In the brain, a pair of neurons often form multiple synaptic contacts, which is called synaptic multiplicity. However, precise examination of synaptic multiplicity requires technically challenging experiments. This protocol describes a simple method for gross estimation of synaptic multiplicity using whole-cell patch-clamp electrophysiology.
This method can be applied to any species and brain area to investigate synaptic multiplicity. This method requires basic skills in whole-cell patch-clamp electrophysiology. Obtaining high-quality recordings with low and stable access resistance is critical for the accurate interpretation of the data.
To obtain whole-cell configuration, place the recording pipette just above the slice and offset pipette current in the voltage clamp mode. Apply slight positive pressure to the pipette, and lock the stopcock. Next, select a healthy cell with an intact membrane, and approach the tissue with the pipette.
The positive pressure should cause a slight disturbance on the tissue. Slowly bring the pipette closer to the cell in a diagonal motion until a small dimple is formed on the cell surface. Then, release the positive pressure lock.
The cell will begin to form a seal, and the resistance will increase above one gigaohm. In voltage clamp, hold the cell at minus 68 millivolts. Subsequently, slightly pull the pipette away from the cell diagonally to remove excess pressure.
Compensate for the fast and slow pipette capacitance. Apply a brief suction through the tube connected to the pipette holder to break through the cell and obtain a whole-cell configuration. Then, switch to cell mode on the membrane test window in an electrophysiology data acquisition and analysis software.
Maintain the temperature of the recording bath at 27 to 30 degrees Celsius and the flow rate at 1.5 to two milliliters per minute for subsequent experiments. In a multiplicity synapse, an action potential synchronizes neurotransmitter release and generates a larger postsynaptic current. Blocking action potential and calcium-dependent vesicular release with TTX and cadmium prevents the postsynaptic current summation and decreases the amplitude.
When there is no multiplicity, blocking action potential will not change the amplitude. In experiment one, to estimate multiplicity, hold the cell at minus 68 millivolts while perfusing it with low-calcium aCSF. Record the spontaneous EPSCs for at least five minutes to ensure a stable baseline.
Next, add 30 micromolars 4-AP to the aCSF to increase action potential-dependent events, and record spontaneous EPSCs for at least 10 minutes to obtain the full drug effect. Then, add 0.5 micromolars TTX and 10 micromolars cadmium to the aCSF with 4-AP, and record the miniature EPSCs for at least 10 minutes. For offline analysis, use the last one minute of baseline immediately before the application of 4-AP, the 10th minute of 4-AP application, and the 10th minute of TTX application.
In this experiment, extracellular calcium is replaced with strontium to desynchronize the release of synaptic vesicles. Therefore, if multiplicity is present, this should decrease the amplitude of postsynaptic currents. In experiment two, record spontaneous EPSCs for at least five minutes while perfusing the cell with normal calcium aCSF.
To desynchronize vesicle release, begin perfusing the cell with strontium aCSF, and record spontaneous EPSCs. For offline analysis, to determine whether the large amplitude spontaneous EPSCs are due to the synchronous release of vesicles, compare the last minute of baseline to the 10th minute of strontium aCSF application. Multiplicity can involve multivesicular release, which causes higher neurotransmitter concentration in the synaptic cleft.
Addition of gamma-DGG, a low-affinity AMPA receptor antagonist, leads to less effective inhibition of larger multiquantal compared to smaller uniquantal postsynaptic currents. Without multivesicular release, gamma-DGG will be equally effective on larger and smaller postsynaptic currents. In experiment three, to test for multivesicular release, record spontaneous EPSCs in low-calcium aCSF for at least five minutes.
Add 30 micromolars 4-AP to the aCSF through the perfusion system. Record the spontaneous EPSCs for at least 10 minutes. Then, add 200 micromolars gamma-DGG to the aCSF with 4-AP, and record the spontaneous EPSCs for at least 10 minutes.
As a control experiment in a separate cell, repeat the procedures, but apply a low concentration of DNQX instead of gamma-DGG. For offline analysis, analyze the last minute of each drug application. Bursts of synaptic activity can transiently increase spontaneous action potential firing and release probability of the stimulated afferents.
If neurons exhibit multiplicity, the increase in action potentials should cause a transient increase in the amplitude of postsynaptic currents. In experiment four, record spontaneous EPSCs in normal calcium aCSF. To increase action potential firing, stimulate the afferents using a monopolar glass electrode filled with aCSF at a rate of 20 hertz for two seconds, and repeat 10 times with an inter-burst interval of 20 seconds.
For analysis, use 5, 000 milliseconds spontaneous EPSCs before the first stimulus as the baseline and compare to the 10 to 300 milliseconds spontaneous EPSCs after the final stimulus. Then, take the average amplitude and frequency change over 10 trials. Analyze spontaneous EPSCs and miniature EPSCs using a program that detects and analyzes synaptic currents.
Use the suggested detection parameters and nonstop analysis function for detecting AMPA receptor-mediated EPSCs. Manually scan each recording to ensure the program is accurately detecting each event. Export the event data by copying it to the clipboard, and paste it into a data management software.
Next, calculate the average frequency and amplitude for each drug treatment, and perform the relevant statistical analyses. In an example shown here, 4-AP increases both the amplitude and frequency of spontaneous EPSCs. Subsequent application of TTX and cadmium decreases both the amplitude and frequency.
Here is the distribution of spontaneous EPSC amplitude from the recording. In the hypothalamic neurons examined here, the amplitude and frequency of the baseline and TTX conditions are the same, suggesting that the baseline spontaneous EPSCs contain very few action potential-dependent EPSCs. Accordingly, subsequent experiments can compare the difference between baseline and 4-AP to measure multiplicity.
The strength of synaptic transmission can be transiently increased by bursts of synaptic activity. To investigate multiplicity under more physiological conditions, afferent stimulation can be used to increase action potential firing and release probability. Here are the summaries of spontaneous EPSC frequency and amplitude changes following synaptic stimulation.
Stable recordings are essential for accurate interpretation of the data. Describe the data if the access resistance changes by more than 20%during the recording, as this could confound the analysis. This protocol offers a simple way to estimate synaptic multiplicity, which is a key determinant of synaptic efficacy and its plasticity in different physiological and pathophysiological conditions.
