Glutamatergic and GABAergic architecture in the rat dorsal raphe nucleus visualized by array tomography, a high-resolution immunofluorescence technique

MARIANO SOIZA REILLY; KATHRYN G. COMMONS

Washington

Congreso; 41st Society for Neuroscience Annual Meeting; 2011

Society for Neuroscience

Serotonin (5-Hydroxytryptamine, 5-HT) neurons located in the dorsal raphe nucleus (DR) are regulated by excitatory and inhibitory drives mediated by a complex network of glutamatergic and GABAergic axons. These networks control forebrain 5-HT function, including the regulation of stress and emotional states. Dysregulation of DR excitability has been linked to the pathophysiology of affective disorders including depression and anxiety. Although behavioral and physiological evidence indicate maladaptive function of the DR during pathological states, whether there could be structural alterations of DR circuitry at the synaptic level remains unknown. In this study, we examined the synaptic organization of the DR using a high-resolution immunofluorescence technique called array tomography (AT). AT is a light microscopy-based method involving immunolabeling and imaging of ultrathin (70 nm) serial sections. Sections may undergo multiple rounds of immunolabeling and resulting images can be rendered in 3D. This allows a more precise localization of antigens but also a quantitative analysis of relationships between multiple antigens in the same 3D space. We analyzed glutamatergic (Vesicular Glutamate Transporters, VGLUT1-3; Postsynaptic Density Protein, PSD95) and GABAergic (enzyme Glutamate Decarboxylase 65, GAD2) specific synaptic markers, together with synapsin I as a general marker for synapses. Also, 5-HT cells were identified by the presence of the enzyme Tryptophan Hydroxylase. Thus, using AT we localized 7 antigens with respect to each other in the same tissue volumes. Analysis of these volumes showed that axon terminals containing any of three types of VGLUT and those labeled for GAD2 were abundantly present within the DR, and frequently associated to 5-HT neurons. VGLUT2-containing boutons were more often found in close apposition with PSD-95 in a likely synaptic association in comparison to those containing VGLUT1 or VGLUT3, either in association or not with 5-HT cells. Consistent results were found when analyzing colocalization of VGLUTs and synapsin. The relationships between axonal boutons containing either VGLUT1-3 and GAD2 revealed organizing characteristics of axoaxonic interactions, which could underlie direct presynaptic modulation of transmitter release. These observations indicate that AT represents a valuable approach to study relationships between different molecular components of synapses. This approach will improve our understanding of the organization of DR circuitry allowing us to further investigate whether there could be fine anatomical alterations in synaptic circuits under pathological conditions.