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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.

Introduction: Injections of botulinum neurotoxin type A (BoNT/A) provide an essential treatment for muscle hyperactivity disorders such as spasticity and dystonias. Recently discovered BoNT/A enzymatic action in motor regions of the central nervous system (CNS) indicate the possible involvement of central action in its therapeutic effect. Centrally transported BoNT/A was shown to be a prerequisite to its antinociceptive activity, while up to now, the functional role of BoNT/A axonal transport in the motor nervous system has been unknown. Here, we studied the central effect of BoNT/A in experimental focal muscular hyperactivity evoked by tetanus toxin (TeNT). Methods: Localized muscle rigidity was induced by low-dose intramuscular (IM) TeNT into rat gastrocnemius (1 to 1.5 ng). Pharmacologic response to intramuscular and intrasciatic injection of BoNT/A (5 U/kg) and intraperitoneal R(+) baclofen (3 mg/kg) was assessed by different motor parameters and tests. BoNT/A transcytosis in the CNS was investigated by intrathecal BoNT/A antitoxin and cleaved synaptosomal-associated protein 25 (SNAP-25) immunohistochemistry. Results: TeNT injected into the calf muscle induced local spastic paralysis. Intramuscular and intrasciatic BoNT/A and baclofen reduced TeNT-evoked hind-paw extension and ankle flexion resistance, while intrasciatic BoNT/A improved rotarod performance. Intrasciatic BoNT/A led to accumulation of toxin enzymatic activity in the CNS without any paralytic effect in normal animals. The effect of intrasciatic BoNT/A on local spasticity and the occurrence of cleaved SNAP-25 in the ventral horn were prevented by intrathecal antitoxin, indicating that the toxin is transcytosed by extracellular movement into second-order synapses. Conclusion: Central effects of BoNT/A do not measurably affect normal motor function ; however, in our experiments, they eliminated local spastic paralysis induced by TeNT. Present results suggest that the central effects of BoNT/A might account for clinically observed improvements of muscle function in spastic conditions and dystonias that are not concurrent with local muscle paralysis

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.

By far, the strongest spinal cord projections to periaqueductal gray (PAG) and thalamus originate from the upper three cervical segments, but their precise organization and function are not known. In the present study in cat, tracer injections in PAG or in thalamus resulted in more than 2400 labeled cells, mainly contralaterally, in the first three cervical segments (C1-C3), in a 1:4 series of sections, excluding cells in the dorsal column and lateral cervical nuclei. These cells represent about 30% of all neurons in the entire spinal cord projecting to PAG and about 45% of all spinothalamic neurons. About half of the C1-C3 PAG and C1-C3 thalamic neurons were clustered laterally in the ventral horn (C1-3v1), bilaterally, with a slight ipsilateral preponderance. The highest numbers Of C1-3v1-PAG and C1-3v1-thalamic cells were found in C1, with the greatest density rostrocaudal ly in the middle part of C 1. A concept is put forward that C1-3v1 cells relay information from all levels of the cord to PAG and/or thalamus, although the processing of specific information from upper neck muscles and tendons or facet joints might also play a role. (c) 2005 Elsevier B.V. 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.

In 1995, we discovered a new pathway in the cat, which originates from the nucleus retroambiguus (NRA) and terminates in a distinct set of lumbosacral hindlimb, axial, and pelvic floor motoneuronal cell groups [VanderHorst VG.JM, Holstege G (1995) Caudal medullary pathways to lumbosacral motoneuronal cell groups in the cat: evidence for direct projections possibly representing the final common pathway for lordosis. J Comp Neurol 359:457-475]. The NRA is a compact group of interneurons located laterally in the caudal medulla oblongata. Its projection to lumbosacral moloneurons is thought to represent the final common pathway for male mounting and for female receptive or lordosis behavior. However, females only display lordosis behavior when they are in estrus, which suggests that the NRA-lumbosacral pathway is only active during estrus. This raised the question of whether estrogen affects this pathway. The effect of estrogen on the NRA-lumbosacral projection was studied light microscopically, using wheat-germ agglutinin horseradish peroxidase (WGA-HRP) as a tracer. The rubrospinal pathway served as control. The density of labeled NRA fibers in their target hindlimb motoneuronal cell groups appeared abundant in estrous and very weak in nonestrous cats. Such differences were not found in the rubrospinal pathway. For electron microscopical study, the NRA projection to the semimembranosus motoneuronal cell group was selected. In this cell group, an almost ninefold increase of labeled profiles was found in estrous versus nonestrous cats. Moreover, the semimembranous motoneuronal cell group contained labeled growth cones in estrous, but not in nonestrous, cats. The present study is the first to show that estrogen induces axonal outgrowth of a precisely identified pathway in the adult mammalian central nervous system. The possible mechanisms underlying this outgrowth are discussed.

Das Ausschalten von Abwehrbewegungen auf Schmerzreize in Narkose (sog. chirurgische Immobilität) wird durch Anästhetika v. a. über ihre Wirkung an Rückenmarksneuronen realisiert. Bislang ist unklar, ob volatile Anästhetika und Propofol Immobilität durch den gleichen oder verschiedene Mechanismen auslösen. Während es für volatile Anästhetika scheint, dass sie Immobilität eher über Wirkungen am Vorderhorn des Rückenmarks als am Hinterhorn hervorrufen, besteht über den Wirkort oder den Mechanismus von Propofol keine Klarheit. Die Studie hatte zum Ziel über den Vergleich der Effekte von Sevofluran und Propofol auf den H-Reflex und den RIII Reflex, zwei unterschiedlich im Vorder- und Hinterhorn des Rückenmarks verschaltete spinale Reflexe, Hinweise auf deren Wirkorte oder Mechanismen für das Auslösen chirurgischer Immobilität zu erhalten. Im Rahmen von Sevofluran- und Propofolmononarkosen konnten bei 13 Probanden die Effekte von Sevofluran und Propofol auf die Amplitude des H-Reflexes, der ausschließlich im Vorderhorn verschaltet ist, und auf die Amplitude des RIII Reflexes, dessen Schaltkreis Vorder- und Hinterhorn-Neuronen integriert, verglichen werden. In jeder Gruppe wurden die Anästhetika-Konzentrationen ausgehend von 1,6 Vol% für Sevofluran bzw. 4,5 μg/ml für Propofol mittels Dixon’s up-and-down Methode in Abhängigkeit der motorischen Reaktion auf einen schmerzhaften tetanischen Stimulus (30 s., 70 mA, 50 Hz) bestimmt. Die Konzentrationen von Sevofluran und die berechneten Propofol-Konzentrationen lagen zwischen 1,2 und 1,6 Vol%, beziehungsweise zwischen 3 und 6 μg/ml. Während unter Sevofluran-Narkose die H-Reflex Amplitude auf 66 ± 17 % der Kontrollwerte signifikant reduziert wurde, erreichte die Reduktion durch Propofol auf 87 ± 14 % der Kontrollwerte keine Signifikanz. Die RIII Reflex Amplitude wurde durch Sevofluran auf 19 ± 10 %, durch Propofol auf 27 ± 12 % der Kontrollwerte signifikant verringert, wobei der Unterschied der Reduktion durch die beiden Medikamente nicht signifikant war. Die Verminderung des RIII-Reflexes, welche die Abschwächung des monosynaptischen H-Reflexes bei weitem übersteigt, ist vermutlich in seiner polysynaptischen Verschaltung begründet. Sevofluran erzeugt eine stärkere Hemmung des H-Reflexes als Propofol, was das Vorderhorn als seinen dominierenden Wirkort bestätigt. Propofol-Effekte am Vorderhorn sind hingegen eher begrenzt. Die Ergebnisse der Studie weisen für Propofol somit indirekt auf eine relativ stärkere Wirkung am Hinterhorn des Rückenmarks hin. Die medikamenten-unabhängige Unterdrückung des RIII-Reflexes, die mit der Immobilität korreliert, könnte den RIII-Reflex zum Monitoring der Immobilität qualifizieren., The suppression of movement responses to noxious stimuli under anesthesia is predominantly realized by effects of anesthetics on spinal cord neurons. Up to now, the question whether volatile anesthetics and propofol produce immobility by different or same actions remains unanswered. Although volatile anesthetics appear to produce immobility by actions on the ventral rather than the dorsal horn, the site of action of propofol remains unclear. The aim of this study was to compare the targets of both propofol and sevoflurane within the human spinal cord that might contribute to the suppression of movement. Two reflex circuits including neurons located in different parts of the spinal cord were employed: the proprioceptive H reflex, including only ventral horn neurons, and the RIII reflex as a component of the nociceptive flexion reflex, which includes ventral as well as dorsal horn neurons. The effects of sevoflurane and propofol on RIII and H reflex amplitudes under anesthesia of 13 volunteers were compared. For each group the concentrations were adjusted according to a Dixon up-and-down approach, depending on movement responses to tetanic stimulation (30 sec., 70 mA, 50 Hz). Initial sevoflurane and propofol concentrations started at 1.6 Vol% and 4.5 μg/ml, respectively and ranged from 1.2 to 1.6 Vol% (sevoflurane) and 3 to 6 μg/ml (propofol). Whereas sevoflurane reduced the H reflex amplitude significantly to 66 ± 17% (mean ± SD) of its control values., propofol did not significantly reduce the H reflex. The reductions under the two drugs differed significantly. The RIII reflex amplitude was significantly reduced to 19 ± 10% and 27 ± 12% (mean ± SD) of the control values by sevoflurane and propofol, respectively. The reductions did not differ between the drugs. Probably because of the polysynaptic relay, the attenuation of the withdrawal reflex exceeds the attenuation of the H reflex. Sevoflurane produces a larger inhibitory effect on the H reflex than propofol, which confirms that the ventral horn is a more important target for volatile anesthetics, whereas effects of propofol on this site of action are rather limited. Our findings indirectly suggest for propofol a relatively stronger effect within the dorsal horn. Being drugtype independent, the suppression of the RIII reflex which correlates with immobility could qualify the RIII reflex as a tool for monitoring immobility.

In 1995, we discovered a new pathway in the cat, which originates from the nucleus retroambiguus (NRA) and terminates in a distinct set of lumbosacral hindlimb, axial, and pelvic floor motoneuronal cell groups [VanderHorst VGJM, Holstege G (1995) Caudal medullary pathways to lumbosacral motoneuronal cell groups in the cat: evidence for direct projections possibly representing the final common pathway for lordosis. J Comp Neurol 359:457-475]. The NRA is a compact group of interneurons located laterally in the caudal medulla oblongata. Its projection to lumbosacral motoneurons is thought to represent the final common pathway for male mounting and for female receptive or lordosis behavior. However, females only display lordosis behavior. However, females only display lordosis behavior when they are in estrus, which suggests that the NRA-lumbosacral pathway is only active during estrus. This raised the question of whether estrogen affects this pathway. The effect of estrogen on the NRA-lumbosacral projection was studied light microscopically, using wheat-germ agglutinin horseradish peroxidase (WGA-HRP) as a tracer. The rubrospinal pathway served as control. The density of labeled NRA fibers in their target hindlimb motoneuronal cell groups appeared abundant in estrous and very weak in nonestrous cats. Such differences were not found in the rubrospinal pathway. For electron microscopical study, the NRA projection to the semi-membranosus motoneuronal cell group was selected. In this cell group, an almost ninefold increase of labeled profiles was found in estrous versus nonestrous cats. Moreover, the semimembranous motoneuronal cell group contained labeled growth cones in estrous, but not in nonestrous, cats. The present study is the first to show that estrogen induces axonal outgrowth of a precisely identified pathway in the adult mammalian central nervous system. The possible mechanisms underlying this outgrowth are discussed.