Selective changes in AMPA receptors in rabbit cerebellum following classical conditioning of the eyelid-nictitating membrane response.

alpha-Amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors are critically involved in several forms of synaptic plasticity proposed to be neural substrates for learning and memory, e.g., long-term potentiation and long-term depression (LTD). The present study was designed to determine changes in cerebellar AMPA receptors following classical conditioning of the eyeblink-nictitating membrane response (NMR) in the rabbit. Quantitative autoradiography was used to assess changes in ligand binding properties of cerebellar AMPA receptors following NMR conditioning elicited by pairing electrical stimulation of the pontine nuclei with an airpuff to the eye. [3H]AMPA and [3H]-6-cyano-7-nitroquinoxaline-2,3-dion (CNQX) binding were determined following preincubation of frozen-thawed brain tissue sections at 0 or 35 degreesC. With 0 degreesC preincubation, no significant differences in [3H]AMPA binding to cerebellar AMPA receptors were seen between any of the experimental groups tested. In contrast, preincubation at 35 degreesC revealed significant decreases in [3H]AMPA binding to the trained side of the cerebellar cortex resulting from paired presentations of the conditioned and the unconditioned stimuli, while unpaired presentations of the stimuli resulted in no significant effect. With 35 degreesC preincubation, there were no significant differences in [3H]CNQX binding between any of the experimental groups and no significant differences in [3H]AMPA binding in the untrained side of the cerebellum. These results indicate that NMR conditioning is associated with a selective modification of AMPA-receptor properties in brain structures involved in the storage of the associative memory. Furthermore, they support the hypothesis that cerebellar LTD, resulting from decreased synaptic efficacy at parallel fiber-Purkinje cell synapses mediated by a change in AMPA-receptor properties, is a form of synaptic plasticity that supports this type of learning.

Screening of receptor antagonists using agonist-activated patch clamp detection in chemical separations.

We present a capillary electrophoresis-patch clamp detection system optimized for screening of antagonists and inhibitors of ligand-gated ion channels. In this system, highly selective receptor agonists are delivered through the electrophoresis capillary to the cell surface where they continuously activate a receptor, resulting in increased steady-state transmembrane currents. Thus, receptor selection and biosensor functionality is simply achieved by selection of an appropriate agonist. The antagonists are fractionated in the same electrophoresis capillary and inhibit the agonist-evoked response, resulting in transiently decreased steady-state transmembrane currents. Specifically, a mixture containing 6-cyano-7-nitroquinoxaline-2,3-dione, that reversibly blocks alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate and kainate receptors, and 6,7-dichloro-3-hydroxy-2-quinoxaline-carboxylate, a broad-spectrum glutamate receptor antagonist, were separated and detected by kainate-activated patch-clamped interneurons freshly dissociated from rat brain olfactory bulb. In addition, Mg2+ that reversibly blocks the N-methyl-D-aspartate receptor in a voltage-dependent way was detected using the same cell detector system when activated by N-methyl-D-aspartate and the co-agonist glycine. The presented method offers new possibilities for drug screening and for identifying endogenous receptor antagonists and to determine their mode of action on any ionotropic receptor system of interest.