Synaptogenesis and amino acid release from long term embryonic rat spinal cord neuronal culture using tissue culture inserts.
In the present study, using tissue culture inserts (TCI) coupled with a primary spinal cord neuronal culture, we characterize a new perfusion system, which permits continuous perfusate collection from cultured neurons. Primary spinal cord neurons were isolated from the lumbar portion of E14 spinal cords of Sprague-Dawley rats, plated on TCI and fed with DMEM/B27/10% FBS. At 1-4 weeks after isolation the development of synapses and neurotransmitter phenotype in cultured neurons was verified using immunofluorescence. A time-dependent development of synapses (Syn) was seen with a dense Syn-positive network identified at 3-4 weeks after plating. A sub-population of plated neurons (35-40%) showed GABA immunoreactivity and expressed NMDAR1 receptor. To measure neurotransmitter release, a chamber accommodating TCI was constructed permitting perfusion of the insert across the membrane. To evoke amino acid release from cultured neurons, NMDA (10 mmol/l) was added into the perfusion buffer. Stimulation with NMDA evoked a significant GABA (4050 +/- 950%) and glutamate release (130 +/- 42%) during first 10 min after exposure. In control non-stimulated cells no significant changes were measured. These data show that by using TCI it is possible to maintain embryonic spinal cord neurons for an extended period and that this system may represent a simple tool to identify neurotransmitter and/or peptides associated with a specific population of cultured brain and/or spinal cord neurons.
Marsala
Martin
M
Anesthesiology Research Laboratory-0818, 9500 Gilman Drive, University of California, San Diego, La Jolla, CA 92093, USA. mmarsala@ucsd.edu
Kakinohana
Osamu
O
Hefferan
Michael P
MP
Cizkova
Dasa
D
Kinjoh
Kiyohiko
K
Marsala
Silvia
S
D000818:Animals; D015415:Biomarkers; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D015194:Diffusion Chambers, Culture / Q000295:instrumentation / Q000379:methods; D005455:Fluorescent Antibody Technique; D018698:Glutamic Acid / Q000378:metabolism; D016202:N-Methylaspartate / Q000494:pharmacology; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D018377:Neurotransmitter Agents / Q000378:metabolism*; D010477:Perfusion / Q000295:instrumentation* / Q000379:methods; D010641:Phenotype; D051381:Rats; D017207:Rats, Sprague-Dawley; D016194:Receptors, N-Methyl-D-Aspartate / Q000378:metabolism; D013116:Spinal Cord / Q000166:cytology / Q000196:embryology / Q000502:physiology*; D013569:Synapses / Q000187:drug effects / Q000378:metabolism; D009435:Synaptic Transmission / Q000187:drug effects / Q000502:physiology; D046509:Tissue Culture Techniques / Q000295:instrumentation* / Q000379:methods; D005680:gamma-Aminobutyric Acid / Q000378:metabolism
D015415:Biomarkers; D018698:Glutamic Acid; D016202:N-Methylaspartate; D009419:Nerve Tissue Proteins; D018377:Neurotransmitter Agents; D016194:Receptors, N-Methyl-D-Aspartate; D005680:gamma-Aminobutyric Acid