Pharmacological characterization of threo-3-methylglutamic acid with excitatory amino acid transporters in native and recombinant systems.

The glutamate analog (+/-) threo-3-methylglutamate (T3MG) has recently been reported to inhibit the EAAT2 but not EAAT1 subtype of high-affinity, Na(+)-dependent excitatory amino acid transporter (EAAT). We have examined the effects of T3MG on glutamate-elicited currents mediated by EAATs 1-4 expressed in Xenopus oocytes and on the transport of radiolabeled substrate in mammalian cell lines expressing EAATs 1-3. T3MG was found to be an inhibitor of EAAT2 and EAAT4 but a weak inhibitor of EAAT1 and EAAT3. T3MG competitively inhibited uptake of D-[(3)H]-aspartate into both cortical and cerebellar synaptosomes with a similar potency, consistent with its inhibitory activity on the cloned EAAT2 and EAAT4 subtypes. In addition, T3MG produced substrate-like currents in oocytes expressing EAAT4 but not EAAT2. However, T3MG was unable to elicit heteroexchange of preloaded D-[(3)H]-aspartate in cerebellar synaptosomes, inconsistent with the behavior of a substrate inhibitor. Finally, T3MG acts as a poor ionotropic glutamate receptor agonist in cultured hippocampal neurons: concentrations greater than 100 microM T3MG were required to elicit significant NMDA receptor-mediated currents. Thus, T3MG represents a pharmacological tool for the study of not only the predominant EAAT2 subtype but also the EAAT4 subtype highly expressed in cerebellum.

Characterization of gamma-aminobutyric acid receptor GABAB(1e), a GABAB(1) splice variant encoding a truncated receptor.

We have identified a splice variant encoding only the extracellular ligand-binding domain of the gamma-aminobutyric acid B (GABA(B)) receptor subunit GABA(B(1a)). This isoform, which we have named GABA(B(1e)), is detected in both rats and humans. While GABA(B(1e)) is a minor component of the total pool of GABA(B(1)) transcripts detected in the central nervous system, it is the primary isoform found in all peripheral tissues examined. When expressed in a heterologous system, the truncated receptor is both secreted and membrane associated. However, GABA(B(1e)) lacks the ability to bind the radiolabeled antagonist [(3)H]CGP 54626A, activate G-protein coupled inwardly rectifying potassium channels, or inhibit forskolin-induced cAMP production when it is expressed alone or together with GABA(B(2)). Interestingly, when co-expressed with GABA(B(2)), not only does the truncated receptor heterodimerize with GABA(B(2)), the association is of sufficient avidity to disrupt the normal GABA(B(1a))/GABA(B(2)) association. Despite this strong interaction, GABA(B(1e)) fails to disrupt G-protein coupled inwardly rectifying potassium activation by the full-length heterodimer pair of GABA(B(1a))/GABA(B(2)).

Selective labeling of embryonic neurons cultured on astrocyte monolayers with 5(6)-carboxyfluorescein diacetate (CFDA).

A method for selectively labeling cultured neurons using the vital dye, 5(6)-carboxyfluorescein diacetate (CFDA), is described. This non-fluorescent membrane-permeant dye is cleaved by cytosolic esterases into the fluorescent anion, 5(6)-carboxyfluorescein (CF). Both astrocytes and neurons exhibit brilliant fluorochromasia within minutes of CFDA loading. However, following a brief rinse in buffered saline in the absence of CFDA, the astrocytes rapidly lose their cellular fluorescence while the neurons retain the dye for several hours. The fluorochromasia is uniformly distributed throughout the soma and processes which greatly facilitates the morphological identification of viable neurons. In addition, this protocol can be used to conveniently quantify neuronal survival in assays of the activities of neurotrophic or neurotoxic substances.

Calcium homeostasis in dissociated embryonic neurons: a flow cytometric analysis.

1. Ca2+ homeostasis in freshly dissociated neurons from embryonic rat hypothalamus, cortex, and brain stem was investigated with flow cytometry. Cells were dissociated from embryonic brain by enzymatic and mechanical means and were incubated with the acetoxymethylester derivative of the Ca(2+)-sensitive dye indo-1. Neurons hydrolyzed and retained the dye as determined by the intensity of fluorescence emission, whereas similarly treated cultured astrocytes gave very low-level fluorescence. 2. The fluorescence of the indo-1 dye was measured at two wavelengths (405 and 485 nm) for each cell. Data were collected only from those cells (presumptive neurons) with high levels of fluorescence. Methods were developed to calibrate the level of intracellular free calcium ([Ca2+]i) as the ratio of fluorescence at 410 and 485 nm. The level of intracellular free Ca2+ was then calculated for each neuron. 3. A wide distribution of resting [Ca2+]i was found, with a median of approximately 90 nM. After addition of ionomycin to cells in Ca(2+)-free medium, there was a transient increase in [Ca2+]i, suggesting that all embryonic neurons had internal Ca2+ stores. The presence of active calcium extrusion mechanisms was demonstrated with the use of ionomycin in Ca(2+)-containing medium and with metabolic inhibitors. Furthermore, incubation in sodium-free medium resulted in a transient increase in [Ca2+]i and a reduced ability to eliminate elevated [Ca2+]i from the cytoplasm, suggesting that calcium homeostasis was dependent on the activity of the Na(+)-Ca2+ exchange mechanism. 4. Depolarization with K+ or veratrine increased [Ca2+]i in approximately 20% of the cells. This increase was blocked by eliminating extracellular free Ca2+ or adding Co2+, nifedipine, or verapamil, suggesting mediation by voltage-sensitive calcium channels. 5. Neurons were sorted on the basis of high [Ca2+]i and placed into dissociated culture. After 24 h, neurons in culture retained indo-1 fluorescence, suggesting that populations of neurons can be collected on the basis of their levels of [Ca2+]i. 6. These results demonstrate that flow cytometric analysis allows the characterization of a variety of Ca(2+)-regulatory mechanisms in populations of freshly dissociated embryonic neurons. Although only a proportion of embryonic day 17 neurons exhibit voltage-sensitive calcium channels, all neurons have developed the ability to sequester and extrude Ca2+.

Basic fibroblast growth factor regulates the ability of astrocytes to support hypothalamic neuronal survival in vitro.

The putative neurotrophic effects of basic fibroblast growth factor (bFGF) were tested on embryonic hypothalamic neurons in dissociated cell culture. Basic FGF dramatically increased the survival of embryonic hypothalamic astrocytes plated on a poly-L-lysine (PLL) substrate. Basic FGF treatment also increased the number of hypothalamic neurons surviving in vitro; however, no neurotrophic effects were observed when astrocyte proliferation was prevented by using serum-free N2 medium or by using the mitotic inhibitor cytosine arabinoside. In contrast to effects when PLL was used as a substrate, bFGF reduced the survival of hypothalamic neurons plated on a confluent, contact-inhibited monolayer of astrocytes. This effect appears to be due to the direct actions of bFGF on astrocytes: treatment of confluent astrocytes with 5 ng/ml bFGF caused the protoplasmic astrocytes to develop a fibrillar morphology and reduced the ability of the astrocyte monolayer to promote neuronal survival after a further 24 hr in bFGF-free medium. It is concluded that in addition to its mitogenic effects, bFGF acts as a differentiation factor for protoplasmic astrocytes in vitro, and these morphological and functional changes may reflect the process of normal astrocytic development and response to brain injury in vivo.

Both survival and development of spontaneously active rat hypothalamic neurons in dissociated culture are dependent on membrane depolarization.

Suppression of endogenous electrical activity was found to have an adverse effect on the survival and bioelectric development of dissociated, embryonic rat hypothalamic neurons in long-term culture. Cultures were treated during the first two weeks in vitro with tetrodotoxin (TTX), a selective blocker of voltage-gated sodium channels, alone and in combination with high extracellular KCl ([K+]o), a membrane depolarizer. Neuron survival was assessed through cell counting experiments, while the development of spontaneous electrical activity was examined with extracellular, patch-electrode recordings. TTX caused both a decrease in cell survival and a decrease in spontaneously active cells; concurrent treatment with K+ protected cells from the adverse effects of TTX. K+ treatment alone increased the fraction of spontaneously active neurons without significantly affecting cell survival. When taken together, these results suggest that the long-term survival of active cells depends on continual membrane depolarization. From these observations, we conclude that there exists two populations of neurons: the electrically active population, whose survival is sensitive to electrical activity, and the quiescent population, whose survival is not.

Astrocyte topography and tenascin cytotactin expression: correlation with the ability to support neuritic outgrowth.

We have observed a heterogeneity in the ability of a monolayer of cultured rat astrocytes to support the attachment and growth of dissociated embryonic hypothalamic neurons in culture. Areas of the monolayer which have an uneven surface ('rocky' astrocytes) are poor substrates for neuronal attachment and neuritic outgrowth, while surrounding areas of the glial monolayer ('flat' astrocytes) support extensive neuronal growth. Astrocytes obtained from both neonatal cerebral cortex or hypothalamus displayed 'rocky' morphology. We utilized immunocytochemical techniques with antibodies directed against putative adhesion molecules to investigate the source of this heterogeneity. Antibodies against tenascin/cytotacin, fibronectin, laminin, N-CAM, thrombospondin, heparan sulfate proteoglycan, and the p185 protein product of the neu oncogene were employed in indirect-immunofluorescence experiments. We found that the difference in the surface properties of astrocytes appears to be correlated with the expression of the extracellular matrix molecule tenascin/cytotacin, but not with any of the other molecules we tested. Our data suggest that tenascin/cytotactin is inhibitory to neuronal attachment and process outgrowth in the developing nervous system.

Development of spontaneous electrical activity by rat hypothalamic neurons in dissociated culture.

The development of spontaneous electrical activity by embryonic rat hypothalamic neurons in dissociated culture was monitored using an extracellular, patch electrode recording method. Embryonic day 17 hypothalamic neurons were plated onto a feeder layer of astrocytes obtained from neonatal rat cerebral cortex, and extracellular electrical activity was monitored beginning the day after plating. The rates and patterns of spontaneous discharge were examined using interpike interval histograms. The percentage of spontaneously active neurons increased steadily with time in culture, from 13% in the first week to 56% during the sixth week in vitro. Although the percentage of spontaneously active cells increased, average firing rates did not change with time in culture. The pattern of electrical discharge was primarily random at all time points, with a small number of cells displaying regular activity while 4 cells were classified as phasic/bursty. In general, spontaneous action potential discharge was not dependent on synaptic transmission, as activity persisted after perfusion with bath solution containing either low Ca2+/high Mg2+ or kynurenic acid. Tetrodotoxin consistently and reversibly abolished spontaneous firing, regardless of culture age. We conclude that spontaneous activity in low density hypothalamic culture develops gradually though steadily, and is generated through an endogenous mechanism, independent of synaptic excitation.