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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
Cell stainNormal Schizophrenia Huntington
Rajkowska et al., Arch Gen Psych (1998) 55: 215-224
Cell stains are useful in determining size,density, and positioning of cells.In this study, a cell stainwas used to examine thedistribution of neuronsand glia in the prefrontalcortex of brains from schizophrenic patients,patients with Huntington’sdisease and normal controls.Schizophrenia is charac-terized by changes in neuronal density, as wellas slight changes in somalsize. Huntington’s diseaseis characterized as aneurodegenerative diseaseby the increase in glialcells and the decrease inneurons.
Cell stain of Schizophrenic HippocampusCONTROLS
SCHIZOPHRENIA
Kovelman et al, Biol Psych (1984) 19: 1601-1621
In this study, a cell stain was used to studycell positioning in the hippocampus. Using thisstaining method, they observed that pyramidalcells in the CA1/CA2 regions were disorganized.
Cell Stain of SZP Entorhinal CortexControl Schizophrenic
Arnold et al, Arch Gen Psych(1991) 48: 625-632
In this study, cell staining showed that in theentorhinal cortex of schizophrenic brain, thereare aberrant invaginations, disruption of corticallayers, 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 comparedto normal controls. The results of this study revealed a significant decrease in AChE activity in thehippocampus of these patients indicating either a loss of cholinergic cells, or a loss of cholinergicactivity in these cells in this region.
NADPH-diaphorase staining
Control Schizophrenia
Akbarian et al., Arch Gen Psych(1993) 50: 169-177
This enzymatic reaction stains nicotinamide-adeninedinucleotide phosphate-diaphorase (NADPH-d) withnitroblue tetrazolium. NADPH-d is present in a smallpopulation of GABAergic neurons in the cortex. In brainsof schizophrenic patients, these cells appear to bemisplaced, indicating a likely failure of migration.
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 hundredneurons, but stains all processes of that neuron. Using this method, it was found that in theprefrontal cortex of postmortem schizophrenic brains, there is a 23% decrease in the numberof spines expressed on the dendrites of pyramidal cells in cortical layer III.
CONTROL
SZP
SZP
DiI Fluorescence
DiI fluorescence is an oil that is placed using a micropipette on the cell soma ofthe cell of interest. Like Golgi impregnation, this method allows the visualizationof the entire neuron and its processes. In this study, this method revealed thatin schizophrenic prefrontal cortex, some pyramidal neurons have a bifurcatedapical dendrite.
Kalus et al., Neuropsychobiology(1999) 40: 1-13
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
Incubate tissue withradioactive ligand
Expose to filmor emulsion
Isotope will emitradiation (usually beta)
Radiation will hit silver grains in emulsion and expose them
Autoradiography of Nicotine Receptors in SmokersPrefrontal Cortex
Temporal Cortex
Hippocampus
Perry et al., JPET (1999) 289: 1545-1552
Using tritiated epibatidine (3H-EB) as a marker of nicotine receptors, autoradiography revealedthat 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
DirectIndirect
AAA
DABD
AB
DA
B
GABA 5-HT
1y antibodyagainst D1
(Biotinylated)
2y antibodyagainst 1y
(Biotinylated)
B
D1 D2
B
1y antibodyagainst D2
Avidin-BiotinComplex
Chromogen:DAB/HRP
AAA
DAB
DA
B
DA
BAvidin-Biotin
Complex
Chromogen:DAB/HRP
Immunostaining for GABA Transporter1Control SZP
Control SZP
C. Pesold
Woo et al., PNAS (1998) 95: 5341-5346
Using an antibody against the neuronal GABAtransporter (GAT1), immunostaining techniquein schizophrenic and control PFC showed adecrease in cartridges (chandelier cell terminalends on pyramidal cell axon initial segment) inSZP patient indicating a specific decrease inGABA function.
Immunostaining for ReelinRELN Nissl
NSP
SZP
100m
I
II
I
II
Reelin is a large glycoprotein involved in neurodevelopment,and likely pays an important rolein synaptic pruning and plasticityin adult brain.In this study, immunostainingusing a reelin-specific antibodyrevealed that schizophrenic (SZP)brains have fewer reelin-expressingcells than normal controls. Thesefindings were compared to a cellstain (right) to show that SZP donot have a decrease in the numberof neurons present, only a decreasein the expression of reelin in cells.
Pesold et al., unpublished
Immunogold
GAD67
GABA
1y antibodyagainst 1
2y antibodyagainst 1y
(Gold-Conjugated)
1
GSilver Enhancement
B
1y antibodyagainst GAD67
2y antibodyagainst 1y
(Biotinylated)
AAA
DAB
DA
B
DA
BAvidin-Biotin
Complex
Chromogen:DAB/HRP
Immunogold Labelling of Serotonin Receptors in Suicide Victims
Control Suicide
5-HT2A
5-HT2C
Pesold et al., unpublished
With immunogold labelling,quantification of the numberof gold particles can give ameasure of the amount ofprotein present in a verydiscrete location. In thisstudy, immunogold labellingwas used to quantify thedensity of 5-HT2A and 5-HT2C subtypes ofserotonin receptors in the PFC of suicide victims andcontrols. It was found thatin suicide victims, there isa significant increase in5-HT2A, but not 5-HT2C
receptors on pyramidal cellsof cortical layer III.
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 interestin cells of a particular neurochemical phenotype. In this study, a decrease in GABAA receptorscontaining 1 subunits (gold particles) was found in GABAergic cells (GAD67-positive orangecells) in the hippocampus of animals that were made tolerant to the benzodiazepine diazepam.
Immunofluorescence
GABA
1y antibodyagainst D1
2y antibodyagainst 1y
(conjugated toFluorescein)
D1 D2
1y antibodyagainst D2
2y antibodyagainst 1y
(conjugated toRhodamine)
Double immunofluorescence for Reelin and GAD67
C. Pesold, unpublished
Two different fluorochromes can be used to determine the colocalization of two differentproteins in the same tissue, cells etc.In this study, the neurochemical phenotype of reelin-containing cells was determined to beGABAergic since it was always found to co-localize with GAD67, the synthesizing enzyme forGABA, 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 probeEnd-labelling (e.g., digoxigenin)Insertion labelling (e.g., biotin)Tagging (e.g., biotin)
In Situ Hybridization using Radiolabelled Probes
5’ AGG CAT TTG CCA TAT GGC 3’(mRNA)
Probe:• must be in reverseorientation to the target• 30-50 bases long•C=G >50%
3’ TCC GTA AAC GGT ATA CCG 5’
35S
Probe is tail-labelledon the 3’ end with
labelled deoxynucleotide(deoxynucleotidyl transferase)
32P 33P 35S 125I 3H
Expose to autoradiographic film
or emulsion
35S 3-15 days3H 6-18 weeks
In-situ Hybridization Using a Radiolabelled Probe for GAD67
Control
Schizophrenic
Volk et al., Arch Gen Psych (2000) 57: 237-245
In this study, in situ hybridization was usedto determine the level of mRNA encoding for GAD67 using an 35S-labelled oligonu-cleotide for GAD67. A 25-35% decrease inGAD67-labelled cells was found in PFC layers III-V of schizophrenic brain ascompared to control brains.
Control
SZP
In Situ Hybridization usingNon-Radiolabelled Probes
5’ AGG CAT TTG CCA TAT GGC 3’(mRNA)
3’ TCC GTA AAC GGT ATA CCG 5’
Probe can be end-labelledwith Digoxigenin or Biotin
Digoxigenin can be visualizedby immunohistochemistry
(1y antibody against Digox)
B
AAA
DAB
DA
B
DA
B
2y antibodyagainst 1y
(Biotinylated)
Avidin-BiotinComplex
Chromogen:DAB/HRP
3’ TCC GTA AAC GGT ATA CCG 5’
Dig
BiotinBiotin
Biotin
Biotin
Fluorescein-conjugatedanti-biotin
Probe can beinserted
with biotin
Double Fluorescent In Situ Hybridization and Immunohistochemistry
C. Pesold, Unpublished
In situ hybridization can be combined with immunohistochemistry. In this study, reelin mRNAwas found to be synthesized in GABAergic cells. Reelin mRNA was detected with adigoxygenin-labelled probe that was then visualized using fluorescence immunohistochemistry(fluorescein: green). GAD67, the synthesizing enzyme for GABA was detected withimmunofluorescence, using an antibody specific to GAD67, and a secondary antibodyconjugated 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