Discharge properties of morphologically identified vestibular neurons recorded during horizontal eye movements in the goldfish.

Computational capability and connectivity are key elements for understanding how central vestibular neurons contribute to gaze-stabilizing eye movements during self-motion. In the well-characterized and segmentally distributed hindbrain oculomotor network of goldfish, we determined afferent and efferent connections along with discharge patterns of descending octaval nucleus (DO) neurons during different eye motions. Based on activity correlated with horizontal eye and head movements, DO neurons were categorized into two complementary groups that either increased discharge during both contraversive (type II) eye (e) and ipsiversive (type I) head (h) movements (eIIhI) or vice versa (eIhII). Matching time courses of slow-phase eye velocity and corresponding firing rates during prolonged visual and head rotation suggested direct causality in generating extraocular motor commands. The axons of the dominant eIIhI subgroup projected either ipsi- or contralaterally and terminated in the abducens nucleus, Area II, and Area I with additional recurrent collaterals of ipsilaterally projecting neurons within the parent nucleus. Distinct feedforward commissural pathways between bilateral DO neurons likely contribute to the generation of eye velocity signals in eIhII cells. The shared contribution of DO and Area II neurons to eye velocity storage likely represents an ancestral condition in goldfish that is clearly at variance with the task separation between mammalian medial vestibular and prepositus hypoglossi neurons. This difference in signal processing between fish and mammals might correlate with a larger repertoire of visuo-vestibular-driven eye movements in the latter species that potentially required a shift in sensitivity and connectivity within the hindbrain-cerebello-oculomotor network. NEW & NOTEWORTHY We describe the structure and function of neurons within the goldfish descending octaval nucleus. Our findings indicate that eye and head velocity signals are processed by vestibular and Area II velocity storage integrator circuitries whereas the velocity-to-position Area I neural integrator generates eye position solely. This ancestral condition differs from that of mammals, in which vestibular neurons generally lack eye position signals that are processed and stored within the nucleus prepositus hypoglossi.

Case report: Disease phenotype associated with simultaneous biallelic mutations in ABCA4 and USH2A due to uniparental disomy of chromosome 1.

Inherited retinal diseases (IRDs) represent a spectrum of clinically and genetically heterogeneous disorders. Our study describes an IRD patient carrying ABCA4 and USH2A pathogenic biallelic mutations as a result of paternal uniparental disomy (UPD) in chromosome 1. The proband is a 9-year-old girl born from non-consanguineous parents. Both parents were asymptomatic and denied family history of ocular disease. Clinical history and ophthalmologic examination of the proband were consistent with Stargardt disease. Whispered voice testing disclosed moderate hearing loss. Next-generation sequencing and Sanger sequencing identified pathogenic variants in ABCA4 (c.4926C>G and c.5044_5058del) and USH2A (c.2276G>T). All variants were present homozygously in DNA from the proband and heterozygously in DNA from the father. No variants were found in maternal DNA. Further analysis of single nucleotide polymorphisms confirmed paternal UPD of chromosome 1. This is the first known patient with confirmed UPD for two recessively mutated IRD genes. Our study expands on the genetic heterogeneity of IRDs and highlights the importance of UPD as a mechanism of autosomal recessive disease in non-consanguineous parents. Moreover, a long-term follow-up is essential for the identification of retinal features that may develop as a result of USH2A-related conditions.

[Serious complications after pulmonary radiofrequency ablation: report of 2 cases]. / Dos casos de complicaciones graves tras radiofrecuencia pulmonar.

Radiofrequency ablation can be used to treat primary or metastatic pulmonary tumors when surgery is not indicated or involves high risk. Although this technique is less invasive than surgical resection, it is not free of risk for complications and adverse events, especially when it is used in patients with serious respiratory disease in whom comorbidity is common. We report 2 cases of serious complications. One was an intractable air leak that led to death. The other was a large hemothorax that was brought under control in the radiology procedure room. We review the literature on this technique as well as recommendations that contribute to making it as safe as possible.

Infrared meibography and molecular assessment of p63 gene mutations in a Mexican patient with EEC syndrome. / Meibografía infrarroja y evaluación molecular de la mutación del gen p63 en un paciente mexicano con síndrome EEC.

OBJECTIVE: To report the finding of infrared meibography in a Mexican patient with EEC syndrome (Ectrodactyly-ectodermal dysplasia-cleft syndrome) confirmed by molecular analysis of the p63 gene. CLINICAL CASE: A 31 year-old male patient was seen due to a history of progressive visual loss in both eyes associated with long-term photophobia. The patient was born with cleft lip and palate, ectrodactyly of right hand, and afterwards, displayed nail dysplasia, anodontia and alopecia, with which ectodermal dysplasia was diagnosed. The ophthalmological findings were limited to the adnexa and the ocular surface. In vivo infrared meibography showed total absence of Meibomian glands in the lower eyelids and severe deficiency in the upper eyelids. In addition, it was shown that the patient was a heterozygous carrier of a missense mutation R304W (C → T) in exon 8 of the p63 gene. DISCUSSION: The R304W mutation in the p63 gene region is definitely related to characteristics such as the absence of Meibomian glands.

Typha latifolia as potential phytoremediator of 2,4-dichlorophenol: Analysis of tolerance, uptake and possible transformation processes.

2,4-Dichlorophenol (2,4-DCP) is considered a priority pollutant due to its high toxicity. Therefore, it is urgent to develop technologies for the disposal of this pollutant. Various remediation processes have been proposed for the elimination of 2,4-DCP in contaminated water, however, most of them involve high costs of operation and maintenance. This study aimed to determine the capacity of remediation of 2,4-DCP in water by Typha latifolia L. wild plants. For that, the tolerance, removal, accumulation and biotransformation of 2,4-DCP by T. latifolia were investigated. The plants were exposed to 2,4-DCP solutions with a concentration range from 1.5 to 300 mgL-1 for 10 days. They exhibited a reduction in chlorophyll levels and growth rate when 2,4-DCP solutions were ≥30 mgL-1 and ≥50 mgL-1, respectively. The removal of contaminant was dose-depended, being 99.7% at 1.5-3 mgL-1, 59-70% at 10-70 mgL-1 and 35-42% at 100-300 mgL-1 of 2,4-DCP in the solution. Studies indicated that 2,4-DCP was mainly accumulated in root tissue rather than in shoot tissue. Acid hydrolysis of biomass extracts suggests 2,4-DCP bioconjugates formation in root tissue as a response mechanism. Additionally, an increment in glutathione S-transferase (GST) activity could indicate a 2,4-DCP conjugation with glutathione as a detoxification mechanism of T. latifolia.

Accumulations of cyclic AMP and inositol phosphates in guinea pig cerebral cortical synaptoneurosomes: enhancement by agents acting at sodium channels.

Activation of alpha 1-adrenergic receptors by norepinephrine in guinea pig cortical synaptoneurosomes augments accumulations of cyclic AMP elicited by 2-chloroadenosine and concomitantly increases formation of inositol phosphates. Various agents that affect calcium channels or sites of action of calcium have little or no effect on cyclic AMP accumulation elicited either with 2-chloroadenosine, or with a 2-chloroadenosine/norepinephrine combination, nor did they markedly affect formation of inositol phosphates elicited by norepinephrine. However, EGTA reduces both cyclic AMP accumulation and inositol phosphate formation. Agents such as batrachotoxin, scorpion (Leiurus) venom and pumiliotoxin B that are active at voltage-dependent sodium channels enhance accumulations of cyclic AMP and inositol phosphates. These effects are blocked by tetrodotoxin. It is proposed that enhanced influx of sodium ions increases phosphatidylinositol metabolism, resulting in formation of diacylglycerols and inositol phosphates, and that the former, through activation of protein kinase, causes an enhancement of cyclic AMP accumulations in brain tissue.

Behaviour of Medial Rectus Motoneurons in the Alert Cat.

The activity of identified medial rectus motoneurons was recorded in alert cats during spontaneous and vestibular induced eye movements. Medial rectus motoneurons fired a burst of spikes slightly preceding adducting saccades and increased their discharge rate linearly with successive eye positions in the adducting direction. Conduction velocity (21.3 - 98.2 m/s), eye position sensitivity (ks, 7.1 +/- 1.5 spikes/s/deg), and eye velocity sensitivity (rs, 1 +/- 0.2 spikes/s/deg/s) during spontaneous eye movements, and time constants calculated from phase lead analysis (To, 135 +/- 36 ms) showed values similar to those described previously for cat abducens motoneurons. The firing rate during repeated fixation of the same eye position was affected significantly by the direction of the preceding saccade and by the animal's level of alertness. Eye velocity sensitivity was not significantly affected by changes in the animal's level of alertness. A weak negative relationship (coefficient of correlation=-0.56) was observed between eye velocity sensitivity (rv) and sinusoidal rotational frequency, with no change in eye position sensitivity (kv) with stimulus frequency. The subsequent changes in the time constant (Tv) calculated as Tv=rv/kv in relation to stimulus frequency suggests that the oculomotor system deviates from a (linear) first-order model.

Discharge characteristics of axotomized abducens internuclear neurons in the adult cat.

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.

Response of abducens internuclear neurons to axotomy in the adult cat.

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 postaxotomy. 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 ( approximately 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.

Effects of botulinum neurotoxin type A on the expression of gephyrin in cat abducens motoneurons.

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.

Localization of parvalbumin, calretinin, and calbindin D-28k in identified extraocular motoneurons and internuclear neurons of the cat.

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.

Influence of the postsynaptic target on the functional properties of neurons in the adult mammalian central nervous system.

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)

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

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.

Synthesis and muscarinic cholinergic receptor affinities of 3-quinuclidinyl alpha-(alkoxyalkyl)-alpha-aryl-alpha-hydroxyacetates.

Seven analogues of 3-quinuclidinyl benzilate (QNB) in which one phenyl ring was replaced by an alkoxyalkyl moiety were synthesized and their affinities for the muscarinic cholinergic receptor determined. An oxygen in the beta-position of the moiety was not well-tolerated. By contrast, an oxygen in the gamma-position did not change the affinity for the muscarinic receptor. However, when a bromine was placed on the remaining phenyl ring, the affinity was significantly reduced in striking contrast to results obtained on halogenation of QNB.

Synthesis and structure-activity relationship of some 5-[[[(dialkylamino)alkyl]-1-piperidinyl]acetyl]-10,11-dihydro-5H- benzo[b,e][1,4]diazepin-11-ones as M2-selective antimuscarinics.

A series of 5-[[[(dialkylamino)alkyl]-1-piperidinyl]acetyl]- 10,11-dihydro-5H-dibenzo[b,e][1,4]-diazepin-11-ones were prepared as potential M2-selective ligands. The compounds were evaluated for their affinity and selectivity for the muscarinic cholinergic receptor. The best M2-selective antimuscarinic agent studied is 5-[[4-[4-diethylamino)butyl]-1- piperidinyl]acetyl]-10,11-dihydro-5H-dibenzo[b,e][1,4]diazepin-11- one, which is approximately 10 times more potent at M2 receptors than previously known compounds such as 11-[[4-[4-(diethylamino)butyl]- 1-piperidinyl]acetyl]-5,11-dihydro-6H- pyrido[2,3-b][1,4]benzodiazepin-6-one (AQ-RA 741).

Grafting of a new target prevents synapse loss in abducens internuclear neurons induced by axotomy.

The loss of afferent synaptic boutons is a prominent alteration induced by axotomy on adult central neurons. In this work we attempted to prove whether synapse loss could be reverted by reconnection with a new target. We severed the medial longitudinal fascicle of adult cats and then transplanted embryonic cerebellar primordia at the lesion site immediately after lesion. As previously shown, the transected axons from abducens internuclear neurons penetrate and reinnervate the graft [J Comp Neurol 444 (2002) 324]. By immunocytochemistry and electron microscopy we studied the synaptology of abducens internuclear neurons under three conditions: control, axotomy and transplant (2 months of survival time). Semithin sections of the abducens nucleus were immunostained against calretinin, to identify abducens internuclear neurons, and either synaptophysin (SF), to label synaptic terminals, or glial fibrillary acidic protein (GFAP) to detect the astrocytic reaction. Optical and linear density of SF and GFAP immunostaining were measured. Data revealed a significant decrease in the density of SF-labeled terminals with a parallel increase in GFAP-immunoreactive elements after axotomy. On the contrary, in the transplant group, the density of SF-labeled terminals was found similar to control, and the astrocytic reaction induced by lesion was significantly reduced. At the ultrastructural level, synaptic coverage and linear density of boutons were measured around the somata of abducens internuclear neurons. Whereas a significant reduction in both parameters was found after axotomy, cells of the transplant group received a normal density of synaptic endings. The ratio between F- and S-type boutons was found similar in the three groups. Therefore, these findings indicate that the grafting of a new target can prevent the loss of afferent synaptic boutons produced by the axotomy.

Intrinsic determinants of synaptic phenotype: an experimental study of abducens internuclear neurons connecting with anomalous targets.

The present experiments investigate the role of postsynaptic neurons in the morphological differentiation of presynaptic terminals that are formed de novo in the adult CNS. Abducens internuclear neurons in the adult cat were chosen as the experimental model. These neurons project onto the contralateral medial rectus motoneurons of the oculomotor nucleus. Abducens internuclear axon terminals were identified by their anterograde labeling with biocytin and analyzed at the electron microscopic level. To promote the formation of new synapses, two different experimental approaches were used. First, after the selective ablation of medial rectus motoneurons with ricin, abducens internuclear neurons reinnervated the neighboring oculomotor internuclear neurons. Second, after axotomy followed by embryonic cerebellar grafting, abducens internuclear axons invaded the implanted tissue and established synaptic connections in both the molecular and granule cell layer. Boutons contacting the oculomotor internuclear neurons developed ultrastructural characteristics that resembled the control synapses on medial rectus motoneurons. In the grafted cerebellar tissue, abducens internuclear axons and terminals did not resemble climbing or mossy fibers but showed similarities with control boutons. However, labeled boutons analyzed in the granule cell layer established a higher number of synaptic contacts than controls. This could reflect a trend towards the mossy fiber phenotype, although labeled boutons significantly differed in every measured parameter with the mossy fiber rosettes found in the graft. We conclude that at least for the abducens internuclear neurons, the ultrastructural differentiation of axon terminals reinnervating novel targets in the adult brain seems to be mainly under intrinsic control, with little influence by postsynaptic cells.

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.].

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.