Neuronal galvanotropism is independent of external Ca2+ entry or internal Ca2+ gradients

The mechanism by which growing neurites sense and respond to small applied electrical fields is not known, but there is some evidence that the entry of Ca²⁺ from the external medium, with the subsequent formation of intracellular Ca²⁺ gradients, is important in this process. We have employed two approaches to test this idea. Xenopus spinal neurites were exposed to electrical fields in a culture medium in which Ca²⁺ was chelated to very low levels compared to the normal extracellular concentration of 2 mM. In other experiments, loading the neurites with the calcium buffer, 1,2‐bis(o‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid (BAPTA), disrupted the putative internal Ca²⁺ gradients, and the effects on the electrical response were determined. Fields of 100 mV/mm were applied for 12 h, and no difference was detected in the cathodal turning response between the treated neurites and the untreated controls. Using the Differential Growth Index (DGI), an asymmetry index, to quantitate the turning response, we recorded DGIs of −0.64, −0.65, and −0.62 for control cells, cells in Ca²⁺‐free medium, and cells preloaded with BAPTA, respectively. Furthermore, we detected an increase in neurite length for those neurons cultured in Ca²⁺‐free medium; they were 1.5–1.7 times as long as neurites from neurons cultured in normal Ca²⁺ medium. Likewise, we found that BAPTA‐loaded neurites were longer than control neurites. Our data indicate that neuronal galvanotropism is independent of the entry of external Ca²⁺ or of internal Ca²⁺ gradients. Both cell‐permeant agonistic and antagonistic analogs of cyclic 3′,5′‐adenosine monophosphate (cAMP) increased the response to applied electrical fields. © 2000 John Wiley & Sons, Inc. J Neurobiol 45: 30–38, 2000     

Product fermented by <Emphasis Type="Italic">Lactobacilli</Emphasis> induces changes in intracellular calcium dynamics in rat brain neurons

In this work we investigated the effect of concentrated metabolic products of lactobacilli (PP) on the dynamics of intracellular calcium concentration ([Ca²⁺]_i) in rat brain neurons. [Ca²⁺]_i was recorded using a fluorescent probe Fura-2 and a ratiometric Ca²⁺ imaging. It was found that PP increased [Ca²⁺]_i, stimulating the intracellular signaling mechanisms. In these processes the activation of ryanodine receptors and protein kinase C are involved at least partially. Continuous application of PP stimulated a sustained release of Ca²⁺ from the endoplasmic reticulum and subsequent entry of Ca²⁺ into the cell. Given that PP is able to stimulate circulation and neurogenesis and is involved in calcium homeostasis in nerve cells in the brain, PP can be regarded as a product for the improvement of psychological parameters and cognitive functions of the brain.     

Store-Dependent Entry of Ca<Superscript>2+</Superscript> into Sensory Neurons of the Rat

We studied store-dependent (activated by depletion of the endoplasmic reticulum, ER, store) entry of Ca²⁺ from the extracellular medium into neurons of the rat spinal ganglia (small- and medium-sized cells; diameter, 18 to 36 μm). Activation of ryanodine-sensitive receptors of the ER in the studied neurons superfused by Tyrode solutions containing Ca²⁺ or with no Ca²⁺ was provided by application of 10 mM caffeine. The decay phase of caffeine-induced calcium transients in a Ca²⁺-containing solution was significantly longer than that in a Ca²⁺-free solution. This fact allows us to suppose that such a phenomenon is determined by Ca²⁺ entry into the neuron from the extracellular medium activated by caffeine-induced depletion of the ER store. Substitution of Ca²⁺-free extracellular solution by Ca²⁺-containing Tyrode solution, after depletion of the ER stores induced by applications of 100 nM ryanodine, 200 μM ATP, or 1 μM thapsigargin, resulted in increases in the concentration of intracellular Ca²⁺. These observations allow us to postulate that store-dependent Ca²⁺ entry into the studied neurons is activated after depletion not only of the inositol trisphosphate-sensitive ER store but also of the ryanodine-sensitive store. This entry also occurs after blocking of ATPases of the ER by thapsigargin. The kinetic characteristics of the rising phase of store-dependent Ca²⁺ entry induced by depletion of the ER stores under the influence of various agents are dissimilar; this can be related to different mechanisms of activation of such signals and/or to a compartmental organization of the ER. Neirofiziologiya/Neurophysiology, Vol. 37, No. 3, pp. 277–283, May–June, 2005.     

Synaptically Activated Ca<Superscript>2+</Superscript> Release From Internal Stores in CNS Neurons

Synaptically activated postsynaptic [Ca²⁺]_i increases occur through three main pathways: Ca²⁺ entry through voltage-gated Ca²⁺ channels, Ca²⁺ entry through ligand-gated channels, and Ca²⁺ release from internal stores. The first two pathways have been studied intensively; release from stores has been the subject of more recent investigations.Ca²⁺ release from stores in CNS neurons primarily occurs as a result of IP₃ mobilized by activation of metabotropic glutamatergic and/or cholingergic receptors coupled to PLC. Ca²⁺ release is localized near spines in Purkinje cells and occurs as a wave in the primary apical dendrites of pyramidal cells in the hippocampus and cortex. The amplitude of the [Ca²⁺]_i increase can reach several micromolar, significantly larger than the increase due to backpropagating spikes.The large amplitude, long duration, and unique location of the [Ca²⁺]_i increases due to Ca²⁺ release from stores suggests that these increases can affect specific downstream signaling mechanisms in neurons.     

A Na+/Ca2+ exchanger of the olive pathogen Pseudomonas savastanoi pv. savastanoi is critical for its virulence

In a number of compatible plant-bacterium interactions, a rise in apoplastic Ca²⁺ levels is observed, suggesting that Ca²⁺ represents an important environmental clue, as reported for bacteria infecting mammalians. We demonstrate that Ca²⁺ entry in Pseudomonas savastanoi pv. savastanoi (Psav) strain DAPP-PG 722 is mediated by a Na⁺/Ca²⁺ exchanger critical for virulence. Using the fluorescent Ca²⁺ probe Fura 2-AM, we demonstrate that Ca²⁺ enters Psav cells foremost when they experience low levels of energy, a situation mimicking the apoplastic fluid. In fact, Ca²⁺ entry was suppressed in the presence of high concentrations of glucose, fructose, sucrose or adenosine triphosphate (ATP). Since Ca²⁺ entry was inhibited by nifedipine and LiCl, we conclude that the channel for Ca²⁺ entry is a Na⁺/Ca²⁺ exchanger. In silico analysis of the Psav DAPP-PG 722 genome revealed the presence of a single gene coding for a Na⁺/Ca²⁺ exchanger (cneA), which is a widely conserved and ancestral gene within the P. syringae complex based on gene phylogeny. Mutation of cneA compromised not only Ca²⁺ entry, but also compromised the Hypersensitive response (HR) in tobacco leaves and blocked the ability to induce knots in olive stems. The expression of both pathogenicity (hrpL, hrpA and iaaM) and virulence (ptz) genes was reduced in this Psav-cneA mutant. Complementation of the Psav-cneA mutation restored both Ca²⁺ entry and pathogenicity in olive plants, but failed to restore the HR in tobacco leaves. In conclusion, Ca²⁺ entry acts as a ‘host signal’ that allows and promotes Psav pathogenicity on olive plants.     

Mechanisms of heterogeneity of calcium response to kainate and neuronal types in rat cortical primary culture

The dynamics of intracellular Ca²⁺ signal in response to NMDA (N-methyl-D-aspartate, 30 μM) or KA (kainite, 30 μM), its dependence on extracellular Ca²⁺ and the mechanisms of KA-triggered Ca²⁺ entry into neurons have been tested in neurons of rat cortical primary cultures. The level of intracellular free Ca²⁺ concentrations ([Ca²⁺] _i ) was evaluated on Leica SP5 MF confocal microscope using Fluo-3 fluorescent dye, which resolves changes in [Ca²⁺] _i in the micromolar range. The dynamics of [Ca²⁺] _i increase in response to NMDA and KA was different but in both cases the [Ca²⁺] _i increase required the presence of Ca²⁺ in the extracellular solution. The neuronal population was found to be heterogeneous, based on the response to KA applied together with either L-type calcium channel blocker nifedipine (3 μM) or IEM-1460 (3 μM), a blocker of Ca²⁺-permeable AMPAR (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor) lacking GluR2 subunit. Experiments exhibited three types of calcium responses, characteristically belonging to interneurons (expressing Ca²⁺-permeable AMPAR), pyramidal neurons (with AMPAR containing GluR2, making them impermeable to Ca²⁺), and intermediate type of cells expressing both AMPAR types. Thus, we have demonstrated the role of AMPAR and L-type calcium channels in KA-triggered Ca²⁺ entry into neurons. The dynamics of [Ca²⁺] _i during the KA treatment was shown to depend on subunit composition of particular AMPAR subtype expressed in neurons. The data suggest that neuronal types existing in adult cortical tissue are probably presented in primary culture, too.     

Dependence of the resting [Ca2+]cyt of RBL-1 cells on Ca2+ entry

The cytoplasmic Ca²⁺ concentration ([Ca²⁺]_cyt) in resting cells in an equilibrium between several influx and efflux mechanisms. Here we address the question of whether capacitative Ca²⁺ entry to some extent is active at resting conditions and therefore is part of processes that guarantee a constant [Ca²⁺]_cyt. We measured changes of [Ca²⁺]_cyt in RBL-1 cells with fluorometric techniques. An increase of the extracellular [Ca²⁺] from 1.3 mM to 5 mM induced an incrase in [Ca²⁺]_cyt from 105±10 nM to 145±8.5 nM. This increase could be inhibited by 10 μM Gd³⁺, 10 μM La³⁺ or 50 μM 2-aminoethoxydiphenyl borate, blockers of capacitative Ca²⁺ entry. Application of those blockers to a resting cell in a standard extracellular solution (1.3 mM Ca²⁺) resulted in a decrease of [Ca²⁺]_cyt from 105±10 nM to 88.5±10 nM with La³⁺, from 103±12 to 89±12 nM with Gd³⁺ and from 102±12 nM to 89.5±5 nM with 2-aminoethoxydiphenyl borate. From these data, we conclude that capacitative Ca²⁺ entry beside its function in Ca²⁺ signaling contributes to the regulation of resting [Ca²⁺]_cyt.     

Early activation of Ca2+‐permeable AMPA receptors reduces neurite outgrowth in embryonic chick retinal neurons

Calcium entry through Ca²⁺‐permeable AMPA/kainate receptors may activate signaling cascades controlling neuronal development. Using the fluorescent Ca²⁺‐indicator Calcium Green 1‐AM we showed that the application of kainate or AMPA produced an increase of intracellular [Ca²⁺] in embryonic chick retina from day 6 (E6) onwards. This Ca²⁺ increase is due to entry through AMPA‐preferring receptors, because it was blocked by the AMPA receptor antagonist GYKI 52466 but not by the N‐methyl‐D ‐aspartic acid (NMDA) receptor antagonist AP5, the voltage‐gated Ca²⁺ channel blockers diltiazem or nifedipine, or by the substitution of Na⁺ for choline in the extracellular solution to prevent the depolarizing action of kainate and AMPA. In dissociated E8 retinal cultures, application of glutamate, kainate, or AMPA reduced the number of neurites arising from these cells. The effect of kainate was prevented by the AMPA/kainate receptor antagonist CNQX and by GYKI 52466 but not by AP5, indicating that the reduction in neurite outgrowth resulted from the activation of AMPA receptors. Blocking Ca²⁺ influx through L‐type voltage‐gated Ca²⁺ channels with diltiazem and nifedipine prevented the effect of 10–100 μM kainate but not that of 500 μM kainate. In addition, joro spider toxin‐3, a blocker of Ca²⁺‐conducting AMPA receptors, prevented the effect of all doses of kainate. Neither GABA, which is depolarizing at this age in the retina, nor the activation of metabotropic glutamate receptors with tACPD mimicked the effects of AMPA receptor activation. Calcium entry via AMPA receptor channels themselves may therefore be important in the regulation of neurite outgrowth in developing chick retinal cells. © 2001 John Wiley & Sons, Inc. J Neurobiol 49: 200–211, 2001     

The actions of the neonicotinoid imidacloprid on cholinergic neurons of Drosophila melanogaster

The neonicotinoid insecticide imidacloprid is an agonist on insect nicotinic acetylcholine receptors (nAChRs). We utilised fura-2-based calcium imaging to investigate the actions of imidacloprid on cultured GFP-tagged cholinergic neurons from the third instar larvae of the genetic model organism Drosophila melanogaster. We demonstrate dose-dependent increases in intracellular calcium ([Ca²⁺]_i) in cholinergic neurons upon application of imidacloprid (10 nM–100 μM) that are blocked by nAChR antagonists mecamylamine (10 μM) and α-bungarotoxin (α-BTX, 1 μM). When compared to other (untagged) neurons, cholinergic neurons respond to lower concentrations of imidacloprid (10–100 nM) and exhibit larger amplitude responses to higher (1–100 μM) concentrations of imidacloprid. Although imidacloprid acts via nAChRs, increases in [Ca²⁺]_i also involve voltage-gated calcium channels (VGCCs) in both groups of neurons. Thus, we demonstrate that cholinergic neurons express nAChRs that are highly sensitive to imidacloprid, and demonstrate a role for VGCCs in amplifying imidacloprid-induced increases in [Ca²⁺]_i.     

Dual role of ryanodine-sensitive calcium stores in central neurons

The ryanodine-sensitive intracellular Ca²⁺ stores are known to play a major role in excitation-contraction coupling in muscles. Although these stores are also abundantly present in central neurons, their functional role in these cells remains unclear. Using fluorometric digital imaging of the intracellular Ca²⁺ concentration ([Ca²⁺] _i ) in rat hippocampal slices, we investigated the dynamic properties of the ryanodine-sensitive Ca²⁺ stores inCA1 hippocampal pyramidal cells. We found that at rest the ryanodine-sensitive Ca²⁺ stores are functioning predominantly as a “sink” for Ca ions responding to an increase in [Ca²⁺] _i with an increase in the amount of Ca ions accumulated inside the stores. If, however, [Ca²⁺] _i increases significantly, as happens during strong neuronal discharges, the ryanodine-sensitive Ca²⁺ stores respond with Ca²⁺ release, thus acting as an amplifier of the intracellular Ca²⁺ signal.     

Hypoxic Modulation of Ca<Superscript>2+</Superscript> Signaling in Human Venous and Arterial Endothelial Cells

Our understanding of vascular endothelial cell physiology is based on studies of endothelial cells cultured from various vascular beds of different species for varying periods of time. Systematic analysis of the properties of endothelial cells from different parts of the vasculature is lacking. Here, we compare Ca²⁺ homeostasis in primary cultures of endothelial cells from human internal mammary artery and saphenous vein and how this is modified by hypoxia, an inevitable consequence of bypass grafting (2.5% O₂, 24 h). Basal [Ca²⁺] _i and store depletion-mediated Ca²⁺ entry were significantly different between the two cell types, yet agonist (ATP)–mediated mobilization from endoplasmic reticulum stores was similar. Hypoxia potentiated agonist-evoked responses in arterial, but not venous, cells but augmented store depletion-mediated Ca²⁺ entry only in venous cells. Clearly, Ca²⁺ signaling and its remodeling by hypoxia are strikingly different in arterial vs. venous endothelial cells. Our data have important implications for the interpretation of data obtained from endothelial cells of varying sources.     

Cation channels in human embryonic kidney cells mediating calcium entry in response to extracellular low glucose

Glucose sensing mechanism has been intensively studied in pancreatic cells and neurons. Depolarization of membrane potential by closure of K_ATP , Kv and TASK channel, and subsequently Ca²⁺ entry via L-type voltage gated Ca²⁺ channel (VGCC) are implicated to mediate the signal transduction in these cells. However, the mechanism of non-excitable cells, which are lacking VGCC, for sensing glucose remains unclear. In this study, we utilized the calcium ratio measurement and patch clamping technique to study the effects of low glucose on [Ca²⁺]_i and currents in the human embryonic kidney epithelial cells (HEK 293). We found low glucose evoked a significant reversible [Ca²⁺]_i elevation in HEK 293 independent of the closure of Kv channels. This increase of [Ca²⁺]_i was mediated by Ca²⁺ entry across plasma membrane and exhibited a dosage dependent behaviour to external glucose concentration. The low glucose-induced entry of Ca²⁺ was characterized as a voltage independent behaviour and had cation permeability to Na⁺ and Ca²⁺. The modulation of PLC, AMPK, tyrosine kinase and cADPribose failed to regulate this glucose-sensitive Ca²⁺ entry. In addition, the entry of Ca²⁺ was insensitive to nifedipine, 2APB, SKF, La³⁺, Gd³⁺, and KBR9743, suggesting a novel signal pathway in mediating glucose sensing.     

Calcium spikes in a leech nonspiking neuron

The NS neurons are nonspiking cells, present as pairs in each midbody ganglion of the leech nervous system, which display a very extensive arborization. They were shown to regulate the coactivation of motoneurons. Here we have investigated the electrophysiological properties of these neurons under the hypothesis that transmission along the extensive neurites requires the aid of voltage-dependent conductances. The results indicate that NS neurons respond to electrical stimulation with a spike-like event, which was not an all-or-none but rather a graded phenomenon that depended on the intensity and duration of the electrical stimulus. The spike-like response was activated at a membrane potential of approximately −50 mV; its amplitude was a logarithmic function of the extracellular Ca²⁺ concentration and was unaffected by a broad range of changes in the extracellular Na⁺ concentration; intracellular application of tetraethylammonium (TEA) caused a large increase in its amplitude and duration. These data indicate that NS neurons bear voltage-dependent low-threshold Ca²⁺ and TEA-sensitive K⁺ conductances that could contribute to shaping synaptic signals, or transmission along the extensive neuritic tree.     

Fluid Flow Modulates Calcium Entry and Activates Membrane Currents in Cultured Human Aortic Endothelial Cells

Human aortic endothelial cells (HAEC) respond to flow with Ca²⁺ entry, activation of a nonselective cation channel, activation of a chloride channel, and activation of a calcium-activated potassium channel. Conversely, human capillary endothelial cells were unaffected by similar flow rates. In HAEC the flow induced cytosolic free calcium increase ([Ca²⁺] _i ) and the ionic currents associated with it were sustained for up to 15 min after perfusion was stopped. In the absence of extracellular Ca²⁺, fluid flow was unable to evoke the [Ca²⁺] _i increase or the increase in membrane currents but the response could be restored by addition of extracellular Ca²⁺. Surprisingly, the flow response was inhibited in 50% of the cells by inhibitors of nitric oxide production. The results suggest that the sustained flow response in HAEC may be partially mediated by nitric oxide production and release. Received: 29 January 1999/Revised: 2 June 1999     

SEPT7 regulates Ca2+ entry through Orai channels in human neural progenitor cells and neurons

Human neural progenitor cells (hNPCs) are self-renewing cells of neural lineage that can be differentiated into neurons of different subtypes. Here we show that SEPT7, a member of the family of filament-forming GTPases called septins, prevents constitutive Ca²⁺ entry through the store-operated Ca²⁺ entry channel, Orai in hNPCs and in differentiated neurons and is thus required for neuronal calcium homeostasis. Previous work in Drosophila neurons has shown that loss of one copy of the evolutionarily-conserved dSEPT7 gene leads to elevated Ca²⁺ entry via Orai, in the absence of ER-Ca²⁺ store depletion. We have identified an N-terminal polybasic region of SEPT7, known to interact with membrane-localized phospholipids, as essential for spontaneous calcium entry through Orai in hNPCs, whereas the GTPase domain of dSEPT7 is dispensable for this purpose. Re-organisation of Orai1 and the ER-Ca²⁺ sensor STIM1 observed near the plasma membrane in SEPT7 KD hNPCs, supports the idea that Septin7 containing heteromers prevent Ca²⁺ entry through a fraction of STIM-Orai complexes. Possible mechanisms by which SEPT7 reduction leads to opening of Orai channels in the absence of store-depletion are discussed.     

Glutamate- and GABA-mediated neuron–satellite cell interaction in nodose ganglia as revealed by intracellular calcium imaging

In the sensory ganglia, neurons are devoid of synaptic contacts, and ganglion neurons surrounded by one of glial cells, satellite cells. Recent studies suggest that neurons and satellite cells interact through neurotransmitters. In the present study, intracellular Ca²⁺ ([Ca²⁺]_i) dynamics of neurons and satellite cells from one of viscerosensory ganglia, nodose ganglion (NG), were investigated by stimulation with glutamate and its agonist and/or the antagonist of the GABA_A receptor bicuculline. In the specimens containing neurons with satellite cells, glutamate and a metabotropic glutamate receptor (mGluR) agonist t-ACPD evoked [Ca²⁺]_i increases in both neurons and surrounding satellite cells. Moreover, bicuculline also induced [Ca²⁺]_i increases in neurons and satellite cells. However, in the isolated neurons, bicuculline did not cause an increase in [Ca²⁺]_i, suggesting that satellite cells are equipped with the ability to release GABA. In the neurons associated with satellite cells, the delay time until the onset of a response was shorter in the case of glutamate stimulation with bicuculline than that without bicuculline (107.3 ± 93.4 vs. 231.8 ± 97.0 s, p < 0.01). Furthermore, immunoreactivities for glutamate transporter, GLAST, and GABA transporter, GAT-3, were observed in both neurons and satellite cells of NG. In conclusion, the levels of [Ca²⁺]_i of NG neurons and surrounding satellite cells are increased by glutamate through at least mGluRs, and endogenous GABA modulates these responses; GABA inhibition is dependent on a close association between neurons and satellite cells. Such neuron–glia interaction in the nodose ganglion may regulate sensory information from visceral organs.     

Peimine inhibits the growth and motility of prostate cancer cells and induces apoptosis by disruption of intracellular calcium homeostasis through Ca2+/CaMKII/JNK pathway

Prostate cancer (PC) is one of the most common malignant tumors in man. Peimine (PM) is a bioactive substance isolated from Fritillaria. Previous studies have shown that PM could inhibit the occurrence of a variety of cancers. However, the roles of PM in PC and its related mechanism have not been elucidated. Calcium (Ca²⁺) is an important intracellular messenger involved in a variety of cell processes. In this study, we found that the appropriate doses of PM (2.5, 5, and 10 μM) significantly inhibited the growth of PC cells (DU-145, LNCap, and PC-3), but has no significant effect on normal prostate cells (RWPE-1). In addition, PM treatment inhibited the invasion and migration of PC-3 cells and blocked the epithelial-mesenchymal transition process. These effects were exhibited a dose-dependent manner. Furthermore, the current results also showed that PM treatment significantly increased the Ca²⁺ concentration, the increased Ca²⁺ promoted the phosphorylation of Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) and c-Jun N-terminal kinase (JNK), further inhibited the growth and invasion of PC-3 cells, and induced its apoptosis. Ca²⁺ chelator BAPTA-AM (1 μM) could counteract the increase of intracellular Ca²⁺ concentration. Similarly, JNK pathway inhibitor SP600125 (10 μM) also inhibited cell growth and invasion and induced apoptosis. In addition, experiments in nude mice showed that PM inhibited tumor formation through Ca²⁺/CaMKII/JNK signaling pathway. In conclusion, our results show that PM inhibits the growth and motility of prostate cancer cells and induces apoptosis by disruption of intracellular calcium homeostasis through Ca²⁺/CaMKII/JNK pathway.     

A two-step model for acetylcholine control of exocytosis via nicotinic receptors

The view that Ca²⁺ entry through voltage-dependent Ca²⁺ channels (VDCC) and through nicotinic receptors for acetylcholine (nAChRs) causes equal catecholamine release responses in chromaffin cells, was reinvestigated here using new protocols. We have made two-step experiments consisting in an ACh prepulse followed by a depolarizing pulse (DP). In voltage-clamped bovine chromaffin cells an ACh prepulse caused a slow-rate release but augmented 4.5-fold the much faster exocytotic response triggered by a subsequent depolarizing pulse (measured with capacitance and amperometry). If the ACh prepulse was given with mecamylamine or in low external Ca²⁺, the secretion increase disappeared. This suggests a two-step model for the effects of ACh: (1) meager Ca²⁺ entry through nAChRs mostly serves to keep loaded with vesicles the secretory machine; and (2) in this manner, the cell is prepared to respond with an explosive secretion of catecholamine upon depolarization and fast high Ca²⁺ entry through VDCC.     

TRPV1 expression and activity during retinoic acid-induced neuronal differentiation

The transient receptor potential vanilloid subtype 1 (TRPV1) is a Ca²⁺-permeable channel primarily expressed in dorsal root ganglion neurons. Besides its function in thermogenic nociception and neurogenic inflammation, TRPV1 is involved in cell migration, cytoskeleton re-organisation and in neuronal guidance. To explore the TRPV1 level and activity during conditions for neuronal maturation, TRPV1-expressing SHSY5Y neuroblastoma cells were differentiated into a neuronal phenotype using all-trans-retinoic acid (RA). We show that RA highly up-regulated the total and cell surface TRPV1 protein expression but the TRPV1 mRNA level was unaffected. The up-regulated receptors were localised to the cell bodies and the developed neurites. Furthermore, RA increased both the basal intracellular free Ca²⁺ concentration by 30% as well as the relative capsaicin-induced Ca²⁺ influx. The results show that TRPV1 protein expression increases during RA-induced differentiation in vitro, which generates an altered intracellular Ca²⁺ homeostasis.     

Synaptic NMDA Receptor-Dependent Ca2+ Entry Drives Membrane Potential and Ca2+ Oscillations in Spinal Ventral Horn Neurons

During vertebrate locomotion, spinal neurons act as oscillators when initiated by glutamate release from descending systems. Activation of NMDA receptors initiates Ca²⁺-mediated intrinsic membrane potential oscillations in central pattern generator (CPG) neurons. NMDA receptor-dependent intrinsic oscillations require Ca²⁺-dependent K⁺ (K_Ca2) channels for burst termination. However, the location of Ca²⁺ entry mediating K_Ca2 channel activation, and type of Ca²⁺ channel – which includes NMDA receptors and voltage-gated Ca²⁺ channels (VGCCs) – remains elusive. NMDA receptor-dependent Ca²⁺ entry necessitates presynaptic release of glutamate, implying a location at active synapses within dendrites, whereas VGCC-dependent Ca²⁺ entry is not similarly constrained. Where Ca²⁺ enters relative to K_Ca2 channels is crucial to information processing of synaptic inputs necessary to coordinate locomotion. We demonstrate that Ca²⁺ permeating NMDA receptors is the dominant source of Ca²⁺ during NMDA-dependent oscillations in lamprey spinal neurons. This Ca²⁺ entry is synaptically located, NMDA receptor-dependent, and sufficient to activate K_Ca2 channels at excitatory interneuron synapses onto other CPG neurons. Selective blockade of VGCCs reduces whole-cell Ca²⁺ entry but leaves membrane potential and Ca²⁺ oscillations unaffected. Furthermore, repetitive oscillations are prevented by fast, but not slow, Ca²⁺ chelation. Taken together, these results demonstrate that K_Ca2 channels are closely located to NMDA receptor-dependent Ca²⁺ entry. The close spatial relationship between NMDA receptors and K_Ca2 channels provides an intrinsic mechanism whereby synaptic excitation both excites and subsequently inhibits ventral horn neurons of the spinal motor system. This places the components necessary for oscillation generation, and hence locomotion, at glutamatergic synapses.