Depolarization-induced Changes Research Articles

Activity-Dependent Transcriptional Program in NGN2+ Neurons Enriched for Genetic Risk for Brain-Related Disorders

BackgroundConverging evidence from large-scale genetic and postmortem studies highlights the role of aberrant neurotransmission and genetic regulation in brain-related disorders. However, identifying neuronal activity–regulated transcriptional programs in the human brain and understanding how changes contribute to disease remain challenging. MethodsTo better understand how the activity-dependent regulome contributes to risk for brain-related disorders, we profiled the transcriptomic and epigenomic changes following neuronal depolarization in human induced pluripotent stem cell–derived glutamatergic neurons (NGN2) from 6 patients with schizophrenia and 5 control participants. ResultsMultiomic data integration associated global patterns of chromatin accessibility with gene expression and identified enhancer-promoter interactions in glutamatergic neurons. Within 1 hour of potassium chloride–induced depolarization, independent of diagnosis, glutamatergic neurons displayed substantial activity-dependent changes in the expression of genes regulating synaptic function. Depolarization-induced changes in the regulome revealed significant heritability enrichment for schizophrenia and Parkinson’s disease, adding to mounting evidence that sequence variation within activation-dependent regulatory elements contributes to the genetic risk for brain-related disorders. Gene coexpression network analysis elucidated interactions among activity-dependent and disease-associated genes and pointed to a key driver (NAV3) that interacted with multiple genes involved in axon guidance. ConclusionsOverall, we demonstrated that deciphering the activity-dependent regulome in glutamatergic neurons reveals novel targets for advanced diagnosis and therapy.

Voltage modulates the effect of μ-receptor activation in a ligand-dependent manner.

Background and PurposeVarious GPCRs have been described as being modulated in a voltage‐dependent manner. Opioid analgesics act via activation of μ receptors in various neurons. As neurons are exposed to large changes in membrane potential, we were interested in studying the effects of depolarization on μ receptor signalling.Experimental ApproachWe investigated potential voltage sensitivity of μ receptors in heterologous expression systems (HEK293T cells) using electrophysiology in combination with Förster resonance energy transfer‐based assays. Depolarization‐induced changes in signalling were also tested in physiological rat tissue containing locus coeruleus neurons. We applied depolarization steps across the physiological range of membrane potentials.Key ResultsStudying μ receptor function and signalling in cells, we discovered that morphine‐induced signalling was strongly dependent on the membrane potential (VM). This became apparent at the level of G‐protein activation, G‐protein coupled inwardly rectifying potassium channel (Kir3.X) currents and binding of GPCR kinases and arrestin3 to μ receptors by a robust increase in signalling upon membrane depolarization. The pronounced voltage sensitivity of morphine‐induced μ receptor activation was also observed at the level of Kir3.X currents in rat locus coeruleus neurons. The efficacy of peptide ligands to activate μ receptors was not (Met‐enkephalin) or only moderately ([D‐Ala2, N‐Me‐Phe4, Gly5‐ol]‐enkephalin) enhanced upon depolarization. In contrast, depolarization reduced the ability of the analgesic fentanyl to activate μ receptors.Conclusion and ImplicationsOur results indicate a strong ligand‐dependent modulation of μ receptor activity by the membrane potential, suggesting preferential activity of morphine in neurons with high neuronal activity.

Equivalent L-type channel (CaV1.1) function in adult female and male mouse skeletal muscle fibers

Loss of total muscle force during aging has both atrophic and non-atrophic components. The former deficit is a direct consequence of reduced muscle mass while the latter has been attributed to a depression of excitation-contraction (EC) coupling. It is well established that age-onset reductions in sex hormone production regulate the atrophic component in both males and females. However, it is unknown whether the non-atrophic component is influenced by sex hormones. Since the non-atrophic component has been linked mechanistically to reduced expression of the skeletal muscle L-type Ca2+ channel (CaV1.1), we recorded L-type Ca2+ currents, gating charge movements and depolarization-induced changes in myoplasmic Ca2+ from flexor digitorum brevis (FDB) fibers of naïve and gonadectomized mice of both sexes. Our first set of experiments sought to identify any basal differences in EC coupling or L-type Ca2+ flux between the sexes; no detectable differences in any of the aforementioned parameters were observed between FDB harvested from either naïve males or females. In the latter segments of the study, ovariectomy (OVX) and orchiectomy (ORX) models were used to assess the possible influence of sex hormones on EC coupling and/or L-type Ca2+ flux. In these experiments, FDB fibers harvested from OVX and ORX mice both showed no differences in L-type Ca2+ current, gating charge movement or depolarization-induced changes in Ca2+ release from the sarcoplasmic reticulum. Taken together, our results indicate L-type Ca2+ channel function and EC coupling are: 1) equivalent between the sexes, and 2) not significantly regulated by sex hormones. Since recent NIH review guidelines mandate the consideration of sex differences as a criterion for review, our work indicates the suitability of either sex for the study of the fundamental mechanisms of EC coupling. Thus, our findings may accelerate the research process by conserving animals, labor and financial resources.

Functional Characterization of Lamina X Neurons in ex-Vivo Spinal Cord Preparation.

Functional properties of lamina X neurons in the spinal cord remain unknown despite the established role of this area for somatosensory integration, visceral nociception, autonomic regulation and motoneuron output modulation. Investigations of neuronal functioning in the lamina X have been hampered by technical challenges. Here we introduce an ex-vivo spinal cord preparation with both dorsal and ventral roots still attached for functional studies of the lamina X neurons and their connectivity using an oblique LED illumination for resolved visualization of lamina X neurons in a thick tissue. With the elaborated approach, we demonstrate electrophysiological characteristics of lamina X neurons by their membrane properties, firing pattern discharge and fiber innervation (either afferent or efferent). The tissue preparation has been also probed using Ca2+ imaging with fluorescent Ca2+ dyes (membrane-impermeable or -permeable) to demonstrate the depolarization-induced changes in intracellular calcium concentration in lamina X neurons. Finally, we performed visualization of subpopulations of lamina X neurons stained by retrograde labeling with aminostilbamidine dye to identify sympathetic preganglionic and projection neurons in the lamina X. Thus, the elaborated approach provides a reliable tool for investigation of functional properties and connectivity in specific neuronal subpopulations, boosting research of lamina X of the spinal cord.

Local Ca2+ coupling between mitochondria and sarcoplasmic reticulum following depolarization in guinea pig urinary bladder smooth muscle cells.

Spatiotemporal changes in cytosolic Ca2+ concentration ([Ca2+]c) trigger a number of physiological functions in smooth muscle cells (SMCs). We previously imaged Ca2+-induced Ca2+ release following membrane depolarization as local Ca2+ transients, Ca2+ hotspots, in subplasmalemmal regions. In this study, the physiological significance of mitochondria on local Ca2+ signaling was examined. Cytosolic and mitochondrial Ca2+ images following depolarization or action potentials were recorded in single SMCs from the guinea pig urinary bladder using a fast-scanning confocal fluorescent microscope. Depolarization- and action potential-induced [Ca2+]c transients occurred at several discrete sites in subplasmalemmal regions, peaked within 30 ms, and then spread throughout the whole-cell. In contrast, Ca2+ concentration in the mitochondria matrix ([Ca2+]m) increased after a delay of ~50 ms from the start of depolarization, and then peaked within 500 ms. Following repolarization, [Ca2+]c returned to the resting level with a half-decay time of ~500 ms, while [Ca2+]m recovered more slowly (∼1.5 s). Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone, a mitochondrial uncoupler, abolished depolarization-induced [Ca2+]m elevations and slowed [Ca2+]c changes. Importantly, short depolarization-induced changes in [Ca2+]m and transmembrane potential in mitochondria coupled to Ca2+ hotspots were significantly larger than those in other mitochondria. Total internal reflection fluorescence imaging revealed that a subset of mitochondria closely localized with ryanodine receptors and voltage-dependent Ca2+ channels. These results indicate that particular mitochondria are functionally coupled to ion channels and sarcoplasmic reticulum fragments within the local Ca2+ microdomain, and thus, strongly contribute to [Ca2+]c regulation in SMCs.

Transmembrane voltage: Potential to induce lateral microdomains

Lateral segregation of plasma membrane lipids is a generally accepted phenomenon. Lateral lipid microdomains of specific composition, structure and biological functions are established as a result of simultaneous action of several competing mechanisms which contribute to membrane organization. Various lines of evidence support the conclusion that among those mechanisms, the membrane potential plays significant and to some extent unique role. Above all, clear differences in the microdomain structure as revealed by fluorescence microscopy could be recognized between polarized and depolarized membranes. In addition, recent fluorescence spectroscopy experiments reported depolarization-induced changes in a membrane lipid order. In the context of earlier findings showing that plasma membranes of depolarized cells are less susceptible to detergents and the cells less sensitive to antibiotics or antimycotics treatment we discuss a model, in which membrane potential-driven re-organization of the microdomain structure contributes to maintaining membrane integrity during response to stress, pathogen attack and other challenges involving partial depolarization of the plasma membrane.This article is part of a Special Issue entitled: The cellular lipid landscape edited by Tim P. Levine and Anant K. Menon.

A role for CaV1 and calcineurin signaling in depolarization-induced changes in neuronal DNA methylation

Direct manipulations of neuronal activity have been shown to induce changes in DNA methylation (DNAm), although little is known about the cellular signaling pathways involved. Using reduced representation bisulfite sequencing, we identify DNAm changes associated with moderate chronic depolarization in dissociated rat hippocampal cultures. Consistent with previous findings, these changes occurred primarily in the vicinity of loci implicated in neuronal function, being enriched in intergenic regions and underrepresented in CpG-rich promoter regulatory regions. We subsequently used 2 pharmacological interventions (nifedipine and FK-506) to test whether the identified changes depended on 2 interrelated signaling pathways known to mediate multiple forms of neuronal plasticity. Both pharmacological manipulations had notable effects on the extent and magnitude of depolarization-induced DNAm changes indicating that a high proportion of activity-induced changes are likely to be mediated by calcium entry through L-type CaV1 channels and/or downstream signaling via the calcium-dependent phosphatase calcineurin.

Closing the Gate in the Limbic Striatum: Prefrontal Suppression of Hippocampal and Thalamic Inputs

Many brain circuits control behavior by integrating information arising from separate inputs onto a common target neuron. Neurons in the ventral striatum (VS) receive converging excitatory afferents from the prefrontal cortex (PFC), hippocampus (HP), and thalamus, among other structures, and the integration of these inputs is critical for goal-directed behaviors. Although HP inputs have been described as gating PFC throughput in the VS, recent data reveal that the VS desynchronizes from the HP during epochs of burst-like PFC activity related to decision making. It is therefore possible that PFC inputs locally attenuate responses to other glutamatergic inputs to the VS. Here, we found that delivering trains of stimuli to the PFC suppresses HP- and thalamus-evoked synaptic responses in the VS, in part through activation of inhibitory processes. This interaction may enable the PFC to exert influence on basal ganglia loops during decision-making instances with minimal disturbance from ongoing contextual inputs.

Nuclear envelope potential: An origin of the first correlated neural activity

Lateral segregation of plasma membrane lipids is a generally accepted phenomenon. Lateral lipid microdomains of specific composition, structure and biological functions are established as a result of simultaneous action of several competing mechanisms which contribute to membrane organization. Various lines of evidence support the conclusion that among those mechanisms, the membrane potential plays significant and to some extent unique role. Above all, clear differences in the microdomain structure as revealed by fluorescence microscopy could be recognized between polarized and depolarized membranes. In addition, recent fluorescence spectroscopy experiments reported depolarization-induced changes in a membrane lipid order. In the context of earlier findings showing that plasma membranes of depolarized cells are less susceptible to detergents and the cells less sensitive to antibiotics or antimycotics treatment we discuss a model, in which membrane potential-driven re-organization of the microdomain structure contributes to maintaining membrane integrity during response to stress, pathogen attack and other challenges involving partial depolarization of the plasma membrane.This article is part of a Special Issue entitled: The cellular lipid landscape edited by Tim P. Levine and Anant K. Menon.

Capacitance Measurements of Regulated Exocytosis in Mouse Taste Cells

Exocytosis, consisting of the merger of vesicle and plasma membrane, is a common mechanism used by different types of nucleated cells to release their vesicular contents. Taste cells possess vesicles containing various neurotransmitters to communicate with adjacent taste cells and afferent nerve fibers. However, whether these vesicles engage in exocytosis on a stimulus is not known. Since vesicle membrane merger with the plasma membrane is reflected in plasma membrane area fluctuations, we measured membrane capacitance (C(m)), a parameter linearly related to membrane surface area. To investigate whether taste cells undergo regulated exocytosis, we used the compensated tight-seal whole-cell recording technique to monitor depolarization-induced changes in C(m) in the different types of taste cells. To identify taste cell types, mice expressing green fluorescent protein from the TRPM5 promoter or from the GAD67 promoter were used to discriminate type II and type III taste cells, respectively. Moreover, the cell types were also identified by monitoring their voltage-current properties. The results demonstrate that only type III taste cells show significant depolarization-induced increases in C(m), which were correlated to the voltage-activated calcium currents. The results suggest that type III, but neither type II nor type I cells exhibit depolarization-induced regulated exocytosis to release transmitter and activate gustatory afferent nerve fibers.

Brought in by force: AMPK, TBC1D1, and contraction-stimulated glucose transport in skeletal muscle

transient intracellular calcium oscillations were the first signaling mediator described to regulate contraction-stimulated glucose transport in skeletal muscle ([6][1]). Thirty years later, the discoveries, that 5′-AMP-activated protein kinase (AMPK) activator 5-aminoimidazole-4-carboxamide 1-β-

Activated Rho/Rho kinase and modified calcium sensitivity in cryopreserved human saphenous veins

Background We have shown previously that cryopreservation of human internal mammary arteries activates protein kinase C and enhances intracellular Ca 2+ [Ca 2+] i. We now present evidence that in human saphenous veins (HSV) cryoinjury is associated with activation of the Rho/Rho kinase signaling pathways and enhanced [Ca 2+] i. Methods HSV were investigated in vitro either unfrozen within 12 h after removal or after storage at −196 °C in a cryomedium containing 1.8 M dimethyl sulfoxide and 0.1 M sucrose as cryoprotectant additives. Results Cryostorage diminished responses to receptor-mediated contractile agonists such as noradrenaline, 5-HT and endothelin-1 by up to 30% whereas responses to KCl were attenuated by about 50%. Concentration–response curves for CaCl 2 on unfrozen and cryopreserved HSV revealed similar inhibitory activities of both blocking 1,4-dihydropyridine derivatives nifedipine and the (−)-( R) enantiomer of SDZ 202-791 whereas the Ca 2+ channel activating (+)-( S) enantiomer of SDZ 202-791 was 10 times less effective at enhancing contractions to CaCl 2 when tested after cryostorage. These functional effects were reflected by changes in [Ca 2+] i as demonstrated by fluorescence of Fluo-3AM loaded veins. The diminished activity of (+)-( S) SDZ 202-791 in cryopreserved HSV was reversed partially when the potassium channel opener pinacidil (1 μM) was present during the freezing/thawing process. Blockade of Rho kinase by HA-1077 proved to be significantly more effective at attenuating contractile responses to both endothelin-1 and KCl after cryostorage. Conclusions Data suggested that cryopreservation modified [Ca 2+] i of venous smooth muscle cells (1) through depolarization-induced changes in Ca 2+ influx and (2) through activation of Rho kinase signaling pathways.

Calcium Response to Retinohypothalamic Tract Synaptic Transmission in Suprachiasmatic Nucleus Neurons

Glutamate released from retinohypothalamic tract (RHT) synapses with suprachiasmatic nucleus (SCN) neurons induces phase changes in the circadian clock presumably by using Ca2+ as a second messenger. We used electrophysiological and Ca2+ imaging techniques to simultaneously record changes in the membrane potential and intracellular calcium concentration ([Ca2+]i) in SCN neurons after stimulation of the RHT at physiologically relevant frequencies. Stimulation of the RHT sufficient to generate an EPSP did not produce detectable changes in [Ca2+]i, whereas EPSP-induced action potentials evoked an increase in [Ca2+]i, suggesting that the change in postsynaptic somatic [Ca2+]i produced by synaptically activated glutamate receptors was the result of membrane depolarization activating voltage-dependent Ca2+ channels. The magnitude of the Ca2+ response was dependent on the RHT stimulation frequency and duration, and on the SCN neuron action potential frequency. Membrane depolarization-induced changes in [Ca2+]i were larger and decayed more quickly in the dendrites than in the soma and were attenuated by nimodipine, suggesting a compartmentalization of Ca2+ signaling and a contribution of L-type Ca2+ channels. RHT stimulation at frequencies that mimicked the output of light-sensitive retinal ganglion cells (RGCs) evoked [Ca2+]i transients in SCN neurons via membrane depolarization and activation of voltage-dependent Ca2+ channels. These data suggest that for Ca2+ to induce phase advances or delays, light-induced signaling from RGCs must augment the underlying oscillatory somatic [Ca2+]i by evoking postsynaptic action potentials in SCN neurons during a period of slow spontaneous firing such as occurs during nighttime.

Membrane potential governs lateral segregation of plasma membrane proteins and lipids in yeast

The plasma membrane potential is mainly considered as the driving force for ion and nutrient translocation. Using the yeast Saccharomyces cerevisiae as a model organism, we have discovered a novel role of the membrane potential in the organization of the plasma membrane. Within the yeast plasma membrane, two non-overlapping sub-compartments can be visualized. The first one, represented by a network-like structure, is occupied by the proton ATPase, Pma1, and the second one, forming 300-nm patches, houses a number of proton symporters (Can1, Fur4, Tat2 and HUP1) and Sur7, a component of the recently described eisosomes. Evidence is presented that sterols, the main lipid constituent of the plasma membrane, also accumulate within the patchy compartment. It is documented that this compartmentation is highly dependent on the energization of the membrane. Plasma membrane depolarization causes reversible dispersion of the H(+)-symporters, not however of the Sur7 protein. Mitochondrial mutants, affected in plasma membrane energization, show a significantly lower degree of membrane protein segregation. In accordance with these observations, depolarized membranes also considerably change their physical properties (detergent sensitivity).

Glutamate pretreatment affects Ca2+ signaling in processes of astrocyte pairs

Simultaneous somatic patch-pipette recording of a single astrocyte to evoke voltage-gated calcium currents, and Ca(2+) imaging, were used to study the spatial and temporal profiles of depolarization-induced changes in intracellular Ca(2+) ([Ca(2+)](i)) in the processes of cultured rat cortical astrocytes existing as pairs. Transient Ca(2+) changes locked to depolarization were observed as microdomains in the processes of the astrocyte pairs, and the responses were more pronounced in the adjoining astrocyte. Considering the functional significance of higher concentrations of glutamate observed in certain pathological conditions, Ca(2+) transients were recorded following pretreatment of cells with glutamate (500 microM for 20 min). This showed distance-dependent incremental scaling and attenuation in the presence of the metabotropic glutamate receptor (mGluR) antagonist, alpha-methyl(4-carboxy-phenyl) glycine (MCPG). Estimation of local Ca(2+) diffusion coefficients in the astrocytic processes indicated higher values in the adjoining astrocyte of the glutamate pretreated group. Intracellular heparin introduced into the depolarized astrocyte did not affect the Ca(2+) transients in the heparin-loaded astrocyte but attenuated the [Ca(2+)](i) responses in the adjoining astrocyte, suggesting that inositol 1,4,5 triphosphate (IP(3)) may be the transfer signal. The uncoupling agent, 1-octanol, attenuated the [Ca(2+)](i) responses in both the control and glutamate pretreated astrocytes, indicating the role of gap junctional communication. Our studies indicate that individual astrocytes have distinct functional domains, and that the glutamate-induced alterations in Ca(2+) signaling involve a sequence of intra- and intercellular steps in which phospholipase C (PLC), IP(3), internal Ca(2+) stores, VGCC and gap junction channels appear to play an important role.

Single and combined effects of carbamazepine and vinpocetine on depolarization-induced changes in Na +, Ca 2+ and glutamate release in hippocampal isolated nerve endings

The single and combined effects of carbamazepine and vinpocetine on the release of the excitatory amino acid neurotransmitter glutamate, on the rise in internal Na + (Na i, as determined with SBFI), and on the rise in internal Ca 2+ (Ca i, as determined with fura-2) induced by an increased permeability of presynaptic Na + channels, with veratridine, or by an increased permeability of presynaptic Ca 2+ channels with high K +, were investigated in isolated hippocampal nerve endings. The present study shows that carbamazepine and vinpocetine, both inhibit dose dependently the release of preloaded [ 3H]Glu induced by veratridine. However, carbamazepine is two orders of magnitude less potent than vinpocetine. The calculated IC 50's for carbamazepine and vinpocetine to inhibit veratridine-induced [ 3H]Glu release are 200 and 2 μM, respectively. Consistently 150 μM carbamazepine and 1.5 μM vinpocetine reduce the veratridine-induced rise in Na i in a similar extent. The single effects of carbamazepine and of vinpocetine on the presynaptic Na + channel mediated responses, namely the rise in Na i and the release of Glu induced by veratridine, are additive. Responses that depend on the entrance of external Ca 2+ via presynaptic Ca 2+ channels, such as the release of [ 3H]Glu and the rise in Ca i induced by high K +, are insensitive to 300 μM carbamazepine and slightly reduced by 5 μM vinpocetine. It is concluded that the additive effects of carbamazepine, which is one of the most common antiepileptic drugs, and vinpocetine that besides its known neuroprotective action and antiepileptic potential is a memory enhancer, may perhaps be advantageous in the treatment of epileptic patients.

Tumor necrosis factor α increases cytosolic calcium responses to AMPA and KCl in primary cultures of rat hippocampal neurons

Acute behavioral effects of tumor necrosis factor α (TNFα) have been previously reported, however the cellular basis for these actions are unknown. To address this issue we examined the effects of TNFα on AMPA- and depolarization-induced changes in cytosolic Ca 2+ in cultured hippocampal neurons. Single cell Ca 2+ levels were determined with the fluorescent calcium indicator fura-2. TNFα caused an up-regulation of AMPA (10 μM)- and depolarization (55 mM KCl)-induced Ca 2+ responses. This effect occurred within a window of concentrations (1 and 10 ng/ml but not 0.1 or 100 ng/ml) and times (3 and 6 h but not 1 and 24 h). The effect was dependent upon protein synthesis (blocked by cycloheximide) and was prevented by the soluble TNF receptor and by a soluble TNF receptor fragment. Treatment with the soluble TNF receptor fragment also caused a decrease in the basal response. The TNFα treatment protocols did not appear to produce any toxicity to the neurons. Results are consistent with the hypothesis that TNFα regulates proteins known to be involved in neuronal communication (AMPA receptors) and cell regulation (voltage-dependent calcium channels) in a relatively rapid period of time (a few hours). These actions may be related to the behavioral effects produced by TNFα that occur within this time frame.

Intracellular calcium signals measured with indo-1 in isolated skeletal muscle fibres from control and mdx mice.

1. Intracellular free calcium concentration ([Ca2+]i) was measured with the fluorescent indicator indo-1 in single skeletal fibres enzymatically isolated from the flexor digitorum brevis and interosseus muscles of control and dystrophic mdx C57BL/10 mice. Measurements were taken from a portion of fibre that was voltage clamped to allow detection of depolarization-induced changes in [Ca2+]i. 2. The mean (+/- s.e.m.) initial resting [Ca2+]i from all control and mdx fibres tested was 56 +/- 5 nM (n = 72) and 48 +/- 7 nM (n = 57), respectively, indicating no significant overall difference between the two groups. However, when comparing a batch of control and mdx fibres obtained from mice older than approximately 35 weeks, resting [Ca2+]i was significantly lower in mdx (16 +/- 4 nM, n = 11) than in control fibres (71 +/- 10 nM, n = 14). 3. Changes in [Ca2+]i elicited by short (5-35 ms) depolarizing pulses from -80 to 0 mV showed similar properties in control and mdx fibres. After a 5 ms duration pulse the mean time constant of [Ca2+]i decay was, however, significantly elevated in mdx as compared to control fibres, by a factor of 1.5-2. For longer pulses, no significant difference could be detected. 4. In response to 50 ms duration depolarizing pulses of various amplitudes the threshold for detection of an [Ca2+]i change and the peak [Ca2+]i reached for a given potential were similar in control and mdx fibres. 5. Overall results show that mdx skeletal muscle fibres are quite capable of handling [Ca2+]i at rest and in response to membrane depolarizations.

Depolarization-Induced Mitochondrial Ca Accumulation in Sympathetic Neurons: Spatial and Temporal Characteristics

Several lines of evidence suggest that neuronal mitochondria accumulate calcium when the cytosolic free Ca(2+) concentration ([Ca(2+)](i)) is elevated to levels approaching approximately 500 nM, but the spatial, temporal, and quantitative characteristics of net mitochondrial Ca uptake during stimulus-evoked [Ca(2+)](i) elevations are not well understood. Here, we report direct measurements of depolarization-induced changes in intramitochondrial total Ca concentration ([Ca](mito)) obtained by x-ray microanalysis of rapidly frozen neurons from frog sympathetic ganglia. Unstimulated control cells exhibited undetectably low [Ca](mito), but high K(+) depolarization (50 mM, 45 sec), which elevates [Ca(2+)](i) to approximately 600 nM, increased [Ca](mito) to 13.0 +/- 1.5 mmol/kg dry weight; this increase was abolished by carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP). The elevation of [Ca](mito) was a function of both depolarization strength and duration. After repolarization, [Ca](mito) recovered to prestimulation levels with a time course that paralleled the decline in [Ca(2+)](i). Depolarization-induced increases in [Ca](mito) were spatially heterogeneous. At the level of single mitochondria, [Ca](mito) elevations depended on proximity to the plasma membrane, consistent with predictions of a diffusion model that considers radial [Ca(2+)](i) gradients that exist early during depolarization. Within individual mitochondria, Ca was concentrated in small, discrete sites, possibly reflecting a high-capacity intramitochondrial Ca storage mechanism. These findings demonstrate that in situ Ca accumulation by mitochondria, now directly identified as the structural correlate of the "FCCP-sensitive store, " is robust, reversible, graded with stimulus strength and duration, and dependent on spatial location.

Protein kinase C in the rat cerebral cortex during spreading depression

Activity and distribution of protein kinase C (PKC) in the rat cerebral cortex was correlaetd with the development of spreading depression. When the ‘waves’ of the slow potential shift, induced by topical application of concentrated KC1 solutions, were allowed to spread over the cerebral cortex for 10 min, both cytosolic and particulate fractions of the enzyme were increased to 169% and 143%, respectively, of the control values obtained from the contralateral, relatively intact hemicortex. When the enzyme activities were correlated with development of a single slow potential shift, it appeared that in the cortical area fully depolarized (under the maximum of negativity), the respective values were 175% and 157%. One mine after recovery of the single wave of spreading depression both cytosolic and particulate fractions continued to rise up to 218% and 239%, respectively. During 5 min of recovery both the cytosolic and particulate fractions fell to 71% and 57%, respectively, of control levels. Even at 10 min the cytosolic enzyme was still decreased to 80%. At 20 min no difference between control and experimental values was found (soluble, 111%; particulate, 96%). The results are discussed in the context of data obtained in a few studies dealing with depolarization-induced changes of PKC in vitro.