Fig 1.
Reduce by 25% Examples of prespikes.
(A-B) Two examples of prespikes in simultaneous pre- and postsynaptic patch-clamp recordings. From top to bottom: calyx of Held AP, which was elicited by afferent stimulation, CC prespike, associated VC prespike. Recordings were made at room temperature in brainstem slices from 7–10 days old Wistar rats [36]. Horizontal scale bar: 0.5 ms. Vertical (from top to bottom): 50 mV, 2 mV, 1 nA. Arrows indicate the amplitudes that were quantified in (C). (C) Relation between the amplitude of the positive peak of the prespike measured in CC and the amplitude of the first negative peak of the prespike measured in VC in the same cell. Slope of the fitted line is -4.6 mV/nA. (D). Six examples of CC prespikes from in vivo recordings of a target neuron of the calyx of Held in 3–5 days old Wistar rats [37]. During this period the calyx of Held expands over the postsynaptic cell. Note the small upward deflection immediately preceding the EPSP in the bottom three prespikes (arrow heads) which, we will argue, arises from the presynaptic calcium current running through the synaptic cleft (see also Fig 5B). Scale bar indicates 0.5 mV and 0.5 ms.
Fig 2.
An electrical circuit for the prespike.
(A) Drawing of the calyx of Held synapse. The calyx of Held terminal (‘pre’) covers a large part of the soma of its target neuron (‘post’). In between is the synaptic cleft (not drawn to scale), a small space where membrane currents will flow during a presynaptic AP. (B) Electrical circuit to reproduce the prespike. Presynaptic membrane currents can leave the synaptic cleft via the cleft conductance (gcl) or through the postsynaptic cell. It is assumed that the conductance from the edge of the cleft to ground is relatively large compared to the cleft conductance itself [38,39].
Fig 3.
Relation between the calyceal AP and the prespike.
(A) An example of a train of presynaptic APs (vpre) elicited by afferent stimulation and the postsynaptic voltage-clamp recording (ipost). A large EPSC is elicited by the first AP, which rapidly depresses during the train. Stimulation artefacts were blanked. The first AP (blue dot) and the last AP (green dot) are shown on an expanded scale in B. Horizontal scale bar: 50 ms. Vertical scale bar: 90 mV, 1.5 nA. (B) The left and right column show the first and last AP of the train, respectively. From top to bottom: the AP recorded in CC from the calyx of Held, its inverted first (-AP’) and second derivative (-AP”), and postsynaptic voltage clamp recording showing the accompanying prespike (ipost). Vertical scale bars (from top to bottom panels): 40 mV, 0.3 kV/s, 2.5 MV/s2, 0.2 nA. Horizontal: 0.5 ms. (C) Relation between the delay between the first negative and positive peak of the prespike and the first two peaks in the first (blue, circle) and second (black, triangle) derivative of the presynaptic AP of the first AP-prespike pair in the train. Dashed line is the identity line. (D) Relation between the amplitude of the first negative and positive peak of the prespike and the first two peaks in the first (blue, circle) and second (black, triangle) derivative of the presynaptic AP of all AP-prespike pairs from an example train. The red lines indicate the linear regression lines. Note how the regression line crosses closer to the origin for the second derivative than for the first derivative. (E) Comparison for each train of Pearson’s r for the regression lines. (F) Comparison of the absolute deviation at 0 V/s (AP’) versus 0 V/s2 (AP”). (G) Cumulative distribution of the regression slope. Paired T-tests: ** p<0.01, *** p<0.001.
Fig 4.
Relation between the calcium current and the prespike.
(A) from top to bottom: The presynaptic voltage-clamp waveform (vpre), the inverted second derivative of the waveform (-vpre”), the presynaptic calcium current (iCa; after P/5 subtraction), its inverted first derivative (-iCa’), the active (black, thick) and passive prespike (ipost; magenta, P/5-scaled), and the calcium prespike which is the difference between the active and the passive prespike (Δipost). The passive prespike is generated by injecting the presynaptic AP at 1/5th of its amplitude. While this generates passive currents across the presynaptic cleft-facing membrane, it does not elicit the presynaptic calcium current. Voltage-gated sodium and potassium currents were blocked (see Materials and Methods). Vertical scale bars: 110 mV, 7 MV/s2, 2 nA, 15 nA/ms, 0.15 nA, 0.1 nA. Horizontal: 0.5 ms. (B) Relation between the inverted first derivative of the presynaptic calcium current and the calcium prespike (black dots). Maximum correlation was obtained by introducing a delay of 60 μs to the first derivative. The slope of the regression line (magenta) is shown in the bottom right corner.
Fig 5.
Impact on the prespike of voltage-gated ion channels located at the release face.
(A) Timing of different voltage-gated currents at the release face during the AP. Note that the amplitudes of each of the currents depend on cleft potential, thus creating a mutual dependency. Vertical scale bar: 60 mV, 2.4 nA, 1 nA, 1 nA, 3 nA. Horizontal: 0.5 ms. (B) The cleft potential (top), the VC prespike (middle), and the CC prespike (bottom) in the presence or absence of different voltage-dependent ion channels at the cleft. All present: homogeneous density for each channel; no VGSCs: no sodium conductance at the release face; only VGCCs: both calcium and capacitive currents at the release face; no channels: the capacitive-only model. The conductances that are present in the model are indicated by their currents at the top. Arrow head indicates the small deflection in the CC prespike that is generated by the calcium prespike. Scale bars: 4 mV, 0.2 nA, 2 mV.; 0.5 ms, except for the homogeneous model where the vertical scale bar corresponds to 1.6 nA.
Fig 6.
Potential impact of the cleft potential on presynaptic calcium currents.
(A) Simulation of the impact of a slow and a fast AP (top; FWHM: left 1.0, right 0.2 ms) on the cleft potential (second row), the transmembrane potential sensed by the calcium channels in the cleft (third row) and the presynaptic calcium current (bottom) at three leak resistances (1/gcl: 0 MΩ, 1 MΩ and 5 MΩ). Scale bar: 200 mV, 40 mV, 50 mV, 1 nA. (B) Relation between peak calcium currents and leak resistance for the two APs. Driving force slightly increases (not illustrated). (C) The change in delay between the calcium peak current and the AP for the two APs (solid line). The dashed line shows the delay after correcting for the decrease in the calcium current shown in B. Calcium conductance density was adjusted to 0.2 nS μm-2 and 2.8 nS μm-2 for the slow and fast AP, respectively, in order to elicit a 2 nA-current at 0 MΩ cleft leak resistance. The apposition area was 1000 μm2. Other conductances were set to 0 nS.
Fig 7.
Synapse geometry determines the profile of the cleft potential.
(A) Drawing of a radially-symmetric synapse with cleft height h and radius r. The capacitive currents will generate a cleft potential v(x) at location x. (B) Cleft voltage profile for synapses with different radii (color-coded) at the first peak in the capacitive current. The horizontal axis depicts the location from the center (0) relative to the edge (1.0). In (B), Rex is 100 Ω cm, Cm is 1 μF cm-2, and h is 30 nm.
Fig 8.
(A) Black traces: Spontaneous, sub- and suprathreshold events recorded during a whole-cell in vivo recording of a principal neuron in the MNTB from a P52 C57BL/6J mouse [58]. Events were aligned on the start of their EPSP and (<1 mV) differences in the resting membrane potential between events were subtracted for the display. Minimum interval between events was 20 ms. Red trace: average of >1000 events. Resting potential was -64 mV. Arrow points at the small prespike. Vertical scale bar: 10 mV. Horizontal: 1 ms. (B) As (A), showing close-up of the prespike (arrow). Vertical scale bar: 0.2 mV. Horizontal: 0.2 ms.
Table 1.
Rate constants for the voltage-gated channels.