Types of Stains for Neurons
Joe C. Adams, Chris Brown
This brief introduction to the neuroanatomy demo describes the
main types of stains used in for revealing different anatomical features of
neurons, and how the different types of cochlear nucleus neurons appear under
these stains. Click on thumbnails to see larger pictures.
 | Nissl stain (e.g., cresyl violet, thionin, azure) stains nuclei
acids (DNA and RNA). This stain is useful for viewing cell sizes and numbers.
In animals with cells as large as cats, the pattern of RNA in the cytoplasm,
seen as free ribosomes and rough endoplasmic reticulum, is helpful for
identifying distinctive neuronal cell classes. Examples include spherical
(Fig. 1) and globular cells in the AVCN, whose names reflect their appearance in
Nissl stains. Multipolar (stellate) cells can usually also be
identified. Octopus cells have little "Nissl
substance" and their shapes must be discerned using other stains. |
 | Reduced silver stains (e.g. Protargol) stains nuclei and
cytoskeletal proteins. This permits visualization of endbulbs in the
AVCN (Fig. 2), the MNTB, and the ventral portion of the VNLL. Boutons terminaux,
which appear as small rings surrounding somata and proximal dendrites are
present on some VCN cells (e.g. octopus cells, Fig. 3) and on MSO somata and proximal dendrites.
Nerve terminals with enough cytoskeleton to be visible using this stain
occur in the brainstem auditory nuclei (excluding the DCN) caudal to the
inferior colliculus. The functional significance of the cytoskeleton
in terminals is not known but it appears to be limited to terminals that
have very "secure" synapses. |

 | Golgi impregnations are not stains, but rather visualization of
cells treated in this way results from a small percentage of cells being
filled with fine black crystals. The advantage of this preparation is that
it enables visualization of the entire cell body and dendrites. Myelinated
axons do not accumulate the salts, although endbulbs, which are not
myelinated, may be filled (Fig. 5). Bushy cells
dendrites (Fig. 4), which have
very few axo-dendritic terminals, have a clearly different appearance from
stellate cells' dendrites (Fig. 6), which may or may not have many axo-dendritic
terminals (Type I and Type II stellate cells). Type II stellate
cells and octopus cells have many axo-dendritic terminals.
DCN pyramidal cells have two dendritic fields, which extend away from
the somata apically and basally. The apical dendrites are covered with
spines, which are specializations for expanding the surface area and are the
sites of synapses from parallel fibers. Parallel fibers are
unmyelinated axons of granule cells which course parallel to the DCN surface
in the uppermost layer and synapse upon many pyramidal cells' dendrites,
which are arrayed in a non-overlapping fashion so that signals from granule
cell axons reach successive pyramidal cells at increasing delays.
Intermingled with pyramidal cell apical dendrites are cartwheel cells,
which have spiny recurved dendrites that also are innervated by parallel
fibers. Cartwheel cells are inhibitory interneurons that innervate
pyramidal cells. |

 | Immunostaining is a means of visualizing the sites in and on cells
where antigens (usually proteins) are situated. The example in this
exercise is brainstem auditory nuclei immunostained for the enzyme glutamate
decarboxylase (GAD). (It cleaves a carboxyl group from glutamate, which
results in production of the inhibitory neurotransmitter GABA, gamma
aminobutyric acid.) There is a high correlation of the presence of
excitatory terminals and GABAergic terminals upon ventral cochlear nucleus
neurons. This demonstration allows one to get a feeling for the
density of nerve terminals on these cells by examining GAD immunostained
tissue. Spherical and globular cells, which receive their
predominant excitatory inputs from auditory nerve fiber endbulbs, are
covered with GAD positive terminals (Fig. 7). Type I stellate cells,
which have few excitatory inputs on somata or dendrites have correspondingly
few GAD positive terminals. Octopus cells, which receive their
excitatory inputs as small terminals from many auditory-nerve fibers, have
fewer and smaller GAD positive terminals than spherical,
globular, or Type II stellate cells. |

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Fos immunostaining
The gene c-fos encodes the transcription factor
Fos. This gene is part of a family of genes that are quickly induced following
sensory stimulation. After the Fos protein has been produced in the
cytoplasm it migrates to the nucleus where it forms heterodimeric
transcription complexes with other proteins and eventually regulates the
transcription of other genes. c-Fos expression can be detected by
immunostaining (i.e. antibodies against the protein) (Fig. 8) or by
in situ hybridization (i.e. detection of mRNA).
Acoustic stimulation leads to the expression of the
gene c-fos in auditory neurons in the dorsal cochlear nucleus, ventral
cochlear nucleus, superior olivary complex, nuclei of the lateral lemniscus,
inferior colliculus, parts of the medial geniculate nucleus, and auditory
cortex. Stimulation of animals with pure tones leads to Fos production
in restricted tonotopic bands/clusters in most nuclei, hence Fos labeling has
been used to study tonotopy without the need for electrophysiology.

Fig. 8:
Fos immunostaining of neurons in the dorsal cochlear (brown reaction product)
with Nissl counterstaining (blue). The stimulus was a 25 kHz tone burst, 85 dB
SPL presented for two hours. Notice that some cells have darker labeling than
others.
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