References

BMC Neurosci. 2005 Mar 17;6(1):19 Open access at:

• http://www.biomedcentral.com/1471-2202/6/19

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

McIlwain DL, Hoke VB.

BACKGROUND: When 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. RESULTS: In 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 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. CONCLUSIONS: We 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. PMID: 15774011 [PubMed – as supplied by publisher]

BMC Neurosci. 2004 Feb 23;5(1):6. Open access at:

• http://www.biomedcentral.com/1471-2202/5/6

Differential inhibition of postnatal brain, spinal cord and body growth by a growth hormone antagonist.

McIlwain DL, Hoke VB, Kopchick JJ, Fuller CR, Lund PK. Department of Cell and Molecular Physiology, University of North Carolina School of Medicine, Chapel Hill, NC 27599, USA. dlmc@med.unc.edu

BACKGROUND: Growth hormone (GH) plays an incompletely understood role in the development of the central nervous system (CNS). In this study, we use transgenic mice expressing a growth hormone antagonist (GHA) to explore the role of GH in regulating postnatal brain, spinal cord and body growth into adulthood. The GHA transgene encodes a protein that inhibits the binding of GH to its receptor, specifically antagonizing the trophic effects of endogenous GH. RESULTS: Before 50 days of postnatal age, GHA reduces spinal cord weight more than brain weight, but less than body weight. Thereafter, GHA ceases to inhibit the increase in body weight, which approaches control levels by day 150. In contrast, GHA continues to act on the CNS after day 50, reducing spinal cord growth to a greater extent and for a longer duration than brain growth. CONCLUSIONS: Judging from its inhibition by GHA, GH differentially affects the magnitude, velocity and duration of postnatal growth of the brain, spinal cord and body. GH promotes body enlargement more than CNS growth early in postnatal life. Later, its CNS effects are most obvious in the spinal cord, which continues to exhibit GH dependence well into adulthood. As normal CNS growth slows, so does its inhibition by GHA, suggesting that reduced trophic effects of GH contribute to the postnatal slowing of CNS growth. GHA is a highly useful tool for studying the role of endogenous GH on organ-specific growth during aging. PMID: 15018641 [PubMed – indexed for MEDLINE]

Brain Res Bull. 2000 Nov 15;53(5):537-46.

Neuron addition and enlargement in juvenile and adult animals.

Farel PB, McIlwain DL. Department of Cell and Molecular Physiology, and Neurobiology Curriculum, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7545, USA.

Locomotion requires bilateral symmetry of neural circuitry in the spinal cord. Although not well understood, the mechanisms responsible for establishing and maintaining this symmetry must balance the numbers, sizes, and connectivity of the neurons on both sides of the spinal cord. Those mechanisms do not cease to function after embryogenesis, since there is substantial evidence that these properties continue to change as juvenile animals grow to adult size. We review the evidence that spinal neuron number and size increase in growing juvenile frogs and mammals. We postulate that these increases are regulated by both local and systemic factors. In addition, we discuss evidence that axotomy of spinal sensory and motor neurons also enlists local and systemic regulatory factors, some of which may also be operative in normal growth and development. Review, Tutorial PMID: 11165789 [PubMed – indexed for MEDLINE]

Exp Neurol. 1999 May;157(1):161-8.

Magnitude, laterality, and uniformity of swelling in axotomized spinal motoneurons: lack of evidence for influence by the distal stump.

McIlwain DL, Hoke VB. Curriculum in Neurobiology, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599, USA.

Injury to frog lumbar motor axons produces a coordinated, allometric enlargement of the nucleolus, nucleus, and cell body of the injured neuron. The mechanisms by which swelling is initiated and sustained are not known. In this study, we have sought evidence for a role of the severed distal stump in the magnitude, laterality, and uniformity of the swelling response in frog spinal motoneurons. We find that swelling of motoneuron nucleoli, nuclei, and perikarya after unilateral spinal nerve transection is exclusively ipsilateral and uniform among motoneurons of different sizes. Removal of the severed distal stump does not affect the magnitude, unilaterality, or uniformity of the swelling responses. Thus, the distal stump appears to play no role in initiating swelling following spinal nerve transection. Copyright 1999 Academic Press. PMID:10222119 [PubMed – indexed for MEDLINE]

J Neurobiol. 1997 Feb;32(2):202-12.

Growth hormone, insulin-like growth factor I, and motoneuron size.

Chen L, Lund PK, Burgess SB, Rudisch BE, McIlwain DL. Department of Physiology and Curriculum in Neurobiology, University of North Carolina at Chapel Hill School of Medicine 27599, USA.

In this study we asked whether growth hormone (GH) and one of its key mediators, insulin-like growth factor I (IGF-I), influence spinal motoneuron size in conjunction with whole body size. We present evidence that GH has such a role, possibly without the mediation of IGF-I. Both lumbar motoneuron and body size were found to be increased relative to littermate controls in transgenic mice overexpressing GH, while body size, but not motoneuron size, was increased in mice overexpressing IGF-I. GH overexpression coordinately increased nucleolar, nuclear, and cell body size in lumbar spinal motoneurons, so that their normal size relationships were preserved in the transgenic mice. In addition, spinal cord and brain weights were significantly increased in both types of transgenic animal. We conclude that GH can regulate motoneuron, central nervous system, and body size in the same animal, and that IGF-I can mimic the effects of GH on at least two of these three parameters. PMID: 9032662 [PubMed – indexed for MEDLINE]

Neuroreport. 1994 Dec 20;5(18):2641-4.

Synchronous, intersegmental responses in motoneurons to lumbar ventral root injury.

Burgess SB, McIlwain DL. Department of Physiology, University of North Carolina, Chapel Hill School of Medicine 27599.

Unilateral transection of lumbar ventral roots in the grass frog, Rana pipiens, causes large, multiphasic transcriptional responses within non-injured motoneurons as far away as the cervical enlargement. The responses, which are not seen after sham surgery, are remarkably similar in their form and timing to those which appear in the injured motoneurons, implying that both axotomized and spared motoneurons are responding to the same external signals. Elucidation of the source, pathway and properties of those signals will help to explain why spared neurons reflect some, but not all, of the features seen within injured motoneurons after motor axon damage. PMID: 7696622 [PubMed – indexed for MEDLINE]

J. Neurochem. 1994 Nov;63(5):1609-15.

Transcription and motoneuron size.

Sato S, Burgess SB, McIlwain DL. Department of Physiology and Curriculum in Neurobiology, University of North Carolina School of Medicine, Chapel Hill 27599.

Nuclear size and total RNA synthesis were compared in single lumbar motoneurons isolated from the grass frog. Transcription was found to correlate significantly, but not exclusively, with nuclear area or volume over a wide range of nuclear size, the largest nuclei having the highest mean transcriptional activity. Flow cytometric analysis of propidium iodide-stained nuclei excluded polyploidy or polyteny as an explanation for the increased transcription, but left open the possibility of a small increase in DNA with increasing nuclear size. Alternatively, motoneurons may increase transcription and nuclear size without increasing their DNA content, possibly by increasing the proportion of dispersed chromatin (euchromatin). These two mechanisms for size-related changes in RNA synthesis in motoneurons present an interesting contrast to mechanisms used by many other large animal cells. PMID: 7523596 [PubMed – indexed for MEDLINE]

J Neurosci Methods. 1994 Jun;52(2):197-202.

Radiolabeling motoneuron proteins in the isolated frog spinal cord preparation.

McIlwain DL, Hoke VB. Department of Physiology, University of North Carolina School of Medicine, Chapel Hill 27599.

An in vitro method for radiolabeling protein in adult frog spinal motoneurons is described, with per cell incorporations which are 2-3 orders of magnitude higher than previously reported for mammalian brain neurons. In the procedure, isolated lumbar spinal cord preparations from Rana pipiens are labeled with 3H-L-leucine, motoneuron cell bodies are recovered and TCA-precipitated protein is analyzed by scintillation counting. The higher levels of labeling (> 90 cpm/cell body) allow one to quantify newly synthesized protein within individual or small groups of identified nerve cell bodies. Motoneuronal labeling correlates directly with cell body size, and other sources of variation in labeling and their control are identified and discussed. PMID: 7967722 [PubMed – indexed for MEDLINE]

Adv Neurol. 1991;56:67-74. In Amyotrophic Lateral Sclerosis and Other Motor Neuron Diseases (Lewis P. Rowland, ed.) Raven Press.

Nuclear and cell body size in spinal motor neurons.

McIlwain DL. Department of Physiology, University of North Carolina, School of Medicine, Chapel Hill 27514.

PMID: 1853783 [PubMed – indexed for MEDLINE]

This study showed positive, linear correlations in nuclear and cell body areas in individual motor neurons isolated from human beings, rats and frogs. Evidence was presented supporting the idea that larger motor neurons are more vulnerable to loss than smalled ones. In 3 cases of sporadic ALS, only 19% of motor neuron cell bodies isolated from ALS patients after autopsy were larger than 3000 sq. um, compared to 66% larger than that size in control patients.

Anal Biochem. 1989 Aug 1;180(2):286-90.

Isolation of motoneuron cell bodies from spinal cord stored at -70 degrees C in ethylene glycol.

Sinicropi DV, Hoke VB, McIlwain DL. Department of Physiology, University of North Carolina School of Medicine, Chapel Hill 27514.

Pretreatment of spinal cord with ethylene glycol permits long-term storage of the tissue at -70 degrees C prior to isolation and biochemical analysis of the cell bodies of spinal motoneurons. The method is useful for storing spinal tissue from laboratory animals, as well as from human post mortem specimens, where aliquots of tissue may then be used for motoneuron isolation over an indefinitely long period. In addition to inhibiting the loss of soluble proteins from the neurons during freezing and thawing, cryoprotection increases the yield and improves the appearance of the isolated cell bodies. The method should aid biochemical studies of many kinds of neuronal subpopulations isolated from small amounts of starting material. PMID: 2817357 [PubMed – indexed for MEDLINE]

Rev Neurol (Paris). 1988;144(11):697-700.

Allocation of newly synthesized proteins in spinal motoneurons.

McIlwain DL, Hoke VB. Department of Physiology, University of North Carolina, Chapel Hill 27514.

When vinblastine sulfate is used in vitro to block slow and fast transport of proteins within frog lumbar spinal motoneurons, the amounts of both newly synthesized protein and total protein increase in motoneuronal perikarya. Analyses of motoneurons isolated from control and vinblastine-treated spinal cords show that 1) about 55 p. 100 of the newly synthesized protein is exported from motoneuron cell bodies during a 4 h incubation period; 2) only about 5 p. 100 of the total perikaryal protein is exported during the same period; and 3) less than 10 p. 100 of the labeled protein is exported by fast axonal transport. Thus, a substantial amount of the newly synthesized protein is quickly and preferentially exported from the cell body. It is not known how much of this exported protein reaches the axon by slow transport. However, when interpreted in conjunction with the studies of Schubert et al.,1972), the above data strengthen the possibility that substantial amounts of the new protein made by a motoneuron may be committed to its dendrites. PMID: 3231958 [PubMed – indexed for MEDLINE]

Neurochem Pathol. 1985 Spring;3(1):1-14.

Relative postmortem stability of spinal motoneuronal proteins detectable by two-dimensional electrophoresis.

Brock TO, McIlwain DL.

The suitability of using spinal tissue several hours after death for analysis by high resolution two-dimensional electrophoresis has been examined. It was found that many of the proteins of bovine spinal motoneurons detectable on two-dimensional polyacrylamide gels appear to be relatively stable in situ at room temperature during the first postmortem day. When extracts of total proteins from ventral roots and motoneuronal cell bodies isolated from 1-d-old tissue were examined, all spots could be matched to control gels. Upon visual inspection of the gels, postmortem changes in the amount of stain associated with a spot were obvious in three of 364 proteins from isolated motoneuronal cell bodies and none of 237 proteins from ventral roots. Other proteins underwent quantitative changes that were detected only after computer-assisted densitometry on the gels, whereas some did not appear to change at all. In the neuropil surrounding the motoneuron cell bodies, more pronounced changes in protein patterns occurred during the postmortem period. We conclude that properly controlled two-dimensional electrophoretic analyses of postmortem spinal tissue can provide reliable qualitative and quantitative information about the antemortem protein composition of spinal motoneurons. PMID: 4040616 [PubMed – indexed for MEDLINE]

J Neuropathol Exp Neurol. 1984 Nov;43(6):609-19.

Astrocytic proteins in the dorsal and ventral roots in amyotrophic lateral sclerosis and Werdnig-Hoffmann disease.

Brock TO, McIlwain DL.

Abnormalities were detected by two-dimensional gel electrophoresis in the protein composition of both the dorsal and ventral roots of three of six patients who succumbed to amyotrophic lateral sclerosis (ALS). The abnormalities consisted of a cascade of acidic protein spots on silver-stained gels which were shown by immunoblotting to react with an antiserum to human glial fibrillary acidic protein (GFAP). They were found distal to the normal central nervous system/peripheral nervous system (CNS/PNS) transition zone and were undetected in cervical and lumbar root segments taken at the same distances from the spinal cord of eight control patients. Similar changes were observed in the dorsal and ventral roots of one patient with Werdnig-Hoffmann disease (WHD), while a second patient with WHD had the changes in only the ventral roots. The abnormalities probably reflect the presence of radicular glial bundles, which are pathological extensions of glial cells into the spinal roots, indicating that subclinical changes occurred in the sensory nerves of the affected ALS and WHD patients. While no other qualitative abnormalities were noted on gels of ALS and WHD spinal roots, some quantitative changes may be present. Publication Types: Case Reports PMID: 6502190 [PubMed – indexed for MEDLINE]

Brain Res. 1983 Jun;284(2-3):275-82.

Cholinergic enzyme activity in neurons of the developing anuran spinal cord.

Farel PB, McIlwain DL.

Cholinergic enzyme activity was investigated over the course of spinal cord development from early larval (tadpole) stages to adult life in bullfrogs (Rana catesbeiana). Acetylcholinesterase (AChE) activity examined histochemically in spinal neurons and AChE and choline acetyltransferase (ChAT) activities were measured biochemically in axons of developing hindlimb motoneurons. At early larval stages, only spinal neurons born during embryonic life (primary neurons) showed histochemical evidence of AChE activity. Hindlimb motoneuron somata did not show AChE activity until mid-larval stages. AChE and ChAT activities were found in hindlimb motoneuron axons at the earliest stages examined, when the hindlimb consists of a small bud of undifferentiated mesenchyme. Activities of both enzymes show steady increases over the course of development until climax, when activities maintain a plateau until metamorphosis is complete. Total activities of both enzymes increase as the adult frog grows, although ChAT activity shows a much greater proportional increase than AChE activity. PMID: 6603253 [PubMed – indexed for MEDLINE]

J Cell Biol. 1983 Jan;96(1):240-7.

Changes in the amounts of cytoskeletal proteins within the perikarya and axons of regenerating frog motoneurons.

Sinicropi DV, McIlwain DL.

Changes in the amounts of tubulin, actin, and neurofilament polypeptides were found in regenerating motoneurons of grass frogs during the period of axonal elongation. Ventral roots 9 and 10 were transected unilaterally about 7 mm from the spinal cord. 35 d later, [3H]colchicine binding had decreased in the proximal stumps to approximately one-half of contralateral control values, well before the regenerating motor axons had reinnervated skeletal muscles of the hind limb. [3H]colchicine binding did not change significantly in the operated halves of the 9th and 10th spinal cord segments over a 75-d period. The relative amounts of actin, tubulin, and neurofilament polypeptides in the operated ventral roots were measured by quantitative densitometry of stained two-dimensional electrophoretic gels. Alpha-tubulin, beta-tubulin, and the 68,000 molecular weight subunit of neurofilaments (NF68) decreased within the transected ventral roots to 78%, 57%, and less than 15% of control values, respectively. The amount of actin increased to 132% of control values within the operated ventral roots, although this change was not statistically significant. Opposite changes were found within motoneuronal cell bodies isolated from the spinal cord. The relative amounts of alpha-tubulin, beta-tubulin and NF68 within axotomized perikarya increased, respectively, to 191%, 146%, and 144% of that in control perikarya isolated from the contralateral side of the spinal cord. Thus, the changes in NF68 and tubulin did not occur uniformly throughout the injured cells. The possible structural and functional consequences of these changes are discussed. PMID: 6402517 [PubMed – indexed for MEDLINE]

J Neurochem. 1982 Apr;38(4):1099-105.

Acetylcholinesterase distribution in axotomized frog motoneurons.

Sinicropi DV, Michels K, McIlwain DL.

The distribution of acetylcholinesterase (AChE; EC 3.1.1.7) activity was examined in the perikarya and proximal axonal stumps of frog motoneurons injured by ventral root transection. Based upon measurements of net AChE accumulation in the proximal stumps of transected ventral roots, and upon orthograde clearances of AChE reported by others, it was determined that an amount of AChE equivalent to at least 0.7-2 times the perikaryal content of this enzyme enters the motor axon each day. A progressive decrease in the rate of AChE accumulation in transected axons during the first 3 days after ventral rhizotomy raised the possibility that excess enzyme might accumulate elsewhere within the axotomized motoneurons. However, AChE accumulation was detected only near the cut ends of the ventral roots and was not appreciably increased within injured motoneuronal cell bodies and proximal dendrites, which were isolated by a new method combining bulk and single-cell isolation techniques. These data suggest that AChE turnover is altered rapidly in response to axonal injury, thereby avoiding large perikaryal accumulations of this enzyme. PMID: 6977618 [PubMed – indexed for MEDLINE]

Comput Biomed Res. 1981 Aug;14(4):314-26.

Television-based densitometric analysis of proteins separated by two-dimensional gel electrophoresis.

Aycock BF, Weil DE, Sinicropi DV, McIlwain DL. PMID: 7261571

A program is described for a computer-aided, television based system which can quantify the densities of stained proteins on two-dimensional electrophoretic gels. The system is designed to detect and graphically reproduce the edges of spots which are often located in nonuniform levels of background stain and to integrate the density of each spot detected. Tests of the system are described which show that it responds linearly to optical densities encountered on the electrophoretic gels and is sufficiently stable and sensitive to permit quantitative analysis of most, if not all, proteins separated on a gel.

J Neurochem. 1981 Jan;36(1):242-50.

Distribution of soluble proteins within spinal motoneurons: a quantitative two-dimensional electrophoretic analysis.

Weil DE, McIlwain DL.

Soluble protein fractions obtained from bovine lumbar spinal motoneuron cell bodies, ventral gray matter, and ventral and dorsal roots were analyzed by two-dimensional gel electrophoresis. Each extract was separated into Coomassie blue-stained patterns of up to 350 polypeptides ranging in isoelectric point from pH 4 to 8 and in molecular weight from 10,000 to 200,000. Visual inspection of the protein pattern of the isolated cell bodies showed it to be substantially different from those of ventral gray matter and the spinal roots, while the patterns obtained from ventral and dorsal roots were indistinguishable. Computer-assisted densitometry of the major soluble proteins from spinal roots showed no quantitative difference between the predominant proteins in ventral and dorsal root extracts. Differences of 10-fold or more were common when the major proteins of the isolated perikarya were compared with those of the other fractions. Since most of the soluble proteins extracted from ventral and dorsal roots were probably derived from the axoplasm of motor and sensory nerves, respectively, these results are interpreted to mean that large differences exist in the distribution of individual soluble proteins between the cell body and axon of spinal motoneurons, while the major soluble proteins of spinal motor and sensory axons are highly similar. PMID: 7463049 [PubMed – indexed for MEDLINE]

Brain Res. 1979 Dec 14;178(2-3):519-28.

Initiation and time course of mitosis of non-neuronal cells after spinal motoneuron axotomy.

McIlwain DL, Farel PB.

The mitotic response of non-neuronal cells following motor axon transection was measured after in vitro incorporation of [3H]thymidine in frog spinal cord. This predominantly ipsilateral response occurs more rapidly and is of greater magnitude when motor axons are unilaterally transected at the ventral root than after sciatic nerve transection. No increase in incorporation occurred when regenerating fibers were transected a second time before reinnervation, but an increase was observed when the second operation was performed after the formation of functional neuromuscular connections had taken place. Autoradiographic studies after dorsal or ventral root transection showed that the distribution of labeled cells approximated the anatomical extent of the injured cellular elements within the spinal cord. These data are discussed in relation to the characteristics of the dividing cells and the nature of the events eliciting mitosis. PMID: 315812 [PubMed – indexed for MEDLINE]

Brain Res. 1978 Dec 22;159(1):41-54.

The plasma membrane of bulk-isolated mature spinal neurons.

Hester S, Capps-Covey P, McIlwain DL.

Large neuronal perikarya isolated in bulk fractions from adult bovine ventral spinal cord and examined by electron microscopy have discontinuities in their plasma membrane. A systematic investigation of the possible causes of the membrane defects indicates that the act of mechanical dissociation of the neurons from the spinal tissue is chiefly responsible for the changes. One likely cause of membrane loss is the avulsion of postsynaptic membrane along with the presynaptic elements, leaving bouton-free cell bodies with many gaps in their plasma membrane. Membrane defects were also demonstrated by the entry of procion yellow into the isolated perikarya. Other studies (Weil et al., 1977) on these neuronal cell bodies have also shown that many soluble proteins can leave the neurons under some, but not all, conditions of isolation. These findings are discussed in relation to the possibilities of tissue culture of mature neurons and future research on bulk-isolated mature neurons. PMID: 728800 [PubMed – indexed for MEDLINE]

J Neurochem. 1977 Nov;29(5):847-52.

Choline acetyltransferase activity in large ventral spinal neurons.

Weil DE, Busby WH Jr, McIlwain DL. PMID: 591962 [PubMed – indexed for MEDLINE]

Up to approx 3 pmol of acetylcholine (ACh)/h/cell body was synthesized by perikarya of large spinal neurons isolated in bulk fractions from bovine ventral spinal cord. Many of the cell bodies are probably derived from motoneurons. A medium of low ionic strength and pH was used to minimize losses of soluble acetyl CoA: choline-o-acetyltransferase (ChAc; EC 2.3.1.6) from the neurons, whose permeability properties were altered. Such a medium also increased the retention of other soluble proteins by the cell bodies. The maximal rate of hydrolysis of ACh by the isolated neurons exceeded that of its synthesis by a factor of at least 100. It was estimated that ChAc and acetylcholinesterase (AChE; EC 3.1.1.7) each represent less than 0.01% by weight of the total protein in these cell bodies and that as little as 10% of each enzzyme in the ventral spinal cord is located within the large neuronal somata and their proximal processes.

J Neurochem. 1976 Jul;27(1):109-12.

The nuclear DNA content of large ventral spinal neurons.

McIlwain DL, Capps-Covey P. PMID: 956819 [PubMed – indexed for MEDLINE]

A nuclear fraction has been obtained from large spinal neurons previously isolated from bovine ventral spinal cord in bulk suspensions. The fraction contained an average of 5.3 +/- 0.9 pg DNA/nucleus, indicating a high incidence of diploid nuclei. This conclusion was confirmed by distribution analysis of DNA in propidium iodide-stained nuclei examined by flow microfluorometry. That technique showed that at least 90% of the nuclei from large spinal neurons are diploid. Mixed, mostly non-neuronal nuclei derived from many types of cells in the ventral spinal cord contained an average of 5.9 +/- 0.6 DNA/nucleus, 19% of which possibly possess more than diploid amounts of DNA. The uniform DNA content in nuclei of large spinal neurons and most other types of cells in the ventral spinal cord contrasts sharply with a wide variation (av 26-fold) in the nuclear volumes of the same cells.

J. Neurochem. 1975 Oct;25(4):517-21.

Bulk isolation of large ventral spinal neurons.

Capps-Covey P, McIlwain DL. PMID: 168314

Cell bodies of spinal neurons can be isolated in bulk fractions, using methods developed for neurons from brain. The technique described here includes sieving steps which allow one to purify and concentrate selectivley the larger neuronal perikarya (50um or more), most of which could be alpha motoneurons. An average of 170,000 large neurons can be obtained from 50g of trimmed bovine ventral enlargements. Roughly 6% of the total large neurons in the ventral grey matter are recovered and 85% of the isolated neurons are larger than 50um in their smallest dia. Non-neuronal contaminants, mostly capillary fragments, account for 19% of the total particles in the fraction. Each isolated neuron containes an average of 9690 pg protein and 1353 pg RNA and hydrolyses 2.2 pmol of acetylthiocholine/min. Pretreatment of the spinal tissue with collagenase before cell isolated is also evaluated here.

Biochem J. 1968 Sep;109(3):389-98.

Choline acetyltransferase binding to and release from membranes.

Fonnum F.

PMID: 5685862 [PubMed – indexed for MEDLINE]

Lab Invest. 1989 Sep;61(3):333-42.

Changes in size of motor axons in hereditary canine spinal muscular atrophy.

Cork LC, Struble RG, Gold BG, DiCarlo C, Fahnestock KE, Griffin JW, Price DL.

Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland.

Hereditary canine spinal muscular atrophy (HCSMA), a dominantly inherited disorder of motor neurons, has three phenotypes: accelerated, intermediate, and chronic. In the accelerated and intermediate phenotypes, axonal sizes in ventral roots were smaller than in controls. Reductions in axonal size occurred primarily in large axons, and the frequency of small-caliber axons was increased. In HCSMA, nerve fiber shape, i.e., circularity, was reduced, and the relative thickness of the myelin sheath as a function of axonal caliber was decreased. The density of fibers in motor nerves was increased, making it unlikely that a selective loss of large-caliber axons explained the increased frequency of small-caliber axons. These observations suggest that, in HCSMA, changes in axonal size in motor nerves are associated with both growth arrest and axonal atrophy.

PMID: 2770248 [PubMed – indexed for MEDLINE]

J Cell Sci. 1978 Dec;34:247-78.

Nuclear volume control by nucleoskeletal DNA, selection for cell volume and cell growth rate, and the solution of the DNA C-value paradox.

Cavalier-Smith T.

The 40,000-fold variation in eukaryote haploid DNA content is unrelated to organismic complexity or to the numbers of protein-coding genes. In eukaryote microorganisms, as well as in animals and plants, DNA content is strongly correlated with cell volume and nuclear volume, and with cell cycle length and minimum generation time. These correlations are simply explained by postulating that DNA has 2 major functions unrelated to its protein-coding capacity: (1) the control of cell volume by the number of replicon origins, and (2) the determination of nuclear volume by the overall bulk of the DNA: cell growth rates are determined by the cell volume and by the area of the nuclear envelope available for nucleocytoplasmic transport of RNA, which in turn depends on the nuclear volume and therefore on the DNA content. During evolution nuclear volume, and therefore DNA content, has to be adjusted to the cell volume to allow reasonable growth rates. The great diversity of cell volumes and growth rates, and therefore of DNA contents, among eukaryotes results from a varying balance in different species between r-selection, which favours small cells and rapid growth rates and therefore low DNA C-values, and K-selection which favours large cells and slow growth rates and therefore high DNA C-values. In multicellular organisms cell size needs to vary in different tissues: size differences between somatic cells result from polyteny, endopolyploidy, or the synthesis of nucleoskeletal RNA. Conflict between the need for large ova and small somatic cells explains why lampbrush chromosomes, nurse cells, chromatin diminution and chromosome elimination evolved. Similar evolutionary considerations clarify the nature of polygenes, the significance of the distribution of haploidy, diploidy and dikaryosis in life cycles and of double fertilization in angiosperms, and of heteroploidy despite DNA constancy in cultured cells, and other puzzles in eukaryote chromosome biology. Eukaryote DNA can be divided into genic DNA (G-DNA), which codes for proteins (or serves as recognition sites for proteins involved in transcription, replication and recombination), and nucleoskeletal DNA (S-DNA) which exists only because of its nucleoskeletal role in determining the nuclear volume (which it shares with G-DNA, and performs not only directly, but also indirectly by coding for nucleoskeletal RNA). Mechanistic and evolutionary implications of this are discussed.

PMID: 372199 [PubMed – indexed for MEDLINE]

J Cell Biol. 1984 Jul;99(1 Pt 2):203s-208s.

Epithelial structure revealed by chemical dissection and unembedded electron microscopy.

Fey EG, Capco DG, Krochmalnic G, Penman S.

Cytoskeletal structures obtained after extraction of Madin-Darby canine kidney epithelial cell monolayers with Triton X-100 were examined in transmission electron micrographs of cell whole mounts and unembedded thick sections. The cytoskeleton, an ordered structure consisting of a peripheral plasma lamina, a complex network of filaments, and chromatin-containing nuclei, was revealed after extraction of intact cells with a nearly physiological buffer containing Triton X-100. The cytoskeleton was further fractionated by extraction with (NH4)2SO4, which left a structure enriched in intermediate filaments and desmosomes around the nuclei. A further digestion with nuclease and elution with (NH4)2SO4 removed the chromatin. The stable structure that remained after this procedure retained much of the epithelial morphology and contained essentially all of the cytokeratin filaments and desmosomes and the chromatin-depleted nuclear matrices. This structural network may serve as a scaffold for epithelial organization. The cytoskeleton and the underlying nuclear matrix intermediate filament scaffold, when examined in both conventional embedded thin sections and in unembedded whole mounts and thick sections, showed the retention of many of the detailed morphological aspects of the intact cells, which suggests a structural continuum linking the nuclear matrix, the intermediate filament network, and the intercellular desmosomal junctions. Most importantly, the protein composition of each of the four fractions obtained by this sequential procedure was essentially unique. Thus, the proteins constituting the soluble fraction, the cytoskeleton, the chromatin fraction, and the underlying nuclear matrix-intermediate filament scaffold are biochemically distinct.

PMID: 6540264 [PubMed – indexed for MEDLINE]

J Cell Biol. 1990 Mar;110(3):569-80.

Core filaments of the nuclear matrix.

He DC, Nickerson JA, Penman S.

Department of Biology, Beijing Normal University, Peoples Republic of China; and Department of Biology 56-535, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

The nuclear matrix is concealed by a much larger mass of chromatin, which can be removed selectively by digesting nuclei with DNase I followed by elution of chromatin with 0.25 M ammonium sulfate. This mild procedure removes chromatin almost completely and preserves nuclear matrix morphology. The complete nuclear matrix consists of a nuclear lamina with an interior matrix composed of thick, polymorphic fibers and large masses that resemble remnant nucleoli. Further extraction of the nuclear matrices of HeLa or MCF-7 cells with 2 M sodium chloride uncovered a network of core filaments. A few dark masses remained enmeshed in the filament network and may be remnants of the nuclear matrix thick fibers and nucleoli. The highly branched core filaments had diameters of 9 and 13 nm measured relative to the intermediate filaments. They may serve as the core structure around which the matrix is constructed. The core filaments retained 70% of nuclear RNA. This RNA consisted both of ribosomal RNA precursors and of very high molecular weight hnRNA with a modal size of 20 kb. Treatment with RNase A removed the core filaments. When 2 M sodium chloride was used directly to remove chromatin after DNase I digestion without a preceding 0.25 M ammonium sulfate extraction, the core filaments were not revealed. Instead, the nuclear interior was filled with amorphous masses that may cover the filaments. This reflected a requirement for a stepwise increase in ionic strength because gradual addition of sodium chloride to a final concentration of 2 M without an 0.25 M ammonium sulfate extraction uncovered core filaments.

PMID: 2307700 [PubMed – indexed for MEDLINE]

Proc Natl Acad Sci U S A. 1995 Jun 6;92(12):5251-7.

Rethinking cell structure.

Penman S.

Department of Biology, Massachusetts Institute of Technology, Cambridge 02139,

USA.

Cell structure, emerging from behind the veil of conventional electron microscopy, appears far more complex than formerly realized. The standard plastic-embedded, ultrathin section can image only what is on the section surface and masks the elaborate networks of the cytoplasm and nucleus. Embedment-free electron microscopy gives clear, high-contrast micrographs of cell structure when combined with removal of obscuring material such as soluble proteins. The resinless ultrathin section is the technique of choice; it is simple and inexpensive, and it uses ordinary electron microscopes. The resulting pictures reveal a world of complex cell structure and function. These images necessarily change our conception of the cytoskeleton, nuclear matrix, mitosis, and the relation of membranes to cytostructure.

PMID: 7777493 [PubMed – indexed for MEDLINE]

J Neurochem. 2002 May;81(3):525-32.

Reduced number of unmyelinated sensory axons in peripherin null mice.

Lariviere RC, Nguyen MD, Ribeiro-da-Silva A, Julien JP.

Department of Neurology and Neurosurgery, McGill University, Montreal, Canada.

Peripherin is a type III intermediate filament (IF) abundantly expressed in developing neurons, but in the adult, it is primarily found in neurons extending to the peripheral nervous system. It has been suggested that peripherin may play a role in axonal elongation and/or cytoskeletal stabilization during development and regeneration. To further clarify the function of peripherin, we generated and characterized mice with a targeted disruption of the peripherin gene. The peripherin null mice were viable, reproduced normally and did not exhibit overt phenotypes. Microscopic analysis revealed no gross morphological defects in the ventral and dorsal roots, spinal cord, retina and gut, but protein analyses showed increased levels of the type IV IF alpha-internexin in ventral roots of peripherin null mice. Whereas the number and caliber of myelinated motor and sensory axons in the L5 roots remained unchanged in peripherin knockout mice, there was a substantial reduction (approximately 34%) in the number of L5 unmyelinated sensory fibers that correlated with a decreased binding of the lectin IB4. These results demonstrate a requirement of peripherin for the proper development of a subset of sensory neurons.

PMID: 12065660 [PubMed – indexed for MEDLINE]

J Neurochem. 1999 Sep;73(3):972-84.

Disruption of type IV intermediate filament network in mice lacking the neurofilament medium and heavy subunits.

Jacomy H, Zhu Q, Couillard-Despres S, Beaulieu JM, Julien JP.

Montreal General Hospital Research Institute, Quebec, Canada.

To clarify the role of the neurofilament (NF) medium (NF-M) and heavy (NF-H) subunits, we generated mice with targeted disruption of both NF-M and NF-H genes. The absence of the NF-M subunit resulted in a two- to threefold reduction in the caliber of large myelinated axons, whereas the lack of NF-H subunits had little effect on the radial growth of motor axons. In NF-M-/- mice, the velocity of axonal transport of NF light (NF-L) and NF-H proteins was increased by about two-fold, whereas the steady-state levels of assembled NF-L were reduced. Although the NF-M or NF-H subunits are each dispensable for the formation of intermediate filaments, the absence of both subunits in double NF-M; NF-H knockout mice led to a scarcity of intermediate filament structures in axons and to a marked approximately twofold increase in the number of microtubules. Protein analysis indicated that the levels of NF-L and alpha-internexin proteins were reduced dramatically throughout the nervous system. Immunohistochemistry of spinal cord from the NF-M-/-;NF-H-/- mice revealed enhanced NF-L staining in the perikaryon of motor neurons but a weak NF-L staining in axons. In addition, axonal transport studies carried out by the injection of [35S]methionine into spinal cord revealed after 30 days very low levels of newly synthesized NF-L proteins in the sciatic nerve of NF-M-/-;NF-H-/- mice. The combined results demonstrate a requirement of the high-molecular-weight subunits for the assembly of type IV intermediate filament proteins and for the efficient translocation of NF-L proteins into the axonal compartment.

PMID: 10461886 [PubMed – indexed for MEDLINE]

Brain Res. 1972 Dec 12;47(2):331-43.

Neuroplasmic transport in dendrites: effect of colchicine on morphology and physiology of motoneurones in the cat.

Schubert P, Kreutzberg GW, Lux HD.

Intracellular application of tritiated amino acids was performed by microelectrophoresis of single lumbar motoneurones in the cat. Incorporation and transport of the radiochemicals were studied in autoradiographs. Simultaneous recording of the electrophysiological activity permitted direct correlation of biochemical, morphological and physiological parameters in the same individual neurone.

This technique was used to test the effect of colchicine. The drug produced severe morphological changes but did not significantly influence amino acid incorporation. But transport of proteins, especially in the dendrites, was impaired. This suggests that dendritic transport depends on the integrity of the neurotubular system. Despite severe morphological changes the neurones retained normal resting potentials and generated spikes when stimulated antidromically. Yet, the majority of the neurones do not respond to orthodromic stimulation. This suggests colchicine-induced structural changes of the presynaptic side.

PMID: 4118532 [PubMed – indexed for MEDLINE]

Nat Neurosci. 1999 Jan;2(1):50-6.

Slowing of axonal transport is a very early event in the toxicity of ALS-linked SOD1 mutants to motor neurons.

Williamson TL, Cleveland DW.

Ludwig Institute for Cancer Research, University of California, San Diego, La Jolla 92093, USA.

Mutations in copper/zinc superoxide dismutase 1 (SOD1), primary causes of human amyotrophic lateral sclerosis (ALS), provoke motor neuron death through an unidentified toxic property. The known neurofilament-dependent slowing of axonal transport, combined with the prominent misaccumulation of neurofilaments in ALS, suggests that an important aspect of toxicity may arise from damage to transport. Here we verify this hypothesis for two SOD1 mutations linked to familial ALS. Reduced transport of selective cargoes of slow transport, especially tubulin, arises months before neurodegeneration. For one mutant, this represents the earliest detectable abnormality. Thus, damage to the cargoes or machinery of slow transport is an early feature of toxicity mediated by mutant SOD1.

PMID: 10195180 [PubMed – indexed for MEDLINE]

Int Rev Neurobiol. 1971;14:49-124.

The axon reaction: a review of the principal features of perikaryal responses to axon injury.

Lieberman AR.

PMID: 4948651 [PubMed – indexed for MEDLINE]

J Neuropathol Exp Neurol. 1987 Jan;46(1):40-9.

Atrophic cell processes of large motor neurons in the anterior horn in amyotrophic lateral sclerosis: observation with silver impregnation method.

Nakano I, Hirano A.

Investigation of silver-stained lumbar anterior horns in four autopsied cases of sporadic amyotrophic lateral sclerosis (ALS) revealed frequent extremely small cell processes originating from large motor neurons. Their perikarya were usually smaller in size than those of normal-looking ones and almost invariably had central chromatolysis-like changes, suggesting an intimate pathomorphological relationship between the perikarya and their processes. Although it was difficult to determine whether these small processes were atrophic dendrites or shrunken axons, some were recognized as dendrites from their multiple branchings and some were identified as axons from their tapering configuration followed by widening of the distal portion. Aggregates of lipofuscin were almost always present in the perikaryal portion from which an atrophic process arose. In addition, the somewhat argentophilic slender cytoplasm which in normal neurons separates lipofuscin from the cell surface and merges with the proximal part of a process, was prominently attenuated. The small processes were more frequently observed in cases with many spheroids and chromatolytic neurons. The change in the proximal portion of the processes may implicate some disturbance of functional connection between the soma and the cell processes in ALS.

PMID: 2432193 [PubMed – indexed for MEDLINE]

“Amyotrophic Lateral Sclerosis” (ed. Japan Medical Research Foundation) University of Tokyo Press Tokyo 1979, pp107-133

Pathology of motor neurons with special reference to amyotrophic lateral sclerosis and related diseases.

Hirano A and Iwata M

Proc R Soc Lond B Biol Sci. 1957 May 7;146(924):416-30.

The spindle and extrafusal innervation of a frog muscle.

Gray EG.

PMID: 13431865 [PubMed – OLDMEDLINE for Pre1966]

Brain Res. 1978 Dec 15;158(2):331-41.

Reflex activity of regenerating frog spinal motoneurons.

Farel PB.

The reflex activity of frog spinal motoneurons whose axons had been sectioned 7 days to 10 months previously was examined. Before the severed axons reinnervated muscle, reflex latency was prolonged, and reflex amplitude was depressed. Examination of input-output relations revealed the presence of a subset of axotomized motoneurons having supranormal excitability. This subset was not present in normal preparations or in preparations tested after reinnervation took place. Following reinnervation of muscle, reflex latency returned to preoperative levels, while amplitude recovery was typically more variable. Decreased conduction velocity of the severed ventral root fibers accounted for the alterations in reflex latency. These and other data form the basis for concluding that motoneurons, even within a particular preparation, show a wide range of responses to section of their axons and will provide a framework for interpretation of more selective intracellular experiments.

PMID: 213172 [PubMed – indexed for MEDLINE]

Acta Neuropathol (Berl). 1969 Sep 9;14(1):62-71.

Effect of actinomycin D on retrograde nerve cell reaction. Further observations.

Torvik A, Heding A.

The retrograde nerve cell reaction was studied in the neurons of the reticular formation and in the facial nucleus of mice after intracerebral injections of actinomycin D. Normally the reticular neurons show a classical type of retrograde reaction with dispersion of the Nissl substance and central chromatolysis. The facial neurons also show a dispersion of the Nissl granules but there is an early increase in the cytoplasmic basophilia instead of chromatolysis. The two types of neurons thus show somewhat different patterns of retrograde reaction. It was shown previously that actinomycin prevented the appearance of retrograde reaction in the facial nucleus when the drug was injected at the time of operation (Torvik and Heding, 1967). The present study showed that actinomycin blocked the retrograde reaction also in the reticular formation when it was given 2h before the reticulospinal fibers were cut. When the drug was given 12h after section of the reticulospinal fibers, the retrograde reaction developed normally in the reticular formation, although the neurons were morphologically normal at the time of injection. In the facial nucleus the retrograde reaction was blocked when the drug was given 9h after the operation but not by 15h. The findings suggest that the morphological nerve cell changes after axon lesions are preceded by the synthesis of new enzymes in the injured cells, which in some way are necessary for the ensuing dispersion of the Nissl substance.

J Neurochem. 1959 Dec;5:43-9.

Ribonucleic acid changes in the motoneurons of the frog during axon regeneration.

Edstrom JE.

The ribonucleic acid (RNA) content and the volume of individual cell bodies of motoneurons of the frog Rana temporaria were measured during chromatolysis after sciatic nerve crush. Preliminary determinations of nucleolar volume and RNA content were also performed. The following results were obtained:

• There was a parallel increase in volume and RNA as early as the fourth day onwards. Maximal values (about 250% of the control side) were reached after about 5 weeks. After this time there was a slow regression in these values, which became normal again after 4-5 months.
• There was never any change in RNA concentration in the cell bodies in contrast to chromatolysed hypoglossal neurons.
• The results indicate that the changes represent a true hypertrophy and are not caused by a temporary arrest in transport of axoplasm. Increased nucleolar volumes and RNA values suggest that the hypertrophy is due to an increased synthesis.

PMID: 13819378 [PubMed – OLDMEDLINE for Pre1966]

J Physiol. 1965 Oct;180(4):741-53.

An autoradiographic study of the incorporation of nucleic-acid precursors by neurones and glia during nerve regeneration.

Watson WE.

1. One hypoglossal or facial nerve was cut, crushed or ligated in rodents, which were permitted to survive fro varying periods. [3H]uridine, [3H]guanosine,[3H]adenosine, [3H]thymidine or [3H]lysine was given by injection into the cerebral ventricles or cisterns. The uptake of tritium by neurones and glia was measured autoradiographically, and its distributions between intracellular compartments determined.

2. In both normal and injured neurones, RNA precursors were incorporated first within the nucleus, and later within the cytoplasm. This change of distribution represented an apparent transfer of RNA from nucleus to cytoplasm.

3. After nerve division the uptake of RNA precursors and of [3H]lysine increased within 48 hr. The rate of apparent transfer of RNA from nucleus to cytoplasm also increased markedly. This change always accompanied cytoplasmic swelling of the neurone.

4. During the period of axon outgrowth preceding maturation of the regenerating nerve the rate of apparent transfer of RNA from nucleus to cytosplasm decreased to a value less than normal, and remained low for the remaining period of maturation.

5. These results are discussed in relation to measurements of the RNA and protein contents of regenerating neurones.

6. Between 2 days and 5 days after crushing, dividing or ligating the nerve the number of perineuronal glial cells increased and they synthesized DNA. This change always accompanied cytoplasmic swelling of the neurones.

7. In the rabbit, endothelial cells of capillaries of the nucleus of the injured nerve synthesized DNA.

8. These results are discussed in relation to the probable changes in metabolic activity of the injured nerve cells.

PMID: 5880359 [PubMed – indexed for MEDLINE]

J Neurochem. 1957;1(4):316-25.

The chemical changes in regenerating neurons.

Brattgard SO, Edstrom JE, Hyden H.

• Nerve regeneration has been studied in the hypoglossal nucleus of the rabbit. Ultramicrochemical and quantitative cytochemical methods were used to determine the content per nerve cell of RNA and dry weight (divided into protein and lipid fractions). The data observed were correlated with observations on the peripheral growth of the axon.
• The weight of the fresh, control hypoglossal cell soma averages 2770ng. The amount of RNA per cell averages 200ng. The RNA concentration expressed as weight per volume is thus 1.5%. Computed on a dry protein basis it averages 9%. The volume of the cells averages 13,300sq.um. During the period of outgrowth of the axons, the nerve cell changes were characterized by an increase in volume and in the lipid and protein content: there was also an uptake of water, causing a decrease in the intracellular substance. The amount of RNA was constant during this period, but the state of aggregation changed, so that it formed small, finely dispersed particles. These changes form the chemical background of chromatolysis in nerve regeneration.
• The change in RNA aggregation during this period of rapid growth is interpreted as a transformation from an active to a more active form of RNA, serving as an initiator of the protein production which follows.
• The maturation period, which starts when contact is re-established between nerve cell and periphery, is characterized by another type of growth process. The most striking features are the 100% increase in RNA content and the volume increase of 100%. The latter is explained by the increase in the amount of proteins and lipids per cell. The original state of aggregation of RNA is eventually restored, as are the original concentration values. The re-establishment of functional contact with the periphery is stressed as a factor of importance for the chemical reconstruction of the neuron.

Mol Biol Cell. 2003 Dec;14(12):5069-81. Epub 2003 Oct 31.

A role for intermediate filaments in determining and maintaining the shape of nerve cells.

Helfand BT, Mendez MG, Pugh J, Delsert C, Goldman RD.

Department of Cell and Molecular Biology, Northwestern University Feinberg School of Medicine, Chicago, Illinois 60611, USA.

To date, the functions of most neural intermediate filament (IF) proteins have remained elusive. Peripherin is a type III intermediate filament (IF) protein that is expressed in developing and in differentiated neurons of the peripheral and enteric nervous systems. It is also the major IF protein expressed in PC12 cells, a widely used model for studies of peripheral neurons. Dramatic increases in peripherin expression have been shown to coincide with the initiation and outgrowth of axons during development and regeneration, suggesting that peripherin plays an important role in axon formation. Recently, small interfering RNAs (siRNA) have provided efficient ways to deplete specific proteins within mammalian cells. In this study, it has been found that peripherin-siRNA depletes peripherin and inhibits the initiation, extension, and maintenance of neurites in PC12 cells. Furthermore, the results of these experiments demonstrate that peripherin IF are critical determinants of the overall shape and architecture of neurons.

PMID: 14595112 [PubMed – indexed for MEDLINE]

J Comp Neurol 1948 89:93-121

A quantitative study of certain morphological changes in spinal motor neurons during axon reaction.

Barr ML and Hamilton JD

• Morphological changes in cells of the dorsolateral group of the cat’s spinal cord were studied quantitatively in a series of 22 animals sacrificed at intervals of 3 to 136 days following unilateral resection of a portion of the sciatic nerve at a constant level.
• The time course of chromatolysis was followed as a background against which other quantitative studies could be evaluated.
• The mean cross sectional area of the cell body, as seen in reduced silver nitrate preparations, increased during the 1 st week, reached a maximum of the order of 20% above the normal value for control cells and returned to an approximately normal value at the end of 4 months. Swelling of the nerve cell body was uniform in all directions.
• An initial increase in the size of the nucleus was followed by a decrease in nuclear size, as seen in cross section. The diminution persisted throughout the period covered by the experiments.
• Nuclear eccentricity was followed quantitatively. The direction of movement was consistently away from the axon hillock, a circumstance related to maximal chromatolysis in the region between the nucleus and axon hillock.
• No significant alteration in the size or position of the nucleolus, or in its structural details as revealed by silver preparations, could be detected.
• Considerable variation in the rate and degree of reaction was encountered among individual cells.
• A dynamic interpretation of the morphological changes, on the basis of known facts regarding the chemistry of the neuron in axon reaction, has been attempted.

J Cell Biol. 1984 Jul;99(1 Pt 2):209s-211s.

Assembly associated with the cytomatrix.

Fulton AB.

Assembly in vivo has been studied both for endogenous cytoskeletal proteins and for several classes of viruses. Autoradiography of cytoskeletal proteins has shown that many associate with the cytoskeletal framework close to the time and place of synthesis. The cytoskeletal proteins rearrange after association with the cytoskeletal framework. Rearrangement in symmetrical giant cells occurs in a centrifugal and coherent pattern. Many of the cytoskeletal proteins associate cotranslationally, as shown by their puromycin resistance in a cell-free translation system. The assembly of several groups of viruses has been shown to be associated with various components of the cytoskeleton; whether such assembly is cotranslational has not yet been addressed directly.

PMID: 6746729 [PubMed – indexed for MEDLINE]

J Comp Neurol. 1958 Apr;109(2):205-19.

Retrograde reaction in motoneuron dendrites following ventral root section in the frog.

Cerf JA, Chacko LW.

Retrograde reaction has been studied in motoneuron dendritic processes of the frog’s spinal cord following ventral root reaction, with the use of thionin-blue staining, silver impregnation and histochemical localization of acid phosphatase. The characteristic topographical pattern and plexus formation of the dendrites in this species permitted the positive identification of retrograde changes, extending to the entire course of the expansions. Decreased affinity for silver and increased acid phosphatase activity in the dendrites were found associated with chromatolysis in the parent cell bodies. Some implications concerning the participation of dendrites in the axon reaction are discussed.

J Cell Biol. 1972 Apr;53(1):24-37.

The response of ventral horn neurons to axonal transection.

Price DL, Porter KR.

The morphological changes induced in the frog ventral horn neurons by axonal transection have been studied with the electron microscope. During the first 2 wk after axotomy, the neuronal nucleus becomes more translucent and the nucleolus becomes enlarged and less compact. The cisternae of the granular endoplasmic reticulum vesiculate and ribosomes dissociate from membranes. Free ribosomes and polysomes are dispersed in the cytoplasmic matrix. Neurofilaments and neurotubules are increased in number. These structures appear to be important in the regeneration of the axon. It is proposed that neurotubules, neurofilaments and axoplasmic matrix are synthesized by the free polyribosomes in the chromatolytic neuron. By the fourth postoperative week, the neurons show evidence of recovery. The cytoplasm is filled with profiles of granular endoplasmic reticulum and many intercisternal polysomes. The substances being manufactured by the newly formed granular endoplasmic reticulum are not clearly defined, but probably include elements essential to electrical and chemical conduction of impulses. The significance of these observations in respect to recent studies of axoplasmic flow is discussed.

Shinkei Kenkyu no Shimpo 1982 26: 737-752

Some aspects of anatomy and pathology of nerve roots.

Ohama E

The cranial and spinal nerve roots have their own peculiar structures which are not seen in the other peripheral nerves. They are summarized as follows.

• The cranial and spinal nerve roots are located within the cranium or vertebral canal and are always bathed in the cerebrospinal fluid throughout their length.
• 2) In contrast to mixed spinal nerves which contain both motor and sensory fibers, spinal nerve roots consist of either motor or sensory nerve fibers alone, although some workers indicate that about 15% of all the anterior root fibers are sensory in nature.
• The human cerebrospinal nerve roots consist of a central glial and a peripheral non-glial segment. The central segment, usually a short distance from the surface of the spinal cord or brainstem, has the structure of a fiber tract of the CNS and the peripheral segment is essentially identical to that of a peripheral nerve. Therefore, the transition from central to peripheral nervous systems occurs in the proximal portion of the roots.
• The cerebrospinal nerve roots have no epineurium and scanty endoneurial connective tissue. Furthermore, they are ensheathed by a peculiar root sheath which differs from any other connective tissue sheaths of peripheral nerves.

Of various pathological changes in the nerve roots, two distinctive lesions were discussed with special reference to their morphopathogenesis; one is those of dystrophic mouse, and the other is the glial bundles. The glial bundles were first described by Hoffmann in the proximal portion of the anterior spinal roots of Werdnig-Hoffmann disease (WHD), and was discovered by Chou and Fakadej to be composed of astrocytes and their cytoplasmic processes. Consequent observations confirmed the constant occurrence of numerous glial bundles in the anterior spinal roots of WHD. Furthermore, the same structures have been also found in the cranial and posterior spinal nerve roots of WHD, and anterior and/or posterior nerve roots of some other pathological conditions in humans and experimental animals.

Many authors have proposed that the glial bundles are a special type of astrocytic reaction at the junction of the central and peripheral nervous systems, secondary to the neuronal and axonal degeneration. However, our results of the examination of the glial bundles in the cranial and spinal nerve roots in ALS (Fig. 6) indicates that the occurrence of the glial bundles is not always related to the lower motor neuron lesions.

Ann Neurol. 1982 Feb;11(2):203-6.

Glial outgrowth along spinal nerve roots in amyotrophic lateral sclerosis.

Ghatak NR, Nochlin D.

Formation of glial bundles in the proximal portion of the ventral nerve roots is described in a 51-year-old patient with the sporadic form of amyotrophic lateral sclerosis (ALS). Although the bundles were relatively fewer, they were identical in morphology and distribution to those consistently found in Werdnig-Hoffmann disease (WHD). The occurrence of glial bundles in ALS, albeit rare, indicates that this phenomenon is not a unique feature of WHD. Similar changes have been observed in several other unrelated conditions, always in association with
degeneration of neurons or axons. Thus, outgrowth of astrocytes in the form of glial bundles should be considered a special type of astrocytic reaction at the interface of the central and peripheral nervous systems.

Case Report: PMID: 7073255 [PubMed – indexed for MEDLINE]

J Neuropathol Exp Neurol. 1981 Nov;40(6):667-75.

Morphometric comparison of the vulnerability of peripheral motor and sensory neurons in amyotrophic lateral sclerosis.

Kawamura Y, Dyck PJ, Shimono M, Okazaki H, Tateishi J, Doi H.

The diameter histograms of cell bodies (cytons) in motor neuron columns at the L5 segment of the spinal cord of adult man reproducibly yield three peaks of increasing height: small (Cs), intermediate (Ci), and large (Cl). Histograms of L5 myelinated axons obtained from the ventral root have two peaks of increasing height: intermediate (Ai) and large (Al). In histograms prepared from seven cases of amyotrophic lateral sclerosis (ALS), the Cl and Al peaks were decreased selectively and severely. This provides evidence for alpha, but not gamma, motor neuron vulnerability. The Cl peak of spinal ganglion neurons and the Al peak of dorsal roots were significantly reduced in number, without a concomitant increase in Ci, Cs, and Ai peaks. This, plus earlier reports of abnormal cutaneous sensation thresholds, abnormal rates of fiber degeneration in cutaneous nerves, and dorsal column demyelination, provides evidence that large afferent neurons are affected in ALS, but to a lesser degree than alpha motor neurons.

PMID: 7299423 [PubMed – indexed for MEDLINE]

Neurology. 1975 Aug;25(8):781-5.

Frequency of nerve fiber degeneration of peripheral motor and sensory neurons in amyotrophic lateral sclerosis. Morphometry of deep and superficial peroneal nerves.

Dyck PJ, Stevens JC, Mulder DW, Espinosa RE.

Among 10 patients with amyotrophic lateral sclerosis who had combined biopsy of muscle and cutaneous nerves, two had a history of paresthesia that suggested involvement of peripheral afferent neurons. Of four patients without paresthesia, two had unequivocal abnormalities of touch-pressure sensation of the toe. On morphometric evaluations of lateral fascicles of deep peroneal nerve, one nerve had an abnormally low myelinated fiber density and seven of 10 had abnormally high frequencies of teased-fiber abnormalities. Teased fibers in which myelin was degenerating into linear rows of myelin ovoids and balls occurred in 10.5 percent of fibers in amyotrophic lateral sclerosis nerves as compared with 1.7 percent in control nerves (0.01 less than p less than 0.025). Estimates of density of myelinated fibers were less sensitive than estimates of the frequency of various changes in teased fibers for detecting abnormality.

PMID: 1171412 [PubMed – indexed for MEDLINE]

Arch Neurol. 1982 Mar;39(3):155-6.

Regular involvement of Clarke’s nucleus in sporadic amyotrophic lateral sclerosis.

Averback P, Crocker P.

Morphometric study of Clarke’s nucleus in 12 cases of sporadic amyotrophic lateral sclerosis and 12 normal control subjects showed an average reduction in the neuronal population in the disease group, with involvement in each case. Histopathologic findings of cell degeneration were definite but less noticeable than in the ventral horns. The process seems likely to be similar to that affecting motor neurons but is less advanced at the time of death.

PMID: 7065933 [PubMed – indexed for MEDLINE]

Ann Neurol. 1990 Mar;27(3):215-25.

Degeneration of spinocerebellar neurons in amyotrophic lateral sclerosis.

Williams C, Kozlowski MA, Hinton DR, Miller CA.

Department of Pathology, School of Medicine, University of Southern California, Los Angeles 90033.

The selective involvement of spinocerebellar neurons in sporadic amyotrophic lateral sclerosis was investigated using two monoclonal antibodies that have neuronal subset specificity in human spinal cord. In normal control subjects, monoclonal antibody 6A2 showed specificity for neurons of the dorsal nucleus of Clarke, the cells of origin of the dorsal spinocerebellar tract. Immunoreactive neurons were also observed in locations corresponding to the central cervical nucleus and spinal border region, containing neurons of the cervicospinocerebellar and ventral spinocerebellar tracts, respectively. The latter two neuronal subsets are indistinguishable from surrounding neurons when conventional histological stains are used. Antigen 6A2 was distributed on surfaces of neuronal somas and proximal neurites and extended into the extracellular space. A second antibody, monoclonal antibody 44.1, labeled the cytoplasm of neuronal somas and neurites, including all monoclonal antibody 6A2-reactive cells and alpha motoneurons. In spinal cords of all 5 patients with amyotrophic lateral sclerosis, monoclonal antibody 6A2 reactivity in the majority of spinocerebellar neurons was absent or localized to the somal cytoplasm, which still stained with monoclonal antibody 44.1. In more severely involved tissues, there was loss of some spinocerebellar neurons and a corresponding loss of monoclonal antibody 44.1 reactivity. These findings confirm involvement of the spinal cord components of the spinocerebellar system at all levels in sporadic amyotrophic lateral sclerosis and suggest that some surface molecules are modified during the degenerative process.

PMID: 2327732 [PubMed – indexed for MEDLINE]

Muscle Nerve. 2001 Nov;24(11):1510-9.

Presymptomatic motor neuron loss and reactive astrocytosis in the SOD1 mouse model of amyotrophic lateral sclerosis.

Feeney SJ, McKelvie PA, Austin L, Jean-Francois MJ, Kapsa R, Tombs SM, Byrne E.

Melbourne Neuromuscular Research Institute, St. Vincent’s Hospital, Fitzroy, Victoria 3065, Australia.

In familial amyotrophic lateral sclerosis (fALS), there is a need to establish more precisely the progression of the disease, particularly whether there is gradual presymptomatic neuronal loss or an abrupt loss coinciding with the symptomatic stage. To elucidate this, we investigated the progression of motor neuron loss through morphological techniques, reactive astrocytosis, and expression of ubiquitin and neurofilament proteins, by immunohistochemistry, in SOD1 G93A mice with a protracted disease course and control mice. Loss of motor neurons in SOD1 G93A mice followed a biphasic progression, with an initial loss at 126 days of age, followed by a gradual loss from onset of symptoms through to end-stage disease. Reactive astrocytosis was first observed at 70 days of age and showed a gradual increase through to end-stage disease. This suggests that there is a need for early detection of fALS cases, and potential therapeutic treatments may be more beneficial if administered at an early stage. Copyright 2001 John Wiley & Sons, Inc.

PMID: 11745954 [PubMed – indexed for MEDLINE]

Mol Cell Neurosci. 1995 Aug;6(4):349-62.

Age-dependent penetrance of disease in a transgenic mouse model of familial amyotrophic lateral sclerosis.

Chiu AY, Zhai P, Dal Canto MC, Peters TM, Kwon YW, Prattis SM, Gurney ME.

Division of Neurosciences, Beckman Research Institute of the City of Hope, Duarte, California 91010, USA.

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.

PMID: 8846004 [PubMed – indexed for MEDLINE]

Neurosci Lett. 2002 Oct 31;332(2):99-102.

Degeneration of corticospinal and bulbospinal systems in the superoxide dismutase 1(G93A G1H) transgenic mouse model of familial amyotrophic lateral sclerosis.

Zang DW, Cheema SS.

Department of Anatomy and Cell Biology, PO Box 13C, Monash University, Victoria 3800, Australia.

In the superoxide dismutase 1 (SOD1)(G93A G1H) transgenic mouse, the primary pathology and disease signs are associated with the degeneration of motor neurons in the lumbar spinal cord. It is unclear if the descending motor pathways from the cortex and brainstem are also compromised. The retrograde tracer Fluorogold was inserted into the T(12) segment of the spinal cord and the number of labelled neurons counted in the sensorimotor cortex and brainstem of 60, 90 and 110 day-old mice. A small loss of corticospinal and bulbospinal projections was detected at 60 days. By 110 days, 53% of corticospinal, 41% of bulbospinal and 43% of rubrospinal neurons were lost. The progressive loss of corticospinal axons was confirmed using the stereological fractionator method. These findings suggest that the expression of the SOD1(G93A G1H) mutant protein results in a disease that resembles the late stages of human motor neuron disease. This involves not only the destruction of lower motor neurons in the spinal cord, but also additional loss of descending cortical and bulbar neurons.

Copyright 2002 Elsevier Science Ireland Ltd.

PMID: 12384220 [PubMed – indexed for MEDLINE]

J Neurol Sci. 1979 May;41(3):287-97.

Morphometric quantification of the cervical limb motor cells in controls and in amyotrophic lateral sclerosis.

Tsukagoshi H, Yanagisawa N, Oguchi K, Nagashima K, Murakami T.

The limb motor cells of the C6 segment of the spinal cord were counted and correlated with quantified histological findings of biceps brachii muscles in controls and in cases of amyotrophic lateral sclerosis (ALS). In 12 controls the motor cells were divided into larger ones with a minimum diameter greater than 20 micron, and smaller cells. Total numbers of the larger motor cells decreased significantly in 11 of 12 cases of ALS and of the smaller cells in 4 cases. In 4 controls most of the constituents of the biceps brachii muscle were normal-sized fibers, while in ALS smaller fibers and interstitial connective tissue increased and hypertrophic fibers decreased in association with a decrease of normal-sized fibers. The correlation coefficients between total numbers of the larger or smaller motor cells and normal-sized fibers in ALS were 0.92 and 0.65 respectively, and the larger motor cells, correlating with muscular atrophy of the upper arm, were considered to be alpha motor cells. Although in ALS the larger motor cells decreased almost diffusely, there were segmental variations, similar to controls, in numbers of the motor cells per 500 micron thickness.

PMID: 438856 [PubMed – indexed for MEDLINE]

Acta Neuropathol (Berl). 1989;77(4):343-9.

Evidence for sequential degeneration of the neurons in the intermediate zone of the spinal cord in amyotrophic lateral sclerosis: a topographic and quantitative investigation.

Oyanagi K, Ikuta F, Horikawa Y.

Center for Materials of Brain Diseases, Niigata University, Japan.

To elucidate the degenerating mechanism of the neurons in the intermediate zone of the spinal cord in classical amyotrophic lateral sclerosis (ALS), the spinal neurons in a patient with ALS, whose muscular strength was fairly well preserved up to death, were examined quantitatively and topographically, and compared with the data of advanced ALS patients and age-matched control subjects reported previously. In advanced ALS patients, anterior horn cells completely disappeared and the medium-sized (nuclear area; 71-150 microns 2) and large (nuclear area; greater than 151 microns 2) neurons in the intermediate zone were severely reduced. In the present case, however, the loss of anterior horn cells was severe but the degree was not equal to that of advanced ALS patients, and the neurons in the intermediate zone were quite well preserved. The finding indicates that the primary degeneration may occur in the anterior horn cells and the neurons in the intermediate zone degenerate sequentially in the spinal gray matter in ALS.

PMID: 2711828 [PubMed – indexed for MEDLINE]

Neurology. 1987 Mar;37(3):529-32.

Size-dependent myelinated fiber loss in the corticospinal tract in Shy-Drager syndrome and amyotrophic lateral sclerosis.

Sobue G, Hashizume Y, Mitsuma T, Takahashi A.

Morphometric evaluation was performed on myelinated fibers of the corticospinal tract at the seventh thoracic spinal cord segment from three patients with Shy-Drager syndrome (SDS), six patients with amyotrophic lateral sclerosis (ALS), and five patients with nonneurologic symptoms. In SDS, small-sized myelinated fibers were nearly completely depleted, while large-sized myelinated fibers were considerably well preserved. In ALS, on the contrary, large myelinated fibers were predominantly decreased. These results suggested that selective vulnerability of axonal loss depends on fiber size and should be considered in interpretation of pathology of corticospinal tracts.

PMID: 3822153 [PubMed – indexed for MEDLINE]

J Neuropathol Exp Neurol. 1994 May;53(3):221-30.

Neurofilament light and polyadenylated mRNA levels are decreased in amyotrophic lateral sclerosis motor neurons.

Bergeron C, Beric-Maskarel K, Muntasser S, Weyer L, Somerville MJ, Percy ME.

Centre for Research in Neurodegenerative Diseases, University of Toronto, Canada.

The presence of large neurofilamentous accumulations in the perikaryon and proximal axon of motor neurons in amyotrophic lateral sclerosis (ALS) suggests that the expression of this abundant cytoskeletal protein may be altered. We performed quantitative in situ hybridization for the low molecular weight neurofilament subunit (NF-L) messenger RNA in six cases of sporadic ALS and six controls. We found a 41% decrease (p < 0.02) in the NF-L mRNA levels in anterior horn cells in ALS, with a 60% decrease (p < or = 0.01) in alpha motor neurons. This alteration may represent a non-specific response to axonal or neuronal injury or, alternatively, reflect the regenerative activity of residual normal motor neurons. NF-L mRNA levels were consistently low (in the third and fourth quartiles) in spheroid-bearing motor neurons, indicating that the neurofilamentous accumulations observed in ALS are not likely the result of overexpression of the NF-L gene. Total neuronal polyadenylated mRNA levels were also 50% lower (p = 0.02) in anterior horn cells and 48% lower (p < or = 0.05) in alpha motor neurons in ALS, possibly reflecting a decrease in selected mRNA species in diseased motor neurons.

PMID: 7909836 [PubMed – indexed for MEDLINE]

Ann Neurol. 1992 Mar;31(3):305-10.

Collagen cross-linking of skin in patients with amyotrophic lateral sclerosis.

Ono S, Yamauchi M.

Dental Research Center, University of North Carolina 27599-7455. Collagen cross-links of skin tissue (left upper arm) from 11 patients with amyotrophic lateral sclerosis (ALS) and 9 age-matched control subjects were quantified. It was found that patients with ALS had a significant reduction in the content of an age-related, stable cross-link, histidinohydroxylysinonorleucine, that was negatively correlated with the duration of illness. The contents of sodium borohydride-reducible labile cross-links, dehydro-hydroxylysinonorleucine and dehydro-histidinohydroxymerodesmosine, were significantly increased and were positively associated with the duration of illness (r = 0.703, p less than 0.05 and r = 0.684, p less than 0.05, respectively). The results clearly indicate that during the course of ALS, the cross-linking pathway of skin collagen runs counter to its normal aging, resulting in a “rejuvenation” phenomenon of skin collagen. Thus, cross-linking of skin collagen is affected in ALS.

PMID: 1637138 [PubMed – indexed for MEDLINE]

Neurology. 1998 Jul;51(1):114-20.

Decreased type IV collagen of skin and serum in patients with amyotrophic lateral sclerosis.

Ono S, Imai T, Takahashi K, Jinnai K, Yamano T, Nagao K, Shimizu N, Yamauchi M.

Department of Neurology, Teikyo University School of Medicine, Ichihara Hospital, Chiba, Japan.

OBJECTIVE: To study type IV collagen of skin and serum in patients with ALS. BACKGROUND: Collagen abnormalities of skin have been reported in ALS patients. However, little is known concerning type IV collagen in ALS. METHODS: We studied type IV collagen immunoreactivity of skin and measured serum levels of the 7S fragment of the N-terminal domain of type IV collagen (7S collagen) in patients with ALS and control subjects. RESULTS: The basement membrane as well as blood vessels of skin in ALS patients was weakly positive for type IV collagen as compared with those of diseased control subjects. This weak immunostaining became more pronounced as ALS progressed. The optical density for type IV collagen immunoreactivity in ALS patients was significantly lower (p < 0.001) than in diseased control subjects and was significantly decreased with duration of illness (r = -0.85, p < 0.01). Serum 7S collagen levels in patients with ALS were significantly decreased (p < 0.01) as compared with those in diseased and healthy control subjects and were negatively and significantly associated with duration of illness (r = -0.81, p < 0.001). There was an appreciable positive correlation between concentrations of serum 7S collagen and the density for type IV collagen immunoreactivity in ALS patients (r = 0.81, p < 0.02). CONCLUSIONS: These data suggest that a metabolic alteration of type IV collagen may take place in the skin of ALS patients and that the decreased levels of serum 7S collagen may reflect a decreased type IV collagen immunoreactivity of skin in patients with ALS.

PMID: 9674788 [PubMed – indexed for MEDLINE]

Cell. 1980 Jul;20(3):849-57.

The spatial distribution of polyribosomes in 3T3 cells and the associated assembly of proteins into the skeletal framework.

Fulton AB, Wan KM, Penman S.

Acridine fluorescence reveals polyribosomes in fibroblasts and Triton-extracted skeletal frameworks; simultaneous phase-contrast images show cellular structure. Polyribosomes appear near nuclei of both intact fibroblasts and skeletal frameworks. Simultaneous autoradiography of cells so examined locates radioactive proteins. After synthesis, most proteins diffuse rapidly through the cytoplasm; intact cells autoradiographed afer a 35S pulse show grains throughout. In sharp contrast, extraction with Triton leaves only radioactive skeletal proteins, which, although released from ribosomes, are near polyribosomes after a pulse. After a chase, skeletal-associated radioactivity is found throughout the framework structure. However, skeletal proteins migrate only if protein synthesis continues. Emetine administered following a pulse block protein migration; skeletal framework radioactivity remains near polyribosomes. This also indicates limited exchange between skeletal framework and soluble cytoplasmic proteins. The fact that proteins insert themselves into the skeletal framework at or near their synthesis site, with limited subsequent exchange, appears to contradict current view of protein self-assembly.

PMID: 6893425 [PubMed – indexed for MEDLINE]

J Comp Neurol. 1980 Sep 1;193(1):165-77.

Axon reaction in dorsal motor vagal and hypoglossal neurons of the adult rat. Light microscopy and RNA-cytochemistry.

Aldskogius H, Barron KD, Regal R.

Qualitative light microscopical observations, morphometric measurements, and cytophotometric values for nucleolar and cytoplasmic RNA were compared in axotomized rat dorsal motor vagal and hypoglossal neurons. These data were correlated with consective cell counts and examination of the peripheral nerves. Vagal neurons showed an early prominent chromatolysis, later accompanied by increased cytoplasmic basophilia. Morphometric data showed a transient slight cytoplasmic enlargement but no nucleolar hypertrophy. Nucleolar RNA was unchanged, but cytoplasmic RNA was elevated 7 to 84 days postoperatively. Cell counts demonstrated a final cell loss of about 70%. Hypoglossal neurons showed a moderate chromatolysis. Nucleolar and cytoplasmic areas were enlarged for a short period. Postoperatively, nucleolar and cytoplasmic RNA were elevated about 3 to 14 days and about 3 to 28 days, respectively. Cell counts demonstrated a loss of 25% at the longest postoperative survival period. The results indicate that axotomized adult mammalian extrinsic neurons–even those destined to die–accumulate RNA. This response contrasts with axon reaction in many axotomized mammalian intrinsic neurons which appear to undergo depletion of RNA.

PMID: 6159377 [PubMed – indexed for MEDLINE]

J Neurol Sci. 1984 Feb;63(2):241-50.

Neurofibrillary axonal swellings and amyotrophic lateral sclerosis.

Delisle MB, Carpenter S.

A series of 22 cases of amyotrophic lateral sclerosis (ALS) and 22 controls have been assessed for the presence of neurofilamentous accumulations in axons and perikarya. Large axonal swellings were seen in the spinal cord of 12 controls and of 13 ALS cases. When they were present in ALS cases they tended to be much more numerous than in controls, and when they were not present in ALS cases there tended to be severe neuronal loss in the cord. Axonal swelling on lower motor neurons appears to be a significant feature of the pathology of ALS. Its implications in terms of etiology are unknown.

PMID: 6538591 [PubMed – indexed for MEDLINE]

Nat Neurosci. 1999 Jan;2(1):50-6.

Slowing of axonal transport is a very early event in the toxicity of ALS-linked SOD1 mutants to motor neurons.

Williamson TL, Cleveland DW.

Ludwig Institute for Cancer Research, University of California, San Diego, La Jolla 92093, USA.

Mutations in copper/zinc superoxide dismutase 1 (SOD1), primary causes of human amyotrophic lateral sclerosis (ALS), provoke motor neuron death through an unidentified toxic property. The known neurofilament-dependent slowing of axonal transport, combined with the prominent misaccumulation of neurofilaments in ALS, suggests that an important aspect of toxicity may arise from damage to transport. Here we verify this hypothesis for two SOD1 mutations linked to familial ALS. Reduced transport of selective cargoes of slow transport, especially tubulin, arises months before neurodegeneration. For one mutant, this represents the earliest detectable abnormality. Thus, damage to the cargoes or machinery of slow transport is an early feature of toxicity mediated by mutant SOD1.

PMID: 10195180 [PubMed – indexed for MEDLINE]

J Neurol. 1999 Jun;246(6):438-42.

Evolution of motor and sensory deficits in amyotrophic lateral sclerosis estimated by neurophysiological techniques.

Theys PA, Peeters E, Robberecht W.

Department of Neurology, University Hospital Gasthuisberg, Leuven, Belgium.

Although amyotrophic lateral sclerosis is a degenerative disease of the upper and lower motor neurons, there is evidence that the disease can affect other systems, including the sensory system. On the other hand, within the motor neuron pool there is possibly a predilection of the degenerative process for the motor neurons fibers with the fastest conduction velocity (MNFCV). We studied these two aspects of the disease in a group of 50 patients by prospectively assessing several sensory indices and by studying the selectivity of the spinal motor neuron loss. At baseline, nerve conduction studies and somatosensory evoked potentials showed abnormal slowing in the peripheral and central sensory pathways. Thermal thresholds for heating were elevated but were normal for cooling. In more than 60% of the patients at least one of the sensory tests studied was abnormal. However, except for a significant decrease in the amplitude of the sensory nerve action potentials of the sural nerves, these afferent dysfunctions were not progressive over the follow-up period of 6 months, in contrast to the marked deterioration in motor functions. Three different statistical models were applied to evaluate the presence of demyelination, selective loss of MNFCV, or the purely random degeneration of fast- and slow-conducting motor neurons. These data indicate a selective loss of the MNFCV and suggest that subclinical abnormalities of the sensory system in ALS are often present but almost nonprogressive. Furthermore, the amyotrophic lateral sclerosis disease process seems preferentially to affect MNFCV.

PMID: 10431767 [PubMed – indexed for MEDLINE]

Muscle Nerve. 1991 Dec;14(12):1242-6.

Abnormalities in the sensory action potential in patients with amyotrophic lateral sclerosis.

Shefner JM, Tyler HR, Krarup C. Division of Neurology, Brigham and Women’s Hospital, Boston, MA 02115.

Sensory function in patients with amyotrophic lateral sclerosis (ALS) is thought to be normal; however, there is convincing morphologic evidence that sensory systems are affected in addition to motor systems. In this study, compound sensory action potentials were recorded with near nerve electrodes from 18 patients with ALS. Up to 1024 responses were averaged at high gain to determine minimum conduction velocity; that is, the conduction velocity of the slowest conducting component of the sensory action potential. Nine of 18 patients had abnormally reduced minimum conduction velocity, even when peak-to-peak amplitude and maximum conduction velocity (calculated from the latency to the initial positive peak) were normal. Only 3 of 18 patients showed abnormalities in peak-to-peak amplitude. Thus, subtle abnormalities in the sensory action potential can be detected in many patients with ALS.

PMID: 1766456 [PubMed – indexed for MEDLINE]

Neurology. 1986 Jun;36(6):796-801.

Abnormal sensory evoked potentials in amyotrophic lateral sclerosis.

Radtke RA, Erwin A, Erwin CW.

We have reviewed sensory evoked potential (EP) findings in 17 patients with amyotrophic lateral sclerosis (ALS). Somatosensory EPs were abnormal in 7 of 16 patients after lower-extremity stimulation and in 2 of 16 patients after upper-extremity stimulation. Brainstem auditory EP abnormalities were found in 2 of 12 patients. No abnormalities were noted on pattern reversal visual EPs in 12 patients. Overall, 47% of all ALS patients studied had at least one EP abnormality. EP evidence of CNS sensory dysfunction in ALS is more frequent than that noted clinically or pathologically and offers further support to previous observations of sensory system involvement in ALS.

PMID: 3703285 [PubMed – indexed for MEDLINE]

Endocrinol Metab Clin North Am. 1987 Dec;16(4):995-1011.

Insulin-like growth factors and aging.

Hammerman MR.

Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri.

Since its proposal three decades ago, the evidence in favor of the somatomedin hypothesis has been compelling. It is clear that somatotrophic actions of growth hormone are mediated through generation of insulin-like peptides and interaction of these peptides with plasma membrane receptors on sensitive cells. It is possible that such actions result from effects of circulating insulin-like peptides and/or insulin-like peptides generated in proximity to their sites of action (autocrine or paracrine effects). Most or all of circulating somatomedin activity in humans can be accounted for by insulin-like growth factors I and II (IFGs I and II). These peptides have considerable structural homology with insulin but, unlike insulin, they circulate in tight, noncovalent association with specific carrier protein. Levels of circulating IGF I and IGF II are affected by growth hormone, but the former peptide is the more sensitive to growth hormone. Levels of circulating IGF I in humans are low at birth, rise progressively during childhood, and peak during midadolescence. The increase in stature that occurs normally during adolescence probably results from this increase in circulating IGF I. Following adolescence, levels of circulating IGF I fall progressively as a function of age. There is good evidence that the reduction in levels of circulating IGF I is related to decreased secretion of growth hormone that accompanies aging. Although it has been suggested that decreased function of the growth hormone-somatomedin axis may cause changes in anabolic indices that accompany the aging process, definitive proof for this hypothesis is lacking. In contrast to IGF I, circulating IGF II reaches “adult” levels early in childhood, and changes are relatively small as a function of increasing age. Counterparts of IGF I and IGF II are present in rats. Dynamics of the growth hormone-somatomedin axis are similar in rats and humans for IGF I. In contrast, levels of IGF II in rat fall precipitously following birth, suggesting a role for rat IGF II in fetal growth and development. The rat has been used as an experimental animal to define the role of the growth hormone-somatomedin axis in aging. As in human studies, no firm relationship between somatomedins and aging has been established in the rat.

PMID: 3322823 [PubMed – indexed for MEDLINE]

J Biol Chem. 1994 Jun 3;269(22):15892-7.

Erratum in:
J Biol Chem 1994 Aug 12;269(32):20806.

In vitro and in vivo studies of antagonistic effects of human growth hormone analogs.

Chen WY, Chen NY, Yun J, Wagner TE, Kopchick JJ.

Department of Biological Sciences, Ohio University, Athens 45701-2979.

A bovine growth hormone (bGH) analog, bGH-G119R, has been shown to act as a unctional antagonist of GH activity both in vitro and in vivo. In the present study, human (h) GH analogs with alterations in the third alpha-helix (G120A, G120R) and N terminus (I4A) were generated. These two regions have been reported to form binding site 2 in hGH, which is involved in in vitro dimerization of the GH receptor (GHR). The biological activities of these hGH analogs were tested in vitro by a radioreceptor assay and an assay to test the ability of the molecules to induce tyrosine phosphorylation of a 93-kDa protein in a human lymphocyte line, IM-9. The growth rate of transgenic mice that express different hGH analogs was used as an in vivo test of the activity of the molecules. The results demonstrated that G120R is a potent hGH antagonist both in vitro and in vivo. Transgenic mice expressing G120R demonstrated a growth-suppressed phenotype. However, I4A, which has been demonstrated to be a potent inhibitor of in vitro GHR dimerization, exhibited full growth promoting activity in transgenic mice. Thus, the ability of hGH analogs to induce GHR dimerization in vitro and the ability to promote growth in vivo are not directly correlated.

PMID: 8195244 [PubMed – indexed for MEDLINE]

Spine. 1994 Sep 15;19(18):2077-81.

Morphometry of the normal cadaveric cervical spinal cord.

Kameyama T, Hashizume Y, Ando T, Takahashi A.

Department of Neurology, Nagoya University School of Medicine, Japan.

STUDY DESIGN. The authors measured the transverse area and diameters of the cadaveric cervical spinal without evidence of nervous system pathology and also investigated factors related to spinal cord size. OBJECTIVES. This study attempted to provide basic data that affords an accurate morphometric analysis of the human cervical spinal cord. SUMMARY OF BACKGROUND DATA. Several postmortem morphometric studies of the spinal cord have been performed, but the measurements are different among the reports, and no authorized standard has been established. METHODS. The authors measured the transverse area and diameters of the C7 segment in 152 cadaveric specimens and investigated the cord size in relation to age, body height, body weight, and brain weight. Of these, 14 cases were selected and their segments from C2 to T1 were measured. RESULTS. There was a considerable individual variation in spinal cord size. The
transverse area of the C7 segment varied from 33.3 mm2 to 74.0 mm2 (mean 49.6 +/- 7.4 mm2). The cord size showed a strong correlation with brain weight, and to a lesser degree with age and body height. Although the size of the spinal cord varied from case to case, the relative ratio of the transverse area of each segment to that of the C3 segment was almost the same among the cases examined. CONCLUSIONS. This large variation in cord size should be taken under consideration in morphometric analyses of the spinal cord. When the pathologic cord conditions such as compression or atrophy exist, the normally expected transverse area of the affected segment in each individual is calculable from measurement of a given single normal segment.

PMID: 7825049 [PubMed – indexed for MEDLINE]

J. Neuropathol. Exp. Neurol. 1949 8: 400-410.

Selective Destruction of Large Motoneurons by Poliomyelitis Virus.

Hodes, R., Peacock, SM, Jr., Bodian, D.

1. The size distribution has been studied of the motoneurons of the anterior horns of 6 normal monkeys and of 3 animals in the chronic stage of experimentally induced poliomyelitis.

2. Cords from infected animals show a greater proportion of small neurons and a smaller proportion of large cells than do normal cords.

3. The relative preponderance of small motoneurons and th paucity of large nerve cells increase pari passu with the severity of the lesion.

4. The abundance of small motor nerve cells and the scarcity of large motoneurons is due to the fact that the virus of poliomyelitis first singles out for destruction the large motoneurons of the population and leaves relatively intact the small cells. As the disease process increases in intensity small motoneurons are also destroyed.

5. The data presented are discussed in the light of some aspects of the pathological physiology of this disease.

J. Comp. Neurol 1902 12: 125-182.

Observations on the medulla spinalis of the elephant with some comparative studies of the intumescentia cervicalis and the neurones of the columna anterior.

Hardesty, I.

Brain Res. 1974 Mar 29;69(1):182-7.

Localization of the acetylcholinesterase-containing membranes in purified myelin fractions.

McIlwain DL.

PMID: 4817912 [PubMed – indexed for MEDLINE]

Biochim Biophys Acta. 1974 Jan 23;337(1):68-78.

The activity of pure phospholipase A2 from Crotalus atrox venom on myelin and on pure phospholipids.

Coles E, Mcilwain DL, Rapport MM.

1. Pure phospholipase A2 was prepared from Crotalus atrox venom, in good yield, by DEAE-cellulose chromatography and preparative acrylamide gel electrophoresis.

2. The hydrolytic activity of the enzyme on different substrates was determined in aqueous and diethyl ether media. In the diethyl ether medium, the relative rates of hydrolysis were phosphatdylcholine>phosphatidalcholine>phosphatidylserine=phosphatidalethanolamine> phosphatidylethanolamine, whereas in the aqueous medium, the relative rates were phosphatdylcholine>phosphatidylethanolamine> phosphatidylserine>phosphatidalethanolamine>phosphatidalcholine.

3. In its action on central nervous system myelin the phospholipase showed a preference for phosphatidylethanolamine over phosphatidalethanolamine. The relative rates of hydrolysis of phosphoglycerides in the membrane were phosphatidylserine> phosphatidylcholine>ethanolamine phosphoglycerides. The products (fatty acid and lysophsphatide) remained associated with the membrane after enzyme action.

PMID: 4433546 [PubMed – indexed for MEDLINE]

Brain Res. 1973 Mar 30;52:97-113.

Non-osmotic swelling in purified bovine myelin.

McIlwain DL.

Purified, lyophilized bovine myelin in aqueous suspension exhibits non-osmotic swelling which is influenced by the pH, ionic strength and temperature of the bulk medium. Trypsin, chymotrypsin, papain or pronase can initiate swelling, as observed by phase contrast microscopy and tubidimetry. Swollen myelin is mechanically fragile and forms protein-deficient vesicles, some of which are not sedimented in water at 100,00 x g for 1h. The swelling process is similar to changes reported for myelinated nerves in several experimental and paathological states and resembles two types of swelling known to occur in unilamellar structures.

PMID: 4573430 [PubMed – indexed for MEDLINE]

J Neurochem. 1971 Dec;18(12):2255-63.

Membrane fragments from myelin treated with different detergents.

McIlwain DL, Graf L, Rapport MM.

Bovine CNS myelin treated with cationic, anionic and non-ionic detergents yielded fragments which did not sediment in water at 56,500 g for 15 min. Substantial chemical and immunological differences were observed among the myelin fragments produced by these detergents. The chemical and immunological characteristics of such membrane products were also depenent upon the concentration of detergent. These and similar findings with other biological membranes emphasize the difficulties in using detergents to obtain and examine proposed subunit structures.

PMID: 5167511 [PubMed – indexed for MEDLINE]

Biochim Biophys Acta. 1971 Jun 8;239(1):71-80.

The effects of phospholipase C (Clostridium perfringens) on purified myelin.

McIlwain DL, Rapport MM.

Fragments of purified myelin, treated with crude phospholipase C (EC 3.1.4.3) from Clostridium perfringens, lost an average of 64% of the lipid-P content, comprising 81% of the sphingomyelin, 92% of the choline phosphoglyceride, 71% of the ethanolamine phosphoglyceride and 14% of the serine phosphoglyceride. The enzyme was capable of penetrating the multi-layered membranes of the myelin fragments. Small quantities of the enzyme were detectable in the treated fragments. At 5 C, substantial quantities of phospholipase C activity became bound to the myelin without showing any hydrolytic action. Most of this enzyme as released into solution on incubation at 38 C for 1h. The ultrastructural morphology of myelin changed little after phospholipase C treatment, except for the appearance of large globules embedded in the membranes. The serological activity of myelin was not appreciably affected by enzyme treatment.

PMID: 4105605 [PubMed – indexed for MEDLINE]

Biochemistry 1966 5: 4054-4061

Characterization of the phospholipids in Pinus ponderosa pollen.

McIlwain, DL, Ballou, CE

Studies on the phospholipids extracted from Ponderosa pine pollen have established the presence of the following: phosphatidylcholine, phosphatidylethanolaine, phosphatidylglycerol, phosphatidylmyoinositol, phosphatidylserine, and bisphosphatidylglycerol. Identification of these lipids was based on group analysis of each component produced by acid and basic hydrolysis, and on chromatographic comparison of each intact phosopholipid with known standards. Palmitic, oleic and linoleic acids are the major fatty acids of each phospholipid.

Brain Res. 1980 May 26;190(2):505-16.

Preferential blockade of the tubulin transport by colchicine.

Komiya Y, Kurokawa M.

L-[35S]Methionine was injected into the dorsal root ganglion (L5) of the adult rat, and migration of the neurofilament polypeptides (the triplet with molecular weights of 200,000, 160,000 and 68,000 daltons), alpha- and beta-tubulins and actin in the sciatic nerve and the dorsal root was quantitatively determined and also examined by fluorography. Colchicine (4 microgram) injected into the ganglion 10 min before methionine preferentially blocked the tubulin transport, with little if any blockade of the triplet and actin. Colchicine at this dose had no effects on the incorporation of L-[14C]leucine into the total protein and also into tubulins. In contrast to colchicine, vinblastine sulphate (4 microgram) injected into the ganglion in a similar way blocked the transport of all thetriplet, tubulins and actin. Cytochalasin D (1 microgram) had no effect on the slow axoplasmic transport.

PMID: 6154510 [PubMed – indexed for MEDLINE]

Proc. Natl. Acad. Sci 1967 57:1239-1245.

Neuronal dynamics and axonal flow, III. Cellufugal transport of labeled neuroplasm in isolated nerve preparations.

Weiss P

PMID: 5231729 [PubMed – indexed for MEDLINE]

FEBS Letters 1979 106: 305-308

Identification and differential distribution of collagen types in the central and peripheral nervous systems.

Shellswell GB, Restall DJ, Duance VC, Bailey AJ

PMID: 387447 [PubMed – indexed for MEDLINE]

J Comp Neurol. 1998 Feb 2;391(1):64-77.

Time course of neuropathology in the spinal cord of G86R superoxide dismutase transgenic mice.

Morrison BM, Janssen WG, Gordon JW, Morrison JH.

Neurobiology of Aging Laboratories, Mount Sinai School of Medicine, New York, New York 10029, USA.

Transgenic mice with a G86R mutation in the mouse superoxide dismutase (SOD-1) gene, which corresponds to a mutation observed in familial amyotrophic lateral sclerosis (ALS), display progressive motor dysfunction leading to paralysis and premature death. In endstage SOD-1 transgenic mice, there is marked loss of spinal motor neurons and interneurons, accumulation of phosphorylated neurofilament inclusions, and reactive astrocytosis. The present study details the time course and ultrastructural appearance of these pathologic changes and correlates the timing of these events with the behavioral symptoms. There is no significant reduction in the number of total neurons, motor neurons, or interneurons in the ventral spinal cord of presymptomatic mice, as compared to age-matched control mice. In contrast, there is a significant reduction in the number of total neurons (-23.5%), motor neurons (-28.9%), and interneurons (-23.5%) in symptomatic SOD-1 transgenic mice. This neuron loss correlates temporally with the onset of reactive astrocytosis and the appearance of phosphorylated neurofilament inclusions. The identical timing of motor neuron and interneuron degeneration in this model of ALS strongly suggests that degeneration in the spinal cord of patients with ALS is not specifically directed at motor neurons, but rather more generally at several populations of neurons in the spinal cord. In addition, the late onset and rapid progression of neuron loss suggest that a toxic property is accumulating while the SOD-1 transgenic mice are presymptomatic, and that this toxic property must reach a threshold level before the onset of neuronal degeneration.

PMID: 9527542 [PubMed – indexed for MEDLINE]