Arterial baroreflex control of muscle blood flow at the onset of voluntary locomotion in mice

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1 J Physiol (2003), 553.1, pp DOI: /jphysiol The Physiological Society Arterial baroreflex control of muscle blood flow at the onset of voluntary locomotion in mice Shizue Masuki and Hiroshi Nose Department of Sports Medical Sciences, Institute on Aging and Adaptation, Shinshu University Graduate School of Medicine, Matsumoto , Japan To assess the role of arterial baroreflex control in muscle blood flow (MBF) and voluntary locomotion, mean arterial pressure (MAP), MBF, and electromyograms (EMGs) were measured in freely moving mice before (CNT) and after blocking the afferent or efferent pathway of arterial baroreflexes, carotid sinus denervation (CSD), or intraperitoneal administration of phentolamine (BLK), respectively. MAP was measured through a catheter placed in the femoral artery. MBF was measured with a needle-type laser-doppler flowmeter and recorded through a low-pass filter with an edge frequency of 0.1 Hz. The frequency and duration of locomotion were judged from EMG recordings in the hindlimb. These probes were implanted at least 2 days before the measurements. Muscle vascular conductance (MVC = MBF/MAP) in all groups started to rise within 1 s after the onset of locomotion, but the increasing rate in CSD and BLK was significantly higher than in CNT for the first 9 s (P < 0.001). MAP in CSD and BLK significantly decreased below the baseline within 1 s and this was highly correlated with the increase in MVC for the first 9 s (R 2 = 0.842, P < 0.001), whereas MAP in CNT increased significantly 8 s after the onset of locomotion. Although the total period of movement in a free-moving state for 60 min was not significantly different between CNT and CSD (P > 0.1), the frequency of movement with a short duration of min was higher in CSD than in CNT (P < 0.001), which was highly correlated with the reduction in MAP accompanying each period of movement (R 2 = 0.883, P < 0.01). These results suggest that arterial baroreflexes suppress vasodilatation in contracting muscle to maintain MAP at the onset of voluntary locomotion, and are necessary to continue a given duration of locomotion in mice. (Received 21 May 2003; accepted after revision 18 August 2003; first published online 22 August 2003) Corresponding author H. Nose: Department of Sports Medical Sciences, Shinshu University Graduate School of Medicine, Asahi Matsumoto , Japan. nosehir@sch.md.shinshu-u.ac.jp The muscular vasodilatation during locomotion is reportedly suppressed by enhanced muscle sympathetic nervous activity (MSNA) (Seals, 1989; Mostoufi-Moab et al. 2000). Since administration of sympathetic ganglionic blockade (Sheriff et al. 1993; Buckwalter & Clifford, 1999) and/or baroreceptor denervation (DiCarlo & Bishop, 1992) are known to decrease mean arterial pressure (MAP), the suppression is probably one of the feedback mechanisms that help maintain MAP by counteracting the vasodilatory effects of local factors (Shepherd & Vanhoutte, 1979) released from contracting muscles. However, there have been no studies to demonstrate the role of arterial baroreflexes in the duration of voluntary locomotion. In the present study, we found in carotid sinus denervated mice that MAP fell by mmhg immediately after the onset of voluntary locomotion, followed by cessation of movement, and the fall in MAP was significantly correlated with the duration of locomotion. Moreover, we recently reported that increased fluctuation of MAP during treadmill exercise was likely to decrease maximal treadmill running speed in genetically calponin-deficient mice (knockout (KO) mice) (Masuki et al. 2003b) in which a-adrenergic vasoconstriction was reduced to half that in the wild-type mice (Masuki et al. 2003a). These results suggest that arterial baroreflexes in mice suppress muscle vasodilatation to continue a given intensity of voluntary locomotion by preventing the fall in MAP. However, there have been no attempts to measure muscle blood flow (MBF) during voluntary locomotion in mice due to their small size. The first aim of the present study was to establish a method to measure the tissue blood flow in contracting muscle in mice, used broadly as a genetically altered animal, using a needle-type laser-doppler flowmeter that has recently become available. The second aim was to examine the hypothesis that arterial baroreflex control of MBF would play an important role in continuing a given duration of voluntary locomotion by maintaining MAP. The measurements were performed before and after carotid

2 192 S. Masuki and H. Nose J Physiol sinus denervation and also after blocking sympathetic nerve terminals in the vascular smooth muscle by administration of a-adrenergic blockade. METHODS Animals Adult male C57BL/6J mice aged 9 30 weeks (n = 24, body weight = 29.2 ± 1.0 g) were housed at 25 C with food and water ad libitum and illuminated from 7:00 to 19:00. The mice were closely monitored to ensure that none experienced undue stress or discomfort. The procedures used here were in accordance with the Guiding Principles in the Care and Use of Animals in the Fields of Physiological Sciences published by the Physiological Society of Japan (1988) with the prior approval of the Animal Ethics Committee of Shinshu University School of Medicine. It was confirmed in a preliminary study that daily activity in mice after the following surgical procedures, counted with locomotion sensors (model LCM-10M, Melquest, Toyama, Japan), recovered to the baseline when the measurements were performed. Surgical procedures Carotid sinus denervation. A carotid sinus denervated (CSD) mouse was anaesthetized with pentobarbital sodium (50 mg (kg body weight) _1 I.P.), a midline incision was made in the anterior neck, and the area of the carotid bifurcation was exposed. The internal, external and common carotid arteries were stripped of connective tissues, and the region was painted with 10 % phenol in ethanol, as reported previously in rats (Krieger, 1964; Van Vliet et al. 1999). The success of the denervation was confirmed by the increased fluctuation in MAP and reduced heart rate (HR) response to the fluctuation, as shown in Table 1. The mice were allowed to recover for 2 days before subsequent implantation of catheters. Implantation of arterial and abdominal catheters. Details of catheterization have been reported elsewhere (Mattson, 1998; Masuki et al. 2003a). Briefly, in the control (CNT1; n = 6, CNT2; n = 6), CSD (n = 6), and a-adrenergic blockade-administered (BLK, n = 6) groups, a polyethylene arterial catheter was inserted into the left femoral artery and the tip placed 5 mm below the left renal artery. In a BLK (CNT2) mouse, a polyethylene catheter for I.P. administration of a-adrenergic blockade was also placed in the abdominal cavity. The arterial and abdominal catheters were secured to the leg and abdominal muscles, respectively, tunnelled subcutaneously, exteriorized between the scapulae and connected to a cannula swivel (model TCS2 21, Tsumura; Tokyo, Japan) with a free moving system (model FM-1121, Tsumura) fixed on a home cage in which a mouse was able to move freely. The arterial catheter was flushed every day with 100 i.u. heparin in 0.2 ml saline. At least 3 days after the catheterization, a flow probe and EMG electrodes were implanted as described below. Implantation of flow probe and EMG electrodes. In the CNT, CSD, and BLK groups, a laser-doppler flow probe and electromyogram (EMG) electrodes were implanted in the right hindlimb. The probe consisted of two glass fibres each of which was covered with a plastic sheet: one for insertion of laser light, and the other for detection of the reflection. The tips of the fibres were stripped of their covers, glued to ensure they were 4 mm in length and 0.5 mm in diameter and inserted into the vastus medialis muscle to a depth of ~2 mm from the fascia. A pair of EMG electrodes was also implanted in the vastus lateralis muscle. The fibres and electric wires from EMG electrodes were secured to the surrounding muscles, tunnelled subcutaneously, and pulled out from between the scapulae. Implantation of tourniquet tube. To remove any artifacts in signals from a laser-doppler flowmeter, MBF was measured before and after occlusion of the iliac artery with a tourniquet tube using another group of mice (n = 6). After induction of anaesthesia, an incision was made on the midline of the abdomen, a 150-mm vinyl tube (BB317 85, i.d mm, o.d mm, Scientific Commodities; Lake Havasu, AZ, USA) for tourniquet was placed around the right common iliac artery at the centre of the tube. Both ends of the tube were passed through the sheath of a 40-mm silicone tube (BB312-S/2, ID 1.5 mm, o.d. 3.0 mm, Scientific Commodities). The sheath was secured to the latissimus dorsi muscles, tunnelled subcutaneously, and pulled out from between the scapulae. The tourniquet tube was left loose without fixation. Protocol MBF measurement before and after occlusion of iliac artery. The measurements were performed in the group implanted with a tourniquet tube, a flow probe, and EMG electrodes, 4 6 h before the measurements. MBF and EMG were continuously measured for 10 min in free-moving mice in their home cages, before and after occlusion of the iliac artery, carried out by pulling the ends of the tourniquet tube. Effects of carotid sinus denervation and a-adrenergic blockade. MAP, HR, MBF and EMG were continuously measured in freemoving mice in their home cages for 60 min in the CNT, CSD and BLK groups. In all the groups, the measurements were performed 2 4 days after the last surgical preparation for the implantation of a flow probe and EMG electrodes, which was done 3 days after the arterial catheterization. In the CSD group, because the carotid sinus denervation was performed 2 days before the catheterization, at least 7 days passed between denervation and taking the measurements. By that time, the elevated MAP, observed immediately after the denervation, had returned to baseline as reported previously (Irigoyen et al. 1995). In the BLK group, a-adrenergic blockade (phentolamine) was administered I.P.15 min before the measurements. The initial dose of blockade was 1 mg (kg body weight) _1, followed by 6 mg (kg body weight) _1 h _1 for continuous administration. The effects of the blockade were confirmed by no increase in MAP by the intraarterial injection of a-adrenergic agonist (phenylephrine). After the injection of 30 mg ml _1 phenylephrine (3.3 ml kg _1 ), MAP increased by 36.3 ± 3.1 mmhg in the CNT2 group (P < 0.001), whereas in the BLK group it decreased by 1.4 ± 1.2 mmhg (P > 0.7). The effect of carotid sinus denervation on muscle vascular conductance (MVC) was compared between the different groups of CNT1 and CSD mice and the effect of the blockade on MVC was compared in the same group of mice before (CNT2) and after the administration (BLK). The measurements were performed between 10:00 and 17:00. Measurements MBF was measured by laser-doppler flowmetry (model FLO-C1 BV, Omegawave; Tokyo, Japan) every 100 ms and recorded after the treatment according to a logic diagram as shown in the Appendix, to remove the artifacts caused by the alteration of a relative angle between the tip of the probe and vascular wall when mice moved. Briefly, the MBF signal during the periods where the EMG burst was above 200 % of the baseline was passed through a

3 J Physiol Baroreflex and behaviour 193 low-pass digital filter of the edge frequency of 0.1 Hz (KC-DF- FIR01, Kissei Comtec; Matsumoto, Japan), whereas the MBF signal during the remaining periods where the EMG burst was below 200 % of the baseline was not passed through the filter, and the signals during the two periods were ultimately combined and recorded every 100 ms by a computer (OptiPlex GX260, Dell; Kawasaki, Japan). The time delay in MBF caused by the measurement of filtering was corrected. EMG was measured through a band-pass filter of Hz (Bioelectric Ampl 4124, NEC; Tokyo, Japan). MAP was measured through an arterial catheter connected to a pressure transducer (model TP-400T, Nihon Kohden; Tokyo, Japan). HR was counted from the arterial pressure pulse with a tachometer (model AT-601G, Nihon Kohden) of which the response time was 100 ms. MAP, HR, MBF, and EMG were continuously recorded with a digital data recorder (Thermal Arraycorder WR 8500, Graphtec; Yokohama, Japan) at 100 ms intervals. MAP, HR, and MVC (as MBF/MAP), were transferred to a computer (OptiPlex GX260) after treatment with a low-pass filter of 0.1 Hz according to the same logic as used in MBF. Integrated EMGs (i EMG ) were determined every 100 ms. Analyses Spectral analyses of MBF. To determine the edge frequency of 0.1 Hz to remove the artifacts from the MBF signals, Fourier power spectral analyses were performed on the MBF signals for 10 min before and after the occlusion, respectively. Frequency and duration of movement. The duration of movement was judged according to the criterion of the i EMG burst being more than 200 % above the baseline. The frequency and duration of each movement were determined in a freemoving state for 60 min from 10:00 to 17:00. The duration was determined every 0.1 min by rounding off the second decimal place. The change in MAP (DMAP) accompanying each movement was determined from the difference in MAP from the baseline just before the cessation of the movement. MVC during voluntary locomotion. The analyses of MVC were performed during locomotion lasting longer than 10 s. The major type of body movement during observation was walking and grooming, and other types of movement standing up, eating, and drinking were rarely observed during the measurements. The changes in MBF, MVC, and i EMG for 20 s after the start of locomotion were expressed as % of the baselines, and those in MAP (DMAP) and HR (DHR) were presented as the differences from the baselines. Since at least two movements lasting longer than 10 s were observed in all mice, the measurements in two trials were averaged at 100 ms intervals after the start of locomotion in each mouse. Means and S.E.M. values for six mice were presented in each group. Statistics Values are expressed as means ± S.E.M. The pairwise comparisons were made in mean and coefficient of variation (CV) values between the groups by using Fisher s least-significant difference test with the post hoc test after confirming the significant differences with a one-way ANOVA (Table 1 (CNT1 vs. CSD), Fig. 2A (CNT1 vs. CSD)), a one-way ANOVA for repeated measures (Table 1 (CNT2 vs. BLK), Fig. 2B (CNT2 vs. BLK), Figs 3A and B (CNT1 vs. CSD), Fig. 4C (before vs. after occlusion), and Fig. 6A (CNT1 vs. CSD)), or a two-way ANOVA for repeated measures (Fig. 6B (CNT2 vs. BLK)). Specific trend analysis for each group was performed with a one-way ANOVA for repeated measures (Figs 3B and 6A and B). The null hypothesis was rejected at P < RESULTS Figure 1 shows typical examples of MAP and EMG recordings in a mouse from the CNT1 group (A) and one from the CSD group (B) in a free-moving state for 60 min. Arterial pressure and EMG in the upper right figures are presented from the parts indicated by the arrows in the lower figures, on an enlarged time scale. MAP in the CNT1 mouse started to increase immediately after the onset of locomotion and sustained a higher level than at rest during the burst of EMG. In contrast, MAP in the CSD mouse Figure 1. Typical examples of mean arterial pressure (MAP) and electromyogram (EMG) for a control mouse (CNT1, A) and a carotid sinus denervated mouse (CSD, B) in the free-moving state for 60 min Arterial pressure (AP) and EMG from the parts indicated by the arrows in the lower figures are presented on an enlarged time scale in the upper right side of each panel.

4 194 S. Masuki and H. Nose J Physiol decreased immediately after the onset of locomotion and returned to the baseline after the cessation of locomotion. The frequency of movement for 60 min was higher and the duration of each movement was shorter in the CSD mouse than in the CNT1 mouse. Figure 2 summarizes the total frequency of movement and the duration of one movement in a free-moving state for 60 min for the CNT1 and CSD groups (Fig. 2A), and those in the CNT2 and BLK groups (Fig. 2B). Although there were no significant differences in the total period of movement for 60 min between the groups (P > 0.1), the frequency of movement was 29 ± 3 times in the CSD group, significantly higher than the value of 13 ± 1 times in the CNT1 group (P < 0.001) and the duration of one Figure 2. Total frequency of movement and duration of one movement in the free-moving state for 60 min Means and S.E.M. bars are presented for the CNT 1(n = 6) and the CSD (n = 6) groups, and for the group before (CNT2, n = 6) and after administration of a- adrenergic blockade (BLK, n = 6). ** and *** Significant differences between the two groups, P < 0.01 and P < 0.001, respectively. Other abbreviations are as in Fig. 1. Figure 3. Frequency distribution of movement (A) and change in MAP (B) with respect to the duration of one movement on a log scale in a free-moving state for 60 min in the CNT1 and CSD groups Means and S.E.M. bars for six mice in each group are presented. *** Significant differences between the two groups, P < Abbreviations are as in Fig. 1.

5 J Physiol Baroreflex and behaviour 195 movement was 0.4 ± 0.1 min in the CSD, significantly shorter (P < 0.01) than the value of 1.3 ± 0.2 min in the CNT1 group. The frequency of movement and the duration of one movement in the BLK group were similar to those in the CSD group, 43 ± 6 times and 0.4 ± 0.1 min, respectively, different from 13 ± 2 times and 1.1 ± 0.2 min in the CNT2 group at P < 0.01 and P < 0.001, respectively, but not different from those in the CSD group (P > 0.07). Figure 3 shows the frequency distribution of movement (Fig. 3A) and the change in MAP (Fig. 3B) with respect to the duration of one movement on a log scale for the CNT1 Figure 4. Typical examples of MBF and EMG in a free-moving state for 10 min before (A) and after (B) occlusion of the iliac artery, respectively MBF signals are shown before (*MBF) and after (MBF) the treatment with a low-pass filter according to the logic diagram in the Appendix. Figures on the right sides are presented on an enlarged time scale from the parts indicated by the arrows in panels A and B, demonstrating that the areas of the artifacts overriding MBF signals were reduced to almost 20 % after the treatment. C, power spectrum of MBF before and after occlusion of the iliac artery. Means (solid lines) and S.E.M.s (shaded areas) are presented for six mice in each group. Significant differences in the power were observed between before and after occlusion in the range of Hz (P < 0.01).

6 196 S. Masuki and H. Nose J Physiol and CSD groups. The frequency was significantly higher in the CSD group than in the CNT1 group in the range of min (P < 0.001), whereas the reduction in MAP for the CSD group was significantly greater than that observed at times longer than 0.8 min of the duration (P < 0.01). In the CSD group, the duration of one movement was highly correlated with the reduction in MAP (R 2 = 0.883, P < 0.01) at times shorter than 0.5 min of the duration, which was highly correlated with the frequency (R 2 = 0.847, P < 0.01). Table 1 shows MAP and HR at rest in the CNT1, CSD, CNT2 and BLK groups. The resting period was % of the total measuring period with no significant differences among the groups (P > 0.1). In the CSD group, neither MAP (P > 0.1) nor HR (P > 0.2) were significantly different from those in the CNT1 group. In contrast, MAP in the BLK group was 12 % lower (P < 0.01) and HR was 13 % higher than in the CNT2 group (P < 0.05). The CV in MAP for the CSD group was 2.5-fold higher than that for the CNT1 group (P < 0.01) but without any significant difference in the CV in HR between the groups (P > 0.05). The CVs in MAP and HR for the BLK group were not significantly different from those in the CNT2 group (P > 0.2). Figure 4 shows typical examples of MBF and EMG signals in a free-moving state for 10 min before (Fig. 4A) and after (Fig. 4B) occlusion of the iliac artery. As shown in the top papel of Fig. 4A, the MBF signal contains two components: the sharp component synchronizing with the burst of EMG and the following dull component. After occlusion, the sharp component remained while the dull component disappeared as shown in the top panel of Figure 4B. After the treatment with a low-pass filter (0.1 Hz) according to a logic diagram shown in the Appendix, the sharp component disappeared and the dull component remained as in the middle panels of Fig. 4A and B. The right side of Fig. 4A and B shows MBF signals before (top) and after (middle) the treatment from the parts indicated by the arrows in each figure, demonstrating that the area of the MBF signal was reduced to almost 20 % after the treatment. Figure 4C shows the power spectrum of MBF signals before and after occlusion of the iliac artery. The power before occlusion was significantly higher than that after occlusion in the range of Hz (P < 0.01), regarded as the main range of frequency of the dull component. Figure 5 shows typical examples of MAP, HR, MBF, MVC and EMG in mice from the CNT1, CSD, and BLK groups in a free-moving state for 5 min. MBF and MVC increased with the burst of EMG in mice from all groups, but the increasing rates in MBF and MVC were higher in the CSD and BLK mice than those in the CNT1 mouse. MAP decreased with the increases in MBF and MVC in the CSD and BLK mice while it increased in the CNT1 mouse. HR increased with the burst of EMG in the CNT1 and BLK mice but remained unchanged in the CSD mouse. Figure 5. Typical examples of MAP, HR, MBF, muscle vascular conductance (MVC) and EMG in a free-moving state for 5 min for a control mouse (CNT1), a carotid sinus denervated mouse (CSD), and a phentolamine-treated mouse (BLK) MAP, HR, MBF and MVC increased with every EMG burst in CNT1, but the increases in MBF and MVC for CSD and BLK were more rapid, and were followed by the rapid falls in MAP.

7 J Physiol Baroreflex and behaviour 197 Figure 6A shows changes in MAP, HR, MBF, MVC and i EMG from their baselines during 20 s after the onset of voluntary locomotion as means and S.E.M. values for six mice in each of the CNT1 and CSD groups. MBF and MVC in the CSD group increased more rapidly after the onset of locomotion than those in the CNT1 group, reaching peak values in the first 4 s in the CSD group while it required 8 s in the CNT1 group. MBF and MVC in the CSD group were significantly higher than in the group during 1 13 and s for MBF (P < 0.001) and during 1 14 and s for MVC (P < 0.001). MAP in the CSD group decreased immediately after the onset of locomotion with the increase in MVC, while MAP in the CNT1 group remained unchanged until 5 s, being significantly lower in the CSD group than in the CNT1 group during 20 s of locomotion (P < 0.001). HR in the CNT1 group increased immediately after the onset of locomotion while that in the CSD group remained unchanged (P > 0.05). HR was significantly higher in the CNT1 group than in the CSD group during 20 s of locomotion (P < 0.001). showed similar patterns to those in the CSD group, being significantly higher in the BLK group than in the CNT2 group during 1 9 s in MBF (P < 0.001), and during 1 11 s and s in the MVC (P < 0.001). MAP in the BLK group decreased immediately after the onset of locomotion with the increase in MVC, being significantly lower in the BLK group than that in the CNT2 group for the first 20 s (P < 0.001). HR in the BLK and CNT2 groups increased immediately after the onset of locomotion with no significant differences between the groups for 20 s (P > 0.8). The increase in MVC was highly correlated with i EMG in the CNT1 (R 2 = 0.661, P < ), CNT2 (R 2 = 0.783, P < ), CSD (R 2 = 0.891, P < ), and BLK (R 2 = 0.893, P < ). The negative correlations between DMAP and DMVC were observed in the CSD (R 2 = 0.961, P < ) and BLK groups (R 2 = 0.914, P < ) while the positive correlations were observed in the CNT1 (R 2 = 0.802, P < ) and CNT2 groups (R 2 = 0.629, P < ). Figure 6-B shows changes in MAP, HR, MBF, MVC and i EMG from their baselines during 20 s after the onset of voluntary locomotion in the CNT2 and BLK groups. In the BLK group, the changes in all parameters except for HR DISCUSSION In the present study, we first measured the tissue blood flow of the contracting muscle during voluntary Figure 6. Changes in MAP, HR, MBF, MVC and EMG after the start of voluntary locomotion in the CNT1 and CSD groups (A), and those before (CNT2) and after administration of a-adrenergic blockade (BLK) (B) Means and S.E.M. bars for six mice in each group are presented at 0.1 s and at 1 s intervals, respectively. * Significant differences between the two groups, P < Abbreviations are as in Figs 1 and 2.

8 198 S. Masuki and H. Nose J Physiol locomotion in mice using laser-doppler flowmetry. The major findings were that the carotid sinus denervation or the administration of a-adrenergic blockade markedly enhanced the increasing rate of MVC at the onset of locomotion, suggesting that muscle vasodilatation was strongly suppressed by arterial baroreflexes at this time. In addition, the reduction in MAP with the increase in MVC after carotid sinus denervation or administration of blockade shortened the duration of each movement. Measurement of tissue blood flow in contracting muscle To assess the baroreflex control of MBF during locomotion, MBF has been measured using an ultrasound Doppler flowmeter in the iliac artery of rats (Martinez-Nieves et al. 2000) and dogs (Buckwalter & Clifford, 1999; Sheriff et al. 2000) during treadmill running, although no attempts have been made in mice due to their small size. In the present study, because a rapid fall in MAP was observed after the onset of voluntary locomotion after carotid sinus denervation or administration of a-adrenergic blockade (Figs 1 and 2), and, in addition, because these results suggested that MSNA through the baroreflex is closely associated with MBF control at the beginning of exercise, it was necessary to use a new technique to measure MBF during voluntary locomotion in mice without disturbing their free movement. The needle-type laser-doppler flowmeter used in the present study has recently become available and has made it possible to measure blood flow in a tissue area of 0.5 mm depth and 1.0 mm diameter from the tip of the probe (Kashima et al. 1996), where small arteries and precapillary arterioles are distributed with a high density of sympathetic nerve fibres (Fuxe & Sedvall, 1965). In addition, this flowmeter made it possible to measure more rapid change in blood flow than could be detected by previous flowmeters by shortening the time constant of the output voltage from 60 to 2 ms. As a result, we were able to measure MBF as rapidly as the arterial pulse pressure though HR in mice was 5 12 Hz, much higher than that in rats or dogs. However, the increased sensitivity made the instrument generate artifacts more easily by bending glass fibres or by dislocation of the tip of the probe in the tissue due to locomotion. To solve this problem, MBF was measured before and after occlusion of the iliac artery and power spectral analyses were performed on the MBF signals to eliminate the artifacts (Fig. 4C). As shown in Fig. 4A and B, the dull component of MBF signals disappeared and the sharp component remained after occlusion of the iliac artery, and, inversely, the sharp component disappeared and the dull component disappeared after passing the signals through the low-pass filter with an edge frequency of 0.1 Hz according to the logic diagram as shown in Appendix. Thus, MBF in the present study was reliable enough to enable discussion of the baroreflex control of MBF. Baroreflex control of MVC at the onset of locomotion In the present study, the increasing rate of MVC in the CSD and BLK groups was higher than that in the CNT groups (Fig. 6A and B). However, the suppression of muscle vasodilatation by MSNA was reported to occur at a delay of 10 s to 5 min after the onset of locomotion (Peterson et al. 1988; Sheriff et al. 1993; Buckwalter & Clifford, 1999). Sheriff et al. (1993) reported in dogs running on a treadmill that an increasing rate of total vascular conductance was not altered until 10 s after the onset of exercise by prior administration of ganglionic blockade. Buckwalter & Clifford (1999) reported similar results in hindlimb conductance in dogs. Peterson et al. (1988) measured tissue blood flow in contracting muscles of rats by the microsphere method and reported that the increased rate in MBF was not altered at 30 s after the start of treadmill running. Thus, these previous findings suggested that the baroreflexes were not involved in MBF control at the onset of exercise, which was different from the observations in the present study. The authors ascribed these results to inhibition of release of noradrenaline (norepinephrine) from sympathetic nerve terminals by local vasodilatory factors released from contracting muscles (Shepherd & Vanhoutte, 1979), known as functional sympatholysis (Laughlin et al. 1996) The discrepancies between the findings of the present and the other studies cited (Peterson et al. 1988; Sheriff et al. 1993; Buckwalter & Clifford, 1999) may be partially caused by the difference in exercise intensity. The oxygen consumption rate at km h _1 of treadmill running in dogs in the aforementioned study was estimated as 30 ml kg _1 min _1 (Ordway et al. 1984), which was equivalent to treadmill running at 5 m min _1 in mice (Rohrer et al. 1998). The increase in HR accompanying each movement in the present study was about 50 beats min _1, which was about 15 % (of 200 beats min _1 ) of the increase in HR observed at 5 m min _1 of treadmill running in mice (Rohrer et al. 1998; Masuki et al. 2003b). Thus, the exercise intensity during voluntary locomotion in the present study was markedly lower than that reported during treadmill exercise in the aforementioned studies (Peterson et al. 1988; Sheriff et al. 1993; Buckwalter & Clifford, 1999). If MVC at the onset of exercise was determined according to the balance between MSNA and the local vasodilatory factors, the increase in MVC would be more suppressed by baroreflexes at such a low intensity of exercise as in the present study, where smaller amounts of the local vasodilatory factors were released. Thus, through arterial baroreflexes, MSNA may play an important role in MBF control in the daily activity of mice, because the increase in HR accompanying each movement observed during a whole day was reported as only beats min _1 (Janssen et al. 2000).

9 J Physiol Baroreflex and behaviour 199 Alternatively, the discrepancy between the findings of the present and previous studies (Peterson et al. 1988; Sheriff et al. 1993; Buckwalter & Clifford, 1999) may be caused by the difference in discharge timing of MSNA due to different styles of exercise: forced treadmill exercise or voluntary locomotion. Matsukawa et al. (1991) found two components of increase in renal sympathetic nerve activity after voluntary static exercise; the first one occurred at or immediately before the onset of exercise and lasted 10 s, whereas the late component gradually increased 14 s after the start of exercise and lasted throughout exercise. They suggested that the initial component was caused by descending input from higher brain centres whereas the late one was caused by feedback signals from contracting muscles. Based on these results, we assumed that if MSNA after voluntary locomotion in the present study increased with a similar pattern to that of the renal sympathetic nerve activity, the first increase in MSNA would be anticipatory control of MBF, which would be unique in voluntary locomotion. However, DiCarlo et al. (1996) measured the activity of lumbar sympathetic nerves innervating the hindlimb muscles in rats running on a treadmill and reported that the nerve activity started to increase at the onset of exercise. By comparing MBF before and after administration of a-adrenergic blockade, Hamann et al. (2002) recently suggested that in rabbits an immediate and sustained increase in sympathetic nerve outflow restrained hindlimb blood flow at the onset of treadmill exercise. These results suggest that MSNA increased at the onset of forced treadmill exercise, which is not unique in voluntary locomotion. Thus, the discrepancy in the results of the MVC response to baroreflexes between the present and previous studies (Peterson et al. 1988; Sheriff et al. 1993; Buckwalter & Clifford, 1999) was not caused by the difference in discharge timing of MSNA between the two styles of exercise but by the difference in the a-adrenergic vasoconstrictive response to MSNA due to exercise intensity. As shown in Table 1, MAP at rest was significantly lower in the BLK group than that in the CSD group despite there being no difference in HR between the groups, suggesting that the muscle vasculature was dilated more in the BLK group than that in the CSD group by abolished a-adrenergic vasoconstriction. However, the difference in the baseline between the two groups did not cause any significant difference in the increasing rates of MVC at the onset of locomotion (Fig 6A and B). These results indicate that the tonicity of muscle vasculature before locomotion did not affect the rates of vasodilatation after the onset of locomotion. Moreover, arterial baroreceptor input was necessary to suppress muscular vasodilatation at the onset of voluntary locomotion because the suppression was abolished in the CSD group. This idea is supported by the results of DiCarlo & Bishop (1992) who reported in rabbits that the increase in renal sympathetic nerve activity at the onset of treadmill running was abolished after sinoaoritic denervation. HR response at the onset of locomotion In the present study, we assessed baroreflex control of MBF at the onset of voluntary locomotion by blocking carotid sinus baroreceptors and sympathetic nerve terminals at the blood vessels, separately. As a result, HR in the BLK group increased by the same degree as in the CNT2 group while HR remained unchanged in the CSD group with the reduction in MAP (Fig 6A and B). The difference in the HR response between the CSD and BLK groups could be explained by the presence or absence of feedback control of arterial baroreflexes to the heart. However, it is not likely. Ludbrook & Potocnik (1986) measured systolic blood pressure and HR in freely moving rabbits and suggested that simultaneous rises in systolic arterial pressure and HR accompanying voluntary locomotion were associated with partial or complete suppression of the reflex effects of arterial baroreceptor input. Recently, Komine et al. (2003) suggested in cats that the bradycardia induced by aortic nerve stimulation was blunted during voluntary static exercise. These results suggest that the increase in HR at the onset of voluntary locomotion for the BLK group was caused not by unloading of arterial baroreceptors but by central command originating from some central sites other than the cerebrum (Matsukawa et al. 1998). However, we found that the arterial baroreceptor input was necessary to activate the system because the increase in HR at the onset of exercise was abolished in the CSD group. Limitation of carotid sinus denervation for assessing baroreflex control of MVC The purpose of carotid sinus denervation in the present study was to block the afferent path of arterial baroreflexes. However, this procedure may remove not only baroreceptor but also chemoreceptor afferents. Franchini et al. (1994) examined the effects of sinoaortic denervation on arterial PJ and MAP, and suggested that arterial PJ was 10 mmhg lower than that in control rats, while MAP remained not different. They also suggested that arterial PJ restoration by increasing inspired PJ elevated MAP by 10 mmhg. These results suggest that the lowered arterial PJ immediately after voluntary locomotion was involved in the reduction of MAP for the CSD mice. However, the reduction in MAP occurred immediately (within 1 s) after the start of locomotion where arterial PJ probably did not decrease. Moreover, the same reduction in MAP was also observed in the BLK mice with intact carotid chemoreceptors. These results suggest that the reduction in MAP for the CSD group was mainly caused by the removal of carotid baroreceptors and the effects of removal of chemoreceptors were minimum.

10 200 S. Masuki and H. Nose J Physiol In the CSD group, arterial baroreflex control of MVC might not be completely eliminated since aortic baroreceptors remained intact. O Leary & Scher (1990) examined the effect of bilateral carotid denervation in dogs and reported that MAP, HR and cardiac output increased, and total peripheral resistance decreased 1 day after the dernervation but they returned to their baselines after 4 7 days of recovery. They ascribed this to the compensatory adaptation of increased aortic baroreceptor sensitivity. However, Nishida et al. (2002) reported in rabbits that MAP, cardiac output, and total periperal resistance at rest returned to baseline 14 days after both carotid and aortic baroreceptor denervation. These results suggest that the restoration of these levels was caused by other mechanisms than compensatory adaptation of aortic baroreceptors. Also in the present study, there was no significant difference in MAP between the CNT1 and CSD groups (Table 1) for at least 7 days after carotid sinus denervation. As shown in Table 1, the coefficient of variation (CV) of MAP at rest in the CSD group increased two-fold compared with that in the CNT1 group. Nishida et al. (2002) also reported in sino-aortic denervated rabbits that the standard deviations of MAP and total peripheral resistance at rest increased two-fold compared with those in intact rabbits. These results suggest that carotid sinus and/or aortic baraoreceptors work to stabilize MAP though they do not alter the level. Moreover, the same increase in the standard deviation of MAP after carotid sinus denervation in the present study and after sinoaortic denervation in the previous study (Nishida et al. 2002) suggests that carotid sinus baroreceptors play an important role in stabilizing MAP against perturbation the rapid increase in MVC after the onset of locomotion. This idea may be supported by the results in the present study showing that the fall in MAP after voluntary locomotion for the CSD group was almost the same as that in the BLK group with abolished sympathetic nerve outflow to the vascular smooth muscle. Reduction in MAP at the onset of locomotion and duration of one movement MAP fell at the onset of locomotion and the duration of one movement was shortened in a mouse from the CSD and BLK groups (Figs 1 and 2). As shown in Fig. 3, the fall in MAP for the CSD group was significantly correlated with the duration of one movement in the range of min, and also with the frequency of movement for 60 min in the range. These results suggest that the fall in MAP was closely associated with the shortened duration of one movement and the increased frequency of movement during a given period of observation. correlation, which significantly correlated with the fall in MAP in the CSD and BLK groups, suggesting that the higher intensity locomotion caused the greater fall in MAP, by the greater increase in MVC. In other words, when the mice intended to move at the higher intensity with the stronger motivation to eat, drink or groom etc, the greater fall in MAP occurred, which might have shortened the duration of one movement at a high intensity. Instead, the mice might learn to increase the frequency of movement at a high intensity and/or to move at a low intensity for a long duration to attain their motivation. The fall in MAP may disturb voluntary locomotion by reducing cerebral blood to match the oxygen demand in the brain (Heistad & Kontos, 1983). Indeed, the blood flow in the cortical motor area and cerebellum have been reported to increase during treadmill running in dogs (Gross et al. 1980). Although there are no available studies on relations between MAP, cerebral blood flow, and brain functions, the rapid and large fall in MAP of 20 mmhg was likely to have affected their behaviour. In summary, these results suggest that arterial baroreflexes play an important role in continuing a given duration of voluntary locomotion by maintaining MAP at the onset of locomotion in mice, which would be partially attained by suppression of muscular vasodilation. APPENDIX A logic diagram for the treatment of MBF, MAP, and HR with a low-pass filter (LPF) X(t)* denotes MBF, MAP, and HR at a time t before being filtered. If i EMG is above the threshold of 200 % of the baseline, f out is 1. If it is below the threshold, f out is 0. If f out is 1, X(t)* is filtered, and if f out is 0, X(t)* is not filtered. The filtered and not filtered components are eventually combined and generated as X(t). The cause and effect relation was unclear. However, MVC in all groups increased with EMG burst rates with high

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