Inhibition of the p44/42 MAP kinase pathway protects hippocampal neurons in a cell-culture model of seizure activity.

Excessive release of glutamate and the subsequent influx of calcium are associated with a number of neurological insults that result in neuronal death. The calcium-activated intracellular signaling pathways responsible for this excitotoxic injury are largely unknown. Here, we report that PD098059, a selective inhibitor of the calcium-activated p44/42 mitogen-activated protein kinase (MAP kinase) pathway, reduces neuronal death in a cell-culture model of seizure activity. Dissociated hippocampal neurons grown chronically in the presence of kynurenate, a broad spectrum glutamate-receptor antagonist, and elevated amounts of magnesium exhibit intense seizure-like activity after the removal of these blockers of excitatory synaptic transmission. A 30-min removal of the blockers produced extensive neuronal death within 24 h as assayed by the uptake of trypan blue and the release of lactate dehydrogenase. Phospho-p44/42 MAP kinase immunoreactivity after 30 min of seizure-like activity was present in many neuronal somata and dendrites as well as some synaptic terminals, consistent with both the presynaptic and postsynaptic effects of this pathway. The addition of PD098059 (40 microM; EC50 = 10 microM) during a 30-min washout of synaptic blockers inhibited the phosphorylation of p44/42 MAP kinase and reduced both the trypan-blue staining (n = 13) and the release of lactate dehydrogenase (n = 16) by 73% +/- 18% and 75% +/- 19% (mean +/- SD), respectively. The observed neuroprotection could be caused by an effect of PD098059 on seizure-like events or on downstream signaling pathways activated by the seizure-like events. Either possibility suggests a heretofore unknown function for the p44/42 MAP kinase pathway in neurons.

Neurologic education for the future: a decade of curricular reform at Harvard Medical School.

The field of neurology is undergoing significant changes to which curricular reform is both responding and contributing. We reflect on a decade of experience at Harvard Medical School with integration of neuroscience, behaviour, pathophysiology and introductory clinical skills. As part of Harvard's "New Pathway" curriculum, this coordinated, pre-clerkship program embraces a "hybrid" form of problem-based learning. A variety of methods are employed synergistically to meet the two broad goals of preparing for competency in neurologic clerkships and for career-long learning in clinically relevant neuroscience. We articulate specific ways of elevating the level of intellectual inquiry, involving multi-disciplinary faculty more productively, and vertically integrating the learning experience through the years of medical school.

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.

Epileptiform activity in microcultures containing small numbers of hippocampal neurons.

1. Microcultures were grown containing small numbers of hippocampal neurons. The neurons grew on glial cells attached to patches of either collagen or palladium. A layer of agarose underlying the microcultures prevented connections from forming between nearby microcultures. 2. Neurons formed strong chemical synaptic connections within each microculture, with monosynaptic fast-excitatory, fast-inhibitory, and slow-inhibitory synaptic actions. 3. Small networks with as few as two neurons generated epileptiform activity that closely resembled the epileptiform activity seen in mass cultures containing thousands of neurons. The epileptiform activity was observed when microcultures that were grown for weeks in blockers of synaptic activity (kynurenate and elevated Mg2+) were washed free of these blockers. 4. Such a microculture technique allows study of epileptiform activity in a simplified system and facilitates analysis of the synaptic actions underlying the epileptiform activity.

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.

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.

Synaptic functions in rat sympathetic neurons in microcultures. IV. Nonadrenergic excitation of cardiac myocytes and the variety of multiple-transmitter states.

In the first 3 papers of this series (Furshpan et al., 1986a, b; Potter et al., 1986), a sensitive microculture procedure was used to show that sympathetic principal neurons, dissociated from newborn or adult superior cervical ganglia and grown singly on cardiac myocytes, display adrenergic, cholinergic, and purinergic functions, sometimes in isolation but more often in combination. In this paper we describe additional effects on cardiac myocytes evoked by these neurons; the effects were excitatory and insensitive to adrenergic blocking agents (and to agents that block the inhibitory effects of acetylcholine and purines). In some of these microcultures, evidence consistent with secretion of serotonin was obtained; the nonadrenergic excitatory effect was diminished or abolished by serotonin blockers or reserpine. Further evidence for serotonergic transmission is presented in the accompanying paper by Sah and Matsumoto (1987). In other cases, an as-yet-unidentified agent "X" also produced a nonadrenergic excitation. The X effect characteristically required a prolonged train of neuronal impulses, had a time course of 50-200 sec, and was insensitive to agents that affected the other transmitters, including serotonin. In addition, we discuss 2 remarkable features of the transmitter repertoire of the microcultured sympathetic neurons: expression of the several transmitters in a variety of combinations, including at-least-quadruple function, and expression of the transmitters within a particular combination in varying relative strengths. The result is a diversity of transmitter release greater than that previously reported for vertebrate or invertebrate neurons.

Synaptic functions in rat sympathetic neurons in microcultures. II. Adrenergic/cholinergic dual status and plasticity.

This is the second in a series of papers that describes the use of a sensitive microculture procedure to investigate the transmitter status of sympathetic neurons. Cultured immature principal neurons, dissociated from the superior cervical ganglia of newborn rats, are known to be plastic with respect to transmitter status; under certain culture conditions, populations of neurons that display (at least) adrenergic properties at the outset can be induced to display a variety of cholinergic properties, including the formation of functional neuron-neuron cholinergic synapses, as adrenergic properties decline. With the microculture procedure described in the preceding paper (Furshpan et al., 1986a), we have examined the transmitter status of individual neonate-derived neurons during this transition. Many such neurons secreted both norepinephrine and ACh (adrenergic/cholinergic dual function); examination of such neurons with the EM revealed a mixed population of synaptic vesicles. Direct evidence for a transition via this dual status was obtained by serial physiological assays of 14 neurons. The neonate-derived neurons were markedly heterogeneous in the rate of change of transmitter status. Principal neurons derived from adult superior cervical ganglia also displayed dual status, but the incidence was lower than in neonate-derived neurons cultured for similar periods. In preliminary serial assays of adult-derived neurons, many of the neurons did not acquire detectable cholinergic function, but in two cases evidence consistent with plasticity was obtained. While it is known that several types of neurons will form functional junctions in the presence of agents that block electrical activity, sympathetic principal neurons have apparently not been tested. In microculture, neuron-neuron synapses and junctions with cardiac myocytes were formed by sympathetic neurons grown chronically in the presence of blocking concentrations of TTX and hexamethonium.

Synaptic functions in rat sympathetic neurons in microcultures. III. A Purinergic effect on cardiac myocytes.

In the first two of this series of papers, a sensitive microculture procedure was used to show that rat sympathetic neurons grown singly on small islands of heart cells release norepinephrine (NE) and/or acetylcholine (ACh). We report here the release of a third transmitter in response to stimulation of these neurons. This agent was recognized by its effect on the cocultured cardiac myocytes: an inhibition of beating or a hyperpolarization that, in contrast to cholinergic inhibition, was unaffected by atropine (up to 5 microM). Evidence described here indicates that this agent was primarily adenosine (or a closely related compound): the atropine-resistant myocyte inhibition was antagonized by adenosine-receptor blockers [8-phenyltheophylline, theophylline, 7-(2-chloroethyl) theophylline] and was attenuated by an enzyme (adenosine deaminase) that hydrolyzes adenosine to pharmacologically inactive inosine. Many of the neurons, whether initially dissociated from ganglia of newborn or adult rats, evoked this purinergic response, almost always in combination with adrenergic and cholinergic responses. In a few cases it was the only detectable response. The relative strength of the adrenergic, cholinergic, and purinergic responses varied widely from neuron to neuron, suggesting that the adrenergic and purinergic or the cholinergic and purinergic agents were not stored at constant stoichiometric ratios.

Synaptic functions in rat sympathetic neurons in microcultures. I. Secretion of norepinephrine and acetylcholine.

This is the first of a series of four papers that describes the use of a sensitive "microculture" procedure for examining the neurotransmitter profile of a neuron by assaying the transmitter(s) it releases. Sympathetic principal neurons isolated from the superior cervical ganglia of neonatal or adult rats were grown for 10 d to several months on small islands of cardiac myocytes (island diameter, ca. 0.5 mm). To assay transmitter status a neuron and a myocyte in the same microculture were impaled with microelectrodes, the neuron was stimulated and the pharmacology of the effect(s) on the group of electrically coupled myocytes, and on the neuron itself, was investigated. Because the growing axonal processes were confined to the island, the innervation of the myocytes became dense; transmission from neuron to myocytes occurred reliably and was often intense. Most experiments were done on islands containing only a single neuron so that the observed effect(s) on the myocytes could be confidently assigned to that neuron. After the physiological assay, the fine structure or cytochemistry of the neuron was often examined. With single-neuron microcultures the physiology and anatomy of the neuron, including the fine structure of its synaptic endings and varicosities, could be correlated unambiguously. During the course of this work, we have observed five pharmacologically distinct effects exerted on the myocytes by either neonate- or adult-derived neurons. Three of these effects, one exerted at least in large part by adenosine and the others by agents still under study (one appears to be 5-HT), are described by Furshpan et al. (1986), Matsumoto et al. (in press), and D. Sah and S. G. Matsumoto (unpublished observations). This paper is concerned with evidence for secretion by these neurons of norepinephrine (NE) and acetylcholine (ACh). The physiological effects of the secretion of these two substances onto the myocytes (excitation and inhibition, respectively) were generally similar to those reported in vivo. The minimal latencies of the responses were short, probably due to the high density of innervation. ACh secreted by a neuron onto itself, at autapses, evoked fast nicotinic EPSPs. We have not detected autaptic effects attributable to the secretion of NE. A minority of the neurons were detectably only adrenergic or only cholinergic. The incidence of these transmitter states was strongly dependent on culture age and culture conditions; in a heterogeneous group of about 300 reasonably well-characterized neurons about 17% (12% of neonate-derived) were apparently purely adrenergic and about 10% (13% of neonate-derived) were apparently purely cholinergic.(ABSTRACT TRUNCATED AT 400 WORDS)

Transmitter status in cultured rat sympathetic neurons: plasticity and multiple function.

Considerable recent study of the development of transmitter status in sympathetic principal neurons, both in vivo and in culture, has produced several surprising findings. In this paper we review work on cultured immature and adult principal neurons dissociated from the superior cervical ganglia of rats. The main points are; 1) Immature principal neurons that display adrenergic properties during the first postnatal week in culture can be shifted to cholinergic status, including formation of functional cholinergic synapses, by coculture with nonneuronal cells (e.g., dissociated heart cells) or by medium conditioned by such cells. Through the use of microcultures that contain only a single neuron grown on heart cells, it has been possible to demonstrate the transition from adrenergic to cholinergic function directly by serial physiological assays of the same neuron at intervals of days or weeks. 2) During this transition, the cultured neurons display adrenergic/cholinergic dual function. This dual function has also been observed in principal neurons isolated from ganglia of adult rats. 3) Some cultured neurons secrete a third transmitter, probably adenosine or a phosphorylated derivative. This purinergic function is expressed with adrenergic or cholinergic function, or with both (triple function). In some cases, the main effect exerted by a neuron on cocultured cardiac myocytes is purinergic.

Adrenergic-cholinergic dual function in cultured sympathetic neurons of the rat.

Sympathetic principal neurons, dissociated from the superior cervical ganglia of newborn rats and put into culture, exhibit plasticity with respect to the choice between noradrenaline (norepinephrine) and acetylcholine as transmitter. The neurons shift from an initial, immature adrenergic state to a cholinergic state in certain culture conditions, e.g in co-culture with a variety of non-neuronal cells or after exposure to a medium conditioned by such cells. To study the transition directly, we have grown single neurons in "microcultures" with cardiac myocytes, which provide a sensitive assay for the transmitters secreted by the neurons. We have shown previously that during the transition from adrenergic to cholinergic status such neurons secrete both transmitters and have terminals of mixed fine structure (dual function). We describe here experiments in which identified neurons were serially assayed over periods of 9-45 days. Partial transitions were observed, always in the direction adrenergic to cholinergic function, and one complete transition was observed from apparently purely adrenergic function to dual function and then to apparently purely cholinergic function. We also report observation of adrenergic-cholinergic dual function, in preliminary single and serial assays, in sympathetic principal neurons from the superior cervical ganglia of adult rats.

Dual function during development of rat sympathetic neurones in culture.

Many sympathetic principal neurones of the superior cervical ganglion of the newborn rat are known to be plastic with respect to the choice between norepinephrine (NE) and acetylcholine (ACh) as transmitter; when the neurones are dissociated and placed in culture, a majority of them can be shifted from an initial, immature, adrenergic state to a cholinergic state by co-culture with a variety of non-neuronal cells or by medium conditioned by such cells. To study this transition it has been helpful to grow single neurones, each in a microculture which also contains cardiac myocytes. The transmitter status of a neurone can be assayed by recording its effect on the myocytes (adrenergic excitation, cholinergic inhibition or dual function); then a fine structural assay of the neurone based on the appearance of the synaptic vesicles can be made and correlated directly with the physiology. In this paper we report the following findings on principal neurones developing in such microcultures. (i) During the transition period, a majority of the neurones were dual in function and in vesicular appearance. (ii) The physiological effects and vesicular appearance varied from mainly adrenergic to mainly cholinergic. (iii) In preliminary attempts to follow the transition by recording at least twice from the same microculture, partial transitions were observed, always in the direction adrenergic-to-cholinergic. (iv) The transitions were not synchronous or fixed in time course even in pairs of neurones grown side by side in the same microculture.