INTRODUCTION

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CHANGING THE LEVEL of activity in peripheral sympathetic nerves in response to periods of physical stress is a primary means by which mammals maintain physiological homeostasis. The direction of change depends on the type of physical stress and on the peripheral nerve from which the activity is being recorded (1, 2, 5, 13-15, 38, 39, 41). Importantly, the dynamic nature of sympathetic neural circuits during physical stress is revealed not only by changes in the level of activity but also by alterations in the pattern of sympathetic nerve discharge (SND) bursts. At least three stress-induced changes in the frequency-domain characteristics of SND bursts have been observed.

First, the pattern of efferent SND bursts is changed during a variety of experimental interventions (e.g., hyperthermia, cerebral ischemia, and asphyxia) (21-24, 26), demonstrating that sympathetic neural circuits are capable of generating different burst frequencies depending on the physiological state of the animal. Second, the sources of synchronized discharges in sympathetic nerve pairs uncouple (i.e., reduce coherence) in response to some types of stress (e.g., asphyxia, cerebral ischemia, and after sustained stimulation of baroreceptor afferents) (4, 21, 24, 26). Uncoupling of the sources of synchronized discharges may be one strategy by which the central nervous system exerts selective control over the activity in different sympathetic nerves during interventions that produce similar directional changes in the level of efferent sympathetic nerve outflow. Third, physical stress can influence the relationship between the sympathetic and respiratory systems, as indicated by changes in the coupling between SND and phrenic nerve discharge (PND) during hyperthermia and severe hypercapnia (23, 43). The results of these studies suggest that altering the pattern of efferent SND bursts is an important regulatory strategy employed by sympathetic neural circuits to respond to physical stress.

Although cold stress activates the sympathetic nervous system, as demonstrated by significant increases in plasma concentrations of norepinephrine and epinephrine (3, 8, 18, 19, 34, 42), activation of preganglionic cervical SND (20), and the formulation of nonuniform peripheral sympathetic responses elicited by spinal cord and hypothalamic cooling (15-17, 40), little information is available concerning the effect of cold stress on the frequency-domain characteristics of SND. This is a significant omission, inasmuch as changes in SND frequency components may provide the key for understanding sympathetic nerve control mechanisms during hypothermia. Importantly, such an understanding may be essential for investigating the suggested involvement of the sympathetic nervous system in hypothermia-related cardiovascular complications (3, 8, 11, 30, 36).

The purpose of the present study was to use autospectral and coherence analyses to determine the influence of cold stress on the frequency components in SND and on the relationships between the discharges in sympathetic nerve pairs. Activity was recorded from the renal, lumbar, splanchnic, and adrenal sympathetic nerves. In addition, the frequency-domain relationships between SND and PND bursts were examined to determine the effect of hypothermia on the respiratory modulation of SND.

	METHODS

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General procedures. The surgical procedures and experimental protocols were approved by the Institutional Animal Use and Care Committee. Experiments were performed on male Sprague-Dawley rats (300 g). Anesthesia was initially induced with methohexital sodium (Brevital, 50-60 mg/kg ip). Two catheters (PE-10 and PE-50) were placed in the femoral vein. The PE-10 catheter was used during the surgical preparation for administration of maintenance doses of methohexital sodium (10-20 mg/kg) and during the experimental protocols for administration of selected pharmacological agents. The PE-50 catheter was used for the administration of an initial dose of -chloralose (50 mg/kg) and for maintenance doses (35 mg · kg¹ · h¹) throughout the surgical preparation and experiment. The trachea was cannulated with a PE-240 catheter. Rats were paralyzed with gallamine triethiodide (5-10 mg/kg iv initial dose, 10-15 mg · kg¹ · h¹ maintenance dose) and artificially ventilated. Femoral arterial pressure and heart rate (HR) were recorded by using standard procedures. Core body temperature (T_c) was measured with a thermistor probe inserted ~5-6 cm into the colon and was kept at 38.0°C during surgery by use of a temperature-controlled table.

Neural recordings. Activity was recorded biphasically with a platinum bipolar electrode after capacity-coupled preamplification (band pass 30-3,000 Hz) from the central end of cut or distally crushed renal, lumbar, splanchnic, and adrenal sympathetic nerves, the phrenic nerve, and the aortic depressor nerve (ADN). The left renal, adrenal, and splanchnic nerves were isolated retroperitoneally or after a midline laparotomy. The left lumbar nerve was isolated from a midline approach. The left phrenic nerve was isolated in the cervical region. The ADN was isolated from a ventral approach ~1 cm caudal to its junction with the superior laryngeal nerve. Baroreceptor nerve activity was recorded from the ADN, because this nerve contains only baroreceptor afferents in the rat (32, 35). The nerve-electrode preparations were covered with a silicone gel to prevent exposure to room air. The nerve potentials were full-wave rectified and integrated (time constant 10 ms), which produced a smooth tracing of the synchronized discharges. Activity in SND, PND, and ADN discharge recordings was quantified as volts times seconds (V · s). The sympathetic nerve recordings were corrected for background noise after administration of the ganglionic blocker trimethaphan camsylate (10-15 mg/kg iv). ADN recordings were corrected for background noise by crushing the distal end of the ADN, whereas phrenic and adrenal nerve activities were corrected for background noise by crushing the central end of these nerves at the end of each experiment.

Experimental protocols. After surgery the chloralose-anesthetized, gallamine-paralyzed, baroreceptor-innervated rats were allowed to stabilize for 30-60 min before initiation of the experimental protocols. At the end of this control period, water (externally cooled to 10°C) was recirculated through a perfusion pad, and T_c was allowed to decrease continuously from 38 to 30°C at a constant rate of ~0.1-0.2°C/min. End-tidal CO₂ was kept at 4.8-5.2% by adjusting the frequency of respiration during hypothermia (control ventilatory rate, 82 ± 2 strokes/min; 30°C ventilatory rate, 52 ± 3 strokes/min). Mean arterial pressure (MAP), HR, and SND bursts were recorded continuously during progressive reductions in T_c. Frequency-domain analyses were performed on SND recordings at T_c of 38, 36, 34, and 30°C. Three protocols were completed. Protocol I determined the effect of cold stress on 1) the level of activity in sympathetic nerves (quantified after integration as V · s; n = 27), 2) the basic pattern of efferent SND bursts (as determined by using autospectral analysis; n = 27), and 3) the frequency-domain relationships (as determined by coherence analysis) between the simultaneously recorded discharges of sympathetic nerve pairs (n = 20). Protocol II determined the effect of hypothermia on the frequency-domain characteristics of ADN discharge bursts (n = 4). Autospectra of renal SND, ADN discharge bursts, and pulsatile arterial pressure were constructed before and during progressive cooling. In three additional experiments we determined renal SND responses to increases in MAP produced by the intravenous administration of phenylephrine hydrochloride (5.0 µg/kg). Phenylephrine was administered during control (38°C) and deep hypothermia (30°C). Protocol III determined the effect of cold stress on the relationships between the simultaneously recorded SND [renal (n = 9) and adrenal (n = 3)] and PND bursts with use of the coherence function. Bilateral vagotomies were completed in five experiments [renal-phrenic (n = 2) and adrenal-phrenic (n = 3)]. Because the coherence values relating the discharges in the renal and phrenic nerves were qualitatively similar in intact (0.63 ± 0.07, 0.78 ± 0.03, and 0.34 ± 0.05 at 38, 36, and 30°C, respectively) and vagotomized (0.41, 0.72, and 0.22, at 38, 36, and 30°C, respectively) animals, the data were combined for presentation.

Data analysis. Autospectral and coherence analyses of the arterial pulse, SND bursts, ADN bursts, and PND bursts were computed by using the methods and programs described earlier (21, 27). Fast Fourier transform was performed on 12-24 contiguous windows of data that were 5 s in duration. The signals were sampled at 200 Hz. Autospectral and coherence functions were computed over a frequency band of 0-15 Hz. The amplitude of the autospectra was autoscaled to the highest peak (21, 27). The frequency resolution was 0.2 Hz/bin. The percentage of total power in individual 2-Hz SND frequency bands was quantified after autospectral analysis with use of the program of Kocsis et al. (27).

	RESULTS

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Protocol I: effect of cold stress on the level of sympathetic nerve activity and the frequency components of SND bursts. The effect of decreases in T_c on HR, MAP, the level of activity in sympathetic nerves, and the frequency-domain characteristics of SND bursts was determined in 27 experiments. In 20 experiments, SND was recorded from sympathetic nerve pairs [renal-splanchnic (n = 7), renal-adrenal (n = 7), and renal-lumbar (n = 6)], and in 7 experiments the activity in a single nerve [lumbar (n = 6) and adrenal (n = 1)] was recorded. Figure 1 summarizes changes in HR, MAP, and renal, lumbar, splanchnic, and adrenal sympathetic nerve activity recorded during cold stress. HR decreased during hypothermia and was reduced significantly from control levels at 30°C. In contrast, MAP increased progressively during cold stress and was significantly elevated from control levels at 36, 34, and 30°C. The sympathetic nerve responses to hypothermia were nonuniform. Specifically, renal SND decreased significantly (22 ± 5, 31 ± 6, and 40 ± 7% at 36, 34, and 30°C, respectively), lumbar SND increased significantly (110 ± 30, 121 ± 32, and 90 ± 25% at 36, 34, and 30°C, respectively), and adrenal and splanchnic SND did not change significantly from control values during cold stress.

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Heart rate (HR), mean arterial pressure (MAP), and renal, lumbar, splanchnic, and adrenal sympathetic nerve discharge (SND) during control (38°C) and hypothermia (36, 34, and 30°C). Tc, core body temperature; bpm, beats/min. * Significantly different from control, P < 0.05.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Traces of integrated SND bursts and pulsatile arterial pressure (AP) from 1 experiment in which simultaneous recordings of renal and splanchnic sympathetic nerves were completed during control (38°C) and hypothermia (34°C). Top and middle traces: SND bursts; bottom traces: pulsatile AP. Mean arterial pressure values for each period are shown below pulsatile AP traces. Horizontal calibration bar, 500 ms.

View larger version (35K): [in this window] [in a new window]   Fig. 3.   Frequency-domain relationships between renal and splanchnic (Spl) SND during control (38°C) and hypothermia [mild (36°C), moderate (34°C) and deep (30°C)]. Top and middle: individual autospectra; bottom: renal SND-to-splanchnic SND coherence functions. Amplitudes of autospectra are autoscaled to highest peak. Frequency band is displayed from 0 to 15 Hz.

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Percentage of total power in individual 2-Hz frequency bands corresponding to low-frequency (LF) and cardiac-related frequency (CF) components in renal, lumbar, splanchnic, and adrenal SND during control (38°C) and hypothermia (36, 34, and 30°C).  CF band significantly different from LF band during control period (P < 0.05).  LF band during hypothermia significantly different from LF band during control (P < 0.05).  CF band during hypothermia significantly different from CF band during control (P < 0.05).

Protocol II: effect of hypothermia on the frequency-domain relationships between the activity in the ADN and renal sympathetic nerve. To determine whether changes in the SND bursting pattern during hypothermia resulted from alterations in the pulse-synchronous discharge characteristics of afferent baroreceptor nerve activity, ADN activity was recorded simultaneously with renal SND in four rats before and during reductions in T_c. Figure 5 shows the results of one representative experiment in which autospectra of renal SND, ADN activity, and pulsatile arterial pressure were constructed during control (38°C) and hypothermia (36, 34, and 30°C). During control the autospectra of renal SND, ADN activity, and arterial pressure contained primary peaks at the frequency of the cardiac cycle. During cooling (36, 34, and 30°C) the primary peak in the renal SND autospectra was contained between 0 and 2 Hz, whereas the primary peak in the ADN autospectra remained at the frequency of the cardiac cycle. Importantly, in the four experiments completed, the renal SND bursting pattern was transformed from one characterized by the presence of cardiac-related bursts during control to one dominated by low-frequency bursts during hypothermia, despite the fact that the ADN discharge bursts remained synchronized to the arterial pulse during the entire cooling protocol.

View larger version (30K): [in this window] [in a new window]   Fig. 5.   Autospectra of simultaneously recorded renal SND (top), aortic depressor nerve (ADN) activity (middle), and pulsatile AP (bottom) during control (38°C) and mild (36°C), moderate (34°C), and deep (30°C) hypothermia. Amplitudes of autospectra are autoscaled to highest peak. Frequency band is displayed from 0 to 15 Hz.

View larger version (15K): [in this window] [in a new window]   Fig. 6.   Traces of integrated renal SND bursts and pulsatile arterial pressure recorded in a baroreceptor-innervated rat during normothermia (38°C) and hypothermia (30°C) before (control) and after (PE) bolus intravenous administration of phenylephrine hydrochloride (5.0 µg/kg). Horizontal calibration bar, 700 ms.

Protocol III: effect of cold stress on the frequency-domain relationships between SND and PND. The influence of hypothermia on the relationships between the discharge bursts in sympathetic and phrenic nerves was determined in 12 experiments: 9 renal-phrenic and 3 adrenal-phrenic. Figure 7 shows the results of one experiment in which renal SND and PND autospectra (top and middle) and the coherence function (bottom) describing the relationships between the activity in these nerves were constructed at 38, 36, and 30°C. During control the renal SND autospectra contained a primary peak at 7.2 Hz (frequency of the cardiac cycle), with a secondary peak at 1.6 Hz. The renal nerve and phrenic nerve coherence function exhibited a prominent peak (0.75) at 1.6 Hz. During mild hypothermia (36°C) the primary peaks in the renal SND and PND autospectra were at 1.4 Hz and the peak coherence value relating the activity in these nerves was increased to 0.92 at 1.4 Hz. At deep hypothermia (30°C) the primary peak in the SND autospectra was at 0.8 Hz, the primary peak in the PND autospectra was at 0.6 Hz, and the peak coherence value relating the renal SND and PND bursts was reduced to 0.46 at 0.6 Hz. In nine experiments involving renal-phrenic nerve recordings, the peak coherence value relating SND and PND bursts was increased (P < 0.05) from 0.58 ± 0.07 at 38°C to 0.77 ± 0.03 at 35-36°C. With additional cooling to 30-31°C, the peak coherence value relating sympathetic and phrenic nerve signals was reduced (P < 0.05) to below control levels (0.32 ± 0.04). In three experiments involving adrenal-phrenic nerve recordings, the peak coherence values relating the discharges in these nerves were reduced from 0.61 during control to 0.23 at 36°C and 0.10 at 30°C.

View larger version (30K): [in this window] [in a new window]   Fig. 7.   Frequency-domain relationships between simultaneously recorded renal SND and phrenic nerve discharge bursts during control (38°C) and mild (36°C) and deep (30°C) hypothermia. Top and middle: individual autospectra for renal SND and phrenic nerve discharge, respectively; bottom: renal SND-to-phrenic nerve discharge coherence functions. Amplitudes of autospectra are autoscaled to highest peak. Frequency band is displayed from 0 to 15 Hz.

	DISCUSSION

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This study examined the effects of cold stress on the frequency-domain relationships between the discharge bursts of sympathetic, sympathetic and aortic depressor, and sympathetic and phrenic nerve pairs in baroreceptor-innervated rats. Our results provide experimental support for four findings that provide insight into the dynamic nature of sympathetic neural circuits and contribute to the understanding of sympathetic regulatory mechanisms during periods of stress. First, hypothermia produced nonuniform changes in the level of activity in regionally selective sympathetic nerves. Second, the fact that the cardiac-related pattern of renal, splanchnic, and lumbar SND was profoundly altered during cold stress, despite the presence of pulse-synchronous activity in arterial baroreceptor afferents, demonstrates changes in the neural circuits responsible for generation of cardiac-related efferent SND bursts. Third, the peak coherence values relating the low-frequency (0-2 Hz) discharges in sympathetic nerve pairs remained unchanged from control levels during hypothermia, indicating that the sources of low-frequency SND bursts remain prominently coupled during progressive reductions in T_c. Fourth, the relationships between PND and renal SND bursts were variable during progressive hypothermia; i.e., the frequency-domain coupling was enhanced during mild hypothermia (as evidenced by significant increases in the peak coherence values relating the discharges in these nerves) but was significantly reduced from control levels during deep hypothermia.

The sympathetic nervous system is capable of producing regionally selective changes in efferent sympathetic nerve outflow (1, 2, 5, 13, 14-17, 38-41). At least two types of nonuniformity have been described. First, directionally opposite changes in the level of activity in nerves innervating different target organs have been demonstrated during a number of experimental interventions, including hemorrhagic hypotension, hypertonic saline infusion, activation of vagal nerve afferents, nitric oxide synthase inhibition, and thermal stress, and after prolonged activation of baroreceptor afferents (4, 13-17, 39-41). The results of the present study extend these observations by demonstrating that cold stress produces regionally nonuniform changes in efferent SND in chloralose-anesthetized rats. Specifically, hypothermia increases lumbar and decreases renal SND without significantly changing the level of activity in the splanchnic and adrenal nerves. Second, the sources of synchronized discharges in different sympathetic nerves can uncouple (i.e., reduce coherence) during various experimental interventions (4, 21, 24, 26), demonstrating stress-induced changes in the frequency-domain relationships between the activity in sympathetic nerve pairs. Consistent with these findings, the peak coherence value relating cardiac-related discharge bursts was reduced from control levels during cold stress in renal-lumbar, renal-splanchnic, and renal-adrenal nerve pairs. Moreover, the peak coherence value relating low-frequency discharge bursts in renal-adrenal nerve pairs was reduced from control levels during hypothermia in five of seven experiments. In contrast, low-frequency SND bursts recorded in renal-lumbar and renal-splanchnic nerve pairs remained prominently coupled during reductions in T_c. Taken together, the present results demonstrate that hypothermia produces complex changes in efferent sympathetic nerve outflow. The results of experiments involving simultaneous recordings of renal and lumbar SND reveal an interesting profile of sympathetic nerve responses to cold stress: nonuniformity in the level of regionally selective sympathetic nerve activity and uniformity in the frequency characteristics of SND bursts. This combination of responses may be an important organizational strategy employed by sympathetic neural circuits to regulate the vast array of physiological changes required to maintain homeostasis in response to environmental challenges.

In baroreceptor-innervated animals the cardiac-related rhythmicity in SND bursts reflects the fundamental organization of sympathetic neural circuits (9, 23, 25). The present results demonstrate that the basic cardiac-related pattern of SND bursts is altered by cold stress. Specifically, the SND bursting pattern during hypothermia is characterized by the loss of cardiac-related bursts, with an increased number of low-frequency bursts that are not coupled to the arterial pulse. Hypothermia-induced changes in the SND bursting pattern are particularly striking, considering that arterial pressure gradually increases during cold stress and ADN discharge bursts remain pulse synchronous. These results suggest that cold stress influences the neural circuits involved in coupling ADN activity and efferent SND bursts. If hypothermia had changed the level of efferent SND without altering the frequency characteristics of the bursting pattern, the autospectra of SND constructed during cold stress would be similar to those constructed during control (i.e., the primary peak in the SND autospectra would be at the frequency of the cardiac cycle). It is interesting to note that activation of the arterial baroreceptors by phenylephrine-induced rapid increases in arterial pressure during deep hypothermia (at a time when the SND bursting pattern was characterized by the presence of low-frequency, non-cardiac-related bursts) reflexly decreased renal SND to levels produced by phenylephrine-induced baroreceptor activation under normothermic conditions. Taken together, these results suggest that the baroreflex control of sympathetic nerve activity and the entrainment of SND bursts to the cardiac cycle by the afferent limb of the baroreceptor reflex can be dissociated under certain experimental conditions. The experimental demonstration of a functional dissociation of the baroreflex control of the level of sympathetic nerve activity and the SND bursting pattern may have an important pathophysiological corollary, inasmuch as the baroreflex regulation of efferent SND is impaired in heart failure, yet SND bursts are characterized by a prominent cardiac-related rhythmicity (6, 7).

In the present study, coupling between renal SND and PND bursts was enhanced during mild hypothermia (as evidenced by significant increases in the peak coherence values relating the discharges in these nerves) but was significantly reduced from control levels during deep hypothermia. The latter observation suggests that respiratory and sympathetic neural networks are independently capable of generating slow periodicities during conditions of deep hypothermia. Similar uncoupling of the slow rhythms in SND and PND bursts has been observed during conditions of extreme hypercapnia in anesthetized cats (43). Variable changes in the coupling between SND and PND bursts in the present study did not result from alterations in respiratory drive, inasmuch as end-tidal CO₂ levels remained constant during reductions in T_c. Moreover, the respiratory-related activity in efferent SND recordings during mild hypothermia was not solely the result of entrainment of sympathetic nerve-related neurons by inputs from pulmonary stretch receptors, inasmuch as renal SND bursts were coupled to the central respiratory cycle in vagotomized and paralyzed animals.

The hypothermia-induced transformation of the SND pattern from cardiac-related to low-frequency bursts was evident in multifiber sympathetic nerves that innervate target organs with heterogeneous functions. Importantly, similar changes in the SND bursting pattern in regionally selective nerves have been observed during a variety of different experimental interventions, including hyperthermia (23), the initial phase of asphyxia (unpublished observation), and after prolonged activation of baroreceptor afferents (4). Thus it appears that pattern transformation is a consistent feature of sympathetic nerve responses to stress in anesthetized rats. It is interesting to note that the low-frequency SND bursts recorded during mild and moderate hypothermia (this study), progressive increases in T_c (23), and the initial phase of asphyxia (unpublished observation) are prominently coupled to PND. This is noteworthy, because respiratory modulation of SND represents an electrophysiological correlate of the cooperation between the cardiovascular and respiratory systems (43). The effect of pattern transformation on target organ function remains to be determined; however, the results of several studies indicate that the impulse pattern of SND bursts can influence important physiological indexes such as vascular responses and transmitter release (12, 28, 31, 33). For example, electrical stimulation of the splenic nerve with burst activity at 2.0 Hz evokes an enhanced vasoconstrictor response compared with stimulation with continuous impulse activity (33). In addition, Stauss and Kregel (37) reported that electrical stimulation of the splanchnic nerve at frequencies between 0.1 and 1.0 Hz significantly increased the power in mesenteric resistance. Importantly, these oscillations were translated to arterial blood pressure (37), suggesting that the frequency range of vasomotor activity in peripheral sympathetic nerves can include that of respiration. Considering the present results, it may be that transformation from cardiac-related to low-frequency SND bursts enhances the effectiveness of efferent nerve activity directed toward selective target organs.

There are several limitations to the present study. First, efferent sympathetic nerves do not contain a functionally uniform population of axons; therefore, the precise functional significance of changes in the SND pattern is difficult to determine. However, similar SND pattern changes were observed in each of the regionally selective sympathetic nerves recorded in this study, supporting the hypothesis that cold stress profoundly affects the organization of neural circuits responsible for regulation of efferent SND. Second, the present study was completed by using a whole body cooling protocol in anesthetized rats, whereas previous studies have used cooling of the anterior hypothalamus or spinal cord in anesthetized rabbits (15-17, 40). These factors may have contributed to differences observed in the SND responses to cold stress. For example, we failed to observe a significant change in the level of splanchnic SND during progressive hypothermia, similar to results observed with anterior hypothalamic cooling in anesthetized rabbits (16). However, Iriki and co-workers (15, 17) and Walther et al. (40) reported significant reductions in splanchnic nerve activity during spinal cord cooling in anesthetized rabbits. Third, anesthesia may influence sympathetic nerve responses to cold stress, such that different responses may be obtained in conscious rats. Although this cannot be discounted as a possibility, the anesthesia regimen used in this study does not alter efferent renal and lumbar SND responses to baroreflex stimuli (29).

Perspectives