Generating brain matrices for zebra finch brain sectioning using three-dimensional printing technology.

BACKGROUND: The demand to sample brain regions in non-model species is increasing as more studies are integrating neurological data into behavioural, ecological or evolutionary analysis. However, the sampling operation is difficult for researchers without neuroscience background. It is also a challenge to collect neuroanatomical regions from animals in the field. NEW METHOD: Here we developed a new brain matrix for guiding researchers to section zebra finches' (Taeniopygia guttata) brains more steadily than by freehand trimming. Based on the 3D printing technology, we produced the zebra finch brain matrix from scratch. We also provided a step-by-step protocol to make brain matrices for any species with a brain size between that of shrews and dogs. RESULTS: The brain matrix could guide us to find the zebra finch's neuroanatomical landmarks, such as the hypothalamus, optic chiasm and occulomotor nerve. The matrix's channels near these landmarks could be used to section brains steadily and rapidly. COMPARISON WITH EXISTING METHODS: Standardized brain sectioning often requires expensive machines that may not be available in most laboratories or in the field, such as microtomes. In addition, machine-based trimming is time-consuming. Although commercial brain matrices can overcome these problems, they are only available for rats and mice. The brain matrices we developed are affordable to most laboratories and can be customised for non-model species in both lab and field experiments. CONCLUSIONS: The matrix-guided approach requires a relatively short training period and can allow researchers to properly and quickly sample brains, and thus will facilitate neuroscience-based interdisciplinary research.

The laryngoscope and nineteenth-century British understanding of laryngeal movements.

The source of the human voice is obscured from view. The development of the laryngoscope in the late 1850s provided the potential to see the action of the vocal folds during speaking for the first time. This new instrument materially contributed to the understanding of vocal fold neuroanatomy, neurophysiology, and neuropathology. The laryngoscope led to elaborated understanding of disorders that previously were determined by changes in sound. The objective of this paper is to detail the consequences of this novel visualization of the larynx, and to trace how it aided in the development of understanding of the movements of the vocal folds. This is demonstrated through an examination of the activities and practices of a group of London clinicians in the second half of the nineteenth century.

Low-Cost Stereoscopic Recordings of Neurologic Surgery Operative Microscopy for Anatomic Laboratory Training.

OBJECTIVE: Stereoscopic video recordings of operative microscopy during neuroanatomic dissections are an important component of surgical training and research in well-financed medical schools and teaching hospitals. However, the high cost of the latest operative microscopes with integrated video recording equipment can be a limiting factor in their worldwide use. The aim of the present work is to provide a simple low-cost 3-dimensional (3D) stereoscopic operative microscope recording system that can be used even in economically and resource-limited locations. This is achieved by using readily available smartphones, smartphone accessories, and computer software. METHODS: Stereoscopic recording is accomplished by attaching and aligning matched or similar smartphones to the eyepieces of an operative microscope using readily available smartphone mounting connectors. Video recordings from the smartphones are then transferred to a personal computer and processed with a video-editing software to generate stereoscopic movies that are viewed on a smartphone using virtual-reality glasses. RESULTS: The setup time to mount and align the smartphone cameras typically requires 15-30 minutes. Video image quality and 3D depth presentation is more than sufficient for surgical training and research purposes. The implementation cost ranges from $1,315-$7,066, or much less if smartphones and a computer are already available. CONCLUSIONS: The 3D video system demonstrated herein can be implemented on any type of operative microscope, including older units for which commercial stereo recording systems are not available. The system and method presented herein can be readily and affordably implemented in low-budget environments for clinical training and research.

Scientists, Instruments, and Even Brains in Transfer: German-Spanish Postwar Networks and the Construction of the Neuroendocrine System (1952-1960).

This article presents the process of relocation of hegemonies and "center-periphery" dynamics in neuroanatomy after World War II through the study of the links between the Spanish anatomical school of José Escolar García and some German institutions. We have analyzed their works on the morphology of the neuroendocrine system as a case study, showing how the first contacts of the Spaniards with the United States started a material transfer process between centers on both sides of the Atlantic Ocean through the mediation-and adaptation-of the periphery. The case also shows how scientific networks in the "new" Europe were reestablished after the Nazi era and how important these systems were for the transfer of knowledge, using them for the circulation of experts, instruments, and even biological samples.

The Rhoton Collection.

The Rhoton Collection is an archive of Dr. Al Rhoton Jr.'s anatomical images and video lectures, as well as an anatomical reference. In an effort to maximize the educational impact of these teaching materials, web-based technologies are used to dynamically format this material for a variety of devices ranging from cellular phones to projectors. Surgical cases are cross-referenced to further enhance the usefulness of this collection, which is available at http://rhoton.ineurodb.org. The features of the Rhoton Collection website are described in this article.

How to prepare neuroanatomical image data.

As image data from a single neuroanatomical study can easily exceed tens of gigabytes, managing, analyzing, and presenting it is not trivial. Careful planning along multiple axes is required and includes the following: (1) Organizational methods developed for images should allow for easy and efficient access, selection, and potential reorganization of images. (2) Experimental information and other metadata should be readily available and accompany image data. (3) Even if a study's entire body of image data is made available, highlighting key results and preparing figures requires selecting image regions and resolutions, creating annotations, and adhering to publishing and community guidelines for image adjustments. Further, it may be necessary to assess Internet accessibility and infrastructure issues and to consider image formats appropriate for Web publishing. Finally, a strategy for robust, long-term, and efficient storage of image data should be developed. This unit provides a guide for preparing neuroanatomical image data.

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Development of a MR-visible compound for tracing neuroanatomical connections in vivo.

Traditional studies of neuroanatomical connections require injection of tracer compounds into living brains, then histology of the postmortem tissue. Here, we describe and validate a compound that reveals neuronal connections in vivo, using MRI. The classic anatomical tracer CTB (cholera-toxin subunit-B) was conjugated with a gadolinium-chelate to form GdDOTA-CTB. GdDOTA-CTB was injected into the primary somatosensory cortex (S1) or the olfactory pathway of rats. High-resolution MR images were collected at a range of time points at 11.7T and 7T. The transported GdDOTA-CTB was visible for at least 1 month post-injection, clearing within 2 months. Control injections of non-conjugated GdDOTA into S1 were not transported and cleared within 1-2 days. Control injections of Gd-Albumin were not transported either, clearing within 7 days. These MR results were verified by classic immunohistochemical staining for CTB, in the same animals. The GdDOTA-CTB neuronal transport was target specific, monosynaptic, stable for several weeks, and reproducible.

Automated analysis of fundamental features of brain structures.

Automated image analysis of the brain should include measures of fundamental structural features such as size and shape. We used principal axes (P-A) measurements to measure overall size and shape of brain structures segmented from MR brain images. The rationale was that quantitative volumetric studies of brain structures would benefit from shape standardization as had been shown for whole brain studies. P-A analysis software was extended to include controls for variability in position and orientation to support individual structure spatial normalization (ISSN). The rationale was that ISSN would provide a bias-free means to remove elementary sources of a structure's spatial variability in preparation for more detailed analyses. We studied nine brain structures (whole brain, cerebral hemispheres, cerebellum, brainstem, caudate, putamen, hippocampus, inferior frontal gyrus, and precuneus) from the 40-brain LPBA40 atlas. This paper provides the first report of anatomical positions and principal axes orientations within a standard reference frame, in addition to "shape/size related" principal axes measures, for the nine brain structures from the LPBA40 atlas. Analysis showed that overall size (mean volume) for internal brain structures was preserved using shape standardization while variance was reduced by more than 50%. Shape standardization provides increased statistical power for between-group volumetric studies of brain structures compared to volumetric studies that control only for whole brain size. To test ISSN's ability to control for spatial variability of brain structures we evaluated the overlap of 40 regions of interest (ROIs) in a standard reference frame for the nine different brain structures before and after processing. Standardizations of orientation or shape were ineffective when not combined with position standardization. The greatest reduction in spatial variability was seen for combined standardizations of position, orientation and shape. These results show that ISSNs automated processing can be a valuable asset for measuring and controlling variability of fundamental features of brain structures.

Changes in SWB following injury to different brain lobes.

UNLABELLED: A neurological substrate for subjective well-being (SWB) has received little research attention. PURPOSE: This study was designed to conduct exploratory investigation into the neuroanatomical correlates of SWB, by monitoring the SWB of a head-injured population over a six-month period. METHOD: Seventy people with head injury (HI), aged 10-65, were studied. The SWB of each participant was measured, and computed tomography (CT) scans were analysed to obtain regional brain injury location (BIL). RESULTS: SWB was associated with BIL. However, the hypothesis that individuals with left frontal injury would report lower SWB was not supported. Instead, it was observed that participants with injury to their right frontal lobe reported higher SWB than individuals with injury to other regions of the brain. CONCLUSIONS: This study provides initial exploration into the neuroanatomical correlates of SWB.

Quantitative assessment of brain volumes in fish: comparison of methodologies.

When correlating brain areas with behavioral and environmental characteristics, a variety of techniques are employed. In fishes (elasmobranchs and teleosts), 2 methods, histology and the idealized ellipsoid and/or half-ellipsoid technique, are primarily used to calculate the volume of a brain area and therefore its relationship to social or ecological complexity. In this study on a perciform teleost, we have quantitatively compared brain volumes obtained using the conventional techniques of histology and approximating brain volume to an idealized ellipsoid (or half ellipsoid) and magnetic resonance imaging, an established clinical tool typically used for assessing brain volume in other vertebrates. Our results indicate that, when compared to brain volumes measured using magnetic resonance imaging of brain regions in situ, variations in brain shape and histological artifacts can lead to significant differences in brain volume, especially in the telencephalon and optic tecta. Consequently, in comparative studies of brain volumes, we advise caution when using the histological and/or ellipsoid methods to make correlations between brain area size and environmental, behavioral and social characteristics and, when possible, we propose the use of magnetic resonance imaging.

Image-guided dissection of human white matter tracts as a new method of modern neuroanatomical training.

Neuronavigation is a kind of image-guided surgery used during neurosurgical procedures. Based on specific equipment which is compatible with the software calculating and processing the patient's data; this method allows the determination of the location of anatomical structures and visualisation of surgical instruments in the operative field. Although standard brain dissection is still the best method of neuroanatomical training, some limitations occur. The most important of these is the inability of conversion from three-dimensional (3D) view to flat pictures of the brain structures, as viewed on computed tomography (CT) and magnetic resonance imaging (MRI), being essential in neuroanatomical training nowadays. The aim of the study was the implementation of a neuronavigating system for brain anatomy training purposes. The study was performed on 10 human brain hemispheres, dissected due to classical methods (standard brain anatomical sections, stepwise ventricular system opening and partial dissection of white matter tracts using Klingler's dissection technique). The material was scanned in a 1.5 T magnetic resonance scanner using a modified neuronavigation protocol. The brains were prepared before dissection as proposed by Klingler. The subsequent steps of the dissection were documented with a digital camera. The progress of the dissection was visualised using the neuronavigation system (Medtronic Stealth Station Treon) with cranial application software. In the course of the study, numerous 3D and 2D images were obtained. The images were related to each other and linked anatomical structures in the specimen with their appearance on CT and MRI scans. The implementation of a neuronavigation system for brain structures dissection facilitates visualization and understanding of their proper location. This new method offers a constant and precise orientation and simplifies understanding of the relation of the 3D view of a specimen to that of the 2D image.

Using diffusion imaging to study human connectional anatomy.

Diffusion imaging can be used to estimate the routes taken by fiber pathways connecting different regions of the living brain. This approach has already supplied novel insights into in vivo human brain anatomy. For example, by detecting where connection patterns change, one can define anatomical borders between cortical regions or subcortical nuclei in the living human brain for the first time. Because diffusion tractography is a relatively new technique, however, it is important to assess its validity critically. We discuss the degree to which diffusion tractography meets the requirements of a technique to assess structural connectivity and how its results compare to those from the gold-standard tract tracing methods in nonhuman animals. We conclude that although tractography offers novel opportunities it also raises significant challenges to be addressed by further validation studies to define precisely the limitations and scope of this exciting new technique.

Evaluation of cerebellar asymmetry with vertigo cases: a stereological study.

AIM: We aimed to evaluate the relevant methods of stereology to estimate cerebellar asymmetry according to gender in both adult right-handed vertigo cases and healthy cases. MATERIAL AND METHODS: The study included 14 adult control subjects and 18 patients with vertigo. The volumes of the cerebellar hemispheres were determined on MRI using the point-counting approach of stereological methods. RESULTS: The mean (+/-SD) of the right cerebellar hemispheres in the patients with vertigo were 52.49+/-5.42 cm3 in males, 50.11+/-4.02 cm3 in females. The mean (+/-SD) of the left cerebellar hemispheres in the patients with vertigo were 53.11+/-3.70 cm3 in males and 49.73+/-4.69 cm3 in females. There was no significant quantitative evidence detected in terms of cerebellar asymmetry between sagittal plane estimates in the cases with vertigo in both genders (p > 0.05). There were no statistically significant differences by genders between vertigo and control subjects (p > 0.05). There was statistical significance only between right and left hemispheres in male control subjects (p=0.039) CONCLUSION: There was no cerebellar asymmetry between control and vertigo cases by gender. The stereological evaluation of cerebellar asymmetry or atrophy in humans in correlation with gender is of importance both for clinicians and anatomists. The technique is simple, reliable, inexpensive and unbiased.

Advanced technique of infrared LED imaging of unstained cells and intracellular structures in isolated spinal cord, brainstem, ganglia and cerebellum.

Light-emitting diodes (LEDs) have recently been used for the imaging of unstained living cells in the whole brain and spinal cord preparations, in which one cut was done to remove the overlying white matter. Here we show that in many cases the neurones can be visualized through the white matter in an intact nervous tissue (rats P0-P36 and mice P0-P2). We used an upright microscope with a water immersion objective and a powerful infrared LED (emission peak, 850 nm; maximum radiant intensity, 270 mW/sr) as a source of oblique illumination. In the isolated spinal cord, we were able to visualize lamina I and II neurones as well as motoneurones. In the brainstem, the neurones from the superficial nuclei were successfully viewed. In the sensory ganglion, we obtained images of unstained cells as well as intracellular structures, like endoplasmic reticulum, nucleus and nucleolus. In isolated cerebellum, parallel fibers, Purkinje and granule cells were viewed. Whole-cell recordings were done to fill spinal lamina I neurones, motoneurones and brainstem neurones with biocytin for detailed 2D-3D reconstruction of their dendritic and axonal arbores. Our imaging technique also allowed labelling individual intact neurones by injecting biocytin through the extracellular cell-attached pipette. This imaging technique opens broad possibilities for functional studies of neurones with completely preserved anatomical structures and synaptic inputs. We also show that the application of oblique infrared LED illumination allows a construction of a simple digital videomicroscope for the high-quality living cell imaging in intact nervous tissue.

Focal macromolecule delivery in neuronal tissue using simultaneous pressure ejection and local electroporation.

Electroporation creates transient pores in the plasma membrane to introduce macromolecules within a cell or cell population. Generally, electrical pulses are delivered between two electrodes separated from each other, making electroporation less likely to be localised. We have developed a new device combining local pressure ejection with local electroporation through a double-barrelled glass micropipette to transfer impermeable macromolecules in brain slices or in cultured HEK293 cells. The design achieves better targeting of the site of pressure ejection with that of electroporation. With this technique, we have been able to limit the delivery of propidium iodide or dextran amine within areas of 100-200 micrometer. We confirm that local electroporation is transient and show that when combined with pressure ejection, it allows local transfection of EGFP plasmids within HEK293 cells or within cerebellar and hippocampal slice cultures. We further show that local electroporation is less damaging when compared to global electroporation using two separate electrodes. Focal delivery of dextran amine dyes within trapezoid body fibres allowed tracing axonal tracts within brainstem slices, enabling the study of identified calyx of Held presynaptic terminals in living brain tissue. This labelling method can be used to target small nuclei in neuronal tissue and is generally applicable to the study of functional synaptic connectivity, or live axonal tracing in a variety of brain areas.

Fluorescent double-labeling with carbocyanine neuronal tracing and immunohistochemistry using a cholesterol-specific detergent digitonin.

The fluorescent carbocyanine dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate) has been widely used for tracing of neuronal pathways. To examine identities of the DiI-labeled neuronal pathways, it is desirable to combine DiI labeling with immunofluorescent staining. However, DiI labeling and immunofluorescent staining are not well compatible, mainly because treatment of DiI-labeled neurons with detergents, which are commonly used for immunohistochemistry, results in high levels of diffusion of the DiI label. In this study, we searched for detergents that are compatible with DiI labeling, and found that a cholesterol-specific detergent digitonin is useful for fluorescent double-labeling with DiI tracing and immunohistochemistry. We show that digitonin treatment, in contrast to Triton X-100, methanol and Nonidet P-40 treatment, preserves DiI labeling, even at higher concentrations. We also show that digitonin also preserves the signal of a DiI derivative CM-DiI. Moreover, we demonstrate that digitonin efficiently increases antibody penetration into brain sections. As a result, immunohistochemical images obtained with digitonin treatment are as good as those obtained with Triton X-100 treatment. In addition, we also try another cholesterol-specific detergent quillaja saponin, but find that it degrades the DiI label. Our simple double-labeling protocol using digitonin should prove useful in enabling detailed examination of the neuronal circuitry of the nervous system.

A computer support system for neurological anatomical diagnosis.

OBJECTIVES: Accurate neurological diagnoses are often difficult to make due to the complexity of the neuroanatomy involved. This study was performed to evaluate the usefulness of a computer system with easily retrievable anatomical information as a support for arriving at more accurate anatomic diagnoses. PATIENTS AND METHODS: Anatomical information from an initial physical examination was programmed into a computer with stored neuroanatomical charts of the brain, spinal cord and peripheral nerves. The information generated a graphic display of possible lesions with suggestions for further examination. These suggestions were then followed and further data entered. This data entry generated a new graphic display with reduced lesion possibilities. Iterations were then followed to narrow the possibilities for diagnosis further, until a final anatomical diagnosis was reached. This method was applied to three hypothetical examples and a number of clinical cases. Here we report three clinical cases in which this method was particularly useful in making a diagnosis. RESULTS: Using computer iterations, the system was able to pinpoint one or more presumptive causative lesions in the CNS or PNS based on known neuronal pathways or nuclei. CONCLUSION: The results indicate that suitably used, computer memory, by virtue of its large capacity, accuracy and fast recall, can supplement human memory in reaching accurate anatomical diagnoses of neurological lesions.

Successes and rewards in sharing digital reconstructions of neuronal morphology.

The computer-assisted three-dimensional reconstruction of neuronal morphology is becoming an increasingly popular technique to quantify the arborization patterns of dendrites and axons. The resulting digital files are suitable for comprehensive morphometric analyses as well as for building anatomically realistic compartmental models of membrane biophysics and neuronal electrophysiology. The digital tracings acquired in a lab for a specific purpose can be often re-used by a different research group to address a completely unrelated scientific question, if the original investigators are willing to share the data. Since reconstructing neuronal morphology is a labor-intensive process, data sharing and re-analysis is particularly advantageous for the neuroscience and biomedical communities. Here we present numerous cases of "success stories" in which digital reconstructions of neuronal morphology were shared and re-used, leading to additional, independent discoveries and publications, and thus amplifying the impact of the "source" study for which the data set was first collected. In particular, we overview four main applications of this kind of data: comparative morphometric analyses, statistical estimation of potential synaptic connectivity, morphologically accurate electrophysiological simulations, and computational models of neuronal shape and development.

Three-dimensional reconstruction of stained histological slices and 3D non-linear registration with in-vivo MRI for whole baboon brain.

The correlation between post-mortem data and in-vivo brain images is of high interest for studying neurodegenerative diseases. This paper describes a protocol that matches a series of stained histological slices of a baboon brain with an anatomical MRI scan of the same subject using an intermediate 3D-consistent volume of "blockface" photographs taken during the sectioning process. Each stained histological section of the baboon brain was first registered to its corresponding blockface photograph using a novel "hemi-rigid" transformation. This piecewise rigid 2D transformation was specifically adapted to the registration of slices which contained both hemispheres. Subsenquently, to correct the global 3D deformations of the brain caused by histological preparation and fixation, a 3D elastic transformation was estimated between the blockface volume and the MRI data. This 3D elastic transformation was then applied to the histological volume previously aligned using the hemi-rigid method to complete the registration of the series of stained histological slices with the MRI data. We assessed the efficacy of our method by evaluating the quality of matching of anatomical features as well as the difference of volume measurements between the MRI and the histological images. Two complete baboon brains (with the exception of cerebellum) were successfully processed using our protocol.