Presynaptic Rac1 in the hippocampus selectively regulates working memory
- Department of Cell Biology, Duke University School of Medicine, United States
- Department of Anatomy and Histology, University of Veterinary Medicine, Hungary
- Department of Psychiatry and Behavioral Sciences, Duke University School of Medicine, United States
- Mouse Behavioral and Neuroendocrine Analysis Core Facility, Duke University School of Medicine, United States
- Department of Neurobiology, Duke University School of Medicine, United States
- Proteomics and Metabolomics Shared Resource and Center for Genomic and Computational Biology, Duke University School of Medicine, United States
Figures
Figure 1
Localization of the presynaptic Rac1 inhibitor construct.
(A) Illustration of the presynaptic Rac1 inhibitor construct. (B) C57BL/6J primary neuron cultures expressing GFP, the negative control construct, or the presynaptic Rac1 inhibitor construct. (C) …
Figure 2
Hippocampal presynaptic Rac1 inhibition impairs spatial working memory.
(A) Schematic of the eight-arm radial maze test (n = 14 mice per group). (B) The total number of arm entries (Scr: 12.86 ± 1.123, W56: 14.29 ± 1.051, p = 0.3615) and (C) latency to the first arm …
Figure 3
Hippocampal presynaptic Rac1 inhibition does not affect other types of learning and memory.
(A) Schematic and timeline of the Morris water maze (MWM) test (Scr: n = 16 mice; W56: n = 15 mice). During the MWM acquisition trials, (B) average swimming speed and (C) escape latency showed no …
Figure 4 with 2 supplements
Hippocampal postsynaptic Rac1 inhibition does not affect hippocampal-dependent learning and memory.
(A) Illustration of the postsynaptic Rac1 inhibitor constructs. (B, C) Fluorescence intensity profiles validated postsynaptic localization of the constructs (Soluble GFP (Homer): n = 12 cells; …
Figure 4—figure supplement 1
Localization of postsynaptic Rac1 inhibitor constructs.
(A) C57BL/6J primary neuron cultures expressing GFP, the negative control construct, or the postsynaptic Rac1 inhibitor construct. The mScarlet-Gephyrin.FingR constructs were immunostained using an …
Figure 4—figure supplement 2
Anxiety and general locomotor activity are not affected by postsynaptic Rac1 inhibition.
In the light/dark transition (n = 16 mice per group), (A) latency to the first transition (Scr: 8.350 ± 1.221 s, W56: 7.588 ± 0.7299 s, p = 0.9612) and (B) time spent in the light box (Scr: 45.60 ± …
Figure 5
EM analysis of presynaptic Rac1 inhibition in hippocampal cells.
Representative electron microscopy (EM) images of the dorsal CA1 stratum radiatum transduced with AAV-hSyn1-Scr-Syn1a (A) and AAV-hSyn1-W56-Syn1a (B). (C) Diameter of axonal boutons (Scr: 493.8 ± …
Figure 6 with 1 supplement
Functional protein interaction network of presynaptic Rac1.
(A) Schematic of the presynaptic Rac1 BioID construct. (B, C) Presynaptic Rac1 BioID constructs are co-localized with a presynaptic marker Bassoon at synaptic boutons in C57BL/6J primary neuron …
Figure 6—figure supplement 1
Synaptic enrichment and additional analysis of the presynaptic Rac1 constitutively active (CA) and dominant negative (DN) proteomes.
(A) Network of biotinylated proteins identified within either CA or DN proteome. Node titles correspond to the gene names. Edges between nodes represent protein–protein interactions previously …
Figure 7
Model of presynaptic Rac1 regulation and its signaling cascades in spatial working memory.
This study suggests this process is driven by kinase-mediated phosphorylation of Guanine nucleotide Exchange Factor (GEF)/GAPs and vesicle proteins, which regulate vesicle cycle and actin dynamics.
© 2024, BioRender Inc. Figure 7 was created using BioRender, and is published under a CC BY-NC-ND 4.0. Further reproductions must adhere to the terms of this license
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Mus musculus) | C57BL/6J | The Jackson Laboratory | Cat# 000664; RRID:IMSR_JAX:000664 | |
| Cell line (Homo sapiens) | HEK293T | ATCC | Cat# CRL-3216; RRID:CVCL_0063 | |
| Biological sample (Mus musculus) | Primary neuron cultures | This paper | Freshly isolated from Mus musculus | |
| Antibody | anti-GFP (Mouse monoclonal) | Synaptic Systems | Cat# 132 011; RRID:AB_887726 | ICC (1:500) |
| Antibody | anti-V5 (Mouse monoclonal) | Thermo Fisher Scientific | Cat# R960-25; RRID:AB_2556564 | IHC (1:500), WB (1:1000) |
| Antibody | anti-Bassoon (Rabbit polyclonal) | Synaptic Systems | Cat# 141 003; RRID:AB_887697 | ICC (1:500) |
| Antibody | anti-RFP (Mouse monoclonal) | Synaptic Systems | Cat# 409 011; RRID:AB_2800533 | ICC (1:500) |
| Antibody | anti-Homer1 (Rabbit polyclonal) | Synaptic Systems | Cat# 160 003; RRID:AB_887730 | ICC (1:500) |
| Antibody | anti-Gephyrin (Rabbit monoclonal) | Synaptic Systems | Cat# 147 008; RRID:AB_2619834 | ICC (1:500) |
| Antibody | anti-Biotin (Goat polyclonal) | Thermo Fisher Scientific | Cat# 31852; RRID:AB_228243 | WB (1:5000) |
| Recombinant DNA reagent | pAAV-hSyn1-W56-EGFP-Linker-Synapsin1a | O’Neil et al., 2021 | See Plasmids construction | |
| Recombinant DNA reagent | pAAV-hSyn1-Scr-EGFP-Linker-Synapsin1a | O’Neil et al., 2021 | See Plasmids construction | |
| Recombinant DNA reagent | pAAV-hSyn1-EGFP-Rac1 | O’Neil et al., 2021 | See Plasmids construction | |
| Recombinant DNA reagent | pAAV-hSyn1-Syanptotagmin1-Linker-V5-UltraID-Linker-Rac1 CA | This paper | See Plasmids construction | |
| Recombinant DNA reagent | pAAV-hSyn1-Syanptotagmin1-Linker-V5-UltraID-Linker-Rac1 DN | This paper | See Plasmids construction | |
| Recombinant DNA reagent | pAAV-hSyn1-V5-UltraID-Linker-Rac1 CA | This paper | See Plasmids construction | |
| Recombinant DNA reagent | pAAV-hSyn1-V5-UltraID-Linker-Rac1 DN | This paper | See Plasmids construction | |
| Recombinant DNA reagent | pAAV-EF1a-W56-Linker-PSD95.FingR-eGFP-CCR5TC | This paper | See Plasmids construction | |
| Recombinant DNA reagent | pAAV-EF1a-Scr-Linker-PSD95.FingR-eGFP-CCR5TC | This paper | See Plasmids construction | |
| Recombinant DNA reagent | pAAV_Ef1a-W56-Linker-mScarlet-Gephyrin.FingR-IL2RGTC | This paper | See Plasmids construction | |
| Recombinant DNA reagent | pAAV_Ef1a-Scr-Linker-mScarlet-Gephyrin.FingR-IL2RGTC | This paper | See Plasmids construction | |
| Chemical compound, drug | Biotin | Millipore Signma | Cat# B4501; CAS: 58-85-5 | |
| Software, algorithm | Cytoscape | The Cytoscape Consortium | RRID:SCR_003032 | Version 3.9 |
