Neuroanatomy and in vivo connectivity

Almut Schüz

Introduction and Scientific Aims

We investigate the relationship between structure and function, using a variety of anatomical and functional approaches. Four different research programs are pursued at present: a) Functional-anatomical studies on the visual system of marmosets. Marmosets like other New World Monkeys show a genetic dichotomy that affects the expression of photoreceptors sensitive for middle to long (M,L) wavelengths as well as the cellular basis of the parvo stream. As a consequence, most male and some female marmosets are colorblind. However, some females show full trichromatic color vision. This makes the marmoset an important model for investigations on the functional segregation of the visual pathways (color, form and motion). We investigate the arrangement of orientation columns in relation to the location of color domains and thalamic input zones such as CO blobs in V1 and V2 in trichromatic female marmosets and compare the results to those on old world monkeys. b) Anatomical investigations on cortical blood vessels, with the aim to improve the interpretation of hemodynamic signals in functional imaging techniques. c) In vivo tract tracing and histology. The chemists in our institute have developed novel tract tracers for visualizing fibre tracts with magnetic resonance imaging (MRI). In our histological laboratory we compare the MRI-results in rat brains with the postmortem histology of these brains. d) Investigations on fibre thickness in the cortical white matter. The aim of this study is to increase knowledge on conduction times and also to enable us to make better estimates of the number of fibres in fibre bundles within the white matter.

Methods

A large variety of techniques is applied in the different projects. a) We use intrinsic optical imaging, microelectrodes and a variety of visual stimuli to map functional domains in the visual system of New World Monkeys. b) The methods to investigate blood vessels are based on fluorescent immunohistochemistry, cytochrome oxidase staining (COX), corrosion cast technique and synchrotron-radiation based X-ray Microscopy (srXTM) with a spatial resolution of 700 nm in a volume of ~1.5 mm³. c) The novel tract tracers for MRI are based on the histological tracer biocytin. Uptake and  transport were investigated in cell cultures (see group by Jörn Engelmann, Dept. High Field MR, Prof. Scheffler) and in histological sections. d) Electron microscopy is used to investigate the thickness of myelinated axons in the white matter of macaques and humans. In particular, we investigate the superior longitudinal fascicle and the uncinate fascicle in both hemispheres, as well as the Corpus callosum.

Results and Preliminary Conclusions

     a. Functional-anatomical investigations on the visual system of New World Monkeys.

We developed a histological protocol that facilitates the alignment of functional and anatomical data (also in Magnetic Resonance Imaging studies [1]) and increases accuracy (Fig.1). Thus, errors in alignments are reduced by more than 50% in comparison to those reported in previous studies [2]. We further developed a stimulus protocol that allows us to map color domains by using non-differential optical imaging in trichromatic marmosets. Our results show that color domains are most effectively activated by red-green (L-M) flicker stimuli. We also showed that color domains in marmosets are colocalized with CO blobs in V1 and thin stripes in V2 [3]. We conclude that the observed color domain activation is primarily triggered by a modulation of the parvo stream ((L-M)-cone axis), thus supporting the notion that CO blobs in New World as well as in Old World trichromatic primates are segregated domains of color processing.

Figure 1:

Functional-anatomical investigations on the primary visual cortex in marmosets. (A) Orientation map obtained by intrinsic optical imaging (IOI), superimposed onto the 3D reconstruction from the histological sections. (B) Pattern of the superficial cortical vasculature (from the same cortical region) labeled with FITC. Scale bar = 500 µm.

    b. Anatomical studies on cortical blood vessels 

Our immunohistochemical approach for visualizing the vascular system resulted in a detailed quantification of vascular densities in layers and areas of the visual cortex [4]. We also could make first estimates of the arterial-venous ratio, derived from corrosion cast preparations (Fig. 2). A tight correlation of vascular density and oxidative metabolism was revealed by immuno­labeling of COX stained sections [5]. The first results of the srXTM approach lead to preliminary 3-dimensional representations of the vasculature, enabling the simulation of changes in cortical blood flow (CBF) [6]. Thus, the methodological portfolio we developed allows for an exact qualitative and quantitative assessment of the cerebral vasculature and can be applied to any brain region of interest.

Figure 2:

Scanning electron micrograph of a vascular corrosion cast preparation from the macaque monkey striate cortex. It shows a view across all cortical layers, covering approximately 1.7 mm from surface to white matter, larger arteries and veins shaded red and blue, resp. Automated mosaic-like scanning enables the investigation of large specimens, though with the superior spatial resolution of the scanning electron microscope

       c. In vivo tract tracing and histology

As a first step, the histological tracer biocytin was modified in such a way as to make it more stable. The degradation of conventional biocytin starts already a few hours after injection. Higher stability is relevant for MRI studies in which one wants to follow the tracer in vivo over time. Our approach was very successful: using aminopropyl-biocytin or serine-biocytin the stain showed no signs of degradation even 4 days after injection [7]. As a next step, a Gd-complex was coupled to biocytin (Fig. 3) and to both of its new modifications in order to make them visible in MRI. Although in the latter two visualization in histology was impaired (due to an impaired reaction with avidin) it could be shown in MRI that all three molecules were transported to the places to be expected (thalamus; contralateral and ipsilateral cortex) [8, 9].

 Figure 3:

Left: Gadolinium complex coupled to biocytin, Ln=Gd³⁺, Tb³⁺. Right: MR-pictures and histological pictures after an injection into the motor cortex of the rat. The injection site can be seen as a blue-purple spot on the horizontal MR-picture and as a white spot on the coronal MR-picture, as well as in the histological coronal section beneath it. The red spots in the MR pictures show the location of the tracer after 24 h. The histological picture on the right shows retrogradely stained neurons in the cortex, in the region corresponding to the red spot lateral to the injection site in the coronal MR-picture. From [8].

        d. Fibre thickness in the cortical white matter

Fibre thickness in the white matter varies enormously, For example, in the superior longitudinate fascicle. the average of the inner diameter (i.e. without the myelin sheet) is only about 0.7 mm, but can range up to about 5 mm in humans and to about 4 mm in monkeys. Thus, a corresponding range of conduction times is to be expected. This study is gained recently much interest in relation with the development of methods visualizing fibre bundles in vivo (Diffusion Magnetic Resonance Imaging): conventional anatomical data provide an indispensable control for these methods which are not yet well understood and prone to misinterpretation.

Supervised students and collaborators

M. F. Valverde Salzmann (PhD thesis on optical imaging in the marmoset)

A. L. Keller (PhD thesis, external supervisor: B. Weber, on blood vessels) 

A. Mishra, R. Mishra, K. Dhingra, J. Engelmann, M. Beyerlein, S. Canals (in vivo tract tracing)

D. Liewald (Diploma thesis on fibre thickness in the white matter)

References

1.         Valverde Salzmann, M.F., N.K. Logothetis, R. Pohmann: High-resolution imaging of vessels in the isolated rat brain. ISMRM: Montreal, Quebec, Canada (2011a).

2.         Valverde Salzmann, M.F., D.J. Wallace, N.K. Logothetis, A. Schüz: Multimodal vessel mapping for precise large area alignment of functional optical imaging data to neuroanatomical preparations in marmosets. Journal of Neuroscience Methods 201, 159-172 (2011b).

3.         Valverde Salzmann, M.F., A. Bartels, N.K. Logothetis, A. Schüz: Color blobs in cortical areas V1 and V2 of the new world monkey Callithrix jacchus, revealed by non-differential optical imaging. Journal of Neuroscience (2011c) (under revision).

4.         Weber B., Keller AL., Reichold J., Logothetis NK. (2008) The microvascular        system of the striate and extrastriate visual cortex of the macaque, Cerebral     Cortex 18 2318-2330.

5.         Keller AL., Schüz A., Logothetis NK., Weber B. (2011) Vascularization of             cytochrome oxidase-rich blobs in the primary visual cortex of squirrel and    macaque monkeys The Journal of Neuroscience 31 1246-1253.

6.         Reichold J., Stampanoni M., Keller AL., Buck A., Jenny P., Weber B (2009)            Vascular graph model to simulate the cerebral blood flow in realistic          vascular networks, Journal of Cerebral Blood Flow & Metabolism 29 1249-      1443.

7.         Mishra A, Dhingra K, Schüz A, Logothetis NK, Canals S (2010) Improved neuronal tract tracing with stable biocytin-derived neuroimaging agents,           ACS Chemical Neuroscience 1 129-138.

8.         Mishra A, Schüz A, Engelmann J, Beyerlein M, Logothetis NK, Canals S (2011)     Biocytin-derived MRI contrast agent for longitudinal brain connectivity     studies  ACS Chem Neurosci Epub ahead DOI: 10.1021/cn200022m.

9.         Mishra A, Dhingra K, Mishra R, Schüz A, Engelmann J, Beyerlein M, Canals S,      Logothetis NK (in press) Biocytin-based contrast agent for molecular

            imaging: an approach to developing new in vivo neuroanatomical tracers   for MRI. In: Neuroimaging / Book 1, (tentative title), Peter Bright  (Ed.), ISBN:      978-953-307-413-9, InTech

Education

Professional activity

Organization of international meetings