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Electrophysiological studies were made on microcultures (300-500 Ism in diameter) in which solitary sympathetic principal neurons from newborn rats grew on previously dissociated rat heart cells. (1,6,7,8). In contrast, when the neurons are cultured in the presence of nonneuronal cells (e.g., from ganglia or heart), or in medium conditioned by such cells, the neuronal population synthesizes both AcCh and NE (2, 9), and many neurons form nicotinic cholinergic synapses on each other (6-8, 10, 11). These effects are graded. A higher proportion of conditioned medium or a greater number of nonneuronal cells gives a higher ratio of AcCh synthesis to catecholamine synthesis (12), a higher incidence of cholinergic transmission, and a higher proportion of synapses which lack small granular vesicles (13 (MacLeish, unpublished). He found that the incidence of detectable neuronmyocyte interaction was too low to be useful, perhaps in part because the endings of each neuron were sparsely distributed in the large field of myocytes; the few cases found all appeared to be cholinergic.It was plausible that the incidence of detectable interaction would increase if the innervation field of a given neuron was concentrated on a few cardiac myocytes. Thus, we made microcultures containing a single neuron and a small number of myocytes in an area only a fraction of a millimeter in diameter. In this paper, we report physiological observations on such microcultures; in the accompanying paper, Landis (15) reports electron microscopic observations on the same microcultures.In two previous studies (16, 17), explants of sympathetic ganglia were found to make functional contacts with explants of heart. METHODSSeveral methods for making microcultures suitable for electrophysiology and microscopy were successful, but none routinely so. The simplest method was, in brief, to apply 25 to 50 equally spaced droplets of dissolved collagen to a nonwetting polystyrene surface. When dried, these produced a grid (ca 50 mm2) of collagen islands, each island 300-500 Atm in diameter.Cardiac cells (myocytes and fibroblasts) were dissociated from hearts of newborn rats by use of collagenase (EC 3.4.24.3) (Worthington Type I; 1 mg/ml), and allowed to settle on the grid for about 2 hr. Almost all cells not adhering to the collagen islands could then be washed away with medium. Proliferation of the cardiac cells was suppressed after 1-2 days by y-irradiation (60Co; 5000 rads in 25-30 sec, where one rad equals 1 X 10-2 J/kg). One to 5 days later, principal neurons were dissociated from superior cervical ganglia of newborn rats (Charles River CD) as previously described (1,18), and plated at a density such that many islands received only one or a few neurons. The cultures were grown in L-15 CO2 medium (1) containing 5% adult rat serum or 10% fetal calf serum (Microbiological Associates, 14-414), but lacking bovine serum albumin and Methocel. Six platings (about 30 dishes per plating) were used in experiments reported here.For electrophysiological recordi...

Excessive release of glutamate and the subsequent inf lux 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 p44͞42 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 glutamatereceptor 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-p44͞42 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 M; EC 50 ‫؍‬ 10 M) during a 30-min washout of synaptic blockers inhibited the phosphorylation of p44͞42 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 p44͞42 MAP kinase pathway in neurons.Stimulation of numerous cell-surface receptors leads to the activation of kinase cascades that integrate and amplify extracellular signals and transmit them to intracellular targets (1). Mitogen-activated protein kinases (MAP kinases), a family of serine͞threonine kinases, are activated by phosphorylation on threonine and tyrosine (2, 3). One subfamily of MAP kinases, known as extracellular signal-regulated kinase (ERK), is activated strongly by mitogens, including growth factors, leading to many cellular responses including proliferation and differentiation (4, 5). Two ERK isoforms, ERK1 (p44) and ERK2 (p42), are highly expressed in the brain; p42 is particularly enriched in the dendrites and somata of discrete neuronal populations including the hippocampus (4, 6). The function of p44͞42 MAP kinase in postmitotic, terminally differentiated neurons is unclear, although recent reports implicate these kinases in synaptic plasticity (7,8).Stimulation of glutamate receptors and influx of Ca 2ϩ are associated with excitotoxic injury (9, 10) and lead to the phosphorylation of p44͞42 MAP kinase in neurons (11-16).Several neurological insults that produce an excessive release of glutamate and neuronal death, including hypoglycemia, ischemia, and kainate-or bicuculline-induced seizures, also result in the phosphoryla...

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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.

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.

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