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Cutaneous sympathetic motor rhythms during a fever-like response induced by prostaglandin E1
PII: S 0 3 0 6 - 4 5 2 2 ( 0 1 ) 0 0 5 7 2 - 3
Neuroscience Vol. 110, No. 2, pp. 351^360, 2002 ß 2002 IBRO. Published by Elsevier Science Ltd All rights reserved. Printed in Great Britain 0306-4522 / 02 $22.00+0.00
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CUTANEOUS SYMPATHETIC MOTOR RHYTHMS DURING A FEVER-LIKE RESPONSE INDUCED BY PROSTAGLANDIN E1 D. R. COLLINS, A. KORSAK and M. P. GILBEY* Department of Physiology, Royal Free and University College Medical School, University College London, Royal Free Campus, London NW3 2PF, UK
AbstractöNeuronal population discharges within the CNS and in somatic and sympathetic motor nerves often display oscillations. Peripheral oscillations may provide a window into central mechanisms, as they often show coherence with population activity of subsets of central neurones. The reduction in heat loss through the cutaneous circulation during fever may be mediated via sympathetic premotor neurones not utilised during normal temperature regulation. Consequently, here we assessed, in anaesthetised rats, whether the frequency signature of population sympathetic discharge observed in neurones innervating the tail (thermoregulatory) circulation changed during a fever-like response induced by intracerebroventricular injection of prostaglandin E1 . We found that when core temperature was raised to 38.8^40.5³C sympathetic activity was abolished. Following administration of prostaglandin (400 ng or 1 Wg per rat), activity was restored to levels seen prior to heating (154 þ 53.5%; n = 10). Injection of vehicle had no e¡ect (n = 7). Prior to heating when most animals were in central apnoea (14/18) two peaks were observed in autospectra of sympathetic activity: one at 0.68^0.93 Hz (T-peak) and another at the frequency of ventilation (2 Hz). Central respiratory drive was recruited during hyperthermia where it was 1:2 locked to the frequency of ventilation and following prostaglandin administration, an additional peak in sympathetic autospectra was seen at this frequency. Time-evolving spectra indicated that this peak resulted from the dynamic locking of the `T-peak' to central respiratory drive. Our data show that during a fever-like response the dominant oscillations in sympathetic activity controlling a thermoregulatory circulation and their dynamic coupling to respiratory-related inputs are similar to those seen under normal conditions. Therefore, during this fever-like response, the neural substrate(s) underlying the oscillations is not recon¢gured and remains capable of sculpturing the pattern of sympathetic neuronal discharge that may be regulated by several descending pathways. ß 2002 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: oscillations, dynamics, synchrony, respiratory, rat, hyperthermia.
ronal assemblies controlling motor output. It has been suggested that synchronisation of discharges could be important in recruitment of motor and antecedent neurones, and therefore the grading of target organ response, and/or re£ect a mechanism for integration and `binding' (Farmer, 1998; Baker et al., 1999; Singer, 1999; McAuley and Marsden, 2000; Gilbey, 2001; Varela et al., 2001). In this study we focus upon the frequency characteristics of sympathetic activity to a thermoregulatory circulation (rat tail) during a fever-like response. The observations of Horn et al. (1994) introduced the idea that fever may recruit central pathways not used in normal temperature regulation. In their experiments, and more recently in the study by Lu et al. (2001), lesions of the hypothalamic paraventricular nucleus (PVN) were observed to reduce a fever-like response induced by injection of a pyrogen, but had no e¡ect on normal body temperature regulation. Consistent with this concept are the following observations. First, there is evidence from physiological studies that sympathetic premotor neurones located in caudal raphe nuclei (pallidus and magnus) and parapyramidal region of the medulla provide a principal excitatory drive to vasoconstrictor neurones regulating cutaneous circulations (Rathner and
Oscillations are a common feature of collective neuronal discharges both throughout the CNS and in somatic and sympathetic motor nerves (e.g., Adrian and Moruzzi, 1939; Green and He¡ron, 1967; Cohen and Gootman, 1970; Malcolm et al., 1970; Gebber, 1990; Lopez da Silva, 1991; Vertes and Kocsis, 1997; Farmer, 1998; Baker et al., 1999; Kocsis et al., 1999; Morrison, 1999; Gilbey, 2001). As central and peripheral oscillations are frequently found to be coherent (Gebber et al., 1990; Baker et al., 1999) and are condition-sensitive (Mayer, 1876; Green and He¡ron, 1967; Polosa et al., 1969; Ninomiya et al., 1989; Gilbey and Spyer, 1996; Welsh and Llina¨s, 1997; Baker et al., 1999; McAuley et al., 1999; McAuley and Marsden, 2000), peripheral oscillations may provide insights into the organisation of neu-
*Corresponding author. Tel. : +44-20-7830-2770; fax: +44-20-78302083. E-mail address: [email protected] (M. P. Gilbey). Abbreviations : BP, arterial blood pressure; DCN, dorsal collector nerve; LPS, lipopolysaccharide; P, partial pressure ; PGE, prostaglandin E; PVN, hypothalamic paraventricular nucleus; RVLM, rostral ventrolateral medulla ; Tc, core temperature; TP, tracheal pressure. 351
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McAllen, 1999; Blessing and Nalivaiko, 2000, 2001). However, following injections of pseudorabies virus into the wall of the caudal ventral artery of the rat tail, Smith et al. (1998) observed putative sympathetic premotor neurones in additional areas of the brain: e.g., in the rostral ventrolateral medulla (RVLM), the A5 cell group, the lateral hypothalamus and PVN. Second, the majority of putative sympathetic premotor neurones demonstrating c-Fos protein activation following lipopolysaccharide (LPS)-induced fever were in the dorsal parvicellular region of the PVN, and RVLM (Zhang et al., 2000). Studies from this laboratory have used a rat tail model of sympathetic control of thermoregulatory circulation. We have demonstrated that during normothermia, population sympathetic activity and single unit activity controlling the tail circulation shows robust rhythmicity in the 0.4^1.2-Hz range (T-rhythm: Gilbey, 2001). We have suggested that a family of centrally located oscillators underlie this rhythmicity and that these can be synchronised dynamically by a range of inputs, including central respiratory drive and lung in£ation cycle related (Johnson and Gilbey, 1998; Chang et al., 1999, 2000; Staras et al., 2001). We hypothesised that if during prostaglandin E (PGE)-evoked fever-like response sympathetic premotor neurones not utilised during normal temperature regulation are recruited and/or neural networks recon¢gured (Marder et al., 1999) these changes might be re£ected in a disruption of the T-rhythm or its dynamics (i.e., peripheral rhythm might provide a window on central organisation). The CNS regulates blood £ow through this circulation via the control of vasoconstrictor sympathetic activity, which during hyperthermia is withdrawn resulting in elevated blood £ow and consequent increase in heat loss (when skin temperature rises above ambient: Raman et al., 1983; O'Leary et al., 1985; Johnson and Gilbey, 1994). The elevation in body temperature (up to 4³C) associated with fever is produced mainly via a reduction in heat loss through cutaneous vasoconstriction (Saper and Breder, 1994). The E group of prostaglandins are regarded generally as important central mediators of the fever response (Stitt, 1986; Boulant, 1997; Scammell et al., 1998). Recent studies indicate that the ventromedial preoptic area is the probable site where prostaglandins activate fever-producing autonomic pathways (Scammell et al., 1996). Furthermore, LPS-induced fever appears to require the production of prostaglandins in the antero-
ventral region of the preoptic area (Scammell et al., 1998) and increased concentrations of prostaglandins have been shown to be present in cerebrospinal £uid during fever produced by administration of leucocytic pyrogen (Coceani et al., 1983). Consequently, the response that follows i.c.v. injection of PGE has become a popular fever model in both anaesthetised and unanaesthetised animals (e.g., Siren, 1982; Malkinson et al., 1988; Saigusa and Iriki, 1988; Komaromi et al., 1994; Monda et al., 1998). Therefore, we examined the e¡ects of i.c.v. injection of PGE1 on cutaneous vasoconstrictor drive. Speci¢cally, we investigated whether the withdrawal of sympathetic activity to the rat tail seen during hyperthermia would be reversed, and tested the hypothesis that the dominant rhythm present during `normal' body temperature regulation would be disturbed.
EXPERIMENTAL PROCEDURES
General preparation and maintenance Experiments were performed on 18 male Sprague^Dawley rats (250^310 g, bred in-house at Royal Free Campus) and carried out in accordance with the UK Animals (Scienti¢c Procedures) Act 1986 and associated guidelines. Experiments were conducted under terminal anaesthesia. Anaesthesia was induced with 4% and initially maintained on 1.5^2.0% halothane. A femoral vein and artery were cannulated to allow i.v. administration of drugs and monitoring of arterial blood pressure (BP), respectively. Anaesthesia was then switched to urethane (1.2^1.5 g/kg i.v., 25% solution). Depth of anaesthesia was assessed using £exor and corneal re£exes, and the stability of BP. Supplementary doses of anaesthetic (12.5^ 25 mg, i.v.) were administered when required. The trachea was cannulated and the animal ventilated arti¢cially with O2 -enriched air. Ventilation rate was set at a frequency of 2 Hz (volume, 1.5^2 ml). Tracheal pressure (TP) was monitored as were arterial blood gases and pH and these were maintained within narrow limits throughout and between experiments (see Results, Table 1). Core temperature (Tc) was recorded using a thermocouple inserted 6 cm into the colon and temperature controlled using a heating blanket. Room temperature was within the range 23^ 26³C. The bladder was cannulated to allow free £ow of urine. Animals were placed in a stereotaxic head frame and a guide cannula (22-gauge stainless steel tube) inserted into the right lateral cerebral ventricle (co-ordinates: 0.8 mm posterior to bregma, 1.5 mm lateral to midline and 3.5 mm ventral to cranial theca). An i.c.v. cannula was then attached to a Hamilton syringe (10 Wl) using ¢ne-bore tubing (0.4 mm inner diameter, 0.8 mm outer diameter) and back-¢lled to prevent inclusion of air bubbles. The i.c.v. cannula (27-gauge stainless steel tubing)
Table 1. Blood pressure, heart rate and blood gas measurements BP (mm Hg)
Control Heat clamp 30 min post i.c.v. Post-cooling
Heart rate (beats/min)
PCO2 (n = 16)
PO2 (n = 16)
Vehicle (n = 10)
PGE1 (n = 7)
Vehicle (n = 10)
PGE1 (n = 7)
102.7 þ 14.0 94.9 þ 16.4 90.3 þ 9.5
99.7 þ 7.0 86.3 þ 12.3 98.3 þ 15.8
360.0 þ 41.2 458.2 þ 42.8 462.9 þ 35.0
349.3 þ 32.3 442.9 þ 47.7 470.0 þ 31.0
23.8 þ 3.7 22.0 þ 3.3 22.9 þ 4.3
159.9 þ 20.8 136.9 þ 16.4 139.0 þ 11.9
95.9 þ 9.6
96.2 þ 16.3
424.5 þ 35.2
420.7 þ 28.7
21.0 þ 3.2
162.6 þ 18.8
Summary data (mean þ S.D.) showing e¡ects of the experimental protocol on various parameters measured.
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Fig. 1. Typical traces recorded during experimental protocol. Standard recordings of TP used to monitor ventilation cycle and central respiratory drive), BP and population DCN activity (recti¢ed and smoothed using a 1-s (upper trace) and 100-ms (lower trace) time constant), from one experimental run taken during the control period (a) and during heat clamp, 30 min post i.c.v. PGE1 injection (b). Power spectrum of TP (c) and DCN activity (d) during control and 30 min post i.c.v. injection (dotted line), note the emergence of the central respiratory drive related component in the TP power spectrum (8), which re£ects the £uctuation shown in the raw trace in b (8). Fluctuating TP (in alternate cycles) indicates the frequency of central respiratory drive (1 Hz). Lowest peak in TP indicates central inspiration and highest peak central expiration.
was then inserted through the guide cannula and positioned to terminate close to the end of the guide cannula tip.
PGE1 (Sigma, St. Louis, MO, USA) was stored as a stock solution of 1 mg/ml in 100% alcohol. Aliquots of the stock were diluted with arti¢cial cerebrospinal £uid to a ¢nal concentration of 400 ng or 1 Wg in 5 Wl and warmed to 40³C prior to i.c.v. injection. Aliquots of 100% alcohol were also prepared and mixed with the stock aliquots. All aliquots were coded for a blind experimental design.
ther 30^40 min prior to cooling during which 500-s data sections were recorded on computer for primary on-line analysis. Post heat clamp levels of sympathetic activity were established following cooling. At the end of an experimental run ganglionic transmission was blocked (i.v. trimetaphan camsylate (Roche, Welwyn Garden City, UK, dose: 50 mg/kg) or hexamethonium bromide (Sigma, dose: 6 mg/kg)) to determine `0-level' for sympathetic activity. Animals were then killed with an overdose of sodium pentobarbital (i.v.) and background electrical noise recorded. 5 Wl of saline containing Pontamine Sky Blue was then injected i.c.v., the brain removed and ¢xed in formaldehyde for later macroscopic con¢rmation of i.c.v. cannula placement.
Sympathetic nerve recording
Data handling and analysis
Population sympathetic activity was recorded from either left or right dorsal collector nerves (DCNs) following transection of the cauda equina to remove somatic motor e¡erents (see Smith et al., 1998). A DCN was exposed, desheathed and the caudal end crushed, and activity recorded using a pair of implanted silver wire electrodes embedded in dental impression material (low viscosity polyvinylsiloxane; Carlisle Laboratories, Rockville Centre, NY, USA). Activity was ampli¢ed (400 K), ¢ltered (band-pass 80 Hz^1 kHz), then recti¢ed and smoothed using an integrator with a time constant of: (a) 100 ms to assess frequency components of activity (0.1^5 Hz; see Chang et al., 1999); (b) 1 s to assess activity levels (see Fig. 1a, b).
Nerve activity, TP and BP were recorded on tape for o¡-line analysis (for details see Chang et al., 1999). Tc was recorded manually to within one decimal place. During each trial 500-s sections of data were acquired on computer for preliminary on-line and o¡-line analysis (sampling rate 100 Hz). Mean BP (diastolic+1/3(systolic minus diastolic)) and heart rate (beats/min) were calculated. Ventilation cycle and central respiratory drive were monitored using TP (see Fig. 1a, b) and their frequency obtained from the power spectrum of TP (Fig. 1c). Central respiratory drive during heat clamp was locked 1:2 with the 2 Hz frequency of the ventilation cycle (see Petrillo et al., 1983). Analysis was performed using Spike2, MatLab and Igor Pro PPC programs running on IBM-compatible computers. All levels of DCN activity were normalised to the level of activity during control (100%, see below). Zero level was taken as that seen following ganglionic blockade (0%). The level following ganglionic blockade was equivalent to that seen following death. Latency of post i.c.v. injection activity onset was calculated from the point of i.c.v. application to the point where a plateau level of activity was reached. The spectral analysis method used to assess rhythmical com-
Drug preparation
Experimental protocol Animals were held at a control temperature of 35.9^36.2³C and nerve activity allowed to stabilise. Following comparable 500-s data sections under control conditions (taken 30 min apart), animals were heated to 40^41³C. The time and Tc at which the nadir of activity occurred were noted. Once the nadir had been established, Tc was held (heat clamp) and stabilised prior to i.c.v. injection. Injections were made over 15^20 s. Following i.c.v. injection the temperature was held for a fur-
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ponents of nerve activity and TP has been described in previous publications (Chang et al., 1999, 2000; Smith and Gilbey, 2000). In brief, a 400-s data set (sampled at 100 Hz) was divided into 50% overlapped subsections and the linear trend removed and windowed by a Hanning taper. The Welch method was used to generate an average autospectrum from these subsections (size of fast Fourier transform, 2048). The frequency resolution of spectra was 0.049 Hz (subsection length, 20.48 s). The autospectrum, plotted as power density against harmonic frequencies, was only displayed between 0 and 5 Hz because the power at frequencies above this level was negligible (see Fig. 1a, b, d). Temporal changes in DCN activity were assessed by generating time-evolving autospectra. Spectral analysis was performed on 20.48-s segments (2048 points, 100 Hz sampling rate) sliding by 50%. Coherence spectra were used to reveal linear correlation between sympathetic activity and TP (see Smith and Gilbey, 2000). Coherence spectra were averaged from the same data sets used to generate autospectra. We used a 95% con¢dence level threshold value of 0.08 for non-zero coherence (Rosenberg et al., 1989; Smith and Gilbey, 2000). All values are expressed as mean þ S.D. Statistical analysis was performed using Student's paired and unpaired t-tests and P 6 0.05 considered signi¢cant.
RESULTS
Of the 18 animals, seven received i.c.v. injections of vehicle only and 11 PGE1 . Of the latter, six received 400 ng and ¢ve 1 Wg (see Malkinson et al., 1988). As no signi¢cant di¡erences were observed between the e¡ects of the two doses (see below) the data are considered together. One animal in the PGE1 group responded in an atypical manner (see below) and therefore is not included in the pooled PGE1 group nerve activity data
set. Examples of vehicle and PGE1 group DCN activity levels during experimental runs are shown in Fig. 2. Level of sympathetic drive during experimental protocol As described in Experimental procedures, 100% activity level was determined when activity had stabilised such that two comparable 500-s data sections taken 30 min apart were obtained under control conditions. Whole body warming was observed to decrease sympathetic activity (n = 18; see Fig. 2) as reported previously (Johnson and Gilbey, 1996; Ha«bler et al., 2000). DCN activity began to decline at Tc of 36.4^38.1³C (vehicle, 37.4 þ 0.6³C (n = 7); PGE1 , 37.5 þ 0.7³C (n = 11)). Nadirs of activity were seen at Tc of 38.6^ 40.5³C (vehicle, 39.8 þ 0.2³C (n = 7); PGE1 , 39.6 þ 0.6³C (n = 11)). Dorsal collector nerve activity during heat clamp prior to i.c.v. injection. During heat clamp (Tc 40^41³C), before i.c.v. injection, normalised activity was 7.6 þ 11.0% of control in the vehicle group (n = 7) and 18.2 þ 30.1% of control in the PGE1 group (n = 10, no signi¢cant di¡erence; see Fig. 2). For both groups these levels of activity were signi¢cantly lower than control (P 6 0.001, Student's paired t-test) but comparable to those seen following ganglionic blockade and following death. E¡ect of i.c.v. prostaglandin E1 on dorsal collector nerve activity during heat-clamp. In all but one animal, during heat clamp, 30 min following i.c.v. injection of PGE1 ,
Fig. 2. Recti¢ed and smoothed (time constant, 1 s) neurogram of population sympathetic activity levels during time course of the experimental protocol. Normalised activity levels showing the e¡ect of i.c.v. injection of vehicle (a) or PGE1 (b). Upper traces in a and b show Tc of the animal throughout the duration of the trial. Asterisks mark the point of ganglion blocker administration (i.v.).
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Fig. 3. Power spectra and DCN^TP coherence during vehicle trials. Autospectra of DCN activity (1a^d), TP (2a^d) and graphs showing their coherence (3a^d, dotted lines mark level where coherence becomes signi¢cant) ; taken during control (a), during heat clamp (b), 30 min post i.c.v. injection (c) and following removal of heat clamp (d). See Experimental procedures for details of analysis. For DCN activity, the control autospectrum has been superimposed on b, c and d (dotted line) to show the changes in power. Note that during heat clamp additional peaks emerge in the TP autospectra, indicative of emerging central respiratory drive and that DCN power is minimal during this period. DCN and TP signals are signi¢cantly coherent at ventilation frequency during control (a) and following removal of heat clamp (d). S indicates the peak at the frequency of T-rhythm, F indicates the peak at the frequency of ventilation and 8 indicates the peak at the frequency of central respiratory drive.
sympathetic activity increased signi¢cantly (from levels prior to i.c.v. PGE1 ) to 154.1 þ 53.3% of control (P 6 0.001, n = 10; see Fig. 2). Such increases in activity had a latency of 913 þ 307 s. Activity of the `non-responder' in this group exhibited a slow return to control levels, outside the 900^1200 s observed in the rest of the PGE1 group. In contrast, following i.c.v. injection of vehicle, activity (19.3 þ 18.8%) did not change signi¢cantly (n = 7, see Fig. 2). A di¡erence in time course of increase in activity was the only observed di¡erence between the two doses of PGE1 . Whereas injection of 1 Wg PGE1 evoked a rapid increase in activity that was sustained for the duration of the heat clamp, the increase in activity that followed 400-ng doses exhibited a slight reduction 30^40 min post injection. Dorsal collector nerve activity post heat clamp. Following termination of the heat clamp (and cooling of Tc to 36.5^38³C) activity was 176.1 þ 31.8% in the vehicle group and 211.4 þ 76.2% in the PGE1 group: these values are not signi¢cantly di¡erent (see Fig. 2). Blood pressures during experimental protocol Coincident with the observed increase in DCN activity during heat clamp, following injection of PGE1 , BP increased compared to pre-injection levels. In contrast,
following injection of vehicle there was a tendency for BP to decrease compared to pre-injection levels (see Table 1). Heart rate changes during experimental protocol There was a tendency for heart rate to increase in both groups when Tc was raised (see Table 1). However, mean post-injection heart rates in vehicle and control groups were similar. Central respiratory drive and blood gases during experimental protocol During the control period in 14 of 18 cases animals were in central apnoea due to hypocapnia and mild hyperoxia (see Table 1 for arterial partial pressure (P) CO2 and arterial PO2 measurements). In four cases, central respiratory drive was present but weak, as indicated by £uctuations in TP. Animals were hyperventilated in order to ensure that central respiratory drive did not become excessive during hyperthermia and to enable the in£uence of recruitment of central respiratory drive to be examined on sympathetic rhythm (see below). In all 18 animals central respiratory drive was present during heat clamp. This was seen as a 1-Hz £uctuation in the tracheal pressure trace (Fig. 1b) as central respiratory drive was locked 1:2 with the lung in£ation cycle (see
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Fig. 4. Power spectra of DCN^TP during PGE1 trials. Autospectra of DCN, TP and coherence as in Fig. 3. Note the emergence of central respiratory drive in TP autospectra (2b and 2c) as in Fig. 3 and the return of DCN activity 30 min post i.c.v. injection (1c), locked to the central respiratory drive frequency. DCN and TP signals show signi¢cant coherence at ventilation frequency (1a, c and d) and at central respiratory drive 30 min post i.c.v. injection (1c). S indicates the peak at the frequency of T-rhythm, F indicates the peak at the frequency of lung in£ation cycle and 8 indicates the peak at the frequency of central respiratory drive.
Petrillo et al., 1983) and was revealed as a 1-Hz peak in the TP power spectrum (Figs. 1c, 32b; c and 42b; c ). No signi¢cant di¡erences were observed in arterial PCO2 or arterial PO2 throughout the experimental protocol (Table 1, n = 16). Rhythmic characteristics of sympathetic activity Control conditions. As reported previously (Chang et al., 1999, 2000; Smith and Gilbey, 2000), during central apnoea (n = 14) and in the presence of weak central respiratory drive (n = 4), autospectra of sympathetic activity exhibited a peak in the T-rhythm frequency range (Figs. 31a , 41a and 5a; T-peak, 0.68^0.93 Hz, n = 18). An additional peak at 2 Hz highly coherent with ventilation cycle frequency was also observed (see Chang et al., 2000) in 15/18 cases (Figs. 31a , 41a and 5a). There was signi¢cant coherence between DCN activity and TP in the 2.05^2.14 Hz range (Figs. 33a and 43a : coherence values were 0.72 þ 0.11 and 0.72 þ 0.16 in PGE1 (n = 10) and vehicle (n = 7) groups, respectively). Heat clamp prior to i.c.v. injection. No peaks were present in autospectra (Figs. 31b , 41b and 5b) and total power was signi¢cantly reduced compared to control (in the PGE1 group total power dropped to 33.21 þ 14.24% of control, in the vehicle group power
decreased to 34.74 þ 19.49% of control; P 6 0.001 for both groups). Heat clamp post i.c.v. prostaglandin E1 . Thirty minutes post i.c.v. injection of PGE1 sympathetic activity had returned; average total power in the PGE1 group was 216.17 þ 107.16% of control (n = 10; P 6 0.005). As seen under control conditions, a peak was present at 2 Hz that was highly coherent with ventilation cycle frequency (coherence values were 0.85 þ 0.07). An additional peak was also present that showed high coherence with the frequency of central respiratory drive (Fig. 41c : range 0.93^1.17 Hz, coherence values were 0.71 þ 0.26). Timeevolving spectra revealed in all cases dynamic locking of the T-rhythm to the frequency of central respiratory drive (Fig. 5c). In contrast, as activity did not recover in the vehicle group during heat clamp 30 min post i.c.v. injection there were no peaks in autospectra (Fig. 31c ) and average total power remained low (42.61 þ 32.85% of control). Post heat clamp. On cooling, power in autospectra of both vehicle and PGE1 groups exceeded control levels (see Figs. 31d and 41d ). Total power for the PGE1 group was 210.57 þ 107.01% of control (P 6 0.05) and for the vehicle group total power was 186.23 þ 51.94% of control (P 6 0.01). Coherence spectra for both groups were com-
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Fig. 5. Dynamic properties of DCN activity during experimental protocol. Upper traces show core temperature recorded throughout the protocol. The middle graph shows a composite image of autospectra from one experimental run (composed of autospectra performed on segments of 2048 points, sliding by 50%). The vertical line indicates the point of i.c.v. PGE1 injection, the asterisk represents the point of ganglion blocker administration. The grey-scale has been maximised to 10 nV2 to enable visualisation of spectral peaks. Examples of power spectra taken from the bracketed points marked on the timeevolving spectrum image (a^d), using a 400-s sample (see Experimental procedures), showing the alterations in power and shift in dominant frequencies (note increased scale in c). Note that during control the power £uctuates around the T-rhythm frequency (0.68 Hz, at arrowhead marked S, (a)) and there is activity locked to the frequency of the ventilation cycle (2 Hz, at arrowhead marked F). During heating the activity decreases (b) and following injection of PGE1 activity initially returns and locks to the frequency of central respiratory drive (1 Hz, at arrowhead marked 8, (c)), activity then £uctuates between T-rhythm and central respiratory drive frequencies (d). S indicates the peak at the frequency of T-rhythm, F indicates the peak at the frequency of lung in£ation cycle and 8 indicates the peak at the frequency of central respiratory drive.
parable to those under control conditions and revealed signi¢cant coherence in the 2-Hz ventilation cycle frequency range (Figs. 33d and 43d ; coherence was 0.75 þ 0.11 for the PGE1 group and 0.78 þ 0.12 for the vehicle group). Furthermore, in the majority of cases (14 out of 17), the central respiratory drive-related peak in the 1 Hz range was no longer present (Figs. 3d, 4d and 5d).
DISCUSSION
In this study we have demonstrated that hyperthermia can cause an extinction of population sympathetic activity recorded from neurones innervating the tail circulation. This con¢rms and extends previous ¢ndings from studies using single- and multi-unit recording techniques (Johnson and Gilbey, 1996; Ha«bler et al., 2000). Novel observations were made with regard to sympathetic activity following i.c.v. PGE1 (400 ng or 1 Wg) administered during heat clamp. First, sympathetic activity returned to or exceeded pre-heat clamp levels. Second, the characteristics of the T-rhythm component of this activity were unchanged from those seen during `normal' body temperature regulation. Thus, the neural substrate generating the T-rhythm is not recon¢gured during the fever-like response to PGE1 .
I.c.v. prostaglandin E1 -induced fever-like syndrome The PGE1 -induced fever-like response appears to share mechanisms common to that produced by exogenous pyrogens. Both can be attenuated by pregnancy (Stobie-Hayes and Fewell, 1996; Eliason and Fewell, 1997) and cortical spreading depression (Monda and Pittman, 1993). Malkinson et al. (1988) ¢rst reported that a fever-like response followed i.c.v. administration of PGE1 (100 ng, 400 ng or 1 Wg) in urethane-anaesthetised rats. Their results are similar to those of Siren (1982) using PGE2 . Increases in the ¢ring rate of nerves innervating intercapsular brown adipose tissue, oxygen consumption and heart rate, and decreases in skin blood £ow were observed (e.g., Siren, 1982; Malkinson et al., 1988; Komaromi et al., 1994; Monda et al., 1995, 1998, 1999). I.c.v. prostaglandin E1 and tail sympathetic activity during hyperthermia In our experiments we examined the e¡ect of i.c.v. PGE1 on tail sympathetic activity during hyperthermia and observed that i.c.v. PGE1 was followed by a restoration of activity. Making the animals hyperthermic before the e¡ects of i.c.v. PGE1 were examined allowed
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us to, ¢rstly, con¢rm that hyperthermia induced extinction of tail sympathetic activity in our anaesthetised animal model and, secondly, determine the Tc at which it occurred. The protocol that we followed does have the potential disadvantage that if hyperthermia precipitates hypotension it can cause activation of sympathetic out£ow to the tail circulation (authors' unpublished observations). However, this did not occur and post vehicle i.c.v., sympathetic activity was not restored. Furthermore, arterial blood gases were kept within a narrow range (mildly hyperoxic and hypocapnic). I.c.v. prostaglandin E1 and rhythmic activity During the control period 14 of 18 animals were in central apnoea. Animals were put in this respiratory state so that a clear T-peak could be observed in sympathetic autospectra (see Chang et al., 1999, 2000; Gilbey, 2001). Consistent with the observation that hyperthermia enhances central respiratory drive through an increase in CO2 sensitivity (Maskrey, 1990), during heat clamp central respiratory drive was recruited although PO2 and PCO2 were changed little from control. As observed in normothermia by Chang et al. (1999), we found that following the return of sympathetic activity post i.c.v. PGE1 , the T-rhythm was entrained dynamically 1:1 with central respiratory drive (with high coherence). Therefore, following i.c.v. PGE1 , we did not detect any robust changes in the rhythmic components of tail sympathetic activity and their dynamic synchronisation by periodic inputs (compared with those seen during `normal' temperature regulation). Our results might appear incompatible with those of Kenney et al. (1998). They remarked that ``enhanced respiratory modulation of sympathetic nerve discharge bursts [that they observed to non-cutaneous targets] during heating did not result from changes in respiratory drive, because end-tidal CO2 levels remained constant during elevations in core body temperature''. Consequently, they reasoned that hyperthermia enhanced the coupling between phrenic nerve discharge and sympathetic nerve discharge. Their conclusions appear unjusti¢ed when the in£uence of hyperthermia on CO2 sensitivity (Maskrey, 1990) is taken into account. Furthermore, data presented in their paper indicate that both phrenic nerve discharge frequency and activity in the phrenic neurogram were increased when core temperature was elevated.
T-rhythm is present and is synchronised dynamically by central respiratory drive and ventilation-related inputs. Therefore, the neural substrate(s) underlying the generation of the T-rhythm are not recon¢gured during fever. Smith and Gilbey (2000) suggested that the substrate giving rise to the T-rhythm was located antecedent to sympathetic preganglionic neurones. Therefore, from our data it might be proposed that the sympathetic premotor neurones in the region of the ventromedial medulla (including the caudal raphe nuclei pallidus and obscurus), which are considered to provide a major excitatory drive to sympathetic preganglionic neurones in£uencing cutaneous (thermoregulatory) vasoconstrictor postganglionic neurones (Rathner and McAllen, 1999; Blessing and Nalivaiko, 2000, 2001), are utilised both during normal body temperature regulation and during fever. The reduction in heat loss (through cutaneous vasoconstriction) associated with fever may result solely from a change of set point in the preoptic area, reducing the drive to GABA-releasing neurones (at raised body temperature) that act to inhibit the ventromedial medullary sympathetic premotor neurones (Zhang et al., 1997; Nagashima et al., 2000). However, this view appears inconsistent with the ¢ndings that LPS-induced fever was associated with the activation of c-Fos protein expression in PVN and RVLM neurones projecting to the upper lumbar level of the spinal cord (Zhang et al., 2000) and that a pathway passing through the PVN appears to be involved in LPS and PGE fever, but not normal body temperature regulation (Horn et al., 1994; Lu et al., 2001). Such observations indicate that additional descending inputs to sympathetic preganglionic neurones are active during fever. An `additional drive' rather than purely a reduction in descending inhibitory drive is indicated further by the ¢nding that although anaesthetised animals are unable to maintain normal body temperature they can raise their core temperature following i.c.v. PGE (e.g., Malkinson et al., 1988). As the T-rhythm remained robust during the fever-like response, it would appear that the rhythmic inputs underlying the T-rhythm are either relayed over multiple descending pathways or that the mechanism underlying the T-rhythm is potent enough to sculpt the discharge patterns of sympathetic neurones receiving high levels of tonic drive (see Gilbey et al., 1986). Clearly, further studies are required to determine the functional contributions of the various descending pathways to tonic drive and oscillatory components of sympathetic neurones regulating cutaneous thermoregulatory circulations.
CONCLUSIONS
The key ¢nding from this study is that the T-rhythm characteristics of cutaneous sympathetic activity seen during normal body temperature regulation and PGE1 induced fever-like syndrome are similar, i.e. the
AcknowledgementsöA.K. and work supported by Wellcome Trust Grant 054584 and D.R.C. by Wellcome Trust Grant 05115.
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Neuroscience Vol. 110, No. 2, pp. 351^360, 2002 ß 2002 IBRO. Published by Elsevier Science Ltd All rights reserved. Printed in Great Britain 0306-4522 / 02 $22.00+0.00
www.neuroscience-ibro.com
CUTANEOUS SYMPATHETIC MOTOR RHYTHMS DURING A FEVER-LIKE RESPONSE INDUCED BY PROSTAGLANDIN E1 D. R. COLLINS, A. KORSAK and M. P. GILBEY* Department of Physiology, Royal Free and University College Medical School, University College London, Royal Free Campus, London NW3 2PF, UK
AbstractöNeuronal population discharges within the CNS and in somatic and sympathetic motor nerves often display oscillations. Peripheral oscillations may provide a window into central mechanisms, as they often show coherence with population activity of subsets of central neurones. The reduction in heat loss through the cutaneous circulation during fever may be mediated via sympathetic premotor neurones not utilised during normal temperature regulation. Consequently, here we assessed, in anaesthetised rats, whether the frequency signature of population sympathetic discharge observed in neurones innervating the tail (thermoregulatory) circulation changed during a fever-like response induced by intracerebroventricular injection of prostaglandin E1 . We found that when core temperature was raised to 38.8^40.5³C sympathetic activity was abolished. Following administration of prostaglandin (400 ng or 1 Wg per rat), activity was restored to levels seen prior to heating (154 þ 53.5%; n = 10). Injection of vehicle had no e¡ect (n = 7). Prior to heating when most animals were in central apnoea (14/18) two peaks were observed in autospectra of sympathetic activity: one at 0.68^0.93 Hz (T-peak) and another at the frequency of ventilation (2 Hz). Central respiratory drive was recruited during hyperthermia where it was 1:2 locked to the frequency of ventilation and following prostaglandin administration, an additional peak in sympathetic autospectra was seen at this frequency. Time-evolving spectra indicated that this peak resulted from the dynamic locking of the `T-peak' to central respiratory drive. Our data show that during a fever-like response the dominant oscillations in sympathetic activity controlling a thermoregulatory circulation and their dynamic coupling to respiratory-related inputs are similar to those seen under normal conditions. Therefore, during this fever-like response, the neural substrate(s) underlying the oscillations is not recon¢gured and remains capable of sculpturing the pattern of sympathetic neuronal discharge that may be regulated by several descending pathways. ß 2002 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: oscillations, dynamics, synchrony, respiratory, rat, hyperthermia.
ronal assemblies controlling motor output. It has been suggested that synchronisation of discharges could be important in recruitment of motor and antecedent neurones, and therefore the grading of target organ response, and/or re£ect a mechanism for integration and `binding' (Farmer, 1998; Baker et al., 1999; Singer, 1999; McAuley and Marsden, 2000; Gilbey, 2001; Varela et al., 2001). In this study we focus upon the frequency characteristics of sympathetic activity to a thermoregulatory circulation (rat tail) during a fever-like response. The observations of Horn et al. (1994) introduced the idea that fever may recruit central pathways not used in normal temperature regulation. In their experiments, and more recently in the study by Lu et al. (2001), lesions of the hypothalamic paraventricular nucleus (PVN) were observed to reduce a fever-like response induced by injection of a pyrogen, but had no e¡ect on normal body temperature regulation. Consistent with this concept are the following observations. First, there is evidence from physiological studies that sympathetic premotor neurones located in caudal raphe nuclei (pallidus and magnus) and parapyramidal region of the medulla provide a principal excitatory drive to vasoconstrictor neurones regulating cutaneous circulations (Rathner and
Oscillations are a common feature of collective neuronal discharges both throughout the CNS and in somatic and sympathetic motor nerves (e.g., Adrian and Moruzzi, 1939; Green and He¡ron, 1967; Cohen and Gootman, 1970; Malcolm et al., 1970; Gebber, 1990; Lopez da Silva, 1991; Vertes and Kocsis, 1997; Farmer, 1998; Baker et al., 1999; Kocsis et al., 1999; Morrison, 1999; Gilbey, 2001). As central and peripheral oscillations are frequently found to be coherent (Gebber et al., 1990; Baker et al., 1999) and are condition-sensitive (Mayer, 1876; Green and He¡ron, 1967; Polosa et al., 1969; Ninomiya et al., 1989; Gilbey and Spyer, 1996; Welsh and Llina¨s, 1997; Baker et al., 1999; McAuley et al., 1999; McAuley and Marsden, 2000), peripheral oscillations may provide insights into the organisation of neu-
*Corresponding author. Tel. : +44-20-7830-2770; fax: +44-20-78302083. E-mail address: [email protected] (M. P. Gilbey). Abbreviations : BP, arterial blood pressure; DCN, dorsal collector nerve; LPS, lipopolysaccharide; P, partial pressure ; PGE, prostaglandin E; PVN, hypothalamic paraventricular nucleus; RVLM, rostral ventrolateral medulla ; Tc, core temperature; TP, tracheal pressure. 351
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McAllen, 1999; Blessing and Nalivaiko, 2000, 2001). However, following injections of pseudorabies virus into the wall of the caudal ventral artery of the rat tail, Smith et al. (1998) observed putative sympathetic premotor neurones in additional areas of the brain: e.g., in the rostral ventrolateral medulla (RVLM), the A5 cell group, the lateral hypothalamus and PVN. Second, the majority of putative sympathetic premotor neurones demonstrating c-Fos protein activation following lipopolysaccharide (LPS)-induced fever were in the dorsal parvicellular region of the PVN, and RVLM (Zhang et al., 2000). Studies from this laboratory have used a rat tail model of sympathetic control of thermoregulatory circulation. We have demonstrated that during normothermia, population sympathetic activity and single unit activity controlling the tail circulation shows robust rhythmicity in the 0.4^1.2-Hz range (T-rhythm: Gilbey, 2001). We have suggested that a family of centrally located oscillators underlie this rhythmicity and that these can be synchronised dynamically by a range of inputs, including central respiratory drive and lung in£ation cycle related (Johnson and Gilbey, 1998; Chang et al., 1999, 2000; Staras et al., 2001). We hypothesised that if during prostaglandin E (PGE)-evoked fever-like response sympathetic premotor neurones not utilised during normal temperature regulation are recruited and/or neural networks recon¢gured (Marder et al., 1999) these changes might be re£ected in a disruption of the T-rhythm or its dynamics (i.e., peripheral rhythm might provide a window on central organisation). The CNS regulates blood £ow through this circulation via the control of vasoconstrictor sympathetic activity, which during hyperthermia is withdrawn resulting in elevated blood £ow and consequent increase in heat loss (when skin temperature rises above ambient: Raman et al., 1983; O'Leary et al., 1985; Johnson and Gilbey, 1994). The elevation in body temperature (up to 4³C) associated with fever is produced mainly via a reduction in heat loss through cutaneous vasoconstriction (Saper and Breder, 1994). The E group of prostaglandins are regarded generally as important central mediators of the fever response (Stitt, 1986; Boulant, 1997; Scammell et al., 1998). Recent studies indicate that the ventromedial preoptic area is the probable site where prostaglandins activate fever-producing autonomic pathways (Scammell et al., 1996). Furthermore, LPS-induced fever appears to require the production of prostaglandins in the antero-
ventral region of the preoptic area (Scammell et al., 1998) and increased concentrations of prostaglandins have been shown to be present in cerebrospinal £uid during fever produced by administration of leucocytic pyrogen (Coceani et al., 1983). Consequently, the response that follows i.c.v. injection of PGE has become a popular fever model in both anaesthetised and unanaesthetised animals (e.g., Siren, 1982; Malkinson et al., 1988; Saigusa and Iriki, 1988; Komaromi et al., 1994; Monda et al., 1998). Therefore, we examined the e¡ects of i.c.v. injection of PGE1 on cutaneous vasoconstrictor drive. Speci¢cally, we investigated whether the withdrawal of sympathetic activity to the rat tail seen during hyperthermia would be reversed, and tested the hypothesis that the dominant rhythm present during `normal' body temperature regulation would be disturbed.
EXPERIMENTAL PROCEDURES
General preparation and maintenance Experiments were performed on 18 male Sprague^Dawley rats (250^310 g, bred in-house at Royal Free Campus) and carried out in accordance with the UK Animals (Scienti¢c Procedures) Act 1986 and associated guidelines. Experiments were conducted under terminal anaesthesia. Anaesthesia was induced with 4% and initially maintained on 1.5^2.0% halothane. A femoral vein and artery were cannulated to allow i.v. administration of drugs and monitoring of arterial blood pressure (BP), respectively. Anaesthesia was then switched to urethane (1.2^1.5 g/kg i.v., 25% solution). Depth of anaesthesia was assessed using £exor and corneal re£exes, and the stability of BP. Supplementary doses of anaesthetic (12.5^ 25 mg, i.v.) were administered when required. The trachea was cannulated and the animal ventilated arti¢cially with O2 -enriched air. Ventilation rate was set at a frequency of 2 Hz (volume, 1.5^2 ml). Tracheal pressure (TP) was monitored as were arterial blood gases and pH and these were maintained within narrow limits throughout and between experiments (see Results, Table 1). Core temperature (Tc) was recorded using a thermocouple inserted 6 cm into the colon and temperature controlled using a heating blanket. Room temperature was within the range 23^ 26³C. The bladder was cannulated to allow free £ow of urine. Animals were placed in a stereotaxic head frame and a guide cannula (22-gauge stainless steel tube) inserted into the right lateral cerebral ventricle (co-ordinates: 0.8 mm posterior to bregma, 1.5 mm lateral to midline and 3.5 mm ventral to cranial theca). An i.c.v. cannula was then attached to a Hamilton syringe (10 Wl) using ¢ne-bore tubing (0.4 mm inner diameter, 0.8 mm outer diameter) and back-¢lled to prevent inclusion of air bubbles. The i.c.v. cannula (27-gauge stainless steel tubing)
Table 1. Blood pressure, heart rate and blood gas measurements BP (mm Hg)
Control Heat clamp 30 min post i.c.v. Post-cooling
Heart rate (beats/min)
PCO2 (n = 16)
PO2 (n = 16)
Vehicle (n = 10)
PGE1 (n = 7)
Vehicle (n = 10)
PGE1 (n = 7)
102.7 þ 14.0 94.9 þ 16.4 90.3 þ 9.5
99.7 þ 7.0 86.3 þ 12.3 98.3 þ 15.8
360.0 þ 41.2 458.2 þ 42.8 462.9 þ 35.0
349.3 þ 32.3 442.9 þ 47.7 470.0 þ 31.0
23.8 þ 3.7 22.0 þ 3.3 22.9 þ 4.3
159.9 þ 20.8 136.9 þ 16.4 139.0 þ 11.9
95.9 þ 9.6
96.2 þ 16.3
424.5 þ 35.2
420.7 þ 28.7
21.0 þ 3.2
162.6 þ 18.8
Summary data (mean þ S.D.) showing e¡ects of the experimental protocol on various parameters measured.
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Fig. 1. Typical traces recorded during experimental protocol. Standard recordings of TP used to monitor ventilation cycle and central respiratory drive), BP and population DCN activity (recti¢ed and smoothed using a 1-s (upper trace) and 100-ms (lower trace) time constant), from one experimental run taken during the control period (a) and during heat clamp, 30 min post i.c.v. PGE1 injection (b). Power spectrum of TP (c) and DCN activity (d) during control and 30 min post i.c.v. injection (dotted line), note the emergence of the central respiratory drive related component in the TP power spectrum (8), which re£ects the £uctuation shown in the raw trace in b (8). Fluctuating TP (in alternate cycles) indicates the frequency of central respiratory drive (1 Hz). Lowest peak in TP indicates central inspiration and highest peak central expiration.
was then inserted through the guide cannula and positioned to terminate close to the end of the guide cannula tip.
PGE1 (Sigma, St. Louis, MO, USA) was stored as a stock solution of 1 mg/ml in 100% alcohol. Aliquots of the stock were diluted with arti¢cial cerebrospinal £uid to a ¢nal concentration of 400 ng or 1 Wg in 5 Wl and warmed to 40³C prior to i.c.v. injection. Aliquots of 100% alcohol were also prepared and mixed with the stock aliquots. All aliquots were coded for a blind experimental design.
ther 30^40 min prior to cooling during which 500-s data sections were recorded on computer for primary on-line analysis. Post heat clamp levels of sympathetic activity were established following cooling. At the end of an experimental run ganglionic transmission was blocked (i.v. trimetaphan camsylate (Roche, Welwyn Garden City, UK, dose: 50 mg/kg) or hexamethonium bromide (Sigma, dose: 6 mg/kg)) to determine `0-level' for sympathetic activity. Animals were then killed with an overdose of sodium pentobarbital (i.v.) and background electrical noise recorded. 5 Wl of saline containing Pontamine Sky Blue was then injected i.c.v., the brain removed and ¢xed in formaldehyde for later macroscopic con¢rmation of i.c.v. cannula placement.
Sympathetic nerve recording
Data handling and analysis
Population sympathetic activity was recorded from either left or right dorsal collector nerves (DCNs) following transection of the cauda equina to remove somatic motor e¡erents (see Smith et al., 1998). A DCN was exposed, desheathed and the caudal end crushed, and activity recorded using a pair of implanted silver wire electrodes embedded in dental impression material (low viscosity polyvinylsiloxane; Carlisle Laboratories, Rockville Centre, NY, USA). Activity was ampli¢ed (400 K), ¢ltered (band-pass 80 Hz^1 kHz), then recti¢ed and smoothed using an integrator with a time constant of: (a) 100 ms to assess frequency components of activity (0.1^5 Hz; see Chang et al., 1999); (b) 1 s to assess activity levels (see Fig. 1a, b).
Nerve activity, TP and BP were recorded on tape for o¡-line analysis (for details see Chang et al., 1999). Tc was recorded manually to within one decimal place. During each trial 500-s sections of data were acquired on computer for preliminary on-line and o¡-line analysis (sampling rate 100 Hz). Mean BP (diastolic+1/3(systolic minus diastolic)) and heart rate (beats/min) were calculated. Ventilation cycle and central respiratory drive were monitored using TP (see Fig. 1a, b) and their frequency obtained from the power spectrum of TP (Fig. 1c). Central respiratory drive during heat clamp was locked 1:2 with the 2 Hz frequency of the ventilation cycle (see Petrillo et al., 1983). Analysis was performed using Spike2, MatLab and Igor Pro PPC programs running on IBM-compatible computers. All levels of DCN activity were normalised to the level of activity during control (100%, see below). Zero level was taken as that seen following ganglionic blockade (0%). The level following ganglionic blockade was equivalent to that seen following death. Latency of post i.c.v. injection activity onset was calculated from the point of i.c.v. application to the point where a plateau level of activity was reached. The spectral analysis method used to assess rhythmical com-
Drug preparation
Experimental protocol Animals were held at a control temperature of 35.9^36.2³C and nerve activity allowed to stabilise. Following comparable 500-s data sections under control conditions (taken 30 min apart), animals were heated to 40^41³C. The time and Tc at which the nadir of activity occurred were noted. Once the nadir had been established, Tc was held (heat clamp) and stabilised prior to i.c.v. injection. Injections were made over 15^20 s. Following i.c.v. injection the temperature was held for a fur-
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ponents of nerve activity and TP has been described in previous publications (Chang et al., 1999, 2000; Smith and Gilbey, 2000). In brief, a 400-s data set (sampled at 100 Hz) was divided into 50% overlapped subsections and the linear trend removed and windowed by a Hanning taper. The Welch method was used to generate an average autospectrum from these subsections (size of fast Fourier transform, 2048). The frequency resolution of spectra was 0.049 Hz (subsection length, 20.48 s). The autospectrum, plotted as power density against harmonic frequencies, was only displayed between 0 and 5 Hz because the power at frequencies above this level was negligible (see Fig. 1a, b, d). Temporal changes in DCN activity were assessed by generating time-evolving autospectra. Spectral analysis was performed on 20.48-s segments (2048 points, 100 Hz sampling rate) sliding by 50%. Coherence spectra were used to reveal linear correlation between sympathetic activity and TP (see Smith and Gilbey, 2000). Coherence spectra were averaged from the same data sets used to generate autospectra. We used a 95% con¢dence level threshold value of 0.08 for non-zero coherence (Rosenberg et al., 1989; Smith and Gilbey, 2000). All values are expressed as mean þ S.D. Statistical analysis was performed using Student's paired and unpaired t-tests and P 6 0.05 considered signi¢cant.
RESULTS
Of the 18 animals, seven received i.c.v. injections of vehicle only and 11 PGE1 . Of the latter, six received 400 ng and ¢ve 1 Wg (see Malkinson et al., 1988). As no signi¢cant di¡erences were observed between the e¡ects of the two doses (see below) the data are considered together. One animal in the PGE1 group responded in an atypical manner (see below) and therefore is not included in the pooled PGE1 group nerve activity data
set. Examples of vehicle and PGE1 group DCN activity levels during experimental runs are shown in Fig. 2. Level of sympathetic drive during experimental protocol As described in Experimental procedures, 100% activity level was determined when activity had stabilised such that two comparable 500-s data sections taken 30 min apart were obtained under control conditions. Whole body warming was observed to decrease sympathetic activity (n = 18; see Fig. 2) as reported previously (Johnson and Gilbey, 1996; Ha«bler et al., 2000). DCN activity began to decline at Tc of 36.4^38.1³C (vehicle, 37.4 þ 0.6³C (n = 7); PGE1 , 37.5 þ 0.7³C (n = 11)). Nadirs of activity were seen at Tc of 38.6^ 40.5³C (vehicle, 39.8 þ 0.2³C (n = 7); PGE1 , 39.6 þ 0.6³C (n = 11)). Dorsal collector nerve activity during heat clamp prior to i.c.v. injection. During heat clamp (Tc 40^41³C), before i.c.v. injection, normalised activity was 7.6 þ 11.0% of control in the vehicle group (n = 7) and 18.2 þ 30.1% of control in the PGE1 group (n = 10, no signi¢cant di¡erence; see Fig. 2). For both groups these levels of activity were signi¢cantly lower than control (P 6 0.001, Student's paired t-test) but comparable to those seen following ganglionic blockade and following death. E¡ect of i.c.v. prostaglandin E1 on dorsal collector nerve activity during heat-clamp. In all but one animal, during heat clamp, 30 min following i.c.v. injection of PGE1 ,
Fig. 2. Recti¢ed and smoothed (time constant, 1 s) neurogram of population sympathetic activity levels during time course of the experimental protocol. Normalised activity levels showing the e¡ect of i.c.v. injection of vehicle (a) or PGE1 (b). Upper traces in a and b show Tc of the animal throughout the duration of the trial. Asterisks mark the point of ganglion blocker administration (i.v.).
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Fig. 3. Power spectra and DCN^TP coherence during vehicle trials. Autospectra of DCN activity (1a^d), TP (2a^d) and graphs showing their coherence (3a^d, dotted lines mark level where coherence becomes signi¢cant) ; taken during control (a), during heat clamp (b), 30 min post i.c.v. injection (c) and following removal of heat clamp (d). See Experimental procedures for details of analysis. For DCN activity, the control autospectrum has been superimposed on b, c and d (dotted line) to show the changes in power. Note that during heat clamp additional peaks emerge in the TP autospectra, indicative of emerging central respiratory drive and that DCN power is minimal during this period. DCN and TP signals are signi¢cantly coherent at ventilation frequency during control (a) and following removal of heat clamp (d). S indicates the peak at the frequency of T-rhythm, F indicates the peak at the frequency of ventilation and 8 indicates the peak at the frequency of central respiratory drive.
sympathetic activity increased signi¢cantly (from levels prior to i.c.v. PGE1 ) to 154.1 þ 53.3% of control (P 6 0.001, n = 10; see Fig. 2). Such increases in activity had a latency of 913 þ 307 s. Activity of the `non-responder' in this group exhibited a slow return to control levels, outside the 900^1200 s observed in the rest of the PGE1 group. In contrast, following i.c.v. injection of vehicle, activity (19.3 þ 18.8%) did not change signi¢cantly (n = 7, see Fig. 2). A di¡erence in time course of increase in activity was the only observed di¡erence between the two doses of PGE1 . Whereas injection of 1 Wg PGE1 evoked a rapid increase in activity that was sustained for the duration of the heat clamp, the increase in activity that followed 400-ng doses exhibited a slight reduction 30^40 min post injection. Dorsal collector nerve activity post heat clamp. Following termination of the heat clamp (and cooling of Tc to 36.5^38³C) activity was 176.1 þ 31.8% in the vehicle group and 211.4 þ 76.2% in the PGE1 group: these values are not signi¢cantly di¡erent (see Fig. 2). Blood pressures during experimental protocol Coincident with the observed increase in DCN activity during heat clamp, following injection of PGE1 , BP increased compared to pre-injection levels. In contrast,
following injection of vehicle there was a tendency for BP to decrease compared to pre-injection levels (see Table 1). Heart rate changes during experimental protocol There was a tendency for heart rate to increase in both groups when Tc was raised (see Table 1). However, mean post-injection heart rates in vehicle and control groups were similar. Central respiratory drive and blood gases during experimental protocol During the control period in 14 of 18 cases animals were in central apnoea due to hypocapnia and mild hyperoxia (see Table 1 for arterial partial pressure (P) CO2 and arterial PO2 measurements). In four cases, central respiratory drive was present but weak, as indicated by £uctuations in TP. Animals were hyperventilated in order to ensure that central respiratory drive did not become excessive during hyperthermia and to enable the in£uence of recruitment of central respiratory drive to be examined on sympathetic rhythm (see below). In all 18 animals central respiratory drive was present during heat clamp. This was seen as a 1-Hz £uctuation in the tracheal pressure trace (Fig. 1b) as central respiratory drive was locked 1:2 with the lung in£ation cycle (see
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Fig. 4. Power spectra of DCN^TP during PGE1 trials. Autospectra of DCN, TP and coherence as in Fig. 3. Note the emergence of central respiratory drive in TP autospectra (2b and 2c) as in Fig. 3 and the return of DCN activity 30 min post i.c.v. injection (1c), locked to the central respiratory drive frequency. DCN and TP signals show signi¢cant coherence at ventilation frequency (1a, c and d) and at central respiratory drive 30 min post i.c.v. injection (1c). S indicates the peak at the frequency of T-rhythm, F indicates the peak at the frequency of lung in£ation cycle and 8 indicates the peak at the frequency of central respiratory drive.
Petrillo et al., 1983) and was revealed as a 1-Hz peak in the TP power spectrum (Figs. 1c, 32b; c and 42b; c ). No signi¢cant di¡erences were observed in arterial PCO2 or arterial PO2 throughout the experimental protocol (Table 1, n = 16). Rhythmic characteristics of sympathetic activity Control conditions. As reported previously (Chang et al., 1999, 2000; Smith and Gilbey, 2000), during central apnoea (n = 14) and in the presence of weak central respiratory drive (n = 4), autospectra of sympathetic activity exhibited a peak in the T-rhythm frequency range (Figs. 31a , 41a and 5a; T-peak, 0.68^0.93 Hz, n = 18). An additional peak at 2 Hz highly coherent with ventilation cycle frequency was also observed (see Chang et al., 2000) in 15/18 cases (Figs. 31a , 41a and 5a). There was signi¢cant coherence between DCN activity and TP in the 2.05^2.14 Hz range (Figs. 33a and 43a : coherence values were 0.72 þ 0.11 and 0.72 þ 0.16 in PGE1 (n = 10) and vehicle (n = 7) groups, respectively). Heat clamp prior to i.c.v. injection. No peaks were present in autospectra (Figs. 31b , 41b and 5b) and total power was signi¢cantly reduced compared to control (in the PGE1 group total power dropped to 33.21 þ 14.24% of control, in the vehicle group power
decreased to 34.74 þ 19.49% of control; P 6 0.001 for both groups). Heat clamp post i.c.v. prostaglandin E1 . Thirty minutes post i.c.v. injection of PGE1 sympathetic activity had returned; average total power in the PGE1 group was 216.17 þ 107.16% of control (n = 10; P 6 0.005). As seen under control conditions, a peak was present at 2 Hz that was highly coherent with ventilation cycle frequency (coherence values were 0.85 þ 0.07). An additional peak was also present that showed high coherence with the frequency of central respiratory drive (Fig. 41c : range 0.93^1.17 Hz, coherence values were 0.71 þ 0.26). Timeevolving spectra revealed in all cases dynamic locking of the T-rhythm to the frequency of central respiratory drive (Fig. 5c). In contrast, as activity did not recover in the vehicle group during heat clamp 30 min post i.c.v. injection there were no peaks in autospectra (Fig. 31c ) and average total power remained low (42.61 þ 32.85% of control). Post heat clamp. On cooling, power in autospectra of both vehicle and PGE1 groups exceeded control levels (see Figs. 31d and 41d ). Total power for the PGE1 group was 210.57 þ 107.01% of control (P 6 0.05) and for the vehicle group total power was 186.23 þ 51.94% of control (P 6 0.01). Coherence spectra for both groups were com-
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Fig. 5. Dynamic properties of DCN activity during experimental protocol. Upper traces show core temperature recorded throughout the protocol. The middle graph shows a composite image of autospectra from one experimental run (composed of autospectra performed on segments of 2048 points, sliding by 50%). The vertical line indicates the point of i.c.v. PGE1 injection, the asterisk represents the point of ganglion blocker administration. The grey-scale has been maximised to 10 nV2 to enable visualisation of spectral peaks. Examples of power spectra taken from the bracketed points marked on the timeevolving spectrum image (a^d), using a 400-s sample (see Experimental procedures), showing the alterations in power and shift in dominant frequencies (note increased scale in c). Note that during control the power £uctuates around the T-rhythm frequency (0.68 Hz, at arrowhead marked S, (a)) and there is activity locked to the frequency of the ventilation cycle (2 Hz, at arrowhead marked F). During heating the activity decreases (b) and following injection of PGE1 activity initially returns and locks to the frequency of central respiratory drive (1 Hz, at arrowhead marked 8, (c)), activity then £uctuates between T-rhythm and central respiratory drive frequencies (d). S indicates the peak at the frequency of T-rhythm, F indicates the peak at the frequency of lung in£ation cycle and 8 indicates the peak at the frequency of central respiratory drive.
parable to those under control conditions and revealed signi¢cant coherence in the 2-Hz ventilation cycle frequency range (Figs. 33d and 43d ; coherence was 0.75 þ 0.11 for the PGE1 group and 0.78 þ 0.12 for the vehicle group). Furthermore, in the majority of cases (14 out of 17), the central respiratory drive-related peak in the 1 Hz range was no longer present (Figs. 3d, 4d and 5d).
DISCUSSION
In this study we have demonstrated that hyperthermia can cause an extinction of population sympathetic activity recorded from neurones innervating the tail circulation. This con¢rms and extends previous ¢ndings from studies using single- and multi-unit recording techniques (Johnson and Gilbey, 1996; Ha«bler et al., 2000). Novel observations were made with regard to sympathetic activity following i.c.v. PGE1 (400 ng or 1 Wg) administered during heat clamp. First, sympathetic activity returned to or exceeded pre-heat clamp levels. Second, the characteristics of the T-rhythm component of this activity were unchanged from those seen during `normal' body temperature regulation. Thus, the neural substrate generating the T-rhythm is not recon¢gured during the fever-like response to PGE1 .
I.c.v. prostaglandin E1 -induced fever-like syndrome The PGE1 -induced fever-like response appears to share mechanisms common to that produced by exogenous pyrogens. Both can be attenuated by pregnancy (Stobie-Hayes and Fewell, 1996; Eliason and Fewell, 1997) and cortical spreading depression (Monda and Pittman, 1993). Malkinson et al. (1988) ¢rst reported that a fever-like response followed i.c.v. administration of PGE1 (100 ng, 400 ng or 1 Wg) in urethane-anaesthetised rats. Their results are similar to those of Siren (1982) using PGE2 . Increases in the ¢ring rate of nerves innervating intercapsular brown adipose tissue, oxygen consumption and heart rate, and decreases in skin blood £ow were observed (e.g., Siren, 1982; Malkinson et al., 1988; Komaromi et al., 1994; Monda et al., 1995, 1998, 1999). I.c.v. prostaglandin E1 and tail sympathetic activity during hyperthermia In our experiments we examined the e¡ect of i.c.v. PGE1 on tail sympathetic activity during hyperthermia and observed that i.c.v. PGE1 was followed by a restoration of activity. Making the animals hyperthermic before the e¡ects of i.c.v. PGE1 were examined allowed
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us to, ¢rstly, con¢rm that hyperthermia induced extinction of tail sympathetic activity in our anaesthetised animal model and, secondly, determine the Tc at which it occurred. The protocol that we followed does have the potential disadvantage that if hyperthermia precipitates hypotension it can cause activation of sympathetic out£ow to the tail circulation (authors' unpublished observations). However, this did not occur and post vehicle i.c.v., sympathetic activity was not restored. Furthermore, arterial blood gases were kept within a narrow range (mildly hyperoxic and hypocapnic). I.c.v. prostaglandin E1 and rhythmic activity During the control period 14 of 18 animals were in central apnoea. Animals were put in this respiratory state so that a clear T-peak could be observed in sympathetic autospectra (see Chang et al., 1999, 2000; Gilbey, 2001). Consistent with the observation that hyperthermia enhances central respiratory drive through an increase in CO2 sensitivity (Maskrey, 1990), during heat clamp central respiratory drive was recruited although PO2 and PCO2 were changed little from control. As observed in normothermia by Chang et al. (1999), we found that following the return of sympathetic activity post i.c.v. PGE1 , the T-rhythm was entrained dynamically 1:1 with central respiratory drive (with high coherence). Therefore, following i.c.v. PGE1 , we did not detect any robust changes in the rhythmic components of tail sympathetic activity and their dynamic synchronisation by periodic inputs (compared with those seen during `normal' temperature regulation). Our results might appear incompatible with those of Kenney et al. (1998). They remarked that ``enhanced respiratory modulation of sympathetic nerve discharge bursts [that they observed to non-cutaneous targets] during heating did not result from changes in respiratory drive, because end-tidal CO2 levels remained constant during elevations in core body temperature''. Consequently, they reasoned that hyperthermia enhanced the coupling between phrenic nerve discharge and sympathetic nerve discharge. Their conclusions appear unjusti¢ed when the in£uence of hyperthermia on CO2 sensitivity (Maskrey, 1990) is taken into account. Furthermore, data presented in their paper indicate that both phrenic nerve discharge frequency and activity in the phrenic neurogram were increased when core temperature was elevated.
T-rhythm is present and is synchronised dynamically by central respiratory drive and ventilation-related inputs. Therefore, the neural substrate(s) underlying the generation of the T-rhythm are not recon¢gured during fever. Smith and Gilbey (2000) suggested that the substrate giving rise to the T-rhythm was located antecedent to sympathetic preganglionic neurones. Therefore, from our data it might be proposed that the sympathetic premotor neurones in the region of the ventromedial medulla (including the caudal raphe nuclei pallidus and obscurus), which are considered to provide a major excitatory drive to sympathetic preganglionic neurones in£uencing cutaneous (thermoregulatory) vasoconstrictor postganglionic neurones (Rathner and McAllen, 1999; Blessing and Nalivaiko, 2000, 2001), are utilised both during normal body temperature regulation and during fever. The reduction in heat loss (through cutaneous vasoconstriction) associated with fever may result solely from a change of set point in the preoptic area, reducing the drive to GABA-releasing neurones (at raised body temperature) that act to inhibit the ventromedial medullary sympathetic premotor neurones (Zhang et al., 1997; Nagashima et al., 2000). However, this view appears inconsistent with the ¢ndings that LPS-induced fever was associated with the activation of c-Fos protein expression in PVN and RVLM neurones projecting to the upper lumbar level of the spinal cord (Zhang et al., 2000) and that a pathway passing through the PVN appears to be involved in LPS and PGE fever, but not normal body temperature regulation (Horn et al., 1994; Lu et al., 2001). Such observations indicate that additional descending inputs to sympathetic preganglionic neurones are active during fever. An `additional drive' rather than purely a reduction in descending inhibitory drive is indicated further by the ¢nding that although anaesthetised animals are unable to maintain normal body temperature they can raise their core temperature following i.c.v. PGE (e.g., Malkinson et al., 1988). As the T-rhythm remained robust during the fever-like response, it would appear that the rhythmic inputs underlying the T-rhythm are either relayed over multiple descending pathways or that the mechanism underlying the T-rhythm is potent enough to sculpt the discharge patterns of sympathetic neurones receiving high levels of tonic drive (see Gilbey et al., 1986). Clearly, further studies are required to determine the functional contributions of the various descending pathways to tonic drive and oscillatory components of sympathetic neurones regulating cutaneous thermoregulatory circulations.
CONCLUSIONS
The key ¢nding from this study is that the T-rhythm characteristics of cutaneous sympathetic activity seen during normal body temperature regulation and PGE1 induced fever-like syndrome are similar, i.e. the
AcknowledgementsöA.K. and work supported by Wellcome Trust Grant 054584 and D.R.C. by Wellcome Trust Grant 05115.
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