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