Laminar specific alterations of thalamocortical projections in organotypiccultures following layer 4 disruption in ferret somatosensory cortex

The developing neocortex influences the growth of thalamocortical projectio ns. Layer 4 in particular receives the majority of input from the thalamus and is important in instructing thalamic afferents to terminate. Previous i n vivo experiments demonstrated that disruption of layer 4 during corticoge nesis in ferret somatosensory cortex by application of methylazoxy methanol acetate (MAM) prevents proper termination of thalamic afferents in appropr iate cortical regions. To further explore the role of layer 4 in thalamocor tical development, we prepared organotypic cocultures consisting of normal gestational day 0 (P0) ferret thalamus paired with normal, embryonic day 33 (E33), or E38 MAM-treated cortex obtained from ferrets at either P0 or P7. Injection of MAM on E33 disrupts layer 4 formation, whereas similar inject ions on E38 interfere with layer 2 formation. The cocultures grew together for a number of days, then discrete injections of either fluorescent dextra ns or 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate ( DiI) were made into the thalamic piece. The labeled thalamic afferents that grew into the cortical slice were analysed and the sites of their terminat ions quantified after 3, 5, or 7-10 days in culture (DIC). Our results vari ed somewhat with the amount of time in culture, but the preponderance of th alamic fibers in normal cortex terminated in layer 4, whereas their counter parts in E33 MAM-treated cortex grew beyond the cortical plate and many fib ers terminated inappropriately within lower cortical layers or white matter . Terminal distribution of thalamic fibers in E38 MAM-treated cortex looked similar to normal. These results demonstrate that the cells of layer 4 pro vide thalamic afferents with important positional and termination cues.