In Vivo Characterization of ExtracellularGABARelease in the Caudate Nucleus andPrefrontal Cortex of the Rhesus Monkey

BHASKAR S. KOLACHANA, RICHARD C. SAUNDERS,* AND DANIEL R. WEINBERGERClinical Brain Disorders Branch, Intramural Research Program, NIMH/NIH,

NIMH Neuroscience Center at St. Elizabeths, Washington, D.C. 20032

KEY WORDS microdialysis; monkey; caudate nucleus; g-aminobutyric acid; highpotassium; veratridine; tetrodotoxin; nipecotic acid; Ca21 dependency

ABSTRACT Extracellular gamma amino butyric acid (GABA) levels were measuredin the caudate nucleus and the prefrontal cortex of the rhesus monkey brain using in vivomicrodialysis under isofluorane gas anesthesia. Evoked GABA release was investigatedfor voltage sensitivity and calcium (Ca21) dependency. There was a multifold increase inextracellular GABA levels following local perfusion with: (1) high potassium (50 mM,KCl), (2) veratridine (10 µM), and (3) the GABA releasing agent and uptake blocker, (2)nipecotic acid (1 mM). Release of GABA was significantly reduced when veratridine or(2) nipecotic acid were coinfused in Ca21-free cerebrospinal fluid (CSF). Coinfusion ofnipecotic acid with TTX (10 µM) also resulted in attenuation of evoked GABA release.These results suggest that GABA levels recovered using in vivo microdialysis, from thecaudate nucleus and the prefrontal cortex in the rhesusmonkey, derive in significant partfrom vesicular pools and the exocytotic process is both Ca21-dependent and voltage-sensitive. Synapse 25:285–292, 1997. r 1997 Wiley-Liss, Inc.†

INTRODUCTION

For several decades, scientists have attempted tostudy the release of neurotransmitters from specificbrain regions. The development of in vivo microdialysishas allowed the examination of the release of neuro-transmitters simultaneously in multiple regions inliving animals (Abercrombie et al., 1989; Kolachana etal., 1994; Moghaddam, 1993; Saunders et al., 1993;Tossman et al., 1986; Ungerstedt, 1984). Measurementof neurotransmitter release has become a powerful toolto study the in vivo relationships of anatomical andchemical neuronal systems (Kolachana et al., 1995;Moghaddam et al., 1990; Youngren et al., 1995). Forinstance, we have shown that in themonkey, pharmaco-logical manipulation of prefrontal cortical dopaminelevels influence caudate dopamine release (Kolachanaet al., 1995).Investigation of cortical-striatal interactions and the

role of dopamine on striatal and prefrontal corticalfunctions necessitate an understanding of amino acidneurotransmitters such as glutamate and gamma ami-nobutyric acid (GABA). GABAergic neurons representone of the main inhibitory output systems of thestriatum (for references see Scheel-Kruger, 1986). Inaddition, in the neocortex GABA interneurons modu-late cortical projection cells and probably influence

glutamatergic corticostriatal projections (Deutch andRoth, 1990; Goldman-Rakic et al., 1992; Santiago et al.,1993; Wilson et al., 1994). Despite the functional rel-evance of GABA as an inhibitory neurotransmitter andits purported role in regulating corticostriatal as wellas striatal output, relatively little information has beenobtained in vivo, particularly in the nonhuman pri-mate.While classical neurotransmitters (e.g., dopamine

and acetylcholine) found in the extracellular space aremainly of neuronal origin, the presence of amino acidssuch as GABA does not necessarily reflect neuronalrelease (Westerink et al., 1987). Their origin may be inpart a result of metabolic or glial pools (Campbell et al.,1993). Although studies in rats have demonstratedrelease of endogenous GABA in response to pharmaco-logical challenge (Girault et al., 1986; Lindefors et al.,1992; Tossman et al., 1986; Tuomisto et al., 1983; Vander Heyden et al., 1980), until now it has not beenfirmly established whether the extracellular GABAlevels in the brain fulfill classic criteria for neurotrans-mitter release, including nerve impulse- and Ca21

*Correspondence to: Dr. Richard C. Saunders, Bldg. 49, Rm. 1B80, 49 ConventRd. MSC 4415, Bethesda, MD 20892-4415.

Received 17 December 1995; Accepted 12 July 1996.

SYNAPSE 25:285–292 (1997)

r 1997 WILEY-LISS, INC. †This article is a US government work and,as such, is in the public domain in the United States of America.

dependency (Katz, 1969). It has been suggested thatthe voltage and Ca21 dependence of neurotransmitteroverflow must be determined for all in vivo prepara-tions before pharmacological effects can be interpretedappropriately (Westerink et al., 1987, 1988). Whilesome in vivo studies in the rat indicate that extracellu-lar GABA in the striatum is a result, at least in part,from exocytotic release by neurons (Bourdelais andKalivas, 1992; Osborne et al., 1990), there are otherstudies that disagree (Bernath and Zigmond, 1988;Westerink and De Vries, 1989). Hence, the presentstudy was designed to determine whether the extracel-lular GABA recovered across the dialysis membrane inthe monkey brain originates from physiologically basedrelease from nerve terminals. Ca21 dependency andvoltage sensitivity of GABA release was examined inthe following manner. Depolarization release of GABAwas demonstrated using high potassium (K1), or veratri-dine (which activates voltage-dependent Na1 channels)infusions. GABA levels were also increased by infusionof the (high affinity) uptake blocker, (2) nipecotic acid.Demonstration of Ca21-dependent GABA release wasexamined by comparing infusion of the artificial cerebro-spinal fluid (aCSF) with Ca21-free aCSF. In addition,we studied the effects of Ca21-free aCSF on evokedGABA release by coinfusing with veratridine or nipe-cotic acid. In experiments where the effects of nerveterminal depolarization on spontaneous release wasassessed, resulting from the influx of Na1 ions, tetrodo-toxin (TTX) was coinfused with nipecotic acid. Further-more, because of our interest in the prefrontal cortex aswell as the striatum, we compared GABA release in theprefrontal cortex with that in the striatum.

MATERIALS AND METHODSSubjects

Four experimentally naive adult rhesus monkeys(Macaca mulatta) weighing 7–9 kg were used in thisstudy. They were housed individually on a 12-h light/dark cycle with access to food and water ad libitum. Allprocedures were carried out following the NIH Guidefor the Care and Use of LaboratoryAnimals.

Dialysis probes and guide holders

For in vivo microdialysis studies, probes were con-structed in-house using fused silica barrels, a shortpiece (4–6 mm) of dialysis membrane (PAN-69, 240 µmi.d., 300 µm o.d., 40,000 Mol Wt. cut off, Hospal MedicalCo. France) and polyethylene tubing (PE 10 & 50, ClayAdams, Parsippany, NJ; for details see Kolachana etal., 1994). Specially designed rectangular polycarbon-ate plastic ‘‘guide holders’’ (15 3 30 3 8 mm) werecustom-made with four to six rows of small holes (,1mm in diameter and 1 mm center-to-center apart)comprising 15 holes in each row. The guide holder fixedto the skull directs the dialysis probes accurately and

stabilizes them in the targeted site (Kolachana et al.,1994). The guide holders are positioned on the skullusing MRI-guided stereotactic coordinates (Kolachanaet al., 1994; Saunders et al., 1990) and fixed in placeusing acrylic bone cement. After surgery, the animalswere allowed to recover for 2 weeks. The targetedregions for dialysis were the caudate nucleus and themedial bank of the principalis sulcus in the prefrontalcortex. The placement and alignment of the guideholders over the target sites were confirmed with asecond set of MR images with several 2.5-cm-long fusedsilica barrels filled with Vitamin E positioned throughthe holes (but without penetrating the dura). Detaileddescription of the surgery and scanning procedureshave been given elsewhere (Kolachana et al., 1994).

In vitro recovery experiments

In vitro evaluations prior to in vivo experimentationwere carried out to determine the percent recovery ofGABA across the dialysis probe membrane. Thesestudies were conducted by immersing the dialysismembranes in an airtight beaker containing 0.5 µMGABA in aCSF at 37°C. The probes were continuouslyperfused with aCSF at 1 µl/min flow rate for 1 hourbefore samples were collected. Three successive 10-µlcollections were analyzed for GABA concentration andcompared with GABA levels directly from the aCSFsolution in the beaker.

Drugs and chemicals

Most of the drugs/pharmacological agents were dis-solved in aCSF with the final pH adjusted to 7.4. (2)Nipecotic acid (1 mM, Research Biochemical Inc.,Natick, MA) and tetrodotoxin (TTX, 10 µM, SigmaChemical Co., St. Louis, MO) solutions were made freshin aCSF just before infusion. Veratridine (ResearchBiochemical Inc.) was dissolved in ethyl alcohol andsubsequent dilutions to 10 µM made with aCSF. Con-stituent chemicals for aCSF and buffers for chromato-graphic separation are of Analytical Reagent Grade(Sigma Chemical Co.; Mallinkrodt Inc., Paris, KY andFischer Scientific Co., Fair Lawn, NJ).

GABAhigh performance liquid chromatography(HPLC) assay

A 10-µl aliquot of dialysate was used to analyzeGABA after precolumn derivatization with o-phthaldi-aldehyde (OPA) and B-mercaptoethanol (BME; Donz-anti and Yamamoto, 1988). The derivatization reagentwas prepared by dissolving 27 mg of OPA in 1 ml 100%methanol, 5 µl of BME and 9 ml 0.1 M sodium tetrabo-rate (pH 9.4). This stock solution was diluted 1:3 with0.1M sodium tetraborate and filtered through a 0.22-µmfilter. A 5-µl aliquot of this reagent was added to 10-µlsample dialysate or standard amino acid solution, and

286 B.S. KOLACHANA ET AL.

derivatization was allowed to proceed for 2 min beforeinjection onto the HPLC column (3 3 100 mm, C-18reversed phase column, ESA Inc., Chelmsford, MA)with the help of a refrigerated autosampler. Separationof the amino acid peaks was accomplished by elutingthe column with 0.05 M disodium phosphate buffer (pH6.8) containing 27%methanol and 50mg/L of EDTAat 1ml/min flow rate. A coulochem detection system (ESAInc.) was operated at 2400 mv and 1600 mv to monitorelectrochemical species and a spectra physics integra-tor was used for quantification of GABAwith the help ofknown external standards. Examples of chromato-grams from theHPLC analysis comparing knownGABAstandards with dialysate samples are shown in Figure1.During a typical dialysis ‘‘session’’ (defined as a

complete dialysis experiment in one animal over aperiod of 6 to 8 h), themonkey was lightly sedated usingketamine hydrochloride (10 mg/kg, im), intubated,placed in a stereotactic head holder and wrapped in aheating blanket (37°C). The animal was then lightlyanesthetized (nonsurgical levels) using isofluorane gaswith vital signs monitored for the duration of theexperiment. The guide holder cap was removed andholes cleaned and flushed with sterile saline. Thedialysis probes were advanced through the dura andpositioned into the caudate nucleus and medial bank ofthe prefrontal cortex. The probe inlet was connected toa 1-ml Hamilton syringe filled with artificial cerebrospi-nal fluid (aCSF: 147mMNa1, 3 mMK1, 1.3 mMCa21, 1mMMg21, 155 mM Cl2 in 1.0 mM phosphate buffer, pH7.4, supplementedwith 0.15mMascorbate) and continu-ously perfused at 1 µl/min flow rate using a microinfu-sion pump (Harvard Apparatus, South Natic, MA). Theprobe outlet was placed in an amber color glass vial andsamples (25 µl) were collected every 25 minutes. Thesamples were split into 15 µl and 10 µl, frozen on dryice, and stored at 280°C until assayed by HPLC withelectrochemical detection.Stable GABA baseline levels were obtained typically

2 to 3 hours after probe insertion; three to four samplesimmediately prior to pharmacological manipulationwere used to calculate the mean baseline level. Localdrug administration was carried out via the dialysisprobe for 25–50 min followed by resumption of theaCSF for an additional 2 hours. Experimental manipu-lations included infusion of either high potassium (50mM), veratridine (10 µM), TTX (10 µM), or (2) nipecoticacid (1 mM). The Ca21 dependency was investigated byomitting Ca21 in the aCSF simultaneously with inclu-sion of veratridine or (2) nipecotic acid, and withincreasing the Mg21 concentration to 12 mM withMgCl2. To maintain isotonicity of Cl2, NaCl was re-duced from (Na1) 147 mM to 136 mM. Ca21-free aCSFalso contained 1 mM EGTA (Sigma) as a Ca21 chelator.In addition, when the concentration of K1 in the aCSFwas increased from 3 mM to 50 mM, the concentration

of Na1 was decreased from 147 mM to 100 mM tomaintain isotonicity.

Statistical analysis

To determine whether evoked changes in GABAlevels were significantly different from baselines orother manipulations, statistical comparisons of the rawdata (ng GABA/10 µl) were made using a one-wayanalysis of variance with repeated measures. Eachmanipulation was carried out in a minimum of threemonkeys at least two times with each probe beingconsidered an individual experiment. For each manipu-lation, it was first determined whether there was anysignificant effect of time, with time being the repeatedmeasure. Time points were compared at which maxi-mum difference was observed (usually time 4 or 5) frombaseline levels (mean of times 1, 2, and 3) using thestudent t-test. The overall effect of time was significant,with P , 0.01, in every manipulation. Thus, presentedbelow are the planned comparisons at time of maxi-mum change with the baseline levels for each pharma-cological manipulation. For the prefrontal cortex, whileeach finding was replicated twice in at least twomonkeys, no statistical analysis was performed. Fig-ures for GABA levels in the prefrontal cortex are from asingle representative probe as this is thought to be themost effective way in presenting the data.

RESULTSIn vitro recovery

For the probes used in these experiments, in vitrorecovery rates varied from 33% to 48% across a 4 to 6mm length dialysis membrane. These recoveries did notdiffer markedly between new probes and probes usedafter two dialysis experiments.

Basal levels of GABA in the caudate nucleusand prefrontal cortex

After implantation of the dialysis probe, extracellularGABA levels tended to be high during the first hour ofsampling and dropped to a stable output averaging0.364 6 0.08 ng/10 µl for the caudate and 0.062 6 0.007ng/10 µl for the prefrontal cortex during the next 2–3 h(Fig. 2). Thus, basal levels in the caudate were found tobe six times that in the prefrontal cortex.

Effects of local infusion of high potassium(K1, 50 mM)

Infusion of 50 mM K1 for 25 min resulted in asignificant increase (F 5 13.3, n 5 4, P , .001; t 5 2.9,P , .02) in GABA overflow during the next collection(Fig. 3A). This 250% increase was short-lived andreturned to baseline within 50 minutes. A similarincrease was seen in the prefrontal cortex after highpotassium infusion (Fig. 3B).

287CORTICAL AND SUBCORTICAL GABA RELEASE IN THE MONKEY

Fig. 1. Representative original chromatograms showing separa-tion of OPA-BME derivative of GABA from: (A) artificial CSF contain-ing 1 ng/10 µl GABA; (B) a 10-µl dialysate sample collected from theprincipal is sulcus of the prefrontal cortexmeasuring 0.08 ng of GABA;(C) an external standard made of artifical CSF containing 1 ng/10 µlGABA; (D) a 10-µl sample collected from the caudate nucleus measur-ing 0.32 ng of GABA. Note that the representative chromatograms for

the prefrontal cortex and the caudate nucleus were recorded atdifferent scales during the analysis. GABA signal for the prefrontalcortex was plotted at attenuation 64 and the caudate nucleus wasrecorded at attenuation 256. Moble phase was made of 0.05 MNa2HPO4, 50 mg EDTA/L, 27% Methanol (v/v), pH 6.8. Detection wasdone using a coulochem detector, 2400 and 1600 mv and chromato-grams were recorded by Spectraphysics integrator.

288 B.S. KOLACHANA ET AL.

Effects of local veratridine infusionand of veratridine in Ca21-free aCSF

Local veratridine (10 µM) infusion produced a robust(1,700%) and significant enhancement in GABA levelsin the caudate (Fig. 4A; F 5 4.2, n 5 4,P , .001; t 5 3.5,P , .01). The multifold increase in GABA was mark-edly reduced (300%) when veratridine (10 µM) wascoinfused with Ca21-free aCSF (n 5 4, t 5 3.6, P , .02).While the reduction was substantial, it did not preventa veratridine-induced increase of GABA levels. In theprefrontal cortex, veratridine infusion also produced amultifold increase in GABA overflow which was nearlyeliminated by exclusion of Ca21 in the aCSF (Fig. 4B).

Effects of local infusion of (2) nipecotic acidand (2) nipecotic acid in Ca21-free aCSF

Nipecotic acid (1 mM), a high affinity GABA uptakeinhibitor, resulted in significant increases in GABAlevels (670%, F 5 6.75, n 5 6, P , .001; t 5 6.7,

P , .001). These levels remained elevated during thenext hour with a gradual decline to basal levels.Nipecotic acid-induced increases were markedly inhib-ited (n 5 6, t 5 9.7, P , .001) when coinfused withCa21-free aCSF, yet were still above (300%) baselinelevels (n 5 6, t 5 16, P , .001; Fig. 5A). A similarpattern of results was seen in the prefrontal cortex (Fig.5B).

Effects of local infusion of (2) nipecotic acidand (2) nipecotic acid plus TTX (10 mM)

TTX, a sodium channel blocker, was coinfused withnipecotic acid to determine the voltage sensitivity of the

Fig. 2. A: Mean (6S.E.) GABA basal levels (ng/10 µl) in thecaudate nucleus of the rhesus monkey. Extracellular GABA levelsstabilized after about 2 h and remained at relatively constant levelsduring the next 4–5 h. The average GABA basal level in the caudatenucleus was 0.364 6 0.08 ng/10 µl (n 5 5).B:GABAbasal levels in themedial bank of the principalis sulcus in the prefrontal cortex. Basallevels stabilized after about 2 h and remained so over the next 3–4 h.GABA overflow in the prefrontal cortex (n 5 5), averaging 0.062 60.007 ng/10 µl, was about one-sixth that in the caudate nucleus.

Fig. 3. A: Infusion of 50mMK1 resulted in a significant increase inGABA overflow (asterisk, n 5 4, t 5 2.9, P , .02). This 250% increasewas short-lived and returned to near basal levels within 25 min afterstopping the K1 administration. B: One example from an experimentin the prefrontal cortex demonstrating the effects of high K1 levels onGABA overflow. As with the caudate nucleus, we observed a multifoldincrease in extracellular GABA.

Fig. 4. A: Administration of veratridine (open triangles) resultedin a significant (asterisk, n 5 4, t 5 3.5, P , .01) multifold (1,700%)increase of extracellular GABA in the caudate nucleus. This robustincrease was significantly (asterisk, n 5 4, t 5 3.6, P , .02) decreasedand no longer enhanced over baseline levels when the veratridine wascoinfused with Ca21-free aCSF (closed triangles).B:An example of theextracellular GABA response in prefrontal cortex to administration ofveratridine (open circles) and coinfusion of veratridine with Ca21-freeaCSF (closed circles). As found in the caudate nucleus, depolarizationstimuli evoked a large and significant increase in GABA followingadministration of veratridine, and near elimination of this evokedresponse by coinfusion of Ca21-free aCSF, suggesting monitored GABAoverflow was dependent on Ca21.

Fig. 5. A: Administration of the high affinity uptake blocker, (2)nipecotic acid in the caudate nucleus on extracellular GABA levels(open triangles). Nipecotic acid evoked a large significant (asterisk,n 5 6, t 5 6.7, P , .001) increase in GABA release (670%) in thecaudate nucleus which was significantly (double asterisk, t 5 9.7,P , .001) attenuated by coinfusion with Ca21-free aCSF (closed tri-angles). Although attenuated, these lower levels were still signifi-cantly above baseline (double asterisk, n 5 6, t 5 16, P , .001). B:Anexample of evoked GABA release in the prefrontal cortex followinglocal administration of nipecotic acid (open circles). As noticed in thecaudate nucleus, this robust response to nipecotic acid was markedlyreduced when coinfused with Ca21-free aCSF (closed circles).

289CORTICAL AND SUBCORTICAL GABA RELEASE IN THE MONKEY

evoked GABA release, as a result of the nipecotic acidinfusion. Inclusion of TTX with the nipecotic acidsignificantly reduced the GABA release induced by thenipecotic acid in the caudate (n 5 6, t 5 7.6, P , .001).While GABAlevels (300%) were reduced, they were stillsignificantly above basal levels (n 5 6, t 5 30.2,P , .001; Fig. 6A). Again, a similar pattern of resultsoccurred in the prefrontal cortex (Fig. 6B).

DISCUSSION

The results of our experiments in the rhesus monkeydemonstrated that the recovered extracellular GABA islargely of neuronal origin. Using in vivo microdialysis,we found extracellular GABAbasal levels in the prefron-tal cortex and the caudate nucleus to stabilize within2–3 hours after probe insertion. These levels remainedstable over at least a 4 to 5 hour period. The basal levelsreported here for the caudate nucleus in the monkeyappear to be much higher than that reported in rats.Basal levels previously reported for the rat appear tohave a large range, 0.18 pmol/10 µl (Osborne et al.,1991) to as large as 3.9 pmol/10 µl (Tossman et al.,1986) with 0.2 pmol/10 µl more often cited (Biggs et al.,1995; Bourdelais and Deutch, 1994; Drew et al., 1989;Osborne et al., 1990). In the monkey, we found GABAlevels to average 3.5 pmol/10 µl which is within therange but appears to be a higher value than generallyreported for the rat.The GABA values reported in this study and in

previous studies are not corrected for probe recoveries.Thus, at least some of the apparent differences betweenthe monkey and rat GABA values may be accounted forby the differences in probe recovery. In the presentstudy, we used probes with in vitro recovery ratesgreater than 30% as compared 16% typically reportedfor the rat (Anderson and DiMicco, 1992; Drew et al.,1989; Osborne et al., 1990). Some important factorsthat may affect recovery include exposed membrane

lengths and perfusion flow rates. In the monkey, weused 4 to 6-mm membranes compared with the moretypical 2 mm used in the rat. Because the caudate ismuch larger in the monkey brain than in the rat, wemade custom probes with longer probe membranes tomaximize recovery. Slower flow rates might also resultin greater recovery, and in the present study the flowrate was 1 µl/min compared with 2 µl/min often usedwith the rat. Thus, it might be difficult to make directcomparisons about basal levels reported here for themonkey with previously reported levels in the rat.The GABA overflow in the caudate nucleus in the

monkey was on average six times that in the prefrontalcortex. The relative differences in GABA basal levelsreported here are consistent in most cases with thecaudate having substantially higher basal concentra-tions than that in the prefrontal cortex in the rat(Anderson and Di Micco, 1992; Tossman et al., 1986).These regional differences of similar magnitude areconsistant with the notion that the GABA levels mea-sured here in the monkey reflect the higher recoveryrates. It should be noted that in one report basal GABAlevels in the prefrontal cortex were higher than thosereported for the striatum (Bourdelais and Deutch,1994). Despite the large difference in basal levels,response to pharmacological manipulations was nearlyidentical in both regions.The aim of the present study was to determine

whether the GABA levels observed were related toimpulse-dependent neurotransmission. Depolarizationinduced by increasing K1 concentration or by activatingvoltage-dependent sodium channels by administeringveratridine significantly increased GABA overflow.Moreover, in both cases and in both regions, the evokedincrease in GABA release was attenuated at least 50%by the removal of Ca21 in the aCSF.The release of GABA was also examined using the

high affinity uptake inhibitor and releasing agent (2)nipecotic acid. Nipecotic acid alone resulted in a multi-fold and long-lasting increase in basal GABA levels inthe caudate nucleus and seemingly in the prefrontalcortex as well. Coinfusion with Ca21-free aCSF or withTTX attenuated the nipecotic acid induced increase by50%. It appears, in the monkey caudate nucleus andmost likely the prefrontal cortex, increased GABAlevels seen after K1, veratridine, or nipecotic acidadministration were both Ca21-sensitive and voltage-dependent. Thus, it could be concluded that the extracel-lular GABA levels recovered using in vivo microdialysisappear in large part to be of neuronal origin. BecauseGABAdepletion was not complete after Ca21-free aCSFor TTX administration, the present data also suggestthat some extracellular GABA may originate eitherfrom metabolic activity or non-Ca21-dependent mecha-nisms. It must be noted, however, that it would benearly impossible to remove all Ca21 from the extracel-lular space as diffusion will replace it to the regions it is

Fig. 6. A: The nipecotic acid-evoked increase (asterisk) in GABAlevels in the caudate nucleus (open triangles) were significantly(double asterisk, n 5 6, t 5 7.6, P , .001) attenuated when nipecoticacid was coinfused with TTX (closed triangles), confirming voltage-dependant release. B: An example of enhanced extracellular GABAlevels in the prefrontal cortex in response to nipecotic acid administra-tion (open circles). As in the caudate nucleus, this response wasmarkedly reduced when coinfused with the Na1 channel blocker TTX.

290 B.S. KOLACHANA ET AL.

being withdrawn. Thus, complete elimination of GABAis not probable.Most of the in vivo studies of GABA neurotransmis-

sion have been conducted in rats using either push-pullcannula (Girault et al., 1986; Tuomisto et al., 1983; Vander Heyden et al., 1980) or microdialysis cannula(Bourdelais and Kalivas, 1992; Drew et al., 1989; Kehrand Ungerstedt, 1988; Osborne et al., 1990; Smith andSharp, 1994a, b; Timmerman et al., 1992; Tossman etal., 1986;Westerink and de Vries, 1989). Similar conclu-sions have been reached from these studies involvingdorsal striatum (Campbell et al., 1993; Kehr andUnger-stedt, 1988; Osborne et al., 1990), n. accumbens (Smithand Sharp, 1994b), caudate nucleus (Westerink andDeVries, 1989), ventral pallidum (Bourdelais and Kali-vas, 1992), and the frontal cortex (Anderson andDiMicco, 1992; Tossman et al., 1986). However, GABAoverflow as described in some earlier reports has notalways been demonstrated to be Ca21-sensitive andvoltage-dependent (e.g., Bernath and Zigmond, 1988;Drew et al., 1989; Westerink and DeVries, 1989). Sensi-tivity to TTX may depend in part on the presence of ananesthetic agent (Drew et al., 1989; Osborne et al.,1990; Smith and Sharp, 1994b). Osborne et al. (1990)reported TTX effects in reducing extracellular GABA inanimals in which probes had been implanted 24 hbefore sampling; in contrast, there were no effects just2.5 h after implantation while still under the effects ofthe anesthetic agent. The lack of an effect of TTX hasbeen corroborated elsewhere (Bourdelais and Kalivas,1992; Campbell et al., 1993). In another study, in bothconscious and halothane-anesthetized rats, TTX com-pletely blocked electrically evoked nigral GABA re-lease, whereas basal release was unaffected by TTXinfusion (Biggs et al., 1995). Not all studies even in theawake rat have been able to show sensitivity of basallevels of GABA to TTX (Timmerman et al., 1992;Westerink and DeVries, 1989).It has been shown that stimulus-evoked efflux of

neurotransmitter release from the GABAergic synapto-somes possessed properties typical of those found forconventional synaptosome preparations and brain slicesand such release has been considered as evidence ofphysiological significance (de Belleroche and Bradford,1972; Docherty et al., 1987; Norris et al., 1988). Asmentioned previously, pharmacological challenges havebeen produced inconsistent results when relying onbasal GABA levels. In the present experiment, wetested the putative sensitivity of GABA release to TTXduring stimulation and demonstrated substantial reduc-tion in GABA as a result of blocking sodium channelfunction. As demonstrated here, the decrease tends notto be absolute but more often in the 50% range. This isin contrast with TTX-induced reduction of 80% to 90%reported with neurotransmitters such as dopamine andacetylcholine (Drew et al., 1989; Saunders et al., 1993;

Smith et al., 1994). This suggests at least some of theGABA pool is of non-neuronal origin. It is possible thatprior inconsistencies reflect low basal GABAlevels froma neuronally comprised pool combined with high vari-ability across animals. To avoid this potential problem,we focussed on pharmacologically evoked GABA levels.In the case of stimulated release, GABA release wasboth Ca21- and TTX-sensitive.It has been noted that GABA neurotransmission, as

well as that of other neurotransmitters, may be sensi-tive to anesthesia. In the present experiments, weinitially used a small dose (,10 mg/kg) of Ketaminehydrochloride to sedate themonkey and throughout thedialysis session the monkey was kept sedated withisofluorane gas. Ketamine affects excitatory amino acidtransmission via NMDA receptor-mediated processes(Desce et al., 1992); however, we used a relativelysmaller sedative dose (10 mg/kg), and observationswere made hours past its maximum effect. Isofluoranegas anesthesia reduces depolarization effects by alter-ing calcium channel function; thus, overall neuronalactivity is reduced (Ries and Puil, 1993; Study, 1993). Itshould be noted that a minimal level of isofluorane gaswas used with stability achieved while above surgicallevels of anesthesia. As noted earlier, there appears tobe some discrepancies in the literature about the effectof anesthesia on the release of GABAwith TTX admin-istration (Girault et al., 1986; Van der Heyden et al.,1980; Westerink and De Vries, 1989). In addition, theremay be some differences in basal levels of differentneurotransmitters that reflect the type of anesthesiaemployed (seeAnderson and DiMicco, 1992; Tossman etal., 1986). While it is unlikely the direction of thepharmacological changewould be different in the awakeanimal, we can not rule out a possible interaction withthe anesthetic agent on GABA release reported here.The results from this study support the use of micro-

dialysis for studying extracellular amino acids in theprimate brain. With the exception of extracellularGABA levels recovered from globus pallidus in parkin-sonian monkeys (Robertson et al., 1991), there are noother published reports of in vivo measurement ofextracellular GABA in the primate brain. Thus, thebasal GABA values observed in the caudate nucleus inthe monkeys in this study are the only normal valuesavailable for comparison. This study also demonstratesthe ability to monitor GABA levels in the prefrontalcortex and thus provides the basis for future studiesinvestigating the interaction of neurotransmitter sys-tems (e.g., GABA and dopamine) in regulating prefron-tal cortical-striatal interactions.

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