On the axonal migration of catecholamines in constricted sciatic nerve of the rat

Summary The radioautographic technique has been used to study the axonal migration of catecholamines in sympathetic fibres of the sciatic nerve of rats after ligature. A first series of experiments aimed at ascertaining the capacity of the proximal portion of adrenergic fibres to take up and store exogenous tritiated catecholamines (³H-DOPA; ³H-DA and ³H-NA) 3 to 22 hours after ligation. The results are qualitatively similar in rats pretreated and non-pretreated with IMAO, but the intensity of the radioautographic reaction is lower in the latter cases. Most of the labeled axons are filled mainly with vesicular and tubular profiles of endoplasmic reticular origin, large dense bodies (probably lysosomes) and/or mitochondria. The silver grains are generally superimposed on the vesicular and/or the tubular profiles, but in some cases on the large dense bodies, suggesting that exogenous catecholamines can be stored in lysosomes. The question whether SGV specifically store catecholamines also in the modified adrenergic fibres has been investigated in KMnO₄ fixed material. These results show that beside a large number of fibres in which there is a strict correlation between labeling and SGV, some fibres containing SGV do not retain the ³H-NA. Conversely some fibres which contain mainly agranular vesicles display radioautographic reaction. Therefore, in case of ligated fibres, SGV cannot be considered the specific organelles for storage of catecholamines.The axonal migration of labeled catecholamines has been studied in animals pretreated with IMAO. A moderate, but selective, labeling is present in the proximal portion of sciatic fibres of rats in which administration of labeled catecholamine preceeded of 2 hours the ligature and this was performed 22 hours before fixation.From these combined types of experiments, it is concluded that despite the presence of all the structures necessary for the storage of a high amount of catecholamines in the modified adrenergic fibres, only a small fraction of catecholamines accumulated above the ligature has been transported by axonal migration. Therefore, the axonal migration of catecholamines appears as an epiphenomenon related to the distal migration of enzymatic and storage proteins from the perikaryon.Part of this work was presented to the 84th Annual Session of American Association of Anatomists (Sotelo and Taxi, 1971).Some of the electron microscopic observations were made at the Unité 106 I.N.S.E.R.M.     

Ultrastructure of catecholamine-containing axons in the intestine of the domestic fowl

Axons in the duodenum, ileum and rectum of the domestic fowl were identified as catecholamine-containing (CA) on the basis of positive reactivity following chromaffin fixation for electron microscopy. CA-axons in association with blood vessels in all regions of the intestine and in non-vascular sites in the small intestine had a 'typical' adrenergic appearance, in that they contained many small granular vesicles (SGV) and variable numbers of large granular vesicles (LGV). In the rectum the non-vascular CA-axon profiles were atypical, in that there were many elongated LGV and few SGV, and the chromaffin reactivity was weak. The nerve profiles in the rectum were dramatically reduced following 6-hydroxydopamine and reserpine treatment and were absent in rectum cultured in the absence of extrinsic ganglia. It was concluded that the profiles, in spite of their low chromaffin reactivity, truely represent CA-axons. The possibility was raised that the atypical morphology and reduced chromaffin reactivity is due to the presence of adrenaline.     

Growth cones of cultured sympathetic neurons contain adrenergic vesicles

The growth cones of dissociated rat sympathetic neurons developing in culture were fixed with potassium permanganate to visualize vesicular stores of norepinephrine through the formation of granular precipitates. It was found that growth cones contain numerous small granular vesicles (SGV) 40-60 nm in diameter. The majority of the SGV was present in the varicosity of the growth cone but SGV also occurred in filopodia. The SGV appeared in clusters or scattered throughout the varicosity. Treatment of the cultured neurons, before fixation, with reserpine, which depletes catecholamine stores by blocking uptake into vesicles, resulted in the presence of small clear vesicles. In contrast, growth cones of nonadrenergic sensory neurons dissociated from dorsal root ganglia and fixed with permanganate lacked SGV and possessed small clear vesicles. These observations indicate that the growth cones of cultured sympathetic neurons contain norepinephrine, suggest that the norepinephrine is stored in synaptic vesicles, and raise the question whether this transmitter plays a role in early axon-target cell interactions during synapse formation.     

P0 and myelin basic protein-like immunoreactivities following ligation of the sciatic nerve in the rat

In this work we analyzed variations in the expression of MBPs and P₀ in ligated sciatic nerves of young and adult rats at 3, 7, and 14 days postligation (PL), by immunohistochemistry and SDS-PAGE of isolated myelin. A protein redistribution was seen in the distal stump of ligated nerves with the appearance of immunoreactive clusters. Using the KS400 image analyzer, immunostained area values were obtained from the different nerves dissected. In adult rats, there was an increase of the immunostained area for MBP from 3 to 7 days PL, coincident with a reorganization of the marker in clusters, followed by a marked decrease at 14 days. P₀ immunolabeling gave similar results without, however, a decrease of the immunostained area at the longer survival time tested. Young animals showed an acceleration in the process of protein redistribution and digestion within ligated nerves, which followed a similar pattern as that of adult animals. Analysis by electrophoresis showed a marked decrease in P₀ and MBP at 7 days PL in young rats and 14 days PL in adult rats. The functional significance of protein clustering within myelin in injured nerves deserves further analysis.     

Ultrastructure of free nerve endings in respiratory and squamous epithelium on the rat nasal septum

The distribution of nerve fibres in the mucosa of the nasal septum of the rat was investigated by means of transmission electron microscopy on transverse and tangential ultrathin sections. Near the basement membrane of respiratory and squamous epithelium, a rather dense network of unmyelinated nerve fibres occurs. Some fibres in the respiratory epithelium ascend between the epithelial cells to reach up to the tight junctions. These fibres appeared in transverse sections to end as hooks or boutons, sometimes with branches. These shapes resemble the free nerve endings that are considered to act as nociceptors. The small intraepithelial fibres, with diameters of about 0.5–1 m, contain both dense granules and clear vesicles comparable to synaptic vesicles. Substance P was found in dense granules in basal fibres; vasoactive intestinal peptide was absent throughout the epithelium. Acetylcholinesterase activity was observed closely associated with the basal fibres; the apical fibres showed little if any activity. Membrane specializations pointing to an efferent function as well as structures usually associated with mechanoreceptive functions were lacking in both respiratory and squamous epithelium.Part of this work was presented at the Annual Conference of the Netherlands Society for Electron Microscopy, 28–29 Nov 1985, Wageningen, The Netherlands. See Spit BJ and Hendriksen EGJ (1986) Ultramicroscopy 19:102–103     

The effect of 17-B-estradiol on the fine structure of the adrenergic axons of the rabbit myometrium

Summary The effects of 17-B-estradiol on the fine structure of the autonomic nerves of the rabbit oviduct and myometrium were studied by means of KMnO₄-fixation. The main changes due to prolonged estrogen treatment were the following: (1) the dimensions of the axons increased, (2) the amount of smooth endoplasmic reticulum in the axons increased, (3) the amounts of both large granular vesicles (LGV) and large agranular vesicles (LAV) in the axons increased and (4) electron dense grains were found within the endoplasmic tubuli of the axons. Furthermore, it seems probable that the amount of small granular vesicles was also considerably higher after two weeks of estrogen treatment.The mechanism of estrogen action on the storage of transmitter within the axon is discussed. In conclusion, the present results suggest that both the axoplasmic transport and the peripheral formation of storage sites for noradrenaline are stimulated by the increased estrogen level.This work was supported by grants from Orion, Helsinki and from Finska Läkaresällskapet.     

Microtubules in myelinated and unmyelinated axons of rat sciatic nerve

Summary Tannic acid in glutaraldehyde was used to stain microtubules in myelinated and unmyelinated axons of rat sciatic nerve. In the majority of areas the tannic acid failed to penetrate the unmyelinated axons whilst penetrating neighbouring myelinated axons, suggesting a difference in the ability of the two types of nerves to exclude tannic acid. Where tannic acid had penetrated the unmyelinated axons the 13 protofilament substructure and size of the microtubules appeared identical to those seen in the myelinated axons.     

Electron-microscopic analysis of adrenergic nerve endings in the cat caudal mesenteric ganglion

The ultrastructure of nerve endings of the cat caudal mesenteric ganglion was studied after fixation of the material with 4% lithium permanganate solution by Richardson's method in the modification of Hökfelt et al. [12]. This fixation method was shown to permit the demonstration of numerous adrenergic as well as cholinergic nerve endings. Four types of adrenergic organelles were distinguished in neurons of the ganglion: small and large granular vesicles 30–50 and 70–90 nm in diameter, respectively, a tubular reticulum with electron-dense contents, and small granular vesicles 15–20 nm in diameter. The localization of the adrenergic endings and their relations with other processes and cells of the caudal mesenteric ganglion were studied in detail. The problem of the origin and physiological role of adrenergic nerve endings in this ganglion is discussed.Institute of Physiology, Academy of Sciences of the Belorussian SSR, Minsk. Translated from Neirofiziologiya, Vol. 12, No. 1, pp. 86–92, January–February, 1980.     

The depolarising action of capsaicin on rat isolated sciatic nerve

The effect of capsaicin on the isolated sciatic nerve of rat was studied by extracellular recording of membrane polarisation. Capsaicin depolarised the sciatic nerve, but desensitization occurred rapidly upon repeated administration. Several other neuroactive substances, including substance P, were inactive. The depolarisation was reduced in nerves depleted of unmyelinated fibres by neonatal capsaicin treatment, suggesting that it occurs mainly in C-fibres. This depolarising action of capsaicin could explain the irritant and acute antinociceptive properties of capsaicin.     

Innervation of the intestine in the bivalve mollusc Chione stutchburyi

Summary The innervation of the gut of the venerid bivalve mollusc, Chione stutchburyi, has been examined by fluorescence histochemistry, electron microscopy and autoradiography. Specific green and yellow varicose fluorescent fibres indicate the presence of dopaminergic and serotonergic axons, respectively. Three different types of axons can be distinguished by the morphological characteristics of their vesicles. Type I axons contain predominantly small granular vesicles (average diameter 65 nm), Type II axons possess large granular vesicles (average diameter 100 nm) and Type III axons contain large opaque vesicles (average diameter 150 nm). The granular vesicles in both Types I and II axons react positively to dichromate, and their granularity is reduced by reserpine indicating that they are monoaminergic. Only Type I axons accumulate tritiated dopamine and are selectively damaged by 6-hydroxydopamine. It is concluded that Type I axons are dopaminergic. Type II axons are serotonergic: they alone take up tritiated 5-hydroxytryptamine, and 5,7-dihydroxytryptamine selectively causes degenerative changes in these axons. Type III axons contain an unidentified neurotransmitter substance. The large opaque vesicles of these axons do not react to dichromate and are unaffected by reserpine, 6-hydroxydopamine or 5,7-dihydroxytryptamine.     

Electron microscopy of neurosecretory nerve fibres in the neural lobe of the embryonic mouse

Summary Nerve fibres of the neurosecretory hypothalamo-hypophyseal tract were studied in embryonic C3H mouse neural lobes; at least four glands at each gestational day 15–19 were examined.Single axons and small bundles of fibres are visible at gestational days 15 and 16. By day 17 large fibre bundles penetrate between glial cells. They increase in number during the next two days.Electron-lucent and electron-dense vesicles are seen in the fibres of the 15th and 16th gestational days. In the 17–19 day-old embryos development is characterized by a successive rise in the number of the two types of vesicles. The mean diameter of the electron-lucent vesicles is approximately unchanged in all the stages examined (50 nm). The electron-dense vesicles increase in size from approximately 80–90 nm at days 15–16 to 140 nm at the 19th gestational day.By day 19 contacts between neurosecretory fibre terminals and the outer basement membrane of internal and peripheral capillaries are occasionally observed. The possibly adrenergic nature of a few terminals contacting peripheral vascular structures in 17 and 18 day-old embryos is suggested.This investigation was supported by grant No. 2180-020 from the Swedish Natural Science Research Council. The skilful technical assistance of Mrs. Ulla Wennerberg is gratefully acknowledged.     

Nervous system of the snailHelix aspersa

Summary The fine structure of neurosecretory nerves and endings associated with the sheath of the infraesophageal ganglion ofHelix aspersa is described. The sheath is a neurohemal organ whose vascularized stroma receives both monoaminergic and peptidergic endings. The latter occur along the surface of the nerves or scattered within the stroma. They include a complex population of vesicular profiles. The granular vesicles (1300–3000 Å in diameter) exhibit structural modifications that may be related to the intra-axonal release of their neurohormones. The agranular vesicles (500–2000 Å in diameter) occur in large numbers and lie mostly adjacent to the axon surface. Synaptoid specializations seem to represent active sites for the extracellular discharge of neurosecretory material. The monoaminergic endings so far studied lack synaptoid specializations and contain small granular (800–1300 Å in diameter) and agranular (700 Å in diameter) vesicles. Two kinds of non-neural cells are associated with the nerves: glial cells and melanocytes.Partly supported by Conicyt (Grant 105) and Comisión de Investigación Científica Universidad de Chile (Grant 48). The technical assistance of Mr. Arnold van Dun is gratefully acknowledged. We also thank the Department of Physics, Faculty of Physical Sciences and Mathematics, University of Chile, for the use of a Philips EM-300 electron microscope.     

Innervation of the ureterovesical junction musculature of man

The ultrastructure of the innervation of the human ureterovesical junction was studied. Three different nerve terminals were distinguished among the smooth muscle cells. 1. Nerve processes containing predominantly small granular vesicles (40--60 nm in diameter). 2. Other nerve fibres contained predominantly small round agranular vesicles (30--50 nm in diameter). 3. Processes with large granulated vesicles (80--120 nm in diameter). The first type may be adrenergic, the second cholinergic and the third may originate from the local nerve cells. The gap between the nerve fibres and muscle cells was 300 to 500 nm wide and no synaptic thickenings were observed. This suggests that the transmitter may influence several muscle cells, and the different nerve fibres may directly innervate the smooth muscle cells.     

Organization and ultrastructural identification of the catecholamine nerve terminals in the neural lobe and pars intermedia of the rat pituitary

Summary The central catecholamine innervation of the pituitary neural lobe and pars intermedia of the rat have been identified ultrastructurally and their organization has been investigated in a combined fluorescence histochemical and electron microscopical study. The dopamine analogues, 5-hydroxydopamine and 6-hydroxydopamine, were used to label the catecholamine terminals, and to enable the direct correlation between the fluorescence microscopical and the electron microscopical pictures.The fibre type that was identified as catecholamine-containing was ultrastructurally chiefly characterized by dense-cored vesicles, 500–1200 Å in diameter, intermingled with varying numbers of small empty vesicles. 5-hydroxydopamine was selectively accumulated in these fibres and caused an increased electron density of the granular vesicles as well as of some small normally agranular vesicles, and systemically administered 6-hydroxydopamine caused a selective degeneration of these fibres, most prominently within the neural lobe. The dopaminergic terminals of the neural lobe showed frequent close contacts (80–120 Å), without real membrane thickenings, to neurosecretory axons and to pituicyte processes. It is suggested that these close contacts might signify a direct dopaminergic influence on the neurosecretory axons and/or on the pituicyte processes. The identified central catecholamine fibres were also found to make common synapse-like contacts on the pars intermedia cells, whereas the innervation by neurosecretory fibres was very rare. This suggests that the direct central nervous control of the rat pars intermedia is exerted by the catecholamine neurons. A very special feature of the catecholamine fibres in the pituitary is the occurrence of peculiar, large dopamine-filled droplet-like swellings. Electron microscopically, such large axonal swellings (more than 2 in diameter) were found to contain, in addition to the characteristic vesicles and organelles, strongly osmiophilic lamellated membrane complexes resembling myelin bodies and multivesicular bodies encircling disintegrated vesicles, suggesting that these droplet fibres represent dilated stumps of spontaneously degenerating dopaminergic axons. It is suggested that the dopaminergic neural lobe fibres are undergoing continuous reorganization through degeneration—regeneration cycles, a phenomenon previously suggested for the neurosecretory axons of the neural lobe.Supported by the Deutsche Forschungsgemeinschaft.Supported by Svenska Livförsäkringsbolags Nämnd för Medicinsk Forskning, by The Medical Faculty, University of Lund and by the Ford Foundation.     

The ultrastructure of the intestinal nerve of Remak in the domestic fowl

Summary An ultrastructural study was made of the neurons, satellite cells and vesiculated axons of the intestinal nerve of the domestic fowl. Broad membrane-to-membrane contacts between adjacent nerve cell bodies were sometimes observed. The cell bodies and processes were not always separated from the extracellular space by a capsule of satellite cells. Following fixation using potassium permanganate, catecholamine (CA)-containing neurons in the intestinal nerve, unlike those in the lumbar parasympathetic ganglia, did not possess any small granular vesicles (SGV). Following exposure to noradrenaline, SGV could be demonstrated in the cell bodies of the juxta-ileal ganglia but not the juxta-rectal ganglia of the intestinal nerve. Non-CA axons were examined in tissue from birds that had been pretreated with 6-hydroxydopamine. Approximately one half of the non-CA axons formed axo-somatic contacts. Most of the non-CA axons contained varying proportions of small clear vesicles, large clear vesicles and large granular vescles. Statistical analysis showed that the non-CA axons could not be subdivided according to their vesicle content. CA-axons contained many SGV and were found in close apposition to neuronal somata and processes, and in the neuropil.     

Laminar ultrastructure of the dorsal cochlear nucleus in rats

The laminar ultrastructure of the dorsal cochlear nucleus was studied in ultrathin wide frontal sections, passing through all layers of the nucleus, placed on blinds with a Formvar film. The ultrastructural characteristics of cells corresponding to the cell types distinguished previously by light microscopy are described. The laminar distribution of the axon terminals was studied. In the surface and middle layers of the neuropil, by contrast with the deep layer, large branching terminals measuring 6–8 µ with spherical synaptic vesicles 40–50 nm in diameter, small terminals measuring 1–3 µ with spherical synaptic vesicles 45–60 nm in diameter, and thin unmyelinated fibers running perpendicularly to the plane of the section were predominant. On transition from the middle to the deep layer there was a corresponding increase in the number of myelinated axons and large oval-shaped terminals measuring 4–6 µ, with central mitochondria and neurofilaments, and also with spherical synaptic vesicles 50–60 nm in diameter, in the neuropil. In the surface and middle layers granular cells also were more numerous than in the deep layer. The functional significance of terminals of each type is discussed.N. A. Semashko Moscow Medical Stomatologic Institute. Translated from Neirofiziologiya, Vol. 10, No. 4, pp. 368–374, July–August, 1978.     

The interaction of homologous series of alkanols with sodium channels in nerve membrane vesicles

Summary The potency of members of the homologous series of alkanols to block²²Na uptake through sodium channels stimulated by veratridine was studied in membrane vesicles obtained from lobster walking leg nerves. A cut-off was revealed at the level of 1-undecanol. However, secondary isomers of inactive primary homologues, such as 5-dodecanol and 5-tridecanol, were able to block ion flux. From the concentration required for an equipotent effect, it was calculated that the standard free energy for adsorption of primary alkanols is –725 cal/mol CH₂. Furthermore, since the concentration required for an equipotent effect for primary isomer was found to be lower than that obtained for secondary isomers, it is concluded that the latter are less potent than the former. The similarity between this set of results and those obtained in intact frog sciatic nerve (J. Requena et al.,J. Membrane Biol. 84:229–238, 1985) offers further support to the notion that the procedure employed to isolate the membrane vesicles does preserve the Na channels. However, the mechanism of alcohol inhibition of the Na channel in isolated membrane vesicles would seem to be somewhat different from that preferred in axons. While in vesicles the block needs to be thought in terms of a reduction in the number of conducting Na channel, in axons this is considered to be the less likely mode of action, mainly because under veratridine it is not possible to invoke a shift in the steady-state activation or inactivation.     

Studies of the guinea-pig epididymis

Summary The innervation of the ductuli efferentes and seven zones of the guinea-pig epididymis was investigated using immunohistochemical, histochemical and electron-micro-scopical techniques. Nerve fibers were localized by use of antibodies against substance P (SP-IR), vasoactive intestinal polypeptide (VIP-IR) and dopamine-beta-hydroxylase (DBH-IR). In the ductuli efferentes and all zones of the epididymal duct, SP-IR is consistently observed in the interstitial tissue and perivascular areas. Histochemistry reveals a significant amount of acetylcholinesterase-containing fibers in the interstitial, perivascular and periductal smooth muscles of the ductuli efferentes and zones V, VI and VII. In contrast to the homogeneous distribution of SP-IR within all zones of the epididymis, VIP-IR is seen only in zones VI and VII. Within these zones, VIP-IR is detected in large amounts in the subepithelial and muscular layers as is a sparse number of SP-IR varicosities. DBH-IR is also seen throughout all zones in the interstitial and perivascular regions with a tendency to increase in zones VI and VII. Transmission electron microscopy (TEM) reveals evidence of a cholinergic (agranular vesicles, AGV), adrenergic (small granular vesicles, SGV) and peptidergic (large granular vesicles, LGV) innervation throughout the interstitial connective tissue of the ductuli efferentes and all epididymal zones. Furthermore AGV are localized in the subepithelial layer, and also co-stored with LGV in the muscular layer of zones VI and VII. No nerve profiles were encountered within the epithelium.Correlation of immunohistochemical findings to TEM counterparts as well as their possible functional role are discussed.Supported by the Deutsche Forschungsgemeinschaft     

Fine structure of the small,granule-containing cells in the superior cervical ganglia of hydrocortisone-treated early postnatal and adult rats

Summary Hydrocortisone injections into rats on postnatal days 3–9 caused an increase in the number of small granulecontaining cells in the superior cervical ganglia. These cells, corresponding to the small, intensely fluorescent cells, showed an extensive rough endoplasmic reticulum, a large Golgi apparatus and a very large number of granular vesicles. In addition to the granular vesicles, 70–160 nm in diameter, in which the dense core filled most of the vesicle, most cells of the hydrocortisone-injected rats contained also larger granular vesicles, up to 350 nm in diameter, in which the dense core was eccentrically located. A minority of the cells contained only granular vesicles 70–100 nm in diameter, which was the only type seen in the saline-treated control rats.Thirty days after discontinuation of the hydrocortisone treatment, most of the cells with large granular vesicles had disappeared, and only two profiles of such cells were seen on day 40. The other small cells contained only granular vesicles 70–160 nm in diameter, and these cells could not be distinguished from the small granule-containing cells of 40-day-old control rats treated early postnatally with saline.Hydrocortisone treatment, first on days 3–9 and subsequently on days 40–46, caused reappearance of the small granule-containing cells with large granular vesicles up to 350 nm in diameter, the dense core of which was eccentrically located. Hydrocortisone treatment on days 40–46 only was not followed by appearance of such cells in rats treated with saline on days 3–9.Abbreviations used in the Text PNMT phenylethanolamine-N-methyltransferase - SIF cell small intensely fluorescent cell - SGC cell small granule-containing cell The author is grateful to Professor Olavi Eränkö and Dr. Seppo Soinila for constructive criticism. Expert technical assistance by Miss Hanna-Liisa Alanen, Mrs. Marja-Leena Piironen and Mrs. Anne Reijula is gratefully acknowledged. This study has been supported by a grant from the Finnish Medical Foundation.     

Effect of hydrocortisone on the ultrastructure of the small,intensely fluorescent,granule-containing cells in cultures of sympathetic ganglia of newborn rats

Summary Sympathetic chain ganglia of newborn rats were cultured in Rose chambers with or without hydrocortisone. After one week, the cultures were examined by light microscopy for formaldehyde-induced catecholamine fluorescence and by electron microscopy after fixation in 5% glutaraldehyde solution and thereafter in 1% osmium tetroxide. Hydrocortisone (10 mg/l) caused a great increase in the number of the small, intensely fluorescent (SIF) cells in the ganglion explants, and the fluorescence intensity of these cells was also increased. The SIF cells corresponded to small, granule-containing (SGC) cells in the electronmicros copic preparations, and in addition to an increase in their number there was also an increase in the size and number of granular vesicles in the presence of hydrocortisone. In control cultures the granular vesicles were round (about 100 nm in diameter) or elongated (40–150 nm in cross section and 150–250 nm in length); both types of vesicles contained electron dense cores. In hydrocortisone-containing cultures round granular vesicles up to 200 nm in diameter were also observed; the cores of these vesicles were of variable electron density. It is concluded that in tissue culture, hydrocortisone causes an increased formation of catecholamine-containing granular vesicles in SIF-SGC cells and their precursors and an increase in the number of these cells.This work was supported by grants from the National Heart Foundation, the Australian Research Grants Committee and the Sigrid Juselius Foundation.University of Melbourne Senior Research Fellow, September, 1971 – August, 1972; present address: Department of Anatomy, University of Helsinki, Siltavuorenpenger, Helsinki, Finland, 00170.Holder of a grant from the National Health and Medical Research Council of Australia.Sunshine Foundation and Rowden White Research Fellow in the University of Melbourne, September, 1971 – August, 1972; present address: Department of Anatomy, University of Helsinki, Siltavuorenpenger, Helsinki, Finland, 00170.