1) Histochemistry
2) Autoradiography
3) Immunohistochemistry
4) In situ Hybridization
HISTOCHEMISTRY
These are basic stains that reveal cellular
elements by colorimetric method
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•
•
•
•
Cell stains/ myelin stains
Acetylcholinesterase staining
NADPH-diaphorase staining
Golgi impregnation
DiI Fluorescent staining
Cell stain
Normal
Schizophrenia
Huntington
Cell stains are useful in
determining size,density,
and positioning of cells.
In this study, a cell stain
was used to examine the
distribution of neurons
and glia in the prefrontal
cortex of brains from
schizophrenic patients,
patients with Huntington’s
disease and normal controls.
Schizophrenia is characterized by changes in
neuronal density, as well
as slight changes in somal
size. Huntington’s disease
is characterized as a
neurodegenerative disease
by the increase in glial
cells and the decrease in
neurons.
Rajkowska et al., Arch Gen Psych (1998) 55: 215-224
Cell stain of Schizophrenic Hippocampus
CONTROLS
SCHIZOPHRENIA
In this study, a cell stain was used to study
cell positioning in the hippocampus. Using this
staining method, they observed that pyramidal
cells in the CA1/CA2 regions were disorganized.
Kovelman et al, Biol Psych (1984) 19: 1601-1621
Cell Stain of SZP Entorhinal Cortex
Control
Arnold et al, Arch Gen Psych
(1991) 48: 625-632
Schizophrenic
In this study, cell staining showed that in the
entorhinal cortex of schizophrenic brain, there
are aberrant invaginations, disruption of cortical
layers, and heterotopic displacement of neurons.
AChE staining
Control
SDAT
Henke & Lang, Brain Res (1983) 267: 281-291
This study used an enzymatic staining technique to reveal the presence of acetylcholinesterase
(AChE) in the brains of patients with senile dementia of the Alzheimer's type (SDAT), as compared
to normal controls. The results of this study revealed a significant decrease in AChE activity in the
hippocampus of these patients indicating either a loss of cholinergic cells, or a loss of cholinergic
activity in these cells in this region.
NADPH-diaphorase staining
This enzymatic reaction stains nicotinamide-adenine
dinucleotide phosphate-diaphorase (NADPH-d) with
nitroblue tetrazolium. NADPH-d is present in a small
population of GABAergic neurons in the cortex. In brains
of schizophrenic patients, these cells appear to be
misplaced, indicating a likely failure of migration.
Control
Schizophrenia
Akbarian et al., Arch Gen Psych
(1993) 50: 169-177
Golgi-impregnation
CONTROL
SZP
SZP
Glantz et al., Arch Gen Psych (2000) 57: 65-75
Golgi impregnation is a method that only randomly labels one out of every several hundred
neurons, but stains all processes of that neuron. Using this method, it was found that in the
prefrontal cortex of postmortem schizophrenic brains, there is a 23% decrease in the number
of spines expressed on the dendrites of pyramidal cells in cortical layer III.
DiI Fluorescence
Kalus et al., Neuropsychobiology(1999) 40: 1-13
DiI fluorescence is an oil that is placed using a micropipette on the cell soma of
the cell of interest. Like Golgi impregnation, this method allows the visualization
of the entire neuron and its processes. In this study, this method revealed that
in schizophrenic prefrontal cortex, some pyramidal neurons have a bifurcated
apical dendrite.
Histochemistry
Advantages:
- relatively simple and quick
- inexpensive
Disadvantages:- Limited Information
- Limited number of
histochemical stains available
- Enzymatic stains cannot easily
be combined
AUTORADIOGRAPHY
Uses :
• Map anatomical location of radiolabelled
ligands to visualize and quantify receptors in
tissue
• Trace neurons by axonal transport of
radioactively labelled amino acids, certain
sugars, or transmitter substances
• Measure DNA production (e.g., 3H-thymidine)
2 Types:
In-vivo autoradiography - receptors are labelled in intact
living tissue by systemic administration of the radioligand (PET)
In-vitro autoradiography - slide-mounted tissue sections
are incubated with radioligand so that receptors are labelled
under very controlled conditions
Autoradiography
Radiation will hit silver grains in emulsion and expose them
Expose to film
or emulsion
Isotope will emit
radiation (usually beta)
Incubate tissue with
radioactive ligand
Autoradiography of Nicotine Receptors
in Smokers
Prefrontal Cortex
Hippocampus
Temporal Cortex
Perry et al., JPET (1999) 289: 1545-1552
Using tritiated epibatidine (3H-EB) as a marker of nicotine receptors, autoradiography revealed
that chronic smokers have a 160-400% increased nicotine binding sites compared to non-smokers
Autoradiography
Advantages:
- Highly specific tool to pharmacologically characterize
receptors in tissue (unlike tissue bath preparations)
- Provides location of receptor (etc) in tissue
- Enables characterization of receptors in different tissues
between different animals or brain regions
- Technically easy
Disadvantages: - Everything binds to everything (easy to misinterpret
results)
- There are no biochemical or physiological criteria to
assess the binding specificity (i.e., to determine whether
the binding site really corresponds to an actual receptor)
- The presence of a high-affinity radiolabelled receptor
does not necessarily imply that the receptor has
physiological significance
- Ligands are not always very specific
IMMUNOHISTOCHEMISTRY
This technique uses antibodies to localize
proteins in tissue sections
Many types of markers:
• Colorimetric
• Gold particles
• Fluorescence
Immunostaining
Indirect
Direct
DAB
A
Chromogen:
DAB/HRP
Avidin-Biotin
Complex
B
DAB
Chromogen:
DAB/HRP
Avidin-Biotin
Complex
2y antibody
against 1y
(Biotinylated)
A
B
1y antibody
against D1
(Biotinylated)
1y antibody
against D2
D1
GABA
D2
5-HT
Immunostaining for GABA Transporter1
Control
SZP
Control
C. Pesold
Using an antibody against the neuronal GABA
transporter (GAT1), immunostaining technique
in schizophrenic and control PFC showed a
decrease in cartridges (chandelier cell terminal
ends on pyramidal cell axon initial segment) in
SZP patient indicating a specific decrease in
GABA function.
Woo et al., PNAS (1998) 95: 5341-5346
SZP
Immunostaining for Reelin
RELN
Nissl
Reelin is a large glycoprotein
involved in neurodevelopment,
and likely pays an important role
in synaptic pruning and plasticity
in adult brain.
In this study, immunostaining
using a reelin-specific antibody
revealed that schizophrenic (SZP)
brains have fewer reelin-expressing
cells than normal controls. These
findings were compared to a cell
stain (right) to show that SZP do
not have a decrease in the number
of neurons present, only a decrease
in the expression of reelin in cells.
I
NSP
II
I
SZP
II
100m
Pesold et al., unpublished
Immunogold
DAB
Chromogen:
DAB/HRP
A
Silver Enhancement
B
G
2y antibody
against 1y
(Gold-Conjugated)
Avidin-Biotin
Complex
2y antibody
against 1y
(Biotinylated)
1y antibody
against 1
1y antibody
against GAD67
1
GAD67
GABA
Immunogold Labelling of Serotonin
Receptors in Suicide Victims
Control
Suicide
With immunogold labelling,
quantification of the number
of gold particles can give a
measure of the amount of
protein present in a very
discrete location. In this
study, immunogold labelling
was used to quantify the
density of 5-HT2A and
5-HT2C subtypes of
serotonin receptors in the
PFC of suicide victims and
controls. It was found that
in suicide victims, there is
a significant increase in
5-HT2A, but not 5-HT2C
receptors on pyramidal cells
of cortical layer III.
5-HT2A
5-HT2C
Pesold et al., unpublished
Combined Immunogold-Immunostaining for
GABAA receptors in GABAergic Neurons
Vehicle
Diazepam
C. Pesold, unpublished
Immunogold can be combined with immunostaining to visualize and quantify a protein of interest
in cells of a particular neurochemical phenotype. In this study, a decrease in GABAA receptors
containing 1 subunits (gold particles) was found in GABAergic cells (GAD67-positive orange
cells) in the hippocampus of animals that were made tolerant to the benzodiazepine diazepam.
Immunofluorescence
2y antibody
against 1y
(conjugated to
Rhodamine)
2y antibody
against 1y
(conjugated to
Fluorescein)
1y antibody
against D1
1y antibody
against D2
D1
D2
GABA
Double immunofluorescence for Reelin
and GAD67
C. Pesold, unpublished
Two different fluorochromes can be used to determine the colocalization of two different
proteins in the same tissue, cells etc.
In this study, the neurochemical phenotype of reelin-containing cells was determined to be
GABAergic since it was always found to co-localize with GAD67, the synthesizing enzyme for
GABA, in the prefrontal cortex of primate brain.
Immunohistochemistry
Advantages:
- Markers are relatively safe to use (do not involve
radioactivity)
- There are many different kinds of markers making
combinations of double and triple labellings possible
- Results can be obtained in a short time (2 days)
- Can also be visualized at the electron microscopy
level
Disadvantages: - The quality of the immunolabelling depends highly on
the specificity and affinity of the primary antibody.
- Primary antibodies are not available for all proteins
of interest and raising a good antibody can be very
difficult, timely and expensive.
IN SITU HYBRIDIZATION
This method utilizes probes to
visualize mRNA in tissue sections
Two types of Probes: Riboprobe - cRNA
Oligoprobe - cDNA
Markers: Radioactively labelled probe
End-labelling (e.g., digoxigenin)
Insertion labelling (e.g., biotin)
Tagging (e.g., biotin)
In Situ Hybridization using
Radiolabelled Probes
Expose to
autoradiographic film
or emulsion
35S
3-15 days
3H 6-18 weeks
Probe:
• must be in reverse
orientation to the target
• 30-50 bases long
•C=G >50%
Probe is tail-labelled
on the 3’ end with
labelled deoxynucleotide
(deoxynucleotidyl transferase) 35S
32P 33P 35S 125I 3H
3’ TCC GTA AAC GGT ATA CCG 5’
5’ AGG CAT TTG CCA TAT GGC 3’
(mRNA)
In-situ Hybridization Using a
Radiolabelled Probe for GAD67
Control
Control
SZP
Schizophrenic
In this study, in situ hybridization was used
to determine the level of mRNA encoding
for GAD67 using an 35S-labelled oligonucleotide for GAD67. A 25-35% decrease in
GAD67-labelled cells was found in PFC
layers III-V of schizophrenic brain as
compared to control brains.
Volk et al., Arch Gen Psych (2000) 57: 237-245
In Situ Hybridization using
Non-Radiolabelled Probes
Chromogen:
DAB/HRP
Avidin-Biotin
Complex
DAB
A
B
Fluoresceinconjugated
anti-biotin
2y antibody
against 1y
(Biotinylated)
Digoxigenin can be visualized
by immunohistochemistry
y
(1 antibody against Digox)
Probe can be end-labelled
with Digoxigenin or Biotin
3’ TCC GTA AAC GGT ATA CCG 5’
Dig
3’ TCC GTA AAC GGT ATA CCG 5’
5’ AGG CAT TTG CCA TAT GGC 3’
(mRNA)
Probe can be
inserted
with biotin
Double Fluorescent In Situ Hybridization
and Immunohistochemistry
C. Pesold, Unpublished
In situ hybridization can be combined with immunohistochemistry. In this study, reelin mRNA
was found to be synthesized in GABAergic cells. Reelin mRNA was detected with a
digoxygenin-labelled probe that was then visualized using fluorescence immunohistochemistry
(fluorescein: green). GAD67, the synthesizing enzyme for GABA was detected with
immunofluorescence, using an antibody specific to GAD67, and a secondary antibody
conjugated to rhodamine (red).
In Situ Hybridization
Advantages:
- Only method to detect mRNA in tissue
- Can determine which cell synthesizes a protein
since many proteins are transported away from the
cell body
- Can be used when no antibody exists for a protein
Disadvantages: - Use of radiolabelled probes requires special training,
handling and can be expensive
- Radiolabelled probes can take weeks to yield results
- In situ hybridization requires very sterile conditions
and is therefore easily subject to error or contamination
- Signal can sometimes be quite weak
- Designing the right probe is critical