Tracing inputs to inhibitory or excitatory neurons of mouse and cat visual cortex with a targeted rabies virus

Abstract

Background: Cortical inhibition plays a critical role in controlling and modulating cortical excitation, and a more detailed understanding of the neuronal circuits contributing to each will provide more insight into their roles in complex cortical computations. Traditional neuronal tracers lack a means for easily distinguishing between circuits of inhibitory and excitatory neurons. To overcome this limitation, we have developed a technique for retrogradely labeling inputs to local clusters of inhibitory or excitatory neurons, but not both, using neurotropic adenoassociated and lentiviral vectors, cell-type-specific promoters, and a modified rabies virus.

Results: Applied to primary visual cortex (V1) in mouse, the cell-type-specific tracing technique labeled thousands of presynaptically connected neurons and revealed that the dominant source of input to inhibitory and excitatory neurons is local in origin. Neurons in other visual areas are also labeled; the percentage of these intercortical inputs to excitatory neurons is somewhat higher (~20%) than to inhibitory neurons (<10%), suggesting that intercortical connections have less direct control over inhibition. The inputs to inhibitory neurons were also traced in cat V1, and when aligned with the orientation preference map revealed for the first time that long-range inputs to inhibitory neurons are well tuned to orientation.

Conclusions: These novel findings for inhibitory and excitatory circuits in the visual cortex demonstrate the efficacy of our new technique and its ability to work across species, including larger-brained mammals such as the cat. This paves the way for a better understanding of the roles of specific cell types in higher-order perceptual and cognitive processes.

Figure 1. Design of the Cell-Type Specific Vectors and Modified Rabies Virus

(A) Schematic representation of the transfer vectors for recombinant adeno-associated virus, AAV/GAD1/YTB (top), and lentivirus, LV/αCaMKII/YTB (bottom). Each vector includes a cell-type specific promoter (magenta), GAD1 (glutamic acid decarboxylase 1) or αCaMKII (α-calcium/calmodulin-dependent protein kinase II), cis-regulatory sequences (hatched; SD/SA = splice donor and acceptor sequence from human β-globin, BGH = bovine growth hormone polyadenylation signal; WPRE = woodchuck hepatitis virus posttranscriptional regulatory element), and the YTB transgene. YTB codes for three gene products using 2A peptide mediated cleavage [52]: the yellow fluorescent protein reporter (YFP, or Y; yellow), the artificial TVA 950 receptor (TVA, or T; blue), and the glycoprotein from the B-19 strain of rabies (RabG, or B; purple). Arrows indicate the start codons for each gene. Inverted terminal repeats (ITR) and long terminal repeats (LTR) are indicated in black. (B) The rabies virus (RV) has been modified in two ways based on [16]: the glycoprotein gene (G) was deleted (ΔG) and replaced with the gene for mCherry, creating, ΔG-RV-mCherry; and it was subsequently pseudotyped with the envelope A glycoprotein, creating EnvA-ΔG-RV-mCherry.

Figure 2. Schematic for Targeting Rabies Virus to Trace the Inputs to Inhibitory or Excitatory Neurons

A simplified diagram of our tracing technique is shown in (A) and described in more detail in (B–E). In the left panel of (A), helper-virus is used to deliver three genes (YFP, TVA, and RabG; see Fig. 1A) to a local population of neurons in mouse V1 at the site of injection. As shown in (B) and (D) the helper-viruses infect directly through the cell soma (magenta lines). The cell-type specificity of gene expression depends on which helper-virus is used; AAV/GAD67/YTB (B) and LV/αCaMKII/YTB (D) vectors are designed to introduce the genes to inhibitory or excitatory neurons, respectively. The three gene products are produced by the single cell-type specific promoter (GAD1 or αCaMKII) using 2A peptide mediated cleavage [52]. Helper-virus infected neurons are depicted as green due to YFP expression. Following a 14–21 day survival period to allow for sufficient gene expression, the modified rabies virus, EnvA-ΔG-RV-mCherry (Fig. 1B), is injected in the same V1 location (middle panel in A). The TVA receptor now embedded in the membrane of the cell soma (blue rectangles on cell bodies in BE) enables direct EnvA infection at the soma as indicated in (C and E). These EnvA-ΔG-RV-mCherry superinfected neurons are depicted as yellow because they will now begin to express mCherry as well as YFP. Because RabG is also co-expressed via the helper-virus, over the next 10 days rabies virions produced in starter cells will incorporate RabG and spread retrogradely from the yellow ‘starter cells’ via the synaptic terminals of presynaptically connected neurons (orange arrows in C and E). Resulting ‘connected cells’ which can be found locally within V1 and at longer-range in other brain regions such as V2 (right panel in A) are depicted as red because they will only express mCherry delivered by the rabies virus. Black crosses (x) in (C and E) illustrate that the ΔG-RV virus cannot spread beyond these directly connected cells because they were not transduced with RabG.

Figure 3. Cell-type Specificity of Helper-Virus Transduction in Mouse Neocortex

Low magnification images show expression of YFP (green) in cortical neurons following AAV/GAD1/YTB (A; AAV) and LV/αCaMKII/YTB (H; LV) helper-virus injections in mouse cortex. Higher magnified black and white images of AAV infected neurons (C and F) co-localize with neurons labeled through GABA immunofluorescence (D and G), as can be seen when shown together in color (B and E; AAV is shown in green and GABA in red). In contrast, higher magnified black and white images of LV infected neurons (J and M) do not co-localize with neurons positive for GABA immunofluorescence (K and N), as can be seen when shown together in color (I and L; LV is shown in green and GABA in red). Scale bars = 100 μm. See also Fig. S1.

Figure 4. Targeting EnvA-Δ G Rabies Virus (RV) infection to and Monosynaptic Retrograde Spread from Inhibitory Neurons in Mouse V1 using the AAV/GAD1/YTB Vector

The local pattern of RV infected inputs to AAV infected inhibitory neurons is shown in coronal sections through V1 in three mice, M11–16 (A–G), M12–17 (H–J) and M11–20 (K). In (A), the AAV and EnvA-ΔG-RV-mCherry (RV) injection sites are indicated by green and red arrow heads, respectively. In (H), injection sites overlap. Starter cells appear yellow due to transduction of the cell first with AAV leading to YFP expression (green) and subsequent infection by EnvA-ΔG-RV-mCherry due to the presence of the TVA receptor, leading to mCherry expression (red; see schematic in Fig. 2BC). Neurons presynaptically connected to starter cells that were retrogradely infected by RV following RabG complementation within starter cells are identified by expression of mCherry-only (red; see Fig. 2C). Higher magnified images of the regions outlined by white and black rectangles in (A and H) are shown in (B–G, and IJ). AAV infected neurons expressing YFP (Green; C, F and I) and RV infected neurons expressing mCherry (red; D, G and J) are shown together in (B and E) and separately in (C, F and I; AAV) and (D, G and J; RV). (K) A representative reconstruction of the brain wide pattern of RV infected neurons (mCherry-expressing; red) providing direct inputs to inhibitory starter cells (green) found in layers 1–6 of V1 is shown for mouse M11–20. The AAV and RV injection sites are located between sections 65 and 76 (see Fig. S2A–C). Sections are ordered from posterior to anterior. Inset images show mCherry expression in long range connected neurons in V2L, LGN, and cingulate cortex (cg). For all sections in (A–K), left is lateral, up is dorsal. Scale bars = 200 μm in (A and H), 50 μm in (E, I and K inset), and 1mm in (K). See also Fig. S2.

Figure 5. Targeting EnvA-Δ G-RV-mCherry Infection to and Retrograde Spread from Excitatory Neurons in Mouse V1 using the LV/α CaMKII/YTB Vector

Examples of the local and long range pattern of RV infected neurons (red) providing inputs to excitatory starter neurons (yellow) are shown in coronal sections through V1 in three mice, M12–15 (A–D), M12–09 (E–H), and M12–10 (I–L). In (A, E, and I), overlapping injection sites of LV/αCaMKII/YTB (green) and EnvA-ΔG-RV-mCherry (red) are shown at low magnification; Higher magnified images of the regions outlined by black or white rectangles are shown in (B, F, and I). Double labeled starter cells (yellow in B, F, and J) co-infected by LV (C, G and K) and RV (D, H and L), express YFP from LV infection and mCherry from RV infection. (M) (K) A representative reconstruction of the brain wide pattern of RV infected neurons (mCherry-expressing; red) providing direct inputs to excitatory starter cells (green) found in layers 2–6 of V1 is shown for mouse M12–09. The LV and RV injection sites are marked by thick black lines in sections 40 and 45. Inset images show RV infected neurons in V2L (sect. 49) and the lateral posterior nucleus (sect. 78). Other conventions are as in Fig. 4. Scale bars = 200 μm in (A, E and I), 50 μm in (B, F, L and M inset), and 1 mm in (M). See also Fig. S3.

Figure 6. Correlating Orientation Preference of Inputs to V1 Inhibitory Neurons in Cat

(A) As shown in the high magnification image of a superficial section of flattened V1 cortex, AAV/GAD1/YTB transduction (green) allowed for initial infection with the EnvA-ΔG-RV-mCherry creating a double-labeled starter cell (yellow; indicated by the white line) and leading to RV spread to presynaptically connected neurons (red). AAV/GAD1/YTB and EnvA-ΔG-RV-mCherry infected neurons are shown separately in (B and C). In (D), the pattern of EnvA-ΔG-RV- mCherry infected neurons is shown in the low magnification image of a superficial section of flattened V1 cortex adjacent to that shown in (A–C). A reconstruction of this pattern is overlaid on the orientation preference map in (E). The numbers of cells present in each colored domain in (E) were counted for short- (within the white circle) and long-range intrinsic distances and displayed in the histograms in (F). Corresponding orientation selectivity index (OSI) values of 0.54 and 0.35 calculated using the formula provided in Supplemental Information are also given. The red line in (D) points to the location of the EnvA-ΔG-RV-mCherry injection site. The white circle in (E) represents a 250 μm radius around the approximate center of the location of the starter cell identified in (A). Scale bars = 100 μm in (A), and 500 μm in (D and E). See also Fig. S5.