Differential responses to sympathetic stimulation in the cerebral and brachial circulations during rhythmic handgrip exercise in humans

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1 Exp Physiol pp Experimental Physiology Research Paper Differential responses to sympathetic stimulation in the cerebral and brachial circulations during rhythmic handgrip exercise in humans Doreen Hartwich, Katherine L. Fowler, Laura J. Wynn and James P. Fisher School of Sport and Exercise Sciences, College of Life and Environmental Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK The sympathetic neural regulation of the cerebral circulation remains controversial. The purpose of the present study was to determine how exercise modulates the simultaneous responsiveness of the cerebral and brachial circulations to endogenous sympathetic activation (cold pressor test). In nine healthy subjects, heart rate (HR) and mean arterial blood pressure (MAP) were continuously measured during cold pressor tests (4 C water) conducted at rest and during randomized bouts of rhythmic handgrip of 10, 25 and 40% of maximal voluntary contraction. Doppler ultrasound was used to examine brachial artery blood flow (FBF) and middle cerebral artery (MCA) mean blood velocity (V mean ), and indices of vascular conductance were calculated for the brachial artery (forearm vascular conductance, FVC) and MCA (cerebral vascular conductance index, CVCi). End-tidal P CO2 (P ET,CO2 ) was evaluated on a breath-by-breath basis. Handgrip evoked increases in HR, FBF, FVC and MCA V mean (P < 0.05 versus rest), while MAP and CVCi were unchanged and P ET,CO2 fell slightly (P < 0.05 versus rest). Increases in MAP and HR during the cold pressor test were similar at rest and during all handgrip trials. Forearm vascular conductance was markedly reduced with the cold pressor test at rest ( 45± 8%), but this vasoconstrictor effect was progressively attenuated with increasing exercise intensity (FVC 17 ± 3% during exercise at 40% of maximal voluntary contraction; P < 0.05). In contrast, the small reduction in CVCi with cold pressor test was similar at rest and during handgrip (approximately 5%). Our data indicate that while the marked vasoconstrictor responses to sympathetic activation in the skeletal muscle vasculature are blunted by handgrip exercise, the modest cerebrovascular responses to a cold pressor test remain unchanged. (Received 25 June 2010; accepted after revision 6 September 2010; first published online 17 September 2010) Corresponding author J. P. Fisher: School of Sport and Exercise Sciences, College of Life and Environmental Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK. j.p.fisher@bham.ac.uk To sustain exercise, skeletal muscle blood flow must be increased to meet the metabolic requirements of the active muscles. The activation of the sympathetic nervous system plays a critical role in increasing cardiac output and redistributing blood flow to the contracting muscles by eliciting vasoconstriction in regions such as renal and splanchnic vascular beds (Rowell, 1993). Notably, sympathetic vasoconstriction is blunted in the exercising muscles of young, healthy individuals in an intensitydependent manner (Remensnyder et al. 1962; Hansen et al. 1996; Tschakovsky et al. 2002; Keller et al. 2003, 2004; Watanabe et al. 2007). This appears to be partly due to the presence of locally produced metabolites acting to attenuate α-adrenergic receptor activation (so-called functional sympatholysis ), thus optimizing nutritive blood flow despite high background levels of sympathetic activation (Remensnyder et al. 1962; Thomas & Segal, 2004). As in the active skeletal muscles, increases in cerebral neuronal activity appear to be coupled with increases in cerebral perfusion during exercise (Secher et al. 2008). However, the role of the sympathetic nervous system in the regulation of cerebral perfusion both at rest and during exercise in humans remains controversial and incompletely understood (Strandgaard & Sigurdsson, 2008; Ogoh & Ainslie, 2009; Ainslie & Tzeng, 2010). The cerebral vasculature is supplied by α-adrenergic nerves (Lowe & Gilboe, 1971; Edvinsson et al. 1976), which DOI: /expphysiol

2 1090 D. Hartwich and others Exp Physiol pp mainly originate in the superior cervical ganglia (Nielsen & Owman, 1967), thus providing the anatomical apparatus for the sympathetic modulation of cerebral perfusion. Indeed, animal studies suggest that direct stimulation of sympathetic nerves leads to vasoconstriction of cerebral blood vessels (Auer et al. 1983; Wagerle et al. 1983), while in humans, Mitchell et al. (2009) recently demonstrated noradrenaline spillover from the brain into the internal jugular vein, indicative of the sympathetic neural activity directed to the cerebral vasculature. Furthermore, there is some evidence from human studies that systemic administration of α-adrenergic agonists (e.g. noradrenaline; Olesen, 1972; Brassard et al. 2009; Lucas et al. 2010) and sympatho-excitatory manoeuvres, such as the cold pressor test (Micieli et al. 1994; Roatta et al. 1998), whole-body heating (Low et al. 2009) and cardiopulmonary baroreceptor unloading with lower body negative pressure (Wilson et al. 2005), can evoke increases in cerebrovascular resistance (vasoconstriction) and/or reductions in cerebral perfusion. Given the exercise-induced increases in cerebral neuronal activity, it is possible that the sympathetic modulation of resting cerebrovascular tone may become blunted during muscular contractions, as it has been previously described for vasculature of the skeletal muscle. Gross et al. (1980) demonstrated that in exercising dogs, the constrictor responses to arterial hypocapnia were blunted in the areas of cerebral vasculature associated with sensorimotor control. However, substantial species differences have been shown in the cerebral vasomotor response to adrenergic activation (Levine & Zhang, 2008). More recently, Brassard et al. (2010) observed that cerebrovascular responses to bolus injections of the α 1 -adrenergic receptor agonist phenylephrine were attenuated during leg cycling. However, it is presently unclear whether exercise modulates the cerebrovascular responses to sympatho-excitatory manoeuvres that may elicit α-adrenergic receptor activation due to local release of endogenous catecholamines. In the present study, we tested the hypothesis that exercise blunts the vasoconstrictor responses to nonpharmacological, endogenous sympathetic activation in the cerebral circulation, as has been previously demonstrated in the exercising skeletal muscle vasculature. Simultaneous measurements of brachial artery blood flow and middle cerebral artery (MCA) mean blood velocity (V mean ) were obtained, and the respective responses to sympathetic stimulation with the cold pressor test compared both at rest and during rhythmic handgrip exercise. The cold pressor test was used because it has been reported to increase vasoconstrictor sympathetic outflow to the skeletal muscles (Victor et al. 1987) and elicit intracerebral vascular changes via a central noradrenergic mechanism (Micieli et al. 1994). Methods Subjects Nine subjects (one woman), with a mean age of 22 ± 5 years (mean ± S.D.), weight 75 ± 8 kg and height 181 ± 7 cm, participated in the present study. All subjects were non-smokers and none had a history or symptoms of cardiovascular, respiratory, metabolic or neurological disease. Participants were recreationally active and did not take any prescribed or over-the-counter medications (except oral contraceptives). The one woman was tested during the early follicular phase of the menstrual cycle to reduce any confounding effects of oestrogen and progesterone, although hormonal status was not directly assessed. All protocols were approved by the College of Life & Environmental Sciences ethical review committee at the University of Birmingham in accordance with the Declaration of Helsinki. Prior to participation and after receiving a detailed verbal and written explanation of the intended experimental procedures and measurements, each subject provided written informed consent. The subjects were requested to abstain from caffeinated beverages for 12 h and from strenuous physical activity and alcohol for a minimum of 24 h before any experimental sessions. All studies were performed at an ambient room temperature of 23 ± 2 C with external stimuli minimized. Experimental measurements Heart rate (HR) was continuously monitored using a three-lead electrocardiogram (Diascope DS 512; S&W Medioteknik AS, Albertslund, Denmark). Beat-to-beat arterial blood pressure (BP) was measured using finger photoplethysmography on the left hand (PortaPres Model-2; TNO Biomedical Instrumentation, Amsterdam, The Netherlands; Imholz, 1996) and verified prior to each trial with an automated sphygmomanometer (SunTech Tango + ; SunTech Medical, Morrisville, NC, USA; Fisher et al. 2008). Mean arterial pressure (MAP) was calculated as the average BP over each cardiac cycle. Stroke volume (SV) was calculated offline from the blood pressure waveform using a Modelflow software program incorporating the BeatScope version 1.0 software (TNOTPD; Biomedical Instrumentation, Amsterdam, The Netherlands;Jansenet al. 2001). Cardiac output (CO) was calculated using the formula: CO = SV HR. The Modelflow method has been shown to estimate rapid changes in CO reliably during a variety of experimental protocols, including exercise (Bogert & van Lieshout, 2005). End-tidal partial pressure of carbon dioxide (P ET,CO2 ) was obtained from a capnogram acquired by means of a nasal cannula connected to a rapid-response

3 Exp Physiol pp Sympathetic regulation of cerebral circulation 1091 infrared CO 2 analyser (Servomex 1440; Crowborough, UK). Forearm blood flow velocity (FBV) from the brachial artery of the right arm was obtained by Doppler ultrasound (Super Dopplex II; Huntleigh Healthcare Ltd, Cardiff, UK). A Doppler transducer with an operating frequency of 8 MHz was placed on the medial aspect of the upper arm approximately 5 8 cm proximal to the antecubital fossa over the brachial artery, and an insonation angle of 60 deg maintained relative to the skin (Dinenno & Joyner, 2003). The diameter of the brachial artery was determined using a linear array Doppler ultrasound probe operating in two-dimensional B-mode (Philips Envisor, Andover, MA, USA). After optimal signals were achieved, the probes were fixed in position using a custom-made probe holder. Diameter was recorded in loops over three cardiac cycles and stored on the ultrasound device for offline analysis. The average of three measurements of arterial diameter made during diastole was then taken as the diameter for that time point (Schrage et al. 2005). During rhythmic handgrip exercise, diastolic cross-sectional measurements were obtained between contractions. Forearm blood flow (FBF; in millilitres per minute) was then calculated using the formula: FBF = FBV π (diameter/2) The FBF was normalized to the lean forearm mass, assessed by measurements of forearm length and circumferences corrected for skinfold thickness using established formulae (Jones & Pearson, 1969; Tschakovsky et al. 1995). Forearm vascular conductance (FVC; in millilitres per minute per kilogram per millimetre of mercury) was calculated using the following equation: FVC = FBF/MAP. Regional cerebral perfusion was assessed by measurements of blood flow velocity (V mean ) in the left middle cerebral artery (MCA). The MCA was insonated through the temporal window, in front of the ear and above the zygomatic arch, using a 2 MHz pulsed-wave transcranial Doppler ultrasound probe (Multidop X; DWL, Sipplingen, Germany) with online spectrum analysis. The MCA was found at a depth ranging from 46 to 63 mm. After the optimal signal was achieved, the probe was attached to an adjustable headband and fixed in position using adhesive ultrasonic gel (Tensive; Parker Laboratories, Orange, NJ, USA). The MCA V mean was expressed in centimetres per second, and the cerebral vascular conductance index (CVCi) was calculated as MCA V mean /MAP. Experimental procedures Rhythmic handgrip exercise. Rhythmic handgrip exercise (right hand) was performed using a customized dynamometer at a duty cycle of 1 s contraction 2 s relaxation (20 contractions min 1 ) to allow blood velocity and diameter measurements during handgrip exercise (Dinenno & Joyner, 2003). The handgrip workloads corresponded to 10 (mild), 25 (low) and 40% (moderate) of maximal voluntary contraction (MVC) in order to minimize the potential for exercise-induced sympathoexcitation (Victor & Seals, 1989). Each exercise bout lasted for 7 min, and the trials were separated by at least 15 min to avoid fatigue and ensure re-establishment of baseline HR and BP before commencing the next trial. The exercise trials were conducted in a counterbalanced order. Ratings of perceived exertion were obtained for each exercise trial using the standard 6 20 Borg scale (Borg, 1998). Cold pressor test. The cold pressor test was employed to stimulate the sympathetic nervous system (Hines & Brown, 1933; Victor et al. 1987). The subject s bare foot was passively placed in ice-cold water (4 C) for 2 min. The cold pressor test was performed twice at rest; however, as previously reported the first test tended to evoke exaggerated increases in MAP compared with subsequent tests and was therefore excluded from the statistical analysis (Schrage et al. 2005). As such, the cardiovascular and haemodynamic changes elicited by the second cold pressor test were used for comparison with the subsequent cold pressor tests during handgrip exercise. The rating of perceived pain was determined for each cold pressor test (Borg, 1998). Experimental protocol Subjects were seated in a semi-recumbent position on a medical examination table with a custom-made handgrip dynamometer held in the right hand while the arm was supported on an adjustable bedside table. The MVC was determined as the highest force produced during three to five efforts, each separated by 1 min. For the experimental protocol, the force exerted by the subject, expressed as a percentage of maximum, was continuously recorded and displayed on a computer screen positioned in front of the subject at eye level. Following instrumentation and assessment of MVC, subjects rested for 15 min. Then two cold pressor tests were applied at rest, each after 5 min of baseline determination of cardiovascular and haemodynamic variables. After a 15 min rest period, thefirsthandgripexercisetrialwasperformed.acold pressor test was applied during the last 2 min of each handgrip exercise bout (5th to 7th minute). Brachial artery diameter measurements were obtained at the fourth minute of both rest and handgrip exercise, and at the first minute of the cold pressor test. Blood flow velocity measurements were averaged over the 30 s spanning the diameter measurements.

4 1092 D. Hartwich and others Exp Physiol pp Table 1. Cardiovascular, haemodynamic and respiratory parameters at rest and during rhythmic handgrip exercise with and without cold pressor test Exercise at Exercise at Exercise at Rest 10% MVC 25% MVC 40% MVC Without Without Without Without Parameter CPT CPT CPT CPT CPT CPT CPT CPT MAP (mmhg) 81 ± 1 90 ± 2 88 ± 2 95 ± 3 86 ± 2 93 ± 4 87 ± 2 95 ± 4 HR (beats min 1 ) 67 ± 3 71 ± 3 68 ± 3 74 ± 3 71 ± 3 75 ± 3 72 ± 3 76 ± 3 FBF (ml min 1 kg 1 ) 52 ± ± 8 66 ± ± ± ± ± ± 17 FVC 0.6 ± ± ± ± ± ± ± ± 0.2 (ml min 1 kg 1 mmhg 1 ) MCA V mean (cm s 1 ) 67 ± 4 70 ± 4 72 ± 3 74 ± 4 72 ± 4 74 ± 4 72 ± 4 73 ± 4 CVCi (cm s 1 mmhg 1 ) 0.83 ± ± ± ± ± ± ± ± 0.04 P ET,CO2 (mmhg) 42 ± ± ± ± ± ± ± ± 0.3 CO (l min 1 ) 6.4 ± ± ± ± ± ± ± ± 0.5 Values are means ± S.E.M. Abbreviations: CO, cardiac output; CPT, cold pressor test; CVCi, cerebrovascular conductance index; FBF, forearm blood flow; FVC, forearm vascular conductance; HR, heart rate; MAP, mean arterial pressure; MCA V mean, middle cerebral artery mean blood velocity; MVC, maximal voluntary contraction; and P ET,CO2, end-tidal partial pressure of carbon dioxide. Main effect of handgrip exercise (P < 0.05); main effect of CPT (P < 0.05). Data reduction and statistical analysis Physiological data were digitized at 1000 Hz (1401plus; Cambridge Electronic Design, Cambridge, UK) and stored on a personal computer. Customized Spike2 script files were used offline to determine beat-to-beat values for MAP,HR,FBF,MCAV mean and P ET,CO2. To assess the magnitude of cold pressor test-mediated vasoconstriction, the percentage change in FVC was calculated as follows (Schrage et al. 2005): % FVC = (FVC CPT FVC SS )/FVC SS 100 where FVC CPT represents FVC during the cold pressor test and FVC SS represents the steady state prior to the cold pressor test (i.e. either at rest or during handgrip exercise). Likewise, the percentage change in CVCi was calculated using the formula: % CVCi = (CVCi CPT CVCi SS )/CVCi SS 100 Cardiovascular data were statistically analysed using a two-way repeated-measures ANOVA in which trial (rest, 10, 25 and 40% MVC) and condition (steady-state rest or handgrip exercise versus cold pressor test) were the main factors. One-way repeated-measures ANOVAs were applied to statistically analyse differences between rest and each handgrip workload (e.g. magnitude of cold pressor test-mediated vasoconstriction). A Student Newman Keuls test was employed post hoc to estimate the significant main effects and interactions. The level of statistical significancewas setatp < All analyses were conducted using SigmaStat for Windows (Jandel Scientific Software; SPSS, Chicago, IL, USA). Data are presented as means ± S.E.M. Results Table 1 shows the cardiovascular, cerebrovascular and respiratory variables measured at rest and during each handgrip exercise workload with and without cold pressor test. Rhythmic handgrip exercise at 10, 25 and 40% MVC evoked exercise intensity-dependent increases in HR, FBF and FVC (Table 1 and Fig. 1A and B; P < 0.01), while MAP remained unchanged during handgrip exercise. Rhythmic handgrip exercise also increased MCA V mean (Fig. 1C; P < 0.01), while CVCi remained unchanged from rest (Fig. 1D; P > 0.05). The P ET,CO2 fell slightly ( 1 mmhg) during each intensity of handgrip exercise compared with rest. Rhythmic handgrip exercise had no effect on CO. The rating of perceived exertion identified a graded increase in effort with increasing handgrip exercise intensity (7.6 ± 0.5, 8.8 ± 0.3 and 10.2 ± 0.5 a.u. for 10, 25 and 40% MVC trials, respectively; P < 0.05). Cold pressor test-mediated sympathetic activation elicited significant increases in MAP (8 ± 1 mmhg; P < 0.05) and HR (4 ± 1 beats min 1 ; P < 0.05) that were not significantly different between rest and each handgrip exercise bout. The cold pressor test evoked significant reductions in FBF and FVC, both at rest and during each handgrip exercise trial (P < 0.01). Notably, the cold pressor test evoked a 45 ± 8% reduction in FVC at rest, but this vasoconstrictor effect was progressively attenuated with increasing handgrip exercise intensity ( 31 ± 7, 20 ± 6and 17 ± 3% for the 10, 25 and 40% MVC trials, respectively; Fig. 2A; P < 0.05). The MCA V mean was unchanged with cold pressor test, while CVCi was slightly but significantly reduced (P < 0.05). The relatively small percentage reduction in CVCi evoked by the cold pressor test at rest ( 5 ± 2%) was similar during each intensity of handgrip exercise ( 5 ± 2, 3 ± 3and 7 ± 2% for 10, 25

5 Exp Physiol pp Sympathetic regulation of cerebral circulation 1093 and 40% MVC trials, respectively; Fig. 2B). Neither P ET,CO2 nor CO was influenced by the cold pressor test. Subjects consistently rated perceived pain during cold pressor test as being moderate (3.2 ± 0.5 a.u.). Discussion The purpose of the present study was simultaneously to determine how the responsiveness of the cerebral and brachial circulations to augmented sympathetic activation (cold pressor test) is modulated during handgrip exercise in young, healthy humans. The major finding of the present study is that while the marked vasoconstrictor responses of the skeletal muscle vasculature to sympathetic activation were blunted by handgrip exercise, the cerebrovascular responses to the cold pressor test remained similarly modest both at rest and during rhythmic handgrip exercise. Our findings highlight the differential regulation of the peripheral and cerebral circulations via the sympathetic nervous system despite handgrip exerciseinduced increases in perfusion in both regions. The regulation of the cerebral circulation via the sympathetic nervous system remains controversial (Strandgaard & Sigurdsson, 2008; Ogoh & Ainslie, 2009; Ainslie & Tzeng, 2010). Although the cerebral vasculature is supplied with α-adrenergic nerves (Lowe & Gilboe, 1971; Edvinsson et al. 1976), and noradrenaline spillover from the human cerebral vasculature has been reported (Mitchell et al. 2009), studies employing direct electrical stimulation of sympathetic nerves in animals (Auer et al. 1983; Wagerle et al. 1983), and pharmacological activation of α-adrenergic receptors (e.g. noradrenaline) in either animals (Edvinsson et al. 1979) or humans (Brassard et al. 2010; Lucas et al. 2010) have reported inconsistent effects on the cerebral vasculature. Despite such equivocal findings, the tonic activity of the autonomic nervous system has been speculated to contribute to the regulation of the cerebral circulation in humans (Zhang et al. 2002), particularly in response to acute perturbations in BP (Ogoh et al. 2008; Hamner et al. 2010). Furthermore, sympatho-excitatory manoeuvres (e.g. cold pressor test, whole-body heating or lower body negative pressure) have been reported to evoke reductions in cerebral perfusion and/or increases in cerebrovascular resistance (Micieli et al. 1994; Roatta et al. 1998; Wilson et al. 2005; Low et al. 2009). Importantly, the cerebrovascular responses to the cold pressor test are attenuated following administration of clonidine, an α 2 -adrenergic receptor agonist with a known ability to inhibit the central actions of noradrenaline, suggesting that the cold pressor A FBF (ml min 1 kg 1 ) B FVC (ml min 1 kg 1 mmhg 1 ) C 80 D 1.0 MCA V mean (cm s 1 ) CVCi (cm s 1 mmhg 1 ) Rest 10% 25% 40% 0.0 Rest 10% 25% 40% Figure 1. Forearm blood flow (FBF; A), forearm vascular conductance (FVC; B), middle cerebral artery mean blood flow velocity (MCA V mean ; C) and cerebrovascular conductance index (CVCi; D) atrestand during rhythmic handgrip exercise at 10, 25 and 40% maximal voluntary contraction P < 0.05 versus rest.

6 1094 D. Hartwich and others Exp Physiol pp test elicits intracerebral vascular changes via a central noradrenergic mechanism (Micieli et al. 1994). Given that the cold pressor test also evokes increases in sympathetic vasoconstrictor outflow to the peripheral vasculature (Victor et al. 1987) it was used in the present study to examine the potential for differential regulation of the peripheral and cerebral circulations via the sympathetic nervous system. Notably, we observed much more pronounced reductions in vascular conductance within the skeletal muscle vasculature ( 45 ± 8%) compared with the cerebral circulation ( 5 ± 2%), indicative of a much greater responsiveness to sympathetic stimulation in the peripheral compared with the cerebral vasculature at rest. In contrast to the peripheral circulation, few studies have examined the neural regulation of the cerebral circulation during exercise in humans (Ide et al. 2000) and its potential interaction with other established regulatory factors (e.g. local metabolism). In a recent study, we compared the cerebrovascular responses elicited by voluntary exercise with those elicited by electrically evoked exercise where the parallel activation of central motor and cardiovascular centres is absent (i.e. central command) but peripheral feedback remains intact (exercise pressor reflex; Vianna et al. 2009). Of note, while cerebral perfusion increased and CVCi was unchanged during volitional calf exercise, during A % ΔFVC B % ΔCVCi Rest 10% 25% 40% Figure 2. Vasoconstrictor responses to cold pressor test at rest and during three levels of rhythmic handgrip exercise (10, 25 and 40% maximal voluntary contraction) expressed as the percentage change in FVC (A) and as the percentage change in CVCi (B) P < 0.05 versus rest. electrically evoked exercise the cerebral blood flow velocity was unchanged and CVCi fell. The reason for these differential responses is unclear, particularly as BP and CO were increased to a similar extent during both exercise regimes. However, a potential explanation may be that during voluntary exercise local dilator influences resulting from increases in cerebral metabolism related to the activation of central command predominated over any potential cerebral vasoconstrictor stimuli elicited by exercise. In support of a metabolic modulation of the sympathetic regulation of cerebrovascular tone during exercise, Brassard et al. (2010) recently demonstrated that phenylephrine-mediated reductions in frontal lobe oxygenation were attenuated during leg cycling. However, it has been suggested, given the influence of heat stress on increases skin blood flow (Davis et al. 2006), that care should be taken when interpreting changes in cerebral oxygenation obtained by nearinfrared spectroscopy from rest to exercise (Rasmussen & Lundby, 2010). Furthermore, the secondary systemic haemodynamic effects of phenylephrine (e.g. transient hypertension, reductions in cardiac output and changes in sympathetic nerve activity) may also complicate the interpretation of these observations (Ainslie & Tzeng, 2010). In the present study, we observed that nonpharmacological, endogenous sympathetic stimulation with the cold pressor test evoked small reductions in CVCi that were consistent from rest to handgrip exercise. These findings are in stark contrast to the simultaneous and marked reduction in the vasoconstrictor responses in the exercising skeletal muscle vasculature (functional sympatholysis) observed by ourselves and others (Remensnyder et al. 1962; Hansen et al. 1996; Tschakovsky et al. 2002; Keller et al. 2003, 2004; Watanabe et al. 2007). While the elucidation of the metabolites responsible for such modulation of sympathetic vasoconstrictor tone is beyond the scope of the present study, these findings indicate that the utilization of the cold pressor test and rhythmic handgrip represents a robust experimental model for examining the neural control of the peripheral circulation. In the present study, rhythmic handgrip exercise evoked expected increases in blood flow to the exercising forearm muscles and also increases cerebral perfusion. We assessed cerebral perfusion using a transcranial Doppler assessment of MCA V mean because the MCA supplies the brain region associated with the cortical representation of the forearm muscles and has previously been shown to increase particularly during contralateral handgrip exercise (Linkis et al. 1995). The magnitude of the increase in MCA V mean reported is similar to that previously observed during handgrip (Pott et al. 1997; Ide et al. 1998) and may be at least partly stimulated by exercise-induced elevations in cerebral neural activity (Secher et al. 2008; Vianna et al. 2009). Changes in BP and CO may also

7 Exp Physiol pp Sympathetic regulation of cerebral circulation 1095 contribute to exercise-induced increases in MCA V mean (Ogoh et al. 2005; Secher et al. 2008); however, in the present study neither BP nor CO significantly increased during handgrip. In contrast to the peripheral circulation, cerebral vascular tone is strongly controlled by arterial P CO2 both at rest and during exercise (Ainslie et al. 2005). In the present study, P ET,CO2 decreased slightly during handgrip and cold pressor tests; thus, changes in MCA V mean and CVCi may have been slightly underestimated during handgrip and slightly overestimated during cold pressor tests. However, the small reductions in P ET,CO2 were consistent between handgrip bouts and cold pressor tests, suggesting that any effect was similar between trials. It has recently been demonstrated that a cholinergic vasodilator mechanism plays a critical role in mediating the exercise-induced increase in cerebral perfusion (Seifert et al. 2010). As such, the contribution of a sympathetic cholinergic mechanism to the regulation of cerebral blood flow during exercise may warrant further investigation (Hamel, 2006). Methodological considerations A potential limitation of the present study may derive from our use of MCA V mean as an indicator of cerebral perfusion. We acknowledge that the transcranial Doppler method only allows velocity measurements and is only reflectiveofelevationsinregionalcerebralbloodflowif the diameter of the insonated vessel remains unchanged. However, MCA V mean and MCA blood flow appear to be closely related (Kirkham et al. 1986), while MCA V mean increases in parallel with the inflow of the internal carotid artery (Hellström et al. 1996) and the initial slope index of the 133 Xenon clearance-determined cerebral blood flow (Jørgensen et al. 1992). We used a cold pressor test to evoke generalized and robust sympathetic nervous system activation because it has previously been reported to elicit elevations in BP, HR (Hines & Brown, 1933), plasma noradrenaline (Halter et al. 1984), muscle sympathetic nerve activity (Victor et al. 1987) and vasoconstriction within the skeletal muscle and cerebral circulations (Micieli et al. 1994). However, given recent work indicating that the α-adrenergic receptors on cerebral blood vessels are located outside the blood brain barrier (Mitchell et al. 2009), it is possible that cold pressor test-induced increases in circulating catecholamines may have contributed to the cerebrovascular responses reported. A further consideration is that it is unclear whether the handgrip exercise intensities employed influenced the findings of the presentstudy.indeed,itispossiblethatrhythmichandgrip exercise did not produce a sufficient increase in cerebral metabolism to modulate sympathetic activation (Secher et al. 2008). Although we were unable to assess cerebral activation and/or metabolism during exercise directly, handgrip exercise has previously been demonstrated to increase neuronal activity in the contralateral primary motor cortex (Sander et al. 2010), and regional increases in perfusion are known to be coupled with the cortical representation of the exercising muscle group (Linkis et al. 1995). Notably, we used low to moderate handgrip exercise workloads to avoid exercise-induced increases in sympathetic nerve activity (Victor & Seals, 1989), which could potentially confound the evaluation of the vasoconstrictor responses to sympathetic stimulation with a cold pressor test between rest and exercise (Hansen et al. 2000). Finally, although data from one female subject were included in the present study, our results were not affected when this subject was excluded. Furthermore, to limit any potentially confounding effects elicited by hormonal status, testing was undertaken in the early follicular phase, when oestrogen and progesterone concentrations are low. 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