The mouse cortico–basal ganglia–thalamic network

The cortico–basal ganglia–thalamo–cortical loop is one of the fundamental network motifs in the brain. Revealing its structural and functional organization is critical to understanding cognition, sensorimotor behaviour, and the natural history of many neurological and neuropsychiatric disorders. Classically, this network is conceptualized to contain three information channels: motor, limbic and associative1–4. Yet this three-channel view cannot explain the myriad functions of the basal ganglia. We previously subdivided the dorsal striatum into 29 functional domains on the basis of the topography of inputs from the entire cortex5. Here we map the multi-synaptic output pathways of these striatal domains through the globus pallidus external part (GPe), substantia nigra reticular part (SNr), thalamic nuclei and cortex. Accordingly, we identify 14 SNr and 36 GPe domains and a direct cortico-SNr projection. The striatonigral direct pathway displays a greater convergence of striatal inputs than the more parallel striatopallidal indirect pathway, although direct and indirect pathways originating from the same striatal domain ultimately converge onto the same postsynaptic SNr neurons. Following the SNr outputs, we delineate six domains in the parafascicular and ventromedial thalamic nuclei. Subsequently, we identify six parallel cortico–basal ganglia–thalamic subnetworks that sequentially transduce specific subsets of cortical information through every elemental node of the cortico–﻿basal ganglia–thalamic loop. Thalamic domains relay this output back to the originating corticostriatal neurons of each subnetwork in a bona fide closed loop.

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Software and code
Policy information about availability of computer code Data collection All epifluorescence images used in the striatal output axonal analyses were collected with the Olympus VS120 fluorescence microscope running Olympus VS-Desktop v2.9. High resolution confocal images were captured using an Andor DragonFly 202 spinning disk confocal microscope running Fusion v2.1.0.81 software. Lightsheet images were captured with a LifeCanvas lightsheet microscope running SmartSPIM Acquisition Software 2019v3. Electrophysiological data were collected using a MultiClamp700B Amplifier (Molecular Devices) running pClamp v. 10.7.

Data analysis
All standard statistical analyses were performed with GraphPad Prism v4.0c for Macintosh, including ANOVA, 2-sided t test with Welch's correction, Fisher's exact test, Pearson's r, and descriptive statistics. The algorithm implementing the Louvain analysis was obtained from the Brain Connectivity Toolbox (available at: https://sites.google.com/site/bctnet/) and executed in Python v2.7.
The Connection Lens v2.5.1 software used to register, threshold, and quantify the striatofugal axonal data was designed in-house. This software has not been released publicly yet, although it has been used in our previously published works (Hintiryan et al. 2016;Benavidez et al. 2021;Hintiryan et al. 2021).
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April 2020
Data Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability Downsampled images of all data used in the striatofugal analyses are shown in Extended Data Figure 2. The quantified data for these cases can be accessed through our B.R.A.I.N. Lab website (http://brain.neurobio.ucla.edu/publications/). Also available are the SNr neuronal reconstructions from Figure 1k, the Supplementary Video, and an application presenting the projection maps of all axonal reconstructions.

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Sample size
The sample size for the striatofugal analysis was determined by the number of domains in the striatum, i.e., 36. This consists of 29 domains in the dorsal striatum as described in Hintiryan et al (2016), as well as 4 additional dorsal striatal domains described in Methods and 3 domains in the ventral striatum: the core, medial shell, and lateral shell. The ventral striatum may or may not contain more subregions, but the core and shell are well-documented sub-compartments of the accumbens, and differences in connectivity patterns of medial and lateral shell have been described ( Data exclusions One and only one injection per striatal domain was used in the striatofugal analysis, as is standard in neuroanatomical research and as is necessary for the kind of community analysis we conducted. The best, most representative injection for each domain was chosen for the analysis. The others were excluded due to off-targeting of the injection site, missing/damaged tissue in the pallidal and nigral regions of interest, and weak tracer labeling of the axons or high background. For electrophysiology experiments, data were excluded from neurons that were recorded outside of the target nuclei (i.e., the GPe, SNr, MOp-m/i, and ACA).

Replication
All striatal domains were targeted with injections multiple times to generate the striatofugal dataset. While the best, most representative cases were chosen for inclusion in the analysis data set, the other injections served as validation cases, demonstrating the replicability and consistency of labeling arising from each domain.
Randomization Randomization is not relevant to the present work since animals were not compared across different conditions.

Blinding
Traditional blinding was not necessary since animals were not compared across different conditions. However, bias in image registration for the striatofugal analysis is the one area where the methods could have affected the results. In that regard, the image registration process, although not technically blinded, was performed by contributors without any a priori knowledge of the pathways under investigation.

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