Presynaptic Ca Influx Research Articles

Closed and open state dependent block of potassium channels cause opposing effects on excitability \u2013 a computational approach

Block of voltage-gated potassium (Kv) channels has been demonstrated to affect neuronal activity described as increasing excitability. The effect has been associated with a closed-state dependent block. However, the block of Kv channels in e.g. local anesthetic and antiarrhythmics, is open state-dependent. Since the reduced excitability in this case mainly is due to sodium channel block, the role of the Kv channel block is concealed. The present investigation aims to analyse the specific role of state-dependent Kv channel block for excitability. Using a computational approach, with introduced blocked states in the Kv channel of the Frankenhaeuser-Huxley axon membrane model, we calculated the effects on threshold, firing and presynaptic Ca influx. The Ca influx was obtained from an N-type Cav channel model linked to the Frankenhaeuser-Huxley membrane. The results suggested that a selective block of open Kv channels decreased the rate of repetitive firing and the consequent Ca influx, thus challenging the traditional view. In contrast, presence of a closed-state block, increased the firing rate and the Ca influx. These findings propose that Kv channel block may either increase or decrease cellular excitability, thus highlighting the importance of further investigating the role of state-specific blocking mechanisms.

The essential oil of Lippia alba and its components affect Drosophila behavior and synaptic physiology.

Lippia alba is a flowering shrub in the verbena family and its essential oil (EO) is known for its sedative, antidepressant and analgesic properties. In the Amazon region of Brazil, it is used in aquaculture to anesthetize fish during transport. Many of the specialized metabolites found in EOs presumably evolved to protect plants from herbivores, especially insects. We used Drosophila to test the behavioral and physiological actions of this EO and its components. We found that a 150 min exposure to the EO vapors resulted in immobilization of adult flies. Gas chromatography-mass spectrometry identified the major components of the EO as the monoterpenes citral (59%), carvone (7%) and limonene (7%). Fly immobilization by the EO was due to citral and carvone, with citral producing more rapid effects than carvone. We tested whether the EO affected synaptic physiology by applying it to the larval neuromuscular junction. The EO delivered at 0.012% (v/v) produced over a 50% reduction in excitatory postsynaptic potential (EPSP) amplitude within 3-4 min. When the EO components were applied at 0.4 mmol l-1, citral and carvone produced a significant reduction in EPSP amplitude, with citral producing the largest effect. Measurement of miniature EPSP amplitudes demonstrated that citral produced over a 50% reduction in transmitter release. Calcium imaging experiments showed that citral produced about 30% reduction in presynaptic Ca2+ influx, which likely resulted in the decrease in transmitter release. Thus, the EO blocks synaptic transmission, largely due to citral, and this likely contributes to its behavioral effects.

α2-Adrenergic Receptor and Isoflurane Modulation of Presynaptic Ca2+ Influx and Exocytosis in Hippocampal Neurons.

Evidence indicates that the anesthetic-sparing effects of α2-adrenergic receptor (AR) agonists involve α2A-AR heteroreceptors on nonadrenergic neurons. Since volatile anesthetics inhibit neurotransmitter release by reducing synaptic vesicle (SV) exocytosis, the authors hypothesized that α2-AR agonists inhibit nonadrenergic SV exocytosis and thereby potentiate presynaptic inhibition of exocytosis by isoflurane. Quantitative imaging of fluorescent biosensors of action potential-evoked SV exocytosis (synaptophysin-pHluorin) and Ca influx (GCaMP6) were used to characterize presynaptic actions of the clinically used α2-AR agonists dexmedetomidine and clonidine, and their interaction with isoflurane, in cultured rat hippocampal neurons. Dexmedetomidine (0.1 μM, n = 10) or clonidine (0.5 μM, n = 8) inhibited action potential-evoked exocytosis (54 ± 5% and 59 ± 8% of control, respectively; P < 0.001). Effects on exocytosis were blocked by the subtype-nonselective α2-AR antagonist atipamezole or the α2A-AR-selective antagonist BRL 44408 but not by the α2C-AR-selective antagonist JP 1302. Dexmedetomidine inhibited exocytosis and presynaptic Ca influx without affecting Ca coupling to exocytosis, consistent with an effect upstream of Ca-exocytosis coupling. Exocytosis coupled to both N-type and P/Q-type Ca channels was inhibited by dexmedetomidine or clonidine. Dexmedetomidine potentiated inhibition of exocytosis by 0.7 mM isoflurane (to 42 ± 5%, compared to 63 ± 8% for isoflurane alone; P < 0.05). Hippocampal SV exocytosis is inhibited by α2A-AR activation in proportion to reduced Ca entry. These effects are additive with those of isoflurane, consistent with a role for α2A-AR presynaptic heteroreceptor inhibition of nonadrenergic synaptic transmission in the anesthetic-sparing effects of α2A-AR agonists.

Endocannabinoids inhibit transmission at granule cell to Purkinje cell synapses by modulating three types of presynaptic calcium channels.

At many central synapses, endocannabinoids released by postsynaptic cells inhibit neurotransmitter release by activating presynaptic cannabinoid receptors. The mechanisms underlying this important means of synaptic regulation are not fully understood. It has been shown at several synapses that endocannabinoids inhibit neurotransmitter release by reducing calcium influx into presynaptic terminals. One hypothesis maintains that endocannabinoids indirectly reduce calcium influx by modulating potassium channels and narrowing the presynaptic action potential. An alternative hypothesis is that endocannabinoids directly and selectively inhibit N-type calcium channels in presynaptic terminals. Here we test these hypotheses at the granule cell to Purkinje cell synapse in cerebellar brain slices. By monitoring optically the presynaptic calcium influx (Ca(influx)) and measuring the EPSC amplitudes, we found that cannabinoid-mediated inhibition arises solely from reduced presynaptic Ca(influx). Next we found that cannabinoid receptor activation does not affect the time course of presynaptic calcium entry, indicating that the reduced Ca(influx) reflects inhibition of presynaptic calcium channels. Finally, we assessed the classes of presynaptic calcium channels inhibited by cannabinoid receptor activation via peptide calcium channel antagonists. Previous studies established that N-type, P/Q-type, and R-type calcium channels are all present in granule cell presynaptic boutons. We found that cannabinoid activation reduced Ca(influx) through N-type, P/Q-type, and R-type calcium channels to 29, 60, and 55% of control, respectively. Thus, rather than narrowing the presynaptic action potential or exclusively modulating N-type calcium channels, CB1 receptor activation inhibits synaptic transmission by modulating all classes of calcium channels present in the presynaptic terminal of the granule cell to Purkinje cell synapse.

Maintenance of High-Frequency Transmission at Purkinje to Cerebellar Nuclear Synapses by Spillover from Boutons with Multiple Release Sites

Cerebellar Purkinje neurons maintain high firing rates but their synaptic terminals depress only moderately, raising the question of how vesicle depletion is minimized. To identify mechanisms that limit synaptic depression, we evoked 100 Hz trains of GABAergic inhibitory postsynaptic currents (IPSCs) in cerebellar nuclear neurons by stimulating Purkinje axons in mouse brain slices. The paired-pulse ratio (IPSC 2/IPSC 1) of the total IPSC was ∼1 and the steady-state ratio (IPSC 20/IPSC 1) was ∼0.5, suggesting a high response probability of postsynaptic receptors, without an unusually high release probability. Three-dimensional electron microscopic reconstructions of Purkinje boutons revealed multiple active zones without intervening transporters, suggestive of “spillover”-mediated transmission. Simulations of boutons with 10–16 release sites, in which transmitter from any site can reach all receptors opposite the bouton, replicated multiple-pulse depression during normal, high, and low presynaptic Ca influx. These results suggest that release from multiple-site boutons limits depletion-based depression, permitting prolonged, high-frequency inhibition at corticonuclear synapses.

Activation of Presynaptic cAMP-Dependent Protein Kinase Is Required for Induction of Cerebellar Long-Term Potentiation

Cerebellar long-term potentiation (LTP) is a persistent increase in the strength of the granule cell-Purkinje neuron synapse that occurs after brief stimulation of granule cell axons at 2-8 Hz. Previous work has indicated that cerebellar LTP induction requires presynaptic Ca influx, stimulation of Ca-sensitive adenylyl cyclase, and activation of PKA. The evidence implicating PKA has come from bath application of drugs during LTP induction, an approach that does not distinguish between PKA activation in the presynaptic or postsynaptic cell. Although bath application of PKA inhibitor drugs (KT5720, Rp-8CPT-cAMP-S) blocked LTP induction in granule cell-Purkinje neuron pairs in culture, selective application to granule cell or Purkinje neuron somata via patch pipettes did not. We hypothesized that presynaptic PKA activation is required for LTP induction but that drugs applied to the granule cell soma cannot diffuse to the terminal within this timescale. To test this hypothesis, we transfected cerebellar cultures with an expression vector encoding a peptide inhibitor of PKA [Rous sarcoma virus (RSV)-protein kinase A inhibitor (PKI)]. Transfection of RSV-PKI into presynaptic granule cells, but not postsynaptic Purkinje neurons or glial cells, blocked LTP induction produced by either synaptic stimulation or an exogenous cAMP analog. An expression vector encoding a control peptide with no PKA inhibitory activity was ineffective. These results show that induction of cerebellar LTP requires a presynaptic signaling cascade, including Ca influx, stimulation of Ca-sensitive adenylyl cyclase, and activation of PKA, and argue against a requirement for postsynaptic Ca signals or their sequelae.

Muscarinic activation reduces changes in [Ca 2+] 0 evoked by stimulation of Schaffer collaterals during blocked synaptic transmission in rat hippocampal slices

The rat hippocampal slice preparation and blockers of postsynaptic glutamate receptors (6-nitro-7-sulphamoylbenzo(f)quinoxaline-2,3-dione (NBQX) 10 μM; dl-2-amino-5-phosphonovaleric acid (APV) 30 μM) were used to study the effects of muscarinic activation on stimulus-induced decreases in [Ca 2+] 0 presumably due to presynaptic Ca-influx. Perfusion with carbachol (100–200 μM) transiently reduced these changes in [Ca 2+] 0. Blockade of this muscarinic effect by pirenzepine (200 μM) and prolongation of this effect after prolonged lithium (1 mM) incubation suggest involvement of the muscarinic M1 receptor subtype. These findings suggest, that activation of M1 receptors might contribute to inhibition of excitatory synaptic transmission by inhibiting presynaptic Ca-influx. In this way, the muscarinic enhancement of excitatory input may be limited to non-deleterious levels.

Detecting changes in calcium influx which contribute to synaptic modulation in mammalian brain slice

The control of neurotransmitter release by modulation of presynaptic calcium influx was investigated at the granule cell to Purkinje cell synapse in rat cerebellar slices. Excitatory post-synaptic currents were measured using whole cell voltage clamp, and changes in presynaptic Ca influx were determined with the Ca-sensitive dye mag-fura-5. Single stimuli of the parallel fibers evoked rapid changes in mag-fura-5 fluorescence which increased from 10 to 90% in 1.4 msec, and then decayed within hundreds of milliseconds to prestimulus levels. These fluorescence changes were unaffected by disruption of internal stores with ryanodine or thapsigargin, and were reduced by 79% by the calcium channel toxin ω-conotoxin-MVIIC. We conclude that these signals result from calcium entry into presynaptic terminals through voltage gated calcium channels opened by action potentials. These fluorescence signals allow us to quantitate changes in calcium influx. We used this approach to study the enhancement of stimulus-evoked synaptic currents by 3-isobutyl-1-methylxanthine (IBMX), a phosphodiesterase inhibitor and antagonist of adenosine receptors. Both enhancement of calcium influx into presynaptic terminals, and reduction in the firing threshold of the parallel fibers, were found to contribute to IBMX-mediated synaptic enhancement. Changes in presynaptic calcium influx were also quantified with a novel method, which is unaffected by changes in fiber threshold. These studies illustrate some of the difficulties encountered when determining the factors responsible for synaptic enhancement and demonstrate how measurements of presynaptic calcium influx can contribute to our understanding of synaptic modulation. The approach described here promises to be widely useful in elucidating the role of calcium influx in the modulation of synapses in brain slice.

Block of multiple presynaptic calcium channel types by omega-conotoxin-MVIIC at hippocampal CA3 to CA1 synapses.

1. The effect of the voltage-dependent Ca channel (VDCC) antagonist omega-conotoxin-MVIIC (omega-CTx-MVIIC) on the presynaptic Ca influx and synaptic transmission was studied in area CA1 of guinea pig hippocampus. The presynaptic Ca transient ([Ca]t) and the field excitatory postsynaptic potential (fEPSP) evoked by a single electrical stimulus were simultaneously recorded at CA3 to CA1 synapses. 2. omega-CTx-MVIIC dose dependently blocked the fEPSP and the presynaptic [Ca]t without affecting the presynaptic fiber volley and the presynaptic resting Ca level. During application of omega-CTx-MVIIC, the decrease of both the fEPSP and the presynaptic [Ca]t had a similar time course, and the initial slope of the fEPSP is proportional to about the fourth power of the amplitude of the presynaptic [Ca]t. These results strongly suggest that omega-CTx-MVIIC inhibits the fEPSP by blocking presynaptic Ca channels at hippocampal CA3 to CA1 synapses. 3. Sequential application of high concentrations of omega-CTx-MVIIC (10 microM) and other VDCC blockers including omega-conotoxin-GVIA (omega-CTx-GVIA, 1 microM) and omega-agatoxin-IVA (omega-Aga-IVA, 1 microM) showed that omega-CTx-MVIIC significantly occludes the effects of omega-CTx-GVIA and omega-Aga-IVA. Combined application of omega-CTx-GVIA (1 microM) and omega-Aga-IVA (1 microM) largely but not completely occluded the effect of omega-CTx-MVIIC.(ABSTRACT TRUNCATED AT 250 WORDS)

Presynaptic calcium is increased during normal synaptic transmission and paired-pulse facilitation, but not in long-term potentiation in area CA1 of hippocampus

We examined the relationship between presynaptic calcium levels and postsynaptic potentials during normal synaptic transmission, paired-pulse facilitation (PPF), and long-term potentiation (LTP) in CA3-CA1 synapses of hippocampus. By selectively loading the presynaptic terminals with the calcium indicator fura-2, we simultaneously recorded a presynaptic calcium (Ca) transient and the corresponding field EPSP evoked by a single stimulus given to the Schaffer collateral-commissural pathway in guinea pig hippocampal slices. A volume average presynaptic Ca influx was obtained by taking the first time derivative of the Ca transient. Our data indicate that the synaptic transmission represented by the initial slope of the field EPSP is approximately proportional to the fourth power of the presynaptic Ca influx, the volume average Ca current. Our results in combination with similar findings at the squid giant synapse (Augustine et al., 1985b; Augustine and Charlton, 1986) suggest that the relationship between Ca influx and transmitter release is well conserved from the molluscan to the mammalian nervous system. A transient increase of the residual Ca level ([Ca]res) is generally thought to be the mechanism underlying PPF (Katz and Miledi, 1968; Charlton et al., 1982); however, the relationship between PPF and the presynaptic [Ca]res had not been examined before. Our results demonstrate that PPF is approximately linearly related to the [Ca]res. This finding further supports the residual Ca hypothesis for PPF. Accumulated evidence from other groups suggests that the presynaptic site contributes to the maintenance of LTP in CA3-CA1 synapses (Bekkers and Stevens, 1990; Malinow and Tsien, 1990); however, our data show that neither an increase of the Ca transient nor a sustained increase of the [Ca]res occurs in the presynaptic terminals during maintenance of LTP. This suggests that the presynaptic mechanism underlying LTP must be downstream to Ca influx.