Determination of the Time Course and Extent of Neurotoxicity at Defined Temperatures in Cultured Neurons Using a Modified Multiwell Plate Fluorescence Scanner

Abstract

Summary:The cellular and molecular mechanisms of hyp oxiclischemic neurodegeneration are sensitive to numerous factors that modulate the time course and degree of neu ronal death. Among such factors is hypothermia, which can dramatically protect neurons from injury. To examine and control for temperature-dependent effects, we devel oped a technique that provides for a high-throughput, accu rate, and reproducible determination of the time course and degree of neurotoxicity in cultured cortical neurons at precisely defined temperatures. We used a fluorescence multi well plate scanner, modified by us to permit the con trol of temperature, to perform serial quantitative measureRecent years have seen a dramatic increase in our understanding of hypoxic/ischemic mechanisms. Much of this knowledge has arisen from in vitro stud ies in cultured neurons in which ischemia-like condi tions are simulated using either anoxic/hypoglycemic conditions or the application of excitatory amino acids (Choi, 1988(Choi, , 1990Lipton and Rosenberg, 1994; Tymianski and Tator, 1996). This approach has helped to unveil some of the molecular mechanisms bridging the onset of an insult and the resultant neu rodegeneration and has contributed to the devel opment of several emerging treatments for stroke (Koroshetz and Moskowitz, 1996 Many in vitro investigations of ischemic mecha nisms consist of simple manipulations of cellular pro cesses presumed to be associated with cell injury, usually by pharmacological means. Recently, it has also become widely appreciated that environmental manipulations, especially temperature, also signifi cantly affect cell survival (Busto et ai., 1987; Bruno et ai., 1994). Evaluation of such manipulations of in vitro preparations exposed to ischemia-like condition requires a precise assessment of cell viability. Also, because the temporal features of cell death may vary, it is important to be able to accurately gauge the time course of cell death. For example, agonists of N methyl-n-aspartate receptors often trigger rapidly oc curring neurodegeneration that can be complete within hours (Tymianski et ai., 1993a,b), whereas the activation of non-NMDA receptors triggers more de layed neurotoxicity (Rothman et ai., 1987; Koh et ai., 1990; Andreeva et ai., 1991;Dubinsky, 1993). It is likely that environmental manipulations, including 456 R. SATTLER ET AL.temperature changes, could also affect the timing of neurodegeneration.Here we describe a high-throughput neurotoxicity assay for cultured neurons that takes into account the temperature sensitivity of neurodegeneration and the need to precisely assay cell viability and that incorporates the ability to monitor the time course of neuronal cell death as a cardinal feature. The utility of this approach is demonstrated in further reports in which it is used to characterize and study the pro tective and toxic effects of moderate and profound hypothermia on excitotoxicity (Tymianski et ai., 1997a,b). The dissociated cells were plated in the precoated culture wells (15-mm d...

Distinct Influx Pathways, Not Calcium Load, Determine Neuronal Vulnerability to Calcium Neurotoxicity

“…Mixed cortical cell cultures were prepared from embryonic Swiss mice at 15 days of gestation as previously described (Sattler et al, 1997), with minor modifications from Choi (1987). In brief, cerebral cortices were incubated for 10-12 min in 0.05% trypsin in EDTA, dissociated by trituration, and plated on poly-L-ornithine-coated 24-well plates (Corning) or 25-mm glass coverslips at a density of 0.43 X l0~cells/well or 0.9 X l0~cells/coverslip.…”

“…Cell death was determined by serial quantitative measurements of FI fluorescence using a multiwell plate fluorescence scanner (Cytofluor II, PerSeptive Biosystems) as described (Sattler et al, 1997). In brief, the culture medium in each tissue culture well was replaced with Fl-containing control solution (50 ‚ug/ml), a baseline fluorescence reading was taken, and subsequent readings were taken at appropriate intervals for 24 h after the experimental manipulation.…”

“…In brief, the culture medium in each tissue culture well was replaced with Fl-containing control solution (50 ‚ug/ml), a baseline fluorescence reading was taken, and subsequent readings were taken at appropriate intervals for 24 h after the experimental manipulation. The fraction of dead cells in each culture at a given time was calculated as: fraction dead = (F, -F0)/FNMDA, where F, is PI fluorescence at time t, F0 is initial Pl fluorescence at time zero, and FNMDA is background-subtracted Pl fluorescence of identical cultures from the same dissection and plating 24 h after a 60-min exposure to i mM NMDA at 37°C.Based on manual observations, this NMDA exposure routinely produced nearly complete neuronal death in each culture, but had no effect on surrounding glia (also see Bruno et al, 1994;David et al, 1996;Sattler et al, 1997). Adding Triton X-100 (0.1%) to cultures treated in this manner produced an additional 10-15% increase in Pl fluorescence due to permeabilization of nonneuronal cell membranes, consis-tent with a 10-15% glial component in the cultures (R. Sattler and M. Tymianski, unpublished observations Cultures on glass coverslips were loaded with fura-2/acetoxymethyl ester (AM; 2 ‚uM) or SBFI/AM (10 ‚uM; Molecular Probes Inc.) and viewed on a Nikon Diaphot with xenon epifluorescence optics through a fluorite oil-immersion lens (Nikon CF UV-F X40, NA = 1.3).…”

“…Columns indicate the mean (+ SE) 45Ca2 accumulation in four cultures exposed to 1 mM NMDA for 60 min at 37°C24 h before exposure to the indicated agonist. The NMDA pretreatment kills >95% of neurons in the plate, leaving only nonneuronal cells (Sattler et al, 1997).…”

“…2B), glutamate and NMDA toxicity was restricted to neurons, leaving glia unaffected (Sattler et al, 1997; see also Koh and Choi, 1987;Bruno et al, 1994;David et al, 1996). To determine the cell type undergoing 45Ca2~accumulation under the same circumstances, neurons were selectively destroyed by a 60-min pretreatment with NMDA (1 mM, 37°C) (Sattler et al, 1997).…”