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Especially for neuroscience and the development of new biomarkers, a direct correlation between in vivo imaging and histology is essential. However, this comparison is hampered by deformation and shrinkage of tissue samples caused by fixation, dehydration and paraffin embedding. We used magnetic resonance (MR) imaging and computed tomography (CT) imaging to analyze the degree of shrinkage on murine brains for various fixatives. After in vivo imaging using 7 T MRI, animals were sacrificed and the brains were dissected and immediately placed in different fixatives, respectively: zinc-based fixative, neutral buffered formalin (NBF), paraformaldehyde (PFA), Bouin-Holland fixative and paraformaldehyde-lysine-periodate (PLP). The degree of shrinkage based on mouse brain volumes, radiodensity in Hounsfield units (HU), as well as non-linear deformations were obtained. The highest degree of shrinkage was observed for PLP (68.1%, P < 0.001), followed by PFA (60.2%, P<0.001) and NBF (58.6%, P<0.001). The zinc-based fixative revealed a low shrinkage with only 33.5% (P<0.001). Compared to NBF, the zinc-based fixative shows a slightly higher degree of deformations, but is still more homogenous than PFA. Tissue shrinkage can be monitored non-invasively with CT and MR. Zinc-based fixative causes the smallest degree of brain shrinkage and only small deformations and is therefore recommended for in vivo ex vivo comparison studies.

Purpose: This work proposes a dual contrast rapid acquisition with relaxation enhancement (RARE) variant (2in1-RARE), which provides simultaneous proton density (PD) and T2 * contrast in a single acquisition., Theory and Methods: The underlying concept of 2in1-RARE is the strict separation of spin echoes and stimulated echoes. This approach offers independent weighting of spin echoes and stimulated echoes. 2in1-RARE was evaluated in phantoms including signal-to-noise ratio (SNR) and point spread function assessment. 2in1-RARE was benchmarked versus coherent RARE and a split-echo RARE variant. The applicability of 2in1-RARE for brain imaging was demonstrated in a small cohort of healthy subjects (n = 10) and, exemplary, a multiple sclerosis patient at 3 Tesla as a precursor to a broader clinical study., Results: 2in1-RARE enables the simultaneous acquisition of dual contrast weighted images without any significant image degradation and without sacrificing SNR versus split-echo RARE. This translates into a factor of two speed gain over multi-contrast, sequential split-echo RARE. A 15% broadening of the point spread function was observed in 2in1-RARE. T1 relaxation effects during the mixing time can be neglected for brain tissue., Conclusion: 2in1-RARE offers simultaneous acquisition of images of anatomical (PD) and functional (T2 *) contrast. It presents an alternative to address scan time constraints frequently encountered during sequential acquisition of T2 * or PD-weighted RARE., (© 2013 Wiley Periodicals, Inc.)

Density gradient centrifugation of native and recombinant gamma-aminobutyric acid, type A (GABA(A)) receptors was used to detect assembly intermediates. No such intermediates could be identified in extracts from adult rat brain or from human embryonic kidney (HEK) 293 cells transfected with alpha(1), beta(3), and gamma(2) subunits and cultured at 37 degrees C. However, subunit dimers, trimers, tetramers, and pentamers were found in extracts from the brain of 8-10-day-old rats and from alpha(1)beta(3)gamma(2) transfected HEK cells cultured at 25 degrees C. In both systems, alpha(1), beta(3), and gamma(2) subunits could be identified in subunit dimers, indicating that different subunit dimers are formed during GABA(A) receptor assembly. Co-transfection of HEK cells with various combinations of full-length and C-terminally truncated alpha(1) and beta(3) or alpha(1) and gamma(2) subunits and co-immunoprecipitation with subunit-specific antibodies indicated that even subunits containing no transmembrane domain can assemble with each other. Whereas alpha(1)gamma(2), alpha(1)Ngamma(2), alpha(1)gamma(2)N, and alpha(1)Ngamma(2)N, combinations exhibited specific [(3)H]Ro 15-1788 binding, specific [(3)H]muscimol binding could only be found in alpha(1)beta(3) and alpha(1)beta(3)N, but not in alpha(1)Nbeta(3) or alpha(1)Nbeta(3)N combinations. This seems to indicate that a full-length alpha(1) subunit is necessary for the formation of the muscimol-binding site and for the transduction of agonist binding into channel gating.

The origin of inter-ictal epileptic activity can be localized from the magnetoencephalogram (MEG) using the Equivalent Current Dipole (ECD) as a source model. One problem with such localizations is that in many patients, the localization of an epileptic spike source becomes inaccurate because the epileptic spikes are superimposed by pathologic brain rhythmic activities. This paper proposes to use the spatial coherence of the measured magnetic field caused by the measured magnetic field to suppress its influence in the ECD localization. In the method proposed here, the covariance matrix, which expresses the spatial coherence of the rhythmic magnetic field, is first calculated using a data portion where no spikes exist and rhythmic slow waves are evident. Then, ECD localization is performed by minimizing the least-squares cost function modified by the covariance matrix. Experiments using a computer generated ECD field and rhythmic slow waves measured from a patient suffering from complex partial epilepsy prove the basic effectiveness of the method proposed. Localizations of actual spike sources in the same clinical data are performed, and results indicating the effectiveness of the proposed method are obtained.

Is MEG source analysis able to precisely locate the primary focal epileptic activity? 22 patients with pharmacoresistant temporal lobe epilepsy were recorded during presurgical evaluation simultaneously with multichannel MEG/EEG and invasive (subdural) electrodes to evaluate the increase of information gained by MEG concerning the localization of focal epileptic activity and lesions. With this systematic study it should become clearer how often MEG can establish a diagnostic bridge between function and morphology. In addition, MEG localization accuracy of focal epileptic activity was to be validated empirically by invasive EEG recordings and postsurgical outcome. Spikes in the MEG were used for magnetic source localization, and the result was combined with magnetic resonance imaging (MRI). All patients definitely suffered from temporal lobe epilepsy and revealed a structural abnormality in MRI. 17 patients with lesions in the temporal lobe were operated meanwhile and became markedly improved or seizure free. In 7 of 8 patients with a tumor and validated operation outcome, a very close correlation of the 3D-magnetic source localization and the border of the tumor in the brain was found (distance less than 10 mm). In 8 of 9 patients with a temporal/hippocampal atrophy and validated operation outcome, dipoles of epileptiform activity were located within the atrophic lobe.(ABSTRACT TRUNCATED AT 250 WORDS)

Equivalent current dipole (ECD) analysis and fully three-dimensional distributed source solutions have been applied to interictal multichannel magnetoencephalographic recordings of a patient with complex partial epilepsy (CPE). Averaged signals were used. At certain instances ECD solutions could be found with a very high cross-correlation coefficient between the measurements and the field, produced by the current dipole solution, typically in excess of 0.97. At these instances a highly localized distribution, very close to the ECD location, was obtained from the distributed source analysis. The ECD solution started to move when the distributed source solution began to develop activity at more than one centre. This clearly contrasted with the way the activity of the distributed source solutions changed, which turned out to be highly stable. Stringent selection criteria for using the different solutions therefore seem mandatory, especially in identifying pathological areas and volumes of the human brain.

Polyclonal antibodies were raised to synthetic peptides having amino acid sequences corresponding with the N- or C-terminal part of the gamma-aminobutyric acidA (GABAA) receptor alpha 5-subunit. These anti-peptide alpha 5(2-10) or anti-peptide alpha 5(427-433) antibodies reacted specifically with GABAA receptors purified from the brains of 5-10-day-old rats in an enzyme-linked immunosorbent assay and were able to dose-dependently immunoprecipitate up to 6.3 or 13.1% of the GABAA receptors present in the incubation, respectively. In immunoblots, each of these antibodies reacted with the same two protein bands with apparent molecular mass of 53 or 57 kDa. After exhaustive treatment of purified GABAA receptors with N-Glycanase, each of these antibodies identified two proteins with apparent molecular masses of 46 and 48 kDa. Additional treatment of GABAA receptors with neuraminidase and O-Glycanase resulted in an apparently single protein with molecular mass of 47 kDa, which again was identified by both the anti-peptide alpha 5(2-10) and the anti-peptide alpha 5(427-433) antibody. These results indicate the existence of at least two different alpha 5-subunits of the GABAA receptor that differ in their carbohydrate content. In contrast to other alpha- or beta-subunits of GABAA receptors so far investigated, at least one of these two alpha 5-subunits contains O-linked carbohydrates.

The authors designed a multichannel system for noninvasive measurement of the extremely weak magnetic fields generated by the brain and the heart. It uses a flat array of 37 superconducting magnetic field-sensing coils connected to sophisticated superconducting quantum interference devices. To prevent interference from external electromagnetic fields, the system is operated inside a shielded room. Complete sets of coherent data, even from spontaneous events, can be recorded. System performance was evaluated with phantom measurements and evoked-response studies. A spatial resolution of a few millimeters and a temporal resolution of a millisecond were obtained. First results in patients with partial epilepsy and investigations of the cardiac conductive pathway indicate that biomagnetism is now ready for a systematic clinical evaluation. Interpretation of measurements was facilitated by highlighting biomagnetically localized electrical activity in three-dimensional digital magnetic resonance images.

gamma-Aminobutyric acid (GABA)/benzodiazepine receptors purified from the brains of young and adult rats were photolabeled by [3H]flunitrazepam or [3H]muscimol. Gel electrophoresis revealed 3 different proteins with apparent molecular weight (Mr) 51,000, 53,000 and 59,000 which were specifically and irreversibly labeled by [3H]flunitrazepam and recognized by the alpha-subunit specific antibody bd-28. Similarly, 3 different proteins with Mrs 51,000, 53,000 and 56,000 were irreversibly labeled by [3H]muscimol and recognized by the beta-subunit-specific antibody bd-17. Comparison of the photolabeling and immunological staining pattern from young and adult rats seems to indicate that proteins labeled by [3H]flunitrazepam and bd-28 are different from those labeled by [3H]muscimol and bd-17.

The presenilin (PS)-dependent site 3 (S3) cleavage of Notch liberates its intracellular domain (NICD), which is required for Notch signaling. The similar gamma-secretase cleavage of the beta-amyloid precursor protein (betaAPP) results in the secretion of amyloid beta-peptide (Abeta). However, little is known about the corresponding C-terminal cleavage product (CTFgamma). We have now identified CTFgamma in brain tissue, in living cells, as well as in an in vitro system. Generation of CTFgamma is facilitated by PSs, since a dominant-negative mutation of PS as well as a PS gene knock out prevents its production. Moreover, gamma-secretase inhibitors, including one that is known to bind to PS, also block CTFgamma generation. Sequence analysis revealed that CTFgamma is produced by a novel gamma-secretase cut, which occurs at a site corresponding to the S3 cleavage of Notch.

The GABAA receptor α2-subunit gene (Gabra2) has a specific spatial and temporal pattern of expression in rat brain. As a first step towards understanding the molecular mechanism underlying this regulation, we have investigated the structural properties of the 5'-flanking region of the rat Gabra2 gene. We identified six α2 transcript isoforms, each of which differs only in the 5'-untranslated region (UTR). Alignment of cDNA and genomic DNA sequences revealed that six 5'-UTRs are generated from three alternative first exons by alternative splicing using internal and terminal 5'-splice donor sites present in these exons. Promoter regions containing multiple transcription initiation sites were identified in the 5' proximity of each first exon. Two of these promoters lack TATA and CCAAT sequences. Finally, we have shown that differential activation of alternative promoters is used for the expression of the α2 mRNA isoforms during brain development, and that the diversity at the 5'-end of these transcripts affects GABAA receptor expression. Taken together, these results suggest that the expression of the Gabra2 gene can be influenced at both the transcriptional and post-transcriptional levels. [ABSTRACT FROM AUTHOR]