Project Summary (Project 1) ! Project One will explore the complementary influences of intrinsic genetic programs and activity to the development of 5HT3aR interneurons. Aim 1 will examine the transcriptional and epigenetic function of Prox1 in 5HT3aR cINs. By examining the targets of Prox1 in 5HT3aR cells we will provide insight in the genetic mechanisms that generate these populations, as well as the basis by which Prox1 directs synaptogenesis,morphology and circuit connectivity (Aim 2). Will examine how activity alters gene expression with 5HT3aR cINs and the role of L-type Ca2+ channels in the activity-dependent development and plasticity of 5HT3aR cINs. This aim will proceed in two subparts: (subaim 2a) Where we will use intersectional genetic methods to target the five L1-3 5HT3aR interneuronal subtypes, by cell autonomously suppressing (Kir2.1) or increasing (NachBac) their activity and use RNA-seq to determine how it alters gene expression within specific subpopulations. And (subaim 2b) Where we will use conditional genetic methods to remove L-Type Ca2+ function (Cav1.2, Cav1.3) from 5HT3aR cINs. This aim will yield insights into the cell autonomous requirement for L-type channels in 5HT3aR cIN populations. , (Aim 3). Will examine the establishment the developmental connectivity of 5HT3aR cINs. This aim will also proceed with two subparts: (subaim 3a) which will examine the development of interneuron/pyramidal cells ensembles within the superficial cortex. This will use a combination of mono-synaptic rabies tracing, genetic encoded Calcium-sensors (gCaMP/rCaMP) and optogenetics to examine the afferent and efferent connectivity of specific 5HT3aR cIN subtypes in L1. Of particular interest will be to compare how this circuitry compares with that seen in the mature auditory and somatosensory cortices and (subaim 2b) which extend our examination of Prox1 and L-Type Ca2+ channels affect the formation of the connectivity of the 5HT3aR cINs during development . Specifically, examination as to how changes in the intrinsic genetic program (Aim1) or activity-mediated maturation (Aim 2) manifest in changes in the circuit function of 5Ht3aR interneurons (as explored by Dr. Rudy in Project 2) and the ability of cholinergic afferents and L1 interneurons to influence plasticity and attention in a Go-No go task where mice are examined for their responses to salient versus non-salient cues (Froemke in Project 3). Together project 1 will provide insight into how assembly of L1-3 circuits that are critically dependent upon 5Ht3aR interneurons impact adult connectivity, plasticity and function.!