The overall goal of this combined analytical methodology is to provide information on how secretory vesicles in cells respond to a physical force, such as extracellular osmotic pressure, and how adjustments of these organelles further affect the exocytosis process. This method can help answer key questions in the neuroscience field such as, how do secretory vesicles directly respond to changes in the extracellular environment or to drug treatment? The main advantage of this approach is to monitor direct effects on vesicular content at live cells and knowing to distinguish alteration in their reh-chemical-ed release before and after exocytosis is treated.
To obtain a flat disk electrode surface, place the electrode in the holder of a microgrinder and bevel each carbon fiber electrode at an angle of 45 degrees. Afterward, mark the capillary for future reference of how to locate the disk electrode surface at a 45 degree angle when placing the electrode near cells for exocytosis measurement. To perform amperometric recording at single cells, mount the freshly beveled and tested carbon fiber disk microelectrode in the electrode holder of the head stage that is used with a potentiostat.
Before use, place each carbon fiber microelectrode in a test solution to monitor the study state current using cyclic voltammetry. For a cyclic voltammetry scan, apply a triangle potential waveform from negative 0.2 volts to positive 0.8 volts against a silver-silver chloride reference electrode at 100 milivolts per second to ensure good reaction kinetics. For amperometry recording of exocytosis, place the Petri dish with cultured chromaffin cells in isotonic buffer on an inverted microscope in a Faraday cage.
Use a microscope heating stage to maintain a temperature of 37 degrees Celsius during the cell experiments. Next, use a low-noise patch clamp instrument to apply a constant potential of positive 700 millivolts at the working electrode. For recording exocytosis of chromaffin cells, digitalize the signal at 10 kilohertz and apply an internal low-pass Bessel filter at two kilohertz to filter the recorded signal.
Note that the oval disk electrode needs to be placed flat on top of the cells and parallel to the surface of the Petri dish. Also, electrodes are beveled the same day as experiments to ensure a clean electrode surface. To stimulate the cell exocytosis, position a glass micropipette filled with five millimolar barium chloride solution at a distance of at least 20 micrometers away from the cell.
Then, apply a five seconds injection pulse of five millimolar barium chloride solution on the cell's surface to stimulate the cell exocytosis. Place the stem pipette in the solution and stimulate the cell exocytosis by applying a barium injection pulse while recording the amperometric current transients for approximately three minutes. To compare the exocytotic responses in isotonic conditions to hypertonic conditions, incubate the cells for 10 minutes in hypertonic buffer solution.
Thereafter, apply a barium injection pulse to stimulate exocytosis, and perform three minutes of amperometric recording. For the reversible response of cells, incubate the cells again for 10 minutes in an isotonic buffer solution. Stimulate the cells by applying a barium injection pulse and perform three minutes of recording of the exocytotic response.
To fabricate the electrodes for vesicular quantal size measurements, prepare a carbon fiber electrode with a diameter of five micrometers. Under a microscope, use a scalpel to cut the carbon fiber that is extending out of the glass tip so that only 30 to 100 micrometers is left. Use a butane flame to prepare a flame-etched tip of the carbon fiber electrode.
To achieve an evenly-etched, cylindrical-shaped electrode tip, hold the cylindrical-shaped carbon fiber electrode while rotating it. And place the carbon fiber extending from the glass into the blue edge of the butane flame until the tip of the carbon develops a red color. After flame etching, place the electrode under a microscope to evaluate the electrode tip.
The etched electrode tip size should be about 50 to 100 nanometers in diameter. Next, insert the cylindrical nanotip microelectrode into the epoxy solution for three minutes followed by a 15 seconds dipping of the electrode tip into the acetone solution. This allow the epoxy to seal the potential gap space between the carbon fiber and the insulating gas capillary wall.
While the acetone clears the epoxy off the etched carbon fiber electrode surface. To cure the epoxy, bake the electrodes in an oven overnight at 100 degrees Celsius. Before use, test the steady state current of each carbon fiber microelectrode using cyclic voltammetry.
For experiments, use only electrodes that display a plateau current around 1.5 to 2.5 nanoamperes. For intracellular amperometry measurements, place the cells under the microscope and use the same experimental settings of the potentiostat. Prevent significant physical damage to the cell.
Insert the nanotip cylindrical microelectrode into the cell by adding a gentle mechanical force. Just enough to push the electrode through the cell plasma membrane and into the cell cytoplasm using a micromanipulator. After insertion, and with the cell membrane sealed around the cylindrical electrode, start the amperometric recording in-situ at the live cell.
At the oxidation potential applied to the electrode, vesicles adsorbed to the electrode surface and stochastically rupture. Hence, there is no need for any kind of stimulus to initiate this process. To determine the osmotic effect on vesicular quantal size, collect the intracellular cytometry measurements from a group of cells that have been incubated in isotonic and in hypertonic buffer using the experimental condition.
The effect of extracellular osmolality on exocytosis activity is presented as the frequency of exocytosis events when chromaffin cells are stimulated with barium solution in isotonic, then hypertonic, and finally in isotonic conditions. This data shows an inhibition in exocytosis activity at cells experiencing osmotic stress and a partial recovery can be achieved after cells returned to an isotonic environment. The control experiment shows the frequency of exocytosis events after three consecutive barium stimulations at chromaffin cells in isotonic conditions.
This shows that multiple barium stimulation within the time frame used in these experiments are causing a reduction in exocytosis activity by consecutive cell stimulation. After watching this video, you should have a good understanding of how to perform these complimentary analytical methods that allow you to compare how secretory vesicles and the exocytosis process are affected by alterations in the extracellular environment.
