K+ channel reorganization and homeostatic plasticity during postembryonic development: biophysical and genetic analyses in acutely dissociated Drosophila central neurons.
ABSTRACT: Intrinsic electric activities of neurons play important roles in establishing and refining neural circuits during development. However, how the underlying ionic currents undergo postembryonic reorganizations remains largely unknown. Using acutely dissociated neurons from larval, pupal, and adult Drosophila brains, we show drastic re-assemblies and compensatory regulations of voltage-gated (IKv) and Ca2+-activated (IK(Ca)) K+?currents during postembryonic development. Larval and adult neurons displayed prominent fast-inactivating IKv, mediated by the Shaker (Sh) channel to a large extent, while in the same neurons IK(Ca) was far smaller in amplitude. In contrast, pupal neurons were characterized by large sustained IKv and prominent IK(Ca), encoded predominantly by the slowpoke (slo) gene. Surprisingly, deletion of Sh in the ShM null mutant removed inactivating, transient IKv from large portions of neurons at all stages. Interestingly, elimination of Sh currents was accompanied by upregulation of non-Sh transient IKv. In comparison, the slo1 mutation abolished the vast majority of IK(Ca), particularly at the pupal stage. Strikingly, the deficiency of IK(Ca) in slo pupae was compensated by the transient component of IKv mediated by Sh channels. Thus, IK(Ca) appears to play critical roles in pupal development and its absence induces functional compensations from a specific transient IKv current. While mutants lacking either Sh or slo currents survived normally, Sh;;slo double mutants deficient in both failed to survive through pupal metamorphosis. Together, our data highlight significant reorganizations and homeostatic compensations of K+?currents during postembryonic development and uncover previously unrecognized roles for Sh and slo in this plastic process.
Project description:We report naturally occurring, systematic variations in synaptic strength at neuromuscular junctions along the dorsal-ventral (D-V) axis of the Drosophila larval body wall. These gradual changes were correlated with differences in presynaptic neurotransmitter release regulated by nerve terminal excitability and in postsynaptic receptor composition influencing miniature excitatory junctional potential (mEJP) amplitude. Surprisingly, synaptic strength and D-V differentials at physiological Ca(2+) levels were not significantly altered in slowpoke (slo) and Shaker (Sh) mutants, despite their defects in two major repolarizing forces, Ca(2+)-activated Slo (BK) and voltage-activated Sh currents, respectively. However, lowering [Ca(2+)](o) levels revealed greatly altered synaptic mechanisms in these mutants, indicated by drastically enhanced excitatory junctional potentials (EJPs) in Sh but paradoxically reduced EJPs in slo. Removal of Sh current in slo mutants by 4-aminopyridine blockade or by combining slo with Sh mutations led to strikingly increased synaptic transmission, suggesting upregulation of presynaptic Sh current to limit excessive neurotransmitter release in the absence of Slo current. In addition, slo mutants displayed altered immunoreactivity intensity ratio between DGluRIIA and DGluRIIB receptor subunits. This modified receptor composition caused smaller mEJP amplitudes, further preventing excessive transmission in the absence of Slo current. Such compensatory regulations were prevented by rutabaga (rut) adenylyl cyclase mutations in rut slo double mutants, demonstrating a novel role of rut in homeostatic plasticity, in addition to its well-established function in learning behavior.
Project description:Rhythmic motor behaviors are generated by networks of neurons. The sequence and timing of muscle contractions depends on both synaptic connections between neurons and the neurons' intrinsic properties. In particular, motor neuron ion currents may contribute significantly to motor output. Large conductance Ca(2+)-dependent K(+) (BK) currents play a role in action potential repolarization, interspike interval, repetitive and burst firing, burst termination and interburst interval in neurons. Mutations in slowpoke (slo) genes encoding BK channels result in motor disturbances. This study examined the effects of manipulating slo channel expression on rhythmic motor activity using Drosophila larva as a model system. Dual intracellular recordings from adjacent body wall muscles were made during spontaneous crawling-related activity in larvae expressing a slo mutation or a slo RNA interference construct. The incidence and duration of rhythmic activity in slo mutants were similar to wild-type control animals, while the timing of the motor pattern was altered. slo mutants showed decreased burst durations, cycle durations, and quiescence intervals, and increased duty cycles, relative to wild-type. Expressing slo RNAi in identified motor neurons phenocopied many of the effects observed in the mutant, including decreases in quiescence interval and cycle duration. Overall, these results show that altering slo expression in the whole larva, and specifically in motor neurons, changes the frequency of crawling activity. These results suggest an important role for motor neuron intrinsic properties in shaping the timing of motor output.
Project description:Calcium-sensitive potassium (K(Ca)) channels have been shown to modulate the diameter of cerebral pial arteries; however, little is known regarding their roles in controlling cerebral parenchymal arterioles (PAs). We explored the function and cellular distribution of small-conductance (SK(Ca)) and intermediate-conductance (IK(Ca)) K(Ca) channels and large-conductance K(Ca) (BK(Ca)) channels in endothelial cells (ECs) and smooth muscle cells (SMCs) of PAs. Both SK(Ca) and IK(Ca) channels conducted the outward current in isolated PA ECs (current densities, ~20?pA/pF and ~28?pA/pF at +40?mV, respectively), but these currents were not detected in PA SMCs. In contrast, BK(Ca) currents were prominent in PA SMCs (~154?pA/pF), but were undetectable in PA ECs. Pressurized PAs constricted to inhibition of SK(Ca) (~16%) and IK(Ca) (~16%) channels, but were only modestly affected by inhibition of BK(Ca) channels (~5%). Blockade of SK(Ca) and IK(Ca) channels decreased resting cortical cerebral blood flow (CBF) by ~15%. NS309 (6,7-dichloro-1H-indole-2,3-dione3-oxime), a SK(Ca)/IK(Ca) channel opener, hyperpolarized PA SMCs by ~27?mV, maximally dilated pressurized PAs, and increased CBF by ~40%. In conclusion, these data show that SK(Ca) and IK(Ca) channels in ECs profoundly modulate PA tone and CBF, whereas BK(Ca) channels in SMCs only modestly influence PA diameter.
Project description:BACKGROUND AND PURPOSE:Anthelmintics are required for treatment and prophylaxis of nematode parasites of humans and domestic animals. Emodepside, a cyclooctadepsipeptide, is a modern anthelmintic that has a novel mode of action involving a Ca-activated K channel (SLO-1) in Caenorhabditis elegans, sometimes mediated by a latrophilin (LAT) receptor. We examined mechanisms of action of emodepside in a parasitic nematode, Ascaris suum. EXPERIMENTAL APPROACH:RT-PCR was used to investigate expression of slo-1 and lat-1 in A. suum muscle flaps, and two-micropipette current-clamp and voltage-clamp techniques were used to record electrophysiological effects of emodepside. KEY RESULTS:Expression of slo-1 and lat-1 were detected. Emodepside produced a slow time-dependent (20 min), 4-aminopyridine sensitive, concentration-dependent hyperpolarization and increase in voltage-activated K currents. Sodium nitroprusside increased the hyperpolarizations and K currents. N-nitro-L-arginine inhibited the hyperpolarizations and K currents. Phorbol-12-myristate-13 acetate increased the K currents, while staurosporine inhibited the hyperpolarizations and K currents. Iberiotoxin reduced these emodepside K currents. The effect of emodepside was reduced in Ca-free solutions. Emodepside had no effect on voltage-activated Ca currents. CONCLUSIONS AND IMPLICATIONS:Asu-slo-1 and Asu-lat-1 are expressed in adult A. suum muscle flaps and emodepside produces slow activation of voltage-activated Ca-dependent SLO-1-like K channels. The effect of emodepside was enhanced by stimulation of protein kinase C and NO pathways. The data are consistent with a model in which NO, PKC and emodepside signalling pathways are separate and converge on the K channels, or in which emodepside activates NO and PKC signalling pathways to increase opening of the K channels.
Project description:The detection of cool temperatures is thought to be mediated by primary afferent neurons that express the cool temperature sensing protein Transient Receptor Potential Cation Channel, Subfamily M, Member 8 (TRPM8). Using mice, this study tested the hypothesis that sex differences in sensitivity to cool temperatures were mediated by differences in neurons that express TRPM8. Ion currents from TRPM8 expressing trigeminal ganglion (TRG) neurons in females demonstrated larger hyperpolarization-activated cyclic nucleotide-gated currents (Ih) than male neurons at both 30° and 18°C. Additionally, female neurons' voltage gated potassium currents (Ik) were suppressed by cooling, whereas male Ik was not significantly affected. At the holding potential tested (-60mV) TRPM8 currents were not visibly activated in either sex by cooling. Modeling the effect of Ih and Ik on membrane potentials demonstrated that at 30° the membrane potential in both sexes is unstable. At 18°, female TRPM8 TRG neurons develop a large oscillating pattern in their membrane potential, whereas male neurons become highly stable. These findings suggest that the differences in Ih and Ik in the TRPM8 TRG neurons of male and female mice likely leads to greater sensitivity of female mice to the cool temperature. This hypothesis was confirmed in an operant reward/conflict assay. Female mice contacted an 18°C surface for approximately half the time that males contacted the cool surface. At 33° and 10°C male and female mice contacted the stimulus for similar amounts of time. These data suggest that sex differences in the functioning of Ih and Ik in TRPM8 expressing primary afferent neurons leads to differences in cool temperature sensitivity.
Project description:Abstract Tethered flies allow studies of biomechanics and electrophysiology of flight control. We performed microelectrode recordings of spikes in an indirect flight muscle (the dorsal longitudinal muscle, DLMa) coupled with acoustic analysis of wing beat frequency (WBF) via microphone signals. Simultaneous electrophysiological recording of direct and indirect flight muscles has been technically challenging; however, the WBF is thought to reflect in a one-to-one relationship with spiking activity in a subset of direct flight muscles, including muscle m1b. Therefore, our approach enables systematic mutational analysis for changes in temporal features of electrical activity of motor neurons innervating subsets of direct and indirect flight muscles. Here, we report the consequences of specific ion channel disruptions on the spiking activity of myogenic DLMs (firing at ?5 Hz) and the corresponding WBF (?200 Hz). We examined mutants of the genes enconding: 1) voltage-gated Ca(2+) channels (cacophony, cac), 2) Ca(2+)-activated K(+) channels (slowpoke, slo), and 3) voltage-gated K(+) channels (Shaker, Sh) and their auxiliary subunits (Hyperkinetic, Hk and quiver, qvr). We found flight initiation in response to an air puff was severely disrupted in both cac and slo mutants. However, once initiated, slo flight was largely unaltered, whereas cac displayed disrupted DLM firing rates and WBF. Sh, Hk, and qvr mutants were able to maintain normal DLM firing rates, despite increased WBF. Notably, defects in the auxiliary subunits encoded by Hk and qvr could lead to distinct consequences, that is, disrupted DLM firing rhythmicity, not observed in Sh. Our mutant analysis of direct and indirect flight muscle activities indicates that the two motor activity patterns may be independently modified by specific ion channel mutations, and that this approach can be extended to other dipteran species and additional motor programs, such as electroconvulsive stimulation-induced seizures.
Project description:Large-conductance calcium-activated potassium (BK(Ca)) channels are composed of the pore-forming alpha-subunit and the auxiliary beta-subunits. The beta4-subunit is dominantly expressed in the mammalian central nervous system. To understand the physiological roles of the beta4-subunit on the BK(Ca) channel alpha-subunit (Slo), we isolated a full-length complementary DNA of rat beta4-subunit (rbeta4), expressed heterolgously in Xenopus oocytes, and investigated the detailed functional effects using electrophysiological means. When expressed together with rat Slo (rSlo), rbeta4 profoundly altered the gating characteristics of the Slo channel. At a given concentration of intracellular Ca(2+), rSlo/rbeta4 channels were more sensitive to transmembrane voltage changes. The activation and deactivation rates of macroscopic currents were decreased in a Ca(2+)-dependent manner. The channel activation by Ca(2+) became more cooperative by the coexpression of rbeta4. Single-channel recordings showed that the increased Hill coefficient for Ca(2+) was due to the changes in the open probability of the rSlo/rbeta4 channel. Single BK(Ca) channels composed of rSlo and rbeta4 also exhibited slower kinetics for steady-state gating compared with rSlo channels. Dwell times of both open and closed events were significantly increased. Because BK(Ca) channels are known to modulate neuroexcitability and the expression of the beta4-subunit is highly concentrated in certain subregions of brain, the electrophysiological properties of individual neurons should be affected profoundly by the expression of this second subunit.
Project description:Homeostatic control of intrinsic excitability is important for long-term regulation of neuronal activity. In conjunction with many other forms of plasticity, intrinsic homeostasis helps neurons maintain stable activity regimes in the face of external input variability and destabilizing genetic mutations. In this study, we report a mechanism by which Drosophila melanogaster larval motor neurons stabilize hyperactivity induced by the loss of the delayed rectifying K+ channel Shaker cognate B (Shab), by upregulating the Ca2+-dependent K+ channel encoded by the slowpoke (slo) gene. We also show that loss of SLO does not trigger a reciprocal compensatory upregulation of SHAB, implying that homeostatic signaling pathways utilize compensatory pathways unique to the channel that was mutated. SLO upregulation due to loss of SHAB involves nuclear Ca2+ signaling and dCREB, suggesting that the slo homeostatic response is transcriptionally mediated. Examination of the changes in gene expression induced by these mutations suggests that there is not a generic transcriptional response to increased excitability in motor neurons, but that homeostatic compensations are influenced by the identity of the lost conductance.NEW & NOTEWORTHY The idea that activity-dependent homeostatic plasticity is driven solely by firing has wide credence. In this report we show that homeostatic compensation after loss of an ion channel conductance is tailored to identity of the channel lost, not its properties.
Project description:Ca2+-activated ion channels shape membrane excitability in response to elevations in intracellular Ca2+. The most extensively studied Ca2+-sensitive ion channels are Ca2+-activated K+ channels, whereas the physiological importance of Ca2+-activated Cl- channels has been poorly studied. Here we show that a Ca2+-activated Cl- currents (CaCCs) modulate repetitive firing in mouse sympathetic ganglion cells. Electrophysiological recording of mouse sympathetic neurons in an in vitro preparation of the superior cervical ganglion (SCG) identifies neurons with two different firing patterns in response to long depolarizing current pulses (1 s). Neurons classified as phasic (Ph) made up 67% of the cell population whilst the remainders were tonic (T). When a high frequency train of spikes was induced by intracellular current injection, SCG sympathetic neurons reached an afterpotential mainly dependent on the ratio of activation of two Ca2+-dependent currents: the K+ [IK(Ca)] and CaCC. When the IK(Ca) was larger, an afterhyperpolarization was the predominant afterpotential but when the CaCC was larger, an afterdepolarization (ADP) was predominant. These afterpotentials can be observed after a single action potential (AP). Ph and T neurons had similar ADPs and hence, the CaCC does not seem to determine the firing pattern (Ph or T) of these neurons. However, inhibition of Ca2+-activated Cl- channels with anthracene-9'-carboxylic acid (9AC) selectively inhibits the ADP, reducing the firing frequency and the instantaneous frequency without affecting the characteristics of single- or first-spike firing of both Ph and T neurons. Furthermore, we found that the CaCC underlying the ADP was significantly larger in SCG neurons from males than from females. Furthermore, the CaCC ANO1/TMEM16A was more strongly expressed in male than in female SCGs. Blocking ADPs with 9AC did not modify synaptic transmission in either Ph or T neurons. We conclude that the CaCC responsible for ADPs increases repetitive firing in both Ph and T neurons, and it is more relevant in male mouse sympathetic ganglion neurons.
Project description:Painful axotomy decreases K(ATP) channel current (IK(ATP)) in primary afferent neurons. Because cytosolic Ca(2+) signaling is depressed in injured dorsal root ganglia (DRG) neurons, we investigated whether Ca(2+)-calmodulin (CaM)-Ca(2+)/CaM-dependent kinase II (CaMKII) regulates IK(ATP) in large DRG neurons. Immunohistochemistry identified the presence of K(ATP) channel subunits SUR1, SUR2, and Kir6.2 but not Kir6.1, and pCaMKII in neurofilament 200-positive DRG somata. Single-channel recordings from cell-attached patches revealed that basal and evoked IK(ATP) by ionomycin, a Ca(2+) ionophore, is activated by CaMKII. In axotomized neurons from rats made hyperalgesic by spinal nerve ligation (SNL), basal K(ATP) channel activity was decreased, and sensitivity to ionomycin was abolished. Basal and Ca(2+)-evoked K(ATP) channel activity correlated inversely with the degree of hyperalgesia induced by SNL in the rats from which the neurons were isolated. Inhibition of IK(ATP) by glybenclamide, a selective K(ATP) channel inhibitor, depolarized resting membrane potential (RMP) recorded in perforated whole-cell patches and enhanced neurotransmitter release measured by amperometry. The selective K(ATP) channel opener diazoxide hyperpolarized the RMP and attenuated neurotransmitter release. Axotomized neurons from rats made hyperalgesic by SNL lost sensitivity to the myristoylated form of autocamtide-2-related inhibitory peptide (AIPm), a pseudosubstrate blocker of CaMKII, whereas axotomized neurons from SNL animals that failed to develop hyperalgesia showed normal IK(ATP) inhibition by AIPm. AIPm also depolarized RMP in control neurons via K(ATP) channel inhibition. Unitary current conductance and sensitivity of K(ATP) channels to cytosolic ATP and ligands were preserved even after painful nerve injury, thus providing opportunities for selective therapeutic targeting against neuropathic pain.