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J Physiol 593.10 (2015) pp 2327–2342

Extensor motoneurone properties are altered immediately before and during fictive locomotion in the adult decerebrate rat C. W. MacDonell1 , K. E. Power1 , J. W. Chopek1 , K. R. Gardiner1 and P. F. Gardiner1,2 1

Spinal Cord Research Centre, Department of Physiology & Pathophysiology, University of Manitoba, Winnipeg, Manitoba, Canada Health Leisure and Human Performance Research Institute, Faculty of Kinesiology and Recreation Management, University of Manitoba, Winnipeg, Manitoba

2

Key points

The Journal of Physiology

r This is the first report, in adult decerebrate rats, to examine intracellular hindlimb motoneurone r r r r

properties during quiescence, fictive locomotion and a tonic period immediately before fictive locomotion that is characterized by increased peripheral nerve activity. It is shown for the first time during fictive locomotion that motoneurones become more responsive in the tonic period, suggesting that the motoneurone pool becomes primed before patterned motor output commences. Spike frequency adaptation exists in quiescence and during fictive locomotion during constant excitation with injected current but not during centrally driven fictive locomotion. Motoneurones within the extensor motor pool show changes in excitability even when they are not directly involved in locomotion. The data show increased responsiveness of motoneurones during locomotion via a lowered threshold for spike initiation and decreased rheobase.

Abstract This study examined motoneurone properties during fictive locomotion in the adult rat for the first time. Fictive locomotion was induced via electrical stimulation of the mesencephalic locomotor region in decerebrate adult rats under neuromuscular blockade to compare basic and rhythmic motoneurone properties in antidromically identified extensor motoneurones during: (1) quiescence, before and after fictive locomotion; (2) the ‘tonic’ period immediately preceding locomotor-like activity, whereby the amplitude of peripheral flexor (peroneal) and extensor (tibial) nerves are increased but alternation has not yet occurred; and (3) locomotor-like episodes. Locomotion was identified by alternating flexor–extensor nerve activity, where the motoneurone either produced membrane oscillations consistent with a locomotor drive potential (LDP) or did not display membrane oscillation during alternating nerve activity. Cells producing LDPs were referred to as such, while those that did not were referred to as ‘idle’ motoneurones. LDP and idle motoneurones during locomotion had hyperpolarized spike threshold (Vth ; LDP: 3.8 mV; idle: 5.8 mV), decreased rheobase and an increased discharge rate (LDP: 64%; idle: 41%) during triangular ramp current injection even though the frequency–current slope was reduced by 70% and 55%, respectively. Modulation began in the tonic period immediately preceding

C. W. MacDonell and K. E. Power contributed equally to this work.  C 2015 The Authors. The Journal of Physiology  C 2015 The Physiological Society

DOI: 10.1113/JP270239

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locomotion, with a hyperpolarized Vth and reduced rheobase. Spike frequency adaptation did not occur in spiking LDPs or firing generated from sinusoidal current injection, but occurred during a sustained current pulse during locomotion. Input conductance showed no change. Results suggest motoneurone modulation occurs across the pool and is not restricted to motoneurones engaged in locomotion. (Resubmitted 23 January 2015; accepted after revision 17 March 2015; first published online 25 March 2015) Corresponding author C. W. MacDonell: 402 Basic Medical Science Building, Spinal Cord Research Centre, Department of Physiology and Pathophysiology, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada R3E 0J9. Email: [email protected] Abbreviations AHP, after-hyperpolarization; AHPpk , peak amplitude of the post-spike after-hyperpolarization; AHPhfd , half-decay time of the post-spike after-hyperpolarization; CPG, central pattern generator; CV, coefficient of variation; ENG, electroneurogram; IC, input conductance; LDP, locomotor drive potential; MAP, mean arterial blood pressure; MLR, mesencephalic locomotor region; SFA, spike frequency adaptation; Vth , voltage threshold.

Introduction The onset of a rhythmic and alternating motor output, such as locomotion, is initiated via descending commands that increase the excitability of spinal circuits known as central pattern generators (CPGs), which excite spinal motoneurones leading to muscle contraction and movement (Jordan et al. 2008). The responsiveness of spinal motoneurones to synaptic input arising from descending pathways, the periphery and/or the spinal CPG thus plays an integral part in whether or not they are recruited to produce movement. Previous work has demonstrated that several electrical properties of spinal motoneurones measured ‘at rest’ in decerebrate adult cat preparations are modulated quickly and reversibly when a fictive motor output is evoked. For example, during fictive locomotion and/or fictive scratch, lumbar motoneurones exhibit a reduced after-hyperpolarization (AHP) amplitude (Brownstone et al. 1992; Power et al. 2010), an increased (Perrault, 2002), unchanged or variable input conductance (a Shefchyk & Jordan, 1985a; Power et al. 2010), an eliminated (Brownstone et al. 1992) or reduced frequency–current (F–I) slope (Fedirchuk et al. 1998), a hyperpolarized spike threshold (Vth ) for spike initiation (Krawitz et al. 2001; Dai et al. 2002; Power et al. 2010) and a reduction in spike-frequency adaptation (Brownstone et al. 2011). Most of these changes would act to facilitate motoneurone recruitment and firing during motor output. Differences in motoneurone properties between the non-locomotor and locomotor states might be different in adult rats with different daily activity patterns (Barnett, 1963) compared to cats, given the increased excitability (Beaumont & Gardiner, 2002, 2003) and decreased rate of adaptation (MacDonell et al. 2012) noted in ‘anaesthetized’ motoneurones in rats after endurance training. Intracellular motoneurone properties during locomotor-like activity have been examined in the neonatal but not the adult rat. Fictive locomotion induced in the

isolated spinal cord of the neonatal rat by electrical brainstem stimulation has induced Vth hyperpolarization for action potential generation (Gilmore & Fedirchuk, 2004), while pharmacologically generated locomotion has led to a large reduction in the AHP (Schmidt, 1994). Although descending inputs to the lumbar spinal cord are present in neonatal rats (Lakke, 1997) the motor system is immature as illustrated by their inability to locomote effectively and explore their environments until the third postnatal week. In addition, the spinal motoneurones are not fully developed morphologically or electrophysiologically (for review see Vinay et al. 2000; Clarac et al. 2004). For example, during the postnatal period motoneurone input resistance decreases and rheobase values increase more than fivefold. Additional changes in motoneurone properties include the continuing development of multiple ionic currents (Vinay et al. 2000). It is currently unclear whether age-dependent differences in spinal motoneurone properties influence the manner in which they are recruited during locomotion. In addition, the serotonergic system, which plays a significant role in the response of the motoneurone to excitation, is still developing in neonatal rats (Ziskind-Conhaim et al. 1993). The aim of this examination of motoneurone properties during fictive locomotion was to determine whether the changes in motoneurone properties during fictive motor outputs in the adult decerebrate cat and neonatal rat also occur during fictive locomotion in the adult rat and at the same time establish baseline data for subsequent experiments. Future experiments will examine the effects of increased and decreased neuromuscular activity on state-dependent changes in rat motoneurones during fictive locomotion. Despite the behavioural differences between rats and cats (rats tend to be more active due to their foraging and predator avoidance behaviour; Barnett, 1963), we hypothesized that the state-dependent changes found in cat motoneurones will occur in extensor hindlimb motoneurones in the adult rat. In addition, based on the observation that Vth becomes hyperpolarized during  C 2015 The Authors. The Journal of Physiology  C 2015 The Physiological Society

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Motoneurone properties during fictive locomotion

fictive locomotion when locomotion commences and the LDP is small in amplitude (Krawitz et al. 2001), the time period marked by increased peripheral nerve signal amplitude before locomotor-like activity (flexor–extensor alternation) may also show state-dependent changes in excitability. This short, pre-locomotor tonic electroneurogram (ENG) period varies in length (between 2 and 10 s after mesencephalic locomotor region (MLR) stimulation begins and before flexor–extensor alternation), and it is expected that modulation will occur during this tonic period and that ongoing modulation will occur during fictive locomotion as well. Extensor motoneurones were chosen to be examined in this experiment due to the specific interest of the lab in changes in their properties. The ankle extensor muscles also exhibit considerably more muscle mass decline than flexors in spinalized cats (Roy et al. 1999), respond differently to 5-HT agonists (Chopek et al. 2013), display different discharge characteristics associated with persistent inward currents (Cotel et al. 2009) and have been shown to respond to training (Beaumont & Gardiner, 2002, 2003; MacDonell et al. 2012) and disuse (Cormery et al. 2000, 2005; Button et al. 2008). Finally, the choice to consider extensor motoneurones was influenced technically by the difficulty of the preparation and the challenge of evoking locomotion and sustaining impalement of the motoneurone during locomotion. From a strategic point of view, it was felt that a focused investigation was the best course. To address these objectives we assessed the electrophysiological properties of rat hindlimb motoneurone properties before, during and immediately after fictive locomotion induced via electrical stimulation of the MLR. Locomotion was identified by alternating peripheral flexor–extensor nerve activity. The results include motoneurones that produced LDPs and those that did not, but were activated by antidromic stimulation of the peripheral nerve innervating extensors. The results demonstrate that state-dependent changes do occur in the motoneurone regardless of whether the motoneurone is directly involved in the motor output; that is, producing a locomotor drive potential. The findings demonstrate that the spinal motor system of rat is primed during locomotion and that motoneurone spike frequency adaptation (SFA) is significantly reduced during LDP-driven discharge, but not when a current pulse is superimposed upon the LDP. Methods Experimental animals

Female Sprague–Dawley rats (280–350 g; n = 26) were used as the experimental group, with each animal acting as its own control. The animal ethics committee for the  C 2015 The Authors. The Journal of Physiology  C 2015 The Physiological Society

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University of Manitoba approved the procedures, which met the guidelines set forth by the Canadian Council of Animal Care. The authors confirm that the present research complies with the policies and regulations outlined by The Journal of Physiology.

Surgical procedures

Induction of anaesthesia occurred in a chamber using a mixture of pure oxygen and isoflurane (5%). Following induction, anaesthesia was maintained (1–2.5% isoflurane), and verified by monitoring heart rate and testing bilateral toe-pinch and eyelash reflexes. Isoflurane delivery occurred until the completion of a precollicular–postmamillary decerebration, after which ventilation of the animal occurred with pure oxygen until the termination of the experiment. Immediately following the induction of anaesthesia, and before any surgical procedures, intraperitoneal administration of atropine (0.05 mg kg−1 atropine within 5% dextrose physiological saline) was administered to minimize airway secretions. Execution of the following procedures occurred on the surgery table post atropine administration: a bilateral peripheral nerve dissection, to expose the peroneal and tibial nerves for antidromic stimulation and ENG recording; insertion of a tracheal tube for ventilation (Harvard Apparatus Canada, Saint-Laurent, QC, Canada; rate: 60–80 strokes min−1 ; tidal volume range 2.0–2.5 ml), and to ensure expired CO2 levels ranged between 3 and 4% (CapStar-100 CO2 analyser, CWE Inc., Ardmore, PA, USA); cannulation of the right carotid artery to monitor mean arterial blood pressure (MAP) and provide an infusion port; and dexamethasone administration (0.2 mgkg−1 ) via the carotid artery cannula to reduce swelling of the brain. In addition, the left carotid artery was tied off with a suture (2-0) immediately following dissection of the back musculature in preparation for a laminectomy (T12–L3). The laminectomy occurred in a stereotaxic frame, and upon completion, the dorsal roots were either brushed aside or cut, and a mineral oil pool formed. Prior to decerebration, the skull of the rat was exposed and the parietal bones excised. In an attempt to reduce excess bleeding, the skull vasculature was cauterized prior to removal. The decerebration procedure used a combination of tissue aspiration and blunt dissection. The tissue was aspirated until the rostral colliculi and hypothalamus were exposed. At this point, the tissue was cut and the hypothalamus, thalamus and forebrain removed and absorbable haemostats controlled bleeding throughout the procedure (Surgicel, Johnson & Johnson, New Brunswick, NJ, USA; Instat, Johnson & Johnson; Gelfoam, Pharmacia and Upjohn, USA;

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and Sugi absorbent swabs, Kettenbach GmbH & Co., Eschenburg, Germany). Typically, MAP decreased during the procedure, but rebounded within minutes. However, if MAP was not restored due to excessive haemorrhaging, a saline–alginate (0.7%) solution was administered I.V. in order to expand plasma volume and restore MAP (Cabrales et al. 2005). Following decerebration, the isoflurane delivery was discontinued and the animal ventilated with pure oxygen for data collection. In order to eliminate movement of the animal from peripheral nerve and mesencephalic locomotor region stimulation (see below), a neuromuscular junction blocker (pancuronium bromide, 0.3 mgkg−1 ) was administered. Lastly, a unilateral pneumothorax helped reduce movement of the spinal cord related to ventilation.

Induction of fictive locomotion

Fictive locomotion was induced through brainstem stimulation of the MLR. A tungsten electrode with a 2 μm tip was advanced through the brainstem at an angle of 37 deg with a stereotaxic micromanipulator (SM-15; Narishige, Narishige International USA, Inc., East Meadow, NY, USA). The angle was calculated in accordance with anatomical landmarks (Paxinos & Watson, 1998), using bregma as the initial position. Advancement of the electrode occurred in steps between 0.1 and 0.5 mm after being positioned 1.5–2.0 mm lateral from the midline. The brainstem was stimulated after electrode advancement at an average current intensity of 80 μA (range: 40–200 μA; 0.5–1 ms duration; 15–30 Hz; the 200 μA was from a single experiment and did not represent the norm for current intensity). ENGs were monitored bilaterally using bipolar hook electrodes, bandpass filtered (10–2000 Hz) and differentially amplified (common-mode rejection ratio (CMRR) > 90 dB at 60 Hz) prior to being A/D converted at 5000 Hz (12 bit A/D board; signals were acquired using the Data Capture and Analysis software, designed in the Spinal Cord Research Centre, University of Manitoba). Fictive locomotion was confirmed by alternating flexor and extensor activity of the mounted peroneal and tibial nerves along with an increase in MAP. Raw ENG signals were collected, monitored, and stored offline for subsequent root mean square (100 ms window) signal conditioning. Three different motoneurone behaviours were evident during alternating ENG activity. In response to MLR stimulation, motoneurones discharged action potentials and displayed rhythmic oscillation in the membrane potential consistent with an LDP (n = 16); displayed voltage dependency whereby rhythmic oscillation occurred without spiking until the membrane was depolarized by a ramp current injection (n = 2); or the

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membrane potential simply depolarized slightly without rhythmic oscillation of the membrane potential (n = 20). The motoneurones that did not have an oscillating membrane potential while ENG signal alteration in peripheral nerves occurred are referred to as idle cells and the neurons with membrane oscillation are referred to as LDP cells. Figure 1 shows examples of idle (Fig. 1A) and LDP (Fig. 1B)-producing cells during ramp current injection. In the absence of an LDP, left and right flexor–extensor nerve alternation and an increase in the amplitude of the ENG activity was used to confirm fictive locomotion. Intracellular recordings

Glass micropipettes (1.0 mm thin-walled; World Precision Instruments, Sarasota, FL, USA) filled with 2 M potassium citrate and formed (Kopf Vertical Pipette Puller, David Kopf Instruments, Tujunga, CA, USA) with tip diameters between 1 and 2 μm and a resistance of approximately 7–12 M. The micropipette was lowered into the cord with an inchworm micro-drive system (Burleigh Instruments Inc., Fishers, NY, USA) in steps of 1–15 μm. The use of bilateral flexor (peroneal) and extensor (tibial) silver-chloride hook electrodes allowed both verification of locomotor-like activity in peripheral nerves and a means to stimulate and identify spinal motoneurones antidromically. Antidromic stimulation of the tibial nerve occurred at a frequency of 2–3 Hz (0.1–0.2 mA for 0.1 ms). Field potentials produced by antidromic stimulation of the tibial nerve were monitored continuously during micropipette advancement through the spinal cord. Evidence of successful motoneurone impalement included a sudden increase in membrane potential to at least −50 mV, an antidromic action potential spike amplitude greater than 55 mV with a positive overshoot and a fixed latency of less than 2.5 ms from the stimulation artifact. Intracellular extensor motoneurone records were collected at 20 kHz by an Axoclamp intracellular amplifier system (Axoclamp 2B, Axon Instruments Inc., Union City, CA, USA) used either in bridge or discontinuous current-clamp mode (DCC; 3–10 kHz switching), with capacitance maximally compensated. Upon completion of data collection from a motoneurone, confirmation of the resting membrane potential occurred by backing the micropipette out of the cell using steps of 5 μm. In accordance with the University of Manitoba animal ethics requirements, animals were killed by an I.V. injection of potassium chloride and a bilateral pneumothorax. Intracellular data

Rheobase (current level at which a 50 ms pulse produces a discharge probability of 0.5), input conductance (IC), spike voltage threshold (Vth ), SFA and  C 2015 The Authors. The Journal of Physiology  C 2015 The Physiological Society

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the frequency–current (F–I) slope were collected in discontinuous current clamp (3–10 kHz switching) mode, while the AHP was recorded in bridge mode after balancing. Signals were collected (1) before locomotion in a quiescent state, (2) during the preparatory period just before locomotion marked by increased ENG signal amplitude prior to flexor–extensor alternation, (3) during fictive locomotion, and (4) after locomotion in a quiescent state, which was the period immediately following and up to 2 min after fictive locomotion. The data are described with reference to LDP (oscillating membrane) and idle cells (no membrane oscillation) during alternating flexor–extensor nerve activity. Figure 2 shows an example of ramp current injection (5 s ascending, 5 s descending) in the quiescent state and during the preparatory tonic ENG period before peripheral nerve alternation. Note the rise in amplitude of the ENG signal (top right panel) during current injection. In this example, discharge from the motoneurone also begins at a reduced rheobase.

Frequency–current relation

Rhythmic motoneurone firing properties were calculated from the relationship between instantaneous discharge rate (reciprocal of the inter-spike interval value) and current amplitude from a ramp injection (5 s ascending, 5 s descending) for both LDP and idle cells. The slope of the F–I curve was calculated from a custom script written in MATLAB (version R2013a, MathWorks Inc., Natick, MA, USA). Frequency–current relationships are presented before locomotion, during the tonic period, during locomotion, and after locomotion, where possible.

Spike threshold measurement

All reported Vth measurements are for the first spike evoked either by current injection or the first spike occurring on an LDP. Action potential voltage thresholds were measured under control conditions from the first spike elicited by either a ramp or rectangular

Figure 1. Rhythmic discharge during ramp current injection Ramp current injection (Ac, Bc) with resulting intracellular motoneurone recording (membrane potential, Em ; Ab, Bb), and alternating flexor/extensor peripheral nerves (Aa, Ba) for an idle (A) and LDP (B) motoneurone. A, recordings from before locomotion, just before the tonic period, and during locomotion for an idle cell. The arrows indicate the rheobase for each ramp from left to right: before, 14.6 nA; tonic, 11.4 nA; locomotion, 8.7 nA. B, recordings from the tonic period, LDP generation during locomotion, and the period following locomotion (two additional ramps are not illustrated) for an LDP cell. The arrows represent the rheobase for the ramps left to right: tonic, 18.5 nA; locomotion, 6.7 nA; and after 14 nA. Note that the current amplitude and ramp duration (Ac, Bc) remain equal for the entire epoch. In B, after an initial response to excitation following locomotion, the motoneurone fails to fire 20 s after locomotion ceased but was able to respond to other measurements of motoneurone properties (IC and rheobase, for example). Ca and b, the after-hyperpolarization period of spikes generated from the motoneurone illustrated in B, in quiescence before locomotion (not shown in B) and during fictive locomotion (spike truncated for illustration purposes). Note the difference in the relative size (denoted by the dashed lines in Ca and b) and the hyperpolarized membrane potential at spike initiation during locomotion compared to before locomotion.  C 2015 The Authors. The Journal of Physiology  C 2015 The Physiological Society

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pulses of intracellular injected depolarizing current. Vth measurements during fictive locomotion were made from spikes generated by current injection or occurring spontaneously during the depolarizing phase of LDP. The Vth of action potentials elicited by ramps and rectangular current injections were not compared due to previous reports (Krawitz et al. 2001; Power et al. 2010) demonstrating more depolarized Vth measurements during ramp-induced spiking when compared to pulses. Ramp-induced depolarization of Vth has been attributed to accommodation of voltage-gated sodium channels resulting from the relatively slow rate of rise of the depolarizing ramps (e.g. Kuo et al. 2006). Comparisons are reported for the Vth of spikes evoked by: (1) current pulses with those elicited with current pulses during fictive locomotion, (2) current pulses with those occurring spontaneously on locomotor cycle depolarizations, or (3) current ramps with those elicited with current ramps occurring during fictive locomotion. Figure 1C (right panels, spikes truncated) shows an example of motoneurone discharge elicited from ramp current injection before and during fictive locomotion with LDP generation.

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Only motoneurones with action potential amplitudes ࣙ55 mV during control conditions elicited by depolarizing current pulses or antidromic stimulation were included in the analysis. Two cells with obvious changes in action potential shape, amplitude, or duration were excluded from the set of 26 motoneurones. Vth was determined prior to the initiation of fictive locomotion using depolarizing current ramps and/or pulses and was compared to the Vth of the same motoneurone during the tonic period and fictive locomotion. Vth was defined as the membrane potential at which depolarization increased at ࣙ10 V s−1 . At the 10 kHz sampling rate used, the reported Vth value was the membrane potential of the data point that was ࣙ1 mV more depolarized than the preceding point. This measure was chosen because it corresponded well to the distinct rising portion of the action potential and was easily selected by independent observers. The same definition of Vth has previously been employed (Krawitz et al. 2001; Power et al. 2010). Changes in Vth < 1 mV were deemed ‘no change.’ In most motoneurones, the Vth values reported for both control and fictive locomotion conditions were from an average of three spikes. The Vth for each motoneurone was measured in the same data file during control conditions and fictive locomotion. Each cell thus served as its own control. Group mean Vth values were calculated from the individual means. All comparisons were tested for statistical significance using Student’s paired t test. After-hyperpolarization

Figure 2. Ramp current injection during the tonic period Motoneurone discharge (B) in response to ramp current injection (C) before locomotion (left) and during the tonic period (right). Note that discharge during the tonic period starts at a lower rheobase. The top panel illustrates the rising ENG amplitude (A) during the lead up to locomotion. The neurograms in the top panel are time locked to the ramps in panels B and C. The ramp before locomotion and that shown during the tonic period are separated by 20 s.

The peak amplitude and half-decay time of the post-spike after-hyperpolarization (AHPpk and AHPhfd , respectively) were measured from an average of at least 30 action potentials generated from a supra-intensity 0.5 ms intracellular current stimulus. For idle cells, these measurements were acquired during quiescence, the tonic phase and during locomotion. For LDP cells, AHPpk and AHPhfd were calculated during quiescence and the tonic phase only. The difference between the resting membrane potential and the point at which the AHP reached its maximum hyperpolarization following the spike determined the AHPpk . The measure AHPhfd was taken as the time between the membrane potential peak and the point the membrane potential reached half the AHPpk value. The baseline for the AHP occurring on the LDP was not possible to ascertain; as such, the analysis for these cells was qualitative. Input conductance

Input conductance (IC) was the averaged response of the membrane potential to several 20 ms, 3 nA hyperpolarizing pulses (at least 20). With reference to  C 2015 The Authors. The Journal of Physiology  C 2015 The Physiological Society

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motoneurones generating LDPs, IC values were evaluated during the trough and peak of the LDP separately, as well as across the entire bout of locomotion regardless of the LDP phase. Since there was no phase associated with the idle cells, the IC was ascertained during flexor–extensor peripheral nerve alternation during locomotion. Spike frequency adaptation

SFA is the time-dependent decrease in motoneurone discharge rate during a prolonged constant current excitation. SFA has also been shown to occur during prolonged intermittent stimulation using an extracellular current pulse (Spielmann et al. 1993). SFA was evaluated during constant current injection before and during locomotion, and during LDP spiking, and during constant current injection with a sinusoidal current pulse superimposed in two motoneurones. In quiescence and fictive locomotion, SFA was tested with a constant-amplitude current stimulus for ࣙ20 s at 1.5× the rhythmic firing threshold of the impaled motoneurone. The number of spikes during the first 5 s of a constant-amplitude current pulse was compared to the number of spikes during the final 5 s of the constant current pulse to test SFA. The method used previously by this lab (Button et al. 2007) was not used in order to ensure consistency (LDP motoneurones had less spikes during the trough phase and this skewed the measure). During LDP-driven firing during locomotion, the definition of the term SFA needed to be readdressed, since a prolonged intracellular current stimulus was not responsible for motoneuron discharge. In the case of the LDP discharging via CPG excitation, evidence of a time-dependent decrease in discharge was determined by evaluating the mean discharge rate and coefficient of variation (CV) for each LDP over time. Given that the discharge pattern from an LDP is quite variable, if SFA occurred across a number of LDPs, then the mean should show a gradual decrease in firing rate and the CV should vary with the mean assuming the variability does not drastically change. LDP discharge was evaluated using the CV and mean for at least 20 s. In order to determine if adaptation was a phenomenon strictly associated with tonic/constant current pulse, in one motoneurone a depolarizing sinusoidal current pulse was injected for 20 s, alone, and superimposed upon a prolonged current step. For the sinusoidal current injection alone, motoneurone discharge was evaluated by looking at the mean and CV of the resulting discharge over time, while the sinusoidal current injection superimposed upon a constant current pulse was evaluated by comparing the number of spikes discharged in the first 5 s compared to the final 5 s.

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Statistics

The differences between the F–I slope, rheobase and IC of extensor motoneurones were compared among the conditions before locomotion, during locomotion and, where possible, during the tonic period and after locomotion using a repeated measures analysis of variance (ANOVA, Statistica, v9; StatSoft, Tulsa, OK, USA) or a Student’s t test with an α level of P ࣘ 0.05 to determine significance. A significant ANOVA F ratio was followed with planned contrasts that determined which means/groups significantly differed from each other. In addition, a trend was said to exist for an α level of P < 0.1 during the tonic period in order to describe ongoing change in the motoneurone. The AHP comparison between no locomotion and locomotion, when a discharging LDP occurred, relied on qualitative judgment due to the inability to accurately define a baseline during locomotion. Data are presented as means ± SD. Results Data from 29 motoneurones are presented. Pooled data for the F–I slope, IC, and Vth , across the conditions are shown in Table 1 for ‘idle cells’ and Table 2 for ‘LDP cells’. In summary, Vth , rheobase, and F–I slope are altered during fictive locomotion. Spike frequency adaptation occurred only when a depolarizing current pulse was delivered for a sustained period of time; SFA did not seem to be present in motoneurones discharging through central pattern-generated activity, or when rhythmic depolarization of the motoneurone occurred via intracellularly injected sinusoidal current, in order to simulate LDPs. The data collected were divided into motoneurones that produced an LDP during fictive locomotion (LDP motoneurones) and motoneurones that did not produce an LDP (idle). The distinction of the idle motoneurones was simply that there was no membrane potential oscillation during locomotion identified by alternating flexor and extensor peripheral nerve activity. Behaviourally, LDP and idle motoneurone properties reacted similarly in terms of enhanced excitability when compared to the resting state. Evidence of enhanced excitability included a reduced rheobase and/or a hyperpolarized Vth in both LDP and idle motoneurones during the tonic period before locomotion initiated and during locomotion. The data therefore suggest that the entire motor pool is primed for movement, regardless of whether the motoneurone being recorded from is participating in locomotion. When the AHP is evaluated after delivering a short 0.5 ms

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Table 1. Motoneurone properties of idle cells Measure

Before nA−1 )

Slope (Hz Rmp-Rh (nA) ࢞Vth from Before ࢞Vth from Tonic IC Rh-50 ms Mean maximum discharge rate (Hz) AHPpk (mV)

Tonic

12.7 ± 10.1 9.4 ± 5.9 N/A

 −3.0 ± 6.1  −1.6 ± 1.0‡ −2.0 ± 2.1∗

0.18 ± 0.08 9.0 ± 4.1 50.7 ± 16.8

0.18 ± 0.08 8.2 ± 4.0† 81.2 ± 13.1†

2.2 ± 1.2

2.3 ± 1.6

Locomotion 6.6 6.2 −5.8 −3.6 0.18 6.8 84.2

± ± ± ± ± ± ±

2.3∗ 4.8∗ 2.2 1.3 0.10 4.1∗ 8.0∗

2.1 ± 2.0

After  −1.7 ± 6.9  0.04 ± 0.6 N/A 0.19 ± 0.09 9.4 ± 4.5 48.1 ± 14.2 1.6 ± 1.1

∗ Significant

difference from all other motoneurone states; between Tonic and Before periods at P < 0.05; ‡ a trend at P < 0.07. The Tonic and After values for Slope and ramp rheobase (Rmp-Rh) are referenced as a change from the Before value because of an unequal n across groups. N/A, not applicable. † difference

Table 2. Motoneurone properties of LDP cells Measure Slope (Hz nA−1 ) Rmp-Rh (nA) ࢞Vth from Before ࢞Vth from Tonic IC Mean maximum discharge rate (Hz) AHPpk (mV)

Before 15.3 ± 6.3 11.1 ± 7.8 N/A

Tonic  −2.0 ± 3.6  −1.7 ± 1.6‡ −2.6 ± 2.4†

0.19 ± 0.07 67.5 ± 19.3

0.17 ± 0.06 108.8 ± 21.1†

2.2 ± 2.0

2.6 ± 2.6

Locomotion 4.5 4.4 −3.7 −3.1 0.19 189.4

± ± ± ± ± ±

N/A

2.1∗ 7.0∗ 2.4 2.5 0.08 15.0∗

After  1.1 ± 5.2  0.6 ± 1.7 N/A 0.18 ± 0.07 66.0 ± 14.8 2.4 ± 2.1

∗ Significant

difference from all other motoneurone states; difference between that value and the Before and After states; ‡ a trend in the data between the Tonic and Before states at P < 0.07. The Tonic and After values for Slope and ramp rheobase (Rmp-Rh) are referenced as a change from the Before value because of an unequal n across groups. † significant

pulse, no measurable differences in AHPpk or AHPhfd were recorded in idle motoneurones or LDP-generating motoneurones during the tonic phase (AHP could not be adequately evaluated during locomotion) compared to quiescence. The AHP appears to be smaller when evaluated on LDP-driven spiking (Fig. 1Cb) compared to that driven by ramp current injection (Fig. 1Ca), a result that is discussed further below. See Table 1 (idle) and Table 2 (LDP) for means (±SD) and significance (where applicable).

No systematic change in input conductance occurred for motoneurones

No systematic change in input conductance (IC) occurred in any state for both LDP (F(5,35) = 1.2, not significant (n.s.)) or idle (F(3,48) = 0.72, n.s.) cells. See Fig. 3A and B for mean and data spread for LDP and idle motoneurones, respectively. When the data are considered for each cell, the change in IC between different states of motoneurone activity is not striking. For LDP-producing motoneurones,

IC changed between 20 and 40% for the LDP cells but the change was not consistent (three increased, two decreased; Fig. 3A) and varied most in the trough phase of the LDP. The IC for idle cells varied little with the exception of a single cell that increased (Fig. 3B).

Vth hyperpolarizes during locomotion for all motoneurones

Vth during fictive locomotion was assessed in 26 antidromically identified extensor motoneurones. The large range of rheobase values (2–20 nA) and AHPhfd (8.5–35.1 ms) suggests that motoneurones innervating both slow (low rheobase; long AHPhfd ) and fast (high rheobase; short AHPhfd ) twitch muscle fibres were included in the sample (Gardiner, 1993). The Vth of all motoneurones became hyperpolarized for both LDP and idle cells. The Vth of motoneurones generating an LDP hyperpolarized between: (1) the quiescent period and the tonic period (−2.6 ± 2.4; t = 4.0, degrees of freedom (d.f.) = 6, P = 0.006); (2) the  C 2015 The Authors. The Journal of Physiology  C 2015 The Physiological Society

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quiescent period and locomotion (−3.7 ± 2.4; t = 5.8, d.f. = 13, P = 0.00005); and (3) the tonic period and locomotion (−3.1 ± 2.5; t = 3.0, d.f. = 4, P = 0.039). Vth hyperpolarization occurred for the first spike during fictive locomotion and was thus not a consequence of previous action potentials. Vth also hyperpolarized in idle motoneurones. Idle motoneurones showed significantly hyperpolarized Vth between the quiescent period and locomotion (−5.8 ± 2.2; t = 7.1, d.f. = 6, P = 0.00039), and between the tonic period and locomotion (−3.6 ± 1.3; t = 7.2, d.f. = 4, P < 0.0019). The before locomotion and the tonic period in idle cells only tended to show hyperpolarized Vth (−2.0 ± 2.1; t = 2.3, d.f. = 5, P < 0.069). Figure 2 illustrates the increased response of the motoneurone during the tonic period. The ENG signal depicted in the top pane (Fig. 2A) illustrates the period before locomotion (left) and during the tonic period (right). Motoneurone discharge (Fig. 2B) is shown to occur at a lower rheobase and Vth in response to ramp current injection (Fig. 2C). This suggests that changes in motoneurone responsiveness begin prior to rhythmic and alternating locomotor output. The mean Vth changes for idle and LDP cells are displayed in Tables 1 and 2, respectively. After-hyperpolarization during locomotion

In accordance with previous investigations (Brownstone et al. 1992; Power et al. 2010) the AHP appears to be reduced during locomotion compared to the AHP during ramp current injection. An example showing the difference in AHP between locomotion and ramp

Figure 3. Motoneurone input conductance Mean and individual input conductance for locomotor drive potential-generating (A) and idle (B) motoneurones. Error bars represent standard deviation. No difference in mean input conductance existed.  C 2015 The Authors. The Journal of Physiology  C 2015 The Physiological Society

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current injection is shown in Fig. 1C. This subjective analysis illustrates the smaller AHP (even at similar firing rates) between locomotion-driven discharge and ramp current-driven discharge (see dashed lines, Fig. 1Ca and b for comparison of AHP amplitudes). However, a definitive conclusion concerning a reduced AHP cannot be firmly established due to a hyperpolarized Vth and possible conductance changes during fictive locomotion.

Frequency–current slope decreases during locomotion

Compared to all other states (quiescence or tonic), a reduction and leftward shift in the F–I slope occurred for all motoneurones involved in locomotion regardless of whether or not an LDP existed (Table 1). LDP-producing motoneurones during locomotion had a 70% reduction in F–I slopes (Table 1; t = −5.5, d.f. = 13, P = 0.00009), discharged at faster firing rates (t = −3.8, d.f. = 13, P = 0.0029), had either a reduced or eliminated rheobase, and showed no change in the frequency of the LDP during ramp current injection. Ramp current injected in LDP-producing motoneurones still maintained a hyperpolarized trough phase without spiking until the injected current reached the level of current excitation that produced a spiking on the ramp before locomotion. See Fig. 1 for an illustration of an LDP and idle cell during ramp current injection. Idle motoneurones discharged at a lower ramp rheobase (49% less current amplitude; t = 4.5, d.f. = 12, P = 0.0007), showed a 55% reduction in the F–I slope (t = 2.4, d.f. = 12, P = 0.036) and discharged at higher firing rates (t =

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5.1, d.f. = 12, P = 0.00014) during locomotion. A trend also existed for these motoneurones to fire at lower ramp current amplitudes (10% lower; t = 2.6, d.f. = 5, P = 0.051) during the tonic period leading up to alternating ENG activity. This behaviour during the tonic period indicates that the locomotor system is slightly primed prior to the commencement of alternating peripheral nerve activity. As seen in the LDP cells as well, the maximal discharge rates for the same peak amplitude of injected ramp current were faster in the tonic period compared to before locomotion (t = 3.1, d.f. = 5, P = 0.024). See Fig. 4 for an illustration of the slopes of F–I relationships for both LDP (Fig. 4A) and idle (Fig. 4B) motoneurones. The F–I slope is variable after locomotion

For each motoneurone sampled, the goal was to collect a series of ramps before, during and after locomotion. However, it was not always possible to collect data following locomotion due to loss of cell penetration. As such, the number of motoneurones analysed before locomotion is greater than that analysed following locomotion. Despite a comparable average F–I slope before and after locomotion (Table 1: idle cells; Table 2: LDP cells), variability in response to ramp current injection was evident. At 20 s after the cessation of MLR stimulation, motoneurones showed evidence of decreased excitability as displayed by: discharge failure, a reduced F–I slope or a reduction in the number of spikes when stimulated with ramp current injections equal in magnitude to those delivered before and during locomotion. Figure 1B illustrates this reduction in excitability in a motoneurone that fired initially after locomotion ceased, but then failed

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to discharge. After locomotion ceased, the motoneurone was excited with five consecutive ramp current injections (5 s up; 5 s down; five are shown), which covered an epoch of 80 s. Although the first ramp current injection after locomotion produced a similar response to that recorded prior to locomotion (before: 5.9 Hz nA−1 ; after: 6.4 Hz nA−1 ), the following four consecutive ramp current injections failed to discharge the cell rhythmically (only 3 of 5 post ramps are illustrated in Fig. 1B). Reduced excitability, as noted by the criteria outlined above, occurred in half of the LDP-generating motoneurones. The variability in F–I slope after locomotion was not limited to LDP cells, as half of the idle motoneurones showed changes in excitability following locomotion. For example, an idle cell responded to the first ramp immediately after fictive locomotion with a slope of 21.9 Hz nA−1 (out of 7 consecutive ramps) compared to a 12.4 Hz nA−1 slope prior to locomotion (43% increase). An interesting observation with this cell was that the third ramp after fictive locomotion (approximately 32 s after fictive locomotion) almost failed to discharge, producing only four spikes (minimum of 15 spikes for other ramps). Spike frequency adaptation is eliminated during locomotion

LDP motoneurones produced the shortest interspike interval often near the peak of the depolarized potential and the mean instantaneous firing rate for each LDP showed no systematic decrease across locomotor episodes. Similarly, the slight variation in the CV appeared to be associated with a shorter duration LDP (Fig. 5A mean and CV). The standard deviation for the CV and mean discharge rate for the LDP train shown in Fig. 5A

Figure 4. Motoneurone frequency–current relationship slope Representative data are illustrated from an LDP (A) and an idle motoneurone (B). The slopes of the frequency–current relationships are shown during Quiescence, before fictive locomotion (continuous line); Quiescence, after fictive locomotion (dashed line); and Fictive Locomotion (dotted line) for an LDP cell. For Idle motoneurones (B), the example also includes the slope of the Tonic period (dash-dot-dash line). Tables 1 and 2 show the mean ± SD for the different groups.  C 2015 The Authors. The Journal of Physiology  C 2015 The Physiological Society

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was 2.2 and 1.8 Hz, respectively. As such, it seems that ‘naturally’ occurring discharge is not associated with adaptation in the rat, which agrees with Brownstone et al. (2011). To determine whether the non-adapting quality was associated with locomotion or the depolarization and hyperpolarization of the membrane, a sinusoidal current that caused rhythmic oscillation and depolarization of the membrane was delivered (different motoneurone). The mean and CV of the resulting motoneurone discharge also showed little change across a 20 s period. Figure 5C shows the mean discharge rate and CV for each burst and the recorded signals (Fig. 5D). Given these results, it seems that the alternating membrane potential is enough to eliminate SFA. However, when sustained constant-amplitude current is delivered intracellularly, SFA was evident, regardless of whether fictive locomotion was occurring. All motoneurones showed adaptation patterns when the motoneurones were excited with a constant-amplitude current pulse. A comparison of the number of spikes discharged in the first 5 s and last 5 s of a sustained constant current step (20 s) revealed that the last 5 s of the current

pulse was associated with fewer motoneurone spikes. In one LDP motoneurone, this comparison showed a 17% reduction in the number of spikes (first 5 s: 307 spikes; last 5 s: 257) during locomotion; a reduction of 16% (first 5 s: 200; last 5 s: 164) during quiescence; and 20% fewer spikes in the last 5 s (first 5 s: 307; last 5 s 244) when sinusoidal current injection was superimposed upon the prolonged current step in quiescence. In the idle motoneurone, a prolonged current step during locomotion resulted in an 18% decrease (first 5 s: 297; last 5 s: 245), while during the quiescence period of this idle cell, the number of spikes decreased by 34% (first 5 s: 303; last 5 s: 197). In summary, SFA is related to the tonic nature of the current pulse and is absent when the membrane is oscillating.

Discussion This is the first investigation to investigate intracellular motoneurone properties in the adult decerebrate rat during fictive locomotion and during the tonic period immediately preceding locomotion. The analysis during

Figure 5. No evidence of spike frequency adaptation in LDPs or discharge from sinusoidal current injection The mean and coefficient of variation of motoneurone discharge (A) from MLR-driven locomotor drive potentials (B) are relatively consistent across the 20 s locomotor bout. The mean and coefficient of variation of motoneurone discharge (C) resulting from intracellular sinusoidal current injection (D) are also relatively consistent. Neither the LDP nor idle motoneurones show spike frequency adaptation.  C 2015 The Authors. The Journal of Physiology  C 2015 The Physiological Society

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the tonic period, in which peripheral nerve signal amplitude increases prior to flexor–extensor alternation, has not previously received significant attention. From a systems perspective, the data provide support for the notion that motoneurones become more responsive to synaptic input before the emerging signal pattern for locomotion. The importance of studying how rat motoneurones respond to a rhythmic motor output is critical in moving forward with increased and decreased physical activity protocols in a behaving animal. In addition, establishing a protocol for ongoing research relating to fictive locomotion seems appropriate given decreased use of the cat model, and the different daily activity patterns of cats versus rats. As such, it is imperative to determine if a significant difference existed in the so-called state-dependent motoneurone properties. The data indicate that many similarities exist between the rat and cat data. The hyperpolarized Vth found in both LDP and idle motoneurones indicates that an increase in excitability occurs across the motoneurone pool regardless of whether an LDP is generated, possibly indicating metabotropic motor pool priming. Furthermore, SFA was not evident in LDP-generating motoneurones when locomotion was driven by descending activation or when driven by an intracellular oscillating current pulse, which suggests that rhythmic excitation is not associated with SFA. This can be implied because the presence of a constant-amplitude current step during a rhythmic output like fictive locomotion did lead to decreased discharge rate. Prior to this investigation, only peripheral nerve activity had been examined during fictive locomotion induced by electrical stimulation of the MLR (Skinner & Gracia-Rill, 1984; Canu et al. 2001 for example) in the rat, while assessment of intrinsic motoneurone properties in the adult rat had not been conducted.

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Power et al. (2010) also present IC data that show no consistent change during scratch drive potentials in the cat. However, Schmidt et al. (1989) found systematic increases/decreases in IC for extensors depending upon the phase and Gosgnach et al. (2000) showed increases in IC during locomotion in the cat, while both Perrault (2002) and Alaburda et al. (2005) show considerable increases (>50%) in IC during fictive scratch in the cat and turtle. From the data in this experiment, and the variability between previous reports, it seems as though the relation between input conductance and motoneurone output during locomotion is not clear. Alaburda et al. (2005) and Berg et al. (2007) argue that balanced excitation and inhibition during scratch drive potentials are responsible for the high-conductance state during fictive scratch in the turtle. Given that the majority of motoneurones in the data presented in Fig. 3A do not exhibit the same large conductance changes as that during the turtle scratch drive potential, the data presented in Fig. 3 are in line with the information presented by Shefchyk & Jordan (1985a,b) in the cat and by Endo & Kiehn (2008) in the neonatal mouse. In these papers, it is shown that excitation/inhibition is not balanced but offset, with a greater amount of inhibition and few IC changes. Even when the Schmidt et al. (1989) IC data are considered, the conductance changes are not in accordance with those shown by Berg et al. (2007). We found no evidence for the coincident inhibition and excitation producing a high conductance state during the depolarizing waves of rhythmic episodes in motoneurons described for the turtle scratch (Berg et al. 2007). As such, the idea of a ‘push–pull’ (Endo & Kiehn, 2008) operation of the CPG network during locomotion in the rat is supported during fictive locomotion.

Locomotion reduces slope of frequency–current relation but increases rate of motoneurone firing Input conductance

The finding of little or no change in input conductance during fictive locomotion is supported by previous reports. The results show that the pooled input conductance did not change amongst idle and LDP motoneurones. However, the response was variable in the LDP-generating motoneurones, with IC increasing in three motoneurones and decreasing in two (Fig. 3A). a Shefchyk & Jordan (1985a) also showed variable conductance changes in hindlimb cat motoneurones during fictive locomotion whereby the majority (54%) of cells showed no change. Of the remaining cells, changes in input conductance (increase/decrease) were limited to less than 20% during brainstem-initiated fictive locomotion. In addition, little difference existed between the peak (depolarized) and the trough (hyperpolarized) phases of the LDP, similar to that shown in the present study (Fig. 3).

In response to ramp current injection motoneurones reached higher firing rates in both the tonic period and during locomotion in both LDP-producing and idle motoneurones (Table 1). The increased discharge rate also occurred during the tonic period leading up to locomotion. The discharge pattern of firing remained linear, but the firing rate versus current data points showed greater variability during the tonic period and during locomotion (mean r < 0.4). In both cat and rat, the relationship between current and frequency has been shown to be linear (Granit et al. 1963; MacDonell et al. 2012) in the resting state in the anaesthetized preparation, while Brownstone et al. (1992) reported an eliminated F–I relationship during locomotor-like activity, and suggested that the AHP does not control rhythmic firing during motor output in the decerebrate cat. The data presented in this manuscript show that fictive locomotion reduces  C 2015 The Authors. The Journal of Physiology  C 2015 The Physiological Society

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the slope of the fitted frequency–current relationship, but it does persist. That being said, the combined results of a decreased F–I slope, apparent reduction in the AHP during locomotion and the lack of SFA when intracellular current is not being injected does support the suggestion the AHP does little to control rhythmic firing in a behaving animal. It is not surprising that the frequency–current slope is reduced when motoneurones are otherwise engaged in a motor behaviour. Modelling studies suggest that increased Na+ conductance reduces the slope of the F–I curve (Dai et al. 2001) and that the F–I slope for a given cell can vary depending upon the type and magnitude of the conductance change (Gardiner et al. 2006). However, the varied rhythmic firing behaviour of the motoneurone to similar amplitude ramp current injections after locomotion in LDP cells is difficult to explain but may be a function of recruitment threshold. In these examples, the resting membrane potential remained consistent with that seen prior to locomotion, but all of these cells (n = 5) had a ramp rheobase > 18 nA. Since the rheobase recruitment thresholds and AHP half-decay durations are consistent with higher threshold motoneurones, the decreasing excitability may be a fatigue mechanism similar to that shown by Cotel and colleagues (2013). In this, serotonin released during bouts of locomotion (Veasey et al. 1995) would be in sufficient concentrations to ‘spill-over’ and activate inhibitory 5-HT-1a receptors located at the initial segment (Cotel et al. 2013), which would reduce spike generation probability. It is possible that higher threshold motoneurones are more susceptible to the effects of this spill-over because of the excitation needed for recruitment.

Spike frequency adaptation

The time-dependent decline in discharge frequency during sustained excitation, a phenomenon known as spike-frequency adaptation (Granit et al. 1963; Kernell & Monster, 1982), appears to be absent during LDP discharging motoneurones activated solely through CPG activation (Fig. 5A and B), which agrees with results shown in the cat (Brownstone et al. 2011). As illustrated in Fig. 5C and D, motoneurones driven to fire from a sinusoidal current impulse also fail to show adaptation (Fig. 5C). Brownstone et al. (2011) compared motoneurone LDP discharge over several minutes and showed that adaptation does not occur in the cat. The duration of adaptation examined here was 20 s and also showed no time-dependent decrease in the mean rate of each bursting LDP. Curiously, a discrepancy in the results between the two experiments occurred with regard to discharge driven by intracellular current injection. Brownstone et al. (2011) show time-dependent decreases in discharge

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in response to 500 ms square-wave pulses delivered over 4 min, whereas no time-dependent decrease in discharge was shown in the present study. Perhaps the difference is due to the sinusoidal pattern of stimulation used in this experiment compared to the square-wave pulse used in the Brownstone et al. (2011) experiment. Another possibility is that a longer stimulation period (4 min vs. 20 s) is the main contributor to the dissimilar result. When an injected intracellular depolarizing pulse is added to the discharging LDP, an initial increase followed by a steady slow decline in peak firing rates from the LDP occurred. An explanation for this behaviour may come from a change in Na+ channel opening probability due to the intracellular current injection. It has been shown in the past that Na+ channel kinetics greatly affect SFA in vitro and that AHP summation probably plays no role in SFA (Miles et al. 2005). Thus, the re-emergence of SFA while engaged in fictive locomotion in both the LDP and idle motoneurones implies that the tonic nature of the constant-amplitude current step may be responsible for changing Na+ channel kinetics. Lastly, superimposing a sine-wave in conjunction with a current step (in order to mimic membrane oscillation similar to that of an LDP) had little effect on SFA except for higher peak firing rates (associated with the peak of the sine-wave).

Locomotor drive potential versus idle motoneurones

Two different motoneurone behaviours were evident during locomotor-like activity. In one case, locomotor-like activity was present and the impaled motoneurone produced LDPs; in the other, locomotor-like activity was present but the impaled motoneurone did not produce an LDP, referred to as an ‘idle’ motoneurone. Idle motoneurones increased in excitability and showed the same increase in excitability as those motoneurones displaying an LDP. With regard to Vth hyperpolarizing in both LDP and idle motoneurones, previous reports show that both the scratch depolarized potential (Power et al. 2010) and LDP (Krawitz et al. 2001) amplitudes were not correlated with Vth hyperpolarization. In addition, Gilmore & Fedirchuk (2004) showed that Vth hyperpolarization was present in spinal neurones in the neonatal rat during electrical stimulation of the medulla that did not produce ventral root activity. Given the hyperpolarization of the Vth in idle motoneurones, it seems as though the Vth change starts to occur when the motor network becomes active. Although it is extremely interesting that idle motoneurones show similar state-dependent changes to those generating LDPs, the difference between these motoneurones is not particularly evident. Both groups showed a similar range of rheobase values, even though the AHP half-decay time indicated that all but two cells

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of the idle motoneurones could be categorized as ‘fast’ motoneurones by the criteria adopted by Gardiner (1993). In addition, the motoneurones in each group (LDP and idle cells) shared a similar range in input resistance, and conformed to the range found in Gardiner (1993). Perhaps the extensors that did not produce LDPs are simply not recruited for the task of locomotion and are used for other motor programmes. However, the data suggest that despite not being recruited for a particular task (locomotion in this example) the motoneurones sampled showed similar ranges of input conductance and AHP parameters to those found previously (Gardiner, 1993). This mechanism may ensure timely and effective recruitment should the need suddenly arise or if a different motor programme needed activation. Although not measured in this study, the concomitant increase in excitability may be a metabotropic response to increased levels of the neuromodulators associated with movement. In the present study, fictive locomotion was preceded in a limited number of experiments by a tonic activation of the motor pools. Interestingly, we demonstrate that during this pre-locomotor tonic activity, motoneurones exhibited Vth hyperpolarization, as occurred during the rhythmic and alternating portion of fictive locomotion. Recently, Power et al. (2010) examined state-dependent changes in motoneurone excitability using the fictive scratch model in the adult decerebrate cat. Briefly, fictive scratch is characterized by an approach phase and a rhythmic phase. During the approach phase there is an initial period of tonic flexion in the ipsilateral hindlimb followed by a series of rhythmic, rapid alternating flexion and extension ‘movements’ (Kuhta & Smith, 1990). Simultaneous activity in the contralateral hindlimb consists of tonic extensor activity to support the body, referred to as body weight support or stance (Perreault, 2002). Power et al. (2010) demonstrated that when a motoneurone was engaged in rhythmic scratching the Vth hyperpolarized and the AHP was reduced. When the same motoneurone was active during stance the Vth depolarized and the AHP amplitude was unchanged, suggesting that there are different neural control mechanisms regulating spinal motoneurone excitability between scratch and stance in the cat. It is currently unclear what this pre-rhythmic tonic activity represented, but it could be a righting reflex or activation of the locomotor network prior to it ‘breaking’ into rhythmic and alternating activity. Either way, it is evident that if locomotion was the goal of the motor system that the motoneurone properties were altered in a manner consistent with locomotion. In the cat, during scratch we see a depolarized Vth during the depolarized phase, while the rat shows hyperpolarization of the Vth during the depolarized phase. As such, the task of stance versus locomotion appears to determine whether excitation or inhibition becomes dominant.

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Comparison to changes in motoneurone properties during rhythmic motor outputs in adult cat and neonatal rat

The changes in motoneurone properties discussed here are similar to those previously described during fictive locomotion (Brownstone et al. 1992; Krawitz et al. 2001) and scratch (Power et al. 2010) in the adult decerebrate cat and fictive locomotion in the neonatal rat (Gilmore & Fedirchuk, 2004). In each of the aforementioned studies, the Vth hyperpolarized for the first action potential, recovered within minutes following motor output cessation and had a high incidence. The mean Vth hyperpolarization in the present study was −5.8 mV and −3.7 mV for idle and LDP cells, respectively. In comparison, the mean Vth hyperpolarization values were −5.8 mV and −8.0 mV during fictive scratch and locomotion in the adult decerebrate cat, respectively, and −6.0 mV in the neonatal rat. It is noteworthy that the incidences of Vth hyperpolarization, though high in each protocol, were not identical (cat locomotion: 100%; cat scratch: 83%; neonatal rat locomotion: 90%; and adult rat locomotion: 100%). There was also a wide range of Vth changes from quiescence to fictive locomotion (range: −1.2 to −10 mV) in the present study, which is in line with those previously reported (Krawitz et al. 2001; Gilmore & Fedirchuk, 2004; Power et al. 2010). It is also worth mentioning that the manner in which cat scratch and neonatal rat locomotion were generated was different from locomotion in the cat and adult rat. In the adult cat and rat, fictive locomotion was induced via electrical stimulation of the MLR (Krawitz et al. 2001) which is functionally defined by its ability to induce locomotor activity. In contrast, cat scratch (Power et al. 2010) was generated via tactile stimulation of the pinnae and neonatal rat locomotion was induced via electrical stimulation of the ventromedial medulla (Gilmore & Fedirchuk, 2004). Thus, the exact pathways activated to induce the motor output were different with MLR stimulation resulting in Vth hyperpolarization in all cells examined whereas not all cells demonstrated Vth hyperpolarization during cat scratch and neonatal rat locomotion. In our study the AHP appeared smaller in LDP motoneurones, a result that would be consistent with the findings of others (Brownstone et al. 1992; Krawitz et al. 2001), although accurate measurement of AHP during locomotor-like activity in our experiments was not possible. It is evident that increasing the excitability of spinal motoneurones is an integral component of CPG-mediated motor outputs. Conclusion

Rat motoneurones show similar state-dependent changes during locomotion to those that have been previously

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reported in the cat and neonatal rat, and thus the state-dependent changes appear to be conserved across species and during development. These state-dependent changes are evident before locomotion begins, suggesting a metabotropic ‘priming’ of the motor system. This occurs in both motoneurones that are involved in the motor output and those that are not involved in the motor output but are part of the motor pool (i.e. extensor motoneurones). Since the rat model has been used often to study changes in the nervous system stimulated by increased/decreased neuromuscular activity, the results of this investigation demonstrate that the rat is an excellent model with which to study these adaptations during motor output. The next step in this research is to determine the extent to which increases and decreases in chronic physical activity alter motoneurone properties during motor output, to supplement and help interpret the changes that have been shown to occur when these motoneurones are measured in the quiescent state.

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Additional information Competing interests None declared.

Author contributions All experiments took place in the Spinal Cord and Neuromuscular Plasticity Lab, Spinal Cord Research Centre, at the University of Manitoba. All authors aided in the design of the experiments. C.W.M. and K.E.P. performed the experiments, analysed the data, interpreted the results and wrote the manuscript. J.W.C. performed the experiments and aided in manuscript preparation. K.R.G. oversaw animal care procedures, devised the surgery protocols and performed the surgical preparation in conjunction with C.W.M., J.W.C. and K.E.P. P.F.G. conceived the study, secured funding and aided in manuscript preparation. All authors approved the final version of the manuscript.

Funding This research was supported by grants from the Canadian Institutes of Health Research (CIHR), including individual operating grants to P.F.G. and a team grant in Physical Activity, Mobility and Neural Health (NERVE). Financial support was also provided by a Manitoba Health Research Council (MHRC) postdoctoral fellowship (C. W. MacDonell) and CIHR Doctoral award (J. W. Chopek).

Acknowledgements The authors thank Dr Larry Jordan for guidance with inducing fictive locomotion, as well as giving valuable insight with regard to the manuscript. The authors also wish to thank Dr Cabrales for generously providing assistance with the alginate solution (Cabrales et al. 2005).

 C 2015 The Authors. The Journal of Physiology  C 2015 The Physiological Society

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