Evolution of cortical neurons supporting human cognition.

Human cognitive abilities are generally thought to arise from cortical expansion over the course of human brain evolution. In addition to increased neuron numbers, this cortical expansion might be driven by adaptations in the properties of single neurons and their local circuits. We review recent findings on the distinct structural, functional, and transcriptomic features of human cortical neurons and their organization in cortical microstructure. We focus on the supragranular cortical layers, which showed the most prominent expansion during human brain evolution, and the properties of their principal cells: pyramidal neurons. We argue that the evolutionary adaptations in neuronal features that accompany the expansion of the human cortex partially underlie interindividual variability in human cognitive abilities.

Verbal and General IQ Associate with Supragranular Layer Thickness and Cell Properties of the Left Temporal Cortex.

The left temporal lobe is an integral part of the language system and its cortical structure and function associate with general intelligence. However, whether cortical laminar architecture and cellular properties of this brain area relate to verbal intelligence is unknown. Here, we addressed this using histological analysis and cellular recordings of neurosurgically resected temporal cortex in combination with presurgical IQ scores. We find that subjects with higher general and verbal IQ scores have thicker left (but not right) temporal cortex (Brodmann area 21, BA21). The increased thickness is due to the selective increase in layers 2 and 3 thickness, accompanied by lower neuron densities, and larger dendrites and cell body size of pyramidal neurons in these layers. Furthermore, these neurons sustain faster action potential kinetics, which improves information processing. Our results indicate that verbal mental ability associates with selective adaptations of supragranular layers and their cellular micro-architecture and function in left, but not right temporal cortex.

Third harmonic generation imaging for fast, label-free pathology of human brain tumors.

In brain tumor surgery, recognition of tumor boundaries is key. However, intraoperative assessment of tumor boundaries by the neurosurgeon is difficult. Therefore, there is an urgent need for tools that provide the neurosurgeon with pathological information during the operation. We show that third harmonic generation (THG) microscopy provides label-free, real-time images of histopathological quality; increased cellularity, nuclear pleomorphism, and rarefaction of neuropil in fresh, unstained human brain tissue could be clearly recognized. We further demonstrate THG images taken with a GRIN objective, as a step toward in situ THG microendoscopy of tumor boundaries. THG imaging is thus a promising tool for optical biopsies.

Unbiased estimation of Langevin dynamics from time series with application to hippocampal field potentials in vitro.

The last decade showed an increased interest in Langevin equations for modeling time series recorded from complex dynamical systems. These equations allow to discriminate between deterministic (drift) and stochastic (diffusion) components of the recorded time series. In practice, the estimation of drift and diffusion is often based on approximations of the models' dynamics that are valid only for high sampling frequencies. Also, model assessment is not or only indirectly performed, potentially leading to false claims. In this study we compare the performance of an asymptotically unbiased estimation method with a generally used approximate method, demonstrating the necessity of using (asymptotically) unbiased estimators. Furthermore, we describe how confidence intervals for the unknown parameters can be constructed and how model assessment can be carried out. We apply the methodology to local field potentials recorded in vitro from mouse hippocampus from eight genetically different strains. The recorded field potentials turn out to be well described by linearly damped Langevin equations with parabolic diffusion. The modeling enables a dynamical interpretation of the spectral power of the field potentials. It reveals that observed spectral power differences in the field potentials across hippocampal regions are associated with differences in the deterministic component of the system, and it reveals transiently active current dipoles, which are not detectable by conventional methods. Also, all estimated parameters have significant heritabilities, which suggests that the Langevin equations capture biological relevant aspects of electrical hippocampal activity.

Slow brain oscillations of sleep, resting state, and vigilance.

The most important quest of cognitive neuroscience may be to unravel the mechanisms by which the brain selects, links, consolidates, and integrates new information into its neuronal network, while preventing saturation to occur. During the past decade, neuroscientists working within several disciplines have observed an important involvement of the specific types of brain oscillations that occur during sleep--the cortical slow oscillations; during the resting state--the fMRI resting state networks including the default-mode network (DMN); and during task performance--the performance modulations that link as well to modulations in electroencephalography or magnetoencephalography frequency content. Understanding the role of these slow oscillations thus appears to be essential for our fundamental understanding of brain function. Brain activity is characterized by oscillations occurring in spike frequency, field potentials or blood oxygen level-dependent functional magnetic resonance imaging signals. Environmental stimuli, reaching the brain through our senses, activate or inactivate neuronal populations and modulate ongoing activity. The effect they sort is to a large extent determined by the momentary state of the slow endogenous oscillations of the brain. In the absence of sensory input, as is the case during rest or sleep, brain activity does not cease. Rather, its oscillations continue and change with respect to their dominant frequencies and coupling topography. This chapter briefly introduces the topics that will be addressed in this dedicated volume of Progress in Brain Research on slow oscillations and sets the stage for excellent papers discussing their molecular, cellular, network physiological and cognitive performance aspects. Getting to know about slow oscillations is essential for our understanding of plasticity, memory, brain structure from synapse to DMN, cognition, consciousness, and ultimately for our understanding of the mechanisms and functions of sleep and vigilance.

Single-shot two-dimensional full-range optical coherence tomography achieved by dispersion control.

We present a full-range Fourier-domain optical coherence tomography (OCT) system that is capable of acquiring two-dimensional images of living tissue in a single shot. By using line illumination of the sample in combination with a two-dimensional imaging spectrometer, 1040 depth scans are performed simultaneously on a sub-millisecond timescale. Furthermore, we demonstrate an easy and flexible real-time single-shot technique for full-range (complex-conjugate cancelled) OCT imaging that is compatible with both two-dimensional as well as ultrahigh-resolution OCT. By implementing a dispersion imbalance between reference and sample arms of the interferometer, we eliminate the complex-conjugate signal through numerical dispersion compensation, effectively increasing the useful depth range by a factor of two. The system allows us to record 6.7 x 3.2 mm images at 5 microm depth resolution in 0.2 ms. Data postprocessing requires only 4 s. We demonstrate the capability of our system by imaging the anterior chamber of a mouse eye in vitro, as well as human skin in vivo.

Reduced 5-HT1A- and GABAB receptor function in dorsal raphé neurons upon chronic fluoxetine treatment of socially stressed rats.

Autoinhibitory serotonin 1A receptors (5-HT(1A)) in dorsal raphé nucleus (DRN) have been implicated in chronic depression and in actions of selective serotonin reuptake inhibitors (SSRI). Due to experimental limitations, it was never studied at single-cell level whether changes in 5-HT(1A) receptor functionality occur in depression and during SSRI treatment. Here we address this question in a social stress paradigm in rats that mimics anhedonia, a core symptom of depression. We used whole cell patch-clamp recordings of 5-HT- and baclophen-induced G-protein-coupled inwardly rectifying potassium (GIRK) currents as a measure of 5-HT(1A)- and GABA(B) receptor functionality. 5-HT(1A)- and GABA(B) receptor-mediated GIRK-currents were not affected in socially stressed rats, suggesting that there was no abnormal (auto)inhibition in the DRN on social stress. However, chronic fluoxetine treatment of socially stressed rats restored anticipatory behavior and reduced the responsiveness of 5-HT(1A) receptor-mediated GIRK currents. Because GABA(B) receptor-induced GIRK responses were also suppressed, fluoxetine does not appear to desensitize 5-HT(1A) receptors but rather one of the downstream components shared with GABA(B) receptors. This fluoxetine effect on GIRK currents was also present in healthy animals and was independent of the animal's "depressed" state. Thus our data show that symptoms of depression after social stress are not paralleled by changes in 5-HT(1A) receptor signaling in DRN neurons, but SSRI treatment can alleviate these behavioral symptoms while acting strongly on the 5-HT(1A) receptor signaling pathway.

NMDA receptors trigger neurosecretion of 5-HT within dorsal raphe nucleus of the rat in the absence of action potential firing.

Activity and calcium-dependent release of neurotransmitters from the somatodendritic compartment is an important signalling mechanism between neurones throughout the brain. NMDA receptors and vesicles filled with neurotransmitters occur in close proximity in many brain areas. It is unknown whether calcium influx through these receptors can trigger the release of somatodendritic vesicles directly, or whether postsynaptic action potential firing is necessary for release of these vesicles. Here we addressed this question by studying local release of serotonin (5-HT) from dorsal raphé nucleus (DRN) neurones. We performed capacitance measurements to monitor the secretion of vesicles in giant soma patches, in response to short depolarizations and action potential waveforms. Amperometric measurements confirmed that secreted vesicles contained 5-HT. Surprisingly, two-photon imaging of DRN neurones in slices revealed that dendritic calcium concentration changes in response to somatic firing were restricted to proximal dendritic areas. This implied that alternative calcium entry pathways may dominate the induction of vesicle secretion from distal dendrites. In line with this, transient NMDA receptor activation, in the absence of action potential firing, was sufficient to induce capacitance changes. By monitoring GABAergic transmission onto DRN 5-HT neurones in slices, we show that endogenous NMDA receptor activation, in the absence of postsynaptic firing, induced release of 5-HT, which in turn increased the frequency of GABAergic inputs through activation of 5-HT(2) receptors. We propose here that calcium influx through NMDA receptors can directly induce postsynaptic 5-HT release from DRN neurones, which in turn may facilitate GABAergic input onto these cells.

Long-term potentiation of excitatory inputs to brain reward areas by nicotine.

Nicotine reinforces smoking behavior by activating nicotinic acetylcholine receptors (nAChRs) in the midbrain dopaminergic (DA) reward centers, including the ventral tegmental area (VTA). Although nicotine induces prolonged excitation of the VTA in vivo, the nAChRs on the DA neurons desensitize in seconds. Here, we show that activation of nAChRs on presynaptic terminals in the VTA enhances glutamatergic inputs to DA neurons. Under conditions where the released glutamate can activate NMDA receptors, long-term potentiation (LTP) of the excitatory inputs is induced. Both the short- and the long-term effects of nicotine required activation of presynaptic alpha7 subunit-containing nAChRs. These results can explain the long-term excitation of brain reward areas induced by a brief nicotine exposure. They also show that nicotine alters synaptic function through mechanisms that are linked to learning and memory.

Regulation of exocytosis in neuroendocrine cells: spatial organization of channels and vesicles, stimulus-secretion coupling, calcium buffers and modulation.

Neuroendocrine cells display a similar calcium dependence of release as synapses but a strongly different organization of channels and vesicles. Biophysical and biochemical properties of large dense core vesicle release in neuroendocrine cells suggest that vesicles and channels are dissociated by a distance of 100-300 nm. This distinctive organization relates to the sensitivity of the release process to mobile calcium buffers, the resulting relationship between calcium influx and release and the modulatory mechanisms regulating the efficiency of excitation-release coupling. At distances of 100-300 nm, calcium buffers determine the calcium concentration close to the vesicle. Notably, the concentration and diffusion rate of mobile buffers affect the efficacy of release, but local saturation of buffers, possibly enhanced by diffusion barriers, may limit their effects. Buffer conditions may result in a linear relationship between calcium influx and exocytosis, in spite of the third or fourth power relation between intracellular calcium concentration and release. Modulation of excitation-secretion coupling not only concerns the calcium channels, but also the secretory process. Transmitter regulation mediated by cAMP and PKA, as well as use-dependent regulation involving calcium, primarily stimulates filling of the releasable pool. In addition, direct effects of cAMP on the probability of release have been reported. One mechanism to achieve increased release probability is to decrease the distance between channels and vesicles. GTP may stimulate release independently from calcium. Thus, while in most cases primary inputs triggering these pathways await identification, it is evident that large dense core vesicle release is a highly controlled and flexible process.

All classes of calcium channel couple with equal efficiency to exocytosis in rat melanotropes, inducing linear stimulus-secretion coupling.

1. The contribution of low voltage-activated (LVA) T-type Ca2+ channels and four different types of high voltage-activated (HVA) Ca2+ channel to exocytosis, and the relationship between calcium influx and exocytosis during action potentials (APs) were studied in pituitary melanotropes. 2. Selective HVA Ca2+ channel blockers reduced exocytosis, monitored by membrane capacitance measurements, proportional to the reduction in Ca2+ influx. The efficacy of Ca2+ in stimulating exocytosis did not change in the presence of the Ca2+ channel blockers, indicating that all HVA Ca2+ channels act together in stimulating exocytosis. 3. The relationship between Ca2+ influx and exocytosis during the AP was examined using APs recorded from spontaneously active melanotropes as command templates under voltage clamp. Under voltage clamp, multiphasic Ca2+ currents were activated over the entire duration of the APs, i.e. during the rising phase as well as the plateau phase. The maximum amplitude of the Ca2+ current coincided with the peak of the AP. 4. The relationship between Ca2+ entry and exocytosis was linear for the different phases of the AP. Also, the influx of Ca2+ through LVA T-type channels stimulated exocytosis with the same efficacy as through the HVA channels. 5. APs of increasing duration ( approximately 50 to approximately 300 ms) evoked increasing amounts of exocytosis. The number of entering Ca2+ ions and the capacitance change were linearly related to AP duration, resulting in a fixed relationship between Ca2+ entry and exocytosis. 6. The results show that Ca2+ ions, entering a melanotrope, couple with equal strength to exocytosis regardless of the channel type involved. We suggest that the linear relationship between Ca2+ entry and secretion observed under physiological conditions (during APs), results from the equal strength with which LVA and HVA channels in melanotropes couple to exocytosis. This guarantees that secretion takes place over the entire duration of the AP.

Calcium channels and the release of large dense core vesicles from neuroendocrine cells: spatial organization and functional coupling.

The release of large dense core vesicles (LDCV) by neuroendocrine cells displays a very similar calcium dependence as found in synapses, yet, the organization of channels and vesicles is quite different. Various biophysical properties of the release process, notably a large delay (>10 ms) between excitation and release and a high impact of mobile calcium buffers, suggest that, generally, vesicles and channels do not co-localize as in synapses, but are separated by a distance of 100-300 nm. This review focuses on the consequences of this organization for the functional coupling of calcium channels to LDCV-release in neuroendocrine cells. The large distance between LDCV and calcium channels in neuroendocrine cells obviates molecular interactions between channels and fusion peptides and implies that each type of calcium channel may be involved in release. Thus, preferential functional coupling of specific calcium channel types to the exocytotic process may be completely lacking, as in melanotropes. Alternatively, it may be present to some extent to induce differences in coupling efficacy between channel types, as in calf chromaffin cells and mouse pancreatic beta-cells. Physiological mechanisms, like recruitment of channels through facilitation processes or suppression of channels through inactivation, may change coupling characteristics during activity. Due to the large distance between channels and vesicles, single action potentials (APs) are usually insufficient to elicit release, and the coupling between individual APs and release is loose. Most neuroendocrine cells are therefore seen to fire in bursts, like pancreatic beta-cells. Furthermore, a large variation in shape and duration of the APs, with APs of up to 300 ms as in melanotropes, acts as another mechanism to enhance stimulus secretion coupling.

Roles of G-protein beta gamma, arachidonic acid, and phosphorylation inconvergent activation of an S-like potassium conductance by dopamine, Ala-Pro-Gly-Trp-NH2, and Phe-Met-Arg-Phe-NH2.

Dopamine and the neuropeptides Ala-Pro-Gly-Trp-NH2 (APGWamide or APGWa) and Phe-Met-Arg-Phe-NH2 (FMRFamide or FMRFa) all activate an S-like potassium channel in the light green cells of the mollusc Lymnaea stagnalis, neuroendocrine cells that release insulin-related peptides. We studied the signaling pathways underlying the responses, the role of the G-protein betagamma subunit, and the interference by phosphorylation pathways. All responses are blocked by an inhibitor of arachidonic acid (AA) release, 4-bromophenacylbromide, and by inhibitors of lipoxygenases (nordihydroguaiaretic acid and AA-861) but not by indomethacin, a cyclooxygenase inhibitor. AA and phospholipase A2 (PLA2) induced currents with similar I-V characteristics and potassium selectivity as dopamine, APGWa, and FMRFa. PLA2 occluded the response to FMRFa. We conclude that convergence of the actions of dopamine, APGWa, and FMRFa onto the S-like channel occurs at or upstream of the level of AA and that formation of lipoxygenase metabolites of AA is necessary to activate the channel. Injection of a synthetic peptide, which interferes with G-protein betagamma subunits, inhibited the agonist-induced potassium current. This suggests that betagamma subunits mediate the response, possibly by directly coupling to a phospholipase. Finally, the responses to dopamine, APGWa, and FMRFa were inhibited by activation of PKA and PKC, suggesting that the responses are counteracted by PKA- and PKC-dependent phosphorylation. The PLA2-activated potassium current was inhibited by 8-chlorophenylthio-cAMP but not by 12-O-tetradecanoylphorbol 13-acetate (TPA). However, TPA did inhibit the potassium current induced by irreversible activation of the G-protein using GTP-gamma-S. Thus, it appears that PKA targets a site downstream of AA formation, e.g., the potassium channel, whereas PKC acts at the active G-protein or the phospholipase.

Diffusion barriers limit the effect of mobile calcium buffers on exocytosis of large dense cored vesicles.

Fast exocytosis in melanotropic cells, activated by calcium entry through voltage-gated calcium channels, is very sensitive to mobile calcium buffers (complete block at 800 microM ethylene glycol bis(beta-aminoethyl ether)-N,N,N'N'-tetraacetic acid (EGTA)). This indicates that calcium diffuses a substantial distance from the channel to the vesicle. Surprisingly, 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), having a similar KD for calcium as EGTA but a approximately 100 times faster binding rate, blocked exocytosis only twice as effectively as EGTA. Using computer simulations, we demonstrate that this result cannot be explained by free diffusion and buffer binding rates. We hypothesized that local saturation of calcium buffers is involved. A diffusion barrier for both calcium and buffer molecules, located 50-300 nm from the membrane and reducing diffusion 1000 to 10,000 times, generated similar calcium concentrations for specific concentrations of EGTA and BAPTA. With such barriers, calcium rise phase kinetics upon short step depolarizations (2-20 ms) were faster for EGTA than for BAPTA, implying that short depolarizations should allow exocytosis with 50 microM EGTA but not with 25 microM BAPTA. This prediction was confirmed experimentally with capacitance measurements. Coupling exocytosis to calcium dynamics in the model, we found that a barrier with a approximately 3000 times reduced diffusion at approximately 130 nm beneath the membrane best explains the experimentally observed effects of EGTA and BAPTA on block and kinetics of release.

The relation of exocytosis and rapid endocytosis to calcium entry evoked by short repetitive depolarizing pulses in rat melanotropic cells.

Melanotropic cells release predocked, large, dense-cored vesicles containing alpha-melanocyte stimulating hormone in response to calcium entry through voltage-gated calcium channels. Our first objective was to study the relationship between exocytosis, rapid endocytosis, and calcium entry evoked by short step depolarizations in the order of duration of single action potentials (APs). Exocytosis and rapid endocytosis were monitored by capacitance measurements. We show that short step depolarizations (40 msec) evoke the fast release of only approximately 3% of the predocked release-ready vesicle pool. Second, we asked what the distance is between voltage-gated calcium channels and predocked vesicles in these cells by modulating the intracellular buffer capacity. Exocytosis and rapid endocytosis were differentially affected by low concentrations of the calcium chelator EGTA. EGTA slightly attenuated exocytosis at 100 microM relative to 50 microM, but exocytosis was strongly depressed at 400 microM, showing that calcium ions have to travel a large distance to stimulate exocytosis. Nevertheless, the efficacy of calcium ions to stimulate exocytosis was constant for pulse durations between 2 and 40 msec, indicating that in melanotropes, exocytosis is related linearly to the amount and duration of calcium entry during a single AP. Rapid endocytosis was already strongly depressed at 100 microM EGTA, which shows that the process of endocytosis itself is calcium dependent in melanotropic cells. Furthermore, rapid endocytosis proceeded with a time constant of approximately 116 msec at 33 degrees C, which is three times faster than at room temperature. There was a strong correlation between the amplitude of endocytosis and the amplitude of exocytosis immediately preceding endocytosis. Both this correlation and the fast time constant of endocytosis suggest that the exocytotic vesicle is retrieved rapidly.

Pattern changes of pituitary peptides in rat after salt-loading as detected by means of direct, semiquantitative mass spectrometric profiling.

We have established a differential peptide display method, based on a mass spectrometric technique, to detect peptides that show semiquantitative changes in the neurointermediate lobe (NIL) of individual rats subjected to salt-loading. We employed matrix-assisted laser desorption/ionization mass spectrometry, using a single-reference peptide in combination with careful scanning of the whole crystal rim of the matrix-analyte preparation, to detect in a semiquantitative manner the molecular ions present in the unfractionated NIL homogenate. Comparison of the mass spectra generated from NIL homogenates of salt-loaded and control rats revealed a selective and significant decrease in the intensities of several molecular ion species of the NIL homogenates from salt-loaded rats. These ion species, which have masses that correspond to the masses of oxytocin, vasopressin, neurophysins, and an unidentified putative peptide, were subsequently chemically characterized. We confirmed that the decreased molecular ion species are peptides derived exclusively from propressophysin and prooxyphysin (i.e., oxytocin, vasopressin, and various neurophysins). The putative peptide is carboxyl-terminal glycopeptide. The carbohydrate moiety of the latter peptide was determined by electrospray tandem MS as bisected biantennary Hex3HexNAc5Fuc. This posttranslational modification accounts for the mass difference between the predicted mass of the peptide based on cDNA studies and the measured mass of the mature peptide.

Dihydropyridine block of omega-agatoxin IVA- and omega-conotoxin GVIA-sensitive Ca2+ channels in rat pituitary melanotropic cells.

High voltage-activated Ca2+ currents in rat melanotropic cells consist of a sustained and an inactivating component. In this study the pharmacological properties of the high voltage-activated Ca2+ channels underlying these components are investigated with whole-cell recordings. We report that melanotropes express four pharmacologically distinct high voltage-activated Ca2+ channels. Non-inactivating L-type channels account for 35% of the total high voltage-activated channel population. These channels have a very high affinity for the dihydropyridine nimodipine (EC50 approximately 3 pM). The cone snail toxin omega-conotoxin GVIA irreversibly blocked an inactivating high voltage-activated component which accounted for 26% of the total whole-cell high voltage-activated Ca2+ current. The spider toxin omega-agatoxin IVA reversibly blocked an additional 31% of the total high voltage-activated current. The current blocked by omega-agatoxin IVA was not homogenous and consisted of a sustained component with a high affinity for omega-agatoxin IVA (< 10 nM) and an inactivating current with a low affinity for omega-agatoxin IVA (> 100 nM). Both the omega-agatoxin IVA and omega-conotoxin GVIA-blocked currents were very sensitive to nimodipine and nitrendipine with a half maximal block at 200-500 nM. 10 microM nimodipine blocked 70% of the omega-conotoxin GVIA-sensitive current and 90% of the omega-agatoxin IVA-sensitive current. Thus, omega-conotoxin GVIA- and omega-agatoxin IVA-sensitive high voltage-activated Ca2+ channels in melanotropes have an unusual high affinity for dihydropyridines compared to N-, P-, and Q-type channels in other preparations.

Voltage gated calcium channels in molluscs: classification, Ca2+ dependent inactivation, modulation and functional roles.

Molluscan neurons and muscle cells express transient (T-type like) and sustained LVA calcium channels, as well as transient and sustained HVA channels. In addition weakly voltage sensitive calcium channels are observed. In a number of cases toxin or dihydropyridine sensitivity justifies classification of the HVA currents in L, N or P-type categories. In many cases, however, pharmacological characterization is still preliminary. Characterization of novel toxins from molluscivorous Conus snails may facilitate classification of molluscan calcium channels. Molluscan preparations have been very useful to study calcium dependent inactivation of calcium channels. Proposed mechanisms explain calcium dependent inactivation through direct interaction of Ca2+ with the channel, through dephosphorylation by calcium dependent phosphatases or through calcium dependent disruption of connections with the cytoskeleton. Transmitter modulation operating through various second messenger mediated pathways is well documented. In general, phosphorylation through PKA, cGMP dependent PK or PKC facilitates the calcium channels, while putative direct G-protein action inhibits the channels. Ca2+ and cGMP may inhibit the channels through activation of phosphodiesterases or phosphatases. Detailed evidence has been provided on the role of sustained LVA channels in pacemaking and the generation of firing patterns, and on the role of HVA channels in the dynamic changes in action potentials during spiking, the regulation of the release of transmitters and hormones, and the regulation of growth cone behavior and neurite outgrowth. The accessibility of molluscan preparations (e.g. the squid giant synapse for excitation release studies, Helisoma B5 neuron for neurite and synapse formation) and the large body of knowledge on electrophysiological properties and functional connections of identified molluscan neurons (e.g. sensory neurons, R15, egg laying hormone producing cells, etc.) creates valuable opportunities to increase the insight into the functional roles of calcium channels.