Meningeal cells can communicate with astrocytes by calcium signaling.

Mechanical stimulation of adult human and rat pia-arachnoid cell cultures (loaded with calcium indicator dye) produced an increase in calcium in the stimulated cell. This change then propagated rapidly among neighboring cells, producing a calcium wave with a maximum distance of propagation and velocity resembling calcium waves in astrocytes. The pia-arachnoid waves were blocked by either octanol or apyrase, suggesting that propagation might occur either by gap junction communication or extracellular movement of ATP. Calcium waves in pia-arachnoid cells could invade contiguous astrocytes, and vice versa. Gap junction coupling between pia-arachnoid cells and astrocytes was shown by dye transfer experiments, in conjunction with immunostaining for connexin43. We infer that calcium signals from cells in the cortical parenchyma may be transmitted to the pia-arachnoid and might then serve in the induction of neurovascular changes, including those postulated to be responsible for the pain of migraine headache.

Cranial meninges of goldfish: age-related changes in morphology of meningeal cells and accumulation of surfactant-like multilamellar bodies.

In the optic tectum of goldfish, the outer, middle and inner layers of the endomeninx were evident in animals ranging in age from 1 month to several years. The outer layer in young animals consisted of closely overlapping cells with intertwined processes, whereas in the older animals it contained large extracellular spaces. The intermediate layer cells were always arranged in a single continuous layer, but in young animals they overlapped extensively with one another toward their edges whereas in the oldest animals they became extremely flat and non-overlapping. The inner layer included an outer tier of cells with their bases adhering to the intermediate layer, and an inner tier of cells detached from both the intermediate layer and the basal lamina overlying the brain parenchyma. Inner layer cells contained many large vacuoles that were in continuity with the extracellular space. With age, the extracellular space and the vacuolar system expanded, and the inner layer evolved into a meshwork of attenuated cytoplasmic processes embedded in the granular extracellular matrix. Another age-related feature was the accumulation adjacent to the basal lamina of uniform disc-shaped membranous structures, resembling multilamellar bodies of lung surfactant. These "disc bodies" were apparently generated by the coalescence of vesicles formed at the surface of the inner layer cells, possibly as a by-product of protein secretion by these cells.

Immunolocalization of exoglycoproteins ("ependymins") in the goldfish brain.

Exoglycoproteins (X-GPs) are a group of very abundant soluble glycoproteins in the goldfish brain. Immunostaining with polyclonal antisera to X-GPs revealed consistent perinuclear staining in the cells of the inner and intermediate layers of the leptomeninx, which is homologous to the pia-arachnoid. Immunolabelling was also prominent in the outer wall of capillaries, and in a variable population of 10-12 microns granular cells that appeared mainly near the ventricles and occasionally within the ventricles or under the meninges. In some cases, small and medium-sized lymphocytes were immunostained. Lymphocytes were sometimes associated with the granular cells, which may be hematogenous cells in transit toward the ventricles. The choroid plexus, saccus dorsalis, the roof of the third ventricle and Reissner's fiber showed strong immunostaining. The localization of the X-GPs suggests that they may contribute to maintenance of the blood-brain barrier or to regulation of immune function within the brain.

Immunochemical studies of extracellular glycoproteins (X-GPs) of goldfish brain.

Exoglycoproteins (X-GPs) are a family of soluble glycoproteins which are the most prominent constituent of the extracellular compartment of goldfish brain. On conventional two-dimensional polyacrylamide gels they typically display two primary molecular weight forms, averaging about 33 and 38 kDa, each appearing as a row of five to seven individual spots. When X-GP antibodies were applied by Western blotting, gels of goldfish brain extract prepared without a reducing agent showed, in addition to the primary molecular weight groups, at least one row of spots of slightly lower molecular weight and a major array of spots in the range of 45-60 kDa. The latter presumably represent dimers of the primary X-GP forms since they gave rise to the primary forms upon treatment with a reducing agent. However, on gradient gels prepared without detergents or reducing agents, X-GPs identified by immunostaining appeared only at 200 kDa and above, indicating that these proteins naturally occur in the form of large particles. Deglycosylation of the brain extract by N-glycosidase F reduced the molecular weight of each primary X-GP form by about 5 kDa, but did not abolish the microheterogeneity, which is at least partly due to minor differences in primary structure among the proteins in individual spots. Both rows of spots in the deglycosylated sample showed a coordinated shift toward the basic side of the gel, and a prominent new spot appeared on the basic end of the lower molecular weight group, which probably represents the fully deglycosylated form of the most abundant X-GP isoform.(ABSTRACT TRUNCATED AT 250 WORDS)

Extracellular proteins of goldfish optic tectum labeled by intraocular injection of 3H-proline.

A prominent group of soluble glycoproteins with a molecular weight of 30K-40K and pI 5.0-5.6 was detected in various parts of the goldfish brain as well as in the optic nerves. Since these proteins are readily liberated from the tissue, we have designated them exoglycoproteins (X-GPs). The X-GPs in the optic tectum were found to be labeled after intraocular injection of radioactive tracers, in a manner consistent with the labeling of proteins transported in the optic axons. However, the labeling of X-GPs was blocked by intracranial injection of a protein synthesis inhibitor, whereas the labeling of axonally transported proteins was unaffected. This shows that the X-GPs can be synthesized locally within the brain. Nevertheless, when protein synthesis in the retina was blocked, the labeling of the X-GPs in the tectum was prevented, like the labeling of axonally transported proteins. Thus precursors for the synthesis of X-GPs can be derived from materials transported in the optic axons. This synthesis can occur in nonneuronal cells, as indicated by the incorporation of labeled amino acid into X-GPs in optic nerves directly exposed to the label. The synthesis of X-GPs was increased in regenerating nerves, suggesting that these proteins may play a role in regeneration. Partial amino acid sequencing of the proteins showed that they are identical to the proteins previously identified as "ependymins," which have been implicated in neuronal plasticity. There are minor differences in amino acid sequence among some individual spots.

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.

Effect of conditioning lesions on regeneration of goldfish optic axons: time course of the cell body reaction to axotomy.

The time course of the cell body reaction to axotomy was determined in goldfish retinal ganglion cells by measuring cell body size and the amount of labelled protein conveyed by fast axonal transport to the optic tectum, both of which increase during regeneration of the optic axons. Following a single testing lesion of the optic nerve, the regenerating axons began to innervate the tectum at about 14 days after the lesion and the cell body reaction began to decline 2-3 weeks thereafter. If the testing lesion had been preceded by a conditioning lesion 2 weeks earlier, the time for the regenerating axons to arrive in the tectum was reduced by a week, because of the faster rate of axonal outgrowth, but the interval between their arrival and the beginning of the decline of the cell body reaction was unchanged. Electrophysiological measurements showed that synaptic transmission was initiated earlier when the axons reached the tectum faster. These results indicate that the mechanisms initiating the recovery of cell body metabolism are independent of those governing the rate of axonal outgrowth. The recovery of the cell body may begin shortly after synapses are established, regardless of whether they are correctly or incorrectly targetted. The correctness of the target may be a separate factor in determining how rapidly and completely the cell body recovers.

Relationship between phosphorylation and synthesis of goldfish optic nerve proteins during regeneration.

After intraocular injection of radiolabeled phosphate and 3H-proline, the labeling of goldfish optic nerve proteins was monitored over a 7 week period of regeneration following a lesion to the optic tract. Labeled phosphate incorporation into total nerve protein increased to a peak value about twice that in normal nerve at 3 weeks after injury, then declined to slightly above normal by 7 weeks. Incorporation of 3H-proline showed a higher rise and a steeper decline, with values still significantly above normal at 7 weeks. Two-dimensional gel electrophoresis revealed that almost all the individual proteins examined underwent an increase in 3H-proline incorporation with a peak at about 3 weeks. However, only 4 proteins showed an increase in incorporation of 32P correlated with the increase in 3H-proline. The closest correlation was seen for protein 4, the equivalent of the growth-associated protein GAP-43; for the other 3 proteins (15, 31, and 38) 32P incorporation remained elevated even when 3H-proline incorporation had declined. Two other proteins (24e and 48) showed increased 32P incorporation not correlated with 3H-proline changes. Several proteins showed a decrease in 32P incorporation, even though 3H-proline incorporation was increased. For example, the phosphorylation of ON2, a neuronal intermediate filament protein, showed a long-lasting decline, which was already evident at 1 week and had not yet returned to normal by 7 weeks. Other proteins in this group (33, 37, and 46) showed a faster recovery.(ABSTRACT TRUNCATED AT 250 WORDS)

Phosphorylation of proteins in normal and regenerating goldfish optic nerve.

Within 6 h after radiolabeled phosphate was injected into the eye of goldfish, labeled acid-soluble and acid-precipitable material began to appear in the optic nerve and subsequently also in the lobe of the optic tectum, to which the optic axons project. From the rate of appearance of the acid-precipitable material, a maximal velocity of axonal transport of 13-21 mm/day could be calculated, consistent with fast axonal transport group II. Examination of individual proteins by two-dimensional gel electrophoresis revealed that approximately 20 proteins were phosphorylated in normal and regenerating nerves. These ranged in molecular weight from approximately 18,000 to 180,000 and in pI from 4.4 to 6.9. Among them were several fast transported proteins, including protein 4, which is the equivalent of the growth-associated protein GAP-43. In addition, there was phosphorylation of some recognizable constituents of slow axonal transport, including alpha-tubulin, a neurofilament constituent (NF), and another intermediate filament protein characteristic of goldfish optic axons (ON2). At least some axonal proteins, therefore, may become phosphorylated as a result of the axonal transport of a phosphate carrier. Some of the proteins labeled by intraocular injection of 32P showed changes in phosphorylation during regeneration of the optic axons. By 3-4 weeks after an optic tract lesion, five proteins, including protein 4, showed a significant increase in labeling in the intact segment of nerve between the eye and the lesion, whereas at least four others (including ON2) showed a significant decrease. When local incorporation of radiolabeled phosphate into the nerve was examined by incubating nerve segments in 32P-containing medium, there was little or no labeling of the proteins that showed changes in phosphorylation during regeneration. Segments of either normal or regenerating nerves showed strong labeling of several other proteins, particularly a group ranging in molecular weight from 46,000 to 58,000 and in pI from 4.9 to 6.4. These proteins were presumably primarily of nonneuronal origin. Nevertheless, if degeneration of the axons had been caused by removal of the eye 1 week earlier, most of the labeling of these proteins was abolished. This suggests that phosphorylation of these proteins depends on the integrity of the optic axons.

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.

In vivo phosphorylation of axonal proteins in goldfish optic nerve during regeneration.

In vivo phosphorylation of axonal proteins was investigated in normal and regenerating optic nerves of goldfish by two-dimensional gel electrophoresis. By 6-24 h after intraocular injection of H3(32)PO4, approximately 20 optic nerve proteins ranging in size from 19 to 180 kilodaltons and in pI from 4.4 to 6.8 were seen to have incorporated radiolabel. Five of these proteins showed a robust increase in incorporation of phosphate during regeneration. Among the latter was an acidic (pI 4.5) 45-kilodalton protein, which has previously been shown to be conveyed by fast axonal transport and to increase dramatically in its rate of synthesis during regeneration of goldfish optic axons.

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.

Intraocular tetrodotoxin reduces axonal transport and transcellular transfer of adenosine and other nucleosides in the visual system of goldfish.

Intraocular injection of tetrodotoxin (TTX) in goldfish, which abolishes physiological activity in the optic axons, decreased by up to about 30% the amount of radioactively labeled adenosine, uridine and guanosine (and their nucleotide derivatives) that was axonally transported in the optic nerve. The amount of labeled nucleoside that reached the optic tectum and became incorporated into RNA in the postsynaptic tectal neurons and glial cells was reduced by up to about 50%. There was no change, however, in the amount of transported nucleoside that became incorporated into RNA in the optic nerve glia. The TTX-induced changes were eliminated when axonal transport was blocked with vincristine, indicating that this change did not involve material moving along the nerve by diffusion. If the TTX injection was delayed until several hours after labeling of the transported materials, the transported labeled nucleoside in the nerve was reduced very little, but the RNA labeling in the tectum was reduced just as much as when TTX was given prior to labeling. This indicates that the labeling of the tectal cells was affected more by the level of activity in the pathway than by the amount of transported nucleoside reaching the optic nerve terminals. It appears likely, therefore, that the process most affected by the decrease in physiological activity is the release of nucleoside from the terminals of the presynaptic neurons or its uptake into postsynaptic tectal neurons and glia. The fact that physiological activity may modify the amount of axonally transported nucleosides made available for metabolism (including RNA synthesis) in postsynaptic neurons may provide an explanation for activity-linked neurotrophic effects.

Changes in axonal transport of phospholipids in the regenerating goldfish optic system.

Changes in axonally transported phospholipids of regenerating goldfish optic nerve were studied by intraocular injection of [2-3H]glycerol 9 days and 16 days after nerve crush at 30 degrees C. The four major glycerophospholipids all showed substantial increases in transported radioactivity above non-regenerating controls at both time points, these being maximal (15- to 35-fold) in the optic nerve-tract at 9 days and about half as great at 16 days. In the contralateral optic tectum transported label increased 6- to 13-fold at 9 days and 10- to 25-fold at 16 days in the various glycerophospholipids. While all glycerophospholipids showed absolute increases in both tissues, PS and PI increased relatively more, especially in the tectum. The regeneration-associated increases in transported label of all glycerophospholipids were larger than those previously demonstrated for gangliosides and glycoproteins in the same system.

Nucleolar changes in goldfish retinal ganglion cells in response to an optic nerve lesion are not perturbed by a second lesion.

The reaction of the goldfish retinal ganglion cells to optic nerve crush, as indicated by an increase in nucleolar incidence 4 to 5 days after this lesion, was not significantly affected if a second nerve crush was made within 3 days after the first. This appears to rule out axon sprouting as the initiating event for the cell body reaction to axotomy.

RNA content of normal and axotomized retinal ganglion cells of rat and goldfish.

The responses of rat and goldfish retinal ganglion cells to axotomy were examined by a quantitative cytochemical method for RNA and by morphometric measurement 1-60 (rat) and 3-90 (goldfish) days after interruption of one optic nerve or tract intracranially. Unoperated control animals were studied also. The RNA content of axotomized neurons of rat fell 7-60 days postoperatively. Additionally, atrophy of the axotomized somas occurred. Over time, neuronal atrophy approximately paralleled the loss of RNA, and mean cell area and RNA content were reduced by about 25% 60 days after axotomy. Incorporation of 3H-uridine by axotomized neurons declined also. Axotomized retinal ganglion cells of goldfish behaved differently from those of the rat and showed increases in RNA content, most conspicuously 14-60 days postoperatively. Enlargement of axotomized fish neurons occurred but was less proportionately than concomitant increases in RNA content. The nonaxotomized ganglion cells of goldfish displayed statistically significant increases in size and RNA content 14-49 days after unilateral optic nerve or tract lesions. In contrast, alterations in rat retinal ganglion cells contralateral to interruption of one optic nerve were of limited and questionable significance. The contrasting reactions to axotomy by the retinal ganglion cells of these two vertebrates, one of which regenerates optic axons and one of which does not, may support the proposition that the somal response to axon injury has an important bearing upon the success or failure of CNS regeneration.(ABSTRACT TRUNCATED AT 250 WORDS)

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.