Techniques to Render Dendritic Spines Visible in the Microscope.

A tiny detail visible on certain neurons at the limit of resolution in light microscopy went in 130 years of neuroscience research through a dazzling career from suspicious staining artifact to what we recognize today as a complex postsynaptic molecular machine: the dendritic spine.This chapter deals with techniques to make spines visible. The original technique, Golgi silver staining, is still being used today.

Neuroanatomical tract-tracing techniques that did go viral.

Neuroanatomical tracing methods remain fundamental for elucidating the complexity of brain circuits. During the past decades, the technical arsenal at our disposal has been greatly enriched, with a steady supply of fresh arrivals. This paper provides a landscape view of classical and modern tools for tract-tracing purposes.

Neurochemical fingerprinting of amygdalostriatal and intra-amygdaloid projections: a tracing-immunofluorescence study in the rat.

Amygdalostriatal and intra-amygdaloid fiber connectivity was studied in rats via injections of one of the tracers Phaseolus vulgaris leucoagglutinin (PHA-L) or biotinylated dextran amine (BDA) into various amygdaloid nuclei. To determine the neurotransmitter identity of labeled fibers we combined tracer detection with immunofluorescence staining, using antibodies against vesicular transporters (VTs) associated with glutamatergic (VGluT1, VGluT2) or GABAergic (VGAT) neurotransmission. High-magnification confocal laser scanning images were screened for overlap: occurrence inside tracer labeled fibers or axon terminals of immunofluorescence signal associated with one of the VTs.

Mesencephalic dopamine neurons interfacing the shell of nucleus accumbens and the dorsolateral striatum in the rat.

Parallel corticostriatonigral circuits have been proposed that separately process motor, cognitive, and emotional-motivational information. Functional integration requires that interactions exist between neurons participating in these circuits. This makes it imperative to study the complex anatomical substrate underlying corticostriatonigral circuits.

Topographic mapping between basal forebrain cholinergic neurons and the medial prefrontal cortex in mice.

The basal forebrain cholinergic innervation of the medial prefrontal cortex (mPFC) is crucial for cognitive performance. However, little is known about the organization of connectivity between the basal forebrain and the mPFC in the mouse. Using focal virus injections inducing Cre-dependent enhanced yellow fluorescent protein expression in ChAT-IRES-Cre mice, we tested the hypothesis that there is a topographic mapping between the basal forebrain cholinergic neurons and their axonal projections to the mPFC.

A fourth generation of neuroanatomical tracing techniques: exploiting the offspring of genetic engineering.

The first three generations of neuroanatomical tract-tracing methods include, respectively, techniques exploiting degeneration, retrograde cellular transport and anterograde cellular transport. This paper reviews the most recent development in third-generation tracing, i.e.

Inhibitory interneuron classes express complementary AMPA-receptor patterns in macaque primary visual cortex.

Glutamate receptors mediate excitatory neurotransmission. A very prevalent type of glutamate receptor in the neocortex is the AMPA receptor (AMPAR). AMPARs mediate fast synaptic transmission and their functionality depends on the subunit composition.

3-D reconstruction of neurons from multichannel confocal laser scanning image series.

A confocal laser scanning microscope (CLSM) collects information from a thin, focal plane and ignores out-of-focus information. Scanning of a specimen, with stepwise axial (Z-) movement of the stage in between each scan, produces Z-series of confocal images of a tissue volume, which then can be used to 3-D reconstruct structures of interest. The operator first configures separate channels (e.

Density gradients of vesicular glutamate- and GABA transporter-immunoreactive boutons in calbindinand μ-opioid receptor-defined compartments in the rat striatum.

Cortical and subcortical inputs to the striatum are functionally highly organized and they obey to some extent striatal patch-matrix topography. Whether this organization is reflected in the density of various glutamatergic endings is unknown. We therefore mapped boutons expressing the vesicular glutamate transporters VGluT1 and VGluT2, together with boutons immunoreactive for vesicular γ-aminobutyric acid (GABA) transporter (VGAT) in patch and matrix throughout the striatum.

A half century of experimental neuroanatomical tracing.

Most of our current understanding of brain function and dysfunction has its firm base in what is so elegantly called the 'anatomical substrate', i.e. the anatomical, histological, and histochemical domains within the large knowledge envelope called 'neuroscience' that further includes physiological, pharmacological, neurochemical, behavioral, genetical and clinical domains.

Neuroanatomical tracing combined with in situ hybridization: analysis of gene expression patterns within brain circuits of interest.

Most of our current understanding of brain circuits is based on hodological studies carried out using neuroanatomical tract-tracing. Our aim is to advance one step further by visualizing the functional correlate in a given circuit. In this regard, we believe it is feasible to combine retrograde tracing with fluorescence, non-radioactive in situ hybridization (ISH) protocols.

What does the anatomical organization of the entorhinal cortex tell us?

The entorhinal cortex is commonly perceived as a major input and output structure of the hippocampal formation, entertaining the role of the nodal point of cortico-hippocampal circuits. Superficial layers receive convergent cortical information, which is relayed to structures in the hippocampus, and hippocampal output reaches deep layers of entorhinal cortex, that project back to the cortex. The finding of the grid cells in all layers and reports on interactions between deep and superficial layers indicate that this rather simplistic perception may be at fault.

Vesicular glutamate and GABA transporters sort to distinct sets of vesicles in a population of presynaptic terminals.

Vesicular glutamate transporters (VGLUTs) 1 and 2 are expressed by neurons generally accepted to release glutamate as a neurotransmitter, whereas VGLUT3 appears in populations usually associated with a different classical transmitter. We now demonstrate VGLUT2 as well as the vesicular GABA transporter (VGAT) in a subset of presynaptic terminals in the dentate gyrus of the rat hippocampal formation. The terminals are distributed in a characteristic band overlapping with the outer part of the granule cell layer and the inner zone of the molecular layer.

Counting contacts between neurons in 3D in confocal laser scanning images.

Study of neuronal networks requires an inventory of the neurons, knowledge of fiber in- and output, and qualitative and quantitative data on the intrinsic connectivity. For this purpose we combined in rat hippocampus fluorescence neuroanatomical tracing and intracellular fluorochrome injection of neurons. Multichannel confocal laser scanning microscopy was followed by computer assisted 3D object- and contact recognition.

3-D reconstruction of neurons from multichannel confocal laser scanning image series.

A confocal laser scanning microscope (CLSM) collects information from a thin, focal plane and ignores out-of-focus information. The operator configures separate channels (laser, filters, detector settings) for each fluorochrome used in a particular experiment. Then, 3-D reconstructions are made from Z-series of confocal images: one series per channel.

Origin of calretinin-containing, vesicular glutamate transporter 2-coexpressing fiber terminals in the entorhinal cortex of the rat.

The entorhinal cortex of the rat (EC) contains a dense fiber plexus that expresses the calcium-binding protein calretinin (CR). Some CR fibers contain vesicular glutamate transporter 2 (VGluT2, associated with glutamatergic neurotransmission). CR-VGluT2 coexpressing fibers may have an extrinsic origin, for instance, the midline thalamic nucleus reuniens.

Convergence of entorhinal and CA3 inputs onto pyramidal neurons and interneurons in hippocampal area CA1--an anatomical study in the rat.

The entorhinal cortex (EC) conveys information to hippocampal field CA1 either directly by way of projections from principal neurons in layer III, or indirectly by axons from layer II via the dentate gyrus, CA3, and Schaffer collaterals. These two pathways differentially influence activity in CA1, yet conclusive evidence is lacking whether and to what extent they converge onto single CA1 neurons. Presently we studied such convergence.

Coexpression of vesicular glutamate transporters 1 and 2, glutamic acid decarboxylase and calretinin in rat entorhinal cortex.

We studied the distribution and coexpression of vesicular glutamate transporters (VGluT1, VGluT2), glutamic acid decarboxylase (GAD) and calretinin (CR, calcium-binding protein) in rat entorhinal cortex, using immunofluorescence staining and multichannel confocal laser scanning microscopy. Images were computer processed and subjected to automated 3D object recognition, colocalization analysis and 3D reconstruction. Since the VGluTs (in contrast to CR and GAD) occurred in fibers and axon terminals only, we focused our attention on these neuronal processes.

Computer-assisted estimation in the CNS of 3D multimarker 'overlap' or 'touch' at the level of individual nerve endings: a confocal laser scanning microscope application.

Presynaptic boutons and associated postsynaptic structures in the CNS express markers that are highly synapse type-specific. In multilabel immunofluorescence imaging, coexpression of such markers appears as overlap of signals in the same structures whereas closely related yet segregated markers, e.g.

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.

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.

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.

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.