HVA, the appearance of this acid in the perfusate was examined. acid (HVA) and 5-hydroxytryptamine (5-HT), at rest and after

Transcription

1 J. Physiol. (1969), 204, pp With 8 text-figure8 Printed in Great Britain RELEASE INTO THE CEREBRAL VENTRICLES OF SUBSTANCES WITH POSSIBLE TRANSMITTER FUNCTION IN THE CAUDATE NUCLEUS BY P. J. PORTIG* AND MARTHE VOGT From the Agricultural Research Council Institute of Animal Physiology, Babraham, Cambridge (Received 16 April 1969) SUMMARY 1. One caudate nucleus of the anaesthetized cat was superfused by perfusing the anterior horn of one lateral cerebral ventricle. The perfusates were examined for their content in acetylcholine (ACh), dopamine, homovanillic acid (HVA) and 5-hydroxytryptamine (5-HT), at rest and after a variety of stimuli. 2. When prostigmine was added to the perfusion fluid, ACh appeared in the effluent; its concentration tended to rise in the course of an experiment. Various afferent stimuli, all of which caused evoked responses recorded from the contra-lateral caudate nucleus, increased the ACh content of the effluent. Effective stimuli were noise and electrical stimulation of afferent nerves or of certain regions of the brain including the ipsilateral substantia nigra. 3. The dopamine content of the effluent was extremely low (of the order of 50 pg/min) at rest, but, on occasion, rose sharply when the substantia nigra was stimulated electrically with trains of pulses repeated once every 3 sec. The results were inconsistent. 4. Since dopamine in tissue is rapidly transformed enzymically into HVA, the appearance of this acid in the perfusate was examined. 5. At rest, HVA was found to appear in the effluent at a rate of 2-8 ng/ min. Its concentration was rapidly depressed by increasing the depth of anaesthesia. 6. Stimulation of the substantia nigra for periods of 3 or 4 min caused an increment in the HVA content of the effluent lasting 1 hr or more. It was frequently seen when two points of the substantia nigra were stimulated * Present address: Institut fuir Toxikologie und Pharmakologie der Universitit Marburg Lahn, Germany.

2 688 P. J. PORTIG AND MARTHE VOGT simultaneously, less regularly with only one stimulating electrode, and rarely if this was placed in the most caudal part of the substantia nigra. 7. These results strongly support the view that there is a dopaminergic nigro-striatal pathway. The following assumption would explain the erratic appearance of dopamine and the long duration of increments in HVA: many of the axons originating in the substantia nigra end either in the putamen or in parts of the caudate nucleus which are far away from the ventricular surface; any dopamine released from these axons will not reach the ventricular surface at all, and HVA will, at best, reach it very slowly. 8. Small amounts of 5-HT appeared in the ventricular perfusate, and the quantity rose after the administration ofmonoamine oxidase inhibitors. It was not increased by the type of stimuli used in this work to elicit the release of ACh or HVA. INTRODUCTION The aim of the present work was to search for substances which might be released from the surface of the caudate nucleus when this structure is activated by a variety of afferent stimuli. Attention was focused on the three substances dopamine, ACh and 5-HT in which the caudate nucleus is richer than most other parts ofthe brain. If any of these were released as a result of increased activity this would support, though it would not prove, that they were acting as transmitters. A special interest attached to dopamine: its accumulation in the caudate nucleus (Carlsson, 1959) and its virtual disappearance from the 'striatum' (caudate nucleus and putamen) in Parkinsonian patients (Ehringer & Hornykiewicz, 1960) suggested an important functional role for this amine which went beyond its ubiquitous role of a precursor of noradrenaline. A role as precursor was unlikely in the striatum which contains hardly any noradrenaline (Vogt, 1954). The demonstration of lesions in the substantia nigra in Parkinsonism (Tretiakoff, 1919; Hassler, 1938), the fall in the dopamine content of the striatum when experimental lesions were made in and around the substantia nigra of monkeys (Poirier & Sourkes, 1965) or rats (Anden et al. 1964; Carman, Faull & Laverty, 1967) incriminate the substantia nigra as the source of striatal dopamine. The absence of major histological lesions in the striatum of Parkinsonian patients (Vogt & Vogt, 1920) would fit the interpretation that the striatal dopamine is synthesized in terminals which have their cells of origin outside that structure. The histochemical visualization of catecholamines by condensation with formaldehyde in freezedried tissue, which readily shows up the terminal parts of noradrenalinecontaining neurones, usually reveals only a diffuse, though intense, fluorescence in the striatum. However, by incubating slices of rat striatum

3 TRANSMITTERS RELEASED FROM CAUDATE NUCLEUS 689 in a medium containing a-methylnoradrenaline and preparing sections 2 /z thick, And6n, Fuxe, Hamberger & H6kfelt (1966) demonstrated very fine fibres and varicosities which they consider to be normally containing dopamine. The suspected cells of origin in the substantia nigra also fluoresce (Dahlstrom & Fuxe, 1964). An ascending nigro-striatal pathway has not been demonstrated by conventional histological methods; since these are not suited for fine, nonmyelinated fibres, one will have to look for evidence with other methods. Llamas & Reinoso-Suarez (1969), using a modified Nauta method, have found degenerating endings in the caudate nucleus after lesions in the substantia nigra of the cat. Carpenter & McMasters had earlier (1964) seen some preterminal degenerations in the putamen of similarly lesioned monkeys. Rosegay (1944) observed retrograde degeneration in the substantia nigra after destruction of the caudate nucleus in the cat, but the possibility of transneural degeneration is not excluded under these conditions. Finally, Hiokfelt & Ungerstedt (1969) incubated rat striatum with oc-methylnoradrenaline and fixed it with permanganate for examination under the electron microscope. After lesion"ing the mid-brain at the site at which any nigro-striatal fibres should converge on to the striatum, they saw a reduction in the number of axons filled with dark-core vesicles which indicate the presence of a monoamine (Richardson, 1966). McLennan (1964, 1965) was the first to seek evidence for the release of substances on activation of the caudate nucleus. He used Gaddum's (1961) push-pull cannula, a pair of concentric needles which allow local perfusion of a small region of an organ. With the cannula in the caudate nucleus, ACh appeared in the effluent when the nucleus ventralis anterior thalami, and dopamine when the nucleus centrum medianum was stimulated. On stimulation of the substantia nigra, dopamine was found when the cannula tip lay in the putamen, but not when it lay in the caudate nucleus. Experiments with push-pull cannulae have, however, one drawback. They cause a lesion at the site of perfusion (see also Rech & Domino, 1959), and there is a risk that any inflow of impulses into this region may cause leakage of substances due to changes in blood flow or cell permeability rather than to activation of transmitter release. Recent experiments (Chase & Kopin, 1968) have shown that this is a real danger; these authors obtained increased release of inert substances such as inulin, urea, and axaminoisobutyric acid into the effluent of a push-pull cannula inserted into the olfactory lobe when they stimulated the afferent pathway by a strong smell. To avoid artifacts which might be caused by lesions of the tissue, the technique employed by us was a 'superfusion' of the caudate nucleus achieved by perfusing the anterior horn of a lateral ventricle of the cat with

4 690 P. J. PORTIG AND MARTHE VOGT artificial cerebrospinal fluid as described by Carmichael, Feldberg & Fleischhauer (1964). The perfusate was tested for various monoamines and the conditions explored which might lead to their release. Preliminary accounts of the results have appeared in the Proceedings of the Society (Portig & Vogt, 1966, 1968). METHODS Cats of either sex and weighing over 2l5 kg were anaesthetized with a mixture of chloroform and ether, a femoral vein was cannulated, and chloralose (70-90 mg/kg) infused as a 0*67 % (w/v) solution. A few experiments were done under pentobarbitone sodium (40 mg/kg intraperitoneally), and for special purposes additional doses of either anaesthetic were given as indicated in the text. When shivering or other muscular movements occurred gallamine triethiodide was given intravenously in 6-10 mg doses. It was sometimes desirable not to interfere with the blood supply to the hind legs. Blood pressure was then recorded from a brachial instead of a femoral artery and the saphenous vein used for injections instead of the femoral vein. Body temperature was kept constant by a battery-powered device described by Krnjevic & Mitchell (1961). Perfusions. The animal's head was fixed in a stereotaxic instrument and, usually, the anterior horn of the left lateral ventricle perfused with the method described by Carmichael et al. (1964). The collection of ventricular perfusates, recording procedures and the composition of the artificial cerebrospinal fluid have been described (Portig, Sharman & Vogt, 1968). In recent experiments, some modifications were made: the motor-driven syringe effecting the perfusion was replaced by a proportioning pump, similar to that used in autoanalysers. The performance of the pump was more reliable than that of the syringe, which was liable to allow fluid to leak past the plunger. A suitable tubing for use with the pump was that made by Technicon (colour code orange/ green). Furthermore, the phosphate of the perfusion fluid, originally added as disodium hydrogen phosphate, was added as equimolar parts of disodium. hydrogen and sodium dihydrogen phosphate, the total amount of phosphate remaining the same. Another innovation was the recording of the pressure in the perfused ventricle. This was done by introducing a side arm into the tubing to the inflow needle and connecting the arm to a Statham pressure transducer (type P 23 BC) which recorded on the Grass polygraph. The zero was set by placing the orifice of the inflow needle at Horsley Clarke co-ordinate H + 5 (where it would be during the experiment) and letting the fluid flow out at the usual speed. The pressure record facilitated positioning of the inflow needle by showing a sudden fall in pressure when the needle reached the ventricular space; a fall was occasionally seen when the needle was still about 5 mm above the correct depth and crossed the cruciate sulcus on the medial surface of the brain. During the perfusion, a rise in the inflow pressure gave a warning of an obstruction forming at the opening of the outflow needle. The last modification was to replace the Collison cannula by a straight steel needle, identical in diameter and bore with the inflow needle, but shorter (36mm long), and clamped in a holder which moved vertically. The advantage of the needle was that clots or tissue aggregates which sometimes formed at the tip of the Collison cannula were hardly ever seen. To avoid irregularities in flow occasioned by gas bubbles which were liable to form in the inflow tubing, a small bubble trap of 1 ml. capacity was made from the barrel of a 2 ml. plastic syringe and fixed immediately above the inflow needle.

5 TRANSMITTERS RELEASED FROM CAUDATE NUCLEUS 691 Stimuli. Trains of three square pulses, and later of three biphasic stimuli, generated by allowing one Grass SD 5 stimulator to modulate the output of a second one, were used in all except the earliest perfusions. It proved necessary to employ a stimulus isolation unit, and for this purpose a transformer Lie B6lin no. TP 86 (used for television equipment) was inserted between stimulator and electrodes. Some distortion of the shape of the stimulus resulted from the use of the transformer, but this was reduced by a capacitance soldered across the output terminals. For most stimulations of the substantia nigra frequency within the train was about 50/sec, and the trains were repeated once every 3 sec. Faster stimuli were tried but appeared to be less effective. They were, however, used for stimulation of the reticular formation or the sciatic nerves. Pulse duration was 1 msec in the early, and changed to 0*5 msec in all later experiments to avoid electrolytic injury (see Rowland, 1966). Except for early experiments when stronger currents were used, the voltage for stimulation within the brain was so chosen that it was sufficient normally to elicit evoked responses either in the caudate nucleus or in the cortex, but to be so low that tissue damage during the prolonged stimulations was avoided or kept to a minimum. To determine the permissible voltage, electrodes were placed in 0 9 % (w/v) sodium chloride solution and the current intensity produced by a known voltage was calculated by observing the voltage drop over a 100 fl resistance placed in series with the electrode. The voltage chosen for stimulation produced a current strength of 2-1 ma in saline. This corresponded to an intensity of 0-7 ma in brain tissue which was found to have three times the resistance of 0 9% sodium chloride solution. With our electrodes, the voltage producing such current was in the region of 4-6 V. Electrodes. In order to stimulate the paws, pairs of straight surgical needles were inserted into the skin. Stimulation of peripheral nerves was by bipolar shielded platinum electrodes. Brain stimulations were carried out with co-axial, stainlesssteel electrodes (type K 6012, Statham) of 0 45 mm external diameter and a core of 0-21 mm. They were insulated except for an outer ring of 0-5 mm and the protruding tip, the bare end of which was at a distance of 0 5 mm from the ring. The bare tip, which was originally blunt and 1mm long, was ground to a point. The electrodes were used both for recording on a dual beam Tektronix type 502A oscilloscope and for stimulating. Single vertical electrodes were inserted into the right caudate nucleus (co-ordinates about A 17, L 5, H + 6), and a pair of vertical electrodes, usually mounted in a sagittal plane at a distance of 1-5 mm were used for stimulations of the substantia nigra. A few experiments in which the two electrodes were placed in a frontal plane will be described in the text. Electrocorticograms were recorded with a Grass polygraph from a steel pin driven into the bone over different parts of the cortex. All leads to electrodes leading from or stimulating the brain were shielded. The substantia nigra is a thin sheet of variable shape. As an aid to finding the correct depth, we followed the advice of Professor Denise Albe-Fessard and observed on the CR0 the potentials evoked in the brain by alternate electrical stimulation of the right and left paws while we gradually lowered the electrodes to the theoretical depth. As long as the electrodes are above the substantia nigra, evoked potentials have latencies of 15 msec or less and the response differs in shape according to whether the ipsi- or contralateral limbs are being stimulated. When, however, the substantia nigra is penetrated, latencies are between 20 and 30 msec and nearly identical responses are evoked from both sides of the body. The shape of the response is a negative deflexion usually followed by a positive wave. If the electrode is in the caudal part of the pars compacta, where large cells lie closely packed, the amplitude of the deflexion is very large, sometimes 300,uV or more. Another characteristic feature of that region is the spontaneous activity which assumes the form of rounded waves of 10-15/sec and which becomes more and more pronounced as the experi-

6 692 P. J. PORTIG AND MARTHE VOGT ment progresses and anaesthesia lightens. In contrast, the spontaneous waves in other parts of the substantia nigra, or in the caudate nucleus, which can also become frequent, are much more irregular in shape and interval. The placement of stimulating electrodes was controlled histologically. Immedi. ately after the perfusion of the ventricles with bromphenol blue to identify the part of the ventricular system from which fluid had been collected (Carmichael et al. 1964) the stimulating electrode pair was lifted out of the brain and reintroduced 3 mm caudal and 1 mm lateral to the original position to produce a pair of 'guide tracks'; a second double track was made after having moved the electrodes 2 mm medially. These tracks were useful in orienting the tissue block on the microtome. Before removal the brain was perfused from the aorta with 0 9 % sodium chloride solution followed by 4% (w/v) formaldehyde in saline. The brain was kept in the fixative overnight, a block containing the electrode tracks cut out, embedded in celloidin and 50 j sections stained with cresyl violet. Analysis of the perfuate Acetyicholine. Neostigmine methylsulphonate, 6 x 10- M, was added to the per. fusion fluid whenever ACh estimations were intended. Assays were carried out on the eserinized dorsal muscle of the leech suspended in a 1 ml. bath. Ten parts of perfusion fluid were diluted with four parts of water before assay. The standards were prepared in perfusion fluid diluted similarly. Samples which had been acidified were neutralized with solid bicarbonate. The choice of a suitable anticholinesterase to preserve the ACh presented some difficulty. Since it was desired to estimate dopamine and ACh in the same perfusates, eserine was ruled out because samples of cerebrospinal fluid containing eserine and treated with ethylene diamine for the determination of dopamine gave an intense fluorescence with the same characteristics as that due to dopamine. Neostigmine caused no such difficulty in a first group of perfusions, but on one occasion was found to form a fluorescent compound which mimicked dopamine. The prostigmine was then replaced by anticholinesterases such as dodecamethylene bisquinolinium bromide, BW 62 C47 (1,5-bis(4-trimethylammoniumphenyl) pentan-3-one diiodide) or BW 284 C 51 (1,5-bis (4-allyldimethylammoniumphenyl) pentan-3-one dibromide) which contained no carbamic ester group. However, the first compound interfered with the ACh assay on the leech, and the other two produced no accumulation of ACh in the perfusate when used in concentrations (2 x 10- m and 5 x 10-4 M) recommended for in vitro work. Since the interference of neostigmine appeared to be due to the formation of a product of disintegration, its use was then resumed but great care taken to renew the drug frequently and to reject it if the crystals were not a pure white. The prostigmine-containing perfusion fluid was then found to produce no more fluorescence thanthe fluid to which no drughad been added. 5-hydroxaytryptamine. This was estimated on the rat stomach strip (Vane, 1957), special care being taken that no trace of blood was present in the perfusates, if necessary, by centrifuging to make certain of the absence of red cells. Any contractions of the stomach were only attributed to 5-HT if they were abolished by bromolysergic acid diethylamide (0.5,sg added for 20 min to 5 ml. bath). Dopamine and homovanillic acid. The methods have been described (Portig et al. 1968). The efficacy of the precautions taken to preserve any dopamine which might appear in the perfusion fluid was tested by pumping a solution of cerebrospinal fluid which contained dopamine 5 ng/ml. through a long piece of plastic tubing immersed in a water bath of 400 C. The effluent was collected for 30 min periods, the collected volume being the same as in real experiments. There was no loss attributable to the long exposure to body temperature. The fluorimetric estimations were not

7 TRANSMITTERS RELEASED FROM CAUDATE NUCLEUS 693 interfered with by probenecid, tetra-ethylammonium chloride or gallamine triethiodide, but neostigmine methylsulphate gave rise to fluorescence when the powder was old, and physostigmine sulphate could not be used because it forms an intensely fluorescing compound. Acetylcholine RESULTS When neostigmine 6 x 10-5 M was added to the perfusion fluid ACh appeared in the effluent in variable quantities with a mean of about 2 ng/ min. Half the experiments were done in atropinized cats (atropine sulphate 2 mg/kg i.v.) since it was known (Mitchell, 1963; Polak, 1965) that release from the brain is increased in the presence of atropine. Atropine about doubled the mean concentration of ACh in the ventricular perfusate. Confirming earlier findings by Bhattacharya & Feldberg (1958), the concentration of acetylcholine in the effluent was found to increase gradually, so that release effected by any stimulus could only be demonstrated if it caused a clear peak on the ascending base line. The gradual rise in basal secretion may have several reasons: a gradual lightening of anaesthesia, shown by MacIntosh & Oborin (1953) to increase acetylcholine release from the surface of the brain; to this might be added the arousal caused by all anticholinesterases, even in the presence of an anaesthetic; finally, the slow penetration, as perfusion proceeds, of neostigmine into regions further removed from the ventricular surface. This may lead to more synapses contributing to the fraction of transmitter which evades destruction and finds its way into the perfusate. Since the work of Bonvallet, Dell & Hugelin (1952), Segundo & Machne (1956) and Albe-Fessard, Oswaldo-Cruz & Rocha-Miranda (1960) it is known that the striatum responds to afferent impulses of many modalities. The stimuli used in this work were noise, electrical stimulation of the paws, of the central ends of the severed sciatic nerves and of several brain structures: the caudate nucleus on the side which was not perfused, the nucleus centrum medianum, some neighbouring thalamic nuclei and the substantia nigra. The electrical stimuli were biphasic pulses (duration 1 msec, frequency 1-10/sec, strength V). All the peripheral stimuli, which were applied for 10 or 30 min, usually increased the acetylcholine content of the effluent. Stimulation of the paws caused the smallest rise in ACh release (mean of 30 %), sciatic stimulation produced, on the average, double that rise and noise gave intermediate results. Chloralose was the anaesthetic in all but three experiments which were done under pentobarbitone; with the barbiturate release of acetylcholine appeared to be smaller. An example is illustrated in Fig. 1; in spite of the gradually rising base line, all four stimuli (applied for 15 min) caused a release of acetylcholine,

8 694 P. J. PORTIG AND MARTHE VOGT the effect being most pronounced on stimulation of the sciatic nerves. Figure 1 also shows that the time course is similar for all four responses; since the columns represent the ACh, in ng/min, appearing in 15 min samples, each stimulation period caused an increment in ACh content of the fluid collected during, and for at least 15 min following, stimulation. E 86 C ~ - -V I i rra m Sk Sci Nol Cd Fig. 1. Each lower column represents, in ng/min, the ACh found in a 15 min perfusate of the anterior horn of the left lateral ventricle of a cat anaesthetized with chioralose, 53 mg/kg. Artificial respiration and occasional intravenous injections of 12 mg gallamine triethiodide. The perfusion fluid contained neostigmine methylsulphonate 6 x 10-5 M. Atropine sulphate, 2 mg/kg, had been injected intravenously 20 mmn before collecting the first sample of perfusate. The signals mark stimulation periods: 1st bar, electrical stimulation of the skin (Sk) of both forepaws (biphasic pulses of 1 msec duration, 2/see, 15 V); 2nd bar, stimulation (same parameters) of central ends of severed sciatic nerves (Sci); 3rd bar, noise (Noi) produced by banging pieces of metal; 4th bar, stimulation of right caudate (Cd) nucleus (co-ordinates A 15, L4-5, H5*5) with biphasic pulses of 1 msec duration, 1 every 3 sec, 20 V. Increments in ACh output emphasized by shading. The response to stimulation of the contralateral caudate nucleus, seen at the end of Fig. 1, was unusual as it failed to occur in two other exrperiments under chloralose and in two decerebrate cats. Among other central structures tried, stimulation of the nucleus centrum medianum did not cause release of ACh, but in one experiment in which the electrode was accidentally placed more anteriorly in the nucleus centralis lateralis, release was seen. The twelve experiments in which the electrodes were correctly placed

9 TRANSMITTERS RELEASED FROM CAUDATE NUCLEUS 695 in the substantia nigra fall into two groups. In the first six, the electrodes were in a frontal plane, 1x5 mm apart, the lateral electrode being aimed at the substantia nigra, and the medial one at the ascending nigro-striatal fibres to be found, according to Dr L. Poirier (personal communication) at co-ordinates A 6-5, L 2-7, H With this positioning, rises in ACh output were nearly always seen and ranged from 40 % to 175 %. However, in that group of experiments, strong stimulating currents ranging from 20 to 50 V were used so that the possibility cannot be excluded that there was spread of current to structures outside the substantia nigra. In the second group, voltage was lower, usually 4, once 5 and once 14 V. The electrodes were exclusively aimed at the substantia nigra, not at the ascending tract. If two electrodes were used, they were placed in a sagittal plane. As in the previous experiments, release of ACh accompanied stimulation, but rarely exceeded 50 % of resting output. With one electrode release was less than with two. Dopamine Tissue content. The assumption that dopamine appearing in the ventricular perfusate would originate in the caudate nucleus was based on the fact that, of the structures bordering on the anterior horn, only the caudate nucleus is known to contain much dopamine. However, figures for the dopamine content of the septum were not available from the literature and since this is the other major grey structure bordering on the anterior horn, the concentration of dopamine was estimated in several structures including the septum (Table 1). TABL3E 1. losses) in some regions of the cat brain. Single estimations Concentration of dopamine (,ug/g fresh tissue, corrected for Septum (excluding most ventral part) 0*14 Nucleus accumbens and ventral part of septum 1*07 Caudate nucleus 105 Cortex (mean of two) 0.12 Olfactory bulb 0*10 It follows from Table 1 that the septum proper contains about the same low amount of dopamine as cerebral cortex or olfactory bulbs, whereas the region where the septum merges into nucleus accumbens and caudate nucleus has a dopamine content amounting to about 10 % of that of pure caudate nucleus tissue. It is thus safe to conclude that dopamine entering the ventricles would have originated in the caudate nucleus. Ventricular perfusates. In most animals, resting values of dopamine in 30 min samples of perfusate were below the threshold of the method. Occasionally they amounted to 1-2 ng, figures so small that one could not

10 696 P. J. PORTIG AND MARTHE VOGT be sure of the specificity of the fluorescence. In early experiments the impression was gained that stimulation of the central ends of the sciatic nerves caused dopamine to appear in the effluent, but it was later discovered that on many of these occasions there had been irregularities of flow which had required adjustments of the position of the cannula. It was found that such manipulations on their own may lead to the appearance of dopamine, and when all experiments were discarded in which the flow had not been constant, there were only six occasions of a total of twenty-five in which dopamine release followed sciatic nerve stimulation, so that release was obviously not a consistent event. In these experiments chloralose, pentobarbitone or ether were used as anaesthetics, and release occurred three times under chloralose and three times under pentobarbitone. The responses to electrical stimulation of the substantia nigra carried out for min were very variable. Sometimes release was absent, sometimes it occurred immediately, and sometimes it was delayed. The quantities varied from 2-20 ng in a 30 min sample and it was quite obvious that another technique had to be devised if more consistent results were to be obtained. Since it was possible that the use of an anaesthetic interfered with neuronal activity, attempts at avoiding anaesthetics by preparing a cerveau isole preparation under ether were made in twelve experiments. Owing to the greater tendency to haemorrhage during the period of ether administration, satisfactory perfusion was only obtained in four cats. They were used to test the effect of electrical stimulation of the contralateral caudate nucleus. The voltage was varied from 5 to 20 V and the frequency from 0 3 to 100/sec. Release of dopamine was not observed. The possibility that entry into the ventricles of a substance released in the tissue might be aided by perfusing with hypertonic cerebrospinal fluid was tested in one cat. The substantia nigra was stimulated before and after change-over to perfusion fluid of 1-5 times normal tonicity. Dopamine was only released before the change-over. On the other hand, intraperitoneal injection of 1 g of the dopamine precursor dihydroxyphenylalanine caused a large quantity of dopamine to appear in the perfusate, but the content rose to a peak and gradually fell off again, not yielding a steady base line. It is also in no way certain whether the dopamine was formed in the walls of brain capillaries (Bertler, Falck & Rosengren, 1963) or inside neurones. Desmethylimipramine inhibits uptake of noradrenaline, either injected into the cerebral ventricles, or newly formed from administered L-dihydroxyphenylalanine, but does not prevent uptake of dopamine similarly injected or formed (Glowinski, Axelrod & Iversen, 1966; Carlsson, Fuxe,

11 TRANSMITTERS RELEASED FROM CAUDATE NUCLEUS 697 Hamberger & Lindqvist, 1966). Increase of noradrenaline overflow by desmethylimipramine was not observed on stimulation of the hypogastric nerves (Vogt, 1965) or the splenic nerves of the cat (Geffen, 1965). However, its action on the release of endogenous dopamine was tested in two cats in which the substantia nigra was stimulated. In one experiment 10 mg/kg was injected intravenously before perfusion started and no dopamine appeared on stimulation; in the second cat the same dose was injected between two stimulation periods, and dopamine appeared during the second stimulation, but the amount did not exceed that seen in other animals without imipramine. One of the probable reasons for the erratic release of dopamine was the presence, in the tissue, of monoamine oxidase and O-methyl transferase which would transform any dopamine released into metabolites, predominantly into HVA. Unless one was fortunate enough to stimulate cells the synapses of which were very near to the ventricular surface, the chances of obtaining unmetabolized dopamine were small. There were two ways open to overcome this difficulty: to inhibit the enzymes or to make use of the appearance of the acid metabolite as an indicator of the release of the amine. Inhibition of enzymes was tried first: iproniazid (100 mg/kg) or nialamide (10-80 mg/kg) was injected on the day before the experiment; in three cats, this treatment was combined, throughout the experiment, with an intravenous infusion of tropolone, an inhibitor of O-methyl transferase (25 mg/kg. hr). There was no increase in the resting release of dopamine, nor in the responses to stimulation of the substantia nigra. That the dose of monoamine oxidase was adequate is shown by the fact that even with the smallest dose of nialamide the amount of 5-HT found in the perfusion fluid was increased. On the other hand, even after nialamide, 75 mg/ kg or iproniazid, 100 mg/kg, the concentration of dopamine in the caudate nucleus was not elevated; similarly, cerebral noradrenaline does not rise in the cat after inhibition of monoamine oxidase (Vogt, 1959; Spector, 1963). The next section deals with experiments in which the main acid metabolite of dopamine, HVA, was estimated instead of the amine itself. Homovanillic acid Basal release. The first striking difference on changing over from estimations of dopamine to estimations of HVA was the high concentration of that acid found in samples of perfusate collected at 'rest'. Instead of 1-2 ng dopamine in effluent collected for 30 min, there were ng HVA. That this difference was not due to a loss of dopamine after it had reached the ventricles was shown in control experiments referred to under 'methods'. Since in the cat the tissue concentration of dopamine is about three times that of HVA, this finding supports the suggestion made in the

12 698 P. J. PORTIG AND MARTHE VOGT previous section that released dopamine does not reach the ventricles because of transformation by the tissue enzymes into acid metabolites. Although there was very little doubt that the HVA came from the brain tissue and not from the blood, control injections of HVA were made intravenously into one cat; neither 100, 250 nor 500 jug affected the HVA content of the perfusate. 159 I.2 E Ln 0-8 Flow 0-4 C 4 E C 2 3' st. Sci Fig. 2. Lower columns represent, in ng/min, the HVA found in a 30 min perfusate of the anterior horn of the left lateral ventricle of a cat given chloralose, 59 mg/kg. Artificial respiration and occasional intravenous injections of 6 mg gallamine triethiodide. The black bar marks a stimulation (st), for 3 min, of the central ends of both sciatic (Sci) nerves (4V, trains of three square pulses of 1 msec duration repeated every second). The upper histogram represents the volume of effluent in ml./15 min; where the outflow falls sharply, a screwclip has been applied to the outflow tubing. In this and subsequent Figures increments in output of HVA elicited by stimuli are shown in black. Flow and perfusion pressure. With the high basal output of HVA the detection of effects of stimulations or other measures depended on a steady base line. The base line, however, was shown to be sensitive to small obstructions of flow such as are liable to develop in the course of long experiments. The importance of unimpeded flow is illustrated in the right half of Fig. 2. The upper histogram represents the flow through the ventricles and is seen to be about 1-2 ml./15 min during collection of the first seven samples. Then the outflow tubing was partially clamped by a screw clip so that pressure rose in the ventricles and the volume collected was reduced to one-

13 TRANSMITTERS RELEASED FROM CA UDATE NUCLEUS 699 half: HVA disappeared from the effluent. The flow was obstructed for shorter periods of time during collection of the next three samples, and this caused less severe reductions of the HVA content of the effluent. During the last sample flow was not obstructed and HVA content of perfusate returned to its initial level. Changes in flow by speeding up or slowing the pump had no effect on HVA production, provided the outflow was unimpeded and perfusion pressure did not rise. E l ~05 hr 1O -A S hr Eq A i C 6 _E 2 < ~~~~~~~~~~~~~~4 2 a + b + SN Fig. 3. Lower columns: HVA content, in ng/min, of 25 min perfusates of the anterior horn of the left lateral ventricle. Cats anaesthetized with chloralose, 70 and 90 mg/kg. Upper columns: volume of effluent in ml./ 25 min. (a) At the arrow, injection of additional chloralose, 37 mg/kg, 4 hr after first dose. Signal: stimulation, with one electrode only, of substantia nigra (SN) at co-ordinates A 4 3, L 3 5, H for four 3 min periods (4 5 V, trains of three biphasic stimuli of 0 5 msec once every 3 sec). Three min rest between stimulation periods. No gallamine or artificial respiration. (b) At the arrow, rapid intravenous injection of additional chloralose, 30 mg/kg (11 ml.) 5 hr after first dose. Signal: loud noise (Noi) for 10 min. Artificial respiration. 10 mg gallamine during third and ninth sample. Note that the scales for HVA in (a) and (b) are different. Spontaneous slowing of flow, accompanied by rises in ventricular pressure, occurred intermittently in some cats by what appeared to be an occlusion of the outflow needle by the chorioid plexus. In other experiments, flow tended to slow down with the passage of time, and this might have been due to narrowing of the ventricles by slight oedema formation. Though adjustment of the position of the outflow needle by rotation or small vertical movements cured most of these troubles temporarily, the Noi

14 700 P. J. PORTIG AND MARTHE VOGT adjustment upset the basal output of HVA, and a new base line had to be established before any stimulation could be carried out. Anaesthesia. The resting release of HVA was influenced by the depth of anaesthesia. With average doses of chloralose (60-95 mg/kg), the range of appearance of HVA was 2-8 ng/min. In a single animal which had been given 113 mg/kg, it fell to 1 ng/min. Figure 3 shows two instances of the fall in resting output of HVA on deepening the anaesthesia. At the arrow on the left (cat 259), a dose of chloralose, 37 mg/kg, was injected intravenously 4 hr after an initial dose of 75 mg/kg: release of HVA fell from 3 to 2-3 ng/min. On the right (cat 231) the fall was from 9 to 6*5 ng/min on rapid injection of chloralose, 30 mg/kg, to a cat which had had 90 mg/kg 5 hr earlier. Spontaneous electrical activity in the caudate nucleus slowed down, and evoked responses elicited by touch were slightly enhanced. The upper histogram shows that the flow of cerebrospinal fluid was constant throughout both experiments, except for a small increase during the rapid injection of chloralose in cat 231. In a third cat subjected to the same treatment the fall in resting release of HVA was from 5 to 2-5 ng/g. Similar experiments carried out with pentobarbitone showed a reduction in resting output of HVA when pentobarbitone (15 mg/kg) was injected into a cat anaesthetized with chloralose; in this animal, effect on e.e.g. and spontaneous activity of caudate nucleus was prolonged and severe, and even evoked responses were unobtainable for a long time. In another cat, in which the same dose of pentobarbitone was superimposed on pentobarbitone anaesthesia, effect on electrical activity of the brain was short-lived and depression of HVA content of perfusate did not ensue. Stimulation of afferent nerves. The left side of Fig. 2 shows an example of a very small rise in the HVA content of effluent after a 3 min stimulation of the sciatic nerves at 1/sec. It lasted for over 1 hr; very slight twitches accompanied the stimulation. Of eleven similar experiments in which stimulus strength was 4 V and evoked responses were elicited in the caudate nucleus, seven showed increments in production of HVA, usually larger than the one of Fig. 2. On the remaining four occasions, HVA production did not rise. In two of those, the initial (resting) release was so high (8-5 and 10-5 ng/min) that this might have been the reason why further increase was not seen. Twitches were not a prerequisite of a positive response. Since the sciatic nerves contain many types of afferent fibres, a series of experiments was performed in an attempt at identifying the nature of the afferent stimulus which enhanced HVA concentrations. When the electrodes were placed on the central end of the saphenous nerve, thus stimulating skin afferents, HVA content of the effluent either remained the same or fell. Of six experiments in which motor branches, containing muscle

15 TRANSMITTERS RELEASED FROM CA UDATE NUCLEUS 701 afferents, were dissected out and stimulated, small releases were observed three times. It appeared possible that muscular twitches involving reflex activation of motor nerves was necessary for the response; therefore in five cats gallamine was withheld and weak stimuli (maximum 1-3 V) were applied to the central ends of the sciatic nerves till reflex movements appeared: no convincing effects on release of HVA were seen. Stimulation of the afferents in the sciatic nerves by currents of moderate intensity causes a fall, and strong, frequent stimuli a rise in blood pressure (Hunt, 1895). Most stimulations had hitherto been carried out with frequencies not exceeding 1/sec, because evoked responses in the caudate nucleus are only seen if at least a second elapses between stimuli, and falls in blood pressure had been the usual response to stimulation. Two experiments were performed in which strong (30 V) stimuli of high frequency (100/sec) were applied to produce a rise in blood pressure; there was no release of HVA. This analysis failed to disclose any single component of the multiple afferents in the sciatic nerve which would satisfactorily account for the frequent appearance of HVA when the whole trunks of the severed nerves were stimulated at low frequency. Arousal. The possibility that stimulation of the sciatic nerves produced effects due to simple arousal was tested in five experiments of which Fig. 4 is representative. A pair of vertical electrodes with anterior-posterior co-ordinates A 5 and A 4 were lowered to H-1 in the lateral plane L 3, and stimulation was carried out with trains of thirteen biphasic shocks of 0*5 msec duration delivered every 2 sec, each train lasting 0-2 sec. There was pupillary dilatation, rise in blood pressure andsome increase in thefrequency of the e.e.g. No rise in HVA output was found. In the experiment of Fig. 4, the electrodes were then lowered to H (arrow) and the stimulation was repeated with the parameters found suitable for the substantia nigra (see Methods); there was some release of HVA. These experiments show that arousal by itself is not sufficient to produce an increase in the content of HVA in the perfusate. Stimulation of the substantial nigra. The first series of experiments was carried out with two electrodes mm apart placed in the substantia nigra in a sagittal plane (approximately L 3). The most frequently used anterior-posterior co-ordinates were A5-5 for the rostral, A for the caudal electrode. Duration of stimulation ranged from 3 to 30 min. Stimulus parameters are described under methods. At the beginning and at the end of the experiment, evoked responses elicited by stimulation of the paws were monitored from the stimulating electrodes; this ascertained whether their shape, which had been used for placement of the electrodes, had persisted unchanged throughout the day.

16 702 P. J. PORTIG AND MARTHE VOGT An early experiment in which stimulation was carried out for 30 min caused a rise by 67 % in HVA output which lasted for 1 hr. It soon became evident, however, that equally sustained increments in HVA followed stimulations as short as 3 or 4 min (Fig. 5). Two occasions when it proved possible to maintain a steady base line (see below) for long enough to observe the results of two stimulations of different duration are shown in Figs. 6 and 7. Figure 6 illustrates stimulations lasting 4 and 8 min; Fig. 7 shows 218 i 05hr 5 4 E C. 12 St. F SN Fig. 4. Columns represent, in ng/min, HVA found in 25 min perfusates of anterior horn of left lateral ventricle of a cat given chloralose, 95 mg/ kg. Artificial respiration and three injections of gallamine triethiodide 10 mg during last hours of experiment. At first bar, 10 min stimulation of reticular formation (RF) with two vertical electrodes (A5 and A4, L3, H - 1); arousal. At arrow, electrodes lowered to H -53. At 2nd bar, 10 min stimulation of substantia nigra (SN). 10 and 20 min periods, and the effect of the 20 min stimulation suggests that prolongation of the period beyond 10 min probably contributes little to the amount released. The duration of increased release was also independent of the length of the stimulation period, thus perhaps indicating that the long duration of the release was due to the slow movement of the acid from its site of production into the ventricles. In twelve stimulations (on nine cats) by means of two electrodes, unequivocal release of HVA was observed; in four cats release failed to occur. In two of those resting secretion was so high it may well have been maximal before stimulation. The third cat in which stimulation produced no effect had a more caudal placement of the electrodes than the others,

17 TRANSMITTERS RELEASED FROM CAUDATE NUCLEUS 703 and this, as will be discussed in the next section, might account for the lack of response. None of these reasons explained the fourth failure. The second set of experiments was carried out by stimulating only one point of the substantia nigra at a time, in an attempt at discovering whether release of HVA into the ventricles occurred on activation of certain points only. The implication was that some regions of the substantia nigra might give rise to axons impinging on ventricle-near parts of the caudate nucleus whereas others sent their fibres elsewhere. The best information of the response to stimulation of different regions of the substantia nigra would have been obtained by stimulating several points in one experiment. Only min Cs 5~~~~~~~~~~~~~'430 min ~~~~~~~~~~~6 E4.E5 E > 34 X~~~~~~~~~~~~~ 2 > X 2 St.' St.' Fig. 5 Fig. 6 Fig. 5. Columns represent, in ng/min, HVA found in 30 min perfusates of anterior horn of left lateral ventricle of cat given chloralose, 52 mg/kg. Gallamine triethiode (6 mg) injected and artificial respiration started during penultimate collection period. Signal indicates 4 min stimulation (st) of substantia nigra with two electrodes at positions A5-7 and A4-5; trains of three 1 msec square pulses every 3 sec, strength 4V. Fig. 6. Columns represent, in ng/min, HVA found in 30 min perfusates of anterior horn of left lateral ventricle of cat given chloralose, 54 mg/kg. Natural respiration. At first signal, 4 min, and at second signal, 8 min stimulation of substantia nigra using two electrodes (positions A5-2 and A4-0) and trains of three 1 msec square pulses every 3 sec, strength 4V. a few such experiments were successful, and the reasons lie in the limitations of the technique, particularly in the effect which changes in flow (see p. 698) have on the appearance of HVA. It is obvious that the difficulties arising from the effect of changes in ventricular pressure on the composition of the effluent were accentuated by the long duration of the increments in HVA produced by even short stimulation periods. This 23 Phy. 204

18 704 P. J. PORTIG AND MARTHE VOGT duration of up to 1.5 hr contrasted with the short-lived appearances of dopamine in the perfusate after stimulation of the substantia nigra. For the response to only two stimuli to be observed, one thus had to allow 1 hr for the effect of setting up the perfusion on HVA output to wear off, then 1-5 hr approximately to establish the resting secretion, and 2-5 hr to observe the effect of a single stimulation and return to base line. Not before 5 hr had elapsed could the effect of a second stimulation be tried, and it was rare that during this long time the perfusion pressure had remained completely constant hr 10 8 E 6 '4 2 st. SN * Fig. 7. Columns represent, in ng/min, HVA found in 30 min perfusates of anterior horn of left lateral ventricle of cats given chloralose, 56 mg/kg. Artificial respiration and several doses of 6 mg gallamine triethiodide. At first signal 10 min, and at second signal 20 min stimulation of substantia nigra (SN) using two electrodes (positions A5*5 and A4.0) and trains of three 1 msec square pulses every 3 sec, strength 4V. Because of these technical difficulties, much of the information on the effect of stimulation of single points of the substantia nigra had to be obtained on different cats. Figure 8 shows an example of release of HVA on stimulation at A5-5, and Fig. 3a on stimulation at A 43. Table 2 illustrates the responses in all experiments carried out under chloralose anaesthesia with the exception of those in which basal secretion had either been erratic so that the results were inconclusive, or so high that further increase was unlikely. The lateral position chosen was always in the

19 TRANSMITTERS RELEASED FROM CA UDATE NUCLEUS 705 vicinity of L 3, and only the co-ordinate in the antero-posterior direction was varied. It will be seen that the best results were obtained at positions A4'5 and A5-5. It is perhaps no accident that the largest release of dopamine ever seen was in an experiment with the anterior electrode in A 55. The failure (twice) to release HVA from position A5 seems to be related _A05 hr C6 4 2 st. *SN Fig. 8. Columns represent, in ng/min, HVA found in 25 min effluent from anterior horn of left lateral ventricle of cat given chloralose, 95 mg/kg. Artificial respiration and one dose of 10 mg gallamine triethiodide. At signal, 10 min stimulation of substantia nigra (SN) with single electrode at A5-5 (trains of three 0*5 msec bipolar 4V stimuli every 3 sec). to accidental placing of the electrode in regions containing very few or very small cells; this histological finding is corroborated by the fact that in these cats only small evoked responses were recorded from the substantia nigra on stimulation of the paws. This contrasts with results in positions A3-A4 in which rises in HVA were also slight or absent but paw stimulation evoked large responses; the electrodes were lying in the vicinity of large cells of the substantia nigra compacta. We suspect that a majority of axons from the posterior portions of the substantia nigra may end in the putamen or parts of the caudate nucleus distant from the ventricles. Control experiments. Many control experiments were provided, often unintentionally, by positioning of the electrodes outside the substantia nigra. Release of HVA was never seen. Of the seventeen placements, two were made deliberately in the nucleus ruber and four in the reticular for- 23-2

20 706 P. J. PORTIG AND MARTHE VOGT mation. The others were in the peduncles or in the vicinity of the nucleus ruber. Another control was provided by comparing shape and size of the responses to paw stimulation recorded from the substantia nigra at the beginning and the end of the experiment. If the response was lost during the experiment, any release of HVA seen earlier in the experiment was also lost. TABLE 2. Release of homovanillic acid on stimulation of single points in the substantia nigra ranging in their anterior-posterior co-ordinates from A 3 to A 7 5. All points lay approximately in the sagittal plane L3. Stimulation (4V, usually trains of bipolar stimuli of 0 5 msec repeated once in 3 sec) for 10 consecutive min or for 5 x 2 or 3 x 4 min interrupted by 3 or 4 min pauses. -, no rise; +, small rise; ++, conspicuous rise in output of HVA A A few experiments were carried out in pentobarbitone anaesthesia. They did not lead to release of HVA but the matter was not pursued, because the lack of typical evoked responses both in the substantia nigra and caudate nucleus with this form of anaesthesia (Albe-Fessard et al. 1960; Denavit, 1963), made it difficult to assess the effectiveness of the stimulation while the experiment was in progress. Hyperventilation did not improve the responses. Effect of ions. Release of acetylcholine and noradrenaline from peripheral nerves is calcium-dependent, but release of 5-hydroxytryptamine, at least from electrically stimulated brain slices, is not (Chase, Katz & Kopin, 1969). A few experiments were carried out to see whether the spontaneous or evoked appearance of HVA in ventricular perfusate was affected by doubling the calcium concentration and omitting the magnesium in the artificial cerebrospinal fluid. Of five experiments in which the flow was satisfactory throughout, output of HVA increased twice and twice remained the same; in one of the latter output was already very high before change-over to high calcium perfusion. The fifth experiment showed a fall in HVA but was rendered invalid by a large mid-brain haemorrhage found on sectioning the brain. The rises obtained were not sufficiently striking to warrant more trials, particularly because it was not clear how far the composition of the perfusate determined the calcium concentration in the depth of the tissue.

21 TRANSMITTERS RELEASED FROM CA UDATE NUCLEUS 707 The effect of injecting KCl into the inflow needle was tried with two doses: 5 and 50 /l. of a 3 75 M solution (0 75 and 7 5 ltg). The first caused an evanescent fall in HVA in the collection period following the injection. The second elicited dramatic suppression of the waves in the e.e.g. and in the oscilloscope record from the caudate nucleus; the pupils dilated and the blood pressure rose. HVA output fell abruptly. In another cat, the effluent was tested for dopamine but none was found when 3,tg KCl was injected into the inflow needle. Thoenen, Haefely & Staehelin (1967) reported a large increase in release of noradrenaline from the cat's perfused spleen when they added tetraethylammonium (1.2 It-mole/ml.) to the arterial inflow. An experiment was therefore performed in which, after collecting three control samples, tetraethylammonium 1 a-mole/ml. was added to the perfusion fluid. This caused striking changes in the shape of evoked responses in caudate nucleus or substantia nigra, the simple bi- or triphasic responses being transformed into a series of irregular peaks; resting output of HVA was, however, unchanged and stimulation ofthe substantia nigra with an electrode in A4 5 produced no rise. The matter was therefore not pursued. 5-Hydroxytryptamine The caudate nucleus has a high content of 5-HT, yet very little of this amine is found in perfusates of the lateral ventricle (Feldberg & Myers, 1966). In resting samples we found a concentration of the order of 0 5 ng/ ml. (35 pg/min) and, in contrast to the findings with ACh and HVA, the amount tended to fall in the course of an experiment. Pre-treatment of the cats with nialamide (10-20 mg/kg injected subcutaneously 18 hr before the perfusion) doubled the concentration in the ventricular effluent. Only stimuli were tried which had been found to release either ACh or HVA into the perfusion fluid, such as electrical stimulation of the four paws, of the central ends of both sciatic nerves or of the substantia nigra; they did not change the amount of 5-HT in the perfusate. Perfusion of the ventricles with D-tubocurarine, which causes dopamine and HVA to appear in the perfusion fluid (Portig et al. 1968), had no such effect on 5-HT. DISCUSSION Methods. Inherent in the use of the push-pull cannula for measuring release of transmitter substances are the restriction of sampling to a small point within the tissue and the danger of artifacts due to tissue damage. The ventricular perfusion technique has other, but equally serious drawbacks. Its sampling is from the ventricular surface only, which is larger than the area tested by the push-pull cannula but cannot be varied. For

22 708 P. J. PORTIG AND MARTHE VOGT ACh, a rapidly appearing transmitter which can be stabilized against enzymic destruction, the method was completely satisfactory. For HVA, which leaves the tissue very slowly, difficulties arose. It became apparent even before estimation of HVA was attempted that completely unobstructed outflow was essential in order to obtain a steady base line for any of the substances searched for. By recording perfusion pressure at the inflow cannula the first signs of developing obstruction could be detected and a new start made by adjusting the position of the outflow needle. By such adjustments the pressure was kept within + 3 mm Hg of 'zero', which was the perfusion pressure with the inflow needle delivering its fluid outside the cat but at the height it would later occupy in the ventricles. Satisfactory perfusion pressure was not obtained in experiments in which the atlanto-occipital membrane was left intact; its opening acted as a safety valve in spite of the fact that, as soon as perfusion was started, outflow from the aqueduct was arrested by stoppering the aqueductual cannula. The fall in resting output of HVA produced by raising the pressure inside the ventricles did not appear to be due to neuronal inhibition since it was not accompanied by changes in the electrical activity in caudate nucleus or cortex. It was therefore considered to be caused by reversing the direction of the movement of fluid, which is normally from tissue into ventricles. The fact that, in long perfusions, spontaneous obstructions to flow are apt to develop, was particularly detrimental to experiments on the release of HVA, when stimuli of only a few minutes duration caused increases in output of HVA which lasted up to 1x5 hr. Levinger & Edery (1968) measured the volume of the lateral ventricle of adult cats and found it to range from 0-2 to 1.0 ml. Such variability explains why obstruction to perfusion is more likely to develop in some cats than in others. They also found the right ventricle nearly always larger than the left so that, unknowingly, we chose the narrower ventricle for perfusion. Release of acetylcholine. HMkfelt (1968) calculated that about 16 % of boutons in rat caudate nucleus might contain monoamines. This leaves 84 % showing 'clear' vesicles under the electron microscope, many or all of which might belong to cholinergic neurones. Since ACh, once protected from break-down by an anticholinesterase, is not subject to reuptake into the terminals, it is not surprising that its release from the caudate nucleus is readily shown. The fact that a variety of stimuli caused both evoked responses and appearance of ACh in the perfusate must mean that cholinergic synapses are activated by the afferent stimuli; the experiments do not give information on whether the impulses arriving from the periphery use cholinergic fibres as their last step in the neuronal chain to the

23 TRANSMITTERS RELEASED FROM CAUDATE NUCLEUS 709 caudate nucleus, or activate cholinergic interneurones within the tissue, or do both. The finding that stimulation of the substantia nigra causes an appearance of ACh agrees with the demonstration (Shute & Lewis, 1967) of cholinergic fibres ascending from cells in the substantia nigra and heading towards the basal ganglia; they are probably interrupted in the pallidum. Release of dopamine. Resting release of dopamine was very low, usually below the threshold of the method, and at most pg/min, or 1-2 ng in half an hour. This is the same order of magnitude as observed for release of 5-HT. It accords well with the observations (McKenzie & Szerb, 1968) made with a push-pull cannula inserted into the caudate nucleus: dopamine was not found to be freely diffusible (< 1 ng/50 min appeared in the effluent), whereas its metabolite dihydroxyphenylacetic acid was. On stimulation of the substantia nigra, dopamine appeared in the perfusate on a number of occasions, but the results were inconsistent. This contrasts with consistent release reported earlier (Portig et al. 1968) when the ventricles were perfused with D-tubocurarine. The difference may well be related to the fact that it was purely accidental whether the small region of the substantia nigra stimulated by the electrodes activated ventriclenear synapses, whereas D-tubocurarine may have caused generalized activation of synapses throughout the corpus striatum, including those from where diffusion into the ventricle was possible before the amine was destroyed. Our failure to increase dopamine release by the use of inhibitors of monoamine oxidase and 0-methyl transferase is unlikely to have been due to the use of doses which were too low. The amount of nialamide used ranged from 10 to 80 mg/kg; Lisch, Aigner & Hornykiewicz (1968) showed that nialamide mg/kg greatly inhibited formation of HVA in rabbit brain, and that inhibition was complete at 43 mg/kg. Furthermore, Delorme (1966) was unable to show any monoamine oxidase in the locus coeruleus of the cat after doses of nialamide as low as 8-10 mg/kg. The dose of tropolone should also have been adequate as judged from the amount required for the disappearance of HVA in the brain of mice (Murphy, Robinson & Sharman, 1969). It has been reported (Costa & Neff, 1966) that inhibitors of monoamine oxidase reduce the turnover of dopamine in rat brain, but if, as the authors suggest, this is a result of end-product inhibition (Spector, Gordon, Sjoerdsma & Udenfriend, 1967), it is difficult to see how this mechanism should be operative in the cat in which the dopamine concentration of tissue does not rise when monoamine oxidase inhibitors are administered. However, Davey, Farmer & Reinert (1963) made an interesting observation which may well have a bearing on our results. They found a sharp

24 710 P. J. PORTIG AND MARTHE VOGT reduction in the release of noradrenaline from the perfused spleen of cats 20 hr after an injection of nialamide 20 mg/kg. In spite of a normal noradrenaline content of the tissue, electrical stimulation of the splenic nerves released less amine than in a normal spleen, and the quantity fell rapidly on repeated stimulation. Desmethylimipramine, known to inhibit uptake of noradrenaline, was also used in an attempt at increasing the amount of dopamine in the perfusate. There might have been a small improvement in one experiment; the limited value ofimipramine isinkeeping with the observation (Glowinski et al. 1966) that this drug does not reduce uptake of [3H]dopamine from the ventricles into the striatum of the rat. Furthermore, Fuxe, Hamberger & Malmfors (1966) found that desmethylimipramine did not interfere with the uptake of dopamine into dopaminergic neurones of the rat's median eminence, and Carlsson et al. (1966) showed that striatal neurones -formed dopamine from injected dihydroxyphenylalanine whether or not the rat had been treated with desmethylimipramine; this contrasts with the fact that noradrenaline formation in adrenergic neurones was inhibited by the drug. Release of HVA. If a metabolite of dopamine instead of the amine itself was to be used as a sign of activity of dopaminergic neurones, one had to ascertain that the metabolite was not solely a product of continuous degradation of tissue dopamine, but that its amount was related to neural dopamine release. The fact that 'resting release' was dependent on the depth of anaesthesia was good evidence in favour of the view that HVA concentration depended on neuronal activity and thus on dopamine release. The high basal release of HVA in 'resting' conditions suggests a continuous activity of striatal dopaminergic neurones in pentobarbitone or chloralose anaesthesia. The amount released per minute in animals anaesthetized with the same dose of chloralose varied considerably, but invariably fell with the administration of an additional dose of chloralose. The fact that ACh release is also reduced with deepening anaesthesia might mean that a balance between the activity of cholinergic and dopaminergic neurones is permanently maintained. With two electrodes in the substantia nigra, stimulation usually elicited a long-lasting increment in the HVA content of the perfusate, provided the electrodes were placed sufficiently rostrally. This finding, in conjunction with the evidence that anaesthesia depressed 'resting' release, are strong support for the existence of a dopaminergic nigro-striatal pathway. However, a handicap to further progress lay in the lack of knowledge of the point-to-point relationship of the stipulated nigro-striatal connexions, because it made the interpretation of negative results doubtful;

25 TRANSMITTERS RELEASED FROM CAUDATE NUCLEUS 711 one could never be sure whether they were due to the procedure or drug used, or the part of the substantia nigra stimulated. However, partial information on the anatomical relationship was obtained in the experiments with single electrodes; these suggested that the most caudal parts of the substantia nigra did not send out many axons which ended in the vicinity of the lateral ventricle. In spite of the fact that HVA was continuously released in the chloralosed cat, anaesthesia remained a complication, as it may have modified the normal responsiveness of the neurones we were trying to activate. The effect of anaesthetics on the response of neurones to monoamines applied electrophoretically is very pronounced (Bloom, Costa & Salmoiraghi, 1965; Johnson, Roberts, Sobieszek & Straughan, 1968), and may indicate that certain neurones are permanently silenced by some anaesthetics. Whether, in the present work, the activation of dopaminergic neurones was seriously depressed or modified by chloralose could only be determined by performing ventricular perfusions on animals with permanently implanted cannulae. Since this work was begun, three electrophysiological papers (Albe- Fessard, Raieva & Santiago, 1967; Frigyesi & Purpura, 1967; Connor, 1968) have reported unit responses in the caudate nucleus of the cat on stimulation of the substantia nigra. Their results are compatible with the existence of fine, slow-conducting axons (mean conduction velocity (1.6 m/ sec) originating in the substantia nigra and terminating in the striatum. Both Albe-Fessard et al. and Connor report depressant and facilitatory effects, but neither of the papers explore the point-to-point relationship of the connexions. However, Connor (1968) stimulated the posterior portion of the substantia nigra compacta only, and rarely encountered responding cells near the ventricle; Albe-Fessard et al. stimulated the rostral substantia nigra at A5 and found responding cells adjacent to the lateral part of the ventricle (D. Albe-Fessard, personal communication). Frigyesi & Purpura (1967) concluded from the long latency that the fibres must be thin to conduct so slowly and are therefore likely to originate from small cells in the pars reticularis of the substantia nigra. Doubling the calcium content of, and omitting the magnesium from, the perfusion fluid did not consistently increase the resting output of HVA. Release of noradrenaline from peripheral neurones is calcium dependent; however, release of cerebral 5-HT is not (Chase et al. 1969), and no information is available on cerebral dopamine. KC1, in the doses used, inhibited release, as it inhibited electrical activity in the brain. These experiments require pursuing with another technique of administering the KC1. The electrical changes seen after adding tetraethylammonium to the perfusion fluid were quite striking. The lack of change in the output of

26 712 P. J. PORTIG AND MARTHE VOGT HVA suggests that the electrical effects were perhaps produced on neurones other than those responsible for releasing dopamine. It appeared to be possible to release dopamine or HVA reflexly by stimulating afferent fibres in the sciatic nerves, but the effect was seen so inconsistently that there may have been unknown contributory causes. One such cause could have been shivering which, on several occasions, was seen to coincide with rises in the perfusate of dopamine, HVA, and, in one cat, ACh concentration. Because of the danger of mechanical disturbance of electrodes or cannulae, even with the head fixed in the stereotaxic instrument, gallamine triethiodide was administered as soon as shivering occurred, and this precluded firm establishment of a correlation between shivering and composition of the perfusate. The rise in dopamine and HVA observed (Portig et al. 1968) during intraventricular administration of D-tubocurarine may have its origin in shivering, since this is one of the many signs elicited when this drug penetrates the brain from the ventricular surfaces. Release of 5-hydroxytryptamine. The high concentration of 5-HT in the striatum suggests that terminals of neurones containing this substance are present in this region, but histological evidence for this is still lacking. The present experiments only show that stimuli found to release either dopamine or ACh or both did not release any 5-HT and that, in all probability, the pathways excited by these stimuli do not involve 5-HT-containing neurones. Experiments are planned in which ventricular fluid is examined while the 5-HT-containing raphe nuclei are stimulated since it is possible that this would cause 5-HT release from the caudate nucleus. When a push-pull cannula was lowered into the caudate nucleus and the ventricles were perfused with tritiated 5-HT (Eccleston, Randic, Roberts & Straughan, 1969), no consistent release of radioactive material was seen to follow stimulation of the raphe nuclei; however, similar stimulation released unlabelled 5-HT from the surface of the brain. We are greatly indebted to Professor Denise Albe-Fessard, Paris, for instruction in electro-physiological techniques. We are grateful to Dr H. T. Openshaw (Wellcome Research Laboratories) for gifts of B.W. 284 C51 and B.W. 62 C47, and to Dr R. Barlow, Edinburgh, for dodecamethylene bisquinolinium. We thank Mr J. E. McEwen, F.I.S.T., for skilful estimations of 5-hydroxytryptamine and Mrs Priscilla Mann for careful technical assistance. This work was made possible by the award to P.J.P. of a Riker Fellowship and, from 1965 to 1968, of a grant from the Mental Health Research Fund. REFERENCES ALBE-FESSARD, D., OSWALD0oCnuz, E. & RocHA-MIRANDA, C. (1960). Activit6s evoqu6es dans le noyau caude du chat en r6ponse a des types divers d'aff6rences. I. Rtude macrophysiologique. Electroenceph. clin. Neurophys-ol. 12,

27 TRANSMITTERS RELEASED FROM CAUDATE NUCLEUS 713 ALBE-FESsARD, D., RAEvA, S. & SANTIAGO, W. (1967). Sur lee relations entre substance noire et noyau caude. J. Phyaiol., Pari8 59, ANDEN, N.-E., CARLSSON, A., DAHLSTROM, A., Fuxa, K., HIL ARP, N.-A. & LARSsoN, K. (1964). Demonstration and mapping out ofnigro-neostriatal dopamine neurons. Life Sci. Oxford 3, ANDEN, N.-E., FuxE, K., HAMBERGER, B. & HOKFELT, T. (1966). A quantitative study on the nigro-neostriatal dopamine neuron system in the rat. Acta phy8iol. 8cand. 67, BERTLER, A., FALCK, B. & ROSENGREN, E. (1963). The direct demonstration of a barrier mechanism in the brain capillaries. Acta pharmac. tox. 20, BHATTACHARYA, B. K. & FELDBERG, W. (1958). Perfusion of cerebral ventricles: assay of pharmacologically active substances in the effluent from cisterna and aqueduct. Br. J. Pharmac. Chemother. 13, BLOOM, F. E., COSTA, E. & SALMOIRAGMI, G. C. (1965). Anesthesia and the responsiveness of individual neurons of the caudate nucleus of the cat to acetylcholine, norepinephrine and dopamine administered by microelectrophoresis. J. Pharmac. exp. Ther. 150, BONVALLET, M., DELL, P. & HUGELIN, A. (1952). Projections olfactives, gustatives, visc6rales, vagales, visuelles et auditives au niveau des formations grises du cerveau anterieur du chat. J. Phy8iol., Parin 44, CARLSSON, A. (1959). The occurrence, distribution and physiological role of catecholamines in the nervous system. Pharmac. Rev. 11, CARLSSON, A., FuxE, K., HAmBERGER, B. & LINDQvIsT, M. (1966). Biochemical and histochemical studies on the effects of imipramine-like drugs and (+ )amphetamine on central and peripheral catecholamine neurons. Acta phyeiol. cand. 67, CARmAN, J. B., FAULL, R. L. M. & LAVERTY, R. (1967). Striatal dopamine levels following nigral lesions in the rat. Proc. Univ. Otago med. Sch. 45, CARMICHAEL, E. A., FELDBERG, W. & FLEISCHHAUER, K. (1964). Methods for perfusing different parts of the cat's cerebral ventricles with drugs. J. Phyeiol. 173, CARPENTER, M. B. & McMASTERs, R. E. (1964). Lesions of the 8ubetantia nigra in the rhesus monkey. Efferent fibre degeneration and behavioural observations. Am. J. Anat. 114, CHASE, T. N., KATZ, R. I. & KoPIN, I. J. (1969). Release of [3H]serotonin from brain slices. J. Neurochem. 16, CHASE, T. N. & KoPIN, I. J. (1968). Stimulus-induced release of substances from olfactory bulb using the push-pull cannula. Nature, Lond. 217, CONNOR, J. D. (1968). Caudate unit responses to nigral stimuli: Evidence for a possible nigro-neostriatal pathway. Science, N.Y. 160, COSTA, E. & NEFF, N.H. (1966). Isotopic and non-isotopic measurements of the rate of catecholamine biosynthesis. In Biochemietry and Pharmacology of the Baeal Ganglia, ed. COSTA, E., COWA, L. J. & YAHR, M. D., pp Hewlett, New York: Raven Press. DAHLsTROM, A. & FuxE, K. (1964). Localization of monoamines in the lower brain stem. Experientia 20, 398. DAVEY, M. J., FARmER, J. B. & REINERT, H. (1963). The effect of nialamide on adrenergic functions. Br. J. Pharmac. Chemother. 20, DELORME, F. (1966). Monoamines et sommeils. Etude polygraphique, neuropharmacologique et histochimique des etats de sommeil chez le chat. These, Lyon. DENAVIT, M. (1963). Action diff6rentielle de quelques anesth6siques sur les r6ponses de structures alimentees par les voies sp6cifiques ou associatives. Aneeth. Anaig. Rdanim. 20,

28 714 P. J. PORTIG AND MARTHE VOGT ECCLESTON, D., RANDI6, M., ROBERTS, M. H. T. & STRAUGHAN, D. W. (1969). In Metabolism of Amines in the Brain, ed. HOOPER, G., pp London: Macmillan. EHRINGER, H. & HORNKEWICZ, 0. (1960). Verteilung von Noradrenalin und Dopamin (3-Hydroxytyramin) im Gehirn des Menschen und ihr Verhalten bei Erkrankungen des extrapyramidalen Systems. Klin. Wschr. 38, FELDBERG, W. & MYERS, R. D. (1966). Appearance of 5-hydroxytryptamine and an unidentified pharmacologically active lipid acid in effluent from perfused cerebral ventricles. J. Physiol. 184, FRIGYESI, T. L. & PURPURA, D. P. (1967). Electrophysiological analysis of reciprocal caudato-nigral relations. Brain Res. 6, FuxE, K., HAMBERGER, B. & MALMFORS, T. (1966). Inhibition of amine uptake in tubero-infundibular dopamine neurones and in catecholamine cell bodies of the area postrema. J. Pharm. Pharmac. 18, GADDUM, J. H. (1961). Push-pull cannulae. J. Physiol. 155, 1-2P. GEFFEN, L. B. (1965). The effect of desmethylimipramine upon the overflow of sympathetic transmitter from the cat's spleen. J. Physiol. 181, 69-70P. GLOWINSKI, J., AxELROD, J. & IVERSEN, L. L. (1966). Regional studies of catecholamines in the rat brain. IV. J. Pharmac. exp. Ther. 153, HASSLER, R. (1938). Zur Pathologie der Paralysis agitans und des postenzephalitischen Parkinsonismus. J. Psychol. Neurol., Lpz. 48, HOEFELT, T. (1968). In vitro studies on central and peripheral monoamine neurons at the ultrastructural level. Z. Zellforsch. mikrosk. Anat. 91, HOEFELT, T. & UNGERSTEDT, U. (1969). Electron and fluorescence microscopical studies on the nucleus caudatus putamen of the rat after unilateral lesions of ascending nigro-neostriatal dopamine neurons. Acta physiol. scand. (In the Press.) HUNT, R. (1895). The fall of blood pressure resulting from the stimulation of afferent nerves. J. Physiol. 18, JOHNSON, E. S., ROBERTS, M. H. T., SOBIESZEK, A. & STRAUGHAN, D. W. (1968). Excitation of cortical neurones by noradrenaline. Br. J. Pharmac. Chemother. 34, P. KRNJEVI6, K. & MITCHELL, J. F. (1961). A simple and reliable device utilizing transistors for the maintenance of a constant body temperature. J. Physiol. 158, 6-8P. LEVINGER, I. M. & EDERY, H. (1968). Casts of the cat cerebro-ventricular system. Brain Res. 11, LIscH, H.-J., AIGNER, A. & HORNYKIEWICZ, 0. (1968). Dopamin-Stoffwechsel im Nucleus caudatus des Kaninchens nach partieller Hemmung der Gehirnmonoaminoxydase mittels Nialamid. Arch. Pharmak. exp. Path. 261, LLAMAS, A. & REINOSO-SUAREz, F. (1969). Connections of the substantia nigra and the ventral tegmental mesencephalic area. Third Symposium on Parkinson's disease. Edinburgh: Livingstone. MAcINTOSH, F. C. & OBORIN, P. E. (1953). Release of acetylcholine from intact cerebral cortex. Abstr. Int. physiol. Congr. XIX, pp MCKENZIE, G. M. & SZERB, J. C. (1968). The effect of dihydroxyphenylalanine, pheniprazine and dextroamphetamine on the in vivo release of dopamine from the caudate nucleus. J. Pharmac. exp. Ther. 162, McLENNAN, H. (1964). The release of acetylcholine and of 3-hydroxytyramine from the caudate nucleus. J. Physiol. 174, McLENNAN, H. (1965). The release of dopamine from the putamen. Experientia 21, MITCHELL, J. F. (1963). The spontaneous and evoked release of acetylcholine from the cerebral cortex. J. Physiol. 165,

29 TRANSMITTERS RELEASED FROM CAUDATE NUCLEUS 715 MURPHY, G. F., ROBINSON, D. & SHARMAN, D. F. (1969). The effect of tropolone on the formation of 3,4-dihydroxyphenylacetic acid and 4-hydroxy-3-methoxyphenylacetic acid in the brain of the mouse. Br. J. Pharmac. Chemother. 36, POIRIER, L. J. & SOURKES, T. L. (1965). Influence of the substantia nigra on the catecholamine content of the striatum. Brain 88, POLAK, R. L. (1965). Effect of hyoscine on the output of acetylcholine into perfused cerebral ventricles of cats. J. Physiol. 181, PORTIG, P. J., SHARMAN, D. F. & VOGT, M. (1968). Release by tubocurarine of dopamine and homovanillic acid from the superfused caudate nucleus. J. Physiol. 194, PORTIG, P. J. & VOGT, M. (1966). Search for substances released on stimulation of the caudate nucleus in the cat. J. Phy8iol. 186, P. PORTIG, P. J. & VOGT, M. (1968). Activation of a dopaminergic nigro-striatal pathway. J. Phyaiol. 197, P. RECH, R. H. & DOMINO, E. F. (1959). Observations on injections of drugs into the brain substance. Arch8 int. pharmacodyn. Th&r. 121, RICHARDSON, K. C. (1966). Electron microscopic identification of autonomic nerve endings. Nature, Lond. 210, 756. ROSEGAY, H. (1944). An experimental investigation of the connections between the corpus striatum and substantia nigra in the cat. J. comp. Anat. 80, ROWLAND, V. (1966). Stereotaxic techniques and the production of lesions. Neuroendocrinology 1, SEGUNDO, J. P. & MACHNE, X. (1956). Unitary responses to afferent volleys in lenticular nucleus and claustrum. J. Neurophysiol. 19, SHUTE, C. C. D. & LEWIS, P. R. (1967). The ascending cholinergic reticular system: neocortical, olfactory and subcortical projections. Brain 90, SPECTOR, S. (1963). Monoamine oxidase in control of brain serotonin and norepinephrine content. Ann. N.Y. Acad. Sci. 107, SPECTOR, S., GORDON, R., SJOERDSMA, A. & UDENFRIEND, S. (1967). End-product inhibition of tyrosine hydroxylase as a possible mechanism for regulation of norepinephrine synthesis. Mol. Pharmacol. 3, THOENEN, H., HAEFELY, W. & STAEHELIN, H. (1967). Potentiation by tetraethylammonium of the response of the cat spleen to postganglionic sympathetic nerve stimulation. J. Pharmac. exp. Ther. 157, TRE'TIAKOFF, C. (1919). Contribution 'a l'etude de l'anatomie pathologique du locus niger de Soemmering. These, Paris. VANE, J. R. (1957). A sensitive method for the assay of 5-hydroxytryptamine. Br. J. Pharmac. Chemother. 12, VOGT, M. (1954). The concentration of sympathin in different parts of the central nervous system under normal conditions and after the administration of drugs. J. Physiol. 123, VOGT, M. (1959). Catecholamines in brain. Pharmac. Rev. 11, VOGT, M. (1965). Transmitter released in the cat uterus by stimulation of the hypogastric nerves. J. Physiol. 179, VOGT, C. & VOGT, 0. (1920). Zur Lehre der Erkrankungen des striaren Systems. J. Psychol. Neurol., Lpz. 25,