Absence of TI-VAMP/Vamp7 leads to increased anxiety in mice

Abstract

Vesicular (v)- and target (t)-SNARE proteins assemble in SNARE complex to mediate membrane fusion. Tetanus neurotoxin-insensitive vesicular-associated membrane protein (TI-VAMP/VAMP7), a vesicular SNARE expressed in several cell types including neurons, was previously shown to play a major role in exocytosis involved in neurite growth in cultured neurons. Here we generated a complete constitutive knock-out by deleting the exon 3 of Vamp7. Loss of TI-VAMP expression did not lead to any striking developmental or neurological defect. Knock-out mice displayed decreased brain weight and increased third ventricle volume. Axon growth appeared normal in cultured knock-out neurons. Behavioral characterization unraveled that TI-VAMP knock-out was associated with increased anxiety. Our results thus suggest compensatory mechanisms allowing the TI-VAMP knock-out mice to fulfill major developmental processes. The phenotypic traits unraveled here further indicate an unexpected role of TI-VAMP-mediated vesicular traffic in anxiety and suggest a role for TI-VAMP in higher brain functions.

Figure 1.

CMV-Cre::Vamp7^flox/flox mice are full Vamp7 knock-out mice. A, Strategy for the CMV-Cre::Vamp7-flox/flox construct. B, TI-VAMP, 3′ fragment (TIV-Cter) and TI-VAMPc (TIVc) cDNAs were used to produced proteins in rabbit reticulocytes (0 = no plasmid), which were immunoprecipitated with pAb TG18 and revealed with 158.2 mAb. C, GFP-TI-VAMPc expressed in Cos cells is recognized by mAb158.2, pAbTG18 and anti-GFP pAbs. D, Extracts of indicated tissues from WT and KO were Western blotted with mAb158.2. E, Immunoprecipitation with TG18 and rabbit IgG (control) of cortex and liver extracts from WT and KO littermates were revealed with mAb158.2. As control, 25 ng of TI-VAMP coiled-coil (aa 119–189, CC) was loaded. SP, Supernatant; IP, immunoprecipitation. F, Extracts of WT and KO cortex (40 μg) were Western blotted for the indicated proteins.

Figure 2.

Gross anatomy of the brain and neurite length in TI-VAMP KO. A, Sagittal sections of WT and KO animals stained with DAPI. Fifteen confocal pictures were manually assembled to generate an image of full brain (scale bar: 500 μm). B, Sagittal sections of WT and KO hippocampus stained with DAPI, Synaptophysin and TI-VAMP. Hippocampal region which harbors the highest expression level of TI-VAMP, is totally deprived of TI-VAMP signal in the KO, whereas the synaptic marker Synaptophysin is expressed at similar level. C, Magnetic resonance imaging experiments were performed using a 7 Tesla Pharmascan magnet (Bruker) on anesthetized mice (4 WT and KO) with high resolution T2 images (rapid acquisition with relaxation enhancement: repetition time = 4 s, echo time = 62 ms, matrix = 384 × 384, slice thickness = 0.5 mm, resolution = 50 × 50 μm, and acquisition time = 38 min). D, Regions of interest were manually outlined in a blinded fashion using ImageJ. The volume of the third ventricle was significantly higher. E, Representative images of 1 DIV cortical explants from WT and KO embryos, grown in plasma matrix (scale bar: 200 μm). Quantification of maximal axonal length of the different genotypes at 1 DIV (means ± SEM) (WT, n = 120; KO, n = 158) (Student's t test p = 0.831). F, Hippocampal neurons from WT and KO embryos were grown with or without astrocyte-conditioned media for 2 DIV and stained for MAP2 and Tau. Scale bar, 20 μm. Dendritic and axonal length of WT and KO are not statistically different (Mann–Whitney test: without astrocytes, axons p = 0.46 and dendrites p = 0.15; with conditioned media, axons p = 0.16 and dendrites p = 0.28).

Figure 3.

TI-VAMP KO show increased anxiety. A–D, KO and WT littermates were analyzed with olfaction cookie test (A), acoustic startle test (B), contextual and cued fear conditioning tests (C), and elevated plus maze (anxiety, D) (KO, n = 11; WT, n = 13; **p < 0.005, Student's t test).