Axonal Non-segregation of the Vesicular Glutamate Transporter VGLUT3 Within Serotonergic Projections in the Mouse Forebrain

Abstract

A subpopulation of raphe 5-HT neurons expresses the vesicular glutamate transporter VGLUT3 with the co-release of glutamate and serotonin proposed to play a pivotal role in encoding reward- and anxiety-related behaviors. Serotonin axons are identifiable by immunolabeling of either serotonin (5-HT) or the plasma membrane 5-HT transporter (SERT), with SERT labeling demonstrated to be only partially overlapping with 5-HT staining. Studies investigating the colocalization or segregation of VGLUT3 within SERT or 5-HT immunolabeled boutons have led to inconsistent results. Therefore, we combined immunohistochemistry, high resolution confocal imaging, and 3D-reconstruction techniques to map and quantify the distribution of VGLUT3 immunoreactive boutons within 5-HT vs. SERT-positive axons in various regions of the mouse forebrain, including the prefrontal cortex, nucleus accumbens core and shell, bed nucleus of the stria terminalis, dorsal striatum, lateral septum, basolateral and central amygdala, and hippocampus. Our results demonstrate that about 90% of 5-HT boutons are colocalized with SERT in almost all the brain regions studied, which therefore reveals that VGLUT3 and SERT do not segregate. However, in the posterior part of the NAC shell, we confirmed the presence of a subtype of 5-HT immunoreactive axons that lack the SERT. Interestingly, about 90% of the 5-HT/VGLUT3 boutons were labeled for the SERT in this region, suggesting that VGLUT3 is preferentially located in SERT immunoreactive 5-HT boutons. This work demonstrates that VGLUT3 and SERT cannot be used as specific markers to classify the different subtypes of 5-HT axons.

A retinal circuit for the suppressed-by-contrast receptive field of a polyaxonal amacrine cell

“…However, it remains to be determined whether there are other amacrine cell types, which receive both glutamatergic and glycinergic inputs from GACs. In the brain, neurons expressing vGluT3 often corelease glutamate and a different classic transmitter (35), such as acetylcholine, GABA, and serotonin (36)(37)(38). While glutamate-GABA cotransmission has been reported at vGluT2-expressing synapses from entopeduncular nucleus to lateral habenula, functional evidence for cotransmission at single vGluT3-expressing synapses is still lacking (37).…”

Sucrose Consumption Alters Serotonin/Glutamate Co-localisation Within the Prefrontal Cortex and Hippocampus of Mice

“…To assess if chronic sucrose consumption alters serotonin innervation, sections containing the PFC (Bregma +2.26 to +2.66) and DG of the hippocampus (Bregma –1.5 to –2.0 mm) were incubated with rat anti-5-HT (Millipore #MAB352, 1:100) for 48 h at room temperature followed by and guinea-pig anti-VGLUT3 (Synaptic System #135204), at 1:500 dilution ( Belmer et al, 2019 ), and phosphor-p44/42 MAPK (Erk 1/2) (Thr 202/Tyr 204) ( Jacques et al, 2019b ) at 1:150, overnight at room temperature (#4370, Cell Signalling Technology, MA, United States). After three washes in the blocking solution, the slices were incubated for 4 h at room temperature with secondary antibodies diluted in the blocking solution: goat anti-rabbit-Alexa 488, goat anti-guinea pig-Alexa 647 (Thermo Fisher Scientific, #A11034 and #A21450, respectively, 1:500) and goat anti-rat biotinylated (Jackson Laboratory # 112-065-003, 1:200).…”

“…Images were deconvolved using Huygens professional v16.10 (Scientific Volume Imaging, The Netherlands) with iteration number set at 100, quality threshold at 0.001, signal to noise ratio at 15 for the 4 channels and converted in. OIF for subsequent quantification for 5-HT, VGLUT3, 5-HT/VGLUT3, and pMAPK in Imaris 9.1.2 using Surface Reconstruction and Spot Detection functions as previously described (Belmer et al, 2017(Belmer et al, , 2019. Using surface rendering, fluorescence thresholding, and masking of unwanted immunolabelling, we obtained 3D objects (structures) of VGLUT3 labelling within 5-HT boutons.…”

Targeting VGLUT Machinery: Implications on mGluR5 Signaling and Behavior

“…The crosstalk between VGLUT3 and mGluR signaling is evident in striatal circuitry ( Figure 1). VGLUT3 mediates the release of glutamate from two neuronal varicosities; striatal TANs and, to lesser extent, serotonergic raphe neurons (El Mestikawy et al, 2011;Belmer et al, 2019).…”

“…Over the recent years, evidence has accumulated on the role glutamate as a second neurotransmitter/neuromodulator in serotoninergic neurons. This VGLUT3-mediated neurotransmission exist in large neuronal populations comprising both dorsal and medial raphe nuclei (Fremeau et al, 2002;Amilhon et al, 2010;Hioki et al, 2010;Wang et al, 2019), which project to different regions across the forebrain including; striatum, hippocampus and lateral septum (Dahlström and Fuxe, 1964;Qi et al, 2014;Belmer et al, 2019). Loss of VGLUT3 signaling attenuates 5-HT 1A autoreceptor-mediated neurotransmission in raphe nuclei, in addition to suppression of 5-HT transmission in projection areas including hippocampus and cerebral cortex (Amilhon et al, 2010).…”