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Journal of Neuroscience, Vol 6, 3044-3060, Copyright © 1986 by Society for Neuroscience
Primary culture of identified neurons from the visual cortex of postnatal rats
JE Huettner and RW Baughman
We have examined the properties of neurons from the visual cortex of
postnatal Long Evans rats in dissociated cell culture. Visual cortex from
rat pups 1-15 d old was subjected to enzymatic and mechanical dissociation
to yield a suspension of single cells. Neurons plated onto collagen or a
feeder layer of astrocytes rapidly extended processes and survived for 4-10
weeks. Antisera to glutamic acid decarboxylase, choline acetyltransferase,
and vasoactive intestinal polypeptide stained 22 +/- 2, 2.3 +/- 0.3, and
2.4 +/- 0.2% of all neurons, respectively, suggesting that different
neuronal classes survived roughly in proportion to their number in vivo. In
order to study a particular identified class of cortical neurons, we
prelabeled cells in vivo by retrograde transport of a fluorescent tracer.
Neurons in layer V of visual cortex that project to the superior colliculus
were labeled after injecting fluorescent latex microspheres into the
colliculus. Retrogradely labeled neurons were readily identified
immediately after dissociation and throughout the period in vitro. After 2
weeks in culture, labeled cells exhibited many ultrastructural features
characteristic of pyramidal neurons in vivo. Intracellular recording
techniques were used to evaluate the response properties of labeled layer V
neurons, as well as other, unlabeled neurons, to excitatory amino acid
agonists and antagonists. Glutamate and aspartate--as well as the synthetic
agonists N-methyl-D-aspartate (NMDA), kainate, and quisqualate--excited
every cortical neuron tested. The antagonist 2- amino-5-phosphonovaleric
acid had no effect on responses to quisqualate and kainate but completely
blocked depolarizations due to NMDA and aspartate and reduced
depolarizations elicited by low concentrations of glutamate. Kynurenic
acid, piperidine dicarboxylic acid, and gamma-D- glutamylglycine
antagonized responses to all 5 of the agonists. These results provide
evidence that corticocollicular neurons in culture express both NMDA-type
and non-NMDA receptors for excitatory amino acids.
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