Anxiety is normal and adaptive. In its pathological form anxiety impairs the ability to optimally respond to the environment and can be debilitating. Anxiety states begin early in life; normal trait anxiety symptoms emerge around age 6, and anxiety disorders may present as early as age 11. Approximately 8% of 13-18 year olds exhibit pathological anxiety (NIMH). The incidence and etiology of anxiety suggest that early life is a critical time for the development of anxiety disorders. Serotonin (5-HT, 5-hydroxytryptamine) is one of the key neurotransmitters involved in anxiety disorders. Direct dysregulation of the midbrain 5-HT system early in development, but not in adulthood, results in a more anxious phenotype in rodents. The 5-HT system is connected to many forebrain areas also implicated in mediating anxiety, leading to the hypothesis that alterations are also occurring in these areas. Our recent studies in mice show that the cellular characteristics, synaptic activity, and 5-HT receptor-mediated responses of 5-HT neurons are immature and develop during the first several weeks of postnatal life. In contrast characteristics recorded from 5-HT1A receptor and Pet-1 knockout mice, that exhibit anxiety phenotype, were similar to each other, immature with enhanced excitability. Unfortunately, the development of raphe-limbic system circuitry is not well understood. Our long- term goal is to elucidate the changes at the cellular level within the 5-HT-limbic system that underlie the development of normal and pathological anxiety. In this proposal our current goal is to focus on the 5-HT- mPFC connection. Our hypothesis is that there is a critical period during the first few weeks of life in which 5- HT innervation of mPFC coordinates the morphological and physiological development of its local circuits. The following Specific Aims (SA) will test this hypothesis. SP1: To determine the developmental profile of layer 5 pyramidal neurons in the mPFC in concert with responses elicited by activation of 5-HT fibers Transgenic Pet-1 ChR2 mice for optogenetic stimulation will be used to test the hypothesis that projection fibers to the mPFC mature between P12-P21 leading to the release of both glutamate and 5-HT and that mPFC neurons mature physiologically between P4 and P21. SP2: To determine the developmental profile of layer 5 pyramidal cells in the mPFC in 5-HT1A and Pet-1 knockout mice. The hypothesis is that the neural development of the layer 5 pyramidal neurons will be disrupted leading to neurons with immature phenotypes. Aim 3: To determine the consequences of losing 5-HT neural activity on behavior and 5-HT-mPFC neural development. We hypothesize that a lack of 5-HT neuron activity in the dorsal raphe between P12-P21 will lead to increased anxiety-related behavior in pups and a higher anxiety in adults. The raphe and mPFC neurons will have immature phenotypes. Mice engineered with a synthetic pharmacologically activated inhibitory receptor located only in dorsal and median raphe neurons will be used. The new technology provides an innovative approach that will provide information at a functional level of 5-HT action on developing circuits. PUBLIC HEALTH RELEVANCE: The cellular mechanisms underlying stress induced behaviors and the development and treatment of stress related psychiatric disorders most likely involve both the 5- hydroxytryptamine (5-HT) cell body containing nuclei of the raphe as well as their projection areas in the limbic forebrain. This project is investigating the postnatal development of the raphe and one its projection areas, the medial prefrontal cortex. This research is relevant to public health because stress at an early age is known to result in mood disorders as adults; knowledge about the neural circuitry of the raphe-limbic system and its development will provide information about where modifications may occur to produce mood disorders.