A distinctive characteristic of the AMPA subset of glutamate receptor channels is their remarkably rapid desensitization. A family of compounds, the benzothiadiazides, is described here that potently inhibit rapid glutamate receptor desensitization. The structure- activity relationships of these compounds are examined and the actions of cyclothiazide (CYZ), the most potent of these compounds, are described in detail. At the macroscopic level CYZ reduced rapid desensitization, enhancing the steady-state and peak current produced by 1 mM quisqualate with EC50 values of 14 and 12 microM, respectively, and shifted the quisqualate peak current concentration-response relation to the left. The slight outward rectification of the steady- state quisqualate current-voltage relationship was reduced by CYZ. At the microscopic level CYZ caused glutamate to induce long bursts of channel openings, and greatly increased the number of repeated openings. At 10 microM CYZ did not have measurable effects on the fast component of deactivation nor did it have statistically significant effects on the distribution of the faster components of glutamate- induced burst duration. In contrast, 10 microM CYZ increased the amplitude and significantly prolonged the duration of the spontaneous miniature EPSCs. The identification and characterization of this new family of gating modifiers may further facilitate the investigation into the mechanisms underlying rapid glutamate receptor desensitization and the physiological roles that it may serve.

Focal activation of glutamate receptors in distal dendrites of hippocampal pyramidal cells triggers voltage-dependent Ca(2+) channel-mediated plateau potentials that are confined to the stimulated dendrite. We examined the role of dendritic K(+) conductances in determining the amplitude, duration, and spatial compartmentalization of plateau potentials. Manipulations that blocked SK-type Ca(2+)-activated K(+) channels, including apamin and BAPTA dialysis, increased the duration of plateau potentials without affecting their amplitude or compartmentalization. Manipulations that blocked Kv4.2 A-type K(+) channels, including a dominant-negative Kv4.2 construct and 4-aminopyridine, increased the amplitude of plateau potentials by allowing them to recruit neighboring dendrites. Prolongation of plateau potentials or block of Kv4.2 channels at branch points facilitated the ability of dendritic excitation to trigger fast action potentials. SK channels thus underlie repolarization of dendritic plateau potentials, whereas Kv4.2 channels confine these potentials to single dendritic branches, and both act in concert to regulate synaptic integration.

The influence of external [Hf] on whole-cell and single-channel currents activated by glutamate agonists was studied in rat hippocampal neurons. In the pH range between 6.6 and 8.0, changes in external [HI] Glutamate is believed to be the neurotransmitter responsible for excitatory synaptic transmission within much of the mammalian central nervous system (1, 2). The N-methyl-Dasparate (NMDA) channel is one of several subsets of neuronal ionic channels activated by glutamate. The NMDA channel is characterized by (i) high permeability to Ca2+ (3),(ii) marked potentiation by glycine (4), and (iii) voltage dependence in the presence of external Mg2+ (5, 6). Activation of the NMDA channel is required for the induction of long-term potentiation (7), one mechanism thought to be involved in learning and memory. Excessive activation may contribute to the generation of seizures (8). Calcium entry through NMDA channels has also been postulated to contribute to the irreversible neuronal lesions associated with hypoxic/ischemic injury (9). receptor-channel complex. In preliminary reports (11), we noted that although the monovalent ion-transporting quisqualate and kainate channels were relatively insensitive to [H+]O, the divalent ion-permeant NMDA channels were highly regulated by [H+]0. In this study we characterize the pH sensitivity of the NMDA-activated current and investigate further the site and mechanism by which H+ modulates the NMDA receptor-channel complex. METHODSRat hippocampal neurons obtained from 21-day embryos and maintained in dissociated cell culture for 2 to 4 weeks were used in these studies. The hippocampus was removed, incubated in trypsin (0.027%; Sigma) in Ca2+-and Mg2+-free medium, tritiated, and filtered to remove clumps. Neurons were plated on polylysine-coated coverslips at 6 x 105 cells per 35-mm tissue-culture dish. Cells were maintained in Dulbecco's minimum essential medium/5% Ham's F-12 medium containing glutamine, 5% Hyclone calf serum, penicillin at 50 units, and 0.05 mg of streptomycin per ml. The medium was changed three times per week. Cytosine arabinoside (10-5 M) was added for 24 hr at -1 week to suppress background cell overgrowth.

The dendritic arbor of pyramidal neurons is not a monolithic structure. We show here that the excitability of terminal apical dendrites differs from that of the apical trunk. In response to fluorescence-guided focal photolysis of caged glutamate, individual terminal apical dendrites generated cadmium-sensitive all-or-none responses that were subthreshold for somatic action potentials. Calcium transients produced by all-or-none responses were not restricted to the sites of photolysis, but occurred throughout individual distal dendritic compartments, indicating that electrogenesis is mediated primarily by voltage-gated calcium channels. Compartmentalized and binary behavior of parallel-connected terminal dendrites can greatly expand the computational power of a single neuron.