Multiple vesicle recycling pathways in central synapses and their impact on neurotransmission

Abstract

Short-term synaptic depression during repetitive activity is a common property of most synapses. Multiple mechanisms contribute to this rapid depression in neurotransmission including a decrease in vesicle fusion probability, inactivation of voltage-gated Ca(2+) channels or use-dependent inhibition of release machinery by presynaptic receptors. In addition, synaptic depression can arise from a rapid reduction in the number of vesicles available for release. This reduction can be countered by two sources. One source is replenishment from a set of reserve vesicles. The second source is the reuse of vesicles that have undergone exocytosis and endocytosis. If the synaptic vesicle reuse is fast enough then it can replenish vesicles during a brief burst of action potentials and play a substantial role in regulating the rate of synaptic depression. In the last 5 years, we have examined the impact of synaptic vesicle reuse on neurotransmission using fluorescence imaging of synaptic vesicle trafficking in combination with electrophysiological detection of short-term synaptic plasticity. These studies have revealed that synaptic vesicle reuse shapes the kinetics of short-term synaptic depression in a frequency-dependent manner. In addition, synaptic vesicle recycling helps maintain the level of neurotransmission at steady state. Moreover, our studies showed that synaptic vesicle reuse is a highly plastic process as it varies widely among synapses and can adapt to changes in chronic activity levels.

Figure 1. Estimation of the time course of vesicle reuse from simultaneous electrical recordings and fluorescence destaining

A, following fusion synaptic vesicles are retrieved and refilled with neurotransmitter (left). Therefore, electrical recordings of neurotransmitter release report the sum of release events originating from fresh vesicles that have not fused before and recycled vesicles refilled with neurotransmitter. In contrast, FM dye-filled vesicles cannot be refilled with dye during recycling (right). Therefore, FM dye destaining can report fusion of a particular vesicle only once as long as all FM dye leaves a vesicle upon fusion. B and C, whole-cell electrical recording of a sucrose response (B) and its instantaneous fluorescence counterpart from multiple boutons on the same neuron (C). D, average fluorescence response (F, grey line) was smoothed by curve fitting (dashed line) to reduce noise, which in turn helped obtain a smooth derivative of the fluorescence signal (dF/dt, smooth line). E, the difference between the rate of dye release and synaptic activity was assessed after alignment of the dF/dt and Current plots with respect to their peaks. Current plot was obtained by integrating current within 1 s intervals. The difference shown in the bottom graph was interpreted as the time course of vesicle reuse (from Sara et al. 2002; copyright 2002 by the Society for Neuroscience).

Figure 2. Effect of synaptotagmin 7 overexpression on synaptic responses detected electrophysiologically

A, the role of synaptotagmin 7 splice variants in directing vesicles towards kinetically distinct recycling pathways. The short splice variant of synaptotagmin 7 (Sy7-B) lacking the two C2 domains directs vesicles towards a fast recycling pathway. The full-length splice variant, synaptotagmin 7-A, targets vesicles towards slower trafficking pathways. These include classical clathrin-mediated endocytosis of individual vesicles, as well as slower pathways uncovered by synaptotagmin 7-A overexpression where vesicles bud off membrane infoldings and/or endosomal cisternae. B, representative whole-cell recordings in neurons from cultures transfected at high efficiency with regular synaptotagmin 7 (Syt7-A), short synaptotagmin 7 (Syt7-B) and two control proteins, synaptotagmin 1 (Syt 1) and ECFP alone. Recordings were made during 10 Hz stimulation from high-density cultures with each signal resulting from multiple synapses per neuron; only the first and last 20 responses during a total of 900 stimuli are shown. C, average normalized response amplitudes from cells stimulated at 10 Hz by field electrodes. Each point represents the average of 10 consecutive responses. Syt7-B overexpression results in a higher steady-state plateau of response amplitudes. In contrast, Syt7-A overexpression results in faster depression of the initial responses (P < 0.02; n = 12) as well as a lower response amplitude at steady-state compared with both controls (P < 0.03; n = 11–12) and Syt7-B (P < 0.001; n = 11, Student's two-tailed t test) overexpression (modified from Virmani et al. 2003; copyright 2003 by the authors).

Figure 3. Spontaneous network activity and the dynamics of presynaptic vesicle trafficking in neocortical and hippocampal cultures

A, spontaneous network activity in neocortical and hippocampal cultures. Sample traces of spontaneous network activity recorded from neurons in neocortical cultures (upper two traces) and hippocampal cultures (lower two traces). B, current-clamp analysis of spontaneous network activity in neocortical and hippocampal cultures. Sample traces of action potentials and excitatory postsynaptic potentials recorded from neurons in neocortical cultures (upper traces) and hippocampal cultures (lower traces). Arrows point to subthreshold synaptic activity. C, sample traces of synaptic depression recorded from hippocampal (upper traces) and neocortical (lower traces) neurons in culture during 10 Hz stimulation (the first 10 responses and the 100th to 110th response are shown). D, traces showing the average synaptic depression from a number of cells normalized to the amplitude of the first response (n = 21 cells and 8 cells for neocortex and hippocampus, respectively). Neocortical neurons depress significantly faster than hippocampal neurons in response to 10 Hz stimulation with significance (P < 0.01) emerging by the 5th response. E, treatment with cyclothiazide (CTZ) to block AMPA receptor desensitization slightly (grey symbols) decreased the time constant of rapid depression in both neocortical (n = 4) and hippocampal (n = 8) cultures but maintained the difference in the rates of depression between the two cultures. F, neocortical and hippocampal synapses have distinct kinetics of synaptic vesicle recycling. Synapses were loaded with FM2-10 using 1200 APs delivered at 10 Hz through field electrodes and after a brief wash were destained by 10 Hz stimulation for 90 s followed by multiple rounds of 90 mm K⁺ to maximally destain the synapses. The total recycling pool size was measured by loading synapses using 1200 APs delivered at 10 Hz and following extracellular dye washout, destaining the synapses using 90 mm K⁺. Hippocampal synapses showed a larger recycling pool size (P < 0.05, n = 5 samples each). G, average dye destaining curves from a number of experiments show that neocortical synapses destain slower than hippocampal synapses that was significant after 20 s of stimulation (P < 0.05 (two tailed t test), n = 5 samples each). The baseline by 90 s was not significantly different (P = 0.2). H, vesicle reuse was measured using a pulse chase experiment. The plot shows the average percentage of reused vesicles for neocortical and hippocampal synapses as a function of the time of continued stimulation (Δt). With this protocol neocortical synapses show significantly faster vesicle reuse than hippocampal synapses (n = 550–800 synapses from 6 to 7 samples for each time point per condition; significance was determined by applying a stringent value of P < 10–8 using the Kolmogorov–Smirnov (K-S) test) (modified from Virmani et al. 2006; copyright 2006 by the Society for Neuroscience).

Figure 4. Folimycin treatment induces frequency-dependent depression of synaptic responses at excitatory synapses in the CA1 region of hippocampal slices

A, bar graphs and representative traces of the average amplitude of the first evoked responses to the stimulation trains. There was a small but statistically insignificant decrease in the amplitudes of the first evoked responses recorded from folimycin-treated cells. B–D, blockade of vesicle refilling evaluated by synaptic depression at 1 Hz (B), 20 Hz (C) and 30 Hz (D) electrical stimulation after 10 min of folimycin exposure. Folimycin treatment hastened synaptic depression at excitatory synapses in higher frequencies with an early onset. Insets show the first and last 5 AP evoked EPSCs at 1 Hz, and the first and last 10 responses for 20 Hz and 30 Hz of continuous stimulation from individual recordings (the top trace for each experimental protocol represents controls and the bottom trace folimycin-treated cells). Folimycin-treated slices respond to stimulation with a continuous decline in neurotransmitter release with a faster rate of depression than that recorded in controls (P < 0.05, n = 5–7 cells for each experimental protocol and group). The decline was more prominent at higher frequencies in the folimycin-treated group. Data shown are mean ± s.e.m. (modified from Ertunc et al. 2007; copyright 2007 by the Society for Neuroscience).

Figure 5. A simple model for the frequency dependence of synaptic vesicle reuse

A, the figure depicts the kinetic model we used to account for our observations. In this model, synapses initially occupy the ‘rest’ state and transition to the ‘reuse’ state in response to stimulation with a rate α. Synapses transition from the reuse state to a state of ‘exhaustion’ with a rate β. The transition rates between the states (α and β) depend on stimulation frequency. B–E, the difference between folimycin-treated and control depression profiles at 1 and 30 Hz stimulations (including data from Fig. 4) plotted with respect to the number of action potentials for excitatory (B and D) and inhibitory (C and E) synapses (^). Continuous lines depict the progression of the reuse state during stimulation estimated by fitting the model to the data by minimizing the mean square of the error. The occupancy of reuse state was calculated by solving the following first order equations: d(rest)/dn =−α(rest), d(reuse)/dn =α(rest) −β(reuse), d(exhaustion)/dn =β(reuse) where n denotes the number of action potentials. F, in the case of excitatory synapses the rates α and β increase monotonically in response to the increase in the frequency of stimulation. G, in inhibitory synapses, the same rates plateau after 10 Hz (modified from Ertunc et al. 2007; copyright 2007 by the Society for Neuroscience).