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Neuroscience 164 (2009) 345–350

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APID REPORTS

OPAMINE D2-CLASS RECEPTOR SUPERSENSITIVITY ASEFLECTED IN Ca2� CURRENT MODULATION IN NEOSTRIATAL

EURONS

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. A. PRIETO, A. PEREZ-BURGOS, T. FIORDELISIO,

. SALGADO, E. GALARRAGA, R. DRUCKER-COLINND J. BARGAS*

nstituto de Fisiología Celular-Neurociencias, Universidad Nacionalutónoma de México

bstract—The loss of dopaminergic neurons followed byopamine (DA) depletion in the neostriatum is a hallmarkf Parkinson’s disease. Among other changes, DA D2-re-eptor class (D2R-class) supersensitivity is a result of stri-tal DA depletion. Pharmacological, biochemical and be-avioral data have documented this phenomenon, butlear electrophysiological-functional correlates are stillacking. This work describes an electrophysiological cor-elate of D2R-class supersensitivity in DA-depleted striatafter unilateral 6-hydroxydopamine (6-OHDA) lesions in theat substantia nigra compacta (SNc). Ca2� current modu-ation mediated by D2R-class activation reflected an alteredensitivity. Thus, while the concentration–response rela-ionship (C–R plot) from control striata was better fit with awo sites model, the C–R plot obtained from DA-depletedtriata was better fit by a three sites model, exhibited aonsiderable leftward shift, and presented an increasedaximal response. Because Ca2� current modulation by2R-class activation is involved in the control of spinyeurons excitability and their synaptic GABA release, theresent findings may help to explain several functionalhanges found in the striatal circuitry after dopaminergicenervation. © 2009 IBRO. Published by Elsevier Ltd. Allights reserved.

ey words: receptor supersensitivity, dopamine receptor,a2� channels, Parkinson’s disease, 6-OHDA, concentration–

esponse plot.

n accepted experimental model of striatal dopamineDA) depletion uses 6-hydroxydopamine (6-OHDA) tonilaterally destroy dopaminergic neurons in the sub-tantia nigra compacta (SNc) (Blandini et al., 2007).opaminergic receptor supersensitivity follows DA de-letion: D2-receptor class (D2R-class) supersensitivityas been correlated with enhancement of turning behav-

or, increased receptor expression, and increases in the

Corresponding author. Tel: �5255-5622-5670; fax: �5255-5622-5607.-mail address: jbargas@ifc.unam.mx (J. Bargas).bbreviations: C–R plots, concentration–response relationships; D2R-lass, D2-receptor class; DA, dopamine; EGTA, ethylene glycol tet-aacetic acid; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic

icid; I–V plots, current–voltage relationships; �-CgTx, �-conotoxinVIA; 6-OHDA, 6-hydroxydopamine.

306-4522/09 $ - see front matter © 2009 IBRO. Published by Elsevier Ltd. All rightoi:10.1016/j.neuroscience.2009.08.030

345

oupling and stimulation of G-proteins and associatedignaling (Creese et al., 1977; Arnt and Hyttel, 1985;erfen et al., 1990; Qin et al., 1994; Schwarting anduston, 1996; Cai et al., 2000, 2002; Hayakawa et al.,001; Newman-Tancredi et al., 2001; Inaji et al., 2005).owever, no physiological function directly related witheuronal excitability has been correlated with D2R-classupersensitivity. Ca2� current modulation by D2R-classOlson et al., 2005; Salgado et al., 2005) controls excit-bility (Hernandez-Lopez et al., 2000) and synapticABA release (Guzman et al., 2003). Therefore, we

nvestigated whether D2R-class supersensitivity can beetected as an altered Ca2� current modulation in neos-

riatal neurons.

EXPERIMENTAL PROCEDURES

tudies were approved by the UNAM Committee of Bioethicsnd followed the National Institutes of Health Guidelines (NIHublications No. 8023, revised 1996). DA depletion after-OHDA has been described (Dunnett et al., 1981). Briefly,ale Wistar rats (180 –200 g) were injected stereotaxically with�g of 6-OHDA (Sigma, St. Louis, MO, USA) into the left

ubstantia nigra (SN: 4.80 mm caudal, 1.6 mm lateral toregma, and 8.2 mm ventral to the skull surface). 6-OHDAolution (4 �g/�l saline with 0.2 mg/ml of ascorbic acid) wasjected at a rate of 0.1 �l/min. After 2 weeks animals wereelected based on their rotational behavior (amphetamine: 4g/kg i.p.; �500 ipsilateral stereotyped turns) (Inaji et al.,005). The number of animals used in the experimental sam-les was the minimal possible to attain statistical significance.ll procedures employed anaesthesia to avoid animal suffering.

Neurons were obtained as previously described (Bargas etl., 1994; Perez-Burgos et al., 2008): the dorsal neostriatumas dissected from 300 �m thick brain slices incubated with 1g/ml of pronase E type XIV (Sigma) at 34 °C. After 20 min,

ells were dissociated using a graded series of fire polishedasteur pipettes (Corning Inc., NY, USA). Cell suspension waslated into a petri dish containing saline (in mM): 0.001 tetro-otoxin (Alomone Laboratories, Jerusalem, Israel), 130 NaCl, 3Cl, 5 BaCl2, 2 MgCl2, 10 HEPES, and 10 glucose (pH�7.4ith NaOH; 300�5 mosM/l with glucose).

Voltage-clamp recordings were performed on medium-sizedeostriatal neurons (10–12 �m main diameter; whole-cell capac-

tance 6–7 pF) (Yan and Surmeier, 1996). Internal saline con-ained (in mM): 180 N-methyl-D-glucamine, 40 HEPES, 10 EGTA,

MgCl2, 2 ATP, 0.4 GTP and 0.1 leupeptin (pH�7.2 with H2SO4;80�5 mOsM/l). Whole-cell recordings were obtained with anxopatch-200 B amplifier (Axon Instruments, Foster City, CA,SA) and monitored with pCLAMP (v8) and a 125 kHz DMA

nterface (Axon Ins.). Series resistance (�10 M�) was compen-

s reserved.

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G. A. Prieto et al. / Neuroscience 164 (2009) 345–350346

ated (70–80%). Ba2� (5 mM) currents were recorded whilelocking Na�-channels (1 �M tetrodotoxin). Current–voltage rela-ionships (I–V plots) were built before and after drug applications.itrendipine, quinelorane (Sigma Aldrich-RBI, St. Louis, MO,SA), �-conotoxin GVIA (�-CgTx) (Alomone Laboratories),-agatoxin TK (Peptides International, Louisville, KY, USA) anddCl2 were applied with a gravity-fed system. Concentration–

esponse relationships (C–R plots) were fitted to the Hill equation:

E��EMAX ⁄ �1�IC50

�L� �n� (1)

(E�effect of ligand (L), EMAX�maximal reduction of Ca2�

urrent, IC50�concentration necessary to obtain a Ca2� currenteduction�0.5EMAX, and “n” is the Hill coefficient). When n�1 wetted the data with a sum of Hill equations with n�1 each:

�i�1

n

Emaxi ⁄ �1 �IC50i

�L� � (2)

(Marquardt algorithm; origin 7, Microcal, Northampton, MA,SA.) Mean�standard error of the mean (SEM) of peak Ca2�

urrents are reported. One-way analysis of variance (ANOVA) and-tests were used to assess significance.

For immunoblotting, dorsal striata were dissected from brainlices and homogenized in 320 mM sucrose containing protease

nhibitors. Homogenate was centrifuged at 2000�g for 5 min; super-atant was recovered and centrifuged at 10000�g for 1 h. Pellet wasesuspended in the same solution and stored at �70 °C. Proteinxtracts (40–60 �g) were submitted to sodium dodecyl sulfate poly-crylamide gel electrophoresis (SDS-PAGE) and resolved proteinsere transferred onto nitrocellulose membranes (Bio-Rad, Hercules,A, USA). Blots were immunostained with the following antibodies:nti-CaV2.1, anti-CaV2.2, anti-CaV1.2, anti-CaV1.3 (1:200 dilution;lomone Labs) and anti-sodium/potassium ATPase alpha-1 (1:250ilution; Affinity BioReagents, Rockford, IL, USA). Immune com-lexes were revealed by using appropriate peroxidase-conjugatedecondary antibodies (Jackson Immuno-Research, West Grove, PA,SA) along with a chemiluminescent reagent (Amersham Bio-ciences, Piscataway, NJ, USA). Densitometric analysis was carriedut by using the quantity one software (Bio-Rad).

RESULTS

o observe whether Ca2� currents were changed after DAepletion, we recorded neurons from non-injured (naïve),ontralateral (control side) and ipsilateral DA-depleted (DA-epleted side) striata of 6-OHDA-lesioned animals (Fig. 1).ig. 1A–C show families of currents evoked with depolarizingoltage commands from �80 to �50 mV in 10 mV incre-ents (on top). Fig. 1D–F illustrate currents evoked by ramp

ommands between the same voltages (top: 180 ms; 0.7V/ms) (e.g., Perez-Burgos et al., 2008). I–V plots resulting

rom both steps and ramp protocols were superimposableFig. 1G–I; symbols, from step commands; lines, from rampommands). For clarity, next figures will only illustrate ramp-btained I–V plots. Average Ca2� current amplitudes were00�31 pA (n�46), 362�27 pA (n�48) and 345�14 pAn�164) in neurons from naïve, control and DA-depletedtriata, respectively (Fig. 1J; NS). Current density (current/hole cell capacitance) showed no significant differences

Fig. 1K), indicating that Ca2� channels numbers do nothange after DA depletion.

But neostriatal neurons express several classes of

a2� channels (CaV1.2, 3 LC,D-, CaV2.1–3–N, P/Q, R) c

Bargas et al., 1994; Salgado et al., 2005; Martella et al.,008). Therefore, we also evaluated whether their relativeroportions change as a result of DA depletion. Fig. 2hows I–V plots from two neurons exposed to three selec-ive channel blockers in sequence: 10 � M nitrendipine tolock L-type (CaV1) Ca2�-channels, 400 nM �-aga-oxin-TK to block P/Q-type (CaV2.1) Ca2�-channels, 1 �M-CgTx to block N-type (CaV2.2) Ca2�-channels, and fi-ally 100 �M Cd2� to block Ca2�-channels which are leftmostly CaV2.3 or R-channels) (Bargas et al., 1994). Theame drug application sequence was administered in sam-les of neurons obtained from both ipsi- and contra-lateraltriata of 6-OHDA-lesioned animals. Percentages of block-ge obtained for the control side (Fig. 2A) were 31�6%,2�6%, 8�1%, and 18�5% after nitrendipine, �-aga-

oxin-TK, �-conotoxin GVIA, and Cd2�, respectivelyn�7). While the corresponding percentages for the DA-epleted striatum were (Fig. 2B): 33�6%, 27�5%,2�3%, and 19�5%, respectively (n�8). No significantNS) differences were found (Fig. 2C). However, becausehis comparison was based on small samples of neuronsommonly exhibiting some variability, as compared to

arger samples in previous studies (Salgado et al., 2005;artella et al., 2008), we also estimated the expression of, N and P/Q types Ca2�-channels by Western blot anal-sis from dissected naïve, control and DA-depleted dorsaltriata (Fig. 2D). Basically, this analysis confirmed thelectrophysiological measurements: DA depletion inducedy 6-OHDA lesion of the SNc does not change the relativexpression of Ca2� channels present in medium-sizedeostriatal neurons.

To examine Ca2� current modulation by D2R-class inA-depleted striata we used quinelorane, a selective D2R-lass agonist (Gackenheimer et al., 1995; Coldwell et al.,999). As expected, not all neurons were sensitive touinelorane: D2R-class-mediated inhibition of Ca2� cur-ent was observed in approximately 70% of the neuronsrom naïve, control and DA-depleted striata. This percent-ge is similar to the proportion of striatal neurons express-

ng D2R-type plus those co-expressing D2 and D1R-type.n addition a few neurons may express the D3 or D4R-ypes (Surmeier et al., 1996). It was observed that 100 nMuinelorane, a concentration that hardly affected Ca2�

urrents in any neuron obtained from naïve/control striataFig. 3A, B), readily reduced Ca2� currents in neuronsbtained from DA-depleted striata (Fig. 3C). C–R plotsere built for D2R-class responsive neurons from naive

Fig. 3D empty circles; n�36) and control side striataFig. 3D filled squares; n�14). C–R plots were best fittedith a two sites model (black line; Hill coefficient

mean�estimation error)�0.57�0.12, r2�0.98) with IC50sf 493�90 nM and 40�17 �M (gray lines; see Experimen-

al Procedures). There were no significant differences be-ween control side and naïve striata (results were pooledogether). In contrast, the C–R plot built with neuronsbtained from DA-depleted striata exhibited a considerable

eftward shift (Fig. 3E gray circles; n�32) and was besttted with a three sites model (black line; Hill coeffi-

ient�0.32�0.04, r2�0.99) with IC50s of 0.8�0.1 nM,

9Pptoiti

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G. A. Prieto et al. / Neuroscience 164 (2009) 345–350 347

0�17 nM, and 9.8�1.5 �M (gray lines) (P�0.0001 and�0.001 comparing the two right sites and P�0.0001 com-aring the Hill coefficients; Student’s t-tests; see Experimen-

al Procedures). The results show that D2R-class modulationf Ca2� current is altered after DA depletion suggesting that

t reflects D2R-class supersensitivity: (1) the leftward shift inhe C–R plot and lower IC s correlated with the described

ig. 1. Ca2� current density is not altered in medium-sized neostriatal ny 20 ms voltage commands (top) from 80 to 50 mV, in 10 mV steps,rrows indicate the times where measurements were taken. (D, E, F)

o 50 mV (top: 180 ms; 0.7 mV/ms) in the same neurons. (G, H, I) Cun (A, B, C) (symbols) and (D, E, F) (continuous lines). Superimpositmplitude (mean�SEM) in samples of neurons (n�40) recorded in eahows no significant change in current density.

50

ncrease in receptor affinity (Chefer et al., 2008), (2) an ad- e

itional very sensitive site of action was unveiled (18% ofodulation; Hill coefficients were significantly different, from0.6 to 0.3, using the best fits in each case; see Experi-ental Procedures), and (3) the maximal reduction of Ca2�

urrent (Emax) was enhanced: 70�3% (n�32 DA-depleted)s. 57�4% (n�50 control/naive; P�0.005), paralleling theeported increase in D R numbers (Creese et al., 1977; Qin

fter DA depletion. (A, B, C) Representative families of currents evokedns dissociated from (A) naive, (B) control and (C) DA-depleted striata.ntative current evoked with a depolarizing ramp command from 80tage relationships (I–V plots) built with corresponding measurementss a match in all three cases. (J) Histogram comparing Ca2� currenttion. (K) Current divided by cell capacitance in each neuronal sample

eurons ain neuroRepreserrent–volion showch condi

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G. A. Prieto et al. / Neuroscience 164 (2009) 345–350348

DISCUSSION

t is demonstrated that Ca2� current modulation by D2R-lass activation reflects D2R-class supersensitivity, indicat-ng that this phenomenon is capable to disrupt intrinsic

echanisms of neostriatal neurons. Similar findings haveeen reported for globus pallidus neurons by using quin-irole (Stefani et al., 2002).

We showed that Ca2� current density and relativeontributions of Ca2�-channel types do not change ineostriatal neurons after DA depletion. As in previous stud-

es, these comparisons did not assume differences amongtriatal neurons. Therefore, they rule out adaptive changesn the molecular effectors (Ca2�-channels) themselves.

ig. 2. Relative expression of Ca2� channel types is not altered by Dntagonists were administered in sequence: 10 �M nitrendipine to b/Q-type (CaV2.1) Ca2�-channels, 1 �M �-CgTx to block N-type (CaV2

eft (mostly CaV2.3 or R-channels) on a sample of neurons from (A) cifferences in the percentages of block between the two samples. (D) Tf Ca2� channels in cell lysates obtained from naive, control and DA-dsed as a loading control.

–R plots built by measuring a function normally regulated f

y DA: Ca2� current modulation in D2R-class responsiveeurons, did exhibit changes compatible with supersensi-ivity: First, a leftward shift of the whole C–R plot after DAepletion demonstrated an increased sensitivity. Sec-ndly, an additional nanomolar sensitive site was unveiledSeeman et al., 2005) after a significantly different Hill coef-cient value. Finally, an increased maximal response waslso detected. Therefore, Ca2� current modulation reveals a

unctional correlate of D2R-class supersensitivity. Becauseelative contributions of Ca2�-channel types were not modi-ed after DA depletion, the differences encountered can bettributed to DA receptor-signaling alterations.

At present, the identity of the different sites of action

n in medium-sized neostriatal neurons. (A, B) Specific Ca2� channelpe (CaV1) Ca2�-channels, 400 nM �-agatoxin-TK (�-AgTx) to block-channels, and finally 100 �M Cd2� to block Ca2�-channels which are

7) and (B) DA-depleted (n�8) striata. (C) There were no significantis supported by Western blots comparing the expression of �-subunitstriata (n, c and d, respectively). Signal from ATPase (110 kDa) was

A depletiolock L-ty.2) Ca2�

ontrol (n�his result

ound is not known. Single neostriatal neurons express

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G. A. Prieto et al. / Neuroscience 164 (2009) 345–350 349

arious types (D2R, D3R, and D4R) of the D2R-classSurmeier et al., 1996). Therefore, a working hypothesisould be that one of these types is particularly involved

n the boosted modulation. Given that the affinity re-orted for quinelorane on D3R-type is about �1 nMGackenheimer et al., 1995; Coldwell et al., 1999), it isuggested that the novel site (IC50�0.8 nM) disclosedfter DA depletion could be related to D3R-type. Prelim-

nary data (unpublished results) using selective antago-ists for D2R-, D3R- and D4R-types are in line with this

dea. Alternatively, different D2R-type affinities resultingrom crosstalk or dimerization of some receptors mayxplain the data.

Ca2� currents are essential for adjusting patterning,

ig. 3. Concentration–response relationships for quinelorane. (A, B, Cn neurons from (A) naive, (B) control-side and (C) DA-depleted striata.triata, which are also affected by smaller quinelorane concentrations.filled squares) striata. Black trace is the best fit for two action sites,eurons from naive and control side striata were pooled together since–R plot obtained from neurons of DA-depleted striata was shifted to tray lines: IC50s: 0.8 nM, 90 nM and 9.8 �M (r2�0.99). P�0.0001 and

he right in the C–R plot from DA-depleted striata, respectively. Maximaas significantly enhanced in neurons obtained from DA-depleted str

xcitability and synaptic transmission in neostriatal neu- a

ons and circuitry (Hernandez-Lopez et al., 1997, 2000;erez-Garci et al., 2003; Vergara et al., 2003; Guzman etl., 2003; Salgado et al., 2005). The present results sug-est a cause of possibly changes found in these variablesfter DA depletion.

cknowledgments—We thank Antonio Laville and Marcela Pal-mero-Rivero for technical support and advice. This work wasupported by a grant from IMPULSA-UNAM 03, and DGAPA-NAM grants numbers: IN-201603 to José Bargas, IN-201507 tolvira Galarraga and CONACyT-Mexico grant 49484 to José Bar-as. G.A. Prieto, A Perez–Burgos and Humberto Salgado hadONACYT (Mexico) fellowships. G.A. Prieto also had a fellowship

rom the Fundación Alberto y Dolores Andrade; A Perez–Burgos

ntative I–V plots before (black) and during 100 nM quinelorane (gray)t 100 nM quinelorane only affects neurons obtained from DA-depletedplots for neurons obtained from naive (empty circles) and control siderepresented by the gray lines: IC50s: 493 nM and 40 �M (r2�0.98;

ere no significant differences; see Experimental Procedures). (E) Thembols) and better fitted with three sites, each one represented by the

1 comparing the two sites in naïve/control striata with the two sites aton of Ca2� current (Emax) obtained from control/naïve striata (57�4%)3%; P�0.005).

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G. A. Prieto et al. / Neuroscience 164 (2009) 345–350350

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(Accepted 12 August 2009)(Available online 20 August 2009)