Biomedical subjects

G D Bittner

Publications and source records attributed to G D Bittner.

At least 55 records · Page 3Linked to original sources

PubMed1992

Effects of ethanol and other drugs on excitatory and inhibitory neurotransmission in the crayfish.

1. Crayfish exposed to 434 mM ethanol (EtOH) showed signs of hyperactivity within 0.5-2 h, at which times crayfish hemolymph EtOH concentration had reached 60-90 mM. 2. A 10-min exposure to 60-90 mM EtOH reduced presynaptic inhibition of excitatory postsynaptic currents (EPSCs) at the crayfish opener neuromuscular junction (NMJ) in vitro but did not significantly alter excitatory neurotransmission. The same concentrations of EtOH did not alter other potentials or currents associated with inhibition at this synapse, such as presynaptic inhibitory potentials (PIPs), inhibitory postsynaptic potentials (IPSPs), and inhibitory postsynaptic currents (IPSCs). 3. Intermediate EtOH concentrations (120-180 mM) applied for 10 min in vitro reduced the amplitude of excitatory postsynaptic potentials (EPSPs) by decreasing the membrane resistance of opener muscle fibers and by reducing the amplitude of EPSCs. 4. High EtOH concentrations (434 mM) applied for 10 min in vitro had yet greater depressive effects on measures of postsynaptic properties described above. The time course of EPSCs was also significantly reduced. In addition, presynaptic properties such as action-potential (AP) amplitude and frequency of spontaneous release of neurotransmitter were reduced by 434 mM EtOH. 5. Presynaptic inhibition, gamma-aminobutyric acid (GABA; 250-500 microM), muscimol (50 microM), and baclofen (75 microM) all reduced the depolarizing afterpotential of APs in the excitor axon and reduced EPSPs in opener muscle fibers. GABA (500 microM) and baclofen (75 microM) significantly reduced presynaptic AP amplitudes, whereas presynaptic inhibition, GABA (250 microM), and muscimol (50 microM) had no effect on AP amplitude. Bicuculline (250-500 microM), a GABAA antagonist, did not entirely eliminate presynaptic inhibition, whereas picrotoxin (50 microM), another GABAA antagonist, completely removed presynaptic inhibition. Thus presynaptic inhibitory mechanisms may involve both GABAA and GABAB receptors on the opener excitor axon. 6. Our data suggest that the behavioral hyperactivity seen at hemolymph EtOH concentrations of 60-90 mM is not accompanied by a change in excitatory synaptic transmission observed at the opener NMJ. Rather, crayfish hyperactivity may be due to depressive effects of EtOH on inhibitory synapses in the CNS similar to the disinhibition evoked by EtOH at the opener NMJ.

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PubMed1991

Long-term survival of severed crayfish giant axons is not associated with an incorporation of glial nuclei into axoplasm.

Glial nuclei have been reported to be incorporated into the axoplasm of surviving distal stumps (anucleate axons) weeks to months after lesioning abdominal motor axons in rock lobsters. We have not observed this phenomenon in crayfish medial giant axons (MGAs) which also survive for weeks to months after lesioning. Glial nuclei were not observed within MGAs perfused with a physiological intracellular saline. However, incorporation of glial nuclei was observed after MGAs were perfused with intracellular salines containing Fast green. From these and previously published data, we confirm that glial incorporation into axoplasm can occur, but we suggest that is is not a common mechanism used by crustaceans to provide for long-term survival of anucleate axons.

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PubMed1991

Rapid artificial restoration of electrical continuity across a crush lesion of a giant axon.

Action potentials never conducted through a crush lesion to the medial giant axon in the earthworm (Lumbricus terrestris) if the axon was exposed to normal or hypotonic salines that did not contain polyethylene glycol. However, action potentials, as well as electrotonic potentials, often conducted through a crush lesion exposed for 1 min to polyethylene glycol in hypotonic saline.

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PubMed1991

Synaptic plasticity at crayfish neuromuscular junctions: facilitation and augmentation.

Simultaneous intracellular recordings from presynaptic nerve terminals and postsynaptic muscle fibers were used to investigate the extent to which changes in presynaptic voltage may contribute to short-term facilitation and augmentation of transmitter release at neuromuscular junctions of the crayfish (Procambarus simulans) opener muscle. Presynaptic nerve terminals have an average resting membrane potential of about -80 mV, single action potentials have an average foot-to-peak amplitude of about 98 mV, and single action potentials are followed by a depolarizing after potential (DAP) of about 10 mV. During stimulus trains of 9-16 impulses at 100 Hz, amplitudes of excitatory postsynaptic potentials (EPSPs) continuously facilitate up to 100-fold. This dramatic facilitation is associated with only slight increases in the peak voltage and duration of APs for the first 2-4 pulses in such a stimulus train. Foot-to-peak total amplitude of APs usually decreases after the first pulse in a stimulus train. The data strongly suggest that short-term facilitation is not due to changes in the amplitude or duration of APs invading the presynaptic terminal. Upon cessation of a longer stimulus train, the presynaptic terminal exhibits a hyperpolarizing after potential (HAP) up to 16 mV in amplitude depending upon the frequency (10-100 Hz) and duration (1-10 sec) of the tetanic stimulation. This post-tetanic HAP decays with a time constant of 10-20 sec, which is approximately equal to the third time constant of decay in EPSP amplitude (augmentation) following tetanic stimulation. Hence, presynaptic voltage changes and/or processes associated with these voltage changes (e.g., accumulation of ions, changes in ionic conductances, etc.) may be partly responsible for augmentation of EPSP amplitudes.

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PubMed1991

Synaptic plasticity at crayfish neuromuscular junctions: presynaptic inhibition.

Intracellular recordings at sites electronically near terminals of the opener excitor axon in the claw of crayfish (Procambarus simulans) show that stimulation of the inhibitor neuron produces hyperpolarizing or depolarizing presynaptic inhibitory potentials (PIPs). GABA applied anywhere along the length of the opener excitor or inhibitor axons also produces hyperpolarizing or depolarizing potentials. The amplitude of action potentials (APs) at recording sites near some excitor terminals is reduced by an average of 6 mV during presynaptic inhibition, which also reduces excitatory postsynaptic potentials (EPSPs) by 50-70%. The time course of AP reduction equals the time course of EPSP reduction and the amount of AP reduction is independent of the sign or amplitude of the PIPs. All these data are consistent with a hypothesis that a conductance increase produced by GABA in these presynaptic terminals of the excitor axon is responsible for presynaptic inhibition. However, the effect of presynaptic inhibition upon the accumulation of short-term facilitation of excitatory transmitter release is not the same in all muscle fibers. In some terminals, the accumulation of short-term facilitation during short, high-frequency trains of action potentials which are presynaptically inhibited often equals the accumulation of facilitation without inhibition. In other terminals, short-term facilitation accumulated during presynaptic inhibition often does not equal facilitation accumulated in the absence of presynaptic inhibition. These data suggest that some other factor which may contribute to presynaptic inhibition, such as a direct effect to decrease calcium currents, may also affect short-term facilitation in some terminals.

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PubMed1991

Long-term survival of anucleate axons and its implications for nerve regeneration.

Severed distal segments of nerve axons (anucleate axons) have now been reported to survive for weeks to years in representative organisms from most phyla, including the vertebrates. Among invertebrates (especially crustaceans), such long-term survival might involve transfer of proteins from adjacent intact cells to anucleate axons. In lower vertebrates and mammals, long-term survival of anucleate axons is more likely attributed to a slow turnover of axonal proteins and/or a lack of phagocytosis by macrophages or other cell types. Invertebrate anucleate axons that exhibit long-term survival are often reactivated by neurites that have grown from proximal nucleate segments. In mammals, induction of long-term survival in anucleate axons might allow more time to use artificial mechanisms to repair nerve axons by fusing the two severed halves with polyethylene glycol, a technique recently developed to fuse severed halves of myelinated axons in earthworms.

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PubMed1991

Calcium-activated potassium conductance in presynaptic terminals at the crayfish neuromuscular junction.

Membrane potential changes that typically evoke transmitter release were studied by recording intracellularly from the excitor axon near presynaptic terminals of the crayfish opener neuromuscular junction. Depolarization of the presynaptic terminal with intracellular current pulses activated a conductance that caused a decrease in depolarization during the constant current pulse. This conductance was identified as a calcium-activated potassium conductance, gK(Ca), by its disappearance in a zero-calcium/EGTA medium and its block by cadmium, barium, tetraethylammonium ions, and charybdotoxin. In addition to gK(Ca), a delayed rectifier potassium conductance (gK) is present in or near the presynaptic terminal. Both these potassium conductances are involved in the repolarization of the membrane during a presynaptic action potential.

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PubMed1991

Presynaptic facilitation at the crayfish neuromuscular junction. Role of calcium-activated potassium conductance.

Membrane potential was recorded intracellularly near presynaptic terminals of the excitor axon of the crayfish opener neuromuscular junction (NMJ), while transmitter release was recorded postsynaptically. This study focused on the effects of a presynaptic calcium-activated potassium conductance, gK(Ca), on the transmitter release evoked by single and paired depolarizing current pulses. Blocking gK(Ca) by adding tetraethylammonium ion (TEA; 5-20 mM) to a solution containing tetrodotoxin and aminopyridines caused the relation between presynaptic potential and transmitter release to steepen and shift to less depolarized potentials. When two depolarizing current pulses were applied at 20-ms intervals with gK(Ca) not blocked, the presynaptic voltage change to the second (test) pulse was inversely related to the amplitude of the first (conditioning) pulse. This effect of the conditioning prepulse on the response to the test pulse was eliminated by 20 mM TEA and by solutions containing 0 mM Ca2+/1 mM EGTA, suggesting that the reduction in the amplitude of the test pulse was due to activation of gK(Ca) by calcium remaining from the conditioning pulse. In the absence of TEA, facilitation of transmitter release evoked by a test pulse increased as the conditioning pulse grew from -40 to -20 mV, but then decreased with further increase in the conditioning depolarization. A similar nonmonotonic relationship between facilitation and the amplitude of the conditioning depolarization was reported in previous studies using extracellular recording, and interpreted as supporting an additional voltage-dependent step in the activation of transmitter release. We suggest that this result was due instead to activation of a gK(Ca) by the conditioning depolarization, since facilitation of transmitter release increased monotonically with the amplitude of the conditioning depolarization, and the early time course of the decay of facilitation was prolonged when gK(Ca) was blocked. The different time courses for decay of the presynaptic potential (20 ms) and facilitation (greater than 50 ms) suggest either that residual free calcium does not account for facilitation at the crayfish NMJ or that the transmitter release mechanism has a markedly higher affinity or stoichiometry for internal free calcium than does gK(Ca). Finally, our data suggest that the calcium channels responsible for transmitter release at the crayfish NMJ are not of the L, N, or T type.

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PubMed1990

Effect of temperature on long-term survival of anucleate giant axons in crayfish and goldfish.

The effect of temperature on the electrophysiology and morphology of anucleate axons was examined following severance of crayfish medial giant axons and goldfish Mauthner axons from their respective cell bodies. Although anucleate segments of each giant axon exhibited long-term survival for weeks to months at 5-25 degrees C in crayfish and 10-30 degrees C in goldfish, the two axons differed in their survival characteristics. All measures of long-term survival in crayfish medial giant axons were independent of animal holding temperature, whereas all measures in Mauthner axons were dependent on holding temperature. Medial giant axons survived for at least 90 days in crayfish maintained at 5-25 degrees C in this and previous studies. Mauthner axons survived for over 5 months in goldfish maintained at 10 degrees C but survived for 1 month at 30 degrees C. Postoperative time had different effects on many single measures of long-term survival (axonal diameter, amplitude of action or resting potentials) in medial giant axons compared to Mauthner axons. For example, resting and action potentials in crayfish medial giant axons remained remarkably constant at all holding temperatures for 0-90 postoperative days. In contrast, resting and action potentials in goldfish Mauthner axons declined abruptly in the first 10-20 postoperative days followed by a slower decline at each holding temperature. We suggest that the mechanism of long-term survival is not necessarily the same in all anucleate axons.

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PubMed1990

Rapid morphological fusion of severed myelinated axons by polyethylene glycol.

We are able to morphologically fuse the severed halves of an invertebrate-myelinated axon by application of polyethylene glycol (PEG) to closely apposed cut ends. Morphological fusion of the medial giant axon (MGA) of the earthworm Lumbricus terrestris is defined as axoplasmic and axolemmal continuity in serial longitudinal sections of MGAs taken through the fusion site as viewed with light or electron microscopes. Morphological continuity is also shown by the transfer of Lucifer yellow dye between apposed MGA segments fused with PEG, but not between apposed MGA segments in normal or hypotonic saline without PEG application. PEG-induced MGA fusion rates can be as high as 80-100% with an appropriate choice of PEG concentration and molecular mass, tight apposition and careful alignment of the cut ends, and treatment with hypotonic salines containing reduced calcium and increased magnesium. A variant of this technique might produce rapid repair of severed mammalian-myelinated axons.

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PubMed1989

Synaptic plasticity at the crayfish opener neuromuscular preparation.

The crayfish opener neuromuscular preparation exhibits most of the plasticities yet described for any synapse, including facilitation, long-term potentiation, presynaptic inhibition, and modulation. Since the presynaptic terminals and postsynaptic muscle fibers can both be intracellularly penetrated, one can now more easily examine the cellular/molecular bases for these plasticities. Data from such studies suggest that facilitation may be influenced by something other than residual free calcium and that presynaptic inhibition is produced by a conductance increase to chloride in the terminals of the excitor axon. Several drugs (ethanol, pentobarbital) have significant effects on these synaptic plasticities over concentration ranges which produce obvious behavioral effects in crayfish and mammals. Hence, this preparation should be a useful model system to determine cellular/molecular bases for various synaptic plasticities and the effects of drugs on these plasticities.

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PubMed1989

Effects of stimulus timing on transmitter release and postsynaptic membrane potential at crayfish neuromuscular junctions.

Different synaptic terminals of the single excitor axon to the opener muscle of crayfish (Procambarus clarkii) often release transmitter in a very different manner when stimulated with the same equal-interval, doublet, or triplet patterns. Compared to synapses that show little facilitation (low Fe synapses), highly facilitating (high Fe) synapses show greater percentage increases in several measures of synaptic efficacy when stimulated with any of these patterns. Low Fe synapses usually show the greater absolute changes in these measures of synaptic efficacy. Changes in the span and pattern of doublets and triplets can independently affect both pre- and postsynaptic measures of synaptic efficacy at either low Fe or high Fe synapses.

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PubMed1988

Effects of pentobarbital on behavioral and synaptic plasticities in crayfish.

Intra-abdominal injections of 90 mg/kg (3.5 x 10(-4) M estimated body concentration) sodium pentobarbital (PB) eliminated the cheliped closing response and eyestalk withdrawal response in crayfish (Procambarus clarkii). Repeated injections produced tolerance to both of these behavioral measures within several days. At glutamatergic synapses of the opener muscle, PB at dosages from 10(-7) to 10(-4) M had no significant effect on non-facilitated transmitter release evoked by 1 Hz stimuli. Facilitated transmitter release at 10 Hz stimuli was significantly decreased by 10(-3) M PB but was not significantly affected at lower concentrations. At 10(-4) M and 10(-3) M, PB significantly reduced the ratio of excitatory postsynaptic potential (EPSP) amplitudes at 10 Hz to those at 1 Hz. The frequency of spontaneous miniature EPSPs (MEPSPs) was reduced by 10(-4) M PB to about half of the control level, while MEPSP amplitudes and time constants were not significantly affected. These results suggest that the ability of PB to depress various crayfish behaviors is due, at least in part, to a presynaptic ability of the drug to depress facilitation of transmitter release at glutamatergic synapses.

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PubMed1988

Protein transport between crayfish lateral giant axons.

Segmental lateral giant axons (SLGAs) in crayfish were used to determine whether functionally intact proteins can move between axons under physiological conditions. Horseradish peroxidase (HRP) was chosen as the tracer protein because its localization requires intact enzymatic activity and because it can be localized in living cells using a non-cytotoxic procedure. Following iontophoretic injection of HRP in a single SLGA, HRP often transferred to adjacent SLGAs. HRP transferred from an injected SLGA to a caudal SLGA with greater frequency than HRP transferred to a rostral SLGA. When HRP transferred between SLGAs, it was ultrastructurally associated with vesicles on both sides of septate junctions between adjacent SLGAs and was also seen in the perijunctional extracellular space. There was no difference between the electrical resistance of synapses at which HRP transferred and those synapses where HRP did not transfer. HRP transfer was significantly reduced when axons were bathed in reduced calcium saline. These and other observations indicate that axon-to-axon transport in this system is accomplished by exocytosis of HRP from injected axons followed by its endocytotic uptake by adjacent, non-injected axons. Similar transfer of endogenous proteins may contribute to the long-term survival for months to years of distal stumps of severed SLGAs.

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PubMed1988

Electrophysiological and behavioral effects of ethanol on crayfish.

The effects of EtOH on the crayfish Procambarus clarkii and P. simulans were examined behaviorally in vivo and electrophysiologically in vitro on pre- and postjunctional mechanisms of synaptic plasticity at opener excitor nerve-muscle junctions. Addition of 75 mM EtOH to the bath water of holding tanks produced 47 to 54 mM EtOH levels in the hemolymph (blood) within 24 hr. These hemolymph EtOH levels were maintained for weeks by daily changes of the bath water containing 75 mM EtOH. After 24 hr of exposure to 75 to 150 mM EtOH in vivo, crayfish showed behavioral signs of intoxication as measured by a significant increase in righting reflex times and a significant decrease in tail-flip escape behavior. After 2 weeks of chronic exposure to 75 mM EtOH, crayfish showed behavioral tolerance as measured by a decrease in righting time and an increase in tail-flip escape behavior to control levels. EtOH applied acutely to opener nerve-muscle preparations in vitro at 10 to 100 mM concentrations produced an increased probability of transmitter release as measured by an increased frequency of spontaneous release of transmitter quanta and an increased amplitude of facilitated synaptic potentials evoked by 10 to 40 Hz stimulation of the excitor axon. Acute application of 300 to 600 mM EtOH resulted in a decreased amplitude of facilitated synaptic potentials due primarily to a decrease in postsynaptic input resistance. These data suggest that EtOH has a concentration-dependent biphasic effect on synaptic transmission.

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PubMed1987

Developmental and other factors affecting regeneration of crayfish CNS axons.

According to histological and ultrastructural criteria, nongiant CNS axons in newly hatched crayfish regenerate more rapidly and with greater frequency than do similar axons in adult crayfish. Regenerative ability is greater in one species (Procambarus clarkii) than in another species (Procambarus simulans), is greater at 20-25 degrees C than at 15-16 degrees C, and is greater in nongiant axons than in giant axons. In contrast to axonal regeneration, nerve cell bodies do not regenerate in newly hatched or adult crayfish of either species. While the ability to regenerate CNS axons differs between newly hatched and adult crayfish, the ultrastructural appearance of the CNS is very similar at any age it is examined.

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PubMed1987

Organization of axoplasm in crayfish giant axons.

Distributions of subcellular organelles in medial giant axons (MGAs) and segmental lateral giant axons (SLGAs) of crayfish were evaluated as part of an ongoing effort to understand and explain differences in distal stump survival following axonotomy. Both axons were able to endocytose tracer proteins placed extracellularly, and horseradish peroxidase injected by cannulation into MGAs could transfer into adaxonal glial cells. Concentrations of tubulovesicular organelles near axonal cell membranes were measured as a possible index of the relative level of axonal endo- and exocytosis, and concentrations in MGAs were found to be twice those in SLGAs. In both axons, microtubule concentrations were highest near the axolemma and lowest in the central core of axoplasm. In thoracic and abdominal regions of MGAs, microtubules and other organelles were located only in a thin layer of subaxolemmal axoplasm. Overall, MGAs contained fewer microtubules per cross-section than did SLGAs, although MGAs are five to ten times as long as SLGAs and support more synapses. Total numbers of microtubules per cross-section varied with distance from the cell body of an MGA, whereas microtubule numbers were similar in proximal and distal cross-sections of SLGAs. In addition to a layer of subaxolemmal mitochondria which was observed in MGAs and SLGAs and which is characteristic of crayfish axons, mitochondria were also concentrated in the central core of SLGA axoplasm.

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PubMed1986

Reconnection of severed nerve axons with polyethylene glycol.

Severed medial giant axons in crayfish can be rejoined in vitro with polyethylene glycol (PEG) to produce axoplasmic continuity and through transmission of action potentials. Severed axon-like processes of a mammalian neuroblastoma/glioma cell line seem to be rejoined to the cell body using PEG in tissue culture. Our data suggest that PEG might be used to rejoin severed axons in vivo in various organisms.

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