Download How to look at the brain:

How to look at the brain:
•1 Possibly: transcardial perfusion of the animal under deep
anesthesia
A: wash out the blood with saline (quick flush)
B: fixative solution:
•10% formalin (4% formaldehyde in water)
•4% paraformaldehyde
•(1-2% glutaraldehyde: good for electron microscopy)
Or
1 Quick brain dissection out from the skull and freezing
Preserves antigens (good for molecular biology;
less good for neurohistology)
Problem of storage in freezers!
(Or
1 Quick brain dissection and fixation by immersion:
Not good to preserve antigens, but can be used for histology)
Rat transcardial perfusion
How to look at the brain:
•2 after perfusion: brain dissection (take it out from the skull carefully;
•3 preparation for cutting the brain into frozen sections:
cryoprotection in sucrose (10-30% sucrose in phosphate buffer, pH 7.4)
overnight (for small pieces) or longer (for larger pieces)
Avoids freezing artifacts
or
•3 The perfused or unperfused brain can be embedded (usually in
paraffin)
Good for histological quality, but may inactivate or mask antigens for
immunohistochemistry
How to look at the brain:
• 4 Cutting the brain in frozen sections (for light microscopy):
•Cryostat (equipment common in pathology labs)
•freezing microtome
or
• 4 Cutting the brain into sections with a vibratome
(allows also to go later to electron microscopy)
Planes of cutting sections:
transverse
or coronal (or frontal)
longitudinal
horizontal
How to look at the brain
•5 Collect sections serially!
•6 Sections can be processed free-floating
or mounted on slides for staining
•7 Dehydration and coverslipping of sections on slides
•8 Study at the microscope:
•Qualitative observations: tissue changes; phenotype cell changes;
cell distribution (keep your question in mind, but do not disregard
serendipitous observations)
•Quantitative observations: cell number, cell density, analysis
of the intensity of the signal
Cell Stains:
visualization of all cells and tissue features (cytoarchitectonics,
nuclear boundaries, layers in the cerebral cortex)
Routine: Nissl staining (cresyl violet)
stains all cells
Cell Stains:
visualization of all cells and tissue features (cytoarchitectonics,
nuclear boundaries, layers in the cerebral cortex)
chemical characterization of a phenotype (immunohistochemistry or
histochemistry)
visualization of a phenomenon: cell death (neurodegeneration)
or cell birth (neurogenesis)
Study of connections: use of substances that are taken up by
axons used as tracers
•Quantitative observations
A cell number, cell density
•To be meaningful (and publishable), very stringent criteria need to be applied
•Criteria need to be established before the analysis
•It allows statistical analysis of the data significance
(qualitative data or semi-quantitative scoring cannot be analyzed
statistically)
B analysis of the intensity of the signal:
densitometric analysis: requires a digital camera
connected to the microscope and an image analysis
system
Characterization of a glial cell type:
Immunohistochemistry for an antigen – protein –
selectively expressed by that cell type
“Chemical characterization of neurons”
Question: where is a given molecule expressed?
mRNA: in situ hybridization
Protein or peptide: immunohistochemistry
•Single labeling (one marker)
•Double labeling
• nNOS is not constitutively expressed by somatic motoneurons
• nNOS induction is part of the repertoire of the response to injury
of mature facial motoneurons
control side
axotomized side
“Functional mapping of neurons”
Question:
which neurons are activated by a given stimulus
(a drug, a task, etc.)?
Induction of c-fos expression (Fos immunohistochemistry)
Proto-oncogenes (immediate early genes, IEGs)
IEGs: transcription factors
in the signal transduction cascade
Expression of IEGs: inducible,
transient
c-fos (gene)
Fos (protein product)
Using Fos expression to map
neurons “functionally activated” by a
given stimulus
•Apply a stimulus
•let the animal survive 1.5 – 3 h (for Fos) Protein synthesis takes about 1.5 h
•run parallel experiments (same conditions but without stimulus)
Histological processing (similar to many other procedures
•perfuse the animal (through the heart, with saline followed by 4%
paraformaldehyde in phosphate buffer pH 7.4)
• cut the brain into sections (30 microns OK) with a cryostat, freezing microtome
(or vibratome)
•collect the sections into ordered serial series in phosphate-buffered saline
run immunohistochemistry (free-floating)
•mount the sections on gelatinized slides and let them dry
•dehydrate and coverlip
•study at the microscope (bright-field illumination)