Article Figures & Data
Figures
- Figure 1.
Antibody amplification of Cre-dependent viral expression. A, Representative images from a TH-Cre mouse injected in the VTA with AAV5-EF1a-DIO-mCherry show a similar pattern of expression between nonamplified and amplified fluorescence (yellow and white arrows). B, Long-range VTA–NAc/DS projections are easier to visualize following mCherry amplification (yellow vs white arrow). C, Similarly, nonamplified fluorescence of VTA to mPFC projections was generally weak (yellow arrows), and the fluorescence signal was significantly improved following mCherry amplification (white arrows). D, E, Representative images from PV-Cre mice injected with AAV5-EF1a-DIO-EYFP (D) or AAV5-EF1a-DIO-mCherry (E). The nonamplified fluorescence signal was similar between eYFP and mCherry constructs. Moreover, fluorescence signal amplification is similar to the nonamplified signal (yellow arrows) but is brighter and easier to visualize (white arrows), especially the dendrites in the ML. F, Representative images from a C57BL/6J mouse injected with AAV5-EF1a-DIO-mCherry show minimal nonamplified fluorescence (yellow arrow). Remarkably, amplification of adjacent sections from the same mouse revealed mCherry expression within the DG (white arrows). Scale bars: Panels A–B: 200 μm; Panels C–F: 100 μm.
- Figure 2.
Nonamplified fluorescence of DIO constructs in WT C57BL/6J mice. A, B, Representative photomicrographs of nonamplified fluorescence signal in C57BL/6J mice injected with AAV5-EF1a-DIO-eYFP (A) or AAV5-EF1a-DIO-mCherry (B). Nonamplified immunofluorescence was generally weak and primarily restricted to the soma (yellow arrows; see insets) of the injected hemisphere only. We hypothesize that the weak nonamplified immunofluorescence in these cells is significantly enhanced after antibody amplification. In addition, a very small number of cells with bright immunofluorescence throughout the cell body and its processes were observed (white arrows; see insets). Scale bars: 10× objective, 100 μm; 20× objective, insets, 25 μm.
- Figure 3.
Amplified expression of DIO-mCherry in the hippocampus of WT C57BL/6J mice. A, B, Experimental design and timeline. AAV5-EF1a-DIO-mCherry was injected into the anterior and posterior hippocampi of C57BL/6J mice (n = 8) and perfused 2–3 weeks later. Brains were sectioned in the coronal plane, and viral signal was amplified with rabbit anti-mCherry and goat anti-rabbit 568 antibodies. C, Representative immunofluorescence of mCherry throughout the relatively dorsal (top) and caudal (bottom) DG. Expression of mCherry was primarily observed in the GCL and dendrites extending into the ML (putative dentate GCs). The amplified mCherry signal also resulted in labeling of mossy fibers and cells in the hilus. D, Quantification of mCherry+ cells indicated that somatic expression was restricted to the injected hemisphere. Female (clear circles) and male (dotted circles) data points are identified, but no sex differences were found. ***p < 0.001. Scale bar, 100 μm. Data for this figure are shown in Extended Data Figure 3-1.
- Figure 4.
Amplified expression of DIO-eYFP in the hippocampus of WT C57BL/6J mice. A, B, Experimental design and timeline. AAV5-EF1a-DIO-eYFP was injected into the anterior and posterior hippocampi of C57BL/6J mice (n = 6) and perfused 2–3 weeks later. The eYFP signal was amplified with chicken anti-GFP and goat anti-chicken 488 antibodies. C, Representative immunofluorescence of GFP throughout the DG. GFP expression was observed primarily in the DG, characterized by robust labeling of putative GCs within the GCL and their dendrites. The hilus also showed bright GFP signal, with expression in mossy fibers and hilar cells. D, Quantification of GFP+ cells revealed that somatic expression was restricted to the injected hemisphere. Female (clear circles) and male (dotted circles) data points are identified, but no sex differences were found. **p < 0.005. Scale bar, 100 μm. Data for this figure are shown in Extended Data Figures 4-1 and 4-2.
- Figure 5.
Amplified expression of DIO-eYFP in the mPFC of WT C57BL/6J mice. A, B, Experimental design and timeline. AAV5-EF1a-DIO-eYFP was injected into left mPFC of C57BL/6J mice (n = 6), and mice were perfused 2–3 weeks later. Viral signal was amplified with chicken anti-GFP and goat anti-chicken 488 antibodies. C, Representative GFP immunofluorescence in the mPFC of two sections from the same mouse. D, Quantification of GFP+ cells in the mPFC showed that expression was primarily restricted to the injected hemisphere, but two mice had sparse expression of GFP+ cells in the noninjected hemisphere, presumably resulting from viral spread because of the close proximity of the left and right mPFC. Female (clear circles) and male (dotted circles) data points are identified, but no sex differences were found. CG, cingulate gyrus; PrL, Prelimbic cortex; IL, infralimbic cortex. **p < 0.005. Scale bar, 200 μm.
- Figure 6.
Amplified expression of DIO-hM3Dq-mCherry in the hippocampi of WT C57BL/6J mice. A, B, Experimental design and timeline. AAV8-hSyn-DIO-hM3Dq-mCherry was injected into the anterior and posterior hippocampus of C57BL/6J mice (n = 8), and mice were perfused 2–3 weeks later. The viral signal was amplified with rabbit anti-mCherry and goat anti-rabbit 568 antibodies and was visualized on an epifluorescence microscope. C, Representative mCherry immunofluorescence in relatively dorsal (top) and caudal (bottom) sections of the DG. Amplified mCherry expression appeared primarily within hilar cells and a sparse number of GCs (yellow arrows). D, Quantification of mCherry+ cells revealed that expression was restricted to the injected hippocampus. Female (clear circles) and male (dotted circles) data points are identified, but no sex differences were found. ***p < 0.001. Scale bar, 100 μm. Data for this figure are shown in Extended Data Figures 6-1 and 6-2.
- Figure 7.
The hM3Dq agonist C21 does not affect fear behavior in C57BL/6J mice injected with DIO-mCherry or DIO-hM3Dq-mCherry in the DG. A, B, Experimental design and timeline. Adult C57BL/6J mice underwent surgery to receive intrahippocampal injections of AAV-EF1a-DIO-mCherry or AAV-hSyn-DIO-hM3Dq-mCherry. After a 2 week recovery period, mice were injected with the hM3Dq agonist C21 1 h before contextual fear training. C, Mice were then placed in a fear-conditioning chamber. Baseline activity was assessed over 2 min, followed by five footshocks (0.5 mA) spaced 1 min apart. D, Minute-by-minute analysis of the training session revealed that freezing behavior did not differ between EF1a-DIO-mCherry or hSyn-DIO-hM3Dq-mCherry groups. E, The average postshock freezing did not differ between the EF1a-DIO-mCherry and hSyn-DIO-hM3Dq-mCherry groups. F, Mice were returned to the same operant chamber 24 h later to test contextual fear memory. Notably, C21 was not administered a second time before the contextual memory test. G, Minute-by-minute analysis revealed that conditioned freezing did not differ between the EF1a-DIO-mCherry or hSyn-DIO-hM3Dq-mCherry groups. H, Average freezing during the memory test did not differ between groups. Female (clear points) and male (dotted points) data points are identified, but no sex differences were found.
- Figure 8.
mCherry and c-Fos immunofluorescence following C21 home-cage challenge. A, B, Experimental design and timeline. Mice underwent surgery for AAV injection and were allowed 2 weeks for recovery. Mice underwent behavioral testing and were then given a 3 d washout period. Mice were then injected with C21 (1.5 mg/kg) in their home cage and were euthanized 90 min later to evaluate the immediate early gene c-Fos. C, The percentage of colocalization of c-Fos+ and mCherry+ cells following C21 challenge was significantly lower in C57BL/6J mice injected with DIO-mCherry (7 c-Fos+mCherry+/497 mCherry+ cells = 1.41%) or DIO-hM3Dq-mCherry (23 c-Fos+mCherry+/1062 mCherry+ cells = 2.17%) compared with PV-Cre+ mice injected with DIO-hM3Dq-mCherry (267 c-Fos+mCherry/367 mCherry+ cells = 72.75%). D–F, Representative images show that C57BL/6J mice lacked the clear elevation of c-Fos (green) in mCherry+ cells seen in PV-Cre+ mice (yellow; white arrows). ****p < 0.0001. Scale bar, 100 μm.
Tables
- Table 1
rAAV information: summary table of the Cre-dependent rAAVs used in the present study
Construct Serotype Titer Injection volume Vendor Figures AAV-EF1a-DIO-eYFP 5 ≥4 × 1012 vg/ml 0.2 μl/site UNC Core 1, 2, 4, 5, Extended Data 4–1,
Extended Data 4–2AAV-EF1a-DIO-mCherry 5 ≥7 × 1012 vg/ml 0.2 μl/site UNC Core 1, 2, 3, 8, 3–1, Extended Data 4–1,
Extended Data 4–2AAV-hSyn-DIO-hM3D(Gq)-
mCherry8 ≥5 × 1012 vg/ml 0.2 μl/site UNC Core 6, 8, Extended Data 6–1 AAV-hSyn-DIO-hM4D(Gi)-
mCherry5 ≥8 × 1012 vg/ml 0.2 μl/site Addgene (catalog
#44362)Extended Data 6–2 Overall, four Cre-dependent constructs were used that differed by promoter (EF1a, hSyn), serotype (AAV5, AAV8), and/or vendor (UNC Core, Addgene). High-titer (≥4 × 1012 vg/ml) stock virus (0.2 μl) was injected into each region of interest.
- Table 2
Antibody information with details of the primary and secondary antibodies used in the present study
Antigen Host Description Dilution Catalog # Vendor RRID# GFP Chicken Polyclonal 1:2000 AB13970 Abcam AB_300798 mCherry Rabbit Polyclonal 1:2000 167453 Abcam AB_2571870 mCherry Rat Monoclonal 1:1000 M11217 Thermo Fisher Scientific AB_2536611 c-Fos Rabbit Polyclonal 1:2500 226003 Synaptic Systems AB_2231974 Anti-rabbit (HRP conjugate) Donkey Polyclonal 1:500 711-036-152 Jackson ImmunoResearch AB_2340590 Anti-rabbit Goat Biotinylated IgG 1:500 BA-1000 Vector Laboratories AB_2313606 Alexa Fluor 488 anti-chicken Goat Fluorescence (488 nm) 1:500 A-11039 Thermo Fisher Scientific AB_2534096 Alexa Fluor 568 anti-rabbit Goat Fluorescence (568 nm) 1:500 A-11011 Thermo Fisher Scientific AB_143157
Figure 3-1
mCherry immunoreactivity in WT C57BL/6J mice injected with AAV5-EF1a-DIO-mCherry. A, Representative photomicrographs of mCherry immunoreactivity in C57BL/6J mice injected with AAV5-EF1a-DIO-mCherry. Overall, mCherry immunoreactivity was comparable to the pattern of expression observed with amplified DIO-mCherry immunofluorescence (Fig. 3). Scale bar, 100 μm. Download Figure 3-1, TIF file.
Figure 4-1
Fluorescence signal amplification of DIO-mCherry and DIO-eYFP is highly specific to the injection site in WT C57BL/6J mice. A, Tile scan of a C57BL/6J mouse injected with AAV5-EF1a-DIO-mCherry. Viral expression was amplified with mCherry antibody. The indent on the top of the left cortex represents a drilling artifact near the injection site. The mCherry expression is primarily restricted to the injected (left) hippocampus, with mCherry+ cells observed in the GCL of the DG. There is also sparse labeling of mCherry+ cells in the CA3. B, Tile-scan of a C57BL/6J mouse injected with AAV5-EF1a-DIO-eYFP. Viral expression was amplified with GFP and observed primarily within the injected (left) DG. Furthermore, GFP+ mossy fiber (MF) axons from dentate GCs were observed projecting to area CA3. Interestingly, commissural GFP+ axons, presumably from mossy cells, were observed within the IML of the contralateral hemisphere. Notably, there were no mCherry+ or GFP+ cells in the noninjected hemisphere. This result indicates that amplified fluorescence signal is highly specific to the target region and the projections of labeled cells. Scale bar, 200 μm. Download Figure 4-1, TIF file.
Figure 4-2
Fluorescence signal amplification in other subfields of the hippocampus. A, B, Viral injections aimed at the DG occasionally resulted in mistargeting, which led to amplified fluorescence signal in other subfields of the hippocampus, such as CA1 or CA2. This finding suggests that amplified viral expression was not unique to the DG, but rather was specific to the injection site. Scale bar, 100 μm. Download Figure 4-2, TIF file.
Figure 6-1
mCherry immunoreactivity in WT C57BL/6J mice injected with AAV8-hSyn-DIO-hM3Dq-mCherry. A, Representative photomicrographs of mCherry immunoreactivity in C57BL/6J mice injected with AAV8-hSyn-DIO-hM3Dq-mCherry. The pattern of mCherry immunoreactivity was comparable to the amplified immunofluorescence of DIO-hM3Dq-mCherry (Fig. 6). Scale bar, 100 μm. Download Figure 6-1, TIF file.
Figure 6-2
Fluorescence signal amplification of AAV5-hSyn-DIO-hM4Di-mCherry in WT C57BL/6J mice. A, C57BL/6J mice were injected in the DG with AAV5-hSyn-DIO-hM4D(Gi)-mCherry, and sections were amplified with mCherry. Interestingly, mCherry+ cells were primarily located in the hilus, but a small number of GCs were also labeled. The pattern of amplified AAV5-hSyn-DIO-hM4Di-mCherry expression is consistent with the AAV8-hSyn-DIO-hM3Dq-mCherry construct shown in Figure 6. Scale bar, 100 μm. Download Figure 6-2, TIF file.
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