INTRODUCTION

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RECENT STUDIES demonstrate that the proinflammatory cytokine interleukin-1 (IL-1) influences sympathetic nerve discharge (SND) regulation (15, 16, 18, 22, 26, 30). For example, the intravenous administration of IL-1 increases splenic and lumbar SND in -chloralose-anesthetized rats (26) and increases splenic and adrenal SND but decreases renal SND (after a transient excitation) in urethane-anesthetized rats (22). In addition, intravenous IL-1 sensitizes interscapular brown adipose tissue SND responses to mild hypothermia as demonstrated by the finding that increases in interscapular brown adipose tissue SND to acute cold stress are significantly higher in IL-1-treated than in saline-treated rats (18). These findings support the idea that IL-1 provides an important signaling pathway to sympathetic neural circuits.

An important unresolved issue concerns what level(s) of the neuraxis is involved in mediating SND responses to peripheral IL-1. At least three possibilities can be considered. First, because intrathecal IL-1 administration increases spinal cord blood flow (24) and because cultured rat sympathetic neurons express IL-1 receptors (1, 11) and activity persists in sympathetic nerves after cervical spinal cord transection (23, 31), spinal and/or ganglionic circuits, in the absence of supraspinal neural circuits, may mediate SND responses to intravenous IL-1. Second, because the brain stem (area postrema and perivascular cells in the ventrolateral medulla) contains IL-1 receptors (9) and is known to play an important role in sympathetic nerve regulation (2, 21, 27, 28), it may be that brain stem and spinal neural circuits, in the absence of forebrain nuclei, are capable of mediating SND responses to intravenous IL-1. Third, because the interruption of ascending projections from the medulla to the forebrain reduces the activation of paraventricular nucleus (PVN) neurons following intravenous IL-1 (4, 8) and because the PVN is considered an integrative center for neuroimmunomodulation (32) and sympathetic nerve regulation (21, 29), forebrain systems, along with brain stem and spinal neural circuits, may be required for mediating SND responses to intravenous IL-1.

In the present study we examined the contributions of spinal, brain stem, and forebrain neural circuits to IL-1-induced increases in splenic and lumbar sympathetic nerve activity. SND responses to intravenous IL-1 were determined in spinal cord-transected (first cervical vertebrae, C1), midbrain-transected (superior colliculus), and sham-transected (midbrain and spinal cord), -chloralose-anesthetized rats.

	METHODS

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General procedures. The Institutional Animal Care and Use Committee approved the surgical procedures and experimental protocols used in the present study. Experiments were performed on male Sprague-Dawley rats (352 ± 8 g). Anesthesia was induced with an intraperitoneal injection of methohexital sodium (Brevital, 50-60 mg/kg) (18-20). Catheters placed in the femoral vein were used for the administration of Brevital (10-20 mg/kg during surgical procedures) and -chloralose (initial dose, 50-60 mg/kg; maintenance dose, 35 mg · kg¹ · h¹) (18-20). The trachea was cannulated with a polyethylene-240 catheter, and rats were paralyzed with gallamine triethiodide (5-10 mg/kg iv, initial dose; 10-15 mg · kg¹ · h¹, maintenance dose) and artificially ventilated (18-20). Femoral arterial pressure and heart rate (HR) were recorded using standard procedures. Colonic temperature was measured with a thermistor probe inserted ~5 cm into the colon and was kept at 38.0°C during the surgery and experimental procedures by a temperature-controlled table. End-tidal CO₂ was kept near 4% during all surgical and experimental interventions.

Neural recordings. Activity was recorded biphasically with a platinum bipolar electrode after capacity-coupled preamplification (band pass 30-3000 Hz) from the central end of cut lumbar and splenic sympathetic nerves. The splenic and lumbar nerves were isolated following a lateral incision. Nerve-electrode preparations were covered with silicone gel. Sympathetic nerve potentials were full-wave rectified and integrated (time constant 10 ms), quantified as volts times seconds (V · s), and corrected for background noise after nerve crush or administration of the ganglionic blocker trimethaphan camsylate (10-15 mg/kg iv) (18-20).

Midbrain transection. Rats were placed in a stereotaxic apparatus, and a small portion of the skull was removed. Performing sequential left and right hemisections through the rostral portion of the superior colliculus completed the midbrain transection (20). The level of transection was verified by visual examination of the brain stem and by evaluation of sagittal sections (40 µm thickness) stained with cresyl violet. A similar portion of the skull was removed, but the brain remained intact for sham midbrain transections.

Cervical spinal cord transection and intrathecal catheter placement. Rats were placed in a stereotaxic apparatus, and a laminectomy was performed. The dura was removed, and a scapel blade was used to transect the spinal cord at C1 (20). Sham C1 transections involved completing a similar laminectomy without subsequent section of neural tissue. At the end of each experiment, the spinal cord was further exposed to allow visual verification of the completeness of the cord transection.

Experimental protocol. After completion of the initial surgical procedures (e.g., isolation of sympathetic nerves, removal of portions of the skull, spinal cord laminectomies, etc.), rats were allowed to stabilize for 30 min before completion of surgical transections (midbrain or spinal cord) or sham transections (midbrain or spinal cord). Preinjection control periods were initiated after the level of SND remained stable for at least 60 min following transection. This occurred within 2-3 h after transection. Levels of mean arterial pressure (MAP) and SND were obtained during preinjection control and at 5, 15, 30, 45, and 60 min after intravenous IL-1 administration (285 ng/kg) in C1- and midbrain-transected (surgical and sham) rats. Percent changes in lumbar and splenic SND in response to IL-1 were calculated from levels recorded during preinjection control. The dose of IL-1 used in this study is similar to that used in studies designed to examine central neural pathways involved in mediating IL-1-induced effects on neuroendocrine neurons (7, 8) and to document the effects of IL-1 on SND (16, 26, 30) and splenic blood flow (25).

Data analysis. Values of SND during the preinjection period were considered as control (0%). Values in this study are means ± SE. Statistical analysis was completed using repeated-measures analysis of variance with Bonferroni post hoc tests. The overall level of statistical significance was P < 0.05.

	RESULTS

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SND responses to intravenous IL-1 after C1 and sham C1 transections. Ten C1 transection and six sham C1 transection experiments were completed. Figure 1 summarizes lumbar and splenic SND responses to intravenous IL-1 in sham-transected and C1-transected rats. Whereas lumbar and splenic SND were significantly increased after IL-1 in sham-transected rats, the level of activity in these nerves did not change after IL-1 in C1-transected rats. SND responses after IL-1 were significantly higher in sham- compared with C1-transected rats at 15, 30, 45, and 60 min for lumbar SND and at 30, 45, and 60 min for splenic SND. Control levels of MAP were significantly lower in C1-transected (82 ± 7 mmHg) compared with sham-transected (104 ± 6 mmHg) rats; however, MAP remained unchanged after IL-1 in C1-transected (Control, 82 ± 7 mmHg; 60 min IL-1, 73 ± 7 mmHg) and sham-transected (Control, 104 ± 6 mmHg; 60 min IL-1, 104 ± 4 mmHg) rats.

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Lumbar and splenic sympathetic nerve discharge (SND) before (control) and for 60 min after intravenous interleukin (IL)-1 (285 ng/kg) in C1-transected and sham C1-transected rats. * Significantly different from control.  Significantly different from C1-transected rats.

View larger version (27K): [in this window] [in a new window]   Fig. 2.   A: traces of integrated lumbar and splenic SND bursts and pulsatile arterial pressure (AP) recorded before (Pre) and after (Post) C1 transection. Horizontal calibration is 500 ms. Amplifier settings were the same for individual nerves in the lumbar-splenic pair. B: splenic SND and pulsatile arterial pressure recorded before and after the intrathecal administration of DL-homocysteic acid (DLH, 10 ng). Horizontal calibration is 5 s.

SND responses to intravenous IL-1 after midbrain and sham midbrain transections. Ten midbrain transection and nine sham midbrain transection experiments were completed. Figure 3 summarizes lumbar and splenic SND responses to intravenous IL-1 in sham midbrain-transected and midbrain-transected rats. Lumbar and splenic SND were progressively and significantly increased after IL-1 in sham-transected rats, whereas the level of activity in these nerves remained unchanged after IL-1 in midbrain-transected rats. SND responses after IL-1 were significantly higher in sham compared with midbrain-transected rats at 30, 45, and 60 min for lumbar SND and at 45 and 60 min for splenic SND. Midbrain transection did not alter the pattern of lumbar and splenic SND bursts (see Fig. 4 for one representative example), demonstrating the integrity of brain stem sympathetic neural circuits after transection. Our previous study demonstrates the responsiveness of brain stem neural circuits after decerebration as acute heat stress changes the pattern of SND bursts in midbrain-transected but not C1-transected rats (20). Control levels of MAP were significantly lower in midbrain-transected (90 ± 5 mmHg) compared with sham- transected (109 ± 7 mmHg) rats. MAP remained unchanged after IL-1 in sham-transected rats (control, 109 ± 7 mmHg; 60 min IL-1, 108 ± 5 mmHg), whereas there was a slight but significant reduction in MAP after IL-1 in midbrain-transected rats (control, 90 ± 5 mmHg; 60 min IL-1, 83 ± 6 mmHg, P < 0.05).

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Lumbar and splenic SND before (control) and for 60 min after intravenous IL-1 (285 ng/kg) in midbrain-transected and sham midbrain-transected rats. * Significantly different from control.  Significantly different from midbrain-transected rats.

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Traces of integrated lumbar and splenic SND bursts and pulsatile AP recorded before (A) and after (B) midbrain transection. Horizontal calibration is 500 ms. Amplifier settings were the same for individual nerves in the lumbar-splenic pairs.

	DISCUSSION

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This study determined lumbar and splenic SND responses to intravenous IL-1 in C1- and midbrain-transected and sham-transected (midbrain and spinal cord), -chloralose-anesthetized rats. The current findings demonstrate that, in contrast to rats with an intact neuraxis, SND remained unchanged after IL-1 in C1- and midbrain-transected rats, indicating that forebrain neural connections are required for producing lumbar and splenic sympathoexcitatory responses to intravenous IL-1. These results provide new information about the organization of neural circuits responsible for mediating sympathetic nervous system-immune system interactions.

Several lines of evidence provided rationales for hypothesizing that spinal neural circuits may be capable of mediating SND responses to intravenous IL-1. First, activity persists in sympathetic nerves after acute cervical spinal transection (20, 23, 31), demonstrating that spinal neural circuits can generate efferent sympathetic nerve activity. Second, acute heat stress, which provides a potent stimulus to the sympathetic nervous system in animals with an intact neuraxis (17, 19, 20), produces renal and splenic sympathoexcitation in spinal cord-transected rats (20), demonstrating that spinal neural circuits are responsive to specific experimental interventions. Third, intrathecal IL-1 administration increases spinal cord blood flow in anesthetized rats (24). Fourth, cultured rat sympathetic neurons from superior cervical ganglia express IL-1 receptors (1, 11). The current results, however, do not support the hypothesis that solely spinal and/or ganglionic neural circuits can mediate SND responses to IL-1 because SND remained unchanged after intravenous IL-1 in C1-transected rats. This was evident despite the fact that asphyxia and intrathecal DLH increased splenic SND in C1-transected rats, demonstrating the responsiveness of spinal and/or ganglionic neural circuits after spinal cord transection.

Buller et al. (4) and Ericsson et al. (7, 8) have demonstrated that brain stem nuclei (including the ventral lateral medulla and the nucleus tractus solitarius) are important anatomic components in the neurocircuitry required for stimulation of hypothalamic neuroendocrine systems. Because the ventral lateral medulla is critically involved in maintaining resting SND and the integrity of the rostral ventral lateral medulla is required for mediating cardiovascular and SND responses to numerous stimuli (2, 27, 28), we hypothesized that, in the absence of forebrain neural structures, the brain stem and spinal cord may contain all essential components of the signaling mechanisms required to mediate SND responses to intravenous IL-1. This was not the case, however, as IL-1 increased lumbar and splenic SND in sham but not midbrain-transected rats, indicating that SND responses to intravenous IL-1 cannot occur in the absence of neural connections between the brain stem and forebrain. Importantly, the current findings do not preclude an involvement of brain stem neural circuits in SND responses to IL-1. For example, surgical interruption of ascending projections from the medulla to the hypothalamus reduces intravenous IL-1-mediated increases in c-fos immunoreactivity and corticotrophin releasing factor in the PVN (8), suggesting that activation of the PVN in response to intravenous IL-1 involves communication from brain stem to forebrain neural circuits. Whether this is the case in the present study or whether IL-1 gains access to forebrain nuclei through circumventricular organs and in turn activates efferent SND by neural projections to brain stem and/or spinal nuclei involved in SND regulation remains to be determined.

Proinflammatory cytokines, such as IL-1, engage the central nervous system, which in turn plays a role in mediating the diverse physiological responses of the acute-phase reaction (5, 6). It is reasonable to expect that an intact neuraxis with functional neural connections among forebrain, brain stem, and spinal neural circuits would be essential for mediating SND responses to IL-1 because of the diverse target organ responses produced by immune activation. This is not the case, however, for all experimental stressors that produce diverse SND and target organ responses. For example, the brain stem contains the essential neural circuitry required for mediating heating-induced changes in SND frequency components and total power (20), despite the fact that the preoptic area of the anterior hypothalamus is considered an important thermointegrative center of the brain (3, 12-14). The current results do not address which forebrain areas are essential for mediating splenic and lumbar sympathoexcitatory responses to systemic IL-; however, Ericcson et al. (8) reported dose-dependent induction of c-fos mRNA expression after intravenous IL-1 in cells in the PVN of the hypothalamus and in several extrahypothalamic nuclei, including the central nucleus of the amygdala and the bed nucleus of the stria terminalis. Importantly, Fos protein was detected after IL-1 administration in autonomic-related parts of the parvocellular division of the PVN (8).

Despite significant increases in lumbar and splenic SND, MAP remained unchanged after IL-1 administration in sham midbrain-transected and sham C1-transected rats. In our previous study (26) we observed that intravenous IL-1 administration in -chloralose-anestheized rats increased splenic and lumbar SND (similar to the current results) but did not change the level of renal and interscalpular brown adipose tissue SND (or MAP), demonstrating that peripheral administration of this cytokine can produce nonuniform SND responses. Similarily, Niijima et al. (22) reported nonuniform SND responses and decreased arterial pressure after intravenous IL-1 in urethane-anesthetized rats. We speculate that nonuniform IL-1-mediated changes in the level of activity in sympathetic nerves innervating different target organs may be one reason that MAP remained unchanged after intravenous IL-1 administration in the current study.