Role of mitochondria in intracellular calcium signaling in primary and secondary sensory neurones of rats

The participation of different calcium-regulated mechanisms in the generation of cytosolic Ca(2+) transients during neuronal excitation has been compared in isolated large and small primary (dorsal root ganglia (DRG)) and secondary (spinal dorsal horn (DH)) rat sensory neurones. As it was shown before in murine primary sensory neurones the application of mitochondrial protonophore CCCP by itself induced only small elevation of [Ca(2+)](i). However, its preceding application substantially increased the peak amplitude of depolarization-induced transients. Application of CCCP immediately after termination of the depolarizing pulse induced in both types of primary neurones a massive release of Ca(2+) from mitochondria into the cytosol. In secondary neurones application of CCCP by itself induced a substantial release of Ca(2+) from the mitochondria, but its preceding application resulted in only an insignificant increase in the peak amplitude of depolarization-triggered calcium transients. Application of CCCP immediately after termination of depolarization elicited a small release of Ca(2+), which became more pronounced when the application was delayed. Preceding application of CCCP increased the amplitude of the transients induced by caffeine-triggered Ca(2+) release from the endoplasmic reticulum in secondary neurones and did not affect those in large primary neurones. These findings may be explained by substantial differences in the density and distribution of mitochondria in the cytosol of primary and secondary sensory neurones. This suggestion was confirmed electronmicroscopically, showing a much lower density of mitochondria near plasmalemma in secondary sensory neurones and predominant clustered location of mitochondria beneath the plasmalemma in the primary cells. The possible functional importance of these differences is discussed.     

Intracellular Calcium Homeostasis Changes Induced in Rat Spinal Cord Neurons by Extracellular Acidification

Changes in intracellular Ca²⁺ induced by extracellular acidification to pH = 6 were studied in isolated rat spinal dorsal horn neurons using indo-1 fluorescent technique. In all neurons such treatment induced a decrease of basal [Ca²⁺]_i level by 20.8%, preceded in some of them by temporary increase. The changes were completely reversible. The depolarization-induced [Ca²⁺]_i transients became strongly and also reversibly depressed. If tested after termination of acidification, they demonstrated substantial prolongation of their decay phase, reaching 310% at 120 sec after the application of depolarization. To analyze the mechanisms of such changes, mitochondrial protonophore CCCP has been applied between the end of acidification and the depolarizing pulse. This completely eliminated the described slowing of the transients' decay. To the contrary, application of caffeine to induce Ca²⁺ release from the endoplasmic reticulum did not show significant changes in the corresponding [Ca²⁺]_i transients. A conclusion is made that in mammalian neurons extracellular acidification, apart from inhibiting voltage-operated Ca²⁺ channels, also substantially alters the Ca²⁺ exchange function of mitochondria responsible for rapid accumulation of ions and their delayed release back into the cytosol.     

Role of Ca2+,Mg2+-ATPases in Diabetes-Induced Alterations in Calcium Homeostasis in Input Neurons of the Nociceptive System

In a rat model of streptozotocin (STZ)-induced diabetes, we earlier showed that under these conditions the concentration of free cytosolic Ca²⁺ in input neurons of the nociceptive system increases, Ca²⁺ signals are prolonged, while Ca²⁺ release from intracellular calcium stores decreases. The aim of our study was to test the hypothesis that changes in the activities of Ca²⁺,Mg²⁺-ATPases of the endoplasmic reticulum (SERCA) and plasmalemma (PMCA) could be responsible for diabetes-induced disorders of calcium homeostasis in nociceptive neurons. We measured the Ca²⁺,Mg²⁺-ATPase activities in microsomal fractions obtained from tissues of the dorsal root ganglia (DRG) and spinal dorsal horn (DH) of control rats and rats with experimentally induced diabetes. The integral specific Ca²⁺,Mg²⁺-ATPase activity in microsomes from diabetic rats was lower than that in the control group. The activity of SERCA in samples of DRG and DH of diabetic rats was reduced by 50 ± 8 and 48 ± 12%, respectively, as compared with the control (P < 0.01). At the same time, the activity of PMCA decreased by 63 ± 6% in DRG and by 60 ± 9% in DH samples (P < 0.01). We conclude that diabetic polyneuropathy is associated with the reduction of the rate of recovery of the Ca²⁺ level in the cytosol of DRG and DH neurons due to down-regulation of the SERCA and PMCA activities.     

Calcium Release From the Internal Stores as a Possible Target for Antinociceptive Treatment in Rats with Experimentally-Induced Diabetes

Distal neuropathy is the most common complication of diabetes mellitus, and it is highly important to reveal the cellular mechanisms leading to its development. In our experiments, neurons of control and streptozotocin-treated diabetic rats were examined. Changes in the intracellular free calcium concentrations ([Ca²⁺] _i ) were fluorometrically measured in primary and secondary nociceptive (dorsal root ganglion, DRG, and dorsal horn, DH, respectively) neurons. The [Ca²⁺] _i elevation was induced by different agents, which can release calcium from the endoplasmic reticulum (ER) calcium stores. The amplitudes of calcium elevation induced by application of caffeine and ionomicine in DRG and DH neurons of diabetic rats were significantly lower, as compared with the control. Application of ATP and glutamate to a Ca-free extracellular solution induced calcium release from the IP₃-sensitive store in DH neurons. Release of calcium from the IP₃-sensitive ER calcium stores became significantly smaller in neurons from diabetic rats. Taken together, these data indicate that significant changes in the calcium regulating mechanisms of the ER develop under diabetes conditions.     

Properties of the caffeine-sensitive intracellular calcium stores in mammalian neurons

Using indo-1- and fura-2-based microfluorometry for measuring the cytoplasmic free calcium concentration ([Ca²⁺] _in ), the properties of caffeine-induced Ca²⁺ release from internal stores were studied in rat cultured central and peripheral neurons, including dorsal root ganglion (DRG) neurons, neurons from then. cuneatus, CA1 and CA3 hippocampal regions, and pyramidal neocortical neurons. Under resting conditions, the Ca²⁺ content of internal stores in DRG neurons was high enough to produce caffeine-triggered [Ca²⁺] _in transients. Prolonged exposure of caffeine depleted the caffeine-sensitive stores of releasable Ca²⁺; the degree of this depletion depended on caffeine concentration. The depletion of the caffeine-sensitive internal stores to some extent was linked to calcium extrusion via La³⁺-sensitive plasmalemmal Ca²⁺-ATPases. Caffeine-induced Ca²⁺ release deprived internal stores in DRG neurons, but they refilled themselves spontaneously within 10 min. Pharmacological manipulation with caffeine-sensitive stores interferred with the depolarization-induced [Ca²⁺] _in transients. In the presence of low caffeine concentration (0.5–1.0 mM) in the extracellular solution, the rate of rise of the depolarization-triggered [Ca²⁺] _in transients significantly increased (by a factor of 2.15 ± 0.29) suggesting the occurrence of Ca²⁺-induced Ca²⁺ release. When the caffeine-sensitive stores were emptied by prolonged application of caffeine, the amplitude and rate of rise of the depolarization-induced [Ca²⁺] _in transients decreased. These findings suggest the involvement of internal caffeine-sensitive calcium stores in generation of calcium signal in sensory neurons. In contrast, in all types of central neurons tested the resting Ca²⁺ content of internal stores was low, but the stores could be charged by transmembrane Ca²⁺ entry through voltage-operated calcium channels. After charging, the stores in central neurons spontaneously lost releasable calcium content and within 10 min they became completely empty again. We suggest that internal Ca²⁺ stores in peripheral and central neurons, although having similar pharmacological characteristics, handle Ca²⁺ ions in a different manner. Calcium stores in sensory neurons are continuously filled by releasable calcium and after discharging they can be spontaneously refilled, whereas in central neurons internal calcium stores can be charged by releasable calcium only transiently. Caffeine-evoked [Ca²⁺] _in transients in all types of neurons were effectively blocked by 10 mM ryanodine, 5 mM procaine, 10 mM dantrolene, or 0.5 mM Ba²⁺, thus sharing the basic properties of the Ca²⁺-induced Ca²⁺ release from endoplasmic reticulum.Neirofiziologiya/Neurophysiology, Vol. 26, No. 1, pp. 16–25, January–February, 1994.     

Calcium signalling in cortical and thalamic neurons of rats with streptozotocin-induced diabetes

Intracellular Ca²⁺ transients were measured with the use of a Ca²⁺-sensitive fluorescent indicator, fura-2, in neocortical and thalamic neurons in brain slices from control rats and rats with uncompensated streptozotocin-induced diabetes. The transients were evoked by high-potassium (50 mM)-induced membrane depolarization. The amplitude of depolarization-induced Ca²⁺ transients demonstrated a tendency to increase under diabetic conditions, beeing more expressed in cortical neurons compared with thalamic ones. The transients in cortical neurons from diabetic animals became also more susceptible to the blocking action of nifedipine (100μM) and less sensitive to Ni²⁺ (50μM), indicating that diabetic changes affect mostly Ca²⁺ transients triggered by high-voltage activated (L-type) calcium channels. The duration of a statistically significant increase was observed in the residual elevation of intracellular Ca²⁺ changes. However, a statistically significant increase was observed in the residual elevation of intracellular Ca²⁺ measured 60 sec after termination of membrane depolarization in both cortical and thalamic neurons, indicating alterations in the mechanisms that restore the resting level of Ca²⁺ in the cytosol. It is concluded that uncomensated insulin-dependent diabetes, which according to earlier data substantially alters calcium signalling in primary sensory neurons, also affects such signalling in the neurons of higher brain structures including the thalamus and cortex.     

Spinorphin inhibits membrane depolarization- and capsaicin-induced intracellular calcium signals in rat primary nociceptive dorsal root ganglion neurons in culture

Abstract

Objective: Spinorphin is a potential endogenous antinociceptive agent although the mechanism(s) of its analgesic effect remain unknown. We conducted this study to investigate, by considering intracellular calcium concentrations as a key signal for nociceptive transmission, the effects of spinorphin on cytoplasmic Ca²⁺ ([Ca²⁺]_i) transients, evoked by high-K⁺ (30?mM) depolariasation or capsaicin, and to determine whether there were any differences in the effects of spinorphin among subpopulation of cultured rat dorsal root ganglion (DRG) neurons. Methods: DRG neurons were cultured on glass coverslips following enzymatic digestion and mechanical agitation, and loaded with the calcium sensitive dye fura-2 AM (1?µM). Intracellular calcium responses in individual DRG neurons were quantified using standard fura-2 based ratiometric calcium imaging technique. All data were analyzed by using unpaired t test, p?<?0.05 defining statistical significance. Results: Here we found that spinorphin inhibited cytoplasmic Ca²⁺ ([Ca²⁺]_i) transients, evoked by depolarization and capsaicin selectively in medium and small cultured rat DRG neurons. Spinorphin (10–300?µM) inhibited the Ca²⁺ signals in concentration dependant manner in small- and medium diameter DRG neurons. Capsaicin produced [Ca²⁺]_i responses only in small- and medium-sized DRG neurons, and pre-treatment with spinorphin significantly attenuated these [Ca²⁺]_i responses. Conclusion: Results from this study indicates that spinorphin significantly inhibits [Ca²⁺]_i signaling, which are key for the modulation of cell membrane excitability and neurotransmitter release, preferably in nociceptive subtypes of this primary sensory neurons suggesting that peripheral site is involved in the pain modulating effect of this endogenous agent.     

Ryanodine inhibits calcium transients evoked by action potentials in a subpopulation of rat dorsal root ganglion neurons

Effects of ryanodine on calcium transients evoked by depolarization of external membrane under voltage clamp conditions or by a train of action potentials under current clamp conditions were studied on isolated dorsal root ganglion neurons of newborn rats. In 70% neurons tested, ryanodine, a blocker of Ca²⁺-induced Ca²⁺ release from endoplasmic reticulum, significantly decreased the amplitude of calcium transients. The data obtained indicate that the Ca²⁺-induced Ca²⁺ release plays an important role for calcium signal generation in a subpopulation of sensory neurons.Neirofiziologiya/Neurophysiology, Vol. 26, No. 6, pp. 420–422, November–December, 1994.     

Inhibition of ATP-Induced Ca<Superscript>2+</Superscript> Influx by Corticosterone in Dorsal Root Ganglion Neurons

In addition to the classic genomic effects, it is well known that glucocorticoids also have rapid, nongenomic effects on neurons. In the present study, the effect of corticosterone (CORT) on ATP-induced Ca²⁺ mobilization in cultured dorsal root ganglion (DRG) neurons were detected with confocal laser scanning microscopy using fluo-4/AM as a calcium fluorescent indicator that could monitor real-time alterations of intracellular calcium concentration ([Ca²⁺]i). ATP, an algesic agent, caused [Ca²⁺]i increase in DRG neurons by activation of P2X receptor. Pretreatment with CORT (1 nM–1 μM for 5 min) inhibited ATP-induced [Ca²⁺]i increase in DRG neurons. The rapid inhibition of ATP-induced Ca²⁺ response by CORT was concentration-dependent, reversible and could be blocked by glucocorticoid receptor antagonist RU38486 (10 μM). Furthermore, the inhibitory effect of CORT was abolished by protein kinase A inhibitor H89 (10 μM), but was not influenced by protein kinase C inhibitor Chelerythrine chloride (10 μM). On the other hand, membrane-impermeable bovine serum albumin-conjugated corticosterone had no effect on ATP-induced [Ca²⁺]i transients. These observations suggest that a nongenomic pathways may be involved in the effect of CORT on ATP-induced [Ca²⁺]i transients in cultured DRG neurons.     

Intracellular calcium mobilization and neurite outgrowth in mammalian neurons

Cultured adult rat dorsal root ganglion (DRG) neurons were used to study depolarization-induced Ca²⁺ mobilization and the effects of intracellular Ca²⁺ depletion on neurite outgrowth. Cytoplasmic and nuclear Ca²⁺ signals were visualized in dissociated DRG neurons using confocal scanning laser microspcopy and the Ca²⁺ indicator dye fluo-3. The depolarization-induced Ca²⁺ signals were highest in neurons during the first few days in culture, prior to neurite extension; during this time nuclear signals exceeded those of the cytoplasm severalfold. After several days in culture, neurons began to arborize, depolarization-induced Ca²⁺ signals became attenuated, and nuclear signals no longer exceeded those of the cytoplasm. Elevated Ca²⁺ signals were dependent upon both Ca²⁺ influx and intact intracellular Ca²⁺ stores, indicating that the signals are generated by calcuim-induced calcium release (CICR). Thapsigargin, an endoplasmic reticulum Ca²⁺ ATPase inhibitor, depleted intracellular Ca²⁺ stores and blocked the induction of the large nuclear Ca²⁺ signals. Treating DRG neurons briefly with thapsigargin (200 nM for 20 min) shortly after plating reduced subsequent neuritogenesis, impyling that intact Ca²⁺ stores are necessery for initiating neurite outgrowth. Immunostaining of DRG neurons with antibodies to Ca²⁺ /calmodulin-dependent kinase II (CaM kinase II) demonstrated that this enzyme is present in the nucleus at early times in culture. These observations are consistent with the idea that CICR triggered by Ca²⁺ entry subsequent to depolarization may elicit neurite outgrowth by activating nuclear enzymes appropriate for such outgrowth. © 1994 John Wile & Sons, Inc.     

Involvement of mitochondria in intracellular calcium sequestration by rat gonadotropes

Intracellular Ca²⁺ ([Ca²⁺]_i) dynamics were studied in identified rat gonadotropes using the whole-cell patch-clamp technique in conjunction with Indo-1 photometry. The kinetics of depolarization-induced [Ca²⁺]_i transients vary with Ca²⁺ load. In addition to a rapid initial decay, large (> 500 nM) [Ca²⁺]_i transients have a slow plateau phase. Application of the mitochondrial inhibitor carbonyl cyanide m-chlorophenylhydrazone (CCCP) significantly slows the decay of [Ca²⁺]_i transients, consistent with stopping uptake of Ca²⁺ by mitochondria. CCCP causes a small increase of [Ca²⁺]_i at rest. After a large Ca²⁺ entry the amount is much larger, consistent with release from a mitochondrial Ca²⁺ pool that fills during cytoplasmic Ca²⁺ loading. The rate of Ca²⁺ uptake by mitochondria is dependent upon [Ca ²⁺]_i. Consistent with previous studies, gonadotropin releasing hormone (GnRH) induces [Ca²⁺]_i oscillations. The mitochondrial inhibitors CCCP and cyanide (CN⁻) terminate these oscillations. The mitochondrial ATP-synthase inhibitor oligomycin reduces the frequency and increases the amplitude of the oscillations. In the presence of ruthenium red (a non-specific blocker of the mitochondrial Ca²⁺-uniporter) in the pipette, GnRH does not induce rhythmic [Ca²⁺]_i oscillations. We suggest that mitochondria play a significant role in the rapid clearance of cytosolic Ca²⁺ loads in gonadotropes and participate in GnRH-induced periodic [Ca²⁺]_i oscillations.     

Chemical signaling from colonic smooth muscle cells to DRG neurons in culture

Transductionmechanisms between target cells within the intestinal wall andperipheral terminals of extrinsic primary afferent neurons are poorlyunderstood. The purpose of this study was to characterize theinteractions between smooth muscle cells from the rat distal colon andlumbar dorsal root ganglion (DRG) neurons in coculture. DRG neuronsvisually appeared to make contact with several myocytes. We show thatbrief mechanical stimulation of these myocytes resulted inintracellular Ca²⁺ concentration([Ca²⁺]_i)transients that propagated into 57% of the contacting neurites. Directmechanical stimulation of DRG neurites cultured without smooth musclehad no effect. We also show that colonic smooth muscle cells expressmultiple connexin mRNAs and that these connexins formed functional gapjunctions, as evidenced by the intercellular transfer of Luciferyellow. Furthermore, thapsigargin pretreatment and neuronal heparininjection abolished the increase in neurite [Ca²⁺]_i,indicating that the neuronal Ca²⁺signal was triggered by inositol 1,4,5-trisphosphate-mediated Ca²⁺ release from intracellularstores. Our results provide evidence for intercellular chemicalcommunication between DRG neurites and intestinal smooth muscle cellsthat mediates the exchange of second messenger molecules betweendifferent cell types.     

Effects of levobupivacaine and bupivacaine on intracellular calcium signaling in cultured rat dorsal root ganglion neurons

Bupivacaine and levobupivacaine have been shown to be effective in the treatment of pain as local anesthetics, although the mechanisms mediating their antinociceptive actions are still not well understood. The aim of this study was to investigate the effects of bupivacaine and levobupivacaine on intracellular calcium ([Ca²⁺]_i) signaling in cultured rat dorsal root ganglion (DRG) neurons. DRG neuronal cultures loaded with 5?μM Fura-2/AM and [Ca²⁺]_i transients for stimulation with 30?mM KCl (Hi K⁺) were assessed by using fluorescent ratiometry. DRGs were excited at 340 and 380?nm, emission was recorded at 510?nm, and responses were determined from the change in the 340/380 ratio (basal-peak) for individual DRG neurons. Data were analyzed by using Student’s t-test. Levobupivacaine and bupivacaine attenuated the KCl-evoked [Ca²⁺]_i transients in a reversible manner. [Ca²⁺]_i increase evoked by Hi K⁺ was significantly reduced to 99.9?±?5.1% (n?=?18) and 62.5?±?4.2% (n?=?15, P?<?0.05) after the application of 5 and 50?µM levobupivacaine, respectively. Bupivacaine also inhibited Hi K⁺-induced [Ca²⁺]_i responses, reduced to 98.7?±?4.8% (n?=?10) and 69.5?±?4.5% (n?=?9, P?<?0.05) inhibition of fluorescence ratio values of Hi K⁺-induced responses at 5 and 50?μM, respectively. Our results indicate that bupivacaine and levobupivacaine, with no significant differences between both agents, attenuated KCl-evoked calcium transients in a reversible manner. The inhibition of calcium signals in DRG neurons by levobupivacaine and bupivacaine might contribute to the antinociceptive effects of these local anesthetics.     

The effects of mitochondrial inhibitors on Ca2+ signalling and electrical conductances required for pacemaking in interstitial cells of Cajal in the mouse small intestine

Interstitial cells of Cajal (ICC-MY) are pacemakers that generate and propagate electrical slow waves in gastrointestinal (GI) muscles. Slow waves appear to be generated by the release of Ca²⁺ from intracellular stores and activation of Ca²⁺-activated Cl⁻ channels (Ano1). Conduction of slow waves to smooth muscle cells coordinates rhythmic contractions. Mitochondrial Ca²⁺ handling is currently thought to be critical for ICC pacemaking. Protonophores, inhibitors of the electron transport chain (FCCP, CCCP or antimycin) or mitochondrial Na⁺/Ca²⁺ exchange blockers inhibited slow waves in several GI muscles. Here we utilized Ca²⁺ imaging of ICC in small intestinal muscles in situ to determine the effects of mitochondrial drugs on Ca²⁺ transients in ICC. Muscles were obtained from mice expressing a genetically encoded Ca²⁺ indicator (GCaMP3) in ICC. FCCP, CCCP, antimycin, a uniporter blocker, Ru360, and a mitochondrial Na⁺/Ca²⁺ exchange inhibitor, CGP-37157 inhibited Ca²⁺ transients in ICC-MY. Effects were not due to depletion of ATP, as oligomycin did not affect Ca²⁺ transients. Patch-clamp experiments were performed to test the effects of the mitochondrial drugs on key pacemaker conductances, Ano1 and T-type Ca²⁺ (Ca_V3.2), in HEK293 cells. Antimycin blocked Ano1 and reduced Ca_V3.2 currents. CCCP blocked Ca_V3.2 current but did not affect Ano1 current. Ano1 and Cav3.2 currents were inhibited by CGP-37157. Inhibitory effects of mitochondrial drugs on slow waves and Ca²⁺ signalling in ICC can be explained by direct antagonism of key pacemaker conductances in ICC that generate and propagate slow waves. A direct obligatory role for mitochondria in pacemaker activity is therefore questionable.     

Voltage-Induced Ca2+ Release in Postganglionic Sympathetic Neurons in Adult Mice

Recent studies have provided evidence that depolarization in the absence of extracellular Ca²⁺ can trigger Ca²⁺ release from internal stores in a variety of neuron subtypes. Here we examine whether postganglionic sympathetic neurons are able to mobilize Ca²⁺ from intracellular stores in response to depolarization, independent of Ca²⁺ influx. We measured changes in cytosolic ΔF/F₀ in individual fluo-4 –loaded sympathetic ganglion neurons in response to maintained K⁺ depolarization in the presence (2 mM) and absence of extracellular Ca²⁺ ([Ca²⁺]_e). Progressive elevations in extracellular [K⁺]_e caused increasing membrane depolarizations that were of similar magnitude in 0 and 2 mM [Ca²⁺]_e. Peak amplitude of ΔF/F₀ transients in 2 mM [Ca²⁺]_e increased in a linear fashion as the membrane become more depolarized. Peak elevations of ΔF/F₀ in 0 mM [Ca²⁺]_e were ~5–10% of those evoked at the same membrane potential in 2 mM [Ca²⁺]_e and exhibited an inverse U-shaped dependence on voltage. Both the rise and decay of ΔF/F₀ transients in 0 mM [Ca²⁺]_e were slower than those of ΔF/F₀ transients evoked in 2 mM [Ca²⁺]_e. Rises in ΔF/F₀ evoked by high [K⁺]_e in the absence of extracellular Ca²⁺ were blocked by thapsigargin, an inhibitor of endoplasmic reticulum Ca²⁺ ATPase, or the inositol 1,4,5-triphosphate (IP₃) receptor antagonists 2-aminoethoxydiphenyl borate and xestospongin C, but not by extracellular Cd²⁺, the dihydropyridine antagonist nifedipine, or by ryanodine at concentrations that caused depletion of ryanodine-sensitive Ca²⁺ stores. These results support the notion that postganglionic sympathetic neurons possess the ability to release Ca²⁺ from IP₃-sensitive internal stores in response to membrane depolarization, independent of Ca²⁺ influx.     

The role of parvalbumin-containing interneurons in the regulation of spontaneous synchronous activity of brain neurons in culture

Subtypes of inhibitory GABAergic neurons containing Ca²⁺-binding proteins play a pivotal role in the regulation of spontaneous synchronous [Ca²⁺]_i transients in a neuronal network. In this study it is shown that: (1) the interneurons that containing Ca²⁺-binding proteins at buffer concentration can be identified by the shape of Ca²⁺-signa1 in response to depolarization or activation of ionotropic glutamate receptors; (2) Ca²⁺-binding proteins are involved in desynchronization of spontaneous Ca²⁺ transients. At low frequencies of spontaneous synchronous [Ca²⁺]_i transients (less than 0.2 Hz) neurons show quasi-synchronous pulsations. At higher frequencies, synchronization of spontaneous synchronous [Ca²⁺]_i transients occurs in all neurons; (3) it is established that several synchronous oscillations with different frequencies coexist in the network and the amplitude of their depolarizing pulse also varies. This phenomenon is apparently the mechanism that selectively directs information in separate neurons using the same network; and (4) in one population of interneurons at high frequencies of spontaneous synchronous [Ca²⁺]_i transients the inversion of Cl^– concentration gradient is observed. In this case, the inhibition of GABA(A) receptors suppresses the activity of neurons in this population and excites other neurons in the network. Thus, the GABAergic neurons that contain Ca-binding proteins show different mechanisms to regulate the synchronous neuronal activities in cultured rat hippocampal cells.     

Calcium Signaling in <Emphasis Type="Italic">Carassius</Emphasis> Cerebellar Neurons: Role of the Mitochondria

We studied the involvement of the mitochondria playing the role of a calcium store in the control of calcium exchange in cerebellar neurons of a fish species tolerant to hypoxia, crucian (Carassius gibelio). In our experiments we used an ionophore, CCCP, that blocked accumulation of calcium by the above organelles. The intracellular concentration of free Ca²⁺ ([Ca²⁺] _і ) was measured using a calcium-sensitive dye, Fura-2AM, and the microfluorescent technique. We found that cerebellar neurons of Carassius gibelio possess a well-expressed system clearing the cytoplasm from excessive Ca²⁺, and the mitochondria are actively involved in this process. Under conditions of suppression of the process of accumulation of calcium by the mitochondria under the action of CCCP, the amplitude of calcium transients increased by about 50%. In addition, the decay phase of depolarization-induced intracellular calcium transients was slowed down considerably. Therefore, our experiments are indicative of the significant role of the mitochondria in the control of calcium dynamics in cerebellar neurons of Carassius gibelio in the course of functional activity of these cells.     

CB<Subscript>1</Subscript> Receptors Mediated Inhibition of ATP-Induced [Ca<Superscript>2+</Superscript>]i Increase in Cultured Rat Spinal Dorsal Horn Neurons

Spinal cannabinoid receptor 1 (CB₁R) and purinergic P2X receptors (P2XR) play a critical role in the process of pathological pain. Both CB₁R and P2XR are expressed in spinal dorsal horn (DH) neurons. It is not clear whether CB₁ receptor activation modulates the function of P2X receptor channels within dorsal horn. For this reason, we observed the effect of CP55940 (cannabinoid receptor agonist) on ATP-induced Ca²⁺ mobilization in cultured rat DH neurons. The changes of intracellular calcium concentration ([Ca²⁺]i) were detected with confocal laser scanning microscopy using fluo-4/AM as a calcium fluorescent indicator. 100 μM ATP caused [Ca²⁺]i increase in cultured DH neurons. ATP-evoked [Ca²⁺]i increase in DH neurons was blocked by chelating extracellular Ca²⁺ and P2 purinoceptor antagonist PPADS. At the same time, ATP-γ-S (a non-hydrolyzable ATP analogue) mimicked the ATP action, while P2Y receptor agonist ADP failed to evoke [Ca²⁺]i increase in cultured DH neurons. These data suggest that ATP-induced [Ca²⁺]i elevation in cultured DH neurons is mediated by P2X receptor. Subsequently, we noticed that, in cultured rat DH neurons, ATP-induced Ca²⁺ mobilization was inhibited after pretreated with CP55940 with a concentration-dependent manner, which implies that the opening of P2X receptor channels are down-regulated by activation of cannabinoid receptor. The inhibitory effect of CP55940 on ATP-induced Ca²⁺ response was mimicked by ACEA (CB₁R agonist), but was not influenced by AM1241 (CB₂R agonist). Moreover, the inhibitory effect of CP55940 on ATP-induced Ca²⁺ mobilization was blocked by AM251 (CB₁ receptor antagonist), but was not influenced by AM630 (CB₂ receptor antagonist). In addition, we also observed that forskolin (an activator of adenylate cyclase) and 8-Br-cAMP (a cell-permeable cAMP analog) reversed the inhibitory effect of CP55940, respectively. In a summary, our observations raise a possibility that CB₁R rather than CB₂R can downregulate the opening of P2X receptor channels in DH neurons. The reduction of cAMP/PKA signaling is a key element in the inhibitory effect of CB₁R on P2X-channel-induced Ca²⁺ mobilization.     

Sensory neurons derived from diabetic rats have diminished internal Ca2+ stores linked to impaired re-uptake by the endoplasmic reticulum

Distal symmetrical sensory neuropathy in diabetes involves the dying back of axons, and the pathology equates with axonal dystrophy generated under conditions of aberrant Ca²⁺ signalling. Previous work has described abnormalities in Ca²⁺ homoeostasis in sensory and dorsal horn neurons acutely isolated from diabetic rodents. We extended this work by testing the hypothesis that sensory neurons exposed to long-term Type 1 diabetes in vivo would exhibit abnormal axonal Ca²⁺ homoeostasis and focused on the role of SERCA (sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase). DRG (dorsal root ganglia) sensory neurons from age-matched normal and 3–5-month-old STZ (streptozotocin)-diabetic rats (an experimental model of Type 1 diabetes) were cultured. At 1–2 days in vitro an array of parameters were measured to investigate Ca²⁺ homoeostasis including (i) axonal levels of intracellular Ca²⁺, (ii) Ca²⁺ uptake by the ER (endoplasmic reticulum), (iii) assessment of Ca²⁺ signalling following a long-term thapsigargin-induced blockade of SERCA and (iv) determination of expression of ER mass and stress markers using immunocytochemistry and Western blotting. KCl- and caffeine-induced Ca²⁺ transients in axons were 2-fold lower in cultures of diabetic neurons compared with normal neurons indicative of reduced ER calcium loading. The rate of uptake of Ca²⁺ into the ER was reduced by 2-fold (P<0.05) in diabetic neurons, while markers for ER mass and ER stress were unchanged. Abnormalities in Ca²⁺ homoeostasis in diabetic neurons could be mimicked via long-term inhibition of SERCA in normal neurons. In summary, axons of neurons from diabetic rats exhibited aberrant Ca²⁺ homoeo<1?show=[fo]?>stasis possibly triggered by sub-optimal SERCA activity that could contribute to the distal axonopathy observed in diabetes.     

Calcium signals activated by ghrelin and D-Lys-GHRP-6 ghrelin antagonist in developing dorsal root ganglion glial cells

Ghrelin is a hormone regulating energy homeostasis via interaction with its receptor, GHSR-1a. Ghrelin activities in dorsal root ganglia (DRG) cells are unknown. Herein we show that ghrelin induces a change of cytosolic calcium concentration in both glia and neurons of embryonic chick DRG. Both RT-PCR and binding studies performed with fluorescent ghrelin in the presence of either unlabeled ghrelin or GHSR-1a antagonist D-Lys³-GHRP-6, indicate that DRG cells express GHSR-1a. In glial cells the response is characterized by a rapid transient rise in [Ca²⁺]_i followed by a long lasting rise. The calcium elevation is dependent on calcium release from thapsigargin-sensitive intracellular stores and on activation of two distinct Ca²⁺ entry pathways, a receptor activated calcium entry and a store operated calcium entry. Surprisingly, D-Lys³-GHRP-6 exerts several activities in the absence of exogenous ghrelin: (i) it activates calcium release from thapsigargin-sensitive intracellular stores and calcium entry via voltage-operated channels in non-neuronal cells; (ii) it inhibits calcium oscillations in non-neuronal cells exhibiting spontaneous Ca²⁺ activity and iii) it promotes apoptosis of DRG cells, both neurons and glia. In summary, we provide the first evidence for ghrelin activity in DRG, and we also demonstrate that the widely used D-Lys³-GHRP-6 ghrelin antagonist features ghrelin independent activities.