Synchronization of Neuronal Activity Promotes Survival of Individual Rat Neocortical Neurons in Early Development

Neural activity is thought to play a significant role during the development of the cerebral cortex. In this study, we examined the effects of global activity block or enhancement and the effects of patterned firing on the ability of cultured rat neocortical neurons to survive during the second week in vitro , beyond the beginning of synaptogenesis. Blockade of neuronal activity by adding tetrodotoxin (TTX) and increasing magnesium concentration in the medium strongly reduced the survival of cortical cells. Increasing neuronal activity by raising the external potassium concentration significantly improved the survival of cortical neurons. We postulated that in a developing neuronal network the survival of nerve cells is regulated by synaptically mediated events that involve changes in the intracellular calcium concentration. To examine this question further, we monitored the activity of the developing network by optically recording the intracellular calcium signals of many neurons simultaneously. These recordings show that in low magnesium neocortical neurons express synchronized oscillation of their intracellular calcium concentration. The ability of a network to synchronize the changes in intracellular calcium of multiple cells appeared gradually during the second week in culture, paralleled by both an increase in the synaptic density and a decline in the number of surviving neurons. By examining the fate of identified cells several days after a recording session, we found that those nerve cells that were co-activated with other neurons had a significantly higher chance to survive than cells that did not participate in synchronized events. These experiments demonstrate that during early cortical network development cortical neurons show synchronized firing activity and that the survival of neurons is at least partially dependent on this pattern of neuronal activity.     

NMDA Receptor Enhances Correlation of Spontaneous Activity in Neonatal Barrel Cortex

Correlated spontaneous activity plays critical role in the organization of neocortical circuits during development. However, cortical mechanisms regulating activity correlation are still elusive. In this study, using two-photon calcium imaging of the barrel cortex layer 4 (L4) in living neonatal mice, we found that NMDA receptors (NMDARs) in L4 neurons are important for enhancement of spontaneous activity correlation. Disruption of GluN1 (Grin1), an obligatory NMDAR subunit, in a sparse population of L4 neurons reduced activity correlation between GluN1 knock-out (GluN1KO) neuron pairs within a barrel. This reduction in activity correlation was even detected in L4 neuron pairs in neighboring barrels and most evident when either or both of neurons are located on the barrel edge. Our results provide evidence for the involvement of L4 neuron NMDARs in spatial organization of the spontaneous firing activity of L4 neurons in the neonatal barrel cortex.SIGNIFICANCE STATEMENT Precise wiring of the thalamocortical circuits is necessary for proper sensory information processing, and thalamus-derived correlated spontaneous activity is important for thalamocortical circuit formation. The molecular mechanisms involved in the correlated activity transfer from the thalamus to the neocortex are largely unknown. In vivo two-photon calcium imaging of the neonatal barrel cortex revealed that correlated spontaneous activity between layer four neurons is reduced by mosaic knock-out (KO) of the NMDA receptor (NMDAR) obligatory subunit GluN1. Our results suggest that the function of NMDARs in layer four neurons is necessary for the communication between presynaptic and postsynaptic partners during thalamocortical circuit formation.     

Tetrodotoxin prevents posttraumatic epileptogenesis in rats.

Severe cortical trauma frequently causes epilepsy that develops after a long latency. We hypothesized that plastic changes in excitability during this latent period might be initiated or sustained by the level of neuronal activity in the injured cortex. We therefore studied effects of action potential blockade by application of tetrodotoxin (TTX) to areas of cortical injury in a model of chronic epileptogenesis. Partially isolated islands of sensorimotor cortex were made in 28- to 30-day-old male Sprague-Dawley rats and thin sheets of Elvax polymer containing TTX or control vehicle were implanted over lesions. Ten to 15 days later neocortical slices were obtained through isolates for electrophysiological studies. Slices from all animals (n = 12) with lesions contacted by control-Elvax (58% of 36 slices) exhibited evoked epileptiform field potentials, and those from 4 rats had spontaneous epileptiform events. Only 2 of 11 lesioned animals and 6% of slices from cortex exposed to TTX in vivo exhibited evoked epileptiform potentials, and no spontaneous epileptiform events were observed. There was no evidence of residual TTX during recordings. TTX-Elvax was ineffective in reversing epileptogenesis when implanted 11 days after cortical injury. These data suggest that development of antiepileptogenic drugs for humans may be possible.     

Bioluminescence calcium imaging of network dynamics and their cholinergic modulation in slices of cerebral cortex from male rats

The activity of neuronal ensembles was monitored in neocortical slices from male rats using wide-field bioluminescence imaging of a calcium sensor formed with the fusion of green fluorescent protein and aequorin (GA) and expressed through viral transfer. GA expression was restricted to pyramidal neurons and did not conspicuously alter neuronal morphology or neocortical cytoarchitecture. Removal of extracellular magnesium or addition of GABA receptor antagonists triggered epileptiform flashes of variable amplitude and spatial extent, indicating that the excitatory and inhibitory networks were functionally preserved in GA-expressing slices. We found that agonists of muscarinic acetylcholine receptors largely increased the peak bioluminescence response to local electrical stimulation in layer I or white matter, and gave rise to a slowly decaying response persisting for tens of seconds. The peak increase involved layers II/III and V and did not result in marked alteration of response spatial properties. The persistent response involved essentially layer V and followed the time course of the muscarinic afterdischarge depolarizing plateau in layer V pyramidal cells. This plateau potential triggered spike firing in layer V, but not layer II/III pyramidal cells, and was accompanied by recurrent synaptic excitation in layer V. Our results indicate that wide-field imaging of GA bioluminescence is well suited to monitor local and global network activity patterns, involving different mechanisms of intracellular calcium increase, and occurring on various timescales.     

Cell-Type-Specific Dynamics of Calcium Activity in Cortical Circuits over the Course of Slow-Wave Sleep and Rapid Eye Movement Sleep

Sleep shapes cortical network activity, fostering global homeostatic downregulation of excitability while maintaining or even upregulating excitability in selected networks in a manner that supports memory consolidation. Here, we used two-photon calcium imaging of cortical layer 2/3 neurons in sleeping male mice to examine how these seemingly opposing dynamics are balanced in cortical networks. During slow-wave sleep (SWS) episodes, mean calcium activity of excitatory pyramidal (Pyr) cells decreased. Simultaneously, however, variance in Pyr population calcium activity increased, contradicting the notion of a homogenous downregulation of network activity. Indeed, we identified a subpopulation of Pyr cells distinctly upregulating calcium activity during SWS, which were highly active during sleep spindles known to support mnemonic processing. Rapid eye movement (REM) episodes following SWS were associated with a general downregulation of Pyr cells, including the subpopulation of Pyr cells active during spindles, which persisted into following stages of sleep and wakefulness. Parvalbumin-positive inhibitory interneurons (PV-In) showed an increase in calcium activity during SWS episodes, while activity remained unchanged during REM sleep episodes. This supports the view that downregulation of Pyr calcium activity during SWS results from increased somatic inhibition via PV-In, whereas downregulation during REM sleep is achieved independently of such inhibitory activity. Overall, our findings show that SWS enables upregulation of select cortical circuits (likely those which were involved in mnemonic processing) through a spindle-related process, whereas REM sleep mediates general downregulation, possibly through synaptic re-normalization.SIGNIFICANCE STATEMENT Sleep is thought to globally downregulate cortical excitability and, concurrently, to upregulate synaptic connections in neuron ensembles with newly encoded memory, with upregulation representing a function of sleep spindles. Using in vivo two-photon calcium imaging in combination with surface EEG recordings, we classified cells based on their calcium activity during sleep spindles. Spindle-active pyramidal (Pyr) cells persistently increased calcium activity during slow-wave sleep (SWS) episodes while spindle-inactive cells decreased calcium activity. Subsequent rapid eye movement (REM) sleep episodes profoundly reduced calcium activity in both cell clusters. Results indicate that SWS allows for a spindle-related differential upregulation of ensembles whereas REM sleep functions to globally downregulate networks.     

Effects of gap junction blockers on human neocortical synchronization

Field potentials and intracellular recordings were obtained from human neocortical slices to study the role of gap junctions (GJ) in neuronal network synchronization. First, we examined the effects of GJ blockers (i.e., carbenoxolone, octanol, quinine, and quinidine) on the spontaneous synchronous events (duration = 0.2-1.1 s; intervals of occurrence = 3-27 s) generated by neocortical slices obtained from temporal lobe epileptic patients during application of 4-aminopyridine (4AP, 50 muM) and glutamatergic receptor antagonists. The synchronicity of these potentials (recorded at distances up to 5 mm) was decreased by GJ blockers within 20 min of application, while prolonged GJ blockers treatment at higher doses made them disappear with different time courses. Second, we found that slices from patients with focal cortical dysplasia (FCD) could generate in normal medium spontaneous synchronous discharges (duration = 0.4-8 s; intervals of occurrence = 0.5-90 s) that were (i) abolished by NMDA receptor antagonists and (ii) slowed down by carbenoxolone. Finally, octanol or carbenoxolone blocked 4AP-induced ictal-like discharges (duration = up to 35 s) in FCD slices. These data indicate that GJ play a role in synchronizing human neocortical networks and may implement epileptiform activity in FCD.     

Spatio-temporal dynamics of oscillatory network activity in the neonatal mouse cerebral cortex

We used a 60-channel microelectrode array to study in thick (600-1000 microm) somatosensory cortical slices from postnatal day (P)0-P3 mice the spatio-temporal properties of early network oscillations. We recorded local non-propagating as well as large-scale propagating spontaneous oscillatory activity. Both types of activity patterns could never be observed in neocortical slices of conventional thickness (400 microm). Local non-propagating spontaneous oscillations with an average peak frequency of 15.6 Hz, duration of 1.7 s and maximal amplitude of 66.8 microV were highly synchronized in a network of approximately 200 microm in diameter. Spontaneous oscillations of lower frequency (10.4 Hz), longer duration (23.8 s) and larger amplitude (142.9 microV) propagated with 0.11 mm/s in the horizontal direction over at least 1 mm. These propagating oscillations were also synchronized in a columnar manner, but these waves synchronized the activity in a larger neuronal network of 300-400 microm in diameter. Both types of spontaneous network activity could be blocked by the gap junction antagonist carbenoxolone. Electrical stimulation of the subplate (SP) or bath application of the cholinergic agonist carbachol also elicited propagating network oscillations, emphasizing the role of the SP and the cholinergic system in the generation of early cortical network oscillations. Our data demonstrate that a sufficiently large network in thick neocortical slice preparations is capable of generating spontaneous and evoked network oscillations, which are highly synchronized via gap junctions in 200-400-microm-wide columns. These via synchronized oscillations coupled networks may represent a self-organized functional template for the activity-dependent formation of neocortical modules during the earliest stages of development.     

Simultaneous Monitoring of Cytosolic Free Calcium and Exocytosis at the Single Cell Level

Quinacrine, a fluorescent basic molecule, accumulates in secretory granules of pituitary cells, as was revealed by its colocalization with immunoreactive prolactin. Thus quinacrine fluorescence may be used to monitor secretory activity at the single cell level. Rat pituitary cells in primary culture were loaded with quinacrine and stimulated with physiological secretagogues, such as thyrotrophin-releasing hormone or bradykinin, which induced a multiphasic lowering of fluorescence, corresponding to the loss of quinacrine contained in exocytosed granules. Quinacrine was further used in combination with the fluorescent calcium probe fura-2, in order to monitor simultaneously exocytosis and variations in the cytosolic free calcium concentration, [Ca²⁺]_i. With an appropriate selection of the excitation wavelengths, in dual excitation microfluorimetry experiments, it was possible to distinguish between fluorescence changes due to altered [Ca²⁺]_i versus quinacrine exocytosis. Transient elevations of [Ca²⁺]_i were provoked in individual pituitary cells by enhancing calcium influx through voltage gated channels. In part of the cells an initial increase in [Ca²⁺]_i coincided with stimulated quinacrine release. The approach was also applied to cells of the neuroblastoma line NCB20, where stimulation with bradykinin caused a transient rise in [Ca²⁺]_i, concomitantly with enhanced exocytosis. No increase in exocytosis was ever detected without an elevation of [Ca²⁺]_i, suggesting that in both cellular systems, an increase in [Ca²⁺]_i, is absolutely necessary, but not sufficient to induce secretion.     

一种观测脑疾病状态下胶质细胞活动的钙成像技术

目的 探讨应用广视野钙成像技术观测神经元和胶质细胞的活动。方法 采用对流强化法对麻醉雄性大鼠的新皮层进行钙染料染色,应用广视野钙成像技术观测药物诱导的急性癫痫模型中神经元和胶质细胞的活动。结果 钙成像的光学脑电图显示癫痫时的神经活动为快速传播的波,其传播范围仅限于致痫灶处,而胶质细胞活动由癫痫起源触发,但是表现为慢速传播的定型波,其传播范围超出神经活动范围。结论 广视野钙成像技术更有效地观测神经元和胶质细胞的活动并区别神经元和胶质细胞的网络活动。     

NR2B antagonists restrict spatiotemporal spread of activity in a rat model of cortical dysplasia

Freeze-lesion-induced focal cortical dysplasia in rats closely resembles human microgyria, a neuronal migration disorder associated with drug-resistant epilepsy. Alterations in expression of N-methyl-D-aspartate receptors (NMDARs) containing NR2B subunits have been suggested to play a role in the hyperexcitability seen in this model. We examined the effect of NMDAR antagonists selective for NR2B subunits (Ro 25-6981 and ifenprodil) on activity evoked by intracortical stimulation in brain slices from freeze-lesioned rat neocortex. Whole-cell voltage-clamp recordings showed that Ro 25-6981 (1 microM) significantly reduced the response area of evoked postsynaptic currents in pyramidal cells from the paramicrogyral area whereas responses were unaffected in slices from control (sham operated) animals. Voltage-sensitive dye imaging was used to examine spatiotemporal spread of evoked activity in lesioned and control cortices. The imaging experiments revealed that peak amplitude, duration, and lateral spread of evoked activity in the paramicrogyral area was reduced by bath application of Ro 25-6981 (1 microM) and ifenprodil (10 microM). Ro 25-6981 had no effect on evoked activity in neocortical slices from control animals. The non-selective NMDAR antagonist d-2-amino-5-phosphonvaleric acid (APV, 20 microM) reduced activity evoked in presence of 50 microM 4-aminopyridine (known to increase excitability by enhancing neurotransmitter release) in neocortical slices from control animals whereas Ro 25-6981 (1 microM) did not. These results suggest that NR2B subunit-containing NMDARs contribute significantly to the enhanced spatiotemporal spread of paroxysmal activity observed in vitro in the rat freeze-lesion model of focal cortical dysplasia.     

Spontaneous network activity of cerebellar granule neurons: impairment by in vivo chronic cannabinoid administration

Synchronized activity of neuronal networks has been proposed to be essential for cerebellar function. To examine the occurrence and organization of spontaneous neuronal activity in the cerebellum in vivo, we imaged mouse cerebellar slices loaded with the intracellular Ca2+ concentration indicator, fura-2. Recordings were then analysed statistically to identify correlated network activity. Ca2+ imaging revealed consistent spontaneous correlated network activity of granule cells (GC), which often occurred in clusters of coactivated GC. The number of spontaneously active GC, their activation frequency and correlation, were controlled by glutamate and GABA ionotropic receptors. These findings indicate that distinctive patterns of correlated activity between GC networks may be relevant for cerebellar circuit function. Cannabinoid antagonist-precipitated delta9-tetrahydrocannabinol (THC) withdrawal impaired motor coordination. Given that the cerebellum has been suggested recently to be a main substrate for cannabinoid withdrawal, we used imaging of spontaneous network activity to examine whether GC, which contain CB1 cannabinoid receptors, respond to chronic THC treatment and withdrawal. Acute administration of THC had no effect on patterns of spontaneous GC network activity. In contrast, chronic THC administration severely impaired GC activity and network coordination. Incubation of cerebellar slices, from chronically THC-treated mice, with the cannabinoid antagonist, SR141716A increased the number and network correlation of active GC. These data provide physiological evidence of the involvement of cerebellar circuits in the adaptive changes occurring during chronic THC exposure and withdrawal.     

Endogenous somatostatin receptors mobilize calcium from inositol 1,4,5-trisphosphate-sensitive stores in NG108-15 cells

Injury and loss of neurons are observed in the center of a cerebral cortical lesion. Mechanisms of early functional reorganization post-lesion involve changes in the strength of synaptic coupling as measured in long-term potentiation (LTP). Since these changes in LTP may depend on the intraneuronal calcium concentration ([Ca²⁺]_I), the present study analyzed the strength of synaptic LTP combined with measurements of the stimulus-induced peak calcium influx in slices from rat visual cortex in vitro. Slices were analyzed 1–7 days post-lesion by use of electrophysiological and calcium fluorescence imaging techniques. A theta-burst stimulus (TBS) was electrically applied to cortical layer IV, while changes in extracellular field potentials (FPs) and in the corresponding peak calcium influx were recorded in layers II/III. Both the strength of LTP and of the FP mediated peak calcium influx were significantly enhanced 1–6 days post-lesion at a distance of 4 mm from the lesion border. Pharmacological experiments revealed that the expression of LTP was dependent on the activation of NMDA receptors. The area of increased stimulus-evoked peak calcium influx correlated with the enhanced LTP, suggesting that changes in [Ca²⁺]_I mediate the strength of long-term synaptic plasticity following a cortical lesion. This mechanism may support synaptic reorganization in the surround of the deafferented region in rat visual cortex.     

NMDA receptor-mediated changes of spontaneous activity patterns in thalamocortical slice cultures

Spontaneous activity is a hallmark of the thalamocortical system in vivo. Up until now, in vitro preparations of this system have been shown to be spontaneously active only when inhibition was reduced or N-methyl-D-aspartate (NMDA) receptor-mediated currents were facilitated via low extracellular magnesium levels. This study investigated the dependence of spontaneous thalamocortical activity patterns on NMDA receptor function via variation of extracellular magnesium levels (0-1 mM) and by the application of the specific NMDA receptor-antagonist D-2-amino-5-phosphonovalerate (AP5) in the absence of magnesium. We used cocultures of rat neocortical and thalamic slices which have been shown to develop reciprocal synaptic connections similar to those in vivo. Multi-site extracellular recordings revealed that the cultures were spontaneously active at all concentrations of magnesium and AP5, albeit with a high variability among cultures. Activity consisted of burst-like events which were largely synchronized within as well as among the neural tissues, and thalamic background activity during periods of neocortical quiescence. Each tissue was capable of triggering activity in the other, indicating that both thalamocortical and corticothalamic synaptic connections were functional. With increasing magnesium concentration, activity rates declined in both tissues and the site of origin of the synchronous, burst-like events shifted from neocortex to thalamus. AP5 in magnesium-free perfusion solution had qualitatively similar effects. We conclude that thalamic activity is not as dependent on the facilitation of NMDA receptor-mediated currents as neocortical activity and consequently, that the thalamus is the pacemaker of thalamocortical synchronized activity in physiological in vitro conditions.     

Changes in neuronal migration in neocortex of connexin43 null mutant mice

To identify a neural phenotype in connexin43 null mutant mice, electrophysiological properties, intercellular communication and neuronal migration were studied in the developing neocortex. In acute slice preparations from newborn mice, electrophysiological characteristics of cortical and hippocampal neurons were not significantly different between wild type and null mutant mice. However, gap junctional coupling as assessed by fluorescence recovery after photobleaching was significantly attenuated in neocortical brain slices of null mutant mice. To assess neuronal migration, dividing cells were labeled with bromodeoxyuridine (BrdU) on embryonic days 12, 14 and 16, respectively, corresponding to the period of cortical neurogenesis, and the neocortex examined 2 or 3 days after the labeling. BrdU-labeled cells were distributed in the neocortical wall with a significant change in the pattern in the neocortex of the null mutant, where labeled cells accumulated in the intermediate zone or in the inner part of the cortical plate. The result suggests a significant delay in neocortical neuronal migration in the connexin43 null mutants, and a possible role of connexin43 in this process through yet unidentified mechanisms.     

Lateral hypothalamus: early developmental expression and response to hypocretin (orexin)

Hypocretin is a recently discovered peptide that is synthesized by neurons in the lateral hypothalamic area (LH) and is believed to play a role in sleep regulation, arousal, endocrine control, and food intake. These functions are critical for the development of independent survival. We investigated the developmental profile of the hypocretin system in rats. Northern blot analysis showed that the expression of hypocretin mRNA increased from postnatal day 1 to adulthood. Both of the identified hypocretin receptor mRNAs were strongly expressed very early in hypothalamic development, and expression subsequently decreased in the mature brain. Immunocytochemistry revealed hypocretin-2 peptide expression in the cell bodies of LH neurons and in axons in the brain and spinal cord as early as embryonic day 19. Whole-cell patch clamp recordings from postnatal P1-P14 LH slices demonstrated a robust increase in synaptic activity in all LH neurons tested (n = 20) with a 383% increase in the frequency of spontaneous activity upon hypocretin-2 (1.5 microM) application. A similar increase in activity was found with hypocretin-1 application to LH slices. Hypocretin-2 evoked a robust increase in synaptic activity even on the earliest day tested, the day of birth. Furthermore, voltage-clamp recordings and calcium digital imaging experiments using cultured LH cells revealed that both hypocretin-1 and -2 induced enhancement of neuronal activity occurred as early as synaptic activity was detected. Thus, as in the adult central nervous system, hypocretin exerts a profound excitatory influence on neuronal activity early in development, which might contribute to the development of arousal, sleep regulation, feeding, and endocrine control.     

Initial expression and endogenous activation of NMDA channels in early neocortical development.

We have made patch-clamp recordings from slices of fetal and postnatal rat neocortex in order to study the initial expression and activation of NMDA channels. Recordings from both whole cells and outside-out patches indicated that functional NMDA channels are expressed on neurons within the cortical plate, but not on younger cells within the ventricular zone. The NMDA channels on cortical plate neurons had a unitary conductance of approximately 40 pS, had a mean open time of approximately 6 msec, required glycine to open, and were blocked in a voltage-dependent manner by magnesium. These precocious channels were present before the appearance of functional synaptic activity, yet like NMDA channels in the mature neocortex, they were spontaneously activated by an agonist within brain slices. These results demonstrate that NMDA channels are initially expressed on neocortical neurons some time between the last mitotic division within the ventricular zone and completion of migration into the cortical plate. These early NMDA channels have properties characteristic of NMDA channels on more mature neurons and are similarly activated by an endogenous agonist in situ. Their early appearance and activation indicate that NMDA channels may play a role during early stages of cortical development.     

Cellular calcium handling in brain slices from calbindin D28k-deficient mice.

Cellular calcium handling was examined in brain slices from transgenic antisense mice with a regional deficiency in the neuronal calcium binding protein calbindin D28k and from their non transgenic wild type litter mate controls. Depolarization of brain slices with NMDA or potassium produced a prolonged elevation of neuronal calcium signal in neurons in brain slices from calbindin D28k-deficient transgenic mice. This effect was selective and was seen only in brain areas where the antisense construct produced a significant depletion of calbindin D28k protein. In other regions where calbindin D28k protein was not modified by the construct and in all glial cells whether from wild type or transgenic mice, cellular calcium handling was normal.     

Modulation of synaptic transmission in neocortex by network activities

Neocortical neurons integrate inputs from thousands of presynaptic neurons that fire in vivo with frequencies that can reach 20 Hz. An important issue in understanding cortical integration is to determine the actual impact of presynaptic firing on postsynaptic neuron in the context of an active network. We used dual intracellular recordings from synaptically connected neurons or microstimulation to study the properties of spontaneous and evoked single-axon excitatory postsynaptic potentials (EPSPs) in vivo, in barbiturate or ketamine-xylazine anaesthetized cats. We found that active states of the cortical network were associated with higher variability and decrease in amplitude and duration of the EPSPs owing to a shunting effect. Moreover, the number of apparent failures markedly increased during active states as compared with silent states. Single-axon EPSPs in vivo showed mainly paired-pulse facilitation, and the paired-pulse ratio increased during active states as compare to silent states, suggesting a decrease in release probability during active states. Raising extracellular Ca(2+) concentration to 2.5-3.0 mm by reverse microdialysis reduced the number of apparent failures and significantly increased the mean amplitude of individual synaptic potentials. Quantitative analysis of spontaneous synaptic activity suggested that the proportion of presynaptic activity that impact at the soma of a cortical neuron in vivo was low because of a high failure rate, a shunting effect and probably dendritic filtering. We conclude that during active states of cortical network, the efficacy of synaptic transmission in individual synapses is low, thus safe transmission of information requires synchronized activity of a large population of presynaptic neurons.     

Calcium transient activity in cultured murine neural crest cells is regulated at the IP3 receptor

In a previous study we have shown that cultured neural crest cells exhibit spontaneous calcium transients and that these events are required for neurogenesis. In this study, we examine the mechanism that generates these calcium transients. Extracellular Ca²⁺ modulates calcium transient activity. Lanthanum (La³⁺), a general calcium channel antagonist and zero extracellular Ca²⁺, reduces the percentage of cells exhibiting calcium transients (26.2 and 40.5%, respectively) and decreases calcium spiking frequency (4.5 to 1.0 and 2.5 to 1.0 spikes/30 min, respectively). Intracellular calcium stores also contribute to the generation of calcium transients. Depleting the calcium stores of the endoplasmic reticulum (ER) reduces the percentage of active cells (15.7%) and calcium spiking frequency (2.8 to 1.5 spikes/30 min). Ryanodine (100 μM), which blocks calcium release regulated by the ryanodine receptor (RyR), had no effect on calcium transient activity. Blocking inositol 1,4,5-triphosphate receptor (IP₃R)-dependent calcium release, with elevated extracellular Mg²⁺ (20 mM), abolished calcium transient activity. Mg²⁺ did not block caffeine-sensitive calcium release (RyR-dependent) or voltage dependent calcium channels. Mg²⁺ also suppressed thimerosal-induced calcium oscillations (IP₃R-dependent). Small increases in the intracellular calcium concentration ([Ca²⁺]_i), increases the percentage of active cells and the calcium spiking frequency, while larger increases in [Ca²⁺]_i block the transients. Reducing intracellular IP₃ levels reduces the percentage of active cells and the calcium spiking frequency. We conclude that the mechanism for generating spontaneous calcium transients in cultured neural crest cells fits the model for IP₃R-dependent calcium excitability of the ER.     

Synchronized population oscillation of excitatory synaptic potentials dependent of calcium-induced calcium release in rat neocortex layer II/III neurons

We examined the roles played by calcium-induced calcium release from ryanodine-sensitive calcium stores in induction of neocortical membrane potential oscillation by using caffeine, an agonist of ryanodine receptors. Intracellular recordings were made from neurons in layer II/III of rat visual cortex slices in a caffeine-containing medium. White matter stimulation initially evoked monophasic synaptic potentials. As low-frequency stimulation continued for over 10 min, an oscillating synaptic potential gradually became evoked, in which a paroxysmal depolarization shift was followed by a 8-10-Hz train of several depolarizing wavelets. This oscillating potential was not induced in a medium containing no caffeine with 2 or 0.5 mM [Mg2+](o). Under blockade of N-methyl-D-aspartate receptors, induction of this oscillating potential failed even with caffeine application. Experiments with the calcium store depletor, thapsigargin, revealed that this oscillating potential is induced in a manner dependent on intracellular calcium release. Dual intracellular recordings revealed that the oscillation was synchronized in pairs of layer II/III neurons. The oscillating potential was detectable by field potential recordings also, suggesting that the present oscillation seems to reflect a network property.