Cell-Type-Specific Afferent Innervation of the Nucleus Accumbens Core and Shell

Abstract

The nucleus accumbens (NAc) is clearly implicated in reward processing and drug addiction, as well as in numerous neurological and psychiatric disorders; nevertheless, the circuit mechanisms underlying the diverse functions of the NAc remain poorly understood. Here, we characterized the whole-brain and monosynaptic inputs to two main projection cell types - D1 dopamine receptor expressing medium spiny neurons (D1R-MSNs) and D2 dopamine receptor expressing medium spiny neurons (D2R-MSNs) - within the NAc core and NAc shell by rabies-mediated trans-synaptic tracing. We discovered that D1R-MSNs and D2R-MSNs in both NAc subregions receive similar inputs from diverse sources. Inputs to the NAc core are broadly scattered, whereas inputs to the NAc shell are relatively concentrated. Furthermore, we identified numerous brain areas providing important contrasting inputs to different NAc subregions. The anterior cortex preferentially innervates the NAc core for both D1R-MSNs and D2R-MSNs, whereas the lateral hypothalamic area (LH) preferentially targets D1R-MSNs in the NAc shell. Characterizing the cell-type-specific connectivity of different NAc subregions lays a foundation for studying how diverse functions of the NAc are mediated by specific pathways.

Keywords: cell-type-specific; nucleus accumbens core and shell; quantitative analyses; rabies virus; whole-brain inputs.

FIGURE 1

Experimental strategy for identification of monosynaptic inputs to D1R- and D2R-MSNs in different NAc subregions. (A) Recombinant AAV strains and RV. (B) Experimental design. (C) Representative coronal brain sections near the injection sites. Blue indicates cell nuclear staining with DAPI. Scale bar, 1000 μm. Inset, enlarged view of the area in white the box showing starter cells (yellow, expressing both eGFP and dsRed, indicated by white arrowheads). Scale bar, 30 μm. AcbC, nucleus accumbens, core (NAc core); aca, anterior commissure, anterior part; AcbSh, nucleus accumbens, shell (NAc shell). (D) Numbers of starter neurons in the individual animals. (E) Proportions of labeled starter neurons in injection regions. LS, lateral septal nucleus; BST, bed nucleus of the stria terminalis. (F) Numbers of transsynaptically labeled neurons, i.e., input neurons in the individual animals. (G) A linear relationship was detected between the number of starter and input neurons.

FIGURE 2

Control experiments for rabies-mediated transsynaptic tracing. (A) Injection of RV without prior AAV injection resulted in no dsRed-labeled neurons, indicating the dependence of the RV infection on AAV-induced expression of TVA. Scale bar, 500 μm. (B) Injecting AAV-DIO-EGFP-TVA, AAV-DIO-RG, and RV into the wild-type mice led to a low level of nonspecific labeled RV neurons that expressed dsRed at the injection site. Scale bar, 500 μm. (C₁) Injection of AAV-DIO-EGFP-TVA and RV without prior AAV-DIO-RG injection in the D1R-Cre mice resulted in no DsRed-labeled input neurons. Scale bar, 500 μm. (C₂) Enlarged view of the boxed region in panel (C₁); all dsRed-positive neurons coexpressed eGFP. Scale bar, 50 μm.

FIGURE 3

Overview of whole brain input to D1R-MSNs and D2R-MSNs in different NAc subregions. (A) Representative coronal sections showing labeling of monosynaptic inputs to D1R-core, D1R-shell, D2R-core, and D2R-shell neurons. Only the side ipsilateral to the injection site is shown. Scale bar, 1 mm. (B) Proportions of total inputs from 11 brain areas. Mean ± SEM (n = 6 mice each for the D1R-core, D1R-shell, D2R-core, and D2R- shell groups). ^∗∗∗p < 0.001, ^∗∗p < 0.01, and ^∗p < 0.05. Only significant differences between the same cell type in different subregions or between different cell types in the same subregions are marked; one-way ANOVA with Bonferroni correction.

FIGURE 4

Quantitative analysis of the proportions of whole-brain input to D1R-MSNs and D2R-MSNs in different NAc subregions. (Left) Monosynaptic inputs to D1R-MSNs in the NAc core (red) and NAc shell (blue). (Right) Monosynaptic inputs to D2R-MSNs in the NAc core (red) and NAc shell (blue). Mean ± SEM (n = 6 mice for each group).

FIGURE 5

Monosynaptic inputs to D1R-MSNs from the cortex and thalamus. (A) Distributions of anterior cortex inputs to D1R-MSNs in the NAc core (upper panels) and shell (lower panels). Scale bar, 200 μm. (B) Proportion of total inputs to D1R-MSNs in the NAc core (red) and shell (blue) from 11 cortical areas that contained relatively large numbers of input neurons (>1% in at least one experimental group). Mean ± SEM (n = 6 mice for each group). ^∗∗∗p < 0.001, ^∗∗p < 0.01, and ^∗p < 0.05, two-tailed unpaired t-test or Mann–Whitney U test. (C) Distributions of thalamic inputs to D1R-MSNs in the NAc core (right) and shell (left). Inset, enlarged view of the area in the white box showing spatially separated labeling. Scale bar, 200 μm.

FIGURE 6

Monosynaptic inputs to D2R-MSNs from the cortex and thalamus. (A) Distributions of anterior cortex inputs to D2R-MSNs in the NAc core (upper panels) and shell (lower panels). Scale bar, 200 μm. (B) Proportion of total inputs to D2R-MSNs in the NAc core (red) and shell (blue) from 11 cortical areas that contained relatively large numbers of input neurons (>1% in at least one experimental group). Mean ± SEM (n = 6 mice for each group). ^∗∗∗p < 0.001, ^∗∗p < 0.01, and ^∗p < 0.05, two-tailed unpaired t-test or Mann–Whitney U test. (C) Distributions of thalamic inputs to D2R-MSNs in the NAc core (right) and shell (left). Inset, enlarged view of the area in the white box showing spatially separated labeling. Scale bar, 200 μm.

FIGURE 7

Comparisons of input distributions across the four groups. (A) Comparison between inputs to D1R-core and D2R-core neurons. (B) Comparison between inputs to D1R-shell and D2R-shell neurons. (C) Comparison between inputs to D1R-core and D1R-shell neurons. (D) Comparison between inputs to D2R-core and D2R-shell neurons. Values are the means of the percent of total inputs from each region. Red circles indicate significant differences (p < 0.05, two-tailed unpaired t-test or Mann–Whitney U test). r, Pearson’s correlation coefficient. (E) Summary of selected prominent monosynaptic inputs to the NAc core (top, red) and shell (bottom, blue), considering D1R-MSNs and D2R-MSNs together. Line thickness represents the number of inputs.