Optimization of freezing, storage, and thawing conditions for the preparation of metabolically active synaptosomes from frozen rat and human brain

Abstract

Samples of rat and human cerebral cortex were frozen, stored, and thawed under a variety of conditions to define further the optimal procedure for storing human brain samples for subsequent metabolic and functional studies that use incubated synaptosomes. Tissue samples were best preserved by immersing them in isotonic sucrose prior to slow freezing, but there was no advantage in first chopping up the material. High concentrations of sucrose, rather than exerting a cryoprotective effect, were detrimental to subsequent synaptosomal performance (oxygen uptake, K+ accumulation, stimulus-induced release of amino acid neurotransmitters). However, good activity was observed in preparations from rat brain frozen in the absence of fluid. This result was confirmed by studies on the uptake of gamma-aminobutyrate (GABA) into an osmotically sensitive compartment in synaptosomes prepared from frozen human autopsy material transshipped from the brain tissue collection ("brain bank") at Harvard Medical School, MA, USA, to Sydney, Australia. Although activity was slowly lost over a 3-mo period in rat tissue samples stored at -20 degrees C, there was little or no such loss at -70 degrees C.

Biochemical and molecular studies using human autopsy brain tissue

“…The protocols differ primarily in the rates of freezing and thawing employed and the nature of the cryoprotectant used (Shirakashi and Tanasawa 1998;Eroglu et al 2000;Furuki 2000). We found the most effective method for the subsequent isolation of synaptosomes to be slow freezing in the presence of an isoö smotic cryoprotectant Dodd et al 1986). In brief, the tissue samples are immersed in sucrose (0.32 M) in polyethylene bags and placed in an insulated container at )80°C.…”

“…The replacement of isoö smotic sucrose by dimethylsulfoxide (a cryoprotectant commonly used in the freezing of tissue culture stocks : Coriell 1979;Danon and Kaminsky 1985;Cai et al 1993;Borderie et al 1998;Draper et al 2002;Pegg 2002) generally failed to yield viable synaptosomal preparations (Haan and Bowen 1981;Haberland and Hetey 1987;Dodd et al 1988). Isoö smotic sucrose remains our preferred cryoprotectant when freezing tissue at slow rates (Haan and Bowen 1981;Dodd et al 1986Dodd et al , 1988.…”

Differential expression of α-synuclein splice variants in the brain of alcohol misusers: Influence of genotype

“…The human brain tissue samples were collected by the Queensland Brain Bank, School of Chemistry and Molecular Biosciences, University of Queensland and the NSW Tissue Resource Centre, University of Sydney. Autopsied tissue samples were immersed in ~10 vol of 0.32 M sucrose and slowly frozen to best preserve the blocks (Dodd et al, 1986). The frozen samples were stored at -80 o C until the RNA isolation outlined below.…”

Examining the GABA_A receptor alpha-subunits in the neonatal pig brain: changes across development and the effect of seizures after hypoxic-ischaemic brain injury

“…The brain was hemisected, right hemisphere slices were fixed in 4% paraformaldehyde/ 0.1 M phosphate buffered saline (PBS, pH 7.4) for 24 h with agitation followed by transfer into in PBS/sodium azide (0.05%) for storage until paraffin-embedding and subsequent microtome sectioning. Regions of interest including four cortical regions, basal ganglia, hippocampus, thalamus and cerebellum were dissected from the left hemisphere and slow frozen in 0.32M sucrose at -80 o C (Dodd et al 1986) until western blot analysis.…”

“…Anatomical regions of interest from the left hemisphere slices were slow frozen in 0.32M sucrose at -80 o C (Dodd et al 1986). These included frontal, parietal, temporal and occipital cortices, hippocampus, basal ganglia, thalamus, cerebellum and brain stem.…”