Whole-Brain Mapping of Specific Orbitofrontal Cortex Neuron Inputs and Outputs in Mice

Research on the mouse brain has always been an important topic in neuroscience, especially studies on the orbitofrontal cortex (ORB). The ORB plays a key role in reward processing, decision making, behavioral flexibility, and emotional regulation. In recent years, it has been discovered that dysfunction of the ORB is often associated with various mental and neurological disorders such as depression, obsessive-compulsive disorder, and substance use disorder. Through the study of ORB, scientists aim to gain a deeper understanding of its functions in both normal and pathological states.

This paper is authored by Yijie Zhang, Wen Zhang, Lizhao Wang, among others, and completed by researchers from institutions including the Center for Excellence in Brain Science and Intelligence Technology of the Chinese Academy of Sciences, the Research Center for Brain Science and Brain-Inspired Intelligence Technology at Fudan University, and the Suzhou Institute of Biomedical Engineering and Technology at Huazhong University of Science and Technology. The study was published in Neurosci. Bull. in December 2023.

Research Background and Motivation

Firstly, the authors point out that ORB neurons project to multiple brain regions, including the cerebral cortex, thalamus, amygdala, striatum, and hypothalamus. Different ORB pathways exhibit significant functional differences. For example, ORB projections to the secondary motor area (MOS) mediate learned action-outcome associations, while projections to the dorsal striatum play an important role in guiding value-based actions.

Despite previous studies using retrograde axonal tracers to examine cortical and thalamic inputs to the ORB, the input patterns to specific projection ORB neurons remain unclear. Moreover, the ORB divides into several subregions such as lateral orbital (ORB-L), middle orbital (ORB-M), and ventrolateral orbital (ORB-VL). Functional heterogeneity across these subregions has been revealed through behavioral, pharmacological, physiological, and optogenetic/chemogenetic methods. However, the distribution of specific inhibitory neurons targeting ORB subregions still requires further research.

Research Methods and Process

Researchers conducted whole-brain mapping in mice to reveal inputs to five types of projection-specific ORB neurons, as well as outputs from ORB to two types of inhibitory neurons.

Animal Models and Viral Injection

The study used adult (2-4 months) male mice, including C57BL/6, PV-Cre, SST-Cre, and hybrid strains. Viral tracing of neuronal connections was administered by injecting viruses into specific brain areas. During surgery, mice were anesthetized and fixed onto a stereotaxic apparatus, and craniotomy was performed at the virus injection site in the left hemisphere.

Viral Strategy

Researchers used retrograde single-synapse tracing technology and anterograde trans-synaptic viral tracing techniques to inject viruses into five brain regions: Visp, VTA, BLA, CP, and MOS. Retrograde viruses and helper viruses were mixed and injected to label specific projection ORB neurons and cause these neurons to express specific receptors and proteins. Subsequently, modified rabies virus was injected, infecting ORB neurons expressing the receptors and spreading retrogradely to presynaptic cells.

To determine the functional connectivity of specific neuronal projections, researchers used optogenetic technology. Laser stimulation of axon terminals in specific brain regions was employed to record the electrophysiological responses of postsynaptic cells.

Data Analysis

Processing and analysis of data included slice preparation, recording, histological processing, and imaging and quantification of whole-brain slices. All data were analyzed using custom software packages, including image registration, signal detection, and quantification/visualization modules.

During mapping, researchers prepared 50μm thick sections post-fixation and imaged them using a microscope. The images were then aligned to the Allen Mouse Brain Atlas, and the locations of labeled cells were manually detected and quantified.

Statistical Analysis

Experimental evidence was analyzed to determine the proportions of inputs and outputs. Statistical analysis was performed using GraphPad Prism, and significance was determined through repeated measures ANOVA and Wilcoxon signed-rank tests.

Main Research Results

Whole-Brain Inputs to Projection-Specific ORB Neurons

The study found that projection-specific ORB neurons mainly received inputs from the brain’s medulla and thalamus. While the inputs to different types of ORB projection neurons were similar, there were quantitative differences in certain regions. Specifically, MOS-projecting neurons received more inputs from MOS, whereas BLA-projecting neurons received more inputs from the primary motor cortex.

ORB Subregion Outputs to Inhibitory Neurons

Using anterograde trans-synaptic viral tracing techniques, researchers mapped the outputs of ORB subregion neurons to PV and SST inhibitory neurons. The study indicated that ORB neurons targeted PV and SST neurons in many brain regions and exhibited significant differences among the three ORB subregions. Notably, ORB-M neurons’ projections to SST were more prominent, especially in the sensory regions, hippocampus, amygdala, and hypothalamic lateral area.

Conclusion and Significance

Through this research, scientists identified the whole-brain inputs to projection-specific ORB neurons and the outputs from neurons in each ORB subregion to inhibitory neurons. This provides an important anatomical foundation for future research into the functional organization of ORB circuits and their role in reward association, learning, and flexible behavior.

Especially, the study highlighted the potential functional heterogeneity of ORB projections to specific inhibitory neurons in regulating neural processing and behavior. For example, the ORB forms direct monosynaptic excitatory connections to PV (or SST) neurons in RT, potentially elucidating the involvement of ORB circuits in perceptual decision-making behavior in future studies.

Research Highlights

The highlights of this study include: 1. Extensive Input and Output Mapping: Revealed the inputs to five types of projection-specific ORB neurons and the outputs from three ORB subregions, providing comprehensive anatomical data. 2. Validation of Neuronal Functional Connectivity: Validated the functional connectivity between ORB neurons and specific inhibitory neurons using whole-brain slice imaging and electrophysiological optogenetics. 3. Diverse Research Methods: Combined viral tracing, histological imaging, data analysis, and optogenetics to provide a comprehensive research process.

Future Research Directions

This study lays a foundation for further exploring the role of ORB circuits in specific tasks and behaviors. Future research can delve deeper into the functional connectivity between ORB and PV and SST neurons in specific behaviors, thereby better understanding the role of ORB in cognitive functions and pathological states. The study provides rich anatomical experimental evidence for the functional organization of the ORB in the mouse brain, holding important scientific and applicative value.