Network for the Generation of Movement The Whisking Rhythm ...

doi: 10.1152/jn.01187.200697:2148-2158, 2007. First published 3 January 2007;J Neurophysiol

Nathan P. Cramer, Ying Li and Asaf KellerNetwork for the Generation of MovementThe Whisking Rhythm Generator: A Novel Mammalian

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The Whisking Rhythm Generator: A Novel Mammalian Network for theGeneration of Movement

Nathan P. Cramer, Ying Li, and Asaf KellerProgram in Neuroscience and the Department of Anatomy and Neurobiology University of Maryland School of Medicine,Baltimore, Maryland

Submitted 8 November 2006; accepted in final form 21 December 2006

Cramer NP, Li Y, Keller A. Whisking rhythm generator: novel mammaliannetwork for the generation of movement. J Neurophysiol 97: 2148–2158,2007. First published January 3, 2007; doi:10.1152/jn.01187.2006. Usingthe rat vibrissa system, we provide evidence for a novel mechanismfor the generation of movement. Like other central pattern generators(CPGs) that underlie many movements, the rhythm generator forwhisking can operate without cortical inputs or sensory feedback.However, unlike conventional mammalian CPGs, vibrissa motoneu-rons (vMNs) actively participate in the rhythmogenesis by convertingtonic serotonergic inputs into the patterned motor output responsiblefor movement of the vibrissae. We find that, in vitro, a serotoninreceptor agonist, �-Me-5HT, facilitates a persistent inward current(PIC) and evokes rhythmic firing in vMNs. Within each motoneuron,increasing the concentration of �-Me-5HT significantly increases theboth the magnitude of the PIC and the motoneuron’s firing rate.Riluzole, which selectively suppresses the Na� component of PICs atlow concentrations, causes a reduction in both of these phenomena.The magnitude of this reduction is directly correlated with the con-centration of riluzole. The joint effects of riluzole on PIC magnitudeand firing rate in vMNs suggest that the two are causally related. Invivo we find that the tonic activity of putative serotonergic premo-toneurons is positively correlated with the frequency of whiskingevoked by cortical stimulation. Taken together, these results supportthe hypothesized novel mammalian mechanism for movement gener-ation in the vibrissa motor system where vMNs actively participate inthe rhythmogenesis in response to tonic drive from serotonergicpremotoneurons.

I N T R O D U C T I O N

The ability to move allows an organism to adapt and respondto its environment. Indeed, a primary goal in processing sen-sory inputs is to select an appropriate motor act in response tothose inputs. Often the patterned activation of muscles requiredto generate a movement is produced subcortically by neuralelements that can operate without patterned inputs from higherbrain centers or sensory feedback. In mammals, these criticalcomponents of motor networks, or central pattern generators(CPGs), are typically composed of groups of interneurons thatgenerate rhythmic drive to motoneurons that innervate thetarget muscles (Arshavsky et al. 1997; Stein et al. 1997). Themotoneurons themselves are not considered part of the rhythm-generating network (Orlovksy et al. 1999). Here, using the ratvibrissa motor system, we provide evidence for an alternativemechanism for the generation of movement in mammals wherethe motoneurons are involved in the rhythmogenesis.

Rats explore their environment by rhythmically palpatingobjects with their mystacial vibrissae (Brecht et al. 1997;

Vincent 1912; Welker 1964). These rhythmic movements, orwhisking, persist after sensory denervation (Welker 1964),cortical ablation (Gao et al. 2003; Semba and Komisaruk1984), or decerebration (Lovick 1972), suggesting that thewhisking motor pattern is produced subcortically by a CPG.We demonstrated previously that serotonin (5-HT) drivesvibrissa motoneurons (vMNs) at whisking frequencies and thatinfusion of 5-HT receptor antagonists onto vMNs suppressesvoluntary whisking (Hattox et al. 2003). On the basis of theseresults, we hypothesized that 5-HT is both necessary andsufficient for rhythmic whisking and that, in this system, themotoneurons are part of the rhythm-generating network. Theexperiments described here were designed to test this hypoth-esis.

In spinal (Perrier and Hounsgaard 2003) and trigeminal(Hsiao et al. 1998) motoneurons, 5-HT acts as a potent facil-itator of inactivation-resistant inward currents. First discoveredin cat spinal motoneurons (Schwindt and Crill 1980), thesepersistent inward currents (PICs) modulate the firing frequen-cies of motoneurons (Heckman et al. 2003; Schwindt and Crill1982). Thus by activating and amplifying PICs, 5-HT is re-ported to modulate firing in motoneurons (Harvey et al. 2006b;Heckman et al. 2005; Hounsgaard and Kiehn 1989; Houns-gaard et al. 1988; Hsiao et al. 1998). Here, we test thehypothesis that 5-HT is sufficient to generate rhythmic firing invMNs through a graded facilitation of PICs. In addition, wetest the prediction that the tonic output from serotonergicpremotoneurons is positively correlated with whisking fre-quencies. Our findings support the hypothesis that vMNsgenerate the rhythmicity of the whisking motor pattern inresponse to a graded facilitation of a PIC by 5-HT. Further-more, they suggest that, in this system, there exists a novelmammalian mechanism for movement generation.

M E T H O D S

In vitro slice preparation

We cut 500-�m-thick coronal brain stem slices from P13 to P18Sprague-Dawley rats (i.e., after the onset of voluntary whiskingWelker 1964). During the isolation of the brain stem and cutting ofslices, the tissue was submersed in ice-cold modified artificial cere-brospinal fluid (ACSF) in which sucrose was substituted for NaCl.The sucrose-ACSF was composed of (in mM) 248 sucrose, 26NaHCO3, 1.25 NaH2PO4, 3 KCl, 5 MgSO4, 1 CaCl2, and 15 glucose.We allowed the slices to recover for 1 h in a holding chambercontaining warmed normal ACSF (32°C), aerated with 95% O2-5%

Address for reprint requests and other correspondence: A. Keller, Dept. ofAnatomy and Neurobiology, University of Maryland School of Medicine, 20Penn St., Rm S251, Baltimore, MD 21201 (E-mail: [email protected]).

The costs of publication of this article were defrayed in part by the paymentof page charges. The article must therefore be hereby marked “advertisement”in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

J Neurophysiol 97: 2148–2158, 2007.First published January 3, 2007; doi:10.1152/jn.01187.2006.

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CO2. The normal ACSF was composed of (in mM) 124 NaCl, 25NaHCO3, 5 N,N-bis(2-hydroxyethyl)-2-aminoethanesul-fonic acid(BES), 3 KCl, 1.3 MgSO4, 2.0 CaCl2, and 15 glucose. For recording,we transferred individual slices to a recording chamber and continu-ously perfused the slice with normal ACSF (3–4 ml/min).

Whole cell recordings

Visually guided whole cell patch-clamp recordings were made frommotoneurons in the lateral subdivision of the facial nucleus with theuse of near-infrared differential interference-contrast microscopy.This region contains motoneurons innervating the intrinsic muscles ofthe vibrissae (Hattox et al. 2003; Klein and Rhoades 1985; Semba andEgger 1986). Recordings were obtained with an EPC-10 amplifier(HEKA Instruments), digitized at 20 kHz with an A/D board (ITC-18;Instrutech, Great Neck, NY) using PatchMaster software (HEKA),and stored on PC. The impedance of patch electrodes was 3–5 M�.The intracellular recording solution contained (in mM) 120 K-glu-conate, 10 KCl, 10 HEPES, 1 MgCl2, 2.5 MgATP, 0.2 Tris-GTP, 0.1bis-(o-aminophenoxy)-N,N,N�,N�-tetraacetic acid (BAPTA), and 5.2biocytin (pH was adjusted to 7.3 with KOH).

The following pharmacological agents were obtained from Sigma-Aldrich (St. Louis, MO): D(�)-2-amino-5-phosphonopentanoic acid(AP5; applied at 50 �M to the perfusate), 6-cyano-7-nitroquinoxaline-2,3-dione disodium (CNQX; 20 �M), gabazine (10 �M), tetrodotoxin(TTX; 1 �M), �-Me-5HT (1–60 �M), and riluzole (2–5 �M).

Cells were filled with biocytin (Molecular Probes, Eugene, OR)through the recording pipette and slices were fixed overnight in abuffered solution containing 4% paraformaldehyde. To visualize la-beled cells, slices were reacted with the Vectastatin ABC kit (1:1000;Vector Labs, Burlingame, CA) and 3–3� diaminobenzidine (DAB; 0.5mg/ml), urea H2O2 (0.3 mg/ml), and CoCl2 (0.2 mg/ml) in 0.05 MTris buffer containing 0.5 M NaCl.

Analysis of PICs

The magnitude and activation voltage of PICs in vMNs weredetermined using a slow triangular voltage ramp command. Theascending phase of the voltage command consisted of a linearlyincreasing ramp (speed: 8–16 mV/s) for 5 s. The descending phasereturned the membrane potential to �90 mV at the same rate. For eachcell, we selected the fastest single ramp speed that minimized theoccurrence of breakthrough action potentials in all conditions (controland drug).

All analyses were performed on leak-subtracted currents, generatedby subtracting the predicted ohmic leak currents from the measuredcurrents. The leak currents were determined by multiplying thesubthreshold membrane conductance (i.e., the membrane conductanceprior to activation of voltage gated channels) by the voltage command.We monitored each cell’s series resistance throughout the experimentsand rejected from analysis cells in which the series resistance changedby �10%.

Surgical procedures

We used nine female Sprague-Dawely rats (225–270 g) for in vivoexperiments. All procedures strictly adhered to Institutional and Fed-eral guidelines. Under halothane (2.5%) anesthesia, a small incisionwas made in the facial skin, and a pair of bipolar EMG electrodes (76�m Teflon-coated stainless steel wire) was tunneled subcutaneouslyinto the deep intrinsic muscles. Prior to insertion, the electrode tipswere bent into a hook to prevent shifts in location during movements.After infusion of local anesthetics to surgical sites, we performedcraniotomies over the vibrissa motor cortex (vMCx) and the lateralparagigantocellularis (LPGi) nucleus. The exposed dura was keptfrom drying by using dental wax to build a saline filled well over the

craniotomies. During recordings, rats were maintained at anestheticlevel III-2, characterized by an ECoG frequency between 5 and 7 Hz,a respiratory rate of �100 breaths/min, and an absence of a with-drawal reflex to noxious stimuli (paw or tail pinch) (Friedberg et al.1999). As the threshold for evoking whisking depended on the depthof anesthesia, care was taken to maintain a consistent and stable levelof anesthesia. Body temperature was maintained at 37°C with aservo-controlled heating blanket.

Cortical stimulation

We evoked rhythmic whisking through intracortical microstimula-tion (ICMS) of the rhythmic subregion (RS) of vMCx as in ourprevious study (Cramer and Keller 2006). Custom-made platinum-in-quartz electrodes (�20 �m tip) were lowered to a depth of 1.5 mm,corresponding to layer V of vMCx. The RS of vMCx was identifiedby applying low-intensity current pulses (50 monophasic pulses at 50Hz, 200-�s pulse width, 25–250 �A) to the area of vMCx identifiedby Haiss and Schwarz (2005). Stimulation in this region evokedrhythmic vibrissae movements, whereas stimulation outside of thisregion either generated small vibrissal retractions or failed to producemovements. RS was reliably located at anterioposterior 1.3, medio-lateral 1.3 relative to bregma, as previously documented (Cramer andKeller 2006; Haiss and Schwarz 2005).

Recording and analyses

We obtained extracellular recordings using a NeuroData IR183Aamplifier (Cygnus Technology, Delaware Water Gap, PA), digitizedat 40 kHz with an A/D board (ITC-18; Instrutech) using softwarecustom written in IGOR (WaveMetrics, Lake Oswego, OR), andstored on PC. Recordings from putative serotonergic premotoneuronswere obtained from pulled borosilicate glass electrodes (�2 �M tip,2–20 M�) stereotaxically guided to the rostral (juxtafacial) LPGi,(anteroposterior 11.4 mm, lateromedial 1.0 mm, relative to bregma).

To analyze the responses of putative premotoneurons to ICMS ofvMCx, we isolated the action potential waveforms of each unit usingOff-line Sorter (Plexon, Dallas, TX). The time stamps of individualunits along with the corresponding stimulus triggers were importedinto IGOR for further analyses.

We defined a premotoneuron response as significant if the numberof action potentials fired during stimulation exceeded the baselineactivity by 2 SD. The baseline activity for each cell was defined as thenumber of spikes fired during the 1-s interval prior to stimulationaveraged over all trials. To measure response latency, we constructedperistimulus time histograms (PSTHs, bin size � 1 ms) from thepremotoneuron timestamps and stimulus triggers. The onset latencywas defined as the first occurrence of two consecutive bins thatexceeded the baseline activity by 2 SD.

Bouts of rhythmic whisking evoked by ICMS of vMCx wererecorded using the implanted vibrissal EMG electrodes. EMG record-ings were amplified (Model 1700 Differential AC Amplifier, A-MSystems, Carlsborg WA) sampled at 40 kHz with an A/D board(ITC-18, Instrutech), filtered (1 Hz to 5 kHz), and stored on acomputer running software written in Igor. We defined the whiskingfrequency as the peak of the power spectral density generated from therectified, filtered EMG data, sub-sampled to 500 Hz.

R E S U L T S

5-HT drive determines vMN firing rates

Using a brain stem slice preparation, we first tested theprediction that individual vMNs generate a range of firingfrequencies in response to varying levels of serotonergic drive.The in vitro preparation is advantageous because it permitsapplication of pharmacological agents with a greater accuracy

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than can be achieved in vivo. To simulate endogenous seroto-nergic drive to vMNs, we bath applied the serotonin type 2(5-HT2) receptor agonist, �-Me-5HT to the brain stem slices.We focused on this receptor subtype because when activated, itproduces rhythmic firing in vMNs, and its antagonist (meter-goline) suppresses rhythmic whisking (Hattox et al. 2003). Weexamined whole cell recordings from 50 vMNs in this exper-iment, all of which lacked spontaneous activity in controlconditions. In support of our prediction, vMNs fired rhythmictrains of action potentials at whisking frequencies (1.5–17 Hz;Figs. 1 and 4) in response to �-Me-5HT. In each cell tested,these effects of �-Me-5HT persisted in the presence of gluta-matergic and GABAergic receptor antagonists (n � 31; seeMETHODS), suggesting that the firing in vMNs did not resultfrom glutamatergic or GABAergic premotoneurons.

To test the effects of different levels of serotonergic drive onvMN firing rates, in 16 vMNs, we applied the agonist at twoconcentrations. The firing frequency evoked in individual neu-rons by any one concentration of �-Me-5HT varied from cellto cell (Fig. 1) possibly due to variable truncation of thedendritic tree during slice preparation, or to differences ineffective drug concentrations near the recorded neurons. Thusfor each cell, we applied a “low” concentration that rangedbetween 1 and 40 �M and a “high” concentration that rangedbetween 2 and 60 �M. We randomized the order of applicationof these drug concentrations.

The response of a vMN to the agonist is shown in therepresentative current-clamp recordings in Fig. 1A. This vMN,which was silent in the absence of the agonist, fired at asignificantly (P � 10�3) higher frequency in response to thehigh agonist concentration (5 �M, 5.5 0.2 Hz) comparedwith the lower agonist concentration (1 �M, 3.3 0.2 Hz).The time course of the neuron’s responses to each agonistconcentration is plotted in Fig. 1B. The delay between the

beginning of drug application and onset of firing may primarilyreflect the time required for the drug to diffuse through thetissue to reach the cell. We computed firing rates from the lastthird of the drug application period (highlighted in red in Fig.1B) by constructing histograms (bin size � 0.25 Hz) ofinstantaneous firing frequencies from this period, which wedefined as the steady state (Fig. 1C). We defined the magnitudeof responses at steady state as the peak of these histograms andidentified the whisking frequency at which this peak occurred.We defined the half-width at half-maximum of each histogramas the SD of the steady-state firing rate. Similar results werefound for 15 of 16 vMNs tested, as shown in Fig. 1D. For thesecells, the higher agonist concentration evoked a significantlyhigher firing rate (Student’s t-test, P �10�3). In one cell only,the firing rates at the two concentrations were not significantlydifferent (P � 1). These findings support the hypothesis thatvMNs generate the whisking frequency in response to a vary-ing degree of drive from serotonergic premotoneurons.

vMNs express PICs

The firing rate of neurons, including motoneurons, is closelyrelated to the magnitude of inward current reaching the soma(Powers and Binder 2001). Neuromodulators that regulatemembrane currents can alter this current-frequency relation-ship and are thus capable of regulating the cell’s firing fre-quency (Powers and Binder 2001; Rekling et al. 2000). Oneimportant neuromodulator is 5-HT, which, acting through5-HT2 receptors, potently facilitates slowly or noninactivatingvoltage-dependent inward currents (Harvey et al. 2006a; Heck-man et al. 2005; Perrier and Hounsgaard 2003). These PICshave a significant impact on the firing rates of some motoneu-rons (Prather et al. 2001). We therefore hypothesized that invMNs, 5-HT produces firing by activating PICs. Because the

A B

C D

FIG. 1. 5-HT2 receptor agonist evokes dose-depen-dent firing in vibrissa motoneurons (vMNs). A: current-clamp recordings from a representative vMN in thepresence of 2 concentrations of the agonist, �-Me-5HT.This neuron was silent in control conditions and fired ata significantly higher rate (P � 10�3) in the presence of5 �M agonist (5.5 0.2 Hz) than in the presence of 1�M agonist (3.3 0.2 Hz). B: time course of theagonist-induced firing for the vMN shown in A. Theinstantaneous firing frequency of the vMN in each ago-nist concentration is plotted as a function of time. Thehorizontal green bar indicates the duration of agonistapplication. We determined steady-state firing rates fromthe final 3rd portion of the drug delivery period (redportions of each trace). C: histograms (1-ms bin size) ofsteady-state firing frequencies for the motoneuronshown in A. The peak in each histogram corresponds tothe mode of the steady-state firing frequency. The half-width at half max of each histogram corresponds to thevariance. D: summary data from 15 vMNs with a sig-nificant dose-dependent response to the agonist. Eachpair of joined data points indicates the mode of thesteady-state firing rate for a single vMN at the individ-ually determined “high” (2–60 �M) and “low” (1–40�M) agonist concentrations. The response of the mo-toneuron shown in A is highlighted in red. In each vMN,increasing the agonist concentration caused a significantincrease in the steady-state firing frequency.

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firing rate of vMNs is determined by the magnitude of 5-HT2receptor activation (see preceding text), we further predictedthat the regulation of vMN firing is achieved through a gradedfacilitation of these PICs.

The presence of a PIC in a vMN is demonstrated by the datadepicted in Fig. 2A. To better isolate the persistent currents, weused protocols with slow voltage ramps that minimize theactivation of potentially confounding transient Na� currents(Lee and Heckman 1998). Starting from a holding potential of�60 mV, we ramped the membrane potential to �49 mV andheld the cell at this potential for three seconds. We examinedthe voltage dependence of the currents activated by this pro-tocol by increasing the holding potential in 3-mV increments.At more depolarized membrane potentials, the increase in netoutward current was reduced (Fig. 2A, middle). Examination ofthe leak subtracted currents (top) reveals that this reductionresulted from a slowly or noninactivating inward current thatlasted for the duration of the voltage command (3 s). Currentswith these kinetics and voltage dependence are defined as PICs(Schwindt and Crill 1982).

To quantify these PICs, we used a slowly increasing (8–16mV/s) triangular voltage command. Like the ramp-and-holdprotocol described in the preceding text, this slow-ramp pro-tocol suppressed activation of fast Na� spikes. In addition, theslow ramp is advantageous in that using a single voltagecommand, it provides information simultaneously about boththe PIC activation voltage and magnitude. A representativerecording using this protocol is shown in Fig. 2B. The bottompanel depicts the triangular voltage command, whereas the toppanel shows the recorded membrane current (gray) and leak-subtracted (see METHODS) current (black). During the ascendingphase of the voltage ramp, the currents were dominated by anoutward leak current; however, at �58 mV, an inward currentwas activated (arrow “1”). As the voltage ramp continued toascend, outward currents dominated. During the descendingphase of the voltage ramp, a second inflection was visible,suggesting that the current had remained active.

We defined the PIC activation voltage as the membranepotential where the slope of leak-subtracted current first be-comes negative (Fig. 2B, arrow “1”). We defined the PICmagnitude as the peak of the leak-subtracted inward currentduring the ascending phase of the voltage command (Fig. 2B,arrow “2”). Cells without a net inward leak-subtracted current

were considered to not have a PIC. We observed a similar PICin 26 of 34 vMNs tested, indicating that most vMNs expressPICs, even in the absence of agonists. The magnitude of thePIC averaged �120 60 pA (median, �150 pA) and had anactivation voltage of �53 6 mV. These PICs persisted in thepresence of antagonists of fast glutamatergic and GABAergicreceptors (n � 20, see METHODS), indicating that the PICs are anintrinsic property of vMNs.

PICs are facilitated by 5-HT2 receptors

To examine the effects of �-Me-5HT on the PICs, wefocused on currents evoked during the ascending phase of thevoltage ramps and compared, in each cell, the leak-subtractedactivation voltage and current magnitude before and after drugapplication (1–30 �M). For the 26 vMNs that expressed a PICin control conditions, the average PIC magnitude in the pres-ence of �-Me-5HT increased from �120 60 to �500 200pA (median, �500 pA). �-Me-5HT also significantly shiftedthe PIC activation voltage to a more hyperpolarized value of�57 5 mV (P � 0.029, Kolmogorov-Smirnov test). In theeight vMNs that did not express a PIC in control conditions,the agonist evoked a PIC of �360 140 pA with an averageactivation threshold of �56 5 mV.

To examine the effects of increases in serotonergic drive onthe facilitation of PIC magnitude, we applied multiple agonistconcentrations (1–30 �M) to 19 of these cells. A representativeexample is shown in Fig. 3A. This vMN had a PIC in controlconditions (black trace) with a peak magnitude of �19 10pA. Application of 1 �M agonist (red trace) enhanced the PICmagnitude to �198 20 pA. Increasing the agonist concen-tration to 5 �M (green trace) further enhanced the PIC mag-nitude to �396 21 pA. The impact of the agonist on the PICmagnitude for all 19 motoneurons is shown in Fig. 3B, wherethe peak PIC magnitude is plotted as a function of agonistconcentration normalized to the lowest concentration appliedto each cell. In every neuron, the increase in PIC magnitudefrom control to low concentration was significant (P � 10�3).Increasing the concentration of the agonist resulted in anadditional, significant increase in PIC magnitude in 18 of 19cells (P � 10�3). These results indicate that, through thegraded facilitation of PICs, 5-HT can modulate the magnitude

FIG. 2. vMNs express persistent inward currents(PICs). A: ramp-and-hold protocol reveals the exis-tence of a PIC in a representative vMN in controlconditions. Note the nearly uniform reduction in therecorded membrane current (Im) at more depolarizedholding potentials. Leak subtracted currents revealthat the observed reduction in Im- resulted from avoltage-dependent PIC. B: protocol for measuringthe PIC magnitude and activation voltage. The mem-brane currents (gray) generated in response to atriangular voltage command (16 mV/s) are domi-nated by an outward current. The downward inflec-tions in the recorded membrane current traces duringboth the ascending and descending phases resultfrom a PIC, seen more clearly in the leak-subtractedcurrent trace (black). The PIC activation voltage wasdefined as voltage where the slope of the leak sub-tracted current trace first becomes negative (1). Thepeak PIC magnitude was measured at the peak of theinward inflection during the ascending phase of thevoltage command (2).



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of inward currents in vMNs, thereby generating the full rangeof whisking frequencies.

PICs are largely composed of sodium currents

PICs in spinal (Harvey et al. 2006a), hypoglossal (Powersand Binder 2003), and trigeminal (Hsiao et al. 1998) motoneu-rons consist of Na� and Ca2� currents. To determine if Na�

currents contribute to PICs in vMNs, we suppressed Na�

conductances with TTX (1 �M). A representative example isdepicted in Fig. 3C. In response to application of 10 �M�-Me-5HT (green trace), this motoneuron had a PIC magni-tude of �741 31 pA. Co-application of 1 �M TTX (redtrace) reduced the PIC magnitude to �210 6 pA. Group datafor 23 motoneurons are shown in Fig. 3D. After application ofTTX, the agonist-facilitated PIC was reduced in magnitude by79 16% (median � 81%). In each case, the reduction wassignificant (P � 10�3, Student’s t-test).

In 13 of these motoneurons, we subsequently applied 50–200 �M cadmium (Cd2�) to determine whether the TTX-insensitive component resulted from Ca2� currents. For the

motoneuron shown in Fig. 3C, application of 50 �M Cd2�

(black trace) completely suppressed the TTX-insensitive com-ponent of the PIC. We recorded a similar complete suppressionof PICs in 10 of 13 vMNs (Fig. 3D). In the remaining threeneurons, Cd2� application significantly (P � 10�3), but notcompletely (38 21%), further suppressed PIC amplitudes.Thus in vMNs, PICs are predominantly composed of sodiumcurrents, suggesting that a persistent Na� current (INaP) playsa central role in generating firing in these cells.

Riluzole modulates PIC magnitudes in vMNs

Because INaP accounted for most of the PIC in the vMNs, wefocused on these currents in the remaining experiments. At lowconcentrations (2–5 �M), riluzole selectively antagonizes thiscurrent without significantly affecting fast Na� currents thatunderlie action potentials (Wu et al. 2005). We took advantageof this specificity to test the hypothesis that INaP underlies theability of vMNs to generate a range of whisking frequencies.Injection of depolarizing current pulses in current-clamp re-

A B

C D

E F

FIG. 3. 5-HT2 receptor agonist facilitates a Na�-based PICin a concentration-dependent manner. A: concentration-depen-dent facilitation of PIC magnitudes in a representative vMN.The magnitude of the PIC in control conditions (black trace,�19 10 pA) was enhanced by addition of 1 �M �-Me-5HT(red trace, �198 20 pA). Application of 5 �M agonistcaused an additional increase in PIC magnitude (green trace,�396 21 pA). B: PIC magnitude as a function of normalizedagonist concentration for 19 vMNs. Agonist concentrationswere normalized to the lowest concentration applied to eachcell. The increase in PIC magnitude from control conditions(concentration of 0) to the lowest agonist concentration wassignificant in each cell. Increasing the agonist concentrationcaused an additional significant increase in 18 vMNs. Here andin similar figures, data are presented as means SD. C: PICsin vMN have a prominent Na� component. Application of 1�M TTX (red trace) caused a significant reduction in theagonist facilitated PIC (green trace) in a representative vMN.Subsequent addition of Cd2�, a calcium channel blocker,completely eliminated the remaining PIC (black trace). D:effect of TTX and Cd2� on facilitated PIC magnitudes, re-corded from 23 vMNs. Application of 1 �M TTX significantlyreduced the PIC magnitude in each motoneuron. The subse-quent application of 50–200 �M Cd2� to 13 vMNs completelysuppressed the remaining PIC in 10 neurons and significantlyreduced it in the remaining 3. E: response of a representativevMN to riluzole. Riluzole (2 �M, blue trace; 5 �M red trace)caused a concentration-dependent reduction in the magnitudeof the agonist facilitated PIC (green trace). Application of TTXcompletely abolished the PIC in this vMN (black trace). F:group data for 13 vMNs demonstrating the concentration-dependent effects of riluzole on the magnitude of the agonistfacilitated PIC. In the 9 vMNs tested at both concentrations,riluzole significantly reduced the PIC magnitude in a concen-tration-dependent manner. A similar reduction was observed inthree of four vMNs tested with only 5 �M riluzole. In the 9vMNs in which it was applied, TTX caused an additionalsignificant reduction in PIC magnitude.



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cordings confirmed that �5 �M riluzole had no effect on thegeneration of fast spikes.

The effects of riluzole are illustrated for a representativemotoneuron in Fig. 3E. Riluzole (2 �M) significantly (Stu-dent’s t-test, P � 10�3) decreased the magnitude of theagonist-facilitated PIC from �396 21 to �143 10 pA.Increasing the concentration of riluzole to 5 �M further sup-pressed the PIC to �50 7 pA. The subsequent addition of 1�M TTX completely abolished the PIC in this motoneuron.Group data for nine neurons tested at both riluzole concentra-tions, and four vMNs tested only at the 5 �M concentration,are shown in Fig. 3F. In all 13 vMNs, riluzole significantlysuppressed PIC magnitudes. The amount of suppression with 5�M riluzole was significantly (P � 10�3, n � 9) greater thanthe suppression in the presence of 2 �M riluzole. Subsequentaddition of TTX suppressed the agonist facilitated PIC by atotal of 80 20% (n � 9), eliminating the majority of the PIC.We obtained similar results in the presence of ionotropicglutamatergic and GABAergic antagonists (see METHODS), sug-gesting that riluzole acted directly on the recorded vMNs.These findings strengthen the premise that INaP is a majorcomponent of PICs in vMNs, and that its activity may underliethe generation of the whisking rhythms by vMNs.

Riluzole modulates firing in vMNs

The postulated role of INaP on regulating the firing rate ofvMNs is demonstrated in the current-clamp recordings shownin Fig. 4A. In control conditions (top), the motoneuron wassilent; however, application of 20 �M �-Me-5HT caused thevMN to fire regularly at �2 Hz. After the addition of 5 �Mriluzole, the firing frequency began to decline and was even-tually completely suppressed. Riluzole’s effects were revers-ible: After washout with artificial cerebrospinal fluid (ACSF)for 22 min, re-application of 20 �M �-Me-5HT evoked firingin the vMN near 2 Hz. Group data for eight vMNs tested atboth 2 and 5 �M riluzole and nine vMNs tested only at 5 �Mare shown in Fig. 4B. In the eight cells tested at both concen-trations, 2 �M riluzole completely suppressed firing in threevMNs and significantly reduced the firing frequency by 28 17% (P � 10�3) in the remaining five neurons. Subsequentapplication of 5 �M riluzole to these five vMNs caused a

complete suppression of firing in four cells and an additionalsignificant 59% reduction in the remaining vMN (P � 10�3).In the nine vMNs tested only at 5 �M riluzole, firing wascompletely suppressed in six cells and significantly reduced by35 21% in the remaining three (P � 10�3). Washout wassuccessful in seven out of eight cells tested.

Injections of current pulses (200 ms) during the peak effectsof riluzole confirmed that the neurons (n � 15) were stillcapable of generating trains of fast action potentials. Thisensured that the fast sodium channels underlying the actionpotentials had not been affected by the riluzole. Together, theeffects of riluzole on the magnitude of INaP and its impact onthe agonist-induced firing in vMNs support the hypothesis thatthe facilitation of a PIC by 5-HT is necessary and sufficient forvMNs to generate whisking over a range of frequencies.

Activity in putative serotonergic premotoneurons iscorrelated with whisking frequency

The results of our in vitro experiments support the hypoth-esis that vMNs generate rhythmic whisking in response to agraded facilitation of a PIC by 5-HT. In the intact animal,serotonergic drive to vMNs originates from a subset of sero-tonergic neurons in the brain stem that both project to vMNs inthe lateral facial nucleus and receive projections from thevibrissa motor cortex (vMCx) (Hattox et al. 2003; Hattox et al.2002). Thus these serotonergic premotoneurons likely serve asthe endogenous source of the 5-HT employed by vMNs togenerate whisking rhythms. As the frequency of the motorpattern generated by vMNs varies with the level of serotoner-gic drive they receive, we predict that the output from theseserotonergic premotoneurons should correlate positively withwhisking frequencies.

To test this prediction, we evoked rhythmic whisking byapplying ICMS to vMCx, as previously described (Cramer andKeller 2006; Haiss and Schwarz 2005), while recording extra-cellularly from putative serotonergic premotoneurons. Our at-tempts to unambiguously identify the premotoneurons byevoking antidromic action potentials after stimulation of thefacial nucleus were unsuccessful. This is most likely due to thedifficulty in activating small-diameter, nonmyelinated fiberscharacteristic of serotonergic neurons (Azmitia and Gannon

FIG. 4. Riluzole causes a concentration-depen-dent reduction in agonist induced firing rates ofvMNs. A: representative example of the effects ofriluzole on agonist induced firing in a vMN. Incontrol conditions, the vMN did not fire spontane-ously. Application of 20 �M �-Me-5HT evokedrhythmic firing that was gradually suppressed bythe addition of 5 �M riluzole. After washout, re-application of 20 �M �-Me-5HT again evokedrhythmic firing in the vMN. The times listed undereach trace indicate the time elapsed after recordingthe control trace. B: group data for 17 vMNs dem-onstrating the concentration-dependent effects ofriluzole on the agonist induced firing in vMNs.Increasing the concentration of riluzole resulted in aconcentration-dependent reduction in the vMN fir-ing frequencies.



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1983) and the close proximity of the recording and stimulationsites. As the electrophysiological properties (e.g., spontaneousactivity and spike shape indices) of serotonergic neurons showconsiderable variation (Zhang et al. 2006), we concluded thatthese criteria cannot be used reliably to identify serotonergicneurons. We therefore accepted for analysis, and defined asputative serotonergic premotoneurons, all cells recorded in therostral lateral (juxtafacial) pargigantocellularis nucleus (LPGi)according to stereotaxic coordinates. This nucleus is composedpredominantly of serotonergic neurons (Bowker and Abbott1990; Darnall et al. 2005) that both receive direct inputs fromvMCx and project to vMNs in the lateral facial nucleus (Hattoxet al. 2002, 2003).

An example of ICMS-evoked whisking, monitored by re-cording EMG activity from the whisker pad, is shown in Fig.5A. As we reported previously, whisking frequency was posi-tively correlated with ICMS intensity (r � 0.98, P � 0.0004;Fig. 5B), enabling us to control whisking frequency in theanesthetized animal. This allowed us to investigate the rela-tionship between whisking frequency and activity in seroto-nergic premotoneurons.

Figure 6A shows the response of putative serotonergic pre-motoneuron to ICMS (50 Hz, 125 �A, 1-s duration) of vMCx.This neuron responded to vMCx stimulation with a latency of6 2 ms (Fig. 6B). Increasing ICMS intensities resulted in anincrease in the number of spikes evoked by this neuron (Fig.6C) without affecting response latency. Group data for 16putative serotonergic premotoneurons are shown in Fig. 6D,where the premotoneuron spike count and stimulus intensityhave been normalized to their threshold values. The activity inthese premotoneurons was positively correlated with the ICMSintensity (r � 0.92, P � 10�8).

The findings that ICMS intensity is positively correlatedwith whisking frequency (Fig. 5) (Cramer and Keller 2006)and that ICMS intensity is correlated also with spike counts ofputative serotonergic premotoneurons support the conjecturethat spiking output of these premotoneurons is causally relatedto whisking frequencies. We were able to test this directly forfour premotoneurons. This small sample size reflects the dif-ficulty in obtaining stable recordings under the relatively lightanesthesia necessary for ICMS-evoked whisking (Cramer andKeller 2006; Haiss and Schwarz 2005). In these neurons, werecorded vibrissal EMG simultaneously with the neuronalrecordings. The relationship between ICMS evoked activity ina representative putative serotonergic premotoneuron and thesimultaneously recorded EMG is shown in Fig. 7A. IncreasingICMS intensity increased both the number of spikes generated

by the premotoneuron and the whisking frequency, revealing apositive correlation between these latter parameters (r � 0.94,P � 0.006). Group data for all premotoneurons/EMG pairs areshown in Fig. 7B where whisking frequency and premotoneu-ron activity were normalized to their threshold values. Whisk-ing frequency was positively correlated with the level ofactivity in the putative premotoneuron (r � 0.89, P � 10�7).These findings support the hypothesis that LPGi premotoneu-rons provide the tonic serotonergic drive employed by vMNs togenerate the whisking rhythm.

D I S C U S S I O N

Using the rodent vibrissa motor system, we provided evi-dence for a novel mammalian mechanism of rhythm generationthat underlies movements. Like CPGs, this system does notrely on cortical inputs (Gao et al. 2003; Lovick 1972; Sembaand Komisaruk 1984) or sensory feedback (Welker 1964) togenerate rhythmic output. However, whereas mammalianCPGs are typically composed of networked interneurons thatgenerate the motor pattern and subsequently drive motoneu-rons, in the vibrissa system, our results suggest that rhythmicmovements are generated by the motoneurons themselves inresponse to tonic input from serotonergic premotoneurons. Theinvolvement of vMNs in the rhythmogenesis, the necessity of5-HT for generating movements, and the sufficiency of 5-HTto generate the motor pattern are unique to this mammalianmodel of motor control.

In support of the postulate that 5-HT is necessary forrhythmic whisking are our findings that 5-HT receptor antag-onists suppress both voluntary whisking (Hattox et al. 2003) aswell as ICMS-evoked whisking (Cramer and Keller 2006). Thefinding that the activity of putative 5-HT premotoneuronsco-varies with whisking frequency (Fig. 7) is also in agreementwith this postulate. Available evidence suggests that 5-HTinteractions with vMNs are also sufficient for generating rhyth-mic whisking: 5-HT (or its receptor agonists) applied at dif-ferent concentrations evoke a range of rhythmic firing rates invMNs (Hattox et al. 2003) (Fig. 1), an effect that is suppressedby 5-HT receptor antagonists (Hattox et al. 2003). Thesepharmacological manipulations are resistant to suppression ofglutamatergic and GABAergic receptors, suggesting that theseserotonergic effects are specific to vMNs. This effect appearsunique to vMNs as an earlier investigation into the effects of5-HT on facial motoneurons, which did not restrict sampling toany particular sub-nucleus, did not observe 5-HT-evokedrhythmic firing (McCall and Aghajanian 1979). Similarly,

FIG. 5. The intensity of intracortical micro-stimulation (ICMS) determines whisking fre-quency. A: example of electromyographic(EMG) activity recorded during ICMS-evokedwhisking. After a significant delay, stimulationof vMCx evoked rhythmic whisking, recordedas bursts of activity in the EMG. The small,regularly spaced spikes that occur throughoutthe stimulation are the stimulus artifacts. Notethat the EMG activity outlasts the stimulus(grey bar). B: frequency of ICMS-evokedEMG activity is positively correlated with theintensity of cortical stimulation.



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5-HT does not evoke rhythmic firing in guinea pig trigeminalmotoneurons (Hsiao et al. 1997). Thus whereas in other motorsystems the actions of 5-HT on motoneurons is described asmodulatory, capable of shaping an on-going rhythm but notresponsible for the rhythm generation itself (Heckman et al.2004), in the vibrissa motor system, 5-HT is both necessaryand sufficient for generating the whisking motor pattern andthus capable of generating rhythmic whisking.

Our results indicate that 5-HT acts through a graded facili-tation of a PIC within vMNs. PICs are voltage-dependentmembrane currents that resist inactivation (Heckman et al.2005; Schwindt and Crill 1980). In many motoneurons, includ-ing vMNs (Figs. 2 and 3) these currents are facilitated by 5-HT,frequently through the activation of 5-HT2 receptors (Harvey etal. 2006a; Heckman et al. 2005; Perrier and Hounsgaard 2003).Although these currents are considered to modulate ongoingfiring in other motoneurons, in vMNs their presence is associ-ated with the generation of rhythmic firing. We found thatconcentrations of a 5-HT2 receptor agonist that produced agraded facilitation in PIC magnitude also generated a progres-sive increase in firing rates in vMNs, suggesting a causalrelationship between the two phenomena. The action of ri-luzole on the agonist-induced firing rates supports this causalrelationship. Riluzole, which selectively antagonizes persistentsodium currents at low concentrations (2–5 �M) (Wu et al.2005) caused a similarly graded suppression in both PICmagnitude and firing rate of vMNs. Together, these datasupport the hypothesis that vMNs generate whisking rhythmsin response to serotonergic drive.

ICMS-evoked whisking displayed a relatively long onsetlatency (Fig. 7). This delay may reflect the kinetics of thetransduction cascades initiated after activation of metabotropic5-HT2 receptors on vMNs. In addition, 5-HT axon terminals inthe facial nucleus, as in other brain regions, are thought not to

form classical chemical synapses. Rather activation of 5-HTreceptors is thought to occur through the slow diffusion of5-HT and the activation of extrasynaptic receptors (De-Migueland Trueta 2005). We have recently reported that increasingICMS intensity significantly decreases whisking onset latency,most likely by increasing the output from 5-HT premotoneu-rons (Cramer and Keller 2006), consistent with the postulatethat extrasynaptic transmission contributes to the delayedwhisking. It is pertinent that the long latencies between corticalactivation and EMG onset are consistent with our previousstudies in behaving rats (Friedman et al. 2006).

The mechanisms responsible for terminating whisking re-main to be determined. Inhibitory inputs are effective inrapidly terminating PICs (Heckman et al. 2005), suggestingthat inhibitory inputs to vMNs may be involved. Indeed, wehave previously found that both the onset and offset of awhisking epoch is preceded by a brief increase in activity invMCx (Friedman et al. 2006), suggesting vMCx sends com-mands to both start and stop a whisking epoch.

Exploratory whisking occurs at frequencies between 5 and15 Hz (Berg and Kleinfeld 2003), a range that is encompassedby the agonist induced firing rates (Figs. 1 and 4). Thecorrespondence between vMN firing rates in vitro and whisk-ing frequencies in vivo suggests that during voluntary whisk-ing, vMNs fire a single action potential per whisk. Previouslywe demonstrated that some vMNs do indeed fire in a one-to-one manner during ICMS-evoked whisking (Cramer and Keller2006). Some vMNs, however, fire bursts of action potentialsper whisk. The failure to evoke a similar bursting firing patternin vMNs in vitro may result from the reduced nature of thispreparation. In particular, the partial truncation of the dendritictree, where the channels that carry PICs are thought to reside(Heckman et al. 2003), might impact the ability of the vMNs toburst in vitro. In addition, nonserotonergic inputs present in the

FIG. 6. The activity of putative serotonergic premo-toneurons is positively correlated with ICMS intensity. A:action potentials (*) evoked in a putative serotonergic pre-motoneuron by ICMS of vMCx. The premotoneuron reli-ably followed cortical stimulation (indicated by arrows).Stimulus artifacts have been truncated for clarity. B: PSTH(1-ms bins) constructed from the response of the premo-toneuron shown in A to repeated ICMS of vMCx. Thedashed grey line depicts the 95.4% confidence interval. Thisneuron responded with a latency of 6 ms. C: like thefrequency of ICMS-evoked whisking, the number of spikesevoked in the premotoneuron was positively correlated withthe stimulation intensity. D: plot of the number of evokedspikes vs. stimulus intensity for 16 putative serotonergicpremotoneurons. These 2 parameters, normalized to theirthreshold values, are positively correlated.



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intact animal but missing in vitro may be essential for burstingin vMNs.

vMNs in the lateral facial nucleus are densely innervated byserotonergic neurons, many of which receive direct projectionsfrom the vMCx (Hattox et al. 2002, 2003). The highest densityof serotonergic inputs to vMNs appears to originate from therostral (juxtafacial) LPGi (Hattox et al. 2002), and stimulationof these neurons produces vibrissa movements (Hattox et al.2003). These observations suggest that serotonergic LPGineurons are strategically placed to act as the source of 5-HTused by vMNs to generate whisking. Indeed our results indi-cate that the activity of neurons within this nucleus is positivelycorrelated with whisking frequency. However, because LPGicontains a subset of non5-HT neurons (Bellintani-Guardia etal. 1996), we cannot conclusively determine the identity of theneurons we recorded from and therefore referred to them asputative serotonergic premotoneurons.

The circuits described in the preceding text suggest that theendogenous source of 5-HT used by vMNs can be activatedand regulated by vMCx. In support of this, we found thatduring rhythmic whisking evoked by ICMS of vMCx, the

activity of putative serotonergic premotoneurons was posi-tively correlated with the whisking frequency (Fig. 7). We haveshown previously that this stimulation-evoked whisking issuppressed by 5-HT receptor antagonists (Cramer and Keller2006), further supporting the hypothesis that voluntary controlof the whisking rhythm is achieved by actions of vMCx on5-HT premotoneurons.

Additional lines of evidence support the role of vMCx inregulating whisking through a 5-HT-dependent mechanism.The activity of vMCx neurons does not co-vary with whiskingfrequency, suggesting that vMCx does not directly drive vMNs(Carvell et al. 1996). Increased vMCx activity does, however,precede both the onset of whisking and changes in whiskingkinematics (Friedman et al. 2006). These observations areconsistent with vMCx modulating the activity of a subcorticalstructure, such as serotonergic premotoneurons, to initiate andmodulate whisking. A role for the motor cortex as a coordina-tor of movement patterns is supported by studies in primates,demonstrating that the motor cortex may control higher-ordermovement parameters, such as ethologically relevant motorbehaviors, rather than activation of individual muscles ormovements (Graziano 2006).

During voluntary whisking, vibrissae often move in unison(Carvell and Simons 1990; Gao et al. 2001). Although thesynchrony between vibrissae in the same whisker pad (Sachdevet al. 2002) and bilaterally (Towal and Hartmann 2006) doesnot occur during all behaviors, the prevalence of such syn-chrony suggests the presence of underlying coordinating mech-anisms. Because the facial nucleus is thought not to containinterneurons (Courville 1966) and because facial motoneuronsdo not have axon collaterals (Fanardjian et al. 1983), unilateralsynchrony may be achieved through electrical coupling. Elec-trical coupling through gap junctions enhances synchronousactivity in neurons (Connors and Long 2004) and is importantfor generating coordinated output from motoneuron pools(Kiehn and Tresch 2002; Tresch and Kiehn 2000). The pres-ence of gap junction proteins in the facial nucleus (Rohlmannet al. 1993) further suggests that the unilateral synchronousmovements of vibrissae results from the coordinated dischargeof electrically coupled vMNs. Such a mechanism is supportedby findings in the developing mouse hindbrain, where 5-HTgenerates widespread synchronous activity that is abolished bygap junction blockers (Hunt et al. 2006). Alternatively, syn-chronous whisking, both unilateral and bilateral, may beachieved through the action of premotoneurons. These mayinclude premotoneurons in LPGi as these project bilaterally tovMNs (Hattox et al. 2002, 2003) or premotoneurons in one ofthe numerous nuclei that project to vMNs (Hattox et al. 2002).

Many of the nuclei that project to vMNs contain nonsero-tonergic neurons the role of which in the regulation of whisk-ing remains to be established (Hattox et al. 2002). Within thesenuclei may reside a more classically composed whisking CPGthat delivers rhythmic inputs to the motoneurons. vMNs alsoreceive direct, albeit sparse, inputs from the vMCx (Grinevichet al. 2005), and although vMCx is not necessary for whisking(Gao et al. 2003; Lovick 1972; Semba and Komisaruk 1984),it has been proposed that vMCx is capable of generating thewhisking rhythm itself on a cycle-by-cycle basis (Berg andKleinfeld 2003). Additionally, in some pathological statesvMCx activity is phased-locked to individual whisks (Castro-Alamancos 2006). In light of these observations, it is unlikely

A

B

FIG. 7. The activity of putative serotonergic premotoneurons is positivelycorrelated with whisking frequency. A: plot of the ICMS-evoked whiskingfrequency vs. the number of spikes evoked in a premotoneuron. The color ofeach data point corresponds to the stimulation intensity. Increasing ICMSintensity resulted in an increased output from the premotoneuron and acorresponding increase in EMG frequency. Data are shown as means SD. B:group data for 4 premotoneuron/EMG pairs. The color of each data pointrepresents the ICMS intensity. All parameters have been normalized to theirrespective values at the threshold intensity. The strong correlation between theICMS-evoked EMG frequency and output from the putative serotonergicpremotoneurons suggests the two phenomena are causally related.



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that any one mechanism operates in isolation to generate thefull range of vibrissal movements. Nevertheless, our findingssupport the hypothesis that vMNs require only serotonergicinputs to generate the whisking motor pattern. The involve-ment of vMNs in rhythmogenesis and the necessary andsufficient role of 5-HT in generating the whisking motorpattern establish this network as a novel mechanism for thegeneration of movements in mammals.

A C K N O W L E D G M E N T S

We are grateful to Drs. Danny Weinreich, Eve Marder, Phillip Zeigler, RadiMasri, and W. Friedman and M. Hemelt for critically reading the manuscript.We are also grateful to Dr. Larisa Sellers for expert technical assistance.

G R A N T S

This work was supported by National Institute of Neurological Disordersand Stroke Grants NS-35360 to A. Keller and National Research ServiceAward NS-053306 to N. P. Cramer.

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