January 10, 2011
PI: Stephen Van Hooser
This report includes:
1. A description of the joint project between the Van Hooser and Paradis lab to characterize the small GTPase Rem2, and
2. A tentative immunohistochemistry (IHC) protocol for Rem2.
Suzanne Paradis and Amy Ghiretti from the Paradis lab are interested in characterizing the small GTPase Rem2. Small GTPases are a family of enzymes that bind to and hydrolyze guanosine triphosphate (GTP) converting it to GDP. Small GTPases are often involved in signal transduction as there are many proteins (e.g. G-proteins) that are active when bound to GTP and inactive when bound to GDP.
Using RNAi to perform a loss-of-function analysis of Rem2 in cultured rat hippocampal neurons, Suzanne and Amy found that knockdown of Rem2 decreases the density and maturity of dendritic spines, and alters the gross morphology of dendritic arborizations by increasing the number of dendritic branches without altering total dendrite length. This implies that Rem2 functions to inhibit dendritic branching and promote the development of dendritic spines and synapses. They also found that binding to the calcium binding protein calmodulin (CaM) is required for Rem2 regulation of dendritic branching, but is not required for synapse development. This suggests that Rem2 regulates dendritic branching and synapse development via dissociable signal transduction pathways. This would make it the first GTPase for which the signal transduction pathways regulating dendritic branching and synapse development have been dissociated.
The work above is described in their paper ‘The GTPase Rem2 regulates synapse development and dendritic morphology.’ I am not sure that it has been published yet, so you may need to ask Amy for a copy.
More recently, and important to the joint project with the Van Hooser lab, they found that Rem2 function is activity dependent, i.e. transcription of the protein is related to the firing of the neuron.
To further characterize the Rem2 and explore its role in living mammalian brain circuits, Suzanne and Amy approached the Van Hooser lab. The proposal, that was recently approved, is to examine whether or not neurons in the developing mouse visual cortex exhibit rapid reorganization of dendritic branching in response to visual stimulation after a period of darkness. Or to rephrase: to look for dendritic changes in the developing visual cortex of mice in response to visual activity.
The protocol actually describes two sets of experiments. The first is to look at the short-term impact of visual activity on protein expression in the developing brain. In this experiment, the expression level of proteins, such as Rem2, are compared between dark reared mice, and mice that are dark reared and then visually stimulated for 1 to 4 hours. In the second set of experiments, the short-term impact of visual activity on the morphology of cortical neurons is imaged using the 2-photon microscope. The general idea is to set up dark reared mice in the 2-photon rig, expose one eye to visual stimuli for up to 12 hours, and look at changes in the morphology of dendrites in each hemisphere.
In a literature search, I found many papers from 1970 on that documented dendritic changes in the visual cortex in response to activity. When scientists were looking at changes in dendrites 40 years ago, they used the Golgi method to stain neurons, counted the length and number of branches on basal and apical dendrites, and used a method called Sholl analysis to characterize their morphology1. Since then, major changes seem to have been in imaging techniques, rather than in what we are looking for. Today we look at:
1. Cumulative length of dendrite
2. Number of primary dendrites
3. Number of higher order branches of each primary dendrite
4. Number of spines / maturity of spines
We also still use Sholl analysis to characterize the morphology of neurons, although methods for interpreting the results seem to have evolved quite a bit2, 3, 4.
As an extension of this joint project, we decided to try and develop an IHC protocol for Rem2. What we have so far is kind of a mash up of the following protocols:
1. The VECTASTAIN Elite ABC and the DAB (3,3’ diaminobenzidine) Peroxidase Substrate Kit which you can find in the fridge or download from vectorlabs.com.
2. The immunocytochemistry methods from Amy and Suzanne’s paper.
3. An ABC protocol Steve used to stain brain slices in wells
4. A standard IHCworld.com ABC protocol
The core of the protocol is from the VECTASTAIN and DAB kits, using Rabbit anti-Rem2 antibody we borrowed from the Paradis lab. We currently have frozen stock solutions (50 ul each) of this antibody in our -20C fridge. The protocols that came with these kits were designed for staining brain slices mounted slides, so I tried to adapt them to brain slices in plastic wells with Steve’s ABC well protocol. The concentration of primary antibody used was 1:500, which is what was used for immunocytochemistry in Amy and Suzanne’s described above. The IHCworld.com protocol was mostly just a sanity check, but I was interested in trying out their washing steps as they took less time. The current protocol is below, followed by Steve’s.
I would also like to mention that the ‘Dako Immunochemical Staining Handbook,’ which you can find at IHCworld.com, proved to be very useful background.
Tentative Rem2 IHC Protocol (all steps performed on shaker unless otherwise noted)
1) 0.1M PBS bath after sectioning
2) 0.1M PBS with 0.5% Triton-X 100 - 30 min
3) Rinse in 0.1M PBS – 5 min
4) Rinse in 0.1M PBS – 5 min
5) Quench peroxidase activity with 0.4% H2O2 in PBS – 5 min
6) Rinse in 0.1M PBS – 5 min
7) Rinse in 0.1M PBS – 5 min
8) Rinse with diluted normal rabbit serum (instructions + material in VECTASTAIN Elite ABC kit) – 30 min
9) Incubate in 1:500 rabbit anti-Rem2 in 0.1M PBS (primary antibody) – over night at 4C
10) Rinse in 0.1M PBS – 5 min
11) Rinse in 0.1M PBS – 5 min
12) Rinse in 0.1M PBS – 5 min
13) Incubate in 1:50 biotinylated anti-rabbit IgG (secondary antibody) (instructions + material in VECTASTAIN Elite ABC kit) – 30 min
14) Rinse in 0.1M PBS – 5 min*
15) Rinse in 0.1M PBS – 5 min*
16) Rinse in 0.1M PBS – 5 min*
17) Incubate in VECTASTAIN Elite ABC Reagent (instructions + material in VECTASTAIN Elite ABC kit) – 30 min
18) Rinse in 0.1M PBS – 5 min*
19) Rinse in 0.1M PBS – 5 min*
Steps 20 and 21 should be performed in the hood without the shaker (shake plates by hand). Diaminobenzidine is a hazardous waste, and needs to be disposed of in a hazardous waste container. Should dilute with equal amounts of 10% bleach in water solution.
20) Incubate in diaminobenzidine (follow instructions in DAB kit) – until desired color is reached, around 5-10 minutes.
21) Rinse in water – 5 min
22) 0.1M PBS until mounting
Steve’s Protocol (use Triton-X 100 instead of trypsin)
1) 0.1M PB bath after sectioning
2) Pre-heat Trypsin solution (0.13g in 50mL 0.1M PB) to 37.5 deg.C
3) Trypsin incubation - 5 minutes at 37.5deg.C
4) Rinse in cold 0.1M PB - 5 minutes
5) Rinse in cold 0.1M PB - 5 minutes
6) Quench peroxidase activity with...
3.3mL of 30% H2O2
22.5mL 0.4M PB
... for 5 minutes at room temperature
7) Rinse in 0.1M PB - 5 minutes
8) Rinse in 0.1M PB - 5 minutes
9) Blocking: 10% Normal Goat Serum in 0.1M PB - 30 minutes at room temperature
10) Primary Antibody incubation: 1:500 calbindin with 1% Normal Goat Serum in 0.1M PB -
overnight at 4deg.C
1:1000 parvalbumin with 1% Normal Goat Serum in 0.1M
PB - overnight at 4deg.C
11) Rinse in 0.1M PB - 20 minutes
12) Rinse in 0.1M PB - 20 minutes
13) Rinse in 0.1M PB - 20 minutes
14) Secondary Antibody incubation: 1:50 biotinylated anti-mouse IgG (gamma-chain specific) -
30 minutes at room temperature
15) Rinse in 0.1M PB - 10 minutes
16) Rinse in 0.1M PB - 10 minutes
17) Rinse in 0.1M PB - 10 minutes
18) Avidin-biotin complex (ABC) incubation - 1 hour at room temperature
19) Rinse in 0.1M PB - 20 minutes
20) Rinse in 0.1M PB - 20 minutes
21) Rinse in 0.1M PB - 20 minutes
22) Diaminobenzidine (DAB) incubation in darkness - 5 minutes at room temperature
23) Rinse in MilliQ
24) Bath in 0.1M PB until mounting
1 Greenough, William T., and Fred R. Volkmar. "Pattern of Dendritic Branching in Occipital Cortex of Rats Reared in Complex Environments." Experimental Neurology 40.2 (1973): 491-504.
2 McAllister, A. K., D. C. Lo, and L. C. Katz. "Neurotrophins Regulate Dendritic Growth in Developing Visual Cortex." Neuron (1995): 791-803.
3 Chang, F. L., and W. T. Greenough. "Lateralized Effects of Monocular Training on Dendritic Branching in Adult Split-brain Rats." Brain Res. (1982): 283-92.
4 Wei-Chung, Allen Lee et. al. "Dynamic Remodeling of Dendritic Arbors in GABAergic Interneurons of Adult Visual Cortex." PLoS Biology (2006)