CONICET | Buscador de Institutos y Recursos Humanos

Neurons located in the dorsal raphe nucleus (DR) represent the main source of forebrain serotonin, regulating stress and emotional states. Dysfunction of DR networks is involved in the pathophysiology of affective disorders including anxiety and depression. Moreover, maladaptive formation/refinement of neural circuits within the DR could contribute to vulnerability to affective disorders later in life. However, the synaptic organization of the developing DR remains unknown. In this study, we examined synaptic innervations of the developing mouse DR using the novel and quantitative high-resolution immunofluorescence technique, array tomography (AT). AT is a light microscopy-based method involving immunolabeling and imaging of ultrathin (70 nm) serial sections. Sections may be subjected to multiple rounds of immunolabeling, and resulting images can be rendered in 3D. This allows to map and quantitatively analyze multiple antigens as well as their relationships to each other in the same tissue volume. We focused on glutamate and GABA synaptic innervations because they represent the main synaptic inputs to the nucleus. Glutamate axons arising from cortical and subcortical structures were detected by immunolabeling for the vesicular glutamate transporters, VGLUT1 and VGLUT2 respectively. GABA axons were labeled against the GABA synthetic enzyme GAD2. Specific presynaptic markers were combined with general markers for synaptic boutons (e.g. Synapsin 1) as well as with postsynaptic markers (e.g. PSD-95). Additionally, serotonin cells were identified by the presence of the enzyme tryptophan hydroxylase. The results showed that at early postnatal ages both excitatory glutamate and inhibitory GABA synaptic innervations are abundantly present in the DR. Additionally, they frequently established direct associations with serotonin neurons. These observations indicate that AT represent a unique approach to quantitatively examine the spatial organization of multiple populations of synapses in the same volume of tissue. This approach will increase our understanding of how the DR circuitry develops, and if possible alterations in the synaptic connectivity could contribute to maladaptive mechanisms underlying affective disorders.

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