Iontophoresis can be used to expel or hold charged particles. Using a glass pipette, we can use iontophoresis to emit a solution into the brain. Because it does not rely on mechanical force, this can be used in conjunction with smaller diameter pipettes than mechanical air pump methods.

The solution#

When preparing a solution to be used with iontophoresis, you must be aware that the particle you wish to emit must be charged, and all particles you wish to emit must thave the same charge. For example, ATP in solution is a weak acid with a pea value of ~6.5, meaning that half of the ATP molecules are de-protonated, and therefore negatively charged, at a pH of 6.5. We therefore also need an acidic fluorophore. Alexa hydrazides, dextrans, and Lucifer yellow are, like de-protonate ATP, anions, so you need to pass a negative current to emit them from the pipette. See here for how to make an ATP solution with an Alex Fluor. Remember to test the pH of the solution. For more information see here.

The machine: Model 260#

In the lab. we have mainly used WPI microiontophoresis dual current generator 260s, manual here. Model 260 is designed for electroiontophoresis of dyes, drugs and charged substances from micropipettes. It is battery operated (two Duracell 9V transistor alkaline batteries, see manual for how to replace, if you cannot reach and exceed full scale on the ammeter, the transistor batteries may need replacement.). The left half of the box provides HOLD current at a constant level. The right half supplies transient EJECT current for ejection. In ordinary use, the two current generators are operated in parallel providing two distinct currents; one for preventing substances in the micropipette from outward diffusion (the retain or hold current) and the second for the active ejection of charged material. For pipettes with submicron tips, a hold current may not be necessary if there is little outward diffusion of pipette material. Here are what some of the knobs on Model 260 accomplish:

  • CURRENT LEVEL – This control varies the current amplitude as indicated on the ammeter. To prevent accidental current flow via the output terminals, place the OUTPUT switch in the PRESET position while adjusting current amplitude.
  • CURRENT RANGE – Selects either 100 or 1000nA current range.
  • MODE SELECT – MANUAL operation allows you to apply a DC current when the OUTPUT switch is changed from PRESET to ENABLE. AUTO operation requires you to apply a +5V command potential to initiate the flow of current. This could be provided via a break out board and MATLAB code.
  • OUTPUT POLARITY – NORMAL indicates that the red output connector is positive with respect to the black connector. INVERT reverses connector polarity.
  • AUTO INPUT – If the MODE SELECT switch is in the AUTO position, the AUTO INPUT connector controls current activation. Five volts positive with respect to the outer connector shell maintains the current for as long as this voltage is applied. The CURRENT LEVEL control continues to dictate the actual current amplitude. Therefore, the exact current delivered cannot be controlled programmatically from Matlab.
  • OUTPUT – In the PRESET position, the red and black current output terminals are disconnected from the current generating circuit. ENABLE connects the output terminals to the current source. If the OUTPUT switch is in the PRESET mode, current cannot flow out of the instrument in either MANUAL or AUTO operating modes.
  • CHAS GND – This green binding post is connected to the chassis or metallic enclosure of the instrument. It should normally be connected to the ground point of your recording system. Ground the CHAS GND green binding post to assure low noise.
  • [Alarm sound] - If the OUTPUT switch is changed from PRESET to ENABLE at any current setting, an audible alarm sounds, and a red lamp on the front panel illuminates to indicate that the output current pathway is an open circuit. This happens in normal operation of the device.

HOLD + EJECT#

  1. The electrophoretic release of charged substances from micropipettes, which often requires the use of a second current to counter the outward diffusion of material from the delivery pipette. This current has been referred to in the literature as “backing, holding or retaining” current. It cause a small current to flow in a direction that offsets the spontaneous leakage of the active agent from the pipette until it is required. To set your device up to use both the HOLD and EJECT halves of the machine in parallel. Place both channels in the PRESET mode to prevent accidental current flow during set up:
  2. Connect a shorting wire or jumper to tie the red OUTPUT terminals of the HOLD channel and the EJECT channel together.
  3. Connect an identical jumper wire between the black OUTPUT terminals. This effectively connects the two current generating channels in parallel. Current does not flow from one channel into the other, but it will sum algebraically in the common electrode pathway, if both channels are operating simultaneously.
  4. Set the polarity and current amplitude on the HOLD channel to retain the active agent in the electrode. Set the polarity and amplitude of the current in the EJECT channel.
  5. Choose one of two operating methods:
  6. Leave the hold current on at all times and effect ejection by superimposing the larger eject current by operating the PRESET/ENABLE switch on the EJECT channel. The ejection current swamps the smaller hold current. Then, algebraically adds the hold current to the eject current to compute the actual ejection current. For example, if the hold current is 5nA subtracted from the eject current of 50nA, the effective ejection current is 45nA. This method has the advantage that you only need to operate a single PRESET/ENABLE switch on the EJECT channel during the experiment.
  7. Alternatively, simultaneously operate the PRESET/ENABLE switches on both channels so that the hold current is shut off at the moment the eject current is applied. In the example above, the eject current would then be +50nA. This could be achieved programmatically from Matlab, which would make it more convenient.

Stimulation paradigm#

  1. Stimulations to try:
  2. Square voltage pulses (500 ms, -0.1 to -15V)
Backward Effectors and perturbations Pressurised ATP delivery Forward