1) Histochemistry

2) Autoradiography

3) Immunohistochemistry

4) In situ Hybridization

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