Super-resolution 2-photon microscopy reveals that the morphology of each dendritic spine correlates with diffusive but not synaptic properties

Abstract

The structure of dendritic spines suggests a specialized function in compartmentalizing synaptic signals near active synapses. Indeed, theoretical and experimental analyses indicate that the diffusive resistance of the spine neck is sufficient to effectively compartmentalize some signaling molecules in a spine for the duration of their activated lifetime. Here we describe the application of 2-photon microscopy combined with stimulated emission depletion (STED-2P) to the biophysical study of the relationship between synaptic signals and spine morphology, demonstrating the utility of combining STED-2P with modern optical and electrophysiological techniques. Morphological determinants of fluorescence recovery time were identified and evaluated within the context of a simple compartmental model describing diffusive transfer between spine and dendrite. Correlations between the neck geometry and the amplitude of synaptic potentials and calcium transients evoked by 2-photon glutamate uncaging were also investigated.

Keywords: 2-photon microscopy; dendritic spine; stimulated emission microscopy; super resolution microscopy; synaptic transmission.

Figure 1

Schematic of microscope. Laser pulses from a femtosecond-pulsed Ti-Sapph laser tuned to 810 nm for two-photon excitation (2PE) are synchronized by an electronic feedback circuit (Synchrolock) with those of a picosecond-pulsed Ti-Sapph laser (pSTED) tuned to 736 nm for stimulated emission. The STED laser can be exchanged by a flip mirror (FM) with the beam from a femtosecond-pulsed Ti-Sapph laser tuned to 720 nm for two-photon laser-induced uncaging (2PLU) of caged compounds, such as caged glutamate. STED pulses are stretched to ~200 ps by dispersion through a 120 m single-mode polarization-maintaining fiber optic (FO) and phase patterned to achieve a helical wavefront by a vortex phase plate (VP). The 2PE and STED lasers are combined by a dichroic (D1). Fluorescence is separated from excitation and depletion light by a dichroic (D2) and collected by photomultiplier tubes (PMT). λ /2 and λ /4 are half- and quarter-waveplates used to adjust the polarization. SL, Scan lens; TL, Tube lens; SM, Scanning mirror; OL, Objective lens; CL, Condenser lens.

Figure 2

Combined FRAP and STED-2P analysis. (A) STED-2P image of a dendritic spine on the apical dendrite of a CA1 hippocampal neurons filled with Alexa Fluor 594 through a somatic whole-cell recording pipette. (B) 2PLSM linescan taken through the spine and dendrite in (A). The photobleaching pulse was delivered on the spine head after a 100 ms delay (yellow arrow). (C) Fluorescence in the spine head over time quantified from (B). Recovery was fit with a decaying exponential (red line) to obtain the recovery time constant. (D–F) Recovery time constants plotted against neck length (D), head volume (E), and neck diameter (F).

Figure 3

Modeling of diffusive transfer across the spine neck. (A) Recovery times from Figure 2 plotted against ζ. (B) STED-2P image of the spine producing the outlying point marked by the arrow in (A).

Figure 4

Functional analysis of STED-2P resolved dendritic spines. (A) STED-2P image of dendritic spines. MNI-glutamate was uncaged at a point located near a target spine (white arrowhead) while line scanning over the spine and dendrite (yellow dashed line). (B) Green fluorescence, indicative of Ca-bound Fluo 5F, measured in line scan. MNI-glutamate uncaging following a 100 ms delay (white arrowhead) produced a transient increase in green fluorescence. (C) Somatic membrane potential recording of the uEPSP elicited from the synapse in (A). (D) Quantification of Fluo 5F fluorescence in the spine head (red) and in the dendrite (black) as imaged in (B).

Figure 5

Lack of correlations between spine dimensions, synaptic potentials, and associated Ca transients. (A,B) uEPSP amplitude plotted against spine neck diameter (A) and length (B). (C,D) Amplitude of uncaging evoked Ca transient plotted against neck diameter (C) and length (D).