ECG/Tachograph preamplifier calibrated to record rates over a range o f 100-300 bpm. The computer sampied HR from the tachograph voltage output of the preamplifier and output average values for each trial during the 5-sec baseline period and for sixty, 1-sec blocks following tone onset.

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FIG. 1 Polygraph tracings dlustratmg the cardiovascular responses of individual animals to the infusion of methoxamlne (100/~g/kg) in Experiment 1. Shown are representative records obtmned during the mmal day of either control (upper panel) or classical conditioning (lower panel) training, as described m the text. The arrow denotes the onset of infusion. BP was recorded for 14 of the 20 ammals on both the first and final days of trmning. Immediately prior to these sessions, a 30-g teflon cannula filled with heparinized saline was inserted into the medial ear artery under local anesthesia (lidocame hydrochloride), sutured to the surrounding fascia, and connected to a Statham strain-gauge pressure transducer coupled to a Grass Model 7P1/E AC preamplifier. The input bridge o f the preamplifier was calibrated with a mercury manometer, and the sensitivity of the preamplifier was set so as to record a range of pressures of 50--130 mmHg. The computer sampled the output voltage of the preamplifier and output averages for each trial as descnbed above.

Procedure Each animal received one day of adaptation to handhng and restraint, followed by five successive days of either Pavlovian conditioning or nonassociative training and a subsequent day of extinction (tones alone), during which BP responses were assessed. On the adaptation day, the animals were restrained and placed within the experimental chamber for a period of at least 30 min, during which time a single, 35-sec tone was presented to assess unconditioned HR changes (orienting responses; ORs). Conditioning training (n=12) revolved pairings of the tone with methoxamine infusion. In particular, each conditioning session consisted of six presentations of the tone at 15-min intervals, and during all but the fifth tone, methoxamine was administered; the interval between tone onset and drug infusion was 25 sec. Nonassociative training (n=8) involved either one of two

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procedures. F o u r animals received explicitly unpaired presentations of the tone and methoxamine infusion; the minimum tone-drug interval was 2.5 min. The remaining four animals were treated exactly as animals in the conditioning group, except that physiological saline was infused in lieu of methoxamine. During the extinction session, animals received six presentations of the tone alone at intervals of 15 min.

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Statistical Analysis The effects of conditioning and nonassociative training on HR and BP responses were evaluated by mixed-effects analyses of variance (ANOVAs); follow-up analyses were performed according to the method of Tukey (see [20]). Initial analyses indicated that the two nonassociative treatments had statistically indistinguishable effects on toneevoked cardiovascular changes across sessions; consequently, in the analyses below, the data for these groups were pooled to form a single contrast control group.

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RESULTS AND DISCUSSION

Methoxamine Effects The dose of methoxamine employed was observed to have consistent and pronounced effects upon BP and HR in both the conditioning and the unpaired control animals. However, the quantitative nature of these effects could be assessed unequivocally only in the control animals receiving unsignaUed drug infusions; consequently, the cardiovascular responses of these animals during the initial training session, when BP was recorded, were statistically evaluated. Methoxamine elicited pressor responses of 7 - > 4 0 mmHg (mean-+S.E.M. =23.4-+4.1 mmHg), as illustrated in the upper panel of Fig. 1; the latency of these responses, measured from the onset of drug infusion, ranged from 5.6-10.2 sec (mean=6.9-+0.5 sec). These drug-induced pressor responses were paralleled by large bradycardiac responses; decelerations of 80-120 bpm were commonly observed. Drug action was relatively prolonged, being quite apparent for 3-5 rain. The drug also produced qualitatively similar cardiovascular responses in the conditioning animals, as shown in the lower panel of Fig. 1.

Training Effects on HR HR ORs to the 35-sec tone were assessed during the adaptation session, as described above. Consistent with previous findings [4, 12, 17], all animals showed unconditioned bradycardiac responses to the novel tone stimulus. Figure 2 illustrates the group mean HR response to this stimulus for animals assigned to the conditioning and control groups. In each case, the tone elicited a short-latency HR deceleration which was maximal during the first 5 sec of stimulation ( m e a n = - 1 7 . 3 bpm) and which persisted until shortly after tone termination. An A N O V A of the OR data using group assignment and 5-sec block as factors revealed a significant effect of block, F(8,126)=7.6, p<0.001, reflecting reliable bradycardiac ORs in these animals. However, the effects of groups and group × blocks were unreliable, suggesting that the groups did not differ with respect to either baseline HR or tone-evoked HR changes prior to training. Associative and nonassociative effects on tone-evoked HR changes were assessed during each of the five training sessions, and differences between the training schedules were, in fact, observed. Specifically, starting with the first

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trial of day 2 training, animals in the conditioning group began to show tachycardiac responses to the tone, although the magnitude of these responses declined across trials within each of the conditioning sessions. In contrast, control animals showed only a progressive habituation of the unconditioned bradycardiac responses (ORs) to the tone, both within and across the nonassociative training sessions. To statistically evaluate schedule effects on HR responses to the tone, all HR profiles were converted to difference scores which reflected changes from baseline levels in bpm during successive 5-sec blocks of the tone. This conversion was restricted to the initial five, 5-sec blocks following tone onset which preceded drug infusion. A mixedeffects A N O V A of these difference scores, with factors of group, day, trial and 5-sec block, revealed significant effects o f d a y , F(4,72)=11.73, p < 0 . 0 0 1 ; block, F(4,72)=19.18, p <0.001; and day x block, F(16,288)=3.60, p<0.001, all reflecting the apparent development of tachycardiac responses in the conditioning group, as well as the attenuation of toneevoked bradycardia in control animals. The effect of group was only marginally reliable, F(1,18)=4.10, p<0.06, however, a finding most likely attributable to the rather substantial within-session response decrements observed in the conditioning animals, as noted above. Further examination of the HR data for individual animals suggested that these within-session response decrements may have simply reflected "trial massing," since these decrements were paralleled by rather substantial declines in baseline H R across trials 1-5 of each conditioning session

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(mean change = - 2 6 . 9 bpm). In contrast, only small and variable decreases in tonic HR were observed in the salinecontrol animals. Thus, the repeated administration of methoxamine at 15-min intervals could have resulted in cumulative, pharmacological effects on cardiovascular responsivity in general. Consequently, additional analyses were undertaken which were restricted to the HR data from the initial trial of each of the five training sessions. Figure 3 illustrates the trial 1 HR response (in bpm) of each group plotted in 5-sec blocks of the tone period for each day of training. As illustrated m the left-hand panel of the figure, the conditioning group showed an initial short-latency bradycardiac response, which became a tachycardiac response over days of training; cardioaccelerations were evident during tone blocks 2-5 and maximal during the final two training sessions. As illustrated in the right-hand panel of Fig. 3, the control group also showed a short-latency bradycardiac response during the initial training trial; however, this response simply showed progressive attenuation across subsequent training sessions. These suggested group differences were confirmed by ANOVA, F(1,18)=8.13, p <0.02. The preceding analysis indicated that repeated pairings of tone and methoxamine infusion led to the acquisition of a conditioned tachycardiac response to the tone. The CR reached maximal levels during days 4 and 5 of conditioning training and was well-developed within 10 sec of tone onset. The temporal characteristics of the HR CR are more clearly revealed in Fig. 4, which plots group mean rate changes from baseline during the first ten, 1-sec blocks of the tone; the data summarized in the figure represent the average HR responses for the initial trial of sessions 4 and 5. It can be seen that the tachycardiac response shown by the conditioning animals occurred with a latency of approximately 4 sec and reached a level of 11.5 bpm during the eighth block of the

tone period. In contrast, control animals showed an mitial, small evoked bradycardla during blocks 2-5, followed by a return to just above the pre-tone baseline level. Within-session changes in baseline HR in the conditioning animals were previously described. In addition to these, consistent changes in baseline HR across sessions were observed in most animals receiving methoxamine infusion, whether paired or unpaired with tone. F o r example, three of the four methoxamine control animals showed consistent elevations in baseline HR prior to the first tone trial of trammg sessions 2-5, relative to the corresponding control rates recorded on day 1; the fourth such control animal also showed similar baseline increases during the final three unpaired training sessions. In addition, nine of the 12 conditioning animals exhibited similar, although smaller and more varruble, basehne HR elevations during sessions 3-5. In each group, changes in basal HR were most prominent during the third training session, for which group mean increases of 27.1---9.1 (,o<0.05) and 10.7---6.5 (p<0.07) bpm were recorded respectively, for the control and conditioning animals. Since the saline control animals showed no consistent changes in baseline HR across sessions, these data suggest the development of a tachycardiac CR to contextual cues associated with the training apparatus. Possible training effects on drug-induced HR changes were also evaluated. F o r example, HR UR magnitudes during either the first signalled (conditioning) or first unsignalled (control) drug infusion of each session were estimated by calculating differences in bpm between (a) HR during the 5-sec period immediately preceding drug infusion and (b) the lowest HR observed across the seven, 5-sec blocks following infusion. Interestingly, examination of these difference scores suggested that unconditioned HR responses to methoxamine infusion declined somewhat over days of both associative and nonassociative training; this effect, however, proved statistically unreliable (ps>0.15).

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1-sec BLOCKS FIG. 4 HR changes from baseline during the first 10 sec of tone in animals receiving either conditioning (COND) or nonassociative (CONT) training in Experiment 1. Illustrated are the group mean responses shown dunng the initial trial of the final two training sessions. Training Effects on B P BP responses were recorded for eight animals in the conditioning group and six animals in the control group during both the first day of training and the extinction test session. (The H R response of these subgroups reflected the group mean data described above.) Examination of the individual records for the initial training day suggested that the groups did not differ from one another with respect to either baseline or tone-evoked BP, consistent with the results of statistical analysis ( F s < l . 0 5 , ps>0.35). However, the analysis yielded a significant block effect, F(5,60)=2.98, p < 0 . 0 2 , which reflected the tone-evoked depressor responses of these animals during day 1 training; this finding is consistent with previous characterization of the BP component of the OR as a depressor response [26]. On the other hand, conditioning and nonassociative training differentially affected these tone-evoked BP changes as assessed during the extinction session. Specifically, seven of the eight animals in the conditioning group showed pressor responses to the tone CS, although these responses were more variable than the conditioned HR changes described above. In contrast, control animals typically showed either no BP response or small (<1-5.6 mmHg) depressor responses to tone presentations. These group differences are illustrated in Fig. 5, which depicts tone-evoked BP changes from baseline during day 1 training (Pre) and extinction (Post). The left-hand panel shows mean evoked changes in BP for the initial training trial, whereas the right-hand panel shows the mean maximum change in the pressor direction observed during extinction training. Small, long-latency (t> 10 sec) depressor responses were elicited by the initial tone presentation o f both the conditioning and control schedules. This depressor response apparently persisted during nonassociative train-

FIG. 5. Tone-evoked BP changes from baseline during the first day of training (PRE) and the extinction test session (POST) in Experiment l, as described in the text. Shown are group mean BP responses for animals subjected to either classical conditwning (COND) or nonassociatIve training (CONT)

ing. By the end of the conditioning training, however, the evoked BP response had assumed distinct pressor characteristics, increasing monotonically during successive 5-sec blocks of the tone. An A N O V A of the BP data depicted in Fig. 5 revealed significant effects of training day, F(1,12) = 4.73, p <0.05; day x block, F(4,48) =3.97, p <0.01; and group x day x block, F(4,48) =2.92, p <0.05, as well as a marginally significant group x day interaction, F(1,12)=4.56, p<0.055. Subsequent Tukey analyses indicated that the groups differed reliably from one another during the extinction day across tone blocks 2-5 (ps<0.05). EXPERIMENT 2 The results of Experiment 1 indicated that the intravenous administration of methoxamine, which specifically influences hemodynamics [1,2], can serve as an effective US for establishing classically conditioned cardiovascular responses to a discrete, exteroceptive stimulus. That is, pairings of a pure tonal CS with methoxamine infusion resulted in the gradual development of a tachycardiac CR; this response was clearly of an associative character, since it did not develop in control animals receiving either explicitly unpaired tones and drug infusions or tones paired with saline infusions. Moreover, tone-evoked BP changes consisting of pressor responses were observed following conditioning, but not nonassociative, training. Finally the elevations in baseline HR observed across sessions in animals receiving dally methoxamine administration suggested the concomitant development of a learned cardiovascular adjustment to the general training context. The present findings are thus consistent with, and extend, the results of previous studies of situation-specific drug tolerance (e.g., [23,31]) and conditioned drug-evoked responses (e.g., [7, 21, 35]), in which various physiological parameters (e.g., core body temperature, blood glucose) have been shown to change as a

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function of pairing drug injections with distinctive contextual cues. On the other hand, the sympathetic character of the presently observed CRs poses a striking contrast to the results of previous studies of cardiovascular conditioning m the rabbit. Specifically, in numerous studies which have employed an aversive (e.g., eye-shock) US, the CRs observed have invariably involved bradycardia and, when measured, hypotension (e.g., [4, 10, 12, 16, 17]); moreover, such CRs have been obtained even at CS-US intervals comparable to that employed in the present study [22]. The differential effects of conditioning and nonassoclative training on tone-evoked HR changes were most apparent during the initial trial of training days 2-5, since withinsession decrements in response magnitude were invariably observed. As noted above, these response decrements were paralleled by substantial and progressive declines in baseline HR within each conditioning session. Since such precipitous changes in tonic HR were not observed in saline-control animals, we reasoned that the 15-mm intertrial interval employed was not sufficient to permit animals to recover completely from the preceding methoxamine infusion(s). Accordingly, a second experiment was undertaken in which animals received classical conditioning involving pairings of the 35sec, 1216-Hz tone CS with methoxamine infusion; however, in this study, the intertrial interval was substantially increased. METHOD

Ammals The animals were one male and five female, experimentally naive New Zealand albino rabbits, each approximately 6 months of age and weighing 2.7-3.2 kg; each was housed and maintained as previously described. Apparatus and Procedure The apparatus, recording procedures and stimulus events were the same as described above. All animals received one

day of adaptation trmnmg, followed by five successive days of Pavlovian conditioning. Each conditioning session consisted of three pairings of the 35-sec tone CS and methoxamine; trials were presented at 30-min intervals. As in Experiment 1, BP responses were recorded during the first day of classical conditioning and during a three-triM extmctlon test session conducted on the day following the conclusion of associative training. RESULTS

AND

DISCUSSION

Trammg Effects on H R As in Experiment 1, all six ammals showed bradycardiac ORs to the initial presentation of the 35-sec tone during adaptation training; this HR response was maximal during the first 5 sec of st]mulation ( m e a n - - - 14.7 bpm). Similarly, the animals continued to show bradycardiac responses to the tone dunng the first conditioning session, as illustrated in the left-hand panel of Fig. 6 However, starting with the first trial of the second training session, the animals began to show tone-evoked tachycardia; this HR CR occurred with a latency of 1-2 sec and was maximal during the second, 5-sec block of the tone period. The reliability of the trmninginduced changes in the HR response to tone were confirmed by A N O V A , which revealed a significant effect of training day, F(4,20) =3.18, p <0.05. On the other hand, the analysis indicated that the main and interactive effects of trial were unreliable ( F s < l . 4 9 , ps<0.15); this outcome is consistent with the absence of w~thln-session response decrements in this instance. Finally, it should be noted that asymptotic response levels were apparently not achieved by the end of training, since examination of the day 5 data Indicated a systematic trend of increasing HR changes across trials. Regarding baseline HR, systematic changes were observed across days of training which were entirely consistent with those previously described. Specifically, relative to day 1 levels, maximal changes in baseline HR of 13.0-+5.2 bpm (,o<0.05) were observed at the beginning of conditioning sessions 3 and 4. Again, this elevation of baseline HR suggested the development of a contextual CR. Training effects on drug-induced HR changes were also assessed according to procedures detailed above. Examination of these data suggested no obvious change or trends in bradycardiac responses to methoxamlne infusion, either within or across the five training sessions. Training Effects on B P The right-hand panel of Fig. 6 illustrates tone-evoked BP changes for the six animals during the first trial of classical conditioning and the first extinction test trial. As clearly suggested in the figure, the initial BP response to tone was a depressor response which occurred with a latency of approximately 5 sec and showed a maximal change from baseline of - 4 . 1 mmHg just prior to methoxamine infusion. In contrast, the BP response following associative training was a shortlatency pressor response; the maximal CS-evoked change in BP (3.1 mmHg) was observed during the fifth, 5-sec block of the tone. A N O V A of these data revealed significant effects of day, F(1,5)=8.78, p<0.05, and day x 5-sec block, F(4,20) = 14.12, p <0.001, confirming the suggested traininginduced change in BP responses. Subsequent analyses indicated that the BP responses during the training and test days differed reliably during the second tone block (p <0.05), as well as blocks 3-5 (ps<0.01).

METHOXAMINE AND PAVLOVIAN CONDITIONING GENERAL DISCUSSION Taken together, the results of the present experiments indicate that methoxamine can serve as an effective US for autonomic conditioning. In particular, each experiment provided clear evidence for the development of learned tachycardiac and pressor responses to a discrete, tonal stimulus, when paired with methoxamine infusion. Moreover, the results of the present studies also clearly suggested the concomitant development of a HR CR to the contextual stimuli involved in training. As such, we feel that the present animal model may well prove advantageous in studying the mechanisms underlying visceral learning. Indeed, the present model would appear to have several analytical advantages over previous animal models of visceral conditioning involving pharmacological USs. First, it involves a discrete-trial procedure that potentially permits explicit control over both the temporal and intensity aspects of the CS-US contingency, thus, making possible a detailed parametric analysis of which aspects of these stimuli control the changes in behavior. Second, the present model is specific to cardiovascular variables. In this regard, convergent data from a number of laboratories have clearly indicated that the development and/or expression of classically conditioned cardiovascular adjustments is mediated by a corticolimbic regulatory system [4, 5, 10, 12, 15, 16, 24]. However, in each instance, an averslve, exteroceptive US has been employed that elicits specific types of somatomotor activity, but that has more generalized ("nonspecific") effects upon hemodynamics. A major quesiton to be answered is whether cardiovascular CRs established with a pharmacological US that specifically influences hemodynamics are also mediated by the limbic forebrain. The present model, combined with central interventions, should make it possible to answer this and related questions. Methoxamine is believed to be a peripherally-acting ~lreceptor agonist ([1]; but see [19]). Thus, it acts directly on vascular tzl-receptors, resulting in vasoconstriction and increased BP. Bradycardia is produced, secondarily, via baroreceptor-activation of vagal tone [2]. Since methoxamine has minimal, if any, central effects [34], it is unlikely that its US properties are the result of central al-stimulation. Instead, a more likely hypothesis is that these properties are attributable to peripheral afferent feedback to the brain from the barroreceptors and/or vagal activity. However, a direct test of this hypothesis awaits further research. Nevertheless, it is of special interest that the learned BP response was in the same direction as the BP UR, but that the conditioned HR response, tachycardia, was opposite in direction to the HR UR. As such, the present findings support neither a compensatory (e.g., [31,32]) nor a traditional stimulus substitution [25] model of classical conditioning. The present data are, however, compatible with a recent suggestion [8] that the direction of a CR to cues predicting drug administration will be compensatory to the observed drug-elicited effect only if the latter is reflexively produced by direct action of the drug on the efferent arm of a response feedback system. If so, the observed drug-induced effect would act as a US that, in turn, leads to the activation of corrective (i.e., compensatory) physiological changes (the "true" UR). On the other hand, CRs resulting from drug action on the afferent arm of a response feedback system should be directionally consistent with the observed drug

183 effect, according to this analysis, because this effect is, in fact, the drug-elicited UR of the system. Although this modified stimulus substitution model, as proposed by Eikelboom and Stewart [8], was meant to apply only to centrally acting drugs, the present results are clearly consistent with their analysis. That is, methoxamine-elicited pressor responses apparently result from drug action on the afferent arm of this response system (i.e., at the vascular al-receptors); thus, the BP CR would also be expected to be a pressor response, as was observed. However, the bradycardiac response to methoxamine would be considered to be produced by drug action on the efferent arm of this system (i.e., in response to baroreceptor activation); accordingly, the HR CR would be expected to be a compensatory, tachycardiac response, as was also observed. While the above model would appear to provide a reasonably coherent framework for interpreting the present data, other plausible mechanisms nevertheless remain. For example, it is entirely possible that the associative effects of methoxamine, when repeatedly administered in the presence of distinctive, exteroceptive cues, may involve traininginduced modification of neuronal activity along the sympathoadrenal axis (cf. [13]). This could, in turn, lead to increases in peripheral catecholamine release that would result in systematic elevations in baseline HR and possibly produce a general sympathetic bias influencing HR and BP responses to the tone CS (cf. [3]). Of course, this hypothesis is readily testable, since it predicts that adrenal demedullation should abolish the development of tachycardiac and pressor CRs, as observed in the present experiments. Final mention should be made of the elevations in baseline HR observed over training, As noted above, such changes in tonic HR occurred during both associative and nonassociative training involving methoxamine administration but were not observed in saline-control animals. Thus, these changes were drug-specific, suggesting the development of a tachycardiac CR to contextual stimuli. Moreover, the elevations in baseline HR were apparent only at the outset of the training sessions when, it could be argued, the contextual stimuli associated with training might be most salient. While such training-induced changes in baseline HR were observed during classical conditioning in both experiments, these changes were much more consistent and of greater magnitude for the methoxamine-control animals of Experiment 1. We would suggest that the relative variability of the baseline changes shown by the conditioning animals might logically reflect a competition for associative significance between the various contextual CSs and the tonal CS employed in the present studies. Indeed, as presently programmed, the initial trial of each conditioning session could be considered to involve a serial compound CS involving an initial, contextual component and a subsequent, tonal component. Based on results of previous studies of stimulus selection during serial compound conditioning [11,18], one would expect to see CRs develop initially to each component stimulus of the compound; however, with extended training, CRs generally tend to be elicited only by the presentation of the most predictive and/or salient component CS [11,18], which would certainly be tone in the present instance. According to this stimulus selection analysis, then, there should be an inverse relationship between conditioned responding to the training context and that to the tone as training progresses. In fact, a post hoc examination of the HR data for the conditioning animals in Experiment 1 uncovered a signif-

184

GIBBS SPENCER, KAO AND POWELL

icant, n e g a t i v e c o r r e l a t i o n b e t w e e n t r a i n i n g - i n d u c e d c h a n g e s in b a s e l i n e H R a n d t o n e - e v o k e d t a c h y c a r d i a c r e s p o n s e s during d a y s 3-5 o f t r a i n i n g ( r = - . 5 7 , p < 0 . 0 5 ) . F u r t h e r , rea s s e s s m e n t o f the H R d a t a for a n i m a l s in E x p e r i m e n t 2 rev e a l e d a similar, a l t h o u g h statistically unreliable, n e g a t i v e c o r r e l a t i o n b e t w e e n e l e v a t i o n s in b a s e l i n e H R a n d tonee v o k e d t a c h y c a r d i a ( r = - . 2 0 ) ; h o w e v e r , it s h o u l d b e n o t e d t h a t s u c h b a s e l i n e c h a n g e s w e r e n o t o b s e r v e d in t h e s e a n i m a l s d u r i n g t h e final training day, at w h i c h time five o f the six a n i m a l s s h o w e d s u b s t a n t i a l H R C R s to the t o n e . T h u s , the p r e s e n t m o d e l w o u l d also a p p e a r to b e p r o m i s m g for t h e

analysis of m o r e c o m p l e x issues r e l e v a n t to visceral learning.

ACKNOWLEDGEMENTS This research was supported by VA Institutional Research Funds awarded to the Wm. Jennlngs Bryan Dorn Veterans' Hospital. We would like to thank the Burroughs Wellcome Co for providing the Vasoxyl used m these studies We would also like to thank Dr. Llnda L Hernandez for her invaluable cntical input, Ms. Elizabeth Hamel for her assistance in manuscript preparaaon, and Ms. Adrienne Walter-Gibbs for her editorial suggesUons.

REFERENCES 1. Anderson, K. E. Classification and function of peripheral vascular a-adrenoceptors. In: a-Adrenoceptor Blockers tn CardlovascularDzsease, edited by H. Refsum and O. D. MjCs New York: Churchill Livingston, 1985, pp 3-18 2 Avlado, D M., Jr and A. L. Wnuck. Mechanisms for cardtac slowing by methoxamme. J Pharmacol Exp Ther 119:99-113, 1957. 3 Borkowskl, K. R. and E. Kelly. The effect of adrenal demedullation on cardiovascular responses to enwronmental sUmulat~on in conscious rats Br J Pharmacol 88: 943-945, 1986 4 Buchanan, S. L. and D. A. Powell. Cmgulate cortex: Its role in Pavlovian conditioning. J Comp Phystol Psychol 96: 755-774, 1982. 5 Cohen, D H. Involvement of the avtan amygdalar homologue (archlstnatum posterior and medlale) m defensively conditioned heart rate change J Comp Neurol 160: 13-35, 1975 6. Cohen, D. H. and D. C. Randall. Classical conditioning of cardiovascular responses Annu Rev Physzol 46: 187-197, 1984 7 Eikelboom, R and J. Stewart. Conditioned temperature effects using morphine as the unconditioned stimulus Psychopharmacology (Berlin) 61: 31-38, 1979 8 Eikelboom, R. and J. Stewart. Conditioning of drug-induced physiological responses Psychol Rev 5: 507-528, 1982 9 Garcia, J , W G. Haskins and K. W Ruslntak. Behavioral regulation of the mlllieu interne in man and rat. Scwnce 185: 824831, 1974. 10 Gentde, C G , T. W. Jarrell, A. Telch, P McCabe and N. Schneiderman. The role of the amygdalold central nucleus m the retention of differential Pavlovian conditioning of bradycardm in rabbits. Behav Brain Res 20: 263-273, 1986. ll. Gibbs, C. M. Serial compound classical conditioning (CS1CS2-UCS): Effects of CS2 intensity and pretramlng on component acquisition. Unpublished doctoral dissertatton, University of Iowa, 1979. 12 Gibbs, C. M. and D. A. Powell. Neuronal correlates of classically conditioned bradycardla m the rabb~t studies of the medml prefrontal cortex. Bratn Res 442: 86--96, 1988 13. Goadsby, P. J. Bramstem activation of the adrenal medulla m the cat. Bram Res 327: 241-248, 1985. 14 Gormezano, I. Classical conditioning. In: Expermwnt Methods and lnstrumentatton tn Psychology, edited by J B. Sldowskl New York. McGraw Hill, 1966, pp. 385-420 15 Iwata, J., J E LeDoux and D. J. Rels. Destruction ofmtnns~c neurons m the lateral hypothalamus disrupts the classzcal condlt~omng of autonomic but not behavioral emotional responses m the rat. Brain Res 368: 161-166, 1986. 16 Kapp, B. S., R. C Frysinger, M. Gallagher and J. R. Haselton Amgydala central nucleus lesions: Effect on heart rate conditioning in the rabbit. Physzol Behav 23:1109-1117, 1979. 17 Kazis, E., W L. Milligan and D. A. Powell. Autonomic-somatic relationships: blockade of heart rate and corneoretinal responses. J Comp Physiol Psychol 84:98-110, 1973.

18. Kehoe, E J , C M Gibbs, E G a r o a and I Gormezano Assoctat~ve transfer and stimulus selection in classical conditioning of the rabbit's mctitating membrane response to serial compound CSs. J Exp Psychol [Atom Behav] 5: 1-18, 1979. 19. Klm, Y I , Y H. Palk, S S. K a n g a n d J H. Kim Effects of a-adrenoreceptor agonlsts administered mtraventncularly on central hypotenslve action of clomdine and on central hypertensive action of methoxamlne in rabbits. Arch lnt Pharmacodyn Ther 257: 66-76, 1982. 20. Kirk, R E Experimental Destgn Procedures for the Behavtotal Scwnces Belmont, CA Brooks/Cole Publisbang Company, 1968 21 Lal, H , S Mtkslc and N. Smith. Naloxone antagonism of condltloned hyperthermla. An evidence for release of endogenous oplold. Life Sct 18: 971-976, 1976 22 Manmng, A. A., N Schne~derman and D S Lordahl. Delay vs trace heart rate classical discrimination condlUonmg m rabbits as a function of ISI. J Exp Psychol 80: 225-230, 1969. 23 Mansfield, J G and C L. Cunnlngham Conditioning and extinction of tolerance to the hypothermlc effect of ethanol in rats. J Comp Phsylol Psychol 94: 962-969, 1980. 24. Pascoe, J. P and B. S. Kapp Electrophysiologlcal characteristics of amygdaloid central nucleus neurons dunng Pavlovlan fear conditioning m the rabbit Behav Bram Res 16:117133, 1985 25. Pavlov, I. P. In Condmoned Reflexe~, edited and translated by G V. Anrep. New York: Dover Press, 1960. (Originally pubhshed, 1927) 26 Powell, D A and E Kazls. Blood pressure and heart rate changes accompanying classical eyebhnk conditioning in the rabbit (Oryctolagus Cumculus). Psychophystology 13: 441-447, 1976 27 Powell, D A , M Llpkln and W L Mdhgan Concomitant changes m classically conditioned heart rate and corneoretmal potentml dlscrimlnatton in the rabbit (Oryctolagus Cumculus) Learn Mottv 87: 978-986, 1974. 28. Prokasy, W. F. Acqmsltlon of skeletal conditioned responses in Pavlovmn conditioning. Psychophysiology 24: 1-13, 1984. 29. Schnelderman, N. Response system dwergencles m averslve classical conditioning In Classwal Condzttonmg 11 Current Theory and Research, edited by A. H Black and W. F. Prokasy New York" Appleton-Century-Crofts, 1972, pp. 341376 30. Schnelderman, N The relationship between learned and unlearned cardiovascular responses In' Cardtovascular Psychophysiology, edited by P. Obnst, A. Black, J Brener and L. V. Dl Cara. Chicago, IL' Aldlne Pubhshmg Company, 1974, pp. 190-210. 31. Siegel, S. Morphine analgesic tolerance Its situation specificity supports a Pavlovian conditioning model Scwnce 193: 323-325, 1976

METHOXAMINE

AND PAVLOVIAN CONDITIONING

32. Siegel, S. The role of conditioning m drug tolerance and addiction. In' Psychopathology tn Ammals, edited by J. D. Keehn. New York: Academic Press, 1979. 33. Wemberger, N. M. Effects of conditioned arousal on the auditory system. In: The Neural Basts of Behavior, edited by A. L. Beckman. New York: Spectrum, 1972.

185 34. Wemer, N. Norepinephrine, epinephnne, and the sympathyometic amines. In: Goodman & Gdman's The PharmacologicalBasis ofTherapeutws, 7th edition, edited by A. G. Goodman, L. S. Goodman, T. W. Rail and F. Murad. New York: McMillan, 1985, pp. 145-180. 35. Woods, S. C. Conditioned hypoglycemia. J Comp Physiol Psychol 90: 1164--1168, 1976.