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Biomedical subjects

J A Freeman

Publications and source records attributed to J A Freeman.

At least 109 records · Page 6Linked to original sources
PubMed1988

Goldfish retinotectal transmission in vitro: component current sink-source pairs isolated by varying calcium and magnesium levels.

Field potentials and radial current source-densities (CSDs) evoked by optic tract stimulation were observed in goldfish tectum in vitro. The effects of different concentrations of calcium and magnesium were studied to improve understanding of component events in retinotectal transmission and interpretations of effects of pharmacological agents. Responses were of 3 forms: 'non-synaptic', 'subthreshold', and 'complex.' Subthreshold responses occurred when amplitudes were less than 40% of their maximal levels. They consisted of a sink-source pair with the sink in the superficial optic neuropil and the source 100-150 micron deeper. They were monophasic, rising in 1-2 ms and decaying as simple exponentials with time constants between 3.3 and 4.5 ms. Several other conditions which reduce amplitudes (besides low [Ca2+] and/or high [Mg2+]) produce responses of the subthreshold form. Complex responses, observed when amplitudes were 40-100% of maximal, were characterized by more rapid rise and decay and included, in the most extreme cases, a late, long, low-amplitude sink-source pair of opposite polarity. We propose that the time course of decay of subthreshold responses is determined by the passive cable properties of bi- or multistratified neurons with one dendritic arbor in the optic neuropil and a second arbor 100-250 micron deeper. Complex responses probably include recurrent inhibition to depolarized dendrites of the optic neuropil, latent by 4 ms to the monosynaptic excitation. Pharmacological assessments of retinotectal transmission may be made more precise by using low [Ca2+]/high [Mg2+] media to isolate monosynaptic activity.

Animals↗
PubMed1988

Fluorescence recordings of electrical activity in goldfish optic tectum in vitro.

Optical methods for recording electrical activity in the goldfish optic tectum were evaluated. Tectal slices, with a short section of the optic nerve attached, were stained with a fluorescent styryl dye. Potential-dependent fluorescence changes following optic nerve stimulation were monitored with a photodiode. We found that large optical signals could be obtained. Experimental manipulations of the slice bathing solution permitted us to identify several events that contributed to the optical response, including activity in afferent fibers, excitatory and inhibitory postsynaptic potentials, and presumptive glial depolarizations. These results suggest that voltage-sensitive dyes can provide a useful alternative method for monitoring synaptic responses in the goldfish tectum, and may prove valuable in studying changes in the functional synaptic organization of the tectum following manipulations of the retinotectal pathway.

Animals↗
PubMed1987

Cloning of complementary DNA for GAP-43, a neuronal growth-related protein.

GAP-43 is one of a small subset of cellular proteins selectively transported by a neuron to its terminals. Its enrichment in growth cones and its increased levels in developing or regenerating neurons suggest that it has an important role in neurite growth. A complementary DNA (cDNA) that encodes rat GAP-43 has been isolated to study its structural characteristics and regulation. The predicted molecular size is 24 kilodaltons, although its migration in SDS-polyacrylamide gels is anomalously retarded. Expression of GAP-43 is limited to the nervous system, where its levels are highest during periods of neurite outgrowth. Nerve growth factor or adenosine 3',5'-monophosphate induction of neurites from PC12 cells is accompanied by increased GAP-43 expression. GAP-43 RNA is easily detectable, although at diminished levels, in the adult rat nervous system. This regulation of GAP-43 is concordant with a role in growth-related processes of the neuron, processes that may continue in the mature animal.

Amino Acid Sequence↗
PubMed1987

An experimental implant for applying a DC electrical field to peripheral nerve.

Several studies have shown that nerve fibers grow preferentially toward the cathode when placed in an electrical field. To study the effects of electrical stimulation on in vivo nerve regeneration, an inexpensive implantable current source has been developed which is able to reliably deliver a minute DC current (0.57 microA) via wick electrodes. The current density was measured directly with a vibrating probe and compared to the delivered current. The mean current density along the normal unoperated rat sciatic nerve was 0.19 microA/cm2, while the value along the nerve near the wick electrodes positioned 15 mm apart with an active in vivo implant was 23.39 microA/cm2. This application promises to provide a potentially useful means of enhancing nerve regeneration clinically.

Animals↗
PubMed1987

Pharmacology of retinotectal transmission in the goldfish: effects of nicotinic ligands, strychnine, and kynurenic acid.

The goal of this study was to evaluate different neurotransmitters and their receptors that might be involved in retinotectal transmission in the goldfish. Sections of tectum were isolated and maintained in vitro while pharmacological agents were administered via the tissue bath. Field potentials were elicited by electrical stimulation of the optic nerve and recorded at 50 micron depth intervals, and profiles of current source densities (CSDs) were computed from the second spatial derivatives of these potentials. The preparations were treated with low [Ca2+]/high [Mg2+] media, various cholinergic agonists and antagonists, eserine, strychnine, or kynurenic acid, via the tissue bath. Prior to treatment, depth profiles of these in vitro field potentials and CSDs closely resembled those previously reported in vivo, including 2 prominent sink-source pairs with their sinks in the superficial optic neuropil, followed by a smaller and more prolonged sink-source pair of opposite polarity. These were rapidly and reversibly eliminated by low [Ca2+]/high [Mg2+] bathing media, and substantially reduced by 0.5 or 1.0 mM kynurenic acid. By contrast, d-tubocurarine (d-TC; up to 0.16 mM) reduced peak response amplitudes by less than 40%, eliminated the third sink-source pair, and more than doubled the duration of decay of sink-source pairs 1 and 2 in a concentration-dependent manner. Strychnine had a similar action to d-TC but was slightly more potent. The time course and amplitudes of responses were not much affected by the following nicotinic agonists or antagonists (concentrations in microM): mecamylamine, 50; dihydro-beta-erythroidine, 50; nicotine, 200; tetramethylammonium, 500; ACh (protected by eserine, 20), 200; alpha-bungarotoxin, 2 microM for 2.5 hr, and 0.4 microM for up to 10.5 hr; and lophotoxin, 32 microM for up to 94 min. Eserine (20 microM) and carbachol (200 microM) increased peak response amplitudes by up to 80% within 5-10 min, and amplitudes remained elevated during 20-33 min of continued treatment. The onset of the effects of d-TC, strychnine, and kynurenic acid began in 5-10 min and was completed in 30 min or less, indicating that test substances could adequately penetrate into the interior of the isolated sections of tectum. The failure of these cholinergic ligands to prevent postsynaptic responses indicates that excitatory retinotectal transmission does not depend on an intact nicotinic (or other cholinergic) system, as previously proposed. The action by kynurenic acid suggests the involvement of an excitatory amino acid neurotransmitter in retinotectal transmission.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1987

Evidence for the coidentification of GAP-43, a growth-associated protein, and F1, a plasticity-associated protein.

GAP-43 is a fast-axonally transported protein whose expression correlates with periods of axon growth both during development and during regeneration. Similarities in molecular weight (43-47 kDa), pI (4.3-4.5), and aberrant behavior in acrylamide gels suggested that GAP-43 might be related or identical to protein F1, a protein kinase C substrate that has been shown to undergo a change in phosphorylation state during long-term potentiation in the hippocampus. Here we show that GAP-43 and protein F1 comigrate by two-dimensional PAGE and that antiserum raised against GAP-43 specifically immunoprecipitates protein F1. More direct evidence that GAP-43 and protein F1 are identical proteins was obtained by performing S. aureus V8 protease digests of a mixture of purified 32P-labeled protein F1 and purified GAP-43. Under these conditions, 2 phosphorylated peptide fragments of protein F1 corresponded exactly to 2 Coomassie-stainable bands from purified GAP-43. We conclude on the basis of these data that GAP-43 and protein F1 are identical proteins. Using light-microscopic immunocytochemistry, we also show that GAP-43/protein F1 immunoreactivity is localized to neuropil areas of the hippocampus consistent with its roles as a protein kinase C substrate in vivo and in long-term potentiation. These findings suggest that nerve growth during development and regeneration, and synaptic plasticity in the adult mammalian brain, may be mediated by a common mechanism involving the phosphorylation of GAP-43/protein F1.

Animals↗
PubMed1986

Antagonists of glutaminergic neurotransmission block retinotectal transmission in goldfish.

The hypothesis that excitatory retinotectal transmission is mediated primarily by a glutamate or glutamate-related transmitter-receptor system was examined by recording extracellular field potentials in isolated sections of goldfish tectum while stimulating the optic tract and applying antagonists of excitatory amino acid (EAA) neurotransmission via the tissue bath. Three antagonists of EAA receptors produced greater than 90% reduction in the postsynaptic components of these evoked potentials. In order of potency, these were (with the concentrations that produced 50% block): kynurenic acid (0.15 mM), gamma-D-glutamylglycine (0.33 mM), and cis-2,3-piperidine dicarboxylic acid (0.47 mM). All 3 log concentration-effect curves were parallel, symmetrically sigmoidal, and somewhat steeper than non-cooperative single-site binding isotherms. All antagonist actions stabilized within 15 min and were completely reversible. An EAA antagonist potent and selective for the N-methyl-D-aspartate (NMDA) subtype of receptor, 2-amino-5-phosphonovalerate, had little or no effect in either normal, low [Ca2+]/high [Mg2+], or Mg2+-free media. These data indicate that an excitatory amino acid receptor not of the NMDA subtype plays an essential role in fast excitatory retinotectal transmission, and would be most consistent with the mediation of most or all excitatory retinotectal transmission by a single class and subtype of glutamate receptor.

2-Amino-5-phosphonovalerate↗
PubMed1986

A protein induced during nerve growth (GAP-43) is a major component of growth-cone membranes.

Growth cones are specialized structures that form the distal tips of growing axons. During both normal development of the nervous system and regeneration of injured nerves, growth cones are essential for elongation and guidance of growing axons. Developmental and regenerative axon growth is frequently accompanied by elevated synthesis of a protein designated GAP-43. GAP-43 has now been found to be a major component of growth-cone membranes in developing rat brains. Relative to total protein, GAP-43 is approximately 12 times as abundant in growth-cone membranes as in synaptic membranes from adult brains. Immunohistochemical localization of GAP-43 in frozen sections of developing brain indicates that the protein is specifically associated with neuropil areas containing growth cones and immature synaptic terminals. The results support the proposal that GAP-43 plays a role in axon growth.

Aging↗
PubMed1986

Success rates of blind orotracheal intubation using a transillumination technique with a lighted stylet.

The technique of guided orotracheal intubation using a lighted stylet depends on the transillumination of the soft tissues of the neck to direct the tube through the glottis and into the trachea. We conducted an operating room study of this technique, recording success rates and intubation times of 50 patients undergoing elective surgery. All patients were intubated successfully, 35 of 50 (70%) on the first attempt, 12 of 15 (80%) on the second attempt, and three of three (100%) on the third attempt. The average time for intubation was 37 seconds. A new design of the lighted stylet method resulted from the experience gained. Intubator training and experience influenced initial success rates, and the cadaver laboratory was of particular value in teaching the technique.

Adolescent↗
PubMed1986

Nerve injury stimulates the secretion of apolipoprotein E by nonneuronal cells.

Nerve trauma initiates significant changes in the composition of proteins secreted by nonneuronal cells. The most prominent of these proteins is a 37-kDa protein, whose expression correlates with the time course of nerve development, degeneration, and regeneration. We now report that the 37-kDa protein is apolipoprotein E (apoE). We produced a specific antiserum against the 37-kDa protein isolated from previously crushed nerves. This antiserum recognizes a 36-kDa protein in rat serum that we have purified and identified as apoE. The anti-37-kDa antiserum also recognizes apoE on electrophoretic transfer blots of authentic samples of high and very low density lipoproteins. The nerve 37-kDa protein comigrates with apoE by two-dimensional electrophoresis, shares a similar amino acid composition, and reacts with an antiserum against authentic apoE. The purified apoE specifically blocks the immunoprecipitation of [35S]methionine-labeled 37-kDa protein synthesized by nonneuronal cells. Thus, on the basis of its molecular mass, isoelectric point, amino acid composition, and immunological properties, we conclude that the 37-kDa protein is apoE. We also used light microscopic immunohistochemistry to localize apoE following nerve injury. In rats with optic nerve lesions, the 37-kDa antiserum bound specifically to the degenerating optic tracts and to the retino-recipient layers of the lateral geniculate nucleus and the superior colliculus. We propose that apoE is synthesized by phagocytic cells in response to nerve injury for the purpose of mobilizing lipids produced as a consequence of axon degeneration.

Amino Acids↗
PubMed1985

Steady growth cone currents revealed by a novel circularly vibrating probe: a possible mechanism underlying neurite growth.

The rate and direction of neurite growth have been shown in a number of studies to be determined by the distribution of adhesive sites on the growth cone. Recent evidence showing that the application of extrinsic electric fields can redistribute membrane molecules and alter both the rate and direction of neurite growth have raised the question whether endogenous electric fields might be produced by steady currents in growth cones. To investigate this question, we have devised a novel circularly vibrating microprobe capable of measuring current densities in the range of 5 nA/cm2 (near the theorectical limit of sensitivity), with a spatial resolution of 2 micron. The design of this device and the development of a novel algorithm for computing current vectors on-line is described. Using this probe we have found that cultured goldfish retinal ganglion cell growth cones generate steady inward currents at their tips. The measured currents, in the range of 10-100 nA/cm2, appear to flow into the filopodia at their tips and back outward near the junctures of the filopodia and the growth cone. The currents appear to be produced only during active growth. Ion substitution experiments support the conclusion that the majority of this current is carried by Ca2+ ions, which we postulate flow through a population of activated voltage-sensitive Ca2+ channels located on the filopodial tips. Calculation of the transmembrane current density (4 X 10(-6) nA/cm2) leads to an estimate of channel density (10 channels/micron2) in close agreement with the measured density of Ca2+ channels in other systems. The assumption that calcium channel proteins are conveyed to nerve terminals by active transport, whereas sodium channel proteins are conveyed passively by a slower somatofugal diffusion process [Strichartz et al, 1984], would explain why developing neurons tend to display Ca2+-sensitive electrogenesis at their growing tips, and Na+-sensitive action potentials later in development. In order to gain some insight into the possible role of these steady growth currents, we estimated the membrane depolarization and axial voltage gradient they produce. It is likely that the currents produce sufficient membrane depolarization (approximately equal to 4 mV) to cause autogenous activation of ion channel permeabilities. Similarly, the axial voltage gradient (approximately equal to 4 mV/cm) would be expected to move intracytoplasmic vesicles by electrophoresis at a rate (20-40 microns/hr) very close to that at which the filopodia are observed to grow.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗