J. Exp. Bid. (1974). 6i, 401-4094 plates and 1 figure

Great Britain

CALCIUM-DEPENDENCE OF NEUROSECRETIONBY EXOCYTOSIS

BY TOM CHRISTIAN NORMANN*

Institute of General Zoology, University of Copenhagen,Universitetsparken 15, DK-2100, Copenhagen 0, Denmark

(Received 18 February 1974)

SUMMARY

Exocytosis of neurosecretory granules in the corpus cardiacum of theblowfly Calliphora erythrocephala was induced both by electrical stimulationand by depolarization with K+. This secretory activity was quantitativelydetermined by the frequency of omega figures and vesicle clusters,which are the ultrastructural indications of two distinct phases of theexocytotic process. Experimentally elicited exocytosis was reduced when Ca2*were omitted from the bathing medium. This result supports the idea thatthe coupling between excitation (depolarization) and exocytosis involvesCa2"1" influx through the axolemma. Barium effectively substitutes forcalcium in the secretory process. High levels of magnesium in the bathingmedium, however, decreased exocytosis, possibly by interfering withcalcium influx.

INTRODUCTION

The corpus cardiacum neurosecretory cells (c.n.c.) of the blowfly Calliphoraerythrocephala release the contents of their granules by exocytosis (Normann, 1965).The presence in thin sections of omega-like figures is the characteristic ultrastructuralindication of this process. Omega figures are formed by fusion of granule membraneswith the axolemma, the contents thus being voided and allowed to dissolve andescape by diffusion.

Clusters of small vesicles (30-40 nm) at the axolemma can also be observed inactively secreting axonal endings. These configurations were previously interpretedas indications of a different release mechanism, viz. intracellular fragmentation ofgranules. However, substantial evidence supports the idea that in neurosecretorycells 'omega figures' and 'vesicle clusters' represent consecutive phases of exocytosisviewed as a dynamic sequence of membrane phenomena (Normann, 1969). Themicrovesiculation provides for membrane retrieval and prevents addition of excessmembrane area to the axolemma during secretion (Normann, 1970).

The same mechanism of neurosecretion operates in other animals (Bunt, 1969; U.Smith, 1970; Nagasawa, Douglas & Schulz, 1970; Santolaya, Bridges & Lederis,1972; and others), and the view that exocytosis is the general release mechanism ofneurohormones and perhaps all neurohumours is gaining acceptance (see A. D.Smith, 1971).

• Present address: Department of Zoology, University of Cambridge, Downing Street, Cambridge,England.

402 T . C. NORMANN

Calcium is known to be essential for stimulus-secretion coupling in neurosecretio^and secretion by neurones (Douglas & Poisner, 1964; Douglas, 1966; Berlind &Cooke; 1968; reviews by Douglas, 1968; Simpson, 1968; A. D. Smith, 1971). How-ever, the precise site of action of calcium remains to be identified (see Discussion).

The purpose of this study is to examine the role of calcium in experimentallyelicited neurosecretion by exocytosis. The effects of increasing the magnesiumconcentration and of substituting barium for calcium in the medium have also beentested.

METHODSElectrical stimulation

Corpora cardiaca of 4-day old blowflies were stimulated in situ as previouslydescribed (Normann, 1970). Prior to stimulation the organs were bathed for 3-4 minin one of the following fluids, which was repeatedly changed to wash away all haemo-lymph: Calliphora-Rxagtv (Normann, 1973) containing 5 mM calcium ('R'), Ringerwithout added calcium ('R-Ca'), or calcium-free Ringer containing 1 mM EGTA('R-Ca EGTA'). EGTA (Ethyleneglycol-bis-^-aminoethyl ether), N,N'-tetra-acetic acid) strongly binds calcium while negligibly affecting magnesium concentration.

The 3 groups ('SR') were subsequently stimulated for 1-2 min whilst being kept inthe appropriate experimental fluid, which (after a total of 5 min) was replaced byfixative for electron microscopy. The controls ('R') were not stimulated, but werebathed in Ringer for 5 min.

Depolarization by high potassium

Three groups of corpora cardiaca were bathed for 3 min in Ringer containing calciumas indicated in the three pairs of histograms on the right of Fig. 1. This medium wassubsequently replaced by a Ringer with similar concentration of calcium, but withpotassium concentration increased from 5 to 100 mM. Glucose was omitted from thepotassium-rich Ringer to avoid hypertonicity. After 1 min in the potassium-richRinger ('R + K'), this was replaced by fixative. One group was treated similarly,except that the magnesium concentration had been increased from 2 to 20 mM(5 mM Ca5^). Another group was treated with a calcium-free Ringer containing 1 mMEGTA, to which 5 mM barium had been added.

Secretory activity was determined by electron microscopy as described in previousstudies (Normann, 1969, 1970). The omega figures (Fig. 4, Plate 3; Fig. 5, Plate 4)and vesicle clusters (Fig. 6, Plate 4) were counted and their occurrence expressed asobserved numbers per 100 axon profiles examined. In each corpus cardiacum atleast 325 profiles of neurosecretory axon endings (400 on average) have been examined.

RESULTS

Some relevant morphological features of the corpus cardiacum are illustrated inFigs. 2 and 3 (Plates 1 and 2). (A more detailed structural analysis has been givenpreviously (Normann, 1965)). Fig. 2, Plate 1, shows a lateral portion of the corpuscardiacum located between the aorta and the oesophagus. The c.n.c. bodies aresituated at the ventral and lateral periphery of the gland, and axonal projectionsextend into the neuropile (Fig. 3, Plate 2).

Calcium-dependence of exocytosis 4°3

25 r

20

15

10

Electrical stimulation High potassium

1i

R5mM Ca

SR

5 ISM CaSR

-CaSR

-CaEGTA

R + KSmMCa

R+K20mMCa

R+K.- C a

EGTA

Fig. i. Counts of omega figures (cross-hatched columns) and of vesicle clusters (opencolumns) per too neurosecretory axon profiles (±s.E.). R: controls, in Ringer, electrodesimplanted, not stimulated (n=i6). SR: in Ringer, electrically stimulated (cf. text) (n = o).SR —Ca: in nominally calcium-free Ringer, electrically stimulated (n = 8). SR —Ca EGTA:in calcium-free Ringer, I mM EGTA added, electrically stimulated (n = 7).

R + K: depolarized, in Ringer (5 mM Ca) with 100 mM potassium (n = 8). R + K 20 mM Ca:depolarized in Ringer with 20 mM calcium and 100 mM potassium (n = 7). R + K — Ca EGTA:depolarized in Ringer without calcium, with 100 mM potassium and 1 mM EGTA (n = 7).

Haemocoel indentations ramify deeply into the neuropile, thus facilitating ex-change of substances between the latter and the blood. However, not all neuro-secretory axon terminals face haemocoel spaces directly; in fact, exocytosis is mostoften observed at the narrow (10-20 nm) intercellular cleft between apposed neuro-secretory axons or opposite a glial cell.

Since exocytosis, apparently, depends on the availability of extracellular Ca+a,these must be present in a more or less diffusible form throughout the intercellularspaces. Not knowing how readily this compartment exchanges Ca2"1" with the incu-bation solution, a short experimental period was chosen to avoid adversely affectingcellular ultrastructure and yet ensure a partial washout of Ca2*.

The corpus cardiacum neuropile contains two main types of neurosecretory axonendings. The majority, the c.n.c. axons, contain granules most of which are oblongand range in size from 180 to 300 nm. According to Knowles (1967) this can be termed'A-type' neurosecretion and is probably peptidergic. Using the traditional term'granule' instead of 'vesicle' appears to be justified in view of the apparent rigidity ofthe contents, which consist in part of rod-like, tubular subunits in a paracrystallinearray (Fig. 4, Plate 4). This substructure is masked in intact intra-axonal granulesby a substance that stains heavily with lead, and disappears quickly at exocytosis.

Axons of 'B-fibre' type, possibly aminergic (Knowles, 1967), from the brain

404 T . C. NORMANN

innervate the c.n.c. axons by synapses (Fig. 4, Plate 3). These extrinsicsynaptic axons contain small (80-120 nm) dense core vesicles and synaptic vesicles(Normann, 1970).

The present study deals with exocytosis from the intrinsic (c.n.c.) axon endings.The omega figures (Fig. 4, Plate 3; Fig. 5, Plate 4) and the vesicle clusters (Fig. 6,Plate 4) were counted and their frequency alculated as numbers per 100 profilesof axon endings.

The left part of the histogram (Fig. 1) shows the results of the quantitative electronmicroscopical analysis of the controls ('R') and of the three groups of electricallystimulated corpora cardiaca ('SR') exposed to different external calcium concentrations.Neurosecretion by exocytosis is obviously reduced at low calcium concentration.The visible cellular events following the establishment of connexion between granulemembranes and plasma membranes are, however, unchanged at low calcium con-centrations. Thus, calcium appears to be involved at the very beginning of exocytosis,either by taking part in establishing the physical connexion between membranes,or perhaps by liquefying the axoplasm and thus increasing the mobility of the granules.

A decreased external calcium concentration is known to reduce membrane stabilityand to increase spontaneous activity of excitable cells (review by Triggle, 1972). Itmay be mentioned that application of calcium-free Ringer, particularly when EGTAhad also been added, led to vigorous trembling of the somatic musculature. Never-theless, the inhibition of secretory activity could be due to a decreased excitability ofneurosecretory neurones, rather than to a dependence on calcium entry of the couplingbetween excitation and exocytosis.

To test this possibility the influence of varying the calcium concentration on theinduction of exocytosis by total depolarization with potassium has been examined(right part of Fig. 1).

The large number of omega figures produced by high potassium medium supportsthe idea that depolarization of the plasma membrane allows it to fuse with granulemembranes, provided calcium ions are present. The different relation between omegafigures and vesicle clusters observed in the depolarization experiments may be partly dueto a more effective induction of exocytosis and to a longer persistence of the 'omegaphase' relative to the duration of the microvesiculation phase (cf. Normann, 1970).

There is some indication, however, that calcium can interfere with microvesicu-lation. With high potassium and with calcium raised to 20 mM the occurrence ofvesicle clusters is significantly lower than at an external calcium concentration of5 mM. And in the 'S' experiments, in which at least some of the calcium must havebeen removed from the intercellular spaces by means of EGTA, the relation betweenvesicle clusters and omega figures appears slightly different from the other groups.

Since some vesicle clusters have been found in nearly all specimens, even after5 min at low external calcium concentration, these vesicles may be the remnants ofan exocytotic activity occurring prior to calcium deprivation.

Magnesium is known to antagonize calcium influx; raising the concentration ofmagnesium can interrupt synaptic transmission (Del Castillo & Engbaek, 1954), andneurohormone release can be antagonized by increased external magnesium con-centration (Douglas & Poisner, 1964; Berlind & Cooke, 1971; and others). A groupof corpora cardiaca was examined after incubation and subsequent potassiur™

Calcium-dependence of exocytosis 405

^Pepolarization in Ringer with 5 mM calcium and with magnesium concentration raisedfrom 2 to 20 mM. The average number of omega figures per 100 axon profiles recordedin 7 specimens was i*6 ± 0-37 (s.E.) which is significantly (P < o-ooi) lower thanin group 'R + K, 5 mM Ca' (Fig. 1).

Certain divalent cations, including barium, can substitute for calcium in neuro-hormone release (Haller et al. 1965; Dicker, 1966; Berlind &Cooke, 1971). Five corporacardiaca were treated with a calcium-free, EGTA-containing Ringer, to which 5 mMbarium had been added. The average frequency of omega figures was found to be8-i ± 1-27 (s.E.) which is significantly higher (P < o-ooi) than in group 'R+K,-Ca, EGTA'(Fig. 1).

DISCUSSION

The corpus cardiacum neurosecretory cells of the blowfly secrete a hyperglycaemicneurohormone (Normann & Duve, 1969; Vejbjerg & Normann, 1974) and its release,when induced by electrical stimulation, can be correlated with exocytotic activity(Normann, 1969, 1970). The c.n.c. are excitable (3-7 msec spikes) and they areinnervated through synapses, the presynaptic axons coming from the brain (Nor-mann, 1973).

Release of neurohormone from the c.n.c. occurs by exocytosis (Normann, 1965).In view of the neuronal characters of the neurosecretory cells and of the occurrenceof Brownian movement of granules in the axon endings, it was suggested that exo-cytosis was regulated by nervous impulses, membrane fusion occurring only duringdepolarization. This scheme was further suggested to be the general mechanismof neurohormone secretion (Normann, 1965).

The products of protein-secreting cells are normally sequestered in membrane-limited vesicles or granules until exocytosis. However, in the case of the vertebrateneurohypophysis the exocytosis theory was for long viewed with scepticism. Thiswas partly because of the failure until more recently to obtain plain ultrastructuralevidence such as omega figures in this tissue. But though substantial evidence nowsupports the extended exocytosis theory (see Introduction) (Nagasawa et al. 1970;Douglas, Nagasawa & Schulz, 1971; Santolaya et al. 1972; and others), referenceshould nevertheless be made to a variant of the still widely held theory of 'moleculardispersion' (for a survey, see Douglas et al. 1971). The 'complex dissociation' hypo-thesis (Thorn, 1970) is based on biochemical studies indicating that calcium ionsmay interfere competitively with the binding of neurohypophysial hormones to theircarrier proteins, and it also assumes that vasopressin-neurophysin complexes canbe located in two distinct compartments within axon endings.

According to the 'complex dissociation' hypothesis, the well-established significanceof calcium for stimulus-secretion coupling lies in its ability to dissociate the carrierprotein from the neurohormone that is present in an 'easily releasable pool' in thecytoplasmic matrix. The hormone would then have to travel through the axolemmalbarrier, which would presumably have been rendered temporarily permeable. Storedneurosecretory material would subsequently have to be conveyed from the granulesthrough their membranes to replenish the hypothetical pool in the cytoplasmiccompartment.

From a cytological point of view this scheme is questionable. Sequestering the neuro-26 I I B 6l

406 T. C. NORMANN

secretory substance within granule membranes, a function of the Golgi apparatuBseems advantageous for the cells for the following reasons, (i) The substance isprotected from destruction by cytoplasmic enzymes. (2) The cell maintains controlover a substance which might interfere with normal cellular function, should it escapeinto the cytoplasm. (3) The substance can be conveniently transported in its nativeform through the axon and stored in its terminal, from which (4) it can be releasedin quantal packets as demanded.

Our results show that the initiation of exocytosis is calcium-dependent, i.e. the ionexerts its function in the coupling between depolarization and membrane attachmentThis is in line with the studies of Douglas and co-workers, of Berlind & Cooke(1968, 1971) and of Uttenthal, Livett & Hope (1971). The latter found thatneurophysin and vasopressin were released in parallel by a calcium-dependentmechanism, that is compatible with exocytosis rather than with release by 'complexdissociation'.

It may be significant that the conditions leading to release are the same in neuro-secretory neurones and in, for example, cholinergic neurones, in which the followingsequence has been suggested: depolarization -> entry of calcium ->• acceleration ofquantal release (Katz, 1971). Recent ultrastructural evidence supports the view thattransmitter release occurs by exocytosis (Nickel & Potter, 1971; Heuser & Reese,1973; Pysh & Wiley, 1974).

The actual site of calcium action remains to be identified; it could be located in or atthe interior surface of the plasma membrane, on the granule membrane, or even inthe axoplasm. Recent findings indicate that calcium having entered a depolarizedaxon terminal can liquefy the axoplasm and induce Brownian movement enablingthe vesicles to reach the axolemma (Shaw & Newby, 1972; Piddington & Sattelle,1974)-

An earlier observation of frequent collisions between granules in vivid Brownianmovement and the axolemma was made on corpora cardiaca which had been dissectedout (Normann, 1965). The observed Brownian movement could therefore result fromcellular damage; nevertheless, the collisions with the axolemma did not lead to attach-ment of the granules to the plasma membrane. In this study the effect of potassiumand calcium was not tested.

The electrophoretic properties of chromaffin granules have been studied byMatthews (1971), who calculated the net number of negative changes on the surface(one for every 11-4 square nm). Assuming a similar potential of the interior plasma-lemmal surface, the granule membrane and the cell membrane would each have aboundary ionic layer about 0-75 nm thick. At a distance of 1-5 nm these boundarylayers would set up a potential barrier which must be overcome for the granule tointeract with the cell membrane. Calcium, however, is effective in decreasing thewidth of the boundary layer (as are other divalent cations). Further, reduction ofthe boundary layer of the granule membrane may not be necessary, because a transi-tory charge reversal from negative to positive of the inner surface of the cell membranemay occur at the same time as calcium influx and so remove the potential energybarrier to interaction (cf. Matthews, 1971).

Calcium ions alone cannot account for the membrane interactions leading tomembrane fusion. Some sort of recognition process is required, perhaps existing

Calcium-dependence of exocytosis 407

the form of specific proteins or 'active sites' on the interacting membranes. Theexistence of such sites was suggested by del Castillo & Katz (1957; see also Katz1969). Electron microscopical evidence for the presence of specific calcium-bindingsites on synaptic vesicles (one site per vesicle) has recently been obtained by Politoffet al (1973).

The available evidence does not permit the identification of membrane specializa-tions and/or site(s) of calcium action in the c.n.c. That an appreciable net influx ofcalcium occurs during secretion is suggested by the following unpublished obser-vation: when c.n.c. of the blowfly and of the locust are forced to secrete continuouslym vivo for several minutes, the mitochondria increase in size and number, and largematrix granules (up to 100 nm), probably indicative of calcium accumulation, develop.The mitochondria may have a buffering function, maintaining a low cytoplasmiccalcium concentration (cf. Baker, 1972). Electron microscope microprobe analysis ofcalcium binding in mitochondria and other cell components is planned and mayyield quantitative data required for further elucidation of the mechanism of secretion.

Part of this work was done at the Department of Zoology, University of Cambridge,during the tenure of a Wellcome-Carlsberg Travelling Research Fellowship.

REFERENCES

BAKER, P. F. (1972). Transport and metabolism of calcium iong in nerve. Prog. Biopkys. Mol. Biol.24, i77-"3-

BERLIND, A. & COOKB, I. M. (1968). Effect of calcium omission on neurosecretion and electrical activityof crab pericardial organs. Gen. Comp. Endocrinol. 11, 458-63.

BERLIND, A. & COOKE, I. M. (1971). The role of divalent cations in electrically elicited release of aneurohormone from crab pericardial organs. Gen. Comp. Endocrinol. 17, 60—72.

BUNT, A. H. (1969). Formation of coated and 'synaptic' vesicles within neurosecretory axon terminalsof the crustacean sinus gland. J. Ultrattruct. Res. a8, 411—21.

DEL CASTILLO, J. & ENGBAEK, L. (1954). The nature of the neuromuscular block produced by mag-nesium. J. Physio!., Lond, 1x4, 370-84.

DEL CASTILLO, J. & KATZ, B. (1957). La base 'quantale' de la transmission neuro-musculaire. In'Microphysiologie comparee des dements excitables', Coll. internat. C.N.R.S. Paris no. 67, 245-58.

DICKER, S. (1966). Release of vasopressin and oxytocin from isolated pituitary glands of adult andnew-born rats. J. Phytiol., Lond. 185, 429-44.

DOUGLAS, W. W. (1966). Calcium-dependent links in stimulus-secretion coupling in the adrenalmedulla and neurohypophysis. In Mechanisms of Release of Biogenic Amines (ed. U. S. von Euler,S. Rosell and B. Uvnfls), pp. 267-89. Oxford, New York, London, Paris. Pergamon Press.

DOUGLAS, W. W. (1968). Stimulus-secretion coupling: the concept and clues from chromamn and othercells. Br.J. Pharmac. 34, 451-74.

DOUGLAS, W. W., NAOASAWA, J. & SCHULZ, R. (1971). Electron microscopic studies on the mechanismof secretion of posterior pituitary hormones and significance of microvesicles ('synaptic vesicles'):Evidence of secretion by exocytosis and formation of microvesicles as a by-product of this process.Mem. Soc. Endocrinol. 19, 353-78.

DOUGLAS, W. W. & POISNER, A. M. (1964). Stimulus-secretion coupling in a neuro-secretory organ:the role of calcium in the release of vasopressin from the neurohypophysis. J. PhysioL, Lond. \yx,1-18.

HALLER, E., SACHS, H., SPERELAKIS, N. & SHARE, N. (1965). Release of vasopressin from isolated guineapig posterior pituitaries. Amer.J. PhysioL 309, 79-83.

HBUSER, J. E. & REESE, T. S. (1973). Evidence for recycling of synaptic vesicle membrane during trans-mitter release at the frog neuromuscular junction. J'. Cell Biol. 57, 315.

KATZ, B. (1969). The release of neural transmitter substances. Liverpool University Press.KATZ, B. (1971). Physiological evidence for quantal transmitter release. Phil. Trans. R. Soc. B 261,

381.KNOWLES, F. G. W. (1967). Neural properties of neurosecretory cells. In 'Neurosecretion', IVth

International Symposium on Neurosecretion (ed. F. Stutinaki), pp. 8-19. Berlin, Heidelberg,New York: Springer-Verlag.

26-2

408 T. C. NORMANN

MATTHEWB, E. K. (1971). The ionogenic nature of the granule surface. Mem. Soc. Endocrmol. 1 ^959-64-

NAGASAWA, J., DOUGLAS, W. W. & SCHULZ, R. (1970). Ultrastructural evidence of secretion by exo-cytosis and of 'synaptic' vesicle formation in posterior pituitary glands. Nature 337, 407-9.

NICKEL, E. & POTTER, A. L. (1971). Synaptic vesicles in freeze-etched electric tissue of Torpedo.Phil. Tram. R. Soc. B 361, 383-5.

NORMANN, T. C. (1965). The neurosecretory system of the adult Calliphora erytkrocephala. I.The fine structure of the corpus cardiacum with some observations on adjacent organs. Z. Zellforich.67, 461-501.

NORMANN, T. C. (1969). Experimentally induced exocytosis of neurosecretory granules. Expl Cell Ret.55. 385-7.

NORMANN, T. C. (1970). The mechanism of hormone release from neurosecretory axon endings in theinsect Calliphora erythrocephala. In Aspects of Neuroendocrmology (ed. W. Bargmann andB. Scharrer), pp. 30-43. Berlin, Heidelberg, New York: Springer-Verlag.

NORMANN, T. C. (1973). Membrane potential of the corpus cardiacum neurosecretory cells of the blow-fly, Calliphora erythrocephala. J. Intect Physiol. 19, 303-18.

NORMANN, T. C. & DUVE, H. (1969). Experimentally induced release of a neurohormone influencinghemolymph trehalose level in Calliphora erythrocephala. (Diptera). Gen. Comp. Endocrmol. 13,449-59-

PIDDINGTON, R. W. & SATTELLE, D. B. (1974). Motion in nerve ganglia detected by light-beatingapectroscopy. (In preparation.)

POLJTOFF, A. L., ROSE, S. & PAPPAS, G. D. (1973). Calcium binding sites in synaptic vesicles of thefrog neuromuscular junction. Biol. Bull. mar. biol. Lab., Woods Hole 145, 449.

PVSH, J. J. & WILEY, R. G. (1974). Synaptic vesicle depletion and recovery in cat sympathetic gangliaelectrically stimulated in vivo. Evidence for transmitter release by exocytosis. .7- Cell Biol. 60, 365-74.

SANTOLAYA, R. C , BRIDGES, T. E. & LEDERIS, K. (197a). Elementary granules, small vesicles andexocytosis in the rat neurohypophysis after acute haemorrhage. Z. Zellfortch. 135, 277-88.

SHAW, T. I. & NEWBY, B. J. (197a). Movement in a ganglion. Biochim. Biophyt. Ada 333, 411-12.SIMPSON, L. L. (1968). The role of calcium in neurohumoral and neurohormonal extrusion processes.

J. Pharm. Pharmac. 30, 889-910.SMITH, A. D. (1971). Summing up: some implications of the neuron as a secreting cell. Phil. Tram.

R. Soc. B 361, 423-37.SMITH, U. (1970). The origin of small vesicles in neurosecretory axons. Tissue and Cell 3, 427-33.THORN, N. A. (1970). Mechanism of release of neurohypophysial hormones. In Aspects of Neuro-

endocrmology (ed. W. Bargmann and B. Scharrer), pp. 140-52. Berlin, Heidelberg, New York:Springer-Verlag.

TRIGGLB, D. J. (1972). Effects of calcium on excitable membranes and neurotransmitter action. InProgress m Surface and Membrane Science (ed. J. F. Danielli, M. D. Rosenberg and D. A. Cadenhead),vol. v, p. 267-331.

UTTHNTHAL, L. O., LrvETT, B. G. & HOPE, D. B. (1971). Release of neurophysin together with vaso-pressin by a Ca^-dependent mechanism. Phil. Trans. R. Soc. B 361, 379-80.

VEJBJKRG, K. & NORMANN, T. C. (1974). Secretion of hyperglycaemic hormone from the corpuscardiacum of flying blowflies, Calliphora erythrocephala. J. Insect Physiol. 30, 1189-1192.

EXPLANATION OF PLATES

PLATE I

Fig. 2. Low magnification electron micrograph of a lateral portion of the corpus cardiacum, which issituated between the aorta and the oesophagus. The bodies of the corpus cardiacum neurosecretorycells, the c.n.c, are located at the ventral and lateral periphery of the ganglion-like gland. Most of thegranule-containing axon profiles in the neuropile belong to the c.n.c The axons that have few particlesand are embedded in darker glioplasm belong to branches of the recurrent nerve. Note the deeplyramifying haemocoel indentations. AW, aorta wall; OES, oesophageal wall; CNC, c.n.c. bodies;HI, haemocoel indentations; NR, recurrent nerve, x 5850.

PLATE 2

Fig. 3. C.n.c. with axonal projection extending into the neuropile. N, nucleus of c.n.c; A, axon, longi-tudinally sectioned; NP, neuropile; HI, haemocoel indentation. X7650.

Journal of Experimental Biology, Vol. 61, No. 2

AW

T. C. NORMANN (Facing p. 408)

Journal of Experimental Biology, Vol. 61, No. 2 Fig. 3, Plate 2

T. C. NORMANN

Journal of Experimental Biology, Vol. 61, No. 2 Fig. 4, Plate 3

T. C. NORMANN

Journal of Experimental Biology, Vol. 61, No. 2 Fig. 5, Plate 4

*' vc~

^

T. C. NORMANN

Calcium-dependence of exocytosis 409

PLATE 3

Fig. 4. Extrinsic microgranular axon of 'B-type' with synapse on a cn.c. axon ('A-type'). Omega figureindicating exocytosis at arrow. In this specimen, Ba** had been substituted for Ca** prior to stimu-lation, x 60000.

PLATE 4

Fig. 5. Omega figures - exocytosis of neurosecretory granules from two adjacent axon terminals.Note the paracrystalline array of rod-like subunits, visible at the very beginning of exocytosis. Thiscannot be seen in intracellular (intact) granules (cf. text), x 98300.

Fig. 6. Vesicle clusters (VC), representing the process of membrane retrieval from the axolemma atrelease sites. Most of such microvesides are 30-40 nm (external diameter), x 154000.