Imaging Dendritic Spines of Rat Primary Hippocampal Neurons using Structured Illumination Microscopy

Abstract

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. Technological advances have identified these structures as key elements in neuron connectivity and synaptic plasticity. The quantitative analysis of spine morphology using light microscopy remains an essential problem due to technical limitations associated with light's intrinsic refraction limit. Dendritic spines can be readily identified by confocal laser-scanning fluorescence microscopy. However, measuring subtle changes in the shape and size of spines is difficult because spine dimensions other than length are usually smaller than conventional optical resolution fixed by light microscopy's theoretical resolution limit of 200 nm.Several recently developed super resolution techniques have been used to image cellular structures smaller than the 200 nm, including dendritic spines. These techniques are based on classical far-field operations and therefore allow the use of existing sample preparation methods and to image beyond the surface of a specimen. Described here is a working protocol to apply super resolution structured illumination microscopy (SIM) to the imaging of dendritic spines in primary hippocampal neuron cultures. Possible applications of SIM overlap with those of confocal microscopy. However, the two techniques present different applicability. SIM offers higher effective lateral resolution, while confocal microscopy, due to the usage of a physical pinhole, achieves resolution improvement at the expense of removal of out of focus light. In this protocol, primary neurons are cultured on glass coverslips using a standard protocol, transfected with DNA plasmids encoding fluorescent proteins and imaged using SIM. The whole protocol described herein takes approximately 2 weeks, because dendritic spines are imaged after 16-17 days in vitro, when dendritic development is optimal. After completion of the protocol, dendritic spines can be reconstructed in 3D from series of SIM image stacks using specialized software.

MicroRNA-124 and -137 cooperativity controls caspase-3 activity through BCL2L13 in hippocampal neural stem cells

“…50 thousand hippocampal NSPC were seeded per well in 24-well plates containing poly-L-lysine and Laminin coated glass coverslips as described before 68 . The next day cells were transfected with FLAG-tagged human BCL2L13, a kind gift from Dr. Jürg Tschopp, Institute of Biochemistry, University of Lausanne (Kataoka et al , 2001), using Attractene (Qiagen) or empty vector and were incubated for 48 h. In the last 7 h of the incubation cells were treated with varying concentrations of KA ranging from 0–300 μM KA or vehicle 46 .…”

“…Subsequently, cells were stained with polyclonal sheep anti-CytC (Sigma-Aldrich, 1:100) in combination with donkey anti-sheep Alexa488 (Invitrogen, 1:1000) and coverslips were mounted in vectashield mounting medium (Vector Labs). SIM was performed using a Nikon Eclipse Ti inverted microscope based SIM system as described before 68 .…”

Determination of dendritic spine morphology by the striatin scaffold protein STRN4 through interaction with the phosphatase PP2A

“…To better define the various spine types, the images of dendritic spines were acquired using Super-resolution Structured Illumination Microscopy (SR-SIM), which has about 2-fold higher axial resolution than confocal microscopy (42) and is suitable for visualization of cellular structures around 200 nm, such as the neck of a dendritic spine (see supplemental Fig. S2) (43). We found that STRN4 expression differed in distinct subtypes of dendritic spines, with the highest STRN4 immunoreactivity present in stubby spines and the spine heads of mushroomshaped spines that contained PSD-95 clusters, whereas STRN4 immunoreactivity was significantly reduced in thin spines and filopodia (Fig.…”

Super-resolution microscopy and its applications in neuroscience

“…59 What's more, Schouten et al also identi¯ed dendritic spines of rat primary hippocampal neurons using SIM and analyzed the spines morphology quantitatively. 60 Because of the characteristics of PALM/STORM algorithm, this super-resolution microscopy is convenient for monitoring the structure of axons. Zhuang et al used STORM to observe the development of axons and revealed the mechanism of actin, spectrin, and associated molecules forming a periodic sub-membrane lattice structure in axons.…”