CircRNA_01477 influences axonal growth via regulating miR-3075/FosB/Stat3 axis.

Circular RNAs (circRNAs) are important for the development and regeneration of the nervous system. We investigated the differential expression profiles of circRNA induced by spinal cord injury and reported that circRNA_01477 facilitates spinal astrocyte proliferation and migration after injury in rats. In this study, we further clarified the function and possible mechanism of action of circRNA_01477 in neurons. Fluorescence in situ hybridization assay revealed that circRNA_01477 is mainly localized in the neuronal cytoplasm. Knockdown of circRNA_01477 significantly increased axonal length. The circRNA_01477/microRNAs (miRNA)/messenger RNA (mRNA) interaction network was investigated using RNA sequencing. miRNA-3075 showed a remarkable increase after circRNA_01477 depletion, and either overexpression of miRNA-3075 or downregulation of its target gene FosB significantly promoted axonal growth. Luciferase reporter assay showed that miRNA-3075 could directly bind to the 3'UTR of FosB and negatively regulated FosB transcription. Dual silencing of circRNA_01477 and miR-3075 revealed that miR-3075 inhibition rescued the increased axon length caused by siCircRNA_01477. Finally, we verified that the Stat3 pathway was activated after FosB protein depletion in rat spinal neurons, while the NF-κB pathway was not altered. In summary, our study is the first to report that circRNA_01477 contributes to axon growth by functioning as miRNA sponge by regulating the miRNA-3075/FosB/Stat3 axis.

Downregulation of microRNA­29c reduces pain after child delivery by activating the oxytocin­GABA pathway.

A significant decrease in the expression of spinal microRNA­29c (miR­29c), which is responsible for the regulation of oxytocin receptor (OXTR) expression, was observed in nerve injury pain during childbirth. The present study investigates whether spinal miR­29c could be a potential target for the treatment of pain, via the oxytocin (OT)­Î³­aminobutyric acid (GABA) pathway. A spared nerve injury (SNI) rat model was established to induce neuropathic pain, simulating hyperalgesia. Spinal neurons were treated with OT to mimic the hormonal changes in the central nervous system after delivery. A change in the neuronal miniature inhibitory postsynaptic currents (mIPSCs) was observed in neurons, following the silencing of miR­29c or OT treatment with or without OXTR antagonist. The Von­Frey apparatus was used to measure the animal behaviors. Molecular biological experiments and electrophysical recordings in vivo and in vitro were performed to reveal the potential analgesic mechanisms. miR­29c was significantly downregulated (more than 8­fold) in the spinal dorsal horn of delivery+SNI rats compared with the SNI rats. The silencing of miR­29c resulted in increased pain threshold in SNI rats. Bioinformatics analysis indicated that OXTR was a potential target gene of miR­29c. The delivery+SNI rats presented with higher levels of OT in the cerebrospinal fluid compared with SNI rats, which indicated that the OT signaling pathway may participate in pain relief response. The increased expression of OXTR and GABA in delivery+SNI rats were observed in the miR­29c­silenced SNI rat model, suggesting that the silencing of miR­29c can mediate pain relief by enhancing the OT­GABA pathway. In addition, an electrophysiology assay was performed to assess the mIPSCs in neurons. The silencing of miR­29c in neurons increased the frequency and amplitude of mIPSCs but there was no influence on the decay time, which suggested that the spinal inhibitory neurons became more active, subsequently reducing the feeling of pain. The inhibition of OXTR reversed the enhanced inhibitory postsynaptic currents, indicating a crucial role for OXTR in the miR­29c­associated pain regulation. Taken together, the results of the present study suggested that spinal oxytocinergic inhibitory control plays an important role in pain relief in the neuropathic pain rat model undergoing vaginal delivery. Silencing spinal miR­29c may be a potential target for pain relief through the OT­GABA pathway.

Spinal glial cell line-derived neurotrophic factor infusion reverses reduction of Kv4.1-mediated A-type potassium currents of injured myelinated primary afferent neurons in a neuropathic pain model.

High frequency spontaneous activity in injured primary afferents has been proposed as a pathological mechanism of neuropathic pain following nerve injury. Although spinal infusion of glial cell line-derived neurotrophic factor reduces the activity of injured myelinated A-fiber neurons after fifth lumbar (L5) spinal nerve ligation in rats, the implicated molecular mechanism remains undetermined. The fast-inactivating transient A-type potassium current (IA) is an important determinant of neuronal excitability, and five voltage-gated potassium channel (Kv) alpha-subunits, Kv1.4, Kv3.4, Kv4.1, Kv4.2, and Kv4.3, display IA in heterologous expression systems. Here, we examined the effect of spinal glial cell line-derived neurotrophic factor infusion on IA and the expression of these five Kv mRNAs in injured A-fiber neurons using the in vitro patch clamp technique and in situ hybridization histochemistry. Glial cell line-derived neurotrophic factor infusion reversed axotomy-induced reduction of the rheobase, elongation of first spike duration, and depolarization of the resting membrane potential. L5 spinal nerve ligation significantly reduced the current density of IA and glial cell line-derived neurotrophic factor treatment reversed the reduction. Among the examined Kv mRNAs, only the change in Kv4.1-expression was parallel with the change in IA after spinal nerve ligation and glial cell line-derived neurotrophic factor treatment. These findings suggest that glial cell line-derived neurotrophic factor should reduce the hyperexcitability of injured A-fiber primary afferents by IA recurrence. Among the five IA-related Kv channels, Kv4.1 should be a key channel, which account for this IA recurrence.

Cav3.2 T-type calcium channels shape electrical firing in mouse Lamina II neurons.

The T-type calcium channel, Cav3.2, is necessary for acute pain perception, as well as mechanical and cold allodynia in mice. Being found throughout sensory pathways, from excitatory primary afferent neurons up to pain matrix structures, it is a promising target for analgesics. In our study, Cav3.2 was detected in ~60% of the lamina II (LII) neurons of the spinal cord, a site for integration of sensory processing. It was co-expressed with Tlx3 and Pax2, markers of excitatory and inhibitory interneurons, as well as nNOS, calretinin, calbindin, PKCγ and not parvalbumin. Non-selective T-type channel blockers slowed the inhibitory but not the excitatory transmission in LII neurons. Furthermore, T-type channel blockers modified the intrinsic properties of LII neurons, abolishing low-threshold activated currents, rebound depolarizations, and blunting excitability. The recording of Cav3.2-positive LII neurons, after intraspinal injection of AAV-DJ-Cav3.2-mcherry, showed that their intrinsic properties resembled those of the global population. However, Cav3.2 ablation in the dorsal horn of Cav3.2GFP-Flox KI mice after intraspinal injection of AAV-DJ-Cav3.2-Cre-IRES-mcherry, had drastic effects. Indeed, it (1) blunted the likelihood of transient firing patterns; (2) blunted the likelihood and the amplitude of rebound depolarizations, (3) eliminated action potential pairing, and (4) remodeled the kinetics of the action potentials. In contrast, the properties of Cav3.2-positive neurons were only marginally modified in Cav3.1 knockout mice. Overall, in addition to their previously established roles in the superficial spinal cord and in primary afferent neurons, Cav3.2 channel appear to be necessary for specific, significant and multiple controls of LII neuron excitability.

The greater occipital nerve and its spinal and brainstem afferent projections: A stereological and tract-tracing study in the rat.

The neuromodulation of the greater occipital nerve (GON) has proved effective to treat chronic refractory neurovascular headaches, in particular migraine and cluster headache. Moreover, animal studies have shown convergence of cervical and trigeminal afferents on the same territories of the upper cervical and lower medullary dorsal horn (DH), the so-called trigeminocervical complex (TCC), and recent studies in rat models of migraine and craniofacial neuropathy have shown that GON block or stimulation alter nociceptive processing in TCC. The present study examines in detail the anatomy of GON and its central projections in the rat applying different tracers to the nerve and quantifying its ultrastructure, the ganglion neurons subserving GON, and their innervation territories in the spinal cord and brainstem. With considerable intersubject variability in size, GON contains on average 900 myelinated and 3,300 unmyelinated axons, more than 90% of which emerge from C2 ganglion neurons. Unmyelinated afferents from GON innervates exclusively laminae I-II of the lateral DH, mostly extending along segments C2-3 . Myelinated fibers distribute mainly in laminae I and III-V of the lateral DH between C1 and C6 and, with different terminal patterns, in medial parts of the DH at upper cervical segments, and ventrolateral rostral cuneate, paratrigeminal, and marginal part of the spinal caudal and interpolar nuclei. Sparse projections also appear in other locations nearby. These findings will help to better understand the bases of sensory convergence on spinomedullary systems, a critical pathophysiological factor for pain referral and spread in severe painful craniofacial disorders.

In vitro effects of Crotalus atrox snake venom on chick and mouse neuromuscular preparations.

The neuromuscular effect of venoms is not a major clinical manifestation shared between rattlesnakes native to the Americas, which showed two different venom phenotypes. Taking into account this dichotomy, nerve muscle preparations from mice and chicks were used to investigate the ability of Crotalus atrox venom to induce in vitro neurotoxicity and myotoxicity. Unlike crotalic venoms of South America, low concentrations of C. atrox venom did not result in significant effects on mouse neuromuscular preparations. The venom was more active on avian nerve-muscle, showing reduction of twitch heights after 120 min of incubation with 10, 30 and 100 µg/mL of venom with diminished responses to agonists and KCl. Histological analysis highlighted that C. atrox was myotoxic in both species of experimental animals; as evidenced by degenerative events, including edematous cells, delta lesions, hypercontracted fibers and muscle necrosis, which can lead to neurotoxic action. These results provide key insights into the myotoxicity and low neurotoxicity of C. atrox in two animal models, corroborating with previous genomic and proteomic findings and would be useful for a deeper understanding of venom evolution in snakes belonging to the genus Crotalus.

Clarification of the Innervation of the Bladder, External Urethral Sphincter and Clitoris: A Neuronal Tracing Study in Female Mongrel Hound Dogs.

Many studies examining the innervation of genitourinary structures focus on either afferent or efferent inputs, or on only one structure of the system. We aimed to clarify innervation of the bladder, external urethral sphincter (EUS) and clitoris. Retrograde dyes were injected into each end organ in female dogs. Spinal cord, mid-bladder, and spinal, caudal mesenteric, sympathetic trunk and pelvic plexus ganglia were examined for retrograde dye-labeled neurons. Neurons retrogradely labeled from the bladder were found primarily in L7-S2 spinal ganglia, spinal cord lateral zona intermedia at S1-S3 levels, caudal mesenteric ganglia, T11-L2 and L6-S2 sympathetic trunk ganglia, and pelvic plexus ganglia. The mid-bladder wall contained many intramural ganglia neurons labeled anterogradely from the pelvic nerve, and intramural ganglia retrogradely labeled from dye labeling sites surrounding ureteral orifices. Neurons retrogradely labeled from the clitoris were found only in L7 and S1 spinal ganglia, L7-S3 spinal cord lateral zona intermedia, and S1 sympathetic trunk ganglia, and caudal mesenteric ganglia. Neurons retrogradely labeled from the EUS were found in primarily at S1 and S2 spinal ganglia, spinal cord lamina IX at S1-S3, caudal mesenteric ganglia, and S1-S2 sympathetic trunk ganglia. Thus, direct inputs from the spinal cord to each end organ were identified, as well as multisynaptic circuits involving several ganglia, including intramural ganglia in the bladder wall. Knowledge of this complex circuitry of afferent and efferent inputs to genitourinary structures is necessary to understand and treat genitourinary dysfunction. Anat Rec, 2018. © 2018 Wiley Periodicals, Inc.

Effects of forced, passive, and voluntary exercise on spinal motoneurons changes after peripheral nerve injury.

After peripheral nerve injury, there are important changes at the spinal level that can lead to disorganization of the central circuitry and thus compromise functional recovery even if axons are able to successfully regenerate and reinnervate their target organs. Physical rehabilitation is a promising strategy to modulate these plastic changes and thus to improve functional recovery after the damage of the nervous system. Forced exercise in a treadmill is able to partially reverse the synaptic stripping and the loss of perineuronal nets that motoneurons suffer after peripheral nerve injury in animal models. The aim of this study was to investigate whether passive exercise, by means of cycling in a motorized bicycle, or voluntary free running in a wheel is able to mimic the effects induced by forced exercise on the changes that axotomized motoneurons suffer after peripheral nerve injury. Partial preservation of synapses and perineuronal nets was observed only in axotomized motoneurons from animals subjected to high-intensity cycling and the ones that freely ran long distances, but not when low-intensity exercise protocols were applied. Therefore, the intensity but not the type of exercise used is the key element to prevent synaptic stripping and loss of perineuronal nets in motoneurons after axotomy.

The distribution of nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) in the medulla oblongata, spinal cord, cranial and spinal nerves of frog, Microhyla ornata.

Nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) enzymatic activity has been reported in few amphibian species. In this study, we report its unusual localization in the medulla oblongata, spinal cord, cranial nerves, spinal nerves, and ganglions of the frog, Microhyla ornata. In the rhombencephalon, at the level of facial and vagus nerves, the NADPH-d labeling was noted in the nucleus of the abducent and facial nerves, dorsal nucleus of the vestibulocochlear nerve, the nucleus of hypoglossus nerve, dorsal and lateral column nucleus, the nucleus of the solitary tract, the dorsal field of spinal grey, the lateral and medial motor fields of spinal grey and radix ventralis and dorsalis (2-10). Many ependymal cells around the lining of the fourth ventricle, both facial and vagus nerves and dorsal root ganglion, were intensely labeled with NADPH-d. Most strikingly the NADPH-d activity was seen in small and large sized motoneurons in both medial and lateral motor neuron columns on the right and left sides of the brain. This is the largest stained group observed from the caudal rhombencephalon up to the level of radix dorsalis 10 in the spinal cord. The neurons were either oval or elongated in shape with long processes and showed significant variation in the nuclear and cellular diameter. A massive NADPH-d activity in the medulla oblongata, spinal cord, and spinal nerves implied an important role of this enzyme in the neuronal signaling as well as in the modulation of motor functions in the peripheral nervous systems of the amphibians.

Spinal Fbxo3-Dependent Fbxl2 Ubiquitination of Active Zone Protein RIM1α Mediates Neuropathic Allodynia through CaV2.2 Activation.

UNLABELLED: Spinal plasticity, a key process mediating neuropathic pain development, requires ubiquitination-dependent protein turnover. Presynaptic active zone proteins have a crucial role in regulating vesicle exocytosis, which is essential for synaptic plasticity. Nevertheless, the mechanism for ubiquitination-regulated turnover of presynaptic active zone proteins in the progression of spinal plasticity-associated neuropathic pain remains unclear. Here, after research involving Sprague Dawley rats, we reported that spinal nerve ligation (SNL), in addition to causing allodynia, enhances the Rab3-interactive molecule-1α (RIM1α), a major active zone protein presumed to regulate neural plasticity, specifically in the synaptic plasma membranes (SPMs) of the ipsilateral dorsal horn. Spinal RIM1α-associated allodynia was mediated by Fbxo3, which abates Fbxl2-dependent RIM1α ubiquitination. Subsequently, following deubiquitination, enhanced RIM1α directly binds to CaV2.2, resulting in increased CaV2.2 expression in the SPMs of the dorsal horn. While exhibiting no effect on Fbxo3/Fbxl2 signaling, the focal knockdown of spinal RIM1α expression reversed the SNL-induced allodynia and increased spontaneous EPSC (sEPSC) frequency by suppressing RIM1α-facilitated CaV2.2 expression in the dorsal horn. Intrathecal applications of BC-1215 (a Fbxo3 activity inhibitor), Fbxl2 mRNA-targeting small-interfering RNA, and ω-conotoxin GVIA (a CaV2.2 blocker) attenuated RIM1α upregulation, enhanced RIM1α expression, and exhibited no effect on RIM1α expression, respectively. These results confirm the prediction that spinal presynaptic Fbxo3-dependent Fbxl2 ubiquitination promotes the subsequent RIM1α/CaV2.2 cascade in SNL-induced neuropathic pain. Our findings identify a role of the presynaptic active zone protein in pain-associated plasticity. That is, RIM1α-facilitated CaV2.2 expression plays a role in the downstream signaling of Fbxo3-dependent Fbxl2 ubiquitination/degradation to promote spinal plasticity underlying the progression of nociceptive hypersensitivity following neuropathic injury. SIGNIFICANCE STATEMENT: Ubiquitination is a well known process required for protein degradation. Studies investigating pain pathology have demonstrated that ubiquitination contributes to chronic pain by regulating the turnover of synaptic proteins. Here, we found that the spinal presynaptic active zone protein Rab3-interactive molecule-1α (RIM1α) participates in neuropathic pain development by binding to and upregulating the expression of CaV2.2. In addition, Fbxo3 modifies this pathway by inhibiting Fbxl2-mediated RIM1α ubiquitination, suggesting that presynaptic protein ubiquitination makes a crucial contribution to the development of neuropathic pain. Research in this area, now in its infancy, could potentially provide a novel therapeutic strategy for pain relief.

The rabbit brachial plexus as a model for nerve repair surgery--histomorphometric analysis.

One of the most devastating injuries to the upper limb is trauma caused by the avulsion. The anatomical structure of the rabbit's brachial plexus is similar to the human brachial plexus. The aim of our study was to analyze the microanatomy and provide a detailed investigation of the rabbit's brachial plexus. The purpose of our research project was to evaluate the possibility of utilizing rabbit's plexus as a research model in studying brachial plexus injury. Studies included histomorphometric analysis of sampled ventral branches of spinal nerves C5, C6, C7, C8, and Th1, the cranial trunk, the medial part of the caudal trunk, the lateral part of the caudal trunk and peripheral nerve. Horizontal and vertical analysis was done considering following features: the axon diameter, fiber diameter and myelin sheath. The number of axons, nerve area, myelin fiber density and minimal diameter of myelin fiber, minimal axon diameter and myelin area was marked for each element. The changes between ventral branches of spinal nerves C5-Th1, trunks and peripheral nerve in which the myelin sheath, axon diameter and fiber diameter was assessed were statistically significant. It was found that the g-ratio has close value in the brachial plexus as in the peripheral nerve. The peak of these parameters was found in nerve trunks, and then decreased coherently with the nerves travelling peripherally.

Expression and functions of ß1- and ß2-adrenergic receptors on the bulbospinal neurons in the rostral ventrolateral medulla.

The expression and effects of ß-adrenergic receptors (ß-ARs) on the neurons of the bulbospinal rostral ventrolateral medulla (RVLM) have been limitedly examined to date. The objective of this study was to examine the expression of ß1- and ß2-ARs on the bulbospinal RVLM neurons electrophysiologically and histologically. To directly investigate whether RVLM neurons display sensitivity to metoprolol (a ß1-AR antagonist), dobutamine (a ß1-AR agonist), butoxamine (a ß2-AR antagonist), and salbutamol (a ß2-AR agonist), we examined changes in the membrane potentials of the bulbospinal RVLM neurons using the whole-cell patch-clamp technique during superfusion of these drugs. During metoprolol superfusion, 16 of the 20 RVLM neurons were hyperpolarized, and 5 of the 6 RVLM neurons were depolarized during dobutamine superfusion. During butoxamine superfusion, 11 of the 16 RVLM neurons were depolarized, and all of the 8 RVLM neurons were hyperpolarized during salbutamol superfusion. These results suggest the expression of ß1- and ß2-ARs on the RVLM neurons. To determine the presence of ß1- and ß2-ARs histologically, immunofluorescence examination was performed. Five metoprolol-hyperpolarized neurons were examined for ß1-AR and tyrosine hydroxylase (TH) immunoreactivity. All of the neurons displayed ß1-AR immunoreactivity, whereas three of the neurons displayed TH immunoreactivity. All of the five RVLM neurons that became depolarized during metoprolol superfusion and hyperpolarized during butoxamine superfusion displayed ß1- and ß2-AR immunoreactivity. Our findings suggest that ß1-AR antagonists or ß2-AR agonists may decrease blood pressure through decreasing the activity of the bulbospinal RVLM neurons.

Generating spinal motor neuron diversity: a long quest for neuronal identity.

Understanding how thousands of different neuronal types are generated in the CNS constitutes a major challenge for developmental neurobiologists and is a prerequisite before considering cell or gene therapies of nervous lesions or pathologies. During embryonic development, spinal motor neurons (MNs) segregate into distinct subpopulations that display specific characteristics and properties including molecular identity, migration pattern, allocation to specific motor columns, and innervation of defined target. Because of the facility to correlate these different characteristics, the diversification of spinal MNs has become the model of choice for studying the molecular and cellular mechanisms underlying the generation of multiple neuronal populations in the developing CNS. Therefore, how spinal motor neuron subpopulations are produced during development has been extensively studied during the last two decades. In this review article, we will provide a comprehensive overview of the genetic and molecular mechanisms that contribute to the diversification of spinal MNs.

Dual-mode operation of neuronal networks involved in left-right alternation.

All forms of locomotion are repetitive motor activities that require coordinated bilateral activation of muscles. The executive elements of locomotor control are networks of spinal neurons that determine gait pattern through the sequential activation of motor-neuron pools on either side of the body axis. However, little is known about the constraints that link left-right coordination to locomotor speed. Recent advances have indicated that both excitatory and inhibitory commissural neurons may be involved in left-right coordination. But the neural underpinnings of this, and a possible causal link between these different groups of commissural neurons and left-right alternation, are lacking. Here we show, using intersectional mouse genetics, that ablation of a group of transcriptionally defined commissural neurons--the V0 population--leads to a quadrupedal hopping at all frequencies of locomotion. The selective ablation of inhibitory V0 neurons leads to a lack of left-right pattern at low frequencies, mixed coordination at medium frequencies, and alternation at high locomotor frequencies. When ablation is targeted to excitatory V0 neurons, left-right alternation is present at low frequencies, and hopping is restricted to medium and high locomotor frequencies. Therefore, the intrinsic logic of the central control of locomotion incorporates a modular organization, with two subgroups of V0 neurons required for the existence of left-right alternating modes at different speeds of locomotion. The two molecularly distinct sets of commissural neurons may constrain species-related naturally occurring frequency-dependent coordination and be involved in the evolution of different gaits.

Age-related changes of neurochemically different subpopulations of cardiac spinal afferent neurons in rats.

This study investigated the effect of aging on cardiac spinal afferent neurons in the rat. A patch loaded with retrograde tracer Fast Blue (FB) was applied to all chambers of the rat heart. Morphological and neurochemical characteristics of labeled cardiac spinal afferent neurons were assessed in young (2 months) and old (2 years) rats using markers for likely unmyelinated (isolectin B4; IB4) and myelinated (neurofilament 200; N52) neurons. The number of cardiac spinal afferent neurons decreased in senescence to 15% of that found in young rats (1604 vs. 248). The size of neuronal soma as well as proportion of IB4+ neurons increased significantly, whereas the proportion of N52+ neurons decreased significantly in senescence. Unlike somatic spinal afferents, neurochemically different populations of cardiac spinal afferent neurons experience morphological and neurochemical changes related to aging. A major decrease in total number of cardiac spinal afferent neurons occurs in senescence. The proportion of N52+ neurons decreased in senescence, but it seems that nociceptive innervation is preserved due to increased proportion and size of IB4+ unmyelinated neurons.

CLP1 links tRNA metabolism to progressive motor-neuron loss.

CLP1 was the first mammalian RNA kinase to be identified. However, determining its in vivo function has been elusive. Here we generated kinase-dead Clp1 (Clp1(K/K)) mice that show a progressive loss of spinal motor neurons associated with axonal degeneration in the peripheral nerves and denervation of neuromuscular junctions, resulting in impaired motor function, muscle weakness, paralysis and fatal respiratory failure. Transgenic rescue experiments show that CLP1 functions in motor neurons. Mechanistically, loss of CLP1 activity results in accumulation of a novel set of small RNA fragments, derived from aberrant processing of tyrosine pre-transfer RNA. These tRNA fragments sensitize cells to oxidative-stress-induced p53 (also known as TRP53) activation and p53-dependent cell death. Genetic inactivation of p53 rescues Clp1(K/K) mice from the motor neuron loss, muscle denervation and respiratory failure. Our experiments uncover a mechanistic link between tRNA processing, formation of a new RNA species and progressive loss of lower motor neurons regulated by p53.

Musculotopic organization of the motor neurons supplying forelimb and shoulder girdle muscles in the mouse.

We identified the motor neurons (MNs) supplying the shoulder girdle and forelimb muscles in the C57BL/6J mouse spinal cord using Fluoro-Gold retrograde tracer injections. In spinal cord transverse sections from C2 to T2, we observed two MN columns (medial and lateral) both with ventral and dorsal subdivisions. The dorsolateral column consisted of the biceps brachii, forearm extensors, forearm flexors, and hand MNs, and the ventrolateral column consisted of the latissimus dorsi, trapezius, teres major, deltoid, and triceps MNs. The supraspinatus muscle MNs were located in the dorsomedial column, and pectoralis major and serratus anterior MNs were located in the ventromedial columns. MNs of the dorsolateral column innervated the biceps brachii in mid-C4 to mid-C7, forearm extensors in caudal C4 to mid-T1, forearm flexors in rostral C5 to mid-T1, and hand muscles in mid-C8 to mid-T2 segments. The MNs innervating the trapezius were located in mid-C2 to mid-C4, triceps brachii in mid-C6 to rostral T1, deltoid in rostral C4 to mid-C6, teres major in rostral C5 to mid-C8, and latissimus dorsi in mid-C5 to caudal C8. In addition, MNs innervating the supraspinatus were located from rostral C4 to caudal C8, pectoralis major in mid-C6 to mid-T2, and serratus anterior in rostral C5 to caudal C7/rostral C8 segments. While the musculotopic pattern of MN groups was very similar to that documented for other species, we found differences in the position and cranio-caudal extent of some MN pools compared with previous reports. The identification of mouse forelimb MNs can serve as an anatomical reference for studying degenerative MN diseases, spinal cord injury, and developmental gene expression.

Interplay between electrical activity and bone morphogenetic protein signaling regulates spinal neuron differentiation.

A gradient of bone morphogenetic proteins (BMPs) along the dorsoventral axis of the spinal cord is necessary for the specification of dorsal neurons. Concurrently, a gradient of calcium-mediated electrical activity is present in the developing spinal cord but in an opposing ventrodorsal direction. Whether BMPs and electrical activity interact in embryonic spinal neurons remains unknown. We show that BMP decreases electrical activity by enhancing p38 MAPK-mediated negative modulation of voltage-gated sodium channels. In turn, electrical activity affects the phosphorylation status and nuclear level of activated Smads, the canonical components of BMP signaling. This interaction between calcium spike activity and BMP signaling regulates the specification of the dorsal commissural spinal neuron phenotype. The present study identifies an unexpected interplay between BMPs and electrical activity that is critical for decoding the morphogen gradient during spinal neuron differentiation.

Constituent ratio of motor fibers from the C5-C7 spinal nerves in the radial nerve is greater in pup rats than in adult rats.

Clinically, injuries of C5-C7 of the brachial plexus cause falling of the wrist and fingers in infants but not in adults unless 4 consecutive spinal nerves are injured. The purpose of this study was to compare the constituent difference of spinal nerves in the radial nerve between pup and adult rats.A group of 16 pup rats and a group of 16 adult rats were each divided into 2 groups of 8 (P1 and A1 groups, C5-C6 were divided; P2 and A2 groups, C5-C7 were divided]). A nerve conduction study and histological examination were performed to evaluate radial nerve innervation to the extensor digitorum communis muscle after dividing the spinal nerves. Retrograde tracing with 5% cholera toxin B for anterior horn motoneurons of the spinal cord innervating the radial nerve was performed in 8 pup rats and 8 adult rats. Results showed that the division of C5-C7 caused more significant damage to radial nerve innervation to the extensor digitorum communis in pups than in adults, although the division of C5-C6 did not. In pups, the percentages (median with interquartile) of anterior horn motoneurons of the spinal cord innervating the radial nerve were 36.4 (28.3-38.5) in C5-C6, 28.1 (24.5-32.5) in C7, and 37.5 (36.5-39.3) in C8-T1. In adults, they were 24.2 (23.6-27.8) in C5-C6, 21.8 (19.5-26.3) in C7, and 50.7 (48.7-55.5) C8-T1.This study implies that C7 innervation in the radial nerve in humans may be more critical to the function of this nerve in infants than in adults.

Retrograde loading of nerves, tracts, and spinal roots with fluorescent dyes.

Retrograde labeling of neurons is a standard anatomical method(1,2) that has also been used to load calcium and voltage-sensitive dyes into neurons(3-6). Generally, the dyes are applied as solid crystals or by local pressure injection using glass pipettes. However, this can result in dilution of the dye and reduced labeling intensity, particularly when several hours are required for dye diffusion. Here we demonstrate a simple and low-cost technique for introducing fluorescent and ion-sensitive dyes into neurons using a polyethylene suction pipette filled with the dye solution. This method offers a reliable way for maintaining a high concentration of the dye in contact with axons throughout the loading procedure.