Constructing an adult orofacial premotor atlas in Allen mouse CCF

Premotor circuits in the brainstem project to pools of orofacial motoneurons to execute essential motor action such as licking, chewing, breathing, and in rodent, whisking. Previous transsynaptic tracing studies only mapped orofacial premotor circuits in neonatal mice, but the adult circuits remain unknown as a consequence of technical difficulties. Here, we developed a three-step monosynaptic transsynaptic tracing strategy to identify premotor neurons controlling vibrissa, tongue protrusion, and jaw-closing muscles in the adult mouse. We registered these different groups of premotor neurons onto the Allen mouse brain common coordinate framework (CCF) and consequently generated a combined 3D orofacial premotor atlas, revealing unique spatial organizations of distinct premotor circuits. We further uncovered premotor neurons that simultaneously innervate multiple motor nuclei and, consequently, are likely to coordinate different muscles involved in the same orofacial motor actions. Our method for tracing adult premotor circuits and registering to Allen CCF is generally applicable and should facilitate the investigations of motor controls of diverse behaviors.

Note that while we aimed our injection at the intrinsic whisker muscles controlling whisker 168 protraction, we could not rule out the possibility of infecting a few extrinsic motoneurons 169 regulating whisker-pad retraction. We did not want to lesion the nerve innerving the extrinsic 170 pad muscle in order to make the tracing more specific for intrinsic muscle, because the mice 171 need to survive into adult (8-9 weeks old) in order to trace adult premotor circuit and lesioning in 172 In the masseter premotor circuit, extensive labeling was also found bilaterally along the anterior-217 posterior axis of the dorsal IRt ( Figure 4A and 4B) (More quantitative analyses of the 218 distribution of labeled masseter premotor cells in individual animals are described below). 219 Interestingly, the majority of labeled dorsal IRt neurons were observed contralaterally in the 220 caudal part of IRt (AP coordinate) ( Figure 4C). Bilateral labeling in PCRt was observed as a 221 lateral continuum of the dorsal IRt neurons at the level of the FN ( Figure 4B). Rostrally, we 222 found a distinct bilateral cluster of large-size neurons with medially directed dendrites situated 223 around PCRt/PrV area immediately caudal to the trigeminal motor nucleus ( Figure 4D). This 224 group of neurons wedged into the dorsomedial and ventrolateral PrV. This area is identified by 225 Nissl staining as containing a distinct cluster of neurons with large size than neighboring cells. 226 Similar to the tongue-protruding circuit but with fewer numbers, cells of very large soma size 227 were labeled ipsilaterally along the anterior-posterior axis spanning Gi/LPGi/LRt areas (AP 228 coordinate) ( Figure 4A). In the pons, numerous labeled masseter premotor neurons were also observed in the supratrigeminal nucleus and peritrigeminal areas ( Figure 4D). In the sensory-230 related areas, labeled cells resided bilaterally in the dorsal PrV ( Figure 4D   Subsequently, the labeled cells were identified and counted semi-automatically or manually, and 249 their coordinates were transformed into CCF coordinates ( Figure 5A). All traced orofacial motor 250 neurons for whisker (n = 4 mice), genioglossus (n = 4 mice), and masseter (n = 4 mice) were 251 registered to the CCF, and their coordinates are accessible from the source file. The cells in CCF 252 coordinates were reconstructed in 2D and 3D spaces using Brainrender (Claudi et al., 2020).  Movie 1). The distribution density plot analysis of each premotor circuit also supports the 268 muscle-specific differential spatial organizations as shown for all three planes: coronal, sagittal, 269 and horizontal ( Figure 5D). All three premotor circuits showed the highest density of labeling in premotor circuit showed highest labeling density in the caudoventral areas of IRt ( Figure 5D, 273 Red). The masseter premotor circuit had densest labeling in the anterodorsal area of IRt ( Figure  274 5D, Yellow). The highest-density area of the genioglossus premotor neurons located in IRt in 275 between the peaks for the whisker and masseter premotor cells (along the A-P axis), although 276 there were shared regions between genioglossus and masseter premotor distributions ( Figure 5D, were also shown as landmarks). Again, these 3D reconstructions revealed partial overlapping and 293 partial segregation of the three premotor circuits ( Figure 6A-6C). Along the A-P axis, the 294 highest density regions of ipsilateral jaw-closing and tongue-protruding premotor neurons in IRt 295 were close to each other but with the peak of jaw premotor neurons shifted rostrally and ventrally 296 ( Figure 6G -6O, jaw peak: AP -6.02 ± 0.18 mm, DV -6.45 ± 0.04 mm; n = 4 mice , tongue 297 peak: AP -6.20 ± 0.23 mm, DV -5.74 ± 0.29; n = 4 mice). Along the D-V axis, while tongue 298 premotor neurons are concentrated to more dorsal IRt than jaw premotor neurons, their 299 distribution spread more to ventral IRt. Notably, the contralateral jaw IRt premotor neurons 300 formed a discernable cluster caudal to the densest area of tongue IRt premotor neurons, 301 displaying a bilaterally asymmetric distribution ( Figure 6H). Whisker premotor neurons were 302 more spatially separated from tongue and jaw premotor neurons in IRt, i.e at more caudal and 303 ventral (AP -6.45 ± 0.19 mm, DV -6.45 ± 0.04 mm; n = 4 mice) locations in IRt ( Figure 6D-6F). 304 Furthermore, the tongue and jaw IRt premotor neurons showed similar densities between the 305 ipsilateral and contralateral side (as licking and chewing generally involve muscles of both 306 sides), the whisker IRt premotor neurons showed biased distribution to the ipsilateral side 307 ( Figure 6D and 6E). Collectively, these results suggest that functionally distinct groups of 308 orofacial premotor neurons occupy the overlapping yet distinct spatial positions within IRt, and 309 there is roughly a ventral-to-dorsal, and caudal-to-rostral gradient of whisker-tongue-jaw 310 premotor neurons. 311 312

Axon collaterals revealed common premotor neurons for distinct motor neurons 313
As mentioned in introduction, orofacial behaviors often require coordinated activity of multiple 314 groups of motoneurons. A premotor neuron that simultaneously innervates distinct motoneurons 315 forms the simplest motor coordinating circuit. We therefore examined whether our premotor  Notably, in addition to VIImiddle and XII, we found tdTomato-positive axon terminals onto motoneurons in VAD (jaw opening) and in the nucleus ambiguus (mostly in semi-compact part), 353 which are known to be involved in swallowing. Thus, VIImiddle-XII common premotor neurons 354 located in SupV and dorsal IRt simultaneously innervate motoneurons controlling tongue 355 protrusion, lower lip, jaw-opening, and throat (through the nucleus ambiguus) ( Figure 7O and 356 7P), suggesting that those common premotor neurons likely represent a fundamental neural 357 substrate for coactivating these muscles. Interestingly, SupV and dorsal IRt were also labeled by 358 the retrograde split-Cre tracing from the left and right sides of VIImiddle (Figure supplement 8). 359 Those neurons also project additional collaterals to V and XII, in addition to VIImiddle. These 360 results indicate that SupV and dorsal IRt regions may be critical brainstem hubs containing 361 common premotor neurons that coordinate multiple groups of motoneurons for orofacial feeding 362 behaviors ( Figure 7P). SupV and dorsal IRt as potential substrates for coordinating multiple distinct orofacial muscles 373 involved in feeding-related behaviors. Since these three groups of motoneurons are involved in 374 three rhythmic orofacial behaviors, whisking, licking, or chewing, we next focus our discussion 375 on the implications of the premotor atlas for rhythm generations. 376 377

Implications for premotor neurons modulating whisking rhythm 378
Among the three orofacial premotor circuits in adult mice that we have mapped, the whisker 379 premotor atlas consists of the most numerous brain structures (Figure 8). This is not surprising

Monosynaptic transsynaptic rabies virus tracing 509
The tracing was performed in three steps. 510

Peripheral tissue injection 511
To label a specific group of orofacial motor neurons, AAV2-retro-CAG-Cre (1000 µl, Harvard 512 University, Boston Children's Hospital Viral Core) was injected into either the whisker pad, 513 genioglossus or masseter muscles at postnatal day 17, using a volumetric injection system (based subcutaneously into the areas around C2 and B2 whiskers (500 nl each). For the genioglossus, 519 the virus was injected directory into the muscle after exposing it by ventral neck dissection. 520 Briefly, the genioglossus muscle was exposed by making a small incision in the mylohyoid 521 can handle a more diverse scenarios of section distortion. Second, the procedures to determine 588 the coordinates of the brain sections after nonrigid transformation and registration were 589 corrected, and this is also critical for 3D reconstruction and visualizations of the results from 590 serial 2D sections. Third, in addition to the manual cell identification by user generated click, an 591 optional automatic cell identification function was developed to recover most identifiable cells, 592 and subsequently users can manually correct mistakes. The automatic cell identification method 593 contains a series of simple filters which balanced the speed and the precision of the 594 approximation. Detailed implementation can be found in the Github repository 595 (https://github.com/wanglab-neuro/Allen_CCF_reg). This site will be freely available upon 596 publication. Coordinates of Bregma in Allen CCF was set at AP, 5400; ML, 5700; DV 0 597 (Shamash et al., 2018). 598

Spatial correlation analysis 600
All 3D coordinates of the identified cells per mouse were concatenated. The spatial distribution 601 was then estimated using multivariate kernel smoothing density function estimation. The 602 estimated multivariate (3D) density functions for each mouse were then vectorized and pairwise 603 cosine similarity was computed for all the mice. The result was shaped to a square matrix which 604 was shown in the figure. 605 606 Visualization of labeled neurons on Allen common coordinate framework 607 Premotor neurons registered in Allen CCF were visualized using Brainrender (Claudi et al., 608 2020) or a custom written code (for density plots). Briefly, the coordinates are converted into 609 Allen CCF coordinates by multiplying 1000 and adding 5400 (for AP) and 5700 (for ML).