Adult Rat Facial Motoneurons Research Articles

Neuroprotective effect of acute prior inflammation with lipopolysaccharide for adult male rat facial motoneurones

Increases in inflammatory cytokines are reported to have both neuroprotective and neurotoxic effects depending on the type and age of neurones studied. This study aimed to determine the effect of experimental inflammation induced by Lipopolysaccharide (LPS) on the survival of injured male adult rat facial motoneurones. Time- and dose-response studies were done to optimise the LPS administration time and dose, to best correlate with inflammatory levels previously reported for aged rats. 12 cytokines were assayed through multiplex analysis. 24 h after intraperitoneal injection of 0.5 mg/kg Lipopolysaccharide in rats, IL-1β, IL-5 and IL-12p70 levels were elevated, with no observed LPS-associated sickness behaviour. In other groups of 5–6 adult rats, the facial nerve was either crushed (as mild injury) or avulsed (as severe injury) after the LPS priming injection. Stereology revealed that most motoneurones survived 28 days after nerve crush only and LPS- or saline-priming preceding nerve crush. Most motoneurones died following nerve avulsion only, whereas over half survived when LPS-priming preceded nerve avulsion. We suggest that elevated levels of experimental inflammation are neuroprotective for severely injured adult male rat facial motoneurones.

Accumulation of glycogen in axotomized adult rat facial motoneurons.

This study biochemically determined glycogen content in the axotomized facial nucleus of adult rats up to 35 days postinsult. The amounts of glycogen in the transected facial nucleus were significantly increased at 5 days postinsult, peaked at 7 days postinsult, and declined to the control levels at 21-35 days postinsult. Immunohistochemical analysis with antiglycogen antibody revealed that the quantity of glycogen granules in the axotomized facial nucleus was greater than that in the control nucleus at 7 days postinjury. Dual staining methods with antiglycogen antibody and a motoneuron marker clarified that the glycogen was localized mainly in motoneurons. Immunoblotting and quantification analysis revealed that the ratio of inactive glycogen synthase (GS) to total GS was significantly decreased in the injured nucleus at about 1-3 days postinsult and significantly increased from 7 to 14 days postinsult, suggesting that glycogen is actively synthesized in the early period postinjury but suppressed after 7 days postinsult. The enhanced glycogen at about 5-7 days postinsult is suggested to be responsible for the decrease in inactive GS levels, and the decrease of glycogen after 7 days postinsult is considered to be caused by increased inactive GS levels and possibly the increase in active glycogen phosphorylase.

Functional down-regulation of axotomized rat facial motoneurons

Functional alterations in injured motoneurons were quantitatively analyzed in axotomized rat facial nuclei. Choline acetyltransferase (ChAT), vesicular acetylcholine transporter (VAchT) and m2 muscarinic acetylcholine receptor (m2MAchR) were chosen as indicators of motoneuron function. Immunoblotting showed that the amounts of ChAT in the ipsilateral facial nucleus significantly decreased to below 20% from 3 to 14 days after transection. The decreased level of ChAT in injured motoneurons was ascertained by immunohistochemical study. However, at 4–5 weeks after transection the level of ChAT was restored to that of control side. The amounts of VAchT in the transected nucleus were observed to decrease to below 20% in the first 14 days after transection. The down-regulated levels of VAchT in injured motoneurons were confirmed by immunohistochemical results. The reduced VAchT levels returned to the control levels at 4–5 weeks following insult. The level of m2MAchR in the ipsilateral nucleus was recognized to decrease to below 10% starting on the 5th day after insult, and the low levels were sustained for 5 weeks. Nissl staining at 5 days and 12 days after insult revealed that facial motoneurons in the transected nucleus were almost all alive. Altogether, these results indicate that transected adult rat facial motoneurons are functionally depressed with down-regulated levels of ChAT, VAchT and m2MAchR during the first 14 days after insult, and during Weeks 4–5 ChAT and VAchT levels are restored while the levels of m2MAchR remain low.

Growth inhibitory factor prevents degeneration of injured adult rat motoneurons.

We examined neuroprotective effects of growth inhibitory factor (GIF) on injured adult rat facial motoneurons. The right facial nerves of adult rats were avulsed and removed from the stylomastoid foramen, and an adenoviral vector encoding rat GIF and Myc epitope (AxCArGIFM) were injected into the facial canal. Animals treated with AxCArGIFM showed intense immunolabeling for GIF/Myc in injured facial motoneurons. Treatment with AxCArGIFM after avulsion significantly prevented the loss of injured facial motoneurons, improved choline acetyltransferase immunoreactivity and prevented the induction of nitric oxide synthase activity in these neurons. These results indicate that GIF may have therapeutic potential against degeneration of motoneurons in adult humans with motoneuron injury and motor neuron diseases.

Adenoviral gene transfer of GDNF, BDNF and TGF beta 2, but not CNTF, cardiotrophin-1 or IGF1, protects injured adult motoneurons after facial nerve avulsion.

We examined neuroprotective effects of recombinant adenoviral vectors encoding glial cell line-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), cardiotrophin-1 (CT1), insulin-like growth factor-1 (IGF1), and transforming growth factor-beta2 (TGFbeta2) on lesioned adult rat facial motoneurons. The right facial nerves of adult Fischer 344 male rats were avulsed and removed from the stylomastoid foramen, and adenoviral vectors were injected into the facial canal. Animals avulsed and treated with adenovirus encoding GDNF, BDNF, CNTF, CT1, IGF1 and TGFbeta2 showed intense immunolabeling for these factors in lesioned facial motoneurons, respectively, indicating adenoviral induction of the neurotrophic factors in these neurons. The treatment with adenovirus encoding GDNF, BDNF, or TGFbeta2 after avulsion significantly prevented the loss of lesioned facial motoneurons, improved choline acetyltransferase immunoreactivity and prevented the induction of nitric oxide synthase activity in these neurons. The treatment with adenovirus encoding CNTF, CT1 or IGF1, however, failed to protect these neurons after avulsion. These results indicate that the gene transfer of GDNF and BDNF and TGFbeta2 but not CNTF, CT1 or IGF1 may prevent the degeneration of motoneurons in adult humans with motoneuron injury and motor neuron diseases.

Transplantation of Olfactory Ensheathing Cells Stimulates the Collateral Sprouting from Axotomized Adult Rat Facial Motoneurons

Axon regrowth after CNS and PNS injury is only the first step toward complete functional recovery which depends largely on the specificity of the newly formed nerve-target projections. Since most of the studies involving the application of glial cells to the lesioned nervous system have focused primarily on the extent of neurite outgrowth, little is known regarding their effects on the accompanying processes of axonal sprouting and pathfinding. In this study, we analyzed the effects of transplanted olfactory ensheathing cells (OECs) on axonal sprouting of adult facial neurons by using triple fluorescent retrograde tracing and biometrical analysis of whisking behavior. We found that 2 months after facial nerve axotomy and immediate implantation of OECs in between both nerve stumps fixed in a silicon tube, the total number of labeled neurons was increased by about 100%, compared to animals with simple facial nerve suture or entubulation in an empty conduit. This change in the number of axon sprouts was not random. The highest increase in axon number was observed in the marginal mandibular branch, whereas no changes were detected in the zygomatic branch. This increased sprouting did not improve the whisking behavior as measured by biometric video analysis. Our results demonstrate that OECs are potent inducers of axonal sprouting in vivo. Hence OEC-filled nerve counduits may be a powerful tool to enforce regeneration of a peripheral nerve under adverse conditions, e.g., after long delay between injury and surgical repair. In mixed nerves, increased axonal sprouting will improve specificity since inappropriate nerve-target connections are pruned off during preferential motor innervation. In pure motor nerves, however, OEC-mediated axonal sprouting may result in polyneuronal innveration of target muscles.

Expression of alpha-synuclein in non-apoptotic, slowly degenerating facial motoneurones.

The discovery that missense mutations in the alpha-synuclein gene represent a rare genetic cause of Parkinson's disease (PD) has had significant impact on the development of research into neurodegenerative disorders. It is becoming increasingly clear that alpha-synuclein plays a central role in the pathological process, which causes Lewy body formation and neurodegeneration in PD. Importantly, there is evidence to suggest that mutated alpha-synuclein is toxic to both nerve cells and glia. However, the regulation and function of wild-type alpha-synuclein are as yet ill defined. Using the facial nerve axotomy model, we have addressed the question whether the expression of alpha-synuclein in nerve cells may change in response to injury. We were particularly interested in testing the hypothesis that the severity of neuronal injury had an effect on alpha-synuclein metabolism. Facial nerve cut and crush, respectively, were performed in adult rats where normal facial motoneurones do not express alpha-synuclein. Following axotomy, a subset of facial motoneurones newly expressed high levels of alpha-synuclein immunoreactivity in their cell body and, occasionally, their nucleus. Significantly more nerve cells were labelled following facial nerve transection than following facial nerve crush. Confocal microscopy revealed a granular pattern of alpha-synuclein aggregation in degenerating nerve cells. Interestingly, the observed cell death phenotype was clearly non-apoptotic and developed over days or weeks rather than hours. Thus, axotomy of adult rat facial motoneurones triggers de novo expression of alpha-synuclein and this expression is associated with a non-apoptotic, slow form a neurodegeneration. In addition, the extent of alpha-synuclein expression is related to the severity of neuronal injury.

Adenoviral vector-mediated GDNF gene transfer prevents death of adult facial motoneurons.

We examined neuroprotective effects of an adenoviral vector encoding glial cell line-derived neurotrophic factor (AxCAhGDNF) on the lesioned adult rat facial motoneurons. After facial nerve avulsion, animals locally injected into the facial canal with AxCALacZ (adenovirus encoding beta-galactosidase gene) or AxCAhGDNF showed expression of beta-galactosidase activity or intense immunolabeling for GDNF in lesioned facial motoneurons, respectively. The treatment with AxCAhGDNF after avulsion significantly prevented the loss of lesioned facial motoneurons, ameliorated choline acetyltransferase immunoreactivity, and suppressed the activity of nitric oxide synthase in these neurons. These results indicate that the adenovirus-mediated gene transfer of GDNF may prevent the degeneration of motoneurons in adult humans with peripheral nerve injury and motor neuron diseases.

The use of brain slices and dissociated neurones to explore the multiplicity of actions of 5-HT in the central nervous system

The cellular actions of 5-hydroxytryptamine (5-HT) on adult and neonatal rat central neurones have been investigated in detail using a combination of in vitro slice and dissociated neurone preparations. Patch-clamp recordings from acutely dissociated adult rat dorsal raphe neurones confirm data obtained using conventional slice preparations that 5-HT activates an inwardly rectifying potassium channel through a 5-HT 1A receptor leading to hyperpolarization of the cell. Single-channel recordings indicate that this pathway requires only the involvement of a pertussis toxin-sensitive G-protein. Adult rat facial motoneurones in conventional slices are depolarized by 5-HT through a combination of mechanisms, closure of potassium channels and enhancement of the hyperpolarization-activated, cationic current, I H. Distinct receptors appear to mediate these two actions. Both mechanisms are present in visually indentified neonatal rat facial motoneurones in thin brain slices. Whole-cell patch-clamp recordings show the action of 5-HT on I H to mediate a caesium-sensitive inward current which can be mimicked by the adenylate cyclase activator, forskolin. The experimental data illustrate how a range of complimentary in vitro electrophysiological techniques can be employed to unravel neurotransmitter mechanisms and pharmacology.

Ionic mechanisms mediating 5-hydroxytryptamine- and noradrenaline-evoked depolarization of adult rat facial motoneurones.

1. The actions of 5-hydroxytryptamine (5-HT) and noradrenaline (NA) on the membrane properties of facial motoneurones in slices from the adult rat brainstem in vitro were examined using intracellular recording techniques. 2. In voltage clamp recording, hyperpolarizing voltage steps (> 20 mV), from holding potentials at or close to the resting potential, induced a slowly activating, voltage-dependent inward current possessing properties similar to the hyperpolarization-activated current (Ih) seen in other cell types. From tail current analysis two groups of facial motoneurones can be distinguished in terms of the activation range for Ih, one with a half-maximal activation at -81 mV and the other at -94 mV but with similar shapes. 3. 5-HT (120/126) and NA (21/21) depolarized facial motoneurones. The reversal potentials (Em) obtained from peak voltage amplitude I-V plots in varying extracellular potassium concentrations suggested mechanisms involving a decrease in K+ conductance. 4. Under voltage clamp, close to the resting potential, both 5-HT (39/41) and NA (13/13) evoked inward currents. 5. I-V plots and plots of 5-HT-sensitive current at different membrane potentials, obtained from currents evoked by voltage steps and measured before the development of Ih (instantaneous current), indicated that the 5-HT-evoked inward current was predominately associated with a decrease in conductance but with a range of reversal potentials for 5-HT (E5-HT) from close to, to much more negative than the reversal potential for a potassium conductance (EK). In some cases no change or increases in instantaneous conductance were observed. 6. Steady-state I-V relationships and plots of 5-HT-sensitive current, measured after development of Ih, indicated a 5-HT-associated conductance increase with a time and voltage dependence close to that of Ih, which could be abolished by extracellular caesium (2-5 mM). 7. The NA-evoked inward current was always associated with a decrease in conductance. Instantaneous and steady-state I-V relationships as well as plots of NA-sensitive current indicated a reversal potential at EK. 8. The activation curve for Ih was shifted to more positive potentials in the presence of 5-HT. The time constant for activation of Ih showed a similar shift. 9. 5-Carboxamidotryptamine (5-CT), a 5-HT receptor agonist, was selective for the enhancement of Ih and only evoked an inward current when the holding potential was within the activation range of Ih.(ABSTRACT TRUNCATED AT 400 WORDS)

Electrophysiology of adult rat facial motoneurones: the effects of serotonin (5-HT) in a novel in vitro brainstem slice

Studies of adult rat motoneurones using in vitro slice preparations are rare. We here describe a novel brainstem slice of the adult rat containing the facial motor nucleus (FMN). Data obtained for facial motoneurones (FM) by intracellular recording indicate that they display several passive and active properties seen in other rat cranial and spinal motoneurones. Bath application of serotonin (5-HT) evokes a reversible depolarization of FMs which is associated with an increase in input resistance due to a reduction in potassium permeability. This effect is unaffected by tetrodotoxin indicating a postsynaptic site of action.

Anomalous rectification in cat spinal motoneurons and effect of polarizing currents on excitatory postsynaptic potential.

Anomalous rectification in cat spinal motoneurons and effect of polarizing currents on excitatory postsynaptic potential.

RECURRENT FACILITATION OF FROG MOTONEURONS.

ArticlesRECURRENT FACILITATION OF FROG MOTONEURONSKisou Kubota, and John M. BrookhartKisou Kubota, and John M. BrookhartPublished Online:01 Nov 1963https://doi.org/10.1152/jn.1963.26.6.877MoreSectionsPDF (2 MB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInWeChat Previous Back to Top Next Download PDF FiguresReferencesRelatedInformationCited ByElectrophysiology of adult rat facial motoneurones: the effects of serotonin (5-HT) in a novel in vitro brainstem sliceJournal of Neuroscience Methods, Vol. 28, No. 1-2Excitatory postsynaptic currents in response to different synaptic inputs of frog spinal motoneuronsNeuroscience Research, Vol. 5, No. 3Effects of changes in osmolality on spinal cord activityExperimental Neurology, Vol. 68, No. 3Intracellular synaptic potentials of primate motor cortex neurons during voluntary movementBrain Research, Vol. 163, No. 1Dual effects of acetylcholine on the spontaneous activity in the isolated perfused spinal cord of the frogNeuropharmacology, Vol. 17, No. 1The pharmacology of the amphibian spinal cordProgress in Neurobiology, Vol. 11, No. 1Electrotonic coupling between frog spinal motoneurons. An electrophysiological and morphological studyBrain Research, Vol. 138, No. 2Dendritic responses of frog motoneurons produced by antidromic activationNeuroscience, Vol. 1, No. 6The morphology of motoneurons and dorsal root fibers in the frog's spinal cordBrain Research, Vol. 103, No. 2Presynaptic modulation of synaptic effectiveness of afferent and ventrolateral tract fibers in the frog spinal cordExperimental Neurology, Vol. 45, No. 3Light and electron microscopy of motoneurons and neuropile in the amphibian spinal cordBrain Research, Vol. 31, No. 1Acetylcholine depolarization of the dorsal root nerve terminals in the amphibian spinal cordInternational Journal of Neuropharmacology, Vol. 8, No. 4Inhibitory interaction between motoneurons of adjacent segments in the frog spinal cordExperimental Neurology, Vol. 23, No. 2Facilitatory and excitatory interaction between motoneurons of adjacent segments in the spinal cord of the frogExperimental Neurology, Vol. 20, No. 4Differences between monosynaptic and polysynaptic excitatory post-synaptic potentials of cat motoneurons during current injectionBrain Research, Vol. 3, No. 4Rubrally evoked unitary potentials in ventro-lateral thalamus of catElectroencephalography and Clinical Neurophysiology, Vol. 21, No. 3 More from this issue > Volume 26Issue 6November 1963Pages 877-893 https://doi.org/10.1152/jn.1963.26.6.877PubMed14084164History Published online 1 November 1963 Published in print 1 November 1963 Metrics