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Confocal Microscopy Image Gallery
Rat Brain Tissue Sections
Dentate Gyrus
The dentate gyrus is one of the components of the hippocampal formation, a telencephalic structure chiefly situated in the temporal lobe that functions in learning and memory consolidation. The other sections of the hippocampal formation include the hippocampus proper and the subiculum. All parts of the formation exhibit a laminar organization, consisting of three main cell layers, collectively referred to as archicortex.
The strata of the dentate gyrus include molecular, granule cell, and polymorphic layers, though the rest of the hippocampal formation features a pyramidal layer in place of the granule cell stratum. The granule cells of the dentate gyrus primarily receive input from the entorhinal cortex, a part of the limbic association cortex that accepts signals from higher-order association areas of the cerebral cortex and other areas of the limbic association cortex. In turn, the axons of granule cells (termed mossy fibers) project to the pyramidal cells located in the CA3 section of the hippocampus.
For many years it was widely assumed that humans and other animals possessed a limited supply of brain cells and no more could be produced in adulthood. Studies carried out in the early 1960s by Joseph Altman suggested that new neurons can be produced in adult rats, but Altman’s research was generally overlooked and the traditional view that adult neurogenesis was impossible prevailed until about three decades later. At that time, new research was reported that clearly confirmed that new neurons are produced in the hippocampal formation of adult rats, as well as adult monkeys and human subjects. The new neurons are now known to be derived from stem cells that reside in a subgranular zone of the hippocampal formation. From this zone, the progenitor cells migrate into the granule cell layer of the dentate gyrus where they mature and form synaptic connections with other cells in the hippocampal formation.
A coronal thin section of rat brain featuring the dentate gyrus (presented above) was immunofluorescently labeled for heavy chain neurofilament subunits (present in neurons) with chicken anti-NF-H antibodies followed by goat anti-chicken secondary antibodies conjugated to Alexa Fluor 488 (green fluorescence). In addition, glial fibrillary acidic protein (GFAP), which is strongly and specifically expressed in various astroglia and neural stem cells, was targeted in the specimen with rabbit anti-GFAP monoclonal antibodies visualized with goat anti-rabbit antibodies conjugated to Alexa Fluor 568 (red emission). Nuclear DNA was counterstained with the red-absorbing probe DRAQ5 (pseudocolored blue). Images were recorded with a 20x objective using a zoom factor of 2.2 and sequential scanning with the 488-nanometer spectral line of an argon-ion laser, the 543-nanometer line from a green helium-neon laser, and the 633-nanometer line of a red helium-neon laser. During the processing stage, individual image channels were pseudocolored with RGB values corresponding to each of the fluorophore emission spectral profiles unless otherwise noted above.
Additional Confocal Images of Rat Dentate Gyrus Tissue Sections
Distribution of Calbindin and Neurofilaments in Dentate Gyrus Tissue - The calcium-binding protein calbindin and heavy chain neurofilament subunits were immunofluorescently labeled in the rat dentate gyrus tissue section (coronal) shown in this confocal image by treating the specimen with a cocktail of mouse anti-calbindin and chicken anti-NF-H primary antibodies followed by goat anti-mouse and anti-chicken secondary antibodies conjugated to Alexa Fluor 488 and Alexa Fluor 568, respectively. DRAQ5, a DNA-interactive agent that exhibits preferential intercalation at AT base pairs, was utilized to target cell nuclei.
Coronal Rat Brain Section Triple Stained with Alexa Fluor 488, Alexa Fluor 568, and DRAQ5 - In a double immunofluorescence experiment, a coronal rat brain section featuring the dentate gyrus was fixed, permeabilized, blocked with 10-percent normal goat serum, and then treated with primary chicken antibodies against NF-H (heavy chain neurofilament subunits) and mouse antibodies against calbindin (calcium-binding protein). The primary targets were then visualized with goat anti-chicken secondary antibodies conjugated to Alexa Fluor 568 and anti-mouse antibodies conjugated to Alexa Fluor 488. Cell nuclei were targeted with DRAQ5.
DNA, NF-H, and Calbindin Visualized in the Dentate Gyrus - Neurofilaments, which are found specifically in neurons and are especially prominent in the axons of the cells, are typically composed of light (NF-L), medium (NF-M), and heavy (NF-H) chain subunits. In the dentate gyrus tissue section presented above, the heavy chain neurofilament subunits were targeted with chicken anti-NF-H antibodies followed by goat anti-chicken secondary antibodies conjugated to Alexa Fluor 568. The calcium-binding protein calbindin was simultaneously targeted in the specimen with mouse anti-calbindin antibodies visualized with goat anti-mouse secondary antibodies conjugated to Alexa Fluor 488. Nuclear DNA was subsequently labeled with the far-red fluorescent dye DRAQ5.
Contributing Authors
Nathan S. Claxton, Shannon H. Neaves, and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.