We investigated to what extent Pitx3 deficiency, causing hyperdopaminergic transmission in the nucleus accumbens microcircuitry, may lead to developmental changes. First, spontaneous firing activity of cholinergic interneurons in the nucleus accumbens was recorded in vitro. Firing patterns in the Pitx3-deficient mice were more variable and intrinsically different from those observed in wild-type mice. Next, to test whether the irregular firing patterns observed in mutant mice affected the endogenous nicotinic modulation of the GABAergic input of medium spiny neurons, we recorded spontaneous GABAergic inputs to these cells before and after the application of the nicotinic receptor blocker mecamylamine. Effects of mecamylamine were found in slices of either genotype, but in a rather inconsistent manner. Possibly this was attributable to heterogeneity in firing of nearby cholinergic interneurons. Thus paired recordings of cholinergic interneurons and medium spiny neurons were performed to more precisely control the experimental conditions of the cholinergic modulation of GABAergic synaptic transmission. We found that controlling action potential firing in cholinergic neurons leads to a conditional increase in GABAergic input frequency in wild-type mice but not in Pitx3-deficient mice. We conclude that Pitx3-deficient mice have neural adaptations at the level of the nucleus accumbens microcircuitry that in turn may have behavioral consequences. It is discussed to what extent dopamine release in the nucleus accumbens may be a long-term gating mechanism leading to alterations in cholinergic transmission in the nucleus accumbens, in line with previously reported neural adaptations found as consequences of repeated drug treatment in rodents. Copyright © 2006 The American Physiological Society.