RESEARCH ARTICLE

Decreased GABAA Receptors and Benzodiazepine Binding Sitesin the Anterior Cingulate Cortex in AutismA. Oblak, T.T. Gibbs, and G.J. Blatt

The anterior cingulate cortex (ACC; BA 24) via its extensive limbic and high order association cortical connectivity toprefrontal cortex is a key part of an important circuitry participating in executive function, affect, and socio-emotionalbehavior. Multiple lines of evidence, including genetic and imaging studies, suggest that the ACC and gamma-amino-butyric acid (GABA) system may be affected in autism. The benzodiazepine binding site on the GABAA receptor complexis an important target for pharmacotherapy and has important clinical implications. The present multiple-concentrationligand-binding study utilized 3H-muscimol and 3H-flunitrazepam to determine the number (Bmax), binding affinity (Kd),and distribution of GABAA receptors and benzodiazepine binding sites, respectively, in the ACC in adult autistic andcontrol cases. Compared to controls, the autistic group had significant decreases in the mean density of GABAA receptorsin the supragranular (46.8%) and infragranular (20.2%) layers of the ACC and in the density of benzodiazepine bindingsites in the supragranular (28.9%) and infragranular (16.4%) lamina. In addition, a trend for a decrease in for the densityof benzodiazepine sites was found in the infragranular layers (17.1%) in the autism group. These findings suggest that inthe autistic group this downregulation of both benzodiazepine sites and GABAA receptors in the ACCmay be the result ofincreased GABA innervation and/or release disturbing the delicate excitation/inhibition balance of principal neurons aswell as their output to key limbic cortical targets. Such disturbances likely underlie the core alterations in socio-emotional behaviors in autism.

Keywords: autistic; anterior cingulate cortex; GABA; post-mortem; ligand binding

Introduction

The anterior cingulate cortex (BA 24; ACC), part of theoriginal Papez circuit of emotion [Papez, 1937], isconsidered a part of the brain’s limbic lobe and isinvolved in attention and the regulation of cognitiveand emotional processing [Allman, Hakeem, Erwin,Nimchinsky, & Hof, 2001; Bush, Luu, & Posner, 2000;Heimer & Van Hoesen, 2006]. Anterior cingulate lesionsare known to cause blunted affect, impulsivity, disinhibi-tion, aggressive behavior, disabling obsessions and com-pulsions, and impaired social judgment including theinability to interpret social cues [Devinsky, Morrell, &Vogt, 1995].Neuroanatomical studies have shown that the ACC

encompasses a number of specialized subdivisions thatsubserve a vast array of cognitive, emotional, motor,nociceptive, and visuospatial functions [Bush et al., 2000;Heimer & Van Hoesen, 2006; Isomura & Takada, 2004;Posner, Rothbart, Sheese, & Tang, 2007; Zhuo, 2006]. TheACC is intimately connected to prefrontal cortex (PFC) aswell as key limbic structures including the hippocampalformation and the amygdala, positioning it as a key

structure for roles in executive function, learning,memory and socio-emotional behavior [Pandya, VanHoesen, & Mesulam, 1981; Petrides & Pandya, 2007;Vogt & Pandya, 1987].In the neurodevelopmental disorder autism, character-

ized by impairments in communication including lan-guage, as well as narrowly focused interests and poorsociability, there is demonstrated neuropathology in theACC in some reported cases. These were first described byBauman and Kemper [1985] and recently quantitativelyconfirmed by Simms, Kemper, Timbie, Bauman, and Blatt[2009], who reported an increased cell packing densityand decreased cell size in several subregions within theACC. Furthermore, positron emission tomography (PET)studies have demonstrated abnormalities in the activa-tion of the ACC in autism during a verbal learning task[Haznedar et al., 1997].Recent neurochemical research has focused on the

gamma-amino-butyric acid (GABA) system and its possi-ble involvement in a number of limbic and cerebellarregions in autistic cases [Blatt et al., 2001; Fatemi et al.,2002; Fatemi, Folson, Reutiman, & Thuras, 2009a;Fatemi, Reutiman, Folson, & Thuras, 2009b; Guptill

INSAR Autism Research 2: 205–219, 2009 205

Received March 12, 2009; revised June 15, 2009; accepted for publication June 21, 2009

Published online 31 July 2009 in Wiley InterScience (www. interscience.wiley.com)DOI: 10.1002/aur.88& 2009 International Society for Autism Research, Wiley Periodicals, Inc.

From the Boston University School of Medicine, Anatomy and Neurobiology, Boston, Massachusetts (A.O., G.J.B.); Boston University School of Medicine,Pharmacology and Experimental Therapeutics, Boston, Massachusetts (T.T.G.)

Address for correspondence and reprints: Adrian Oblak, Boston University School of Medicine, Department of Anatomy and Neurobiology and,Department of Pharmacology and Experimental Therapeutics, 715 Albany Street, Boston, MA 02118. E-mail: aoblak@bu.eduGrant sponsor: National Institutes of Health; Grant numbers: NIH U54 MH66398; NIH NINDS NS38975.

et al., 2007; Yip, Soghomonian, & Blatt, 2007, 2008,2009]. GABA plays a crucial role in normal corticalfunctioning, information processing, and the formationof brain cytoarchitecture during development [Conti,Minelli, & Melone, 2004; Di Cristo, 2007; Fritschy, Benke,Johnson, Mohler, & Rudolph, 1997; Mohler, Benke,Benson, Luscher, & Fritschy, 1995a]. One of the proposedcauses of impaired information processing and socialbehavior in autism is an altered balance betweeninhibition and excitation in the brain [Rubenstein &Merzenich, 2003]. The distribution, electrophysiology,and molecular characteristics of GABA receptors changemarkedly during development, leaving the formationof the cortex vulnerable to aberrations in neurotrans-mission at key developmental periods. Seizures arefairly common in individuals with autism, occurringin approximately 25–33% of individuals [Olsson,Steffenburg, & Gillberg, 1988; Volkmar & Nelson, 1990]and may result from abnormal inhibitory control in keycortical areas. Genetic studies have also implicated theGABA system in autism. For example, Schroer et al.[1998] found abnormalities on chromosome 15q11-q13,which includes a cluster of three GABAA receptor genes(GABRa5, GABRb3, and GABRg3). Several genetic studieshave proposed the involvement of multiple GABAA

receptor subunits and suggest that, through complexinteractions, these subunits are involved in autism[Ashley-Koch et al., 2006; Ma et al., 2005].The GABAA receptor has binding sites for multiple

modulators, including benzodiazepines (BZDs), making ita target for pharmacological intervention. BZDs enhanceGABAA receptor mediated neurotransmission, and classi-cally act as sedative/hypnotics to reduce anxiety andsuppress seizure activity and panic attacks [Low et al.,2000]. In clinical studies, BZDs have been reported toelicit paradoxical behavioral responses in some autisticindividuals, producing increased anxiety and aggressivebehavior [Marrosu, Marrosu, Rachel, & Biggio, 1987].This variable response could be due to an alteration inBZD-sensitive GABAA receptors in the specific subset ofcases investigated.Taken together, there is compelling evidence that the

GABA system is impacted in autism and alterations in theGABAA receptor system likely play a role in the etiology ofthe disorder during development and may contribute tothe abnormal phenotype and the variable response topharmacotherapy. Changes in these key neural substratesin the ACC could disrupt critical circuits involved in socio-emotional behavior as well as other high-order associativefunctions especially via its abundant prefrontal corticalconnectivity. The aim of this study was to determine if thenumber, density, and distribution of GABAA receptors andBZD binding sites in the ACC are altered in postmortemadult autistic cases, and to explore how such alterationsmight impact individuals with the disorder.

Methods and MaterialsBrain Tissue and Case Data

Fresh frozen brain tissue from the ACC was obtainedfrom the Harvard Brain Tissue Resource Center (HBTRC)in Belmont, Massachusetts. All clinical cases werediagnosed as autistic and had Autism Diagnostic Inter-views (ADI) completed either pre- or post-mortem. Alltissue was provided by The Autism Research Foundation,Autism Tissue Program, and Harvard Brain TissueResource Center. A total of 17 blocks were obtained(7 autism and 10 controls) from the rostrum of theACC and stored at !801C. One autism case, ]3845, wasused for the muscimol study but not for the benzodia-zepine study due to limited availability of tissue sectionsas this case has been used for many of our previousstudies. A summary of the case details is seen in Table I.There was no significant difference in age or post-morteminterval (PMI) between autism and control groups(student t-test). Four of the seven autism cases had ahistory of at least one seizure (1078, 1484, 2825, and3845). Of those four cases, three had received treatmentwith anticonvulsant therapy (1078, 2825, and 3845). Allcases used in the study had an autism diagnosis ofmoderate to severe.

Multiple-Concentration Binding Assay

All tissue blocks were sectioned coronally at 20 mm usinga Hacker/Brights motorized cryostat at !201C. Sectionswere then thaw mounted on 2"3 inch gelatin coatedglass slides. For each of the seven concentrations, twosections from each case were used to determine totalbinding and one section from each case was used todetermine non-specific binding. Non-specific bindingwas measured by adding a high concentration of acompetitive displacer (see Table II) to the tritiated ligandand buffer solution. The ligand used for GABAA receptorswas [3H]-Muscimol (specific activity 36.6Ci/mmol; NewEngland Nuclear) and the ligand for BZD binding siteswas [3H]-flunitrazepam (specific activity 85.2Ci/mmol;New England Nuclear). To eliminate variability inbinding conditions, the selected sections from all caseswere assayed in parallel. Slides were then dried under astream of cool air overnight and loaded into X-raycassettes with a tritium standard (Amersham), apposedto tritium-sensitive film (3H-Hyperfilm, Kodak) andincubated for a period of time (see Table II). The exposedfilms were developed for 4 minutes with Kodak D19developer, fixed with Kodak Rapidfix (3min) at roomtemperature, and allowed to air dry. Slides were stainedwith thionin to determine cytoarchitecture and laminardistribution of the ACC (Fig. 1). Supragranular layerscorresponded to layers I–III and infragranular layers tolayers V–VI as layer IV is absent in the ACC [Vogt,Nimchinsky, Vogt, & Hof, 1995].

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Data Analysis

The film autoradiograms were digitized using an Inquirydensitometry system (Loats Associates) to gather quantita-tive measurements of optical density. The supragranularand infragranular layers were sampled from each case. The3H standards that were exposed with the sections were

used to calibrate the autoradiograms to quantify theamount of ligand bound per milligram of protein in thetissue sections. Optical density for the standards as afunction of specific activity (corrected for radioactivedecay) was fitted by nonlinear least squares regression tothe equation, optical density5B1" (1–10k1#specific activity$$1B3, by adjusting the values of the parameters k1, B1, andB3 using the Solver tool of Excel (Microsoft Office XPProfessional) to construct a standard curve, which was usedto convert the measured optical densities into nCi/mg.Binding in femtomoles per milligram of protein (fmol/mg)was calculated based on specific activity of the ligand.

Statistical Analyses

Specific binding of the tritiated ligands in the supragra-nular and infragranular layers from each subject was fittedindependently with a hyperbolic binding equation toestimate binding affinity (Kd) and number of receptors(Bmax) for each region. Examples of binding curves areshown in Figure 2. Least-squares nonlinear regression wascarried out using the Microsoft Excel Solver tool. Bmax datais reported as mean1standard error of the mean (SEM). Kd

is reported as the geometric average 10mean#log#Kd$$ % SEMav,calculated as SEMav5 (((10#x&s$–10x)1(10x–10#x!s$))/2),where x5mean(logKd) and s is the SEM of the log (Kd)

Table II. Multiple Concentration Binding Conditions for GABAA Receptors and Benzodiazepine Binding Sites

Ligand Target Concentration (nM) Displacer Exposure time

3[H]-Muscimol GABAA receptor [0.3, 1.5, 3, 8, 30, 60, 110] 100mM GABA 6–360 days3[H]-Flunitrazepam Benzodiazepine binding site [0.3, 1.5, 3, 5, 15, 30, 120] 100mM Clonazepam 10–180 days

Table I. Information for Cases in the Multiple Concentration Binding Studies

CASE Diagnosis Age PMI (hr) Medication history Cause of death Gender

1078!! Autism 22 14.3 Dilantin, Tegretol, Theodur, Phenobarbital Drowning Male1401 Autism 21 20.6 None Pneumonia, Sepsis Female1484! Autism 19 15 None Burns Male2825!! Autism 19 9.5 Klonopin, Mysoline, Phenobarbital, Thorazine Heart attack Male3845!! Autism 30 28.4 Dilantin, Mellaril, Phenobarbital Cancer Male4099 Autism 19 3 None Conj. Heart Failure Male5754 Autism 20 30.0 None Unknown MaleMean 21.4 17.34103 Control 43 23 None Heart attack/disease Male4104 Control 24 5 None Gun shot Male4188 Control 16 13 None Gun Shot Male4267 Control 26 20 None Accidental Male4268 Control 30 22 None Heart attack/disease Male4269 Control 28 24 None Heart disease Male4271 Control 19 21 None Epiglotitus Male4275 Control 20 16 None Accidental Male4364 Control 27 27 None Motor Vehicle Accident MaleMean 25.9 19.0

Cases with at least one asterisk (!) had a history of at least one seizure. Cases with two asterisks (!!) were being treated with an anticonvulsant at timeof death. All autism cases were considered mentally retarded.

Figure 1. A Nissl stained section from a control case (4103)demonstrates the cytoarchitecture of the human ACC (Area 24a-c).The box at left illustrates our sampling scheme dividing the cortexinto supragranular (I–III) and infragranular (V–VI) layers. [Colorfigure can be viewed online at www.interscience.wiley.com]

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values. Significance testing for differences in Kd was carriedout using log(Kd) values. Two-Way Analysis of Variance(ANOVA) was used to determine if there was a significantdifference in receptor or binding site number betweenautistic and control cases, between supragranular andinfragranular layers, or if an interaction existed. Non-parametric Mann–Whitney U tests were used to determineif seizures or anticonvulsant therapy in the autism caseshad an effect on receptor binding.

Results

Table III reports specific binding of [3H]-muscimol and[3H]-flunitrazepam in the ACC from autistic and controlcases. Overall, the supragranular layers of the ACC ofboth autistic and control cases contained a higher densityof GABAA receptors and benzodiazepine binding sitesthan the infragranular layers, as measured by the Bmax

values (P50.0004, P5 0.0003, respectively). We founddecreases in the density of both GABAA receptors andBZD binding sites in the supra- and infragranular layers ofautistic cases. Binding affinity (Kd) did not differsignificantly between autistic and control cases for eitherligand (Fig. 3; Table IV). Binding data for both autisticand control cases were well-fitted by a single-componenthyperbolic binding equation, consistent with a single,homogeneous class of binding sites (Fig. 3). Figures 5 and7 show the specific binding curves for each of the cases.Figures 4 and 6 show typical examples of binding of asingle concentration (15nM) of [3H]-muscimol and [3H]-flunitrazepam, respectively, in typical sections from anautistic and a control case. The autoradiograms havebeen pseudocolored using the Inquiry program to bettervisualize binding density, with red corresponding to highbinding (optical density) and purple to low binding. TheNissl stained section in Figure 1 was used to identify theregion of interest as well as the division of the region into

Figure 2. Sample binding curves from two cases (autistic on left, control on right) demonstrating specific binding in the supragranularlayers from each concentration of tritiated ligand. Data from [3H]muscimol is in the upper panel and data from [3H]flunitrazepam is in thebottom panel. [Color figure can be viewed online at www.interscience.wiley.com]

208 Oblak et al./Decreased GABAA receptors in autism INSAR

supragranular (layers I–III) and infragranular (layersV–VI) layers. The distribution of Bmax values across layersfor GABAA receptors and benzodiazepine binding sites isgiven in Table III. Sample binding curves demonstratingspecific binding in the supragranular layers from each ofthe seven concentrations of tritiated ligand can be foundin Figure 2.

GABAA Receptor Binding

Multiple concentration binding experiments revealedthat the mean Bmax value for all autistic cases fell belowthat for control cases. The results from the seven-concentration binding study indicate a significant reduc-tion of GABAA receptors (Bmax) in both the supragranular(P55.2"10!6) and the infragranular (P5 0.04) layers ofthe ACC in the autistic group (n57) when compared toage- and PMI matched controls (n59). The Bmax valuesdemonstrate that there was a 46.8% reduction inGABAA receptors in the supragranular layers of theACC and a 20.2% decrease in the number of GABAA

receptors in the infragranular layers in autism. The threecases with the lowest Bmax values in the supragranularlayers did have a history of seizure. However, the casedemonstrating the highest binding also had a history ofseizure. In the infragranular layers, two of the cases with ahistory of seizure fell below the mean Bmax values of theautistic group. In contrast, there was no difference inbinding affinity comparing autistic and control cases(Fig. 2).

Figure 3. Summary of mean Kd values for GABAA (a) and BZD (b)receptor binding in autistic and control cases. There was nosignificant difference between autistic and control cases in eitherligand or layers. Geometric means for Kd are reported here.

Table III. Summary of Bmax Values for GABAA and BZD Receptor Binding in Autistic and Control Cases

Muscimol specific binding (fmol/mg protein) Flunitrazepam specific binding (fmol/mg protein)

Case Diagnosis Supragranular Infragranular Supragranular Infragranular

1078 Autism 361.21 428.58 261.17 229.801401 Autism 497.67 473.07 219.45 162.491484 Autism 574.10 446.28 321.89 181.762825 Autism 371.43 525.64 306.07 198.433845 Autism 432.83 345.314099 Autism 558.68 424.41 381.36 217.175754 Autism 480.17 387.30 337.30 222.49Mean1SEM 468.01131.80 432.94121.95 304.54121.65 202.02110.664103 Control 830.26 493.42 353.76 204.554104 Control 1030.31 522.53 335.13 231.794188 Control 873.65 328.05 453.82 288.034267 Control 712.05 448.30 447.37 214.594268 Control 741.45 510.97 391.17 234.534269 Control 827.73 742.58 542.08 215.164271 Control 1104.62 554.65 509.70 313.104275 Control 1003.04 635.73 384.67 192.154364 Control 788.97 694.93 420.54 279.98Mean1SEM 879.11147.14 542.35140.49 427.03122.90 241.54114.01P-value (t-test) 5.16" 10!6 0.035 0.0028 0.043

Table IV. Summary of Muscimol and Flunitrazepam BindingAffinity in the Anterior Cingulate Cortex

Muscimol bindingaffinity (nM)

Flunitrazepam bindingaffinity (nM)

Group Supragranular Infragranular Supragranular Infragranular

Autism 13.511.26 9.6911.70 4.4410.61 4.7910.19Control 1311.55 9.3211.60 6.1011.45 5.7610.82

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Benzodiazepine Binding Sites

With respect to the benzodiazepine binding sites, allbut one autistic case (in the infragranular layers) fellbelow the average Bmax value of the control group.The results from the seven concentration bindingstudy indicated a significant decrease in the Bmax

values in the supragranular layers (P50.003) andinfragranular layers (P50.04) of the ACC in autism.The multiple concentration binding study revealed a28.7% and 16.4% reduction in benzodiazepine bindingsites in the supragranular layers and infragranular layers,respectively, in autistic cases. In the supragranularlayers, one of the two autistic cases that fell below themean Bmax value for autistic cases had a history of seizure.In the infragranular layers, two of the cases that fellbelow the mean Bmax value had a history of seizure. Therewas no significant difference observed in binding affinity(Fig. 2).

Effect of Seizure on Binding

Four of the cases in this study had a history of having atleast one seizure. In the muscimol binding study, therewas no significant difference in binding between autisticcases with seizure (n54) and autistic cases withoutseizure (n53). Furthermore, in the flunitrazepam bind-ing study no significant difference in binding wasobserved between autistic cases with seizure (n53) andautistic cases with no seizure history (n54).

Effect of Pharmacotherapy on Binding

The cases were divided into two groups (autism with ahistory of anticonvulsant therapy and autism with nohistory of anticonvulsant therapy) to determine if themedication had a significant effect on binding. In themuscimol binding experiments, three of the seven caseswere taking anticonvulsants at the time of death. Using a

Figure 4. Examples of pseudocolored images of [3H]-muscimol binding (15 nM) in autistic (a) and control (b) cases. Solid black arrowsdemonstrate the location of sampling in the supragranular layers; arrows with dashed lines indicate sampling in the infragranular layers.In the [3H]muscimol binding experiments, the number of GABAA receptors in the supragranular (P5 5.16" 10!6) and infragranular layers(P5 0.04) was significantly (!!) decreased (c). [Color figure can be viewed online at www.interscience.wiley.com]

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Mann–Whitney U non-parametric test, a trend (P50.06)was observed in the supragranular layers, such that thosereceiving anticonvulsant therapy had a trend towardsdecreased GABAA receptors. There was no effect ofanticonvulsant therapy on the decrease in receptorbinding in the infragranular layers (P5 0.99 andP50.113, respectively). In the flunitrazepam bindingstudy, half (n53) of the cases were receiving antic-onvulsant therapy at the time of death. There was nosignificant difference in the number of benzodiazepinebinding sites in the supra- or infragranular layers in theautistic group receiving anticonvulsants compared to theautistic group not receiving anticonvulsants.

DiscussionNormal Functions of the ACC

The ACC (Brodmann area 24; BA 24) has been shownto participate in a variety of functions including executive,evaluative, and cognitive functions and emotion [Devinskyet al., 1995; Lane et al., 1998; Vogt, Finch, & Olson, 1992].

Processing of high order sensory and multimodal informa-tion occurs via connections with the PFC and parietalcortex as well as the motor system and the frontal eye fields[Allman et al., 2001; Calzavara, Mailly, & Haber, 2007;Kaitz & Robertson, 1981; Robertson & Kaitz, 1981; Vogtet al., 1987]. When effort is needed to carry out a task suchas in early learning and problem solving [Allman et al.,2001], the ACC is recruited, and many studies attributefunctions such as error detection and anticipation of tasks,motivation, andmodulation of emotional responses to thisregion [Bush et al., 2000; Nieuwenhuis, Yeung, van denWildenberg, & Ridderinkhof, 2003; Posner, Pothbart, &Digirolamo, 1999; Posner et al., 2007]. The ACC is anintegral part of many higher order functions. Disruptionsof this region, at any level, cellular or chemical, could leadto social, communication, and other behavioral deficits.

The Role of GABA in the ACC: Implications for Connectivity

GABAergic neurons are essential to information proces-sing in virtually every brain region, and thus analteration in GABAergic function, of any nature, in aregion such as the ACC, with widespread corticalconnections, may result in impairments of informationprocessing, a feature considered to be central to autism[Belmonte et al., 2004]. Information processing in thebrain occurs via feed-forward and feedback projections.Long-range cortico-cortical input is provided by ‘‘feed-back’’ projections from higher order regions that primar-ily target the distal dendritic tufts of pyramidal cells fromall layers that terminate in layer I [Rockland & Drash,1996]. Layer II pyramidal axons make local connectionswithin layers II and III, and layer III pyramidal axonsramify most extensively in layers II/III and V [Thomson,West, Wang, & Bannister, 2002]. Layer V pyramidal cellsproject to all other layers of the cortex and represent amajor route of excitatory feedback projections to thesuperficial laminae [Burkhalter, 1989]. Abnormalities inthe integrity of the layers through cell loss, abnormalcytoarchitecture or receptor changes, as observed in thisstudy, may interfere with cortical processing.The present observations demonstrate the highest

binding to GABAA receptors and benzodiazepine bindingsites in the supragranular layers of the anterior cingulatecortices in agreement with previous autoradiographic andimmunohistochemical data [Fritschy et al., 1997; Mohleret al., 1995a,b]. Previous receptor binding experimentshave reported that [3H]-muscimol labeled GABAA recep-tors and [3H]-flunitrazepam-labeled benzodiazepine bind-ing sites were significantly reduced in the hippocampus ofadult autistic individuals [Blatt et al., 2001; Guptill et al.,2007]. This study found significant reductions in GABAA

receptors and benzodiazepine binding sites in the supra-granular and infragranular layers of the ACC. These resultssuggest abnormal connectivity between the ACC and the

Figure 5. Examples of individual [3H]muscimol binding curves.Specific binding of [3H]-muscimol to the supragranular (a) andinfragranular (b) layers of the ACC in seven autistic and ninecontrol subjects. Smooth curves indicate fits to the hyperbolicbinding equation. [Color figure can be viewed online atwww.interscience.wiley.com]

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regions of the cortex to which the ACC projects (e.g. PFC)and also abnormal reciprocal connections between thehigher order association cortices and the ACC. This studyfurther suggests that the limbic system is abnormal inautism, which may contribute to the social and emotionalaspects of the disorder. In autism, decreases in inhibitoryreceptors on pyramidal neurons or interneurons mayrestrict normal connectivity within the ACC and betweencortical regions. Laminar and receptor-specific decreases inthe infragranular layers may support a role of these layersin a loss of normal signal transmission and a decrease inthe information relaying to and from the thalamus [Kaitz& Robertson, 1981; Yakovlev & Locke, 1961].Inadequate inhibitory damping of excitatory projec-

tions to other excitatory cells further downstream couldresult in undesirable positive feedback loops, and coulddisrupt signaling from the thalamus as it passes through

cortical columns. Excitatory projections from pyramidalcells to interneurons would help control the excitatoryflow through the cortex by inhibiting cells responsible fortheir inputs as soon as appropriate outputs to the nextstage of processing have been attained [Burkhalter, 1989].However, abnormal receptors in this region woulddecrease the likelihood of controlling the excitatory flow.This abnormal connectivity may result in changes incognition and in the proper understanding of socio-emotional interactions that are common in autism.

GABAA Receptors During Development and RelatedNeuropathology of the Autistic Brain

Aberrant development of GABAergic circuits is not uniqueto autism and has been implicated in other neurodevelop-mental disorders such as schizophrenia [Benes, Vincent,

Figure 6. Examples of autistic (a) and control (b) pseudocolored images of [3H]-flunitrazepam (15 nM) binding in the ACC. Solid blackarrows demonstrate the location of sampling in the supragranular layers; arrows with dashed lines indicate sampling in the infragranularlayers. There was a significant decrease (!!) in benzodiazepine binding sites in the supragranular layers (P5 0.003) and infragranularlayers (P5 0.04) in autism (c). [Color figure can be viewed online at www.interscience.wiley.com]

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Alsterberg, Bird, & SanGiovanni, 1992] and Tourette’ssyndrome [Kalanithi et al., 2005]. Compelling evidenceindicates that GABA and GABA receptors play a role inseveral developmental processes. During development,GABAA receptors participate in proliferation, migration,and differentiation of precursor cells that orchestrate thedevelopment of the embryonic brain [Akerman & Cline,2007; Barker et al., 1998; Conti et al., 2004; Di Cristo, 2007;Mohler, Fritschy, Crestani, Hensch, & Rudolph, 2004].Despite its known effects in adults as a strong inhibitoryneurotransmitter, during development GABAA mediatedneurotransmission plays an excitatory role in the develop-ing nervous system, shifting to an inhibitory function inthe mature nervous system as a consequence of changes inCl! gradients [Ben-Ari, Gaiarsa, Tyzio, & Khazipov, 2007].Eliminating excitatory GABA action between postnataldays 4 and 6 in rats resulted in severe impairment of themorphological maturation of cortical neurons [Cancedda,

Fiumelli, Chen, & Poo, 2007; Heck et al., 2007]. Further-more, modulation of GABAA receptor-mediated mechan-isms in the immature cerebral cortex of the rat has alsobeen shown to impair neuronal migration in vitro and invivo [Conti et al., 2004].In autism, pathological alterations in the cortical cytoarch-

itecture in autism resemble neuronal migration disorderspreviously described in the human cerebral cortex [Menget al., 2005; Corfas, Roy, & Buxbaum, 2004]. Simms et al.[2009] has shown irregular lamination and increased densityof neurons in the subcortical white matter of the ACC in fiveof nine cases examined, potentially occurring from adisrupted GABAergic receptor system. Furthermore, Baileyet al. [1998] demonstrated abnormal cytoarchitecture in thefrontal cortex, demonstrating changes in migrational beha-vior of the neurons to the cortical layers, and Casanova,Buxhoevden, Andrew, Roy, and Roy [2002], Casanova,Buxhoeveden, and Gomez [2003], and Casanova et al.[2006] has shown a reduced intercolumnar width ofminicolumns in dorsal lateral PFC as well as an abnormalnumber of minicolumns. Again, these results are suggestiveof a abnormality in the GABAergic receptor system duringdevelopment resulting in abnormal cytoarchitecture in theadult autistic brain. The changes observed in a limbic systemarea (ACC) and a region that it is highly connected with(PFC) suggests further a circuitry/connectivity problem. Thiscould potentially lead to abnormalities in cognitive proces-sing and higher order functioning, a theory central to autism.Bauman and Kemper [1985] first observed qualitatively

a decrease in cell volume in limbic structures of autisticbrain. In the ACC, a significant decrease in cell area wasobserved in the supragranular and infragranular layers ofarea 24b (a subregion of the ACC) as well as a significantdecrease in cell volume in the supragranular and infra-granular layers of the same region [Simms et al., 2009].More recently, Schumann and Amaral [2006] found aquantitative decrease in the volume of neurons in thelateral nucleus of the amygdala, a region with connectionsto the anterior cingulate [Porrino, Crane, & Goldman-Rakic, 1981]. An abnormal GABA receptor system duringdevelopment, such as one that is delayed in the switchfrom excitatory to inhibitory or one that retains the fetalcircuitry [Bauman & Kemper, 1985], could form the basisfor abnormal processing and decreased neuron volume inthe adult anterior cingulate in autism. These changescould interfere with socio-emotional and cognitive pro-cessing similar to what has been found in imaging studies[Calzavara et al., 2007; Haznedar et al., 2000; Kana, Keller,Minshew, & Just, 2007; Kennedy, Redcay, & Courchesne,2006; Shafritz, Dichter, Baranek, & Belger, 2008; Silk et al.,2006; Vollm et al., 2006]. A developmental deficiency inany of these roles would adversely affect the temporalordering of neurogenesis and synaptogenesis, therebyaffecting maturation of circuits that are later involved incomplex behaviors associated with the ACC.

Figure 7. Examples of individual [3H]-flunitrazepam bindingcurves. Specific binding of [3H]-flunitrazepam to the supragra-nular (a) and infragranular (b) layers of the ACC in six autistic andnine control subjects. Smooth curves indicate fits to thehyperbolic binding equation. [Color figure can be viewed onlineat www.interscience.wiley.com]

INSAR Oblak et al./Decreased GABAA receptors in autism 213

Effects of Seizure/Anti-convulsant Treatment on Bmax and Kd

of GABAA and Benzodiazepine Binding

Approximately, 80% of all GABAA receptors contain theclassical benzodiazepine binding site and are character-ized largely by the subunit combinations a1b2g2, a2b3g2,and a3b3g2 [Speth et al., 1980]. The benzodiazepine site,located at the interface between a and g subunits, isstructurally homologous to the GABA binding sitelocated in the a-b subunit interface. Epileptogenic seizureactivity is likely a result of a deficit in GABA-mediatedinhibition, and seizures are common in approximatelyone-fourth to one-third of autistic individuals [Baileyet al., 1998; Gillberg, Steffenburg, & Jakobsson, 1987;Olsson et al., 1988; Rutter, 1970; Volkmar & Nelson,1990]. In a PET imaging study, the binding of[11C]-flumazenil to benzodiazepine binding sites inepileptogenic foci was found to be reduced with nochange in binding affinity [Savic et al., 1988]. In a morerecent imaging study using [125I]-iomazenil, reductionsin central benzodiazepine binding sites were found in48% of individuals studied in medial temporal loberegions [Matheja et al., 2001], although this may be aconsequence of localized neuronal damage or neuronalloss in the regions with temporal lobe epileptogenicactivity. It should be noted that these changes wereoccurring in the hippocampus and may not be reflectiveof the changes occurring in the cortex.Four of the autistic cases studied had a history of seizure,

raising the question of whether the changes in [3H]fluni-trazepam and [3H]-muscimol binding could be a conse-quence of seizures. Alterations in GABAA receptor subunitexpression and composition in epilepsy are well documen-ted in human [Loup et al., 2006; Loup, Wieser, Yonekawa,Aguzzi, & Fritschy, 2000] and in animal models [Gilby,DaSilva, & McIntyre, 2005; Li, Wu, Huguenard, & Fisher,2006; Nishimura et al., 2005; Peng, Huang, Steil, MOdy, &Houser, 2004; Roberts et al., 2005]. In particular, there isevidence that seizures can induce a shift of GABAA receptorsubunit mRNA expression such that a4 subunit expressionis increased, whereas a1 subunit expression is decreased[Brooks-Kayal, Raol, & Russek, 2009]. An increase in a4-containing GABAA receptors at the expense of a1-contain-ing receptors could result in a decreased number of [3H]-flunitrazepam binding sites, as a4-containing receptors donot contain a [3H]-flunitrazepam binding site, but because[3H]-muscimol binding was also decreased argues that theresults reflect an overall deficit in GABAA receptors.In addition to the potential effect of seizures on GABAA

receptor expression, three subjects had a history oftreatment with anticonvulsants, including the barbitu-rate phenobarbital. Phenobarbital is a GABAA receptorpositive modulator that has been reported to reduceGABAA receptor binding in mice [Weizman, Fares, Pick,Yanai, & Gavish, 1989]. Phenobarbital withdrawal is

sometimes associated with seizures, suggesting thatchronic phenobarbital use alters GABAA receptor systems.There was no statistically significant difference betweenthe cases with and without seizures or those with andwithout a history of antiseizure medication, arguing thatdecreases in binding of GABAA receptor ligands is not aconsequence of antiseizure drug treatment, although thisconclusion is tentative given the limited number of casesin both groups. In particular, there was a trend towardlower [3H]-muscimol binding in the supragranular layerof patients receiving anticonvulsant therapy. It is difficultto conclude whether autism, seizures, drug therapy, asmall group, or a combination of any of these could beresponsible for the reduced binding in one particularlayer. Therefore it will be important in future studies tocontinue to control for seizures, anticonvulsant therapy,and to obtain a larger sample size if possible.Taken together with previous studies [Guptill et al.,

2007; Blatt et al., 2001; Yip et al., 2007, 2008, 2009], thepresent results support the hypothesis that deficits inGABAA receptor systems are widespread in autistic brain.Polymorphisms in the genes for the a4 and b1 GABAA

receptor subunits have been found to be significantlyassociated with autism, raising the possibility thatdecreases in binding of GABAA receptor ligands in autisticbrain may reflect a primary defect in GABAA receptorsubunit expression. Alternatively, it is possible thatdown-regulation of GABAA receptors occurs as a com-pensatory response to increased GABA release associatedwith increased numbers, activity, or dendritic branchingof GABAergic interneurons. In the hippocampus, in-creased density of interneurons was found in subfields ofthe hippocampus [Lawrence, Kemper, Bauman, & Blatt,2009] in which reductions in GABAA and BZD receptorswere found [Blatt et al., 2001; Guptill et al., 2007].Alterations in GAD67 and GAD65mRNA levels have beendetected in specific neuronal subpopulations in autisticcerebellum [Yip et al., 2007, 2008, 2009], suggestingenhanced GABA utilization by these cells. If the decreasein GABAA receptors in ACC is a consequence of excessiveGABAergic release by interneurons, then it is likely thatinterneuron numbers and/or GAD expression will simi-larly be up-regulated in ACC of autistic cases. Suchstudies may help to clarify the role of GABAA receptorswith respect to disturbances of executive function thathave been described in Autism Spectrum Disorders (ASD).ACC hypoactivity has been reported in functionalimaging studies of ASD subjects during some executivefunction dependent tasks [Kana, Keller, Cherkassky,Minshew, & Just, 2006; Shafritz et al., 2008], whichwould be consistent with excessive release of GABA byACC interneurons, but abnormally increased ACC activa-tion has also been reported for some tasks [Schmitz et al.,2008; Thakker et al., 2008]. A more sophisticated under-standing of the role of GABAA receptor mediated

214 Oblak et al./Decreased GABAA receptors in autism INSAR

inhibition with respect to executive function deficits willlikely require identification of the specific GABAA

receptors affected and their localization with respect toneuronal circuits.

The Role of GABAA Receptors in Anxiety Disorders

Pharmacological evidence suggests that GABAA receptorsplay a role in anxiety disorders. Classical BZDs and otherGABAA receptor positive modulators reduce anxiety,whereas competitive and non-competitive GABAA receptorantagonists and benzodiazepine inverse agonists areanxiogenic [Belzung, Misslin, Vogel, Dodd, & Chapouthier,1987; Dalvi & Rodgers, 1996], and altered GABAA receptorbinding in the limbic system has been correlated withincreased anxiety in both humans [Sundstrom, Ashbrook,& Backstrom, 1997] and animals [Rainnie, Asprodini, &Shinnick-Gallagher, 1992]. Patients with panic disorderhave reduced benzodiazepine binding [Cameron et al.,2007; Hasler et al., 2008] and a reduced sensitivity tobenzodiazepine drugs [Roy-Byrne, Wingerson, Radant,Greenblatt, & Cowley, 1996]. Individuals with ASDs havehigh levels of anxiety disorders [Gillott & Standen, 2007;Juranek et al., 2006; Kuusikko et al., 2008; Sukhodolskyet al., 2008; Towbin, Pradella, Gorrindo, Pine, & Leibenluft,2005] and Gillott, Furniss, and Walter [2001] suggests thatadults with autism are almost three-times more anxiousthan their non-autistic peers.Despite the high frequency of anxiety disorders in

patients with ASD, use of BZDs in this group of patients islimited in practice; in 2002, only 4.2% of autistic patientsreceived at least one prescription for a benzodiazepine[Oswald & Sonenklar, 2007], suggesting that the effec-tiveness of this class of drugs for treatment of anxietyassociated with ASD is not high. Marrosu et al. [1987]found that some individuals in a small group of childrenwith autism displayed a paradoxical reaction to BZDs, inwhich half of the autistic individuals receiving thistreatment showed improvement in behavior and theother half exhibited anxiogenic and aggressive response.We hypothesize that the reduction in GABAA receptorsleads to increased anxiety in autism. GABAA receptorscontaining the a2 and a3 subunits have been shown tomediate anxiolytic effects of benzodiazepine site ligandsin mice, suggesting that these receptors play a role inregulating anxiety [Morris, Dawson, Reynolds, Atack, &Stephens, 2006]. Moreover, these subunits have beenfound to be reduced in regions of the brains ofindividuals with autism [Fatemi et al., 2009b], whichcould result in both increased susceptibility to anxietyand reduced anxiolytic efficacy of BZDs. Paradoxicalreactions to BZDs could reflect different alterations ofGABAA receptor subtype or neuronal distribution insubgroups of individuals with autism. Following up within situ hybridization studies, immunohistochemical

studies, or Western blotting experiments would conclu-sively determine whether a2 and a3 subunits are reducedin these cases.

Concluding Remarks and Future Experiments

Through many recent neurochemical studies in autism, itis becoming clear that multiple transmitter systems areinvolved in the neuropathology of the disorder. Interest-ingly, the system with the most consistent findings is theGABAergic system and specifically the GABAA andbenzodiazepine sites in limbic and cerebellar structures.This study further elucidated that decreases in thesereceptor subtypes in the ACC potentially disruptsinformation processing to high order association corticesin the frontal cortex likely contributing to the core socio-emotional behavior deficits in autistic individuals. Asnoted in Table I, all cases used in the study had a historyof mental retardation. A subnormal IQ has been observedin most cases of individuals with ‘‘classic autism,’’ similarto all cases within this study. It is unlikely that thepresence of mental retardation is the cause of a decreaseddensity of GABAA receptors and BZD binding sites andspecific to autism. However, it would be important toexamine Asperger’s cases as well to determine conclu-sively that the decreased density of GABAA receptors andBZD binding sites is specific to ‘‘classic autism’’ and notto mental retardation or to high-functioning autism.However, these findings do lend further evidence that theGABAA receptors should be considered to be a core neuralsubstrate in autism and its alterations in different brainareas in autism are in support of Rubenstein andMerzenich’s [2003] hypothesis of an altered inhibi-tory:excitatory imbalance directly affecting the autismphenotype.

Acknowledgment

This work was supported by a ‘‘Studies to Advance AutismResearch and Treatment’’ grants from the NationalInstitutes of Health (NIH U54 MH66398) and NIH NINDSNS38975. Brain tissue was provided by the Harvard BrainTissue Resource Center (HBTRC), the Autism TissueProgram, and the Autism Research Foundation. We thankSandy Thevarkunnel for her help in receptor bindingautoradiography methodology.

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