Facial Neurons Research Articles

Differential effects of axotomy on immature and mature hamster facial neurons: a time course study of initial nucleolar and nuclear changes.

The early nuclear and nucleolar responses at 0.5, 1, 2 and 4 days after axotomy were observed in neurons just before and after the completion of nuclear maturation. Axotomy of hamster facial motor neurons at a postnatal age of 15 days did not produce any changes within the nucleus that were significantly different from those of control cells. In addition, no significant changes were evident in the adults at 0.5 and 1 day after axotomy. At postoperative days 2 and 4, however, the adult neurons showed enlargement of the nucleus and nucleolus. Nucleolonemal strands became more rounded and distinct, and the large cluster of granules located centrally in the nucleolus disaggregated. The irregularly distributed clumps of nucleolus-associated chromatin dispersed to form a thin shell about the nucleolar periphery. In adults at postoperative day 4, the nucleoplasmic granules became more homogeneous and less distinctly outlined than normal. The peak of both nucleolar and nuclear responses coincided at 2 days after injury in the adult, i.e. 2 days before the previously documented chromatolytic peak at 4 days after injury. These studies on the ultrastructural level support our previous hypothesis that the 15-day neurons are synthesizing at peak capacity related to their rapid growth phase and cannot be stimulated further by axotomy. The adult neurons, however, do undergo a metabolic reorganization for regenerative synthesis, and the nucleolar and nuclear changes observed are indicative of transcriptive alterations involving the underlying genome.

Somatopetal axonal transport of fluorescent lectins: Distribution pattern and cytophotometric quantification in mouse peripheral neurons

Six FITC-labeled lectins (Con A, WGA, SBA, DBA, UeA 1, PNA) with different carbohydrate specificities were injected into the snout region of 12-day-old mice. WGA, and to a lesser extent Con A, accumulated in trigeminal and facial neurons as a result of somatopetal axonal transport. Only an occasional trigeminal neuron was labeled after injection of SBA and DBA. None of the other lectins were incorporated. WGA and Con A were used for cytophotometric quantification of neuronal accumulation of transported FITC-lectins in isolated neurons from trigeminal ganglia. This technique makes it possible to study changes in neuronal lectin binding sites and adsorptive endocytosis during different physiological and pathological states.

Effect of chromatolysis upon the incorporation of L-4,5-(3H)-leucine into axotomized neurons

Autoradiography was used to compare the rate of protein synthesis in neurons which showed different types of neuronal change after axotomy. Different types of axon reaction were obtained after crush lesion of the facial nerve in rats and young rabbits and after evulsion of the facial nerve in rats. The incorporation of (3H)-leucine into protein was determined after intraperitoneal injection by counting silver grains over facial neurons in autoradiograms prepared from paraffin sections. Crush lesion of the facial nerve in rats was followed by barely discernible changes in the neurons, no chromatolysis, and full functional recovery. The lesion caused a significant transitory increase in the protein synthesis. After nerve evulsion, the same neurons showed a severe central chromatolysis and ultimate nerve cell disintegration. The protein synthesis was unchanged during the early chromatolytic phase after evulsion and decreased at later stages. Crush lesion of the facial nerve in young rabbits was followed by severe central chromatolysis, full functional recovery, and no loss of neurons. There was a moderate increase in the protein synthesis during chromatolysis and a further increase during the phase of recovery. It is concluded that regeneration after axotomy is accompanied by an increase in protein synthesis, regardless of the type of morphologic change in the neurons. Chromatolysis is not a prerequisite for this increase, and the chromatolytic response per se seems to be of minor importance for the extent of incorporation of (3H)-leucine into neuronal proteins.

Long-term effects of regeneration and prevention of regeneration on nucleolar morphology after facial nerve injury during development

The effect of regeneration and prevention of regeneration on nucleolar morphology was examined in developing hamster facial neurons. Specifically, the presence or absence of an intranucleolar body (INB), a large, Feulgen-negative accumulation of ribonucleoprotein, was characterized after either nerve crush, which allows functional return, or after ligature-axotomy, which does not allow functional reconnection. Surgery was carried out at selected, critical ages in the maturation of the reaction to injury of facial neurons at 15, 20, and 25 days postnatal age. After crush injury at any of the selected postnatal ages, nucleolar morphology returned to normal by postoperative day 50. When functional reconnection was not permitted, a differential recovery was seen which depended on the age at the time of injury. After ligature-axotomy at 15 days postnatal age, formation of INBs to normal adult values did not occur by postoperative day 200. After ligature-axotomy at 20 and 25 days postnatal age, adult INB percentages were attained by postoperative day 100. It appears that age and degree of nucleolar maturity at the time of operation correlate with the morphologic recovery of the nucleolus to its mature configuration when functional regeneration is not permitted. These differences in recovery from injury in developing neurons have several possible metabolic bases.

Neuronal uptake of iron: somatopetal axonal transport and fate of cationized and native ferritin, and iron-dextran after intramuscular injections.

Mice were injected into the muscles of the vibrissae with native ferritin (NF), cationized ferritin (CF) and iron-dextran. CF adsorbed on to the surface of the axon terminal at the neuromuscular junction, while NF did not. Both CF and NF were incorporated into vesicles and vacuoles at the synapse, but CF uptake was detected after injections at much lower concentrations than NF. In contrast to NF, CF was also found histochemically in cell bodies of facial neurones after a single i.m. injection, showing that the electrical charge of a molecule is one factor of importance for its potential to be incorporated in axons and transported somatopetally. Repeated i.m. injection of iron-dextran into suckling mice resulted in a marked iron load of Schwann cells and nerve cell bodies. This produced no signs of toxicity and the nerve fibre developed normally. Iron had disappeared from the nerve cell bodies after 25 days, while in Schwann cells it still persisted after 223 days.

Peripheral nerve repair.

The neuron is remarkable as a cell with a prolonged process, the axon. Various substances and organelles are transported up and down the axon. Axotomy profoundly affects this axonal transport. The proximal stump of the axon seals off and organelles accumulate. Terminal sprouts appear and move towards the distal stump. Soon after reinnervation neurotransmission reappears. Initially polyneuronal innervation occurs but this is then eliminated. Somatopetal axonal transport has been studied using tracer macromolecules in the mouse facial neuron model. Transport was blocked by axotomy from terminals in muscle but persisted at the injured axonal membrane. This stopped when the axonal membrane grew over the stump. Transport returned after reinnervation. Such transport may carry the signals that govern neuronal response to axotomy.

Hallucinogens potentiate responses to serotonin and norepinephrine in the facial motor nucleus

The present investigation was designed to determine the effect of hallucinogens on the facilitating action of serotonin (5-HT) and norepinephrine (NE) in the facial nucleus. Intravenous administration of d-lysergic acid diethylamide (LSD, 5–10 μg/kg), mescaline (0.5–1.0 mg/kg), or psilocin (0.5–1.0 mg/kg) had no effect by themselves on the glutamate-induced excitation of facial motoneurons. In contrast, the facilitation of facial neuron excitation by iontophoretically applied 5-HT and NE was enhanced 6–10 fold by these hallucinogens. The LSD-enhanced responses to 5-HT and NE continued for at least 4 hours after administration of the hallucinogen. Iontophoretic application of LSD or mescaline (low currents) also markedly potentiated the facilitating effect of 5-HT and NE. Higher currents of LSD (15–40 nA) temporarily antagonized the response to 5-HT. The nonhallucinogen ergot derivatives lisuride and methysergide failed to potentiate the facilitating effects of 5-HT or NE. These observations suggest that hallucinogens potentiate the effect of monoamines on facial motoneurons by increasing the sensitivity of 5-HT and NE receptors. A novel mechanism regarding the psychedelic effects of hallucinogens is discussed.

Cytophotometric quantification of retrograde axonal transport of a fluorescent tracer (primuline) in mouse facial neurons

A method for cytophotometric quantifications of retrograde axonal transport of a fluorescent tracer in tissue sections is described. As a fluorescent tracer the anionic vital stain primuline proved to be suitable since it resulted in a strong yellow-green fluorescence, which faded very slowly permitting localization of cells during illumination with UV-light. Primuline injected into the muscles of the vibrissae in mice was transported to the corresponding nerve cell bodies in the facial nucleus, where it appeared as fluorescent granules 9 h after the injection. The fluorescence intensity increased with increasing exposure times and concentrations of the injected tracers. The motor endplates showed no ultrastructural changes after the tracer injections. With this method a substantial increase in tracer accumulation in facial neurons could be revealed during nerve regeneration 11 days after crushing the facial nerve. Small alterations in neuronal tracer accumulation could be measured after intoxication of the mice with botulinum and tetanus toxins. Since these toxins should cause a decrease or increase in the degree of synaptic activity the amount of retrograde axonal transport may to a certain extent be dependent on the synaptic function. The findings with this new technique therefore indicate that quantitative changes occur in axonal transport in materials from the periphery during different pathological and physiological conditions, which may be important for an understanding of how a nerve cell body is dependent on its peripheral field of innervation.

Structural alterations in normal and axotomized facial nucleus neurons after treatment with morphine.

Chronic morphine administration induced ultrastructural alterations in neurons of the facial nucleus and increased the incidence of cell death after axotomy. These findings may correlate with the significant depression of neuronal metabolism known to occur after opiate treatment.

The effects of acute morphine treatment on the incorporation of [ 3H] l-lysine by normal and regenerating facial nucleus neurons

Although opiates significantly alter RNA and protein synthesis in a variety of neuronal cell types, their effect on the biosynthetic activity of regenerating neurons has not been investigated. In the present study, the effect of morphine on the incorporation of [ 3H] l-lysine into proteins of facial nucleus neurons was examined by light microscopic radioautography. Silver grains present within various compartments of normal and regenerating (3-, 7-, 14- and 21 days post-axotomy) neurons from saline-treated Wistar rats were compared with the amount present in similar cells from animals receiving 40 mg/kg morphine sulfate i.v. At 14- and 21-days post-axotomy, regenerating neurons were larger and the grain count in the emulsion over these cells was greater than that observed in normal (unoperated) neurons. In normal facial neurons, the accumulation of lysine into the nucleus and nucleolus was significantly lower 60 min after morphine administration. However, morphine's inhibition of lysine incorporation was even more pronounced in regenerating neurons. In these cells, nuclear lysine uptake was depressed at 3 and 7 days, while maximum inhibition of cytoplasmic incorporation occurred at 14-days post-axotomy. Morphine administration decreased nucleolar lysine incorporation at all survival intervals.

Uptake and retrograde axonal transport of horseradish peroxidase in regenerating facial motor neurons of the mouse.

Uptake and retrograde axonal transport of intravenously injected horseradish peroxidase (HRP) was studied during regeneration after a crush injury of the facial nerve of the mouse. The circulation time of HRP was 12 to 24 h. HRP injected immediately after the crush diffused into injured axons in the crushed region and accumulated subsequently in perikarya of facial neurons in the brain stem. After a time interval of 1 h or 5 days between the crush and the injection only a faint HRP accumulation occurred in a few facial neurons. After an interval of 7 days a moderate number of neurons had incorporated the tracer, while after more than 9 days the HRP activity in the regenerating neurons was more pronounced than in the contralateral neurons. Ultrastructurally, muscles of the vibrissae showed denervated subneural apparatuses 6 days after the crush. 8 days after the crush regenerating axon terminals containing small clusters of synaptic vesicles, dense cored vesicles and some HRP-labelled vesicles, were found over some gutters and after 10 to 13 days all examined gutters contained axon terminals with large numbers of synaptic vesicles and some HRP-containing vesicles. More than one axon terminal profile was seen in the same synaptic gutter. 32 and 64 days after the crush the neuromuscular junctions had regained a more mature appearance. The calibre spectra of the crushed facial nerves still showed a shift towards smaller diameters 134 days after the crush, at a time when a slight increase in HRP activity in the facial neurons persisted.

Synaptic activation of facial afferents on the facial neurons of the cat

Synaptic activation of facial afferents on the facial neurons of the cat

The facial motor nucleus of the opossum: Synaptic endings on dendrites

The diameters of dendrites of large, medium and small neurons (Falls and King, '76) were measured from Golgi impregnations of the opossum facial motor nucleus in order to classify dendritic profiles sectioned in the transverse plane in electron micrographs. Three categories of dendrites are described: (1) proximal (4-7 mu in diameter); (2) intermediate (2-4 mu in diameter) and (3) distal (0.5-2 mu in diameter). The distribution of axodendritic synaptic endings was determined, recognizing that the neuronal source of individual dendritic profiles when seen in the transverse plane of section cannot be absolutely determined in view of the overlap in size of the dendrites issuing from the three types of neurons. Presynaptic terminals were categorized according to vesicle shape (spherical, pleomorphic or ellipsoidal), vesicle size, terminal size, junctional characteristics and post synaptic distribution. The vesicle size is expressed as a mean area (nm2) and was determined by using a cybergraphic tablet and a PDP-12 computer system. In any given plane of section, synaptic terminals cover most of the membrane of proximal dendrites and decrease in number as intermediate and distal dendrites are encountered. In Golgi impregnations four classes of afferent fibers which ramify among the dendrites of facial neurons can be distinguished. As yet, their sources have not been identified. Possible sites of origin for presynaptic profiles are discussed in the context of previous light microscopic findings.

Changes in an intranucleolar body in hamster facial neurons following axotomy

Facial motor neurons of adult golden hamsters were studied at frequent intervals up to 25 days after severance of the right facial nerves at their exit from the stylomastoid foramen. All brains were perfusion-fixed and prepared for light microscopy; sections were stained with either thionine, Feulgen reagent, or mercuric bromphenol blue. The intranucleolar body was observed as a rounded, intensely basophilic, Feulgen-negative structure located centrally in the nucleolus of normal adult facial neurons. After axotomy, chromatolysis resulted within 1 day and peaked at about 4 days, with gradual restoration of stainable Nissl substance thereafter. The intranucleolar body in severed neurons was apparently unaffected at 1 and 2 days, but by 4 days was represented by three or four densely staining particles forming a ringlike configuration, which persisted even after complete return of Nissl substance. Only after a postoperative period of 25 days did the intranucleolar body again appear as a single, densely staining structure comparable to that in normal cells. The disappearance of the intranucleolar body during chromatolysis coincided with what is known to be a period of heightened nucleolar synthetic activity. Delay in returning to its definitive appearance, despite a rather prompt reconstitution of Nissl substance, may reflect a persistently high level of protein synthesis in neurons which are prevented from reestablishing their original connections. Evidence indicates that the existence of the body does not depend upon the establishment of terminal connections per se, even in the developing neuron.

Afferent projections in the hypoglossal nerve to the facial neurons of the cat

Afferent projections in the hypoglossal nerve to the facial neurons of the cat

Developmental protein changes in normal and chromatolytic facial nerve nuclear regions

Facial neurons were severed at the stylomastoid foramen in immature (15- and 20-day-old) and adult hamsters. Four days later animals were killed under Nembutal, in accordance with either of two approaches. In one series, after perfusion with physiological saline, the medullas were immediately removed, quickfrozen in liquid nitrogen, and sectioned in a cryostat. Facial nerve nuclear regions from the experimental and control sides were dissected free from the dehydrated sections and analyzed for protein differences by micro disc electrophoresis. In another series, 3H-leucine was injected intraperitoneally 24 hr before killing by perfusion-fixation. Isotope uptake in experimental and control facial neurons was determined by autoradiography. The pattern of protein changes and the uptake of the amino acid on the chromatolytic side became progressively more dissimilar from the normal, the more mature the neurons were at the time of operation. This involved an increase in both the number of separated proteins and the uptake of labeled leucine on the experimental side. We suggest that these reactive differences may reflect two different types of metabolism, present, respectively, in immature and mature nuclear regions. The first type, present in immature and chromatolytic regions, may be concerned with the synthesis of structural proteins. The second type, present in adult normal nuclear regions, may be concerned with the synthesis of proteins for cell maintenance and function.