The formation of functional synapses is a late milestone of neuronal differentiation. The establishment of functional synapses can be used to assess neuronal characteristics of different cell lines. In the present study, we examined the in vitro conditions that influence the ability of human neurons derived from the NT2 cell line (NT2N neurons) to establish synapses. The morphologic, immunologic, and electrophysiologic characteristics of these synapses was examined. In the absence of astrocytes, NT2N neurons rarely formed synapses and their action potentials were weak and uncommon. In contrast, when plated on primary astrocytes, NT2N neurons were able to form both glutamatergic excitatory (71%) and GABAergic inhibitory (29%) functional synapses whose properties (kinetics, ion selectivity, pharmacology, and ultrastructure) were similar to those of synapses of neurons in primary cultures. In addition, coculture of NT2N neurons with astrocytes modified the morphology of the neurons and extended their in vitro viability to more than 1 year. Because astrocyte‐conditioned medium did not produce these effects, we infer that direct contact between NT2N neurons and astrocytes is required. These results suggest that NT2N neurons are similar to primary neurons in their synaptogenesis and their requirement for glial support for optimal survival and maturation. This system provides a model for further investigations into the neurobiology of synapses formed by human neurons. J. Comp. Neurol. 407:1–10, 1999. © 1999 Wiley‐Liss, Inc.

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Temporal summation at dendrites of cultured rat hippocampal neurons was examined as a function of the interval separating two dendritic inputs. A novel method that relies on single-mode optical fibers to achieve rapid photorelease of glutamate was developed. Dendritic excitation achieved with this approach resembles that associated with miniature excitatory postsynaptic currents (mEPSCs), but the strengths, sites, and timing of the inputs can be precisely controlled. Dendritic summation deviated markedly from behavior predicted by passive cable theory. Subthreshold temporal summation varied as a triphasic function of the interpulse interval. As the interpulse interval decreased, local dendritic Na+ conductances were recruited to generate a marked transition from sublinear to supralinear summation. These results suggest that active dendritic conductances acting in concert with passive cable properties may serve to boost coincident synaptic inputs and attenuate noncoincident inputs.

The introduction of parallel patch clamp instruments offers the promise of moderate-throughput, high-fidelity voltage clamp for drug screening assays. One such device, the IonWorks HT (Molecular Devices, Sunnyvale, CA), was evaluated and compared to conventional human ethera- go-go-related gene (hERG) patch clamp data and an alternative functional screen based on rubidium flux. Data generated by the IonWorks HT and rubidium assays were compared to determine if either offered superior predictive value compared to conventional patch clamp. Concentration-effect curves for a panel of known hERG blockers were shifted to higher concentrations on the IonWorks HT compared to conventional voltage clamp determinations. The magnitude of the potency shifts was compound-specific and ranged from no shift (e.g., quinidine) to over 200-fold (astemizole). When the extreme value for astemizole was disregarded, the potency shift for 13 other known reference standards was 12-fold or less, with an average shift of fivefold. The same subset of compounds in the rubidium efflux assay exhibited an average potency shift of 12-fold. To provide a simulation of how the IonWorks HT assay might perform in a single concentration screening mode, a panel of test compounds was evaluated. The IonWorks HT screen did not outperform the rubidium efflux screen in predicting conventional voltage clamp measurements. The most likely explanation appears to rest with variable and compound-specific potency shifts in the IonWorks HT assay. The variable potency shifts make it difficult to select a screening concentration that meets the criterion of a high positive predictive value while avoiding false-positives.

We report the synthesis, the physicochemical characterization, and biological evaluation of a new caged glutamate, N-(o-nitromandelyl)oxycarbonyl-L-glutamic acid (Nmoc-Glu), that liberates free glutamate on photolysis. The low affinity of certain glutamate receptors and their rapid entry into desensitization have effectively prevented the creation of an ideal caged glutamate. In the absence of an ideal compound, Nmoc-Glu was designed to resist spontaneous hydrolysis while maintaining reasonable photorelease yield and kinetics. Chemical and physiological analyses reveal that NmocGlu, indeed, has exceptionally low residual activity and high chemical stability. The quantum yield of Nmoc-Glu is 0.11. Photolytic uncaging and release of free glutamate occur in two steps, consisting of an initial lightinduced cleavage that proceeds on the sub-millisecond time scale, and a subsequent light-independent, pH-dependent decarboxylation step that proceeds on the millisecond time scale. The low residual activity and high chemical stability of Nmoc-Glu are important advantages in applications where pre-photolysis Glu receptor activation and desensitization must be minimized.Non-NMDA 1 glutamate receptor (GluR) channels are the molecular entities that mediate the majority of the fast excitatory synaptic transmissions in the mammalian central nervous system (1). Studies aimed at improving understanding of the properties of synaptic non-NMDA GluR channels by direct application of glutamate are severely limited by poor access in the intact preparation. A potential solution to this problem is the use of "caged" compounds. A caged compound is an effector molecule whose activity is temporarily masked by the attachment of a photosensitive masking, or caging, group (2,3,16). Cleavage of the caging group by flash photolysis rapidly liberates the fully bioactive molecule to cause a "jump" in the concentration of the effector molecule. This feature, coupled with the fact that photolysis can be achieved with highly focused light beams, means that photorelease of caged molecules can afford excellent spatial and temporal control over reagent delivery to biological preparations. In situ photorelease of caged glutamate offers a potentially powerful means for studying the properties of synaptic GluRs, their distribution, and for eliciting action potentials from afar in a specifically targeted neuron (4, 5). However, a number of distinctive properties of GluRs present formidable challenges to the design of caged glutamate reagents. The non-NMDA subset of GluRs requires Ͼ1 mM glutamate for full activation, yet Ͻ10 M glutamate can induce significant desensitization in these same GluRs (6, 7). Furthermore, 10 M glutamate is sufficient to activate the NMDA subset of GluRs (1). An ideal caged glutamate should, therefore, give high yield of free glutamate on photolysis and should have minimal pre-photolysis activity and high chemical stability. Moreover, because entry into desensitization occurs on the millisecond time scale, photorelease must be suffi...