MH/MHS/RnD Cerebellum

MHS 289 Cerebellum

The cerebellar cortex is composed of the outer, hypocellular molecular layer and an inner, densely populated granular cell layer. At the interface of these two layers are large neurons known as Purkinje cells.

At higher magnification, examine Purkinje cells stained with hematoxylin and eosin (H&E).

• Their nuclei are much larger compared to those in the inner, densely populated granular layer.
• Their cell bodies and dendrites are intensely acidophilic. This indicates they contain large amounts of proteins.

Only the nuclei of neurons in the inner, densely populated granular layer are visible.

MH 003 Toluidine Blue

Toluidine blue is a basic dye that binds nucleic acids but preferentially stains RNA.

At higher magnification in the cerebellum, examine Purkinje cells stained with toluidine blue.

• Their large nuclei contain lightly stained euchromatin.
• Their nucleoli are intensely stained because they contain negatively charged RNA involved in ribosome assembly.
• Nissl (i.e., chromophil) substance appears dark blue due to the staining of ribosomal RNA, giving the cytoplasm a mottled appearance.
• The presence of Nissl substance suggests these cells synthesize large amounts of protein.

MHS 284 Brain

Golgi's method randomly stains a limited number of neurons and their processes (axon and dendrites). This allows the morphology of individual neurons to be investigated.

At higher magnification in the cerebellum, examine the morphology of Purkinje cells (#1, #2, #3):

• Perikaryon (cell body) - large diameter
• Dendrites - highly branched in the outer molecular layer of the cerebellum
• Axon - a single axon extends from each Purkinje cell into the inner granular layer of the cerebellum

RnD 002 Cerebellum

Purkinje cells can also be identified by using immunohistochemistry for proteins that are highly expressed in these cells.

In this case, HSPH1 was detected using the following procedure:

1. Incubation with an HSPH1 goat primary antibody
2. Incubation with an HRP-conjugated anti-goat secondary antibody
3. HRP was visualized by its conversion of soluble DAB to a brown precipitate
4. Counter-stained with hematoxylin (blue/purple)

HSPH1 is found in the cell bodies and larger dendrites of the Purkinje cells (#1, #2, #3). The light brown staining of the outer molecular layer of the cerebellum is non-specific.

Abbreviations:

• HSPH1: heat shock protein family H member 1
• HRP: horse radish peroxidase
• DAB: diaminobenzidine

Courtesy of Alexander E. Kalyuzhny (R&D Systems, Minneapolis, MN).

MHS 290 Cerebellum

Purkinje cells can also be identified by using immunofluorescence for specific proteins. Secondary antibodies conjugated with fluorescent dyes are used to detect the primary antibodies.

• Purkinje cells (green)
• Glia (non-neuronal cells; red)
• Nuclei (blue)

Courtesy of Thomas Deerinck (National Center for Microscopy and Imaging Research, University of California, San Diego, CA).

(This image is licensed under CC BY-NC-SA 3.0 US.)

MHS 291 Purkinje Cell

The morphology of individual neurons can also be examined by using a small electrode to inject a fluorescent dye.

A Purkinje cell in the cerebellum injected with Lucifer Yellow. This projection was generated from a series of optical sections acquired with a laser scanning confocal microscope.

• Perikaryon (cell body) - large diameter
• Dendrites - highly branched in the outer layer of the cerebellum
• Axon - a single axon extends into the inner layer of the cerebellum

Courtesy of Maryann Martone and Eric Bushong (University of California, San Diego, CA).

(This image is licensed under CC BY 3.0.)