Angel M. Pastor

Effects of botulinum neurotoxin type A on abducens motoneurons in the cat: ultrastructural and synaptic alterations

The synaptic alterations induced in abducens motoneurons by the injection of 3 ng/kg of botulinum neurotoxin type A into the lateral rectus muscle were studied using ultrastructural and electrophysiological techniques. Motoneurons identified by the retrograde transport of horseradish peroxidase showed a progressive synaptic stripping already noticeable by four days post-injection which increased over the study period. By 35 days post-injection, the normal coverage of motoneurons by synaptic boutons (66.4 +/- 4.0%) significantly decreased to 27.2 +/- 4.0%. Synaptic boutons detached by a widening of the subsynaptic space but remained apposed by synaptic contacts and desmosomes to the motoneuron. Detachment did not affect equally flat and round vesicle-containing boutons. The control motoneuron had almost equal numbers of both types of boutons, but after 35 days post-injection the ratio of round to flat vesicle-containing boutons was 1.20 +/- 0.01. Synaptic boutons impinging on motoneurons showed signs of alterations in membrane turnover, as indicated by an increase in the number of synaptic vesicles and a decrease in the number of coated vesicles and synaptic vesicles near the active zone. Abducens motoneurons had a transient increase in soma size by 15 days that returned to normal at 35 days, but no signs of chromatolysis or organelle degeneration were seen. Accompanying the swelling of motoneurons, a 15-fold increase in the number of spines, very infrequent in controls, was observed. Spines located in the soma and proximal dendritic trunk received synaptic contacts from both flat and round vesicle-containing boutons that could be either partly detached or completely attached to the motoneuron. An increased turnover of the plasmatic membrane of the motoneuron was observed, as indicated by a four-fold increase in the number of somatic coated vesicles. Animals were implanted with bipolar electrodes in the ampulla of both horizontal semicircular canals for evoking contralateral excitatory and ipsilateral inhibitory postsynaptic potentials. Motoneurons were antidromically identified from the lateral rectus muscle. Synaptic potentials of vestibular origin were recorded in abducens motoneurons. In the period between two and six days post-injection, a complete abolition of inhibitory synaptic potentials was observed. By contrast, excitatory synaptic potentials remained, but were reduced by 82%. The imbalance between excitatory and inhibitory inputs to motoneurons induced a progressive increase of firing frequency within a few stimuli applied to the contralateral canal. Between 7 and 15 days post-injection, both excitatory and inhibitory postsynaptic potentials were virtually abolished and remained so up to the longest time checked (105 days). Some motoneurons recorded beyond 60 days post-injection showed signs of recovery of excitatory postsynaptic potentials. During the whole time-span studied, presynaptic wavelets were present, indicating no affecting of the conduction of afferent volleys to the abducens nucleus. Taken together, these data indicate that botulinum neurotoxin at high doses causes profound synaptic alterations in motoneurons responsible for the effects seen in the behavior of motoneurons recorded in alert animals.

Complementary Actions of BDNF and Neurotrophin-3 on the Firing Patterns and Synaptic Composition of Motoneurons

Neurotrophins, as target-derived factors, are essential for neuronal survival during development, but during adulthood, their scope of actions widens to become also mediators of synaptic and morphological plasticity. Target disconnection by axotomy produces an initial synaptic stripping ensued by synaptic rearrangement upon target reinnervation. Using abducens motoneurons of the oculomotor system as a model for axotomy, we report that trophic support by brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) or a mixture of both, delivered to the stump of severed axons, results in either the prevention of synaptic stripping when administered immediately after lesion or in a promotion of reinnervation of afferents to abducens motoneurons once synaptic stripping had occurred, in concert with the recovery of synaptic potentials evoked from the vestibular nerve. Synaptotrophic effects, however, were larger when both neurotrophins were applied together. The axotomy-induced reduction in firing sensitivities related to eye movements were also restored to normal values when BDNF and NT-3 were administered, but discharge characteristics recovered in a complementary manner when only one neurotrophin was used. This is the first report to show selective retrograde trophic dependence of circuit-driven firing properties in vivo indicating that NT-3 restored the phasic firing, whereas BDNF supported the tonic firing of motoneurons during eye movement performance. Therefore, our data report a link between the synaptotrophic actions of neurotrophins, retrogradely delivered, and the alterations of neuronal firing patterns during motor behaviors. These trophic actions could be responsible, in part, for synaptic rearrangements that alter circuit stability and synaptic balance during plastic events of the brain.

Limits to the capacity of transplants of olfactory glia to promote axonal regrowth in the CNS.

Olfactory bulb ensheathing cell (OBEC) transplants promoted axonal regeneration in the spinal cord dorsal root entry zone and in the corticospinal tract. However, OBECs failed to promote abducens internuclear neuron axon regeneration when transplanted at the site of nerve fibre transection. In experiments performed in both cats and rats, OBECs survived for up to 2 months, lining themselves up along the portion of the regrowing axons proximal to the interneuron cell body. However, OBECs migrated preferentially towards abducens somata, in the direction opposite to the oculomotor nucleus target. OBECs seem to promote nerve fibre regeneration only where preferred direction of glial migration coincides with the direction of axonal growth towards its target.

Dose-dependent, central effects of botulinum neurotoxin type A A pilot study in the alert behaving cat

Article abstract-We investigated, in alert behaving cats, the long-term effects of botulinum neurotoxin (BoNT) type A injected into the lateral rectus muscle of the eye. We studied orthodromic field potentials recorded in the injected muscle, eye movements, and the discharge characteristics of the innervating abducens motoneurons. Single BoNT injections at doses from 0.01 to 0.3 ng/kg reduced, or even completely eliminated, eye movements in the abducting direction for up to 2 months without affecting the motoneuron discharge profile that remained related to actual eye movements of the contralateral unparalyzed eye. This result indicates that abducens motoneurons were still under the influence of the ocular motor central control system regardless of their ineffective action on lateral rectus muscle fibers. We also conclude that paralysis per se is not enough to initiate axotomy-like neural responses in ocular motoneurons. The injection of BoNT at a dose of 3 ng/kg produced significant changes in the discharge pattern of abducens motoneurons lasting up to 3 months-the maximum time checked. This finding was probably due to retrograde and, perhaps, transneuronal effects of BoNT when injected in a high dose. The results give some indications of the maximum allowable dose that can be used without the induction of unwanted side effects in the motoneuronal pool innervating the injected muscle. NEUROLOGY 1997;48: 456-464

Role of GABA in the extraocular motor nuclei of the cat : a postembedding immunocytochemical study

The GABAergic innervation of the extraocular motor nuclei in the cat was evaluated using postembedding immunocytochemical techniques. The characterization of GABA-immunoreactive terminals in the oculomotor nucleus was carried out at the light and electron microscopic levels. GABA-immunopositive puncta suggestive of boutons were abundant in semithin sections throughout the oculomotor nucleus, and were found in close apposition to somata and dendrites. Ultrathin sections revealed an extensive and dense distribution of GABA-immunoreactive synaptic endings that established contacts with the perikarya and proximal dendrites of motoneurons and were also abundant in the surrounding neuropil. GABAergic boutons were characterized by the presence of numerous mitochondria, pleiomorphic vesicles and multiple small symmetrical synaptic contacts. The trochlear nucleus exhibited the highest density of GABAergic terminations. In contrast, scarce GABA immunostaining was associated with the motoneurons and internuclear neurons of the abducens nucleus. In order to further elucidate the role of this neurotransmitter in the oculomotor system, retrograde tracing of horseradish peroxidase was used in combination with the GABA immunostaining. First, medial rectus motoneurons were identified following horseradish peroxidase injection into the corresponding muscle. This was carried out because of the peculiar afferent organization of medial rectus motoneurons that contrasts with the remaining extraocular motoneurons, especially their lack of direct vestibular inhibition. Semithin sections of the oculomotor nucleus containing retrogradely labeled medial rectus motoneurons and immunostained for GABA revealed numerous immunoreactive puncta in close apposition to horseradish peroxidase-labeled somata and in the surrounding neuropil. At the ultrastructural level, GABAergic terminals established synaptic contacts with the somata and proximal dendrites of medial rectus motoneurons. Their features and density were similar to those found in the remaining motoneuronal subgroups of the oculomotor nucleus. Second, oculomotor internuclear neurons were identified following the injection of horseradish peroxidase into the abducens nucleus to determine whether they could give rise to GABAergic terminations in the abducens nucleus. About 20% of the oculomotor internuclear neurons were doubly labeled by retrograde horseradish peroxidase and GABA immunostaining. A high percentage (80%) of the oculomotor internuclear neurons projecting to the abducens nucleus showed immunonegative perikarya. It was concluded that the oculomotor internuclear pathway to the abducens nucleus comprises both GABAergic and non-GABAergic neurons and, at least in part, the GABA input to the abducens nucleus originates from this source. It is suggested that this pathway might carry excitatory and inhibitory influences on abducens neurons arising bilaterally.

Chapter 29 Involvement of cerebellar cortex and nuclei in the genesis and control of unconditioned and conditioned eyelid motor responses

The eyelid motor system of the cat was used here for the study of the kinetic properties of reflex and conditioned lid movements, and of the role played by the cerebellum in the acquisition and/or performance of both types of motor responses. Spontaneous blinks, eyelid reflex responses, eye-guided lid movements and conditioned lid responses were recorded in alert cats in simultaneity with unitary and field electrical activity of cerebellar cortex and nuclear zones related to the eyelid motor system. Results indicate that nuclear unitary activity does not precede unconditioned or conditioned lid responses, but that cerebellar nuclei are directly involved in the performance of the late components of reflex lid movements and in the acquisition of conditioned lid responses.

Effects of target depletion on adult mammalian central neurons: Morphological correlates

The morphological sequelae induced by target removal were studied on adult cat abducens internuclear neurons at both the somata and terminal axon arborization levels. The neuronal target--the medial rectus motoneurons of the oculomotor nucleus--was selectively destroyed by the injection of toxic ricin into the medial rectus muscle. Retrograde labeling with horseradish peroxidase demonstrated the survival of the entire population of abducens internuclear neurons up to one year after target removal. However, soma size was reduced by about 20% three months postlesion and maintained for one year. At the ultrastructural level, a considerable deafferentation of abducens internuclear neurons was observed at short intervals (i.e. 10 days after lesion). Large regions of the plasmalemma appeared devoid of presynaptic boutons but were covered instead by glial processes. The detachment of synaptic endings was selective on abducens internuclear neurons since nearby motoneurons always showed a normal synaptic coverage. By one month, abducens internuclear neurons recovered a normal density of receiving axosomatic synapses. Anterogradely biocytin-labeled axon terminals of abducens internuclear neurons remained in place after the lesion of medial rectus motoneurons, although with a progressive decrease in density. Ultrastructural examination of the oculomotor nucleus 10 days after the lesion revealed numerous empty spaces left by the dead motoneurons. Targetless boutons were observed surrounded by large extracellular gaps, still apposed to remnants of the postsynaptic membrane or, finally, ensheathed by glial processes. At longer intervals (> one month), the ultrastructure of the oculomotor nucleus was re-established and labeled boutons were observed contacting either unidentified dendrites within the neuropil or the soma and proximal dendrites of the oculomotor internuclear neurons, that project to the abducens nucleus. Labeled boutons were never found contacting with the oculomotor internuclear neurons either in control tissue or at short periods after ricin injection. These results indicate that the availability of undamaged neurons close to the lost target motoneurons might support the long-term survival of abducens internuclear neurons. Specifically, the oculomotor internuclear neurons, which likely suffer a partial deafferentation after medial rectus motoneuron loss, constitute a potential new target for the abducens internuclear neurons. The reinnervation of a new target might explain the recovery of synaptic and firing properties of abducens internuclear neurons after medial rectus motoneuron lesion, which occurred with a similar time course, as described in the accompanying paper [de la Cruz R. R. et al. (1994) Neuroscience 58, 81-97.].

Regulation of Gephyrin Cluster Size and Inhibitory Synaptic Currents on Renshaw Cells by Motor Axon Excitatory Inputs

Renshaw cells receive a high density of inhibitory synapses characterized by large postsynaptic gephyrin clusters and mixed glycinergic/GABAergic inhibitory currents with large peak amplitudes and long decays. These properties appear adapted to increase inhibitory efficacy over Renshaw cells and mature postnatally by mechanisms that are unknown. We tested the hypothesis that heterosynaptic influences from excitatory motor axon inputs modulate the development of inhibitory synapses on Renshaw cells. Thus, tetanus (TeNT) and botulinum neurotoxin A (BoNT-A) were injected intramuscularly at postnatal day 5 (P5) to, respectively, elevate or reduce motor axon firing activity for ∼2 weeks. After TeNT injections, the average gephyrin cluster areas on Renshaw cells increased by 18.4% at P15 and 28.4% at P20 and decreased after BoNT-A injections by 17.7% at P15 and 19.9% at P20. The average size differences resulted from changes in the proportions of small and large gephyrin clusters. Whole-cell recordings in P9-P15 Renshaw cells after P5 TeNT injections showed increases in the peak amplitude of glycinergic miniature postsynaptic currents (mPSCs) and the fast component of mixed (glycinergic/GABAergic) mPSCs compared with controls (60.9% and 78.9%, respectively). GABAergic mPSCs increased in peak amplitude to a smaller extent (45.8%). However, because of the comparatively longer decays of synaptic GABAergic currents, total current transfer changes after TeNT were similar for synaptic glycine and GABAA receptors (56 vs 48.9% increases, respectively). We concluded that motor axon excitatory synaptic activity modulates the development of inhibitory synapse properties on Renshaw cells, influencing recruitment of postsynaptic gephyrin and glycine receptors and, to lesser extent, GABAA receptors.

Effects of target depletion on adult mammalian central neurons: Functional correlates

The physiological signals and patterns of synaptic connectivity that CNS neurons display after the loss of their target cells were evaluated in adult cats for one year. Abducens internuclear neurons were chosen as the experimental model because of their highly specific projection onto the medial rectus motoneurons of the oculomotor nucleus. Selective death of medial rectus motoneurons was induced by the injection into the medial rectus muscle of ricin, a potent cytotoxic lectin that leaves the presynaptic axons intact. The electrical activity of antidromically identified abducens internuclear neurons was recorded in chronic alert animals, during both spontaneous and vestibularly induced eye movements, before and after target removal. During the three weeks that followed ricin injection, abducens internuclear neurons exhibited several firing-related abnormal properties. There was an overall reduction in firing rate with a corresponding increase in the eye position threshold for recruitment. In addition, neuronal sensitivities to eye position and velocity were significantly decreased with respect to control data. Bursting activity was also altered since low-frequency delayed burst accompanied the saccades in the on-direction and, occasionally, internuclear neurons exhibited low-frequency discharges associated with off-directed saccades. Intracellular recordings carried out seven and 15 days after ricin injection demonstrated no significant changes in their electrical properties, although a marked depression of synaptic transmission was evident. The amplitude of both excitatory and inhibitory postsynaptic potentials of vestibular origin was reduced by 60-85% with respect to controls. However, postsynaptic potentials recorded one month after ricin injection showed normal amplitude values which persisted unaltered one year after target loss. Recovery of synaptic transmission occurred at the same time as the re-establishment of normal eye-related signals in the discharge pattern of abducens internuclear neurons recorded in alert cats from days 25-30 post lesion. The functional restoration of firing properties was maintained in the long term (one year). Conversely, abducens motoneurons showed normal firing and synaptic patterns at all time intervals analysed. These results demonstrate that, after an initial period of altered physiological properties, abducens internuclear neurons survive the loss of their target motoneurons and regain a normal discharge pattern and afferent synaptic connections.

Effects of botulinum neurotoxin type A on abducens motoneurons in the cat: alterations of the discharge pattern

The discharge characteristics that abducens motoneurons exhibit after paralysis of the lateral rectus muscle with botulinum neurotoxin type A were studied in the alert cat. Antidromically identified motoneurons were recorded during both spontaneous and vestibularly induced eye movements. A single injection of 0.3 ng/kg produced a complete paralysis of the lateral rectus muscle lasting for about 12-15 days, whereas after 3 ng/kg the paralysis was still complete at the longest time checked, three months. Motoneurons recorded under the effect of the low dose showed differences in their sensitivities to both eye position and velocity according to the direction of the previous and ongoing movements, respectively. These directional differences could be explained by post-saccadic adaptation of the non-injected eye in the appropriate direction for reducing ocular misalignment. Thus, backward and forward post-saccadic drifts accompanied on- and off-directed saccades, respectively. The magnitude of the drift was similar to the magnitude of changes in eye position sensitivity. The discharge of the high-dose-treated motoneurons could be described in a three-stage sequence. During the initial 10-12 days, motoneuronal discharge resembled the effects of axotomy, particularly in the loss of tonic signals and the presence of exponential-like decay of firing after saccades. In this stage, the conduction velocity of abducens motoneurons was reduced by 21.4%. The second stage was characterized by an overall reduction in firing rate towards a tonic firing at 15-70 spikes/s. Motoneurons remained almost unmodulated for all types of eye movement and thus eye position and velocity sensitivities were significantly reduced. Tonic firing ceased only when the animal became drowsy, but was restored by alerting stimuli. In addition, the inhibition of firing for off-directed saccades was more affected than the burst excitation during on-directed saccades, since in many cells pauses were almost negligible. These alterations could not be explained by adaptational changes in the movement of the non-injected eye. Finally, after 60 days the initial stages of recovery were observed. The present results indicate that the high dose of botulinum neurotoxin produces effects on the motoneuron not attributable to the functional disconnection alone, but to a direct effect of the neurotoxin in the motoneuron and/or its synaptic inputs.