The leaner P/Q-type calcium channel mutation renders cerebellar Purkinje neurons hyper-excitable and...

The leaner P ⁄Q-type calcium channel mutation renderscerebellar Purkinje neurons hyper-excitable and eliminatesCa2+-Na+ spike bursts

Saak V. Ovsepian and David D. FrielDepartment of Neurosciences, School of Medicine, Case Western Reserve University, 10900, Euclid Avenue, Cleveland, OH 44106,USA

Keywords: ataxia, calcium current, channelopathy, dendrites, development, excitability

Abstract

The leaner mouse mutation of the Cacna1a gene leads to a reduction in P-type Ca2+ current, the dominant Ca2+ current in Purkinjecells (PCs). Here, we compare the electro-responsiveness and structure of PCs from age-matched leaner and wild-type (WT) mice inpharmacological isolation from synaptic inputs in cerebellar slices. We report that compared with WT, leaner PCs exhibit lowercurrent threshold for Na+ spike firing, larger subthreshold membrane depolarization, rapid adaptation followed by complete block ofNa+ spikes upon strong depolarization, and fail to generate Ca2+-Na+ spike bursts. The Na+ spike waveforms in leaner PCs haveslower kinetics, reduced spike amplitude and afterhyperpolarization. We show that a deficit in the P-type Ca2+ current caused by theleaner mutation accounts for most but not all of the changes in mutant PC electro-responsiveness. The selective P-type Ca2+ channelblocker, x-agatoxin-IVA, eliminated differences in subthreshold membrane depolarization, adaptation of Na+ spikes upon strongcurrent-pulse stimuli, Na+ spike waveforms and Ca2+-Na+ burst activity. In contrast, a lower current threshold for eliciting repetitiveNa+ spikes in leaner PCs was still observed after blockade of the P-type Ca2+ current, suggesting secondary effects of the mutationthat render PCs hyper-excitable. Higher input resistance, reduced whole-cell capacitance and smaller dendritic size accompanied theenhanced excitability in leaner PCs, indicative of developmental retardation in these cells caused by P ⁄ Q-type Ca2+ channelmalfunction. Our data indicate that a deficit in P-type Ca2+ current leads to complex functional and structural changes in PCs,impairing their intrinsic and integrative properties.

Introduction

In mammalian neurons Ca2+ influx is mediated via several voltage-dependent calcium channels, which include five high-threshold (L, N,P, Q and R) and low-threshold (T) types (Bean, 1989; Tsien et al.,1991; Catterall, 2000). The P ⁄ Q-type Ca2+ channel mediates thepredominant fraction of high voltage-activated Ca2+ current (ICa) incerebellar Purkinje cells (PCs; Mintz et al., 1992; Mintz & Bean,1993). Largely dendritic (Tank et al., 1988; Westenbroek et al., 1995;Rancz & Hausser, 2006), the P-type current is responsible for thegeneration of Ca2+ spikes, driving Na+-Ca2+ burst firing in PCs (Llinas& Sugimori, 1980a,b; Mori et al., 2000). It also mediates Ca2+ influxduring somatic Na+ action potentials, activating big- (BK) and small-conductance (SK) Ca2+-activated K+ currents, which stabilize mem-brane depolarization and drive afterhyperpolarizing potentials (AHPs;Gruol et al., 1991; Edgerton & Reinhart, 2003). These importantP-type ICa functions determine the high vulnerability of PC physiologyto channel aberrations, with implications for cerebellar performance.Indeed, as shown recently, several neurological conditions withcerebellar symptoms in mice (Green & Sidman, 1962; Fletcher et al.,1996; Campbell et al., 1999; Katoh et al., 2007) and humans (Ophoffet al., 1996; Zhuchenko et al., 1997; Ducros et al., 1999) are linked to

inherited defects in P ⁄ Q-type Ca2+ channels. A direct relation betweenpoor motor performance and erratic PC firing in P ⁄ Q-type channelmutant mice has been shown recently (Hoebeek et al., 2005; Walteret al., 2006). Whether the abnormal cerebellar motor performance is aconsequence of compromised synaptic inputs (Matsushita et al., 2002;Zhou et al., 2003) or PC intrinsic bioelectrical properties (Edgerton &Reinhart, 2003; Womack et al., 2004), or as yet unknown changescaused by P ⁄ Q-type channel malfunctions remains to be determined.The leaner mouse carries a recessive mutation in the Cacna1a gene,

which encodes the a1A pore-forming subunit of P ⁄ Q-type Ca2+

channels (CaV2.1). As a consequence of this mutation, two abnormalsplice variants of CaV2.1 with novel carboxyl terminal tails areexpressed in homozygous leaner PCs (Fletcher et al., 1996; Doyleet al., 1997), leading to �70% reduction in P-type ICa (Dove et al.,1998; Lorenzon et al., 1998; Wakamori et al., 1998). To examine howsuch a large decline in P-type ICa affects PC electro-responsiveness,we compared depolarization-induced responses in PCs from leanerand age-matched wild-type (WT) mice in acute cerebellar slices. Wereport that leaner and WT PCs show several distinctive differences intheir firing, elicited by depolarizing current-pulse injection, whichcannot be accounted for exclusively by a reduction of P-type ICa. Ourdata indicate that in addition to immediate effects of P ⁄ Q-type Ca2+

channel malfunction on PC electro-responsiveness, a chronic deficit inthe P-type ICa caused by the mutation leads to retarded PCdevelopment, altering their passive electrical properties. The physio-logical consequences of the P ⁄ Q-type Ca2+ channel defect describedhere have been unrecognized previously and predict abnormal output

Correspondence: Dr S.V. Ovsepian, Center for Molecular and Behavioral Neuroscience,

197 University Avenue, The State University of New Jersey Rutgers, Newark, NJ 07102,USA.E-mail: [email protected]

Received 23 August 2007, revised 19 October 2007, accepted 15 November 2007

European Journal of Neuroscience, Vol. 27, pp. 93–103, 2008 doi:10.1111/j.1460-9568.2007.05998.x

ª The Authors (2007). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd

from PCs, which may contribute to the aberrant motor phenotypecharacteristic of leaner mutant mice.

Materials and methods

Animals

C57BL ⁄ 6J WT (++ ⁄ ++) and heterozygous (Os+ ⁄ +tgla) mice with aC57BL ⁄ 6J background were purchased from the Jackson Laboratory(Bar Harbor, ME, USA) and maintained at the Animal ResourceCenter at Case Western Reserve University. All animals were providedwith food and water ad libitum, and maintained at 22 ± 2 �C with a12 : 12 h light : dark cycle. Homozygous leaner (tgla+ ⁄ tgla+) micewere obtained by Os+ ⁄ +tgla inbreeding. Because Os+ ⁄ Os+ homozy-gous mice die early in embryogenesis (Green & Sidman, 1962), onlyOs+ ⁄ +tgla and tgla+ ⁄ tgla+ are born. The tgla+ ⁄ tgla+ mice(tgla ¼ leaner) were distinguished from their Os+ ⁄ +tgla littermatesbased on normal paw structure and motor disability, which becomeevident by postnatal day 10 (P10). We limited our investigation toP16–P21 mice, as within this developmental period leaner miceexpress severe motor abnormalities, yet without PC loss (Herrup &Wilczynski, 1982).

Slice preparation

Experimental procedures conformed to guidelines approved by theInstitutional Animal Care and Use Committee at Case WesternReserve University. Mice were deeply anesthetized with ketamine(150 mg ⁄ kg) and killed by decapitation. The cerebellum was rapidlyremoved and placed in ice-cold aerated (95% O2, 5% CO2) sucrose-based, high-Mg2+, low-Ca2+ artificial cerebrospinal fluid (ACSF)containing (in mm): sucrose, 75; NaCl, 85; KCl, 2.5; NaH2PO4, 1.25;NaHCO3, 25; CaCl2, 0.5; MgCl2, 4; glucose, 25 (Aghajanian &Rasmussen, 1989). Sagittal slices (300 lm thick) were cut from thecerebellar vermis on a Vibrotome Series 1000 (St Louis, MO, USA) inthe same medium and transferred to a solution with the samecomposition, except that sucrose was omitted and the NaCl concen-tration was increased to 125 mm, for 30 min incubation at 32 �Cunder continuous aeration with 95% O2, 5% CO2. Subsequently, sliceswere transferred to recording ACSF (in mm): NaCl, 125; KCl, 2.5;NaH2PO4, 1.25; NaHCO3, 25; CaCl2, 2; MgCl2, 2; glucose, 25; andmaintained at room temperature with continuous aeration (95% O2,5% CO2) until their use within 6–7 h. For recordings, individual sliceswere transferred to a chamber fixed to the microscope stage and weresuperfused with aerated ACSF at a rate of �4 mL ⁄ min.

Electrophysiological recordings

Whole-cell voltage- and current-clamp recordings (Hamill et al., 1981)were made at 32–34 �C from PCs in lobules III–V, which werevisually identified based upon their location, size and distinctivedendrites using an upright IR-DIC microscope (Leica DM-LFSA). Wefocused on the anterior cerebellar vermis (Fig. 1A) as this area exhibitsthe highest susceptibility to late-onset PC neurodegeneration in leaner(from P30–P40; Herrup & Wilczynski, 1982).For all current-clamp recordings and Ih measurements under

voltage-clamp, patch pipettes were filled with a K-methyl sulfate-based internal solution containing (in mm): KCH3O3S, 140; KCl, 10;NaCl, 5; MgATP, 2; EGTA, 0.01; HEPES, 10; pH 7.3. Voltageresponses in PC soma were monitored using an Axoclamp 2Bamplifier (Axon Instruments, Foster City, CA, USA). Bridge balance

was employed throughout experiments, and recordings were discardedif changes in series resistance (Rs) during a recording session exceeded15% of the initial value. Blockers of fast synaptic currents (kynurenicacid 5 mm; picrotoxin 200 lm) were routinely included in recordingmedia to eliminate background fast synaptic activity. Neurons weretested with depolarizing current-pulses from a hyperpolarized mem-brane potential (Vm) of approximately )65 mV, which was maintainedby injection of a steady holding current. Membrane voltages were notcorrected for liquid junction potentials. All Purkinje neurons includedin the analysis showed robust repetitive action potentials uponsufficient depolarizing current-pulse injection with spike amplitudesexceeding 55 mV. The initial firing rate (Finit) during evoked spike-trains was estimated as the mean frequency over the first five spikes.The spike-train duration was measured based on the time differencebetween the first and last spikes (Fig. 1B). For measurements of theaction potential voltage excursions (over- and undershoots, corre-sponding to spike amplitude and AHPs, respectively), a referencevoltage was defined during the upstroke where dV ⁄ dt exceeds 5 V ⁄ s.This reference voltage was also taken as a voltage-threshold for thegeneration of Na+ spikes in response to depolarizing current injection(Fig. 1C). The Na+ spike width was evaluated as the width of

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Fig. 1. Overview of PC evoked response parameters. (A) Schematic illus-tration of a cerebellar vermis in sagittal plane showing the region (lobules III–V,anterior vermis, thicker dotted line) in which PC recordings were made.(B) Somatic depolarizing current-pulses of constant length (1 s) used to evokespike-train responses. Spike-train duration was defined as the difference in timebetween the first and last action potentials. The dash preceding the spike-trainindicates )60 mV. (C) Measurement of action potential amplitude andafterhypolarizing potential (AHP) were made relative to reference ⁄ thresholdvoltage (top, dotted line), where the magnitude of dV ⁄ dt first exceeds 5 V ⁄ s(bottom, dotted line). (D) Subthreshold membrane depolarization estimatedempirically based on inflection point voltage level (VIP), where –dV ⁄ dtmagnitude is minimal (bottom) between the first and second spikes in the train(top). Note that positive deflections of –dV ⁄ dt are truncated.

94 S. V. Ovsepian and D.D. Friel

ª The Authors (2007). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing LtdEuropean Journal of Neuroscience, 27, 93–103

Na+ spike waveform at half-amplitude, measured from the samereference voltage. To estimate subthreshold Vm charging in response todepolarizing current injection (D’Angelo et al., 1998), we measuredthe voltage during the first interspike interval where the magnitude ofdV ⁄ dt is minimal (inflection point voltage, VIP; Fig. 1D).

Input resistance (Rin), membrane capacitance (Cm) and Ih weremeasured under voltage-clamp using an Axopatch 200A (AxonInstruments). To measure Rin and Cm we used hyperpolarizing voltagesteps ()10 mV) delivered at 0.2 Hz from a holding potential of)70 mV. Rin was estimated based on the change in steady membranecurrent produced by given voltage steps. Cm was measured from thechange in membrane charge, determined from the integrated capacitytransients. To improve voltage control in these experiments, we used acesium-based internal solution (in mm): CsCl, 130; MgCl2, 4.6;HEPES, 10; EGTA, 5; Na-GTP, 0.3; adjusted to pH 7.3 with CsOH.Rin and Cm were estimated at hyperpolarized potentials ()70 mV) tominimize interference from voltage-activated currents. In addition,synaptic and remaining voltage-activated currents were blocked withkynurenic acid (5 mm), picrotoxin (200 lm), tetrodotoxin (TTX,0.5 lm) and ZD7288 (10 lm), which were included in the recordingsolution. To minimize Rs voltage error, we initially measured Rs undercurrent-clamp conditions based on responses to negative current-pulses (100 pA) in cells maintained at a holding potential close to)70 mV. Then the amplifier was switched to voltage-clamp mode andRs compensation was set using the measured value. Correction was setat 85–95%. Rin was estimated based on the change in steady current atthe end of a 1-s, )10-mV voltage step.

Instead of using the capacitance compensation circuitry of theAxopatch 200A to estimate Cm, which over the developmental rangewe studied can exceed 1000 pF (Llano et al., 1991; Roth & Hausser,2001), we set the Cm compensation at 100 pF (the compensationlimit for the CV 201A head-stage used in the current study) andmeasured the uncompensated capacitance as the ratio of theintegrated capacity transients to the size of the voltage step. Thetotal capacitance was then calculated as the sum of the compensatedand uncompensated capacitance components. Prediction was setbetween 70 and 80% in an effort to achieve rapid membranepotential charging, which increased the peak and decay rate of thecapacity transients, but did not measurably alter the total chargetransferred during these transients. Whole-cell patch pipettes werepulled from borosilicate glass (WPI, Sarasota, FL, USA) and showeda tip resistance of 4–8 MW.

Morphometric measurements

Fluorescence images were used to compare size and dendritic structureof WT and tgla PCs. Cells were loaded with bis-fura-2 (K6 salt) underwhole-cell recording conditions, using patch electrodes filled withKCH3O3S-based internal solution supplemented with fluorophore(100 lm). After loading for at least 15 min, images of fluorescent PCswere acquired using a cooled charge-coupled device (12 bit,535 · 512 pixels) camera (Quantix 57, Roper Scientific) and savedfor subsequent analysis. Images provided a spatial resolution of1.3 lm ⁄ pixel and made it possible to resolve difference in fluores-cence intensity of 1 part in 4096 (0.024%). Bis-fura-2 was excited at360 ± 10 nm and fluorescence intensity was measured at510 ± 40 nm using a band-pass filter (Chroma Technology, Brattle-boro, VT, USA). Image acquisition was controlled with softwarewritten in IgorPro (Wavemetrics, Lake Oswego, OR, USA) usingSIDX camera control XOPs (Bruxton, Seattle, WA, USA). A fixedfluorescence intensity threshold was set to distinguish between cellular

and background fluorescence, and to estimate projected somatic anddendritic areas. As PC dendrites are sagitally planar (Ramon y Cajal,1995), the projected area measurements can be taken as a reliableestimate of dendritic tree size. Other parameters such as thickness andlength of the primary dendrite from the soma to the first dendriticbifurcation point, major cellular axis length, maximum width andheight of dendritic trees were estimated based on the distance betweentwo cursors.

Data analysis

Signals were filtered at 5–10 kHz, digitized at 10–20 kHz andanalysed off-line, using software written in IgorPro. Data are reportedas mean ± SE, and statistical significance was assessed using paired orunpaired Student’s two-tailed t-tests, with P < 0.05 defining asignificant difference.

Drugs

All chemicals and drugs were obtained from Sigma, except ZD7288(Tocris, Ellisville, MO, USA), x-agatoxin-IVA (Peptide International,Louisville, KT, USA) and K6 bis-fura-2 (Invitrogen, Carlsbad, CA,USA). Stocks of 100 lm x-agatoxin-IVA were prepared in dH2O andstored at )20 �C. Before use an aliquot was thawed and added tothe recording solution in order to prepare a 150 nm concentration ofx-agatoxin-IVA for bath application purposes.

Results

Depolarization induced active membrane responses in WTand leaner PCs

To evaluate the effect of the leaner mutation on intrinsic electricalproperties of PCs, we compared depolarizing current-pulse-inducedvoltage responses in WT and leaner Purkinje neurons from a negativeholding potential in pharmacological isolation from fast synapticinputs (Konnerth et al., 1990; Perkel et al., 1990). Figure 2A and Bshows representative recordings of evoked activity from normal andmutant PCs. Sufficient depolarization drives robust spike firing in bothWT and leaner Purkinje neurons. Comparison of the evoked spike-trains from WT and leaner PCs revealed several differences betweenthese two groups. First, the minimal depolarizing current required toinitiate repetitive Na+ spike firing in leaner PCs is significantlyreduced compared with WT (P < 0.05), with no differences in thevoltage threshold for initiating the spike-trains (P ‡ 0.05; Fig. 2C) andthe threshold Finit of Na+ spike-trains (P ‡ 0.05; data not shown).Second, leaner PCs responded to weak supra-threshold depolarizingcurrent injection (< 0.35 nA), with higher Finit compared with WTneurons (P < 0.05, Fig. 2D). Third, subthreshold membrane depolar-ization estimated from the inflection point voltage between the firsttwo spikes of evoked spike-trains (VIP, see Materials and methods) waslarger in leaner PCs compared with WT cells over the entire stimulusrange (P < 0.05, Fig. 2E). Fourth, upon strong depolarizing currentinjection, the interspike membrane potential level in leaner PCsbecame progressively depolarized, causing adaptation of Na+ spikesfollowed by complete block of firing and the appearance of plateaupotentials (Fig. 2B right panels, and F). Fifth, leaner PCs did notexhibit Ca2+-Na+ spike bursts, in contrast to > 90% WT PCs (10 ⁄ 11cells) that responded to strong depolarizing current injection(> 1.0 nA) by generating robust burst firing (compare Fig. 2A andB, right panels).

Leaner mutation renders PC hyper-excitable 95


Effects of x-agatoxin-IVA on depolarization-inducedresponses in WT and leaner PCs

The differences observed between WT and leaner PCs are likely to bethe result of a reduced P-type ICa in mutants. Hence, the leanerelectrophysiological phenotype should be mimicked in WT PCs bypartial blockade of the P-type Ca2+ channel. As the prevailing level ofthe P-type Ca2+ channel function in intact leaner PCs is unknown, weused an alternative approach examining whether observed differencesbetween evoked responses in leaner and WT PCs are eliminated bycomplete blockade of the P-type ICa using a saturating concentrationof x-agatoxin-IVA (150 nm; Mintz & Bean, 1993; McDonough et al.,1997).Figure 3 shows representative responses from WT and leaner PCs

(left and right, respectively) elicited by injection of weak and strongdepolarizing current-pulses before and during PC exposure to x-aga-toxin-IVA (Fig. 3, A1–A2 and B1–B2). Collected results fromx-agatoxin-IVA-treated WT and leaner PCs are summarized inFig. 3C–E. Of the five differences between WT and leaner PCsdescribed above, four were abolished by x-agatoxin-IVA. Specifically,blockade of the P-type ICa rendered similar Finit and subthresholdmembrane depolarization ofWTand leaner PCs over the entire range ofinjected currents the elicited spiking (Fig. 3C and D). x-Agatoxin-IVAalso eliminated differences in Na+ spike-train duration between thesetwo groups (Fig. 3E). Finally, as with leaner PCs, in the presence of

x-agatoxin-IVA,Ca2+-Na+ spike bursts could not be elicited inWTcells.Instead, in both cell populations, depolarizing current injection(> 0.4 nA) induced rapidly adapting Na+ spikes followed by spikefailure and appearance of plateau depolarization (Fig. 3, A2 and B2,right panels).Surprisingly, a lower current threshold for repetitive Na+ spike

discharges was still observed in leaner PCs after blockade of theP-type channel (P < 0.05; Fig. 3C). This finding indicates that anacute reduction in P-type ICa cannot account for all differencesbetween evoked voltage responses in WT and leaner Purkinjeneurons, and suggests secondary changes in mutant PCs that renderthem hyper-excitable. Of note, x-agatoxin-IVA increased the currentthreshold for initiation of Na+ spike-trains in both WT and leaner PCs(29.8% vs 43.3%, tgla and WT, respectively, P < 0.05), and slowedthe velocity of subthreshold membrane depolarization, delaying theonset of Na+ spike-trains (Fig. 3F), consistent with a depolarizingfunction of x-agatoxin-IVA-sensitive ICa in PCs, which withinsubthreshold voltage ranges accelerates PC membrane depolarization.

Comparison of Na+ spikes and spike-trains between WTand leaner PCs

We next focused upon a comparison of Na+ spikes and spike-trainsbetween leaner and WT PCs, and revisited the effect of x-agatoxin-

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Fig. 2. Comparison between electro-responsiveness of wild-type (WT) and leaner PCs. (A and B) Representative voltage responses evoked in WT and leaner PCs,respectively, by depolarizing current-pulses of increasing size (left to right). The dash preceding the spike-trains indicates )60 mV. Compared with WT (A), leanerPCs fired action potentials in response to smaller injected current (B, far left), failed to maintain high-frequency spike firing (B, center, right) and did not show slowCa2+-Na+ spike bursts (B, far right). (C) The collected results show average current and voltage thresholds for eliciting Na+ spike-trains from WT (n ¼ 11) andleaner (n ¼ 12) PCs. (D) Summary plot showing Finit vs injected current in WT (n ¼ 10) and leaner (n ¼ 10) PCs. (E and F) Collected data from the same cellpopulations showing VIP and the spike-train duration plotted vs injected current, respectively. *P < 0.05.



IVA on Na+ spikes. In order to ensure similar starting voltageconditions, spike-trains were elicited from a holding potential near)65 mV in all groups with the current-pulse intensity adjustedto trigger spike-trains with comparable Finit. Figure 4 comparesNa+ spikes and spike-trains from WT, leaner and x-agatoxin-IVA-treated WT PCs at low and high Finit.

These experiments highlighted that Na+ spike amplitude, AHP andmembrane charging ⁄ discharging rates, including the initial Na+ spikesin leaner and WT + x-agatoxin-IVA Purkinje neurons, were consid-erably reduced compared with control (WT PCs; Fig. 4, A1–C2;Table 1). Of note, at low Finit leaner and WT + x-agatoxin-IVA PCsdischarge irregularly, unlike WT, which show a regular firing pattern(Fig. 4, A1–C1; Table 1). Upon comparison of Na+ spike-trains withhigher Finit (> 150 Hz), leaner and WT + x-agatoxin-IVA PCsexhibited a prominent time-dependent increase in spike dischargerates throughout the spike-trains (28% and 19%, respectively)accompanied by a steep spike amplitude decline followed by completeblock of Na+ spike firing (Fig. 4, B3–C3). In contrast, Na+ spike-trainsin WT PCs at a comparably high Finit maintain a steady discharge rateand exhibit little or no decrease in spike amplitude during prolongedfiring (Fig. 4, A3).

Steady depolarizing potentials in WT and leaner PCs

Purkinje neurons do not fire at low rates but rather as the stimulusreaches a threshold level they fire steady repetitive Na+ spike-trains,typically at 30–50 Hz (Llinas & Sugimori, 1980a). Several currents,including Ca2+-activated K currents (IK(Ca)) contribute to this high-ratepersistent firing state (Llinas & Sugimori, 1980a; Edgerton &Reinhart, 2003; Khaliq et al., 2003). We have shown that subthresholddepolarization in leaner PCs is enhanced, which in the absence ofCa2+-Na+ spikes leads to adaptation of Na+ spike firing and theappearance of plateau potentials. To test whether the larger depolar-izing response in mutant PCs is fully attributable to reduced Ca2+-activated outward currents, we compared depolarizing plateauresponses between WT and leaner PCs in the presence ofx-agatoxin-IVA (150 nm). If the P-type Ca2+-independent current isnot changed in leaner PCs, comparable steady depolarizing potentialswould be expected in leaner and WT cells after the blockade of theP-type ICa. As can be seen in Fig. 5, x-agatoxin-IVA shifted VIP

towards more depolarized voltages and rendered an appearance ofsteady plateau responses in both WT and leaner PCs. Notably, for agiven depolarizing current-pulse stimulus the amplitude of the plateau

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Fig. 3. Blockade of P-type Ca2+ current partially eliminates differences in depolarization-induced voltage responses in wild-type (WT) and leaner PCs. (A and B)Comparison between representative voltage responses evoked in WT and leaner PCs by weak and strong depolarizing current steps (left and right, respectively)before (A1, B1) and after (A2, B2) exposure to x-agatoxin-IVA. The dash preceding the spike-trains indicates )60 mV. Note that the toxin abolished Ca2+-Na+ spikebursts, shortened Na+ spike-train duration and increased the latency to the first spike during responses to weak depolarizing current (0.4 and 0.1 nA in WT andleaner, respectively). (C and D) Collected results show Finit and subthreshold membrane depolarization in WT (n ¼ 8) and leaner (n ¼ 6) PCs treated withx-agatoxin-IVA, respectively. (E) Summary plot showing the spike-train duration vs injected current in the presence of x-agatoxin-IVA. (F) Effect of x-agatoxin-IVA (black arrow) on the subthreshold depolarization and latency to first spike in WT PC evoked by 0.4 nA current injection on an expanded scale. *P < 0.05.



response was indistinguishable between x-agatoxin-IVA treated WTand leaner PCs (P > 0.05; Fig. 5, A2 and B2).An all-or-none regimen of plateau potential operation in PCs indicates

the activation of a voltage-dependent, presumably persistent, Na+

current (Llinas & Sugimori, 1980b; Kay et al., 1998), which may alsoaffect PC subthreshold depolarization. We therefore compared thesteady depolarizing membrane responses in WT and leaner PCs afteradditional Na+ current blockade, using TTX (0.5 lm). As shown inFig. 5, A3 and B3, the steady plateau potential dropped considerablyupon addition of TTX in both groups. Notably, the overall decrease inplateau potential amplitude in leaner within the lower range of injectedcurrent (0.1–0.3 nA) was significantly smaller compared with WT PCs(P < 0.05), unmasking a larger passive depolarizing response in mutantPCs (Fig. 5C–E). Consequently, the V–I relation in mutant cells within

the lower injected current range in the presence of x-agatoxin-IVA andTTX displayed a steeper slope compared with that in WT (Fig. 5F),suggesting higher input resistance ofmutant PCs (fitted slope ofV–I, Rin:59.2 MW WT, compared with 83.8 MW leaner). The differences in V–Irelation for both leaner and WT PCs became non-linear with largerstimuli, presumably due to activation of voltage-dependent outwardrectifier K+ currents (Etzion & Grossman, 2001).

Secondary effects of reduced P-type Ca2+ current in leaner PCs

The lower current threshold for eliciting Na+ spike-trains in leanerPCs, which was still observed after blockade of P-type ICa, indicatesthat the impact of the leaner mutation on PC electro-responsivenesscannot be attributed entirely to acute reduction in P-type ICa. Indeed,

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60

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p(m

V)

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z)

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160

Time (sec)

Time (sec)

40

50

60

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V)

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z)

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Fig. 4. Comparison of Na+ spikes in wild-type(WT) and leaner PCs. (A1, B1 and C1)Representative recordings of early spike-train inWT, leaner and x-agatoxin-IVA-treated WT PCsat lower (left) and higher (right) initial firing rate(Finit). The dash preceding the spike-trainsindicates )60 mV. Note that leaner and WT +x-agatoxin-IVA PCs at higher initial firing rateshow progressive attenuation of spike amplitudefollowed by an abrupt Na+ spike failure.Corresponding Finit indicated above traces. Blackarrows specify initial Na+ spikes expanded in A2,B2 and C2 along with their negative first derivative(–dV ⁄ dt), which is offset in time for clarity.(B2 and C2) Initial spikes from leaner andWT + x-agatoxin-IVA PCs, respectively, at lowerFinit (thick line) along with WT spike forcomparison (thin line). The dash preceding spikesindicates )40 mV. Note the smaller spikeamplitude and AHP in leaner and WT +x-agatoxin-IVA PCs compared with WT cells.(A3, B3 and C3) Summary comparison betweenNa+ spike amplitude and firing rate changes in WT(n ¼ 7), leaner (n ¼ 6) and WT + x-agatoxin-I-VA PCs (n ¼ 6). Note the time-dependent changein both spike amplitude and firing rate in leanerand WT + x-agatoxin-IVA PCs compared withWT controls.

Table 1. Na+ spike parameters in WT, tgla and WT + x-aga-IVA PCs

n Peak (mV) AHP (mV) HW(ms) –dV ⁄ dt(V ⁄ s) +dV ⁄ dt(V ⁄ s) CV

WT 17 66.2 ± 3.1 8.2 ± 1.1 0.42 ± 0.04 331 ± 9 288 ± 12.1 0.09 ± 0.02tgla 13 55.1 ± 2.7* 3.8 ± 1.9* 0.64 ± 0.04* 230 ± 44* 178 ± 16.3* 0.18 ± 0.03*WT + x-Aga-IVA 6 54.2 ± 2.3* 5.1 ± 1.2* 0.54 ± 0.04* 254 ± 15* 237 ± 11* 0.17 ± 0.02*

Na+ spike amplitude and afterhyperpolarizing potential (AHP), respectively, measured from the reference voltage defined during the Na+ spike upstroke where dV ⁄ dtexceeds 5 V ⁄ s. CV, coefficient of variation of interspike intervals; –dV ⁄ dt and +dV ⁄ dt, peak magnitude of first time derivatives during upstroke and repolarization,respectively; HW, width of Na+ spike at ½ peak amplitude measured from the same reference voltage. *P < 0.05 compared with wild-type (WT).



the steeper V–I relation in leaner PCs compared with WT (Fig. 5F)after blockade of voltage-activated Na+ and P-type Ca2+ currents indi-cates their higher Rin, which would render these cells hyper-excitable.To identify the underlying mechanisms contributing to differences inRin between these two groups, we first compared background currentsin WT and leaner PCs at hyperpolarized voltages. Experiments wereconducted in the presence of Ih and fast synaptic current blockers(10 lm ZD7288, 0.5 lm TTX, 5 mm kynurenic acid and 200 lm

picrotoxin). Figure 6A illustrates representative current responses

elicited by )10 mV hyperpolarizing voltage-steps from a holdingpotential of )70 mV. Steady current responses evoked by 1-s voltagesteps were used to measure Rin, while Cm was estimated based on theintegrated capacity transient (see Materials and methods). Theseexperiments revealed significantly larger Rin in leaner PCs comparedwith WT (363 ± 28 MW vs 249 ± 23 MW, leaner n ¼ 12, and WTn ¼ 12, respectively, P < 0.05), which corresponds to 31.5 ± 5%smaller input conductance Gin (¼ 1 ⁄ Rin) in mutants. Cm was similarlyreduced in leaner PCs (n ¼ 12) compared with normal PCs (n ¼ 12;

ControlControl

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TT

X (

ΔV

m,

%)

100

75

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*

EWT tg la

Fig. 5. Steady depolarizing potentials in wild-type (WT) and leaner PCs. (A and B) Typical Na+ spike-trains from WT and leaner PCs elicited by low-rangedepolarizing current-pulse injection under: (1) control conditions; (2) after blockade of P-type ICa by +x-agatoxin-IVA; and (3) subsequent addition of tetrodotoxin(TTX). Superimposed traces (bottom) compare current-pulse-induced responses in WT and leaner PCs after blockade of P-type current and after further blockade ofNa+ current (2 + 3). Note the upward shift of VIP in both WT and leaner PCs upon blockade of P-type ICa by x-agatoxin-IVA and a greater decrease in steadydepolarizing potential in WT PCs upon TTX application (black arrows). The dash preceding the spike-trains indicates )60 mV. (C and D) Representative recordingsof depolarizing current-pulse-evoked responses of increasing size from WT and leaner PCs in the presence of x-agatoxin-IVA and TTX. Note larger depolarizingmembrane responses in leaner PCs. (E) Collected results comparing the TTX-sensitive steady depolarizing potentials from WT (n ¼ 7) and tgla (n ¼ 5) PCs in thepresence of x-agatoxin-IVA (0.4 nA depolarizing current-pulse). The results are expressed relative to the TTX-sensitive component in WT PCs. (F) V–I relations inWT (n ¼ 6) and leaner (n ¼ 5) PCs in the presence of x-agatoxin-IVA and TTX (average voltage over the last 100 ms of the current-pulse stimuli). *P < 0.05.



487 ± 72 pF vs 770 ± 84 pF; leaner and WT, respectively, P < 0.05;37 ± 6% reduction; Fig. 6B). Given that previous studies of disso-ciated PC soma reported no difference in Cm between leaner and WTPCs (Dove et al., 1998; Wakamori et al., 1998), it is likely that thesmaller Cm and higher Rin in intact leaner PCs in slices is due to theirundersized dendrites. Consistent with this, we found that Ih, which inPCs is predominantly dendritic (Raman & Bean, 1999; Williams et al.,2002), is significantly smaller in leaner PCs compared with WTneurons (P < 0.05; Fig. 6C and D).To evaluate differences in dendritic size and structure between WT

and leaner PCs, we compared PC projected area and dendriticmorphology using fluorescence microscopy. Figure 7A and B illustratestypical images of WTand leaner Purkinje neurons at a comparable age.Table 2 summarizes collected data characterizing morphometricparameters of WT (n ¼ 13) and leaner (n ¼ 10) PCs. While nosignificant difference was revealed when somatic areas were comparedbetween these two groups (P > 0.05), the whole-cell projected areas ofleaner PCs were significantly reduced (�32%; P < 0.05), consistentwith their undersized dendrites (Fig. 7C andD; Table 2). The decrease indendritic area was accompanied by an atrophic appearance of first- andsecond-order PC dendrites as well as poor development of third- andhigher-order dendritic branches (Fig. 7A and B).

Discussion

The leaner mouse mutation occurs in the Cacna1a gene, whichencodes the a1A pore-forming subunit of P ⁄ Q-type Ca2+ channels(Fletcher et al., 1996). This mutation causes a �70% decrease in the

P-type Ca2+ current in dissociated cerebellar PCs (Dove et al., 1998;Lorenzon et al., 1998; Wakamori et al., 1998). The present study is thefirst to address the impact of the leaner mutation on PC electricalproperties and dendritic morphology. Analysis of the effects ofx-agatoxin-IVA, a potent and selective P-type channel blocker, oncurrent-pulse-evoked responses showed that the P-type ICa only partlyaccounts for the observed electrophysiological differences betweenWT and mutant Purkinje neurons. Comparison of PC structurebetween these two groups revealed that the dendrites in mutantneurons were considerably undersized, which most likely accounts forx-agatoxin-IVA-resistant residual difference in electro-responsivenessbetween these two groups of neurons.

Differences that can be attributed to an acute reductionin P-type Ca2+ current

Compared with WT PCs, leaner cells fire Na+ spikes at higher rates inresponse to weak current-pulse stimuli, show larger subthresholdmembrane depolarization, robust attenuation of Na+ spike amplitudefollowed by spike failure, and do not generate Ca2+-Na+ spike bursts.Na+ spike waveforms in leaner PCs exhibited slower kinetics, smalleramplitude and AHP. These distinctive mutant PC electrophysiologicalfeatures, largely mimicked in WT neurons upon treatment withx-agatoxin-IVA, are expected consequences of reduced ICa and IK(Ca).Larger subthreshold membrane depolarization in response to current-pulse stimuli in leaner PCs provides evidence in nature (i.e. withoutthe use of pharmacological manipulations) for the net hyperpolarizingeffect of Ca2+-dependent currents in intact Purkinje neurons, shown

-70 mV -70 mV

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*WT tg la

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Gm

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m (

%)

100

125

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WT tg la

Fig. 6. Comparison of Ileak, Cm and Ih between wild-type (WT) and leaner PCs. (A) Leak (Ileak) and capacity (Cm) currents elicited by 10-mV hyperpolarizingvoltage steps from a holding potential of )70 mV in WT (left), leaner (center), and WT and leaner superimposed (right). The small, sustained inward current thatoutlasted the capacity transient was taken as a measure of the Ileak. Cm was estimated by integration of capacity transient. (B) Collected results showing smallerwhole-cell membrane conductance (1 ⁄ Rin, left) and Cm (right) in WT and tgla (n ¼ 12, n ¼ 12, respectively) PCs. Measurements were made after 15 min treatmentwith kynurenic acid, picrotoxin and ZD7288. (C) Family of Ih currents (lower traces) elicited by hyperpolarizing voltage steps (1 s) in WT (left) and leaner (right)PCs. The Ih was estimated by subtraction of the ZD7288-insensitive component from total current. Holding potential: )70 mV; voltage increments: 5 mV.(D) ‘Steady-state’ Ih activation curves. Measurements represent the average ZD7288-sensitive current over the last 100 ms of the voltage step in WT (n ¼ 7) andleaner (n ¼ 7) PCs. *P < 0.05.



previously through pharmacological means (Llinas & Sugimori,1980a; Crepel & Penit-Soria, 1986; Raman & Bean, 1999). As PCsexpress IK(Ca) (Gruol et al., 1991; Stocker & Pedarzani, 2000;Cingolani et al., 2002), which regulate the interspike voltage duringrepetitive Na+ action potential discharges (Edgerton & Reinhart,2003), a decrease in this stabilizing outward IK in mutants wouldpromote interspike voltage depolarization, affecting Na+ spikefrequency and amplitude during prolonged spike-trains. The predom-

inant expression of P-type ICa in PC dendrites and their activecontribution to the generation of dendritic Ca2+ spikes (Llinas &Sugimori, 1980b; McKay & Turner, 2004) predicts impairedCa2+ ⁄ Na+ spike bursts, which are completely eliminated in leanerPCs. We suggest that reduced IK(Ca) and lack of Ca2+ spikes in mutantPCs lead to rapid depolarization block and Na+ spike failure uponlarge current-pulse injection. A similar phenotypic change has beenreported in Rolling Nagoya, another CaV2.1 mutant mouse strain(Mori et al., 2000). Remarkably, tottering PCs, that carry a mutation inthe same Cacna1a gene (Fletcher et al., 1996), show fully preservedCa2+ ⁄ Na+ spike bursts (S.V. Ovsepian, J.S. Stahl and D.D. Friel,unpublished data). These findings are in agreement with previouswork, demonstrating that the P-type current density in tottering PCs ismodestly reduced, compared with that in leaner and Rolling Nagoya(Wakamori et al., 1998; Mori et al., 2000).As P-type ICa regulates BK and SK IK(Ca) in PCs (Womack &

Khodakhah, 2002; Edgerton & Reinhart, 2003; Womack et al., 2004),a deficit in this channel function would affect Na+ spike length andAHPs. Decline in Na+ spike amplitude and slower upstroke in mutantPCs, including the first action potential in evoked spike-trains, weresurprising. While several earlier reports showed a reduction in Na+

spike amplitude in PCs upon blockade of Ca2+ currents (Llinas &Sugimori, 1980a; Hounsgaard & Midtgaard, 1988; Edgerton &Reinhart, 2003), the underlying mechanisms of this effect remainunclear. It seems unlikely that the reduction in the first Na+ spikeamplitude during spike-trains in WT PCs upon blockade of P-typeCa2+ current is due to changes in Na+ channel functions, given thatx-agatoxin-IVA is a highly selective blocker for the P-type Ca2+

current (Mintz & Bean, 1993; McDonough et al., 1997). Rather, areduced first spike amplitude in both leaner and x-agatoxin-IVA-treated WT PCs during spike-trains indicates the active contribution ofP-type Ca2+ current in the generation of Na+-dependent spikes. Thefact that x-agatoxin-IVA slows down the action potential upstroke(dV ⁄ dt) and subthreshold membrane depolarization in PCs is consis-tent with a depolarizing role of ICa during the generation of Na+ spikesin these neurons. Notably, similar effects of ICa on Na+-dependentspikes have been proposed in cerebellar granule cells (D’Angelo et al.,1998), midbrain dopamine neurons (Puopolo et al., 2007) and cardiacsinoatrial node pace-making cells (Sanders et al., 2006).

Differences arising from chronic defects in P ⁄ Q-typeCa2+ channel activity

The lower current threshold for eliciting Na+ spikes from negativeholding potentials in leaner PCs was unexpected, given the highactivation voltage for P-type ICa (Llinas et al., 1989; Mintz et al., 1992;Mintz & Bean, 1993). The enhanced responsiveness of leaner PCs todepolarizing current-pulse stimuli is unlikely to be an immediateconsequence of reduced P ⁄ Q-type Ca2+ channel function as it was alsoobserved in the presence of x-agatoxin-IVA. This implicates secondary

BA

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WT tg la

0.00 0.2 0.4

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tic A

rea (

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20 μm20 μm

10 μm

D

Whole

-cell

Are

a (

%)

0

100

125

50

25

75

*

Fig. 7. Morphometric characteristics of wild-type (WT) and leaner PCs.(A and B) Fluorescence images of typical WT and leaner PCs loaded with bis-fura-2 (100 lm) through the patch pipette. Note the less expanded dendritictrees with smaller dendritic branching and atrophic appearance of leanerdendrites compared with WT. (C) Estimated projected area of the PC dendrites(dendritic size) plotted against PC projected somatic area (somatic size). (D)Summary histogram of whole-cell projected area of WT (n ¼ 13) and leaner(n ¼ 10) PCs. *P < 0.05.

Table 2. Morphometric characteristics of WT and tgla PCs

nSPA(lm2)

WC PA(lm2)

MaxW(lm)

MaxH(lm)

PDL(lm)

WT 13 305.1 ± 20 4470.8 ± 336 119.8 ± 6 119.6 ± 5 22.9 ± 2tgla 10 245.1 ± 23 2748.3 ± 310* 73.3 ± 5* 80.1 ± 6* 18.4 ± 3

MaxW and MaxH, maximal width and height of Purkinje neuron dendritic tree;PDL, the primary dendritic length from the soma to the first bifurcation point;SPA, somatic projected area; WC PA, whole cell projected area.*P < 0.05 compared with WT.



changes in leaner Purkinje neurons, perhaps due to chronic deficiency inP-type ICa that ultimately render mutant PCs hyper-excitable.As we have shown, mutant PCs exhibit higher Rin, which may

account for enhanced PC excitability. We examined the potentialsource of higher Rin in mutant PCs at negative voltages, by comparingIh and Ileak currents between WT and mutant PCs, given that these arethe dominant currents in Purkinje neurons at hyperpolarized voltages(Llano et al., 1991; Williams et al., 2002). Both Ih and Ileak wereconsiderably reduced in mutants compared with WT PCs. Althoughour experiments cannot exclude the possibility of altered PC Rin due toreduced Ih and Ileak channel density, the smaller whole-cell Cm anddecreased whole-cell projected area found in mutant PCs suggests thatthe higher Rin is a consequence of reduced cell size. Remarkably, thedifference in the whole-cell projected area between WT and leanerPCs was predominantly due to undersized dendrites in mutants. This isin line with several previous reports, which indicate the importance ofP-type Ca2+ channel function in regulating PC growth and dendriticpatterning (Rhyu et al., 1999; Zwingman et al., 2001; Miyazaki et al.,2004). Given the rapid expansion of PC dendrites in the juvenilecerebellum (McKay & Turner, 2005), abnormal P ⁄ Q-type Ca2+

channel function in leaner most likely results in developmentalaberrations, ultimately affecting PC passive properties that account forthe enhanced electro-responsiveness of mutant PCs. Importantly, inagreement with previous reports showing similar Cm in WT and leanerdissociated PC somata (Dove et al., 1998; Wakamori et al., 1998), thePC somatic projected areas in these two groups were not distinguish-able in the current study.

Implications for cerebellar function

We have shown that a defect in P ⁄ Q-type Ca2+ channel functioncaused by the leaner mutation renders PCs hyper-excitable andeliminates Ca2+-Na+ spike bursts. In keeping with the central positionthat PCs occupy in the cerebellum, alterations in their intrinsicelectrophysiological properties would profoundly affect cerebellarcomputation and motor control.From the perspective that PC intrinsic activity encodes time-variant

information relayed via synaptic inputs, the enhanced responsivenessof these neurons will degrade their firing precision and impairintegration of synaptic inputs, most effectively within low, physio-logical activation ranges (< 300 pA; Konnerth et al., 1990; Hausser &Clark, 1997; Walter & Khodakhah, 2006). A higher Rin would convertsubthreshold synaptic noise into signal, resulting in reduction ofsignal-to-noise ratio, rendering PC intrinsic firing erratic and compu-tation unreliable. The absence of Ca2+-Na+ spikes upon strongdepolarizing current-pulse injection (> 500 pA, equivalent to activa-tion of climbing fiber input), the second major defect in PC intrinsicelectrical properties caused by the leaner mutation, would also impaircerebellar functioning, as dendritic Ca2+ spikes are presumed to beinvolved in complex climbing fiber response generation (Schmoleskyet al., 2002; McKay et al., 2007). Given that Ca2+ influx duringdendritic spikes plays an essential role in governing PC slow voltagedynamics (Cerminara & Rawson, 2004; McKay et al., 2007), synapticinput integration and plasticity (Ito, 2001; Evans, 2007), the lack ofCa2+-Na+ spikes would also impair these essential calcium-dependentprocesses in PCs.In conclusion, the present study is the first to address the impact of

the CaV2.1 leaner mutation on PC intrinsic properties. We show thatP ⁄ Q-type Ca2+ channel dysfunction affects Purkinje neurons in acomplex manner, involving their electro-responsiveness and structuraldevelopment. Changes in PC intrinsic functions that are described hereneed to be considered along with compromised synaptic functions in

order to fully comprehend the importance of P-type ICa for normal PCphysiology and development of neurological conditions of cerebellarorigin.

Acknowledgements

This work was supported by a grant from the National Institutes ofHealth ⁄ National Institute of Neurological Disorders and Stroke (NS 33514)to Dr David D. Friel. We thank Dr B. Strowbridge and Prof. Karl Herrup forhelpful discussions. S.O. is indebted to Dr V.B. O’Leary and Dr P. Chaddertonfor reading this manuscript in its preliminary form.

Abbreviations

ACSF, artificial cerebrospinal fluid; AHP, afterhyperpolarizing potential; BK,big conductance; P, postnatal day; PC, Purkinje cell; SK, small conductance;TTX, tetrodotoxin; WT, wild-type.

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The leaner P/Q-type calcium channel mutation renders cerebellar Purkinje neurons hyper-excitable and...

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