Neurosecretory granule release and endocytosis during prolonged stimulation of the rat neurohypophysis in vitro

Nruroscience Vol. 5, pp. 651 to 659 Pergamon Press Ltd 1980. Printed in Great Britam &I IBRO

NEUROSECRETORY GRANULE RELEASE AND ENDOCYTOSIS DURING PROLONGED STIMULATION THE RAT NEUROHYPOPHYSIS IN VITRO

OF

HUGUE~~E LESCUREand J. J. NORDMANN Laboratoire de Neurophysiologie, Universite de Bordeaux II, and Unitt de Recherche de Neurobiologie des Comportements, U. 176, Domaine de Carreire, rue Camille Saint-SaEns, 33077-Bordeaux-Cedex, France Abstract-The nature of the readily releasable pool of neurohypophysial hormone and the recapture of membrane which occurs after hormone release have been investigated using a radioimmunoassay and stereological analysis of electron micrographs. Prolonged stimulation of the rat neurohypophysis in vitro with high potassium ion concentration (high-K*) gives rise to hormone release which declines with time. A second increase in hormone release is observed when veratridine (an agent which increases intracellular Ca2+ concentration independent of K+-produced depolarization) is added to the high-K+ containing medium at the time when the decline in hormone release has occurred. There is a depletion of neurosecretory granules from the nerve endings associated with both phases of hormone release and the time course of granule release and hormone release are similar. There is no quantitative change in the microvesicle population either during the high-K+- or the veratridine-stimulated release, There is, however, a large increase in the volumetric density occupied by endocytotic vacuoles associated with both phases of release, and the time course of the appearance of vacuoles closely parallels that of the decrease in the granules. These findings indicate that the hormone in both the ‘readily releasable’ pool mobilized by high-K+ and that in the pool released by veratridine is contained within granules and that therefore the decline in hormone release during prolonged stimulation is unlikely to be due to the exhaustion of a readily releasable pool of granules defined by their position within the nerve endings. They also indicate that, in the neurohypophysis, vacuoles are the major route for membrane retrieval after hormone release and that microvesicles are unlikely to arise from the division of vacuoles.

THERE is now considerable evidence that release of neurohormones occurs by exocytosis (see reviews by DOUGLAS, 1973; DREIFUSS, 1975; MORRIS, NORDMANN & DYBALL, 1978; NORDMANN, 1979; NORMANN, 1976). This mechanism involves the fusion of the neurosecretory granule with the plasmalemma and the formation of an aperture through which the granule content of hormone escapes into the extracellular space. Some authors, however still insist on the possibility that hormone release occurs by other means, from a soluble pool of hormone (PICARD, BOUDIER & TASSO, 1979). It has been known for some time that prolonged stimulation of hormone release results in first a rise, and then a decline in the amount of hormone released (SACHS, SHARE, OSINCHAK & CARPI, 1967; SACHS & HALLER, 1968; THORN, 1966), and the hormone released during the initial rise is known as the ‘readily releasable pool’. There have been a number of suggestions concerning the origin of the readily releasable pool, one of which is that it comprises granules in a certain position in relation to the plasmalemma of the nerve endings (SACHS et al., 1967). It has also been known for some time that a rise in free calcium ions within the nerve endings is the key event without which hormone release does not occur. The decline in hormone release that characterizes exhaustion of the ‘readily releasable pool’ has been shown to be associated with a reduction of calcium entry into

the tissue (NORDMANN, 1976). Veratridine, which is known to depolarize excitable cells (STRAUB, 1956; ULBRICHT, 1969) by holding the sodium channels in an open state (OHTA, NARAHASHI & KEELER, 1973) gives rise to a large amount of neurohormone release even when neurohypophyses have been depolarized by potassium ions for long periods of time (DYBALL & NORDMANN, 1977; NORDMANN& DYBALL, 1978). The fusion of the granule membrane with the cell membrane in exocytosis leads to an increase in the total surface of the nerve ending. However, there is evidence for membrane retrieval which regulates the size of the nerve terminal (NAGASAWA, DOUGLAS & SCHULZ, 1970; MORRIS & NORDMANN, 1978; NORDMANN, DREIFUSS, BAKER, RAVAZZOLA, MALAISSELAGAE & ORCI, 1974; NORDMANN & MORRIS, 1976; THEO~SIS, DREIFUSS, HARRIS & ORCI, 1976). Membrane retrieval after release has been demonstrated in many types of neurone (HOLTZMAN, 1977). In some nerve terminals retrieval seems to occur immediately after the release of neurotransmitter (HEUSER& REESE, 1973) or neurohormone (NORDMANN & MORRIS, 1976). In the superior cervical ganglion, however, endocytosis occurs during the hour which follows secretion of neurotransmitter (PYSH & WILEY, 1974). While it has been suggested that, in neurosecretory endings, membrane reuptake occurs through the formation of microvesicles (BUNT, 1969; NORMANN,

651

652

HLIGUETT~ LESTUREand .I. J.

NORDMANN

TABI.I.I. COMPOSITION UFTHF.MEDIAustn FORTHI;INCUBA- In one group (S3) the glands

were incubated fol- the liml I5 min in fresh high-K ’ medium containing vcratridine (0. I5 mM). Sodium was replaced bq choline in media B and C (Tables I and 2) because sodium-free solution potcnLiates the potassium-induced release of lleurt)hqpoph)si;~I hormones (DOUGLAS & POISKLH. 1964). The composition of the media are given in Table I and the experimental protocols are summarized in Table 7. At the end of the incubation period the neural lobe5 were divided into four blocks, fixed for 120min in a mixture of 2.5”:; glutaraldehyde and I”,, formaldehyde tn 0. I M sodium phosphate buffer. pH 7.2; washed in the phosphate buffer for 30min: postfixed in I”,, osmium tetroxldc for 30 min; washed m distilled water for 2 min, and ctained LT,! bloc in 70”” ethanol containing l.S”,, uranyl acetate for 30min in the dark. Blocks were then dehydrated in ;t series of ethanol concentrations and propylene oxldc and embedded in Epon resin. Thin sections were stained with uranyl acetate and lead citrate and examined using a Philips EM 300 electron microscope. Electron micrographs were printed to a final magnification of 43.000. The nerve endings were selected from their palisade on blood vessels by a systematic random procedure (WEIBI:L, 1969) according to their position on the grid square. The populations of ncurosecrctorq grsnules, microvesicles. vacuoles and mitochondria (Fig. I j within the ncurosecretorq axon endings were assessed h) stereological techniques (W~IREL. 1969) as described in the preceding paper (MOKKIS& NOWI~MANK. 1980). Most data are expressed as mean i S.E.M.However, 111 some cases the change in overall volumetric density (Vv) of total points intercepting organelle was an organelle total points ~~~--~ intercepting nerve --ending, i-------- -~~~~~-~~~~I

TION OF THE NEURAL LOBES

Concentration Incubation medium NaCl KC1 CaC&

MgC& Glucose Choline-Cl Hepes Veratridine _

A

of solute (mM)

C

H

150.0 5.6 2.2 I .o IO.0

5.6 2.2 1.0 10.0 150.0

56.0

2.2 I.0 10.0 loo.0

10.0

10.0

IO.0

D

E

lOO.0

loo.0

56.0

2.2 1.0 10.0 ~-

IO.0

56.0 2.2 I.0 10.0 -~10.0 0.15

1969; NAGASAWAut ul., 1971) recent experiments on the neurohypophysis suggest that it occurs by the formation of large electron-lucent vacuoles after the release of neurohormone (MORRIS & NORDMANN. 1980; NORDMANNet al., 1974; NORDMANN& MORRIS, 1976; THEODOSISet al., 1976). In the present paper we have studied whether the decline of hormone release observed during a prolonged stimulus is due to the exhaustion of a readily releasable pool composed of granules having a particular position within the nerve endings, by seeing whether hormone release, the loss of neurosecretory granules and vacuole formation can all be reactivated by the exposure to veratridine of tissue in which the readily releasable pool had been depleted by prolonged stimulation with high potassium ion concentration.

EXPERIMENTAL

used, because this figure takes into account the differences in the size of profiles of the different nerve terminals (see MORRIS & NOKDMANN, 1980). In parallel experiments vasopressin was measured using a radioimmunoassay (LFGKOS, STEWART. NOHDMANN,

PROCEDURES

Wistar albino rats of 25@3OOg body weight were decapitated, their neurohypophyses isolated, and stabilized by incubation for 30 min in saline which, like all the incubation media, was continuously gassed with 5% CO, in 0, (solution A. Table 1). Thereafter the medium was changed every 15min for both experimental and control groups. Hormone release was triggered from stimulated glands (Sl. S2. S3; Table 2) by an increase in the external potassium ion concentration for periods of 15 min, controls being incubated in media of normal potassium ion concentration.

DREIFUSS & FRANCHIMONT. 1971). Differences mean data were assessed by the Student’s

between

All chemicals were of AnalaR grade. Veratridine was obtained from Serva (Heidelberg, Germany), Hepes (4-(2-hydroxyethyl)-I-piperazine-ethanesulphonic acid) from Sigma (St Louis, Missouri. U.S.A.), choline chloride from Fluka (Buchs. Switzerland). other chemicals were obtained from Merck (Darmstadt. Germany).

TABLE 2. DETAILS OF THL EXPERIMENTALPROWCOLS USIXIFOR THE INCYHAIIOU ok THE NEUKAL L.OBES. Time (min)

Group Cl SI s2 s3

Number of glands per group 3 6 4 4

Prestimulation -45-(15) - 15-O A A A A

a-15

B

_

B B B

C C C

A. B, C. D and E are the media, the composition

FIG. 1. Electron

the

t-test.

micrograph of rat neurohypophyses vesicles (mv), vacuoles (V). (a) control gland; (b) nerve containing solution (S3; see Experimental Procedures). unstimulated glands.

Stimulation 15-30 30-45 45-60 ~~ .-.

.~

C C

C C

_

6@75

~~

~~

D D

E

of which is given in Table

I

showing neurosecretory granules (NSG), microterminal after exposure to high-K+ veratridine Note that a vacuole can occasionally be seen in Mag. x 54,600.

FIG. 1.

653

Neurohypophysial TABLE

Time of incubation

3. MORPHOMETRIC ANALYSIS OF PITUITARY

Area bm2) 3.41 +0.21 4.14 kO.35

45 min 105 min

655

granule release and endocytosis NERVE ENDINGS

Control Mitochondria NSG (Vv) (Vv) 0.169 kO.013 0.168 kO.014

0.071 (118); + 0.007 0.056 (87)* f 0.007

Stimulated Area

Mitochondria

(pm’)

(Vv)

3.61 *0.17 3.72 kO.37

0.070(121)* * 0.004 0.068 (60)* kO.007

* Number of endings studied. Surface area of nerve endings and volumetric density (Vv) occupied by neurosecretory granules (NSG) and mitochondria at different time of incubation. Results are given as mean f S.E.M.Four glands (four blocks/gland) were analysed for each experimental condition. The glands used for control studies were incubated according to protocol Cl ; stimulated glands were incubated according to protocol S3 (Table 2).

RESULTS

the volume that they occupy within the nerve endings

Prolonged incubation of the neural lobe for up to 105 min does not significantly alter the area of nerve ending profiles. In control experiments the volumetric density occupied by the granule population of the nerve endings of unstimulated glands was also unchanged by this procedure. In addition, there was no change in the volumetric density occupied by mitochondria in the nerve endings of either control or stimulated glands (Table 3). The lack of change of the size of nerve ending profiles means that the organelle populations can be assessed by direct comparison of

56mM

of glands incubated

for different periods of time.

Hormone release during prolonged stimulation Incubation of the neural lobe in a medium containing 56 mM Kf gives rise to an increase of vasopressin release which declines during prolonged depolarization. The addition of veratridine, which promotes calcium uptake in the neural lobe (NORDMANN & DYBALL, 1978), gives a further increase in release (Fig. 2; DYBALL& NORDMANN,1977). The mean vasocontent of a gland being equal to pressin

KCL kratrldd 0

.

30 Time (min)

FIG. 2. Effects of prolonged depolarization on hormone release, depletion of neurosecretory granules and vacuole formation. The evoked hormone release (0; 3 $ n < 6; S.E.M.< 17% of the mean) and vacuole formation (m) were calculated by substracting from the value observed during depolarization that of control experiments. The changes in the neurosecretory granule (0) and vacuole population are expressed as o/o of the volume of the endings occupied by these organelles. Potassium was increased to 56m~ at time zero. The heavy bar indicates the period during which the glands were incubated in high-K+, veratridine-containing solution (Protocol S3). The stereological data are given as the overall volumetric density (see Experimental Procedures).

HMXETTE LESCURE and J. J. NOKDMANP\!

6.56

a

b 56mM

KCI

1

I 0

I 30

I 90

I 90

Ttme (min)

$ >

10

I

0

-

I

30

I

60

I

90

Time tmtn)

FIG. 3. Effects of prolonged depolarization on the volumetric density of (a) neurosecretory granules and (b) microvesicles found in endings abutting the basal membrane. The results are expressed as y0 of the volume nerve of the endings (mean k s.E.M.; 36 i )I ,< 124). Potassium was increased to 56 mM at time zero. Veratridine was present during the period indicated by the heavy bar (Protocol S3). The vertical

bars represent the standard error of the mean. 42X k 1 1.6 mU (S.E.M.: II = 5) it can be calculated that the neural lobe has released 122Y;, of its content during prolonged stimulation. Depletion of neurosecretory stimulation

granules during prolonged

Figure 2 shows the effect of prolonged depolarization on the overall volumetric density of the neurosecretory granules found in nerve endings. At the onset of stimulation there is a sharp decrease in the granule population but the rate of disappearance of neurosecretory granules diminishes during sustained depolarization. After 60 min incubation in a high-K* solution, when the rate of hormone release has fallen to a low level, veratridine provokes a further loss of granules and release of hormone. Figure 3a shows the decrease in volumetric density of neurosecretory granules found in nerve ending profiles abutting the basal membrane. Its time course closely fits that obtained using the overall volumetric density change measurement (see Experimental Procedures) suggesting that there is a considerable homogeneity of response in the granule populations of the nerve endings. It is noteworthy that the time course of the decrease in the granule population is very similar to the ‘mirror image’ of the time course for hormone release (Fig. 2). The microvesicle population prolonged stimulation

qf‘ nerve endings during

Despite the large depletion in the population of neurosecretory granules (Fig. 3a) there is no significant change in the overall volumetric density occupied by microvesicles at the onset of stimulation of hormone release. Furthermore, the microvesicle population remains constant throughout the 75 min period of stimulation of the neural lobe (Fig. 3b).

The appearance of vacuoles during prolonged lation, and its time course

stimu-

At the onset of stimulation there was a large and significant increase in the volumetric density of the vacuole population. The rate of appearance of vacuoles declined during the prolonged stimulation. but was further increased by the addition of veratridine. The time course closely fits that observed for hormone release (Fig. 2).

DISCUSSION Hormone release and granule depletion If exocytosis results in the release of the contents of neurosecretory granules, hormone secretion must result in a net loss of neurosecretory granules from the neurohypophysis during in vitro experiments because in the isolated neural lobe no new granules can be transported from the cell bodies in the hypothalamus. As Fig. 2 demonstrates there is, associated with both potassium-stimulated and veratridinestimulated hormone release, a depletion of neurosecretory granules from the nerve endings. The time course of this granule depletion closely parallels the time course for hormone release suggesting that neurosecretory granules are the main source for the hormone released both by potassium and veratridine. The loss of neurosecretory granules during K ‘-induced depolarization confirms earlier work where a relatively short period of stimulation was used (NORDMANN & MORRIS, 1976; MORRIS & NORDMANN, 1980). In that experiment, the loss of neurosecretory granules was shown to occur from nerve endings, but not from the nerve swellings or the undilated axons. The accelerated decrease in the population of ncuro-

Neurohypophysial granule release and endocytosis

651

1969) fail to produce measurable increases in the microvesicle population. I/acuoles. Vacuoles in neurohypophysial nerve endings were first mentioned in studies on the rat (NORDMANN et al., 1974) and mouse (CASTEL,1974). The Size of the vacuole population in the nerve endings depends largely on the release of neurohormone. Both the release of hormone and the uptake of extracellular marker were abolished when neurohypophyses were depolarized in a solution containing Ca2+ uptake blocking agents (BAKER,MEVES & RIDGWAY, 1973; DREIFUSS,GRAU & NORDMANN,1973) in order to uncouple depolarization from release (NORDMANN et al., 1974). Using stereology as a quantifying method (WEIBEL, 1969) a large increase in the vacuole population associated with hormone release can be demonstrated after exposure of neurohypophyses for 15 min to high-K+ stimulation (NORDMANN& MORRIS, 1976; MORRIS& NORDMANN, 1980), electrical stimulation of the pituitary stalk or haemorrhage (THEODOSIS et al., 1976). The present paper extends these findings. It shows that, on prolonged stimulation for from 15 to 105 min there is a continuous rise in the volumetric density occupied by vacuoles (Fig. 2). The time course of their appearance is very similar to that of hormone release indicating that, in this experiment, endocytosis is tightly coupled to exocytosis. A similar tight coupling occurs during electrical stimulation- and haemorrhage-induced release (THEODOSIS et al., 1976). Retrieval of membrane in the neurohypophysis is thus similar to that in the neuromuscular junction (HEUSER & REESE,1973; HEUSER,1976) in terms of coupling between endo- and exocytosis, but differs from that seen in the superior cervical ganglion (PYSH & WILEY, 1974). The physiological relevance of this distinction may not be as marked as it would at first appear. The coupling between exo- and endocytosis may depend on parameters such as the frequency of stimulation, the temperature and composition of the incubation media (CECCARELLI,HURLBUT & MAURO, 1973; HOLTZMAN,1977; HOLTZMANN,SCHACHER,EVANS& TEICHBERG,1977; SILVERSTEIN, STEINMAN& COHN, 1977). In the neural lobe, at least, haemorrhage, electrical stimulation, potassium stimulation and veraMembrane reuptake into the nerve endings tridine stimulation all appear to produce membrane Microvesicles. Neurosecretory nerve endings are retrieval tightly coupled to exocytosis. characterized by their prominent content of microVacuoles could arise either (a) by coalescence of vesicles (MORRIS,1976b) of approximately 50nm dia- microvesicles or (b) by direct uptake from the plasma meter (PALAY, 1957; THEODOSIS,DREIFUSS& ORCI, membrane. Although the two possibilities are difficult 1977). Their role has been the subject of debate which to distinguish experimentally the first seems to us unhas been reviewed recently (MORRISet al,, 1978) and is likely for the following reasons. Glands which have considered in detail in the previous paper (MORRIS& not been subject to stimulation contain large numbers NORDMANN, 1980). We will merely note here that pro- of microvesicles. If microvesicles were continuously longed stimulation of the gland associated with two converted to vacuoles these organelles would already phases of hormone release and granule loss causes no have coalesced to form vacuoles. Vacuoles are, howoverall increase in the microvesicle population. A ever, very scarce in unstimulated tissue. Furthermore, whole range of hormone releasing stimuli applied in reduction of hormone release from the neurohypovitro and in vivo from periods ranging from 5 min physes of Brattleboro rats, which would presumably (THEODOSIS et al., 1976) through 105 min (the present reduce the number of extindocytotic events causes study) to 3 days (REINHARDT,HENNING & ROHR, no reduction of the microvesicle population (MORRIS

secretory granules in the presence of veratridine points to two conclusions. Firstly, since both phases of hormone release are associated with loss of neurosecretory granules it seems even more unlikely that a non-granular pool of hormone is available for release as some authors suggest (PICARD et al., 1979). Secondly, it strengthens the hypothesis (NORDMANN, 1976) that the decrease in hormone release observed during prolonged stimulation (SACHS et al., 1967; SACHS& HALLER,1968; THORN, 1966) is not due to the exhaustion of a readily releasable pool composed of granules in some particular location within the nerve ending (Sachs et al., 1967; THORN, 1970). Rather, the stimulation by veratridine of both the rate of depletion of neurosecretory granules and of hormone release suggests that the size of the ‘readily releasable pool’ depends primarily on the type of stimulus used. Both stimuli used in this experiment produced loss of neurosecretory granules from the endings and so all the neurosecretory granules in the endings might be releasable by an appropriate stimulus. This would not reflect physiological release accurately, however, since in uioo the granule population of the nerve endings would be repleted both from the perikaryon (via the axon) and from the nerve swellings where many granules are stored. Twenty-eight per cent of neurosecretory granules in the neurohypophysis are found in the nerve endings (NORDMANN,1977) and granule depletion on acute stimulation occurs only from the nerve endings (NORDMANN& MORRIS, 1976). Making the assumption that the releasable vasopressin and oxytocin are located exclusively in neurosecretory granules, it can be calculated that the nerve endings have released 44% of their hormone content after the prolonged stimulation used in this experiment. The stereological data (Fig. 3) shows a 46% depletion of the population of neurosecretory granules in the nerve endings. There is thus a very close relationship between the observed decrease in the population of neurosecretory granules measured stereologically and the expected loss of granules derived from the measured hormone release.

65X

Hucu~r~b

LESCL’REand

& NORDMANN, 1978). Furthermore, the present experiments demonstrate that, although the rates of depletion of neurosecretory granules and of vacuole formation decrease on prolonged stimulation (Fig. 2) there is no increase or decrease in the population of microvesicles at this time (Fig. 3b). This suggests, once again, that vacuoles do not arise from microvesicles, and also that microvesicles do not arise as the result of the division of vacuoles. It therefore seems to us that the most plausible explanation for the continuous parallelism between hormone release, granule depletion and vacuole formation is the direct uptake of vacuoles from the plasma membrane. Although it would be almost impossible to demonstrate vacuole

J. J.

NORDMANN

formation in the neurohypophysis after such short periods of stimulation (because of the small amount of hormone released and thus membrane recaptured), it is noteworthy that, in the neuromuscular ,junction. vacuoles can be observed only I s after the onset 01 stimulation (HEUSER, 1976). It is therefore most likely that vacuoles in the neural lobe represent membrane retrieved directly from the plasmalemma. Ackuowkdgements-This work was supported by grants D.G.R.S.T. 78.7.3072 and C.N.R.S. ERA 493. We thank Professor A. BARETSfor the use of the Philips EM 300 electron microscope. We are indebted to Dr J. F. MORRIS for many helpful suggestions concerning the manuscript.

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CECCARELLI B., HURLBUTW. P. & MAUROA. (1973) Turnover of transmitter and synaptic vesicles at the frog neuromuscular junction. J. Cell Biol. 57, 499-524. DOUGLASW. W. (1973) HOW do neurones secrete peptides? Exocytosis and its consequences, including synaptic vesicle formations in the hypothalamo-neurohypophyseal system. Progr. Brain Res. 39, 2lL38. DOUGLASW. W. & POISNERA. M. (1964) Stimulus secretion coupling in a neurosecretory organ and the role of calcium in the release of vasopressin from the neurohypophysis. J. Physiol., Land. 172, l-18. DREIFUSS J. J. (1975) A review on neurosecretory granules: their contents and mechanisms of release. Ann. N. y Acad. Sci. 248, 184-20 1. DKEIFUSS J. J., GRAU J. D. & NORDMANN J. J. (1973) Effects on the isolated neurohypophysis of agents which affect the membrane permeability to calcium. J. Physiol.. Lond. 231, 9th98P. DYAALLR. E. J. & NORDMANN J. J. (1977) Reactivation by veratridine of hormone release from the K +-depolarized rat neurohypophysis. J. Physiol., Lond. 269, 65-66P. HEUSERJ. (1976) Morphology of synaptic vesicle discharge and reformation at the frog neuromuscular junction. In Mote, Innervation ofMuscle (ed. THESLEFF S.), pp. 51--l 15. Academic Press, London. HEUSERJ. E. & REESET. S. (1973) Evidence for recycling of synaptic vesicle membrane during transmitter release at the frog neuromuscular junction. J. Ce/[ Biol. 57, 315-344. HOLTZMAN E. (1977) The origin and fate of secretory packages. especially synaptic vesicles. Neuroscience 2. 327 355. HOLTZMAN E., SCHACHER S., EVANSJ. & TEICHBERG S. (1977) Origin and fate of secretion granules and synaptic vesicles: membrane circulation in neurons, gland cells and retinal photoreceptors. In Ceil Surfacr RecGrws. Vol. 4 (eds POSTI-G. 8~ NICOL~ON G. L.), pp. 165-246. Elsevier, North Holland. LEGROSJ. J., STEWARTU., NORDMANNJ. J., DREIFUSSJ. J. & FRANCHIMONT P. (1971) Libkration de vasopressine. d’oxytocine et de neurophysine appr&ci&e par mithode radioimmunologique lors de la stimulation in tritro de neurohypophyses de Rat. C.r. S&c. Sot. Biol. 165, 2443-2447. LIVIN(~STON A. (1975) Morphology of the perivascular regions of the rat neural lobe in relation to hormone release. Cc>// Tissur Res. 159, 551.-561. MORRISJ. F. (1976a) Hormone storage in individual neurosecretory granules of the pituitary gland: a quantitative ultrastructural approach to hormone storage in the neural lobe. J. Endocr. 68, 209-224. MORRISJ. F. (19766) Distribution of neurosecretory granules among the anatomical compartments of the neurosecretory processes of the pituitary gland: a quantitative ultrastructural approach to hormone storage in the neural lobe. J. Endocr. 68, 225.-234.

MORRISJ. F. & NORDMANN J. J. (1978) Membrane recapture after hormone release from the neural lobe. J. Anat. 127, 210. MORRISJ. F. & NORDMANN J. J. (1980) Membrane recapture after hormone release from nerve endings in the neural lobe of the rat pituitary gland. Neuroscience 5, 639-649. MORRIS J. F., NORDUNN J. J. & DYBALL R. E. J. (1978) Structure-function correlation in mammalian neurosecretion. In International Review of Experimental Pathology (eds RICHER G. W. & EPSTEINM. A.), Vol. 18, Pp. t-97. Academic Press. New York. NAGASAWA J., DOLIGLAS W. W. & SCHULZ R. A. (1970) Ultrastructural evidence of secretion by exocytosis and of ‘synaptic vesicle’ formation in posterior pituitary glands. Nature, Lond. 227, 407409.

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granule release and endocytosis

659

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principles for morphometry

in electron microscopic cytology. Inr. Rev. Cytol. 26,

(Accepted 7 October 1979)