Unraveling the developing synaptic neuropil of the mouse dorsal raphe nucleus using array tomography

MARIANO SOIZA REILLY; PATRICIA GASPAR

Milan

Congreso; FENS Forum 2014; 2014

Federation of European Neuroscience Societies

Neurons located in the dorsal raphe nucleus (DRN) represent the main source of forebrain serotonin, regulating stress and emotional states. Dysfunction of DRN networks is involved in the pathophysiology of affective disorders including anxiety and depression. Although maladaptive formation/refinement of DRN neural circuits could contribute to vulnerability to affective disorders, the synaptic organization of the developing DRN remains unknown. In this study, we explored this using a quantitative high-resolution immunofluorescence technique called array tomography (AT). AT is a light microscopy-based method involving multiple rounds of immunolabeling and imaging of ultrathin (70 nm) serial sections, and 3D visualization. This allows mapping and quantitative analysis of multiple antigens and their relationships to each other in the same tissue volume. We focused this study on glutamate and GABA innervations since they represent the main synaptic inputs to the DRN. We identified glutamate synaptic innervations to the DRN arising from cortical and subcortical structures by immunolabeling against the vesicular glutamate transporters 1 and 2, respectively. GABA axons were immunolabeled against the glutamate decarboxylase 65. Synaptic boutons and serotonin cells were identified using immunolabeling for synapsin 1 and tryptophan hydroxylase, respectively. The results showed that at postnatal day 7 glutamate and GABA synaptic innervations are abundantly present in the DRN, often associated with serotonin neurons. These observations indicate that AT represents a unique approach to increase understanding of how the DRN circuitry develops, and if possible alterations in synaptic connectivity could contribute to maladaptive mechanisms underlying affective disorders.