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Neurotrophic factors delivered from target muscles are essential for motoneuronal survival, mainly during development and early postnatal maturation. It has been shown that the disconnection between motoneurons and their innervated muscle by means of axotomy produces a vast neuronal death in neonatal animals. In the present work, we have evaluated the effects of different neurotrophic factors on motoneuronal survival after neonatal axotomy, using as a model the motoneurons innervating the extraocular eye muscles. With this purpose, neonatal rats were monocularly enucleated at the day of birth (postnatal day 0) and different neurotrophic treatments (NGF, BDNF, NT-3, GDNF and the mixture of BDNF+GDNF) were applied intraorbitally by means of a Gelfoam implant (a single dose of 5 μg of each factor). We first demonstrated that extraocular eye muscles of neonatal rats expressed these neurotrophic factors and therefore constituted a natural source of retrograde delivery for their innervating motoneurons. By histological and immunocytochemical methods we determined that all treatments significantly rescued extraocular motoneurons from axotomy-induced cell death. For the dose used, NGF and GDNF were the most potent survival factors for these motoneurons, followed by BDNF and lastly by NT-3. The simultaneous administration of BDNF and GDNF did not increase the survival-promoting effects above those obtained by GDNF alone. Interestingly, the rescue effects of all neurotrophic treatments persisted even 30 days after lesion. The administration of these neurotrophic factors, with the exception of NT-3, also prevented the loss of the cholinergic phenotype observed by 10 days after axotomy. At the dosage applied, NGF and GDNF were revealed again as the most effective neuroprotective agents against the axotomy-induced decrease in ChAT. Two remarkable findings highlighted in the present work that contrasted with other motoneuronal types after neonatal axotomy: first, the extremely high efficacy of NGF as a neuroprotective agent and, second, the long-lasting effects of neurotrophic administration on cell survival and ChAT expression in extraocular motoneurons., (Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.)

Tetanus neurotoxin (TeNT) is a blocker of synaptic vesicle exocytosis in central synapses with preferential affinity for inhibitory neurotransmission. Following its intramuscular injection, TeNT is retrogradely and trans-synaptically transported towards the premotor terminals. Therefore, we have used TeNT as a tool to study the consequences of functional deafferentation on motoneurons following its peripheral administration. For this, we injected the toxin into the lateral rectus muscle at doses of 5 or 0.5 ng/kg and recorded the discharge activity of abducens motoneurons and internuclear neurons in the alert cat. Our results showed that: (i) TeNT blocked selectively the afferent inhibitory signals on abducens neurons only when used at a low dose, whereas both excitatory and inhibitory synaptic drive was lost after the high dose treatment; (ii) all effects were reversible within one month; and (iii) strikingly, the internuclear neurons of the abducens nucleus showed similar discharge alterations to the motoneurons, suggesting a TeNT action on shared common afferences.

In this study, we investigated the effects of long-term synaptic blockade on postsynaptic receptor clustering at central inhibitory glycinergic synapses. High doses of botulinum neurotoxin type A injected in the lateral rectus muscle completely abolishes inhibitory postsynaptic potentials onto abducens motoneurons within 2 days postinjection, and transmission remains blocked for at least 2 months. Using this model, we analyzed the expression of gephyrin, a glycine receptor clustering protein, on the membrane of motoneuron somata after botulinum neurotoxin type A injection in their target muscle. Immunofluorescence or electron microscopy immunohistochemistry revealed gephyrin-immunoreactive clusters (most < 0.5 microm in diameter) densely covering the surface of control abducens motoneurons. Ultrastructurally, presynaptic terminals containing flattened synaptic vesicles (F terminals) were found associated with multiple gephyrin-immunoreactive postsynaptic densities (average 1.24 gephyrin clusters/F+ profile). No significant changes in gephyrin-immunoreactive clusters were observed at 5 days postinjection, but we found significant reductions (25-40%) in the density of gephyrin clusters 19 and 35 days postinjection. Hence, the physiological alterations reported in this model precede structural changes on postsynaptic receptor cluster density. The decrease in gephyrin-immunoreactive clusters was paralleled by reductions in synaptic covering (F+ terminals per 100 microm of membrane). Presumed inactive F+ terminals that remained attached to the motoneuron surface displayed normal gephyrin-immunoreactive clusters; however, the pre- and postsynaptic membranes in between synaptic active zones frequently appeared separated by enlarged extracellular spaces. We concluded that postsynaptic receptor cluster dissolution seemed more directly related to terminal retraction than to inactivity alone.

Calcium-binding proteins have been shown to be excellent markers of specific neuronal populations. We aimed to characterize the expression of calcium-binding proteins in identified populations of the cat extraocular motor nuclei by means of immunohistochemistry against parvalbumin, calretinin, and calbindin D-28k. Abducens, medial rectus, and trochlear motoneurons were retrogradely labeled with horseradish peroxidase from their corresponding muscles. Oculomotor and abducens internuclear neurons were retrogradely labeled after horseradish peroxidase injection into either the abducens or the oculomotor nucleus, respectively. Parvalbumin staining produced the highest density of immunoreactive terminals in all extraocular motor nuclei and was distributed uniformly. Around 15-20% of the motoneurons were moderately stained with antibody against parvalbumin, but their axons were heavily stained, indicating an intracellular segregation of parvalbumin. Colchicine administration increased the number of parvalbumin-immunoreactive motoneurons to approximately 85%. Except for a few calbindin-immunoreactive trochlear motoneurons (1%), parvalbumin was the only marker of extraocular motoneurons. Oculomotor internuclear neurons identified from the abducens nucleus constituted a nonuniform population, because low percentages of the three types of immunostaining were observed, calbindin being the most abundant (28.5%). Other interneurons located within the boundaries of the oculomotor nucleus were mainly calbindin-immunoreactive. The medial longitudinal fascicle contained numerous parvalbumin- and calretinin-immunoreactive but few calbindin-immunoreactive axons. The majority of abducens internuclear neurons projecting to the oculomotor nucleus (80.7%) contained calretinin. Moreover, the distribution of calretinin-immunoreactive terminals in the oculomotor nucleus overlapped that of the medial rectus motoneurons and matched the anterogradely labeled terminal field of the abducens internuclear neurons. Parvalbumin immunostained 42% of the abducens internuclear neurons. Colocalization of parvalbumin and calretinin was demonstrated in adjacent semithin sections, although single-labeled neurons were also observed. Therefore, calretinin is proven to be a good marker of abducens internuclear neurons. From all of these data, it is concluded that parvalbumin, calretinin, and calbindin D-28k selectively delineate certain neuronal populations in the oculomotor system and constitute valuable tools for further analysis of oculomotor function under normal and experimental conditions.

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.

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.

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.

In this review we have attempted to summarize present knowledge concerning the regulatory role of target cells on the expression and maintenance of the neuronal phenotype during adulthood. It is well known that in early developmental stages the survival of neurons is maintained by specific neurotrophic factors derived from their target tissues. Neuronal survival is not the only phenotype that is regulated by target-derived neurotrophic factors since the expression of electrophysiological and cytochemical properties of neurons is also affected. However, a good deal of evidence indicates that the survival of neurons becomes less dependent on their targets in the adult stage. The question is to what extent are target cells still required for the maintenance of the pre-existing or programmed state of the neuron; i.e., what is the functional significance of target-derived factors during maturity? Studies addressing this question comprise a variety of neuronal systems and technical approaches and they indicate that trophic interactions, although less apparent, persist in maturity and are most easily revealed by experimental manipulation. In this respect, research has been directed to analyzing the consequences of disconnecting a group of neurons from their target-by either axotomy or selective target removal using different neurotoxins-and followed (or not) by the implant of a novel target, usually a piece of embryonic tissue. Numerous alterations have been described as taking place in neurons following axotomy, affecting their morphology, physiology and metabolism. All these neuronal properties return to normal values when regeneration is successful and reinnervation of the target is achieved. Nevertheless, most of the changes persist if reinnervation is prevented by any procedure. Although axotomy may represent, besides target disconnection, a cellular lesion, alternative approaches (e.g., blockade of either the axoplasmic transport or the conduction of action potentials) have been used yielding similar results. Moreover, in the adult mammalian central nervous system, neurotoxins have been used to eliminate a particular target selectively and to study the consequences on the intact but target-deprived presynaptic neurons. Target depletion performed by excitotoxic lesions is not followed by retrograde cell death, but targetless neurons exhibit several modifications such as reduction in soma size and in the staining intensity for neurotransmitter-synthesizing enzymes. Recently, the oculomotor system has been used as an experimental model for evaluating the functional effects of target removal on the premotor abducens internuclear neurons whose motoneuronal target is destroyed following the injection of toxic ricin into the extraocular medial rectus muscle. The functional characteristics of these abducens neurons recorded under alert conditions simultaneously with eye movements show noticeable changes after target loss, such as a general reduction in firing frequency and a loss of the discharge signals related to eye position and velocity. Nevertheless, the firing pattern of these targetless abducens internuclear neurons recovers in parallel with the establishment of synaptic contacts on a presumptive new target: the small oculomotor internuclear neurons located in proximity to the disappeared target motoneurons. The possibility that a new target may restore neuronal properties towards a normal state has been observed in other systems after axotomy and is also evident from experiments of transplantation of immature neurons into the lesioned central nervous system of adult mammals. It can be concluded that although target-derived factors may not control neuronal survival in the adult nervous system, they are required for the maintenance of the functional state of neurons, regulating numerous aspects of neuronal structure, chemistry and electro-physiology.(ABSTRUCT TRUNCATED)

1. The effects of botulinum neurotoxin (BoTx) injected into the lateral rectus muscle were examined in alert cats by recording the extracellular activity of abducens motoneurons during spontaneous eye movements. 2. A single high dose (3 ng/kg) of BoTx produced a complete paralysis of abduction that lasted for more than 2 mo. In addition, changes were found in the discharge pattern of abducens motoneurons. Motoneurons discharged steadily at a low firing rate (15-50 spikes/s), which in some instances showed a complete independence of eye position. Their increases in activity during ON-directed saccades were markedly reduced with respect to controls. The loss of inhibitory signals for OFF-directed saccades was even more evident. 3. A low dose (0.3 ng/kg) of BoTx also produced a paralysis of the lateral rectus muscle that lasted for approximately 1 mo. In this case, only minor modifications in the firing characteristics of abducens motoneurons were observed. 4. The present findings indicate that the effects of BoTx observed in the discharge pattern of abducens motoneurons might be due not only to target disconnection, but also to a central action of the neurotoxin on the motoneuron.

The fate of abducens internuclear neurons was studied after the loss of their target medical rectus motoneurons of the oculomotor nucleus in adult cats. Target motoneurons were killed by injecting ricin, a toxin lectin, into the medial rectus muscle of the eye. The entire population of abducens internuclear neurons survived the loss of target for a long time (1 year), although they exhibited an initial critical period after target loss characterized by a marked reduction of afferent synaptic transmission. One month later, the normal parameters of synaptic transmission resumed. In contrast, the density of axonal terminals of abducens internuclear neurons in the oculomotor nucleus progressively decreased during the year following target loss. Evidence is presented for the reinnervation of a new neuronal target within the oculomotor nucleus that might support both the long-term survival and the recovery of physiological characteristics in the abducens internuclear neurons.

The electrical activity of antidromically identified abducens internuclear neurons selectively deprived of their target motoneurons was recorded in chronic alert cats. Target motoneurons were killed by the injection of the cytotoxic lectin of Ricinus communis into the medial rectus muscle. Following target removal, the discharge pattern of abducens internuclear neurons showed an overall decrease in firing rate, a significant reduction in their sensitivity to eye position and velocity, and the presence of anomalous responses such as bursts of spikes associated with off-directed saccades. The decreased excitability of abducens internuclear neurons correlated well with a marked reduction in the synaptic efficacy of their inputs. Thus, both excitatory and inhibitory synaptic potentials of vestibular origin showed a noticeable decrease in amplitude. The alterations in firing properties and synaptic transmission were only observed during an initial period of 3 weeks following ricin injection. Within 1 month the electrophysiological parameters returned to control values and remained unaltered for 1 year. Retrograde labelling of abducens internuclear neurons revealed that no cell death occurred after target loss. The anterograde axonal labelling of these neurons showed a progressive decrease in the density of their axonal terminals, and no sign of redistribution to other areas was found. These findings indicate that abducens internuclear neurons are not dependent on the presence of their natural target cells, either for the survival or for the maintenance of appropriate physiological signals.

The aim of this investigation was to study the behavior of identified abducens motoneurons in the chronic cat following a single injection of toxic ricin into the lateral rectus muscle. Lateral rectus electromyographic potentials induced by VIth nerve stimulation disappeared, and abducens antidromic field potentials decreased by 90% 3 days following ricin injection. Several abnormalities and a significant decrease in eye position and velocity sensitivities were observed in motoneuron activity up to 8-10 days following ricin injection. Contrary to a previous report for axotomized abducens motoneurons, no functional sign of recovery was observed. Histological analysis showed a survival of 10-15% of the abducens motoneuron population 10 days following ricin injection. From this time on, recorded motoneurons behaved like controls, but showed a specific retraction signal suggesting an exclusive projection onto the retractor bulbi muscle. Although intermingled in the nucleus with motoneurons, no recorded abducens internuclear interneuron was affected by the ricin during one month following the injection.

The morphological and physiological integrity of abducens internuclear interneurons was evaluated following chemical removal of their almost exclusive target, i.e. the contralateral medial rectus motoneuron population. Motoneuron death was induced by toxic ricin injection into the ipsilateral medial rectus muscle. The main advantage of this method is that target removal is not associated with presynaptic axon damage. The activity of identified abducens internuclear interneurons was recorded before and after target removal during spontaneous eye movements in the chronic cat preparation. Several abnormalities and a remarkable decrease in eye position and velocity sensitivities were observed in the discharge pattern of these neurons for a period of 15-20 days following target removal. After that time all recorded interneurons behaved normally. A parallel morphological study showed the absence of any abducens internuclear interneuron death. These results indicate that, after a critical period, the abducens internuclear interneuron population recovers its normal behavior following target removal with no evidence of neuronal loss.