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

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.

Neuron addition and enlargement in juvenile and adult animals.

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.

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

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.

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

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.

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

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.

Transcription and motoneuron size.

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.

Radiolabeling motoneuron proteins in the isolated frog spinal cord preparation.

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.

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

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.

Allocation of newly synthesized proteins in spinal motoneurons.

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, Kreutzberg and Lux (Brain Res. 47, 331-343, 1972), the above data strengthen the possibility that substantial amounts of the new protein made by a motoneuron may be committed to its dendrites.

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

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.

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

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.

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

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.

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

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.

Acetylcholinesterase distribution in axotomized frog motoneurons.

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.

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

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.

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

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.

The plasma membrane of bulk-isolated mature spinal neurons.

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 studies29 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.