Automatic quantification of mitochondrial fragmentation from two-photon microscope images of mouse brain tissue.

The morphology of mitochondria can inform about their functional state and, thus, about cell vitality. For example, fragmentation of the mitochondrial network is associated with many diseases. Recent advances in neuronal imaging have enabled the observation of mitochondria in live brains for long periods of time, enabling the study of their dynamics in animal models of diseases. To aid these studies, we developed an automatic method, based on supervised learning, for quantifying the degree of mitochondrial fragmentation in tissue images acquired via two-photon microscopy from transgenic mice, which exclusively express Enhanced cyan fluorescent protein (ECFP) under Thy1 promoter, targeted to the mitochondrial matrix in subpopulations of neurons. We tested the method on images prior to and after cardiac arrest, and found it to be sensitive to significant changes in mitochondrial morphology because of the arrest. We conclude that the method is useful in detecting morphological abnormalities in mitochondria and, likely, in other subcellular structures as well.

[Effect of alpha-conotoxin MII and its N-terminal derivatives on Ca2+ and Na+ signals induced by nicotine in neuroblastoma cell line SH-SY5Y].

Nicotinic acetylcholine receptors (nAChRs) are implicated in the regulation ofintracellular Ca2+-dependent processes in cells both in normal and pathological states, alpha-Conotoxins isolated from Conus snails venom are a valuable tool for the study of pharmacological properties and functional role of nAChRs. In the present study the alpha-conotoxin MII analogue with the additional tyrosine attached to the N terminus (Y0-MII) was prepared. Also we synthesized analogs with the N-terminal glycine residue labeled with the Bolton- Hunter reagent (BH-MII) or fluorestsein isothiocyanate (FITC-MII). Fluorescence microscopy studies of the neuroblastoma SH-SY5Y cells loaded with Ca2+ indicator Fura-2 or with Ca2+ and Na+ indicators Fluo-4 and SBFI were performed to examine effect of MII modification on its ability to inhibit nicotin-induced increases in intracellular free Ca2+ and Na+ concentrations ([Ca2+] and [Na+]i respectively). Monitoring of individual cell [Ca2+]i and [Na+]i signals revealed different kinetics of [Ca2+]i and [Na+]i rise and decay in responses to brief nicotine (Nic) applications (10-30 microM, 3-5 min), which indicates to different mechanisms of Ca2+ and Na+ homeostasis control in SH-SY5Y cells. MII inhibited in concentration-dependent manner the both [Ca2+]i and [Na+]i increase induced by Nic. Additional tyrosine in the Y0-MII or, especially, more sizeable label in FITC-MII significantly reduced the inhibitory effect of MII. Whereas the efficiency of the Ca2+ response inhibition by BH-MII was found to be close to the efficiency of its inhibition by natural alpha-conotoxin MII, radioiodinated derivatives BH-MII can be used in radioligand assay.

Chemical and photonic interactions in vitro and in vivo between fluorescent tracer and nanoparticle-based scavenger for enhanced molecular imaging.

We hereby present a concept of scavenging excess imaging agent prior to a diagnostic imaging session, consequently allowing for enhanced contrast of signals originating from the tissue area of interest to the signals originating from systemic imaging agent residues. In our study, a prospective silica core-shell nanoparticle-based scavenger was designed and explored for its feasibility to scavenge a specific imaging agent (tracer) in the bloodstream. The developed tracer-scavenger system was first investigated under in vitro conditions to ensure proper binding between tracer and scavenger is taking place, as confirmed by Förster/fluorescence resonance energy transfer studies. In vivo, two-photon imaging was utilized to directly study the interaction of the scavenger particles and the tracer molecules in the vasculature of mice. To our knowledge, a methodological solution for in vivo differentiation between signals, originating from tissue and blood, has not been presented elsewhere.

Local calcium release in dendritic spines required for long-term synaptic depression.

We have used rats and mice with mutations in myosin-Va to evaluate the range and function of IP3-mediated Ca2+ signaling in dendritic spines. In these mutants, the endoplasmic reticulum and its attendant IP3 receptors do not enter the postsynaptic spines of parallel fiber synapses on cerebellar Purkinje cells. Long-term synaptic depression (LTD) is absent at the parallel fiber synapses of the mutants, even though the structure and function of these synapses otherwise appear normal. This loss of LTD is associated with selective changes in IP3-mediated Ca2+ signaling in spines and can be rescued by photolysis of a caged Ca2+ compound. Our results reveal that IP3 must release Ca2+ locally in the dendritic spines to produce LTD and indicate that one function of dendritic spines is to target IP3-mediated Ca2+ release to the proper subcellular domain.

Cerebellar defects in Ca2+/calmodulin kinase IV-deficient mice.

The Ca(2+)/calmodulin-dependent protein kinase CaMKIV was first identified in the cerebellum and has been implicated in nuclear signaling events that control neuronal growth, differentiation, and plasticity. To understand the physiological importance of CaMKIV, we disrupted the mouse Camk4 gene. The CaMKIV null mice displayed locomotor defects consistent with altered cerebellar function. Although the overall cytoarchitecture of the cerebellum appeared normal in the Camk4(-/-) mice, we observed a significant reduction in the number of mature Purkinje neurons and reduced expression of the protein marker calbindin D28k within individual Purkinje neurons. Western immunoblot analyses of cerebellar extracts also established significant deficits in the phosphorylation of cAMP response element-binding protein at serine-133, a proposed target of CaMKIV. Additionally, the absence of CaMKIV markedly altered neurotransmission at excitatory synapses in Purkinje cells. Multiple innervation by climbing fibers and enhanced parallel fiber synaptic currents suggested an immature development of Purkinje cells in the Camk4(-/-) mice. Together, these findings demonstrate that CaMKIV plays key roles in the function and development of the cerebellum.

A novel CGRP-neutralizing Spiegelmer attenuates neurogenic plasma protein extravasation.

BACKGROUND AND PURPOSE: Calcitonin gene-related peptide (CGRP) plays an important role in the pathology of migraine, and recent clinical trials suggest the inhibition of CGRP-mediated processes as a new therapeutic option in migraine. In this study, we describe the generation of NOX-L41, a CGRP-neutralizing mirror-image (L-)aptamer (Spiegelmer) and investigate its in vitro and in vivo function. EXPERIMENTAL APPROACH: A CGRP-binding Spiegelmer was identified by in vitro selection. Binding studies were performed using surface plasmon resonance (SPR), and the inhibitory activity was determined in cell-based assays. The pharmacokinetic profile comparing i.v. and s.c. dosing was analysed in rats. Intravital two-photon microscopy was employed to follow extravasation from meningeal vessels. Finally, in vivo efficacy was tested in a model of electrically evoked meningeal plasma protein extravasation (PPE) in rats. KEY RESULTS: We identified NOX-L41, a novel CGRP-neutralizing Spiegelmer. SPR studies showed that NOX-L41 binds to human and rat/mouse CGRP with sub-nanomolar affinities and is highly selective against related peptides such as amylin. In vitro, NOX-L41 effectively inhibited CGRP-induced cAMP formation in SK-N-MC cells. In rats, NOX-L41 had a plasma half-life of 8 h. Pharmacodynamic studies showed that NOX-L41 extravasates from blood vessels in the dura mater and inhibits neurogenic meningeal PPE for at least 18 h after single dosing. CONCLUSIONS AND IMPLICATIONS: This is the first description of the CGRP-neutralizing Spiegelmer NOX-L41. Preclinical studies confirmed a role for CGRP in neurogenic PPE and provided proof-of-concept for the potential use of this new drug candidate for the treatment or prevention of migraine.

Rat nicotinic acetylcholine receptor alpha2beta2 channels: comparison of functional properties with alpha4beta2 channels in Xenopus oocytes.

Rat hippocampal interneurons express diverse subtypes of nicotinic acetylcholine receptors (nAChRs), including alpha7- and non-alpha7-containing receptors. Although the major subtype of non-alpha7 nAChRs in the hippocampus is thought to be composed of alpha4beta2 subunits, the molecular makeup of these non-alpha7 receptors is likely to be more complicated. Previously, we reported a high level of expression of the alpha2 nAChR subunit in individual rat hippocampal CA1 stratum oriens interneurons. In addition, the non-alpha7 nAChRs from these neurons are less sensitive to block by dihydro-beta-erythroidine (DHbetaE; the broad spectrum non-alpha7 nAChR antagonist) than that expected for alpha4beta2 receptors. We studied the functional properties of rat alpha2beta2 channels expressed in Xenopus oocytes using two-electrode voltage-clamp, and compared these to those properties of the more widely expressed and studied alpha4beta2 channels. Dose-response curves for both receptor subtypes indicated that there are at least two different affinity sites for ACh, the fractional contribution of which depended on the ratio of injected RNA. DHbetaE blocked both receptor subtypes, although the sensitivity to block of alpha4beta2 channels was significantly higher than that for alpha2beta2. Finally, the current-voltage (I-V) relationship for the alpha2beta2 channels more strongly rectified than for the alpha4beta2 channels. These data suggest that functional properties, in particular the sensitivity to block by DHbetaE, might be useful indicators to differentiate between native alpha4beta2 and alpha2beta2 channels. In addition, these data suggest that the relative balance between the high- and low-affinity components being determined by the relative levels of the alpha and beta subunits might be a general property of the heteromeric non-alpha7 nAChRs. By comparing the properties of expressed nAChRs with those of the native channels, we might be able to learn what role alpha2-containing nAChRs may be playing in forming functional channels in the hippocampus.

Fading and rebound of currents induced by ATP in PC12 cells.

1. Patch clamp recording (whole cell configuration) was used to study the action of ATP on rat phaeochromocytoma (PC12) cells usually held at -70 mV and rapidly superfused with buffered saline. ATP (0.5, 1 or 5 mM), applied from micropipettes by pressure application with brief (< or = 50 ms) pulses, induced inward currents with rapid onset and decay. ADP and alpha, beta-methylene ATP were ineffective. 2. ATP (5 mM) applied with pulses > 200 ms long elicited a complex current response characterized by a rapid peak which faded and was followed by a strong current rebound (lasting several s) as soon as the application was terminated. This type of response was readily replicated as long as ATP applications were spaced at 2-3 min intervals. The amplitude of peak and rebound currents was dependent on the length of pressure pulse and was similarly depressed by bath application of a threshold dose (25 microM) of ATP. Rapid fading and rebound of ATP-induced membrane currents were also observed when the Y-tube method was used for applying this agonist. 3. The reversal potential for peak and rebound currents was the same while the time constant values for peak fading and rebound onset were insensitive to changes in membrane potential between -70 and -40 mV. When ATP was applied to a cell clamped at depolarized potential, no current was observed but rapid return of the membrane potential to -70 mV immediately at the end of ATP application was associated with a large rebound current. 4. Brief (20 ms) application of ATP during the onset of the rebound current strongly and transiently suppressed it. The same application performed during the gradual decay of the rebound wave elicited a transient inward current which was much smaller and shorter than the one observed when the cell was in its resting state. Application of 2 s ATP pulses at 20 s intervals equally reduced the initial peak and rebound currents which recovered at the same rate. 5. The present data are interpreted according to a scheme which suggests two types of ATP receptor desensitization. The first one (D1) would be characterized by fast kinetics and low agonist affinity; rapid recovery from D1 would then be manifested as current rebound presumably due to receptor reactivation. The second desensitized state (D2) has slow kinetics and high affinity for the agonist: it is therefore typically seen with sustained application of a low dose of ATP. It is proposed that desensitization and its recovery can influence the time course of membrane responses mediated by purinoceptors.

The effect of the neuropeptide substance P on desensitization of ATP receptors of PC12 cells.

1. Patch clamp recording (whole cell configuration) was employed to investigate the modulatory action of substance P on inward currents elicited by adenosine 5'-triphosphate (ATP, focally applied via a pressure pipette) from phaechromocytoma (PC12) cells usually held at -70 mV. 2. Bath-applied substance P (0.2-20 microM) had no effect on baseline membrane current but reversibly reduced ATP peak currents in a concentration-dependent fashion. The depressant effect was not associated with a change in the ATP current reversal potential. 3. Equiamplitude peak responses induced by 50 microM or 5 mM ATP were differentially affected by substance P which preferentially reduced currents evoked by 5 mM ATP. In the presence of substance P a conditioning pulse of ATP evoked a stronger depression to subsequent test pulses of the same agonist. 4. Combined patch clamping and confocal laser imaging of intracellular Ca2+ ([Ca2+]i) of single PC12 cells showed that substance P (applied by a pressure pipette) per se had no effect on [Ca2+]i or current baseline, although it reduced the inward current and associated [Ca2+]i rise elicited by ATP. 5. These results are interpreted as due to facilitation by substance P of desensitization of ATP-gated P2x2 receptors of PC12 cells. It is proposed that the novel modulation by this peptide of ATP responses may serve as a model for further studies aimed at elucidating the action of substance P on purinergic neurotransmission.

Role of intracellular calcium in fast and slow desensitization of P2-receptors in PC12 cells.

1. Combined whole-cell patch clamp recording and confocal laser scanning microscopy of [Ca2+]i transients were performed on single PC12 cells to study any correlation between membrane currents induced by ATP and elevation in [Ca2+]i. ATP was applied by pressure from micropipettes near the recorded PC12 cells continuously superfused at a fast rate. 2. Brief (20 ms) pulses of ATP elicited monophasic inward currents and [Ca2+]i increases. Long applications (2 s) of ATP (5 mM) evoked peak currents which rapidly faded during the pulse and were followed by a large rebound current, interpreted as due to rapid desensitization and recovery of P2-receptors. The associated [Ca2+]i increase grew monotonically to a peak reached only after the occurrence of the current rebound, indicating that it is unlikely this cation has a role in fast desensitization. 3. Both membrane currents and [Ca2+]i transients were linearly dependent on holding membrane potential, suggesting that Ca2+ influx is the predominant cause of [Ca2+]i elevation. This view was supported by experiments carried out in Ca(2+)-free solution. 4. Brief pulses of ATP applied after a desensitizing pulse (2 s) of the same elicited smaller inward currents and [Ca2+]i rises indicating a role for [Ca2+]i in controlling slow desensitization of P2-receptors. 5. This notion was confirmed in experiments with various [Ca2+]i chelators which differentially affected slow desensitization in relation to their buffering capacity, while sparing fast receptor desensitization. 6. These results suggest a role for [Ca2+]i in slow rather than fast desensitization of P2-receptors, thus proposing this divalent cation as an intracellular factor able to provide an efficient and reversible control over receptor activity induced by ATP.

Imaging of intracellular calcium during desensitization of nicotinic acetylcholine receptors of rat chromaffin cells.

1. The possible role of intracellular Ca2+ levels ([Ca2+]i) in desensitization of nicotinic acetylcholine receptors (AChRs) was investigated in rat cultured chromaffin cells by use of combined whole-cell patch clamping and confocal laser scanning microscopy with the fluorescent dye fluo-3. 2. On cells held at -70 mV, pressure-application of nicotine elicited inward currents with associated [Ca2+]i rises mainly due to influx through nicotinic AChRs. These responses were blocked by (+)-tubocurarine (10 microM) but were insensitive to alpha-bungarotoxin (1 microM) or Cd2+ (0.1 mM). 3. Pressure applications of 1 mM nicotine for 2 s (conditioning pulse) evoked inward currents which faded biexponentially to a steady state level due to receptor desensitization and were accompanied by a sustained increase in [Ca2+]i. Inward currents evoked by subsequent application of brief test pulses of nicotine were depressed but recovered with a time course reciprocal to the decay of the [Ca2+]i transient induced by the conditioning pulse. 4. Omission of intracellular Ca2+ chelators or use of high extracellular Ca2+ solution (10 mM) lengthened recovery of nicotinic AChRs from desensitization while adding BAPTA or EGTA intracellularly had the opposite effect. When the patch pipette contained fluo-3 or no chelators, after establishing whole cell conditions the rate of recovery became progressively longer presumably due to dialysis of endogenous Ca2+ buffers. None of these manipulations of external or internal Ca2+ had any effect on onset or steady state level of desensitization. 5. High spatial resolution imaging of [Ca2+]i in intact cells (in the presence of 0.1 mM Cd2+) showed that its level in the immediate submembrane area decayed at the same rate as in the rest of the cell, indicating that Ca2+ was in a strategic location to modulate (directly or indirectly) AChR desensitization. 6. The present data suggest that desensitized nicotinic AChRs are stabilized in their conformation by raised [Ca2+]i and that this phenomenon retards their recovery to full activity.

ALS IgGs suppress [Ca2+]i rise through P/Q-type calcium channels in central neurones in culture.

Confocal laser scanning microscopy (with the fluorescent calcium dye fluo-3) was used to test the effect of IgG obtained from patients with amyotrophic lateral sclerosis (ALS) on the KCl-induced [Ca2+] rise in rat hippocampal neurones in culture. In the presence of tetrodotoxin and ionotropic glutamate receptor antagonists, ALS IgGs depressed (by 30-40%) Ca2+ transients evoked by influx of Ca2+ through voltage-activated channels; such an effect did not occur with IgG obtained from healthy donors. The depressant action of ALS IgG was selectively prevented by the inhibitor of P/Q-type Ca2+ channels, omega-agatoxin IVA (which alone reduced Ca2+ transients by 40%). The reduced Ca2+ transients might impair Ca(2+)-dependent glutamate receptor desensitization and thus facilitate excitotoxic damage.

Changes in intracellular calcium induced by NMDA in cultured rat hippocampal neurons require exogenous glycine.

Confocal laser scanning microscopy was used to study changes in intracellular free calcium concentration ([Ca2+]i) at the level of the soma of cultured hippocampal neurones following pressure application of glutamate or N-methyl-D-aspartate (NMDA). [Ca2+]i was imaged in the presence of tetrodotoxin after loading cells with the fluorescent dye indicator fluo-3/AM. Responses to glutamate were potently antagonized by 6-cyano-7 nitroquinoxaline-2,3-dione (CNQX: 20 microM). They were also strongly and reversibly depressed by 3-((+/-)-2-carboxypiperazine-4-yl)-propyl-1-phosphonic acid (CPP; 20 microM), leaving a small CNQX-sensitive component. Responses to NMDA were also blocked by CNQX. In the presence of saturating concentrations of glycine (100 microM), the depression of glutamate or NMDA responses by CNQX was greatly reduced. Exogenously applied glycine also potentiated the NMDA response. These data indicate that the glycine binding site of the NMDA receptor channel is not saturated in cultured hippocampal neurones and thus is susceptible to the action of agonists or antagonists.

Quantification of spread of cerebellar long-term depression with chemical two-photon uncaging of glutamate.

Localized, chemical two-photon photolysis of caged glutamate was used to map the changes in alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptors caused by long-term synaptic depression (LTD) in cerebellar Purkinje cells. LTD produced by pairing parallel fiber activity with depolarization was accompanied by a decline in the response of Purkinje cells to uncaged glutamate that accounted for both the time course and magnitude of LTD. This depression of glutamate responses was observed not only at the site of parallel fiber stimulation but also at more distant sites. The amount of LTD decreased with distance and was half-maximal 50 microm away from the site of parallel fiber activity. Estimation of the number of parallel fibers active during LTD induction indicates that LTD modified glutamate receptors not only at active synapses but also at 600 times as many inactive synapses on a single Purkinje cell. Therefore, both active and inactive parallel fiber synapses can undergo changes at a postsynaptic locus as a result of associative pre- and postsynaptic activity.

Recovery from desensitization of neuronal nicotinic acetylcholine receptors of rat chromaffin cells is modulated by intracellular calcium through distinct second messengers.

The mechanisms through which changes in intracellular Ca2+ concentration ([Ca2+]i) might influence desensitization of neuronal nicotinic receptors (nAChRs) of rat chromaffin cells were investigated by simultaneous patch-clamp recording of membrane currents and confocal microscopy imaging of [Ca2+]i induced by nicotine. Increases in [Ca2+]i that were induced by membrane depolarization or occurred spontaneously did not influence inward currents elicited by focally applied test pulses (10 msec) of nicotine, indicating that raised [Ca2+]i per se did not trigger desensitization of nAChRs. Desensitization of nAChRs, evoked by 2 sec focal application of nicotine, which largely raised [Ca2+]i, was not affected by intracellular application of agents that activate or depress protein kinase C (PKC) or A (PKA) or inhibit phosphatase 1, 2 A and B. Conversely, recovery from desensitization was facilitated by the phorbol ester phorbol 12-myristate 13-acetate (PMA) or the phosphatase 2 B inhibiting complex of cyclosporin A-cyclophilin A, whereas it was impaired by the broad spectrum kinase inhibitor staurosporine. The effects of PMA or staurosporine were prevented by the intracellularly applied Ca2+ chelator BAPTA. The adenylate cyclase activator forskolin accelerated recovery, whereas the selective PKA antagonist Rp-cAMPS had an opposite effect. The action of staurosporine and Rp-cAMPS on recovery from desensitization was additive. It is proposed that when nAChRs are desensitized, they become susceptible to modulation by [Ca2+]i via intracellular second messengers such as serine/threonine kinases and calcineurin. Thus, the phosphorylation state of neuronal nAChRs appears to regulate their rate of recovery from desensitization.

Calcitonin gene-related peptide rapidly downregulates nicotinic receptor function and slowly raises intracellular Ca2+ in rat chromaffin cells in vitro.

Although calcitonin gene-related peptide (CGRP) modulates muscle-type nicotinic acetylcholine receptors (nAChRs) via intracellular second messenger-mediated phosphorylation, the action of this peptide on neuronal-type nAChRs remains unknown. Using neuronal nAChRs of rat chromaffin cells in vitro we studied the effect of CGRP, which is physiologically present in adrenal medulla, on membrane currents and [Ca2+]i transients elicited by nicotine. Our main novel observation was that CGRP (either bath-applied or focally applied for a few seconds or even co-applied with nicotine for a few milliseconds) selectively and rapidly blocked nAChRs (a phenomenon unlikely caused by intracellular messengers in view of its speed) without affecting GABA receptors. The inhibitory effect of CGRP was independent of [Ca2+]i or membrane potential and not accompanied by baseline current changes. Like the competitive antagonist N,N,N-trimethyl-1-(4-trans-stilbenoxy)-2-propilammonium, CGRP induced a rightward, parallel shift of the nicotine dose-response curve; during co-application of these blockers the nicotine dose-ratio value was the sum of the values obtained with each antagonist alone. The block by CGRP was insensitive to the receptor antagonist hCGRP8-37 but mimicked by CGRP1-7. Persistent application of CGRP slowly increased [Ca2+]i, a phenomenon independent from external Ca2+, thus implying Ca2+ release from internal stores, and suppressed by hCGRP8-37. CGRP1-7 had no significant effect on [Ca2+]i. We propose that the 1-7 amino acid sequence of CGRP was responsible for the direct, rapid block of nAChRs, whereas the full-length peptide molecule was necessary for the delayed rise in internal Ca2+ potentially able to trigger phosphorylation-dependent modulation of nicotinic receptor function.