Synaptic activity, induced rhythmic discharge patterns, and receptor subtypes in enriched primary cultures of embryonic rat motoneurones

Long-term cultures of ventral horn neurones from embryonic rat spinal cord were established, after enrichment using density gradient centrifugation, to give a high proportion of cells (&gt;82%) with motoneurone characteristics. Neurones were grown on spinal cord glial monolayers for 4-83 days and investigated using whole-cell patch clamp. Synaptic activity interrupted by periods of quiescence increased in frequency with culture age and was suppressed by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and strychnine. However, strychnine (10 µM) or bicuculline (10-30 µM) or removal of Mg2+ alone induced patterned rhythmic bursting. Glutamate (3-300 µM), alpha -amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA, 0.3-30 µM), and kainate (1-300 µM) evoked inward currents, as did N-methyl-D-aspartic acid (NMDA, 100 µM) in the absence of Mg2+ and presence of glycine (3-10 µM). Inward currents carried by Cl- were elicited by glycine (10-300 µM) and GABA (1-300 µM), while adenosine (1-10 µM) and cyclopentyladenosine (10 nM - 1 µM) evoked a K+-dependent hyperpolarization. 5-HT, GABAB, purine A, and metabotropic glutamate receptors modulated synaptic excitation of presumed motoneurones. The results suggest that long-term cultures, containing more than 82% developing motoneurones, are able to generate rhythmic bursting; they respond to many of the neurotransmitters that are likely to be released onto motoneurones developing in vivo.Key words: embryonic rat motoneurones, culture, amino acid receptors, adenosine, spinal cord. </jats:p

Detection of a novel mutation in X-linked amelogenesis imperfecta

Amelogenesis imperfecta (AI) is a heterogeneous group of inherited disorders of defective enamel formation. The major protein involved in enamel formation, amelogenin, is encoded by a gene located at Xp22.1-Xp22.3. This study investigated the molecular defect producing a combined phenotype of hypoplasia and hypomineralization in a family with the clinical features and inheritance pattern of X-linked amelogenesis imperfecta (XAI). Genomic DNA was prepared from buccal cells sampled from family members. The DNA was subjected to the polymerase chain-reaction (PCR) in the presence of a series of oligonucleotide primers designed to amplify all 7 exons of the amelogenin gene. Cloning and sequencing of the purified amplification products identified a cytosine deletion in exon VI at codon 119. The deletion resulted in a frameshift mutation, introducing a premature stop signal at codon 126, producing a truncated protein lacking the terminal 18 amino acids. Identifying mutations assists our understanding of the important functional domains within the gene, and finding another novel mutation emphasizes the need for family-specific diagnosis of amelogenesis imperfecta