Presynaptically Silent Synapses Studied with Light Microscopy

Summary

Glutamatergic synapses can switch from an active mode to a silent mode. We demonstrate that presynaptic activity status in dissociated culture of rodent neurons is visualized using a fixable form of the FM1-43 dye to visualize active synapses and immunostaining with vGluT-1 antibody to visualize all glutamate synapses.

Abstract

Synaptic plasticity likely underlies the nervous system's ability to learn and remember and may also represent an adaptability that prevents otherwise damaging insults from becoming neurotoxic. We have been studying a form of presynaptic plasticity that is interesting in part because it is expressed as a digital switching on and off of a presynaptic terminal s ability to release vesicles containing the neurotransmitter glutamate. Here we demonstrate a protocol for visualizing the activity status of presynaptic terminals in dissociated cell cultures prepared from the rodent hippocampus. The method relies on detecting active synapses using staining with a fixable form of the styryl dye FM1-43, commonly used to label synaptic vesicles. This staining profile is compared with immunostaining of the same terminals with an antibody directed against the vesicular glutamate transporter 1 (vGluT-1), a stain designed to label all glutamate synapses regardless of activation status. We find that depolarizing stimuli induce presynaptic silencing. The population of synapses that is silent under baseline conditions can be activated by prolonged electrical silencing or by activation of cAMP signaling pathways.

Protocol

Culture preparation

1. Prepare dissociated cell cultures of rat or mouse hippocampal cells from postnatal day 0 to 3 animals¹. Our neurons adhere to an underlying astrocyte monolayer, which in turn adheres to a collagen layer spread on a number 0 thickness coverslip. Plate the neurons at a density of approximately 500 cells per square centimeter.
2. Allow the cultures to grow for 10-14 days to allow synaptic development and maturation. Treat the cultures at day in vitro 4 with the antimitotic AraC to arrest further glial growth. Feed them at day in vitro 5 with a half medium exchange with Neurobasal medium plus B27 supplement to enhan....

Discussion

Significance

1. Typically synapses are thought to operate by releasing transmitter with a measurable probability. Work by ourselves and others makes it evident that some synapses are refractory to releasing neurotransmitter, despite a full complement of vesicular neurotransmitter transporter and other key presynaptic markers^{2, 6-8}. We have shown previously that the basal network activity in neurons is critical to maintain this population of inactive synapses.

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Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
FM1-43FX	Invitrogen	F-35355	A red-shifted FM4-64FX is also available, but has not proven as amenable to assays of presynaptic silencing.
Advasep-7	CyDex	AR-0A7-001
vGluT-1	EMD Millipore	AB5905
Alexa 647-conjugated anti-GP	Invitrogen	A-21450
Fluoromount-G	SouthernBiotech	0100-01