The Journal of Physiology Neuroscience

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1 J Physiol 594. (26) pp The Journl of Physiology Neuroscience Aerrnt regultion of synchronous network ctivity y the ttention-deficit/hyperctivity disorder-ssocited humn dopmine D4 receptor vrint D4.7 in the prefrontl cortex Ping Zhong,2,WenhuLiu,3 nd Zhen Yn,2 Deprtment of Physiology & Biophysics, Stte University of New York t Bufflo, School of Medicine nd Biomedicl Sciences, Bufflo, NY, USA 2 VA Western New York Helthcre System, Bufflo, NY, USA 3 School of Lifescience, Zhoqing University, Zhoqing, Chin Key points The vrint hs een linked to ttention-deficit/hyperctivity disorder (ADHD); however, the underlying mechnism is unknown. We found tht ctivtion of induced over-suppression of glutmtergic excittory network ursts nd under-suppression of GABAergic inhiitory network ursts in the prefrontl cortex (PFC) circuitry. Methylphenidte, psychostimulnt drug used to tret ADHD, normlized the effects of on synchronous network ursts in PFC pyrmidl neurons. The findings of the present study suggest tht the errnt regultion of PFC synchronous network ctivity y my underlie its involvement in ADHD. Astrct A unique feture of the humn D 4 receptor (hd 4 R) gene is the existence of lrge numer of polymorphisms in exon 3 coding for the third intrcellulr loop, which consists of vrile numer of tndem repets. The hd 4 R vrints with long repets hve een linked to ttention-deficit/hyperctivity disorder (ADHD); however, the underlying mechnism is unknown. Emerging evidence suggests tht selective ttention is controlled y the rhythmic synchroniztion in the prefrontl cortex (PFC) nd its connected networks. In the present study, we exmined the role of hd 4 R vrints in regulting PFC synchronous network ctivity. D 4 R knockout mice with virl infection of or in the medil PFC were used. Whole-cell ptch-clmp recordings were performed to exmine the effects of ctivting hd4.x on the spontneous lrge scle correlted ctivity in PFC pyrmidl neurons. We found tht, compred to the norml four-repet vrint, the ADHD-linked vrint induces more suppression of glutmtergic excittory network ursts nd less suppression of GABAergic inhiitory network ursts in the PFC circuitry. Methylphenidte, psychostimulnt drug used to tret ADHD, normlized the effects of on synchronous network ursts in PFC pyrmidl neurons. These results revel the differentil effects of hd 4 R vrints on the integrted excitility of PFC circuits. It is suggested tht the errnt regultion of PFC network ctivity y my underlie its involvement in ADHD. The methylphenidte-induced normliztion of synptic circuitry regultion my contriute to its effectiveness in ADHD tretment. C 25 The Authors. The Journl of Physiology C 25 The Physiologicl Society DOI:.3/JP2737

2 36 P. Zhong nd others J Physiol 594. (Resumitted 7 July 25; ccepted fter revision 29 Octoer 25; first pulished online 6 Novemer 25) Corresponding uthor Z. Yn: Deprtment of Physiology nd Biophysics, Stte University of New York t Bufflo, 24 Shermn Hll, Bufflo, NY 424, USA. Emil: zhenyn@ufflo.edu Arevitions CSF, rtificil cererospinl fluid; ADHD, ttention-deficit/hyperctivity disorder; AMPAR, α-mino-3-hydroxy-5-methyl-4-isoxzolepropionic cid receptor; APV, DL-2-mino-5-phosphonovleric cid; DNQX, 6,7-dinitroquinoxline-2,3-dione; GABA A R, GABA A receptor; GFP, green fluorescent protein; hd 4 R, humn dopmine D 4 receptor; NMDAR, NMDA receptor; PFC, prefrontl cortex; PSD, postsynptic density. Introduction Attention-deficit/hyperctivity disorder (ADHD) is prevlent nd deilitting disorder dignosed on the sis of overctivity, inttention nd impulsivity. A met-nlysis suggests tht ADHD is ssocited with significnt weknesses in severl key executive functions, including response inhiition, vigilnce, working memory nd plnning (Willcutt et l. 25), ll of which re controlled y the prefrontl cortex (PFC). Although the pthophysiologicl mechnisms underlying ADHD remin to e clrified, genetic studies hve linked the dopmine D 4 receptor, which is highly enriched in the PFC (Mrzljket l. 996; Wedzony et l. 2), to ADHD (Bo et l. 25; Grdy et l. 23; Swnson et l. 27). ThegeneencodinghumnD 4 receptor (hd 4 R) contins lrge numer of polymorphisms in the coding region for the third intrcellulr loop, which consists of vrile numer (2 ) of 48 p tndem repets (Vn Tol et l. 992). The three most common vrints contin two, four nd seven repets (D4.2, D4.4, D4.7), with glol frequency of 8%, 64% nd 2%, respectively (Chng et l. 996). These proline-rich repets provide the inding sites for other proteins contining the SH3 domin. So fr, the moleculr mechnism nd functionl significnce of the remrkle vrile numer tndem repet polymorphism of hd 4 R is poorly understood. Importntly, severl studies hve reported significnt ssocition of ADHD with the D4R gene seven-repet llele (LHoste et l. 996; Rowe et l. 998; Smlley et l. 998; Frone et l. 999; El-Fddgh et l. 24; Li et l. 26). However, the underlying mechnism is unknown. Emerging evidence suggests tht the control of ttention rises from interctions etween widespred corticl nd sucorticl networks regulted vi their rhythmic synchroniztion (Buschmn & Miller, 27; Gregoriou et l. 29. Womelsdorf et l. 2; Miller nd Buschmn, 23). The synchronized network ctivity is n importnt intrinsic feture of corticl circuits (Opitz et l. 22; Buzsáki & Drguhn, 24) nd plys significnt role in controlling rin functions (Vrel et l. 2; von der Mlsurg et l. 2). Key fctors involved in synchronous network ctivity include the strength of glutmtergic input from the recurrent excittory connections etween pyrmidl neurons (Stoop et l. 23; Pnuccio et l. 29) nd the strength of GABAergic input from interneurons (Korn et l. 987; Tru et l. 989). Excittory nd inhiitory synptic conductnces my e exquisitely lnced in norml neuronl ctivity (Borg-Grhm et l. 998; Hider et l. 26), which is criticl for complex ehviours (Kluserger & Somogyi 28; Womelsdorf et l. 2; Yizhr et l. 2). An understnding of how the synchronized corticl network ctivity is regulted in norml nd ADHD conditions is lcking. In the present study, we exmined the role of hd 4 R vrints in regulting the integrted excitility of corticl circuits y mesuring their impct on spontneous lrge scle correlted ctivity in PFC pyrmidl neurons. We show tht the synchronized network ctivity in the PFC is more prominently suppressed y. These results provide potentil pthophysiologicl sis for the frontl hypoctivity found in the dignosis of ADHD (Dickstein et l. 26; Fernández et l. 29). Methods The genertion of hd4.x viruses The HA-tgged humn D4.4 nd D4.7 plsmids (Rondou et l. 28) were kindly provided y Dr Kthleen Vn Crenenroeck t the Lortory of Eukryotic Gene Expression nd Signl Trnsduction (LEGEST), BELGIUM. The genertion of Sindis virus used the sme procedure s tht descried previously (Liu et l. 2). Briefly, the cdnas encoding green fluorescent protein (GFP) or GFP-hD4.x were sucloned to psinrep5 vector (Invitrogen, Crlsd, CA, USA) in ccordnce with the mnufcturer s instructions. Recominnt GFP-pSinRep5 or GFP-hD4.x-pSinRep5 ws linerized with NotI. The DH26S plsmid ws linerized using XhoI. The linerized templtes were trnscried in vitro using the mmessge Mchine SP6 kit (Amion, Austin, TX, USA) nd the RNAs were electroported into y hmster kidney cells. The extrcellulr medium contining the recominnt viruses ws hrvested fter h. The medium ws concentrted on discontinuous sucrose grdient (55% nd 2% sucrose) using ultrcentrifugtion (6, g for 9 min t 4 C). C 25 The Authors. The Journl of Physiology C 25 The Physiologicl Society

3 J Physiol 594. Aerrnt regultion of PFC synchronous network ctivity 37 In vivo delivery of hd4.x viruses All niml experiments were performed with the pprovl of the Institutionl Animl Cre nd Use Committee of the Stte University of New York t Bufflo. D 4 Rknockout mice (Ruinstein et l., 997, 2) were originlly provided y Dr Dvid Grndy t Oregon Helth & Science University nd lter red nd mintined in our own lortory for the experiments. In vivo virus-sed gene delivery into medil PFC ws performed s descried previously (Duffney et l. 25). In rief, D 4 Rknockout mice (2 3 months old) were nesthetized y n I.P. injection of pentoritl (5 mg kg ) nd plced on stereotxic pprtus (Dvid Kopf Instruments, Tujung, CA, USA). The Sindis virl suspension (.5 μl) ws injected with Hmilton syringe (needle guge 3) t speed of.2 μl min,ndtheneedlewskeptin plce for n dditionl 5 min. The virus ws delivered ilterlly to the medil prelimic re of mice using the co-ordintes: 2. mm nterior to regm nd.5 mm lterl. The needle ws extended to depth of.3 mm elow the tissue surfce, nd the virus ws injected to ech side. Animls were llowed to recover for h fter virl injection, nd nlgesi ws provided postopertively during the recovery. Slice preprtion Mice (2 3 months old) were deeply nesthetized using isoflurne nd killed y decpittion. The rin ws removed quickly nd plced into the ice-cold sucrose solution contining (in mm): 234 sucrose, 4 MgSO 4, 2.5 KCl, NH 2 PO 4,.CCl 2,5Hepesndglucose (ph 7.4, 3 mosm). Coronl slices (35 μm) were cut with virtome (VPS; Leic Microsystems, Wetzlr, Germny). Slices were incuted t C for h in rtificil cererospinl fluid (CSF) (in mm: 3 NCl, 26 NHCO 3,CCl 2,2MgCl 2, 3 KCl, glucose nd.25 NH 2 PO 4 ) uled with 95% O 2 nd 5% CO 2. Then slices were kept t room temperture (22 24 C) in modified CSF solution (in mm: 2CCl 2,.MgCl 2 nd 3.5 KCl) (Ardi & Mccferri, 24; Juuri et l. 2; Ziurkus et l. 23) for 2 h efore recordings. Electrophysiologicl recordings The whole-cell voltge-clmp technique (Zhong & Yn, 24, 24; Yuen et l. 22) ws used to record the neuronl network currents. Slices were plced in recording chmer nd superfused with the modified CSF solution contining low (. mm) MgCl 2, high (2 mm) CCl 2 nd slightly higher (3.5 mm) KCl to keep the slices in n ctive stte. Neurons infected with GFP-conjugted or Sindis virus in lyer V of the medil PFC were visulized with fluorescence microscope nd selected for recordings. A Multuclmp 7A mplifier (Moleculr Devices, Sunnyvle, CA, USA) nd Digidt322A (Moleculr Devices) were used. Ptch electrodes were filled with the internl solution contining (in mm): 3 Cs-methnesulphonte, 4 NCl, Hepes,.5 EGTA, 2 QX-34, 2 phosphocretine, 4 ATP nd.5 GTP (ph , mosm). Tight sels (2 G ) were otined y pplying negtive pressure. The memrne ws disrupted with dditionl suction nd the whole-cell configurtion ws otined. Neurons were voltge clmped t 7 mv or 2 mv throughout the recordings. Series resistnces were 2 M nd were not compensted. Sttisticl nlysis Dt cquisition ws crried out using Clmpex, version 9 (Moleculr Devices). The smpling rte ws 5 khz nd the low-pss filtering frequency ws khz. Ech neuron ws continuously recorded for min, nd the verged mplitude nd frequency of network ursts within 2 min timefrme t the stle stte efore nd fter drug ppliction were clculted. Dt nlyses were performed with Clmpfit (Moleculr Devices), Mini- Anlysis (Synptosoft Inc., Fort Lee, NJ, USA) nd KleidGrph (Synergy Softwre, Reding, PA, USA) softwre. All dt re expressed s the men ± SEM. Mnn Whitney U tests were used to determine the significnce of the effects of vrious gents on network ursts. Experiments with two niml groups were nlysed sttisticlly using unpired Student s t tests. Experiments with more thn two niml groups were sujected to one-wy ANOVA, followed y post hoc Bonferroni tests. Results The synchronous network ctivity in the PFC Neurons in the PFC re not highly ordered like those in hippocmpus, nd the loss of the integrity of inhiitory nd excittory synptic trnsmissions in in vitro preprtions hs often led to difficulty in uncovering synchronous network ctivity in PFC slices. To elevte the neuronl ctivity in slices to thein vivo level, we performed whole-cell ptch clmp recordings in the modified CSF with lower Mg 2+ concentrtion (. mm) ndhigher C 2+ concentrtion (2 mm), s estlished previously (Ardi & Mccferri, 24; Juuri et l. 2; Ziurkus et l. 23). In corticl slices (35 μm), the slowly rhythmic synchronous ursting of lrger numers of neurons in the network gve rise to the spontneous lrge scle correlted ctivity. As shownin Fig. A, spontneous network ursts occurred on ckground of synchronous unitry postsynptic currents in lyer V PFC pyrmidl neurons (held C 25 The Authors. The Journl of Physiology C 25 The Physiologicl Society

4 38 P. Zhong nd others J Physiol 594. t 7 mv). The network ursting-ssocited inwrd current hd men ± SEM mplitude of 8 ± 72 pa (n = 8) nd men ± SEMintervlof8.3± 2. s (n = 8). To exmine the synptic origin of synchronous network ctivity, we pplied α-mino-3-hydroxy-5- methyl-4-isoxzolepropionic cid receptor (AMPAR), NMDA receptor (NMDAR) nd GABA A receptor (GABA A R) ntgonists, respectively. As shown in Fig. A, the lrge-scle network current ws locked y AMPAR ntgonist 6,7-dinitroquinoxline-2,3-dione (DNQX) (5 μm) (n = 5) or NMDAR ntgonist DL-2-mino-5-phosphonovleric cid (APV) (5 μm) (n = 6). Inhiition of GABA A receptors with icuculline (2 μm) did not result in the lockde of synchronous network ctivity ut led to the modultion of urst mplitude (4.2 ± 5.3% increse, n = 5, P <.5) nd frequency (43 ± 4% decrese, n = 5, P <.). A 7mV PFC B 2mV PFC I inh. DNQX 4pA 4pA s DNQX pa I exc. pa s APV 4pA APV pa BIC 4pA BIC pa 4pA pa C 7mV PFC (μm) D 7 mv Stritum 2pA 2min pa Figure. The synchronous network ctivity in the PFC is dependent on AMPAR- nd NMDAR-medited glutmtergic trnsmission Network ursts recorded in PFC pyrmidl neurons held t 7 mv (A) or 2 mv (B) in the sence or presence of DNQX (AMPAR ntgonist), APV (NMDAR ntgonist) or icuculline (GABAAR ntgonist). Enlrged view of the network ursting-ssocited currents. At 7 mv, the network urst is mde of n inwrd excittory current. At 2 mv, the network urst is composed of inwrd excittory nd outwrd inhiitory currents. C nd D, synptic currents recorded in PFC pyrmidl neurons (held t 7 mv) from thin slices ( µm) or medium spiny neurons (held t 7 mv) from stritl slices. C 25 The Authors. The Journl of Physiology C 25 The Physiologicl Society

5 J Physiol 594. Aerrnt regultion of PFC synchronous network ctivity 39 To revel inhiitory synptic currents in the mesurement of network ctivity, we held the memrne potentil t more depolrized level ( 2 mv). Under this condition, the rhythmic synchronous network ursting-ssocited current ws composed of two prts: n inhiitory outwrd current (mplitude: 28 ± 7 pa, n = 7) nd n excittory inwrd current (mplitude: 37 ± 2 pa, n = 7) (Fig. B). The frequency of synchronous network ctivity ws not influenced y holding potentils (intervl: 9.4 ± 2. s, n = 2). Appliction of DNQX (5 μm) or APV (5 μm) locked oth excittory inwrd network current nd inhiitory outwrd network current (n = 6) (Fig. B). Appliction of icuculline locked the inhiitory outwrd network current, nd drmticlly ltered the excittory inwrd network current (mplitude: 6 ± 4% increse; A GFP Figure 2. Activtion of induces stronger suppression of PFC synchronous network ctivity thn A, confocl imges of PFC slices from D 4 R knockout mice with stereotxicl injection of GFP or GFP-conjugted or Sindis virus. Network urst mplitudes (B) nd frequencies (C) show the effect of th pplied D 4 R gonist PD6877 (2 µm) in PFC pyrmidl neurons (held t 7 mv) of D 4 R knockout mice with or without virl infection of humn D4.4 or D4.7 vrints. D, representtive network ursts in the sence or presence of PD6877 in PFC pyrmidl neurons from different groups. E, percentge reduction of the mplitude nd frequency of network ursts y PD6877 in different groups. P <., ANOVA, vs.. F, mplitude or frequency of seline network ursting currents (held t 7 mv) in different groups. B Norm. Amp. of Network Curr. (@ 7 mv) D +KO +KO D4KO.2.8 PD KO.2 +KO D4KO Time (min) (control) (PD6877) 4pA 3s C E Norm. Freq. of Network Curr. (@ 7 mv) Reduction y PD (%) (@ 7 mv) F Amplitude (na) (@ 7 mv).2.8 PD KO +KO D4KO Time (min) Amp Frequency (Hz) (@ 7 mv) D4KO Freq D4KO C 25 The Authors. The Journl of Physiology C 25 The Physiologicl Society

6 4 P. Zhong nd others J Physiol 594. frequency: 46 ± 5% decrese, n = 8). This suggests tht glutmtergic trnsmission, which is medited y AMPA nd NMDA receptors, is required for synchronous network ctivity, wheres GABAergic trnsmission modultes oscilltory network ctivity in the PFC. Thin PFC slices (2 μm), which hd severe loss of neuronl circuit integrity, filed to produce the lrge scle rhythmic synchronous network ursts (n = 8) (Fig. C). No gint network ursts were oserved in medium spiny neurons from stritl slices (n = 9) (Fig. D). This suggests tht the synchronized network ctivity requires intct neuronl connections in corticl circuits nd occurs in rin region-specific mnner. The effect of nd on synchronous network ctivity in PFC neurons To clrify the role of humn D 4 R vrints in the corticl network, we exmined the effects of the specific D 4 R gonist PD6877 on spontneous network ctivity in the PFC of D 4 R knockout mice with virl infection of or. The rodent D 4 R only contins two repets, nd wild-type mice were lso used for comprison. To void high overexpression of the hd4.x Sindis virus, mice were killed within 2 dys fter virl delivery. PFC neurons expressing the GFP-conjugted or exhiited norml morphologicl structures (Fig. 2A). In -expressing PFC pyrmidl neurons (held t A PD6877 B PD6877 Norm. Amp. of Network Curr. (@ 2 mv) C +KO +KO E Amplitude (pa) (@ 2 mv) (control) +KO +KO I excittory I excittory (inwrd) Time (min) (PD6877) pa 2s I inhiitory Norm. Amp. of Network Curr. (@ 2 mv) pa 2 s D KO +KO I inhiitory (outwrd).2 +KO +KO Time (min) Reduction of Amp. y PD (%) (@ 2 mv) Frequency (Hz) (@ 2 mv) +KO +KO I exc. I inh Figure 3. Activtion of induces n over-suppression of excittory network ctivity nd n under-suppression of inhiitory network ctivity in the PFC Excittory or inhiitory network urst mplitudes (A) nd frequencies (B) showing the effect of PD6877 (2 µm) inpfc pyrmidl neurons (held t 2 mv) of D 4 R knockout mice with virl infection of humn D4.4 or D4.7 vrints. C, representtive excittory nd inhiitory network ursts in the sence or presence of PD6877 in PFC pyrmidl neurons from different groups. D, percentge reduction of the mplitude of excittory or inhiitory network ursts y PD6877 in different groups. P <., ANOVA. E, mplitude or frequency of seline excittory nd inhiitory network ursts (held t 2 mv) in different groups. C 25 The Authors. The Journl of Physiology C 25 The Physiologicl Society

7 J Physiol 594. Aerrnt regultion of PFC synchronous network ctivity 4 7 mv), PD6877 (2 μm) decresed the mplitude of network ursts y 4.7 ± 2.5% (n = 7), which ws significntly (P <.) lrger thn the reducing effect of PD6877 in -expressing PFC pyrmidl neurons (2.8 ±.4%, n = 7) (Fig. 2B, D nd E) orin PFC pyrmidl neurons from wild-type mice tht endogenously express D4.2 (2.4 ±.5%, n = 6) (Fig. 2D nd E). PD6877 reduced the frequency of network ursts to similr level in D4.x-expressing neurons (: 3.9 ± 2.4%, n = 7; : 3.3 ± 2.%, n = 7) (Fig. 2C, D nd E) nd neurons from wild-type mice (3.7 ± 2.5%, n = 6) (Fig. 2D nd E). PD6877 hd no effect on network ctivity in D 4 R knockout mice (mp: 3.7 ±.7%, freq: 3.2 ±.6%, n = 5) (Fig. 2D nd E), confirming the medition y D 4 Rs. The seline network ursts (held t 7 mv) were similr mong different groups (wild-type: 76 ± 59 pa,.54 ±.5 Hz; D 4 KO: 88 ± 8 pa,.6 ±.6 Hz; : 67 ± 45 pa,.52 ±.4 Hz; : 7 ± 52 pa,.5 ±.4 Hz) (Fig. 2F). This suggests tht hd 4 R ctivtion results in sustined decrese of urst occurrence without disrupting the ptterned nture of ctivity, nd ctivtion of the ADHD-linked vrint results in more prominent hypoexcitility of the PFC network. The recordings descried ove were performed t room temperture (23 C). We lso exmined the network ursting current nd its regultion y D 4 Rctivtiont more physiologicl temperture (32 C). We found tht the rise in temperture incresed the network current mplitude nd frequency (mplitude: 28.9 ± 2.2%, frequency: 23.6 ± 2.7%, n = 5), lthough the reducing effect of PD6877 on network ursts ws similr t different tempertures (23 C, mplitude: 2 ±.5%, frequency: 3.7 ± 2.5%, n = 6; 32 C, mplitude: 2.6 ±.9%, frequency: 28.8 ± 2.6%, n = 5). Next, we compred the effect of hd4.x on excittory nd inhiitory network ursts in PFC pyrmidl neurons held t depolrizedlevel ( 2 mv). In -expressing neurons, PD6877 (2 μm) decresed the excittory inwrd network current mplitude y 24. ±.9% (n = 8) (Fig. 3A, C nd D), wheres it hd no effect on the inhiitory outwrd network current mplitude (.8 ±.2%, n = 8) (Fig. 3B to D). By contrst, in -expressing neurons, PD6877 (2 μm) hd no effect on the excittory inwrd network current mplitude (2.8 ±.4%, n = 7) (Fig. 3A, C nd D) ut decresed the inhiitory outwrd network current mplitude y 22.8 ±.7% (n = 7) (Fig. 3B to D), which ws similr to the effect of PD6877 in the PFC pyrmidl neurons from wild-type mice (inwrd-mplitude:.8 ±.6%, outwrd-mplitude: 2.7 ± 2.% reduction) (Fig. 3C nd D). The reducing effects of PD6877 on network urst frequency were similr in D4.x-expressing neurons (: 32.6 ± 3.5%, n = 8; : 3.3 ± 2.8%, n = 7) nd those from wild-type mice (3. ± 2.9%, n = 5). The seline network ursts (held t 2 mv) were similr mong different groups (wild-type, inwrd: 289 ± 9 pa, outwrd: 23 ± 5 pa,.55 ±.4 Hz;, inwrd: 272 ± 3 pa, outwrd: 25 ± 6 pa,.53 ±.4 Hz;, inwrd: 3 ± 2 pa, outwrd: 93 ± 7 pa,.5 ±.4 Hz) (Fig. 3E). This suggests tht, compred to the norml vrint, the ADHD-linked vrint induces more suppression of glutmtergic excittory trnsmission nd less suppression of GABAergic inhiitory trnsmission in the synptic circuitries, which my collectively result in PFC network hypoctivity. A control L74587 B +KO +KO 4pA.5min Chnge y L745 (%) (@ 7mV) Amplitude +KO +KO Frequency Figure 4. Appliction of D4R ntgonist does not chnge network ursting currents A, representtive network ursts ( 7 mv) in the sence or presence of the D 4 R ntgonist L (2 µm) in PFC pyrmidl neurons from wild-type mouse or D 4 R knockout mice with virl infection of humn D4.4 or D4.7 vrints. B, effect of L on the mplitude nd frequency of network ursts. C 25 The Authors. The Journl of Physiology C 25 The Physiologicl Society

8 42 P. Zhong nd others J Physiol 594. To determine whether the virlly expressed hd 4 R vrints re ctivted y spontneous dopmine relese, we exmined the effect of D 4 Rntgonistonnetwork ctivity. As shown in Fig. 4A nd B,lockingD 4 Rwiththe ntgonist L (2 μm) did not significntly lter network ursting currents in hd4.x-expressing neurons (wild-type, mplitude: 7.6 ±.8%, frequency: 8.2 ± 2.4%, n = 6;, mplitude: 2. ±.3%, frequency: 5. ±.2%, n = 5;, mplitude: 2.5 ±.4%, frequency: 4.2 ±.7%, n = 5, P >.5, ANOVA), suggesting tht hd 4 R vrints re not constitutively ctive. The normliztion of regultion of PFC network ctivity y methylphenidte (MPH) To determine whether the errnt regultion of PFC network ctivity y is relted to its role in ADHD, we exmined whether MPH, n effective gent for ADHD tretment, could normlize the effects of on synchronous network ursts in PFC pyrmidl neurons. MPH (.5 mg kg ) or sline control ws I.P. injectedto -infected D 4 R knockout mice nd, h lter, nimls were killed efore slicing (Cheng et l. 24). A Norm. Amp. of Network Curr. (@ 7 mv) D Norm. Amp. of Network Curr. (@ 2 mv) F MPH -sline PD Time (min) (control) -MPH -sline Time (min) PD6877 I excittory (inwrd prt) -sline -MPH B -MPH -sline (PD6877) pa 4s (control) E Norm. Amp. of Network Curr. (@ 2 mv) pa 2 s (PD6877) G Reduc. of Amp. y PD (%) (@ 2mV) 4pA 3s 2 3 C Reduction y PD (%) PD I excittory sline -MPH Amp I inhiitory (outwrd prt) -MPH -sline -MPH -sline I inhiitory Figure 5. The regultion of PFC network ctivity is normlized y MPH injection A, D nd E, network urst mplitudes showing the effect of PD6877 (2 µm) in PFC pyrmidl neurons of -infected, D 4 R knockout mice injected with MPH or sline control. B nd F, representtive network ursts in the sence or presence of PD6877 in -expressing neurons with MPH or sline injection. C nd G, percentge reduction of the mplitude of network ursts y ctivtion in PFC neurons from MPH- or sline-injected mice. Cell memrnes were held t 7 mv (A to C) or 2 mv (D to G). P <., t test, -MPH vs. -sline. Freq C 25 The Authors. The Journl of Physiology C 25 The Physiologicl Society

9 J Physiol 594. Aerrnt regultion of PFC synchronous network ctivity 43 As shown in Fig. 5A to C, in -expressing PFC pyrmidl neurons (held t 7 mv) from MPH-injected nimls, PD6877 (2 μm) induced significntly smller reducing effect on network urst mplitude thn in neurons from sline-injected mice (MPH: 22.3 ±.7%, n = 6; sline: 38.4 ± 2.2%, n = 4, P <.). Moreover, in -expressing PFC pyrmidl neurons (held t 2 mv) from MPH-injected nimls (Fig. 5D to G), PD6877 hd no effect on the excittory inwrd network current mplitude (MPH: 2.2 ±.2%, n = 5; sline: 22.3 ± 2.%, n = 4), wheres it decresed the inhiitory outwrd network current mplitude (MPH: 22. ±.7%, n = 5; sline:.6 ±.%, n = 4). The reducing effect of PD6877 on network current frequency ws not ffected y MPH ( 7 mv, MPH: 3.7 ± 2.6%, n = 6; sline: 32.7 ± 3.2%, n = 4; 2 mv, MPH: 3. ± 2.9%, n = 5; sline: 29.4 ± 2.7%, n = 4). These dt suggest tht in vivo dministrtion of MPH switches the regultory effects of on PFC network ctivity to levels similr to those of (Figs 2 nd 3). In vitro ppliction of MPH (2 μm) decresed network urst mplitude (26.7 ± 2.9%, n = 6) ut incresed network urst frequency (3.3 ± 4.%, n = 6) in -expressing neurons. We further exmined the impct of MPH in -expressing neurons. Compred to sline-injected mice, MPH injection (I.P.) did not lter the effect of PD6877 on network ctivity in D4.4-expressing PFC pyrmidl neurons recorded t 7 mv (MPH, mplitude: 9.8 ±.5% reduction, frequency: 29.3 ± 2.3% reduction, n = 6; sline, mplitude: 2.9 ±.6% reduction, frequency: 3.9 ± 2.4% reduction, n = 6, Fig. 6A nd C) or t 2 mv (MPH, inwrd-mplitude: 2.8 ±.7%, outwrd-mplitude: 22.9 ±.4% reduction, frequency, 34.5 ± 3.% reduction, n = 6; sline, inwrd-mplitude: 3.7 ±.5%, outwrd-mplitude: 2. ±.7% reduction, frequency, 33.3 ± 2.3%, n = 5) (Fig. 6B nd D). Discussion A unique primte-specific feture of D 4 R is the dditionl 2 proline-rich repets locted in the third intrcellulr loop (Wng et l. 24), which llows more complex simultneous interctions with other proteins contining thesh3domin.thehd 4 R vrints with long repets hve een linked to deficiencies in executive control processes in ADHD (LHoste et l. 996; Swnson et l. 998; Tlkowski et l. 28; Gizer et l. 29; Brnes et l. 2). To understnd the potentil mechnism, we hve exmined their impct on synchronized network ursts A -MPH -sline control PD6877 4pA.5min B Chnge y PD (%) (@ 7mV) Amp -sline -MPH Freq C -MPH -sline control pa 2s PD6877 pa 2 s D Chnges y PD (%) (@ 2mV) I exc. -sline -MPH I inh. Figure 6. The regultion of PFC network ctivity is not ltered y MPH injection A nd C, representtive network ursts in the sence or presence of PD6877 in -expressing neurons with MPH or sline injection. B nd D, percentge chnges of the mplitude or frequency of network ursts y ctivtion in PFC neurons from MPH- or sline-injected mice. Cell memrnes were held t 7 mv (A nd B) or 2 mv (C nd D). C 25 The Authors. The Journl of Physiology C 25 The Physiologicl Society

10 44 P. Zhong nd others J Physiol 594. originting from the lrge scle correlted ctivity of interconnected neurons, which controls PFC-medited cognitive function, such s ttention (Buschmn & Miller, 27; Miller & Buschmn, 23). Synchronized network ctivity in cortex hs een suggested to co-opertively support temporl representtion nd long-term consolidtion of informtion (Buzsáki & Drguhn, 24). Locl nd long-rnge rhythmic synchroniztion determines neuronl interctions nd selective ttention (Womelsdorf & Fries, 27; Womelsdorf et l. 27). Spontneous synchrony in neurl network is expected to depend on the finely tuned interply of excittory nd inhiitory neuronl popultions, which gives rise to the precisely timed nd dynmiclly lnced excittory nd inhiitory conductnces (Tru et l. 989). The dt otined in the present study (Fig. ) indicte tht the synchronous network ctivity requires oth AMPA nd NMDA receptors, wheres GABAergic trnsmission minly modultes oscilltory network ctivity. Anorml synchrony of spontneous network ctivity hs een ssocited with vrious neurologicl nd psychitric disorders, rnging from epileptic seizures (Steride, 23; Grci Dominguez et l. 25) nd Prkinson s disese (Borud et l. 25; Uhlhs & Singer, 26) to schizophreni (Spencer et l. 23; Uhlhs & Singer, 2) nd utism (Wilson et l. 27; Yizhr et l. 2). An imlnce fvouring excittion my underlie the excessive synchrony in epilepsy (Dichter & Ayl, 987). The dt otined in the present study (Figs 2 nd 3) indicte tht ctivtion of reduced excittory nd inhiitory network ctivity, similr to the effects of D 4 receptor ctivtion in wild-type mice. However, ctivtion of the ADHD-linked induces more suppression of the excittory network ursts nd less suppression of the inhiitory network ursts in the PFC circuitry, suggesting tht it shifts the neuronl excittion inhiition lnce towrds inhiition in postsynptic neurons, which my explin the significnt frontl hypoctivity detected in ADHD ptients (Dickstein et l. 26; Fernández et l. 29). Little is known out the mechnisms underlying the functionl differences of hd 4 R vrints. Using trnsfected cell lines, no mjor discrepncies in phrmcologicl profiles or the ilities to lock camp production hve een found mong hd4.x isoforms (Asghri et l. 995; Jovnovic et l. 999). Using knock-in mice tht crry in the third intrcellulr loop of D 4 R, it ws found tht D4.7 does not form functionl heteromers with the dopmine D2S receptor, which is ssumed to ffect presynptic dopminergic control of corticostritl glutmte relese (Gonzlez et l. 22). Our previous studies in rodents hve found tht ctivtion of D 4 receptors in PFC pyrmidl neurons produces significnt reduction of NMDAR-medited currents (Wng et l., 23, 26) nd n ctivity-dependent, homeosttic regultion of AMPAR-medited trnsmission (Yuen et l. 2; Yuen & Yn, 2) vi mechnisms dependent on CMKII nd receptor chnnel trfficking. In ddition, rodent D 4 R ctivtion in PFC pyrmidl neurons leds to significnt decrese of GABA A R currents (Wng et l. 22) nd GABA A R memrne trfficking vi n ctin/cofilin/myosin-dependent mechnism (Grzine et l. 29). These effects re similr to those of (norml vrint) on excittory nd inhiitory network ursts. The dditionl proline-rich repets on (ADHD-linked vrint) enle the inding of more proteins contining the SH3 domin, such s mny scffolding proteins in the postsynptic density (PSD). The inding of dopmine to my chnge the receptor conformtion, disrupting the inding of to these SH3-contining PSD proteins. Thus, the stronger suppression of glutmtergic trnsmission-medited excittory network ursts y ctivtion my e ttriutle to the lrger inhiitory effect of on the memrne trfficking or mintennce of AMPA nd NMDA receptors t PSD. Moreover, lost the regultion of GABA A R-medited inhiitory network ursts, which my e result of the ttenuted regultion of ctin dynmics nd the myosin-sed trnsport of GABA A Rs to the surfce. The detiled mechnisms wit further investigtion. To determine whether the errnt regultion of PFC network ctivity y is relted to its role in ADHD, we further exmined whether MPH, dopmine reuptke inhiitor pproved for ADHD tretment, could normlize the effects of on synchronous network ursts in PFC pyrmidl neurons. The dt otined in the present study (Fig. 5) indicte tht MPH reduced the effect of on excittory network ursts, nd restored the effect of on inhiitory network ursts, ringing it close to in the regultion of PFC synchronized network ctivity. The mechnisms underlying MPH-induced chnges in regultion of network ctivity wit clrifiction. In sum, the MPH-induced normliztion of synptic circuitry regultion could contriute to its effectiveness in ADHD tretment. References Ardi I & Mccferri G (24) Cell type-specific synptic dynmics of synchronized ursting in the juvenile CA3 rt hippocmpus. JNeurosci24, Asghri V, Snyl S, Buchwldt S, Pterson A, Jovnovic V & Vn Tol HH (995) Modultion of intrcellulr cyclic AMP levels y different humn dopmine D4 receptor vrints. JNeurochem65, Brnes JJ, Den AJ, Nndm LS, O Connell RG & Bellgrove MA (2) The moleculr genetics of executive function: role of monomine system genes. Biol Psychitry 69, e C 25 The Authors. The Journl of Physiology C 25 The Physiologicl Society

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13 J Physiol 594. Aerrnt regultion of PFC synchronous network ctivity 47 Additionl informtion Competing interests The uthors declre tht they hve no competing interests. Author contriutions ZY nd PZ conceived nd designed the experiments. PZ nd WL collected, nlysed nd interpreted the dt. ZY drfted the rticle nd revised it criticlly for importnt intellectul content. All uthors hve pproved the finl version of the mnuscript nd gree to e ccountle for ll spects of the work. All persons designted s uthors qulify for uthorship, nd ll those who qulify for uthorship re listed. Funding This work ws supported y NIH grnt DA3768 nd VA merit wrd IBX633 to ZY, NSFC grnt (32724) nd Gungdong Nturl Science Foundtion (S23662) grnt to WL. Acknowledgements We would like to thnk Xioqing Chen for her excellent technicl support. C 25 The Authors. The Journl of Physiology C 25 The Physiologicl Society