Adult spinal V2a interneurons show increased excitability andserotonin-dependent bistability

Andreas Husch, Shelby B. Dietz, Diana N. Hong, and Ronald M. Harris-WarrickDepartment of Neurobiology and Behavior, Cornell University, Ithaca, New York

Submitted 26 September 2014; accepted in final form 21 November 2014

Husch A, Dietz SB, Hong DN, Harris-Warrick RM. Adult spinalV2a interneurons show increased excitability and serotonin-dependentbistability. J Neurophysiol 113: 1124–1134, 2015. First publishedDecember 17, 2014; doi:10.1152/jn.00741.2014.—In mice, most studiesof the organization of the spinal central pattern generator (CPG) forlocomotion, and its component neuron classes, have been performedon neonatal [postnatal day (P)2-P4] animals. While the neonatal spinalcord can generate a basic locomotor pattern, it is often argued that theCPG network is in an immature form whose detailed propertiesmature with postnatal development. Here, we compare intrinsic prop-erties and serotonergic modulation of the V2a class of excitatoryspinal interneurons in behaviorally mature (older than P43) mice tothose in neonatal mice. Using perforated patch recordings fromgenetically tagged V2a interneurons, we revealed an age-dependentincrease in excitability. The input resistance increased, the rheobasevalues decreased, and the relation between injected current and firingfrequency (F/I plot) showed higher excitability in the adult neurons,with almost all neurons firing tonically during a current step. The adultaction potential (AP) properties became narrower and taller, and theAP threshold hyperpolarized. While in neonates the AP afterhyper-polarization was monophasic, most adult V2a interneurons showed abiphasic afterhyperpolarization. Serotonin increased excitability anddepolarized most neonatal and adult V2a interneurons. However, in�30% of adult V2a interneurons, serotonin additionally elicitedspontaneous intrinsic membrane potential bistability, resulting inalternations between hyperpolarized and depolarized states with adramatically decreased membrane input resistance and facilitation ofevoked plateau potentials. This was never seen in younger animals.Our findings indicate a significant postnatal development of theproperties of locomotor-related V2a interneurons, which could altertheir interpretation of synaptic inputs in the locomotor CPG.

locomotion; maturation; neuromodulation; spinal cord

THE EXISTENCE OF CENTRAL PATTERN generator (CPG) neuronalnetworks in the spinal cord that coordinate locomotion was firstdemonstrated and studied in adult cats and dogs (Brown 1914;Eccles et al. 1956; Sherrington 1910). Nonmammalian modelorganisms such as the lamprey and zebrafish have providedimportant advances in understanding the cellular compositionand function of the locomotor CPG (Ausborn et al. 2012;Buschges et al. 2011; Fetcho et al. 2008; Grillner 2003;McLean et al. 2008). The combination of molecular genetics,electrophysiology, and imaging techniques in recent years hasmade the mouse spinal cord an important model system tostudy the organization of CPGs for mammalian locomotion(Arber 2012; Grossmann et al. 2010; Guertin 2009; Kiehn2006, 2011).

Several interneuron (IN) classes have been identified by theexpression of unique combinations of transcription factors(Goulding 2009; Goulding and Pfaff 2005), allowing scientiststo label, record, and manipulate the activity of each classduring fictive locomotion (Gosgnach et al. 2006; Lanuza et al.2004; Talpalar et al. 2013; Vallstedt and Kullander 2013). Thiscombined approach has shown that the V2a class of Chx10-expressing ipsilaterally projecting glutamatergic INs plays anumber of roles in the spinal cord. They contribute to thedescending inputs that normally drive locomotion (Crone et al.2008; Kimura et al. 2013), and cervical V2a INs help to controlskilled reaching (Azim et al. 2014). In the zebrafish, stimula-tion of V2a INs is sufficient to initiate locomotion in thezebrafish (Ljunggren et al. 2014), and distinct subsets of theseneurons are activated with increasing locomotor speed torecruit additional motoneurons for faster swimming (Ampatziset al. 2014; Fetcho and McLean 2010). In the mouse lumbarspinal cord (where the hindlimb locomotor CPG is located),these neurons are functionally heterogeneous (Dougherty andKiehn 2010; Zhong et al. 2010); one population shows nolocomotor-related activity while other locomotor-related pop-ulations synapse on motoneurons or commissural INs (CINs;Crone et al. 2008; Zhong et al. 2012). These neurons areincreasingly recruited with locomotor speed (Zhong et al.2011); V2a ablation has little effect on locomotion at lowspeeds but disrupts locomotion at intermediate speeds andresults in left-right synchrony at high speeds (Crone et al.2009). This suggests that at least a subset of V2a INs functionsto maintain left-right alternation at high locomotor speeds.(Al-Mosawie et al. 2007; Crone et al. 2008, 2009; Lundfald etal. 2007; Zhong et al. 2011).

The locomotor-related V2a INs receive descending activa-tion from the brain (Crone et al. 2008), including serotonergicinnervation. Serotonin (5-HT) plays a key role in spinal cordreflex and locomotor pattern generation (for review see Caza-lets et al. 1990; MacLean et al. 1998; Schmidt and Jordan2000). Distinct components of the motor pattern are controlledby multiple serotonin receptor subtypes (Al-Mosawie et al.2007; Landry et al. 2006; Liu et al. 2009; Madriaga 2004;Musienko et al. 2011; Pearlstein et al. 2005; Slawinska et al.2012), which modulate different cellular properties and ionicconductances in locomotor-related INs (Abbinanti and Harris-Warrick 2012; Dai and Jordan 2010; Diaz-Rios et al. 2007;Zhong et al. 2006a). In neonatal [postnatal day (P)1-P4] V2aneurons, 5-HT depolarizes and increases neuronal excitabilityand changes action potential (AP) properties (Dietz et al. 2012;Zhong et al. 2010).

To understand the role of 5-HT during rodent locomotion, itis important to determine its effects on adult as well as neonatal

Address for reprint requests and other correspondence: R. M. Harris-Warrick, Dept. of Neurobiology and Behavior, Seeley Mudd Hall, CornellUniv., Ithaca, NY 14853 (e-mail: ron.harris-warrick@cornell.edu).

J Neurophysiol 113: 1124–1134, 2015.First published December 17, 2014; doi:10.1152/jn.00741.2014.

1124 0022-3077/15 Copyright © 2015 the American Physiological Society www.jn.org

spinal neurons. Serotonergic processes reach the lumbar spinalcord by embryonic day 16, but the innervation pattern is stilldiffuse after birth and only achieves a more restricted adultpattern by P21 (Ballion et al. 2002; Rajaofetra et al. 1989).Most studies of spinal locomotor networks have been per-formed in neonates (typically P0-P5). These studies havefurthered our understanding of the neuronal composition of thelocomotor CPG, since it is possible to record from identifiedspinal INs in the isolated intact neonatal spinal cord duringfictive locomotion. However, the question remains whether theproperties of the neonatal INs remain the same as the networksmature to the fully locomoting adult. Postnatal changes incellular properties during the first 2–3 postnatal weeks, such asnarrowing of AP shape and emergence of plateau potentials,have been shown in CINs (Abbinanti et al. 2012) and inmotoneurons (Carlin et al. 2008; Gao and Ziskind-Conhaim1998). By developing a method to record electrophysiologicalactivity from spinal INs in adult spinal cord slices of any age(Husch et al. 2011), we can now answer this question morefully. In this article we demonstrate marked developmentalchanges in the electrophysiological properties of spinal V2aINs between neonatal (P2 to P4) mice and behaviorally matureadult (�P43) mice.

MATERIALS AND METHODS

Animals. Experiments were performed using two age groups:neonatal mice (P2–P4) and adult, locomotor mature mice (P43–P93).Chx10::cyan fluorescent protein (CFP) heterozygotes, with one re-combined Chx10::CFP allele and one wild-type Chx10 allele (Croneet al. 2009) were generated by Drs. Steven Crone and Kamal Sharmaat the University of Chicago (Crone et al. 2008). The animal protocolwas approved by the Institutional Animal Care and Use Committee atCornell University and was in accordance with National Institutes ofHealth guidelines.

Immunocytochemistry and confocal microscopy. Neonatal andadult mice were anesthetized with ketamine/xylazine intraperitoneally(150 mg/kg ketamine; 15 mg/kg xylazine) and perfused transcardiallywith 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer for 20min. The spinal cord was removed, and the rostral portion of the upperlumbar enlargement was postfixed in 4% PFA, 0.1 M phosphatebuffer solution for 1.5 h at 4°C, and then transferred to 30% sucrosefor 12 h at 4°C. A cryostat was used to make 15-�m transversesections of the upper lumbar enlargement. At room temperature, slideswere rinsed in phosphate-buffered saline (PBS) bath for 5 min. Slideswere incubated in 0.2% Triton X-100 for 10 min and then rinsed withthree changes of PBS over 15 min. Slices were incubated in peroxi-dase quenching buffer for 60 min at room temperature and then 1%blocking reagent for 60 min at room temperature. Slices were incu-bated with the primary antibody [goat polyclonal to green fluorescentprotein (GFP); 1:250; Immunostar] for 16 h at 4°C. Slides were rinsedin 4°C PBS for 15 min and then incubated in secondary antibody(donkey polyclonal to goat DyLight 488) for 45 min at room temper-ature and rinsed in PBS for 15 min. Slides were then mounted withFluor-Gel. A Leica TCS SP2 confocal microscope was used forimaging using the 488-nm laser line. Z-series were taken through the15-�m tissue in 1-�m steps.

Spinal cord slice procedure in adult mice. The adult mouse spinalcord slice procedure has been described previously in detail (Husch etal. 2011). Animals were deeply anesthetized with ketamine (1.5mg/10 g body wt) and xylazine (0.15 mg/10 g body wt) and placed onice. Pure oxygen was administered via a custom-built air mask. Thesacral to midthoracic spinal cord was exposed by a dorsal laminec-tomy. Once exposed, the cord was superfused with ice-cold (0–4°C)oxygenated (95% O2-5% CO2), glycerol-based modified artificial

cerebrospinal fluid (GACSF; Ye et al. 2006), which contained thefollowing (in mM): 222 glycerol, 3.08 KCl, 1.18 KH2PO4, 1.25MgSO4, 2.52 CaCl2, 25 NaHCO3, and 11 D-glucose. The ventral anddorsal roots and the meninges were removed. The spinal cord wastransferred to low melting point agarose, and 250-�m transversesections of the upper lumbar cord were made with a vibrating blademicrotome (HM-650 V; Thermo Scientific). The slices were imme-diately transferred for 45 min at 35°C in oxygenated ACSF (in mM:111 NaCl, 3.08 KCl, 1.18 KH2PO4, 1.25 MgSO4, 2.52 CaCl2, 25NaHCO3, and 11 D-glucose). The slices were allowed to recover atroom temperature for 1 h. Fluorescent INs were visualized with afixed-stage upright microscope (BX51WI; Olympus) using a �60water-immersion objective.

Spinal cord slice procedure in neonatal mice. Neonatal (P2–P4)male and female Chx10::CFP mice were euthanized by decapitation,and their spinal cords were removed by ventral laminectomy (Jiang etal. 1999; Kudo and Yamada 1987) in ice-cold (4°C), oxygenated(95% O2-5% CO2) DACSF. The meninges were removed, and theupper lumbar cord was embedded in agarose. Transverse slices (250�m) were prepared using a vibrating microtome (HM 650 V; ThermoScientific). The slices were maintained in oxygenated ACSF.

Perforated patch recordings. A detailed description of the perfo-rated patch procedure in adult mouse spinal cord slices has beenprovided previously (Husch et al. 2011). Slices were continuouslysuperfused with oxygenated ACSF at a flow rate of �2 ml/min. Toblock most synaptic input in the slices, neurons were isolated fromrapid synaptic inputs with a combination of D,L-2-amino-5-phospho-nopentanoic acid (AP-5; 10 �M) and 6-cyano-7-nitroquinoxaline-2,3-dione disodium salt hydrate (CNQX; 10 �M) to block glutamatergicsynapses; picrotoxin (10 �M) to block GABAergic synapses; andstrychnine (10 �M) to block glycinergic synapses. Patch pipettes weremade with thick wall borosilicate glass (WPI) on a vertical puller(Narishige) with low resistances of 3–5 M�. The tip of the pipette wasfilled with intracellular solution containing the following (in mM):135 K-gluconate, 10 KCl, 10 HEPES, 0.1 EGTA, and 2 MgCl2(adjusted to pH 7.2 with KOH). To prepare for the perforated patchrecording procedure (Rae et al. 1991), the pipette was backfilled witha combination of intracellular solution, amphotericin B (1.2 mg/ml),and pluronic F127 (1 mg/ml). All chemicals were obtained fromSigma-Aldrich Chemical (St. Louis, MO).

Data acquisition and analysis. Current-clamp recordings weremade with a Multiclamp 700B amplifier controlled by Clampex(pClamp 9; Molecular Devices, Sunnyvale, CA). Data were sampledat 10 kHz (20 kHz for AP shape analysis) and low-pass filtered at 2kHz. For AP analysis, the membrane potential was adjusted with aholding current (Ihold) to set the spontaneous firing frequency at 1 Hzor lower to elicit temporally isolated APs. The voltage threshold forAP generation was measured as the peak of the second derivative ofvoltage with time during the rising phase of the AP. The spikeamplitude was measured from the peak of the AP to the peakafterhyperpolarization (AHP). The AP half-width was established atthe voltage halfway from the spike threshold to the peak of the AP. Tomeasure the membrane input resistance and rheobase, all neuronswere held below threshold at �60 mV with Ihold. Input resistance wasestimated by averaging the responses to small hyperpolarizing currentpulses. The minimal amount of current necessary for spike generationfrom �60 mV was defined as the rheobase. To generate current-frequency (I–V) plots, the neuron was stimulated by a series ofincreasing current steps; instantaneous firing frequency was the in-verse of the first interspike interval, while the average firing frequencywas calculated by dividing the number of evoked APs by the stimu-lation duration. To measure the spontaneous firing rate, the meanfiring rate of twelve 10-s bins was averaged. To keep our datacomparable to previous work on V2a INs, we did not correct for theliquid junction potential.

Statistics. All data were analyzed using Excel (Microsoft), Clampfit(Axon Instruments), JMP (SAS), and MATLAB (MathWorks). Wil-

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coxon rank sum tests were used to compare adult vs. neonate param-eters and Wilcoxon rank sum to compare control vs. 5-HT parametersin the same cell. A significance level of 0.05 was accepted for all tests.To compare the response to 5-HT in adult and neonatal neurons weused regression analysis, with the absolute difference in a parameterunder control conditions and during 5-HT as the dependent variableand both age group and initial value of the parameter in controlconditions as independent variables. If the initial value variable wasnot significant, then that independent variable was removed and theregression re-run with the age group as the only independent variable.To make the regression more correct, data with non-normal distribu-tions were transformed to make the residuals of least-squares best fitnormally distributed (Neter et al. 1996). F–I regressions used arandom coefficient model to correct for multiple measurements fromeach cell. In the box plots, the extent of the box indicates the 25th and75th percentiles, and the central line represents the median. Whiskersextend to maximum and minimum values within q3 � w(q3 � q1)and q1 � w(q3 � q1), where q1 and q3 are the 25th and 75thpercentiles, respectively, and w � 1.5. Outliers outside these bound-aries are plotted as black dots and are included in all analysis. All errorbars represent the SD.

RESULTS

Morphological differences between neonatal and adult V2aspinal INs. Mice display dramatic developmental changes inbody size, body mass, and behavior repertoire over the first 4wk of life. They double their length and body weight in the first4–5 wk and change their locomotor behavior from nonmovingto normal rapid running. With body growth, the spinal cordincreases in diameter (Fig. 1A). While in neonates manyCFP-labeled V2a INs (�50) were visible in each lumbarsection of the cord, in adults only about 5–10 V2a INs werevisible. To confirm and quantify the decrease in the number ofvisible somata per slice, we conducted immunocytochemistryto amplify fluorescence of enhanced CFP-labeled neurons inthe lumbar enlargement of the spinal cord (Fig. 1, B and C). Inneonatal slices we found 30.6 3.7 labeled somata per slice(n � 12), while in adult mice we found 14.5 2.5 somata (n �12; P � 0.001; Fig. 1D). The other obvious age-dependentdifference in the appearance of the V2a INs was the soma size.Even though the diameter of the neonatal spinal cord was muchsmaller (Fig. 1A), the neonatal V2a somata appeared to belarger in diameter than the adult somata. This difference wasconfirmed by measuring the cell capacitance during patchrecordings. The neonatal V2a INs showed a significantlyhigher capacitance compared with adult neurons (neonateCm � 17.9 1.3 pF, n � 19; adult Cm � 12.2 0.6 pF, n �24; P � 0.002; Fig. 1E), indicating smaller somata in adults.

Intrinsic properties of V2a INs in neonatal and adult ages.Previous work in neonatal V2a INs using whole cell recordingsrevealed three classes of V2a neurons with distinct firingproperties in response to depolarizing square pulse currentinjections (Zhong et al. 2010). Using perforated patch-clamprecordings from neonatal V2a INs, we found a similar distri-bution of firing types: 45% of the neurons fired tonically duringa depolarizing current pulse (tonic class: Fig. 2A), 45% werephasic with an initial burst of APs that terminated during thestep (phasic class), and 10% of the cells fired tonically duringcurrent injection but with a marked delay of �200 ms beforefiring onset (delay class). In contrast to this neonatal distribu-tion, the majority of adult V2a neurons (90%) showed tonicfiring with no delay during current steps (Fig. 2A), whereas

only 3% were phasic and 7% showed a delayed onset of tonicfiring. Since almost all the adult V2a INs were in the tonicallyfiring class, we decided to compare only the tonic firing V2a INproperties between neonatal and adult ages.

Using a series of increasing depolarizing step current injec-tions (5-pA increments, 1-s duration, Fig. 2A), we measuredthe relation of firing frequency to injected current (F/I plot, Fig.2A). A regression analysis with a random coefficient model tocorrect for multiple measurements from each cell revealed thatthe slopes of the mean firing rates (P � 0.0001; n � 10–14;Fig. 2B) and the instantaneous initial firing rates (P � 0.01;Fig. 2C) with increasing current were significantly larger inadult V2a cells compared with neonatal neurons. The excit-ability was increased in adults, as reflected in lower rheobasevalues compared with neonatal V2a INs (n � 20–28; P �0.003, Fig. 2D). When holding the neurons at Vhold � �60mV, the membrane input resistance was significantly higher inadult neurons compared with neonatal neurons (n � 20–25;P � 0.01; Fig. 2E). The neonatal V2a neurons were slightlymore depolarized, as seen by significantly greater negativeholding current needed to hold them at �60 mV (n � 20–26;P � 0.04; Fig. 2F). This finding was confirmed by measuring

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Fig. 1. Morphological changes of V2a interneurons (INs) in the lumbar spinalcord with age. A: schematic coronal lumbar spinal cord slices at neonate andadult ages indicating the V2a IN somata location. The rectangles indicate theareas shown in B and C. B and C: cyan fluorescent protein (CFP) immunore-activity from neonatal (B) and adult mice (C). D: with age there is a significantdecrease in the number of immunoreactive somata per slice. E: somata of V2aINs in adult mice have significantly smaller whole cell capacitance than inneonatal mice. ***P � 0.001.

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the membrane potential of all cells that did not exceed a 1-Hzspontaneous firing: neonatal V2a cells were more depolarized(neonate Em � �44.4 5.6 mV; n � 16) than adult V2a INs(adult Em � �52.8 9.3 mV; n � 18; P 0.0001).

One of the most strikingly different features with age wasthe AP shape. In adult V2a INs, the AP width decreased to halfthat of neonatal V2a APs (n � 20–22; P 0.0001; Fig. 3, Aand B). In addition, in adult V2a INs, the AP threshold wassignificantly more negative (P 0.01; Fig. 3C), and the meanAP amplitude was increased by �10 mV (P � 0.0001; Fig.3D). While the AP AHP in all neonatal V2a INs was mono-phasic with a slow afterhyperpolarization peak at �50 ms afterthe AP, in most adult V2a INs the postpeak kinetics were morecomplex (Fig. 3E). Most (76%) adult neurons showed a bipha-sic AHP, with a fast AHP that showed a time to peak of �4 msafter the spike peak (Fig. 3F), followed by a repolarization anda second slower AHP. The time between AP peak and the slowAHP peak was similar to that seen in neonatal V2a INs (P �0.4; n � 20, Fig. 3G), and the amplitude of the slow AHP wassimilar at both ages (P � 0.1; n � 20–21; Fig. 3H). Theremaining 24% showed APs with a monophasic slow AHP

similar to the neonatal AP AHP. The adult APs showed ahigher variability in these parameters reflected by a remarkableincrease in the SD (Fig. 3G).

Effects of serotonin on intrinsic firing properties. To com-pare 5-HT responses in neonatal and adult V2a INs, we applied10 �M 5-HT in current-clamp mode, initially without holdingcurrent, and recorded spontaneous membrane potential andfiring changes (Fig. 4, A and B); this 5-HT concentrationevokes a strong response at all ages (Husch et al. 2012). Sincemany neurons fired tonically before 5-HT application (whichhinders an accurate measurement of the membrane potential),we also measured the membrane potential indirectly by mon-itoring the holding current at �60 mV; this serves as a proxyfor the membrane potential in tonically active as well as silentneurons. In a previous study (Dietz et al. 2012), we introducedamphotericin B perforated patch recordings in neonatal V2aINs and compared whole cell to perforated patch measure-ments of neuronal activity in the response to 5-HT. NeonatalV2a INs responded to 5-HT with depolarization, as measuredby the holding current at �60 mV (n � 20; P 0.0001; Fig.4C) and initiation or acceleration of firing (n � 20; P

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Fig. 2. V2a INs become more excitable with postnatalmaturation. A: responses of neonatal and adult V2a INsto increasing amplitude current pulses from a holdingpotential of �60 mV. B: current and firing frequency(F/I) plots of the average firing frequency during squarepulse current injections (grey circles: adult, n � 16;open squares: neonate, n � 10). AP, action potential. C:F/I plot of the instantaneous firing frequency (inverse offirst interspike interval; n same as in B). D: significantlylower rheobase value in adult V2a INs. E: input resis-tance measured at �60 mV is higher in adult V2a INs.F: holding current (Ihold) needed to maintain the mem-brane potential at �60 mV is smaller in adult V2aspinal INs. *P 0.05.

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0.0001; Fig. 4D). Adult V2a INs showed a similar response to5-HT, with depolarization (n � 16; P � 0.0004; Fig. 4C) andan increase in firing rate (n � 11; P � 0.03, Fig. 4D). Inneonatal V2a INs, input resistance was increased with 5-HTapplication (n � 20; P � 0.002; Fig. 4E). In 8 of 16 adult V2aINs, 5-HT clearly increased the input resistance at �60 mV,often with a clear washout (n � 8; P � 0.001). While most ofthe remaining cells had a stable input resistance during 5-HTapplication, one adult cell responded with a decreased mem-brane resistance on 5-HT application with a partial washout(control: 1.9 0.1 G�; 5-HT: 1.6 0.1 G�; wash: 1.7 0.1G�). The magnitude of the change in response to 5-HT waslarger in neonates for the holding current (P � 0.0001; Fig. 4C)and firing rate (P � 0.03; Fig. 4D) but not the input resistance(Fig. 4E). Both neonatal and adult V2a INs showed signifi-cantly increased excitability during 5-HT application. As a firstexcitability measure, we sought a reduction in the rheobase,calculated as the minimal current injected from �60 mV toreliably elicit an AP. In both neonatal and adult V2a INs, 5-HTreduced the rheobase (neonates: n � 20, P 0.0001; adults:n � 17, P 0.0001; Fig. 4F). The relative change of therheobase during 5-HT was not significantly different betweenneonates and adults (neonates: �Irheo � �28 24%; adults:�Irheo � �34 21%; P � 0.46; Fig. 4F).

We then measured the effect of 5-HT on the shape ofspontaneous low-frequency APs (Fig. 4, G–K) and comparedthe magnitude of response between the age groups. In neonatalV2a IN recordings, 5-HT increased the AP amplitude (n � 20;P � 0.049; Fig. 4G), prolonged the AHP peak time (n � 20;P � 0.0003; Fig. 4K), and lowered the AP threshold voltage(n � 20; P � 0.0001; Fig. 4H). There was no effect of 5-HTon AP width (Fig. 4I) or AHP amplitude (Fig. 4J) (Dietz et al.2012). In adult V2a INs, 5-HT reduced the AP threshold (n �11; P � 0.001; Fig. 4H). Interestingly, 5-HT did reduce theamplitude of the slow component of the AHP in adult V2a INs(n � 11; P � 0.02; Fig. 4J) as has previously been reported inmouse CINs (Zhong et al. 2006b) and cat motoneurons (Whiteand Fung 1989). The AP amplitude, AP width, and AP AHPkinetics were not significantly affected by 5-HT (Fig. 4, G, I,and K). For AP amplitude, the magnitude of the change inresponse to 5-HT was larger in neonates than adults (P �0.046; Fig. 4G); there was no difference in the magnitude ofthe change in any other measured AP parameter (Fig. 4, H–K).

Effects of serotonin on membrane potential bistability. Inneonatal V2a INs, serotonin (5-HT) did not evoke significantbistable activity (Zhong et al. 2010; Dietz et al. 2012). Inmarked contrast, in �30% of the adult 5-HT-sensitive V2a INs,5-HT introduced periodic membrane potential alterations, causingpronounced irregular firing and shifts in average firing frequencywith time (Fig. 5A). Eventually, a neuron would accelerate itsfiring frequency and depolarize to a state of depolarization block,where no spontaneous AP firing occurred (Figs. 5B and 6A). Themembrane potential then dropped below AP threshold and firingresumed during subsequent spontaneous depolarizations (Fig. 5,B1 and B2). In some recordings (2/7), the APs disappearedaltogether and it became obvious that the oscillations were notdirectly mediated by the APs, as they continued in the absence ofAPs (Fig. 5B3). These membrane potential oscillations (0.1–0.2Hz) disappeared upon removal of 5-HT (Fig. 5B4).

During longer 5-HT applications, some adult V2a INs re-mained in depolarization block for long periods of time (Fig.

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Fig. 3. Action potential AP shape narrows dramatically from neonatal to adultage. A: example traces from an adult (grey) and a neonatal (black) V2a IN. APthreshold, width, and amplitude measurements are indicated. B: AP widthnarrows significantly from neonatal to adult ages. C: AP threshold is hyper-polarized in adult V2a INs. D: AP amplitude increases in adult V2a INs. E:example traces (average of 10–12 APs) of a neonatal and an adult AP, showingafterhyperpolarization (AHP) kinetics. For the adult AP, the fast (AHPf) andslow (AHPs) AHP components are indicated. In neonatal APs there is only aslow AHP. F: AHPf time and the amplitude are measured from the APthreshold to the first peak of the AHP. G: mean time to the slow AHP peak isnot significantly different in neonates and adults. H: amplitude of the slowAHP is similar in neonatal and adult V2a spinal INs. **P 0.01 ***P 0.001.

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6A). With negative current injection, it was possible to restorespontaneous firing of overshooting APs upon postinhibitoryrebound (Fig. 6B, beginning). Releasing the negative holdingcurrent elicited a brief bout of high-frequency firing that onceagain resulted in a silent depolarization block. Small hyperpo-larizing current pulses revealed a dramatic decrease in mem-brane input resistance from 1.3 G� in the hyperpolarized state

to values as low as 0.2 G� during the depolarization block(Fig. 6B). In cases where 5-HT established bistable activitywith alternating down and up states, the silent depolarizationblock was typically terminated by a rather sudden repolariza-tion to a nonfiring hyperpolarized state (Fig. 6C). During theseprolonged periods of depolarization block (Fig. 6D, grey area),the membrane input resistance increased gradually until the

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Fig. 4. Comparison of the magnitude of serotonin (5-HTz) responses of V2a INs in neonatal and adult mice. A: spontaneous firing activity of a neonatal V2aIN under control conditions, during application of 10 �M 5-HT, and after a 30-min washout of 5-HT. B: spontaneous firing activity of an adult V2a IN before,during, and after 5-HT application. C–K: in the box plots, the extent of the box indicates the 25th and 75th percentiles, and the central line represents the median.Whiskers extend to maximum and minimum values within q3 � w(q3 � q1) and q1 � w(q3 � q1), where q1 and q3 are the 25th and 75th percentiles,respectively, and w � 1.5. Outliers outside these boundaries are plotted as black dots. Significance levels for 5-HT-induced changes in neonatal and adult neuronsare indicated above the box-plots. Differences in the magnitude of change between neonatal and adult V2a INs are indicated above the thick horizontal lines.C: increase of the holding current with 5-HT at �60 mV is significant for both neonatal and adult cells but is larger in neonatal cells (neonate: n � 20; adult:n � 16). D: AP firing frequency increases significantly during 5-HT application in neonatal and adult V2a INs (neonate: n � 20; adult: n � 11), with a strongerresponse in neonatal neurons. E: in both age groups input resistance increases with 5-HT application (neonate: n � 20; adult: n � 8). F: rheobase decreases during5-HT in neonatal and adult V2a INs (neonate: n � 20; adult: n � 17). G: AP amplitude increases in neonatal but not adult neurons upon 5-HT application(neonate: n � 20; adult: n � 11). H: AP threshold becomes more negative in neonatal and in adult V2a Ins (neonate: n � 20; adult: n � 11). I: AP width isnot influenced by 5-HT application (neonate: n � 20; adult: n � 11). J: amplitude of the slow AHP is decreased in adult V2a INs (neonate: n � 20; adult:n � 11). K: response of AHP delay increases in neonatal V2a spinal neurons (neonate: n � 20; adult: n � 11). *P 0.05, **P 0.01 ***P 0.001.

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sudden repolarization, where the input resistance reached apeak (Fig. 6D).

In six of the V2a INs that showed 5-HT-induced bistability,5-HT application facilitated the appearance of plateau poten-tials upon depolarizing current injections from �60 mV, withaccelerating spike frequency that often culminated in depolar-ization block (Fig. 7). This plateau state outlasted the currentstep by several seconds before it spontaneously repolarized toa more negative potential than before the current injection (Fig.7). In neonatal V2a INs we have never been able to elicitplateau potentials with depolarizing current injections. Takentogether, these findings suggest that the V2a INs show morecomplex intrinsic properties in locomotor-mature adults.

DISCUSSION

In the present study, we show that spinal V2a INs undergoextensive maturation in their intrinsic properties from neonatalto adult ages. The results clearly show that the V2a intrinsicproperties in neonates differ from those of adults, which mayaffect their function during locomotion. Until recently, it wasvery difficult to record for long times from spinal INs of adult

mice, so estimates of the cellular mechanisms for locomotorgeneration in adults had to be inferred from studies in neonatalanimals. Our recent development of methods to record fromadult spinal neurons without age restrictions (Husch et al.2011) enabled us to directly compare the intrinsic properties aswell as the response to locomotor relevant modulatory stimulisuch as 5-HT in V2a INs from neonatal and adult mice.

Postnatal neuronal differentiation. In transverse lumbar spi-nal cord slices, the number of detectable CFP-labeled V2asomata per slice decreases significantly with age (Fig. 1).Immunofluorescent amplification of the CFP signal using aGFP antibody revealed additional somata, suggesting that theCFP protein signal is falling with time. CFP synthesis in thesetransgenic mice occurs only during a limited embryonic time,when the Chx10 promoter is active (Al-Mosawie et al. 2007;Liu et al. 1994); thus the apparent reduction in V2a neuronnumber may be an artifact, and many other V2a neurons couldbe present in adults but with no remaining CFP label. Analternative explanation for the decreased number of adultneurons is V2a cell death during development. Apoptotic cellshave been observed in the dorsal and ventral horn from late

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embryonic development to P12; however, the number of suchcells is very low (Lowrie and Lawson 2000). These points raisethe question of whether in the adult, we are recording from asubset of the neonatal V2a INs. The only evidence in supportof this is the fact that almost all adult V2a INs show tonic firingproperties while neonatal neurons show varied tonic, phasic, ordelayed responses to stimulation (Zhong et al. 2010; Dough-erty and Kiehn 2010). However, this could also arise fromdevelopmental maturation of the neurons, which eventuallymainly become tonic firing neurons, instead of a preferentialloss of CFP label in the phasic and delayed firing classes. As

described in the Introduction, the neonatal V2a class is alsofunctionally heterogeneus; even when restricted to the lumbarspinal cord, only half of V2a INs are rhythmically activeduring fictive locomotion, and these appear to subdivide intoclasses that synapse onto motoneurons or onto CINs (Dough-erty and Kiehn 2010; Zhong et al. 2012). Some of the hetero-geneity in adult neuron properties (for example, bistability inthe presence of 5-HT) may arise from the heterogeneity of theV2a class. In our slice preparations we are unable to distin-guish these functional classes.

V2a INs show increased excitability in adulthood. Withmaturation to adult INs, the increase in V2a input resistance leadsto a marked increase in membrane excitability, resulting in asteeper I/V relationship as well as a significantly decreased rheo-base in F/I plots (Fig. 2). This increased excitability has functionalconsequences for the input-output characteristics of the locomotorCPG. Increased V2a excitability and higher input resistance canlead to a lower threshold for activation within the CPG network,because similar synaptic input would have a higher impact on theneurons’ membrane potential. When the synaptic input is mainlyexcitatory, AP generation is facilitated. In parallel, however,inhibitory input is also amplified by the increased membraneresistance and might be strong enough to shunt V2a firing activityand affect the neuronal network. This increased responsiveness toall synaptic inputs is appropriate when the locomotor network isactively used for behavior and the neurons must turn on and off atprecise and appropriate times.

AP properties change dramatically with age. Narrowing ofAPs during postnatal development (Fig. 3) has been describedin a number of other systems. Mouse spinal CINs showsignificant AP narrowing during the first 2 wk of life (Ab-binanti et al. 2012). Motoneuron APs in developing tadpolesbecome narrower with age, mainly due to a shift from calcium-driven to sodium-driven APs (Spitzer and Lamborghini 1976).In rat MNs, spike narrowing with development has beenrelated to changes in the inactivation properties of voltage-gated sodium channels (Carlin et al. 2008) and to a change inionic channel densities of already existing voltage-gated ionchannels rather than the expression of new channels (Gao and

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control

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Fig. 7. 5-HT facilitates plateau potentials in adult V2a spinal INs. Square pulsecurrent injections of 10 and 20 pA elicit tonic firing under control conditions.During application of 10 �M 5-HT, accelerating spiking leading to long-lasting depolarization block outlasting the current step is seen. This effect isreversible with time.

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Ziskind-Conhaim 1998). The faster repolarization in adult APscan contribute to the increased firing rate we saw duringdepolarizing current injections (Fig. 2). The lower AP thresh-old probably reflects changes in the set of voltage activated orleak currents expressed with age in these neurons. These couldinclude persistent sodium currents, which influence the APthreshold in CINs (Lamas et al. 2009; Zhong et al. 2007); theonset of bistability often appears to arise from an increase inpersistent inward currents (Bouhadfane et al. 2013; see below).All of these properties would contribute to increase V2a spikefrequency during locomotion in the adult.

Role of 5-HT-induced bistability for the locomotor control-ling network. In the neonatal isolated spinal cord preparation,the V2a INs did not reveal bistable membrane properties(Dougherty and Kiehn 2010; Zhong et al. 2010). In our studyon adult V2a INs, we found a subpopulation of V2a INs thatshows bistable firing properties, with the potential for rhythmicfiring activity, in the presence of 5-HT (Fig. 6). The inputresistance is significantly reduced during the plateau state; thissuggests activation of tonic inward currents such as the L-typecalcium current, which has been shown to underlie the appear-ance of bistability in older adolescent mouse spinal CINs(Abbinanti et al. 2012). Such bistability could change thefunctional significance of synaptic input to the V2a INs withage. In the neonate, the firing pattern of these neurons is shapeddirectly by the pattern of synaptic input, while in the adultsufficient initial synaptic input could instead act as a trigger toinitiate intrinsically activated firing independent of synapticexcitation, until terminated by sufficiently strong inhibitorysynaptic input. Thus the computational function of synapticdrive to these neurons could change with age. The fact thatmany of our neurons were sufficiently excited to go intodepolarization block may reflect the saturating concentrationsof serotonin we used in these experiments. The cells that didnot reveal bistability had a 5-HT response similar to that seenin neonatal V2a INs (Dietz et al. 2012), mainly characterizedby an increase in excitability and depolarization (Fig. 5).

Bistability is a critical cellular mechanism for the productionof stable rhythms in many CPGs (Harris-Warrick 2002; Marderand Bucher 2001). During development, functional adult CPGsare constructed by the establishment of correct synaptic con-nections simultaneously with the emergence of intrinsic mem-brane properties and responses to neuromodulation in the CPGINs (Fenelon et al. 1998). In the mouse locomotor network,bistability has been observed to develop in CINs, at P14–16(Abbinanti et al. 2012), close to the crucial age for the begin-ning of mature locomotion at P14–16 (Clarac et al. 1998). Thisstudy demonstrates that a subset of V2a INs also acquiresbistability during postnatal development. This property altersthe function of synaptic excitation from driving postsynapticactivity to triggering intrinsic postsynaptic activity, which isthen regulated by inhibitory input. Such a change would alterthe basic rules of network function in the adult locomotor CPG.

ACKNOWLEDGMENTS

We thank B. McGlaughon for outstanding technical assistance. We alsothank Dr. Kamal Sharma at the University of Chicago for kindly providing theChx10::CFP mouse line and Bruce Johnson for valuable comments on earlierversions of the manuscript.

Present addrees of A. Husch: University of Bonn, Institute of Genetics,Karlrobert-Kreiten-Str. 13, 53115 Bonn, Germany.

GRANTS

This work was supported by National Institute of Neurological Disordersand Stroke Grants NS-057599 and NS-17323 (to R. M. Harris-Warrick) andDeutsche Forschungsgemeinschaft Grant HU-1963-1/1 (to A. Husch).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

Author contributions: A.H., S.B.D., and R.M.H.-W. conception and designof research; A.H., S.B.D., and D.N.H. performed experiments; A.H., S.B.D.,and D.N.H. analyzed data; A.H., S.B.D., D.N.H., and R.M.H.-W. interpretedresults of experiments; A.H. prepared figures; A.H. and R.M.H.-W. draftedmanuscript; A.H., S.B.D., and R.M.H.-W. edited and revised manuscript;A.H., S.B.D., D.N.H., and R.M.H.-W. approved final version of manuscript.

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