AbstractOrganotypic cerebellar cultures derived from neonatal mice were exposed to the DNA synthesis inhibitor, cytosine arabinoside, or to cytosine arabinoside plus picrotoxin, an anti‐GABA agent that increased neuronal activity, for the first five daysin vitro. The group treated with cytosine arabinoside alone was subsequently maintained in standard nutrient medium, while the group exposed to both cytosine arabinoside and picrotoxin was continuously maintained in medium with incorporated picrotoxin. Granule cells were destroyed and astrocytes were functionally compromised in both culture groups, and both groups exhibited Purkinje cell axon collateral sprouting, with projection of sprouted inhibitory terminals to unensheathed Purkinje cell somata and to Purkinje cell dendritic spines in equal numbers. Spontaneous cortical discharge rates were the same in both groups, and antidromic stimulation of Purkinje cell axons induced inhibition of cortical activity. These results differed from those of a previous study in which chronic exposure of otherwise untreated cerebellar cultures to anti‐GABA agents increased the complement of inhibitory terminals on glially ensheathed Purkinje cell somata and resulted in a reduction of spontaneous cortical discharge rates. These differences were attributed to the failure of picrotoxin (1) to alter the plastic changes consequent to exposure to cytosine arabinoside, in which Purkinje cells had excess inhibitory projections, and (2) to extend inhibitory synaptogenesis in a system in which inhibitory synapse development was already enhanced.