Inhibitory Postsynaptic Currents Induced by Activation of Interneurons in Cultured Rat Hippocampal Neurons

Using the patch-clamp technique in the whole-cell configuration, we studied the characteristics of a series of action potentials (APs) induced by a 500-msec-long current pulse applied to a pre-synaptic unit, as well as the kinetic characteristics of post-synaptic currents (PSCs) evoked by the APs in a post-synaptic unit, in synaptically connected pairs of cultured hippocampal neurons. Presynaptic inhibitory units were identified as GABA-ergic interneurons; they were divided into two groups according to the size of the soma and the number of processes. The kinetic characteristics of PSCs, which were induced in the post-synaptic neuron by a series of the APs generated in the pre-synaptic cell, demonstrated a certain dependence on the morphological characteristics of these cells. In interneurons with large-sized somata, the kinetics of the currents were more fast, and the reversal potential was close to the equilibrium Cl potential. In interneurons with small-sized somata, currents were slower, and the reversal potential was shifted. We conclude that under conditions of culturing, a pre-synaptic cell not only directly provokes the development of PSC in a post-synaptic neuron and determines the amplitude of this current but also significantly influences the kinetics of this current. Neirofiziologiya/Neurophysiology, Vol. 37, No. 2, pp. 116–123, March–April, 2005.     

Rapid and Long-Lasting Increase in Sites for Synapse Assembly during Late-Phase Potentiation in Rat Hippocampal Neurons

Long-term potentiation in hippocampal neurons has stages that correspond to the stages of learning and memory. Early-phase (10–30 min) potentiation is accompanied by rapid increases in clusters or puncta of presynaptic and postsynaptic proteins, which depend on actin polymerization but not on protein synthesis. We have now examined changes in pre- and postsynaptic puncta and structures during glutamate-induced late-phase (3 hr) potentiation in cultured hippocampal neurons. We find that (1) the potentiation is accompanied by long-lasting maintenance of the increases in puncta, which depends on protein synthesis, (2) most of the puncta and synaptic structures are very dynamic, continually assembling and disassembling at sites that are more stable than the puncta or structures themselves, (3) the increase in presynaptic puncta appears to be due to both rapid and more gradual increases in the number of sites where the puncta may form, and also to the stabilization of existing puncta, (4) under control conditions, puncta of postsynaptic proteins behave similarly to puncta of presynaptic proteins and share sites with them, and (5) the increase in presynaptic puncta is accompanied by a similar increase in presumably presynaptic structures, which may form at distinct as well as shared sites. The new sites could contribute to the transition between the early and late phase mechanisms of plasticity by serving as seeds for the formation and maintenance of new synapses, thus acting as local “tags” for protein synthesis-dependent synaptic growth during late-phase plasticity.     

c-Fos Expression by Dopaminergic Receptor Activation in Rat Hippocampal Neurons

While dopamine is likely to modulate hippocampal synaptic plasticity, there has been little information about how dopamine affects synaptic transmission in the hippocampus. The expression of IEGs including c-fos has been associated with late phase LTP in the CA1 region of the hippocampus. The induction of c-fos by dopaminergic receptor activation in the rat hippocampus was investigated by using semiquantitative RT-PCR and immuno-cytochemistry. The hippocampal slices which were not treated with dopamine showed little expression of c-fos mRNA. However, the induction of c-fos mRNA was detected as early as 5 min after dopamine treatment, peaked at 60 min, and remained elevated 5 h after treatment. Temporal profiles of increases in c-fos mRNA by R(+)-SKF-38393 (50 M) and forskolin (50 M) were similar to that of dopamine. An increase in [cAMP] was observed in dopamine-, SKF-, or forskolin-treated hippocampal slices. By immunocytochemical studies, control hippocampal cells showed little expression of c-Fos immunoreactivity. However, when cells were treated with dopamine, an increase in the expression of c-Fos immunoreactivity was observed after treatment for 2 h. The treatment of hippocampal neurons with R(+)-SKF38393 (50 M) or forskolin (50 M) also induced a significant increase in c-Fos expression. These results indicate that the dopamine D1 receptor-mediated cAMP dependant pathway is associated with the expression of c-Fos in the hippocampal neurons. These data are consistent with the possible role of endogenous dopamine on synaptic plasticity via the regulation of gene expression. Furthermore, these results imply that dopamine might control the process of memory storage in the hippocampus through gene expression.     

原代大鼠海马神经元的高效转染

用原代大鼠海马神经元为模型,对新型电转染方法Nucleofector^TM与脂质体DOTAP和Lipofectaimine^TM的转染效率和转染前后细胞存活率进行比较研究,探讨Nucleofector^TM的高效性与可靠性。从E18胎鼠海马中取出神经元进行体外培养,并用神经微丝(NF)抗体进行免疫细胞化学染色鉴定细胞类型。分别用DOTAP,Lipofectamine^TM and Nucleofector^TM包裹pCMV-eGFP质粒转染原代大鼠海马神经元。神经元的存活率用流式细胞仪检测。实验结果表明：DOTAP和Lipofectamine^TM的基因转染效率仅为1．55％和2．45％,而Nucleofector^TM的转染效率则超过20％;细胞转染前后的存活率在DOTAP组分别为98．37％和88．35％,Lipofectamine^TM组分别为98．37％和90．11％,而在Nucleofector^TM组中分别为98．37％和51．82％。上述实验数据表明：Nucleofector^TM转染技术能高效并安全地转染原代大鼠海马神经元,但死亡率较高。     

Regulation of GABA Equilibrium Potential by mGluRs in Rat Hippocampal CA1 Neurons

The equilibrium potential for GABA-A receptor mediated currents (E_GABA) in neonatal central neurons is set at a relatively depolarized level, which is suggested to be caused by a low expression of K⁺/Cl^- co-transporter (KCC2) but a relatively high expression of Na⁺-K⁺-Cl^- cotransporter (NKCC1). Theta-burst stimulation (TBS) in stratum radiatum induces a negative shift in E_GABA in juvenile hippocampal CA1 pyramidal neurons. In the current study, the effects of TBS on E_GABA in neonatal and juvenile hippocampal CA1 neurons and the underlying mechanisms were examined. Metabotropic glutamate receptors (mGluRs) are suggested to modulate KCC2 and NKCC1 levels in cortical neurons. Therefore, the involvement of mGluRs in the regulation of KCC2 or NKCC1 activity, and thus E_GABA, following TBS was also investigated. Whole-cell patch recordings were made from Wistar rat hippocampal CA1 pyramidal neurons, in a slice preparation. In neonates, TBS induces a positive shift in E_GABA, which was prevented by NKCC1 antisense but not NKCC1 sense mRNA. (RS)-a-Methyl-4-carboxyphenylglycine (MCPG), a group I and II mGluR antagonist, blocked TBS-induced shifts in both juvenile and neonatal hippocampal neurons. While blockade of mGluR1 or mGluR5 alone could interfere with TBS-induced shifts in E_GABA in neonates, only a combined blockade could do the same in juveniles. These results indicate that TBS induces a negative shift in E_GABA in juvenile hippocampal neurons but a positive shift in neonatal hippocampal neurons via corresponding changes in KCC2 and NKCC1 expressions, respectively. mGluR activation seems to be necessary for both shifts to occur while the specific receptor subtype involved seems to vary.     

Neurotoxicity Induced by Glutamate in Glucose-Deprived Rat Hippocampal Slices is Prevented by GMP

Guanosine-5-monophosphate (GMP) was evaluated as a neuroprotective agent against the damage induced by glutamate in rat hippocampal slices submitted to glucose deprivation. In slices maintained under physiological conditions, glutamate (0.01 to 10 mM), Kainate, alpha-amino-3-hydroxi-5-methylisoxazole-propionic acid (AMPA), N-methyl-D-aspartate (NMDA), 1S,3R-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD), or L-2-amino-4-phosphonobutanoic acid (L-AP4) (100 M) did not alter cell membrane permeability, as evaluated by lactate dehydrogenase (LDH) release assay. In slices submitted to glucose deprivation, GMP (from 0.5 mM) prevented LDH leakage and the loss of cell viability induced by 10 mM glutamate. LDH leakage induced by Kainate, AMPA, NMDA or 1S,3R-ACPD was fully prevented by 1 mM GMP. However, glutamate uptake was not altered in slices submitted to glucose deprivation and glutamate analogues. Glucose deprivation induced a significant decrease in ATP levels which was unchanged by addition of glutamate or GMP. Our results show that glucose deprivation decreases the energetic charge of cells, making hippocampal slices more susceptible to excitotoxicity and point to GMP as a neuroprotective agent acting as a glutamatergic antagonist.     

Initial Characterization of the Nicotinic Acetylcholine Receptors in Rat Hippocampal Neurons

Abstract

The properties of the neuronal nicotinic acetylcholine receptor in primary cultures of hippocampal cells from fetal rats (17–18 days gestation) were studied using the whole-cell patch-clamp technique in Na⁺-external, Cs⁺-internal and nominally Mg²⁺-free solutions. The nicotinic agonists acetylcholine, (+)anatoxin-a, and (-) and (+)nicotine all evoked inward whole-cell currents in hippocampal neurons that were voltage clamped near their resting potentials. Sensitivity to (+)anatoxin-a was first detected at around day 6, and thereafter the magnitude of the response increased as a function of number of days in culture up to about 40 days. The whole-cell current waveforms consisted of more than one peak whose relative amplitude depended on the agonist concentration. These currents were reversibly blocked by micromolar concentrations of d-tubocurarine, mecamylamine, and dihydro-β-erythroidine. At nanomolar concentrations, neuronal bungarotoxin, α-bungarotoxin and α-cobratoxin caused an irreversible blockade of the currents but they were unaffected by tetrodotoxin, atropine, DL-2-amino-5-phosphonovaleric acid, Mg²⁺, and 6,7-dinitroquinoxaline-2,3-dione. In addition, the currents were also blocked in a reversible manner by methyllycaconitine at picomolar concentration. The current-voltage plots elicited by both (+)anatoxin-a and acetylcholine revealed larger inward currents and smaller or no outward currents. The present results demonstrate the existence of an inwardly rectifying, snake neurotoxin-sensitive functional nicotinic acetylcholine receptor ion channel in rat hippocampal neurons.     

Wnt-5a Ligand Modulates Mitochondrial Fission-Fusion in Rat Hippocampal Neurons

The Wnt signaling pathway plays an important role in developmental processes, including embryonic patterning, cell specification, and cell polarity. Wnt components participate in the development of the central nervous system, and growing evidence indicates that this pathway also regulates the function of the adult nervous system. In this study, we report that Wnt-5a, a noncanonical Wnt ligand, is a potent activator of mitochondrial dynamics and induces acute fission and fusion events in the mitochondria of rat hippocampal neurons. The effect of Wnt-5a was inhibited in the presence of sFRP, a Wnt scavenger. Similarly, the canonical Wnt-3a ligand had no effect on mitochondrial fission-fusion events, suggesting that this effect is specific for Wnt-5a alone. We also show that the Wnt-5a effects on mitochondrial dynamics occur with an increase in both intracellular and mitochondrial calcium (Ca²⁺), which was correlated with an increased phosphorylation of Drp1(Ser-616) and a decrease of Ser-637 phosphorylation, both indicators of mitochondrial dynamics. Electron microscope analysis of hippocampal tissues in the CA1 region showed an increase in the number of mitochondria present in the postsynaptic region, and this finding correlated with a change in mitochondrial morphology. We conclude that Wnt-5a/Ca²⁺ signaling regulates the mitochondrial fission-fusion process in hippocampal neurons, a feature that might help to further understand the role of Wnt-related pathologies, including neurodegenerative diseases associated with mitochondrial dysfunction, and represents a potentially important link between impaired metabolic function and degenerative disorders.     

艾芬地尔对异氟烷所致发育期海马神经元毒性的保护作用

目的:通过体外实验探讨艾芬地尔对异氟烷所致发育期海马神经元毒性的保护作用。方法:从出生一天的大鼠海马获取神经元并体外培养5天。这些神经元被随机分入4组,包括对照组(control组)、异氟醚组(Iso组)、艾芬地尔(Ifen组)和艾芬地尔+异氟烷组(Ifen+ISO组)。使用MTT法检测细胞活力及细胞损伤程度。使用TUNEL染色法检测细胞凋亡。使用Western blot法检测神经元中NMDA受体亚基NR2B和活化caspase-3的表达水平。结果:与对照组比较,在2.4%异氟烷暴露后1小时神经元的细胞活力显著下降(P0.05)。同时,在2.4%异氟烷暴露后神经元的凋亡指数也显著升高(P0.05)。Western blot结果显示,异氟烷暴露可显著升高神经元活化caspase-3和NR2B的表达水平(P0.05)。然而,使用NR2B拮抗剂艾芬地尔(20μM)不仅可显著减少异氟烷所致的NR2B表达水平增高,也可缓解异氟烷造成的神经元凋亡和细胞损伤(P0.01)。结论:异氟烷可导致发育期神经元NR2B表达水平增高,而使用NR2B受体拮抗剂艾芬地尔可有效抑制NR2B的表达水平从而减少异氟烷所致神经元毒性。     

Activation of Mitogen-Activated Protein Kinase in Cultured Rat Hippocampal Neurons by Stimulation of Glutamate Receptors

Abstract: Mitogen-activated protein kinase (MAP kinase) was activated by stimulation of glutamate receptors in cultured rat hippocampal neurons. Ten micromolar glutamate maximally stimulated MAP kinase activity, which peaked during 10 min and decreased to the basal level within 30 min. Experiments using glutamate receptor agonists and antagonists revealed that glutamate stimulated MAP kinase through NMDA and metabotropic glutamate receptors but not through non-NMDA receptors. Glutamate and its receptor agonists had no apparent effect on MAP kinase activation in cultured cortical astrocytes. Addition of calphostin C, a protein kinase C (PKC) inhibitor, or down-regulation of PKC activity partly abolished the stimulatory effect by glutamate, but the MAP kinase activation by treatment with ionomycin, a Ca²⁺ ionophore, remained intact. Lavendustin A, a tyrosine kinase inhibitor, was without effect. In experiments with ³²P-labeled hippocampal neurons, MAP kinase activation by glutamate was associated with phosphorylation of the tyrosine residue located on MAP kinase. However, phosphorylation of Raf-1, the c- raf protooncogene product, was not stimulated by treatment with glutamate. Our observations suggest that MAP kinase activation through glutamate receptors in hippocampal neurons is mediated by both the PKC-dependent and the Ca²⁺-dependent pathways and that the activation of Raf-1 is not involved.     

Curcumin Rescues Aging-Related Loss of Hippocampal Synapse Input Specificity of Long Term Potentiation in Mice

Curcumin has neuroprotective effect and could enhance memory. However, the mechanisms underlying the protection of curcumin on aging-related memory decline are not well understood. In this study, high frequency stimulation (HFS)-induced long term potentiation (LTP) was evaluated by a cellular model of memory formation. A two-input stimulation paradigm was used to record the potentiation as well as synapse input specificity. The data suggested that an N-Methyl-d-aspartate receptors (NMDAR) -dependent LTP was inducible in adult hippocampal slices with a characteristic of synapse input specificity. It also indicated that aging resulted in a reduction in LTP but more importantly a loss of synaptic input specificity. The reason behind the above conclusions is that LTP induction is more dependent on the calcium channel. This is due to a switch of the dependence of LTP induction to voltage-dependent calcium channel (VDCC) compared to NMDA receptors. Curcumin administration recovers input specificity by re-establishing NMDA receptor dependence of induction. In addition, curcumin administration ameliorated aging-related increase of brain thiobarbituric acid-reactive substances and elevated aging-related decrease of glutathione in hippocampus. It is then concluded that curcumin modulates hippocampal redox status and restores aging-related loss of synapse input specificity of HFS-induced LTP by switching VDCC calcium source into NMDA receptor-dependent one.     

Endocannabinoids Differentially Modulate Synaptic Plasticity in Rat Hippocampal CA1 Pyramidal Neurons

Background

Hippocampal CA1 pyramidal neurons receive two excitatory glutamatergic synaptic inputs: their most distal dendritic regions in the stratum lacunosum-moleculare (SLM) are innervated by the perforant path (PP), originating from layer III of the entorhinal cortex, while their more proximal regions of the apical dendrites in the stratum radiatum (SR) are innervated by the Schaffer-collaterals (SC), originating from hippocampal CA3 neurons. Endocannabinoids (eCBs) are naturally occurring mediators capable of modulating both GABAergic and glutamatergic synaptic transmission and plasticity via the CB1 receptor. Previous work on eCB modulation of excitatory synapses in the CA1 region largely focuses on the SC pathway. However, little information is available on whether and how eCBs modulate glutamatergic synaptic transmission and plasticity at PP synapses.

Methodology/Principal Findings

By employing somatic and dendritic patch-clamp recordings, Ca²⁺ uncaging, and immunostaining, we demonstrate that there are significant differences in low-frequency stimulation (LFS)- or DHPG-, an agonist of group I metabotropic glutamate receptors (mGluRs), induced long-term depression (LTD) of excitatory synaptic transmission between SC and PP synapses in the same pyramidal neurons. These differences are eliminated by pharmacological inhibition with selective CB1 receptor antagonists or genetic deletion of the CB1 receptor, indicating that these differences likely result from differential modulation via a CB1 receptor-dependent mechanism. We also revealed that depolarization-induced suppression of excitation (DSE), a form of short-term synaptic plasticity, and photolysis of caged Ca²⁺-induced suppression of Excitatory postsynaptic currents (EPSCs) were less at the PP than that at the SC. In addition, application of WIN55212 (WIN) induced a more pronounced inhibition of EPSCs at the SC when compared to that at the PP.

Conclusions/Significance

Our results suggest that CB1 dependent LTD and DSE are differentially expressed at the PP versus SC synapses in the same neurons, which may have an impact on synaptic scaling, integration and plasticity of hippocampal CA1 pyramidal neurons.     

Glucose Deprivation Activates Diversity of Potassium Channels in Cultured Rat Hippocampal Neurons

1. Glucose is one of the most important substrates for generating metabolic energy required for the maintenance of cellular functions. Glucose-mediated changes in neuronal firing pattern have been observed in the central nervous system of mammals. K⁺ channels directly regulated by intracellular ATP have been postulated as a linkage between cellular energetic metabolism and excitability; the functional roles ascribed to these channels include glucose-sensing to regulate energy homeostasis and neuroprotection under energy depletion conditions. The hippocampus is highly sensitive to metabolic insults and is the brain region most sensitive to ischemic damage. Because the identity of metabolically regulated potassium channels present in hippocampal neurons is obscure, we decided to study the biophysical properties of glucose-sensitive potassium channels in hippocampal neurons.2. The dependence of membrane potential and the sensitivity of potassium channels to glucose and ATP in rat hippocampal neurons were studied in cell-attached and excised inside-out membrane patches.3. We found that under hypoglycemic conditions, at least three types of potassium channels were activated; their unitary conductance values were 37, 147, and 241 pS in symmetrical K⁺, and they were sensitive to ATP. For K⁺ channels with unitary conductance of 37 and 241, when the membrane potential was depolarized the longer closed time constant diminished and this produced an increase in the open-state probability; nevertheless, the 147-pS channels were not voltage-dependent.4. We propose that neuronal glucose-sensitive K⁺ channels in rat hippocampus include subtypes of ATP-sensitive channels with a potential role in neuroprotection during short-term or prolonged metabolic stress.     

Investigating Sub-Spine Actin Dynamics in Rat Hippocampal Neurons with Super-Resolution Optical Imaging

Morphological changes in dendritic spines represent an important mechanism for synaptic plasticity which is postulated to underlie the vital cognitive phenomena of learning and memory. These morphological changes are driven by the dynamic actin cytoskeleton that is present in dendritic spines. The study of actin dynamics in these spines traditionally has been hindered by the small size of the spine. In this study, we utilize a photo-activation localization microscopy (PALM)–based single-molecule tracking technique to analyze F-actin movements with ∼30-nm resolution in cultured hippocampal neurons. We were able to observe the kinematic (physical motion of actin filaments, i.e., retrograde flow) and kinetic (F-actin turn-over) dynamics of F-actin at the single-filament level in dendritic spines. We found that F-actin in dendritic spines exhibits highly heterogeneous kinematic dynamics at the individual filament level, with simultaneous actin flows in both retrograde and anterograde directions. At the ensemble level, movements of filaments integrate into a net retrograde flow of ∼138 nm/min. These results suggest a weakly polarized F-actin network that consists of mostly short filaments in dendritic spines.     

Membrane Potential Dependent Duration of Action Potentials in Cultured Rat Hippocampal Neurons

(1) Fluctuations of the membrane potential states are essential for the brain functions from the response of individual neurons to the cognitive function of the brain. It has been reported in slice preparations that the action potential duration is dependent on the membrane potential states. (2) In order to examine whether dependence of action potential duration on the membrane potential could happen in isolated individual neurons that have no network connections, we studied the membrane potential dependence of the action potential duration by artificially setting the membrane potentials to different states in individual cultured rat hippocampal neurons using patch-clamp technique. (3) We showed that the action potential of individual neurons generated from depolarized membrane potentials had broader durations than those generated from hyperpolarized membrane potentials. (4) Furthermore, the membrane potential dependence of the action potential duration was significantly reduced in the presence of voltage-gated K⁺ channel blockers, TEA, and 4-AP, suggesting involvement of both delayed rectifier I _K and transient I _A current in the membrane potential dependence of the action potential duration. (5) These results indicated that the dependence of action potential duration on the membrane potential states could be an intrinsic property of individual neurons. Bo Gong and Mingna Liu contributed equally to this work.     

Loss of Hippocampal Neurons after Kainate Treatment Correlates with Behavioral Deficits

Treating rats with kainic acid induces status epilepticus (SE) and leads to the development of behavioral deficits and spontaneous recurrent seizures later in life. However, in a subset of rats, kainic acid treatment does not induce overt behaviorally obvious acute SE. The goal of this study was to compare the neuroanatomical and behavioral changes induced by kainate in rats that developed convulsive SE to those who did not. Adult male Wistar rats were treated with kainic acid and tested behaviorally 5 months later. Rats that had experienced convulsive SE showed impaired performance on the spatial water maze and passive avoidance tasks, and on the context and tone retention tests following fear conditioning. In addition, they exhibited less anxiety-like behaviors than controls on the open-field and elevated plus-maze tests. Histologically, convulsive SE was associated with marked neuron loss in the hippocampal CA3 and CA1 fields, and in the dentate hilus. Rats that had not experienced convulsive SE after kainate treatment showed less severe, but significant impairments on the spatial water maze and passive avoidance tasks. These rats had fewer neurons than control rats in the dentate hilus, but not in the hippocampal CA3 and CA1 fields. Correlational analyses revealed significant relationships between spatial memory indices of rats and neuronal numbers in the dentate hilus and CA3 pyramidal field. These results show that a part of the animals that do not display intense behavioral seizures (convulsive SE) immediately after an epileptogenic treatment, later in life, they may still have noticeable structural and functional changes in the brain.     

Long-Term Primary Culture of Highly-Pure Rat Embryonic Hippocampal Neurons of Low-Density

In order to develop a simplified method for long-term primary culture of highly-pure rat embryonic hippocampal neurons of low-density (10³ cells/cm²), we optimized and modified conventional culturing methods. The modifications of our simplified method include: (1) combinational application of two growth substrates, tail collagen and poly-L-lysine, to coat plastic culture dishes and coverslips for a better neuronal attachment; (2) dissociation of hippocampal tissues with combinational use of two milder enzymes (collagenase and dispase) and trypsin of a lower concentration to minimize enzymatic damages to cultured neurons; (3) a cell pre-plating step to preliminarily eliminate the contaminating non-neuronal cells; (4) a modified culture medium as a critical step to promote highly pure neurons of low-density for a long term; and (5) appropriately reduced frequency and volume of refreshment of the culture medium. Using our modified method, the β-tubulin III-immunostained and Hoechst 33342 counterstained neurons harvested a steady and healthy growth with a longer culture time of over 35 days, and a clear distinction between TAU-1- and MAP2-immunoreactive neurites was apparent at the early culturing period. In addition, the purity of neurons was over 95% at the different time points in comparison with the control culture using conventional serum-free method in which most neurons degenerated and died within 5 days. Thus, our modified method proved to be a simple, feasible as well as time- and resource-saving approach for a long-term survival of pure rat embryonic hippocampal neurons of low-density.     

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.     

Intracellular Calcium Spikes in Rat Suprachiasmatic Nucleus Neurons Induced by BAPTA-Based Calcium Dyes

Background

Circadian rhythms in spontaneous action potential (AP) firing frequencies and in cytosolic free calcium concentrations have been reported for mammalian circadian pacemaker neurons located within the hypothalamic suprachiasmatic nucleus (SCN). Also reported is the existence of “Ca²⁺ spikes” (i.e., [Ca²⁺]_c transients having a bandwidth of 10∼100 seconds) in SCN neurons, but it is unclear if these SCN Ca²⁺ spikes are related to the slow circadian rhythms.

Methodology/Principal Findings

We addressed this issue based on a Ca²⁺ indicator dye (fluo-4) and a protein Ca²⁺ sensor (yellow cameleon). Using fluo-4 AM dye, we found spontaneous Ca²⁺ spikes in 18% of rat SCN cells in acute brain slices, but the Ca²⁺ spiking frequencies showed no day/night variation. We repeated the same experiments with rat (and mouse) SCN slice cultures that expressed yellow cameleon genes for a number of different circadian phases and, surprisingly, spontaneous Ca²⁺ spike was barely observed (<3%). When fluo-4 AM or BAPTA-AM was loaded in addition to the cameleon-expressing SCN cultures, however, the number of cells exhibiting Ca²⁺ spikes was increased to 13∼14%.

Conclusions/Significance

Despite our extensive set of experiments, no evidence of a circadian rhythm was found in the spontaneous Ca²⁺ spiking activity of SCN. Furthermore, our study strongly suggests that the spontaneous Ca²⁺ spiking activity is caused by the Ca²⁺ chelating effect of the BAPTA-based fluo-4 dye. Therefore, this induced activity seems irrelevant to the intrinsic circadian rhythm of [Ca²⁺]_c in SCN neurons. The problems with BAPTA based dyes are widely known and our study provides a clear case for concern, in particular, for SCN Ca²⁺ spikes. On the other hand, our study neither invalidates the use of these dyes as a whole, nor undermines the potential role of SCN Ca²⁺ spikes in the function of SCN.