Orexin-A potentiates glycine currents by activating OX1R and IP3/Ca2+/PKC signaling pathways in spinal cord ventral horn neurons.

Orexin-A/B modulates multiple physical functions by activating their receptors (OX1R and OX2R), but its effects in the spinal cord motor control remain unknown. Using acute separation (by digestive enzyme) of cells and patch-clamp recordings, we aimed to investigate the effect and mechanisms of orexin-A on the glycine receptors in the spinal cord ventral horn neurons. Orexin-A potentiated the glycine currents by activating OX1R. In Ca2+-free extracellular solution, orexin-A still increased the glycine currents. While, the orexin-A-induced potentiation was blocked when Ca2+ was chelated by internal infusion of BAPTA, and the orexin-A effect was abolished by the IP3 receptor antagonists heparin and Xe-C. The PKC inhibitor Bis-IV nullified the orexin-A effect. In addition, orexin-A did not cause a further enhancement of the glycine currents after bath application of the PKC activator PMA. In conclusion, after OX1R is activated, a distinct IP3/Ca2+-dependent PKC signaling pathway, is likely responsible for the orexin-A potentiation on glycine currents in the spinal cord ventral horn neurons.

Histamine H3 Receptors Expressed in Ventral Horns Modulate Spinal Motor Output.

Motoneuron activity is modulated by histamine receptors. While H1 and H2 receptors have been widely explored, H3 histamine receptors (H3Rs) have not been sufficiently characterized. This paper targets the effects of the selective activation of H3Rs and their expression on the membranes of large ventral horn cells. The application of selective pharmacological agents to spinal cords isolated from neonatal rats was used to identify the presence of functional H3Rs on the membrane of physiologically identified lumbar motoneurons. Intra and extracellular recordings revealed that H3R agonist, α-methylhistamine, depolarized both single motoneurons and ventral roots, even in the presence of tetrodotoxin, an effect prevented by H3R antagonist, thioperamide. Finally, immunohistochemistry located the expression of H3Rs on a subpopulation of large cells in lamina IX. This study identifies H3Rs as a new exploitable pharmacological target against motor disturbances.

Delayed short-term tamoxifen treatment does not promote remyelination or neuron sparing after spinal cord injury.

The tamoxifen-dependent Cre/lox system in transgenic mice has become an important research tool across all scientific disciplines for manipulating gene expression in specific cell types. In these mouse models, Cre-recombination is not induced until tamoxifen is administered, which allows researchers to have temporal control of genetic modifications. Interestingly, tamoxifen has been identified as a potential therapy for spinal cord injury (SCI) and traumatic brain injury patients due to its neuroprotective properties. It is also reparative in that it stimulates oligodendrocyte differentiation and remyelination after toxin-induced demyelination. However, it is unknown whether tamoxifen is neuroprotective and neuroreparative when administration is delayed after SCI. To properly interpret data from transgenic mice in which tamoxifen treatment is delayed after SCI, it is necessary to identify the effects of tamoxifen alone on anatomical and functional recovery. In this study, female and male mice received a moderate mid-thoracic spinal cord contusion. Mice were then gavaged with corn oil or a high dose of tamoxifen from 19-22 days post-injury, and sacrificed 42 days post-injury. All mice underwent behavioral testing for the duration of the study, which revealed that tamoxifen treatment did not impact hindlimb motor recovery. Similarly, histological analyses revealed that tamoxifen had no effect on white matter sparing, total axon number, axon sprouting, glial reactivity, cell proliferation, oligodendrocyte number, or myelination, but tamoxifen did decrease the number of neurons in the dorsal and ventral horn. Semi-thin sections confirmed that axon demyelination and remyelination were unaffected by tamoxifen. Sex-specific responses to tamoxifen were also assessed, and there were no significant differences between female and male mice. These data suggest that delayed tamoxifen administration after SCI does not change functional recovery or improve tissue sparing in female or male mice.

Astrocytes and Microglia-Mediated Immune Response in Maladaptive Plasticity is Differently Modulated by NGF in the Ventral Horn of the Spinal Cord Following Peripheral Nerve Injury.

Reactive astrocytes and activated microglia are the key players in several pathophysiologic modifications of the central nervous system. We used the spared nerve injury (SNI) of the sciatic nerve to induce glial maladaptive response in the ventral horn of lumbar spinal cord and examine its role in the remodeling of the tripartite synapse plasticity. Imaging the ventral horn revealed that SNI was associated with both an early microglial and astrocytic activation, assessed, respectively, by analysis of Iba1 and GFAP expression. Microglia, in particular, localized peculiarly surrounding the motor neurons somata. Perineuronal astrocytes, which play a key role in maintaining the homeostasis of neuronal circuitry, underwent a substantial phenotypic change following peripheral axotomy, producing reactive gliosis. The gliosis was associated with the reduction of glial aminoacid transporters (GLT1 and GlyT1) and increase of neuronal glutamate transporter EAAC1. Although the expression of GABAergic neuronal marker GAD65/67 showed no change, glutamate increase, as demonstrated by HPLC analysis, shifted the excitatory/inhibitory balance as showed by the net increase of the glutamate/GABA ratio. Moreover, endogenous NGF levels were altered in SNI animals and not restored by the intrathecal NGF administration. This treatment reverted phenotypic changes associated with reactive astrocytosis, but failed to modify microglia activation. These findings on one hand confirm the correlation between gliopathy and maladaptive plasticity of the spinal synaptic circuitry, on the other hand add new data concerning the complex peculiar behavior of different glial cells in neuronal degenerative processes, defining a special role of microglia in sustaining the inflammatory response.

Calbindin-D28K is increased in the ventral horn of spinal cord by neuroprotective factors for motor neurons.

Slow glutamate-mediated neuronal degeneration is implicated in the pathophysiology of motor neuron diseases such as amyotrophic lateral sclerosis (ALS). The calcium-binding proteins calbindin-D28K and parvalbumin have been reported to protect neurons against excitotoxic insults. Expression of calbindin-D28K is low in adult human motor neurons, and vulnerable motor neurons additionally may lack parvalbumin. Thus, it has been speculated that the lack of calcium-binding proteins may, in part, be responsible for early degeneration of the population of motor neurons most vulnerable in ALS. Using a rat organotypic spinal cord slice system, we examined whether the most potent neuroprotective factors for motor neurons can increase the expression of calbindin-D28K or parvalbumin proteins in the postnatal spinal cord. After 4 weeks of incubation of spinal cord slices with 1) glial cell line-derived neurotrophic factor (GDNF), 2) neurturin, 3) insulin-like growth factor I (IGF-I), or 4) pigment epithelium-derived factor (PEDF), the number of calbindin-D28K -immunopositive large neurons (>20 µm) in the ventral horn was higher under the first three conditions, but not after PEDF, compared with untreated controls. Under the same conditions, parvalbumin was not upregulated by any neuroprotective factor. The same calbindin increase was true of IGF-I and GDNF in a parallel glutamate toxicity model of motor neuron degeneration. Taken together with our previous reports from the same model, which showed that all these neurotrophic factors can potently protect motor neurons from slow glutamate injury, the data here suggest that upregulation of calbindin-D28K by some of these factors may be one mechanism by which motor neurons can be protected from glutamate-induced, calcium-mediated excitotoxicity.

More than at the neuromuscular synapse: actions of botulinum neurotoxin A in the central nervous system.

Botulinum neurotoxin type A (BoNT/A) is a metalloprotease that produces a sustained yet transitory blockade of transmitter release from peripheral nerve terminals. Local delivery of this neurotoxin is successfully employed in clinical practice to reduce muscle hyperactivity such as in spasticity and dystonia, and to relieve pain with long-lasting therapeutic effects. However, not all BoNT/A effects can be explained by an action at peripheral nerve terminals. Indeed, it appears that BoNT/A is endowed with trafficking properties similar to the parental tetanus neurotoxin and thus be able to directly affect the CNS. In this review, we present and discuss novel compelling evidence for a direct central effect of BoNT/A in both dorsal and ventral horns of the animal and human spinal cord after peripheral injection of the neurotoxin, with important consequences on pain and motor control. This new knowledge is expected to radically change the approach to the use of BoNT/A in the future. As BoNT/A central action appears to also contribute to functional improvement, for instance in human spastic gait, the challenge will be to develop new subtypes or BoNT derivatives with deliberate, cell-specific central effects in order to fully exploit the spectrum of BoNT/A therapeutic activity.

Effects of adenosine monophosphate-activated kinase in the ventral horn of rabbit spinal cord after transient ischemia.

OBJECTIVE: To investigate the effect compound C, an adenosine monophosphate-activated kinase (AMPK) inhibitor, has on motor neurons of rabbit spinal cord after ischemia/reperfusion. DESIGN: Compound C (30 mg/kg) was administered intraperitoneally to rabbits 30 minutes before ischemia and the animals were sacrificed at 15 minutes after ischemia/reperfusion to measure lactate levels and at 72 hours after ischemia/reperfusion for morphological study. RESULTS: The administration of compound C did not produce any significant changes in physiological parameters such as pH, arterial blood gas (PaCO(2) and PaO(2)), and blood glucose in rabbit either at 10 minutes before ischemia or at 10 minutes after reperfusion. However, the administration of compound C did significantly ameliorate lactate acidosis at 15 minutes after reperfusion. In addition, the administration of compound C significantly improved the neurological scores of the rabbits and reduced the neuronal death seen in the ventral horn of their spinal cords at 72 hours after ischemia/reperfusion. CONCLUSIONS: Inhibition of AMPK can ameliorate the ischemia-induced neuronal death in the spinal cord via the reduction of early lactate acidosis.