The overall goal of this procedure is to measure gabaa receptor surface localization and endocytosis in hippocampal neurons. This is accomplished by first transecting hippocampal neurons with the tagged gabaa receptor subunit and allowing the cultured neurons to develop in the incubator. The second step is to label surface gabaa receptors with alpha bungalow toin coupled fluorescent Alexa die.
Next, the neurons are incubated for various time points in order to allow for labeled receptors to be endocytose. The final step is to fix the specimens at the appropriate time points and to perform immuno staining using anti GFP to observe the total tagged receptor pool. Ultimately, confocal microscopy is used to show changes in surface localization and endocytosis of the tagged gabaa receptors using fixed or live imaging approaches.
This method can help us answer key questions in the field of receptor trafficking, such as what effect ligand binding can have on receptor endocytosis and insertion rates, and whether different ligands and modulators can affect receptor recycling and degradation. Although this method has been used to study ligated ion channels an G-protein coupled receptors primarily in the context of the nervous system, it can also be applied to study the dynamic localization and trafficking of other membrane proteins in cell culture and primary cells of diverse tissues. Generally, individuals new to this method will struggle for two reasons.
First, to create functional constructs and second practice and experiences needed to process several time points simultaneously. To begin, prepare four to five round poly de lysine coated glass cover slips inside each 3.5 centimeter tissue culture dish as described in the text protocol. Then transfect freshly dissociated neurons on the day of culturing with one to four micrograms of maxi prepped construct DNA plate.
The transfected neurons on the poly de lycine coated cover slips at a final density of approximately 200, 000 neurons per 3.5 centimeter dish. Replace the medium four to 24 hours after preparation of neuronal cultures. Then allow the neurons to develop in the incubator until 14 to 17 days in vitro or the desired stage to perform the endocytosis assay.
First set a benchtop cooling heating device to 16 degrees Celsius. Cool extracellular heaps, buffered saline or HBS to 16 degrees Celsius in a water bath. Transfer the neuron dishes to the aluminum plate at 16 degrees Celsius and cool for five minutes.
Remove the medium and replace with one milliliter of 16 degrees Celsius HBS plus 150 Micromolar two Bo Rine for two minutes. Next, remove the medium to a labeled waste container and replace it with one milliliter of 16 degrees Celsius HBS plus two bo kerine plus alpha bungalow toxin. Alexa 5 9 4 incubating at 16 degrees Celsius for 15 minutes.
Following incubation, transfer the medium to a labeled waste container and wash the dishes three times with two milliliters of 16 degrees Celsius HBS for live imaging time series experiments following receptor endocytosis using a glass bottomed 3.5 centimeter culture dish. Transfer the dish to a heated stage on the microscope and begin imaging with a confocal or turf microscope. Set up for fixed cover slips.
Transfer T equals zero time point cover slips into a dish of room temperature 4%para formaldehyde, 4%sucrose for 20 minutes for other time points. Replace HBS with conditioned equilibrated 37 degrees Celsius medium, and return to the incubator for endocytosis at 37 degrees Celsius at time points needed. Take dishes from the incubator and wash quickly twice with two milliliters of room temperature, delcos phosphate, buffered saline or DPBS.
Then fix the specimen in 4%paraform aldehyde, 4%sucrose for 20 minutes. After a 20 minute fixation, remove 4%paraform aldehyde, 4%sucrose to the waste container, and wash the dishes twice with two milliliters of room temperature DPBS. Next, incubate the cover slips at room temperature for 10 minutes.
An immunofluorescence block solution containing 0.2%Triton X 100 to perme the neurons and enable anti GFP immuno staining of the intracellular receptor pool and or labeling of other proteins of interest. Then remove permeable block solution and incubate the cover slips in immunofluorescence block solution without Triton X 100 for 20 to 30 minutes. Perform primary antibody incubations of cover slips in block for several hours at room temperature or overnight at four degrees Celsius following incubation.
Wash the cover slips by soaking in DPBS for five minutes for a total of three washes. Then incubate the cover slips with secondary antibodies in block solution for one hour at room temperature after one hour. Wash the cover slips three times with DPBS as before.
Next mount each cover slip by removing excess liquid from the back of the cover slip with lab tissue and then inverting the cover slip onto four microliters of mounting medium on a glass slide. Perform image acquisition with confocal microscopy blind to experimental condition using a 60 x oil numerical aperture. 1.49 immersion objective.
Using the same image acquisition settings, acquire single Z section images with the neuronal cell body and several dendritic processes in focus quantify alpha bungalow toxin, Alexa fluorescent signal and GFP immuno staining along 20 microns of three to four proximal dendrites per neuron using the same threshold for analysis of all endocytosis data. Analyze data from 10 to 12 neurons for each time point. Repeating the experiment with several independent neuronal cultures.
GABAergic axons make inhibitory contacts onto pyramidal neuron dendrites. Pre-synaptic inhibitory terminals are identified by the vesicular inhibitory amino acid transporter that loads GABA and glycine into synaptic vesicles. GABA a receptors are closely opposed to the neurotransmitter release sites tethered by the post-synaptic inhibitory scaffold Protein gein shown here are alpha two phg F-P-B-B-S expressing neurons with surface gabaa receptors labeled with Alexa 5 9 4 bungalow toxin followed by immuno staining for total receptor population with anti GFP antibody shown in green, and either the post-synaptic inhibitory scaffold protein gein or the vesicular inhibitory amino acid transporter.
Here the endocytosis of alpha two containing GABA A receptor and hippocampal neurons at 15 days in vitro is shown alpha bungalow toxin. Fluorescence Measurements of individual dendritic processes as shown in the panels are normalized to the level of construct expression via the GFP antibodies. Staining final comparison of time points is made by normalization to the T equals zero measurement.
Quantification of internalization of receptors over a 60 minute period from fluorescent signal changes over time was best described by a single exponential. An alternative approach is to follow receptor endocytosis in a single neuron by live imaging time-lapse confocal microscopy. The punctate and overlapping fluorescence of P-H-G-F-P tagged GABA A receptor and surface alpha bungalow toxin labeled receptors is visible at the start of the experiment over the time course alpha bungalow toxin labeled GABA A receptor signal decreases as receptors endocytose.
While the P-H-G-F-P signal remains high due to new receptor insertion, the minimal decrease in P-H-G-F-P signal is due to cell surface receptor distribution changes and photobleaching Following this procedure. Other methods like bungalow toxin insertion assays can be performed to answer additional questions like receptor insertion rates with different treatments. This technique allows one to assess how cellular activity, specific protein domains, agonists, and other modulators can modify receptor turnover in live neurons, other cells and tissues.
After watching this video, you should have a good understanding as to how to use alpha bungalow toxin coupled Alexa dyes in combination with standard immunofluorescent techniques to quantify surface receptor localization in hippocampal neurons.
