Cranial nerve fasciculation and Schwann cell migration are impaired after loss of Npn-1.

Interaction of the axon guidance receptor Neuropilin-1 (Npn-1) with its repulsive ligand Semaphorin 3A (Sema3A) is crucial for guidance decisions, fasciculation, timing of growth and axon-axon interactions of sensory and motor projections in the embryonic limb. At cranial levels, Npn-1 is expressed in motor neurons and sensory ganglia and loss of Sema3A-Npn-1 signaling leads to defasciculation of the superficial projections to the head and neck. The molecular mechanisms that govern the initial fasciculation and growth of the purely motor projections of the hypoglossal and abducens nerves in general, and the role of Npn-1 during these events in particular are, however, not well understood. We show here that selective removal of Npn-1 from somatic motor neurons impairs initial fasciculation and assembly of hypoglossal rootlets and leads to reduced numbers of abducens and hypoglossal fibers. Ablation of Npn-1 specifically from cranial neural crest and placodally derived sensory tissues recapitulates the distal defasciculation of mixed sensory-motor nerves of trigeminal, facial, glossopharyngeal and vagal projections, which was observed in Npn-1(-/-) and Npn-1(Sema-) mutants. Surprisingly, the assembly and fasciculation of the purely motor hypoglossal nerve are also impaired and the number of Schwann cells migrating along the defasciculated axonal projections is reduced. These findings are corroborated by partial genetic elimination of cranial neural crest and embryonic placodes, where loss of Schwann cell precursors leads to aberrant growth patterns of the hypoglossal nerve. Interestingly, rostral turning of hypoglossal axons is not perturbed in any of the investigated genotypes. Thus, initial hypoglossal nerve assembly and fasciculation, but not later guidance decisions depend on Npn-1 expression and axon-Schwann cell interactions.

BDNF-induced synaptic delivery of AMPAR subunits is differentially dependent on NMDA receptors and requires ERK.

Previous studies using an in vitro model of eyeblink classical conditioning in turtles suggest that increased numbers of synaptic AMPARs supports the acquisition and expression of conditioned responses (CRs). Brain-derived neurotrophic factor (BDNF) and its associated receptor tyrosine kinase, TrkB, is also required for acquisition of CRs. Bath application of BDNF alone induces synaptic delivery of GluR1- and GluR4-containing AMPARs that is blocked by coapplication of the receptor tyrosine kinase inhibitor K252a. The molecular mechanisms involved in BDNF-induced AMPAR trafficking remain largely unknown. The aim of this study was to determine whether BDNF-induced synaptic AMPAR incorporation utilizes similar cellular mechanisms as AMPAR trafficking that occurs during in vitro classical conditioning. Using pharmacological blockade and confocal imaging, the results show that synaptic delivery of GluR1 subunits during conditioning or BDNF application does not require activity of NMDARs but is mediated by extracellular signal-regulated kinase (ERK). In contrast, synaptic delivery of GluR4-containing AMPARs during both conditioning and BDNF application is NMDAR- as well as ERK-dependent. These findings indicate that BDNF application mimics AMPAR trafficking observed during conditioning by activation of some of the same intracellular signaling pathways and suggest that BDNF is a key signal transduction element in postsynaptic events that mediate conditioning.

Differential organization of gamma-aminobutyric acid type A and glycine receptors in the somatic and dendritic compartments of rat abducens motoneurons.

Premotor inhibitory neurons responsible for the decrease in the firing discharge during fast or slow eye movements selectively target the cell bodies and the dendrites of abducens motoneurons. Gamma-aminobutyric acid (GABA) and glycine, the main inhibitory synaptic neurotransmitters in the central nervous system, act via glycine and GABAA receptors, assembled from various types of subunits, which determine the kinetics of the currents mediated. Therefore, our hypothesis was that the expression of the inhibitory receptors on the somatic and the dendritic compartments, involved in different functions, may differ. In this study, we compared the subcellular patterns of expression of the main GABAA receptor subunits (GABAARalpha1, alpha2, alpha3, alpha5), glycine receptors (GlyRalpha1), and gephyrin in the somatic and dendritic compartments of rat abducens motoneurons, using double or triple immunocytochemical experiments with confocal microscopy. Significant differences exist in the patterns of organization and the synaptic expression of the GlyR and GABAAR subunits in the cell bodies and dendrites of abducens motoneurons. In the somata, only the GABAARalpha1 subunit was expressed, whereas both GABAARalpha1 and GABAARalpha3 were present in the dendrites. The GlyRalpha1 to GABAARalpha1 density ratio was reversed in the somatic and dendritic compartments (0.9 vs. 2.3). A quantitative electron microscopy study showed that the modes whereby gephyrin reaches its postsynaptic inhibitory synaptic target differ between the somata and the dendrites. Therefore, our results support the idea that a structure-function adaptation occurs at the single-neuron level.

A comparative study of oculomotor, trochlear and abducens nerves in Arabian foals.

We investigated the microscopic structure of transverse sections of the oculomotor, trochlear and abducens nerves of Arabian foals using stereological methods. Bilateral nerve pairs from 2-month-old female Arabian foals were analyzed. The tissues were embedded in plastic blocks, then 1 µm thick sections were cut and stained with osmium tetroxide and methylene blue-azure II. Stereology was performed using light microscopy. Morphometry showed that the right and left pairs of nerves were similar. The transverse sectional areas of the oculomotor, trochlear and abducens nerves were 1.93 ± 0.19 mm2, 0.32 ± 0.06 mm2 and 0.70 ± 0.08 mm2, respectively. The oculomotor nerve exhibited a significantly greater number of myelinated axons (16755 ± 1279) and trochlear (2656 ± 494) and the abducens nerves (4468 ± 447). The ratio of the axon diameter to myelinated nerve fiber diameter was 0.58, 0.55 and 0.55 for the oculomotor, trochlear and abducens nerves, respectively. Of the three nerves studied, the abducens nerve exhibited the greatest nerve fiber area, myelin area, nerve and axon diameters, and myelin thickness. The ratio of small myelinated nerve fibers was greatest in the oculomotor nerve.

Neurochemically defined cell columns in the nucleus prepositus hypoglossi of the cat and monkey.

Many studies have shown that the nucleus prepositus hypoglossi (PH) participates with the vestibular nuclear complex, the cerebellum and the oculomotor nuclei in the control of eye movements. We have looked at the neurochemical organization of PH in the cat and monkey using a recently developed antibody, 8B3, that recognizes a chondroitin sulfate proteoglycan. In the cat, immunoreactivity to 8B3 labels a set of cells in PH. On frontal sections, these cells form a cluster that is seen over the entire anterior-posterior (A-P) extent of PH, but the number of cells in the cluster changes with A-P level. Earlier studies have identified an A-P cell column in PH of the cat whose neurons synthesize nitric oxide. We have used both single- and double-label protocols to investigate the relation between the two cell groups. Single-label studies show spatial overlap but that the cells immunoreactive to nitric oxide synthase (nNOS) are more numerous than cells immunoreactive to 8B3. Double-label studies show that all cells immunoreactive to 8B3 were also immunoreactive to nNOS, but, as suggested by the single-label data, there are many nNOS-immunoreactive cells not immunoreactive to 8B3. Populations of 8B3 and nNOS-immunoreactive cells are also found in PH of squirrel and macaque monkeys. The results suggest that nNOS-immunoreactive cells in PH may consist of two functionally different populations.

Parvalbumin and calbindin D-28k immunoreactivity in transgenic mice with a G93A mutant SOD1 gene.

Immunohistochemical study was performed to examine if calcium-binding proteins are involved in the degeneration of motor neurons in the brain stems and the spinal cords of transgenic mice carrying a G93A mutant human SOD1 gene. Specimens from age-matched non-transgenic wild-type mice served as controls. In the spinal cord of the controls, the density of parvalbumin-immunoreactive neurons was highest in the large anterior horn neurons and lower in the posterior horn neurons in the spinal cord. On the other hand, calbindin D-28k immunoreactivity was much less apparent than that observed with parvalbumin antisera. Rexed's lamina II was densely immunostained for calbindin D-28k, whereas, in the anterior horn, calbindin-D-28k-positive small neurons were barely dispersed in a scattered pattern. In transgenic mice, parvalbumin-positive anterior horn neurons were severely reduced, even at the presymptomatic stage, whereas calbindin-positive neurons were largely preserved. At the symptomatic stage, both parvalbumin and calbindin D-28k immunoreactivity markedly diminished or disappeared in the anterior horn. Immunoblotting analysis revealed a significant reduction of immunoreactivity to parvalbumin antibody in transgenic mice compared with the controls. In the brain stem, parvalbumin-positive oculomotor and abducens neurons and the calbindin D-28k-positive sixth nucleus were well-preserved in transgenic mice as well as in the controls. Thus, the diffuse and severe loss of parvalbumin immunoreactivity of large motor neurons even at early stages in SOD1-transgenic mice and the absence of calbindin D-28k immunoreactivity of normal large motor neurons suggest that these calcium-binding proteins may contribute to selective vulnerability and an early loss of function of large motor neurons in this SOD1-transgenic mouse model.

Isolated abducens nerve palsy as a regional variant of Guillain-Barré syndrome.

The authors reviewed clinical profiles and laboratory findings for 100 cases of abducens nerve paresis without impairment of the other cranial nerves, limb weakness, and ataxia throughout the clinical course. Review of the medical records of 9300 patients referred to our neuoroimmunological laboratory for serum anti-ganglioside antibody testing. Information was obtained from each primary physician on symptoms of preceding infection; initial symptoms; neurological signs during the illness; the clinical course; treatment provided; and outcome. Isolated abducens nerve paresis was present in 100 patients and bilateral paresis in 29. Tentative diagnoses made by the primary physicians on request of anti-ganglioside antibody testing were abducens nerve palsy (n = 68), Fisher syndrome (n = 14), acute ophthalmoparesis without ataxia (n = 14). Symptoms of infection anteceded in 63. Tendon reflexes were absent or decreased in 27. Distal paresthesias were experienced by seven. Serum anti-GQ1b antibody was positive in 25. These findings suggest that some cases of isolated abducens nerve palsy can be categorized as a regional variant of Guillain-Barré syndrome or mild form of Fisher syndrome.

Transynaptic effects of tetanus neurotoxin in the oculomotor system.

The question whether general tetanus arises from the independent sum of multiple local tetani or results from the actions of the transynaptic tetanus neurotoxin (TeNT) in higher brain centres remains unresolved. Despite the blood-borne dissemination of TeNT from an infected wound, the access to the central nervous system is probably prevented by the blood-brain barrier. However, several long-term sequelae (e.g. autonomic dysfunction, seizures, myoclonus, and sleep disturbances) present after the subsidence of muscle spasms might be indicative of central actions that occur farther away from lower motoneurons. Subsequently, the obvious entry route is the peripheral neurons followed by the transynaptic passage to the brain. We aimed at describing the pathophysiological correlates of TeNT translocation using the oculomotor system as a comprehensive model of cell connectivity and neuronal firing properties. In this study, we report that injection of TeNT into the medial rectus muscle of one eye resulted in bilateral gaze palsy attributed to firing alterations found in the contralaterally projecting abducens internuclear neurons. Functional alterations in the abducens-to-oculomotor internuclear pathway resembled in part the classically described TeNT disinhibition. We confirmed the transynaptic targeted action of TeNT by analysing vesicle-associated membrane protein2 (VAMP2) immunoreactivity (the SNARE protein cleaved by TeNT). VAMP2 immunoreactivity decreased by 94.4% in the oculomotor nucleus (the first synaptic relay) and by 62.1% presynaptic to abducens neurons (the second synaptic relay). These results are the first demonstration of physiological changes in chains of connected neurons that are best explained by the transynaptic action of TeNT on premotor neurons as shown with VAMP2 immunoreactivity which serves as an indicator of TeNT activity.

P2, P1, and P0 myelin protein expression in developing rat sixth nerve: a quantitative immunocytochemical study.

Myelination and the expression of myelin proteins P2, P1, and P0 were studied quantitatively in the rat sixth cranial nerve during development. The postnatal development and growth of all myelin sheaths in this nerve have been studied morphometrically in a companion paper. Epon-embedded blocks with closely matched topography in the transverse plane were selected from rats perfused at ages 1-4, 8, 15, and 20 days. From each block, serial semithin sections were cut, etched, and immunostained according to the peroxidase-antiperoxidase method with well-characterized polyclonal antisera that reacted specifically with P0 glycoprotein and the basic proteins P1 and P2. The immunoreactivities of individual myelin sheaths were measured by densitometry. Numbers of compact myelin lamellae, myelin spiral lengths, and axon diameters were determined on electronmicrographs of adjacent thin sections. At birth anti-P0 immunoreactivity was found on sheaths with two and more compact lamellae; neither P1 nor P2 immunoreactivity was observed. On day 2, myelin sheaths with five and eight lamellae were stained respectively by anti-P1 and anti-P2. On day 3 the percentages of myelin sheaths stained were substantially higher: P0 95%, P1 78%, P2 15%. By day 4, anti-P0 and anti-P1 immunoreactivity was present in 95% of myelin sheaths; 35% were stained by anti-P2. For P2, staining intensity and percentage of myelin sheaths stained continued to increase and by day 20, 85% were anti-P2-positive. The density of immunoreactivity was not uniform in all myelin sheaths. At young ages staining varied with all three proteins. The variability decreased as myelin sheaths thickened; it persisted longest for anti-P2. We conclude that the density and distribution of immunoreactivities of P0, P1, and P2 reflect their relative concentrations during myelin sheath development and growth. We attribute lack of detectable anti-P2 immunoreactivity in some small sheaths at 20 days to their early stage of myelination and also to limitations of the method. We infer from our observations that all myelin-forming Schwann cells express P2 basic protein.

Correlation between CGRP immunoreactivity and firing activity in cat abducens motoneurons.

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 betaCGRP gene expression, was analyzed during these periods of altered firing activity. The number of CGRP-immunofluorescent abducens motoneurons increased to approximately 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.

Development of oculomotor axon projections in the chick embryo.

The pattern of innervation of the extraocular muscles is highly conserved across higher vertebrate species and mediates sophisticated visuomotor processes. Defects in oculomotor development often lead to strabismus, a misalignment of the eyes that can cause partial blindness. Although it has been intensively studied from a clinical perspective, relatively little is known about how the system develops embryonically. We have therefore mapped the development of the oculomotor nerve (OMN) in chick embryos by using confocal microscopy. We show that OMN development follows a series of stereotyped steps that are tightly regulated in space and time. The OMN initially grows past three of its targets to innervate its distal target, the ventral oblique muscle, only later forming branches to the more proximal muscles. We have also investigated spatiotemporal aspects of the unusual contralateral migration of a subpopulation of oculomotor neurons by using molecular markers and have found the semaphorin axon guidance molecules and their receptors, the neuropilins, to be expressed in discrete subnuclei during this migration. Finally, we have created an embryological model of Duane retraction syndrome (DRS), a form of strabismus in which the OMN is believed to innervate aberrantly the lateral rectus, the normal target of the abducens nerve. By ablating rhombomeres 5 and 6 and hence the abducens, we have mimicked a proposed oculomotor deficit occurring in DRS. We find that the absence of the abducens nerve is not sufficient to produce this inappropriate innervation, so other factors are required to explain DRS.

Morphological identification of nitric oxide sources and targets in the cat oculomotor system.

Nitric oxide (NO) production by specific neurons in the prepositus hypoglossi (PH) nucleus is necessary for the correct performance of eye movements in alert cats. In an attempt to characterize the morphological substrate of this NO function, the distribution of nitrergic neurons and NO-responding neurons has been investigated in different brainstem structures related to eye movements. Nitrergic neurons were stained by either immunohistochemistry for NO synthase I or histochemistry for reduced nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase. The NO targets were identified by cyclic guanosine monophosphate (cGMP) immunohistochemistry in animals treated with a NO donor immediately before fixation of the brain. Connectivity between cells of the NO-cGMP pathway was analyzed by injections of the retrograde tracers horseradish peroxidase or fast blue in different structures. The motor nuclei commanding extraocular muscles did not contain elements of the NO-cGMP pathway, except for some scattered nitrergic neurons in the most caudal part of the abducens nucleus. The PH nucleus contained the largest number of nitrergic cell bodies and a rich neuropil, distributed in two groups in medial and lateral positions in the caudal part, and one central group in the rostral part of the nucleus. An abundant cGMP positive neuropil was the only NO-sensitive element in the PH nucleus, where no cGMP-producing neuronal cell bodies were observed. The opposite disposition was found in the marginal zone between the PH and the medial vestibular nuclei, with a large number of NO-sensitive cGMP-producing neurons and almost no nitrergic cells. Both nitrergic and NO-sensitive cell bodies were found in the medial and inferior vestibular nuclei and in the superior colliculus, whereas the lateral geniculate nucleus contained nitrergic neuropil and a large number of NO-sensitive cell bodies. Some of the cGMP-positive neurons in the marginal zone and medial vestibular nucleus projected to the PH nucleus, predominantly to the ipsilateral side. These morphological findings may help to explain the mechanism of action of NO in the oculomotor system.

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.

Targeting of GLUR4-containing AMPA receptors to synaptic sites during in vitro classical conditioning.

The synaptic delivery of GluR4-containing AMPA receptors during in vitro classical conditioning of a neural correlate of an eyeblink response was examined by fluorescence imaging of punctate staining for glutamate receptor subunits and the presynaptic marker synaptophysin. There was a significant increase in GluR4-containing AMPA receptors to synaptic sites after conditioning as determined by colocalization of GluR4 subunit puncta with synaptophysin. Moreover, the trafficking of these receptor subunits requires NMDA receptor activation as it was blocked by D,L-2-amino-5-phosphonovaleric acid (AP-5). In contrast, colocalization of NR1 subunits with synaptophysin was stable regardless of whether the preparations had undergone conditioning or had been treated by AP-5. The enhanced colocalization of GluR4 and synaptophysin was accompanied by an increase in both the total number and size of puncta for both proteins, suggesting greater synthesis and aggregation during conditioning. Western blot analysis confirmed upregulation of synaptophysin and GluR4 following conditioning. These data support the hypothesis that GluR4-containing AMPA receptors are delivered to synaptic sites during conditioning. Further, they suggest coordinate presynaptic and postsynaptic modifications during in vitro classical conditioning.

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.

Differential regulation of alpha- and beta-CGRP mRNAs within oculomotor, trochlear, abducens, and trigeminal motoneurons in response to axotomy.

Spinal and cranial motoneurons express alpha- and beta-calcitonin gene-related peptide (CGRP) mRNAs constitutively at variable ratios, and these two mRNAs are differentially regulated following axotomy in spinal, facial, and hypoglossal motoneurons. The purpose of this study was to investigate the change in CGRP mRNA expression following nerve injury in oculomotor, trochlear, abducens, and trigeminal motor nuclei in which beta-CGRP mRNA is predominantly expressed under normal conditions. Using male Sprague-Dawley rats, either the left eyeball and the orbital contents including the bulbar muscles were removed, or the left masseter nerve was ligated and transected. The rats were allowed to survive for 1, 3, 7, 14, 28, 56 days following these procedures. The levels of mRNAs for alpha- and beta-CGRP and growth-associated protein (GAP)-43 were analyzed by in situ hybridization histochemistry using 35S-labeled oligonucleotide probes. Following nerve injury, the expression of alpha-CGRP mRNA rapidly increased on the directly-injured side in all of these nuclei. Thereafter, it gradually decreased and returned to about the control level at postoperative day 56 within oculomotor, trochlear, and abducens motoneurons, but it sustained at a high level within trigeminal motoneurons. The expression of beta-CGRP was quite variable among these nuclei, and significant changes were also seen on the side contralateral to the directly-injured side. These data indicate that the up-regulation of alpha-CGRP mRNA may be a common response of cranial motor neurons following axotomy even if the constitutive expression of beta-CGRP mRNA exceeds that of alpha-CGRP mRNA in these neurons.

Transneuronal transport in the vestibular and auditory systems of the squirrel monkey and the arctic ground squirrel. I. Vestibular system.

Transneuronal transport of [3H]proline, [3H]fucose, and [3H]leucine in various combinations from pledgets implanted in the ampulla of a single semicircular duct was studied in the squirrel monkey and arctic ground squirrel after long survival periods. Tritiated amino acids implanted in any single ampulla resulted in labeling of nearly all vestibular and auditory receptors, nearly all cells of the vestibular and spiral ganglia and central transport via nearly all root fibers of both nerves. Primary vestibular fibers were distributed to the vestibular nuclei (VN) and specific parts of the cerebellum in the pattern previously described. Transneuronal transport of [3H]proline by vestibular neurons was present in all known secondary pathways, except those projecting to thalamic nuclei. Observations were similar in both species, except for small differences in commissural vestibular projections. Major commissural transport was to all parts of the opposite medial vestibular nucleus (MVN) and to peripheral parts of the superior vestibular nucleus (SVN), but some transport was present in all contralateral VN, including ventral cell group y. Descending transneuronal transport was evident in vestibulospinal tract (VST) ipsilaterally and in the medial longitudinal fasciculus (MLF) bilaterally. Both [3H]proline and [3]fucose were transported transneuronally to the ipsilateral abducens nucleus (AN); with long survivals [3H]proline was transported peripherally via the ipsilateral abducens nerve root. Ascending transport in the MLF was bilateral, asymmetric and greatest contralaterally. Fibers entered the contralateral MLF near the AN and the lateral wing of the ipsilateral MLF rostral to most of the VN. Terminals in the trochlear nuclei (TN) were bilateral and greatest contralaterally. In the monkey terminals in ipsilateral oculomotor complex (OMC) were distributed uniformly in all subdivisions, except for the medial rectus subdivision (MRS), where terminals were more numerous. The greatest density of terminals was present contralaterally in the superior rectus subdivision (SRS) of the OMC; only sparse terminals were present in the MRS on that side. Transport in the ipsilateral abducens nerve roots in the monkey and the virtual absence of transport to the MRS of the contralateral OMC suggested transneuronal transport to abducens motor neurons, but not to internuclear neurons (AIN). The AIN project only to the MRS of the contralateral OMC and do not appear to receive vestibular input. Comparable observations were made in the AN, TN and OMC of the ground squirrel, although the representation of the extraocular muscles in the OMC is unknown.(ABSTRACT TRUNCATED AT 400 WORDS)

Changes in brainstem calcitonin gene-related peptide after VIIth and VIIIth cranial nerve lesions in guinea pig.

The present study investigated the effect of seventh and eight cranial nerve lesions on the prominence of calcitonin gene-related peptide in the hypoglossal (XII), facial (VII), abducens (VI), and oculomotor (III) cranial nerve nuclei. Guinea pigs were anesthetized and subjected to unilateral cochlear removal, vestibular end organ ablation, and seventh nerve transection. After a survival period ranging from 4 h to 5 days, each animal was anesthetized and perfused intracardially. Frozen sections were collected through the brainstem and stained immunohistochemically for calcitonin gene-related peptide using a polyclonal antibody with the Vectastain ABC kit and protocol. Positive cells were counted in each nucleus bilaterally and analyzed for side to side differences. Nuclei XII and III showed no significant difference in the numbers of cells staining positively for calcitonin gene-related peptide between the ipsilateral and the contralateral sides to the lesion. However, nuclei VII and VI showed elevated numbers ipsilateral to the lesion on some days, but not all. For VII, there was no significant difference before 24 h, but there were significant differences 1-5 days after the lesion. Similarly, in VI, there was no difference before 24 h, but differences were significant beginning with day 1 and continuing through day 3, and finally disappearing by day 4. Changes in the numbers of CGRP positive cells in VII measurable 24 h after the lesion and continuing for at least 5 days afterward indicate a central nervous system retrograde response to peripheral motor nerve injury.(ABSTRACT TRUNCATED AT 250 WORDS)

Anterograde axonal transport of [125I]wheat germ agglutinin by motor neurons innervating the rat lateral rectus muscle.

Despite widespread evidence of the affinity of wheat germ agglutinin (WGA) for sensory, preganglionic autonomic and other CNS neuron cell bodies, evidence of the uptake and anterograde transport of WGA by motor neurons innervating skeletal muscle is lacking. It was hypothesized that these cells might lack plasma membrane moieties with N-acetylglucosamine for selective uptake and transport of WGA. To test this, rats were anesthetized and injected intracisternally with affinity-purified [125I]-WGA in order to label abducens motor neurons. Based on results of light and electron microscopic autoradiography, it can be concluded that abducens motoneurons, and presumably other motoneurons, are capable of uptake and anterograde axonal transport of [125I]WGA.

Localization of axonally transported [125I]wheat germ agglutinin in rat abducens motoneuron axons and terminals after intracisternal injection.

Iodinated wheat germ agglutinin (WGA) was taken up and transported by rat abducens motoneurons to nerve terminals in the lateral rectus muscle. Five days after intracisternal injection of lectin, axon terminals wee found to be the most radioactive source, based on the density of labeling. In axons the radioactive label was concentrated in an annular region about 1.26 microns wide beneath the plasma membrane. We hypothesize that the WGA was associated with smooth vesicular and tubular structures in the axoplasm but not limited to organelles immediately beneath the axon plasma membrane. No evidence of intercellular transfer to muscle cells was found.