Article Figures & Data
Figures
- Figure 1.
Viral genetic targeting of hilar mossy cells for rabies-based mapping of circuit connections in vivo. A, The schematic illustrates our projection-based mossy cell targeting strategy (Gradinaru et al., 2010). The WGA-Cre AAV was unilaterally injected into one DG (1) in the wild-type C57BL/6 mouse to express Cre in some mossy cells in the contralateral DG, while the Cre-dependent helper AAV for rabies tracing was injected into the contralateral DG of the same mouse (2). The construct design is shown for WGA-Cre (top) and Cre-dependent (bottom) AAV vectors. At three weeks following the AAV injection, the genetically modified rabies was injected (3) for monosynaptic tracing of direct inputs to the targeted mossy cells. B, Example starter mossy cells (expressing both nuclear EGFP and mCherry) and their presynaptic neurons (only labeled by rabies mCherry expression) in the DG and CA3 at the helper AAV and rabies injection site. The arrows point to starter neurons. The sale bar (200 μm) in B applies to both B and F. C, The starter neurons have GFP expression (top) from the helper AAV genome and mCherry expression (bottom) from the rabies genome. Scale bar, 50 μm. D, The starter neurons show mossy appearances that defines hilar mossy cells. The white arrows point to numerous thorny excrescences at the cell’s proximal dendrites. Scale bar, 25 μm. E, The starter cell pointed by the green arrowhead is also immunopositive for CR staining. Scale bar, 25 μm. F, Another example showing starter mossy cells and their presynaptic neurons in the DG and CA3 (with some labeled cells in CA3b). The arrows point to putative inhibitory neurons labeled in both DG molecular layer and CA3 nonpyramidal layer. G, I, rabies-labeled neurons in SFi. The scale bar in G (200 μm) applies to both G and H; the scale bar in I (25 μm) applies to both I and J. H, J, Rabies-labeled neurons in the MS-DB. K, L, Only one labeled entorhinal neuron identified in the example case. Scale bars, 200 μm (K) and 50 μm (L). M, The graph shows quantification of average strengths of specific circuit connections to targeted mossy cells. The input CSI is defined as the ratio of the number of labeled presynaptic neurons in a specified structure versus the number of starter mossy neurons. Data are presented as mean ± SE. See Table 1 for more information.
- Figure 2.
Example section images showing the ipsilateral DG with the WGA-Cre AAV injection, and spatially restricted starter neurons in the dentate hilus in the contralateral DG. A, The AAV-mCherry-IRES-WGA-Cre strongly labels the cells (mCherry fluorescence, red) in the granule cell layer and the hilus, delineated by DAPI fluorescence (blue) in coronal sections at the septal, intermediate, and temporal levels. Scale bar, 200 μm. B, WGA-Cre activated helper AAV expression (nuclear EGFP fluorescence) is confined in the contralateral hilus of six different coronal sections at the septal to temporal levels. Arrows point to the helper AAV and rabies double-labeled starter neurons in the hilus. Scale bar, 200 μm.
- Figure 3.
GABA immunostaining of rabies-labeled neurons indicate that mossy cells receive inputs from both GABAergic and non-GABAergic neurons in local circuits. A, Rabies virus-labeled neurons (mCherry expression only, red) in the DG are direct presynaptic neurons of targeted hilar mossy cells. B, DAPI staining (blue) shows the anatomic structure of the DG, and nuclear EGFP label (small white box) shows a mossy starter neuron with AAV helper virus infection. C, GABA immunostaining (gray) shows GABAergic cells. D, The overlay image shows rabies-labeled hilar cells that are immunopositive for GABA (indicated by the white arrows). The white arrowhead points to a rabies-labeled neuron that is GABA immunopositive from the dentate granule cell layer. The small white box shows the hilar mossy starter neuron that lacks GABA staining. Scale bar, 200 μm.
- Figure 4.
Cholinergic neurons in the MS-DB provide direct synaptic inputs to hilar mossy cells. Rabies virus-labeled neurons in the MS-DB are consistently immunopositive for ChAT but not GABA. A, An example MS-DB slice shows rabies-labeled neurons (red) and DAPI staining (blue). Scale bar, 500 μm. B–D, Enlarged views of the white box region shown in A with ChAT immunostaining (green). The arrows point to rabies-labeled neurons that are immunopositive for ChAT. Scale bar, 200 μm.
- Figure 5.
Cre-dependent rabies tracing of circuit connections to dentate granule cells. A, The schematic illustrates the use of Cre dependent helper AAV and a D1-Cre transgenic mouse, in which the dentate granule cell layer expresses Cre recombinase (Lemberger et al., 2007) for Cre-dependent monosynaptic rabies tracing. B, Spatially restricted iontophoretic injection of the helper AAV (AAV-DIO-HTB) in the temporal DG of the D1-Cre mouse (left), followed by EnvA-SADΔG-mCherry rabies infection (right). The short white lines indicate restricted GFP-expressing helper AAV infection in the granule cell layer. Scale bar, 200 μm. C–L, Dentate granule cells receive very strong input from lateral and medial EC (C, F–H), extensive input from mossy cells at the temporal hilus (F), and inputs from medial septum (MS-DB) (L). Other long range inputs come from the subiculum, supramammillary nucleus (SuM), median raphe nucleus (D, E, I) as well as contralateral mossy cells (J and K). Scale bars in C (500 μm) applies to G, J, L; in D (200 μm) applies to E, H, I; in F, 500 μm; in K, 20 μm. M, The graph shows quantitative strengths of specific circuit connections to targeted dentate granule cells. The input CSI is defined as the ratio of the number of labeled presynaptic neurons in a specified structure versus the number of starter dentate granule neurons. Data are presented as mean ± SE. See Table 2 for more information.
- Figure 6.
Dentate granule cells tend to receive inputs from GABAergic hilar neurons around the tracer injection site and non-GABAergic hilar mossy neurons from more distal sites. A–C, Example images of GABA immunostaining of rabies-labeled hilar neurons around the injection site of a D1-Cre tracing case. White arrows point to GABA-immunopositive rabies-labeled neurons. D–F, GABA immunostaining of hilar rabies-labeled neurons from a more temporal position to the injection site. There is no colocalization between rabies-labeled neurons and GABA immunostaining. Small white boxes indicate representative non-GABA+ rabies-labeled neurons. G–I, GABA immunostaining of hilar rabies-labeled neurons from a more septal position to the injection site. There is no colocalization between rabies-labeled neurons and GABA immunostaining. Scale bar in B (100 μm) applies to all the panels. AP numbers indicate the positions of the coronal sections relative to the bregma landmark.
- Figure 7.
Monosynaptic rabies tracing reveals back projections from CA3 and CA1 to dentate granule cells. A–F, The targeted dentate granule cells receive extensive noncanonical inputs from CA3 in the septal directed hippocampus. AP numbers indicate the positions of the coronal sections relative to the bregma landmark. The example shows that putative CA3a/b excitatory neurons in the pyramidal cell layer and inhibitory neurons outside the pyramidal cell layer provide extensive inputs to dentate granule cells. The fimbria (indicate by the white bar) divides the distal CA3 (CA3a) and the middle CA3 subfield (CA3b). Panels on the right show enlarged views of the white box regions in the left panels. Scale bars, in A (500 μm) applies to A, C, E, G.; in B (100 μm) applies to B, D, F. G, H, Putative CA1 inhibitory neurons are also found projecting to dentate granule cells. Scale bar in H, 50 μm.
- Figure 8.
Cholinergic and GABAergic MS-DB inputs to dentate granule cells. A, An example MS-DB section image from a D1-Cre rabies tracing case. The red shows rabies-labeled neurons that are presynaptic to dentate granule cells, the green shows ChAT immunostaining, while the blue shows PV immunostaining. Scale bar, 200 μm. B–D, Enlarged view of the white box region at the top of A with arrows pointing to ChAT+ rabies-labeled septal neurons. E–G, Enlarged view of the white box region at the bottom of A with arrows pointing to a PV+ rabies-labeled septal neuron. The scale bar in B (50 μm) applies to B–G.
- Figure 9.
Functional circuit mapping with fast VSD imaging of neural activity supports anatomic rabies tracing results. A, Schematic of living brain slice preparation and the VSD imaging set up. B–E, Spatially restricted photostimulation in EC evokes wide-spread activation in DG (full extent), but weak hilar neuronal activation. B, D, Time series data from VSD imaging of photostimulation-evoked neural activation in the hippocampal formation circuitry. The purple dots (laser stimulation artifact) indicates the photostimulation site. Color-coded activity is superimposed on the background slice image. The color scale codes VSD signal amplitude expressed as SD multiples above the mean baseline. The stronger activation is indicated by the warmer color. Scale bar, 500 μm. C, E, Time course plots of VSD signal from the regions of interest (EC, DG granule cell layer, hilus) indicated by the colored rectangles (red, green, and blue) in the first image frame in B, respectively, starting from the baseline of 22 ms preceding the photostimulation onset. No baseline drift was corrected. F, Spatially restricted photostimulation of dentate granule cells activate hilar and CA3 responses, but hilar photostimulation does not cause significant DG activation. The imaging of individual stimulation at nine different sites (including one hilus stimulation, number 9) was performed; the peak response image frames are plotted at 44 ms after photostimulation. Scale bars: 250 μm (first panel) and 250 μm (second panel), for both F and G. G, Time series data from VSD imaging of photostimulation-evoked neural activation in proximal CA3b with simultaneous stimulation of three sites (purple dots). Compared with weaker CA3 activation in the lower panels, the stronger CA3 activation in upper panels causes more CA3 collateral activation and feedforward signal propagation to CA1. In later time points, CA3 activation also leads to some hilar activation.
- Figure 10.
Local functional circuit mapping identifies strong DG versus weak CA3 inputs to hilar mossy cells. A, Genetic label guided recordings of hilar mossy neurons in the horizontal hippocampal slices. Given that hilar mossy cells are the only glutamatergic cell type in the hilus, they are targeted by visualizing neurons with large somata and with nonred fluorescent expression (i.e., the white square in A) in the dentate hilus of the Gad2-Cre; Ai9 mouse which expresses red fluorescent proteins (tdTomato) in GABAergic neurons (i.e., the arrowhead). B, The targeted neuron in A shows regular adapting spiking in response to intrasomatic depolarizing current injection. C, Post hoc identification of the recorded neuron as a mossy cell via intracellular biocytin staining. Scale bar, 50 μm. D–J, LSPS allows for extensive and quantitative analysis of synaptic inputs to recorded mossy cells from local hippocampal circuits in a relatively large region. D, A hippocampal slice image superimposed with photostimulation sites (cyan circles) spaced at 90 × 90 µm. The red circle indicates the tip of a recording electrode and the cell body location of the recorded mossy neuron. Scale bar, 200 µm. E, The plot of EPSC responses from the recorded cell at the corresponding sites in response to photostimulation via glutamate uncaging. The response traces are plotted for 200 ms beginning at the photostimulation onset. F, The two types of responses can be distinguished based on their waveforms, amplitudes, and response latencies. An example of direct uncaging responses is shown in red, and examples of synaptically mediated responses from presynaptic neuronal spiking are shown in green. The photostimulation (1 ms) is indicated by the vertical dashed blue line. The direct responses are excluded for synaptic input analysis. The raw data as shown in E are quantified and used for construction of a color-coded quantitative input map. G, The input response sites are overlaid on the bright field image to show anatomic position. Scale bar, 250 μm. H, The plot of IPSC responses from the recorded cell at the corresponding sites in response to photostimulation. The raw data as shown in H are quantified and used for construction of a color-coded quantitative input map. I, Example IPSC responses from the recorded mossy cell. J, The input response sites are overlaid on the bright field image to show anatomic position. K, L, Summary data of average total EPSC and IPSC inputs to the recorded mossy neurons measured from the DG granule cell and molecular layers (labeled as DG), hilus, and CA3.
Tables
A. Individual case summary Starter neurons DG GCL DG ML DG Hilus CA3c SP CA3c non-SP CA3b SP EC MS-DB SFi Case 1561 (total 365 neuron counts) Number of neurons 36 227 15 59 16 12 5 0 17 10 CSI 6.31 0.42 1.64 0.44 0.33 0.14 0.00 0.47 0.28 % of total labeling 62.19 4.11 16.16 4.38 3.29 1.37 0.00 4.66 2.74 Case 1562 (total 431 neuron counts) Number of neurons 39 262 15 79 22 15 4 1 12 11 CSI 6.72 0.38 2.03 0.56 0.38 0.10 0.03 0.31 0.28 % of total labeling 60.79 3.48 18.33 5.10 3.48 0.93 0.23 2.78 2.55 Case 2543 (total 1247 neuron counts) Number of neurons 70 660 33 366 78 20 21 0 20 3 CSI 9.43 0.47 5.23 1.11 0.29 0.30 0.00 0.29 0.04 % of total labeling 52.93 2.65 29.35 6.26 1.60 1.68 0.00 1.60 0.24 Case 2544 (total 1333 neuron counts) Number of neurons 62 702 59 369 49 22 27 2 31 6 CSI 11.32 0.95 5.95 0.79 0.35 0.44 0.03 0.50 0.10 % of total labeling 52.66 4.43 27.68 3.68 1.65 2.03 0.15 2.33 0.45 B. Summary data of all 4 cases DG GCL DG ML DG Hilus CA3c SP CA3c non-SP CA3b SP EC MS-DB SFi CSI 8.44 0.56 3.71 0.73 0.34 0.24 0.01 0.39 0.17 SE 1.18 0.13 1.10 0.15 0.02 0.08 0.01 0.06 0.06 % of total labeling 57.14 3.67 22.88 4.85 2.51 1.50 0.10 2.84 1.50 SE 2.53 0.39 3.30 0.55 0.51 0.23 0.06 0.65 0.67 As every one in three sections across the whole brain series was used for mapping rabies-labeled presynaptic neurons of targeted dentate granule cells, the actual total number of labeled neurons in each case would be estimated three times of the total neuron counts listed in the table. The labeled neurons are predominantly distributed in the ipsilateral hemisphere. The neuronal count for each structure in each case is pooled from both hemispheres if there are any labeled neurons in the contralateral hemisphere. The percentage of total labeled neurons is calculated as the percentage of the number of labeled presynaptic neurons in a specified structure versus the overall total presynaptic neuron count in each case. The input CSI is defined as the ratio of the number of labeled presynaptic neurons in a specified structure versus the number of starter neurons.
DG GCL, DG granule cell layer; DG ML, DG molecular layer; CA3b-c SP, CA3b-c pyramidal cell layer; CA3c non-SP, CA3c nonpyramidal cell layer.
Starter neuron DG GCL DG ML DG Hilus CA1 non-SP CA3a SP CA3a non-SP CA3b SP CA3b non-SP CA3c SP CA3c non-SP PRh LEnt mEnt MS-DB Subicu-lum SuM Raphe A. Individual case summary Case 1711 (a total of 2421 neuron counts) Number of neurons 128 23 10 261 13 125 24 80 24 33 7 40 1285 286 182 8 15 1 CSI 0.18 0.08 2.04 0.10 0.98 0.19 0.63 0.19 0.26 0.05 0.31 10.04 2.23 1.42 0.06 0.12 0.01 % of total labeling 0.95 0.41 10.78 0.54 5.16 0.99 3.30 0.99 1.36 0.29 1.65 53.08 11.81 7.52 0.33 0.62 0.04 Case 1713 (a total of 1810 neuron counts) Number of neurons 119 18 7 168 18 48 8 34 24 12 9 73 1048 165 161 8 7 2 CSI 0.15 0.06 1.41 0.13 0.40 0.07 0.29 0.20 0.10 0.08 0.61 8.81 1.39 1.35 0.07 0.06 0.02 % of total labeling 0.99 0.39 9.28 1 2.65 0.44 1.88 1.33 0.66 0.50 4.03 57.90 9.12 8.90 0.44 0.39 0.11 Case 1868 (a total of 3192 neuron counts) Number of neurons 228 35 16 421 29 147 29 96 35 53 17 77 1669 315 196 10 35 5 CSI 0.15 0.07 1.85 0.13 0.64 0.13 0.42 0.15 0.23 0.07 0.34 7.32 1.38 0.86 0.04 0.15 0.02 % of total labeling 1.10 0.50 13.19 0.91 4.61 0.91 3.01 1.10 1.66 0.53 2.41 52.29 9.87 6.14 0.31 1.10 0.16 Case 1867 (a total of 1441 neuron counts) Number of neurons 88 15 11 137 28 63 29 39 24 32 6 42 693 198 106 4 7 0 CSI 0.17 0.13 1.56 0.30 0.72 0.33 0.44 0.27 0.36 0.07 0.48 7.88 2.25 1.20 0.05 0.08 0.00 % of total labeling 1.04 0.76 9.51 1.94 4.37 2.01 2.71 1.67 2.22 0.42 2.91 48.09 13.74 7.36 0.28 0.49 0.00 Case 1864 (a total of 509 neuron counts) Number of neurons 32 5 7 45 8 17 5 10 4 7 2 9 269 66 52 1 2 0 CSI 0.16 0.22 1.41 0.25 0.53 0.16 0.31 0.13 0.22 0.06 0.28 8.41 2.06 1.63 0.03 0.06 0.00 % of total labeling 0.98 1.38 8.84 1.57 3.34 0.98 1.96 0.79 1.38 0.39 1.77 52.85 12.97 10.22 0.20 0.39 0.00 B. Summary data of all 5 cases DG GCL DG ML DG Hilus CA1 non-SP CA3a SP CA3a non-SP CA3b SP CA3b non-SP CA3c SP CA3c non-SP PRh LEnt mEnt MS-DB Subicu-lum SuM Raphe CSI (Mean) 0.16 0.11 1.65 0.18 0.65 0.17 0.42 0.19 0.23 0.07 0.40 8.49 1.86 1.29 0.05 0.09 0.01 SE 0.01 0.03 0.13 0.04 0.10 0.04 0.06 0.03 0.04 0.00 0.06 0.46 0.20 0.13 0.01 0.02 0.00 % of total labeling 1.01 0.69 10.32 1.19 4.03 1.07 2.57 1.17 1.46 0.43 2.56 52.84 11.50 8.03 0.31 0.60 0.06 SE 0.03 0.18 0.79 0.25 0.45 0.26 0.28 0.15 0.25 0.04 0.43 1.56 0.88 0.70 0.04 0.13 0.03 As every one in three sections across the whole brain series were used for mapping rabies-labeled presynaptic neurons of targeted dentate granule cells, the actual total number of labeled neurons in each case would be estimated three times of the total neuron counts listed in the table. The labeled neurons are predominantly distributed in the ipsilateral hemisphere. The neuronal count for each structure in each case is pooled from both hemispheres if there are any labeled neurons in the contralateral hemisphere. The percentage of total labeled neurons is calculated as the percentage of the number of labeled presynaptic neurons in a specified structure versus the overall total presynaptic neuron count in each case. The input CSI is defined as the ratio of the number of labeled presynaptic neurons in a specified structure versus the number of starter neurons.
DG GCL, DG granule cell layer; DG ML, DG molecular layer; CA1 non-SP, CA1 nonpyramidal cell layers; CA3a-c SP, CA3a-c pyramidal cell layer; CA3a-c non-SP, CA3a-c nonpyramidal cell layer; LEnt, lateral EC; mEnt, medial EC; SuM, supramammillary nucleus; Raphe, raphe nuclei.
In this issue
