Whole-cell recordings were obtained from retinal ganglion cells of the tiger salamander (Ambystoma tigrinum) in a superfused slice preparation to evaluate contributions of NMDA (N-methyl-D-aspartate) and KA/AMPA (kainate/α-amino-3-hydroxy-5-methyl-4-isoxalone propionic acid) receptors to excitatory postsynaptic potentials (EPSPs) of retinal ganglion cells. Synaptic activation of retinal ganglion cells was achieved through the use of a brief pressure pulse of hyperosmotic Ringer (Ringer + sucrose) delivered through a microelectrode visually placed in the inner plexiform layer while whole-cell recordings were obtained from adjacent cells in the ganglion cell layer. Separation of NMDA and KA/AMPA excitatory postsynaptic currents (EPSCs) was achieved through the application of the antagonists NBQX and D-AP7, while inhibitory currents were blocked by strychnine and picrotoxin. Simple addition of the two independent EPSCs showed, most often, that the sum of the KA/AMPA and NMDA currents was less than the control response, but in some cases the sum of the two currents exceeded the magnitude of the control response. Neither result was consistent with expectations based on voltage-clamp principles and the assumption that the two currents were independent; for this reason, we considered the possibility of nonlinear interactions between KA/AMPA and NMDA receptors. Computer simulations were carried out to evaluate the summation experiments. We used both an equivalent cylinder model and a more realistic, compartmental model of a ganglion cell constrained by a passive leakage conductance, a linear KA/AMPA synaptic current, and a nonlinear NMDA current based on the well-known, voltage-sensitive Mg2+ block. Computer simulation studies suggest that the hypo- and hyper-summation of NMDA and KA/AMPA currents, observed physiologically, can be accounted for by a failure to adequately space clamp the neuron. Clamp failure leads to enhanced NMDA currents as the ion channels are relieved of the Mg2+ block; their contribution is thus exaggerated depending on the magnitude of the conductance change and the spatial location of the synaptic input.

Effects of the major glutamate receptor agonists, kainate (KA), α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), quisqualate (QA), N-methyl-D-aspartate (NMDA), L-α-amino-4-phosphonobutyrate (L-AP4), and trans-l-aminocyclopentane-1,3-dicarboxylic acid (ACPD) on horizontal cells (HCs) were studied in superfused larval tiger salamander retina. 20 μM of KA, AMPA, and QA mimicked the action of 3 mM glutamate in the absence and presence of 1 mM Co2+-20 μM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) blocked the actions of KA and AMPA, but not those of QA and glutamate, indicative of the existence of CNQX-resistant QA receptors in the tiger salamander HCs. Prolonged application of ACPD hyperpolarized the HCs and enhanced the light responses, probably by shifting the resting HC voltage (Er) to a more hyperpolarized position. It is possible that the KA, AMPA, and CNQX-resistant QA receptors are involved in mediating the postsynaptic light responses in HCs, and ACPD receptors are involved in sensitivity adjustment of the HC responses.

The seminal in vivo work of Mircea Steriade and his colleagues has, as is always the case with the most insightful and creative scientists, raised many serious questions: questions, for example, of the detailed cellular and molecular mechanisms of the phenomena they discovered. In our tribute to this great investigator, we shall present some examples, based on our in vitro and modeling work, that shed light on some of his remarkable breakthroughs: the slow oscillation of sleep (<1 Hz); the several types of fast oscillations that are superimposed on the intracellular depolarizing, or active, phases of the slow oscillation; and the transition from slow oscillation to seizure via a period of very fast (>70 Hz) oscillations.

The superfused retinal slice preparation was used to examine the morphology and glutamate-activated whole-cell currents of rabbit bipolar cells. There were six morphologically distinct types of cone bipolar cells and a rod bipolar cell that had axon terminals stratifying in stratum 3 to 5 of sublamina-b. All of these bipolar cell types exhibited an outward current in response to the application of the metabotropic glutamate receptor, mGluR6, agonist AP-4 (APB), and had I/V curves indicative of membrane channel closure. Conversely, there were no currents activated during the application of kainate, the AMPA/kainate receptor agonist. These data demonstrate they were on-bipolar cells. In addition, there were six morphologically distinct cone bipolar cells that stratified in sublamina-a. Every cell with axonal arborizations in stratum 1 and 2 exhibited an inward current when the ionotropic glutamate receptor agonist kainate was applied. This current was blocked by application of the AMPA/kainate receptor antagonist CNQX. These cells also decreased their membrane resistance in response to kainate, a characteristic of the opening of channels within the plasma membrane. Without exception, no cells stratifying in sublamina-a responded to the mGluR6 agonist AP-4, further identifying them as off-bipolar cells.

The effects of kainate receptor-preferring glutamate ligands were tested on the electroretinogram (ERG) of the Xenopus retina. Kainate, domoic acid, and 5-iodowillardiine (20–100 μM) acted similarly in every respect. They increased peak amplitudes of the ERG a-, b-, and d-waves significantly over controls. The AMPA-specific antagonist, GYKI 53655, prevented a kainate-induced increase in ERG a- and d-waves, but was without effect on an increase in the b-wave. Once the effect of agonist on the b-wave had peaked, the ERG began to subside, leading to its nearly complete disappearance within 20 min. Prior exposure to GYKI followed by a combination of GYKI + agonist did not significantly slow the rate of b-wave disappearance. Our results indicate that (1) AMPA receptors contribute to ERG a- and d-waves. (2) The kainate-evoked increase in ERG a-, b-, and d-waves probably results, in part, from an excitotoxic swelling of inner retinal processes. (3) The inner retina has a population of GYKI-resistant, kainate-sensitive receptors which may contribute to b-wave generation.