Tongue and hypoglossal morphology after intralingual cholera toxin B-saporin injection.

INTRODUCTION: We recently developed an inducible model of dysphagia using intralingual injection of cholera toxin B conjugated to saporin (CTB-SAP) to cause death of hypoglossal neurons. In this study we aimed to evaluate tongue morphology and ultrastructural changes in hypoglossal neurons and nerve fibers in this model. METHODS: Tissues were collected from 20 rats (10 control and 10 CTB-SAP animals) on day 9 post-injection. Tongues were weighed, measured, and analyzed for microscopic changes using laminin immunohistochemistry. Hypoglossal neurons and axons were examined using transmission electron microscopy. RESULTS: The cross-sectional area of myofibers in the posterior genioglossus was decreased in CTB-SAP-injected rats. Degenerative changes were observed in both the cell bodies and distal axons of hypoglossal neurons. DISCUSSION: Preliminary results indicate this model may have translational application to a variety of neurodegenerative diseases resulting in tongue dysfunction and associated dysphagia.

Endogenous Rho-kinase signaling maintains synaptic strength by stabilizing the size of the readily releasable pool of synaptic vesicles.

Rho-associated kinase (ROCK) regulates neural cell migration, proliferation and survival, dendritic spine morphology, and axon guidance and regeneration. There is, however, little information about whether ROCK modulates the electrical activity and information processing of neuronal circuits. At neonatal stage, ROCKα is expressed in hypoglossal motoneurons (HMNs) and in their afferent inputs, whereas ROCKß is found in synaptic terminals on HMNs, but not in their somata. Inhibition of endogenous ROCK activity in neonatal rat brainstem slices failed to modulate intrinsic excitability of HMNs, but strongly attenuated the strength of their glutamatergic and GABAergic synaptic inputs. The mechanism acts presynaptically to reduce evoked neurotransmitter release. ROCK inhibition increased myosin light chain (MLC) phosphorylation, which is known to trigger actomyosin contraction, and reduced the number of synaptic vesicles docked to active zones in excitatory boutons. Functional and ultrastructural changes induced by ROCK inhibition were fully prevented/reverted by MLC kinase (MLCK) inhibition. Furthermore, ROCK inhibition drastically reduced the phosphorylated form of p21-associated kinase (PAK), which directly inhibits MLCK. We conclude that endogenous ROCK activity is necessary for the normal performance of motor output commands, because it maintains afferent synaptic strength, by stabilizing the size of the readily releasable pool of synaptic vesicles. The mechanism of action involves a tonic inhibition of MLCK, presumably through PAK phosphorylation. This mechanism might be present in adults since unilateral microinjection of ROCK or MLCK inhibitors into the hypoglossal nucleus reduced or increased, respectively, whole XIIth nerve activity.

Differential toxicities of intraneurally injected mercuric chloride for sympathetic and somatic motor fibers: an ultrastructural study.

BACKGROUND/PURPOSE: Mercury is a well-known neurotoxin but the susceptibility of autonomic nerves to mercury poisoning in vivo has seldom been studied. Our previous studies have shown that the hypoglossal nerve in hamsters contains somatic motor and postganglionic sympathetic fibers. The aim of this study was to investigate the ultrastructural changes in the nervous system following intraneural injection of mercuric chloride into the hypoglossal nerve in hamsters. METHODS: Six adult hamsters were used in this study. After anesthesia, the digastric muscle on the right side was removed and the trunk of the hypoglossal nerve was exposed. Two microliters of mercuric chloride aqueous solution was injected into the main trunk of the hypoglossal nerve at the bifurcation. The contralateral hypoglossal nerve was kept intact and used as the normal control. Animals were allowed to survive for 1 or 3 days and were prepared for ammonium sulfide histochemistry and electron microscopy. RESULTS: Three days after injection of mercuric chloride solution, almost all unmyelinated sympathetic fibers in the hypoglossal nerve trunk were lost, whereas myelinated somatic axons were spared. Although mercury deposition in the myelin sheaths of neuronal processes was observed in the hypoglossal nucleus, the neuronal somas were intact. By contrast, degenerated neuronal processes and mercury deposition in neuronal somas were frequently found in the superior cervical ganglia. CONCLUSION: This study demonstrated an undue susceptibility of sympathetic fibers to mercury intoxication. The mechanisms that underlie the selective reaction of sympathetic fibers to mercury warrant further investigation.

Crossing dendrites of the hypoglossal motoneurons: possible morphological substrate of coordinated and synchronized tongue movements of the frog, Rana esculenta.

Application of different fluorescent tracers to the right and left hypoglossal nerve of the frog revealed the extent of dendrites crossing the midline into the territory of contralateral hypoglossal motoneurons. By using confocal microscopy, a large number of close appositions were detected between hypoglossal motoneurons bilaterally, which formed dendrodendritic and dendrosomatic contacts. The distance between the neighboring profiles suggested close membrane appositions without interposing glial elements. Application of neurobiotin to one hypoglossal nerve resulted in labeling of perikarya exclusively on the ipsilateral side of tracer application, suggesting the absence of dye-coupled connections with contralateral hypoglossal motoneurons. At the ultrastructural level, the dendrodendritic and dendrosomatic contacts did not show any morphological specialization; the long membrane appositions may provide electrotonic interactions between the neighboring profiles. We propose that dendrites of hypoglossal motoneurons that cross the midline subserve one of the morphological substrates of co-activation, synchronization and timing of bilateral activity of tongue muscles during prey-catching behavior of the frog.

Ultrastructural observations on the progress of nerve degeneration and regeneration at the suture site following vagal-hypoglossal nerve coaptation in cats.

Nerve degeneration and regeneration have been investigated at the suture site following proximal-to-distal vagal-hypoglossal nerve coaptation (VHC) in cats at different time points (from 3 to 315 days postoperatively; dpo). Massive axonal degeneration and myelin breakdown and removal of degraded neural debris were observed during the first 2 weeks postoperatively. This was followed by active Schwann cell multiplication and inflammatory cell invasion at 14 dpo. Schwann cells appeared mobile, and were guided to the newly developed growth cones, dividing them into axonal sprout clusters. At 18 dpo, the migrating Schwann cells were confined to the preexisting basal lamina scaffolds, forming bands of Bungner. It is suggested that the latter may play a key role in navigating the regenerating axons to their newly acquired target organ at 22 dpo. Remyelination of axons was not observed till 46 dpo. Compared with the rapid axonal reaction in other models of nerve injury, the degeneration process in VHC was protracted and, furthermore, regeneration and remyelination were delayed. The subtle remodeling of the nerve in cross-coaptation may be far greater than previously recognized, and this may have clinical importance since patients undergoing nerve crossover microsurgery exhibit delayed motor rehabilitation, apparently as a direct result of a change in target innervation. Defining the mechanisms underlying the neuroplastic program could thus potentially improve the prognosis of crossover of two different peripheral nerves.

Quantitative analysis of multivesicular bodies (MVBs) in the hypoglossal nerve: evidence that neurotrophic factors do not use MVBs for retrograde axonal transport.

Multivesicular bodies (MVBs) are defined by multiple internal vesicles enclosed within an outer, limiting membrane. MVBs have previously been quantified in neuronal cell bodies and in dendrites, but their frequencies and significance in axons are controversial. Despite lack of conclusive evidence, it is widely believed that MVBs are the primary organelle that carries neurotrophic factors in axons. Reliable information about axonal MVBs under physiological and pathological conditions is needed for a realistic assessment of their functional roles in neurons. We provide a quantitative ultrastructural analysis of MVBs in the normal postnatal rat hypoglossal nerve and under a variety of experimental conditions. MVBs were about 50 times less frequent in axons than in neuronal cell bodies or dendrites. Five distinct types of MVBs were distinguished in axons, based on MVB size, electron density, and size of internal vesicles. Although target manipulations did not significantly change MVBs in axons, dystrophic conditions such as delayed fixation substantially increased the number of axonal MVBs. Radiolabeled brain- and glial-cell derived neurotrophic factors (BDNF and GDNF) injected into the tongue did not accumulate during retrograde axonal transport in MVBs, as determined by quantitative ultrastructural autoradiography, and confirmed by analysis of quantum dot-labeled BDNF. We conclude that for axonal transport, neurotrophic factors utilize small vesicles or endosomes that can be inconspicuous at transmission electron microscopic resolution, rather than MVBs. Previous reports of axonal MVBs may be based, in part, on artificial generation of such organelles in axons due to dystrophic conditions.

Ultrastructural changes of posterior lingual glands after hypoglossal denervation in hamsters.

Posterior lingual glands consist of two sets of minor salivary glands that serve important functions in oral physiology. To investigate the hypothesis that the hypoglossal nerve provides sympathetic innervation to the posterior lingual glands, we examined ultrastructural changes in the glands following hypoglossal denervation. In the posterior deep lingual glands (of von Ebner), the serous acinar cells showed a decrease in the number of secretory granules and an increase in lipofuscin accumulation. The ratios of cells containing lipofuscin granules were 11.39, 36.49 and 50.46%, respectively, of the control, 3- and 7-day post-axotomy glands (P < 0.001). Intraepithelial phagocytotic activity was increased. The mucous acinar cells in the posterior superficial lingual glands (of Weber) also showed degenerative changes after hypoglossal denervation. One week after nerve transection, marked cytoplasmic vacuolation and fragmentation of organelles were frequently observed. Degenerative changes were also found in unmyelinated axons associated with the glands. We provide the first evidence of the structural and functional connections between the sympathetic component of the hypoglossal nerve and posterior lingual glands.

Aspectos morfométricos do nervo hipoglosso humano em adultos e idosos / Morphometric aspects of the human hypoglossal nerve in adults and the elderly

OBJETIVO: Realizar análise morfométrica das fibras mielínicas do nervo hipoglosso direito, em dois grupos etários, com a finalidade de verificar modificações quantitativas decorrentes do processo de envelhecimento. FORMA DE ESTUDO: anatômico. MATERIAL E MÉTODO: Foi coletado fragmento de 1cm do nervo hipoglosso direito de 12 cadáveres do sexo masculino, sem antecedentes para doenças como diabetes, alcoolismo e neoplasia maligna. A amostra foi dividida em dois grupos: grupo adulto (idade inferior a 60 anos), composto por seis cadáveres; grupo idoso (idade igual ou superior a 60 anos), composto por seis cadáveres. O material foi fixado em solução contendo 2,5 por cento de glutaraldeído e 2 por cento de paraformaldeído; pós-fixado em tetróxido de ósmio 2 por cento; desidratado em concentrações crescentes de etanol e incluído em resina epóxi. Cortes semifinos de 0,3»m de espessura foram obtidos, corados com azul de toluidina a 1 por cento e avaliados em microscópio de luz acoplado a sistema analisador de imagens. Os seguintes dados morfométricos foram quantificados: área de secção transversal intraperineural, número e o diâmetro das fibras mielínicas. RESULTADOS: A área intraperineural do nervo hipoglosso foi semelhante nos dois grupos etários (p=0,8691). A média da área no grupo adulto foi de 1,697 mm2, e no grupo idoso foi de 1,649 mm2. O número total de fibras mielínicas do nervo hipoglosso foi semelhante nos dois grupos etários (p=0,9018). O grupo adulto apresentou média de 10.286 ± 2308 fibras mielínicas e o grupo idoso apresentou média de 10.141 ± 1590 fibras mielínicas. Foi observada distribuição bimodal das fibras mielínicas, com pico acentuado nas fibras de 9»m e outro menor nas fibras de 2»m. CONCLUSÃO: A área intraperineural e o número total de fibras mielínicas do nervo hipoglosso direito é semelhante nos dois grupos etários.

Ultrastructural identification of a sympathetic component in the hypoglossal nerve of hamsters using experimental degeneration and horseradish peroxidase methods.

We employed experimental degeneration, tract-tracing with wheatgerm agglutinin conjugated with horseradish peroxidase (WGA-HRP) and electron microscopy to explore the postganglionic sympathetic fibers in the hypoglossal nerve of hamsters. Quantitative results of normal untreated animals at the electron microscopic level showed the existence of unmyelinated fibers, which made up about 20% of the total fibers in the nerve, being more numerous on the left side. The nerve fibers were preferentially distributed at the periphery of the nerve. Following superior cervical ganglionectomy, most of the unmyelinated fibers underwent degenerative changes. Tract-tracing studies showed that some of the unmyelinated fibers were labeled by WGA-HRP injected into the superior cervical ganglion (SCG). It is suggested that the unmyelinated fibers represent the postganglionic sympathetic fibers originated from the SCG.

Morphometric aspects of the human hypoglossal nerve in adults and the elderly.

AIM: Perform a morphometric analysis of the myelinic fibers of the right hypoglossal nerve, in two age groups; to verify quantitative changes as a result of the aging process. STUDY DESIGN: Anatomic. MATERIAL AND METHOD: A 1 cm fragment of the right hypoglossal nerve was collected from 12 male corpses without any medical history of diseases such as: diabetes, alcoholism, and malignant neoplasia. The sample was divided in two groups: group with six corpses under sixty years old (adult), and another group with six corpses sixty years old or above (elderly). The material was fixed at 2.5% glutaldehyde and 2% paraformaldehyde solution; post-fixed at 2% osmium tetroxide; dehydrated with increasing ethanol concentrations, and included in epoxy resin. Semi-thin sections of 0.3 microm were obtaining, colored in 1% toluidine blue, and evaluated with light microscope combined with image analyzing system. The following morphometric data were quantified: intraperineural transversal section area, number, and diameter of the myelinic fibers. RESULTS: The intraperineural area of the hypoglossal nerve was similar in both age groups (p=0.8691). The average area in the adult group was 1.697 mm2 and in the elderly group it was 1.649 mm2. The total number of myelinic fibers of the hypoglossal nerve was similar in both age groups (p=0.9018). The adult group presented an average of 10,286 +/- 2,308 myelinic fibers, and the elderly group presented an average of 10,141 +/- 1,590 myelinic fibers. A bimodal distribution of the myelinic fibers was observed, with a significant peak on the 9 microm fibers, and another smaller peak on the 2 microm fibers. CONCLUSION: The intraperineural area and the total number of myelinic fibers of the right hypoglossal nerve are similar in both age groups.

Changes of high-affinity choline transporter CHT1 mRNA expression during degeneration and regeneration of hypoglossal nerves in mice.

The high-affinity choline transporter CHT1 works for choline uptake in the presynaptic terminals of cholinergic neurons. We examined its expression in the hypoglossal nucleus after unilateral hypoglossal nerve transection in mice by fluorescent in situ hybridization. One week after axotomy, CHT1 mRNA expression was lost in all hypoglossal motoneurons in the lesioned side. Two weeks after axotomy, CHT1 mRNA started to be re-expressed in a few motoneurons that recovered connections to tongue muscles as revealed by retrograde labeling with Fast Blue. After 4 weeks, most of axotomized hypoglossal motoneurons were reconnected and re-expressed CHT1 mRNA as strongly as control neurons, and the regenerating cholinergic axons established mature neuromuscular junctions. These results suggest that the establishment of motor innervation is critical for CHT1 mRNA expression in hypoglossal neurons after axotomy.

Synaptic remodeling in the nucleus ambiguus following vagal-hypoglossal nerve anastomosis in the cat.

We reported recently the occurrence of a massive and selective elimination of synaptic boutons on motoneurons in the dorsal motor nucleus of the vagus (DMV) in the cat following vagal-hypoglossal nerve anastomosis (VHA) [J. Comp. Neurol. 458 (2003) 195]. This study was aimed to explore the synaptic reorganization in the other major nucleus associated with the vagus, namely, the nucleus ambiguus (NA) following the same treatment. In view of the tremendous difference in function, the NA and DMV are considered to be two ideal nuclei for explanatory studies seeking to elucidate how VHA could induce different plasticity of brainstem neurons influenced by the newly reestablished neural pathway. The present results showed that the vagal efferent neurons in the NA had responded to VHA in a different manner compared with those in the DMV. Firstly, the numbers of axon terminals containing round (R), round with dense-cored (R+D), pleomorphic (P) or flattened (F) synaptic vesicles contacting the NA motoneurons were markedly increased at 500-day postoperation, the longest reinnervation interval. The percent increases in the synapse frequency for R, R+D, P and F boutons were 8.6%, 274.4%, 238.3% and 400.0%, respectively. Secondly, the formation of astroglial ensheathment around the motoneurons in the DMV following VHA was not evident in the NA. Another striking difference was the extensive dendritic sprouting of the NA neurons as opposed to the dendritic retraction of the DMV neurons as shown by a significant increase in distal dendrites of NA motoneurons. The different modes of neural remodeling between NA and DMV may be attributed to the unique nature of the two nuclei to structures they normally supply and their different compatibility with the newly innervated target, viz. tongue skeletal musculature.

Genioglossal hypoglossal motoneurons contact substance P-like immunoreactive nerve terminals in the cat: a dual labeling electron microscopic study.

This study investigated the synaptic interactions between hypoglossal motoneurons that project to the genioglossus muscle and substance P (SP) containing immunoreactive nerve terminals. Cholera toxin B conjugated to horseradish peroxidase (CTB-HRP) was injected into the right half of the genioglossus muscle in four anesthetized cats. Two days later, the animals were perfused with acrolein fixative. Tetramethylbenzidine (TMB) was the chromogen used to detect retrogradely labeled cells containing CTB-HRP. The tissues were then processed for immunocytochemistry using an antiserum raised against SP with diaminobenzidine (DAB) as the chromogen. At the light microscopic level, labeled cells were observed primarily ipsilaterally in ventral and ventrolateral subdivisions of the hypoglossal nucleus. The majority of these labeled cells were observed at the level of the area postrema. At the electron microscopic level, SP-like immunoreactive nerve terminals formed synaptic contacts with retrogradely labeled dendrites and perikarya. Nineteen percent of the terminals that contacted retrogradely labeled cells contained SP. These are the first ultrastructural studies demonstrating synaptic interactions between protruder hypoglossal motoneurons and SP terminals. These studies demonstrate that hypoglossal motoneurons which innervate the major protruder muscle of the tongue, the genioglossus muscle, may be modulated by SP. Thus, SP may play a role in the control of protrusive movements of the tongue acting via neurokinin receptors.

Ultrastructural features of synapse from dorsal parvocellular reticular formation neurons to hypoglossal motoneurons of the rat.

The dorsal parvocellular reticular formation (PCRt) receives projection of the trigeminal mesencephalic nucleus neurons. It contains the dorsal group of interneurons that integrate and coordinate activity of the oral motor nuclei. Ultrastructural features of synaptic connection from the dorsal PCRt neurons to the motoneurons of the hypoglossal nucleus (XII) were examined at both the light and electron microscopic levels in rats. Biotinylated dextran amine (BDA) was initially iontophoresed into the dorsal part of PCRt unilaterally. Seven days later horseradish peroxidase (HRP) was injected into the body of the tongue. After histochemical reaction for visualization of HRP and BDA, the BDA-labeled fibers and terminals were seen distributing bilaterally in XII with ipsilateral predominance. BDA-labeled terminals were closely apposed upon HRP retrogradely labeled somata and dendrites of the XII motoneurons. A total of 1408 BDA-labeled boutons were examined ultrastructurally, which had mean size of 1.22+/-0.37 microm in diameter. Five hundred-ninety three of these boutons in both the ipsilateral (n=401) and contralateral (n=192) XII were seen to synapse on both the dendrites and somata of HRP-labeled motoneurons. The vast majorities of synapses were axodendritic (98%, 580/593), while 2% of them were axosomatic. Of the 1408 BDA-labeled boutons, 69.6% of them were S-type boutons containing small clear and spherical synaptic vesicles and 30.4% of them were PF-type boutons containing pleomorphic and flattened synaptic vesicles. Approximately 64% of synapses between BDA-labeled boutons and HRP-labeled motoneurons were asymmetric, and 33% of synapses were symmetric. No axoaxodendritic or axoaxosomatic synaptic triad was observed. The present study illustrated the anatomical pathway and synaptological characteristics of neuronal connection between the dorsal PCRt premotor neurons and the XII motoneurons. Its functional significance in coordinating activity of XII motoneurons during oral motor behaviors has been discussed.

Morphological changes in frog mast cells induced by nerve stimulation in vivo.

We investigated at both histochemical and ultrastructural levels the effects of unilateral electrical stimulation in vivo of the frog hypoglossal nerve on the mast cells (MCs) within the nerve fascicles and among the axon terminals. The right ventral root of the hypoglossal nerve in different experiments was stimulated respectively for 1, 3, 5, 10 min with over-threshold stimuli (10 Hz; 2 ms duration). The stimulations at 3, 5 and 10 min caused a progressive degranulation and histochemical and ultrastructural changes of the MCs at the stimulated side. The morphological changes consisted of the loss of Alcian Blue secretory content and of a progressive release of safranin+ secretory granules, depending upon duration of stimulation. The ultrastructural study showed that granules are discharged whole into the microenvironment or may release their content through exocytosis. A functional relationship between nerve and MCs is also suggested by the close anatomical association between MCs and pre-terminal axons observed following 10 min of hypoglossal stimulation. No changes in MC morphology occurred after 1 min of electrical stimulation. The results suggest that active cholinergic fibres can modulate MC secretion.

Axonal projections and synapses from the supratrigeminal region to hypoglossal motoneurons in the rat.

Neural circuits from the supratrigeminal region (Vsup) to the hypoglossal motor nucleus were studied in rats using anterograde and retrograde neuroanatomical tracing methodologies. Iontophoretic injection of 10% biotinylated dextran amine (BDA) unilaterally into the Vsup anterogradely labeled axons and axon terminals bilaterally in the hypoglossal nucleus (XII) as well as other regions of the brainstem. In the ipsilateral XII, the highest density of BDA labeling was found in the dorsal compartment and the ventromedial subcompartment of the ventral compartment, where BDA labeling formed a dense, patchy distribution. Microinjection of 20% horseradish peroxidase (HRP) ipsilaterally or bilaterally into the tongue resulted in retrograde labeling of XII motoneurons confined to the dorsal and ventral compartments of the hypoglossal motor nucleus. Under light microscopical examination, BDA-labeled terminals were observed closely apposing the somata and primary dendrites of HRP-labeled hypoglossal motoneurons. Two hundred and sixty-five of these BDA-labeled terminals were examined at the ultrastructural level. One hundred and twelve BDA-labeled axon terminals were observed synapsing with either the somata (39%, 44/112) or the large or medium-size dendrites (61%, 68/112) of retrogradely labeled hypoglossal motoneurons. Axon terminals containing spherical vesicles (S-type) formed asymmetric synapses with HRP-labeled hypoglossal motoneuron dendrites. In contrast to this, F(F)-type axon terminals, containing flattened vesicles, formed symmetric synapses with both the somata and dendrites of HRP-labeled hypoglossal motoneurons with a preponderance of the contacts on their somata. Axon terminals containing pleomorphic vesicles (F(P)-type) were noted forming both symmetric and asymmetric synapses with HRP-labeled hypoglossal motoneuron somata and dendrites. The present study provides anatomical evidence of neuronal projections and synaptic connections from the supratrigeminal region to hypoglossal motoneurons. These data suggest that the supratrigeminal region, as one of the premotor neuronal pools of the hypoglossal nucleus, may coordinate and modulate the activity of tongue muscles during oral motor behaviors.

Different astroglial reaction between the vagal dorsal motor nucleus and nucleus ambiguus following vagal-hypoglossal nerve anastomosis in cats.

The dorsal motor nucleus of the vagus (DMV) and nucleus ambiguus (NA) were both traced with horseradish peroxidase (HRP) retrograde labelling technique after vagal-hypoglossal nerve anastomosis (VHA). By light microscopy, reinnervation of the new target, viz. tongue skeletal musculature, by DMV and NA was established at 22 days postoperation (dpo) as shown by the neuronal labelling with HRP. Ultrastructurally, signs of retrograde degeneration occurred in some DMV and NA neurons between 3 and 25 days after VHA. The incidence of darkened dendrites, an early sign of dendritic loss, was more common in the DMV compared to the NA. Accompanying the neuronal alteration were drastic astrocytic reactions in the DMV, but not in the NA. Between 3 and 7 dpo, the astrocytes in the DMV showed extensively hypertrophied processes and by 22 dpo, the somata and dendrites of HRP-labelled DMV neurons, but not NA's, appeared to be delineated by the increased lamellar astrocytic processes. Such a feature was sustained throughout the remaining postoperative intervals up to 500 dpo. It is concluded that the DMV motoneurons being autonomic in nature are probably not conducive to the newly acquired target organ. Hence, the insulation of the regenerating DMV motoneurons by the astroglial ensheathment would be vital in the neuronal remodelling and reconstruction of the vagal-hypoglossal pathway.

Ultrastructural evidence of calcium involvement in experimental autoimmune gray matter disease.

Experimental studies have suggested that increased calcium and inappropriate calcium handling by motoneurons might have a significant role in motoneuron degeneration. To further define the involvement of calcium in motoneuron loss we used the oxalate-pyroantimonate technique for calcium fixation and monitored the ultrastructural distribution of calcium in spinal motoneurons in experimental autoimmune gray matter disease (EAGMD). In cervical and hypoglossal motoneurons from animals with relatively preserved upper extremity and bulbar function, increased calcium precipitates were present in the cytoplasm as well as in mitochondria, endoplasmic reticulum and Golgi complex without significant morphologic alterations. In surviving lumbar motoneurons of animals with hindlimb paralysis, however, there was massive morphological destruction of intracellular organelles but no significant accumulation of calcium precipitates. These findings suggest that altered calcium homeostasis is involved in motoneuron immune-mediated injury with increased calcium precipitates early in the disease process and decreased to absent calcium precipitates later in the pathogenesis of motoneuron injury.

Substance P in the hypoglossal nucleus of the rat.

The distribution of substance P (SP)-containing synaptic terminals in the hypoglossal nucleus (XII) of adult rats was examined by retrograde peroxidase labelling and immunocytochemistry. From the location of peroxidase injections into the tongue and of labelled neurones in the ventral lamina of XII, motor neurones that supply intrinsic vertical, longitudinal and transverse fibres as well as the extrinsic muscle genioglossus appear to have been labelled. SP-containing terminals were found making contact, and sometimes dual synapses, with unlabelled neuronal dendrites but not with retrogradely labelled somata or dendrites. These findings suggest that SP terminals may contact dendrites of interneurones or of neurones supplying other extrinsic muscles located in the anterior part of the tongue. Dual SP-containing synapses between XII motor neurones may be the means by which tongue muscle fibres are recruited and their function synchronized.

Quantitative analysis of the dendritic architectures of cat hypoglossal motoneurons stained intracellularly with horseradish peroxidase.

Little is known about the dendritic architecture of cat hypoglossal motoneurons. Thus, the present study was done to provide quantitative descriptions of hypoglossal motoneurons and to determine correlations between dendritic size parameters by using the intracellular horseradish peroxidase (HRP) injection technique in the cat. Twelve hypoglossal motoneurons stained with HRP were antidromically activated by stimulation applied to the medial branch of hypoglossal nerve. Eight (type I) and four (type II) of the 12 motoneurons were located in the ventral and dorsal parts of the ventromedial subnucleus of hypoglossal nucleus, respectively. The somatodendritic morphology of the two types of neurons was remarkably different, especially in the dendritic arborization pattern. The type I neurons established an egg-shaped dendritic tree that was distributed through the nucleus to the reticular formation; the type II dendritic tree was confined within the nucleus and presented a rostrocaudally oriented, mirror-image, fan-shape appearance. The total dendritic area and length and the number of terminations and branch points were significantly larger for type I than for type II neurons. For the two types of neuron, there was a positive correlation between stem dendritic diameter and several dendritic size parameters. Although the slopes of the regression lines were slightly different between the two, these were not statistically significant. The present study provides evidence that hypoglossal motoneurons located in the ventromedial subnucleus could be divided into two types according to the dendritic arborization pattern and quantitative analysis of the dendritic tree and according to neuronal location and suggests that the two types of hypoglossal motoneurons can be viewed as intrinsically distinct cell types: type I and type II, which innervate extrinsic and intrinsic muscles, respectively. In addition, the morphometric analysis made it possible to estimate the size of the dendritic tree by measuring the stem dendritic diameter.