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It is well known that the CaVα2δ auxiliary subunit regulates the density of high voltage-activated Ca2+ channels in the plasma membrane and that alterations in their functional expression might have implications in the pathophysiology of diverse human diseases such as neuropathic pain. However, little is known concerning the transcriptional regulation of this protein. We previously characterized the promoter of CaVα2δ, and here we report its regulation by the transcription factor Egr-1. Using the neuroblastoma N1E-115 cells, we found that Egr-1 interacts specifically with its binding site in the promoter, affecting the transcriptional regulation of CaVα2δ. Overexpression and knockdown analysis of Egr-1 showed significant changes in the transcriptional activity of the CaVα2δ promoter. Egr-1 also regulated the expression of CaVα2δ at the level of protein. Also, functional studies showed that Egr-1 knockdown significantly decreases Ca2+ currents in dorsal root ganglion (DRG) neurons, while overexpression of the transcription factor increased Ca2+ currents in the F11 cell line, a hybrid of DRG and N18TG2 neuroblastoma cells. Studying the effects of Egr-1 on the transcriptional expression of CaVα2δ could help to understand the regulatory mechanisms of this protein in both health and disease.

Albiflorin (AF), one of important bioactive constituents of Paeonia lactiflora Radix, possesses neuro-protective effect. The present study aims to investigate the antinociceptive activities of AF and possible mechanisms. AF suppressed acetic acid-caused writhing, lengthened the latency period of mouse in hot plate test, and reduced the licking and biting response time of the injected mouse paw during phase I and phase II, and it suggested that AF exerted the antinociceptive activity mainly through central nervous system. Nimodipine, a commonly used calcium channels blocker, strongly lengthened AF-enhanced latency period of mouse in hot plate test. Compared with control group, AF reduced the levels of euronal nitric oxide synthase (nNOS), and enhanced the levels of serotonin (5-HT) in serum and/or hypothalamus before and after 30-s thermal stimuli. The reduced activation of calmodulin-dependent protein kinase II and c-Jun N-terminal kinase in hypothalamus was observed in AF-treated mice. Collectively, AF-mediated antinociceptive activities were, at least partially, related to calcium channels.

Mitophagy plays an important role in mitochondrial quality control and cell survival during the process of ischemia/reperfusion (I/R) injury. Mitochondrial calcium uniporter (MCU) is the most important channel responsible for Ca(2+) influx into mitochondria and Ca(2+) signal plays a potential role in modulating mitophagy. However, the effect of MCU on mitophagy during the process of I/R injury remains unknown. This study constructed an in vitro I/R model by subjecting oxygen and glucose deprivation/reperfusion (OGD/RP) model to SH-SY5Y cells to mimic the cerebral I/R injury and aimed to explore the exact effect of MCU on I/R induced mitophagy. The results showed that OGD/RP induced autophagy and mitophagy in SH-SY5Y cells. Ru360, the inhibitor of MCU, improved mitochondrial morphology and fuctional stability as well as cell viability, significantly reduced OGD/RP induced mitophagy as evidenced by the decrease in Beclin-1 and the increase in Tom20 and P62 expression. Whereas spermine, the agonist of MCU, had no significant impact on the expression of those mitophagy related proteins compared with OGD/RP group. This study indicates that inhibition of MCU can inhibit excessive mitophagy and protect the neurocytes from I/R injury.

GABAA receptors are targets of different pharmacologically relevant drugs, such as barbiturates, benzodiazepines, and anesthetics. In particular, benzodiazepines are prescribed for the treatment of anxiety, sleep disorders, and seizure disorders. Benzodiazepines potentiate GABA responses by binding to GABAA receptors, which are mainly composed of α (1-3, 5), β2, and γ2 subunits. Prolonged activation of GABAA receptors by endogenous and exogenous modulators induces adaptive changes that lead to tolerance. For example, chronic administration of benzodiazepines produces tolerance to most of their pharmacological actions, limiting their usefulness. The mechanism of benzodiazepine tolerance is still unknown. To investigate the molecular basis of tolerance, we studied the effect of sustained exposure of rat cerebral cortical neurons to diazepam on the GABAA receptor. Flunitrazepam binding experiments showed that diazepam treatment induced uncoupling between GABA and benzodiazepine sites, which was blocked by co-incubation with flumazenil, picrotoxin, or nifedipine. Diazepam also produced selective transcriptional down-regulation of GABAA receptor α1 subunit gene through a mechanism dependent on the activation of L-type voltage-gated calcium channels. These findings suggest benzodiazepine-induced stimulation of calcium influx through L-type voltage-gated calcium channels triggers the activation of a signaling pathway that leads to uncoupling and an alteration of receptor subunit expression. Insights into the mechanism of benzodiazepine tolerance will contribute to the design of new drugs that can maintain their efficacies after long-term treatments.

Previous study has shown that the co-localization of calbindin (CB) with L-type voltage dependent Ca 2+ channel (VDCC) alpha 1C subunit (Ca v 1.2) in the rat insulinoma 1046-38 (RIN) beta cells may play an important regulatory role in Ca 2+ influx and exocytosis of insulin granules. In the present study, L-type voltage dependent Ca 2+ channel (VDCC) and calbindin (CB) were demonstrated in different regions of the mouse central nervous system (CNS). Double labeling immunofluorescence staining showed a co-localization of Ca v 1.3 and CB. The co-localization of Ca v 1.3 and CB in certain brain regions such as the hippocampus suggests their important roles in neuroplasticity. The relative high percentages of co-localization of Ca v 1.3 with CB in the laminae II of the dorsal horn of the spinal cord indicate that the regulation mechanism of nociceptive transmission may be related with both VDCC and Ca 2+ binding protein.

Nurr1 is an orphan nuclear transcription factor expressed in the brain. While Nurr1 is assumed to be an immediate early gene, it is not fully understood how Nurr1 expression is regulated in an activity-dependent manner in the central nervous system. Here, we investigated the molecular mechanisms underlying the regulation of Nurr1 expression in cultured hippocampal and cortical neurons. We found that upregulation of neural activity by high KCl and bicuculline enhances Nurr1 levels, while blockade of its activity by tetrodotoxin reduces Nurr1 levels. The induction of Nurr1 expression was mediated by voltage-dependent calcium channels (VDCCs), as shown by cadmium and VDCC-specific inhibitors. Furthermore, calcineurin, but not calcium/calmodulin-dependent protein kinase (CaMK) was critical for the induction. Thus, Nurr1 expression is regulated by VDCC and calcineurin in a cell-autonomous, neural activity-dependent manner.

Calcium influx through N-methyl-D-aspartate receptors (NMDAR) and voltage-gated calcium channels (VGCC) play major roles in postsynaptic signaling mechanisms. NMDAR subunit GluN2B is phosphorylated at Ser1303. Phosphorylation at this site is a prominent event in cell culture systems as well as in vivo. However, the functional significance of phosphorylation at this site is not completely understood. In this study, we compared the effect of calcium signaling through NMDAR and VGCC on the phosphorylation status of GluN2B-Ser1303 in the rat in vivo. VGCC was activated by intraperitoneal (IP) injection of the activator, BayK8644 and NMDAR was activated by intracerebroventricular (ICV) injection of NMDA in separate experimental groups. We found that the level of phospho-GluN2B-Ser1303 in the cortex and in the hippocampus increased in response to activation of either channel. The effects could be prevented by prior ICV administration of the specific blockers of these channels such as MK-801 for NMDAR and nifedipine for VGCC. The effect was also blocked by pretreatment with ICV administration of KN-93 indicating that it is mediated through CaM kinase. Both during NMDAR activation and VGCC activation, cell survival associated signals such as phospho-AKT and phospho-CREB showed decrease, consistent with activation of cell death pathways during these treatments. We conclude that under in vivo conditions, calcium influx through either NMDAR or VGCC activates CaM kinase, which in turn phosphorylates GluN2B-Ser1303.

The medial prefrontal cortex (mPFC) is a key regulator of neurocognition. The glutamatergic pyramidal neurons are the predominant component of neurons in the mPFC. Aging and HIV profoundly alter the structure and function of mPFC pyramidal neurons, including, but are not limited to, dysregulation of NMDA receptors and voltage-gated calcium channels. Here we assessed the impact of aging and in vivo HIV exposure on the functional activity (firing) of mPFC pyramidal neurons mediated by voltage-gated K+ (Kv) channels and inwardly-rectifying K+ (Kir) channels using patch-clamp recording in rat brain slices ex vivo. We found that aging and HIV significantly affect firing in different manners by altering the activity of Kv and likely Kir channels, associated with changes in membrane properties and the mRNA levels of specific Kv channels. Evoked firing was significantly decreased in mPFC neurons of older (12 month, 12 m) rats compared to younger (6/7 week, 6/7wk) rats, regardless of HIV status. In contrast, firing was significantly increased in neurons from Tg rats compared to non-Tg rats, regardless of age. Aging/HIV-induced alterations in firing were mediated by dysfunctional Kv channels and Kir channels, which exhibit significant changes in their activity and/or expression induced by aging and HIV exposure in vivo. Collectively, these novel findings demonstrate that aging is associated with a significant decline of mPFC neuronal activity; while long-term HIV exposure in vivo could drive mPFC neurons from over-activation to loss of firing, which could ultimately exacerbate the decline of mPFC neuronal activity.

The inflammatory response is a natural response of the body that occurs immediately following tissue damage, which may be due to injury, infection or disease. The acute inflammatory response is an essential mechanism that promotes healing and a key aspect is the ensuing pain, which warns the subject to protect the site of injury. Thus, it is common to see a zone of primary sensitization as well as consequential central sensitization that generally, is maintained by a peripheral drive from the zone of tissue injury. Inflammation associated with chronic pain states, such as rheumatoid and osteoarthritis, cancer and migraine etc. is deleterious to health and often debilitating for the patient. Thus there is a large unmet clinical need. The mechanisms underlying both acute and chronic inflammatory pain are extensive and complex, involving a diversity of cell types, receptors and proteins. Among these the contribution of voltage gated sodium and calcium channels on peripheral nociceptors is critical for nociceptive transmission beyond the peripheral transducers and changes in their distribution, accumulation, clustering and functional activities have been linked to both inflammatory and neuropathic pain. The latter has been the main area for trials and use of drugs that modulate ion channels such as carbamazepine and gabapentin, but given the large peripheral drive that follows tissue damage, there is a clear rationale for blocking voltage gated sodium and calcium channels in these pain states. It has been hypothesized that pain of inflammatory origin may evolve into a condition that resembles neuropathic pain, but mixed pains such as low back pain and cancer pain often include elements of both pain states. This review considers the therapeutic potential for sodium and calcium channel blockers for the treatment of chronic inflammatory pain states.

This study describes the expression of the voltage operated calcium channels (VOCCs) subunits α1A (typical of the P/Q family) and α1D (of the L family) in the bovine hypothalamus. The expression of both P/Q and L families has been characterized in the brain of adult mammals. However, their distribution and expression during foetal neuronal differentiation have not yet been determined. The expression profile of the α1A and α1D pore-forming subunits was investigated during four embryonic stages in bovine foetuses. Our data suggest that the expressions of α1A and α1D are correlated during development, with an increase only in males that peaks on the last period of gestation. Bovine male hypothalami showed significantly higher α1A and α1D expression values in comparison to female ones during the whole developmental period. In the females, the expression profiles of both genes were constant during all the developmental time. Immunohistochemical studies confirmed the presence of the α1A and α1D protein subunits in foetal hypothalamic neurones starting from the third foetal stage. Our data provide new information on the hypothalamic expression of α1A and α1D subunits during development in a mammal with a long gestation period and a large and convoluted brain.

In decerebrated rats, we determined the dose of A803467, a NaV 1.8 antagonist, needed to attenuate the reflex pressor responses to femoral arterial injections of lactic acid (24 mM; ~0.1 ml) and capsaicin (0.1 μg), agents which stimulate thin fiber afferents having NaV 1.8 channels. We also determined whether the dose of A803467 needed to attenuate these reflex responses affected the responses of muscle spindle afferents to tendon stretch and succinylcholine (200 μg). Spindle afferents are not supplied with NaV 1.8 channels, and consequently their responses to these stimuli should not be influenced by A803467. Pressor responses to lactic acid and capsaicin were not altered by 500 μg of A803467 (n=6). A803467 in a dose of 1mg, however, significantly reduced (p

Background and objectives The non-depolarizing muscle relaxant vecuronium inhibits contraction by competitive inhibition of postsynaptic acetylcholine receptors (AchRs), which decreases the number of quanta released per impulse in response to 50 Hz stimulation. The specific role of calcium influx through L-type calcium channels is the promotion of endocytosis and vesicle recycling during high-frequency stimulation. Vecuronium also induces four pulse tetanic fade, a proxy measure of decreased quanta release. We examined whether vecuronium suppresses neuromuscular transmission during high-frequency stimulation by inhibiting presynaptic L-type calcium channels. Methods Fifty male Sprague-Dawley rats were divided into five treatment groups: unstimulated control group, α-bungarotoxin (BTX) group, nifedipine group, vecuronium group, and nifedipine plus vecuronium group. Rat phrenic nerve–diaphragm neuromuscular juctions were stimulated at 50 Hz and field excitatory post-synaptic potentials (fEPSPs) were recorded. Expression levels of the presynaptic Ca2+-binding, protein synaptotagmin 1, and the presynaptic plasma membrane protein, syntaxin 1, were measured by Western blots. Results The fEPSPs evoked by 50 Hz stimulus trains were decreased by vecuronium, nifedipine, and by vecuronium plus nifedipine. Nifedipine, an L-type calcium channel blocker, reduced the expression of synaptogamin and syntaxin and blocked the suppressive effect of vecuronium, suggesting that both agents inhibit presynaptic L-type calcium channels. Conclusions Vecuronium which blocked L-type calcium channels may suppress activity of the α3β2 nAChR subunit, which exists in the presynaptic membrane and enhances quantal release. This α3β2 nAChR-mediated positive feedback effect may be facilitated by L-type Ca2+ channel activity under high-frequency stimulation. Vecuronium may disrupt this positive feedback cycle, leading to suppression of fEPSPs. Vercuronium may reduce neuromuscular transmission through presynaptic and postsynaptic mechanisms.

Memory reconsolidation and maintenance depend on calcium channels and on calcium/calmodulin-dependent kinases regulating protein turnover in the hippocampus. Ingestion of a jellyfish protein, apoaequorin, reportedly protects and/or improves verbal learning in adults and is currently widely advertised for use by the elderly. Apoaequorin is a member of the EF-hand calcium binding family of proteins that includes calmodulin. Calmodulin-1 (148 residues) differs from Apoaequorin (195 residues) in that it contains four rather than three Ca2+-binding sites and three rather than four cholesterol-binding (CRAC, CARC) domains. All three cholesterol-binding CARC domains in calmodulin have a high interaction affinity for cholesterol compared to only two high affinity CARC domains in apoaequorin. Both calmodulin and apoaequorin can form dimers with a potential of eight bound Ca2+ ions and six high affinity-bound cholesterol molecules in calmodulin with six bound Ca2+ ions and a mixed population of eight cholesterols bound to both CARC and CRAC domains in apoaqueorin. MEMSAT-SVM analysis indicates that both calmodulin and apoaqueorin have a pore-lining region. The Peptide-Cutter algorithm predicts that calmodulin-1 contains 11 trypsin-specific cleavage sites (compared to 21 in apoaqueorin), four of which are potentially blocked by cholesterol and three are within the Ca-binding domains and/or the pore-lining region. Three are clustered between the third and fourth Ca2+-binding sites. Only calmodulin pore-lining regions contain Ca2+ binding sites and as dimers may insert into the plasma membrane of neural cells and act as Ca2+ channels. In a dietary supplement, bound cholesterol may protect both apoaequorin and calmodulin from proteolysis in the gut as well as facilitate uptake across the blood-brain barrier. Our results suggest that a physiological calmodulin-cholesterol complex, not cholesterol-free jellyfish protein, may better serve as a dietary supplement to facilitate memory maintenance.

The repressor element-1 silencing transcription factor (REST) has emerged as a key controller of neuronal differentiation and has been shown to play a critical role in the expression of the neuronal phenotype; however, much has still to be learned about its role at specific developmental stages and about the functional targets affected. Among these targets, calcium signaling mechanisms are critically dependent on the developmental stage and their full expression is a hallmark of the mature, functional neuron. We have analyzed the role played by REST in GN11 cells, an immortalized cell line derived from gonadotropin hormone releasing hormone (GnRH) neurons at an early developmental stage, electrically non-excitable and with a strong migratory activity. We show for the first time that functional voltage-dependent calcium channels are expressed in wild type GN11 cells; down-regulation of REST by a silencing approach shifts these cells towards a more differentiated phenotype, increasing the functional expression of P/Q-type channels and reducing their migratory potential.

Intracochlear application of exogenous or transgenic neurotrophins, such as neurotrophin-3 (NT-3) and brain derived neurotrophic factor (BDNF), could promote the resprouting of spiral ganglion neuron (SGN) neurites in deafened animals. These resprouting neurites might reduce the gap between cochlear implant electrodes and their targeting SGNs, allowing for an improvement of spatial resolution of electrical stimulation. This study is to investigate the impact of electrical stimulation employed in CI on the extension of resprouting SGN neurites. We established an in vitro model including the devices delivering charge-balanced biphasic electrical stimulation, and spiral ganglion (SG) dissociated culture treated with BDNF and NT-3. After electrical stimulation with varying durations and intensities, we quantified neurite lengths and Schwann cell densities in SG cultures. Stimulations that were greater than 50μA or longer than 8h significantly decreased SG neurite length. Schwann cell density under 100μA electrical stimulation for 48h was significantly lower compared to that in non-stimulated group. These electrical stimulation-induced decreases of neurite extension and Schwann cell density were attenuated by various types of voltage-dependent calcium channel (VDCC) blockers, or completely prevented by their combination, cadmium or calcium-free medium. Our study suggested that charge-balanced biphasic electrical stimulation inhibited the extension of resprouting SGN neurites and decreased Schwann cell density in vitro. Calcium influx through multiple types of VDCCs was involved in the electrical stimulation-induced inhibition.

Kenyon cells (KCs), which are present in the mushroom bodies (MBs) of the insect brain, play an important role in olfactory information processing and associative learning. However, the intrinsic electrophysiological properties of KCs in silkmoth (Bombyx mori) MBs remain unknown. Here, we use whole-cell patch-clamp recordings to elucidate the functional parameters of membrane voltage and voltage-activated ionic currents of KCs in silkmoth MBs. KCs generated action potentials in response to stepping pulses of depolarizing current, and application of GABA-receptor blocker abolished inhibitory synaptic inputs and depolarized resting membrane potential. Pharmacological isolation of KC voltage-gated ionic currents revealed that KCs express a range of voltage-activated channels, including transient and non-inactivating potassium, sodium, and calcium channels. Our results provide the first electrophysiological characterization of KCs in silkmoth MBs and represent an important step toward understanding neuronal computation that underlies olfactory information processing in silkmoths.

The predominant calcium current in nodose sensory neurons, including the subpopulation of baroreceptor neurons, is the N-type channel, Cav2.2. It is also the primary calcium channel responsible for transmitter release at their presynaptic terminals in the nucleus of the solitary tract in the brainstem. The P/Q channel, Cav2.1, the other major calcium channel responsible for transmitter release at mammalian synapses, represents only 15–20% of total calcium current in the general population of sensory neurons and makes a minor contribution to transmitter release at the presynaptic terminal. In the present study we identified a subpopulation of the largest nodose neurons (capacitance > 50 pF) in which, surprisingly, Cav2.1 represents over 50% of the total calcium current, differing from the remainder of the population. Consistent with these electrophysiological data, anti-Cav2.1 antibody labeling was more membrane delimited in a subgroup of the large neurons in slices of nodose ganglia. Data reported in other synapses in the central nervous system assign different roles in synaptic information transfer to the P/Q-type versus N-type calcium channels. The study raises the possibility that the P/Q channel which has been associated with high fidelity transmission at other central synapses serves a similar function in this group of large myelinated sensory afferents, including arterial baroreceptors where a high frequency regular discharge pattern signals the pressure pulse. This contrasts to the irregular lower frequency discharge of the unmyelinated fibers that make up the majority of the sensory population and that utilize the N-type channel in synaptic transmission.

Transgenic Drosophila melanogaster carrying the human gene for alpha synuclein is an animal model for the study of Parkinson's Disease. Climbing activity in these flies is reduced as a result of the effect of this protein on the locomotor activity of the transgenic fly. l -DOPA and gamma amino butyric acid (GABA) reverse the loss of this activity when placed in the food fed to these flies. While muscimol, a GABA A receptor agonist has no effect in this system, baclofen and the allosteric agonists CG 7930 and GS 39783 which affect the GABA B receptor reverse this activity. This latter effect is eliminated when these compounds are fed in conjunction with the GABA B receptor antagonist 2-hydroxysaclofen. In addition, fendiline which is a Ca ++ receptor blocker also reverses the loss of climbing ability. Because there is a calcium channel close to the GABA B receptor on the cell surface, these data are indicative of a relationship between the roles of the GABA B receptor, the calcium channel and the effect of alpha-synuclein on the motor activity of the transgenic fly.

Neuronal L-type Ca(2+) channels play pivotal roles in regulating gene expression, cell survival, and synaptic plasticity. The Ca(V)1.2 and Ca(V)1.3 channels are 2 main subtypes of neuronal L-type Ca(2+) channels. However, the specific roles of Ca(V)1.2 and Ca(V)1.3 in L-type Ca(2+) channel-mediated neuronal responses and their cellular mechanisms are poorly elucidated. On the basis of our previous study demonstrating a physical interaction between the Ca(V)1.3 channel and GABA(B) receptor (GABA(B)R), we further examined the involvement of Ca(V)1.2 and Ca(V)1.3 in the GABA(B)R-mediated activation of ERK(1/2), a kinase involved in both CREB activation and synaptic plasticity. After confirming the involvement of L-type Ca(2+) channels in baclofen-induced ERK(1/2) phosphorylation, we examined a specific role of Ca(V)1.2 and Ca(V)1.3 channels in the baclofen effect. Using siRNA-mediated silencing of Ca(V)1.2 or Ca(V)1.3 messenger, we determined the relevance of each channel subtype to baclofen-induced ERK(1/2) phosphorylation in a mouse hippocampal cell line (HT-22) and primary cultured rat neurons. In the detailed characterization of each subtype using HEK293 cells transfected with Ca(V)1.2 or Ca(V)1.3, we found that GABA(B)R can increase ERK(1/2) phosphorylation and Ca(V)1.3 channel activity through direct interaction with Ca(V)1.3 channels. These results suggest a functional interaction between Ca(V)1.3 and GABA(B)R and important implications of Ca(V)1.3/GABA(B)R clusters for translating synaptic activity into gene expression alterations.

The calcium control hypothesis posits that postsynaptic calcium increases are required to trigger synaptic plasticity, with large increases inducing LTP and small increases inducing LTD. In CA1 of the hippocampus, however, LTD induced by chemical activation of metabotropic glutamate receptors (agonist-LTD) is independent of increases in postsynaptic calcium. Here we tested whether LTD induced by pairing of presynaptic stimulation with postsynaptic depolarization (synaptic-LTD) is similarly calcium-independent. This protocol induced an NMDA-dependent LTP when paired at 0 mV, which was converted to mGluR-dependent LTD when paired at −20 mV. The LTD was not blocked by calcium chelation, blockers of L- or T-type voltage-dependent calcium channels, or hyperpolarization to −70 mV. We conclude that synaptically induced mGluR-dependent LTD, like agonist induced mGluR LTD, does not require calcium influx for its induction.

Measles virus (MV) infection may lead to severe chronic CNS disease processes, including MV-induced encephalitis. Because the intracellular Ca(2+) concentration ([Ca(2+)](i)) is a major determinant of the (patho-)physiological state in all cells we asked whether important Ca(2+) conducting pathways are affected by MV infection in cultured cortical rat neurons. Patch-clamp measurements revealed a decrease in voltage-gated Ca(2+) currents during MV-infection, while voltage-gated K(+) currents and NMDA-evoked currents were unaffected. Calcium-imaging experiments using 50mM extracellular KCl showed reduced [Ca(2+)](i) increases in MV-infected neurons, confirming a decreased activity of voltage-gated Ca(2+) channels. In contrast, the group-I metabotropic glutamate receptor (mGluR) agonist DHPG evoked changes in [Ca(2+)](i) that were increased in MV-infected cells. Our results show that MV infection conversely regulates Ca(2+) signals induced by group-I mGluRs and by voltage-gated Ca(2+) channels, suggesting that these physiological impairments may contribute to an altered function of cortical neurons during MV-induced encephalitis.

The expression of Arc is tightly coupled to synaptic activities. Recent studies suggested the functional relevance of Arc translation in group I metabotropic glutamate receptor (mGluR)-mediated long-term depression. The present study investigated the transcription-dependent changes of Arc in response to the activation of group I mGluR by (R, S)-3, 5-dihydroxyphenylglycine (DHPG) in cultured cortical neuron. The increase in Arc mRNA did not require de novo protein synthesis, indicating that Arc is an immediate early gene upon DHPG stimulation. We further examined the major pathways involved in group I mGluR signaling, and found that DHPG-induced Arc up-regulation depended on CaMK, PLC, and ERK1/2 activity. Moreover, the activity of NMDA receptors, but not L-type voltage gated calcium channels (L-VGCC), was required for Arc transcription. Interestingly, blocking CaMK, PLC, and NMDAR, but not L-VGCC, suppressed DHPG-stimulated ERK1/2 activation. These data suggest the central role of ERK1/2 in group I mGluR-mediated Arc transcription.

We have previously shown that P7 rat pups injected with the N-methyl- d -aspartate receptor (NMDAR) blocker MK801 displayed robust apoptotic injury within hours after injection. Further studies from our lab suggest that loss of calcium cannot be compensated for when vulnerable neurons lack calcium buffering capabilities. Thus, to elevate calcium in these neurons prior to MK801 exposure, we injected P7 rats with the calcium channel agonist BayK 8644. Whereas BayK 8644 did not induce apoptosis by itself, it was found to block MK801-induced injury in a dose-dependent manner. Reversal of MK801 toxicity was complete in the caudate–putamen, partial in the somatosensory cortex but was not observed in the retrosplenial cortex. These results suggest that postnatal brain injury resulting from agents that block the NMDAR, which include commonly used anesthetics as well as drugs of abuse, may be prevented in vulnerable neurons by compensatory increases in calcium prior to exposure to these antagonists.

Presenilin-1 is required for gamma-secretase activity, which participates in Notch receptor processing, the pathogenesis of Alzheimer's disease and the modulation of Ca(2+) signaling. We tested the hypothesis that gamma-secretase proteolytic activity modulates store-operated Ca(2+) entry (SOCE) in rat dorsal root ganglion (DRG) neurons. Depletion of intracellular Ca(2+) stores by blocking the endoplasmic reticulum (ER) Ca(2+) pump with cyclopiazonic acid (CPA) evoked a transient increase in [Ca(2+)](i) but no sustained Ca(2+) influx. However, in cells expressing a dominant negative presenilin-1 mutant (PS1-D257A), gamma-secretase activity was inhibited and treatment with CPA evoked sustained Ca(2+) influx. Similarly, pharmacologic inhibition of gamma-secretase with DAPT for 48h enhanced SOCE. SKF96365, an inhibitor of store-operated channels, blocked SOCE in cells expressing PS1-D257A. Thus, gamma-secretase proteolytic activity regulates a SOCE pathway in sensory neurons.

Ecto-peptidases hydrolyze peptides in the extracellular fluid of the brain. This process is critical for defining the strength of peptidergic communication. A few studies suggest that brain ecto-peptidase activities are regulated by brain function but the extracellular messengers involved are generally unknown. Pyroglutamyl peptidase II (PPII) is specific for thyrotropin releasing hormone (TRH), a tripeptide with multiple homeostatic functions in brain. The purpose of this study was to identify regulators of brain PPII activity. Electrical stimulation (multiple tetani) did not change PPII activity in cortical or hippocampal slices. However, in hippocampal slices, blockade of calcium channels with high magnesium, or of L-type calcium channels (LTCC) or NMDA receptors, decreased PPII activity, while blockade of AMPA or GABA A receptors did not. Blockade of NMDA receptors did not change PPII mRNA levels but decreased PPII levels. The activity of another ecto-peptidase, aminopeptidase N, was also down regulated by a magnesium blockade, not regulated by NMDA receptor blockade and increased by LTCC blockade. The data show a differential regulation of the activity of ecto-peptidases by that of Ca 2+ channel and that synaptic activity, through the NMDA receptor, specifically regulates that of pyroglutamyl peptidase II.

International audience; Two distinct native alpha-bungarotoxin (alpha-Bgt)-insensitive nicotinic acetylcholine receptors (nAChRs), named nAChR1 and nAChR2, were identified in the cockroach Periplaneta americana dorsal unpaired median (DUM) neurons. They differed in their electrophysiological, pharmacological properties and intracellular regulation pathways. nAChR2 being an atypical nicotinic receptor closed upon agonist application and its current-voltage relationship resulted from a reduction in potassium conductance. In this study, using whole-cell patch-clamp technique, we demonstrated that calcium modulated nAChR2-mediated nicotine response. Under 0.5 microM alpha-Bgt and 20 mM d-tubocurarine, the nicotine-induced inward current amplitude was strongly reduced in the presence of intracellularly applied BAPTA or bath application of calcium-free solution. In addition, using cadmium chloride, we showed that nicotine response was modulated by extracellular calcium through plasma membrane calcium channels. Moreover, extracellular application of caffeine and thapsigargin reduced nAChR2-mediated response. Together these experiments revealed a complex calcium-dependent regulation of nAChR2.

Most cold-sensitive subnucleus caudalis (Vc) neurons are also excited by the TRPM8 agonist menthol and the TRPA1 agonist cinnamaldehyde (CA). We investigated how interactions among menthol, CA and noxious cooling and heating of the tongue affected responses of superficial Vc units recorded in thiopental-anesthetized rats. Units responded to 1% CA which enhanced cold- and heat-evoked responses 5 min later. They responded more strongly to 10% CA which initially depressed cold responses, followed by enhancement at 5 min without affecting responses to heat. Following 10% CA, the mean response to 1% menthol was significantly lower than when menthol was tested first. After menthol, the subsequent response to CA was significantly weaker compared to the mean CA-evoked response when it was tested first. These results demonstrate mutual cross-desensitization between CA and menthol. The response to CA was enhanced following prior application of 10% ethanol (menthol vehicle). Prior application of menthol did not prevent the biphasic effect of 10% CA on cold-evoked responses, nor did prior application of CA prevent menthol enhancement of cold-evoked responses. Responses to noxious heat were unaffected by 10% CA and menthol regardless of the order of chemical presentation. These data indicate that superficial Vc neurons receive convergent input from primary afferents expressing TRPM8 and TRPA1. The mutual cross-desensitization between CA and menthol, and differential modulation of cold- vs. heat-evoked responses, suggests a direct inhibition of TRPM8 and TRPA1 expressed in peripheral nerve endings by CA and menthol, respectively, rather than a central site of interaction.

The sphenopalatine ganglion (SPG) is the main parasympathetic ganglion that is involved in regulating cerebral vascular tone and gland secretion. SPG neurons have been implicated in some types of migraine headaches but their precise role has yet to be determined. In addition, very little information is available regarding ion channel modulation by neurotransmitters that are involved in the parasympathetic drive of SPG neurons. In this study, acute isolation of adult rat SPG neurons was developed in order to begin the electrophysiological characterization of this ganglion. Under our dissociation conditions, the average number of neurons obtained per ganglion was greater than 1200. Immunofluorescence imaging results showed positive labeling with acetylcholinesterase (AChE), confirming the parasympathetic nature of SPG neurons. On the other hand, weak tyrosine hydroxylase immunostaining was observed in these neurons. Whole-cell patch-clamp recordings revealed that most of the Ca(2+) current is carried by N-type (53%) and SNX-482 resistant R-type (30%) Ca(2+) channels. In addition, Ca(2+) currents were inhibited in a voltage-dependent manner following exposure to oxotremorine-M (Oxo-M), norepinephrine and ATP via muscarinic acetylcholine receptor 2 (M(2) AChR) subtype, adrenergic and P2Y purinergic receptors, respectively. The peptides VIP and angiotensin II failed to modulate Ca(2+) currents, suggesting that these receptors are not present on the SPG soma or do not couple to Ca(2+) channels. In summary, our data suggest that the Ca(2+) current inhibition mediated by Oxo-M, NE and ATP in adult rat SPG neurons plays an integral part in maintaining parasympathetic control of cranial functions.

High-voltage-activated Ca 2+ channels on presynaptic nerve terminals are known to play an important role in neurotransmitter release at both excitatory and inhibitory synapses. Whereas there is currently debate over the contribution of L-type voltage dependent Ca 2+ channels (L-type VDCCs) on the short-term presynaptic plasticity which is a defining feature of neuronal activity, the underlying mechanisms are poorly understood. In the present study, the L-type VDCCs chronically was inhibited with different doses of verapamil (10, 20 and 50 mg/kg; orally) to evaluate hippocampal dentate gyrus (DG) inhibitory interneuron function and its involvement on short-term plasticity using paired pulse stimulation in perforant path-DG of hippocampus. Our data show that chronic oral treatment of verapamil at dose of 50 mg/kg but not at lower doses, facilitated the excitability of DG cells at inter-stimulus intervals 20, 30 and 50 ms (P < 0.03, 0.01 and 0.001; respectively) in population spike amplitude ratio, which is indicative of paired pulse potentiation in perforant path-DG synapses. While there are no significant differences in field excitatory postsynaptic potential slope ratio at all doses. We suggest that DG neurons facilitation is caused by inhibition of inhibitory interneurons directly and/or indirectly via inhibition of glutamate release in hippocampal DG. Therefore, these experiments indicate that chronic use of verapamil has effect on short-term presynaptic plasticity. © 2007 Published by Elsevier Ireland Ltd.

This study addressed the effect of sulfur dioxide (SO(2)) derivatives on high-voltage-activated calcium currents (HVA-I(Ca)) in somatic membrane of freshly isolated rat dorsal root ganglion (DRG) neurons by using the whole-cell configuration of patch-clamp technique. High-threshold Ca(2+) channels are highly expressed in small dorsal root ganglion neurons. SO(2) derivatives increased the amplitudes of calcium currents in a concentration-dependent and voltage-dependent manner. The 50% enhancement concentrations (EC(50)) of SO(2) derivatives on HVA-I(Ca) was about 0.4 microM. In addition, SO(2) derivatives significantly shifted the activation and inactivation curve in the depolarizing direction. Parameters for the fit of a Boltzmann equation to mean values for the activation were V(1/2)=-17.9+/-1.3 mV before and -12.5+/-1.1 mV after application 0.5 microM SO(2) derivatives 2 min (P0.05). The half inactivation of HVA-I(Ca) was shifted 9.7 mV to positive direction (P0.05). Furthermore, SO(2) derivatives significantly prolonged the slow constant of inactivation, slowed the fast recovery but markedly accelerated the slow recovery of HVA-I(Ca) from inactivation. From HP of -60 mV 0.5 microM SO(2) derivatives increased the amplitude of HVA-I(Ca) with a depolarizing voltage step to -10 mV about 54.0% in small DRG neurons but 33.3% in large DRG neurons. These results indicated a possible correlation between the change of calcium channels and SO(2) inhalation toxicity, which might cause periphery neurons abnormal regulation of nociceptive transmission via calcium channels.

Inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R) acts as a ligand-gated channel that mediates neuronal signals by releasing Ca(2+) from the endoplasmic reticulum. The three-dimensional (3D) structure of tetrameric IP(3)R has been demonstrated by using electron microscopy (EM) with static specimens; however, the dynamic aspects of the IP(3)R structure have never been visualized in a native environment. Here we attempt to measure the surface topography of IP(3)R in solution using atomic force microscopy (AFM). AFM revealed large protrusions extending approximately 4.3 nm above a flat membrane prepared from Spodoptera frugiperda (Sf9) cells overexpressing mouse type 1 IP(3)R (Sf9-IP(3)R1). The average diameter of the large protrusions was approximately 32 nm. A specific antibody against a cytosolic epitope close to the IP(3)-binding site enabled us to gold-label the Sf9-IP(3)R1 membrane as confirmed by EM. AFM images of the gold-labeled membrane revealed 7.7-nm high protrusions with a diameter of approximately 30 nm, which should be IP(3)R1-antibody complexes. Authentic IP(3)R1 immuno-purified from mouse cerebella had approximately the same dimensions as those of the IP(3)R-like protrusions on the membrane. Altogether, these results suggest that the large protrusions on the Sf9-IP(3)R1 membrane correspond to the cytosolic domain of IP(3)R1. Our study provides the first 3D representation of individual IP(3)R1 particles in an aqueous solution.

The ability of nitrendipine, nisoldipine, verapamil and gabapentin to inhibit the development of CNS excitation induced by spermine was assessed in mice. Injection of an excitotoxic dose of spermine (100 microg, i.c.v.) in mice results in worsening tremor that culminates in the development of a fatal tonic convulsion within 8 h of spermine administration. The dihydropyridines, nitrendipine and nisoldipine, which are L-type calcium channel antagonists acting at the alpha1 subunit, inhibited the development of spermine-induced effects. Verapamil, which also acts at the alpha1 subunit of the L-type calcium channel, also inhibited the development of spermine-induced CNS excitation. Gabapentin, a postulated L-type calcium channel antagonist interacting at the alpha2delta subunit, did not inhibit the development of spermine-induced effects. These results show that antagonists of the alpha1 subunit of L-type calcium channels can effectively inhibit the effects of spermine in vivo. This may highlight the importance of L-type calcium channels in spermine action.

Increased oxyradical production and membrane lipid peroxidation (MLP) occur under physiological and degenerative conditions in neurons. We investigated whether 4-hydroxynonenal (4HN), one of the membrane lipid peroxidation products, affects long-term potentiation (LTP) in the rat dentate gyrus in vitro. Treatment of hippocampal slices with 4HN (10 microM) enhanced LTP without affecting basal evoked potentials. The enhancement was completely inhibited by 2 microM nifedipine, a blocker of L-type Ca2+ channels. In cultured dentate gyrus neurons, treatment of the cells with 4HN for 24 h resulted in a significant amount of cell death that was detoxified by glutathione, whereas short-term treatment with 4HN (< or = 6 h) had no effect. Nifedipine partially but significantly suppressed the 4HN-induced cell death. These results suggest that 4HN modulates LTP and induces delayed cell death through L-type Ca2+ channel activation in the dentate gyrus. 4HN thereby plays an important role in both physiological and pathophysiological events in the hippocampus.

We investigated the N-type voltage-dependent calcium channel blocking action of pranidipine, a novel dihydropyridine (DHP) derivative. Pranidipine significantly suppressed KCl-induced intracellular calcium changes ([Ca 2+ ] i ) in a dose-dependent fashion in dorsal root ganglion neurons. A patch-clamp investigation revealed a dose-dependent blocking effect on N-type currents. Intrathecal injection of pranidipine significantly shortened the licking time in the late phase of the formalin test, as occurs with cilnidipine and amlodipine, which act on L- and N-type channels. Conversely, nicardipine, which acts exclusively on L-type channels, had no antinociceptive effect. Our results indicate that pranidipine inhibits N-type calcium channels. Furthermore, it exerts an antinociceptive effect, which might be related to an attenuation of synaptic transmission by nociceptive neurons due to the blocking effect of pranidipine on N-type calcium channels in primary nociceptive afferent fibers.

International audience; Voltage-gated calcium currents are important for the survival and growth of embryonic cockroach brain neurons in primary culture. In the present experiments, we have studied, using the patch-clamp technique, the evolution with time in culture of the voltage-dependency and of the pharmacological properties of the calcium conductance of these neurons during the formation of a network. We have observed a progressive increase of the high-voltage-activated calcium conductance and a 10mV shift of the voltage-dependency of activation towards more negative potentials. The proportion of the R-like calcium current component increased during network formation. At the same time, the highly omega-AgaTxIVA-sensitive P-like component of the current is progressively replaced by a component which is less sensitive to the toxin. The origin and functional implications of these modifications are discussed.

An in vitro mouse hemisected spinal cord was used to characterize the γ-aminobutyric acid receptor type B (GABA B ) modulation of the ventral root potential (VRP) in response to electrical stimulation of the dorsal root (DR). Low-intensity (LI) and high-intensity (HI) stimulation induced VRPs with progressively higher amplitude and duration. Repetitive HI-stimulation of the DR (1–10 Hz) produced windup. The selective GABA B receptor agonist, CGP35024, inhibited the VRPs in a dose-dependent manner. The inhibitory action of CGP35024 was blocked by CGP52432, a potent GABA B receptor antagonist. Following washout of the GABA B receptor agonist, VRPs and windup were significantly enhanced. The rebound increase of the VRP following removal of CGP35024 was also blocked by the GABA B receptor antagonist, CGP52432. This phenomenon is not linked to receptor desensitization, but rather due to GABA B receptor-induced hyperactivity of N-, P/Q-type Ca 2+ channels, as ω-CgTx GVIA and MVIIC abolished/prevented the increase. The ‘rebound’ enhancement of the spinal transmission after exposure to GABA B agonists sheds light on the possible mechanism of the severe withdrawal effects after abrupt termination of baclofen treatment in patients suffering from multiple sclerosis.

We examined directional sensitivities in the dendritic activity of the identified giant interneurons (GIs) in the cricket, using in vivo Ca(2+) imaging during different directional air-current stimuli. Air current stimulus evoked action potential burst and quick Ca(2+) increase in GI. The stimulus direction of the maximal Ca(2+) responses corresponded to that of the maximal voltage response. However, the shapes of the directional tuning curves based on the Ca(2+) responses for each dendritic branch were different from the overall tuning curve based on spike counts for the cell. Moreover, different dendritic branches displayed distinct directional sensitivity profiles to the air-current stimuli. We propose that postsynaptic activities will influence the local Ca(2+) signals in the distal dendrites, and produce the difference in directional sensitivity of the dendritic Ca(2+) response.

Modification of voltage-gated Ca(2+) channels by hydrogen peroxide, a membrane-permeable form of reactive oxygen species, in cultured dentate granule cells was examined using the whole cell patch clamp technique. Pretreatment with hydrogen peroxide (1 and 10 microM) for 2 h enhanced the Ca(2+) current without affecting its voltage dependence. The enhancement was completely cancelled by 1 mM glutathione, an antioxidant, and 2 microM nifedipine, an L-type Ca(2+) channel blocker. In contrast, the enhancement of the Ca(2+) current was not mimicked by pretreatment with 10 microg/ml tunicamycin, an endoplasmic reticulum stressor. These results suggest that oxidative stress induced by hydrogen peroxide selectively regulates the activity of L-type Ca(2+) channels.

Activation (phosphorylation) and the possible mechanism of extracellular signal-regulated kinase 5 (ERK5) were evaluated after cerebral ischemia-reperfusion (I/R) in the hippocampus in a four-vessel occlusion model of Sprague-Dawley rats. Western blotting showed that ERK5 was strongly activated from 10 min to 1 day and peaked at 30 min of reperfusion after 15 min ischemia. Pretreatment with N-acetylcysteine, a free radical scavenger, effectively inhibited ERK5 activation in a dose-dependent manner. Consistently, ERK5 activation was significantly suppressed by genistein (protein-tyrosine kinase inhibitor), PP2 (specific inhibitor of Src family kinases), nifedipine (L-VGCC blocker) and dextromethorphan (NMDA receptor antagonist), but not 6,7-dinitroquinoxaline-2, 3(1H, 4H)-dione (AMPA receptor antagonist). These results suggested that ERK5 could be significantly activated by I/R, which might be mediated by NMDA receptor and L-VGCC through Src kinase pathway involving oxidative stress in rat hippocampus.

Cerebral ischemia induces rapid efflux of glutamate into the extracellular space contributing to excessive activation of glutamate receptors in postsynaptic cells, particularly N-methyl-D-aspartate (NMDA) receptors, which triggers the neuron lesion through calcium overload. Our studies indicated that cerebral ischemia stimulated the rapid activation of nonreceptor tyrosine kinases proline-rich tyrosine kinase 2 (Pyk2) and Src and the binding to Pyk2 activated the latter. Pyk2 activation significantly depends on the increase of the intracellular calcium level; blockage of both calcium ion channel NMDA receptors and L-type voltage-gated Ca2+ channel (L-VGCC), respectively, could effectively inhibit phosphorylation of Pyk2 in early ischemia episodes. Moreover, pretreatment with the protein kinase C inhibitor (chelerythrine chloride) reduced the ischemia-induced activation of Pyk2. Noticeably, CaMKII, a family of calcium/calmodulin-dependent kinases, also may be involved in the regulation of Pyk2 activity because its inhibitor KN62 attenuated Pyk2 phosphorylation during ischemia. Together with previous studies, these results indicate that calcium influx elicited by active NMDA receptors and L-VGCC triggers the Pyk2-Src signaling pathway mediated by PKC, which aggravates cerebral ischemia lesions through up-regulating the function of NMDA receptors after the onset of ischemia, and also could be regulated partly by CaM-dependent kinases like CaMKII.

Calcium influx and the resulting increase in intracellular calcium concentration [Ca2+]i can induce enhanced sensitivity to temperature increases in nociceptive neurons. Using the patch-clamp technique and simultaneous calcium microfluorimetry we show that experimental elevation of [Ca2+]i using the calcium ionophore ionomycin resulted in a significant potentiation of heat-activated currents. This was not the case when rises in [Ca2+]i were elicited by depolarization of the cell membrane by current injection via the patch pipette. Our data provide first, however, indirect evidence that in sensory neurons calcium ions may be guided into different intracellular microdomains depending on the type of ion channel or pore through which they enter the cell. We conclude that the compartmentalization of sensory neurons for calcium ions may be decisive on further signalling cascades accounting, for example, for neuronal plasticity.

Previous studies on sporadic amyotrophic lateral sclerosis (SALS) have detected IgG within motoneurons of patients and these IgGs have been shown to alter voltage dependent calcium channel activity in various cell types. The current study investigates whether IgG from categorized SALS patients alter voltage dependent calcium currents in rat motoneurons in culture. Patients were categorized based on onset and progression pattern. IgG yields were 38% higher in SALS patients compared to control subjects. Incubation with 1 mg/ml IgG from SALS patients did not cause visible toxicity, alter input resistance, capacitance or the maximal calcium conductance in rat motoneurons when compared to motoneurons incubated with control IgG. However, the activation curve of calcium current was shifted to the left in motoneurons treated with SALS IgG compared to control IgG.

Blockers of L-, N-, and P/Q-type voltage-gated calcium channels (VGCCs) were topically applied to the cortical surface of anaesthetized adult rats to study their role in cortical spreading depression (CSD), a correlate of the migraine aura. By pricking the brain, single CSD could still be elicited after blockade of the three different types of VGCCs as in the untreated brain. Topical KCl application to the untreated cortex resulted in repetitive CSD. However, after application of blockers at either L-, or N-, or P/Q-type VGCCs to the cortical surface, application of KCl elicited only one or very few CSD, and their repetition rate was dramatically reduced. The results suggest that cortical excitability resulting in repetitive CSD is markedly influenced by N- and P/Q-type VGCCs and less by L-type VGCCs.

To investigate the role of P/Q type Ca(2+) channels in determining the firing pattern of Purkinje cells (PCs) we compared the somatically evoked discharge of action potentials (APs) in PCs from 3 to 4 week old cerebellar slice cultures obtained with ataxic mice lacking alpha(1A)-subunit (alpha(-/-)) and with normal mice (non-ataxic alpha(+/-) or alpha(+/+)) using the whole-cell configuration of the patch-clamp recording method. Whereas evoked responses of PCs in normal mice were mainly fast APs, those of PCs from ataxic mice were mainly low-threshold Ca(2+) spikes (LTS). Furthermore, a sustained plateau potential due to the activation of cadmium sensitive Ca(2+) conductances was not observed in PCs from ataxic mice by blocking K(+) channels. These results confirm that P/Q Ca(2+) channels elicit Ca(2+)-dependent plateau potentials and control the propagation of the dendritic LTS to the soma.

Ca2+ channels on growth cones of cultured rat dorsal root ganglion (DRG) neurons were functionally characterized with an optical method using Fura-2. An increase in intracellular calcium concentration ([Ca2+](i)) of the growth cone was induced in response to electrical stimulation to the DRG cell body. The ([Ca2+](i))-increase was partly inhibited by either of omega - conotoxin GVIA (omega - CgTx, 3 microM) or omega - agatoxin IVA (omega - aga IVA, 300 nM) and completely blocked by both present at the same time, but was not affected by nicardipine (30 microM). The omega - CgTx - as well as omega - aga IVA - sensitive Ca2+ channels were immunologically localized on the growth cones using field emission scanning electron microscopy. It is concluded that the omega - CgTx - as well as omea - aga IVA - sensitive Ca2+ channels are involved in the ([Ca2+](i))-increase in the growth cones of the cultured DRG neurons, leading to glutamate release before synapse formation.

Manganese-enhanced magnetic resonance imaging (MEMRI) has been extensively used in studying the structural and functional features of the central nervous system (CNS). Divalent manganese ion (Mn 2+ ) not only enhances MRI contrast, but also enters cells via voltage-gated calcium channels or ionotropic glutamate receptors, which represents an index of neural activities. In the current mouse model, following the repeated amphetamine (Amph) treatment, a reduction of reactivity to thermal pain stimulus was noticed. Since the spinal dorsal horn is the first relay station for pain transmission in CNS, we examined the changes of neural activity in the dorsal spinal cord, particularly the superficial dorsal horn, by analyzing manganese-enhanced T1-weighted MR images (T1WIs). Our data revealed a temporal correlation between reduced pain sensitivity and increased MEMR signals in the spinal dorsal horn subsequent to repeated Amph treatments.

Voltage gated calcium channels (VGCC) are sensitive to oxidative stress, and their activation or inactivation can impact cell death. Although these channels have been extensively studied in expression systems, their role in the brain, particularly in the substantia nigra pars compacta (SNc), remain controversial. In this study, we assessed 6-hydroxydopamine (6-OHDA) induced transformation of firing pattern and functional changes of calcium channels in SNc dopaminergic neurons. Application of 6-OHDA (0.5-2mM) evoked a dose-dependent, desensitizing inward current and intracellular free calcium concentration ([Ca(2+)]i) rise. In voltage clamp, ω-conotoxin-sensitive Ca(2+) current modulation mediated by 6-OHDA reflected an altered sensitivity. Furthermore, we found that 6-OHDA modulated Ca(2+) currents through PKA pathway. These results provided evidence for the potential role of VGCCs and PKA involved in oxidative stress in degeneration of SNc neurons in Parkinson's disease (PD).