RESEARCH ARTICLE

Decreased GABAA Receptors and Benzodiazepine Binding Sites in the Anterior Cingulate Cortex in Autism A. 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 to prefrontal cortex is a key part of an important circuitry participating in executive function, affect, and socio-emotional behavior. 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 complex is an important target for pharmacotherapy and has important clinical implications. The present multiple-concentration 3 3 ligand-binding study utilized H-muscimol and H-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 and control cases. Compared to controls, the autistic group had significant decreases in the mean density of GABAA receptors in the supragranular (46.8%) and infragranular (20.2%) layers of the ACC and in the density of benzodiazepine binding sites in the supragranular (28.9%) and infragranular (16.4%) lamina. In addition, a trend for a decrease in for the density of benzodiazepine sites was found in the infragranular layers (17.1%) in the autism group. These findings suggest that in

the autistic group this downregulation of both benzodiazepine sites and GABAA receptors in the ACC may be the result of increased GABA innervation and/or release disturbing the delicate excitation/inhibition balance of principal neurons as well 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 structure for roles in executive function, learning, memory and socio-emotional behavior [Pandya, Van The anterior cingulate cortex (BA 24; ACC), part of the Hoesen, & Mesulam, 1981; Petrides & Pandya, 2007; original Papez circuit of emotion [Papez, 1937], is Vogt & Pandya, 1987]. considered a part of the brain’s limbic lobe and is In the neurodevelopmental disorder autism, character- involved in attention and the regulation of cognitive ized by impairments in communication including lan- and emotional processing [Allman, Hakeem, Erwin, guage, as well as narrowly focused interests and poor Nimchinsky, & Hof, 2001; Bush, Luu, & Posner, 2000; sociability, there is demonstrated neuropathology in the Heimer & Van Hoesen, 2006]. Anterior cingulate lesions ACC in some reported cases. These were first described by are known to cause blunted affect, impulsivity, disinhibi- Bauman and Kemper [1985] and recently quantitatively tion, aggressive behavior, disabling obsessions and com- confirmed by Simms, Kemper, Timbie, Bauman, and Blatt pulsions, and impaired social judgment including the [2009], who reported an increased cell packing density inability to interpret social cues [Devinsky, Morrell, & and decreased cell size in several subregions within the Vogt, 1995]. ACC. Furthermore, positron emission tomography (PET) Neuroanatomical studies have shown that the ACC studies have demonstrated abnormalities in the activa- encompasses a number of specialized subdivisions that tion of the ACC in autism during a verbal learning task subserve a vast array of cognitive, emotional, motor, [Haznedar et al., 1997]. nociceptive, and visuospatial functions [Bush et al., 2000; Recent neurochemical research has focused on the Heimer & Van Hoesen, 2006; Isomura & Takada, 2004; gamma-amino-butyric acid (GABA) system and its possi- Posner, Rothbart, Sheese, & Tang, 2007; Zhuo, 2006]. The ble involvement in a number of limbic and cerebellar ACC is intimately connected to prefrontal cortex (PFC) as regions in autistic cases [Blatt et al., 2001; Fatemi et al., well as key limbic structures including the hippocampal 2002; Fatemi, Folson, Reutiman, & Thuras, 2009a; formation and the amygdala, positioning it as a key Fatemi, Reutiman, Folson, & Thuras, 2009b; Guptill

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.) Received March 12, 2009; revised June 15, 2009; accepted for publication June 21, 2009 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: [email protected] Grant sponsor: National Institutes of Health; Grant numbers: NIH U54 MH66398; NIH NINDS NS38975. 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.

INSAR Autism Research 2: 205–219, 2009 205 et al., 2007; Yip, Soghomonian, & Blatt, 2007, 2008, Methods and Materials 2009]. GABA plays a crucial role in normal cortical Brain Tissue and Case Data functioning, information processing, and the formation Fresh frozen brain tissue from the ACC was obtained of brain cytoarchitecture during development [Conti, from the Harvard Brain Tissue Resource Center (HBTRC) Minelli, & Melone, 2004; Di Cristo, 2007; Fritschy, Benke, in Belmont, Massachusetts. All clinical cases were Johnson, Mohler, & Rudolph, 1997; Mohler, Benke, diagnosed as autistic and had Autism Diagnostic Inter- Benson, Luscher, & Fritschy, 1995a]. One of the proposed views (ADI) completed either pre- or post-mortem. All causes of impaired information processing and social tissue was provided by The Autism Research Foundation, behavior in autism is an altered balance between Autism Tissue Program, and Harvard Brain Tissue inhibition and excitation in the brain [Rubenstein & Resource Center. A total of 17 blocks were obtained Merzenich, 2003]. The distribution, electrophysiology, (7 autism and 10 controls) from the rostrum of the and molecular characteristics of GABA receptors change ACC and stored at 801C. One autism case, ]3845, was markedly during development, leaving the formation À used for the muscimol study but not for the benzodia- of the cortex vulnerable to aberrations in neurotrans- zepine study due to limited availability of tissue sections mission at key developmental periods. Seizures are as this case has been used for many of our previous fairly common in individuals with autism, occurring studies. A summary of the case details is seen in Table I. in approximately 25–33% of individuals [Olsson, There was no significant difference in age or post-mortem Steffenburg, & Gillberg, 1988; Volkmar & Nelson, 1990] interval (PMI) between autism and control groups and may result from abnormal inhibitory control in key (student t-test). Four of the seven autism cases had a cortical areas. Genetic studies have also implicated the history of at least one seizure (1078, 1484, 2825, and GABA system in autism. For example, Schroer et al. 3845). Of those four cases, three had received treatment [1998] found abnormalities on chromosome 15q11-q13, with anticonvulsant therapy (1078, 2825, and 3845). All which includes a cluster of three GABAA receptor genes cases used in the study had an autism diagnosis of (GABRa5, GABRb3, and GABRg3). Several genetic studies moderate to severe. have proposed the involvement of multiple GABAA receptor subunits and suggest that, through complex Multiple-Concentration Binding Assay interactions, these subunits are involved in autism [Ashley-Koch et al., 2006; Ma et al., 2005]. All tissue blocks were sectioned coronally at 20 mm using The GABAA receptor has binding sites for multiple a Hacker/Brights motorized cryostat at 201C. Sections À modulators, including benzodiazepines (BZDs), making it were then thaw mounted on 2 3 inch gelatin coated  a target for pharmacological intervention. BZDs enhance glass slides. For each of the seven concentrations, two

GABAA receptor mediated neurotransmission, and classi- sections from each case were used to determine total cally act as sedative/hypnotics to reduce anxiety and binding and one section from each case was used to suppress seizure activity and panic attacks [Low et al., determine non-specific binding. Non-specific binding 2000]. In clinical studies, BZDs have been reported to was measured by adding a high concentration of a elicit paradoxical behavioral responses in some autistic competitive displacer (see Table II) to the tritiated ligand individuals, producing increased anxiety and aggressive and buffer solution. The ligand used for GABAA receptors behavior [Marrosu, Marrosu, Rachel, & Biggio, 1987]. was [3H]-Muscimol (specific activity 36.6 Ci/mmol; New This variable response could be due to an alteration in England Nuclear) and the ligand for BZD binding sites 3 BZD-sensitive GABAA receptors in the specific subset of was [ H]-flunitrazepam (specific activity 85.2 Ci/mmol; cases investigated. New England Nuclear). To eliminate variability in Taken together, there is compelling evidence that the binding conditions, the selected sections from all cases GABA system is impacted in autism and alterations in the were assayed in parallel. Slides were then dried under a

GABAA receptor system likely play a role in the etiology of stream of cool air overnight and loaded into X-ray the disorder during development and may contribute to cassettes with a tritium standard (Amersham), apposed the abnormal phenotype and the variable response to to tritium-sensitive film (3H-Hyperfilm, Kodak) and pharmacotherapy. Changes in these key neural substrates incubated for a period of time (see Table II). The exposed in the ACC could disrupt critical circuits involved in socio- films were developed for 4 minutes with Kodak D19 emotional behavior as well as other high-order associative developer, fixed with Kodak Rapidfix (3 min) at room functions especially via its abundant prefrontal cortical temperature, and allowed to air dry. Slides were stained connectivity. The aim of this study was to determine if the with thionin to determine cytoarchitecture and laminar number, density, and distribution of GABAA receptors and distribution of the ACC (Fig. 1). Supragranular layers BZD binding sites in the ACC are altered in postmortem corresponded to layers I–III and infragranular layers to adult autistic cases, and to explore how such alterations layers V–VI as layer IV is absent in the ACC [Vogt, might impact individuals with the disorder. Nimchinsky, Vogt, & Hof, 1995].

206 Oblak et al./Decreased GABAA receptors in autism INSAR Table I. Information for Cases in the Multiple Concentration Binding Studies

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

1078ÃÃ Autism 22 14.3 Dilantin, Tegretol, Theodur, Phenobarbital Drowning Male 1401 Autism 21 20.6 None Pneumonia, Sepsis Female 1484Ã Autism 19 15 None Burns Male 2825ÃÃ Autism 19 9.5 Klonopin, Mysoline, Phenobarbital, Thorazine Heart attack Male 3845ÃÃ Autism 30 28.4 Dilantin, Mellaril, Phenobarbital Cancer Male 4099 Autism 19 3 None Conj. Heart Failure Male 5754 Autism 20 30.0 None Unknown Male Mean 21.4 17.3 4103 Control 43 23 None Heart attack/disease Male 4104 Control 24 5 None Gun shot Male 4188 Control 16 13 None Gun Shot Male 4267 Control 26 20 None Accidental Male 4268 Control 30 22 None Heart attack/disease Male 4269 Control 28 24 None Heart disease Male 4271 Control 19 21 None Epiglotitus Male 4275 Control 20 16 None Accidental Male 4364 Control 27 27 None Motor Vehicle Accident Male Mean 25.9 19.0

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

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] 100 mM GABA 6–360 days 3[H]-Flunitrazepam Benzodiazepine binding site [0.3, 1.5, 3, 5, 15, 30, 120] 100 mM Clonazepam 10–180 days

used to calibrate the autoradiograms to quantify the amount of ligand bound per milligram of protein in the tissue sections. Optical density for the standards as a function of specific activity (corrected for radioactive decay) was fitted by nonlinear least squares regression to k specific activity the equation, optical density 5 B1 (1–10 1ð Þ 1 Â Þ B3, by adjusting the values of the parameters k1, B1, and B3 using the Solver tool of Excel (Microsoft Office XP Professional) to construct a standard curve, which was used to 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.

Figure 1. A Nissl stained section from a control case (4103) Statistical Analyses demonstrates the cytoarchitecture of the human ACC (Area 24a-c). The box at left illustrates our sampling scheme dividing the cortex Specific binding of the tritiated ligands in the supragra- into supragranular (I–III) and infragranular (V–VI) layers. [Color nular and infragranular layers from each subject was fitted figure can be viewed online at www.interscience.wiley.com] independently with a hyperbolic binding equation to

estimate binding affinity (Kd) and number of receptors Data Analysis (Bmax) for each region. Examples of binding curves are shown in Figure 2. Least-squares nonlinear regression was

The film autoradiograms were digitized using an Inquiry carried out using the Microsoft Excel Solver tool. Bmax data densitometry system (Loats Associates) to gather quantita- is reported as mean1standard error of the mean (SEM). Kd mean log K tive measurements of optical density. The supragranular is reported as the geometric average 10 ð ð dÞÞ SEMav, x s x x Æx s and infragranular layers were sampled from each case. The calculated as SEMav 5 (((10ð þ Þ–10 )1(10 –10ð À Þ))/2), 3 H standards that were exposed with the sections were where x 5 mean(log Kd) and s is the SEM of the log (Kd)

INSAR Oblak et al./Decreased GABAA receptors in autism 207 Figure 2. Sample binding curves from two cases (autistic on left, control on right) demonstrating specific binding in the supragranular layers from each concentration of tritiated ligand. Data from [3H]muscimol is in the upper panel and data from [3H]flunitrazepam is in the bottom panel. [Color figure can be viewed online at www.interscience.wiley.com]

values. Significance testing for differences in Kd was carried values (P 5 0.0004, P 5 0.0003, respectively). We found out using log(Kd) values. Two-Way Analysis of Variance decreases in the density of both GABAA receptors and (ANOVA) was used to determine if there was a significant BZD binding sites in the supra- and infragranular layers of difference in receptor or binding site number between autistic cases. Binding affinity (Kd) did not differ autistic and control cases, between supragranular and significantly between autistic and control cases for either infragranular layers, or if an interaction existed. Non- ligand (Fig. 3; Table IV). Binding data for both autistic parametric Mann–Whitney U tests were used to determine and control cases were well-fitted by a single-component if seizures or anticonvulsant therapy in the autism cases hyperbolic binding equation, consistent with a single, had an effect on receptor binding. homogeneous class of binding sites (Fig. 3). Figures 5 and 7 show the specific binding curves for each of the cases. Figures 4 and 6 show typical examples of binding of a Results single concentration (15 nM) of [3H]-muscimol and [3H]- flunitrazepam, respectively, in typical sections from an Table III reports specific binding of [3H]-muscimol and autistic and a control case. The autoradiograms have [3H]-flunitrazepam in the ACC from autistic and control been pseudocolored using the Inquiry program to better cases. Overall, the supragranular layers of the ACC of visualize binding density, with red corresponding to high both autistic and control cases contained a higher density binding (optical density) and purple to low binding. The of GABAA receptors and benzodiazepine binding sites Nissl stained section in Figure 1 was used to identify the than the infragranular layers, as measured by the Bmax region of interest as well as the division of the region into

208 Oblak et al./Decreased GABAA receptors in autism INSAR 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 361.21 428.58 261.17 229.80 1401 Autism 497.67 473.07 219.45 162.49 1484 Autism 574.10 446.28 321.89 181.76 2825 Autism 371.43 525.64 306.07 198.43 3845 Autism 432.83 345.31 4099 Autism 558.68 424.41 381.36 217.17 5754 Autism 480.17 387.30 337.30 222.49 Mean1SEM 468.01131.80 432.94121.95 304.54121.65 202.02110.66 4103 Control 830.26 493.42 353.76 204.55 4104 Control 1030.31 522.53 335.13 231.79 4188 Control 873.65 328.05 453.82 288.03 4267 Control 712.05 448.30 447.37 214.59 4268 Control 741.45 510.97 391.17 234.53 4269 Control 827.73 742.58 542.08 215.16 4271 Control 1104.62 554.65 509.70 313.10 4275 Control 1003.04 635.73 384.67 192.15 4364 Control 788.97 694.93 420.54 279.98 Mean1SEM 879.11147.14 542.35140.49 427.03122.90 241.54114.01 P-value (t-test) 5.16 10 6 0.035 0.0028 0.043 Â À

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

Muscimol binding Flunitrazepam binding affinity (nM) affinity (nM)

Group Supragranular Infragranular Supragranular Infragranular

Autism 13.511.26 9.6911.70 4.4410.61 4.7910.19 Control 1311.55 9.3211.60 6.1011.45 5.7610.82

GABAA Receptor Binding

Multiple concentration binding experiments revealed

that the mean Bmax value for all autistic cases fell below that for control cases. The results from the seven- concentration binding study indicate a significant reduc-

tion of GABAA receptors (Bmax) in both the supragranular 6 (P 5 5.2 10À ) and the infragranular (P 5 0.04) layers of  the ACC in the autistic group (n 5 7) when compared to Figure 3. Summary of mean K values for GABA (a) and BZD (b) d A age- and PMI matched controls (n 5 9). The B values receptor binding in autistic and control cases. There was no max significant difference between autistic and control cases in either demonstrate that there was a 46.8% reduction in GABAA receptors in the supragranular layers of the ligand or layers. Geometric means for Kd are reported here. ACC and a 20.2% decrease in the number of GABAA receptors in the infragranular layers in autism. The three

cases with the lowest Bmax values in the supragranular supragranular (layers I–III) and infragranular (layers layers did have a history of seizure. However, the case

V–VI) layers. The distribution of Bmax values across layers demonstrating the highest binding also had a history of for GABAA receptors and benzodiazepine binding sites is seizure. In the infragranular layers, two of the cases with a given in Table III. Sample binding curves demonstrating history of seizure fell below the mean Bmax values of the specific binding in the supragranular layers from each of autistic group. In contrast, there was no difference in the seven concentrations of tritiated ligand can be found binding affinity comparing autistic and control cases in Figure 2. (Fig. 2).

INSAR Oblak et al./Decreased GABAA receptors in autism 209 Figure 4. Examples of pseudocolored images of [3H]-muscimol binding (15 nM) in autistic (a) and control (b) cases. Solid black arrows demonstrate the location of sampling in the supragranular layers; arrows with dashed lines indicate sampling in the infragranular layers. 3 6 In the [ H]muscimol binding experiments, the number of GABA receptors in the supragranular (P 5 5.16 10À ) and infragranular layers A Â (P 5 0.04) was significantly (ÃÃ) decreased (c). [Color figure can be viewed online at www.interscience.wiley.com]

Benzodiazepine Binding Sites Effect of Seizure on Binding

With respect to the benzodiazepine binding sites, all Four of the cases in this study had a history of having at but one autistic case (in the infragranular layers) fell least one seizure. In the muscimol binding study, there below the average Bmax value of the control group. was no significant difference in binding between autistic The results from the seven concentration binding cases with seizure (n 5 4) and autistic cases without study indicated a significant decrease in the Bmax seizure (n 5 3). Furthermore, in the flunitrazepam bind- values in the supragranular layers (P 5 0.003) and ing study no significant difference in binding was infragranular layers (P 5 0.04) of the ACC in autism. observed between autistic cases with seizure (n 5 3) and The multiple concentration binding study revealed a autistic cases with no seizure history (n 5 4). 28.7% and 16.4% reduction in benzodiazepine binding sites in the supragranular layers and infragranular layers, Effect of Pharmacotherapy on Binding respectively, in autistic cases. In the supragranular layers, one of the two autistic cases that fell below the The cases were divided into two groups (autism with a mean Bmax value for autistic cases had a history of seizure. history of anticonvulsant therapy and autism with no In the infragranular layers, two of the cases that fell history of anticonvulsant therapy) to determine if the below the mean Bmax value had a history of seizure. There medication had a significant effect on binding. In the was no significant difference observed in binding affinity muscimol binding experiments, three of the seven cases (Fig. 2). were taking anticonvulsants at the time of death. Using a

210 Oblak et al./Decreased GABAA receptors in autism INSAR Processing of high order sensory and multimodal informa- tion occurs via connections with the PFC and parietal cortex 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; Vogt et al., 1987]. When effort is needed to carry out a task such as in early learning and problem solving [Allman et al., 2001], the ACC is recruited, and many studies attribute functions such as error detection and anticipation of tasks, motivation, and modulation of emotional responses to this region [Bush et al., 2000; Nieuwenhuis, Yeung, van den Wildenberg, & Ridderinkhof, 2003; Posner, Pothbart, & Digirolamo, 1999; Posner et al., 2007]. The ACC is an integral part of many higher order functions. Disruptions of this region, at any level, cellular or chemical, could lead to 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 an alteration in GABAergic function, of any nature, in a region such as the ACC, with widespread cortical connections, may result in impairments of information processing, a feature considered to be central to autism [Belmonte et al., 2004]. Information processing in the brain occurs via feed-forward and feedback projections. 3 Figure 5. Examples of individual [ H]muscimol binding curves. Long-range cortico-cortical input is provided by ‘‘feed- 3 Specific binding of [ H]-muscimol to the supragranular (a) and back’’ projections from higher order regions that primar- infragranular (b) layers of the ACC in seven autistic and nine ily target the distal dendritic tufts of pyramidal cells from control subjects. Smooth curves indicate fits to the hyperbolic all layers that terminate in layer I [Rockland & Drash, binding equation. [Color figure can be viewed online at www.interscience.wiley.com] 1996]. Layer II pyramidal axons make local connections within layers II and III, and layer III pyramidal axons ramify most extensively in layers II/III and V [Thomson, Mann–Whitney U non-parametric test, a trend (P 5 0.06) West, Wang, & Bannister, 2002]. Layer V pyramidal cells was observed in the supragranular layers, such that those project to all other layers of the cortex and represent a receiving anticonvulsant therapy had a trend towards major route of excitatory feedback projections to the decreased GABAA receptors. There was no effect of superficial laminae [Burkhalter, 1989]. Abnormalities in anticonvulsant therapy on the decrease in receptor the integrity of the layers through cell loss, abnormal binding in the infragranular layers (P 5 0.99 and cytoarchitecture or receptor changes, as observed in this P 5 0.113, respectively). In the flunitrazepam binding study, may interfere with cortical processing. study, half (n 5 3) of the cases were receiving antic- The present observations demonstrate the highest onvulsant therapy at the time of death. There was no binding to GABA receptors and benzodiazepine binding significant difference in the number of benzodiazepine A sites in the supragranular layers of the anterior cingulate binding sites in the supra- or infragranular layers in the cortices in agreement with previous autoradiographic and autistic group receiving anticonvulsants compared to the immunohistochemical data [Fritschy et al., 1997; Mohler autistic group not receiving anticonvulsants. et al., 1995a,b]. Previous receptor binding experiments 3 have reported that [ H]-muscimol labeled GABAA recep- 3 Discussion tors and [ H]-flunitrazepam-labeled benzodiazepine bind- Normal Functions of the ACC ing sites were significantly reduced in the hippocampus of adult autistic individuals [Blatt et al., 2001; Guptill et al.,

The ACC (Brodmann area 24; BA 24) has been shown 2007]. This study found significant reductions in GABAA to participate in a variety of functions including executive, receptors and benzodiazepine binding sites in the supra- evaluative, and cognitive functions and emotion [Devinsky granular and infragranular layers of the ACC. These results et al., 1995; Lane et al., 1998; Vogt, Finch, & Olson, 1992]. suggest abnormal connectivity between the ACC and the

INSAR Oblak et al./Decreased GABAA receptors in autism 211 Figure 6. Examples of autistic (a) and control (b) pseudocolored images of [3H]-flunitrazepam (15 nM) binding in the ACC. Solid black arrows demonstrate the location of sampling in the supragranular layers; arrows with dashed lines indicate sampling in the infragranular layers. There was a significant decrease (ÃÃ) in benzodiazepine binding sites in the supragranular layers (P 5 0.003) and infragranular layers (P 5 0.04) in autism (c). [Color figure can be viewed online at www.interscience.wiley.com] regions of the cortex to which the ACC projects (e.g. PFC) cortical columns. Excitatory projections from pyramidal and also abnormal reciprocal connections between the cells to interneurons would help control the excitatory higher order association cortices and the ACC. This study flow through the cortex by inhibiting cells responsible for further suggests that the limbic system is abnormal in their inputs as soon as appropriate outputs to the next autism, which may contribute to the social and emotional stage of processing have been attained [Burkhalter, 1989]. aspects of the disorder. In autism, decreases in inhibitory However, abnormal receptors in this region would receptors on pyramidal neurons or interneurons may decrease the likelihood of controlling the excitatory flow. restrict normal connectivity within the ACC and between This abnormal connectivity may result in changes in cortical regions. Laminar and receptor-specific decreases in cognition and in the proper understanding of socio- the infragranular layers may support a role of these layers emotional interactions that are common in autism. in a loss of normal signal transmission and a decrease in the information relaying to and from the thalamus [Kaitz GABAA Receptors During Development and Related & Robertson, 1981; Yakovlev & Locke, 1961]. Neuropathology of the Autistic Brain Inadequate inhibitory damping of excitatory projec- tions to other excitatory cells further downstream could Aberrant development of GABAergic circuits is not unique result in undesirable positive feedback loops, and could to autism and has been implicated in other neurodevelop- disrupt signaling from the thalamus as it passes through mental disorders such as schizophrenia [Benes, Vincent,

212 Oblak et al./Decreased GABAA receptors in autism INSAR 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 also been shown to impair neuronal migration in vitro and in vivo [Conti et al., 2004]. In autism, pathological alterations in the cortical cytoarch- itecture in autism resemble neuronal migration disorders previously described in the human cerebral cortex [Meng et al., 2005; Corfas, Roy, & Buxbaum, 2004]. Simms et al. [2009] has shown irregular lamination and increased density of neurons in the subcortical white matter of the ACC in five of nine cases examined, potentially occurring from a disrupted GABAergic receptor system. Furthermore, Bailey et al. [1998] demonstrated abnormal cytoarchitecture in the frontal 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 of minicolumns in dorsal lateral PFC as well as an abnormal number of minicolumns. Again, these results are suggestive of a abnormality in the GABAergic receptor system during development resulting in abnormal cytoarchitecture in the adult autistic brain. The changes observed in a limbic system area (ACC) and a region that it is highly connected with (PFC) suggests further a circuitry/connectivity problem. This could 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 autistic 3 Figure 7. Examples of individual [ H]-flunitrazepam binding brain. In the ACC, a significant decrease in cell area was curves. Specific binding of [3H]-flunitrazepam to the supragra- observed in the supragranular and infragranular layers of nular (a) and infragranular (b) layers of the ACC in six autistic and area 24b (a subregion of the ACC) as well as a significant nine control subjects. Smooth curves indicate fits to the hyperbolic binding equation. [Color figure can be viewed online decrease in cell volume in the supragranular and infra- at www.interscience.wiley.com] granular layers of the same region [Simms et al., 2009]. More recently, Schumann and Amaral [2006] found a quantitative decrease in the volume of neurons in the Alsterberg, Bird, & SanGiovanni, 1992] and Tourette’s lateral nucleus of the amygdala, a region with connections syndrome [Kalanithi et al., 2005]. Compelling evidence to the anterior cingulate [Porrino, Crane, & Goldman- indicates that GABA and GABA receptors play a role in Rakic, 1981]. An abnormal GABA receptor system during several developmental processes. During development, development, such as one that is delayed in the switch

GABAA receptors participate in proliferation, migration, from excitatory to inhibitory or one that retains the fetal and differentiation of precursor cells that orchestrate the circuitry [Bauman & Kemper, 1985], could form the basis development of the embryonic brain [Akerman & Cline, for abnormal processing and decreased neuron volume in 2007; Barker et al., 1998; Conti et al., 2004; Di Cristo, 2007; the adult anterior cingulate in autism. These changes Mohler, Fritschy, Crestani, Hensch, & Rudolph, 2004]. could interfere with socio-emotional and cognitive pro- Despite its known effects in adults as a strong inhibitory cessing similar to what has been found in imaging studies neurotransmitter, during development GABAA mediated [Calzavara et al., 2007; Haznedar et al., 2000; Kana, Keller, neurotransmission plays an excitatory role in the develop- Minshew, & Just, 2007; Kennedy, Redcay, & Courchesne, ing nervous system, shifting to an inhibitory function in 2006; Shafritz, Dichter, Baranek, & Belger, 2008; Silk et al., the mature nervous system as a consequence of changes in 2006; Vollm et al., 2006]. A developmental deficiency in ClÀ gradients [Ben-Ari, Gaiarsa, Tyzio, & Khazipov, 2007]. any of these roles would adversely affect the temporal Eliminating excitatory GABA action between postnatal ordering of neurogenesis and synaptogenesis, thereby days 4 and 6 in rats resulted in severe impairment of the affecting maturation of circuits that are later involved in morphological maturation of cortical neurons [Cancedda, complex behaviors associated with the ACC.

INSAR Oblak et al./Decreased GABAA receptors in autism 213 Effects of Seizure/Anti-convulsant Treatment on Bmax and Kd sometimes associated with seizures, suggesting that of GABAA and Benzodiazepine Binding chronic phenobarbital use alters GABAA receptor systems. There was no statistically significant difference between

Approximately, 80% of all GABAA receptors contain the the cases with and without seizures or those with and classical benzodiazepine binding site and are character- without a history of antiseizure medication, arguing that ized largely by the subunit combinations a1b2g2, a2b3g2, decreases in binding of GABAA receptor ligands is not a and a3b3g2 [Speth et al., 1980]. The benzodiazepine site, consequence of antiseizure drug treatment, although this located at the interface between a and g subunits, is conclusion is tentative given the limited number of cases structurally homologous to the GABA binding site in both groups. In particular, there was a trend toward located in the a-b subunit interface. Epileptogenic seizure lower [3H]-muscimol binding in the supragranular layer activity is likely a result of a deficit in GABA-mediated of patients receiving anticonvulsant therapy. It is difficult inhibition, and seizures are common in approximately to conclude whether autism, seizures, drug therapy, a one-fourth to one-third of autistic individuals [Bailey small group, or a combination of any of these could be et al., 1998; Gillberg, Steffenburg, & Jakobsson, 1987; responsible for the reduced binding in one particular Olsson et al., 1988; Rutter, 1970; Volkmar & Nelson, layer. Therefore it will be important in future studies to 1990]. In a PET imaging study, the binding of continue to control for seizures, anticonvulsant therapy, [11C]-flumazenil to benzodiazepine binding sites in and to obtain a larger sample size if possible. epileptogenic foci was found to be reduced with no Taken together with previous studies [Guptill et al., change in binding affinity [Savic et al., 1988]. In a more 2007; Blatt et al., 2001; Yip et al., 2007, 2008, 2009], the recent imaging study using [125I]-iomazenil, reductions present results support the hypothesis that deficits in in central benzodiazepine binding sites were found in GABAA receptor systems are widespread in autistic brain.

48% of individuals studied in medial temporal lobe Polymorphisms in the genes for the a4 and b1 GABAA regions [Matheja et al., 2001], although this may be a receptor subunits have been found to be significantly consequence of localized neuronal damage or neuronal associated with autism, raising the possibility that loss in the regions with temporal lobe epileptogenic decreases in binding of GABAA receptor ligands in autistic activity. It should be noted that these changes were brain may reflect a primary defect in GABAA receptor occurring in the hippocampus and may not be reflective subunit expression. Alternatively, it is possible that of the changes occurring in the cortex. down-regulation of GABAA receptors occurs as a com- Four of the autistic cases studied had a history of seizure, pensatory response to increased GABA release associated raising the question of whether the changes in [3H]fluni- with increased numbers, activity, or dendritic branching trazepam and [3H]-muscimol binding could be a conse- of GABAergic interneurons. In the hippocampus, in- quence of seizures. Alterations in GABAA receptor subunit creased density of interneurons was found in subfields of expression and composition in epilepsy are well documen- the hippocampus [Lawrence, Kemper, Bauman, & Blatt, ted in human [Loup et al., 2006; Loup, Wieser, Yonekawa, 2009] in which reductions in GABAA and BZD receptors Aguzzi, & Fritschy, 2000] and in animal models [Gilby, were found [Blatt et al., 2001; Guptill et al., 2007]. DaSilva, & McIntyre, 2005; Li, Wu, Huguenard, & Fisher, Alterations in GAD67 and GAD65 mRNA levels have been 2006; Nishimura et al., 2005; Peng, Huang, Steil, MOdy, & detected in specific neuronal subpopulations in autistic Houser, 2004; Roberts et al., 2005]. In particular, there is cerebellum [Yip et al., 2007, 2008, 2009], suggesting evidence that seizures can induce a shift of GABAA receptor enhanced GABA utilization by these cells. If the decrease subunit mRNA expression such that a4 subunit expression in GABAA receptors in ACC is a consequence of excessive is increased, whereas a1 subunit expression is decreased GABAergic release by interneurons, then it is likely that [Brooks-Kayal, Raol, & Russek, 2009]. An increase in a4- interneuron numbers and/or GAD expression will simi- containing GABAA receptors at the expense of a1-contain- larly be up-regulated in ACC of autistic cases. Such 3 ing receptors could result in a decreased number of [ H]- studies may help to clarify the role of GABAA receptors flunitrazepam binding sites, as a4-containing receptors do with respect to disturbances of executive function that not contain a [3H]-flunitrazepam binding site, but because have been described in Autism Spectrum Disorders (ASD). [3H]-muscimol binding was also decreased argues that the ACC hypoactivity has been reported in functional results reflect an overall deficit in GABAA receptors. imaging studies of ASD subjects during some executive

In addition to the potential effect of seizures on GABAA function dependent tasks [Kana, Keller, Cherkassky, receptor expression, three subjects had a history of Minshew, & Just, 2006; Shafritz et al., 2008], which treatment with anticonvulsants, including the barbitu- would be consistent with excessive release of GABA by rate phenobarbital. Phenobarbital is a GABAA receptor ACC interneurons, but abnormally increased ACC activa- positive modulator that has been reported to reduce tion has also been reported for some tasks [Schmitz et al.,

GABAA receptor binding in mice [Weizman, Fares, Pick, 2008; Thakker et al., 2008]. A more sophisticated under- Yanai, & Gavish, 1989]. Phenobarbital withdrawal is 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 will studies, or Western blotting experiments would conclu- likely require identification of the specific GABAA sively determine whether a2 and a3 subunits are reduced receptors affected and their localization with respect to in these cases. neuronal circuits. Concluding Remarks and Future Experiments

The Role of GABAA Receptors in Anxiety Disorders Through many recent neurochemical studies in autism, it is becoming clear that multiple transmitter systems are Pharmacological evidence suggests that GABA receptors A involved in the neuropathology of the disorder. Interest- play a role in anxiety disorders. Classical BZDs and other ingly, the system with the most consistent findings is the GABAA receptor positive modulators reduce anxiety, GABAergic system and specifically the GABAA and whereas competitive and non-competitive GABA receptor A benzodiazepine sites in limbic and cerebellar structures. antagonists and benzodiazepine inverse agonists are This study further elucidated that decreases in these anxiogenic [Belzung, Misslin, Vogel, Dodd, & Chapouthier, receptor subtypes in the ACC potentially disrupts 1987; Dalvi & Rodgers, 1996], and altered GABA receptor A information processing to high order association cortices binding in the limbic system has been correlated with in the frontal cortex likely contributing to the core socio- increased anxiety in both humans [Sundstrom, Ashbrook, emotional behavior deficits in autistic individuals. As & Backstrom, 1997] and animals [Rainnie, Asprodini, & noted in Table I, all cases used in the study had a history Shinnick-Gallagher, 1992]. Patients with panic disorder of mental retardation. A subnormal IQ has been observed have reduced benzodiazepine binding [Cameron et al., in most cases of individuals with ‘‘classic autism,’’ similar 2007; Hasler et al., 2008] and a reduced sensitivity to to all cases within this study. It is unlikely that the benzodiazepine drugs [Roy-Byrne, Wingerson, Radant, presence of mental retardation is the cause of a decreased Greenblatt, & Cowley, 1996]. Individuals with ASDs have density of GABAA receptors and BZD binding sites and high levels of anxiety disorders [Gillott & Standen, 2007; specific to autism. However, it would be important to Juranek et al., 2006; Kuusikko et al., 2008; Sukhodolsky examine Asperger’s cases as well to determine conclu- et al., 2008; Towbin, Pradella, Gorrindo, Pine, & Leibenluft, sively that the decreased density of GABAA receptors and 2005] and Gillott, Furniss, and Walter [2001] suggests that BZD binding sites is specific to ‘‘classic autism’’ and not adults with autism are almost three-times more anxious to mental retardation or to high-functioning autism. than their non-autistic peers. However, these findings do lend further evidence that the Despite the high frequency of anxiety disorders in GABAA receptors should be considered to be a core neural patients with ASD, use of BZDs in this group of patients is substrate in autism and its alterations in different brain limited in practice; in 2002, only 4.2% of autistic patients areas in autism are in support of Rubenstein and received at least one prescription for a benzodiazepine Merzenich’s [2003] hypothesis of an altered inhibi- [Oswald & Sonenklar, 2007], suggesting that the effec- tory:excitatory imbalance directly affecting the autism tiveness of this class of drugs for treatment of anxiety phenotype. associated with ASD is not high. Marrosu et al. [1987] found that some individuals in a small group of children with autism displayed a paradoxical reaction to BZDs, in Acknowledgment which half of the autistic individuals receiving this This work was supported by a ‘‘Studies to Advance Autism treatment showed improvement in behavior and the Research and Treatment’’ grants from the National other half exhibited anxiogenic and aggressive response. Institutes of Health (NIH U54 MH66398) and NIH NINDS We hypothesize that the reduction in GABA receptors A NS38975. Brain tissue was provided by the Harvard Brain leads to increased anxiety in autism. GABA receptors A Tissue Resource Center (HBTRC), the Autism Tissue containing the a2 and a3 subunits have been shown to Program, and the Autism Research Foundation. We thank mediate anxiolytic effects of benzodiazepine site ligands Sandy Thevarkunnel for her help in receptor binding in mice, suggesting that these receptors play a role in autoradiography methodology. regulating anxiety [Morris, Dawson, Reynolds, Atack, & Stephens, 2006]. Moreover, these subunits have been found to be reduced in regions of the brains of References individuals with autism [Fatemi et al., 2009b], which Akerman, C.J., & Cline, H.T. (2007). Refining the roles of could result in both increased susceptibility to anxiety GABAergic signaling during neural circuit formation. Trends and reduced anxiolytic efficacy of BZDs. Paradoxical in Neuroscience, 30, 382–389. reactions to BZDs could reflect different alterations of Allman, J.M., Hakeem, A., Erwin, J.M., Nimchinsky, E., & Hof, P. GABAA receptor subtype or neuronal distribution in (2001). The anterior cingulate cortex. The evolution of an subgroups of individuals with autism. Following up with interface between emotion and cognition. Annals of the New in situ hybridization studies, immunohistochemical York Academy of Science, 935, 107–117.

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