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B Grafstein

Publications and source records attributed to B Grafstein.

At least 37 records · Page 2Linked to original sources
PubMed1985

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.

Actins↗
PubMed1985

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.

Animals↗
PubMed1984

Intraocular injection of tetrodotoxin in goldfish decreases fast axonal transport of [3H]glucosamine-labeled materials in optic axons.

When physiological activity in goldfish visual system was abolished by repeated intraocular injection of tetrodotoxin (TTX), the fast axonal transport of radioactive amino acid-labeled protein in the optic axons was unaltered. However, the TTX treatment reduced the amount of [3H]glucosamine-labeled glycolipids that were axonally transported to the optic tectum, and may have decreased their rate of turnover in the tectum. A similar though smaller effect was observed for glucosamine-containing glycoproteins. These alterations in axonal transport may be the basis for at least some of the deleterious effects of TTX on axonal regeneration in this system.

Animals↗
PubMed1984

Ganglioside changes in the regenerating goldfish optic system: comparison with glycoproteins and phospholipids.

Axonally transported radioactivity in sialoglycoconjugates, labeled by intraocular injection of [3H]N-acetylmannosamine, increased significantly during regeneration of goldfish optic axons at 30 degrees C. Ganglioside radioactivity showed the largest increase--approximately eightfold--in the optic nerve tract at 8 days after optic nerve crush while sialoglycoprotein radioactivity increased fourfold under the same conditions. As regeneration proceeded the magnitude of the increase in the nerve tract diminished for both glycoconjugates. In the optic tectum, however, transported radioactivities remained approximately twofold higher than controls between 15 and 25 days postcrush. The zwitterionic fraction of glycerophospholipids, labeled by intraocular injection of [14C]glycerol, also showed large increases during regeneration, but the acidic glycerophospholipids showed only modest increases. Thus while membrane components in general were elevated during the early stages of regeneration, the most pronounced increases occurred in gangliosides and certain glycerophospholipids. The significance of these changes in the regeneration process remain to be determined.

Animals↗
PubMed1984

Antibodies to gangliosides inhibit goldfish optic nerve regeneration in vivo.

Intraocular injection of antiserum to mixed ganglioside or to GM1 inhibited the regeneration of goldfish optic axons following an optic nerve crush. For example, injections of antiserum on 5 consecutive days beginning the day before the crush resulted in a decrease of about 40% in the axonal outgrowth distance measured at 10 days after the crush. The inhibition was observed even when the treatment was begun a few days after the lesion, and greater degrees of inhibition were observed when the treatment was given later in regeneration. This indicates that the antiganglioside serum interfered with axonal elongation more than with the initial sprout formation. The antiganglioside treatment did not impair the enlargement of the cell bodies and nucleoli that accompanies regeneration, nor did it affect fast axonal transport of protein, glycoprotein, or glycolipid in regenerating nerves. Thus inhibition of outgrowth by antiganglioside treatment was not mediated by a gross change in the metabolism of the regenerating neurons. Treatment of normal neurons with the antiserum produced a 20-30% increase in the amount of 3H-glucosamine-labeled glycoproteins and glycolipids conveyed by fast axonal transport. These results suggest that membrane gangliosides may normally influence the supply of axonally transported glycosylated macromolecules. However, the effect of antiganglioside on axonal transport of glycosylated molecules and on axonal outgrowth are not necessarily related to each other.

Animals↗
PubMed1984

Local application of calcium-modulating agents to a crushed goldfish optic nerve modifies visual recovery.

The effects of various Ca2+-modulating agents on regeneration in the optic nerve of goldfish were determined by assaying recovery of visual function. One to three daily applications of the agents were made at the site of an optic nerve crush beginning within 3 days after the lesion. Application of calcium ionophore A-23187 significantly shortened the time required for reappearance of the startle reaction to a bright light. Some shortening of recovery time was also observed with application of high-Ca2+ Ringer's solution. A significant effect was obtained with 1% and 6% dimethylsulfoxide (DMSO). When A-23187 was combined with DMSO, a further enhancement was seen if the original DMSO effect had been weak, whereas a strong DMSO effect was reduced in the presence of A-23187. The effect of DMSO alone or DMSO in combination with A-23187 was blocked by the calcium-chelating agent EGTA. These results indicated that increased entry of Ca2+ into the regenerating axons or supporting cells may be responsible for the enhanced rate of recovery. There was no histologic evidence that the faster recovery was due to accelerated axon outgrowth, but the packing density of the regenerating axons was increased. We postulate that the recovery-enhancing agents may act by promoting axonal interactions leading to the reestablishment of the correct retinal projection, or by facilitating the function of the regenerating synaptic terminals.

Animals↗
PubMed1983

Effect of acetoxycycloheximide and dibutyryladenosine cyclic 3':5'-monophosphate on axonal regeneration in the goldfish optic nerve.

Acetoxycycloheximide (AXM) or dibutyryl cyclic AMP (dbcAMP) was injected unilaterally into the vitreous humor of the eye beginning 5-6 days after bilateral optic nerve crush. Injections were repeated every 12-24 h for a total of 3-5 days; goldfish were sacrificed 10 days after lesioning the nerves. At a low dosage of AXM (0.1 microgram daily for 5 days), the mean outgrowth distance in treated neurons was 60% less than in contralateral control neurons. At a high dosage (0.3 microgram daily for 4 days), outgrowth was immediately blocked in both treated and contralateral control axons. Dibutyryl cyclic AMP, in a dose of 5 microM every 12 h for 3 days, produced a 38% reduction in outgrowth distance, associated with a 30% reduction in protein synthesis by the retinal ganglion cells and a 73% reduction in the amount of protein carried by the fast component of axonal transport.

Animals↗
PubMed1983

Intraocular tetrodotoxin in goldfish hinders optic nerve regeneration.

Repeated intraocular injection of tetrodotoxin (TTX) was used to produce a maintained block of neural activity in goldfish optic axons which were regenerating following a crush of the optic nerve. The recovery of visual function was delayed in the TTX-treated fish, as demonstrated by delays in the return of the startle reaction to sudden illumination, the dorsal light reflex and food pellet localization. A single injection of TTX at the time of optic nerve crush delayed recovery of the startle reaction but not of food localization. There was no loss of ganglion cells in the TTX-treated animals, but the number of regenerated axons detectable by light microscopy was reduced. Also, axonal transport of radioactively labeled protein in some of the regenerating axons showed a deficit at 21-28 days after the lesion, i.e., during innervation of the tectum. Incorporation of labeled amino acid into protein in the retinal ganglion cells was depressed during the same period, but both the transport and incorporation had returned to normal by 36 days after the lesion. These results, together with the results of the accompanying electrophysiological analysis of the effects of TTX58,59, suggest that TTX treatment interferes with two separate events in regeneration, one occurring soon after the nerve lesion and the other during innervation of the tectum. During at least the first of these events the effect of TTX treatment may be to reduce the number of size of the regenerating axon branches. We propose that the TTX effect may be mediated by a reduction in the axonal transport of certain materials, including gangliosides, nucleosides, or proteins specifically involved in regeneration.

Animals↗
PubMed1982

Protein synthesis and axonal transport in goldfish retinal ganglion cells during regeneration accelerated by a conditioning lesion.

Axonal outgrowth in goldfish retinal ganglion cells following a testing lesion of the optic axons is accelerated by a prior conditioning lesion. Changes in protein synthesis and axonal transport were examined during the accelerated regeneration. The conditioning lesion was an optic tract cut made 2 weeks prior to the testing lesion, which consisted of a tract cut at the chiasma, so that nerves subjected to either a conditioning lesion ('conditioned nerves') or a sham operation ('sham-conditioned nerves') could be examined in the same animal. In the retinal ganglion cells of conditioned nerves, the incorporation of [3H]proline into protein began to increase between 1 and 8 days after the testing lesion. The amount of fast-transported labeled protein was elevated to about 8 X normal by 1 day after the testing lesion but had decreased to about 3-5X normal at 8 and 22 days. The 8 and 22 day values were not significantly different from those in sham-conditioned nerves or nerves that had received a testing lesion alone. For slow protein transport, the instantaneous amount transported was 15-16 X normal in the conditioned nerves at 1 and 8 days after the testing lesion, and the velocity of slow transport, which was already elevated above normal by 1 day after the testing lesion, was elevated still further by 8 days--to a value in excess of 1.5 mm/day (compared to 0.2-0.4 mm/day in normal animals). We believe that the enhanced outgrowth resulting from the conditioning lesion is due to a transient increase in the amount of fast transport (possibly responsible for a decreased delay in the initiation of sprouting), and a sustained increase in the amount and velocity of slow transport (which may account for an increased rate of elongation).

Animals↗
PubMed1982

Perikaryal routing of newly synthesized proteins in regenerating neurons: quantitative electron microscopic autoradiography.

Intracellular transport of newly synthesized proteins through organelles in the perikarya of regenerating goldfish retinal ganglion cells was studied using electron microscopic autoradiography. Retinas were removed 14 or 30 days after optic tract cut or sham operation, pulse-labeled in [3H]proline-containing medium for 5 min, and then chase-incubated in medium containing unlabeled proline for various times up to 55 min before fixation. Fourteen days after axotomy, during rapid growth of the regenerating axons, the time course of change of relative grain density (% grains/% area) in the rough endoplasmic reticulum in regenerating cells was almost identical to that in control cells. However, the grain distribution analysis revealed an increased delivery of newly synthesized proteins to the Golgi apparatus, perikaryal plasma membrane and nucleus in regenerating cells. Thirty days after axotomy, during synaptogenesis, Golgi apparatus labeling in the regenerating cells became significantly higher than control, but the increase was delayed compared to the increase seen 14 days after axotomy. Labeling of the plasma membrane and nucleus did not rise above control in 30-day regenerating cells chase-incubated for up to 55 min. Thus the pattern of intracellular transport of newly synthesized proteins varies with the stage stage of axonal regeneration.

Animals↗
PubMed1982

Effect of nerve growth factor on regeneration of goldfish optic axons.

Axonal outgrowth following a crush of the goldfish optic nerve was enhanced if nerve growth factor (NGF) was administered by intraocular injection or by local application to the lesion site. Various forms of NGF (beta, 2.5S and 7S) were effective, producing a 20-40% decrease in the time required for recovery of the startle reaction to a bright light. A corresponding increase in axonal outgrowth was revealed by histological examination of the optic nerves. The effect produced by a single intraocular injection given at the time of the lesion was not further increased by subsequent injections. Up to 14 days after the lesion, the size of the retinal ganglion cell bodies and the incidence of nucleoli detectable by light microscopy were not affected by the NGF treatment.

Animals↗
PubMed1982

Protein synthesis and fast axonal transport in regenerating goldfish retinal ganglion cells.

To characterize the fast component of axonal transport in regenerating goldfish optic axons, the incorporation of L-2,3-[3H]proline into newly-synthesized proteins in the cell bodies of the retinal ganglion cells and the amount of transported labeled protein were determined at 2-36 days after cutting the optic tract. Both the incorporation and the amount of transported protein had doubled by 10 days after the lesion and continued to increase to about 5 times normal at 15 days, a time when a large proportion of the regenerating axon population had reached the optic tectum. Near-normal levels were recovered by 36 days. In contralateral control neurons, the incorporation of L-2,3-[3H]proline was unchanged from normal throughout, whereas the amount of labeled transported protein entering control axons was decreased by 55% at 2 and 10 days after the testing lesion, returning to normal by 15 days. An increase in fast transport velocity was seen in the regenerating axons beginning at 10 days after the lesion. However, a similar velocity increase was also seen in the contralateral control axons and in undamaged axons following removal of the cerebral hemispheres. Therefore, the velocity increase was not a specific consequence of axotomy.

Amino Acids↗