Excitatory and Inhibitory Descending Commissural Interneurons Differentially Integrate Supraspinal and Segmental Sensory Signals.

The limited information about how descending inputs from the brain and sensory inputs from the periphery use spinal cord interneurons (INs) is a major barrier to understanding how these inputs may contribute to motor functions under normal and pathologic conditions. Commissural interneurons (CINs) are a heterogeneous population of spinal INs that has been implicated in crossed motor responses and bilateral motor coordination (ability to use the right and left side of the body in a coordinated manner) and, therefore, are likely involved in many types of movement (e.g., dynamic posture stabilization, jumping, kicking, walking). In this study, we incorporate mouse genetics, anatomy, electrophysiology, and single-cell calcium imaging to investigate how a subset of CINs, those with descending axons called dCINs, are recruited by descending reticulospinal and segmental sensory signals independently and in combination. We focus on two groups of dCINs set apart by their principal neurotransmitter (glutamate and GABA) and identified as VGluT2+ dCINs and GAD2+ dCINs. We show that VGluT2+ and GAD2+ dCINs are both extensively recruited by reticulospinal and sensory input alone but that VGluT2+ and GAD2+ dCINs integrate these inputs differently. Critically, we find that when recruitment depends on the combined action of reticulospinal and sensory inputs (subthreshold inputs), VGluT2+ dCINs, but not GAD2+ dCINs, are recruited. This difference in the integrative capacity of VGluT2+ and GAD2+ dCINs represents a circuit mechanism that the reticulospinal and segmental sensory systems may avail themselves of to regulate motor behaviors both normally and after injury.SIGNIFICANCE STATEMENT The way supraspinal and peripheral sensory inputs use spinal cord interneurons is fundamental to defining how motor functions are supported both in health and disease. This study, which focuses on dCINs, a heterogeneous population of spinal interneurons critical for crossed motor responses and bilateral motor coordination, shows that both glutamatergic (excitatory) and GABAergic (inhibitory) dCINs can be recruited by supraspinal (reticulospinal) or peripheral sensory inputs. Additionally, the study demonstrates that in conditions where the recruitment of dCINs depends on the combined action of reticulospinal and sensory inputs, only excitatory dCINs are recruited. The study uncovers a circuit mechanism that the reticulospinal and segmental sensory systems may avail themselves of to regulate motor behaviors both normally and after injury.

Segmental motor neuron dysfunction in amyotrophic lateral sclerosis: Insights from H reflex paradigms.

INTRODUCTION/AIMS: In amyotrophic lateral sclerosis (ALS), the role of spinal interneurons in ALS is underrecognized. We aimed to investigate pre- and post-synaptic modulation of spinal motor neuron excitability by studying the H reflex, to understand spinal interneuron function in ALS. METHODS: We evaluated the soleus H reflex, and three different modulation paradigms, to study segmental spinal inhibitory mechanisms. Homonymous recurrent inhibition (H'RI ) was assessed using the paired H reflex technique. Presynaptic inhibition of Ia afferents (H'Pre ) was evaluated using D1 inhibition after stimulation of the common peroneal nerve. We also studied inhibition of the H reflex after cutaneous stimulation of the sural nerve (H'Pos ). RESULTS: Fifteen ALS patients (median age 57.0 years), with minimal signs of lower motor neuron involvement and good functional status, and a control group of 10 healthy people (median age 57.0 years) were studied. ALS patients showed reduced inhibition, compared to controls, in all paradigms (H'RI 0.35 vs. 0.11, p = .036; H'Pre 1.0 vs. 5.0, p = .001; H'Pos 0.0 vs. 2.5, p = .031). The clinical UMN score was a significant predictor of the amount of recurrent and presynaptic inhibition. DISCUSSION: Spinal inhibitory mechanisms are impaired in ALS. We argue that hyperreflexia could be associated with dysfunction of spinal inhibitory interneurons. In this case, an interneuronopathy could be deemed a major feature of ALS.

Basic principles of processing of afferent information by spinal interneurons.

Integrative functions of spinal interneurons are well recognized but the relative role of different interneuronal populations in this process continues to be investigated. It therefore appeared useful to review the principles of integration of afferent information by the interneurons analyzed so far as these principles should apply also to those remaining to be analyzed. Considering the results of both functional and morphological studies of spinal interneurons and of the morphology and immunochemistry of afferent fibers that provide input to them, the following five basic principles of processing of afferent information by them will be outlined; 1) afferent information of any origin is forwarded to several neuronal populations, 2) information from any sources of input is distributed unevenly, 3) input from several sources is integrated by individual neurons as well as by their populations, 4) specific combinations of input are integrated by different neuronal populations, and 5) afferent input to spinal interneurons is only one of the features distinguishing their functional populations. As the spinal neuronal organization and properties of neurons and afferent fibers in the so far investigated species (cat, rodents, and primates) have been found to resemble, future studies using molecular techniques in the mouse should allow the new data to integrate with those of the preceding studies and the principles outlined earlier as well as any new ones should apply also in humans.

Rhythmogenic networks are potently modulated by activation of muscarinic acetylcholine receptors in the rodent spinal cord.

Electrical stimulation of the spinal cord is a potent means for activating mammalian stepping in the absence of the descending control from the brain. Previously, we have shown that stimulation of pain delivering (Aδ) sacrocaudal afferents (SCA) has a powerful capacity to activate the sacral and lumbar rhythmogenic networks in the neonatal rodent spinal cord. Relatively little is known about the neural pathways involved in activation of the locomotor networks by Aδ afferents, on their mechanism of action and on the possibility to modulate their activity. We have shown that elevation of the endogenous level of acetylcholine at the sacral cord by blocking cholinesterase could modulate the SCA-induced locomotor rhythm in a muscarinic receptor-dependent mechanism. Here, we review these and more recent findings and report that controlled stimulation of SCA in the presence of muscarine is a potent activator of the locomotor network. The possible mechanisms involved in the muscarinic modulation of the locomotor rhythm are discussed in terms of the differential projections of sacral relay neurons, activated by SCA stimulation, to the lumbar locomotor rhythm generators, and to their target motoneurons. Altogether, our studies show that manipulations of cholinergic networks offer a simple and powerful means to control the activity of locomotor networks in the absence of supraspinal control. Cover Image for this issue: https://doi.org/10.1111/jnc.15079.

Three-dimensional induction of dorsal, intermediate and ventral spinal cord tissues from human pluripotent stem cells.

The spinal cord contains more than 20 distinct subclasses of neurons that form well-organized neural circuits capable of sensing the environment and generating motor behavior. Although recent studies have described the efficient in vitro generation of spinal motor neurons, the induction of the spinal cord as a whole tissue has not been achieved. In the present study, we demonstrate three-dimensional (3D) induction of dorsal spinal cord-like tissues from human pluripotent stem cells. Our 3D spinal cord induction (3-DiSC) condition recapitulates patterning of the developing dorsal spinal cord and enables the generation of four types of dorsal interneuron marker-positive cell populations. By activating Shh signaling, intermediate and ventral spinal cord-like tissues are successfully induced. After dissociation of these tissues, somatosensory neurons and spinal motor neurons are detected and express neurotransmitters in an in vivo manner. Our approach provides a useful experimental tool for the analysis of human spinal cord development and will contribute to research on the formation and organization of the spinal cord, and its application to regenerative medicine.

Neural basis for hand muscle synergies in the primate spinal cord.

Grasping is a highly complex movement that requires the coordination of multiple hand joints and muscles. Muscle synergies have been proposed to be the functional building blocks that coordinate such complex motor behaviors, but little is known about how they are implemented in the central nervous system. Here we demonstrate that premotor interneurons (PreM-INs) in the primate cervical spinal cord underlie the spatiotemporal patterns of hand muscle synergies during a voluntary grasping task. Using spike-triggered averaging of hand muscle activity, we found that the muscle fields of PreM-INs were not uniformly distributed across hand muscles but rather distributed as clusters corresponding to muscle synergies. Moreover, although individual PreM-INs have divergent activation patterns, the population activity of PreM-INs reflects the temporal activation of muscle synergies. These findings demonstrate that spinal PreM-INs underlie the muscle coordination required for voluntary hand movements in primates. Given the evolution of neural control of primate hand functions, we suggest that spinal premotor circuits provide the fundamental coordination of multiple joints and muscles upon which more fractionated control is achieved by superimposed, phylogenetically newer, pathways.

Shaping the Output of Lumbar Flexor Motoneurons by Sacral Neuronal Networks.

The ability to improve motor function in spinal cord injury patients by reactivating spinal central pattern generators (CPGs) requires the elucidation of neurons and pathways involved in activation and modulation of spinal networks in accessible experimental models. Previously we reported on adrenoceptor-dependent sacral control of lumbar flexor motoneuron firing in newborn rats. The current work focuses on clarification of the circuitry and connectivity involved in this unique modulation and its potential use. Using surgical manipulations of the spinal gray and white matter, electrophysiological recordings, and confocal microscopy mapping, we found that methoxamine (METH) activation of sacral networks within the ventral aspect of S2 segments was sufficient to produce alternating rhythmic bursting (0.15-1 Hz) in lumbar flexor motoneurons. This lumbar rhythm depended on continuity of the ventral funiculus (VF) along the S2-L2 segments. Interrupting the VF abolished the rhythm and replaced it by slow unstable bursting. Calcium imaging of S1-S2 neurons, back-labeled via the VF, revealed that ∼40% responded to METH, mostly by rhythmic firing. All uncrossed projecting METH responders and ∼70% of crossed projecting METH responders fired with the concurrent ipsilateral motor output, while the rest (∼30%) fired with the contralateral motor output. We suggest that METH-activated sacral CPGs excite ventral clusters of sacral VF neurons to deliver the ascending drive required for direct rhythmic activation of lumbar flexor motoneurons. The capacity of noradrenergic-activated sacral CPGs to modulate the activity of lumbar networks via sacral VF neurons provides a novel way to recruit rostral lumbar motoneurons and modulate the output required to execute various motor behaviors. SIGNIFICANCE STATEMENT: Spinal central pattern generators (CPGs) produce the rhythmic output required for coordinating stepping and stabilizing the body axis during movements. Electrical stimulation and exogenous drugs can reactivate the spinal CPGs and improve the motor function in the absence of descending supraspinal control. Since the body-stabilizing sacral networks can activate and modulate the limb-moving lumbar circuitry, it is important to clarify the functional organization of sacral and lumbar networks and their linking pathways. Here we decipher the ascending circuitry linking adrenoceptor-activated sacral CPGs and lumbar flexor motoneurons, thereby providing novel insights into mechanisms by which sacral circuitry recruits lumbar flexors, and enhances the motor output during lumbar afferent-induced locomotor rhythms. Moreover, our findings might help to improve drug/electrical stimulation-based therapy to accelerate locomotor-based rehabilitation.

Phrenic motoneurons: output elements of a highly organized intraspinal network.

pontomedullary respiratory network generates the respiratory pattern and relays it to bulbar and spinal respiratory motor outputs. The phrenic motor system controlling diaphragm contraction receives and processes descending commands to produce orderly, synchronous, and cycle-to-cycle-reproducible spatiotemporal firing. Multiple investigators have studied phrenic motoneurons (PhMNs) in an attempt to shed light on local mechanisms underlying phrenic pattern formation. I and colleagues (Marchenko V, Ghali MG, Rogers RF. Am J Physiol Regul Integr Comp Physiol 308: R916-R926, 2015.) recorded PhMNs in unanesthetized, decerebrate rats and related their activity to simultaneous phrenic nerve (PhN) activity by creating a time-frequency representation of PhMN-PhN power and coherence. On the basis of their temporal firing patterns and relationship to PhN activity, we categorized PhMNs into three classes, each of which emerges as a result of intrinsic biophysical and network properties and organizes the orderly contraction of diaphragm motor fibers. For example, early inspiratory diaphragmatic activation by the early coherent burst generated by high-frequency PhMNs may be necessary to prime it to overcome its initial inertia. We have also demonstrated the existence of a prominent role for local intraspinal inhibitory mechanisms in shaping phrenic pattern formation. The objective of this review is to relate and synthesize recent findings with those of previous studies with the aim of demonstrating that the phrenic nucleus is a region of active local processing, rather than a passive relay of descending inputs.

Diversity of molecularly defined spinal interneurons engaged in mammalian locomotor pattern generation.

Mapping the expression of transcription factors in the mouse spinal cord has identified ten progenitor domains, four of which are cardinal classes of molecularly defined, ventrally located interneurons that are integrated in the locomotor circuitry. This review focuses on the properties of these interneuronal populations and their contribution to hindlimb locomotor central pattern generation. Interneuronal populations are categorized based on their excitatory or inhibitory functions and their axonal projections as predictors of their role in locomotor rhythm generation and coordination. The synaptic connectivity and functions of these interneurons in the locomotor central pattern generators (CPGs) have been assessed by correlating their activity patterns with motor output responses to rhythmogenic neurochemicals and sensory and descending fibers stimulations as well as analyzing kinematic gait patterns in adult mice. The observed complex organization of interneurons in the locomotor CPG circuitry, some with seemingly similar physiological functions, reflects the intricate repertoire associated with mammalian motor control and is consistent with high transcriptional heterogeneity arising from cardinal interneuronal classes. This review discusses insights derived from recent studies to describe innovative approaches and limitations in experimental model systems and to identify missing links in current investigational enterprise.

Ascending pathways that mediate cholinergic modulation of lumbar motor activity.

Deciphering neuronal pathways that reactivate spinal central pattern generators (CPGs) and modulate the activity of spinal motoneurons in mammals in the absence of supraspinal control is important for understanding of neural control of movement and for developing novel therapeutic approaches to improve the mobility of spinal cord injury patients. Previously, we showed that the sacral and lumbar cholinergic system could potently modulate the locomotor CPGs in newborn rodents. Here, we review these and our more recent studies of sacral relay neurons with lumbar projections to the locomotor CPGs and to lumbar motoneurons and demonstrate that sacral and lumbar cholinergic components have the capacity to control the frequency of the locomotor CPGs and at the same time the motor output of the activated lumbar motoneurons during motor behavior. A model describing the suggested ascending sacro-lumbar connectivity involved in modulation of the locomotor rhythm by sacral cholinergic components is proposed and discussed. This is an article for the special issue XVth International Symposium on Cholinergic Mechanisms.

Inhibition of Parkinsonian tremor with cutaneous afferent evoked by transcutaneous electrical nerve stimulation.

BACKGROUND: Recent study suggests that tremor signals are transmitted by way of multi-synaptic corticospinal pathway. Neurophysiological studies have also demonstrated that cutaneous afferents exert potent inhibition to descending motor commands by way of spinal interneurons. We hypothesize in this study that cutaneous afferents could also affect the transmission of tremor signals, thus, inhibit tremor in patients with PD. METHODS: We tested this hypothesis by activating cutaneous afferents in the dorsal hand skin innervated by superficial radial nerve using transcutaneous electrical nerve stimulation (TENS). Eight patients with PD having tremor dominant symptom were recruited to participate in this study using a consistent experimental protocol for tremor inhibition. Resting tremor and electromyogram (EMG) of muscles in the upper extremity of these subjects with PD were recorded, while surface stimulation was applied to the dorsal skin of the hand. Fifteen seconds of data were recorded for 5 s prior to, during and post stimulation. Power spectrum densities (PSDs) of tremor and EMG signals were computed for each data segment. The peak values of PSDs in three data segments were compared to detect evidence of tremor inhibition. RESULTS: At stimulation intensity from 1.5 to 1.75 times of radiating sensation threshold, apparent suppressions of tremor at wrist, forearm and upper arm and in the EMGs were observed immediately at the onset of stimulation. After termination of stimulation, tremor and rhythmic EMG bursts reemerged gradually. Statistical analysis of peak spectral amplitudes showed a significant difference in joint tremors and EMGs during and prior to stimulation in all 8 subjects with PD. The average percentage of suppression was 61.56% in tremor across all joints of all subjects, and 47.97% in EMG of all muscles. The suppression appeared to occur mainly in distal joints and muscles. There was a slight, but inconsistent effect on tremor frequency in the 8 patients with PD tested. CONCLUSIONS: Our results provide direct evidence that tremor in the upper extremity of patients with PD can be inhibited to a large extent with evoked cutaneous reflexes via surface stimulation of the dorsal hand skin area innervated by the superficial radial nerve.

Putative spinal interneurons mediating postural limb reflexes provide a basis for postural control in different planes.

The dorsal-side-up trunk orientation in standing quadrupeds is maintained by the postural system driven mainly by somatosensory inputs from the limbs. Postural limb reflexes (PLRs) represent a substantial component of this system. Earlier we described spinal neurons presumably contributing to the generation of PLRs. The first aim of the present study was to reveal trends in the distribution of neurons with different parameters of PLR-related activity across the gray matter of the spinal cord. The second aim was to estimate the contribution of PLR-related neurons with different patterns of convergence of sensory inputs from the limbs to stabilization of body orientation in different planes. For this purpose, the head and vertebral column of the decerebrate rabbit were fixed and the hindlimbs were positioned on a platform. Activity of individual neurons from L5 to L6 was recorded during PLRs evoked by lateral tilts of the platform. In addition, the neurons were tested by tilts of the platform under only the ipsilateral or only the contralateral limb, as well as during in-phase tilts of the platforms under both limbs. We found that, across the spinal gray matter, strength of PLR-related neuronal activity and sensory input from the ipsilateral limb decreased in the dorsoventral direction, while strength of the input from the contralateral limb increased. A near linear summation of tilt-related sensory inputs from different limbs was found. Functional roles were proposed for individual neurons. The obtained data present the first characterization of posture-related spinal neurons, forming a basis for studies of postural networks impaired by injury.

The role of spinal neurons targeted by corticospinal neurons in central poststroke neuropathic pain.

BACKGROUND: Central poststroke pain (CPSP) is one of the primary sequelae following stroke, yet its underlying mechanisms are poorly understood. METHODS: By lesioning the lateral thalamic nuclei, we first established a CPSP model that exhibits mechanical and thermal hypersensitivity. Innocuous mechanical stimuli following the thalamic lesion evoked robust neural activation in somatosensory corticospinal neurons (CSNs), as well as in the deep dorsal horn, where low threshold mechanosensory afferents terminate. In this study, we used viral-based mapping and intersectional functional manipulations to decipher the role of somatosensory CSNs and their spinal targets in the CPSP pathophysiology. RESULTS: We first mapped the post-synaptic spinal targets of lumbar innervating CSNs using an anterograde trans-synaptic AAV1-based strategy and showed these spinal interneurons were activated by innocuous tactile stimuli post-thalamic lesion. Functionally, tetanus toxin-based chronic inactivation of spinal neurons targeted by CSNs prevented the development of CPSP. Consistently, transient chemogenetic silencing of these neurons alleviated established mechanical pain hypersensitivity and innocuous tactile stimuli evoked aversion linked to the CPSP. In contrast, chemogenetic activation of these neurons was insufficient to induce robust mechanical allodynia typically observed in the CPSP. CONCLUSION: The CSNs and their spinal targets are required but insufficient for the establishment of CPSP hypersensitivity. Our study provided novel insights into the neural mechanisms underlying CPSP and potential therapeutic interventions to treat refractory central neuropathic pain conditions.

Lumbar V3 interneurons provide direct excitatory synaptic input onto thoracic sympathetic preganglionic neurons, linking locomotor, and autonomic spinal systems.

Although sympathetic autonomic systems are activated in parallel with locomotion, the neural mechanisms mediating this coordination are incompletely understood. Sympathetic preganglionic neurons (SPNs), primarily located in the intermediate laminae of thoracic and upper lumbar segments (T1-L2), increase activation of tissues and organs that provide homeostatic and metabolic support during movement and exercise. Recent evidence suggests integration between locomotor and autonomic nuclei occurs within the brainstem, initiating both descending locomotor and sympathetic activation commands. However, both locomotor and sympathetic autonomic spinal systems can be activated independent of supraspinal input, in part due to a distributed network involving propriospinal neurons. Whether an intraspinal mechanism exists to coordinate activation of these systems is unknown. We hypothesized that ascending spinal neurons located in the lumbar region provide synaptic input to thoracic SPNs. Here, we demonstrate that synaptic contacts from locomotor-related V3 interneurons (INs) are present in all thoracic laminae. Injection of an anterograde tracer into lumbar segments demonstrated that 8-20% of glutamatergic input onto SPNs originated from lumbar V3 INs and displayed a somatotopographical organization of synaptic input. Whole cell patch clamp recording in SPNs demonstrated prolonged depolarizations or action potentials in response to optical activation of either lumbar V3 INs in spinal cord preparations or in response to optical activation of V3 terminals in thoracic slice preparations. This work demonstrates a direct intraspinal connection between lumbar locomotor and thoracic sympathetic networks and suggests communication between motor and autonomic systems may be a general function of the spinal cord.

Neuroplasticity and regeneration after spinal cord injury.

Spinal cord injury (SCI) is a debilitating condition with significant personal, societal, and economic burden. The highest proportion of traumatic injuries occur at the cervical level, which results in severe sensorimotor and autonomic deficits. Following the initial physical damage associated with traumatic injuries, secondary pro-inflammatory, excitotoxic, and ischemic cascades are initiated further contributing to neuronal and glial cell death. Additionally, emerging evidence has begun to reveal that spinal interneurons undergo subtype specific neuroplastic circuit rearrangements in the weeks to months following SCI, contributing to or hindering functional recovery. The current therapeutic guidelines and standards of care for SCI patients include early surgery, hemodynamic regulation, and rehabilitation. Additionally, preclinical work and ongoing clinical trials have begun exploring neuroregenerative strategies utilizing endogenous neural stem/progenitor cells, stem cell transplantation, combinatorial approaches, and direct cell reprogramming. This review will focus on emerging cellular and noncellular regenerative therapies with an overview of the current available strategies, the role of interneurons in plasticity, and the exciting research avenues enhancing tissue repair following SCI.

c-Maf-positive spinal cord neurons are critical elements of a dorsal horn circuit for mechanical hypersensitivity in neuropathy.

Corticospinal tract (CST) neurons innervate the deep spinal dorsal horn to sustain chronic neuropathic pain. The majority of neurons targeted by the CST are interneurons expressing the transcription factor c-Maf. Here, we used intersectional genetics to decipher the function of these neurons in dorsal horn sensory circuits. We find that excitatory c-Maf (c-MafEX) neurons receive sensory input mainly from myelinated fibers and target deep dorsal horn parabrachial projection neurons and superficial dorsal horn neurons, thereby connecting non-nociceptive input to nociceptive output structures. Silencing c-MafEX neurons has little effect in healthy mice but alleviates mechanical hypersensitivity in neuropathic mice. c-MafEX neurons also receive input from inhibitory c-Maf and parvalbumin neurons, and compromising inhibition by these neurons caused mechanical hypersensitivity and spontaneous aversive behaviors reminiscent of c-MafEX neuron activation. Our study identifies c-MafEX neurons as normally silent second-order nociceptors that become engaged in pathological pain signaling upon loss of inhibitory control.

Neural Network Models for Spinal Implementation of Muscle Synergies.

Muscle synergies have been proposed as functional modules to simplify the complexity of body motor control; however, their neural implementation is still unclear. Converging evidence suggests that output projections of the spinal premotor interneurons (PreM-INs) underlie the formation of muscle synergies, but they exhibit a substantial variation across neurons and exclude standard models assuming a small number of unitary "modules" in the spinal cord. Here we compared neural network models for muscle synergies to seek a biologically plausible model that reconciles previous clinical and electrophysiological findings. We examined three neural network models: one with random connections (non-synergy model), one with a small number of spinal synergies (simple synergy model), and one with a large number of spinal neurons representing muscle synergies with a certain variation (population synergy model). We found that the simple and population synergy models emulate the robustness of muscle synergies against cortical stroke observed in human stroke patients. Furthermore, the size of the spinal variation of the population synergy matched well with the variation in spinal PreM-INs recorded in monkeys. These results suggest that a spinal population with moderate variation is a biologically plausible model for the neural implementation of muscle synergies.

Harnessing Spinal Interneurons for Spinal Cord Repair.

Interest in spinal interneurons (SpINs), their heterogeneity in the naive spinal cord and their varying responses to central nervous system injury or disease has been steadily increasing. Our recent review on this topic highlights the vast phenotypic heterogeneity of SpINs and the efforts being made to better identify and classify these neurons. As our understanding of SpIN phenotype, connectivity, and neuroplastic capacity continues to expand, new therapeutic targets are being revealed and novel treatment approaches developed to harness their potential. Here, we expand on that initial discussion and highlight how SpINs can be used to develop advanced, targeted cellular therapies and personalized medicines.

Long-Term Cultures of Spinal Cord Interneurons.

Spinal cord interneurons (SpINs) are highly diverse population of neurons that play a significant role in circuit reorganization and spontaneous recovery after spinal cord injury. Regeneration of SpIN axons across rodent spinal injuries has been demonstrated after modification of the environment and neurotrophin treatment, but development of methods to enhance the intrinsic regenerative ability of SpINs is needed. There is a lack of described in vitro models of spinal cord neurons in which to develop new regeneration treatments. For this reason, we developed a new model of mouse primary spinal cord neuronal culture in which to analyze maturation, morphology, physiology, connectivity and regeneration of identified interneurons. Isolated from E14 mice, the neurons mature over 15 days in vitro, demonstrated by expression of maturity markers, electrophysiological patch-clamp recordings, and formation of synapses. The neurons express markers of SpINs, including Tlx3, Lmx1b, Lbx1, Chx10, and Pax2. The neurons demonstrate distinct morphologies and some form perineuronal nets in long-term cultivation. Live neurons in various maturation stages were axotomized, using a 900 nm multiphoton laser and their fate was observed overnight. The percentage of axons that regenerated declined with neuronal maturity. This model of SpINs will be a valuable tool in future regenerative, developmental, and functional studies alongside existing models using cortical or hippocampal neurons.

A transgenic mouse embryonic stem cell line for puromycin selection of V0V interneurons from heterogenous induced cultures.

BACKGROUND: Spinal interneurons (INs) relay sensory and motor control information between the brain and body. When this relay circuitry is disrupted from injury or disease, it is devastating to patients due to the lack of native recovery in central nervous system (CNS) tissues. Obtaining a purified population of INs is necessary to better understand their role in normal function and as potential therapies in CNS. The ventral V0 (V0V) INs are excitatory neurons involved in locomotor circuits and are thus of interest for understanding normal and pathological spinal cord function. To achieve scalable amounts of V0V INs, they can be derived from pluripotent sources, such as mouse embryonic stem cells (mESCs), but the resultant culture is heterogenous, obscuring the specific role of V0V INs. This study generated a transgenic mESC line to enrich V0V INs from induced cultures to allow for a scalable, enriched population for future in vitro and in vivo studies. METHODS: The transgenic Evx1-PAC mESC line was created by CRISPR-Cas9-mediated insertion of puromycin-N-acetyltransferase (PAC) into the locus of V0V IN marker Evx1. Evx1 and PAC mRNA expression were measured by qPCR. Viability staining helped establish the selection protocol for V0V INs derived from Evx1-PAC mESCs inductions. Immunostaining was used to examine composition of selected inductions. Cultures were maintained up to 30 days to examine maturation by expression of mature/synaptic markers, determined by immunostaining, and functional activity in co-cultures with selected motor neurons (MNs) and V2a INs on microelectrode arrays (MEAs). RESULTS: V0V IN inductions were best selected with 4 µg/mL puromycin on day 10 to 11 and showed reduction of other IN populations and elimination of proliferative cells. Long-term selected cultures were highly neuronal, expressing neuronal nuclear marker NeuN, dendritic marker MAP2, pre-synaptic marker Bassoon, and glutamatergic marker VGLUT2, with some cholinergic VAChT-expressing cells. Functional studies on MEAs showed that co-cultures with MNs or MNs plus V2a INs created neuronal networks with synchronized bursting. CONCLUSIONS: Evx1-PAC mESCs can be used to purify V0V IN cultures for largely glutamatergic neurons that can be used in network formation studies or for rodent models requiring transplanted V0V INs.