Biomedical subjects

D L Eng

Publications and source records attributed to D L Eng.

11 recordsLinked to original sources

PubMed1997

Expression of glutamate uptake transporters after dibutyryl cyclic AMP differentiation and traumatic injury in cultured astrocytes.

Our findings indicate that differentiation of primary astrocytes by dibutyryl cyclic adenosine monophosphate (dBcAMP) and scratch injury together resulted in increased glutamate transporter gene expression. Confluent primary cultures were prepared from cerebral cortex of normal new born rat pups. The primary cultures were then divided into four groups each: control and scratch-injured, and dBcAMP-treated control and scratch-injured cultures. Total RNA was extracted at 0, 1, 2, 4, and 7 days after injury. Expression of the electrogenic glutamate transporters, GLAST, GLT-1, and EAAC-1, was quantitated by the reverse transcriptase-polymerase chain reaction method (RT-PCR) and slot blot hybridization followed by densitometric scanning. Triplicate cultures were analyzed for each time-point. Our studies indicate that all these astrocyte cultures expressed the two glial transporters, GLAST and GLT-1, while none of the cultures expressed the neuronal transporter, EAAC-1. The expression of the two transporters in the dBcAMP-treated primary cultures were markedly increased from the non-treated cultures. The dBcAMP-treated cultures had 2- to 4-times increase in levels of GLAST and GLT-1-mRNA expression both before and after scratch injury, as compared to untreated non-injured and injured primary cultures. All of the cultures expressed GLAST in greater proportion than GLT-1.

ATP-Binding Cassette Transporters↗

PubMed1995

Enhancement of GABAA receptor-mediated conductances induced by nerve injury in a subclass of sensory neurons.

1. The effects of axotomy on the electrophysiologic properties of adult rat dorsal root ganglion (DRG) neurons were studied to understand the changes in excitability induced by traumatic nerve injury. Nerve injury was induced in vivo by sciatic nerve ligation with distal nerve transection. Two to four weeks after nerve ligation, a time when a neuroma forms, lumbar (L4 and L5) DRG neurons were removed and placed in short-term tissue culture. Whole cell patch-clamp recordings were made 5-24 h after plating. 2. DRG neurons were grouped into large (43-65 microns)-, medium (34-42 microns)-, and small (20-32 microns)- sized classes. Large neurons had short duration action potentials with approximately 60% having inflections on the falling phase of their action potentials. In contrast, action potentials of medium and small neurons were longer in duration and approximately 68% had inflections. 3. Pressure microejection of gamma-aminobutyric acid (GABA, 100 microM) or muscimol (100 microM) onto voltage-clamped DRG neurons elicited a rapidly desensitizing inward current that was blocked by 200 microM bicuculline. To measure the peak conductance induced by GABA or muscimol, neurons were voltage-clamped at a holding potential of -60 mV, and pulses to -80 mV and -100 mV were applied at a rate of 2.5 or 5 Hz during drug application. Slope conductances were calculated from plots of whole cell current measured at each of these potentials. 4. GABA-induced currents and conductances of control DRG neurons increased progressively with cell diameter. The mean GABA conductance was 36 +/- 10 nS (mean +/- SE) in small neurons, 124 +/- 21 nS in medium neurons, and 527 +/- 65 nS in large neurons. 5. After axotomy, medium neurons had significantly larger GABA-induced conductances compared with medium control neurons (390 +/- 50 vs. 124 +/- 21; P < 0.001). The increase in GABA conductance of medium neurons was associated with a decrease in duration of action potentials. In contrast, small neurons had no change in GABA conductance or action potential duration after ligation. The GABA conductance of large control neurons was highly variable, and ligation resulted in an increase that was significant only for neurons > 50 microns. The mean action potential duration in large neurons was not significantly changed, but neurons with inflections on the falling phase of the action potential were less common after ligation. There was no difference in resting potential or input resistance between control and ligated groups, except that the resting potential was less negative in small cells after axotomy.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1992

Three types of sodium channels in adult rat dorsal root ganglion neurons.

Several types of Na+ currents have previously been demonstrated in dorsal root ganglion (DRG) neurons isolated from neonatal rats, but their expression in adult neurons has not been studied. Na+ current properties in adult dorsal root ganglion (DRG) neurons of defined size class were investigated in isolated neurons maintained in primary culture using a combination of microelectrode current clamp, patch voltage clamp and immunocytochemical techniques. Intracellular current clamp recordings identified differing relative contributions of TTX-sensitive and -resistant inward currents to action potential waveforms in DRG neuronal populations of defined size. Patch voltage clamp recordings identified three distinct kinetic types of Na+ current differentially distributed among these size classes of DRG neurons. 'Small' DRG neurons co-express two types of Na+ current: (i) a rapidly-inactivating, TTX-sensitive 'fast' current and (ii) a slowly-activating and -inactivating, TTX-resistant 'slow' current. The TTX-sensitive Na+ current in these cells was almost completely inactivated at typical resting potentials. 'Large' cells expressed a single TTX-sensitive Na+ current identified as 'intermediate' by its inactivation rate constants. 'Medium'-sized neurons either co-expressed 'fast' and 'slow' current or expressed only 'intermediate' current. Na+ channel expression in these size classes was also measured by immunocytochemical techniques. An antibody against brain-type Na+ channels (Ab7493)10 labeled small and large neurons with similar intensity. These results demonstrate that three types of Na+ currents can be detected which correlate with electrogenic properties of physiologically and anatomically distinct populations of adult rat DRG neurons.

Action Potentials↗

PubMed1992

Intranuclear Ca2+ transients during neurite regeneration of an adult mammalian neuron.

Depolarization-induced increases in cytoplasmic and intranuclear Ca2+ were visualized in adult mammalian dorsal root ganglion (DRG) neurons during different stages of neurite extension by using confocal laser scanning microscopy and the long-wavelength Ca2+ indicator dye fluo 3-AM (acetoxymethyl ester of fluo 3). In neurons beginning to extend neurites, depolarization led to pronounced increases in nuclear and nucleolar Ca2+ levels severalfold greater than corresponding increases in the cytoplasm. The nucleolar Ca2+ signal often exceeded that of the nucleus, indicating regional heterogeneity of the nucleus. The subcellular calcium transients were dependent on extracellular Ca2+ and the level of depolarization, indicating the importance of transmembrane Ca2+ fluxes in triggering the nuclear events. After neurite extension, the nuclear Ca2+ signals were attenuated and never exceeded cytoplasmic levels. These results indicate that activity-dependent modulation of intranuclear Ca2+ levels is greater in DRG neurons during early neurite extension. Given the importance of Ca2+ in gene expression, the results may be relevant to Ca(2+)-dependent nuclear events responsible for axonal regeneration.

Animals↗

PubMed1990

Current-clamp analysis of a time-dependent rectification in rat optic nerve.

1. Rat optic nerves were studied using intra-axonal and whole-nerve recording techniques in a sucrose-gap chamber. Constant-current pulses were applied across the outer compartments of the chamber to achieve a current clamp. 2. The nerves displayed a prominent time-dependent conductance increase elicited by a hyperpolarizing constant-current pulse, as evidenced by a relaxation or 'sag' in membrane potential towards resting potential. The inward current began at about 80 ms and reached a steady level over the next 100-200 ms. Its magnitude progressively increased with increasing levels of hyperpolarization. 3. The inward current elicited by hyperpolarization was reduced, but not abolished, when Na+ was reduced from the normal bath concentration of 151 mM to 0 mM. In Na(+)-free solutions the bath K+ concentration, [K+]o, was varied between 0 and 5 mM; the inward current was greatest when [K+]o was 5 mM and was abolished when [K+]o was zero. 4. The inward current was not abolished by tetrodotoxin (TTX), tetraethylammonium (TEA) or 4-aminopyridine (4-AP) suggesting that conventional voltage-dependent sodium and potassium channels do not underlie the time-dependent conductance increase. Low concentrations of Cs+ completely blocked the inward current, and Ba2+ induced a partial block. External application of divalent cations (Cd2+ and Mg2+) did not block the inward current. These properties are similar to the inwardly rectifying conductance observed in a central nervous system neurone. 5. Stimulus-response curves obtained during the hyperpolarization pulse, before and during the conductance increase, indicate that excitability is increased during the conductance increase. This along with the intra-axonal recordings demonstrates that the origin of the increased conductance is axonal and not glial. 6. It is concluded that central nervous system myelinated fibres in rat optic nerve display a prominent time-dependent conductance increase in response to hyperpolarization that depends on both Na+ and K+ and is blocked by Cs+. This conductance is similar to an inward rectifier described for a variety of neurone types. The increased axonal excitability observed during the conductance increase suggests that its functional role may be to maintain or stabilize axonal excitability during periods of intense action potential activity.

4-Aminopyridine↗

PubMed1988

Development of 4-AP and TEA sensitivities in mammalian myelinated nerve fibers.

1. The sensitivities of mammalian myelinated axons to potassium channel blockers was studied over the course of development using in vitro sucrose gap and intra-axonal recording techniques. 2. Application of 4-aminopyridine (4-AP; 1.0 mM) to young nerves led to a delay in return to base line of the sciatic nerve compound action potential and to a postspike positivity (indicative of hyperpolarization) lasting for tens of milliseconds. These effects were very much attenuated during the course of maturation. 3. Tetraethylammonium chloride (TEA; 10 mM) application alone had little effect on the waveform of the compound action potential at any age. However, the 4-AP-induced postspike positivity was blocked by TEA, Ba/+, and Cs+. This block was observed in Ca2+-free electrolyte solutions containing EGTA (1.0 mM). 4. Immature sciatic nerves (approximately 3 wk postnatal) were incubated in a potassium-free electrolyte solution containing 120 mM CsCl for up to 1 h in an attempt to replace internal potassium with cesium. When the nerves were tested in the sucrose gap chamber using solutions containing 3.0 mM CsCl substituted for KCl, the compound action potential was broadened and a prolonged depolarization appeared, but there was no postspike positivity; the CsCl effect was similar to the combined effects of 4-AP and TEA. 5. Intra-axonal recordings were obtained to study the effects of 4-AP and TEA on individual axons. In the presence of 4-AP a single stimulus led to a burst of action potentials followed by a pronounced afterhyperpolarization (AHP) in sensory fibers. The AHP was blocked by TEA. In motor fibers 4-AP application resulted in action potential broadening with no AHP. 6. Repetitive stimulation (200-500 Hz; 100 ms) was followed by a pronounced AHP in both sensory and motor fibers at all ages studied. This activity-elicited AHP was sensitive to TEA at all ages. 7. The results indicate that 4-AP and TEA sensitivity change over the course of development in rat sciatic nerve. The effects of 4-AP are much more pronounced in immature nerves than in mature nerves, suggesting that 4-AP-sensitive channels become masked as they are covered by myelin during maturation. However, the TEA-sensitive channels, demonstrable after repetitive firing, remain accessible to TEA after myelination. These channels therefore may have a nodal representation.

4-Aminopyridine↗

PubMed1987

Functional differences between 4-aminopyridine and tetraethylammonium-sensitive potassium channels in myelinated axons.

Intracellular recordings from rat sciatic nerve fibers showed that the potassium channel blocking agents 4-aminopyridine (4-AP) and tetraethylammonium (TEA) had different effects on action potential waveform. When applied alone, TEA did not appreciably alter the waveform of an individual action potential, whereas 4-AP application resulted in action potential broadening and, in some axons, repetitive firing. A prolonged afterhyperpolarization which was blocked by TEA occurred subsequent to repetitive firing. These results indicate the presence of at least two pharmacologically defined potassium channels in mammalian peripheral nerve fibers. The 4-AP-sensitive potassium channels are important for rapid action potential repolarization and the TEA-sensitive potassium channels may serve to modulate axonal excitability during repetitive firing.

4-Aminopyridine↗

PubMed1987

Activity-dependent changes in extracellular potassium and excitability in turtle olfactory nerve.

The excitability properties of turtle olfactory nerve (o.n.) were studied in vitro using potassium-sensitive microelectrodes (KSM), a modified sucrose gap chamber, and a standard nerve chamber to measure conduction velocity. A pronounced supernormal period (SNP), as indicated by increased conduction velocity of the o.n. fiber volley, lasting up to several seconds, was observed following a single stimulus. The compound action potential recorded in the sucrose gap chamber showed a prolonged depolarization with a similar time course to the SNP. When stimulation intensity was submaximal the response amplitude, and the extracellular potassium concentration [K+]o, continuously increased during repetitive stimulation. In contrast, when supramaximal stimuli were applied, the amplitude of the o.n. fiber volley was reduced during a high-frequency stimulus train for all responses after the initial one even though latency was maximally reduced, i.e., during supernormal conduction. Superfusion with various levels of K+ elicited changes in the excitability of the o.n. fibers. Small increases in [K+]o above the resting concentration of 2.6 mM led to an increase in resting excitability, whereas larger increases resulted in decreased excitability and conduction block. The SNP was eliminated when extracellular potassium was elevated between 3 and 4 mM above resting levels. Microstimulation of a small bundle of o.n. fibers led to an increase in [K+]o along the bundle but also around adjacent nonactivated fibers. The excitability of these neighboring nonactivated fibers was increased, further indicating the importance of activity-dependent changes in [K+]o in modulating axonal excitability. These results demonstrate the importance of activity-dependent increases in extracellular potassium in modulating nonmyelinated o.n. fiber excitability. They also indicate that increases in [K+]o and an associated membrane depolarization contribute to the increased excitability observed during fiber recruitment and the supernormal period.

Animals↗

PubMed1985

Ligature-induced injury in peripheral nerve: electrophysiological observations on changes in action potential characteristics following blockade of potassium conductance.

The effects of the potassium channel blocking agent 4-aminopyridine (4-AP) on action potential properties were studied in chronically injured rat sciatic nerves. In normal, mature myelinated fibers, application of 4-AP does not lead to any significant change in action potential waveform or firing pattern in response to single stimuli. In contrast, application of 4-AP to nerves injured by the placement of loose ligatures results in the appearance of late rippled components in the compound action potential. This alteration in waveform is present at the injury site, but not at nerve segments proximal or distal to this region. Paired stimulation experiments demonstrate that this oscillation of the whole nerve response reflects repetitive firing in response to single stimuli following application of 4-AP. Intra-axonal recording following 4-AP application demonstrates bursts of action potentials, with several spikes of reduced amplitude arising from a depolarizing potential following the initial spike. Refractory period for the late spike is greater than that of the primary action potential. These results demonstrate that potassium channels are present and functional in chronically injured nerves, where blockage of these channels results in repetitive firing in response to single stimuli.

4-Aminopyridine↗

PubMed1984

Adenosine selectively blocks parallel-fiber-mediated synaptic potentials in rat cerebellar cortex.

Electrophysiological techniques were used to study the efficacy of adenosine in modulating synaptic transmission mediated from convergent parallel- and climbing-fiber inputs to Purkinje cells. Our results indicate that adenosine application leads to selective blocking of parallel fiber-mediated synaptic activity but not of climbing fiber activity. Adenosine does not alter the action-potential excitability properties of the parallel fibers. However, application of gamma-aminobutyric acid (GABA), which directly affects Purkinje cell dendritic membranes [Malenka, R. C. & Kocsis, J. D. (1982) J. Neurophysiol. 48, 608-621], leads to reduction of both parallel- and climbing-fiber synaptic activity. These results support the proposals that adenosine receptors in the cerebellar cortex are selectively localized on the nonmyelinated parallel fibers and that the blocking action of adenosine is the result of a mechanism other than direct alteration of axon excitability.

Adenosine↗

PubMed1980

Immunocytologic analyses of 10 nm intermediate filaments in the nervous system of Myxicola.

Antibodies prepared in rabbits against Myxicola infundibulum neurofilaments have been employed to stain neurofilaments immunohistochemically in intact Myxicola infundibulum nervous tissue. Paraffin-embedded and frozen sections (5--6 mu) were examined at the light microscopic level with Sternberger's peroxidase-antiperoxidase method, and Vibratome (20--40 mu) sections were studied at the ultrastructural level with Nakane's conjugated peroxidase method. The neurofilament antibody stained only neurons and axons at the light microscopic level. The staining pattern at the electron microscopic level corresponded to the neurofilaments within axons and neurons. Glial cells, which surround the axons, contain large bundles of filaments that resemble astrocytic filaments in mammalian astrocytes. These filaments do not stain with the anti-neurofilament antibody. Neurons, neurofilaments, glial cells, glial filaments, and nonnervous tissue showed no peroxidase staining when specific antiserum absorbed with neurofilaments was used. These structures were also unstained when antiserum to the glial fibrillary acidic protein of mammalian central nervous system astrocytes was substituted for the neurofilament antiserum. Therefore, in Myxicola infundibulum, the antigenic determinants of the neurofilament protein, as recognized immunohistochemically by anti-neurofilament protein antibodies, are not shared with those of glial filaments.

Animals↗