GABA is the main inhibitory neurotransmitter in adults. Depolarizing GABA responses have been well characterized at neuronal-population average level during typical neurodevelopment and partially in brain disorders. However, no investigation has specifically assessed whether a mosaicism of cells with either depolarizing or hyperpolarizing/inhibitory GABAergic responses exists in animals in health/disease at diverse developmental stages, including adulthood. Here, we showed that such mosaicism is present in WT and Down syndrome (DS) neuronal-networks, as assessed at increasing scales of complexity (cultures, brain-slices, behaving mice). Nevertheless, WT mice presented a much-lower percentage of cells with depolarizing GABA than DS mice. Restoring the mosaicism of hyperpolarizing and depolarizing GABA-responding neurons to WT levels rescued anxiety behaviour in DS mice. Moreover, we found heterogeneous GABAergic responses in developed control and trisomic human iPSC-derived neurons. Thus, a heterogeneous subpopulation of GABA-responding cells exists in physiological/pathological conditions in mouse and human neurons, possibly contributing to disease-associated behaviours.