Constructing an adult orofacial premotor atlas in Allen mouse CCF
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- Neuroscience (eLife)
Abstract
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.
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Author Response:
Reviewer #1 (Public Review):
Facial muscles control the execution of essential tasks like eating, drinking, breathing and (in most mammals) tactile exploration. The activity of motor neurons targeting different muscles are coordinated by premotor regions distributed throughout brainstem. The precise identity of these cells and regions in adults is presently unclear, largely due to technical challenges. In the current work, Takaoh and colleagues develop an elegant strategy to label premotor neurons that target select muscles and register these cells on a common digital atlas. Their work confirms and also extends previous studies in neonates and provides a useful resource for the field.
We thank Reviewer 1 for the positive evaluation.
Reviewer #2 (Public Review):
The authors describe a variant of retrograde …
Author Response:
Reviewer #1 (Public Review):
Facial muscles control the execution of essential tasks like eating, drinking, breathing and (in most mammals) tactile exploration. The activity of motor neurons targeting different muscles are coordinated by premotor regions distributed throughout brainstem. The precise identity of these cells and regions in adults is presently unclear, largely due to technical challenges. In the current work, Takaoh and colleagues develop an elegant strategy to label premotor neurons that target select muscles and register these cells on a common digital atlas. Their work confirms and also extends previous studies in neonates and provides a useful resource for the field.
We thank Reviewer 1 for the positive evaluation.
Reviewer #2 (Public Review):
The authors describe a variant of retrograde monosynaptic rabies tracing from skeletal muscle. They make use of AAV2-retro-Cre to infect brainstem motoneurons projecting to muscles involved in regulation of orofacial movements (whisking, genioglossus, masseter motoneurons). The strategy that worked most efficiently and with specificity was to inject AAV2-retro-Cre intramuscularly at P17, followed 3 weeks thereafter by central injection of Cre-dependent AAVs expressing TVA and oG, and 2 weeks thereafter followed by central injection of EnvA(M21)-ΔG-RV-GFP. Five days after this final injection, experiments were terminated to analyse the distribution of premotor neurons. This allowed the authors to reconstruct and compare the distribution of premotor neurons to the whisking (lateral 7N), tongue protruding genioglossus (12N), and jaw-closing masseter (5N) motoneurons. To do so, they used the Allen Brain Atlas as a reference for 3D reconstruction, into which they integrated all data. Notably, the authors found that for all three injection types, the highest density of neurons was found in the IRt and PCRt, but the precise peak of highest density was consistently distinct for the three different injection types. The peak for whisker premotor neurons was most caudal-ventral, for masseter premotor neurons most rostro-dorsal, and jaw-closing genioglossal premotor neurons in between these. The authors also make use of the strong expression of fluorescent proteins through rabies virus to analyse collateralization to other motor nuclei. Interestingly, they found cross-talk to other motor nuclei in selective patterns, supporting a model whereby some premotor neurons to one brainstem motor pool also interact with other output circuits, perhaps to coordinate orofacial behaviors. Using a split-Cre retrograde approach from motor nuclei, dual-projecting premotor neurons were identified to be located in dorsal IRt and SupV.
This is a high-quality study making use of several methods not previously brought together in one study. Particularly interesting is the 3-way virus strategy in wild-type mice allowing visualization of premotor neurons in the adult. Second, alignment in a common reference brain is also very useful. And finally, the beginning of understanding dynamics of premotor circuit distribution between development and adult is also a value of this paper. Overall, the study is very interesting for the field.
We thank Reviewer 2 for the positive evaluation.
Reviewer #3 (Public Review):
Orofacial actions show exquisite coordination among many muscles, yet the pools of motor neurons exciting each of these muscles is specific to that muscle. The coordination of activity across muscles therefore relies on circuits of premotor neurons that excite the motor neurons. Work by the authors and others has produced major progress in delineating these complex premotor circuits. Recent work using transsynaptic viral tracing has overcome limitations associated with traditional retrograde tracing methods, such as a lack of adequate specificity. However, these transsynaptic viral methods have been unsuccessful in animals older than approximately postnatal day 8 (P8). This is a problem because circuits continue to develop far beyond P8 in mice. Here, the authors overcome this limitation by introducing a novel viral transsynaptic tracing method that can be applied in adult mice.
The authors apply their method to trace premotor circuits for whisking, licking, and jaw movements. They align their anatomical data to the Allen Mouse Brain Common Coordinate Framework and make it available with the manuscript, greatly facilitating its quantitative use by other laboratories. The authors find premotor circuits in adult mice that are almost entirely consistent with results from younger mice, with some important exceptions that they highlight and discuss. The authors quantify overlap of premotor circuits for whisking, licking and jaw movements and discuss the implications of interactions among these circuits.
The experiments and analysis are carefully performed, and the results put into proper context. Overall, this is a straightforward and valuable contribution to our knowledge of the premotor circuits that coordinate orofacial behaviors. It will be of wide interest to neuroscientists.
Suggestions:
-The methods applied in neonatal mice (Takatoh et al. 2013; Stanek et al. 2014), while obviously different, are similar enough that it may be worth including discussion of any possible ways that differences between the neonatal and adult results could be due to methods, rather than age. I defer to the authors about whether such discussion is worthwhile, but readers may benefit from knowing what was considered.
Now we added the technical considerations that may cause the difference in the tracing patterns: Line 505-517.
-Spatial correlation in Figure 5C. To interpret this properly it's important to know the degree of smoothing. I could not find this in the relevant methods section describing the kernel density estimation or elsewhere.
Same as the above: The cells detected in each mouse were first registered into the standard three-dimensional brain model. The (x, y, z) coordinates of each cell were then extracted, and the multivariate kernel smoothing density estimation was applied (bandwidth = 1). The resulting kernel density estimation was then vectorized, and the cosine similarity between any two of the mice were calculated to form the correlogram.
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Reviewer #2 (Public Review):
The authors describe a variant of retrograde monosynaptic rabies tracing from skeletal muscle. They make use of AAV2-retro-Cre to infect brainstem motoneurons projecting to muscles involved in regulation of orofacial movements (whisking, genioglossus, masseter motoneurons). The strategy that worked most efficiently and with specificity was to inject AAV2-retro-Cre intramuscularly at P17, followed 3 weeks thereafter by central injection of Cre-dependent AAVs expressing TVA and oG, and 2 weeks thereafter followed by central injection of EnvA(M21)-ΔG-RV-GFP. Five days after this final injection, experiments were terminated to analyse the distribution of premotor neurons. This allowed the authors to reconstruct and compare the distribution of premotor neurons to the whisking (lateral 7N), tongue protruding …
Reviewer #2 (Public Review):
The authors describe a variant of retrograde monosynaptic rabies tracing from skeletal muscle. They make use of AAV2-retro-Cre to infect brainstem motoneurons projecting to muscles involved in regulation of orofacial movements (whisking, genioglossus, masseter motoneurons). The strategy that worked most efficiently and with specificity was to inject AAV2-retro-Cre intramuscularly at P17, followed 3 weeks thereafter by central injection of Cre-dependent AAVs expressing TVA and oG, and 2 weeks thereafter followed by central injection of EnvA(M21)-ΔG-RV-GFP. Five days after this final injection, experiments were terminated to analyse the distribution of premotor neurons. This allowed the authors to reconstruct and compare the distribution of premotor neurons to the whisking (lateral 7N), tongue protruding genioglossus (12N), and jaw-closing masseter (5N) motoneurons. To do so, they used the Allen Brain Atlas as a reference for 3D reconstruction, into which they integrated all data. Notably, the authors found that for all three injection types, the highest density of neurons was found in the IRt and PCRt, but the precise peak of highest density was consistently distinct for the three different injection types. The peak for whisker premotor neurons was most caudal-ventral, for masseter premotor neurons most rostro-dorsal, and jaw-closing genioglossal premotor neurons in between these. The authors also make use of the strong expression of fluorescent proteins through rabies virus to analyse collateralization to other motor nuclei. Interestingly, they found cross-talk to other motor nuclei in selective patterns, supporting a model whereby some premotor neurons to one brainstem motor pool also interact with other output circuits, perhaps to coordinate orofacial behaviors. Using a split-Cre retrograde approach from motor nuclei, dual-projecting premotor neurons were identified to be located in dorsal IRt and SupV.
This is a high-quality study making use of several methods not previously brought together in one study. Particularly interesting is the 3-way virus strategy in wild-type mice allowing visualization of premotor neurons in the adult. Second, alignment in a common reference brain is also very useful. And finally, the beginning of understanding dynamics of premotor circuit distribution between development and adult is also a value of this paper. Overall, the study is very interesting for the field.
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Reviewer #1 (Public Review):
Facial muscles control the execution of essential tasks like eating, drinking, breathing and (in most mammals) tactile exploration. The activity of motor neurons targeting different muscles are coordinated by premotor regions distributed throughout brainstem. The precise identity of these cells and regions in adults is presently unclear, largely due to technical challenges. In the current work, Takaoh and colleagues develop an elegant strategy to label premotor neurons that target select muscles and register these cells on a common digital atlas. Their work confirms and also extends previous studies in neonates and provides a useful resource for the field.
-
Reviewer #3 (Public Review):
Orofacial actions show exquisite coordination among many muscles, yet the pools of motor neurons exciting each of these muscles is specific to that muscle. The coordination of activity across muscles therefore relies on circuits of premotor neurons that excite the motor neurons. Work by the authors and others has produced major progress in delineating these complex premotor circuits. Recent work using transsynaptic viral tracing has overcome limitations associated with traditional retrograde tracing methods, such as a lack of adequate specificity. However, these transsynaptic viral methods have been unsuccessful in animals older than approximately postnatal day 8 (P8). This is a problem because circuits continue to develop far beyond P8 in mice. Here, the authors overcome this limitation by introducing a …
Reviewer #3 (Public Review):
Orofacial actions show exquisite coordination among many muscles, yet the pools of motor neurons exciting each of these muscles is specific to that muscle. The coordination of activity across muscles therefore relies on circuits of premotor neurons that excite the motor neurons. Work by the authors and others has produced major progress in delineating these complex premotor circuits. Recent work using transsynaptic viral tracing has overcome limitations associated with traditional retrograde tracing methods, such as a lack of adequate specificity. However, these transsynaptic viral methods have been unsuccessful in animals older than approximately postnatal day 8 (P8). This is a problem because circuits continue to develop far beyond P8 in mice. Here, the authors overcome this limitation by introducing a novel viral transsynaptic tracing method that can be applied in adult mice.
The authors apply their method to trace premotor circuits for whisking, licking, and jaw movements. They align their anatomical data to the Allen Mouse Brain Common Coordinate Framework and make it available with the manuscript, greatly facilitating its quantitative use by other laboratories. The authors find premotor circuits in adult mice that are almost entirely consistent with results from younger mice, with some important exceptions that they highlight and discuss. The authors quantify overlap of premotor circuits for whisking, licking and jaw movements and discuss the implications of interactions among these circuits.
The experiments and analysis are carefully performed, and the results put into proper context. Overall, this is a straightforward and valuable contribution to our knowledge of the premotor circuits that coordinate orofacial behaviors. It will be of wide interest to neuroscientists.
Suggestions:
-The methods applied in neonatal mice (Takatoh et al. 2013; Stanek et al. 2014), while obviously different, are similar enough that it may be worth including discussion of any possible ways that differences between the neonatal and adult results could be due to methods, rather than age. I defer to the authors about whether such discussion is worthwhile, but readers may benefit from knowing what was considered.
-Spatial correlation in Figure 5C. To interpret this properly it's important to know the degree of smoothing. I could not find this in the relevant methods section describing the kernel density estimation or elsewhere.
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