Correlative light and electron microscopic study on 3D reconstruction of lateral habenular axon terminals with distinct synapses releasing glutamate and GABA

Authors

• S Zhang
• A Shevelkin
• K Yu
• H Wang
• M Morales

Abstract

The lateral habenula (LHb) is involved in reward, aversion, addiction, and depression through descending interactions with several brain structures, including the ventral tegmental area (VTA). We recently discovered a subset of VTA neurons that co-express vesicular glutamate transport 2 (VGluT2) and vesicular GABA transporter (VGaT), and co-release glutamate and GABA in the LHb. To selectively target the dual VGluT2-VGaT neurons, we developed a dual recombinase transgenic mouse lines to inject INTRSECT2.0 vectors into the VTA. To determine the LHb ultrastructural properties of axon terminals from dual VTA VGluT2-VGaT neurons, we first injected the Con/Fon-ChR2-eYFP viral vector in the VTA of vglut2-Cre/vgat-Flp mice to tag VGluT2-VGaT neurons. By confocal microscopy we collected fluorescent Z-stack images, followed by ultrathin serial section collection with the “Automated Tape Collecting Ultramicrotome” (ATUMtome), and carbon coated on the “wafer”. We collected scanning electron microscopic (SEM) serial images, which were correlated with the fluorescent Z-stack images and applied 3D imaging software Amira and Dragonfly to reconstruct VGluT2-VGaT axon terminals. As a follow up study, we applied super-resolution microscopy to investigate within microdomains of VGluT2-VGaT axon terminals the spatial distribution of (a) presynaptic VGluT2 in relation to PSD95 and asymmetric synapses and (b) presynaptic VGaT in relation to gephyrin and symmetric synapses. By combining selective viral transfection of VGluT2-VGaT neurons, CLEM, super-resolution microscopy and 3D reconstruction, we visualized the compartmentalization of glutamate-vesicles and GABA-vesicles within the LHb, in which single axon terminal from VTA glutamate-GABA axons made asymmetric synapses on dendritic spines and symmetric synapses on different dendrites.

Scientific Focus Area: Neuroscience

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