Cryoloading: introducing large molecules into live synaptosomes

Abstract

Neurons communicate with their target cells primarily by the release of chemical transmitters from presynaptic nerve terminals. The study of CNS presynaptic nerve terminals, isolated as synaptosomes (SSMs) has, however, been hampered by the typical small size of these structures that precludes the introduction of non-membrane permeable test substances such as peptides and drugs. We have developed a method to introduce large alien compounds of at least 150 kDa into functional synaptosomes. Purified synaptosomes are frozen in cryo-preserving buffer containing the alien compound. Upon defrosting, many of the SSMs contain the alien compound presumably admitted by bulk buffer-transfer through the surface membranes that crack and reseal during the freeze/thaw cycle. ~80% of the cryoloaded synaptosomes were functional and recycled synaptic vesicles (SVs), as assessed by a standard styryl dye uptake assay. Access of the cryoloaded compound into the cytoplasm and biological activity were confirmed by block of depolarization-induced SV recycling with membrane-impermeant BAPTA (a rapid Ca2+-scavenger), or botulinum A light chain (which cleaves the soluble NSF attachment protein receptor (SNARE) protein SNAP25). A major advantage of the method is that loaded frozen synaptosomes can be stored virtually indefinitely for later experimentation. We also demonstrate that individual synaptosome types can be identified by immunostaining of receptors associated with its scab of attached postsynaptic membrane. Thus, cryoloading and scab-staining permits the examination of SV recycling in identified individual CNS presynaptic nerve terminals.

Synaptic vesicle tethering and the CaV2.2 distal C-terminal

“…This hypothesis was tested in the chick SSMs using our novel “cryoloading” method (Nath et al, 2014) that permits the introduction of large (up to at least 150 kD) membrane-impermeable alien compounds into functional SSMs. Briefly, fresh SSMs were frozen in a buffer containing the test compound, a 3 kD dextran-FITC loading marker and a cryoprotectant.…”

Differential roles of hippocampal glutamatergic receptors in neuropathic anxiety-like behavior after partial sciatic nerve ligation in rats

“…At 28 days after PSNL, one set of rats (n = 4 or 6 for each group) was sacrificed by an overdose of chloral hydrate (80 mg/kg). Immunohistochemical staining for GluR1, GluR2, NR1, NR2A, NR2B, and p-ERK1/2 was performed as described in previous studies [ 15 , 29 - 31 ] with a slight modification. Briefly, brains were fixed for 4 h in 4% paraformaldehyde after perfusion and cryoprotection by immersion in 20% sucrose in phosphate buffer (pH = 7.4) overnight.…”

The Calcium Channel C-Terminal and Synaptic Vesicle Tethering: Analysis by Immuno-Nanogold Localization

“…The first concern was that a significant fraction of the synaptosomes may have resealed after the osmotic shock step, occluding antibody access to the luminal face of the AZ. This concern was solved by freeze-thaw-loading of the antibodies into the resealed synaptosomes using a protocol that we term cryoloading as described recently in detail (Nath et al, 2014). To improve labeling efficiency, we used a cocktail of L4569 and L4570, termed here L45, that is in effect a more heterogeneous polyclonal antibody than either alone.…”

“…To improve our signal to noise ratio, we made two key improvements to the gold labeling method. First, we used cryoloading (Nath et al, 2014) to freeze-load the synaptosome ghosts with the antibodies. Second, we replaced the conventional colloidal gold secondary antibody with Fab fragments that are covalently tagged with nanogold particles.…”

“…Cryoloading is described in detail in a previous publication (Nath et al, 2014). SSM ghost pellets were resuspended in sucrose/EDTA/Tris buffer (“SET”: 320 mM sucrose, 1 mM EDTA, 5 mM Tris at pH 7.4) with 5% DMSO at room temperature.…”