Whole-Brain Map of Long-Range Monosynaptic Inputs to Different Cell Types in the Amygdala of the Mouse

Abstract

The amygdala, which is involved in various behaviors and emotions, is reported to connect with the whole brain. However, the long-range inputs of distinct cell types have not yet been defined. Here, we used a retrograde trans-synaptic rabies virus to generate a whole-brain map of inputs to the main cell types in the mouse amygdala. We identified 37 individual regions that projected to neurons expressing vesicular glutamate transporter 2, 78 regions to parvalbumin-expressing neurons, 104 regions to neurons expressing protein kinase C-δ, and 89 regions to somatostatin-expressing neurons. The amygdala received massive projections from the isocortex and striatum. Several nuclei, such as the caudate-putamen and the CA1 field of the hippocampus, exhibited input preferences to different cell types in the amygdala. Notably, we identified several novel input areas, including the substantia innominata and zona incerta. These findings provide anatomical evidence to help understand the precise connections and diverse functions of the amygdala.

Keywords: Basolateral amygdala; Central amygdala; GABAergic; Glutamatergic; Mouse; Rabies virus retrograde tracing.

Fig. 1

Virus injection strategy. A Schematic of viral vectors and rabies virus-mediated trans-synaptic retrograde tracing of amygdala inputs. B–E Ratios of input cells to starter cells in BLA VGLUT2-Cre mice (B), BLA PV-Cre mice (C), CeA PKC-δ-Cre mice (D), and CeA SST-Cre mice (E) (n = 4/group, mean ± SEM).

Fig. 2

Long-range inputs to BLA VGLUT2⁺ neurons. A Representative images of starter cells restricted to the BLA. Starter cells expressed both GFP and dsRed fluorescent proteins were marked by arrowheads. Scale bars, 100 μm. B Representative images of RV-labeled input neurons to BLA VGLUT2⁺ neurons from selected nuclei. Scale bars, 100 μm. C Whole-brain distribution of input nuclei to BLA VGLUT2⁺ neurons with input percentages > 1% (mean ± SEM). D Schematic summary of brain regions providing the largest average fractional inputs to BLA VGLUT2⁺ neurons. Abbreviations are shown in Table 1.

Fig. 3

Long-range inputs to BLA PV⁺ GABAergic neurons. A Representative images of starter cells restricted to the BLA. Starter cells expressed both GFP and dsRed fluorescent proteins were marked by arrowheads. Scale bars, 100 μm. B Representative images of RV-labeled input neurons to BLA PV⁺ neurons from selected nuclei. Scale bars, 100 μm. C Whole-brain distribution of input nuclei to BLA PV⁺ neurons with input percentages > 1% (mean ± SEM). D Schematic summary of regions providing the largest average fractional inputs to BLA PV⁺ neurons. Abbreviations are shown in Table 1.

Fig. 4

Long-range inputs to CeA PKC-δ⁺ GABAergic neurons. A Representative images of starter cells restricted to the CeA. Starter cells expressed both GFP and dsRed fluorescent proteins were marked by arrowheads. Scale bars, 100 μm. B Representative images of RV-labeled input neurons to CeA PKC-δ⁺ neurons from selected nuclei. Scale bars, 100 μm. C Whole-brain distribution of input nuclei to CeA PKC-δ⁺ neurons with input percentages > 1% (mean ± SEM). D Schematic summary of regions providing the largest average fractional inputs to CeA PKC-δ⁺ neurons. Abbreviations are shown in Table 1.

Fig. 5

Long-range inputs to CeA SST⁺ GABAergic neurons. A Representative images of starter cells restricted to the CeA. Starter cells expressed both GFP and dsRed fluorescent proteins were marked by arrowheads. Scale bars, 100 μm. B Representative images of RV-labeled input neurons to CeA SST⁺ neurons from selected nuclei. Scale bars, 100 μm. C Whole-brain distribution of input nuclei to CeA SST⁺ neurons with input percentages > 1% (mean ± SEM). D Schematic summary of regions providing the largest average fractional inputs to CeA SST⁺ neurons. Abbreviations are shown in Table 1.

Fig. 6

Comparison of input percentages of different nuclei among different cell types. A Comparison of input percentages from different nuclei in VGLUT2-IRES-Cre and PV-IRES-Cre mice (n = 4, ***P < 0.001, mean ± SEM). B Comparison of input percentages in PKC-δ-IRES-Cre and SST-IRES-Cre mice (n = 4, **P < 0.01, ***P < 0.001). C Percentage of total inputs from selected regions for BLA glutamatergic and PV⁺ neurons (n = 4, *P < 0.05, ****P < 0.0001). D Percentage of total inputs from selected regions for CeA PKC-δ⁺ and SST⁺ neurons (n = 4, *P < 0.05, ****P < 0.0001). Abbreviations are shown in Table 1.