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In JoVE (1)
Other Publications (14)
- The Journal of Neuroscience : the Official Journal of the Society for Neuroscience
- Archives of Biochemistry and Biophysics
- Journal of Neurophysiology
- Neuron
- Proceedings of the National Academy of Sciences of the United States of America
- The Journal of Neuroscience : the Official Journal of the Society for Neuroscience
- Biomaterials
- Developmental Dynamics : an Official Publication of the American Association of Anatomists
- The Journal of Neuroscience : the Official Journal of the Society for Neuroscience
- Lasers in Surgery and Medicine
- The Journal of Neuroscience : the Official Journal of the Society for Neuroscience
- Development (Cambridge, England)
- Experimental and Molecular Pathology
- British Journal of Pharmacology
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Articles by Luis Polo-Parada in JoVE
JoVE General
Mikrosirkülasyon intravital Floresans Görüntüleme Microiontophoresis ve Mikromanipülasyon
1Department of Medical Pharmacology and Physiology, University of Missouri, 2Dalton Cardiovascular Research Center, University of Missouri
Microiontophoresis mikropipet içi ve dışı arasındaki elektriksel potansiyel bir fark yanıt mikropipet iyonların hareketi gerektirir. Biyolojik aktif molekülleri böylece elektrik akımı ile orantılı olarak teslim edilir. Biz mikrosirkülasyon endotel bağımlı vazodilatasyon çalışma mikromanipülasyon ile birlikte asetilkolin microiontophoresis göstermektedir.
Other articles by Luis Polo-Parada on PubMed
Distinct Roles of Different Neural Cell Adhesion Molecule (NCAM) Isoforms in Synaptic Maturation Revealed by Analysis of NCAM 180 KDa Isoform-deficient Mice
Mice that lack all three major isoforms of neural cell adhesion molecule (NCAM) (180 and 140 kDa transmembrane, and 120 kDa glycosylphosphatidylinositol linked) were previously shown to exhibit major alterations in the maturation of their neuromuscular junctions (NMJs). Specifically, even by postnatal day 30, they failed to downregulate from along their axons and terminals an immature, brefeldin A-sensitive, synaptic vesicle-cycling mechanism that used L-type Ca2+ channels. In addition, these NCAM null NMJs were unable to maintain effective transmitter output with high-frequency repetitive stimulation, exhibiting both severe initial depression and subsequent cyclical periods of total transmission failures that were of presynaptic origin. As reported here, mice that lack only the 180 kDa isoform of NCAM downregulated the immature vesicle-cycling mechanism on schedule, implicating either the 140 or 120 kDa NCAM isoforms in this important maturational event. However, 180 NCAM-deficient mice still exhibited many functional transmission defects. Although 180 NCAM null NMJs did not show the severe initial depression of NCAM null NMJs, they still had cyclical periods of complete transmission failure. In addition, several presynaptic molecules were expressed at lower levels or were more diffusely localized. Thus, the 180 kDa isoform of NCAM appears to play an important role in the molecular organization of the presynaptic terminal and in ensuring effective transmitter output with repetitive stimulation. Our results also suggest that PKC and MLCK (myosin light chain kinase) may be downstream effectors of NCAM in these processes. Together, these results indicate that different isoforms of NCAM mediate distinct and important events in presynaptic maturation.
Action Potential Stimulation Reveals an Increased Role for P/Q-calcium Channel-dependent Exocytosis in Mouse Adrenal Tissue Slices
Chromaffin cells of the adrenal medulla receive cholinergic input from the splanchnic nerve. Upon sympathetic activity, chromaffin cells fire action potentials that open voltage-gated calcium channels and evoke the exocytic release of catecholamines. Catecholamines then regulate homeostatic processes such as cardiac output and vascular tone. Thus control of the Ca(2+) influx in chromaffin cells represents a target for the regulation of multiple physiological functions. Previous reports utilized square pulse depolarizations to quantify the proportional exocytic response as a function of Ca(2+) channel subtype. In this study, we use perforated patch voltage clamp and action potential waveforms to depolarize cells in situ. We analyze Ca(2+) current components under conditions that match the dynamic native cell behavior. This approach revealed a greater role for P/Q-type calcium channels in evoked exocytosis than previously reported. Thus, the P/Q-type channels represent a more important control point for the regulation of catecholamine-dependent processes than previously believed.
Adrenal Chromaffin Cells Exhibit Impaired Granule Trafficking in NCAM Knockout Mice
Neural cell adhesion molecule (NCAM) plays several critical roles in neuron path-finding and intercellular communication during development. In the clinical setting, serum NCAM levels are altered in both schizophrenic and autistic patients. NCAM knockout mice have been shown to exhibit deficits in neuronal functions including impaired hippocampal long term potentiation and motor coordination. Recent studies in NCAM null mice have indicated that synaptic vesicle trafficking and active zone targeting are impaired, resulting in periodic synaptic transmission failure under repetitive physiological stimulation. In this study, we tested whether NCAM plays a role in vesicle trafficking that is limited to the neuromuscular junction or whether it may also play a more general role in transmitter release from other cell systems. We tested catecholamine release from neuroendocrine chromaffin cells in the mouse adrenal tissue slice preparation. We utilize electrophysiological and electrochemical measures to assay granule recruitment and targeting in wild-type and NCAM -/- mice. Our data show that NCAM -/- mice exhibit deficits in normal granule trafficking between the readily releasable pool and the highly release-competent immediately releasable pool. This defect results in a decreased rate of granule fusion and thus catecholamine release under physiological stimulation. Our data indicate that NCAM plays a basic role in the transmitter release mechanism in neuroendocrine cells through mediation of granule recruitment and is not limited to the neuromuscular junction and central synapse active zones.
NCAM 180 Acting Via a Conserved C-terminal Domain and MLCK is Essential for Effective Transmission with Repetitive Stimulation
NCAM 180 isoform null neuromuscular junctions are unable to effectively mobilize and exocytose synaptic vesicles and thus exhibit periods of total transmission failure during high-frequency repetitive stimulation. We have identified a highly conserved C-terminal (KENESKA) domain on NCAM that is required to maintain effective transmission and demonstrate that it acts via a pathway involving MLCK and probably myosin light chain (MLC) and myosin II. By perfecting a method of introducing peptides into adult NMJs, we tested the hypothesized role of proteins in this pathway by competitive disruption of protein-protein interactions. The effects of KENESKA and other peptides on MLCK and MLC activation and on failures in both wild-type and NCAM 180 null junctions supported this pathway, and serine phosphorylation of KENESKA was critical. We propose that this pathway is required to replenish synaptic vesicles utilized during high levels of exocytosis by facilitating myosin-driven delivery of synaptic vesicles to active zones or their subsequent exocytosis.
CD24 is Expressed by Myofiber Synaptic Nuclei and Regulates Synaptic Transmission
The genes encoding several synaptic proteins, including acetylcholine receptors, acetylcholinesterase, and the muscle-specific kinase, MuSK, are expressed selectively by a small number of myofiber nuclei positioned near the synaptic site. Genetic analysis of mutant mice suggests that additional genes, expressed selectively by synaptic nuclei, might encode muscle-derived retrograde signals that regulate the differentiation of motor axon terminals. To identify candidate retrograde signals, we used a microarray screen to identify genes that are preferentially expressed in the synaptic region of muscle, and we analyzed one such gene, CD24, further. We show that CD24, which encodes a small, variably and highly glycosylated, glycosylphosphatidylinositol (GPI)-linked protein, is expressed preferentially by myofiber synaptic nuclei in embryonic and adult muscle, and that CD24 expression is restricted to the central region of muscle independent of innervation. Moreover, we show that CD24 has a role in presynaptic differentiation, because synaptic transmission is depressed and fails entirely, in a cyclical manner, after repetitive stimulation of motor axons in CD24 mutant mice. These deficits in synaptic transmission, which are accompanied by aberrant stimulus-dependent uptake of AM1-43 from axons, indicate that CD24 is required for normal presynaptic maturation and function. Because CD24 is also expressed in some neurons, additional experiments will be required to determine whether pre- or postsynaptic CD24 mediates these effects on presynaptic development and function.
Selective Targeting of Different Neural Cell Adhesion Molecule Isoforms During Motoneuron Myotube Synapse Formation in Culture and the Switch from an Immature to Mature Form of Synaptic Vesicle Cycling
Characterization of neuromuscular junction formation and function in mice lacking all neural cell adhesion molecule (NCAM) isoforms or only the 180 isoform demonstrated that the 180 isoform was required at adult synapses to maintain effective transmission with repetitive stimulation whereas the 140 and/or 120 isoform(s) were sufficient to mediate the downregulation of synaptic vesicle cycling along the axon after synapse formation. However, the expression and targeting of each isoform and its relationship to distinct forms of synaptic vesicle cycling before and after synapse formation was previously unknown. By transfecting chick motoneurons with fluorescently tagged mouse 180, 140 and 120 isoforms, we show that before myotube contact the 180 and 140 isoforms are expressed in distinct puncta along the axon which are sites of an immature form (Brefeldin A sensitive, L-type Ca2+ channel mediated) of vesicle cycling. After myotube contact the 140 and 180 isoforms are downregulated from the axon and selectively targeted to the presynaptic terminal. This coincided with the downregulation of vesicle cycling along the axon and the expression of the mature form (BFA insensitive, P/Q type Ca2+ channel mediated) of vesicle cycling at the terminal. The synaptic targeting of exogenously expressed 180 and 140 isoforms also occurred when chick motoneurons contacted +/+ mouse myotubes; however only the 180 but not the 140 isoform was targeted on contact with NCAM-/- myotubes. These observations indicate that postsynaptic NCAM is required for the synaptic targeting of presynaptic 140 NCAM but that the localization of presynaptic 180 NCAM occurs via a different mechanism.
Preferential Cell Attachment to Nitrogen-doped Diamond-like Carbon (DLC:N) for the Measurement of Quantal Exocytosis
Electrochemical measurement of transmitter or hormone release from individual cells on microchips has applications both in basic science and drug screening. High-resolution measurement of quantal exocytosis requires the working electrode to be small (cell-sized) and located in immediate proximity to the cell. We examined the ability of candidate electrode materials to promote the attachment of two hormone-secreting cell types as a mechanism for targeting cells for to recording electrodes with high precision. We found that nitrogen-doped diamond-like carbon (DLC:N) promoted cell attachment relative to other materials tested in the rank order of DLC:N>In(2)O(3)/SnO(2) (ITO), Pt>Au. In addition, we found that treating candidate electrode materials with polylysine did not increase attachment of chromaffin cells to DLC:N, but promoted cell attachment to the other tested materials. We found that hormone-secreting cells did not attach readily to Teflon AF as a potential insulating material, and demonstrated that patterning of Teflon AF leads to selective cell targeting to DLC:N "docking sites". These results will guide the design of the next generation of biochips for automated and high-throughput measurement of quantal exocytosis.
Cardiac Cushions Modulate Action Potential Phenotype During Heart Development [corrected]
The extracellular matrix plays an important role in cardiac function. Its role in the generation and modulation of electrical activity in the early stages of heart development has not been studied extensively. Our study demonstrates that the extracellular matrix in cardiac cushions can alter the action potential phenotype by direct contact with cardiomyocytes from different regions of the heart. We also demonstrate that fibronectin, an important and abundant component of the cardiac extracellular matrix, partially mimics the effects of the cushion tissue in altering the changes in action potential. Fibronectin increases I(Ca) (2+) and acutely increases cytosolic calcium. These findings suggest that the composition of the cardiac extracellular matrix during development plays an important role in defining patterns of electrical activity in the developing heart.
Characterization of Rhythmic Ca2+ Transients in Early Embryonic Chick Motoneurons: Ca2+ Sources and Effects of Altered Activation of Transmitter Receptors
In the nervous system, spontaneous Ca(2+) transients play important roles in many developmental processes. We previously found that altering the frequency of electrically recorded rhythmic spontaneous bursting episodes in embryonic chick spinal cords differentially perturbed the two main pathfinding decisions made by motoneurons, dorsal-ventral and pool-specific, depending on the sign of the frequency alteration. Here, we characterized the Ca(2+) transients associated with these bursts and showed that at early stages while motoneurons are still migrating and extending axons to the base of the limb bud, they display spontaneous, highly rhythmic, and synchronized Ca(2+) transients. Some precursor cells in the ependymal layer displayed similar transients. T-type Ca(2+) channels and a persistent Na(+) current were essential to initiate spontaneous bursts and associated transients. However, subsequent propagation of activity throughout the cord resulted from network-driven chemical transmission mediated presynaptically by Ca(2+) entry through N-type Ca(2+) channels and postsynaptically by acetylcholine acting on nicotinic receptors. The increased [Ca(2+)](i) during transients depended primarily on L-type and T-type channels with a modest contribution from TRP (transient receptor potential) channels and ryanodine-sensitive internal stores. Significantly, the drugs used previously to produce pathfinding errors altered transient frequency but not duration or amplitude. These observations imply that different transient frequencies may differentially modulate motoneuron pathfinding. However, the duration of the Ca(2+) transients differed significantly between pools, potentially enabling additional distinct pool-specific downstream signaling. Many early events in spinal motor circuit formation are thus potentially sensitive to the rhythmic Ca(2+) transients we have characterized and to any drugs that perturb them.
Detection of Melanoma Cells in Vitro Using an Optical Detector of Photoacoustic Waves
Circulating tumor cells have been shown to correlate positively with metastatic disease state in patients with advanced cancer. We have demonstrated the ability to detect melanoma cells in a flow system by generating and detecting photoacoustic waves in melanoma cells. This method is similar to flow cytometry, although using photoacoustics rather than fluorescence. Previously, we used piezoelectric films as our acoustic sensors. However, such films have indicated false-positive signals due to unwanted direct interactions between photons from the high laser fluence in the flow system and the film itself. We have adapted an optical detection scheme that obviates the need for piezoelectric films.
Frequency-dependent Modes of Synaptic Vesicle Endocytosis and Exocytosis at Adult Mouse Neuromuscular Junctions
During locomotion, adult rodent lumbar motoneurons fire in high-frequency (80-100 Hz) 1-2 s bursts every several seconds, releasing between 10,000 and 20,000 vesicles per burst. The estimated total vesicle pool size indicates that all vesicles would be used within 30 s; thus, a mechanism for rapid endocytosis and vesicle recycling is necessary to maintain effective transmission and motor behavior. However, whether such rapid recycling exists at mouse neuromuscular junctions (NMJs) or how it is regulated has been unclear. Here, we show that much less FM1-43 dye is lost per stimulus with 100 Hz stimulation than with 10 Hz stimulation even when the same number of vesicles undergo exocytosis. Electrophysiological data using folimycin show this lesser amount of dye loss is caused in part by the rapid reuse of vesicles. We showed previously that a myosin light chain kinase (MLCK)-myosin II pathway was required for effective transmission at 100 Hz. Here, we confirm the activation of MLCK, based on increased nerve terminal phospho-MLC immunostaining, with 100 Hz but not with 10 Hz stimulation. We further demonstrate that activation of MLCK, by increased extracellular Ca(2+), by PKC (protein kinase C) activation, or by a MLCK agonist peptide, reduces the amount of dye lost even with 10 Hz stimulation. MLCK activation at 10 Hz also resulted in more vesicles being rapidly reused. Thus, MLCK activation by 100 Hz stimulation switches the mechanism of vesicle cycling to a rapid-reuse mode and is required to sustain effective transmission in adult mouse NMJs.
Knockdown of Embryonic Myosin Heavy Chain Reveals an Essential Role in the Morphology and Function of the Developing Heart
The expression and function of embryonic myosin heavy chain (eMYH) has not been investigated within the early developing heart. This is despite the knowledge that other structural proteins, such as alpha and beta myosin heavy chains and cardiac alpha actin, play crucial roles in atrial septal development and cardiac function. Most cases of atrial septal defects and cardiomyopathy are not associated with a known causative gene, suggesting that further analysis into candidate genes is required. Expression studies localised eMYH in the developing chick heart. eMYH knockdown was achieved using morpholinos in a temporal manner and functional studies were carried out using electrical and calcium signalling methodologies. Knockdown in the early embryo led to abnormal atrial septal development and heart enlargement. Intriguingly, action potentials of the eMYH knockdown hearts were abnormal in comparison with the alpha and beta myosin heavy chain knockdowns and controls. Although myofibrillogenesis appeared normal, in knockdown hearts the tissue integrity was affected owing to apparent focal points of myocyte loss and an increase in cell death. An expression profile of human skeletal myosin heavy chain genes suggests that human myosin heavy chain 3 is the functional homologue of the chick eMYH gene. These data provide compelling evidence that eMYH plays a crucial role in important processes in the early developing heart and, hence, is a candidate causative gene for atrial septal defects and cardiomyopathy.
A Comparative Evaluation of Microarray Slides As Substrates for the Development of Protease Assay Biosensors
The application of commercially available microarray slides as substrates for fluorogenic protease assays has been explored in terms of binding efficiency, stability, and activity. A fluorescent, biotinylated substrate for botulinum neurotoxin A (BoNTA) was attached via self-assembled monolayer of Streptavidin to amine-reactive aldehyde, epoxy, hydrogel, and polymer slides. Nexterion Slide P® was found to have optimal protein binding efficiency and stability of the slides examined. Addition of glycerol to the printing buffer improved spot morphology significantly and polyvinylpyrrolidone provided long-term stability, allowing chips to be stored for up to 1 month with good viability. Detection of a recombinant BoNTA light chain was then carried out at 37°C and a sub-lethal dose was detected in 2 hours.
Electrical Conduction Along Endothelial Cell Tubes from Mouse Feed Arteries: Confounding Actions of Glycyrrhetinic Acid Derivatives
Background and purpose: Electrical conduction along endothelium of resistance vessels has not been determined independent from the influence of smooth muscle, surrounding tissue or blood. Two interrelated hypotheses were tested: (1) Intercellular conduction of electrical signals is manifest in endothelial cell (EC) tubes; (2) Inhibitors of gap junction channels (GJCs) have confounding actions on EC electrical and Ca(2+) signaling. Experimental approach: Intact EC tubes were isolated from abdominal muscle feed arteries of C57BL/6 mice. Hyperpolarization was initiated with indirect (acetylcholine) and direct (NS309) stimulation of intermediate- and small-conductance Ca(2+) -activated K(+) channels (IK(Ca) /SK(Ca) ). Remote membrane potential (V(m) ) responses to intracellular current injection defined the length constant (λ) for electrical conduction. Dye coupling was evaluated following intracellular microinjection of propidium iodide. Intracellular Ca(2+) dynamics were determined using Fura-2 photometry. Carbenoxolone (CBX) or β-glycyrrhetinic acid (βGA) were used to investigate the role of GJCs. Key results: Steady-state V(m) of ECs was ∼-25 mV. Acetylcholine and NS309 hyperpolarized ECs by ∼-40 mV and ∼-60 mV, respectively. Electrical conduction decayed monoexponentially with distance (λ∼1.4 mm). Propidium iodide injected into one EC spread into surrounding ECs. CBX or βGA inhibited dye transfer, electrical conduction and EC hyperpolarization reversibly. Both agents elevated resting Ca(2+) while βGA inhibited responses to ACh. Conclusions and implications: Individual cells are well-coupled to each other within EC tubes. Inhibiting GJCs with glycyrrhetinic acid derivatives coincides with loss of hyperpolarization mediated by IK(Ca) /SK(Ca) irrespective of Ca(2+) signaling, obviating use of these agents in resolving key determinants of electrical conduction along the endothelium.
