The Cholinergic Blockade of Both Thermally and Non-Thermally Induced Human Eccrine Sweating

7/25/2019 The Cholinergic Blockade of Both Thermally and Non-Thermally Induced Human Eccrine Sweating

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930 Exp Physiol97.8 (2012) pp 930942

Research PaperResearchPaper

The cholinergic blockade of both thermally and

non-thermally induced human eccrine sweatingChristiano A. Machado-Moreira1, Peter L. McLennan2, Stephen Lillioja2,3, Wilko van Dijk1,

Joanne N. Caldwell1 and Nigel A. S. Taylor1

1Centre for Human and Applied Physiology, School of Health Sciences,2Graduate School of Medicine and3 Illawarra Health and Medical Research Institute, University of Wollongong, Wollongong, NSW 2522, Australia

Thermally induced eccrine sweating is cholinergically mediated, but other neurotransmitters

have been postulated for psychological (emotional) sweating. However, we hypothesized

that such sweating is not noradrenergically driven in passively heated, resting humans. To

test this, nine supine subjects were exposed to non-thermal stimuli (palmar pain, mental

arithmetic and static exercise) known to evoke sweating. Trials consisted of the following four

sequential phases: thermoneutral rest; passive heating to elevate (by1.0C) and clamp mean

body temperature and steady-state sweating (perfusion garment and footbath); an atropine

sulphateinfusion(0.04 mg kg1)withthermalclampingsustained;andfollowingclampremoval.

Sudomotor responses from glabrous (hairless) and non-glabrous skin surfaces were measured

simultaneously (precursor and discharged sweating). When thermoneutral, these non-thermal

stimuli elicited significant sweating only from the palm (P< 0.05). Passive heating induced

steady-state sweating ranging from 0.20 0.04 (volar hand) to 1.40 0.14 mg cm2 min1

(forehead), with each non-thermal stimulus provoking greater secretion (P< 0.05). Atropine

suppressed thermal sweating, and it also eliminated the sudomotor responses to these non-

thermal stimuli when body temperatures were prevented from rising (P> 0.05). However,

when the thermal clamp was removed, core and skin temperatures became further elevated andsweating was restored (P< 0.05), indicating that the blockade had been overcome, presumably

through elevated receptor competition. These observations establish the dependence of both

thermal and non-thermal eccrine sweating from glabrous and non-glabrous surfaces on

acetylcholine release, and challenge theories concerning the psychological modulation of

sweating. Furthermore, no evidence existed for the significant participation of non-cholinergic

neurotransmitters during any of these stimulations.

(Received 5 February 2012; accepted after revision 5 April 2012; first published online 11 April 2012)

Correspondingauthor N. A. S. Taylor: Centre forHuman and Applied Physiology, School of HealthSciences, University

of Wollongong, Northfields Avenue, Wollongong, NSW 2522, Australia. Email: nigel [email protected]

Human eccrine sweat secretion during thermal loading ismodulated by sympathetic, cholinergic neurons (Dale &Feldberg, 1934; Chalmers & Keele, 1952; Landis, 1990;Kimura et al. 2007) and controlled by the preopticanterior hypothalamus (Hardyet al.1964; Boulantet al.1989). However, individual eccrine glands are innervatedby several neurons (Kennedy et al. 1994) and possessreceptors for bothacetylcholine and noradrenaline(Reddyet al. 1992; Weihe et al. 2005). Moreover, Uno (1977)and Donadio et al. (2006) reported the existence of

noradrenergic neurons in close proximity to these glands,albeit less dense in their distribution, while others haveshown that noradrenergic agonists can elicit sweating(Haimovici, 1950; di SantAgnese et al. 1953; Allen &Roddie, 1972; Wolf & Maibach, 1974; Sato & Sato, 1981).Collectively, these observations form the backgroundfrom which some have suggested that human eccrinesweat glands may be innervated by both cholinergic andnoradrenergic neurons that are capable of independentglandular activation (Robertshaw, 1977; Noppen et al.

DOI: 10.1113/expphysiol.2012.065037 C2012 The Authors. Experimental Physiology C2012 The Physiological Society


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Exp Physiol97.8 (2012) pp 930942 Cholinergic modulation of non-thermal sweating 931

1997; Nakazato et al. 2004). The functional significanceof these interpretations was evaluated within this study.

Altered affective states can also induce eccrine sweating(Harrison & MacKinnon, 1966; Homma et al. 2001;Kobayashiet al.2003), and such sweating occurs withouttissue temperatures changing. This form of non-thermal(psychological or emotional) sweating in thermoneutralindividuals is thought by some to be induced via separateefferent pathways (List & Peet, 1938; Chalmers & Keele,1952), which are possibly of a noradrenergic phenotype(Robertshaw, 1977; Noppen et al. 1997; Nakazato et al.2004). Moreover, others have suggested that psychologicalsweating is controlled by a different, possibly cortical,centre (Darrow, 1937; Kuno, 1956; Ogawa, 1975) and thatthe autonomic efferents innervate only the glabrous (non-hairy) skin of the hands and feet (Darrow, 1937; Kuno,1956; Ogawa, 1975). Indeed, Kuno (1956) proposed thatthermal stimulation will inhibit psychological sweating,whilst psychological provocation acts to suppress thermal

sweating. The current authors have long consideredsome aspects of these hypothetical control mechanismsto be imprecise. Indeed, recent research from ourlaboratory has demonstrated psychological sweating to beubiquitous in its distribution, and certainly not restrictedto the glabrous skin surfaces, when evoked either inpassively heated (Machado-Moreira & Taylor, 2012a)or in thermoneutral individuals (Machado-Moreira &Taylor, 2012b). Therefore, the regional distribution ofpsychological sweating and the inhibitory nature ofpsychological stimuli upon thermal sweating were re-evaluated across glabrous and non-glabrous skin surfaces,

with and without systemic cholinergic blockade. As thecholinergic pathways dominate sudomotor control, it washypothesized that residual psychological sweating, in thepresence of this blockade, would reflect the participationof a non-cholinergic mechanism.

Cholinergic sudomotor blockades have been usedfor decades (Chalmers & Keele, 1951), but severalinvestigators have described either an incomplete ora transient suppression of both thermal (Rodman,1952; Cummings & Craig, 1967; Gibinski et al. 1973;Shastry et al. 2000) and non-thermal (psychological)sweating (Allen et al. 1972; Wolf & Maibach, 1974).Such evidence has been used to support the premise that

neurotransmitters other than acetylcholine participatein the control of eccrine sweating. However, as thesecompetitive blockades also reduce evaporative heat loss,then, in the continued presence of a fixed externalthermal load, one may observe a further elevationin body temperature, as reported by Clark & Lipton(1985). In this circumstance, there will be increasedthermoreceptor feedback and an elevated acetylcholinerelease from the sudomotor neurons, possibly leadingto a blockade breakthrough. This will re-establish sweatsecretion (Rodman, 1952) and it may, perhaps incorrectly,

be interpreted to reflect the participation of an alternativemode of sweat gland activation.

With this possibility in mind, the present experimentwas designed using a whole-body thermal clamp (water-perfusion suit and footbath) to stabilize core temperature(Cotter & Taylor, 2005) after it had been passivelyelevated, and to sustain that clamped state during asystemic cholinergic blockade. This technique establishesconditions in which both the whole-body thermal energycontent and thermoreceptor feedback are increasedand held constant, thereby ensuring that sympatheticsudomotor drive is elevated and stable (open-loopcontrol). The aim was to clamp thermoafferent activityso that the sweating responses arising from changes inthe efferent neural activity accompanying several non-thermal stimulations could be isolated. These methodsprovided the experimental conditions necessary to testthe hypothesis that psychological sweating is not of anoradrenergic origin, either at the glabrous or at the non-

glabrous skin surfaces. This was achieved through therepeated application of standardized, non-thermal stimuliknown to provoke sweat secretion in thermoneutralindividuals (Kuno, 1956; Abramet al.1973; Amanoet al.2011; Machado-Moreira & Taylor, 2012b). Finally, todemonstrate the importance of this clamping procedurewithin the experimental design, the thermal clamp wasremoved to induce a blockade breakthrough.

Methods

Ethical approval

The procedures for this research were approved bythe Human Research Ethics Committee (University ofWollongong) in accordance with the regulations of theNational Health and Medical Research Council (Australia)and in compliance with the Declaration of Helsinki. Allparticipants provided written, informed consent.

Subjects

Nine healthy males (mean SD: 29.7 9.0 years old,179.5 9.8 cm tall and weighing 73.54 7.22 kg)

participated in a single, resting trial composed of foursequential phases. Participants were not taking anymedication, nor did they have a history of cardiovascularor thermal illness.

Procedures

Subjects wore a swimming costume and water-perfusionsuit (for details see Thermal stimulation and clamping)and remained supine during both preparation and testing.Every trial was conducted within a climate-controlled

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932 C. A. Machado-Moreira and others Exp Physiol97.8 (2012) pp 930942

chamber using the same, four-phase sequence (Fig. 1), asfollows: thermoneutral rest (control); passive heating withthermal clamping to establish a constant thermal load andto elicit steady-state thermal sweating; atropine-inducedcholinergic blockade with the thermal clamp sustained;and removal of the thermal clamp with a concomitantelevation in mean body temperature.

Firstly, to establish baseline responses, sweating wasevaluated in thermoneutral conditions using three non-thermal stimuli. These included two psychological stresses(acute pain for 15-s and cognitive challenge for 2-min) and static, hand-grip exercise (for 2-min). Eachstimulus was separated by a 3-min recovery period torestore the pretreatment sweating baseline. This wasthe control phase, and a climate chamber (27.528C)and water-perfusion suit (33C) were used to clampthis thermoneutral state. Secondly, passive heating wasinitiated(airtemperature, 27.528C; water temperatures,perfusion suit 48C and footbath 42C), with the aim

of elevating core and mean body temperatures about 0.5and 1C above their respective thermoneutral baselinesfor each individual. This was important, because thesetemperatures are beyond the thresholds for sweating.In combination with a skin temperature increase of2.5C, thermal sweating is primed, and a whole-body sweat rate of about 0.30 mg cm2 min1 couldbe anticipated (Machado-Moreira & Taylor, 2012a).Moreover, as noradrenergic sudomotor activation hasbeen postulated within thermoneutral individuals, it

was essential to minimize overall thermal strain andensure that systemic adrenergic influences were avoided.Passive heating established steady-state, thermal sweatingover 2036 min, after which core temperature wasclamped (changing


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Pilot testing. Extensive pilot research led to the useof several strategies necessary to test the workinghypothesis. Preliminary work involved determining themost appropriate blocking agent (intravenous atropine inpreference to oral hyoscine), the atropine dose infused(0.04 mg kg1), the non-thermal treatments and theirapplication durations, the order of these treatmentsand the interstimulus recovery period (Machado-Moreira& Taylor, 2012a,b). These are discussed briefly insubsequent sections. However, perhaps the most criticalpilot testing concerned the use of whole-body thermalclamping techniques (Cotter & Taylor, 2005). When firstused, atropine sulphate fully suppressed sweat secretion.However, body temperature soon began to rise further,followed by a return of sweating, albeit at a lower level,as others had noted (Rodman, 1952). When the threenon-thermal stimulations were superimposed upon thisslowly increasing secretion, clear sudomotor responseswere evident, and whilst sweating declined after each

stimulus, it did not return to a steady-state baseline, butcontinuedto rise. Thus,it wasunclear whether or nottheseresponses reflected a non-cholinergic influence or merelya breakthrough of the blockade. As core temperatures rosesoon after the blockade was administered, as previouslyreported (Clark and Lipton, 1985), the latter possibilitywas suspected, and it was deemed essential to prevent thisand the accompanying elevation in thermoafferent feed-back. This required thermal clamping.

Thermal stimulation and clamping. The climate chamberwas regulated at 27.528C throughout every trial.

A water-perfusion suit and footleg bath heated andclamped skin and core temperatures. This thermal clampwas used during the control, passive heating and blockadephases of each trial. The perfusion suit was constructedfrom a network of tubing (180 m Tygon

Rtubing;

i.d.= 1.58 mm; o.d.= 3.0 mm) arranged in parallel, 1-mlengths to form anterior and posterior jackets and trousers(Paul Webb Associates, Yellow Springs, OH, USA), but thehead, hands and feet were not covered. Adjacent tubeswere linked to form a diamond pattern (8 cm 4 cm)such that the temperature of approximately 90% of theskin surface could be modified and clamped (Cotter &Taylor, 2005). These linkages optimized skin contact andskin temperature control, but the suit contained no othercomponents, nor was it covered with other garments. Thispermitted precise temperature control while optimisingevaporative cooling. Two stirred water baths (38 litre;Grant Instruments (Cambridge) Ltd, Shepreth, UK)regulated the temperature of water delivered to thisgarment. A third bath was used for immersing the feetand lower calves. The water temperature for each bathwas independently and thermostatically regulated, withsmall volumes of water at slightly different temperatureadded to facilitate fine control of the clamp using core

temperature feedback. As a consequence, at the onset ofpassive heating, subjects were exposed to sudden heating.However, once clamping was achieved, subtle and gradualwater temperature adjustments were used, with thesechanges always being


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Thus, to reveal the possibility for even a low levelof non-cholinergically driven sweating, this backgroundsweating first needed to be removed, and this was alsobest achieved using a systemic blockade. Under doserestrictions placed upon this investigation, hyoscine wasunable to suppress thermal sweating fully (pilot testing).Therefore, atropine sulphate was chosen and it wasadministered intravenously [dorsal hand; 0.04 mg kg1;average dose, 2.94 mg (SD 0.29); AstraZeneca Pty Ltd,North Ryde, NSW, Australia]. This dose was based onprevious research (Cummings & Craig, 1967; Gibinskiet al.1973; Sawkaet al.1984), with its efficacy evaluatedthrough pilot testing. To standardize and maximize drugdelivery, a timed infusion rate was used (1-min) with asterile saline (0.9%) flush (5 ml) following delivery. As thisexperiment was designed to evaluate fully the possibilitythat these non-thermal stimuli might elicit sweating, evenin the presence of this blockade, two further (local)blockades were planned. Firstly, bretylium tosylate was to

be used to block local noradrenaline release. Secondly, ifnon-thermal sweating still persisted, botulinum toxin wasto be administered locally to exclude the cotransmissionof other sudorific agents. However, in our hands,systemic atropine totally suppressed both thermal andnon-thermal sweating in every subject during thermalclamping, obviating the need for these supplementaryblockades.

Standardization. Subjects acted as their own controlswithin a single trial, with ambient conditions stable

throughout. Subjects were instructed to refrain fromstrenuous exercise on the day preceding testing, notto consume caffeine or alcohol for 12 h preceding theexperiment, and to present in a well-hydrated state,following a high-carbohydrate breakfast. On arrival, urinespecific gravity was measured (Clinical Refractometer,model 140; Shibuya Optical, Tokyo, Japan) to confirmhydration state [mean= 1.016 (SD 0.007)]. Neither foodnorliquid wasconsumed during testing, and subjects wereblindfolded throughout the experiment, except duringthe cognitive and exercise challenges. The use of 3-min interstimulus recovery periods ensured that thesudorific effect of each non-thermal treatment was notcarried through into the subsequent treatments. This wasestablished throughpilot testing, and verified within everytrial by the return to pretreatment sweating baselines(where present). It was therefore felt that a randomizedpresentation order of these stimuli was not required,and as the time between subsequent reapplications ofany one stimulus was at least 30 min, the residualeffects would not be carried into the next experimentalphase. At the completion of every experiment, recoverywas medically supervised, and subjects were drivenhome.

Measurements

Auditory canal temperature was monitored using an ear-moulded plug with a thermistor protruding 1 cm (EdaleInstruments Ltd, Cambridge, UK), and positioned withinthe external auditory meatus and insulated with cottonwool. The water-perfusion garment did not contact facial

tissues. These procedures isolate the auditory canal fromthermal artefacts, permitting auditory canal temperatureto track oesophageal temperature faithfully and rapidly inthese conditions (Cotter et al. 1995). Skin temperatureswere measured from eight skin sites (forehead, chest,scapula, upper arm, forearm, dorsal hand, thigh and calf;Type EU; Yellow Springs Instruments Co. Ltd, YellowSprings, OH, USA). All thermistors were calibratedagainsta certified reference thermometer in a stirred water-bath(Dobros total immersion; Dobbie Instruments, Sydney,NSW, Australia). These data were collected at 5-s intervals(1206 Series Squirrel; Grant Instruments (Cambridge)Ltd). Mean skin temperature was derived from a weightedsummation of the eight local temperatures (ISO 9886,1992), with mean body temperature taken as 80% of thecore plus 20% of the mean skin temperature (Hardy &DuBois, 1938). Heart rate was monitored continuously(5-s intervals) from ventricular depolarization (VantageNV, Polar Electro Sport Tester; Kempele, Finland).

Local surface sweat rates (discharged sweat) weremeasured simultaneously from two glabrous [hairlesssites; forehead and volar (palmar) surface of the righthand] and three non-glabrous skin surfaces (hairy sites;dorsal surface of the right forearm, dorsal surface of theright hand and upper medial surface of the right calf).

These measurements were performed using ventilatedsweat capsules (3.16 cm2) glued to the skin to preventleakage and pressure-induced artefacts (Collodion USP;Mavidon Medical Products, FL, Lake Worth, USA). Low-humidity air was obtained by passing room air overan enclosed, saturated, lithium chloride solution housedoutside the chamber, with local air temperature measured.Air collected above this solution remains at 12% relativehumidity over a broad range of temperatures. This air waspumped through each sweat capsule at a flow that ensuredcomplete evaporation (600 ml min1), and through tubeslong enough to guarantee thermal equilibration with

the internal air temperature. Postcapsular (exhaust) airtemperatures (thermistors) and humidities (capacitancehygrometers) were continuously sampled downstream(1-m) as part of an integrated sweat monitor system(Clinical Engineering Solutions, NSW, Sydney, Australia).Temperature and humidity sensors were equilibrated withambient conditions prior to each trial. Data were recordedat 1-s intervals (DAS1602; Keithley Instruments, Inc.,Cleveland, OH, USA) and used to derive local sweatrates (Taylor et al. 1997). Hygrometer calibration, usingsaturated solution standards, preceded experimentation.



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Sweat capsules can only be used to measure surface(discharged) sweat, and low production rates of precursor(primary) sweat can result in complete fluid reabsorptionwithin the sweat duct (Bullard, 1971). Such sweat wouldnot be evident at the skin surface, nor could it be detectedgravimetrically. As it was also necessary to evaluatesweat production during non-thermal stimulations inthermoneutral conditions, which are known to elicit low-level sudomotor responses, a technique was requiredthat could detect precursor sweat secretion. Changes inskin conductance have long been used for this purpose(Veraguth, 1908; Thomas & Korr, 1957; Machado-Moreiraet al.2009), and this method was particularly importantduring the thermoneutral and systemic blockade phases.In the latter phase, an absence of surface (discharged)sweat would not have been sufficient evidence to justify aclaim for complete sweat suppression (Machado-Moreiraet al. 2009). A pair of AgAgCl surface electrodes (1081FG) was positioned adjacent to the sweat capsule on

the volar (glabrous) surface of the right hand, becausethe probability of detecting psychological sweating inthermoneutral individuals was much greater from thatsite (Machado-Moreira & Taylor, 2012b). An electrolyteof 0.05 M sodium chloride in an inert ointment baseprovided the conductive medium, and a constant voltageof 0.5 V was applied across each electrode pair. Data wererecorded at 10 Hz (UFI Bioderm model SC2000/4-SCLSimple Scope Data Collection System; UFI, Morro Bay,CA, USA; connected to a computer).

Analysis

The effect of each stimulus was evaluated by expressingsweat flow and skin conductance as change scoresrelative to prestimulus baselines, and averaged across eachstimulation. The dynamics of the atropine-induced sweatsuppression were derived assuming exponential decays(Machado-Moreira et al. 2010). One-way ANOVA, withrepeated measures, was used to evaluate interconditiondifferences (control, passive heating and blockade).Tukeys honestly significant differencepost hoctests wereused to isolate sources of significant differences. Withinthe same condition, Students paired ttests were used tocompare sweat rates immediately prior to, andduringeachnon-thermal stimulus, and also sweating and temperaturedata before and after removing the thermal clamp. For allanalyses, was set at the 0.05 level. Data are presented asmeans SEM and standard deviations (SD).

Results

Phase one: control (thermoneutral) state

During the control phase, the baseline thermal status ofeach participant was clamped within the thermoneutral

range, with the core, skin and mean body temperaturesaveraging 36.6 (SD 0.2), 33.7 (SD 0.3) and 36.0C (SD0.3), respectively. The corresponding mean pretreatmentheart rate was 58 beats min1 (SD 8), which increasedto average 78 beats min1 across the three non-thermalstimuli (SD 11;P< 0.05). However, none of the thermalvariables changed significantly during these challenges(P> 0.05). It was therefore assumed that all sudomotorresponses during this phase could be attributed to neuralevents associated with these stimulations, and were notassociated with an altered thermal state.

When subjects were thermoneutral, the application ofeach non-thermal stimulus resulted in significant sweatsecretion from the volar (glabrous) surface of the hand(P< 0.05; Fig. 2), as evidenced by changes in both skinconductance and discharged sweat. However, minimalsudomotor activity was apparent from each of the othersites (P> 0.05; Fig. 2).

Phase two: passively heated state

Passive heating [26 min (SD 6)] resulted in significantelevations in core [0.5C (SD 0.2);P< 0.05], mean skin[2.5C (SD 0.4);P< 0.05] and mean body temperatures[0.9C (SD 0.2); P< 0.05]. As the feet and lower legswere immersed in hot water, the calf skin temperatureincreased from 33.1 (SD 0.5; phase one) to 36.7C (SD 2.0;phase two; P< 0.05). Thermal clamping sustained thisstate, with core temperatures changing non-significantlythereafter ( 0.05). Heart rate increased from

itsthermoneutral,pretreatment baselineto 76 beats min1

(SD 12; P< 0.05) after the thermal clamp had beenestablished. In this state, discharged sweating was evidentfrom every glabrous and non-glabrous site, whichdisplayed steady-state baselines of 1.40 0.14 (forehead),0.78 0.18 (calf), 0.74 0.08 (dorsal hand), 0.20 0.04(volar hand) and 0.33 0.05 mg cm2 min1 (forearm).From these data, it was concluded that the thermalclamp was successful, and it was further assumed thatsubsequent sudomotor responses would be the resultof superimposing the non-thermal stimuli onto thisbackground of steady-state (primed) thermal sweating.

Significantly more sweat was discharged from mostsites in all subjects, relative to both the control andprimed steady-state conditions, when exposed to the threenon-thermal challenges (P< 0.05; Fig. 2). Thus, none ofthese stimulations inhibited thermal sweating (P> 0.05;Fig. 2), although discharged sweat from the calf duringmental arithmetic, and from the volar hand during eachstimulation, did not differ significantly from the controlphase (P> 0.05). Sweating and the electrodermal changesin response to these stimulations, measured from the volar(glabrous) hand surfaces, were large, but were equivalentto responses observed within the control phase (P> 0.05;



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Figure 2. Changes in local sweat rates and skin conductance (volar hand) accompanying a painful

stimulus (A; 15 s), mental arithmetic (B; 2-min) and static exercise (C; 2-min)

These non-thermal stimuli were applied in the thermoneutral (control) state, following passive heating and finally

after establishing a systemic cholinergic blockade (atropine) in the presence of a thermal clamp. Baseline sweat

rates (in milligrams per square centimetre per minute) appear in parentheses next to the abscissa labels of Fig. 2 C. Significantly different from the control and blockade conditions (P< 0.05); and significantly different from the

blockade phase (P< 0.05). Data are means SEM, with the sample size being nine for all variables except for

forehead sweating (n = 7) and volar hand skin conductance (n = 8).



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Fig. 2). Therefore, thermal loading did not inhibit thesenon-thermal sudomotor responses (P> 0.05; Fig. 2).Heart rate was again significantly elevated in responseto these stimuli (P< 0.05), averaging 94 beats min1

(SD 12) across these challenges. Following the ensuingstimulus, baseline sweating was restored during eachrecovery period, to be not significantly different from theimmediate pretreatment levels (P> 0.05). This was animportant standardization strategy.

Phase three: systemic blockade condition

Application of the systemic cholinergic blockade almostimmediately released the parasympathetic brake on heartrate (Fig. 3A). As a consequence, heart rate increasedby 40 4 beats min1 (P< 0.05) relative to its heatedand clamped baseline, prior to the first non-thermalstimulation, and now averaged 119 beats min1 (SD 12).

It took 510 min to suppress sweating from all sites

completely, which, on average, returned to preheated

baselines 4.9 0.2-min after injection (Fig. 3B). Indeed,the time constant of this decay was 2.4 0.1 min acrosssites, with no significant intersite variations (P> 0.05).These changes verified the whole-body nature of theblockade. Throughout the non-thermal stimulations, thethermal clamp prevented body temperature elevation,with core temperature changes kept 0.05;Fig. 3A). Likewise, skin and mean body temperaturesvaried by 0.05). Collectively, theseprocedures established the physiological conditionsnecessary to test the working hypothesis.

From this state, it was assumed that non-thermalsudomotor responses, if present, could be attributableto non-cholinergic mechanisms. However, each stimulusfailed to elicit significant precursor or discharged sweatfrom either the glabrous or the non-glabrous skin surfaces(P> 0.05; Fig. 2). Nevertheless, the chronotropic responseto each non-thermal stimulus remained and was againsignificantly elevated, albeit less so. When averaged across

these stimuli, the heart rate was now 130 beats min1

Figure 3. Core temperature, heart rate (A) and local sweating responses (B) during thermoneutral

rest (control baseline) and for the 5 min of thermal clamping prior to initiating a systemic cholinergic

blockade (atropine infusion at time 0), and for 15 min afterwards

Data are mean curves, with means and SEMs indicated by the symbols.



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(SD 12; P< 0.05). That is, even in the presence of anatropine-induced tachycardia, these psychological andexercise challenges were able to elicit cardiac, but notsudomotor responses.

Phase four: thermal clamp removedAt the end of each trial, water-bath temperatures wereincreasedtoequalthoseusedduringpassiveheating(phasetwo). Restoring these temperatures, in conjunction withreduced evaporation due to the blockade, led to increasesin core (0.4 0.1C; P< 0.05), mean skin (1.1 0.2C;P< 0.05) and mean body temperatures (0.5 0.1C;P< 0.05) over the next 15 min. In these conditions, calfskin temperatureincreased from 36.0 (SD0.9; phase three)to 37.7C (SD 1.0; phase four; P< 0.05). These changeswere accompanied by significant increases in dischargedsweating from every site (Fig. 4; P< 0.05) except for

the volar surface of the hand (P> 0.05), even thoughsubjects were still under the influence of atropine. Thiswas consistent with the breakthrough of a competitiveblockade, and it was hypothesized to be due to theincreased sympathetic drivethat accompanied the gradualelevation in mean body temperature. This temperaturedependence was evident in every subject (Fig. 5).

Discussion

Firstly, this four-phase experiment has confirmedobservations that, when each of these non-thermal

treatments is applied to passively heated individuals,

increments in eccrine sweating are not restricted to theglabrous skin surfaces (Kuno, 1956; Kennard, 1963; Allenet al.1973; Machado-Moreira & Taylor, 2012a). Secondly,these data show no evidence for a reciprocal inhibitionbetween the thermal and non-thermal drives for sweating,as had been postulated by Kuno (1956) and accepted byothers (Ogawa, 1975; Ogawa et al. 1977; Quinton, 1987;Iwaseet al. 1997). Thirdly, during a systemic cholinergicblockade, it was demonstrated that none of the threenon-thermal stimuli could evoke either precursor ordischarged sweating when body tissue temperatures wereprevented from rising. These observations are, in thefirst instance, consistent with the universally acceptedcholinergic mediation of thermal sweating (Chalmers &Keele, 1951, 1952; Foster & Weiner, 1970). Moreover, theynow comprehensively extend this sudomotor control toinclude the non-thermal sudomotor responses from boththe glabrous and non-glabrous skin surfaces, as noted byAllen et al. (1973). This evidence rebuts the postulated

participation of noradrenergic efferents in the control ofpsychological sweating from the glabrous skin surfaces(Robertshaw, 1977; Noppen et al. 1997; Nakazato et al.2004; Weiheet al. 2005), even in the absence of thermalsweating. Finally, thermal clamping has revealed that,when a competitive blockade of this form is employed,residual sudomotor activity is not necessarily evidencefor the existence of other participating neurotransmitters.Indeed, it may simply represent an increased thermal loadleading to a blockade breakthrough.

Atropine crosses the bloodbrain barrier, making itimperfect for evaluating sudomotor function, because

one cannot exclude the possibility of central interactions

Figure 4. Local sudomotor and average core temperature responses prior to, and following the removal

of the thermal clamp, but with subjects under the influence of atropine

The vertical line indicates removal of the clamp (0 min). Data are mean curves, with means and SEMs indicated by

the symbols.



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in the control of sweating, or thermoregulation ingeneral. Central muscarinic receptor activation canmodify thermoregulation (Takahashi et al. 2001).However, the small drug-induced body temperaturedisplacements in animals (Gordon, 1994) are preventable(thermal clamping). Moreover, the systemic effectsof atropine match changes observed following itsintramuscular administration (Matthew et al. 1988).Indeed, Craig (1970) demonstrated, using three systemicinfusions (atropine sulphate, atropine methyl nitrate andscopolamine hydrobromide), that the sudomotor effectswere indistinguishable. Thus, the antagonistic thermalactions were almost exclusively peripheral in nature,because only atropine could cross the bloodbrain barrier.This stands in contrast to their different central nervoussystem potencies (Cheshire & Fealey, 2008). It is alsonotable that the inhibition of sweating by atropine istransient (Mirakhur & Dundee, 1980; Ellinwood et al.1990), in contrast to its prolonged inhibitory effects

on cognition and psychomotor performance (Ellinwoodet al. 1990). Furthermore, the acute, centrally mediatedtachycardia during each non-thermal stimulus wasunimpeded, revealing that only the peripheral sudomotorcomponent of the sympathetic response was blocked. Thepotential for a peripheral ganglionic blockade can equallybe excluded, because atropine did notinhibit theheart rateresponses. Therefore, whilst a central sudorific influencecannot absolutely be excluded, no evidence was found tosuggest such an action, andseveral piecesof circumstantialevidence strongly suggest that the inhibition of sweatingwas peripherally mediated, and at the end-organ level.

Several groups have previously reported incompletesweat suppression during cholinergic blockades applied

to resting, heated subjects (Rodman, 1952; Cummings& Craig, 1967; Allen et al. 1972; Gibinski et al. 1973;Shastryet al.2000) and also during exercise (Craig, 1952;Goldsmith et al. 1967; Sawka et al. 1984; Kolka et al.1986, 1989). Some of these studies had methodologicallimitations, leading one to question the veracity of theblockade. Forinstance, thefollowing four factors may leadto an incomplete or transient blockade: the choice of a lesseffective blocking agent (e.g. hyoscine; Goldsmith et al.1967); the use of local, rather than systemic, drug delivery(e.g. electrophoresis; Gibinskiet al.1973); intramuscularinjection (Wolf & Maibach, 1974; Kolkaet al.1986, 1989);and the use of an insufficient dose (Gibinski et al. 1973;Wolf & Maibach, 1974; Shastry et al. 2000). However,one cannot dispute either the reported reappearance orthe residual presence of discharged sweat during suchblockades within experiments that do not have theselimitations. Why is it, therefore, that some previousresearchers have failed to achieve complete and sustained

sweat suppression during a cholinergic blockade?Based on the present evidence (Figs 4 and 5), it is

now clear that residual sweating, or its reappearance,as seen within the pilot trials, was dependent uponthe concomitant rise in tissue temperatures in theabsence of thermal clamping. This increased the thermalstimulus and facilitated a blockade breakthrough. Indeed,such a systemic blockade transposes an otherwisephysiologically compensable thermal load into anuncompensable state, simply by reducing heat loss.Therefore, without a thermal clamp, one has no controlover thermoreceptor feedback, with the ensuing elevation

in body temperature stimulating sympathetic activity andincreasing acetylcholine release. As competitive receptor

Figure 5. The relationship between dorsal hand sweat secretion and mean body temperature for

atropinized individuals before and after removal of the thermal clamp



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binding is a dynamic process, the probability of eitherthe agonist (acetylcholine) or its antagonist (atropine)interacting with these muscarinic receptors is a simplefunction of their affinity for the receptor and theirrelative concentrations. With the atropine concentrationapproximating a state of equilibrium, at least over the timeframe in question, heightened sympathetic activity willincrease the probability of acetylcholine activating sweatgland receptors, and if this body temperature elevationpersists, then blockade breakthrough must eventuate.Whilst the present atropine dose (0.04 mg kg1) wassufficient to block steady-state sweating totally duringpassive heating with superimposed non-thermal stimuli,it is not a maximal blockade. This is illustrated in thefourth phase of every trial by the rapid rise in core(Fig. 4) and mean body temperatures (Fig. 5) whenparticipants were heated further. As this can readilyoccur in resting conditions, it is certainly possible duringexercise. However, systemic adrenaline release, which may

occur during exercise and can stimulate eccrine sweatglands (Sato & Sato, 1981; Mapleet al.1982), will increasethe probability of a second (humoral) agonist activatingthe sweat gland. For these reasons, one must be cautiouswhen attempting to interpret the cause of eccrine sweatingduring a cholinergic blockade alone.

The present experimental procedures resulted in thecomplete and sustained suppression of eccrine sweatingwhen each non-thermal stimulus was applied in thepresence of a whole-body thermal clamp. Indeed,clamping permitted the magnitude of the thermalstimulus to be fixed and controlled, thereby isolating the

sudomotor responses to these non-thermal stimulations.These conditions were critical to test the hypothesis thatpsychological sweating is not of a noradrenergic (neural)origin.

It is well recognized that eccrine sweat glands respondto catecholamines (Allen & Roddie, 1972; Sato &Sato, 1981; Maple et al. 1982; Reddy & Bell, 1996).This is not contested. However, it has been suggestedthat different neuroendocrine controls exist for thethermal (cholinergic) and non-thermal [cholinergic andadrenergic (both neural and humoral)] modulation ofsweating (Robertshaw, 1977; Macketal. 1986; Shields et al.1987; Noppen et al. 1997; Nakazato et al. 2004; Weihe et al.

2005). The present observations refute this hypothesis.Indeed, these data demonstrate that sweating during thesethermal, psychological and static exercise stimulationsappeared to be exclusively of a cholinergic origin. Whilstwe were prepared to block local noradrenaline release,had sweating persisted, there was no evidence for theexistence of either primary or discharged sweat followingsystemic atropine administration. Therefore, the presentobservations challenge the existence of functionallyrelevant noradrenergic efferents for the control of humaneccrine sweating in general, at least in resting participants

who have experienced a mean body temperature elevationof1C. More specifically, these data refute the possibilitythat psychological stimulationprovokes noradrenergicallymediated sweating from the glabrous skin surfaces(Robertshaw, 1977).

Conclusion

Thermal clamping and a systemic cholinergic blockadewere combined to evaluate the neurotransmitter controlof eccrine sweating responses from glabrous and non-glabrous skin during non-thermal stimuli (palmarpain, mental arithmetic and static exercise). Noevidence was found to support the physiologicallysignificant participation of neurotransmitters other thanacetylcholine. It is therefore concluded that, withinresting and mildly hyperthermic humans (mean bodytemperature elevation 1C), both thermally and

non-thermally mediated sweating are dependent uponcholinergic mechanisms at the sweat gland. This evidencechallenges existing theories concerning the psychologicalmodulation of sweating. Nevertheless, the capacity ofprogressively higher atropine doses to continue to blocksuch sweating with increments in body temperature hasnot been demonstrated. No doubt within hyperadrenergicstates (neural and humoral) that may obtain in moreextreme conditions (Rowell, 1990), atropine may becomeless effective, regardless of its dose. Therefore, untilsuch experiments have been performed, one cannotabsolutely exclude the possibility of noradrenergic neuralpathways influencing eccrine sweating in more stressfulstates.

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Acknowledgements

C.A.M.-M. was supported by a doctoral scholarship fromCoordenacao de Aperfeicoamento de Pessoal de Nvel Superior

CAPES (Ministry of Education, Brazil). J.N.C. was supported by

an Australian Postgraduate Award (Department of Innovation,

Industry, Science and Research, Australia).

http://http//dx.doi.org/10.1159/000210594http://http//dx.doi.org/10.1159/000210594

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The Cholinergic Blockade of Both Thermally and Non-Thermally Induced Human Eccrine Sweating

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