ACTIVITY-DEPENDENT REGULATION OF THE DOPAMINE TRANSPORTER

IS MEDIATED BY CAM KINASE II SIGNALING

By

Shalini Padmanabhan

Submitted to the Faculty of the School of Graduate Studies of the Medical

College of Georgia in partial fulfillment of the Requirements of the Degree of

Doctor of Philosophy

January

2009 ACTIVITY-DEPENDENT REGULATION OF THE DOPAMINE TRANSPORTER

IS MEDIATED BY CAM KINASE II SIGNALING

This dissertation is submitted by Shalini Padmanabhan and has been. examined and approved by an appointed committee of the faculty of the School of Graduate Studies of the Medical College of Georgia.

The signatures which appear below verify the fact that all required changes have been incorporated and that the dissertation has received final approval with reference to content, form, and accuracy of presentation.

This dissertation is therefore in partial fulfillment of the requirements for the degree of Doctor of Philosophy.

Date - . · sor - Balakrishna M. Prasad, PhD

Dept. Chair - R. William Caldwell, P D

Dean, School of Gr ate Studies - Gretchen Caughman, PhD SHALINI PADMANABHAN Activity-dependent Regulation of the Dopamine Transporter is Mediated by CaM Kinase II Signaling (Under the direction of BALAKRISHNA M. PRASAD, Ph.D.)·

Dopamine signaling in the brain governs a variety of functions such as locomotor activity, reward, attention and working memory. The dopamine transporter (DAT) plays a crucial role in the clearance of extracellular dopamine and thus helps terminate dopamine neurotransmission. DAT is also the target for psychostimulant drugs of abuse and therapeutic agents. Changes in DAT expression occur in neuropsychiatric disorders such as attention deficit hyperactivity disorder (ADHD) and chro_nic psychostimulant use, and variability in

DAT abundance is associated with differences in working memory. However, mechanisms regulating DAT expression are poorly understood. We tested the hypothesis that neuronal activity is one of the non-genetic determinants of DAT abundance. Chronic perturbations in neuronal firing, caused by pharmacological agents, significantly altered DAT expression and function in primary cultures of mesencephalic neurons. Pharmacological experiments showed that calcium entry through L-type voltage-gated calcium channels and calcium/calmodulin­ dependent protein kinase II (CaMKII) activity played a role in activity-dependent changes in DAT expression. In order to further evaluate the role of CaMKII in

DAT regulation, the effect of sustained depolarization, a stimulus often used to study activity-dependent changes in gene expression, on DAT expression was tested. Surprisingly, chronic KCl-induced depolarization decreased DAT expression and function. Measurement of CaMKII activity in dopam·ine neurons showed that chronic depolarization led to a decrease in CaMKI I activity, even in the presence of elevated intracellular calcium, due to activation of the serine/threonine protein phosphatase 2A. Moreover, increasing CaMKII activity in dopamine neurons by introducing a constitutively active CaMKII mutant caused a significant increase in DAT abundance while inhibiting CaMKII activity in dopamine neurons using a dominant-negative CaMKII mutant decreased DAT abundance suggesting that CaMKII activity is both sufficient and required to cause changes in DAT expression in a cell autonomous fashion. Taken together, our data demonstrate that CaMKI I activity can govern DAT expression and may play an important role in dopamine neurotransmission in the brain.

INDEX WORDS: Monoamine, Transporter, Action potential, Depolarization,

Neuronal activity, Calcium, CaMKII, protein phosphatase 2A, Mesencephalic ACKNOWLEDGMENTS

I express my heartfelt gratitude to my advisor and mentor, Dr. Balakrishna

Prasad for his enthusiasm, patience and perseverance in training and educating me. Everything I know about doing research, I have learnt from him. He has played an important role in molding me as a scientist and in doing so he has built a solid foundation for my future career in biomedical research.

Many thanks to_ the School of Graduate Studies (Dr. Gretchen Caughman) and the Department of Pharmacology (Dr. William Caldwell) for their financial support through the years. I also thank Marvis Baynham, Dora Walden, Jennifer

Nunn and Wendy Cliett for all their help, without which timely completion of administrative work would not have been possible. Their assistance is greatly appreciated.

I am also grateful to all members of my thesis committee: Dr. Lori

Redmond, Dr. Clare Bergson, Dr. Nevin Lambert and Dr. Wendy Bollag, for taking a keen interest in my project. Their valuable input, guidance, support and encouragement through the years have been instrumental in my growth as a scientist. I also thank Dr. Lynnette McCluskey and Dr. Krishnan Dhandapani for readily agreeing to serve as readers for my thesis defense.

V vi

I am indebted to my many student colleagues for providing a fun and intellectually stimulating environment during the last five years. I specially thank the members of Redmond, Lambert, Bergson, Browning, Ganapathy and

Bieberich labs for providing me with lab reagents, supplies and technical assistance when I was in desperate need of them.

I am grateful for the support I received from the wonderful people I met in

Augusta - Kavita Natarajan, Bindu Pillai, Anil Pillai and Muralidharan

Jagadeesan who made my stay truly fun and memorable and whose friendship I will cherish for life.

I thank my husband, Kannan Krishnamurthy, for all his help during the last five years- for patiently going over and over the basic concepts in Biochemistry and Cell Biology, for his help in proofreading every scientific document that I wrote and above all for his moral support. He was a second mentor to me and above all my greatest friend. It truly would not have been easy without him.

Lastly, and most importantly, I thank my family, especially my parents,·for always standing by me and for cheerfully supporting me financially as well as . emotionally, for always encouraging me to do my best and for constantly checking on me. Without them, none of this would have been possible. ACKNOWLEDGMENTS

Acknowledgment of Contributions to Manuscripts:

I would like to acknowledge Dr. Nevin Lambert for his help with electrophysiological recordings of 4-AP, TTX and KCl-treated primary mesencephalic cultures.

I, Nevin Lambert, agree with the above assessment of my individual contribution to the manuscript within Shalini Padmanabhan's doctoral dissertation.

Signed Date

vii TABLE OF CONTENTS

Page

ACKNOWLEDGMENTS ...... v

LIST OF FIGURES ...... ix

I. INTRODUCTION

A. Statement of Problem and Specific Aims ...... 1

B. Review of Related Literature ...... 5

II. PUBLISHED MANUSCRIPT

A. Activity-dependent regulation of the dopamine transporter is mediated by calcium/calmodulin-dependent protein kinase signaling ...... 37

Ill. UNPUBLISHED MANUSCRIPTS

B. Sustained depolarization decreases calcium/calmodulin-dependent protein kinase II activity and gene expression in dopamine neurons ...... 66

C. Calcium/calmodulin-dependent protein kinase II activity governs dopamine transporter expression in primary mesencephalic cultures ..... 94

IV. DISCUSSION ...... : ...... 113

V. SUMMARY ...... 122

VI. REFERENCES OF LITERATURE CITED ...... 123

APPENDIX ...... : ...... 139

viii LIST OF FIGURES

Figure Page

1 Rationale for experiments proposed...... 2

2 Schematic of the saggital section of the brain indicating the maior dopaminergic pathways ...... 7

3 Dopamine signaling at the nerve terminals ...... 12

4 An overview of the maior afferent inputs to midbrain dopaminergic neurons ...... 27

5 RT-PCR assay validation ...... 58

6 Specificity and utility of the new DAT antibody...... 59

7 Chronic changes in neuronal activity alter dopamine uptake in mesencephalic cultures ...... 60

8 Chronic changes in neuronal activity alter DAT protein and mRNA abundance without influencing dopamine neuronal survival ...... 61

9 Neuronal activity regulates calcium influx into dopamine neurons via L-type calcium channels ...... 62

10 Changes in neuronal activity cause sustained changes in CaMK/1 activity in dopamine neurons ...... 63

11 Activity-dependent changes in DAT function are mediated by calcium and CaMK...... 64

12 Sensitivity of western blot assays to detect changes in TH and 8-actin ...... 65

ix X

Figure Page

13 Depolarization-induced changes in CaMK/1- and ERK-mediated phosphorylation in dopamine neurons ...... 86

14 Chronic depolarization decreases CaMK/1 activity in dopamine neurons ...... 87

15 KCl-induced increases in intracellular calcium are sustained for 2 days...... 88

16 PP2A mediates the sustained depolarization-induced decrease in CaMKII activity...... 89

17 Chronic depolarization selectively decreases DAT mRNA and protein abundance ...... 90

18 Chrqnic depolarization decreases DAT-mediated uptak_e ...... ·... :. 91

19 Chronic depolarization does not alter survival or morphology of dopamine neurons ...... 92

20 Chronic depolarization does not increase the activity of PP2B ...... 93

21 CaMK//o and CaMKl/y are the predominant isoforms in dopamine neurons in culture ...... 11 0 \

22 CaMK/1 activity is both sufficient and required for causing changes in DAT expression ...... ·...... 111

23 CaMKl/a and CaMKIIB antibody validation in immunocytochemical assays ...... 112

24 Schematic representation of the molecular mechanisms involved in activity-dependent regulation of DAT expression and function ...... 116

25 Acute .changes in CaMK/1 activity do not alter DAT-mediated dopamine uptake or efflux ...... ~ ...... 119 ......

. . . . :l INTRODUCTION

......

...... - A" $tatemer1t: of the.·problem ari~-specific_:aims ofthe-overan:project --

. . . · ·:Dopamine·signalin,g· in the· b.rain ·play~ a· crucia_Lrole inar, -array o(functions:_such -

a~ loc~mo~':)r activity, a~tentio~, working_ memory and:rew~~d~related behavio~s : :·: .

. . (Kell~y·_arid--~erridge;·-~~02; Sch~~~z.,_ ~002)'.:_The.·dop~ry,ine-frar:,~p~rter (PAT) IS:·a: .·

...... ·:key ·presynaptlc protein .th_at·limits:~opam!rieneuro~rarisnii&s1on .(J·ones· e~ at,:· __ --

...... 1_998}. Hence,_·chan_ges_in_DAT·apundan_c~ are exp~ct~d-to modify_.d~pamin~~ ·

associated behaviors~ A number.of studies.have· demonstrated.that.

...... p~ych.ostfm~lants,·:ep~ictied·:er1viror.111i:en_t~. andtestricted:·fe_eding are_:~apabi~ qt, : :......

...... __ altering- QAT abun~ance ·(s~·nnon:et -~'!:; 2002; __ Be.llo-·et a.L; ·2003-; _Bezard_et_ at, · ..

...... ' ' . . 200:3}.: Ho_wevet,·the nie:chanism biWhichtt1es·e dive_rse·phant1acologicar and.

...... : . . . . ' ...... envJron_r:n~ntal. si~~als :re:gul~te-DAT_ expres:si_on. is ·not known .. At_ t~e. ceUular. - .. -:ley~f~ :the.se':f«fo~ors co~id-corivE!rg~·to· aite·r· the:acti_vity of doparriine· n¢urons.· ...... - ...... Qop_a.-~i-ne.neu'rons·_fi.re_:~ctio,:i p·otentia_l.s ~ricUh~ir acth~'ity can_:~e a!ter~_d ~y- __ .. · . . . behavlo.raUy' re.levaril s:timuli -~s·wefl-:as· dopa'minetgi_c._drugs. (Mercuri et.a•i.:, 1992;_._ -·-: .. . .·. . .· ..· .. ·-...... '.. . .·-.. Sch~lti:, '1.~~~r Thusithe:maj_~~:goal ·of this:study:~a~. to".t~~~ if :neur~~al :activ_i~y : :. ·. __reg.ulate~the-e)(pression _an_d·functior1·ofDAT. __ Un_derst~~di_ng·tn~_m~chimi_~-ms .·.·:·_-.

i_nvC>lved iD- atti_yity~dependen·t ·oAJ reg.ul.at'i_on· njay:p_rovide ~oyel th.erap·~utic. ·_: ··:·: -· __ . . .~tress/Environmental factors/Drugs of abuse

• 1 Neuronal activity J ··· · ·

. . . ?' ··. . ·1. ·.... .

. . .. . ·.. ·. -1·...... · . '......

Behavior : ...... ' ' . ·strategies·.for.ttie freatm.ent of dopamine:~Unkedne.urological':and psychiatric.·.:.

disorders... ··

·sp~~.i:fic .Airn•.~::·To.test the.ro1e:·of·~~uro·~.a:1·a~tivity.!n" m.ediatfr1g chang.e·s: in·DA! .. ·.- .

·:expres·~ion ih_. primary mese~cephafic ·cultu.re.s ·:·.

■ ... To test.the effects of chronic changes in.-neuro.nalfiring, caused. by .. ·

.. -.pharmacoiogica!'ag.en.ts, ·011 DAT exp·ression··ancJ..funcfion.·. - .·

...... '...... ■ ·T~ ide~.t.ify the ·mute .of calGitJm .entry and.cal~iu.m-activa~e~ sig~.alin.g ..

. . . . · · .mechanisms_•i'1volved in .. activity.:.d.ependentDATregtJlation.

-· ...... ·~pe~.~~i~ Ahn .2:Toinve.stigate·th~··~ole of 9aM.Kll.sig.naiing .:in :the.teg:ulat.iori·o(.:

...... ' . ■.-. _T9·t~st_.the.~ffect of susta~ned depola.rization., a· GOmm~mly.·used paradigm.to ..

. . ·study activity ...dependent g·erieexpression; on·caMKU·activity and DAT . ·· -

... expression .

■.· · ..To.te~tthe··role .of.phosp.hatase$ in· regl.il'ati.ng ·CaMKlf.activ:i'ty .i.rid.op~m.ine .. · .·.

neurons .·

·:S.pecific.:Ain1· 3: :To d.eten~ine :if CaM~li:·ac~ivityis·gufficiefrit fo: c·ause·_chahges in:.·.

DA(.ex:pressi9n.i"n ·a .ceH:autono~o.us··fa~hio_n· in_pr1~:ary.m.es:en6e.phali~:c'i.11ture~:.

. . .. · ·:Ghten: the.i.~portari~e:of DAT.i.n:maintairiing :dop~n~itie ho~:~ostasis :a~ well.as.:-

the.alteratio~s.in.it~.expr~ssion.·observed· in.·certai.n.disease. states,. ~n.·.·. . _1.-_- ·_ ... :-_4-

...... ·:unde_rsta.nding_ of the.mecha~isms· blwh.ich DAT .abundance. can b_e.Tegulated_·js.

. . . required _in orderto p"rovide_new·insights-irito"the· proces.ses leading to these··

...... n~uropsych_iatric -d is~rde·rs: :I. INTRODUCTION ' . . . .

. - ...... - . • • • • • > • B. Literatu·re· review·· .

.. 1~ The.dopaminergic systern·.and··its··role: in behavior.-·

. -Alth:ough dopamine neurons·_coristitute orily:o:ne in:every".milii•on ·neurons in the ' ...... - . . - ...... brain,: they· nev~rtheless :con_trihute· ·sig·nif1cantly_ to· ·several· aspects_· of. behav_ior --:· .. - ...... ·. ·:(Banrion .et aL; :2001°): .The cell:bodi:es·_of'p.opan1·ine, ne:ur~n·s _are.clustered inth~-- ..

midbrai,n largely .in tw~ _areas:. the:s~bsta_ti,tia.nig_ra·(s_N) and·'tl:i_e_vent~al:tegmen.tal ..

...... ' ...... a·rea"(VT A).' Neuron·s· ~ram.the -~N-projectto.·the ·striatum (riigmstriataLpathway) .... ·

- ...... ·:while net.fro_ns arising: ir.i the.VTA proJeq(to the·:nt1cleus ac_q.umberi~ (n1esoli'r1~bic : ...... - . . . - ......

...... · .. Pathway)._and the p·refrontal __ cortex·_(n1.es_ocortic.al pathwa.y): .Dopary,ine -rele~sed·-·_· ·

at thenerrninal$:birids to:its receptors "to elicitdifferent behaviots: The:n_igrostriata·1 .

...... ' . . . . .pathway ·is- vital for locomotor -a¢tivity;· the: niesolimbi~ -~ystem. is· important_ for the .

-:fee!i"ngs ofplea:sure··aJJd rewcfrd;'while dqpaniine neurotrarismission":.in.tne -:· .·......

. . . . mesocortical° pathway-plays an fmportanfroie in _co.griition, .attention .aricf workin·g:: ...... - ......

. . . · d~parriin·e ~ignaHng ~xecutes s~ch:cruci·a:I functio_n~, :it isConceivab_l~ th~t ·:· _

_dysre·g_ul~tion-·o_fd()pamirie ~eurofr~n_s-missicm _Gan.re~ul~ in sever~I ·n_eur:ological· -_· ·

...... and psychiatric disorders:. -: ·:For:example~:-degeneration ·of dopam1ne.neurons.·of_the ;n_igrostriat~l.p~thway .is-_·.·

. . . . . )mplicated··in the· pathogenesis.of Par;ki'rison's 9isease·'(p.D) (Dawson and ·:· ·· · - -·

. . . . . Dawson, 2_00:3):whil~ dysfunctio:~s: in d()pamihe s_ig:~aling ir:,-the_me~oiirri~icand :·

.mesocqrticaLpathways· qccu_r in:disorderisuch ·as--d~l:J~ ~buse; -~~hizop~renia-.an~---- .

· atten.t1o·n:9efi~it hype.radivity ctis_ord:er"(/-\DHD) (Yang:e·t al.,J999;·:Everifran~ ·:· ...... Wolf, 2002;. Staller and Faraone, 2007) (Figure 2f .

.. 2~ ·oopamine·•signaling: ·.

Although them·idbrain.:dopamine· neurons ·e•xtiib.it anatomicai heterogeneity,·_they: · .·

...... - . . . - . · a. Oopa~in_,f synthe$is: -_· ·:·_·

...... Qopamine is• ~_ynthesiz~d .fror:n th~ amin~ ~cid pr.ecu_rs.or tyro~ine. Tyrq.s_ine is._·.··

converted··to LlJOPA by.tyrosine-Jiydroxytase (THfand L~DOPA is'then--:

·:c9nverted ~9 _dopam_iq~ by ttie e~zynie arom.atrc _amino acid: 9ecarb~xylase: .

. . · ..(MDC) (Misu-et-aL,· 2002).Jn this: set of.reacti.9m~, the-c_onversior1_ oftyr:osi_~e-to ·:

...... L-DOPA by TH':is the rate~limitirig:·step· in.:ci9paniine·):>iosynthe,sis (Mis·u·:efal_:, _: ··:·:

. .. .. " ...... ·_19·9:sr ·Ttius;TH ~~tivity :g~l'i~rn·~ ~opam·ine: synthesis-and is:b~lieved:to be·_ .. ·-·

. pa_rticuiarly ·s_Lfaceptib1e·'tc:,"physiol9g·icai ·r~·gulatiorr~Y phosp~orylatiofl ·at,d .:· .·.

. . ~ep~_~sph9ry·1_atkin.-_S~y~ral reaid~es hav~ beem_id~:~_tified. o~J~~ (~er1_s:,_:$er;31 _ imd $er4o)·th~t_.G~nbe:pho.sphory_la'ted by'qifferent :ki_riases r~sultirig· in_ c1ctivation,.,

¢al.cilim/c_almodulin~depe·ndent_.prt?~eirj.k~na·se ll'(CaMKll),· S·er4·0·bY-P.rot~.in ~in~se·_·...... Figure 2. Schematic of the sagr,ital section of the brain indicating the major dopaminergic pathways

Represented in the figure is the location of the dopamine neuron cell bodies (SN and VTA). Also demonstrated are the three majordopaminergic pathways in the brain.

1. Nigrostriatal pathway (SN to striatum)

2. Mesolimbic (VTA to nucleus accumbens)

3. Mesocortical (VTA to PFC)

(Taken from www.drugabuse.gov) 7 . . . . -A (PKA);:protein- k1~ase_ G (PKG) _and protein ki~as.e_ C (PKC), and·_Ser~1-by .· .· · ...... '. ·:ext~acellylar:·sigria!~regulated·kinase V2-(ERK1./2)- (Hayco·c_k,··199_0)'.' .Of theJhtee :_.·. _. ··

-: ary,irto: ~cid _re~idues_,. p~6~'ph(?ry.lation 0~ S~?fr46 has-the_·gre_~te:st ¢ffe_~t-on: !H .. _: ..

...... -- ·.·:- -: .·_.· actiy_i~yin_vivo_.(C_arj,pbeli ·et a1.:--1986).·.oeph'o:spho~~:atio_n ofTH_ by .

. . . . . ·:serine!thr~oil_ine_.ph_os·phatases·_can.-al~o:·r~gyla~e :lJ{~ctiyity. Of th_e majo·r ·:

...... serine/threo~ine.phos_phatases,· protein phosphata·se. .:.2A {P_P2A) hasbeen shown .

. . . -: to.cause dephosphorylation ofTHat Se'r40 ·1eadingto-.its inactivation-(Haavik-·et_: -_._

...... ·:al._;J 989).·_. __ -.

__ b-■ -DopalJline·release .·.·:·. ·

-: Withfrt the· dop.amirie ne.u:ron·,· ve:si:cu·1ar morioari'iine tra'nsporter (VMAT2): is ...... 'respo:n~i~le'for:p~ckagi~~-.dcipamine i~to'syriaptic-v~~i~l~s-·usi~~ a·.pro~Clri_:

·:grgdien't-(Sc~uldiner et· al,' 1~9_sr The .fi.ri.~g Of.:an:·~·cticin:·poten:ti_al:fol_lowed by a_n·: · .

...... · .. increc1se in .i~tr,aqellular qalcium ,n_ -the ·pre~yn_aptic •n~rye term_inal c_aus.e_s _

:: dopamine·c~htaining·~es.ic.les_ tc{fuse with the. pli:i's_ma mem~rane an~:release

· 1 ·:do.parriine (Pete:r et. af.-,_ t995) ..Plasm·a merribrane-do'r,amirie_receptors (02·: ...... ' . . . .

receptors) play_~n important role in-exerting fe~dbacl< regulatory effects on

-: doparni·ne_rgic·tr_a·ns.mis·sion. ·

. . . -·_. --->. --~a_sed-on·ttieir-furictional:effe_cts,-D2 re_ceptqrs can.-be·c_lassified as_ .

:synthesis-r~gul:ating· ~~d impul~e~regula~i~g -autor~_c~pto~s-_(~qt~ et' at.·; 2~00}>_ .. : _-...... j.\ctiy~ti~m-of-Q~: recepttjf~_:locatefat· th_e. m~-rve terni_i~a:1·s ·is_invol_ve·~- ind~preasirig: ·_ -

...... ·:a~~ibuted.to· inh~bitiorfofadenyiyl cycla~e.and a ~ubsequ~~~-de:creas~_ ir\.TH ...... ·:receptors·(Stoof·and .K~babian,·:1981}.lmpu.lse~regul:atin.g autoreteptors:are. _:_ ...

...... ·. ·:associated with sQniatodendritictegions-as:well'as hetve·termina1s·. Activation-of:.·.

. . . . ' ...... D2 rece'pto_rs In'the·somat6de~dritiC:re9iohs can _incre~se potassium_

conductarices_-to_hyperpe>l'ari_ze·th~·cell·and_.inhibit c~U-firing·(~wm.eieretal.~; ..

. ·:200_7). On the 'other.hand·, 'activation of D2· recepto·~s· at th_e ter~_i.na_ls:~arr ·:·_

. '. decrease ca_lcium currents to.prevent dopamine_.release aqhe·nerve_termina1s·. · . . ' . ' . . .

. . ' . . ' . . . ·:tur11.modulate the 'firi11g of. s·omc;itoderid,rjtic_ dopamine neur9ns :Vic:ifeedb.ack' .· _: ·.

· pathway~. (discuss:ed.in section 7b}.·

c. Dopamine -~Je:arance: ·.

...... Like· with ~tlier :n·eurotransm\tter:systems,: prolong.ed dopamrne sighaling··is·.- ··. ·. ·:p.reyented'by the' existe·n:c·e-of s~veral· neLiro(ran·sn,itter clearance:me~h~nisrr1s.···

The_si.mpiest rr1e~h-~n!sr11: for·trans:mitter. cleara~ce•·is passive:_diffu~io~_:9t.the_ . ·· · ·

neu:rotransriiitter away)rom re1e:ase:sites/.Other·mechanisi11s·.invoive :cl.earance ·.·. . . ·:~Y:m~no·anii:ne :oxida~e _(MAO},:-a_ degr~tj~~ive ·e~?'.yme p:rese.nt :mai_n_ly:,i_n the. cmt~r: . . . ·. _mitoch.on_drial-·rri~n,brane inJhe presynaptic:·ne~ron; an~_.by ·reuptc:1ke .. int.o-th..e ·_.- -··

presynapt1c rieqron_.by high affinity transpmters'.

3. T~e dopamfr1e iram~porter ancUts role :in term.ina'ting dC>pamine' signalins( _·.

The·cioparr,in:e transportefr(DAT).-is .~ memo.er of the.sodium ctnd chloric;te~

·:d~·ileliderit_ tran·sporters thatal~o iriclude:theJwo ~ther ni~~:~amin•e _trans·pod~rs_- :

_the· norepin~phrin~_·a~ct-.s·~ro_ton·in -~ransport~rs (M~ss_on et al.·,J 9~:9). - □AT-... · · . . . ·. Jhe tr~nspo~. of soµiu_~--~nd:ch_loride i:ohs (for every molecu1e··of-dopaniine_:· · ·

' tr_an:sp:~rted_, ~-~•a+ a·nd:1'cr a~~ 6o~t~arisp6rte_d) 'thus:nlaking this :process -:

ele~~~~g_enic·(~onders :e~:-~~-, ·.19~7). Bes1de~--~~_rvin~ l~S: primciry'furictio_~.()f .. · .·'

. . ·:i_nfluencing th_e spatiotempor~I :p_ropertjes _of c;jopamine neurotra~smission', DATis ·.·

also.t'tie· primary targe(for.psych.osti.mu1a·ri.cfrug,s. of abuse.suc.has:cocaine·and:.:_: ..

amphetamh,es-(Ama'ra· and s·oriders, .f998;-.Swans:6n,et at,'t9.98) .. ' This· '

...... ' . . transporter is.p,resent,in the:p~·ri.syna'pti9_reg:ions (HoWella_nq Kimmel;. 2008), _:_

·... exclusive.ly ·on·pre_synaptic q.op.amine-:-synthesi~ing neurcms (Cilia}( et.at,.·1~95). ·

The critical role:played by.DAT hterminatirir{the a:~~~o·n of rele~sed dopamine.:· .

...... was' d:emonstrated. by 'studi'es· using' pharmacological: b_lockade or ·genetic. deletlon .... '

. . . - ...... ' ...... ·:Of this ~rarisp~rte'r.· ·_Blockers·_otDAT,-iric.1u·ding-c·ocaine and_-rnethylphenidat~, :· ..

increc1se the ·e)(tracellular .concentration of,dopamiqe_(Church_·et aL, .19~7; Prascld.

. . . ' ...... - efal., .1999): ]n.micefacking. the dopamineJrans·porter,. dopa.mfne. ·

...... ' ...... - . - ...... :n~u_rotrans_n,issiori:·i~.:~ugment~d and: d(?pamirie J>~rs:ists-~t least 1OO_tinies·10Mger......

PA.T IJ/oc_kers: and sLib$trates : · ·

·_Both.DAT.blockers_ and su_bstra,es can.act as. psychos~imulants_by.increasi.~g·.· ..

·. ·:dopa·mrne_ sighal:ing· in the:·me·soco~ic~iin,_bic:sy~te_~-: While·_DAT ble>ckers (~uch·.: -:·

. . . a$ C()caine a~d ~ethylp_~enidate) ·_incre.ase_ :extracell.ular d_oparni~e ·by· preventing_· · .

dop:aroine··clea.riince '(2~h-nisenind Sorkin; 2004),·the·mecha_nism·ofsµbstrate :· ·

'. . ·:a~tiori :is ·m~re c9mpl~~- Amph_etamines :~eltjngto :the. group :of. psy~h:C?,strmtilants : :.

th~t increase dopaminel_evels·bycompetitively_inhibi~ing_DAT.mediated·_·. :-11-·

'dopa_mine_uptake. ln.addition_, by acting.as substrates·fotDAT;.amphe~ami'nes·:­

·:get_ti"arispo~~d:·i'nto·the'·cytqplasm'aqd'eng'age.fr1:.a-heteroexchange·.methariisni

-: whith:~esul~si~:t~e. ~~Ver~~ tr_an:s:p:oit -~f dbpatni~e-from the 6ytoplasm to the : ··:··

extr~9~_l_lu_lar-~pace_·(VV~i~e-:and ~~_l_i¥as-,· ·19_~_8). By ~c:tinj_ng--a~~~s-~ to th~---·-: _·.- · · :·.·_._.--:

· · ·.· in :cytop'iasmic.dopam_ine: conce·ntration (Sulzer ·et _ai.:,: 1993): .T.hus;. urilike. ·.... ··_:_:_: ·.·

-_- -: blockers that:de.pend o·ri.-impulse.·activity'of.the dop~mine•·rieurons to·cause- ari :·.·_._· -:

...... ·:inc,re~se·:,n _extraceUul~r dopamin_e:1ev~I_S,:.the ~ictjcm of DAT:S_Ubstrat~.s is ..

. . . . . · )ndepenclent o(ac~ion- poterJ.t°ial fir1ng _(Figure.·3).· ......

...... ·:4. Reg~lation' of the·dop~m-ine· transporter

...... _ ~ecc1us_e tt,e· PAT is. ~·crjti~aldete_rmin_ant_-9qhe_.dur~tion·_o.fd()pam_ine .s_igna!in_g,_

...... · ._ -: changes iri the .. expression:or'flii1.ction-6fthe. tran·sp:orter wou:id-have 'ii significant.-.

...... ·: 1~pacf on. _tti~. presy~_~pttc ahf p~stsynciptic :respo~ses to_ _released_ ~()pam in~::_·:

a. Regulation :of DA r fu,nction: · ·

., .. .. Numerous.-stud_i~~ have d~mon~trat~d.th~t DAT..trafficki~g-to .and.from .th~ .

·:Pi~S~a· mem.~r~~e in:{Ei~p-ons~-_tcfsever~rc:ellul~r-r:>roc·esses··ca·n -~ffe?t _DAT

L Regulatiorr'by membrane pt>tential:. ·· · · .·

...... Because dopamine -~ptake through.DAT i$ electr9g¢nic, :,hvas·spequlated that:...... _·_. ~en:ibr~ne .vo_ltage_might i_hfluehce the.ki'n_etics-of-th_is pro.cess. l_i1 e·le~,rically· .·. . ..

...... Figure 3. Dopamine signaling at the nerve terminals

Dopamine is packaged into vesicles in the presynaptic neuron via VMA T2. A presynaptic intracellular calcium rise causes dopamine to be released into the synapse. Dopamine can bind to postsynE;Jptic dopamine receptors, including the

D1 and D2 dopamine receptors and presynaptic D2 autoreceptors (that act as a feedback mechanism to regulate dopamine release). DAT is located : perisynaptical/y, exclusively on presynaptic dopamine-synthesizing neurons, and functions to terminate the· actions of dopamine. Psychostimulants target DAT to cause an· increase in synaptic levels of dopamine. Cocaine blocks DAT thereby inhibiting uptake of dopamine into the presynaptic nerve terminal.

Methylphenidate, used in the treatment of ADHD, also inhibits DAT function (not indicated in the graph). On the other hand, amphetamines get taken up by DAT and promote release of dopamine. 12

• • Postsynaptic neuron

Dopamine neuron

Dopamine Transporter Amphetamine (DAT) Dopamine (DA) Dopamine receptor • (D2) Coe Cocaine

' Dopamine receptor Vesicular Monoamine (D1) Transporter (VMAT ) ' '' 2 . . . · voltage~dependent:- DAT ·uptake .increas·ed·at hyperpo:larized -rn~mbran·e_:· ·· ·

·. _potential~ wherea~ me~brane: depol~rizati()ri. \JVa:s :s~:own to be ass6~iated: Y"ith: _·:

-red_~Jce~--tr~·n_~pmt-vel_q~i-~y_(Z~h~i~~-r-·and_Poolen;·_2001}.-How~ver, -s~udi~$- .·.·:·.-.·

...... ·__ changes in .membrane_ potential.do not alter the.uptake_ki_netics of~opamine _. ·.· ._

...... -(Prasad·a~d.Amara,:2001):.

iL. Re_gulaii_on by:.substrates -~_nd bi°ock~rs:-.

. - . . . . -· In addition to: pr9moting dopamine release·,· OAT subs_trates· su9.h·as·dopc3mine: _·...... and :a.niphetarrtiries are believed to "regulate cellSutface exptession of the

.· .. ·.- fra~sp~rter:.-.In· HEK293-9~11s· ov~t~expressing·DAT, ·addition of DATsub_sttates .·

...... --: · . ·:· . $µch ·as ·dop-amine and ·amph~famine c~used ·gr~·ate·r DAT k1t~r~al_iiati6n -.

...... ·_. re~ulting. in _decreas_e_d d_opami_ne._up~ak~_($aun~ers et ~1.,· ~.000)·._.I.~· qontra_st tq·_ .

. . . ·. the. effect observed -with :DAT :substrates~ .. DAT blockers· ·such. as. cc>"cairie and: .. · .

·:. _.m.ethylph~nidate \IVe~e-shb\fY_n-Jo increc1se· DAT tr-?tnsp6'1 _by i"ricrea~i_ng .the _ceU · ...... - . . . · surf:c;tct3. expre$s_\on. of OAT-(l.ittl~ et. al_., -2002,);· Although the$e ~tudies su_ggesf _·-

...... that:altered ·OATtraffickii,g· in :respo·nse: tp su~strat~s a_nd.ir,:~ibitprs:c9uld·_· · · · · ·

- .· cause cha~ges in--extrac_e.llul·ar .dopamine· levelsJhe mole¢ular -~e·chanis:ms >...... ' . ' . . . ' . . . . . _. involved·i·n this pro·ces~itemain· unkriown.: · .. · .·...... iiL. Re_gulation· _by autorec~ptors: -

...... The.·o_bservatjcm .thaf_dopamihe .. cl~aranc~·i~ m~n.k~~:Uy .redLJced in rr,iqe .that.

...... __lc3ckthe lJ2:_re¢eptC>r-(Dickin~ol'.l et aL! _1~99) J>-~~~pted:s~~~ie$ to_ ~~":~stigat_e:~he: .

...... ' . . . . . ' . . . ' ...... ' ...... '. . ' . . . . . ·:· .. been proposed forD2_-mediated· regulation·ofDAr function .. Asl~2agonists .can.·

...... · activat~· invvard"".re·ctifyin·g .potassiuq, channels ·Ieadingto· .hyperpolarization, ·it.·.

...... ' . ' . . . ·.. was· ~pe~ulated thatautore~eptor~niedi·ated r~gulation·.of DAT function ·occurs:· .

·. dueto altera.tkms.·in:niembran~.POtenti·a_LQ.2 receptor agohi~ts were_~hpwn'to.: .·

.· .increase ·oATtrans·p'6rtcapadty·and thiS:p.rocess·was sh9wn to b·e voltage~· ...

...... ·.· .dependen.t .in-oocytC3s.·over~expressing_'DAT and D2 receptors.(Sonders et.al_:, .. ··

. ·1_997). Another stud_y.·conducted.in oocytes:stror:igly arg·ued.against02-:

·:· ... rece:pt6r~rn_ediated.reg'uIat'iori.·ormemtJrane PC?.t~ntial as.a.·mechan,~m:

· underlying the ·enhanced OATactiv=ity. lnstec1d,'.the: au~hors··oftt1is·.study :·

. . . proposed that 02. receptor act1vatiori enhance.s DAT ·tu·nctiori:by increasing the-·: - . . . ·. ni.Jm~erof cell surface: t~_arisporters _arid that this :effect is· independent ·of_·.···.

...... ' ' ...... ' . . . . ' ' c.han.ges i.n.·m~mbra.ne potentia'l(Mayfie·ld ~nd· Zahn!ser, 2901).-: .H9wever,· a ..

. . . . .· sutJs.equent study conducted_•in.02 knockout mice_dem9nst~ated·that dopa_m.ine.

·: . . . ·: . . . ·: . . . ·: . . . ·: . . . ·: . . ·: . . . ·: '' ·: .· ·ciearan~e· by DAT is:·urialtered. in these: mtce .(Rb.uge~Poht.et al.; ·2002).:

...... ·:· . _A:nothei~:tudy.c6~~.u.~ted:i~ _ratdoparn_ine neur~ns:(n culttJre :sho\JVe9 that __ : -

. . . . . ' . . ·.- neither,.aGtivatio~·n.or-inhibition-of. 02 recepto_r-activity _had an effect on.·.

. dopamine .uptake (Piasadand Amara, .2Q01 }.furtti~r

. ·.· .r~gulatiori by 02. rec·~ptors: ... :· .. ·· . . . . .

. . . ·. . .. · th~..lh.ird·intr~_cellular_lpop ofDAT.(Kilty:et aL, 199_1}triggerE3d a larg~.ri~mbe(qf:

. ·regLJlat,on.· N_umerqus·~tudies have demonst_rated.that.DATc:a~b~ regulated.· ...... '' .. ·· PKC.by·phorbotesters .w~s· shoWnJo ·inducetthe phosphorylatiqn· and:.· .

. . _dow~reg_l:lla~ion of_ D~T _ceHsLHface·~xpressior:1. (2;ahni'se_r-and-:So_rkin,:·2·00~)-: ..

.. · c;o~verseiy·,_. _incr~asi:n~_-phosp:he>rylatio~ _of' ~erine/th~eo'nin~- _r_e.~idue~_ -~i~h: ·a· · :·

. p~osphata~e: inhi_bitor mim_icked: ~.he_eff~ctofPKQ_ ori DAT, sufface:-'expression : ·...... and function-(Vaughan et a1.,_·1997):.However, '1'ihen potential. PKG .. ·.·.·-.

.· ·p_hosphoryfation sif~s·in-DAi·wei-e.mutate·d, the· effect-of pho.rbol·e~ters: ori

· ·other proteins.•in_.this·effeqt;-A recerit'study ir:,dicated·that.mitog~n..;activated ... ·.: .· .

. pro.tefn 'kinase '(MAP.K): phosphata:se· 3 .cc>uld ·serve.to downregulate{ DA t in _: ··:·:

·_ .. · response'fo.PKc· acti~:atio_r1·(Mort~nsen· etaL; .. 20~~) .. "A1th9~9~ these.~tudies> ..

·:'. ·s.u~m.ested. a :r~le for ph~sph~_~l~tion' in 'ihe' regulatio'n:of.pAT furictioii; th~ :· ...... ·.. up.s_tream prqteins {r~qeptors/iqn .chann~l_s) th~t ·activate .these sig~·~l_ir,g

.· ·cascades fri·.dopamine.·n.eurorfa.are. riot'knowri ·and ..need to·be. ide'ritified in'the·_._·

future.·:·_

-··.:. Activati6_i, -of the-MAPK ·ca~cad_e i_ri ·HEK2-93 cells was- shov1in ·to· fri_cr~ase- ·- -:.. ·- .· .DAT trans.port ·capacity by_i~creasing -c~il:s•urfa~e-·e_xpr~~~io_n ofU,-~:trarisp~rte'r~:- :

. . . . : . . . ·_. -w:her~as i'ri_hibition of-this ~ascade_ "Yas·:shown tcrh~vethe:opp_o.site·:effe~t_'.- -·

.· ·cM~ron-_et:·~L;-2003): -·-~oreov~fr\ an.otfte·r· ~tudy d_em?nsfrclted-'that -t~e :1)2::· .·.

re.9~ptor~ind~ced-_ir:1cr~ase-in DJ\T cell s_yrface_ expre_ssion·!~: rrie~_iate~- py·-_ .. · ...... _··: ·activatioh "C~f.the MAP.K ..ERK1/2_.in HEK293 cells'(Bql~·n ·et.~l-,: 2007)~. _Recent ..

...... _studies .h~~e:~1so -~~m-onst~~t~d :a ro~~:-fo(Ca~-~~I i~ the ~~gulati.~n:-9f DAT __ :: ...... ' ...... :··.· .capabie,.of:activ~tiri~ CaM~II~ .. This_activatio.n, 'in turn; was s·hown.to resGltin.: .

. . ·· the :phosphorylation of DAT ·and the ·promotion ·or dop_ariiine :release thro.ugh ·

- ...... _the·trarisporter· (F()g· et al.;. 2006).·.

v> -Recnilation·~y ghicosylation -and ·ubiquitination: · ·

. . . - . . . ' . . . . '' ...... D,ATalso:has·consens~s·:glycosy·lat_iori_sites:in the:second· extracellular loop._

. . . . - . . . . . · .. and· it hasbeen shown that t~e.se site~· do .become ·glycosyl.ated;_DAT ·.

...... ' ...... - ...... · ·g_lycosylation.is thou·ghtto maintain activity-arid -~tability· of the:trarisporteraf

.· . . . .· . ' . _· . . . .· . . . _· . . . .· . . . .· . . . ," . . . .· _the cell -~lJrface ·(Torres-et ~1.,:20.03). Non-.glyc_osylated PAT is less :stable c1t.

. . . . . ·· the:surfaceand lack ofglycosylatie>n- results.,in a decr~ase in.DATtransport .. · ' . . . . capacity (Li et ·aL, 2004): .

~s. ·ublquiti.natioh. of_ protein~· ·is. _known to. serve as· an· i:ntetnalizatio~. ~tndior: · .·. lysosomal,~arg'etingsigrial,·'st.udies.investigatedthe·:role bfubiquitination-.in •...

...... ' . · . Pl

.· ·endocytosis•.and PKC>•induced. DAT ·enctocytosiswere .botfrdeperident on DAT

' ...... · ·neuro~s; it was _demon·strated tb~t_Neod4~2 _was the· ~.biquitifr-liga&e·fo,r [)AT,·.

· · · . as.Nedd4-2·.interacted:With DAT: and drarnaUc deubiqu_itinaUon of DAT: oc·curreci -:

.. · .in· c'ells that were depleted .of Nedd4-2':(Mira'nda:et aL, 2005): ..

vL ·Regulation ·by oligomerizadon a·n·d .protein~brotein· interactions: :· .·.

. - ...... ' . . .. ·., .DAT.can .exi~t_as_.a hornod~mer:~_nd·DAJ.oligome:dz~tion is. ~elieved:to._be_ ... ·

. . . . . ' ...... " ...... · ·impqrtarit fqr.D.AT fUnqtion·at,:d_stability ~fthe··surface (Ha~trup etaL, 2001f :· ·

·.· -~au_ses·asig~ificant re~uctiori.in' wil_d-typeDJ\T fu~~tio_n.-_Lik¢wise, .expressi9n. :· · •· ...... · ·_. of.~moth_errDutantlac~ing·criticalresidue_s.for-membra·n_e.-targetin_g·in~ibits_DAT .· . . .

...... · function·by·interferingwith the·normal.proce~s·ing·and_.localizati.pn·otthe.wild-··

.~ype fransportet (Torres_·et.aL-,: 2003)~ _ : : :· . .

...... signaling complexes) th_at·~an. interacfwit~ PI_CK_1 (a PDZ coritai_ning protein ..

...... •.·...... '...... ·. that'aids. in the. targeting a~d cluste~in~· of_receptors.and ion.ch~nn~ls).

. . .- Mutation· of the:PDZ:domain. in.DAT·abolishes·its interadion-.with.PfCK1 and:.

. . ·: ·. _ifllpaired tt,e ceU Sl)_rface targ_et_ing o~. OAT .(Torr~~ et aL_, .~001-) .. Alth.ough: ·

. . ·· oligom_erization 9f' DAT.w~s shown=to·a1terQ:AT-surfa9.e· express.ion,··· understandin'g ·the mechai1lsnis:·by which:'these interactioris:·can·.be.: ··:·: .· ...... ·.. stre:n·gt.hen·ed ~r ~eakeri_ed.·in·:n~u.ro_naf:sys~.ems-"w,ould_·provide alter:nat~ ......

. . app:r~a~h~s t:o_·a1teriri~i DAT furict_ion_: : ._- · ·

. . ... ·...... ' .... Most of the .a~qve studies ..that.e}.(c1mined ·t_he._regulat_ion of. DAT fun9tio:1Jwere. ' . . .

·:o~t:airied·w~r~ corifr~-~!ctor)id.eperidin·g- the syst~rn utilizetj::t_o addre~~ . . qr:, ·m_9~ef .

.: warra:rJted_. .: -··

.. -. b.-Regulation_ofDA-T:expression .... ·. Alth6'ug·h:reglilatlo1i'of DAT.fLfrictlon· may represenfa t'rans,entmech~nism ·=· .·. . . . ' ...... ·...... u_tilized._by.cei~fto alter dopamine_:~ign~lirif1~· resp~:~_se t(?. ce~_ain ~tirri_u.li,._1t i$. -·.:: ..

...... ·_-_· -: imponant.to understanq·_how-DATexpress_ior1: isreg_ul_ated as.c,hange$Jn· OAT·:·_._

...... ~ :. ·:a~uDd~nc~ -~ay:.con_t_r~bute to _t_he etiolog}(ofvari~us doparn:ine~relat~d. di_so·rd~:r~.: :.

.·.·:·.·. Discu'sse~-·belo\iy. are _so111e f~ctor~·_kn9~n-t9,-regulate the·.e?(pr~_ssi_on .. of_DAT~ ·...... · · t. Regulation:by psvchostini:ulants· .·

. . . . . - .· ~vidence :for reg_ulaticm.·of .DJ.\Texpressicm··by· p_~ycho~timulants, ·such as:.-··

_co~a_ine,_ met~ylp~enid:ate ·and_:~mph_~ta_~ines_,. _co_rnes pred?:~inantly from in-: . .

. . . ' . . . : ·: ·_. ·viv~rs:t4dies_-9qn<;tuct~d_·i_n rode~ts.:a.nd··fr_~:m: ~rain•_ir~naging/_po_st-mort~m·.

. . .. ar:,alysis of_liuman drug_ad_dicts.: Rats t~at·self-administe(~oc~in_e f9r three :·......

' . ·.· we.eks showed a ·30%·.decr.ease. in DAT.abundance .in the· ntJcleus accumbens:.·.

·.·Another st~dy in-rats .demoris_trated-a·tje·crease 'i_n·DATmRNA: leve1s·.in-the -~N ...... ' . ' . . ·:·_. _and VTA _in_.respon~~ to chror,ic:co'cain~ treatn,en{{Burc_t)ett arid_.~-~nnon", .. :

...... ·199.7).: _P·o.st~moftem analysis- of :Co9aine· abus'ers reve_~led that _striata·I. DAT .. ' '

...... protein ·levels: are marke.dly redu:ced (Wilso'n et aL,:":199.6b}. ·Us1n·g in: situ

...... - ...... ' ...... _· l_ev~I~ a_re:decreased significa_ntly in_the s~~iatum __ of human co_cai_ne users-as· __ ·.:·.

...... - ... ·_ . .cornparedtq·_age-, .Se?<;- ~nd race"."m~tch_ed _control_-subjec~s.{Uttle.et_al.,_ 1998;_._ ·

' ' ·· Chen:et·a1.:,_t999}.' Cons:iste·rit with the ab.ove·studies, .B~frinon efal/.have .· ·

. . . - ·: .. ~hown·:th~t.niRNA _i~veis of_ p~t:are _greatly"di_~i_nished i_n :the mid~rafri of. :·.

. . . -· cocain_e-apw~ers (Bannon .et.aL,: 2002}. However,- other studies have-reported··: ·.

,' ~ither: no ·ch~·nge_~·r.an:increa~e:in DAT·apunda,ricC3 foll.~~irig chro-~ic:c;:ocai~~--:·: ,·

.,' .. ,.· .. · ...... -· .. ·.· e·~p"osure"(Zahniser-'arid _Dool~n-,·?001);-Th~se dis

' ' ' .contiJ:1U(?.US exp·osu.re :versus. b_f~_ge t~eatment, co-preserycee~i.stence. of. other ..

. . - . . . - ...... ' . . - . . . ' . . . . . ·.· dr-~g·s, the·type:Of racJiql,igand :~s.eo to·;ne3~sure· oAT.~bund,ance'a·nd.·th'e model_.·.· .· .. ·. .· ... ·. ·. .· ... · .. ·. .· .. · .. ·. .· ... · .. ·. .· ... ·. ·. .· ... · .. ·. .· ... · .. ·. .· .·. : Chroni~ treat_nie~t wi.th. another _DAT ·blocker, methylphenidate.,: ~hawed ...... · more· c:or1sistehtdecreases in DAT.abundance ·"across .studies. Most" of the data.·-

with: hlet_hyiphe:nidate were ob~ained _f_rom hu~~~ :subjects: (either COhtml ·o_r _:: :·· _·

. . . _·_. AD_HQ-~ffe~t~9) _and·DA!-:sel~-~~iv:e,radio_ligarids :~ere use~ tom·easu_re:

·:·_· trari~po~e'r_-~bundan_ce·py·po"~-ifron·:emissio·n:tom_o·graphy(PET)·~nd single :· --..

...... ·_ photon emis_s·ion ·co_mputed. tomography ..(SPECT)_techniques. Afte~ three-_ ..

.· weeks on ·methylphenidate·; ·a:study- reported a.52% reduction-:in DAT-.·-· -__aburidanc~among_:c1~0-leiic~mts:(Szobot et aL,_ ~008).-Tnre~:md~ths of-

.· methyl_phenidat~_ treatme~t·causesa 74%.d~.crease -ir1. DAT abtmdance i_n -

- chHdren (D.resel et aL, 200.0) .. fntere·stingiy, disco'ntinuation of methylp'heriidate:·: ...... ·_ reverses the de~rease: imj_icating·that-methyiphet,id~t~~ind_uceddown_-_ .

- : _-- ·re·gulation _of DA~~--~b-ur,dam:~_-i.s: riot d·u_e-·to deathq(dopamine neurons·

_. (_M~dra~ e~_aL,_2005). -· .

. . . . ·. .-C~hroriic.admiriis'tration ofoAr sub~trates· s·uchas ·arhphetamines.. has· also ...

: .. _been sho'Nn to decrease 'bAT.abu'rid_ar,ce:in:prir:nates·:ar,d:.hum·an subJects._::.: .

. · PET·studi_es and_-post~_rnortem·analysis from methamphetam·in~_-abus·e_rs ---__ --- -

- demonstrated de.creased DAT-binding .si.tes in the· SN,·nucle~s accumbens a·nd: .

-·__ striatum.-(Wilson-_ef al.,: -1 g·gat:1; McCanrl et ~I., .1998)~ Sim'ilar: r~ductioris __irr.DAT -· ...... - ·e_xpn~ssio_n Were aiso· obse·rved \,vittt •iong~term· r:r)et_hamphetaminet_reafrnent_in: -­

.. baboons .(ViU_e~a_gne_etaL, .19~8)·. However, -othe(reports_have-i_ndi¢ated _th~f: _-

...... · de_creases :in. _DAT expressiorr"cire: accon,p·anied PY reductions in·· oth~r :

_-~opamine:~~u~ohc1l:-n1~:r·ken;::cWi!son e:ta1.:, 19~_6a): Henc~: dopan:'1ne:. - ·: · . neurofoxici_ty· coL1l_d·.at_ least __ partly- contribute fo_ am_phetami.n_e~induced: ch~nge~: :. ··:·

· ·in-DAT-abundance.- -·

...... ' ...... ' . . : · :·: h1terestingly, studies have :also sugg·este~ that fndividual":differen·ces in the -:

.· bc1sat exprE:}SSiori- ofl)AT _gov~rns~sceptibili_ty-'tCJJh_e~e ·psychostimLJ:lants; wit~---:

. lower DAT-express_frig . individuals b~frig more.: susceptlble ·:·to:·_such:· drugs·

· . (Bri.egleb. ~tal., 2094; ·Zahni_ser and Sorkin, ·2004). Consistent wi_th.-the above...... · finding,- tepeated ·exposure to: psychostirnularits can-:lead to. diffeteritbehavi

: -. _effects( _iri-: different indivicl°ua1s:~ ·:In _::r:oderit~,_::.:both ·:· b~havio"ral ::•sensiUzation: .

. "(increa.sed ·sens.itivity· to ~he. effects· of -the cjrug)"-anc(drug· tOIC3.rance (rC3dUced· . serisitivi.ty .to drug effectsfhave :been demoristrat~d-:after :repeated:exposure to .. ' . . .

...... , ...... · · -psychostimulants;: A: tra.nsi·ent: ·increase ·in _DAT. abundan:ce, _consistent-with·_ ..

. ·rapid. doparnine·_ ·c.1~·ar~·nc_e JrO:m -thef synapse,···rs:· believed to ':c_ontdbute_to .

...... · .. behavioral _tol(3rance. -(Zahnise_r_ and ..~q_rkin, 2004) .. On t~~ .cor:1tr_a_ry; ._rec;tuc~c;t ._

. · DAT abUridarice;•·cons1sterit with-:prolo:ng.ed dopamine· signaling;· is._speculated.

...... ' ...... '...... __ t~. lead. t() behavioral :se'r1sitizatiori (Gas:s et _a:L, 1:99.3).:•This ·p~rsistent. drug~· ...... ·induced· behaviorai' -sensitizatio"n ha~ ·81$0 ,been: _suggested to ·coritri~ute ·to.

. . . .. - ...... - - - ...... craving·and·the·high: relapse· rate _of .psychostim,ulant addi,cts·_·(Robi_n·son ancl .

...... , ...... ··_.-Berridge,-. :2_~0_3).- .· H~weve1\:· .different. :exposure _paradi~ms_ .·have-: yiel~~~- _. ·

..ctisparateJe$tHts ··f~_ the above: in .Yi~o. ~tudJes _suggesting: thaf qth~r·· external·...... ·- .. _fa_ctors.m.ig_htcontribLJte:·to·_the:C>bserved_:discreparicies_.in·the:regulation of .DA+.:· . ' '

... .( . " :21 .

. . · .. ·. .· ..· .. ·. .· .. ·. - ·, .· ..· . . . . ii .. Regulation:by:·other·factors: _: .

. . -· Genetic and ehyironmehtal'factors.have·been·proposed to play_.a to1·e i'n .. · ·

. mediating the i'nter-individual diffem:~nces ·in basa{DAT.expressicm (Briegl~b et:: .· .·' . . '...... ·...... ·...... ' ......

.. · aL_,._2004).··ln~e:ed; re·cent ·evid~~ce· in··ro_d:ents de~o·nstratedthaterivironment_a)_··_ ..

. and metabolic_facto'rs. playar.61e·_1n:feguiat1ng DAT ·expression.·An·enriched·._

...... ·_ e·nv.ironme·ntdecreases DAT_expression·in. rats _and.mice {Bezard.et.al._, 2003):_· .

.· 'R'e·stricted feeding 'irirats increases bATin:the VTA-and' m.icieu~f accumberis.

...... - . . ' ...... : _(B~llo et_aL,_2003_),·while chron_ic-stres_~-!n.niic~·_was foynct.to.decrease DJ\T·:

-· abundcmce in the striatuni. (EI-Khotjor.and.Hoksa;-200,2):.. · ...... - ...... ' ...... - ...... - ...... - ...... Bas:ed onJhe lite·rature p'resented:·above, :it:i:s· evident:that environmerita·1 factors·:· .·

·(such as enrich:ed envfronments:·a:nd stress);·_aswell as· pharmacologk:al agents,:-·

. -are._capable·~f altering_'DAT:expres~ion in' vivo.-·_·

·:s. DAT regulatiorfin ne·uropsych.iatric disorders

·. ..Jh_e stror1ge_st-evid_ence-lfr1k_irigDAT to a_ neuropsychiatric disorder.com~$ from·

-: studies·on.ADHD. ADHD: is a childhood psychiatric dis•orde(ch_aracterized ~y : ---- -. . .

...... -· ...... ,. .. inattention; ir\,p·u1s1vity'arid_ hyp·eractivity (Wilens-et:al.·,· 2°002}. Although ADHD-·:· ..

-:ha~ been describedas·a: childhood.dis·or~er;··symptoms.such ·as inattentkm-can··· ..

. . --_ .' ...... · - . . .. -.· - . . . _. - - . - .. · . . - - - _- . . ' ... · . . ' ... · ...... persi~tinto ad~_ithood (l<~~use ef_aL,' 2006)_;_:s·oth _ge~etic-and environmental_ .. · .. .

. ' ' ...... -: etiologies hay~._b~en·p:rqposed 'to_account_for:the· d_ysfunctiori~ in dopan,ine -· ·

·. .· ..· .. ·. .· ..- .. ·. .· - .·. - ·_ .· .· .. ·. .· ..· ... ·. .· ..· .. ·. .·' .·.' ·. .· .... ·. . . · n~umtran·s_mi~sion:·1e~dirig to t~e sympt()mS :in•A_l?HD :cswc1nso~ et ctL, 2007). :· .

. . · .. since:th~ ·mosfconimonl_y·.prescribed _the'rapeutics·for ADH_D.ta_rgetDAT,·th_is .· ...... ' protein-has .received substahtial:~tteriti_on for-its-possible contribution to the-_._-·

. . . .. ·. ·:disorder.Jn adults.and children-with AD~D, D~T levels are:·~ign·ifipantly :higher- in:_·.

the basal 9anglia:as co~pare_d t

.. . .. ,...... -.·: DAT_a_bun·da~c;~ is_elevc1tedapp~oxim_ately-70%-in.;L\l)HD adults (Dou9h~rty-et: _: .·

·:ai.-;J999·;: _Kraus~ etaL,-2000).-:Anoihe_r group re.plicated:this finding kl chj.ldren·......

. ' ...... , ...... -- as cdmpared:·tClCOntroi"subjects:·(cheori etaL:, 2003):.-Three_:r.nonths after: --.·.

tr~atment-of _ADHD ct1Udrerfwi_th the DAT.blockerrnethylphenidate(~reatmentfor .

. .

- - 1 · _ADHD.pc1tients),·-pAT-levels_ decreasced by74% in-the.striatum and-this: ·

...... -: corresporidecf-to ·a positive:cl_inicalrespon:se (Dresel :e(al., 2000~: Cheon·:et al.,.·· . . . . - · _·2003): ·ryl(?re--intetestingly,--a ·recent ·s_tudy in~icated)hat_theref is a sigrMi~a_nt_"inter-: _:

- -:indiv1du·a1·varfabHityin· the ·expre_ssion·of:DAT·, and_that a~undance(>f.PAT-iri U1e·: ---

. . . . striatµm is.negatively cqrrelate~ witt:, rnea~_ures -of attention _and _affect _in- not only- · .

- : ADH.D~affected .individuals:but aiso healthy_ individLfals (Volkow·et al; 2007).·· .

.. 6. Regulation of DAr:gene· expression : -

. . · - _ Thef hu'Tlan DAT gene:spans.about 60.-kb·and con~ists_of.-1S exons-separ~ted-by ·

-_ :14:i_ntro·ns.-theDAT p_romoter isnot weil·_charad~rized .. lmn1edi-at~ly·upstrea·rD_6f:

' ...... ' ...... th~·~tart si_te,· ~:conse~e_d_TATA-~i°ke TAAGAseq.ue:n:ce is_ locc1ted ·(at-~32bp}_ aqd

. . ' · -: is sp~culated:"to_·serve ~~_'the-prorn_oterfor_DAT.(Bannon .et al.-, _2001).:f~ecent ·

·:ci_~~jng. of.8_1<~ of the_:?'.- flanking_ regio~ _of the D/.\i-gene.h~s aided_ injhe ·.

identificat_ior:i·of two·.regul~tors of-DATtr~ns¢ription.The orpha_n·.nuclear·re·ceptor.· · · -.. :23-·

:Nurr1 •is transcription factor that •is critical .for· the. development cind: maintenance a· , ......

:ofthe: mi.dbraih:dopamine cell phen·otype. Nurr1 is heavJIY expressed irrmidbrain .. ·

...... - ...... de>p:amfr1e neUi"b_hs·.~nd: is thm1ghfto drive :DATtranscripti~n: (Sacc~~tti :efal., __ : ..

·20·0:11._··1naddition, the·:sp1: famHy- of protei~s can ·bin_d.to_GC ~!Ch sequence·s ...

. . . . . ·:pro~imal-to t~e:putative transcription ·s_tart site to prom_ot~ DAT transcription

... (Wang and .Bannon,. 2005.). Although proximal promoter regions. could .increase_._·

OAT.transcription, successive ·addition· of distal- se·quences· of the DAT.5'--flanking .

...... · . - . .· . .· . . . .· . . .· . . . .· . - . .· . . .· ...... r~gions wa~ found to_·inhibif DAT trariscript_ion ·sugg~stirig_ that-th es~_ r~gioris_ niay· .

·___ contain-g_ene-silencing elem.ents (Sac:.chetti:et _al:; -1999t. Further ~tudies.ai_med-at.·- - ......

' ...... ' : detecting the: exad sequences involved in en·hancing or silencing DAT : .·

...... -- .. "transcription wHl"aid in :detennining the. factor~ thafcan regu·1ate_DAT.gerie ... ·

·. -.expression.-•The:·3.':·untranslated __ reQion··contains·vc:1riab·1e·_n~·111ije·r-~f t_a_ndem_·

...... repeats (VNT_R) .. _Th_is -~~quen.ce _can vary from 3~13._d~pendin_g on-the number ·of-_-.

repeatswiththe 9 and:to repeats.-occlirrihg_most commonly (Bannon:etal.; · · ·: _-.

. - ...... - ...... :2_091 f .ltha~-beeri:su·g_gested_ fhafthere may. be a: l_ink.bet\Jy~en:the _o~curre·nc~·:of ..

. · a parti·cular·_repeatnumber c:1nd .the_·i~cidence ()f neurop~ychiatric_disorders __such:- ·

as ADHDor tti~ response toADHD·tr~atme_nt withmethylphen_idate. However,: ·_.

.. .. -- ...... ·the :inco_nsistency of result~ ~rom several studies- attempt_ing ·to· examine the effect ..

...... - . . . regio_n may_n9(b:e a&so~iated whh: DAT.ab_Lm_dance:(~ad_ras_:et _al.,_2095)~ - . . - - - . . ·:7 ..Electrophysiolt>gical· .properties .e:>fmidbrain: do:parnine neu:rons _:.

·__ popamine·neuron~ in-vivo fire action potential~, ·and their·activity pattetn.·i$:- -· ·

...... - . . . . . thought to conel~te withbehavio{:Midbrain:dopamine·:neu_rbrts mainly exi:st.i~ the .

...... folio\Jvi_ri_g threefiring st~te~ (Grace_ and- Bunney;.·1981t

· i. · _pacem.akei- firing/ton1cfiring.cliaracter1ze9 by single spike firing·

·ii. _B.urst firing/phasicfiring characterized .by.clusters_·of 2~asp_ikes .·. ·

...... - . . . ' . . . iiL ·_si_lent/hyperpolarized- state· due to -activation ·of _local GABA interrieu·rons .·.

V\Jhil~:tonic firing can :p~ :driven" d~·e to-th~ intrins_ic_·membr_~·n_e_ properties:oftn~se::

. . . . . - · _neurons, phasi.c.-fir.ing deperids-heavi_ly·on affe_rent inputs>· . .

- . . . . - . . . . . ' . . . . . a. IV/idbrain dopamine· neuronal firing as. a· resuit :of iiJtrinsic•prop•erties

_Pacemaker or.tonic.ffri:ng·of_dopami_ne,neuro·ns·was.demoristrated.to·occur_even •. ·

·. -:in "the:p"rese_n_ce ·of:inhibitors:to}\MPAreceptors_;· N_MDAreceptors:and".GABA: ...

...... ' . . . ' ...... ' ...... ' . . chan.ne:ls .indicating· thafintrinsic_p:ro·p~rtie~::contribute to the··9bs_erved _to~,c- _.. · ..

activity of the:se. neu·rons-.{Atherton·and Bevan, -2005).-1:t was:·showri that.SN-·.

:d_opa_rriine __n~_urons ~ciiv~:a tetr()dotoxin_ (TTX)-s.en~_i_t~ve sod~um·currer,t_(calle~::th_e: ...

. . · __ persistent so.di~rn_current) a.otivated c1tvoltages p_ositiveto·_~a2:sn:iVthat_ca,rr ·.. . . drivethese cells to•fire action pote_ntial·s (Atherton and Bevan:: 2005) .. Hqwever, . ·

-~ven. inthe presence of TTX,.-th<3s:ene.urons··~ere.found· to·existi~ .a·relatively. · ..

. :depolarized state suggesUng"that :midbrairl dopam_in"e neu"roris: poss~ss a

' . - . . . ' . . . . . ' . background catk?.nic conductance.that is cap_able of bringing· these ne'urons.to a:

. . . . . ' ...... ' . . . . volt~g~ range a_t .which ~ction potentials· can be obs~rved {Ran,~n. eta_l._,_-20O0).:"_·_ ..

·Alt~oughth~)d~ntity_of the cha:~_nel c"bntr!bu~ing t_o.this background co_nd:ucta~ce:

_is ·nofknqwn~ -st·udi_¢s_haye ~uggested_.th_at $od_iun:t iori_s co~tribute _in a.way to-this·_·...... '...... '...... ·backgrodn_d:condu:ctan~e~- Calciu~ entry through N--type ·cav2~2 channels during:.

...... ·. Jhe depolarizing. phase ofao action·potential"causes activatio·n otSK-cha·noels .·. : ·

·. (small:¢onductance ~aicfu_rti~a

. .. . . suggested to: be·. r~sponsible "for th:e _long. and _deep :aft~r~hyperpolarization . . . , . • • . . , . . • , . . . . • • • . . • . . , . I . • • . • • • . •

...... b .. Midbrain dopamine. neuronal.firing by_·afferents .

. ' ...... ' ...... ' . -: The:firing ofmidbrain dopamirie:.neurons· is.also governed by.·severaf receptors:··· .

.Pr~sen·t dn _p_resynapt1c. dopam_1ne Jieu rqns _and. po~tsynaptic: neu rm,s ·that ..

·. feedback_.into·the~emidbrain·dopami_ne·neuro11s·.· ·

·. i. Reg:ulati.on: by excitator/afferents: The :phasic fir1ng paitem:ofdopamin·e ._·

: ... neuro.n:s ·is. :contri_buted largely: byg ldtamatergic ·affere~t-in.pu:ts, .and :d(?pamine. : .

.· n_e.urons.·inc~lture ~.nd: i~ ·S_lic~s qom_pietely'lack:"this forni:·of fir"ingpattern;·_· .

, . . . . ' ...... ' . . . . : .. -: . /. .. ;· .. : .. ·. : . . ... ,-: . : .. -: . : .. · -: . : .. ·. : : . . : · . .GI uta_materg 1c. _afferents. _arise n1a1_n ly .froni ·the prefrontal. cortex (PFC),. .

. subthala.rrii~·nucl~u~·(STN)', ·the.peduriculopontihe. .tegnie·ntalnucle8s-.(PPTg) ...... · ·_ and the.laterodo:rsal thalamic-nucleiJs:(LDTg). W.hiie giutama:te·:,s the major:·._ ...... ' ...... - ...... ' ......

·_. neu_r_ot~~nsmi_ttef..in_the· PfC·andthe-STN.afferents:,.P_PTg. and ~DTg·-~fferents-·: _· .

. also: contain :ac·etylcholin·e. (ACh). Midbra,r:i dopam.ir,e neuronal activity i•s ..

. ·. increased.on· stimulation of PFC and-STN due to the action. of released

.· g_luta.matero'rf postsynaptic AMPA, NMDA andmGhiR1/5 n!ice·ptcfrs. Stfrnulatic>'ri · . . ·...... ·_ . - - ...... - ......

. . ' . . . - - ...... ' ' ...... · cues):also qause exc_itation· ofoopamin~· rieurohs_cj_u~ to·a:Gtivati9n qf_NMDA:

...... - . . . . . - - ...... ' - . . . . : . _c1nd AMPJ\recept~r~ (when glutamate: is ·releas·ed) ~nd due fo activation:of :· .

.. · n·ico{in_i_c ·(a4~2. and 01). _and_.~ usc·arin"ic ..(M1lMq} ·r~c~ptors ·(when ACh ·i$_ ·. .. ··-i ·.

' ' .i .. .. 'F ·:· released); PPTglLDTg afferents :cohtain _M2/M3_re~eptors:while .PF.C/STN ·_. _:.

. . . · ·afferents co·ntain mGluR213receptors. These:receptors act·as·a.utoreceptprs- --

...... , .. -· - - n·eurotransrnitters-glutc1mate an_dACh. (<3_fil!ner an_d_Jyl~rcuri,_:2002)>· --

...... ' . ' -ii. Re.gu.lat1on by inh.ibitory.-afferents: GABAergic affere_nts· aris·e ·ma1nly fro·m _the_ .. . . . - ... ·. striatum, n·ucle_us accumbens, pallidum _or from local_ GABA. interneurons. · . · ......

...... · Striatal-GABA ne_uro:n·s co~fain~ithet D1or-D2 rec~ptors. _Tonic-dopamine{ -

...... -_~ig_naling .m~inlyactiv~te~{~he hi.gh ·:affinit_y D{~~Geptors whil_e: phas:i_c relea~e_-qf :.

...... ·_. dopam:ine activa.tes the-iow affin.ity Dfreceptors-. The D1 ·receptor-coritair1i.ng ·.

...... ' ...... GABA neuro:hs: project:directly·to the SN :(drrect pathway) while theD2 .

. -contain_ing·:GABAn·e:umns_ of.therstriatum)>rojectt6·the.SN by_~ay ofthe·_-- -- .

.· gl_obus:p.a:ll!d~s exte_ma1:·(G·P~)-.and:STN-,(indirect·pathwayf' Activ~tion:ofU1~_-.- .

. dire_c1 and_indirect pathways. result in n~(jnhi~iti~r,-_of midbr~in dop~rn_ir,e -_ . ·

...... ' ...... -· neuronal firing_.(Surmeier et ·al.,.2007}. :Local GABA-interheurons exhibit ari.

·:· __ 1nhibitoryJnfliience:9n :some_:~idbrain_d_opamin~-neuronsJhus caLJsing thern:to -_

-· remain_ ina hyp~_rpolar:i;ze9 or non-firing-·mode; The inhibi_tory_ a~tion -of_ Gf.\BA-i$ _·-

. eli~ited·due tQ activati.on:of· ioriot_rop"ic GABAAchlori_de ·currents o·r by ctctivatiorf .

·. -of- potassium. conductan9es mediated-·by metabo_tropic ·GABA receptors .· . . - . - 8 .

. . - (Kaliva~·, 1_99:3).-:GABAergic c:lff~rents ~ohtain mGl"t.iR213·receptors ~ricf M2/M~_ -

- _receptors. afautorec~ptors_; .(i::igure 4) ,:_:: Figure 4. An overview of the major afferent inp11ts to midbrain dopaminergic neurons

Glutamatergic neurons are indicated in red while GABAergic neurons are indicated in blue. Activation of the prefrontal cortex (PFC), laterodorsal thalamic nucleus (LDTg), pedunculopontine tegmental nucleus (PPTg) and subthalamic nucleus (STN) increase midbrain dopamine neuronal activity while activation of the striatum and nucleus accumbens afferents via 01 and 02 receptors exerts a net inhibitory effect on substantia nigra (SN)lventral tegmental area (VTA) neuronal firing. 27

PFC

STR/nucleus accumbens ~~

LDTg/PPTg

~I GPe I

mGluR/

GluR3

SNNTA ·a. Regulatio~ of mid brain dopamine neurorial firing 'i>y external stimuli· .

·:Th_e·~bove·ctiscussed-niodes of dopamine neuronal'firing·cari'be altered in_:·· ·

-: s~ve:ral_\vays:as discussed below:·.

-: -~~ ·-Relli.!l~tic,11 _by beha~iq:rally relevant· ~t~muli- · .

·Heh_aviorally.relevarit:stim_uli·_c~.. n cause atransi~ntincreas~ ._in :qop_amirie .- ......

...... newonal firing and pr~mote dopami_ne releas.e {Schultz, 19~8); .An.increase. i~ .-.-.·

-: dopamine ·c~U-activity}s·.thought to code -~he. reward.·p.redi6tf6n-error sig·nal, which. - ...... ·:is d~fined-a~·the diff~rence ·b~tween preo:icted:r~ward-a'hd _o_btaineg _re~ard -.- .·

. -· .. (Schultz, .. 20.07).'.F9rexarnpl~,· 'it. was ~hown:.th~t-an unpredicted r~wa~d.Cartelicif_ ·

-: greater'activity:\flihile ttie:·lcfok ofa:·pred.icted :reward inhibits activity .. of dopamine··:·: . . . . ' . . . . . '...... neurons .. This fuhction·:of s_ig_n-aling.-reward w~_salso t~qughfto: be· reievari~ t_o · -·:· --

·. :Other rriotivated behaviors· such_ as drug.:See~ing; -f~eding ·and' ~exu'al beh:avior in':.· .

. rria_le_ra_ts (Pf~u_s _et al'.~ )_990;_.Tar.tda' ar:id_·btc·hiarc(1_998). M_ore re,centiy, a ~tu~Y: ..

--: coriducted'i'ri :monkeys:·demoristrated that'fidng ofdopamirie{neurons :a1s·o -.·. . . . · encodes the magnitude of the reward (Tobler et aL., 2005). -For .examp.le,_ ·: . -...... · - . . . . , . . .·... . _ ...

...... - ...... , - ...... : juice· fE3ward .. :TQg:et.her:, -~nese studies demo·nstrate.:thaf dopamine ·neurqnal 'firing:·: .

:noforily_ encodes_the·reward·predic~jon err~r·~ignalbut-_a_lso·~h~- reward.

·_ Jn~g-nitude: :·: ·

. .- • . • • . . • . . . . . _·1 . ~~ _R_egf.!la(;on :~f-fi,:ing _by p$y~iJo_~timulants ·..

.·.: Adtn_in.is_tration-_ofpsychostimulant.drugs ·suGh as coc:c;iine;· m~tbylpheni.dat_e ~n~·--_ .··

...... ·:~unphetamin'.~s~ :decre:~.ses dopc1mine:ne~roriaVa~tiyity (R~~~c: ~nd -~€3gal, 1'9?,~.; : :. · - ...... · . ·:blockers;: t_hese· psychostimu.lants increase the\~xtracellu.lar concentration of ...... - ...... doparnin~·--·This.leads-to·e·nhaliced .. sti.mulaUon.ofD2·autoi-eceptorsthat·i~·turh .· ...... : inhih"its ·the firihg ·of mid brain ·.dopamlne neurons. by: different. mech~nisni"s.: .·. · .. ·. .·. . ·......

. . . · hyperpolarizi~g: 9urretits Jn r~spo·n:~e -~9 amphet~niin.es· in_.mi9b~~in_·s·li~es}ro_m:·._··· ...... dopamine~deficien~-~!ce:~ugg~_st.~~at_rel_e.ased dop.amin~. play~.a critical role iii_._

: coritrolling -d~pamine· ~-~ui-onaf ffring:(Paladini:efal_.:;.2003).· ·_The-role -~fI>2

...... - ...... :r~c-~ptors in·.inhibiting· the.activ_ity _of dopcUJl_ine ·n~µr:ons i·s_~xempli_fi~~;Lin·studje~ .

·. __ where a ~elective P2 ·recept9r antagonist, and _not.a·D.1 ~eceptor-~ptagonist_,:· -· ·_. · ·: .·

: : -: completely reversed the:in"hibition:·produced:by·amphetamine treatmenfirfrats.: · .

: .·_ ..(Shi·eta1:_,-.-2·0:09r Taken :tagetner, :these·studje~.s·ugge_st_that co_nt!hued ~ .· ...... ' ......

...... ·:psychostirrn.;il~rit ·use ~au_ses· a.oe·crea~e:·1~· t~e:~irinfi of m_idprai~- dop~mine: . . . . .

neurons...... ·

. -: c~ Regulatiofr of:firing. by: other-factor:;:° . ..

. . . . · · · · neµrons,_ numerous agerits _cind dr~gs_·actin.gat.gluta·r-nate; ·GAl3A,_dopamine; ·.

. . . . . '...... norepi_nephrin~·:and_. serotonin re·c~pto~s can: also niociulate firing: of these· .. ' ...... ·neurons. ·1t is:how known thatafcohof (.Sh~ri-et aL;.·2001j;.ati~ipsychoti_c.s-(tliat.a:ct -· ·. -:at·p2·receptClrs)-an.d·c:1ru·gs acH~_g··afthe ·n_orepinephri~e trar:isporter··c~orcin":Gates _·...... - ...... ~t.aL,)2909) c_~~-~lter d_opami_ne _n·eurcmal activity, ··1ht~i-e.~tin9Iy,:·record~·ng·s from_::: ..

. . . dop~rnine ·heµrons. ih t~t~· have revealed·thcd.chroni_c antipsycl'.lotic·dttig.

...... ' ...... ·tn~atment.res_ult~ iri. c1 foLirth:stat~ of firing: a time~depend.e~:ririactiv_ati9ti :oF . :· dopamine _neuronid firing due. to:-0\/er~~xcitation·_leadi.ng ·10 a-:qepoiarization.hlock

. Jhat-c~ah ber~lieved only:·by membrane hy·perpolarization·(Grace et al.; .1997)> · .·.

: ··:: fncr~ases·:in· d_op:~niin·e _neuto:ni:d finnQ_"are _aiso thought:to pla; a· roiein .the: :

··addictive· effects. of opiates and. nicotine (Britt .~ndMcGehee;· 2008)~ ·lnfusi.on·of µ-· .•....

·:receptor agonists increases the. firihg:·of :mid brain dopam·ine.neurons·and opiate......

antagonists are·_capable·:C?f reversjng this effect_in_.rat~-(Walker'et al.; 19~7)._·. - .

...... Rather than a-~irect effect:ofthes~ drugs:on do_pamine newo1:1s;: it_wasthe .

·inhibition interneurons:that contributesto":the opiate~mediated increas.e. · ofGABA . . ·. Jri ·dopamine neuronal firing.{Johnson.and Nort_h; ·1992) .. Midbraindopamine · ·

n~urc>rlS are ~.1s·(?-:thought to be targ_ets for riicoti~e's stihlu_latoiy effe9ts :si_nce_ th:ey -:

. . . - ...... ·. .· ..., . . . . . pos~ess nicotink~: a_cetylcholinereceptors:('r'in a·nd French,-2990). _Usitig ··-:

.. i_ntracellular recording:.techniq_u~s in VTl\n.eu·rons:in vitro,_ it.was ®~en,onstrated._ .

thatnicotine ·exerts a stimulatory.action .yia nicotinicrecepto"ractivation.· .. . ,· . . ... · . ' .. · . . ... · . . .. · - . ' .. · . . .. · . . ... · ...... ' . . . . . - ...... (Caiabresi"efal·., 1989) .. Also,·n·icotine infused locaily.in the nucleus accumbens .·

... orVTA of.adult rafa_is_·capable_of promoting dop~rnine rel_ease,.an-effecttha, i·s: .. ·

·:blocked py nicotinlc recepto:r antagon.ist.(Mifsud et aI.,·-1_989; Clar~e;.-1990t · ·

...... Moreover,

• • • ' ' r • • • • mid brain dopamine .neur9nsthat was arifagoniiecLby ·a nicotini~. r~ceptor

:a.ritagonist'{.Fa et at, 2_0·00·);:furtheffvalidathig' the effects·.of:nicotine cm th:e-: .·

... activity of qopamine. n_eurons. Tc;J~en together, thesE! _studies _demonstrate_ that ·

...... nicotine: increases the firing of niidbrairi ·aopamirie_neurons irr.vitro ·a·n_d'in.vivb.·_: .· -.

...... : Studi.es have indicate·d that environmental :factors·-such :as·:enriched .

...... __ enviro·nm.ents lead to de·cre~sed·sen~itivity·to cfrug abuse·(Xu-et c1.L, 200·1-). __ .Since·_·. .tpe firing· of mid bra.in .dopamine ·neurons:.i~ _alsa'-implk;at~d in.susceptibmty to .. :.

·. :psycho·stimulants .(Marinelli.and Whit.e,-·2000), .it-is pos·si.blethats_uch-an _·.

...... ·. ~- . .· .... erivirohhlerit-alters·dopam:im~ cellactiv_ity~: Other factors such :as·:str~ss cah 'also •...

. .. .. " ...... alte~. dopami'?~:_neu'ronalactivity: -rvl~lt_iple:s(udies h:aye

· expos~d-acutely to_stressors_, s·~chas_tajf sh9ck~ foot shqcKo'r restrain.t,: exhibit_

.. increases·in_extracelllilar dopamine. levels in.the. PFC, .. nucleus acc.umbens an~. ·

strfatum, suggesting enhanced.activation_ of SN and VTA ·dopamine ·neurons· with. ·

...... ·:aq~te:stre~~.f1:"hie.rry ~t _aL, l~76; Rothet:al.·,. 19~8;.Aber~_rqm_bie 'et ell.,_ 198~t O_n:.

)he cont~~ry; 'chm111c·stressJn rodent~ was· sh9wn.to .deqrease: sppntaneo~;,-· ·

activity of SNI\/TA dopamine ne:urons·_(Moo're: et al., 20·01). Howev.er, ..the·precise·:

·:mechani~rns invol~ed--in .the acute·.an·cf-chronk.effects·of stress··on•niidbra_in·.· -·.

:dopamine m3u·rons ar~··not clear. Takentcigether,Jh·e above_studies.qe·mon~tra_te: .

· the st1sceptibil_ity_ ofmktbrain·dopc1mine ne:Uronsto· r~g_ula_ti.~n by a ·varj~~Y of_ .. ·

exte.mal factcfrs.· -:

9. Second rriessengers•·involved in activity•-dependent plasticity i'n·neuroris·: .·

There are .numerous examples ofactivity.:depe~denf changes :in_ stru~tu:r~, _·.·

. Junction andgene:expressiori in·neuro·ns_.· Different ~ign.al trahsducti_qh pathw~ys ·.

thattra_nslijte .ri~u ro~aljictivi.ty_ int_o ~ltere(g.ene. expression -h~ve b_e.en. i_d.ent_i_fied_:.: .. (ltnp~y et al./Hl98;·Kornhause(efal., 2002)..: lnflU)( qfcalcium·either Vici. liga·nd~·-·.

· g~ted: i_on-.c~:a.nn:e1s (~fylDA an_~-AMPA receptors for volta~e~gated ca_l_ci~m·.

channels _often·:$erve~· a~_the in1ti~ltrigger·fo_r a¢tivaticm of the _kin~se·pathways. . ., ..

. .. ,\ · ___ 32-·

:(West ef~_l.,:_29·01Y,._.T.h~:pattem.c;ine~rona{activity, mode_:of_calcium:entry.and : ...... , . . ' . . . . : . . ' ' . _:d iff~reriti_al. activati_ori -of fr1tracell u·lar- kinases· can -all· influ_erice -gene· ·express:iciri - -· -

-: (Vyest:~t aL_, 2_0"0_1; Fi_eids: et ~L, -200"5). _E-ievated_ in:tracellul_ar:caldum :lea~s"to the: .·

...... cyt9sol._in ·so_luble form _or bound _to cytoskeletal pr9te_in com_plexes:.(Schulman; _-_- -

-: 2004)·.· CaMKIV, :ordhe·.other" hand,-:is ·predomiriantly ioca"li~ed.-in the-nucleus a·nd.- -:

. .· . . . ,· . . . .· . . . .· . . . .· . . . .· . . . .· . . . .· . . . .· .. ·:cc1n:be-actlvc1ted- by _in_creased _cytosoliq _calcium Vici. acth,~tio_n by ·c~l_rnodulin and -_-

. . . . ·. _CaMKK (En.slen-etal., -19.94; NakamlJra.et-aL, J 995"; To~umitsu·-et al., -1999.f ·__ --- - . .

...... ' . . . ' ...... Neuronal·actlvity~depend:e·nt.stimlilation of CaMKII, -CaMKIV,·PKA_and- ERKs _: · :: .-

_- _·could :evehtua11y·_1_ead·fo_ ph_osphorylati_on-·of :c~MP-r:esponse·eleme·nf-binding.- --

...... -protein(CR~B) and CREB-meqiated-trans¢ription·_(West_et: ~L; ·2001)~_- CRE_B :·

.... phosphorylatiqn .can .o·qc;:~~ at .sev€3ral diffe_r€3n_t resi_d_ue~, ~ut'p~o_sp~orylatjonat _. -·

. . . . Sei"133_.is criticalfor.trans.criptiorial.activatit:m.(Enslen .et:aL; '1994). Alth.ough -- --

...... - - __ mediated,_transcript"ion_ (Sun et_aL-, t994), it _is- r1ow. kri_own-to b~_inyolved.in _gene;.··_._

...... -: specif~c:trans·cription through_ its:reg(1latioi1 qfCREB'~ interaction with:it~ bin_ding :·: -:

. -· _·partner~ (Kornha~ser.~t aL, ·2002) Activated CRE~ .cannot.ini~i~_te-.transc~iptiori­

·:ori: its :own·bufneed~ :t9'-~ind· fo:the·coactivator.c"RE'B-bindi'r1~f prote,rr(PBP) .· ·:·_ .· . . (~ha~I~ et aL~_:_1998}. · QBP its.ei(can also)eregulated by p~~sphoryi~tion by_·_::_: ...... -: neu:rqrial activity ~nd· CaMKIV-(H_~_et at, 1999; .lmp~y ~t.aL; :2002).·· ·

...... ·: __ ::•:- : :in :a~cHtio:n to_cdt~ring:tra:11scriptio~factor.a~tivity, ki~~se$_ and_::

.. ·. phosphat~ses-¢an also·r~gulat_e tr~ns_cription. by-chrqmatin_rerri_odeling. :- · . .. .. ,. .. .. ·- .. ·:Phosphotylatiqn·of.histone d~ac~tylas~s:•(HDAC.s}:can ,-~~d:-to:thei~. export from .. :

·. ·:the_riublem,·,-resulting.-iri··ehh~t"riced·.tr~nscription·-medi~ted by·tr~nstriptic>ri" factor~:-

. . . ' . . . . SLJCh as: my~cyte·:enhahcer fa~tor~2_·(M~F-2):(We~t-ef~"I., -~001:):._ :CaMKIVand_ -

-·-: CaMK_ILi~ofornishave:~~e_n shown·to"phosp~orylate:_HDACfor its bindi~g-:

. · part~ers-and._iea~ t9. cippo~ite· effects·o'n:t~e ~eg_ulafion"ot_"MEF2:·me.d:i~iecf -

...... tra_risc~ipt!o~_{EH_is ~t aL,·_200_3k Thus,· CaM.K.11 an~ .the. p~edominantly nuclear_·.···

· Ca~KIV ma}/ ~~ve- differential -~ffects-cm·_gene tran~~riptiori> Furthermore; the _.

...... ·:different ls_pfqrms o~.C~MKll G~n. select_iv~ly influ~nce ge~~-_transcri_pt_ipn by: acting.-

. . ·. __ on.differemt"down~~ream :effE3ctors :{Nc:tkagawa et aL;· 20Q6; ·Uttle-efal,·2007): ·. · . . . . .

...... - . · · · · .·. Hence, ·studying activity~dependeritchanges ·of .the:se: calcium~:activated·signaling· .·

proteins i·ri--dop~mfrie·neurons-is·notonly"iniportant-for·uride"r:sfanding> .. -· ......

' ...... mechanism$ _re:g_ulating ·oAT_e~pressionJi1Jalso fo·r prov_idir:,g :info~rnatiori on :·._

. signc1i_:tian_sdu:9tion -patti_wa·ys.thaf mediate_11europ_lasticity_.o~·cfop.ar,:1i~•e:_11eurons:_:_: ._· . . . . in ·general:· ·

...... 10 .. Struct.lire··and ·tun·c·tion·of CaMKt°I

...... :caMKII _includes· a·farnily o_f multifun_ctional. pr9tein·:kinases-involyed -iri·a wide- ---

. :array of functions in· the·nervous·system·,· such._as-iort channel' arid-i"ece·ptor:·. -· ......

. . . . . · ·::. · function, calci_um home_ostasis,-gen.e e?(pres$_io~, learning__ and- memory -·

...... -: (Yaro~uchi",·2005). CaMKH is·higtily._enric~_~d-:in the ~rain,--ahd_ !rl:SOITie_-r.~gions :·_ ...

·:su_

. . . ~erinedy,_ 1~85)..-.c~~klr_is a.family. of .fo.ur"cliff~'re_rit-i~_ofo'rms-ehcoded.·by.di_stiricf .. " . -· ·• .. . .. " .. :34.·

...... ·:genes :Ca:·-~,j) _andy).This_ fa~11ily_ is ~tu:ther_ expanded due to: aiterhat~: sp.lidng::of:.

·.. each· gene· thus .re.sultin·g i"n .severals_ub.:.isoforms.·.These.differeht_isofornis_.vaty··in · . - ...... : their:tissLie~specific ~xpres:sion atid_·subcellul_ar"lo_calizatio~_ (Hudmon -arid-:

Schulrri_ari;· 2092)_. _·_. ·

...... ~ ...... : -the protoiyp1caf CaMKll".subunit consists· of a :c_at~'lytic.ch:>m~iri_at theN~ ... - . . . .

...... terminus, an·a.utoregulatory domain and _an.association do~ain.atthe_C~-- ...... -: termfnus.-ihe.·assocfadion-domain-.is necessary-for:the-form"ation arid_assembly. Of -:

··thE3 qaMKl_l_ho_loerizyrne; .the-_native fo~m-of-the en;zyme.(Hqdmorf ancl. _· .·

...... ·.. Schulm~nJ.·.The.·aµtoregula~ory. dom~in ·can interact-with_.the ·catalytic doma.in to·- . . .

. . inhibit:thekinas:e· a·nd maintain it·in an.inactive state.:·calciurn :ari:d·calmodulin

. . . -:pe_rmits· kiria~e auto-ph_osphoryl_atkm and: ·activation._. Autophosphorylaticm of· -

CaMKII_ occurs at threonine 28? (Thr2a6) _in a.or Thr2a7 .in J3, o anq y.is_oforms'.

...... ---: When Ca~+/CaM binds.to two· ad.jacenfsUbunits·of the--holoenzyme; ihtersubu"nit -. -·

-Phosphorylation .can occur resul_ting in. the_ CaM.-t~apped s~c1t~ of the_.kina:sE{

.. · _(highfy_·increased c:1ffinity:forCa2+/Carv1-b_inding) -(Strack ~t·ai°.;·-·1991).-.This-c~uses:· ·.

: the.k1na'se· to:rerriain act_ive everiwhen calcium.level~ fallto.basal values.

...... :Ca~: /C~M. dissociates. from .the. kiriase·,. other inhibitory. pho~;phory:l_atio:n sites.. in": ..

-:the_~iutoregulato"ry:doniain are.:revealed°:(Thr;os13oe·in· ~--a~d-Thr3oe1301:in-~,:·o:a~d-·v: -:·

·.. . - .· . . ' .· . . .· . . ' .· . . .. · . . .. · . . .. · . . .. · . ·isoform~).-_Ph9sphorylati~n at_ these inhibi~o_ry· res.id~_e$_.pr~verits_-additi9.rial

...... Ca2+/CaM··b·ind_lng resul_ting irl a:CaM-capped:state: (Ha_nso·n:~nd: Schulm~n,·-· .·.

···1 ~92}. The _kinase:can:_orily be_:c1ctivate~ :further.'ftlheri :the _i~~ibi_tory __ :

,· phosphorylatio"n sites _uncJergo dephosphorylation_ by: protein phosphatas_es. (P_Ps) .... :(~olbran>19_9~)--S~ver~I: different phosphatases_ (PP1-, P~2A_andPP2C)-h~ve·:- ......

...... ·,· . . '...... '...... ·. ·:been· shown-to :dephOsphorylate·CaMKll-at all _three·-phosphorylation.-sites in-Vitro: ·. . . . . ·. (Fukunag8 ~t ~1.,··19_~3:}.:Whil~_ PP-1dep_hosp_ho'rylates_·carv1Kffthat _is-ass_odated: .

· . with'tt)e. pos.tsym:iptic ·de~$ity-(F>SD), PP2A !~_.thought fo ·be··r.espon·sible :tor·_.·.·.

· · ·:dep_~o~phorylati~g:·CaMK_II in the cytosoL(S.track et aL,· 1~-9:7_; Yos~-i-m.tJraetal.:,

...... - ...... ·:k~n~se -act!vc;1tion and_·functkm_._T~e.-au~9pt,osphory!atiorf of-G.aMKII _in-vitro \,vc1s:.

...... ·. __ observeq_.to.be:ser.tsitive:to 1he.-frequemcy ·of .ca,lcium .spikes-. At.lo~.rfrequer:t_cy .· -· ...... -: firing;: Ca2+/CaM can diss:ociate.frdm ihdividual subunits between caldum·sp·ikes·:·: .·

.. preventing-in-tersuburiit phos_phoryl~tion;··qn the.other-hand; :during.-high: calcium.:-· ...... -':Spike frequend~s,:C~2~,c~M-ca_n·not c_ompl~telyd_isspcia~e-:fro~· CaMKl!-su~units:.·:·

This_increase~-~he p.r~bc1bjlity.that_s~ver~I subunits _get ph.osphorylated_with-_ .. _· ._·_·...... : succ.essive spikes resulting in· a :rmissive increase· in.CaMKII° activity (Sdu.ilmii"n·.-.· .. -

...... ·:e~_aL, :199?f1he abillty_of Cafv1Kli to n~t~_in activ_ity:-~ver{ i_n_:the :abse_nce or· .. _:_.:-

·. __ Ca~+/Caryfb_inding _and to·furycti.on·as _a-frequen_cy·_detec~9r mak~s_ item attrc1otive-·:· ·

-: candiqate_ for: linking activity-dependent changes to al_terations•:iri synaptic:

. . ' plasticity ·and: gene ·expres~ion.'. 'Indeed.; studi~s haye. indicat~d that .post~' ' ...... ·. .translation·a1·modificatiohs·ofs~ve:ral proteins in·the{P$D by_ CaMKII :and··· . - ......

...... ' . . . . . phosphatas_es: are i~volved in. sy~~ptic plas:t,dty unde"r_lying lorig~term' potentiation: .. '

...... ' . . . . (L TP.)._and ·tcm·g~_term depression (~ T_DY(NiG_oU: and· M~lenk·a~ :1~9~;. Beln,eguenai.-.

·:~md _Hanse1,:·20QS} .. CaMKII activity has :ah~o been-.iniplicated. in altered gene :·

. ' ~xpres_sio_h i_n. response· tq· actiyitf-_dependerit. change~-. in_. neurc;jrial_. m_orphology· .· .. - ...... 36·'

{Gaudimere :et _al., ·2004; Zhou .et aL, 2006).: Ad.diUonally;·-studies have. s:ugg-~s-ted .

· Jhat CaMKII. -niay· c:ontributeJo a -r1umberof neuroadaptations· in· d.opaniinergic ··

. . . . . - ...... ·: sy_stem_s r~suUing: from chroni~ e~·po:sur~-to:·amph.etarnines_ (Loweth_ et :aL;:2'0_08).:·: .·

...... fiowever,_the:·~ownstre~m:ta'rg.et~:_o.f carv119r.!n.dopc1n,,ine· neurons-that_c9nfribute_·

...... to t~es~ neuro.adaptations, and.thus alte'red ~e~avior, have·notyet' been . . examined; .. ·

SUMMARY .. ·

.·... . .· .. - . .·... . _·... . .·... . .·... . .· ... ' .·'.. . _- ...... Taken: together·,: results·presented·:above from the available .iiterature .underscore:· .·

.. ., ...... ·. the 'impmtarice:of DAT:·iri ~egulatingdopamirte .neurotr~n.smi$sion~_'Aifholigh.· .·. . -:ger1etic.fa~tors :p:lay·animportar,frole:·inthe: de~eiqpm~nt·ofADHD. and ~rng.

~bus~; ~eve~a! _studies _have suggested t~c1t- non~g_enorr,ic,factors could_also· ..

. . corifrihute tc>' the development and. ·severity. of:the· disease: Additionally,· the above.

...... stu~ies alsC>: ~.ug·ges~ _t_~_at :alteration:s in [)_AT :abun_qarice·:co~l_d pla'y. ct: role.irt.the:. . . . .

...... __ pathophysiology- o_(these· ne.uropsych_iatric -dlso.rders>H~nce,-foarn'ing how DAT· -..

. ' ...... : abunda'i,ce is r~gUlated will further our und~rstand.ing of'its ro,e in·_normai

A. First Manuscript

Activity-dependent regulation of the do.pamine transporter is mediated by calcium/calmodulin-dependent protein kinase signaling

Shalini Padmanabhan, Nevin A. Lambert and Balakrishna M. Prasad

Department of Pharmacology and Toxicology

Medical College of Georgia, Augusta, GA 30912

Corresponding Author: Balakrishna M. Prasad

Address: 1459 Laney Walker Blvd., Medical College of Georgia, Augusta, GA

30912, Tel: 706-721-7641, Fax: 706-721-2347, Email: [email protected]

Running Title: Activity-dependent regulation of the dopamine transporter

Keywords: Mesencephalic, Action potential, Calcium, Uptake, Monoamine

Abbreviations Used: DAT, dopamine transporter; CaM kinase,

Ca2+/Calmodulin-dependent protein kinase signaling; TH, tyrosine hydroxylase;

TTX, tetrodotoxin; 4-AP, 4-aminopyridine

Acknowledgments: The DAT antibody used in this study was generated in the laboratory of Susan G. Amara, University of Pittsburgh, PA.

37 . ' . . . ·Abstract -

. . . · ·:Th_e·mec_~anisms regulating:expression ·onhe -~opamihe.transpoqet.are pQorly ---

...... : uriderstood'. -we·tes_ted:the hypothesis_thaf~eurcmal'acti~ity: is:o:ne_oUhe :non--:::·· .·

·genl3t!c_d-eterrn~na11ts·of-~qpami.ne:_tra.nsp·o_~er: abun~ar:ice·. ---~-~:stai_necJ·~ra~ges:i_n_

:· .. : ·: :. ·:neuronal:activity ca_used :by·tetrodotoxjn.:and:4-ami~apyridiri~ ·a~ter~d dopam.ine· · ...... up~ake,_ aburidance·_ofdopamine .transpo_rter and its.mRNA i·n.rat mesencephalic __ ·.

...... -: cu·1~ures:. -lhe.altered ~euronal ·activity-c~used by ~hese two_drugs- is.:.

- ...... ' ...... ' ...... ' ...... a~qompa:ni_~q.by-changes-iri: in_tr~cellular_calciu:m _cc;mcent_r~tions an_d_:

. . . . . calcium/c:almoduli11-dependE:mt protein ·kinase I_I. (CaMKll) ·activity i_n·dopam_ine_

...... -: neurons. ·chronic treatment" witfr ari: L~type:calciurri channel.blocker (nifedip_ine) ·: · .·

. -·. ~r Ca~_Kinh1bi~of. (KN93)·de_creased dopa~inetransp·o~er~med_i_ated·uptake-and: · .· -

...... -~cch.1oed: ~he_ effect~ Qftetrodotoxin ·and 4~aniin6pyridine~ -These data ·suggest"_ .. : · . .., . ttia_t _n~l:Jron~I -~ct_ivity.can- regµl~te:d_opc1mi_ne.transporter fur:rction ~n~· c1bundance_-.

. . -: via· calciurri/CaMKII ·signaling:·

. . . .-. Introduction·

. -- _°The:dopamine. transporter _(DAT) .plays.a.criticalrole. in ttle-cleararrce-of -·

lead_s-to iricre~sed ~OP?Ir:nine sig_naling __ th~t is_"pr~.ma.~i_ly_ respe>flsible __ for_ ~rug~_-._·_ -

'...... irtduc~ci: chang~S: in·beha_vi_or (Gkos :et-aL; .1~86)'. -Th~·re is· a~igoificant j_nter~ · ...... '. . .. •:ind.ividual".v~fri_a~ility· iri- the.expression: of PAT, and ~b~ndari·c_e:of DAT_iri :basaf. __ ...... gangli_~-is_·negative!y correla,ed.-with m·easlires of-~tt~t1tio·n-and_aff~cfin :he~lthy.- .. ., ...... ·:individuals (Newbe.rg_:et aL, ·2.001; :vo·ikow.etaL,-_2007). ·some:i·m~ging _stuclies·:

...... · :ha~eshoWri:·that-drlig~na·ive:.A~H □ ·p~ti~~ts .have.increa~ed DAT .in basal· ga·nglia _·.

ce>mpa~ed~o thatofhe:~lt~:y VC>IU~~eers __ (D~u~·he_~t~~:a{,._1_99:9:: _Spenc_~:r: et a_L, _:: :·· .·

·2007t··s1mtl~r-~s~9dati9ns_be~e_e~--DAT_-~xpres·si·o_n·a~d-o~~-er:psychi~tric_·.- .·.·:·_.: .·

:illne~se's •~uch as obs:e_s.siye:coinpulsive: dis~rd~r and depre~sro_n ·~a~~- also· b~en: .'

...... reported (Brunswick. et.al.,. 2003;.van der·wee et.aL,·2004)._· Howeve_r,._·_· ..

mechanismsthat: re·g'ul~te- DAlexpress"i~ri' are -not:well unde,rstood: ...

'' . . A number ofs~udies hav_e _repor:t~q.~n as~oci~tion-_between' ''

polymorphisms in _DATg.en~·and·fr1d_dence:of ADHD:(Purper~()u~kil.et'aL, __2005;·:·.·.

: Asherson.et aL~: 2007)..: · Most· oftt,-ese·_stud.ies: examined the:relationstiip: between .

...... -.number of t$_nde_n1-rep_eat~ i~ the 31~untrc1ns_late~ r~gi:o_n-~f OATmRNA.: l_n-.- · :·._ ., ...

...... ' ... additjon, a_.few-.r~ce_nt ~tud_ies. suggested ar,· ~sso~iatio_n betw.~_en ADHO.· and_ .. polymorphisrns-.ln the promotet region ·or those.· in intron-ff oHh.e DAT:gene . . ...

...... (Bro.ekes: et_:c11.,: 2006_;:_ Friedel _et:aL,. 2007;. Geriro _et aL., 2·00!). _: Alth9ugli :vari~~~s: :. · ...... of-_intron-8h_av~·-been.shown·-to.·influence· th~-e~press'ion_'of_.rej:iorter COrl:$tr~_ct$. -·-':·.·.

:_ (Guindalini et aL,:2006)/af present there.is:no comp~lling evicferic~ tharthe_

.. obsery~d polymo_rph isms· contribute·_ t

. . . , of OAT-: Fat ~~~r:nple; methylphen_iqate·tr~c1tmentc~n .dec~eas_e:DAT:~pundanc~- ..

...... ••i~ _p~ti~nts wi~hADH~--(D~esel e~ al_., 2.q~:qr This effect is reve'r~ibl~ upon ·.

~e~sa_t~o~_-of.drug t~e~tm·~nt_, __ -indic~~ing.thaf _m~~hy_lph¢nidat~ effects are·:due.to ·. ··40

...... •.. :regulatiohof'DAT ·expression rather th?tn·a result.of dop'amine neurotoxicity-

. :(Feron.·etaL; 2005).-· Several·studies_in· human~ ·and animal models ·have showrt · . . .

- ...... -: that DAT mRNA abundance is lowe·r in subjects exposed. to-psychostfrnularit : ... · .. · .. : . ' .. · .. · .· .. _· ... · .. · .. : _- ... · .· _· .· .. _· ... · .· .· _·_: _· ... · .· .· .·.: · ... · .' ·_ .. ·.: _- ... · .· -.- .·.: · ...... ·: drugsJCerruti_e~ _aL; 1994;-:Bannorletal.; :2002f- ·E~i~ence in-rodents:also ·.. ··

·:indicat~s:th~t-exposure.to:·enriched:·en_vi.roriments:or restricted:feedin·g.·canalter·.

DATexpression .(Bello .et al.,'2903;.Bezard _et al., 20.03). ·.How.the diverse

-: pharmacological and' environmental:signa·ls-.mediate.altered_:DAT eipression· is:. ·

. . . . - . . . ·npt-known.. At_a ceuu,a~ :level_, _these factqrs· cou!cfconverge_ to:alter_ c1ct_ivity:o_f_··.

' ...... ·. dopamin~· neurons, .. Dopamine neurons-.in-vivq.are to'nically activ~/and·:·.·.

: emotiorfally rele.varit stimufi can. 81ter the firing of these

·su·ri:ney, 1984; Schultz:, 2007). Differentdopaminetgiccfrugs, iricludil'i:g: ··

. methylphe'n.idate'and other psychos'timulants; can.alter the activity of these:· ..

...... neurons, (Eint")o,rn et.a_l., .. ·1988; Feqe_rici.et c1L, 2005) .... _Thus, wetested the.

· hypoth·e_sis· that .sustai'rie.d changes in n·eu'i-onal activity can· regulate the .. ·

. . . . . ·:a~undan·ce_~f DAT. in.:dopamin.e:neur6ns;:•

...... ·Materials and Methods···

.. .Mesencephalic ·cultures.

. . .- .· ...... _- . . .. · . . ' .· . . .. · . . .. · . . .. · . . . AU an.imal·car~ and-_h.aridli.rig-proced·ure$ w,~r~ approved by the MCG·1_nstitut_i.onc1i_: ..

. . . . ' . . . ' ...... : ...... ' ...... · . animal care· anq use committee :and:performe.d.in qon,plianc~ ,with the A.ni.mal · ·

...... - ...... :Welfare Acfand.other.fedral're·gulations:(uSDA# 57-~-0002).: Time~ma(ed·. :· ...... ' . . . . . - ...... female-rats wereobtatned.from.Harlari·SpragueDaw_ley,_ ln_c .. ·(l.ndianapolis, _IN,·.· \JSA).: :-Mes_enceph~lic:~ultures.w~re "i:>repared :f~om post-nata_l da·y-_2~4 Sprague · . . . . . - . . . .

. . . . . :Dawley q1t pups·as ·previ_ous1y·descri~ed-(Prasad:~nd.Amara·,· 2001}.- Briefly;·_. -·

...... : VE3nUaJ _n,i~~rain tissue ~i~sected fr~m. k_etam_ihe-i_nJected.(~ mg/pu"p_,_ :

...... ,...... , . .intraperitoneal):rat.pups _was·dis:sociated fo_r·fyvo·tiou_rs_:in•ttie pres.ence_-9f_20·units··_:

·. -/rr1l_ac~1vate:c(pa.pai_n::·._o,s:sociated ·cells.wer~· plated· at-a density of approxini.ate.ly·:

...... 1.50,000 cells.per well_"in-.48-we!I _tissue:-culture dis~e.s· coatedwith 100_µg/ml p~ly-:-· ._ . . D~lysine and ·s-.µg/niL!"aminin.·· Ne·uronaf medium· conditioned .over-glia was us·ed· .. -·

. . . . - . . . . ·:to _rn~intain _the mese_ncephal!q·_qultures. · Cells·were plated ·in _24-w~U.·plates-~t_a ·.

· ·:density-of 300;000:_cells-p.e~_well forw,estern.blot assay~: For-imniunostainjng_. -·

· and electrophysiolo_gical:"recordings;· cells·were plated on glass: coverslips~ After:·· · . . . .

preinGuba~in~ .~ultures in_different·m_edia _fo_r_-~O n,inqte.s befor~. ~pt~ke assays.·

.. C~lls-cult_~red·qn-glass-cpve.rslips":for_7.;.10·d~y~ invitio "."'ere-u~ed. fo_.deterrnine-·-·.· ·

: t_he.effeds ofTTX, ·4-AP: and elev~tedpofa~s·ium on :f_iring of dopamine _11e·urons~ :·: .·

.. · .. ..· Ring_et·~ solution was· useq a-s -external solution while th.e pipe~te was "filled ~ith: a -··

...... : pertorm~d-wi(h_ .Axopatch~200H ~n,-plifier (Mole_cula:r.-Oevices:,_ -s·u.nnyvale, .CA,· ·

·USA).: :.Men:,~_rabe·,v~l~ag·e dat~ collecb:~d:-~r,o:k~~-""~s·fil_t~~ed:.with_l:ow-pass_:·:-:. _

Bessel_·fil~er_at.2·k~z-~ncfdigitized:·by National l_hs_fru'!}ents·_I/0-9ar_d.-_Voltage ·_ .· .. ' .. .. '' ...... - _trac:es we~e-r~corded·and analyzed hytt1e_WinWCP whol_e cell program:

·. (University of Strathclyde, Glasgow; UK)-. Firing·frequen.cy.and membrane. · ·

. ·:, .· ... ·.·:- .. · ... _·:- .· ' ... ·:- .. · .. _·_·:- .. · .. _·_·:- .. · ... _·_·:- .· ... _-_·:- .· ... . VC>ltag~· data -obtained during ·d_rug. :perfusion: wer~ compar~d: to: the ·ctverage· .. .

contro_ldata·b~fo_re_·and_-ctfter· drug_:applicati9n. A-b:2-_au_toreceptor-_me~_ia_ted_-- -

. ·: hyperpola_rization response to dopamine· .appiic~tion was·_ u~ecf to id~n~ify: ·

dopamine·_neurons.-_.

D9_p~mi11e uptake a!5says:

.. ·. ·:After·diff~rent trea_tments·, ·cµ.ltuies·w~_re· washe.ct·~ice·with.Ringer's ·solutiqnand·:

d~pami"ne_-~ptake w~·s.:initiat~d .by_·additi~~- of1 Q(). ~M [2, s,· 63 H]~dop.amirie "(GE:: .·

-·Healthcare,--Piscataway,:NJ:·usA)~---six-minlites-later·,--uptake.,}ias_te·rminated-by.: -­

·. -:twpwashes:of ice~coldRingeri~ solution an:dradio_activityw".1s:extraGted int63%: _·.

trichle>roacet_ic._acid.-_. For-satu_ra~ie>n .~nalys_es, upta_k~ _of five _concentratiqns of ·

...... - ...... - ...... dopaniine·rangirig frorr10:05-4.05-µM was.performed for.two minutes (uptake· of.. ·

4.95 µiVl dopamine -i~:1inear"i1p:to 4 minutes;datc1:r1ot sho\JVn) .. bniy _20o/~-ofthe:

doparnine-at different. concentrations was -radiofabeled-for saturatiqn.analys1s:_. ---- -

...... ' ...... ' ...... Non~$peeific ·uptake was·:detemiin•ed in the:presen.ce of 1_0 µM GBR12Q09. ·_

-_ DAT"antibo_dy :generation and characterizatiofr .

A G~T~_fusion_protein c9ntaini.ng _f\J-terrninc:11 -amino·a_cid resid~e~ of_.rat_pAT (G3_9_ .

. . - ~- Rf30) was· en,~lsified ·in Freund's _adjuvant and ihj~cted .subcµt~neously i_nto ·fou( -:

...... '...... ·:Ne\N Zealan·~_White r~bbits (Covarice·.1_mmunolo~y:_Services,_ Denver, PA, USA). : .

Th_is·r~gio·n-of-DAT_ was previou·sly"used fo.·generate ~ntipeptid~- antibodies _by .. ·...... ·:assessed by western.blots of rafstriataLand.·cqrtical· samples:• One of-the :· ·

a~tisera (8~f~as signi~icantly_bette"r th_an._ ~heoth~t~;:h,•se_lectiveiy".rem>gnizin_g -• · · · .·

PAT -irf striat~I- -~·amples.- ___ T_h is· anti~eru·m was_ f~rther c~arad~rizedari~--~seq- in:- -

-weste:rn b_lotand immunocyt9chemistry analyses.:· __

. . . . : Western-bl.ots-. -·

...... :qlJJtures·g_rown in ·2fwell-pla_te~_were ~a~hed·=twi9e_ in Ring~(s-s"olµt_ion,.-after-_·:

...... _appropric:1tetreatm_ents~· :C-e!ls were·lysed -and extractedJnto -200 µIHIPA-~~fferat

-: 4°C: for"30 miqutes·.with.gentle agitation .. "celf debris:and.nuclei were .re:moved .by .

. -- _ ~eritrifugation at_3q00X :g _for-1·0· mfnutes> .Western blo_ts•of.2~ µ.I ·1ysatesarriples: · ...... ·. -were· probe<;i __ with r~boff polycloral ·pAT an"tibod_y (8X}.: T_h~hlots-wete.treatecf

...... with_~_-comme{9ial ~tripping buffer (Pie_rce, _R9ckfo_rd_; IL) and s~quentia_lly

.. reprobed with.TH (clo·~·e.TH16,· Sig.ma-~Aldric.h, St. L.ouis,--MO,·.USA) and-~-acun· . . . . .

_Phospho~CaMKII (Th.r2a6i1) ~nc(CaM~H anti_bogie~·we_re__ fro_nl-Cel_l __ Sign~ling;-- ·_.· .-.·.· ·.

-: while:pho·spho~TH -(sehs) ·an_tibod_y wa~ 06t~ined.frorn ·1nyit~ogeri (Carl~bad~. CA;·::--: ...... · _·usA). · o·ptical. derisity:of ~a-nd~fon·the.-im_a~i11g ·film w~s· measur~d-Lis_in~·_NIH ___ _

-:im~ge.-

...... '• Quantitative:·RT ~PCR

. . . . . · · ·T~t~I RNA frC?,ni:cult~-~~s:grown:-!n :12~~eit_plates_w~s:extra~t~d :info_~:-9 ml Triz~_I : ·

...... reage_nt p_erwell_-(lnvitrog~n).> ~b~A ~a~ synthesizeq_fr

·:using :su·pers.cript °Iii. reverse{tran~criptase (inv1trogen)-arid_:o.ligo-dr. primers·;. . .in-a. : :.

. . . . . ·.. volurne of 20 µ1..· Four µl:of .RT.reaction ·product was used to ·amplify DAT ontctin:

·( . . ', -: cpNA::l:isinQ SX~R® _Green P~Rst~permix:(~QOn,Nl Kc1,·.49rnM_.Tris~HCI,_ OAmM:·: _-:

. . .. ·:~tabiliz~rs_; Bi_o-:R.ad_ La_boratqrie.s,· Hercules,· CA,· USA). RT:~~arr:iples:"Ye:re ·:·_ :· _· ..

...... analyzed in _duplicate for.e.ac~ .t_ra.~~cript._ Bio-Ra~_-i_Q·i.Cycl~rwas ~sed.to. amplify

. -: cDNA-.with.in_itiai denaturation· fori min.utes at 95°C.·followed.by-4o·cycles:ofPC.R. -·

·:(9~°C; :57~_G: ~nd-72°C ~ _30 seconds e~c:h:step).·_fluoresce3nce "measLJrement~:•:-

- . . . ·. :during th_e·extensi9n ·step ·ofPCR cycl_e·s.were -~sed to·c~lculate·-tn.reshold c.ycle-· · ·

. . . - ...... - . . . . . -: (CT} vali1es. -Maximum c·urvature :offluorescence traces was calculated"from

. . - ...... ' ...... - ...... -:DAT mRNA·aburidanc::e were· calculated:bft~e·¢o~parati"ve. Cr rnethod: ·.

...... · · (S.chr:ni~tg~.n and_Livak ~008).usir.,g -?ct_in fTl_RNA_as _an._internc;1_l_c9ntml in_·each. · · ·

.: s·amp.le;: The: underlying. assu·mptions- of -~he. comp·arative -C~:method, ·that.:

. . . . . •imiplification-eff1ciency. of the two c:DNAs-are.sfrniiar and.close· to·100%,: wefre:·._ ...... ·...... ·. . . .·... . .·... . .·... . .·... . .·... . .· ...

__c:tirectl_y-validated- ~sing standard-·cu~~--analys~s·(.Figure_.5} .. · PCR __ reactionfof_. -·

-: RNA-~amples·that werEHJOt subjeqted_to revers~ transcription:demonstr~ted that:: -:

. . ~ ......

. . . . · _ there. was·ncfgenomic·nNA·coritami_natio·n.: M~lt-c-~rve_and-agaros.e·gel ~nalyse~ --

. : --_ ... ofpCRprod~.ic~s-sho~eq-thatoriY. one p~oq~·ct-~fpredicted_siz~ wa~·amplifi_ed __ i1f --­

each. ~eactiori_.:.:. Pri~e~s: _ll~_eci to. a~plify DAr. ~nd .actir,.. c~NA: 'Ne.re-;_ ..

. . . DAT{F) ~ 5':.J(JGGCCTCAATGACACCTTT-3', -· · · .

. . . ,...... -D~r (R) -~. 5_'~AGCAGMCAATC3ACCA~_CAG.CA~3'-,_ . . ···:·_. __ AcUn--(F)_~-~'.~TrG_CTGAQA_~GATC3CAGMG(3.AGA~~-'- c1nd_...... ·Acti.n_ (R) -~- 5'_.. TAGMGCATTTGQGGTGCACGAIG_-·3-' . .

. . . . h11mun·ost~ini"ng an~l"~lu~re~c~rice i~agh~g ·__

. . . _Cult~-~~~ Wer~_ -f!~e~. wit~_ -~o/o _·paraf prm_alde_hyqe_ ·in -P~S: for ·15_ -~,~~tes: -~~ ·t~om

...... :tempe~at~·re._ After_two w~shes._in: PB~, :t~e i_mmunost~in_ing_.proce_du"r_e was_

...... init_iated wit~_-a.-~0-r:ni~ut~_ in~ub_ation in-~BS·_conta_ining 4_% _horse ser_um~_-1 %.. · ...... -,._· -: bovine seri..im.alburriiri:~nd: o.-1"%.Triton· X~1.00. -For DAT-and TH: double. -: -· . '. . . . ' ...... ' .. · ·:·iryim~nost~i_ni~g·,. cinti6o~ies·:a)(a~d TH_1-~. were-u~ed· in ·c9mb!natibn_-wi~h-AIE:?~c;l:- '.

· ·:·_-. __ F·luor)l8~-and-Ale)(a Flo.ur 933-conjug.ated--secondary.artibod1es._:··1mages Y"ere· ..

_: ··:·: -: c;aptuh~d by la~er-s.carin:ing c·onfocal micro.scopy (Catl~Z~iss:,.·Thornwood;: NY,_:··:·: .·

USA) :~sf~g-·o~?.( .?Wiml:n~r~ion-·objective·wit~·-a11u·mefica~·.ap~1u.re·or1 .·f-: qpti9ai .·

- -:seqtions-(2.0 ·µm· th_i'cKr1e~~): i_n the:widest_ p~rt of th_~- c~ll:som·a:"Yere imaged fot_ ...... each·n~urqn·:_:_i_n._ou_r .cLil_t_u~es,. app_rqximat~iy.40% o(neur9~·~:_(N_euN · _.·.· ·. ·_ .. ·

...... immunoreactive· cells) w.ere found-to be· dopaminergic (TH.positive).·-. Dopamine_·_._ -: ...... ·:neu_rons tn_ iy.ltifre•:~r~·:e_ither.~_i-p_olar or_rnu~tipolar_h:1 theifappearaiic~ a_nd th~_::•:-_::.

. . . of two: diagona_llf o·ppo·s·i_t~{proces_ses in eaph:neurcm~ ·soma -~_nd proc~$Se~- wer~ .·

...... outl_ined_ by-ii~itig_TH-irnmunostairiing for fh.io~escence· iritensity-·rneas·wen:-tents;· ...... - ·: ln"p_r~limiria~{~xpe_rinierit~,-:we·compare~· backgro_Lind~s~btract~dJlu_(?r~s-ce~c~ .. : · - -:·.

. . . int~·n_s,tie_s_.of PAl in 2-~9:_µ_m optl(~al __ sectioh~ _(Meta~:._2: sortwa_r~_;_· C~r1~·?ei~s) _a~d_:_: ..

the :int_ensiUes ·of _30:.r~Gonstructed -image:s _from· Z ~s_eries. of optical -se~tions. (bY: -_·...

_intensities-of DJ.\T analy~ed µy_Meta 3.21n ·2;0 µm.optical.-s_ect!~m~fwere·~s _-- · . . . . . - . . . ·:charigesJri DATa.bundanc~ between neurons ~ithin :a· treatment .fllid between

: tr~a:tmehts_ (Correlat_ion: c~effi~ie~t:of flt:1~r~s~erice-interisities: m¢aslJred':by th~-: ..

...... ·. two:n,et_hodsJr9m the·~~m_e.neuro_ri_s was: q:._9~ for·[lf\T a·nd-·p .. _99 for-TH_)_:' :All··>_.:

immun9fluorescen~e :intensity data_preser,te~ in this r,:ianuscript were_ fro_m:~.0 .

. . . . µm·optical. sections· ar:,d analyzed·;by Meta .. 3.2 software. Argon or helium/neon . ·

lasers.were used:to··excite:Alexa.dyes ·at 488 and 633 nnf ~1rid-emissionwas

.:fi_lt_ered -by u_sing: band~pass. 50Ei~53:o anq _long-pass. 650. filt~rs, •retfp~·ctiyeli -..Th~ : :-

· .. same:settings·of-lciser pow~r;· detectC>rgain:an_d·amplifier·offsetwere-us.ed_for.aU: :··

. . . image: acquisition with1n:ari experiment.

. : ·l_mages.of_T_H-immu.nostain:e·d 'neurons. acquire~_With'a.,tqx-obje~tive·were .

. ·:Us~d- tci m_easi1re' pa.rameter~· of _dopamin~··neuronar morp't-1ology. ·: Fifty neuron$·'·

. - - . . - - . . . . r~nqc;>rnly ~ele~t~d fro_m.e~ch.tre_atment.group (1Ope_r.experirnent,·_5. ~.iffe.rent _· .

experiments}were an'alyzed.·· The.diameter.·of the· dopamine·_neuroriaf soma'was ... ·

...... ·:m~asured ctt-the.-wide~t regron_:qf e:ach _6~11.: tength_s:and. riumbe(of hra:riches:_of: :. . .

. . . - . . - . . the lori_gest,. second -longes~ .and-short.est pr_oc~sses-were mea_$U~ed _by' :LISi_n'g ...

Meta.:3.2 softwc1re .. Numpers' ofT:H~'pq.sitiv~: rieuron~·wer~ ana_lyz~d by:counting·:··

·all'neurons-on.a·coverslip in each:e~periment (total· of-five exper_imentsJ.·· A11 ... ·> ...... jm~ging anal_yses .wer~:{perforn:,¢d blincH~f t~e .trea~me'nt-parameters~·- .· ......

. . . . . Ace:toxyni~t~yl {AM) ~ster form~. of_the_ccilcium·inclicator'dy~~ fluo~4 imdJura-red: :.

. . ·. __(ln_vitr

...... · .· ·:were loaded. with fluo~4 ·and:fura.-red dyes. (4;5-:µM·each-:in .Ringer;s_ solution) .for . . . - ...... ' . . . - ......

. , .. ·. .20.-30: mi.nutes·.: Tin,e.-lapse_:ct>"nfocal _iniages··o.f. 4:0 µni optical se9tions· were

-: cc1ptur~·d u~ing:·a·4ox achropl~n: ~bjectiye ~t _1 second_.interv~ls._·: B(?th: fluorescent -:

probeswere·~~ci~e_d wi~h_il 488--h~--~rgori:·la_ser,·-an~--th~ em1~t~_d:fluores_~·e:nce·-·._

. . . ·:was· re9orded· between· s:oo-s·so·nm· fo'r~ nu·o~4- and·betwe~n:t?.42~71_7 nm:for:ft.ira~-: ·.

- . . . . . '...... red~ .The fluore.scenc~ _of fluo.-4; .fura-red_·and. their"ratio w.ere_.analyz~d-.in-.

...... - .... -: selected re~ii_ons-of friterest. . hi ·order to ·account for .variabiHty-.in-bas·e_lirie :

·:flu_orescenc~:vaiue~, -~II _fluor~~c.encEtratiQ _valde~_we"re nqrn1aiizecHq :a base_li_r)f3_:.

...... · __ ratio•valu:e calcula~ed.-from ~Meast .1Q.data-poi_~ts.just.prior.to·batti -application.of:.·

· drugs:: Fluorescence ratio values:during the drug application-were_ compared to ·..

. . those :of baseline_ to.-calc~lc.;tte-average .effects_ for diffetent-treat~erits> ..

S.t~ti~ti_cal. an~_lyses. _· ... · ·

. . Be~ause ofthe-.limited :number of .ce.lls·obiained ·in" each bafoh..of culture, we·we·re.·.

__ culture;-e.xperim.er:-ts were-designed to p_air.dr~g-treatrne.nts.-with c1pp_ropdat¢ ·__ · -· ..

. . . _: · :·: -: vehicl~~tr~ated·~ontrols·. ·Paired: Student's Oest_ was:used to" asse$s th~:effects: of .·

_·:drugs·on-:diff~rent ·param·ete~s st_udied .. -GrapnPad-"P.ris~ {V."4.0) ~as·.use~ "to.· . . ·. :determtne-Vm.ax··and:Kt:val_ue·s_ of dopa.rnfn:e··upt~~e.: --- . . "Results·

-_ ·:Characterization pfthe: do:pamine transport~r ·antiserum.·.

S_evera_(cormnercially avai'lable DA! antibodies.\ft/e:re':f6u·n_~ to:be un_suitable for:·· ·

. ··-: west~rn blot-'a_~~-imm.Lin9cytoch~rr1ipa.l-·ana_ly~es·o(qAT i'n-rat_m_esericeph_alic·

-- ...... ' ...... ' ...... ·:primary c~ltures~· Rabbi°f polyclqnai_" a~tiseru~ ·(8X}wa.s·g·eri~ratedag~irrst-a :· .. - ...... - ...... GST~fusion. protein ·containing .an.N:-t~r~inal_.p~ptide·Jegion_·otrat DJ\T.(G.39_,-< .· · . ..

. . . ·:im_rnunoc~o~h_emical_· c3nalyse_~·._)_n :we~~~r~ :blofa _qf. sanipl~s:-f_rorrf different -b~ai_n. · regions·, ~·broadband that qorresponds-.to· bAJ°w.as--de~e'cted 'at ctpproxinu~tely -

...... - ...... 80 k:Da·in_strfatum and nucle.us.accumben-s·; the two:braih regions with highly .·'en'rictied 'dopamineoterm!nals-and 'DAT(F':ig~~e·_6Af. ~o 'sig'r1:ificantDAT" · ....

...... -:imni'l.in6r~a¢t~vity w~~f~bs_erved in: brafn :regi(?ns th~fd_o-notexpress ~_ppreciat)le·: ·.

...... amqunts qf.DAT.. A_ntil).ody ax detecte_d th~·transporte_r in. lysa_tes of rQAT- ·_ .. -_._·_· ._ . . . .

. . . . . : trarlsfected HEK293 cells· bufnot'.in untransfectea· c.elis (Figure·asr · More . -- .. :·_-_._·.

·:important'ly, :this: ar1tibody :primarily re·cognized ·:a b·road band _correspondi:n~{to:· ·._ .. '...... ' .. . . . - . , ......

. - ...... PATw.ith negligible··cros·s~reactivit-y·to_·other·proteins :in l_ysates:·obtained]rom ·.· .·. •·

. . . . .·...... mesenc·ephalic·:cult_ures :(Figure_:6G); Prein_cubation- ()fDAT .antise·run:i With 5

·:µg/ml:Gs!-DATp.mtein.elimina~ed··~he. ban~-c~_rrespo~di_ng.,o :o_AT. --In.: . ·. -:imrniinoc_yt~chemicai:~·ssays,· aritipody-~x··recog'r1_ized·a.Jfroteih.thafis.present __ ...... only _in -~opam.ine· (tyros_i_~~ hydroxyl~s~Hmr:J1unoreaptive)·_neyror:ts ~nd_·_· _

. . . .. pre~dsorptiori_tci.GST~OAT prot~_in_-l)lo·cked_.this.sta:ining (Figµre:.p □): ·These·datc3._ .. - ...... ·:m_Li~trate·t~e:~p~.cificity_ahd tlti!ity of an~i-~9dy. BX in ~nalysing_ DAT.abundance!::i~ : .

mesericephalic. cultures.· .. ·...... , ...... _: D~par:nine· ·neu rans ·in· mesencephalic: .c~ ltu res .fire· spontaneous· ·action· potentials : .· -

: arid expre~s f~tnctionai DAT (pra:sad ar:i~?\mara, _200:1). ·.}:\ctiy.ity cif_these-:

.. ·: dopctrr,i_n~·-ne:~r~ns. was -~bolistied_._by tetro~otoxin. (:fJX) fre.atrt)~nt;.wh!l~-it was:·

...... ·:~ignifican~iy:if1Cr~asedby 4-a·minopyridine .(4-AP, :Figure 7 At. Treatni~nt,'of . . . .

. . . . . cul~ures wit~ these two .. compou·ndsfor Sdays, but notfor.30. .minute~·,_a.ltered _ ......

. . . : dopamine ·uptake (Figure 78)~ ·r~dicating ~hat-sustained changes: in.·rieuronar

...... ' . - . . . - . . . . - . . . '. - . - - - . ·:~qt_ivi_ty leact·_to_ alter~ct.·pATf~.n~_tion. ·:~_aturatidn __ analysis-9.f_·ctopamin~. ~ptake __ ·:

. . . . . ·. __indicatedthat'TTX_.caused ~- significant decre~~e•in•the xnaximal y.elocity. of_-· .· · ··: · ......

...... uptake '{Vmax):'with nd change in :apparerit:affiri.ity (Figure 7C):.: A decre·ase.in _:

. ' ' ...... - ; ...... - . - . . . .·-: Vn;ax._c.ould refJ_e.cta ·c~~~ge -in- ~a.talytic ~~~ivity'or ~- decrea~~)n_· the n·um_be·r of: __ .

·:fun.ctio:nal DATmolec~les. ·short-term -~h_ariges. iri -neurona1·.activity are ~nlikely to: ...

alter-DAT. catal~ic_.rate.·as.n_either:pre.tre~tm·entfo,r. 30_mi_nu_tes:·nor'co~in¢ubation.·: ·

-: with ~rrx ·c1ltet~d-0Ai-~m.~diatecf uptake .(.Figure· 78.; -(Pra~aci.ar1d:An,ara;: 200·1·).: ··:

. . . . . ·:Chronie.:eh~riges}r1:n·euro:na_lactivitfalter DAT_ proteiri:a·n~··mRN_A: _· .. · .· . . abundance ·· ..

• • • • • # • • • • • • • - • • • • • • • • • • .·-: We:u~e9 western blob{an~ semi_-qi1_arititat_iy_e.:immun_9cytoc~~rni~al· as·sc1ys to··>.-.·.·

:directiy'test :the: ~ffe?~~::of sustained· cha_~ge:~_-in·.neur6nal.acti:vity on OAT,prbtein : :.·

( . abundan~e; __ ·D~T abundanc~ was·_signifi.ca·~tly :de_cre_ased by-chmnic.·rrx

-: t_re~t~ent~··wtier-eas 'it was·incre~sed by·4~AP<(FigLfr~~ -a~·and 8Bf Abunda_iice-·.

:qftyrosiri:e_ hyqioxylase _(TH; -the rate iimiting.·enzyme· in 'dopamine biosyntties.is:- .. . . - . . .

. . - ...... ·:ah~ ·a:rn~rker·for·dopaminepe·~-rtms ip these·c'-1l~ures)ar-,d'J3.::actir:J were: noi .· ...... -:~ignificaritly·al~ered-_by these ~rea~ments_;_demcin_s.trating-:t~at:t~e efl'.ects -of·:- ___ -_·:- - ...... --- - ·:sustain·ed cha·ng·es. ih.-neuropal.-activity·on-OATare telatively·speqific-to· this.. -- ·__ --- -- ·

.••• > . ' . --- -. p~ot~in.· Further~cire,-(?A! mRNA: abundartce assessed.by-qli'antitative:RT~PCR: -

...... ·:lmmunocyto·chemical:analysis i~dicated:t~atthe decr~ase ih DAT~bundance:·_ · ·

. . - ...... ' . . caused_byTTX.treatm·ent-in somaand_.proc_esses of-neurons.was comparable in-·._ ' ...... · . ' ...... ·

·:morp_holdgyqf culfu_re:d ~ells-including--thos_e:of~_opamirie_neurons--($ctlthud~_- _:·_

·___ Lassalle -~t aL~--2004}. Howe_ver, in our experim_ental·co~qitions, ·to.tal numb~r of-·-: · . . . . .

. . . . . -:lerlgth ~nd brari~h:nu_rrjb~rS:~~te· n~tait~red:byT:l~X:treatm~nt· T~-e$_e· d~t~- ...

. . . ' ...... qemonstr~te·,~a~ alter~d_-s_urvival_qr_morphqlogy_.of _qopamine neur~ms_.is -~ot. · _- - ' . . .

. . . ' ...... ' - - ...... responsible· for .activity;depende:nt chari~jes_ -fn DAT ·protein -or. ni RNA ·abundance~ -.- --

' ...... _Neuro_n~_l'-firing ·c~·n-cause_$~stai_n~d cha:ng,s ·jn·-C_al~ll"KII activijty_.in --·

.. . . -'Neuron~i activity _could·reg_u·1ate·:0AT function_ and-'abundariqe 'vi~·sev~ral ...... · -:intr?ic~llutar:sigh~ding:p·a~hway~_that hav~ been- pr:eviously sho~·n-:io·~e- iniport~nf -· . . .

. . ..· . - ..· ' - .. · . , ... ,- ...· . . ..· . . ' .· . . .. · i~ atjti_v_i_ty-dep¢_ndent. pl~sUcity' (V~m_ant_ et a_l. '· ·2q.02·~: -~~Ue~er 'e3t _al. j' _2094f ......

...... - ...... ' . . - . . Calcivm influx-~·n_d .sut:)se3quent'~c:tivation:ofCaMKll_s·ignaling-_often· s~rves as th_e_· -: . -·

...... ·: in i_t!~I stimu ~u~ fQr ac~iyi_ty~dep~·n:det,t ·g~~~ expre_~-si_on:. in -~e:~_ro~s· {~!~.Ids: .et ·al_.:!.:_

2005):.- Thus·,-~~ a~a_lyz~d iritr~·ce_l~ular ~.alc_i~m ·dynarnics-~nd:CaMKil.-~ctiv_ity_in-·> .. .. ---5.1 .·

.... ·dopa~Tline·_neurons.-_. Fo~ cal°ci_um. imaging·:expetim:e.n_ts; dopamine·:neurons w_e.re.: :.

. . . . · :· .. ·. · .· Jde~tified:b}':a~ecrea_~e- ~rt intracellular calcium:.concentratio_n··~au~e~_ b~ 0 2,.-· ·

...· . . ..· . ' .. ·· . . ..· ' ...· . . .. · . . .. · . . .. · .....- . -·::: .. ~µtor_e·cep~or··adivati_ori (Figur~s-9A_ and_ 9~l--_Applicationpf ·T~X d~Gre:as_ed;. . ..

. . ' ...... · : whil'3 _4-AP- inqr~~sed fri_tracellular_.calcium·_in· doparn_ine: neurpn~_(Figur~s· 9C·a~d

...... ·:9p)._ F_urth~-~more;:"L~tXpEfcalcium:~hanri·e_ls ~re. irivolved·_in:·mediatingat lea·s~_: -.

part of the.activity-dependent-calcium inffux.·into.dopamine-rietirons-as-nifedipioe·.· ·......

- ...... ·- . ·• (an_L~~w>e·caiciurn dia:nn_el blocker)-•signi.fic~ritly.decreased·_in~race·i1u1~frcal(?iUrn·:·:

...... ·co.n·c~mtration-,n-con.trol _culture·s~- These.data indicate-that .altered neuronal· .. ·.·.

...... · .ac~ivity caLis~d by TT~Umd A~AP•is·acc~~panied:bY ~hanges· in" intr~cellular ·

-: caltilim-:ccmc~ritr~tlon~: :·· ......

. . •· -. yYe-ne~-~ncdyze~_CaMK!I_ ~-c~ivity in _dopamin,f~e_Liro~_s._by _me~~1:fring ·

...... ' ...... ·:Ca~KII and:TH phospt,orylation_. :qaMKI.~ s:~_bunits __ are autophosphoiylated·_af._

. . . . -: provides a· bipchemical measiir~-of kinas~-·activatiori.(Loli and-Schulman,·•1989).-·......

...... · · · Jts·.activity)"and:is not.select_i:ve·.tor:dopamine·neurons.··TH ·is·pho~phorylated at···:_·.

. . . · · : Ser~ 9:s~h3c_t~vely by'.Ca-~Ks and:thus provides a direct measure oft~e:activity of':·: .·

. . . . ·: - ·: . . . . . --: ~hisJa~ily"-ofk,i~a:ses in_

...... ·:Tra~sfedion·_of:con~titutively_ ac~hieGaMKff cion_strticts· (~2aso· or· T2a1PY but:riot._'.: :.

...... ' .. wild~typ_e constru.cts.i~creased ~.er1s ptioshorylation._in._MN9_D..doparn!he.rgic ·ce_ll~--- ._

'...... - ...... '. . .· . . . .· . . . .· . . . .· . . . .· . . . .· . . . .. · ! . . .· . . . .· ·:ph9shorylat_icm_ in me~enceph~li~ _culti.J~e~., indicatin~ that TH.. phosp.~9.rylatio11 __ •

...... · .. :52-

. . . - . . .. - Jhat ·is consistent vyith-the.·effects··ofthe·se drugs·on ·neuronal·firihg. rate··and:- -- ·

CcilbiUm: irif_l~x :(Figure -1 o·s). ·.rv1me·:imp.~rtantly, C_aMKll":an~ t~: pho~phorylati_on:

- . . . :(Figure_ 10C)._ The~e:data d~monst_rate t~at -~ct!vity-depe_ndent·changes ·in·:·_ ...... CaMKH sign~_ling are sustained-for at least 5 daysin-.dopa~·ine -neurons:.

. . "l~-type:-cal<:i_~m:chan_n~i-s anc;i GaMK !;ignaling are: inv~lved :in:ac:tivity-·:·

. . .. __ depende.nt.changes.-in DAT·functio_n· ·

. . ·we n·ext ·sought to·_deterniin·e the:to.le_of cal_dum and: CaMKII :signaling :in··activity~ ·.

· · _ depend_ent- regulation of DAT~m-edi~te_d tipt~~e._·.Calci~m·entry via_L~typ~·_voltage: -- - . . . . .

...... , ' ...... - -:gated cal~"iu_fTi -Gh-annel~- and·_NMDA receptors play~ ~n inipqrtaf:it-role in: acti~iti ·: ·.

' dependent regulation _o(gen~. expressi_on_in:qeu_ro_n_s:_(Badin_g:_~tal., 1993)_: Ip.

...... ' ...... - ...... , ...... - . . ' . . . . . - -: order.to:determine-the:·relative· roles:ofthese two ·modes-of calcium·en"try in--· -

·a~tivity,.dep~ndenfre_gulation _ofDATf~~~ti_on, ·w~ :~ssessetj-the effe~t_s of ·:· _

__ pharrnac_ologica_l-i~hibitors- C?fr dopam_ine_ uptak~~-- ·Chronic treatrne_rit with--­

nifed1piriebut n·ot an NMDA antagonist (AP5f d~Grea·seddop~mine upt~ke i_n _: ..

cultur~~- ~ith"norrr,·a1 a¢tivity· (Figure 1tA)> Jn ~ddiUo.ri;· ~ifedipine abolished.the>. -

- -:sti"r~u1:atory e~e~t of ·c~_fonicA~J\P treatment and· occluded· the effeciqf TTX·o~

dopa_mine _uptake, ind_icati_ng·-~hat_L-_type c:a_ici_um __ chca~nels_ ar~::i"o:vol_v.ed_·i:n:ac,ivity~: _-­

: dep~ndent regulatioti ·of.DAT.· ·wetestedJhe.:roles:ofd_ifferen_t_.protein:k_in~ses · :· __ ..

- th~tare lik~ltt~:be.st,mulate.d _by. n:euro~al· ~ctivi~(: KN93_, _but: ~-ot.ir1h_i:bitor~u)f - . . ' ·:Similar·to. the·effeqts ·otnifedipine,: KN93·o·ccluded the··e.ffect~fof chronic changes:.·.

i~ _n~urona_l_activity".cause~: by 4~AP.and TTK: T~e~e:data _irtdfcate ~hat:calcium · .

. -~ntry. th_ro_ug·h·.~~type calciqm· cha_n:n~ls-a·n:~ _CaMK·~-c~iyation:a_~e:requ1r~d_fo_r-· -·

. ·:activity~dependent regµia~,o~. of _bAT·funcHon. ·: ·..

. ' . . . ·-ois~ussfo_r)_·:-

Based.-ori the·findi_ngs pres~nted·aboye;.we. pr9pose :th~:t-neuron~I firing rate·is ··

. . . one of the majo(determihants of DAT.abundan·ce fn :dopamfne neurons·:in vitro. :· ......

prope_n~ity .t~ _self-admini~ter ·psychosti_mul~_nts (M~ui_n~lli ~n_d .. \/\/hite,. 20.00). ·

Perrnanenfdifferences in.dop·amine:ne"t.ironal firing: rate are· likely to ·ccmtribute to. .-

·th~- _reportect~nter-i"nd_iyiduarvc1riability in_·_dopamiri~:transpe>~er exp~es_sio.n in .. ' ...... nu.mans. _·1n_.adctition,_activity~d~pendent_.regulation of.DAT.might be.respo~sible;·:·.·.

~t lea~thl_.par( for the.cn~nges in:DAT exp_ression:c~use_d by.:chro_nic:exposure ··:·:

·-:to.psychostifl:lul~mts; antipsychotics o:r.str~ssfu:I erwiron~en~ar s~imuli",:all of which·· ......

. . . . required t(?.-a~~~$-S if n~:ur9.na_l _activity playsa_·role iri_invivo c~af:ige$ 6fD/\T ...

. . ... abu:no.ance·c~l)sed bymethylphen_idate·a:no_·other-d_opaminergic:drugs_. These ·_ ...

:d~t~ also.indicate tha:~•:stimuli _t_h~t :1ead to sustain~d. charige·s in:.CarylK)I :activity.in::.

dopa111ine·.n~urqns_·can ~lterDAT.functiqn·and abuncfan~·e; __ - ...... :_ .. : :optimal :dopamim~:sfg~ali~g:·in:·t_he:~rairi: is_requireg for_ rnafntaini_ng ·:· .· _:_.:-.: :.

...... ·. ·:appropriatt:fbehavioral tesponses:to .erivironmeritai--st,mulL This principle i_s -- ·

9?(eh·1plifie~. by:coniprornised \JVdtki~g metnory p~rform'arice-ca~Sed by a_·:

. ' qevia~iq_n _in- e.i~~-e~ direct!

. . . . . ·:prefron_tal _cortex_ (too :muc_h ·qr too :li~tle d:oi>af'!lin_erg_16:t~me) :(Y1jayragh~van -et aL, : -

...... 2007)..· .Dop~mine_ ~eliro~~an_sn:,is~i~n _is-_con~rolle~_-bY:· several..~~m_e~static

. . . mechanism·s_:such as "DAT~med_iated dop_amine upfake; fee~:-back- inhibition by_:_- __

...... ·:t~~-s~riafo~igrai :pathwat, :and _rE3QUlatfo_n of :nelirc>r)ctl :firin.g_ ~r:,d dop_~.rni~e

. · ·:synth·e.si~_-by D~~au.torecept9-i-s·.- DAT_regulatiopby fidng: rate-·repr_e-sents"a novel·-

h~~eo~ta~ic -mechan_ist)fthat _i;s-i_i_~~_ly t~ _op~t-~te _on: a::~iffe_ren~:time~scale tha~ _:: :·: _-:

...·. . . . ' .. - . . . previq_u~ly-descri_bed·dopam_ine regulatory-mechanism$._ -In- a_~-~i~ion·to-controlling. _-

.... ·:the _extrac_ellulat:_dopamine levels, :alte_redDAT·ab:u~d~:mGe •might also:~e~ermiri~ :

the·extent.of"paracrin~_signaling.by._dopa·mine (Floresca et~L; ·2003):" .-In our- - ...... ' ~xpe"rimerita!: conditions·,· acti~ity-deperiderit reg_ulati~n-w~s to·u_nd to be· :specific to

. . . . ·: DAT as the abundance of TH wa~ not regylated by ~hron_ic chang~s in neuronal

...... _firing·.: .F~ture-·stud_ies.will .ne,ed.-to- ex:8:mine- ifother prot~ins involv~d ·in·dop_~rnine: _-

- ~i~n~~i~g -~_u

...... ·:fo(amino·_~cid n~urotran~mitt~r~ '#as:·show~_to _be._reg_ulate?:-iri :an·_actiyity-·:·_

-· - · de_p_endent ~ashi_on _(0€3. ,Gois_ ·et_ aL ,· _2005}.. -.. -

. -·_: -----Alth·o~:g-~ survhial:·~f: p~st_~riatal-dopamin~rg-i_c-·neuron$ i"n culture-h~s been·--_ -:

-previ9usiy_sho_wn to.be ~lte·r~d-_by·nei1rona_l"actiyity:,. we fa_iled _to see.any.effect-of --

...... --- : --- ·:activity··orrdopamine·neuronal"nu'!1be.r"or morpho_logy.~- put culture··co~ditions, - · ... :55.

...... ' ...... ' . . . . ·:such as us.e.of-glial~con~itidned:me&um.and aHowi~g--neuron~ to·:develop for.a

· :week before altering their activity; :were ·optimi?;ed.-to miqimize :any effects qf ·_ · ·

·a~tivUy"oti _neuronal su:rvival ·or morpho_logy.·: _Act!yity-~dependert~ch~ng:e·s_"in.

dopc1n1ine· ne;uro·nal v1a~_ility could_:~~ n,ask~d:by suppl~men~~t_k>:n.with_~lial_celf -: .·

·:lfne.~deriv~d :·qeurotrop~ic ·fac~or.(GPNf) :(Salthu_n~Lass·a11~ et al._, 2904)".: -~eyeral : ·

.. - ...... - ...... trophic factors_presen_t .in conditio.ned medi_um and· addition _otG.DNF _are .likely_to·.·.

eii~ute maxf~ai" survh,.alof dopami.ne-iieurons iri ·out-cultures:.-. Indeed, · -:

. . ·:withdrawal_ qf.ccmditio_ned rrieclitmi :ca:USE!d a:dran,~tic:dEfor~ase- irisµrvival ·of

__ dopamin_e· neurom, .. (Figure 1.2),· sugg~sting thaUrophic factors ·pr~s·ent irr .. ·......

...... conditioned medium are:"likely to ·have. offset the effects of activity on rle·uronal _:

suivivaL _It is: po~.s~ble)h:at. a_ctiv1ty:-de.per1dent release· ·9f heurtjt~ar:ism_itt~rs .9r-. ·

. ·:Ott)~r :molecQ_!"es _into the :medi.um m:ight i_nflLience ~bundanG~· of _DAT::. ·H~w~v~r;.: .·

a~y--~~ects .ofthe.se_.fac~ors wo~ld_have to pe med_iated by L~type cal_du"rn_·

. channels -~nd .Ca MK as .. ph:armabological i"nhibitiori i,f. eith.ei". of. these· p:roteins . . .

. ·. j1s_cultur~s frorii·kr:,ockout-animalfor_use of_co_nstitut:ive_ly ~ctive·a..nd. dornir1ant · .

negative expre_ssio·n constructs.are "likely.to·pfovide·a.dditi·onai:·,nformatiqn: on the:·: .·

·role·of-CaM~II _in __ the.·reg:u1_at_io1i"of DATexpress•ion:and_ the·cell··~utollomo_us.- .

. -:ria~ure-·ofthis ·regula.ti()n·.: ---

: ::· Regulatio.ri of :oJ\T abund~rice ~y _r1eu~onaJ a:ctivity __ ari

...... niight.occurdu_e.to.Cha"ng·es.in·th~.rate·oftran_scripti_on,:mRNA.stability_qr.protein_ . .

alterectneuronal·-~ctivity·~ugge_st-that biq.syrith~si~_-of:DAT protei~_.Pl~ys·:~· role_.1n.·:·.·...... ·_ ... :55.·

...... · ·:between.UATprotein-andmRNA mig_hrbe· rel~ted tc>:po.~si~_le_ adivity-dependent: ,·-

effe~ts _ontransl~tion_ a".'d!Orprot~i~-:ha!~-life: "ActhAtfdepend:e·nf reg_uiaticm 0~ tt,:e: -:

~bu-~d_ance of a~amina.:.3_-~yqroxy~5~.methylisoxazole-+~Prop[ona~e_·(AMPA) ·_. · · :· -

. ·:receptor·sub.~ni~s and.~e".'eral other prote_~ns_.in-the :pdst-~yn.apti_c derisitY:.w~re·.:

...... shown .to bedue.to·_changes·.in_·proteosomal degradation·a~d proteinbalrli~es_·

(Ehlers;: 2003)> .. Howeyer., -acfivi_ty~depen~ent-regtilation of brain~derived.· -: ·.

. . . - - . . . ·:ne.umtrop~i.cJa·ctor-(B.DNFfexpr~ssion is:ca:useq)~y chang~s. in fra.n.scriptio~ _t_h~t :.- .

· · · · · Preiimina"ry evidenc:e indicates th.at"bAT mRNA.1s·.decreased.wtiile Bb°NF mRNA:

·i·s-in?re~sed-·b_y:_sustaine_d·.?e·pol~riz~tion-~~-~t~ -~ot-·s_hown),-·s:~g_ge~tirig_~_-possi~I_~:.·

·:d.ive·rgenc~ ~etwee~ °qaMKIJ_"and· c:a~k~y~m:ediatecf :chan.ge_s· in ger,~ ·expressi_on

i~. m_e~enceph~lic cultu_res_." Expre_s~ion .of :yesicular. transp~r_t~_i-s· fo_r .~xcifatory _· ...

· a·nlinhibitoryamind ·acids:showed.·oppos1te··regulatio.n:by· rieuronal" a6tiv.ity .. ·

. . - - cuJtur~s (.De. Go.is _et aL;--200$} ... s·e_veral_pO$t-t~im~lation~I- modific~tiomi'of OAT·

. . ' ...... or assodated molecules·a·re· invo·lved ·,·n 0Ar-:1ocalization ·and.function.·.·

...... (Morten,senand Ama·ra, ~9O3f.:For exafop~e!_ the.phosp~oryla:ti~:ni_·of.PAT_ ~Y .

. . . . CaMKlla:was showh to ·be involved.in amphetam.ine~induced ·reverse transp·ort ......

w.og_~t_aL_.2oq~>:. ~.owever, -t.he$0 mechariis_ms_.are unlikely t~·play ~l in)o·ng~ ·. . . f.~i~'

...... term_-~ctivity~cJepende·ntregulation_·of DATfunction:·a_s· acute :Ghanges :in.firing ·r~d~.- .·

...... ·:d_id-_notalt~r_DAT-m~diat~d upt~ke'.. Furf~_ermor~/9h~nges::ir:t ~bun~~nce of (?~T:

pa_rall~led -thos·e_ ·of_ upta~~- ind icatir,g that. different_i.al. s_urf~c~ expression: ·of l;)A t. ·is·_·...... ·:demonstrate that activitt-dependent regulation:.of.DATi~ mediated·by-CaMKs.-

. a11d: m~y play :c1n_.important role -in:·d_opamine _sig~~li~g _in brair( _FutLJre:-st~dies .·:: ·

. -·-: ·wii"l--~ave fo-v~l!~at~-the_-~~!~tive -~~1~_:of.tti1~-for~·-of :n~_u.mplasti_9ity._in ·th~:.-··: .. . ·:reg~latjon_ of.PAT in vivd.

......

...... ·· Figure 5. RT-PCR assay validation We tested the assumptions that amplification efficiencies of DAT and actin cDNAs in PCR reactions are similar and are close to 2.0 (100%). Different amounts of pooled cDNA sample from cultures that correspond to 0. 05, 0. 1, 0.2, 0. 4 and 0. 8 µg of total RNA were used to generate standard curves of DAT and actin threshold cycles (CT)- Standard curve graphs were plotted with RNA (µg) concentration and threshold cycles. Insets in these graphs are curves of relative fluorescence units (of cDNA samples corresponding to 0. 05, 0. 1, 0. 2, 0. 4 and 0. 8 µg of RNA) plotted against PCR cycle numbers. The regression coefficients for DAT and actin standard curves are greater than 0. 99. The data are best fit to the following equations; DAT: CT = -3. 189 • Jog (µg RNA) + 20. 98 Actin: CT= -3.172 • log (µg_RNA) + 10.56

Amplification efficiencies, defined as product increase per cycle, were calculated by 10(-tlslopeJ_ PCR efficiency for DAT and actin were 2.058 and 2.066. These data demonstrate that amplification efficiency for DAT and actin are similar and are close to 100% (102.9% and 103.3% respectively). :Figure·5·~ ·. _· . . .

· · · :· DATRT-PCR _Actin RT ".PCR .

... · 26 · · 15_·

· 25_ · · · .·. 14·

·_13:·_ . 24-. i.,i i) t . IP ~ i · v e 151,' _,., kl~~ II . : : 12 . ,...~,- .. , '•!,JI . !I.~ ~ ~,'/ ~ ·_'."' · I,' 1 ,v.• ,'' 1),i );;I#[] ·, I,• · -1f ~

· · .10 .:&,·__ .....______...... 21 ------0.01- : 0.1- · . .1 0.01:. Figure 6. Specificity and utility of the new DAT antibody Antibody BX generated using a GST-fusion protein containing N-terminal amino acid residues of rat DAT (G39 - R60) was tested in western blot and immunocytochemistry analyses. (A) In western blots of samples from different brain regions, a broad band that corresponds to DAT was detected by antibody BX at approximately 80 kDa in striatum (2) and nucleus accumbens (6). No significant DAT immunoreactivity was observed in brain regions that do not express appreciable amounts of DAT. Different brain regions are; 1 - Prefrontal Cortex, 2 - Striatum, 3 - Amygdala, 4 - Parietal Cortex, 5 - Thalamus, 6 - Nucleus Accumbens, 7 - Cerebellum, 8 - Hippocampus and 9 - Hypothalamus. Coomassie blue staining showed approximately equal loading of proteins in different blots presented. (BJ Antibody BX detected the transporter in lysates of rDA T-transfected HEK293 cells but not in untransfected-cells (UNT). ~ (CJ Western blots of lysates obtained from mesencephalic (Mes) and cortical (Cor) cultures were successively probed with antibody BX, TH and B-actin antibodies. The western blot image shows that BX primarily recognized dopamine transporter with negligible cross-reactivity to other proteins in mesencephalic cultures. Bands corresponding to TH and B-actin showed selective presence of TH in mesencephalic cultures and presence of abundant B­ actin in cortical cultures. Preincubation of DAT antiserum with 5 µglml GST-DA T peptide eliminated the band corresponding to DAT (Blot 2). (DJ In immunocytochemical assays, antibody BX recognized a protein that is present only in dopamine (TH-positive) neurons and preadsorption to GST-DA T peptide blocked this staining. Non-dopamine cell bodies are indicated by arrows and did not show any detectable DAT staining. 59

Figure 6.

A Tissue Samples

123456789 123456789 115 115 ·r 82 82 · - ·•· ••• 64· • 64

B HEK293 cells 82 ·181UNT rDAT 82·UNT rDAT

64 • ~ 64 •

C Neuronal Cultures Blot 1 Blot2 Mes Cor Mes Cor Mes Cor 180 • 180 -

115 • 115 -

82 - DAT 82

64 - 64 •

TH 64 ll•Actin -

D Neuronal Cultures Figure 7. Chronic changes in neuronal activity alter dopamine uptake in mesencephalic cultures (A) Representative current-clamp recordings show the effects of TTX (voltage­ gated sodium channel blocker) and 4-AP (potassium channel blocker) on dopamine neuronal firing. Exposure to 1 µM TTX abolished neuronal firing (n = 5), while 1 mM 4-AP increased firing rate to 209 ± 5.8% of control (n = 5). (BJ Uptake of 100 nM 3H-dopamine in cultures treated with 1µM TTX or 1 mM 4- AP medium for 30 minutes or 5 days is shown as the percentage of respective vehicle-treated controls (n = 3- 6 as indicated). (CJ Saturation analysis of dopamine uptake showed that 5-day TTX treatment caused a decrease in Vmax (control: 1.17 ± 0.09, TTX: 0.87 ± 0.10; P < 0.05) without a significant change in apparent affinity (control: 0.46 ± 0.12 µM, TTX: 0.57 ± 0.19 µM; P = 0.61). Uptake data at different dopamine concentrations were normalized to those at the highest (4.05 µM) concentration in control cultures within each experiment (n = 4). All errorbars represent SEM. * P < 0.05, compared with control. A-

... J'

···-•4-AP "k. ·. c=:J Control .. -me··

··30 minutes ·:5 days

c. . ·1.2 · Cl Control . ■ TTX· • Cl) .1.0 . . ~ca . -a::::,· .o;s . ·.,:,· . · .:·.~- ca . 0.6 E ·o.4 -~- z· · ,0.2 · · ·o.o------~------... 0.01 .. 0.1, f 1Q. · Dop~mine [mM]

,I· Figure 8. Chronic changes in neuronal activity alter DAT protein and mRNA abundance without influencing dopamine neuronal survival (A) Representative images of western blots from control, TTX or 4-AP treated cultures that were sequentially probed with DAT, TH and B-actin antibodies. (BJ Intensities of bands corresponding to DAT, TH and B-actin ·were analyzed by NIH image and normalized to respective control values. (n = 5). (CJ Changes in DAT mRNA abundance caused by 5-day treatment of TTX or 4- AP were assessed by quantitative RT-PCR. Data were normalized to actin mRNA and expressed as fold change compared to control cultures. N values are shown in the graph. (DJ Representative confocal images of DAT- and TH-immunostained neurons from control and 5-day TTX treated cultures are shown. Scale bar: 20 µm. (E) Normalized data· for the number of dopamine neurons, different morphologic parameters of dopamine neurons, immunofluorescence intensities of DA Tand TH are presented (P = 0. 004 ·for soma DAT and 0. 002 for process DAT, a total of 50 neurons per treatment group were analyzed as described in Materials and Methods section). P1L, P2L and P3L are lengths and P1B, P2B and P3B are number of branches of longest, second longest and shortest processes of TH positive neurons, respectively. All error bars represent SEM. *P < 0. 05, compared with control cultures. The sensitivity and dynamic range of TH and B-actin analyses by western blots are shown in Figure 12. 61

Figure 8.

A B C C:::::, 4-AP

c:::::, Control 3 Control TTX Control 4-AP * -TIX 8, C * ,.. .r:"' DAT ...... (J 2 - * - "C ~

0 ~ 4 0 DAT TH Actln ~l~ ~lvo

D Control TTX E

160 140 D Control -TT X DAT 12 0 1 0 0 c~ 0 80 ...." 60 * 40 TH 20 Figure 9. Neuronal activity regulates calcium influx into dopamine neurons via L-type calcium channels (A) Confocal images of fluo-4 and fura-red fluorescence in the presence or absence of 0.2µM TTX are shown. A lower concentration of TTX was used in the calcium imaging experiments in order to achieve faster washout of the drug effect. A presumed dopamine neuron used for fluorescence measurements shown in B and C is indicated by arrows. Scale bar: 20 µm. (BJ Changes in the raw fluorescence values of fluo-4 and fura-red in response to 1 µM dopamine and 0.2µM TTX application are shown. TTX decreased fluo-4 fluorescence and increased fura-red fluorescence indicating a decrease in intracellular calcium concentration. (CJ Normalized fluo-4:fura-red fluorescence ratio values calculated from data in B are presented. The change in the fluorescence ratio at each time point from that of baseline (F-F0) is normalized to baseline (F0) values. Baseline ratios were calculated from average of at least 10 data points prior to drug application. (D, E) Representative changes in fluo-4:fura-red fluorescence ratio caused by 1mM 4-AP or 10 µM nifedipine (an L-type calcium channel blocker) are shown. (F) Average changes in fluorescence ratio caused by 4-AP, TTX or nifedipine are shown as means ± SEM. N values shown in the graph represent the total number of dopamine neurons analyzed. All three drugs caused a significant change in fluorescence ratio from the baseline values (P < 0. 05) 62

Figure 9.

A Fura red Fluo-4 B

2250 DA TIX 2000 c::::::J i::::::::i ~ ·;;; 1750 Control C .!! 1500

-=QI 1250 u C QI 1000 u ., 750 I!! 0 :, 500 TTX ii: 250 --Fluo-4 0 0 50 100 150 200 250 300 Time (seconds) C D

1.00 1.00

0.75 0.75

0.50 0.50 4-AP 0 0 !!:: u. c:::i 0 u. 0.25 u.~ 0.25 ~ 0.00 ~ 0.00

-0.25 -0.25 Tlme (seconds)

-0.50 -0.50 Time (seconds)

E F

1.00 0.50

0.75 Nifedipine 0.25 0.50 i i 0 0 u. u. ~ 0.25 ~ 0.00 u. u. 51 ~ 0.00 ~ -0.25 4-AP TTX Nifedipine -0.25

-0.50 -0.50 Tlme (seconds) Figure 10. Changes in neuronal activity cause sustained changes in CaMK/1 activity in dopamine neurons (A) Representative western blots of cultures treated with vehicle or 10 µM KN93 for 30 minutes are shown. Ratios of phospho-CaMK/1:CaMKII and phospho TH: TH were normalized to control cultures in each experiment. KN93 caused a significant decrease in phospho-CaMK/1:CaMKII ratio (a measure of CaMK/1 activation) and phospho TH:TH (a measure of CaMK/1 activity in dopamine neurons). N values are shown in the graph. (BJ The effects of a 30-minute treatment with 1 µM TTX or 1mM 4-AP on CaMK/1. and TH phosphorylation state are presented. TTX and 4-AP had opposite effects on the phosphorylation state of these two proteins (N = 4). (CJ The effects of a 5-day treatment with 1 µM TTX or 1mM 4-AP on Ca MK/I and TH phosphorylation state are shown. The effects of TTX and 4-AP on CaMK and TH phosphorylation were sustained for 5 days (N = 4). No. significant effect of chronic treatments was observed on the total abundance of CaMK/1 or TH when normalized to actin abundance. * P < 0. 05, compared with control cultures. p, phospho. _: · c::::i Control.: · · . ·con·· -_ KN93- .... KN93· - ·_p~Ca~KU ·tuLJ%~- :· Ca~_KII i-/------~"_:_>·•·

. > . P·f: f'.-i~Z.iit:

8

·"4:,,AP: ··cQntrol · ·TTX .~4-AP. · * c:JContr<>I '.'!"~/-~'?e>-. .-me .. ... p~::z: J!l 150 'i:. J ·. :~ ~-· . ::::,, ·P:-TH :S .100 GI. *- TH e. "-·-: ·- ·--~ :~;~:? . ~ _ 50 c.UI ~-

Cor:1trol ··4-AP. .. : Control · _.TtX . . ::::-250 - .E:=4-AP· ·_p-C~!'JIKI_I ~=--s~-- - l-. ~- w ._ .. . C::::IC.ontrol__me:· ·CaMKII .·, _ __,-_, -!1. -~200 , . ~- ·*' p.:A~t•n -~=;!!] ...

,t'.'AP · .· Contro_l :. TTX :p-TH ·. ::~·~_jt1;x·'.~_::.<~x.~:: .·.··~,A::i-~-- ·3:;:'.;• .. ·. Figure 11. Activity-dependent changes in DAT function are mediated by calcium and CaMK (A) The effects of a 5-day treatment with nifedipine (an L-type calcium channel blocker, 10 µM) and AP5 (an NMDA receptor blocker, 50 µM) on dopamine uptake are presented. Data from figure 7 (4-AP and TTX alone) are also shown as a reference to illustrate the effects of nifedipine on activity-dependent modulation of dopamine uptake. (BJ The effects of a 5-day treatment with KN93 (Ca MK inhibitor, 10 µM), PD98059 (MEK inhibitor, 50 µM) or KT5720 (PKA inhibitor, 10 µM) on DAT­ mediated uptake are shown. The effect of KN93 on dopamine uptake in 4-AP and TTX treated cultures is also presented. N values are indicated in the graph. *P < 0.05, compared with control cultures. :Figure-1·1-._ . . .

140 ·e - ·i120 u 100 ~·- .. ·:~. 80 :a.·.so . :::,_. ·e~ ..·40 ··-a· 20 . :8 . o..u.__. ...·.:_3_· ,__.__...... _..=:L...l=:&....11--

). Figure 12. Sensitivity of western blot assays to detect changes in TH and 8- actin

(A, BJ Western blot analysis of different volumes (3 - 25 µI) of a sample from control cultures is presented. These data show the sensitivity and range of densitometric analysis of TH and B-actin.

(CJ To directly test if western blot analysis is able to detect decreased survival of · dopaminergic neurons, we used growth factor deprivation to cause neurotoxicity.

Unconditioned neuronal medium that is not supplemented with GDNF caused at least 50% dopamine neurotoxicity in 24 hours (data not shown). Cultures maintained in conditioned neuronal medium (Control) or in the presence of unconditioned medium (Unc: Medium) for 18 or 24 hours prior to sample · extraction, were analyzed for the abundance of DAT, TH and B-actin.

(D) Densitometric analysis showed that the decrease in TH abundance caused by neurotoxicity is at least as much as that of DAT. Similar results were obtained in a second experiment. · These data also indicate that use of conditioned medium supplemented with GDNF promotes dopamine neuronal survival and is likely to have minimized any activity-dependent effects on dopamine neuronal survival. 65

Figure 12.

A B

180000 160000 140000 .f! C ::, 120000 .II :, 100000 25µ1 12.5µ1 6µ1 3µ1 • .I 80000 ~ 60000 0• 40000 20000 0 0 5 10 15 20 25 30 Sample Volume (microliters) C D 120

--DAT 100 ll DAT ·c:::: 80

ii 60 TH I E o ·! C ~-Actin --1 40 Control 18h 24h ! 20 Unc. Medium 0 Conll'ol 18h 24h Ill. UNPUBLISHED MANUSCRIPTS ..

8. Second Manuscript

Sustained depolarization decreases calcium/calmodulin-dependent protein kinase II activity and gene expression in dopamine neurons

Shalini Padmanabhan and Balakrishna M. Prasad

Department of Pharmacology and Toxicology

Medical College of Georgia, Augusta, GA 30912

Corresponding Author: Balakrishna M. Prasad

Address: 1459 Laney Walker Blvd., Medical College of Georgia, Augusta, GA

30912, Tel: 706-721-7641, Fax: 706-721-2347, Email: [email protected]

Running Title: Depolarization Decreases CaMKII Activity

Keywords: CaMK, ERK, PP2A, Calcium, Depolarization, Dopamine, Transporter

Abbreviations Used: DAT, dopamine transporter; CaMK, calcium/calmodulin­ dependent protein kinase; TH, tyrosine hydroxylase; KCI, potassium chloride; v' PP2A, protein phosphatase 2A

Acknowledgments: The DAT antibody used in this study was generated in the laboratory of Susan G. Amara, University of Pittsburgh, PA.

66 ... .·a.1-·

. . . . ·Abstract'

. . . . . :Activitt~dependentplasticitt.underlie~ several forms· of l~arning ·and mem·o~y in · · ·

·. the-rtta~malian·:rie~ous:·system:. · :Alte·red:gerie expression mediated by:'CaMKli · :·: .·

. ·, .. : and:o_th~r.·intrc1qeUular~ig_rial_ing'~9lecule~. plays·an_irnportarit_ro,le_in···:· .. :: .·

• • • • • • • • • • • • ' • > • • • • • • • • • • • • • • • ' • • • ·:r1europl'asticity'.: ·we· discovered.~tiat s~st~'in:ed·d·epoiadz~tiorj fr1ducedby kCI, . . :a ..

...... ~.ory,monly. used paradig~. for studying. a~tivity-dependent .g~ne .ex_pression, ·... ·

...... - ...... ' ·: s'i.1rprisingly. caused ·a decrease·. in. CaMKI_ I .activity in. mesencephalic dopamine'.: n_~urons. This dec·re~_~e 'fr, C?IMKII activity after 4 days of depolarization. occufred,.in the:pr8:sence._of_,a·cont1nu~d.elevation -in-fr1tr~celluiar Galciurn.·

: conce•nfratiori: :An increase in cai°yculin-sensitive phcisphatas·~:activity was ~t

·. ·:clSS.~ys:revecded:that'the:c1ctivity_~·1~u·t.not·tt,e·:ab~ndarice;·_ofprqtei~··p~osphatas_Ei-: ·:·

2.A_w~s .. incre·as_ed by.~·ust~in~d_·~~pqlari~at1_on. De9r~ased c:aMkl~_ac~jyity was_:_:_:.

: acc_ompanied bya sel~ctive decrease ih DAT:mR~A; whil'etyrosine hydroxylas:e .. -:

·:and ..actirt mR:NA ab~ndance was unaUered~ _.On t~e other h_~nd, BDNF mRNA·· .. · .· ·:.

. . ..abundan_~e wa~·_in.9reased· b.Y·~ustained ,depola,.rizatioo, ·rurt.her:·o~rnonsfrat!rig·_the:·.· .

. ·. > s·pecifi,city_of .ch·~ng·es .. : Sust~·ined:depolarization also:causea-·~ signific~tlf . . . . ·1ntroduction:- · .·

. . . . CaMKU- includes·a_.family .of multifunctional· proieih kinases .involved in ·a Wi.def .· -·

· : ar~ay:of function~ in _the fo~rv~~s ·s·y~ten, such a~ _ibr{cha·n_neI: and ·receptm .

...... ·:{Yamauchi, :2005)~:·_ The critic;al:"role_ of _CaMKII in leaming_ an_d rl)en:,ory p~ocesses ·_

... is i!lustrated _by deficie·ncie_s in spatial learning, cerebellar.motor.learning and the·_·._

. -: formation ofocular-dorrtinance ·columns· in c·aMKlla.knockout a·nd/or ·rnutanfrrifce.· -:

·:(S_,lvRet ·a1~,J992;"Tatia et aL, 2002; Hct~sefefaL:•:2006k Converse.ly deleti_qn of:.

. . . . _protein-phosphata_se·.1 (PP1_);·which 9an-decrease CaM_Kllactivity,-·can fa~ilitate·: ·

learning and memory (Geno·ux et:aL, 2002r: -: Different CaMKll 1soforins can _:

... tra·ri:slat~ neu:ronal firing. fr~que·ri:cy informatiof) ~ht<>:kinase- activi,y(De:·~of.!i~ck :· ·. -

-:ancl_ s:c:hulman·,: 1·9~8;:·.Esh:ete··aqd Fields; __ 2001):.- Post-translationa_r·mod_ificafi6n

of AM PA re.ceptors ·_and. ottier postsyn~ptic_ density- proteins_ qy_ CaM Kl I. and PP 1_ ...... are :involved· in. syna·ptic plasUcity underlying· Ion~f term. potefritiation "(LTP).:

...... ·:(F_uku"riag·a ~t aL, f9_9?; Usma~. and Zhab_ofinsky,_ :~001 )ar,d:.lorig~terr1:1

. . __ d.epre$siqn-(LT.O) _(.Belmegu_enai-a.nd_Hansel_;-2_005;-Han$el_.et-al-.,_2006):."_·-A,,tered:·_-.

• • • • - • • • . ' • • I • . • • • • -: gene :expressic;m·:caused:by CaMKff is_ at.least partly:tesponsib_le for activity~

. . . · · _ ~eperident c~a~ges iri:neu_ronal:nio_rphology· (Zho·u:·et-al.,20O6)._ ·_. -- ·:·. · -· _

. : -The· n1esc>"cortI~0Ii:mbic· q9pamirie:·~y$tem .plays-·an ·1'TI_Pqrtanf ~(?le: in·: _·...... motivated- _behctv!or~,- atte~tion. .mo,or· ~-ctivity .(~e_fley ~nd_: -~~rriqge_,:. ~0·02_; . ·... . a~d .

, ...... -: Schult_z,: 200ZL.~Lon·g~la~_ting·ch~ng~s-in·th~se .dopaniinergic_pathway~_~re. · · · .

...... · a~s9ciated_ ~ith dru~--~-~u~e-~-p~t_hogeries:i~ andth_~r_ap:y of_~ultipIe·.n1·e:r1fa~I · ·

disorders .. ~or"_~xa_mp.le,: differ~ntforms·of -~ctivity~·dependent. ·plas_tic_ity. i_il -the -_. · ...... ' ·_·mesoiimbic_:dopam·i~e·p_athWayundediepsycho~timularit-addi_ction_(Eve:r·itt·~.nd:-

.. . _Wolf,· 2002;·Robbi.r;1s ·eraL; 2ooar: BothLTP·and"LTD.in.tt1es·encephalic.··

: dopamine rieuro_ris have· bee~ _irtipli_cated-:in neu~og~aptation:s :to drug-:usef ......

' ...... Cocaine, nicoti_~e: q·r-alcohol tre~t~enfcan-_1:ead·to··~T_P: in·-ver,tra! tegm~nt_al_are~-- _.·

. - . . · dop_amine_ ne_Lfrons·_(Mansvelde·r_ and McGehee;_2opo;· Ungles·s ·et:ciil.;·.2001-;:·_Melis: ·

...... etal.,·_2.002)._· .On the.other hand,._exposure to amphetamin~· can cause.LTD _in·

...... syn_apses o·n :dopamirie.·neurons (Gutler·n~t-et aL,· 2002).· H~wever; unlike: in· .

...... ·:tor~brairf neu_rons;:t_he·role of CarylKlr ir1 these form~ of pl~~t_icity in qop_ami"n~. _·:-

. . . - . __ neurons _is•unclear. -Protein kinase.·c_.orproteiri kinase t\,· but·not __CaMKII; t,ave-··:

...... , . . . , . . , . bee·n :implicated in L TP ·and LTD ·in dopaminef neurons;: respectively (Gutlerher.et: .·

...... ' ...... ' ...... : -CaMKff has lon_g-been· known to regulat~-th~-bi~synthesis-of dopamine· ..

...... ~n~ .9th_er·mor1oamines _qy_ itsability to _phc;>~pt,orylat~·tyro_sin~- andtryptophan~ _._· · ·

hYdroxylas·e~{(Ehret et"aL,-:1989~ .Bevilaqtfa-et aL; 2°0.0·1:). We-receritl}/s_howed :· ·

...... ·:t~c1t.CaMKU:_c;an ah,o:regulate _gene exp_re.ssiorf~n: dopami_ne:_neuron~·:i.n:an·:·

. . . ·. _activity~d_ependent_.fa~hio"n (Paom·a.naphan-·et c1L-; -2008). __ .-lnari:.attempt-to··fLJ_rth_er-·:·.-.

: explo_re th_e role: of CaMKJHn_.activity-d_ependent plast_icity· and:gen·~ express.ion: in:

...... ·mesehc_epha!ic· d9pani:inef rieur~ns;·~e-tested the-~ffects·ofsust~1ried:· ...... · :depol~rizatio_ri"on Carv1KII- activ_itY:: Chan_ges in:CaMKfl acti'lity: in -dopamine··

neu·rons caused _by-_alte'.red· neuro_na:r Wing_:were-sustairied. fo"r: as.long -~~:five_.days: .:·

...... (Padma_nabh~n_ et at,· 2.008). ·However,· KCl.~in_ducect.d~polariz~tion,· a _commonly_-.·

...... ·:u~ed paradigm:to ·sttJdy activity~depend~nt ~han~e~ iD gen_e:expression::

(V\(ayh)arf eta[; ·2006; Shimi_zu_ et~I., 20_07);_ca_us_ed·~n Linexp~cted·decrease in.._·· ·:caMKII ·activity·an·d_g·ene.expres~ion·.: -Her~,-we describe the mechanisms·:

. '. . . . ·:uhderlying·the·observed :paradoxical _pecrease:in'CaMKll·activity _caused by-·· · · ·

.: s~stained d_ep:c>lciization: .

Mate.rials· _and_ ·M_ethod.s _· .

. - . . . . IVlesenceph~lic culfo_res:-: ... . . - . ·Mesencept,_a.lic· cultures_ were _pre_pared_ fro~.-pos_t~natal d~y 2~4 Spr~gue .Daw_ley_ · . . . . . · · · __ rat-pups -~s previo~sly described·(Pra.sad and f\mara-,.20.01.).· -Brie,fly,. ve.ntr~I-· ·_. -·- · · . . .

. . . . . - ...... ' . . midbrarn tissue dissected from ketamine .. 1njected (3 mg/pup; intraperito:neal) rat--:·· - . . . ' ,pups. was ·dis$ociated for two.hours· intheiresence C?f _20 uri:it~ lml-acti'{a_te~- -- -

-_ -papain:~md :plated-:~t a_de~si~y°:ofappro~irr,a~ely_·1·09·,ooo-ceUs ·per wen in:4a~wen: ---

- tiss~:e_:Gultµre_~i-s_hes cq~te·d withjOO µg/rr{poly~~~-iy_si_ne and :5 µg/ml l~rninin. · _.:: ..

. . . Neuronal rriedium co"riditi.oned·over glia Was· used lo.maintain-the mesencephallC-...... ) . . . ·:culture_s.·: Cell.s were plated.in 24-well plates.at a density of-300,000 cells per well_ . . · . . .·... . .·... . .·... . .·... . - . . . . '...... - ..

__ forweste_r"n·blotas$ays:-·:For·imag_i.ng __ .an_d-e!"ec~rophy~io!ogi_cal:rec,ordings; sell.s-·-·:·_-.

-: wereplat~-d ~n-gla~s cov~rslip~.: .Air exp~rirr,ent~I .treatments ·were perfqrme·d ·_: ··:: -:

·after ?·q~ys·in. vifo:. To. i_ncr~ase extracel_lula_~ ·pota:ssi~~ .co·nc~ntrati~n to_ 30 -···:·;.

-rnrvl, sterile ~-~q mM ~~I :Ringe_r'_s s?lution·lJYcis.adqe~ toJh~_culture llledium.·

: treatments· w~re done PY ·preincubating cu_ltµres in differentmedia for :30 n,inute~---_ -:

...... ' . . . . ;b~fore:upt~~~ assay~:9rby bri~f perfusi~n of KCra~ indicateq. :.. · · ... 71 ·

·. . . ,· .. ·. .· ..· .. '_ .· ..· ... _ .·' .· .. ', . . . . ' ...... ··e1ectrophy.siolog1cal·:recoi"dings:

. . . . . Ringer's solution-was used as the external solution-while the··pipette was fil_led · · ·

-: wi~h• ~-:buffer-contai·n_i~g ('irin1Mf~28: K-glucorfate, 10.KCI, 10HEPES; 1: :MgCl2;: · .

- ...... - ·:perfc>rmed wi~ti:Axopatch~~OOBamplifier'(rvtolecular Devices, ·sun~yvale,_GA,: .

USA)~.--.M~~~rane voltage_data_·collected·at.·10 kHz.was filteted with._lo.w.:.pa~s- __

: Bes·sel filter af2 kHz:'a.nd. digitized.by National lri.strumenti 1/0.card.-Voltage ..

...... - ...... ' ...... ' :t~cices were __recordeq and-analyzed bytl1e WinWOP_ whol_e_-9ell progrc1m .·

· __ (University-of-S:tra~hclyde,·. G.lasgow,. lJK). ·Firing-frequer1cy.andm_embrane_. -

...... ' -: volta·ge·data obtafr1ed during drug perfusion:were compared•tothe average.

-- _ controldata.before and .after·drug :application_. A D2~autorecepto.r-_mediated ·.- --

. :hyperp61arizati6~ respons:e to d_~-p~min~:~·pplic~tipn·was ti$ed•to iderttify :·

d_opan,ine _neurons._ . · . .

__ Cultures_growri:.in_24~weli.·pl~tes-wer~_washed_fyiice-in-R.ingefs·s9·1ution;atter_.

: ~ppropriate.trecitme.nt~ .. Cells ~~~e·1ys-~d:and e~racted into.:200: µI.RIPA bu_ff~i ~t -:

---- 4~C•-f~r_30.mi_n.utes w~th .g~ntle a:gitati~n.' ·ceud~bris. and nuclei-were-·removed-·~y- ··

·. -:cenJrifugatio~{~(30_00_X g:for.tO'.mi:nutes'._ .The:different.a·n·ti_bo:dies u~ed ~ere:· .. ·

. . -: Sigma_, .St.-'Lq~_is,: MO),:'J3~actirt (goat polyclonal, -Sa:ntc;1 .Cruz E3iotechn·o1ogy, .Sarita_·.

...... ·:~r~~' :CA)_ cl~~_ibodi•es/phq.sph_(?~CaMKII (fhr2as).(~e1i:SignaHr:Jg,:Dan\/ers,. MA),::-

QaMKI_I. (Ce!!° Sign~lin_g;·-l;}a~yers;-~A);.pho$pho•:~H-(Se_~1s)..(lnvitrogen,·:san· · . ·uieg~l,.CA),'p~ospho.~ERK (~eU:SignaHng, Danyers,. MA)·, . .E:RK (Cell.·~.ign,ali,ng·~· ..

. . . ·. ·:Danvers,:.MA);pho.~pho.::TH .(Ser31') (lnvitrogen,.:San-Dieg_o; CA),.phospho~PP2A · -

-: (Tyrao1f(Santa C'ruz_ ~i~techn()logy, ·sarita: Cruz, :~Afattd _PP?A(Up~tate,-: . . ..

. . . ·:using NIH. iniage. ·:

.... -: Cal_cium imaging: ·

...... Ace~oxymetbyl {AM). ~~t.ei-- for_rn~ o(the_.c~lciunf i_n.di~ator:'dyC3S fiud~f~.nct: fLira-fed: :.

...... Neurons were· loaded with .fluo-4 ·and fura:-red dyes: (4.5 µM eacli in R1nge:r's

-·-·:-. --- _·soiution) for.-20~30-miriutes.. -Time~lapse·confocal--images·.of4..C(µrn: optica_l --.- ···":- -- ....

...... - ...... ·:S·eptio:n_s .wer~ ·¢apture~ using aA0X achrpp:lan:~bjediye:·at:f s:ecorid·inte~a.ls;

,/ .. · ...... Both._-flu_orescentpro.bes.were. e_x_cited with.cl 488. nm_-argon la~er a11d th_e ~m~tteq_-.-

...... : fh.iores·cence :was recorded betwe.en 500 and.:550 ·nm-for fluo~4- and between· 642-_·.

...... •iu~~:.71:7 ·nrnJor-fura~red.: :F1·u(?r~_scen·ce -~f fiuo~~, _fy.ra~red -~r,d :thefr~atio :.

. . . ·... rnultipliefby a- fac~or·_of--10~9-were _ar,alyzed ·in_ selected._region·s- (?.f-interest_.. -In. -- -· . .

...... ' . --· ..fluof~scence :ratio val"t.ies were· n?_r_malized .to _a· ba~eline· ratio .valu~; .·wastl~s;···:-. - ...... · .. dy~ fo~di~g-:and ·imag_ing:of c_uitu're~· m_ai~~ai.ned_-hr?hr?n.i~ ~GI; ·were pefforniecp~· ...... 30 mM_ K~.1.Ring~r's_ s9lution. T~e ~aselin~. fluorescence ratio was calculated

-: duriilg.·normal.Ringer's·vv~sh ·in' ¢.hro_nically·_KCl~tte~ted:cultUrC3~--: Phosphatas·e. . . . assay:·:

...... ·.. PP2A/PP2C-activitywas:as_sessed.by measurin~fthe dephosphorylation .ofth~_ · · ·

. . p~o~phop~ptide_RRJ\(pT)VA ~sing _the_f!lariufacturer;~·protocol (Prom:ega; -

. . . Madison, _WI): Br_i:efly·, ·"ne_u·rorial··~-u_lt~res··t~~twe·re··m~inta·ined in_30-mM _Ker and- .. ·

...... ' ... ' ...... ext_racted_intq.-t90 µI 9f.:p~o~p~at~se lysi$.b:~ffer-(40.-~M_•imida~ol~~~-Cl~:-2 ~M_· .·.

. . ~D°JJ\ 2 mtvl_:EGTA: ·p_H}.-0 wi.tH-proteas-~-i-~hib!tor:cC>cktail)".: Phosph~t~se a_ssay~ · ...... ·:were perform_ed-in·~_ total-~olume_of _25.µl:in ~P2A·b~ffer{5)~-buffer:.2~0mM. _· ·

...... ·. )midazole. pH 72, 1·mM EGTA;· o·.1-%:~-mercaptt>"etharml a·nd·0."5.mg/ml: BSA) to: --- .. . ..

...... ~ea~ure ·p~2A·:activity.: PP2A _spec_1fic cictiy1ty w~s-·cal¢ulated_ by· deductin·g ·_

. - ...... ·. -· ...... pho~ph~tase;a_ctivity·iri _t_~e: pres~nc~ of ·590nM"okad_aic acid/.100 nM ~~lyculin- :·_._

...... ·:from phosph~ta~e-:adiyity_in·the_absence __of these-.i~hipitqrs.for:contro_l a~d:·_ . . . '

...... chronically- ~Cl:~re~te_d. c~ ltu re~: .In_ order_ to detect_ PP2C _a~~ivity, rea_ction.s yve_re. ·: ·_ -.

. . perlormed.inthe-sarrie.buffer··with.-the-ex·c~ption· being-the a_ddition·o(2·s·mM ......

...... MgCl2 (P.P2C 5X buffer). PP2C specifi~ activity was determined as phosphata$e

...... ·:activity-in.PP2C b~ffer minu~·p.hosph_~tase· activity ih:PP:2A buffer.·

. . ·. we·us~d ·a~ot_her p~~sph~peptide:K-R-pT~l~R-R (Ups~ate, Billerica, MA):to d~te-~t: · ......

...... PP-1" activity:--PP·t reactions we.re carried--ou"t"in PPt buffer (5X buffer:·:iso.mrvf:- .. -· ...... ' ...... - ...... ' . . .

. ' . . . . . - ...... ·:tiEPES,-500._mM N_ac1_; 1d mM))tT,-.0·.5-_niM"E~TA; :o:1-~·s%·t~ee·n~~-0; ·pH_"7;5·

...... ~upplement~dwi_th ·5.rnM MnCl~).-~nd PP1_._sp_ecific•c1ctivity. ~a$.·ca1Gul_at~d. by .. _

. . : determining ·inhibitor~~:se.nsitive: (N.e.w E~glan.d-Biolabs·, Ipswich;: MAf .·.·. -: · · .

...... ' ·:phosphata~-~:~diviiy,_:w~·ichwe_·obtain~d_-by ded_~-c~i~g- ptio~phatase -~ctivitfiq:~~e :......

...... ·_ .. ·.: .·. ·:Sephadex·G-25. columns .(Sig·ma·; :St..:Louis,: MO) prior'to.the ·assayto rem9vEfany.·· ... · · ... : · .·. : free Phosphate~ AH reactiohs YJe~Ei ihcu_~a~ed_.at. room: temperatu·re.for 1:0 mi~utes: _-: ......

...... measuring the· absorbance at 600: n·m in:-~ ·m,crqtit~·r· plate reader ·(Bio.Rad .. · .

microplate reader Model.3550r .·. ·.

. . . :Qu_antitati~~:.RT-PGR:- ·

·.. The· abu.rdancer·ofdifferenfmRNAs_. was· .anc:1iyzed: by_.quantita.~ive· RT~pcR· as ....

· · · · : previously described (F>a:dmanabhan ·et al'.;·:2ooa).:Ptimers used to amplity·_cDNA -: .. . - . . .. ' . · were;-·

. -DAT.(F) ~ 51-~!GGGCCTCAATGA~ACCTTT-3'.; . .

...... OAT._(R) -·_5.'?:•J;,CAGAACMT~A<;,CAf3CACGA.~3~ .. ·.·.

. . . . . : .·.·. -: Actrn.(F) _; S'~TTGCTGACAGGATGCAGAAG.GAGA:-3'-:

. . . . . ·Actin (R):~ 5'~TAGAfo..GCATt,--<3c:GGT~CACGA!G-3~

TH (F).-- $'·-.T-lCACTGTOGAATTCGGGCT~3' _·.· ·_.· .· ..

...... : TH (R) ~ 5'-TGAGGTTGTCCTTGGCGTCATT~_3' _: · :·· ·

BDNF -(_F)_· :-- ·5, -TqTCAG.~qAAG_G9~TGGGAGTATT~3_' .

. .:BDNF -(R) ._ :5'-TGGAGGTTTGGTTCTTf:CATG.GGC~3i·

...... , Dopiunine 11pt~~e as$_ay::

. . . . - . . . . . :~ft~r differ~~~ treatme~ts,:. cultures were_ washed twice. wit~.: :Ringer's solution and ...

. . . . doparr1ine·.upta~e was· iriitiated_.bf~d9itio·n.of.1 qo-_nMJ2,_ s~_·a ~H]-~oparr,_ine·.(GE·:· · . . . ' "ttealthcai~,.Piscafaway, :NJ·;·-usA).: Six.minutes_ later,:·uptake,wasterm_inated.by_:.

. . . . . ·. JWo washes of ice~cc>"ldRinger's solutiort-ahd radioa"ctivity·~as:extracted .into-·3%·: _·.

-: tri~hlor6acetic··acid~-- Choline· chic.ride at a:n :equi~o-1a·r con~e~tr~ticm was used frr: .·

. . ' . . . . ' . repl~9e,:KCl·i·n-9ontrol·cuitures; ·N_mJ-spe~if!~-.uptak~was_·de~ermined·in_presen~~:.

Statistical analyses·:·.

. ' . ' . . . . ' ·To_contrc>l_fqr.~he vari.a:biiity in ~iffererltpcirameters:~tudied petween different__ :·

. . . . __ batches 9fculture~, all ·comparisons \Nere·made·within· t?.atches: paired·StLJdent's.·- .. ·

. . . t test .was used"to assess the effects of KCl":and drug":treatments With .appro·priate·: ·

· Res_ults ·.:

·.. CaMKU cl_nd .. E-RK_.ctivify-i_n·-d_opa_m_ine.ne_ure>ns can-h,~· mea$u_red by_th_e·.

phosphorylation of tyrpsine hyd_roxylas~: .·

Dopami_ne.-rieurons iri-·mesencephalic cultures fire-spontanec;>Us action· po~entials · . . . :(P·r~1sad aritj·_A~ara; 2O0:f).: Depo_larizatim;:of neur6"~s· byel_evated.·potassiurrl .. ·......

. _can·c:au·se_.calcium entry via v.olta_g·e_~g-~_te~:_caicium_·_~-~annels 1:_~~d this._st1~ulus·is_: .. . . . ' ..

. . . ·ofte:n .used. to: ·s_tudy a·ctiyity~dep·en_dent gen~ .. ~xpress_ion and: ·n.europla~_t_icity. (Hu .. ·...

. . . :et_a_L,:199_9;:~himizu et_ aL, 2007).: :As-.e~pected., dopamine:·n_euroris ~ere ·:· :·. __ ..... · .. -.:]6·

. . . . . - . . . after _a: transient in.crease :in :firi.ng rate;- due_ to a~: appareht dep:olarizatiqn-hio_ck· :-

. . . · ·:(Figure· t3A). ·caMKU subunits- are·autophosp_horylated_:af!hr2asq upo·n ·

stimulation_ ~Y: caiciu_m/calmod~i"in"hind!ng,: a~d "th_is phbsp~orylation _prov!des _a ::· .

...... , •· ·• ...... biochernicai"-rneaswe·-of :ki_nase·aGtiva_tio·ri: (Louarid· Schulman,: 1B89).: Tyrosiriei:: .·_ .

. . ·:hydroxylase·(TH) is ph_osphorylateq at S~r1g·sel~ctively by CaMKs_arid th_us_ . . .

. . provides a dire-ct. measure_ of .the_.activity otthis family of kinases in dopamine. · . . ' ......

. . . ' ...... neuions (Haycock a·nd Haycoc~,-"1.991).- ·_nepolarization of-~esence·p_halic

:ctJitures by -~:-1_~minute:KCI fr~~tmenfcaused a: signif1cant_i_ncrease _ir, Thr2at,-_. _· ·

. . ' ·___ ph.ospho_cylation·ofCaMKII ~nd Ser19 _phosphocylation ofTH (Figu_reo13B)~ __ -

. . . . . - . . . . - ...... Pretreafrnentwitli 10 µM:KN93,:a:•caMK.1nhibitor, significantly":decrease:d:CaMkll -

··andTH_ phosptio_rylation hi control cui~ure:s. and blocked the-in~rease·-tn.: .· ..

...... ·:phq_sph.orylation·_caus:ed :by acute· KCl-_treatment. Ac~te-KCl_treatm·ent al~o · -

\. '. ,', . . .. · ... ' . sign_ificantly _in~reased l=RK phosphorylatiqn .~nd ?e.r31_ phospho_rylation of TH . . . . (Figure-:13C),:biochemical-measures of ERK activation:and ERK activi-ty.in . -

...... : : _·:d(?.p_amine ~~_urcmi( rt3spective_iy, A MEK:inhibito_rPD9805~·:(t0 µMf s_ignificartt_l_y: .· . . .

...... - __ de_crease,d· Sers1-·phosphorylation-·of T..H ·in ·oon~_rol·_cultun~_s and:.pr~ve_nted-t~.e KCk-

induc_~d in-crea~e: in. TH phosphory_latiQn. _:fhese data: show thc1t"protein: .·

. - ...... -ph6sph_orylatjon·c~n be.~s~d.tc:>":m;easure:bas~°l_"and .sti_mulated activities of ·.. ·

·. -:Ca~~n and:·E'.RK in"·m_e·s~·ncep_halic dop~mi_ne.n·eu_rons.-:· _. · ... 7.7-

·caMKII actiV:ity-is: dec~eased.·by ch"ronic_ depolariza~io~,-_whil~: ERK_·

... -(, -: Although a~ute·dep.olarizaticm c~Usedthe: expec~~d:inc:rease :in Ca~Kil: _-:

. ---: phos_phmylaU_C?~;-_it-~as· surprisi.ngly·d~·crec1~ed .by-~·-2-day·trec1tment-vvi_t~··~oml\il

...... - . . . . . _·. decreased.by a·2-dax_KCl.tre.at_ment. Tim_e· .. -course _study .s~owed ~h~t.CaMKII _-. _- ·

.. ·. -: activity in -dopamine· n~Lirons ·re"tl.frned-fo ~elow baseline b{~tie day' of:KCI · .· ..

. . ' . . . . . _- .- -fr~atf'!lent {qc1ta not-sho~n),·:with. ~ sign.ifi.c~nt decrE!ase by twC? days._·_The ..

· decreas~)n•.kinas~_activity· \JVas-specific forGa!YlKII as.·K;C-l~induceq ·increase-in --- : _-- _. · · ......

. . : ERi( arid TH-{Ser31) ptiosphorylati·on was: sustained- for two days. Total": .·

. . . . - -treatrrie.nt _Ftfr:ther_mote; the· nu_mp~r-~n_d_· m~rp~ql_~g:y_-ot_dopaniine neiirons

.. we~e. not s_ignificantly.affected by._ch,ronic_-~Cl_treat.rri~n~ (Figure .19).~. Taken-_.

. . - ...... ' . . . . - ...... -: together, thes·e-data· show-that chronic'KCl".exposure-sele·ctively.dec·reases. -·.

. . ·cafvlKU actiyity-fn dop_~rriine ~~-~rans.:. . ' .

......

...... -: Decrease_d Ca_MKI' activity•_is-observed: in. the pre~ence of:elevated::

..

intracellular:cidciurri-·:-. .

. . proajpted ·~s·to_.in·ve_stig~~~-the_u~_derlying·_fj,·e_chani~:rn._:we.firs{t~sted·_i:f.c_~lci_uni_:_:_:

...... ' . . . · : hanc:Jlirig meGhc:1nisms· w~re altet~.d by sw~ta_ined depolarization._-: Acut~·.KCI. -- · · :·_ .. _- -:

...... ·trea~rrient.c~:~_sed a. ~ignificant {~_crease in_ iritracell.ular.calcium: corice-ntr~tion in._

~opam_ine·neurcms,(F_igliff3 1_5A),.·-Neuroris.rnai~tainect·.in_·30· m,vl p_otassium_· · ...... me.di.um for.2:.days showed ·a .. s:ignificantelevat1onin. intra~e.llular c~lcium: as·.· .

. . ·. :demonstrated.by a.decrease·in calciurri-conce':1tration·in response to saline · ·

·• washes·(Figure: 158). -The: maghitudeofthe_i·ncrecise ih ·c_alcium levels :observed:·: .

aftefr.ch.ro.nic-KCl_treat~erit is·smaller tha·~. that caus~d.:by· ac~.te.expo~~r~ to·K~1.:_··

. ·:(F.ig.ure· 1s·c}._.··However: •~· dim-ihlshed _c~lcium response:aftersusta.inecf ·:·

. . depo.larization is .unlike.ly_to be. responsible. for the _decreas~d-CaMK. activity;_ as . · . . .

...... ' . · the_·magnitude·.ot:increase-:in lntracellular •calcium· levels,-caused: by 4-AP ·

...... ' ...... :tre~tmerit (which inc~e~ses ·cciM~ adivi.ty), ·is muqh small<3r·than that-obse·rv(3c;I:.

·... aft~r·chrcu1ic· KCl· treatme'nt .(Padmant::tbhan':et .a°L;.20·0.at·Thus,· s.ustained .....

elevation ·of intracellular:calcium lev·els, in thef absence of possible oscillations,.:· .

...... Cal\llKII: activity caused.by .chron.ic depolarization.·.·

·w.e:hypothesized thc1t activatie>n .. ofa phq~phatasE3·mighf~E3-_respon~i~le to(

·... decrease.d· CaMKIJadivity i.n-the pre~ence· of ~ ..SLJSt~ined·elevaticm of ...

i_ntracelfula~ ~alcium·~~n.ce·ntr~t-icm; .:E~p~sure to· 1·oo·nM·.~~lyculin~a .PP1/PP2A· ::·

. . . - . . inhibito~, ·caU~e~ a· significant in9rea~e.i1i':Ca~Kll'and·TH.(S~r19) phosphorylatk;m ·· ..

. .jn ·~o~trol·~ul~ur~s,:·Showi~g.thata •calycy!~·n.:sensitive( phosphata·se(s)h~s tonic_ . . . . i~h.il?!to~ acti'o_n on ·C~MK.1_1 acti_vlfr .in rpese~ceph~-IJ~:culture$_:(figu.re .16~)._· .In · ·

' , . . . . . - . . ._ ...... _ . . - . - additi_on, calycvlin caU$~d a sig·n·ifiG~ntly ·gr(3ateri'ncr~ase· in •ph,ospho-TH -:

...... "abund~nce in 2~day KGl~treat~·d._cultures'-than coritrol cultures.:The in9rease _ill-., . . . phosphor.:TH .was detected in terms of both absolute ·abundance and· relative· .· .· . .. . - . ..· . . . '.· - . . ..· . . . ..· . ' . ,.· . . ' .. · ...... · ... 79.·

·magnitude of:.chadg_e:_cornpared:.to :untreat~d cultures. Calyculin-tndu~ed

·. __ changesJh the :phosphorylation state:of .CaMKlh,vere .qualitatively.similar-to those.·

...... of phospho~TH:: _These:data·.suggest th~i i~creased_ activity-ofpho~phatase(s)-if: .·

. .. ,. .. .. -· .. •· .. . . -·-: ·resp()r,sible-·fo_r:th_e_decrea~e·-in-'qct~Kll ·a~t.iyity_an~·- □-AT_·expre_ssion·caLJSed-6y:·_._:_··_.

·:~us.tain_ed. depolarization~·. A~ot~er ·pP_1 /P_P2A inhibito~ o~adi_ac ·acid,: but not the : ·. . .

...... PP_2.B.·inhibit~r-F.K5O6_,· ha_d sim_ilar.differe·ntial. e~ects·.on Ca~Kll_acti~ity:in control·.

...... ' ...... arid KCl-treafed cultures-.(Figure 20). These data ·sugges·t that.PP1 or.PP2A iS: .. ·.. ·

...... ·:r~~ponsibleJor:the ch_roniC depqlar.izat!on.~indu·ced ·changes_·in :caM_Kll activity; :

. To.-deterrnine the identit}i.of:this phosphatase, _we directly measured the ac~ivity .

. . - ...... of these two phosphatases ih lysa:tes of me·sen·cephalic cultures: PP2A:activity,·: · .

but not PP1 activity, was found to be significantly nigher in 2-day KGl~treated

·. ·:CUltu.res comp~red to:·contro1_·cu!t.ur~s:·(Fig'ure 1:613).'. ·1ncre·ased PP2A ~ctivity

.... GOUlct·_r~su_lt fr.om. an_.i~crease_.in• PP2A_aburidance_·or_activity. __ VVe foun<;lthat.

sustained,dep.olari~atibn-.did-notalter the :abundah6e-.of PP2A.b.ut sig'ri.ificantly . . . .

d~creased_p~_osphorylation·of _P_P2A ·at_a.n _inhibitory s1te'tfyr30~) (F.igure• 16C)::· .

. . ·. __ Phospt,~rylation·at.Tyr301.is __ as~·o6iat~.d with ..a qecrea~e __i.n c;1·ctivity_ of this·_·.

...... pho~phatase:(Qhen efaJ;, :1~92); :Thus, increa~ed:PP2A activity is resp9risible: · -· . . . . . · . ·for th~ ~el·ective·de_crease in su~taine·d-depolarizatiori-induced· d~crease ·in.

-:CaM-~li activ_iiy: in me~e~cephal_ic cultur~~---: _·...... ' S.ustained. depolarizati_on selectively. de~reases: .CaMKll·".'mediated. gene ... · ·

·. ·:expressi:oii.- .·.: _·.

-: Calcium entryvia: L~~ype: v6It~9e~g~ted _calci:u·m·.~hani1els.~_nd NMD~ r~c~pt~~S·:

-·-: ·with·~u~seq·u~rjf _activ~~i9rt ~f-di~~re_nt· secontj me·s~~_rig~"r· systems-play~--~D·-·

...... - . . . . . - ...... ·:iri,p9rta.ntrole fr-1_.activity~d~p·en:d_erit_.regulatio_n ·of gene_·expres.si9n-Jn:l'.'!·euron_s : . . .

, ...... (Bading e_t aL ,-.1993). For. e~af!l pl·e,. CREB. acti~ati_on. is. responsible ~or.·:· .

. -: depolarization~induced increas~:-inBDN'F:tr.anscrip_tion-(Shieh.et:aL; 1_998). -We· .. ·.·

...... '...... ' ...... h~ve show_n_th_at CaMKI_I- cahr~gulate dqpamirie transporter:.(DAT) e}(p_ression_·in· .·

. . . . an.activity-d.ependent.fashicm (Padmc:1nabhan ~tal.·,··200.8);_. Thus_,. in order Jo·.· . . ' . . . . -: evaluate functiorial"corisequences:of decreased CaMKII for gene express.ion, we: .·

. · . ~easuted.the ab_undance·otdiffer~nt transcripts;· Sustained:depolarization ·_ .·

.... ·:Sigt)ifican~ly:~ecreas~fDAT_mRNAabt.mdance!·whlle· BqN~·mRNA:a_b~ridance··:

was_sig_nificantly_incre_ased (F.igqre 17A). ·_on_the.oth~~ hand,._TH ~R_NA- .

. : abundance was ·nofaltered by" c:hronic depolari~ation .. Th·e de_c.rease· in.DAT .. :· .. _.·-:

·:mRNAwa~ acconipclniecf.by ~-:::,ignificant de:creas~_in:proteln _abLindance, whii~

.. · _TH arid-~ctio ab_undan.ce.wc1s·not~ffe,otedby-2_~day-·KCI treatrnenqFigur~ 178). -·

. -·. ~listai~ed d~po_larization_, ·bu(notacu·te.de_polariz·at_ion, -al.tars dopa·m_irle-· ·. -:transporter'fuilction-_ .· .

. we·rJext t~_stefif·a 9ha_n·ge in_ oAf: ~bundah·ce is. reflected. in:~AT-med~ated-_ ... ·.

uptake.·after sqstained:d~p_olarization .. ··lnc;J~~cj, 2--d-ay._KC.1-treatment ·cause)d.·a·

. . ·:sigriificant ~ecr~ase i~pAT~-m~diated.~ptake (Fig·ure: :1 a)._ -~_his_.decre~s~ is·.m·osf :...... ·:cause an:y_.si~nific~ntchan~e.,in-:u.pt~ke ... :.F~:rth~~niore:• ~b~te:.~hanges: in Ca.MK~I.: :.

. . . . . ·. ·:activity"-c~used :by.neuronatactivity."alsoJailed .~o' show·any·.change··i~. DAT-:-:····.· .·

. ' ...... mediated .uptake.{Pa~ma~abh_ati: efaL,. 2008) ..

Dis·cussion · .. · ·

Sustained ·dep·oiarizati.oh.·caused·a· selective· decre.ase·in CaMKII acti'vity· and.

·:OAT .exp'ression: irim_ese.nceph.~lic :dop~rnine de_µro~s:; ln.. ~:c;ldItion;.we .icieriti_fi.~d. : .a novel· ~9Ie.for PP2A.in'regulating·.c~MKll-°signaling· i·n q.op.~nitn~.ne.urons._.the·in......

. . vivo relevance:ofthis sig·nalihg me:chanisrrtneeds .to:·be expiored in the:futu·.re .. :It: .·

...... pha~n,acological:anq. p~t.h9logi?~.l·_situations· might'c~use .su~tained. .. · .·.· · ..

depolarizaUon. of. neiJ°rti.ns~.: Depbla~iza.tic>'n·. bfo.ck ·of :dopamiri~. ·neu ran~. caused .by ..

...... ·:e)(posure{.~~:.~~tipsy9hotic.drug~:mi'ght.. ~.ediate·~h.e:.therap~·u~i~ :effe_~t~.of.thes¢·.

. . . . ·: Qf ba$~I ganglic:t riucleH.ised to treaf Parkin.s:o'i'-1'$ dis~·ase·.or el~ctrog·raptlic ·.·

·seizure~ ca11·:cau~e·.dep.oladzaU9n: b.lock·.of n~uronal fi~!ri~. (Biksqri ·et.al., ·2003·;.·:· ...... ·.: ·. ·:Shin· et'al•.;· 2007) .. Jn)cfo~;··eIev~te~·exti-~9ellular·pot~s·sium: cohce.n·trc1t1on· may·_be ...... ·.·.·_.. pa~ly.responsi~I.~ for th~·depolar!~~tion.block seen_.in ~he.l~tte_r two. e~~mpl~s.. ··.:.: ...

...... · · · :·.· .... KCHnqucE;!d depola.rization.Js ofte•r;'_ ~$E;!d ·as:a. s~rrog·ate.for. neuronal.····:· ... ·.

...... ·:ac:tiyity. to stuc;ty :activ!fy.-d~p·e~~~nt: pl·as~i~ity :and ~~ne expre:~si:on. (~~. et .aL, .

.19.99.~:·~hi.mi.zu.·E3t aL; ·2007) .. ifDplied ~ssi.Jmpti9n.of.these studies_js.,~at .. · · .. An· ...... · .. ·.·a2-·

:c_hanges·fr~. intraceliular signaling caus~d·by neu_ro.nal depolarizaticm_:mimic·:the·:- . .

. . . . · · · ·:effects.·otaltered·n~utonal·activity~ .·Although·a.cute depolarization.can-activate .·.

...... ·: :·: .·. c_~M_Kff'sig_~al~ri:~;i~ we, pre~erit evid~nce thcd:~ust_~ined depol~r.i~ati'o_n for:two~ .. · .. ·

_·.· · · :· · .. ·. day~_-9~~~ea8:e~_·C~MKll-:~9tiy-ity;: ar:,· effect' op·po_site>to that cau~ed _by· increa_sed· .. · .• 1,·.

...... ' . . . :r:ieuronaH_iring:: This e.ffec_t is_ rel_atiyely· s:e_iectiv~ for CaMKll'.as stimulation of :·......

...... ' ...... ER_K..signalin·g:_pathw~yis .sust~ined for two.days -t?Y depolarizati.on under.our _· .· ·

. . . -: ex'perimenfaLconditio~s>. Furthermore, effects ofa_ltered -neuronal-firing.rate ·o~: .·.· .. ·

·c~rvlKI.I ac_th,ity·can-_b_e:~ustain~d for fi~_e d~ys in vitro (Padrn_a_nabh~rl:~t aL; .· · ·

...... ·. __ 2008); .Tt,ese·find_i_ngs unde_rscore th~ importa.~ce-of:act_ion-po.ten~ials ·in ·.

maintaining CaMKU activity on atinie~scale·:of days: :There are several :examples . .. .. ·.'.> .. _:. 'ttiaf:derno·nsfrate .th·e-1mpo_rt~nce of p~·asfo.~h_ahgefirt'membr~n.e.poten~ial to the ·

. ·:activation-ofqlffe're~t _$e·cond ·m·esseng'er.systems::. 'Spac~d-~e:p·o1~rizing ·sfim~li_···: _·.

...... ·...... ·.- . . . . . are rr,19re ~ffic_iemt th.an _q911tin_u.ous_ depoJari_za_tion .fo_r·s~st~in~d.· E;RK a9tiv~ti9n · ... · . . . , ...... -: and:de.ndritic:growth (\Nu.et at,· 20.01)..·· CaMKII- can.be:activated: in-ai{action· .. :· ... -:

. . ·:~l9t_~ntial 'fr~_quericy·.,~-~pende~(f~shion, __a~d·this -~'-~_ase is -~uggeste~:_t9 be fi~ir.ig :. . a . . . __frequency·d~co,Qer..inneu_ro_n·s -(Eshete-a.ndJ:i~lds,. 200-1)..·· Sev_er~lf~cto.r~ ~.ucn .· . . . _: ··:·: -: $S magnitude ~nd d·urat1qn: of calci_um·_1nflux; kin_ase·a_ctivatio·n :~inetics and ·_·

.. . . intr~neu_ronaHocal1zati9ri ·cf diff~ren~ kina~e· i~oforms· have. b~en. implicated. in-the· ·· ...... :freq uericy~d~p~ridenf: re·gu·latio~ ·of ·gerie: -~xpressf9_n·. (Fields· et :a·1.,: -2·0_05). ·. · - . . . . .

. . . . . -:· nec~~$~rily"lirniteq to· m~~enceph~.lic cult~_r~si .Post.;._n.ata1·n~u.rona1·cultL1res fron:1.-.·.

·:h.ip~mcampu8:.a~d ·ce~C3pral CO~C3?( ~IS'O. ~~9~ed:'~ -~i:griifican.f:de¢re·as~j~: Ga~~II : :.

. __ac~ivatio~_-st~te:foll~wing:a 2.~day·qe3pola_dzatio~-(data·_no,t.sbo\iyri). _·Tbe-·:·.· .

. . . ( . . . . . ·:cuUutes are differ~nt..- CaMKII ·alpha ls·the·m·ajor isoform express_ed in·post-riatal ·

. hippocampal and· co_rtical: cultu_res·,·:whil~ rnesericephaHc cultures· expressdel_ta :

" ...... -- .. " . -·-: and:·gamma ·isofomis·o~ c·aMKi[(qatanofs_hown):- :l'"hus,·the_F'P_2A-me9iated···:. : .

·. ·decrease·i·n·:caMKUactivatio_n maybea cormnon:adaptiye:tDe~hanism· th.at1s:· ·...... independent_·ot.the l_oc·ati.on of neurons_.in the.brain and the. specific_ isoforms_of _._·_·.

...... ~ . . . -: CaMKII :expressed~ -·rhis.mech~nism is 'observed in the abs~nce of sighificanL ..

. . . . :c_h_anges in_ n~urori:a_~ \liability-or morphol_ogy .. Alt~rn~tively,_the· pre~_en_c_e.of -_. ·_· . . .

...... ·. __trophic.-factors·in·glial~conditioned m~_dium-us~d-in all .otJ_r-experim,ents-·may.have: _·...... ·. contributed to ·optimal viability of n·eurtms.: · :·· ·

·- ·.: -. _CaM KIi plays an .important :ro_le ·in· -activ_ity~dependent .pl~sticity- ·and_ gene:· .-. -

. . . -.exp_re~sion. :

regulc1tors:_of CaMKll. locatedinthe post-synaptic ~ensity·and_cytosol; .· ·

__ -_· -: resp-e.ctively (Strack· eta1 .. ; :1997;:°Mullassefril .et al., "2007). Thes·e two·:

·:phosphatas_es have als_o:been Jmplicated-in regulat_i,ng. Ca~:KII actlvity:_durin·g LTP :.

...... · _(Bl_itzei-·e_faL, 1~98-;.F_uktmaga·_et~!:-, _2.000}<_ln_addition, these:twgcl_as&es _of·_. - ..

...... ' ...... '...... -: phosphatases--play-_an.irnp·ortant rq-le in neuroplasticity,-:in_clud1rig:that in : ...... - : -~op~~inoceptiveneu·ron:s_(Genoux_et_"aL/2002;.Sti_panovich:et-~l.-:·200af. Data. -

- -:J~r~~ented_.lrr_thisman~s~riptshow ~hat"Pp·2A c_ari:regulate ·GaM:Ku a~ti-vity u_nd~r: . . c_o:nd!tk~ns ..of depolarization block.:: Ho~. s\j_~tain~d ·q~p_olariza~i_o_n lead~ -to

...... ~ ...... · -: activcttionofP.P2Ais not.dear-at_p_reseht/)t is poss_ib_le that :a.sustaint3d_e:levaUon.· -:

...... •:in _i11tra:cellula_r caldurn jn the.absence· of po~sible_phasic-~h~nti:es=·1i_ke_ly fo be_

~ause_d-by.-~ctiq·n pot~nti~ls may b_e a _trigge_ring·factor.· ~owev~r, nodir~ct . .. -- .. .. ·J

.relationship_ ·between :int_racellular. calcium -~oncentra~ions ·_and. PP2A .activity has ..

...... ·. ;been· reported·yet;. Although the ·activity.of PP?B ·is ·regylated· by yalcium ·and·.· · · ·

-: cc1lmod_onn_ (Kl_ee

. : -·.· ~y-su~t~iriec:f:~~polarizat1on in oLffexped~~rit~lcorldmons; · Of tJ,emultiple. - ··

. · sig~a1frig:mdle·cu·1es·'th.~t :can_be._act:ivated_.by:dep0l?trl~ati~n; _protein .kin·a~e C is_··· ..

. ' . . _one _possible upstream regulator.of .PP2A._._-Physic~l..i:~terac~ion betw~en protei_n.. ·

. . . . kinase C ctrid P.P2A.can· reciprocally: reg·u_late-the ·activities ·ofthese··twc>'.protefr1~.--·.

__ int~ractiq_n·betwe~r.i·PP2A~.rid.PKC· was-obs·erved· in: coimmun"opredpitatiq_ri· · .

studies·,n·.mes.encephalic cul.tures":(data riot:shown);·:·:Equ·a11y·Ukely.are··:· -: ._·

··. ·coritribu.ti.ons':of ottier'l~ina·seand .p~osphatas_e.signal:i~g-_mo"l_ecu_les·and :the . -·

. ' .. JTILflti'tude.·of:i.riteracfion_s· betweem· the~·~ ·:The:·pho$pho~TH ~naJysisu.sed i'n-:t_his __ .·.: .·

. . ' . . . . , ...... s_tudy_prov_ide~ a_reliabl_e_andser:,~itjve_ass~y_to·_expl_ore the ·l:Jpstream ~ignal,ng .·

niolecules··ih mesencephalic primary cultLire.s:orimmortaliied .do.paminergic cell·_._ .

. .· ·: :: s~·sta,ined depo·1arizati6n_;:we.·pres_ent evidence for a'funct.ion·a, _consetju~:m:ce' of.:...... - .

. ' ' -:presehfed·above· m·erely shows that CaMKII activit{arid:-1::>At·expression ·show . . . ·...... ·. . . . . ·...... · para1_r~1. ch~ng:~_s_._ We h~ve p_rev~o_u.sly-_dern~nstra~~fa. re_l.a~i9_n:s_hip__be1"v~en_ .. ·

...... CaMKll.:activi_ty._and·DAT.expres:si_qn_ using_·pharrna¢ologic•a1 :rnc;inipulatlqns . -· · ...... · (P_adm~mab_hari :et:a! .. ~_-?OQ.8): .. ~yrthermor~, :¢oristitutively.actjv~_arid .~:ortjina~r-'.-

~egatjye CaMKl_l.constru_qts _can .al~er PAT--apurida'nc~ in m_es~·nce'phalic_cu_ltur.es:·.·...... ' . . . , ...... ' ...... ' ·:(data nofshown) s:u_ggesting·that_this:kinase·is:n.e·ce_ssaryand sufficientto ·c~use· .·

...... ·. :changesJn· DAre~pres·sion .. Thus, ·sustained depolarizaUontnay:ptovide· a · ·

·. valuable fool-to dissect CaMKll-mediated:e:ve:nts from other-kinase activation ih.:: .

...... ·...... ·\ ...... ·< dopa~ine neurons>Activity~depenq.ent ch_ariges in-C~aMKs and phosph~t.ases· :· · .. · . . . ·:~re._like_ly:to provide_ afran:iework for futur~ st_udies.on ·synaptic: plasticity: in·:· . . .

...... dopamine·.neurons associated.with exposu~~.t~·.stres:~ful -stimuli.,

...... -: psy~hostimLi~~mts: o{ a·ritipsyctio~icS; · Figure 13. Depolarization-induced changes in CaMK/1- and ERK-mediated phosphorylation in-dopamine neurons

(A) Representative current clamp recording shows that a dopamine neuron was depolarized by 30mM KC/ perfusion but neuronal firing was abolished after a transient increase in frequency. The average depolarization caused by 30 mM

KC/ is 27 ± 2.9 mV (n = 4).

(BJ Representative immunoblots showing the effects of a 30-minute pretreatment with KN93 (10 µM) in control and 1-minute KCl-treated cultures.

Images of blots probed with phospho-CaMK/1 (p-CaMK/1), CaMK/1 (CaMK/1), phospho-TH (p-TH, CaMK/1 phosphorylation site) or TH are shown. The abundance of phospho-proteins was normalized to the total abundance of . respective proteins by densitometric. analysis.

(CJ The effect of PD98059 (PD, 10 µM) pretreatment on ERK and TH phosphorylation (ERK phosphorylation site) in control and KCl-stimulated cultures is shown.

N values are shown in the graph. * P < 0. 05 compared to respective control cultures. A-· ·.-KCI. · j:

c· c:::::J Co ntro 1. · · . p'.E~1nf;, PD ··He~ :=•o~K~II r::: -PD . ERK1/21~:~1-1~-~---~---~,~~'_.·c. ~, ~ 150 . . . . . r---:'-----'---..;..__--"---, ::::, ~-TH(S«31)1~r1•-.•1 ·l!.1iici.

·.·· .. TH.1:"t8 ..J1~ ....:;~:l ·c=_; 50 .. o.;J;:·....L,...&.._._.._L-L-~..;..I... p-ERK/ERK . · p~TH(Se r31 )/TH · Figure 14. Chronic depolarization decreases CaMK/1 activity in dopamine

neurons

(A) Representative immunob/ots showing the effects of a 2-day treatm·ent with 30

mM KC/ on the phosphorylation state and abundanqe of different proteins.

(BJ Densitometric analyses of phospho-CaMK/1, CaMK/1, phospho-TH and TH

are presented. Adundance of phospho-:proteins_is normalized to their respective proteins. Abundance of kinases is normalized to that of actin. N values are

shown in the graph. * P < 0. 05 compared to respective control cultures. 87

Figure 14.

Control KCI

p-CaMKII c=::JControl - 175 2 -KCl2days g 150 * CaMKII I u ~ 125 * p-TH(Ser19) I~ ======j1 ~ 100 ::, ::=_=_=, 75 * * 50 ERK1/2 I I 25

p-TH(Sar31) I I TH ::==I===jl

Jl-Actin :,======:, Figure 15. KCl-induced increases in intracellular calcium are sustained for

2days

(A) Confocal images of fluo-4, fura-red fluorescence and their ratio in the presence or absence of 30 mM KC/ are shown. Cultures maintained in elevated .

KC/ for 2 days were imaged in the continued presence of 30 mM KC/ and the · effects of brief perfusion with control Ringer's (3 mM KC/) on fluorescence ratio were analyzed. Scale bar: 20 µm.

(BJ Acute and chronic effects of 30 mM KC/ treatment on fluo-4: fura-red fluorescence ratio in individual neurons (identified by arrows in A) are shown.

Changes in the fluorescence ratio at each time point from that of baseline (F-F0) is normalized to baseline (F0) values. · Baseline ratios were calculated from the average of at least 10 data points prior to exposure of KC/ (in the case of the acute experiment) or during the nadir of the Ringer's wash (in the case of the chronic experim~nt).

(CJ Changes in the fluorescence ratio caused by exposure to acute KC/ or chronic KC/ are presented as means ± SEM. N values shown in the graph represent the total number of neurons analyzed from at least two different batches of cultures. * indicates a significant difference from baseline calcium values (P < 0.05). 88

Figure 15.

A B

Fluo-4 Fura Red Fluo-4/Fura Red 1.75 Acute KCI 1.50 □ 1.25

Control 0 u. 1.00 ~ 0.75 u. ~

Acute KCI -0.25 25 50 75 100 125 Time (seconds)

Fluo-4 Fura Red Fluo-4/Fura Red

1.5 KCI

Chronic KCI 1.0

Saline wash

50 1 00 150 200 250 300 -0.5 Time (seconds)

C 1.25 * 1.00 0 u. 0.75 ~ u. !b. 0.50 * 0.25 54 0.00 c,' c,' .,._e~ .,..c,~ c," 0~ \>' C,~ Figure 16. PP2A mediates the sustained depolarization-induced decrease in CaMK/1 activity

(AJ Representative blots showing the effect of ca/yculin (100 nMJ on Ca MK/I and

TH phosphorylation. Densitometric analyses of phospho-CaMK/1, CaMK/1, phospho-TH and TH are shown: The abundance of phospho-proteins is normalized to the total abundance of the respective proteins. N values indicate the number of experiments from different batches of cultures.

(BJ PP1 and PP2A activities in Jysates of 2-day KCl-treated cultures are expressed as percentage of respective control cultures. N values indicate the number of experiments from different batches of cultures.

(CJ Representative immunoblots and densitometric analysis of PP2A and phospho-PP2A abundance in control and 2-day KC/ treated cultures (N = 5J.

* P < 0. 05 compared to respective control cultures. . . . Figure·16~ ·

__ A .. * -KCI .· .. Control KCl+Caly·. -Caly. -.. - · · · · · :::::- .300. .•.r,.• ·._ ·p-TH:1 ·: _:>:s~;,~:~~~~1 .. ·J-· . c:::::::J Co~trol+Calyculin . u -KCI 2days+Calyculin ·rn 1~~~,~~I ~ 200 . -=s .. :::,· ·:Control--·- t

·C.· -* .~ Control . -:Kcl 2days - Figure 17. Chronic depolarization selectively decreases DAT mRNA and protein abundance

(A) Changes in the abundance of DAT, BDNF and TH mRNA levels in 2-day KC/­

treated cultures were assessed by quantitative RT-PCR. Data are normalized to

actin mRNA and expressed as % of control cultures (N=4).

(B) Representative western blots of DAT, TH and actin in control and 2-day KC/­

treated cultures. Densitometric analyses of DAT, TH and actin are· shown as %

of control cultures (N = 4).

* P < 0. 05 compared to respective control cultures. 90

Figure 17.

A 550 * c:::::JControl 500 -KCI 2days 450 =2 400 g 350 u 300 ~ 0 250 z< 200 a: 150 E 100 so 0 DAT BDNF TH

B Control KCI

DATI

TH I Actin P- I c:::::JControl 120 -KCI 2days e 110 'E 0 100 u 90 ~ 80 !! ·c 70 ::> 60 u ·c so ai 40 E 30 ·.s;;; 20 C a, 10 C 0 Figure 18. Chronic depolarization decreases DAT- mediated uptake

2-day KC/ treatment significantly decreased DAT-mediated uptake in

I mesencephalic cultures (N = 5). Acute KC/ pretreatment for 30-minutes did not affect DAT-mediated uptake (N = 5). * P < 0.05 compared to respective control cultures. F.igure-18. __-_·:- _· ......

c;::::::J Coritr9!" _·: - ..Kc1· *

· 2days Figure 19. Chronic depolarization does not altersurvival or morphology of dopamine neurons

Representative confocal images of TH-immunostained neurons from control and

2-day KCl-treated cultures. Scale Bar: 50 µm. Quantitative graph representing the total number of dopamine neurons and different parameters assessing dopamine neuronai morphology in 2-day KCl-treated cultures as a percentage of control-treated cultures is indicated (N=3). PL 1, PL2 and PL3 are the lengths and P1 B, P2B and P3B are number of branches of the longest, second longest and shortest processes of dopamine neurons, respectively. 92

Figure 19.

150 c:::::J Control -KCI 2days

_ 100 e c 0 u ~ 50

o...... L....J...... ,_ ...... _.._.. __ ...... ___ _,__,_....._..,. .... Figure 20. Chronic depolarization does not increase the activity of PP2B lmmunoblots showing the effects of a PP2B-selective inhibitor (FK506, 1µM) on phospho-TH abundance in control and 2-day KCl-treated cultures. Densitometric analysis of the effect of FK506 on 2-day KC/ vs. control-treated cultures is indicated to the right. N values are shown in the graph. * P < 0. 05 compared to . respective control cultures. .· ... · .. ·. .·. . ·:Figure 20~. _:: ..

: .... _KCI . ·co~ti'~_I . _FK506. : _KCl+_FK506 ...

·: p-TH- . . . :· _i1f ·uN.Pl)sUsHE □ tv1AN_us_cR_iPts.. · ··

C.. Third Ma~uscript . . . . .

. . · :Calcium/calmodu.lin-dependent: pr9tein•·kim1se. n·a~ti:vity ·gov~.rns ·dop~mine: .·.

. . . . transporter expression in primary mesencephalic: cu'ltures ·

Shalini' Padmanabhar1:and Balakristina M. Prasacf ..

:~ep.artmentpf Pharrn·a·colQgy and Toxlcofogy ::· .·

. . ' . . MeqiqaJ Coll.ege.of(3eorgi~, Augu.sta, ·~J\.30~12. ·

·:corresp·on.ding.Autllor: :sala.krishna M.·Prasad _:·

.. ·... Address: .. ··14fi9.. Lct.ney WalkerHlvd.:, .Medical· C,ollege·of ..Georg1a, .ftugusta,. GA · .. . .

. . . . 30912., 'Tel: 706-721-7641,: F.ax::706-721-2347,.Eniail:·[email protected]:edu·

·Running· Titl.e:· QaMKll.activity.'governs·OAT.expression·.· ···> ..

·. ·:~eywords:.:CaMKII: ·1·soforms,·-oop·amine;Transporter, T287 □ ,··K~3A .·

~b~r~yia~io~~.:~sed:· PA!, dopc1mine.. tra.n~p·o~er;·_~aMK,.ca_lci.um/cal~o.dulin~ ... ·...... dep:endent protein .kihase;.TH,· tyros.ine hydroxylase; .. a,: alpha;_.~,: beta(6, delta;y .·

.. -g~rnm~ .... ·.·:· .. · .... ·.·:-

. . Ac;kn-~wy:,d_gm~n-~·:· .The. ·DAT. ariti.boqy. us~q i~. t~is- ·¢_tuqy was· .Qene_rat~d ilj. the .·: · ·

...... · · · · 1.abor~tory· ofS_~san· G: f\mara, Ur:iiversity: of: Pit~sburgh; ~A..:·:·: -: ·· .. 94. 95

Abstract

Calcium/calmodulin~dependent protein kinase II (CaMKII) is required for the

regulation of dopamine transporter (DAT) expression by neuronal actiyity. We

used a genetic approach to determine if CaMKII activity is sufficient to promote

DAT expression in neurons. CaMKII delta and gamma were found to be the

major isoforms expressed in dopamine neurons in culture. Constitutively active

and dominant negative CaMKI I expression constructs significantly altered

CaMKII activity in dopamine neurons. Expression of constitutively active CaMKII

increased while the dominant negative construct decreased DAT expression in

the soma and processes two days after transfection. These data indicate that

CaMKII activity is required and sufficient to regulate DAT expression in a cell

autonomous fashion.

Introduction

Dopamine-transporter (DAT) is the primary mechanism of clearance for

dopamine in the central nervous system. Differential expression of DAT can lead

to alterations in dopamine-mediated behaviors such as attention, motor activity

and drug seeking (Giros et al., 1996; Zahniser and Sorkin, 2004; Volkow et al.,

2007). Multiple lines of evidence suggest that DAT expression in vivo can be

regulated by environmental and pharmacological factors (Wilson et al., 1996;

Dresel et al., 2000; Bezard et al., 2003). We recently found that dopamine

neuronal activity is a major determinant of DAT expression. Additionally, our 96 studies showed that calcium/calmodulin-dependent protein kinase II (CaMKII) plays an important role in this activity-dependent regulation of DAT

(Padmanabhan et al., 2008). Thus, DAT expression parallels CaMKII activity during sustained depolarization of mesencephalic neurons. Furthermore, inhibitors of CaMKII, but not blockers of other activity-dependent kinases alter

DAT expression in neurons.

However, several questions regarding the role of CaMKII in the regulation

..J of DAT expression have yet to be answered. For example, it is not clear if activity-dependent changes in DAT expression occur in a cell autonomous fashion. It is conceivable that release of different factors by neuronal activity could alter DAT expression. In this regard, a few reports suggested that some

DAT substrates can alter CaMKII activity and DAT-surface expression (Saunders et al., 2000; Fog et al., 2006). CaMKII was reported to interact with the C­ terminal region of DAT, and phosphorylation of residues in the N-terminus of

DAT by this kinase was shown to alter dopamine efflux (Fog et al., 2006).

Dopamine or other molecules released by neuronal activity could activate multiple receptors and sig~aling mechanisms in dopamine neurons. Although pharmacological studies suggest that CaMKII is required, it is not clear if the activity of this kinase is sufficient to regulate DAT expression in neurons.

CaMKII functions as a multimer and phosphorylation of a key threonine residue (threonine 286 in a and threonine 287 in ~, o and y isoforms) .results in activation of the kinase (Lou et al., 1986). Transfer of phosphate moieties from

ATP (ATP binding site at lysine 42 in a and lysine 43 in ~' o and y isoforms) 97 plays an important role in threonine autophosphorylation and kinase activation

(Hanson et al., 1994). It has been demonstrated that a phosphomimetic mutant

(T286D/T287D) can render constitutive activity to the kinase while an ATP­ binding mutant (K42A/K43A) can exert a dominant-negative effect and inhibit kinase activation (Rich and Schulman, 1998). We used these two genetic tools to test if CaMKII activity is sufficient to cause changes in DAT expression in neurons in a cell autonomous fashion.

Materials and Methods

Primary mesencephalic cultures:

Mesencephalic cultures were prepared from post-natal day 2-4 Sprague Dawley rat pups as described previously (Prasad and Amara, 2001 ). Briefly, rat pups

· were anesthetized by intraperitoneal injection of ketamine HCI (3 mg/pup) (Fort

Dodge, IA) and ventral mid-brain tissue was dissected and dissociated for 2 hours in the presence of 20 Units/ml activated papain (Worthington

Biochemicals, Lakewood, NJ). For immunoblotting and immunocytochemistry experiments, cells were plated at a density of 300,000 cells per well in 24-well tissue culture dishes. Mesencephalic cultures were maintained in neuronal medium conditioned over cultured cortical glia. lmmunoblotting experiments to measure CaMKII activity in KCI- and TTX-treated cultures were performed at six days in vitro. 98

For measuring CaMKII activity and DAT expression following transfection of the various CaMKII constructs, mesencephalic cultures were transfected at 4 days in vitro by a modified calcium phosphate transfection procedure (Threadgill et al., 1997). Briefly, the culture media was removed and replaced with

Dulbecco's Modified Eagle Medium (DMEM) 1 hr prior to transfection. The calcium phosphate/DNA precipitate was formed in HEPES buffered saline (pH

7 .11 ). The precipitate (50 µI) was added dropwise to the cells in 700 µI of DMEM.

Following a 20-30 min transfection, during which a fine sandy precipitate covered the cells, the cultures were washed in DMEM and returned to the original culture media. The transfection efficiency was about 1% of the total number of cells and there was no apparent toxicity to the cells.

HEK293 and MN9D cell cultures:

HEK293 cells were maintained i~ minimum essential medium (MEM) containing

10% horse serum, 0.45% D-glucose, 5 pg/ml insulin, 100U/ml penicillin and 100

µg/ml streptomycin. MN9D cells were a kind gift from Dr. A. Heller, University of

Chicago, IL. These cells were maintained at 37°C, with 5% CO2 in DMEM

(Sigma, St. Louis, MO) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin. Both HEK293 cells and MN9D cells were plated at a density of 250,000 cells per well in poly-D-lysine (100 µg/ml, Sigma,

St. Louis, MO) coated 24-well plates. 99

A day after plating Lipofectamine 2000 (lnvitrogen, San Diego, CA) was used to transfect 0.3 µg of DNA per well. Cells were extracted for western blotting two days post-transfection.

Generation of CaMKII constructs:

For the construction of GFP-tagged wild-type CaMKII (CaMKllwt), CaMKllo was cloned from rat brain cDNA using sense primer 5'-

GCTCAAGCTTCGAATTCTATGGCTTCGACCACCACC-3' and antisense primer

5'-CCCGCGGTACCGTCGACTTTTCAGATGTTTTGCCA-3'. The PCR product and the pEGFP-C1 vector were digested with EcoR1 and Sal1 and the digested products were ligated using the In-fusion dry-down PCR cloning kit (Clontech,

Mountain View, CA). For the construction of constitutively active (CaMKlh2a10) and dominant-negative CaMKII (CaMKIIK43A) constructs, threonine 287 and lysine

43 in CaMKll8wt were .replaced with aspartic acid and alanine, respectively, and full length mutant plasmids were generated by PCR. Mutant plasmids were also inserted into the pEGFP-C1 vector using the above described protocol. All plasmids were verified by sequencing.

lmmunoblotting:

Cells were washed once with Ringer's solution and then lysed and extracted into

150 µI RIPA buffer (50 mM Tris, pH 6.8, 150 mM NaCl, 4 mM EDTA, protease inhibitor cocktail, 100 mM NaF, 1 mM Na orthovanadate, 1% Na deoxycholate,

0.1 % SOS and 1% triton) for 30 minutes at 4°C with gentle agitation. The 100 samples were then centrifuged at 3000Xg for 10 ·minutes to remove cell debris.

Western blots of 20 µI lysate samples were probed with the different antibodies.

Membranes probed with phosphorylated TH antibody (Ser19) were stripped with a commercial stripping buffer (Pierce, Rockford, IL) and re-probed with TH antibody. All bands were visualized with Supersignal West Pico

Chemiluminescent Substrate (Pierce, Rockford, IL). Optical density of bands on film was measured using the Scion Image software. TH (Ser19), TH (clone TH16),

CaMKlla, CaMKII~, GFP and ~-actin antibodies were from lnvitrogen (San

Diego, CA). CaMKII (pan) antibody was from Cell Signaling Technology

(Danvers, MA) and CaMKllo and CaMKlly antibodies were from Santa Cruz

Biotechnology (Santa Cruz, CA).

Quantitative RT-PCR:

Quantitative RT-PCR was performed as described previously (Padmanabhan et al., 2008). Briefly, Total cellular RNA was extracted using TRlzol reagent

(lnvitrogen, San Diego, CA) following the manufacturer's protocol. 2 µg of total

RNA was reverse-transcribed using the Superscript Ill first-strand synthesis kit

(lnvitrogen, San Diego, CA). Real-time PCR was performed using the BioRad iQ iCycler Detection System with SYBR green fluorophore. Reactions were performed in a total volume of 25 µI that included 12.5 µI 2X SYBR Green PCR

Master Mix (BioRad Labs, Hercules, CA), 1 µI of the reverse-transcribed cDNA template and 1 µI of each primer at 20 µM concentration. The protocol used was as follows: Denaturation at 95°C for 5 minutes and amplification repeated 40 101 times atan annealing temperature of 57°C. A melt curve analysis was performed.

All reactions were carried out in duplicate. The ~-actin threshold cycle (CT) value was subtracted from the threshold cycle values for the different CaMKII isoforms.

Hence, lower threshold cycle values indicate greater mRNA abundance. The following were the primers used:

CaMKlla (F)- 5'- TTCTCCGGAGGGMGAGTGGA-3'

CaMKlla (R)- 5'-ATMTTTCCTGTTTGCGCACTTTG -3'

CaMKll(3 (F)- 5'- TCTCAGCAGCCMGAGTTTACTCA-3'

CaMKII~ (R)- 5'-TCCACAGCCCCTTCTCAC -3'

CaMKllo (F)- 5'-TTCAGCAGCCMGAGTTTGTTGMG-3'

CaMKllo (R)- 5'-ATGATCTCTTGCTTTCGTGCTTTC -3'

CaMKlly (F)- 5'- GCTTGTCTCCAGGMCTTTTCAGTTG -3'

CaMKlly (R)- 5'- CACGGTGGTTTGTGGCTCCA-3'

TH (F)- 5'-TTCACTGTGGMTTCGGGCT-3'

TH (R)-: 5'-TGAGCTTGTCCTTGGCGTCATT-3'

(3-Actin (F)- 5' -TTGCTGACAGGATGCAGMGGAGA-3'

(3-Actin (R)- 5' -TAGMGCATTTGCGGTGCACGATG-3'

lmmunocytochemistry and fluorescence imaging:

Two days post-transfection, mesencephalic cultures were washed once in

Ringer's solution and fixed for 15 minutes in 4% paraformaldehyde in PBS at room temperature. Fixed cells were washed with PBS and incubated in blocking buffer (PBS containing 4% horse serum, 1% bovine serum albumin and 0.1 % 102

Triton X-100). The cells were then incubated in primary antibody at 4°C overnight. Alexa Fluor 488- and Alexa Fluor 555-conjugated secondary antibodies (lnvitrogen, San Diego, CA) were used to visualize the protein of interest. Fluorescence images were acquired using 488 nm or 543 nm excitation wavelengths and 500-550 or 560-720 emission filters. Images were captured by laser-scanning confocal microscopy (Carl-Zeiss) using a 63X oil immersion objective with a numerical aperture of 1.4. Optical sections (2 µm thickness) in the widest part of the cell soma were imaged for each neuron. All immunofluorescence data were analyzed by Meta 3.2 software. The same settings of laser power, detector gain and amplifier offset were used for all image acquisition within an experiment. Phospho-TH and DAT fluorescence intensities were analyzed in selected regions of interest. DAT fluorescence intensity measurements in the soma and the processes could not always be taken from the same neuron because in some cases the neurons formed part of a cluster and tracing the processes in such a scenario was difficult. Phospho-TH and DAT fluorescence intensities from dopamine neurons transfected with the various

CaMKII plasmids were normalized to the average phospho-TH and DAT fluorescence intensities from at least 20 untransfected dopamine neurons within the same batch of cultures.

Statistical analyses: Paired Student's t test was u-sed to assess the effects of the different constructs on DAT expression. N values indicate the number of 103 neurons. All error bars represent SEM. * indicates a significant difference from control cultures (P<0.05).

Results

Characterization of CaMKII isoforms in dopamine neurons in culture

CaMKII is encoded by four genes, a, J3, o and y. Each gene encodes a protein that possesses an N-terminal kinase domain, a regulatory domain containing the threonine autophosphorylation site (Thr2ae for a and Thr2a1 for J3, y and o). The regulatory domain is followed by an association domain responsible for assembling the different subunits into homomeric or heteromeric holoenzymes

(Griffith et al., 2003). The CaMKII family is expanded further due to alternative splicing of a, J3, 8 and y genes resulting in several sub isoforms (Hudmon and

Schulman, 2002). Although a and J3 isoforms are the predominant isoforms in the brain, a recent study demonstrated that certain splice variants of the o and y isoforms were prevalent in midbrain dopamine neurons (Kamata et al., 2006).

We first tested the specificity of the commercially available CaMKII antibodies. HEK293 cells were transfected with GFP-tagged CaMKlla, J3, o and y. While CaMKlla and J3 antibodies were highly selective, the o CaMKII antibody recognized the o and y isoforms (Figure 21A). The CaMKlly antibody showed high selectivity to CaMKIIJ3 and the pan CaMKII antibody recognized all isoforms except Ca~Klla. lmmunoblotting with GFP confirmed that all proteins were expressed at their expected sizes. We next analyzed the expression of the 104 different CaMKII isoforms in primary midbrain cultures. CaMKlla and J3 were not detected but the CaMKllo antibody (that recognizes o and y) and the CaMKH pan antibody (that recognizes ~' o and y) detected two bands at 53 and 58 kilodaltons

(Figure 21 B). To further evaluate the expression of CaMKII isoforms in dopamine neurons, we performed double immunostaining for tyrosine hydroxylase (TH, the rate limiting enzyme in dopamine biosynthesis and a marker for dopamine neurons) and CaMKlla, J3, o and y. CaMKlla and J3 were not detected but dopamine neurons showed increased expression of CaMKllo and y isoforms

(Figure 1 B panels). The lack of CaMKlla and J3 in these neurons was not due to the inability of the antibodies to detect these prot~ins by immunocytochemistry as both isoforms were readily identified after transfection of GFP-CaMKlla/(3 (Figure

23). Furthermore, in quantitative RT-PCR assays, the o and y isoforms were found to be as enriched as TH (approximately 30 fold higher expression) as compared to the a and ~ isoforms (Figure 21 C). These data demonstrate that

CaMKllo and y are the predominant CaMKII isoforms in dopamine neurons in culture.

CaMKII activity governs DAT expression

Using pharmacological tools to alter CaMKII activity, we had previously demonstrated that CaMKII activity is required for activity-dependent DAT regulation (Padmanabhan et al., 2008). We sought to test if CaMKII activity was sufficient to cause changes in DAT expression. We cloned one of the major 105

CaMKII isoforms in dopamine neurons (CaMKllo) and generated constitutively active (CaMKIIOT2a70) and dominant.:.negative (CaMKlloK43A) mutants.

lmmunoblotting with GFP indicated that the proteins were expressed at the expected size; about 84 kilodaltons for CaMKllo, consistent with a previous study (Kamata et al., 2006). In order to confirm that these mutant proteins altered

CaMKII activity in the expected manner, we first tested CaMKII activity in an immortalized dopaminergic cell line (MN9D cells) by measuring the phosphorylation of TH. Phosphorylation of TH at serine 19 (Ser19) has been shown to occur selectively by CaMKII (Haycock and Haycock, 1991). We found that CaMKIIOwt caused no change in phosphorylated TH abundance. In contrast,

CaMKIIOr2s10 increased the phosphorylation of TH whUe CaMKlloK43A caused a decrease in the levels of phosphorylated TH as compared to untransfected

MN9D cells, further validating the utility of TH phosphorylation as readout for

CaMKII activity in dopamine neurons (Figures 22A and· 228). We next tested the effect of wild-type and mutant CaMKII proteins in altering CaMKII activity in dopamine neurons in culture. Mesencephalic cultures were transfected with the different plasmids and 2 days post-transfection immunostained for phosphorylated TH to assess CaMKII activity. Consistent with our observation in

MN9D cells, CaMKIIOwt showed no change in the levels of phosphorylated TH,

CaMKIIOr2s10 showed a significant increase in TH phosphorylation while

CaMKlloK43A showed a significant decrease in the abundance of phosphorylated

TH as compared to untransfected d-opamine neurons within the same batch of culture (Figure 228). Thus, the results in both MN9D cells and primary 106 mesencephalic cultures indicated that the different plasmids altered CaMKII activity in dopamine neurons in the expected manner. Using the same approach, we next examined the effects of CaMKIIOwt, CaMKlloT2a10 and CaMKlloK43A on

DAT expression. CaMKIIOwt expression in dopamine neurons did not affect DAT expression as assessed by immunostaining for DAT. Augmenting CaMKII activity with CaMKlloT2a10 significantly increased DAT expre$sion while inhibiting CaMKII activity with CaMKlloK43A led to a significant decrease in DAT expression in the cell soma as well as in the processes (Figure 22C). Taken together, our results confirm that CaMKII activity can regulate DAT expression and that CaMKll­ induced DAT regulation occurs in a cell autonomous fashion.

Discussion

Regulation of gene expression by calcium signaling is an integral part of processes underlying several important neuronal functions (Colbran, 2004). A key molecule linking changes in intracellular calcium to altered gene expression is CaMKII (Dupont et al., 2003). Using pharmacological agents, we have previously shown that CaMKII activity is required for DAT expression

(Padmanabhan et al., 2008). In this study, we provide further evidence for DAT regulation by CaMKII. We show that CaMKII is not only required but also sufficient for causing changes in DAT expression and that these changes occur in a cell autonomous fashion. Unlike several reports that demonstrated alterations in DAT function within minutes of CaMKII phosphorylation (Hayashi et 107 al., 2000; Mauceri et al., 2007), we find that CaMKll-induced regulation of DAT expression occurs on a longer time-scale. This suggests that this form of regulation may play a significant role in adaptation of the dopaminergic system in response to external stimuli or agents that alter intracellular calcium levels in dopamine neurons.

Four major isoforms of CaMKII with different subcellular localization are thought to mediate several important processes within a cell. In our study,

CaMKllo was used as it was one of the more abundant CaMKII isoforms in our cultures. CaMKll-mediated changes in DAT expression could be due to alterations in transcriptional. or post-translational mechanisms. We have previously shown that changes in DAT mRNA levels at least partly contribute to the observed changes in DAT expression in response to CaMKII activation

(Padmanabhan et al., 2008). Numerous reports have also shown that CaMKII activation can alter the activity of different DNA modifying proteins such as histone deacetylases (HDAC) (Mejat et al., 2005), myocyte enhancer factor-2

(MEF2) (Flavell et al., 2006), methyl CpG binding protein-2 (MeCp2) (Chen et al.,

2003) and neuroD (Gaudilliere et al., 2004), thus providing evidence for transcriptional regulation by CaMKII. It would be of interest to learn if any of these DNA modifying proteins play a role in linking cytosolic CaMKII activity in dopamine neurons to DAT expression.

We had previously shown that neuronal activity plays a role in regulating

DAT expression (Padmanabhan et al., 2008). Changes in neuronal activity could cause release of neurotransmitters (such as dopamine/glutamate/GABA) or other 108 factors (such as BDNF/TNFa). These released factors, in turn, could activate several signaling molecules that could eventually alter DAT gene expression.

The effects of these factors, if any, would have to be mediated by CaMKII as pharmacological inhibition of CaMKII occluded the effects of neuronal activity on

DAT expression. However, it is unlikely that these released factors contribute to altering DAT expression as KCI treatment mimicked the effects of TTX rather than 4-AP. Alternatively, it is possible that, a molecule whose release is dependent on action potential firing regulates DAT expression. In any case, our results with constitutively active and dominant negative CaMKII provide evidence for cell autonomous regulation of DAT expression by CaMKII.

Multiple signaling pathways are triggered in response to increc;ises in intracellular calcium and could play a role in linking neuronal activity to gene expression. For example, neuronal activity could lead to activation of CaMKs, protein kinase A (PKA) and extracellular signal-regulated kinases (ERKs) that, in · turn, could alter the activity of several downstream effectors to alter gene transcription (West et aL, 2001). We have previously demonstrated that sustained changes in neuronal activity cause alterations in DAT expression. Of the major calcium-activated signaling pathways, CaMKII appears to be important for regulating DAT expression, as pharmacological inhibition of

CaMKII, but not PKA or MAPKs, abolished the effect of neuronal activity on

DAT expression. It is likely that activation of other signaling proteins or co­ activati.on of multiple signaling cascades may be required for altering gene expression. For example, gene transcription mediated by CREB requires both · --1:09··

·:cREB :activation by. ER_K:·s1gn.alin:g· and:co~activation:·of· CBP {CR-E.8-.bind1ng:_ ..

-· ·:protein) by CaMKIV signali.n{f(lrnpey et aL,."2.002):·The:.possibili.ty oftequiring··­

multiple: sign~ling cascadesOto ~Uer DJ\T gene.~_xp:res:si"o_r:i tH~eds to-be:_-:

jnv~·s~igated· in __ the_fut~~~.'_Howey~r, Lisfng ~~n.stitutiyely"acti_v_eand ~·omi_nant:

...... ' ...... - ...... - . . . ', . . . - . . ·:~eg_adve· CaMkl_i cons_tru~ts·, we show that: Ca~K!I. i:s: at_'least 9ne· signaiing : ._ .

. . protein.that rs. not only .required .but also s.ufficien_t.for.causing changes in D~T.·.·.

-: expression. : .· -

· Althqugh.a ·a~d-J3 are: th_e:majdrQctMKff:isoforms·i~_ ~everal oth_er brain··

__ regions·, ~argeting _CaMKllo _or y isofo_rms woulcl provide _uswith.a11 opportu_nity.to: ·. . . . .

...... - ...... -: alter·DATexpression by:·altering -C:aMKII .activity in:dor:famim~- neurons.- It wilrbe ·: -

...... - i"nter~~t~ng :to~ l_e_~rn _if the other isofor111s -th~t ar~· .loca{i_zed p"re~o·ryii_na·ritly in the- :· -.

...... - ...... - ...... ' ...... - -Jiuc!eus and·_en:ri_ched _in· o_oparn_ine neuro_ns;, such:·~s:~s-(Kary,·ata et aL,· 200~),:" ..

. ' . . . . . alter.DAT~xpr~s_si~n ~_ifferently._<3enetic n,anipulat_icm~ in. vi\19 have. fo9u~ed on. --_

. . - . . . - ...... CaMKila a·rid-its role ·in-_hippocampal syn·apti·c plasticity:(Elge.rsma.et -~L;.2004).:. _._· ·

...... ·nopamirie _neuron~specific Cat\/lKfl :trans·g_eriic tn~c~. will prC>vide:u~f\JVith .a:.tool te>.

·___ better:·u~dersta_nd __DAT·r~g~lation·:bY_,h~se·ca_MKi'l-is·ofe>rm.s-ih_vivo .. · Figure 21. CaMK/16 and CaMi

(A) lmmunoblots of HEK293 cells transfected with GFP-tagged CaMK/1 isoforms and probed with commercially available CaMK/1 isoform specific (a/{3/olr), non­ specific (pan), GFP and {3-actin antibodies.

(BJ lmmunoblots of mesencephalic cultures were probed with antibodies to the different Ca MK/I isoforms and· TH. Representative confocal images of mesencephalic cultures that were immunostained for TH (red channel) and

CaMKII isoforms (green channel) are indicated in the panels below. Scale bar: 10

µm.

(CJ Real-time quantitative PCR data of mesencephalic cultures at indicating the levels of expression of the different Ca MK/I isoforms and TH. The {3-actin threshold cycle (Cr) value was· subtracted from the threshold.cycle values for the different CaMK/1 isoforms. (Note that lower threshold cycle value indicates greater mRNA ·abundance). 110

Figure 21.

A B

CaMKII IB

GFP.CaMKII a ll 6/y pan Tli EV a II 6 y a 18 72 -

1118 12- - 618

72 -

y l8

72

CaMKII (pan) 12- -. ..

12-

11-Actln EJI I

C

20

15 C il ~ t.?. 10 i g 0 5

alpha beta delta gamma TH Figure 22. CaMK/1 activity is both sufficient and required for causing changes in DAT expression

(A) Representative immunoblots of MN9D cells transfected with the various

CaMK/1 plasmids and probed sequentially with GFP, phospho-TH, TH and {3- actin antibodies. Densitometric analysis indicates the effect of various CaMK/1 plasmids on the -abundance of phospho-THflH compared to untransfected (UT) dopamine neurons. N indicates the total number of experiments.

(BJ Representative confocal images of.mesencephalic cultures that were transfected with the· different CaMK/1 plasmids (green channel) and immunostained for phospho-TH (red channel). Shown on the right is the quantifation of phospho-TH abundance in CaMKllow/CaMKllor2a1olCaMKl/oK43A­ transfected dopamine neurons relative to UT dopamine neurons.

(CJ Representative confocal images of mesencephalic cultures that were transfected with the different CaMK/1 plasmids (green channel) and immunostained for DAT (red channel). DAT abundance in the processes of UT and CaMK/1-transfected dopamine neurons is indicated in the adjacent panels.

Shown· to the right is the quantitation of DAT abundance in the cell soma and processes from CaMK/1-transfected dopamine neurons relative to untransfected dopamine neurons.

(B, CJ CaMK/1-transfected dopamine neurons are indicated by arrows. Scale bar:

1O µm. N values indicated in the graph are representative of the total number of neurons analyzed from at least two different batches of cultures.

* indicates significant difference-from UT dopamine neurons (P<0.05). 111

Figure 22.

A CaMKllo UT WT T287D K43A i=- - 95 ::::> 300 ...,_ C GFP - 72 .'!J 250 * 'i: ~ 200 - 55 ·c: .; 150 E .9 - 43 -~ 100 Cl * p-TH 0 50 TH E 4 5 i!: 0 13-Actln a. UT WT T287D K43A B

§'° 200 0~ * ~ 150 "'C: ..Cl -= Clu 100 C: Clu "'e 50 0 * :::, u:: z 4 5 I- 0 a. UT WT T287D K43A

C

§'° 200 ~ * ~ -~ 150 * i ~ 100 C: Cl u * * "'e 50 0 :::, u:: 3 3 9 4 15 11 ~ o...... _ ...... _...,_.....__._...,___._.__&..J_....._.,__._ 0 Figure 23. CaMKl/a and CaMKl/fJ antibody validation in immunocytochemical assays

Representative confocal images of mesencephalic cultures transfected with either GFP-CaMKl/a or GFP-CaMK/1{3 and immunostained for Ca MK/la and {3- specific antibodies are presented. CaMKl/a and f3 antibodies readily recognized

GFP-CaMKl/a and {3, respectively, thus validating their use in immunocytochemistry assays. Scale bar: 10 µm. 112

Figure 23. : IV.· DISCUSSION:.. . . .

In several"neuropsychiatric conditions;.neuroadap~ations ·in the dopamine.rgic- _.· ·

...... -: . s·ys~em -occi.ir rn response to-external stfr~uli. However;: the mechanism·s. involved-. -·

in .t.his· process arenot:cleariy_un~erstood. _ln-a:series ofstucties, we _aimed·:~L·:­

·:understanding·the_:cellular·events inv9lved in·-r~gulating _the-abunqa·nce of~-· ·

: dopamine. neuron-specific prote1n,:DAT, which plays:·a critical role.in rnairitainin:g:: .· ......

...... · . dopamine·.homeostasis.-\f'/e have provided :eviderice:s~pportirigthree:ma)or.· -- .

...... · findings i~. these studies:: .

...... ·L Sustained al_terations._in dop~n,_ine.neuronal.a_ctivity can lead to ch~_nges in __ ·

... : DAT expiessiori ·and.function- ..

·: ii. · .PP2A pl~ys .ai1irnp~rta"r1t_ r()le· in ·reg~_lating CaMKII acti\/it_y .in·d()pamine . · ·

-neurons and

iii. _: CaMKII activity· in.dopamine·n:eurons can govern DAT expression ~.nd ·.

. . . :-function.> ......

Although: the focus· of this· stud}t was DA,-;. tne -mle. of CaMJq1 ·si~naifn~. ip.

reg~i~ting the:c1bm1dari_c·e.of other:proteim~_:involved _lflrTIOdu·1_ath~g-dop~~i-ne ....

...... neu:rotransm i~sion such. as D2. receptors ·and .vesicu_lar. monoamine: transporters·,· ...

·=sh_ould.be i_nvestigate!d)n :the fu:ture. · .

. .·.·: _In _thefirststuqy; -We _u·se.d·phar_macologi_cal.agemt~-to· bi~direct_ion~lly __alt_er-

.... · . . ... · . . ... · . . . _· ' .... · . . ... · . . .. ,·· . . ... · . . ... neuronal firing>4~AP increased dopam.ine -neur9nal ·firing while: TTX c.o~pletely: . ·_.11:3. · ... ·1:-14-·

...... inhibited the firing ·of dopaniine.neuron_s.: ·C_hronic treatmentw_ith these. drugs.was·.·

. . . .. :found:to .significantlY-alter·-oAT-expressiortand}linction ..in pri·mary · ·

. . , . ' ...... i:nesencephaii:c":cultures.··whil~ 4~AP in~reasedDf\T:expressi_oh·and furict.ion, __ : · ·

treatment w1t~_TTXde~r~·ased.Df\T.:expression·and f~nc_tion~_-lJs_in_g i.nhibitors to_-_:··_.

. . . . . ·:various signaling cascades;: we demonst~ated -thatcalcium :entry thro·ugh L..-type

...... voltage~gated._calciu~ channels .and C.af1/1.Kll.a~tivation were .re~ui_red for.the .. ·

. - ...... - ...... activity-depe_ndent -reg_ulation ·of: DAT exp_ressior;". ..

...... : ·:-.: Jn.th~ subsequent .stuqy,·we-flfrth~r_evalua·ted theHole _of Carvl_KU 1n _·; .. · regulating DAT: expression. __ To.-study __ the effect.of increased calcitJm signal_ing on: · ......

□At- expression arid functfon, we:·used potassium chloride to· trigg·er neuron.al

de·p:ola~ization. :surpris:ingly,chroriic. KCf-tre:atm_ent:m:imi(?ked· t~~ effect.ofTT)(:· ..

· ·:i_nstead of_.4~AP.·:· Th·u_$_,·sustain~d depola_rization· With KC(cau·s~d:a· si_griificant··

. . . decrease in Ga_MKll activity in dopamine_ nem_rons a_lthough _c~l_ci_um. levE3ls

...... remained elevated.. Thfa..finding :suggeste:c:Uhat different patterns of dopamin·e : .· ..

·:·n~·uro·narti_r!·ng couid i~ad tc{differential c3Ctivation_:~f CaMt

. . ·. _previc>usly sug~~jes_t~d that.PP-1.acfivation· iri:·re~po_nsetocalciu_m ~ntrythroygh .·.·.

NMDA recepto~s-:could d~phosphqrylate and in•activ~te: CaMKII fn·the:p_ost-·_·

synaptic.density (She~·eta(_; -200~)-. b.urresu_lts-indicate_that, inresponse_ to:.·...... ·. .sustained depolarizatiorf, -elevated .PP2A activity· 1s capable· ofdephosphorylatirig: · ...... ~ ......

. . . ·. _cyto~ol_ic CaMJ

- ...... actiyity .in.dopc1mine· i'l~U_rons·in· GUlture: ·Fqrth~r studies identi_fying. th~·mode· of:·_ .. '

·c~l~ium en_try_ irito ·dop~mine .n~~rcins: in_r~spon·s~::to chronicXCI tr.ecdmentwi_ll:. I.· -t1.5·

...... - ...... ' ...... ' .. . provi~e ad_ditional ·,nformation. on the ·molecula·r play~rs m_ediating: PP2A .

. . ·. :activation in.dopamine neurons (Figure 24);. ·

- . . . . ' ...... : :Calcium.:.induc:ed ·a1_terations ih "DAT expressionco~ld. oc~ur.d_ue to·

...... · .. chariges in the_:rate· onranscription·, m_RNft s~~bility, -9r_protein degrad~Uon rate ...

·. ·Since:qha_ng·~·s :in DAT mRNA paraUelecn_he chang_e·s ·9bservedant1e ·protei_n·: ·.. · . . .

... - level,· it .is po_ssibl.e that _altered. PAT. transcription contribute~ .(at _least· in _part). to .... · . . . . its· aitered ·abundance, :in .response· to cha:n·ges in CaMKII ·a~tiv-ity. However,- the··

·:effectof p~rsistenfalt~rat1or,s_ in:.r:ieuronc3l_.acthiity_·~n_ DAT_rnRf\J°A ·ancl·protei·n :

- ·:stability r~quires fLJrther·inv~stigation_.:_Studies _by severa;I other gr~>Ups hav~ -- ·_ --

...... ' ' ' ...... deniohstrated·that post~ttanslatiorial mod1fications.inDAT are:crucial :

...... ~eterminants:·of -OAT function; -these studies were·:performed 111 _non-native .

. model syste.ms:and·s11owed·thc1tprote_in kinases· ~-~·ch:aSPJ

. . . Cafv'.IKH_a Ga~ pt,osphoryl_ate DAT,·_ lead_ing_.to .alterat_icms in its_.functi_on· (Vaugt,an._-_- .

. . . :·_._-_ -: et" af.; __1997; Moron-efaL; ·2003;· Fog:efaL;_2b06) .. --

. . . . ' . . - : Jn·:ord_er to test_if DAT phospho"rylation by ~aMKII coLJld coht_r_i"~_ute. to-~~~-

...... - - __ regulatio_n-of DAT _expres$ion; we·te~ted_.the effect ofac_yte_.changes in Ca~KII - --

a~tivi"ty ,·:caused: by TTX- -~nd KCI treatments·; on_ -DAT fu·nction~: Treat~eni of. -

-· _ cultures with ~lev_ated-·extra~elh.ilai" p~tassiurn (30'-mM)_~au·sed-a significa~L- -

-:increase-:in 'c~arylKII ~ictivity in"dopamine":neurons ~hi!e"TTX·treatmeDt had the:·_' ...... opposite effect{figure·_i5A).-Althe>ugti-_thes"e~_~r~gs-_pr9duced:~:ramati~:~hanges:_:·: . . . . .

...... ' . ' ' ...... - . . . . in ·CaMKll ·acfivity in-d(ip_a-mine h~_u·rons, ne_ith_er-KClnor TT)(CalJsed•t;iny.Ghahg~--

' ...... , . . - . . . - ...... - ...... ·:jn the:ma~im~i v~lo~ity:of.dopa~i-n-~ u·ptake:(V~ax) qr:apparent:~ffinity (l

We have shown that in response to alterations in neuronal firing caused by chronic treatments with 4-AP, TTX and KC/, intracellular calcium levels and consequently CaMK/1 activity is altered. 4-AP increases intracellular calcium and

CaMKJI activity while TTX has the opposite effect. Although chronic KC/ leads to sust~ined elevations in intracellular calcium, it causes a decrease in CaMK/1 activity due to activation of PP2A (not indicated). CaMK/l activity in dopamine neurons can govern DAT expression and function. Hence, 4-AP is capable of increasing DAT expression and function while TTX and KC/ decrease DAT expression and function in primary mesencephalic cultures. . ' . . . . '. . . . . ·. . Pharmac~l~gical arid environm~~tal fac~ors ..

. . . . . : . . .

. · 1·......

~c, •.&aM'.,r __• ,r;{ __ I · Dopamme neuron · . . · ·. .· · ·. · · · · . · ···DAT protem .· ··: .·Cytoplasm. · . ·· · • • · ··· ·:.... · ·.·1 · -· ··: .· · .· ·· · · . . . . . · ...

...... '' ·.. KCl/4-AP >trt TTX .. - -..

.. c~~i:c~~xp t p-CaMKII l g~~~~:~ ~~ •.. ! . ·-:~·?···. . ■: . .... Nucleus: .···· ·1 .·· . . : ......

.. DAT mRNA ______, ...... ' ...... ' ...... ·:lTX. treatmentfaiiedjo alter.substrate:-ind_uced·(dopam·,n_e:_or ampheta_min.e). _: ·

·. :DAT~m·ediated efflu:>( (Figure 2?_C): .. These·data .in~icate that acute changes: in· · ·

~aM_KWactivity leading to alterations in DATphosph?rylation: m_~y'.not play a_ rol~: .

. . . ' i'n ·regulatjng .tr~n.~porte~.fu~~tion:• .

. . . .. : Amphetar:nines.were :~ho~ri'_to:·9ecre:~se·. DAT ~mediated dopamfr1:~ upta~e: ·

...... ' ...... (Wei etaL,. 2007.). Thi~.d.ecre.as·e.was mediated·by activatioil.of.CaMKU,· as·.·. ·

...... ' ...... pharmacological inhibition :ofCaMKII prevented the.amphetamine-induced · ..

. . , ...... , ·:d~crease in·DAT funqtion; C~MKII was. c1lso.shown to.ph(?.SPh_orylate.and

. . . .. ·:promote DAT~mec;t:iated·dopamine .efflux.in re~ponse to _amphetamines· (Fog ·et ··

. . . ' ...... al., -2006}. Usin·g ·the DAI C~termimfa ·as bait 1na yeast two-hybrid.screen; the.•· .

·alpha:isoforrn ofCaMK11:was.f6'un:d_to·in.teract with:and phosphorylate DA.T: In··.

•:adcHtioti, these auth.orsdemon$b~ated·ttiat·activate~ CaMKll.a·butnot_heat-:· ..

. . . inactjvated Ca_MKllq .inGreased d.opam.ine.~ffluxthr9_ug_h DAT.._·_These ·studies .. ·

irnpiy.thaU:1'r11phetam·iriescause/an .. incre~s.e.in .exttacellu.lar dopamirie_lev.e1s· by.· ..

. . . d~creasirig b.At .-med~~ted uptc1ke and -~Y: _pronio-*19 bAt~r11ediated_ tjop.aniine. ·. _

·.. release-. aotti•rnec~an.isni.s 9f- regi.il_at_ion.of.QAJfuncti.onhave·:beE:)n proposed.to-: ·

requi~e ·C$MKH:acti_vatio:n.·: ·.

...... However;· our data··so_gg~st that.CaMKII may not play:a role•_in·reg~l~tirig ..

:IJATfu.nctiori.Quuintj .. other studies in.dicatethat:CaMKllcx:is ·not.pr~serifin. . ..

dopamine neu,ro.ns-in c,uuu·re ,orin:d~pamh,·e neuro·~.s: i~ vivd(Kamata _et al.:.· ..

200E5) .. 'Rather,•.Ct,iMKIR>.and'ya~~-the·p·r~d.Orninant·caMKU-isQforms·jn.doparnii1~··.·

. . . . ·:n~·~ro'ris •in ~:u.lhfre·:~mcUri :vivo_ (~amata_ ~tal,, 2096). It. is: p~~sible that

i'ntroduction_ofGaMKUa.·¢ause~· changes in:theactivity. of d_op~min·e·_ne(iron_· ... · -. t18··

·enriched·qaMKII isoforms (as the diff~re~t c~M_Kll:-isoformsJorm multimers) to:

. . . Jegulate _DATfunctioh. The.effect obsetved: on:DATfunction-could then:be: -- ·

attributed.t_o a_lris6forn1s:·of CaMKII pre~enfin: dopamine neu~o~s. ·If C:aM_KII. ___ _

·acthJity·_did· ir,~e~d contri_~ute·-to·~1~~ration~--in_DAT.fu_nc~ion-,"·V!~.-should"_hay~·-· .·.

:ob:se"rved·dram:ajic _ch~rig·es:·i·~ :DAT function•wit_h. ~Cl and. TTX treatments. ·:~·c1_:·· ...... an~ .T.TX treatments. c_aused significant a_lterations_·in.C.aMKllactivity i"n dopa.m!"ne .

neurons (~79% .. in efftier. directicm),· but ne.ither KClnor:TlX caused any-change.·.·.

...... ' . ' . . . . - . . . ' ...... :i~_QAT-niediated uptc1ke~ Moreqver, the_-~ignificant:decreas~s in C~IV1Kllactiyity_.

. . . · ob.served. with-TT)( failed to__ alterDAT efflux. T~ken·tog~ther;··our __studies suggest ·. . - . . . . .

that CaMKII activity may":n·ot.be :invohied 1n:"re:gulation ofDAT function:·:: .

...... - _·: .. ·Cohsist~n_t with:p~eviousreports from adulfSN··tissu·es,:o_urresults.indicat~ _

...... - . . . . . - . . . . ' . . :that CaMKll:o_"an~ CaM.Klly a_relhe·predoniinant· isofor_ms.in ourcu_itutes (Kamata · .

. . . . etaL;.2006). Introducing a con_s.titutively" active·form of CaMKllo intodopamine.-. · . .

...... ' . rieu·rons was:sufficientto prom~te-DAT ·e_xpression: while cfominant~negative . ' ...... '' . . .. :CaMK116s.upp_ressed·.D~T e~pression-. _Qur.dat~ show that.C_aMKHo is a

·· .. cytoplasrpic. -isofor_~ ·that: is c;apable· ·of i·ncreasi_r:,g ·DAT expression, ·ind icatin.g ·that: .· ·

other:"protei_ns link the-activity of .GaMKII to :~AT _expregsi()n~: !dentificat_io:~-:of .. . - . . ' . . . . . thes~:_protefn~-vvill revectl n:ew reg_ul~t9rs··o_f_PATand_:shouId:bE:te~am"in~~:i~-the_._ .

. . . . •futi.Jre:.:Ad~iti~mally;_ stqdies·assess:i.rig_the_ role·:af C~MKlly in.:regulating q,AT

expre~sion wilLbe usefuI:·to determ_ine•ifqifferent isof()rms qfCaMKll_.reg~la_te-_ ·.

DAT~xpr~ssion differEmtly. Figure 25. Acute changes in CaMK/1 activity do not alter DAT-mediated dopamine uptake or efflux

(AJ Representative immunoblots indicating the effect of a 15-minute treatment

with 1µm TTX and 30mM KC/ on phosphorylation of TH (Ser19J as a measure of

, CaMK/1 activity in ·dopamine neurons as well as abundance of TH are shown.

Densitometric analysis of the ratio of phospho-TH to TH is presented normalized to control is presented.

(BJ Saturation analysis of dopamine uptake showed that TTX did not elicit a change in Vmax (Con: 110. 7±4.127, TTX: 110.9±8.329J or KT values (Con:

0.666±0.096, TTX: 0. 743±0.2130J. Similarly, KC/ did not induce alterations in

Vmax (Con: 109. 7±5.424, KC/: 112. 7±11.39J or KT values (Con: 0.693±0.132, KC/:

0.683±0.267J. Uptake data at different dopamine concentrations were normalized

to those at the highest concentration (12. 15 µMJ in control cultures and expressed as a percentage withirJ each experiment (N=3J.

(CJ Dopamine release experiments indicated that TTX did not induce a change in either dopamine-induced release (Vmax: Con: 156.2±17.49, TTX: 170.7±17.59;

KT: Con: 0.090±0.071, TTX: 0.121±0.077J·oramphetamine-induced release

(Vmax: Con: 138. 7±6.99, TTX: 141.2±8. 724; KT: Con: 0.439±11.89, TTX~_

0.272±9.65J (N=3J. ·::::-e- 250 1: 8. _20Q .. · Controi : . me . . Control . . 1

. _: 1_40· "")_ ····o. :'e.·-120 ) . O· . .CJ· 1·00 .. · __ ) ---~- . :~ . 80 l . ca- .. 60 · :>a -' )_ . Cl). ., · ·.c · _-40 ·3 --:·e · ■ -· co,.trol · .· - Iii · Contrc>I .·t 20. . .._.. KCI ··_ g· - •: me )_ O ➔:-----r,------....,...------. .:l-t----...------.-----r----. 0.01_· . - 0.1 · . -1 .. - 10 · ...100 · 0.01. · 0.1 . 10 ·_ 100-· : · DA·concentration (micr_omolar). . DA concentration·(micromolar)

C ..... -l:1go · .155

s:1aoCJ ·: . :~·1-10 . -·145 :';-.160 · iS tso · _135 ur 140 '' '_·_·:-aruo· · .·125 : g.120 . -g ' ■. Control· 115 ";-110 -_■- .Control. ~:100 · TTX . -·e ··go · _105- · · •.TIX : it· 8:0-t------....----. gs~.------....------8 _· . 0.01 · - - . 0.1 .·.1 : · _10 .. 10() 0_.001 1: · 1_0 · · Aft1'f PH _Cone (micromolar). - . . . . ·. Jliodel system andJ>harmac:ological· tools were: used to modulate .neuronal .firing·. :

. . Althou9h our study proyides .valuable d~ta concerning the_ molecular mechan!sms. ·

...... ' ...... ' ...... rnvol_ved in- DAT"regui~~i9n_, futu.re_:st_uqies: c1ssessing )he .role ofCaM.Kl_l)n:

...... ' ...... ·:regul·aung_·oAt ~bqndan:ce1n vivo WOl:Jld._help validate_.our findings:

-:Clinical Implications:-

. . . .. _As-discu~sed·prev_iously; .se_veral-behavi.oral.states have.been:as~ociated '1Vith .·.

...... change·s in dopamine .neurotran:smission.: Discussed.below are the iniplic·ations · :·· .

...... of.this study-to :tliyo ·m·ajor·tjisorders·.in whicfrdo_pamine __ sjgrialing is·.kno~~- t~·be· ..

...... · •:alt~red.· ·

a.. Chronic .d,:t1g_ abusfJ .·

Chronic e~posure td drugs: of abuse: such as amph~tamines:-an'd CC>C~lne .. resulHn' .·

...... ·:increased. dopamine signaling ·in the ·nucleus accumben·s. An fricrease in. . . . ' . . . .· . ·......

. . ·.. extraqellylardopar11ine-levels resultsjn activation.of0·1 ~ecepto,r-~ontaining_ · ·

GABA neurons :iri the accum_beris::: This, inturn~_-inhibi_ts SN dqpamine: n_~uronar --

·firing via ·inhibitio~ :of.t~e· gl_utarriater neurons ·of the STN that:project to the_ ...... Jn1qbr~l°n.-Additionaliy;_ these .p-~yc~ostiniu1a:nts:ca_n act dire<;tiy: on:·dopamine .. . . neuio_n_s locat~d._i·n the-SNNTA·to_·p_romof~ d_opa.mi'ne release_.:_:R·eleas~d .. ·

dop:aroine· can-.b1nd.to p~·-receptors.in-the·midbrain:·c111d ultirri~t.el_y.inhibit.·.­

·:d_op~mim~i ~eurqnal a·~tivity. Hen:ce:, chro:nic drug _e~p·osu"re :ieads to_ ~eci"eased: : :.

midbr~in ne_uro_~al_ ~cUvity-th_atin-tqrn _shoul~· decrea~e. DAT- abundance.·. Indeed-,.- -- · -.·.1-21-·

.analysis ·of .DA le~el.s: in diion.ic drug· abuse p:atients:st."ow.that DAT expression: :. . . f . . . . - ......

:atthe mRNA·and protein.levels is: markedly .re.duced:(Wilson··etal.; 1-996a;_: -· ·

V\/ilscfn_·et.al.;: :1996b; sa·nn:on eU1l, 200_2)~ :· .

_b~- Attention :CJ~ficit ·Hypera·ctivity_Disorc!er

- ' ' ' . . ' . '...... - ...... - . . ·:A posiUvecofrelation: 9f ievels :o_fhomova_n.iili_c acid, a dopamine· m_eta_bolite ·i·n :· ..

cerebrospinal.flu.id,·.and.ADHD s_ymptoms_supports.the view that excess._

dop~mine·h,_the brairi_:rnight co"ntribute to: hyperactivity:(Madras-et ·aL_,:·2005):.

·:·lnqr:eased __ dppamine_n_eurofran~misslonwOuld: in_turr,: lead_:to incre~~_ed: DAT__ :_.:-

...... abunda11ce. Indeed, studies have shown that ADHD-affected·adu_lts and children

have:in·creased: DAT aburidance·in the bas:algangiia· as compared to.·co:ntrol · · · ·

subjects (Doug_herty et aL,_ 1_999; .Cheon et aL, 20·0:a;_ Kra·use· eraL;.-2006). ·_. --·

...... : Taken. together,· our dat~. SU~Jgests· that-activity-:depEmdentcha~ges :iri : ...

...... CaM_KI.I ar:e lit

··progre:ssio_~-:9f several:_dopan,ine-relate~·neuropsychiattic disorders_~:-~ased. 9r:1:-

·.. _ou_r studi¢s;_.it a"pp_ears-that targeting .P-P2A.or GaMKH a~tiv_ity--in-d_opamfoe __ ·_. · -

neurons ~i-tt1erdirect.ly: (qs•ing. i.sofqrm~~p-~c.ific m~du·l_ators). Jr"throu.gh recept~~s- :·: .·

...... , ...... erfr~c~e~· in-·midbra_in-dppamine·neurons·, could provide us wUh atoortoalter .

. ·: do_parr,ine ·ri_~urotransmi$siori. : · · · V .. SUM_MARY

...... ' : We,:have shown thahri .primary' niesen·c'ephalic·CuHLires, ·eleyated c·alcii.Jm leve,l:s· ...·

and CaM~II signalir:tg.can promote DAT exp.ression. ·counteracting this pathway

...... ·. ..is the :act.ivation .of.PP.2A thc1t'can ptevent-CaM,Kll-induced·.DATepcpressior:,. ·Our:

. . . : data indicate that the changes observed in:DAT _proteiri abundance could:be_ due·

. . .. - .· ...... · ...... · . - .. . _· ...... · ...... · . .. . .· ...... · . . . . .· .. . · _·to·altered transcription, sinc~.- □AT mRNAlevel.swere··a1tered. The·.relative role·s ... · ·. . - . . . .. · ·. . ' . ·. . .·. ·...... ·. ·. . . .·. ·. . . .. ·. ·. . . . _·

...... to theobsE3ryect· chang~~· in DAT.protein _an~ _mRNA abun_danGe, in,.response to_ .. ·

. ' ' . . ' ...... neuronal adivity,.:wiff r.equire future investigation~ Add1tionaln1echariisms such as.·•

·:alt~ratloris_i_n:posttrar:i~latlorial_n,odific~t.19ns:in':~AT niay'.n9t.play·:a,ro_1.e:fn ·:·.

...... - ...... ' . ' . . . ' ...... $Ubsequent alterations ·in Ca.MKll'activitiin :dopamine neuron$ did. nol.alterDAT': .

· ·. function_. Takeri tog·ether·, our.resu'lts dem:oristrate.that ~ctivity~deperident. - . . - . - ......

. .reguration 'ofDAT-expressiori' i·s. m:ediated_ byCaMKll s'ignali,ng: and m,ay play ari ..: .

. . . . . ' ...... ' . ' ...... ' . importarirole .. in. controlling d_opa~ine· signaling· _in the .prain:· _Fut:ure. stu_dies · . ··

...... "...... - . .: should exam~n~· the··role .pf CaMKH i.n .regulating·DAT.a.bundance in Vlvo. in ord~r .. ·

.. · . pathol9g.i_ca_l.cor1di.tions._. ·.

. ·_.122. : ·. VI; REFERENCES·:

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APPENDIX

Dopamine uptake and saturation. analysis Cells were washed with Ringer's solution and uptake was initiated by addition of 300 nM tritiated dopamine (Amersham, Piscataway, NJ). After 6 minutes, uptake was terminated by two washes of ice-cold Ringer's solution and radioactivity was extracted into 3% trichloroacetic acid. Non-specific uptake was determined in the presence of 10 µM GBR12909. For saturation analysis, uptake of six different concentrations of dopamine ranging from 0.05-12.15 µM was performed for two minutes on cultures maintained in conditioned media for six days. Only 20% of dopamine at different concentrations was radiolapeled. GraphPad Prism (V 4.0) was used to determine Vmax and Kr values of dopamine uptake. TTX was co­ incubated with dopamine during the uptake and 30mM choline chloride was used as control buffer for experiments in which KCI was used to determine dopamine uptake.

Efflux assay ,. Mesencephalic cultures were incub~ted with 800 nM tritiated dopamine for 15 minutes at 37°C. Following the incubation, cells were washed twice with warm Ringer's solution after which 200 µI fractions were collected to measure dopamine released at 5-minute intervals. For all three fractions, cells were incubated in the presence or absence of TTX. Unlabeled dopamine-stimulated efflux (concentrations ranging from 0.05-12.15 µM) was measured in the third fraction. In the final step, radioactivity remaining in the cells was extracted into 3% trichloroacetic acid. Dopamine released at each 5-minute interval was first calculated as a ratio of total radioactivity. Dopamine release is represented as the percentage of efflux in the presence of TTX (ratio of dopamine released in the third fraction to that in the second fraction) compared to vehicle treated cultures.