Neurexins cluster Ca2+ channels within the presynaptic active zone

Abstract

To achieve ultrafast neurotransmission, neurons assemble synapses with highly organized presynaptic and postsynaptic nanomachines that are aligned by synaptic adhesion molecules. How functional assembly of presynaptic active zones is controlled via trans-synaptic interactions remains unknown. Here, we conditionally deleted all three neurexin adhesion molecules from presynaptic neurons of the calyx of Held in the mouse auditory system, a model synapse that allows precise biophysical analyses of synaptic properties. The pan-neurexin deletion had no effect on synapse development or the basic release machinery, but dramatically impaired fast neurotransmitter release. The overall properties of presynaptic calcium ion channels appeared normal, as reflected by the similar characteristics of calcium currents recorded at the nerve terminals. However, the pan-neurexin deletion significantly impaired the tight coupling of calcium influx to exocytosis, thereby suppressing neurotransmitter release. Furthermore, the pan-neurexin deletion reduced the function of calcium-activated BK potassium channels, whose activation depends on their tight association with presynaptic calcium channels. Together, these results suggest that neurexins perform a major function at the calyx synapse in coupling presynaptic calcium channels to release sites.

Keywords: active zone; adhesion molecules; calcium channels; neurexins; synapse formation.

Figure 1. Neurexins are not required for synapse formation at the calyx of Held

1. A

   Diagram of the calyx of Held synapse.

2. B

   Strategy for selective deletion of all neurexins at the calyx of Held by crossing PV‐Cre driver mice with neurexin‐1/2/3 (Nrxn123) triple conditional knockout (TKO) mice (Zhang et al, 2017; Chen et al, 2017).

3. C

   Representative images of calyx synapses from littermate control and Nrxn123 TKO mice. Brainstem sections were labeled with antibodies to vGluT1 (red) and Syt2 (green). Scale bar, 10 μm.

4. D

   Summary graphs of the synaptic vGluT1 and Syt2 immunostaining intensity (normalized to control).

5. E–H

   Representative traces of spontaneous EPSCs (sEPSCs) (E), and summary graphs of the sEPSC frequency (F), amplitude (G), and kinetics (H) recorded from littermate control and Nrxn123 TKO mice.

Data information: Data in (D, F–H) are means ± SEM with individual data points. Number of image sections/mice (D) or of cells from at least three mice per group (F–H) analyzed are indicated in the bars. No statistical differences were found by Student's t‐test.

Figure EV1. Neurexins are not required for synapse formation at the calyx of Held as evidenced by a lack of change in the signals for presynaptic vesicle markers (vGluT1 and vGAT) or postsynaptic AMPA‐type glutamate receptors (GluA1) after complete deletion of all neurexins in conditional Nrxn123 triple KO mice (TKO) using a Pv‐Cre driver line (related to Fig 1)

1. Representative brainstem sections containing the MNTB from littermate conditional Nrxn123 TKO mice that either do not express Cre‐recombinase (control), or that harbor a parvalbumin‐Cre (Pv‐Cre) allele driving expression of Cre‐recombinase in both pre‐ and postsynaptic neurons of the calyx of Held synapse (Nrxn123 TKO; see Zhang et al, 2017). Sections were labeled with antibodies to vGluT1 (red) and GluA1 (green). Scale bar, 10 μm.

2. Summary of vGluT1 and GluA1 immunostaining intensity (normalized to control).

3. Brainstem sections containing the MNTB from control and Nrxn123 TKO mice that were labeled with antibodies to vGluT1 (red) and vGAT (green). Scale bar, 10 μm.

4. Summary of vGluT1 and vGAT immunostaining intensity (normalized to control).

Data information: Data in (B) and (D) are means ± SEM. Number of image sections/mice analyzed are indicated in the bars (B and D); no statistically significant differences were observed as assessed by Student's t‐test.

Figure EV2. Deletion of all neurexins using Pv‐Cre driver mice has no major impact on the expression of key synaptic proteins in the dissected MNTB (related to Fig 1)

1. Diagram of dissecting MNTB nucleus for immunoblot analysis.

2. Example images of immunoblots of synaptic proteins.

3. Summary graphs of protein levels (normalized to Tuji of control mice).

Data information: Data are means ± SEM (n = 4 mice/group); no statistically significant differences were observed as assessed by Student's t‐test.

Figure 2. Pan‐neurexin deletion severely impairs evoked synaptic transmission at calyx synapses

1. A

   Representative traces of EPSCs evoked by paired stimuli separated by 10 ms and repeated every 20 s, recorded in a standard bath solution containing 2 mM Ca²⁺.

2. B

   Pan‐neurexin deletion suppresses synaptic transmission. Summary graphs show the amplitude (left) and charge transfer (right) of the first EPSC recorded in response to the paired stimuli.

3. C

   Pan‐neurexin deletion more than doubles the paired‐pulse ratio (PPR), suggesting a large decrease in release probability.

4. D

   Pan‐neurexin deletion decelerates the EPSC time course. Summary graphs show the rise time (left) and decay time constants (right) of the first EPSC in response to the paired stimuli.

5. E, F

   Pan‐neurexin deletion does not significantly alter the cumulative EPSC amplitude during a high‐frequency stimulus train (100 Hz for 0.5 s). Left, representative ESPC traces; right, cumulative summary plot of the EPSC amplitudes during the train (dotted lines show linear regression fits for estimating the cumulative EPSC amplitude by back‐extrapolation to zero time, which is used to correct for vesicle replenishment during the train).

6. G

   Pan‐neurexin deletion does not decrease the readily releasable pool of vesicles, but lowers the initial release probability during a high‐frequency stimulus train. Summary graphs show the cumulative EPSC amplitudes extrapolated to time zero as an estimate of the readily releasable pool size (left), and the ratio of the first EPSC amplitude divided by the cumulative EPSC amplitudes extrapolated to time zero as an estimate of the initial release probability (right).

Data information: Data are means ± SEM with individual data points. Number of cells (from at least three mice per group) analyzed are indicated in the bars (B, D, and G) or in the graph (F) and apply to all graphs in a series; statistical differences were assessed by Student's t‐test (**P < 0.01;***P < 0.001; non‐significant differences are not marked).

Figure EV3. Deletion of all neurexins in conditional Nrxn123 TKO mice using a Pv‐Cre driver line has no impact on inhibitory synaptic inputs onto MNTB neurons, presumably because Pv‐Cre drives expression only in postsynaptic MNTB neurons but not in the inhibitory presynaptic input neurons (as opposed to excitatory presynaptic calyx neurons; see Zhang et al, 2017; related to Fig 2)

1. At P12–14, inhibitory synapses on the MNTB are primarily glycinergic. Inhibitory postsynaptic currents (IPSCs) evoked by afferent fiber stimulation were recorded from MNTB neurons in acute brainstem slices from control mice at P12–P14, with sequential addition of 25 μM strychnine (Str) and 50 μM picrotoxin (PTX). Left, example traces of IPSCs; right, summary graph of the percent inhibition of the IPSC amplitude after sequential addition of strychnine and picrotoxin.

2. Sample traces of IPSCs from littermate control and Pv‐Cre conditional Nrxn123 TKO mice at P12–P14.

3. Summary graphs of the amplitude and integrated charge of IPSCs.

4. Summary graphs of the rise times and decay time constants of IPSCs.

Data information: Data in (A, C, and D) are means ± SEM. Number of cells (from three mice per group) analyzed are indicated in the bars (A, C and D); no statistically significant differences were observed as assessed by Student's t‐test.

Figure EV4. Deletion of neurexins has no major effect on presynaptic action potentials measured in patched terminals of calyx of Held synapses (related to Fig 2)

1. Example traces of presynaptic action potentials in littermate control and Nrxn123 TKO mice monitored in presynaptic calyx terminals.

2. Summary graphs of the action potential amplitude and duration. Data are means ± SEM. Number of cells (from three mice per group) analyzed are indicated in the bars; no statistically significant differences were observed as assessed by Student's t‐test

3. Example traces of presynaptic APs firing at 100 Hz, showing that there are no action potential failures.

Figure 3. Pan‐neurexin deletion renders high sensitivity of evoked synaptic transmission to reduced Ca²⁺‐signals at calyx synapses

1. A–D

   Same as Fig 2A–D, except that ACSF contained only 1 mM Ca²⁺, which is close to the estimated physiological concentration of extracellular Ca²⁺ (Forsberg et al, 2019).

2. E–H

   Same as Fig 2A–D, except that the slice was incubated with 0.2 mM EGTA‐AM for 30 min before recording, which introduces EGTA into the presynaptic terminals.

Data information: Data are means ± SEM with individual data points. Number of cells (from at least three mice per group) analyzed are indicated in the bars (C, G) and apply to all graphs in a series; statistical differences were assessed by Student's t‐test (**P < 0.01;***P < 0.001; non‐significant differences are not marked).

Figure 4. Deletion of all neurexins from the calyx of Held synapse does not alter the size or properties of presynaptic Ca²⁺‐currents

1. Example traces of presynaptic Ca²⁺‐currents recorded in patched calyx terminals as induced by a 50‐ms step depolarization from −60 mV to +40 mV in 10 mV increments at a holding potential of −80 mV.

2. Summary graphs of the I–V relationship for the peak Ca²⁺‐current amplitude (left) and of the peak Ca²⁺‐current density (right).

3. Example traces (left) and summary graphs (right) of the Ca²⁺‐current activation and deactivation kinetics. Currents were induced by a 50‐ms step depolarization from −80 mV to +10 mV.

4. Example traces of Ca²⁺‐currents evoked by an action potential‐equivalent depolarization (AP_e, from −80 mV to +17 mV for 1 ms; left), and summary graph of the AP_e‐evoked Ca²⁺‐current peak amplitude and charge (right).

5. Example traces of presynaptic Ca²⁺‐currents induced by a 50‐ms step depolarization from −60 mV to +40 mV in 10 mV increments at a holding potential of −80 mV before and after perfusion of 200 nM agatoxin (Agtx), a selective P/Q‐type Ca²⁺‐channel blocker.

6. Summary graph of the relative contribution of P/Q‐type Ca²⁺‐channels to the total presynaptic Ca²⁺‐currents, as quantified by the relative reduction in Ca²⁺‐currents evoked by step depolarization to +10 mV, in control and Nrxn123 TKO synapses.

Data information: Data in (B–D, F) are means ± SEM. Number of cells (from three mice per group) analyzed are indicated in the graph (B) or in the bars (C, D, and F); no statistically significant differences were observed as assessed by Student's t‐test.

Figure 5. Pan‐neurexin deletion renders depolarization‐induced synaptic vesicle exocytosis sensitive to the slow Ca²⁺‐chelator EGTA without affecting total Ca²⁺‐influx into the nerve terminal, Ca²⁺‐triggering of release, or the size of the readily releasable pool

1. A

   Diagram of direct simultaneous measurements of the presynaptic capacitance jump (C_m) and Ca²⁺‐currents (I_Ca) evoked by depolarization of a patched nerve terminal.

2. B

   Example traces of the presynaptic capacitance and Ca²⁺‐currents at the calyx terminals induced by a 20‐ms step depolarization from −80 mV to +10 mV with an extracellular solution containing 2 mM Ca²⁺, and an internal pipette solution containing a standard 0.05 mM BAPTA.

3. C

   Summary graphs of the depolarization‐induced capacitance jump that monitors synaptic vesicle exocytosis (left), and the peak Ca²⁺‐current density (right).

4. D, E

   Same as (B, C), except that 0.5 mM EGTA was added in addition to 0.05 mM BAPTA to the patch pipette internal solution.

5. F, G

   Same as (B, C), except that 1.0 mM EGTA was added in addition to 0.05 mM BAPTA to the patch pipette internal solution.

6. H, I

   Same as (B, C), except that 10.0 mM EGTA was added in addition to 0.05 mM BAPTA to the patch pipette internal solution.

7. J, K

   Summary plots of the relationship between the total capacitance jump (J) or between the Ca²⁺‐current density (K) and the concentration of EGTA in the patch pipette.

Data information: Data are means ± SEM with individual data points. Number of cells (from at least three mice per group) analyzed are indicated in the bars (C, E, G, and I) or shown in the graph (J and K); statistical differences were assessed by Student's t‐test (*P < 0.05; **P < 0.01).

Figure 6. Pan‐neurexin deletion depletes K⁺‐currents carried by presynaptic BK‐channels in the calyx of Held synapse

1. Representative traces of Ca²⁺‐currents and 4‐AP insensitive K⁺‐currents evoked by step depolarizations (from −50 mV to +10 mV in 10 mV increments), recorded from the calyx terminals in acute slices from littermate control and Nrxn123 TKO mice at P12–P14.

2. Same as (A) but after addition of 200 nM iberiotoxin (IbTx) by perfusion.

3. Representative traces of presynaptic BK‐currents, which are calculated by subtracting currents shown in (B) from currents shown in (A).

4. Summary graphs of the capacitance, Ca²⁺‐current density, and BK‐current density.

Data information: Data are means ± SEM with individual data points. Number of cells (from at least three mice per group) analyzed are indicated in the bars (D); Statistical differences were assessed by Student's t‐test (**P < 0.01).

Figure 7. Pan‐neurexin deletion depletes Ca_V2.1‐type Ca²⁺‐channels and Bassoon from the presynaptic active zone of the calyx of Held

1. Representative images of brainstem cryosections stained by triple immunofluorescence labeling for vGluT1 (green), Ca_V2.1‐type Ca²⁺‐channels (red), and the active zone protein Bassoon (purple). Cryosections were obtained from littermate control and Nrxn123 TKO mice at P12–14. Scale bar, 10 μm.

2. Summary graphs of the vGluT1, Ca_V2.1, and Bassoon immunostaining intensity (normalized to control; Ca_V2.1 and Bassoon signals were quantified over the vGluT1‐positive area to measure active zone‐localized proteins).

3. Summary graphs of the diameter and total size of the vGluT1‐positive presynaptic area of the calyx of Held.

Data information: Data are means ± SEM with individual data points. Number of brain sections and mice analyzed are indicated in the bars (B and C); statistical differences were assessed by Student's t‐test (***P < 0.001).

Figure EV5. Deletion of all neurexins modestly but significantly reduces the levels of the active zone protein Bassoon in the presynaptic terminal of the calyx of Held synapse (related to Fig 7)

1. Representative brainstem sections containing the MNTB from littermate control and Nrxn123 TKO mice, stained with antibodies to vGluT1 (green) and Bassoon (red). Scale bar, 10 μm.

2. Summary graph of vGluT1 and Bassoon immunostaining intensity (normalized to control).

Data information: Data are means ± SEM. Number of image sections/mice analyzed are indicated in the bars; whereas no statistically significant differences were observed for the vGluT1 staining intensity as assessed by Student's t‐test, the Nrxn123 TKO did induce a significant decrease in Bassoon staining intensity (**P < 0.01).