Ultrastructure and Pharmacological Studies of Nerve Endings in the Pineal Organ

389

Ultrastructure and Pharmacological Studies of Nerve Endings in the Pineal Organ A. PELLEGRINO D E IRALDI, L. M. ZIEHER

AND

E. D E ROBERTIS

Instituto de Anatomia General y Embriologia, Facultad de Ciencias Midicas, Buenos Aires (Argentina)

In recent years interest in the pineal gland has been increased considerably by the demonstration that this tissue is rich in biogenic amines (Giarman and Day, 1958; Giarman et al., 1959; Giarman and Freedman, 1960). Histamine, catecholamines and 5-hydroxytryptamine (5-HT) are exceptionally concentrated in all species studied, and in simian and human glands 5-HT has the highest concentration ever reported for neural structures (Giarman and Freedman, 1960). In addition to this amine there is the pineal hormone melatonin (Lerner et al., 1958), which derives from 5-HT by 0-methylation, and N-acetylation and glomerulotrophin, which recently has also been considered as a derivate of 5-HT (Farrell and McIsaac, 1961). From the structural viewpoint one must consider two main components that may be involved in the metabolism of biogenic amines in this tissue. One is represented by the pinealocyte, the specific cell of the gland, and the other by its sympathetic innervation. In a previous paper @e Robertis and Pellegrino de Iraldi, 1961a) some of the nerve fibres and endings within the pineal gland of the rat were investigated electron microscopically. In these as well as in other sympathetic nerve fibres of the splenic nerve (De Robertis and Pellegrino de Iraldi, 1961b), we found aplurivesicular material consisting of homogeneous vesicles plus some heterogeneous ones containing a dense granule of reduced osmium. These granular vesicles were considered as specific for adrenergic nerves and endings, and supporting evidence was obtained by the finding of similar vesicles in the hypothalamus (Pellegrino de Iraldi et al., 1963), in invertebrate nerve tissue (Pellegrino de Iraldi and De Robertis, 1962; Gerschenfeld, 1962) and in vertebrate nerve tissue (Taxi, 1961; Grillo and Palay, 1962; Richardson, 1962). These findings and interpretations have been reinforced by the recent studies of Wolfe et al. (1962) who have shown that these granular vesicles may bind the injected tritiated noradrenaline (NA). The present work was initiated with the idea of investigating the origin of the nerve fibres innervating the pineal gland and particularly the nature of the granular vesicles present in these nerve endings. For this purpose two principal approaches were used : Some rats were submitted to the bilateral extirpation of the superior cervical ganglia, and others were submitted to the action of drugs that may alter the metabolism of References p . 4191421

390

A. P E L L E G R I N O D E I R A L D I

et al.

catechol and indolamines. In both cases the glands were fixed and studied under the electron microscope, and the changes occurring in the nerve fibres and endings were specially recorded. Preliminary determinations of 5-HT content were also carried out in normal and denervated glands and in some of the rats treated with some of the agents that may change the content in this indolamine. In previous preliminary reports (Pellegrino de Iraldi and De Robertis, 1961; Pellegrino de Iraldi and De Robertis, 1963) it was found that after a single injection of reserpine there is a rapid and almost complete disappearance of the granular vesicles while the recovery is rather slow and takes several days. It was also found that a M A 0 inhibitor, iproniazid, induces an increase in the concentration and size of granulated vesicles and that this drug is able to counteract the action of reserpine on the nerve endings. In addition, it was noted that pyrogallol, an inhibitor of 0-methyltransferase, has much less effect on nerve endings and does not influence the action of reserpine. In the present paper these results will be reported in an extended form. The action of other drugs such as DOPA (dopamine) 5-HTP, a-methylmetatyrosine, aramine and nialamide on the ultrastructure of nerve endings, and on the 5-HT content of the gland will be mentioned. METHODS A N D OBSERVATIONS

Electron microscope observations on nerve fibres and endings in normal pineal glands Most investigators agree on the sympathetic origin of the nerve fibres innervating the pineal gland. According to Cajal (1904) it receives exclusively sympathetic perivascular nerve fibres. This view, which excludes the possibility of central control of the gland, is supported by Herring (1927) in regards to the rat. On the other hand Ikuta (1937), in experiments on pinealectomy in the rat, finds experimental evidence of a nerve tract between this body and the habenula. The same type of investigation in the cat gave negative results (Martin, 1941). Gardner (1953), in the hooded rat, finds nerve fibres of central and sympathetic origin, the former coming mainly by way of the habenular commissure and ending among the pinealocytes, away from the blood capillaries. The sympathetic fibres, exclusively unmyelinated, accompany and innervate the pial blood vessels penetrating into the gland. Kappers (1960), in an extensive study of the development and innervation of the epiphysis of the rat, gives conclusive evidence that the innervation is autonomic and supplied bilaterally by the superior cervical ganglia. Extirpation of these sympathetic ganglia produces degeneration of the innervating fibres. The author reaches the conclusion that the innervation is mainly, if not exclusively, orthosympathetic, and that ‘neither nervous nor vascular relations have been observed pointing to the existence of an epithalamo-epiphyseal or habenulo-epiphyseal complex that, in any way, could,be compared with the hypothalamo-hypophyseal complex’. In thin sections observed under the electron microscope, bundles of nerve fibres

NERVE E N D I N G S I N THE P I N E A L O R G A N

391

Fig. 1 . Nerve bundle found near the capsule of the pineal containing mainly unmyelinated nerve fjbres included within Schwann cells (Sch. c.). Ax = axon, rn.Ax. = myelinated axon, Sch. c. N. = Schwann cell nucleus, x 18,500. (I 5 .

n

Figs. 1-18 are electron microgiaphs of rat pineal glands fixed in osmium tetroxide and in most cases embedded in methacrylate. References p. 419-421

N E R V E E N D I N G S I N THE P I N E A L O R G A N

393

Smaller bundles of unmyelinated fibres, invested by Schwann cytoplasm, are seen more frequently in the proximity of large blood vessels. This relationship is lost in the finer bundles where some of the fibres become denuded and free of Schwann cell investment (Fig. 2). In these finer axons, in addition to the mitochondria, neurotubules and neurofilaments that are the characteristic fibrillar structures of the axon, and

Fig. 3. Typical nerve endings (n.end) in the pericapillary space (p. cap). The capillary (cap) lumen is surrounded by endothelial cells (end. c). gv = granular vesicles, d.mi = degenerated mitochondria, L = lipid droplet, N = nucleus, p = pinealocyte, x 32,000. References p. 419-421

394

A. P E L L E G R I N O D E I R A L D I

et al.

groups of vesicles, similar to those found in larger amounts at the nerve endings are observed. This vesicular material was first designated by us as plurivesicular material (De Robertis and Pellegrino de Iraldi, 1961a, b). It is composed of clear vesicles similar in size and shape to the synaptic vesicles observed in central and peripheral synapses

Fig. 4. Nerve ending (n. end) adjacent to pinealocytes and to the pericapillary space (p. cap). The axons (Ax) appear as thin pedicles of the endings containing neurotubules and some plurivesicular material. Granular vesicles (gv) are conspicuous but in lesser concentration than the clear vesicles. d.mi = degenerated mitochondria, p = pinealocyte, x 36,000.

N E R V E E N D I N G S I N THE P I N E A L O R G A N

395

(De Robertis and Bennett, 1954, 1955), and of granulated vesicles of similar size but containing a dense granule of reduced osmium (Fig. 2). The nerve endings are mainly found in the large perivascular spaces that surround the blood capillaries of the pineal gland (Fig. 3). They are completely denuded of Schwann investment, and frequently they appear as claviform expansions of thinner axons in the form of particles in which neurotubules are apparent. The relationship of these nerve endings to the pineal tissue varies. While most of them seem to end free in the perivascular space as if the transmitter could be directly released there, other endings are seen near the pinealocytes adjacent to the space and in a rather intimate contact (Fig. 4). However, images that could be interpreted as a definite functional contact with pinealocytes are not readily observed. Only in rare cases endings are found in between pinealocytes without apparent relationship to the perivascular spaces. The plurivesicular material composed of homogeneous and granular vesicles is the most conspicuous component of the ending and fills the larger part of it. The vesicles are round or oval in shape, and have a mean diameter of 425 A. In normal endings the percentage of granular vesicles varies between 31 and 40% of the total. The mean diameter of the dense granule is 210 A. The size distribution of this vesicular material, and the percentage and size of the granules, are shown in the histogram of Fig. 18. Another constant component of the ending is several mitochondria, some of which undergo a typical alteration of the inner structure with loss of crests and decrease in electron opacity (Fig. 3). Electron microscope observations of pineal glands after bilateral gangliectomy The cervical superior ganglia of adult rats were extirpated under ether anesthesia, and pineal glands were observed at different intervals between a few minutes and 4 months after bilateral gangliectomy. As shown in Fig. 5, at 21 h after gangliectomy the changes of the nerve endings are still not very conspicuous, although in comparison to the normal ones they show a general retraction with a denser matrix and certain degree of clumping of the vesicles (Fig. 3). Definite alterations of the nerve fibres and endings were found 66 h after gangliectomy. As observed in Fig. 6 there is a total disappearance of the plurivesicular material from the ending, vacuolization of the axoplasm, and mitochondria1 alterations. Endings appear swollen as ghost-like elements still surrounded by a membrane and with a clear watery content (Fig. 6). After 5 days all nerve endings containing the plurivesicular material have disappeared. The perivascular spaces are free of nerve terminals and, in contrast with the normal tissue, look empty (see Fig. 7 in comparison with Fig. 3). Near the pinealocytes it is possible to recognize rests of altered nerve structures and some debris apparently phagocytized by the Schwann cells. The degeneration of the nerve endings makes it easier to recognize some true processes of the pinealocytes, which may have vesicles of various sizes, but lack the granular vesicles and the typical mitochondria of the nerve endings. In Fig. 8 two of these cellular processes, which correspond to a denervation of 9 References p.:419-421

396

A. P E L L E G R I N O DE I R A L D I

et al.

days, can be observed and the similarity with the apical cytoplasm of the pinealocytes is evident. In no case it has been possible to see granular vesicles in these processes even after treatments such as the injection of dopamine, which produces an increase in concentration of granular vesicles in the normal endings. This condition persists

Fig. 5. Similar to Fig. 4 but 21 h after bilateral gangliectomy. Some nerve endings (n. end) are retracted with a dense matrix and clumping of the vesicular material (arrows). p = pinealocyte, p. cap. =.pericapillary space, x 60,000.

NERVE ENDINGS I N THE PINEAL O R G A N

397

for a period of 4 months, which is the:longest studied, and it is probably irreversible. These results show clearly that nerve fibres and endings of the pineal gland of the rat originate from the superior cervical ganglion, as was demonstrated previously at the optical level by Ariens Kappers (1960). They also demonstrate that the pluri-

Fig. 6. Degenerating nerve fibres and endings 66 h after gangliectomy. The endings (n. end) are completely altered with swelling and loss of the plurivesicular component. The axons (Ax) show vacuolization and destruction of neurofibrillar material. N = nucleus, p. cap = large pericapillary space with collagen fibres, x 19,000. References p. 419-421

398

A. P E L L E G R I N O D E I R A L D I

et al.

vesicular material with the granular vesicles found within sympathetic nerve fibres and endings is probably related to the adrenergic transmitter. These findings also show that our previous interpretation of the so-called secretory processes of the,pinealocytes was incorrect @e Robertis and Pellegrino de Iraldi,

Fig. 7. Pericapillary space (p. cap) devoid of nerve endings 5 days after gangliectomy. Cap = capillary lumen. Some rests of degenerated nerves are indicated with arrows. p = pinealocyte, x 16,800.

NERVE ENDINGS I N THE PINEAL ORGAN

399

196la) and that all the plurivesicular material belongs to the sympathetic nerve fibres and terminals. Pinealocytes have some processes, but they only have clear vesicles of different sizes which are found in the cytoplasm. This experimental demonstration simplifies the matter considerably since we now

Fig. 8 Pericapillary space (p. cap) 9 days after gangliectomy. Two cellular processes (cp) containing clear vesicles of different sizes are observed. Similar material is found in the pinealocyte. mi = mitochondria, x 60,OOO. Refermcrs p . 419421

400

A. P E L L E G R I N O DE I R A L D I

et al.

can attribute all those changes induced physiologically or pharmacologically in the plurivesicular material to the sympathetic nerve endings. This is then an excellent object for studying the action of agents that may act on sympathetic nerve endings at a submicroscopic level. Some examples of this are shown below. Action of reserpine, iproniazid, aramine, nialamide, 5-HTP, DOPA and dopamine on the ultrastructure of nerve endings of the pineal gland It is known that reserpine produces a depletion of 5-HT (Shore et al., 1955; Pletscher et al., 1955, and also Shore et al., 1956), NA (Carlsson et al., 1957), and dopamine (Bertler and Rosengren, 1959) in the nervous tissue, but the mechanism of action of this drug is little understood. According to Carlsson et al. (1957) and Carlsson (1963) the reserpine syndrome is largely caused by blockage of the transmission mechanism in noradrenergic peripheral nerves and by the lack of fixation of the synthesized NA on the granules. In a recent study of the time course of action of reserpine on the ‘free’ and ‘bound’ 5-HT of the brain, Giarman (1963) postulates a multiple site of action of reserpine on the permeability of the cell membrane, on the particle containing the inactivating enzyme MAO, and on the storage granule. Zeller et al. (1952) and Zeller and Barsky (1952) have shown that iproniazid has a powerful and prolonged influence on MAO. Brodie et al. (1956) and Shore et al. (1957) observed that this drug increases the 5-HT and NA content of the brain and that it can counteract the depletion induced by reserpine. Similar findings were reported by Green and Erickson (1960) and Green and Sawyer (1960) in studies of the content of free and bound amines. However, there are different species and a different effect on the levels of 5-HT and NA (Spector et al., 1960b). Aramine is a sympathomimetic amine that produces depletion of the catecholamine stores (Udenfriend and Zaltzman-Nirenberg, 1962; Gessa et al., 1962). Recently Bertler et al. (1963) reported that aramine depletes 5-HT from the pineal gland, reducing it considerably in a few hours. Nialamide is a powerful M A 0 inhibitor of prolonged effect which may double the catecholamine content of the mouse brain in 3 h. NA, dopamine and their methylated derivatives increase with this treatment. Given after reserpine, nialamide is not able to restore the content of these catecholamines. However, 6 h after the injection of nialamide (following the reserpine treatment) the values of 5-HT in the brain have not only recovered but are higher than in the normal brain (Carlsson et al., 1960, 1962). Results Reserpine. In our experiments we injected reserpine in a dose of 5 mg/kg intraperitoneally. Under the electron microscope it was observed that between 2 and 48 h after a single injection of reserpine there is an almost complete disappearance of the dense grains. The size of the vesicles is also decreased and there are many crosses of oblique sections of neurotubules within the endings (Fig. 9, see also Fig. 18).

NERVE E N D I N G S I N THE PINEAL O R G A N

40 1

The action of reserpine can be noted within only 10 min, and it reaches its maximal effect at 24 h. The replacement of the dense grains becomes evident after 3 days and reaches the normal range after 8 days (see Pellegrino de Iraldi and De Robertis, 1961). The recovery follows a curve that is strikingly similar to that observed by Shore and Brodie (1957) for NA and 5-HT in the brains of rabbits after reserpine.

Fig. 9. Nerve endings (n. end) 2 h after injection of reserpine. Practically all dense granules have disappeared. The vesicles are smaller and some tubulai structures are observed (arrows). Cap = capillary end. c = endothelial cell, p. cap = pericapillary space, mi = mitochondria, x 100,OOO. References p . 419421

402

A. P E L L E G R I N O D E I R A L D I

et al.

Iproniazid. The injection of iproniazid at the single dose of 100 mg/kg for 8-16 h or of 25 mg/kg daily intraperitoneally for 9-16 days produced a moderate increase in the proportion of granular vesicles (Fig. 11) in all cases and also in the size of the vesicles and granules (Fig. 18). After 8 h the concentration of granular vesicles rose

Fig. 10. Nerve endings (n. end) in a large pericapillary space (p. cap) 29 h after reserpine and 5.3 h after nialamide. Note the lack of dense grains. er = erythrocyte, cap = capillary. Embedded in Epon 812, x 24,000.

N E R V E E N D I N G S I N THE P I N E A L O R G A N

403

to 62 %, while after 17 h it was 55 %, and with chronic administration of the drug it was 53 %. The mean size of the vesicles in the chronic experiments reached 520 8, as opposed to 425 8, in the normal. Iproniazid and reserpine. The combined effect of iproniazid and reserpine was even

Fig. 1 1 . Nerve endings (n. end) 1 h after iproniazid. Note the increase in size and concentration of granular vesicles as compared with the normal (Fig. 3). Some dense granules of large size are indicated with arrows. Ax = axon, mi = mitochondria, gv granular vesicles, p. cap = pericapillary space, x 100,OOO. References p.1419-421

404

A. P E L L E G R I N O D E I R A L D I

et al.

more significant. The M A 0 inhibitor given previously prevented the effect of reserpine on the release of the granular vesicles. In Fig. 12, corresponding to a rat injected with iproniazid 15 h prior to reserpine and sacrificed 2 h later, there is a considerable proportion of granular vesicles. In this case it was 50% (Fig. 18), while after 4 h of reserpine (after 13 h of iproniazid) the proportion of granular vesicles was

Fig. 12. Nerve endings, 17 h after iproniazid and 2 h after reserpine. Note that the granular vesicles Cpv) have persisted, and compare with Fig. 9 in which only reserpine was administered. With arrow: one neurotubule, x 100,OOO.

NERVE E N D I N G S I N T H E P I N E A L O R G A N

405

52%. In one case (Fig. 13), sacrificed at 40 h after iproniazid and 24 h after reserpine, the protection was still evident and the concentration of granular vesicles was 30%. The protective effect of iproniazid on the granular vesicles could not be observed when the reserpine was administered 1 h prior to iproniazid or even simultaneously.

Fig. 13. Nerve endings (n. end) in a pericapillary space (p. cap) 40 h after iproniazid (and 24 h after reserpine. The granular vesicles (gv) represent 30% of the total. Ax = axon, x 60,000. References p . 419-421

406

A. P E L L E G R I N O D E I R A L D I

et al.

Aramine. Aramine given intraperitoneally (25-30 mg/kg) in 6 h produced complete depletion of the granular vesicles (Fig. 14) but the size of the vesicles is not reduced as with reserpine.

Fig. 14. Nerve endings (n. end) after administration of aramine for 6 h. Note disappearance of dense grains with the exception indicated by an arrow. dmi = degenerated mitochondria, mi = mitochondria, p = pinealocyte, p. cap = pericapillary space, x 60, OOO.

NERVE E N D I N G S I N THE PINEAL ORGAN

407

Reserpine and nialarnide. The results of the combined action of reserpine and nialamide were of considerable interest in view of the findings of Carlsson et al. (1960 and 1962) reported above on the content of brain amines. Reserpine (5 mg/kg) was given intraperitoneally for 29 h, and nialamide (500 mg/kg intraperitoneally) was

Fig. 15. Nerve endings after treatment with pyrogallol (50 mg/kg in 3 injections). The concentration of granular vesicles Cpv) is about normal. cf = collagen fibres, p. cap = pericapillaryspace, d.mi = degenerated mitochondria, mi = mitochondria, x 60,000. References p . 419-421

408

A. P E L L E G R I N O D E I R A L D I

et al.

given for 5+ h. The latter treatment did not reverse the effect of reserpine in relation to the granular vesicles, which were completely lacking. However, some of the clear vesicles were oflarger size than those in the gland which was only reserpinized (Fig. 10). 5-HTP. 50 mg/kg of 5-HTP were administered intravenously and the rats were

Fig. 16. Nerve endings treated with pyrogallol and reserpine. Note the lack of protection and disappearance. of all dense grains. The vesicles (arrow) are larger than in controls. mi = mitochondria, d.mi = degenerated mitochondria, p. cap = pericapillary space., X 80,000.

NERVE E N D I N G S I N THE PINEAL O R G A N

409

sacrificed after 60 min. This treatment, which according to the pharmacological data increases the 5-HT content of the gland by 400% (see below), produces a slight decrease in granular vesicles. Their concentration was 25 %. Iproniazid and 5-HTP. The association of 5-HTP with the prior administration of iproniazid (100 mg/kg iproniazid i.p. for 16 h, followed by 50 mg/kg intravenously

Fig. 17. Nerve endings (n. end) 21 h after injection of 50 mg/kg of a-MMT. Note the marked reduction in concentration and size of dense grains (arrows). p. cap = pericapillary space, x 60,ooO. References p . 419-421

410

A. P E L L E G R I N O DE I R A L D I

et al.

injected 5-HTP for 30-40 min) counteracts the effect of iproniazid alone, and there is no increase in the number of granular vesicles. The total concentration found was within the normal range (30 %). The administration of 5-HTP to gangliectomized rats did not produce morphological changes in the pineal glands. a-Methyl-3-hydroxyphenylalanine,a-MMT is an inhibitor of dopadecarboxylase which determines the depletion of catecholamines and, in larger doses, also the depletion of 5-HT. The mechanism of this effect is probably more complex and it may interfere with the binding of these amines. According to Hess etal. (1961), with 400 mg/kg a-MMT there is a marked decrease of NA and less 5-HT than in the brain and heart, but after 18 h the 5-HT has reached normal levels while the depression of NA persists longer. Brodie and Costa (1961), using 50 mg/kg, found disappearance of NA without change in 5-HT content in the mesencephalon of the rabbit after 13 h. In preliminary studies we gave 50 mg/kg a-MMT intravenously and observed the pineals after 21 h. We also gave 400 mg/kg and studied the pineals after 21 and 48 h. In the first case (Fig. 17) the almost complete disappearance on the dense grains was found, an effect which was even more marked with 400 mg. With this last dose, and at 48 h, the recovery of granular vesicles was already observed. DOPA. DOPA was given intravenously at 100 mg/kg and the material was fixed at 30 and 60 min. This treatment was associated with iproniazid and pyrogallol in some cases. After 30 min there was a moderate increase in granular vesicles (45%) which was more marked after 60 min (48 %). The mean diameter of the vesicles increased (468 A) without notable changes in the grains. With the prior administration of iproniazid (100 mg/kg i.v. for 17 h) and DOPA (100 mg/kg for 60 min) the concentration of grains rose to 57%. With pyrogallol prior to DOPA in 3 injections i.v. of 50 mg/kg every 30 min, the concentration of granular vesicles was lower than with DOPA alone (39 %). The injection of DOPA to gangliectomized rats did not produce apparent changes in the pineal. The administration of dopamine in conjunction with iproniazid (iproniazid 100 mg/kg for 16 h, dopamine 10 mg/kg for 30 min) gave the maximal increase in granular vesicles (73%) and a considerable increase in diameter of the vesicles (500 A) and grains (283 A). Action of pyrogallol and reserpine on the ultrastructure of nerve endings

Axelrod and Laroche (1959) demonstrated that pyrogallol inhibits the methylation of adrenaline and NA by the enzyme catechol-0-methyltransferase (COMT), and prolongates the physiological action of these amines. Crout et al. (1961), in a comparative study of COMT and M A 0 in different tissues, reached the conclusion that M A 0 is more active in the brain and heart, but much less than COMT in the liver. They found that several M A 0 inhibitors increased the endogenous catecholamines in the brain and heart, but pyrogallol either did not change them or else prodyed a slight decrease. Furthermore, Spector et al. (1960a) have shown that pyrogallol does not counteract the sedation of the animal and the depletion of the brain of NA and 5-HT produced by reserpine. They have reached the conclusion that while M A 0

N E R V E E N D I N G S I N THE P I N E A L O R G A N

41 1

is responsible for the inactivation of 5-HT and NA in tissues, COMT would act on catecholamines after their release into the circulation. In our experiments, after 9 intravenous injections of pyrogallol (50 mg/kg) in 44 h there was little or no change in the proportion of granular vesicles. The concentration observed, 29 %, is within the lower range of the controls (see Fig. 18). Similar findings 4. 60 Reserpine

40

20 0

20 lproniazid - reserpine

40t 60

n

-

Pyrogalloi reserpine

40

20 0

0grains

20 0

vesicies

300

700

0

300

700 A

Fig. 18. Histograms of the distribution of sizes of vesicles and dense grains, and of the concentration of granular vesicles in control animals and in those treated as indicated.

were obtained after 2, 3 and 5 injections of pyrogallol (Fig. 15). The most striking result was observed when the above treatment was accompanied by reserpine (for 2 and 4 h). In all cases (as shown in Fig. 16) the depletion of granular vesicles was almost complete. In spite of this effect, the size of the vesicles increased (mean diameter of 500 A) while with reserpine alone it decreased (350 A) below the normal level of 425 A (see Fig. 18).

5-HT content of normal and denervated pineal gland

5-HT was assayed biologically in single pineal glands using a modification of the rat stomach strip method of Vane (Zieher and De Robertis, 1963). The acetone extraction References p . 419421

412

A. P E L L E G R I N O D E I R A L D I

et al.

was according to Costa’s method (1960), and included incubation with chymotrypsin to inactivate substance P and other polypeptides. The normal content of the adult rat pineal generally varied between 10 to 20 ng per pineal. The mean of 55 determinations was 14.5 f 1.3. Considering the weight of the pineal in approximately 1.3 mg, there is the high concentration of 1 1.1 pg/g 5-HT in this tissue. This is more than 30 times higher than it was in the whole rat brain, where it was approximately 0.28 pg/g. Our values agree with those of Giarman et a/. (1959), Milin et al. (1959) and Giarman and Freedman (1960), whose values were also obtained by bioassay, but they are definitely lower than those of Bertler et al. (1963) (74-90 ng/p) and Quay and Halevy (1962) (80-128 ng/mg) in which a fluorimetric method was used. These higher figures are probably due to contamination with other amines as indicated by Quay and Halevy (1962). The results of the bioassay technique are probably more reliable for this specific indolamine. TABLE I 5-HT

CONTENT I N DENERVATED PINEAL GLANDS

Normal pineal 5-HT in ng/p*

*

15.4 4~1.4 (10) 12.2 2.1 (5) 10.3 f 0.7 (3) 16.8 & 2.3 (5)

Days of denervation

10 15 20 30

Denervated pineal E H T in nglp* % of control

8.4 f 0.9 (10) 6.7 z t 2.1 (5) 5.0 f 0.2 (3) 8.5 f 1.5 (6)

-45.7 -45.0 -51.4 -48.3

* Data are expressed in ng per pineal (mean f standard error) of 5-HT base. Number of cases in parentheses. In Table I the effect of denervation on the 5-HT content is indicated. The time after bilateral gangliectomy varied between 5 and 30 days, periods during which our previous electron microscope observations showed complete degeneration of the sympathetic fibres and endings. It can be observed that there was a decrease in content of about 50% in all of them, as compared with the controls carried out in parallel. This result, which agrees with the recent report of Bertler et a/. (1963), indicates the existence of two pools of 5-HT in the pineal of approximately similar size: one is located in the nerve fibres and endings and the other in the pinealocytes. 5-HT content of normal and denervated pineal glands after injection of the precursor 5-HTP

Normal rats and rats after 20 days of gangliectomy were injected intravenously with 25 mg/kg 5-HTP and sacrificed after 30 min. The 5-HT was assayed as indicated above in the pineal gland and in the whole brain. The results of Table I1 and Fig. 19 show that in the normal pineal the content of 5-HT increases about 400 % after that treatment, while in the brain it only increases about200 %.The most striking results were found in the denervated glands, in which 5-HT only increased 100%‘abov{the’control level. In other words the denervated glands, in this period of 30 m h , were only able

NERVE E N D I N G S I N THE P I N E A L O R G A N

413

T A B L E I1 5-HT

CONTENT AFTER 5 - H T P INJECTION I N N O R M A L P I N E A L GLAND A N D BRAIN A N D I N DENERVATED PINEAL GLANDS

Normal pineal gland* * Denervated 6 pineal gland** Brain* * * ~

Untreated

5-HTP i. v.*

% of control

10.3 f 0.7 (3) 5.0 f 0.17 (3) 0.231 f 0.007 (6)

51.9 f 10.9 (3) 10.0 f 1.47 (3) 0.731 f 0.092 (6)

+ 403.8 + 100.0 + 216.4

~~

* 25 mg/kg 5-HTP injected i. v. 30 min prior to sacrifice. ** Data are expressed as ng per pineal (mean fstandard error) of

5-HT base. Number of cases in parentheses. *** Data are expressed as pg per g wet weight (mean f standard error) of 5-HT base. Number of cases in parentheses. 5 Bilateral cervical superior gangliectomy was done 20 days before. 5-HT ngip

50

5-HT

1

-

"" 0U n t r w t e d

40 -

0.81

30 -

06-

20 -

04-

aS-HTP

07-

05-

10-

0Expt.

1

d 2

030201-

-

3

Fig. 19. Histograms showing the content of 5-HT in normal pineal (Expt. l), denervated pineals for 20 days (Expt. 2) and in brain (Expt. 3) after injection of 5-HTP (see description in the text and data from Table JID.

to synthesize 5-HT (or to bind) to about 1/5 of the extent of the normal pineal. This result indicates that the formation of 5-HT in the nerves of the pineal is probably much greater or more rapid than in the pinealocytes. 5-HT content of the rat pineal after reserpine and aramine

The effect of reserpine on the 5-HT content of both pineal and whole brain was studied using an acute and a chronic treatment (Table I11 and Figs. 20 and 21). In the first case reserpine was injected intraperitoneally at 5 mg/kg (Expt. 1) or at 2.5 mg/kg intravenously (Expt. 2) and the rats sacrificed after 24 h. In the second case 1 mg/kg was given daily intraperitoneally for I days. Aramine (metaraminol) was injected intraperitoneally in 25 mg/kg and the pineal References p . 419-421

TABLE 111 EFFECT OF R E S E R P I N E A N D A R A M I N E ON

5-HT

CONTENT OF PINEAL G L A N D A N D B R A I N

Pineal gland Treatment

Brain Treated

Control

Treated

Control nglpineal*

ng/pineal*

% of control

Wk?**

Nk**

% of control

1 Reserpine 5 mg/kg i.p. 24 h

13.0 f 1.20 (18)

12.7 f 0.92 (18)

-2.35

0.278 f 0.015 (7)

0.145 f0.013 (6)

-47.8

2 Reserpine 2.5 mglkg i.v. 24 h

12.3 i0.77 (9)

10.0 f 0.87 (9)

-18.35

0.275 f 0.013 (7)

0.103 f 0.015 (7)

-62.9

3 Reserpine 1 mg/kg i.p. daily during 7 days

13.6 f 1.00 ( 5 )

3.4 f 0.61 (3)

-74.8

0.209 f 0.021 ( 5 )

0.066 f0.010 (3)

-68.4

4 Aramine 25 mg/kg i.p. 6h

12.3 f 1.72 (4)

7.5 f 0.61 (4)

-39.9

0.260 f 0.016 (4)

0.202 i0.015 (4)

-22.65

* Data are expressed as ng per pineal (mean f- standard error) of 5-HT base. Number of cases in parentheses.

**

Data are expressed as ng per g wet weight (mean f.standard error) of 5-HT base. Number of cases in parentheses.

5 Not significant at P 0.05

NERVE ENDINGS I N THE PINEAL O R G A N

I 4 1 t Pineal

15

10-

5-

-

0-

415

Ucontrol

at reated

1

4

2

Ocontrol

Brain

0.3000.2000.100 -

mtreated

4

1

Fig. 20. Histograms showing the different effect of reserpine and aramine on the 5-HT content of the pinealgland and brain of the rat. Expt. 1 = 5 mg/kg reserpine i.p. for 24 h. Expt. 2 = 2.5 mg/kg reserpine i.v. for 24 h. Expt. 3 = 1 mg/kg reserpine daily for 7 days. Expt. 4 = 25 mg/kg aramine for 6 h. In the pineal the 5-HT content has been expressed as ng/pineal, in the brain as ,ug/g. (Data from Table 11.)

5-HT% o f normal

100 -

Opineal abrain

40

20

0 Expt.

1

4

Fig. 21. The same experiment as in Fig. 20, but expressed as percent variation of 5-HT content, considering thecontrol pineal and brain as 100%.Note the different effect of acute reserpine (Expts. 1 and Z), chronic reserpine (Expt. 3) and acute aramine (Expt. 4). References p . 419-421

416

A. P E L L E G R I N O D E I R A L D I

et al.

and brain studied after 6 h (Expt. 4). The acute treatment with reserpine had only a slight or else an insignificant effect on the 5-HT of the pineal (Expts. 1 and 2). The depletion of 5-HT is remarkable in the brain. The chronic treatment (Expt. 3) reduced the 5-HT considerably, both in the brain and the pineal. Another interesting finding is that given by aramine (Expt. 4), in which the acute effect is higher in the pineal than in the brain. Our findings with acute reserpine are at variance with those of Bertler et al. (1963), who report a 50 % decrease in the 5-HT assayed fiuorimetrically. The difference between the pineal gland and brain with acute reserpine is very interesting and points toward a more refractory pool of this amine in the pineal. However, with aramine the result is different and the pineal 5-HT is as sensitive or even more so than the brain 5-HT towards this drug. DISCUSSION

Nerve endings in the pineal gland of the rat The interpretation of nerve endings of the pineal gland presented here differs with the one previously advanced (De Robertis and Pellegrino de Iraldi, 1961a; Pellegrino de Iraldi and De Robertis, 1961). Previously most of them were interpreted as secretory processes on the pinealocytes because of a few apparent continuities with the cells. However the experiments on bilateral excision of the superior cervical ganglia have convinced us that our interpretation was faulty and that all the granular vesicles present in the plurivesicular material are located within nerve endings, which may be either free in the perivascular space or adjacent to the pinealocytes. This finding simplifies the matter considerably, since we now can attribute all the neuropharmacological changes observed in the plurivesicular material to the sympathetic nerve endings. This is then an excellent subject for studying at a submicroscopic level such complex phenomena as those involved in the transport of precursors, synthesis, binding, storage, selective release and inactivation of biogenic amines, and the pharmacological action of drugs on these different metabolic processes. Nature of the granular vesicles The fact that both indol- and catecholamines reduce osmium tetroxide makes a finer cytochemical discrimination of the granular vesicles of the pineal nerve endings difficult for the moment. In some test-tube experiments we observed that NA has much more of a reducing effect on osmium tetroxide than 5-HT, and that melatonin also reduces more strongly than 5-HT. The action of drugs may be helpful in solving this problem. It is known that in the CNS reserpine induces the liberation of NA, dopamine and 5-HT. However, in the experiments reported here 5-HT is not liberated from the pineal gland at times (24 h) in which there is depletion from the CNS. Several other findings are against the interpretation of the dense grains containing 5-HT : (a) The administration of 5-HTP slightly reduces the concentration of granular vesicles although the content in 5-HT of the pineal increases about 400%; (b) The

N E R V E E N D I N G S IN T H E P I N E A L O R G A N

417

association of reserpine with nialamide, which according to Carlsson et al. (1962) re-establishes the content in 5-HT (of the brain) with depletion of catecholamines, does not modify the total depletion of granular vesicles determined by reserpine. All these findings suggest that the dense grains do not contain 5-HT, but the possibility that they may have melatonin cannot be discarded for the moment. In favour of the interpretation that the granular vesicles contain NA are the following facts: (a) The experiments of gangliectomy show the degeneration of nerve fibres and endings and the disappearance of all granular vesicles; (b) The granular vesicles increase in concentration with treatments that are known to increase the catecholamine deposits in brain (DOPA, dopamine, iproniazid) ; (c) These vesicles diminish with drugs that are known to deplete the adrenergic stores such as reserpine, aramine and a-methylmetatyrosine; (d) This interpretation is also supported by the findings of Wolfe et al. (1962) on the fixation of H3-NA on the granular vesicles. However, this last property cannot be considered decisive by itself because different amines can be interchanged at the sites of binding (Bertler et al., 1960). Also along these lines is the fact that similar granular vesicles have been found in other nervous territories rich in catecholamines. However, the site of storage of melatonin and glomerulotrophin in the pineal gland remains open to further investigations. Different pools of 5-HT in the pineal gland The experiments of denervation demonstrate that there are two different pools of 5-HT of similar magnitude in the pineal gland: one is probably in the pinealocytes and the other in the nerve fibres and endings. These findings agree with those recently reported by Bertler et al. (1963). Both pools show marked differences toward the administration of the precursor 5-HTP. In fact, while there is an increase of 400% in the normal gland, in the denervated one the increase is only 100%. From these data it can be calculated that the synthetic capacity is 5 times greater or more rapid in nerves than in the pinealocytes. The 5-HT stores in the pineal gland show some differences with those in the brain toward the administration of reserpine and aramine. Although chronic treatment with reserpine depletes the store in both tissues, acute treatment induces slight or no decrease in the pineal while in the brain this is very marked. On the contrary, the acute administration of aramine induces more depletion of 5-HT in the pineal than in the brain (see Table 111). Mechanism of action of reserpine, iproniazid and pyrogallol on the adrenergic nerve endings

Our electron microscope observations showing the rapid depletion of the granular vesicles determined by reserpine, which can be observed as early as after 10 min, point toward an initial effect on the permeability of the vesicular wall leading to the loss of the content of reducing amines. A secondary and most lasting effect could be the blocking of the binding of the newly synthesized amine into the vesicle. References p . 419-421

418

A. PELLEGRINO D E I R A L D I

et al.

The fact that iproniazid increases the 5-HT and NA content of the brain, and that it can counteract the depletion induced by reserpine, can be correlated with the increase in concentration and size of granular vesicles observed after administration of iproniazid, and especially with the fact that this MA0 inhibitor can almost completely counteract the effect of reserpine on the granular vesicles. The mechanism of this protective action of iproniazid is difficult to explain at the present time. It would appear that the prolonged and complete inhibition of M A 0 produced by iproniazid alters the regulation of the intracellular amine level within the nerve ending. The amine liberated by reserpine cannot be inactivated and is fixed and stored again within the vesicle. However, other sites of action of iproniazid at the level of the cell and vesicular membranes cannot be discarded. The importance of 0-methylation on the inactivation of catecholamines has been postulated, and the enzyme catechol-0-methyltransferasehas been found in the CNS and in other tissues (see Axelrod, 1959). Axelrod and Laroche (1959) demonstrated that pyrogallol inhibits the methylation of adrenaline and NA, and Wylie et al. (1 960) found that pyrogallol prolongates the physiological action of these amines. Our observations showing that the intravenous administration of pyrogallol does not change the proportion of granular vesicles, nor protect the nerve ending from the action of reserpine, point toward a different effect of the COMT as compared to the MA0 at the nerve ending. While M A 0 located within the mitochondria (see Rodriguez de Lores Arnaiz and De Robertis, 1962) would act within the adrenergic ending regulating the intracellular level of amines, COMT probably has a different subcellular topography and cannot protect the amine within the ending proper. However, a possible physiological action of COMT in the brain cannot be ruled out. SUMMARY

The pineal gland of the rat was studied under the electron microscope in the normal condition, after gangliectomy, and after treatment with drugs that may change the metabolism of indole- and catecholamines. The content in 5-HT was determined in the denervated glands, and in the pineal and the brain of animals treated with drugs acting on indolamines. From this combined morphological and pharmacological study the following general conclusions can be drawn: (1) The pineal gland has an orthosympathetic innervation coming bilaterally from the superior cervical ganglia. Nerve fibres mainly follow the perivascular spaces and end free around the capillaries or in loose association with the pinealocytes. These nerve endings contain a plurivesicular material with clear and granular vesicles which are characteristic of adrenergic terminals (De Robertis and Pellegrino de Iraldi, 1961a, b) . (2) After bilateral gangliectomy degeneration of these nerve endings takes place and the plurivesicular material disappears. Only a few cellular processes, containing a variety of clear vesicles, can be observed in these denervated glands. (3) Reserpine produces an almost complete depletion of granular vesicles between

NERVE E N D I N G S I N THE P I N E A L O R G A N

419

2 and 48 h and the recovery is rather slow. Aramine has a similar acute effect. Nialamide or iproniazid after reserpine do not counteract the action of reserpine on the granular vesicles. Iproniazid increases the concentration and size of the granular vesicles, and, given prior to reserpine, it has a protective effect. Pyrogallol has no effect on the granular vesicles and does not protect the effect of reserpine on the depletion of granular vesicles. 5-HTP produces a slight decrease of granular vesicles. DOPA alone or associated with iproniazid increases the concentration of granular vesicles. Highest effects are obtained with the association of dopamine plus iproniazid. On the contrary, a-MMT decreases the concentration of dense granules. All these findings are discussed and interpreted within the concept that the granular vesicles contain the adrenergic transmitter. (4) The normal content of 5-HT varies between 10-20 ng per pineal (14 f 1.3 mean of 55 determinations). This corresponds to the high concentration of 11.1 ,ug/g which in the whole brain is only 0.28 pg/g. Denervation causes a 50% decrease in 5-HT content. With 5-HTP there is a 400 % increase in 5-HT of the pineal against 200 % in the brain. In denervated glands the synthesis or fixation of 5-HT is reduced to 115 of the normal. Reserpine in acute treatment (24 h), in opposition to the brain, has no effect on the 5-HT content of the pineal. In chronic administration there is a similar depletion in the brain and the pineal. Aramine has an acute effect that is more prominent in the pineal than in the brain. Some of these findings demonstrate the existence of two pools of 5-HT in the pineal, one located in the nerves and the other in the pinealocytes. (5) The most general conclusion is that the innervation of the pineal gland is both tryptaminergic and adrenergic, and that the granular vesicles are related to the adrenergic transmitter. ACKNOWLEDGEMENTS

Iproniazid was kindly supplied by Roche Argentina and aramine, and a-methylmetatyrosine by Merck, Sharp & Dohme, USA. This work has been supported by grants from the Consejo Nacional de Investigaciones Cientificas y TCcnicas, Buenos Aires (Argentina), and the Air Force Office of Scientific Research of the USA. REFERENCES J., (1959); Metabolism of epinephrine and other sympathomimetic amines. Physiol. Rev., AXELROD, 39,751-776. AXELROD, J., AND LAROCHE, M. J., (1959); Inhibitor of 0-methylation of epinephrine and norepinephrine in vitro and in vivo. Science, 130, 800. BERTLER, A., FALCK,B., AND OWMAN,C., (1963); Cellular localization of 5-hydroxytryptamine in the rat pineal gland. Kungl. Fysiografiska Sallskapets Lund Forhandlingar, 33, 13-16. A., AND ROSENGREN, E., (1959); Occurrence and distribution of dopamine in brain and BERTLER, other tissues. Experientia, 15, 10-11. BERTLER, A., ROSENGREN, A. M., AND ROSENGREN, E., (1960); In vivo uptake of dopamine on 5hydroxytryptamine by adrenal medullary grains. Experientia, 16, 41 8 4 1 9 . BRODIE, B. B., AND COSTA, E., (1961); Some current views on brain monoamines. Monoamines et SystPme nerveux central. J. De Ajuriaguerra, Editor. Genbve, Georg & Co. Pans, Masson et Cie. (p. 13).

420

A. P E L L E G R I N O D E I R A L D I

et al.

BRODIE, B. B., PLETSCHER, A., AND SHORE, P. A., (1956); Possible role of serotonin in brain function and in reserpine action. J. Pharmacol. exp. Ther., 116, 9. CAJAL,S., RAMONY, (1904); Textura del Sistema nervioso del Hombre y de 10s Vertebrados. 11. Madrid, Nicolas Moya (p. 758). CARLSSON, A., (1963); Functional significance of drug-induced changes in brain monoamine levels. Progress in Brain Research, Vol. 8, Biogenic Amines. H. E. and W. A. Himwich, Editors. Conf. at the International Symposiumon problems of the brain, Galesburg. Amsterdam, Elsevier (pp. 9-27). CARLSSON, A., FALCK, B., AND HILLARP,N. A., (1962); Cellular localization of brain monoamines. Acta physiol. s c a d . , 56, Suppl. 196, 1-28. CARLSSON, A., LINDQMST,M., AND MAGNUSSON, T., (1960); On the biochemistry and possible functions of dopamine and noradrenaline in brain. Ciba Found. Symp. Adrenergic Mechanisms (pp. 432-445). CARLSSON, A., ROSENGREN, E., BERTLER, A., AND NILSSON,J., (1957); Effect of reserpine on the metabolism of catecholamines.Psychotropic Drugs. S. Garattini and V. Ghetti, Editors. Amsterdam, Elsevier (pp. 363-372). COSTA,E., (1960); The role of serotonin in neurobiology. Int. Rev. Neurobiol., 2, 175-227. CROUT,J. R., GREVELING, C. R., AND UDENFRIEND, S., (1961); Norepinephrine metabolism in rat brain and heart. J. Pharmacol. exp. Ther., 132, 269-277. DE ROBERTIS, E., AND BENNETT, H. S., (1954); Fed. Proc., 13, 35. DE ROBERTS, E., AND BENNETT,H. S., (1955); Some features of the submicroscopic morphology of synapses in frog and earthworm. J. biophys. biochem. Cytol., 1, 47-58. DE ROBERTIS, E., AND PELLEGRINO DE IRALDI, A., (1961a); Plurivesicular secretory processes and nerve endings in the pineal gland. J. biophys. biochem. Cytol., 10, 361-372. DE IRALDI, A., (1961b); A plurivesicular component in adrenergic DE ROBERTIS, E., AND PELLEGRINO nerves. Anat. Rec., 139, 299. FARRELL, G., AND MCISAAC, W. M., (1961); Adrenoglomerulotropine. Arch. Biochem., 94,543-544. GARDNER, J. H., (1953); Innervation of pineal gland in hooded rat. J. comp. Neurol., 99, 319-329. GERSCHENFELD, H. M., (1962); Submicroscopicbases of synaptic organization in gastropod nervous system. Fifth Intern. Congr. Electron Microscopy, V. 5. GESSA, G. I., HIRSCH, C., KUNTZMAN, R., COSTA,E., AND BRODIE, B. B., (1962); On the mechanism of norepinephrine release by a-methyl-meta-tyrosine. Life Sci., 8, 353-360. GIARMAN, N. J., (1963); Drug-induced changes in the subcellular distribution of serotonin in rat brain with special reference to the action of reserpine. Progress in Brain Research, Vol. 8, Biogenic Amines. H. E. and W. A. Himwich, Editors. Conf. at the Intern. Symp. on problems of the brain, Galesburg. Amsterdam, Elsevier (pp. 72-80). GIARMAN, N. J., AND DAY, M., (1958); Presence of biogenic amines in the bovine pineal body. Biochem. Pharmacol., 1, 235. GIARMAN, N. J., DAY,M., AND PEPEN, G., (1959); Presence of neurohumors in bovine pineal glands. Fed. Proc., 18, 394. GIARMAN, N. J., AND FREEDMAN, D. X.,(1960); Serotonin content of the pineal glands in man and monkey. Nature (Lond.), 186, 4 8 M 8 1 . GREEN, H., AND ERICKSON, R. W., (1960); Effect of trans-2-phenylcyclopropylamineupon norepinephrine concentrations and monoamine oxydase activity of rat brain. J. Pharmacol. exp. Ther., 129, 236-242. GREEN,H., AND SAWYER, J., (1960); Intracellular distribution of norepinephrine in rat brain. I. Effect of reserpine and the monoamine oxydase inhibitors trans-2-phenylcyclopropylamineand isonicotinyl-2-isopropylhydrazine. J. Pharmacol. exp. Ther., 129, 243-249. GRILLO,M. A., AND PALAY, S. L., (1962); Granule containing vesicles in the autonomic nervous system. Fifth Intern. Congr. EIectron Microscopy, V. 1. HERRING,P. T., (1927); The pineal region of m;immalian brain; its morphology and histology in relation to function. Quart. J. exp. Physiol., 17, 125. HESS,S. M., CONNAMACHER, R. H., AND UDENFRIEND, S., (1961); Effect of a-methyl amino acids on catecholamines and serotonin. Fed. Proc., 20, 344. IKUTA,H., (1937); Uber experimentelle Studien der Zirbeldruse. I. Veranderungen des Zentralnervensystems bei Extirpation derselben. Trans. SOC.Pathol. Jap., 27, 498. KAPPERS, J. ARIENS,(1960); The development, topographical relations and innervation of the epiphysis cerebri in the albino rat. Z. ZellforJch., 52, 163-215. LERNER, A. B., CASE,J. D., TAKAHASHI, Y.,LEE,T. H., AND MORI,W. J., (1958); Isolation of melatonin, the pineal gland factor that lightens melanocytes. J. Amer. chem. SOC.,80,2587.

NERVE ENDINGS I N THE P I N E A L O R G A N

42 1

J., (1941); Experimental and clinical observations concerning the results of destruction of the Pineal gland. Summaries Doct. Diss., Northwest Univ., 9, 314. R.* STERN, p., AND HUKOVIC,s., (1959); SUr ki pr6sence de la drotonine dans la glande Pinkale. Bull. Sci. Cons. Acad. R.P.F. Yougosl., 4, 75. PELLEGRINoDEIRALDr, A.7 AND DEROBERTIS, E., (1961); Action of reserpine on the submicroscopic morphology of the pineal gland. Experientia, 17, 122-123. PELLEGRINo DE IRALDI, A.9 AND DE ROBERTIS, E., (1962); Electron microscope study of a special neurosecretory neuron in the nerve cord of the earthworm. Fifth Iniern. Congr. Electron Microscopy, 2, v. 7. PELLEGRINO DE IRALDI, A., AND DEROBERTIS, E., (1963); Action of reserpine, iproniazid and pyrogallol on nerve endings of the pineal gland. Znt. J. Neuropharmacol. In the press, PELLEGRINO DE IRALDI, A., FARMIDUGGAN, H., AND DEROBERTIS, E., (1963); Adrenergic synaptic vesicles in the anterior hypothalamus of the rat. Anat. Rec., 145, 521-531. PLETSCHER, A., SHORE, P. A., AND BRODIE, B. B., (1955); Serotonin release as a possible mechanism of reserpine action. Science, 122, 374-375. QUAY, W. B., AND HALEVY, A., 11962); Experimental modification of the rat pineal content of serotonin and related indolamines. Physiol. Zool., 35, 1-7. RICHARDSON, K. C., (1962); The fine structure of autonomic nerve endings in smooth muscle of the rat vas deferens. J. Anat., 96, 427-442. RODRIGUEZ DE LORES ARNAIZ, H., AND DE ROBERTIS,E., (1962); Cholinergic and non-cholinergic endings in the rat brain. 11. Subcellular localization of monoamine oxydase and succinate dehydrogenase. J. Neurochem., 9, 503-508. SHORE, P. A., AND BRODIE, B. B., (1957); Influence of various drugs on serotonin and norepinephrine in the brain. Psychotropic Drugs. S. Garattini and V. Ghetti, Editors. Amsterdam, Elsevier (pp. 423-427). SHORE, P. A., MEAD,J. A. R., KUNTZMAN, R. G., SPECTOR, S., AND BRODIE,B. B., (1957); On the physiologic significance of monoamine oxidase in brain. Science, 126, 1063-1064. SHORE,P. A., PLETSCHER, A., TOMICH, E. G., CARLSSON, A., KUNTZMAN, R., AND BRODIE, B. B., (1956); Role of brain serotonin in reserpine action. Ann. N. Y. Acad. Sci., 66, 609-617. SHORE,P. A., SILVER,S. L., AND BRODIE,B. B., (1955); Interaction of reserpine, serotonin and lysergic acid diethylamide in brain. Science, 122, 284-285. SPECTOR. S., KUNTZMAN, R., SHORE, P. A., AND BRODIE, B. B., (1960a); Evidence of release of brain amines by reserpine in presence of monoamine oxidase inhibitors : implication of monoamine oxidase in norepinephrine metabolism in brain. J. Pharmacol. exp. Ther., 130, 256-261. SPECTOR, S., SHORE,P. A., AND BRODIE,B. B., (1960b); Biochemical and pharmacological effects of the monoamine oxidase inhibitors iproniazid, 1-phenyl-2-hydrazinopropane(JB 516) and 1-phenyl-3-hydrazinobutane(JB 835). J. Pharmacol. exp. Ther., 128, 15-21. TAXI,J., (1961) ; Etude de I’ultrastructure des zones synaptiques dans Ies ganglions sympathiques de la grenouille. C. R . Acad. Sci., 252, 174-176. UDENFRIEND, S., AND ZALTZMAN-NIRENBERG, P., (1962); On the mechanism of the norepinephrine release by a-methyl-meta-tyrosine. J. Pharmacol. Exptl. Therap., 138, 194199. WOLFE, D. E., AXELROD, J., POTTER, L. T., AND RICHARDSON, K. C . , (1962); Localization of norepinephrine in adrenergic axons by light and electron microscopic autoradiogaphy. Fifih Intern. Congr. Electron Microscopy, 2, 1-12. WYLIE,D. W., ARCHER, S., AND ARNOLD, A., (1960); Augmentation of pharmacological properties of catecholamines by 0-methyltransferase inhibitors. J. Pharmacol. exp. Ther., 130, 239-244. ZELLER, E. A., AND BARSKY, J., (1952); In vivo inhibition of liver and brain monoamine oxidase by 1-isonicotinyl-2-isopropylhydrazide. Proc. SOC.exp. Biol. N . Y., 81, 459461. ZELLER, E. A., BARSKY, J., FONTS, J. R., KIRCHHEIMER, W. F., AND VAN ORDEN,L. S., (1952); Influence of isonicotinic and hydrazide (INH) and 1-isonicotinyl-2-isopropylhydrazide (IlH) on bacterial and mammalian enzymes. Experientia, 8, 349-350. ZIEHER, L. M., AND DE ROBERTIS, E., (1963); Subcellular localization of 5-hydroxytryptamine in rat brain. Biochem. Pharmacol., In the press.

422

DISCUSSION

DISCUSSION

THIEBLOT: Au sujet de la communication de Mme Pellegrino de Iraldi nous rappelons que nous avons montr6 il y a 10 ans que l’excitation r6pttCe du ganglion cervical supkrieur chez le chat entraine une augmentation importante de la pigmentation de la glande pinkale. DE IRALDI: I worked only with the rat, not with the cat. The epiphysis of PELLEGRINO the rat does not show pigmentation. But I thank you for this interesting remark.