Fast neurotransmitter release regulated by the endocytic scaffold intersectin

Abstract

Sustained fast neurotransmission requires the rapid replenishment of release-ready synaptic vesicles (SVs) at presynaptic active zones. Although the machineries for exocytic fusion and for subsequent endocytic membrane retrieval have been well characterized, little is known about the mechanisms underlying the rapid recruitment of SVs to release sites. Here we show that the Down syndrome-associated endocytic scaffold protein intersectin 1 is a crucial factor for the recruitment of release-ready SVs. Genetic deletion of intersectin 1 expression or acute interference with intersectin function inhibited the replenishment of release-ready vesicles, resulting in short-term depression, without significantly affecting the rate of endocytic membrane retrieval. Acute perturbation experiments suggest that intersectin-mediated vesicle replenishment involves the association of intersectin with the fissioning enzyme dynamin and with the actin regulatory GTPase CDC42. Our data indicate a role for the endocytic scaffold intersectin in fast neurotransmitter release, which may be of prime importance for information processing in the brain.

Keywords: endocytosis; synaptic transmission; synaptic vesicle recruitment.

Fig. 1.

Intersectin 1 is localized at and around AZs. (A) STED microscopy image of a mature mouse calyx of Held synapse (age P50–P70) immunostained for endogenous bassoon (BSN; green) and intersectin 1 (ITSN1; red). The postsynaptic neuron is surrounded by the calyx terminal immunopositive for the AZ marker bassoon. Note that bassoon staining is also detectable at noncalycial terminals. (Inset) Confocal image of bassoon immunopositive puncta. For the scale bar, see the line scan profiles in C–E. (B) Magnified images of the data shown in A (from line C). (B, i) ITSN1; (B, ii) BSN; (B, iii) merged image. (Insets) Intersectin-immunopositive puncta confined to the active zones. (Scale bars: 750 nm; 350 nm in Insets.) (C–E) Spatial intensity profiles of bassoon (green) and intersectin 1 (red). The regions of interest (lines C, D, and E in A) were selected where the presynaptic compartment showed a finger-like structure.

Fig. 2.

SV replenishment in intersectin 1 KO mice. (A) Simultaneous recordings of the presynaptic and postsynaptic compartments at the calyx of Held synapse. A pair of depolarizing pulses (0 mV for 50 ms, after a prepulse to +70 mV for 2 ms) was applied to the presynaptic terminal with an interval of 500 ms. During a 0-mV period, presynaptic Ca current was elicited. Evoked postsynaptic EPSCs (dotted line, first stimulation; solid line, second stimulation with an interval of 500 ms) and the cumulative release are shown. Cumulative release in response to the first pulse showed a double-exponential time course. (Upper) WT. (Lower) Intersectin 1 KO littermates. (B and C) Recovery of the fast (FRP; B) and the slow (SRP; C) components of release plotted against the stimulation interval. Dotted and solid circles represent data from control (four WT cells and four +/− cells) and KO (eight cells) mice, respectively. (D) The capacitance trace in response to a 50-ms pulse was normalized to the peak value and averaged among cells. Open and filled circles represent data from control and KO mice, respectively (n = 6 cells each). (E) The pulse duration increased to 500 ms. Note that for the open circle data, only downward error bars are displayed. Upward error bars are shown for the closed circle data for clarity.

Fig. 3.

Acute perturbation of intersectin 1 SH3A domain function causes short-term synaptic depression. Simultaneous recordings of the presynaptic and postsynaptic compartments at the calyx of Held synapse (rats, P8–P11). A train of AP-like stimuli (depolarization to +40 mV for 1.5 ms) was applied (20 pulses), at 10, 50, and 100 Hz, and then at 10 Hz. Presynaptic Ca currents (Top), EPSCs (Middle), and magnified EPSCs (with initial EPSC peaks truncated; Bottom) are shown. (A and B) Data under control conditions (A) and in the presence of SH3A domain (5 μM) (B). (C) Normalized time course of the EPSC amplitudes plotted over time. Red and blue symbols indicate the data under control and in the presence of the SH3A domain, respectively. (D and E) Confocal images of a calyx terminal (rats, P8–P11) preloaded with Alexa Fluor 488 (200 μM; D) and the Atto647N-conjugated SH3A domain of intersectin (10 μM; E). (F) STED images of magnified data outlined by the box in E, illustrating the localization of the AZ markers bassoon (F, i; green), and Atto-SH3A (F, ii; red). (F, iii) Composite image, with colocalizing puncta in yellow. (Scale bar: 500 nm.)

Fig. 4.

Acute perturbation of the intersectin 1 SH3A domain slows SV replenishment. A pair of depolarizing pulses (0 mV for 50 ms after a prepulse to +70 mV for 2 ms) was applied to the presynaptic terminal with an interval of 500 ms. (A–C) EPSCs (dotted line, first stimulation; solid line, second stimulation, with an interval of 500 ms) and cumulative release (cum rel) are shown. (A) Control conditions. (B and C) Terminals dialyzed with SH3A domain (5 μM) (B) or antibodies directed against intersectin 1 SH3A (2,030 μg/mL) (C). (D) Similar experiments as in A–C, but with the stimulus interval of the two pulses varied. Recovery of the FRP (Left) and the SRP (Right) after RRP depletion was plotted against the ISI. Data from control conditions (open circles) and obtained in the presence of SH3A (filled circles), mutant SH3A (filled squares), or antibodies against the intersectin 1 SH3A domain (filled triangles) are shown. (E) Recovery of the fast-releasing component is plotted against ISI. The proline-rich domain of dynamin (filled triangles), a dynamin 1-derived proline-rich peptide (filled circles), or the proline-rich domain of N-WASP (filled squares) were introduced into the terminal (ISI = 0.5 and 1 s). Open circles, control condition. (F) Same as in E, except that the SH3E domain of intersectin 1 was introduced into the terminal (filled circles). Open circles, control condition.

Fig. 5.

Blocking the activity of the intersectin binding partner CDC42 impairs recruitment of release-ready SVs. (A) Simultaneous recordings of the pre- and postsynaptic compartments at the calyx of Held synapse (as depicted in Figs. 2 and 4). The DH-PH domain of intersectin 1 was introduced into the terminal. (Left) panel is similar to Fig 4 and the traces from one cell pair are shown (dotted: first stimulation, solid: second stimulation with an interval of 500 ms). Right) Plot of recovery of the fast component (FRP) against the stimulus interval. Filled circles, Dbl homology-pleckstrin homology (DH-PH) domain; open circles, control condition. (B) Same as in A, except with 1 μM toxin B introduced into the presynaptic terminal. Toxin B potently inhibits Rho family small G proteins. Open and filled circles represent the data from controls and in the presence of toxin B, respectively. (C) Same as in A, except that 20 μM secramine A (filled circles), a specific small-molecule inhibitor of CDC42, was applied to the terminal.