Cervical Motor Neurons Research Articles

Functional respiratory rhythm generating networks in neonatal mice lacking NMDAR1 gene.

N-methyl-D-aspartate (NMDA) receptor-mediated synaptic transmission is implicated in activity-dependent developmental reorganization in mammalian brain, including sensory systems and spinal motoneuron circuits. During normal development, synaptic interactions important in activity-dependent modification of neuronal circuits may be driven spontaneously (Shatz 1990b). The respiratory system exhibits substantial spontaneous activity in utero; this activity may be critical in assuring essential and appropriate breathing movements from birth. We tested the hypothesis that NMDA receptors are necessary for prenatal development of central neural circuits underlying respiratory rhythm generation by comparing the responsiveness of control mice and mutant mice lacking the NMDA receptor R1 subunit (NMDAR1) gene to glutamate receptor agonists and antagonists and comparing endogenous respiratory-related oscillations generated in vitro by brain stem-spinal cord and medullary slice preparations from control and mutant mice. In control mice, local application of NMDA and the non-NMDA receptor agonist, (R,S)-alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid hydrobromide (AMPA), over the pre-Bötzinger Complex, the C4 cervical motor neuron pool, and the hypoglossal motor nucleus produced profound increases in inspiratory frequency, tonic discharge on C4 ventral nerve roots, and inward currents in inspiratory hypoglossal motoneurons, respectively. Responses of mutant mice to AMPA were similar. However, mutant mice were completely unresponsive to NMDA applications. Preparations from mutant mice generated a respiratory rhythm virtually identical to control. Results demonstrate that NMDA receptors are not essential for respiratory rhythm generation or drive transmission in the neonate. More importantly, they suggest that NMDA receptors are not obligatory for the prenatal development of circuits producing respiratory rhythm.

The diagnostic utility of cerebrospinal fluid creatine kinase activity in the horse.

We evaluated creatine kinase (CK) activity in the cerebrospinal fluid (CSF) of 128 horses with various neurological disorders. No association was found between CSF CK activity and CSF red blood cell count, CSF nucleated cell count, CSF total protein concentration, or serum CK activity. The sensitivity and specificity of CSF CK activity as a diagnostic test for protozoal myelitis in horses (61% and 56%, respectively) was higher than for cervical stenotic myelopathy, degenerative myelopathy, or motor neuron disease, but was considered to be inadequate to be of use diagnostically. Contamination of CSF with whole blood, hemolyzed red blood cells, or serum did not substantially contribute to increases in CSF CK activity. Addition of epidural fat or dura to CSF significantly increased CSF CK activity in all cases. We suggest that the use of CSF CK activity as a diagnostic indicator of neurological disease in the horse in unreliable, and that CSF CK activity may be falsely increased by contamination of CSF with epidural fat or dura during CSF collection.

Age-Dependent Penetrance of Disease in a Transgenic Mouse Model of Familial Amyotrophic Lateral Sclerosis

The mutation gly93 → ala of Cu,Zn superoxide dismutase (SOD) is found in patients with familial amyotrophic lateral sclerosis and causes motor neuron disease when expressed in transgenic mice. The progression of clinical and pathological disease was studied in a line of mice designated G1H. Clinical disease started at 91 ± 14 days of age with fine shaking of the limbs, followed by paralysis and death by 136 ± 7 days of age. Pathological changes begin by 37 days of age with vacuoles derived from swollen mitochondria accumulating in motor neurons. At the onset of clinical disease (90 days), significant death of somatic motor neurons innervating limb muscles has occurred; mice at end-stage disease (136 days) show up to 50% loss of cervical and lumbar motor neurons. However, neither thoracic nor cranial motor neurons show appreciable loss despite vacuolar changes. Autonomic motor neurons also are not affected. Mice that express wild-type human Cu,Zn SOD remain free of disease, indicating that mutations cause neuron loss by a gain-of-function. Thus, the age-dependent penetrance of motor neuron disease in this transgenic model is due to the gradual accumulation of pathological damage in select populations of cholinergic neurons.

Dose-Dependent Selective Suppression of Light (NFL) and Medium (NFM) but Not Heavy (NFH) Molecular Weight Neurofilament mRNA Levels in Acute Aluminum Neurotoxicity

We inoculated 5- to 6-week old New Zealand white rabbits intracisternally with either 100, 250, 500, 750, or 1000 μg of AlCl3 or 0.9% NaCl and correlated the extent of cervical motor neuron neurofilamentous inclusion formation at 48 h postinoculation with alterations in neurofilament (NF) mRNA levels. RNA was isolated from cervical spinal cord by the guanidine isothiocyanate method and individual RNA samples were normalized for poly(A+) content. Northern blot analysis was performed with cDNA probes for light (NFL), medium (NFM), and heavy (NFH) neurofilament subunit protein or with oligonucleotide probes for α-tubulin or actin. No significant alteration in the levels of α-tubulin, actin, or NFH mRNA were observed, regardless of the aluminum dose. In contrast, dose-dependent reductions in NFL and NFM mRNA levels occurred in direct proportion to the extent of neurofilamentous inclusion formation. While inoculums of NaCl or 100 or 250 μg AlCl3 induced neither inclusion formation or alterations in mRNA levels, both inclusion formation and reductions in the levels of NFL and NFM mRNA occurred thereafter, becoming maximal with inoculums of 1000 μg AlCl3. These experiments indicate that intracisternally administered AlCl3 acutely suppresses NFL and NFM mRNA levels without affecting those of NFH. This pattern is in distinct contrast to the uniform reductions of all NF mRNA transcript levels during neurogenesis or following axotomy, indicating a specific effect of aluminum upon steady-state levels of NF mRNA that correlates with the induction of neurofilamentous aggregates.

Mutant mouse models of ALS

THERE are three major mouse models of ALS in the current literature. Two early onset models show vacuolar pathology, while the late-onset motor neuron degeneration (mnd) shows inclusions, increased lysosomal, and nonlysosomal hydrolases and rearrangement of neurofilaments. All models may be useful in determining motor neuron-specific vulnerability. Additional late-onset models may be particularly valuable for examination of delayed or gradual neuronal degenerations. Animal models of neurodegenerative disease offer opportunities for the study of aspects of the etiology that are not available from purely clinical studies. Such studies can then progress readily to investigation of drug efficacy and mechanisms. Mutant mouse models are particularly attractive in that they are backed by strong genetics. There are also several highly inbred backgrounds available, plus several wild mouse strains which have been bred to genetic homogeneity. These should allow the study of modulators of the primary effects of genes for neurological disease. Because mice are small and age quickly, it is economically feasible to keep substantial numbers of them. In a well-defined genetic disease, the gene acts as a reproducible perturbant; thus, it is possible to study each stage of the disorder from presymptomatic on. This is useful for developmental disorders and is especially valuable for delayed neuronal degenerations. Identification of cognitive defects in mice presents difficulty; however, a large number of motor diseases are readily recognized by progressive neurological dysfunction (3). Two early onset autosomal recessive motor neuron diseases in mouse mutants have been studied in detail, wobbler (wb) and wasted (wst) rev. in (6). Both show vacuolar pathology in motor neurons and onset during the first 3 weeks after birth. The former shows forelimb involvement, affecting predominantly soma of the cervical motor neurons. The rate of axonal transport and the number of neurofilaments are greatly reduced in affected cells (11) and there is a selective reduction in the regenerative capacity of these cells as well (12). In addition to vacuolar pathology, the wst mutants show accumulations of phosphorylated 200 kDa neurofilamerit immunoreactive protein in motor neurons in the anterior horn of the spinal cord and the brainstem cranial nuclei by 30 days of age (5). Tremor appears to involve all four limbs, although the paralysis was reported to affect mainly the hindlimbs, with wasting and death by Day 31. This mutant strain also develops reduced secretory immune responses and shows abnormal DNA repair mechanisms. (An endogenous retrovirus has been postulated as a mechanism of action for both the immune and neurological defects, based on preliminary data (5).) While wst, wb, and a new nonvacuolar early onset recessive motor neuron disease (an insertional mutation; M. Meisler, pers. comm.) may seem irrelevant to adult-onset ALS, it is probably worthwhile to examine aged and stressed heterozygotes of these recessives for signs of a semidominant effect, or delayed neuronal degeneration. The late-onset mutation Motor neuron degeneration (mnd)

Establishing brain death: the potential role of nuclear medicine in the search for a reliable confirmatory test

Establishing the diagnosis of brain death has never been easy for most physicians. Recently it has become even more complex as modern technology allows doctors to prolong almost indefinitely cardiac and respiratory function in comatose patients. In addition, because of advances in transplant medicine, it is more crucial than ever to make a diagnosis of brain death quickly and accurately in order to harvest transplant organs before they are damaged. The field of nuclear medicine could play an important part in this rapidly evolving area by providing a proven, sensitive and specific test to help make the diagnosis. Most Western European countries except Denmark now agree that 'brain death' equals 'death' (Danish Ethics Council 1990). Gone are the days when 'death' meant 'cessation of all organ systems'. Some writers prefer the more cumbersome term 'irreversible cessation of brain function' (ICBF) to remind doctors exactly what they are deciding when they form the diagnosis (George et al. 1990). At present, the diagnosis of brain death is largely clinical. Numerous professional committees have met and proposed guidelines in this area (Beecher 1968; Mohandas and Chou 1971; President's Commission 1981; Task Force 1987). The key elements in an adult are: (1) the absence of cerebral functions; (2) the absence of brain stem functions including spontaneous respirations for a period of at least 6 h; and (3) an irreversible state (Adams and Victor 1985). Cerebral function is evaluated by looking for volitional activity and checking the patient's reaction to auditory (shout), visual (wave) and cutaneous (pinch) stimuli (Adams and Victor 1985). Some movements, particularly spinal reflexes, may occur in a brain-dead patient (Ropper 1984; Jordan et al. 1985). There is even a 'Lazarus sign', where patients, 5-10 min after the ventilator has been stopped, show sudden movements of the arms and shoulders (Jordan et al. 1985). One patient, 5 min after respiratory support was stopped and cardiac function ceased, crossed both arms over his chest and began to sit up (Ropper 1984). Mechanical and hypoxic stimuli probably trigger upper cervical cord motor neurons to produce this sign. Brain stem functions are commonly evaluated by testing pupillary reaction to light, reaction to corneal stimulation, and the oculocephalic, oculovestibular, oropharyngeal and gag reflexes (Plum and Posner 1982). The most essential test is the apnea test, to see whether or not high levels of carbon dioxide can stimulate the respiratory portion of the brain stem. If the patient does not breathe after 10 min with documented levels of PCO2>60 mmHg, then the respiratory drive function of the brain stem is absent (Outwater and Rockoff 1984). In addition to searching for possible cerebral and brain stem function, one should try and establish an aetiology of the coma. If the cause is known and irreversible, one examination alone in an adult is sufficient to declare brain death. If the cause of the coma is unknown, then one must exclude possible reversible causes. Common examples of reversible coma include sedative-hypnotic agents (benzodiazepines, phenobarbital), paralytic agents, toxic and metabolic disorders (hepatic encephalopathy, uraemia, porphyria), hypothermia and hypotension (Plum and Posner 1982). Most authors recommend that for an adult, if the cause of a coma is unknown, reversible causes should be excluded and two examinations should be performed 12 h apart before making the diagnosis (Mohandas and Chou 1971; President's Commission 1981 ; Task Force 1987; George et al. 1990). The essential problem in establishing brain death, after showing that no cerebral or brain stem function exists, lies in determining irreversibility. In an adult with a known cause of coma this is not a problem. However, in newborns, infants and adults with coma of undetermined aetiology, it is often difficult to prove irreversibi-

Early axon and dendritic outgrowth of spinal accessory motor neurons studied with dii in fixed tissues

We have utilized lateral diffusion of DiI in fixed tissues (Godement et al., '87: Development 101: 697-713) to study early axon and dendritic outgrowth of spinal accessory motor neurons in embryonic rats. Crystals were placed in the central canal of the cervical spinal cord near the ventral commissure in order to label growing accessory axons anterogradely and on the spinal accessory nerve to label somata and dendrites retrogradely. Animals were studied on E11-E13. We show here that it is possible to stain axonal and dendritic processes from the earliest stages of motor neuron differentiation by using DiI. Our results demonstrate that, unlike axons of other cervical motor neurons, accessory axons traverse the lateral region of the embryonic cord, which consists of neuroepithelial endfeet. Thus an affinity for neuroepithelial endfeet could partially explain their unusual intraspinal trajectory. We also show that morphology of the spinal accessory growth cones differs according to position along the accessory nerve pathway. Finally, we show that accessory motor neuron axons are in the region of their target precursors prior to the initiation of dendritic arborization. Use of DiI in fixed tissue allows study of process outgrowth in mammalian spinal cord with detail previously obtainable only in nonmammalian vertebrates.

Experimental morphological studies on central projection of snout proprioceptive afferents in moles

It had been provedt hat the facial nerve of molesc ontaineda proprioceptivefi ber element. But its projectiona reas remainedu nknown. They were here investigatedb y the Nauta method and I-IRPla bellingm ethoda nd by electronm icroscopy. The proprioceptivaef ferentf ibers, a fter entering the pons, are dividedr ostro-caudallyi nto the shorter ascending and the longer descending roots, both running closely dorsal to the trigeminals pinal tract nucleus (TSTN. ) The ascending root takes the vertical course adjacently dorsal to the TSTN, giving off manyt erminalf iberst o it and the other dorso-medialp ontine nuclei. At the levelo f the inner genu, it turns medially to meet the facial motor root and runs to the facial motor nucleus cells bilaterally. The descendingro ot takes the vertical coursec audallya t least downt o C3, g iving off many terminal fibers to the TSTN, and the nuclei of IXth, Xth and XIIth cranial nerves. At the level of the spinal cord, the root is divided into two and they descend bilaterally through the posterior fasciculi, p rojectingt o the posteriora nd anteriorh ornc ells. Fromt hese results, it can bee mphasizetdh at the snoutp roprioceptivfeib ersp rojectb ilaterally to the facialm otorn ucleusa nd the cervicalm otorn eurons, t he latter composinga mono-synaptic reflex system between the snout muscles and neck muscles

Impaired slow axonal transport in wobbler mouse motor neuron disease.

We studied slow axonal transport and morphometry of forelimb axons in wobbler mice and controls. In wobbler mice, the total radioactivity migrating with the slow transport was decreased by 50%. The velocity of transport also appeared to be reduced; 15 days following administration of a radioisotope, polypeptides migrating with slow component a of transport did not form a peak and remained mostly 2 mm from the spinal cord, while in controls slow component a was distributed as a peak which was located 4 mm from the cord. The ratios of the 68-kDa neurofilament subunit to tubulin and actin were significantly decreased (p less than 0.01 and p less than 0.005, respectively). This finding is consistent with a preferential reduction of the radioactivity migrating with neurofilament proteins in wobbler mice. Moreover, both the size and number of myelinated axons were markedly diminished, but their length was not significantly different, indicating that dying-back does not take place in axons of wobbler mice up to 12 mm from the spinal cord. The reduction in axonal transport may be due to the reduction in number and caliber of the axons and/or to reduced protein synthesis in cervical lower motor neurons; however, the abnormal distribution of the radioactive substance definitely results from impairment of the slow transport in the axons of the forelimb roots of wobbler mice. The transport impairment is not related to the presence of morphological changes in the perikaryon of wobbler mouse lower motor neurons, as it is much more widespread than would be expected if only altered neurons were involved.(ABSTRACT TRUNCATED AT 250 WORDS)