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Monosynaptic trans-collicular pathways link mouse whisker circuits to integrate somatosensory and motor cortical signals [1]

['Jesús Martín-Cortecero', 'Medical Biophysics', 'Institute For Physiology', 'Pathophysiology', 'Heidelberg University', 'Emilio Ulises Isaías-Camacho', 'Berin Boztepe', 'Katharina Ziegler', 'Rebecca Audrey Mease', 'Alexander Groh']

Date: 2023-05

The superior colliculus (SC), a conserved midbrain node with extensive long-range connectivity throughout the brain, is a key structure for innate behaviors. Descending cortical pathways are increasingly recognized as central control points for SC-mediated behaviors, but how cortico-collicular pathways coordinate SC activity at the cellular level is poorly understood. Moreover, despite the known role of the SC as a multisensory integrator, the involvement of the SC in the somatosensory system is largely unexplored in comparison to its involvement in the visual and auditory systems. Here, we mapped the connectivity of the whisker-sensitive region of the SC in mice with trans-synaptic and intersectional tracing tools and in vivo electrophysiology. The results reveal a novel trans-collicular connectivity motif in which neurons in motor- and somatosensory cortices impinge onto the brainstem-SC-brainstem sensory-motor arc and onto SC-midbrain output pathways via only one synapse in the SC. Intersectional approaches and optogenetically assisted connectivity quantifications in vivo reveal convergence of motor and somatosensory cortical input on individual SC neurons, providing a new framework for sensory-motor integration in the SC. More than a third of the cortical recipient neurons in the whisker SC are GABAergic neurons, which include a hitherto unknown population of GABAergic projection neurons targeting thalamic nuclei and the zona incerta. These results pinpoint a whisker region in the SC of mice as a node for the integration of somatosensory and motor cortical signals via parallel excitatory and inhibitory trans-collicular pathways, which link cortical and subcortical whisker circuits for somato-motor integration.

Funding: This work was supported by the German Research Foundation (DFG Grants GR3757/3-1, GR3757/4-1 to AG), the Heidelberg Graduate Academy completion grant through the Landesgraduiertenförderung program with funds allocated by the German Ministry of Science, Research and Arts (salary grant to EIC), the Chica and Heinz Schaller Stiftung (salary grant to RM) and the Brigitte-Schlieben-Lange Programm by the Ministry of Science, Research and the Arts Baden-Württemberg (salary grant to RM). We acknowledge the data storage service SDS@hd and high-performance computing initiative bwHPC, supported by the Ministry of Science, Research and the Arts Baden-Württemberg (SDS@hd and bwHPC) and the German Research Foundation (DFG) through grant INST 35/1314-1 FUGG and INST 35/1503-1 FUGG (SDS@hd). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data Availability: All relevant data are within the paper and its Supporting Information files. Data files for the figures and electrophysiology data are available from the public repository heiDATA: https://doi.org/10.11588/data/DNOSZG .

Copyright: © 2023 Martín-Cortecero et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Here, we mapped the input–output connectivity of the mouse SC on the single-cell level with trans-synaptic tracing, intersectional viral approaches, and optogenetic-assisted electrophysiology. In awake animals, we identify a whisker-sensitive region in the SC, which receives trigeminal and cortico-collicular input from motor and somatosensory cortex. We then targeted specific subsets of input-defined pathways from the brainstem and motor and somatosensory cortices to the SC and traced their axonal outputs to downstream targets, revealing direct trans-collicular pathways, which provide disynaptic long-range links between cortical pathways and SC target regions. Trans-synaptic labeling in combination with optogenetic input mapping reveals long-range input convergence on the level of individual SC neurons, with approximately one-third of the cortical recipient SC neurons receiving convergent input from both motor and somatosensory cortex. We find that long-range input pathways extensively target inhibitory SC neurons, which, in turn, give rise to long-range GABAergic projections to thalamic nuclei and the zona incerta (ZI). In sum, this study pinpoints a “whisker SC,” in which converging cortical and brainstem inputs innervate GABAergic and non-GABAergic SC neurons, which, in turn, provide parallel trans-collicular pathways to downstream whisker circuits in the diencephalon and brainstem. These results suggest that this cortical control of SC directly affects the brainstem-SC-brainstem sensory-motor arc, as well as the outputs from SC to diencephalic stations.

(3) Do pathways from motor and somatosensory cortices converge at the level of individual SC neurons? The multisensory nature of the SC has motivated a large body of studies demonstrating the integration of visual, auditory, somatosensory, and motor signals in the SC [ 17 , 18 , 21 – 25 ]. Nevertheless, whether sensory and motor cortex pathways converge in individual SC neurons remains untested ( Fig 1 , right panel). The lateral SC (LSC) has been identified as a putative point of convergence of somatosensory and motor cortical efferents [ 16 ], making it a promising target to test this hypothesis.

(2) Are cortical-recipient SC neurons excitatory or inhibitory? The SC comprises excitatory and inhibitory neurons; for example, in the visual SC, approximately one-third of the neurons are GABAergic [ 15 , 20 ]. However, very little is known about GABAergic neurons in the somatosensory SC ( Fig 1 , center panel), including their proportions, their inputs and outputs, and whether they are classic local interneurons or projections neurons. Determining how cortical pathways engage specific excitatory or inhibitory SC circuits and identifying which downstream pathways emanate from these circuits is indispensable to make circuit-mechanistic predictions about recently discovered top-down modulation of SC-mediated innate escape and defense behaviors [ 4 – 6 ].

(1) To where do cortical-recipient SC neurons project? Cortico-collicular signals may be routed directly to SC downstream targets (i.e., in the diencephalon and brainstem) via monosynaptic trans-collicular pathways and/or more indirectly, via intracollicular connections to SC output neurons [ 2 ] ( Fig 1 , left panel). While intracollicular circuits are well described [ 13 ], evidence for monosynaptic trans-collicular pathways is limited because relatively few studies have delineated the precise input–output connectivity of defined subpopulations in SC [ 2 , 14 ]. Indeed, input–output connectivity of the SC is mostly considered at the level of SC layers [ 2 , 15 ] and radial cortico-collicular input zones [ 16 ]. However, these mesoscopic modules comprise intermingled populations of input and output neurons as well as input neurons with different input identities [ 16 – 18 ], for example, retinal and cortical inputs [ 19 ]. Therefore, dissection of input-defined cell types and analysis of their outputs is critical to unambiguously link specific SC input pathways to specific SC downstream targets and to determine whether cortical input signals can be transformed directly into SC output to downstream SC target circuits.

The superior colliculus (SC) is part of a phylogenetically ancient brain network that directs quick motor actions in response to ascending sensory signals [ 1 , 2 ]. As such, the SC is a central hub for multiple sensory-motor arcs linking sensory information to motor actions. From an evolutionary perspective, much of the SC’s function in mammalian brains has been taken over by the neocortex, via parallel, cortically controlled sensory-motor arcs [ 3 ]. Intriguingly, recent work in the visual and auditory systems demonstrate that SC-mediated behaviors are modulated by cortical inputs [ 4 – 6 ], raising the question of how cortex- and SC-mediated sensory-motor arcs interact to organize desirable behavior. More specifically, it is not well understood how cortico-collicular pathways engage with SC microcircuitry and, in turn, generate SC output signals to the brainstem and diencephalon (including thalamus). This information is essential not only to understand how cortico-collicular pathways may coordinate between the “new” cortical and the “old” collicular arcs, but also to answer principal questions concerning the identity of cortical-recipient (i.e., targeted by cortex)–SC neurons: to where do they project, are they excitatory or inhibitory, and do they receive sensory and/or motor signals? SC circuits have mostly been studied in relation to vision [ 2 , 6 – 8 ], i.e., looking at functions of the “visual SC.” In contrast, somatosensory functions of the SC are less studied and less understood, even though somatosensory functions such as whisker sensation are vital for rodents and other animals [ 9 – 12 ]. This study set out to address the involvement of “somatosensory SC” circuits in the whisker system and focused on 3 principal questions about the organization of trans-collicular pathways that mediate cortical input to SC downstream targets ( Fig 1 ).

Results

Cellular organization of somatosensory and motor pathways in the lateral SC We next sought to directly target MC-, BC-, and Bs-recipient neurons (RNs) in the LSC to study their organization, cellular identity, and projection patterns. To do so, we employed a trans-synaptic anterograde approach, which is based on the ability of the AAV1 serotype to trans-synaptically jump to postsynaptic neurons [4]. We injected AAV1-Cre in combination with AAV2-DIO-mCherry into MC, BC, and Bs to simultaneously label the injection sites and their axonal projections with mCherry (red) and to express cre in the synaptically connected postsynaptic target neurons in LSC. Finally, Cre-expressing RNs were revealed by injecting AAV2-DIO-EGFP into LSC (green) (Figs 4A, 4B, S3 and S9). This strategy allowed us to visualize and reconstruct the 3 RN populations (MC-RNs, 6 mice; BC-RNs, 6 mice; Bs-RNs, 7 mice) and register them to standard anatomical borders within SC. This revealed that the 3 whisker-related input pathways predominantly target neurons in the intermediate layer of the LSC (Fig 4C). The RN types largely overlap along the antero-posterior axis (Fig 4D) but are more segregated in the dorsal-ventral and medial-lateral dimensions. Here, the organization shows a lateral Bs-recipient zone, adjacent to a medial cortical-recipient zone with considerable overlap between MC- and BC-RNs (Fig 4E and 4F). We overlaid these recipient zones on the approximate outlines of the radial zones determined by Benavidez and colleagues [16]. The comparison shows that recipient zones fall into the radial zones as follows: BS → lateral / centrolateral; BC → centrolateral; MC → centrolateral / centromedial (Fig 4E). PPT PowerPoint slide

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TIFF original image Download: Fig 4. Whisker-related sensory-motor RNs are organized into overlapping zones within the intermediate layer the LSC. (A) Trans-synaptic labeling of MC-, BC-, and Bs-RNs in SC (left). Cocktails of AAV1-Cre + AAV-DIO-mCherry were injected into projecting sites (MC, BC, or Bs) and AAV2-DIO-EGFP in SC. Example fluorescent pictures (right) of mCherry expression (red) in the projecting sites. (B) Top left: Schematic of trans-synaptically labeled RNs (green). Top right: Example confocal image of SC showing mCherry-expressing BC axons (red) and Cre-dependent expression of EGFP in BC-RNs (green). Bottom: Fluorescent images of EGFP expression (green) in MC-, BC-, and Bs- RNs. (C) Example reconstructions of the 3 RN populations along the rostro-caudal axis (red: MC-RNs; blue: BC-RNs; green: Bs-RNs), registered to standard anatomical borders within SC. (D) Anterior–posterior distributions of RNs. Data points show mean RN counts per 100 μm bin (red: MC-RNs, 6 mice; blue: BC-RNs, 6 mice; green: Bs-RNs, 7 mice) normalized to their maximum count. Box plots show medians (line in box) and IQRs (first to third quartile) in mm (boxes), ([median, Q1, Q3 in mm] MC-RNs: 3.58, 3.38, 3.88; BC-RNs: 3.68, 3.48, 3.88; Bs-RNs: 3.68, 3.48, 3.88). (E) Comparison of recipient zones in the DV and ML dimensions in SC. Fluorescent thresholded representative slices of each RN population, registered at a similar AP coordinate and to approximate radial SC zones according to [16]. (Centroids: [DV, ML, μm] MC: 1,123, 1,505; BC: 1,035, 1,297; Bs: 1,276, 993). (F) Colabeling experiment of cortical (red, MC-RNs + BC-RNs) and Bs-RNs (green). Top right: Example fluorescent image showing cortical and peripheral RNs. Middle: Fluorescence thresholded RN signals from 15 consecutive images (1 brain). Bottom: Histograms show the thresholded pixel gray value probability for cortical-RNs and Bs-RNs in the DV axis and in the ML axis. ([DV, median and IQR, relative to SC dorsal surface] Cortical-RN [μm]: 1,115, 147; Bs-RN: 1,365, 281; [ML, median and IQR, μm relative to SC midline] Cortical-RN: 1,472, 211; Bs-RN: 1,637, 287, p < 0.001). * represents p < 0.01; D: Kruskal–Wallis, F: Wilcoxon rank-sum; exact p-values in S1 Table, exact N in S2 Table. Data are shown as mean ± SEM. The data for Fig 4D–4F can be found at: https://doi.org/10.11588/data/DNOSZG. BC, barrel cortex; Bs, brainstem; DV, dorsal-ventral; icp, inferior cerebellar peduncle; IQR, interquartile range; LSC, lateral SC; LV, lateral ventricle; MC, motor cortex; ML, medial-lateral; Pia, pia mater; RN, recipient neuron; SC.m, medial superior colliculus; SC.cm, superior colliculus centromedial; SC.cl, centrolateral superior colliculus; SC.l, lateral superior colliculus; sp5, spinal trigeminal tract; Sp5, spinal trigeminal nucleus; WM, white matter; 7N, facial nucleus. https://doi.org/10.1371/journal.pbio.3002126.g004 Together, the anterograde and retrograde tracing experiments identify a region within the intermediate layers of the LSC, which spans approximately 1.2 mm in the rostro-caudal axis (approximately 85% of SC’s extent) and which receives input from main whisker circuits in the cortex and brainstem. This region is highly whisker sensitive (Fig 2), suggesting that the intermediate layer of the LSC is a “whisker SC” located ventrally to the “visual SC.”

Cortical and brainstem pathways directly target GABAergic neurons in the LSC Are the neurons targeted by cortical and brainstem long-range input to the LSC excitatory or inhibitory? To address this question, we first estimated the proportion of GABAergic neurons in the LSC. Using a GAD-GFP mouse, in which GABAergic neurons express GFP, we colabeled all neurons using the pan-neuronal marker NeuN-Alexa 647 (Fig 5A) and estimate that approximately 23% of the LSC neurons in the intermediate layer are GABAergic (Figs 5F and S6), which is slightly lower than in the superficial SC (approximately 30%; [15]). PPT PowerPoint slide

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TIFF original image Download: Fig 5. Cortical and brainstem pathways directly target GABAergic neurons in LSC. (A) The proportion of GABAergic neurons in the LSC was determined in a GAD-GFP mouse by calculating the proportion of GFP-expressing neurons (GABA, yellow) and all neurons labeled with pan-neuronal marker NeuN-Alexa 647 (red). From left to right: Example single rostro-caudal section; corresponding fluorescence image of GFP and NeuN signals; reconstruction of soma locations; pie chart of GABA/NeuN proportions for this section (84 GABA/384 NeuNs, 21.9%). The proportion for all analyzed sections (n = 30, 10 slices, 1 brain) was 23.1 ± 1.2% (see also S6 Fig). (B) Intersectional labeling of RNs and iRNs for each pathway in GAD-cre mice. AAV1-Flpo + AAV-fDIO-mCherry was injected in the projecting sites (MC, BC, and Bs), and AAV8-Con/Fon-EYFP + AAV2-fDIO-mCherry in LSC of GAD-Cre mice to label iRNs and RNs, respectively. (C) Top: Schematic of trans-synaptically labeled RNs (red) and iRNs (green). Bottom: Confocal images of BC-iRNs and BC-RNs. (D) Distribution of iRNs and RNs. Left: Example fluorescence images of iRNs and RNs in different SC sections for MC, BC, and Bs pathways, respectively. Right: Example reconstructions of MC-, BC-, and Bs-iRN and RN populations along the rostro-caudal axis (red: RNs, yellow: iRNs), registered to standard anatomical borders within SC. (E) Example for quantification of iRN/RN ratio for the MC-SC pathway. Fluorescence image, reconstruction, and pie chart summary of iRNs/RNs proportion (41 iRNs/119 RNs, 34%). (F) Summary of iRN/RN proportion for all 3 input pathways (MC red, BC blue, Bs green) along the rostro-caudal SC axis and GABA/NeuN proportion (grey dashed line). (G) Means and SEMs of iRN/RN proportions and GABA/NeuN for all 3 input pathways (same colors as in F); [mean per slice ± SEM, iRN/RN or GABA/NeuN, n mice]; MC: 36.4 ± 2.4%, 2; BC: 35.6 ± 2.4%, 3; Bs: 33.2 ± 1.5%, 3; GABA/NeuN: 23.1% ± 1.2, 1). * represents p < 0.01; Kruskal–Wallis; exact p-values in S1 Table, exact N values in S2 Table. Data are shown as mean ± SEM. The data for Fig 5A, 5E and 5F can be found at: https://doi.org/10.11588/data/DNOSZG. BC, barrel cortex; Bs, brainstem; Int, Intermediate layers; LSC, lateral SC; MC, motor cortex; RN, recipient neuron; Sup, Superficial layers. https://doi.org/10.1371/journal.pbio.3002126.g005 We then tested for monosynaptic innervation of GABAergic LSC neurons by employing a trans-synaptic intersectional strategy that allowed us to separate GABAergic RNs (iRNs) from the population of RNs, for each long-range input pathway (Fig 5B). In GAD-Cre mice, which express Cre in GABAergic neurons [34], we injected anterograde virus (AAV1-Flpo) into MC, BC, or Bs, as well as conditional reporter viruses (AAV-ConFon EYFP, AAV-fDIO mCherry) into LSC. This intersectional approach differentially labeled RNs with mCherry and iRNs with EYFP, demonstrating direct innervation of GABAergic neurons by MC, BC, and Bs input pathways (Figs 5C and S4 and S5 for controls). Reconstructions of the 2 populations show that iRNs and RNs are intermingled for all 3 input pathways (Fig 5D). Our estimates of the iRN/RN proportions suggest that each pathway targets between 34% and 37% GABAergic neurons along the full extent of the recipient antero-posterior axis (Figs 5E–5G and S11). In summary, long-range input pathways from MC, BC, and Bs extensively target GABAergic neurons in the LSC. The proportion of iRNs exceeded the proportion of GABAergic neurons (>34% versus 23%; Fig 5G), suggesting a preferential targeting for GABAergic neurons by these long-range input pathways.

Convergence of somatosensory and motor cortex in LSC neurons The considerable overlap between recipient zones in LSC observed in Fig 4 suggests possible convergence of long-range input pathways on the level of individual LSC neurons. To test for monosynaptic input convergence in single LSC neurons (CVG-RN), we employed an intersectional strategy as follows: In the same mouse, we injected 2 projection sites—for example, MC and BC—each with a different trans-synaptic variant—AAV1-Cre or AAV1-Flpo. Injections of the double-conditional (Cre- and Flpo-dependent) reporter virus in SC indeed revealed CVG-RNs for all 3 pathways (Fig 6A and 6B). MC and Bs convergence is highest in the anterior LSC, while MC and BC and BC and Bs convergences peak in the center and posterior SC, respectively (Fig 6C). PPT PowerPoint slide

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TIFF original image Download: Fig 6. Whisker-related input pathways converge in subpopulations of LSC neurons. (A) Left: Targeting convergence neurons (CVG-RNs) in LSC. AAV1-Cre + AAV2-DIO-mCherry and AAV1-Flpo + fDIO-mCherry were injected into 2 projecting sites and AAV8-Con/Fon-EYFP + AAV2-DIO-mCherry into LSC to reveal CVG-RNs and RNs, respectively. Right: Example fluorescent images of CVG-RNs. (B) Example reconstructions of CVG-RNs along the rostro-caudal axis, registered to standard anatomical borders within SC. (C) Normalized distributions of CVG-RNs along the rostro-caudal axis (bin size 100 μm). Box plots above show medians (white lines), and IQR (first to third quartile, boxes). MC and BC, MC and Bs, BC and Bs n = 4, 3, 2 mice, respectively. (D) Left: Z-scored mean number of CVG-RNs for each input pair. Right: Fold difference between mean number of CVG-RNs per 100 μm bin for cortex-cortex (4 mice) and brainstem-cortex (5 mice). (E) Proportions of CVG-RNs to RNs for each convergence input pair. Box plots show the median (line in box), mean (square in box), and IQR (first to third quartile, boxes). ([median, mean, IQR] MC and BC: 0.20, 0.22, 0.01; BC and Bs: 0.08, 0.01, 0.09; MC and Bs: 0.09, 0.01, 0.06). (F) The two-component Gaussian Mixture Model shows the area of MC and BC convergence zone in the intermediate layer of the LSC (11 slices, 2 mice, 222 neurons). The black dot indicates the location of the highest probability of finding MC and BC CVG-RNs (1,126 μm from SC surface and 1,492 μm from the midline). * represents p < 0.01; C, E: Kruskal–Wallis; exact p-values in S1 Table, exact N in S2 Table. Data are shown as mean ± SEM. The data for Fig 6C–6F can be found at: https://doi.org/10.11588/data/DNOSZG. BC, barrel cortex; Bs, brainstem; Int, intermediate layer; IQR, interquartile range; LSC, lateral SC; MC, motor cortex; RN, recipient neuron; SC, superior colliculus; Sup, superficial layer. https://doi.org/10.1371/journal.pbio.3002126.g006 To quantify the degree of convergence, we compared (1) z-scored mean CVG-RN counts per input pair and (2) the proportion of CVG-RNs with respect to RNs between all input pairs. Both analyses revealed that MC and BC convergence stands out compared to the other convergence pairs. Notably, the z-scored MC and BC CVG-RN estimates a 2.33-fold higher deviation compared to Bs and cortex convergence (Fig 6D). To estimate the proportion of convergence neurons relative to all recipient neurons, we counted mCherry-labeled RNs and EYFP-labeled CVG-RNs (Figs 6A and S12). Cortico-cortical convergence was estimated to be about 2.3-fold higher than brainstem-cortical convergence (approximately 23% CVG-RN for the MC and BC input pair versus approximately 10% CVG-RN for both the BC and Bs and MC and Bs input pairs) (Fig 6E). Fitting a two-component Gaussian Mixture Model to the coordinates of MC and BC CVG-RNs localized the cortico-cortical convergence zone to the dorsal portion of the intermediate layer (Fig 6F). Thus, motor and barrel cortex pathways converge on single neurons in the LSC, highlighting the whisker SC as a node for the integration of somatosensory and motor cortical signals. This convergence motif could potentially support fast, temporally precise integration of signals from different cortical regions; therefore, we next tested this possibility at the functional level.

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