Motor Neuron Cell Bodies Research Articles

Distinct Roles for Secreted Semaphorin Signaling in Spinal Motor Axon Guidance

Neuropilins, secreted semaphorin coreceptors, are expressed in discrete populations of spinal motor neurons, suggesting they provide critical guidance information for the establishment of functional motor circuitry. We show here that motor axon growth and guidance are impaired in the absence of Sema3A-Npn-1 signaling. Motor axons enter the limb precociously, showing that Sema3A controls the timing of motor axon in-growth to the limb. Lateral motor column (LMC) motor axons within spinal nerves are defasciculated as they grow toward the limb and converge in the plexus region. Medial and lateral LMC motor axons show dorso-ventral guidance defects in the forelimb. In contrast, Sema3F-Npn-2 signaling guides the axons of a medial subset of LMC neurons to the ventral limb, but plays no major role in regulating their fasciculation. Thus, Sema3A-Npn-1 and Sema3F-Npn-2 signaling control distinct steps of motor axon growth and guidance during the formation of spinal motor connections.

Hox Genes: The Instructors Working at Motor Pools

Motor neurons are assigned unique subidentities preceding their axon navigation. This ensures proper innervation of muscle targets and is accompanied by a stereotypical clustering of motor neuron cell bodies into "motor pools" within the spinal cord. However, the mechanisms that drive motor neuron diversification have been poorly understood. A new study by Dasen et al. (2005) in this issue of Cell shows that a network of Hox genes is responsible for instructing motor pool development.

Somatodendritic accumulation of misfolded SOD1-L126Z in motor neurons mediates degeneration: αB-crystallin modulates aggregation

Mice expressing variants of superoxide dismutase-1 (SOD1) encoding C-terminal truncation mutations linked to familial amyotrophic lateral sclerosis (FALS) have begun to define the role of misfolding and aggregation in the pathogenesis of disease. Here, we examine transgenic mice expressing SOD1-L126Z (Z = stop-truncation of last 28 amino acids), finding that detergent-insoluble mutant protein specifically accumulates in somatodendritic compartments. Soluble forms of the SOD1-L126Z were virtually undetectable in spinal cord at any age and the levels of accumulated protein directly correlated with disease symptoms. Neither soluble nor insoluble forms of SOD1-L126Z were transported to distal axons. In vitro, small heat shock protein (Hsp) alphaB-crystallin suppressed the in vitro aggregation of SOD1-L126Z. In vivo, alphaB-crystallin immunoreactivity was most abundant in oligodendrocytes and up-regulated in astrocytes of symptomatic mice; neither of these cell-types accumulated mutant SOD1 immunoreactivity. These results suggest that damage to motor neuron cell bodies and dendrites within the spinal cord can be sufficient to induce motor neuron disease and that the activities of chaperones may modulate the cellular specificity of mutant SOD1 accumulation.

Altered axonal architecture by removal of the heavily phosphorylated neurofilament tail domains strongly slows superoxide dismutase 1 mutant-mediated ALS

Eliminating assembled neurofilaments (NFs) from axons or misaccumulating NFs in motor neuron cell bodies strongly slows disease in mouse models of mutant superoxide dismutase 1 (SOD1)-induced amyotrophic lateral sclerosis. One proposal for how reducing axonal NFs can increase survival is that the multiphosphorylated tail domains of the two larger NF subunits act in motor neuron cell bodies as phosphorylation sinks where they mitigate cyclin-dependent kinase 5 dysregulation induced by mutant SOD1. Elimination by gene targeting in mice of the NF medium and NF heavy tail domains and their 58 known phosphorylation sites accelerates aberrant phosphorylation of other neuronal substrates while leaving overall NF content unaltered. However, disease onset is significantly delayed and survival is extended, inconsistent with the ameliorative property of altered NF content protecting by serving as substrates for dysregulation of any NF kinase. Moreover, at comparable disease stages significantly more surviving motor neurons and axons were found in SOD1 mutant mice deleted in the NF tails than in similar mice with wild-type NFs. This finding supports noncell autonomous toxicity in SOD1 mutant-mediated amyotrophic lateral sclerosis: removal of the NF tails slows damage developed directly within motor neurons, but SOD1 mutant damage within nonneuronal supporting cells reduces motor neuron functionality.

Modulation of the glutamatergic receptors (AMPA and NMDA) and of glutamate vesicular transporter 2 in the rat facial nucleus after axotomy

Facial nerve axotomy is a good model for studying neuronal plasticity and regeneration in the peripheral nervous system. We investigated in the rat the effect of axotomy on the different subunits of excitatory glutamatergic AMPA (GLuR1-4), NMDA (NR1, NR2A-D) receptors, post-synaptic density 95, vesicular glutamate transporter 2, β catenin and cadherin. mRNA levels and/or protein production were analyzed 1, 3, 8, 30 and 60 days after facial nerve axotomy by in situ hybridization and immunohistofluorescence. mRNAs coding for the GLuR2-4, NR1, NR2A, B, D subunits of glutamatergic receptors and for post-synaptic density 95, were less abundant after axotomy. The decrease began as early as 1 or 3 days after axotomy; the mRNAs levels were lowest 8 days post-lesion, and returned to normal or near normal 60 days after the lesion. The NR2C subunit mRNAs were not detected in either lesioned or intact facial nuclei. Immunohistochemistry using specific antibodies against GLuR2-3 subunits and against NR1 confirmed this down-regulation. There was also a large decrease in vesicular glutamate transporter 2 immunostaining in the axotomized facial nuclei at early stages following facial nerve section. In contrast, no decrease of NR2A subunit and of post-synaptic density 95 could be detected at any time following the lesion. β Catenin and cadherin immunoreactivity pattern changed around the cell body of facial motoneuron by day 3 after axotomy, and then, tends to recover at day post-lesion 60 days. Therefore, our results suggest a high correlation between restoration of nerve/muscle synaptic contact, synaptic structure and function in facial nuclei. To investigate the mechanisms involved in the change of expression of these proteins following axotomy, the facial nerve was perfused with tetrodotoxin for 8 days. The blockade of action potential significantly decreased GLuR2-3, NR1and NR2A mRNAs in the ipsilateral facial nuclei. Thus, axotomy-induced changes in mRNA abundance seemed to depend partly on disruption of activity.

The role of the cytoskeleton in cell body enlargement, increased nuclear eccentricity and chromatolysis in axotomized spinal motor neurons

BackgroundWhen spinal motor axons are injured, the nucleolus, nucleus and cell body of the injured cell transiently increase in size, the nucleus becomes more eccentrically placed, and the organization of polyribosomes into Nissl bodies is temporarily disrupted. The mechanisms for these classical morphological responses to axotomy have not been satisfactorily explained.ResultsIn this study we address the role of the cell body cytoskeleton in these structural changes. We show that the cytoskeleton of uninjured lumbar motor neuron cell bodies maintains nucleolar, nuclear and cell body size and nuclear position. When isolated, the relatively insoluble cell body cytoskeleton contains Nissl bodies and lipofuscin granules. After axotomy, protein labeling increases markedly and the cytoskeleton enlarges, increasing nucleolar, nuclear and cell body size, as well as nuclear eccentricity. Nearly all of the protein mass that accumulates in the cell body after axotomy appears to be added to the cytoskeleton.ConclusionWe conclude that axotomy causes the conjugate enlargement of the nucleolus, nucleus and cell body and increases nuclear eccentricity in spinal motor neurons by adding protein to the cytoskeleton. The change in nuclear position, we propose, occurs when cytoskeletal elements of the axon cannot enter the shortened axon and "dam up" between the nucleus and axon hillock. As a consequence, we suggest that Nissl body-free axonal cytoskeleton accumulates between the nucleus and axon, displaces Nissl body-containing cytoskeleton, and produces central chromatolysis in that region of the cell.

Anatomic effect of electrical stimulation on the reinnervation of the paralyzed canine larynx

Problem: The larynx relies on correct innervation to mediate its normal functions. The aberrantly reinnervated larynx is compromised in its vocalization and airway-protective functions. Methods: Six canines were implanted with electrical stimulation devices, and their recurrent laryngeal nerve sectioned and re-anastomosed on one side. The effects of interrupted chronic muscle stimulation of experimental animals were compared with nonstimulated control animals. Physiologic measures of electromyography of thyroarytenoid (TA) and posterior cricoarytenoid (PCA) muscles demonstrated enhanced native reinnervation and reduced foreign reinnervation at 180 days in experimental animals. Fluorescent retrograde tracers true blue (TB) and fluorogold (FG) were injected into the PCA and TA, respectively. The identity and relative locations of labeled motoneurons in the nucleus ambiguus were analyzed for segregation and compared to normal animals. Results: PCA and TA muscles that appeared to become selectively reinnervated based on physiologic measurements retained the normal localization of motoneuron cell bodies in the nucleus ambiguus. Significant segregation was observed across the mediolateral axis. Animals with greater foreign reinnervation based on physiologic measurements showed more random distribution of PCA and TA motoneurons, with variable double-labeling of some neurons. Conclusion: This study provides the first direct anatomic evidence that stimulation of a denervated laryngeal muscle promotes selective reinnervation by native over foreign reinnervation. The native reinnervation appears to be from the original motoneuron projections to the PCA or TA located within the nucleus ambiguus of the brainstem. Significance: Laryngeal paralysis is a significant problem with decreased function and increased morbidity for affected individuals. This study demonstrates anatomically that stimulation of the larynx by an implantable device leads to improved reinnervation. Thus, implantable devices may be useful in the rehabilitation of patients with laryngeal paralysis. Support: Supported by NIH grant R01-DC001149.

Characterization of Ighmbp2 in motor neurons and implications for the pathomechanism in a mouse model of human spinal muscular atrophy with respiratory distress type 1 (SMARD1).

Spinal muscular atrophy with respiratory distress type 1 (SMARD1) is caused by recessive mutations of the IGHMBP2 gene. The role of IGHMBP2 (immunoglobulin mu-binding protein 2) in the pathomechanism of motor neuron disease is unknown. We have generated antibodies against Ighmbp2 and showed that low levels of Ighmbp2 immunoreactivity are present in the nucleus of spinal motor neurons and high levels in cell bodies, axons and growth cones. Ighmbp2 protein levels are strongly reduced in neuromuscular degeneration (nmd) mice, the mouse model of SMARD1. Mutant mice show severe motor neuron degeneration before first clinical symptoms become apparent. The loss of motor neuron cell bodies in lumbar spinal cord is followed by axonal degeneration in corresponding nerves such as the femoral quadriceps and sciatic nerve and loss of axon terminals at motor endplates. Motor neuron degeneration and clinical symptoms then slowly progress until the mice die at the age of 3-4 months. In addition, myopathic changes seem to contribute to muscle weakness and especially to respiratory failure, which is characteristic of the disorder in humans. Cultured motor neurons from embryonic nmd mice did not show any abnormality with respect to survival, axonal growth or growth cone size, thus differing from motor neurons derived from, e.g. Smn (survival motor neuron) deficient mice, the model of spinal muscular atrophy (SMA). Our data suggest that the pathomechanism in SMARD1 is clearly distinct from other motor neuron diseases such as classic SMA.

Mitogen-Activated Protein Kinase p38 Mediates Reduced Nerve Conduction Velocity in Experimental Diabetic Neuropathy

This study examined the role of p38 mitogen-activated protein (MAP) kinase in transducing high glucose into deficits in nerve conduction velocity (NCV) that are characteristic of diabetic neuropathy. p38 activation and NCV were measured in streptozocin-induced diabetic rats treated with a p38 inhibitor, an aldose reductase inhibitor, and insulin. Dorsal root ganglia (DRG) from diabetic animals showed marked activation of p38 at 12 weeks of diabetes. Insulin treatment for the last 4 of 12 weeks of diabetes normalized p38 activation. Furthermore, activation was completely prevented by 12 weeks' treatment with the aldose reductase inhibitor, fidarestat. Immunocytochemistry localized activation of p38 to the nuclei of virtually all sensory neuronal phenotypes in the DRG, and activation was clear in diabetes, as was inhibition by fidarestat and by the p38 inhibitor SB 239063. In the ventral horn of the spinal cord, p38 was present in motoneuron cell bodies; and again, activation in diabetes and fidarestat inhibition was clear. Treatment of diabetic animals with a specific inhibitor of p38 (SB 239063), fidarestat, or insulin also prevented reductions in both motor and sensory NCV. These findings suggest that increased polyol pathway flux in diabetic animals leads to the activation of p38. This activation can mediate changes in gene transcription and cellular phenotype that are likely to underlie the NCV deficits. Insulin and aldose reductase inhibitors can prevent excess polyol pathway flux, and hence these agents may prevent NCV deficits by preventing p38 MAP kinase activation.

Necessity and redundancy of guidepost cells in the embryonic Drosophila CNS

Guidepost cells are specific cellular cues in the embryonic environment utilized by axonal growth cones in pathfinding decisions. In the embryonic Drosophila CNS the RP motor axons make stereotypic pathways choices involving distinct cellular contacts: (i) extension across the midline via contact with the axon and cell body of the homologous contralateral RP motoneuron, (ii) extension down the contralateral longitudinal connective (CLC) through contact with connective axons and longitudinal glia, and (iii) growth into the intersegmental nerve (ISN) through contact with ISN axons and the segmental boundary glial cell (SBC). We have now ablated putative guidepost cells in each of the CNS pathway subsections and uncovered their impact on subsequent RP motor axon pathfinding. Removal of the longitudinal glia or the SBC did not adversely affect pathfinding. This suggests that the motor axons either utilized the alternative axonal substrates, or could still make filopodial contact with the next pathway section’s cues. In contrast, RP motor axons did require contact with the axon and soma of their contralateral RP homologue. Absence of this neuronal substrate frequently impeded RP axon outgrowth, suggesting that the next cues were beyond filopodial reach. Together these are the first direct ablations of putative guidepost cells in the CNS of this model system, and have uncovered both pathfinding robustness and susceptibility by RP axons in the absence of specific contacts.

Delayed nerve repair is associated with diminished neuroenhancement by FK506.

The immunosuppressive agent FK506 has been shown in many studies to enhance nerve regeneration and to accelerate functional recovery after immediate nerve repair. However, in clinical practice the diagnosis and treatment of patients with peripheral nerve injuries is often delayed. The study investigated whether FK506 would retain its neuroregenerative properties when nerve repair and initiation of FK506 therapy were delayed for 7 days. In vivo laboratory study. Thirty-two Lewis rats underwent tibial nerve transection and were randomly assigned to four experimental groups: immediate repair with FK506 treatment, immediate repair without FK506 treatment, 7-day delayed repair with FK506 treatment, and 7-day delayed repair without FK506 treatment. Treated animals received daily subcutaneous injections of 2 mg/kg FK506. Serial walking track measurements were performed at 14, 16, and 18 days after nerve repair. On day 18 after repair, peripheral nerves were injected with a fluorescent tracer for retrograde labeling. On day 21, peripheral nerves and spinal cords were harvested for histomorphometric analysis and motor neuron cell body counts, respectively. Animals that underwent immediate repair with FK506 had significantly higher fiber counts and percentages of nerve than the other three groups (P <.05) but did not show statistically significant earlier functional recovery. The remaining three groups had intermediate levels of nerve regeneration that were not significantly different. Retrograde abled motor neurons counts were decreased in animals with delayed nerve repair that received no FK506 (P <.05). In a rat tibial nerve transection model, the neuroregenerative effects of FK506 diminished markedly when repair and initiation of FK506 therapy were delayed by 7 days.

Progressive and selective degeneration of motoneurons in a mouse model of SMA.

Spinal muscular atrophy (SMA), an autosomal recessive disorder characterized by the degeneration of motoneurons of the spinal cord and brainstem, results from loss-of-function mutations in the survival motor neuron gene (smn). The goal of these experiments was to analyse axons and cell bodies of motoneurons in different regions of the CNS during disease progression in a mouse model of SMA carrying a deletion of the exon 7 directed to neurons. These experiments demonstrate a progressive loss of motor axons and of motoneurons in the CNS. This is the first study that describes a selective neurodegeneration in this line of mice and underlines the importance of exon 7 in some populations of motoneurons for survival in vivo.

In vivo application of mitochondrial pore inhibitors blocks the induction of apoptosis in axotomized neonatal facial motoneurons.

Axotomy induces apoptosis in motoneurons of neonatal rodents. To identify the key players in motoneuron apoptosis, we assessed the progression of apoptosis at 4 h intervals following facial motoneuron axotomy. The mitochondrial release of cytochrome c, caspase-3 activation and nuclear condensation were first observed in the motoneuron cell bodies 16 h postaxotomy. In vivo application of inhibitors of the mitochondrial permeability transition pore, Bongkrekic acid and cyclosporin A prevented cytochrome c release as well as caspase-3 activation and attenuated motoneuron apoptosis. Similarly, in vivo application of RU360, an inhibitor of the mitochondrial calcium uniporter, also protected axotomized motoneurons from apoptosis. Taken together, our results show that cytochrome c release and subsequent caspase-3 activation are critical events that precipitate the apoptotic death of axotomized neonatal motoneurons in vivo. In addition, these results provide evidence that application of mitochondrial pore inhibitors in vivo can block the induction of apoptosis following motoneuron axotomy.

Single neuron activity in the Drosophila larval CNS detected with calcium indicators

Although the Drosophila larva has been extensively used for genetic studies of synaptic transmission and locomotion, neurophysiological studies have lagged because it is difficult to investigate circuitry and synaptic function in the larval central nervous system (CNS). Here we introduce an optical technique to monitor neuronal activity in the intact Drosophila larval CNS. We loaded neurons retrogradely through cut axons with dextran-conjugated calcium indicators. Fluorescence responses to changes in the concentration of intracellular calcium are sufficiently fast and large to monitor electrical activity in single neurons. Responses to action potentials were detected in motor neuron cell bodies, axons, neurites, dendrites and sensory neuron afferents identified by genetically targeted green fluorescent protein expression. Our findings provide an experimental procedure for testing synaptic function and connectivity within the intact larval CNS.

Therapeutic benefits of cardiotrophin-1 gene transfer in a mouse model of spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a recessive autosomal disorder characterized by degeneration of lower motor neurons caused by mutations of the survival motor neuron gene (SMN1). No curative treatment is known so far. Mutant mice carrying homozygous deletion of Smn exon 7 directed to neurons display skeletal muscle denervation, moderate loss of motor neuron cell bodies and severe axonal degeneration. These features, similar to those found in human SMA, strongly suggest the involvement of a dying back process of motor neurons and led us to test whether neurotrophic factors might have a protective role in SMA. We report here the therapeutic benefits of systemic delivery of cardiotrophin-1 (CT-1), a neurotrophic factor belonging to the IL-6 cytokine family. Intra-muscular injection of adenoviral vector expressing CT-1, even at very low dose, improves median survival, delays motor defect of mutant mice and exerts protective effect against loss of proximal motor axons and aberrant cytoskeletal organization of motor synaptic terminals. In spite of the severity of SMA phenotype in mutant mice, CT-1 is able to slow down disease progression. Neuroprotection could be regarded as valuable therapeutic approach in SMA.

Architectonics of crayfish ganglia.

The central nervous system of crayfish consists of a chain of segmental ganglia that are linked by cables of intersegmental axons. Each ganglion contains a highly-ordered core of longitudinal tracts, vertical tracts, commissures, and synaptic neuropils. We review from a technical perspective the history of the description of these ganglia, and recognize four episodes of progress. Each major innovation in anatomical methods has led to new insight into the structure and function of this nervous system, and new awareness of the structural patterns that are common to the CNS of all arthropods. Ganglia in different segments of the body differ in size, and appear to differ in anatomy. From a comparison of the structures of the cores of abdominal, thoracic, and subesophageal ganglia, we argue that this apparent difference is illusory. Rather, each of these ganglia is organized on the same plan, a plan also found in insect segmental ganglia. The apparent differences follow from longitudinal compression during development and from allometric growth of particular neuropils associated with innervation of the walking legs. Different authors have described the internal organization of ganglia in different segments, so we provide a cross-reference to the nomenclatures they have introduced. We compare the locations of cell bodies of motor neurons and accessory neurons that innervate different peripheral structures, and demonstrate double-labeling of certain GABAergic peripheral inhibitory neurons. Finally, we describe the construction of digital movies of serial sections of these ganglia, and discuss their utility.

Enhancement of BDNF and activated-ERK immunoreactivity in spinal motor neurons after peripheral administration of BDNF

Brain-derived neurotrophic factor (BDNF) shows neurotrophic effects on adult motor neurons when given systemically, But it is unknown whether systemically administered BDNF is transported to central cell bodies to affect them directly. Here we used immunohistochemistry to investigate the transport of peripherally injected BDNF to spinal motor neurons and the subsequent activation of a signaling pathway. We first injected BDNF into the flexor digitorum brevis (FDB) and analyzed the motor nucleus that projects to the FDB for BDNF immunoreactivity (BDNF-ir) and phosphorylated extracellular signal-regulated kinase (ERK) 1/2 immunoreactivity (pERK1/2-ir). Both immunoreactivities were observed in the motor neuron cell bodies. Next, BDNF was injected subcutaneously (s.c.) into rats with a unilaterally axotomized sciatic nerve. pERK1/2-ir was detected in motor neurons of the lesioned side. BDNF-ir and pERK1/2-ir were also observed on the unlesioned side when a high dose of BDNF was injected. Therefore, we examined BDNF-ir and pERK1/2-ir after injecting BDNF s.c. into normal rats. Both immunoreactivities were observed in motor nuclei on both sides. Finally, we examined pERK1/2-ir after a lower dose of BDNF was injected, which prevents the decrease in choline acetyl transferase that occurs in the motor neuron upon axotomy. Spinal motor nuclei contained a few cell bodies with pERK1/2-ir. These findings represent the first direct evidence that subcutaneously injected BDNF is transported to motor neurons and that it activates a signaling pathway in the spinal cord and exhibits neurotrophic effects in vivo.

Evolution of the arthropod neuromuscular system. 1. Arrangement of muscles and innervation in the walking legs of a scorpion: Vaejovis spinigerus (Wood, 1863) Vaejovidae, Scorpiones, Arachnida

(1) The musculature of the walking legs is analysed with regard to both morphology and function in the scorpion, Vaejovis spinigerus (Wood, 1863) (Vaejovidae, Scorpiones, Arachnida), and selected other species. Conspicuous features are multipartite muscles, muscles spanning two joints, and partial lack of antagonistic muscles. The muscle arrangement is compared to that in the walking limbs of other Arthropoda and possible phylogenetic implications are discussed. (2). Histochemical characterisation of selected leg muscles indicates that these are composed of layers of slow, intermediate and fast muscle fibres. Anti-GABA immunohistochemistry shows that mainly the intermediate fibres receive innervation from putative inhibitory motoneurons. (3). Intracellular recording from muscle fibres reveals both excitatory and inhibitory muscle innervation. Individual muscle fibres may receive input from more than one inhibitory motoneuron, as indicated by different IPSP amplitudes. (4). The motoneuron supply of the leg muscles is analysed by retrograde fills of motor nerves. The general arrangement of leg motoneurons in the central nervous system and motoneuron anatomy conforms to the situation in pterygote insects and decapod crustaceans. For example, there are an anterior and a posterior group of leg motoneurons in each hemineuromere, and two contralateral somata near the ganglion midline. Between 12 and 20 motoneurons are found to supply each muscle. Most motoneuron cell bodies supplying a given muscle are arranged in a single cluster with a specific location.

Neurofilament accumulation at the motor endplate and lack of axonal sprouting in a spinal muscular atrophy mouse model.

Mutations of survival of the motor neuron gene (SMN1) are responsible for spinal muscular atrophy (SMA), a common genetic cause of death in childhood. The cellular mechanism by which mutations of SMN1 are responsible for the selective neuromuscular defect and motor neuron cell degeneration observed in SMA has not been described. We have previously generated mice carrying a homozygous deletion of Smn exon 7 directed to neurons. We report here that these mutant mice display a dramatic and progressive loss of motor axons involving both proximal and terminal regions, in agreement with the skeletal muscle denervation process and disease progression. Moreover, we found massive accumulation of neurofilaments, including phosphorylated forms, in terminal axons of the remaining neuromuscular junctions. This aberrant cytoskeletal organization of synaptic terminals was associated with reduction of branched structures of the postsynaptic apparatus and defect of axonal sprouting in mutant mice. Together, these findings may be responsible for severe motor neuron dysfunction, and suggest that loss of motor neuron cell bodies results from a 'dying-back' axonopathy in SMA. Smn mutant mice should represent a valuable model for elucidating the pathway linking Smn to cytoskeletal organization.

The distribution of respiration-related and swallowing-related motoneurons innervating the rat genioglossus muscle

The present study was an attempt to identify the location of genioglossal respiratory and swallowing motoneuron cell bodies within the hypoglossal (XII) nucleus using both electrophysiological and morphological studies. The genioglossus muscle is innervated by the genioglossal branch of the medial XII nerve. At the entrance to the muscle, the genioglossal branch divides in the directions of the mandible and tongue. Five of five rats displayed both respiratory-related and swallowing-related bursts in the medial XII branch towards the mandible. All five rats also displayed swallowing-related bursts in the medial XII branch towards the tongue. In addition, horseradish peroxidase conjugated to wheatgerm agglutinin (HRP:WGA) was injected into the proximal cut ends of each branch. When HRP:WGA was injected into the branch in the direction of the mandible, HRP-labeled cells were detected in the lateral region of the ventromedial subnucleus in the XII nucleus, extending from 0.7 to 1.2 mm rostral to the obex. On the other hand, after injection into the branch in the direction of the mandible, HRP-labeled cells were detected in the ventromedial subnucleus of the XII nucleus, extending from 0.3 to 1.2 mm rostral to the obex. These results provide evidence that genioglossal respiration-related and swallowing-related motoneurons are located in different portions within the ventromedial subnucleus of the XII nucleus.