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REPORT
Department of Neurology, Wheeler Center for the Neurobiology of Addiction and the Ernest Gallo Clinic and Research Center, University of California San Francisco, Emeryville, California
Submitted 1 November 2005; accepted in final form 6 December 2005
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ABSTRACT |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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; Rahamimoff and Yaari 1973). Asynchronous release is enhanced after bursts of spikes, presumably because of a build-up of residual calcium in the synaptic terminal. However, many questions persist about the mechanisms of asynchronous release as well as its relationship to synchronous release, including whether the two processes share the same readily releasable pool (RRP) of vesicles and whether asynchronous release interacts with short-term plasticity. These issues are especially pertinent in light of recent data indicating the spontaneous release and synchronous release use separate pools of vesicles (Sara et al. 2005).
Many studies investigating the mechanisms of asynchronous release at excitatory central synapses have been performed on cultured hippocampal neurons (Goda and Stevens 1994; Hagler and Goda 2001; Otsu et al. 2004), in part because they show a greater degree of asynchronous release than excitatory synapses in neurons recorded from these same synapses in situ. To explore these issues in situ, we have looked at the interaction between asynchronous release and short-term depression at the corticostriatal synapses in the nucleus accumbens slice, which show considerable asynchronous release after a spike train (Lape and Dani 2004). At this synapse, synchronous release and asynchronous release are positively correlated and do not seem to compete for a common set of vesicles.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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3°C) containing (in mM) 119 NaCl, 2.5 KCl, 1.3 MgSO4, 1.0 NaH2PO4, 2.5 CaCl2, 26.2 NaHCO3 and 11 glucose saturated with 95% O2-5% CO2. Coronal slices (350 µm thick) containing the nucleus accumbens (NAc) were cut using a vibratome (Leica Instruments). Slices were submerged in Ringer solution and allowed to recover for >1 h.
Individual slices were transferred to a poly-D-lysine-coated coverslip and visualized under an Olympus Optical (Melville, NY) upright microscope with differential interference contrast optics and infrared illumination. Slices were perfused with Ringer solution at 27°C. Whole cell patch-clamp recordings were made from medium spiny neurons using 2.5- to 4-M
pipettes containing (in mM) 123 Cs-gluconate, 10 HEPES, 0.2 EGTA, 8 NaCl, 2 MgATP, and 0.3 Na3GTP (pH 7.2, osmolarity adjusted to 280). Neurons were voltage-clamped at –80 mV, and trains (25 Hz) of excitatory postsynaptic currents (EPSCs) were evoked each 15 s with a bipolar stimulating electrode placed in the NAc shell dorsal to the recording site.
Recordings were made using an Axopatch 1-D (Axon Instruments, Foster City, CA) amplifier and were filtered at 2 kHz and collected at 5 kHz using Igor Pro (Wavemetrics, Lake Oswego, OR). Series resistance was monitored on-line by measuring the peak of the capacitance transient in response to a –4 mV voltage step applied before each stimulus. Synchronous release was calculated by measuring the integrated current for a 20-ms period after the onset of the EPSC compared with baseline calculated from a 1-ms period measured just before the stimulus artifact. Asynchronous release was calculated as by measuring the integrated current for the period 50–100 ms after the final EPSC of the train. For determining the proportion of asynchronous release during an EPSC, asynchronous release was calculated as the total charge transfer during the 20-ms period – the synchronous release. Responses in drug conditions were calculated 10–15 min after bath application. Some of the data in this paper was obtained from recordings used for previously described experiments (Hjelmstad 2004). Unless otherwise noted, statistical analyses were performed using the Student's t-test, and significance was defined at P < 0.05. Results are presented as means ± SE.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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). In addition, trains of stimulation also produce asynchronous or delayed release. At low stimulus strengths, individual quantal events can clearly be distinguished (Fig. 1A). Increasing the stimulus strength, which recruits more synapses, obscures the individual quanta (Fig. 1B). However, the magnitude of asynchronous release can be measured and compared with synchronous release by measuring total charge transfer either during an EPSC or after the end of the train. Figure 1C shows that increasing the stimulus intensity produces a linear increase in asynchronous release (n = 4). Likewise, this same increase in stimulus strength had no effect on the short-term depression of synchronous release (Fig. 1D). These data argue against a significant role for any stimulus intensity–dependent heterosynaptic processes on either short-term depression or asynchronous release at this synapse.
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40% of the total release that occurred during the first 20 ms after the eighth stimulus (42 ± 2%, n = 26). If both synchronous and asynchronously released vesicles arise from the same release pool, the two processes should compete for the same vesicles. That is, more asynchronous release should produce more depletion resulting in more short-term depression (i.e., a smaller synchronous response). This was tested by applying the membrane-permeant slow calcium buffer EGTA-AM (200 µM; Fig. 2A). EGTA-AM decreases asynchronous release by buffering the presynaptic calcium transient, but, because of its kinetics, has only a small effect on the probability of synchronous release (Borst and Sakmann 1996; Castillo et al. 1996). Interestingly, despite a significant decrease in asynchronous release (30 ± 10%, P < 0.05), the eighth EPSC decreased after the application of EGTA-AM, and short-term depression was unaffected by the buffer (Fig. 2B).
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The relationship between synchronous and asynchronous release was studied further by examining the effects of changing release probability on the two processes. Lowering external calcium from 2.5 to 1.0 mM caused a 33 ± 10% reduction in the probability of release (determined by the change in charge transfer of the initial EPSC; Fig. 3A). On the other hand, the size of the eighth EPSC was unaffected by lowering calcium (2 ± 3%). Although the steady-state synchronous release at the end of the train was unaffected, asynchronous release measured after the train was inhibited by 27 ± 4%.
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). Again, asynchronous release paralleled the overall change in release probability (41 ± 4 vs. 43 ± 7%; Fig. 3C). Thus a number of manipulations provide similar results: asynchronous release correlates with the change in the initial response but not with the change in the steady-state synchronous response (Fig. 3, D and E). |
DISCUSSION |
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There is sufficient asynchronous release after a stimulus train to expect significant interactions between synchronous and asynchronous release, assuming that they indeed share the same readily releasable pool. Studies from cultured hippocampal neurons indicate that synchronous and asynchronous release compete for a common set of vesicles. Specifically, EGTA-AM decreases asynchronous release and at the same time increases synchronous release—that is, decrease the magnitude of short-term depression (Hagler and Goda 2001; Otsu et al. 2004). Here, EGTA-AM had no significant effect on short-term depression, although asynchronous release was decreased after application of the buffer. While this discrepancy between the NAc slice and hippocampal cultures could be caused by a difference in the mechanisms for asynchronous release, it might instead reflect different processes underlying the short-term depression at these two synapses. Consistent with this, high-frequency stimulation reduces synchronous release to nearly zero in hippocampal cultures, but not in the NAc slice. In addition, other calcium-sensitive mechanisms, such as calcium-dependent reloading of the RRP (Dittman and Regehr 1998) might counteract the effects of reducing asynchronous release with EGTA-AM.
Lowering probability of release had little effect on the steady-state synchronous responses while inhibiting asynchronous release to a degree similar to the overall probability of release. Based on the depletion model of short-term depression, the lack of effect on steady-state responses has been attributed to a larger available RRP, because vesicles are not being released during earlier stimuli (Zucker and Regehr 2002). However, if this is the case, and if asynchronous release shares the same pool, one might expect asynchronous release to also be unaffected. Previous data at the parallel fiber-stellate cell synapse in the cerebellum showed that the overall magnitude of asynchronous release is less sensitive than synchronous release to changes in extracellular Ca2+ (Atluri and Regehr 1998). However, these data imply that, if one accepts the depletion model of depression and assumes a common vesicle pool for both synchronous and asynchronouse release, the actual probability of release for an asynchronous vesicle has a greater sensitivity to both changes in calcium influx as well as to dopamine, which acts downstream of calcium entry at this synapse (Nicola and Malenka 1997). This is because the change in asynchronous release would occur despite a larger vesicle pool. Alternatively, if asynchronous release arises from an independent vesicle pool or if short-term depression is not be caused by depletion, these results are not unexpected.
The positive correlation between the release during a train and the asynchronous release after the train strengthens the evidence that synchronous and asynchronous release do not compete for a common set of vesicles, which would predict a negative correlation. Importantly, these data also argue that the two processes are coupled—that is, if they were independent, one would expect no correlation at all.
There are two plausible explanations for this positive correlation. First, asynchronous release may arise from a separate, but linked pool of vesicles, such as a "reloading" pool. Under this scenario, trials with greater release will produce a larger pool of reloading vesicles, resulting in more asynchronous release. This scenario is supported by the synaptotagmin knock-out mouse, where synchronous release is absent but slow asynchronous release and spontaneous release remains (Geppert et al. 1994; Nishiki and Augustine 2004). Thus asynchronous release might be accounted for by vesicles that have docked but have not yet bound to synaptotagmin.
Alternatively, if the variability in the response to the train is caused by variability in calcium influx, we would expect to see more asynchronous release during trials with high calcium influx (corresponding to more release during the train). The hypothesis is appealing because of a recent report at the Calyx of Held arguing that short-term depression is caused by calcium channel inactivation as opposed to depletion (Xu and Wu 2005).
What is the functional impact of asynchronous release? Often, asynchronous release is dismissed as a cost of having a highly tuned synapse. However, recent reports point to a role for asynchronous GABA release in smoothing synaptic responses during high-frequency transmission (Lu and Trussell 2000) and may be involved in gain-control mechanisms (Hefft and Jonas 2005) In addition, asynchronous release can act as a memory trace, keeping a synapse active for hundreds of milliseconds after a spike train. In depolarized neurons, this asynchronous activity would activate N-methyl-D-aspartate (NMDA) receptors, and thus could influence long-term synaptic plasticity such as long-term potentiation (LTP) and long-term depression (LTD).
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GRANTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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FOOTNOTES |
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Address for reprint requests and other correspondence: G. Hjelmstad, Ernest Gallo Clinic and Research Ctr., 5858 Horton St., Suite 200, Emeryville, CA 94608 (E-mail: gregh{at}egcrc.net)
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REFERENCES |
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Barrett EF and Stevens CF. The kinetics of transmitter release at the frog neuromuscular junction. J Physiol 227: 691–708, 1972.
Borst JG and Sakmann B. Calcium influx and transmitter release in a fast CNS synapse. Nature 383: 431–434, 1996.[CrossRef][Medline]
Castillo PE, Salin PA, Weisskopf MG, and Nicoll RA. Characterizing the site and mode of action of dynorphin at hippocampal mossy fiber synapses in the guinea pig. J Neurosci 16: 5942–5950, 1996.
Dittman JS and Regehr WG. Calcium dependence and recovery kinetics of presynaptic depression at the climbing fiber to Purkinje cell synapse. J Neurosci 18: 6147–6162, 1998.
Geppert M, Goda Y, Hammer RE, Li C, Rosahl TW, Stevens CF, and Südhof TC. Synaptotagmin I: a major Ca2+ sensor for transmitter release at a central synapse. Cell 79: 717–727, 1994.[CrossRef][ISI][Medline]
Goda Y and Stevens CF. Two components of transmitter release at a central synapse. Proc Natl Acad Sci USA 91: 12942–12946, 1994.
Hagler DJ Jr and Goda Y. Properties of synchronous and asynchronous release during pulse train depression in cultured hippocampal neurons. J Neurophysiol 85: 2324–2334, 2001.
Hefft S and Jonas P. Asynchronous GABA release generates long-lasting inhibition at a hippocampal interneuron-principal neuron synapse. Nat Neurosci 8: 1319–1328, 2005.[CrossRef][ISI][Medline]
Hjelmstad GO. Dopamine excites nucleus accumbens neurons through the differential modulation of glutamate and GABA release. J Neurosci 24: 8621–8628, 2004.
Lape R and Dani JA. Complex response to afferent excitatory bursts by nucleus accumbens medium spiny projection neurons. J Neurophysiol 92: 1276–1284, 2004.
Lu T and Trussell LO. Inhibitory transmission mediated by asynchronous transmitter release. Neuron 26: 683–694, 2000.[CrossRef][ISI][Medline]
Nicola SM and Malenka RC. Dopamine depresses excitatory and inhibitory synaptic transmission by distinct mechanisms in the nucleus accumbens. J Neurosci 17: 5697–5710, 1997.
Nishiki T and Augustine GJ. Synaptotagmin I synchronizes transmitter release in mouse hippocampal neurons. J Neurosci 24: 6127–6132, 2004.
Otsu Y, Shahrezaei V, Li B, Raymond LA, Delaney KR, and Murphy TH. Competition between phasic and asynchronous release for recovered synaptic vesicles at developing hippocampal autaptic synapses. J Neurosci 24: 420–433, 2004.
Rahamimoff R and Yaari Y. Delayed release of transmitter at the frog neuromuscular junction. J Physiol 228: 241–257, 1973.
Sara Y, Virmani T, Deak F, Liu X, and Kavalali ET. An isolated pool of vesicles recycles at rest and drives spontaneous neurotransmission. Neuron 45: 563–573, 2005.[CrossRef][ISI][Medline]
Xu J and Wu LG. The decrease in the presynaptic calcium current is a major cause of short-term depression at a calyx-type synapse. Neuron 46: 633–645, 2005.[CrossRef][ISI][Medline]
Zucker RS and Regehr WG. Short-term synaptic plasticity. Annu Rev Physiol 64: 355–405, 2002.[CrossRef][ISI][Medline]
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