Intravital Imaging of Neocortical Heterotopia Reveals Aberrant Axonal Pathfinding and Myelination around Ectopic Neurons.

Neocortical heterotopia consist of ectopic neuronal clusters that are frequently found in individuals with cognitive disability and epilepsy. However, their pathogenesis remains poorly understood due in part to a lack of tractable animal models. We have developed an inducible model of focal cortical heterotopia that enables their precise spatiotemporal control and high-resolution optical imaging in live mice. Here, we report that heterotopia are associated with striking patterns of circumferentially projecting axons and increased myelination around neuronal clusters. Despite their aberrant axonal patterns, in vivo calcium imaging revealed that heterotopic neurons remain functionally connected to other brain regions, highlighting their potential to influence global neural networks. These aberrant patterns only form when heterotopia are induced during a critical embryonic temporal window, but not in early postnatal development. Our model provides a new way to investigate heterotopia formation in vivo and reveals features suggesting the existence of developmentally modulated, neuron-derived axon guidance and myelination factors.

Isoflavone supplementation, via red clover hay, alters the rumen microbial community and promotes weight gain of steers grazing mixed grass pastures.

Biochanin A, an isoflavone present in the pasture legume red clover (Trifloium pratense L.), alters fermentation in the rumen of cattle and other ruminants. Biochanin A inhibits hyper-ammonia-producing bacteria and promotes cellulolytic bacteria and fiber catalysis in vitro and ex vivo. Consequently, biochanin A supplementation improves weight gain in grazing steers. Red clover contains biologically active isoflavones that may act synergistically. Therefore, the objective was to evaluate the effect of two levels of red clover hay on growth performance and the microbial community in growing steers grazing mixed grass pastures. A grazing experiment was conducted over 2 early growing seasons (2016 and 2017) with 36 cross-bred steers and twelve rumen-fistulated, growing Holstein steers for evaluation of average daily gain and rumen microbiota, respectively. Steers were blocked by body weight and assigned to pastures with one of four treatments: 1) pasture only, 2) pasture + dry distillers' grains (DDG), 3) pasture + DDG + low level of red clover hay (~15% red clover diet), or 4) pasture + DDG + high level of red clover hay (~30% red clover diet). DDG were added to treatments to meet protein requirements and to balance total protein supplementation between treatments. All supplementation strategies (DDG ± red clover hay) increased average daily gains in comparison to pasture-only controls (P < 0.05), with a low level of red clover supplementation being the most effective (+0.17 kg d-1 > DDG only controls; P < 0.05). Similarly, hyper-ammonia-producing bacteria inhibition (10-100-fold; P < 0.05), fiber catalysis (+10-25%; P < 0.05) and short chain fatty acid concentrations were greatest with the low red clover supplement (+~25%; P < 0.05). These results provide evidence that lower levels or red clover supplementation may be optimal for maximizing overall microbial community function and animal performance in grazing steers.

Motor learning requires myelination to reduce asynchrony and spontaneity in neural activity.

Myelination increases the conduction velocity in long-range axons and is prerequisite for many brain functions. Impaired myelin regulation or impairment of myelin itself is frequently associated with deficits in learning and cognition in neurological and psychiatric disorders. However, it has not been revealed what perturbation of neural activity induced by myelin impairment causes learning deficits. Here, we measured neural activity in the motor cortex during motor learning in transgenic mice with a subtle impairment of their myelin. This deficit in myelin impaired motor learning, and was accompanied by a decrease in the amplitude of movement-related activity and an increase in the frequency of spontaneous activity. Thalamocortical axons showed variability in axonal conduction with a large spread in the timing of postsynaptic cortical responses. Repetitive pairing of forelimb movements with optogenetic stimulation of thalamocortical axon terminals restored motor learning. Thus, myelin regulation helps to maintain the synchrony of cortical spike-time arrivals through long-range axons, facilitating the propagation of the information required for learning. Our results revealed the pathological neuronal circuit activity with impaired myelin and suggest the possibility that pairing of noninvasive brain stimulation with relevant behaviors may ameliorate cognitive and behavioral abnormalities in diseases with impaired myelination.

Visualization of myelinated fiber bundles orientation during brain slice preparation by reflection polarized light microscopy.

Polarized light imaging (PLI) enables detecting the orientation of myelinated axon bundles in brain slices at microscopic resolution without histological staining. However, standard PLI requires labor-intensive procedures such as mounting brain cryosections on slide glasses. We developed an optical system that does not require a mounting procedure for PLI. Specifically, we developed an optical system to perform PLI in reflection mode (rPLI) instead of employing transmitted light as in standard PLI. We integrated this rPLI system with a conventional vibratome slicer whose cutting blade surface is a mirror. This combination allowed PLI measurements directly during the slicing procedure at room temperature. Thus, mounting procedure for PLI is not necessary. As a proof-of-concept experiment, a perfusion-fixed brain of a mouse was embedded in gelatin-containing agar and cut serially at 40~200 µm intervals. The slicing procedure was temporarily halted after each cut to capture the PLI images of the slice on the reflecting blade surface while the slice was still held up by the agar block. The orientation of the fiber bundle estimated with this method agreed with the results obtained from previous reports. Combination of a popular vibratome slicer and our rPLI system that uses versatile and inexpensive optical components would increase popularity of PLI and facilitates connectome studies at microscopic resolution. RESEARCH HIGHLIGHTS: Polarized light imaging (PLI) of brain slices was realized by using reflected light (rPLI) instead of transmitted light. The rPLI method allows detecting the myelinated fiber bundle orientation during slice preparation.

Myelin structure in unfixed, single nerve fibers: Scanning X-ray microdiffraction with a beam size of 200nm.

Previous raster-scanning with a 1µm X-ray beam of individual, myelinated fibers from glutaraldehyde-fixed rat sciatic nerve revealed a spatially-dependent variation in the diffraction patterns from single fibers. Analysis indicated differences in the myelin periodicity, membrane separations, distribution of proteins, and orientation of membrane lamellae. As chemical fixation is known to produce structural artifacts, we sought to determine in the current study whether the structural heterogeneity is intrinsic to unfixed myelin. Using a 200nm-beam that was about five-fold smaller than before, we raster-scanned individual myelinated fibers from both the peripheral (PNS; mouse and rat sciatic nerves) and central (CNS; rat corpus callosum) nervous systems. As expected, the membrane stacking in the internodal region was nearly parallel to the fiber axis and in the paranodal region it was perpendicular to the axis. A myelin lattice was also frequently observed when the incident beam was injected en face to the sheath. Myelin periodicity and diffracted intensity varied with axial position along the fiber, as did the calculated membrane profiles. Raster-scanning with an X-ray beam at sub-micron resolution revealed for the first time that the individual myelin sheaths in unfixed nerve are heterogeneous in both membrane structure and packing.

Field inhomogeneity correction for gradient echo myelin water fraction imaging.

PURPOSE: Recently, the multi-echo gradient echo (MGRE) sequence has been proposed for multicomponent T2* (MC T2*) based myelin water fraction (MWF) mapping. This approach has appeal because it can provide fast whole-brain coverage, has low specific absorption rate, and short echo spacing. However, the MGRE signal requires correction for accurate MWF mapping, because of its sensitivity to magnetic field inhomogeneities (ΔB0 ). We propose a ΔB0 correction method for 2D MGRE data obtained for MWF mapping. THEORY AND METHODS: Latter-echo MGRE data were fit to estimate B0 gradients in the slice-select direction ( Gz). The decay signal was corrected for the effects of Gz, and MC T2* analysis was performed using nonnegative least-squares fitting. The method was evaluated using simulations and its performance demonstrated in healthy volunteers. RESULTS: Simulations showed that MWFs are significantly biased in the presence of Gz and that our correction method leads to accurate MWF estimates. In vivo MWF maps obtained from corrected data showed recovery of MWF estimates in areas of high ΔB0, and overall good agreement with literature values obtained with the reference MC T2-based method. CONCLUSION: A new algorithm was presented for ΔB0 correction of 2D MGRE echo data acquired for MWF imaging. Simulations and in vivo data showed an improvement in MWF estimates. Magn Reson Med 78:49-57, 2017. © 2016 International Society for Magnetic Resonance in Medicine.

The Transcription Factors EBF1 and EBF2 Are Positive Regulators of Myelination in Schwann Cells.

Myelin formation by Schwann cells is tightly controlled by multiple pathways and regulatory molecules. The Ebf2 gene, belonging to the Ebf family of transcription factors regulating cell development and differentiation, is expressed in Schwann cells, and Ebf2 knockout mice show peripheral nerve defects. We also found that Ebf1 is expressed in Schwann cells. To investigate Ebf function in myelination, we silenced Ebf genes in myelinating dorsal root ganglia cultures. Combined downregulation of Ebf genes leads to a severe impairment of myelin formation that is completely rescued by their specific overexpression, suggesting that the expression level of Ebf genes strongly influences axon myelination. In addition, by profiling Ebf target genes, we found several transcripts belonging to pathways actively involved in peripheral myelination, including Gliomedin, a gene with a role in the formation of the nodes of Ranvier and recently implicated in the pathogenesis of the nodo-paranodopathies. Our results suggest that Ebf genes act as positive regulators of myelination and directly regulate the promoter of Gliomedin.

Septin/anillin filaments scaffold central nervous system myelin to accelerate nerve conduction.

Myelination of axons facilitates rapid impulse propagation in the nervous system. The axon/myelin-unit becomes impaired in myelin-related disorders and upon normal aging. However, the molecular cause of many pathological features, including the frequently observed myelin outfoldings, remained unknown. Using label-free quantitative proteomics, we find that the presence of myelin outfoldings correlates with a loss of cytoskeletal septins in myelin. Regulated by phosphatidylinositol-(4,5)-bisphosphate (PI(4,5)P2)-levels, myelin septins (SEPT2/SEPT4/SEPT7/SEPT8) and the PI(4,5)P2-adaptor anillin form previously unrecognized filaments that extend longitudinally along myelinated axons. By confocal microscopy and immunogold-electron microscopy, these filaments are localized to the non-compacted adaxonal myelin compartment. Genetic disruption of these filaments in Sept8-mutant mice causes myelin outfoldings as a very specific neuropathology. Septin filaments thus serve an important function in scaffolding the axon/myelin-unit, evidently a late stage of myelin maturation. We propose that pathological or aging-associated diminishment of the septin/anillin-scaffold causes myelin outfoldings that impair the normal nerve conduction velocity.

Study of the Peripheral Nerve Fibers Myelin Structure Changes during Activation of Schwann Cell Acetylcholine Receptors.

In the present paper we consider a new type of mechanism by which neurotransmitter acetylcholine (ACh) regulates the properties of peripheral nerve fibers myelin. Our data show the importance of the relationship between the changes in the number of Schwann cell (SC) acetylcholine receptors (AChRs) and the axon excitation (different intervals between action potentials (APs)). Using Raman spectroscopy, an effect of activation of SC AChRs on the myelin membrane fluidity was investigated. It was found, that ACh stimulates an increase in lipid ordering degree of the myelin lipids, thus providing evidence for specific role of the "axon-SC" interactions at the axon excitation. It was proposed, that during the axon excitation, the SC membrane K+- depolarization and the Ca2+-influx led to phospholipase activation or exocytosis of intracellular membrane vesicles and myelin structure reorganization.

Simple, Scalable Proteomic Imaging for High-Dimensional Profiling of Intact Systems.

Combined measurement of diverse molecular and anatomical traits that span multiple levels remains a major challenge in biology. Here, we introduce a simple method that enables proteomic imaging for scalable, integrated, high-dimensional phenotyping of both animal tissues and human clinical samples. This method, termed SWITCH, uniformly secures tissue architecture, native biomolecules, and antigenicity across an entire system by synchronizing the tissue preservation reaction. The heat- and chemical-resistant nature of the resulting framework permits multiple rounds (>20) of relabeling. We have performed 22 rounds of labeling of a single tissue with precise co-registration of multiple datasets. Furthermore, SWITCH synchronizes labeling reactions to improve probe penetration depth and uniformity of staining. With SWITCH, we performed combinatorial protein expression profiling of the human cortex and also interrogated the geometric structure of the fiber pathways in mouse brains. Such integrated high-dimensional information may accelerate our understanding of biological systems at multiple levels.

Ex vivo peripheral nerve detection of rats by spontaneous Raman spectroscopy.

Nerve-sparing surgery is increasingly being applied to avoid functional deficits of the limbs and organs following surgery. Peripheral nerves that should be preserved are, however, sometimes misidentified due to similarity of shape and color to non-nerve tissues. To avoid misidentification of peripheral nerves, development of an in situ nerve detection method is desired. In this study, we report the label-free detection of ex vivo peripheral nerves of Wistar rats by using Raman spectroscopy. We obtained Raman spectra of peripheral nerves (myelinated and unmyelinated nerves) and their adjacent tissues of Wistar rats without any treatment such as fixation and/or staining. For the identification of tissue species and further analysis of spectral features, we proposed a principal component regression-based discriminant analysis with representative Raman spectra of peripheral nerves and their adjacent tissues. Our prediction model selectively detected myelinated nerves and unmyelinated nerves of Wistar rats with respective sensitivities of 95.5% and 88.3% and specificities of 99.4% and 93.5%. Furthermore, important spectral features for the identification of tissue species were revealed by detailed analysis of principal components of representative Raman spectra of tissues. Our proposed approach may provide a unique and powerful tool for peripheral nerve detection for nerve-sparing surgery in the future.

Comparison of myelin water fraction from multiecho T2 decay curve and steady-state methods.

PURPOSE: Myelin water fraction is conventionally measured from the T2 decay curve. Recently, a steady-state approach entitled multicomponent-driven equilibrium single pulse observation of T1 /T2 (mcDESPOT) was employed for myelin water fraction mapping. However, no direct comparison between the established multiecho T2 relaxation method and mcDESPOT has been performed. METHODS: Gradient and spin echo (GRASE) acquired T2 decay curve and mcDESPOT measurements were acquired from 10 healthy volunteers using a 3T MRI. We compared myelin water fraction, transmit radio frequency field (B1 ), and T2 's of intra- and extracellular water obtained from both methods. RESULTS: For all brain regions examined, myelin water fractions from mcDESPOT were significantly higher than those from multiecho GRASE. B1 maps were qualitatively similar between GRASE and mcDESPOT, but multicomponent T2 times were significantly different. To investigate the effect of exchange, mcDESPOT data were analyzed with and without exchange. When exchange was turned off, intra- and extracellular T2 times from mcDESPOT were roughly consistent with GRASE results; however, myelin water fractions derived from mcDESPOT were still significantly higher than those derived from GRASE. CONCLUSION: Myelin water fraction values derived from mcDESPOT cannot be considered to be equivalent to those derived from T2 decay curve approaches.

Positive symptoms and water diffusivity of the prefrontal and temporal cortices in schizophrenia patients: a pilot study.

The development of diffusion tensor imaging (DTI) has provided information about microstructural changes in the brain. Most DTI studies have focused on white matter (WM). Few DTI studies have examined the gray matter (GM) in schizophrenia and, to date, there has been no attempt to identify the relationship between water diffusivity and symptom severity in schizophrenia. The present study aimed to examine microstructural deficits in the dorsal prefrontal cortex (DPFC) and temporal cortex in schizophrenia patients using fractional anisotropy (FA) and water diffusivity. This study also explored the relationship between DTI measurements and psychotic symptoms. Magnetic resonance imaging (MRI) and DTI were used to study 19 schizophrenia patients and 19 healthy controls. Fractional anisotropy, axial diffusivity, radial diffusivity, and regional volumes were measured in the prefrontal cortex and temporal cortex. On DTI measurements, patients showed increased axial and radial diffusivities in the prefrontal cortex and temporal cortex, but they did not demonstrate any difference in fractional anisotropy and regional volumes. Additionally, axial and radial diffusivities were significantly correlated with positive symptom scores in all regions of interest. These results indicate that water diffusivity measurements, including axial and radial diffusivities, can be used to identify microstructural changes in the gray matter in schizophrenia that may be related to symptom severity.

Diffusion-weighted imaging and magnetic resonance proton spectroscopy following preterm birth.

AIM: To study the associations between magnetic resonance proton spectroscopy (MRS) data and apparent diffusion coefficients (ADC) from the preterm brain with developmental outcome at 18 months corrected age and clinical variables. MATERIALS AND METHODS: A prospective observational cohort study of 67 infants born before 35 weeks gestational age who received both magnetic resonance imaging of the brain between 37 and 44 weeks corrected gestational age and developmental assessment around 18 months corrected age. RESULTS: No relationships were found between ADC values and MRS results or outcome. MRS ratios involving N-acetyl aspartate (NAA) from the posterior white matter were associated with "severe" and "moderate to severe" difficulties, and fine motor scores were significantly lower in participants with a visible lactate doublet in the posterior white matter. The presence of a patent ductus arteriosus (PDA) was the only clinical factor related to NAA ratios. CONCLUSION: Altered NAA levels in the posterior white matter may reflect subtle white matter injury associated with neuro-developmental difficulties, which may be related to a PDA. Further work is needed to assess the longer-term neuro-developmental implications of these findings, and to study the effect of PDAs on developmental outcome in later childhood/adolescence.

Stabilization of the dystroglycan complex in Cajal bands of myelinating Schwann cells through plectin-mediated anchorage to vimentin filaments.

Previous studies have unmasked plectin, a uniquely versatile intermediate filament-associated cytolinker protein, to be essential for skin and skeletal muscle integrity. Different sets of isoforms of the protein were found to stabilize cells mechanically, regulate cytoskeletal dynamics, and serve as a scaffolding platform for signaling molecules. Here, we investigated whether a similar scenario prevails in myelinating Schwann cells. Using isoform-specific antibodies, the two plectin variants predominantly expressed in the cytoplasmic compartment (Cajal bands) of Schwann cells were identified as plectin (P)1 and P1c. Coimmunoprecipitation and immunolocalization experiments revealed complex formation of Cajal band plectin with ß-dystroglycan, the core component of the dystrophin glycoprotein complex that in Schwann cells is crucial for the compartmentalization and stabilization of the myelin sheath. To study the functional implications of Schwann cell-specific plectin-ß-dystroglycan interaction, we generated conditional (Schwann cell-restricted) plectin knockout mice. Ablation of plectin in myelinating Schwann cells (SCs) was found not to affect myelin sheath formation but to abrogate the tight association of the dystroglycan complex with the intermediate filament cytoskeleton. We show that the disruption of this association leads to the destabilization of the dystroglycan complex combined with increased myelin sheath deformations observed in the peripheral nerve during ageing of the animal.

A case of severe progressive early-onset epileptic encephalopathy: unique GABAergic interneuron distribution and imaging.

Early-onset epileptic encephalopathies include various diseases such as early-infantile epileptic encephalopathy with suppression burst. We experimentally investigated the unique clinicopathological features of a 28-month-old girl with early-onset epileptic encephalopathy. Her initial symptom was intractable epilepsy with a suppression-burst pattern of electroencephalography (EEG) from 7 days of age. The suppression-burst pattern was novel, appearing during sleep, but disappearing upon waking and after becoming 2 months old. The EEG showed multifocal spikes and altered with age. Her seizures demonstrated various clinical features and continued until death. She did not show any developmental features, including no social smiling or head control. Head MRI revealed progressive atrophy of the cerebral cortex and white matter after 1 month of age. (123)IMZ-SPECT demonstrated hypo-perfusion of the cerebral cortex, but normo-perfusion of the diencephalon and cerebellum. Such imaging information indicated GABA-A receptor dysfunction of the cerebral cortex. The genetic analyses of major neonatal epilepsies showed no mutation. The neuropathology revealed atrophy and severe edema of the cerebral cortex and white matter. GAD-immunohistochemistry exhibited imbalanced distribution of GABAergic interneurons between the striatum and cerebral cortex. The results were similar to those of focal cortical dysplasia with transmantle sign and X-linked lissencephaly with ARX mutation. We performed various metabolic examinations, detailed pathological investigations and genetic analyses, but could not identify the cause. To our knowledge, her clinical and pathological courses have never been described in the literature.

Center-out echo-planar spectroscopic imaging with correction of gradient-echo phase and time shifts.

A procedure to prevent the formation of image and spectral Nyquist ghosts in echo-planar spectroscopic imaging is introduced. It is based on a novel Cartesian center-out echo-planar spectroscopic imaging trajectory, referred to as EPSICO, and combined with a correction of the gradient-echo phase and time shifts. Processing of homogenous sets of forward and reflected echoes is no longer necessary, resulting in an optimized spectral width. The proposed center-out trajectory passively prevents the formation of Nyquist ghosts by privileging the acquisition of the center k-space line with forward echoes at the beginning of an echo-planar spectroscopic imaging dwell time and by ensuring that all k-space lines and their respective complex conjugates are acquired at equal time intervals. With the proposed procedure, concentrations of N-acetyl aspartate, creatine, choline, glutamate, and myo-inositol were reliably determined in human white matter at 3 T.

Synaptic innervation to rat hippocampus by vasopressin-immuno-positive fibres from the hypothalamic supraoptic and paraventricular nuclei.

The neuropeptide arginine vasopressin (AVP) exerts a modulatory role on hippocampal excitability through vasopressin V(1A) and V(1B) receptors. However, the origin and mode of termination of the AVP innervation of the hippocampus remain unknown. We have used light and electron microscopy to trace the origin, distribution and synaptic relationships of AVP-immuno-positive fibres and nerve terminals in the rat hippocampus. Immuno-positive fibres were present in all areas (CA1-3, dentate gyrus) of the whole septo-temporal extent of the hippocampus; they had the highest density in the CA2 region, strongly increasing in density towards the ventral hippocampus. Two types of fibres were identified, both establishing synaptic junctions. Type A had large varicosities packed with immuno-positive large-granulated peptidergic vesicles and few small clear vesicles forming type I synaptic junctions with pyramidal neuron dendrites, dendritic spines and with axonal spines. Type B had smaller varicosities containing mostly small clear vesicles and only a few large-granulated vesicles and established type II synaptic junctions mainly with interneuron dendrites. The AVP-positive axons in stratum oriens appeared to follow and contact metabotropic glutamate receptor 1α (mGluR1α)-immuno-positive interneuron dendrites. Fluoro-Gold injection into the hippocampus revealed retrogradely labelled AVP-positive somata in hypothalamic supraoptic and paraventricular nuclei. Hypothalamo-hippocampal AVP-positive axons entered the hippocampus mostly through a ventral route, also innervating the amygdala and to a lesser extent through the dorsal fimbria fornix, in continuation of the septal AVP innervation. Thus, it appears the AVP-containing neurons of the magnocellular hypothalamic nuclei serve as important sources for hippocampal AVP innervation, although the AVP-expressing neurons located in amygdala and bed nucleus of the stria terminalis reported previously may also contribute.

Diffusely abnormal white matter in multiple sclerosis: further histologic studies provide evidence for a primary lipid abnormality with neurodegeneration.

Although multiple sclerosis (MS) lesions have been studied extensively using histology and magnetic resonance imaging (MRI), little is known about diffusely abnormal white matter (DAWM). Diffusely abnormal white matter, regions with reduced mild MRI hyperintensity and ill-defined boundaries, show reduced myelin water fraction, and decreased Luxol fast blue staining of myelin phospholipids, with relative preservation of myelin basic protein and 2',3'-cyclic-nucleotide 3'-phosphohydrolase. Because DAWM may be important in MS disability and progression, further histologic characterization is warranted. The MRI data were collected on 14 formalin-fixed MS brain samples that were then stained for myelin phospholipids, myelin proteins, astrocytes and axons. Diffusely abnormal white matter showed reduced myelin water fraction (-30%, p < 0.05 for 13 samples). Myelin phospholipids showed the most dramatic and consistent histologic reductions in staining optical density (-29% Luxol fast blue and -24% Weil's, p < 0.05 for 13 and 14 samples,respectively) with lesser myelin protein involvement (-11% myelin-associated glycoprotein, -10% myelin basic protein, -8% myelin-oligodendrocyte glycoprotein, -7% proteolipid protein, -5% 2',3'-cyclic-nucleotide 3'-phosphohydrolase, p < 0.05 for 3, 3, 1, 2, and 3 samples, respectively). Axonal involvement was intermediate. Diffusely abnormal white matter lipid and protein reductions occurred independently. These findings suggest a primary lipid abnormality in DAWM that exceeds protein loss and is accompanied by axonal degeneration. These phenomena may be important in MS pathogenesis and disease progression, which is prominent in individuals with DAWM.

Automated determination of axonal orientation in the deep white matter of the human brain.

The wide-spread utilization of diffusion-weighted imaging in the clinical neurosciences to assess white-matter (WM) integrity and architecture calls for robust validation strategies applied to the data that are acquired with noninvasive imaging. However, the pathology and detailed fiber architecture of WM tissue can only be observed postmortem. With these considerations in mind, we designed an automated method for the determination of axonal orientation in high-resolution microscope images. The algorithm was tested on tissue that was stained using a silver impregnation technique that was optimized to resolve axonal fibers against very low levels of background. The orientation of individual nerve fibers was detected using spatial filtering and a template-matching algorithm, and the results are displayed as color-coded overlays. Quantitative models of WM fiber architecture at the microscopic level can lead to improved interpretation of low-resolution neuroimaging data and to more accurate mapping of fiber pathways in the human brain.