John A. Freeman

Effect of α-bungarotoxin on retinotectal synaptic transmission in the goldfish and the toad

Abstract Alpha-bungarotoxin has been used extensively to characterize the acetylcholine receptor at the myoneural junction and in the electric organ, where it irreversibly blocks the response to acetylcholine. α-Bungarotoxin has also been employed in numerous biochemical studies of presumed acetylcholine receptors in the brain, although physiological studies demonstrating its effectiveness in blocking the response to acetylcholine are lacking, and α-bungarotoxin is ineffective in blocking the response to acetylcholine in sympathetic ganglia neurons. In-this study α-bungarotoxin was applied to the optic tectum of the goldfish and of the marine toad in dilute concentration (10−7 m ) either directly to the pial surface, or to the tectal neuropil by a micropipette. The latter method of application resulted in the rapid irreversible abolition of excitatory postsynaptic potentials generated in dendrites of tectal neurons by optic nerve activation, as shown by analysis of the laminar current source density. Responses to nonretinal (thalamic) inputs were unaffected, indicating specificity of action. Topical application of α-bungarotoxin was ineffective in the goldfish, and of considerably reduced effectiveness in the toad, due to a substantial diffusion barrier posed by the pia-arachnoid. A mathematical method based on the solution of the differential equation describing simultaneous diffusion and chemical reaction was developed to determine both the diffusion coefficient and the association rate constant for α-bungarotoxin from physiological measurements. We estimate the diffusion coefficient and the Stokes radius of α-bungarotoxin to be approx1.7 × 10−6 cm2/s and 12A˚respectively, and the association rate constant to be 5.1 × 104 m −1s−1, in reasonable agreement with values obtained by biochemical methods. Possible reasons for the differences in effectiveness of α-bungarotoxin in blocking the response to acetylcholine at different synapses are discussed in terms of possible differences in subunit composition of the oligomer complex of the acetylcholine receptor and in its dissociation kinetics.

Electrical stimulation of nerve regeneration in the rat: The early effects evaluated by a vibrating probe and electron microscopy

This study examines the effect of applied d.c. electric fields on nerve regeneration following injury to the rat sciatic nerve using the circularly vibrating probe and electron microscopy. The transected and treated nerve which received a d.c. electrical stimulator (0.6 mu A) was compared with untreated transected and crushed nerves. At one week postoperative, the probe was used to measure in vivo the current density along the nerve length. All nerves studied had a proximal peak at the lesion site and a second peak at varying distal locations: crushed/untreated (13.3 mm), transected/untreated (9.7 mm) and transected/treated (16.3 mm). A significant difference (69%) between the distal peak distances in the two transection groups suggests that the electrical treatment enhanced the progress of nerve regeneration. There were no significant differences between the mean peak amplitudes (1.6-2.2 mu A/cm2). Applied verapamil reduced the peaks, suggesting they are associated in part with a calcium-dependent current. Electron microscopy at selected nerve regions indicated that the peaks correspond to regenerating axonal growth cones. The results suggest the potential clinical application of d.c. electric fields in the treatment of nerve injuries.

Factors influencing GAP-43 gene expression in PC12 pheochromocytoma cells

We have studied factors controlling message levels for the neuronal growth- and plasticity-associated protein, GAP-43. Following exposure of PC12 cells to various effectors, cytoplasmic RNA was isolated and analyzed by Northern transfer and autoradiography using a GAP-43 cDNA probe. Induction by NGF is apparent after 3 hr exposure and reaches maximal levels at 24 hr. Beyond 24 hr, levels remain constant in the continued presence of NGF. Induction is insensitive to variations in culture conditions, such as plating density or substrate, which influence NGF-induced neurite outgrowth. Other inducers, in order of decreasing efficacy, are FGF, dBcAMP, TPA, K+, and EGF. Insulin and retinoic acid are ineffective. Dexamethasone partially inhibited basal expression as well as induction by NGF, FGF, dBcAMP, and TPA. The methyltransferase inhibitor 5′-S-(2-methyl-propyl)adenosine completely inhibited induction by NGF, FGF, and dBcAMP. Inhibition of protein synthesis by cycloheximide partially decreased induction by NGF, FGF, and TPA but slightly enhanced dBcAMP induction. Complete down- regulation of protein kinase C by chronic TPA treatment completely eliminated the TPA response but slightly enhanced induction by NGF. These findings and the results of additivity experiments in which cells were stimulated with various combinations of NGF, dBcAMP and TPA suggest that NGF induction of GAP-43 RNA (1) does not involve activation of protein kinase C but (2) may be mediated partially via activation of protein kinase A.

Synaptic organisation of the pigeon's optic tectum: a golgi and current source-density analysis.

Abstract This paper presents an analysis of the synaptic organisation of the pigeons optic tectum, with special reference to its retinal input. The structure of the tectum was studied in Golgi-impregnated material. The manner of termination of the retinal afferent fibres to the tectum and the morphology of the neurones of the tectum are described. The afferent fibres terminate in one or more columnar plexus. The plexuses are closely packed, forming a compact layer of afferent terminals at a constant depth in the tectum. It is suggested that the tectal neurones be classified according to the orientation of their dendritic processes into two broad groups, radial and non-radial cells, and the characteristics of and variations within each group are described. The field potential profile generated in the tectum by optic nerve stimulation was subjected to a current source-density analysis to determine the location and sequence of sources and sinks of current produced by an optic nerve volley. The results suggest that the radial cells are the principal cells involved in processing retinal input. The postsynaptic events generated by an optic nerve volley are described and the functional importance of these events is discussed.

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.

Electrophysiologic evidence that retinotectal synaptic transmission in the goldfish is nicotinic cholinergic

A previous study identified, by conduction velocity following optic nerve shock, 3 classes of retinal fibers which project to 3 distinct laminae of the goldfish optic tectum. In the present study, the effect of various pharmacological agents on the synaptic efficacy of each of the 3 classes of retinal fibers was assessed by the use of current source-density analysis. All 3 classes of optic fibers appear to be nicotinic cholinergic. Six different nicotinic antagonists were tested. All 6 were effective in decrementing the responses of all 3 classes to a criterion level: alpha-bungarotoxin (10-8 M), alloferin (10-5 M), curare (10-4 M), metocurine (10-4 M), hexamethonium (10-4 M) and gallamine (10-3 M). Atropine, a muscarinic antagonist, had only a slight effect even at 10-3 M. Five nicotinic agonists tested also decremented synaptic responses: nicotine (10-5 M), carbamylcholine (10-4 M), acetylcholine (10-4 M), succinyl choline (10-4 M) and decamethonium (10-3 M), presumably via cellular depolarization and receptor desensitization. Two inhibitors of acetylcholinesterase prolonged the response at 10-4 M and decremented it as well at 10-3 M. Hemicholinium 3, an inhibitor of the high affinity uptake of choline, produced a gradual activity-dependent decrement in the responses. Beta-bungarotoxin, a presynaptically-acting toxin, abolished not only the postsynaptic components but also the presynaptic components at 10-6 M. In all other cases the presynaptic deflections were generally unaffected, and with the exception of the toxins, a return to at least 90% of the control value was achieved. In contrast, GABA (10-3 M) and bicuculine (10-4 M) both produced no discernible effect on the 3 classes of responses, and glutamate (10-3 M) produced only a slight decrement, which probably represents a non-specific effect.

Localization of α-bungarotoxin binding sites to the goldfish retinotectal projection

Abstract The optic tectum of the goldfishCarassius auratus is a rich source of α-bungarotoxin (α-Btx) binding protein. In order to determine whether some fraction of these receptors is present at retinotectal synapses, we have compared the histological distribution of receptors revealed by the use of [125Iα-Btx radioautography to the distribution of optic nerve terminals revealed by the use of cobalt and horseradish peroxidase (HRP) techniques. The majority of α-Btx binding is concentrated in those tectal layers containing primary retinotectal synapses. The same layers contain high concentrations of acetylcholinesterase (AChE), revealed histochemically. Following enucleation of one eye, there is a loss of α-Btx binding in the contralateral tectum, observed both by radioautography and by a quantitative binding assay of α-Btx binding. Approximately 40% of the α-Btx binding sites are lost within two weeks following enucleation. By contrast, no significant change in AChE activity could be demonstrated up to 6 months enucleation. These results are discussed in light of recent studies which show that the α-Btx binding protein and the nicotinic acetylcholine receptor are probably identical in goldfish tectum. We conclude that the 3 main classes of retinal ganglion cells projecting to the goldfish tectum are nicotinic cholinergic and that little or no postdenervation hypersensitivity due to receptor proliferation occurs in tectal neurons following denervation of the retinal input.

Regulation of specific neuronal and nonneuronal proteins during development and following injury in the rat central nervous system.

Publisher Summary This chapter presents studies on growth-associated protein, GAP-43, to further characterize this protein using both anatomical and biochemical methods. It discusses a specific antibody to the rat GAP-43 and this antibody is used as a probe to examine the developmentally regulated pattern of the expression of GAP-43 in the rat hippocampus. The most prominent neuronal protein whose synthesis and transport is selectively enhanced during nerve growth is a protein designated as GAP-43 for its apparent molecular weight by two-dimensional polyacrylamide gel electrophoresis. It was initially reported that the synthesis of this protein was enhanced 15- to 20-fold during the regeneration of the optic nerve of the toad as compared to the levels of this protein found in normal adult nerves. The increased transport of a newly synthesized protein of the same approximate molecular weight and isoelectric point was subsequently identified during the regeneration of the hypoglossal nerve in the rabbit and corticospinal tract in neonatal hamsters and during the development of the optic nerve in rabbits. The synthesis of a similar protein is also increased during the regeneration of the optic nerve in goldfish and during the development of the optic nerve in rats. Taken together, these results suggest that GAP-43 may play a special role in neuronal growth.

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.

Chapter 5: Expression of the growth- and plasticity-associated

Publisher Summary This chapter summarizes observations on nerve growth factor (NGF) induction of GAP-43 in PC12 cells. It characterizes this NGF response in terms of its time course, dose–response relationship, sensitivity to variations in culture conditions, which affect neurite outgrowth, dependence on protein synthesis, and sensitivity to methyltransferase inhibitors and glucocorticoids. Many of these parameters have also been examined for a number of other agents that were found to increase GAP-43 expression. To determine whether GAP-43 in PC12 cells is also primarily localized to neurites, NGF-treated cells were stained immunohistochemically for GAP-43. Specific immunoreactivity is present throughout the neurites, but is not evident in cell bodies. Recent immunogold labeling experiments have shown that the very low levels of GAP-43 in unstimulated PC12 cells is mostly associated with lysosomal structures and Golgi apparatus. In stimulated cells, the much higher GAP-43 content is localized mostly to the plasma membrane, especially around processes, and to membranous structures in neurites.