E. J. Furshpan

Electrical Transmission at an Excitatory Synapse in a Vertebrate Brain

A type of excitatory synaptic transmission which is novel for the vertebrate brain has been found in the ner neuron (M-cell) by means of the passive spread of their action currents across the synaptic membrane. After stimulating the ipsilateral eighth cranial nerve, an excitatory postsynaptic potential (EPSP) appears in the M-cell with a latency which is very brief ( about 0.1 msec) and which proba; bly represents a negligible synaptic delay. This response is attributed to the club endings: there were steep gradients of potential along the lateral dendrite of the M-cell during activity and the early EPSP was maximal in the distal part of the dendrite where the club endings predominate. Potential changes in the M-cell spread (passively) backwards into certain eighth-nerve fibers (probably club endings) indicating the presence of special low-resistance connections between them and the M-cell.

Seizure-like activity and cellular damage in rat hippocampal neurons in cell culture

Neurons dissociated from the hippocampal formations of neonatal rats were grown in medium containing kynurenic acid (a glutamate receptor antagonist) and elevated Mg2+. Such chronically blocked neurons, when first exposed to medium without blockers (after 0.5-5.0 months), generated intense seizure-like activity. This consisted of bursts of synchronous electrical responses that resembled paroxysmal depolarization shifts and sustained depolarizations that, in some neurons, nearly abolished the resting potential. Sustained depolarizations were usually reversed by timely application of kynurenate or 2-amino-5-phosphonovalerate, indicating that continuous activation of glutamate receptors was required for their maintenance. Prolonged periods of intense seizure-like activity usually killed most neurons in the culture. This system allows seizure-related cellular mechanisms to be studied in long-term cell culture.

Studies on rat sympathetic neurons developing in cell culture. I. Growth characteristics and electrophysiological properties.

In this series of three papers, we describe electrophysiological and pharmacological studies on sympathetic principal neurons developing in cell culture. This paper is concerned with the methods for growing and recording from the neurons and with observations on some of their electrical properties. The succeeding papers are concerned with functional synapses which the neurons form with one another. Superior cervical ganglia of newborn rats were dissociated into single cells and small cell clusters, and the resulting cell suspension of principal neurons and a much smaller number of non-neuronal cells was cultured at low density in medium containing nerve growth factor (D. Bray, 1970, Proc. Nat. Acad. Sci. USA. 65, 905–910; R. E. Mains and P. H. Patterson, 1973a, J. Cell Biol. 59, 329–345). As in the previous studies the multiplication of the non-neuronal cells could be controlled so that the neurons grew in the presence of an increasing number of non-neuronal cells or in the virtual absence of other cell types. Another method for obtaining mixed cultures was to plate the initial cell suspension onto a preexisting layer of cells dissociated from some other tissue (e.g., heart). Neurons grown for 3 weeks or longer in the presence of non-neuronal cells had resting potentials, passive electrical properties, and action potentials generally similar to those reported for principal neurons of the superior cervical ganglia of adult rats. Through the use of tetrodotoxin, tetraethylammonium, and cobalt, evidence was obtained for the presence of potential-sensitive sodium, potassium, and calcium channels. Frequently the action potential was followed by a prolonged after-hyperpolarization whose properties suggested the presence of potassium channels controlled by calcium ions. When the neurons were grown in the absence of non-neuronal cells, the action potentials were similar, but the prolonged after-hyperpolarization was rarely seen, and the neurons usually discharged repetitively in response to a steady depolarization.

THE MECHANICS OF FLIGHT MOVEMENTS IN DIPTERA

1. The mechanics of insect flight are usually studied by squeezing the thorax so as to imitate the action of the indirect flight muscles. In flies certain elements of the articulation are not properly set in such experiments, so the interpretations made are not accurate. That CCl4 sets the articulations of flies as in normal flight is shown by the dramatic wing movements easily produced in these insects. This report is based on the study of CCl4-treated flies and outlines the main features of the mechanics of wing movement. Much additional physiological evidence for the interpretations given here has been accumulated and will be reported separately. The abstracts referred to state some of the experimental results.2. The secret of the peripheral control of wing rate certainly lies in the indirect flight muscles. These muscles are the power plants of the wings, operating in essentially an all-or-none fashion. It is shown here that they play little part in steering or in amplitude changes. They operate betwe...

The effects of osmotic pressure changes on the spontaneous activity at motor nerve endings.

Fatt & Katz (1952) described a particularly striking effect on the frequency of the spontaneous miniature end-plate potentials (e.p.p.s) of frog muscle, which resulted from changing the osmotic pressure of the external fluid. In one experiment, for example, a 50% increase in osmotic pressure resulted in a 45-fold rise in the frequency of the random potentials. A converse experiment was cited in which a 50% decrease in osmotic pressure reduced the frequency by a factor of more than 30. Since the normal e.p.p. may be considered as a large, synchronized burst of miniature e.p.p.s evoked by the nerve impulse (del Castillo & Katz, 1954 a, b), the study of an agent which induces large changes in the frequency of these random discharges was of interest. The nature of this osmotic effect has so far remained obscure, and in the present experiments an attempt has been made to determine if its size and time course are related to properties of the solute molecules and to the permeability of the nerve endings. The procedure was, therefore, to increase the molarity of the external fluid by adding substances like glycerol and ethanol, which are known to penetrate cell membranes easily, or alternatively like sucrose which may not penetrate at all (cf. Overton, 1902).

Studies on rat sympathetic neurons developing in cell culture: III. Cholinergic transmission

Abstract Principal neurons were dissociated from the superior cervical ganglia of newborn rats and grown in culture with several types of non-neuronal cells. As described in the second paper of this series, the neurons in such mixed cultures formed two types of excitatory synapses with each other, electrical and chemical. Evidence is presented here that transmission at the chemical synapses was cholinergic. Four nicotinic ganglionic blocking agents (curare, hexamethonium, tetraethylammonium, and mecamylamine) strongly attenuated or eliminated the excitatory postsynaptic potentials (e.p.s.p.s) at moderate concentrations; atropine at relatively high concentrations also blocked transmission. Iontophoretic application of acetylcholine (ACh) to the surface of the neurons gave rise to depolarizations that could be made to resemble the e.p.s.p.s in size and time course; the ACh potentials and the e.p.s.p.s were then similarly affected by nicotinic blocking agents. The sensitivity to ACh was often distributed nonuniformly on the neuronal surface; it was common to find small, sharply localized regions of high sensitivity. Catecholamines (norepinephrine, epinephrine, and dopamine) had only inhibitory actions; in a few experiments adrenergic blocking agents (phenoxybenzamine, propranolol) were found to have no effect on the e.p.s.p.s. These observations leave no doubt that the neurons released ACh and had ganglionic, nicotinic ACh receptors on their surfaces. The significance of the fact that a high proportion of the sympathetic neurons in mixed cultures formed cholinergic synapses is discussed.

Studies on rat sympathetic neurons developing in cell culture: II. Synaptic mechanisms

Abstract Sympathetic neurons dissociated from superior cervical ganglia of newborn rats were grown in culture either alone or with non-neuronal cells, as described in the preceding paper. In the presence of the non-neuronal cells, but rarely in their absence, neurons formed functional synapses with each other de novo. The synapses were of two types, both excitatory. One type operated by nonrectifying electrical transmission and comprised only a few percent of the interactions; it was characterized by negligible synaptic delay and the transfer of steady depolarizations or hyperpolarizations from one cell to the other. At the second type of synapse which was chemical, there was a synaptic delay (minimum, 1 msec) and the amplitudes of the chemically mediated postsynaptic potentials (e.p.s.p.s) were dependent on the concentrations of Ca2+ and Mg2+ in the extracellular medium. As described in the following paper, the e.p.s.p.s were sensitive to nicotinic-cholinergic blocking agents. The incidence of chemical transmission increased markedly with age in culture. This increase was associated with the formation of networks in which the neurons were extensively connected to each other. In such cultures an action potential evoked in one neuron often gave rise in other neurons and in the stimulated neuron to a volley of synaptic activity (“complex wave”) which occurred nearly synchronously, though not identically, in each neuron. The complex waves depended on chemical transmission since they, like the simple e.p.s.p.s, were abolished by nicotinic blocking agents.

5. Seizure-like activity in cell culture

I will describe experiments with neurons in long-term culture that display seizure-like electrical activity. The neurons are dissociated from the hippocampal formation of newborn rats and then chronically exposed to agents that block synaptic transmission, especially glutamatergic transmission. Seizure-like behavior of the neurons develops as the cultures mature and is revealed when the blocking agents are withdrawn. This spontaneous electrical behavior of the culture has many of the characteristics of seizure activity in intact cortex. It can be very intense and can lead to the death of many neurons. This system allows the familiar experimental advantages of dissociated-cell culture to be applied to the study of seizure-like activities. The experiments to be described were all done with mass cultures containing hundreds or thousands of neurons. However, many of the seizure-like events observed in mass cultures can also be seen in microcultures containing only a few neurons.

Transmitter status in cultured sympathetic principal neurons: plasticity, graded expression and diversity.

Publisher Summary This chapter summarizes the recent investigations of the transmitter status of sympathetic principal neurons derived from neonatal or adult rats and grown singly in microcultures with cardiac cells. The work began out of an interest in the status of individual neonatal sympathetic neurons during a transition from an initial (at least) adrenergic state to a predominantly cholinergic state, under the influence of non-neuronal cells. Under the influence of nerve growth factor, neurites grow progressively over the microculture but not beyond its borders. Many microcultures survive for 1–3 months; after such periods, the density of neurites over the myocytes is often greater than that of the normal innervation of sympathetic target tissues in vivo . The classical view of transmitter status in adult mammalian sympathetic principal neurons is that two transmitters—norepinephrine and acetylcholine—are expressed. Each neuron secretes only one transmitter (monofunction), and that transmitter is expressed approximately full-on; once the appropriate transmitter is adopted, the neuron does not change status.

Seizure-like activity and glutamate receptors in hippocampal neurons in culture

Hippocampal neurons that were grown for prolonged periods in the continuous presence of agents that interfere with synaptic transmission, especially excitatory synaptic transmission, appeared to become seizure-prone. Washout of the synaptic blocking agents, that had been continuously present for several weeks to several months, caused the population of neurons to produce an abnormal and intense electrical activity. This consisted of two major components: spontaneously arising phasic responses that closely resembled paroxysmal depolarization shifts and, less frequently, slowly rising depolarizations similar to the sustained depolarizations observed during ictus-like episodes in intact cortex or cortical slices. We describe here observations on the role of the N-methyl-D-aspartate (NMDA) and non-NMDA types of glutamate receptors in the generation of these activities.