Genetic targeting of adult Renshaw cells using a Calbindin 1 destabilized Cre allele for intersection with Parvalbumin or Engrailed1.

Renshaw cells (RCs) are one of the most studied spinal interneurons; however, their roles in motor control remain enigmatic in part due to the lack of experimental models to interfere with RC function, specifically in adults. To overcome this limitation, we leveraged the distinct temporal regulation of Calbindin (Calb1) expression in RCs to create genetic models for timed RC manipulation. We used a Calb1 allele expressing a destabilized Cre (dgCre) theoretically active only upon trimethoprim (TMP) administration. TMP timing and dose influenced RC targeting efficiency, which was highest within the first three postnatal weeks, but specificity was low with many other spinal neurons also targeted. In addition, dgCre showed TMP-independent activity resulting in spontaneous recombination events that accumulated with age. Combining Calb1-dgCre with Parvalbumin (Pvalb) or Engrailed1 (En1) Flpo alleles in dual conditional systems increased cellular and timing specificity. Under optimal conditions, Calb1-dgCre/Pvalb-Flpo mice targeted 90% of RCs and few dorsal horn neurons; Calb1-dgCre/En1-Flpo mice showed higher specificity, but only a maximum of 70% of RCs targeted. Both models targeted neurons throughout the brain. Restricted spinal expression was obtained by injecting intraspinally AAVs carrying dual conditional genes. These results describe the first models to genetically target RCs bypassing development.

Differential Loss of Spinal Interneurons in a Mouse Model of ALS.

Amyotrophic lateral sclerosis (ALS) leads to a loss of specific motor neuron populations in the spinal cord and cortex. Emerging evidence suggests that interneurons may also be affected, but a detailed characterization of interneuron loss and its potential impacts on motor neuron loss and disease progression is lacking. To examine this issue, the fate of V1 inhibitory neurons during ALS was assessed in the ventral spinal cord using the SODG93A mouse model. The V1 population makes up ∼30% of all ventral inhibitory neurons, ∼50% of direct inhibitory synaptic contacts onto motor neuron cell bodies, and is thought to play a key role in modulating motor output, in part through recurrent and reciprocal inhibitory circuits. We find that approximately half of V1 inhibitory neurons are lost in SODG93A mice at late disease stages, but that this loss is delayed relative to the loss of motor neurons and V2a excitatory neurons. We further identify V1 subpopulations based on transcription factor expression that are differentially susceptible to degeneration in SODG93A mice. At an early disease stage, we show that V1 synaptic contacts with motor neuron cell bodies increase, suggesting an upregulation of inhibition before V1 neurons are lost in substantial numbers. These data support a model in which progressive changes in V1 synaptic contacts early in disease, and in select V1 subpopulations at later stages, represent a compensatory upregulation and then deleterious breakdown of specific interneuron circuits within the spinal cord.

Positional Strategies for Connection Specificity and Synaptic Organization in Spinal Sensory-Motor Circuits.

Proprioceptive sensory axons in the spinal cord form selective connections with motor neuron partners, but the strategies that confer such selectivity remain uncertain. We show that muscle-specific sensory axons project to motor neurons along topographically organized angular trajectories and that motor pools exhibit diverse dendritic arbors. On the basis of spatial constraints on axo-dendritic interactions, we propose positional strategies that can account for sensory-motor connectivity and synaptic organization. These strategies rely on two patterning principles. First, the degree of axo-dendritic overlap reduces the number of potential post-synaptic partners. Second, a close correlation between the small angle of axo-dendritic approach and the formation of synaptic clusters imposes specificity of connections when sensory axons intersect multiple motor pools with overlapping dendritic arbors. Our study identifies positional strategies with prominent roles in the organization of spinal sensory-motor circuits.

Molecular Logic of Spinocerebellar Tract Neuron Diversity and Connectivity.

Coordinated motor behaviors depend on feedback communication between peripheral sensory systems and central circuits in the brain and spinal cord. Relay of muscle- and tendon-derived sensory information to the CNS is facilitated by functionally and anatomically diverse groups of spinocerebellar tract neurons (SCTNs), but the molecular logic by which SCTN diversity and connectivity is achieved is poorly understood. We used single-cell RNA sequencing and genetic manipulations to define the mechanisms governing the molecular profile and organization of SCTN subtypes. We found that SCTNs relaying proprioceptive sensory information from limb and axial muscles are generated through segmentally restricted actions of specific Hox genes. Loss of Hox function disrupts SCTN-subtype-specific transcriptional programs, leading to defects in the connections between proprioceptive sensory neurons, SCTNs, and the cerebellum. These results indicate that Hox-dependent genetic programs play essential roles in the assembly of neural circuits necessary for communication between the brain and spinal cord.

A Role for Sensory end Organ-Derived Signals in Regulating Muscle Spindle Proprioceptor Phenotype.

Proprioceptive feedback from Group Ia/II muscle spindle afferents and Group Ib Golgi tendon afferents is critical for the normal execution of most motor tasks, yet how these distinct proprioceptor subtypes emerge during development remains poorly understood. Using molecular genetic approaches in mice of either sex, we identified 24 transcripts that have not previously been associated with a proprioceptor identity. Combinatorial expression analyses of these markers reveal at least three molecularly distinct proprioceptor subtypes. In addition, we find that 12 of these transcripts are expressed well after proprioceptors innervate their respective sensory receptors, and expression of three of these markers, including the heart development molecule Heg1, is significantly reduced in mice that lack muscle spindles. These data reveal Heg1 as a putative marker for proprioceptive muscle spindle afferents. Moreover, they suggest that the phenotypic specialization of functionally distinct proprioceptor subtypes depends, in part, on extrinsic sensory receptor organ-derived signals.SIGNIFICANCE STATEMENT Sensory feedback from muscle spindle (MS) and Golgi tendon organ (GTO) sensory end organs is critical for normal motor control, but how distinct MS and GTO afferent sensory neurons emerge during development remains poorly understood. Using (bulk) transcriptome analysis of genetically identified proprioceptors, this work reveals molecular markers for distinct proprioceptor subsets, including some that appear selectively expressed in MS afferents. Detailed analysis of the expression of these transcripts provides evidence that MS/GTO afferent subtype phenotypes may, at least in part, emerge through extrinsic, sensory end organ-derived signals.

Subtype Diversification and Synaptic Specificity of Stem Cell-Derived Spinal Interneurons.

Neuronal diversification is a fundamental step in the construction of functional neural circuits, but how neurons generated from single progenitor domains acquire diverse subtype identities remains poorly understood. Here we developed an embryonic stem cell (ESC)-based system to model subtype diversification of V1 interneurons, a class of spinal neurons comprising four clades collectively containing dozens of molecularly distinct neuronal subtypes. We demonstrate that V1 subtype diversity can be modified by extrinsic signals. Inhibition of Notch and activation of retinoid signaling results in a switch to MafA clade identity and enriches differentiation of Renshaw cells, a specialized MafA subtype that mediates recurrent inhibition of spinal motor neurons. We show that Renshaw cells are intrinsically programmed to migrate to species-specific laminae upon transplantation and to form subtype-specific synapses with motor neurons. Our results demonstrate that stem cell-derived neuronal subtypes can be used to investigate mechanisms underlying neuronal subtype specification and circuit assembly.

Sox14 Is Required for a Specific Subset of Cerebello-Olivary Projections.

We identify Sox14 as an exclusive marker of inhibitory projection neurons in the lateral and interposed, but not the medial, cerebellar nuclei. Sox14+ neurons make up ∼80% of Gad1+ neurons in these nuclei and are indistinguishable by soma size from other inhibitory neurons. All Sox14+ neurons of the lateral and interposed cerebellar nuclei are generated at approximately E10/10.5 and extend long-range, predominantly contralateral projections to the inferior olive. A small Sox14+ population in the adjacent vestibular nucleus "Y" sends an ipsilateral projection to the oculomotor nucleus. Cerebellar Sox14+ and glutamatergic projection neurons assemble in non-overlapping populations at the nuclear transition zone, and their integration into a coherent nucleus depends on Sox14 function. Targeted ablation of Sox14+ cells by conditional viral expression of diphtheria toxin leads to significantly impaired motor learning. Contrary to expectations, associative learning is unaffected by unilateral Sox14+ neuron elimination in the interposed and lateral nuclei.SIGNIFICANCE STATEMENT The cerebellar nuclei are central to cerebellar function, yet how they modulate and process cerebellar inputs and outputs is still primarily unknown. Our study gives a direct insight into how nucleo-olivary projection neurons are generated, their projections, and their function in an intact behaving mouse. These neurons play a critical conceptual role in all models of cerebellar function, and this study represents the first specific analysis of their molecular identity and function and offers a powerful model for future investigation of cerebellar function in motor control and learning.

Role of muscle spindle feedback in regulating muscle activity strength during walking at different speed in mice.

Terrestrial animals increase their walking speed by increasing the activity of the extensor muscles. However, the mechanism underlying how this speed-dependent amplitude modulation is achieved remains obscure. Previous studies have shown that group Ib afferent feedback from Golgi tendon organs that signal force is one of the major regulators of the strength of muscle activity during walking in cats and humans. In contrast, the contribution of group Ia/II afferent feedback from muscle spindle stretch receptors that signal angular displacement of leg joints is unclear. Some studies indicate that group II afferent feedback may be important for amplitude regulation in humans, but the role of muscle spindle feedback in regulation of muscle activity strength in quadrupedal animals is very poorly understood. To examine the role of feedback from muscle spindles, we combined in vivo electrophysiology and motion analysis with mouse genetics and gene delivery with adeno-associated virus. We provide evidence that proprioceptive sensory feedback from muscle spindles is important for the regulation of the muscle activity strength and speed-dependent amplitude modulation. Furthermore, our data suggest that feedback from the muscle spindles of the ankle extensor muscles, the triceps surae, is the main source for this mechanism. In contrast, muscle spindle feedback from the knee extensor muscles, the quadriceps femoris, has no influence on speed-dependent amplitude modulation. We provide evidence that proprioceptive feedback from ankle extensor muscles is critical for regulating muscle activity strength as gait speed increases. NEW & NOTEWORTHY Animals upregulate the activity of extensor muscles to increase their walking speed, but the mechanism behind this is not known. We show that this speed-dependent amplitude modulation requires proprioceptive sensory feedback from muscle spindles of ankle extensor muscle. In the absence of muscle spindle feedback, animals cannot walk at higher speeds as they can when muscle spindle feedback is present.

Nuclear Organization in the Spinal Cord Depends on Motor Neuron Lamination Orchestrated by Catenin and Afadin Function.

Motor neurons in the spinal cord are found grouped in nuclear structures termed pools, whose position is precisely orchestrated during development. Despite the emerging role of pool organization in the assembly of spinal circuits, little is known about the morphogenetic programs underlying the patterning of motor neuron subtypes. We applied three-dimensional analysis of motor neuron position to reveal the roles and contributions of cell adhesive function by inactivating N-cadherin, catenin, and afadin signaling. Our findings reveal that nuclear organization of motor neurons is dependent on inside-out positioning, orchestrated by N-cadherin, catenin, and afadin activities, controlling cell body layering on the medio-lateral axis. In addition to this lamination-like program, motor neurons undergo a secondary, independent phase of organization. This process results in segregation of motor neurons along the dorso-ventral axis of the spinal cord, does not require N-cadherin or afadin activity, and can proceed even when medio-lateral positioning is perturbed.

Motor Cortex Embeds Muscle-like Commands in an Untangled Population Response.

Primate motor cortex projects to spinal interneurons and motoneurons, suggesting that motor cortex activity may be dominated by muscle-like commands. Observations during reaching lend support to this view, but evidence remains ambiguous and much debated. To provide a different perspective, we employed a novel behavioral paradigm that facilitates comparison between time-evolving neural and muscle activity. We found that single motor cortex neurons displayed many muscle-like properties, but the structure of population activity was not muscle-like. Unlike muscle activity, neural activity was structured to avoid "tangling": moments where similar activity patterns led to dissimilar future patterns. Avoidance of tangling was present across tasks and species. Network models revealed a potential reason for this consistent feature: low tangling confers noise robustness. Finally, we were able to predict motor cortex activity from muscle activity by leveraging the hypothesis that muscle-like commands are embedded in additional structure that yields low tangling.

Balance Control Mediated by Vestibular Circuits Directing Limb Extension or Antagonist Muscle Co-activation.

Maintaining balance after an external perturbation requires modification of ongoing motor plans and the selection of contextually appropriate muscle activation patterns that respect body and limb position. We have used the vestibular system to generate sensory-evoked transitions in motor programming. In the face of a rapid balance perturbation, the lateral vestibular nucleus (LVN) generates exclusive extensor muscle activation and selective early extension of the hindlimb, followed by the co-activation of extensor and flexor muscle groups. The temporal separation in EMG response to balance perturbation reflects two distinct cell types within the LVN that generate different phases of this motor program. Initially, an LVNextensor population directs an extension movement that reflects connections with extensor, but not flexor, motor neurons. A distinct LVNco-activation population initiates muscle co-activation via the pontine reticular nucleus. Thus, distinct circuits within the LVN generate different elements of a motor program involved in the maintenance of balance.

Origin and Segmental Diversity of Spinal Inhibitory Interneurons.

Motor output varies along the rostro-caudal axis of the tetrapod spinal cord. At limb levels, ∼60 motor pools control the alternation of flexor and extensor muscles about each joint, whereas at thoracic levels as few as 10 motor pools supply muscle groups that support posture, inspiration, and expiration. Whether such differences in motor neuron identity and muscle number are associated with segmental distinctions in interneuron diversity has not been resolved. We show that select combinations of nineteen transcription factors that specify lumbar V1 inhibitory interneurons generate subpopulations enriched at limb and thoracic levels. Specification of limb and thoracic V1 interneurons involves the Hox gene Hoxc9 independently of motor neurons. Thus, early Hox patterning of the spinal cord determines the identity of V1 interneurons and motor neurons. These studies reveal a developmental program of V1 interneuron diversity, providing insight into the organization of inhibitory interneurons associated with differential motor output.

Behaviorally Selective Engagement of Short-Latency Effector Pathways by Motor Cortex.

Blocking motor cortical output with lesions or pharmacological inactivation has identified movements that require motor cortex. Yet, when and how motor cortex influences muscle activity during movement execution remains unresolved. We addressed this ambiguity using measurement and perturbation of motor cortical activity together with electromyography in mice during two forelimb movements that differ in their requirement for cortical involvement. Rapid optogenetic silencing and electrical stimulation indicated that short-latency pathways linking motor cortex with spinal motor neurons are selectively activated during one behavior. Analysis of motor cortical activity revealed a dramatic change between behaviors in the coordination of firing patterns across neurons that could account for this differential influence. Thus, our results suggest that changes in motor cortical output patterns enable a behaviorally selective engagement of short-latency effector pathways. The model of motor cortical influence implied by our findings helps reconcile previous observations on the function of motor cortex.

Divergent Hox Coding and Evasion of Retinoid Signaling Specifies Motor Neurons Innervating Digit Muscles.

The establishment of spinal motor neuron subclass diversity is achieved through developmental programs that are aligned with the organization of muscle targets in the limb. The evolutionary emergence of digits represents a specialized adaptation of limb morphology, yet it remains unclear how the specification of digit-innervating motor neuron subtypes parallels the elaboration of digits. We show that digit-innervating motor neurons can be defined by selective gene markers and distinguished from other LMC neurons by the expression of a variant Hox gene repertoire and by the failure to express a key enzyme involved in retinoic acid synthesis. This divergent developmental program is sufficient to induce the specification of digit-innervating motor neurons, emphasizing the specialized status of digit control in the evolution of skilled motor behaviors. Our findings suggest that the emergence of digits in the limb is matched by distinct mechanisms for specifying motor neurons that innervate digit muscles.

Tectal-derived interneurons contribute to phasic and tonic inhibition in the visual thalamus.

The release of GABA from local interneurons in the dorsal lateral geniculate nucleus (dLGN-INs) provides inhibitory control during visual processing within the thalamus. It is commonly assumed that this important class of interneurons originates from within the thalamic complex, but we now show that during early postnatal development Sox14/Otx2-expressing precursor cells migrate from the dorsal midbrain to generate dLGN-INs. The unexpected extra-diencephalic origin of dLGN-INs sets them apart from GABAergic neurons of the reticular thalamic nucleus. Using optogenetics we show that at increased firing rates tectal-derived dLGN-INs generate a powerful form of tonic inhibition that regulates the gain of thalamic relay neurons through recruitment of extrasynaptic high-affinity GABAA receptors. Therefore, by revising the conventional view of thalamic interneuron ontogeny we demonstrate how a previously unappreciated mesencephalic population controls thalamic relay neuron excitability.

Spinal Inhibitory Interneuron Diversity Delineates Variant Motor Microcircuits.

Animals generate movement by engaging spinal circuits that direct precise sequences of muscle contraction, but the identity and organizational logic of local interneurons that lie at the core of these circuits remain unresolved. Here, we show that V1 interneurons, a major inhibitory population that controls motor output, fractionate into highly diverse subsets on the basis of the expression of 19 transcription factors. Transcriptionally defined V1 subsets exhibit distinct physiological signatures and highly structured spatial distributions with mediolateral and dorsoventral positional biases. These positional distinctions constrain patterns of input from sensory and motor neurons and, as such, suggest that interneuron position is a determinant of microcircuit organization. Moreover, V1 diversity indicates that different inhibitory microcircuits exist for motor pools controlling hip, ankle, and foot muscles, revealing a variable circuit architecture for interneurons that control limb movement.

Bayesian Sparse Regression Analysis Documents the Diversity of Spinal Inhibitory Interneurons.

Documenting the extent of cellular diversity is a critical step in defining the functional organization of tissues and organs. To infer cell-type diversity from partial or incomplete transcription factor expression data, we devised a sparse Bayesian framework that is able to handle estimation uncertainty and can incorporate diverse cellular characteristics to optimize experimental design. Focusing on spinal V1 inhibitory interneurons, for which the spatial expression of 19 transcription factors has been mapped, we infer the existence of ~50 candidate V1 neuronal types, many of which localize in compact spatial domains in the ventral spinal cord. We have validated the existence of inferred cell types by direct experimental measurement, establishing this Bayesian framework as an effective platform for cell-type characterization in the nervous system and elsewhere.

Muscle-type Identity of Proprioceptors Specified by Spatially Restricted Signals from Limb Mesenchyme.

The selectivity with which proprioceptive sensory neurons innervate their central and peripheral targets implies that they exhibit distinctions in muscle-type identity. The molecular correlates of proprioceptor identity and its origins remain largely unknown, however. In screens to define muscle-type proprioceptor character, we find all-or-none differences in gene expression for proprioceptors that control antagonistic muscles at a single hindlimb joint. Analysis of three of these genes, cadherin13 (cdh13), semaphorin5a (sema5a), and cartilage-acidic protein-1 (crtac1), reveals expression in proprioceptor subsets that supply muscle groups located at restricted dorsoventral and proximodistal domains of the limb. Genetically altering the dorsoventral character of the limb mesenchyme elicits a change in the profile of proprioceptor cdh13, sema5a, and crtac1 expression. These findings indicate that proprioceptors acquire aspects of their muscle-type identity in response to mesenchymal signals expressed in restricted proximodistal and dorsoventral domains of the developing limb.

Rabies Virus CVS-N2c(ΔG) Strain Enhances Retrograde Synaptic Transfer and Neuronal Viability.

Virally based transsynaptic tracing technologies are powerful experimental tools for neuronal circuit mapping. The glycoprotein-deletion variant of the SAD-B19 vaccine strain rabies virus (RABV) has been the reagent of choice in monosynaptic tracing, since it permits the mapping of synaptic inputs to genetically marked neurons. Since its introduction, new helper viruses and reagents that facilitate complementation have enhanced the efficiency of SAD-B19(ΔG) transsynaptic transfer, but there has been little focus on improvements to the core RABV strain. Here we generate a new deletion mutant strain, CVS-N2c(ΔG), and examine its neuronal toxicity and efficiency in directing retrograde transsynaptic transfer. We find that by comparison with SAD-B19(ΔG), the CVS-N2c(ΔG) strain exhibits a reduction in neuronal toxicity and a marked enhancement in transsynaptic neuronal transfer. We conclude that the CVS-N2c(ΔG) strain provides a more effective means of mapping neuronal circuitry and of monitoring and manipulating neuronal activity in vivo in the mammalian CNS.

Simultaneous Denoising, Deconvolution, and Demixing of Calcium Imaging Data.

We present a modular approach for analyzing calcium imaging recordings of large neuronal ensembles. Our goal is to simultaneously identify the locations of the neurons, demix spatially overlapping components, and denoise and deconvolve the spiking activity from the slow dynamics of the calcium indicator. Our approach relies on a constrained nonnegative matrix factorization that expresses the spatiotemporal fluorescence activity as the product of a spatial matrix that encodes the spatial footprint of each neuron in the optical field and a temporal matrix that characterizes the calcium concentration of each neuron over time. This framework is combined with a novel constrained deconvolution approach that extracts estimates of neural activity from fluorescence traces, to create a spatiotemporal processing algorithm that requires minimal parameter tuning. We demonstrate the general applicability of our method by applying it to in vitro and in vivo multi-neuronal imaging data, whole-brain light-sheet imaging data, and dendritic imaging data.