The initial single concentration ligand binding studies were carried out in the hippocampus as a pilot study effectively to screen a number of different transmitter receptor types from four major systems: 5-HT, glutamate, GABA and acetylcholine (Blatt et al., 2001
). Surprisingly, the results from this study found that most measures in adult autism cases were at normal receptor density levels including 5-HT1aR, 5-HT2R, muscarinic cholinergic type 1 and 2 (M1, M2), High Affinity Choline Uptake Sites (HACU), kainate and NMDAR in all major subfields of the hippocampus. The only two that were not at normal levels were the higher binding hippocampal subfields for 3
receptors and 3
[H]flunitrazepam labeled benzodiazepine (BZD) binding sites (on the GABAA
receptor) that showed a significant decrease in receptor density. A follow up study from this laboratory, further demonstrated that the 3
[H]flunitrazepam labeled BZD binding reduction in the autism cases was due to a decrease in the Bmax, or number of receptors and not due to the Kd or binding affinity which was at normal levels (Guptill et al., 2007
). A more recent study from our laboratory further demonstrated that there is an increased density of GABAergic hippocampal interneurons labeled with specific types of antibodies to calcium binding proteins. Lawrence et al., (2010)
found a selective increase in calbindin-immunopositive interneurons in the dentate gyrus, parvalbumin-immunopositive interneurons in the CA1 and CA3 subfields and calretinin-immunopositive neurons in CA1. This agrees with the qualitative observations of Bauman and Kemper (1985)
that the hippocampus gave a flattened appearance with smaller neurons and increased packing density. Taken together, these data infer increased density of neurons and interneurons, reduced neuropil, and decreased GABA receptors in select but key subfields that could collectively contribute to altered hippocampal circuitry, synaptogenesis, and ultimately function.
In the cerebellum, recently completed (article in preparation) studies by Thevarkunnel et al. have found in adult autism cases compared to controls, significant decreases in 3[H]flunitrazepam labeled BZD binding sites in the molecular layer and reduced 3[H]muscimol-labeled GABAA receptors in the PC layer in the posterior lateral cerebellar hemisphere. This cerebellar region receives fronto-pontine projections (via the granular layer) as well as olivocerebellar climbing fibers from the principal olive. These studies show that the GABAA receptor changes appear not to be limited to the hippocampus and, that there is another change in the GABAergic synaptic regulation in the cerebellum of the autism brain.
The next series of ligand binding studies was in the cerebral cortex and found a reduction of 46.8% in the mean density of 3
receptors in the superficial (supragranular layers I-III) and a reduction of 20.2% in the mean density in the deep (infragranular layers V-VI) of anterior cingulate cortex (ACC) in adult autism subjects compared to matched controls (Oblak et al., 2009
). A similar significant decrease in the supragranular layers (28.9%) and in the infragranular layers (16.4%) for 3
[H]flunitrazepam labeled BZD binding sites in the same cases (Oblak et al., 2009
; ) was also demonstrated. These results demonstrate that the GABA changes are widespread throughout many areas and regions in the autism brain suggesting that selective and significant neurochemical changes may underlie the observed neuropathology as well as to likely contribute to social-emotional deficits.
Figure 1 3[H]flunitrazepam labeled BZD binding sites in the anterior cingulate cortex in a control case compared to an individual with autism. The binding density has been pseudocolored to show differences in binding in the superficial and deep layers. The red (more ...)
Current studies are underway investigating the role of the 5-HT and glutamate systems in these areas as well as extending the studies to additional cortical areas. The changes in GABAA receptors using on-the-slide ligand binding autoradiography itself does not provide information regarding protein expression of this receptor type. In the sections that follow, studies by Fatemi and colleagues address this issue and demonstrate that protein expression does indeed follow the receptor changes for both GABAA and GABAB receptors in a variety of structures in the autism brain.