The effects of artificial calcium buffers on calcium responses and glutamate-mediated excitotoxicity in cultured hippocampal neurons.

After loading cultured rat hippocampal neurons with teh acetoxymethyl ester of the Ca2+ buffer BAPTA, or its dimethyl analogue DMB, the magnitudes of transient (20-25 s) depolarization- or excitatory amino acid-induced Ca2+ responses were reduced, as were the rates of increase and recovery of [Ca2+]i. In contrast, during prolonged (3-30 min) stimulation, the magnitudes of the Ca2+ responses were not reduced in buffer-loaded neurons, even though the rates of increase and recovery were still much slower compared to neurons loaded with the control molecule half-BAPTA-AM. The potential consequences of this action of BAPTA and DMB were then examined in an in vitro model of excitotoxicity in which we found that, in both fetal and postnatal cultures, glutamate-induced excitotoxicity was enhanced, rather than reduced. An additional and unexpected observation was that during exposure of neurons to solutions containing BAPTA-AM, dimethyl-BAPTA-AM, or half-BAPTA-AM, we observed a rapid but reversible increase in intracellular [Ca2+] that appeared to be mediated via an activation of voltage-operated Ca2+ channels; most probably due to a direct depolarizing effect. We suggest that the presence of artificial Ca2+ buffers interferes with the normal Ca(2+)-dependent mechanisms for limiting Ca2+ entry during stimulation and thereby leads to an enhanced net Ca2+ influx. One consequence of this action is to enhance the potency of glutamate as an excitotoxic agent. These results agree with previous observations that excitotoxicity is better correlated with the total net flux of Ca2+, rather than measurements of intracellular ionic Ca2+. Our results do not support a potential use of artificial Ca2+ buffers as neuroprotective agents.

Mechanisms and effects of intracellular calcium buffering on neuronal survival in organotypic hippocampal cultures exposed to anoxia/aglycemia or to excitotoxins.

Neuronal calcium loading attributable to hypoxic/ischemic injury is believed to trigger neurotoxicity. We examined in organotypic hippocampal slice cultures whether artificially and reversibly enhancing the Ca2+ buffering capacity of neurons reduces the neurotoxic sequelae of oxygen-glucose deprivation (OGD), whether such manipulation has neurotoxic potential, and whether the mechanism underlying these effects is pre- or postsynaptic. Neurodegeneration caused over 24 hr by 60 min of OGD was triggered largely by NMDA receptor activation and was attenuated temporarily by pretreating the slices with cell-permeant Ca2+ buffers such as 1, 2 bis(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid acetoxymethyl ester (BAPTA-AM). This pretreatment produced a transient, reversible increase in intracellular buffer content as demonstrated autoradiographically using slices loaded with 14C-BAPTA-AM and by confocal imaging of slices loaded with the BAPTA-AM analog calcium green-acetoxymethyl ester (AM). The time courses of 14C-BAPTA retention and of neuronal survival after OGD were identical, indicating that increased buffer content is necessary for the observed protective effect. Protection by Ca2+ buffering originated presynaptically because BAPTA-AM was ineffective when endogenous transmitter release was bypassed by directly applying NMDA to the cultures, and because pretreatment with the low Ca2+ affinity buffer 2-aminophenol-N,N,O-triacetic acid acetoxymethyl ester, which attenuates excitatory transmitter release, attenuated neurodegeneration. Thus, in cultured hippocampal slices, enhancing neuronal Ca2+ buffering unequivocally attenuates or delays the onset of anoxic neurodegeneration, likely by attenuating the synaptic release of endogenous excitatory neurotransmitters (excitotoxicity).

A novel use for a carbodiimide compound for the fixation of fluorescent and non-fluorescent calcium indicators in situ following physiological experiments.

The inability to determine the precise intracellular location of non-fluorescent organic calcium chelators such as BAPTA is a persistent problem which has precluded much detailed analysis of the chelators' spatial or temporal dynamics in live cells. Similarly, following physiological experiments with fluorescent indicators like Fura-2, it has often been desirable to maintain the dye within the cell for later analysis by additional histological techniques. Based on chemical considerations, and its prior use in tissue fixation, we examined the water soluble reagent 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) as a potential fixative for diverse calcium chelators. The utility of EDC, but not other common fixatives, was confirmed through electrophysiological means, through a novel ELISA, which exploits anti-BAPTA antibodies to assess the extent and kinetics of fixation; by autoradiography of neurons loaded with [14C]-BAPTA, and by immunocytochemistry and imaging of intracellular BAPTA or Calcium Green in neurons. At concentrations > 0.1 mg/ml, EDC caused virtually instantaneous, irreversible, fixation of > 95% of BAPTA free acid. Fixation of intracellular BAPTA was confirmed in hippocampal brain slices loaded with BAPTA/AM ester, and showed biphasic kinetics consistent with rapid loading and subsequent extrusion of the chelator. Immunocytochemistry on neurons microinjected with BAPTA free acid and the dye Lucifer Yellow showed BAPTA-specific staining which was distributed in the cell similarly to that of the accompanying marker dye. Application of EDC also efficiently fixed in situ analogs of BAPTA such as Calcium Green (a fluorescent Ca2+ indicator) as shown by confocal imaging of EDC-fixed brain slices loaded with this indicator. Taken together, these data show that EDC is an effective, inexpensive and versatile fixative for calcium chelators in diverse cells. The availability of a suitable fixative now makes it possible to determine the distributions of such chelators at both the light and, possibly, the electron microscope level. Two important features of EDC, arise from its specificity for free carboxyl groups. First, the ability to fix, selectively, the chelators but not their AM esters; and, second, its enormous potential as a fixative for the numerous other carboxyl-containing chelators, dyes and pH indicators currently available.