Importantly, these new data also raise essential questions with regard to the debate about the safety and cognitive consequences of administering anesthetics to young infants. To examine the role of the general anesthetics on spine dynamics, we carried out both in vivo and in vitro experiments using different pharmacological tools including anesthetics which either enhance GABAergic transmission or interfere with excitatory NMDA dependent responses. For in vivo analyses, spine density and morphology were analyzed using the transgenic H-line mice expressing the yellow fluorescent protein in distinct subsets of cortical and hippocampal neurons from the second postnatal week. Mice were subjected to a 5 h anesthesia at different ages and then sacrificed, fixed through perfusion and spine characteristics analyzed. These data indicate that midazolam-induced increase in protrusions density is primarily due to a rapid increase in spinogenesis Bafilomycin A1 followed by an enhanced stabilization of newly formed protrusions. These combined, but transient effects probably account for the fact that the midazolaminduced increase in spine density lasted for several days. We next tested if new spines formed following midazolam application could be functional. After bolus loading of cells with the calcium indicator Fluo-4 AM, functionality of the new spines detected after midazolam application was tested by stimulation of Schaffer collaterals and recordings of calcium signals in individual spines. Out of 43 new spines tested, 22 SR9243 responded to stimulation by an increase in intracellular calcium. Spines that did not show calcium responses were thus either silent, non-functional or more simply, the presynaptic axon could not be stimulated through the depolarizing current pulses. Under control conditions, the same proportion of spines also failed to show calcium responses, indicating that newly formed spines have the same probability to be functional as preexisting spines. Furthermore, the responses of newly formed spines were comparable in amplitude with responses of neighboring preexisting spines, and were abolished by blocking fast glutamatergic transmission with AP5 and NBQX.