The pharmacology of synapses formed by identified corticocollicular neurons in primary cultures of rat visual cortex

Abstract

Primary cultures of neurons from the visual cortex of 7-10-d-old Long Evans rats were used to study the pharmacology of synaptic transmission. Dissociated cells were grown either in mass cultures, which contained 8000-10,000 neurons, or in miniature island cultures of 50-100 cells. Prior to dissociation, cells in layer V of visual cortex that project to the superior colliculus were labeled in vivo by retrograde transport of fluorescent latex microsphereas - a permanent fluorescent marker. After 2 d to 8 weeks in culture, labeled neurons were identified by epifluorescent illumination, and electrophysiological recordings were obtained from a labeled cell and, simultaneously, from a nearby unlabeled neuron in the same field of view. The 2 neurons were stimulated sequentially by current injection and the pharmacology of evoked postsynaptic potentials (PSPs) was investigated. In mass cultures, relatively few pairs of neurons from which we recorded were synaptically connected, although nearly every cell exhibited abundant spontaneous EPSPs and IPSPs. Neurons grown on island cultures generally did not exhibit spontaneous synaptic activity; however, stimulation of one of the cells in a pair frequently elicited a short-latency PSP in the follower neuron. Retrogradely labeled corticocollicular neurons produced only excitatory PSPs in follower cells, while unlabeled neurons were either excitatory or inhibitory. Three antagonists of excitatory amino acid receptors, kynurenic acid, piperidine dicarboxylic acid, and γ-D-glutamylglycine, completely blocked EPSPs produced by labeled corticocollicular neurons, as well as EPSPs produced by nearly all of the unlabeled excitatory cells. We have previously shown that these compounds block both N-methyl-D-aspartate (NMDA)-type and non-NMDA receptors on cultured cortical neurons (Huettner and Baughman, 1986). The specific NMDA receptor antagonist 2-amino-5-phosphonovaleric acid (APV) did not alter short-latency EPSPs recorded in 1 mM Mg2+, but did reduce longer-latency polysynaptic activity. Since responses mediated by the NMDA receptor are known to be antagonized by Mg2+ (Mayer and Westbrook, 1985), we perfused cultures with Mg2+-free medium and found that the falling phase of some monosynaptic EPSPs was prolonged. Addition of APV to Mg2+-free medium reduced the duration of the falling phase of EPSPs such that they returned to the time course obtained in 1 mM Mg2+. Finally, we found that IPSPs were completely blocked by 10-20 μM bicuculline methiodide and that the action potentials of inhibitory cells were significantly narrower (width at half-height=1.0±0.14 msec; n=18) than were action potentials of excitatory neurons (2.0±0.47 msec; n=51). Our results provide evidence that excitatory synaptic transmission between cortical neurons in culture is mediated by receptors for excitatory amino acids. In solutions containing physiological levels of Mg2+, EPSPs are largely due to non-NMDA receptors, but in Mg2+-free medium, most EPSPs displayed a slow, APV-sensitive component attributable to the NMDA receptor.