A robust statistics-based global energy function for the alignment of serially acquired autoradiographic sections

Autoradiographic analysis of the functional changes occurring in the rat brain are most often performed on coronal sections that allow a good insight into the events occurring at the structural level but lacks the 3D context which is necessary to fully understand the involvement of the brain structures in specific situations like focal seizures with or without generalization. Therefore a robust, fully-automated algorithm for the registration of serially acquired autoradiographic sections is presented. The method accounts for the main difficulties of autoradiographic alignment: corrupted data (cuts and tears), dissimilarities or discontinuities between slices, non parallel or missing slices. The approach relies on the minimization of a global energy function based on robust statistics. The energy function measures the similarity between a slice and its neighborhood in the 3D volume. No particular direction is privileged in the method, so that global offsets, biases in the estimation or error propagations are avoided. The method is evaluated qualitatively and quantitatively on real autoradiographic data. Rat brain autoradiographic volumes are reconstructed with registration errors less than 1 degree in rotation and less than 1 pixel in translation.     

A low temperature embedding and section registration strategy for 3D image reconstruction of the rat brain from autoradiographic sections

In studies on animal models of human brain pathologies, three-dimensional reconstruction from histological sections is particularly useful when assessing the morphologic, functional and biochemical changes induced by pathology. It allows assessing lesion heterogeneity in planes different from the cutting plane and allows correlating the histology with images obtained in vivo, such as by means of magnetic resonance imaging. To create a 3D volume from autoradiographic sections with minimal distortion, both cryosectioning as well as section registration need to be optimal. This paper describes a strategy whereby four external fiducial markers are positioned outside the rat brain with the use of a low temperature brain embedding procedure. The fiducial markers proposed here can be rapidly added to any frozen tissue block with no impact on the subsequent histological operations. Since embedding is performed at a low temperature, no tissue degradation occurs due to sample heating. The markers enable robust and almost error free registration, even in the presence of missing sections and poor image quality. Furthermore, the markers may be used to partially correct for global distortions.     

Autoradiographic maps of regional brain glucose consumption in resting, awake rats using (14C) 2-deoxyglucose

The 2-deoxy-D-[14C]-glucose (2-DG) autoradiographic method for determining regional brain glucose consumption has been applied successfully by a number of workers for mapping the alterations of brain glucose consumption which occur in association with experimental alterations of brain functional activity. This paper provides a framework for the interpretation of these and further studies by presenting: (1) the pattern of regional brain glucose consumption in the normal, resting, awake rat; (2) the anatomical identities of brain structures which on autoradiographs appear only as regional variations of optical density. For this purpose, a series of 2-DG autoradiographs of coronal brain sections from an injected animal is compared with adjacent labeled Nissl sections.     

Distribution of cytochrome oxidase in rat brain: studies with diaminobenzidine histochemistry in vitro and [14C]cyanide tissue labeling in vivo

Studies in experimental animals and post-mortem studies in humans have indicated that the level of the mitochondrial enzyme cytochrome oxidase within brain anatomical pathways is regulated by the long-term functional use of those pathways. To study this relationship, we have measured cytochrome oxidase spectrophotometrically in punch biopsies from different brain regions of rat. We compared these assays against results from the diaminobenzidine histochemical technique. We found a high degree of correlation (r = 0.90) between the density of diaminobenzidine reaction product and enzyme activity. This validates the usefulness of the diaminobenzidine technique for anatomical localization and measurement of this enzyme. To study the feasibility of using radioactive cyanide as an in vivo ligand of cytochrome oxidase, we performed quantitative autoradiographic analysis of rat brains of animals given an intravenous bolus injection of [14C]cyanide. Analysis of the arterial blood curve indicated a complex redistribution of cyanide between red blood cells, plasma, and tissues. Brain labeling reached peak levels at 1 min and then fell despite rising concentrations of free plasma cyanide. Analysis of autoradiographic images revealed good anatomical resolution. The density of labeling in individual structures over time failed to show a strong correlation with cytochrome oxidase activity or diaminobenzidine reaction product.     

A method for the automatic segmentation of autoradiographic image stacks and spatial normalization of functional cortical activity patterns

This paper introduces two new methods for the automatic anatomical and functional analysis of neurobiological autoradiographic image stacks, such as 2-fluoro-deoxyglucose (2FDG) images. The difficulty in the evaluation of these "2(1/2)D" datasets is that they do not inherently represent a continuous 3D data volume (as generated by MRI or CT), but consist of a stack of images from single tissue slices, suffering from unavoidable preparation artifacts. In the first part of the paper, a semi-automatic segmentation method is presented which generates a 3D surface model of certain brain structures and which is robust against different cutting directions with respect to the brain coordinate system. The method saves man-hours compared to manual segmentation and the results are highly reproducible. In the second part, a fully automatic method for the extraction, analysis and 3D visualization of functional information is described, which allows not only a more accurate localization of activation sites, but also greatly enhances the comparability of different individuals. Results are shown for 2FDG autoradiographs from rat brains under acoustical stimulation.     

A new method for receptor autoradiography: [H]Opioid receptors in rat brain

Opioid receptors can be labeled with [³H]ligands in lightly fixed tissue sections mounted on microscope slides. The preparation of these sections does not seem to alter any of the known characteristics of opioid receptors. The light microscopic autoradiographic distribution of these binding sites can be observed if one attaches emulsion-coated coverslips to these slides to obtain autoradiograms. The distribution of [³H]diprenorphine binding sites determined by this in vitro method is identical to the distribution found in earlier studies utilizing in vivo labeling of opioid receptors. In addition, [³H]opioid peptide binding sites and [³H]dihydromorphine binding sites may be similar, perhaps identical, to those for [³H]diprenorphine, an opiate antagonist.

This method has several important advantages over earlier methods for determining the autoradiographic localization of receptors. It is possible, by washing, to reduce nonspecific binding to low levels. Since receptor labeling is performed with single tissue sections mounted on slides, one can examine different receptors in different but adjacent sections. One can use ligands that are not entirely suitable for in vivo labeling (For example, one can use [³H]peptides which do not normally cross the blood-brain barrier). One can perform autoradiographic studies after laveling tissues under a wide variety of conditions (For example, one can examine the effects of various ions and nucleotides on ligand binding distributions). This technique seems to be free of various artifacts, such as edge artifacts, which were common by techniques used in our laboratory earlier. Since one does not have to load an entire animal with radioactive ligand as one must with in vivo labeling, this approach is economically advantageous. One can use postmortem tissue, including that from humans to study receptor distribution with a high anatomical resolution.     

BAX: A Toolbox for the Dynamic Analysis of Functional MRI Datasets

We developed a toolbox called BAX (brain activation explorer) for the dynamic analysis of functional magnetic resonance imaging (fMRI) datasets using the general linear model. The toolbox provides a graphical user interface where several routines can be accessed to extract different sets of information from a given series of functional images. The dynamic analysis can be implemented using either an incremental approach or a sliding window approach. In particular, BAX can be used to construct dynamic activation maps that can be used to assess the contribution of newly added volumes in the final activation map, detect problematic segments in the dataset, or localize in time dynamic changes in brain activity. Consistency maps, which graphically represent the number of times voxels are consecutively detected as active in a given analysis, can also be constructed using either incremental or sliding window analysis. BAX runs under Matlab (MathWorks, Inc.) and requires some routines from SPM2 (Wellcome Department of Cognitive Neurology, London, UK) for its operation. It can be freely downloaded at website.     

Methods for analysis of brain connectivity: An IFCN-sponsored review

The goal of this paper is to examine existing methods to study the “Human Brain Connectome” with a specific focus on the neurophysiological ones. In recent years, a new approach has been developed to evaluate the anatomical and functional organization of the human brain: the aim of this promising multimodality effort is to identify and classify neuronal networks with a number of neurobiologically meaningful and easily computable measures to create its connectome. By defining anatomical and functional connections of brain regions on the same map through an integrated approach, comprising both modern neurophysiological and neuroimaging (i.e. flow/metabolic) brain-mapping techniques, network analysis becomes a powerful tool for exploring structural–functional connectivity mechanisms and for revealing etiological relationships that link connectivity abnormalities to neuropsychiatric disorders. Following a recent IFCN-endorsed meeting, a panel of international experts was selected to produce this current state-of-art document, which covers the available knowledge on anatomical and functional connectivity, including the most commonly used structural and functional MRI, EEG, MEG and non-invasive brain stimulation techniques and measures of local and global brain connectivity.     

Broca’s region: linking human brain functional connectivity data and non‐human primate tracing anatomy studies

Brodmann areas 6, 44 and 45 in the ventrolateral frontal cortex of the left hemisphere of the human brain constitute the anterior language production zone. The anatomical connectivity of these areas with parietal and temporal cortical regions was recently examined in an autoradiographic tract‐tracing study in the macaque monkey. Studies suggest strong correspondence between human resting state functional connectivity (RSFC) based on functional magnetic resonance imaging data and experimentally demonstrated anatomical connections in non‐human primates. Accordingly, we hypothesized that areas 6, 44 and 45 of the human brain would exhibit patterns of RSFC consistent with patterns of anatomical connectivity observed in the macaque. In a primary analysis, we examined the RSFC associated with regions‐of‐interest placed in ventrolateral frontal areas 6, 44 and 45, on the basis of local sulcal and gyral anatomy. We validated the results of the primary hypothesis‐driven analysis with a data‐driven partitioning of ventrolateral frontal cortex into regions exhibiting distinct RSFC patterns, using a spectral clustering algorithm. The RSFC of ventrolateral frontal areas 6, 44 and 45 was consistent with patterns of anatomical connectivity shown in the macaque. We observed a striking dissociation between RSFC for the ventral part of area 6 that is involved in orofacial motor control and RSFC associated with Broca’s region (areas 44 and 45). These findings indicate rich and differential RSFC patterns for the ventrolateral frontal areas controlling language production, consistent with known anatomical connectivity in the macaque brain, and suggest conservation of connectivity during the evolution of the primate brain.     

A toolbox for brain network construction and classification (BrainNetClass)

Brain functional network has been increasingly used in understanding brain functions and diseases. While many network construction methods have been proposed, the progress in the field still largely relies on static pairwise Pearson's correlation‐based functional network and group‐level comparisons. We introduce a “Brain Network Construction and Classification (BrainNetClass)” toolbox to promote more advanced brain network construction methods to the filed, including some state‐of‐the‐art methods that were recently developed to capture complex and high‐order interactions among brain regions. The toolbox also integrates a well‐accepted and rigorous classification framework based on brain connectome features toward individualized disease diagnosis in a hope that the advanced network modeling could boost the subsequent classification. BrainNetClass is a MATLAB‐based, open‐source, cross‐platform toolbox with both graphical user‐friendly interfaces and a command line mode targeting cognitive neuroscientists and clinicians for promoting reliability, reproducibility, and interpretability of connectome‐based, computer‐aided diagnosis. It generates abundant classification‐related results from network presentations to contributing features that have been largely ignored by most studies to grant users the ability of evaluating the disease diagnostic model and its robustness and generalizability. We demonstrate the effectiveness of the toolbox on real resting‐state functional MRI datasets. BrainNetClass (v1.0) is available at https://github.com/zzstefan/BrainNetClass .     

Multimodal vessel mapping for precise large area alignment of functional optical imaging data to neuroanatomical preparations in marmosets

Imaging technologies, such as intrinsic optical imaging (IOI), functional magnetic resonance imaging (fMRI) or multiphoton microscopy provide excellent opportunities to study the relationship between functional signals recorded from a cortical area and the underlying anatomical structure. This, in turn, requires accurate alignment of the recorded functional imaging data with histological datasets from the imaged tissue obtained after the functional experiment. This alignment is complicated by distortions of the tissue which naturally occur during histological treatment, and is particularly difficult to achieve over large cortical areas, such as primate visual areas. We present here a method that uses IOI vessel maps revealed in the time course of the intrinsic signal, in combination with vascular casts and vascular lumen labeling techniques together with a pseudo three dimensional (p3D) reconstruction of the tissue architecture in order to facilitate alignment of IOI data with posthoc histological datasets. We demonstrate that by such a multimodal vessel mapping approach, we are able to constitute a hook in anatomical-functional data alignment that enables the accurate assignment of functional signals over large cortical regions. As an example, we present precise alignments of IOI responses showing orientation selectivity of primate V1 with anatomical sections stained for cytochrome-oxidase-reactivity.     

Registration of in vivo magnetic resonance T1-weighted brain images to triphenyltetrazolium chloride stained sections in small animals

Signal changes observed in high-resolution in vivo magnetic resonance (MR) images acquired during cerebral ischemia in small animal models must be correlated to molecular indicators of tissue damage obtained from digitized histological brain sections. An effective image registration technique that incorporates both a linear and non-linear thin plate spline transform was developed to compensate the distortions that occur in the brain during the extraction, fixation, and staining process. Features in different layers of the brain were utilized in conjunction with a radial guideline-assisted landmark selection method to register tissue layers with few distinguishing characteristics. Quantitative analysis using simulated data demonstrated average registration error of 400 microm (corresponding to approximately 2.5 pixels in the MR images) when > or =50 landmark points are used. Visual agreement was obtained between T(1)-weighted MR images and 2,3,5-triphenyltetrazolium chloride stained histology. These methods will allow accurate registration of in vivo images with histology to correlate in vivo surrogate markers of tissue damage with specific histological indicators of disease.     

Gene expression profiling of neurochemically defined regions of the human brain by in situ hybridization-guided laser capture microdissection

Laser capture microdissection (LCM) permits isolation of specific cell types and cell groups based upon morphology, anatomical landmarks and histochemical properties. This powerful technique can be used for region-specific dissection if the target structure is clearly delineated. However, it is difficult to visualize anatomical boundaries in an unstained specimen, while histological staining can complicate the microdissection process and compromise downstream processing and analysis. We now introduce a novel method in which in situ hybridization (ISH) signal is used to guide LCM on adjacent unstained sections to collect tissue from neurochemically defined regions of the human postmortem brain to minimize sample manipulation prior to analysis. This approach was validated in nuclei that provide monoaminergic inputs to the forebrain, and likely contribute to the pathophysiology of mood disorders. This method was used successfully to carry out gene expression profiling and quantitative real-time PCR (qPCR) confirmation from the dissected material. When compared to traditional micropunch dissections, our ISH-guided LCM method provided enhanced signal intensity for mRNAs of specific monoaminergic marker genes as measured by genome-wide gene expression microarrays. Enriched expression of specific monoaminergic genes (as determined by microarrays and qPCR) was detected within appropriate anatomical locations validating the accuracy of microdissection. Together these results support the conclusion that ISH-guided LCM permits acquisition of enriched nucleus-specific RNA that can be successfully used for downstream gene expression investigations. Future studies will utilize this approach for gene expression profiling of neurochemically defined regions of postmortem brains collected from mood disorder patients.     

SPECT imaging of the benzodiazepine receptor: autoradiographic comparison of receptor density and radioligand distribution.

SPECT imaging of benzodiazepine (BZ) receptor using the antagonist [123I]iomazenil in nonhuman primates was correlated with ex vivo autoradiography and in vitro receptor autoradiography of postmortem tissue sections taken at the time of maximal brain uptake of the radiotracer. Cryostat sections prepared from brain tissue blocks at the orientation of the SPECT imaging plane showed high activity in gray matter regions, with gray to white matter ratios of greater than 30:1. After at least one week decay of 123I, these same tissue sections were used to localize and quantify the distribution of BZ receptors with the standard technique of in vitro receptor autoradiography using [125I]iomazenil. The ex vivo autoradiographic distribution of activity was highly correlated (r = 0.89) with the distribution of BZ receptors, although a few brain regions showed reproducible discrepancies between ex vivo and in vitro results. The ex vivo autoradiograms provided quantitative data from a realistic "biological phantom," which may be used to assess the accuracy of image reconstruction and to investigate differences between the distribution of an intravenously (i.v.) administered tracer and that of its target in brain tissue.     

Topographical study of supratentorial brain tumors

The topographic analysis of supratentorial brain tumors must answer the following questions: location of the tumor, its extension, its being infiltrative or well localized and especially the relationships with the main functional areas such as the speech areas, the sensori-motor area, and the visuel areas. Also the topographic analysis may help to approach the diagnosis of nature. The neuroradiological procedures include the CT, the angiography and especially the MRI in sagittal and coronal sections, with gadolinium enhancement. The anatomical accuracy is obtained using the bicommissural plane and superimposition techniques according to the Talairach's Atlas and anatomical sections and MRI superimposition with angiography. 3D volumic reconstruction is of considerable contribution for the topographic analysis of a cerebral tumor.     

Analysis of galactocerebrosidase activity in the mouse brain by a new histological staining method

Gene therapy of galactocerebrosidase (GALC) deficient mice (Twitcher mutants) requires a fast and sensitive assay to detect transduced cells in vitro and in vivo. We have developed a new rapid histochemical method that specifically detects GALC activity in situ in neural cells using 5-Br-3Cl-beta-galactopiranoside (X-Gal) in the presence of taurodeoxycholic and oleic acids to enhance suspension of the substrate at low pH. Using this method, we observed robust X-Gal staining in diverse neuronal populations and interfascicular oligodendrocytes in sections from normal mouse brain. In contrast, sections of Twitcher brain did not show a specific staining pattern in neurons or glial cells. The availability of this new sensitive and rapid in situ detection assay is fundamental for the follow-up of Twitcher mice under gene or cellular therapies to correct central GALC deficiency.     

Ventral visual cortex in humans: cytoarchitectonic mapping of two extrastriate areas

The extrastriate visual cortex forms a complex system enabling the analysis of visually presented objects. To gain deeper insight into the anatomical basis of this system, we cytoarchitectonically mapped the ventral occipital cortex lateral to BA 18/V2 in 10 human postmortem brains. The anatomical characterization of this part of the ventral stream was performed by examination of cell-body-stained histological sections using quantitative cytoarchitectonic analysis. First, the gray level index (GLI) was measured in the ventral occipital lobe. Cytoarchitectonic borders, i.e., significant changes in the cortical lamination pattern, were then identified using an observer-independent algorithm based on multivariate analysis of GLI profiles. Two distinct cytoarchitectonic areas (hOC3v, hOC4v) were characterized in the ventral extrastriate cortex lateral to BA 18/V2. Area hOC3v was found in the collateral sulcus. hOC4v was located in this sulcus and also covered the fusiform gyrus in more occipital sections. Topographically, these areas thus seem to represent the anatomical substrates of functionally defined areas, VP/V3v and V4/V4v. Following histological analysis, the delineated cytoarchitectonic areas were transferred to 3D reconstructions of the respective postmortem brains, which in turn were spatially normalized to the Montreal Neurological Institute reference space. A probabilistic map was generated for each area which describes how many brains had a representation of this area in a particular voxel. These maps can now be used to identify the anatomical correlates of functional activations observed in neuroimaging experiments to enable a more informed investigation into the many open questions regarding the organization of the human visual cortex.     

Visualization of mitochondrial oxygen fixation in brain slices by gas-tissue autoradiography.

We have developed a novel autoradiographic method of visualizing oxygen fixation with sufficient delivery of [(15)O]O(2)/O(2). Brain slices (400 microm) were preincubated in Krebs-Ringer phosphate buffer and exposed to [(15)O]O(2) in a chamber. Fixation of [(15)O]O(2) correlated with the polarographically measured oxygen consumption among tissue slices from various organs (r=0.84). The fixation of [(15)O]O(2) by brain slices was significantly reduced (7. 2% of the control) by heat-treatment or dose dependently by NaCN (18. 2% of the control on 50 mM NaCN pretreatment). The (15)O radioactivity in the brain slices prepared from rotenone injected rats was also reduced compared to the control (56.8% of the control side). In an autoradiographic study, (15)O radioactivity showed a heterogeneous distribution both in coronal and sagittal sections. Autoradiography of young and senescent rat brain sections showed reduction of oxygen uptake with aging in the cerebrum, the senescent being 77.4% of the young. This method provides information regarding basic oxygen consumption of tissue slices under condition of sufficient O(2) delivery, which reflects mitochondrial electron transport.     

Unravelling the intrinsic functional organization of the human lateral frontal cortex: a parcellation scheme based on resting state FMRI

Human and nonhuman primates exhibit flexible behavior. Functional, anatomical, and lesion studies indicate that the lateral frontal cortex (LFC) plays a pivotal role in such behavior. LFC consists of distinct subregions exhibiting distinct connectivity patterns that possibly relate to functional specializations. Inference about the border of each subregion in the human brain is performed with the aid of macroscopic landmarks and/or cytoarchitectonic parcellations extrapolated in a stereotaxic system. However, the high interindividual variability, the limited availability of cytoarchitectonic probabilistic maps, and the absence of robust functional localizers render the in vivo delineation and examination of the LFC subregions challenging. In this study, we use resting state fMRI for the in vivo parcellation of the human LFC on a subjectwise and data-driven manner. This approach succeeds in uncovering neuroanatomically realistic subregions, with potential anatomical substrates including BA 46, 44, 45, 9 and related (sub)divisions. Ventral LFC subregions exhibit different functional connectivity (FC), which can account for different contributions in the language domain, while more dorsal adjacent subregions mark a transition to visuospatial/sensorimotor networks. Dorsal LFC subregions participate in known large-scale networks obeying an external/internal information processing dichotomy. Furthermore, we traced "families" of LFC subregions organized along the dorsal-ventral and anterior-posterior axis with distinct functional networks also encompassing specialized cingulate divisions. Similarities with the connectivity of macaque candidate homologs were observed, such as the premotor affiliation of presumed BA 46. The current findings partially support dominant LFC models.     

Mixed models and multiple comparisons in analysis of human neurochemical maps

Examination of brain regional neurochemistry in disease states reveals differences among brain regions. Knowing where alterations in brain function are located is crucial to understanding the disease effect. The anatomical distribution of neurotransmitter receptors is now often studied using quantitative autoradiography, but the large number of brain regions involved raises serious problems for statistical analysis of such data. Due to the dependence among the subjects in case control designs, statistical analysis based on a 'mixed model' is useful. Such an analysis is illustrated using a small autoradiographic data set. The Bonferroni method, the method of Holm, and two 'False Discovery Rate'-controlling methods for adjusting P-values for multiple comparisons are compared.