Research paper
Cryopreservation of freshly isolated synaptosomes prepared from the cerebral cortex of rats

https://doi.org/10.1016/0165-0270(93)90081-2Get rights and content

Abstract

In the present study, we established a cryopreservation method for freshly isolated synaptosomes prepared from the cerebral cortex of rats. Freshly prepared synaptosomes were either shock-frozen or frozen under temperature-controlled conditions using a programmable temperature controller. Each group was resuspended in iso-osmotic or hyperosmotic sucrose buffer prior to freezing, resulting in 4 different preservation protocols. The viability of the frozen synaptosomes was estimated by the recovery of basal and stimulated respiration after short-term storage (1 h) in liquid nitrogen. With regard to basal, FCCP- and veratridine-induced respiration, best recovery revealed controlled-frozen synaptosomes resuspended in iso-osmotic sucrose buffer (con/iso group). Basal respiration of this group recovered completely, whereas veratridine- and FCCP-induced oxygen uptake was decreased to 87.7% and 82.4% of control, respectively. Further investigations performed with the con/iso group revealed complete recovery of anaerobic and aerobic lactate synthesis, and unaffected synaptosomal integrity, as judged by the amount of released l-lactate dehydrogenase before and after the cryopreservation procedure. Long-term storage of the con/iso group in liquid nitrogen up to 88 days did not have any influence on synaptosomal viability, as evaluated by the recovery of anaerobic lactate production and synaptosomal respiration. Therefore, based on the results of respiration, synaptosomal integrity, and lactate synthesis, metabolically active synaptosomes could be obtained after cryopreservation and storage in liquid nitrogen for at least 88 days.

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    Citation Excerpt :

    Following homogenization and fractionation (125), synaptosomal preparations are enriched in isolated pre- and postsynaptic components retaining their in vivo functions, including synaptic mitochondrial bioenergetics and respiration (126,127), neurotransmitter release (including glutamate), and endocytosis activities (123,128,129), and contain synaptic nucleotides including noncoding circular RNAs (130) and microRNAs (131). In addition, synaptosomal preparations can be cryopreserved while retaining their activity (132) to be banked as a ready-to-use reagent for downstream assays. Synaptosomes derived from postmortem patients have been used to model schizophrenia for the study of synaptic-specific protein levels and RNA expression specifically in synapses (131,133), aberrant synaptic protein content (134,135), synaptic proteomic studies (136,137), glutamate release (138,139), and complement deposition (140).

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