Types Of Voltage-gated Calcium Channels Research Articles

Alterations in the Expression of Calcium Channels and Neurotrophic Factors in the Cerebellum Are Linked to the Induction of Morphine Dependence and Withdrawal

Background: Recent studies have shown that the cerebellum directly interacts with the ventral tegmental area, a critical component of the reward system. Objectives: This study aimed to explore potential changes in the expression of neurotrophic factors and various types of voltage-gated calcium channels in the cerebellum following morphine dependence and withdrawal. Methods: This study involved three groups of male Wistar rats. For ten consecutive days, the second and third groups were administered morphine (10 mg/kg), while the first group received saline (1 mL/kg). Analgesic responses were assessed using a hotplate test on days 1 and 10 of the repeated injections and after a 30-day withdrawal period. Rats were sacrificed on day 10 of the injections or on day 30 of withdrawal, and their cerebella were dissected for analysis. Gene expression was analyzed using the real-time PCR method. Results: The study found that morphine analgesia decreased during the 10 days of repeated injections but partially recovered after a 30-day withdrawal period. Morphine dependence led to a decrease in the expression of Cav1.1, which increased after withdrawal. The expression of Cav1.2 in the cerebellum consistently rose after both morphine dependence and withdrawal. There were no significant changes in the expression of Cav2.2 due to morphine dependence or withdrawal. An increase in the expression of Cav3.1 was observed following morphine dependence, which decreased after withdrawal. There were significant reductions in the mRNA levels of neurotrophic factors (BDNF, GDNF, and NGF) and their receptors (TrkB, GFRA1, and NGFR) following morphine dependence. However, the expression of almost all neurotrophic factors increased after morphine withdrawal. Conclusions: The findings suggest that changes in neurotrophic factors, their receptors, and specific types of voltage-gated calcium channels in the cerebellum play roles in the processes of morphine dependency and withdrawal.

Blood-brain barrier breakdown and autoimmune cerebellar ataxia

Autoimmune cerebellar ataxia is a disease entity that affects the cerebellum and is induced by autoimmune mechanisms. The disease is classified into several etiologies, including gluten ataxia, anti-glutamate decarboxylase (GAD) ataxia, paraneoplastic cerebellar degeneration, primary autoimmune cerebellar ataxia and postinfectious cerebellar ataxia. The autoimmune response in the periphery cross-reacts with similar antigens in the cerebellum due to molecular mimicry. Breakdown of the blood‒brain barrier (BBB) could potentially explain the vulnerability of the cerebellum during the development of autoimmune cerebellar ataxia, as it gives rise to the entry of pathogenic autoantibodies or lymphocytes into the cerebellum. In this review, the maintenance of the BBB under normal conditions and the molecular basis of BBB disruption under pathological conditions are highlighted. Next, the pathomechanism of BBB breakdown in each subtype of autoimmune cerebellar ataxia is discussed. We recently identified glucose-regulated protein (GRP) 78 antibodies in paraneoplastic cerebellar degeneration and Lambert-Eaton myasthenic syndrome, and GRP78 antibodies induced by cross-reactivity with tumors can disrupt the BBB and penetrate anti-P/Q type voltage-gated calcium channel (VGCC) antibodies into the cerebellum, thus leading to cerebellar ataxia in this disease.

Betulinic acid analogs inhibit N- and T-type voltage-gated calcium channels to attenuate nerve-injury associated neuropathic and formalin models of pain

Over the past three decades, there has been a significant growth in the use of natural products, with approximately 80% of individuals using them for some aspect of primary healthcare. Our laboratories have identified and studied natural compounds with analgesic effects from dry land plants or their associated fungus during the past ten years. Here, we isolated and characterized thirteen betulin analogs and fifteen betulinic acid analogs for their capacity to prevent calcium influx brought on by depolarization in sensory neurons. The in vitro inhibition of voltage-gated calcium channels by the top drugs was then assessed using whole cell patch clamp electrophysiology. In vivo experiments, conducted at two sites, evaluated the best compound in acute and tonic, neuropathic, inflammatory, post-operative and visceral models of pain. We found that the betulinic acid analog 8 inhibited calcium influx in rat dorsal root ganglion neurons by inhibiting N- (CaV2.2) and T- (CaV3) type voltage-gated calcium channels. Moreover, intrathecal delivery of analog 8 had analgesic activity in both spared nerve injury model of neuropathic pain and acute and tonic pain induced by formalin. The results presented herein highlight the potential antinociceptive properties of betulinic acid analog 8 and set the stage for the development of novel non-opioid pain therapeutics based on the triterpenoid scaffold of betulinic acid.

Calcium channels control tDCS-induced spontaneous vesicle release from axon terminals

BackgroundTranscranial direct current stimulation (tDCS) is a subthreshold neurostimulation therapeutic method that ameliorate neuropsychiatric impairments. The most sensitive subcellular compartment for tDCS are the axons that polarize. However, how these relatively small polarizations significantly alter synaptic dynamics is still unknown.Objective/Hypothesis: We hypothesized that tDCS-induced axonal polarization modulates calcium channel activity at the presynaptic compartment, thus playing a crucial role in synaptic vesicle release. MethodsFor this aim, we examined how different DCS conditions and orientations affect the spontaneous excitatory post synaptic currents (sEPSCs) recorded from hippocampal CA1 pyramidal neurons. Since P/Q-type calcium-channels are the main presynaptic voltage-dependent calcium-channels in the hippocampus, we further examined the DCS effects while applying a P/Q-type calcium channels blocker, ω-agatoxin. Additionally, to explain the DCS-induced calcium channel-regulated vesicle release dynamics, we developed a simplified model to complement our experimental results. ResultsWe demonstrated that anodal-DCS application in a dorso-ventral orientation, similar to that of in-vivo experiments, enhanced the sEPSCs frequency, while cathodal-DCS was ineffective. Moreover, DCS application in parallel to the Schaffer collaterals (medio-lateral orientation), showed both anodal and cathodal significant effects. Furthermore, the ω-agatoxin application occluded the DCS-induced modulation of sEPSC frequencies in any orientation. The model showed the interaction between DCS-induced membrane polarization, calcium channel activation and presynaptic vesicle release. ConclusionUsing experiments and modeling we show that DCS induces a small variation in terminal membrane potential sufficient to activate P/Q type voltage-gated calcium channels, and that this is sufficient to modify presynaptic calcium concentration, subsequently altering spontaneous vesicle release.

Role of L-Type Voltage-Gated Calcium Channels in Epileptiform Activity of Neurons.

Epileptic discharges manifest in individual neurons as abnormal membrane potential fluctuations called paroxysmal depolarization shift (PDS). PDSs can combine into clusters that are accompanied by synchronous oscillations of the intracellular Ca2+ concentration ([Ca2+]i) in neurons. Here, we investigate the contribution of L-type voltage-gated calcium channels (VGCC) to epileptiform activity induced in cultured hippocampal neurons by GABA(A)R antagonist, bicuculline. Using KCl-induced depolarization, we determined the optimal effective doses of the blockers. Dihydropyridines (nifedipine and isradipine) at concentrations ≤ 10 μM demonstrate greater selectivity than the blockers from other groups (phenylalkylamines and benzothiazepines). However, high doses of dihydropyridines evoke an irreversible increase in [Ca2+]i in neurons and astrocytes. In turn, verapamil and diltiazem selectively block L-type VGCC in the range of 1–10 μM, whereas high doses of these drugs block other types of VGCC. We show that L-type VGCC blockade decreases the half-width and amplitude of bicuculline-induced [Ca2+]i oscillations. We also observe a decrease in the number of PDSs in a cluster and cluster duration. However, the pattern of individual PDSs and the frequency of the cluster occurrence change insignificantly. Thus, our results demonstrate that L-type VGCC contributes to maintaining the required [Ca2+]i level during oscillations, which appears to determine the number of PDSs in the cluster.

The CACNA1A Mutant Disrupts Lysosome Calcium Homeostasis in Cerebellar Neurons and the Resulting Endo-Lysosomal Fusion Defect Can be Improved by Calcium Modulation.

Mutations in P/Q type voltage gated calcium channel (VGCC) lead severe human neurological diseases such as episodic ataxia 2, familial hemiplegic migraine 1, absence epilepsy, progressive ataxia and spinocerebellar ataxia 6. The pathogenesis of these diseases remains unclear. Mice with spontaneous mutation in the Cacna1a gene encoding the pore-forming subunit of P/Q type VGCC also exhibit ataxia, epilepsy and neurodegeneration. Based on the previous work showing that the P/Q type VGCC in neurons regulates lysosomal fusion through its calcium channel activity on lysosomes, we utilized CACNA1A mutant mice to further investigate the mechanism by which P/Q-type VGCCs regulate lysosomal function and neuronal homeostasis. We found CACNA1A mutant neurons have reduced lysosomal calcium storage without changing the resting calcium concentration in cytoplasm and the acidification of lysosomes. Immunohistochemistry and transmission electron microscopy reveal axonal degeneration due to lysosome dysfunction in the CACNA1A mutant cerebella. The calcium modulating drug thapsigargin, by depleting the ER calcium store, which locally increases the calcium concentration can alleviate the defective lysosomal fusion in mutant neurons. We propose a model that in cerebellar neurons, P/Q-type VGCC maintains the integrity of the nervous system by regulating lysosomal calcium homeostasis to affect lysosomal fusion, which in turn regulates multiple important cellular processes such as autophagy and endocytosis. This study helps us to better understand the pathogenesis of P/Q-type VGCC related neurodegenerative diseases and provides a feasible direction for future pharmacological treatment.

CACHD1-deficient mice exhibit hearing and balance deficits associated with a disruption of calcium homeostasis in the inner ear

CACHD1 recently was shown to be an α2δ-like subunit that can modulate the activity of some types of voltage-gated calcium channels, including the low-voltage activated, T-type CaV3 channels. CACHD1 is widely expressed in the central nervous system but its biological functions and relationship to disease states are unknown. Here, we report that mice with deleterious Cachd1 mutations are hearing impaired and have balance defects, demonstrating that CACHD1 is functionally important in the peripheral auditory and vestibular organs of the inner ear. The vestibular dysfunction of Cachd1 mutant mice, exhibited by leaning and head tilting behaviors, is related to a deficiency of calcium carbonate crystals (otoconia) in the saccule and utricle. The auditory dysfunction, shown by ABR threshold elevations and reduced DPOAEs, is associated with reduced endocochlear potentials and increased endolymph calcium concentrations. Paint-fills of mutant inner ears from prenatal and newborn mice revealed dilation of the membranous labyrinth caused by an enlarged volume of endolymph. These pathologies all can be related to a disturbance of calcium homeostasis in the endolymph of the inner ear, presumably caused by the loss of CACHD1 regulatory effects on voltage-gated calcium channel activity. Cachd1 expression in the cochlea appears stronger in late embryonic stages than in adults, suggesting an early role in establishing endolymph calcium concentrations. Our findings provide new insights into CACHD1 function and suggest the involvement of voltage-gated calcium channels in endolymph homeostasis, essential for normal auditory and vestibular function.

Calcium Channels Genes and Their Epilepsy Phenotypes

AbstractCalcium (Ca2+) channel gene mutations play an important role in the pathogenesis of neurological episodic disorders like epilepsy. CACNA1A and CACNA1H genes are involved in the synthesis of calcium channels. Mutations in the α1A subunit of the P/Q type voltage-gated calcium channel gene (CACNA1A) located in 19p13.13, which encodes for the transmembrane pore-forming subunit of CAV2.1 voltage-dependent calcium channel, have been correlated to a large clinical spectrum of epilepsy such as idiopathic genetic epilepsy, early infantile epilepsy, and febrile seizures. Moreover, CACNA1A mutations have been demonstrated to be involved in spinocerebellar ataxia type 6, familiar hemiplegic migraine, episodic ataxia type 2, early-onset encephalopathy, and hemiconvulsion–hemiplegia epilepsy syndrome. This wide phenotype heterogeneity associated with CACNA1A mutations is correlated to different clinical and electrophysiological manifestations. CACNA1H gene, located in 16p13.3, encodes the α1H subunit of T-type calcium channel, expressing the transmembrane pore-forming subunit Cav3.2. Despite data still remain controversial, it has been identified as an important gene whose mutations seem strictly related to the pathogenesis of childhood absence epilepsy and other generalized epilepsies. The studied variants are mainly gain-of-function, hence responsible for an increase in neuronal susceptibility to seizures. CACNA1H mutations have also been associated with autism spectrum disorder and other behavior disorders. More recently, also amyotrophic lateral sclerosis has been related to CACNA1H alterations. The aim of this review, other than describe the CACNA1A and CACNA1H gene functions, is to identify mutations reported in literature and to analyze their possible correlations with specific epileptic disorders, purposing to guide an appropriate medical treatment recommendation.

長期経過を観察できた抗voltage–gated calcium channel抗体陽性傍腫瘍性小脳変性症の1例

長期経過を観察できた抗voltage–gated calcium channel抗体陽性傍腫瘍性小脳変性症の1例

Lambert-Eaton myasthenic syndromes

Lambert Eaton Myasthenic Syndrome (LEMS) is a rare disorder of the neuromuscular junction characterized by a presynaptic defect in neuromuscular transmission. It can either be paraneoplastic in about half of the cases, or a primary autoimmune disease. It is caused by autoantibodies directed against the P/Q type voltage gated calcium channels (VGCC), which results in reduced influx of calcium during depolarization, interfering with the calcium dependent release of acetylcholine in the synaptic cleft. LEMS is usually a disease of adults, with the clinical features of weakness, more so in the lower limbs but also affecting upper limbs, hypo or areflexia, and autonomic dysfunction such as dry mouth. Besides the typical clinical features, the diagnosis is dependent on electrodiagnostic findings, characterized by low compound muscle action potentials (CMAP) with prominent post exercise potentiation, a CMAP amplitude decrement on low frequency repetitive stimulation, but with an increment on high frequency repetitive stimulation. Serum anti-P/Q-type VGCC can be detected in about 50% of cases. However, about 90% of paraneoplastic LEMS cases associated with small cell lung cancer (SCLC), test positive for the antibody. Sixty-four percent of LEMS patients with SCLC also test positive for antibodies against SOX1, which may play a role as an early marker for predisposition to LEMS/SCLC. Anti- SOX1 antibodies are 95% specific and about 65% sensitive for LEMS with SCLC. Once LEMS is diagnosed, the patient should be screened for cancer. If cancer is not detected initially, the patient should continue to be screened every 3 to 6 months for at least two years. Treatment of LEMS consists of treating the primary cancer in paraneoplastic cases. The drug of choice for symptomatic treatment of LEMS is 3,4- diaminopyridine. Immunosuppressive therapy with azathioprine and corticosteroids might be helpful in patients who do not respond to symptomatic treatment.

Ankyrin B and Ankyrin B variants differentially modulate intracellular and surface Cav2.1 levels

Ankyrin B (AnkB) is an adaptor and scaffold for motor proteins and various ion channels that is ubiquitously expressed, including in the brain. AnkB has been associated with neurological disorders such as epilepsy and autism spectrum disorder, but understanding of the underlying mechanisms is limited. Cav2.1, the pore-forming subunit of P/Q type voltage gated calcium channels, is a known interactor of AnkB and plays a crucial role in neuronal function. Here we report that wildtype AnkB increased overall Cav2.1 levels without impacting surface Cav2.1 levels in HEK293T cells. An AnkB variant, p.S646F, which we recently discovered to be associated with seizures, further increased overall Cav2.1 levels, again with no impact on surface Cav2.1 levels. AnkB p.Q879R, on the other hand, increased surface Cav2.1 levels in the presence of accessory subunits α2δ1 and β4. Additionally, AnkB p.E1458G decreased surface Cav2.1 irrespective of the presence of accessory subunits. In addition, we found that partial deletion of AnkB in cortex resulted in a decrease in overall Cav2.1 levels, with no change to the levels of Cav2.1 detected in synaptosome fractions. Our work suggests that depending on the particular variant, AnkB regulates intracellular and surface Cav2.1. Notably, expression of the AnkB variant associated with seizure (AnkB p.S646F) caused further increase in intracellular Cav2.1 levels above that of even wildtype AnkB. These novel findings have important implications for understanding the role of AnkB and Cav2.1 in the regulation of neuronal function in health and disease.

Involvement of Voltage-Gated Calcium Channels in Inflammation and Inflammatory Pain.

Voltage-gated calcium channels (VGCCs) are classified into high-voltage-activated (HVA) channels and low-voltage-activated channels consisting of Cav3.1-3.3, known as T ("transient")-type VGCC. There is evidence that certain types of HVA channels are involved in neurogenic inflammation and inflammatory pain, in agreement with reports indicating the therapeutic effectiveness of gabapentinoids, ligands for the α2δ subunit of HVA, in treating not only neuropathic, but also inflammatory, pain. Among the Cav3 family members, Cav3.2 is abundantly expressed in the primary afferents, regulating both neuronal excitability at the peripheral terminals and spontaneous neurotransmitter release at the spinal terminals. The function and expression of Cav3.2 are modulated by a variety of inflammatory mediators including prostanoids and hydrogen sulfide (H2S), a gasotransmitter. The increased activity of Cav3.2 by H2S participates in colonic, bladder and pancreatic pain, and regulates visceral inflammation. Together, VGCCs are involved in inflammation and inflammatory pain, and Cav3.2 T-type VGCC is especially a promising therapeutic target for the treatment of visceral inflammatory pain in patients with irritable bowel syndrome, interstitial cystitis/bladder pain syndrome, pancreatitis, etc., in addition to neuropathic pain.

The Lambert-Eaton Myasthenic Syndrome — an Overview

AbstractThe Lambert Eaton myasthenic syndrome (LEMS) has a prevalence of around 5/100 0000 and is around 10–20 times rarer than myasthenia gravis (MG). Although LEMS does have a number of similarities to MG, there are important differences. The syndrome is characterized by a mostly proximally localised exercise induced muscle weakness that can lead to respiratory failure often accompanied by autonomous dysfunction. Disease symptoms are caused by autoantibodies directed against P/Q type voltage gated calcium channels (VGCC) that are expressed in the presynaptic motoric nerve terminals. The diagnosis of LEMS is based on the detection of the pathogenic anti-VGCC antibodies as well as the observation of an increment of at least 60% in the electrophysiological examination of an affected muscle. An increment is defined by an increase of the at rest reduced compound muscle action potential (CMAP) either after voluntary maximal innervation or after high frequent (≥20 Hz) stimulation. In almost one third LEMS is of paraneoplastic origin. Therefore an intensive tumor screening is necessary after diagnosis.There are some differences in the clinical presentation between paraneoplastic (pLEMS) and the exclusively autoimmune (aiLEMS) form of LEMS. With respect to this the DELTA-P-Score and the detection of SOX1-antibody are important. The most frequent tumor associated with LEMS is small cell lung carcinoma (SCLC). Therapy is based on the initial distinction between paraneoplastic and autoimmune ethiology. pLEMS necessitates therapy of underlying neoplasia. Usually, aiLEMS- as well as pLEMS patients respond well to 3,4 diaminopyridine (3,4 DAP) often augmented by pyridostigmine. Similar to treatment of myasthenia gravis long-term immunosuppressive treatment is usually required to control symptoms effectively. Myasthenic crisis in LEMS can be controlled by intensive care and immunoglobulins, plasmaphereses or immunoadsorption. Based on case reports more specific immunomodulatory treatment approaches such as the B-cell depleting therapeutic antibody rituximab should be considered in therapy refractory courses of LEMS. Long-term prognosis of autoimmune LEMS with respect to clinical stabilization with (pharmacological) remission is good, although in around 75% of patients significant reductions in quality of life remain. Prognosis of tumor-associated LEMS is largely determined by the tumor and its effective therapy. Curative treatment of the tumour as well as complete remission of pLEMS are possible.

Responsiveness of voltage-gated calcium channels in SH-SY5Y human neuroblastoma cells on micropillar substrates

In this study, poly-L-lactic acid micropillar substrates were fabricated to evaluate the influence of topographic substrates on cell morphological and functional characteristics, such as spreading area, voltage-gated calcium channels (VGCCs) and membrane potential. The proliferation, spreading area, perimeter and circularity of SH-SY5Y cells interfaced with different substrates were first investigated. In addition, the cytoskeleton and focal adhesion of a cell as important manifestations of cell morphology were analyzed by immunofluorescence. VGCC responsiveness was evaluated by measuring the dynamic changes in intracellular Ca2+ evoked by 50 mM extracellular K+. To determine study whether the differences in VGCC responsiveness were caused by the differences in VGCC gene expression, the expression of N/L- type VGCCs was determined by qPCR and fluorescence staining. Notably, improved measurement of the membrane potential with potentiometric fluorescent dye TMRM was applied to determine the membrane potential of SH-SY5Y cells. Results indicated that the SH-SY5Y cells were deformed significantly to adapt to the substrates; however, no distinct effect on the proliferative ability of SH-SY5Y cells was observed. The micropillar substrates markedly influenced VGCC responsiveness, which correlated strongly with cell spreading but not with VGCC expression. The resting membrane potential of SH-SY5Y cells cultured on different substrates also changed, but no effect on responsiveness of VGCC was observed. These results suggest that the effect of the micropillar substrates on cell VGCC responsiveness may be attributed to changes in the functionality of the ion channel itself. Thus, topographic substrates can be used to engineer cell functionality in cell-based drug screening.

3-2-11. Peripheral nerve excitability changing in spinocerebellar ataxia type 6 (SCA6)

Abstract Motor nerve excitability testing in the median nerve with QtracS program (©Professor H. Bostock, UCL) was compared between 15 spinocerebellar ataxia type 6 (SCA6) patients and 22 age-matched healthy controls. Motor nerve excitability testing in tail nerve of 2 male mice was compared between before and after intraperitoneal injection of ω -agatoxin-IVA. Compared with normal controls, SCA6 showed statistically significant decrease of depolarizing threshold electrotonus (p ω -agatoxin-IVA. The present study indicates that motor nerves of SCA6 increase slow potassium currents derived from dysfunction of mutant protein, P/Q type voltage gated calcium channels.

Short-Term Facilitation at a Detonator Synapse Requires the Distinct Contribution of Multiple Types of Voltage-Gated Calcium Channels.

Neuronal calcium elevations are shaped by several key parameters, including the properties, density, and the spatial location of voltage-gated calcium channels (VGCCs). These features allow presynaptic terminals to translate complex firing frequencies and tune the amount of neurotransmitter released. Although synchronous neurotransmitter release relies on both P/Q- and N-type VGCCs at hippocampal mossy fiber-CA3 synapses, the specific contribution of VGCCs to calcium dynamics, neurotransmitter release, and short-term facilitation remains unknown. Here, we used random-access two-photon calcium imaging together with electrophysiology in acute mouse hippocampal slices to dissect the roles of P/Q- and N-type VGCCs. Our results show that N-type VGCCs control glutamate release at a limited number of release sites through highly localized Ca2+ elevations and support short-term facilitation by enhancing multivesicular release. In contrast, Ca2+ entry via P/Q-type VGCCs promotes the recruitment of additional release sites through spatially homogeneous Ca2+ elevations. Altogether, our results highlight the specialized contribution of P/Q- and N-types VGCCs to neurotransmitter release.SIGNIFICANCE STATEMENT In presynaptic terminals, neurotransmitter release is dynamically regulated by the transient opening of different types of voltage-gated calcium channels. Hippocampal giant mossy fiber terminals display extensive short-term facilitation during repetitive activity, with a large several fold postsynaptic response increase. Though, how giant mossy fiber terminals leverage distinct types of voltage-gated calcium channels to mediate short-term facilitation remains unexplored. Here, we find that P/Q- and N-type VGCCs generate different spatial patterns of calcium elevations in giant mossy fiber terminals and support short-term facilitation through specific participation in two mechanisms. Whereas N-type VGCCs contribute only to the synchronization of multivesicular release, P/Q-type VGCCs act through microdomain signaling to recruit additional release sites.

Flexible and age-dependent modulation of pacemaker activity of substantia nigra dopaminergic neurons by distinct types of voltage-gated calcium channels

Flexible and age-dependent modulation of pacemaker activity of substantia nigra dopaminergic neurons by distinct types of voltage-gated calcium channels

A case of encephalomyeloradiculopathy in a non-carcinomatous patient associated with P/Q type voltage gated calcium channel antibodies

The presence of P/Q type voltage gated calcium channel (VGCC) antibodies has been strongly correlated with Lambert Eaton Syndrome (LES), present in 90% of non-immunocompromised patients with LES. However, there have been case reports which have shown its association between paraneoplastic syndrome affecting both central nervous system and the peripheral nervous system causing encephalomyelitis and sensory neuronopathy/neuropathy. We present a case of a young man, who presented with encephalomyelitis, and was further noted to have superimposed cervical polyradiculopathy associated with P/Q type VGCC antibodies.

Verapamil - L type voltage gated calcium channel inhibitor diminishes aggressive behavior in male Siamese fighting fish.

Verapamil is a L-type voltage gated calcium channels inhibitor (VGCCI), which is a highly prescribed drug used in the treatment of hypertension, angina pectoris, cardiac arrhythmia and cluster headaches. Its common use caused its appearance in water environment. VGCC inhibit epinephrine release and cause many neuro-hormonal changes influencing also fish behavior. Siamese fighting fish was chosen to study the influence of verapamil given to the water on the beginning of experiment in 3 different concentrations of 0 (control), 8 and 160 μg · L-1, on aggressive behavior in these fish. The experimental fish were placed in individual glass containers for 3 weeks and the mirror test was used. The highest concentration led to a significant modulation of fish behavior after 1 week and the lower dose caused statistically significant behavioral changes after 2 weeks of verapamil treatment. Siamese fighting fish males exposed to verapamil had longer latencies to the first chase - 12.6 s (8 μg · L-1 of verapamil) and 18.8 s (160 μg · L-1 of verapamil) compared to 5.6 s in the control group, decreased attack frequency and shorter duration of these attacks. The number of attacks within 10 min was decreased from 38.3 in the control group to 27.1 and 16.1, respectively. Also the total duration of these attacks decreased from 354.8 (control) to 326.4 (decrease statistically insignificant) and to 194.8 s in verapamil treated groups. It was shown, that even relatively low concentrations of verapamil in water may have adverse effects on fish and probably other living organisms.

Cell stimulation and calcium mobilization by picosecond electric pulses

We tested if picosecond electric pulses (psEP; 190kV/cm, 500ps at 50% height), which are much shorter than channel activation time, can activate voltage-gated (VG) channels. Cytosolic Ca2+ was monitored by Fura-2 ratiometric imaging in GH3 and NG108 cells (which express multiple types of VG calcium channels, VGCC), and in CHO cells (which express no VGCC). Trains of up to 100psEP at 1kHz elicited no response in CHO cells. However, even a single psEP significantly increased Ca2+ in both GH3 (by 114±48nM) and NG108 cells (by 6±1.1nM). Trains of 100psEP amplified the response to 379±33nM and 719±315nM, respectively. Ca2+ responses peaked within 2–15s and recovered for over 100s; they were 80–100% inhibited by verapamil and ω-conotoxin, but not by the substitution of Na+ with N-methyl-D-glucamine. There was no response to psEP in Ca2+-free medium, but adding external Ca2+ even 10s later evoked Ca2+ response. We conclude that electrical stimuli as short as 500ps can cause long-lasting opening of VGCC by a mechanism which does not involve conventional electroporation, heating (which was under 0.06K per psEP), or membrane depolarization by opening of VG Na+ channels.