MICROELECTRODE METHOD OF THE RESEARCH — a method of assignment of the bioelectric potential generated by excitable body tissues of animals and the person by means of microelectrodes to-rye can be shipped in depth of fabric without its essential damage.
Distinguish three main ways microelectrode assignments: assignment from group of cells (focal extracellular assignment); assignment from a separate cell at an arrangement of a tip of a microelectrode near it (single extracellular assignment); assignment from a separate cell at an arrangement of a tip of a microelectrode in it (intracellular assignment). In all cases the source of electric potentials (in this case a cell) is located in the thickness of fabric, edges are represented by the volume conductor. Thereby the peculiar conditions of assignment which are absent at usual registration of electric reactions of a nerve or muscle superficial are created electrodes (see).
Apply two main types of microelectrodes — metal and glass. Metal microelectrodes represent the needle from solid metal covered throughout except for the tip, a layer of the isolating material. In some cases several microelectrodes fasten together, having their tips at various depth that creates an opportunity for simultaneous assignment of a set of bioelectric potential. Metal microelectrodes differ in low electric resistance. However because diameter of their tip cannot be made less than several microns, such electrodes use only for extracellular assignment. The first the amer began to use metal microelectrodes. the researcher R. Laurent de No (V. of Lorente de No, 1935), and in the USSR — I. S. Beritashvili and sotr., A. B. Kogan, etc.
The microglass electrode represents the micropipet from glass filled with electrolyte. Diameter of a tip of a pipette can be equal to the several tenth shares of micron. Therefore the microglass electrode can be entered in a separate cell without essential disturbance of its functions. A lack of microglass electrodes is the big fragility excluding a possibility of their prolonged use, especially in conditions hron, experience and also high electric resistivity (to several tens Megohms). Resistance is reduced by filling of microglass electrodes with the concentrated salt solutions (more often potassium chloride); however at the same time the probability of diffusion of electrolyte from an open tip in a cell increases that can lead to undesirable effects. Microglass electrodes were for the first time used an amer. the researcher Gerrard (V. W. Gerard) with sotr. (1946), and in the USSR — P. G. Kostiuk, etc. Production of multichannel microglass electrodes was widely adopted, to-rye are extended from several glass tubules which are previously fastened among themselves; at the same time the part of canals can be used not for registration of electric activity, and for leading to a cell or introductions in its these or those substances by them diffusions (see) or microionophoresis (see).
In all cases of microelectrode assignment a source of bioelectric potential is the superficial membrane of an excitable cell with the transmembrane potential difference existing on it created by uneven distribution of the main inorganic ions between protoplasm of a cell p the extracellular environment (see. Bioelectric phenomena , Bioelectric potential ). At extracellular assignment existence of potential difference can be revealed only in that case when its size in a certain site of a cell is changed under the influence of the factors changing ion permeability of a membrane. At the same time there are ring electric (ionic) currents between the based and activated site of a cell. As indifferent (i.e. relative) the electrode of the big area located on removal is used (e.g., on the surface of fabric); such electrode can be considered as the having constant zero potential. Respectively the microelectrode located in the field of an exit of currents from a cell (area of «source» of current) will register positive potential in relation to a relative electrode, and the microelectrode located in the field of their entrance (area of «drain» of current) — negative potential. The leading of a microelectrode is closer to a source of current, the potential difference more registered. The provision of a relative electrode provided that it is removed to the area where density of the passing currents practically is insignificant, does not affect results of assignment. Amplitude of the taken-away fluctuations will depend not only on quantity of the excited cells and size of the site captured by the corresponding electric change but also on distance from a source of biopotentials to a tip of a microelectrode.
At intracellular assignment the source of EMF (i.e. a superficial cell membrane) turns out between a micro and relative electrode what leads to assignment of constant potential difference in several tens of millivolts. Spasmodic emergence of such difference is the main criterion of penetration of a tip of a microelectrode in a cell. Emergence of active reaction in the taken-away cell is registered as change of constant potential difference towards its reduction or a perversion (the phenomenon of depolarization) or increases (hyperpolarization).
Focal microelectrode assignment finds application during the studying of spread of activation within separate brain structures. Especially successful it is in case of the correct orientation of the relevant neural structures (e.g., layered) as the electric fields created in this case by separate cells are summed up and respectively amplify. Therefore at focal assignment even the weak effects created by synoptic influences can be registered (see. Synapse ). Especially widely focal assignment is used at a research of bark of big cerebral hemispheres, allowing to separate to some extent the processes proceeding in dendrites of the pyramidal neurons (forming top coats of gray matter) from the processes proceeding in their bodies. In a trunk of a brain and in a spinal cord there is also rather correct orientation of motor-neurons and their axons therefore focal assignment was used for establishment of features of distribution of process of excitement in various parts of a cell (the myelinized and not myelinized parts of an axon, som and partly dendrites) here. The method of focal assignment was of great importance during the development of detailed maps of distribution of potentials on a certain section of a brain in various timepoints after receipt in a brain of the afferent alarm system. Such cards allow to draw a number of conclusions on distribution of processes of excitement within this area of a brain.
Single extracellular microelectrode assignment found application at assignment of bioelectric potential from muscles, nek-ry receptors (a retina of an eye) etc. In the first case usually study electric activity of group of the muscle fibers innervated by one motive fiber and forming functional neuromotor unit (see. Electromyography ). At assignment of biopotentials from a retina registration of activity of separate ganglionic elements by applying of a microelectrode to an inner surface of a retina is possible (without immersion in a cell). Extracellular assignment of activity of separate neurons of a brain is widely made from all its sites both in an experiment, and in a wedge, conditions now during neurosurgical operations. The main criterion of assignment of activity of a separate cell is at the same time registration of the category of impulses of constant amplitude. Especially successful such assignment is, naturally, in a case rhythmic - active cells that creates convenient conditions for comparison of amplitudes of consecutive categories. Extracellular assignment with success is used for a research of the mechanism of process of convergence of the exciting and braking synoptic influences, changes of activity of neurons in various fiziol, conditions, under action pharmakol, factors etc. Extracellular assignment of synaptic potentials of separate cells is much less effective, than assignment of pulse activity as the electric fields created by them are many times weaker than the fields created by action potentials. Besides, it is almost impossible to confirm authentically the fact of assignment of activity from one cell as synaptic potentials do not submit to the rule «everything or nothing» (see « Everything or nothing » the law) and their size is very variable in the same neuron. Extracellular assignment of potentials of separate neurons is still unique method of the thin analysis of activity of a nervous system. At special methods of immersion of microelectrodes perhaps rather long extracellular assignment on animals without anesthesia at the kept higher nervous activity and even in the conditions of free behavior.
Intracellular assignment is the main method of studying of internal mechanisms of functioning of excitable cells, including the ionic mechanisms of generation of action potential which are exciting and the braking postsynaptic potentials etc. By means of intracellular microelectrode assignment communication of postsynaptic braking with hyperpolarization of a postsynaptic membrane was for the first time shown. Intracellular assignment found broad application during the studying of activity of receptors, in particular during the studying of an origin and functional value of generator potentials.
Along with studying of electric reactions of excitable cells the microelectrode technics are applied also to local irritation and polarization of various structures, and also to leading to them or introductions in cells of physiologically active agents. At the same time for leading of physiologically active agents not only the method of a microionophoresis, but also a way of administration of solution by means of creation in a microelectrode of supertension is used (in several atmospheres).
The microelectrode equipment
Applied in M. by m and. the equipment includes different types of microelectrodes, devices for their production, the device for fixing, manipulations, assignments of the registered electric parameters etc. Very small sizes of electrodes allow to perform measurements at the minimum damage of fabrics and cells.
The microglass electrodes called still by liquid consist of a micropipet (diameter them depends on the purpose and object of studying) and the filling electrolyte. For successful use of microelectrodes properties of its narrow part have essential value, edges shall be rather flexible not to damage a cell, and rather strong to puncture fabrics and surfaces of cells. Also simplicity and ease of production, reliability of isolation, a possibility of use for intracellular injections are characteristic of microglass electrodes. Shortcomings them: small durability, a possibility of diffusion of ions from electrolyte of a microelectrode on Wednesday, existence of own potential of a tip (page of the item to.). For assignment of bystry capacities of the village of the item to. nesushchestven. At assignment of constant capacities of the village of the item to. an electrode it is summed up with the taken-away difference that can be a serious hindrance to a research since own potential of a tip can change upon transition of a microelectrode from one electrolytic solution in another.
Microglass electrodes make generally of hard glass a pyrex. There are manual also automatic ways of production of microelectrodes. At a manual way the site of preparation representing a glass tubule is warmed on a flame of a microburner to a softening and quickly stretched. This operation can be made also in several stages, More thin-walled capillaries turn out at bystry stretching. At strict selection only apprx. 5% of the micropipets prepared in such a way it is possible to recognize suitable for work. Use various automatic and semi-automatic devices to simplification of work and standardization of pipettes. The simplified device for an extract of micropipets consists of the holder of preparation, heating element and an element extending preparation with the determined, in advance chosen force; it is possible to watch process of production in a microscope. The schematic diagram of one of such devices is shown on rice, 1.
Ready micropipets fill with solution of electrolyte. There is a number of ways of filling of microelectrodes: boiling in electrolyte with the lowered atmospheric pressure; a pulling of micropipets from the glass tubes which are in advance filled with electrolyte; with use of a difference of pressure of vapors of two not adjoining liquids (i.e. electrolyte and water); by means of a glass needle; under the influence of supertension, etc. Many laboratories apply own ways of filling of microelectrodes.
Metal microelectrodes differ from glass in the bigger durability, durability, small resistance. It is necessary to carry a possibility of polarization to their shortcomings that involves emergence on a tip of unstable constant potential.
Apply the following metals to production of metal microelectrodes: nichrom, constantan, tungsten, platinum, antimony, lead, tin. The thinnest microelectrodes receive from tungsten. The piece of a thin metal wire is sharpened on the end on thin sandpaper or a grinding stone or an electrolytic way. Regulating depth and time of immersion of a tip, a tilt angle to a surface, try to obtain the necessary form of a microelectrode. At a thermal way of processing the tip grinds off the heated platinum thread. Ground, microelectrodes isolate various dielectrics: varnishes, enamels, plexiglas, etc. Sometimes produce metal microelectrodes in glass isolation.
Various holders apply to fixing and accession of electrodes to the entrance amplifier. Delivery of a microelectrode to the place of measurement and a manipulation to them carry out by means of various micromanipulators (see. Mikromanipulyator ). In fig. 2 the scheme of the simple micromanipulator for elektrofiziol, researches is submitted.
See also Micrurgy .
Bibliography: Kostiuk P. G. Microelectrode technics, Kiev, 1960, bibliogr.; Meshchersky R. M. Analysis of neural activity, M., 1972, bibliogr.; E to l with J. Fiziologiya of nervous cells, the lane with English, M., 1959; Hubbard J. I., L linas R. Q u as-tel D. M of J. Electrophysiological analysis of synaptic transmission, Baltimore, 1969.
P. G. Kostiuk; Yu. V. Agibalov, A. B. Tsypin (tekhn.).