3. Results
3.2. Morphological heterogeneity of the docked vesicle pool in mossy fiber synapses
3.2.6. Mossy fiber giant vesicles are the morphological correlate of giant mEPSCs
Although my data provide the first unequivocal evidence that giant vesicles dock in physical contact with mossy fiber active zones, their existence has been previously reported (Figure 16 A) (Borges-Merjane et al., 2020; Henze et al., 2002b; Laatsch and Cowan, 1966;
Rollenhagen et al., 2007). These observations became of particular importance upon the demonstration that large amplitude (giant) mEPSCs can be recorded from CA3 pyramidal neurons, and that the giant mEPSCs are monoquantal and of mossy fiber origin (Henze et al., 1997, 2002b). Based on the assumption that giant vesicles contain neurotransmitter and are capable of fusing at mossy fiber active zones to generate giant mEPSCs, I rationalized that the
81 relative distribution of mEPSC amplitudes should correlate with the relative proportion of docked giant vesicles visualized and quantified in electron tomograms. To this end, I collaborated with Dr. Bekir Altas, who used patch-clamp electrophysiology to isolate and record mEPSCs from CA3 pyramidal cells in cultured hippocampal slices at DIV14 (Figure 16 B). Initial baselines of all mEPSC events were recorded for a 5-minute period in the presence of TTX, to block sodium-propagated action potentials, and the GABA receptor blocker bicuculline, to exclude contributions from GABAergic transmission (Figure 16 B; black trace).
Subsequently, DCG-IV was applied to the bath solution and all DCG-IV-insensitive mEPSCs were recorded for the next 5-minute period (Figure 16 B; gray trace; 5-minute epoch was between the 10th and 15th minute after DCG-IV wash-on). Inhibition of mossy fiber synaptic transmission with DCG-IV reduced the frequency of mEPSC events regardless of amplitude (Figure 16 C). To test whether docked giant vesicles are neurotransmitter-filled, I next compared the frequency of giant mEPSCs to the proportion of docked giant vesicles. To correlate the proportion of giant mEPSC events with the relative proportion of docked giant vesicles at mossy fiber active zones, I subtracted the amplitudes of DCG-IV-insensitive events from the baseline mEPSCs to get an estimate of the DCG-IV-sensitive amplitudes (Figure 16 D, purple line). I then rationalized that the most frequently observed DCG-IV-sensitive mEPSC amplitudes (statistical mode of DCG-IV-sensitive mEPSC amplitudes = 10 pA) reflected fusion and transmitter release from docked vesicles with the most frequently observed dimensions (the statistical mode of docked synaptic vesicle diameters = 44 nm). I calculated the inner lumenal volume of a synaptic vesicle with an outer diameter of 44 nm, accounting for the lipid bilayer (~4 nm of radius measured from tomograms; ~24,400 nm3 lumenal volume). I then calculated the theoretical mEPSC amplitude that would arise from the fusion of a vesicle at the lower threshold for classification as a giant vesicle (Ø=60 nm; ~ 30 pA; Figure 16 D dotted line). Approximately 27% of DCG-IV-sensitive mEPSCs were larger than 30 pA, in agreement with my finding that approximately 20% of all docked vesicles are giant vesicles in age-matched mossy fiber synapses (Figure 16 C). It is important to note that my calculations are based on several assumptions: i) DCG-IV-sensitive mEPSC events originated from docked
82 synaptic vesicles at mossy fiber synapses; ii) synaptic vesicle filling was proportional to the size of a given vesicle (Bruns et al., 2000); and iii) postsynaptic receptor saturation was negligible.
The amplitudes of the remaining DCG-IV-insensitive mEPSCs were reduced compared to amplitudes of all mEPSCs (Figure 16 D). DCG-IV caused a significant reduction in the mEPSC frequency in CA3 pyramidal neurons compared to all mEPSC events before the application of DCG-IV (Figure 16 E; 1.376 ± 0.273 events/sec, TTX/BIC; 0.60 ± 0.186 events/sec, TTX/BIC/DCG-IV; p<0.001). The median amplitude of mEPSC events recorded in CA3 pyramidal neurons was significantly reduced after DCG-IV application (Figure 16 F; 16.25 ± 1.031 pA, TTX/BIC; 13.0 ± 0.524 pA, TTX/BIC/DCG-IV; p=0.007). The specific inhibition of mossy fiber synaptic transmission with DCG-IV demonstrates that 41% of mEPSCs onto CA3
Figure 16. Electrophysiological and morphological analysis of giant vesicles in mossy fiber synapses.
(A) Tomographic subvolume of a mossy fiber synapse with a giant vesicle docked directly at the active zone membrane. (B-G) Effects of DCG-IV on mEPSC events recorded in CA3 pyramidal neurons in hippocampal slice cultures at DIV14. (B) Example traces of mEPSCs recorded from CA3 pyramidal neurons in the presence of 1 µM TTX and 10 µM bicuculline (BIC) before (black trace) and after the wash on of 2 µM DCG-IV (grey trace). (C) Frequency distribution of mEPSC amplitudes recorded in CA3 pyramidal neurons before (black) and after (gray) application of DCG-IV (2 pA bins from 8 pA until 100 pA and all events greater than 100 pA are pooled in one bin). (D) Cumulative distribution of mEPSC amplitude before and after application of DCG-IV. (E) Before-after scatter plot of mEPSC frequency before (T/B) and after (T/B/D) application DCG-DCG-IV. (F) Before-after scatter plot of median mEPSC amplitude before and after the application of DCG-IV. (G) Relative changes in the median amplitude and frequency after the application of DCG-IV normalized to TTX/BIC.
Statistical significance is represented as *, p<0.05; **, p<0.005; ***, p<0.001. N=2 cultures; n=28 cells. Scale bar: 100 nm, A. See Table 29 for full statistical analysis.
83 pyramidal neurons are insensitive to DCG-IV (Figure 16 G; 40.75 ± 3.20%), and likely arise from excitatory collaterals from other CA3 pyramidal neurons described to form in rat slice cultures (Frotscher and Gähwiler, 1988). Furthermore, the median amplitude of DCG-IV-insensitive events was reduced to about 82% of all mEPSC events measured prior to the application of DCG-IV (Figure 16 G; 82.43 ± 3.939%), meaning that many but not all giant mEPSC events were sensitive to DCG-IV. It is unclear the extent at which DCG-IV inhibits spontaneous vesicle fusion at hippocampal mossy fiber synapses however these results are in agreement with the reduction in mEPSC events after gamma-irradiation of hippocampal granule cells (Henze et al., 1997).
My findings indicate a close correlation between the relative proportions of DCG-IV-sensitive giant mEPSCs and docked giant vesicles at mossy fiber synapses, thereby supporting the hypothesis that giant vesicles contribute to glutamatergic signaling at mossy fiber-CA3 synapses. A potential contribution of multivesicular release events to the larger amplitude mEPSCs recorded in CA3 pyramidal neurons cannot be completely excluded. My data support the notion that giant vesicles indeed contain neurotransmitter and that they have the potential to profoundly influence synaptic transmission at mossy fiber-CA3 synapses (Henze et al., 2002b). I therefore investigated whether changes in synaptic strength correlate with corresponding changes in the numbers of docked, and presumably fusion-competent, giant vesicles.