Biomedical subjects

Floris G Wouterlood

Publications and source records attributed to Floris G Wouterlood.

6 recordsLinked to original sources

PubMed2005

Random or selective neuroanatomical connectivity. Study of the distribution of fibers over two populations of identified interneurons in cerebral cortex.

We present a neuroanatomical tracing method in a stereological approach to study the proportional distribution of fibers of a particular projection over two chemically different populations of neurons. The fiber projection from the presubiculum to the medial division of the entorhinal cortex of the rat serves as a model projection. Potential target interneurons express calcium binding proteins, either parvalbumin or calretinin. The three markers were simultaneously stained in one and the same histological section. The procedure is according to a three-phase procedure, i.e., in vivo tracer injection phase, histology phase, laserscanning phase. Steps involved are: (1) Surgical application to the presubiculum (injection) of the neuroanatomical tracer, biotinylated dextran amine (BDA), with the purpose of labeling fibers innervating the entorhinal cortex. After surgery, transport of the tracer takes place during the one-week survival period; (2) Fluorescence detection of the labeled fibers through staining with fluorochromated avidin (avidin-Alexa Fluor 488 [green fluorescence]); (3) Simultaneous Immunofluorescence detection of two interneuron markers (using the appropriate primary antibodies and secondary antibodies conjugated to the fluorochromes Alexa Fluor 594 [red fluorescence] and Alexa Fluor 633 [infrared fluorescence]); (4) Acquisition of low-magnification images in a confocal laserscanning microscope and the preparation on a computer of a montage image covering the entire entorhinal cortex; (5) Overlaying this montage with a sampling grid; (6) Acquisition at high magnification of Z-series of confocal images in a statistical valid way based on this grid. Each marker was visualized in its own laser excitation/emission channel: 488, 568 and 647 nm; (7) Image processing and 3D reconstruction followed by evaluation of the results. The present approach can be used to examine whether or not a particular class of chemically identified neurons receives preferential innervation by a particular fiber projection.

Animals↗

PubMed2004

Input from the presubiculum to dendrites of layer-V neurons of the medial entorhinal cortex of the rat.

The entorhinal cortex (EC) and the hippocampus are reciprocally connected. Neurons in the superficial layers of EC project to the hippocampus, whereas deep entorhinal layers receive return connections. In the deep layers of EC, pyramidal neurons in layer V possess apical dendrites that ascend towards the cortical surface through layers IIII and II. These dendrites ramify in layer I. By way of their apical dendrites, such layer-V pyramidal cells may be exposed to input destined for the superficial entorhinal neurons. A specific and dense fiber projection that typically ends in superficial entorhinal layers of the medial EC originates in the presubiculum. To investigate whether apical dendrites of deep entorhinal pyramidal neurons indeed receive input from this projection, we injected the anterograde tracer PHA-L in the presubiculum or we lesioned the presubiculum, and we applied in the same experiments the tracer Neurobiotin trade mark pericellularly in layer V of the medial EC of 17 rats. PHA-L labeled presubiculum axons in the superficial layers apposing apical segments of Neurobiotin labeled layer-V cell dendrites were studied with a confocal fluorescence laserscanning microscope. Axons and dendrites were 3D reconstructed from series of confocal images. In cases in which the presubiculum had been lesioned, material was investigated in the electron microscope. At the confocal fluorescence microscope level we found numerous close contacts, i.e. appositions of boutons on labeled presubiculum fibers with identified dendrites of layer-V neurons. In the electron microscope we observed synapses between degenerating axon terminals and spines on dendrites belonging to layer-V neurons. Hence we conclude that layer-V neurons receive synaptic contacts from presubiculum neurons. These findings indicate that entorhinal layer-V neurons have access to information destined for the superficial layers and eventually the hippocampal formation. At the same time, they have access to the hippocampally processed version of that information.

Animals↗

PubMed2003

Synaptic contacts between identified neurons visualized in the confocal laser scanning microscope. Neuroanatomical tracing combined with immunofluorescence detection of post-synaptic density proteins and target neuron-markers.

The axons of neurons in the CNS with their delicate ramification patterns and terminal boutons can be visualized with conventional neuroanatomical techniques with a high degree of accuracy. Whether identified terminal boutons form synaptic contacts with target neurons identified by a second and different marker needs resolution beyond that offered by conventional light microscopy. The morphological elements associated with synaptic connectivity consist of specialized pre- and post-synaptic junctional complexes known as the pre- and post-synaptic densities. Electron microscopy of these junctional complexes consumes much time and resources. In an attempt to increase the speed with which we can analyze networks of neurons we developed a high-resolution triple-fluorescence approach including neuroanatomical tracing, immunofluorescence, confocal laserscanning and 3D-computer reconstruction to pinpoint at the light microscopic level the three elements involved in synaptic connectivity: afferent fibers and their terminal boutons, close apposition with neurons identified by the presence of a fluorescent marker, and sandwiched in between a post-synaptic density marker. We used morphological criteria for the detection of axon terminals (swellings on fibers). Antibodies against ProSAP2/Shank3, a post-synaptic density-associated scaffolding protein, were used to pinpoint the location of the synaptic junctions. The results show the existence of sandwich-like configurations: pre-synaptic fiber, ProSAP2/Shank3, post-synaptic neuron. Thus we feel that we can minimize (and perhaps completely eliminate) the need for electron microscopy and hence dramatically increase the overall efficiency of neuroanatomical tracing and network analysis.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Morphological and numerical analysis of synaptic interactions between neurons in deep and superficial layers of the entorhinal cortex of the rat.

Neurons providing connections between the deep and superficial layers of the entorhinal cortex (EC) constitute a pivotal link in the network underlying reverberation and gating of neuronal activity in the entorhinal-hippocampal system. To learn more of these deep-to-superficial neurons and their targets, we applied the tracer Neurobiotin pericellularly in layer V of the medial EC of 12 rats. Labeled axons in the superficial layers were studied with light and electron microscopy, and their synaptic organization recorded. Neurobiotin-labeled layer V neurons displayed "Golgi-like" staining. Two major cell types were distinguished among these neurons: (1) pyramidal neurons with apical spiny dendrites traversing all layers and ramifying in layer I, and (2) horizontal neurons with dendrites confined to the deep layers. Labeled axons ramified profusely in layer III, superficially in layer II and deep in layer I. Analysis of labeled axon terminals in layers I-II and III showed that most synapses (95%) were asymmetrical. Of these synapses, 56% occurred with spines (presumably belonging to principal neurons) and 44% with dendritic shafts (presumably interneurons). A small fraction of the synapses (5%) was of the symmetrical type. Such synapses were mainly seen on dendritic shafts. We found in two sections a symmetrical synapse on a spine. These findings suggest that the deep to superficial projection is mainly excitatory in nature, and that these fibers subserve both excitation and feed-forward inhibition. There is an additional, much weaker, inhibitory component in this projection, which may have a disinhibitory effect on the entorhinal network in the superficial layers.

Animals↗

PubMed2002

Double-label confocal laser-scanning microscopy, image restoration, and real-time three-dimensional reconstruction to study axons in the central nervous system and their contacts with target neurons.

The current double tracing-double confocal laser-scanning method was developed to reconstruct identified nerve fibers and their contacts with identified target neurons in the rat brain in three dimensions. It intends to fill the gap between conventional light microscopic and electron microscopic neuroanatomic tracing. The steps involved are as follows: (1) injection of two neuroanatomic tracers--Phaseolus vulgaris leucoagglutinin (PHA-L) to label fibers innervating a particular brain area and Neurobiotin to label prospective target neurons in that area; (2) immunofluorescence detection of the labeled fibers (fluorophore Cy5, infrared emission), together with fluorochromated avidin detection of the taken-up Neurobiotin (Cy2 or Alexa 488; green emission); (3) acquisition of Z-series of confocal images at high magnification with a laser-scanning microscope using the laser lines 488 nm and 647 nm; and (4) computer-processing and three-dimensional reconstruction of the labeled fibers and the presumed target dendrites. Rotation on the computer of the three-dimensional reconstructed fibers and dendrites along all three spatial axes enabled the authors to determine whether "true" or "false" contacts occur. In a true contact no space was present between the apposing structures, whereas a false contact consisted of two differently stained structures close to each other but separated by a narrow, optically empty space. One important phenomenon in the three-dimensional reconstruction of double-stained structures that needed correction was "twin image mismatch"--i.e., the observation that a three-dimensional reconstruction of a small test object (double-stained on purpose) produced two slightly shifted objects, each associated with its particular fluorochrome. To measure the actual twin image mismatch of the confocal instrument and to obtain accurate correction factors the authors took in each session in which they obtained image series of the real experiments, with both laser wavelengths Z-series of images of multifluorescent microspheres (500-nm diameter) and of thin, double-stained fibers. Given the small dimensions of the structures of interest, i.e., synaptic contacts, it is necessary in this type of research that the optical characteristics of the imaging system--e.g., the alignment errors and chromatic aberration that produce twin image mismatch--be precisely known.

Animals↗

PubMed2002

Tracing tools to resolve neural circuits.

In this paper we summarize neuroanatomical tracing methods, in particular combinations of methods designed to achieve the combined goals of tracing connectivity and extracting extra information from the projection or the target neurons. These combinations include techniques that identify projection fibres together with the neurons from which they originate, methods which establish the morphological or chemical identity of the target neurons and techniques to verify the presence of contacts between the terminal boutons on fibres and prospective target neurons. In the second part of this paper we describe a method recently developed in our laboratory, which consists of fluorescence tracing (introducing marker #1) combined with immunofluorescence with different fluorochromes (markers #2 and #3). This method enables us to observe the distribution of terminals of a particular set of projection fibres in contact with neurons belonging to two chemically different populations of interneuron. Multifluorescence confocal laser scanning is used for image acquisition of fibres and processes of presumed target neurons, and follow-up is by three-dimensional computer reconstruction. These reconstructions are used to determine that the contacts between the differentially labelled structures are real, i.e. with no optically empty space in between regardless of the angle of inspection. The specific experiments reported in this paper comprise the tracing with biotinylated dextran amine of fibres in the rat brain running from the presubiculum to layer III of the medial division of the entorhinal cortex. We studied the possibility of contacts in the latter area between the terminal boutons of these projecting fibres in association with presumed target neurons, i.e. parvalbumin and calretinin expressing interneurons.

Animals↗