Recent anterograde and retrograde studies in the rat have provided detailed information on the origin and termination of the interconnections between the amygdaloid complex and the hippocampal formation and parahippocampal areas (including areas 35 and 36 of the perirhinal cortex and the postrhinal cortex). The most substantial inputs to the amygdala originate in the rostral half of the entorhinal cortex, the temporal end of the CA1 subfield and subiculum, and areas 35 and 36 of the perirhinal cortex. The amygdaloid nuclei receiving the heaviest inputs are the lateral, basal, accessory basal, and central nuclei as well as the amygdalohippocampal area. The heaviest projections from the amygdala to the hippocampal formation and the parahippocampal areas originate in the lateral, basal, accessory basal, and posterior cortical nuclei. These pathways terminate in the rostral half of the entorhinal cortex, the temporal end of the CA3 and CA1 subfields or the subiculum, the parasubiculum, areas 35 and 36 of the perirhinal cortex, and the postrhinal cortex. The connectional data are summarized and the underlying principles of organization of these projections are discussed.

The amygdaloid complex and hippocampal formation mediate functions involving emotion and memory. To investigate the connections that regulate the interactions between these regions, we injected the anterograde tracer Phaseolus vulgaris-leucoagglutinin into various divisions of the lateral, basal, and accessory basal nuclei of the rat amygdala. The heaviest projection to the entorhinal cortex originates in the medial division of the lateral nucleus which innervates layer III of the ventral intermediate and dorsal intermediate subfields. In the basal nucleus, the heaviest projection arises in the parvicellular division and terminates in layer III of the amygdalo-entorhinal transitional subfield. In the accessory basal nucleus, the parvicellular division heavily innervates layer V of the ventral intermediate subfield. The most substantial projection to the hippocampus originates in the basal nucleus. The caudomedial portion of the parvicellular division projects heavily to the stratum oriens and stratum radiatum of CA3 and CA1. The accessory basal nucleus projects to the stratum lacunosum-moleculare of CA1. The subiculum receives a substantial input from the caudomedial parvicellular division. The parasubiculum receives dense projections from the caudal portion of the medial division of the lateral nucleus, the caudomedial parvicellular division of the basal nucleus, and the parvicellular division of the accessory basal nucleus. Our data show that select nuclear divisions of the amygdala project to the entorhinal cortex, hippocampus, subiculum, and parasubiculum in segregated rather than overlapping terminal fields. These data suggest that the amygdaloid complex is in a position to modulate different stages of information processing within the hippocampal formation.

α-Synuclein is the major protein associated with Lewy body dementia, Parkinson's disease and multiple system atrophy. Since α-synuclein is present in the brain in physiological conditions as a presynaptic protein, it is crucial to characterize disease-associated modifications to develop an in vivo biomarker. With the aim to develop antibodies showing high specificity and sensitivity for disease-associated α-synuclein, synthetic peptides containing different amino acid sequences were used for immunization of mice. After generation of α-synuclein aggregates, ELISA and immunoblotting were used to test the specificity of antibodies. Tissue microarray sections originating from different human α-synucleinopathies were used to compare immunostaining with other, commercially available antibodies. Immunization of mice with the peptide TKEGVVHGVATVAE (amino acid 44-57 of α-synuclein) resulted in the generation of a monoclonal antibody (5G4), which was able to bind aggregated α-synuclein preparation in sandwich ELISA or coated on magnetic beads. 5G4 proved to be superior to other antibodies in comparative immunohistochemical studies by revealing more widespread and distinct α-synuclein pathology. Immunoblotting of human brain tissue revealed an additional band seen in dementia with Lewy bodies, whereas the band representing monomeric α-synuclein was very weak or lacking. In summary, the 5G4 antibody is most promising for re-evaluation of archival material and may offer new perspective for the development of in vivo diagnostic assays for detecting disease-associated α-synuclein in body fluids.

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β-Amyloid (Aβ) related pathology shows a range of lesions which differ both qualitatively and quantitatively. Pathologists, to date, mainly focused on the assessment of both of these aspects but attempts to correlate the findings with clinical phenotypes are not convincing. It has been recently proposed in the same way as ι and α synuclein related lesions, also Aβ related pathology may follow a temporal evolution, i.e. distinct phases, characterized by a step-wise involvement of different brain-regions. Twenty-six independent observers reached an 81% absolute agreement while assessing the phase of Aβ, i.e. phase 1 = deposition of Aβ exclusively in neocortex, phase 2 = additionally in allocortex, phase 3 = additionally in diencephalon, phase 4 = additionally in brainstem, and phase 5 = additionally in cerebellum. These high agreement rates were reached when at least six brain regions were evaluated. Likewise, a high agreement (93%) was reached while assessing the absence/presence of cerebral amyloid angiopathy (CAA) and the type of CAA (74%) while examining the six brain regions. Of note, most of observers failed to detect capillary CAA when it was only mild and focal and thus instead of type 1, type 2 CAA was diagnosed. In conclusion, a reliable assessment of Aβ phase and presence/absence of CAA was achieved by a total of 26 observers who examined a standardized set of blocks taken from only six anatomical regions, applying commercially available reagents and by assessing them as instructed. Thus, one may consider rating of Aβ-phases as a diagnostic tool while analyzing subjects with suspected Alzheimer’s disease (AD). Because most of these blocks are currently routinely sampled by the majority of laboratories, assessment of the Aβ phase in AD is feasible even in large scale retrospective studies.

Amyloid-beta-protein (Abeta) is generally assessed by neuropathologists in diagnostics. This BrainNet Europe ( http://www.brainnet-europe.org/ ) (15 centres and 26 participants) study was carried out to investigate the reliability of such an assessment. In the first part of this trial, tissue microarray sections were stained with the antibody of each centre's choice. Reflecting the reality, seven antibodies and a plethora of pretreatment strategies were used. Ninety-two percent of the stainings were of good/acceptable quality and the estimation of presence of Abeta aggregates yielded good results. However, a poor agreement was reached particularly regarding quantitative (density) and qualitative (diffuse/cored plaques) results. During a joint meeting, the clone 4G8 was determined to label best the fleecy/diffuse plaques, and thus, this clone and the formic acid pretreatment technique were selected for the second part of this study. Subsequently, all stained sections were of good/acceptable quality and again a high level of concordance of the dichotomized (presence/absence) assessment of plaques and CAA was achieved. However, even when only one antibody was used, the type of Abeta-aggregates (diffuse/cored), type of vessel and Vonsattel grade, were not reliably assigned. Furthermore, the quantification of lesions was far from reliable. In line with the first trial, the agreement while assessing density (some, moderate and many) was unimpressive. In conclusion, we can confirm the utility of immunohistochemical detection of Abeta-protein in diagnostics and research. It is noteworthy that to reach reproducible results a dichotomized assessment of Abeta-immunoreactivity rather than quantification and assignment of various types of lesions should be applied, particularly when comparing results obtained by different neuropathologists.

This interlaboratory study evaluated the reproducibility of the assessments of neuritic plaques and neurofibrillary tangles (NFTs)--the hallmark lesions of Alzheimer disease--and compared the staining between the BrainNet Europe centers. To reduce the topography-related inconsistencies in assessments, we used a 2-mm tissue microarray (TMA) technique. The TMA block included 42 core samples taken from 21 paraffin blocks. The assessments were done on Bielschowsky and Gallyas silver stains using an immunohistochemical (IHC) method with antibodies directed to beta-amyloid (IHC/Abeta) and hyperphosphorylated tau (IHC/HPtau). The staining quality and the assessments differed between the participants, being most diverse with Bielschowsky (good/acceptable stain in 53% of centers) followed by Gallyas (good/acceptable stain in 57%) and IHC/Abeta (good/acceptable stain in 71%). The most uniform staining quality and assessment was obtained with the IHC/HPtau method (good/acceptable stain in 94% of centers). The neuropathologic diagnostic protocol (Consortium to Establish a Registry for Alzheimer Disease, Braak and Braak, and the National Institute of Aging and Reagan [NIA-Reagan] Institute) that was used significantly influenced the agreement, being highest with NIA-Reagan (54%) recommendations. This agreement was improved by visualization of NFTs using the IHC/HPtau method. Therefore, the IHC/HPtau methodology to visualize NFTs and neuropil threads should be considered as a method of choice in a future diagnostic protocol for Alzheimer disease.

Genetic, clinical, and neuropathologic heterogeneity have been observed in frontotemporal lobar degeneration with ubiquitin (Ubq)-positive inclusions (FTLD-U) and FTLD-U with motor neuron disease. Here, the distribution and morphologic features of neuronal and glial inclusions in the brains of 20 FTLD-U and 2 FTLD-U/motor neuron disease cases were assessed using immunohistochemistry for Ubq-binding protein p62. Eighteen cases displayed TAR DNA-binding protein 43-immunoreactive lesions and were classified as Types 3 (neuronal cytoplasmic inclusions and neurites; 72%), 2 (primarily neuronal cytoplasmic inclusions; 17%), or 1 (primarily neurites; 11%) FTLD-U. The distribution of p62-immunoreactivity varied considerably in each type. Of 4 unclassifiable cases, 2 displayed p62-immunoreactive lesions suggestive of FTLD-U with a mutation in the charged multivesicular body protein 2B gene; 1 suggested basophilic inclusion body disease, and 1 was of a type not previously described. By immunohistochemistry for Ubq-binding protein p62, the distribution of abnormalities was wider than expected; in approximately half of the cases, there were p62-positive but TAR DNA-binding protein 43-negative inclusions in the cerebellum, a region not previously considered to be affected. In other regions, TAR DNA-binding protein 43-, Ubq-, and Ubq-binding protein p62 labeling of inclusions was variable. Whether variations in inclusion morphologies, immunoreactivity, and topographic distribution are due to methodologic factors, different stages of inclusion and disease evolution, different disease entities or biologic modifications of the same disease are presently unclear.