K.C. Marshall

P19 cells differentiate into glutamatergic and glutamate-responsive neurons in vitro.

The neurotransmitter L-glutamate has been associated with a number of developmental events within the central nervous system including synaptogenesis and the refinement of topographically ordered neural maps. As a model for studying such events at the molecular level, we have examined the expression of glutamate and glutamate receptors in neurons that develop from P19 cells in response to retinoids. We report here that many P19-derived neurons do contain glutamate in secretory vesicles and that this glutamate appears to function as a neurotransmitter. The neurotransmitter GABA is also present in these cultures and both glutamate and GABA appeared to co-localize in some neuronal processes. Both neurotransmitters were released from the neurons in response to membrane depolarization. These neurons also express various glutamate receptor subunits including GluR1, GluR4 and NMDAR1 as detected by immunological methods. Using whole-cell patch-clamping, we have recorded spontaneous postsynaptic potentials which increase in both amplitude and frequency with time in culture and which are sensitive to the glutamate antagonist kynurenic acid Thus, P19-derived neurons mature in culture and form electrically active neural networks involving glutamate and glutamate receptors.

Axonal projection patterns visualized with horseradish peroxidase in organized cultures of cerebellum

Abstract Neurons of the cerebellum and brain stem region of organotypic cultures were injected intracellularly with horseradish peroxidase. The axonal pattern was analyzed for the three cell types studied—Purkinje neurons, deep cerebellar neurons, and brain stem neurons. There was a consistent pattern for each cell type. The axon of the Purkinje neuron was uniform in diameter throughout, sometimes with one recurrent collateral, and within the deep nuclear region branched to produce a terminal field with many axon bulbs measuring 2–4 μm in diameter. The deep cerebellar neuron gave rise to a single axon which branched into a number of major axons, most of which coursed for long distances along the margin of the cortical region. Where the cortical region was separated off from the deep nuclear area, the axon of the deep cerebellar neuron branched and many axons coursed through the intervening zone to the cortex. The terminals in the cortical region were seen either as expansions along the fibre or as grape-like clusters on small side branches of the axons. The brain stem neuron had multiple axons which branched profusely and produced a system of fine-calibre varicose fibres, many of which coursed far into the outgrowth region. This study characterized the distribution of the axons of each neuron type. The results corroborate the fibre patterns seen in the living, stained and fluorescence studies of these cultures. The axon pattern for each neuron type resembles that of the corresponding neuron in the animal. These results will be correlated with the electrophysiological studies of the connections formed by the same neurons in sister cultures.

Electrophysiological and pharmacological studies of the inhibitory projection from the cerebellar cortex to the deep cerebellar nuclei in tissue culture

Abstract In explant cultures of mouse cerebellum, short latency inhibitions can be recorded from neurones in the deep cerebellar nuclei after electrical stimulation in the cortical region. The responsible projection appears to originate from Purkinje cells and to be monosynaptic to cells in deep nuclei. In many cases, the inhibition is followed by a rebound excitation which has been attributed to a disinhibition. Known blockers of synaptic inhibition (bicuculline, bicuculline methiodide, picrotoxin and strychnine) were added to the perfusing solution, for study of their effects on the evoked inhibition and on depressions of activity by iontophoretically applied glycine and γ-aminobutyrate. The results indicate that γ-aminobutyrate is probably the transmitter responsible for the synaptic inhibition. The experiments demonstrate the reproducibility of a central nervous system pathway in tissue culture and the ease of pharmacological manipulation of such a model system. The model has promising applicability for the study of synaptic mechanisms and as a test substrate for the interaction of drugs with a γ-aminobutyrate mediated pathway, particularly where the study of such substances is difficult in vivo.

Patterns of functional synaptic connections in organized cultures of cerebellum

Abstract Organized cultures of mouse cerebellum with separated regions containing cortical, deep nuclear neurons and brain stem neurons from the peduncular zone were used for electrophysiological studies of axonal projections and synaptic interactions. Responses of single neurons of each of the regions were recorded extracellularly and intracellularly during localized electrical stimulation of other parts of the explant, and indicated extensive synaptic interactions. Cortical stimulation inhibited deep nuclear neurons, apparently monosynaptically, and frequently caused antidromic activation of these cells. Synaptic responses of brain stem neurons to cortical stimulation were usually excitatory, but these were often succeeded by inhibitory potentials. Since brain stem cells were often antidromically activated, the excitatory synaptic responses may be mediated by collaterals of antidromically stimulated brain stem axons. Stimulation of the deep nuclear region could evoke inhibitory or excitatory potentials in cortical neurons, the most frequent response being an excitatory postsynaptic potential which was followed in about 2 ms by an inhibitory potential. Most excitatory and some inhibitory postsynaptic potentials followed high frequency stimulation with constant latencies. The results indicate that within these cultures there are rich synaptic interconnections, many of which follow patterns resembling those seen in the intact brain. The monosynaptic inhibitory projection from the cortex to the deep nuclei and collateral inhibition by Purkinje cell axons appear to be features of cerebellar function that are reproduced in this culture model. In addition, a projection from the deep nuclei to the cortex recently described in the intact cerebellum is also present in the cultures and gives postsynaptic potential responses typical of excitatory afferents to the cerebellar cortex. Such cultures appear useful as an experimental model for the study of synaptic mechanisms or the effects of drugs in the mammalian CNS.

Catecholamine Neurons of the Central Nervous System in Organotypic Culture

A standardized dissection has been designed to produce explants from the locus coeruleus of the newborn mouse brain. This area, termed the peduncular region, may be explanted with or without cerebellar neurons. Organotypic cultures with peduncular cells contain a cluster of 20-50 (or more) closely-packed large neurons, characterized by intracellular refractile granules in the living state. When treated with the glyoxylic acid method, somata of the granule-containing neurons exhibited catecholamine fluorescence, and a profuse plexus of varicose fluorescent fibers permeated the explant and its outgrowth. Intracellular staining of these cells with horseradish peroxidase demonstrated bipolar and multipolar neurons (25-30 micrometers), with stout dendrites and varicose axons. Granule-containing neurons stained for acetylcholinesterase. Electron microscopically, large dense granules were seen, which appear to correspond to the granules seen in living neurons. Cultures with these cells also have a high concentration of catecholamines in the medium. It is concluded that the granule-bearing neurons of the living culture are in fact neurons of the locus coeruleus.

Synchronous bursting of locus coeruleus neurons in tissue culture

Synchronous bursts of firing of locus coeruleus neurons have been observed in unanesthetized rats, particularly in response to various sensory stimuli. In explant tissue cultures, synchronous bursting activity of locus coeruleus neurons was also observed and the possible mechanisms responsible for this synchronous activation have been investigated. Barrages of depolarizing events apparently initiated and continued throughout spontaneous bursts of spikes in the cultured neurons. Simultaneous intracellular recordings from pairs of neurons show a very high degree of synchrony of such barrages between cells. On the basis of tests for electrical coupling in simultaneously recorded cell pairs, and tests for dye coupling with Lucifer Yellow, it was concluded that the synchrony is not due to electrical coupling of locus coeruleus neurons. Small non-synaptic interactions between cell pairs that may reflect elevated extracellular potassium levels have been observed on some occasions. Spontaneous and evoked depolarizations similar to those initiating the bursts appear to be synaptically mediated events, suggesting that locus coeruleus neurons are synchronously activated by a common excitatory input. It was concluded that the neurons providing this common excitation are located within or very close to the locus coeruleus, at least at birth. The synchronization of activation of many locus coeruleus neurons could result in almost simultaneous release of neurotransmitter in the widespread target areas of locus coeruleus projections.

GROWTH AND MYELINATION OF EXPLANT CULTURES IN DEFINED MEDIUM

SummaryThe purpose of this study was to compare the development of organotypic cultures in defined medium versus nutrient containing serum and embryo extract (EE). Explant cultures of cerebellum with or without locus ceruleus were grown in the Maximow system and monitored in the living state and with histological stains. Thinner explants, fibronectin and a more frequent feeding schedule were required to overcome the growth differences encountered using a defined medium. The final medium formulation was arrived at by evaluation of living cultures and consisted of a basal medium (Dulbeccos minimal essential medium), a number of hormones and other supplements, and a final glucose concentration of 750 mg %. Using a Golgi stain and histofluorescence, it was shown that the three major types of neurons—Purkinje, deep nuclear, and locus ceruleus—developed similarly in the defined medium and in serum-EE cultures. Myelination occurred in virtually all cerebellar cultures in defined medium and the onset was earlier than in serum-EE cultures. These results indicate that differentiation of oligodendroglia and maturation of neurons occur in a defined medium. Elimination of thyroid hormone delayed the maturation of the cultures, both neurons and myelin, by 3–4 days.