No abstract

The functional state of denervated muscle is a critical factor in the ability to restore movement after injury- or disease-related paralysis. Here we used peripheral optogenetic stimulation and transcriptome profiling in the mouse whisker system to investigate the time course of changes in neuromuscular function following complete unilateral facial nerve transection. While most skeletal muscles rapidly lose functionality after lower motor neuron denervation, optogenetic muscle stimulation of the paralyzed whisker pad revealed sustained increases in the sensitivity, velocity, and amplitude of whisker movements, and reduced fatigability, starting 48 h after denervation. RNA-seq analysis showed distinct regulation of multiple gene families in denervated whisker pad muscles compared with the atrophy-prone soleus, including prominent changes in ion channels and contractile fibers. Together, our results define the unique functional and transcriptomic landscape of denervated facial muscles and have general implications for restoring movement after neuromuscular injury or disease. NEW & NOTEWORTHY Optogenetic activation of muscle can be used to noninvasively induce movements and probe muscle function. We used this technique in mice to investigate changes in whisker movements following facial nerve transection. We found unexpectedly enhanced functional properties of whisker pad muscle following denervation, accompanied by unique transcriptomic changes. Our findings highlight the utility of the mouse whisker pad for investigating the restoration of movement after paralysis.

SUMMARYTo achieve smooth motor performance in a rich and changing sensory environment, motor output must be constantly updated in response to sensory feedback. Although proprioception and cutaneous information are known to modulate motor output, it is unclear whether they work together in the spinal cord to shape complex motor actions such as locomotion. Here we identify the medial deep dorsal horn (mDDH) as a “hot zone” of convergent proprioception and cutaneous input for locomotion. Due to increased responsiveness to sensory input, inhibitory interneurons in the mDDH area are preferentially recruited in locomotion. To study inhibitory interneurons in this area, we utilize an intersectional genetic strategy to isolate and ablate a population of parvalbumin-expressing glycinergic interneurons in the mDDH (dPVs). Using histological and electrophysiological methods we find that dPVs integrate proprioceptive and cutaneous inputs while targeting ventral horn motor networks, suggesting a role in multimodal sensory processing for locomotion. Consistent with this, dPVs ablation alters step cycle parameters and kinematics in a speed and phase dependent manner. Finally, we use EMG muscle recordings to directly show that dPVs are part of a cutaneous-motor pathway. Our results indicate that dPVs form a critical node in the spinal sensorimotor circuitry.HighlightsInhibitory interneurons in the medial deep dorsal horn (mDDH), a “hot zone” of convergence cutaneous and proprioceptive inputs, are preferentially recruited during locomotion.We identified an inhibitory population of Glycinergic deep dorsal horn parvalbumin-expressing interneurons (dPVs) which are active during locomotion, integrate multimodal sensory inputs, and target motor networks.Ablation of dPVs reveals a state and phase-dependent role in modulation of locomotion parameters and kinematics.Electromyogram recordings demonstrate that dPVs modulate the cutaneous-evoked response in hindlimb muscles, establishing them as the first genetically identified inhibitory neurons in a cutaneous-motor pathway.

SUMMARYImprovements in the speed and cost of expression profiling of neuronal tissues offer an unprecedented opportunity to define ever finer subgroups of neurons for functional studies. In the spinal cord, single cell RNA sequencing studies1,2support decades of work on spinal cord lineage studies3–5, offering a unique opportunity to probe adult function based on developmental lineage. While Cre/Flp recombinase intersectional strategies remain a powerful tool to manipulate spinal neurons6–8, the field lacks genetic tools and strategies to restrict manipulations to the adult mouse spinal cord at the speed at which new tools develop. This study establishes a new workflow for intersectional mouse-viral strategies to dissect adult spinal function based on developmental lineages in a modular fashion. To restrict manipulations to the spinal cord, we generate a brain-sparingHoxb8FlpOmouse line restricting Flp recombinase expression to caudal tissue. Recapitulating endogenousHoxb8gene expression9, Flp-dependent reporter expression is present in the caudal embryo starting day 9.5. This expression restricts Flp activity in the adult to the caudal brainstem and below.Hoxb8FlpOheterozygous and homozygous mice do not develop any of the sensory or locomotor phenotypes evident in Hoxb8 heterozygous or mutant animals10,11, suggesting normal developmental function of the Hoxb8 gene and protein inHoxb8FlpOmice. Compared to the variability of brain recombination in available caudal Cre and Flp lines12,13Hoxb8FlpOactivity is not present in the brain above the caudal brainstem, independent of mouse genetic background. Lastly, we combine theHoxb8FlpOmouse line with dorsal horn developmental lineage Cre mouse lines to express GFP in developmentally determined dorsal horn populations. Using GFP-dependent Cre recombinase viruses14and Cre recombinase-dependent inhibitory chemogenetics, we target developmentally defined lineages in the adult. We show how developmental knock-out versus transient adult silencing of the same RORβlineage neurons affects adult sensorimotor behavior. In summary, this new mouse line and viral approach provides a blueprint to dissect adult somatosensory circuit function using Cre/Flp genetic tools to target spinal cord interneurons based on genetic lineage.In briefWe describe the generation of aHoxb8FlpOmouse line that targets Flp-recombinase expression to the spinal cord, dorsal root ganglia, and caudal viscera. This line can be used in intersectional Cre/Flp strategies to restrict manipulations to the caudal nervous system. Additionally, we describe an intersectional genetics+viral strategy to convert developmental GFP expression into adult Cre expression, allowing for modular incorporation of viral tools into intersectional genetics. This approach allows for manipulation of a developmentally determined lineage in the adult. This strategy is also more accessible than traditional intersectional genetics, and can adapt to the constantly evolving available viral repertoire.Highlights-A newHoxb8FlpOmouse line allows Flp-dependent recombination in the spinal cord, dorsal root ganglia, and caudal viscera.-We observed no ectopic brain expression across mouse genetic backgrounds with theHoxb8FlpOmouse line.-Combining this new mouse line for intersectional genetics and a viral approach, we provide a novel pipeline to target and manipulate developmentally defined adult spinal circuits.

14The functional state of denervated muscle is a critical factor in the ability to restore movement 15 after injury-or disease-related paralysis. Here we used peripheral optogenetic stimulation and 16 transcriptome profiling in the mouse whisker system to investigate the time course of changes in 17 neuromuscular function following complete unilateral facial nerve transection. While most 18 skeletal muscles rapidly lose functionality after lower motor neuron denervation, optogenetic 19 muscle stimulation of the paralyzed whisker pad revealed sustained increases in the sensitivity, 20 velocity, and amplitude of whisker movements, and reduced fatigability, starting 48 h after 21 denervation. RNA-seq analysis showed distinct regulation of multiple gene families in 22 denervated whisker pad muscles compared to the atrophy-prone soleus, including prominent 23 changes in ion channels and contractile fibers. Together, our results define the unique functional 24 and transcriptomic landscape of denervated facial muscles, and have general implications for 25 restoring movement after neuromuscular injury or disease. 26 27New & Noteworthy: Optogenetic activation of muscle can be used to non-invasively induce 31 movements and probe muscle function. We used this technique in mice to investigate changes 32 in whisker movements following facial nerve transection. We found unexpectedly enhanced 33 functional properties of whisker pad muscle following denervation, accompanied by unique 34 transcriptomic changes. Our findings highlight the utility of the mouse whisker pad for 35 investigating the restoration of movement after paralysis. 36 37 Haidarliu et al 2010, Park et al 2016), collectively referred to here as "whisker pad muscles". In 61 the present study, we used ChAT-ChR2 and Emx1-ChR2 mice to evoke whisker movements via 62 stimulation of the facial motor nerve (cranial nerve VII) or the whisker pad muscles, respectively. 63 This allowed us to investigate the functional changes that occur in nerve and muscle after the 64 paralysis of whisker movements caused by facial nerve transection. 65 66 One recent study used optogenetic muscle stimulation in the hindlimb triceps surae after sciatic 67 nerve lesion to demonstrate dramatic atrophy and loss of function (Magown et al 2015), 68consistent with classic studies in this system (Nelson 1969), that could be attenuated by daily 69 optogenetic activation. We considered it possible that whisker pad muscles undergo distinct 70 denervation-induced changes compared to other muscle types, in part because whisker pad 71 position of the nerve in ChAT-ChR2 mice, the buccal branch of the facial nerve (cranial nerve 126 VII) was targeted with the light spot at a position between the stylomastoid foramen, ventral to 127 the ear canal and caudal to the whisker pad. Different illumination positions in this region were 128 tested for each subject to optimize the evoked whisker protraction. To test effects on 129 fasciculations, dantrolene (1 mM) was applied subcutaneously to the lesioned (right-...