INTRODUCTION

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MUCH OF THE RESEARCH in our laboratory is intended to elucidate the autonomic nervous control of the heart and circulation under a variety of behavioral states. For example, we have described a stereotypic, patterned change in sympathetic nerve activity (SNA) and arterial blood pressure (BP) evoked by classical (i.e., Pavlovian) conditioning in the Sprague-Dawley rat (8, 9). The procedure involves presenting a 15-s pulsed tone, the conditional stimulus (CS+), followed by a 0.5-s tail shock. The discriminative stimulus, or CS, is a tone of the same frequency and duration, but which is not pulsed (i.e., is sounded continuously) and is never followed by shock.

Presentation of both the CS+ and CS evokes a sudden, intense burst of SNA that is followed by a rapid increase in BP (8). We have termed this the "initial component" (i.e., C1) of the conditional pressure response. The magnitudes of the sudden burst in SNA and the associated C1 BP increase evoked by the CS+ are significantly larger than are evoked by CS (7-9). There are later sustained, but smaller magnitude, increases in SNA and BP during the CS+ that do not occur in response to CS. Therefore, this second component of the conditional response (i.e., C2) is a sensitive discriminator between the physiological responses to the two tones. Delivery of the short shock at the end of the CS+ tone is associated with a sharp unconditional pressor response. Likewise, there is a much smaller, but definite, increase in BP at the termination of the CS. Heart rate (HR) is essentially unchanged during the behavioral stress in the Sprague-Dawley rat (9), despite the increase in sympathetic nervous activity. The changes in BP described above must be associated with underlying adjustments in cardiac output (CO) and total peripheral resistance (TPR).

The obvious next step in our exploration of these relationships is to quantify the changes in SV, CO, and TPR that occur during both CS+ and CS trials. Each of these variables is strongly influenced by changes in sympathetic nervous activity, so an effort of this nature should not only advance our general understanding of the way in which the body marshals its resources to respond to an acute behavioral stress but should also clarify how the autonomic nervous system orchestrates the BP response pattern to an acute behavioral challenge. Therefore, the present experiment was conducted to determine the relative changes in CO and total peripheral vascular resistance temporally associated with the changes in arterial BP during acute conditioning. An abstract of this study was reported previously (6).

	METHODS

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Subjects. Ten male Sprague-Dawley rats, purchased from Harlan Sprague Dawley at body weight of 200-240 g, were used in this experiment. The standards for the use and care of experimental animals of the American Physiological Society were observed at all stages of this study.

Apparatus. The rats were housed three per cage, under a 12:12-h light-dark cycle in a temperature-controlled room. All animals had free access to water and normal rat chow.

Surgery. Rats were anesthetized with pentobarbital sodium (65 mg/kg ip) in preparation for surgery. Aseptic rat surgical procedures were used. A Teflon arterial catheter (ID 0.029 mm) was inserted into the upper abdominal aorta via the left femoral artery. A polyethylene catheter (ID 0.58 mm) was also inserted into the right jugular vein for use in another study not reported here. The animal's trachea was intubated, and the animal was artificially respired at 50 breaths/min, with tidal volume of 20 ml and inspiration-to-expiration ratio at 1:2. A 3S Transonic flow probe (Transonic System, Taconic, NY) was implanted around the ascending aorta as described by Smith (13). Briefly, the chest was opened at the third intercostal space to expose the heart. A small section (1 cm long) of the ascending aorta was freed from connective tissue. The flow probe was then implanted around the root of the ascending aorta. The chest incision was closed, and a negative intrathoracic pressure was restored. The distal end (connector) of the probe cable was tunneled subcutaneously to the nape of the neck where it and the catheters exited the body. They were stabilized with a plastic saddleback cuff (Transonic, AAPC103) and sutured to the skin. The animal was returned to a recovery cage with free access to water and food after the surgery.

Conditioning protocol. A soft, conical, terry cloth sock was used for mild restraint of the rat during the training and experimental sessions (9). Each animal was allowed 2 days postoperative recovery before any behavioral training was instigated. Starting on the 3rd day they were habituated to a 15-s pulsed tone (eventually to become the CS+) and a nonpulsed, but otherwise identical, tone (eventually to become a nonreinforced CS). No shock was given in any trial during these habituation sessions. The five CS+ and five CS tones were delivered in pseudorandom pairs (e.g., CS+, CS; CS, CS+; etc.) with at least a 5-min interval between presentations. A 486-based microcomputer was used to control the onset and offset of the tones and to trigger the two-pole shocker (Coulbourn Instruments). An animal was trained in the behavioral paradigm over the next 3 or 4 days. All CS+ tones were reinforced during the training sessions with a 0.5-s-long electrical tail shock delivered at the end of each pulsed tone (CS+ trials). The intensity of the shock was adjusted for each rat to the lowest value (usually 0.3-0.4 mA) that caused it to flinch. None of the CS tones was ever followed by shock for the whole experiment. We recorded the physiological data during these training sessions, but the results reported here were taken only from a final set of five CS+ and five CS trials taken on a 4th (n = 3) or 5th (n = 7) experimental day (i.e., the 7th or 8th day after surgery).

Data acquisition and analysis. BP was measured using a calibrated transducer (Cobe model CDX-III) connected to a Grass 7-channel polygraph (model 7H2560). Aortic blood flow was also recorded on the polygraph during the conditioning trials using an ultrasonic blood flowmeter (Transonic T101). The BP and blood flow data were digitally sampled at 500 Hz using an analog-to-digital converter and a 486-based microcomputer. Each conditioning trial lasted 30 s. The first 9 s of the recording comprised the resting period; the ensuing 15 s comprised the tone-on period. The last 6 s constituted the recovery period.

Data for each animal were analyzed across five CS+ or five CS trials using computerized ensemble averaging of multiple trials, as is described in detail elsewhere (8). Measurements were based on the average over each specific period [i.e., baseline, C1, C2, and the unconditional response (UR)]. The CO was calculated from the aortic blood flow, and the TPR index was calculated by dividing mean aortic BP by CO (i.e., neglecting possible changes in right atrial pressure). An analysis of variance (ANOVA) was carried out for mean arterial BP (mBP), CO, HR, stroke volume (SV), and TPR to test the significance of difference between responses to CS+ and CS. Post hoc t-tests were performed when justified by the results of the ANOVA. Statistical significance was accepted for P < 0.05. All data are expressed as means ± SE.

	RESULTS

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Figure 1 is an ensemble average of the mBP, TPR, HR, SV, and CO computed over 50 CS+ trials (10 rats × 5 CS+ trials each). Because Fig. 1 is based on multiple trials from all rats, it provides a reliable summary of the magnitude and timing of the conditional cardiovascular response. The dark bar on the time axis indicates when the tone was sounded. BP increased to an initial peak, the "first component" (C1) of the conditional response, at 1.5 s after onset of the conditional stimulus. The thin vertical lines allow for comparisons across variables of the changes from the beginning of the tone to this initial BP peak. BP then dropped transiently before rising again as part of the smaller, but more sustained, "second component" (C2). The C2 was followed by an UR in CS+ trials or an offset response in the nonshock CS trials (not shown; Fig. 1). The rising phase of the C1 pressor response was closely linked with an increased resistance. Conversely, SV and CO were essentially unchanged until after the initial peak in mBP. SV and CO started to rise as pressure declined and were sustained at an elevated level throughout the latter seconds of the CS+ tone that corresponds to the second component of the conditional BP response. During this same period, TPR was modestly depressed compared with control, but fluctuations in resistance were generally mirrored in changes in BP. Finally, HR was essentially unchanged during the 15-s tone. The UR to tail shock consisted of a dramatic increase of mBP associated with a sharp drop in SV and CO and an equally sharp increase in TPR. HR increased after the shock delivery.

View larger version (27K): [in this window] [in a new window]   Fig. 1.   An ensemble average over 10 rats of the conditional response in mean arterial blood pressure (mBP), total peripheral resistance (TPR), heart rate (HR), stroke volume (SV), and cardiac output (CO). Filled bar on time scale indicates the period of tone presentation. Thin vertical lines denote the beginning of the tone and the peak of the "first component" (C1) blood pressure response. Blood pressure increased to an initial peak, C1, at 1.5 s after onset of conditional stimulus. Pressure then dropped transiently before rising again to a small, but sustained, "second component" (C2) conditional blood pressure increase. Pressor responses were associated with TPR increase during C1, whereas CO increased during C2. bpm, Beats/min; UR, unconditional response.

Figures 2-6 summarize the average changes from baseline for mBP, CO, HR, SV, and TPR for C1, C2, and the UR for both stimuli. The values for C2 are averages over the last 10 s of the tones. Statistical significance is shown relative to baseline and for comparisons between CS+ and CS. For example, the mBP data in Fig. 2 confirm that the C1 and C2 mBP increases during CS+ and for the UR were significantly greater than the pretone baseline and were larger for CS+ than for CS.

View larger version (14K): [in this window] [in a new window]   Fig. 2.   mBP changes from baseline for conditioned stimulus tone followed by tail shock (CS+) and tone with no tail shock (CS) trials during the C1 and C2 components of the conditional response and unconditional response (UR) periods. Notice that the responses were greater for CS+ in C1, C2, and UR periods compared with CS trials. mBP during all phases, except C2 during CS trials, was greater than baseline. t-Test: * P < 0.05 vs. baseline; # P < 0.05 between CS+ and CS trials.

Figure 3 depicts the changes of CO from baseline. CO was not significantly changed during C1 for either type of trial. These data confirm that CO was sustained at a significantly higher level during C2 compared with the baseline for CS+. Conversely, there was little change in CO during C1, C2, or at the tone off (i.e., UR) in CS trials. Note also that the CO was significantly decreased during the UR compared with the offset response of the CS trials. In dissecting the CO into HR and SV components, there was a rather uniform, but small (5 beats/min), trend toward bradycardia, especially for CS+ trials (Fig. 4). On the other hand, the SV (Fig. 5) was significantly increased during C2 (but not C1) for CS+. Finally, Fig. 6 shows that there were no significant changes in TPR during C2 for either CS+ or CS trials. Note also that TPR was significantly greater for CS+ than for CS trials during the C1 and UR phases.

View larger version (13K): [in this window] [in a new window]   Fig. 3.   CO changes from baseline for CS+ and CS trials during C1 and C2 components of the conditional response and UR. Notice that the responses were greater in C2 and smaller in UR periods for CS+ compared with CS trials. t-Test: * P < 0.05 vs. baseline; # P < 0.05 between CS+ and CS trials.

View larger version (13K): [in this window] [in a new window]   Fig. 4.   HR changes from baseline for CS+ and CS trials during C1 and C2 components of the conditional response and UR. There was a modest bradycardia, but no significant differences between the changes for CS+ and CS  trials. t-Test: * P < 0.05 vs. baseline.

View larger version (13K): [in this window] [in a new window]   Fig. 5.   SV changes from baseline for CS+ and CS trials during C1 and C2 components of the conditional response and UR. Notice that, similar to the CO changes, the responses were greater in C2 and smaller in UR periods for CS+ compared with CS trials. t-Test: * P < 0.05 vs. baseline; # P < 0.05 between CS+ and CS trials.

View larger version (14K): [in this window] [in a new window]   Fig. 6.   TPR changes from baseline for CS+ and CS trials during C1 and C2 components of the conditional response and UR. Notice that the changes were greater during C1 and UR and identical during C2 for CS+ compared with CS trials. t-Test: * P < 0.05 vs. baseline; # P < 0.05 between CS+ and CS trials.

	DISCUSSION

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Previous experiments have revealed a pattern in the arterial BP response to a classical conditioning paradigm in rat (7-9) that is closely associated with underlying changes in renal SNA (2, 3, 8). For example, we believe the initial part of the C1 response results from an "open loop" relationship between changes in SNA and BP, rather than from a biofeedback relationship (8). The present experiments expand on this earlier work by showing that the C1 pressor response is closely coupled with an equally rapid increase in TPR and that the sustained pressor response of C2 is coupled with a maintained elevation in CO secondary to increased SV. We believe that these experiments reveal important principles of the organization of the cardiovascular response to an acute behavioral stress.

The basis for early (i.e., C1) changes in TPR and later (i.e., C2) changes in CO may be examined from two perspectives. The first of these concerns the physiological capabilities and limitations of the cardiac and vascular effector mechanisms in response to the external stressor. These particularly include the "time constants" for each. The onset latency of the nervous response (i.e., the time between the beginning of the tone and the initial burst in SNA that is responsible for C1) is extremely short, only slightly >0.1 s. (8). A simple mathematical model predicts that the efferent time delay between a change in SNA and a change in mBP is ~0.5 s (3, 4). This value was computed purely on the relationship between changes in SNA and BP, so it cannot discriminate between the effector response of the heart (i.e., a major determinant of SV) versus the vascular smooth muscle (i.e., a major determinant of TPR). Therefore, one possible explanation for the apparent primacy of TPR in mediating C1 is that, although the change in the SNA driving function probably occurs synchronously at each effector, the increases in vasomotor tone/resistance occur more rapidly than those of the inotropic state of the heart. Another important physiological consideration is that other factors beside SNA affect SV. Specifically, increases in afterload, as represented in part by the C1 pressor response, tend to depress SV, at least until elevated myocardial contractility allows the myocardium to maintain or increase muscle shortening despite the increased afterload. Therefore, another possible physiological explanation for the intimate early coupling of changes in TPR and changes in BP is that increases in SV and CO are initially limited by the increase in afterload as represented by the C1 increase in mBP.

The second perspective concerns the etiology of C1 compared with C2. We have speculated that different regions of the brain mediate these two phases of the conditional response (11). We based this supposition in part on the fact that the initial part of C1 is under open loop control, although the peak of C1 may be changed by learning and the later part of C1 may be regulated by the baroreflex; C2 appears to be mostly under the control of the baroreflex (8). If so, it may be that the supposed first neural pathway converges primarily on vascular smooth muscle, whereas the second preferentially targets the heart. Further work will obviously be required to confirm or reject these possibilities.

In another vein, the C2 pressor response probably contains multiple hemodynamic mechanisms and results from causes that differ from those for C1. For example, the plasma catecholamines may have contributed more to the CO increase during C2, as was reported at the end of a 30-min classical conditioning on both borderline hypertensive and Wistar-Kyoto rats (5). In addition, we have reported in the spontaneously hypertensive rat (SHR) that SNA and, by inference, parasympathetic activity were both increased compared with baseline and maintained at a relatively higher level during the C2 period (7).

We have also focused attention on the changes in SV, CO, and TPR associated with the large unconditional BP response. Note, in this regard, that the large pressor response to tail shock was associated with an increased TPR and HR but a decreased SV and CO. This differs from a report on dogs (1) where HR and CO increased, but the TPR decreased, during a 30-min continuous aversive-conditioning period; this disparity is probably primarily attributable to the considerable difference between the two behavioral situations. Likewise, there are a few descriptions of changes in CO and TPR during a conditional stimulus. In particular, an increase in CO and concomitant decrease in TPR were reported in monkey (10) and in the borderline hypertensive rat (5) during a period similar to the C2 in the current study. However, neither of these experiments described the pattern of the change in cardiac and vascular function, as is the case in the present study.

Some limitations of this experiment should be noted. First, our value for TPR must be regarded as an index of this important variable, because the computation was based only on mean arterial pressure and not on the difference between aortic and right atrial pressure. However, given that the downstream pressure changes must be quantitatively small, we believe the index is quite reliable. The second qualification concerns a related issue: our data provide no information on cardiac preload, another major determinant of SV. For this reason and because the circulation is a closed system, it is difficult to attribute the various BP changes to changes in SV or peripheral resistance, although it seems safe to attribute the C1 pressor response to the early rise in TPR. One could argue on sound physiological principles, for example, that the elevated CO during C2 "resulted" from a sympathetic-induced venoconstriction that mobilized venous return. We do not believe these considerations detract, however, from the important new insights into the cardiovascular response patterns described here.

Perspectives