A concerted action of L- and T-type Ca2+ channels regulates locus coeruleus pacemaking

A concerted action of L- and T-type Ca2+ channels regulates locus coeruleus pacemaking
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  A concerted action of L- and T-type Ca 2+ channels regulates locuscoeruleus pacemaking Lina A. Matschke a , Mirjam Bertoune b , Jochen Roeper c , Terrance P. Snutch f  , Wolfgang H. Oertel d,e ,Susanne Rinné a , Niels Decher a, ⁎ a Institut für Physiologie und Pathophysiologie, Abteilung Vegetative Physiologie, Universität Marburg, 35037 Marburg, Germany b Institut für Anatomie und Zellbiologie, Medizinische Zellbiologie, Universität Marburg, 35037 Marburg, Germany c Institut für Neurophysiologie, Neuroscience Center, 60590 Frankfurt, Germany d Klinik für Neurologie, Philipps Universität Marburg, 35037 Marburg, Germany e Charitable Hertie Foundation, Frankfurt/Main, Germany f  Michael Smith Laboratories, Djavad Mowafaghian Centre for Brain Health, University of British Columbia, Vancouver, BC V6T 1Z4, Canada a b s t r a c ta r t i c l e i n f o  Article history: Received 9 March 2015Revised 18 August 2015Accepted 21 August 2015Available online 25 August 2015 Keywords: Locus coeruleusL- and T-type voltage gated Ca 2+ channelsIsradipineMibefradilZ944Parkinson's disease Dysfunctionofnoradrenergiclocuscoeruleus(LC)neuronsisinvolvedinpsychiatricandneurodegenerativedis-eases and is an early hallmark of Parkinson's disease (PD). The analysis of ion channels underlying the autono-mous electrical activity of LC neurons, which is ultimately coupled to cell survival signaling pathways, can leadtoabetterunderstandingof thevulnerabilityof theseneurons.InLCneuronssomatodendriticCa 2+ oscillations,mediated by L-type Ca 2+ channels, accompany spontaneous spiking and are linked to mitochondrial oxidantstress. However, the expression and functional implication of low-threshold activated T-type Ca 2+ channels inLCneuronswerenotyetstudied.TothisendweperformedRT-PCRexpressionanalysisinLCneurons.Inaddition,weutilizedslice patchclamp recordingsofinvitrobrainstemslicesincombinationwithL-type andT-type Ca 2+ channel blockers. Wefoundtheexpression ofa distinct set of L-type and T-typeCa 2+ channel subtypesmediat-ingapronouncedlow-thresholdactivatedCa 2+ currentcomponent.Analyzingspiketrains,werevealedthatnei-ther L-type Ca 2+ channel nor T-type Ca 2+ channel blockade alone leads to a change in  fi ring properties. Incontrast, a combined application of antagonists signi fi cantly decreased the afterhyperpolarization amplitude,resulting in an increased  fi ring frequency. Hence, we report the functional expression of T-type Ca 2+ channelsin LC neurons and demonstrate their role in increasing the robustness of LC pacemaking by working in concertwith Cav1 channels.© 2015 Elsevier Inc. All rights reserved. 1. Introduction The spontaneously active noradrenergic locus coeruleus (LC) is in-volved in various adaptive behaviors and its projections innervate alarge number of brain regions (Benarroch, 2009). Dysfunction of theLCnoradrenergic systemisimpliedtodistinctpsychiatricandneurode-generative disorders, such as depression or Parkinson's disease (PD)(Berridge and Waterhouse, 2003; Gesi et al., 2000). Loss of LC neuronscorrelates with prodromal, i.e. premotor symptoms in PD, that precedeup to several decades the onset of motor symptoms (Lang, 2011). Themechanism that renders these neurons vulnerable to the pathogenesisof PD during aging still remains unclear. At present it is proposed thatactivity-dependent oxidant stress is a common feature of LC andsubstantia nigra pars compacta (SNpc) neurons. In both LC and SNpcmitochondrial oxidant stress is attributed to Ca 2+ entry through L-type Ca 2+ channels (Chan et al., 2007; Sanchez-Padilla et al., 2014).The existence of an intrinsic pacemaker mechanism in LC neuronshas been shown in various studies both in vivo (Foote et al., 1980)and in brainstem slices (Alreja and Aghajanian, 1991; Williams et al.,1984). Firing rate of LC neurons is increased in response to noxious orstressfulstimuliorduringperiodsofarousalandincreasedwakefulness(Aston-Jones and Cohen, 2005; Gompf et al., 2010; Takahashi et al.,2010). On the other hand, in certain stages of sleep or in situations of low vigilance or drowsiness  fi ring rate is decreased (Foote et al.,1980). On a cellular basis  fi ring frequency is modulated by the actionof neurotransmitters, the pH or the cAMP concentration (Alreja andAghajanian, 1991; D'Adamo et al., 2011; Murai and Akaike, 2005).Various ionic conductances, mediating inward and outward cur-rents,havebeencharacterizedinLCneurons.However,itisnoteworthythatmostofthese studies were performed intherat, whileinmice,de-spite the predominant role of these rodents for generating transgenicanimal models, LC neurons are only poorly characterized. Outward po-tassium currents described in LC neurons comprise a transient K + ( I  A ), Molecular and Cellular Neuroscience 68 (2015) 293 – 302 ⁎  Corresponding author at: Institut für Physiologie, Vegetative Physiologie, Philipps-Universität Marburg, Deutschhausstraße 1-2, 35037 Marburg, Germany. E-mail address: (N. Decher).© 2015 Elsevier Inc. All rights reserved. Contents lists available at ScienceDirect Molecular and Cellular Neuroscience  journal homepage:  apersistentK + ( I  K ),aCa 2+ activatedK + ( I  SK / I  BK ),anATPdependentK + ( I  KATP )andaninwardrecti fi erK + ( I  Kir )(MuraiandAkaike,2005;Muraiet al., 1997; Nieber et al., 1995; Osmanovi ć  and Shefner, 1993; Zhanget al., 2010). Inward currents include a tetrodotoxin (TTX)-sensitiveand-insensitiveNa + current,aninwardnon-speci fi ccationcurrentcar-ried primarily by Na + and L-, N-, P- and Q-type Ca 2+ conductances(Chieng and Bekkers, 1999; Murai and Akaike, 2005; van den Polet al., 2002; Zhang et al., 2010). The speed of the spontaneous depolar-ization during the interspike interval of mouse LC neurons was sug-gested to be primarily determined by a combination of a TTX-sensitiveNa + and a TEA-sensitive K + current(deOliveira et al.,2010). Althoughlow-threshold L-type and T-type Ca 2+ currents are known to regulatepacemaking in distinct central neurons (Deleuze et al., 2012; Guzmanetal.,2009;WolfartandRoeper,2002),thereisalackofstudiesanalyz-ingthe functional role of these channels for thespontaneous activity of LCneurons.InarecentstudybySanchez-Padillaetal.thelow-thresholdactivated L-type Ca 2+ channel Cav1.3 was shown to be expressed in LCneurons and may therefore contribute to the Ca 2+ currents phase-locked to spontaneous  fi ring. Nevertheless, a direct in fl uence on  fi ringfrequency was not found (Sanchez-Padilla et al., 2014).In the present study we performed RT-PCR expression analysis of collected LC neurons in combination with slice patch clamp experi-mentstoidentifylow-thresholdactivatedL-typeandT-typeCa 2+ chan-nels. We furthermore elucidated the potential role of these channels inthe pacemaking mechanism of spontaneously active LC neurons. Wetherefore provide an important novel insight for the understanding of LCneuronactivitywhichmightbeultimatelycoupledtothevulnerabil-ity of these neurons during degenerative diseases. 2. Results  2.1. LC neurons are autonomous pacemakers The region of the LC was  fi rst identi fi ed by immunohistochemistry,as an accumulation of tyrosine hydroxylase (TH) positive neurons atthe border of the fourth ventricle (Fig. 1A). A typical LC neuron duringapatchclamprecordingisillustratedinFig.1B.Toverifytheirnoradren-ergic nature, each neuron was  fi lled with neurobiotin (NB) duringrecording and later co-stained with an anti-TH antibody (Fig. 1C). Au-tonomousactivityofLCneuronswasrecordedinacutebrainstemslicesatroomtemperatureinthewhole-cellcon fi gurationwithglutamatergicand GABAergic blockers added to the bath (Williams et al., 1984). The fi ring frequency was 3 – 4 Hz and action potentials were broad (1.8 ±0.4 ms duration at half amplitude) with a mean overshoot of 32 ±2.4 mV (n = 23) (Fig. 1D, E), which is consistent with previous studies(Sanchez-Padilla et al., 2014). LC neurons displayed a strongafterhyperpolarization (AHP) with a mean amplitude of 32 ± 2 mV (n = 23) (Fig. 1F) reaching hyperpolarized membrane voltages of  − 70 ± 2.5 mV (n = 23) (Fig. 1G).  2.2. LC neurons display a low-threshold activated Ca  2+ conductance To elucidate whether LC neurons express functional voltage depen-dentCa 2+ channels,weinitiallyperformedwhole-cellvoltageclampre-cordings in acute brainstem slices perfused with ACSF. Voltage stepsfrom − 80 mV to +20 mV, for 500 ms, followed by repolarization to − 40 mV elicited an outward current consisting of a fast inactivatingand a persistent current component (Fig. 2A), mediated by voltage-gated potassium channels with so far unknown molecular identities.In addition, at membrane potentials of  − 60 mV to − 30 mV an inwardCa 2+ currentwithapeakamplitudeof375±68pAwasevident(n=7)(Fig. 2A, zoom-in). Plotting of the peak-current voltage relationship (I/V) (Fig. 2A, arrow) revealed that the Ca 2+ inward current had a maxi-mum at negative membrane voltages of  − 50 mV (Fig. 2B). Previousstudies reported the functional expression of low-threshold activatedCav1.3 and Cav1.2 channels in LC neurons (Imber and Putnam, 2012;Sanchez-Padilla et al., 2014). However, these channels are known tohave a maximal conductance at membrane potentials between − 30and − 10mV(Koschaketal.,2001;XuandLipscombe,2001).Althoughthese experiments were, due to the presence of K + conductances, notdesigned to isolate speci fi c Ca 2+ current components, they suggestedthe presence of low-threshold activated T-type Ca 2+ channels in LCneurons.Toprovidefurtherevidenceforsuchalow-thresholdactivatedCa 2+ conductance,wenextappliedvoltagestepsto − 40mVfrommorehyperpolarized potentials, as T-type Ca 2+ channels might be alreadyinactivated to a large fraction when holding at  − 80 mV. Using Fig.1. Locuscoeruleusneuronsareautonomouspacemakers.(A)BrainstemsectionofajuvenileC57BL/6jmouse.LCneuronsreactiveforthemonoaminergicmarkerenzymetyrosinhy-droxylase(TH)arestainedblack.(B)Patchpipette(blackarrow)approachingaLCneuron(asterisk).Scalebar:20 μ  m.(C)Neurobiotin(NB) fi lledneuronco-stainedwith Alexa488con- jugatedstreptavidin(green)andanti-TH/anti-rabbitAlexa568(magenta).DashedlinesindicatetheoutlinesofthreeTH-positiveLCneurons.Scalebars:20 μ  m.(D)ExamplerecordingofaspontaneouslyactiveLCneuroninthewhole-cellcurrentclampcon fi guration.ToisolateautonomousspikingACSFwascomplementedwithGABAergic(CGP,gabazine)andglutamatergic(AP-5,NBQX)blockers.(E – G)BasicpropertiesofLCspiking(n=23neurons):(E) fi ringfrequency,(F)afterhyperpolarization(AHP)amplitudeand(G)mostnegativepotentialreachedduring AHP (V  max  hyperpol.).294  L.A. Matschke et al. / Molecular and Cellular Neuroscience 68 (2015) 293 –  302  hyperpolarizedpre-stepsintherangeof  − 140mVto − 90mVrevealedCa 2+ currentswithincreasedamplitudes(n=8)(Fig.2C,blacktraces).Plotting the normalized peak currents at the test pulse of  − 40 mV (Fig. 2C, arrow), we derived the voltage of half-maximal inactivation(V  1/2 inact ) with − 73.9 ± 1.5 mV (n = 8) (Fig. 2D). These experimentsshow that when holding at − 80 mV about 25% of the Ca 2+ channelswere already inactivated. The simultaneous presence of activated andinactivated channels at hyperpolarized potentials is a hallmark of T-typeCa 2+ channels.This,togetherwiththehyperpolarizedV  1/2 ofinac-tivation strongly suggests that, in addition to L-type Ca 2+ channels,functional Cav3 channels might be expressed in autonomously spikingLC neurons.  2.3. LC neurons express Cav1.3, Cav1.2 and low-threshold activated Cav3channels To further investigate the idea that LC neurons functionally expresslow-voltage activated T-type Ca 2+ channels RT-PCR analysis were per-formed.Therefore,mRNAwasisolatedfromLCneuronswhichwerecol-lected out of acute brainstem slices via a patch pipette. These RT-PCR experiments con fi rmed the expression of the low-voltage activated L-type Ca 2+ channel subunit Cav1.3. In addition, we found transcriptsfor the high voltage activated L-type Ca 2+ channel subunit Cav1.2,while Cav1.1 and Cav1.4 were not detected (Fig. 3A). As a control,Cav1.1 and Cav1.4 could be ampli fi ed from murine skeletal muscle oreye, respectively (Fig. 3B). In addition, we also found expression of other high-voltage activated (Cav2) channels (Fig. 3C). Most impor-tantly, expression was also revealed for the low-threshold activated T-type Ca 2+ channel subunits Cav3.1, Cav3.2 and Cav3.3 (Fig. 3C).  2.4. Identi  fi cation of L-type and T-type Ca  2+ currents in LC neurons Next, we aimed at characterizing the relative contribution of L-typeand T-type Ca 2+ channels to the total Ca 2+ current of LC neurons. ToisolateCa 2+ currentsforfurtheranalysisweperformedwhole-cellvolt-ageclamprecordingsinabathsolutionsupplementedwithTTXtoblocksodiumcurrentsandcesiumtoblockpotassiumcurrents. Ba 2+ (2mM)was used as charge carrier to resolve small currents mediated by thesechannels.ThedihydropyridineisradipinethatblocksL-typeCa 2+ chan-nels with an IC 50  of 12 nM was used to isolate currents mediated byCav1 channels (Ito et al., 1997). In our current study we used racemicisradipine which has due to the inactive ( − ) enantiomer an IC 50  of 24 nM. The T-type Ca 2+ channel antagonist mibefradil that blocksCav3 channels with an IC 50  of 200 nM was used to isolate currents me-diated by Cav3 channels (Klugbauer et al., 1999). By stepping from aholding potential at − 80 mV to membrane potentials of  − 70 mV to+20 mV in 500 ms inward Ba 2+ currents with a peak amplitude of  − 564 ± 58 pA at − 10 mV were recorded under control conditions(n = 9) (Fig. 4A). As demonstrated in the representative trace(Fig. 4A, upper panel) and the I/V of the normalized peak currents(Fig. 4B), addition of 120 nM isradipine ( fi ve times IC 50 ) to the bathcaused a signi fi cant current reduction in the voltage range from − 20to +10 mV (n = 6). In contrast, application of 2  μ  M mibefradil didnot signi fi cantly decrease current amplitudes (n = 4) (Fig. 4A, lowerpanel and Fig. 4C). Similar results were obtained using 1  μ  M of thehighly T-type Ca 2+ channel speci fi c blocker Z944 (Tringham et al.,2012) (Fig. 4D). In our recordings performed in ACSF, we found about 25% of the Ca 2+ channels to be inactivated at a holding potential of  − 80 mV (Fig. 2D). Thus, the voltage protocol described afore with aholding potential of  − 80 mV might not be suitable to properly detect Fig.2. LCneuronsdisplayalow-thresholdactivatedCa 2+ conductance.(A)RepresentativecurrentrecordingofaLCneuronperfusedwithACSF.Theupperpartshowsthevoltageprotocol.Theinsetdepicts azoominofinward-currents thatemergefromstepstonegativepotentials.(B)Current – voltagerelationships(I/V)(n=9)derivedfrom thestartof thetest potentials(seearrowinA).Currentwasnormalizedtothehighestinwardcurrentforeachmeasurement.(C)Representativecurrentrecordingofaneuronexposedtothevoltageprotocolshownintheupperpanel.ColorsindicatetestpotentialsandcorrespondingcurrenttraceswhereCa 2+ currentsbecomeinactivated.(D)InactivationcurvecalculatedwiththeBoltzmannequation.Currentswereanalyzedatthestepto − 40mV(seearrowinC)andnormalizedtothepeakcurrent(n=8).V  1/2 inactivation(inact.)indicatesthemembranepotentialatwhichhalfofthechannels are inactivated. Fig. 3.  LC neurons express Cav1, Cav2 and Cav3 Ca 2+ channel subunits. Reverse transcription (RT) PCR expression analysis of mRNA isolated from LC neurons collected out of acutebrainstem slices. Intron-spanning primers were designed to generate PCR products of 110 base pairs. The housekeeping enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH)andthenoradrenergicmarkerenzymedopamine-beta-hydroxylase(Dbh)servedaspositivecontrols.(Cav:voltagegatedCa 2+ channel;n.c.:negativecontrol).(A)Cav1channelexpres-sion pattern in the locus coeruleus. (B) Positive controls for Cav1.1 and Cav1.4 using murine skeletal muscle or eye cDNA. (C) Cav2 and Cav3 channel expression in the locus coeruleus.295 L.A. Matschke et al. / Molecular and Cellular Neuroscience 68 (2015) 293 –  302  currents mediated by T-type Ca 2+ channels. Thus, we proceeded withrecording Ba 2+ currents following a protocol with a hyperpolarizingpre-step to  − 100 mV (see inset in Fig. 4F). Applying this protocolagain resulted in a pronounced inward current peaking near − 10 mV (Fig. 4E – H). However, after the pre-pulse to − 100 mV, we found ablock with both isradipine and mibefradil when stepping to − 30 mV (Fig. 4E versus A). Plotting the I/Vs of the normalized peak currents re-vealed that isradipine primarily reduced a Ba 2+ current component atdepolarized membrane potentials of   − 20 to +10 mV (n = 6)(Fig. 4F). In contrast, mibefradil caused a signi fi cant current reductionat more hyperpolarized membrane potentials in the range from − 40to − 20 mV. The mibefradil-sensitive difference current (gray) peakedat a membrane potential of  − 30 mV (n = 6) (Fig. 4G). In accordancewith the results obtained with mibefradil, application of Z944 also pri-marily blocked a current component at more negative membrane po-tentials (Fig. 4H).Subsequentlywedeterminedthekineticsofthedrug-sensitivecom-ponents recorded in Fig. 4E. Therefore, the currents after drug applica-tion were subtracted from control currents. These analyses revealedthat the isradipine-sensitive current (blue) inactivates slowly, whereasthe mibefradil-sensitive current (red) is fully inactivated within100 ms (Fig. 4I). In a further attempt to isolate L- and T-type Ca 2+ Fig. 4. L-andT-typemediatedCa 2+ currentscan bepharmacologicallyisolated.RecordingsofCa 2+ currents inabathsolution containing2mMBaCl 2  and1mMTTX.(A)Representativecurrenttracesderivedfromavoltagestepto − 10mVfromaholdingpotentialof  − 80mVbefore(black)andafteradding120nMisradipine(upperpanel,gray)or2 μ  Mmibefradil(lowerpanel,gray)tothebath.(B – D)I/Vsderivedfromthestartofthetestpotentials(forvoltageprotocolseeinsetinB).Currentwasnormalizedtothepeakinwardcurrentforeachmeasure-ment. Control I/Vs are shown in black, and dark gray lines display I/Vs after applying isradipine (B, n = 6), mibefradil (C, n = 4) or Z944 (D, n = 5). Light gray lines depict the I/Vs of difference currents before and after drug application. (E) Example current recordings at voltage steps to − 30 mV from a hyperpolarizing prestep to − 100 mV before (black) and afterapplication of isradipine (upper panel, gray) or mibefradil (lower panel, gray). (F) I/V of normalized peak currents derived from the voltage protocol shown in the inset before andafter isradipine application. The difference current peaked at a membrane potential of  − 10 mV (n = 6). (G – H) I/V of Ca 2+ currents before and after mibefradil or Z944 application.The mibefradil (n = 6) and Z944 (n = 5) sensitive difference currents peaked at − 30 mV (light gray). (I) Representative isradipine-sensitive (blue) and mibefradil-sensitive (red) dif-ference currents derived from E. Currents afterdrugapplication were subtracted from control currents, andsubsequentlynormalized to thepeak value.(J – K)Representativecurrent re-cordings measured by voltage ramps running from − 90 mV to +10 mV in 0.2 s. Control currents show two current components with two peaks (arrows). (J) Display of isradipinereducing both current components (n = 7). (K) Demonstration of mibefradil reducing only the low-threshold activated current component (n = 5). (A – K) *, indicates  P   b  0.05 usingWilcoxon signed-rank test.296  L.A. Matschke et al. / Molecular and Cellular Neuroscience 68 (2015) 293 –  302  channel mediated current components, we used a fast voltage rampprotocol from hyperpolarized potentials of   − 90 mV (Fig. 4 J, leftpanel). The shape of the current response under control conditions al-ready indicated the presence of a  ‘ shoulder ’  near  − 40 mV, whichcould represent low-threshold activated Ca 2+ channels, while themainpeakwasnear − 10mV(Fig.4 JandFig.4K,seearrows).Isradipine reduced both the peak current near − 10 mV and currents at morehyperpolarized potentials (n = 7) (Fig. 4 J, dark gray). Using thesevery stable voltage ramp protocols, we were able to isolate anisradipine-sensitive current component which peaked near − 40 mV (Fig. 4 J, light gray), which is consistent with a contribution of low-threshold activated L-type Ca 2+ channels to the difference current. Incontrast, mibefradil did not block the peak current near − 10 mV andselectively blocked a small current component (Fig. 4K, light gray)which activated at more hyperpolarized membrane potentials (n = 5)(Fig. 4K). Thus, the data con fi rm that both L-type and Cav3 channelscontribute to the low-threshold activated Ca 2+ conductance in LCneurons.  2.5.SimultaneousblockofL-andT-typeCa  2+ channelsincreasesspontane-ous  fi ring frequency of LC neurons To elucidate whether low-threshold activated Ca 2+ conductancesplayaroleinregulatingtheintrinsic fi ringpropertiesofjuvenileLCneu-rons,spiketrainsinthewhole-cellcurrentclampcon fi gurationwerere-corded. After 2 min of stable spontaneous spiking, control ACSF wasswitched to either isradipine, mibefradil or isradipine and mibefradilcontaining ACSF. The relative change of the different action potential(AP) parameters was analyzed 2 min later. To exclude that drug effectsare only caused by artifacts of the solution switch, control measure-ments were performedwitha switchto anothervialcontainingcontrolACSF. Application of 120 nM isradipineor 2  μ  M mibefradil did not alter fi ring frequency (Fig. 5A – B), whereas a simultaneous application of bothdrugsacceleratedAPrate(Fig.5C).Quantifyingtherelativechangeof   fi ring frequency revealed a signi fi cant, 1.6-fold, acceleration of APratecausedbysimultaneousdrugapplicationcomparedtocontrolmea-surements (Fig. 5D). An elevated  fi ring frequency can result from Fig. 5.  Simultaneous blockade of L-type and T-type Ca 2+ channels increases fi ring frequency. (A – C) Representative spike trains recorded in the whole-cell current clamp con fi guration.ACSF was supplemented with (A) 120 nM isradipine, (B) 2  μ  M mibefradil or (C) both drugs simultaneously. (D) Quanti fi cation of changes in  fi ring frequency. For each measurementthespikingfrequencyat120minafterapplicationofdrugorcontrolsolutionwassetrelativetothefrequencydirectlybeforeswitchingsolutions.(E)Relativechangesofactivationthresh-old after drug application. Threshold was de fi ned as the membrane voltage at which action potentials at higher resolution illustrate an abrupt rise. (F) Overlay of representative currentclamp recordings before(black) and aftersimultaneous isradipine/mibefradilapplication.Theinsetdisplaysthedruginduced changeoftheafterhyperpolarization amplitude (AHP) (ar-rows).(G)RelativedruginducedchangesofAHP.AconcertedblockadeofL-andT-typechannelssigni fi cantlyalleviatedAHP.(H)RelativechangeofthemostnegativepotentialreachedduringAHP(V  max hyperpol.).V  max hyperpol.wassigni fi cantlyshiftedtomorepositivemembranevoltageaftersimultaneousdrugapplication.(I)Relativechangesofspikeheight,calcu-latedfromthepeaktotheactivationthreshold(control,n=5;isradipine,n=6;mibefradil,n=7;isradipine+mibefradil,n=5).n.s.:notsigni fi cantusingunpairedStudent'st-testorMann – Whitney U test for not normally distributed data. *, indicates  P   b  0.05; **,  P   b  0.01 using unpaired Student's t-test, as these data followed a normal distribution.297 L.A. Matschke et al. / Molecular and Cellular Neuroscience 68 (2015) 293 –  302
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