Memantine increases NMDA receptor level in the prefrontal cortex but fails to reverse apomorphine-induced conditioned place preference in rats

Studies have shown that inflammation and neurodegeneration may accompany the development of addiction to apomorphine and that the glutamate NMDA receptor antagonist, memantine, may be neuroprotective. The similarity between apomorphine and dopamine with regard to their chemical, pharmacological and toxicological properties provided a basis for investigating the mechanism of action of the former agent. In this study, we investigated whether memantine would suppress apomorphine-seeking behavior in rats subjected to apomorphine-induced place preference conditioning, through modulation of NMDA receptors in the prefrontal cortex. Repeated apomorphine (1 mg/kg) treatment induced conditioned place preference (CPP) and had no significant effect on NMDA receptor levels in the prefrontal cortex. Prior treatment with memantine (5 mg/kg or 10 mg/kg) increased the levels of NMDA receptors in the prefrontal cortex but did not suppress CPP induced by apomorphine. These data give further support to the addictive effect of apomorphine and demonstrate that blockade of NMDA receptors by memantine is unable to suppress apomorphine-seeking behavior

Preparation, characterization and utility of a novel antibody for resolving the spatial and temporal dynamics of the calcium chelator BAPTA

In spite of its importance as a tool to manipulate cell calcium, the versatility of the octadentate chelator BAPTA in cell physiological and diverse other applications is limited by the difficulty with which it can be quantified and its cell and tissue distributions determined. Conventional approaches, such as HPLC analysis or autoradiography, are of limited sensitivity and resolution and have attendant biohazard risks. We now describe a versatile, facile and inexpensive means for quantifying and determining the distribution of BAPTA which exploits an immunological approach based on our generation of novel antibodies to BAPTA. Antibodies to BAPTA were prepared by immunizing rabbits with BAPTA conjugated to keyhole limpet hemocyanin via a zero-order cross-linking reagent - EDC. The ability of anti-BAPTA IgGs to recognize free or conjugated BAPTA was confirmed using enzyme-linked and immunoblotting assays made possible by our introduction of a BAPTA-BSA adduct. Using such assays, we show that the anti-BAPTA antibodies possess marked selectivity for BAPTA compared to several structurally-related BAPTA analogs. The utility of the anti-BAPTA antibodies in cell calcium research has been confirmed in two ways. First, by determining the spatial distribution of BAPTA through immunocytochemistry and confocal microscopy of cortical neurons loaded with BAPTA/AM and, second, by determination of the kinetics of loading and efflux of BAPTA through enzyme-linked cell immunoassays (ELISA) and immunocytochemistry. Together, these data demonstrate that anti-BAPTA antibodies are a powerful new tool with which to quantify BAPTA and to define the spatial and temporal distribution of this important calcium chelator in live cells. Such information should greatly aid the design of cell physiological experiments, the development of new chelators and the identification of sources of chelator selectivity in emerging therapeutic applications

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\u27 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 \u3e 0.1 mg/ml, EDC caused virtually instantaneous, irreversible, fixation of \u3e 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