Hemispheric Lateralization of Pain Processing by Amygdala Neurons

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1 Hemipheric Lateralization of Pain Proceing by Amygdala Neuron Guangchen Ji and Volker Neugebauer J Neurophyiol 12:2-226, 29. Firt publihed 22 July 29; doi:1.12/jn You might find thi additional info ueful... Supplemental material for thi article can be found at: Thi article cite 8 article, 28 of which can be acceed free at: Thi article ha been cited by other HighWire hoted article Role of Extracellular Signal-Regulated Kinae in Synaptic Tranmiion and Platicity of a Nociceptive Input on Capular Central Amygdaloid Neuron in Normal and Acid-Induced Mucle Pain Mice Sin-Jhong Cheng, Chien-Chang Chen, Hiu-Wen Yang, Ya-Ting Chang, Shin-Wen Bai, Chih-Cheng Chen, Chen-Tung Yen and Ming-Yuan Min J. Neuroci., February, 9 211; 1 (6): [Abtract] [Full Text] [PDF] Mitochondrial Reactive Oxygen Specie Are Activated by mglur through IP and Activate ERK and PKA to Increae Excitability of Amygdala Neuron and Pain Behavior Zhen Li, Guangchen Ji and Volker Neugebauer J. Neuroci., January, ; 1 (): [Abtract] [Full Text] [PDF] Reactive Oxygen Specie Are Involved in Group I mglur-mediated Facilitation of Nociceptive Proceing in Amygdala Neuron Guangchen Ji and Volker Neugebauer J Neurophyiol, July, 21; 1 (1): [Abtract] [Full Text] [PDF] Activation of Metabotropic Glutamate Receptor in the Amygdala Modulate Pain-Like Behavior Benedict J. Kolber, Michael C. Montana, Yarimar Carraquillo, Jian Xu, Stephen F. Heinemann, Loui J. Muglia and Robert W. Gereau IV J. Neuroci., June, 16 21; (2): [Abtract] [Full Text] [PDF] Cognitive Impairment in Pain through Amygdala-Driven Prefrontal Cortical Deactivation Guangchen Ji, Hao Sun, Yu Fu, Zhen Li, Miguel Pai-Vieira, Vaco Galhardo and Volker Neugebauer J. Neuroci., April, 1 21; (): 1-6. [Abtract] [Full Text] [PDF] Downloaded from jn.phyiology.org on March 9, 211 Updated information and ervice including high reolution figure, can be found at: Additional material and information about Journal of Neurophyiology can be found at: Thi infomation i current a of March 9, 211. Journal of Neurophyiology publihe original article on the function of the nervou ytem. It i publihed 12 time a year (monthly) by the American Phyiological Society, 96 Rockville Pike, Betheda MD Copyright 29 by the American Phyiological Society. ISSN: 22-77, ESSN: Viit our webite at

2 J Neurophyiol 12: 2 226, 29. Firt publihed July 22, 29; doi:1.12/jn Hemipheric Lateralization of Pain Proceing by Amygdala Neuron Guangchen Ji and Volker Neugebauer Department of Neurocience and Cell Biology, The Univerity of Texa Medical Branch, Galveton, Texa Submitted February 29; accepted in final form 21 July 29 Ji G, Neugebauer V. Hemipheric lateralization of pain proceing by amygdala neuron. J Neurophyiol 12: 2 226, 29. Firt publihed July 22, 29; doi:1.12/jn Recent biochemical and behavioral data ugget right-hemipheric lateralization of amygdala function in pain. Our previou electrophyiological tudie howed pain-related neuroplaticity in the latero-capular diviion of the central nucleu of the amygdala (CeLC) in the right brain hemiphere. Here we determined difference in the proceing of pain-related ignal in right veru left CeLC neuron. Individual CeLC neuron were recorded extracellularly before and after induction of an arthriti pain tate in anethetized rat. Brief innocuou and noxiou tet timuli were applied to peripheral tiue ipi- and contralateral to the recording ite. A monoarthriti wa induced in the ipi- or contralateral knee by intraarticular injection of kaolin and carrageenan. Under normal condition, CeLC neuron in the left amygdala had maller receptive field than thoe in the right, but the magnitude of background and evoked activity wa not ignificantly different. After arthriti induction, neuron in the right, but not left, CeLC developed increaed background activity and evoked repone, irrepective of the location of the arthriti (ipi- or contralateral to the recording ite). A protein kinae A (PKA) inhibitor decreaed the activity of right CeLC neuron after arthriti induction but had no effect in the left amygdala. Forkolin, however, increaed the activity of left and right CeLC neuron under normal condition. The reult how for the firt time laterality of pain-related electrophyiological activity change in individual amygdala neuron. Wherea both left and right amygdala neuron receive nociceptive input and can become enitized in principle, a yet unknown mechanim prevent PKA activation and pain-related change in the left amygdala. INTRODUCTION Hemipheric lateralization in emotional proceing i now well documented, but it remain to be determined if brain aymmetrie are baed on right hemipheric dominance, poitive veru negative valence, appetitive approach veru defenive withdrawal, or behavioral activation veru inhibition ytem (Atchley et al. 2; Davidon et al. 2; Demaree et al. ; Stephan et al. 27). Hemipheric pecialization for emotion involve not only the cerebral cortex but alo ubcortical area uch a the amygdala, a key player in emotion (Adolph 22; Davidon 22; Maren ; Pare et al. 2; Phelp and Ledoux ). Lateralization of amygdala function in emotional proceing ha been uggeted to depend on valence, gender, and other factor uch a level of awarene, actuality of experience, and temporal activation dynamic. Predominant activation or involvement of the right amygdala in averive behavior and negative emotion wa found in animal model (Baker and Kim 2; Coleman-Meche and McGaugh 199a,b; Coleman-Meche Addre for reprint requet and other correpondence: V. Neugebauer, Dept. of Neurocience and Cell Biology, The Univerity of Texa Medical Branch, 1 Univerity Blvd., Galveton, TX ( voneugeb@utmb.edu). et al. 1996; Lalumiere and McGaugh ) and in human (Angrilli et al. 1996; Canli et al. 1998; Funayama et al. 21; LaBar et al. 1998; Lee et al. 2; Yohimura et al. 28). There i alo evidence to ugget the preferential involvement of the right amygdala in emotional repone and emotional memory in men and of the left amygdala in women (ee Cahill 26 for review). The underlying principle of hemipheric lateralization of amygdala function in emotion remain unclear and need to be determined for different emotion and condition. Pain ha a trong emotional-affective component. The amygdala play a critical role in the emotional repone to pain and in pain modulation (Carraquillo and Gereau 27; Field 2; Gauriau and Bernard 2; Heinricher and McGaraughty 1999; Ikeda et al. 27; Neugebauer et al. 2, 26; Pederen et al. 27; Rhudy and Meagher 21). Our previou tudie focued on the right amygdala and howed central enitization and ynaptic platicity in neuron of the latero-capular diviion of the central nucleu (CeLC) in an animal model of arthriti pain (Bird et al. ; Fu and Neugebauer 28; Han et al. b; Ji and Neugebauer 27; Neugebauer and Li 2; Neugebauer et al. 2). The localized arthriti wa induced in the contralateral (left) knee only. It remain to be determined if neuronal change depend on the ide of injury (ipi- or contralateral knee) and if they occur in the left amygdala a well. Thi quetion i important becaue recent tudie howed that pain i aociated with biochemical change predominantly in the right amygdala. Pain-related ERK activation wa oberved in the right but not left CeLC, irrepective of the ide of a formalin injection in the hind paw (Carraquillo and Gereau 27, 28). Accordingly, blockade of ERK activation in the right but not left CeLC ignificantly decreaed formalininduced mechanical hyperenitivity in both the injected and the uninjured contralateral hind paw (Carraquillo and Gereau 27, 28). Evidence for pain-related lateralization i pare and controverial. Pychophyical tudie have uggeted a functional aymmetry toward the right hemiphere for pain perception baed on higher pain rating for timuli applied to the left ide, independently of handedne (Lugo et al. 22; Merkey and Waton 1979; Schiff and Gagliee 199). Other tudie found no uch difference in pain enation (Coghill et al. 21; Hall et al. 1981; Seltzer et al. 1992). More direct evidence for right hemipheric lateralization in pain come from a neuroimaging (PET) tudy that oberved right lateralized activation of everal brain area, regardle of the ide of peripheral timulation (Coghill et al. 21). Patient with chronic complex regional pain yndrome (CRPS) howed ign of gray matter atrophy in the right hemiphere but decreaed white matter connectivity in the left (Geha et al. 28). Right amygdala activation wa een Downloaded from jn.phyiology.org on March 9, /9 $8. Copyright 29 The American Phyiological Society 2

3 2 G. JI AND V. NEUGEBAUER in an fmri tudy in repone to painful viceral (gatric) timulation (Lu et al. 2). The preent tudy teted the hypothei that functional propertie (reponivene) of neuron in the right but not left CeLC are altered in a pain tate, uggeting right-hemipheric lateralization of pain proceing in a ubcortical brain area. We alo ought to determine if the lack of pain-related functional change in left CeLC neuron correlate with failure to activate PKA in thee neuron and if CeLC neuron in both hemiphere are capable of PKA-mediated enitization. Our previou tudie identified PKA activation a a critical mechanim of pain-related enitization and platicity in the amygdala (Bird et al. ; Fu et al. 28; Ji and Neugebauer 28). The preent electrophyiological tudy i the firt to how hemipheric difference in the reponivene of individual amygdala neuron to noxiou timuli in a pain model. The reult further ugget that PKA activation i neceary and ufficient for increaed reponivene of CeLC neuron but doe not occur in the left CeLC in the arthriti pain model. METHODS Adult male Sprague Dawley rat ( g) were houed in a temperature-controlled room and maintained on a 12-h day/night cycle. Water and food were available without retriction. All experimental procedure were approved by the Intitutional Animal Care and Ue Committee at the Univerity of Texa Medical Branch and conform to the guideline of the International Aociation for the Study of Pain and of the National Intitute of Health. Animal preparation and anetheia On the day of the electrophyiological experiment, the animal wa anethetized with pentobarbital odium ( mg/kg ip). A cannula wa inerted into the trachea for artificial repiration and to meaure end-tidal CO 2 level. A catheter wa placed in the jugular vein for continuou adminitration of anethetic and for fluid upport ( ml kg 1 h 1 lactated Ringer olution, adminitered intravenouly). Depth of anetheia wa aeed by teting the corneal blink, hindpaw withdrawal, and tail-pinch reflexe and by continuouly monitoring the end-tidal CO 2 level (kept at..2%), heart rate, electrocardiogram (ECG) and breathing pattern. Core body temperature wa maintained at 7 C by mean of a homeothermic blanket ytem. Animal were mounted in a tereotaxic frame, paralyzed with pancuronium (induction:.. mg iv; maintenance:. mg/h iv) and artificially ventilated (. ml; 6 troke/min). Contant level of anetheia were maintained by continuou intravenou infuion of pentobarbital ( mg kg 1 h 1 ). A craniotomy wa performed at the utura frontoparietali level for the recording of neuron in the latero-capular diviion of the central nucleu of the amygdala (CeLC) and for the adminitration of drug into the central nucleu. The dura mater wa opened and reflected; the pia mater wa removed over the recording and drug-adminitration ite to allow mooth inertion of the recording electrode and microdialyi probe, repectively. Electrophyiological recording A decribed previouly (Han et al. b; Ji and Neugebauer 27; Li and Neugebauer 2a,b, 26; Neugebauer and Li 2), long-term extracellular recording were made from ingle neuron in the CeLC with gla-inulated carbon filament electrode ( 6 M ) uing the following tereotaxic coordinate (Paxino and Waton 1998): mm caudal to bregma;.8. mm lateral to midline; depth 7 9 mm. The recorded ignal were amplified and diplayed on analog and digital torage ocillocope. Signal were alo fed into a window dicriminator the output of which wa proceed by an interface (CED 11 Plu) connected to a Pentium PC. Spike2 oftware (CED, verion ) wa ued to create peritimulu rate hitogram on-line and to tore and analyze digital record of ingleunit activity off-line. Identification of amygdala neuron An individual CeLC neuron wa identified by it background activity and by it repone to brief mechanical timuli applied to the ipi- and contralateral knee with a calibrated forcep (ee Mechanical timuli). Spike ize and configuration were continuouly monitored on the torage ocillocope and with the ue of Spike2 oftware. Spike were detected and recorded by the waveform ignal that croed a trigger level and matched a preet hape or template that wa created for the individual neuron at the beginning of the recording period. Included in thi tudy were only thoe neuron the pike configuration of which remained contant (matching the template) and could be clearly dicriminated from activity in the background throughout the experiment, indicating that the activity of one and the ame one neuron wa meaured. Receptive field Neuron were elected that had a receptive field in the knee. Size and threhold of the receptive field in deep tiue and kin were mapped uing graded mechanical timuli of innocuou and noxiou intenitie (ee Mechanical timuli). Cutaneou input wa ditinguihed from deep tiue input by elective timulation of kin fold gently raied from the underlying deep tiue. Mechanical timuli were conidered to activate deep tiue (joint and mucle) if the timulation of overlying kin evoked no or a clearly ditinct repone. The focu of thi tudy wa on the proceing of nociceptive information from the deep tiue. Standard diagram of the rat body were ued to record the location and ize of the receptive field. Mechanical timuli Mechanical timuli were applied to the deep tiue by mean of a forcep equipped with a force tranducer the calibrated output of which wa amplified, digitized, and recorded on a Pentium PC for onand off-line analyi. Stimulu-repone function were generated uing brief ( ) graded mechanical tet timuli of increaing intenitie (1,, 1,, 1,, and 2, g/ mm 2 at - intertimulu interval). Stimulu intenitie of 1 and g/ mm 2 applied to the knee and other deep tiue are conidered innocuou becaue they do not evoke hind limb withdrawal reflexe in awake rat and are not felt to be painful when teted on the experimenter. An intenity of 1, g/ mm 2 repreent a firm but nonpainful timulu that doe not evoke a hind limb withdrawal reflex. Preure timuli 1, g/ mm 2 are noxiou becaue they evoke hind limb withdrawal reflexe and vocalization in awake rat and are ditinctly painful when applied to the experimenter (Han et al. a,b; Han and Neugebauer ; Ji et al. 27). Background activity before timulation wa ubtracted from the total repone during timulation to calculate the net repone evoked by a particular timulu. Claification and repone threhold All neuron elected for thi tudy were multireceptive (MR) neuron according to our claification of CeLC neuron with deep tiue input (Han et al. b; Ji and Neugebauer 27; Li and Neugebauer 2a,b, 26; Neugebauer and Li 22, 2) MR neuron repreent the predominant type of neuron in the CeLC. Thi claification i primarily baed on the repone to mechanical timulation of the knee joint and other deep tiue. MR neuron repond conitently to innocuou timuli ( g/ mm 2 ) but are Downloaded from jn.phyiology.org on March 9, 211 J Neurophyiol VOL 12 OCTOBER 29

4 LATERALIZED AMYGDALA FUNCTION 2 more trongly activated by noxiou timuli ( g/ mm 2 ). Mechanical threhold wa defined a the minimum timulu intenity that evoked an excitatory repone (pike frequency higher than the upper 9% confidence interval of background activity). Experimental protocol In each experiment, one CeLC neuron wa recorded in the left or right CeLC before and for everal hour after arthriti induction in the ipi- or contralateral knee joint (ee Arthriti). Background activity, evoked repone, and receptive-field ize were meaured repeatedly before and after induction of arthriti and before and during drug adminitration into the CeLC (ee Drug). Background activity wa recorded for 1 min to calculate mean SE and 9% confidence interval (CI; GraphPad Prim.). Before arthriti induction and during the development of arthriti, mechanical tet timuli were applied to the knee and other deep tiue in the receptive field at regular interval of min. Before and during drug application, interval between the tet timuli were 1 min. Number of timulation wa kept at a minimum to avoid any enitization that might be produced by repeated timulation. Sufficiently long control period were ued to etablih conitent baeline repone before arthriti induction and drug application. A paired paradigm wa ued to determine arthriti pain-related change. Rather than comparing neuronal activity in arthriti with aline-injected control group, each neuron erved a it own control and wa recorded continuouly before and after arthriti induction in the ame animal. A previou tudy found no difference between aline-injected and untreated normal rat on ynaptic tranmiion and excitability in CeLC neuron (Neugebauer et al. 2). Arthriti Arthriti wa induced a decribed in detail previouly (Neugebauer et al. 27; Schaible and Schmidt 199). A kaolin upenion (%, 1 l) wa lowly injected into the joint cavity through the patellar ligament with the ue of a yringe and needle (1 ml, gauge, /8 in). After repetitive flexion and extenion of the knee for min, a carrageenan olution (2%, 1 l) wa injected into the knee joint cavity, and the leg wa flexed and extended for another min. Thi treatment paradigm reliably lead to inflammation and welling of the knee within 1 h and the inflammation perit for week (Neugebauer et al. 27). Drug and drug adminitration by microdialyi KT72, a potent and elective membrane-permeable PKA inhibitor (Bird et al. ; Cabell and Audeirk 199), and forkolin, a membranepermeable activator of adenylyl cyclae (Awad et al. 198; Laurenza et al. 1989), were purchaed from Tocri Biocience, Elliville, MO. Drug were adminitered into the CeLC by microdialyi at a rate of l/min for min. Several hour before the tart of the electrophyiological recording a microdialyi probe (CMA11; CMA/Microdialyi; membrane diameter: m; membrane length: 1 mm) wa poitioned tereotaxically in the left or right CeLC, uing the following coordinate: 1.6 mm caudal to bregma;. mm lateral to midline; depth of tip 9. mm (Han et al. b; Ji and Neugebauer 27; Li and Neugebauer 2a,b, 26). Uing PE- tubing, the microdialyi probe wa connected to an infuion pump (Harvard) and perfued with artificial cerebropinal fluid (ACSF) containing (in mm) 1. NaCl, 2.6 KCl, 2. NaH 2 PO,1. CaCl 2,.9MgCl 2,21.NaHCO,and.glucoe;oxygenatedand equilibrated to ph 7.. Before the recording, ACSF wa pumped through the fiber for 1 htoetablihequilibriuminthetiue.acsf wa preent throughout the experiment and alo erved a a vehicle control. KT72 and forkolin were diolved in ACSF on the day of the experiment at a concentration of 1 time that predicted to be needed baed on data from our previou tudie (Bird et al. ; Fu et al. 28; Han et al. b; Ji and Neugebauer 28). Drug concentration in the tiue i 1 time lower than in the microdialyi probe a a reult of the concentration gradient acro the dialyi membrane and diffuion in the tiue (Fu et al. 28; Ji and Neugebauer 28). The number given in thi article refer to the drug concentration in the microdialyi fiber. Hitology At the end of each experiment, the recording ite in the CeLC wa marked by injecting DC ( Aformin)throughthecarbonfilament recording electrode. The brain wa removed and ubmerged in 1% formalin and potaium ferrocyanide. Tiue were tored in 2% ucroe before they were frozen-ectioned at m. Section were tained with Neutral Red, mounted on gel-coated lide, and cover-lipped. The boundarie of the different amygdala nuclei were eaily identified under the microcope. Leion/recording ite were verified hitologically and plotted on tandard diagram adapted from Paxino and Waton (1998) (ee Fig. 1). The poition of the microdialyi probe in the CeLC were alo verified hitologically (not hown; they were virtually identical and the ame tereotaxic coordinate were ued in every experiment). Data analyi Extracellularly recorded ingle-unit action potential were analyzed off-line from peritimulu rate hitogram uing Spike2 oftware (CED, verion ). Repone to mechanical timuli were meaured and expreed a pike per econd (Hz). Background activity wa ubtracted from the total activity during the timulu to obtain the net timuluevoked activity. A two-way ANOVA with Tukey pottet (SigmaStat.1) wa ued to evaluate tatitically the effect of lateralization (left v. right amygdala) and treatment (arthriti v. normal) on neuronal activity (data in Fig. ). A paired t-tet wa ued to compare in four different experimental paradigm (left CeLC/right knee; left CeLC/left knee; right CeLC/left knee; left CeLC/right knee; data in Fig. ) each neuron activity in arthriti with prearthriti normal control value (paired experimental paradigm; Prim., GraphPad Software). A paired t-tet wa alo ued to determine ignificant difference of neuronal activity before and during drug adminitration (Prim., GraphPad Software). Statitical analyi wa performed on raw data (firing rate meaured a pike per econd). Statitical ignificance wa accepted at the level P.. For quantification of the receptive field ize, the body map wa divided into 21 different area. A core of 1 wa aigned to each area that wa part of the total receptive field. Addition of the core yielded a value for the total receptive field of a neuron. Averaged value for left veru right amygdala neuron were compared uing a Mann- Whitney U tet (unpaired experimental paradigm; Prim., Graph- Pad Software). Averaged value for normal veru arthriti tate were compared uing a Wilcoxon igned-rank tet (paired experimental paradigm; Prim., GraphPad Software). For multiple comparion in nonparametric tet, the alpha level wa adjuted and tatitical ignificance accepted at the level P.. RESULTS Extracellular ingle-unit recording were made from 17 neuron in the left and neuron in the right CeLC of anethetized adult male rat (Fig. 1). Only one neuron wa recorded in each rat. Neuron were elected that had a receptive field in the knee joint a in our previou tudie. Neuron were multireceptive (MR, ee Claification)andrepondedmoretronglytobriefnoxiouthan innocuou timuli applied to the knee and other part of the receptive field. In thi tudy, we included only MR neuron becaue our previou tudie howed that they conitently and reliably become enitized in the arthriti pain model (Han et al. b; Ji and Neugebauer 27; Li and Neugebauer 2a,b, 26; Neugebauer and Li 2) and are believed to integrate Downloaded from jn.phyiology.org on March 9, 211 J Neurophyiol VOL 12 OCTOBER 29

5 26 G. JI AND V. NEUGEBAUER L e f t C e A R i g h t C e A n = n = CeLC CeL CeM bregma -2. mm CeM CeL CeLC n=8 n= CeLC CeL CeM bregma -2.6 mm CeM CeL CeLC n= n=7 FIG. 1. Hitologically verified recording ite of 2 neuron in the laterocapular diviion of the central nucleu of the amygdala (CeLC) in the left and right brain hemiphere., F, Œ, the location of neuron that were recorded before and after arthriti induction;, E,, neuron that were recorded only under normal condition. Symbol alo differentiate between the different type of receptive field (ee Fig. 2):,, contralateral hindlimb only; F, E, bilateral hindlimb; Œ,, whole body. Diagram (adapted from Paxino and Waton 1998) how coronal brain ection at different level poterior to bregma ( 2. to 2.8). Next to each ection i hown in detail the central nucleu and it medial (CeM), lateral (CeL), and latero-capular (CeLC) ubdiviion. Calibration bar are 1 mm. Downloaded from jn.phyiology.org on March 9, 211 CeLC CeL CeM bregma -2.8 mm CeM CeL CeLC nociceptive and affective information (Neugebauer 26; Neugebauer et al. 2). Continuou recording before and after arthriti induction were made from 11 neuron in the left and 9 neuron in the right CeLC. The remaining neuron (6 in the left and 6 in the right CeLC) were only recorded under normal condition to determine the effect of forkolin alone and in the preence of a PKA inhibitor (KT72). Propertie of left and right CeLC neuron under normal condition RECEPTIVE FIELD SIZE. All neuron were activated by mechanical timulation (compreion) of the knee joint, which erved a the earch timulu. The receptive field of neuron in the left CeLC (n 17) wa maller than that of neuron in the right CeLC (n ; ee Fig. 2). Receptive field of left CeLC neuron were either confined to the contralateral hindlimb (n 9) or included an additional high-threhold receptive field in the ipilateral hindlimb (n 8; Fig. 2B, normal). In contrat, receptive field of right CeLC neuron were alway (n ) bilateral and ymmetrical in the deep tiue of the hindlimb and tail; ome of thee neuron (n 8) had additional receptive field in the forepaw and trunk (Fig. 2A, normal). In an attempt to quantify the difference, we divided the body map into 21 different ector (ee Fig. 2). The total number of area that contained part of the receptive field of a neuron wa calculated and averaged for neuron in the left and for thoe in the right CeLC. The comparion (Fig. 2C) howed that the average receptive field ize of right CeLC neuron wa ignificantly larger than that of left CeLC neuron under normal condition (P., Mann-Whitney U tet). BACKGROUND ACTIVITY AND EVOKED RESPONSES. No evidence of lateralization wa found for background activity and repone to innocuou and noxiou timuli under normal condition. Figure how the background activity and evoked J Neurophyiol VOL 12 OCTOBER 29

6 LATERALIZED AMYGDALA FUNCTION 27 A Right amygdala B C normal arthriti normal arthriti Left amygdala normal arthriti normal arthriti # Receptive field ize 2 receptive field core 1 left right left right normal arthriti FIG. 2. Receptive field of neuron in the right (A) and left (B) CeLC. Receptive field in the deep tiue are hown before (normal) and h potinduction of arthriti. Change of receptive field ize were oberved in right, not left, CeLC neuron. A and B: all neuron howed increaing repone to graded innocuou and noxiou timulation of area colored black ( multireceptive neuron, ee METHODS). 1, high-threhold receptive field, timulation of which activated the neuron weakly. i.l., ipilateral; c.l., contralateral to recording ite. A: under normal condition, receptive field of right CeLC neuron (n ) were ymmetrical in the deep tiue of both hindlimb and the tail (n 7, left) or covered the whole body (n 8, right). B: receptive field of left CeLC neuron (n 17) were either confined to the contralateral hindlimb (n 9) or included an additional high-threhold receptive field in the ipilateral hindlimb (n 8). C: emi-quantitative analyi of the receptive field ize in neuron that were recorded continuouly before and after arthriti induction. The body map wa divided into 21 area (A and B, - - -). The total number of area that contained part of the receptive field wa calculated for each neuron and averaged for left (n 11) and right (n 9) CeLC neuron (ee Data analyi). Only neuron that were recorded before and after arthriti induction are included in the analyi. In the graph, each box extend from the th to the 7th percentile, with a line at the median (th percentile). The whiker extend above and below the box to how the highet and lowet value., P. (receptive field ize of right compared with left CeLC neuron; Mann-Whitney U tet), #, P. (receptive field ize after arthriti compared with normal; Wilcoxon igned-rank tet), alpha level adjuted for multiple comparion. Downloaded from jn.phyiology.org on March 9, 211 repone of individual CeLC neuron in the left (A) and right (B) CeLC before arthriti induction (and change after arthriti; ee Propertie of left and right CeLC neuron in the arthriti pain model). The averaged value for the ample of left (n 11) and right (n 9) CeLC neuron under normal condition are hown in Fig. C. The analyi only include neuron that were recorded before and after arthriti induction to allow the direct comparion. Statitical analyi revealed no ignificant difference between left and right CeLC neuron under normal condition (P., Tukey tet). J Neurophyiol VOL 12 OCTOBER 29

7 G. JI AND V. NEUGEBAUER Propertie of left and right CeLC neuron in the arthriti pain model RECEPTIVE FIELD SIZE. After the induction of a knee joint arthriti (ee METHODS) the ize of the receptive field of neuron in the right CeLC expanded (Fig. 2A). Thi change wa oberved in the majority of right CeLC neuron (6 of 9 neuron); the receptive field of the remaining right CeLC neuron covered the whole body before arthriti and no apparent increae wa detected. In contrat, the receptive field ize of neuron in the left CeLC did not change (Fig. 2B). The A pike / B pike / C pike / Left CeLC g/ mm 2 pike/ Pre (g) Event Pre (g) Event 7 6 Right CeLC g/ mm 2 pike/ noxiou innocuou background noxiou innocuou background left right normal arthriti arthriti arthriti 1 normal arthriti g/ mm 2 pike/ g/ mm 2 pike/ Pre (g) Event Pre (g) Background Innocuou Noxiou 2 left 2 left right right 2 Event normal arthriti quantitative analyi of the receptive field ize on the body map (Fig. 2C) alo revealed ignificant increae for right (P.) but not left (P.) CeLC neuron in the arthriti pain model (Wilcoxon igned-rank tet). The receptive field ize of right CeLC neuron wa ignificantly greater than that of left CeLC neuron (P., Mann-Whitney U tet). The analyi only included neuron that were recorded before and after arthriti induction to allow the direct comparion of the receptive field ize. BACKGROUND ACTIVITY AND EVOKED RESPONSES. Activity of right but not left CeLC neuron increaed in the arthriti pain model. Figure B how an individual example of a neuron in the right CeLC. In agreement with our previou tudie (Han et al. b; Ji and Neugebauer 27; Li and Neugebauer 2a,b, 26; Neugebauer and Li 2), background activity and repone to innocuou and noxiou timulation of the knee (ee Mechanical timuli) increaed after arthriti induction and reached a plateau at h. In contrat, background activity and evoked repone of a neuron in the left CeLC did not change for everal hour after arthriti induction (Fig. A). In both cae, arthriti wa induced in the knee contralateral to the recording ite becaue our previou tudie howed enitization of right CeLC neuron when arthriti wa induced in the contralateral (left) knee (Han et al. b; Ji and Neugebauer 27; Li and Neugebauer 2a,b, 26; Neugebauer and Li 2). Both neuron were recorded continuouly before and after arthriti induction. Figure C how ignificant right-hemipheric lateralization of arthriti pain-related change. The activity of left (n 11) veru right (n 9) CeLC neuron are compared under normal condition and in the arthriti pain model. Two-way ANOVA revealed ignificant main effect of lateralization on background activity [P., F(1,6).6] and on repone to innocuou [P.1, F(1,6) 8.28] and noxiou [P., F(1,6).76] timuli. Tukey pottet howed ignificant difference between left and right CeLC neuron in the arthriti pain model (background, P.; innocuou, P.1; noxiou, P.1) but not under normal condition (P.). Two-way ANOVA alo revealed ignificant main effect of treatment on the repone to innocuou [P., F(1,6).2] and noxiou ([P., FIG.. Right-hemipheric lateralization of arthriti pain-related change. A: unchanged background and evoked activity of 1 left CeLC neuron in arthriti. B: increaed background and evoked activity of 1 right CeLC neuron after arthriti induction. A and B: line graph how the time coure of extracellularly recorded repone (number of pike per econd) to brief ( ) innocuou ( g/ mm 2 ) and noxiou (2, g/ mm 2 ) timulation of the knee and background activity. Symbol how the mean activity during a - period before timulation ( background activity) or the difference between mean activity during and before - timuli ( net activity evoked by noxiou or innocuou timuli; ee METHODS). Peritimulu time hitogram (inet) how individual repone (pike per econd) before and h after induction of arthriti. Top trace how recording of the force (g/ mm 2 ) applied to the knee joint with a calibrated forcep (ee METHODS). C: comparion of left v. right CeLC neuron. Under normal condition before arthriti, there wa no ignificant difference of background and evoked activity between left (n 11) and right (n 9) CeLC neuron (P., Tukey tet). Five hour after arthriti induction, activity of right CeLC neuron wa ignificantly higher than that of left CeLC neuron (P.1., Tukey tet). Bar hitogram how background activity and repone to innocuou ( g/ mm 2 ) and noxiou (2, g/ mm 2 ) timulation of the knee averaged for the ample of neuron (mean SE). Single aterik, P.; double aterik, P.1 (Tukey tet, comparing value in right v. left CeLC neuron). Downloaded from jn.phyiology.org on March 9, 211 J Neurophyiol VOL 12 OCTOBER 29

8 LATERALIZED AMYGDALA FUNCTION 29 F(1,6).] timuli but not on background activity [P., F(1,6) 2.97]. Importantly, the differential effect of arthriti pain on right and left CeLC neuron were independent of the ide of the monoarthriti. Significant (P., paired t-tet performed on raw data) change of background and evoked activity were oberved in right CeLC neuron after arthriti wa induced in the contralateral (left) knee (n neuron;fig.c) oripilateral(right)knee(n neuron; D). Left CeLC neuron howed no change after arthriti wa induced either in the right knee (n ; A) orleftknee(n 6; B). Thee reult ugget that pain-related lateralization i independent of the ide of arthriti. Effect of a PKA inhibitor Our previou tudie howed an important contribution of PKA to central enitization and ynaptic platicity in the right CeLC (Bird et al. ; Fu et al. 28). Here we addreed the hypothei that lack of PKA activation in the left CeLC contribute to pain-related right-hemipheric lateralization. A elective PKA inhibitor (KT72, KT; 1 M, concentration in the microdialyi fiber; min; ee Drug and drug adminitration by microdialyi) wa adminitered into the CeLC 6 h potinduction of arthriti. The poition of the microdialyi probe in the CeLC were verified hitologically. Adminitration of KT72 into the left CeLC had no effect on CeLC neuron in the left hemiphere. An individual example i A % of pre-arthriti C % of pre-arthriti Arthriti contralateral normal arthriti backgr innoc noxiou Arthriti contralateral normal arthriti Left CeLC B % of pre-arthriti % of pre-arthriti 2 1 Right CeLC D 2 1 hown in Fig. A. Figure B ummarize the lack of effect of KT72 on background and evoked activity in the ample of left CeLC neuron (n ) after arthriti induction in the contralateral knee. Adminitration of KT72 into the right CeLC ignificantly (P., paired t-tet) inhibited the increaed activity of neuron in the right CeLC (n ; ee individual example in Fig. C and ummary of data in D). The data ugget that PKA i not activated endogenouly in the left CeLC in the arthriti pain model. Effect of forkolin Next we ought to determine if the exogenou activation of ignal tranduction pathway could enitize neuron in the left CeLC. A widely ued cell-permeable activator of adenylyl cyclae (forkolin, 1 mm, concentration in microdialyi fiber; min) wa adminitered into the left or right CeLC under normal condition (no arthriti). The poition of the microdialyi probe in the CeLC were verified hitologically. Forkolin increaed background activity and evoked repone of left and right CeLC neuron. The receptive field ize did not change. Figure 6 how individual neuron in the left (A) and right(c) CeLC and ummarize the ignificant effect of forkolin on the ample of neuron in the left (n, B) andright(n, D) CeLC.Inthe preence of a PKA inhibitor (KT72, 1 M, concentration in the microdialyi fiber; min), forkolin had no ignificant effect (P., compared with predrug control value; paired t-tet; Arthriti ipilateral normal arthriti backgr innoc noxiou Arthriti ipilateral normal arthriti FIG.. Pain-related lateralization i independent of the ide of arthriti. A and B: there wa no ignificant change of background and evoked activity of left CeLC neuron after induction of arthriti in the right (contralateral, A; n ) or left (ipilateral, B; n 6) knee. C and D: background and evoked activity of CeLC neuron in the right hemiphere increaed h after arthriti wa induced in the left (contralateral, C; n ) or right (ipilateral, D; n ) knee. Bar hitogram how background activity and repone to brief ( ) innocuou ( g/ mm 2 )andnoxiou(2,g/mm 2 )timulation of the knee expreed a percent of prearthriti value (et to 1%). Data were averaged for the ample of neuron (mean SE)., P. (paired t-tet comparing value in arthriti with prearthriti value under normal condition; tatitical analyi wa performed on raw data). Downloaded from jn.phyiology.org on March 9, 211 backgr innoc noxiou backgr innoc noxiou J Neurophyiol VOL 12 OCTOBER 29

9 G. JI AND V. NEUGEBAUER Left CeLC A pike/ pike/ pike/ g/ mm Pre (g) 2 noxiou innocuou Event background Pre (g) Event - Right CeLC C noxiou innocuou background pike/ g/ mm Pre (g) Event KT time (min) KT Pre (g) Event - time (min) 2 1 Pre (g) Event Pre (g) Event % of predrug control D KT72 % of predrug control B KT72 1 backgr innoc noxiou 1 backgr innoc noxiou backgr innoc noxiou FIG.. Lateralized effect of a protein kinae A (PKA) inhibitor in the arthriti pain model. A: adminitration of KT72 (1 M, concentration in the microdialyi probe; min) into the left CeLC had no effect on background and evoked activity of a neuron in the left CeLC. B: normalized data ummarize the lack of effect of KT72 on background activity and evoked repone of left CeLC neuron (n ) 6 h potinduction of arthriti in the contralateral (right) knee. C: KT72 (1 M) adminitered into the right CeLC inhibited the increaed background activity and evoked repone of a neuron in the right CeLC. D: normalized data ummarize the ignificant inhibition of background activity and evoked repone right CeLC neuron (n ) 6 h potinduction of arthriti in the contralateral (left) knee. A and C: ymbol how background activity (mean activity during a - period before timulation) and repone to innocuou ( g/ mm 2 ) and noxiou (2, g/ mm 2 ) timuli before [in arificial cerebropinal fluid (ACSF)], during and after KT72 adminitration (ee - - -). Evoked repone are calculated a the difference between mean activity during and before - timuli (ee METHODS). Inet: individualrepone(pike/,peritimulu-time hitogram, bin width: 1 ) and recording of the force (g/ mm 2, top trace) applied to the knee joint with a calibrated forcep (ee METHODS) before, during, and after adminitration of KT72 into the CeLC. B and D: barhitogram how averaged value (mean SE) during drug adminitration normalized to predrug control value (in ACSF, et to 1% a indicated, - - -)., P.;, P.1 (paired t-tet). Downloaded from jn.phyiology.org on March 9, 211 n neuron;supplementaryfig.s1 1 ). The data demontrate that in principle the activity of left CeLC neuron can be modulated (increaed) like that of right CeLC neuron, uggeting that PKA activation i ufficient for increaed reponivene of CeLC neuron but doe not occur in the left CeLC in the arthriti pain model. DISCUSSION The key finding of thi tudy are a follow. Unlike CeLC neuron in the right amygdala, neuron in the left CeLC do not develop increaed reponivene in a rodent model of arthriti pain. Thi hemipheric lateralization i independent of the ide of the peripheral injury (ipi- or contralateral to the recording ite). No ignificant difference wa found in the magnitude of the repone of left and right CeLC neuron to brief phyiological noxiou timuli under normal condition, indicating that individual input have comparable effect on neuron in the left and right CeLC. 1 The online verion of thi article contain upplemental data. The maller receptive field ize of left compared with right CeLC neuron may ugget the tonic control of effective input. The contribution of PKA in the right but not left amygdala to pain-related change i in agreement with the finding of another group (Carraquillo and Gereau 27, 28) that pain-related lateralization involve difference in the endogenou activation of ignaling pathway in the CeLC. Importantly, the exogenou activation of intracellular effector in the left or right CeLC by forkolin produced activity change that reembled thoe oberved in the arthriti pain model. The reult ugget that PKA activation i neceary and ufficient for increaed reponivene of CeLC neuron but doe not occur in the left CeLC in the arthriti pain model. The reult how that nociceptive information reache both left and right amygdala (CeLC), but it i the right CeLC that play a major role in the proceing of prolonged nociceptive input and develop enitization. Neuron in the left CeLC are capable of activity change when intracellular igning pathway are timulated directly (forkolin experiment), but an J Neurophyiol VOL 12 OCTOBER 29

10 Left CeLC A forkolin pike/ C 2 1 pike/ g/ mm Pre (g) Event noxiou innocuou Pre (g) Event background - Right CeLC pike/ 2 pike/ g/ mm Pre (g) Event time (min) forkolin Pre (g) Event 2 1 noxiou 1 innocuou background - time (min) Pre (g) Event 2 Pre (g) Event LATERALIZED AMYGDALA FUNCTION B forkolin % of predrug control D forkolin % of predrug control backgr innoc noxiou 1 backgr innoc noxiou 2261 FIG. 6. Forkolin effect are not lateralized. A: adminitration of forkolin (1 mm, concentration in the microdialyi probe; min) into the left CeLC increaed background activity and evoked repone of a neuron in the left CeLC. B: normalized data ummarize the ignificant effect of forkolin in the ample of left CeLC neuron (n ). C: forkolin (1 mm) adminitered into the right CeLC had imilar facilitatory effect on a right CeLC neuron. D: normalized data ummarize the ignificant facilitatory effect of forkolin in the right CeLC (n ). A and C: ymbolhowbackgroundandevoked net activity before, during, and after forkolin adminitration (ame diplay a in Fig., Aand C). B and D: barhitogram how averaged value (mean SE) during drug adminitration normalized to predrug control value (ame diplay a in Fig., B and D). Recording were made under normal condition (no arthriti)., P.;, P.1 (paired t-tet). Downloaded from jn.phyiology.org on March 9, 211 unknown mechanim prevent their activation in the arthriti pain model. Thi reult i novel and ignificant becaue there ha been little evidence for hemipheric lateralization of brain function related to pain (ee INTRODUCTION). Two recent biochemical and behavioral tudie (Carraquillo and Gereau 27, 28) were the firt to how lateralization of amygdala function in pain. Activation of the MAP kinae ERK wa oberved in the right, but not left, CeLC in the formalin pain model. Converely, blockade of ERK activation in the right, but not the left, CeLC inhibited pain behavior. Reult from the preent tudy ugget that hemipheric lateralization i not retricted to the function of a ingle molecule (ERK) but alo involve PKA and poibly other effector. Failure to activate thee ignaling pathway appear to prevent the left CeLC from developing pain-related activity increae, therefore contributing to right-hemipheric lateralization. A conequence of pain-related change in the right, but not left CeLC, would be aymmetric output from the amygdala to target tructure uch a the periaqueductal gray (PAG) (Heinricher and McGaraughty 1999; Neugebauer et al. 2; Rizvi et al. 1991; Shipley et al. 1991; Tracey and Mantyh 27). The PAG i an important brain tem center for the decending modulation of pain and other behavior (Heinricher and McGaraughty 1999; Maon ; Tracey and Mantyh 27). Importantly, increaed neural tranmiion in thi largely ipilateral output pathway during tre-induced anxiety wa oberved in the right but not left hemiphere (Adamec et al. a,b). Conitent with thi finding, pain behavior wa modified by manipulating ERK activation in the right but not left CeLC (Carraquillo and Gereau 27, 28). Relatively few tudie have pecifically addreed or decribed lateralization of amygdala function in rodent. Predominant activation or involvement of the right amygdala wa found in averively motivated learning and memory (Coleman- Meche and McGaugh 199a,b; Coleman-Meche et al. 1996; Lalumiere and McGaugh ) and contextual fear J Neurophyiol VOL 12 OCTOBER 29

11 2262 G. JI AND V. NEUGEBAUER conditioning (Baker and Kim 2) in rat. In human, right hemipheric lateralization of amygdala function wa aociated with negative emotion (Angrilli et al. 1996; Canli et al. 1998; Funayama et al. 21; Lee et al. 2; Yohimura et al. 28), fear extinction (LaBar et al. 1998), ubconciou emotional learning (Morri et al. 1998), rapid automatic timulu detection and repone (Cotafreda et al. 28; Sergerie et al. 28), and pain (Lu et al. 2). Poitive emotion tend to be lateralized to the left amygdala in human (Canli et al. 1998; Lee et al. 2; Yohimura et al. 28). However, greater right than left amygdala activation wa reported for viewing happy face and greater left amygdala activation for fearful face (Hardee et al. 28; Killgore and Yurgelun-Todd 21). The left rather than right amygdala ha been implicated in perceived or anticipated but not actually experienced fear (Funayama et al. 21; Phelp et al. 21). Sex-related hemipheric difference of amygdala activation include the preferential involvement of the right amygdala in emotional repone and emotional memory in men and of the left amygdala in women (ee Cahill 26 for review). Sexrelated difference, however, may be valence dependent becaue they were oberved for happy but not fearful face (Killgore and Yurgelun-Todd 21). Thee data trongly upport the concept of hemipheric lateralization of amygdala function in emotion, but the underlying principle and mechanim remain to be determined. Pain-related right hemipherical lateralization of amygdala function i conitent with the predominant activation or involvement of the right amygdala in emotion. One tudy uggeted that left ided pain, experimental or chronic, produced greater emotional diturbance and anxiety (Schiff and Gagliee 199). Pain ha a negative emotional-affective component and i cloely related to anxiety and depreion (Gallagher and Verma 2; Grachev et al. 21; Rhudy and Meagher 2; Tracey and Mantyh 27), but the role of hemipheric lateralization in thi relationhip remain to be determined. Poible mechanim of pain-related lateralization of amygdala function include difference in nociceptive input, neuronal propertie, and control by other brain area. The amygdala receive nociceptive information through anatomically and functionally ditinct line of input (Braz et al. ; Neugebauer 26; Neugebauer et al. 2). Purely nociceptive information reache the CeLC directly from the pinal cord and brain tem (parabrachial area), thu bypaing the thalamu (Bernard and Beon 199; Cliffer et al. 1991; Gauriau and Bernard 2). Polymodal enory, including nociceptive, input from thalamu (poterior area) and cortex (inula and aociation cortice) reach primarily the lateral amygdala (Pare et al. 2; Phelp and Ledoux ; Shi and Davi 1999). Aociative proceing in the lateral-baolateral amygdala network generate affect-related information that i tranmitted to the central nucleu, a major output nucleu for amygdala function (Maren ; Pare et al. 2; Phelp and Ledoux ). The preent tudy how that CeLC neuron on the left and right repond to brief innocuou and noxiou timuli with imilar magnitude under normal condition, arguing againt a major difference in nociceptive and nonnociceptive input to the CeLC. Difference in the neuronal population of the left and right CeLC are unlikely to account for lateralized amygdala function. The two principal type of CeLC neuron are nociceptive-pecific (NS) neuron, which receive excluively nociceptive input, and multireceptive (MR) neuron, which repond to innocuou and noxiou timuli and integrate nociceptive ignal with affective information from the lateral-baolateral circuitry (Neugebauer 26; Neugebauer et al. 2). MR neuron, but not NS neuron, undergo central enitization in the arthriti pain model (Neugebauer and Li 2). The preent tudy did not ytematically analyze the proportion of NS and MR neuron, but MR neuron were identified in the left CeLC and did not how the pain-related change of MR neuron in the right CeLC. Biochemical and pharmacological data ugget that lateralized amygdala function in pain involve difference in the activation of cellular ignaling pathway (ERK, Carraquillo and Gereau 27, 28; PKA, preent tudy). The effect of forkolin in the preent tudy how that effector ytem can be activated in left CeLC neuron and produce increaed neuronal activity. Thee data ugget that a yet unknown mechanim prevent the pain-related endogenou activation of ignaling mechanim and enitization of left CeLC neuron. The maller receptive field ize of neuron in the left compared with the right CeLC alo argue for the preence of a control mechanim of effective input and cellular effector. Expanion of the receptive field of CNS neuron i well documented in the arthriti pain model and i generally taken a evidence for central enitization that render normally ineffective input functional (Neugebauer and Li 2; Neugebauer and Schaible 199). The difference in receptive field ize between left and right CeLC neuron and the lack of pain-related change in left CeLC neuron may ugget a tonic inhibitory mechanim, which could involve the well-known cortical control of amygdala function (ee Bank et al. 27). The amygdala i reciprocally connected with prefrontal cortical area (ee Ghahghaei et al. 27) that exert a top-down inhibitory influence on the amygdala (Carmichael and Price 199; Mc- Donald et al. 1996; Quirk and Beer 26; Roenkranz and Grace 22). Invere coupling of prefrontal cortex and amygdala wa oberved in imaging tudie in human in aociation with averive and other emotional timuli, poibly repreenting a neural ytem for cognitive regulation of emotion (Kim et al. 2; Ochner et al. 22; Urry et al. 26; but ee Bank et al. 27). Pertinent to the current tudy, greater left than right prefrontal cortical electroencephalographic (EEG) activity predicted an attenuated phyiological repone to averive timuli (Jackon et al. 2). In concluion, the preent tudy demontrate difference in the proceing of nociceptive information by neuron in the left veru right amygdala in a model of arthritic pain. Pain-related enitization of left CeLC neuron i prevented by a mechanim that remain to be determined but may involve prefrontal cortical inhibition of the amygdala. Right hemipheric lateralization of pain proceing in the amygdala i conitent with a predominant role of the right amygdala in negative emotion. ACKNOWLEDGMENTS We thank Dr. William D. Willi Jr., for critical reading of and helpful comment on thi manucript. We alo thank Dr. N. Bradley Keele for valuable aitance with the tatitic and uggetion on the manucript. Downloaded from jn.phyiology.org on March 9, 211 J Neurophyiol VOL 12 OCTOBER 29