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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.

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., (Copyright © 2015 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.)

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.

Background: Strychnine-sensitive glycine receptors are activated by glycine and facilitate chloride influx into neurons. Glycinergic transmission might be either mediated by synaptic or extrasynaptic glycine receptors. While phasic neurotransmission is provided by a synaptic pathway, activation of extrasynaptic glycine receptors induces tonic inhibition. The glycine transporter 2 (GlyT2) regulates the uptake of glycine into presynaptic boutons. It is not determined yet, whether inhibition of GlyT2 by ALX 1393 can produce inhibition of spinal motoric networks and, whether phasic or tonic glycinergic inhibition is mostly enhanced., Methods: We investigated the effect of ALX 1393 on spontaneous action potential firing activity by extracellular recordings in the ventral horn area of organotypic spinal cultures. Additionally, using the whole-cell patch-clamp technique, we defined the influence of GlyT2 inhibition on tonic and phasic glycinergic transmission in commissural interneurons of the ventral horn., Results: GlyT2 inhibition by ALX 1393 potently reduced neuronal action potential activity in a concentration-dependent manner (n=211). The half maximal effect of ALX 1393 was observed at 100 ± 31 nM. Moreover, 88.3 ± 2.6% of the action potential activity was suppressed at 1 μM. Whole-cell patch-clamp recordings unveiled that ALX 1393 (200 nM) induced a tonic current (-45.7 ± 11.6 pA, n=5) that was significantly reversed by application of the competitive glycine receptor antagonist strychnine. Contrastingly, phasic glycinergic transmission was not augmented by GlyT2 inhibition (charge transferred per time period for control conditions: 1.1 ± 0.1 pC, n=7, for ALX 1393: 0.9 ± 0.2 pC, n=7, p>0.05)., Conclusion: GlyT2 inhibition induced glycinergic tonic currents, which might be the underlying mechanism for the observed suppression of spontaneous action potential activity by ALX 1393 in the spinal ventral horn. Silencing neuronal action potential activity by blocking GlyT2 might be a novel principle to inhibit locomotor circuits in the ventral horn area and to induce muscle relaxation., (Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.)

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]

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]

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]

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]

Abstract: γ-aminobutyric acid (GABA) is utilized in the peripheral as well as central nervous system. In this study, fibers immunoreactive for 67 kDa isoform of glutamic acid decarboxylase (GAD67), an enzyme which synthesizes GABA, were found to terminate in the intercapsular region of muscle spindles of the upper limb. GABA-containing fibers were also found in the ventral roots of C5 to T5 spinal segments, brachial plexus, and radial nerve. These fibers were thin and immunoreactive for choline-acetyl transferase (ChAT). After transection of the brachial plexus, GABA immunoreactivity disappeared completely in the ipsilateral triceps brachii muscle (TBM). After the injection of fluorogold into the TBM, some retrogradely labeled medium-sized neurons were positive for GAD67, but not VGAT mRNA. All these observations clearly indicate that GABA-containing γ-motoneurons in the lower cervical spinal cord send their fibers to muscle spindles in the upper extremities. Since we detected neither GABAA nor GABAB receptors in the TBM by RT-PCR, the function of the GABA-containing γ-motoneurons remains unclear. [Copyright &y& Elsevier]

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Abstract: One class of spinal interneurons, the Renshaw cells, is able to discharge at very high frequencies in adult mammals. Neuronal firing at such high frequencies requires voltage-gated potassium channels to rapidly repolarize the membrane potential after each action potential. We sought to establish the pattern of expression of calbindin and potassium channels with Kv3.1b and Kv3.2 subunits in Renshaw cells at different developmental stages of postnatal mice. The pattern of expression of calbindin changed dramatically during early postnatal development. An adult pattern of calbindin reactive neurons started to emerge from postnatal day 10 to postnatal day 14, with cells in laminae I and II of superficial dorsal horn and the ventral lamina VII. Renshaw cells were identified immunohistochemically by their expression of calbindin and their location in the ventral horn of the spinal cord. Western blot results of the lumbar spinal cord showed that Kv3.1b expression became faintly evident from postnatal day 10, reached a maximum at postnatal day 21 and was maintained through postnatal day 49. Double labeling results showed that all Renshaw cells expressed Kv3.1b weakly from postnatal day 14, and strongly at postnatal day 21. Western blot results showed that Kv3.2 expression became detectable in the lumbar cord from postnatal day 12, and increased steadily until reaching an adult level at postnatal day 28. In contrast to the Kv3.1b results, Kv3.2 was not expressed in Renshaw cells, although some neurons located at laminae VIII and VI expressed Kv3.2. We conclude that Renshaw cells express Kv3.1b but not Kv3.2 from postnatal day 14. [Copyright &y& Elsevier]

Acetylcholine is the neurotransmitter of somatic and autonomic motor systems of the spinal cord. However, there are also intrinsic cholinergic systems which have modulatory functions. Modulatory functions have also been assigned to nitric oxide (NO). Acetylcholine is synthesized by choline acetyltransferase and NO by nitric oxide synthase, which is a NADPH diaphorase. The distribution of both enzymes in the mammalian spinal cord is well known. However, there is a lack of comparative data in avian species. Therefore, the distribution of both enzymes in the spinal cord of the pigeon was studied using histochemical and immunohistochemical methods. Aside from somatic motor neurons and autonomic preganglionic neurons choline acetyltransferase-immunoreactivity was found throughout the spinal cord in lamina III of the superficial dorsal horn and near the central canal. The location of choline acetyltransferase-positive preganglionic neurons in the centrally located column of Terni and the lack of an intermediolateral column typical of the mammalian spinal cord can be confirmed. In lumbosacral segments the axons of centrally located cholinergic neurons crossed to the contralateral side to form a tract in the ventral funiculus, which then innervates the contralateral grey substance. A dense band of NADPH diaphorase staining was found in lamina II and in centrally located neurons of all segments. Part of the centrally located neurons double-labelled for choline acetyltransferase and NADPH diaphorase. In contrast to mammals, preganglionic neurons labelled only weakly for NADPH diaphorase. Altogether, despite the divergent evolution of both classes of vertebrate intrinsic modulatory choline acetyltransferase and NADPH diaphorase systems of birds seem to be largely similar to those of the mammalian spinal cord. [ABSTRACT FROM AUTHOR]

The primary motor cortex of cats, monkeys, and humans has distinct rostral and caudal subregions. In the cat, projections from the caudal subregion terminate predominantly in laminae 4–6 and, from the rostral subregion, in laminae 6–8. The purpose of this study was to determine if these distinctive termination patterns are present during early postnatal development, when corticospinal axons are establishing connections with spinal neurons, or if there was postnatal refinement of the distribution of terminations. We used the anterograde tracer biotinylated dextran amine (BDA) to label selectively projections from the two subregions in immature (postnatal days 25, 35 at time of analysis) and mature animals. We compared the distribution of spinal gray matter labeling from caudal and rostral motor cortex. In immature animals, substantial bilateral terminations were present after tracer injection into either subregion. Partial axon reconstructions revealed that individual axons terminated bilaterally. The dorso-ventral laminar distribution of contralateral labeling after caudal motor cortex injections was significantly more extensive for immature than mature animals. In immature animals, most of the labeling was present in laminae 5–7 (dorsal portion), with lesser amounts in laminae 1–4 and 7 (ventral portion), 8, and 9. In mature animals, there were significant reductions in the amount of label in laminae 7–9, resulting in contraction of the labeled territory. The distribution of dorsal horn–ventral horn labeling shifted from 41% and 59% in immature animals to 77% and 23% in maturity. The distribution of contralateral labeling after rostral motor cortex injections also was different in immature and mature animals, but the changes were less extensive than for the caudal motor cortex. In immature animals, the distribution of labeling was similar to that after caudal motor cortex injections in animals of the same age. In mature animals, there was a significant reduction in the amount of labeling in laminae 1–4 and a smaller reduction in 7 (ventral)–9. The overall dorsal horn–ventral horn distribution, however, remained largely unchanged, from 40% and 60% in immature animals to 44% and 56% in maturity. In immature animals after rostral motor cortex injection, corticospinal terminations were present within the lateral motor nuclei. Thus, the distinctive spinal termination patterns of caudal and rostral motor cortex in maturity each reflected postnatal refinement of the distribution of axon terminations. It is plausible that the dorso-ventral refinement of corticospinal terminations relies on activity-dependent competition between caudal and rostral motor cortex, similar to activity-dependent refinement of the laterality of terminations. Our results, however, suggest that the developmental program for achieving corticospinal connectional specificity by the two subregions is different because there was large-scale refinement of the terminations of caudal motor cortex but only local refinement of rostral motor cortex terminations. [ABSTRACT FROM AUTHOR]

We used anterograde transport of WGA-HRP to examine the topography of corticospinal projections from the forelimb areas within the rostral and caudal motor cortex subregions in the cat. We compared the pattern of these projections with those from the somatic sensory cortex. The principal finding of this study was that the laminar distribution of projections to the contralateral gray matter from the two motor cortex subregions was different. The rostral motor cortex projected preferentially to laminae VI-VIII, whereas caudal motor cortex projected primarily to laminae IV-VI. Confirming earlier findings, somatic sensory cortex projected predominantly to laminae I-VI inclusive. We found that only rostral motor cortex projected to territories in the rostral cervical cord containing propriospinal neurons of cervical spinal segments C3-4 and, in the cervical enlargement, to portions presumed to contain Ia inhibitory interneurons. We generated contour maps of labeling probability on averaged segmental distributions of anterograde labeling for all analyzed sections using the same algorithm. For rostral motor cortex, heaviest label in the dorsal part of lamina VII in the contralateral cord was consistently located in separate medial and lateral zones. In contrast, no consistent differences in the mediolateral location of label was noted for caudal motor cortex. To summarize, laminae I-III received input only from the somatic sensory cortex, while laminae IV-V received input from both somatic sensory and caudal motor cortex. Lamina VI received input from all cortical fields examined. Laminae VII-IX received input selectively from the rostral motor cortex. For motor cortex, our findings suggest that projections from the two subregions comprise separate descending pathways that could play distinct functional roles in movement control and sensorimotor integration. [ABSTRACT FROM AUTHOR]

It has been thought that the ventral motor column in the rat spinal cord is virtually free of dopaminergic fibers. However, a new dopaminergic terminal plexus was visualized at all spinal levels in the ventral horn using electron as well as light microscopic immunohistochemistry. [ABSTRACT FROM AUTHOR]

Substance P (SP)-and 5-hydroxytryptamine (5-HT)-containing presynaptic boutons in the cervical ventral horn were studied in chicken, hamster, rat and monkey spinal cords, using PAP and protein A-gold double-labeling techniques in conjunction with monoclonal antibodies. In the chicken, the PAP method demonstrated that SP-immunoreactive boutons contained large spherical dense-cored vesicles (DCVs) whereas 5-HT-immunoreactive boutons displayed both elongated and spherical DCVs. Using the protein A-gold double-labeling technique, 10-nm gold particles for SP were localized over the spherical DCV-containing boutons whereas 15-nm gold particles for 5-HT were localized on elongated DCV-containing boutons. On the other hand, in the other species investigated, both SP-and 5-HT-immunoreactive boutons had similar morphological features as shown by the PAP method; both contained elongated and spherical DCVs. The two differentsized gold particles, each of which labeled either 5-HT or SP, were found together over DCVs in a single bouton. These results indicate that 5-HT and SP are contained in different presynaptic boutons in the chicken, although in the hamster, rat and Japanese macaque, the two neurotransmitters/modulators coexist in the same DCVs in a single bouton. Species differences have thus been demonstrated for the coexistence of 5-HT and SP in the spinal ventral horn. [ABSTRACT FROM AUTHOR]

In our previous in situ hybridization study in the adult rat spinal cord we demonstrated that only neurons express mRNAs for acetylcholinesterase (AChE; EC 3.1.1.7.) and butyrylcholinesterase (BuChE; EC 3.1.1.8.) in this CNS region; no staining was detected for these two messages in the glia cells. Here we used immunocytochemical staining for choline acetyltransferase (ChAT; EC 2.3.1.6.) to answer the, as yet only briefly approached, question whether the cells expressing the messages for both cholinesterases are cholinergic. All neurons expressing mRNAs for AChE and BuChE were found to be ChAT positive. This finding supports the previous proposal that BuChE modulates cholinergic transmission in AChE knockout mice. [ABSTRACT FROM AUTHOR]

Although the role of developmental apoptosis in shaping the complement and connectivity of sensory and motoneurons is well documented, the extent to which cell death affects the 13 cardinal classes of spinal interneurons is unclear. Using a series of genetic manipulations in vivo, we demonstrate for the first time a differential pattern of developmental apoptosis in molecularly identified spinal interneuron populations, and implicate the adhesion molecule family encoded by the 22-member protocadherin-γ (Pcdh-γ) gene cluster in its control. In constitutive Pcdh-γ null mouse embryos, many interneuron populations undergo increased apoptosis, but to differing extents: for example, over 80% of En1-positive V1 neurons are lost, whereas only 30% of Chx10-positive V2a neurons are lost and there is no reduction in the number of V1-derived Renshaw cells. We show that this represents an exacerbation of a normal, underlying developmental pattern: the extent of each population's decrease in Pcdh-γ mutants is precisely commensurate both with the extent of its loss during normal embryogenesis and with the extent of its increase in Bax-/- mice, in which apoptosis is genetically blocked. Interneuron apoptosis begins during the first wave of synaptogenesisis in the spinal cord, occurring first among ventral populations (primarily between E14 and E17), and only later among dorsal populations (primarily after P0). Utilizing a new, conditional Pcdh-γ mutant allele, we show that the γ-Pcdhs can promote survival non-cell-autonomously: mutant neurons can survive if they are surrounded by normal neurons, and normal neurons can undergo apoptosis if they are surrounded by mutant neurons. [ABSTRACT FROM AUTHOR]