Perturbed Ca2+-dependent signaling of DYT2 hippocalcin mutant as mechanism of autosomal recessive dystonia.

A recent report of autosomal-recessive primary isolated dystonia (DYT2 dystonia) identified mutations in HPCA, a gene encoding a neuronal calcium sensor protein, hippocalcin (HPCA), as the cause of this disease. However, how mutant HPCA leads to neuronal dysfunction remains unknown. Using a multidisciplinary approach, we demonstrated the failure of dystonic N75K HPCA mutant to decode short bursts of action potentials and theta rhythms in hippocampal neurons by its Ca2+-dependent translocation to the plasma membrane. This translocation suppresses neuronal activity via slow afterhyperpolarization (sAHP) and we found that the N75K mutant could not control sAHP during physiologically relevant neuronal activation. Simulations based on the obtained experimental results directly demonstrated an increased excitability in neurons expressing N75K mutant instead of wild type (WT) HPCA. In conclusion, our study identifies sAHP as a downstream cellular target perturbed by N75K mutation in DYT2 dystonia, demonstrates its impact on neuronal excitability, and suggests a potential therapeutic strategy to efficiently treat DYT2.

Persistent calcium current in rat suprachiasmatic nucleus neurons.

The suprachiasmatic nuclei contain the primary circadian clock, and suprachiasmatic nuclei neurons exhibit a diurnal modulation of spontaneous firing rate. The present study examined the voltage-gated persistent Ca(2+) current, in acutely isolated rat suprachiasmatic nuclei neurons using a ramp-type voltage-clamp protocol. Slow triangular voltage-clamp commands from a holding potential of -85 mV to +5 mV elicited inward current (100-400 pA) that was completely blocked by Cd(2+). This current showed little or no hysteresis, and was identified as persistent Ca(2+) current. The threshold for persistent Ca(2+) current ranged between -60 and -45 mV, and it was maximal at about -8 mV. Nifedipine at 10-20 microM blocked 80-100%. To assess the role of persistent Ca(2+) current in the generation of spontaneous action potentials in both acutely isolated and intact suprachiasmatic nuclei neurons, the effect of Cd(2+) and nifedipine on firing rate was studied using on-cell recording. Application of Cd(2+) exerted a weak excitatory effect and nifedipine had no significant effect on the spontaneous firing rate of isolated suprachiasmatic nuclei neurons. In all intact suprachiasmatic nuclei neurons in slice preparations (n=15), Cd(2+) slowly inhibited spontaneous firing; in high-frequency firing cells (four of 15), a transient increase of firing rate preceded inhibition. No significant effect of nifedipine on firing rate of intact suprachiasmatic nuclei neurons was found. Therefore, persistent Ca(2+) current itself (as carrier of charge) does not appear to contribute significantly to spontaneous firing of suprachiasmatic nuclei neurons. A slowly developing inhibitory effect of Cd(2+) on spontaneous firing of intact suprachiasmatic nuclei neurons in slice preparations may be due to penetration of Cd(2+) through Ca(2+) channels, and its subsequent effect on intracellular mechanisms, while the transient increase of firing rate in high-frequency firing neurons is probably due to inhibition of Ca(2+)-activated K(+) current.

Persistent subthreshold voltage-dependent cation single channels in suprachiasmatic nucleus neurons.

The hypothalamic suprachiasmatic nucleus (SCN) contains the primary circadian pacemaker in mammals, and transmits circadian signals by diurnal modulation of neuronal firing frequency. The ionic mechanisms underlying the circadian regulation of firing frequency are unknown, but may involve changes in membrane potential and voltage-gated ion channels. Here we describe novel tetrodotoxin- and nifedipine-resistant subthreshold, voltage-dependent cation (SVC) channels that are active at resting potential of SCN neurons and increase their open probability (P(o)) with membrane depolarization. The increased P(o) reflects changes in the kinetics of the slow component of the channel closed-time, but not the channel open-time or fast closed-time. This study provides a background for investigation of the possible role of SVC channels in regulation of circadian oscillations of membrane excitability in SCN neurons.

Role of the axodendritic tree in the functioning of helix bursting neurons: generation of pacemaker activity and propagation of action potentials along the axon.

The purpose of this study was to determine the role of the axodendritic tree in the generation of bursting pacemaker activity in the identified Helix RPa1 neuron, which is homologous to the Aplysia R15 cell, and propagation of action potentials along the axons. In doing so, I used recording of RPa1 neuron electrical activity after cutting off the right or left parietovisceral connections, the two-electrode voltage-clamp technique, registration of electrical activity of visceral nerve containing RPa1 axon branches, isolation of the RPa1 neuron and puff application of oxytocin on it. Cutting of the right (but not left) parietovisceral connection in all cases (more than 15 preparations) evoked complete disappearance of bursting pacemaker activity in the RPa1 neuron and hyperpolarization of its membrane potential up to -65 to -67 mV. Such silent state of the RPa1 neuron was maintained after its complete isolation from the ganglion. The described cutting did not result in a change of bursting activity of the pacemaker neuron V7 located in the visceral ganglion, although isolation of the V7 neuron also eliminated its own activity. Puff application of oxytocin (10 microM in a micropipette) on to the RPa1 neuron both after cutting the right parietovisceral connection or isolation of the neuron from the ganglion resulted in all cases (more than 10 cells) in transient depolarization with development of beating, oscillatory or bursting activity. Voltage clamping of RPa1 soma in the intact ganglion at a level close to zero membrane current sometimes, and, as a rule, at a more depolarized level, revealed bursting-like oscillations of membrane current, reflecting electrical bursting activity in the unclamped remote region of a neuron, most likely in the dendritic tree. Voltage clamping of RPa1 soma possessing bursting activity reveals bursting-like oscillation of membrane current and prevents propagation of corresponding axon action potentials in the visceral nerve. Controversially, clamping of RPa1 soma possessing beating activity exhibits a beating-like oscillation of membrane current and does not prevent propagation of corresponding action potentials in the nerve. Within the framework of the developed hypothesis that persistent bursting pacemaker activity of the RPa1 neuron is due to a constant activation of its peptidergic synaptic inputs [Kononenko N. I. (1993) Comp. Biochem. Physiol. 106A, 135-147], the experimental results were interpreted in the manner that these synapses and, correspondingly, the locus of electrical bursting activity generation, are localized on the dendritic tree of the RPa1 neuron mainly or possibly exclusively in the visceral ganglion. It is hypothesized that bursting and beating neuronal activities are due to functioning of different loci of the dendritic tree, regarding their electrical relations with axon branches.

Ion regulation of the kinetics of potential-dependent potassium channels.

We apply a theoretical approach developed earlier. The interaction ofions that permeate a channel with slowly relaxing charged channel-forminggroups (ion-conformational interaction - ICI) is addressed by thisapproach. One can describe the ion concentration influence (ion regulation)on channel functioning in this manner. A patch-clamp method in a'whole-cell' configuration is used to study the ICI. For this purpose theinfluence of an external concentration of potassium ions on thepotential-dependent potassium current (I(A)) in the externalmembrane of GH(3) cells was studied. The increase of[K(+) (out)] from 5 mM to 100 mM causes anon-monotonous shift of current-voltage dependencies. The dependence of bothan activation time constant tgr(n) and a steady-state activation(n(∞)) on [K(+)](out) have a minimum andmaximum respectively. The analysis of the results suggests that the observedeffects are caused by ICI. A physical model is developed to describe thedependence of the potassium channel kinetics on the external concentrationof the ions and the membrane potential. The 'deformation' of the closedstate of the gate and the corresponding energy shifts cause the observednon-monotonous dependencies due to ICI. Thus, the general theoreticalapproach has an experimental confirmation and is applied to concreteexamples. Formulas for concentrational dependencies of the channel kineticsare given for practical uses.

Mathematical model of pacemaker activity in bursting neurons of snail, Helix pomatia.

On the basis of experimental data we have developed a mathematical model of pacemaker activity in bursting neurons of snail Helix pomatia which includes a minimal model of membrane potential oscillation, spike-generating mechanism, voltage- and time-dependent inward calcium current, intracellular calcium ions, [Ca2+]in, their fast buffering and accumulation, stationary voltage-dependent [Ca2+]in-inhibited calcium current. A resulting model of bursting pacemaker activity reproduces all experimental phenomena which were mimicked on the minimal model for membrane potential oscillation including (a) the effect of polarizing current on bursting activity, (b) an increase of input resistance during depolarizing phase, (c) induced hyperpolarization, etc. This model demonstrates adaptation of bursting activity to both the polarizing current and changes in the stationary sodium or potassium conductances. The model also reproduces the behavior of the transmembrane ionic current at membrane potentials clamped in different phases of slow-wave development; the calculated current-voltage relationships of the model neuronal membrane using a slow ramp potential clamp demonstrate hysteresis properties. Relationships between the model of bursting activity and the properties if intact bursting neurons are discussed.

Deterministic chaos in mathematical model of pacemaker activity in bursting neurons of snail, Helix pomatia.

Chaotic regimes in a mathematical model of pacemaker activity in the bursting neurons of a snail Helix pomatia, have been investigated. The model includes a slow-wave generating mechanism, a spike-generating mechanism, an inward Ca current, intracellular Ca ions, [Ca2+]in, their fast buffering and uptake by intracellular Ca stores, and a [Ca2+]in-inhibited Ca current. Chemosensitive voltage-activated conductance, gB*, responsible for termination of the spike burst, and chemosensitive sodium conductance, gNa*, responsible for the depolarization phase of the slow-wave, were used as control parameters. These conductances in intact snail bursting neuron are regulated by neuropeptides. Time courses of the membrane potential and [Ca2+]in were employed to analyse different regimes in the model. Histograms of interspike intervals, autocorrelograms, spectral characteristics, one-dimensional return maps, phase plane trajectories, positive Lyapunov exponent and especially cascades of period-doubling bifurcations demonstrate that approaches to chaos were generated. The bifurcation diagram as a function of gB* and the ([Ca2+]in-V) phase diagram of initial conditions reveal fractal features. It has been observed that a short-lasting depolarizing current of elevation of [Ca2+]in may evoke transformation of chaotic activity into a regular bursting one. These kinds of transitions do not require any changes in the parameters of the model. The results demonstrate that chaotic regimes of neuronal activity modulated by neuropeptides may play a relevant role in information processing and storage at the level of a single neuron.

Electron cytochemical study of carbohydrate components in different types of cultured glial cells of snail Helix pomatia.

Using a variety of colloidal gold-labelled lectins with different sugar specificities, the structure and topography of carbohydrate determinants of the surface membrane of in vitro cultured glial and nerve cells of the snail Helix pomatia have been electron cytochemically studied. Heterogeneity of carbohydrate pools among different types of glial cells and between glial and nerve cells was established. It was found that satellite glial cells having the ultrastructural signs of cells with high metabolic level (type II cells) selectively bind GNA which is specific to terminal alpha-D-mannose residues and do not bind other mannose-specific lectins, Con A and LCA. GNA determinants are absent in satellite type I glial cells, fibrous glial cells, microglia and neurons. It has been found that glial cells (satellite type I and II glial cells, filamentous glial cells and microglial cells) do not bind PVA and LABA. LTA did not bind to any glial cells and binds weakly to neurons. Con A and WGA determinants which are abundant on the neurons are completely absent on satellite type II glial cells but present on satellite type I glial cells and filamentous glial cells. Microglial cells contain Con A and LCA determinants and the density of PNA determinants on these cells is the highest compared to other types of glial cells or neurons. It is concluded that some lectin determinants (for RCA-1, PNA, LPA) are present on all types of glial cells, while another determinant (GNA) is specific for a definite type of glial cells and can serve as a marker of these cells. The role of specific carbohydrate determinants in the functioning of a neuron-glial complex is discussed.

Excitatory and inhibitory monosynaptic peptidergic transmissions in the Cns of the snail Helix pomatia.

The results of an investigation of the properties of excitatory and inhibitory monosynaptic peptidergic transmission between identified nerve cells of the snail Helix pomatia are presented in this paper. It was demonstrated that stimulation of the presynaptic interneuron activates stationary and potential-and time-dependent ionic channels in the postsynaptic cells.

Newly identified nerve cells of the snail, Helix pomatia, associated with the generation of pacemaker activity.

Data on new, previously unidentified nerve cells of the snail Helix pomatia are presented in this paper. The identified neurons described may serve as a convenient model for the investigation of the cellular mechanisms of pacemaker activity, the role of neuropeptides in the generation and regulation of pacemaker activity, peptidergic transmission, and the functional role of the inward calcium current.

Patch-clamp recording of cAMP-induced membrane current noise in Helix pomatia neurons.

Membrane current noise evoked by intracellular cAMP injection was studied in isolated Helix pomatia neurons with the patch-clamp technique. Fluctuation analysis was used to estimate the elementary current amplitude (i) and the single channel conductance. It was found that i decreased linearly with cell depolarization and the extrapolated reversal potential was approximately -12 mV. The calculated single-channel conductance was 0.9 +/- 0.14 pS, a value quite different from those obtained for cAMP-activated channels in Pleurobranchaea neurons.

[Excitatory and inhibitory monosynaptic peptidergic transmissions in the CNS of the edible snail Helix pomatia]. / Vozbuzhdaiushchaia i tormoziashchaia monosinapticheskie peptidergicheskie peredachi v TsNS vinogradnoi ulitki Helix pomatia.

Dual action of peptidergic interneuron stimulation was described in the CNS of Helix, being excitatory on bursting neurons and inhibitory on beating ones. The properties of postsynaptic responses were studied.

Dopamine inhibits transmission between the interneuron initiating pacemaker activity in a bursting neuron and the bursting neuron on the snail Helix pomatia.

1. The effect of external application of dopamine on connection between a peptidergic interneuron initiating bursting pacemaker activity in non-active burster RPa1 and the bursting neuron itself of the snail Helix pomatia has been investigated under clamp conditions. 2. External application of dopamine in a dose-dependent manner (Kd 15 microM) produces a reversible inhibition of slow inward current (SIC) in the RPa1 neuron evoked by stimulation of peptidergic interneuron. At the same time, an increase of duration and amplitude of interneuronal action potential was observed. 3. Stimulation of the anal nerve evokes in RPa1 neuron postsynaptic current consisting of at least three components: two fast ones with reversal potentials -50 and -70 mV and a long-lasting one with no reversal potential between -50 and -95 mV. 4. It is concluded that inhibition of SIC by dopamine is due to the processes occurring in the postsynaptic RPa1 neuron. 5. Based on the hypothesis that bursting activity of RPa1 neuron results from the activation of presynaptic unidentified peptidergic interneuron(s) with persistent activity and on data presented it is suggested that inhibition of bursting activity evoked by application of dopamine or anal nerve stimulation is a consequence of decreased efficiency of synaptic transmission between the interneuron initiating bursting activity and the burster.

[Electron-cytochemical study of carbohydrate components in the surface membrane of cultured Helix pomatia neurons]. / Elektronno-tsitokhimicheskoe issledovanie karbogidratnykh komponentov poverkhnostnoi membrany kul'tiviruemykh neironov vinogradnoi ulitki.

The structure and topography of carbohydrates on the surface of nerve cells of snail Helix pomatia cultured in vitro have been characterized with a series of colloidal gold-labelled lectins of different sugar specificity. The analysis of the lectin binding has shown substantial differences in the carbohydrate pattern between the soma of monoaminergic and peptidergic neurons. It has been found that the surface of monoaminergic and peptidergic neurons contains N-acetylglucosamine (WGA+) and N-acetyllactosamine (RCA-1-) determinants and does not contain neuraminic acid (LPA-) and complex branched N-glycosyl chains (PVA-). At the same time N-acetylgalactosamine (HPA+) was detected on the peptidergic neuron membrane only. It has been concluded that terminal residues of sialic acid are absent on the most of snail nerve cells. Differences in lectin binding between monoaminergic and peptidergic neurons can serve as a basis for formation of specific connections of cells by different types in the developing brain.

Inhibition by oxytocin of voltage-activated calcium influx in cultured PC12 pheochromocytoma cells.

1. Voltage-activated dihydropyridine-sensitive Ca2+ influx was measured in PC12 pheochromocytoma cells using 45Ca. 2. It has been found that oxytocin inhibits voltage-activated dihydropyridine-sensitive Ca2+ influx with ED50 about 0.30 x 10(-6) M. 3. Tolbutamide (1.3 x 10(-3) M) has no visible effect on both Ca2+ influx itself and on the inhibitory oxytocin effect. 4. External application of Li+ (10 mM) causes a slight shift of ED-curve to lower oxytocin concentrations. 5. It is suggested that the hydrolysis of phosphoinositides may play a role in oxytocin action on Ca2+ influx in PC12 cells.

The effect of oxytocin on potential-dependent calcium current in snail neurons, Helix pomatia.

1. The effect of external application of oxytocin on inward calcium current in dialyzed snail neurons has been investigated under clamp conditions. 2. External application of oxytocin in a dose-dependent manner (Kd 0.9 microM) inhibits inward calcium current in dialyzed neurons of the snail, Helix pomatia. 3. Inhibition of calcium current developed with the time constant of about 2 min. The degree of restoration of calcium current after oxytocin washout depends on duration of oxytocin action. 4. It has been suggested that inhibition of calcium current by oxytocin occurs in two stages, the initial one is more fast and reversible and the second one--more slow and irreversible. The participation of soluble second messengers in the inhibitory effect of oxytocin on calcium current is discussed.

Inhibition of an identified snail postsynaptic neuron evoked by stimulation of a peptidergic interneuron initiating bursting pacemaker activity in another neuron.

Beating neuron V8, whose activity is abolished upon interneuronal stimulation, has been found and identified in the CNS of the snail, Helix pomatia. It was found earlier that stimulation of the same interneuron initiates bursting pacemaker activity in another neuron. Under voltage clamp conditions interneuronal stimulation evoked a slow outward current in the V8 neuron with a latency of about 2 s. Intracellular injection of tetraethylammonium or Cs+ ions into the interneuron greatly increased the slow outward current amplitude. External application of CdCl2 (1 mM), LaCl3 (5 mM) or replacement of Ca2+ by Mg2+ ions reversibly abolished transmission between the interneuron and the V8 neuron. The neuronal membrane conductance increased by about three times on the maximum of the slow outward current development. Computer fitting showed that a decay of the slow outward current can be approximated by the sum of two exponentially decaying components with time constants of about 1 s and 16 s. Extrapolated reversal potential for a fast (1 s) component of the current was about -70 mV. A slow (16 s) component asymptotically decreased upon hyperpolarization and it was observed at even more negative potential than Ek. Theophylline (1 mM) and 3-isobutyl-1-methylxanthine (0.1 mM) reversibly increased the slow outward current amplitude, whereas imidazole (5 mM) and tolbutamide (5 mM) caused its reversible decrease. It is concluded that inhibitory monosynaptic transmission exists between the interneuron and the beating V8 neuron. Interneuronal simulation evokes an increase in the membrane potassium conductance and probably a decrease in stationary potential-dependent sodium conductance of the postsynaptic V8 neuron. The cellular cyclase system seems to be involved in the postsynaptic response.