In a long-term neuromuscular blocked (NMB) rat preparation, tetanic stimulation of the aortic depressor nerve (ADN) enhanced the A-fiber evoked responses (ERs) in the cardiovascular region, the nucleus of the solitary tract (dmNTS). The potentiation persisted for at least several hours and may be a mechanism for adaptive adjustment of the gain of the baroreflex, with functional implications for blood pressure regulation. Using a capacitance electrode, we selectively stimulated A-fibers and acquired a stable 10-h “A-fiber only” ER baseline at the dmNTS. Following baseline, an A+C-fiber activating tetanus was applied to the ADN. The tetanus consisted of 1,000 “high current” pulses (10 trains; 300 μs, 100 Hz, 1 s), with intertrain interval of 9 s. A 10-h A-fiber only posttetanic test phase repeated the stimulus pattern of the baseline. Fourteen tetanus experiments were done in 12 rats. Compared with the baseline before tetanus, the A-fiber ER magnitudes of posttetanus hours were larger [F(13, 247) = 3.407, P < .001]; additionally, the 10-h posttetanus magnitude slopes were more positive than during 10 h before tetanus (df = 13; t = −3.47; P < 0.005); thus, an ADN A+C fiber-activating tetanus produced increases in the magnitude of the A-fiber ERs in the dmNTS that persisted for several hours. In an additional rat, application of an NMDA receptor antagonist, prior to the tetanus, blocked the potentiation effect. The stimulus protocols, magnitude and duration of the effect, and pharmacology resemble associative long-term potentiation (LTP).
- long-term potentiation
- aortic depressor nerve
- nucleus of the solitary tract
the baroreflex is a negative feedback control system, and the loop gain of the system relates the degree of baroreceptor deformation to the strength of the cardiovascular compensations. To protect against vascular rupture or tissue ischemia, baroreflex action must be smooth, rapid, and accurate. Appropriate gain is crucial to efficient blood pressure stabilization, but the mechanisms that adjust baroreflex gain are not known. The particular roles of the A- and C-fiber coupled baroreceptors in the operation of the baroreflex are also not known: The aortic depressor nerve (ADN) is composed of ∼25% myelinated A-fibers, and ∼75% unmyelinated C-fibers (13, 15, 16); compared with A-fibers, C-fiber coupled receptors are activated primarily at very high pressures, have lower firing frequencies, irregular discharge patterns, and very steep, almost vertical, characteristic curves (14).
Using a stimulus sequence on the ADN similar to that used for inducing and testing long-term potentiation (LTP) in the CA1 region of the hippocampus, we found that following simultaneous A-fiber and C-fiber tetanus (A+C), the A-fiber evoked responses in the cardiovascular region of the dorsal-medial nucleus of the solitary tract (dmNTS) are increased. We propose that this mechanism could optimize blood pressure stability by adjusting the gain of the A-fiber baroreflex (1).
Twelve female Sprague-Dawley rats, (230–270 g), were studied in the experiments. All acute surgery or any possibly irritating manipulation, such as replacement of the bladder cannula or removal of feces during experimental periods, was done with accurately controlled and carefully monitored deep isoflurane anesthesia. Rats were studied one at a time, monitored, and attended around the clock. Details about the general surgical procedures, ADN preparation, and extracellular multiunit recordings at the dmNTS are found in Refs. 2 and 12. The experimental protocol was supervised and certified to be in compliance with National Institutes of Health and American Physiological Society guidelines by the Pennsylvania State University College of Medicine Institutional Animal Care and Use Committee.
Surgery required 2 days; during all procedures, the anesthetic level was >1.5%, ensuring that 1) the EEG was synchronized and dominated by high-voltage slow-wave activity (4a), 2) mean blood pressure (BP) was <100 mmHg, and heart rate (HR) was <420 beats/min, and 3) there were no evident EEG, BP, or HR responses to surgical manipulations. In the first day of surgery, to record the EEG, silver wires were attached to two 0–80 screws inserted into the left parietal bone. For the ECG, two precordial silver wires were implanted subcutaneously. For the arterial blood pressure, an abdominal aortic catheter (28-gauge, Teflon) was inserted via the right femoral artery. Finally, a silicone (0.025 in. ID × 0.047 in. OD) catheter was threaded into the inferior vena cava, from the right femoral vein, for administering parenteral solutions and venous blood pressure recording. After induction of paralysis with a 75 μg iv bolus of α-cobrotoxin, the rats were mechanically ventilated, via a per os coaxial tracheal cannula, with positive pressure at 72 breaths/min (I:E 1:2), and a minute volume of 180–200 ml of 48.5% O2, 47% N2, and 3% CO2, and 1.5% isoflurane. Core temperature was measured by a thermistor inserted in the vagina and servo-regulated at 37°C.
During the second day of surgery, an ∼3-mm segment of the left ADN was carefully dissected free from the surrounding tissues, placed on an anodized Ta-Ta2O5 capacitance electrode (3), and embedded in a silicone compound (Kwik-Sil WPI, Sarasota, FL). The nerve was stimulated by an optically isolated constant current unit (CCIU-8, FHC, Bowdoinham, ME) controlled by Spike2 software (Cambridge Electronic Design, Cambridge, UK). The anodized Ta-Ta2O5 capacitance electrode enabled thousands of stimulations without damage to the ADN (2, 3).
Chronic ADN preparation required that the NTS be approached ventrally via the foramen magnum. Recording was with 1–2 MΩ glass insulated Tungsten microelectrodes (Alpha-Omega, Alpharetta, GA), mounted in a custom modified FHC hydraulic probe drive (50-16-1, FHC), attached to the modified Benchmark Deluxe Digital stereotaxic instrument (MyNeurolab.com, St. Louis, MO) (see Ref. 12 for details of the preparation setup). The baroreflex area in the NTS was located by atlas coordinates and by reference to somatotopic features of the gracile nucleus, which is between 100 and 200 μm dorsal to the baroreflex cells in the NTS. For evoked responses (ERs), the preamplifier (XCELL-3 X 4, 40-#40-8B, FHC) gain was 20 k, and the signals were bandpass filtered at 0.3–1.0 kHz, and digitized at 10 kHz using Spike2 and a Power 1401 data acquisition system (Cambridge Electronic Design, Cambridge, UK).
Effect of A+C-fiber tetanic stimulation on A-fiber ERs.
Fig. 1 shows the pattern of stimuli used for measuring the effect of A+C-fiber tetanic stimulation on the amplitude of A-fiber ERs. A 10-h “A-fiber only” baseline phase consisted of 6,000 pseudo-random (interpulse interval = 6 ± 1 s, 300 μs) “low-current” pulses; from the responses, the average ER magnitude was calculated for each hour (see Ref. 12 for calculation procedure), and a line was fit to the 10 average values. If the absolute value of the slope of the line was >0.36 z-units/h (see data analysis section for the z-score calculation), the baseline was deemed unstable, and the baseline procedure continued until the absolute value of the slope of the 10 most recent ERs was <0.36 z-units/h; at which time, the A+C-fiber activating tetanus was applied. Criterion stability was ordinarily achieved in <24 h. The tetanus consisted of 1,000 “high-current” pulses [10 trains (300 μs, 100 Hz, 1 s), with intertrain interval of 9 s]. The entire tetanus procedure required 100 s. A 10-h A-fiber only posttetanic test phase used the same pulse pattern as the baseline phase.
The A-fiber low current was established by an explicit transduction curve analysis as the current that elicited maximal A-fiber with no or minimal high-threshold late (HTL) ERs. The high current was always the lowest intensity that fully saturated the HTL complex. See details of the definition of A-fiber and HTL complexes and the transduction curve analysis in Ref. 12. In 10 rats the high and low current ranges did not overlap, but in the other two, it was necessary to find a compromise. Then, we chose a low current that produced a stable A-fiber response and minimal HTL complex activation, rather than optimizing the A-fiber response stability. This strategy was conservative, because, although more strongly driving the A-fibers improves A-fiber ER stability, inadvertent C-fiber activation during the baseline and test phases might have obviated the key differential condition of the experimental design.
Effects of an NMDA antagonist.
The NMDA receptor antagonist 3-(2-carboxypiperazin-4-yl)-propyl-1-phosphoric acid (CPP) blocks the induction of NMDA-dependent LTP (8, 9). In a supplementary rat, CPP was administered before the tetanus. The protocol was as follows: control, 6-h A-fiber only baseline (interpulse interval = 6 ± 1 s, 300 μs); A+C-fiber tetanus of 1,000 high-current pulses [10 trains; (300 μs, 100 Hz, 1 s), with an intertrain interval of 9 s] 6-h A-fiber only test. For the CPP test, the same procedure was used, except CPP (2.5 mg = 1.25 mg/cc × 0.2 cc/min × 10 min iv) was infused 30 min before the tetanus.
To evaluate the effect of the A+C-fiber tetanic stimulation on the A-fiber ER magnitudes, the A-fiber magnitude was determined from the 1-h stimulus triggered ensemble-averaged ER. After removing the mean, the integral of the absolute value of the A-fiber complex and the corresponding symmetrical area, before the trigger, were calculated, and the pretrigger value was subtracted from the A-fiber complex (see Ref. 12). The calculation covered 20 h, from 10 h before, to 10 h after the tetanus. To average across rats, for each rat, the magnitudes were standardized to z-units, zER = (ER − μ)/σ, where the mean (μ) and standard deviation (σ) were of the 10 pretetanus ERs. The resulting values in σ units are the change from the mean of the 10 pretetanus hours. With a z-score-averaging procedure, rats having more stable baselines more strongly influence the overall mean.
Effect of the tetanus on the slope of the ER curve.
For each trial, a line was fit to the 10 z-scored pretetanus ERs, and another to the 10 posttetanus ERs (starting from the last hour in the baseline, to the 9th h after the tetanus). The reliability, over trials, of the slope change, produced by A+C-fiber tetanus, was evaluated using a two-tailed, paired Student's t-test.
Effect of the A+C-Fiber Tetanic Stimulation on the A-Fiber ER Magnitudes
Figure 2 shows the blood pressure effects of four typical A+C-fiber tetanus applications from four different rats. To evaluate the tetanus effect on the magnitude of A-fiber ERs, 14 experiments were done on 12 rats. Fig. 3 shows the average effect, in z-units, of all 14 experiments (means ± SE), beginning from 10 h before to 10 h after the tetanus. The 10-h pretetanus and the 10-h posttetanus A-fiber magnitudes were reliably different [one-way repeated-measures ANOVA: F(13, 247) = 3.407, P < 0.001]. Post hoc analyses using Student-Newman-Keuls tests showed that the A-fiber magnitudes during post-LPT hours 6 and 7 were larger than the baseline (P < 0.05 for each hour); thus, the A+C-fiber tetanus reliably increased the magnitude of A-fiber ERs. The right ordinate of Fig. 3 is an approximate microvolt scale, which was generated by multiplying the z-values on the left ordinate by the grand average σ over all rats. The μV values were not used in any analysis but do give a useful sense of the physical size of the ER change, which also can be appreciated in Figs. 1 and 4. The shape of the curve in Fig. 3 resembles the curves of most of the individual tetanus experiments: The mean correlation coefficient between the average response curve and the individual curves of the six most similar trials was 0.86.
Using the same ER data, we calculated the response curve slope change following the A+C-fiber tetanus. For each trial, a line was fit to the 10 z-scored 1-h average pretetanus ERs and to the 10 posttetanus ERs (starting from the last hour in the baseline, to the 9th h after the tetanus), the corresponding slopes, characterized as the “preslope” and “postslope,” respectively, were calculated for each line. Over the 14 slope pairs (Table 1), the postslope was larger than the preslope (df = 13; t = −3.47; P < 0.005), showing that there was a reliable time-dependent increase in the magnitudes of A-fiber ERs following the A+C-fiber tetanus.
Effects of an NMDA Antagonist
In one additional rat, we used CPP, a NMDA receptor antagonist, prior to the A+C-fiber tetanus (see Fig. 4). The CPP procedure (bottom) was done on day 7 of a neuromuscular blocked (NMB) preparation that was functional for 12 days. With CPP, the A-fiber magnitude was not augmented by an A+C-fiber tetanus. On day 8, the procedure was repeated without CPP (top): In contrast to the result with CPP, the A-fiber magnitude was augmented by an A+C-fiber tetanus.
Our results show that tetanic stimulation of the ADN produces sustained increases in the magnitude of the A-fiber-evoked response in the NTS. This conclusion is based on two assumptions that were evaluated in Ref. 12: 1) that A-fibers are selectively stimulated by currents that are below the C-fiber threshold, and 2) that the A-fiber ER can be discriminated by latency and independently measured. To set the test and tetanus currents, we constructed detailed transduction curves for both the A and HTL complexes; for the tetanus, we used a current strength adequate to fully activate the HTL complex, but not greater. Following tetanus, the A-fiber ERs increased and continued to grow for several hours, with the larger magnitude sustained for at least several additional hours.
A possibility that would relegate the result to artifact is that it is somehow due to A-fiber damage caused by tetanic stimulation; however, this is extremely unlikely for the following reasons: 1) We have found no report of an effect of this kind, electrolytic damage usually causes dimunition of function, and the time course of the potentiation effect is not what would be expected from damage. 2) We used a noncorrosive, Ta-Ta2O5, capacitance electrode that has no electrochemical interaction with the nerve; previously, we reported that using 40-Hz pulse trains of 30-s duration, at C-fiber-activating currents, this electrode could elicit thousands of cardiovascular responses over many days without any noticeable change in threshold or response magnitude (cf. Fig. 1 in Ref. 3). 3) To stabilize the baseline ERs, the A-fiber test stimulus was set so as to maximally activate the A-fibers, without activating C-fibers; thus, damage would have to have had increased transmitter output of active fibers rather than sensitizing and recruiting inactive fibers. Therefore, we conclude that tetanic A+C-fiber activation was probably necessary to the potentiation effect and that a synaptic interaction most likely was involved. To our knowledge, there is no prior report of sustained synaptic potentiation of ERs in the cardiovascular NTS via the natural baroreceptor afferents; in fact, there have been only occasional sporadic reports of synaptic potentiation of any kind in the NTS.
The test and induction procedures that we chose were specifically intended to emulate those used for associative LTP in the CA1 region of the hippocampus (11). The onset delay and persistence of the effect (see Fig. 3) resembles the late form of LTP (L-LTP), and the within-subject control experiment using the NMDA receptor antagonist CPP is consistent with an LTP-related effect. In a hippocampus slice study, Harris et al. (9) demonstrated that LTP of Schaffer synaptic responses was elicited by high-frequency stimulation (100 pulses at 100 Hz, 3 times) of the Schaffer collateral system. Bath application of 10 μM CPP reversibly blocked the induction of the LTP. In another in vivo study (8), in the control situation, high-frequency stimulation of the fimbria resulted in LTP. When the high-frequency trains were delivered 40–60 min after CPP administration (CPP, 10 mg/kg ip), LTP was not induced; however, 180 min after CPP administration, LTP could again be induced.
Additional controls, which might have reinforced the LTP interpretation, were not done: Showing that simultaneous weak and strong pathway activation was both necessary and sufficient was not practical, because both the “strong” and “weak” inputs travel in the same nerve. A proper “sensitization” control would have required a special procedure, such as, anodal block of the A-fibers, during the tetanus (7). Doing this, in the chronic NMB preparation, would be problematical, and because the block itself is complicated, it is not clear that the result, whatever it was, would have been convincing. Finally, applying capsaicin to the nerve might have shown that C-fiber in distinction to A-fiber, tetanus was required; however, because the foramen magnum lies only millimeters below the nerve, and it was essential that the recording microelectrode be not in any way disturbed, this would have been precarious. Furthermore, from a regulatory perspective, it is actually immaterial whether the potentiation is due to A-fiber or C-fiber tetanus: high-frequency A-fiber activation, as well as C-fiber activation, elicits a powerful depressor effect (Refs. 3, 5–7); both A and C-fibers are activated by elevated blood pressure, and either might produce sufficient postsynaptic depolarization to unblock NMDA receptors. In preliminary observations, we found no evidence of potentiation by 100-Hz A-fiber, stimulation; however, we did not do a full parametric analysis.
Unquestionably, much additional work is needed to define the mechanism; however, attributing the potentiation to LTP or to C-fiber activation is not essential to the cardiovascular implications of the finding that brief strong activation of the ADN potentiates its A-fiber-elicited evoked responses in the dmNTS. The putative regulatory scheme is presented below.
A-fiber-coupled receptors have lower thresholds and more stable and more proportional firing patterns than C-fiber receptors, most of which have very steep transduction curves, with erratic firing, which increases from near zero to a maximum rate over a narrow range of pressures (13–15). Thus, as components of a conventional linear control system, the C-fiber-coupled receptors would likely detract from the system's stability and accuracy. In fact, the C-fiber contribution to moment-to-moment, closed-loop blood pressure control, except possibly to help blunt a sudden spike, appears to be negligible: When electrically stimulated, C-fibers produce large depressor effects; but, within the ordinary arterial pressure range, the absolute gain of the intact aortic reflex is similar with or without C-fibers [1.7 vs. 1.8 in Table 5 of (4) and Fig. 3 in (5)]; nonetheless, C-fiber receptors are good candidates for “out-of-range” sensors, similar to those that are incorporated in “adaptive” control systems (10). Because the transduction properties of C-fiber receptors fit the theoretical scheme that motivated these experiments (1), we intentionally used high current for the tetanus, and we refer to C-fiber receptor activation as the tetanic device, but substituting “high-threshold receptors” would not change the logic.
The core of the hypothesis is as follows: A- and C-fiber receptors, because they are collocated and exposed to the same pressure, could interact through the potentiation mechanism that we have described to optimize blood pressure control. Specifically, occasional activation by high pressure would adjust baroreflex parameters by incrementing the gain of the A-fiber reflex. In the intact natural baroreflex, C-fiber activation occurs primarily at a very high pressure; if the A-fiber baroreflex gain is adequate to produce a prompt and sufficient depressor response, the pressure does not reach the C-fiber activating level; only when A-fiber gain is insufficient does the blood pressure rise to where the C-fibers are activated. Because the A-fiber receptors have lower thresholds and the A- and C-fiber receptors have the same receptive fields in the intact system, A-fiber activation necessarily precedes, and continues, during C activation; hence, the arrangement is inherently associative, and the stimulus sequence fits the LTP mechanism well. The concept is illustrated in Fig. 5, which shows a hypothetical blood pressure time series: as a pressure rise begins, low threshold A-fiber receptors are activated; if the A-fiber depressor response is inadequate, the pressure continues to rise, and, then, the higher threshold, C-fiber, receptors are also activated. The C-fiber activation has two effects: conventionally, it augments the depressor response, but also, in accord with our new observations, it increases, for several hours, the depressor effectiveness (gain) of the A-fiber reflex. We have not yet shown that the effects of successive C-fiber activations are cumulative; however, assuming that, as with LTP, they are, the A-fiber reflex would eventually develop sufficient strength that blood pressure peaks would be truncated before the C-fiber threshold is reached. Eventually, the A-fiber pathway will habituate, the A-fiber reflex gain will decrease, C-fiber receptors will again be occasionally activated, and the potentiation process will be repeated. Fig. 5 is conceptual, but mathematical models predict that the kind of interaction described converges in a way that optimizes regulation (1).
The studies were supported by Grant HL-40837 (to B. R. Dworkin) from the National Heart, Lung, and Blood Institute, Division of Heart and Vascular Diseases.
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