1. 1) Histochemistry 2) Autoradiography 3) Immunohistochemistry 4) In situ Hybridization

2. 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

3. Cell stain NormalSchizophreniaHuntington 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 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 charac- terized 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.

4. Cell stain of Schizophrenic Hippocampus CONTROLS SCHIZOPHRENIA Kovelman et al, Biol Psych (1984) 19: 1601-1621 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.

5. Cell Stain of SZP Entorhinal Cortex ControlSchizophrenic Arnold et al, Arch Gen Psych (1991) 48: 625-632 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.

6. AChE staining ControlSDAT 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.

7. NADPH-diaphorase staining ControlSchizophrenia Akbarian et al., Arch Gen Psych (1993) 50: 169-177 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.

8. 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. CONTROL SZP

9. DiI Fluorescence 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. Kalus et al., Neuropsychobiology(1999) 40: 1-13

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

11. 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., 3 H-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

12. Autoradiography Incubate tissue with radioactive ligand Expose to film or emulsion Isotope will emit radiation (usually beta) Radiation will hit silver grains in emulsion and expose them

13. Autoradiography of Nicotine Receptors in Smokers Prefrontal Cortex Temporal Cortex Hippocampus Perry et al., JPET (1999) 289: 1545-1552 Using tritiated epibatidine ( 3 H-EB) as a marker of nicotine receptors, autoradiography revealed that chronic smokers have a 160-400% increased nicotine binding sites compared to non-smokers

14. 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

15. IMMUNOHISTOCHEMISTRY This technique uses antibodies to localize proteins in tissue sections Many types of markers: Colorimetric Gold particles Fluorescence

16. Immunostaining Direct Indirect A A A DAB GABA 5-HT 1 y antibody against D 1 (Biotinylated) 2 y antibody against 1 y (Biotinylated) B D1D2 B 1 y antibody against D 2 Avidin-Biotin Complex Chromogen: DAB/HRP A A A DAB Avidin-Biotin Complex Chromogen: DAB/HRP

17. Immunostaining for GABA Transporter 1 Control SZP Control SZP C. Pesold Woo et al., PNAS (1998) 95: 5341-5346 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.

18. Immunostaining for Reelin RELNNissl NSP SZP 100  m I II I 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

19. Immunogold GAD 67 GABA 1 y antibody against  1 2 y antibody against 1 y (Gold-Conjugated) 11 G Silver Enhancement B 1 y antibody against GAD 67 2 y antibody against 1 y (Biotinylated) A A A DAB Avidin-Biotin Complex Chromogen: DAB/HRP

20. Immunogold Labelling of Serotonin Receptors in Suicide Victims Control Suicide 5-HT 2A 5-HT 2C Pesold et al., unpublished 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-HT 2A and 5-HT 2C 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-HT 2A, but not 5-HT 2C receptors on pyramidal cells of cortical layer III.

21. Combined Immunogold-Immunostaining for GABA A receptors in GABAergic Neurons VehicleDiazepam 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 GABA A receptors containing  1 subunits (gold particles) was found in GABAergic cells (GAD 67 -positive orange cells) in the hippocampus of animals that were made tolerant to the benzodiazepine diazepam.

22. Immunofluorescence GABA 1 y antibody against D 1 2 y antibody against 1 y (conjugated to Fluorescein) D1D2 1 y antibody against D 2 2 y antibody against 1 y (conjugated to Rhodamine)

23. Double immunofluorescence for Reelin and GAD 67 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 GAD 67, the synthesizing enzyme for GABA, in the prefrontal cortex of primate brain.

24. 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.

25. 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)

26. In Situ Hybridization using Radiolabelled Probes 5 ’ AGG CAT TTG CCA TAT GGC 3 ’ (mRNA) Probe: must be in reverse orientation to the target 30-50 bases long C=G >50% 3 ’ TCC GTA AAC GGT ATA CCG 5 ’ 35 S Probe is tail-labelled on the 3 ’ end with labelled deoxynucleotide (deoxynucleotidyl transferase) 32 P 33 P 35 S 125 I 3 H Expose to autoradiographic film or emulsion 35 S 3-15 days 3 H 6-18 weeks

27. In-situ Hybridization Using a Radiolabelled Probe for GAD 67 Control Schizophrenic Volk et al., Arch Gen Psych (2000) 57: 237-245 In this study, in situ hybridization was used to determine the level of mRNA encoding for GAD 67 using an 35 S-labelled oligonu- cleotide for GAD 67. A 25-35% decrease in GAD 67 -labelled cells was found in PFC layers III-V of schizophrenic brain as compared to control brains. Control SZP

28. In Situ Hybridization using Non-Radiolabelled Probes 5 ’ AGG CAT TTG CCA TAT GGC 3 ’ (mRNA) 3 ’ TCC GTA AAC GGT ATA CCG 5 ’ Probe can be end-labelled with Digoxigenin or Biotin Digoxigenin can be visualized by immunohistochemistry ( 1 y antibody against Digox) B A A A DAB 2 y antibody against 1 y (Biotinylated) Avidin-Biotin Complex Chromogen: DAB/HRP 3 ’ TCC GTA AAC GGT ATA CCG 5 ’ Dig Biotin Fluorescein- conjugated anti-biotin Probe can be inserted with biotin

29. 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). GAD 67, the synthesizing enzyme for GABA was detected with immunofluorescence, using an antibody specific to GAD 67, and a secondary antibody conjugated to rhodamine (red).

30. 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