Immunohistochemical evidence for the existence of adrenaline neurons in the rat brain

Brain Research, 66 (1974) 235-251 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

235

IMMUNOHISTOCHEMICAL EVIDENCE FOR THE ADRENALINE NEURONS IN THE RAT BRAIN*

OF

EXISTENCE

T. HOKFELT, K. FUXE, M. GOLDSTEIN AND O. JOHANSSON Department of Histology, Karolinska Institute, 104 O1 Stockholm (Sweden) and Department of Psychiatry, Neurochemistry Laboratories, New York Medical Center, New York, N.Y. 10016 (u.s.A.)

(Accepted July 20th, 1973)

SUMMARY With the help of immunohistochemical studies using antibodies against bovine adrenal phenylethanolamine-N-methyltransferase (PNMT), the enzyme converting noradrenaline (NA) to adrenaline (A), the cellular localization of P N M T in the rat central nervous system has been demonstrated. The brains were fixed by perfusion with ice cold 4 % formalin and sectioned on a cryostat, after which the sections were stained using the indirect immunofluorescence technique. Preimmune serum and P N M T antiserum adsorbed with P N M T served as control sera. Specific immunofluorescence was localized in two groups of reticular nerve cell bodies in the medulla oblongata and in nerve terminals in special nuclei of the brain stem and the spinal cord. A P N M T positive axon bundle was also observed in the reticular formation of the pons-medulla oblongata. The distribution and morphology of these P N M T containing neurons was such that they probably are identical with some catecholamine nerve terminals and cell bodies previously demonstrated with the Falck-HiUarp technique (see Dahlstr/Sm and Fuxe18). The hypothesis is therefore given that the P N M T containing neurons represent A containing neurons, and that A may act as a neurotransmitter in the rat brain. The reticular A neurons appear to have similar morphological characteristics as the N A neurons with long ascending and descending fibers to the brain stem and spinal cord. The A terminals are mainly found in certain visceral afferent and efferent nuclei of the lower brain stem, in the locus coeruleus, in certain nuclei or the hypothalamus and in the periventricular grey. The well defined distribution o f the A net-

* Part of these results were reported at a symposium on 'Dynamic Aspects of the Synapse' in Boldern, Switzerland, April 11-13, 1973 and at the XIV Nordic Congress for Physiology and Pharmacology in Bergen, Norway, August 5-8, 1973. A preliminary report has appeared 53.

236

T. H/3KFELTet al.

works to these nuclei underline the view that the A neuron may participate in the control of oxytocin secretion, food and water intake, body temperature, gonadotrophin secretion, blood pressure and respiration and sleep and wakefulness.

INTRODUCTION

The formaldehyde fluorescence method (Falck-Hillarp technique) demonstrates the cellular localization of certain biogenic monoamines like dopamine (DA), noradrenaline (NA), adrenaline (A) and 5-hydroxytryptamine (5-HT) and thus offers the possibility to trace cell systems containing these putative neurotransmitter substances z4,z6. In the central nervous system the distribution of DA, NA and 5-HT neurons has been extensively studied, and there has always been a good correlation between histochemical and biochemical estimations of brain NA, DA and 5-HT levels both as to regional variations in normal animals as well as changes in amine levels after various experimental procedures 1-3,11,1s,29,30,33-35,76. However, the levels of mammalian brain A are only 5-10 ~ of total NA and A levels in brain23,4a,56,62,69,Ta,77 (see also review by Holzbauer and SharmanS7). With a fluorimetric assay method, Gunne 45 found after chromatographic separation that in rat brain stem the A brain levels were 4.5 ~ of total NA and A levels. Ever since the discovery of the central catecholamine (CA) pathways it has not been possible to exclude that a few of the CA pathways may contain A and not N A as a neurotransmitter. This is due to the fact that using the Falck-Hillarp technique it is difficult to differentiate between NA and A, since they have the same excitation and emission spectraT,15, a6. However, A requires more severe reaction conditions than NA, since the formaldehyde condensation reaction in the former case results in the formation of a quaternary nitrogen. This difference has been utilized to localize A in nerve terminals in the frog heart 25, in which high amounts of A can be found, but has not been found sensitive enough for differentiating NA and A in the mammalian central nervous system. The biochemical findings on the presence of A in mammalian brain are supported by the demonstration ofphenylethanolamine-N-methyltransferase (PNMT) activity in rat brainla, 71 and especially in the brain stem. New possibilities to identify and differentiate between various types of monoamine neurons arose with the findings of Gibb et al. 3s that dopamine-fi-hydroxylase (DBH), the enzyme converting DA to NA, possesses antigenic activity and with the subsequent introduction ofimmunohistochemistry into neurotransmitter research a7,46. Thus, not only DBH but also two other catecholamine synthesizing enzymes, dopadecarboxylase (DDC) and P N M T could be demonstrated in specific neuron systems in the central n e r v o u s system12,31,32,40,47,51,53, 54. In the present paper a detailed description of the localization of P N M T in the rat brain is given and evidence is presented that this enzyme is localized to special reticular neurons and thus provides the first morphological evidence that A neurons may exist in the mammalian central neuron system.

ADRENALINE NEURONS IN RAT BRAIN

237

MATERIAL AND METHODS

P N M T was purified from bovine adrenal glands 39,43,~8. Antiserum to this enzyme was obtained from rabbits and the specificity was tested by immunoelectrophoresis (for details, see Goldstein et al.41). Male Albino rats (Sprague-Dawley, body weight 150-200 g) were anesthetized with Nembutal, and a tracheal cannula for artificial respiration was inserted. After opening the chest, ice cold 4 % formalin in 0.1 M phosphate buffer, prepared from paraformaldehyde powder according to Pease 7°, was perfused via the ascending aorta for 30 min-2 h. Immediately after the start of perfusion the rat was immersed in a mixture of ice and water. All together about 20 rats were used. The brain was rapidly dissected out, divided into 5 pieces by frontal sections and rinsed overnight in 0.1 M phosphate buffer (pH 7.2-7.4). Ten # m thick sections were cut on a Leitz cryostat and every tenth section mounted an on object slide pretreated with chrom-alum. The sections were mostly stored in a deep freezer for 1-14 days before staining. The indirect immunofluorescence technique (see refs. 14 and 67) was used in this study. After rinsing in phosphate buffered saline (PBS) (pH 7.4) at r o o m temperature, the sections were incubated with P N M T antibodies in dilutions from 1 : 4 to 1 : 32 in PBS with added Triton (0.3 %)47 for 30 min at r o o m temperature or for 20 min at ÷ 3 7 °C. After rinsing with PBS for 30-120 min at r o o m temperature, incubation with fiuorescein isothiocyanate (FITC) conjugated rabbit anti-sheep antibodies commercially available from Statens Bakteriologiska Laboratorium, SBL, Stockholm, Sweden 6) diluted 1 : 4 in PBS was performed for 30 min at room temperature or for 20 min at + 3 7 °C. This serum had mostly been shaken with mouse brain or liver powder (Miles Laboratories, U.S.A.). After rinsing for 30 rain-2 h the sections were mounted in a mixture of glycerin and PBS (4:1). Sections incubated in preimmune serum or in P N M T antiserum adsorbed with P N M T served as controls. The specific antiserum was in addition also tested on the adrenal gland: as previously shown only a certain population of gland cells, probably corresponding to the A containing cells 2°-22,4s-5° were stained with P N M T antiserumZ~,4z, ~2 whereas, e.g., anti-DBH and anti-DDC stain all gland cells32,z7,42,a6,sL The sections were examined in a Zeiss fluorescence microscope equipped with a dark-field oil condenser and a HBO 200 high pressure mercury lamp. As activation filter a 3 or 4 m m Schott BG12 or a Tal filter was used and as secondary filter a Zeiss 50 or 47 was used. The atlas o f K6nig and Klippe161 and the book Craigie's Neuroanatomy of the Rat 78 were used for localizing the P N M T containing neurons and for schematic drawings (Figs. 1 and 2). RESULTS

After incubation with P N M T antiserum specifically fluorescent nerve cell bodies, fibers and dot-like structures could be identified in certain parts of the rat brain. Their structural characteristics strongly indicate that they represent neurons and we will

¢D

03

ILl

Fig. 1. A - G : schematic diagrams of frontal sections of the rat central nervous system showing the distribution of P N M T positive cell bodies, axons and nerve terminals as revealed with immunofluorescence microscopy after staining with bovine adrenal P N M T antiserum. The diagrams are based on the KiSnig and Klippel atlas ~1 (A-C) and Craigie's NeuroanatomyTM (D-F). Explanations for symbols and abbreviations are found on page 248. The frontal sections were taken at the level of the anterior hypothalamus (A, Kbnig and Klippel A 5340 #m), at the level of the middle hypothalamus (B, Kbnig and Klippel A 4230/~m), at the level of the caudal mesencephalon (C, Kbnig and Klippel P 100 #m), at the level of the locus coeruleus (D, Craigie A7), at the level of the rostral medulla oblongata (E and F, Craigie A6 and A5, respectively) and a transverse section through the thoracic spinal cord (G). In this and subsequent figures, large black dots represent cell groups (with name alongside), small black dots represent nerve terminals, and diagonal shading represents axons. For further explanations see text and Table I.

ADRENALINE NEURONS IN RAT BRAIN

239 PERIVENTRICLIEAR AREAS

\

Fig. 2. Schematic diagram of a sagittal section of the rat brain based on the Kbnig and Klippel atlas 61. The two PNMT positive cell groups (CI and C2) and hypothetical adrenergic connections are demonstrated. Both ascending and descending axons are seen giving rise to axon terminals in the brain stem and spinal cord. therefore term these structures nerve cell bodies, axons and nerve terminals (varicosities, nerve endings, boutons), respectively. N o such fluorescent nerve cell systems could be identified after incubation with preimmune serum or P N M T antiserum adsorbed with P N M T . The distribution o f P N M T positive neurons is shown in the schematic Figs. 1 and 2. Fig. 1 A - G represents frontal sections at various levels of the central nervous system and Fig. 2 is a sagittal section indicating schematically possible adrenergic connections.

Nerve cell bodies Morphology. The P N M T positive cell bodies (Figs. 3-6) were multipolar with large cell processes, notably dendrites, which could be traced in the frontal plane for long distances up to 1 mm. The cell bodies varied in size mainly from 15 to 25/~m and the shape was usually spindle shaped or oval. Distribution. Two nerve cell groups could be distinguished and are termed C1 and C2 in analogy with the nomenclature of Dahlstrdm and Fuxe is (Figs. 1 and 2). Nerve cell group C1 (Figs. 1F, 2-4). This group is the largest of the two and found in the rostral medulla oblongata lateral to the olivary complex and caudal to the nucleus nervi facialis. These nerve cell bodies are located within the rostral area of the nucleus reticularis lateralis in the same way as the rostral part of the CA cell group A118. The distribution and morphology of the C1 cells are in fact very similar to the rostrally located CA cells of group A1. Nerve cell group C2 (Figs. 1E, F, 2, 5 and6). The cell bodies o f this comparatively small group were present at the same level o f the long axis o f the brain as cell group C1, i.e. at the rostral level of the medulla oblongata. The cells were located close to the midline in the dorsal part of the reticular formation ventral and medial to the vestibular nuclei. At a more caudal level some cell bodies were found immediately

240

T. HOKFELT et al.

Fig. 3. immunoftuorescence micrograph of the ventrolateral reticular formation of the rostra1 medulla oblongata. Specific P N M T fluorescence is observed in oval and spindle shaped nerve celi bodies and their processes (group C1). Note the extensive dendritic network. Magnification × 160. Fig. 4. Higher magnification of Fig. 3. Magnification × 400.

Fig. 5. lmmunolluo~'e>cence lllicl ogKaph t~' Lhc dol~,LL] )'L~phe FegiOll Lll"t/lC I O'[FLTtl illcdLI]IL~ o h h m g a t a . Specilic P N \ I T flLiore,,ccuce i,, observed in o\ al ,,ha}~ed nor\ e cell bod,e~ and thew procc~,,e,, {al-ro\~-,) (group C21 locMized clo',e to the midJine. V f o u r t h xcntricic with plexu~ choroidcus. MagnllicatloIa 140. Fig. 6. lmmunofklore~cence micrograph of the nuclcn~ tractu~ ~olitarii (NTS). Specific fluore~ccuce i~ observed in nerve terminaN f o r m i n g a plexu~ i~l the most do~,omedial part ~,f NTS. A te\~ ( a r r o \ ~ ) o~al shaped nerve cell bodie~ belonging to group C2 arc fouud. \ fourth xcntricle. Mz~gnil]cation 150, Fig. 7. l m m u n o f l u o r e s c e n c e micrograph of the medial reticular formation o f the pons. A x\eak, specific P N M T fluorescence is observed in trans\ersely cut axons ( a r r o a s ) r u n n i n g immediatel_~ xentral to genu nuclei facialis. Magnification " 400.

242

T. HOKFELTet al.

ventromedial to the nucleus tr. solitarii. Thus, cell group C2 surrounded the medial part of the ventral surface of the fourth ventricle. A few cell bodies were found more ventrally in between group C1 and C2. It may be pointed out that scattered CA nerve cell bodies have been found in this region is. At least one axon bundle from these two cell groups has been discovered (Figs. 2 and 7). This bundle appears to ascend in the reticular formation lying medially to the outgoing fibers of nervus facialis and ventrally to the genu nervi facialis. The bundle was round and well defined with a diameter of 100-150 #m. A CA bundle has previously been described in this area of the reticular formation 3,76. This CA bundle belongs to the ventral NA bundle 6s,76. The pathways in Fig. 2 only represent hypothetical adrenergic connections since the sensitivity of the technique is too low to obtain a complete picture of the A axons. Lesion experiments are now in progress to map the A pathways. Nerve terminals Morphology. As described above the fluorescent terminals had a dot-like appear-

ance, the diameter of the dots (varicosities) being mainly between 0.5-1 # m (Figs. 813) as seen in the fluorescence microscope. The intervaricose parts of the terminals could sometimes be observed but in these cases the so-called terminals may have represented preterminal axons, since it is sometimes difficult to differentiate between axons and axon terminals. Distribution. So far P N M T positive terminals have only been found in the brain stem. The distribution of these terminals has been summarized in schematic drawings of 7 frontal sections at various levels of the central nervous system (Fig. 1A-G). The density of the P N M T positive terminals in the various areas and nuclei has been roughly estimated and divided into 3 categories with a high, moderate and low density, respectively. These results are summarized in Table I. It should be underlined that the P N M T positive terminals are mainly found in certain visceral afferent or efferent nuclei of the brain and spinal cord, in the ventral periventricular grey of the lower brain stem and in certain nuclei of the hypothalamus. DISCUSSION

The evidence for the existence of DA, NA and 5-HT pathways in the mammalian central nervous system is well accepted. However, the significance of the small amounts of A present in the mammalian brain (5 ~ of both N A and A in rat brain 45) has been questioned, although Gunne 45 could identify A with a fluorimetric assay method. The demonstration of enzymatic synthesis of A in mammalian brain 5,13,71 with the highest enzyme activity in the diencephalon supports the view that A in brain is not derived from the periphery but from the brain tissue itself. Due to the difficulties with the Falck-Hillarp technique to differentiate between A and N A (see ref. 15) previous morphological studies have failed to either exclude or prove the existence of separate A pathways in brain. The present investigation therefore gives the first morphological evidence that reticular A containing neurons exist in the

ADRENALINE NEURONS IN RAT BRAIN

FI~. N, {IIInlLIIIO[]LIOF~C~[1CC

llIiCrO~Fa[lh 0~" [[I@ nuclcu~ Jol ~ah'~ 111oIoI ILlx llOI~ i \ a~I ( ~\

243

) ai1d IILICICLI'~

tract~i~ xohtal li tNTS). P N M T l~t.ioFe~,CellCCi'~ o b , e r \ o d in llCF\C tcl illlnal,~ ol'lhc~e nuclei The dcil,q[} l~ highest in NV btlt there h, a to~al lack of-,pecific fluore-~cencc 111 nucleu, hF poglo~',u> I N H ) \ ['OLII'[]I \ cntricle. \lagnif~cation 160. Fig. 9. Higher magnification of part of [zig. S denlon,trating the high den>it3 of P N \ | T p o q t i \ e terminals in the medial part of nucletl~ dor~ali,, motoi it> ncr~i \agl. N H nLIclcL> 11_~poglo~Ll~,. \ fourth ~entriclc. Magmfication 400.

244

x. HOKFELT et al.

TABLE I D E N S I T Y V A R I A T I O N S OF

PNMT

POSITIVE TERMINALS IN VARIOUS AREAS OR NUCLEI OF THE BRAIN STEM

AND SPINAL CORD OF RAT

High density

1. 2. 3. 4. 5.

Nucleus motorius dorsalis n. vagi (Figs. 8 and 9). Nucleus tr. solitarii (dorsal and rostral part) (Figs. 6 and 8). Nucleus paraventricularis magnocellularis. Nucleus paraventricularis rotundocellularis (Fig. 10). Nucleus sympathicus lateralis (Fig. 12).

Moderate density

1. The periventricular grey surrounding the ventral floor of the fourth ventricle and the aqueductus Sylvii. 2. The locus coeruleus, ventral part. 3. The periventricular grey surrounding the posterodorsal part of the third ventricle. 4. Nucleus dorsomedialis hypothalami. 5. Perifornical area (Fig. 11). From the lateral part of this area networks extend both dorsally up to the subthalamus and ventrally to the basal surface of the hypothalamus. Low density

1. Nucleus rhomboideus. 2. Posterolateral hypothalamus and medial subthalamus. Some or all of these terminals, however, can represent fibers of passage. 3. The periventricular area of the hypothalamus. 4. Nucleus arcuatus, ventrolateral part. 5. The grey matter surrounding the central canal of the cervical and thoracic part of the spinal cord (Fig. 13).

m a m m a l i a n brain with morphological characteristics similar to those in the D A , N A and 5-HT neuron systems. Furthermore, the findings strongly support the view that A m a y serve as a neurotransmitter in the m a m m a l i a n brain. It should be pointed out that the present immunohistochemical evidence on the cellular localization o f P N M T only gives indirect evidence for the existence o f A neurons, since the antibodies to P N M T m a y cross-react with other methylating enzymes. Furthermore, other phenylethanolamine derivatives like octopamine and metanephrine are methylated by P N M T 4 and therefore the P N M T positive neuron system could contain any o f these amines. These alternatives, however, are made unlikely by the following facts indicating that the P N M T positive neurons also exhibit a C A induced fluorescence when studied with the Falck-Hillarp technique. (1) G r o u p C1 seems to be identical with the rostral part o f the C A group A1 according to Dahlstr6m and Fuxe is and part o f group C2 identical to the rostral p a r t o f C A group A2 according to D a h l s t r 6 m and Fuxe is. (2) The ascending P N M T positive p a t h w a y in the reticular formation appears to be identical with part o f the ascending ventral C A pathway 6s,76. (3) M o s t o f the P N M T positive terminals are present in areas rich in C A nerve terminals according to the Falck-Hillarp technique. The available information therefore strongly supports the view that the P N M T containing neurons store C A and most p r o b a b l y A. Final evidence, however, must

ADRENALINE NEURONS IN RAT BRAIN

245

Fig. 10. lmmunofluorescence micrograph of the nucleu, paraxentricularis rotundocellulari~ thalami (NPR) close to the third ventricle (V). PNNIT fluorescence it observed in nerxe terminal~ in NPR \~hich is roughly outlined by arrow he:tds. H habenula. Magnification 1(~0. Fig. 11. lmmunofluorescence micrograph of the perifornical area. Specific PNMT fluorescence is observed in nerve terminals. Magnification 250.

await biochemical determinations of A in the areas rich in PNMT positive terminals. Furthermore, direct correlations between CA and PNMT fluorescent structures should be performed on adjacent sections of the same tissue (for techniques, see refs. 52, 55 and 59). In the following, however, the P N M T containing neurons will be described as the A containing neurons. In connection with the discussion of the specificity and the significance of the present results obtained with antibodies to P N M T it should be pointed out that the conversion of NA to A is the last step in the CA synthesis and that in the hypothetical A neuron s, in addition to PNMT, 3 other enzymes should be p r e s e n t - tyro sine hydroxylase, D D C and DBH. Thus, it cannot be taken for granted that with antibodies, e.g., to DBH, it is possible selectively to stain N A neurons in the rat brain as D B H is also present in A neurons (cf refs. 32 and 47). The possibilities to differentiate between various types of monoamine neurons using different CA synthesizing enzymes, partly

246

T. HOKFELT et al.

Fig. 12. Immunoftuorescence micrograph of the thoracic spinal cord. The nucleus sympathicus lateralis (NSL) contains a dense network of PNMT fluorescent nerve terminals. FL funiculus lateralis. Magnification ' 360. Fig. 13. Immunofluorescence micrograph of the intermediate part of the thoracic spinal cord. PNMT fluorescence is seen in nerve terminals (arrou s) in the dorsal commissure behind the central canal (C), possibly crossing and projecting towards the nucleus sympathicus lateralis. FD = funiculus dorsalis. Magnification × 150.

ADRENALINE NEURONS IN RAT BRAIN

247

in combination with the Falck-Hillarp technique, will be discussed elsewhere 54,59. The distribution of the A cell bodies, axons and terminals demonstrate that they are reticular neurons originating from the medulla oblongata innervating specific nuclei in the brain stem, most of which have a periventricular localization. It should be underlined, that the sensitivity of the technique may not be sufficient to reveal all PNMT positive neurons and therefore the present mapping must be regarded as preliminary. On the other hand, the comparatively few nuclei of A terminals discovered, seem roughly to agree with the low biochemical level of A found by GunnO ~. On the basis of the adrenergic innervation pattern shown in this paper, important central adrenergic mechanisms could be assumed to exist in the control of the brain functions mentioned below. Earlier studies have in fact demonstrated a number of pharmacological and physiological effects of A on various central functions many of which have been summarized by Rothballer 72 and Marley and Stephenson64. It might be interesting to compare some of these results with the present findings. (1) Oxytocin secretion. Dense innervation of nucleus paraventricularis (of. physiological studies of Cross16,17). (2) Food and water intake. Adrenergic innervation of e.g., the perifornical area and nucleus dorsomedialis hypothalami (el, physiological studies of Grossman44, Miller et al. 65, Booth 1°, Leibowitz63). (3) Body temperature. Adrenergic innervation of various parts of the hypothalamus (el, physiological studies of Feldberg and Lotti 27, Myers and Yaksh 66 and Schmidt74). (4) Gonadotrophin secretion. Adrenergic innervation of e.g., parts of the nucleus arcuatus. A has been found more effective than DA and NA in triggering ovulation in proestrus pentobarbital blocked rats (see Rubinstein and Sawyer73). (5) Blood pressure and respiration. Adrenergic innervations of nucleus dorsalis motorius nervi vagi, parts of nucleus tr. solitarii and the sympathetic lateral column of the spinal cord. These visceral afferent and efferent nuclei are well-known relay stations in vasomotor and respiratory control. The role of A in sleep and wakefulness regulation is complex and therefore deserves special attention. It is well-known that A can act as a central depressant or a central excitant depending upon dose and mode of administration (see review of Rothballer 72 and Marley and Stephenson64). Several explanations have been given for these opposite effects. Thus, the possibility of a toxic paralysis of excitatory A synapses has been mentioned to explain the depressant action (also described as stupor-analgesia, decreased locomotor activity), since this action is mainly observed with high doses of A (see ref. 72). On the other hand, the alerting effect after intravenous A may be due to peripheral effects of A and not to a direct action on the reticular activating system, although some workers have shown that the arousal after A is probably not caused by the hypertension induced by A 9,72. The present discovery of probable A pathways in brain offers, however, a possible additional explanation. The alerting action of A could be mediated via activation of the NA receptor belonging to coerulo-cortical NA pathways which is of importance for maintenance of wakefulness (see refs. 8 and 60). The depressant action of A could be mediated via the A receptors in the hypothala-

248

T. HOKFELTet al.

mus and periaqueductal grey substances. These areas have, in fact, already been suggested to be involved in the mediation of the behavioral depression and analgesia found after A19,2s. It should be emphasized that the locus coeruleus, which gives rise to the cortical NA pathways, is innervated by A terminals demonstrating the possibility of an interaction between A and NA neurons in brain. Adrenergic synapses can, thus, directly control activity in the NA 'arousal' system. Somnolence and sleep induced by intracerebral injections of A could thus, partly be obtained by the inhibition of nervous activity in the locus coeruleus. In conclusion, the present findings give morphological evidence for the existence of reticular A neurons in the rat central nervous system with ascending and descending projections to the brain stem and spinal cord, respectively. I N D E X O F SYMBOLS A N D ABBREVIATIONS

C1

~!~

Axons

Nerve termnnals

• o• l"e

AC CAI CER F FL FLM FM FMT FP GNF HA HP HI HV LM

Cellgroupwith name

= = = = = = =

a q u e d u c t u s c e r e b r i (Sylvii) capsula interna cerebellum columna fornicis fasciculus longitudinalis fasciculus longitudinalis medialis nucleus paraventricularis, pars magnocellularis = fasciculus mammillothalamicus = fibrae pyramidales = genu nuclei facialis = nucleus anterior hypothalami = nucleus dorsalis hypothalami = hippocampus = nucleus ventromedialis hypothalami = lemniscus medialis

ND NO

= =

NOS NT NTS PCI PVR

= = ~ = =

RH RNF TC TO 4.V ¥H

= = = = = =

nucleus dentatus nucleus olivaris (inferior, accessorius m e d i a l i s et d o r s a l i s ) nucleus olivaris superior radix tractus spinalis nuclei trigemini nucleus tractus solitarii pedunculus cerebellaris inferior nucleus periventricularis rotundocellularis nucleus rhomboideus radices nuclei facialis tractus corticospinalis tractus opticus fourth ventricle ventral horn

ACKNOWLEDGEMENTS

This work has been supported by grants from the Swedish Medical Research Council (14X-2887, 14P-3262 and 04X-715), by grants from Karolinska Institutets Forskningsfonder, the Population Council (M73.73), Stiftelsen Margarethahemmet, from Magn. Bergvalls Stiftelse and 'Svenska livf6rs~kringsbolags n~mnd f6r medicinsk forskning', by USPHS Grant MH-02717 and by NSF Grant GB-8465. The skilful technical assistance of Mrs. K. Andreasson and Miss A.-C. Swensson is gratefully acknowledged.

ADRENALINE NEURONS IN RAT BRAIN

249

REFERENCES 1 AND~N, N.-E., CARLSSON, A., DAHLSTRGM, A., FUXE, K., HILLARP, N.-A., AND LARSSON, K., Demonstration and mapping out of nigro-neostriatal dopamine neurons, Life Sci., 3 (1964) 523-530. 2 ANDrSN, N.-E., CORRODI,H., AND FUXE, K., Turnover studies using synthesis inhibition. In A. HOOPER (Ed.), Metabolism of Amines in the Brain, MacMillan, London, 1969, pp. 38-47. 3 ANDI~N,N.-E., DAHLSTROM,A., FUXE, K., LARSSON,K., OLSON, L., AND UNGERSTEDT,U., Ascending monoamine neurons to the telencephalon and diencephalon, Acta physiol, scand., 67 (1966) 313-326. 4 AXELROD,J., Metabolism of epinephrine and other sympathomimetic amines, Physiol. Rev., 39 (1959) 751-776. 5 BARCHAS,J. D., CIARANELLO,R. D., AND STEINMAN,A. M., Epinephrine formation and metabolism in mammalian brain, Biol. Psychiat., 1 (1969) 31-48. 6 BERGQUIST,N. R., AND SCHILLING,W. G. E. E., Preparation of anti-human immunoglobulin for indirect fluorescent tracing of auto-antibodies. In E. J. HOLBOROW (Ed.), Standardization in Immunofluorescence, Blackwell, Oxford, 1968, pp. 171-176. 7 BJ()RKLUND, A., FALCK, B., AND OWMAN, CH., Fluorescence microscopic and microspectrofluorometric techniques for the cellular localization and characterization of biogenic amines. In J. E. RALL AND I. J. KOPIN (Eds.), The Thyroid and Biogenic Amines, North-Holland Publ., Amsterdam, 1972, pp. 318-368. 8 BOLME,P., FUXE, K., AND LIDBRINK,P., On the function of central catecholamine neurons - their role in cardiovascular and arousal mechanisms, Res. Commun. Chem. Path. Pharmacol., 4 (1972) 657-697. 9 BONVALLET,M., DELL, P., ETHIEBEL,G., Tonus sympatique et activit6 61ectrique cortical, Electroenceph, clin. Neurophysiol., 6 (1954) 119-144. 10 BOOTH,D. A., Mechanisms of action of norepinephrine in eliciting an eating response on injection into the rat hypothalamus, J. Pharmacol. exp. Ther., 160 (1968) 336-348. 11 CARLSSON,A., FALCK,B., AND HILLARP,N.-~., Cellular localization of brain monoamines, Acta physiol, scand., 56, Suppl. 196 (1962) 1-28. 12 CHEAH,T. B., AND GEEFEN,L. B., Immunofluorescent staining of dopamine-fl-hydroxylase in the central nervous system, Proc. Aust. Phys. Pharm. Soc., 1 (1970) 1. 13 CIARANELLO,R. D., BARCHAS,R. E., BYERS,G. S., STEMMLE,D. W., AND BARCHAS,J. D,, Enzymatic synthesis of adrenaline in mammalian brain, Nature (Lond.), 221 (1969) 368-369. 14 COONS,A. H., Fluorescent antibody methods. In J. F. DAmELLI (Ed.), General Cytochemical Methods, Academic Press, New York, 1958, pp. 399-422. 15 CORRODI, H., AND JONSSON, G., The formaldehyde fluorescence method for the histochemical demonstration of biogenic monoamines. A review on the methodology, J. Histochem. Cytochem., 15 (1967) 65-78. 16 CROSS,B. A., Sympathetico-adrenal inhibition of the neurohypophysial milk-ejection mechanism, J. Endocr., 9 (1953) 7-18. 17 CROSS,B. A., Emotional inhibition of the milk-ejection reflex, J. Physiol. (Lond.), 125 (1954) 43P. 18 DAHLSTROM,A., AND FUXE, K., Evidence for the existence of monoamine containing neurons in the central nervous system. I. Demonstration of monoamines in the cell bodies of brain stem neurons, Acta physiol, scand., 62 (1964) 1-55. 19 DOWER, F. R., AND FELDBERG,W., Some central actions of adrenaline and noradrenaline when administered into the cerebral ventricles. In J. R. VANE (Ed.), Adrenergic Mechanisms, Churchill, London, 1960, pp. 386-392. 20 ER.~NK6, O., Fluorescing islets, adrenaline and noradrenaline in the adrenal medulla of some common laboratory animals, Ann. Med. exp. Fenn., 33 (1955) 278-290. 21 ER~NK/3,O., On the histochemistry of the adrenal medulla of the rat, with special reference to acid phosphatase, Acta anat. (Basel), 16, Suppl. 17 (1952) 1-60. 22 ER~NK6, O., Histochemistry of noradrenaline in the adrenal medulla of rats and mice, Endocrinology, 57 (1955) 363-368. 23 EtJLER, U. S. YON, A specific sympathomimetic ergone in adrenergic nerve fibers (sympathin) and its relations to adrenaline and noradrenaline, Acta physiol, scand., 12 (1946) 73-96. 24 FALCK, B., Observations on the possibilities of the cellular localization of monoamines by a fluorescence method, Acta physiol, scand., 56, Suppl. 197 (1962) 1-25. 25 FALCI¢,B., H.~GGENDAL,J., AND OWMAN, CIq,, The localization of adrenaline in adrenergic nerves in the frog, Quart. J. exp. Physiol., 48 (1963) 253-257.

250

T. HOKFELT et al.

26 FALCK,B., HILLARP,N.-A., THIEME,G., ANDTORP, A., Fluorescence of catecholamines and related compounds condensed with formaldehyde, J. Histochem. Cytochem., 10 (1962) 348-354. 27 FELDBERG, W., AND LOTTI, V. J., Temperature responses to monoamines and an inhibitor of MAD injected into the cerebral ventricles of rat, Brit. J. Pharmacol., 31 (1967) 152-161. 28 FELDBERG,W., AND SHERWOOD,S. L., Injections of drugs into the lateral ventricle of the cat, J. Physiol. (Lond.), 123 (1954) 148. 29 FUXE, K., Evidence for the existence of monoamine neurons in the central nervous system. III. The monoamine nerve terminal, Z. Zellforsch., 65 (1965) 573-596. 30 FUXE, K., Evidence for the existence of monoamine neurons in the central nervous system. IV. The distribution of monoamine nerve terminals in the central nervous system, Acta physiol, scand., 64, Suppl. 247 (1965) 39-85. 31 FUXE, K., GOLDSTEIN,M., HOKFELT, T., AND JOH, T. H., Immunohistochemical localization of dopamine-fl-hydroxylase in the peripheral and central nervous system, Res. Commun. chem. Path. Pharmacol., 1 (1970) 627-636. 32 FUXE, K., GOLDSTEIN,i . , HOKFELT,T., AND JOH, T. H., Cellular localization of dopamine-flhydroxylase and phenylethanolamine-N-methyl transferase as revealed by immunohistochemistry. In O. ERXNKG(Ed.), Histochemistry of Nervous Transmission, Progress in Brain Research, Vol. 34, Elsevier, Amsterdam, 1971, pp. 127-138. 33 FuxE, K., HOKFELT,T., JONSSON,G., AND UNGERSTEDT,U., Fluorescence microscopy in neuroanatomy. In W. J. H. NAUTAAND S. O. E. EBBESSON(Eds.), Contemporary Research Methods in Neuroanatomy, Springer, Berlin, 1970, pp. 275-314. 34 FuxE, K., HOKFELT,T., ANDUNGERSTEDT,U., Localization of indolealkylamines in CNS, Advanc. Pharmacol., 6A (1968) 235-251. 35 FUXE, K., HOKFELT,T., AND UNGERSTEDT,U., Morphological and functional aspects of central monoamine neurons, hzt. Rev. Neurobiol., 13 (1970) 93-126. 36 FUXE, K., AND JONSSON, G., The histochemical fluorescence method for the demonstration of catecholamines. Theory, practice and application, J. Histochem. Cytochem., 21 (1973) 293-311. 37 GEFEEN,L. B., LIVETT, D. G., AND RUSH, R. k., Immunohistochemical localization of protein components of catecholamine storage vesicles, J. Physiol. (Lond.), 204 (1969) 593-605. 38 GIBB,J. W., SPECTOR,S., ANDUDENFRIEND,S., Production of antibodies to dopamine-fl-hydroxylase of bovine adrenal medulla, Molec. Pharmacol., 3 (1967) 473478. 39 GOLDSTEIN, i . , Enzymes involved in the catalysis of catecholamine biosynthesis. In R. N. UBELL (Ed.), Methods in Neurochemistry, Vol. 1, Plenum Press, New York, 1972, pp. 317-340. 40 GOLDSTEIN,M., ANAGNOSTE,B., FREEDMAN,L. S., ROFFMAN,M., EBSTEIN,R. P., PARK, n . H., FUXE, K., AND HOKFELT, T., Characterization, localization and regulation of catecholamine synthesizing enzymes, Proc. II. Catecholamine Symposium, Strasburg, May 1973, In press. 41 GOLDSTEIN,M., FUXE,K., AND HOKFELT,T., Characterization and tissue localization of catecholamine synthesizing enzymes, PharmacoL Rev., 24 (1972) 293-309. 42 GOLDSTEIN,M., FUXE,K., HOKFELT,T., ANDJOH, T. H., Immunohistochemical studies on phenylethanolamine-N-methyltransferase, dopadecarboxylase and dopamine-fl-hydroxylase, Experientia (Basel), 27 (1971) 951-952. 43 GOLDSTEIN,M., GANG, H., AND JOH, T. H., Subcellular distribution and properties of bovine phenylethanolamine-N-methyltransferase (PNMT), Fed. Proc., 28 (1969) 287. 44 GROSSMAN,S. P., Eating or drinking elicited by direct adrenergic or cholinergic stimulation of hypothalamus, Science, 132 (1960) 301-302. 45 GUNNE,L.-M., Relative adrenaline content in brain tissue, Aetaphysiol. scand., 56 (1962) 324-333. 46 HARTMAN,B. K., AND UDENFRIEND,S., Immunofluorescent localization of dopamine-/3-hydroxylase in tissues, Molec. Pharmacol., 6 (1970) 85-94. 47 HARTMAN,B. K., ZIDE, D., ANDUDENFRIEND,S., The use of dopamine-fl-hydroxylase as a marker for the noradrenergic pathways of the central nervous system in the rat, Proc. nat. Acad. Sci. (Wash.), 69 (1972) 2722-2726. 48 HILLARP,N.-/~., AND H~KFELT,B., Evidence of adrenaline and noradrenaline in separate adrenal medullary cells, Acta physiol, scand., 30 (1953) 55-68. 49 HILLARP,N.-/~., AND H6KrELT, B., Cytological demonstration of noradrenaline in the suprarenal medulla under conditions of varied secretory activity, Endocrinology, 55 (1954) 255-260. 50 HILLARP,N.-/~., ANDH6KFELT,B., Histochemical demonstration of noradrenaline and adrenaline in the adrenal medulla, J. Histochem. Cytochem., 3 (1955) 1-5. 51 H6KrELT, T., FUXE, K., AND GOLDSTEIN, M., Immunohistochemical localization of aromatic

ADRENALINE NEURONS IN RAT BRAIN

52 53 54

55

56 57 58 59 60 61 62 63 64 65 66

67 68 69

70 71 72 73 74 75 76 77 78

251

L-amino acid decarboxylase (DOPA decarboxylase ) in central dopamine and 5-hydroxytryptamine nerve cell bodies of the rat, Brain Research, 53 (1973) 175-180. H6Kr~LT, T., FtJXE, K., GOLDSTEIN, M., AND JOH, T. H., Immunohistochemical studies of three catecholamine synthesizing enzymes: Aspects on methodology, Histochemie, 33 (1973) 231-254. HOKFELT,T., FUXE, K., GOLDSTEIN,M., AND JOHANSSON,O., Evidence for adrenaline neurons in the rat brain, Acta physioL scand., 89 (1973) 286-288. H/)KFELT, T., FUXE, K., GOLDSTEIN, M., AND JOHANSSON,O., On the possibility to identify and differentiate between monoamine neurons with immunohistochemical studies on catecholamine synthesizing enzymes, In preparation. H/)KFELT, T., AND LJUNGDAHL,/~k., Modification of the Falck-Hillarp formaldehyde fluorescence method using the Vibratome®: Simple, rapid and sensitive localization of catecholamines in sections of unfixed or fixed brain tissue, Histochemie, 29 (1972) 324-339. HOLTZ, P., Uber die sympathicomimetische Wirksamkeit von Gehirnextrakten, Acta physioL scand., 20 (1950) 354-362. HOLZBAUER, M., AND SHARMAN, D. F., The distribution of catecholamines in vertebrates. In H. BLASCHKOAND E. MUSCHOLL(Eds.), Catecholamines, Springer, Berlin, 1972, pp. 110-185. JOH. T. H., AND GOLDSTEIN, M., Isolation and characterization of multiple forms of phenyl ethanolamine N-methyltransferase, Molec. Pharmacol., 9 (1973) 117-129. JOHANSSON,O., H~3KEELT,T., AND GOLDSTEIN, M., Combined immunofluorescence and formaldehyde fluorescence studies on monoamine cell systems, In preparation. JOUVET, M., The role of monoamines and acetylcholine-containing neurons in the regulation of the sleep-waking cycle, Rev. Physiol., 64 (1972) 166-307. KONIG, J. F. R., AND KLIPPEL, R. A., The Rat Brain: A Stereotaxic Atlas of the Forebrain and Lower Parts of the Braht Stem, Williams and Wilkins, Baltimore, Md. 1963. KOVACS,K., UND FAREDIN,I., Untersuchungen fiber die Beeinflussung des Catecholamingehaltes des Hypothalamus der Ratte, Acta neuroveg. (Wien), 22 (1961) 184-191. LEmOWITZ, S. F., Hypothalamic fl-adrenergic 'satiety' system antagonizes an a-adrenergic 'hunger' system in the rat, Nature (Lond.), 226 (1970) 963-964. MARLEY,E., AND STEPHENSON,J. D., Central actions of catecholamines. In H. BLASCHKOAND E. MUSCt~OLL(Eds.), Catecholambzes, Springer, Berlin, 1972, pp. 463-537. MILLER, N. E., GOTTESMAN,K. S., AND EMERY, N., Dose response to carbachol and norepinephrine in rat hypothalamus, Amer. J. PhysioL, 206 (1964) 1384-1388. MYERS,R. D., AND YAKSH, T. L., Feeding and temperature responses in the unrestrained rat after injections of cholinergic and aminergic substances into the cerebral ventricles, Physiol. Behav., 3 (1968) 917-928. NAIRN, R. C., Fluorescent Protein Tracing, 3rd ed., Livingstone, Edinburgh, 1969. OLSON,L., AND FUXE, K., Further mapping out of central noradrenaline neuron systems: Projections of the 'subcoeruleus' area, Brain Research, 43 (1972) 289-295. PAASONEN,M. K., AND DEWS, P. B., Effects of raunescine and isoraunescine on behaviour and on the 5-hydroxytryptamine and noradrenaline contents of brain, Brit. J. PharmacoL, 13 (1958) 84-88. PEASE,D. C., Buffered formaldehyde as a killing agent and primary fixative for electron microscopy, Anat. Rec,, 142 (1962) 342. POHORECKY,L. A., ZIGMOND, M., KARTEN, H., AND WURTMAN, R. J., Enzymatic conversion of norepinephrine to epinephrine by the brain, J. Pharmacol. exp. Ther., 165 (1969) 190-195. ROTnBALLER,A. B., The effects of catecholamines on the central nervous system, Pharmacol. Rev., 11 (1959) 494-547. RUBINSTEIN,L., AND SAWYER,C. n., Role of catecholamines in stimulating the release of pituitary ovulating hormone(s) in rats, Endocrinology, 86 (1970) 988-995. SCrIMIOT,J., Die Abh~ngigkeit der Temperaturbeeinflussung yon Ratten durch biogene Amine yon der Applikationsart und der Umgebungstemperatur, Acta biol. reed. germ., 10 (1963) 350-356. STOVEEL,M., ET ROEF~,J., Action de l'anoxie et de diff6rents taux de gas carbonique sur le contenu en noradr6naline et en adr6naline de cerveau de rat, C.R. Soc. Biol. (Paris), 155 (1961) 237-240. UNGERSTEDT,U., Stereotaxic mapping of the monoamine pathways in the rat brain, Acta physiol. scand., 82, Suppl. 367 (1971) 1-48. VOGT, M., The concentration of sympathin in different parts of the central nervous system and normal conditions and after the administration of drugs, J. Physiol. (Lond.), 123 (1954) 451-481. ZEMAN,W., AND INNES, J. R. M., Craigie's N~troanatomy of the Rat, Academic Press, New York, 1963.