"Kiss and run" exocytosis at hippocampal synapses

Abstract

We have combined electrophysiology and imaging to measure the release of neurotransmitter and fluorescent dye at synapses of cultured hippocampal neurons. These experiments have revealed a "kiss and run" mode of exocytosis in which synaptic vesicles release glutamate normally but do not permit dye to enter or escape from the vesicle. During "kiss and run," the vesicle interior may be exposed very transiently (<6 ms), or a special configuration of the fusion pore may prevent dye exchange. We estimate that about 20% of the vesicles normally use this "kiss and run" pathway, and that the fraction of "kiss and run" events can be increased to over 80% by superfusing the synapses with hypertonic solution.

Figure 1

Increased osmolarities permit less dye release. (A) Percent of FM1–43 that escapes per quantum of transmitter released by hypertonic stimulation, relative to dye released per quantum with action potentials, as a function of hypertonicity. Figure presents data from 780 synapses in 8 experiments on conventional and microdot cultures; error bars indicate one SEM. The smooth line is given by where h is the hypertonicity in osmolars. See text for discussion of equation. Dye release was determined for hypertonic and action potential stimulation in the same synapses. (B) Image of field after loading as described in text (Left) and after prolonged stimulation to release all dye in synaptic vesicles (Right). Synapses appear as small black dots (Left) that are no longer visible (Right).

Figure 2

Two modes of exocytosis exist. (A) Percent FM2–10 and FM1–43 released per quantum released (open circles) and percent FM1–43 taken up per quantum released (filled circles) with hypertonicity of solution as a parameter (Left to Right), the hypertonicities are 1.5, 1.0, 0.8, 0.6, 0.5, and 0.0 (nerve impulse stimulation) osM. For example, the left-most point is derived from experiments in which the release was produced by a 1.5-osM solution; the left ordinate value is the percentage dye release from FM2–10 loaded synapses, and the abscissa value is the percent dye released from FM1–43 loaded synapses. All percentage uptake and release are relative to the dye released or taken up per quantum of glutamate release produced by action potentials. Data for the abscissa is from experiments of Fig. 1. FM2–10 dye release data from 365 synapses in 5 experiments and FM1–43 dye uptake data, from 520 synapses in 5 experiments; error bars indicate one SEM, with the larger of the overlapping error bars associated with the filled circles (dye uptake measurements have more error). The solid line has a slope of one (equality for ordinate and abscissa), and the dotted line, with a slope of 1.05, is the least squares fit to the data. (B) Example of three synapses loaded with FM1–43 by 1 osM hypertonic solution application (Left) and then (after prolonged stimulation to release dye) by action potential stimulation (Right); much less dye was loaded with hypertonic stimulation, although in each case the preparation had been stimulated to release the same amount of neurotransmitter. The images were thresholded and false colored after subtraction of corresponding nonspecific fluorescence remaining after prolonged stimulation. The color map ranges from blue to red for increasing fluorescence and is intended to show the relative difference between dye uptake with hypertonic and action potential stimulation.

Figure 3

Neurotransmitter release is unchanged in “kiss and run.” Cumulative histograms of peak mEPSC amplitudes in the presence of bathing medium with hypertonicities of 0.0 osM (spontaneous mEPSCs, dotted line, mean amplitude, 25.67 ± 1.13 pA, n = 172), 0.5 osM (dashed, mean amplitude, 25.8 ± 1.15 pA, n = 166) and 1.0 osM (solid, mean amplitude, 26.09 ± 0.98 pA, n = 182). Inset shows averaged mEPSCs from the three conditions superimposed. (Bar = 10 pA and 10 ms.)

Figure 4

“Kiss and run” in normal synaptic transmission. (A) Predicted and observed percentage of “kiss and run” events as a function of hypertonicity. Theoretical function is , where h is the hypertonicity in osmolars, with a = 2.3 and h₀ = 0.65. Experimental points are derived from those in Fig. 1. Note that p(0) = 18.3%, which corresponds to the percentage of action potential-evoked exocytotic events that are “kiss and run.” (B) Cartoon of three pathways. (1) “Kiss and run,” in which the fusion pore opens and then closes to return the vesicle to its docked state intact; (2) rapid endocytosis that requires dynamin to pinch the vesicle membrane off from the surface membrane, but not clathrin; and (3) the classical endocytic pathway that requires both dynamin and a clathrin coat that is removed after endocytosis.