Synaptic structure quantification in cultured neurons

Abstract

Behavioral problems (e.g., learning and memory) following developmental exposure to toxicants suggests that dysregulation of the process of synapse formation and function may occur. The ability to assess these changes is thus of value. This unit describes a method to investigate toxicant-induced changes to synaptic structure formation in primary hippocampal neurons using immunocytochemical labeling of the pre- and post-synaptic markers synaptophysin and PSD-95, confocal imaging, and three-dimensional object analysis. Protocols for the long-term culturing of primary hippocampal neurons and of primary cortical astrocytes, as well as their co-culture, are included. While the described methods focus on how astrocytes influence synapse formation and how toxicants may interfere in this process, modifications to the experimental plan can easily be implemented. This would allow for the investigation of the effects of toxicants after treating neurons alone, or both astrocytes and neurons in co-culture. With the common endpoint of synapse structure formation, differences between varying treatment paradigms can expand the understanding of the influence of particular toxicants on these diverse cell types and provide insight into potential mechanisms of effect and the contributions of each to synapse formation.

Keywords: 3-dimensional analysis; PSD-95; astrocytes; sandwich co-culture; synaptogenesis; synaptophysin.

Figure 1. Immunocytochemical Labeling

Hippocampal neurons (13 DIC) were co-cultured for 24 hours with astrocytes that were pre- treated with carbachol (1 mM) or ethanol (75 mM). Neurons were fixed, immunocytochemically labeled for the pre-synaptic protein, synaptophysin, and the post-synaptic protein, PSD-95. Shown are maximum intensity projected images after confocal imaging. (Physin: synaptophysin, green; PSD-95, red; merge, yellow.)

Figure 2. Effect of Deconvolution

Hippocampal neurons (13 DIC) were directly treated with high-density lipoprotein (10 µg/mL cholesterol) for 24 hours, immunocytochemically labeled for the synaptic protein synaptophysin, and imaged using confocal microscopy. Prior to deconvolution (A) the signal perimeter appears cloudy and somewhat blurred. After deconvolution (B) the signal is refined and refocused without a loss of relative intensity differences. Shown is the greyscale representation of a portion of the maximum intensity z-projected image taken from the green channel and enlarged to 150%.

Figure 3. Comparison of deconvolved image vs. surface rendering after thresholding

The method used to generate a threshold for each channel results in a three-dimensional surface rendering of puncta (B) that is representative of punctate structures evident in the deconvolved image (A). Shown is one slice of a deconvolved confocal z-series channel (A) of hippocampal neurons grown in culture for 13 days and treated for 24 hours with high density lipoproteins (10 µg/mL cholesterol), and immunocytochemically labeled for synaptophysin. A total of 450 puncta per channel were manually selected from 15 images and the threshold value was determined by averaging the mean intensities. Selecting a representative sample of puncta results in a threshold and a rendering (B) that accurately reflects the deconvolved image (A). Setting the threshold too high (D), or too low (C), results in an inaccurate three-dimensional rendering.