Micro-Iontophoretic Studies on Cortical Neurons

MICRO-IONTOPHORETIC STUDIES ON CORTICAL NEURONS By K . Krnjevic A.R.C. Institute of Animal Physiology. Babroham. Cambridge. England

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I Introduction . . . . . . . . . . . . . I1. Technical Considerations . . . . . . . . . A . Preparation of Micropipettes . . . . . . . B . Quantitative Aspects of Micro-iontophoresis . . . C . Current Artifacts . . . . . . . . . . I11. Actions of Amino Acids . . . . . . . . . . A . Excitatory Amino Acids . . . . . . . . . B . Inhibitory Amino Acids . . . . . . . . . IV Actions of Various Amines . . . . . . . . . A. Phenethylamine Derivatives . . . . . . . . B . Tryptamine Derivatives . . . . . . . . . C . Derivatives of Lysergic Acid . . . . . . . D. Some Other Amines . . . . . . . . . V . Effects Produced by Acetylcholine and Related Substances A . Pharmacology of Cortical Cholinoceptive Neurons . B . Distribution of Cortical Cholinoceptive Neurons . . C . Characteristic Features of Cholinoceptive Cells . . D . Cholinergic Innervation of Ccrebral Cortex . . . VI . Conclusions and Summary . . . . . . . . . References . . . . . . . . . . . . .

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I. Introduction

Nerve cells in vertebrate animals are hardly ever situated conveniently in isolation . They are found mostly in the central nervous system where large numbers of them are packed within a relatively small volume of tissue . To study their properties therefore requires the use of some special technique. such as recording with microelectrodes capable of distinguishing the electrical responses of a single cell. either from inside the cell or from its immediate vicinity ( Amassian. 1961) . As it is generalIy believed that excitation or inhibition of a 41

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neuron usually depends upon the synaptic release of a transmitter substance (Eccles, 1961; McLennan, 1963) it is important for the analysis of neuronal activity and synaptic transmission to analyze the action of various chemical agents on central neurons. The effects of various substances can be observed after administering them to the tissue either directly or through its blood supply. But this is often rather unsatisfactory, because too many cells are affected at the same time and the responses of the cell under observation may be greatly modified by changes in the activity of other neurons from which it receives excitatory or inhibitory signals. Furthermore, many substances do not readily penetrate nervous tissue from the blood stream. A more direct method is to release minute amounts of substances by iontophoresis from a micropipette whose tip lies very close to a cell. This technique was developed by Nastuk (1953) and del Castillo and Katz (1955) for the application of acetylcholine to single muscle fibers. In their experiments, glass micropipettes were filled with a concentrated solution of acetylcholine ( ACh) chloride. When the tip of the pipette was brought close to the surface of a muscle, positive ions of ACh were released in a controllable manner from the tip by causing an electrical current to flow through the pipette. If the pipette has a very fine tip ( <1 p in diameter) and its opening is extremely close to a sensitive region of the membrane, one can use very short pulses of current and then the effect produced becomes comparable with what is seen during the normal transmission of an impulse (del Castillo and Katz, 1955; Krnjevib and Miledi, 1958). Micropipettes suitable for iontophoresis may have two or more barrels, with closely adjacent openings at the common tip, each barrel being filled with a different agent; studies can then be made of the interactions between various synergists or antagonists on a minute scale, which justifies the expression micropharmacology ( del Castillo and Katz, 1957). Iontophoresis was first used systematically for investigations on neurons in the central nervous system by Curtis and R. M. Eccles (1958) in their analysis of the cholinergic synapses on Renshaw cells in the spinal cord. With this technique, Curtis and his collaborators have made an extensive survey of the chemical sensitivity of spinal neurons (viz., Curtis et al., 1959, 1960a, 1961; Curtis and

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Watkins, 1960) and more recently they have examined neurons in the brain stem (Curtis and Koizumi, 1961; Curtis and Davis, 1962, 1963). The present article is principally concerned with observations made in the author's laboratory on cortical neurons in mammals. 11. Technical Considerations

A. PREPARATION OF MICROPIPETTES 1. With a Single Opening Micropipettes for iontophoresis are similar to those used in single unit or intracellular recording. They are prepared by pulling out hard glass tubing in a flame or by means of a suitable device, as described for instance by Fatt (1961). The tip diameter of a single pipette should not exceed about 1-2 ,u, otherwise it may be impossible to prevent a large spontaneous output by diffusion and bulk flow. Outward diffusion, which is approximately proportional to the tip diameter, can be neutralized by a current flow in the opposite direction, by applying an appropriate %raking voltage (del Castillo and Katz, 1955). Bulk flow, on the other hand, increases so rapidly with larger tips that even a hydrostatic pressure of only a few centimeters of water may cause the outfiow to become embarrassingly large ( Krnjevii: et al., 1963a). Release by bulk flow can be an alternative to iontophoresis, using the same solution and a similar micropipette, or even different barrels of the same pipette, to eliminate artifacts arising from current flow. This may also be suitable for dealing with solutions of substances that do not ionize or which are very impure. 2. With Multiple Openings

If one starts with two or more glass tubes fused together side by side, it is not difficult to pull out multibarrelled micropipettes, such as those described by del Castillo and Katz (1957) and Curtis and R. M. Eccles (1958). With five-barrelled pipettes it is usually preferable to have an outside diameter of 6-10 p, although some authors (e.g., Spehlmann, 1963) believe that the disadvantages of a very fine tip are outweighed by finer discrimination of single cells and a more IocaIized appIication of drugs.

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3. With Concentric Openings

If one wishes to record the changes in membrane potential caused by an external iontophoretic application of a substance, this can be done by inserting a fine pipette inside a coarser one. The protruding tip of the inner pipette can then enter a cell, while the outer one remains outside; if the concentric tubes have been carefully selected and fit properly together, there is little or no leakage of fluid from the outer barrel, but substances can be applied to the outer surface of the cell by iontophoresis in the usual manner while recording the membrane potential through the inner barrel. This method was pioneered by Curtis et al. (1959) in their studies of spinal motoneurons. B.

QUANTITATIVE

ASPECTSOF MICRO-IONTOPHORESIS

1. Effective Transport Number During applications lasting more than a few milliseconds the over-all movement of ions into or out of the tip must bear a simple relation to the total current flow through the pipette, 1 gm-equiv. of ions being transported for every 96,500 coulombs. Hence if one measures the rate of current flow on a galvanometer, one can estimate the rate of displacement of ions. However, in an electrolyte, the total current is not shared equally between the various ions. The fraction carried by any one ion, i.e., the transport number of that ion species, depends upon its mobility, as well as certain other factors such as the total ionic concentration. In the tip of a micropipette, the transport number might be affected by the close proximity of fixed negative charges in the glass wall or by charged particles obstructing the lumen. Direct estimations have been made of the amounts of substances actually released from micropipettes by currents comparable with those used in iontophoretic studies on neurons (Krnjevib et al., 1963a). For a number of micropipettes filled with ACh chloride, the mean transport number of ACh+was 0.42 ( n = 65, SE 2 0.042), which is only a little greater than might be expected from previous measurements of the conductivity of solutions of AChCI. This means that if a potential difference is applied between the pipette

IONTOPHORETIC STUDLES ON CORTICAL NEURONS

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and the preparation, making the AChCl solution inside the lumen relatively positive, on the average 42%of the resulting current flow is carried by ACh ions released from the tip, One must point out, however, that there was a good deal of variation between individual pipettes, and the behavior of some departed considerably from the average.

Electrical Charge (yC)

FIG. 1. Relation between quantity of adrenaline released from tip and electrical charge passed through three similar micropipettes containing identical solution of adrenaline acid tartrate (Krnjevii. et al., 1963b).

Similar experiments with pipettes filled with adrenaline, noradrenaline and 5-hydroxytryptamine ( 5HT) indicated in most cases a simple proportional relation between the current flow and the rate of release (Fig. l ) ,but the transport numbers of the same substance varied appreciably in different pipettes, as shown by the unequal slopes in Fig. 1. In the experiments with noradrenaline some pipettes allowed the passage of substantial currents without

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releasing any corresponding amounts of drug. This probably happens when the tip is obstructed by a charged particle of dust, which acts as a semipermeable membrane, allowing the passage of only smaller ions such as C1- or H+. It is clear from these observations that one cannot safely draw any general conclusions from data obtained using only one pipette.

2. Electroosmotic Transport When there is a potential gradient between the two ends of a glass tube containing an aqueous solution the latter may tend to flow along the tube, at a rate determined among other factors, by the electrokinetic or zeta potential between the glass wall and the fluid. Although some electroosmotic flow probably occurs from micropipettes, as some previous observations have suggested (Taylor, 1953; Rubio and Zubieta, 1961), it cannot be of much importance since the observed transport numbers were not much above the expected values, perhaps because zeta potentials become very small in the presence of a high concentration of electrolytes. In certain cases, however, electroosmosis probably does become significant. For instance, a neutral amino acid such as y-aminobutyric acid (GABA) is least ionized at its isoelectric point near pH 7. Hence solutions of GABA are usually acidified to pH 4 where it is largely dissociated, as a cation, and therefore in a suitable form for iontophoresis (Curtis and Watkins, 1960). Yet neutral GABA can be released from pipettes, presumably by electroosmosis owing to a marked zeta potential (Krnjevih and Phillis, 1963a). This zeta potential extends much further on the alkaline than on the acid side of pH 7, and its presence may explain the greater difficulty of demonstrating a release of GABA from alkaline solutions by an inward current than its release from an acid solution by an outward current ( Curtis and Watkins, 1960). Although electroosmosis may thus cause some interference with iontophoresis, under certain conditions it may be a useful way of applying substances which are poorly ionized in solution. 3. Concentration of Substances in Tissue

One can readily give an approximate estimate of the amount of substance probably released from a micropipette by iontophoresis, but it is much more diflicult to predict the concentration attained

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in the region of a particular nerve cell. If the tissue is imagined to be an infinite medium, permitting free diffusion in all directions, the concentration in the tissue is theoretically described by a set of error function complement curves, whose shape changes continually with time after the beginning of the application (cf. Carslaw and Jaeger, 1959; del Castillo and Katz, 1955; Curtis et al., 1960b). From the curves illustrated by Curtis et al. (1960b) one can see that there is a very rapid fall in the concentration, which probably becomes relatively negligible beyond some 40 p from the tip, i.e., at distances where neuronal responses are not likely to be detected by a micropipette. Within the useful range, the final Concentration produced by an iontophoretic current of about 0.1 PA would probably be greater than 0.1 mM. Because of the uncertainty about the real distance between an observed cell and the microelectrode tip, it is more profitable to estimate the tissue concentration obtained after very short pulses of iontophoretic current. One can then assume that the release from a point source has been instantaneous and the corresponding theoretical concentration has a peak value which can be calculated from the latency without having to make any assumptions about the probable distance (cf. del Castillo and Katz, 1955). In this way, one can show that the concentration of a substance such as L-glutamate is probably in the order of 0.1 mM when it excites cerebral cortical neurons (Krnjevib and Phillis, 1963a). C. CURRENT ARTIFACTS Cerebral neurons can be excited or depressed by polarization in an electrical field (Burns and Salmoiraghi, 1960; von Euler and Green, 1960; Strumwasser and Rosenthal, 1960). Since iontophoresis requires that an appreciable current Bows into the tissue from the tip of the micropipette, some nonspecific, purely electrotonic effects can be expected to occur. Their magnitude and direction depends largely upon the proximity of the electrode to the neuron, and, therefore, on the size of the tip, because relatively large tips cannot be brought so close to the surface of a cell. With relatively large tips (5-10 p ) outward currents tend to have the expected anodal depressant effect, and the reverse is seen with inward currents (cf. Burns and Salmoiraghi, 1960; Curtis and Koizumi, 1961; Krnjevib and Phillis, 1963a).

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With fine tips (1 p or less) the largest responses are probably obtained when the tip is pushed right up against the cell membrane; in this position the recorded potentials are mainly positive, presumably because the membrane is inactive at the point of contact, and the electrode is effectively connected to the inside of the cell. Under these conditions outward currents can have no action at the point of entry into the cell, but they depolarize the membrane as they flow out and thus tend to produce excitation (Strumwasser and Rosenthal, 1960; Spehlmann, 1963) . Although current effects can usually be distinguished by their instantaneous onset and termination, followed by appreciable rebound effects in the opposite direction (Krnjevib and Phillis, 1963a), if there is any doubt with respect to a particular drug action it is advisable to perform controls by passing similar currents through neutral barrels containing NaCl or some other ions presumed inactive. One method which very much limits interference by current flow is to neutralize the current passed through a given barrel with a similar but opposite current through an indifferent barrel, thus keeping the over-all flow of current through the pipette constant (Salmoiraghi and Steiner, 1963). The release of the same solution by bulk flow under pressure is another useful control which has already been mentioned. Ill. Actions of Amino Acids

The first descriptions of marked excitatory and inhibitory actions of amino acids on central neurons were made by Hayashi (1954,

1956), but they received very little attention until the later studies of Purpura et al. (1959). The effects produced by amino acids on spinal neurons have been studied extensively by Curtis and his collaborators (Curtis et al., 1959, 1960a; Curtis and Watkins, 1960, 1963). The actions on cortical neurons have been the subject of a micro-iontophoretic investigation ( Krnjevib and Phillis, 1963a) which provides the main material for the present description.

A. EXCITATORY AMINO ACIDS 1. L-Glutamate The most interesting amino acid in this group is L-glutamate, because of its strong action and because it is present in high con-

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centration in the brain [about 10 pmoles/gm according to Berl and Waelsch (1958), Tower (1960), and Singh and Malhotra (1962)l. Several features of its action are particularly impressive. It usually causes a very rapid excitation of a cell, which is maintained during a prolonged release, but ceases almost instantaneously whenever the application stops (Fig. 2). As shown in the figure, one can usually control the rate of evoked firing by varying the

FIG.2. Excitation by L-glutamate of a unit in cerebellar cortex of cat. Extracellular spikes were recorded by the central saline-containing barrel of the multibarrelled micropipette, and L-glutamate was applied iontophoretically by inward currents (making tip relatively negative) through another barrel filled with strong solution of sodium L-glutamate. Duration of iontophoretic release is indicated by horizontal white lines below traces. Effects produced by 3 different rates of release are shown in A to C; D is a control showing minimal excitant action of much larger negative current carrying out of another barrel C1 ions instead of L-glutamate (Krnjcvib and Phillis, 1963a).

strength of the iontophoretic current of L-glutamate; if the rate of release is excessive, the cell fires maximally, then its responses fail, no doubt because the membrane has become overdepolarized, and it is now in a state of cathodal block. However, this is quickly reversible, and there is no evidence that the cell suffers any injury as a result, nor does it appear to be desensitized by a prolonged application. One can easily demonstrate the excitatory action of ce glut am ate reIeased from a micropipette by pressure, and so the effect is clearly not dependent in any way upon current flow. For example, in

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Fig. 3 two different units are shown (above and below, respectively) both of which were excited by liberating L-glutamate by iontophoresis or by pressure from different barrels of the same micropipette. This powerful and quick action is seen especially well when applying short pulses of L-glutamate. Figure 4 shows the responses of two cortical neurons evoked by a very short application of L-glutamate. The large initial deflections at the left of each trace

FIG.3. Comparing the action of L-glutamate applied to a cortical unit by iontophoresis from one barrel and by pressure injection from another barrel of same micropipette, both barrels being filled with nearly saturated solutions of sodium L-glutamate. Times of application are indicated by horizontal white lines underneath traces, which show different units above and below.

is the electrical artifact produced by a 2-msec pulse of inward current. After a latency of some 2 0 3 0 msec, the neurons gaveea sharp discharge, the full duration of one of which is visible in Fig. 4B. Such sharp effects are only seen when the tip of the micropipette is very close to the neuron in question. Under these conditions, the amount of L-glutamate required for excitation is probably not more than mole. The short latency of the response suggests that the action of L-glutamate is instantaneous, since the time is approximately what would be needed for diffusion between the tip of the micropipette and the cell (as calculated according to Section II,B,3 above).

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When a micropipette is inserted into the cortex, large numbers

of units can be excited with L-glutamate. Not all of them respond

in the same way: some give a steady, easily graded discharge while others react in short bursts, in a much more “all-or-none” manner.

FIG. 4. Cortical units excited by transient applications of L-glutamate. Rupetitive discharge of first unit is shown on fast and slow time base in A and B; and the actual iontophoretic pulse used, in C. D is similar discharge of another unit, while E shows that an identical pulse of current through an indifferent barrel had no effect ( Krnjevik, 1963 ).

The required amounts of L-glutamate also vary considerably, but all the cells which can be identified extracellularly clearly respond to the application. This leads one to believe that all neurons are probably being excited. This is not the case. If a coaxial micropipette is used, so that even totally quiescent cells can be identified by the internal resting potential, one finds some cells which respond to L-glutamate by a partial depolarization (Fig. 5 ) but do not fire impulses, even when large amounts of g glut am ate are released. Some of these cells have comparatively large and stable resting potentials and, therefore, resemble similar potentials seen by other authors (Phillips, 1956; Li, 1959), who have ascribed them to glial cells. This

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suggestion is given support by the fact that glial cells in tissue cultures have relatively high and stable resting potentials (Hild and Tasaki, 1962). An alternative explanation may be that some very large nerve cells are not easily depolarized to the point of firing by the localized application of L-glutamate: although such large cortical neurons as the Betz cells can be excited with L-glutamate, they often have a relatively high threshold, and certain other cells, such as spinal A

11

GLUT SOOnA

J-

B

11 6GLUT 40nA

3OSEC I

FIG. 5. L-Glutamate action on a cortical cell's membrane potential recorded with inner barrel of coaxial micropipette. First deflection indicates resting potential as tip enters the cell. Between two arrows, L-glutamate was released iontophoretically from opening of outer barrel which remained outside cell. ( A ) First application and ( B ) effect of second application recorded with higher gain (K. Krnjevik and J. W. Phillis, unpublished observations, 1962).

motoneurons in cats (Curtis et al., 1960a) and Mauthner cells in fish ( Diamond, 1963), are only depolarized subliminally. It has been reported that most nerve cells in the olfactory bulb fail to show any effects of glutamate (von Baumgarten et al., 1963). Therefore one cannot ignore the possibility of substantial variations between neurons in their sensitivity to this amino acid. 2. S:gnificance of Glutamate Action a. Mechanism. The important part played by glutamate in cerebral metabolic exchanges is so well-known that it needs no emphasis ( Weil-Malherbe, 1936; Braunstein, 1947; Stern et at., 1949; Tower, 1960). As a result all actions of applied glutamate

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have in the past tended to be ascribed to change in cellular metabolism (Elliott, 1960; Hillman and McIlwain, 1961). But Curtis and Watkins (1960) have shown that glutamate and other excitatory amino acids probably cause a large increase in the membrane permeability of spinal neurons, by an interaction between the amino acid and a membrane “receptor” with relatively well-defined characteristics. Moreover, an intracellular release of glutamate produces little or no change in membrane potential (Coombs et al., 1955; Araki et al., 1961; Takeuchi and Takeuchi, 1963). The remarkably quick effects observed on cortical neurons (cf. Fig. 3) suggest that a similar surface mechanism works in the cortex. It is we11 known that cortical slices readily absorb glutamate from surrounding fluid (Stern et al., 1949; Takagaki et al., 1959; Tsukada et QZ., 1963). This seems a remarkably useful, nonenzymatic mechanism for the removal of glutamate from the extracellular spaces, which would prevent an excessively prolonged depolarization. The same molecule may be able to act as both excitatory receptor and carrier for the uptake if its positive charge is so placed in the surface membrane as to prevent normally the influx of cations such as Na’. This charge would be neutralized during the uptake of glutamate, Na’ would enter the cell freely and thus depolarize the membrane. Although L-glutamate has no effect on nerve trunks (Robbins, 1959) there is some evidence that afferent fibers near their central termination may be depolarized by its action (Schmidt, 1963); but it is unlikely that such easily graded effects as those shown in Fig. 2 could be elicited by a purely presynaptic excitation. The chelating properties of glutamate cannot be of much importance for its excitatory effects because of the marked difference in potency between the L- and D-enantiomorphs (see Section III,A,3 below); much stronger chelators such as ethylenediaminetetraacetic acid are by comparison only weak excitants. b. Possible Function a r Transmitter. The presence of large amounts of L-glutamate in the brain and the remarkably quick and reversible excitatory action of even minute quantities inevitably raise the question whether L-glutamate normally acts as a transmitter of activity at cortical synapses. There is plenty of evidence that glutam am ate excites crustacean muscle ( Robbins, 1959; Van Harreveld and Mendelsohn, 1959) and there is a close parallel

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between its action there and that of ACh on vertebrate muscle (Takeuchi and Takeuchi, 1963). However, its release from nerve endings remains to be demonstrated, in crustacean muscle and in the mammalian brain. Some preliminary experiments by J. F. Mitchell (personal communication, 1962) failed to show any definite liberation from the cat's cortex, possibly because the mechanism for the removal of glutamate from the surroundings of the cells is too efficient to permit an appreciable accumulation in the tissue. This problem may not be solved until an agent is discovered which is as efficient in stopping this process as is eserine in preventing the hydrolysis of ACh. Curtis et al. (1960a) have argued that glutamate is unlikely to function as a transmitter in the spinal cord, because its excitatory action could not be modified by various substances known to interfere with glutamate metabolism. However, if glutamate is removed from its site of action at the surface by absorption into the cell rather than by immediate metabolic destruction, enzymatic inhibitors would not have much effect. c. Spreading Depression. Van Harreveld (1959) has shown that spreading depression is readily elicited in the cerebral cortex of rabbits by a topical application of glutamate. In view of the large amount of glutamate normally present in the brain, he suggested that some of it may leak out of depolarized neurons, thus contributing to the spread of this phenomenon, whose advancing wave is associated with a transient phase of excitation (Grafstein, 1956). The powerful blocking effect produced by an excessive application of L-glutamate (cf. Fig. 2C) lends some support to Van Harrevelds hypothesis, but here again one must emphasize that no direct evidence has yet been obtained that glutamate is actually released during spreading depression. 3. Other Excitatory Amino Acids

a. Amino Acids Related to L-Glutamate. Cortical cells, like crustacean muscle ( Robbins, 1959), are much less readily excited by D-glutamate than L-glutamate (Fig. 6). The difference between the activity of the two enantiomorphs is greater than in the spinal cord, where they appear to be almost equipotent (Curtis and Watkins, 1960). On the other hand, D-glutamate is more effective

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in producing spreading depression when applied to the surface of the cortex (Van Harreveld, 1959); perhaps because it is only metabolized rather slowly by the brain ( Weil-Malherbe, 1936; Stern et al., 1949), though it is said to be taken up by brain slices as quickly as L-glutamate (Takagaki et al., 1959).

FIG. 6. Comparing actions of L- and D-isomers of glutamate and aspartate on same cortical unit; all were released iontophoretically by identical current [60 nanoamps( nA)] from near-saturated solutions at pH 8.5 (Kmjevii: and Phillis, 1963a).

Both L- and D-aspartate are quite effective excitants of cortical neurons, their action being very simiIar to that of L-glutamate, though consistently weaker as can be seen in Fig. 6. An interesting effect is produced by n-methybaspartate; that is, strong excitation, but with a somewhat slower onset and longer persistence than is usually observed with L-glutamate. Moreover there is some interference with the action of glutamate which may be less effective than normal for some 20 seconds after the end of an application of n-methybaspartate. As suggested by Curtis and Watkins (1960) it seems that the reaction between this substance and the postulated receptor is only comparatively slowly reversible. In general, dicarboxylic amino acids having a molecule not very different in length from that of glutamate tend to excite cortical neurons presumably because they all fit in some degree the same

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receptor molecule in the surface membrane. Curtis and Watkins ( 1960), who have made a detailed analysis of the relation between the structure and the excitatory actions on spinal neurons of a large number of amino acids have shown that one of the carboxyl group can be replaced with another acidic group, especially a sulfonic radical. The sulfonated derivatives are particularly strong excitants of cortical, as well as of spinal neurons. This is clearly visible in Fig. 7: cysteic acid was at least as potent as L-glutamate, whereas homocysteic acid had such a strong depolarizing effect that the fast discharge stopped almost immediately, no doubt owing to a cathodal block.

FIG.7. Sulfonated derivatives exciting a cortical unit even more effectively than L-glutamate. Homocysteic acid has an especially powerful action ( Krnjevii: and Phillis, 1963a).

b. Other Amino Acids. Monocarboxylic w-amino acids tend to depress neuronal activity, as will be shown below (Section 111,B); however, as Hayashi (1956) has pointed out, these amino acids also have some excitatory properties, which are more pronounced the longer the chain of carbon atoms in the molecule. Thus a-aminocaproic and o-aminocaprylic acids produce rather mixed effects when tested directly on cortical neurons (cf. Fig. 13). Neither L- nor n-glutamine have any excitatory action comparable with that of L-glutamate, but L-asparagine often causes some quite substantial excitation (Fig. 13).

IONTOPHORETIC

STUDIES ON

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XEURONS

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B. INHIBITORY AMINO ACIDS 1. 7-AminobutyricAcid (GARA)

The depressant action of GABA on cortical neurons is as remarkable as the excitation caused by L-glutamate. An example is given in Fig. 8; the two cortical neurons illustrated here were firing spontaneously at a depth of 1.1 mm in the posterior sigmoid gyrus of a cat anesthetized with Dial; the two units being distinguishable by the different sizes of their respective spikes. A steady iontophoretic release of GABA from one barrel of the recording micropipette (horizontal signal) caused a disappearance of the larger spikes after a delay of about 2 seconds, and they did not reappear until some 2 seconds after the end of the application. The smaller spikes were apparently not affected, probably because this cell was too far from the micropipette’s tip. It will be noted that GABA blocked the discharge of the first unit without a gradual change in the amplitude of its spikes (see also Figs. 9, 10 and 18). In this

FIG. 8. Cortical unit found at depth of 1.1 mm in posterior sigrnoid gyms of cat under Dial; the spontaneous discharge was blocked by iontophoretic application of CABA. This small relcase of CABA had no effect on another more distant unit, whose spikes are also visible (K. Krnjevib and J. W. Phillis, unpublished observations, 1962).

respect its action is quite different from that of certain other depressant agents such as procaine and atropine to be described below (Section IV,D). The most characteristic features of the action of GABA are thus its great swiftness and potency, and quick reversibility. GABA can block most types of activity in the cortex including spontaneous

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discharges, responses elicited by stimulating various nervous pathways, and firing initiated by a local application of exciting substances such as glutamate and ACli. Thus in Fig. 9A, a neuron was made to fire by a steady release, between two arrows, of L-glutamate from one barrel of a micropipette, but the discharge was stopped rapidly by a short additional iontophoretic application

FIG.9. Blocking by GABA of discharge of a cortical unit induced by iontophoretic release of L-glutamate ( between arrows). White lines below traces signal applications of GABA by iontophoresis ( A ) , and by pressure injection ( B ) , from other barrels of same micropipette.

of GABA from another barrel (during signal below trace). Under suitable conditions, when the tip of a micropipette is very close to a cell, short pulses of GABA may be sufficient to cause a temporary depression corresponding to the quick effects produced with short pulses of glutamate (cf. Fig. 4).For instance, the steady discharge illustrated in Fig. 10 was interrupted by a regular series of short pulses of GABA. The lower half of Fig. 9 shows that one does not have to apply GABA by iontophoresis to demonstrate its blocking action. The same cell was again excited by an application of L-glutamate (as in Fig. 9A), but GABA was expelled by pressure from a third barrel of the 5-barrelled micropipette, during the three periods indicated by horizontal white lines; this barrel had been filled with a strong (approximateIy molar) soIution of GABA at a pH of about 7. For iontophoresis, GABA is usually prepared in acid solutions, in which it tends to have a positive charge: some authors have, therefore, suggested that strong depressant actions may be peculiar to ionized

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GABA (Kuno, 1961; Muneoka, 1961). However, it is evident from Fig. 9 that there is no qualitative difIerence between the effects produced by GABA in its two forms. Although GABA can maintain a prolonged state of block of cortical neurons, in some cases its effectiveness appears to diminish with time, as found previously with some other preparations (Kuffler and Edwards, 1953; Curtis et al., 1959).

FIG. 10. Excitation of a cortical unit by iontophoretic release of L-@amate (between two arrows), temporarily blocked by 9 transient iontophoretic applications of GABA; the 20-msec pulses of outward current are visible as vertical artifacts (Krnjevib and Phillis, 19G3a).

Changes in the membrane of cortical cells produced by GABA have been studied in some unpublished experiments by Krnjevii: and Phillis. These were of some interest for an eventual comparison with synaptic inhibitory potentials. The membrane potentials were recorded by the inner barrel of a coaxial micropipette, which was filled with a strong solution of K citrate to avoid any possible interference by leakage of C1 ions from the tip such as occurs in spinal motoneurons (Coombs et al., 1955) and in crustacean muscle (Boistel and Fatt, 1953). GABA was applied by iontophoresis from the outer barrel whose opening remained outside the impaled cell. Some observations made by this technique are shown in Fig. 11. A Betz cell was impaled, and its resting membrane potential recorded continuously on a paper recorder ( E ) . The cell was excited antidromically by stimulating the pyramidal tract at the ventral surface of the medulla, initially giving a spike such as that shown in trace A. When a large amount of GABA was applied, the excitability of the cell became so depressed that only partial antidromic invasion was possible ( B ) . After the end of the release of GABA, the excitability rapidly returned toward the normal, allow-

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ing full antidromic invasion ( C and D ) . Although record E might at first sight suggest that GABA caused a substantial depolarization, a similar release of GABA outside the cell ( F ) shows that most of the observed deflection was an artifact due to electrical

JJ----lT*J IMSEC

E

CABA

I

1

360nA

30SEC

FIG. 11. Action of GABA, released from outer barrel of coaxial micropipette, on membrane potentials of Betz cell recorded intracellularly with inner barrel. ( A-D ) Spike evoked antidromically by stimulating medullary pyramid, before ( A ) , during ( B ) , and after (C, D ) application of GABA; note partial block of antidromic invasion. ( E ) A continuous trace of resting potential recorded during above sequence. ( F ) Trace recorded outside cell to determine amount of electrical coupling between inner and outer barrels; it shows that most of the potential change seen in E during application of GABA was a coupling artifact (Krnjevib, 19L3).

coupling between the inner and the outer barrels of the coaxiaI pipette. Neither in this nor in other similar experiments was there any evidence that GABA produces large changes in resting potential. 2. Significance of GABA

Like glutamate, GABA occurs in relatively large amounts in the brain, where it probably plays a substantial role in general metabolism (Roberts and Eidelberg, 1960; McKhann et al., 1960). There is good reason to believe that most of the depressant actions of Factor I (the inhibitory factor extracted from the mammalian brain) can be ascribed to GABA (Levin et al., 1961). On the other hand, all cortical neurons appear to be depressed by GABA when tested individually by iontophoresis. This is evident what-

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ever the method of testing the excitability, whether by indirect synaptic or direct electrical stimulation, or by excitation with L-glutamate. I t is, therefore, clear that GABA does not merely block cortical excitatory synapses, as suggested by Purpura et al. ( 1959), and the possibility must therefore be considered that GABA is an inhibitory transmitter. What evidence have we from other species or from other regions of the nervous system? It is quite likely that GABA is an inhibitory transmitter in crustacea. Its action on crustacean neurons and muscle fibers is very similar to that of inhibitory volleys (Kuffler and Edwards, 1958; Boistel and Fatt, 1958; Furshpan and Potter, 1959; Hagiwara et al., 1960), and large amounts of it are found in crustacean inhibitory nerve fibers but not in excitatory fibers (Kravitz et al., 1963). These facts are strongly suggestive, though not conclusive without evidence that GABA is actually released by inhibitory volleys. GABA has been tested rather thoroughly on mammalian spinal neurons by Curtis et al. (1959). Although there was a strong depressant action, which in most respects appears to be similar to that seen in the cortex, these authors concluded that GABA was not the spinal inhibitory transmitter for two main reasons. First, GABA applied iontophoretically to motoneurons reduced all synaptic potentials whether excitatory or inhibitory. However, it is by no means clear that the membrane shunting effect of the transmitter would necessarily be any different, nor is it really certain that GABA may not depress the activity of the spinal presynaptic endings, as it evidently does in crustacea and mammalian muscle (Dudel, 1962; Hofmann et al., 1962). The second objection was based on the lack of a clear hyperpolarizing effect of GABA on spinal motoneurons which might correspond to the hyperpolarizing inhibitory synaptic potentials. But this discrepancy may have been due to an excessive application of GABA. Kuffler and Edwards (1958) noticed that, whereas GABA in relatively low concentrations gave a good imitation of the effects of neural inhibition of crayfish receptor neurons, at a concentration 10-15 times greater the hyperpolarizing tendency disappeared and the neuron was actually depolarized, though still remaining depressed. When applying GABA by iontophoresis from a coaxial micropipette, it may be difficult to avoid releasing

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an excessive amount; the observations made by this technique on spinal and cortical neurons are, therefore, not wholly conclusive. For these various reasons it seems premature to dismiss the possibility that GABA may function as an inhibitory transmitter in the mammalian central nervous system, especially as there is no other likely candidate for this role.

3. Other Inhibitory Amino Acids a. Derioatiues of GABA. Several other monocarboxylic w-amino acids with a comparable chain of carbon atoms are approximately as potent as GABA itself, the activity diminishing more or less regularly with changes in the length of the molecule. This agrees

FIG. 12. Excitation of cortical unit by iontophoretic release of L-glutamate (between arrows), suppressed by additional iontophoretic release of p-alanine and y-guanidinopropionic acid indicated by white lines (K. Kmjevii. and J. W. Phillis, unpublished observations, 1962).

in general with the structure-activity relationship observed by Robbins (1959) and Curtis and Watkins (1960); the latter, in particular, stressed the fact that removal of the a-carboxyl group from excitatory amino acids tends to produce compounds with a corresponding inhibitory activity. In several respects cortical neurons differ somewhat from spinal

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neurons. They are blocked especially by 8-aminovaleric acid, as well as GABA and 7-aminohydroxybutyric acid (“Gabob),whereas p-alanine, though quite effective (Fig. 12) is distinctly less potent. The peak of efficiency, therefore, occurs with a slightly greater separation between the positive and negative charges on the molecule. Cortical cells are strongly depressed by guanidinoacetic and guanidinopropionic acids (Fig. 12), but only relatively slightly by taurine. In general, the spectrum of activity is very similar to that of various amino acids which depress crayfish stretch receptors ( Edwards and Kuffler, 1959). yAminobutyrylcholine has no obvious inhibitory action (cf. Takahashi et al., 1958).

FIG. 13. ( A , B ) Some actions of long chain a-amino acids on cortical neurons. (-4) Unit in cerebellar cortex of cat excited by L-glutamate (between arrows) ; firing was stopped by two iontophoretic applications of w-aminocaprylic acid. ( B ) Two units in rat’s cerveau kolk strongly excited by c-aminocaproic acid; first unit fired almost immediately, second one only after 12 seconds. ( C ) Excitation of another cerebellar unit by L-asparagine released as a cation (Krnjevib and Phillis, 1963a).

b. Longer-chain Monocat boxylic co-Amino Acids. As already mentioned above some of these compounds have both weak excitatory and inhibitory properties (cf. Hayashi, 1956). Some inhibitory effects obtained with w-aminocaprylic acid are illustrated in Fig. 13.

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IV. Actions of Various Amines

The effects produced on cortical neurons by many amines of biological interest have been described in some detail in two articles by Krnjevi6 and Phillis (1963a,d), the second of these giving a systematic survey of derivatives of phenethylamine and of indole compounds. The results obtained will therefore not be enumerated here; only a general survey of the principal compounds and types of action being presented. One can say at the outset that the predominant effect observed was neuronal depression, which was either a relatively quick and reversible reduction in firing frequency (not unlike that produced by GABA), or a progressive diminution of the spike amplitude, with little change in firing frequency before the onset of complete block; the second effect was only slowly reversible. Large applications of several of the depressant compounds sometimes also caused a rather sudden and sharp excitation, while a few substances had a predominantly excitant action.

A. PHENETHYLAMINE DERIVATIVES This group shows rather clearly the two main types of depressant actions. Catecholamines such as dopamine, adrenaline, isoprenaline, and noradrenaline (in descending order of effectiveness) slow the neuronal discharge with relatively little change in spike height. For instance, in Fig. 14 a neuron was excited by regular 5-second applications of L-glutamate, signalled by the white lines below traces. The resulting discharge was very much depressed by an additional steady release of dopamine or adrenaline (between the two arrows ), but this did not cause any progressive reduction in spike height and the effect disappeared within a few seconds after the end of the release of each catecholamine. Spontaneous activity and responses to indirect stimulation could also be blocked (as shown in Fig. 15). Although these compounds act somewhat like GABA, they are never as effective and their potency is much more variable. These results therefore confirm at the cellular level previous observations by Marrazzi of a depression of cortical potentials by adrenaline (Marrazzi, 1953, 1961; Marrazzi and Hart, 1957) and they give some support to the suggestion that dopamine may have

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FIG. 14. Comparison of depressant action of dopamine and of adrenaline on excitation of a cortical unit by L-glutamate (30 nanoamps) applied at times indicated by white lines. Catceholamincs were released from other barrels of same pipette during periods between arrows ( KrnjeviC: and PhilIis, 1963d).

FIG.15. Unit responses, evoked in soinatosensory cortex of cat by peripheral stimulation, blocked by iontophoretic release of dopamine, LSD, and 5HT. Traces were recorded before and during each application, and also after recovery (Kmjevi6 and Phillis, 1963d).

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an inhibitory function in the central nervous system ( McLennan, 1961). Unlike dopamine, dopa only caused some moderate degree of excitation. Some other related compounds, likely to be of interest, such as tyramine and mescaline, did not reveal anything more than a weak depressant effect when tested by this method. Another subgroup of potent phenethylamine compounds consisted of various forms of ephedrine and close derivatives. Their action was quite different from that of the catecholamines; there was a gradual depression of spike height, with relatively little slowing of the rate of discharge before the neurons ceased firing, followed by a slow recovery (Figs. 16 and 1SC). Amphetamine

FIG. 16. ( A ) Spontaneous activity of cortical unit depressed by iontophoretic release of ( - )-ephedrine; note gradual change in spike amplitude, and slow recovery. ( B ) Control record during release of Na' by large iontophoretic current from another barrel ( Krnjevii: and Phillis, 1963d).

was among the compounds having this kind of action, but it was much weaker than ephedrine, and it also differed in tending to excite when applied in large amounts. In this respect it resembled adrenaline.

B. TRYPTAMINE DERIVATIVES 5-Hydroxytryptamine ( 5HT) was quite an effective blocking agent, whether tested on activity initiated by glutamate (Fig. 17) or by indirect stimulation (Fig. 15C). It is evident from Fig. 17 that the effect produced was somewhat like that of GABA, that is, a quick and rapidly reversible change in the frequency of firing. The

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figure shows that this effect cannot be ascribed to the creatinine sulfate with which commercial preparations of 5HT are usually associated, as a similar effect was produced by releasing 5HT from a solution of 5HT bimaleinate. Both compounds also produced the same kind of strong but delayed discharge when they were released more rapidly (Fig. 17C and D ) . The action of SHT is probably more potent than might appear at first sight, as it is released ioiitophoretically in relatively small quantities from solutions of SHT creatinine sulfate ( KrnjeviC: et al., 1963b), presumably because a substantial fraction of current is carried by creatinine (cf. Curtis and Davis, 1962). The blocking effects of 5HT seen in these experiments confirm some previous observations, such as those of Marrazzi (1961) on the transcallosal response.

FIG. 17. Cortical unit showing Iioth depression and excitation by 5HT. ( A ) Unit was excited throughout by continuous application of L-glutamate; discharge was blocked by 5HT relcasecl from n solution of 5HT bimaleinate in one barrel (first signal) or a solution of 5HT creatinine sulfatc in another barrel of micropipette (second signal). ( B ) Control outward currents of Na’ had no such effect, the initial reductions in spike size being artifacts; at arrow, glutamate release was stopped. In C and D, there was delayed excitation by larger iontophoretic currents of 5HT; further applications of L-glutamate in D indicate a rapid recovery of excitability (Krnjevii: and Phillis, 1963a).

Several closely related compounds, including tryptamine itself had rather similar properties, though the excitatory actions were not always so evident. S-Hydroxytryptophan, on the other hand, was very much less effective as a depressant, nor did it cause much excitation. Two related psychotomimetics, psilocin and bufotenin produced only rather weak depression. It appears that 5HT may have substantially different actions in

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various parts of the central nervous system. Curtis (1962) could not detect any effects on spinal neurons, while in the lateral geniculate it caused a more discrete type of block, affecting only synaptic transmission (Curtis and Davis, 1962); this observation led to the suggestion that 5HT may interfere specifically with the release or the action of the synaptic excitatory transmitter, perhaps because of some structural resemblance between the two compounds. The rather general actions seen in the cortex probably involve a much less specific mechanism. C. DERIVATIVES OF LYSERGIC ACID The predominant effects were depressant (Fig. 15B), causing a large reduction in spike size, somewhat akin to that observed with ephedrine. But larger doses tended to excite, as in the case of 5HT. The potency of the strongest compound, ergometrine, exceeded substantially that of D-lysergic acid diethylamide ( LSD ). On the other hand, methyl ergometrine had such marked excitatory properties that cells were blocked by excessive depolarization.

D. SOMEOTHERAMINES 1. lmiduzole Derivatives Neither histamine nor histidine had more than a weak depressant action on cortical neurons, but another related compound, imidazolylacetic acid proved very potent indeed. When it was compared directly with GABA, by testing on the same neuron as in Fig. 18A and B, it appeared to be almost as effective, with a similar type of quick and easily reversible action. The third trace in Fig. 18 shows, for contrast, the slowly reversible type of block produced by ephedrine, and the accompanying reduction in the size of the spike. Imidazolylacetic acid was probably the strongest depressant of the various amines tested on cells in the cortex, judging by the amount of iontophoretic current needed for a gil-en effect. This criterion, of course, is not infallible; as already pointed out, different compounds may have substantially different transport numbers, so that the actual amounts of substance released in the tissues by a given iontophoretic current could differ by a factor of 2 (KrnjeviL et al., 1963b). However, unless there is a complete failure of

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FIG. 18. Comparing blocking actions of GABA ( A ) , imidazolylacetic acid ( B ) , and "( - )-ephedrine" ( C ), applied (between arrows) on a cortical unit excited by L-glutamate ( 80 nanoamps ) released during periods indicated by

white lines. Note prolonged action of ephedrine ( Krnjevii. and Phillis, 1963d ) .

release, the iontophoretic current should give a reasonable indication of approximate relative potencies. 2. Procaine and Atropine Procaine is particularly interesting because its characteristic properties, causing a relatively gradual and prolonged neuronal depression, with marked changes in spike amplitude, throw some light on the mechanism of this kind of block which has already been encountered with several other compounds. A further example is shown in Fig. 19, which compares the actions of procaine and of atropine; the latter depresses a little more slowly than procaine, but its effect persists even longer after the end of an application (Fig. 19B). This action of atropine (also seen with hyoscine) does not bear any relation to cholinergic mechanisms in the cortex. It is similar to the nonspecific l h c k observed in the spinal cord by Curtis and Phillis (1960) and is probably due to the local anesthetic properties of atropine. Since some other substances that produce the same kind of block of corticaI cells, including ephedrine and LSD, are also known to act as local anesthetics (Schultz, 1940;

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Di Carlo, 1961), it seems likely that all compounds with similar properties will tend to depress central neurons in this manner. The mechanism by which local anesthetics block conduction interferes particularly with the initiation of spikes (Shanes, 1958). It clearly differs from the effects produced by GABA and other depressant @-aminoacids, which probably tend to maintain the membrane

FIG. 19. Depressant actions of procaine and atropine (released between two arrows) on a cortical unit. Excitability was tested repeatedly by identical short applications of L-glutamate (Krnjevib and Phillis, 1963a).

potential near its resting level by a specific change in ionic permeability (Boistel and Fatt, 1958; Hagiwara et al., 1960). The positive and negative charges at each end of the molecule of the @-amino acids probably interact with corresponding charged groups on a “two-point” receptor component of the cell membrane (cf. Curtis and Watkins, 1960). The comparable depressant activity of substances such as imidazolylacetic acid, 5HT, and dopamine may be due to their ability to react with the same two-point receptor. V. Effects Produced by Acetylcholine a n d Related Substances

When large numbers of mammalian cortical neurons were tested with ACh released iontophoretically from micropipettes, it was observed that a certain proportion of them could be excited in a characteristic manner ( Krnjevib and Phillis, 1961, 1963b). The effect was relatively slow and prolonged, being quite different from the sharp excitation seen when ACh is applied in the same way to Renshaw ceIls in the spinal cord [the latter were the only central neurons hitherto known to be readily stimulated by ACh (Eccles et al., 1956; Curtis and Eccles, 1958)l. The contrast between the

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two effects is illustrated in Fig. 20 where B is the response of a Renshaw cell and C that of a cortical Betz cell to iontophoretic applications of ACh during the periods indicated by white lines below the traces; the discharge of the cortical cell went on for

FIG. 20. Comparison of ACh action on Reiishaw cell ( B ) in spinal cord and Betz cell in cerebral cortex ( C ) of cats. Renshaw cell was identified by characteristic high-frequcncy response to antidromic volley in 7th lumbar ventral root ( A ) . In both cases ACh was applied iontophoretically from a micropipette during period indicated by horizontal white line.

another 20 secs after the end of the record. Before considering the distribution and significance of these cortical cholinoceptive neurons, a description will be given of their pharmacological characteristics ( KrnjeviL and Phillis, 1 9 6 3 ~ ) . A. PHARMACOLOGY OF CORTICAL CHOLINOCEPTIVE NEWROXS 1. Cholinomimetic Excitation These cells differ from Renshaw cells as much in their pharmacological properties as in their way of responding to ACh. Renshaw cells are in several ways similar to skeletal muscle fibers; this is not surprising, of course, since they are innervated by other S receptors are of a branches of the same motor axons. T ~ Utheir strongly nicotinic type, being activated particularly well by nicotine itself, but only relatively poorly by muscarinic agents such as acetylP-methylcholine and arecoline (Eccles et al., 1956; Curtis and Eccles, 1958). Cortical cells by contrast are particularly well

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excited by strong muscarinic agents, such as muscarine, muscarone, and acetyl-p-methylcholine ( Fig. 21). Several other cholinesters are also quite effective, usually in proportion to their muscarinic potency. For instance, propionyl choline causes a significant exci-

FIG.21. Excitation of cortical units by ACh and by acetyl-p-methylcholine ( Mecholyl), released iontophoretically from same micropipette ( K. Krnjevib

and J. W. Phillis, unpublished observations 1963).

tation, whereas butyryl choline is largely ineffective (Fig. 22). Some other compounds with muscarinic properties, such as pilocarpine, arecoline, and oxotremorine (Cho et al., 1962) also excited cortical cholinoceptive cells though their action is usually rather

FIG.22. Unlike ACh, butyryl choline causes little or no excitation of cortical cholinoceptive cells, even when applied for much longer time, as in above example (Krnjevi6 and Phillis, 1 9 6 3 ~ ) .

slow. Nicotinic agents typically caused little or no excitation; for example, nicotine, tetramethylammonium and dimethyl phenyl piperazinium (Chen et aL, 1951) were tested on many cortical cholinoceptive neurons without showing any specific effects. Nicotine itself tends to behave somewhat like 5HT; moderate amounts

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slightly depress activity but prolonged applications by large iontophoretic currents may lead to a sudden paroxysmal discharge, which continues for some time after the end of the release. These effects are quite unlike those produced by ACh and they occur independently of any sensitivity to ACh. Although carbamyl choline was first reported to be less effective than ACh (Krnjevik and Phillis, 1963c), it was found in later experiments to have a substantially longer action. 2. Antagonists of ACh The muscarinic nature of cortical cliolinoceptive receptors is confirmed by their susceptibility to block by substances such as atropine, hyoscine, and benactyzine. Both atropine and hyoscine can also produce an unspecific depression of all types of cells by virtue of their local anesthetic properties. This action, which has already been described (Section IV,D), is relatively quickly reversible, seldom lasting more than a minute even after a large application. When atropine is applied to cholinoceptive cells, there is an additional, specific effect, that is a very prolonged block of responsiveness to ACh. An example of this is shown in Fig. 23; it can be seen that, whereas the iontophoretic application of atropine had no prolonged effect on discharges caused by L-glutamate, there was no full response to ACh for about 45 minutes. A comparable block can also be produced by injecting atropine intravenously in doses of about 1mg/kg (Fig. 24). Apart from the three substances mentioned aboi-e, the only other antagonist tested which was sometimes clearly effecti1.e was gallamine; its action is relatively quick and it does not persist like that of atropine or hyoscine. Although gallamine blocks peripheral nicotinic synapses, it does have an atropine-like action on the heart (Riker and Wescoe, 1951; Laity and Garg, 1962). The neuronal depressant effect described here may be peculiar to cortical neurons; in some other parts of the brain, such as the thalamus (Curtis and Davis, 1963) and the medulla (Salmoiraghi and Steiner, 1963), gallamine has been found to produce excitation only. Other antagonists of the nicotinic actions of ACh were mostly very ineffective. Those tested included tubocurarine, dimethyl tubocurarine, dihydro-P-erythroidine, toxiferin, and hexamethonium.

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FIG. 23. Prolonged effect of atropine on response of a cortical unit to thalamic stimulation and to ACh applied iontophoretically. Control records show characteristic repetitive discharge after single shocks to ventroposterolateral thalamic nucleus ( A ) and the excitatory effect of ACh applied for 38 seconds ( B ) . The next pair of records (C, D ) were taken 4 minutes after the end of a %minute iontophoretic application of atropine (80 nA); there was a reduction in responses to thalamic stimulation, and ACh produced no obvious excitation, though L-glutamate released from another barrel of micropipette had its usual affect. Full responses had not returned at 17 minutes (E, F), even though ACh was applied for 54 seconds; there was no real recovery until about 45 minutes after the end of release of atropine, when sensitivity to ACh and better repetitive discharges reappeared at same time (G, H ) . In B, D, F, and H initial horizontal white line shows last part of ACh release (K. Krnjevib and J. W. Phillis, unpublished observations, 1962).

Mecamylamine, another strong ganglionic blocking agent, which depresses Renshaw cells selectively like dihydro-p-erythroidine (Ueki et al., 1961), had a general depressant effect on all cortical neurons tested, but it did not interfere specifically with the response to ACh. Tubocurarine, as well as gallamine and dihydro-p-erythroidine, sometimes tended to excite cells; this effect was also not related to ACh sensitivity. It confirms previous observations of excitatory changes produced by curare in the cerebral cortex (Funderburk and Case, 1951; Chang, 1953; Rech and Domino, 1960; Morlock and Ward, 1961).

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FIG.24. Block by intravenous atropine of responses of cortical cholinoceptive unit to acetyl-~-mcthylcholine (Mecholyl). Upper traces ( A ) were recorded before, and lower ( B ) 15 minutes after giving to cat injection of atropine sulfate ( 1 mg/kg). Note much longer application of acetyl-p-methylcholine in B. There was no olwious change in the excitation caused by L-glutamate or in early responses to periplte~tl stimulation ( Krnjevik ant1 Phillis, 1 9 6 3 ~ ) .

Neither diphenylhydantoin nor imipramine interfered with the action of ACh in the cortex. 3. Potentiation by Anticholinestclmses

The relatively prolonged excitation produced by ACh suggests that ACh is not extremely rapidly removed from its site of action, either because it remains firmly attached to the receptor, out of reach of tissue cholinesterase, or because the latter is not very plentiful in the cortex. The first of these possibilities is probably the more correct, since the cortex shows a substantial level of cholinesterase activity in vitro ( Burgen and Chipman, 1951; Pope, 1952), in histochemical preparations ( Koelle, 1950; Krnjevii: and Silver, 1963a,b), and in situ (Mitchell, 1963). Inhibition of this cholinesterase is presumably responsible for the moderate, but clear potentiation of the effects of ACh by the three anticholinesterase agents which have been tested iontophoretically-eserine, Prostigmine (see also Spehlmann, 1963), and Tensilon. All three coinpounds tend to facilitate the “spontaneous” activity of cholinoceptive cells, and these respond more forcibly and for a longer period to a given application of ACh. This kind of effect is shown in Fig. 25, where a release of Tensilon was started shortly before the beginning of trace B, causing the observed potentiation. Both

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FIG. 25. Tensilon, an anticholinesterase agent, potentiates ACli firing of cortical unit in rabbit. ( A ) Control record; ( B , C ) continuous trace beginning 12 seconds after starting iontophoretic release of Tensilon (80 nA); this ceased at arrow. Iontophoretic release of ACh is indicated by horizontal white lines (Krnjevib and Phillis, 1 9 6 3 ~ ) .

Prostigmine and Tensilon could excite cholinoceptive cells if relatively large amounts were applied.

4. Actions of Other Substances Cholinoceptive neurons do not appear to differ significantly from other cortical neurons with respect to the excitatory and inhibitory actions of various amino acids and amines already described in Sections I11 and IV. They are all excited by L-glutamate, though perhaps somewhat less effectively than many other neurons; and GABA blocks their activity, whether initiated by ACh or by L-glutamate. Substances such as adrenaline and 5HT have their usual depressant effects; only atropine and hyoscine have an obvious selective action on cholinoceptive neurons.

5. Some Comments There have been many previous suggestions that cortical neurons are excited when ACh is injected into the carotid blood stream or applied directly on to the exposed surface of the brain (e.g., Bonnet

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and Bremer, 1937; Miller et al., 1940; Bornstein, 1946; Infantellina, 1955; Monnier and Romanowski, 1962). These effects were reproduced by other muscarinic agents (Riehl and Unna, 1960; Herz, 1962) and prevented by atropine (e.g., Miller ct ul., 1940; Chatfield and Dempsey, 1942; Bornstein, 1946; Arduiiii and Machne, 1949; Funderburk and Case, 1951; Infantellina, 1955). The direct demonstration by micro-iontophoresis that ACh can excite some cortical cells with rather clear muscarinic-type receptors is thus in broad agreement with much of the previous evidence. Apart from some observations of excitatory effects of ACh on neurons in the visual cortex (Spehlmann, 1963), most of the other iontophoretic studies on the brain have dealt with other regions. The most complete are those of Curtis and Andersen (1962) concerning thalamic cells; these usually show some excitation by ACh, comparable in time course with that seen in the cortex, but apparently mostly much weaker 5ince ACh does not readily cause a discharge. Judging by the actions of various cholinesters and antagonistic agents, the ACh receptors of thalamic cells do not appear to be of a clearly drfined muscarinic or nicotinic type. Cholinoceptive neurons in the medulla, on the other hand, behave with regard to the actions of antagonists much more like the spinal Renshaw cells since the responses to ACh are blocked more readily by dihydro-P-erythroidine and hexamethonium than by atropine ( Salmoiraghi and Steiner, 1963, Bradley and Wolstencroft, 1963). It has been suggested that there are two cholinergic systems in the cortex-one inhibiting and one enhancing cortical activity (Chatfield and Purpura, 1954; Chatfield and Lord, 1955). This idea arose mainly from the observation of a dual action of atropine, first facilitating and then depressing evoked potentials. However, if there is a substantial amount of cholinergic activity going on spontaneously (see below), evoked potentials would be reduced by occlusion and any agent which tends to diminish the background discharges, such as atropine, will make them appear larger. But stronger doses of atropine or a more prolonged application would produce a general nonspecific depression of all activity. This seems the most probable explanation for the findings of Chatfield and his co-workers, as it is very difficult to show any convincing inhibitory action of ACh on cortical cells. Such an effect apparently occurs when neurons are tested in the brain stem (Sal-

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moiraghi and Steiner, 1963; Bradley and Wolstencroft, 1962), in the olfactory bulb (17on Baumgarten et al., 1963), and in the hypothalamus (Bloom et al., 1963) although Curtis found no significant depression of neurons in the midbrain and the thalamus (Curtis and Koizumi, 1961; Curtis and Davis, 1963). In the cortex, weak depressant effects produced by iontophoresis cannot be easily distinguished from anodal current artifacts which are probably responsible for most of the apparent depressions caused by an iontophoretic application of ACh. Nevertheless it seems that a small number of cortical cells may be inhibited by ACh (Spehlmann, 1963; Randid et al., 1964), but there is not enough evidence at present to decide whether this is a specific action of ACh, perhaps related to the inhibitory system postulated by Chatfield and Purpura (1954) or an indirect effect caused by the excitation of adjacent, inhibitory interneurons. The marked neuronal inhibition produced by cortical stimulation is not sensitive to atropine ( Krnjevid et al., 1964).

B. DISTRIBUTION OF CORTICAL CHOLINOCEPTIVE NEURONS Most of the material given here has already been presented in greater detail elsewhere ( Kmjevid and Phillis, 1963b).

1. Distribution in Diferent Areas of Neocortex of Cat Initial experiments suggested that cholinoceptive cells are more likely to occur in or near primary afferent regions, particularly in the visual and the primary somatosensory areas (Krnjevid and Phillis, 1961). But as similar cells were later found in most areas they are certainly not sharply restricted in their regional localization. If one considers the vast number of units in the cortex, and the minute proportion which can be tested in any one experiment, remembering also such complicating factors as individual variations between animals and different degrees of interference by anesthesia, it will not be surprising that no definite statement can yet be made about their distribution. Within any one area, the sensitive cells seem to occur in small groups, several being often found together; a horizontal displacement of only 1mm may be sufficient to leave a zone containing such a group.

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2. Distribution in Depth One of the clearest characteristics of the cholinoceptive cells is that they occur very rarely within about 0.8 mm of the surface; they are especially concentrated in a relatively narrow zone between 0.8 and about 1.3 mm, corresponding approximately to the fourth and fifth cortical layers (Li and Jasper, 1953), but many are also seen deeper. The characteristic depth distribution is also evident when cholinoceptive cells are examined in a definite population. For instance the Betz cells in the pericruciate region, which can be identified by antidromic activation from the pyramidal tract and most of which are excited by ACh ( a s described below), have a very similar distribution in depth when compared with cholinoceptive cells in general. On the other hand, units excited by transcallosal volleys are found at all depths throughout the cortex, but those which are cholinoceptive (Fig. 26) are concentrated in a narrow band at a depth of about 1.0 mm, like other cholinoceptive cells.

FIG.26. Cortical unit 0.9 mm deep in suprasylvian gyrus of cat, discharging spontaneously ( A ) , and in response to transcallosal volleys from symmetrical area of opposite hemisphere ( B ) . It was strongly excited by ACh (Krnjevi6 and Phillis, 1963b).

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C. CHARACTERISTIC FEATURES OF CHOLINOCEFTIVE CELLS 1. What Kind of Cells are Excited by ACh? The responses evoked by ACh are probably neuronal in origin. They are unlikely to come from glial cells, because there is evidence that glial cells do not give electrical discharges (Hild and Tasaki, 1962). Many of the units studied could also be excited by the usual methods of eliciting unit activity in the cortex, that is by electrical stimulation of peripheral structures or of various parts of the brain. It has always been assumed that the unit discharges seen under these conditions when recording with microelectrodes indicate neuronal activity (Renshaw et al., 1940; Adrian, 1941; Amassian, 1961) . Moreover, many responses came from Betz cells, that is the cells from which originate the axons of the pyramidal tracts; they can be identified with a high degree of certainty using the standard criteria followed by several previous authors (Woolsey and Chang, 1948; Landau, 1956; Phillips, 1956, 1959; Patton and Amassian, 1960). A good example of Betz cells excited by ACh is given in Fig. 27. They were identified by their short-latency antidromic activation from the bulbar pyramid (A, B) and they gave a typical prolonged discharge in response to an iontophoretic application of ACh ( J ) . Like many other cholinoceptive cells (see below) they could be activated by stimulating some other neural pathways. To account for the relatively slow action of ACh it might be argued that ACh only acts on some glial cells which then stimulate adjacent neurons. But this idea is based on so many unknowns, that it is surely preferable to accept the no more improbable, but simpler hypothesis that ACh has a relatively slow depolarizing effect on the neurons themselves; this would be in keeping with the generally slow and prolonged character of muscarinic actions of ACh ( Dale, 1938) . There is also good reason to believe that the cholinoceptive responses originate from the cell body and not from nerve fibers. Unit responses can be obtained from single fibers, but the recording conditions are relatively unstable and units cannot be studied for long periods, unlike typical cholinoceptive responses. The latter are not seen in fiber tracts in subcortical white matter. Moreover, the

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depolarizing effect of ACh on peripheral nerve fibers (which is usually not associated with a discharge of impulses) is a typical nicotinic action ( Armett and Ritchie, 1961).

FIG. 27. Two Betz cells behaving like typical cholinoceptive units. They were found at depth of 1.1 mm in lateral precruciate cortex of cat, and were identified as Betz cells by short-latency antidromic activation from medullary pyramids ( A ) , responding up to frequency of dOO/second ( B ) . They were spontaneously active ( C ) ; gave early responses to stimulation of contralateral forepaw ( D ) and afferent relay nucleus in thalamus ( E ) , and also repetitive afterdischarges ( F , G ) . Both early and late responses were also elicited transcallosally (H, I ) . Units were excitcd vigorously by ACh applied from micropipette ( J ) (Krnjevib and Phillis, 1963b).

It is, therefore, most likely that ACh excites the perikarya of cholinoceptive neurons. The relath-ely deep region in which responses are obtained might suggest that the apical dendrites are not affected since they often extend most of the way to the surface (Cajal, 1911). But if the dendrites are incapable of producing

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spikes, excitatory effects may not be readily detectable by extracellular recording. It is conceivabIe that the soma and initial segment where spikes are initiated (cf. Coombs et al., 1957) are not depolarized directly, being only affected by electrotonic spread from relatively distant areas on the basal or apical dendrites where cholinergic synapses are perhaps situated. This might be an alternative explanation for the delayed action of ACh; it is supported by electronmicroscopic and histochemical studies of synaptic fragments, which have detected a cholinesterase reaction in some axodendritic but not in axosomatic, cerebral synapses (de Lorenzo, 1961; Torack and Barrnett, 1962). However, there is some discrepancy between these observations and the microscopic distribution of cholinesterase staining in the cortex, which seems to indicate a cholinergic innervation for cell bodies rather than dendrites (Krnjevib and Silver, 1963a,b). One can conclude by saying that the cells excited by ACh are probably nerve cells, and, in view of the high sensitivity of Betz cells, whose distribution in depth was similar to that of cholinoceptive units in general, it is likely that these neurons are mostly deep pyramidal cells. 2. Spontaneous Discharges The cholinoceptive units are usually spontaneously active. Their discharge makes a characteristic, irregular, shuffling sound, which is quite unlike the regular rhythm of a spindle or burst, though the latter is not infrequently superimposed. Its general character agrees with the description of the so-called projection type of spontaneous activity given by Dempsey and Morison ( 1943). Although Dempsey and Morison, who recorded the activity from the surface, believed that it was mainly associated with primary sensory areas, cellular discharges of this type can be found in most regions, nearly always at the characteristic depth, and in relation to cholinoceptive neurons. By contrast spindles or bursts may occur at any depth, but especially in the more superficial layers, and they do not seem to be particularly closely associated with the sensitivity to ACh. Although most cholinoceptive units tend to fire spontaneously, this is not an invariable feature: some of them are initially com-

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pletely silent. On the other hand, superficial cells do not show any excitant action of ACh even against a background of activity evoked by applying L-glutamate. 3. W h i c h Neural Pathways Innerzjate Cholinoceptioe Cells?

It is evident that the peculiar properties of cortical cholinoceptive cells might result from their being innervated by cholinergic nerve endings, the great differences in the sensitivity of neurons in various regions to ACh possibly being due to corresponding variations in the distribution of cholinergic synapses. There have been many previous suggestions that cholinergic fibers play a significant part in cerebral function (Feldberg, 1945, 1951; Stone, 1957) . All the usual main factors required for cholinergic transmission are certainly present in the cortex. As already mentioned, acetylcholinesterase is not lacking (Burgen and Chipman, 1951; Pope, 1952), and there is an adequate supply of cholineacetylase (Feldberg and Vogt, 1948; Hebb and Silver, 1956). In the presence of eserine and other anticholinesterases, large amounts of ACh accumulate in the cortex and can be collected by diffusion from its surface ( MacIntosh and Oborin, 1953; Mitchell, 1963; Szerb, 1963a). The rate of release is related to the level of spontaneous activity and to the amount of sensory input from the periphery. It vanishes during chloralose anesthesia, but atropine increases the resting rate considerably, possibly by its vasoconstrictor action, which tends to pre\,ent the removal of ACh by the blood stream (Szerb, 1963b). These observations strongly support the hypothesis that a system of cholinergic fibers is present in the cortex. To identify these it is, therefore, of particular interest to examine the various pathways by which cholinoceptive cells can be activated. a. Indifferent Pathways. Several pathways do not appear to bear any special relation to cortical cholinoceptive neurons because, although some of the excited cells may be sensitive to ACh, many other cells are quite insensitive. They include the short-latency, primary, afferent pathway excited by stimulating peripheral structures or the thalamic relay in the specific thalamic nuclei; the transcallosal system ( Fig. 26) ; the nonspecific thalamocortical connections which generate cortical recruiting responses during lowfrequency stimulation of medial thalamic nuclei, and the neural

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network involved in the superficial response to direct stimulation of the brain surface (Adrian, 1937). Although a cholinoceptive neuron may be innervated by one or more of these pathways, like the Betz cell illustrated in Fig. 27, such connections do not seem to be an essential feature. These synapses are, therefore, probably not cholinergic. b. A Possibly Significant Pathway. A characteristic feature of cholinoceptive units is the tendency to a prolonged afterdischarge in response to single shock excitation of the specific afferent pathway, especially when the shock is applied to the thalamic relay. This may or may‘not be preceded by an early response but, as shown by the illustrations in Fig. 27 ( F , G ) the afterdischarge typically begins after some 100 msec in a series of repetitive bursts which may go on for a second or more (see also Fig. 23). This kind of rhythmic activity is seen widely in the cortex and the thalamus. It appears to result from a rapidly alternating sequence of excitatory and inhibitory volleys, which can be triggered by a strong stimulus in many parts of the brain, or which is generated “spontaneously” in the form of barbiturate spindles or bursts. Such rhythmic variations between excitatory and inhibitory inputs are shown l-ery clearly by thalamic neurons and they may well originate in various parts of the thalamus (Adrian, 1941; Morison and Dempsey, 1943), but similar effects can be detected when most cortical units are carefully examined presumably because they are all subjected to powerful volleys originating in the thalamus. What seems peculiar to cholinoceptive neurons is the greatly heightened excitability, leading to a strong afterdischarge, whereas with most other neuron5 there is only a weak or subthreshold facilitation. Clearly the afferent volley must activate a pathway capable of producing a relatively long-lasting excitation, perhaps by the local release of ACh. A cholinergic mechanism is suggested by the fact that the repetitive afterdiwharges are much reduced by systemic atropine; even local ( iontophoretic ) applications of atropine have a marked depressant effect (e.g., Fig. 23), although this is neither so strong nor so prolonged as the block of the sensitivity to locally applied ACh (Fig. 23). A substantial difference between blocking effects on local applications of ACh and on normal transmission is not an unusual feature of cholinergic synapses (cf. Dale

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and Gaddum, 1930; Ursillo and Clark, 1956; Ambache, 1955; Curtis and Eccles, 1958). The repetitive responses and spontaneous activity of cholinoceptive neurons are also much depressed by chloralose (cf. Adrian, 1941), in doses which have little or no effect on the sensitivity of the cells to ACh. In view of previous evidence (MacIntosh and Oborin, 1953; Mitchell, 1963) that chloralose prevents the release of ACh from the cortex, it was suggested that chloralose may stop the release of ACh from central cholinergic nerve endings ( KrnjeviL and Phillis, 1963c) ; but later experiments on spinal Renshaw cells have not shown any interference by chloralose with cholinergic transmission in the spinal cord (Biscoe and KrnjeviL, 1963). One must presume therefore that chloralose depresses specifically activity or transmission in the cerebral cholinergic pathways; in view of the possibly cholinergic nature of the reticular, ascending, activating system (see below), it is significant that chloralose blocks cortical arousal elicited by stimulation of the midbrain reticular formation (Bremer and Stoupel, 1959). D. CHOLINERGIC INNERVATION OF CEREBRAL CORTEX 1. Histochemical Evidence Although the repetitive afterdischarges probably originate in the thalamus, the pathway by which they reach the cortex is unknown. All that is known is that this pathway is not identical with the direct thalamocortical link (Dempsey and Morison, 1943). In an attempt to define further a possible cholinergic pathway innervating cholinoceptive cells, studies have been made of nerve fibers in the cortex after staining by a modification of Koelle’s method for demonstrating acetylcholinesterase ( KrnjeviL and Silver, 1963b). Although cholinesterase staining is not an infallible criterion for the identification of cholinergic fibers, it can be of great value, as shown by the recent experiments of Sjoqvist (1963), who found a strikingly good agreement between cholinesterase staining and independent evidence of the cholinergic nature of certain sympathetic neurons. In the absence of any more reliable technique, this method may be considered as a useful general indication of cholinergic neurons. Cerebral nerve fibers which give a

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FIG.28. Frontal section of cat’s brain, treated by a modification of Koelle’s method for acetylcholinesterase. Notc lack of staining in corpus callosum ( A ) ,

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positive acetylcholinesterase reaction will be described here as being “cholinergic.” The first important feature of cholinergic pathways, clearly visible in Fig. 28, is that they are conspicuously absent in the great connecting links of white matter, such as the internal capsule, and the two main commiswres, the corpus callosum and the anterior commissure. Furthermore, nowhere do they stand out as a really compact bundle which would be at all obvious to the naked eye. As can be seen in Fig. 29 one finds instead a relatively diffuse system of fibers which characteristically join adlacent gyri, forming so-called U-shaped or arcuate bundles around the bottoms of intervening sulci. I n the core of each gyrus, they are visible mainly at the edge of the gray matter, h i t near the top of the gyrus, they spread out and ramify over the whole core, in a network that penetrates the gray matter, principally in its deeper portion (Fig. 28). The cholinergic U fibers thus belong to a complex system of association fibers linking all regions of the cortex (Meynert, 1867; Kaes, 1891; Dejerine, 1895). The only substantial coiinections between this system and the brain stem occur along the medial margin and in the lower lateral region of the hemisphere (Fig. 28). On the medial side, the cholinergic U fibers link tip with similar fibers in the cingulate gyrus, the cingulum, and the supracallosal gray matter, which are themselves probably continuous with cholinergic elements in the septum. On the lateral side, the cortex is supplied by a rich projection of fibers from the putamen via the external capsule. Most of these fibers are probably afferent to the cortex, as they tend to disappear above the level of a chronic lesion, whereas below, the fibers show a marked swelling and accumulation of stain. Moreover, if the cortex is undercut there is a large reduction in cortical cholineacetylase activity ( H e b b ct al., 1963); this would be internal capsule ( B ) , and anterior coininissure ( C ) . U fibers in white matter around sulci are clearIy stained and form a continuous network from supraca1losal gray matter and cingulum at medial margin of hemisphere ( D ) , to lower ectosylvian ( E ) and orbital region ( F ) laterally, where it becomes continuous with fibers emerging from putamen ( C , ) via external capsule ( H ) . Calibration mark indicates 5 mm ( K . Krnjevib and A. Silver, unpublished observations. 19E3).

FIG. 29. U fibers sweeping around bottom of marginal sulcus (upper left-hand corner) and into suprasylvian gyrus. Similar U fibers coming from adjacent ectorylvian gyrus are in lower right-hand corner. Fibers were stained by a modification of Koelle’s method for acetylcholinesterase. Note lack of staining in middle of core of gyrus, where radiation fibers are situated. Calibration mark indicates 0.5 mm (K. Krnjevii, and A. Silver, unpublished observations, 1963).

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difficult to understand if the cholinergic elements were wholly intracortical (like Golgi I1 cells) or if they were mostly cortical eff erents. The substantial staining of some deep pyramidal cells (Fig. 30) appears to be due to their innervation by cholinergic fibers, presumably by diffusion from the synaptic areas on their surfaces. These are probably the cholinoceptive cells. The cholinergic projections from the septum and the putamen described here agree well with cholinergic septal and striatal radiations observed in the forebrain of the rat by Shute and Lewis ( 1963). According to these authors, the cholinergic fibers are an extension of the brain stem reticular formation. It has been shown that in cats, the midbrain reticular formation has important projections to the medial septal nuclei and the corpus striatum (Nauta and Kuypers, 1958); but how the reticular influence reaches the cortex itself has never been clear. The most probable route is evidently via the cholinergic radiations from the putamen and the septum, the effects being disseminated over the cortex by the system of U fibers. 2. Other Observations The histological evidence, insofar as cholinesterase staining is a specific index of cholinergic fibers, points to a widespread cholinergic system in the cortex, apparently continuous with, and perhaps similar in nature to, the reticular formation of the brain stem. This is in keeping with many previous suggestions, based mainly on pharmacological evidence, that the mechanism of cortical arousal is probably cholinergic (e.g., Funderburk and Case, 1951; Rinaldi and Himwich, 1955a,b; Longo and Silverstrini, 1957; Bremer and Stoupel, 1959; Monnier and Romanowski, 1962); but it was not clear from earlier experiments whether ACh and cholinomimetic agents actually excited cortical neurons, or whether their effects were produced indirectly via the ascending activating system (cf. Rinaldi and Himwich, 1955b; Bremer and Stoupel, 1959). Much emphasis has been placed upon the cortical desynchronization produced by high-frequency stimulation of nonspecific thalamic nuclei, although it was clear from the original observations of Moruzzi and Magoun (1949) that these nuclei could not

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be an essential relay station and that similar effects could be elicited by stimulation in the region of the specific thalamic nuclei. The secondary role played by the medial thalamic nuclei in arousal has been emphasized recently by Schlag and Chaillet ( 1963). The effects produced by stimulating thalamic nuclei may be mediated by direct links with the striatum (cf. Powell and Cowan, 1954), by thalamoreticular connections ( Schlag and Chaillet, 1963), or by antidromic firing of primary sensory collaterals supplying the reticular formation ( Starzl et al., 1951 ). Attempts have been made to demonstrate direct excitation of cortical cholinoceptive cells by the postulated reticular system ( K . Krnjevih and M. Randik, unpublished observations, 1963). Although these cells could be activated by stimulating the midbrain tegmentum, it was not possible to evoke short-latency responses either from that region or from other relevant areas, such as the putamen or the septum. This is not contrary evidence, since the whole cholinergic pathway apparently consists of very fine myelinated fibers, and many synapses may well be present; moreover, if ACh is indeed, liberated by these fibers, its normal action may be comparatively slow, like that of ACh applied iontophoretically. However, the lack of really quick responses makes a direct electrophysiological confirmation of the histochemical data much more difficult and uncertain. 3. General Significance of Cortical Cholinergic Actizjitzj

Apart from the possibility that the ascending activating system may produce its effects by liberating in the cortex ACh, which would excite a certain number of sensitive neurons (mostly deep pyramidal cells ) , the presence of these cholinoceptive cells is probably of interest in some other respects. For instance, the great sensitivity of Betz cells to ACh suggests that a derangement of the normal cholinergic innervation could be a significant factor in the genesis of Parkinsonian tremor. It is well known that the impulses responsible for the tremor are conducted to the spinal cord over the pyramidal pathway (Bucy, 1944, 1958; Cordeau, 1961; Hassler, 1962 ) ; whether the pathological process is the result of pyramidal overactivity, or is due to an imbalance between pyramidal and other, possibly inhibitory, influences, the

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beneficial effects of pyramidal section (Bucy, 1944) and of drugs such as atropine (Anosov, 1960; Vernier and Unna, 1963) are readily understood. There have been several claims that thought processes and learning are markedly influenced by cholinomimetic or cholinolytic drugs. Thus cholinomimetic drugs, anticholinesterases and their antagonists may promote or inhibit the development of conditioned responses, while established responses may be disturbed ( Herz, 1960). Some correlation has been found between the learning ability of rats and the cholinesterase contents of their brains (Rosenzweig et al., 1958). In man, cholinomimetic drugs can have strong effects on mental processes (Grob et al., 1950); the marked interference with sustained thinking caused by the central cholinolytic drug, benactyzine (which acts rather like atropine), seems particularly suggestive ( Larsen, 1955; Dallies, 1956).

VI. Conclusions and Summary

Ionized substances are easily applied to nerve cells in situ by a controlled iontophoretic release from micropipettes. By using multibarrelled pipettes, it is possible to record the extracellular responses of a neuron, while applying to it one or more substances, in turn or in various combinations. One can thus analyze in detail the effects produced on individual neurons, as well as interactions between different antagon'stic or potentiating compounds. With this method, a comprehensive study has been made of the chemical properties of cortical neurons in mammals, leading to the following findings. Certain amino acids ha\.e very potent actions on neuron excitability. L-Glutamate and related dicarboxylic (and some sulfonic ) acids strongly excite most cortical neurons; the action is very rapid and stops immediately on ceasing the application. Further doses do not cause desensitization, nor do they appear to be injurious in any way. Under optimal conditions, only a minute amount of mole) is needed to produce clear excitation. In L-glutamate ( view of the Iarqe content of L-glutamate in the brain, the possibility must be considered that this substance is an excitatory transmitter at cortical synapses. o-Monocarboxylic amino acids, such as y-aminobutyric acid (GABA) and several derivatives, have such a potent inhibitory

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action, which is so general and so quickly reversible, that, in spite of some contrary evidence, one cannot a t present dismiss the possibility that GABA is a central inhibitory transmitter. Most amines of biological interest tend to depress neurons; though large doses may sometimes cause a sudden excitation, probably by a nonspecific effect on the cell membrane. Thus, dopamine, adrenaline, and 5HT all depress activity somewhat like GABA, though they are very much weaker; the strongest blocking agent with this type of action is probably imidazolylacetic acid, which is sometimes comparable with GABA in its effectiveness. Certain other amines before blocking activity markedly reduce the spike amplitude; this effect, which is only relatively slowly reversible, is likely to be produced by all compounds having a local anesthetic action, as it is shown particularly clearly by procaine, atropine, ephedrine, and LSD. The majority of cortical cells are neither excited nor depressed by ACh and cholinomimetic agents. However, a proportion of deep neurons, found mostly below the third Iayer (including most Betz cells) are readily excited with ACh; the action is relatively slow and prolonged. The ACh receptors on these cells are characteristically of a muscarinic type, being affected particularly well by muscarine and acetyl-p-methylcholine, and not by nicotine; the sensitivity to ACh is abolished by atropine and hyoscine, but is not greatly altered by nicotinic antagonists. These cholinoceptive cells are often spontaneously active and they tend to give prolonged afterdischarges in response to sensory volleys or to stimulation of specific thalamic nuclei. From a variety of evidence, including the histochemical demonstration of a system of cholinesterase-staining fibers that link all regions of the neocortex and are connected with subcortical projections from the striatum and the septum, it is concluded that the cortical cholinoceptive cells are probably innervated by cholinergic radiations related to the brain stem reticular formation. The postulated cholinergic fibers, which may be identical with the ascending activating system, are likely to play an important role in controlling the excitability of the cerebral cortex. REFERENCES Adrian, E. D. (1937).J . PhysioE. (London) 88, 127. Adrian, E. D. (1941). 1. Physiol. ( London) 100, 159.

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Amassian, V. E. (1961). Intern. Rev. Neurobiol. 3, 67. Ambache, N. (1955). Phamiacol. Reu. 7, 467. Anosov, N. N. (1960). Korsakou J. Neurol. Psychiat. ( U S S R ) (English Transl.) 1, 25. Araki, T., Ito, M., and Oscarsson, 0. (1961). J. Physiol. ( L o n d o n ) 159, 410. Arduini, A., and Machne, X. (1949). Archiu. Fisiol. 48, 152. Armett, C. J., and Ritchie, J. M. ( 1961). J. Physiol. ( L o n d o n ) 155, 372. Berl, S., and Waelsch, H. (1958). J. Neiirochem. 3, 161. Biscoe, T. J., and Krnjevih, K. (1963). Exptl. Nettrol. 8, 395. Bloom, F. E., Oliver, A. P., and Salmoiraghi, G. C. (1963). Intern. J. Neuropharmacol. 2, 181. Boistel, J., and Fatt, P. (1958). j . Physiol. ( L o n d o n ) 144, 176. Bonnet, V., and Bremer, F. (1937). Compt. Rend. Soc. B i d . 126, 1271. Bornstein, M. B. (1946). J. Neurophysiol. 9, 347. Bradley, P. B., and Wolstencroft, J. H. (1962). Nature 196, 840. Bradley, P. B., and Wolstencroft, J. H. (1963). Personal communication. Braunstein, A. E. (1947). Adsan. Protein C h e m . 3, 1. Bremer, F., and Stoupel, N. (1959). Arch. Intern. Pharmacodyn. 122, 234. Bucy, P. C. ( 1944). I n “The Precentral Frontal Cortex” ( P . Bucy, ed. ), p. 397. Univ. Illinois Press, Urbana, Illinois. Bucy, P. C. ( 1958). In “Pathogenesis and Treatment of Parkinsonism” ( W . S. Fields, e d . ) , p. 271. C. C Thomas, Springfield, Illinois. Burgen, A. S. V., and Chipman, L. M. ( 1951). J. Physiol. ( L o n d o n ) 114, 296. Burns, B. D., and Salmoiraghi, G. C. (1960). J. Neurophysiol. 23, 27. Cajal, S. R. (1911). “Histologie du systkme nerveux de l’homme et des vertAbrAs,” Vol. 2, p. 566. A. Maloine, Paris. Carslaw, H. S., and Jaeger, J. C. (1959). “Conduction of Heat in Solids,” 2nd ed., p. 257. Clarendon Press, Oxford, England. Chang, H. T. (1953). J. Neurophysiol. 16, 221. Chatfield, P. O., and Dempsey, E. W. (1942). Am. J. Physiol. 135, 633. Chatfield, P. O., and Lord, J. T. (1955). Electroencephalog. Clin. Neurophysiol. 7, 553. Chatfield, P. O., and Purpura, D. P. (1954). Electroencephalog. Clin. Neurophysiol. 6, 287. Chen, G., Portman, R., and Wickel, A. (1951). J. Pharmacol 103, 330. Cho, A. K., Haslett, W. L., and Jenden, D. J. (1962). J. Pharmncol. 138, 249. Coombs, J. S., Eccles, J. C., and Fatt, P. (1955). J. Physiol. ( L o n d o n ) 130, 326. Coombs, J. S., Curtis, D. R., and Eccles, J. C. (1957). J. Physiol. ( L o n d o n ) 139, 198. Cordeau, J. P. (1961). Rev. Can. B i d . 20, 147. Curtis, D. R. (1962). Nature 194, 292. Curtis, D. R., and Andersen, P. (1962). Nature 195, 1105. Curtis, D. R., and Davis, R. (1962). Brit. J. Phamacol. 18, 217. Curtis, D. R., and Davis, R. (1963). J. Physiol. ( L o n d o n ) 165, 62. Curtis, D. R., and Eccles, R. M. (1958). J. Physiul. ( L o n d o n ) 141, 435. Curtis, D. R., and Koizumi, K. (1961). J. Neurophysiol. 24, 80.

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