A ´ lvarez, and A ´ ngel M. Pastor. Functional alterations of cat abducens neurons after peripher... more A ´ lvarez, and A ´ ngel M. Pastor. 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 neurotrans-mitter 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 ex-citation 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.
A relationship between motoneuron activity and calcitonin gene-related peptide (CGRP) expression ... more 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 CGRP gene expression, was analyzed during these periods of altered firing activity. The number of CGRP-immunofluorescent abducens motoneurons increased to 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.
Tetanus neurotoxin (TeNT) is a blocker of synaptic vesicle exocytosis in central synapses with pr... more Tetanus neurotoxin (TeNT) is a blocker of synaptic vesicle exocytosis in central synapses with preferential af®nity 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.
A low or a high dose of tetanus neurotoxin (TeNT) injected in the lateral rectus muscle of the ca... more 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. Neu-rophysiol. 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-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 neu-rons (calretinin). Seven days after high-dose treatment, the number of gephyrin-immunoreactive (IR) clusters per 100 m of membrane perimeter was reduced on the soma of abducens motoneu-rons 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.
The discharge characteristics that abducens motoneurons exhibit after paralysis of the lateral re... more 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. 1997 IBRO. Published by Elsevier Science Ltd.
Calcium-binding proteins have been shown to be excellent markers of specific neuronal populations... more 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 aim of the present work was to characterize the axotomy-induced changes in the discharge prop... more The aim of the present work was to characterize the axotomy-induced changes in the discharge properties of central nervous system neurons recorded in the alert behaving animal. The abducens internuclear neurons of the adult cat were the chosen model. The axons of these neurons course through the contralateral medial longitudinal fascicle and contact the medial rectus motoneurons of the oculomotor nucleus. Axotomy was carried out by the unilateral transection of this fascicle (right side) and produced immediate oculomotor deficits, mainly the incapacity of the right eye to adduct across the midline. Extracellular single-unit recording of abducens neurons was carried out simultaneously with eye movements. The main alteration observed in the firing of these axotomized neurons was the overall decrease in firing rate. During eye fixations, the tonic signal was reduced, and, on occasion, a progressive decay in firing rate was observed. On-directed saccades were not accompanied by the high-frequency spike burst typical of controls; instead, there was a moderate increase in firing. Similarly, during the vestibular nystagmus, neurons hardly modulated during both the slow and the fast phases. Linear regression analysis between firing rate and eye movement parameters showed a significant reduction in eye position and velocity sensitivities with respect to controls, during both spontaneous and vestibularly induced eye movements. These firing alterations were observed during the 3 month period of study after lesion, with no sign of recovery. Conversely, abducens motoneurons showed no significant alteration in their firing pattern. Therefore, axotomy produced long-lasting changes in the discharge characteristics of abducens internuclear neurons that presumably reflected the loss of afferent oculomotor signals. These alterations might be due to the absence of trophic influences derived from the target.
The goal of this work was to compare the distribution and morphology of neurons projecting to the... more The goal of this work was to compare the distribution and morphology of neurons projecting to the oculomotor nucleus in goldfish with those preüously des"iib"¿ n áth". vertebrate groups. Afferent neurons were revealed by retrográde hbetittg with horseradish peroxidase. The tracer was electrophoretically injected intó the oculomlotor nucleus. The location of the injection site was determined by the antidromic field potential elicited in the oculomotor nucleus by electrical stimulation of the oculomotor nervé. Labeled axons whose trajectories could be reconstructed were restricted to the medial longitudinal fasciculus. In order of quantitative importance, the afferent areas to the oculomotoi nucleus were: (1) the ipsilateral anterior nucleus and the contralateral tangential and descenüng nuclei ;Í;h; octaval column. Furthermore, a few labeled cells were found dorsomedially to the caudal pole of the unlabeled anterior octaval nucleus; (2) the contralateral abducens nucleus. The übeled internuclear neutons were arranged in two groups within and 500 pm behind the caudal subdiüsion of the abducens nucleus; (B) a few hbéled cells were observed in the rhombence_ phalic reticular formation near the abducens nucleus, most of which were contralateral to the injection site. Specifically, stained cells were found in the caudal pole ofthe superior reticular nucleus, throughout the medial reticular nucleus and in the rostral area ofthe inferior retlcutar nucleus; (4) eurydendroid cells of the cerebellum, located close to the contralateral eminentia granularis pars lateralis, were also labeled; and (5) a small and primarily ipsilateral grorrp oi labeled cells was located at the mesencephalic nucleus of the mediál longitldinal fascicrllus.' _ The similarity in the structures projecting to the oculomotor nucleüs in goldfish to those in other vertebrates suggests that the neural network involved in the oculomofor system is quite conservativ^e throughout phylogeny. Nevertheless, in goldfish these projections áppeare¿ wittr some specific peculiarities, such as the cerebellar and mesencepha[c Jffeients to thliculomotor nucleus. o 1992 Wiley-Liss, Inc.
The synaptic alterations induced in abducens motoneurons by the injection of 3 ng/kg of botulinum... more 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 (66.44.0%) significantly decreased to 27.24.0%. 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 1.200.01. 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. 1997 IBRO. Published by Elsevier Science Ltd.
The highly specific projection of abducens internuclear neurons on the medial rectus motoneurons ... more The highly specific projection of abducens internuclear neurons on the medial rectus motoneurons of the oculomotor nucleus constitutes an optimal model for investigating the effects of axotomy in the central nervous system. We have analyzed the morphological changes induced by this lesion on both the cell bodies and the transected axons of abducens internuclear neurons in the adult cat. Axotomy was performed by the transection of the medial longitudinal fascicle. Cell counts of Nissl-stained material and calretinin-immunostained abducens internuclear neurons revealed no cell death by 3 months postaxo-tomy. Ultrastructural examination of these cells at 6, 14, 24, and 90 days postaxotomy showed normal cytological features. However, the surface membrane of axotomized neurons appeared contacted by very few synaptic boutons compared to controls. This change was quantified by measuring the percentage of synaptic coverage of the cell bodies and the linear density of boutons. Both parameters decreased significantly after axotomy, with the lowest values at 90 days postlesion (70% reduction). We also explored axonal regrowth and the possibility of reinnervation of a new target by means of anterograde labeling with biocytin. At all time intervals analyzed, labeled axons were observed to be interrupted at the caudal limit of the lesion; in no case did they cross the scar tissue to reach the distal part of the tract. Nonetheless, a conspicuous axonal sprouting was present at the caudal aspect of the lesion site. Structures suggestive of axonal growth were found, such as large terminal clubs, from which short filopodium-like branches frequently emerged. Similar findings were obtained after parvalbumin and calretinin immunostaining. At the electron microscopy level, biocytin-labeled boutons originating from the sprouts appeared surrounded by either extracellular space, which was extremely dilated at the lesion site, or by glial processes. The great majority of labeled boutons examined were, thus, devoid of neuronal contact, indicating absence of reinnervation of a new target. Altogether, these data indicate that abducens internuclear neurons survive axotomy in the adult cat and show some form of axonal regrowth, even in the absence of target connection.
In this study, we investigated the effects of long-term synaptic blockade on postsynaptic recepto... more 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 µm in diameter) densely covering the surface of control abducens motoneurons. Ultrastructur-ally, 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 µm 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 postsynap-tic receptor cluster dissolution seemed more directly related to terminal retraction than to inactivity alone.
The motoneurons of the extraocular motor nuclei represent the final site of convergenco of premot... more The motoneurons of the extraocular motor nuclei represent the final site of convergenco of premotor signals related to the control of eye movements. Premotor afferents to the abducens, trochlear and oculomotor nuclei comprise both excitatory and in-hibitory synaptic connections that arise mainly from VeStibular,5'17'23'24'21'33'se'6w10 retiCglar,lT'2e'31'53'54 pre-positus hypoglossirT'ae and interstitial nucleus of tTo whom correspondence should be addressed. Abbreoiations'. ABC, avidin-biotin-HRP complex; BSA, bovine serum albumin; DAB, 3,3'-diaminobenzidine; HRP, horseradish peroxidase; NGS, normal goat serum; PBS, phosphate-buffered saline; IPSP, inhibitory post-synaptic potential. Abstract-The GABAergic innervation of the ext¡aocula¡ motor nuclei in the cat was evaluated using postembedding immunoJytochemical techniques. The characterization ol 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 oculomoior nuclárs, and we¡e found in close apposition to somata and dendrites. Ultrathin sections revealed an extensivá and dense distribution of GABA-immunoreactive synaptic endings that established óontacts with the perikarya and proximal dendrites of motoneurons and were also abundant in the surrounding ne,r.opl. CAÍlA"rgic boutons were characterized by the presence olnurnerous mitochondria, pleiomorphlc vesic-les and multiple small symmetrical synaptic contacts. The trochlear nucleus exhibited ih. high".t d.nrity of GABAergii 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-motoneu.on, *it" 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 vestibula¡ 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 peroxidaseJabeled somata and in the surrounding neuropil. At the ultrastruc-tuüt tevel, GABAergic terminals established synaptic contacts with the somata and proximal dendrites of medial rectus motoneurons. Their features and density were similar to those lound in the remaining motoneuronal subgroups of the oculomotor nucleus' Second, oculom-otoi internuclear neurons were identified following the injection ol horseradish peroxidase into the abducens nucleus to determine whether they could give rise to GABAergic ierminations in the abducens nucleus. About 20Vo ol the oculomotor internuclear neurons were doubly labeled by retrograde horseradish peroxidase and GABA immunostaining. A high percentage (80%) of the oculomoior internuclear neurons projecting to the abducens nucleus showed immunonegative perikarya. It was concluded that the oculómótor 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 originateí from this source. II is suggested that this pathway might carry excitatory and inhibitory influences on abducens neurons arising bilaterally. 0306-4522192 $5.00 + 0.oo Pergamon Press Ltd @) 1992 IBRO Cajalst'st'sz neurons. The medial rectus motoneurons of the oculomotor nucleus constitute the only exception to the excitatory/inhibitory connectivity since direct vestibular inhibition has been shown to be absent.4 Electrophysiological, immunocytochemical and pharmacological studies have provided evidence on the nature of the inhibitory neurotransmitters involved in the oculomotor system and their relative participiation in each premotor subsystem. GABA has been funüionally implicated as the source of vestibular inhibition of oculomotor and trochlear motoneurons. Systemic administration of the GABA antagonists, bicuculline and picrotoxin, blocks the inhibitory postsynaptic potentials recorded in these motoneurons after vestibular nerve
A ´ lvarez, and A ´ ngel M. Pastor. Functional alterations of cat abducens neurons after peripher... more A ´ lvarez, and A ´ ngel M. Pastor. 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 neurotrans-mitter 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 ex-citation 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.
A relationship between motoneuron activity and calcitonin gene-related peptide (CGRP) expression ... more 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 CGRP gene expression, was analyzed during these periods of altered firing activity. The number of CGRP-immunofluorescent abducens motoneurons increased to 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.
Tetanus neurotoxin (TeNT) is a blocker of synaptic vesicle exocytosis in central synapses with pr... more Tetanus neurotoxin (TeNT) is a blocker of synaptic vesicle exocytosis in central synapses with preferential af®nity 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.
A low or a high dose of tetanus neurotoxin (TeNT) injected in the lateral rectus muscle of the ca... more 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. Neu-rophysiol. 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-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 neu-rons (calretinin). Seven days after high-dose treatment, the number of gephyrin-immunoreactive (IR) clusters per 100 m of membrane perimeter was reduced on the soma of abducens motoneu-rons 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.
The discharge characteristics that abducens motoneurons exhibit after paralysis of the lateral re... more 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. 1997 IBRO. Published by Elsevier Science Ltd.
Calcium-binding proteins have been shown to be excellent markers of specific neuronal populations... more 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 aim of the present work was to characterize the axotomy-induced changes in the discharge prop... more The aim of the present work was to characterize the axotomy-induced changes in the discharge properties of central nervous system neurons recorded in the alert behaving animal. The abducens internuclear neurons of the adult cat were the chosen model. The axons of these neurons course through the contralateral medial longitudinal fascicle and contact the medial rectus motoneurons of the oculomotor nucleus. Axotomy was carried out by the unilateral transection of this fascicle (right side) and produced immediate oculomotor deficits, mainly the incapacity of the right eye to adduct across the midline. Extracellular single-unit recording of abducens neurons was carried out simultaneously with eye movements. The main alteration observed in the firing of these axotomized neurons was the overall decrease in firing rate. During eye fixations, the tonic signal was reduced, and, on occasion, a progressive decay in firing rate was observed. On-directed saccades were not accompanied by the high-frequency spike burst typical of controls; instead, there was a moderate increase in firing. Similarly, during the vestibular nystagmus, neurons hardly modulated during both the slow and the fast phases. Linear regression analysis between firing rate and eye movement parameters showed a significant reduction in eye position and velocity sensitivities with respect to controls, during both spontaneous and vestibularly induced eye movements. These firing alterations were observed during the 3 month period of study after lesion, with no sign of recovery. Conversely, abducens motoneurons showed no significant alteration in their firing pattern. Therefore, axotomy produced long-lasting changes in the discharge characteristics of abducens internuclear neurons that presumably reflected the loss of afferent oculomotor signals. These alterations might be due to the absence of trophic influences derived from the target.
The goal of this work was to compare the distribution and morphology of neurons projecting to the... more The goal of this work was to compare the distribution and morphology of neurons projecting to the oculomotor nucleus in goldfish with those preüously des"iib"¿ n áth". vertebrate groups. Afferent neurons were revealed by retrográde hbetittg with horseradish peroxidase. The tracer was electrophoretically injected intó the oculomlotor nucleus. The location of the injection site was determined by the antidromic field potential elicited in the oculomotor nucleus by electrical stimulation of the oculomotor nervé. Labeled axons whose trajectories could be reconstructed were restricted to the medial longitudinal fasciculus. In order of quantitative importance, the afferent areas to the oculomotoi nucleus were: (1) the ipsilateral anterior nucleus and the contralateral tangential and descenüng nuclei ;Í;h; octaval column. Furthermore, a few labeled cells were found dorsomedially to the caudal pole of the unlabeled anterior octaval nucleus; (2) the contralateral abducens nucleus. The übeled internuclear neutons were arranged in two groups within and 500 pm behind the caudal subdiüsion of the abducens nucleus; (B) a few hbéled cells were observed in the rhombence_ phalic reticular formation near the abducens nucleus, most of which were contralateral to the injection site. Specifically, stained cells were found in the caudal pole ofthe superior reticular nucleus, throughout the medial reticular nucleus and in the rostral area ofthe inferior retlcutar nucleus; (4) eurydendroid cells of the cerebellum, located close to the contralateral eminentia granularis pars lateralis, were also labeled; and (5) a small and primarily ipsilateral grorrp oi labeled cells was located at the mesencephalic nucleus of the mediál longitldinal fascicrllus.' _ The similarity in the structures projecting to the oculomotor nucleüs in goldfish to those in other vertebrates suggests that the neural network involved in the oculomofor system is quite conservativ^e throughout phylogeny. Nevertheless, in goldfish these projections áppeare¿ wittr some specific peculiarities, such as the cerebellar and mesencepha[c Jffeients to thliculomotor nucleus. o 1992 Wiley-Liss, Inc.
The synaptic alterations induced in abducens motoneurons by the injection of 3 ng/kg of botulinum... more 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 (66.44.0%) significantly decreased to 27.24.0%. 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 1.200.01. 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. 1997 IBRO. Published by Elsevier Science Ltd.
The highly specific projection of abducens internuclear neurons on the medial rectus motoneurons ... more The highly specific projection of abducens internuclear neurons on the medial rectus motoneurons of the oculomotor nucleus constitutes an optimal model for investigating the effects of axotomy in the central nervous system. We have analyzed the morphological changes induced by this lesion on both the cell bodies and the transected axons of abducens internuclear neurons in the adult cat. Axotomy was performed by the transection of the medial longitudinal fascicle. Cell counts of Nissl-stained material and calretinin-immunostained abducens internuclear neurons revealed no cell death by 3 months postaxo-tomy. Ultrastructural examination of these cells at 6, 14, 24, and 90 days postaxotomy showed normal cytological features. However, the surface membrane of axotomized neurons appeared contacted by very few synaptic boutons compared to controls. This change was quantified by measuring the percentage of synaptic coverage of the cell bodies and the linear density of boutons. Both parameters decreased significantly after axotomy, with the lowest values at 90 days postlesion (70% reduction). We also explored axonal regrowth and the possibility of reinnervation of a new target by means of anterograde labeling with biocytin. At all time intervals analyzed, labeled axons were observed to be interrupted at the caudal limit of the lesion; in no case did they cross the scar tissue to reach the distal part of the tract. Nonetheless, a conspicuous axonal sprouting was present at the caudal aspect of the lesion site. Structures suggestive of axonal growth were found, such as large terminal clubs, from which short filopodium-like branches frequently emerged. Similar findings were obtained after parvalbumin and calretinin immunostaining. At the electron microscopy level, biocytin-labeled boutons originating from the sprouts appeared surrounded by either extracellular space, which was extremely dilated at the lesion site, or by glial processes. The great majority of labeled boutons examined were, thus, devoid of neuronal contact, indicating absence of reinnervation of a new target. Altogether, these data indicate that abducens internuclear neurons survive axotomy in the adult cat and show some form of axonal regrowth, even in the absence of target connection.
In this study, we investigated the effects of long-term synaptic blockade on postsynaptic recepto... more 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 µm in diameter) densely covering the surface of control abducens motoneurons. Ultrastructur-ally, 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 µm 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 postsynap-tic receptor cluster dissolution seemed more directly related to terminal retraction than to inactivity alone.
The motoneurons of the extraocular motor nuclei represent the final site of convergenco of premot... more The motoneurons of the extraocular motor nuclei represent the final site of convergenco of premotor signals related to the control of eye movements. Premotor afferents to the abducens, trochlear and oculomotor nuclei comprise both excitatory and in-hibitory synaptic connections that arise mainly from VeStibular,5'17'23'24'21'33'se'6w10 retiCglar,lT'2e'31'53'54 pre-positus hypoglossirT'ae and interstitial nucleus of tTo whom correspondence should be addressed. Abbreoiations'. ABC, avidin-biotin-HRP complex; BSA, bovine serum albumin; DAB, 3,3'-diaminobenzidine; HRP, horseradish peroxidase; NGS, normal goat serum; PBS, phosphate-buffered saline; IPSP, inhibitory post-synaptic potential. Abstract-The GABAergic innervation of the ext¡aocula¡ motor nuclei in the cat was evaluated using postembedding immunoJytochemical techniques. The characterization ol 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 oculomoior nuclárs, and we¡e found in close apposition to somata and dendrites. Ultrathin sections revealed an extensivá and dense distribution of GABA-immunoreactive synaptic endings that established óontacts with the perikarya and proximal dendrites of motoneurons and were also abundant in the surrounding ne,r.opl. CAÍlA"rgic boutons were characterized by the presence olnurnerous mitochondria, pleiomorphlc vesic-les and multiple small symmetrical synaptic contacts. The trochlear nucleus exhibited ih. high".t d.nrity of GABAergii 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-motoneu.on, *it" 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 vestibula¡ 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 peroxidaseJabeled somata and in the surrounding neuropil. At the ultrastruc-tuüt tevel, GABAergic terminals established synaptic contacts with the somata and proximal dendrites of medial rectus motoneurons. Their features and density were similar to those lound in the remaining motoneuronal subgroups of the oculomotor nucleus' Second, oculom-otoi internuclear neurons were identified following the injection ol horseradish peroxidase into the abducens nucleus to determine whether they could give rise to GABAergic ierminations in the abducens nucleus. About 20Vo ol the oculomotor internuclear neurons were doubly labeled by retrograde horseradish peroxidase and GABA immunostaining. A high percentage (80%) of the oculomoior internuclear neurons projecting to the abducens nucleus showed immunonegative perikarya. It was concluded that the oculómótor 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 originateí from this source. II is suggested that this pathway might carry excitatory and inhibitory influences on abducens neurons arising bilaterally. 0306-4522192 $5.00 + 0.oo Pergamon Press Ltd @) 1992 IBRO Cajalst'st'sz neurons. The medial rectus motoneurons of the oculomotor nucleus constitute the only exception to the excitatory/inhibitory connectivity since direct vestibular inhibition has been shown to be absent.4 Electrophysiological, immunocytochemical and pharmacological studies have provided evidence on the nature of the inhibitory neurotransmitters involved in the oculomotor system and their relative participiation in each premotor subsystem. GABA has been funüionally implicated as the source of vestibular inhibition of oculomotor and trochlear motoneurons. Systemic administration of the GABA antagonists, bicuculline and picrotoxin, blocks the inhibitory postsynaptic potentials recorded in these motoneurons after vestibular nerve
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Papers by Angel Pastor