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The spinal cord is home to the intrinsic networks for locomotion. An animal in which the spinal cord has been fully severed from the brain can still produce rhythmic, patterned locomotor movements as long as some excitatory drive is provided, such as physical, pharmacological, or electrical stimuli. Yet it remains a challenge to define the underlying circuitry that produces these movements because the spinal cord contains a wide variety of neuron classes whose patterns of interconnectivity are still poorly understood. Computational models of locomotion accordingly rely on untested assumptions about spinal neuron network element identity and connectivity. In this review, we consider the classes of spinal neurons, their interconnectivity, and the significance of their circuit connections along the long axis of the spinal cord. We suggest several lines of analysis to move toward a definitive understanding of the spinal network. [ABSTRACT FROM AUTHOR]

Brachial plexus root avulsions cause debilitating upper limb paralysis. Short-term neuroprotective treatments have reported preservation of motor neurons and function in model animals while reports of long-term benefits of such treatments are scarce, especially the morphological sequelae. This morphological study investigated the long-term suppression of c-Jun- and neuronal nitric oxide synthase (nNOS) (neuroprotective treatments for one month) on the motor neuron survival, ultrastructural features of lower motor neurons, and forelimb function at six months after brachial plexus roots avulsion. Neuroprotective treatments reduced oxidative stress and preserved ventral horn motor neurons at the end of the 28-day treatment period relative to vehicle treated ones. Motor neuron sparing was associated with suppression of c-Jun, nNOS, and pro-apoptotic proteins Bim and caspases at this time point. Following 6 months of survival, neutral red staining revealed a significant loss of most of the motor neurons and ventral horn atrophy in the avulsed C6, 7, and 8 cervical segments among the vehicle-treated rats (n = 4). However, rats that received neuroprotective treatments c-Jun JNK inhibitor, SP600125 (n = 4) and a selective inhibitor of nNOS, 7-nitroindazole (n = 4), retained over half of their motor neurons in the ipsilateral avulsed side compared. Myelinated axons in the avulsed ventral horns of vehicle-treated rats were smaller but numerous compared to the intact contralateral ventral horns or neuroprotective-treated groups. In the neuroprotective treatment groups, there was the preservation of myelin thickness around large-caliber axons. Ultrastructural evaluation also confirmed the preservation of organelles including mitochondria and synapses in the two groups that received neuroprotective treatments compared with vehicle controls. Also, forelimb functional evaluation demonstrated that neuroprotective treatments improved functional abilities in the rats. In conclusion, neuroprotective treatments aimed at suppressing degenerative c-Jun and nNOS attenuated apoptosis, provided long-term preservation of motor neurons, their organelles, ventral horn size, and forelimb function.

It is hypothesized that the central integrative state (CIS) of the spinal cord's ventral horn depends upon myriad inputs from primary afferentation secondary to gravitational perturbation of joint mechanoreceptors. Further, the current perspective of manual muscle testing (MMT) and diagnostic muscle testing (DMT) research—each being universially accepted by the medical community—tends to focus exclusively on the conditional state of the muscle and/or the α-motoneuron affecting that muscle. Diagnostic muscle testing, however, is mainly an assessment tool for functional disorders. Moreover, since the human brain is receptor dependent, the entirety of the human neurological environment extends from the primary peripheral receptor and its subsequent action potential to the dosral root ganglion, dorsal horn of the spinal cord, and rostral neuroaxial centers like the cerebellum, midbrain, thalamus, and cortex—and ultimately to the effector, including all components in between. The human nervous system concerns itself with whole body reactions, not individual muscles. The premise here is that the highest primary afferentation is secondary to mechanoreceptor deformation after gravitational perturbation. The goal of treatment is to facilitate the patient's highest level of human performance by recognizing and treating discrete pathology while avoiding an iatrogenic effect. Merely focusing on the α-motoneuron and/or its influence on the effector generates a myopic understanding that loses appreciation of efferent autonomic drive and structural performance. An individual muscle focus does not recognize the complexity of homologous involvement. [ABSTRACT FROM AUTHOR]

Dendrites play irreplaceable roles in the nerve conduction pathway and are vulnerable to various insults. Peripheral axotomy of motor neurons results in the retraction of dendritic arbors, and the dendritic arbor can be re-expanded when reinnervation is allowed. RhoA is a target that regulates the cytoskeleton and promotes neuronal survival and axon regeneration. However, the role of RhoA in dendrite degeneration and regeneration is unknown. In this study, we explored the potential role of RhoA in dendrites. A line of motor neuronal RhoA conditional knockout mice was developed by crossbreeding HB9 Cre+ mice with RhoA flox/flox mice. We established two models for assaying dendrite degeneration and regeneration, in which the brachial plexus was transection or crush injured, respectively. We found that at 28 days after brachial plexus transection, the density, complexity, and structural integrity of dendrites in the ventral horn of the spinal cord of RhoA conditional knockout mice were slightly decreased compared with that in Cre mice. Dendrites underwent degeneration at 7 and 14 days after brachial plexus transection and recovered at 28-56 days. The density, complexity, and structural integrity of dendrites in the ventral horn of the spinal cord of RhoA conditional knockout mice recovered compared with results in Cre mice. These findings suggest that RhoA knockout in motor neurons attenuates dendrite degeneration and promotes dendrite regeneration after peripheral nerve injury., Competing Interests: None

Motor neurons are the site of action for several neurological disorders and paralytic toxins, with cell bodies located in the ventral horn (VH) of the spinal cord along with interneurons and support cells. Microelectrode arrays (MEAs) have emerged as a high content assay platform for mechanistic studies and drug discovery. Here, we explored the spontaneous and evoked electrical activity of VH cultures derived from embryonic mouse spinal cord on multi-well plates of MEAs. Primary VH cultures from embryonic day 15–16 mice were characterized by expression of choline acetyltransferase (ChAT) by immunocytochemistry. Well resolved, all-or-nothing spontaneous spikes with profiles consistent with extracellular action potentials were observed after 3 days in vitro, persisting with consistent firing rates until at least day in vitro 19. The majority of the spontaneous activity consisted of tonic firing interspersed with coordinated bursting across the network. After 5 days in vitro, spike activity was readily evoked by voltage pulses where a minimum amplitude and duration required for excitation was 300 mV and 100 μs/phase, respectively. We characterized the sensitivity of spontaneous and evoked activity to a host of pharmacological agents including AP5, CNQX, strychnine, ω-agatoxin IVA, and botulinum neurotoxin serotype A (BoNT/A). These experiments revealed sensitivity of the cultured VH to both agonist and antagonist compounds in a manner consistent with mature tissue derived from slices. In the case of BoNT/A, we also demonstrated intoxication persistence over an 18-day period, followed by partial intoxication recovery induced by N- and P/Q-type calcium channel agonist GV-58. In total, our findings suggest that VH cultures on multi-well MEA plates may represent a moderate throughput, high content assay for performing mechanistic studies and for screening potential therapeutics pertaining to paralytic toxins and neurological disorders.

Motor neurons are the site of action for several neurological disorders and paralytic toxins, with cell bodies located in the ventral horn (VH) of the spinal cord along with interneurons and support cells. Microelectrode arrays (MEAs) have emerged as a high content assay platform for mechanistic studies and drug discovery. Here, we explored the spontaneous and evoked electrical activity of VH cultures derived from embryonic mouse spinal cord on multi-well plates of MEAs. Primary VH cultures from embryonic day 15-16 mice were characterized by expression of choline acetyltransferase (ChAT) by immunocytochemistry. Well resolved, all-or-nothing spontaneous spikes with profiles consistent with extracellular action potentials were observed after 3 days in vitro, persisting with consistent firing rates until at least day in vitro 19. The majority of the spontaneous activity consisted of tonic firing interspersed with coordinated bursting across the network. After 5 days in vitro, spike activity was readily evoked by voltage pulses where a minimum amplitude and duration required for excitation was 300 mV and 100 ms/phase, respectively. We characterized the sensitivity of spontaneous and evoked activity to a host of pharmacological agents including AP5, CNQX, strychnine, &-agatoxin IVA, and botulinum neurotoxin serotype A (BoNT/A). These experiments revealed sensitivity of the cultured VH to both agonist and antagonist compounds in a manner consistent with mature tissue derived from slices. In the case of BoNT/A, we also demonstrated intoxication persistence over an 18-day period, followed by partial intoxication recovery induced by N- and P/Q-type calcium channel agonist GV-58. In total, our findings suggest that VH cultures on multi-well MEA plates may represent a moderate throughput, high content assay for performing mechanistic studies and for screening potential therapeutics pertaining to paralytic toxins and neurological disorders. [ABSTRACT FROM AUTHOR]

Calcitonin gene-related peptide (CGRP) is a 37-amino-acid neuropeptide, synthesized by alternative splicing of calcitonin gene mRNA. CGRP is characteristically distributed in the nervous system, and its function varies depending on where it is expressed. To reveal developmental formation of the CGRP network and its function in neuronal maturation, we examined the immunohistochemical localization of CGRP in the developing mouse cervical spinal cord and dorsal root ganglion. CGRP immunolabeling (IL) was first detected in motor neurons on E13, and in ascending axons of the posterior funiculus and DRG neurons on E14. CGRP-positive sensory axon fibers entered Laminae I and II on E16, and Laminae I through IV on E18. The intensity of the CGRP-IL gradually increased in both ventral and dorsal horns during embryonic development, but markedly decreased in the ventral horn after birth. These results suggest that CGRP is expressed several days after neuronal settling and entry of sensory fibers, and that the CGRP network is formed in chronological and sequential order. Furthermore, because CGRP is markedly expressed in motor neurons when axons are vastly extending and innervating targets, CGRP may also be involved in axonal elongation and synapse formation during normal development. [ABSTRACT FROM AUTHOR]

The aim of the present work was to study the topography and structure of 28-kDa calbindin (CB)-containing interneurons in the ventral horn and intermediate zone of the gray matter at levels T and L of the spinal cord (SC). Experiments were performed in adult female Wistar rats ( n = 4) using immunohistochemical and morphometric methods. Cryostat transverse sections of the SC of thickness 14 μm were studied. The intermediate zone of the SC was found to contain two subpopulations of CB-immunoreactive (IR) interneurons in lamina VII in both segments: sympathetic preganglionic neurons and 'partition' cells. The ventral horn also contained two subpopulations of CB-IR interneurons: Renshaw cells in lamina IX in T and lamina VII in L and large interneurons in lamina V in T and lamina VII in L. The only differences between segments were in the larger numbers of Renshaw cells and partition cells in L. The mean cross-sectional areas of CB-IR Renshaw cells and autonomic preganglionic neurons were greater in T, while large interneurons of the ventral horn and partition cells were larger in L. [ABSTRACT FROM AUTHOR]

Introduction Hyperglycemia is the main cause of diabetic complications, contributing to a widespread degeneration of the nervous system. Nevertheless, the main focus has been the sensory neurons because of neuropathic pain, while the impairments associated with the spinal cord and motor deficits, mostly of those initiated at early stages of the disease, have been poorly investigated. In this way, the present study used the nonobese diabetic mouse model to evaluate the microenvironment around motoneurons at ventral horn of the spinal cord, following prolonged hyperglycemia. Methods Adult female mice were divided into two groups: spontaneously diabetic ( n = 33) and nondiabetic ( n = 26). Mice were considered hyperglycemic when blood glucose surpassed 400 mg/ dL. Following 2 weeks from that stage, part of the animals was euthanized and the lumbar intumescences were obtained and processed for immunohistochemistry and transmission electron microscopy. For immunohistochemistry, the antibodies used for integrated density of pixels quantification were anti-synaptophysin, anti- GFAP, and anti-Iba1. The functional analysis was monitored with the walking track test (CatWalk system) during 4 weeks. Results The results revealed significant motor impairment in diabetic animals in comparison to the control group. Such loss of motor control correlated with a significant reduction in presynaptic terminals apposed to the motoneurons. Nevertheless, there were no significant changes in glial reaction in the spinal cord. Conclusion Overall, the results herein revealed central nervous system changes at early stages of the disease that may in turn contribute to the motor deficit. Such changes open a new window of investigation in early stages of diabetes to better comprehend motor impairment as a long-term complication of the disease. [ABSTRACT FROM AUTHOR]

Nicotinic acetylcholine receptors (nAChRs) are distributed widely in the central nervous system and play important roles in higher brain functions, including learning, memory, and recognition. However, functions of the cholinergic system in spinal motoneurons remain poorly understood. In this study, we investigated the actions of presynaptic and postsynaptic nAChRs in spinal ventral horn neurons by performing whole-cell patch-clamp recordings on lumbar slices from male rats. The application of nicotine or acetylcholine generated slow inward currents and increased the frequency and amplitude of spontaneous excitatory postsynaptic currents (sEPSCs). Slow inward currents by acetylcholine or nicotine were not inhibited by tetrodotoxin (TTX) or glutamate receptor antagonists. In the presence of TTX, the frequency and amplitude of miniature excitatory postsynaptic currents (mEPSCs) were also increased by acetylcholine or nicotine. A selective α4β2 nicotinic receptor antagonist, dihydro-β-erythroidine hydrobromide (DhβE), significantly decreased nicotine-induced inward currents without affecting the enhancement of sEPSCs and mEPSCs. In addition, a selective α7 nicotinic receptor antagonist, methyllycaconitine, did not affect either nicotine-induced inward currents or the enhancement of sEPSCs and mEPSCs. These results suggest that α4β2 AChRs are localized at postsynaptic sites in the spinal ventral horn, non-α4β2 and non-α7 nAChRs are located presynaptically, and nAChRs enhance excitatory synaptic transmission in the spinal ventral horn. [ABSTRACT FROM AUTHOR]

Highlights: [•] GlyT2 inhibition potently reduced spontaneous action potential firing activity in the spinal ventral horn. [•] The efficacy of GlyT2 inhibition to depress network activity of ventral horn neurons reached approximately 90%. [•] GlyT2 inhibition by ALX 1393 induced a strychnine-sensitive glycinergic tonic current. [•] Silencing neuronal action potential activity by GlyT2 inhibition might be a novel principle to inhibit locomotor circuits. [ABSTRACT FROM AUTHOR]

Highlights: [•] The TARP isoforms γ-2, γ-3, γ-4, γ-7 and γ-8 were detected in the rat spinal cord. [•] γ-4, γ-7 and γ-8 showed distinct laminar distribution in the spinal gray matter. [•] γ-8 and γ-7, and to a lesser extent γ-2, were observed in primary afferent synapses. [•] γ-4 and γ-7 but not γ-8 were expressed in astrocytes and oligodendrocytes. [ABSTRACT FROM AUTHOR]

Abstract: Embryonic spinal cord motor neurons (MNs) can be maintained in vitro for weeks with a cocktail of trophic factors and muscle-derived factors under serum-containing conditions. Here we investigated the beneficial effects of muscle-derived factors in the form of muscle-conditioned medium (MCM) on the survival and neurite outgrowth of adult rat spinal cord MNs under serum-free conditions. Ventral horn dissociated cell cultures from the cervical enlargement were maintained in the presence of one or more of the following factors: brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), a cell permeant cyclic adenosine-3′,5′-monophosphate (cAMP) analog and MCM. The cell cultures were immunostained with several antibodies recognizing a general neuronal marker the microtubule-associated protein 2 (MAP2) and either one or more motor neuronal markers: the non-phosphorylated neurofilament heavy isoform (SMI32), the transcription factors HB9 and Islet-1 and the choline acetyl transferase. We found that treatment with MCM together with the cAMP analog was sufficient to promote selective survival and neurite outgrowth of adult spinal cord MNs. These conditions can be used to maintain adult spinal cord MNs in dissociated cultures for several weeks and may have therapeutic potential following spinal cord injury or motor neuropathies. More studies are necessary to evaluate how MCM and the cAMP analog act in synergy to promote the survival and neurite outgrowth of adult MNs. [Copyright &y& Elsevier]