Whole-brain Mapping of Inputs and Outputs of Specific Orbitofrontal Cortical Neurons in Mice

Abstract

The orbitofrontal cortex (ORB), a region crucial for stimulus-reward association, decision-making, and flexible behaviors, extensively connects with other brain areas. However, brain-wide inputs to projection-defined ORB neurons and the distribution of inhibitory neurons postsynaptic to neurons in specific ORB subregions remain poorly characterized. Here we mapped the inputs of five types of projection-specific ORB neurons and ORB outputs to two types of inhibitory neurons. We found that different projection-defined ORB neurons received inputs from similar cortical and thalamic regions, albeit with quantitative variations, particularly in somatomotor areas and medial groups of the dorsal thalamus. By counting parvalbumin (PV) or somatostatin (SST) interneurons innervated by neurons in specific ORB subregions, we found a higher fraction of PV neurons in sensory cortices and a higher fraction of SST neurons in subcortical regions targeted by medial ORB neurons. These results provide insights into understanding and investigating the function of specific ORB neurons.

Keywords: Anterograde transsynaptic virus tracing; Inhibitory neurons; Orbitofrontal cortex; Projection specific; Rabies virus retrograde tracing.

Fig. 1

Virus strategy for input mapping and data processing. A Virus vectors and injection procedure for RV-mediated monosynaptic retrograde tracing of the inputs of projection-specific ORB neurons. B Main steps for data processing. C Example fluorescence images showing BFP in the downstream targets of ORB and starter cells (expressing both GFP and mCherry) in the ORB, for different projection-specific ORB neurons. Scale bars, 200 μm for the left column and 100 μm for the right column. D Convergence index (the ratio between the number of input cells and starter cells) for each brain sample grouped by projection cell type. E Fractions of stater cells in the ORB and its adjacent region ILA. Data are represented by the mean ± SEM.

Fig. 2

Whole brain inputs to five types of projection-specific ORB neurons. A Representative fluorescence images in selected brain regions showing RV-labeled input cells to CP-projecting ORB neurons. Scale bars, 1 mm. B Illustration of the anatomical location of the sections shown in A. C Whole-brain distributions of input neurons to five types of projection-specific ORB neurons. *P < 0.05, **P < 0.01, n = 3 mice for each group, two-way repeated measures ANOVA followed by Tukey’s multiple comparisons test. Data are represented by the mean ± SEM.

Fig. 3

Percentage of input cells in different subdivisions within a cortical region. A Percentages of input cells in MOp and MOs. B Percentages of input cells in SSp and SSs. C Percentages of input cells in ACAd and ACAv. D Percentages of input cells in AId, AIp, and AIv. E Representative fluorescence image showing RV-labeled input cells to CP-projecting ORB neurons in different layers of SS, MO, and ACA. Scale bar, 500 μm. F Percentages of input cells in different layers of limbic, motor, and sensory areas. Limbic area: ACA, PL, ILA, and AI. Motor area: FRP and MO. Sensory area: SS, AUD, and VIS. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Two-way repeated measures ANOVA followed by Tukey’s multiple comparisons test. Data are represented by the mean ± SEM.

Fig. 4

Thalamic inputs to five types of projection-specific ORB neurons. A Representative fluorescence images showing RV-labeled input cells in MD, SMT, VAL, and VM, which project to CP-projecting ORB neurons. Scale bars, 200 μm. B Percentages of input cells in different thalamic regions. C Percentages of input cells in MDc, MDl, and MDm. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Two-way repeated measures ANOVA followed by Tukey’s multiple comparisons test. Data are represented by the mean ± SEM.

Fig. 5

Functional inputs from MD to CP-projecting ORB neurons. A Virus strategy for slice recording of CP-projecting ORB neurons in response to photostimulation of MD axon terminals. B Upper, representative fluorescence image showing ChR2-expressing MD axon terminals (red) surrounding CP-projecting ORB neurons (green). Blue, DAPI. Scale bar, 25 μm. Lower, EPSC of an example cell in response to laser stimulation (blue). C. The number and percentage of recorded cells showing light-evoked EPSCs. D Latency and peak amplitude of EPSCs from 8 CP-projecting ORB neurons. In each box, the central line is the median, the edges of the box are the 25^th and 7^th percentiles, and the cross indicates the outlier. E Example traces of EPSCs from a CP-projecting ORB neuron under different conditions. Blue, laser stimulation of MD axon terminals. F The peak amplitudes of EPSCs of CP-projecting ORB neurons under different conditions.

Fig. 6

Virus strategy for mapping ORB outputs to PV or SST neurons. A Virus vector for anterograde transsynaptic tracing of ORB outputs to PV or SST neurons. The injection site was marked by co-injection of CTB-555 with the virus. B Representative fluorescence images showing CTB-555 (red) and labeled cells (green) in injection sites of ORBl, ORBm, and ORBvl in SST-Cre mice. Scale bars, 200 μm. C Representative fluorescence images showing labeled cells in selected brain regions after injecting scAAV1-hSyn-FLEX-EGFP in the ORBm of a PV-Cre mouse. Scale bars, 1 mm. D Illustration of the anatomical location of the sections shown in C.

Fig. 7

The distribution of PV or SST neurons innervated by neurons in specific ORB subregions. A Whole-brain distributions of labeled PV neurons targeted by neurons in ORBl, ORBm, or ORBvl. B Whole-brain distributions of labeled SST neurons targeted by neurons in ORBl, ORBm, or ORBvl. C Comparison of the percentages of labeled PV and SST neurons targeted by neurons in ORBl (upper panel), ORBm (middle panel), or ORBvl (lower panel). D Comparison of the percentages of labeled PV and SST neurons targeted by neurons in ORBm. Upper, labeled neurons in sensory areas (SS, AUD, and VIS) and association areas (RSP, PTLp, TEa, PERI, and ECT). Lower, labeled neurons in selected regions outside the isocortex and thalamus. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, two-way repeated measures ANOVA followed by Tukey’s multiple comparisons test. Data are represented by the mean ± SEM.

Fig. 8

Functional inputs from ORB to PV or SST neurons in RT. A Virus strategy for slice recording from transsynaptically labeled PV or SST neurons in the RT in response to photostimulation of ORB axon terminals. B Upper, representative fluorescence image showing ChR2-expressing ORB axon terminals (red) surrounding PV neurons (green) in the RT. Blue, DAPI. Scale bar, 25 μm. Lower, EPSC of an example PV neuron in response to laser stimulation (blue). C Latency and peak amplitude of EPSCs from 7 PV neurons in RT. D Latency and peak amplitude of EPSCs from 4 SST neurons in RT. E Example traces of EPSCs from a PV neuron (left) and an SST neuron (right) in the RT under different conditions. Blue, laser stimulation of ORB axon terminals. F The peak amplitudes of EPSCs of RT neurons under different conditions.