INTRODUCTION

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CHANGING THE LEVEL OF ACTIVITY in sympathetic nerves in response to acute environmental stress is a primary means by which mammals maintain physiological homeostasis. In young Sprague-Dawley rats, elevated internal body temperature (Tc) produced by acute heat stress increases efferent sympathetic nerve activity (9, 12, 13, 15) and alters the pattern of sympathetic nerve discharge (SND) bursts (12, 13, 15). With respect to the SND bursting pattern, increased Tc transforms cardiac-related SND bursts to low-frequency bursts that contain more power than cardiac-related bursts, demonstrating an important role for pattern transformation in mediating heating-induced sympathoexcitatory responses (13).

Aging is known to alter sympathetic nerve responses to various acute stressors, including hypoxia (30, 31), footshock (23), and 2-deoxy-D-glucose administration (23). In addition, senescent rats demonstrate altered norepinephrine turnover rates in selected organs during heating (18), suggesting age-related changes in sympathetic nerve responses to heating. However, our understanding of the effect of age on directly recorded SND responses to heating is limited in at least three ways. First, the effect of age on renal sympathetic nerve responses to increased Tc is not well established. Stauss et al. (32) reported that the level of renal SND remained unchanged in response to hyperthermia in 12-mo-old (mature) and 24-mo-old (senescent) Fischer 344 (F344) rats. However, renal SND responses to heating in young F344 rats were not reported. Therefore, it is not clear whether the lack of renal sympathoexcitation to heating in mature and senescent F344 rats is age related or whether F344 rats, regardless of age, fail to demonstrate renal sympathoexcitation to heating. Second, although the sympathetic innervation to the splanchnic vasculature plays a key role in mediating cardiovascular responses to heating (19, 21) and hyperthermia increases splanchnic SND in young rats (9, 12), the effect of age on splanchnic SND responses to increased Tc is not well established. Third, the effect of hyperthermia on SND frequency components in aged rats is not known. This is an important omission because SND pattern changes reveal the dynamic nature of sympathetic neural circuits (4, 13) and contribute to heating-induced increases in SND (13).

The first aim of this study was to determine the effect of hyperthermia on the level of activity and the frequency components of renal and splanchnic SND in young (3-6 mo old), mature (12 mo old), and senescent (24 mo old) baroreceptor-innervated (BRI) F344 rats. We hypothesized that the responsiveness of sympathetic neural circuits to increased Tc would be altered, as demonstrated by the absence of hyperthermia-induced sympathoexcitatory responses and SND bursting pattern alterations, in senescent and mature but not in young F344 rats.

As the results reveal, renal and splanchnic SND responses to heating are markedly attenuated in senescent compared with young and middle-aged F344 rats. Although it is known that baroreflex regulation of renal SND is impaired in senescent rats (10), the fact that the cardiac-related discharge pattern and the level of renal and splanchnic SND remained unchanged from control during heating in senescent rats, despite little or no hyperthermia-induced increase in mean arterial pressure (MAP), suggests that baroreflex control of SND might be enhanced during acute heat stress in senescent rats. If this were the case, then SND responses to heating in sinoaortic-denervated (SAD) senescent rats would be higher than in BRI senescent rats. Similarly, it may be that heating-induced sympathoexcitatory responses in young and mature F344 rats are opposed by activation of the arterial baroreceptors secondary to increases in MAP that occur during heating in these rats. The second aim of this study was to determine renal and splanchnic SND responses to heating in SAD young, mature, and senescent F344 rats.

Despite significantly higher renal and splanchnic SND responses to heating in young compared with mature F344 rats, heating-induced increases in MAP at 41°C were similar in these groups. Because activation of efferent adrenal SND increases the release of epinephrine from the adrenal medulla (2, 25) and because circulating epinephrine can bind to peripheral ₂-adrenergic receptors and reduce peripheral vascular resistance (16), it may be that hyperthermia-induced activation of adrenal SND is higher in young compared with mature rats. The third aim of this study was to determine adrenal SND responses to heating in BRI young and mature F344 rats.

	METHODS

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General procedures. The Institutional Animal Care and Use Committee approved the experimental procedures and protocols used in the present study. Experiments were performed on male 3- to 6-mo-old (291 ± 9 g, n = 33), 12-mo-old (413 ± 5 g, n = 25), and 24-mo-old (411 ± 9 g, n = 23) F344 rats. Anesthesia was initially induced with methohexital sodium (50-60 mg/kg ip Brevital) (13-15). Catheters were placed in the femoral vein for the administration of maintenance doses of methohexital sodium (10-20 mg/kg) during surgical interventions and for the administration of -chloralose (50 mg/kg, initial dose; 35-50 mg · kg¹ · h¹, maintenance dose) (13-15). The trachea was cannulated with a polyethylene-240 catheter. Femoral arterial blood pressure and heart rate (HR) were recorded using standard procedures. Tc was measured with a thermistor probe inserted 5-6 cm into the colon and was kept at 38.0°C during surgery by a temperature-controlled table.

Neural recordings. Activity was recorded biphasically (band pass 30-3,000 Hz) with a platinum bipolar electrode from the central end of cut or distally crushed renal, splanchnic, and adrenal sympathetic nerves. Sympathetic nerves were isolated retroperitoneally. The nerve-electrode preparations were covered with silicone gel, and sympathetic nerve activity was full wave rectified and integrated (time constant 10 ms). The level of activity was quantified as volts × seconds (V · s), and sympathetic nerve recordings were corrected for background noise after administration of the ganglionic blocker trimethaphan camsylate (10-15 mg/kg iv) or nerve crush (13-15).

Sinoaortic denervation. Bilateral denervation of the aortic arch was completed by 1) cutting the superior laryngeal nerve near its junction with the vagus nerve and 2) removing the superior cervical ganglion (22). Bilateral carotid sinus denervation was completed by removing the adventitia from the area of the carotid sinus bifurcation (22). Denervation was considered complete by the loss of coherence between the arterial pulse and SND (17) and/or the absence of a reflex change in SND from control levels during increases in arterial pressure produced by the administration of phenylephrine hydrochloride (3-5 µg/kg iv) and during decreases in arterial pressure produced by the administration of sodium nitroprusside (3-5 µg/kg iv).

Experimental protocol. After completion of the nerve-electrode preparations, the anesthetized rats were allowed to stabilize for 60 min before initiation of the heating protocol. At the end of this control period, Tc was increased at a rate of ~0.1°C/min from 38 to 41°C using a heat lamp. Heating experiments were completed in BRI and SAD rats. MAP, HR, and SND were recorded continuously before and during heating. The rate of rise in Tc during heating did not differ between BRI young (n = 16), mature (n = 17), and senescent (n = 12) F344 rats (38-39°C: young, 11 ± 1 min; mature, 12 ± 2 min; senescent, 10 ± 1 min; 39-40°C: young, 11 ± 2 min; mature, 9 ± 2 min; senescent, 11 ± 1 min; and 40-41°C: young, 15 ± 5 min; mature, 15 ± 5 min; senescent, 13 ± 1 min) or between SAD young (n = 17), mature (n = 8), and senescent (n = 11) F344 rats (38-39°C: young, 13 ± 1 min; mature, 15 ± 2 min; senescent, 13 ± 1 min; 39-40°C: young, 12 ± 1 min; mature, 9 ± 1 min; senescent, 10 ± 1 min; and 40-41°C or peak change in SND: young, 11 ± 1 min; mature, 10 ± 1 min; senescent, 9 ± 1 min).

Data analysis. Autospectral analysis of SND bursts was completed as previously described (17). Fast Fourier transform was performed on 12 contiguous windows of data that were 5 s in duration. Autospectra were computed over a frequency band of 0-15 Hz. The frequency resolution was 0.2 Hz/bin. The autospectrum of a signal shows the relative power present at each frequency.

	RESULTS

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Control levels of MAP (young, 132 ± 3 mmHg; mature, 113 ± 3 mmHg; senescent, 113 ± 2 mmHg) and HR (young, 454 ± 13 beats/min; mature, 417 ± 4 beats/min; senescent, 376 ± 13 beats/min) were higher (P < 0.05) in BRI young compared with BRI mature and senescent F344 rats. Control levels of HR were higher (P < 0.05) in BRI mature compared with senescent rats (mature, 417 ± 4 beats/min; senescent, 376 ± 13 beats/min). Figure 1 summarizes changes from control (38°C) for MAP (A), HR (B), renal SND (C), and splanchnic SND (D) during increases in Tc in BRI young, mature, and senescent F344 rats. Each experimental variable was significantly increased from control during heating in young and mature rats, whereas only HR was significantly increased in senescent rats. At 41°C, MAP, HR, renal SND, and splanchnic SND were significantly higher in young compared with senescent rats, MAP and renal SND were significantly higher in mature compared with senescent rats, and HR and renal SND were significantly higher in young compared with mature rats. Renal and splanchnic SND responses to a short bout of asphyxia (20-25 s) were determined after Tc had been increased to 41°C in seven senescent rats. Asphyxia significantly increased SND (renal and splanchnic combined) 85 ± 12% from levels recorded at 41°C. Adrenal SND responses to heating were determined in eight BRI F344 rats (young, n = 4; mature, n = 4). Heating-induced increases in adrenal SND were significantly higher in young than in mature rats at 40.5°C (young, 114 ± 36%; mature, 45 ± 18%; P < 0.05) and 41°C (young, 148 ± 36%; mature, 65 ± 23%; P < 0.05).

View larger version (30K): [in this window] [in a new window]   Fig. 1.   Mean arterial pressure (MAP; A), heart rate (HR; B), renal sympathetic nerve discharge (SND; C), and splanchnic SND (D) responses during progressive increases (39, 40, 40.5, and 41.0°C) in internal body temperature (Tc) in young, mature, and senescent baroreceptor-innnervated Fischer 344 (F344) rats. Values at 38°C were considered as control. bpm, Beats/min. * Significantly different from 38°C. Significantly different from senescent rats. Significantly different from mature rats.

Figure 2 shows traces of simultaneously recorded renal and splanchnic SND bursts and pulsatile arterial pressure during control (38°C, left) and after heating to 41.0°C (right) in representative young (A), mature (B), and senescent (C) BRI F344 rats. During control, the majority of renal and splanchnic SND bursts were coupled to the arterial pulse, regardless of age. At 41°C, sympathetic recordings were characterized by the presence of low-frequency bursts in the young rat (A) but remained coupled to the arterial pulse in the mature (B) and senescent (C) rats.

View larger version (45K): [in this window] [in a new window]   Fig. 2.   Traces of integrated SND bursts (renal and splanchnic) and pulsatile arterial pressure (AP) recorded during control (38°C) and after increasing internal Tc to 41.0°C in young (A), mature (B), and senescent (C) F344 rats. Horizontal calibration is 500 ms.

SND autospectra constructed at 38°C (control) and 41°C in representative young (A), mature (B), and senescent (C) BRI rats are shown in Fig. 3. During control, SND autospectra in each rat contained primary peaks at the frequency of HR (consistent with the presence of cardiac-related bursts) with secondary peaks located in the 0- to 3-Hz frequency band. During heating at 41°C, the primary peaks in the SND autospectra in the young rats were shifted to 1.8 Hz (consistent with the presence of low-frequency discharge bursts) (Fig. 3A), whereas they remained at the cardiac frequency in the mature (Fig. 3B) and senescent (Fig. 3C) rats. Group results (renal and splanchnic SND combined) demonstrated that in young rats hyperthermia increased the relative power in the 0- to 3-Hz frequency band and reduced the relative power at the cardiac frequency, whereas heating did not significantly change the SND frequency profile in mature and senescent rats (Table 1).

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Renal and splanchnic (Spl) SND autospectra constructed during control (38°C; left) and after increasing internal Tc to 41.0°C (right) in young (A), mature (B), and senescent (C) F344 rats. Amplitudes of autospectra are autoscaled to the highest peak. Frequency band is displayed from 0 to 15 Hz.

Figure 4 summarizes changes from control (38°C) for MAP, HR, renal SND, and splanchnic SND during increased Tc in BRI and SAD young (left), mature (middle), and senescent (right) F344 rats. Peak increases in MAP, HR, renal SND, and splanchnic SND occurring in the 40.5-41°C temperature range were used for the final data point. Each experimental variable was significantly increased from control during heating in BRI and SAD young and mature rats. SAD young rats had significantly higher MAP, HR, renal SND, and splanchnic SND responses to heating than did BRI young rats. SAD mature rats had significantly higher MAP, HR, and splanchnic SND (not renal SND) responses to heating than did BRI mature rats. HR was significantly increased from control during heating in BRI and SAD senescent rats, and splanchnic SND was increased from control during heating in SAD senescent rats. In contrast, MAP and renal SND were unchanged from control during heating in BRI and SAD senescent rats, and splanchnic SND was unchanged from control during increased Tc in BRI senescent rats. MAP, renal SND, and splanchnic SND responses to heating did not differ between BRI and SAD senescent rats, whereas HR responses were significantly higher in SAD compared with BRI senescent rats.

View larger version (28K): [in this window] [in a new window]   Fig. 4.   MAP (A), HR (B), renal SND (C), and splanchnic SND (D) responses during progressive increases (39, 40, 40.5-41.0°C) in internal Tc in baroreceptor-innervated (Intact) and sinoaortic-denervated (SAD) young (left), mature (middle), and senescent (right) F344 rats. Values at 38°C were considered as control. * Significantly different from 38°C. Significantly different from intact rats.

Figure 5 summarizes changes from control (38°C) for MAP (A), HR (B), renal SND (C), and splanchnic SND (D) during increases in Tc (39 to 41°C) in SAD young, mature, and senescent F344 rats. MAP, renal SND, and splanchnic SND responses to heating were significantly higher in SAD young and mature F344 rats compared with SAD senescent F344 rats. At 41°C, renal SND but not splanchnic SND responses were higher in SAD young compared with mature F344 rats. HR responses to heating were significantly higher in SAD young compared with SAD mature and senescent rats, whereas HR responses to increased Tc did not differ between SAD mature and senescent rats. Control levels of MAP did not differ between SAD young and senescent rats (young SAD, 99 ± 5 mmHg; senescent SAD, 88 ± 6 mmHg) or SAD young and mature rats (young SAD, 99 ± 5 mmHg; mature SAD, 111 ± 7 mmHg) but were significantly higher in SAD mature than in senescent rats (mature SAD, 111 ± 7 mmHg; senescent SAD, 88 ± 6 mmHg; P < 0.05). Control levels of HR were significantly (P < 0.05) higher in SAD young and mature rats compared with SAD senescent rats (young SAD, 428 ± 6 beats/min; mature SAD, 419 ± 10 beats/min; senescent SAD, 342 ± 9 beats/min).

View larger version (30K): [in this window] [in a new window]   Fig. 5.   MAP (A), HR (B), renal SND (C), and splanchnic SND (D) responses during progressive increases (39, 40, 40.5-41°C) in internal Tc in SAD young, mature, and senescent F344 rats. Values at 38°C were considered as control. Significantly different from senescent rats. Significantly different from mature rats.

	DISCUSSION

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We present four new findings concerning sympathetic nerve regulation to acute heating in chloralose-anesthetized young, mature, and senescent F344 rats. First, unlike BRI young F344 rats that demonstrate progressive hyperthermia-induced increases in renal and splanchnic SND, the level of activity in these nerves remained unchanged during heating in BRI senescent F344 rats. Second, in BRI rats, renal SND responses to heating in mature F344 rats were higher than those in senescent rats, but they were less than those in young rats. Third, hyperthermia changed the cardiac-related discharge pattern of renal and splanchnic SND bursts in BRI young, but not in BRI mature and senescent, F344 rats. Fourth, MAP and SND (renal and splanchnic) responses to heating did not differ between BRI and SAD senescent rats, whereas SND responses to heating were significantly higher in SAD compared with BRI young F344 rats. These results support the hypothesis that aging alters sympathetic nerve regulation to heat stress.

What factors may be responsible for the diminished renal and splanchnic SND responses to heat stress in senescent rats? Four ideas are considered, each central to SND regulation. First, the basal level of SND in chloralose-anesthetized senescent rats may represent the physiological maximum. That is, increasing the level of SND in response to acute stress may be limited by a ceiling effect. This is not the case, however, as asphyxia significantly increased renal and splanchnic SND from levels recorded at 41°C in senescent rats. Second, senescent rats may be unable to activate thermoregulatory effectors. This is unlikely because in BRI and SAD senescent rats, HR was significantly increased from control during heating, demonstrating that these animals are capable of activating selective thermoregulatory responses to increased Tc. Third, hyperthermia may sensitize the arterial baroreceptor reflex in senescent rats, which would oppose heating-induced sympathoexcitation. This is not the case, however, as renal and splanchnic SND responses to acute heating did not differ between BRI and SAD senescent rats. In contrast, SND responses to heating were significantly higher in SAD compared with BRI young and mature rats. Fourth and based on the findings that senescent rats are capable of increasing SND (asphyxia response) and activating selected thermoregulatory responses (increased HR), aging appears to alter the responsiveness of sympathetic neural circuits to acute heat stress. This is supported by the findings that SND responses to heating in senescent F344 rats are attenuated in multiple sympathetic nerves, and senescent rats do not alter the SND bursting pattern in response to increased Tc.

The current results do not address central neural mechanisms by which aging alters SND regulation to heat stress. However, our recent study may provide some clues (14). Similar to the current results (attenuated SND responses to heating in senescent compared with young F344 rats), renal SND responses to heating are significantly attenuated in heart failure compared with sham heart failure rats (14). Interestingly, after ibotenic acid-induced lesions of the paraventricular nucleus, heart failure and sham heart failure rats exhibit similar renal sympathoexcitatory responses to heating, suggesting that this hypothalamic nucleus plays a key role in suppressing renal SND responses to hyperthermia in heart failure rats (14). These results suggest that the central neural pathways regulating renal SND responses to heating are different in heart failure compared with sham heart failure rats (14). Whether this is the case in senescent compared with young rats remains to be determined.

Several discharge patterns are evident in recordings from peripheral sympathetic nerves, including cardiac- and respiratory-related oscillations (1, 4, 7, 8, 13, 33). The cardiac-related pattern in efferent sympathetic nerve outflow is considered the signature output of central sympathetic neural circuits in BRI animals (1, 7, 8). The respiratory-related pattern in efferent sympathetic nerve outflow results from the interaction of central neural circuits involved in the regulation of sympathetic and phrenic nerve discharges, providing the neural substrate for central cooperation between the sympathetic and respiratory systems (33). Transformation of the SND bursting pattern from cardiac- related to low-frequency bursts, which occurred during heating in the young F344 rats, is a consistent feature of sympathetic nerve responses to acute stress in young chloralose-anesthetized rats (4, 11, 13). In contrast to young F344 rats, the SND bursting pattern remained unchanged during heating in senescent and mature F344 rats, supporting the hypothesis that aging alters the response characteristics of sympathetic neural circuits during increased Tc. Heating-induced, low-frequency SND bursts contribute to increasing the level of activity in efferent sympathetic nerves (13) and are prominently coupled to phrenic nerve outflow (13), suggesting an enhanced cooperation between the sympathetic neural and respiratory systems during acute heat stress in young rats (33). In addition, the pattern of SND bursts, and not necessarily changes in the level of activity, may be physiologically important (6, 28, 29). For example, the pattern of electrical stimulation of sympathetic nerves influences renal function (vasoconstriction and urinary sodium excretion) (6), neurotransmitter release from sympathetic nerves (29), and contractile responses of mesenteric arteries (28).

What is the functional importance of the attenuated SND responses to heating in senescent rats? Heating-induced sympathoexcitation in young rats plays an important role in mediating physiological changes to hyperthermia (13, 19, 21). For example, celiac ganglionectomy abolishes increases in mesenteric resistance to increased Tc (19), and loss of splanchnic vasoconstriction contributes to cardiovascular changes in heat stroke (21). In addition, blockade of autonomic ganglionic transmission during heating reduces arterial pressure to values less than those produced by ganglionic blockade at control (13), suggesting that sympathetic activation is important for counteracting vasodilatory influences during hyperthermia. Relative to the current study, several lines of evidence demonstrate altered cardiovascular responses to heating in senescent humans and F344 rats. Minson et al. (24) reported reduced skin blood flow and less redistribution of blood flow from the splanchnic and renal circulations during heating in older than in young men. Renal blood flow is reduced during heating in 12- but not 24-mo-old F344 rats (32), and senescent rats demonstrate increased internal and decreased tail temperature responses to heating compared with young rats (5). The altered blood flow profiles to heating in senescent humans and rats may be mediated, at least in part, by age-related changes in SND regulation.

MAP responses to heating were significantly higher in BRI and SAD young and mature rats compared with BRI and SAD senescent rats, likely due in part to the enhanced hyperthermia-induced SND responses in young and mature rats. However, despite higher SND and HR responses to heating in young compared with mature rats, increases in MAP at 41°C were similar between BRI young and mature rats and were higher in SAD mature compared with young rats. One explanation for these findings may involve differences in hyperthermia-induced activation of adrenal SND in young and mature F344 rats. As stated previously, activation of efferent adrenal SND increases the release of epinephrine from the adrenal medulla (2, 25), and it is known that circulating epinephrine can bind to peripheral ₂-adrenergic receptors and reduce peripheral vascular resistance (16). The present results demonstrate significantly higher heating-induced increases in adrenal SND in young than in mature F344 rats, providing neural evidence for the idea that activation of adrenal SND may limit MAP responses to heating in young F344 rats.

There are at least three limitations to the present study. First, anesthesia may adversely affect SND responses to increased Tc. Although this possibility cannot be entirely discounted, the fact that heating increases SND in conscious humans (27), conscious rats (20), and young, chloralose-anesthetized rats (9, 12-15, and the current study) suggests this is not the case. Moreover, SND remains unchanged from control during heating in conscious (32) and chloralose-anesthetized (current study) senescent F344 rats, suggesting that the present findings are not the result of age-associated changes in the sensitivity to anesthesia. In addition, SND responses to increased Tc can be altered by behavioral modifications (3). Therefore, we studied SND regulation to heating in anesthetized rats to eliminate this influence. Second, differences in body weight between young and mature or senescent rats may contribute to the observed differences in SND responses to heating. This seems unlikely, however, because the rate of rise in Tc during heating did not differ between age groups. In addition, despite similar body weights, increases in MAP and renal SND to acute heating were significantly higher in mature than in senescent rats. Third, because the sympathetic nervous system is capable of generating nonuniform changes in efferent nerve outflow (26), the present findings address only adrenal, renal, and splanchnic SND responses to heating in young and mature F344 rats and renal and splanchnic SND in senescent F344 rats.