doi: 10.1152/jn.00336.2012108:2191-2202, 2012. First published 25 July 2012;J Neurophysiol 

Matthew D. Abbinanti, Guisheng Zhong and Ronald M. Harris-Warrickspinal cordpotentials in commissural interneurons of the mouse Postnatal emergence of serotonin-induced plateau

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Postnatal emergence of serotonin-induced plateau potentials in commissuralinterneurons of the mouse spinal cord

Matthew D. Abbinanti, Guisheng Zhong, and Ronald M. Harris-WarrickDepartment of Neurobiology and Behavior, Cornell University, Ithaca, New York

Submitted 23 April 2012; accepted in final form 18 July 2012

Abbinanti MD, Zhong G, Harris-Warrick RM. Postnatal emer-gence of serotonin-induced plateau potentials in commissural in-terneurons of the mouse spinal cord. J Neurophysiol 108: 2191–2202,2012. First published July 25, 2012; doi:10.1152/jn.00336.2012.—Most studies of the mouse hindlimb locomotor network have usedneonatal (P0–5) mice. In this study, we examine the postnatal devel-opment of intrinsic properties and serotonergic modulation of in-tersegmental commissural interneurons (CINs) from the neonatalperiod (P0–3) to the time the animals bear weight (P8–10) and beginto show adult walking (P14–16). CINs show an increase in excitabil-ity with age, associated with a decrease in action potential halfwidthand appearance of a fast component to the afterhyperpolarization atP14–16. Serotonin (5-HT) depolarizes and increases the excitabilityof most CINs at all ages. The major developmental difference is thatserotonin can induce plateau potential capability in P14–16 CINs, butnot at younger ages. These plateau potentials are abolished by nifed-ipine, suggesting that they are mediated by an L-type calciumcurrent, ICa(L). Voltage-clamp analysis demonstrates that 5-HTincreases a nifedipine-sensitive voltage-activated calcium current,ICa(V), in P14 –16 CINs but does not increase ICa(V) in P8 –10 CINs.These results, together with earlier work on 5-HT effects onneonatal CINs, suggest that 5-HT increases the excitability of CINsat all ages studied, but by opposite effects on calcium currents,decreasing N- and P/Q-type calcium currents and, indirectly, cal-cium-activated potassium current, at P0 –3 but increasing ICa(L) atP14 –16.

modulation; development; central pattern generator; bistability; cal-cium current

MOST RESEARCH ON THE MOUSE HINDLIMB locomotor network hasused spinal cords from postnatal day 0–5 (P0–5) mice, whichcannot walk. This work is motivated by the assumption that thelocomotor central pattern generator (CPG) is reasonably welldeveloped at birth, so neonatal studies can help us understandthe adult locomotor network. We address this assumption bystudying changes in the properties and neuromodulation ofCPG interneurons during the time that mice begin to bearweight (P8–10) and develop mature locomotion (P14–16)(Clarac et al. 1998).

Neonatal spinal cords are more resistant to hypoxia, improv-ing the viability of neurons in slices and enabling fictivelocomotion in the isolated cord (Wilson et al. 2003). However,evidence suggests that there are important maturation eventsoccurring postnatally. Ion channel and receptor expressionvaries during the first postnatal week (Jakowec et al. 1995;Stegenga and Kalb 2001). Expression of two L-type calciumchannel transcripts begins at P7–10 (Jiang et al. 1999). Know-

ing this, how is the locomotor CPG changing as the mousedevelops toward mature locomotion?

To understand how locomotion is generated, we must un-derstand the intrinsic properties and modulation of CPG com-ponent neurons (Marder and Bucher 2001). The locomotorCPG is located primarily within the lumbar spinal cord, butdescending modulatory input, particularly serotonergic (5-HT)input, is critically important for its function (Kjaerulff andKiehn 1996; Schmidt and Jordan 2000). Serotonergic neuronsprojecting to the spinal cord increase firing during locomotion(Fornal et al. 1985). Serotonergic antagonists block brain stemstimulation-induced and NMDA-induced fictive locomotion(Liu and Jordan 2005; MacLean et al. 1998). Descending 5-HTinputs reach the lumbar spinal cord by birth in the mouse butcontinue to mature over several weeks (Ballion et al. 2002).Thus it is important to determine whether the effects of 5-HTon CPG neurons change during postnatal development.

In this article we focus on developmental maturation of theintrinsic properties and serotonin neuromodulation of knowncomponents of the hindlimb CPG, the ascending and descend-ing intersegmental commissural interneurons (CINs). CINs arerhythmically active during fictive locomotion and provideexcitatory and inhibitory input to motor neurons and interneu-rons in the opposite hemicord (Birinyi et al. 2003; Butt et al.2002; Butt and Kiehn 2003; Quinlan and Kiehn 2007; Zhonget al. 2006a, 2006b). Genetic deletion of a subset of CINsresults in poor left-right alternation (Lanuza et al. 2004). AtP0–3, 5-HT excites intersegmental CINs (Carlin et al. 2006;Zhong et al. 2006a, 2006b), in part by reducing N- andP/Q-type calcium currents and indirectly reducing calcium-activated potassium currents (Abbinanti and Harris-Warrick2012; Diaz-Rios et al. 2007).

We show how the intrinsic properties and serotonergicmodulation of CINs change during postnatal development. Weexamine three ages, P0–3 (neonates, unable to walk or bearweight; Zhong et al. 2006a, 2006b), P8–10 (beginning to bearweight), and P14–16 (emergence of mature walking). We findthat the spike properties and excitability change over postnataltime. Most importantly, although 5-HT increases the excitabil-ity of CINs at all ages, it has opposing effects on differentcalcium currents at different ages and evokes bistability only inP14–16 CINs.

MATERIALS AND METHODS

Slice preparation. Experiments were performed on P0–3, P8–10,and P14–16 C57BL/6 mice. The animal protocol was approved by theCornell University Institutional Animal Care and Use Committee andis in accordance with National Institutes of Health guidelines. Inaccordance with our animal protocol, P1–3 and P8–10 mice werekilled by rapid decapitation, whereas P14–16 mice were anesthetized

Address for reprint requests and other correspondence: R. M. Harris-Warrick, Dept. of Neurobiology and Behavior, Mudd Hall, Cornell Univ.,Ithaca, NY 14853 (e-mail: rmh4@cornell.edu).

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with 100% CO2 before decapitation. The spinal cord was dissected outof the animal by vertebrectomy (also called ventral laminectomy)under ice-cold (4°C), oxygenated (95% O2-5% CO2), sucrose-basedlow-calcium artificial cerebrospinal fluid (aCSF; in mM: 188 sucrose,25 D-glucose, 26 NaHCO3, 25 NaCl, 10 MgSO4, 1.2 NaH2PO4, and1.9 KCl, pH � 7.4). The isolated spinal cord was pinned ventral sideup, and CINs in the lumbar 2 (L2) spinal segment were fluorescentlyretrograde labeled by making small slits in the contralateral hemicordrostral and caudal to the L2 region and placing crystals of fluorescentdextran amines in the slits (3,000 MW Texas red dextran amine rostralto L2 and 3,000 MW fluorescein dextran amine caudal to L2;Invitrogen, Carlsbad, CA) (Zhong et al. 2006a, 2006b). Differentiallylabeling the neurons sending axons rostrally or caudally by more thanone segment from L2 assured that only intersegmental CINs werelabeled.

Preparations were incubated in ice-cold sucrose-based aCSF for20–45 min to allow the dyes to diffuse to the contralateral cell bodies.Transverse spinal cord slices (250 �m) were made with a vibratingmicrotome (Leica Microsystems, Wetzlar, Germany). Slices wereplaced in 30°C normal mouse aCSF (in mM: 111 NaCl, 3 KCl, 25NaHCO3, 1.2 KH2PO4, 1.25 MgSO4, 2.5 CaCl2, and 11 D-glucose,pH � 7.4) and allowed to come to room temperature (�20–22°C)over 1 h before use.

Drugs. The AMPA receptor antagonist 6-cyano-7-nitroquinoxa-line-2,3-dione disodium salt (CNQX), glycine antagonist strychnine,and sodium channel antagonist tetrodotoxin (TTX) were purchasedfrom Tocris Bioscience (Ellisville, MO). The following drugs werepurchased from Sigma (St. Louis, MO): NMDA and AMPA/kainateantagonist kynurenic acid, NMDA receptor antagonist D-(-)-2-amino-5-phosphopentanoic acid (APV), GABA antagonist picrotoxin, tran-sient potassium current antagonist 4-aminopyridine (4-AP), potassiumchannel antagonist tetraethylammonium chloride (TEA-Cl), L-typecalcium channel antagonist nifedipine, and serotonin creatinine sulfate(5-HT).

Whole cell current-clamp recordings. Slices were transferred to therecording dish on the stage of a Zeiss Axioskop 2 FS Plus uprightmicroscope and perfused with oxygenated normal aCSF at 3 ml/minat room temperature (21–22°C). CINs were identified as Texas red- orfluorescein-labeled neurons contralateral to the dye application sitesunder epifluorescent illumination and visualized using differentialinterference contrast (DIC) optics. Only single-labeled cells, repre-senting either ascending (aCINs) or descending neurons (dCINs),were recorded. Double-labeled, bifurcating adCINs (ascending anddescending) were eliminated from our study. Electrodes were pulledfrom thick-wall borosilicate glass (WPI, Sarasota, FL) on a Narishigevertical puller (Narishige International USA, East Meadow, NY) andhad resistances of 5–8 M�. The pipette solution contained (in mM)128 K-gluconate, 10 HEPES, 5 ATP-Mg, 0.3 GTP-Li, and 0.0001CaCl2 (pH � 7.4 with KOH). A minimum 1-G� seal resistance wasobtained before all recordings. The extracellular recording solutiondesigned to block fast synaptic transmission contained, in addition tothe normal aCSF described above, (in �M) 20 APV, 9 strychnine, 30CNQX, and 10 picrotoxin. Kynurenic acid (1 mM) was substituted forAPV and CNQX in some experiments. Recordings were made with aMulticlamp 700A amplifier (Axon Instruments, Foster City, CA) andDigidata 1322A digitizer (Axon Instruments) using Clampex (frompClamp 9; Axon Instruments). Data were filtered at 2 kHz anddigitized at 5 or 10 kHz.

Whole cell voltage-clamp recordings. Procedures for voltage-clamp experiments were similar to those described above except forthe following. Slices were incubated in at room temperature (21–22°C) in a solution of (in mM) 111 NaCl, 3.1 KCl, 25 NaHCO3, 1.2KH2PO4, 1 MgCl2, 2.5 CaCl2, and 11 D-glucose (pH � 7.4). Toisolate calcium currents, the voltage-clamp experiment intracellularsolution contained (in mM) 100 CsCl, 30 TEA-Cl, 1 MgCl2, 10EGTA, 5 NaCl, 1 CaCl2, 10 HEPES, 3 ATP-Mg, 0.5 GTP-Li, and 10sucrose (pH � 7.3). For voltage-clamp experiments, a sulfate-free

extracellular solution was used which contained (in mM) 111 NaCl,3.1 KCl, 25 NaHCO3, 1.2 KH2PO4, 1 MgCl2, 2.5 CaCl2, 7 D-glucose,10 TEA-Cl, 2 4-AP, 2 CsCl, and 0.001 TTX (pH � 7.4). In someexperiments, BaCl2 was substituted for CaCl2. To block any sponta-neous synaptic events, the same concentrations of fast synaptic trans-mission blockers described above were included in the recordingsolutions. Patch electrodes had resistances of 3–5 M�. Series resis-tance and capacitance were compensated �70%. Leak currents werecorrected either online using P/4 leak subtraction or offline by sub-traction of a scaled current determined by the average response to�10-mV hyperpolarizing pulses from �60 mV.

Data analysis. Ascending and descending CINs were held at �60mV with bias current to assure uniformity in current-clamp measure-ments. Only cells with action potentials (APs) overshooting 0 mVwere included. We measured the threshold for AP generation as thepeak of the second derivative of voltage vs. time during the risingphase of the AP. The width of the AP halfway between the thresholdand the peak was measured as the half-width of the AP. The amplitudeof the spike afterhyperpolarization (AHP) was measured relative to 0mV. The time between the peak of the AP and the AHP minimum wasdefined as the AHP time. In many P14–16 CINs, there were two localminima during the AHP; these are reported as the fast and slowcomponents of the AHP. We measured the input resistance by appli-cation of small hyperpolarizing current pulses from �60 mV.

To determine the frequency-current (f-I) relationship, 1- to 2-sdepolarizing current steps of increasing amplitude were delivered andthe average spike frequency during the first 1,000 ms of the step wasdetermined and plotted against the amplitude of the injected current.The slope of the plot was linearly fitted. We determined the “averagef-I value” by calculating the ratio of the firing frequency during thefirst 1,000 ms of each step divided by the amplitude of the injectedcurrent for that step; we then averaged this ratio over all the steps foreach f-I run. The average integral of poststep afterpotentials wasdetermined by averaging the area under the membrane potential andabove the baseline voltage over 4 s after the termination of the currentstep. We averaged this value for all the steps of an f-I run.

Peak currents in response to voltage steps in voltage clamp werefitted to the Boltzmann equation in Clampfit (Clampex) using theequation

f�V� � Imax ⁄ �1 � exp��Vmid � V� ⁄ Vc�� � C

where Imax is the maximal current, Vmid is the voltage of half-activation, and Vc is the slope of the curve.

Data are means � SD. One-way analysis of variance and applicableposttests were used to compare data between the three age groups andwhen more than two conditions were tested. The appropriate Student’st-tests were used to compare two treatments within age groups.Chi-square tests were used to compare the proportions of cellsdisplaying a given intrinsic property where appropriate to analyzestatistical significance. An analysis of covariance (ANCOVA) wasused to analyze the statistical significance of changes in the f-Irelationships between ages and treatments.

RESULTS

Baseline electrophysiological properties of CINs. Our label-ing method allowed us to differentiate aCINs and dCINs fromadCINs by their pattern of fluorescent labeling. Because adCINsare not rhythmically active during fictive locomotion and do notrespond to 5-HT at P0–3 (Zhong et al. 2006b), we eliminatedthis class of neurons from our analysis. The remaining aCINsand dCINs showed similar properties and responses to 5-HT atP8–10 and P14–16, and their data were combined for thisstudy. Our first goal was to determine the postnatal develop-ment of the electrophysiological and firing properties of CINs.

2192 POSTNATAL SEROTONERGIC MODULATION OF CINs

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Our hypothesis was that the intrinsic properties of the CINs areimmature at P0–3 and that their properties change with time.Using whole cell patch-clamp recordings after blocking fastsynaptic inputs, we measured AP and passive electrical prop-erties of CINs from three age groups, P0–3, P8–10, andP14–16 (Table 1, Fig. 1; Zhong et al. 2006a, 2006b). At P0–3,only 20% of synaptically isolated CINs were spontaneouslyactive without the addition of bias current. However, by P8–10and P14–16, most CINs (67% and 63%, respectively) werespontaneously active, firing on average at 5 and 7 Hz, respec-tively. A significantly greater proportion of CINs were active atlater ages than at P0–3 [C2(2, n � 104) � 21.75, P � 0.001].The resting potential of silent cells and the input resistance ofall cells were not significantly different between age groups.There was a significant decrease in the membrane capacitanceof CINs between P0–3 and P8–10 (F2,98 � 5.599, P � 0.01;Table 1), which remained unchanged between P8–10 andP14–16.

The properties of the APs in these neurons changed duringpostnatal development (Fig. 1, Table 1). The recordings for ourmeasurements of AP properties were taken during tonic injec-tion of bias current to just below threshold, where APs oc-curred spontaneously (�2 Hz). CIN APs narrowed during thefirst two postnatal weeks; by P14–16, their halfwidth was onlyone-half that of neonatal APs (F2,98 � 173.44, P � 0.001; Fig.1B). Action potential amplitude, defined as the membranepotential difference between the peak of the AP and the troughof the AHP, increased by 33% from P0–3 to P8–10, butdecreased by 19% between P8–10 and P14–16 (F2,98 � 37.94,P � 0.001; Fig. 1A). The threshold for AP generation wasslightly depolarized at P8–10 compared with P0–3 (F2,98 �4.027, P � 0.05). The P14–16 AP threshold, although alsoslightly depolarized, was not significantly different from theother age groups (Fig. 1B).

At all ages, CINs had a significant AHP following the AP.The minimum AHP voltage following a spike was not signif-

Table 1. Properties of CINs from different postnatal age groups

P0–3 P8–10 P14–16

Spontaneous firing at zero current, Hz 3.0 � 0.6 (11/56) 4.5 � 4.7 (14/21) 6.7 � 7.0 (17/27)Rest potential of silent cells, mV �60.0 � 4.3 (45/56) �56.1 � 6.0 (7/21) �56.9 � 6.3 (10/27)Input resistance, M� 618.4 � 250.9 (31) 480 � 282 (25) 630 � 333 (19)Mean capacitance, pF 67.0 � 13.2 (56) 51.9 � 34.8* (22) 51.7 � 26.5* (19)AP halfwidth, ms 3.0 � 0.5 (57) 1.6 � 0.2* (25) 1.2 � 0.4*† (19)AP threshold, mV �39.7 � 6.6 (57) �35.6 � 5.6* (25) �37.7 � 5.1 (19)AP amplitude, mV 62.3 � 7.8 (57) 80.4 � 9.8* (25) 65.0 � 10.0† (19)Minimum AHP voltage, mV �56.6 � 5.8 (57) �58.5 � 6.4 (25) �56.8 � 5.7 (19)AHP time (slow), ms 25.3 � 7.5 (31) 34.7 � 8.4* (25) 35.3 � 12.0* (16)AHP time (fast), ms 3.2 � 1.4 (8)

Values are means � SD in different postnatal (P) age groups; numbers in parentheses indicate no. of commissural interneuons (CINs). AP, action potential;AHP, afterhyperpolarization. *P � 0.05, significantly different from P0–P3. †P � 0.05, significantly different from P8–P10.

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Fig. 1. Commisural interneuron (CIN) actionpotential (AP) properties change during post-natal development. A: representative traces ofAPs from postnatal day 2 (P2; light gray), P8(dark gray), and P14 (black) CINs. B: averagemeasurements of AP halfwidth, threshold,and afterhyperpolarization (AHP) minimumfor P0–3, P8–10, and P14–16 CINs. *P �0.05. C: expanded view of the AHP of 2different P14–16 CINs demonstrating a fastand slow AHP.

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icantly different between any ages studied (Fig. 1B). Wemeasured the AHP onset time as the time from spike peak tominimum voltage during the AHP. P0–3 and P8–10 CINs hada single-component slow AHP, whereas P14–16 CINs showedheterogeneity in AHP components, with 42% of cells showingboth a slow and a fast component of the AHP (Fig. 1C). Theslow component of the AHP was significantly slower in P8–10and P14–16 CINs compared with P0–3 CINs [P0–3: onsettime 25.3 � 7.5 ms (n � 31); P8–10: 34.7 � 8.4 ms (n � 25);P14–16: 35.3 � 12.0 ms (n � 16); F2,69 � 10.24, P � 0.001].In those P14–16 CINs that had a fast component of the AHP,its average time to peak was 3.2 � 1.4 ms (Fig. 1C). We alsoexamined the AHP halfwidth as a measure of AHP durationand found no difference between age groups (P � 0.5). Thus,by P14–16, a significant number of the CINs displayed APsshaped by the interaction of a fast and slow AHP component,mediated by currents with different activation and deactivationrates; this was not seen at earlier ages.

Baseline excitability of CINs. We next examined the base-line excitability of CINs by holding the membrane potential at�60 mV and applying increasing current steps, and thenplotting the average firing frequency during the step vs. in-jected current. These f-I plots illustrate how a given neuronresponds to current and are useful for comparing the CINexcitability at different ages or in response to a neuromodulatorsuch as serotonin. Zhong et al. (2006 a, 2006b) found that the

large majority of P0–3 ascending and descending CINs firetonically in response to current injection. All P8–10 andP14–16 CINs fired tonically in response to a sufficiently largecurrent pulse.

We developed a measure to compare f-I plots between thethree CIN ages by averaging the instantaneous slope for eachpoint of the f-I plots. The ratio of the average AP frequency tothe injected current was calculated for each step and averagedover the current range to yield an average f-I value. An increasein the average f-I value quantitatively demonstrates an “upwardshift” of the f-I plot, even when there is no change in the slopeof the f-I relation (Diaz-Rios et al. 2007; Zhong et al. 2006a,2006b). The average f-I value between P0–3 and P8–10 CINsdid not change significantly, but there was a significant in-crease between P8–10 and P14–16 (P0–3: 0.13 � 0.03 Hz/pA;P8–10: 0.18 � 0.11 Hz/pA; P14–16: 0.31 � 0.14 Hz/pA;F2,50 � 13.27, P � 0.001; Fig. 2C). The slopes of the f-I plotsincreased from P0–3 to P8–10, as well as from P8–10 toP14–16 (P0–3: 0.13 � 0.02 Hz/pA; P8–10: 0.22 � 0.09Hz/pA; P14–16: 0.34 � 0.17 Hz/pA; F2,50 �15.81, P � 0.001;Fig. 2F). The minimal measured current to evoke firing from�60 mV was not different between the three ages (P0–3: 38 �23 pA; P8–10: 29 � 27 pA; P14–16: 28 � 25 pA). These datademonstrate that CINs become more excitable with age andthat the increase in firing with larger depolarizations becomesgreater with age.

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Fig. 2. Effects of 5-HT on excitability of P0–3, P8–10, and P14–16 CINs. A: representative responses to 60-pA depolarizing current injections in P2, P9, andP14 CINs before and during application of 9 �M 5-HT. B: representative frequency-current (f-I) plots from individual P2, P9, and P14 CINs before and during5-HT application. Average f-I values are shown for P0–3, P8–10, and P14–16 CINs (C), P8–10 CINs before and during 5-HT (D), and P14–16 CINs beforeand during 5-HT (E). f-I slope values are shown for P0–3, P8–10, and P14–16 CINs (F), P8–10 CINs before and during 5-HT (G), and P14–16 CINs beforeand during 5-HT (H). *P � 0.05.

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Postnatal changes in effects of serotonin on CIN firingproperties. Serotonin (5-HT) plays a critical role in enablingrhythmic output from the mouse spinal locomotor network (Liuand Jordan 2005; MacLean et al. 1998; Nishimaru et al. 2,000),so we studied the postnatal maturation of CIN responses to5-HT. As previously noted by Diaz-Rios et al. (2007), at allages tested, we found that 5-HT’s effects are difficult to washout after longer application times. Even with application timesas short as 5 min, it often took more than 45 min to see anappreciable reversal of its effects.

We found that 9 �M 5-HT depolarized and increased theexcitability of neonatal (P0–3) CINs, as we reported earlier(Zhong et al. 2006a, 2006b). CINs were depolarized by anaverage of 9.2 � 2.5 mV, and most silent cells began to fireAPs. Serotonin also depolarized P8–10 and P14–16 CINs. Inall silent P8-P10 CINs and most silent P14–16 CINs, theserotonin-evoked depolarization was strong enough for theneuron to begin to fire (7/7 silent P8–10, 4/5 silent P14–16CINs). We quantified this depolarization by determining thebias current needed to hold the cell at �60 mV before, during,and after the application of 5-HT. At P8–10, the bias current at�60 mV increased from �33 � 25 to �60 � 41 pA in 9 �M5-HT (P � 0.005, n � 15). The average firing frequency oftonically firing P8–10 CINs with no bias current increasedfrom 6.9 � 4.8 Hz in control to 11.7 � 6.3 Hz during 5-HTapplication (P � 0.005, n � 11). The P8–10 CINs that weresilent under control conditions began to fire during 5-HTapplication, with an average frequency of 6.8 � 5.7 Hz.Serotonin also increased neuronal excitability of P14–16 CINs.Application of 9 �M 5-HT depolarized the older neurons, asindicated by an increased bias current at �60 mV (�31 � 22pA in control to �51 � 28 pA during 5-HT; P � 0.001, n �17). The average frequency of tonically firing P14–16 CINswith no bias current increased from 10.0 � 6.3 to 16.8 � 7.8Hz (P � 0.001, n � 14). There was no significant difference inthe increase of firing frequency by 5-HT between the P8–10and P14–16 CINs (F1,32 � 1.891, P � 0.179).

Serotonin increased the excitability of a majority of CINs atall ages, as measured by the f-I relation (Fig. 2, A and B). AtP0–3, serotonin shifted the f-I plot of both aCINs and dCINs inthe hyperpolarizing direction, without changing the slope(Zhong et al. 2006a, 2006b). Using our method of averagingthe f-I plots discussed above, 9 �M 5-HT increased the averagef-I value from 0.13 � 0.03 to 0.17 � 0.04 Hz/pA (n � 21). Inthe P8–10 CINs that depolarized in response to 5-HT, weobserved a 45% decrease in the minimum current step to evokespiking from �60 mV (32 � 34 vs. 18 � 18 pA in 5-HT, P �0.05, n � 11). Serotonin caused a hyperpolarizing shift in thef-I relation in 11/15 P8–10 CINs, with no significant change inthe average slope (Fig. 2, B and G); this resulted in anincreased average f-I value from 0.18 � 0.13 to 0.23 � 0.14Hz/pA (P � 0.03, n � 11; Fig. 2D). Although there was norepeatable change in the average slope of the f-I relationship atthis age, ANCOVA revealed that 5 of the 11 cells in which ahyperpolarizing shift was observed had a significant change inthe slope of the regressions, precluding further analysis ofthese cells by ANCOVA. Of the remaining 6 cells that re-sponded to 5-HT, 5 showed a significant (P � 0.05) hyperpo-larizing shift in the f-I relationship with an average increase inthe y-intercept of 2.6 � 1.5 Hz during 5-HT. This increase in

the y-intercept represents an average increase in firing rate of2.6 � 1.5 Hz for a given current step during 5-HT.

Serotonin also excited the P14–16 CINs: 9 �M 5-HT de-creased the minimum current step to evoke spiking from �60mV by 39% in the 15 CINs excited by 5-HT (28 � 29 pA incontrol vs. 17 � 15 pA in 5-HT, P � 0.05; n � 15). In 15/17P14–16 CINs, 5-HT shifted the f-I plots in the hyperpolarizingdirection and increased the average f-I value from 0.29 � 0.13Hz/pA in control to 0.40 � 0.24 Hz/pA (P � 0.02, n � 15; Fig.2E). In contrast to the younger CINs, in P14–16 CINs excitedby 5-HT, the average slopes of the f-I plots also increasedslightly but significantly during 5-HT application (0.34 � 0.18Hz/pA in control vs. 0.39 � 0.19 Hz/pA during 5-HT, P �0.005, n � 15; Fig. 2H). These results were supported byANCOVA, which demonstrated that 8 of the 15 P14–16 CINsthat responded to serotonin had a significant increase in slope;the remaining 7 demonstrated a significant (P � 0.05) increasein the y-intercept of their regressions (4.3 � 3.3 Hz), againshowing the increased excitability of the CINs during 5-HT. Insummary, 5-HT increases CIN excitability at all ages studied.

Serotonin affects AP shape at all ages. Serotonin also affectedthe AP shape at older ages (Fig. 3, A and B). In P0-P3 CINs(Zhong et al. 2006a, 2006b), 5-HT increased the AP halfwidthby 16% in aCINs (control: 33 � 10 ms, 5-HT: 36 � 10 ms) butnot in dCINs, reduced the AHP amplitude by 14% and 20% inaCINs and dCINs, respectively (aCIN control: 18.3 � 5.5 mV;5-HT: 15.7 � 4.7 mV; dCIN control: 16.1 � 3.7 mV; 5-HT:12.7 � 3.1 mV), and hyperpolarized the AP threshold by 5%and 8% in aCINs and dCINs, respectively (aCIN control:�30.5 � 7.5 mV; 5-HT: �32.1 � 8.3 mV; dCIN control:�38.2 � 4.3 mV; 5-HT: �41.2 � 4.0 mV; Zhong et al. 2006a,2006b). At P8–10 and P14–16, both aCINs and dCINs re-sponded similarly to 5-HT, with no significant differencesbetween the groups; as a result we combined their responses.At P8–10, 9 �M 5-HT reversibly increased the AP by 24%(1.6 � 0.3 to 2.0 � 0.4 ms, P � 0.005, n � 12; Fig. 3C). TheAHP minimum voltage was depolarized by 3.3 mV (�58.6 �4 to �55.3 � 5 mV, P � 0.01, n � 12; Fig. 3D). Interestingly,in contrast to the younger P0–3 neurons, the AP threshold atP8–10 was not affected by 5-HT application (�35.5 � 5.0 vs.�35.1 � 5.6 mV; Fig. 3E). At P8–10, a significant rundown ofthe AP parameters such as amplitude, halfwidth, threshold, andAHP minimum voltage during prolonged recordings made itdifficult to obtain significant washout of 5-HT’s effects. Toaddress this problem, we performed a series of experiments onP8–10 CINs in which AP properties were recorded every30–60 s for the duration of the experiment. By using thismethod, the time course of 5-HT’s effects could be observed,superimposed on the rundown observed before and after 5-HTapplication. We observed an effect of 5-HT on both APhalfwidth and AHP minimum in most of these experiments(n � 4/5 for both), whereas we observed no effect on otherparameters measured (AP amplitude, threshold, and AHPtime). These experiments confirmed our earlier results andshowed that the 5-HT effect was not simply an artifact ofrundown.

Serotonin’s effects on the AP shape of P14–16 CINs were ingeneral similar to thonse on both younger groups of CINs,although the AP properties showed more rapid rundown at thisage. For example, 5-HT reversibly reduced the AHP amplitudefrom �57.1 � 4.9 to �52.3 � 5.4 mV (P � 0.02). These

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results suggest that 5-HT has similar effects to depolarizeCINs, change the AP shape, and enhance the response todepolarizing inputs during the first two postnatal weeks.

Serotonin evokes plateau potentials in P14–16 CINs. Oneimportant developmental difference that arose from our studieswas the emergence of bistability during 5-HT application inolder CINs. Bistability and plateau potentials allow a neuron torespond to a brief input with a shift from a silent to a stabledepolarized and active firing state. We found that 5-HT caninduce plateau potential capability in synaptically isolatedP14–16 CINs, but not in P0–3 or P8–10 CINs (Fig. 4, A andB). We defined plateau potential capability as prolonged firingor a prolonged afterdepolarization (ADP) following the termi-nation of a 500- to 2,000-ms current step; prolonged ADPshave been observed as a subthreshold poststimulus response inother bistable cells (Bennett et al. 2001; Hounsgaard and Kiehn1989). This phenomenon was never observed in P8–10 orP0–3 CINs (Fig. 4B; n � 21 P0–3 CINs, n � 15 P8–10 CINs).To quantify plateau capability in P14–16 CINs, we integratedthe area under the membrane potential for 4 s beginning at theend of the current step during f-I plots and averaged across allsteps given. Fifteen of 27 P14–16 CINs showed prolongedexcitability after a current step in the presence of 9 �M 5-HT.For these 15 neurons, the average integrated poststep depolar-ization increased nearly fivefold in response to 5-HT (2.4 �12.2 V·s in control vs. 11.7 � 13.1 V·s in 5-HT, P � 0.03, n �15; Fig. 4C). Eight of those 15 CINs demonstrated full bista-bility, defined as continued spiking after the termination of theinjected current step (Fig. 4B). Six of these 8 neurons showed

no bistable properties under control conditions and developedbistability only during the application of 5-HT (Fig. 4, A andB). The other two neurons demonstrated a distinct ADP undercontrol conditions, which was further enhanced by 5-HT in oneneuron. The remaining 7 of the 15 CINs showed prolongedADPs after termination of the current step but did not fire APsduring the ADP (Fig. 4D). Of these seven, three CINs had anADP under control conditions which grew larger during 5-HT,whereas the other four developed an ADP only in the presenceof 5-HT. These ADPs were never observed in the younger agegroups studied (for example, Fig. 4B, left panels).

Nifedipine-sensitive current underlies serotonin-evoked pla-teau potentials in P14–16 CINs. We next sought to determinewhich ionic currents mediate 5-HT’s ability to enable plateaupotential capability in P14–16 CINs. In other preparations,bistability requires the activation of persistent calcium and/orsodium currents (Housgaard and Kiehn 1989; Lee and Heck-man 1996; Li and Bennett 2003; Li et al. 2007). In all P14–16CINs tested (n � 4), 10 �M nifedipine, an L-type calciumchannel blocker, eliminated the plateau potential capability(Fig. 5A). Nifedipine reduced the average poststep integratedADP by 90% (14.9 � 7.9 V·s during serotonin before nifedi-pine vs. 1.4 � 2.1 V·s with added nifedipine, P � 0.03, n � 4;Fig. 5B), which was not significantly different from the pre-5-HT control value. These results suggest that a nifedipine-sensitive L-type calcium current sustains the bistability inP14–16 CINs.

Effect of 5-HT on calcium currents in CINs. Because 5-HTacts to enhance plateau potential capability in P14–16 CINs,

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Fig. 3. 5-HT modulates P8–10 AP shape. A: representative traces of P8–10 AP shape in control (black), 9 �M 5-HT (dark gray), and washout (light gray). B: aslower time course view of the same APs to demonstrate 5-HT’s increase of AP halfwidth. C: 5-HT increased the AP halfwidth in P8–10 CINs. D: 5-HTdecreased the absolute (Abs.) AHP amplitude in P8–10 CINs. E: 5-HT had no effect on the AP threshold of P8–10 CINs (*P � 0.05).

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and the 5-HT-enabled plateaus are blocked by nifedipine, wehypothesized that 5-HT enhances an L-type calcium current inP14–16 CINs. In whole cell voltage-clamp, with sodium andpotassium currents blocked, a distinct inward current wasobserved in all P14–16 cells examined (n � 21 in calcium-containing aCSF). The current was also observed with externalcalcium replaced by barium (n � 7; Fig. 6A). The threshold ofactivation of calcium currents was around �40 mV, and thevoltage at peak calcium current was between �20 and �10mV (Fig. 6C). First-order Boltzmann analysis of calciumcurrents showed that the average Imax value was highly vari-able between neurons (600 � 376 pA), with an average voltagefor half-activation (Vmid) at �26.5 � 5.2 mV (n � 21) andslope (Vc) of 5.5 � 1.5 mV/e-fold. Serotonin increased the totalcurrent in almost one-half of these neurons (Fig. 6A). Boltz-mann analysis showed that Imax was increased by 11 � 5% in43% of cells tested (756 � 574 pA in control vs. 832 � 615 pAin 5-HT, P � 0.02, 6/14 CINs in Ca2�; Fig. 6B). There was noeffect of 5-HT on Vmid or slope Vc (P � 0.60 and 0.40,respectively). In the other 8/14 CINs, 5-HT did not signifi-cantly increase Imax, consistent with the inability of 5-HT toevoke bistability in about one-half of P14–16 CINs.

Next, we examined whether nifedipine would block theserotonin-induced increase in calcium current, since it blocks5-HT-evoked bistability. Because we were unable to holdindividual cells long enough to test 5-HT’s effects on voltage-activated calcium current, wash it out, and then add nifedipinealong with 5-HT, we performed parallel experiments by addingnifedipine before 5-HT to see whether it could occlude 5-HT’seffects. Nifedipine (10 �M) reduced the calcium current in allP14–16 cells tested (n � 7). The Boltzmann fit showed thatnifedipine caused a 34 � 18% reduction in Imax (265 � 119 pAin control, 172 � 74 pA in nifedipine, n � 5, P � 0.05; Fig. 7, Aand B). After nifedipine blockade of L-type calcium current,5-HT did not increase the remaining nifedipine-insensitivecalcium current (Imax : 180 � 83 pA in nifedipine, 155 � 72pA in nifedipine and 5-HT, n � 4; Fig. 7, A and B).

Serotonin’s effects on calcium currents in P8–10 CINs.These results present an interesting question. Previous work inour laboratory (Abbinanti and Harris-Warrick 2012; Diaz-Rioset al. 2007) showed that 5-HT increases excitability in P0–3CINs (but does not induce plateau potential capability) byreducing P/Q and N-type calcium currents and indirectly decreas-ing calcium-activaetd potassium current; thus the sign of the effect

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Fig. 4. 5-HT imparts plateau potential capability in P14–16 CINs. A: increasing steps of current injected into a P16 CIN under control conditions and during9 �M 5-HT. Note that during 5-HT, firing continued after the termination of the current pulse at a number of steps. B: an example of a P10 (left) and P16 (right)CIN firing in response to an injected current step in control conditions as well as during 9 �M 5-HT. Only P14–16 CINs demonstrated plateau-potentialcapability. C: average integral of poststep afterpotentials in P14–16 CINs was increased by 5-HT (*P � 0.05). D: a P16 CIN that demonstrated anafterdepolarization (ADP) in 5-HT.

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of 5-HT on calcium currents changed during the first two post-natal weeks. We next examined serotonergic modulation of cal-cium currents at the intermediate age, P8–10. In experiments withbarium replacing calcium, a barium current was observed with anaverage Imax of 376 � 329 pA (n � 5; Fig. 8A). The threshold for

activation of P8–10 currents was near �40 mV, and the voltageof peak current was around �10 mV. Serotonin did not increasethe total barium or calcium current in P8–10 CINs (Fig. 8, A andB; 4/5 CINs measured with calcium, 5/5 CINs measured withbarium). Serotonin did not produce any significant effects onBoltzmann parameters such as Imax, Vmid, or slope at this age.Typically, the calcium current decreased with 5-HT, but rundownmade it difficult to determine whether 5-HT was in fact decreasingthe current. To resolve this issue, we monitored barium currentwith voltage steps at 30-s intervals while washing in 5-HT. In 4/5P8-P10 CINs, we did not observe any deflection from the pro-gressive barium current rundown when 9 �M 5-HT was added(Fig. 8, C and D), suggesting that any decrease seen during 5-HTat this age was an artifact of the slow rundown of the current withtime.

DISCUSSION

We examined the changes in the intrinsic properties andserotonergic modulation of CINs in the lumbar spinal cordduring postnatal development. We sought to examine changesin the properties of intersegmental CINs as an indication ofqualitative, as well as quantitative, changes in the overallnetwork with age.

Postnatal changes of intrinsic properties of CINs. At P8–10and P14–16, about two-thirds of CINs were spontaneouslyactive in the absence of synaptic input, compared with only20% of P0–3 CINs. In addition, the AP narrowed, as has beenseen during development in many neuronal types. This couldarise from changes in potassium and/or sodium currents. Songet al. (2006) showed that expression of the Kv3.1 voltage-gatedpotassium channel involved in repolarization was stronglyincreased between P10 and P21 in mouse Renshaw cells.Spatial clustering of Kv2.1 delayed-rectifier channels occursbetween P2 and P14 in mouse motor neurons (Wilson et al.2004). Increasing density of voltage-gated sodium current,INa(V), coupled with increased delayed-rectifier potassium cur-rent, IK(V), and calcium-activated potassium current, IK(Ca),between embryonic day 16 and P1–3 in rat motor neuronsresults in a narrowing of the embryonic AP (Gao and Ziskind-Conhaim 1998). This trend may continue into the postnatalperiod for CINs.

In addition, we found a decrease in capacitance between theneonatal and the two older CIN groups. Several factors couldexplain this result. Although no studies have examined poten-tial morphological changes to these cells during postnataldevelopment, a reduction in overall cell size, such as a sim-plification of the dendritic arborization or a smaller cell body,could account for the capacitance reduction. Additionally, ashas been found in motor neurons (Chang et al. 1999), areduction in gap junctional coupling to other unidentifiedneurons could be responsible. Zhong et al. (2006a) observed5-HT-induced membrane oscillations that were blocked by thegap junction blocker carbenoxelone and are truncated APsfrom electrically coupled neurons. We observed these oscilla-tions in older (P8–10 and P14–16) cells as well (data notshown), but this does not preclude a reduction in overallcoupling strength.

The CIN AP threshold depolarized slightly between P3 andP8. We and others have shown that the persistent sodiumcurrent (INaP) plays a role in setting the AP threshold in

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Fig. 5. Nifedpine abolishes plateaus in P14–16 CINs. A: a P16 CIN fired inresponse to an injected current pulse in control, 9 �M 5-HT, and 5-HT � 10�M nifedipine (NIF). Note that 5-HT induced a plateau potential (middle,arrow) and that addition of NIF abolished the plateau and ADP (bottom,arrow). B: the average integral of afterpotentials in P14–16 CINs was in-creased by 5-HT and abolished by NIF (*P � 0.05). There was no differencebetween the control and 5-HT � NIF conditions.

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different cell types (Lamas et al. 2009; Zhong et al. 2007). Gaoand Ziskind-Conhaim (1998) suggested that hyperpolarizingchanges in threshold of INa(V) activation help hyperpolarize theAP threshold in neonatal rat spinal motor neurons. Cooperationbetween multiple mechanisms may produce the changes in APthreshold.

The shape of the spike AHP changed between P10 and P14,with a rapidly deactivating component appearing only inP14–16 CINs. This is likely due to the appearance of newcurrent(s) contributing to the AHP. An apamin-sensitive small-conductance calcium-activated potassium (SK) channel is re-sponsible for most of the slow AHP in P0–3 CINs (Dias-Rios

et al. 2007). In P15–30 rat Purkinje neurons, large-conductancecalcium-activated potassium (BK) channels were responsiblefor the fast-inactivating component of the AHP (Haghdoost-Yazdi et al. 2008). Additionally, enhancement of the delayed-rectifier current, IK(V), could contribute to the fast AHP. There-fore, increases in BK or delayed-rectifier currents during post-natal development may lead to the emergence of the fast AHP.

A number of changes could cause CINs to become moreexcitable with age, as indicated by the increased average f-Ivalue and slope. Interestingly, we found no significant changein the baseline input resistance at different ages, suggestingthat voltage-activated currents are responsible for the increased

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excitability. Increased INaP, as discussed above, could increasethe baseline excitability by amplifying the depolarizing driveof a given current injection step. Decreasing the SK currentwith apamin caused a similar upward shift in the f-I plot andsteeper slope in neonatal CINs, suggesting this current mightdecrease over time (Diaz-Rios et al. 2007). The emergence ofnew persistent inward currents, as discussed in more detailbelow, could also be responsible for this increase in excitabilitywith age (Carlin et al. 2000; Li and Bennett 2003; Li et al.2004). The increase in excitability would allow older cells tobe intrinsically more responsive to excitatory synaptic current,perhaps affecting the necessary balance of excitation andinhibition for proper functioning of the network.

Serotonin induces plateau potential capability in older CINs.In some ways, serotonergic excitation of CINs at older ages issimilar to that at P0–3. At all ages, 5-HT evokes a leftwardshift of the f-I plot, a reduction in current required for the cellto spike, and a concurrent increase in average f-I value (Zhonget al. 2006a, 2006b). Interestingly, P0–3 aCINs showed anincrease in AP halfwidth during 5-HT application, whereasdCINs did not (Zhong et al. 2006a, 2006b). 5-HT caused anincrease in halfwidth in both aCINs and dCINs at P8–10 andP14–16. In contrast to P0–3 and P8–10 CINs, P14–16 CINsshowed two qualitative differences in excitability. First, in

older CINs, 5-HT induced a small but significant increase inthe slope of the f-I plot. The second and major difference in5-HT modulation with age was the emergence of plateaupotential capability in about 50% of P14–16 CINs during 5-HTapplication. Some of these neurons continued to fire APs afterthe end of a depolarizing current step, whereas others simplyshowed a prolonged ADP. Critically, we never observed 5-HT-induced plateau potential capability or ADPs in CINs at P10 oryounger. Plateau potentials have been found in motor neuronsof the adult turtle, cat, and P8–15 mouse spinal motor neurons(Carlin et al. 2000; Hounsgaard and Kiehn 1989; Lee andHeckman 1998). This result suggests that serotonin, a criticalneuromodulator for locomotion, causes a qualitative change inthe firing properties of older CINs compared with neonatalneurons.

Plateau potentials in network neurons like CINs could mod-ify network function. First, 5-HT-induced bistability couldprovide flexibility within the network by altering the manner inwhich a cell will respond to a given synaptic input. The outputof bistable neurons can become binary, depending on whetherthe cell is in the depolarized or resting state (Loewenstein et al.2005). Second, bistability could change the functional role ofsynaptic inputs to the CINs. Bistable neurons respond to a brief“trigger” of synaptic input with prolonged firing, instead of

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being driven to fire by the temporal pattern of synaptic input.A third possibility is that the persistent inward currents (PICs)that sustain bistability in our experiments may serve a differentrole in vivo, as a dendritic postsynaptic amplification system.PICs, in the form of persistent sodium or calcium currents, canprovide a voltage-dependent boost to synaptic currents in thedendrites (Elbasiouny et al. 2006; Lee and Heckman 2000; Liand Bennett 2003; Li et al. 2004). 5-HT activation of thesedendritic currents could change the cell’s output through se-lective amplification of synaptic inputs. These possible actionsof 5-HT are not mutually exclusive.

Plateaus are sustained by an L-type calcium current. TheL-type calcium channel blocker nifedipine blocked the 5-HT-induced plateau potentials in P14–16 CINs. L-type calciumcurrent underlies plateau potentials in P9–16 mouse motorneurons (Carlin et al. 2000). New L-type calcium channelsubunits are expressed around P8–10 in presumptive mousemotor neurons, reaching their maximal expression at P18(Jiang et al. 1999). The time course of this expression roughlyparallels our results in CINs, suggesting that plateau potentialcapability appears around P14 due to the upregulation ofL-type calcium channels, ICa(L), at this time. We used voltage-clamp analysis to show that 5-HT increases a nifedipine-sensitive calcium current in about one-half of P14–16 CINs;5-HT evokes bistability in a similar fraction. There are severalpossible reasons why 5-HT excites a majority of CINs at allages but induces bistability in only 50% of P14–16 CINs. First,there may be different 5-HT receptors mediating the increase inexcitability and the ICa(L)-mediated bistability, which can bedifferentially expressed among CINs. Second, P14–16 may bean intermediate age for development of CIN bistability; at laterages, perhaps all CINs become bistable with 5-HT.

These results expose an unusual plasticity in the mechanismsof 5-HT modulation of CINs at different ages. Serotoninincreases CIN excitability at all ages, but apparently throughopposing effects on voltage-activatred calcium currents, ICa(V).In P0–3 CINs, 5-HT acts in part by decreasing IK(Ca) indirectlythrough a conductance decrease in ICa(V) (Diaz-Rios et al.2007). We have recently shown that 5-HT decreases N- andP/Q-type calcium currents in P0–5 CINs (Abbinanti and Har-ris-Warrick 2012). We show here that by P14–16, 5-HTexcites neurons at least in part by enhancing ICa(L), an effectthat was not seen at earlier ages. Therefore, 5-HT increases theexcitability of CINs at different ages through opposite effectson different calcium currents. SK-type calcium-activated po-tassium channels, whose current appears to be reduced by5-HT in P0–3 CINs, are activated by different calcium channelsubtypes in different preparations, providing a mechanism bywhich L-type calcium current can be increased while N- andP/Q-type calcium current coupled with IK(Ca) can be concur-rently reduced by 5-HT (Abbinanti and Harris-Warrick 2012;Goldberg and Wilson 2005; Kasten et al. 2007; Perez-Roselloet al. 2005; Protti and Uchitel 1997; Sah 1995; Vilchis et al.2000; Wikstrom and El Manira 1998). At P8–10, we saw nonet effect of 5-HT on total calcium current. Given that Jianget al. (1999) demonstrated that new CaV1.2 and CaV1.3 L-typecalcium channel transcripts begin to be expressed in the ventralhorn around P8, perhaps some L-type calcium current ispresent at this time. If 5-HT was having opposing effects onN-, P/Q-, and L-type ICa(V) at P8–10, it may be difficult toresolve these effects due to the small net change.

Conclusion. Our results show that CINs in the mouse spinallocomotor CPG are not fully mature at birth and that theirneonatal firing properties do not fully reflect those of adultanimals. Both the baseline firing properties of CINs and theirresponses to 5-HT are qualitatively different in young adultscompared with newborn mice. This suggests that CPG maymature as the animals learn to walk and may not be identical inneonates and adults. Therefore, we must be careful drawingconclusions on the function of the adult locomotor networkfrom neonatal studies. Even though it has not yet been possibleto evoke fictive locomotion in an isolated adult mouse spinalcord, it is important to continue to examine the intrinsicproperties of network components in slices of the mature adultcord (Husch et al. 2011; Mitra and Brownstone 2012) inconjunction with neonatal studies using the intact spinal cord.Future investigations should focus on developing a function-ally mature intact spinal cord preparation to determine theextent of changes in network properties during postnataldevelopment.

ACKNOWLEDGMENTS

We thank Bruce Johnson, Andreas Husch, and Shelby Dietz for usefuldiscussions and comments on the manuscript.

Present address of G. Zhong: Department of Chemistry and ChemicalBiology, Harvard University, Cambridge, MA 02138.

GRANTS

This work was supported by National Institutes of Health (NIH) GrantsNS17323 and NS057599 (to R. M. Harris-Warrick) and NIH Training GrantT32 GM007469.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

M.D.A. and R.M.H.-W. conception and design of research; M.D.A. andG.Z. performed experiments; M.D.A. and G.Z. analyzed data; M.D.A., G.Z.,and R.M.H.-W. interpreted results of experiments; M.D.A. prepared figures;M.D.A. and R.M.H.-W. drafted manuscript; M.D.A. and R.M.H.-W. editedand revised manuscript; M.D.A., G.Z., and R.M.H.-W. approved final versionof manuscript.

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