'Functional' neuroanatomical tract tracing: analysis of changes in gene expression of brain circuits of interest

Neuroanatomical tracing when considered as an isolated method produces relatively straightforward answers. Although single-, double- or even triple-tracing paradigms produce valuable data on the organization of brain circuits, the final outcome often is too simplistic since it is not possible to elucidate the activity of these circuits. In this regard, emerging technologies contribute with additional information about the status of neuronal circuits. The laser-guided capture microdissection microscope (LCM) allows the accurate dissection of small brain areas under the microscope that could be further analyzed for gene expression or proteomics. In order to elucidate the gene expression of a given circuit of interest, we have developed a combination of methods comprising (i) fluorescent non-radioactive in situ hybridization for the detection of vGLUT2 mRNA expression combined with retrograde tracing with Fluoro-Gold (FG; analysis performed under the confocal microscope) and (ii) laser-guided capture microdissection of brain areas containing neurons retrogradely labeled with FG followed by the measurement of changes in mRNA levels encoding for vGLUT2 by real-time PCR. Our goal was to detect changes in gene expression of the thalamostriatal pathway in unilaterally 6-OHDA lesioned rats. Taking advantage of this procedure, we found a three-fold increase in vGLUT2 mRNA expression within thalamic neurons projecting to the dopamine-depleted striatum when compared with the activity of the thalamic neurons innervating the control striatum.     

Anterograde neuroanatomical tract tracing with central nervous system injections of immunoglobulin G: A Light and electron microscopic evaluation

Normal rabbit serum (NRS) was pressure injected into the forebrain of rats to be tested as an anterograde neuroanatomical tracing substance. Undiluted NRS was stereotaxically injected into the bed nucleus of the stria terminalis (BST) with a 1-microliter Hamilton syringe. Postinjection survival times ranged from 24 h to 14 days. An immunohistochemical method utilizing goat anti-rabbit IgG antibody was used to detect the rabbit IgG within vibratome sections. Visualization of the final reaction product (diaminobenzidine, DAB) was enhanced by a silver/gold postintensification (SGI) method. Rabbit IgG-containing neural structures were examined at both light and electron microscopic (EM) levels. At the injection site neuronal soma, dendrites and axons were filled homogeneously with the SGI-DAB at 24 h, 48 h, 7 days and 14 days indicating local neuronal uptake, storage and transport of rabbit IgG. In the hypothalamus many anterogradely filled axons were present and displayed short collateral branches and terminals. EM examination revealed synaptic terminals containing IgG, without signs of transsynaptic transport after 14 days. Signs of retrograde transport of IgG were never observed. A propensity of neurons to take up, sequester and anterogradely transport immunoglobulin G is indicated.     

A new method for the in vivo volumetric measurement of the human hippocampus with high neuroanatomical accuracy

Accurate and reproducible in vivo measurement of hippocampal volumes using magnetic resonance (MR) imaging is complicated by the morphological complexity of the structure. Additionally, separation of certain parts of the hippocampus from the adjacent brain structures on MR images is sometimes very difficult. These difficulties have led most investigators to either use arbitrary landmarks or to exclude certain parts of the structure from their measurements. Based on three-dimensional MR data, we have developed a reliable in vivo volumetric measurement of the human hippocampus. In contrast to most of the previously described volumetric MR-based methods, we aimed to sample the entire hippocampal formation using its true anatomical definition. This was accomplished by relying on the capacity of the BRAINS software to simultaneously visualize in multiple planes, to "telegraph" tracings or cursor position from one plane to another, and to simultaneously rely on multispectral data from three different image sets (T1, T2, and tissue classified). The methods for identifying boundaries and measuring the hippocampal volume are described. The method has excellent reliability, sensitivity, and specificity. The method may be of use in studies of structure-function relationships in neuropsychiatric disorders such as schizophrenia, temporal lobe epilepsy, and Alzheimer's disease. Future work will use these measurements as training data for a neural net-based technique to identify the anatomical boundaries automatically.     

The habenulo-interpeduncular and mammillothalamic tracts: early developed fiber tracts in the human fetal diencephalon

Purpose

The habenulo-interpeduncular (HI) and mammillothalamic (MT) tracts are phylogenetically ancient. The clinical relevance of these tracts has recently received attention. In this work, we map the anatomy the developing HI and MT.

Methods

To investigate the topographical anatomy of developing fiber tracts in and around the diencephalon, we examined the horizontal, frontal, and sagittal serial paraffin sections of 28 human fetuses at 8–12 weeks of gestation.

Results

In all specimens, eosinophilic early fiber bundles were limited to the bilateral HI and MT tracts in contrast to pale-colored later developing fibers such as the thalamocortical projections and optic tract. The HI and MT tracts ran nearly parallel and sandwiched the thalamus from the dorsal and ventral sides, respectively. The nerve tract course appeared to range from 5–7 mm for the HI tract and 3–5 mm for the MT tract in 15 specimens at 11–12 weeks. The HI tract was embedded in, adjacent to, or distant from the developing parvocellular red nucleus.

Conclusions

In early human fetuses, HI and MT tracts might be limited pathways for primitive cholinergic fiber connections between the ventral midbrain and epithalamic limbic system.     

Atrophy in white matter fiber tracts in multiple sclerosis is not dependent on tract length or local white matter lesions

The pathogenesis of tissue injury outside the white matter (WM) plaques of multiple sclerosis (MS) has not yet been clearly defined. To better understand the pathogenesis of this injury and the associated atrophy, we investigated volume loss over time in 20 WM fiber tracts. We defined two main aims: (1) to examine whether certain fiber tracts were more prone to atrophy, and to test the possible relation of tract atrophy to tract length and selected MS-specific variables; and (2) to investigate the possible relation of atrophy to lesion load (whole brain and in the specific tract). Local volume change was assessed between two distant time points for each MS patient studied. Fiber tracts were segmented automatically using a tractography-based atlas. Results demonstrate volume loss in all fiber tracts. The uncinate fasciculus and anterior-thalamic radiation had the greatest yearly percentage atrophy. Disease type, duration, median expanded disability status scale, total lesion load, and gender exhibited significant effects on atrophy in at least one tract. Together, these data are more consistent with a pathogenesis for the degeneration related to diffuse inflammation rather than the secondary effects of focal lesions.     

Anterograde tracing method using DiI to label vagal innervation of the embryonic and early postnatal mouse gastrointestinal tract

The mouse is an extremely valuable model for studying vagal development in relation to strain differences, genetic variation, gene manipulations or pharmacological manipulations. Therefore, a method using 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) was developed for labeling vagal innervation of the gastrointestinal (GI) tract in embryonic and postnatal mice. DiI labeling was adapted and optimized for this purpose by varying several facets of the method. For example, insertion and crushing of DiI crystals into the nerve led to faster DiI diffusion along vagal axons and diffusion over longer distances as compared with piercing the nerve with a micropipette tip coated with dried DiI oil. Moreover, inclusion of EDTA in the fixative reduced leakage of DiI out of nerve fibers that occurred with long incubations. Also, mounting labeled tissue in PBS was superior to glycerol with n-propyl gallate, which resulted in reduced clarity of DiI labeling that may have been due to DiI leaking out of fibers. Optical sectioning of flattened wholemounts permitted examination of individual tissue layers of the GI tract wall. This procedure aided identification of nerve ending types because in most instances each type innervates a different tissue layer. Between embryonic day 12.5 and postnatal day 8, growth of axons into the GI tract, formation and patterning of fiber bundles in the myenteric plexus and early formation of putative afferent and efferent nerve terminals were observed. Thus, the DiI tracing method developed here has opened up a window for investigation during an important phase of vagal development.     

Postnatal development of cytochrome oxidase activity in fiber tracts of the rat brain

This paper describes postnatal changes in cytochrome oxidase (C.O.) activity in developing fiber tracts. Quantitative histochemistry was used to measure changes in C.O. activity in nine white matter regions at postnatal days (P) 7, 12, 17, 30, and 60 in the rat. At P7, enzyme activity was maximal in the spinal trigeminal tract, medial longitudinal fasciculus, and cerebellar white matter. At P12, maximal levels were measured in the medial lemniscus and cerebral peduncle. C.O. activity increased from low levels at P7 to maximal levels by P17 in the hippocampal commissure, posterior and anterior corpus callosum, and anterior commissure. In all nine regions, C.O. activity decreased by P60. Thus, peaks in C.O. activity shifted as a function of postnatal age in a caudo-rostral direction. The regional heterogeneity in the age of onset in C.O. fluctuations suggests that vulnerability to injury and metabolic dysfunction during the perinatal period will differentially affect white matter structures, depending on the age of onset of such disruptions.     

The use of kainic acid for tracing neuroanatomical connections in the septohabenvilointerpeduncular system of the rat

Intracerebral injections of the neuron-destroying agent, kainic acid, and electron microscopic examination of the injected and the target areas, have been used in order to confirm some controversial anatomical connections in the Septohabenulointerpeduncular system. Kainic acid injections in the lateral habenula (LHb) caused the selective degeneration of several LHb neurons, sparing the neurons of the medial habenula (MHb). Degenerating terminals were found in well-known target areas of the LHb (dorsal and median raphe nuclei) and, in addition, in the interpeduncular nucleus (IPN), thus confirming the existence of a small LHb-IPN projection. Kainic acid injections in the nucleus of the diagonal band of Broca (NDBB) resulted in severe ncuronal degeneration in the nucleus itself and in terminal degeneration in the LHb and MHb as well as, to a lesser extent, in the IPN. No evidence of distant neuronal damage was apparent by light (conventional and cupric-silver stain) and electron microscopic examination, in nuclei receiving afferent inputs from the injected areas and projecting in turn to the target areas. Furthermore, terminal degeneration was not observed in the Hb and IPN after injections of kainic acid in areas not projecting toward these nuclei (hippocampus and striatum). In the present study, therefore, problems of interpretation due to the possible occurrence of distant neuronal damage could be overcome by control tests. On the basis of the selectivity and sensitivity of its action, kainic acid seems particularly useful in the study of small projections and in attempts to discriminate between differential connections of adjacent neuronal populations.     

Navigation expertise and the human hippocampus: a structural brain imaging analysis

Grey matter volume in the posterior hippocampus of London taxi drivers is greater than in age-matched controls, and the size of this increase correlates positively with time spent taxi driving (E.A. Maguire et al., 2000. Proc Natl Acad Sci USA 97: 4398-4403). This change suggests that increased posterior hippocampal grey matter volume is acquired in response to increased taxi driving experience, perhaps reflecting their detailed representation of the city. However, an alternate hypothesis is that the difference in hippocampal volume is instead associated with innate navigational expertise, leading to an increased likelihood of becoming a taxi driver. To investigate this possibility, we used structural brain imaging and voxel-based morphometry (VBM) to examine a group of subjects who were not taxi drivers. Despite this group showing a wide range of navigational expertise, there was no association between expertise and posterior hippocampal grey matter volume (or, indeed, grey matter volume throughout the brain). This failure to find an association between hippocampal volume and navigational expertise thus suggests that structural differences in the human hippocampus reflect the detail and/or duration of use of the spatial representation acquired, and not innate navigational expertise per se.     

Autometallographic tracing of bismuth in human brain autopsies.

For decades, drugs containing bismuth have been used to treat gastrointestinal disorders. Although a variety of adverse effects, including neurological syndromes, have been recorded, the biological/toxicological effects of bismuth ions are far from disclosed. Until recently, only quantitative assessments were possible, but resent research has made histochemical tracing of bismuth possible. The technique involves silver enhancement of bismuth crystallites by autometallography (AMG). In the present study, the localization of bismuth was traced by AMG in sections of paraffin-embedded brain tissue obtained by autopsy from 6 patients suffering from bismuth intoxication in a period ranging from 1975 through 1977. Tissue was analyzed at light and electron microscopical levels, and the presence of bismuth further confirmed by proton-induced x-ray emission (PIXE). Clinical data and bismuth concentrations in blood, cerebellum, and thalamus were measured by atomic absorption spectrophotometry (AAS) and are reported here. Histochemical analyses demonstrate that bismuth accumulated in neurons and glia cells in the brain regions examined (neocortex, cerebellum, thalamus, hippocampus). Cerebellar blood vessels stained most intensely. The PIXE and AAS data correlated with the histochemical staining patterns and intensities. At the ultrastructural level, bismuth was found to accumulate intracellularly in lysosomes and extracellularly in the basement membranes of some vessels.     

Acetylcholinesterase fiber staining in the human hippocampus and parahippocampal gyrus

The AChE fiber distribution within the human hippocampus and parahippocampal gyrus was studied in order to provide normative data for the examination of cholinergic fiberarchitecture in human pathology and to clarify the cytoarchitectonic organization of these structures. A modification of the Koelle method was used to stain temporal lobe serial sections from 6 neurologically normal human brains collected at autopsy. The hippocampal formation contains some of the densest staining of any cortical area. Regions with the heaviest concentrations of AChE fibers include a thin band along the inner edge of the molecular layer of the dentate gyrus (ml-DG) and parts of the CA2, CA3, and CA4 sectors of Ammon's horn. Staining is of intermediate intensity in the CA1 region. The subiculum (S) is more lightly stained than the CA fields. Staining in the parahippocampal gyrus is generally less dense than in the hippocampal formation. The most conspicuous feature of the human entorhinal cortex (EC) is the AChE-rich fiber patches seen overlapping the stellate cell islands in layer II. An additional band of relatively dense AChE staining is identified in layers IV-V. Prominent AChE-rich polymorphic neurons are present within the hilum of the dentate gyrus. The CA1/subiculum transition in Nissl preparation is characterized by an oblique interdigitation of CA1 cells. The transition from EC to prorhinal cortex occurs along the medial bank of the rhinal sulcus and is characterized by a band of AChE staining, which slopes obliquely away from layer II until it joins an intermediate pyramidal cell layer. Some comparisons with AChE staining in the monkey were made. The monkey has a similar pattern except in DG, where the intensely AChE staining band along the inner ml-DG is thicker and much more prominent. In comparison to the human, the monkey has more conspicuous AChE staining in the parasubicular region.     

The evolutionary origin of the language areas in the human brain. A neuroanatomical perspective

The capacity to learn syntactic rules is a hallmark of the human species, but whether this has been acquired by the process of natural selection has been the subject of controversy. Furthermore, the cortical localization of linguistic capacities has prompted some authors to suggest a modular representation of language in the brain. In this paper, we rather propose that the neural device involved in language is embedded into a large-scale neurocognitive network comprising widespread connections between the temporal, parietal and frontal (especially prefrontal) cortices. This network is involved in the temporal organization of behavior and motor sequences, and in working (active) memory, a sort of short-term memory that participates in immediate cognitive processing. In human evolution, a precondition for language was the establishment of strong cortico–cortical interactions in the postrolandic cortex that enabled the development of multimodal associations. Wernicke's area originated as a converging place in which such associations (concepts) acquired a phonological correlate. We postulate that these phonological representations projected into inferoparietal areas, which were connected to the incipient Broca's area, thus forming a working memory circuit for processing and learning complex vocalizations. As a result of selective pressure for learning capacity and memory storage, this device yielded a sophisticated system able to generate complicated utterances (precursors of syntax) as it became increasingly connected with other brain regions, especially in the prefrontal cortex. This view argues for a gradual origin of the neural substrate for language as required by natural selection.     

Neuronal fiber tracts connecting the brain and ventral nerve cord of the early Drosophila larva

By using a combination of dye injections, clonal labeling, and molecular markers, we have reconstructed the axonal connections between brain and ventral nerve cord of the first‐instar Drosophila larva. Out of the approximately 1,400 neurons that form the early larval brain hemisphere, less than 50 cells have axons descending into the ventral nerve cord. Descending neurons fall into four topologically defined clusters located in the anteromedial, anterolateral, dorsal, and basoposterior brain, respectively. The anterolateral cluster represents a lineage derived from a single neuroblast. Terminations of descending neurons are almost exclusively found in the anterior part of the ventral nerve cord, represented by the gnathal and thoracic neuromeres. This region also contains small numbers of neurons with axons ascending into the brain. Terminals of the ascending axons are found in the same basal brain regions that also contain descending neurons. We have mapped ascending and descending axons to the previously described scaffold of longitudinal fiber tracts that interconnect different neuromeres of the ventral nerve cord and the brain. This work provides a structural framework for functional and genetic studies addressing the control of Drosophila larval behavior by brain circuits. J. Comp. Neurol. 515:427–440, 2009. © 2009 Wiley‐Liss, Inc.     

Axonal projections between fetal spinal cord transplants and the adult rat spinal cord: a neuroanatomical tracing study of local interactions.

Three neuroanatomical tracers have been employed to map the axonal projections formed between transplants of fetal spinal cord tissue and the surrounding host spinal cord in adult rats. Solid pieces of embryonic day 14 (E14) rat spinal cord were placed into hemisection aspiration cavities in the lumbar spinal cord. Injections of either (1) a mixture of horseradish peroxidase and wheat germ agglutinin- conjugated horseradish peroxidase, (2) Fluoro-Gold, or (3) Phaseolus vulgaris leucoagglutinin (PHA-L) were made into the transplants or the neighboring segments of the host spinal cord at 6 weeks to 14 months post-transplantation. Injections of anterograde and retrograde tracers into the transplants revealed extensive intrinsic projections that often spanned the length of the grafts. Axons arising from the transplants extended into the host spinal cord as far as 5 mm from the host-graft interface, as best revealed by retrograde labeling with Fluoro-Gold. Consistent with these observations, iontophoretic injections of PHA-L into the transplants also produced labeled axonal profiles at comparable distances in the host spinal cord, and in some instances elaborate terminals fields were observed surrounding host neurons. The majority of these efferent fibers labeled with PHA-L, however, were confined to the immediate vicinity of the host-graft boundary, and no fibers were seen traversing cellular partitions between host and transplant tissues. Host afferents to the transplants were also revealed by these tracing methods. For example, the injection of Fluoro-Gold into the grafts resulted in labeling of host neurons within the spinal cord and nearby dorsal root ganglia. In most cases, retrogradely labeled neurons in spinal gray matter were located within 0.5 mm of the graft site, although some were seen as far as 4-6 mm away. The distance and relative density of ingrowth exhibited by host axons into the grafts, however, appeared modest based upon the results of HRP and Fluoro-Gold retrograde labeling. This was further confirmed with the PHA-L anterograde method. Whereas some host fibers were seen extending into the transplants, the majority of PHA-L containing axons formed terminal-like profiles at or within 0.5 mm of the host-graft interface. The comprehensive view of intrinsic connectivity and host-graft projections obtained in these studies indicates that intraspinal grafts of fetal spinal cord tissue can establish a short-range intersegmental circuitry in the injured, adult spinal cord. These observations are consistent with the view that such grafts may contribute to the formation of a functional relay between separated segments of the spinal cord after injury.(ABSTRACT TRUNCATED AT 400 WORDS)     

MRI identification of dorsal hippocampus homologue in human brain

We investigated hippocampal substructure in the rat, cat, dog, and human by means of magnetic resonance imaging to elucidate phylogenetic differences in longitudinal organization. Multidirectional high-resolution images obtained with a 3 T scanner revealed that the dorsal part of the hippocampus was well developed in the rat, cat, and dog brain, and was homologous to the hippocampal tail, a poorly-developed posterior part, in the human. We conclude that the dorsal hippocampus of laboratory animals corresponds to the hippocampal tail in the human brain, which is considered to be hypoplastic and of less importance clinically than more anterior regions. These data may help in understanding phylogenetic, and in correlating results from animal experiments with clinical findings on the functions and pathologies of the human hippocampus.     

Sprouting of crossed entorhinodentate fibers after a unilateral entorhinal lesion: Anterograde tracing of fiber reorganization with Phaseolus vulgaris-leucoagglutinin (PHAL)

Fibers from the contralateral entorhinal cortex (EC) to the dentate gyrus partially replace the input lost after an ipsilateral EC lesion. To study the morphology and course of single sprouted crossed entorhinodentate fibers, the anterograde tracer Phaseolus vulgaris-leucoagglutinin (PHAL) was used. Rats that survived for 4 to 8 weeks after a unilateral entorhinal lesion received PHAL deposits into the entorhinal cortex contralateral to the lesion. Control animals received a similar PHAL deposit. Single PHAL-labeled fibers in the molecular layer of the contralateral (EC lesion) fascia dentata were drawn with a camera lucida, and an axon-branching index (branch points/100 μm axon length) was calculated for these crossed entorhinodentate fibers in controls and operated animals. In animals with EC lesions, the density of PHAL-labeled crossed entorhinodentate fibers had increased remarkably. Single crossed entorhinodentate axons showed significantly more axon branch points in experimental than in control animals. In addition, some axon segments displayed high densities of small axonal extensions. Frequently, tanglelike structures were observed in the denervated outer molecular layer. These tangles consisted of one or more PHAL-labeled axons that intertwined and formed an axon tangle filled completely with branches, extensions, and boutons. Our data indicate that crossed EC fibers sprout by forming additional collaterals, axonal extensions, and tangles. Abnormal neurite formations are a characteristic feature of plaques in Alzheimer's disease. Future studies must be done to show whether or not there is a close relationship between axonal tangles and plaques in Alzheimer's disease, which, like the present lesion paradigm, severely affects entorhinal projection neurons. © 1996 Wiley-Liss, Inc.     

Neural tract tracing using Di-I: a review and a new method to make fast Di-I faster in human brain

The use of Di-I in tract-tracing is briefly reviewed and a novel delayed-fixation approach to neural tract-tracing in the postmortem human adult brain is reported. Using the new approach, fast Di-I, a highly lipophilic fluorescent dye was injected into a particular region or nucleus and labelled tracts were followed for distances of some 20-40 mm. The procedure required approximately 36 h, yielding dye penetration rates of 1.0 mm/h or more. This contrasts with previous Di-I, silver impregnation, and horseradish peroxidase protocols, where the tracer penetration rate is typically 0.003 mm/h or less, and the distance traversed amounts to only a few mm even after months of incubation. The new method hinges on the simple consideration that aldehyde fixation, which is normally employed prior to administration of the marker, crosslinks membrane proteins and impedes dye diffusion. The short postmortem samples used in our protocol permit delaying fixation until after the dye has had time to penetrate, dramatically increasing the length and scope of neural circuits that can be traced. Using these methods, for example, we have confirmed the presence of an ipsilateral olivocerebellar climbing fiber projection in the human.