High-threshold Calcium Spikes Research Articles

Anoctamin 2-chloride channels reduce simple spike activity and mediate inhibition at elevated calcium concentration in cerebellar Purkinje cells.

Modulation of neuronal excitability is a prominent way of shaping the activity of neuronal networks. Recent studies highlight the role of calcium-activated chloride currents in this context, as they can both increase or decrease excitability. The calcium-activated chloride channel Anoctamin 2 (ANO2 alias TMEM16B) has been described in several regions of the mouse brain, including the olivo-cerebellar system. In inferior olivary neurons, ANO2 was proposed to increase excitability by facilitating the generation of high-threshold calcium spikes. An expression of ANO2 in cerebellar Purkinje cells was suggested, but its role in these neurons remains unclear. In the present study, we confirmed the expression of Ano2 mRNA in Purkinje cells and performed electrophysiological recordings to examine the influence of ANO2-chloride channels on the excitability of Purkinje cells by comparing wildtype mice to mice lacking ANO2. Recordings were performed in acute cerebellar slices of adult mice, which provided the possibility to study the role of ANO2 within the cerebellar cortex. Purkinje cells were uncoupled from climbing fiber input to assess specifically the effect of ANO2 channels on Purkinje cell activity. We identified an attenuating effect of ANO2-mediated chloride currents on the instantaneous simple spike activity both during strong current injections and during current injections close to the simple spike threshold. Moreover, we report a reduction of inhibitory currents from GABAergic interneurons upon depolarization, lasting for several seconds. Together with the role of ANO2-chloride channels in inferior olivary neurons, our data extend the evidence for a role of chloride-dependent modulation in the olivo-cerebellar system that might be important for proper cerebellum-dependent motor coordination and learning.

Biophysical properties and computational modeling of calcium spikes in serotonergic neurons of the dorsal raphe nucleus

Serotonergic neurons of the dorsal raphe nuclei, with their extensive innervation of nearly the whole brain have important modulatory effects on many cognitive and physiological processes. They play important roles in clinical depression and other psychiatric disorders. In order to quantify the effects of serotonergic transmission on target cells it is desirable to construct computational models and to this end these it is necessary to have details of the biophysical and spike properties of the serotonergic neurons. Here several basic properties are reviewed with data from several studies since the 1960s to the present. The quantities included are input resistance, resting membrane potential, membrane time constant, firing rate, spike duration, spike and afterhyperpolarization (AHP) amplitude, spike threshold, cell capacitance, soma and somadendritic areas. The action potentials of these cells are normally triggered by a combination of sodium and calcium currents which may result in autonomous pacemaker activity. We here analyse the mechanisms of high-threshold calcium spikes which have been demonstrated in these cells the presence of TTX (tetrodotoxin). The parameters for calcium dynamics required to give calcium spikes are quite different from those for regular spiking which suggests the involvement of restricted parts of the soma-dendritic surface as has been found, for example, in hippocampal neurons.

Presynaptic calcium signalling in cerebellar mossy fibres

Whole-cell recordings were obtained from mossy fibre terminals in adult turtles in order to characterize the basic membrane properties. Calcium imaging of presynaptic calcium signals was carried out in order to analyse calcium dynamics and presynaptic GABA B inhibition. A tetrodotoxin (TTX)-sensitive fast Na+ spike faithfully followed repetitive depolarizing pulses with little change in spike duration or amplitude, while a strong outward rectification dominated responses to long-lasting depolarizations. High-threshold calcium spikes were uncovered following addition of potassium channel blockers. Calcium imaging using Calcium-Green dextran revealed a stimulus-evoked all-or-none TTX-sensitive calcium signal in simple and complex rosettes. All compartments of a complex rosette were activated during electrical activation of the mossy fibre, while individual simple and complex rosettes along an axon appeared to be isolated from one another in terms of calcium signalling. CGP55845 application showed that GABA B receptors mediated presynaptic inhibition of the calcium signal over the entire firing frequency range of mossy fibres. A paired-pulse depression of the calcium signal lasting more than 1 s affected burst firing in mossy fibres; this paired-pulse depression was reduced by GABA B antagonists. While our results indicated that a presynaptic rosette electrophysiologically functioned as a unit, topical GABA application showed that calcium signals in the branches of complex rosettes could be modulated locally, suggesting that cerebellar glomeruli may be dynamically sub-compartmentalized due to ongoing inhibition mediated by Golgi cells. This could provide a fine-grained control of mossy fibre-granule cell information transfer and synaptic plasticity within a mossy fibre rosette.

Modulation of bursts and high-threshold calcium spikes in neurons of rat auditory thalamus

Neurons in the ventral partition of the medial geniculate body are able to fire high-threshold Ca 2+-spikes. The neurons normally discharge such spikes on low-threshold Ca 2+-spikes after the action potentials of a burst. We studied membrane mechanisms that regulate the discharge of high-threshold Ca 2+-spikes, using whole-cell recording techniques in a slice preparation of rat thalamus. A subthreshold (persistent) Na +-conductance amplified depolarizing inputs, enhancing membrane excitability in the tonic firing mode and amplifying the low-threshold Ca 2+-spike in the burst firing mode. Application of tetrodotoxin blocked the amplification and high-threshold Ca 2+-spike firing. A slowly inactivating K + conductance, sensitive to blockade with 4-aminopyridine (50–100 μM), but not tetraethylammonium (2–10 mM), appeared to suppress excitability and high-threshold Ca 2+-spike firing. Application of 4-aminopyridine increased the low-threshold Ca 2+-spike and the number of action potentials in the burst, and led to a conversion of the superimposed high-threshold Ca 2+-spike into a plateau potential. Application of the Ca 2+-channel blocker Cd 2+ (50 μM), reduced or eliminated this plateau potential. The tetrodotoxin sensitive, persistent Na +-conductance also sustained plateau potentials, triggered after 4-aminopyridine application on depolarization by current pulses. Our results suggest that high-threshold Ca 2+-spike firing, and a short-term influx of Ca 2+, are regulated by a balance of voltage-dependent conductances. Normally, a slowly inactivating A-type K +-conductance may reduce high-threshold Ca 2+-spike firing and shorten high-threshold Ca 2+-spike duration. A persistent Na +-conductance promotes coupling of the low-threshold Ca 2+-spike to a high-threshold Ca 2+-spike. Thus, the activation of both voltage-dependent conductances would affect Ca 2+ influx into ventral medial geniculate neurons. This would alter the quality of the different signals transmitted in the thalamocortical system during wakefulness, sleep and pathological states.

Calcium conductances and their role in the firing behavior of neonatal rat hypoglossal motoneurons

1. The role of calcium conductances in action potential generation and repetitive firing behavior of hypoglossal motoneurons (HMs) was investigated using intracellular recording and patch-clamp techniques in a brain stem slice preparation of neonatal rats (0-15 days old). 2. The action potential was followed by an afterdepolarization (ADP). The ADP was voltage dependent, increasing with membrane hyperpolarization. Raising the extracellular Ca2+ concentration or replacing Ca2+ with Ba2+ increased the ADP amplitude, whereas replacement of Ca2+ with Mn2+ blocked it. The ADP was partially reduced by amiloride and low concentrations of Ni2+. 3. The firing behavior of individual neonatal HMs was influenced by membrane potential. From depolarized potentials, HMs fired tonically in response to a depolarizing current pulse, whereas from more hyperpolarized membrane potentials (more negative than -70 mV), a subset of HMs fired an initial burst of action potentials followed by a prolonged afterhyperpolarization and tonic firing. The incidence of burst-firing behavior was highest among young motoneurons and disappeared by the tenth postnatal day. In addition, prominent rebound depolarizations characterized the response of neonatal motoneurons to hyperpolarizing prepulses. 4. Pharmacological characterization of the rebound depolarization demonstrated that it was calcium dependent. Its amplitude was insensitive to tetrodotoxin and it was eliminated by replacement of Ca2+ with Mn2+ or addition of Ni2+. Amiloride (1-1.5 mM) had no effect on the rebound response or burst firing. 5. The presence of high-threshold calcium spikes was detected at all postnatal ages, but only after blockade of outward currents with intra- or extracellular tetraethylammonium. The high-threshold calcium spikes were greatly enhanced when Ba2+ replaced Ca2+. 6. Calcium currents of neonatal HMs were characterized in whole-cell patch-clamp recordings of thin medullary slices under conditions that minimized voltage-dependent Na+ and K+ currents. Low voltage-activated (LVA) and a high voltage-activated (HVA) calcium current components were identified on the basis of their voltage thresholds for activation, kinetics of inactivation, and pharmacological sensitivity. 7. The LVA calcium current began to activate at around -60 mV and inactivated nearly completely within 100 ms. Complete steady-state inactivation occurred at potentials more positive than -60 mV. The LVA current was selectively reduced by 1 mM amiloride (31%). 8. A larger-amplitude calcium current activated at potentials around -35 mV. Inactivation of this HVA current was slower than that of the LVA current and incomplete. About 1/3 of this current was sensitive to 1 microM omega-conotoxin GVIA, whereas a smaller fraction was blocked by 10 microM nifedipine.(ABSTRACT TRUNCATED AT 400 WORDS)

Physiological and morphological properties of accumbens core and shell neurons recorded in vitro.

The morphology and electrophysiological properties of neurons in the nucleus accumbens were studied using intracellular recording techniques in rat brain slices maintained in vitro. Neurons were subdivided according to their location in the shell or core region of the nucleus accumbens. Most of the cells in both regions had small to medium-sized (15.8 +/- 2.8 microns) somata with densely spinous dendrites, somewhat similar to the striatal medium spiny neuron. However, minor morphological differences between neurons from accumbens core and shell regions were found, such as fewer primary dendrites in shell neurons than in the core (3.8 +/- 0.8 vs. 4.4 +/- 1.0) and the spatial organization of their dendritic trees. In general, the passive membrane properties of neurons in each region were similar. However, shell neurons appeared to be less excitable in nature, as suggested by (1) a faster time constant, (2) the absence of TTX-insensitive events resembling low-threshold spikes, and (3) the lower probability of evoking spikes in shell neurons by stimulation of amygdaloid or cortical afferents in comparison to the responses of core neurons to cortical afferent stimulation. In most nucleus accumbens neurons the action potentials evoked by membrane depolarization were preceded by a slow Ca(2+)-dependent depolarization and showed firing-frequency adaptation. Following TTX administration, all-or-none spike-like events resembling high-threshold calcium spikes were observed in both regions. In summary, except for minor differences, most of the properties of core and shell neurons are similar, supporting their characterization as subdivisions of a single structure. Therefore, differences in the functional properties of these neuronal populations are likely to be due to their distinct connectivity patterns.

Medial vestibular nucleus in the guinea-pig. II. Ionic basis of the intrinsic membrane properties in brainstem slices.

In the preceding paper, medial vestibular nuclei neurones (MVNn) were shown to belong to two main classes, A MVNn and B MVNn, depending on their membrane properties in brainstem slices. In the following study we attempted to confirm this segregation by studying some of the ionic conductances that these cells are endowed with. Type A MVNn demonstrated small high threshold calcium spikes that could be potentiated by barium, a 4-AP resistant A-like conductance and a calcium-dependent afterhyperpolarization. Type B MVNn, in contrast, had large high threshold calcium spikes and prolonged calcium-dependent plateau potentials. In addition, they had a calcium-dependent afterhyperpolarization as well as a subthreshold persistent sodium conductance. A subpopulation of B MVNn had also low threshold calcium spikes that gave them bursting properties. These data confirm the segregation of MVN neurones into two main classes and will be discussed with respect to the firing characteristics of vestibular neurones in vivo.

Electrophysiology and Lucifer yellow injection of nucleus gigantocellularis neurones in an isolated and perfused guinea pig brain in vitro

Intracellular recordings from nucleus gigantocellularis (NGC) neurones were obtained in isolated and perfused whole brains of guinea pigs in vitro. A majority of cells (90%) were characterized by an action potential of short duration (0.3 ms) followed first by a fast and then by a slower afterhyperpolarization (AHP). Their firing pattern was mostly irregular. These cells were shown to have high threshold calcium spikes and plateau potentials. The other cell type represented only 10% of the recorded cells in the NGC. It was characterized by a wider (0.6 ms) action potential, a large single AHP, the presence of a transient rectification presumably due to an A-current and a rather regular resting discharge. Using Lucifer yellow injections in brainstem slices, both cell types were shown to correspond to gigantocellular neurones.

Electrophysiological properties of hypoglossal motoneurons of guinea-pigs studied in vitro

Intracellular recordings were made from the hypoglossal nuclear complex in brain slices from guinea-pigs. Retrograde transport of horseradish peroxidase from the tongue confirmed the identity of the visually identified hypoglossal nucleus. Eighteen neurons were stained by intracellular electrophoresis of Lucifer Yellow through the recording pipette. Two types of neurons were encountered, motoneurons with maximal discharge rates of 90 Hz and another type with maximal discharge rates of 250 Hz. Motoneurons were prevalent in the hypoglossal nucleus and the other type prevailed in the adjoining nucleus prepositus hypoglossi. In both nuclei the two types were mixed. Antidromic spikes elicited from hypoglossal root fibres had initial segment and somatodendritic components. Electrical stimulation of the reticular matter dorsolateral to the hypoglossal nucleus elicited excitatory postsynaptic potentials and strychnine sensitive inhibitory postsynaptic potentials. Motoneurons responded to depolarizing current pulses with a train of spikes. The initial spike interval was much shorter than the rest and fast adaptation occurred over three to four intervals. Slow adaptation was most prominent when the neuron was depolarized and discharged at a high rate. High threshold calcium spikes were evoked by depolarizing pulses when sodium spikes were blocked by tetrodotoxin and the potassium conductance reduced by tetraethylammonium bromide. Motoneurons discharged in a single range, inflections on the frequency-current plot being absent. Spikes and spike trains evoked by depolarizing pulses were followed by afterhyperpolarizations with fast and slow parts. The fast phase was eliminated by tetraethyl-ammonium bromide, possibly because the delayed rectifier was involved. A calcium dependent potassium conductance was probably involved in the slow phase, because it was sensitive to inorganic calcium blockers. The amplitude of the afterhyperpolarization following trains of spikes depended on the frequency of the preceding spikes. At constant frequency, the amplitude depended, in addition, on the strength of stimuli arising from different hyperpolarized potentials. Afterdepolarizing potentials were absent. Lissajous plots of double ramp current stimulation showed anomalous rectification between resting potential and spike threshold. The rectification was sensitive to inorganic calcium blockers. Subthreshold responses showed initial sags and rebound responses in all healthy cells and these were eliminated by caesium. Barium, substituted for calcium, unleashed a depolarizing plateau potential sensitive to tetrodotoxin, indicating the presence of a persistent sodium conductance. The membrane of hypoglossal motoneurons contains voltage dependent conductances in the voltage range around resting potential and spike threshold. These conductances provide for effective switching between the discharging and the non-discharging state of the motoneuron.

Effect of D890 on membrane properties of neocortical neurons

D890, a derivative of the Ca 2+ channel antagonist D600, was intracellularly applied from conventional microelectrodes into pyramidal neurons of neocortical slices. The effects of D890 were ascertained by evaluating alterations in membrane properties following drug administration and by comparing these neurons to untreated controls. The amplitude of action potentials (APs) evoked by depolarizing current pulses was attenuated by up to 30% within about 15 min after impalement with D890-containing electrodes. AP rate of rise was reduced by up to 80% and duration was increased. These effects were dependent upon the rate of stimulation. When depolarizing pulses were delivered at low rates of stimulation (e.g. 0.1 Hz), the overshoot of evoked APs declined by about 10%. At higher frequencies (2Hz) the AP overshoot decreased by up to 90%. These effects were mostly reversible on decreasing the frequency of stimulation. A half maximal effect was attained at about 1 Hz, when APs of control neurons were unaltered. In neurons impaled with D890-containing electrodes, depolarizing current pulses delivered in the presence of tetraethylammonium (TEA) and tetrodotoxin elicited high threshold calcium spikes which had a duration between 20 and 200 ms. In the early phase of D890 application, the duration of Ca 2+ spikes decreased in a reversible frequency-dependent manner; after prolonged application, however, the recovery was incomplete. On the average, Ca 2+ spike amplitude and duration decreased by 20% and 50%, respectively, suggesting that D890 usually produces an incomplete blockade of the underlying CA current. The duration of the slow envelope of orthodromically evoked epileptiform paroxysmal depolarizing shifts (DSs), induced by bath application of 10 −5 M bicuculline, was frequency dependent and consistently increased from about 20 ms to 150 ms (half amplitude width) at frequencies above 0.5 Hz. In the presence of D890, the action potentials superimposed on the slow envelope of the DS were attenuated, but neither the amplitude nor the frequency-dependent progressive prolongation of the DS was altered. Application of TEA in the presence of bicuculline (10 −5 M) increased the amplitude and duration of the DS in neurons impaled with D890-containing electrodes. Under these conditions, the durations of DSs evoked by low frequency orthodromic stimulation (0.5Hz) were still progressively prolonged, while, in the same neuron, directly evoked Ca 2+ spikes progressively decreased in amplitude and duration. During DSs, the membrane potential depolarized to levels beyond those required for directly eliciting high threshold Ca 2+ spikes, however, a Ca 2+-spike component was not obvious during the DS. A high conductance state of the membrane at the peak of the DS may limit Ca 2+ spike electrogenesis. The results suggest that D890 affects fast Na + currents and the conventional Ca 2+ conductance underlying the Ca 2+ spike. The absence of effects of D890 on the DS slow envelope suggests that Ca 2+ conductances do not make a large contribution to the latter in the neurons studied, or that Ca 2+ flows through channels with different pharmacological properties during the DS and the Ca 2+ spike.