R1-5 Mutant

Restriction sites for seven hexanucleotide-specific endonucleases were located on the map of the conjugative IncN plasmid R15 (SmrSurHgr, 62.3 kb). The distribution of the cleavage sites is strongly asymmetric. Twenty-eight of thirty-four sites for BamHI, EcoRI, HindIII, SalI, SmaI, and PstI were located close to or within the sequences of an IS5-like element and the transposons Tn2353 and Tn2354. By analysis of R15::Tn1756 deletion derivatives and recombinant plasmids harboring R15 fragments, the genetic determinants for the streptomycin, sulfonamide, and mercury resistances were mapped, as well as the regions necessary for EcoRII restriction-modification and for plasmid replication and conjugation. The features of physical and genetic structures of the plasmid R15 and other IncN plasmids are discussed.

Abstract

We have improved and extended an equivalent circuit model of the bursting neuron R15 in Aplysia and have simulated the neuromodulatory effects of serotonin (5-HT) and dopamine (DA), incorporating previously proposed mechanisms. The model produces dynamic model behavior consistent with the observed behavior of R15 in vitro, demonstrating the sufficiency of the proposed mechanisms. Responses of the model to extrinsic stimuli reveal that some of the seemingly paradoxical results can be attributed to the increase in conductance of two opposing currents, providing insight into the complex effects of the neuromodulation of multiple currents by a single neurotransmitter. These simulation results also suggest that neuromodulatory agents and second messengers act not only directly upon channel conductances, but also indirectly through the subsequent regulation of intracellular Ca2+ concentrations. A nullcline and bifurcation analysis of reduced fast and slow subsystems of the model is employed to investigate the mathematical mechanism of bursting and neuromodulation. The slow processes define a steady-state manifold upon which the burst trajectory lies upon during the silent phase of the burst. The active (spiking) phase of the burst occurs as the slow system variables sweep into an oscillatory branch of the solution space of the fast subsystem via a homoclinic transition. The neuromodulatory effects of 5-HT and DA are examined by analyzing their effects upon the slow subsystem. Changes in behavioral modes (silence, bursting, beating) of the model are related to the occurence of subcritical Hopf bifurcations in the slow subsystem. Theses bifurcations are caused by the modulation of the shape of both the nullclines and manifold of the slow subsystem by 5-HT and DA.The inhibitory response to dopamine (DA) of theAplysia burster R15 has been attributed previously either to a K+ conductance increase3 or to a decrease of the slow inward current (SIC) which is responsible for the bursting activity of R1524. The present study re-examines the main points which are at the origin of this controversy. It is shown that the response to short applications of DA is actually sensitive to the extracellular K+ concentration and that this response may always be inverted by hyperpolarization of the cell below EK (at least in a high-K+ solution). The results suggest that short applications of DA induce an increase in K+ conductance rather than a blockade of the SIC. This conclusion is strengthened by the effects of intracellular Cs+ injections and by Ca2+-removal experiments.

Prolonged DA perfusions may occasionally induce a second slow response in addition to the initial K+ conductance increase. This slow component of the DA response, which may imply modifications of the synaptic activity, does not seem to result either from a K+ conductance increase or from a blockade of the SIC.Voltage-clamp methods were employed to study the effects of serotonin (5-HT) and dopamine on the pharmacologically isolated calcium current in the identified Aplysia neuron R15 grown in cell culture. Neurons were obtained from juvenile animals and had not yet developed the bursting pacemaker pattern of activity characteristic of R15 in mature animals. In R15 5-HT elicits a biphasic response consisting of excitatory depolarization followed by an inhibitory hyperpolarization and dopamine elicits an inhibitory hyperpolarization. 5-HT increased the Ca2+ current without affecting its voltage dependence. The 5-HT effect persisted when Ba2+ was employed to carry current through Ca2+ channels. 5-HT did not affect the rate of Ca2+-dependent Ca2+ current inactivation other than through its effect on the magnitude of the Ca2+ current. The adenylate cyclase activator forskolin, in the presence of a phosphodiesterase inhibitor, also increased the magnitude of the Ca2+ or Ba2+ current. This result suggested that the 5-HT-induced enhancement of Ca2+ current was mediated by cAMP. Dopamine inhibited Ca2+ current when either Ca2+ or Ba2+ was employed as the current carrier. Dopamine did not affect the rat of Ca2+-dependent inactivation of Ca2+ current other than through its effect on the magnitude of the Ca2+ current. Intracellular injection of the Ca2+ chelator EGTA inhibited serotonergic modulation of the Ca2+ current but not dopaminergic modulation. These results indicated that the putative neurotransmitters 5-HT and dopamine may regulate bursting activity in mature R15 neurons through modulation of Ca2+ current.

Keywords

Aplysia

Calcium current

Serotonin

Dopamine

Forskolin

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An abdominal ganglion of the molluscAplysia california was found to contain 3 neurons in the place normally occupied by a single R15 cell. The 3 neurons exhibited properties characteristic of R15 neurons including spontaneous bursts. The bursts appeared asynchronously in spite of electrotonic coupling between them. The coupling function approximated a low pass filter with a cut-off frequency between 0.02 and 0.05 Hz in accordance with a measured coupling time-constant of 5–10 sec. Coupling measured in the cell body was found to be stronger for hyperpolarizing currents than for depolarizing currents injected into any of the 3 cells. This 'symmetrical rectification' can be explained by a rectifying axonal membrane interposed between the site of coupling and the site of recording.

All 3 cells were found to have dopamine receptors and to receive common synaptic inputs. Since the coupling efficiency was found to vary depending on the direction of current flow, depolarizing synaptic inputs and spike burst generation remain autonomous.

Spectrins belong to repetitive three-helix bundle proteins that have vital functions in multicellular organisms and are of potential value in nanotechnology. To reveal the unique physical features of repeat proteins we have studied the structural and mechanical properties of three repeats of chicken brain α-spectrin (R15, R16 and R17) at the atomic level under stretching at constant velocities (0.01, 0.05 and 0.1 Å·ps−1) and constant forces (700 and 900 pN) using molecular dynamics (MD) simulations at T = 300 K. 114 independent MD simulations were performed and their analysis has been done. Despite structural similarity of these domains we have found that R15 is less mechanically stable than R16, which is less stable than R17. This result is in agreement with the thermal unfolding rates. Moreover, we have observed the relationship between mechanical stability, flexibility of the domains and the number of aromatic residues involved in aromatic clusters.Brain Research

Volume 222, Issue 2, 19 October 1981, Pages 383-387

Development of intrinsic burst generation in identified neuron R15 ofAplysia

Author links open overlay panelHarunoriOhmoriab**

Outline

Abstract

The development of endogenous bursting pattern of identified neuron R15 ofAplysia was studied, in animals ranging from juvenils weighing 27 mg to adult animals weighing 350 g. In the youngest animals studied, R15 only fires irregularly, somewhat later it begins firing in brief bursts, and only in early adulthood does it acquire its characteristic burst firing pattern.We have concentrated our review on the single identified neurone R15 from the abdominal ganglion of aplysia. With the exception of the squid giant axon, R15 is probably the most widely studied cell in neurobiology. It has long attracted attention because of its bursting activity, its production of peptides, and its possible role in osmoregulation. More recently, a numeber of studies have focused on the synaptic inputs to R15 and on the mediation by second messengers of its response to neurotransmitters.

A central feature of the functioning of R15 is its rhythmic activity. This activity is generated endogenously by R15, but is subject to modulation by synaptic input and by hormonal Influences. Our account of the burst cycle emerges from a consideration of currents experimentally measurable in R15. Three elements not emphasized in previous descriptions of neuronal bursting activity are very important in our interpretation: (a) individual currents appear to have dual functions taking part both in the burst cycle and in its modulation by external input; (b) very small currents (nanoampere range) can have profound effects on activity in a cell producing action currents that are measured in microamperes; (c) axonal action potential conduction and hence cell topology play a crucial role in the generation of somal activity. These properties reveal a delicate balance of channel thresholds, passive membrane properties and anatomy. This is not engineer's solution to the problem of endogeneous burst production, but a suprrisingly complicated harmony of the diverse elements available. This became increasingly clear as we examined some of the modulatory mechanism present in R15. Many cases are known of synaptic or neurotransmitter modulation of voltage-dependent currents which contribute to endogenous neuronal activity (Siegelbaum and Tsien, 1983), and R15 provides one of the most complex examples (Wilson, 1980). In R15 known the synaptic inputs, as well as two putative neurotransmitters, directly alter the currents which we have shown are intimately involved in the generation of burst activity.

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