Kainic acid responses and toxicity show pronounced Ca dependence

Responses of pyramidal neurons to ionophoretic kainate, quisqualate and N-methyl aspartate were studied in a submerged rat piriform cortex slice as a function of Ca²⁺ and Mg²⁺ concentrations. The results suggest that (a) the channel activated by kainate is unusually influenced by Ca²⁺, (b) excitotoxicity is Ca²⁺-dependent and a function of Ca²⁺ concentration, and (c) the excitotoxic actions of various amino acid agonists are correlated with the Ca²⁺ dependence of their responses.     

Kainic acid induction of mossy fiber sprouting: dependence on mouse strain

After seizures caused by kindling or kainic acid (KA), hippocampal granule-cell axons, the mossy fibers, sprout into the supragranular layer of the rat. The mechanisms underlying this phenomenon remain elusive, but excitotoxic loss of hilar cells, which project to this supragranular layer, is suspected to be a critical determinant. Consistent with this hypothesis, we previously reported that while rats show mossy fiber sprouting after kainate, ICR mice do not. This may be associated with the observation that ICR mice, unlike rats, do not appear to show hilar cell death after KA (McNamara et al., Mol Brain Res 1996;40:177-187). Other strains of mice, however, such as 129/SvEMS, do show hilar cell death after KA (Schauwecker and Steward, Proc Natl Acad Sci USA 1997;94:4103-4108). We examined the possibility that the 129/SvEMS mouse strain would show granule-cell sprouting, in contrast to ICR mice. After administration of KA, mossy fiber sprouting was indeed observed in strain 129/SvEMS, but only in animals displaying evident hilar cell death. In contrast, neither hilar cell death nor mossy fiber sprouting was observed in ICR mice, confirming previous results. Both mouse strains demonstrated comparable behavioral seizures. These results strengthen the view that hilar cell death, together with epileptogenesis, triggers reactive synaptogenesis and mossy fiber sprouting.     

Heterogeneity in low voltage-activated Ca2+ channel-evoked Ca2+ responses within neurons of the thalamic paraventricular nucleus

Low voltage-activated Ca2+ channels (LVA or T-type Ca2+ channels) are crucial to burst firing and oscillations in thalamocortical relay cells and are exhibited by neurons in the paraventricular nucleus of thalamus (PVT), a dorsal midline nucleus deemed important in the neural representation of motivational behaviours. We used a functional approach (whole-cell patch-clamp electrophysiology combined with confocal laser scanning microscopy) to analyse the spatial distribution of LVA Ca2+ channel-evoked Ca2+ transients in PVT neurons. We observed that the magnitude of LVA Ca2+ channel-evoked Ca2+ transients was significantly greater in proximal dendrites (located up to 20 microm from the soma) than in the soma. In addition, the magnitudes of these Ca2+ transients varied significantly not only among different dendrites of the same cell but also within individual dendrites. These findings suggest that LVA Ca2+ channels are expressed (i) predominantly on the proximal dendrites and (ii) heterogeneously within individual dendrites of PVT neurons. The spatial characteristics of dendritic LVA Ca2+ channels in PVT neurons suggest that these channels may regulate burst firing by modulating dendritic afferent inputs.     

Fura-2 measurements of cultured rat Purkinje neurons show dendritic localization of Ca2+ influx

The specific objectives of this study were the following: (1) to characterize the types of calcium currents in cultured PCs using whole-cell voltage-clamp techniques; (2) using fura-2 imaging techniques, to monitor intracellular Ca2+ levels during application of high potassium, glutamate, or glutamate analogs; and (3) to evaluate the types of calcium channels contributing to the calcium fluxes using pharmacological blocking agents. Voltage-clamp analysis of calcium currents proved to be difficult due to space-clamping problems. The latter was presumably due to the unfavorable geometry of cultured PCs. Nevertheless, we found no evidence for inward currents in cells bathed in TTX-TEA-BaCl2 saline. On the other hand, fura-2 measurements demonstrated that free Ca2+ levels were elevated in PCs following local application of either high-potassium saline or glutamate. When individual cells were injected with fura-2 and analyzed in TTX-containing saline, the Ca2+ elevation was usually greater in the dendrites. Since Ca2+ levels were not elevated in all dendrites of the same cell, the smaller responses in the soma wre not simply due to volumetric differences. Together with the voltage-clamp results, the fura-2 data indicate that calcium channels were localized to certain dendrites. Using selective calcium channel blockers, we found evidence for 2 types of calcium conductances in the dendrites of cultured PCs. The Ca conductance induced by high potassium was reduced in a dose-dependent manner by nifedipine (ED50 = 5 X 10(-7) M), indicating that a high-threshold voltage-dependent calcium channel was present. The Ca response to glutamate (or NMDA) was reduced by 2-amino-5-phosphonovaleric acid (ED50 = 10(-4) M), as well as by nifedipine or 10(-4) M LaCl3, indicating that both voltage-dependent and glutamate-coupled channels were opened by glutamate application.     

Galactocerebroside and sulfatide independently mediate Ca2+ responses in oligodendrocytes.

Galactocerebroside (GalC) and sulfated galactocerebroside (sulfatide) are sphingolipids highly enriched in myelin. The binding of antibodies reactive with either sulfatide or GalC to cultured oligodendrocytes causes a Ca2+ influx, followed by microtubule depolymerization; however, antisulfatide is less effective than anti-GalC in altering cytoskeleton. Typical Ca2+ responses are delayed for both antibodies but are transient for sulfatide-reactive antibodies in contrast to the sustained responses previously reported for anti-GalC (Dyer and Benjamins, J Cell Biol 111: 625-633, 1990). Approximately one-half as many oligodendrocytes respond to sulfatide-reactive antibodies (about 39%) as to anti-GalC (about 75%). Subpopulations of oligodendrocytes were identified that responded to neither antibody, only one antibody, or both antibodies, indicating that sulfatide and GalC independently mediate Ca2+ responses. These results suggest that sulfatide and GalC have different physiologic roles in regulating elaboration of myelin membrane by oligodendrocytes in vivo and support the possibility that viral or immune attack via GalC or sulfatide on oligodendrocytes may mimic normal signals in a manner that disrupts the sequence of events that coordinates myelination or maintenance of myelin in vivo.     

Synaptotagmin 1 is necessary for the Ca2+ dependence of clathrin-mediated endocytosis

The role of Ca2? in synaptic vesicle endocytosis remains uncertain due to the diversity in various preparations where several forms of endocytosis may contribute variably in different conditions. Although recent studies have demonstrated that Ca2? is important for clathrin-mediated endocytosis (CME), the mechanistic role of Ca2? in CME remains to be elucidated. By monitoring CME of single vesicles in mouse chromaffin cells with cell-attached capacitance measurements that offer millisecond time resolution, we demonstrate that the dynamics of vesicle fission during CME is Ca2? dependent but becomes Ca2? independent in synaptotagmin 1 (Syt1) knock-out cells. Our results thus suggest that Syt1 is necessary for the Ca2? dependence of CME.     

Alpha-synuclein potentiates Ca2+ influx through voltage-dependent Ca2+ channels

Alpha-synuclein localized in synaptic terminals plays an important role in the pathogenesis of neurodegenerative diseases. The central domain of the protein, the nonamyloid component, is probably responsible for alpha-synuclein toxicity. Here, we report that alpha-synuclein and its nonamyloid component induced Ca2+ influx in rat synaptoneurosomes. The effect of alpha-synuclein was eliminated by the N-type specific Ca2+ channel blocker, omega-conotoxin GVIA. The antioxidant, resveratrol, and the nitric oxide synthase inhibitor, Nomega-nitro-L-arginine, did not prevent alpha-synuclein-induced Ca2+ influx. Our findings indicate that alpha-synuclein stimulated Ca2+ influx through N-type voltage-dependent Ca2+ channels by a mechanism other than free radicals. A direct interaction between alpha-synuclein and N-type Ca2+ channels could be responsible for their effects on Ca2+ influx through voltage-dependent Ca2+ channels.     

Nerve growth factor-induced neurite outgrowth of rat pheochromocytoma PC 12 cells: dependence on extracellular Mg2+ and Ca2+

Dependence of neurite outgrowth on extracellular Mg2+ and Ca2+ was studied in nerve growth factor-responsive pheochromocytoma PC 12 cells under assay conditions in which neurite formation was independent of both RNA synthesis and protein synthesis. NGF-induced neurite formation occurred maximally in the presence of extracellular Mg2+ at concentrations greater than 1.0 mM. However, extracellular Ca2+ alone did not stimulate the neurite formation, and inhibited this process at higher concentrations (greater than 10 mM). These data are consistent with the fact that NGF-mediated neurite extension occurred in assay medium containing either 1.0 mM EGTA or 0.5 mM LaCl3. Other divalent cations so far tested proved to be negative, suggesting that this phenomenon appears to be specific to Mg2+. Moreover, quantitative analysis revealed that the length and thickness of neurites formed were controlled by the presence of extracellular Ca2+. Thus, neurites formed at lower concentrations of Ca2+ in the presence of 1.0 mM Mg2+ and NGF were found to be thinner and longer than those formed at higher concentrations of Ca2+, suggesting that Ca2+ and Mg2+ have separate regulatory functions in the formation of neurites of PC 12 cells.     

Toluene inhibits muscarinic receptor-mediated cytosolic Ca2+ responses in neural precursor cells

Toluene is widely used as a component in industrial solvents and many toluene-containing products are abused via inhalation. While many studies have demonstrated its inhibitory effects on neuronal activity, the effects of toluene on receptor signaling in proliferating and differentiating neural precursor cells are presently unclear. Here, using digital video microscopy and Ca2+ imaging, we investigated the effects of acute exposure to toluene on the function of muscarinic acetylcholine receptors (mAChRs) expressed in neural precursor cells. The neural precursor cells were isolatedfrom embryonic day 13 (E13) rat cortex and expanded in serum-free medium containing basic fibroblast growth factor (bFGF). We found that the acetylcholine (ACh) analog carbachol (CCh) induced a dose-dependent increase in cytosolic Ca2+, which was blocked by the muscarinic receptor antagonist atropine in a reversible manner. Toluene was added to the perfusion medium and concentrations of toluene in the medium were determined by gas chromatographic analysis. Following imaging, the cells were fixed and processed for 5-bromo-2'-deoxyuridine (BrdU, cell proliferation marker) and beta-tubulin (TuJ1, neuronal marker) immunostaining. In the 5 day culture, most cells continued to divide (BrdU+), while afew cells differentiated into young neurons (TuJ1-). The CCh-induced Ca2+ elevations in proliferating (BrdU+TuJ1-) neural precursor cells were significantly reduced by acute exposure to 0.15 mM toluene and completely blocked by 10 mM toluene. Toluene's inhibition of muscarinic receptor-mediated Ca2+ signaling was rapid, reversible and dose-dependent with an IC50 value 0.5 mM. Since muscarinic receptors mediate cell proliferation and differentiation during neural precursor cell development, these results suggest that depression of muscarinic signaling may play a role in toluene's teratogenic effect on the developing nervous system.     

Ca2+-permeable and Ca2+-impermeable AMPA receptors coexist on horizontal cells

Fura-2 fluorescent calcium imaging was used for analyzing the subtype of AMPA receptors in freshly dissociated horizontal cells of carp retina. Exogenous application of AMPA induced an increase of intracellular concentration of free Ca2+ ([Ca2+]i) in horizontal cells, while the [Ca2+]i increase was partly inhibited by nifedipine. The residual [Ca2+]i increase was completely eliminated by joro spider toxin-3, a blocker of Ca2+-permeable AMPA receptors. On the other hand, the application of pentobarbital, which blocked Ca2+-impermeable AMPA receptors, could also partly inhibit the increase of [Ca2+]i, implying that the application of AMPA induced the activation of both Ca2+-permeable and Ca2+-impermeable AMPA receptors and the consequent activation of voltage-gated Ca2+ channels. Taken together, these results suggested that Ca2+-permeable and Ca2+-impermeable AMPA receptors were coexpressed on horizontal cells.     

AMPA receptors shape Ca2+ responses in cortical oligodendrocyte progenitors and CG-4 cells

Intracellular calcium signals triggered by glutamate receptor activation were studied in primary cortical oligodendrocyte lineage cells and in the oligodendrocyte cell line CG-4. Glutamate, kainate, and AMPA (30-300 μM) increased [Ca ²⁺]_i in both types of cells at the stage of oligodendrocyte progenitors (O-2A; GD3⁺) or pro-oligodendroblasts (04⁺). The peak amplitude of Ca²⁺ responses to glutamate receptor agonists was significantly larger in cortical cells. In CG-4 and in cortical cells, the majority (more than 90%) of bipolar GD3⁺ or multipolar 04⁺ cells responded to kamate. In all the cells analyzed, kainate was more efficacious than AMPA and glutamate. The percentage of bipolar or multipolar cells responding to glutamate was significantly lower in the CG-4 cell line than in primary cultures. Cellular responses typical of metabotropic glutamate receptor activation were observed in 20% of the cortical O-2A progenitors, but in none of the CG-4 cells. The AMPA-selective antagonist GYKI 52466 blocked kainate-induced Ca²⁺ responses in cortical O-2A cells. The selective AMPA receptor modulator cyclothiazide (30 μM) greatly potentiated the effects of AMPA (30-100 μM) on [Ca ²⁺]_i in cortical and CG-4 cells. Our findings indicate that Ca²⁺ responses in cells of the oligodendrocyte lineage are primarily shaped by functional AMPA receptors. © 1995 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Effects of Na+ and Ca2+ gradients on intracellular free Ca2+ in voltage-clamped Aplysia neurons

Selected neurons of the abdominal ganglion of Aplysia californica were voltage-clamped and intracellular free Ca [( Ca2+]i) and Na [( Na+]i) concentrations were monitored with ion selective microelectrodes. Reducing [Na+]o from 500 mM (normal seawater, NSW) to 5 mM resulted in a decrease of the potential measured by the Ca electrode (VCa). Increasing [Ca2+]o from 10 to 50 mM increased [Ca2+]i two-fold, keeping [Ca2+]o at 50 mM and decreasing [Na+]o to 5 mM still led to a decrease in VCa. With 100 mM [Ca2+]o, which also increased [Ca2+]i, decreasing [Na+]o increased VCa in two of the eight cells tested. This indicates that in normal or moderately high resting [Ca2+]i, Ca2+ extrusion by Na/Ca exchange (forward mode) is not essential for [Ca2+]i buffering. [Na+]i was 12.9 +/- 3.6 mM (S.E.M., n = 7) in NSW; reducing [Na+]o to 5 mM decreased [Na+]i to 2.0 +/- 1.1 mM (S.E.M.). Keeping [Na+]o at 5 mM and increasing [Ca2+]o from 10 to 20 mM further decreased [Na+]i to about 1.0 mM, evidence of Na/Ca exchange operating in the reverse mode. Attempts to increase [Ca2+]i by bath application of the Ca ionophores A23187, X537A, ionomycin or ETH 1001 resulted in no measurable change of the resting [Ca2+]i. Application of Ouabain caused an apparent increase in [Ca2+]i in two of the six cells tested. In cells injected with the metallochromic indicator arsenazo III (AIII), the rate of the falling phase of the AIII absorbance increase, following a voltage-clamp pulse, was significantly slower in 5 mM [Na+]o. This indicates that in its forward mode Na-Ca exchange is active in clearing large submembrane increases in [Ca2+]i.     

The effects of trimethyltin on the Ca+2, Mg+2 and Ca+2 + Mg+2-dependent ATPases of human neuroblastoma GM 3320

Data presented here indicate neuroblastoma GM 3320 tissue homogenates exhibit ouabain insensitive Ca+2-dependent, Mg+2-independent, Mg+2-dependent, Ca+2-independent and Ca+2 + Mg+2-dependent ATPase activities. Inclusion of trimethyltin in homogenate preparations of these cells appears to discriminate between these various ATPase activities. At low concentrations (25 microM), trimethyltin preferentially stimulated the Ca+2-dependent, Mg+2-independent ATPase activity while inhibiting the Ca+2 + Mg+2-ATPase activity approximately 70%. At 75 microM trimethyltin, the Ca+2 + Mg+2-dependent ATPase activity is inhibited greater than 95% while the Ca+2-dependent, Mg+2-independent activity is essentially unchanged from control activity and the Mg+2-dependent, Ca+2-independent activity is inhibited approximately 50%. At concentrations greater than 75 microM, trimethyltin significantly inhibits the Ca+2-dependent, Mg+2-independent ATPase activity. Thus, at trimethyltin concentrations of 50-75 microM, preferential inhibition of the Mg+2-dependent, Ca+2-independent and Ca+2 + Mg+2-dependent ATPase activities of neuroblastoma GM 3320 is achieved.     

Cycle length dependence of the chronotropic effects of adrenaline, acetylcholine, Ca2+ and Mg2+ in the Guinea-pig sinoatrial node

Ca (1.1-5.5 mM) has a positive chronotropic action on isolated right atria of the guinea-pig. The magnitude of the response depends on the cycle length. Magnitude and cycle length dependence of the Ca response are independent of beta-blockade by propranolol. Mg (0.6-6.0 mM) has a negative chronotropic action. At 6.0 mM it interferes with responses to adrenaline and acetylcholine by preventing pacemaker shifts. Adrenaline has a positive chronotropic action in a cycle length dependent manner. A shift of pacemaker dominance under the influence of adrenaline to an identical site in all preparations (as in the rabbit) was not observed. However, pacemaker shifts in the presence of adrenaline do occur and they are always directed towards the inferior part of the node. Acetylcholine has a negative chronotropic action, independent of cycle length. Acetylcholine also induces pacemaker shifts. Contrary to the pacemaker shifts caused by adrenaline, the new, acetylcholine-induced pacemaker center, has an identical site in all preparations. This was previously observed in the rabbit too. The acetylcholine-induced center is located about 1 mm inferior from the primary center. During exposure to acetylcholine different action potentials may be recorded at the epi- and endocardial side of the preparation, but only close to the Ach-induced center. The acetylcholine-induced center is located at the epicardial side. The response to acetylcholine predominates over the response to adrenaline. All results are discussed in comparison with our previous findings in the rabbit.     

Intercellular Ca2+ waves induce temporally and spatially distinct intracellular Ca2+ oscillations in glia

Mechanically induced intercellular Ca²⁺ waves propagated for approximately 300 μm in primary glial cultures. Following the wave propagation, 34% of the cells displayed Ca²⁺ oscillations in a zone 60–120 μm from the stimulated cell. The initiation, frequency, and duration of these Ca²⁺ oscillations were dependent on the cells' distance from the wave origin but were not dependent on the cell type nor on the magnitude of the Ca²⁺ wave. When an individual cell propagated two sequential intercellular Ca²⁺ waves originating from different sites, the characteristics of the Ca²⁺ oscillations initiated by each wave were determined by the distance of the cell from the origin of each wave. Each Ca²⁺ oscillation commonly occurred as an intracellular Ca²⁺ wave that was initiated from a specific site within the cell. The position of the initiation site and the direction of the intracellular Ca²⁺ wave were independent of the orientation of the initial intercellular Ca²⁺ wave. Because initiation and frequency of Ca²⁺ oscillations are dependent on the intracellular inositol trisphosphate concentration ([IP₃]_i), we propose that the zone of cells displaying Ca²⁺ oscillations is determined by an intercellular gradient of [IP₃]_i, established by the diffusion of IP₃ through gap junctions during the propagation of the intercellular Ca²⁺ wave. Exposure to acetylcholine, a muscarinic agonist that initiates IP₃ production, shifted the zone of oscillating cells about 45 μm farther away from the origin of the mechanically induced wave. These findings indicate that a glial syncytium can resolve information provided by a local Ca²⁺ wave into a distinct spatial and temporal pattern of Ca²⁺ oscillations. GLIA 28:97–113, 1999. © 1999 Wiley‐Liss, Inc.     

Kainic acid upregulates uncoupling protein-2 mRNA expression in the mouse brain

Intraperitoneal injection of kainic acid (KA) in C57BL/6J and 129T2SvEmsJ mice led to a transient induction of uncoupling protein-2 (Ucp2) mRNA expression in several brain regions, which included the CA1 subfield of the hippocampus, the dorsal endopiriform nucleus and the piriform cortex in both strains. In all those regions, levels of Ucp2 mRNA expression, as determined by in situ hybridization, peaked at 24 h and returned to basal levels within 72 h post-injection. The increase in mRNA expression was mainly observed in neurons, with microglial cells displaying only scattered expression of the gene. The neuronal induction of Ucp2 in response to KA was stronger in 129T2SvEmsJ mice than in C57BL/6J, which suggests a role for Ucp2 in excitotoxic challenges and neuroprotection.     

Probable Ca2+-mediated inactivation of Ca2+ currents in mammalian brain neurons

In rat hippocampal neurons, current- and single-electrode voltage-clamp analyses revealed a pronounced inactivation of probable Ca2+ currents (ICa), which was dependent on the amount of Ca2+ influx. Studies were conducted in cesium-loaded, tetrodotoxin-treated brain slice neurons in which known contaminating currents were blocked. These results therefore provide the first clear evidence that apparent Ca2+-mediated inactivation of ICa is an important mechanism with which mammalian brain neurons limit Ca2+ influx.     

Synaptic vesicle Ca2+/H+ antiport: dependence on the proton electrochemical gradient.

Synaptic vesicles isolated from sheep brain cortex accumulate Ca2+ by a mechanism of secondary active transport associated to the H(+)-pump activity. The process can be visualized either by measuring Ca(2+)-induced H+ release or DeltapH-dependent Ca2+ accumulation. We observed that the amount of Ca2+ taken up by the vesicles increases with the magnitude of the DeltapH across the membrane, particularly at Ca2+ concentrations (approximately 500 microM) found optimal for the antiporter activity. Similarly, H+ release induced by Ca2+ increased with the magnitude of DeltapH. However, above 60% DeltapH (high H(+)-pump activity), the net H+ release from the vesicles decreased as the pump-mediated H+ influx exceeded the Ca(2+)-induced H+ efflux. We also observed that the Ca2+/H+ antiport activity depends, essentially, on the DeltapH component of the electrochemical gradient (approximately 3 nmol Ca2+ taken up/mg protein), although the Deltaphi component may also support some Ca2+ accumulation by the vesicles (approximately 1 nmol/mg protein) in the absence of DeltapH. Both Ca(2+)-induced H+ release and DeltapH-dependent Ca2+ uptake could be driven by an artificially imposed proton motive force. Under normal conditions (H+ pump-induced DeltapH), the electrochemical gradient dependence of Ca2+ uptake by the vesicles was checked by inhibition of the process with specific inhibitors (bafilomycin A(1), ergocryptin, folymicin, DCCD) of the H(+)-pump activity. These results indicate that synaptic vesicles Ca2+/H+ antiport is indirectly linked to ATP hydrolysis and it is essentially dependent on the chemical component (DeltapH) of the electrochemical gradient generated by the H(+)-pump activity.     

Fast Ca2+ responses in astrocyte end‐feet and neurovascular coupling in mice

Cerebral blood flow (CBF) is regulated by the activity of neurons and astrocytes. Understanding how these cells control activity‐dependent increases in CBF is crucial to interpreting functional neuroimaging signals. The relative importance of neurons and astrocytes is debated, as are the functional implications of fast Ca²⁺ changes in astrocytes versus neurons. Here, we used two‐photon microscopy to assess Ca²⁺ changes in neuropil, astrocyte processes, and astrocyte end‐feet in response to whisker pad stimulation in mice. We also developed a pixel‐based analysis to improve the detection of rapid Ca²⁺ signals in the subcellular compartments of astrocytes. Fast Ca²⁺ responses were observed using both chemical and genetically encoded Ca²⁺ indicators in astrocyte end‐feet prior to dilation of arterioles and capillaries. A low dose of the NMDA receptor antagonist (5R,10s)‐(+)‐5‐methyl‐10,11‐dihydro‐5H‐dibenzo[a,d]cyclohepten‐5,10‐imine‐hydrogen‐maleate (MK801) attenuated fast Ca²⁺ responses in the neuropil and astrocyte processes, but not in astrocyte end‐feet, and the evoked CBF response was preserved. In addition, a low dose of 4,5,6,7‐tetrahydroisoxazolo[5,4‐c]pyridin‐3‐ol (THIP), an agonist for the extrasynaptic GABA_A receptor (GABA_AR), increased CBF responses and the fast Ca²⁺ response in astrocyte end‐feet but did not affect Ca²⁺ responses in astrocyte processes and neuropil. These results suggest that fast Ca²⁺ increases in the neuropil and astrocyte processes are not necessary for an evoked CBF response. In contrast, as local fast Ca²⁺ responses in astrocyte end‐feet are unaffected by MK801 but increase via GABA_AR‐dependent mechanisms that also increased CBF responses, we hypothesize that the fast Ca²⁺ increases in end‐feet adjust CBF during synaptic activity.