Evolution of peripheral nerve function in humans: novel insights from motor nerve excitability

J Physiol 591.1 (2013) pp 273–286 273

Evolution of peripheral nerve function in humans: novelinsights from motor nerve excitability

Michelle A. Farrar1,2, Susanna B. Park1,3, Cindy S.-Y. Lin1,4 and Matthew C. Kiernan1,3

1Neuroscience Research Australia, Randwick, Sydney, Australia2Department of Neurology, Sydney Children’s Hospital and School of Women’s and Children’s Health, University of New South Wales, Sydney, Australia3Prince of Wales Clinical School, University of New South Wales, Randwick, Sydney, Australia4School of Medical Sciences, University of New South Wales, Randwick, Sydney, Australia

Key points

• The evolution of human peripheral nerve function after birth to facilitate more complex neuraltasks has not been fully elucidated.

• The present study has established the changes that occur in nerve function in developinghumans using specialized non-invasive excitability techniques in infants, children, adolescentsand young adults for the first time.

• The activity of axonal K+ conductances reduces with formation of the axo-glial junction.This occurs simultaneously with alterations in passive membrane properties and conductance(axonal diameter and myelination).

• These functional alterations serve to enhance the efficiency and speed of impulse conduction,whilst maintaining membrane stability, concurrent with the acquisition of motor skills inchildhood.

• Significantly, these findings bring the dynamics of axonal development to the clinical domainand serve to further illuminate pathophysiological mechanisms that occur during development.

Abstract While substantial alterations in myelination and axonal growth have been describedduring maturation, their interactions with the configuration and activity of axonal membraneion channels to achieve impulse conduction have not been fully elucidated. The present studyutilized axonal excitability techniques to compare the changes in nerve function across healthyinfants, children, adolescents and adults. Multiple excitability indices (stimulus–response curve,strength–duration time constant, threshold electrotonus, current–threshold relationship andrecovery cycle) combined with conventional neurophysiological measures were investigated in57 subjects (22 males, 35 females; age range 0.46–24 years), stimulating the median motornerve at the wrist. Maturational changes in conduction velocity were paralleled by significantalterations in multiple excitability parameters, similarly reaching steady values in adolescence.Maturation was accompanied by reductions in threshold (P < 0.005) and rheobase (P = 0.001);depolarizing and hyperpolarizing electrotonus progressively reduced (P < 0.001), or ‘fanned-in’;resting current–threshold slope increased (P < 0.0001); accommodation to depolarizing currentsprolonged (P < 0.0001); while greater threshold changes in refractoriness (P = 0.001) andsubexcitability (P < 0.01) emerged. Taken together, the present findings suggest that passivemembrane conductances and the activity of K+ conductances decrease with formation of theaxo-glial junction and myelination. In turn, these functional alterations serve to enhance theefficiency and speed of impulse conduction concurrent with the acquisition of motor skillsduring childhood, and provide unique insight into the evolution of postnatal human peripheral

C© 2012 The Authors. The Journal of Physiology C© 2012 The Physiological Society DOI: 10.1113/jphysiol.2012.240820

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274 M. A. Farrar and others J Physiol 591.1

nerve function. Significantly, these findings bring the dynamics of axonal development to theclinical domain and serve to further illuminate pathophysiological mechanisms that occur duringdevelopment.

(Received 13 July 2012; accepted after revision 19 September 2012; first published online 24 September 2012)Corresponding author M. C. Kiernan: Neuroscience Research Australia, Barker St, Randwick, Sydney, NSW 2031,Australia. Email: [email protected]

Abbreviations CMAP, compound motor action potential; GBB, Barrett and Barrett conductance; GKfi, internodalfast K+ channels; T SD, strength–duration time constant; TE, threshold electrotonus; TEd(10–20), depolarizing changeat 10–20 ms in threshold electrotonus; TEd(90–100), depolarizing change at 90–100 ms; TEh(10–20), hyperpolarizingchange at 10–20 ms; TEh(90–100), hyperpolarizing change at 90–100 ms.

Introduction

The intricate structure and molecular organization of thehuman myelinated axon is important in determining theefficient conduction of nerve impulses. Development ofthe peripheral nerve commences between 4 and 6 weeksgestation in humans, with nerve fibres growing out fromneuroblasts and neural crest cells in the spinal cord anddorsal root ganglia (Sadler, 1990). Schwann cells migratefrom the neural crest to sites adjacent to nerve fibres, andmyelination begins at about 15 weeks of gestation.

Histopathological studies have described the postnataldevelopment of peripheral nerves, with a doubling ofaxonal diameter between 5 months and 5 years (Schroderet al. 1978; Jacobs & Love, 1985). While myelin thicknessis related to axonal diameter, it increases proportionatelymore than axonal caliber, by a factor of 2.5 times initialpostnatal values until between 5 and 14 years old (Gutrecht& Dyck, 1970; Schroder et al. 1978; Jacobs & Love,1985). After myelination has commenced, some peripheralnerves elongate by more than a factor of four, with inter-nodal lengths increasing until the second decade (Jacobs& Love, 1985).

As the speed of impulse conduction is related to thediameter of the largest myelinated fibres (Rushton, 1951;Waxman, 1980), these morphological changes coincidewith marked increases in conduction velocities. Themotor conduction velocities in a full-term infant areapproximately half those of adult values, increasing toapproach adult values at slightly over the age of 4 years(Gamstorp, 1963; Baer & Johnson, 1965; Wagner &Buchthal, 1972; Lang et al. 1985). A further gradualincrease until 16 years has been variably observed (Baer& Johnson, 1965).

Molecular interactions between myelinating glial cells,axonal membrane and cytoskeletal proteins have recentlybeen shown to have prominent roles in the molecularspecialization of the axon (Susuki & Rasband, 2008;Thaxton et al. 2011). Distinct nodal and juxtaparanodalregions are produced, physically separated by axo-glialjunctions at the paranode. Importantly, the clustering ofvoltage-gated Na+ channels at nodes of Ranvier enablesefficient and rapid propagation of impulses (Hille, 1972;

Scholz et al. 1993; Waxman & Ritchie, 1993). In maturemyelinated axons, fast K+ channels are concentratedunder the myelin at the juxtaparanode and may limitre-excitation of the node following conduction of an actionpotential or participate in the generation of the internodalresting potential (Baker et al. 1987; Waxman & Ritchie,1993).

The configuration and activity of a variety of ionchannels, exchangers and pumps activated during impulseconduction in maturing human peripheral axons and theirinteractions with myelination and axonal growth havenot yet been elucidated. While nerve conduction studiesprovide very limited information about the physiologyand function of such ion channels, axonal excitabilitytechniques have been established in adults that provideinsights into axonal ion channel function in health anddisease (Krishnan et al. 2009). As such, the presentstudy utilized in vivo axonal excitability studies in healthyinfants, children, adolescents and young adults for thefirst time to provide insight into the maturation of axonalbiophysical properties that develop with growth.

Methods

Conventional nerve conduction and specialized axonalexcitability studies were undertaken in 57 subjects (22males, 35 females; age range 0.46–24 years). All subjectsgave written informed consent, assent or parental consentto the procedures, in accordance with the Declarationof Helsinki, which were approved by the South EasternSydney and Illawarra Area Health Service Human ResearchEthics Committee.

No subject had a history of illness known to beassociated with neurological dysfunction, or medicationuse known to potentially affect axonal excitability;for example, anticonvulsants, local anaesthetics oranti-arrhythmics (Krishnan et al. 2009). Neuro-physiological studies were undertaken at the conclusionof planned medical procedures in 22 children (age range0.46–4.9 years) admitted to the Sydney Children’s Hospitalday unit for sedation. The majority of admissions (78%)were for follow up of previous urinary tract infection.

C© 2012 The Authors. The Journal of Physiology C© 2012 The Physiological Society

J Physiol 591.1 Evolution of peripheral nerve function in humans 275

Serum electrolytes and creatinine and renal functionwere normal on laboratory measurement. Childrenwere sedated with oral chloral hydrate ± intramuscularmorphine and droperidol according to standard protocol.These medications are not known to affect peripheralnerve function or excitability. A further 35 healthy sub-jects (age range 4.5–24 years) were recruited from thecommunity, and studies undertaken whilst consciouswith distraction and play therapy. Mean temperature was32.9 ± 0.2◦C and did not demonstrate significant changesacross different cohorts.

Axonal excitability studies were performed using pre-viously described threshold tracking protocols appliedto the median nerve in adults (Kiernan et al. 2000).The compound motor action potential (CMAP) wasrecorded using surface electrodes (4620M; UnomedicalLtd, Birkerød, Denmark) positioned over the abductorpollicis brevis muscle, with the active electrode at themotor point and the reference electrode distal over thetendon insertion. An electrosurgical neutral earth plate(2406M; Unomedical Ltd) was placed in the palm, withRedux electrolyte creme (Parker Laboratories, Fairfield,USA). Bipolar electrodes were utilized to locate theoptimal stimulation site of the median nerve at thewrist for each patient. The cathode was located overthe median nerve at the wrist and the anode 5–10 cmproximally, depending on arm size. Median nerve motorconduction velocities were calculated following proximalstimulation at the elbow (Medelec Synergy System, OxfordInstruments, Oxfordshire, UK; Kimura, 1983). Skin wasprepared with Nuprep abrasive skin prepping gel (Weaverand Company, Aurora, USA).

Data acquisition and stimulation delivery werecontrolled by QTRACS software (Institute of Neurology,UK). Recordings of CMAP were amplified and filtered(3 Hz–3 kHz) using a Medelec Sapphire 4ME amplifier(Medelec AA6 MK III, Surrey, UK), with electronic noiseremoved (Hum Bug 50/60 Hz Noise Eliminator, QuestScientific Instruments, North Vancouver, Canada) anddigitized by computer via a data acquisition device (DAQPCI-6221; Shielded connector block BNC-2110; CableSHC-68-68-EPM; National Instruments, Austin, USA).Electrical stimulation was converted to current usingan isolated linear bipolar constant current simulator(maximal output 50 mA; DS5, Digitimer, Welwyn GardenCity, UK). Temperature was measured with a surface probeat the wrist (Digitech, Jaycar, Rydalmere, Australia). Theamplitude of the CMAP was measured from baselineto negative peak and the threshold tracking target setto 40% of the maximum, utilizing the area of steepestslope of the stimulus–response curve. Threshold trackingfollows the changes in the intensity of a test stimulusrequired to produce the target potential. Excitabilitytesting incorporated a number of assessments designedto reflect different nodal and internodal properties, from

which various excitability measures could be extracted foranalysis. These included the following.

Stimulus–response relationship To commence theprotocol, a stimulus–response curve was generated byincreasing the stimulus intensity in a stepwise fashionfrom zero until maximal CMAP amplitude was achieved.The following parameters were measured: (i) the stimulusintensity (mA), defined as the current required to elicita target response set to 50% of maximal CMAP for astimulus of 1 ms duration; (ii) stimulus/response slope.

Strength–duration relationship The strength–durationrelationship was recorded by tracking the threshold toa stimulus as it is reduced from 1 ms to 0.2 ms duration.The measurements generate the charge–duration plot, inwhich the points are almost linear, of which the slopeindicates the rheobase and the intercept on the x-axisindicates the strength–duration time constant (TSD). TSD

is an apparent membrane time constant representingthe relationship between stimulus intensity and width,as described by Weiss’ formula (Weiss, 1901; Bostock,1983; Mogyoros et al. 1996). Rheobase is defined as thethreshold current required to produce a target responsefor a stimulus of infinitely long duration (Bostock et al.1998).

Threshold electrotonus (TE) and current–threshold (I–V)relationship In this part, threshold tracking was used torecord the changes in excitability in response to prolonged100 ms subthreshold polarizing currents, set to ±40% ofthe control (1 ms) threshold current (Bostock et al. 1998;Kiernan et al. 2000). The changes in threshold at differenttime intervals before, during and after the conditioningstimuli were measured. TE is used to provide informationabout internodal properties and conductances in additionto an estimate of resting membrane potential. Therecorded TE was plotted in conventional format asthreshold reduction, so that the response to depolarizingcurrent was plotted upwards (Bostock & Baker, 1988;Bostock et al. 1998). Depolarizing TE threshold changewas assessed at multiple intervals, in particular, as theaverage between 10 and 20 ms (TEd (10–20 )), and 90 and100 ms (TEd (90–100 )). The parameter accommodationhalf-time was measured as the time from the start of the40% depolarizing current until the threshold reductionreturned to half way between the peak and plateau levels.The threshold change to the 40% hyperpolarizing currentwas also assessed at multiple intervals, as the averagebetween 10 and 20 ms (TEh (10–20 )), and 90 and 100 ms(TEh (90–100 )).

The current–threshold relationship is comparable toTE and reflects the rectifying properties of the axon. TheI–V relationship was assessed by measuring the change



in threshold following the injection of polarizing currentsof 200 ms duration, the strength of which was altered in10% steps from +50% (depolarizing) to −100% (hyper-polarizing) of the control 1 ms threshold. Similarly, theI–V relationship was plotted in conventional format asthreshold reduction, so that the response to depolarizingcurrent was plotted upwards. From the I–V graph, thefollowing parameters were recorded: (i) resting I–V slope,calculated from polarizing currents between +10% to−10%; (ii) hyperpolarizing I–V slope, calculated frompolarizing current between 0 and 100%.

Recovery cycle. This protocol records the excitabilitychanges occurring at various interstimulus intervals,decreasing from 200 ms to 2.5 ms, after a supramaximalconditioning stimulus (Kiernan et al. 1996, 2000). Threestimulus combinations were recorded: (i) the control1 ms threshold; (ii) supramaximal conditioning stimulusalone; (iii) conditioning and tracking test stimuli incombination. The response in (ii) was subtracted on-linefrom the response in (iii) so that only the response tothe second stimulus was measured. The recovery cyclenormally includes three phases: refractoriness; super-excitable period; and the late subexcitable phase. Thefollowing parameters were measured: (i) refractoriness,which reflects the inactivation of nodal voltage-gatedsodium (Na+) channels following an impulse, wasmeasured as the percentage threshold increase at the 2.5 msinterstimulus interval; (ii) superexcitability (%), definedas the largest mean reduction in threshold of three adjacentpoints, peaking at a conditioning-test interval of 5–15 ms;(iii) subexcitability (%), defined as the largest increase inthreshold after an interstimulus interval of 15 ms.

Data analysis

Neurophysiological results were summarized across fourgroups: late infancy and early childhood, 0.5–3 years;late childhood, 3–8 years; adolescence, 8–15 years; andyoung adulthood, 15–25 years. These groups and ageranges were selected as they were similar to pre-vious conventional neurophysiological studies on theevolution of nerve conduction velocity (Gamstorp, 1963;Lang et al. 1985). All results were expressed asmean ± standard error of the mean (SEM). Regressionanalyses and curve estimation were performed using SPSS20 for Windows XP (SPSS Inc., Chicago, IL, USA) todetermine whether age-related changes were best fittedby a linear or logarithmic function. Where more thanone function was significant, the one with the higherR2 value has been reported. The one-way ANOVA testwas used to compare mean differences between groups.Correlations between excitability measures and motorconduction velocity were analysed by Spearman’s rank

correlation coefficient. A probability (P) value of <0.05was considered statistically significant.

Mathematical computer model of excitability

To model the excitability changes in motor axons withmaturation and the effects of altered axonal conductancesand passive membrane properties, mathematicalsimulations were undertaken using a model of thehuman axon (Bostock et al. 1991, 1995; Kiernan et al.2005). Transient Na+ channels were modelled using thevoltage-clamp data (Schwarz et al. 1995), and persistentNa+ currents were added (Bostock & Rothwell, 1997). Theequations for a single node and internode, representing aspatially uniform axon, were assessed by integration oversuccessive small time steps (Euler’s method; Press et al.1992; Kiernan et al. 2005; Lin et al. 2008; Boland et al.2009; Farrar et al. 2011). At times corresponding to thosein human nerve excitability recordings, the excitabilityof the model nerve was tested repeatedly to determinethreshold with an accuracy of 0.5%. The discrepancybetween the thresholds determined for the model andthose determined from a sample of real nerves was scoredas the weighted sum of the error terms: [(xm − xn)/sn]2,where xm is the threshold of the model, xn the meanand sn the standard deviation of the thresholds for thereal nerves. The weights were the same for all thresholdmeasurements of the same type (e.g. recovery cycle), andchosen to give an equal total weight to the different types ofthreshold measurement: current–threshold relationship,TE and the recovery cycle. The standard model wasobtained by minimizing the discrepancy between themodel and the young adulthood data with an iterativeleast squares procedure, so that alteration of any of theabove parameters would make the discrepancy worse.

Results

Conventional neurophysiological assessment

Motor conduction velocity increased from40.6 ± 2.3 m s−1 during late infancy and early childhoodto reach mean maximal values of 55.6 ± 0.9 m s−1 at8–15 years, and remained constant thereafter (Table 1;Fig. 1; logarithmic function, R2 = 0.74, P < 0.0001). Themost rapid increase in motor conduction velocity wasobserved during late infancy and early childhood, whilesmaller increases occurred during late childhood. Adultvalues (greater than 50 m s−1) were reached between 3and 5 years. Similarly, CMAP amplitude increased from4.3 ± 0.3 mV during late infancy and early childhoodto 8.4 ± 0.5 mV in adolescence, then became relativelyconstant with age (Fig. 2; logarithmic function, R2 = 0.43,P < 0.0001). The mean temperature was 32.9 ± 0.2◦C anddid not significantly differ between groups (P = 0.2).



Table 1. Demographic and conventional neurophysiological measures

Late infancy Late Young Changeand early childhood childhood Adolescence adulthood with age

Age range (years) 0.5–3 3–8 8–15 15–25Number 13 12 19 13Mean age ± SEM 1.6 ± 0.2 4.4 ± 0.3 10.3 ± 0.4 19.4 ± 1.1Sex ratio M:F 4:9 6:6 11:8 5:8CMAP amplitude (mV) 4.3 ± 0.3 6.4 ± 0.7 8.4 ± 0.5 8.6 ± 0.6 Logarithmic R2 = 0.43, P < 0.0001Median nerve MCV (m s−1) 40.6 ± 2.3 50.9 ± 1.9 55.6 ± 0.9 57.2 ± 0.4 Logarithmic R2 = 0.74, P < 0.0001Temperature (◦C) 32.7 ± 0.4 32.5 ± 0.3 33.2 ± 0.3 33.2 ± 0.3 NS

Values are represented as mean ± SEM. CMAP, compound motor action potential; MCV, motor conduction velocity.

Figure 1Median nerve motor conduction velocity changes with age

Measures of axonal excitability

Multiple measures of excitability showed significantchanges with age, paralleling the maturation of motorconduction velocity, with the greatest changes overyounger ages (Table 2).

Changes in stimulus–response and strength–durationrelationships with maturation. The stimulus thresholdreduced significantly with age, becoming relativelyconstant during adolescence, indicating that more currentwas required to excite axons at younger ages (Table 2;Fig. 2). In addition, the stimulus–response slope did notsignificantly change with age, confirming a consistentreduction in threshold with age for axons of both lowand high thresholds (late infancy and early childhood,5.3 ± 1.1; late childhood, 4.4 ± 1.1; adolescence, 4.6 ± 1.1;young adulthood, 5.5 ± 1.1, P = 0.9). The reduction incurrent threshold was associated with a reduction in

Figure 2Mean stimulus–response relations for healthy subjects grouped bychronological age

rheobase with age (Table 2). Again, the greatest reductionin rheobase was during late infancy and early childhood,becoming steady by adolescence. While TSD, an indirectmeasure of nodal persistent Na+ conductances (Bostock& Rothwell, 1997), did not significantly change with age(P = 0.09), the previously established negative correlationwith rheobase was maintained (R = −0.4, P < 0.005;Mogyoros et al. 1998).

Changes in TE and current–threshold relationship withmaturation. TE waveforms showed significant changeswith age, and again the most prominent occurred overyounger ages (Table 2; Fig. 3). Early and late depolarizingelectrotonus responses progressively reduced untiladolescence, and accommodation to depolarizing currentswas markedly faster at younger ages (accommodationhalf-time). In the same way, hyperpolarizing electro-tonus recordings at younger ages demonstrated greaterthreshold changes. Together the more prominent changesin TE waveforms at younger ages have previously been



Table 2. Measures of axonal excitability for each age group

Late infancy andearly childhood Late childhood Adolescence Young adulthood Change

(0.5–3 years) (3–8 years) (8–15 years) (15–25 years) with age

Mean threshold 50% CMAP (mA) 4.1 ± 1.1 3.6 ± 1.1 3.2 ± 1.0 3.1 ± 1.1 Logarithmic R2 = 0.14,P < 0.005

TSD (ms) 0.33 ± 0.02 0.42 ± 0.03 0.39 ± 0.01 0.39 ± 0.03 Logarithmic R2 = 0.05,P = 0.09

Rheobase (mA) 3.1 ± 1.1 2.4 ± 1.1 2.2 ± 1.1 2.1 ± 1.1 Logarithmic R2 = 0.2,P = 0.001

TEd40 (10–20 ms) (%) 74.0 ± 1.3 72.1 ± 1.4 66.0 ± 1.0 65.0 ± 1.6 Logarithmic R2 = 0.32,P < 0.0001

Accommodation half-time (ms) 31.8 ± 0.7 35.3 ± 1.0 42.8 ± 0.7 43.0 ± 1.3 Logarithmic R2 = 0.61,P < 0.0001

TEd40 (90–100 ms) (%) 50.2 ± 1.3 47.7 ± 1.0 42.4 ± 0.7 43.9 ± 1.1 Logarithmic R2 = 0.29,P < 0.0001

TEh40 (10–20 ms) (%) −99.8 ± 3.1 −86.6 ± 2.9 −73.4 ± 1.6 −68.4 ± 2.2 Logarithmic R2 = 0.62,P < 0.0001

TEh40 (90–100 ms) (%) −181.0 ± 10.7 −145.8 ± 8.1 −108.8 ± 3.8 −108.0 ± 5.0 Logarithmic R2 = 0.50,P < 0.0001

Resting I–V slope 0.50 ± 0.03 0.53 ± 0.02 0.66 ± 0.02 0.64 ± 0.03 Logarithmic R2 = 0.33,P < 0.0001

Hyperpolarizing I–V slope 0.43 ± 0.02 0.42 ± 0.04 0.38 ± 0.01 0.31 ± 0.01 Logarithmic R2 = 0.24,P < 0.001

RRP (ms) 2.4 ± 1.0 2.6 ± 1.1 3.0 ± 1.0 3.0 ± 1.0 Logarithmic R2 = 0.27,P < 0.0001

Refractoriness at 2.5 ms (%) −10.2 ± 2.0∗ 2.7 ± 5.6 24.3 ± 4.6 21.6 ± 3.6 Logarithmic R2 = 0.27,P = 0.001

Peak superexcitability (%) −25.7 ± 0.9 −24.0 ± 1.4 −24.8 ± 1.0 −25.8 ± 1.0 NSSubexcitability (%) 8.8 ± 1.1 11.0 ± 1.1 12.8 ± 0.7 13.0 ± 1.0 Logarithmic R2 = 0.13,

P < 0.01

Values are mean ± SEM. CMAP, compound motor action potential; TSD, strength–duration time constant; RRP, relative refractoryperiod; TEd(10–20), depolarizing change at 10–20 ms in threshold electrotonus; TEd(90–100), depolarizing change at 90–100 ms;TEh(10–20), hyperpolarizing change at 10–20 ms; TEh(90–100), hyperpolarizing change at 90–100 ms. ∗Refractoriness was notpresent at the 2.5 ms interstimulus interval as a mean threshold reduction was observed.

described as ‘fanning-out’, related to their resemblanceto a Japanese fan (Kaji, 1997). In addition, the ratio ofTEd(10–20) to TEh(10–20) increased with maturation(logarithmic function, R = 0.79, P < 0.0001; Fig. 3F),suggesting simultaneous alterations in several distinctbiophysical properties of the axon (see Discussion).

Similar to the ‘fanning-in’ of TE waveforms with age, theI–V relationship showed reductions in threshold changesfor both depolarization and hyperpolarization with age(Table 2; Fig. 4). Additionally, the slope of the I–V curve atlow current strengths, termed resting I–V slope, increasedwith age.

Changes in recovery cycle with maturation. The recoverycycle showed significant changes in membrane excitabilityin response to a supramaximal conditioning stimulus withage (Table 2; Fig. 5). Notably refractoriness, the initialphase of the recovery cycle in which threshold increases

related to the inactivation of transient Na+ channels, wasnot observed at the 2.5 ms interstimulus in one-third of theyoungest subjects during late infancy and early childhoodwith threshold reductions occurring. Refractoriness andsubexcitability similarly showed significant increases withage, while peak superexcitability did not significantlychange. The greatest changes occurred during late infancyand early childhood, and excitability measures stabilizedduring adolescence.

Correlations with conduction velocity. Combiningmeasures of axonal excitability and motor conductionvelocity, it was evident that specific excitability parameterswere significantly associated with increases in conductionvelocity (Fig. 6); including reductions in TEh(10–20)(R = 0.67, P < 0.0001) and TEh(90–100) (R = 0.63,P < 0.0001), and increases in accommodation half-time(R = 0.56, P < 0.0001).



Mathematical modelling of altered excitabilityproperties with maturation

To assist in interpreting the complex changes observed inclinical nerve excitability with maturation, a mathematicalmodel of the human motor axon was first adjustedto provide a close match to the recordings from theyoung adulthood group. The model was then used to

explore whether changes in any membrane parametercould reproduce the changes seen in each of the youngerage groups. Taken in isolation, no single parameterchange could account for the younger subject recordingssatisfactorily. The best match was an increase in inter-nodal fast K+ channels (GKfi) with immaturity; from100 to 172 units during late infancy and early childhood,which reduced the discrepancy by 25.3%. The best fit

Figure 3. Comparison of TE measures with ageA, depolarizing TE curves for healthy subjects grouped by age. B, hyperpolarizing TE curves for healthy subjectsgrouped by age. C, hyperpolarizing changes at 10–20 ms (TEh(10–20 ms)) reduced with age, P < 0.0001. D,depolarizing changes at 10–20 ms (TEd(10–20 ms)) reduced with age, P < 0.0001. E, accommodation half-timeincreased with age, P < 0.0001. F, the ratio of TEd(10–20) to TEh(10–20) increased with age, P < 0.0001.



and most plausible model was determined by changingtwo parameters, including GKfi and Barrett and Barrettconductance (GBB), and was consistent for the twoyounger age groups. Simulating the axonal excitabilityrecordings with this model is depicted in Fig. 7. In lateinfancy and early childhood GKfi increased from 100to 309 units and GBB increased from 33.9 to 48.2 units,which reduced the discrepancy by 83.1%. Importantly themagnitude of change for these parameters reduced withmaturation. In late childhood, GKfi increased from 100 to218 units and GBB increased from 33.9 to 40.4 units, whichreduced the discrepancy by 79.6%. In contrast, modellingthese parameter changes in adolescence produced a poormatch with subject recordings, GKfi increased from 100to 194 units and GBB increased from 33.9 to 36.3 units,which reduced the discrepancy by 48.3%. As such,mathematical simulations support the hypothesis thatmaturation produces changes in passive cable conductanceand axonal ion channel function to reach steady valuesduring adolescence, with a reduction in GKfi as the mostimportant channel alteration with maturation.

Discussion

The present study has identified striking changes inmotor axonal membrane properties associated withmaturation of the peripheral nerve in humans, utilizingin vivo nerve excitability testing for the first time.Specifically, the present findings suggest that the activityof K+ conductances decreases with formation of the

Figure 4Comparison of current–threshold relationship for healthy subjectsgrouped by chronological age

axo-glial junction, and myelination reduces the GBB.Importantly, concurrent assessment of peripheral nervemotor conduction velocities established that alterationsthat were paralleled by significant changes across multipleexcitability parameters, similarly reached steady valuesduring adolescence. Taken in total, these functionalalterations serve to enhance the efficiency and speed ofimpulse conduction concurrent with the acquisition ofmotor skills during childhood.

A distinctive pattern of changes occurred in peri-pheral axonal excitability with maturation, typified byreductions in threshold, rheobase and TE, with wave-forms ‘fanning-in’, while resting current–threshold slopeincreased, all indicative of a relative reduction incurrent to excite the axon and evolution to a moreefficient state. Overall, this pattern of excitability changeswith development may reflect interactions betweenpassive structural factors, such as axonal diameter andmyelination, and active variables, including ion channelfunction and membrane potential. Significantly, changesin motor conduction velocities were in general agreement

Figure 5. Comparison of recovery cycle of excitabilitymeasures with ageA, recovery cycle of excitability curves. B, mean group dataillustrating that refractoriness at 2.5 ms significantly increased withage; refractoriness was not present for the youngest group as amean threshold reduction was observed.



with previous studies, indicating the present cohort wasrepresentative of the normal population (Gamstorp, 1963;Baer & Johnson, 1965; Wagner & Buchthal, 1972; Langet al. 1985).

Maturation in passive axonal properties

Mathematical models examining the effects of passivemembrane properties on TE have previously confirmedthat the degree of early ‘fanning-out’ increases withreduced axon diameter or internodal resistance throughand under the myelin sheath, as occurs with thin myelinor immaturity of the axo-glial junction (GBB, confirmedby modelling in the present study; Yang et al. 2000;Nodera et al. 2004). Accordingly, excitability changes withmaturation may reflect alterations in passive membraneproperties related to smaller axonal diameter, shortenedinternodes and thin myelin at younger ages, and aresimilar to previous axonal excitability studies in immatureanimal nerves (Yang et al. 2000; Boerio et al. 2009).However, not all elements predicted by alteration of passivemembrane properties in in vivo animal models havebeen observed in the present study, suggesting additionalalterations in axonal biophysical properties with growth.

Figure 6. The relationships between axonal excitabilityparameters and motor conduction velocityA, threshold changes to subthreshold 40% hyperpolarizing currentsat 10–20 ms (TEh(10–20 ms)). B, threshold changes to subthreshold40% hyperpolarizing currents at 90–100 ms (TEh(90–100 ms)). C,accommodation half-time.

Further, while animal models have attempted to outlineand provide an understanding of these changes, the pre-sent studies using intact human ‘preparations’ providedefinitive information and indicate that animal modelsmay be inaccurate.

Alterations in membrane potential and conductances– K+ conductances

The present results also suggest modulation of ion channelfunction with maturation, with faster accommodationat younger ages indicating increased activation of fastK+ conductances. Altered organization of fast K+

conductances at the axonal membrane and immaturityof the axo-glial junction during development may enabletheir activation in response to nodal depolarization.Increased activation of fast K+ conductances alone maybe expected to ‘clamp’ the ‘fanning-out’ of depolarizingTE, yet in the present study this may have been masked bythe added effect of passive axonal properties. In support ofthis, the ratio TEd(10–20) to TEh(10–20), which is relatedto nodal K+ channel activity, increased with maturation.Insights into the developmental changes of fast K+

channels and the effects of myelination have been providedby studies that have demonstrated that the sensitivity of4-aminopyridine, a blocker of fast K+ channels, attenuatedduring development (Ritchie, 1982; Eng et al. 1988; Yanget al. 2000). These findings suggest that fast K+ channelsgradually shift from the node to the juxtaparanodeduring maturation, where they are covered by myelinand segregated from the effects of 4-amiopyridine. Recentstudies have demonstrated that the correct clustering ofjuxtaparanodal K+ channels depends on several distinctinteractions between myelinating glial cells and axons(Rasband, 2011). Specifically, juxtaparanodal K+ channelsare part of a larger protein complex comprising theglial and axonal cell adhesion molecules contactin2(TAG-1) and contactin-associated protein-2 (caspr2) andthe cytoskeletal protein 4.1B (Bhat et al. 2001; Boyleet al. 2001; Poliak et al. 2003; Horresh et al. 2010).In addition, the development of paranodal axo-glialjunctions containing the axonal proteins contactin andcaspr with glial neurofascin-155 function as a physicalbarrier to lateral diffusion (Einheber et al. 1997;Menegoz et al. 1997; Rios et al. 2000; Tait et al.2000). These molecular studies provide an understandingof the simultaneous maturation of passive membraneproperties and K+ conductances shown in the presentstudy, which are inextricably linked. Taken together thesefactors influence increases in motor conduction velocity asreflected by correlations with the appropriate excitabilityparameters.

In addition, a nodal representation of fast K+ channelsin immature nerves may influence action potential



repolarization, as occurs in the Hodgkin–Huxley giantsquid axon model (Hodgkin & Huxley, 1952); theiractivation may reduce action potential duration and therefractory period, thereby advancing the ensuing periodof increased membrane excitability, also known as super-excitability. A striking finding in the present study wasthe leftwards shift of the recovery cycle of excitability, suchthat at younger ages superexcitability was more prominentat short interstimulus intervals and its peak earlier, yet thepeak magnitude was not significantly altered. Molecularand electrophysiological studies in the developing rathave confirmed a dynamic distribution of fast K+

channels, functioning to ensure stable and secure impulsepropagation during development. Specifically, duringearly development nodal fast K+ channels were directlyinvolved in speeding action potential repolarization, to

allow trains of impulses at high frequency (Vabnick etal. 1999). At intermediate stages of myelination, fast K+

channels were shown to have a role in the prevention ofrepetitive discharges.

Taken together it may be expected that changes inpassive and active (voltage-dependent) axonal membraneproperties will produce complex changes in the recoverycycle of excitability, as indicated by the modelling. Pre-vious animal and computer models have establishedthat smaller diameter axons along with lower inter-nodal resistances generated greater depolarizing after-potentials, as measured by superexcitability (David et al.1995; McIntyre et al. 2002). In contrast, the activation of‘nodal’ fast K+ channels limits the amplitude and durationof the depolarizing afterpotential and superexcitability(Stephanova & Mileva, 2000).

Figure 7. Simulation of the excitability changes in clinical nerve excitability with maturation using themathematical modelGrey lines represent the model generated by the young adulthood group. Green lines were generated by themodel by increasing GBB from 33.9 to 48.2 units and GKfi from 100 to 309 units, which reduced the discrepancyin late infancy and early childhood by 83.1%. Red lines were generated by the model by increasing GBB from33.9 to 40.4 units and GKfi from 100 to 218 units, which reduced the discrepancy in late childhood by 79.6%. A,recovery cycles. B, charge–duration plot based on stimuli of 0.2 ms and 1 ms duration, with the negative intercepton the x-axis equating to TSD, and the slope equal to the rheobase. C, TE for 100 ms polarizing currents ±40%of the resting threshold. D, current–threshold relationship.



Alterations in membrane potential and Na+

conductances

The shape of the recovery cycle is also dependent onthe kinetics of Na+ conductances (McIntyre et al. 2002;Kiernan et al. 2005), and previous studies have suggestedthat nodal Na+ channel density may be a factor involvedin maturation (Ritchie, 1982; Boerio et al. 2009). Thechanges in the recovery cycle are difficult to interpret inthe present study and not solely in keeping with alterationsin passive membrane properties, membrane potential orion conductances alone. TSD, an indirect measure ofpersistent Na+ conductances, did not significantly changewith maturation, and suggested that the time frame ofnodal Na+ channel maturation is early. The early maturityof persistent Na+ conductances at nodes of Ranviercritically enables saltatory conduction of nerve impulsesalong myelinated fibres (Hille, 1972; Scholz et al. 1993;Waxman & Ritchie, 1993). Recent molecular studies haverevealed that nodal organization occurs independentlyof paranodes, with several molecules enriched at thenode regulating voltage-gated Na+ channel localization,stabilization and function, including neurofascin 186,Ankyrin-G and βIV-spectrin (Rasband & Trimmer, 2001;Girault & Peles, 2002; Komada & Soriano, 2002; Thaxtonet al. 2011). Accordingly, it may be expected that theconfiguration and activity of nodal Na+ conductancesmay occur independently of myelination and developmentof the axo-glial junction (Utzschneider et al. 1993), assuggested in the present study.

In addition to assessing voltage-independent propertiesof nerve fibres, TE also provides information aboutvoltage-dependent internodal conductances and anestimate of axonal membrane potential (Baker & Bostock,1989; Kiernan & Bostock, 2000). The raised thresholds,fanning out of TE, together with significantly reducedrefractoriness and subexcitability at younger ages inthe present study are consistent with the alterationsin excitability established in axonal membrane hyper-polarization (Kiernan & Bostock, 2000; Kiernan etal. 2002). However, this seems unlikely given thatother excitability parameters were not appropriatelyaltered. Furthermore, mathematical modelling clarifiedthe complex interpretation of the excitability data anddid not predict shifts in membrane potential.

Excitability changes in developing nerves in the pre-sent study share some similarities to those in regeneratingaxons, explained by changes in fibre morphology andfunctional organization of K+ channels, yielding increasedconductances (Kocsis et al. 1982; Kocsis & Waxman,1983; Moldovan & Krarup, 2004b, 2007; Sawai et al.2008). Importantly, during development nerve growth isachieved by increasing the internodal length with pre-servation of the number of nodes (Vizoso & Young, 1948).In contrast, following regeneration/remyelination the

number of nodes per unit length, where voltage-gated Na+

channels are located in highest concentration, increases byup to a factor of four, and there is a corresponding increasein the Na+ channel content (Rosenbluth, 1976; Ritchie,1982; Nakata et al. 2008). In response to the greater Na+

load there is increased Na+/K+ pump activity, which inturn distinctly produces membrane hyperpolarization inregenerated nerves (Moldovan & Krarup, 2004a,b, 2007;Sawai et al. 2008).

Clinical implications

The changes of multiple excitability measures with agein the present study illustrate the time course of post-natal axonal growth, myelination and maturation of theparanodal axo-glial junction, together with its effect onthe organization and activity of fast K+ conductances.Together these changes induced increases in conductionvelocity and provide unique insight into the evolutionof postnatal human peripheral nerve function, servingto ensure stable and secure impulse propagation, whilstenhancing efficiency. Altogether these processes weresimultaneous, concurrent with the acquisition of motorskills in childhood. Changes were greatest during lateinfancy and early childhood, and while adult valueswere reached between 3 and 5 years, small alterationscontinued during late childhood to reach constant valuesin adolescence.

Unlike conventional diagnostic nerve conductionstudies, usually including several nerves, axonalexcitability testing focuses on a single nerve and maybe completed in less than 7 minutes. Similar to under-taking routine diagnostic procedures in young children,patients may not be able to cooperate with axonalexcitability testing, being unable to sit still or tolerate milddiscomfort during the procedure. Consequently carefulpreparation utilizing an arm board and securing electro-des with surface taping to reduce movement, togetherwith distraction play therapy or sedation may assist withsuccessful completion of studies. Understanding normalmaturation through infancy and childhood may providea platform for developing a further understanding ofthe pathophysiology of various childhood-onset neuro-muscular conditions throughout the disease course(Farrar et al. 2010). As such, the present study hasestablished the feasibility and potential importance ofnovel nerve excitability testing in children.

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Author contributions

The studies were undertaken at Sydney Children’s hospital,Randwick, Australia. M.A.F.: conception and design of thestudy, data collection, data analysis, data interpretation anddrafting of the manuscript; S.B.P.: data collection, data analysis,data interpretation and revision of the manuscript; C.S.-Y.L.:data collection, data analysis, data interpretation and revisionof the manuscript; and M.C.K.: conception and design ofthe study, data analysis, data interpretation and revision of

the manuscript. All authors approved the final version of themanuscript.

Acknowledgement

M.A.F. received grant support from the National Health andMedical Research Council of Australia: Medical PostgraduateScholarship, ID568915. The authors wish to thank James Howellsfor his assistance with mathematical modelling.


Evolution of peripheral nerve function in humans: novel insights from motor nerve excitability

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