41 research outputs found
Neurotrophic actions of dopamine on the development of a serotonergic feeding circuit in Drosophila melanogaster
<p>Abstract</p> <p>Background</p> <p>In the fruit fly, <it>Drosophila melanogaster</it>, serotonin functions both as a neurotransmitter to regulate larval feeding, and in the development of the stomatogastric feeding circuit. There is an inverse relationship between neuronal serotonin levels during late embryogenesis and the complexity of the serotonergic fibers projecting from the larval brain to the foregut, which correlate with perturbations in feeding, the functional output of the circuit. Dopamine does not modulate larval feeding, and dopaminergic fibers do not innervate the larval foregut. Since dopamine can function in central nervous system development, separate from its role as a neurotransmitter, the role of neuronal dopamine was assessed on the development, and mature function, of the 5-HT larval feeding circuit.</p> <p>Results</p> <p>Both decreased and increased neuronal dopamine levels in late embryogenesis during development of this circuit result in depressed levels of larval feeding. Perturbations in neuronal dopamine during this developmental period also result in greater branch complexity of the serotonergic fibers innervating the gut, as well as increased size and number of the serotonin-containing vesicles along the neurite length. This neurotrophic action for dopamine is modulated by the D<sub>2 </sub>dopamine receptor expressed during late embryogenesis in central 5-HT neurons. Animals carrying transgenic RNAi constructs to knock down both dopamine and serotonin synthesis in the central nervous system display normal feeding and fiber architecture. However, disparate levels of neuronal dopamine and serotonin during development of the circuit result in abnormal gut fiber architecture and feeding behavior.</p> <p>Conclusions</p> <p>These results suggest that dopamine can exert a direct trophic influence on the development of a specific neural circuit, and that dopamine and serotonin may interact with each other to generate the neural architecture necessary for normal function of the circuit.</p
Contribution of potential EF hand motifs to the calcium-dependent gating of a mouse brain large conductance, calcium-sensitive K+ channel
The large conductance, calcium-sensitive K+ channel (BKCa channel) is a unique member of the K+-selective ion channel family in that activation is dependent upon both direct calcium binding and membrane depolarization. Calcium binding acts to dynamically shift voltage-dependent gating in a negative or left-ward direction, thereby adjusting channel opening to changes in cellular membrane potential.We hypothesized that the intrinsic calcium-binding site within the BKCa channel α subunit may contain an EF hand motif, the most common, naturally occurring calcium binding structure. Following identification of six potential sites, we introduced a single amino acid substitution (D/E to N/Q or A) at the equivalent of the -z position of a bona fide EF hand that would be predicted to lower calcium binding affinity at each of the six sites.Using macroscopic current recordings of wild-type and mutant BKCa channels in excised inside-out membrane patches from HEK 293 cells, we observed that a single point mutation in the C-terminus (Site 6, FLD923QD to N), adjacent to the ‘calcium bowl’ described by Salkoff and colleagues, shifted calcium-sensitive gating right-ward by 50–65 mV over the range of 2–12 μm free calcium, but had little effect on voltage-dependent gating in the absence of calcium. Combining this mutation at Site 6 with a similar mutation at Site 1 (PVD81EK to N) in the N-terminus produced a greater shift (70–90 mV) in calcium-sensitive gating over the same range of calcium. We calculated that these combined mutations decreased the apparent calcium binding affinity ˜11-fold (129.5 μm vs. 11.3 μm) compared to the wild-type channel.We further observed that a bacterially expressed protein encompassing Site 6 of the BKCa channel C-terminus and bovine brain calmodulin were both able to directly bind 45Ca2+ following denaturation and polyacrylamide gel electrophoresis (e.g. SDS-PAGE).Our results suggest that two regions within the mammalian BKCa channel α subunit, with sequence similarities to an EF hand motif, functionally contribute to the calcium-sensitive gating of this channel
β2 and β4 Subunits of BK Channels Confer Differential Sensitivity to Acute Modulation by Steroid Hormones
Specific distribution of sodium channels in axons of rat embryo spinal motoneurones
The distribution of Na+ channels and development of excitability were investigated in vitro in purified spinal motoneurones obtained from rat embryos at E14, using electrophysiological, immunocytochemical and autoradiographical methods.One hour after plating the motoneurones (DIV0), only somas were present. They expressed a robust delayed rectifier K+ current (IDR) and a fast-inactivating A-type K+ current (IA). The rapid neuritic outgrowth was paralleled by the emergence of a fast-activating TTX-sensitive sodium current (INa), and by an increase in both K+ currents.The change in the three currents was measured daily, up to DIV8. The large increase in INa observed after DIV2 was accompanied by the onset of excitability. Spontaneous activity was observed as from DIV6.The occurrence of axonal differentiation was confirmed by the fact that (i) only one neurite per motoneurone generated antidromic action potentials; and (ii) 125I-α-scorpion toxin binding, a specific marker of Na+ channels, labelled only one neurite and the greatest density was observed in the initial segment. Na+ channels therefore selectively targeted the axon and were absent from the dendrites and somas.The specific distribution of Na+ channels was detectable as soon as the neurites began to grow. When the neuritic outgrowth was blocked by nocodazole, no INa developed.It was concluded that, in spinal embryonic motoneurone in cell culture, Na+ channels, the expression of which starts with neuritic differentiation, are selectively addressed to the axonal process, whereas K+ channels are present in the soma prior to the neuritic outgrowth
Molecular determinants of emerging excitability in rat embryonic motoneurons
Molecular determinants of excitability were studied in pure cultures of rat embryonic motoneurons. Using RT-PCR, we have shown here that the spike-generating Na+ current is supported by Nav1.2 and/or Nav1.3 α-subunits. Nav1.1 and Nav1.6 transcripts were also identified. We have demonstrated that alternatively spliced isoforms of Nav1.1 and Nav1.6, resulting in truncated proteins, were predominant during the first week in culture. However, Nav1.6 protein could be detected after 12 days in vitro. The Navβ2.1 transcript was not detected, whereas the Nav β1.1 transcript was present. Even in the absence of Navβ2.1, α-subunits were correctly inserted into the initial segment. RT-PCR (at semi-quantitative and single-cell levels) and immunocytochemistry showed that transient K+ currents result from the expression of Kv4.2 and Kv4.3 subunits. This is the first identification of subunits responsible for a transient K+ current in spinal motoneurons. The blockage of Kv4.2/Kv4.3 using a specific toxin modified the shape of the action potential demonstrating the involvement of these conductance channels in regulating spike repolarization and the discharge frequency. Among the other Kv α-subunits (Kv1.3, 1.4, 1.6, 2.1, 3.1 and 3.3), we showed that the Kv1.6 subunit was partly responsible for the sustained K+ current. In conclusion, this study has established the first correlation between the molecular nature of voltage-dependent Na+ and K+ channels expressed in embryonic rat motoneurons in culture and their electrophysiological characteristics in the period when excitability appears