Copyright © 1999 Massachusetts Institute of Technology
One of the most useful tools for the study of individual neurons in the nervous system is the microelectrode. A variety of specific techniques have evolved using this tool which enable investigators to determine how neurons act, how they communicate with each other, what kinds of NEUROTRANSMITTERS they use, what molecular mechanisms underlie their excitability, how they respond to sensory inputs, how they generate signals to activate muscles, and what the relationship is between their physiology and morphology.
In trying to understand the various uses to which microelectrodes have been put, the first distinction that needs to be made is between extracellular and intracellular recordings. In the case of extracellular recording, the tip of the microelectrode is not intended to enter the cell by penetrating its membrane; instead, electrical events are recorded in the immediate vicinity of each neuron. This method limits investigators mostly to the study of action potentials. A variety of electrode types have been developed for this kind of recording. These include fine-tipped glass pipettes filled with electrolytes and electrolytically sharpened metal wires coated with insulating material. Metal electrodes, especially those coated with a thin layer of molten glass except for the tip, are very strong and can penetrate without breaking even relatively tough tissue like dura. This makes it possible to record individual cells in alert animals, often for many hours, while they engage in various behavioral tasks. Using such methods, extensive sampling of neurons allows investigators to infer the functional characteristics of different brain structures. Such studies investigate how neurons respond to sensory inputs, how they discharge when various motor acts are executed, and even how higher-level mental events, such as ATTENTION, are represented at the neuronal level.
Intracellular recording methods enable investigators to study not only action potentials but also the graded potentials that reflect the excitatory and inhibitory inputs to the cell (excitatory and inhibitory postsynaptic potentials). Injection of current and pharmacological agents can provide information about the neurotransmitters and neuromodulators involved as well as the properties of the cell membrane at the molecular level.
Intracellular recording in the living organism is fraught with difficulties: the cells and axons are small and the movement artifact produced by heartbeat and breathing tends to dislodge the tip of the pipette from inside the cell. To combat this problem a variety of refinements have been made. Considerable improvement in recording stability can be realized in in vitro preparations: nerve tissue or individual cells are maintained outside of the body in an artificial medium, thereby eliminating movement due to heartbeat and breathing.
Three refinements in intracellular recording are noteworthy: Voltage clamping is a procedure that enables investigators to measure the flow of current across the membrane of single cells while the intracellular voltage is maintained at a constant value. This procedure has made a major contribution to our understanding of the mechanisms responsible for the electrical excitability of neurons. Patch clamping is also a technique for studying the flow of current through cell membranes. As in the case of voltage clamping, voltage across the membrane is typically controlled and the resultant current flow is measured. Mild suction is applied to the microelectrode when the tip comes in apposition with the cell membrane, which thereby creates a very tight and reliable connection. A variant of this technique, called whole-cell clamping, involves applying enough suction to rupture the cell membrane within the small opening of the pipette tip. Whole-cell clamping has the benefit of ease of fluid exchange. In general, these last two methods, compared with other intracellular recording approaches, can provide a more detailed description of the characteristics of membrane channel proteins; the experimenter has better control of the experimental situation and can study smaller cells than with previous methods.
To establish the relationship between function and morphology, investigators have carried out intracellular recordings using dye-filled glass pipettes; first, the response characteristics of the cell are studied, after which the dye is injected into the cell. Some dyes, such as Lucifer yellow, diffuse through the entire dendritic network of the cell so that subsequently, using a variety of anatomical procedures, the morphology of the cell can be disclosed in detail. Such studies have succeeded in establishing the functional characteristics of the major cell types identified in the mammalian RETINA, in the CEREBELLUM, and in several other neural structures.
Lastly, microelectrodes can also be used to eclectically stimulate various brain regions. When areas involved in the execution of motor acts are stimulated in alert animals with brief trains of 60- to 500-Hz pulses, the responses elicited provide important clues about the role these structures play in the control of eye, head, limb, and body movement.
Articles in Journal of Neuroscience Methods.
Smith, T. G., Jr., Ed. (1985). Voltage and Patch Clamping with Microelectrodes. Bethesda, MD: American Physiological Society.
Copyright © 1999 Massachusetts Institute of Technology