Premotor Interneurons Research Articles

A comparative distribution of parvalbumin-immunopositive neural elements in the spinal cord of newborn and adult cats

We analyzed characteristic features of the parvalbumin-immunostaining within the lumbosacral spinal cord of newborn kittens. In contrast to the adults, parvalbumin-immunostaining was mainly revealed for the sensory fibers located within the dorsal horns and in the medial part of the intermediate gray matter. The location of these fibers partially resembles the location of Clarke's nuclei, but lasted throughout the total length of the lumbar spinal cord and merged with the presumptive Stilling’s nuclei in the sacral region. Therefore, in newborns, in contrast to adults, the parvalbumin-immunostaining proprioceptive fibers seem like a single unit. We propose that with maturation, this system is restructured because of the spread of the neuronal and neuropil elements of the lumbar enlargement responsible for the locomotor control. As a result, two local nuclear complexes: Clarke’s and Stilling’s are retained. A single population of parvalbumin-immunostaining neurons in newborns are premotor interneurons located around the lamina IX. These neurons are characterized by the low or absent NeuN-immunostaining. We believe that this neurochemical feature may be inherent for these cells.

Hierarchical regulation of functionally antagonistic neuropeptides expressed in a single neuron pair

Neuronal communication involves small-molecule transmitters, gap junctions, and neuropeptides. While neurons often express multiple neuropeptides, our understanding of the coordination of their actions and their mutual interactions remains limited. Here, we demonstrate that two neuropeptides, NLP-10 and FLP-1, released from the same interneuron pair, AVKL/R, exert antagonistic effects on locomotion speed in Caenorhabditis elegans. NLP-10 accelerates locomotion by activating the G protein-coupled receptor NPR-35 on premotor interneurons that promote forward movement. Notably, we establish that NLP-10 is crucial for the aversive response to mechanical and noxious light stimuli. Conversely, AVK-derived FLP-1 slows down locomotion by suppressing the secretion of NLP-10 from AVK, through autocrine feedback via activation of its receptor DMSR-7 in AVK neurons. Our findings suggest that peptidergic autocrine motifs, exemplified by the interaction between NLP-10 and FLP-1, might represent a widespread mechanism in nervous systems across species. These mutual functional interactions among peptidergic co-transmitters could fine-tune brain activity.

Arid3c identifies an uncharacterized subpopulation of V2 interneurons during embryonic spinal cord development.

Motor activity is organized by neuronal networks composed of motor neurons and a wide variety of pre-motor interneuron populations located in the brainstem and spinal cord. Differential expression and single-cell RNA sequencing studies recently unveiled that these populations subdivide into multiple subsets. However, some interneuron subsets have not been described yet, and the mechanisms contributing to this neuronal diversification have only been partly deciphered. In this study, we aimed to identify additional markers to further describe the diversity of spinal V2 interneuron populations. Here, we compared the transcriptome of V2 interneurons with that of the other cells of the embryonic spinal cord and extracted a list of genes enriched in V2 interneurons, including Arid3c. Arid3c identifies an uncharacterized subset of V2 that partially overlaps with V2c interneurons. These two populations are characterized by the production of Onecut factors and Sox2, suggesting that they could represent a single functional V2 unit. Furthermore, we show that the overexpression or inactivation of Arid3c does not alter V2 production, but its absence results in minor defects in locomotor execution, suggesting a possible function in subtle aspects of spinal locomotor circuit formation.

STEERING FROM THE REAR: COORDINATION OF CENTRAL PATTERN GENERATORS UNDERLYING NAVIGATION BY ASCENDING INTERNEURONS.

Understanding how animals coordinate movements to achieve goals is a fundamental pursuit in neuroscience. Here we explore how neurons that reside in posterior lower-order regions of a locomotor system project to anterior higher-order regions to influence steering and navigation. We characterized the anatomy and functional role of a population of ascending interneurons in the ventral nerve cord of Drosophila larvae. Through electron microscopy reconstructions and light microscopy, we determined that the cholinergic 19f cells receive input primarily from premotor interneurons and synapse upon a diverse array of postsynaptic targets within the anterior segments including other 19f cells. Calcium imaging of 19f activity in isolated central nervous system (CNS) preparations in relation to motor neurons revealed that 19f neurons are recruited into most larval motor programmes. 19f activity lags behind motor neuron activity and as a population, the cells encode spatio-temporal patterns of locomotor activity in the larval CNS. Optogenetic manipulations of 19f cell activity in isolated CNS preparations revealed that they coordinate the activity of central pattern generators underlying exploratory headsweeps and forward locomotion in a context and location specific manner. In behaving animals, activating 19f cells suppressed exploratory headsweeps and slowed forward locomotion, while inhibition of 19f activity potentiated headsweeps, slowing forward movement. Inhibiting activity in 19f cells ultimately affected the ability of larvae to remain in the vicinity of an odor source during an olfactory navigation task. Overall, our findings provide insights into how ascending interneurons monitor motor activity and shape interactions amongst rhythm generators underlying complex navigational tasks.

Potential Roles of Specific Subclasses of Premotor Interneurons in Spinal Cord Function Recovery after Traumatic Spinal Cord Injury in Adults.

The differential expression of transcription factors during embryonic development has been selected as the main feature to define the specific subclasses of spinal interneurons. However, recent studies based on single-cell RNA sequencing and transcriptomic experiments suggest that this approach might not be appropriate in the adult spinal cord, where interneurons show overlapping expression profiles, especially in the ventral region. This constitutes a major challenge for the identification and direct targeting of specific populations that could be involved in locomotor recovery after a traumatic spinal cord injury in adults. Current experimental therapies, including electrical stimulation, training, pharmacological treatments, or cell implantation, that have resulted in improvements in locomotor behavior rely on the modulation of the activity and connectivity of interneurons located in the surroundings of the lesion core for the formation of detour circuits. However, very few publications clarify the specific identity of these cells. In this work, we review the studies where premotor interneurons were able to create new intraspinal circuits after different kinds of traumatic spinal cord injury, highlighting the difficulties encountered by researchers, to classify these populations.

Drosophila larval motor patterning relies on regulated alternative splicing of Dscam2.

Recent studies capitalizing on the newly complete nanometer-resolution Drosophila larval connectome have made significant advances in identifying the structural basis of motor patterning. However, the molecular mechanisms utilized by neurons to wire these circuits remain poorly understood. In this study we explore how cell-specific expression of two Dscam2 isoforms, which mediate isoform-specific homophilic binding, contributes to motor patterning and output of Drosophila larvae. Ablating Dscam2 isoform diversity resulted in impaired locomotion. Electrophysiological assessment at the neuromuscular junction during fictive locomotion indicated that this behavioral defect was largely caused by weaker bouts of motor neuron activity. Morphological analyses of single motor neurons using MultiColour FlpOut revealed severe errors in dendrite arborization and assessment of cholinergic and GABAergic projections to the motor domain revealed altered morphology of interneuron processes. Loss of Dscam2 did not affect locomotor output, motor neuron activation or dendrite targeting. Our findings thus suggest that locomotor circuit phenotypes arise specifically from inappropriate Dscam2 interactions between premotor interneurons and motor neurons when they express the same isoform. Indeed, we report here that first-order premotor interneurons express Dscam2A. Since motor neurons express Dscam2B, our results provide evidence that Dscam2 isoform expression alternates between synaptic partners in the nerve cord. Our study demonstrates the importance of cell-specific alternative splicing in establishing the circuitry that underlies neuromotor patterning without inducing unwanted intercellular interactions.

Potential contribution of spinal interneurons to the etiopathogenesis of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) consists of a group of adult-onset fatal and incurable neurodegenerative disorders characterized by the progressive death of motor neurons (MNs) throughout the central nervous system (CNS). At first, ALS was considered to be an MN disease, caused by cell-autonomous mechanisms acting specifically in MNs. Accordingly, data from ALS patients and ALS animal models revealed alterations in excitability in multiple neuronal populations, including MNs, which were associated with a variety of cellular perturbations such as protein aggregation, ribonucleic acid (RNA) metabolism defects, calcium dyshomeostasis, modified electrophysiological properties, and autophagy malfunctions. However, experimental evidence rapidly demonstrated the involvement of other types of cells, including glial cells, in the etiopathogenesis of ALS through non-cell autonomous mechanisms. Surprisingly, the contribution of pre-motor interneurons (INs), which regulate MN activity and could therefore critically modulate their excitability at the onset or during the progression of the disease, has to date been severely underestimated. In this article, we review in detail how spinal pre-motor INs are affected in ALS and their possible involvement in the etiopathogenesis of the disease.

Inhibitory Components in Muscle Synergies Factorized by The Rectified Latent Variable Model from Electromyographic Data.

Non-negative matrix factorization (NMF), widely used in motor neuroscience for identifying muscle synergies from electromyographical signals (EMGs), extracts non-negative synergies and is yet unable to identify potential negative components (NegCps) in synergies underpinned by inhibitory spinal interneurons. To overcome this constraint, we propose to utilize rectified latent variable model (RLVM) to extract muscle synergies. RLVM uses an autoencoder neural network, and the weight matrix of its neural network could be negative, while latent variables must remain non-negative. If inputs to the model are EMGs, the weight matrix and latent variables represent muscle synergies and their temporal activation coefficients, respectively. We compared performances of NMF and RLVM in identifying muscle synergies in simulated and experimental datasets. Our simulated results showed that RLVM performed better in identifying muscle-synergy subspace and NMF had a good correlation with ground truth. Finally, we applied RLVM to a previously published experimental dataset comprising EMGs from upper-limb muscles and spike recordings of spinal premotor interneurons (PreM-INs) collected from two macaque monkeys during grasping tasks. RLVM and NMF synergies were highly similar, but a few small negative muscle components were observed in RLVM synergies. The muscles with NegCps identified by RLVM exhibited near-zero values in their corresponding synergies identified by NMF. Importantly, NegCps of RLVM synergies showed correspondence with the muscle connectivity of PreM-INs with inhibitory muscle fields, as identified by spike-triggered averaging of EMGs. Our results demonstrate the feasibility of RLVM in extracting potential inhibitory muscle-synergy components from EMGs.

Action of Serotonin Precursor Synthesis 5-Oxytryptophan on EPSP Recorded in Premotor Interneurons of Snail after Formation of Conditioned Defensive Reflex

Action of Serotonin Precursor Synthesis 5-Oxytryptophan on EPSP Recorded in Premotor Interneurons of Snail after Formation of Conditioned Defensive Reflex

Inhibitory feedback from the motor circuit gates mechanosensory processing in Caenorhabditis elegans.

Animals must integrate sensory cues with their current behavioral context to generate a suitable response. How this integration occurs is poorly understood. Previously, we developed high-throughput methods to probe neural activity in populations of Caenorhabditis elegans and discovered that the animal's mechanosensory processing is rapidly modulated by the animal's locomotion. Specifically, we found that when the worm turns it suppresses its mechanosensory-evoked reversal response. Here, we report that C. elegans use inhibitory feedback from turning-associated neurons to provide this rapid modulation of mechanosensory processing. By performing high-throughput optogenetic perturbations triggered on behavior, we show that turning-associated neurons SAA, RIV, and/or SMB suppress mechanosensory-evoked reversals during turns. We find that activation of the gentle-touch mechanosensory neurons or of any of the interneurons AIZ, RIM, AIB, and AVE during a turn is less likely to evoke a reversal than activation during forward movement. Inhibiting neurons SAA, RIV, and SMB during a turn restores the likelihood with which mechanosensory activation evokes reversals. Separately, activation of premotor interneuron AVA evokes reversals regardless of whether the animal is turning or moving forward. We therefore propose that inhibitory signals from SAA, RIV, and/or SMB gate mechanosensory signals upstream of neuron AVA. We conclude that C. elegans rely on inhibitory feedback from the motor circuit to modulate its response to sensory stimuli on fast timescales. This need for motor signals in sensory processing may explain the ubiquity in many organisms of motor-related neural activity patterns seen across the brain, including in sensory processing areas.

Central projections from Johnston’s organ in the locust: Axogenesis and brain neuroarchitecture

Johnston’s organ (Jo) acts as an antennal wind-sensitive and/or auditory organ across a spectrum of insect species and its axons universally project to the brain. In the locust, this pathway is already present at mid-embryogenesis but the process of fasciculation involved in its construction has not been investigated. Terminal projections into the fine neuropilar organization of the brain also remain unresolved, information essential not only for understanding the neural circuitry mediating Jo-mediated behavior but also for providing comparative data offering insights into its evolution. In our study here, we employ neuron-specific, axon-specific, and epithelial domain labels to show that the pathway to the brain of the locust is built in a stepwise manner during early embryogenesis as processes from Jo cell clusters in the pedicel fasciculate first with one another, and then with the two tracts constituting the pioneer axon scaffold of the antenna. A comparison of fasciculation patterns confirms that projections from cell clusters of Jo stereotypically associate with only one axon tract according to their location in the pedicellar epithelium, consistent with a topographic plan. At the molecular level, all neuronal elements of the Jo pathway to the brain express the lipocalin Lazarillo, a cell surface epitope that regulates axogenesis in the primary axon scaffold itself, and putatively during fasciculation of the Jo projections to the brain. Central projections from Jo first contact the primary axon scaffold of the deutocerebral brain at mid-embryogenesis, and in the adult traverse mechanosensory/motor neuropils similar to those in Drosophila. These axons then terminate among protocerebral commissures containing premotor interneurons known to regulate flight behavior.

Differences in responses of premotor interneurons to serotonin and 5-hydroxytryptophan, a precursor for serotonin synthesis, in naive and trained snails

This study investigated the responses of premotor interneurons LPa3 and RPa3 of the snails to applications of serotonin and 5-hydroxytryptophan (5-HTP), a precursor for serotonin synthesis, as ingredients of the broth that bathes the central nervous system of naive snails and snails after the formation of long-term sensitization. Measurements of the electrical characteristics have shown that a membrane potential of interneurons LPa3 and RPa3 in naive snails was significantly depolarized (3.7 mV) in the presence of serotonin, while the threshold potential was increased (unsupported hypothesis). The similar pattern was observed in the presence of 5-hydroxytryptophan: true depolarization to 3.1 mV occurred in interneurons until reaching the unreliable rise in the threshold potential inducing an action potential. It was found that application of serotonin causes a significant decrease in the membrane potential of interneurons LPa3 and RPa3 of trained snails (depolarization to 4.6 mV) and the unreliable increase in the threshold potential of premotor interneurons (0.9 mV). In contrast, application of 5-hydroxytryptophan causes an unreliable increase (2.5 mV) of the membrane potential and also an unreliable increase (0.8 mV) in the threshold potential.

Differences in Responses of Premotor Interneurons to Serotonin and the Precursor of Its Synthesis 5-Hydroxytryptophan in Naive and Sensitized Snails

Differences in Responses of Premotor Interneurons to Serotonin and the Precursor of Its Synthesis 5-Hydroxytryptophan in Naive and Sensitized Snails

Spinal Basis of Direction Control during Locomotion in Larval Zebrafish.

Navigation requires steering and propulsion, but how spinal circuits contribute to direction control during ongoing locomotion is not well understood. Here, we use drifting vertical gratings to evoke directed "fictive" swimming in intact but immobilized larval zebrafish while performing electrophysiological recordings from spinal neurons. We find that directed swimming involves unilateral changes in the duration of motor output and increased recruitment of motor neurons, without impacting the timing of spiking across or along the body. Voltage-clamp recordings from motor neurons reveal increases in phasic excitation and inhibition on the side of the turn. Current-clamp recordings from premotor interneurons that provide phasic excitation or inhibition reveal two types of recruitment patterns. A direction-agnostic pattern with balanced recruitment on the turning and nonturning sides is primarily observed in excitatory V2a neurons with ipsilateral descending axons, while a direction-sensitive pattern with preferential recruitment on the turning side is dominated by V2a neurons with ipsilateral bifurcating axons. Inhibitory V1 neurons are also divided into direction-sensitive and direction-agnostic subsets, although there is no detectable morphologic distinction. Our findings support the modular control of steering and propulsion by spinal premotor circuits, where recruitment of distinct subsets of excitatory and inhibitory interneurons provide adjustments in direction while on the move.SIGNIFICANCE STATEMENT Spinal circuits play an essential role in coordinating movements during locomotion. However, it is unclear how they participate in adjustments in direction that do not interfere with coordination. Here we have developed a system using larval zebrafish that allows us to directly record electrical signals from spinal neurons during "fictive" swimming guided by visual cues. We find there are subsets of spinal interneurons for coordination and others that drive unilateral asymmetries in motor neuron recruitment for direction control. Our findings suggest a modular organization of spinal premotor circuits that enables uninterrupted adjustments in direction during ongoing locomotion.

Effects of motor cortex neuromodulation on the specificity of corticospinal tract spinal axon outgrowth and targeting in rats

BackgroundNeural activity helps construct neural circuits during development and this function is leveraged by neuromodulation protocols to promote connectivity and repair in maturity. Neuromodulation targeting the motor cortex (MCX) strengthens connections for evoking muscle contraction (MEPs). Mechanisms include promoting local MCX and corticospinal tract (CST) synaptic efficacy and also axon terminal structural changes. ObjectiveIn this study, we address the question of potential causality between neuronal activation and the neuronal structural response. MethodsWe used patterned optogenetic activation (ChR2-EYFP), daily for 10-days, to deliver intermittent theta burst stimulation (iTBS) to activate MCX neurons within the forelimb representation in healthy rats, while differentiating them from neurons in the same population that were not activated. We used chemogenetic DREADD activation to produce a daily period of non-patterned neuronal activation. ResultsWe found a significant increase in CST axon length, axon branching, contacts targeted to a class of premotor interneuron (Chx10), as well as projections into the motor pools in the ventral horn in optically activated but not neighboring non-activated neurons. A period of 2-h of continuous activation daily for 10 days using DREADD chemogenetic activation with systemic clozapine N-oxide (CNO) administration also increased CST axon length and branching, but not the ventral horn and Chx10 targeting effects. Both patterned optical and chemogenetic activation reduced MCX MEP thresholds. ConclusionOur findings show that targeting of CST axon sprouting is dependent on patterned activation, but that CST spinal axon outgrowth and branching are not. Our optogenetic findings, by distinguishing optically activated and non-activated CST axons, suggests that the switch for activity-dependent axonal outgrowth is neuron-intrinsic.

Cerebrospinal fluid-contacting neuron tracing reveals structural and functional connectivity for locomotion in the mouse spinal cord.

Cerebrospinal fluid-contacting neurons (CSF-cNs) are enigmatic mechano- or chemosensory cells lying along the central canal of the spinal cord. Recent studies in zebrafish larvae and lampreys have shown that CSF-cNs control postures and movements via spinal connections. However, the structures, connectivity, and functions in mammals remain largely unknown. Here we developed a method to genetically target mouse CSF-cNs that highlighted structural connections and functions. We first found that intracerebroventricular injection of adeno-associated virus with a neuron-specific promoter and Pkd2l1-Cre mice specifically labeled CSF-cNs. Single-cell labeling of 71 CSF-cNs revealed rostral axon extensions of over 1800 μm in unmyelinated bundles in the ventral funiculus and terminated on CSF-cNs to form a recurrent circuitry, which was further determined by serial electron microscopy and electrophysiology. CSF-cNs were also found to connect with axial motor neurons and premotor interneurons around the central canal and within the axon bundles. Chemogenetic CSF-cNs inactivation reduced speed and step frequency during treadmill locomotion. Our data revealed the basic structures and connections of mouse CSF-cNs to control spinal motor circuits for proper locomotion. The versatile methods developed in this study will contribute to further understanding of CSF-cN functions in mammals.

Changes in the Electrical Characteristics of Premotor Interneurons and Serotonin-Containing Modulator Snail Neurons upon Developing a Contextual Conditioned Reflex and Its Reconsolidation

Changes in the Electrical Characteristics of Premotor Interneurons and Serotonin-Containing Modulator Snail Neurons upon Developing a Contextual Conditioned Reflex and Its Reconsolidation

Spinal premotor interneurons controlling antagonistic muscles are spatially intermingled.

Elaborate behaviours are produced by tightly controlled flexor-extensor motor neuron activation patterns. Motor neurons are regulated by a network of interneurons within the spinal cord, but the computational processes involved in motor control are not fully understood. The neuroanatomical arrangement of motor and premotor neurons into topographic patterns related to their controlled muscles is thought to facilitate how information is processed by spinal circuits. Rabies retrograde monosynaptic tracing has been used to label premotor interneurons innervating specific motor neuron pools, with previous studies reporting topographic mediolateral positional biases in flexor and extensor premotor interneurons. To more precisely define how premotor interneurons contacting specific motor pools are organized, we used multiple complementary viral-tracing approaches in mice to minimize systematic biases associated with each method. Contrary to expectations, we found that premotor interneurons contacting motor pools controlling flexion and extension of the ankle are highly intermingled rather than segregated into specific domains like motor neurons. Thus, premotor spinal neurons controlling different muscles process motor instructions in the absence of clear spatial patterns among the flexor-extensor circuit components.

Extrasynaptic signaling enables an asymmetric juvenile motor circuit to produce symmetric undulation

In many animals, there is a direct correspondence between the motor patterns that drive locomotion and the motor neuron innervation. For example, the adult C.elegans moves with symmetric and alternating dorsal-ventral bending waves arising from symmetric motor neuron input onto the dorsal and ventral muscles. In contrast to the adult, the C.elegans motor circuit at the juvenile larval stage has asymmetric wiring between motor neurons and muscles but still generates adult-like bending waves with dorsal-ventral symmetry. We show that in the juvenile circuit, wiring between excitatory and inhibitory motor neurons coordinates the contraction of dorsal muscles with relaxation of ventral muscles, producing dorsal bends. However, ventral bending is not driven by analogous wiring. Instead, ventral muscles are excited uniformly by premotor interneurons through extrasynaptic signaling. Ventral bends occur in anti-phasic entrainment to activity of the same motor neurons that drive dorsal bends. During maturation, the juvenile motor circuit is replaced by two motor subcircuits that separately drive dorsal and ventral bending. Modeling reveals that the juvenile's immature motor circuit is an adequate solution to generate adult-like dorsal-ventral bending before the animal matures. Developmental rewiring between functionally degenerate circuit solutions, which both generate symmetric bending patterns, minimizes behavioral disruption across maturation.

Electrophysiological Validation of Monosynaptic Connectivity between Premotor Interneurons and the aCC Motoneuron in the Drosophila Larval CNS.

The Drosophila connectome project aims to map the synaptic connectivity of entire larval and adult fly neural networks, which is essential for understanding nervous system development and function. So far, the project has produced an impressive amount of electron microscopy data that has facilitated reconstructions of specific synapses, including many in the larval locomotor circuit. While this breakthrough represents a technical tour de force, the data remain underutilized, partly because of a lack of functional validation of reconstructions. Attempts to validate connectivity posited by the connectome project, have mostly relied on behavioral assays and/or GFP reconstitution across synaptic partners (GRASP) or GCaMP imaging. While these techniques are useful, they have limited spatial or temporal resolution. Electrophysiological assays of synaptic connectivity overcome these limitations. Here, we combine patch-clamp recordings with optogenetic stimulation in male and female larvae, to test synaptic connectivity proposed by connectome reconstructions. Specifically, we use multiple driver lines to confirm that several connections between premotor interneurons and the anterior corner cell motoneuron are, as the connectome project suggests, monosynaptic. In contrast, our results also show that conclusions based on GRASP imaging may provide false-positive results regarding connectivity between cells. We also present a novel imaging tool, based on the same technology as our electrophysiology, as a favorable alternative to GRASP imaging. Finally, of eight Gal4 lines tested, five are reliably expressed in the premotor interneurons they are targeted to. Thus, our work highlights the need to confirm functional synaptic connectivity, driver line specificity, and use of appropriate genetic tools to support connectome projects.SIGNIFICANCE STATEMENT The Drosophila connectome project aims to provide a complete description of connectivity between neurons in an organism that presents experimental advantages over other models. It has reconstructed hundreds of thousands of synaptic connections of the fly larva by manual identification of anatomic landmarks present in serial section transmission electron microscopy (ssTEM) volumes of the larval CNS. We use a highly reliable electrophysiological approach to verify these connections, providing useful insight into the accuracy of work based on ssTEM. We also present a novel imaging tool for validating excitatory monosynaptic connections between cells and show that several genetic driver lines designed to target neurons of the larval connectome exhibit nonspecific and/or unreliable expression.