Bioscience Reports, Vol. 8, No. 5, 1988
Release of Neuropeptides does not only
Occur at Nerve Terminals
Jean J. Nordmann and Govindan Dayanithi 1
Received March 10, 1988
Neurohypophysial hormones are packed in secretory granules which are stored in nerve endings and
in dilatations called nerve swellings. Although it was originally believed that the nerve swellings were
storage compartments and that release occurred solely from the nerve terminals, the present paper
demonstrates that secretion can occur to the same extent from both nerve endings and nerve
swellings.
KEY WORDS: exocytosis; secretion; neuropeptides; vasopressin; neurohypophysis.
INTRODUCTION
One of the most intriguing questions when studying the mechanism of secretion in
nervous tissue, is: does exocytosis occur at specialized sites such as discrete
domains on the surface of nerve terminals, or can n e u r o h o r m o n e or neurotransmitter be released all along the surface of a neuron? Although one would think,
at first, that nerve endings would be the preferential release site, there is evidence
that secretion can also occur from other regions of the neuron. For instance, the
release of amines from dendrites has been observed in the substantia nigra (for
review see Cheramy et al., 1981). In the present study we have tried to define if,
m a peptidergic neuron, release occurs at specific sites of the plasmalemma at the
distal part of the neuron.
The neural lobe is a convenient model for studying the release of neuropepfides
since it contains the distal parts of the oxytocin- and vasopressin-producing
neurones (for review see Morris et al., 1978). These two peptides are synthesized
in the magnocellular neurones of the hypothalamus; they are packaged into
neurosecretory granules (NSG) which are transported along the axons to the
nerve terminals. The neurosecretory processes in the neural lobe can be
subdivided into three morphological compartments, namely the undilated axons,
the nerve endings or nerve terminals and the nerve swellings (Morris, 1976). The
Centre de Neurochimie, 5 rue'Blaise Pascal, 67084 Strasbourg Cedex, France.
1 Present address: Department of Human Anatomy, South Park Road, Oxford OX1 3QX, England.
471
0144-8463/88/1000-0471506.00/0 (~ 1988 Plenum Publishing Corporation
Nordmann and Dayanithi
472
nerve endings contain, beside neurosecretory granules and mitochondria, microvesicles that resemble classical synaptic vesicles. The nerve swellings, on the other
hand, lack significant numbers of microvesicles but do contain NSG and
mitochondria. The mean diameter of the endings and swellings fixed in situ is 1.6
and 2.7/tm respectively (Nordmann, 1977). Following acute stimulation of
hormone release there is a depletion of the granules in the nerve endings whereas
there is no significant change in the number of NSG in the swellings (Morris and
Nordmann, 1980; Lescure and Nordmann, 1980). This led us to the conclusion
that release occurs from the nerve endings and that the swellings mostly represent
a storage compartment. In another study it was found that during dehydration
depletion also occurs in the swellings but this depletion can only be determined
quantitatively at a time later than that observed in the endings (Nordmann,
1985). These results could be interpreted in two ways. First, exocytosis occurs
only in the nerve endings, and depletion of the NSG in the swellings is due to the
movement of NSG from this compartment to the endings from where hormones
are actually secreted. The second possibility is that hormone release can occur
from both the nerve endings and the nerve swellings. In this paper we present
evidence for release from both the nerve endings and the nerve swellings.
Preliminary results have been communicated at the 10th International Symposium
on Neurosecretion (Nordmann et al., 1988).
MATERIALS
AND
METHODS
Male Wistar rats of 260-290 g body weight were decapitated and the neural lobe
was dissected free of pars intermedia. After being rinsed in physiological saline
(Cazalis et al., 1987a) they were homogenized in a medium containing (mM):
sucrose, 270; HEPES, 10, pH 7.2; EGTA, 0.01. The resulting homogenate was
centrifuged at 100g for 4 min and the resulting pellet was either loaded and
perfused on Millipore filters (see below) or fixed and processed for electron
microscopy or lysed in 0.1 N HC1 for further hormone determination (see below).
The supernatant was centrifuged at 300g and the resulting pellet was processed as
described above. The same protocol was used for the pellets obtained after
centrifugations at higher speed (see Results).
H o r m o n e Release
Vasopressin (AVP) release was studied as already described (Cazalis et al.,
1987a). Briefly, the different pellets were loaded onto a 0.22 #M Millipore filter
and perfused with saline. The perfusate (I00 #l/min) was collected by a fraction
collector and evoked hormone release was triggered by increasing the external
potassium concentration to 100 mM. In order to lyze the neurosecretosomes the
filters were soaked in 0.5% Triton X-100 at the end of the experiment. The
vasopressin in the different fractions was measured by radioimmunoassay.
Release of Neuropeptides
473
Electron Microscopy and Stereology
The different pellets were fixed with 2.5% gluteraldehyde in 100raM
Na-cacodylate, pH 7.2, for 90 rain. They were postfixed in 1% osmium tetroxide
in the sodium cacodylate buffer for 60 min and dehydrated in a graded series of
ethanol concentrations, immersed in propylene oxide, and embedded in Spurr
resin. Following sections of the pellets electron micrographs were taken at
random as already described. Using conventional stereology (for details see
Morris and Nordmann, 1980) we measured the mean surface area Ss of the
sectioned neurosecretosomes. The mean diameters of the endings and the
swellings were calculated from the equation d = 2VS/)x. Assuming that the
structures (endings and swellings) are nearly spherical and that the distribution of
their size is approximately normal, the true mean diameter D can be calculated
from the equation D = 4d/~t (Weibel, 1969). Finally the mean surface area and
mean volume of the structures were calculated as follows: S = 4 r t R 2 and
V = 4:rtR33.
RESULTS
Figure 1 is a montage of electron micrographs of swellings and endings in the
pellets resulting from differential centrifugation. On these micrographs the
structures were chosen in such a way that their diameter would be close to the
mean calculated diameter of the endings or swellings of the different pellets.
Note, however, that some swellings in the heavier fraction had a diameter of up
to 7/~m. Figure 2 illustrates the mean volume of the endings and swellings and
the ratio of the number of swellings compared to that of endings in the different
fractions. Note that the 300g pellet is very much enriched in swellings. The mean
diameter of the structures found in the 100 and 300g pellets was 2.7 J: 0.3 #m,
whereas the mean diameter of those found in the 1200 and 2400g pellets was
1.9 5:0.2/~m. These values are extremely close to those calculated from fixed
tissue in which the mean diameters of the swellings and the endings are 2.7 J: 0.1
and 1.7+0.04/~m respectively (Nordmann, 1977). Compared to the in situ
situation, where there are about 5 times more endings than swellings (Nordmann,
1977), this fraction is enriched by a factor of ca. 40 times in swellings compared to
endings. Figure 2 also shows that there is no significant difference in one given
pellet between the mean size of the swellings and that of the endings. From the
data described above and those concerning the volumetric density of the
neurosecretory granules (see below), one can conclude that following differential
centrifugation the heaviest fractions contain mostly nerve swellings whereas most
of the endings are located in the lighter fractions.
Figure 3 illustrates the volumetric density of the neurosecretory granules in the
swellings and the endings in the different pellets. Note that, beside a slight but
not significant increase in the volumetric density of the NSG in both the endings
and the swellings in the 300g pellet, the percent of the volume occupied by the
granules, in a given compartment, does not change significantly in the different
474
Nordmann and Dayanithi
Fig. 1. Electron micrographs of the different pellets obtained by
differential centrifugation. These structures have been chosen so as
to be representative of the mean size of the endings and swellings in
each pellet. A, 100g; B, 300g; C, 600g; D, 1200g; E, 2400g and F,
3400g. The bar represents 1/*m. Note the presence of microvesicles
only in the nerve endings.
pellets. The values obtained are very similar to those observed f r o m neural lobes
fixed in situ (Morris and N o r d m a n n , 1980) and show a significant difference
between the m e a n volume occupied by the N S G in the nerve endings and in the
nerve swellings. The volumetric density of the mitochondria in the nerve endings
was also very similar to that m e a s u r e d in tissue fixed in situ (Morris and
N o r d m a n n , 1980). In the 600, 1200, 2400 and 3400g pellets the volumetric density
of the mitochondria in the nerve terminals was 0.058, 0.065, 0.068 and 0.059
respectively. This compares well with the value of 0.068 measured in situ. Thus
one can conclude that the nerve endings and nerve swellings do not lose
significant amounts of organelles during their isolation. Note, however, that the
475
Release of Neuropeptides
-8
I
16(~L/
\\
(3r
TT
I
E
0
II
~ 4 ~
Q.
O}
O-
-0
f
0
I1000
!
2000
centrifugatien
I
3000
force
|
4000
(g)
Fig. 2. Volume of the nerve endings and the nerve swellings and the ratio of
these two compartments in the different pellets obtained after differential
centrifugation. The closed and open circles represent the mean volume of the
endings and the swellings respectively. The dashed line represents the ratio
between the number of swellings and endings in the different pellets. The
results are expressed as mean • S.E.M. (4 < n < 147). Only standard errors of
the mean larger than the symbols used are shown.
0.4-
I
v
0
/'\
Z
I\
II
"~ 0 . 2 o.
i
Y ~ v
A
_§
0
E
>o
OI
0
I
1000
t
2000
I
3000
]
4000
centrifugation f o rce (g)
Fig. 3. Volumetric density of the neurosecretory granules in the nerve
endings (closed circles) and the swellings (open circles). The results are
given as percent of the volume of the two compartments occupied by the
NSG ( 4 < n <42). Only standard errors of the mean larger than the
symbols used are shown.
476
Nordmann and Dayanithi
1.0--
9
/I
/
/
/
r
t
/
~ 0.5E
0
0
n
OI
0
I
1000
I
2000
I
3000
7
4000
c e n t r i f u g a t i o n f o r c e (g)
Fig. 4. Vasopressin content of the pellets obtained after differential
centrifugation of the bomogenate of 2-3 neural lobes. The value obtained
for a given pellet has been added to those found in the heavier fractions.
The results in the different fractions have been calculated as amount of
AVP per single gland. The open circle represents the total amount of AVP
obtained after addition of the content of each pellet and of the final
supernatant. Only standard errors of the mean larger than the symbols
used are shown(n = 6).
mitochondria in the large nerve endings found in the 100g and 300g pellets had a
significantly lower volumetric density (0.010 and 0.023 respectively).
In another series of experiments the amount of vasopressin in the different
pellets was determined with a radioimmunoassay. The results were obtained after
centrifugation of homogenates from 2-3 neural lobes. They are shown in Fig. 4
and expressed as the mean amount of AVP per single neural lobe. The amount of
AVP found in the 300g pellet was added to that of the 100g pellet and this
procedure was repeated for each of the values obtained from the different pellets.
Note that most of the nerve endings and swellings are sedimented after
centrifugation at 3400g as judged from electron micrographs and the plateau
illustrated in Fig. 4. It is worth mentioning here that a significant number of
isolated NSG can be found in the pellet obtained after centrifugation at 3400g and
thus the "3400g" pellet represents a mixture of nerve endings and nerve swellings
and of isolated neurosecretory granules.
In another series of experiments we measured the amount of AVP released
from the different pellets following K+-induced depolarization. The pellets were
perfused with saline as described in the Materials and Methods section and the
AVP content of the perfusate and the filters was measured. Figure 4 shows the
release of AVP from endings and swellings isolated by differential centrifugation.
The results are expressed as fractional release (rate constant of release) which
was calculated as follows: rate constant (min -1) = AVP(At - AVPt) -1 where AVP
represents the amount of AVP released in the time interval At, and AVPt the
tissue content of vasopressin at the midpoint of interval At. The rate constant
thus represents the fractional release of hormone. The main conclusion which can
be drawn from the results illustrated in Fig. 5 is that the time course of release is
477
Release of Neuropeptides
-
;:\
T\.,
"~ 2 . 5 -
of'
/-o j '~,6\
J
,
i
m
OI
0
I
15
time (min}
t
30
Fig. 5. AVP release from mixed isolated nerve endings and swellings.
The fractions were perfused as described in the Materials and Methods
section and an augmentation in hormone secretion was triggered by
increasing the external K + concentration to 1130mM during the period
indicated by the heavy bar. The results are expressed as rate constant of
release from the 100g (open circles), 1200g (triangles) and 2400g (closed
circles) pellets. Only standard errors of the mean larger than the symbols
used are shown (n = 4).
I
5.0-
o
,/;
2.5-
,//'
v
o
!
Q.
O-g
0
i
1000
I
2000
- - T
3000
- - 1
4000
centrifugation f o r c e (g)
Fig. 6. Percent of vasopressin released from the different pellets
obtained after differential centrifugation. AVP release was triggered with
100 mM K + and the total amount of the depolarization-induced peptide
secretion was expressed as percent of the amount of hormone loaded
onto the filters (see Materials and Methods). Only standard errors of the
mean larger than the symbols used are shown (n -~ 4).
478
Nordmann and Dayanithi
very similar whatever the size of the nerve processes. Figure 6 illustrates the
percent of the A V P content released upon K§
depolarization. Whereas
the 100g pellet, which contains mostly large nerve swellings, releases only
1.4 + 0.3% (mean + S.E.M., n = 4) of its content, 4.7 + 0.6% of the vasopressin
content of the 2400g pellet is secreted. An interesting observation is that the
3400g pellet releases proportionally less A V P than the 2400g pellet. This can be
explained by the presence of isolated NSG in the 3400g pellet (see above) which
will increase the amount of h o r m o n e on the filter but will also tend to decrease
the fractional depolarization-induced A V P release.
Finally we measured the amount of A V P released from the 300g and 2400g
pellets isolated from rats dehydrated for a period of 5 days. Under these
conditions the neural lobe has a reduced hormonal content and the volumetric
Fig. 7. Unstained electron micrograph of a nerve
swellings and a nerve ending following potassiuminduced depolarization in the presence of the extracellular marker horseradish peroxydase. Labelled
endocytotic vacuoles can be observed in both swellings
(A) and endings (B). The bar represents 0.2/~m.
Release of Neuropeptides
479
density of the NSG in the nerve endings is less than 10% of that in controls. We
found that the 2400g pellet released only 1.0 • 0.2% (n = 6), whereas the 300g
pellet still released 2.0 + 0.2% (n = 6) of its content following K+-depolarization.
Similar experiments were performed on pellets isolated from rats dehydrated
during 3 days, i.e. a situation in which the hormonal content is only partially
depleted. Under these conditions the 300g and 3400g pellets released 1.3 and
3.3% respectively of their AVP content following potassium-induced depolarization. These last values are extremely close to those obtained from control pellets
and highlight the above data, namely that release also occurs from those fractions
containing mostly nerve swellings. Evidence for the occurrence of exoendocytosis in both nerve swellings and nerve endings are presented in Fig. 7.
The fractions were obtained by cumulating the 100 and 300g pellets on one hand
and the 1200 and 2400g pellets on the other. The samples were incubated in
normal saline during a period of 60 rain. The medium was then replaced by
normal saline containing 0.5% horseradish peroxide which was used as an
extracellular marker, hence as a marker for the endocytosis following the release
by exocytosis. After a period of 10 rain release was triggered by increasing the
extracellular potassium concentration to 100mM. Figure 7 shows that exoendocytosis occurs in both the nerve endings and the nerve swellings as judged by
the presence of labeled endocytotic vacuoles.
Calculation of the Number of Granules Released per gm 2 of Plasma
Membrane of Nerve Endings and Nerve Swellings.
Table 1 gives the mean surface area and the volume of the two compartments
in the different pellets. Knowing (i) the mean volume of a single granule in a
nerve ending or in a swelling (2.6 x 10 .3/~m 3 and 3.6 x 10 .3 Nm 3 respectively;
Nordmann et al., 1979), (ii) the mean volume of the endings and of the swellings
and (iii) the volumetric density of their granules, one can calculate the mean
number of granules in the nerve terminals and in the dilations in the different
pellets. The results are given in Table 2. The large majority of the values
obtained in this study (hormone content and release, volumetric densities, surface
area and volume, etc.) have a standard error of the mean lower than 10% of the
Table 1
Area (/~m2)
Volume (/~m3)
Pellets
Endings
Swellings
Endings
Swellings
lOOg
300g
60(0
1200g
2400g
3400g
22.6 • 9.6 (4)
22.6•
18.2•
16.4 • 1.7 (59)
8.8•
5.9•
25.6•
29.9 • 2.7 ( 7 9 )
17.3•
16.1 • 1.1 (143)
10.7 :k 2.1 ( 3 7 )
5.5+0.9(24)
10.1•
10.1+2.8(10)
7.3 • 1.0 ( 2 7 )
6.2 • 0.7 (59)
2.4•
1.3•
12.2•
15.4•
6.8:t:0.3(47)
6.1 • 0.4 (143)
3.3•
1.2•
Mean surface area and volume of nerve terminals and nerve swellingsisolated by differential
centrifugation. The results are given as mean • standard error of the mean. The number of
determinations are given in parenthesis.
480
Nordmann and Dayanithi
Table 2
Pellet
100g
300g
600g
1200g
2400g
3400g
Number of NSG
Endings
Swellings
650
842
454
315
119
85
880
1292
430
380
214
86
Number of NSG released per/tin 2
Endings
Swellings
0.40
0.84
0.65
0.76
0.56
0.42
0.48
0.93
0.67
0.91
0.82
0.45
Mean number of neurosecretory granules in nerve endings and nerve swellings
and number of NSG released per/~m 2 of plasma membrane from nerve endings
and nerve swellings isolated by differential centrifugation and depolarized during
a period of 10 min with 100 mM potassium.
mean. Thus in the following calculations we only give the mean values. When the
pellets obtained after relatively high speeds of centrifugation are considered, the
number of granules in the endings is very similar to that in the swellings.
However, there is a large difference between the number of granules in the large
endings and that found in the largest swellings (see Table 2).
Knowing the mean number of granules in the endings and the swellings, the
mean surface area of these structures and the percent of granules released upon
potassium-induced depolarization, one can calculate the mean number of
granules released per # m 2 of plasma membrane. The results are presented in
Table 2. There is generally a good agreement between the number of granules
released per unit of membrane of endings and swellings in the same fraction. The
mean number of granules released per/~m 2 of m e m b r a n e of endings and swellings
is 0.60 + 0.07 and 0.70 + 0.08 respectively. Note that the n u m b e r of granules
released per /tm 2 is somewhat smaller in the 100g and 3400g pellets. As a
plausible explanation one can suggest that part of the larger structures do indeed
release less granules than the average sized swellings and endings; the lower value
obtained for the structures found in the 3400g pellet can easily be explained by
the presence of isolated granules (see above).
DISCUSSION
The present study shows that neurohypophysial h o r m o n e secretion can occur
from both the isolated nerve endings and the nerve swellings. Furthermore our
data demonstrate that the number of granules which release their content per unit
of surface area of plasma m e m b r a n e is very similar in the two compartments. This
suggests that the membrane of the nerve terminals is not different from that of
the swellings nor are the components of the membrane, involved in exocytosis, of
the NSG found in the endings v s . those located in the swellings. In favour of this
interpretation is the fact that electrophysiological recordings using the patch
clamp technique of different types of Ca 2§ channels in large structures (presumably nerve swellings) and those recorded from smaller structures (presumably
Release of Neuropeptides
481
the nerve endings) do not show any significant difference in the properties of the
channels (J. R. Lemos and M. C. Nowycky, personal communication). Furthermore, Buma and Nieuwenhuys (1987) and Morris et aL (1988) have observed in
both nerve endings and nerve swellings of the neural lobe in situ exocytosis via
the tannic acid technique (Buma and Roubos, 1987). Moreover, isolated swellings
stimulated with depolarizing concentrations of potassium in the presence of
dextran or horseradish peroxidase contain labelled endocytotic vacuoles (Nordmann and Shaw, 1984; Morris et al., 1988; the present study); this suggests that
exo-endocytosis has indeed occurred in the swellings. Although one could
interpret the present data by saying that the release observed in the fractions
containing mostly nerve swellings is actually secreted by the "contaminating"
endings, the observation of endocytotic vacuoles in the isolated nerve swellings
strongly argue against this interpretation. Another alternative to explain our data
is that during homogenization there would be an abnormal detachment of the
NSG from microtubules in the swellings leaving them free to be released.
However this hypothesis seems to us unlikely, for agents which affect the structure
of the cytoskeleton do not affect the amount of hormone released from the
neurohypophysis in vivo (S. Barlier and J. J. Nordmann, unpublished). One
question however remains: do the nerve swellings, which in situ are distant from
the basal membrane, release, in vivo, neurohormone under normal conditions?
One can estimate the proportion of granules which in vivo would be released
from the nerve endings versus the nerve swellings. One neural lobe contains, on
average, 3.4 x 107 nerve endings and 7.1 x 106 nerve swellings (Nordmann, 1977).
From their mean surface areas and the data presented in Table 1 one can
calculate that K+-induced depolarization for a period of 10 rain would release
1.6 x 108 and 1.2 x 108 NSG from the endings and the swellings respectively. Thus
there is about 1.3 more granules released from the endings than from the
swellings.
Although the present paper demonstrates that release occurs from both the
endings and the swellings one should keep in mind that these two compartments
are somewhat arbitrary. Beside their average diameters, their only difference is
the presence of microvesicles solely in the endings. Furthermore, preliminary
data (Newcomb, Hartline, Lorentz and Nordmann, unpublished) on the peptide
content of the endings and the swellings show apparently no difference between
the composition of the NSG in the two compartments. This would argue against
the concept of the endings mostly containing newly synthesized material and
being the release site, and the swelling containing only the "aged" granules and
being solely the storage site. Recently, Chapman and Morris (see Morris et aL,
1988) have found that often nerve endings are excrescences of the swellings and
thus it is perhaps not too surprising that secretion from both compartments can
occur. Furthermore, release of OT and AVP from neuronal varicosities has been
observed in the median eminence (Buma and Nieuwenhuys, 1987). Thus our
present knowledge suggests that neuropeptide release does not only occur at the
nerve terminals but also at other sites along the plasma membrane of the neuron.
Finally, one point which deserves comment is why in the neural lobe one
anatomical compartment (the endings) contains large amounts of "synaptic-like"
482
Nordmann and Dayanithi
microvesicles, whereas the swellings and the undilated axons do not. Data
obtained from morphometric analysis of stimulated tissues have shown that they
are not responsible for the process of endocytosis which follows exocytosis
(Morris and Nordmann, 1980; Lescure and Nordmann, 1980). Ultrastructural
(Shaw and Morris, 1980) and biochemical (Nordmann and Chevallier, 1980;
Torp-Pedersen et al., 1980) data suggest strongly that they play a role in the
calcium homeostasis of the nerve endings. However an enigma remains: why
swellings do not contain these organelles. The present study shows that they are
not directly coupled with the process of exocytosis of AVP and OT since this
process occurs from the swellings which do not contain microvesicles. This lack of
microvesicles has also been observed in many invertebrate neurosecretory
systems where exocytosis can be visualized as occurring anywhere along the
plasma membrane of large dilatations (for review see Normann, 1976). Secretion
from the isolated neurohypophysial nerve endings is half-maximal at a Ca 2+
concentration at the site of release of c.a. 1.7/~M (Cazalis et al., 1987b).
Combining these data with those of, on one hand, Zucker and Stockbridge
(1983), who analyzed in the squid giant synapse the cytoplasmic calcium
concentration during and after an action potential, and on the other hand on
models on the calcium homeostasis in neurons (Fogelson and Zucker, 1985; Nasi
and Tillotson, 1985; Simon and Llinas, 1985), one can estimate that the calcium
concentration necessary to trigger secretion in the neural lobe is only reached
within a few tenths of a micron away from the plasma membrane. Thus the lack
of microvesicles in the swellings and other neurosecretory systems suggests that
the plasma membrane of these structures has the necessary properties to control
the cytoplasmic calcium concentration.
Although we have demonstrated that neurosecretion does not only occur at the
level of the nerve endings but also from the nerve swellings, one can still wonder
what are the reasons for the clear ultrastructural differences (Morris, 1976)
between the endings and the swellings.
ACKNOWLEDGEMENTS
We are in debt to Jean-Claude Artault for the electron micrographs. We
thank Malou Dreyer and Marlyse Kretz-Zaepfel for much help with the
radioimmunoassay and Jose Lemos for helpful suggestions concerning the
manuscript. The AVP antiserum was a kind gift from Ruud Buijs. Supported by
grants from CNRS and INSERM.
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Release of Neuropeptides
483
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