The intact central nervous system of the newborn opossum in long-term culture: fine structure and GABA-mediated inhibition of electrical activity.
1. The entire central nervous system (CNS) of the newly born, South American opossum (Monodelphis domestica) was isolated and maintained in basal medium, Eagle's (BME) with 0.2% foetal calf serum and antibiotics. Isolated CNS preparations remained electrically excitable for up to 10 days. The fine structure of the spinal cord was normal after 5 days in culture: axons, synapses, dendrites and glia were virtually unchanged. Signs of degeneration were evident only in dorsal areas of the spinal cord, which had been denervated by removal of the dorsal root ganglia during dissection. 2. Amino acid transmitters such as glycine, glutamate, N-methyl-D-aspartate (NMDA) and gamma-aminobutyric acid (GABA), applied to the bathing fluid, rapidly and reversibly inhibited synaptic transmission in cervical segments of the spinal cord. GABA (10-100 mumol l-1) produced a dose-dependent reduction in the magnitude of ventral root responses evoked by dorsal root stimulation. GABA also inhibited synaptically activated compound action potentials produced by spinal cord stimulation. Dose-response curves for GABA obtained in different preparations were highly reproducible. 3. Both GABAA and GABAB receptors were reversibly activated by selective agonists and inhibited by specific antagonists. The actions of GABA were potentiated by benzodiazepines, competitively antagonised by bicuculline (a selective GABAA antagonist) and mimicked by muscimol (a GABAA agonist). Baclofen (a specific GABAB agonist) also inhibited electrical activity and was competitively antagonised by the GABAB antagonist, CGP 35348. 4. After 5 days of culture in BME or minimal essential medium (MEM), GABA dose-response curves were unchanged from those observed immediately after removal of the CNS. The inhibitory potency of baclofen was also unaffected by culture in BME. By contrast, after 5 days of culture in MEM, baclofen no longer inhibited electrical activity. This difference between BME and MEM could be attributed to the higher content of L-histidine in MEM. Thus, addition of 150 mumol l-1 L-histidine to BME produced similar results to culture in MEM: the inhibitory action of baclofen was virtually abolished after 3-5 days. L-Histidine had no effect on freshly dissected preparations. Chronic application of L-histidine did not affect glycine or glutamate responses after 5 days. Addition of D-histidine or other amino acids, such as arginine, to BME did not abolish the responses to baclofen. 5. These results show that the isolated CNS of the newborn opossum survives well in long-term culture and that it provides a useful preparation to study receptor development and plasticity of an intact mammalian CNS in vitro.(ABSTRACT TRUNCATED AT 400 WORDS)
Stewart
R R
RR
Department of Pharmacology, Universität Basel, Switzerland.
Zou
D J
DJ
Treherne
J M
JM
Møllgård
K
K
Saunders
N R
NR
Nicholls
J G
JG
D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D002470:Cell Survival; D002490:Central Nervous System / Q000254:growth & development* / Q000502:physiology; D003470:Culture Media; D004594:Electrophysiology; D006639:Histidine / Q000378:metabolism; D009893:Opossums; D009924:Organ Culture Techniques; D011963:Receptors, GABA-A / Q000378:metabolism; D013116:Spinal Cord / Q000254:growth & development / Q000502:physiology; D013997:Time Factors; D005680:gamma-Aminobutyric Acid / Q000378:metabolism* / Q000494:pharmacology
D000596:Amino Acids; D003470:Culture Media; D011963:Receptors, GABA-A; D006639:Histidine; D005680:gamma-Aminobutyric Acid