(Received 29 October 1966)

Transcription

1 J. Phy8iol. (1967), 189, pp With 1 plate and 7 text-figsre8 Printed in Great Britain MONOAMINES AND THEIR METABOLITES IN THE AVIAN BRAIN BY A. V. JUORIO AD MARTHE VOGT From the Agricultural Research Council Institute of Animal Physiology, Babraham, Cambridge (Received 29 October 1966) SUMMARY 1. In the avian brain, a high concentration of dopamine was found in a sharply contoured region of the nucleus basalis which may or may not have included the nucleus entopeduncularis, and therefore lay within the palaeostriatum of the nomenclature of Crosby and Huber. This was thus the only region which may be considered biochemically homologous to the mammalian corpus striatum. For purposes of macroscopic identification only, the region is described here as the 'anterior part of the nucleus basalis'. The concentration of dopamine was 3 /tg/g in the pigeon, about the same in the duck and chicken, and 75,ug/g in the finch. In the pigeon this region also contained some noradrenaline; the quantity of 5-hydroxytryptamine (1.4,ug/g) and 5-hydroxyindolylacetic acid (.6,ug/g) was larger than in any other part of the brain. 2. In the brain of the pigeon and the chicken, the highest concentrations of noradrenaline (1.5 and 1 4,ug/g) were found in the hypothalamus. 3. The concentration of adrenaline was higher in the avian than in the mammalian brain. In the hypothalamus, it ranged from 4,tg/g in the pigeon to 1,ug/g in the chicken. 4. Fluorescence microscopy, using the formaldehyde condensation method, showed, in the anterior part of the nucleus basalis, a large area of diffuse green-yellow fluorescence, similar in appearance to the fluorescence of the striatum of the rat. In addition this part of the brain contained a small region of fluorescent fibres and varicosities. It is suggested that the diffuse fluorescence was produced by dopamine. It was absent from brains of reserpine-treated pigeons. 5. In the pigeon, reserpine, tetrabenazine and prenylamine produced a decrease in the concentration of brain monoamines, an effect which was comparable to that seen in mammals. Yet, none of these drugs raised the Present address: Catedra de Farmacologia Experimental, Facultad de Farmacia y Bioquinica, Buenos Aires, Argentina.

2 49 A. V. JUOBIO AND MARTHE VOGT concentration of homovanillic acid, but they increased that of 5-hydroxyindolylacetic acid; these drugs raise the concentration of both acids in mammalian brain. 6. In the pigeon /,-tetrahydronaphthylamine decreased the concentration of all monoamines and their metabolites, an action quite different from that produced in the mammalian brain. 7. The main effect of morphine and of M99 (6,14-endoetheno-7-(2- hydroxy-2-pentyl)-tetrahydro-oripavine hydrochloride) was a lowering of the noradrenaline concentration. 8. As in mammals, chlorpromazine affected only the dopamine metabolism. 9. In the guinea-pig and the pigeon, the administration of a-methyl- DOPA led to a substitution of much of the cerebral noradrenaline by a-methyl-noradrenaline, sometimes in excess of the lost noradrenaline. However, although the loss of dopamine was severe in both pigeon and guinea-pig, only little a-methyl-dopamine accumulated in the pigeon brain, so that it did not consitute a replacement for the lost dopamine; in the guinea-pig, ac-methyl-dopamine was found in quantities similar to, or exceeding those, of the lost dopamine. INTRODUCTION The distribution of catecholamines, i.e. dopamine (3,4-dihydroxyphenylethylamine), noradrenaline and adrenaline, and of 5-hydroxytryptamine has been extensively studied in the mammalian brain (Bertler & Rosengren, 1959; Vogt, 1954; Amin, Crawford & Gaddum, 1954). Some of the acid metabolites of these amines (homovanillic acid formed from dopamine and 5-hydroxyindolylacetic acid derived from 5-hydroxytryptamine) have also been estimated (Sharman, 1963; Anden, Roos & Werdinius, 1963; Ashcroft & Sharman, 1961). Much less is known about these compounds in the avian brain. The presence of dopamine was shown by Montagu (1957) and confirmed with different techniques (Bertler, Falck, Gottfries, Ljunggren & Rosengren, 1964; Aprison & Takahashi, 1965, and Pscheidt & Haber, 1965). Noradrenaline and 5-hydroxytryptamine were found in the brain of the bird by a number of workers (Correale, 1956; Aprison, Wolf, Poulos & Folkerth, 1962; Bogdanski, Bonomi & Brodie, 1963; Pscheidt & Himwich, 1963; Bertler et al and Aprison & Takahashi, 1965). In the brain of mammals the caudate nucleus and putamen, the so-called 'striatum', contains the highest concentration of dopamine. In birds a great part of the cerebral hemispheres has a histological similarity to the mammalian striatum. Therefore it was of interest to show the precise

3 MONOAMINES IN AVIAN BRAIN 491 distribution of dopamine and its metabolite homovanillic acid in the brain of the bird with the object of determining which part corresponded chemically to the mammalian striatum. In addition, an investigation was made of the distribution of noradrenaline, adrenaline 5-hydroxytryptamine and its metabolite 5-hydroxyindolylacetic acid in avian brain. Furthermore, the microscopical localization of some of the monoamines was examined by fluorescence microscopy. Finally, changes in amine concentration were produced by drugs and the responses of the avian brain compared with those of mammalian brains. Some of these findings were demonstrated to the Physiological Society (Juorio, 1966). METHODS Most experiments were done on adult pigeons of both sexes, the body weight ranging from 3 to 5 g. For comparison four adult cocks (two Rhode Island Red and two Plymouth Rock, body weight kg), one Muscovy duck (body weight 4-5 kg), twelve spice finches, Lonchura punctuata (body weight 1 g) and some adult male white rats and a few guinea-pigs were used. Drugs used The drugs used were: reserpine (Serpasil, Ciba), 2-5 mg/ml.; tetrabenazine, dissolved in a little glacial acetic acid and diluted with -9% sodium chloride solution; prenylamine gluconate (Segontin gluconate, Hoechst), 5 %; morphine hydrochloride, dissolved in -9 % sodium chloride solution; M99 hydrochloride (6,14-endoetheno-7-(2-hydroxy-2-pentyl)- tetrahydro-oripavine hydrochloride), Reckitt; chlorpromazine hydrochloride, dissolved in -9% sodium chloride solution;,b-tetrahydronaphthylamine hydrochloride (2-aminotetralin hydrochloride, 1,2,3,4-tetrahydro-naphthylamin-(2)-hydrochloride) dissolved in 9% sodium chloride solution; DL-or L-a-methyl-3,4-dihydroxyphenylalanine (DL or L-amethyldopa, Merck, Sharp & Dohme), dissolved in -9 % sodium chloride solution (all doses are given as L-a-methyl-3,4-dihydroxyphenylalanine); DL-3,4-dihydroxyphenylalanine (DL-DOPA), a suspension prepared in -9 % sodium chloride solution. Injections were made either into the wing vein or into the breast muscle. Estimations of amines and related compounds Dopamine. The tissues were weighed, homogenized in twice the weight of -1 N hydrochloric acid, and the proteins precipitated with perchloric acid (final concentration -4 N). The catecholamines were adsorbed to a column of Dowex 5X 8 essentially as described by Bertler, Carlsson, Rosengren & Waldeck (1958). Dopamine was eluted from the column with 8 ml. of 2 N hydrochloric acid. The eluates were acetylated and condensed with ethylene diamine and the fluorescent product was estimated in a Locarte fluorimeter (Laverty & Sharman, 1965). Pooling of tissue from several birds was only required when parts of the brain containing little dopamine were analysed. a-methyl-dopamine. The procedure for forming a fluorescent compound was similar to that for dopamine, but deproteinization was with 1 N sulphuric acid followed by neutralization with 2 N sodium hydroxide; the supernatant was acetylated, extracted with dichloromethane and the concentrated dichloromethane extract subjected to ascending paper chromatography (Laverty & Sharman, 1965). The paper was cut into strips of no more than 1 cm width which were eluted in water, and condensation with ethylene diamine was performed in each of the eluates. The acetylated derivative of c-methyl-dopamine travelled farther than the derivative of dopamine, but the two bands very often touched. Recovery of

4 492 A. V. JUORIO AND MARTHE VOGT dopamine and a-methyl-dopamine was found to be the same. The anterior parts of the nucleus basalis of two or three birds were pooled for a single estimation. In some experiments, dopamine and a.-methyl-dopamine were not separated but analysed together using the method employed for dopamine, both amines being eluted simultaneously from the column. The results were expressed as dopamine (Table 11). Attempts were made to estimate a-methyl-dopamine after separation on Dowex 5X8 using the oxidation with ferricyanide which does not give a fluorescent compound with dopamine but does so with a-methyl-dopamine. It was found that when the estimations were done up to 4 hr after the administration of the a-methyl-dopa, there was enough of this compound in the eluate to interfere with the chemical estimation. Though small amounts of a-methyl-dopa are not retained by the column, the resin gets contaminated when the concentrations are high and erroneous results are obtained. The interference disappears when a long interval such as a day is allowed between administration of a-methyl-dopa and analysis of the tissue. Homovanillic acid. Two methods were used. (1) The deproteinized tissue extract was acidified, saturated with sodium chloride, extracted with ethyl acetate and subjected to paper chromatography (Sharman, 1963). The development of fluorescence was carried out by the method described by And6n et al. (1963). The fluorescence was read in a Locarte (filter) fluorimeter. (2) This consisted of adsorption of homovanillic acid from the deproteinized extract to a column of anion exchange resin, elution with -1 N hydrochloric acid and formation of the fluorescent compound as before (Juorio, Sharman & Trajkov, 1966). The fluorescence readings had to be made in the Aminco-Bowman spectrophotofluorimeter. The minimum amount of tissue (anterior part of nucleus basalis) needed for method 1 required two birds, and for method 2 one bird. 5-Hydroxyindolyl-compound8. Basic and acidic compounds, corresponding mainly to 5-hydroxytryptamine and 5-hydroxyindolylacetic acid, were always analysed separately (Ashcroft & Sharman, 1962), 5-hydroxytryptamine by extracting the deproteinized tissue extract at ph 1 with n-butanol (Bogdanski, Pletscher, Brodie & Udenfriend, 1956), 5-hydroxyindolylacetic acid by extracting with ether at ph 1-2 (Udenfriend, Titus & Weissbach, 1955). An Aminco-Bowman spectrophotofluorimeter was used for the estimations. The cerebral hemispheres of one pigeon were sufficient for a single estimation. Noradrenaline, adrenaline and ac-methyl-noradrenaline Fluorimetric estimations. The homogenization, deproteinization and separation by ionexchange chromatography on Dowex 5X8 was carried out as for dopamine. Noradrenaline was eluted with 1 ml. of 4 N hydrochloric acid and estimated by oxidation with ferricyanide (Euler & Lishajko, 1961) to form a fluorescent trihydroxyindole; the details of the method have been published (Sharman, Vanov & Vogt, 1962). Bioassay. The methods were based on those previously published (Vogt, 1952, 1953, 1954). The tissue was dissected, weighed and immersed into 1 ml. of acid ethanol (.1 ml. conc. hydrochloric acid: 1 ml. ethanol) cooled in a mixture of carbon dioxide and acetone. The tissue was homogenized and extracted with acid ethanol, the extracts were purified, chromatographed on paper and the regions containing the individual amines were eluted separately. The a-methyl-noradrenaline spot was situated between those of noradrenaline and adrenaline and was well separated from either of them (Text-fig. 1; see also Lindmar & Muscholl, 1965). The amount of amine in the eluates was determined by their press or effects on the pithed rat (Shipley & Tilden 1947); pronethanol was administered before the assay of adrenaline (Vanov & Vogt, 1963). Under these conditions it was possible to estimate amounts of noradrenaline, adrenaline and z-methyl-noradrenaline ranging from 5 to 3 ng in less than 2 mg of tissue. When given intravenously to a pithed rat ac-methyl-noradrenaline produced a pressor response which was somewhat smaller than that of noradrenaline (range , both amines calculated as base). On the other hand, a-methyl-dopamine has only 1/1 of the pressor activity of noradrenaline in the pithed rat.

5 MONOAMINES IN AVIAN BRAIN 493 When bioassay and fluorimetric estimations were performed in the same sample, two or three hypothalami were homogenized in acid ethanol and divided into two aliquots. Before applying one of them to the ion exchange column it was deproteinized with perchloric acid and the acid removed with potassium carbonate. The ifiter set used gave the same intensity of fluorescence for noradrenaline and adrenaline, so that the sum of the two compounds only was estimated. F c'-m-da A ONA a-m-na 'a-m-dopa Text-fig. 1. Regions to which some catechol derivatives travel on paper in the phenol-hydrochloric acid system. NA, noradrenaline; a-m-dopa, a-methyl- DOPA; a-m-na, a-methyl-noradrenaline; A, adrenaline; a-m-da, a-methyldopamine; F front; x origin. Recoveries Recoveries were checked at frequent intervals by adding to a portion of the homogenate amounts of the different compounds approximating the quantities present in the tissue. For means and standard deviations see Table 1. Fluorescence micro8copy The technique proposed by Falck & Owman (1965) was used. Pieces of brain were removed immediately after killing the pigeons with an overdose of pentobarbitone. The tissue was freeze-dried for 3 days, and treated during 1 hr at 8C with paraformaldehyde stored previously at X relative humidity of 15 %. After vacuum-embedding in paraffin the block was sectioned, and the wax was removed by warming and quick washing with xylol. Then the sections were mounted in liquid paraffin, illuminated with U.V. light and the fluorescence examined under the microscope and photographed.

6 494 A. V. JUORIO AND MARTHE VOGT Dis8etiont In the present work the nomenclature of Kuhlenbeck (1938) for the nuclei of the striatum of birds is used. It is based on an embryological study. The validity of Kuhlenbeck's subdivisions was fully substantiated by the subsequent work of Jones & Levi-Montalcini (1958). The skull or the spinal canal were opened quickly. The dura mater was removed and the brain or spinal cord were taken out. The tissues were kept in the cold for periods up to 1 h before grinding up in a glass homogenizer. The brain of all bird species used was divided into hemispheres, thalamus, hypothalamus, optic lobes, the medulla together with the remaining mid-brain, the cerebellum and the spinal cord. The hemispheres were further subdivided into the epibasalis complex of nuclei and the nucleus basalis (palaeostriatum TABLE 1. Percentage recovery + S.D. Number of experiments in parentheses Dopamine (using Dowex 5X8) 754 (1) Dopamine (paper chromatographic separation) 511(8) a-methyl-dopamine (paper chromatographic separation) 45 ± 8 (9) Homovanillic acid (paper chromatographic separation) 58, 45 (2) Homovanillic acid (using Dowex 1) 563 (11) Noradrenaline (paper chromatographic separation followed by 67 ±24 (16) Adrenaline y (pbioasmay) 652(9) a-methyl-noradrenaline 714 (5) Noradrenaline (fluorimetry) 74±7 (3) 5-Hydroxytryptamine (fluorimetry) 574 (9) 5-Hydroxyindolylacetic acid (fluorimetry) 47±1 (12) augmentatum of the old nomenclature). The latter was then divided into an anterior and a posterior part. There was no ambiguity about the identification of the different parts of the diencephalon, mesencephalon and rhombencephalon, but the macroscopic separation of the nucleus basalis and the epibasalis complex cannot be made with complete certainty. The method used was to cut along the lamina medullaris dorsalis (Text-figs. 3 and 4) on the ventral surface of the brain, starting laterally; when the cut reached the medial surface of the brain it was continued along that surface in a semicircle (see cut 1, Text-fig. 2) so as to remove a large part of the hemispheres, which consists mainly of the epibasalis complex; finally the diencephalon was also cut off (cut 2, Text-fig. 2). The small remaining ventromedial piece of tissue contains, but does not consist exclusively of, the nucleus basalis. It was further divided into an anterior and a posterior part, cutting along the tractus cortico-septomesencephalicus, which is visible macroscopically (cut 3, Text-fig. 2). A little of the adjoining nucleus epibasalis centralis and much of the nucleus entopeduncularis may be included in either portion of the 'nucleus basalis'. The 'hypothalamus' of the bird was the translucent tissue situated under the thalamus between the anterior and the posterior commissures (Text-fig. 2). The optic tract was removed. The portion of spinal cord used contained the cervical and lumbar enlargements. The term 'striatum' used in experiments on rat and guinea-pig brains refers to a block of tissue which includes the caudate nucleus, putamen and part of the parietal cortex; the approximate weight was 45 g for the rat and 4 g for the guinea-pig. Single rat brains from which cerebellum and olfactory lobes had been removed were used for the estimation of the 5-hydroxyindoles. RESULTS Distribution of amines and metabolites in the brain of birds Dopamine. In preliminary experiments it was found that most of the dopamine of the pigeon brain was present in the cerebral hemispheres. Analysis of different parts of the hemispheres showed that dopamine was

7 MONOAMINES IN AVIAN BRAIN 495 A Tractus cortico-septo mesencephalicus Optic chiasma 1 cm Text-fig. 2. Medial sagittal section of the brain of the pigeon. A.C., anterior commissure; P.C., posterior commissure; A, plane of the section represented in Fig. 4. Interrupted lines 1, 2, 3: successive cuts used in the dissection. Lamina medullaris dorsalis A I c I cm Text-fig. 3. Ventral view of the brain of the pigeon. A, plane of the section represented in Fig. 4. Interrupted line: cut along the basal surface of the brain. 32 Physiol. I89

8 496 A. V. JUORIO AND MABTHE VOGT localized in the ventrally situated nucleus basalis. On further division of the nucleus basalis (Table 2, rows 4-6) the highest concentration of dopamine was found in its anterior part. Much less was present in its posterior part. In an attempt to localize the dopamine more precisely, the anterior part of the nucleus basalis was further subdivided into an anterior and posterior, a dorsal and ventral, or a medial and lateral portion. Table 3 shows that the highest concentrations were found in the anterior, ventral and lateral portions. The statistical analysis of the results indicated that only the difference between anterior and posterior portion was significant. These further subdivisions were not easily reproducible, and most of the work has been carried out using the whole anterior part of the nucleus basalis as the 'dopamine-rich' region. Optic lobe Lamina medullaris dorsalis Optic chiasma 1 cm Text-fig. 4. Transverse section of the brain of the pigeon at plane A (Text-figs. 2 and 3). The interrupted line indicates the cut separating nucleus basalis from epibasalis complex. Within the posterior part of the nucleus basalis, it was also the anterior region which contained most of the dopamine (Table 3). Small amounts of dopamine appeared to be present in the remaining part of the telencephalon (epibasalis complex) and in the hypothalamus. On subdividing the ' epibasalis complex' into a dorsal and a ventral part and on analysing the nucleus diffusus (cerebral cortex) separately, the dopamine concentration found was the same as for the whole complex. In the cerebellum and the spinal cord the amount of dopamine was below the limit of sensitivity of the method. The hypothalamic dopamine, which amounts to 1 % of the noradrenaline content, may well be just a precursor of noradrenaline. When dopamine was estimated in the anterior part of the nucleus basalis after separation of its acetylated derivative by paper chromatography

9 MONOAMINES IN AVIAN BRAIN 497 o) W 1 So C) 4.1 ho g 4) X,j:.-4-1 m r- 2 I- Cq m 1 - O IF In - _ 2 m "- xo O 2 N1 1 1 m 6 r- N 1 1l 11 1 lo C XO CS O 6 6 CD ci d4 N O O +l +l t- to O f - bo d o2 3 od ~4-4 " o ; E4o -4.5 e j 6 o - 1 to ~'- o 1O 1 SR - -I - r- C5O 6 m Co O O- - n m. 1 +l +l - C e 1 6 I- co to 1 6 1o P- to 6 14 m - C> C? 6 aco P 1 o1 ICI +l - v 4 - N 16 a4-4 -ec - C 6. O 1C 6 o 2 O O +l N1 Mt c o r C)oooP- -I v 6 6 v v m aq v v I- - 6 r- - 6 o C) CD to o 32-2

10 498 A. V. JUOBIO AND MARTHE VOGT (row 1, Table 1), the quantities measured were the same as those given in Table 2. Furthermore, Text-fig. 5 shows that the amine estimated in brain extracts by the fluorescence of its acetyl derivative occupies the same region on a paper chromatogram as authentic triacetyldopamine. In the chicken, duck and finch, as in the pigeon, most of the brain dopamine was found in the nucleus basalis, and the highest concentration in its anterior part. In the chicken and the duck, the concentration of dopamine in this nucleus was of the same order as in the pigeon but in the finch it was twice as high (Table 4). The distribution of homovanillic acid in the brain of pigeons follows the same pattern as that of dopamine (Table 2). Only the two parts of the nucleus basalis contain amounts which can be estimated accurately. For TABEim 3. Distribution of dopamine in the nucleus basalis of the pigeon telencephalon. Values are means (± s.e.) in ptg/g of fresh tissue and are corrected for recovery. Number of experiments in parentheses Nucleus basalis, anterior part Anterior portion (3) Posterior portion (3) Dorsal portion (3) Ventral portion (3) Medial portion (4) Lateral portion (4) Nucleus basalis, posterior part Anterior portion (3) Posterior portiond -14+O1 (3) Significantly different from anterior portion (P <.1). C C C U_ Tissue blank fluorescence Solvent flow Text-fig. B. Distribution of acetylated material giving rise to fluorescence in I cm strips cut from the middle portion of a chromatogram of an extract of pigeon brain (anterior part of the nucleus basalis). The black bar on top indicates the region to which authentic triacetyl-dopamine travels.

11 4 jd- P4 E4. 4 Q, - 2O -4~ 2 4.,s 1 14 r4 bo 2,.,._ 54 v rw O - I - r- M bxobobob O O 1 O ceo o eno o - cro t a ca o ObO v I,. - bobo E. v v O ) _ P4a w 14 ) rt (D b C4-1 co O 14. CD rs DO E Z 2 M P- 1 xo C) w m2 me O 1-.5 r- M 2 O 1 x.5 r- - A co O + C1) m SCD rto "-. 71 C e+ "% $4 ""Cs 4 14 CD 19 fv O D XO F-4 Cs ee4,^.h IK DCD CD o.14 MONOAMINES IN AVIAN BRAIN Q r 11 E+ 1 Co O O C9 v v to b ;4 4 b O 4

12 53 A. V. JUORIO AND MARTHE VOGT identification of the fluorescent compound formed from homovanillic acid, the anterior part of the nucleus basalis taken from eight pigeons was extracted (see Methods, procedure 1) and subjected to paper chromatography. The whole strip of paper was cut into pieces of 1 cm width, each piece was eluted and the eluates were analysed. The only material found to give fluorescence (see Text-fig. 6) was located in the region corresponding to authentic homovanillic acid. Homovanillic acid. F- _ ~~~~~~~~~~~~Tissue blank fuorescence Sovn flw Solvent front Text-fig. 6. Distribution of material giving rise to fluorescence on a paper chromatogram of an extract of pigeon brain (anterior part of the nucleus basalis). The peak coincides with the region to which authentic homovanillic acid travels (black bar).- Noradrenaline and adrenaline. As shown in Table 2, noradrenaline was found in all parts of the brain of the pigeon. In the cerebral hemispheres, the highest concentration (-4,ug/g) was in the anterior part of the nucleus basalis. Within the remaining brain, the concentrations were highest in the hypothalamus (I -5 /,cg/g), thalamus ( 75 /,g/g) and medulla ( 4,ug/g). As with dopamine, the concentrations of noradrenaline were lowest in the cerebellum and spinal cord. All estimations of adrenaline and the majority of those of noradrenaline were done by bioassay. Some discrepancies were found when the results of fluorimetric estimations were compared with those of bioassays. Table 5 shows that, in the hypothalamus, estimations on the rat's blood pressure yielded figures which were only three quarters of those estimated by fluorimetry. The difference is caused by interfering substances; these are not eliminated in the procedure which uses adsorption to a resin, but can be separated off by acetylating the noradrenaline and chromatographing

13 MONOAMINES IN AVIAN BRAIN 51 the acetate on paper (Sharman & Vogt, 1965). Where the discrepancy is small, as here, this complicated procedure is not necessary. In some tissues, such as the rabbit's caudate nucleus, the error is very large. The distribution of adrenaline followed a similar pattern to that of noradrenaline. Twenty per cent or more of the sum (adrenaline plus noradrenaline) were in the methylated form. This is a higher percentage than in most mammalian brains. TABLE 6. The distribution of noradrenaline and adrenaline in the central nervous system of the chicken. Values are means and s.e. in Itg/g of fresh tissue and are corrected for recovery. Number of experiments in parentheses Weight of the Methylated tissue amine (g) Noradrenaline Adrenaline (%) Nucleus basalis, anterior (1) - - part Nucleus basalis, pos (1) terior part Epibasalis complex (1) Thalamus (3) (3) 42 Hypothalamus 5 1'42+-9 (3) 1-1±-16 (3) 42 Medulla 47 45, -42 (2) 35 (1) 44 Optic lobes 5-23, -23 (2) -7 (1) Cerebellum 5-21, -12 (2) <-2 (1) The distribution of noradrenaline and adrenaline was also studied in the brain ofthe chicken (Table 6). The concentration ofnoradrenaline was of the same order as in the pigeon, but that of adrenaline was higher. The percentage of methylation was above 4, the highest value found so far in birds or mammals. This fact has previously been reported by Callingham & Cass (1965). A single estimation on a pool of six finch hypothalami gave a noradrenaline concentration of 2,tg/g and an adrenaline content of 5,tg/g. 5-Hydroxytryptamine and 5-hydroxyindolylacetic acid. Table 2 shows the distribution of these substances. The cerebral hemispheres contained much of the indole compounds and the highest concentrations were found in the anterior part of the nucleus basalis. The smallest amounts were in the cerebellum, in which they were only just above the threshold of the methods. Fluorescence microscopy. The dopamine-rich region (anterior part of the nucleus basalis) of the pigeon brain was examined for formaldehydeinduced fluorescence. Serial sections were cut either in the sagittal or the frontal plane. Two fluorescent regions were found, a large area of diffuse yellow-green fluorescence (P1. 1, fig. 1) and a small area of distinct fluorescent fibres and varicosities which were slightly more yellow (P1. 1, fig. 2). The contrasting appearance of the two areas is best seen in sections showing the two types of fluorescence side by side (P1. 1, figs. 3 and 4).

14 52 A. V. JUOBIO AND MARTHE VOGT In one of the blocks of which sagittal sections were made, and which had a width of 4-8 mm, the diffuse green fluorescence started as a small patch 4 mm from the midsagittal plane, it increased in size up to about 3 mm lateral to that plane, then decreased and had nearly disappeared at 4-1 mm. A series of frontal sections was cut of another block of the anterior part of the nucleus basalis. The piece of tissue was 5X3 mm long. The fluorescent area began at a distance of 5 mm from the posterior end of the tissue, it then increased in size, reaching its greatest area between 1-8 and 2-7 mm, and had nearly disappeared 4-5 mm from the starting plane. Thus most of the fluorescent area was inside the piece of tissue dissected out as 'anterior part of the nucleus basalis', but dorso-laterally the fluorescence sometimes reached the surface of the block so that a little fluorescent tissue might have been left behind in the remaining part of the brain. The area of discrete fluorescence seen in the anterior part of the nucleus basalis was comet-shaped in some sagittal sections, quite small and very bright. In a block cut sagittally this fluorescent region was found in all sections between 9 and 2- mm from the mid line. Outside of the two fluorescent areas occasional fluorescent cells were seen (P1. 1, fig. 5). The specificity of the fluorescence was checked in three ways. (1) Pigeons were treated with reserpine (1 mg/kg) and killed 24 hr later when most of the dopamine and noradrenaline had disappeared from the brain (see Table 7). In sections of the anterior part of the nucleus basalis prepared as usual, the area of diffuse fluorescence was absent and the small area of fluorescent fibres was exceedingly faint. (2) Fluorescent sections of normal animals were floated on water, and this removed all fluorescence. When a drop of water was placed on the section and allowed to dry off, the diffuse fluorescence disappeared. The discrete fluorescence disappeared completely on some occasions, and was very much reduced on others. It might be that the fluorescence which proved a little more resistant to water was due to 5-hydroxytryptamine. Part of the epibasalis complex, a region of the brain of the pigeon in which the concentration of dopamine was very low (Table 2), was also examined after exposure to formaldehyde. A very faint fluorescence was observed which was only seen with a dark field condenser. (3) Treatment of the sections with borohydride removed all fluorescence. In order to compare the diffuse fluorescent region seen in pigeons with a part of the brain of a mammal in which a high concentration of dopamine was known to be present, the corpus striatum of a rat was dried and exposed to formaldehyde: throughout this structure, the grey matter showed a yellow-green diffuse fluorescence which was similar in appearance to that observed in the pigeon (P1. 1, fig. 6).

15 MONOAMINES IN AVIAN BRAIN 53 Effect of drugs on the concentration of brain amines and metabolites Reserpine. When given to pigeons in an intramuscular injection the drug produced regurgitation from the crop, miosis, closure of the palpebral fissure and tranquillization. These signs appeared 2-4 hr after the administration of the drug with the exception of the regurgitation which was seen during the first hour. Some of the pigeons are resistant in this respect and do not vomit even after a high dose of reserpine. In the brain of the pigeon, reserpine, 1 mg/kg, produced a severe loss of noradrenaline, dopamine and 5-hydroxytryptamine (Table7). A tenth ofthat dose reduced the dopamine within less than 24 hr but not the noradrenaline. The latter, however, was somewhat reduced 4 hr after 3 mg/kg. With 1 mg/kg the depleting effect reached its maximum between 1 and 3 days after drug administration; at this time the loss of noradrenaline was about 86 %, that of dopamine 97 %. The return to normal concentrations was slow, and for noradrenaline not quite complete in 19 days. The effect of reserpine on the concentration of dopamine in the brain was not only earlier in onset, but recovery was also quicker than for noradrenaline. In spite of the substantial loss of dopamine produced by reserpine, the concentration of the acid metabolite of dopamine, homovanillic acid, was not increased 2, 4 or 6 hr after drug administration. The concentration of 5-hydroxytryptamine was decreased 1 hr and had reached a minimum 2 hr after the injection of reserpine (1 mg/kg). Depletion lasted at least 11 days and had disappeared on the 19th day. The amount of the acid metabolite of 5-hydroxytryptamine, 5-hydroxyindolylacetic acid, increased in the brain of pigeons 1 or 2 hr after the administration of the drug and then returned to its control value. Tetrabenazine. When administered to pigeons, it elicited signs similar to those produced by reserpine. The biochemical effects were also reserpinelike. There was a decrease in the concentration of noradrenaline, dopamine and 5-hydroxytryptamine which was observed as early as 1 hr after drug administration. Like reserpine, the drug did not modify the concentration of homovanillic acid, but it increased that of 5-hydroxyindolylacetic acid (Table 7). Prenylamine. Though this substance is not chemically related to reserpine it produced the same signs of regurgitation, closure of the palpebral fissure and tranquillization in pigeons as did reserpine. Effects on the eye or sedation did not occur in rats after subcutaneous injection of doses up to 1 mg/kg. Prenylamine reduced the content of noradrenaline, dopamine and 5-hydroxytryptamine in the pigeon brain (Table 7). With small doses (5-15 mg/kg) there was a fall in dopamine only, with 5 mg/kg noradrenaline and 5-hydroxytryptamine were also low; recovery was

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17 MONOAMINES IN AVIAN BRAIN 55 slow and required about 5 days for 5-hydroxytryptamine to return to normal, and over 11 days for noradrenaline. It is interesting to note that rats which are hardly sedated by the drug show no fall in brain 5-hydroxytryptamine (Juorio & Vogt, 1965). In spite of the large fall in dopamine concentration the concentration of homovanillic acid in the pigeon brain was not changed 2, 4 or 6 hr after the administration of prenylamine (5 mg/kg). Both species, however, respond with an increase in the concentration of 5-hydroxyindolylacetic acid. /3-Tetrahydronaphthylamine. On injection into pigeons this drug produced regurgitation, ataxia and sometimes mydriasis. When handled, the injected pigeons struggled a little more than normal animals. In contrast, rats exhibited a high degree of sympathetic excitation, the animals showed exophthalmos, piloerection, increased motor activity, tremor and salivation.,-tetrahydronaphthylamine was also more toxic to rats than to pigeons. When the drug was given in a dose of 5 mg/kg, 7-8 % of the rats died in the first 2 hr whereas pigeons survived under the same conditions. Rectal temperature in pigeons fell slightly.,/-tetrahydronaphthylamine caused moderate falls in the concentration of noradrenaline, dopamine and 5-hydroxytryptamine in the brain of the pigeon (Table 8). The content of noradrenaline was decreased 2 hr after the administration of the drug and was back to normal 24 hr later. The concentration of dopamine reached its minimum earlier than that of noradrenaline. The concentration of homovanillic acid, the metabolite of dopamine, also fell and so did that of 5-hydroxyindolylacetic acid. The concentration of 5-hydroxytryptamine was back to normal at 4 hr, whereas that of its acid metabolite was still low at that time. fl-tetrahydronaphthylamine had a very different action on the brain of rats (Table 9). The drug increased the concentration of dopamine, an effect which appeared 1 hr after the administration of 3 mg/kg; neither the concentration of homovanillic acid nor that of 5-hydroxytryptamine was modified, but there was a fall in that of 5-hydroxyindolylacetic acid. Brain noradrenaline is known to be lowered by fl-tetrahydronaphthylamine in many mammals. Morphine. In a dose of 5 mg/kg morphine occasionally produced central stimulation or regurgitation in the pigeon; the birds showed some ataxia and had the beak slightly open, probably a sign of respiratory disturbance. The only change produced in the brain amines was a decrease in the concentration of noradrenaline (Table 8), an effect which lasted for at least 4 hr and has previously been observed in cats, dogs and rats. This drug did not modify the concentration of either homovanillic acid or 5-hydroxyindolylacetic acid. M99. The administration of this very potent morphine-like substance

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19 MONOAMINES IN AVIAN BRAIN 57 produced an immediate change in the behaviour of the pigeons at doses as low as O 1 mg/kg. The animals started to run about the cage, flapped their wings, lost control over their legs and -became unable to walk normally. After the first hour the activity subsided, and the pigeons remained resting on their breast with the beak slightly open. Four hours after the largest dose (-8 mg/kg) the only chemical change found was a low concentration of noradrenaline (Table 8). There was a short-lived rise in the concentration of 5-hydroxyindolylacetic acid for the first 2 hr after.1 and 4 mg/kg. TABLE 9. The effect of a single subcutaneous injection of f8-tetrahydronaphthylamine on amines and their metabolites in rat brain. Values are means and S.E. in jug/g of fresh tisues and are corrected for recovery. Number of experiments in parentheses Time Whole brain Striatum,A 5-Hydroxy- Dose (mg/kg) interval (hr) Dopamine Homovanillic acid 5-Hydroxytryptamine indolylacetic acid Control (1) (4) ' (5) (3) (3) ) (8) (4) (3) (4) (4) (4) (6) (3) P <.1, P <.1, P < 5. Chlorpromazine. Doses of this drug up to 5 mg/kg did not elicit any detectable change in the pigeons' behaviour. Chlorpromazine produced a small decrease in the concentration of dopamine, and simultaneously a marked increase in that of homovanillic acid (Table 8). The concentrations of noradrenaline and 5-hydroxyindolylacetic acid remained unaltered. DL-3,4-dihydroxyphenylalanine (DOPA). When pigeons were given an intramuscular injection of DOPA, the aminoacid precursor of dopamine, the animals did not, as do mammals, show mydriasis or any other sign of hyperexcitability or autonomic stimulation. Yet the content of dopamine in the nucleus basalis was raised by 57 % (Table 8). ac-methyl-dopa. Small doses of oa-methyl-dopa, a competitive antagonist of DOPA, produced little visible effect except regurgitation in pigeons, but doses of over 4 mg/kg sedated the birds heavily. The animals sat quietly, with eyes closed, but always awoke immediately at any noise or touch. Daily doses of 1 mg/kg did not produce sedation. Table 1 summarizes the biochemical effects on the brain. Noradrenaline and ac-methyl-noradrenaline Small doses of a-methyl-dopa (up to 25 mg/kg) did not change the concentration of noradrenaline, and did not give rise to the formation of

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21 MONOAMINES IN A VIAN BRAIN 59 more than traces of the 'false transmitter' a-methyl-noradrenaline (Table 1). When 5 mg/kg was injected, there was a loss of noradrenaline within i hr, and the concentration remained low for 3 days. The formation' of z-methyl-noradrenaline lagged behind the loss in noradrenaline: it was hardly detectable i hr after the injection, when the noradrenaline was already low, but reached values equivalent to those of the noradrenaline deficit during a period lasting from 4 to 24 hr. After 2 mg/kg (Table 1 and Text-fig. 7), there was a greater fall in the noradrenaline concentration, and it took again about 4 hr for the a-methyl-derivative to rise to a value equivalent to the deficit in noradrenaline. With this higher dose, there was a period between the fifth and the seventh day after the injection 6X1 mg/kg Dopamine 11 Noradrenaline O a-methyl-catecholamines a-methyl-dopa, 2 mg/kg 2x4 mg/kg I I Time (hr) Text-fig. 7. The amine concentrations (calculated on a molar basis) are expressed as percentage of control figures for noradrenaline in the hypothalamus (black coluimns), and for dopamine in the anterior part of the nucleus basalis (shaded columns). The amount of methylated derivative is drawn above the parent compound, so that 1 % is reached whenever normal and methylated amine add up to the normal molar tissue content. when noradrenaline values were back to normal, but a-methyl-noradrenaline was still present in significant amounts. When a-methyl-dopa was administered repeatedly in six daily injections of 1 mg/kg and the pigeons were killed 24 hr after the last injection, the amount of noradrenaline left was only 3% of that present in normal conditions and the concentration of a-methyl-noradrenaline exceeded by 4 % that of noradrenaline in control animals (Text-fig. 7). In a further series of experiments a higher dose of ac-methyl-dopa was given twice at intervals of

22 51 A. V. JUORIO AND MARTHE VOGT 25 hr. When the pigeons were killed 5 hr after the first injection the concentration of noradrenaline in the hypothalamus was reduced by 3-5% and ac-methyl-noradrenaline was present in an amount approximately equivalent to the lost noradrenaline. If the pigeons were given a similar treatment, but killed 24 hr after the first injection (Table 1, last row), the decrease in the concentration of noradrenaline was similar to that observed before, but the amount of c-methyl-noradrenaline accumulated was larger. Dopamine and ac-methyl-dopamine The effect of injections of a-methyl-dopa on the dopamine concentration in the brain of pigeons is also shown in Table 1. One hour after the administration of a dose of 2 mg/kg the concentration of dopamine was low, and the depletion lasted for at least 4 hr. The next day the concentration of dopamine had returned to the control value. ac-methyl-dopamine, the decarboxylation product of a-methyl-dopa, was also found to be present, but its concentration was low and was far from equivalent to the lost dopamine. When a-methyl-dopa, 1 mg/kg, was given intra- TABlE 11. The effect of injections of a-methyl-dopa on the content of dopamine plus a-methyl-dopamine estimated together in the anterior part of the nucleus basalis of the pigeon. Values are means and s.e. in /zg/g of fresh tissue and are corrected for recovery. Number of experiments in parentheses Time interval (hr) after injection and mode of Dopamine +a-methyl- Dose (mg/kg) administration dopamine Control (dopamine) (9) 2 1 i.v. 2'22+13 (7) 2 2 i.v (4) 2 4 I.v (6) 1 x 6 Daily injectiont I.M. 2-61, 242 (2) 4x2$ 5 i.v (4) P < 1, P <.1. t Killed 24 hr after the last injection. 4 Interval of 2-5 hr between injections. muscularly for 6 successive days and the pigeons were killed 24 hr after the last injection, the dopamine content of the brain was found to be only moderately decreased, but when 8 mg/kg of the drug was given intravenously in two doses with an interval of 21 hr, and the pigeons were killed 5 hr after the first injection, the concentration of dopamine was reduced to 18 % of its normal value (Text-fig. 7). Yet, after both these treatments the content of oc-methyl-dopamine was less than 1 % of the normal concentration of dopamine. Table 11 summarizes experiments in which cx-methyl-dopa was given

23 MONOAMINES IN AVIAN BRAIN 511 to pigeons and the dopamine and a-methyl-dopamine were estimated together. The results obtained are in perfect agreement with those shown in Table 1 in which dopamine and ac-methyl-dopamine were estimated separately. Homovanillic acid and 5-hydroxyindoles The content of homovanillic acid in the brain of the pigeon (Table 1) was decreased after an intravenous injection of a-methyl-dopa, 2 mg/ kg; this effect was observed 2 and 4 hr after the administration of the drug. The concentrations had nearly returned to normal in 24 hr. Five hours after the administration of 8 mg/kg in two doses given at an interval of 25 hr there was a decrease in the concentration of homovanillic acid to about 2 % of its normal value, but there was no significant change when pigeons were injected with 1 mg/kg daily during 6 days and killed 24 hr after the last injection. With 8 mg/kg, but not with smaller doses, there was a large decrease in the concentrations of 5-hydroxytryptamine and of 5-hydroxyindolylacetic acid in the brain of pigeons killed 21 hr after the second injection (Table 1). Experiments on guinea-pigs. The amount of a-methyl-dopamine found in these experiments on pigeons was much lower than that reported by Schumann & Grobecker (1965) after giving ac-methyl-dopa to guineapigs. In order to ascertain that genuine species differences were involved and that differences in methods were not responsible, some experiments were carried out on guinea-pigs (Table 12). When the drug (1 mg/kg) was given subcutaneously daily for 6 days and the animals were killed 24 hr after the last injection, the amount of dopamine left in the brain amounted to 64 % of its normal concentration and the increase in a- methyl-dopamine was slightly in excess of the lost dopamine. The administration of a-methyl-dopa in a single dose of 2 mg/kg reduced the concentration of dopamine to about 25 % of normal and there was twice as much a-methyl-dopamine present as dopamine. When two large doses of a-methyl-dopa were given and the guinea-pigs were killed after 5 hr, the concentration of dopamine was reduced to 4 % of its normal value, and that of c-methyl-dopamine was found to be far in excess of the lost dopamine. In one experiment, noradrenaline and ac-methyl-noradrenaline were also estimated. After six daily doses of 1 mg/kg the concentration of noradrenaline fell below the limit of the sensitivity of the method. Simultaneously the concentration of oc-methyl-noradrenaline rose to an amount which surpassed that of noradrenaline in control animals by 11%. 33 Physiol. i89

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25 MONOAMINES IN AVIAN BRAIN 513 DISCUSSION On studying the distribution of dopamine in the brain of several avian species only a small region of the forebrain designated as the anterior part of the nucleus basalis was found to contain a high concentration of this amine. As pointed out under Methods, the region called 'anterior part of the nucleus basalis' is only defined by macroscopic criteria. It is not possible to separate by dissection the nucleus basalis from the nucleus entopeduncularis. This is a small nucleus which contains large cells (see Rose, 1914, who called it J) and is enclosed by the nucleus basalis with which it forms the palaeostriatum of the old nomenclature. From the extent of the diffuse fluorescence it is certain that the nucleus entopeduncularis is too small to account for the whole fluorescent area, but it is impossible to tell whether it forms part of it. This can only be decided by histological study which would also show whether the region, defined macroscopically as ' posterior part of the nucleus basalis' and containing but little dopamine, is truly a portion of the nucleus basalis (and nucleus entopeduncularis) or encroaches on adjacent regions. Since little or no dopamine was found outside the nucleus basalis (and perhaps entopeduncularis), it is only this part of the brain which may be considered homologous with the ' striatum' of mammals, at least from a biochemical point of view. Such a view is not in agreement with the old nomenclature according to which the term 'neostriatum' was used for the mammalian striatum (Kappers, Huber & Crosby, 1936) as well as (Huber & Crosby, 1929) for what Kuhlenbeck (1938) later called the nucleus epibasalis centralis. Kuhlenbeck (1938) and Jones & Levi-Montalcini (1958) have shown that the whole epibasal complex has the same embryological origin and that there are no anatomical grounds for singling out the nucleus epibasalis centralis as the equivalent of the mammalian neostriatum. The present biochemical findings lead to the same conclusion and furthermore suggest that the avian palaeostriatum or, more precisely, the nucleus basalis with or without the nucleus entopeduncularis, is homologous to the neostriatum in mammals. Of other workers who studied the distribution of dopamine in the avian brain, Bertler et al. (1964) found dopamine 1.5,ug/g in the combined 'palaeostriatum' and 'neostriatum' but did not subdivide further, whereas Spooner & Winters (1966), in an abstract which has just appeared, mention that the palaeostriatum of the chicken was found to contain the highest concentration of dopamine. In contrast to the mammalian corpus striatum, the dopamine-rich part of the pigeon brain also contained more noradrenaline than other parts of the telencephalon, about one third of the amount found in the hypo- 33-2

26 514 A. V. JUORIO AND MARTHE VOGT thalamus. In the same region the concentration of 5-hydroxytryptamine was also high, and higher than anywhere else in the brain. When the dopamine-rich region was examined by fluorescence microscopy using the condensation with formaldehyde to visualize the monoamines, a diffuse fluorescence greatly resembling that shown by the mammalian caudate nucleus was seen to occupy a large part of the excised region. In addition, a clump of fluorescent nerve terminals suggesting the presence of fibres containing noradrenaline or 5-hydroxytryptamine appeared to be one of the structures in which the noradrenaline and 5- hydroxy-tryptamine determined chemically were localized. Other peculiarities of the avian brain were the high proportion of adrenaline in the noradrenaline-rich regions, and the relatively high amount of noradrenaline in telencephalon and optic lobes. The second part of the paper deals with the biochemical changes produced by drugs in the brain constituents examined in the first part, with a view to discovering whether these constituents, and particularly the dopamine, react similarly in avian and mammalian brain. The most striking differences were seen after reserpine-like drugs. Following their administration the dopamine concentration in the avian brain fell as sharply as in other species, but there was at no time a rise in the concentration of homovanillic acid. This may either mean that in the bird the mechanism by which this acid is removed from the tissue is particularly efficient, or that dopamine released by reserpine is not metabolized in the same way in bird and mammal. Even within mammals there are large species differences in the degree to which homovanillic acid accumulates after a dose of reserpine (Juorio et al. 1966). A second difference is that the reserpine-like action of prenylamine is very slight in the rat (Juorio & Vogt, 1965) but quite severe in the pigeon; there is heavy sedation and a sharp fall not only in the catecholamines, but also in 5-hydroxytryptamine which remains unchanged in the rat. It is tempting to relate the greater sedation of the bird to this release of 5-hydroxytryptamine. A correlation between sedation and disturbance of 5-hydroxytryptamine metabolism is also suggested by the observations with ac-methyl-dopa in the bird; profound sedation was caused only with doses which lowered the concentration of 5-hydroxytryptamine. These facts, however, do not exclude the possibility that changes in catecholamine metabolism participate in bringing about sedation. Another drug which affected brain amine metabolism in the bird quite differently from that of the mammal was,/-tetrahydronaphthylamine. In the pigeon brain it caused a moderate decrease in the concentrations of all monoamines and their metabolites. This suggests that this drug may block

27 MONOAMINES IN AVIAN BRAIN 515 an early step in the synthesis of all monoamines, such as ring-hydroxylation. In the mouse (Sharman, 1966), and probably in the rat,,5-tetrahydronaphthylamine does not decrease, but increases the brain concentration of dopamine and of homovanillic acid. In contrast to the stimulation of sympathetic centres produced in mammals treated with,-tetrahydronaphthylamine, there were no certain signs of such stimulation in the pigeon, and the drug was also much less toxic. DOPA, too, was tolerated by the pigeon without the signs of motor restlessness seen in the mammal. Finally, there were differences in the way in which the avian brain disposed of the DOPA analogue ac-methyl-dopa. Though decarboxylation to a-methyl-dopamine took place, this compound did not accumulate in the pigeon brain, even when large doses or repeated injections were used. On the other hand, the guinea-pig brain, as already shown by Schumann & Grobecker (1965), was found capable of accumulating ac-methyldopamine to concentrations equal to the normal concentrations of dopamine. This suggests that a different mechanism is involved in storage or utilization of that substance in pigeon and guinea-pig brain. There has been some controversy (see Muscholl, 1966) on the question whether the amount of a-methyl-noradrenaline found in tissues after the administration of oa-methyl-dopa replaces the lost noradrenaline stoichiometrically. The experiments on pigeons show that, soon after an injection of a-methyl-dopa, noradrenaline is low but very little of the methylderivative has yet been formed; conversely, in the late phase, the methylated amine may still linger on when the noradrenaline values have returned to normal; furthermore, after prolonged treatment, the methylated compound may accumulate in excess of normal noradrenaline concentrations. This was seen in the brain of both pigeons and guinea-pigs, and confirms similar findings in the rabbit heart (Muscholl & Maitre, 1963) and the guinea-pig brain (Schumann & Grobecker, 1965). It is obvious, and is illustrated in Text-fig. 7, that at certain times the sum of noradrenaline and ca-methyl-noradrenaline passes through a phase at which it adds up to the normal concentration of noradrenaline. The work was made possible by the award to one of us (A. V. J.) of a Riker Fellowship and of a grant from the Wellcome Trust. We are very grateful to Mr J. E. McEwen, F.I.S.T., for invaluable help throughout the work and for carrying out the bioassays. Our thanks are also due to Dr C.. Hebb who put her equipment for fluorescence microscopy at our disposal and to Mr S. P. Mann, from whose experience with this technique we greatly benefited and who helped in much of the actual work. We wish to thank Ciba Laboratories for a gift of reserpine, Professor G. Kroneberg for a-methyl-dopamine and ac-methyl-noradrenaline, Priv. Doz. Dr E. Lindner (Farbwerke Hoechst) for Segontin, Dr D. R. Maxwell (May & Baker) for chlorpromazine, Merck, Sharp and Dohme for a-methyl-dopa, and Messrs Reckitt and Sons Ltd. for M 99.

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29 MONOAMINES IN AVIAN BRAIN 517 KUHLENBECK, H. (1938). The ontogenetic development and phylogenetic significance of the cortex telencephali in the chick. J. comp. Neurol. 69, LAVERTY, R. & SHARMAN, D. F. (1965). The estimation of small quantities of 3,4-dihydroxyphenylethylamine in tissues. Br. J. Pharmac. Chemother. 24, LINDMAR, R. & MUSCHOLL, E. (1965). Die Aufnahme von a-methylnoradrenalin in das isolierte Kaninchenherz und seine Freisetzung durch Reserpin und Guanethidin in vivo. Arch. exp. Path. Pharmak. 249, MONTAGU, K. A. (1957). Catechol compounds in rat tissues and in brains of different animals. Nature, Lond. 18, MUSCHOLL, E. (1966). Autonomic nervous system: newer mechanisms of adrenergic blockade. A. Rev. Pharmac. 6, MUSCHOLL, E. & MAITRE, L. (1963). Release by sympathetic stimulation of a-methylnoradrenaline stored in the heart after administration of a-methyldopa. Experientia 19, PSCHEIDT, G. R. & HABER, B. (1965). Regional distribution of dihydroxyphenylalanine and 5-hydroxytryptophan decarboxylase and of biogenic amines in the chicken central nervous system. J. Neurochem. 12, PSCHEIDT, G. R. & HIMwIcH, H. E. (1963). Chicken brain amines, with special reference to cerebral norepinephrine. Life Sci. Oxford 2, ROSE, M. (1914). Uber die cytoarchitektonische Gliederung des Vorderhirns der Vogel. J. Psychol. Neurol., Lpz. 21, Erg. Heft 1, SCHUMANN, H. J. & GROBECKER, H. (1965). Uber die Wirkung von a-methyl-dopa auf den Brenzcatechinamingehalt von Meerschweinchenorganen. Arch. exp. Path. Pharmak. 251, SHARMAN, D. F. (1963). A fluorimetric method for the estimation of 4-hydroxy-3-methoxyphenylacetic acid (homovanillic acid) and its identification in brain tissue. Br. J. Pharmac. Chemother. 2, SHARMAN, D. F. (1966). Changes in the metabolism of 3,4-dihydroxyphenylalanine (dopamine) in the striatum of the mouse induced by drugs. Br. J. Pharmac. Chemother. 28, SEARMAN, D. F., VANOV, S. & VOGT, M. (1962). Noradrenaline content in the heart and spleen of the mouse under normal conditions and after administration of some drugs. Br. J. Pharmac. Chemother. 19, SHARMAN, D. F. & VOGT, M. (1965). The noradrenaline content of the caudate nucleus of the rabbit. J. Neurochem. 12, 62. SHIFPLEY, R. E. & TILDEN, J. H. (1947). A pithed rat preparation suitable for assaying pressor substances. Proc. Soc. exp. Biol. Med. 64, SPOONER, C. E. & WINTERS, W. D. (1966). Distribution of monoamines and regional uptake of DL-norepinephrine-7-H3 and dopamine-1-h3 in the avian brain. Pharmacologist 8, 189. UDENFRIEND, S., TITUS, E. & WEISSBACH, H. (1955). The identification of 5-hydroxy-3- indolacetic acid in normal urine and a method for its assay. J. biol. Chem. 216, VANOV, S. & VOGT, M. (1963). Catecholamine-containing structures in the hypogastric nerves of the dog. J. Physiol. 168, VOGT, M. (1952). The secretion of the denervated adrenal medulla of the cat. Br. J. Pharmac. Chemother. 7, VOGT, M. (1953). Vasopressor, antidiuretic and oxytocic activities of extracts of the dog's hypothalamus. Br. J. Pharmac. Chemother. 8, 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. Phy8iol. 123,

30 518 A. V. JUORIO AND MARTHE VOGT EXPLANATION OF PLATE Fig. 1. Pigeon brain, anterior part of nucleus basalis, frontal section. Area of diffuse fluorescence. White bar 1 pt. Fig. 2. Pigeon brain, anterior part of nucleus basalis, sagittal section. Discrete area of fluorescence; many fibres and varicosities. White bar 1,u. Fig. 3. Pigeon brain, anterior part of nucleus basalis, frontal fluorescence and area of discrete fluorescence. Black bar 1,u. section. Area of diffuse Fig. 4. Pigeon brain, anterior part of nucleus basalis, sagittal fluorescence and area of discrete fluorescence. White bar 1 gcb. section. Area of diffuse Fig. 5. Pigeon brain, anterior part of nucleus basalis, sagittal section. Fluorescent nerve cells and fibres, found at some distance of the area of discrete fluorescence. White bar 1 at. Fig. 6. Rat brain, corpus striatum. Black bar 1,u.

31 The Journal of Physiology, Vol. 189, No. 3 Plate 1 -MEMWPW71, A. V. JUORIO AND MARTHE VOGT (Facing p. 518)