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Overexpression of synaptophysin enhances neurotransmitter secretion at Xenopus neuromuscular synapses.
Alder J
,
Kanki H
,
Valtorta F
,
Greengard P
,
Poo MM
.
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Previous studies have suggested the importance of synaptophysin (p38), a major integral membrane protein of the synaptic vesicle, in transmitter secretion, but few have directly addressed its functional role at intact synapses. In the present study, injection of synthetic mRNA for synaptophysin into one of the early blastomeres of a Xenopus embryo resulted in elevated synaptophysin expression in 1 and 2 d embryos and in cultured spinal neurons derived from the injected blastomere, as shown by immunocytochemistry. At neuromuscular synapses made by neurons overexpressing synaptophysin [p38(+)] in 1 d cell cultures, the spontaneous synaptic currents (SSCs) showed a markedly higher frequency, as compared to control synapses. This increase in frequency was not accompanied by a change in the mean amplitude or the amplitude distribution of the SSCs, suggesting that synaptophysin is not involved in determining the size of transmitter quanta. The impulse-evoked synaptic currents (ESCs) of synapses made by p38(+) neurons showed increased amplitude as well as reduced fluctuation and delay of onset of ESCs. Under high-frequency tetanic stimulation at 5 Hz, the rate of tetanus-induced depression was faster for p38(+) neurons. Taken together, these results suggest a role for synaptophysin in the late steps of transmitter secretion, affecting the probability of vesicular exocytosis and/or the number of synaptic vesicles initially docked at the active zone.
Figure 1. Plasmid used for in vitro transcription of synaptophysin
mRNA. Rat svnantoohvsin cDNA (Buckley et al.. 1987) was subcloned
into the HindI site of pSP64(polyk). The-plasmid was then linearized
with FspI and in vitro transcribed using SP6 RNA polymerase.
Figure 2. Overexpression of synaptophysin in Xenopus embryos injected with synaptophysin mRNA. Embryos were injected at the two-cell stage,
and synaptophysin expression was examined by immunocytochemical staining using alkaline phosphatase-conjugated secondary antibodies. A and
D, Dorsal view of 1 -d-old embryos showing positive staining for synaptophysin (purple) on one half only. B and C, E and F, Lateral views of 2-dold
embryos, expressing exogenous synaptophysin only on one side. Left and right photographs are opposite lateral views of the same embryo. GI,
Same as A-C, except that the embryos were not injected with synaptophysin mRNA. In the 2-d-old embryo (Hand I), weak staining was visible
in the spinal cord and other neuronal structures, but no difference in staining intensity was found between the two sides of the embryo. J and K,
Nerve-muscle cultures prepared from l-d-old embryos injected with synaptophysin mRNA. Some neurons and their processes (marked by solid
arrows) are more darkly stained than others (open arrows). Staining is also visible in some myocytes. L, Culture prepared from a control, uninjected
embryo. All neurons (open arrow) stained with the same low intensity, and no myocytes showed significant staining. M and N, Myocytes in nervemuscle
cultures prepared from embryos injected with synaptophysin mRNA. Some myocytes (marked by arrowheads) expressed synaptophysin
while others (unmarked) are unstained. 0, Nerve-muscle culture from an mRNA-injected embryo stained with preimmune serum instead of
synaptophysin antibodies. No background staining is visible in either the neurons (open arrow) or myocytes. Scale bars: 0.5 mm for 1 d embryos,
0.7 mm for 2 d embryos, and 30 pm for cultures.
Figure 3. Overexpression of synaptophysin enhances spontaneous synaptic current (SSC) frequency. A, Continuous trace depicts membrane current
recorded from whole-cell voltage-clamped ( Vh = - 70 ImV) myocytes that were innervated either by a control neuron (top) or by a neuron
overexpressing synaptophysin [p38 (+)I (bottom). Inward currents are shown as downward deflections at a slow (left) and a fast (right) time scale.
The right panels show superimposed traces of the events recorded during a 3 min period. Calibration: 0.25 nA, 40 set, and 0.25 nA, 10 mse-c for
the slow andfast traces, respectively. B, SSC frequency for p38(+) synapses and control synapses (left) and @-globin and control synapses (right).
Each data point (top) represents the SSC frequency at a single synapse during a 10 min recording period, and the scattering of the data along the
abscissa is for display only. Mean SSC frequency of all synapses + SEM is shown in the bar graphs below. The mean SSC frequency of p38(+)
synapses (open bars) is higher than that of control synapses (soLid bars) (p < 0.05, t test), but that of &globin(+) synapses is not significantly
different from the controls in the same culture @ > 0.05, t test).
Figure 4. Overexpression of synaptophysin has no effect on SSC amplitudes
or rise times: SSC amplitude (A) and rise time (B) distributions
for p38(+) (solid line) and control (dashed he) synapses. Curves represent
averaged amplitude or rise time distributions from 15 p38(+)
and 1 I control synapses. The cumulative frequency refers to the proportion
of total events with amplitudes or rise times smaller than a
given value. For clarity, error bars (?SEM) are shown only for some
points along the solid lines. No significant difference was found between
the p38(+) and control synapses (p > 0.05, t test) at any point along
the amplitude or rise time distributions.
Figure 5. Enhancede vokeds ynapticc urrents(E SCs) resultingfr om overexpressioonf synaptophysiinn presynapticn euronsA. , Continuoustr ace
depictst he membranec urrent recordedf rom myocytest hat were innervatede ither by a control neuron( fop) or by a neurono verexpressing
synaptophysi[np 38 (+)] (bottom).T he neuronsw eree xtracellularlys timulated(0 .5m secd uration,0 .05 Hz) to generataec tionp otentialsR. ecorded
ESCsa re showna sd ownwardd eflectionss paceda t regulari ntervalsa mongr andomlyo ccurringS SCsa, t a slow( left) anda fast (right) time scale.
Sampleosf oscilloscoptera ceso f three ESCsa re showno n the right. Calibration:0 .5 nA, 40 set, and 1 nA, 10m secf or the slowa ndf ast truces,
respectivelyB. , Evokedr esponsefos r p38(+) synapseasn d control synapse(sle ft) and fi-globin and control synapse(srig ht). Eachd ata point
(top) representtsh e averageE SCa mplitudea t a singles ynapsea nd the scatteringo f the data alongt he abscissias for displayo nly. Mean ESC
amplitudeo f all synapse(+s SEM) is shownin the bar graphsb elow.T he meanE SCa mplitudeo f p38(+) synapse(sop en bars) is highert han that
of control synapse(sso lid bars) (p < 0.01, f test) but the meanE SCa mplitudeo f @-globin synapseis not significantlyd ifferentf rom that r
controlsin the samec ulture (p > 0.05, t test).
Figure 6. Effecto f synaptophysionv erexpressioonn tetanus-induced
depressionA.m plitudeso f ESCsd uring a 1 min tetanusa t 5 Hz were
averagedin 6 set bins. Eachd ata point (GEM) represenths e value
averagedfo r eight p38(+) (solid line) and eight control (dashed line)
synapsesA. significandt ifferencew asf ound during the first 48 set (p
< 0.05, t test).
