Radioprotective effect of a pan-caspase inhibitor in a novel model of radiation injury to the nucleus of the abducens nerve.

There is increasing evidence that neuronal cell death occurs via extrinsic (death receptors) and intrinsic (mitochondria) pathways. Radiation induces caspase activation fundamentally via the mitochondrial pathway. Caspases are the key regulators of apoptosis. Healthy male Sprague­Dawley rats were used in the present study to examine the radioprotective effect of a type of pan-caspase inhibitor, z-VAD-fmk, following radiation, to investigate the effects of caspase blockade in a model of the nucleus of the abducens nerve. z-VAD-fmk was injected intracerebroventricularly as a bolus injection (0.2 µg/h for 1 h) into rats prior to exposure to radiation. Irradiation was conducted at room temperature at a dose of radiation of 4 Gy. The present study performed immunohistochemistry, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and western blot analysis and identified no significant changes in the expression of the X-linked inhibitor of apoptosis protein (XIAP) following radiation (P>0.05). As compared with the radiation alone group, the quantification of TUNEL-positive neurons was reduced in z-VAD­fmk-treated animals following radiation (P<0.01). Inhibition of caspase induced by z-VAD­fmk reduced the expression and activation of caspase-3, -8 and -9 (P<0.01). z-VAD-fmk effectively prevented radiation-induced apoptosis and this caspase inhibitor may be a potential therapeutic target in the treatment of brain radiation injury. The nucleus of the abducens nerve may be used as a radiation injury model, providing visual information and data on the apoptotic morphology of the abducens nucleus.

Eye movements and abducens motoneuron behavior after cholinergic activation of the nucleus reticularis pontis caudalis.

STUDY OBJECTIVES: the aim of this work was to characterize eye movements and abducens (ABD) motoneuron behavior after cholinergic activation of the nucleus reticularis pontis caudalis (NRPC). METHODS: six female adult cats were prepared for chronic recording of eye movements (using the scleral search-coil technique), electroencephalography, electromyography, ponto-geniculo-occipital (PGO) waves in the lateral geniculate nucleus, and ABD motoneuron activities after microinjections of the cholinergic agonist carbachol into the NRPC. RESULTS: unilateral microinjections of carbachol in the NRPC induced tonic and phasic phenomena in the oculomotor system. Tonic effects consisted of ipsiversive rotation to the injected side, convergence, and downward rotation of the eyes. Phasic effects consisted of bursts of rhythmic rapid eye movements directed contralaterally to the injected side along with PGO-like waves in the lateral geniculate and ABD nuclei. Although tonic effects were dependent on the level of drowsiness, phasic effects were always present and appeared along with normal saccades when the animal was vigilant. ABD motoneurons showed phasic activities associated with ABD PGO-like waves during bursts of rapid eye movements, and tonic and phasic activities related to eye position and velocity during alertness. CONCLUSION: the cholinergic activation of the NRPC induces oculomotor phenomena that are somewhat similar to those described during REM sleep. A precise comparison of the dynamics and timing of the eye movements further suggests that a temporal organization of both NRPCs is needed to reproduce the complexity of the oculomotor behavior during REM sleep.

Eye movements and abducens motoneuron behavior during cholinergically induced REM sleep.

STUDY OBJECTIVES: The injection of cholinergic drugs in the pons has been largely used to induce REM sleep as a useful model to study different processes during this period. In the present study, microinjections of carbachol in the nucleus reticularis pontis oralis (NRPO) were performed to test the hypothesis that eye movements and the behavior of extraocular motoneurons during induced REM sleep do not differ from those during spontaneous REM sleep. METHODS: Six female adult cats were prepared for chronic recording of eye movements (by means of the search-coil technique) and electroencephalography, electromyography, ponto-geniculo-occipital (PGO) waves at the lateral geniculate nucleus, and identified abducens motoneuron activities after microinjections of the cholinergic agonist carbachol into the NRPO. RESULTS: Unilateral microinjections (n = 13) of carbachol in the NRPO induced REM sleep-like periods in which the eyes performed a convergence and downward rotation interrupted by phasic complex rapid eye movements associated to PGO waves. During induced-REM sleep abducens motoneurons lost their tonic activity and eye position codification, but continued codifying eye velocity during the burst of eye movements. CONCLUSION: The present results show that eye movements and the underlying behavior of abducens motoneurons are very similar to those present during natural REM sleep. Thus, microinjection of carbachol seems to activate the structures responsible for the exclusive oculomotor behavior observed during REM sleep, validating this pharmacological model and enabling a more efficient exploration of phasic and tonic phenomena underlying eye movements during REM sleep.

Upper lip blanching and diplopia associated with local anaesthesia of the inferior alveolar nerve.

A case of transient left lateral rectus nerve palsy, following an inferior alveolar nerve block to enable the surgical removal of a permanent mandibular left third molar tooth, is reported. The anatomy related to this case is considered together with suggestions for management of such patients.

Transient diplopia following maxillary local anesthetic injection.

A 36-year-old female patient developed diplopia and an ipsilateral lateral rectus paresis following local anesthetic administration to remove a left maxillary second molar. Complete resolution occurred within 3 hours. The clinical examination and management plan are reviewed for this uncommon occurrence. The relevant anatomical pathways are discussed and illustrated with photographs.

Transynaptic effects of tetanus neurotoxin in the oculomotor system.

The question whether general tetanus arises from the independent sum of multiple local tetani or results from the actions of the transynaptic tetanus neurotoxin (TeNT) in higher brain centres remains unresolved. Despite the blood-borne dissemination of TeNT from an infected wound, the access to the central nervous system is probably prevented by the blood-brain barrier. However, several long-term sequelae (e.g. autonomic dysfunction, seizures, myoclonus, and sleep disturbances) present after the subsidence of muscle spasms might be indicative of central actions that occur farther away from lower motoneurons. Subsequently, the obvious entry route is the peripheral neurons followed by the transynaptic passage to the brain. We aimed at describing the pathophysiological correlates of TeNT translocation using the oculomotor system as a comprehensive model of cell connectivity and neuronal firing properties. In this study, we report that injection of TeNT into the medial rectus muscle of one eye resulted in bilateral gaze palsy attributed to firing alterations found in the contralaterally projecting abducens internuclear neurons. Functional alterations in the abducens-to-oculomotor internuclear pathway resembled in part the classically described TeNT disinhibition. We confirmed the transynaptic targeted action of TeNT by analysing vesicle-associated membrane protein2 (VAMP2) immunoreactivity (the SNARE protein cleaved by TeNT). VAMP2 immunoreactivity decreased by 94.4% in the oculomotor nucleus (the first synaptic relay) and by 62.1% presynaptic to abducens neurons (the second synaptic relay). These results are the first demonstration of physiological changes in chains of connected neurons that are best explained by the transynaptic action of TeNT on premotor neurons as shown with VAMP2 immunoreactivity which serves as an indicator of TeNT activity.

Non-traumatic sixth nerve palsy in a young patient.

Non-traumatic sixth nerve palsy in the 20-50 years old group is unusual and a detailed history is often needed to identify serious and potentially reversible causes. We present a case of sixth sense nerve palsy in a young female with eczema for which she was treated with low-dose cyclosporin. We review the indications for and ocular side effects of cyclosporin, a drug with which most ophthalmologists will be unfamiliar.

Capsaicin administration inhibits the abducent branch but excites the thyroarytenoid branch of the recurrent laryngeal nerves in the rat.

Our recent study showed that both inspiratory and expiratory activities of the recurrent laryngeal nerve (RLN) were enhanced by capsaicin administration in the rat (Lu IJ, Ku LC, Lin JT, Lee KZ, and Hwang JC. Chin J Physiol 45: 143-154, 2002). There are two intralaryngeal branches of the RLN: one innervates the thyroarytenoid (TA) muscle and the other innervates the abductor (Abd) muscles. To examine whether these two intralaryngeal branches respond similarly to capsaicin administration, their discharges as well as activities of the phrenic nerve (PNA) and the superior laryngeal nerve (SLNA) were monitored in anesthetized and ventilated rats at normocapnia in hyperoxia. The low dose of capsaicin (0.625 microg/kg) produced a cardiopulmonary chemoreflex, showing apnea, a decrease in PNA, hypotension, and bradycardia, and significant decreases in SLNA and the activity of the Abd branch. Concurrently, there was an increase in the intralaryngeal TA activity during both apnea and the recovery from apnea. The high dose of capsaicin (1.25 microg/kg) evoked larger chemoreflexive responses and laryngeal nerve activities. In addition, both doses of capsaicin initiated a similar delay in the onset of Abd activity and SLNA but an earlier onset for the TA branch to commence during inspiration. A bilateral vagotomy abolished the laryngeal responses to capsaicin administration. However, PNA and blood pressure were enhanced with capsaicin administration after the vagotomy. These results suggest that laryngeal adduction in response to capsaicin administration is vagal afferent dependent and that it may also represent reflexive protection for the airway and lungs.

Acute sixth-nerve palsy after vincristine therapy.

The chemotherapeutic effectiveness of vincristine was first reported in 1962, and with its increased use the incidence of reported neurologic side effects has also increased. We reported a case of acute sixth-nerve palsy occurring soon after administration of vincristine for acute lymphoblastic leukemia (ALL), a previously unreported side effect.

Isolated abducens nerve palsy induced by vincristine therapy.

We report a 28-year-old man with non-Hodgkin's lymphoma who presented with acute onset of diplopia 3 weeks after the completion of combination chemotherapy with vincristine. He had a left esotropia with marked decrease in abduction. Magnetic resonance imaging scan of the brain showed thickening and enhancement of the left abducens nerve. Lymphomatous and other intracranial pathologies were excluded, and vincristine neurotoxicity was considered as the possible etiology of the abducens nerve palsy. His diplopia improved gradually, then completely resolved 4 weeks after the cessation of vincristine therapy. We concluded that isolated ocular muscle paresis can be the presenting sing of a toxic neuropathy associated with vincristine use.

Synaptic structural modification following changes in activity induced by tetanus neurotoxin in cat abducens neurons.

A low or a high dose of tetanus neurotoxin (TeNT) injected in the lateral rectus muscle of the cat causes respectively, functional block of inhibitory synapses only or of both inhibitory and excitatory synapses simultaneously in abducens neurons (González-Forero et al. [2003] J. Neurophysiol. 89:1878-1890). As a consequence, neuronal firing activity increases (at low dose) or decreases (at high dose). We investigated possible structural modifications of inhibitory synapses in response to these activity alterations induced by TeNT. We used immunofluorescence against postsynaptic (gephyrin) and presynaptic (vesicular gamma-aminobutyric acid [GABA] transporter [VGAT]) markers of inhibitory synapses in combination with cell type markers for abducens motoneurons (calcitonin gene-related peptide or choline acetyltransferase) or internuclear neurons (calretinin). Seven days after high-dose treatment, the number of gephyrin-immunoreactive (IR) clusters per 100 microm of membrane perimeter was reduced on the soma of abducens motoneurons by 55.3% and by 60.1% on internuclear neurons. Proximal dendritic gephyrin-IR clusters were also significantly altered but to a lesser degree. Partial synaptic re-establishment was observed 15 days post injection, and complete recovery occurred after 42 days. Coverage by VGAT-IR terminals was reduced in parallel. In contrast, a low dose of TeNT caused no structural alterations. With electron microscopy we estimated that overall synaptic coverage was reduced by 40% in both types of neurons after a high dose of TeNT. However, F-type terminals with postsynaptic gephyrin were preferentially lost. Thus, the ratio between F and S terminals diminished from 1.28 to 0.39 on motoneurons and from 1.26 to 0.47 on internuclear neurons. These results suggest that the maintenance of proximal inhibitory synaptic organization on central neurons is best related to neuronal activity and not to the level of inhibitory synaptic function, which was equally blocked by the high or low dose of TeNT.

Recruitment order of cat abducens motoneurons and internuclear neurons.

Abducens neurons undergo a dose-dependent synaptic blockade (either disinhibition or complete blockade) when tetanus neurotoxin (TeNT) is injected into the lateral rectus muscle at either a low (0.5) or a high dose (5 ng/kg). We studied the firing pattern and recruitment order in abducens neurons both in control and after TeNT injection. The eye position threshold for recruitment of control abducens neurons was exponentially related to the eye position and velocity sensitivities. We also found a constancy of recruitment threshold for different eye movement modalities (spontaneous, optokinetic, and vestibular). Exponential relationships were found, as well, for eye velocity sensitivity during saccades and for position and velocity sensitivities during the vestibulo-ocular reflex. Likewise, inverse relationships were found between recruitment threshold or position sensitivity with the antidromic latency in control abducens neurons. These relationships, however, did not apply following TeNT treatment. Neuronal firing after TeNT appeared either disinhibited (low dose) or depressed (high dose), but the relationships between neuronal sensitivities and recruitment still applied. However, the pattern of recruitment shifted toward the treated side as more inputs were blocked by the low- and high-dose treatments, respectively. Nonetheless, although the recruitment-to-sensitivity relationships persisted under the TeNT synaptic blockade, we conclude that synaptic inputs are determinant for establishing the recruitment threshold and recruitment spacing of abducens motoneurons and internuclear neurons.

Abducens conditioning in in vitro turtle brain stem without cerebellum requires NMDA receptors and involves upregulation of GluR4-containing AMPA receptors.

Previous work showed that in vitro abducens eyeblink classical conditioning of turtle brain stem-cerebellum preparations involved NMDA-mediated mechanisms and redistribution of GluR4-containing AMPA receptors in the abducens motor nuclei. Since conditioning can be obtained in brain stem preparations without the cerebellum, we examined whether similar mechanisms were involved during conditioning of the brain stem alone. The results showed that conditioning could not be induced in the presence of the NMDA receptor antagonist dl-2-amino-5-phosphonovaleric acid (AP-5) and that abducens nerve conditioned responses, once initiated in normal saline, were significantly attenuated in the presence of AP-5. The effects of AP-5 did not generally depress physiological responsiveness of preparations because some abducens nerve reflexes were not significantly reduced by the compound. GluR4-containing AMPA receptors in the abducens motor nuclei were significantly upregulated and positively correlated with the levels of conditioning similar to that of preparations having an intact cerebellum. Furthermore, increased GluR4 subunits after brain stem conditioning was confirmed by Western blot analysis. These results suggest that NMDA receptor-mediated mechanisms and GluR4 upregulation may mediate in vitro abducens eyeblink classical conditioning and that these mechanisms reside in the brain stem eyeblink circuitry.

Functional alterations of cat abducens neurons after peripheral tetanus neurotoxin injection.

Tetanus neurotoxin (TeNT) cleaves synaptobrevin, a protein involved in synaptic vesicle docking and fusion, thereby preventing neurotransmitter release and causing a functional deafferentation. We injected TeNT into the lateral rectus muscle of adult cats at 0.5 or 5 ng/kg (low and high dose, respectively). In the periphery, TeNT slightly slowed motor axon conduction velocity, and at high doses, partially blocked neuromuscular transmission. TeNT peripheral actions displayed time courses different to the more profound and longer-lasting central actions. Central effects were first observed 2 days postinjection and reversed after 1 mo. The low dose induce depression of inhibitory inputs, whereas the high dose produce depression of both inhibitory and excitatory inputs. Simultaneous recordings of eye movement and neuronal firing revealed that low-dose injections specifically reduced inhibition of firing during off-directed saccadic movements, while high-dose injections of TeNT affected both inhibitory and excitatory driven firing patterns. Motoneurons and abducens interneurons were both affected in a similar way. These alterations resulted in modifications in all discharge characteristic analyzed such as background firing, threshold for recruitment, and firing sensitivities to both eye position and velocity during spontaneous movements or vestibulo-ocular reflexes. Removal of inhibition after low-dose injections also altered firing patterns, and although firing activity increased, it did not result in muscle tetanic contractions. Removal of inhibition and excitation by high-dose injections resulted in a decrease in firing modulation with eye movements. Our findings suggest that the distinct behavior of oculomotor and spinal motor output following TeNT intoxication could be explained by their different interneuronal and proprioceptive control.

Short-term effects of botulinum toxin on the lateral rectus muscle of the cat.

Botulinum toxin type A (BTX) is often used as an alternative to surgery for the treatment of strabismus and many other motor or cosmetic problems. Although numerous studies established BTX as a powerful transmission-blocking agent at the neuromuscular junction, no evaluation of extraocular muscle (EOM) contractile properties after administration of BTX exists. Some anatomical studies on EOM fiber types suggested a long-term preferential effect of BTX on orbital layer, singly innervated muscle fibers. In this study, we examined the short-term effects of BTX on the contractile properties of normal lateral rectus muscle to determine the functional effect of BTX on muscle-force output over time. Measurements of muscle tension and the corresponding EMG evoked by stimulation of nerve VI were made hourly for up to 18 h following BTX administration. An intramuscular BTX injection of 2 U caused a dramatic decrease in maximum twitch and tetanic tension of the muscle in response to different frequencies of stimulation. This suppression developed gradually over time, with a concomitant reduction of EMG amplitude. No significant changes in muscle-speed-related characteristics (e.g., twitch contraction time, fusion frequency) were found. The results suggest a functional effect of BTX on all muscle fiber types, although, with the dose used, we did not observe complete muscle paralysis within the time of recording. The time course of muscle tension suppression by BTX also was frequency dependent, with the lower stimulation frequencies being more affected, suggesting that implementation of higher frequencies could still produce adequate eye movements.

Correlation between CGRP immunoreactivity and firing activity in cat abducens motoneurons.

A relationship between motoneuron activity and calcitonin gene-related peptide (CGRP) expression was previously suggested based on indirect inferences. We show here a positive correlation between CGRP immunoreactivity and firing activity in an experimental model that used tetanus neurotoxin (TeNT) to alter basal firing levels. A low dose (0.5 ng/kg) of TeNT injected in the lateral rectus muscle raised the basal firing rate of ipsilateral abducens motoneurons, estimated as the firing rate at straight-ahead gaze (F(0)); the firing rate returned to control values after 2 weeks. In contrast, a high dose (5 ng/kg) of TeNT decreased basal firing, which recovered slowly over a 7-week period. Expression of CGRP immunoreactivity by abducens motoneurons, preferentially related to betaCGRP gene expression, was analyzed during these periods of altered firing activity. The number of CGRP-immunofluorescent abducens motoneurons increased to approximately 120% by 7 days after low-dose TeNT, to include all available motoneurons in the nucleus. In addition, the average CGRP immunofluorescence optical density inside motoneurons almost doubled after 4 days and returned toward control values in the following 2 weeks. In contrast, a high-dose injection of TeNT reduced the number of CGRP-immunofluorescent motoneurons to 5.4% of control 7 days post injection, and the number returned to 77.6% after 42 days. CGRP immunofluorescence intensity inside motoneurons was also reduced. Regression analysis of F(0) values with either the number of CGRP-immunolabeled motoneurons, their average immunofluorescence intensity, or both factors combined resulted in positive correlations with regression coefficients of 0.87 or higher. Therefore, CGRP expression and firing activity in abducens motoneurons are positively correlated.

Influence of afferent synaptic innervation on the discharge variability of cat abducens motoneurones.

The discharge variability of abducens motoneurones was studied after blocking inhibitory synaptic inputs or both excitatory and inhibitory inputs by means of an intramuscular (lateral rectus) injection of either a low (0.5 ng kg(-1)) or a high dose (5 ng kg(-1)) of tetanus neurotoxin (TeNT), respectively. Motoneuronal firing increased after low-dose TeNT. High-dose treatment, however, produced a firing depression, and in some cells, a total lack of modulation in relation to eye movements. Firing became increasingly more regular with larger TeNT doses as shown by significant reductions in the coefficient of variation after low- and high-dose treatments. Similarly, autocorrelation histograms of interspike intervals increased the number of resolvable peaks twofold in low-dose-treated motoneurones and sevenfold in high-dose-treated motoneurones. The plots of standard deviation versus the mean instantaneous firing frequency showed an upward deflexion with low firing frequencies. The upward deflexion occurred in controls at 39.9 +/- 4.9 ms, an interval similar to the mean afterhyperpolarisation (AHP) duration (48.4 +/- 8.8 ms). Low-dose TeNT treatment shifted the deflexion point to 20.9 +/- 3.9 ms, whereas the high dose increased it to 60.7 +/- 6.1 ms, in spite of the fact that no differences in AHP parameters between groups were found. The density of synaptophysin-immunoreactive boutons decreased by 14 % after the low-dose treatment and 40.5 % after the high-dose treatment, indicating that protracted synaptic blockade produces elimination of synaptic boutons. It is concluded that abducens motoneurone spike variability during spontaneous ocular fixations depends largely on the balance between inhibitory and excitatory synaptic innervation.

In vitro effects of acetyl-DL-leucine (tanganil) on central vestibular neurons and vestibulo-ocular networks of the guinea-pig.

For 40 years, the amino acid acetyl-DL-leucine (or isoleucine/Tanganil) has been used in clinical practice to reduce the imbalance and autonomic signs associated with acute vertigo crises. In animal models, acetyl-DL-leucine was shown to accelerate vestibular compensation following unilateral labyrinthectomy, while having only minor effects on normal vestibular function. However, the underlying mechanisms are unknown. In this study, the effect of acetyl-DL-leucine on the activity of central vestibular neurons of the medial vestibular nucleus (MVN) and/or the overall activity of vestibular-related networks was electrophysiologically measured in brainstem slices and in the isolated, in vitro whole brain (IWB) of guinea-pig. Only moderate effects were obtained in normal animals, where both excitatory and inhibitory actions of acetyl-DL-leucine were obtained. However, intracellular recordings from MVN neurons revealed that the nature of the response depended on the resting membrane potential. The neurons excited by acetyl-DL-leucine were significantly hyperpolarized compared to nonsensitive cells, whereas the neurons inhibited by this compound tended to display higher than normal membrane potentials. In accordance with these data, acetyl-DL-leucine reduced the prominent asymmetry characterizing the vestibular-related networks of IWBs taken from previously labyrinthectomized animals, by decreasing the activity of the abnormally depolarized neurons on the hyperactive side. Altogether, our results suggest that acetyl-DL-leucine might act mainly on abnormally hyperpolarized and/or depolarized MVN neurons, by bringing back their membrane potential towards a mean value of -65 to -60 mV. Since in animal models, acute vestibular disorders are associated with asymmetrical spontaneous activities of MVN neurons, this study suggests how acetyl-DL-leucine may reduce acute, vestibular-related imbalances in humans.