In Chlamydomonas reinhardtii, expression of the cabII-1 gene increases dramatically in response to light (cabII-1 encodes one of the light-harvesting chlorophyll a/b-binding proteins of photosystem II). We have used a region upstream of the cabII-1 gene in translational fusions to the bacterial uidA gene (encodes beta-glucuronidase) and transcriptional fusions to the Chlamydomonas nitrate reductase gene (nit1). Chlamydomonas transformants carrying intact copies of the chimeric uidA gene do not express beta-glucuronidase at the level of enzyme activity or mRNA accumulation. Methylation in the cabII-1 promoter region of the introduced gene is extensive in these strains, suggesting that newly introduced foreign genes may be recognized and silenced by a cellular mechanism that is correlated with increased methylation. Transformants that express the chimeric cabII-1/nit1 gene have been recovered. In contrast to the endogenous nit1 gene, the chimeric cabII-1/nit1 gene is expressed in ammonium-containing medium. Moreover, nit1 mRNA accumulation is dramatically stimulated by light, with a time course that is indistinguishable from that of the endogenous cabII-1 gene. The cabII-1/nit1 gene has been used to select transformants in a nit1- nit2- Chlamydomonas strain (CC400G) and should be useful for transformation of the large number of mutants in the Ebersold-Levine lineage, which carry the same mutations.

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1. Large neurons in the pedal ganglia of Aplysia californica were examined for their potential to modulate motoneuron-evoked contractions in foot and body-wall muscle. These neurons lie near the medial border of each pedal ganglion and have peripheral axons but no detectable motor effect. 2. Neurons in this region fire in rhythmic bursts during fictive escape crawling, and their action potentials resemble those recorded from parapodial opener-phase (POP) neurons in the swimming species, A. brasiliana. 3. Firing these neurons in conjunction with pedal motoneurons potentiates the force of contractions and increases their relaxation rate. Similar effects are produced by the serotonin (5-HT) analogue, bufotenine. 4. The modulatory neurons can be stained in vivo by 5,7-dihydroxytryptamine (5,7-DHT), suggesting they are serotonergic. 5-HT immunoreactivity is present in axons associated with foot and body-wall muscle. 5. Bath-applied 5-HT causes rhythmic bursting in the modulatory neurons. It appears likely that they are homologous to the POP cells of A. brasiliana.

1. We describe a group of serotonergic neurons in the pedal ganglia of Aplysia brasiliana and characterize their modulatory effects on the motoneuron input to swimming muscles of the parapodia. Each pedal ganglion contains one cluster of large neurons near its dorsomedial surface that fires in phase with opening (downstroke) of the parapodia; these have been designated parapodial opener-phase (POP) cells. 2. POP cells are large, number 15-20 per ganglion, have peripheral axons in parapodial nerves, have distinctively shaped action potentials, and fire in bursts phasically with motoneurons during the opening, or downstroke portion, of parapodial movement during fictive swimming. Firing individual POP cells with intracellular current indicates that they have no direct detectable effect on muscle, causing neither junction potentials nor contractions. 3. 5,7-Dihydroxytryptamine (5,7-DHT) staining, immunocytochemistry using serotonin (5-HT) antibodies, and direct biochemical measurements revealed that POP cells are serotonergic. Serotonergic nerve endings were also seen in parapodial muscle. 4. Simultaneous intracellular recordings and use of altered divalent concentrations revealed that no detectable direct synaptic interactions exist between POP cells and motor neurons. 5. When POP cells and motoneurons were simultaneously recorded while measuring muscle contractions, it was found that POP cell activity enhances motoneuron-induced tension by 120-900%, averaging around 300%. Variability in the efficacy of individual POP cells suggests that they may influence specific regions or groups of muscle fibers. 6. POP cell activity also significantly increased the rate of relaxation of parapodial muscle contractions, averaging about a 40% reduction in the time required to relax to one-half peak tension. Increased relaxation rate implies a postsynaptic change in muscle behavior. 7. The effectiveness of POP cells to increase contraction tension and relaxation rate was positively correlated with POP cell spike frequency. These effects were slow (seconds) in onset and could persist for a minute or more after cessation of POP firing. Concurrent motoneuron activity is not required for modulation by POP cells. 8. Simultaneous intracellular recording from a POP cell, motoneuron, and muscle fiber revealed that POP cell activity enhanced the amplitude of motoneuron-induced excitatory junction potentials (EJPs). Activity of POP cells did not alter muscle fiber membrane potential, but the experiments left open the possibility that EJP enhancement is presynaptic, postsynaptic, or a combination.(ABSTRACT TRUNCATED AT 400 WORDS)

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