M

Microiontophoresis and Related Methods. Fig. 1. Examples of different types of multibarrel micropipette assemblies used in microiontophoretic experiments. (a) Standard 7-barrel assembly in most common use, introduced first by Curtis. (b) Twin, or parallel micropipette. (c) Co-axial assembly. (d) Staggered tip multibarrel. (From Hicks, 1984.)

layer'' within the barrel tip. When an aqueous solution is in contact with glass, negative ions are tightly adsorbed on the glass surface, leaving the bulk of solution carrying a net positive charge. The passage of positive (or outward) current then causes the ejection of a small volume of solution containing the compound of interest (Fig. 2). It should be noted, however, that this mechanism has nothing to do with the osmotic pressure of a solution or the establishment of any osmotic gradient. The term electro-osmosis derives simply from the fact that the driving force is the movement of the solvent, not the solute, just as the case of osmotic movements across a semipermeable membrane. An alternative method of applying both ionized and nonionized compounds from micropipettes is the use of pressure. A suitable source of pressure, usually a cylinder of compressed gas, is connected to the open end of a micropipette barrel. Pressure usually up to 20 pounds per square inch (p.s.i.) will eject fluid from a 1 mm pipette tip. One advantage of micropressure ejection is that it can be applicable to all compounds; however, it is not devoid of problems and artifacts and is unlikely to replace micro-iontophoresis as a microapplication method.

Principles and Role in Psychopharmacology

General Principles

Each barrel of a micropipette assembly to be used for drug ejection is filled with a solution of the ionized compound and the solution is connected to the iontophoresis machine by a suitable lead, which is in contact with the drug solutions. The establishment of a potential difference

Microiontophoresis and Related Methods. Fig. 2. Schematic diagram of a micropipette that contains a salt X+Y , showing the direction of current necessary to eject (a) and retain (b) the ion X+. (From Hicks, 1984.)

between the drug solution and the medium surrounding the barrel tip will then cause the movement of ions through the solution and out of the pipette tip (Fig. 2). A chief advantage of the microiontophoretic method is that it is possible to examine the effects of drugs on single neurons in vivo without affecting the whole nervous system or other physiological responses, such as those that may occur when drugs are administered systemically (Aghajanian 1972). If a voltage is applied to a solution, ions and charged molecules will migrate toward and away from the source of the imposed electrical field depending on the sign of their net charge. This phenomenon is the fundamental principle of microiontophoresis: the desired charged particles are ejected from the mouth of one barrel of a multipipette assembly by appropriately charging the interior of that barrel (Fig. 2). An outward current will cause the "ejection" of positively charged ions, and an inward current flow, the ejection of negatively charged particles. If the pipette assembly is positioned close to a neuron, so that the recordings of its activity can be made through another electrolyte-filled barrel, drugs may be ejected and their pharmacological effects are inferred by the resulting changes in the rate and/or ► firing pattern.

The Transport Number and the T50 Value

An important technical consideration for experiments employing microiontophoresis is the transport number. The transport number is a measure of the amount of drug released from the micropipette by iontophoretic expulsion and it is important, because it helps one to evaluate dose-response relations between different compounds, and it can also provide some indication of the absolute potency of compounds. The transport number varies for individual compounds and is based on the interaction of the following variables: their solubility, the extent of their dissociation in solution, their polarity, and the nature of the external medium into which the drugs are administered. The transport number may be formally described by the following equation:

n = RjZFi1, where n = apparent transport number of the drug ion,

F = Faraday's constant, in Coulombs i = intensity of ► ejecting current, in nanoamperes, and

R = rate of microiontophoretic release (which is equivalent to total release minus the sum of the rate of steady-state spontaneous release and where applicable, the release due to electro-osmosis).

During microiontophoresis, the total number of ions transported is related in a direct manner to the amount of current applied to the solution, according to Faraday's law. However, only a certain proportion of the charge imposed is carried by the ion species of interest. This value, which is "n" the transport number, is not constant for a given material but will vary not only from pipette to pipette, but also, to a lesser extent, between different barrels of the same micropipette assembly containing identical solutions. Despite these inconsistencies, it remains valid that under steady-state conditions, drug release from micropipettes conforms to Faraday's law: the amount of drug released is proportional to the magnitude of current passed (Hicks 1984).

Another important parameter to consider when interpreting microiontophoretic data is the T50 value, which is the time taken for a response to reach 50% of its maximum (Fig. 3). The basis for this procedure is the hypothesis that each individual response to an agonist may be considered as a cumulative dose-response relationship

Microiontophoresis and Related Methods. Fig. 3. Time courses of inhibition of neuronal firing following microiontophoretic application of GABA with four different currents, 20 nA (filled circle), 10 nA (o), 5 nA (x), and 2 nA (open triangle). Each curve was obtained from the same neuron at a depth of 957 mm in the middle suprasylvian gyrus of the cat cortex. The neuron was driven by continuous microiontophoretic application of L-glutamate (20 nA). Each of the points for 20, 10, and 5 nA applications of GABA is the mean ± SEM of three values obtained from three separate applications of the same current of GABA. The values of T50 shown are the times taken to achieve 50% inhibition of neuronal firing. (From Hill and Simmonds (1973) Br J Pharmacol 48:1-11.)

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