Biomedical subjects

G D Bittner

Publications and source records attributed to G D Bittner.

At least 19 recordsLinked to original sources

PubMed2000

Plasmalemmal repair of severed neurites of PC12 cells requires Ca(2+) and synaptotagmin.

Ca(2+) and synaptotagmin (a Ca(2+)-binding protein that regulates axolemmal fusion of synaptic vesicles) play essential roles in the repair of axolemmal damage in invertebrate giant axons. We now report that neurites of a rat pheochromocytoma (PC12) cell line transected and maintained in a serum medium form a dye barrier (exclude an external hydrophilic fluorescent dye) and survive for 24-hr posttransection (based on morphology and retention of another hydrophilic dye internally loaded at 6-hr posttransection). Some (25%) transected neurites that form a dye barrier regrow. Most (83%) neurites transected in a saline solution containing divalent cations (PBS(++)) also exclude entry of an externally placed hydrophilic fluorescent dye at 15-min posttransection. In contrast, only 14 or 17% of neurites maintained in a divalent cation-free solution (PBS(=)) or in PBS(=) + Mg(2+), respectively, form a dye barrier. Neurites that do not form a dye barrier do not survive for 24 hr. When PC12 neurites are loaded with an antibody to squid synaptotagmin, most (81%) antibody-loaded neurites do not form a dye barrier, whereas most (>/=81%) neurites loaded with heat-inactivated antibody or preimmune IgG do form a barrier. These data show that: 1) transected neurites of PC12 cells have mechanism(s) for plasmalemmal repair (dye barrier formation and survival); 2) Ca(2+) is necessary for dye barrier formation, which occurs minutes after transection and is necessary for survival and regrowth; and 3) synaptotagmin is an essential mediator of barrier formation. The similarity in the requirements for plasmalemmal repair in this mammalian cell preparation with those reported previously for invertebrate axons suggests that mechanisms necessary for plasmalemmal repair have been conserved phylogenetically.

Animals↗

PubMed2000

Axolemmal repair requires proteins that mediate synaptic vesicle fusion.

A damaged cell membrane is repaired by a seal that restricts entry or exit of molecules and ions to that of the level passing through an undamaged membrane. Seal formation requires elevation of intracellular Ca(2+) and, very likely, protein-mediated fusion of membranes. Ca(2+) also regulates the interaction between synaptotagmin (Syt) and syntaxin (Syx), which is thought to mediate fusion of synaptic vesicles with the axolemma, allowing transmitter release at synapses. To determine whether synaptic proteins have a role in sealing axolemmal damage, we injected squid and crayfish giant axons with an antibody that inhibits squid Syt from binding Ca(2+), or with another antibody that inhibits the Ca(2+)-dependent interaction of squid Syx with the Ca(2+)-binding domain of Syt. Axons injected with antibody to Syt did not seal, as assessed at axonal cut ends by the exclusion of extracellular hydrophilic fluorescent dye using confocal microscopy, and by the decay of extracellular injury current compared to levels measured in uninjured axons using a vibrating probe technique. In contrast, axons injected with either denatured antibody to Syt or preimmune IgG did seal. Similarly, axons injected with antibody to Syx did not seal, but did seal when injected with either denatured antibody to Syx or preimmune IgG. These results indicate an essential involvement of Syt and Syx in the repair (sealing) of severed axons. We suggest that vesicles, which accumulate and interact at the injury site, re-establish axolemmal continuity by Ca(2+)-induced fusions mediated by proteins such as those involved in neurotransmitter release.

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PubMed2000

Structural changes at cut ends of earthworm giant axons in the interval between dye barrier formation and neuritic outgrowth.

We describe structural changes at the cut ends of invertebrate myelinated earthworm giant axons beginning with the formation of a dye barrier (15 minutes posttransection or postcalcium addition) and ending with the formation of a neuritic outgrowth (2-10 days posttransection). The morphology of the cut end, and the location and morphological configuration of the dye barrier, were assessed by time-lapse confocal, fluorescence microscopy and by electron microscopy. During the interval from 15 to 35 minutes postcalcium addition, the dye barrier continuously migrated away from a cut axonal end; the dye barrier then remained stable for up to 5 hours. The size, packing density, and arrangement of membranous structures were correlated with changes in the dye barrier from 15 to 35 minutes postcalcium addition. During this interval, uptake of an externally placed hydrophilic dye by these membranous structures was also variable. After 35 minutes postcalcium addition, the membranous structures remained stable until they completely disappeared between 1 and 2 days posttransection. The disappearance of membranous structures always preceded neuritic outgrowth, which only arose from cut axonal ends. These results demonstrate that the dye barrier and associated membranous structures, which form after transection of earthworm giant axons, are very dynamic in the short term (35 minutes) with respect to their location and morphological configuration and suggest that axolemmal repair must be completed before neuritic outgrowth can occur.

Animals↗

PubMed2000

Barrier permeability at cut axonal ends progressively decreases until an ionic seal is formed.

After axonal severance, a barrier forms at the cut ends to rapidly restrict bulk inflow and outflow. In severed crayfish axons we used the exclusion of hydrophilic, fluorescent dye molecules of different sizes (0.6-70 kDa) and the temporal decline of ionic injury current to levels in intact axons to determine the time course (0-120 min posttransection) of barrier formation and the posttransection time at which an axolemmal ionic seal had formed, as confirmed by the recovery of resting and action potentials. Confocal images showed that the posttransection time of dye exclusion was inversely related to dye molecular size. A barrier to the smallest dye molecule formed more rapidly (<60 min) than did the barrier to ionic entry (>60 min). These data show that axolemmal sealing lacks abrupt, large changes in barrier permeability that would be expected if a seal were to form suddenly, as previously assumed. Rather, these data suggest that a barrier forms gradually and slowly by restricting the movement of molecules of progressively smaller size amid injury-induced vesicles that accumulate, interact, and form junctional complexes with each other and the axolemma at the cut end. This process eventually culminates in an axolemmal ionic seal, and is not complete until ionic injury current returns to baseline levels measured in an undamaged axon.

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PubMed1999

Calcium entry initiates processes that restore a barrier to dye entry in severed earthworm giant axons.

After severance, axons can restore structural barriers that are necessary for recovery of their electrical function. In earthworm myelinated axons, such a barrier to dye entry is mediated by many vesicles and myelin-derived membranous structures. From time-lapse confocal fluorescence and DIC images, we now report that Ca2+ entry and not axonal injury per se initiates the processes that form a dye barrier, as well as the subsequent structural changes in this barrier and associated membranous structures. The time required to restore a dye barrier after transection also depends only on the time of Ca2+ entry.

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PubMed1999

Rapid induction of functional and morphological continuity between severed ends of mammalian or earthworm myelinated axons.

The inability to rapidly restore the loss of function that results from severance (cutting or crushing) of PNS and CNS axons is a severe clinical problem. As a novel strategy to help alleviate this problem, we have developed in vitro procedures using Ca2+-free solutions of polyethylene glycol (PEG solutions), which within minutes induce functional and morphological continuity (PEG-induced fusion) between the cut or crushed ends of myelinated sciatic or spinal axons in rats. Using a PEG-based hydrogel that binds to connective tissue to provide mechanical strength at the lesion site and is nontoxic to nerve tissues in earthworms and mammals, we have also developed in vivo procedures that permanently maintain earthworm myelinated medial giant axons whose functional and morphological integrity has been restored by PEG-induced fusion after axonal severance. In all these in vitro or in vivo procedures, the success of PEG-induced fusion of sciatic or spinal axons and myelinated medial giant axons is measured by the restored conduction of action potentials through the lesion site, the presence of intact axonal profiles in electron micrographs taken at the lesion site, and/or the intra-axonal diffusion of fluorescent dyes across the lesion site. These and other data suggest that the application of polymeric fusiogens (such as our PEG solutions), possibly combined with a tissue adherent (such as our PEG hydrogels), could lead to in vivo treatments that rapidly and permanently repair cut or crushed axons in the PNS and CNS of adult mammals, including humans.

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PubMed1998

Anomalies associated with dye exclusion as a measure of axolemmal repair in invertebrate axons.

After axonal injury, dye exclusion is often used as a measure of the re-establishment of a structural barrier. We now report that this use of dye exclusion is equivocal in two situations. (1) When a negatively-charged hydrophilic fluorescent dye (HFD) was placed in the physiological saline (PS) surrounding a crayfish medial giant axon (CMGA) before transection, this dye did not readily diffuse into the cut ends after transection whereas uncharged or neutralized dyes did do so. (2) When axoplasm flowed out of the cut ends of a transected squid giant axon (SGA), this outflow markedly slowed hydrophilic fluorescent dyes from diffusing into the cut ends. These anomalies suggest that dye exclusion by an injured axon does not always indicate that a structural barrier has formed. Therefore, dye assessments of axonal repair require control experiments that rule out anomalous exclusion due to dye interactions (biochemical and fluid dynamics) with components (axoplasm, axolemma, glial sheath, etc.) of the particular axon under study.

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PubMed1998

Heat-shock proteins in axoplasm: high constitutive levels and transfer of inducible isoforms from glia.

To characterize heat-shock proteins (HSPs) of the 70-kDa family in the crayfish medial giant axon (MGA), we analyzed axoplasmic proteins separately from proteins of the glial sheath. Several different molecular weight isoforms of constitutive HSP 70s that were detected on immunoblots were approximately 1-3% of the total protein in the axoplasm of MGAs. To investigate inducible HSPs, MGAs were heat shocked in vitro or in vivo, then the axon was bathed in radiolabeled amino acid for 4 hours. After either heat-shock treatment, protein synthesis in the glial sheath was decreased compared with that of control axons, and newly synthesized proteins of 72 kDa, 84 kDa, and 87 kDa appeared in both the axoplasm and the sheath. Because these radiolabeled proteins were present in MGAs only after heat-shock treatments, we interpreted the newly synthesized proteins of 72 kDa, 84 kDa, and 87 kDa to be inducible HSPs. Furthermore, the 72-kDa radiolabeled band in heat-shocked axoplasm and glial sheath samples comigrated with a band possessing HSP 70 immunoreactivity. The amount of heat-induced proteins in axoplasm samples was greater after a 2-hour heat shock than after a 1-hour heat shock. These data indicate that MGA axoplasm contains relatively high levels of constitutive HSP 70s and that, after heat shock, MGA axoplasm obtains inducible HSPs of 72 kDa, 84 kDa, and 87 kDa from the glial sheath. These constitutive and inducible HSPs may help MGAs maintain essential structures and functions following acute heat shock.

Action Potentials↗

PubMed1998

Effects of fibrin micromorphology on neurite growth from dorsal root ganglia cultured in three-dimensional fibrin gels.

The effect of fibrin matrix micromorphology on neurite growth was investigated by measuring the length of neurites growing in three-dimensional fibrin gels with well characterized micromorphologies. Dorsal root ganglia (DRGs) from 7-day chick embryos were entrapped and cultured in gels made from varying concentrations of fibrinogen (5-15 mg/mL) or calcium (2-10 mM). The length of growing neurites was measured with light videomicroscopy, and the number and diameter of fibrin fiber bundles were measured from scanning electron micrographs. An increase in fibrinogen concentration caused a decrease in the average fiber bundle thickness, an increase in the number of fiber bundles, and a marked decrease in neurite length. Gels made with different calcium concentrations had a similar range of variation in fibrin fiber bundle number or diameter, but these variations had little effect on neurite and associated nonneuronal cell outgrowth. These results provide insights into the process of neurite advance within fibrin and may be useful in the design of fibrin-based materials used for peripheral nerve regeneration. Furthermore, this study provides the first detailed experimental data on the micromorphology of fibrin matrices made from more than 5 mg/mL of fibrinogen and indicates that existing kinetic models of fibrin polymerization do not accurately predict fibrin structure at these higher concentrations.

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PubMed1998

Endocytotic formation of vesicles and other membranous structures induced by Ca2+ and axolemmal injury.

Vesicles and/or other membranous structures that form after axolemmal damage have recently been shown to repair (seal) the axolemma of various nerve axons. To determine the origin of such membranous structures, (1) we internally dialyzed isolated intact squid giant axons (GAs) and showed that elevation of intracellular Ca2+ >100 microM produced membranous structures similar to those in axons transected in Ca2+-containing physiological saline; (2) we exposed GA axoplasm to Ca2+-containing salines and observed that membranous structures did not form after removing the axolemma and glial sheath but did form in severed GAs after >99% of their axoplasm was removed by internal perfusion; (3) we examined transected GAs and crayfish medial giant axons (MGAs) with time-lapse confocal fluorescence microscopy and showed that many injury-induced vesicles formed by endocytosis of the axolemma; (4) we examined the cut ends of GAs and MGAs with electron microscopy and showed that most membranous structures were single-walled at short (5-15 min) post-transection times, whereas more were double- and multi-walled and of probable glial origin after longer (30-150 min) post-transection times; and (5) we examined differential interference contrast and confocal images and showed that large and small lesions evoked similar injury responses in which barriers to dye diffusion formed amid an accumulation of vesicles and other membranous structures. These and other data suggest that Ca2+ inflow at large or small axolemmal lesions induces various membranous structures (including endocytotic vesicles) of glial or axonal origin to form, accumulate, and interact with each other, preformed vesicles, and/or the axolemma to repair the axolemmal damage.

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PubMed1997

Repair of plasmalemmal lesions by vesicles.

Crayfish medial giant axons (MGAs) transected in physiological saline form vesicles which interact with each other, pre-existing vesicles, and/or with the plasmalemma to form an electrical and a physical barrier that seals a cut axonal end within 60 min. The formation of this barrier (seal) was assessed by measuring the decay of injury current at the cut end; its location at the cut end was determined by the exclusion of fluorescent hydrophilic dye at the cut end. When a membrane-incorporating styryl dye was placed in the bath prior to axonal transection and a hydrophilic dye was placed in the bath just after axonal transection, many vesicles near the barrier at the cut axonal end had their limiting membrane labeled with the styryl dye and their contents labeled with the hydrophilic dye, indicating that these vesicles originated from the axolemma by endocytosis. This barrier does not form in Ca2+-free salines. Similar collections of vesicles have been observed at regions of plasmalemmal damage in many cell types. From these and other data, we propose that plasmalemmal lesions in most eukaryotic cells (including axons) are repaired by vesicles, at least some of which arise by endocytosis induced by Ca2+ inflow resulting from the plasmalemmal damage. We describe several models by which vesicles could interact with each other and/or with intact or damaged regions of the plasmalemma to repair small (1-30 microm) plasmalemmal holes or a complete transection of the plasmalemma.

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PubMed1997

Calpain activity promotes the sealing of severed giant axons.

A barrier (seal) must form at the cut ends of a severed axon if a neuron is to survive and eventually regenerate. Following severance of crayfish medial giant axons in physiological saline, vesicles accumulate at the cut end and form a barrier (seal) to ion and dye diffusion. In contrast, squid giant axons do not seal, even though injury-induced vesicles form after axonal transection and accumulate at cut axonal ends. Neither axon seals in Ca2+-free salines. The addition of calpain to the bath saline induces the sealing of squid giant axons, whereas the addition of inhibitors of calpain activity inhibits the sealing of crayfish medial giant axons. These complementary effects involving calpain in two different axons suggest that endogenous calpain activity promotes plasmalemmal repair by vesicles or other membranes which form a plug or a continuous membrane barrier to seal cut axonal ends.

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PubMed1997

Delaminating myelin membranes help seal the cut ends of severed earthworm giant axons.

Transected axons are often assumed to seal by collapse and fusion of the axolemmal leaflets at their cut ends. Using photomicroscopy and electronmicroscopy of fixed tissues and differential interference contrast and confocal fluorescence imaging of living tissues, we examined the proximal and distal cut ends of the pseudomyelinated medial giant axon of the earthworm, Lumbricus terrestris, at 5-60 min post-transection in physiological salines and Ca2+-free salines. In physiological salines, the axolemmal leaflets at the cut ends do not completely collapse, much less fuse, for at least 60 min post-transection. In fact, the axolemma is disrupted for 20-100 microm from the cut end at 5-60 min post-transection. However, a barrier to dye diffusion is observed when hydrophilic or styryl dyes are placed in the bath at 15-30 min post-transection. At 30-60 min post-transection, this barrier to dye diffusion near the cut end is formed amid an accumulation of some single-layered and many multilayered vesicles and other membranous material, much of which resembles delaminated pseudomyelin of the glial sheath. In Ca2+-free salines, this single and multilayered membranous material does not accumulate, and a dye diffusion barrier is not observed. These and other data are consistent with the hypothesis that plasmalemmal damage in eukaryotic cells is repaired by Ca2+-induced vesicles arising from invaginations or evaginations of membranes of various origin which form junctional contacts or fuse with each other and/or the plasmalemma.

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PubMed1997

Cyclosporin A retards the wallerian degeneration of peripheral mammalian axons.

The distal (anucleate) segments of mammalian peripheral axons typically undergo complete Wallerian degeneration within 1-3 days after severance from their cell bodies, unlike invertebrates and lower vertebrates, where anucleate axons do not degenerate for weeks to months. This rapid Wallerian degeneration in mammals could be due to a more efficient immune system and/or to differences in calcium-dependent pathways relative to invertebrates and lower vertebrates. To suppress the immune system and to inhibit calcium-dependent pathways in axons, we gave daily subcutaneous injections of cyclosporin A (CsA: 10 mg/kg) to Sprague-Dawley rats for 7 days before, and 5 days after, severing their right ventral tail nerves. To confirm that CsA suppressed the immune system, white blood cell density was measured in CsA-treated and in non-treated rats. Our data showed that the number of surviving anucleate myelinated axons at 5 postoperative days in CsA-treated rats was significantly higher than the number in non-treated rats. Anucleate unmyelinated axons in the ventral tail nerve also exhibited better survival in CsA-treated rats than in nontreated rats. These results are consistent with the hypothesis that the immune response and/or calcium-dependent pathways play important roles in the rapid Wallerian degeneration of anucleate mammalian axons.

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PubMed1996

Presynaptic calcium currents at voltage-clamped excitor and inhibitor nerve terminals of crayfish.

1. A two-electrode voltage clamp was used to record calcium currents from the excitatory and inhibitory nerve terminals that innervate the crayfish (Procambarus spp.) opener muscle. Other voltage-dependent currents were blocked with tetrodotoxin, 3,4-diaminopyridine, 4-aminopyridine and tetraethylammonium. 2. The presynaptic calcium current at both excitatory and inhibitory synapses was blocked by cadmium and omega-agatoxin IVA but was not affected by omega-conotoxin GVIA, omega-conotoxin MVIIC or nifedipine, suggesting that the calcium currents flow through P-type calcium channels. 3. Current-voltage (I-V) relations at both excitatory and inhibitory synapses are similar, with current activation near -40 mV, peak current near -10 mV and current reversal at membrane potentials greater than +25 mV. I-V relations were scaled along the current axis by partial calcium current blockade with cobalt, suggesting that series resistance and space-clamp errors were small. 4. A subset of terminals on one muscle fibre was locally superfused with a physiological saline containing barium; the rest of the preparation was superfused with a physiological saline containing calcium channel antagonists. Under such conditions the characteristics of the I-V relation were very similar to the I-V relations recorded when the entire preparation was bathed in physiological levels of calcium, suggesting that the space clamp was adequate. 5. Calcium channel activation, as determined from tail current analyses, was similar when the entire preparation was bathed in physiological levels of calcium or if terminals on one muscle fibre were locally superfused with barium. 6. During a 30 ms depolarization, calcium currents inactivated to a greater extent in inhibitory than in excitatory terminals. The inactivation was of small magnitude (< 20%) and was eliminated by intracellular injection of the calcium chelator BAPTA, suggesting that the inactivation was calcium dependent. 7. These data show that biophysical and pharmacological properties of calcium currents at crayfish neuromuscular junctions resemble those found at stellate synapses in squid.

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PubMed1996

Calcium currents, transmitter release and facilitation of release at voltage-clamped crayfish nerve terminals.

1. The presynaptic terminals at crayfish (Procambarus spp.) opener neuromuscular junctions were voltage clamped. Calcium currents were measured during (ICa) and following (tail ICa) presynaptic depolarizations; EPSPs or IPSPs were simultaneously recorded from the (postsynaptic) muscle fibre directly beneath the presynaptic impalement. 2. For short (< or = 6 ms) presynaptic depolarizations, most of the transmitter release occurred during the tail ICa. EPSP or IPSP amplitudes at the end of the 6 ms pulse (end EPSP or end IPSP) increased monotonically with the integral of the ICa ([symbol: see text]ICa). The suppression potential for transmitter release was near the apparent reversal potential for ICa. 3. When the end EPSP or end IPSP amplitude was plotted against the peak ICa elicited during a presynaptic pulse (peak ICa), large and small depolarizations which evoked the same peak ICa evoked different amounts of transmitter release. The differences in transmitter release were eliminated when end EPSP amplitude was plotted against [symbol: see text] ICa, suggesting that transmitter release during a depolarization depends only upon calcium current and not upon a subsequent voltage-dependent step. 4. The synaptic transfer function of various measurements of EPSP or IPSP amplitude vs. [symbol: see text]ICa evoked during a presynaptic depolarization was a power function having an exponent of about 3. Similar measurements of EPSP amplitude vs. [symbol: see text]tail ICa evoked following a presynaptic depolarization had an exponent of about 2. 5. Facilitation of an EPSP or IPSP was not due to increases in calcium current at the test depolarization. 6. When the conditioning depolarization was increased and the test depolarization remained constant, EPSP amplitude at the test depolarization and facilitation increased . When the conditioning depolarization remained constant and the test depolarization was increased, EPSP amplitude at the test depolarization increased, while facilitation decreased. 7. Our data suggested that transmitter release at crayfish neuromuscular junctions is a non-linear function of calcium influx, and that facilitated release utilizes intracellular calcium differently from non-facilitated release. These data contradict simple models of facilitation which combine the residual calcium hypothesis with the calcium co-operativity hypothesis of non-facilitated release.

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PubMed1996

Medium weight neurofilament mRNA in goldfish Mauthner axoplasm.

Although axons are generally considered to lack the ability to synthesize proteins, the Mauthner axon (M-axon) of the goldfish has been reported to contain some of the basic components of the translational machinery, such as transfer RNA (tRNA), ribosomal RNA (rRNA), and ribosomes. To determine if the M-axon also contains mRNA, we isolated samples of M-axoplasm free of glial contamination as demonstrated by the absence of glial-specific mRNA and protein. Reverse transcription-polymerase chain reaction (RT-PCR) of M-axoplasmic cDNA in the presence of primers for the goldfish medium-weight neurofilament (NF-M) gene produced a single product of the expected length for RT-PCR amplification of goldfish NF-M mRNA. This mRNA might direct protein synthesis of NF-M within the M-axoplasm.

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PubMed1996

Mechanisms for the maintenance and eventual degradation of neurofilament proteins in the distal segments of severed goldfish mauthner axons.

Cellular mechanisms that might affect the degradation of neurofilament proteins (NFPs) were examined in the distal segments of severed goldfish Mauthner axons (M-axons), which do not degenerate for more than 2 months after severance. Calpain levels, as determined by reactivity to a polyclonal antibody, remained constant for 80 d postseverance in distal segments of M-axons and then declined from 80 to 85 d postseverance. Calpain activity in rat brain, as determined by a spectrophotometric assay, was much higher than calpain activity in control and severed goldfish brain, spinal cord, muscle, or M-axons. Calpain activity was extremely low in M-axons compared with that in all other tissues and remained low for up to 80 d postseverance in distal segments of M-axons. Phosphorylated NFPs, as determined by Stains-All treatment of SDS gels, were maintained for up to 72 d postseverance and then decreased noticeably at 75 d postseverance when NFP breakdown products appeared on silver-stained gels. By 85 d post-severance, phosphorylated NFPs no longer were detected, and NFP breakdown products were the most prominent bands on silver-stained gels. These results suggest that the distal segments of M-axons survive for months after severance, because NFPs are maintained in a phosphorylated state that stabilizes and protects NFPs from degradation by low levels of calpain activity in the M-axon; the distal segments of severed M-axons degenerate eventually when NFPs no longer are maintained in a phosphorylated state and become susceptible to degradation, possibly by low levels of calpain activity in the M-axon.

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