Dynamin and Activity Regulate Synaptic Vesicle Recycling in Sympathetic Neurons
2008; Elsevier BV; Volume: 284; Issue: 3 Linguagem: Inglês
10.1074/jbc.m803691200
ISSN1083-351X
AutoresWenbo Lu, Huan Ma, Zu‐Hang Sheng, Sumiko Mochida,
Tópico(s)Neuroscience and Neuropharmacology Research
ResumoNeurotransmission in central neuronal synapses is supported by the recycling of synaptic vesicles via endocytosis at different time scales during and after transmitter release. Here, we examine the kinetics and molecular determinants of different modes of synaptic vesicle recycling at a peripheral neuronal synapse formed between superior cervical ganglion neurons in culture, via acute disruption of endocytosis with Dynasore, an inhibitor of dynamin activation, or a dynamin peptide (P4) that perturbs linkage of dynamin to clathrin coats through amphiphysin. When paired action potentials are generated to produce excitatory postsynaptic potential responses, the second response was reduced after application of Dynasore but not P4. In addition, graded reduction in synaptic transmission during a train of action potentials was accelerated by Dynasore but enhanced by P4. After full depletion of releasable vesicles, P4 delayed the recovery of synaptic transmission while Dynasore limited recovery to 10%. In control neurons, synaptic transmission is stable for more than 1 h under low frequency presynaptic stimulation (0.2 Hz), but was reduced gradually by P4 and rapidly but incompletely blocked by Dynasore at a much lower stimulation frequency. These results suggest two essential modes of dynamin-mediated synaptic vesicle recycling, one activity-dependent and the other activity-independent. Our findings extend the current understanding of synaptic vesicle recycling to sympathetic nerve terminals and provide evidence for a physiological and molecular heterogeneity in endocytosis, a key cellular process for efficient replenishment of the vesicle pool, and thus for synaptic plasticity. Neurotransmission in central neuronal synapses is supported by the recycling of synaptic vesicles via endocytosis at different time scales during and after transmitter release. Here, we examine the kinetics and molecular determinants of different modes of synaptic vesicle recycling at a peripheral neuronal synapse formed between superior cervical ganglion neurons in culture, via acute disruption of endocytosis with Dynasore, an inhibitor of dynamin activation, or a dynamin peptide (P4) that perturbs linkage of dynamin to clathrin coats through amphiphysin. When paired action potentials are generated to produce excitatory postsynaptic potential responses, the second response was reduced after application of Dynasore but not P4. In addition, graded reduction in synaptic transmission during a train of action potentials was accelerated by Dynasore but enhanced by P4. After full depletion of releasable vesicles, P4 delayed the recovery of synaptic transmission while Dynasore limited recovery to 10%. In control neurons, synaptic transmission is stable for more than 1 h under low frequency presynaptic stimulation (0.2 Hz), but was reduced gradually by P4 and rapidly but incompletely blocked by Dynasore at a much lower stimulation frequency. These results suggest two essential modes of dynamin-mediated synaptic vesicle recycling, one activity-dependent and the other activity-independent. Our findings extend the current understanding of synaptic vesicle recycling to sympathetic nerve terminals and provide evidence for a physiological and molecular heterogeneity in endocytosis, a key cellular process for efficient replenishment of the vesicle pool, and thus for synaptic plasticity. The cycling of synaptic vesicles (SVs) 3The abbreviations used are: SV, synaptic vesicle; SCG, superior cervical ganglion; EPSP, excitatory postsynaptic potential; RRP, readily releasable pool; RP, reserve pool; ISI, inter-stimulus interval.3The abbreviations used are: SV, synaptic vesicle; SCG, superior cervical ganglion; EPSP, excitatory postsynaptic potential; RRP, readily releasable pool; RP, reserve pool; ISI, inter-stimulus interval. through repetitive episodes of exocytosis and endocytosis is fundamental to synaptic transmission (1LoGiudice L. Matthews G. Neuron. 2006; 51: 676-677Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). The classic endocytic cycle consists of vesicle exocytosis from a readily releasable pool (RRP) (2Birks R.I. Macintosh F.C. Br. Med. Bull. 1957; 13: 157-161Crossref PubMed Scopus (37) Google Scholar) followed by retrieval via a clathrin-mediated endocytotic pathway (3Heuser J.E. Reese T.S. J. Cell Biol. 1973; 57: 315-344Crossref PubMed Scopus (1616) Google Scholar) that passes through a reserve pool (RP) (4Elmqvist D. Quastel D.M. J. Physiol. 1965; 178: 505-529Crossref PubMed Scopus (403) Google Scholar) en route to the RRP (5Harata N.C. Aravanis A.M. Tsien R.W. J. Neurochem. 2006; 97: 1546-1570Crossref PubMed Scopus (153) Google Scholar, 6Sudhof T.C. Nature. 1995; 375: 645-653Crossref PubMed Scopus (1766) Google Scholar). The classic pathway, well studied at the frog neuromuscular junction (3Heuser J.E. Reese T.S. J. Cell Biol. 1973; 57: 315-344Crossref PubMed Scopus (1616) Google Scholar), also functions in brain central synapses (7Granseth B. Odermatt B. Royle S.J. Lagnado L. Neuron. 2006; 51: 773-786Abstract Full Text Full Text PDF PubMed Scopus (491) Google Scholar, 8Fernandez-Alfonso T. Ryan T.A. Neuron. 2004; 41: 943-953Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). In addition, other non-traditional modes of recycling such as "kiss-and-run" (9Ceccarelli B. Hurlbut W.P. Mauro A. J. Cell Biol. 1973; 57: 499-524Crossref PubMed Scopus (631) Google Scholar) and fast recycling, which bypasses the RP (10Artalejo C.R. Henley J.R. McNiven M.A. Palfrey H.C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8328-8332Crossref PubMed Scopus (247) Google Scholar), have been described (11Gandhi S.P. Stevens C.F. Nature. 2003; 423: 607-613Crossref PubMed Scopus (373) Google Scholar, 12Aravanis A.M. Pyle J.L. Tsien R.W. Nature. 2003; 423: 643-647Crossref PubMed Scopus (342) Google Scholar, 13Klingauf J. Kavalali E.T. Tsien R.W. Nature. 1998; 394: 581-585Crossref PubMed Scopus (344) Google Scholar, 14Stevens C.F. Williams J.H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12828-12833Crossref PubMed Scopus (204) Google Scholar). Endocytic pathways have also been categorized in terms of their kinetics, as fast or slow (1LoGiudice L. Matthews G. Neuron. 2006; 51: 676-677Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar, 15Royle S.J. Lagnado L. J. Physiol. 2003; 553: 345-355Crossref PubMed Scopus (108) Google Scholar). Together, it seems likely that various forms of SV recycling pathways function under different conditions of synaptic activity and in a cell type-specific manner (15Royle S.J. Lagnado L. J. Physiol. 2003; 553: 345-355Crossref PubMed Scopus (108) Google Scholar, 16Wu L.G. Trends Neurosci. 2004; 27: 548-554Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Although the precise mechanisms of synaptic reformation remain a matter of debate (3Heuser J.E. Reese T.S. J. Cell Biol. 1973; 57: 315-344Crossref PubMed Scopus (1616) Google Scholar, 4Elmqvist D. Quastel D.M. J. Physiol. 1965; 178: 505-529Crossref PubMed Scopus (403) Google Scholar, 5Harata N.C. Aravanis A.M. Tsien R.W. J. Neurochem. 2006; 97: 1546-1570Crossref PubMed Scopus (153) Google Scholar, 6Sudhof T.C. Nature. 1995; 375: 645-653Crossref PubMed Scopus (1766) Google Scholar, 7Granseth B. Odermatt B. Royle S.J. Lagnado L. Neuron. 2006; 51: 773-786Abstract Full Text Full Text PDF PubMed Scopus (491) Google Scholar), the GTPase dynamin has a key role for this process (17Klingauf J. Neuron. 2007; 54: 857-858Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar, 18Cremona O. De Camilli P. Curr. Opin. Neurobiol. 1997; 7: 323-330Crossref PubMed Scopus (194) Google Scholar, 19Ferguson S.M. Brasnjo G. Hayashi M. Wolfel M. Collesi C. Giovedi S. Raimondi A. Gong L.W. Ariel P. Paradise S. O'Toole E. Flavell R. Cremona O. Miesenbock G. Ryan T.A. De Camilli P. Science. 2007; 316: 570-574Crossref PubMed Scopus (403) Google Scholar). Dynamin oligomerization in endocytic pits mediates neck constriction and scission (20Takei K. McPherson P.S. Schmid S.L. De Camilli P. Nature. 1995; 374: 186-190Crossref PubMed Scopus (654) Google Scholar). As a component of the clathrin coat, amphiphysin interacts with dynamin and links clathrin-coats to dynamin (21Takei K. Slepnev V.I. Haucke V. De Camilli P. Nat. Cell Biol. 1999; 1: 33-39Crossref PubMed Scopus (513) Google Scholar). Dynamin binds to the Src homology 3 (SH3) domain of amphiphysin I via the PSRPNR sequence in the dynamin polyproline domain near its C terminus (22Grabs D. Slepnev V.I. Songyang Z. David C. Lynch M. Cantley L.C. De Camilli P. J. Biol. Chem. 1997; 272: 13419-13425Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 23Wigge P. Kohler K. Vallis Y. Doyle C.A. Owen D. Hunt S.P. McMahon H.T. Mol. Biol. Cell. 1997; 8: 2003-2015Crossref PubMed Scopus (209) Google Scholar). A myristoylated peptide derived from this sequence called P4 (QVPSRPNRAP) is able to competitively block dynamin binding to amphiphysin I and II in vitro (24Marks B. McMahon H.T. Curr. Biol. 1998; 8: 740-749Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar) and inhibits SV endocytosis, thus resulting in the depression of transmitter release (25Shupliakov O. Low P. Grabs D. Gad H. Chen H. David C. Takei K. De Camilli P. Brodin L. Science. 1997; 276: 259-263Crossref PubMed Scopus (401) Google Scholar). Dynasore, a specific cell-permeable dynamin inhibitor (26Macia E. Ehrlich M. Massol R. Boucrot E. Brunner C. Kirchhausen T. Dev. Cell. 2006; 10: 839-850Abstract Full Text Full Text PDF PubMed Scopus (1504) Google Scholar), completely blocks SV endocytosis, suggesting an essential role for dynamin in all forms of compensatory SV endocytosis, including "kiss-and-run" events (27Newton A.J. Kirchhausen T. Murthy V.N. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 17955-17960Crossref PubMed Scopus (192) Google Scholar). However, in the calyx of Held, a dynamin-independent endocytosis was detected in the presence of Dynasore (28Xu J. McNeil B. Wu W. Nees D. Bai L. Wu L.G. Nat. Neurosci. 2008; 11: 45-53Crossref PubMed Scopus (67) Google Scholar). The mechanism of SV recycling in sympathetic neurons remains an open question. To investigate this issue, we studied the cholinergic synapse formed between rat SCG neurons in culture (29Ma H. Mochida S. Neurosci. Res. 2007; 57: 491-498Crossref PubMed Scopus (28) Google Scholar). In this model system, it is possible to introduce reagents directly into presynaptic terminals by microinjection, and their effect on acetylcholine release evoked by action potentials can be monitored by recording EPSPs from neighboring neurons (29Ma H. Mochida S. Neurosci. Res. 2007; 57: 491-498Crossref PubMed Scopus (28) Google Scholar, 30Mochida S. Kobayashi H. Matsuda Y. Yuda Y. Muramoto K. Nonomura Y. Neuron. 1994; 13: 1131-1142Abstract Full Text PDF PubMed Scopus (133) Google Scholar). By perturbing dynamin function with either P4 peptide or Dynasore, we examine both activity-dependent and -independent modes of endocytosis, as well as dynamin-dependent and -independent pathways for refilling of the RRP in sympathetic neurons. Cultured SCG neurons were prepared as described previously (29Ma H. Mochida S. Neurosci. Res. 2007; 57: 491-498Crossref PubMed Scopus (28) Google Scholar, 30Mochida S. Kobayashi H. Matsuda Y. Yuda Y. Muramoto K. Nonomura Y. Neuron. 1994; 13: 1131-1142Abstract Full Text PDF PubMed Scopus (133) Google Scholar). For immunocytochemistry, SCG neurons in culture (8 weeks) were fixed and stained as described previously (30Mochida S. Kobayashi H. Matsuda Y. Yuda Y. Muramoto K. Nonomura Y. Neuron. 1994; 13: 1131-1142Abstract Full Text PDF PubMed Scopus (133) Google Scholar) with polyclonal anti-dynamin 1 and anti-amphiphysin II antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) or monoclonal anti-synaptophysin antibody (Sigma-Aldrich) (supplemental Fig. S1). For electrophysiology, SCG neurons 6-8 weeks in culture were studied. EPSP recording and injection of peptides were performed as described previously (29Ma H. Mochida S. Neurosci. Res. 2007; 57: 491-498Crossref PubMed Scopus (28) Google Scholar, 30Mochida S. Kobayashi H. Matsuda Y. Yuda Y. Muramoto K. Nonomura Y. Neuron. 1994; 13: 1131-1142Abstract Full Text PDF PubMed Scopus (133) Google Scholar). To measure the replenishment of the RRP with readily releasable SVs, either a paired-pulse protocol or 5-30 Hz stimulation for 2 s was applied. For each neuron pair, three recordings were performed every 2 min for each interval of stimuli, and the EPSP peak amplitudes were averaged to account for variations in transmitter release following repetitive action potentials. For the depletion of synaptic vesicles, action potentials were applied at 5 Hz for 4 min, and replenishment of readily releasable SVs was monitored by tracking the recovery of baseline EPSP via recording every 1 s. The peak amplitudes of EPSP were averaged, and the resultant values were smoothed by an eight-point moving average algorithm. To measure the change in readily releasable SVs during prolonged repetitive activity, EPSPs were recorded at either 0.2 or 0.05 Hz. The peak amplitudes were averaged and plotted against recording time with t = 0 corresponding to the presynaptic injection of P4 (QVPSRPNRAP), a scrambled control peptide (QPPASNPRVR), or bath application of Dynasore. 1 mm peptide in the injection pipette was applied as this is the concentration producing a maximum reduction of EPSP amplitude, whereas 5 mm peptide showed no further reduction. 4W. Lu, H. Ma, Z.-H. Sheng, and S. Mochida, unpublished data. For Dynasore bath application, 100 μl of 1 mm Dynasore dissolved in 5% DMSO was drop-applied to a 1.25-ml bath. A final concentration of 80 μm (0.4% DMSO) Dynasore was used to achieve maximum inhibition of endocytosis, because this concentration completely blocked all forms of endocytosis in hippocampal neurons (28Xu J. McNeil B. Wu W. Nees D. Bai L. Wu L.G. Nat. Neurosci. 2008; 11: 45-53Crossref PubMed Scopus (67) Google Scholar). As a control, 5% DMSO was drop-applied, producing a bath concentration of 0.4%. To reach the final concentration it takes a few minutes after bath superfusion was stopped (31Mochida S. Westenbroek R.E. Yokoyama C.T. Itoh K. Catterall W.A. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 2813-2818Crossref PubMed Scopus (63) Google Scholar). Before recording EPSPs at 20 min after the Dynasore application, cultured SCG neurons showed no spontaneous synaptic activity (32Mochida S. J. Physiol. Paris. 1995; 89: 83-94Crossref PubMed Scopus (38) Google Scholar), suggesting that reduction of SVs in the RRP with the treatment is unlikely. Error bars shown in the text and figures represent mean ± S.E. A two-tailed Student t test was applied as indicated. Readily Releasable SVs after Evoked Transmitter Release—To examine the readily releasable SVs after single action potential-evoked transmitter release, a paired-pulse protocol was applied under acute disruption of endocytosis by P4 or Dynasore (Fig. 1). Synaptic responses induced by two consecutive action potentials showed a depression of the second response (paired-pulse depression) with inter-stimulus interval (ISI) of 20-100 ms. In contrast, at longer ISIs (200-2000 ms) the amplitudes of the second response were similar to the first (Fig. 1A). Paired-pulse responses were subsequently recorded 20 min after either P4 (Fig. 1A) or Dynasore application (Fig. 1B). The amplitude and the ratio of the EPSPs did not change with P4 (Fig. 1A). In contrast, with Dynasore the amplitude of the second EPSP decreased more than the first EPSP (with ISI = 50 ms: 14.7 ± 0.9 mV1st and 11.2 ± 0.7 mV2nd before Dynasore and 8.8 ± 0.5 mV1st and 5.0 ± 0.7 mV2nd after Dynasore; with ISI = 120 ms: 13.2 ± 0.9 mV1st and 14.7 ± 0.9 mV2nd before Dynasore and 9.3 ± 0.5 mV1st and 6.6 ± 1.0 mV2nd after Dynasore, mean ± S.E., n = 5; p < 0.05, paired Student t test) (Fig. 1B, panel b). Thus the paired-response ratio decreased with Dynasore (with ISI = 50 ms, from 0.62 ± 0.03 to 0.45 ± 0.06; with ISI = 120 ms, from 1.1 ± 0.03 to 0.70 ± 0.08; p < 0.05, paired t test) (Fig. 1B, panel c). These results suggest that dynamin dysfunction prevented replenishment of readily releasable SVs with an ISI of <120 ms, although the clathrin-mediated pathway might not function in replenishment of readily releasable SVs with an ISI of 10 Hz, in addition to dynamin-mediated endocytosis seen after a single action potential (Fig. 1B). It should be noted that, in addition to recycling from the plasma membrane, Dynasore will inhibit vesicle budding from sorting endosomes that supply de novo synaptic vesicles to the RRP (35Praefcke G.J. McMahon H.T. Nat. Rev. Mol. Cell. Biol. 2004; 5: 133-147Crossref PubMed Scopus (1112) Google Scholar). Interestingly, the train-evoked decrease in EPSP amplitude in the presence of Dynasore returned to the initial value 2 min after cessation of each train. Normalized first EPSP amplitudes recorded at 10, 20, and 30 Hz were ∼0.2 (Fig. 2, C, J, and L), suggesting a possible dynamin-independent pathway for replenishment of readily releasable SVs through the transport route from the RP to the RRP during the 2-min cessation of each train. We note that an incomplete effect of Dynasore or an effect of dynamin 3 at this synapse cannot be excluded with these data. However, inhibition of available dynamin 1 and 2 and most if not all components of endocytosis would be consistent with prior work employing similar concentrations of Dynasore (27Newton A.J. Kirchhausen T. Murthy V.N. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 17955-17960Crossref PubMed Scopus (192) Google Scholar, 28Xu J. McNeil B. Wu W. Nees D. Bai L. Wu L.G. Nat. Neurosci. 2008; 11: 45-53Crossref PubMed Scopus (67) Google Scholar). Replenishment of Readily Releasable SVs after Depletion of Releasable SVs—To test whether both dynamin-dependent and -independent pathways can replenish readily releasable SVs, SVs in the presynaptic terminals were depleted with 4-min trains of 5-Hz action potentials, and the recovery of EPSP amplitude was measured every 1 s. At the end of the train, the EPSP amplitude was within baseline noise levels (Fig. 3, A and B), and subsequently recovered at two distinct rates: fast and slow (see arrows in Fig. 3A, panel a). A control scrambled P4 peptide or DMSO did not show any reduction in the recovery rate (Fig. 3, A (panel b) and B (panel b)). In contrast, both P4 and Dynasore inhibited fast recovery (Fig. 3, A (panel a), B (panel a), and C). At 20 s after the train, the EPSP amplitude was significantly smaller than that before reagent applications (Fig. 3C, panel b). These results indicate that releasable SVs are depleted, while SVs in the RP may persist at the end of a 4-min train of action potentials. Furthermore, SVs in the RP may refill the RRP through the dynamin-dependent and clathrin-mediated pathway. In addition, readily releasable SVs may also be replenished through a dynamin-independent pathway in the presence of Dynasore. At 5 min after the train, the EPSP amplitude recovered to 41.4 ± 4.5% with P4, but remained at 12.3 ± 2.1% with Dynasore (Fig. 3C, panel b). The slow recovery could be described by a linear relationship (Fig. 3C, panel a). The slopes before and after P4 injection were 8.1 ± 1.7%/min and 6.7 ± 1.6%/min (p = 0.63, unpaired t test), whereas the slope with Dynasore was 0 ± 0.4%/min (Fig. 3C, panel a). These results demonstrate that the slow recovery rate was not significantly affected by P4 but blocked completely by Dynasore, suggesting the replenishment of readily releasable SVs through dynamin-dependent recycling (18Cremona O. De Camilli P. Curr. Opin. Neurobiol. 1997; 7: 323-330Crossref PubMed Scopus (194) Google Scholar) or de novo sorting via an endosomal pool (35Praefcke G.J. McMahon H.T. Nat. Rev. Mol. Cell. Biol. 2004; 5: 133-147Crossref PubMed Scopus (1112) Google Scholar). Together, the results suggest that the fast replenishment of the readily releasable SVs may involve SV transport from the RP via dynamin-mediated and non-dynamin-mediated pathway, whereas the slow replenishment of the readily releasable SVs may be achieved solely through dynamin-mediated endocytosis. Readily Releasable SVs during Low Frequency Repetitive Transmitter Release—To examine the role of the dynamin-mediated pathway in replenishment of readily releasable SVs during low frequency repetitive transmitter release, changes in the amplitude of EPSPs evoked by presynaptic action potentials at 0.2 or 0.05 Hz were measured (Fig. 4). P4 gradually reduced the EPSP amplitude at 0.2 Hz (Fig. 4A, panels a and c), but not at 0.05 Hz (Fig. 4A, panels b and c). At 40 min after P4 injection, reduction of EPSP amplitude was -47.9 ± 9.4% at 0.2 Hz (n = 7), and -11.7 ± 6.4% at 0.05 Hz (n = 4). This value was similar to the control value with the scrambled P4 peptide (-8.9 ± 4.9% at 0.2 Hz; n = 6) (Fig. 4A, panel d) In contrast, Dynasore reduced the EPSP amplitude at 0.2 and 0.05 Hz (Fig. 4B). The reduction rate was more rapid than that of P4 with 0.2 Hz stimuli (Fig. 4, A (panel a) versus B (panel a)). The decay time constant of the EPSP amplitude in the presence of Dynasore was 4.8 ± 0.12 min at 0.2 Hz, and 13.2 ± 0.17 min at 0.05 Hz (p < 0.01, unpaired t test) (Fig. 4B, panel d), whereas it was 28 ± 1.0 min at 0.2 Hz in the presence of P4. These results suggest that dynamin also mediates replenishment of readily releasable SVs during low frequency firing. Surprisingly, the EPSP amplitude was very small at 60 min after Dynasore application, but not completely blocked. The amplitudes were 6.8 ± 1.6% (at 0.2 Hz) and 6.8 ± 0.9% (at 0.05 Hz) of the initial value before Dynasore application, suggesting that non-dynamin-mediated processes may function in replenishing readily releasable SVs, although further experiments will be necessary to address this issue in future. In this study, we demonstrate that sympathetic neurons maintain synaptic transmission via the recycling of SVs through dynamin-mediated pathways during and after action potential activity. In addition, we provide evidence for a non-dynamin-mediated endocytic pathway, assuming that P4 and Dynasore blockade of dynamin-mediated recycling is complete. Refilling of the RRP via dynamin-mediated endocytic pathways was dependent on both rate and number of action potential firing (Figs. 2, 3, 4), in accord with activity-dependent recycling of synaptic vesicles observed at other synapses. In contrast, another mode of the RRP refilling through a dynamin-mediated endocytic pathway was activated independently of action potential firing rate and number (Figs. 1, 2, 3, 4), consistent with an activity-independent pathway. The third pathway, not affected by dynamin dysfunction, was also activated at all rates or numbers of firing tested (Figs. 3 and 4) and was thus activity-independent; it is estimated that 10% of SVs in readily releasable SVs were replenished via this pathway to maintain efficient synaptic vesicle recycling with long lasting repetitive firing of the SCG neuron. Compared with neurons in the central nervous system, sympathetic nerve fibers show relatively low firing activity in the 0.5- to 7.5-Hz range in vivo (36Janig W. Schmidt R.F. Pflugers Arch. 1970; 314: 199-216Crossref PubMed Scopus (45) Google Scholar). Thus, evidence for activity-dependent refilling of the RRP observed in this study may reflect physiological synaptic transmission in autonomic neurons in vivo. The kinetics of endocytosis is variable at different presynaptic terminals (15Royle S.J. Lagnado L. J. Physiol. 2003; 553: 345-355Crossref PubMed Scopus (108) Google Scholar, 16Wu L.G. Trends Neurosci. 2004; 27: 548-554Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). In hippocampal neurons, imaging studies have shown a wide range of time constants for endocytosis from a τ = 0.1-6 s for fast components (13Klingauf J. Kavalali E.T. Tsien R.W. Nature. 1998; 394: 581-585Crossref PubMed Scopus (344) Google Scholar, 15Royle S.J. Lagnado L. J. Physiol. 2003; 553: 345-355Crossref PubMed Scopus (108) Google Scholar) and τ = 4-90 s for slow clathrin-mediated endocytosis (15Royle S.J. Lagnado L. J. Physiol. 2003; 553: 345-355Crossref PubMed Scopus (108) Google Scholar). In Drosophila neuromuscular synapses, two pathways of vesicle recycling (37Koenig J.H. Ikeda K. J. Neurosci. 1989; 9: 3844-3860Crossref PubMed Google Scholar) and two or three SVs pools, the RRP and the RP (38Kuromi H. Kidokoro Y. Neuron. 1998; 20: 917-925Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar) or "immediately releasable pool" (39Delgado R. Maureira C. Oliva C. Kidokoro Y. Labarca P. Neuron. 2000; 28: 941-953Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar), were documented. During short period or low frequency presynaptic activity, SVs in the RRP, including the immediately releasable pool but not those in the RP, participate in transmitter release, whereas vesicles in the RP are required during intense neuronal activity (>10 Hz). A dynamin mutant, shibire, exhibits rapid synaptic fatigue within 0.02 s of repetitive stimulation, a phenotype that cannot be explained by vesicle depletion, suggesting that dynamin is required for rapid replenishment of the RRP with synaptic vesicles (40Kawasaki F. Hazen M. Ordway R.W. Neuron. 2000; 3: 859-860Google Scholar). In the present study, reduction of the second of two consecutive EPSPs with an ISI of 0.05 s by Dynasore (Fig. 1B) suggests that in sympathetic neurons dynamin is also required for rapid replenishment of readily releasable SVs, in addition to its role in slow clathrin-mediated endocytosis, which contributes to SVs recycling via the RP. Our data suggest approximate time constants for endocytosis in cultured sympathetic neurons. The dynamin-mediated pathway is able to mobilize SVs to the RRP in 10 Hz) stimulation, but not after cessation of the stimulus train. Dynamin 3 appears to share a similar role to dynamin 1. In contrast, ubiquitously expressed Dynamin 2 may play a role in slow activity-independent constitutive replenishment of clathrin-coated vesicles. Here we report that presynaptic terminals of a sympathetic neuron have two dynamin-mediated SV replenishment pathways, which differ in activity dependence. The relationship of these two recycling modes to specific dynamin isoforms will require further investigation. The differential effects of P4 peptide and Dynasore may be accounted for different steps in which they are likely to participate in the endocytic pathway. P4 disrupts the interaction of dynamin with amphiphysin (24Marks B. McMahon H.T. Curr. Biol. 1998; 8: 740-749Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar), which may interfere with formation of the clathrin-coat (21Takei K. Slepnev V.I. Haucke V. De Camilli P. Nat. Cell Biol. 1999; 1: 33-39Crossref PubMed Scopus (513) Google Scholar) or fission complex. On the other hand, Dynasore may act at a downstream step involving dynamin activity and subsequently provide constitutive inactivation of dynamin across the endocytic cycle. It is also possible that, by targeting different parts of the dynamin endocytic complex, P4 peptides and Dynasore may act on different time scales and efficacies and thus account for their differential effects. Clearly the two reagents provide unique insight into distinct roles of dynamin in endocytosis with respect to synaptic vesicle pool recovery during different patterns of action potential stimulation. We should caution that one cannot exclude incomplete inhibition of dynamin function by injected P4 peptides, which may be unable to fully target preassembled dynamin endocytic complexes due to steric hindrance and lower concentrations at nerve terminals. Because it was technically unfeasible to collect enough injected neurons for in vitro biochemical co-immunoprecipitation study, we are unable to provide experimental evidence to verify the assumption that injected P4 peptide could completely block the dynamin-amphiphysin interaction in our SCG neurons. Although the amphiphysin I/II knockout mice exhibit some defects in synaptic vesicle recycling (41Di Paolo G. Sankaranarayanan S. Wenk M.R. Daniell L. Perucco E. Caldarone B.J. Flavell R. Picciotto M.R. Ryan T.A. Cremona O. De Camilli P. Neuron. 2002; 33: 789-804Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar), its essential role in clathrin-mediated endocytosis remains further investigation. It is possible that P4 might target not only to amphiphysin but also to other SH3 domain-containing proteins (24Marks B. McMahon H.T. Curr. Biol. 1998; 8: 740-749Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar), thus playing a less specific role in blocking endocytosis than that by Dynasore. However, previous work in the SCG neuron system has indicated that injected peptides at even lower concentrations can disassemble other preformed synaptic protein complexes (42Mochida S. Sheng Z.H. Baker C. Kobayashi H. Catterall W.A. Neuron. 1996; 17: 781-788Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar). Furthermore, P4 peptides have the efficacy to block clathrin-dependent endocytosis at intracellular concentrations (25Shupliakov O. Low P. Grabs D. Gad H. Chen H. David C. Takei K. De Camilli P. Brodin L. Science. 1997; 276: 259-263Crossref PubMed Scopus (401) Google Scholar) that are comparable to those used in the current study, although it was injected into different neuronal types and the effect was measured by different readouts. Likewise, one must interpret the Dynasore inhibition data with caution. Dynasore is a fast-acting cell-permeable small molecule that inhibits the GTPase activity of dynamin 1, dynamin 2, and Drp1, the mitochondrial dynamin (43Kirchhausen T. Macia E. Pelish H.E. William E. Balch C.J.D. Alan H. Methods in Enzymology. 2008; (Academic Press, New York, pp.): 77-93Crossref PubMed Scopus (310) Google Scholar). Thus, it remains possible that in SCG neurons Dynasore may inhibit endocytosis dependent on dynamins 1 and 2 but not dynamin 3. Incomplete inhibition can also not be excluded without an independent measure of endocytosis inhibition. To address these issues, investigation of the role of dynamin isoforms in synaptic vesicle endocytic pathways in SCG neurons is under further study. However, it should be noted that robust inhibition of dynamin 1 and 2 and most if not all kinetic components of endocytosis is consistent with prior work employing similar concentrations of Dynasore (27Newton A.J. Kirchhausen T. Murthy V.N. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 17955-17960Crossref PubMed Scopus (192) Google Scholar, 28Xu J. McNeil B. Wu W. Nees D. Bai L. Wu L.G. Nat. Neurosci. 2008; 11: 45-53Crossref PubMed Scopus (67) Google Scholar). The results in the present study suggest a dynamin-independent pathway for SV recycling in a sympathetic neuron synapse. Recent studies at other synapses support this conclusion. SV endocytosis at the large presynaptic terminal of the calyx of Held is partially independent of dynamin (29Ma H. Mochida S. Neurosci. Res. 2007; 57: 491-498Crossref PubMed Scopus (28) Google Scholar). Endocytosis activated during intense stimulation persists in the calyx synapse dialyzing with Dynasore and PQVPSRPNRAP (called pp11, one amino acid longer than P4) (29Ma H. Mochida S. Neurosci. Res. 2007; 57: 491-498Crossref PubMed Scopus (28) Google Scholar). In peripheral dorsal root ganglion neurons, a calcium- and dynamin-independent form of rapid endocytosis, which is controlled by protein kinase A-dependent phosphorylation, has been described (44Zhang C. Xiong W. Zheng H. Wang L. Lu B. Zhou Z. Neuron. 2004; 42: 225-236Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). It will be of interest to further examine the molecular and physiological basis of vesicle fission via dynamin-independent endocytosis and its relationship to kiss-and-run and other proposed fast, non-classic modes of synaptic vesicle recycling. The synaptic short-term depression observed during repetitive neuronal firing may be attributed to multiple mechanisms, including a decrease in vesicle fusion probability, inactivation of voltage-gated Ca2+ channels (34Mochida S. Few A.P. Scheuer T. Catterall W.A. Neuron. 2008; 57: 210-216Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar), or use-dependent inhibition of the vesicle release machinery (33Kavalali E.T. J. Physiol. 2007; 585: 669-679Crossref PubMed Scopus (31) Google Scholar). In hippocampal or neocortical neurons, rapidly recycled SVs in the RRP are capable of rapid reuse (45Pyle J.L. Kavalali E.T. Piedras-Renteria E.S. Tsien R.W. Neuron. 2000; 28: 221-231Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar) and slow the rate of synaptic depression (46Ertunc M. Sara Y. Chung C. Atasoy D. Virmani T. Kavalali E.T. J. Neurosci. 2007; 27: 341-354Crossref PubMed Scopus (43) Google Scholar). Our study shows that synaptic transmission in cultured SCG neurons also decreases rapidly in response to repetitive action potential firing (Fig. 2). The decrease was strongly accelerated in the presence of Dynasore, suggesting that a rapid reduction in the number of vesicles available for fast release may contribute to synaptic depression. These results may suggest that dynamin-mediated pathways are critical to maintain baseline levels of neurotransmission. Together, our results along with other results in the literature indicate that distinct endocytic pathways may be engaged under distinct patterns of synaptic activity history, short- and long-term neuromodulation and cell type. For example, the release probability of presynaptic terminals (11Gandhi S.P. Stevens C.F. Nature. 2003; 423: 607-613Crossref PubMed Scopus (373) Google Scholar), Ca2+ and protein kinase activity may regulate the relative engagement of endocytotic pathways as a function of firing history and modulation (5Harata N.C. Aravanis A.M. Tsien R.W. J. Neurochem. 2006; 97: 1546-1570Crossref PubMed Scopus (153) Google Scholar, 13Klingauf J. Kavalali E.T. Tsien R.W. Nature. 1998; 394: 581-585Crossref PubMed Scopus (344) Google Scholar, 16Wu L.G. Trends Neurosci. 2004; 27: 548-554Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). In addition, dynamic engagement of multiple modes of recycling may be useful in homeostatic maintenance of a working amplitude and gain of synaptic transmission under different stimulation patterns. In summary, the present data characterize the fundamental modes of SV recycling and refilling of the RRP in SCG neurons during and after single or sustained firing of action potentials, supporting the involvement of activity-dependent and -independent pathways with distinct molecular requirements for synaptic vesicle endocytosis. We thank Dr. Tom Kirchhausen for the kind gift of Dynasore and Dr. Charles T. Yokoyama and Dr. Gary J. Stephens for comments on the manuscript. Download .pdf (.29 MB) Help with pdf files
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