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.