Lisa Foster

In Vitro Characterization of Pittsburgh Compound-B Binding to Lewy Bodies

Dementia with Lewy bodies (DLB) is pathologically characterized by the presence of α-synuclein-containing Lewy bodies within the neocortical, limbic, and paralimbic regions. Like Alzheimers disease (AD), Aβ plaques are also present in most DLB cases. The contribution of Aβ to the development of DLB is unclear. [11C]-Pittsburgh compound B ([11C]-PIB) is a thioflavin-T derivative that has allowed in vivo Aβ burden to be quantified using positron emission tomography (PET). [11C]-PIB PET studies have shown similar high cortical [11C]-PIB binding in AD and DLB subjects. To establish the potential binding of PIB to α-synuclein in DLB patients, we characterized the in vitro binding of PIB to recombinant human α-synuclein and DLB brain homogenates. Analysis of the in vitro binding studies indicated that [3H]-PIB binds to α-synuclein fibrils but with lower affinity than that demonstrated/reported for Aβ1–42 fibrils. Furthermore, [3H]-PIB was observed to bind to Aβ plaque-containing DLB brain homogenates but failed to bind to DLB homogenates that were Aβ plaque-free (“pure DLB”). Positive PIB fluorescence staining of DLB brain sections colocalized with immunoreactive Aβ plaques but failed to stain Lewy bodies. Moreover, image quantification analysis suggested that given the small size and low density of Lewy bodies within the brains of DLB subjects, any contribution of Lewy bodies to the [11C]-PIB PET signal would be negligible. These studies indicate that PIB retention observed within the cortical gray matter regions of DLB subjects in [11C]-PIB PET studies is largely attributable to PIB binding to Aβ plaques and not Lewy bodies.

Localization of NMDA receptor subunits and mapping NMDA drive within the mammalian retina

Glutamate is a major neurotransmitter in the retina and other parts of the central nervous system, exerting its influence through ionotropic and metabotropic receptors. One ionotropic receptor, the N-methyl-D-aspartate(NMDA) receptor, is central to neural shaping, but also plays a major role during neuronal development and in disease processes. We studied the distribution pattern of different subunits of the NMDA receptor within the rat retina including quantifying the pattern of labelling for all the NRI splice variants, the NR2A and NR2B subunits. The labelling pattern for the subunits was confined predominantly in the outer two-thirds of the inner plexiform layer. We also wanted to probe NMDA receptor function using an organic cation, agmatine (AGB); a marker for cation channel activity. Although there was an NMDA concentration-dependent increase in AGB labelling of amacrine cells and ganglion cells, we found no evidence of functional NMDA receptors on horizontal cells in the peripheral rabbit retina, nor in the visual streak where the type A horizontal cell was identified by GABA labelling. Basal AGB labelling within depolarizing bipolar cells was also noted. This basal bipolar cell AGB labelling was not modulated by NMDA and was completely abolished by the use of L-2-amino-4-phosphono-butyric acid,which is known to hyperpolarize retinal depolarizing bipolar cells. AGB is therefore not only useful as a probe of ligand-gated drive, but can also identify neurons that have constitutively open cationic channels. In combination,the NMDA receptor subunit distribution pattern and the AGB gating experiments strongly suggests that this ionotropic glutamate receptor is functional in the cone-driven pathway of the inner retina.

Localization and expression of the glutamate transporter, excitatory amino acid transporter 4, within astrocytes of the rat retina

Mechanisms for the removal of glutamate are vital for maintaining normal function of the retina. Five excitatory amino acid transporters have been characterized to date from neuronal tissue, all of which are expressed within the retina except excitatory amino acid transporter 4 (EAAT4). In this study we examined the expression and localization of the glutamate transporter EAAT4 in the rat retina using RT-PCR and immunocytochemistry. RT-PCR using rat EAAT4 specific primers revealed a prominent 296-bp product in the retina, cortex and cerebellum. The identity of the EAAT4 fragment was confirmed by DNA sequencing. We examined the tissue expression levels of EAAT4 in cortex, retina and cerebellum using real-time PCR. The highest expression level was found in the cerebellum. Expression in the cortex was approximately 3.1% that of the cerebellum and the retina was found to have approximately 0.8% the total cerebellar EAAT4 content. In order to examine the specific cell types within the retina that express EAAT4, we performed immunocytochemistry using a rat EAAT4 specific antiserum. Cellular processes within the nerve fibre layer of the retina were intensely labelled for EAAT4. Double labelling EAAT4 with glial fibrillary acidic protein (GFAP) revealed extensive colocalization indicating that EAAT4 is localized within astrocytes within the retina. Double labelling of EAAT4 and the glutamate transporter EAAT1 (GLAST) revealed extensive colocalization suggesting that astrocytes in the retina express at least two types of glutamate transporters. These results suggest that astrocytes within the retina are well placed to provide mechanisms for glutamate removal as well as controlling cellular excitability.

Mapping photoreceptor and postreceptoral labelling patterns using a channel permeable probe (agmatine) during development in the normal and RCS rat retina.

The aim of this study was to determine whether agmatine, a channel permeable probe, can identify photoreceptor dysfunction in the Royal College of Surgeons (RCS) retina at an earlier stage to that shown by apoptosis or anatomical markers, and also characterize the neurochemical development of the inner retina in the normal and degenerating rat. We used isolated retinas at different ages incubated in physiological media containing agmatine. Subsequently, postembedding immunocytochemistry was used to determine the number of labelled photoreceptors and the labelling pattern within postreceptoral neurons. Agmatine labelling patterns revealed a sequential development of retinal neurons beginning at postnatal day (PND) 11/12 with most horizontal cells, a few ganglion and amacrine cells, showing a strong signal. The neurochemical development progressed rapidly, and reflects to a large part the known distribution of glutamate receptors, with inner nuclear labelling being evident by PND14, continuing with the same pattern of labelling in adulthood for the control retina. The RCS retina showed markedly reduced agmatine labelling in the inner retina at PND20. A rapid increase in photoreceptor AGB labelling was evident during the degeneration phase. Multiple samples at PND14 and PND16 confirmed a significant increase of labelled photoreceptors in the RCS retina.

P2-317: In vitro characterization of PIB binding to α-synuclein

years, 4 women, education 12 years) and 7 age, gender and education matched normal Controls (including 4 non-affected siblings) received complete clinical, neuropsychological, MRI and FDG-PET examinations. Regions of interest (ROIs) including the hippocampus (Hip), the entorhinal cortex (EC), the posterior cingulate cortex (PCC), the inferior parietal lobule (IPL), and the superior temporal gyrus (STG) were drawn bilaterally on the MRI scans of all subjects. The ROIs were used to measure volume loss (atrophy) on MRI, and MRglc from the MRI-coregistered, atrophy corrected PET scans. The whole-brain (WB) MRglc was also examined. Results: All presymptomatic FAD cases were comparable to Controls for neuropsychological measures. On MRI, after correcting for the total intracranial volume, none of the FAD cases showed significant volume reductions in any ROI. Conversely, after correcting for pons MRglc, MRglc reductions were found with PET in FAD as compared to Controls in the WB (13%), bilaterally in the IPL (14-16%) and in the STG (11-12%), and in the left EC (21%), PCC (20%), and Hip (12%) (p .05). Conclusions: These results show that FAD individuals in a presymptomatic stage already show widespread MRglc reductions consistent with the typical AD PET pattern in the absence of structural brain atrophy. These data further suggest that PET MRglc measures may serve as biomarkers in the preclinical diagnosis of AD.

P-066: Characterization of PIB binding to white matter

Michelle T. Fodero-Tavoletti, Catriona McLean, Paul A. Adlard, Laura Leone, Lisa E. Foster, Christopher C. Rowe, Colin L. Masters, Roberto Cappai, Victor L. Villemagne, University of Melbourne, Melbourne, VIC, Australia; The Mental Health Research Institute of VIctoria, Melbourne, VIC, Australia; Alfred Hospital, Melbourne, VIC, Australia; Austin Hospital, Melbourne, VIC, Australia. Contact e-mail: villemagne@petnm.unimelb.edu.au