Muscarinic Enhancement of Persistent Sodium Current Synchronizes Striatal Medium

doi: 10.1152/jn.00455.200594:3771-3787, 2005. First published 24 August 2005;J Neurophysiol

Enrique Perez-Garci, Elvira Galarraga and Jose BargasHumberto Salgado, Fatuel Tecuapetla, Tamara Perez-Rosello, Azucena Perez-Burgos,Neostriatal Neurons

Modulation in Developing2Its Dopaminergic D Channel Current and2+2 CaVA Reconfiguration of Ca

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A Reconfiguration of CaV2 Ca2� Channel Current and Its Dopaminergic D2

Modulation in Developing Neostriatal Neurons

Humberto Salgado, Fatuel Tecuapetla, Tamara Perez-Rosello, Azucena Perez-Burgos, Enrique Perez-Garci,Elvira Galarraga, and Jose BargasDepartamento de Biofısica, Instituto de Fisiologıa Celular, Universidad Nacional Autonoma de Mexico, Mexico City, Mexico

Submitted 3 May 2005; accepted in final form 17 August 2005

Salgado, Humberto, Fatuel Tecuapetla, Tamara Perez-Rosello,Azucena Perez-Burgos, Enrique Perez-Garci, Elvira Galarraga,and Jose Bargas. A reconfiguration of CaV2 Ca2� channels currentand its dopaminergic D2 modulation in developing neostriatalneurons. J Neurophysiol 94: 3771–3787, 2005. First published August24, 2005; doi:10.1152/jn.00455.2005. The modulatory effect of D2

dopamine receptor activation on calcium currents was studied inneostriatal projection neurons at two stages of rat development:postnatal day (PD)14 and PD40. D2-class receptor agonists reducedwhole cell calcium currents by about 35% at both stages, and thiseffect was blocked by the D2 receptor antagonist sulpiride. Nitrendi-pine partially occluded this modulation at both stages, indicating thatmodulation of CaV1 channels was present throughout this develop-mental interval. Nevertheless, modulation of CaV1 channels wassignificantly larger in PD40 neurons. �-Conotoxin GVIA occludedmost of the Ca2� current modulation in PD14 neurons. However, thisocclusion was greatly decreased in PD40 neurons. �-Agatoxin TKoccluded a great part of the modulation in PD40 neurons but had anegligible effect in PD14 neurons. The data indicate that dopaminer-gic D2-mediated modulation undergoes a change in target duringdevelopment: from CaV2.2 to CaV2.1 Ca2� channels. This changeoccurred while CaV2.2 channels were being down-regulated andCaV2.1 channels were being up-regulated. Presynaptic modulationmediated by D2 receptors reflected these changes; CaV2.2 type chan-nels were used for release in young animals but very little in matureanimals, suggesting that changes took place simultaneously at thesomatodendritic and the synaptic membranes.

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

Neostriatal projection neurons express a diverse array ofvoltage-gated Ca2� channels (Bargas et al. 1994). The differenttypes of Ca2� channels exhibit striking dissimilar roles duringrepetitive firing and transmitter release (Perez-Garci et al.2003; Tecuapetla et al. 2005; Vilchis et al. 2000). For example,L-type Ca2� (CaV1) channels activate near spike threshold andcontribute to set the range of frequencies for evoked dis-charge—the dynamic range (Hernandez-Lopez et al. 1997,2000; Perez-Garci et al. 2003). By this token, dopaminergic D1receptor activation facilitates neuronal excitability by enhanc-ing Ca2� current through L-type Ca2� channels (Hernandez-Lopez et al. 1997; Surmeier et al. 1995). Activation of dopa-minergic D2 receptors reduces L-type Ca2� currents, leading toa decrease in firing (Hernandez-Lopez et al. 2000; Olson et al.2005). These actions partially explain the so-called facilitatoryand repressing actions of dopamine on striatal output at themolecular level (Prescott et al. 2003).

However, not only CaV1 Ca2� channels but also CaV2 Ca2�

channels may be controlled by dopamine. Consequently, thisstudy asked if D2 receptor activation also regulates CaV2 Ca2�

channels in striatal neurons. The dopaminergic modulation ofCaV2 Ca2� channels may be important given the roles of thesechannels in spiny cells (Perez-Garci et al. 2003; Tecuapetla etal. 2005; Vilchis et al. 2000). Moreover, because the contribu-tion of diverse CaV2 Ca2� channels may change during devel-opment (Chameau et al. 1999), and because medium spinyneurons have a protracted development (Tepper et al. 1998)with respect to other neurons, it is important to see whathappens with dopaminergic modulation during possible chan-nel reconfigurations.

The relevance of this question becomes evident whenrecalling that CaV2 channels supply the Ca2� necessary toactivate Ca2�-dependent K� channels (Vilchis et al. 2000)in mature neurons. In turn, these K� channels generate thepostspike afterhyperpolarization (AHP) that makes up theinterspike interval. The AHP controls the firing of spinycells by setting the gain for a given stimulus (Bargas et al.1999; Perez-Garci et al. 2003; Pineda et al. 1992; Vilchis etal. 2000). In addition, CaV2 channels are in charge of GABArelease at the synaptic terminals of spiny neurons (Tecu-apetla et al. 2005). In this place too, there is evidence thatdopamine exerts a presynaptic modulation of GABA release(Guzman et al. 2003). Thus theoretically, dopamine couldcontrol neostriatal output at two different levels through theregulation of CaV2 Ca2� channels: first, at the somatoden-dritic membrane through the regulation of the firing mech-anism, and second, at the synaptic terminals (Cooper andStanford 2001; Guzman et al. 2003) through the regulationof transmitter release. Accordingly, the main objective ofthis study was to find evidence of this dual regulation. Inrelation to this, it was recently shown that activation ofmuscarinic M1 receptors controls both the interspike intervaland the transmitter release at the synaptic terminals of spinyneurons (Perez-Rosello et al. 2005). Because muscarinic M1

receptors and dopaminergic D2 receptors seem to be linked tothe same signaling cascade (Hernandez-Lopez et al. 2000;Rakhilin et al. 2004), it seems interesting to ask if thesereceptors share some physiological actions in neostriatal cells,and if not, why. This work has been published in abstract form(Salgado et al. 2004).

Address for reprint requests and other correspondence: J. Bargas, Institutode Fisiologıa Celular, UNAM, Mexico City DF 04510, Mexico (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 94: 3771–3787, 2005.First published August 24, 2005; doi:10.1152/jn.00455.2005.

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M E T H O D S

Preparation of slices

The protocols followed the National University of Mexico andNational Institutes of Health guidelines for the use of animals inbiomedical experiments. Briefly, and as described elsewhere (Bargaset al. 1999), male Wistar rats of postnatal days 14 (PD14) and 40(PD40), from our animal house, were deeply anesthetized, and theirbrains were quickly removed into ice-cold saline (4°C) containing (inmM) 126 NaCl, 3 KCl, 1 MgCl2, 2 CaCl2, 25 NaHCO3, and 11glucose (pH 7.4 with NaOH, 298 mOsm/l with glucose; aerated with95% O2-5% CO2). Parasagittal neostriatal slices (300 �m thick) werecut in 4°C saline using a vibratome (Ted Pella, Reading, CA). Sliceswere transferred to room temperature saline (23–25°C) and left torecover for 1 h.

Voltage-clamp recordings of calcium currents

Neostriatal neurons from PD14 or PD40 rats were acutely dissoci-ated using procedures similar to those previously described (Bargas etal. 1999). Briefly, after a 1- to 6-h incubation, slices were removedinto HEPES-buffered saline, and the dorsal striatum was dissected.Neostriatal slices were placed in the same HEPES solution, nowcontaining 1 mg/ml of pronase E type XIV (Sigma, St. Louis, MO) at32°C. After about 20 min, the slices were removed into a low-Ca2�

HEPES-buffered saline. Slices were rinsed and dissociated mechani-cally with a graded series of fire-polished Pasteur pipettes. The cellsuspension (2 ml) was plated into a 35-mm Lux petri dish mounted onthe stage of an inverted microscope containing 1 ml of the followingrecording saline (in mM): 0.001 TTX, 140 NaCl, 3 KCl, 5 BaCl2, 2MgCl2, 10 HEPES, and 10 glucose (pH 7.4 with NaOH; 298 mOsm/lwith glucose). After allowing the cells to settle, superfusion began atabout 1 ml/min with saline of the same composition. Recordings weremade only from medium-sized neurons (10–12 �m soma diam).Whole cell currents recordings used standard techniques. Electrodeswere pulled from borosilicate glass (WPI, Sarasota, FL) in a Flaming-Brown puller (Sutter Instrument Corp., Novato, CA) and fire polishedbefore use. The internal saline contained (in mM) 180 N-methyl-D-glucamine (NMDG), 40 HEPES, 4 MgCl2, 10 EGTA, 2 Na2ATP, 0.2Na3GTP, and 0.1 leupeptin (pH � 7.2 with H2SO4, 265–270 mOsm/l). Electrode DC resistances were 4–8 M� in the bath. Recordingswere obtained with an Axopatch-1D patch-clamp amplifier (AxonInstruments, Foster City, CA), and controlled and monitored with aPC clone running pClamp (version 5) with a 125-kHz DMA interface(Axon Instruments). After seal rupture, the series resistance (�15M�) was compensated (70–80%) and monitored before and afterdrug application. Current-voltage relationships (I-V plots) before andduring drug applications were built from currents evoked with both20-ms step voltage commands, from –80 to 50 mV in 10-mV steps,and with responses to voltage ramp commands (0.7 mV/ms) from –80to 50 mV. Because the results from both methods coincided (see Fig.1), most figures only show representative I-V plots built from theresponses to voltage ramps.

In addition, a double pulse protocol was performed to explore thevoltage dependency of the action of D2-class receptor activation (Fig.8E). Voltage commands to 0 mV were given in pairs to compareevoked Ca2� current before and after a depolarizing command to 80mV. This command preceded the second pulse of a pair to 0 mV.Drugs were applied with a gravity-fed system that positioned a glasscapillary tube 100 �m from the recorded cell in the direction of flowsuperfusion. This method allowed reversible drug applications (seeFig. 5). Solution changes were performed with a DC-controlledmicrovalve system (Lee Co., Essex, CT).

Voltage-clamp recordings of synaptic currents

Neostriatal slices from juvenile (PD14) or mature young rats(PD40) were transferred to a custom plexiglass recording chamber

and superfused with oxygenated saline (3–6 ml/min) as above. Indi-vidual neurons were visualized (�40 water immersion objective)under differential interference contrast (DIC) enhanced visual guid-ance using infrared videomicroscopy in an upright microscope (Dia-phot, Nikon, Melville, NY) adapted with a CCD camera (CCD-100,Dage-MTI, Michigan City, IN). Synaptic events were evoked with abipolar concentric tungsten electrode (12 �m at the “pencil shaped”tip; FHC, Bowdoinham, ME) located at the globus pallidus to stim-ulate antidromically the axons of spiny cells (Guzman et al. 2003;Tecuapetla et al. 2005). Paired shock stimulation (45–50 ms ofinterstimulus interval; 0.2- to 0.4-ms duration; 1- to 4-V strength;frequency of 0.1 Hz) was delivered with a computer interface. Isola-tion units (Digitimer, Hertfordshire, UK) between the computer andthe stimulating electrodes were used to adjust stimulus parametersduring the experiment. Distance between recording and stimulatingelectrode was about 1 mm. Synaptic responses in these conditionswere of moderate amplitude, but some experiments were performedwith either strong or weak stimulation strength to check if D2-classreceptor actions were present. Thus evoked inhibitory postsynapticcurrents (eIPSCs) had amplitude variation but without exhibitingfailures except at the weaker stimulation strengths. Traces shown arethe average of �4-min recordings (24 traces) taken once the ampli-tude had been stabilized in a given condition. A hyperpolarizingvoltage command (10 mV) continuously monitored input conduc-tance. Internal solution was (in mM) 72 KH2PO4, 36 KCl, 2 Mg Cl2,10 HEPES, 1.1 EGTA, 2 Na2ATP, 0.2 Na3GTP, 5 QX 314 (to preventneuronal firing and enhance input resistance), and 0.5% biocytin(pH � 7.2, 275 mOsM/l). Bath solution contained 10 �M 6-cyano-2,3-dihydroxy-7-nitro-quinoxaline disodium salt (CNQX) and 50 �MD-(�)-2-amino-5 phosphonovaleric acid (AP5) to block glutamatergiccurrents so that synaptic responses were eIPSC sensitive to bicucul-line. Cells with resting potential more negative than –70 mV (at 0current), input resistance about 100–200 M�, and holding current (involtage clamp mode) �0.02 nA to maintain a holding potential nearthe resting potential of the cell were chosen. Whole cell recordingswere made using an Axoclamp 2B amplifier (Axon Instruments).Whole cell access resistances were in the range of 5–20 M�. Accessresistance was continuously monitored, and experiments were aban-doned if changes �20% were encountered. No cell capacitance, seriesresistance, or liquid junction potential (2 mV) compensations weremade. All recordings were filtered at 1–3 KHz and digitized with anAT-MIO-6040E interface and a DAQ (NI-DAQ) board (NationalInstruments, Austin, TX) in a PC clone. On-line data acquisition usedcustom programs made by L. Carrillo in the LabVIEW environment(National Instruments). The NI-DAQ board was used to save the dataon binary files in the computer hard disk for further off-line analysis.

eIPSCs obtained with different simulation strengths (weak tostrong; see intensity-amplitude plots in Tecuapetla et al. 2005) anddifferent ages were chosen to have an array of different amplitudes foreIPSCs. These were used to perform a mean-variance analysis asdescribed in Clements and Silver (2000).

Intracellular recordings

Slices obtained as above (see Preparation of slices), from PD40rats, were also recorded in a submerged chamber and superfused withthe same saline at 1 ml/min (34–36°C). Intracellular recordings wereperformed with sharp microelectrodes filled with 3 M K-acetate (DCresistances: 80–120 M�) and the help of an active bridge electrom-eter (Neuro Data, Cygnus Tech, DWG) (Pineda et al. 1992). Recordswere digitized and saved on VHS tapes (40 KHz) and analyzedoff-line in a PC clone. Stimulation consisted of intracellular injectionsof constant current steps to evoke the AHP after a single actionpotential (Pineda et al. 1992). Stimulus were of suprathreshold inten-sity and given at a holding potential of –55 to –60 mV by adjustingconstant current. Bridge balance as well as recovery periods (withoutDC current) were monitored between sample records. After recording,

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some neurons were injected with biocytin as previously described(Galarraga et al. 1999). All neurons identified in this study weremedium-sized spiny projection neurons.

Drugs used

Drugs were dissolved in the bath saline from daily made stocksolutions using a gravity-driven superfusion system. Equilibratedconcentrations of the drugs were achieved in 4–5 min. L-�-amino-3-hydroxy-5-methyl-isoxazolepropionate and 2-carboxy-3-carboxymethyl-4-isopropenylpyrrolidine/kainate (AMPA/KA) antagonist, CNQX (10�M), N-methyl-D-aspartate (NMDA) antagonist, AP5 (50 �M), ni-trendipine, bicuculline, QX-314, TTX, sulpiride, quinelorane, andquinpirole were all purchased from Sigma-RBI (St. Louis, MO).Calcium channels antagonists �-conotoxin GVIA (�-CgTx) and�-agatoxin TK (�-AgaTK) were obtained from both Peptides inter-national (Louisville, KY) and Alomone Labs (Jerusalem, Israel).Almost all drugs were dissolved in water to get stock solutions andadded to the superfusate to reach the final concentration. Nitrendipinewas disolved in dimethylsulfoxide (DMSO; 0.01%).

Data analysis

Digitized data were imported for analysis and graphing into com-mercial software (origin v.6. Microcal, Northampton, MA). Mean SE of all ICa

2� and IPSCs are reported. Free-distribution statisticaltests were used to assess statistical significance; Mann-Whitney U testor Wilcoxon’s t-test (depending on nonpaired or paired samples) andone-way ANOVA with post hoc Tukey’s test were used to assesssignificance between multiple samples in which quinelorane modula-tion was tested in the presence or the absence of Ca2� channelblockers (for details see Vilchis et al. 2000).

To approximate the contribution for each type of Ca2� channel, wetook the amount of Ca2� current blocked by a given antagonist(nitrendipine for L-type; �-AgaTK for P/Q-type; and �-CgTx forN-type) as the contribution of a given channel type to the whole cellcalcium current. Current through R-type Ca2� channels was calcu-lated as the amount of current left after all three blockers were addedtogether. The sum was normalized to 100% so that L � N � P/Q �R � 100%. To determine the amount of quinelorane modulation foreach type of Ca2� channel, we compared quinelorane modulation on

FIG. 1. Ca2� current density does not change between PD14and PD40 developmental stages. A: representative family ofcurrents evoked by 20-ms step voltage commands from –80 to50 mV, in 10-mV steps, in a PD14 cell. B: family of currentsobtained with the same protocol in a PD40 cell. C: currentevoked with a depolarizing ramp command from �80 to 50 mVand 180-ms duration (0.7-mV/ms depolarizing rate) in a PD14cell (same cell as in A). D: similar current response evoked withthe same ramp protocol in a PD40 cell (same cell as in B). E:current-voltage relationship of the PD14 cell (I-V plot) builtwith measurements from both steps (filled circles) and ramp(continuous line) evoked responses. There is close agreementbetween both measurements. For clarity, next figures only showI-V plots built after ramp commands. F: I-V plot of a PD40 cellbuilt as in E. G: histogram comparing averaged current ampli-tudes (means SE) of PD14 and PD40 neurons. H: current wasdivided by cell capacitance to report a quantity proportional tocurrent density. Histogram shows that current density does notchange between these stages (PD14 and PD40). Currents werecarried by 5 mM Ba2�. All records were from medium-sizedneostriatal neurons.

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Ca2� currents in the absence and presence of the different Ca2�

channels blockers and introduced the data into a system of linearequations (for details, see Vilchis et al. 2002).

R E S U L T S

Ca2� currents expressed in dissociated neostriatal neuronsat two different developmental stages

Figure 1A shows a family of Ca2�currents obtained inresponse to depolarizing step voltage commands (see METHODS)from a PD14 neuron. Ba2� was the charge carrier. Figure 1Bshows a similar experiment done in a PD40 neuron. Figure 1Cshows the Ca2� current evoked in the same PD14 neuron (Fig.1A) after a depolarizing ramp command from –80 to 50 mV. Asimilar experiment is shown in Fig. 1D for the PD40 neuron.Figure 1, E and F, shows the current-voltage relationships (I-Vplots) obtained from the experiments depicted in Fig. 1, A–D.Filled circles are measurements from the currents depicted inFig. 1, A and B, measured at the end of the voltage step.Continuous traces are taken from currents depicted in Fig. 1, Cand D, plotted as a function of ramp voltage. Note thatramp-evoked I-V plots look as the “fits” of the I-V plotsobtained with step commands; indicating a virtual agreementbetween both recording methods. For the sake of clarity, mostfigures will show I-V plots obtained with ramp commandsonly. However, most experiments were done with both proto-cols. Histogram in Fig. 1G compares mean current amplitudesat both developmental stages. In PD14 neurons, peak currentamplitude was 234 14 (SE) pA (n � 49), whereas it was254 12 pA in PD40 neurons [n � 54; not significant (NS);Mann-Whitney U test]. Because current density could changeduring development, this parameter was compared in Fig. 1Hby dividing ionic current over whole cell capacitance (CN). CNin PD14 cells was 7.3 0.2 pF, whereas it was 7.5 0.3 pFin PD40 cells (NS; Mann-Whitney U test), indicating thatsomatic surface does not change between these developmentalstages. Accordingly, normalized currents were 32 2.2 pA/pFin PD14 and 34 1.4 pA/pF in PD40 cells (Fig. 1H; NS;Mann-Whitney U test). These measurements made us confi-dent that the total number of Ca2� channels does not increasebetween these two developmental stages. Experiments to doc-ument possible changes in kinetics or voltage dependence werenot done in this work (cf., Fig. 1, A and B). Instead we focusedon the channel types that contribute to the whole cell current,because, even if channel numbers do not increase, the channeltypes that compose the current may change. To test thisexpectation, a pharmacological dissection of Ca2� currents atboth stages, PD14 and PD40, was done with the help of knownCa2� channel antagonists.

Figure 2A shows that, in PD14 neurons, 10 �M nitrendipineblocked 29 3% (n � 15) of the Ca2� current, whereas inPD40 neurons, the percentage of Ca2� current blocked bydihydropyridine was 23 2% (n � 24; this difference was notstatistically significant; Mann Whitney U test; Fig. 2C). There-fore it was concluded that L-type Ca2� channels do not changetheir numbers between these developmental stages. However,further study is needed to see if the subtypes of L-type channels(�1D and �1C) do change (Olson et al. 2005).

Similar experiments were done using the CaV2.1 (P/Q-type)channel blocker, �-AgaTK (400 nM). This peptide reducedCa2� current in PD14 cells by 24 3% (n � 7; Fig. 2D),

whereas it reduced Ca2� current in PD40 cells by 40 1%(n � 15; Fig. 2E). This difference was statistically significant(Fig. 2F; P � 0.0001; Mann-Whitney U test); indicating thatP/Q-type Ca2� channels increase their numbers and theirrelative contribution to the whole cell Ca2� current betweenthese developmental stages.

Equivalent experiments were done using the CaV2.2 (N-type) channel blocker, �-CgTx (1 �M). This peptide reducedCa2� current in PD14 cells by 43 2% (n � 11; Fig. 2G),whereas it reduced Ca2� current in PD40 cells by 28 2%(n � 8; Fig. 2H). This difference was statistically significant(Fig. 2I; P � 0.002; Mann-Whitney U test), indicating thatN-type channels decrease their numbers and their relativecontribution to the whole cell Ca2� current during the PD14 toPD40 developmental window. Comparable findings have beenreported in cultured pyramidal neurons (Chameau et al. 1999).

Finally, to isolate the CaV2.3 (R-type) current, we added thethree blockers together (nitrendipine, �-AgaTK, and �-CgTx)at the above concentrations and measured the current that wasleft. Less than 15% of the initial current was left in both PD14and PD40 cells (Fig. 2, J–L), indicating that, as L-typechannels, R-type channels do not change their numbersduring this developmental period (Bargas et al. 1994; Foehringet al. 2000).

In PD14 cells, the percentage that each current type contrib-utes to the whole cell current was (see METHODS and Vilchis etal. 2002) as follows: L-type, 27%; P/Q-type, 22%; N-type,39%; R-type, 12% (Fig. 3A). In PD40 cells, the percentageswere as follows: L-type, 22%; P/Q-type, 38%; N-type, 26%;R-type, 14% (Fig. 3B). Therefore the results show that expres-sion of CaV2.2 (N-type) channels diminishes, whereas that ofCaV2.1 (P/Q-type) channels augments during this developmen-tal interval. Because one of the main functional roles of N- andP/Q-type Ca2� channels in spiny neurons is the triggering oftransmitter release (Tecuapetla et al. 2005), we chose to startsearching into the functional significance of these changes byhypothesizing that GABA released from the terminals of ma-ture spiny neurons should be mainly triggered by P/Q-typeCa2� channels.

Reassignment of the Ca2� channels that trigger transmitterrelease at the synaptic terminals of medium spiny neurons

To test the above hypothesis, we observed eIPSCs elicitedby the activation of axon collaterals that interconnect spinyneurons. These were obtained by electrically stimulating theglobus pallidus to antidromically activate striofugal axons and,therefore axon collaterals that stay inside the neostriatum andinterconnect spiny neurons (Guzman et al. 2003). At the PD14stage, both N- and P/Q-types Ca2� channels trigger GABArelease from these terminals (Tecuapetla et al. 2005) (Fig. 4, Aand D): �-CgTx and �-AgaTK reduced the amplitude ofantidromically eIPSCs by 65 7 (n � 10) and 90 5% (n �8), respectively (Tecuapetla et al. 2005)—both peptides wereused at saturating concentrations (1 �M and 400 nM, respec-tively).

In contrast, the blocking effect of �-CgTx on antidromi-cally eIPSCs was significantly less important in PD40 neu-rons compared with PD14 neurons: in PD40 neurons,�-CgTx blocked 18 7% of the current (n � 5; Fig. 4B;P � 0.009; Mann Whitney U test; Fig. 4C). On the other

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hand, the P/Q-type Ca2� channel blocker, �-AgaTK, virtu-ally blocked all eIPSCs in PD40 cells: 98 1% (n � 5; Fig.4E). Thus the effect of �-AgaTK was not significantlydifferent when comparing both developmental stages (Fig.4F; Mann Whitney U test); it is only the participation ofN-type Ca2� channels in synaptic transmission that is de-creased during maturation. The participation of P/Q-type Ca2�

channels becomes the most important. These experimentsshow that the reconfiguration of Ca2� current types at theterminals parallels that occurring at the soma. A similar devel-opmental change has been reported in other synaptic terminals,some of them, inhibitory (Iwasaki and Takahashi 1998;Iwasaki et al. 2000; Verderio et al. 1995). Antidromicallyevoked IPSCs from spiny cells axon collaterals were com-pletely blocked by 10 �M bicuculline (data not shown, but seeTecuapetla et al. 2005).

Dopaminergic D2 receptor modulation of Ca2� currents inyoung and mature neurons

An important characteristic of medium spiny neurons is theirhaving D2 dopaminergic receptors whose activation modulatesCa2� currents. However, only the modulation of CaV1 Ca2�

channels has been extensively studied (Hernandez-Lopez et al.2000; Olson et al. 2005; Rakhilin et al. 2004). Here we reportthat modulation of CaV2 Ca2� channels is also very important.Figure 5 confirms the D2 modulation of Ca2� current inneostriatal cells, showing that this modulation is reversibleduring the time course of a typical experiment. This modula-tion reduces the slower tail currents recorded after a stepcommand (Fig. 5B, inset), finding that has been taken asevidence of L-type current modulation (Olson et al. 2005;Rakhilin et al. 2004). D2 modulation of Ca2� current was

FIG. 2. CaV2.1 (P/Q-type) Ca2� chan-nels increase, whereas CaV2.2 (N-type)Ca2� channels decrease in neostriatal neu-rons between the PD14 and the PD40 devel-opmental stages. A and B: sensitivity ofCa2� current to dihydropyridine (10 �Mnitrendipine) was not significantly differentwhen comparing PD14 and PD40 cells. C:histogram summarizing results in 2 neuronalsamples. D and E: sensitivity of Ca2� cur-rent to �-agatoxin TK (�-AgaTK; 400 nM)is significantly different when comparingPD14 and PD40 cells. More current isblocked by �-AgaTK in PD40 neurons. F:histogram summarizing results in 2 neuronalsamples (P � 0.0001). G and H: sensitivityof Ca2� current to �-conotoxin GVIA (�-CgTx; 1 �M) is significantly different whencomparing PD14 and PD40 cells. More cur-rent is blocked by �-CgTx in PD14 neurons.I: histogram summarizing results in 2 neu-ronal samples (P � 0.002). J and K: amountof Ca2� current left: CaV2.3 or R-type cur-rent, after all 3 blockers (nitrendipine, �-AgaTK, and �-CgTx) were administered to-gether to PD14 (J) and PD40 (K) neurons,was not significantly different. L: histogramsummarizing results in 2 neuronal samples.




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observed in 80% of the recorded dissociated neurons at eachdevelopmental stage.

Figure 6, A–C, shows that the D2 modulation of Ca2�

current (10 �M quinelorane) is present in both juvenile (PD14)and mature (PD40) neurons. This modulation comprises aboutthe same amount of current reduction at both developmentalstages (Fig. 6C; NS). Quinelorane acted in a concentration-dependent fashion (data not shown) at both stages. Thus 10�M quinelorane reduced the current in PD14 by 35 2% (n �12) of neurons and by 36 3% (n � 12) in PD40 neurons (NS;Mann-Whitney U and ANOVA tests). All the modulationproduced by quinelorane and other D2 receptor agonists (e.g.,quinpirole) was virtually blocked by 1 �M of the unselectiveD2 receptor class antagonist, sulpiride (1 �M), at both stages(n � 12; data not shown).

The D2 receptor modulation extends to the synaptic termi-nals and not only resides at the somatodendritic membrane. Apresynaptic modulation of GABA release form the synapticterminals of medium spiny neurons has been described inyoung animals (�PD14) (Cooper and Stanford 2001; Guzmanet al. 2003; see also: Floran et al. 1997). Figure 6, D–F, showsthat about the same amount of D2 modulation of synaptictransmission is present in both young (PD14) and mature(PD40) neurons. Quinelorane (1 �M) reduced eIPSCs ampli-tude in both cases: 50 8% (n � 12) in PD14 cells and 42 5% (n � 6) in PD40 cells (Fig. 6F; NS; Mann Whitney U test).Therefore dopaminergic modulation of the axon collaterals that

FIG. 3. Contribution of different Ca2� channel types to the whole cell Ca2�

current. CaV2.2 channels decline during development, whereas CaV2.1 channelsincrease with age. Other channel types remain constant during development.

FIG. 4. �-CgTx sensitivity of synaptic terminals declines with development. A: 1 �M �-CgTx greatly reduced (�65%) inhibitory postsynaptic potentials(IPSCs) in PD14 neurons. Note transition from paired pulse depression to paired pulse facilitation during �-CgTx action (inset). B: IPSC sensitivity to �-CgTxwas greatly reduced in PD40 neurons (�18%). In the case shown, �-CgTx had no effect. Note paired pulse depression in the control (inset). C: histogramsummarizes effect of �-CgTx in 2 neuronal samples (P � 0.009). D and E: sensitivity of IPSCs to �-AgaTK (400 nM) was strong at both developmental stages.Blockage of P/Q-type channels enhanced paired pulse ratio at both stages. Also note that the same concentration of �-AgaTK had a faster action in PD40 neurons.F: histogram summarizes effect of �-AgaTK in 2 neuronal samples (NS). IPSCs were evoked by electrically stimulating the globus pallidus to antidromicallyactivate striofugal axons and therefore axon collaterals that interconnect spiny neurons (Guzman et al. 2003); 10 �M cyano-2,3-dihydroxy-7-nitro-quinoxalinedisodium salt (CNQX) and 50 �M D-(�)-2-amino-5 phosphonovaleric acid (AP5) were present in the bath saline during the experiment. Antidromically evokedIPSCs were completely blocked by 10 �M bicuculline (data not shown, but see Tecuapetla et al. 2005).

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interconnect spiny neurons is preserved throughout this devel-opmental interval. This presynaptic response was observed inall cells recorded from mature slices (n � 6) and was notobserved in 2 of 12 cells from young animals (17%), suggest-ing that not all terminals posses D2-class receptors.

In juvenile animals (PD14), the mean paired pulse ratio(PPR) of eIPSCs after a pair of stimulus (Fig. 6D, inset) in thecontrol condition was 1.3 0.16 and in the presence of D2

receptor agonists was 1.9 0.26 (n � 12; P � 0.002;Wilcoxons t-test). In mature animals (PD40; Fig. 6E, inset),control PPR was 0.82 0.1 and in the presence of D2 receptoragonists it was 0.97 0.08 (n � 6; P � 0.05; Wilcoxonst-test). Because the PPR value is linearly proportional to theprobability of release, the paired pulse stimulation has been anaccepted protocol to search for presynaptic actions (e.g.,Baldelli et al. 2005). Thus the modulation is suggested to bepresynaptic in both juvenile and mature animals (Guzman et al.2003). However, to further support this inference, a mean-variance analysis was performed (Clements and Silver 2000)and accompanied with an independent analysis of amplitudehistograms.

Figure 7A shows an amplitude histogram of spontaneousIPSCs (sIPSCs) recorded in a neostriatal neuron (Fig. 7B).These events may come from interneurons or spiny cell termi-nals and fitted to Gaussian functions with a first modal ampli-tude of 8 pA (range, 5–12 pA), similar to that reported for otherGABAergic synapses (e.g., Ling and Benardo 1999). Thesecond mode was 16 pA (range, 12–20 pA) (cf., Paulsen andHeggelund 1994). Thereafter, some eIPSCs were recordedafter applying quasi-minimal stimulation strength. The ampli-

tudes histogram of these eIPSCs was multimodal (Fig. 7, C andD), obtaining values of 9 and 17 pA for the first modes; theinterval between peaks was 8–9 pA (Ling and Benardo 1999).Similarity between sIPSCs and eIPSCs histograms supports thenotion that terminals that interconnect spiny neurons are fairlyhomogeneous (Tecuapetla et al. 2005), and their modal valuescan now be used to independently ascertain the results of themean-variance analysis (Clements and Silver 2000). eIPSCswere obtained from a range of eIPSCs amplitudes from differ-ent experiments (see intensity-amplitude plots in Tecuapetla etal. 2005). Mean eIPSCs amplitudes were graphed against theirpeak variance (Fig. 7E, black circles) and the resultant plotcould be fitted wit a parabola (see Clements and Silver 2000)that yielded a weighted quantal amplitude, Qw � 8 2 pA,notably similar to the first modal amplitude in the histograms.In the presence of the D2-class receptor agonist, quinelorane (1�M), the cases appeared intermingled, with control cases oflower mean amplitudes at the initial portion of the parabola(Fig. 7E, gray circles), and linear fits of the two samples werenot significantly different. Qw was 9 2 pA for the D2 cases(NS; Mann-Whitney U test; also see amplitude histograms).Therefore mean-variance analysis supported the view that D2receptor actions are presynaptic (i.e., initial parabola slopes donot diverge; Clements and Silver 2000).

Because maximal eIPSCs amplitudes in the presence of D2agonists did not give a reliable fit of a parabola, it was notpossible to distinguish if D2 actions were caused by a reductionin mean release probability (Pw) or a reduction in the numberof active sites (Clements and Silver 2000). Nonetheless, Fig.7F directly suggests that D2 action may in part result from a

FIG. 5. Activation of dopaminergic D2 receptors reversibly modulates Ca2� currents in both young and adult neurons. A: time-course showing that 2successive applications of the dopaminergic D2 receptor agonist quinelorane (10 �M) reduced whole cell Ca2� current (gray symbols). It is also shown that thesecurrent reductions were reversible. Bars indicate time of drug application and wash off. Stability of current amplitude when no drug was applied is also shown(empty symbols; measurements taken from another neuron). B: representative records of experiment in A. Note that both steady-state amplitude and tail currentamplitude were decreased by the D2 agonist. Numbers correspond to labels in A. C: box plot showing distribution of percentage of run down found in Ca2�

current when no drug was applied (n � 15). PD40 neuron. Similar results were obtained in PD14 neurons.




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decrease in Pw, although a simultaneous decrease in N couldnot be discarded. Thus P can be ascertained directly as thenumber of recorded eIPSCs over the number of trials (1stresponse). P was reduced from 0.6 to �0.1 after quinelorane inthis experiment. The average of individual trials (Fig. 7,bottom) showed an augmented PPR after quineloreane (seeBaldelli et al. 2005). In conclusion, both PPR changes andmean-variance analysis suggested that the actions of the D2-class receptor agonists were presynaptic.

D2-class receptor–mediated actions on other ion channelshave been linked to intracellular Ca2� mobilization and cal-cineurin (CaN) (Hernandez-Lopez et al. 2000; Hu et al. 2005;Rakhilin et al. 2004). However, it was shown (Fig. 8, A–D) thatthe percentage of D2-mediated modulation on Ca2� currentswas not significantly different when the neurons were inter-nally perfused with either 1 or 10 mM EGTA: 34 2 and 36 3%, respectively (NS; n � 6; Mann-Whitney U test). Never-theless, D2-class receptor–mediated actions were virtuallyabolished when 15 mM BAPTA were perfused instead (Fig. 8,C and D), suggesting that D2 receptor actions were mediatedby a Ca2�-dependent diffusible pathway (Dargan and Parker2003; Stefani et al. 2002). Figure 8F shows Ca2� currentevoked with the double pulse protocol (depicted in Fig. 8E; seeMETHODS) in a PD14 neuron. It can be seen that the amount ofcurrent modulated by the D2 receptor agonists did not changesignificantly (Fig. 8H; Wilcoxons t-test). The same result wasfound on PD40 neurons (Fig. 8, G and I) (Hernandez-Lopez etal. 2000). The results suggest that the signaling pathway usedby D2-class receptors to modulate CaV2 Ca2� channels is adiffusible Ca2�-sensitive cascade, as is the case of CaV1 Ca2�

channels (Hernandez-Lopez et al. 2000; Hu et al. 2005; Olson

et al. 2005; Rakhilin et al. 2004). In view of these results, thenext question was whether this modulation was being mediatedby the same Ca2� channels at both developmental stages.

Ca2� channels mediating dopaminergic D2 receptormodulation change during development

Because the amount of D2 modulation on the Ca2� currentwas the same in juvenile (PD14) and mature (PD40) neurons,it was decided to use different Ca2� channel blockers inconjunction to the D2-class receptor agonist, quinelorane (10�M), to see if the channels being modulated were the same injuvenile and mature neurons. The calcium channel blockers,nitrendipine (10 �M), �-AgaTK (400 nM), and �-CgTx (1�M) were used, the rationale being that, if one channel typemediates the D2-induced modulation, its blockage would oc-clude a part of this modulation (Perez-Rosello et al. 2005;Vilchis et al. 2002). However, if a channel type does notmediate most of the D2-induced modulation, its blockagewould not occlude the modulation significantly because per-centage of modulation is calculated from the current left afterthe action of a given blocker. Nonetheless, the percentages ofcurrent components vary somewhat from cell to cell, andmultiple samples were compared with the same control sam-ples (quineloranes effect without channel blockers). Thereforeboth parametric and nonparametric (Kruskal-Wallis) ANOVAtests were used to compare all samples altogether taken intoaccount intrasampling variance. Both tests yielded significantdifferences when the D2-class agonist was administered (F �15.2; K-W: 53.8; P � 0.0001 in both cases). Therefore posthoc (Tukey’s test) pair to pair comparisons were used to

FIG. 6. Amount of D2 receptor modulation of Ca2� current was the same in young and mature neurons. A and B: amount of Ca2� current reduction by D2

receptor agonists (10 �M quinelorane in this case) is about the same in juvenile (PD14: A) and mature (PD40: B) neurons. C: histogram summarizes results fromjuvenile and mature neuronal samples (NS). D and E: amount of IPSC reduction by D2 receptor agonists (1 �M quinelorane in this case) is not significantlydifferent in juvenile (PD14) and mature (PD40) synaptic terminals. Note that the paired pulse ratio (Guzman et al. 2003) was enhanced by quinelorane in bothcases. F: histogram summarizes results from juvenile and mature samples (NS). IPSCs were evoked as in Fig. 4.

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contrast different groups. At the PD14 stage, pretreatment withnitrendipine did not completely block quinelorane action butreduced it from 35 2 to 26 2%; (n � 11; Fig. 9A; �9%occlusion; P � 0.05; ANOVA with post hoc Tukey’s test), asexpected after a block of a modulated channel. This shows thatD2 receptor activation modulates CaV1 Ca2� channels at thisstage, but also shows that there is a remaining modulation onCaV2 Ca2� channels. Still, a more important occlusion of theD2 agonist effect by the L-type channel blocker was observedin PD40 neurons: quinelorane action was reduced from 36 3to 17 1% (n � 11; Fig. 9B; �19% occlusion; P � 0.0001;ANOVA with post hoc Tukey’s test). When the degree ofocclusion by the L-type channel blocker was compared be-tween both developmental stages, the difference was statisticalsignificant (P � 0.04; ANOVA and post hoc Tukey’s test).These experiments show that the dopaminergic D2 modulationof L-type Ca2� channels is not only preserved but enhancedsignificantly with age. This suggests that the modulation of the

dynamic firing range (Perez-Garci et al. 2003) enhances itsimportance with network development.

In contrast, modulation of P/Q-type channels with �-AgaTKin PD14 neurons was not enough to significantly occlude theD2 agonist modulation in PD14 neurons: �-AgaTK reducedquinelorane effect from 35 2 to 27 2%; (n � 6; Fig. 9C;P � 0.3; ANOVA with post hoc Tukey’s test). However, inPD40 neurons, the P/Q-type channel blocker produced a sig-nificant occlusion of the D2 agonist effect: quinelorane actionwas reduced from 36 3 to 20 1% (n � 6; Fig. 9D; �44%;P � 0.0001; ANOVA with post hoc Tukey’s test). When thedegree of occlusion by P/Q-type channel blockers wascompared between both developmental stages, the differ-ence was statistical significant (P � 0.05; ANOVA withpost hoc Tukey’s test). This means that modulation ofP/Q-type channels by D2 receptor activation is small injuvenile (PD14) neurons but becomes important in mature(PD40) neurons.

FIG. 7. Mean-variance analysis indicates thataction of dopaminergic D2-class receptor agonistson synaptic transmission is presynaptic. A: ampli-tude histogram of spontaneous IPSCs (sIPSCs)recorded in a neostriatal neuron. Two modes areevident: 8 and 16 pA. B: sample records fromwhich the histogram for sIPSCs was built. C:amplitude histogram of an evoked IPSC (eIPSC)recorded in the same neostriatal neuron. Histo-gram is multimodal, 1st 2 modes are 9 and 17 pA.Interval between modes is 8–9 pA. eIPSCs wereevoked by electrically stimulating the globus pal-lidus to antidromically activate striofugal axonsand therefore axon collaterals that interconnectspiny neurons. D: sample records of eIPSCs foreach mode, including failures. E: mean peak am-plitudes of eIPSCs taken from different experi-ments in which different stimulation strengthswere used were graphed against their correspond-ing peak variances (black circles). A parabola(continuous line) of the form y � Ax � Bx2

(Clements and Silver 2000) was fitted to thesedata. From this fit, an approximation of quantalamplitude (Qw) could be obtained: 8 pA. Note thesimilarity of values with values obtained withamplitude histograms (A–D). Mean peak ampli-tudes of eIPSCs recorded in the presence ofquinelorane (1 �M) were also graphed againsttheir peak variances (gray circles), Qw � 9 pA(NS). Because data from both control and testsamples share initial slope of parabola, analysissupports that the action of D2-class receptors ispresynaptic (Clements and Silver 2000). F: fam-ily of eIPSCs obtained with quasi-minimal stim-ulation. Observe few failures in the control con-dition (1st event), and many more failures duringquinelorane. Experiments as this one allow adirect assessment of P. Bottom: average recordingof individual trials: note that PPR increases whenP decreases.




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A completely different outcome was obtained with theN-type channel blocker �-CgTx. In the presence of thisblocker, the D2 agonist action on Ca2� current of PD14neurons was reduced from 35 2 to 12 2% (n � 8; Fig. 9E;�66% occlusion; P � 0.0001; ANOVA with post hoc Tukey’stest). Therefore �-CgTx resulted in the most potent agentcapable to occlude the dopaminergic modulation in juvenileneurons, suggesting that modulation of CaV2 Ca2� channels bydopamine is more important than modulation of CaV1 Ca2�

channels at this stage. In contrast, �-CgTx was unable tosignificantly decrease the action of D2 agonists on the Ca2�

current of PD40 neurons. Ca2� current reduction by quinelo-rane in the presence of �-CgTx in PD40 neurons was from36 3 to 31 3% (n � 8; Fig. 9F; P � 0.9; ANOVA and posthoc Tukey’s test). When the degree of occlusion by the N-typechannel blocker was compared between both developmentalstages, the difference was statistical significant (P � 0.0001;ANOVA and post hoc Tukey’s test). This means that modu-lation of N-type channels by D2 receptor activation is large or

very important in juvenile (PD14) neurons, but it is muchsmaller in mature (PD40) neurons.

A summary of all samples compared can be seen in Fig. 10.It was observed that the most potent blocker of the D2 agonistaction in young (PD14) neurons was �-CgTx, followed bynitrendipine. However, in mature neurons (PD40), both nitren-dipine and �-AgaTK were potent blockers of the D2-agonistaction on Ca2� currents, but �-CgTx did not longer occludedopaminergic actions. In summary, a considerable switch intarget for D2 receptor signaling occurs in medium spiny neu-rons during development.

In PD14 neurons, the solution of a system of equations (seeVilchis et al. 2002) showed what percentage of each channeltype was modulated to explain the 35% total modulation: 35 �L (0.25) � P/Q (0.17) � N (0.63) � R (0), where the L, P/Q,N, and R coefficients denote the percentage that each channeltype contributes to the whole cell Ca2� current in PD14neurons (see Fig. 3A), and numbers in parentheses are thesolved unknowns. The same system of equations applied for

FIG. 8. Action of dopaminergic D2-class receptorson Ca2� currents are mediated by a voltage-indepen-dent, Ca2�-dependent, diffusible pathway. A: D2-classreceptor agonists reduce Ca2� current in the presenceof 1 mM EGTA. B: D2-class receptor agonists reduceCa2� current in the presence of 10 mM EGTA. Amountof modulated current did not change when A and Bwere compared. C: action of D2-class receptor agonistson Ca2� current were virtually abolished in the pres-ence of 15 mM BAPTA; suggesting that these actionsare mediated by a Ca2�-dependent diffusible pathway.D: histogram summarizing these results. Differenceswere significant only when comparing EGTA andBAPTA samples (P � 0.001; ANOVA with post hocTukey tests). E: double pulse protocol to 0 mV. Notethat the 2nd pulse is preceded by a depolarizing com-mand to 80 mV. F: current evoked after the depolariz-ing commands to 0 mV before and after the depolariz-ing prepulse to 80 mV. Note that both control (blacktraces) and modulated current (gray trace; quinelorane)were enhanced in the same proportion, indicating thatthere is a constitutive voltage-dependent modulationbut that quinelorane does not change it significantly inPD14 neurons. G: similar results were obtained inPD40 neurons. H and I: histograms summarizing sam-ples of experiments (n � 6) as in F and G, respectively.

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PD40 neurons explained the 36% modulation at this stage:36 � L (0.81) � P/Q (0.45) � N (0.04) � R (0) (see Fig. 3B).This analysis helped to approximate a quantitative assessmentof the results described above: 1) L-type channel D2 modula-tion increased during development from 25 to 81% becomingthe most important Ca2� channel modulation with age, 2)P/Q-type channel modulation increased during development to45%, becoming the second most important channel beingmodulated by D2-class receptors, and 3) N-type channels arethe most modulated in juvenile neurons (63%); however, theirmodulation tends to disappear in mature neurons.

If the above data are correct, the following predictions canbe made to further corroborate these findings: first, the con-joined administration of nitrendipine (10 �M) and �-AgaTK(400 nM) will virtually abolish quinelorane action on the Ca2�

current of PD40 neurons, but not in PD14 neurons (cf., Fig. 11,A and B). Conversely, the conjoined administration of nitren-dipine (10 �M) and �-CgTx (1 �M) will almost totally blockthe action of the D2 receptor agonist on the Ca2� current ofPD14 neurons, but not in the case of PD40 neurons (cf., Fig.11, D and E). Thus after L-type and P/Q-type Ca2� channelswere blocked, quinelorane was still capable to reduce theremaining current by 19 1% (n � 6) in PD14 neurons (Fig.11C). In contrast, when L-type and P/Q-type Ca2� channelswere blocked in PD40 neurons, quinelorane action was virtu-

ally abolished to 3 1% (n � 6: Fig. 11C; P � 0.0001;ANOVA with post hoc Tukey’s test). Conversely, when L-typeand N-type channels were blocked in PD14 neurons, almost noquinelorane action was left: 6 0.7% (n � 6; Fig. 11, D andF). The opposite findings were observed at the PD40 stage:quinelorane was still capable to modulate the remaining Ca2�

current by 18 2% in the same conditions (n � 6; Fig. 11F;P � 0.0001; ANOVA with post hoc Tukey’s test). Globally,both parametric and nonparametric (Kruskal-Wallis) ANOVAtests were significant (F � 50.7; K-W: 18.5; P � 0.0001 forboth tests). In summary, besides modulating CaV1 Ca2� chan-nels, D2 receptor activation also modulates CaV2 Ca2� chan-nels. However, the modulation is mainly on CaV2.2 Ca2�

channels (N-type) in juvenile striatal neurons (PD14), whereasit is mainly on CaV2.1 Ca2� channels (P/Q-type) in maturestriatal neurons (PD40).

Developmental changes are reflected at the synapticterminals of medium spiny neurons

To study whether the change of target affects presynapticinhibition (Guzman et al. 2003), the effect of the D2 receptoragonist, quinelorane (1 �M), in the presence of �-CgTx (1�M) was examined on the eIPSCs generated by antidromicactivation of the axon collaterals. Figure 12A shows that

FIG. 9. Ca2� currents modulated by D2 receptor activationchange during development. A: previous blockage of CaV1Ca2� channels (L-type) by nitrendipine (10 �M) in PD14neurons does not impede the action of the D2-receptor agonist,quinelorane (10 �M), on Ca2� current, indicating an importantD2-modulation on CaV2 Ca2� channels. However, a partialocclusion of D2 modulation was present and statistically sig-nificant. B: similarly, blockage of L-type channels partiallyoccludes quinelorane action on the Ca2� current of PD40neurons. However, partial occlusion was significantly larger inPD40 neurons (P � 0.04). C: quinelorane can act on Ca2�

current of a PD14 neuron after block of CaV2.1 Ca2� channels(P/Q-type) by �-AgaTK (400 nM). In fact, the action of the D2

receptor agonist was not significantly different with and without�-AgaTK. D: �-AgaTK significantly occluded the action ofquinelorane on the Ca2� current of a PD40 neuron. Occlusionof the D2 receptor agonist action by �-AgaTK was significantlydifferent when comparing both developmental stages (P �0.05). E: quinelorane had a much reduced action on the Ca2�

current of a PD14 neuron after blocking CaV2.2 Ca2� channels(N-type) with �-CgTx (1 �M). F: �-CgTx does not occludequinelorane action on the Ca2� current of PD40 neurons.Occlusion of the D2 receptor agonist action by �-CgTx wassignificantly different when comparing both developmentalstages (P � 0.001).




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�-CgTx (1 �M) almost abolished the D2 agonist mediatedreduction of IPSCs in PD14 terminals: from a 50 8%reduction in the control (Guzman et al. 2003) to only 10 2%

reduction in the presence of �-CgTx (n � 3; Fig. 12A; P �0.02; Mann-Whitney U test). A posterior application of �-AgaTK (400 nM) blocked the remaining eIPSC (Fig. 12A),confirming that P/Q-type channels were also present at theterminals and that quinelorane only had its full action if themain target, N-type channels, were present in juvenileterminals.

In contrast, the presence of 1 �M �-CgTx did not impede D2agonist actions in PD40 terminals. Reduction of IPSCs was42 5% in the controls and 40 8% in the presence of�-CgTx (n � 5; Fig. 12B; NS; Mann-Whitney U test). Thisshows that presynaptic N-type channels were no longer thetarget for D2 receptors at this stage. These results are summa-rized in the histogram of Fig. 12C (P � 0.009; Mann-WhitneyU test), which shows the effect of quinelorane in the presenceof �-CgTx at this stage. Thus the dopaminergic modulationthat distinguishes these terminals is present at both devel-opmental stages but mediated by a different Ca2� channeltype.

AHP in medium spiny neurons is negligibly affected by D2receptor agonists

Because P/Q-type Ca2� channels are a main source of Ca2�

to activate the Ca2� dependent K� channels that generate theAHP in medium spiny neurons (Vilchis et al. 2000), D2receptor agonists were tested on the AHP of mature spinyneurons after a single action potential. Two D2 receptor selec-tive agonists were used: 5 �M quinelorane and 5 �M quinpi-

FIG. 10. Action of D2 receptor activation on different components of theCa2� current. A: most potent blocker of D2 receptor agonist action on the Ca2�

current of PD14 neurons was �-CgTx (***P � 0.0001), indicating thatdopaminergic modulation of N-type channels is very important at juvenilestages of development. Regulation of L-type channels is also present atjuvenile stages (*P � 0.05). B: modulation of both L- and P/Q-types Ca2�

channels becomes very important with neuronal maturation (***P � 0.0001).However, modulation of N-type channels, important at juvenile stages, be-comes virtually nonexisting at a mature age (PD40).

FIG. 11. Different pairs of Ca2� channels explain most dopaminergic D2 modulation of Ca2� current in young and mature neurons. A: D2 receptor agonist,quinelorane (10 �M), still had a substantial action on the Ca2� currents of young (PD14) neurons after L-type and P/Q-type Ca2�channel types were blocked (with 10�M nitrendipine plus 400 nM �-AgaTK). B: action of the D2 receptor agonist on the Ca2� currents of mature (PD40) neurons was virtually abolished after L-type andP/Q-type Ca2�channel types were blocked (with 10 �M nitrendipine plus 400 nM �-AgaTK). C: histogram summarizes quinelorane action on the Ca2� current of youngand mature neurons in the presence of L- and P/Q-type Ca2� channels blockage (*P � 0.0001). D: action of the D2 receptor agonist on the Ca2� currents of young(PD14) neurons was almost abolished after L-type and N-type Ca2�channel types were blocked (with 10 �M nitrendipine plus 1 �M �-CgTx). E: D2 receptoragonist still had a substantial action on the Ca2� currents of mature (PD40) neurons after L-type and N-type Ca2�channel types were blocked. F: histogramsummarizes quinelorane action on the Ca2� current of young and mature neurons in the presence of L- and N-type Ca2� channels blockage (*P � 0.0001).

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role. Figure 13 shows that, in mature spiny neurons, no changein AHP was observed in n � 6/18 neurons (33%; Fig. 13,A–C), and a mild change was observed in n � 12/18 neurons(66%; Fig. 13, D–F) for a mean overall reduction of 1.3 0.26mV (20%). Note that even when D2-mediated modulation ofCa2� currents was seen in 80% of dissociated cells, only about60% of intracellularly recorded (PD40) neurons exhibited AHPmodulation. This is a number more in agreement with the

expected number of cells presenting D2-type responses,probably suggesting that not all members of the D2 receptorfamily (D2, D3, D4) participate in the same specific cellularfunctions.

D I S C U S S I O N

This study shows that total current density through voltage-activated Ca2� channels does not change between stages PD14to PD40 of rat development. However, the Ca2� channel typesthat carry this current do change. In particular, CaV2.2 (N)channels are down-regulated, whereas CaV2.1 (P/Q) channelsare up-regulated during this period. On the other hand, CaV1(L) and CaV2.3 (R) currents remain constant. That is, thechange is arranged as though an increasing channel “replaces”a decreasing one. This study also shows that channel recon-figuration occurs in parallel at both the somatodendritic mem-brane and the synaptic terminals. Thus the contribution ofN-type channels to trigger GABA release appears to decreaseduring this period, whereas the contribution of P/Q-type chan-nels becomes the most important. This “replacement” of N-type channels by P/Q-type channels has been observed in otherCNS synapses, some of them inhibitory (Iwasaki and Taka-hashi 1998; Iwasaki et al. 2000; Verderio et al. 1995). How-ever, this change seems to occur at later times in the synapsesarising from spiny neurons (cf., Iwasaki et al. 2000; see Tepperet al. 1998), suggesting that these synapses undergo a some-what late maturation (Colwell et al. 1998; Tepper et al. 1998)compared with other neurons. Because channel reconfigu-ration was observed at both the somatodendritic membraneand the synaptic terminals, the view that field stimulation inthe globus pallidus preferentially activates striofugal axonsfrom spiny neurons (Tecuapetla et al. 2005) is thereforesupported.

Dopaminergic modulation of Ca2� currents undergoes ashift in target during development

Previous studies have shown that medium spiny neuronsexpress functional D2-class receptors (e.g., Aizman et al. 2000;Surmeier et al. 1993, 1996). These receptors increase theirnumber between the third and fourth postnatal weeks (Broad-dus and Bennett 1990), when an important escalation in thenumbers of dopaminergic synaptic terminals occurs (Antono-poulos et al. 2002).

This work reports that N and P/Q-type channels are modu-lated in neostriatal projection neurons by D2-class receptoractivation. If only the whole cell Ca2� current is observed, nodifference could be seen in the amount of current modulatedwhen comparing young and mature neurons. Moreover, inhib-itory transmission between spiny neurons was similarly mod-ulated in young (Guzman et al. 2003) and mature neurons.Nonetheless, a more careful look showed that the type ofchannels being modulated change with age.

In young neurons, a great part of the D2-mediated modula-tion is on N-type channels, whereas the modulation of P/Q-type channels is small at this stage (PD14). At a mature age(PD40), however, the modulation of N-type channels virtuallydisappears, and the modulation of P/Q-type channels becomesvery important. This change in target for D2 receptor signalingoccurs at a time when channel expression is being reconfig-

FIG. 12. The D2-mediated modulation of IPSCs is occluded by CaV2.2channel blockage in young but not in mature neurons. A: time-course of IPSCamplitude recorded in the presence of 1 �M �-CgTx. �-CgTx virtuallyabolished the action of quinelorane (1 �M) on the IPSCs recorded from PD14neurons (cf., Fig. 6D). Insets 1 and 2 show representative records in thepresence of �-CgTx. A subsequent addition of �-AgaTK shows that P/Q-typechannels are present in terminals because the peptide blocked all the IPSCs;thus the dopaminergic agonist does not act on P/Q-type channels at thisdevelopmental stage. B: time-course of IPSC amplitude recorded in thepresence of 1 �M �-CgTx. �-CgTx did not affect the action of quinelorane (1�M) on the IPSCs recorded from PD40 neurons (cf., Fig. 6E). Insets 1 and 2show representative records in the presence of �-CgTx. Addition of quinelo-rane reduced the paired pulse depression. It is concluded that the dopaminergicagonist does not act on N-type channels at this developmental stage. C:histogram shows different degree that �-CgTx has on dopaminergic D2 effectsat these 2 different developmental stages (P � 0.009).




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ured, as though D2 dopamine signaling were favoring thechannel type that increases its expression (P/Q) and discard-ing the channel type that decreases its expression (N). Themodulation of L-type channels also increases with age. Inbrief, the order of potency for D2-class receptor mediatedmodulation in PD14 neurons is N � L �� P/Q, and in PD40neurons it is L � P/Q �� N. Both a change in target formodulation and channel reconfiguration occur, but at the end,the same current density and the same amount of D2 modula-tion were observed.

As expected, presynaptic modulation mediated by D2-classreceptors was occluded by the N-type Ca2� channel blocker,�-CgTx, in young neurons (PD14), but it was not occluded bythis peptide in mature neurons (PD40). This is evidence that D2

receptor–mediated modulation of synaptic transmission alsoendures a shift of target at the synaptic terminals. Because inmature synaptic terminals �-AgaTK virtually blocks all syn-aptic transmission, occlusion experiments of D2 agonist actionswith saturating concentrations of �-AgaTK could not be done.However, this property of mature terminals makes it logical to

FIG. 13. Activation of D2 receptors negligibly affect the afterhyperpolarization (AHP) in medium spiny neurons. A: action potential was evoked by a briefdepolarization (top) from a holding potential of –55 mV (bottom). B: addition of a D2-selective agonist, in this case quinpirole (5 �M), does not reduce or modifyAHP. C: superimposition of records in A and B. D: similar experiment in another spiny neuron held at �60 mV. E: In this case, addition of D2 receptor agonistproduced a small reduction of the AHP. F: superimposition of C and D. Inset: behavior of a sample of mature spiny neurons: one-half of the neurons had noAHP modification after D2 agonists and one-half had a minor change.

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think that P/Q-type channels mediate D2 agonist actions inmature synaptic terminals, because 1) L-type channels are notlikely to participate in transmitter release (Tecuapetla et al.2005; Wu and Saggau 1997), 2) P/Q-type channels are themain channels mediating synaptic release at these terminals,and 3) P/Q-type channels are the Ca2� channels modulated byD2 receptor activation at the somatodendritic membrane ofmature neurons. Presynaptic modulation by D2 receptors dis-tinguishes neostriatal inhibition (Centonze et al. 2003; Cooperand Stanford 2001; Guzman et al. 2003; Momiyama 2003)from the inhibitory inputs arising from pallidal efferents; whichare insensitive, and not presynaptically modulated, by selectiveD2 receptor ligands (Cooper and Stanford 2001; Shin et al.2003). To summarize, N-type Ca2� channels cease to be atarget for D2 receptor signaling at both the soma and theterminals of mature neostriatal projection neurons during net-work development. It would be interesting to know if theneurons in organotypic cultures undergo these developmentalchanges (Plenz and Kitai 1998) and if this is a function ofdopaminergic transmission.

What would be the functional impact of this channel switchat the synaptic terminals? N- and P/Q-type Ca2� channels areknown to underlie different forms of short-term plasticity:N-type channels favor short-term facilitation, whereas P/Q-type channels favor short-term depression (Scheuber et al.2004). Interestingly, moderate stimulation preferentially showspaired pulse facilitation for PD14 terminals, whereas it exhibitspaired pulse depression in PD40 terminals (see RESULTS and cf.,insets in Fig. 6, D and E), the expected outcome if P/Q-typechannels are the main controllers of GABA release in matureterminals. Therefore these changes suggest a change in thefunctional properties of lateral (or feedback) inhibition (Plenz2003; Scheuber et al. 2004) during development. Furthermore,the dopaminergic modulation of P/Q-type channels may indi-cate that these functional properties could be finely tuned(Baimoukhametova et al. 2004; Tecuapetla et al. 2004) andthus are ideal for being regulated by dopamine.

Functional significance of dopaminergic modulation of Ca2�

currents in the firing pattern

To modulate the AHP that follows the action potential ofmedium spiny neurons, both CaV2 Ca2� channel types, N andP/Q, have to be blocked simultaneously (Perez-Garci et al.2003). Muscarinic M1 receptor agonists reduce Ca2� currentthrough both channels (N and P/Q) and thus greatly reduce theAHP (70–90%), shorten the interspike interval, and enhanceevoked discharge in mature and young neurons (Perez-Roselloet al. 2005). This adds to the action of muscarinic receptoractivation on inward and delayed rectifications (Figueroa et al.2002; Shen et al. 2005). In contrast, D2 receptor activation iscapable of reducing current only through one of the CaV2 Ca2�

channels, P/Q, which is not enough to greatly affect the AHPas muscarine does (Perez-Garci et al. 2003; Perez-Rosello et al.2005). Reduction is about 20% in mature spiny cells andoccurs in about 60% of the neurons. Interestingly, although D2and M1 receptors are linked to the same phospholipase-Csignaling cascade (Hernandez-Lopez et al. 2000; Rakhilin et al.2004), they have opposing actions on evoked discharge; D2receptor agonists decrease firing, whereas M1 receptor agonistsincrease firing. These data partially explain this opposition: in

the somatodendritic membrane, a main action of M1 receptorsis the reduction of the AHP to increase firing, whereas the maineffect of D2 receptors is the reduction of L-type current todecrease firing (Hernandez-Lopez et al. 2000); the lack ofaction on N-type channels at this level impedes the action of D2class receptors on the AHP. Thus even if linked to the samePLC signaling cascade, M1 and D2 receptors have oppositeeffects in the integration of neuronal firing. Nonetheless, theyhave cooperative actions in controlling transmitter release,which depends only on P/Q-type channels in mature neurons(Calabresi et al. 2000; Guzman et al. 2003; Perez-Rosello et al.2005). Therefore opposition and cooperativity depend on theneuronal region and function being modulated.

Because the D2 class receptor agonists used in this study,quinelorane and quinpirole, hardly distinguish among differentmembers of the D2 receptor class (D2, D3, D4), we do not thinkthat the pharmacological responses at high saturating concen-trations were caused exclusively by responses from the D2receptor type (Gerfen et al. 1995; Le Moine and Bloch 1995;but see Aizman et al. 2000). Thus the D2 modulation of Ca2�

current observed in this work was found in 80% of the recordeddissociated neurons (n � 24/29; young and mature) (Surmeieret al. 1996). Nonetheless, when looking at specific functions, asthe AHP, percentage of cells modulated decreased to about60%, suggesting that, although the actions on currents is thesame, different members of the D2 class receptors may couplethese currents to different functions.

Finally, because field stimulation used to antidromicallyactivate striofugal axons was not intended to isolate singleaxons or terminals (i.e., minimal stimulation), it did not seg-regate dopaminergic D2 type receptor responses. Thus thepresynaptic D2 response was observed in almost all cellsrecorded from either young or mature slices and not observedin only in 2 of 18 cases. Terminals with any of the receptorclasses, D1 or D2, may make synaptic contacts (feedbackinhibition) with either substance P or enkephalinergic contain-ing neostriatal neurons (Guzman et al. 2003).

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

We thank D. Tapia and A. Laville for technical assistance, L. Carrillo-Reidfor software development, and J. N. Guzman for recordings shown in Fig. 7F.

G R A N T S

This work was supported by the following grants: Stem Cell ResearchGroup UNAM/Universidad Nacional Autonoma de Mexico 02, DireccionGeneral de Asuntos del Personal Academico, Universidad Nacional Autonomade Mexico Grants IN201603 to J. Bargas and IN200803 to E. Galarraga, andConsejo Nacional de Ciencia y Tecnologıa Grant 42636 to E. Galarraga.

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Muscarinic Enhancement of Persistent Sodium Current Synchronizes Striatal Medium

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