omega-agatoxin-iva and Disease-Models--Animal

On trigeminal ganglion neurons, pain-sensing P2X3 receptors are constitutively inhibited by brain natriuretic peptide via its natriuretic peptide receptor-A. This inhibition is associated with increased P2X3 serine phosphorylation and receptor redistribution to non-lipid raft membrane compartments. The natriuretic peptide receptor-A antagonist anantin reverses these effects. We studied whether P2X3 inhibition is dysfunctional in a genetic familial hemiplegic migraine type-1 model produced by introduction of the human pathogenic R192Q missense mutation into the mouse CACNA1A gene (knock-in phenotype). This model faithfully replicates several properties of familial hemiplegic migraine type-1, with gain-of-function of CaV2.1 Ca(2+) channels, raised levels of the algogenic peptide calcitonin gene-related peptide, and enhanced activity of P2X3 receptors in trigeminal ganglia.. In knock-in neurons, anantin did not affect P2X3 receptor activity, membrane distribution, or serine phosphorylation level, implying ineffective inhibition by the constitutive brain natriuretic peptide/natriuretic peptide receptor-A pathway. However, expression and functional properties of this pathway remained intact together with its ability to downregulate TRPV1 channels. Reversing the familial hemiplegic migraine type-1 phenotype with the CaV2.1-specific antagonist, ω-agatoxin IVA restored P2X3 activity to wild-type level and enabled the potentiating effects of anantin again. After blocking calcitonin gene-related peptide receptors, P2X3 receptors exhibited wild-type properties and were again potentiated by anantin.. P2X3 receptors on mouse trigeminal ganglion neurons are subjected to contrasting modulation by inhibitory brain natriuretic peptide and facilitatory calcitonin gene-related peptide that both operate via complex intracellular signaling. In the familial hemiplegic migraine type-1 migraine model, the action of calcitonin gene-related peptide appears to prevail over brain natriuretic peptide, thus suggesting that peripheral inhibition of P2X3 receptors becomes insufficient and contributes to trigeminal pain sensitization.

Topics: Animals; Calcitonin Gene-Related Peptide Receptor Antagonists; Disease Models, Animal; Gene Knock-In Techniques; Mice; Migraine with Aura; Models, Biological; Natriuretic Peptide, Brain; omega-Agatoxin IVA; Peptides, Cyclic; Phenotype; Purinergic P2X Receptor Antagonists; Receptors, Atrial Natriuretic Factor; Receptors, Calcitonin Gene-Related Peptide; Receptors, Purinergic P2X3; Sensory Receptor Cells; Trigeminal Ganglion; TRPV Cation Channels

Voltage-gated Ca(2+) channels (VGCCs) mediate calcium entry into neuronal cells in response to membrane depolarisation and play an essential role in a variety of physiological processes. In Amyotrophic Lateral Sclerosis (ALS), a fatal neurodegenerative disease caused by motor neuron degeneration in the brain and spinal cord, intracellular calcium dysregulation has been shown, while no studies have been carried out on VGCCs. Here we show that the subtype N-type Ca(2+) channels are over expressed in G93A cultured cortical neurons and in motor cortex of G93A mice compared to Controls. In fact, by western blotting, immunocytochemical and electrophysiological experiments, we observe higher membrane expression of N-type Ca(2+) channels in G93A neurons compared to Controls. G93A cortical neurons filled with calcium-sensitive dye Fura-2, show a net calcium entry during membrane depolarization that is significantly higher compared to Control. Analysis of neuronal vitality following the exposure of neurons to a high K(+) concentration (25 mM, 5h), shows a significant reduction of G93A cellular survival compared to Controls. N-type channels are involved in the G93A higher mortality because ω-conotoxin GVIA (1 μM), which selectively blocks these channels, is able to abolish the higher G93A mortality when added to the external medium. These data provide robust evidence for an excess of N-type Ca(2+) expression in G93A cortical neurons which induces a higher mortality following membrane depolarization. These results may be central to the understanding of pathogenic pathways in ALS and provide novel molecular targets for the design of rational therapies for the ALS disorder.

Topics: Amyotrophic Lateral Sclerosis; Animals; Animals, Newborn; Calcium Channel Blockers; Calcium Channels, N-Type; Cell Survival; Cells, Cultured; Cerebral Cortex; Cytophotometry; Disease Models, Animal; Electric Stimulation; Gene Expression Regulation; Humans; Membrane Potentials; Mice; Mice, Transgenic; Motor Neurons; omega-Agatoxin IVA; omega-Conotoxin GVIA; Patch-Clamp Techniques; Sodium Channel Blockers; Superoxide Dismutase; Tetrodotoxin

Developing central white matter is subject to ischemic-type injury during the period that precedes myelination. At this stage in maturation, central axons initiate a program of radial expansion and ion channel redistribution. Here we test the hypothesis that during radial expansion axons display heightened ischemic sensitivity, when clusters of Ca(2+) channels decorate future node of Ranvier sites.. Functionality and morphology of central axons and glia were examined during and after a period of modeled ischemia. Pathological changes in axons undergoing radial expansion were probed using electrophysiological, quantitative ultrastructural, and morphometric analysis in neonatal rodent optic nerve and periventricular white matter axons studied under modeled ischemia in vitro or after hypoxia-ischemia in vivo.. Acute ischemic injury of central axons undergoing initial radial expansion was mediated by Ca(2+) influx through Ca(2+) channels expressed in axolemma clusters. This form of injury operated only in this axon population, which was more sensitive to injury than neighboring myelinated axons, smaller axons yet to initiate radial expansion, astrocytes, or oligodendroglia. A pharmacological strategy designed to protect both small and large diameter premyelinated axons proved 100% protective against acute ischemia studied under modeled ischemia in vitro or after hypoxia-ischemia in vivo.. Recent clinical data highlight the importance of axon pathology in developing white matter injury. The elevated susceptibility of early maturing axons to ischemic injury described here may significantly contribute to selective white matter pathology and places these axons alongside preoligodendrocytes as a potential primary target of both injury and therapeutics.

Topics: Age Factors; Animals; Animals, Newborn; Apoptosis; Astrocytes; Axons; Disease Models, Animal; Dizocilpine Maleate; Glucose; Green Fluorescent Proteins; Hypoxia; Hypoxia-Ischemia, Brain; Mice; Mice, Transgenic; Myelin Sheath; Nerve Degeneration; Nerve Fibers, Myelinated; Neuroprotective Agents; Oligodendroglia; omega-Agatoxin IVA; Optic Nerve; Organ Culture Techniques; Rats; Rats, Sprague-Dawley; Recovery of Function; Thy-1 Antigens

Inherited errors in ion channel genes comprise the largest subset of monogenic causes of idiopathic epilepsy, and pathogenic variants contribute to genetic risk in the complex inheritance of this common disorder. We generated a digenic mouse model of human idiopathic epilepsy by combining two epilepsy-associated ion channel mutations with mutually opposing excitability defects and overlapping subcellular localization. We found that increasing membrane excitability by removing Shaker-like K(+) channels, which are encoded by the Kcna1 gene, masked the absence epilepsy caused by a P/Q-type Ca(2+) channelopathy due to a missense mutation in the Cacna1a gene. Conversely, decreasing network excitability by impairing Cacna1a Ca(2+)-channel function attenuated limbic seizures and sudden death in Kcna1-null mice. We also identified intermediate excitability phenotypes at the network and axonal levels. Protective interactions between pathogenic ion channel variants may markedly alter the clinical expression of epilepsy, highlighting the need for comprehensive profiling of this candidate gene set to improve the accuracy of genetic risk assessment of this complex disease.

Topics: 2-Amino-5-phosphonovalerate; Age Factors; Animals; Calcium Channel Blockers; Calcium Channels, N-Type; Calcium Channels, P-Type; Calcium Channels, Q-Type; Disease Models, Animal; Electric Stimulation; Electroencephalography; Epilepsy; Excitatory Amino Acid Antagonists; Hippocampus; In Vitro Techniques; Kv1.1 Potassium Channel; Membrane Potentials; Mice; Mice, Inbred C57BL; Mice, Transgenic; Mutation; Nerve Net; omega-Agatoxin IVA; Potassium Chloride

The tottering (tg) mice have a mutation in the CaV2.1 (P/Q-type) voltage-dependent Ca2+ channel alpha(1)2.1 subunit gene. tg mice show not only cerebellar ataxia but also absence epilepsy, which begins at approximately 3 weeks of age and persists throughout life. Similarities in EEG and sensitivity to antiepileptic drugs suggest that tg mice are a good model for human absence epilepsy. Although imbalance between excitatory and inhibitory activity in the thalamocortical network is thought to contribute to the pathogenesis of absence epilepsy, the effect of the mutation on thalamocortical synaptic responses remains unknown. Here we showed imbalanced impairment of inhibitory synaptic responses in tg mice using brain slice preparations. Somatosensory thalamocortical projection makes not only monosynaptic glutamatergic connections but also disynaptic GABAergic connections, which mediate feedforward inhibition, onto layer IV neurons. In tg mice, IPSC amplitudes recorded from layer IV pyramidal cells of the somatosensory cortex in response to thalamic stimulation became disproportionately reduced compared with EPSC amplitudes at later developmental stages (postnatal days 21-30). Similar results were obtained by local stimulation of layer IV pyramidal neurons. However, IPSC reduction was not seen in layer V pyramidal neurons of epileptic tg mice or in layer IV pyramidal neurons of younger tg mice before the onset of epilepsy (postnatal days 14-16). These results showed that the feedforward inhibition from the thalamus to layer IV neurons of the somatosensory cortex was severely impaired in tg mice and that the impairment of the inhibitory synaptic transmission was correlated to the onset of absence epilepsy.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Afferent Pathways; Age Factors; Animals; Calcium Channel Blockers; Calcium Channels, N-Type; Disease Models, Animal; Electric Stimulation; Electroencephalography; Epilepsy, Absence; Evoked Potentials; Excitatory Postsynaptic Potentials; Homeostasis; Lidocaine; Mice; Mice, Inbred C57BL; Mice, Neurologic Mutants; omega-Agatoxin IVA; omega-Conotoxin GVIA; Patch-Clamp Techniques; Picrotoxin; Pyramidal Cells; Somatosensory Cortex; Synaptic Transmission; Thalamic Nuclei; Valine

Cerebral aneurysm rupture and subarachnoid hemorrhage (SAH) inflict disability and death on thousands of individuals each year. In addition to vasospasm in large diameter arteries, enhanced constriction of resistance arteries within the cerebral vasculature may contribute to decreased cerebral blood flow and the development of delayed neurological deficits after SAH. In this study, we provide novel evidence that SAH leads to enhanced Ca2+ entry in myocytes of small diameter cerebral arteries through the emergence of R-type voltage-dependent Ca2+ channels (VDCCs) encoded by the gene CaV 2.3. Using in vitro diameter measurements and patch clamp electrophysiology, we have found that L-type VDCC antagonists abolish cerebral artery constriction and block VDCC currents in cerebral artery myocytes from healthy animals. However, 5 days after the intracisternal injection of blood into rabbits to mimic SAH, cerebral artery constriction and VDCC currents were enhanced and partially resistant to L-type VDCC blockers. Further, SNX-482, a blocker of R-type Ca2+ channels, reduced constriction and membrane currents in cerebral arteries from SAH animals, but was without effect on cerebral arteries of healthy animals. Consistent with our biophysical and functional data, cerebral arteries from healthy animals were found to express only L-type VDCCs (CaV 1.2), whereas after SAH, cerebral arteries were found to express both CaV 1.2 and CaV 2.3. We propose that R-type VDCCs may contribute to enhanced cerebral artery constriction after SAH and may represent a novel therapeutic target in the treatment of neurological deficits after SAH.

Topics: Animals; Blood; Calcium; Calcium Channel Blockers; Calcium Channels, L-Type; Calcium Channels, R-Type; Cerebral Arteries; Cisterna Magna; Dihydropyridines; Diltiazem; Disease Models, Animal; Drug Resistance; Injections; Ion Transport; Male; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Nifedipine; omega-Agatoxin IVA; omega-Conotoxin GVIA; Patch-Clamp Techniques; Rabbits; Spider Venoms; Subarachnoid Hemorrhage; Vasoconstriction; Vasospasm, Intracranial

Neuropathic pain, due to peripheral nerve damage, can include allodynia (perception of innocuous stimuli as being painful), hyperalgesia (increased sensitivity to noxious stimuli) and spontaneous pain, often accompanied by sensory deficits. Plasticity in transmission and modulatory systems are implicated in the underlying mechanisms. The Kim and Chung rodent model of neuropathy (Kim and Chung, Pain 50 (1992) 355) employed here involves unilateral tight ligation of two (L5 and L6) of the three (L4, L5, and L6) spinal nerves of the sciatic nerve and reproducibly induced mechanical and cold allodynia in the ipsilateral hindpaw over the 14 day post-operative period. In vivo electrophysiological techniques have then been used to record the response of dorsal horn neurones to innocuous and noxious electrical and natural (mechanical and thermal) stimuli after spinal nerve ligation (SNL). Activation of voltage-dependent calcium channels (VDCCs) is critical for neurotransmitter release and neuronal excitability, and antagonists can be antinociceptive. Here, for the first time, the effect of N- and P-type VDCC antagonists (omega-conotoxin-GVIA and omega-agatoxin-IVA, respectively) on the evoked dorsal horn neuronal responses after neuropathy have been investigated. Spinal omega-conotoxin-GVIA (0.1-3.2 microg) produced prolonged inhibitions of both the electrically- and low- and high-intensity naturally-evoked neuronal responses in SNL and control rats. Spinal omega-agatoxin-IVA (0.1-3.2 microg) also had an inhibitory effect but to a lesser extent. After neuropathy the potency of omega-conotoxin-GVIA was increased at lower doses in comparison to control. This indicates an altered role for N-type but not P-type VDCCs in sensory transmission after neuropathy and selective plasticity in these channels after nerve injury. Both pre- and post-synaptic VDCCs appear to be important.

Topics: Animals; Behavior, Animal; Calcium Channel Blockers; Calcium Channels, P-Type; Disease Models, Animal; Dose-Response Relationship, Drug; Electric Stimulation; Electrophysiology; Ligation; Male; Neuralgia; Nociceptors; omega-Agatoxin IVA; omega-Conotoxin GVIA; Posterior Horn Cells; Rats; Rats, Sprague-Dawley; Spinal Nerves

We investigated the effects of intrathecally administered N-type and P-type voltage-sensitive calcium channel (VSCC) blockers on the level of thermal hyperalgesia in two neuropathic pain models: the chronic constriction injury (CCI) model and the partial sciatic nerve injury (PSNI) model. N-type, but not P-type, VSCC blockers attenuated the level of thermal hyperalgesia induced by CCI in a dose-dependent manner. In the PSNI model, both N-type and P-type VSCC blockers had no effect on thermal hyperalgesia. This suggests that some types of neuropathic pain may be treatable with N-type VSCC blockers.

Topics: Analysis of Variance; Animals; Calcium Channel Blockers; Constriction; Disease Models, Animal; Injections, Spinal; Male; Membrane Potentials; Neuralgia; Neuroprotective Agents; omega-Agatoxin IVA; omega-Conotoxin GVIA; omega-Conotoxins; Peptides; Rats; Rats, Sprague-Dawley; Sciatic Nerve; Spider Venoms

This study investigated the behavioural and anticonvulsant effects of voltage-sensitive calcium channel blockers in DBA/2 mice. Omega-Conotoxin MVIIC (0.1, 0.3 micrograms ICV/mouse) and omega-agatoxin IVA (0.1, 0.3, 1 micrograms ICV), which act predominantly at P- and/or Q-type calcium channels, prevented clonic and tonic sound-induced seizures in this animal model of reflex epilepsy (ED50 values with 95% confidence limits for protection against clonic sound-induced seizures were 0.09 (0.04-0.36) micrograms ICV and 0.09 (0.05-0.15) micrograms ICV respectively and against tonic seizures 0.07 (0.03-0.16) micrograms ICV and 0.08 (0.04-0.13) micrograms ICV, respectively). The N-type calcium channel antagonists omega-conotoxin GVIA and omega-conotoxin MVIIA were also tested in this model. Omega-Conotoxin GVIA was anticonvulsant in DBA/2 mice, but only at high doses (3 micrograms ICV prevented tonic seizures in 60% of the animals; 10 micrograms ICV prevented clonic seizures in 60% and tonic seizures in 90% of the animals), whereas omega-conotoxin MVIIA did not inhibit sound-induced seizures in doses up to 10 micrograms ICV. Both omega-conotoxin GVIA and omega-conotoxin MVIIA induced an intense shaking syndrome in doses as low as 0.1 microgram ICV, whereas omega-conotoxin MVIIC and omega-agatoxin IVA did not produce shaking at any of the doses examined. Finally, omega-conotoxin GI (0.01-1 microgram ICV) and alpha-conotoxin SI (0.3-30 micrograms ICV), which both act at acetylcholine nicotinic receptors, were not anticonvulsant and did not induce shaking in DBA/2 mice. These results confirm that blockers of N- and P-/Q-type calcium channels produce different behavioural responses in animals. The anticonvulsant effects of omega-conotoxin MVIIC and omega-agatoxin IVA in DBA/2 mice are consistent with reports that P- and/or Q-type calcium channel blockers inhibit the release of excitatory amino acids and are worthy of further exploration.

Topics: Acoustic Stimulation; Animals; Behavior, Animal; Calcium Channel Blockers; Disease Models, Animal; Male; Mice; Mice, Inbred DBA; omega-Agatoxin IVA; omega-Conotoxin GVIA; omega-Conotoxins; Peptides; Seizures; Spider Venoms