Distribution of GFAP+ astrocytes in adult and neonatal rat brain.

Astrocytes can proliferate as a result of trauma to the brain, such as occurs in a variety of diseases. Understanding the normal distribution of astrocytes is necessary before the extent of astrogliosis can be clearly determined. However, little is known about the normal distribution of GFAP+ astrocytes especially during development. This study examined distribution of GFAP+ astrocytes in regions of the cortex, cerebellum, and brainstem of adult and rat pup brains by immunocytochemistry using antibodies against GFAP. The findings showed a differential distribution of GFAP+ astrocytes in the rat brain. A paucity of GFAP expression was found in most regions of the normal adult rat brainstem, whereas GFAP+ astrocytes were abundantly distributed in all areas of the cortex and cerebellum. A similar regional heterogeneity in the distribution of GFAP+ astrocytes was seen in the neonatal rat brain. These findings suggest that the development of the differential pattern of GFAP+ astrocytes seen in the rat brain does not occur postnatally, but instead is present at birth and appears to be determined during fetal development.

Modulation of microglial form and immune function by factors released from goldfish optic nerves.

Activation of microglia is associated with neural damage and may aid repair of the CNS. To begin to investigate their role, microglia purified from mouse brain were grown in media conditioned (CM) by goldfish optic nerve (GFON), optic tectum (GFOT), vagal lobe, telencephalon and cerebellum, and medium conditioned by rat optic nerves (RON). Microglia maintained in GFON- or GFOT-CM assumed an ameboid morphology, whereas microglia grown in media conditioned by the other neural tissues produced long, crenellated processes that resembled the ramified microglial form. Microglia maintained in all types of CM functioned as antigen presenting cells in a MHC-restricted manner when tested on conalbumin-specific Thelper (Th) cells, except for microglia maintained in GFON- and GFOT-CM. These studies suggest that GFON, in contrast to RON, produces a substance(s) that affects microglial morphology and immune reactivity, and may promote the vigorous regeneration seen in GFON after damage.

Fetal hemoglobin levels in sudden infant death syndrome.

OBJECTIVE: The aim of this study was to determine and compare fetal hemoglobin levels from infants dying of the sudden infant death syndrome (SIDS) with aged-matched control infants dying of other causes. Similar previous studies have reported both elevated and normal levels of fetal hemoglobin in whole blood samples from infants dying of SIDS. DESIGN: Triton-acid-urea gel electrophoresis and densitometry were used to determine fetal hemoglobin levels in postmortem whole blood samples from infants dying of SIDS and from appropriately age-matched control infants. Whole blood samples were analyzed blindly and matched for postgestational age. Infant ages at death ranged from birth to less than 1 year. MAIN OUTCOME MEASURES: Fetal hemoglobin in whole blood from infants dying of SIDS and control infants. RESULTS: During the period of postnatal development most associated with SIDS cases (2 to 6 months after birth), fetal hemoglobin levels were found to be significantly elevated in postmortem whole blood samples from SIDS infants compared with gestational age-matched control infants dying of causes other than SIDS. CONCLUSION: We conclude that levels of fetal hemoglobin are elevated in postmortem whole blood of SIDS infants compared with controls. Furthermore, the apparent conflict in the literature regarding fetal hemoglobin levels in SIDS infants and controls is most likely due to variability in the control data of some studies.

Differential regulation of fast axonally transported proteins during the early response of rat retinal ganglion cells to axotomy.

We have found that the early response of axotomized rat retinal ganglion cells is characterized by the differential regulation of a number of fast axonally transported proteins. The abundance of 23 radiolabeled fast transported proteins was analyzed at 2 and 5 days after axotomy using two-dimensional gel electrophoresis. Corresponding changes in retinal GAP-43 mRNA were measured using northern analysis. Within 2 days of injury, > 40% of the transported proteins analyzed, including GAP-43, showed increased labeling above control levels. Approximately 13% of transported proteins decreased below control levels, whereas the remainder did not change. Five days after axotomy, only GAP-43 and another fast transported protein, C3, continued to sustain measurable increased labeling above control levels; all previously elevated proteins appeared to have been down-regulated by this time, which corresponds to the onset of cell death. These differential changes were accompanied by parallel increases in GAP-43 mRNA. These results suggest that the molecular changes within rat retinal ganglion cells are differentially regulated within two stages subsequent to damage, initial regenerative growth followed by cell death.

Sudden infant death syndrome: a theory.

A hypothesis, and supporting evidence, is presented for the sudden infant death syndrome (SIDS). Our model is as follows. Fetal hemoglobin levels are abnormally elevated in SIDS infants, which contributes to a state of chronic hypoxia. Chronic hypoxia produces pronounced depressive effects on the respiratory system during slow wave sleep (a state of normal respiratory depression). These depressive effects are particularly manifest during that period of development in which slow wave sleep begins to occupy a very large percentage of total sleep time in infants -2-4 mo of age. A downward spiral is initiated in slow wave sleep such that hypoxia-induced decreases in ventilation produce more extreme hypoxia leading to further respiratory depression and ultimately death due to a cessation of respiration.

Effect of target removal on goldfish optic nerve regeneration: analysis of fast axonally transported proteins.

How is axonal transport in regenerating neurons affected by contact with their synaptic target? We investigated whether removing the target (homotopic) lobe of the goldfish optic tectum altered the incorporation of 3H-proline into fast axonally transported proteins in the regenerating optic nerve. Regeneration was induced either by an optic tract lesion (to reveal the changes in the original axon segment that remained connected to the cell body) or by an optic nerve lesion (to reveal the changes in the newly formed axon segment). Of 26 proteins analyzed by 2-dimensional gel electrophoresis and fluorography, all but one showed increased labeling as a result of tectal lobe ablation. By 2 d after the lesion, significantly increased labeling of some proteins was seen with a 6-hr labeling interval, but not with a 24-hr labeling interval. This is probably indicative of an increased velocity of transport, which may have been a nonspecific consequence of the surgery. Otherwise, tectal lobe removal had relatively little effect until 3 weeks, when there was a transitory increase in labeling of transported proteins in the new axon segments of the tectum-ablated animals. Beginning at 5 weeks, tectal lobe ablation caused considerably higher labeling of many of the proteins in the original axon segments. Because this was seen with both 6-hr and 24-hr labeling intervals, it is probably indicative of increased protein synthesis. The increased synthesis lasted until at least 12 weeks, though some proteins were beginning to show a diminished effect at this time. In the late stages of regeneration (8-12 weeks), there was also increased labeling of proteins in the new axon segments as a result of the absence of the target tectal lobe. This included a disproportionately large increase in the relative contribution of cytoskeletal proteins and of protein 4, which is the goldfish equivalent of the growth-associated protein GAP-43 (neuromodulin). We conclude that, after the regenerating axons begin to innervate the tectum, the expression of most of the proteins in fast axonal transport is down-regulated by interaction between the axons and their target. However, the changes in expression may be preceded by a modulation of the turnover and/or deposition of proteins in the newly formed axon segment.

Some hypotheses concerning axon regeneration.

We review evidence related to several hypotheses concerning the mechanism of axon regeneration and present new data addressing one hypothesis. That one hypothesis concerns the signal that initiates changes in the cell bodies of neurons after axon damage. We identify a molecule that has a number of the properties expected of such a signal. We also review the hypothesis that induction of some genes is tightly correlated with nerve regeneration, and conclude that such a correlation is not so 'tight'. Nevertheless, proteins whose rate of synthesis or transport is increased in some systems are good candidates for playing important roles in regrowth. A third hypothesis, that mammalian CNS neurons fail to regenerate because of a failure to induce growth-associated proteins, is probably not true. Growth-associated proteins appear to be induced, at least transiently, in some cases where regeneration is abortive. The state of the neuron undoubtedly is important in regeneration, but many neurons, even in the CNS, appear to be able to support axon regrowth given the proper environment. Thus, support seems stronger for the view that the environment at the site of damage (including surfaces and growth factors) determines whether significant regrowth occurs in most cases.

A comparison of fluorographic methods for the detection of 35S-labeled proteins in polyacrylamide gels.

Eight different methods of fluorographic enhancement of sensitivity to 35S decay after gel electrophoresis were compared. Using Kodak X-Omat AR X-ray film, we found that some fluors were about equivalent to 2,5-diphenyloxazole/dimethyl sulfoxide embedding, whereas several other fluors were not quite as effective, but still were significantly more sensitive than control autoradiography. The most sensitive procedures can yield a detectable darkening of film with less than 1 dpm/mm2 of 35S after a 1-week exposure.

Effects of a conditioning lesion on bullfrog sciatic nerve regeneration: analysis of fast axonally transported proteins.

We have shown that bullfrog sciatic nerves respond to a conditioning lesion similarly to goldfish optic nerve and rat or mouse sciatic nerve; that is, following a crush the rate of regeneration is faster in nerves that have received a conditioning lesion compared to nerves that have not. Also, damaged nerve fibres show initial growth or sprouting earlier in a previously conditioned nerve compared to nerves that have not received a prior conditioning lesion. We have not detected changes in the transport of fast axonally transported proteins with the conditioning lesion paradigm, other than those changes seen in regenerating nerves after receiving a single lesion. However, more label was present in a few fast axonally transported proteins at the lesion site in conditioned nerves compared to non-conditioned nerves, and this difference is not apparently due to increased transport. It seems that changes in fast axonally transported proteins probably do not contribute directly to the mechanism underlying the conditioning lesion effect of higher out growth rates, although some of the fast transported proteins may be involved in functions, possibly at the growing tip of damaged fibres, which promote or result from the conditioning effect.

Fast axonally transported proteins in regenerating goldfish optic axons.

Fast axonal transport of protein was examined in regenerating goldfish optic axons after a lesion of either the optic tract or optic nerve, which revealed changes in the original intact optic axon segments or in the newly regenerated axon segments, respectively. In animals killed either 6 or 24 hr after injection of 3H-proline into the eye, labeling of total fast-transported protein in the original axon segments was increased by 2 d after the lesion, reached a peak of nearly 20 X normal at 2 weeks, and then declined to a level somewhat above normal at 12 weeks. When the labeling of individual transported proteins was examined by 2-dimensional gel electrophoresis, it was found that no new labeled proteins appeared during regeneration, but all proteins examined showed an increase in labeling. Among the various proteins, there was great variation in the magnitude and time course of the labeling increase. The largest increase, to nearly 200 X normal with 6 hr labeling, was seen in a protein with a molecular weight of 45 kDa and a pl of about 4.5, resembling a protein that has previously been designated a "growth-associated protein" (GAP-43; Skene and Willard, 1981a). The proteins showing increased labeling included a small fraction of cytoskeletal proteins (alpha-tubulin, beta-tubulin, and actin) that was apparently transported at a much faster rate than is usually expected of these constituents. In the new axon segments, the total protein labeling was increased by 1 week after the lesion, remained elevated at a nearly constant level of about 7 X normal from about 2 to 5 weeks, and then declined to levels somewhat above normal by 12 weeks. The 45 kDa protein again showed the largest increase, and became the single most prominently labeled constituent in the new axons. On the basis of the time course of labeling in both original and new axon segments during regeneration, the fast-transported proteins were tentatively separated into 5 classes that may represent groups of proteins that are coregulated during regeneration. They may conceivably correspond to different functional or structural entities within the neuron.

Fast axonally transported proteins in regenerating goldfish optic nerve: effect of abolishing electrophysiological activity with TTX.

Blocking neural activity with intraocular tetrodotoxin (TTX) hinders regeneration of goldfish optic axons, and prevents the refinement of the retinotopic map that is formed in the optic tectum. The latter effect is not observed with TTX treatment confined to the first two weeks of regeneration, but is produced when the TTX treatment is delayed until after this time. In the present study, 2-dimensional gel electrophoresis was used to analyse the effects of two different schedules of TTX treatment (0-9 days or 14-32 days) on incorporation of [3H]proline into individual proteins conveyed by fast axonal transport in the optic nerve. The labelling of many of these proteins was somewhat reduced by either schedule of TTX treatment, but a number of proteins showed a larger reduction as a result of the delayed treatment. These included some glycoproteins, as well as a protein of about 45 kDa and pI 4.5, which shows greatly increased synthesis during regeneration, and which is probably identical to the 'growth-associated protein' GAP-43. By contrast, cytoskeletal proteins (alpha- and beta-tubulin and actin) were unaffected by the delayed TTX treatment. It is possible that the differential effects of the early and delayed TTX treatments on various transported proteins may account for differences in the effect of these treatments on the retinotectal projection.

Changes in protein content of goldfish optic nerve during degeneration and regeneration following nerve crush.

After the goldfish optic nerve was crushed, the total amount of protein in the nerve decreased by about 45% within 1 week as the axons degenerated, began to recover between 2 and 5 weeks as axonal regeneration occurred, and had returned to nearly normal by 12 weeks. Corresponding changes in the relative amounts of some individual proteins were investigated by separating the proteins by two-dimensional gel electrophoresis and performing a quantitative analysis of the Coomassie Brilliant Blue staining patterns of the gels. In addition, labelling patterns showing incorporation of [3H]proline into individual proteins were examined to differentiate between locally synthesized proteins (presumably produced mainly by the glial cells) and axonal proteins carried by fast or slow axonal transport. Some prominent nerve proteins, ON1 and ON2 (50-55 kD, pI approximately 6), decreased to almost undetectable levels and then reappeared with a time course corresponding to the changes in total protein content of the nerve. Similar changes were seen in a protein we have designated NF (approximately 130 kD, pI approximately 5.2). These three proteins, which were labelled in association with slow axonal transport, may be neurofilament constituents. Large decreases following optic nerve crush were also seen in the relative amounts of alpha- and beta-tubulin, which suggests that they are localized mainly in the optic axons rather than the glial cells. Another group of proteins, W2, W3, and W4 (35-45 kD, pI 6.5-7.0), which showed a somewhat slower time course of disappearance and were intensely labelled in the local synthesis pattern, may be associated with myelin. A small number of proteins increased in relative amount following nerve crush. These included some, P1 and P2 (35-40 kD, pIs 6.1-6.2) and NT (approximately 50 kD, pI approximately 5.5), that appeared to be synthesized by the glial cells. Increases were also seen in one axonal protein, B (approximately 45 kD, pI approximately 4.5), that is carried by fast axonal transport, as well as in two axonal proteins, HA1 and HA2 (approximately 60 and 65 kD respectively, pIs 4.5-5.0), that are carried mainly by slow axonal transport. Other proteins, including actin, that showed no net changes in relative amount (but presumably changed in absolute amount in direct proportion to the changes in total protein content of the nerve), are apparently distributed in both the neuronal and nonneuronal compartments of the nerve.

Protein synthesis and rapid axonal transport during regrowth of dorsal root axons.

Damage to the sciatic nerve produces significant changes in the relative synthesis rates of some proteins in dorsal root ganglia and in the amounts of some fast axonally transported proteins in both the sciatic nerve and dorsal roots. We have now analyzed protein synthesis and axonal transport after cutting the other branch of dorsal root ganglia neurons, the dorsal roots. Two to three weeks after cutting the dorsal roots, [35S]methionine was used to label proteins in the dorsal root ganglia in vitro. Proteins synthesized in the dorsal root ganglia and transported along the sciatic nerve were analyzed on two-dimensional gels. All of the proteins previously observed to change after sciatic nerve damage were included in this study. No significant changes in proteins synthesized in dorsal root ganglia or rapidly transported along the sciatic nerve were detected. Axon regrowth from cut dorsal roots was observed by light and electron microscopy. Either the response to dorsal root damage is too small to be detected by our methods or changes in protein synthesis and fast axonal transport are not necessary for axon regrowth. When such changes do occur they may still aid in regrowth or be necessary for later stages in regeneration.

Production of monoclonal antibodies against calmodulin by in vitro immunization of spleen cells.

Monoclonal antibodies against the highly conserved ubiquitous calcium-binding protein, calmodulin (CaM), were produced by immunization of mouse primary spleen cell cultures. Dissociated spleen cells were cultured for 5 d in the presence of mixed thymocyte culture conditioned media (TCM) and purified bovine testes CaM (50 ng-1 mg). Following immunization, cells were fused with mouse myeloma cells (SP2/0, Ag 8.653) and cultured for 2-3 wk before initial screening for antibody. In five independent immunizations there was a range of 25-44% of the initial polyclonal cultures which produced antibodies reacting with purified CaM as determined by immunoassay. 80% of the cloned hybridoma produced IgM immunoglobulins while the remaining clones were IgG producers. This ratio was changed to 50% IgM and 50% IgG by subsequent extension of the in vitro immunization periods and reduced amounts of antigen and extended in vitro culturing. In vitro immunization introduces a new dimension to monoclonal antibody production where limited antigen or poorly antigenic proteins are of interest. The monoclonal antibodies produced in this study have enabled us to to selectively localize CaM in association with distinct subcellular structures, mitochondria, stress fibers, centrioles, and the mitotic spindle.

On the identification of alpha- and beta-tubulin subunits.

Confusion appears to have arisen in the literature regarding the designation of alpha- and beta-tubulin in polyacrylamide gels. The presence or absence of 8 M-urea in sodium dodecyl sulfate (SDS) polyacrylamide gels leads to different patterns for unalkylated tubulin subunits (and other proteins), making difficult the designation of the alpha and beta subunits by original definition using electrophoretic mobility in the molecular weight dimension. The specific biochemical property of posttranslational tyrosylation of the alpha subunit has been used to identify further this subunit. Under all conditions tested, the beta subunit has been found to be more acidic than the alpha subunit, with isoelectric point differences that agree with theoretical and published values. If the tubulin subunits are reduced and alkylated, the beta subunit migrates more rapidly in SDS polyacrylamide gels, with or without urea present. However, unalkylated tubulin subunits can comigrate or even reverse their relative mobility if 8 M-urea-SDS polyacrylamide gels are used for subunit separation. The results also confirm the earlier reports that the post-translational tyrosylation of protein appears exclusively restricted to alpha-tubulin and can be demonstrated in an in vivo situation. In addition, the results suggest that only the alpha 2 subunit of tubulin is tyrosylated.

Protein synthesis and axonal transport during nerve regeneration.

Protein synthesis and axonal transport have been studied in regenerating peripheral nerves. Sciatic nerves of bullfrogs were unilaterally crushed or cut. The animals were killed 1, 2, or 4 weeks later, and 8th and 9th dorsal root ganglia removed together with sciatic nerves and dorsal roots. The ganglia were selectively labeled in vitro with [35S]-methionine. Labeled proteins, in dorsal root ganglia and rapidly transported to ligatures placed on the sciatic nerves and dorsal roots, were analyzed by two-dimensional polyacrylamide gel electrophoresis. Qualitative analysis of protein patterns revealed no totally new proteins synthesized or rapidly transported in regenerating nerves. However, quantitative comparison of regenerating and contralateral control nerves revealed significant differences in abundance for some of the proteins synthesized in dorsal root ganglia, and for a few of the rapidly transported proteins. Quantitative analysis of rapidly transported proteins in both the peripheral processes (spinal nerves) and central processes (dorsal roots) revealed similar changes despite the fact that the roots were undamaged. The overall lack of drastic changes seen in protein synthesis and transport suggests that the neuron in its program of normal maintenance synthesizes and supplies most of the materials required for axon regrowth.

Are axonally transported proteins released from sciatic nerves?

A recent report by Hines and Garwood claimed a significant release of axonally transported proteins from frog sciatic nerve into a surrounding solution. In the present study no significant release of axonally transported protein from frog sciatic nerves was detected with more stringent control for non-axonal sources of released protein.

Effect of visual experience on tubulin synthesis during a critical period of visual cortex development in the hooded rat.

1. In some species, restriction of visual experience in early life may affect normal functional development of visual cortical cells. The purpose of the present study was to determine if visual deprivation during post-natal development in the hooded rat also affects the production in brain cells of certain molecular components such as tubulin, that are needed for growth and maintenance of synapses and neurites. 2. Norwegian black hooded rats were reared under a variety of conditions of visual deprivation. At various stages of development the animals were killed and the rate of synthesis of tubulin in visual and motor cortex determined. Tritiated colchicine was used to assay tubulin and L-[14C]leucine injected into the brain ventricles 2 hr before death was used to measure rate of tubulin synthesis. 3. In rats reared in normal light there is a marked elevation in visual cortex tubulin synthesis that spans the period from eye-opening (13 days) until approximately 35 days. This elevation in tubulin synthesis is absent in animals reared in darkness from birth or deprived of pattern vision by eyelid suture. Also the effect of visual deprivation on tubulin synthesis was specifically confined to visual cortex and was not found for the motor cortex. Similarly, the incorporation of L-[14C]leucine into total protein in visual cortex was unaffected by dark rearing. Hence the stimulation of tubulin synthesis by visual experience in rat visual cortex is not attributable to a general non-specific stimulation of protein synthesis. 4. Rats that were dark-reared from birth and then exposed to a lighted environment for 24 hr during a certain critical period that extends from eye-opening (13 days) until approximately 35 days, displayed a significant increase in visual cortex tubulin rats that were brought into the light later than 35 days showed no significant increase in tubulin synthesis when compared with their continuously dark-rearer controls. 5. It is suggested that the number of synapses and cytoplasmic processes that a developing cell can maintain depends on the size of the tubulin pool available to that cell. Tubulin in brain only has a half-life of about 4 days, so when the level of tubulin drops this could result in competition between different synapses for the limited supply of tubulin needed for their maintenance, a factor which may contribute to the structural plasticity of the visual cortex during the critical period.