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Release of putative transmitters from the corpus striatum
Pharmac. Therap. B, 1975, Vol. 1, No. I, pp. 39-47.
Pergamon Press.
Printed in Great Britain
Specialist Subject Editor: O. HORNYKIEWICZ
RELEASE OF PUTATIVE FROM THE CORPUS
TRANSMITTERS STRIATUM
MARTHE VOGT Institute of Animal Physiology, Babraham, Cambridge, England
1. INTRODUCTION The corpus striatum--a term which comprises the caudate nucleus and putamen of the telencephalon---differs from other parts of the brain in its content of the so-called 'monoamines' epinephrine, norepinephrine, dopamine (DA) and 5-hydroxytryptamine (5°HT). Only DA and 5-HT are present in what can be considered physiologically important concentrations; thus the cat caudate nucleus contains 10/xg DA per g fresh tissue (Laverty and Sharman, 1965), and approximately 0.37/xg 5-hydroxytryptamine. The striatum is rich in a third base, acetylcholine (ACh), considered by most workers to be a transmitter. The fact that the monoamines can be visualized within neurones, at least in the cells and in the terminals (Dahlstr6m and Fuxe, 1964; Fuxe, 1965) has greatly strengthened the suspicion that their function in the brain is also that of transmitting impulses. Neither DA nor 5-HT are known to act as transmitters in peripheral mammalian nerves, but they play this role in the invertebrate nervous system. The desire to have direct rather than circumstantial evidence for the transmitter nature of monoamines has prompted experiments which would show that appropriate stimuli cause the release of these compounds. In the brain, there are essentially two ways of demonstrating release of substances in response to stimuli, the local perfusion of a small area of tissue with the push-pull cannula (Gaddum, 1961), and the collection of fluid bathing either the external (Macintosh and Oborin, 1953) or the internal brain surfaces (Carmichael et al., 1964). The advantage of the push-pull cannula is that it can be inserted into almost any part of the brain and allows a strictly localized perfusion to be carried out. Its drawback is the production of some local tissue damage which may result in the escape of any tissue constituent when there is an inflow of impulses into the region. Such leakage may be due to changes in blood flow or cell permeability rather than to activation of transmitter release. The possibility of such artefacts has been demonstrated (Chase and Kopin, 1968). It is thus desirable to obtain confirmation by other methods of any results based on the use of the push-pull cannula. The simplest way to obtain fluid which has been in contact with the surface of part of the striatum is to perfuse the anterior horn of the lateral ventricle; such a perfusion is, in fact, a 'superfusion' of the grey masses of the caudate nucleus and the septum. A more elaborate way of superfusing a restricted region of the caudate nucleus has recently been devised (Besson et al., 1971); it necessitates removal of the overlying cerebral cortex in order to apply a perspex cup to the exposed caudate. 2. RESULTS OBTAINED WITH T H E P U S H - P U L L C A N N U L A In the earlier experiments, the output of endogenous DA was studied by fluorimetric estimation. McLennan (1964, 1965) placed a push-pull cannula into the caudate nucleus and saw a release of ACh into the effluent when the nucleus ventralis anterior thalami, and of DA when the nucleus centrum medianum was stimulated. Stimulation of the substantia nigra caused the appearance of DA when the cannula was inserted into the putamen, not when it lay in the caudate nucleus. The author observed a resting release of DA of about 1 ng/min; in contrast, McKenzie and Szerb (1968), also using
MARTHE VOGT
fluorimetry, found no DA (< 1 ng/50 min) under resting conditions. They were, however, able to obtain DA in the effluent when dextroamphetamine was added to the perfusing fluid; this effect was explained by inhibition of uptake. Quite recently, Riddell and Szerb (1971) have labelled the DA in the caudate nucleus by perfusing L-['4C]tyrosine through the push-pull cannula. Labelling always poses a problem of equilibration with different 'pools', but overcomes the difficulty of the insufficient sensitivity of the fluorimetric estimations. On stimulating the rostral substantia nigra, the authors obtained an increase of ['4C]DA in the effluent; no such increase occurred when the stimulation was carried out more caudally. These results are in good agreement with those obtained (Portig and Vogt, 1969) on endogenous DA, which are discussed below. 3. R E S U L T S O B T A I N E D W I T H V E N T R I C U L A R P E R F U S I O N Cats anaesthetized with chloralose (60-90 mg/kg) were used in this work. The perfusion technique is illustrated in Fig. 1; the shaded area represents the perfused anterior horn of the lateral ventricle. Perfusion pressure is measured by connecting the inflow tubing to a transducer. In order to get a valid baseline of 'resting release', it is essential that perfusion pressure remains absolutely steady. If any small obstruction causes a rise in pressure and necessitates adjustment of the outflow needle, new control samples have to be taken before any stimulus can be applied to the brain.
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J FIG. 1. Position of cannulae for perfusion of the anterior horn of the lateral cerebral ventricle of the cat. Perfused area shaded. (Modified from Carmichael et al. (1964) by permission of jr. Physiol. Lond.)
3.1 ACETYLCHOLINE The suitability of the perfusion technique for the detection of transmitters is shown in experiments in which the release of ACh was examined. In order to preserve the ACh, neostigmine (as methylsulphate, 6 x 10 5 M) was added to the perfusion fluid. This causes, at first, a steep rise in resting concentrations of ACh in the effluent, but after several hours a plateau is reached. The amount of ACh in the perfusate may be increased further by injecting the cats with atropine sulphate (3 mg/kg, either subcutaneously or intravenously) at the start of the experiment (see Mitchell, 1963). On the average, the amount of ACh released 'at rest' was of the order of 3 ng]min when the cats had been treated with atropine. There are a great number of stimuli capable of increasing this basal release (Portig and Vogt, 1966). Effective sensory stimuli include electrical stimulation of the paws, or of the central ends of the severed sciatic nerves, and loud noise. The same effect is seen on stimulation of many regions of the brain, such as the nucleus central lateralis thalami, the substantia nigra and (not consistently) the contralateral caudate nucleus.
Release of putativetransmitters from the corpus striatum
41
To these sites may be added certain areas of the frontal cortex (Mitchell and Szerb, 1962) and the nucleus ventralis anterior thalami (McLennan, 1964), the stimulation of which caused release of ACh into the effluent of a push-pull cannula inserted into the caudate nucleus. Figure 2 (Portig and Vogt, 1969) illustrates an experiment in which four different stimuli were applied for 15 min each, and increments in ACh content of the perfusate were observed during, and for 15 min following each stimulation. It is also evident that the resting release increased throughout the observation period, yet this did not obscure the sudden rises initiated by each stimulation period. The upper set of columns (Fig. 2) indicates the flow of perfusate during the experiment. It remained constant throughout, thus ruling out the possibility that the changes in ACh concentration were artefacts due to irregularities in the perfusion. .£
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FIG. 2. 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 chloralose. The perfusion fluid contained neostigmine methylsulphate 6 x 10-~ M. Atropine sulphate, 2 mg/kg, had been injected intravenously 20 min before collecting the first sample of perfusate. The signals mark stimulation periods: 1st bar, electrical stimulation of the skin (Sk) of both forepaws; 2nd bar, stimulation 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 AI5, L4.5, H5.5) with biphasic pulses of 1 msec duration, 1 every 3 sec, 20 V. Increments in ACh output emphasized by shading. By permission of J. Physiol. Lond.
Anatomical and electrophysioiogical facts are in agreement with the result that ACh can be released from the caudate nucleus by a multitude of stimuli. Acetylcholine and choline acetyl transferase concentrations in the striatum are among the highest in the central nervous system; much of the ACh is situated in the neuropil and not in cells (Krnjevi6 and Silver, 1965, 1966). Evoked responses in the striatum can be elicited by afferent stimuli of any modality. The ACh released into the lateral ventricle thus probably originates from terminals of either a final cholinergic pathway used by the afferent stimuli or from cholinergic interneurones within the tissue. The septum is not likely to contribute to this release, or only to a small extent, because its ACh is mainly situated in cells giving rise to axons ending outside the septum (Krnjevi6 and Silver, 1965, 1966). 3.2. DOPAMINE The observation (Ehringer and Hornykiewicz, 1960) that the striatum of Parkinsonian patients, while histologically fairly intact (Vogt and Vogt, 1920), contains only a small fraction of the normal concentration of DA, might have been interpreted as a 'biochemical lesion'. However, when this finding was coupled with the observation made in 1919 by Tr6tiakoff (and confirmed since) that the main anatomical lesion in Parkinson's disease is in the substantia nigra, and, further, with the demonstration of the presence of DA, but of hardly any noradrenaline, in the cells of the substantia nigra (Bertler, 1961), another interpretation of the loss of DA suggested itself: the existence of a dopaminergic nigro-striatal pathway was postulated, and its destruction by
42
MARTHE VOGT
infection or degeneration b e c a m e the most likely explanation of P a r k i n s o n ' s disease. Much work which is outside the scope of this article has been done in the last decade in support of the existence of this pathway, and the attempt at releasing DA from the striatum by stimulation of the substantia nigra is but one a p p r o a c h to the problem. In spite of the very intense fluorescence seen when striatal tissue is freeze-dried and exposed to f o r m a l d e h y d e vapour, H 0 k f e l t (1968) calculated that only about 16 per cent of boutons in rat caudate nucleus might contain monoamines. Since some of these terminals belong to tryptaminergic neurones, the n u m b e r of terminals containing DA must be smaller still. This alone might contribute to the fact that it is more difficult to d e m o n s t r a t e a release of DA than of ACh, but there are more fundamental differences between the two situations. In the p r e s e n c e of an anticholinesterase, ACh is not only protected f r o m destruction, but there is no re-uptake of transmitter, since only free choline can re-enter the neurone. To achieve the same for DA, it would be necessary to employ three drugs, two (an inhibitor of m o n o a m i n e oxidase and one of Omethyltransferase) for the prevention of enzymatic destruction, and one to prevent re-uptake. Though this is possible, and the n u m b e r of drugs can be reduced by using inhibitors of m o n o a m i n e oxidase which also prevent uptake, the conditions created are highly unnatural. Our first experiments (Portig and Vogt, 1966, 1969) were therefore carried out without the use of inhibitors. The method of estimation of DA was fluorimetry of the acetylated derivative ( L a v e r t y and Sharman, 1965). The 'resting' effluent contained less DA than could be estimated with the very sensitive method (<1 ng in 30 min perfusate), and afferent stimuli of the kind which release ACh did not induce the a p p e a r a n c e of DA. H o w e v e r , when the DA-containing cells of the substantia nigra were stimulated, DA was found in the effluent in some experiments but not in others; the amount released varied b e t w e e n 2 and 20 ng in 30 min and did not spill over into the post-stimulation sample. There were obvious reasons which might explain the inconsistency of the results: one could only hope for any DA to reach the ventricles if it was released f r o m terminals which were so near the ventricular surface that the DA could diffuse into the perfusate before it had been destroyed by the metabolizing enzymes. Since, however, there was no anatomical guidance indicating which part of the substantia nigra might contain cells which gave rise to ventricle-near terminals, it was left to chance whether or not a particular electrode placement could release DA at a site f r o m which it could enter the ventricles before being destroyed. There is, at present, no solution to the anatomical problem, but there were two ways in which the difficulty presented by the metabolic destruction could be o v e r c o m e . One was the inhibition of m o n o a m i n e oxidase and O-methyltransferase, the other was to e m p l o y a metabolite as an index of DA release. The first way was not successful, possibly because the e n z y m e inhibitors interfered with transmitter release, but the second one was: it consisted in using the increased formation of the main metabolite of DA, homovanillic acid (HVA) to detect the release of DA. The estimation was b y fluorimetry (Portig et al., 1968). Figures 3-5 illustrate some of the results. There is a high resting release of H V A into the effluent, the amount ranging a p p r o x i m a t e l y f r o m 2-8 ng/min. Figure 3 shows that this resting outflow falls as the depth of anaesthesia is increased. In both halves of Fig. 3, additional chloralose was injected intravenously at the arrow, and this reduced the basal release by about 25 per cent. This finding suggests that the height of the basal release depends on continous neuronal activity, which is depressed by additional anaesthetic. As can also be seen in Fig. 3, stimulation of the substantia nigra, but not a period of continuous loud noise, increased the release of H V A . The increase outlasted the stimulation period, as is even more clearly shown in Figs. 4 and 5, where brief stimuli were applied, lasting 10 (Fig. 4) and 4 (Fig. 5) min only. Following the short stimuli, increased output continued for 50 min (Fig. 4) or 90 min (Fig. 5). This long duration is what one might expect if the H V A reaching the ventricles was released at different depths within the caudate nucleus: the first 25 min-sample would contain the metabolite of DA released f r o m terminals near the surface, and the later samples H V A f o r m e d at greater depth. N o t only did sensory stimuli usually fail to release any H V A , but electrical stimulation of the reticular f o r m a t i o n caused no release either.
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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 of substantia nigra (SN) at co-ordinates A4.3, L3.5, H-4.2 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. (b) At the arrow, rapid intravenous injection of additional chloralose, 30 mg/kg, 5 hr after first dose. Signal: loud noise (NOI) for l0 min. Note that the scales for HVA in (a) and (b) are different. By permission of J. Physiol. Lond.
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mSn FIG. 4. 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. At signal, l0 rain stimulation of substantia nigra (Sn) with electrode at AS.5,L3,H-5 (trains of three 0.5 msec biphasic 4V stimuli every 3 sec). By permission of J. Physiol. Lond.
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ST' FIG. 5. Columns represent, in rig/rain, HVA found in 30 min perfusates of anterior horn of left lateral ventricle of cat given chloralose, 52 mg/kg. Signal indicates 4 min stimulation (ST) of substantia ni~ra with two electrodes at nositions A5.7 and A4.5.1,3. l-l-5: trains of three 1 msec
44
MARTHE VOGT
A little information was obtained on the point, raised earlier, whether an identifiable part of the substantia nigra sends its terminals to ventricle-near regions of the caudate nucleus. Positioning of the electrodes in the same parasagittal plane of L3, but in frontal planes ranging f r o m A3 to A7, showed fairly consistent release f r o m A5.5 to A7, and frequent failures b e t w e e n A3 and A4. It thus appears that the cells in the more caudal regions send their terminals either into the p u t a m e n or into deep layers of the caudate nucleus, f r o m where H V A cannot reach the ventricle. Recently, Besson et al., (1971) tried to determine release of endogenous D A with their perspex cup method discussed above. T h e y were not successful and, therefore, labelled the D A b y superfusing the tissue with [3H]tyrosine. Application of KCl increased the amount of [3H]DA appearing in the cup. So did superfusion with an inhibitor of m o n o a m i n e oxidase or with a m p h e t a m i n e ; these effects were attributed to inhibition of re-uptake of DA. Similarly, Voigtlander and Moore (1971a, b) labelled striatal DA by injecting [3H]DA into the ventricles and, 1 hr later, perfusing the entire ventricular system. Stimulation of the substantia nigra caused an increment in [3H]DA content of the effluent amount to 15 per cent; a m u c h large release was obtained when the nigro-striatal fibres were stimulated in the lateral hypothalamus. 3.3. 5-HYDROXYTRYPTAMINE As previously found b y Feldberg and Myers (1966), basal release of 5-HT f r o m the anterior horn of the lateral ventricle is v e r y low, on the average (in our hands) <0.5/xg in 25 min. After nialamide, usually given subcutaneously 16 hr before the experiment in a dose of 25 mg/kg, this figure rose to 0.5, and, frequently, to 1 ng in 25 min. Assay of these small quantities has to be done biologically, on the rat stomach strip (Vane, 1957). On employing stimuli which had been shown to release either A C h or H V A , no increment in the outflow of 5-HT was seen (Portig and Vogt, 1969). H o w e v e r , it was likely that release might be obtained if it was possible to find and stimulate cells which give rise to tryptaminergic neurones terminating in nuclei adjacent to the lateral ventricle. Cells containing 5-HT are present in most, if not all, raphe nuclei (Dahlstr6m and Fuxe, 1964); furthermore, ascending efferents to the striatum were seen in one cat to originate f r o m the nucleus linearis rostralis raphe (Brodal et al., 1960). Electrodes were, therefore, placed ( H o l m a n and Vogt, 1970, 1972) into the two m o s t anterior nuclei of the raphe, i.e., the pars rostralis and pars intermedia of the nucleus linearis raphe (for nomenclature see T a b e r et al., 1960). The cats were pretreated with nialamide as described above, and stimulation of one of the divisions of the nucleus linearis was carried out for 15 min with biphasic stimuli at frequencies varying b e t w e e n 0.5 and 2 0 H z . Voltage was between 3 and 4 V . W h e n e v e r the electrode was in, or in the immediate vicinity of, one of the two nuclei, there was a release of 5-HT. When stimulation occupied the first 15 min of a 25 min collection period, the increment did not spill over into the next sample of perfusate. The average increase was 1 ng in 25 min, and represented usually a doubling of the basal 5-HT concentration. N o release was seen when the electrodes were positioned in structures other than the nucleus linearis, such as the red nucleus, the habenulointerpeduncular bundle, the decussation of the superior cerebellar peduncles, the interpeduncular nucleus, the third nucleus, the fasciculus longitudinalis medialis or the reticular formation. In several experiments, the electrode was lowered stepwise and stimulation carried out at different depths. Only when the electrode had reached the surface of the nucleus linearis did 5-HT release occur. Thus in one cat, the electrode passed just lateral to the nucleus linearis intermedius, and identical stimuli were applied at three heights, - 3.5, - 4 . 2 5 and - 5 . 0 ram. The first position made the electrode tip level with the upper border of the nucleus, and stimulation caused the release of 0.5 ng 5-HT. The second position brought the tip in contact with the bulk of the cells, and release a m o u n t e d to 1.0 ng 5-HT. The third position lowered the electrode into the interpeduncular nucleus, and no 5-HT was released. Figures 6 and 7 give an example each of stimulation of the nucleus linearis intermedius (Fig. 6) and the nucleus linearis rostralis (Fig. 7). When
Release of putative transmitters from the corpus striatum
45
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5 O~ 20/sec FIG. 6. Perfusate from anterior horn of lateral ventricle in cat anaesthetized with chloralose 65 mg/kg. Nialamide injected twice s.c., 30 mg/kg 3 days, and 20 mg/kg 1 day before the experiment. Columns represent 5-HT content (in ng) of consecutive 25 rain samples of perfusate. Stimulation of nucleus linearis intermedius (A2, L0, H - 3.5) during the first 15 min of collection periods 3, 6 and 9 with biphasic pulses of 0.5 msec duration and 4 V. The three frequencies used are indicated at the foot of the column. Increments indicated by shading. Note that release per stimulus is highest at the lowest frequency. By permission of Jr. Physiol. Lond.
41
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FIG. 7. Perfusate from anterior horn of right lateral ventricle in cat anaesthetized with chloralose 60 mg/kg. Nialamide 30 mg/kg injected subcutaneously on day preceding experiment. Columns represent 5-HT content (in ng) of successive 25 min samples of perfusate; stimulation of nucleus linearis rostralis at position A4.0, LI.0, H -4.5 for the first 15 rain of samples 3 and 6, with biphasic pulses of 0.5 msec and 3 V; frequencies 5 and 20/sec. Increments indicated by shading. There is little difference between the effect of the two frequencies. By permission of J. Physiol. Lond. t h r e e d i f f e r e n t f r e q u e n c i e s w e r e u s e d , e a c h f o r 15 min (Fig. 6), 20 H z w a s f o u n d to b e slightly m o r e e f f e c t i v e t h a n 5 H z , a n d 0.5 H z r e l e a s e d less t h a n either. A b o v e 20 H z , r e l e a s e d e c l i n e d s h a r p l y . W i t h i n t h e r a n g e o f 0.5 to 20 H z , t h e h i g h e s t r e l e a s e p e r s h o c k w a s at t h e l o w e s t f r e q u e n c y (Fig. 6). This w a s p a r t i c u l a r l y s t r i k i n g in e x p e r i m e n t s , in w h i c h t h e s a m e n u m b e r o f stimuli w e r e a p p l i e d at d i f f e r e n t f r e q u e n c i e s : thus, on o n e o c c a s i o n , 750 stimuli at 0 . 5 H z r e l e a s e d 1 . 6 n g 5 - H T , b u t at 5 H z no r e l e a s e w a s d e t e c t a b l e . T h e t w o p a r t s o f t h e n u c l e u s linearis r e a c t e d in t h e s a m e w a y to d i f f e r e n t frequencies. R e l e a s e o f 5 - H T w a s f o u n d to b e h i g h l y d e p e n d e n t on b o d y t e m p e r a t u r e . A l t e r n a t e cooling and warming of the cat while perfusate was being collected throughout the day, c a u s e d s w i n g s in t h e b a s a l r e l e a s e o f 5 - H T w h i c h f o l l o w e d t h e b o d y t e m p e r a t u r e w i t h a lag of a b o u t half an h o u r . I n o n e cat, f o r e x a m p l e , r e l e a s e w a s 0.66 ng/25 min at a b o d y t e m p e r a t u r e o f 33°C, a n d r o s e to 2.7 ng/25 min w h e n t h e t e m p e r a t u r e h a d r e a c h e d 40.2°C. T h i s p h e n o m e n o n in t h e a n a e s t h e t i z e d a n i m a l h a s p r o b a b l y n o t h i n g to d o w i t h t e m p e r a t u r e r e g u l a t i o n b u t m a y be a sign o f g e n e r a l i z e d i n c r e a s e in n e u r o n a l a c t i v i t y with cerebral temperature.
46
MARTHE VOGT
It has been shown in the preceding sections that stimulation of the substantia nigra m a y release ACh and DA, a finding suggesting that both dopaminergic and cholinergic neurones originate in that region. It seemed of interest to see whether stimulation of the nucleus linearis released ACh in addition to 5-HT. The results of experiments, in which ACh and 5-HT were either determined in the same control and stimulation samples, or in two successive sets of such samples (which included a stimulation period), were quite unambiguous: w h e n e v e r the electrode was inside the nucleus linearis and 5-HT was released, there was no increment in the ACh content of the samples (Ashkenazi et al., 1972). There is thus no indication that the release of 5-HT is caused by, or linked with, simultaneous release of ACh. The cells in the nucleus linearis appear to give rise to tryptaminergic neurones only. The terminals in the vicinity of the anterior horn of the lateral ventricle which are activated by stimulation of the raphe nuclei m a y lie in two grey structures, the caudate nucleus or the septum. The septum has the smaller ventricular surface, but the higher concentration of 5-HT. Since in both tissues the 5-HT might be localized in terminals, it is possible that both regions contribute to the release. This interpretation is supported b y the results of 5-HT estimations after destruction of the putative ascending pathways. When lesions were placed in the t e g m e n t u m (Poirier et al., 1967), the 5-HT content of the striatum fell; but so did that of the septum (Heller and Moore, 1968) after section of the medial forebrain bundle in which aminergic fibres originating in the midbrain are said to travel to the telencephalon. T r y p t o p h a n hydroxylase, too, was reduced in both septum and caudate after ventromedial tegmental lesions (Poirier et al., 1969). Thus the demonstration that stimulation of the nucleus linearis raphe releases 5-HT from the surface of the grey structures bordering on the anterior horn of the lateral ventricle are evidence for the existence of tryptaminergic p a t h w a y s originating in the nucleus linearis and probably terminating in both caudate nucleus and septum. 4. S U M M A R Y The e x p e r i m e n t s reported in this article d e m o n s t r a t e the release of ACh, DA and 5-HT f r o m striatal tissue. The technique used was the perfusion of the anterior horn of the lateral ventricle. The ACh and DA appearing in the perfusate must h a v e originated f r o m the caudate nucleus only, whereas 5-HT was probably released f r o m terminals in the septum as well. The results support the view that the m o n o a m i n e s act as transmitters of impulses, and that the cells in the midbrain which contain DA and 5-HT send uninterrupted axons into different parts of the telencephalon. ADDENDUM During the two years which have elapsed since this review was written, progress has been made in releasing labelled DA f r o m the caudate nucleus into cerebrospinal fluid. Von Voigtlander & Moore (1973) added [3H]DA to ventricular perfusates to label the caudate nucleus, and found that a m p h e t a m i n e increased the a p p e a r a n c e of labelled c o m p o u n d , and that the combination of electrical stimulation of the nigro-striatal p a t h w a y with a m p h e t a m i n e produced even greater effects. Chiueh and Moore (1973) showed that [14C]DA f o r m e d in the caudate nucleus by perfusion with ['4C]tyrosine could also be liberated with a m p h e t a m i n e or by electrical stimulation. Release of ACh from the caudate nucleus in the cat has been studied with the push-pull cannula (Stadler et al., 1973) and the interesting observation made that chlorpromazine, k n o w n to increase the turnover of DA, causes an enhanced a p p e a r a n c e of ACh; a correlation between release of the two transmitters does, therefore, not only hold when release is dependant on the depth of anaesthesia (see p. 4). REFERENCES ASHKENAZI,R., HOLMAN,R. B. and VOGT,M. (1972) Release of transmitters on stimulation of the nucleus linearis raphe in the cat. J. Physiol. Lond. 223: 255-259. BERTLER, ,~. (1961) Occurrence and localization of catecholamines in the human brain. Acta physiol, scand.
Release of putative transmitters from the corpus striatum
47
BESSON, M.-J., CHERAMY, A., FELTZ, P. and GLOW/NSKI, J. (1971) Dopamine: spontaneous and drug-induced release from the caudate nucleus in the cat. Brain Res. 32: 407-424. BRODAL,A. E., TABER,E. and WALBERG,F. (1960) The raphe nuclei of the brain stem in the cat. II. J. comp. Neurol. 114: 239-259. CARMICHAEL, E. A., FELDBERG,W. and FLEISCI-IHAUER,K. (1964) Methods for perfusing different parts of the cat's cerebral ventricles with drugs. J. Physiol. Lond. 153: 354-367. CHASE, T. N. and KOPtN, I. J. (1968) Stimulus-induced release of substances from olfactory bulb using the push-pull cannula. Nature, Lond. 217: 466-467. CI-IIUEH, C. C. and MOORE, K. E. (1973) Release of endogenously synthesized catechols from the caudate nucleus by stimulation of the nigro-striatal pathway and by the administration of d-amphetamine. Brain Res. 50: 221-225. DAHESTROM, A. and FUXE, K. (1964) Evidence for the existence of monoamine-containing neurons in the central nervous system. I. Acta physiol, scand. 62: suppl. 232. ErlRINGER, H. and HORNYKIEWlCZ, O. (1960) Verteilung von Noradrenalin und Dopamin (3-Hydroxytyramin) im Gehirn des Menschen und ihr Verhalten bei Erkrankungen des extrapyramidalen Systems. Klin. Wschr. 38: 1236-1239. FEEDBERG, W. and MYERS, R. D. (1966) Appearance of 5-hydroxytryptamine and an unidentified pharmacologically active lipid acid in effluent from perfused cerebral ventricles. J. Physiol. Lond. 184: 837-855. FUXE, K. (1965) Evidence for the existence of monoamine neurons in the central nervous system. IV. Acta physiol, scand. 64, suppl. 247: 37-85. GADDUM, J. H. (1961) Push-pull cannulae. J. Physiol. Lond. 155: 1-2P. HEELER, A. and MOORE, R. Y. (1968) Control of brain serotonin and norepinephrine by specific neural systems. Adv. Pharmac. 6A: 191-206. HOKFELT, T. (1968) In vitro studies on central and peripheral monoamine neurons at the ultrastructural level. Z. Zellforsch. mikrosk. Anat. 91: 1-74. HOEMAN, R. B. and VOGT, M. (1970) Release of 5-hydroxytryptamine (5-HT) from septum and caudate nucleus. J. Physiol. Lond. 210: 163P. HOE~AN, R. B. and VOGT, M. (1972) Release of 5-hydroxytryptamine from caudate nucleus and septum. J. Physiol. Lond. 223: 243-254. KRNJEVIt~, K. and SILVER, A. (1965) A histochemical study of cholinergic fibres in the cerebral cortex. J. Anat. 99: 711-759. KRNJEVI~, K. and Sn.v ER, A. (1966) Acetylcholinesterease in the developingforebrain. J. Anat. 100: 63-89. LAVERTY, R. and SHARMAN,D. F. (1965) The estimation of small quantities of 3,4-dihydroxyphenylethylamine in tissues. Br. J. Pharmac. Chemother. 24: 538-548. MACINTOSH, F. C. and OBOR1N, P. E. (1953) Release of acetylcholine from intact cerebral cortex. Abstr. Int. physiol. Congr. XIX, pp. 580-581. MCKENZIE, G. M. and SZERI3, 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: 302-308. MCLENNAN, H. (1964) The release of acetylcholine and of 3-hydroxytyramine from the caudate nucleus. J. Physiol. Lond. 174: 152-161. MCLENNAN, H. (1965) The release of dopamine from the putamen. Experientia 21: 725-726. MITCHELL, J. F. (1963) The spontaneous and evoked release of acetylcholine from the cerebral cortex. J. Physiol. Lond. 165: 98-116. MITCHELL, J. F. and SZERB, J. C. (1962) The spontaneous and evoked release of acetylcholine from the caudate nucleus. Excerpta Medica, Series No. 48, Leiden, Abstr. No. 819. POmlER, L. J., MCGEER, E. G., LAROCrtEELE, L., MCGEER, P. L., BEDARD, P. and BOUCHER, R. (1969) The effect of brain stem lesions on tyrosine and tryptophan hydroxylases in various structures of the telencephalon of the cat. Brain Res. 14: 147-155. PO1RIER, L. J., SINGH, P., BOUCHER, R., BOUVlER, G., OHVlER, A. and LAROCHELLE, P. (1967) Effect of brain lesions on striatal monoamines in the cat. Arch. Neurol. 17: 601-608. PORTIG, P. J., SHARMAN, D. F. and VO~3T, M. (1968) Release by tubocurarine of dopamine and homovanillic acid from the superfused caudate nucleus. J. Physiol. Lond. 194: 565-572. PORTIG, P. J. and VOGT, M. (1966) Search for substances released on stimulation of the caudate nucleus in the cat. J. Physiol. Lond. 186: 131-132P. PORTIG, P. J. and VOGT, M. (1969) Release into the cerebral ventricles of substances with possible transmitter function in the caudate nucleus. J. Physiol. Lond. 2114: 687-715. RIDDELL, D. and SZERB, J. C. (1971) The release in vivo of dopamine synthesized from labelled precursors in the caudate nucleus of the cat. J. Neurochem. 18: 989-1006. STADEER, H., LLOYD, K. G., GADEA-CmIA, M. and BARTHOEINI, G. (1973) Enhanced striatal acetylcholine release by chlorpromazine and its reversal by apomorphine. Brain Res. 55: 476-480. TABER, E., BRODAL, A. and WALBERG, F. (1960) The raphe nuclei of the brain stem in the cat. I. J. comp. Neurol. 114: 161-187. TR~TIAKOFF, C. (1919) Contribution ~ l'6tude de I'anatomie pathologique du locus niger de Soemmering. Th~se, Paris. VANE, J. R. (1957) A sensitive method for the assay of 5-hydroxytryptamine. Br. J. Pharmac. Chemother. 12: 344-349. VOGT, C. and VOGT, O. (1920) Zur Lehre der Erkrankungen des stri~iren Systems. J. Psychol. Neurol., Lpz. 25: 627-846. VOIGTLANDER, P. F. VON and MOORE, K. E. (1971a) The release of H3-dopamine from cat brain following electrical stimulation of the substantia nigra and caudate nucleus. Neuropharmacology 10: 733-741. VOIOTLANDER,P. F. VON and MOORE, K. E. (1971b) Nigro-striatal pathway: stimulation-evoked release of [3H] dopamine from caudate nucleus. Brain Res. 35: 580-585. VOIGTLANDER, P. F. VON and MOORE, K. E. (1973) Involvement of the nigro-striatal neurons in the in vivo release of dopamine by amphetamine, amantadine and tyramine. J. Pharmac. exp. Ther. !84: 542-552.
Pergamon Press.
Printed in Great Britain
Specialist Subject Editor: O. HORNYKIEWICZ
RELEASE OF PUTATIVE FROM THE CORPUS
TRANSMITTERS STRIATUM
MARTHE VOGT Institute of Animal Physiology, Babraham, Cambridge, England
1. INTRODUCTION The corpus striatum--a term which comprises the caudate nucleus and putamen of the telencephalon---differs from other parts of the brain in its content of the so-called 'monoamines' epinephrine, norepinephrine, dopamine (DA) and 5-hydroxytryptamine (5°HT). Only DA and 5-HT are present in what can be considered physiologically important concentrations; thus the cat caudate nucleus contains 10/xg DA per g fresh tissue (Laverty and Sharman, 1965), and approximately 0.37/xg 5-hydroxytryptamine. The striatum is rich in a third base, acetylcholine (ACh), considered by most workers to be a transmitter. The fact that the monoamines can be visualized within neurones, at least in the cells and in the terminals (Dahlstr6m and Fuxe, 1964; Fuxe, 1965) has greatly strengthened the suspicion that their function in the brain is also that of transmitting impulses. Neither DA nor 5-HT are known to act as transmitters in peripheral mammalian nerves, but they play this role in the invertebrate nervous system. The desire to have direct rather than circumstantial evidence for the transmitter nature of monoamines has prompted experiments which would show that appropriate stimuli cause the release of these compounds. In the brain, there are essentially two ways of demonstrating release of substances in response to stimuli, the local perfusion of a small area of tissue with the push-pull cannula (Gaddum, 1961), and the collection of fluid bathing either the external (Macintosh and Oborin, 1953) or the internal brain surfaces (Carmichael et al., 1964). The advantage of the push-pull cannula is that it can be inserted into almost any part of the brain and allows a strictly localized perfusion to be carried out. Its drawback is the production of some local tissue damage which may result in the escape of any tissue constituent when there is an inflow of impulses into the region. Such leakage may be due to changes in blood flow or cell permeability rather than to activation of transmitter release. The possibility of such artefacts has been demonstrated (Chase and Kopin, 1968). It is thus desirable to obtain confirmation by other methods of any results based on the use of the push-pull cannula. The simplest way to obtain fluid which has been in contact with the surface of part of the striatum is to perfuse the anterior horn of the lateral ventricle; such a perfusion is, in fact, a 'superfusion' of the grey masses of the caudate nucleus and the septum. A more elaborate way of superfusing a restricted region of the caudate nucleus has recently been devised (Besson et al., 1971); it necessitates removal of the overlying cerebral cortex in order to apply a perspex cup to the exposed caudate. 2. RESULTS OBTAINED WITH T H E P U S H - P U L L C A N N U L A In the earlier experiments, the output of endogenous DA was studied by fluorimetric estimation. McLennan (1964, 1965) placed a push-pull cannula into the caudate nucleus and saw a release of ACh into the effluent when the nucleus ventralis anterior thalami, and of DA when the nucleus centrum medianum was stimulated. Stimulation of the substantia nigra caused the appearance of DA when the cannula was inserted into the putamen, not when it lay in the caudate nucleus. The author observed a resting release of DA of about 1 ng/min; in contrast, McKenzie and Szerb (1968), also using
MARTHE VOGT
fluorimetry, found no DA (< 1 ng/50 min) under resting conditions. They were, however, able to obtain DA in the effluent when dextroamphetamine was added to the perfusing fluid; this effect was explained by inhibition of uptake. Quite recently, Riddell and Szerb (1971) have labelled the DA in the caudate nucleus by perfusing L-['4C]tyrosine through the push-pull cannula. Labelling always poses a problem of equilibration with different 'pools', but overcomes the difficulty of the insufficient sensitivity of the fluorimetric estimations. On stimulating the rostral substantia nigra, the authors obtained an increase of ['4C]DA in the effluent; no such increase occurred when the stimulation was carried out more caudally. These results are in good agreement with those obtained (Portig and Vogt, 1969) on endogenous DA, which are discussed below. 3. R E S U L T S O B T A I N E D W I T H V E N T R I C U L A R P E R F U S I O N Cats anaesthetized with chloralose (60-90 mg/kg) were used in this work. The perfusion technique is illustrated in Fig. 1; the shaded area represents the perfused anterior horn of the lateral ventricle. Perfusion pressure is measured by connecting the inflow tubing to a transducer. In order to get a valid baseline of 'resting release', it is essential that perfusion pressure remains absolutely steady. If any small obstruction causes a rise in pressure and necessitates adjustment of the outflow needle, new control samples have to be taken before any stimulus can be applied to the brain.
f
J FIG. 1. Position of cannulae for perfusion of the anterior horn of the lateral cerebral ventricle of the cat. Perfused area shaded. (Modified from Carmichael et al. (1964) by permission of jr. Physiol. Lond.)
3.1 ACETYLCHOLINE The suitability of the perfusion technique for the detection of transmitters is shown in experiments in which the release of ACh was examined. In order to preserve the ACh, neostigmine (as methylsulphate, 6 x 10 5 M) was added to the perfusion fluid. This causes, at first, a steep rise in resting concentrations of ACh in the effluent, but after several hours a plateau is reached. The amount of ACh in the perfusate may be increased further by injecting the cats with atropine sulphate (3 mg/kg, either subcutaneously or intravenously) at the start of the experiment (see Mitchell, 1963). On the average, the amount of ACh released 'at rest' was of the order of 3 ng]min when the cats had been treated with atropine. There are a great number of stimuli capable of increasing this basal release (Portig and Vogt, 1966). Effective sensory stimuli include electrical stimulation of the paws, or of the central ends of the severed sciatic nerves, and loud noise. The same effect is seen on stimulation of many regions of the brain, such as the nucleus central lateralis thalami, the substantia nigra and (not consistently) the contralateral caudate nucleus.
Release of putativetransmitters from the corpus striatum
41
To these sites may be added certain areas of the frontal cortex (Mitchell and Szerb, 1962) and the nucleus ventralis anterior thalami (McLennan, 1964), the stimulation of which caused release of ACh into the effluent of a push-pull cannula inserted into the caudate nucleus. Figure 2 (Portig and Vogt, 1969) illustrates an experiment in which four different stimuli were applied for 15 min each, and increments in ACh content of the perfusate were observed during, and for 15 min following each stimulation. It is also evident that the resting release increased throughout the observation period, yet this did not obscure the sudden rises initiated by each stimulation period. The upper set of columns (Fig. 2) indicates the flow of perfusate during the experiment. It remained constant throughout, thus ruling out the possibility that the changes in ACh concentration were artefacts due to irregularities in the perfusion. .£
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FIG. 2. 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 chloralose. The perfusion fluid contained neostigmine methylsulphate 6 x 10-~ M. Atropine sulphate, 2 mg/kg, had been injected intravenously 20 min before collecting the first sample of perfusate. The signals mark stimulation periods: 1st bar, electrical stimulation of the skin (Sk) of both forepaws; 2nd bar, stimulation 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 AI5, L4.5, H5.5) with biphasic pulses of 1 msec duration, 1 every 3 sec, 20 V. Increments in ACh output emphasized by shading. By permission of J. Physiol. Lond.
Anatomical and electrophysioiogical facts are in agreement with the result that ACh can be released from the caudate nucleus by a multitude of stimuli. Acetylcholine and choline acetyl transferase concentrations in the striatum are among the highest in the central nervous system; much of the ACh is situated in the neuropil and not in cells (Krnjevi6 and Silver, 1965, 1966). Evoked responses in the striatum can be elicited by afferent stimuli of any modality. The ACh released into the lateral ventricle thus probably originates from terminals of either a final cholinergic pathway used by the afferent stimuli or from cholinergic interneurones within the tissue. The septum is not likely to contribute to this release, or only to a small extent, because its ACh is mainly situated in cells giving rise to axons ending outside the septum (Krnjevi6 and Silver, 1965, 1966). 3.2. DOPAMINE The observation (Ehringer and Hornykiewicz, 1960) that the striatum of Parkinsonian patients, while histologically fairly intact (Vogt and Vogt, 1920), contains only a small fraction of the normal concentration of DA, might have been interpreted as a 'biochemical lesion'. However, when this finding was coupled with the observation made in 1919 by Tr6tiakoff (and confirmed since) that the main anatomical lesion in Parkinson's disease is in the substantia nigra, and, further, with the demonstration of the presence of DA, but of hardly any noradrenaline, in the cells of the substantia nigra (Bertler, 1961), another interpretation of the loss of DA suggested itself: the existence of a dopaminergic nigro-striatal pathway was postulated, and its destruction by
42
MARTHE VOGT
infection or degeneration b e c a m e the most likely explanation of P a r k i n s o n ' s disease. Much work which is outside the scope of this article has been done in the last decade in support of the existence of this pathway, and the attempt at releasing DA from the striatum by stimulation of the substantia nigra is but one a p p r o a c h to the problem. In spite of the very intense fluorescence seen when striatal tissue is freeze-dried and exposed to f o r m a l d e h y d e vapour, H 0 k f e l t (1968) calculated that only about 16 per cent of boutons in rat caudate nucleus might contain monoamines. Since some of these terminals belong to tryptaminergic neurones, the n u m b e r of terminals containing DA must be smaller still. This alone might contribute to the fact that it is more difficult to d e m o n s t r a t e a release of DA than of ACh, but there are more fundamental differences between the two situations. In the p r e s e n c e of an anticholinesterase, ACh is not only protected f r o m destruction, but there is no re-uptake of transmitter, since only free choline can re-enter the neurone. To achieve the same for DA, it would be necessary to employ three drugs, two (an inhibitor of m o n o a m i n e oxidase and one of Omethyltransferase) for the prevention of enzymatic destruction, and one to prevent re-uptake. Though this is possible, and the n u m b e r of drugs can be reduced by using inhibitors of m o n o a m i n e oxidase which also prevent uptake, the conditions created are highly unnatural. Our first experiments (Portig and Vogt, 1966, 1969) were therefore carried out without the use of inhibitors. The method of estimation of DA was fluorimetry of the acetylated derivative ( L a v e r t y and Sharman, 1965). The 'resting' effluent contained less DA than could be estimated with the very sensitive method (<1 ng in 30 min perfusate), and afferent stimuli of the kind which release ACh did not induce the a p p e a r a n c e of DA. H o w e v e r , when the DA-containing cells of the substantia nigra were stimulated, DA was found in the effluent in some experiments but not in others; the amount released varied b e t w e e n 2 and 20 ng in 30 min and did not spill over into the post-stimulation sample. There were obvious reasons which might explain the inconsistency of the results: one could only hope for any DA to reach the ventricles if it was released f r o m terminals which were so near the ventricular surface that the DA could diffuse into the perfusate before it had been destroyed by the metabolizing enzymes. Since, however, there was no anatomical guidance indicating which part of the substantia nigra might contain cells which gave rise to ventricle-near terminals, it was left to chance whether or not a particular electrode placement could release DA at a site f r o m which it could enter the ventricles before being destroyed. There is, at present, no solution to the anatomical problem, but there were two ways in which the difficulty presented by the metabolic destruction could be o v e r c o m e . One was the inhibition of m o n o a m i n e oxidase and O-methyltransferase, the other was to e m p l o y a metabolite as an index of DA release. The first way was not successful, possibly because the e n z y m e inhibitors interfered with transmitter release, but the second one was: it consisted in using the increased formation of the main metabolite of DA, homovanillic acid (HVA) to detect the release of DA. The estimation was b y fluorimetry (Portig et al., 1968). Figures 3-5 illustrate some of the results. There is a high resting release of H V A into the effluent, the amount ranging a p p r o x i m a t e l y f r o m 2-8 ng/min. Figure 3 shows that this resting outflow falls as the depth of anaesthesia is increased. In both halves of Fig. 3, additional chloralose was injected intravenously at the arrow, and this reduced the basal release by about 25 per cent. This finding suggests that the height of the basal release depends on continous neuronal activity, which is depressed by additional anaesthetic. As can also be seen in Fig. 3, stimulation of the substantia nigra, but not a period of continuous loud noise, increased the release of H V A . The increase outlasted the stimulation period, as is even more clearly shown in Figs. 4 and 5, where brief stimuli were applied, lasting 10 (Fig. 4) and 4 (Fig. 5) min only. Following the short stimuli, increased output continued for 50 min (Fig. 4) or 90 min (Fig. 5). This long duration is what one might expect if the H V A reaching the ventricles was released at different depths within the caudate nucleus: the first 25 min-sample would contain the metabolite of DA released f r o m terminals near the surface, and the later samples H V A f o r m e d at greater depth. N o t only did sensory stimuli usually fail to release any H V A , but electrical stimulation of the reticular f o r m a t i o n caused no release either.
Release of putative transmitters from the corpus striatum
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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 of substantia nigra (SN) at co-ordinates A4.3, L3.5, H-4.2 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. (b) At the arrow, rapid intravenous injection of additional chloralose, 30 mg/kg, 5 hr after first dose. Signal: loud noise (NOI) for l0 min. Note that the scales for HVA in (a) and (b) are different. By permission of J. Physiol. Lond.
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mSn FIG. 4. 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. At signal, l0 rain stimulation of substantia nigra (Sn) with electrode at AS.5,L3,H-5 (trains of three 0.5 msec biphasic 4V stimuli every 3 sec). By permission of J. Physiol. Lond.
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ST' FIG. 5. Columns represent, in rig/rain, HVA found in 30 min perfusates of anterior horn of left lateral ventricle of cat given chloralose, 52 mg/kg. Signal indicates 4 min stimulation (ST) of substantia ni~ra with two electrodes at nositions A5.7 and A4.5.1,3. l-l-5: trains of three 1 msec
44
MARTHE VOGT
A little information was obtained on the point, raised earlier, whether an identifiable part of the substantia nigra sends its terminals to ventricle-near regions of the caudate nucleus. Positioning of the electrodes in the same parasagittal plane of L3, but in frontal planes ranging f r o m A3 to A7, showed fairly consistent release f r o m A5.5 to A7, and frequent failures b e t w e e n A3 and A4. It thus appears that the cells in the more caudal regions send their terminals either into the p u t a m e n or into deep layers of the caudate nucleus, f r o m where H V A cannot reach the ventricle. Recently, Besson et al., (1971) tried to determine release of endogenous D A with their perspex cup method discussed above. T h e y were not successful and, therefore, labelled the D A b y superfusing the tissue with [3H]tyrosine. Application of KCl increased the amount of [3H]DA appearing in the cup. So did superfusion with an inhibitor of m o n o a m i n e oxidase or with a m p h e t a m i n e ; these effects were attributed to inhibition of re-uptake of DA. Similarly, Voigtlander and Moore (1971a, b) labelled striatal DA by injecting [3H]DA into the ventricles and, 1 hr later, perfusing the entire ventricular system. Stimulation of the substantia nigra caused an increment in [3H]DA content of the effluent amount to 15 per cent; a m u c h large release was obtained when the nigro-striatal fibres were stimulated in the lateral hypothalamus. 3.3. 5-HYDROXYTRYPTAMINE As previously found b y Feldberg and Myers (1966), basal release of 5-HT f r o m the anterior horn of the lateral ventricle is v e r y low, on the average (in our hands) <0.5/xg in 25 min. After nialamide, usually given subcutaneously 16 hr before the experiment in a dose of 25 mg/kg, this figure rose to 0.5, and, frequently, to 1 ng in 25 min. Assay of these small quantities has to be done biologically, on the rat stomach strip (Vane, 1957). On employing stimuli which had been shown to release either A C h or H V A , no increment in the outflow of 5-HT was seen (Portig and Vogt, 1969). H o w e v e r , it was likely that release might be obtained if it was possible to find and stimulate cells which give rise to tryptaminergic neurones terminating in nuclei adjacent to the lateral ventricle. Cells containing 5-HT are present in most, if not all, raphe nuclei (Dahlstr6m and Fuxe, 1964); furthermore, ascending efferents to the striatum were seen in one cat to originate f r o m the nucleus linearis rostralis raphe (Brodal et al., 1960). Electrodes were, therefore, placed ( H o l m a n and Vogt, 1970, 1972) into the two m o s t anterior nuclei of the raphe, i.e., the pars rostralis and pars intermedia of the nucleus linearis raphe (for nomenclature see T a b e r et al., 1960). The cats were pretreated with nialamide as described above, and stimulation of one of the divisions of the nucleus linearis was carried out for 15 min with biphasic stimuli at frequencies varying b e t w e e n 0.5 and 2 0 H z . Voltage was between 3 and 4 V . W h e n e v e r the electrode was in, or in the immediate vicinity of, one of the two nuclei, there was a release of 5-HT. When stimulation occupied the first 15 min of a 25 min collection period, the increment did not spill over into the next sample of perfusate. The average increase was 1 ng in 25 min, and represented usually a doubling of the basal 5-HT concentration. N o release was seen when the electrodes were positioned in structures other than the nucleus linearis, such as the red nucleus, the habenulointerpeduncular bundle, the decussation of the superior cerebellar peduncles, the interpeduncular nucleus, the third nucleus, the fasciculus longitudinalis medialis or the reticular formation. In several experiments, the electrode was lowered stepwise and stimulation carried out at different depths. Only when the electrode had reached the surface of the nucleus linearis did 5-HT release occur. Thus in one cat, the electrode passed just lateral to the nucleus linearis intermedius, and identical stimuli were applied at three heights, - 3.5, - 4 . 2 5 and - 5 . 0 ram. The first position made the electrode tip level with the upper border of the nucleus, and stimulation caused the release of 0.5 ng 5-HT. The second position brought the tip in contact with the bulk of the cells, and release a m o u n t e d to 1.0 ng 5-HT. The third position lowered the electrode into the interpeduncular nucleus, and no 5-HT was released. Figures 6 and 7 give an example each of stimulation of the nucleus linearis intermedius (Fig. 6) and the nucleus linearis rostralis (Fig. 7). When
Release of putative transmitters from the corpus striatum
45
3.0l 14 .c: E to 2'0 I i t13 I-O
5 O~ 20/sec FIG. 6. Perfusate from anterior horn of lateral ventricle in cat anaesthetized with chloralose 65 mg/kg. Nialamide injected twice s.c., 30 mg/kg 3 days, and 20 mg/kg 1 day before the experiment. Columns represent 5-HT content (in ng) of consecutive 25 rain samples of perfusate. Stimulation of nucleus linearis intermedius (A2, L0, H - 3.5) during the first 15 min of collection periods 3, 6 and 9 with biphasic pulses of 0.5 msec duration and 4 V. The three frequencies used are indicated at the foot of the column. Increments indicated by shading. Note that release per stimulus is highest at the lowest frequency. By permission of Jr. Physiol. Lond.
41
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5
20/sec
FIG. 7. Perfusate from anterior horn of right lateral ventricle in cat anaesthetized with chloralose 60 mg/kg. Nialamide 30 mg/kg injected subcutaneously on day preceding experiment. Columns represent 5-HT content (in ng) of successive 25 min samples of perfusate; stimulation of nucleus linearis rostralis at position A4.0, LI.0, H -4.5 for the first 15 rain of samples 3 and 6, with biphasic pulses of 0.5 msec and 3 V; frequencies 5 and 20/sec. Increments indicated by shading. There is little difference between the effect of the two frequencies. By permission of J. Physiol. Lond. t h r e e d i f f e r e n t f r e q u e n c i e s w e r e u s e d , e a c h f o r 15 min (Fig. 6), 20 H z w a s f o u n d to b e slightly m o r e e f f e c t i v e t h a n 5 H z , a n d 0.5 H z r e l e a s e d less t h a n either. A b o v e 20 H z , r e l e a s e d e c l i n e d s h a r p l y . W i t h i n t h e r a n g e o f 0.5 to 20 H z , t h e h i g h e s t r e l e a s e p e r s h o c k w a s at t h e l o w e s t f r e q u e n c y (Fig. 6). This w a s p a r t i c u l a r l y s t r i k i n g in e x p e r i m e n t s , in w h i c h t h e s a m e n u m b e r o f stimuli w e r e a p p l i e d at d i f f e r e n t f r e q u e n c i e s : thus, on o n e o c c a s i o n , 750 stimuli at 0 . 5 H z r e l e a s e d 1 . 6 n g 5 - H T , b u t at 5 H z no r e l e a s e w a s d e t e c t a b l e . T h e t w o p a r t s o f t h e n u c l e u s linearis r e a c t e d in t h e s a m e w a y to d i f f e r e n t frequencies. R e l e a s e o f 5 - H T w a s f o u n d to b e h i g h l y d e p e n d e n t on b o d y t e m p e r a t u r e . A l t e r n a t e cooling and warming of the cat while perfusate was being collected throughout the day, c a u s e d s w i n g s in t h e b a s a l r e l e a s e o f 5 - H T w h i c h f o l l o w e d t h e b o d y t e m p e r a t u r e w i t h a lag of a b o u t half an h o u r . I n o n e cat, f o r e x a m p l e , r e l e a s e w a s 0.66 ng/25 min at a b o d y t e m p e r a t u r e o f 33°C, a n d r o s e to 2.7 ng/25 min w h e n t h e t e m p e r a t u r e h a d r e a c h e d 40.2°C. T h i s p h e n o m e n o n in t h e a n a e s t h e t i z e d a n i m a l h a s p r o b a b l y n o t h i n g to d o w i t h t e m p e r a t u r e r e g u l a t i o n b u t m a y be a sign o f g e n e r a l i z e d i n c r e a s e in n e u r o n a l a c t i v i t y with cerebral temperature.
46
MARTHE VOGT
It has been shown in the preceding sections that stimulation of the substantia nigra m a y release ACh and DA, a finding suggesting that both dopaminergic and cholinergic neurones originate in that region. It seemed of interest to see whether stimulation of the nucleus linearis released ACh in addition to 5-HT. The results of experiments, in which ACh and 5-HT were either determined in the same control and stimulation samples, or in two successive sets of such samples (which included a stimulation period), were quite unambiguous: w h e n e v e r the electrode was inside the nucleus linearis and 5-HT was released, there was no increment in the ACh content of the samples (Ashkenazi et al., 1972). There is thus no indication that the release of 5-HT is caused by, or linked with, simultaneous release of ACh. The cells in the nucleus linearis appear to give rise to tryptaminergic neurones only. The terminals in the vicinity of the anterior horn of the lateral ventricle which are activated by stimulation of the raphe nuclei m a y lie in two grey structures, the caudate nucleus or the septum. The septum has the smaller ventricular surface, but the higher concentration of 5-HT. Since in both tissues the 5-HT might be localized in terminals, it is possible that both regions contribute to the release. This interpretation is supported b y the results of 5-HT estimations after destruction of the putative ascending pathways. When lesions were placed in the t e g m e n t u m (Poirier et al., 1967), the 5-HT content of the striatum fell; but so did that of the septum (Heller and Moore, 1968) after section of the medial forebrain bundle in which aminergic fibres originating in the midbrain are said to travel to the telencephalon. T r y p t o p h a n hydroxylase, too, was reduced in both septum and caudate after ventromedial tegmental lesions (Poirier et al., 1969). Thus the demonstration that stimulation of the nucleus linearis raphe releases 5-HT from the surface of the grey structures bordering on the anterior horn of the lateral ventricle are evidence for the existence of tryptaminergic p a t h w a y s originating in the nucleus linearis and probably terminating in both caudate nucleus and septum. 4. S U M M A R Y The e x p e r i m e n t s reported in this article d e m o n s t r a t e the release of ACh, DA and 5-HT f r o m striatal tissue. The technique used was the perfusion of the anterior horn of the lateral ventricle. The ACh and DA appearing in the perfusate must h a v e originated f r o m the caudate nucleus only, whereas 5-HT was probably released f r o m terminals in the septum as well. The results support the view that the m o n o a m i n e s act as transmitters of impulses, and that the cells in the midbrain which contain DA and 5-HT send uninterrupted axons into different parts of the telencephalon. ADDENDUM During the two years which have elapsed since this review was written, progress has been made in releasing labelled DA f r o m the caudate nucleus into cerebrospinal fluid. Von Voigtlander & Moore (1973) added [3H]DA to ventricular perfusates to label the caudate nucleus, and found that a m p h e t a m i n e increased the a p p e a r a n c e of labelled c o m p o u n d , and that the combination of electrical stimulation of the nigro-striatal p a t h w a y with a m p h e t a m i n e produced even greater effects. Chiueh and Moore (1973) showed that [14C]DA f o r m e d in the caudate nucleus by perfusion with ['4C]tyrosine could also be liberated with a m p h e t a m i n e or by electrical stimulation. Release of ACh from the caudate nucleus in the cat has been studied with the push-pull cannula (Stadler et al., 1973) and the interesting observation made that chlorpromazine, k n o w n to increase the turnover of DA, causes an enhanced a p p e a r a n c e of ACh; a correlation between release of the two transmitters does, therefore, not only hold when release is dependant on the depth of anaesthesia (see p. 4). REFERENCES ASHKENAZI,R., HOLMAN,R. B. and VOGT,M. (1972) Release of transmitters on stimulation of the nucleus linearis raphe in the cat. J. Physiol. Lond. 223: 255-259. BERTLER, ,~. (1961) Occurrence and localization of catecholamines in the human brain. Acta physiol, scand.
Release of putative transmitters from the corpus striatum
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