Water for Electrophysiology Studies

Overview of Electrophysiology Techniques

Many animal and plant tissues have electrical properties, which can be measured with electrodes. Electrophysiology can be used to study sensory organs, muscles (in particular the heart), cells of the endocrine system, and the central nervous system. Recording of voltage change or electric current may be measured on anything from an ion channel protein to an entire organ. These methods are used extensively to study ion channels, which have become important targets for drug discovery. Ion channels play a critical role in nerve and muscle function. Drugs that modulate ion channels have been investigated in therapeutic areas such as neuropathic pain, cardiac arrhythmia, hypertension, local anesthesia, stroke, Parkinson’s disease, obesity, epilepsy, diabetes and depression.

Extracellular recordings can also be performed using techniques such as single unit recording, a method used to measure the activity of a single neuron in the brain of animals in vivo. Brain slices may also be used for the study of electrophysiology of neurons and local brain circuits. In this case, artificial cerebrospinal fluid (ACSF) must be used in order to keep them viable.

Classical electrophysiology techniques involve placing electrodes into cells or biological tissue. In optical electrophysiology, voltage sensitive dyes and fluorescent proteins are used, which allow the measurement of changes in potential without disrupting the tissue studied.

Intracellular Recording Techniques

Various techniques are being used in electrophysiology for intracellular recording, such as:

• Voltage clamp: The voltage clamp is used to measure the ion currents across a cell membrane while holding the membrane voltage at a constant level. The membrane voltage can be manipulated independently of the ionic currents, allowing the current-voltage relationships of membrane channels to be studied.
  
• Current clamp: The current clamp records the voltage difference across a cell membrane by injecting current into the cell through the recording electrode (usually a sharp micropipette).
  
• Patch-clamp: Unlike conventional intracellular recordings, which involve impaling a cell with a fine electrode, the patch clamp uses a relatively large microelectrode that is placed next to the cell. Gentle suction is applied on the small area of membrane ("patch") covered by the pipette. This technique allows the study of single or multiple ion channels in cells, and is often used to study the mechanism of action of drugs.

Many variations of these methods are being used. Certain technologies offer higher throughput than conventional techniques, such as planar patch clamp technology (instead of positioning the pipette on an adherent cell, a cell suspension is pipetted on a chip containing a microstructured aperture). In addition, the development of automated electrophysiology platforms has accelerated the capacity and speed that information-rich assays can be performed.

Impact of Water Quality on Electrophysiology Assays

When using electrophysiology techniques such as patch-clamp recording, all the solutions and equipment coming in contact with the cells must be sterile and of the highest purity available. Water is used for rinsing and to prepare the buffered solutions used to mimic the physiological conditions the cells were accustomed to (the cell culture media in the case of cultured cells). An extracellular solution (ECS) is used to bathe the cells, and an intracellular solution (ICS) is used to fill the pipettes. The ionic composition, pH and osmolarity of these solutions must be carefully controlled.

If brain slices are used, artificial cerebrospinal fluid (ACSF) or a similar solution is used to mimic in vivo conditions. This solution is used both to prepare the brain slice and to perfuse it during the experiment. It may also be used as the extracellular solution during patch clamp measurement. The ACSF solution must be freshly prepared and pH, osmolarity and ionic composition must also be carefully controlled. The water used to prepare the ACSF solution must be free of compounds that may affect the tissue or interfere with the experiment.¹

Ultrapure water may also be used to wash and rinse coverslips before their use to culture neuronal cells.²

The following water contaminants should be avoided:

• Ions: Since the osmotic pressure of all solutions used in electrophysiology assays must be carefully controlled, the water used to prepare them should contain the lowest amount of ions possible. In addition, many ion channels are sensitive to ions, especially cations, which should be controlled. Water with a resistivity of 18.2 MΩ.cm ensures very low concentration of ionic species in the water.

• Heavy metals: Heavy metals may also affect cell function. In addition, many ion channels are sensitive to heavy metals, in particular calcium channels. Therefore, the water used for ion channel studies must be free of heavy metals.

• Bacteria and endotoxins: Bacteria can release a variety of ions and organic compounds into the solution. Bacteria contamination can also cause sudden changes in pH. In addition, most Gram-negative bacteria release endotoxins, a complex lipopolysaccharide (LPS). Endotoxins, which have pyrogenic properties, may lead to changes in cell growth and function. The use of ultrafiltration ensures water is free of endotoxins and bacteria.

• Organic compounds: Small organic molecules are commonly present in raw water supplies (humic acids, tannins, pesticides, endocrine disrupters, etc.) and may remain in tap water, which is then purified to generate lab water. These organic molecules may affect cell function and experimental results.

Selecting the Optimal Lab Water Solution for Electrophysiology

Freshly purified ultrapure water is recommended for electrophysiology experiments. A resistivity of 18.2 MΩ.cm ensures very low concentration of all ionic species, and a low total oxidizable carbon (TOC) value (<5 ppb) assures that organic molecules are not present in water. Using an ultrafiltration cartridge (Biopak^® polisher) at the point of use of the water purification system eliminates the risk of bacteria and endotoxin contamination.

A range of solutions adapted to the needs of scientists performing electrophysiology studies are available.