AUTORADIOGRAPHY

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Cellular Neurochemistry
1) Histochemistry 2) Autoradiography 3)
Immunohistochemistry 4) In situ Hybridization
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HISTOCHEMISTRY

• These are basic stains that reveal cellular
  elements by colorimetric method
• Cell stains/ myelin stains
• Acetylcholinesterase staining
• NADPH-diaphorase staining
• Golgi impregnation
• DiI Fluorescent staining

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Cell stain
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 Huntingtons disease and
normal controls. Schizophrenia is charac- terized
by changes in neuronal density, as well as
slight changes in somal size. Huntingtons
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
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Cell stain of Schizophrenic Hippocampus
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
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Cell Stain of SZP Entorhinal Cortex
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.
Arnold et al, Arch Gen Psych (1991) 48 625-632
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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 (AC
hE) 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.
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NADPH-diaphorase staining
This enzymatic reaction stains nicotinamide-adenin
e 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.
Akbarian et al., Arch Gen Psych (1993) 50 169-177
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Golgi-impregnation
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.
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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.
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Histochemistry

• Advantages - relatively simple and quick
• - inexpensive
• Disadvantages- Limited Information
• - Limited number of
• histochemical stains available
• - Enzymatic stains cannot easily
• be combined

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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

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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
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Autoradiography of Nicotine Receptors in Smokers
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
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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
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IMMUNOHISTOCHEMISTRY

• This technique uses antibodies to localize
  proteins in tissue sections
• Many types of markers
• Colorimetric
• Gold particles
• Fluorescence

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Immunostaining
Indirect
Direct
Chromogen DAB/HRP
Avidin-Biotin Complex
Chromogen DAB/HRP
2y antibody against 1y (Biotinylated)
Avidin-Biotin Complex
1y antibody against D1 (Biotinylated)
1y antibody against D2
D1
D2
GABA
5-HT
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Immunostaining for GABA Transporter1
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
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Immunostaining for Reelin
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.
Pesold et al., unpublished
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Immunogold
Chromogen DAB/HRP
Silver Enhancement
Avidin-Biotin Complex
2y antibody against 1y (Gold-Conjugated)
2y antibody against 1y (Biotinylated)
1y antibody against ?1
1y antibody against GAD67
?1
GAD67 GABA
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Immunogold Labelling of Serotonin Receptors in
Suicide Victims
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.
Pesold et al., unpublished
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Combined Immunogold-Immunostaining for GABAA
receptors in GABAergic Neurons
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.
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Immunofluorescence
2y antibody against 1y (conjugated to Fluorescein)
2y antibody against 1y (conjugated to Rhodamine)
1y antibody against D1
1y antibody against D2
D1
D2
GABA
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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.
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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.

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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)

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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
• CG gt50

Probe is tail-labelled on the 3 end
with labelled deoxynucleotide (deoxynucleotidyl
transferase) 32P 33P 35S 125I 3H
3 TCC GTA AAC GGT ATA CCG 5
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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 oligonu- cleotide 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
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In Situ Hybridization usingNon-Radiolabelled
Probes
Chromogen DAB/HRP
Avidin-Biotin Complex
Fluorescein- conjugated anti-biotin
2y antibody against 1y (Biotinylated)
Digoxigenin can be visualized by
immunohistochemistry (1y antibody against Digox)
Probe can be inserted with biotin
3 TCC GTA AAC GGT ATA CCG 5
Probe can be end-labelled with Digoxigenin or
Biotin
3 TCC GTA AAC GGT ATA CCG 5
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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 immunofluorescen
ce, using an antibody specific to GAD67, and a
secondary antibody conjugated to rhodamine (red).
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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