A CNS catecholaminergic cell line, Cath.a, was established by targeted oncogenesis in transgenic mice. Cath.a cells express neuronal properties but lack neuronal morphology. Here, we describe a variant of Cath.a, called CAD (Cath.a-differentiated), in which reversible morphological differentiation can be initiated by removal of serum or exogenously added protein from the medium. In serum- or protein-free media, CAD cells stop proliferating and extend long processes. Differentiated CAD cells can be maintained without serum or protein for at least 6 weeks. CAD cells are distinct from Cath.a cells; most significant, the original immortalizing oncogene, SV40 T antigen, was spontaneously lost. By immunostaining or immunoblotting, we show that CAD cells express neuron-specific proteins, such as class III beta-tubulin, GAP-43, SNAP-25, and synaptotagmin, but not GFAP. Ultrastructurally, processes from differentiated CAD cells have abundant parallel microtubules and intermediate filaments, and bear varicosities that contain both large dense-core vesicles/granules (120-160 nm) and smaller clear vesicles (60-80 nm). Additionally, CAD cells express enzymatically active tyrosine hydroxylase and accumulate L-DOPA. CAD cells exhibit biochemical and morphological characteristics of primary neurons and provide an unique tool for studying neuronal differentiation.

We report here the results of a chemical genetic screen using small molecules with known pharmacologies coupled with a cortical brain slice-based model for ischemic stroke. We identified a smallmolecule compound not previously appreciated to have neuroprotective action in ischemic stroke, the cardiac glycoside neriifolin, and demonstrated that its properties in the brain slice assay included delayed therapeutic potential exceeding 6 h. Neriifolin is structurally related to the digitalis class of cardiac glycosides, and its putative target is the Na ؉ ͞K ؉ -ATPase. Other cardiac glycoside compounds tested also showed neuroprotective activity, although with lower apparent potencies. In subsequent whole-animal studies, we found that neriifolin provided significant neuroprotection in a neonatal model of hypoxia͞ischemia and in a middle cerebral artery occlusion model of transient focal ischemia. The neuroprotective potential of Na ؉ ͞K ؉ -ATPase is of particular interest because of its known ''druggability''; indeed, Food and Drug Administration-approved, small-molecule compounds such as digitoxin and digoxin have been in clinical usage for congestive heart failure and arrhythmias for several decades. Thus, an existing cardiac glycoside or closely related compound could provide an accelerated path toward clinical trial testing for ischemic stroke. Our findings underscore the important role that hypothesis-neutral, high-content, tissue-based screens can play in the identification of new candidate drugs and drug targets for the treatment of diseases for which validated therapeutic pathways are not currently available.biotechnology ͉ drug discovery ͉ high-content screening ͉ translational medicine

The specific binding of benzodiazepines (BZDs) has been observed in many tissues and cell types and can be pharmacologically separated into two classes. The first and most studied are the central-type receptors in brain that are thought to mediate the known clinical effects of the BZDs (1-3). The other class is the peripheral-type binding site found in a variety of tissues and cells, including kidney (4, 6), heart (5, 6), platelets (7), mast cells (8), lymphocytes (9), many cell lines (10), and brain (11,12). BZD binding to this site has no known physiological consequences. Several pharmacological effects of the BZDs have been reported (13-15); however, none has been supported by a positive correlation between the peripheral-type binding and the biological response. We now report the presence of specific, (New England Nuclear, 101.0 Ci/mmol) and unlabeled 0.5 ,uM thymidine. After 2 hr, the cells were harvested and washed over glass fiber filters by a Microharvester (Bellco Glass). The filters were dried and placed in 4 ml of Aquasol (New England Nuclear) in minivials for scintillation spectroscopy.Binding Studies. The thymnoma cells were centrifuged at 100 x g and washed once in balanced glucose salt buffer (5.0 mM KCI/120.0 mM NaCI/5.0 mM Na2HPO4/5.0 mM Tris/0.6 mM CaCl2/1.0 mM MgSO4/5.5 mM glucose, final pH = 7.4). Binding of [3H]Ro5-4864 (New England Nuclear, 73.8 Ci/mmol) to the cells was carried out in this buffer at 0°C for 40 min with the indicated concentrations of labeled ligand in a total volume of 250 ,ul.[3H]Diazepam (New England Nuclear, 87.6 Ci/mmol) binding was also carried out at 0°C but the incubation time was 15 min. Binding reaction was terminated by the addition of 3 ml of ice-cold Dulbecco's phosphate-buffered saline and rapid filtration over Whatman GF/B glass fiber filters, followed by two washes of 3 ml of ice-cold phosphate-buffered saline. The filters were air dried and placed in 4 ml of Aquasol for liquid scintillation spectroscopy. Specific binding was defined as the difference between total binding (in the absence of unlabeled ligand) and nonspecific binding (in the presence of unlabeled 10 ,M diazepam).The benzodiazepine compounds were obtained from Hoffmann-La Roche, Inc. All reagents were obtained from commercial sources. RESULTSThe specific binding of [3H]Ro5-4864, a BZD selective for peripheral-type sites (4), was saturable, whereas nonspecific binding increased linearly with increasing concentrations of the labeled ligand (Fig. 1). Scatchard analysis of the specific binding showed a single class of sites with a Kd (mean ±

The wide-ranging neuronal actions of glutamate are thought to be mediated by postsynaptic N-methyl-D-aspartate (NMDA) and non-NMDA receptors. The present report demonstrates the existence of presynaptic glutamate receptors in isolated striatal dopaminergic nerve terminals (synaptosomes). Activation of these receptors, by NMDA in the absence of Mg2+ and presence of glycine and by non-NMDA agonists in the presence of Mg2+, results in Ca(2+)-dependent release of dopamine from striatal synaptosomes. The release stimulated by NMDA is blocked by Mg2+ and by selective NMDA antagonists, whereas the release stimulated by selective non-NMDA agonists is blocked by a non-NMDA antagonist but not by Mg2+ or NMDA antagonists. Thus, these presynaptic glutamate receptors, localized on dopaminergic terminals in the striatum, appear to be pharmacologically similar to both the NMDA and the non-NMDA postsynaptic receptors. By modulating the release of dopamine, these presynaptic receptors may play an important role in transmitter interactions in the striatum.

The wide-ranging neuronal actions of excitatory amino acids, such as glutamate, are thought to be mediated mainly by postsynaptic N-methyl-D-aspartate (NMDA) and non-NMDA receptors. We now report the existence of presynaptic glutamate receptors in isolated nerve terminals (synaptosomes) prepared from hippocampus, olfactory bulb, and cerebral cortex. Activation of these receptors by NMDA or non-NMDA agonists, in a concentration-dependent manner, resulted in Ca(2+)-dependent release of noradrenaline from vesicular transmitter stores. The NMDA-stimulated release was potentiated by glycine and was blocked by Mg2+ and selective NMDA antagonists. In contrast, release stimulated by selective non-NMDA agonists was blocked by 6-cyano-7-nitroquinoxaline-2,3- dione, but not by Mg2+ or NMDA antagonists. Our data suggest that the presynaptic glutamate receptors can be classified pharmacologically as both the NMDA and non-NMDA types. These receptors, localized on nerve terminals of the locus ceruleus noradrenergic neurons, may play an important role in interactions between noradrenaline and glutamate.