The overall goal of this procedure is to understand the integration of inputs along the very complex structure of the neuronal dendritic tree, which finally leads to the neuronal output of action potentials at the axon. This is accomplished by first pulling the appropriate ion aporetic pipettes. The second step is to test the ion aporetic pipettes and to correctly compensate their capacitance.
Next, establish the whole cell configuration. The final step is to approach the dendrite and to evoke post-synaptic potentials in the recorded cell. Ultimately, fast micro iontophoresis allows investigating the integration of multiple inputs, which can also belong to different neurotransmitter systems.
This method can help to answer key questions in the neuroscientific field, like how inhibitory and excitatory inputs are integrated, and under which conditions. This can lead to neuronal output. Begin this procedure by pulling pipettes with very small tips for controlled neurotransmitter iontophoresis.
With a horizontal or vertical pipette puller, small fine tips can be achieved with several pulling steps. However, the tip should still be rigid enough to penetrate into the tissue. Next, set up the ion aporetic amplifier to test the pipette performance in a chamber without tissue.
Fill the pipette with neurotransmitter and dye containing solution. Then place it into the A CSF. Subsequently compensate the capacitance with the amplifier.
Usually very sharp pipettes will have a higher capacitance than the blunt ones after their capacitance is compensated correctly. Check the resistance of the pipette with the built-in feature of the micro iontophoresis amplifier. The pipette should have a resistance between 25 and 90 mega ohms.
Afterward, focus on the pipette tip with a 60 x or 40 x water immersion objective. Then switch to fluorescent imaging. If fluorescent D leaks out of the pipette tip, apply a small positive retain current in the case of glutamate or negative retain current in the case of gaba.
If that doesn't help to cancel the leakage, change the pipette. Next, apply a strong step current to the pipette or use the manual trigger Monitor the fluorescent image of the pipette tip to see if the solution can be ejected. If there is no visible leakage and test ejection is successful, check the capacitance compensation and start the experiment.
In general, ion aporetic events can be evoked at defined locations depending on the desired experiment. For example, at a spiny dendrite for glutamate micro phoresis, or at the dendritic shaft, soma or axon initial segment for GABA micro phoresis. After establishing the whole cell configuration, place the ion aporetic pipette tip to approximately one micrometer distance from the dendrite or other compartment of interest.
Make sure that no neurotransmitter is leaking out and that the pipette and the capacitance are compensated correctly. If approaching the cell with a glutamate filled ion aporetic pipette causes detectable depolarization of the membrane, potential glutamate is leaking out of the pipette. Try to adjust the retain current or change the ion aporetic pipette.
Next, apply short negative pulses to the ion aporetic pipette for glutamate current starting from zero and increase the current systematically. This helps to find out the range in which the ion aporetic current evokes the desired responses in the specific experimental setup. If there is no detectable response, lift the ion aporetic pipette several hundred micrometers, and apply a strong current larger than 0.1 microamps to clean the tip.
Then adjust the capacitance compensation. Again, approach the cell and try again. If there is still no response, try to reduce the retain current.
However, be very careful with the reduction of the retain current since this can cause uncontrolled neurotransmitter release. Therefore, constantly monitor the recording to detect respective changes in membrane potential. If glutamate leaks out of the pipette, reject the pipette for gaba micro iontophoresis.
Fill a new pipette with GABA solution. It is very important to have the correct polarity for the ejection current when changing the transmitter in the pipette. For GABA ejection, change the settings to normal resulting in a positive ejection current to detect the GABAergic events more easily.
Inject long current steps resulting in membrane potential changes from around negative 100 millivolts to negative 48 millivolts. With this protocol, the chloride ion driving force can be increased, making the events more prominent, and the equilibrium of the event is revealed. This schematic drawing shows single glutamate micro iontophoresis.
The EPSP shown here is evoked in a CA one pyramidal neuron with glutamate iontophoresis, and the dendritic sodium calcium spike depicted here is evoked at a dendrite of a CA one pyramidal neuron. The lower trace shows the slope of the voltage trace, and the peak indicates the peak slope of the dendritic spike. When increasing current applied to the ionophore pipette, the EPSP amplitude increases until it crosses the action potential threshold.
The schematic drawing here shows double glutamate micro iontophoresis at different locations on the dendritic tree. This ion retic EPSP is evoked at a proximal dendri of a CA one pyramidal neuron, and this one is evoked at a distal dite. This schematic drawing shows GABA micro phoresis.
The retic IPSP shown here is evoked at a proximal dendrite of a CA one pyramidal neuron and long current injection with different amplitudes applied to a CA one pyramidal neuron via the patch pipette helps to determine the reversal potential of the evoked event the evoked event reverses at approximately negative 70 millivolts. Lastly, this schematic drawing shows the simultaneous glutamate and GABA iontophoresis that investigates the integration of excitation and inhibition. The dendritic spikes evoked by retic application of glutamate alone.
In subsequent sweeps are shown here. The peaks of the lower trace showing the slope of the voltage trace indicate the occurrence of the dendritic spikes, and here is ionically evoked GABAergic IPSP alone. Lastly, both the glutamatergic and GABAergic events together are evoked, which leads to the inhibition of dendritic spikes.
After watching this video, it should be very easy to establish fast micro resis in your lab and to gain insight how synaptic inputs are integrated in neurons.
