Biomedical subjects

V Lev-Ram

Publications and source records attributed to V Lev-Ram.

At least 19 recordsLinked to original sources

PubMed2001

Spatiotemporal dynamics of guanosine 3',5'-cyclic monophosphate revealed by a genetically encoded, fluorescent indicator.

To investigate the dynamics of guanosine 3',5'-cyclic monophosphate (cGMP) in single living cells, we constructed genetically encoded, fluorescent cGMP indicators by bracketing cGMP-dependent protein kinase (cGPK), minus residues 1-77, between cyan and yellow mutants of green fluorescent protein. cGMP decreased fluorescence resonance energy transfer (FRET) and increased the ratio of cyan to yellow emissions by up to 1.5-fold with apparent dissociation constants of approximately 2 microM and >100:1 selectivity for cGMP over cAMP. To eliminate constitutive kinase activity, Thr(516) of cGPK was mutated to Ala. Emission ratio imaging of the indicators transfected into rat fetal lung fibroblast (RFL)-6 showed cGMP transients resulting from activation of soluble and particulate guanylyl cyclase, respectively, by nitric oxide (NO) and C-type natriuretic peptide (CNP). Whereas all naive cells tested responded to CNP, only 68% responded to NO. Both sets of signals showed large and variable (0.5-4 min) latencies. The phosphodiesterase (PDE) inhibitor 3-isobutyl-1-methylxanthine (IBMX) did not elevate cGMP on its own but consistently amplified responses to NO or CNP, suggesting that basal activity of guanylate cyclase is very low and emphasizing the importance of PDEs in cGMP recycling. A fraction of RFL cells showed slowly propagating tides of cGMP spreading across the cell in response to delocalized application of NO. Biolistically transfected Purkinje neurons showed cGMP responses to parallel fiber activity and NO donors, confirming that single-cell increases in cGMP occur under conditions appropriate to cause synaptic plasticity.

Amino Acid Sequence↗

PubMed2000

Optical imaging of calcium transients in neurons and pharyngeal muscle of C. elegans.

Electrophysiology and optical indicators have been used in vertebrate systems to investigate excitable cell firing and calcium transients, but both techniques have been difficult to apply in organisms with powerful reverse genetics. To overcome this limitation, we expressed cameleon proteins, genetically encoded calcium indicators, in the pharyngeal muscle of the nematode worm Caenorhabditis elegans. In intact transgenic animals expressing cameleons, fluorescence ratio changes accompanied muscular contraction, verifying detection of calcium transients. By comparing the magnitude and duration of calcium influx in wild-type and mutant animals, we were able to determine the effects of calcium channel proteins on pharyngeal calcium transients. We also successfully used cameleons to detect electrically evoked calcium transients in individual C. elegans neurons. This technique therefore should have broad applications in analyzing the regulation of excitable cell activity in genetically tractable organisms.

Animals↗

PubMed1997

Synergies and coincidence requirements between NO, cGMP, and Ca2+ in the induction of cerebellar long-term depression.

Parallel fiber synapses onto Purkinje neurons in acute cerebellar slices undergo long-term depression (LTD) when presynaptic activity coincides with postsynaptic depolarization. These electrical inputs can be respectively replaced by nitric oxide (NO) and Ca2+ photolytically released inside the Purkinje neuron, showing that these two messengers are sufficient for LTD induction. NO acts via cGMP production because inhibitors of guanylate cyclase prevent LTD but can be circumvented by photoreleased cGMP combined with Ca2+ elevation. Three inhibitors of cGMP-dependent protein kinase, Rp-8Br-PET-cGMPS, KT5823, and a novel pseudosubstrate peptide, all block LTD. LTD induction permits <10 ms gap between NO release and Ca2+ elevation, whereas 200-300 ms is allowed between uncaged cGMP and Ca2+ increase. This surprising difference in timing precision can be explained either by tighter localization and faster decay of cGMP when generated by NO rather than uncaging, or by two independent coincidence detectors in series.

Animals↗

PubMed1997

A new caged Ca2+, azid-1, is far more photosensitive than nitrobenzyl-based chelators.

BACKGROUND: Photolabile chelators that release Ca2+ upon illumination have been used extensively to dissect the role of this important second messenger in cellular processes such as muscle contraction and synaptic transmission. The caged calcium chelators that are presently available are often limited by their inadequate changes in Ca2+ affinity, selectivity for Ca2+ over Mg2+ and sensitivity to light. As these chelators are all based on nitrobenzyl photochemistry, we explored the use of other photosensitive moieties to generate a new caged calcium with improved properties. RESULTS: Azid-1 is a novel caged calcium in which a fluorescent Ca2+ indicator, fura-2, has been modified with an azide substituent on the benzofuran 3-position. Azid-1 binds Ca2+ with a dissociation constant (Kd) of approximately 230 nM, which changes to 120 microM after photolysis with ultraviolet light (330-380 nm). Mg2+ binding is weak (8-9 mM Kd) before or after photolysis. Azid-1 photolyzes with unit quantum efficiency, making it 40-170-fold more sensitive to light than caged calciums used previously. The photolysis of azid-1 probably releases N2 to form a nitrenium ion that adds water to yield an amidoxime cation; the electron-withdrawing ability of the amidoxime cation reduces the chelator's Ca2+ affinity within at most 2 ms following a light flash. The ability of azid-1 to function as a caged calcium in living cells was demonstrated in cerebellar Purkinje cells, in which Ca2+ photolytically released from azid-1 could replace the normal depolarization-induced Ca2+ transient in triggering synaptic plasticity. CONCLUSIONS: Azid-1 promises to be a useful tool for generating highly controlled spatial and temporal increases of Ca2+ in studies of the many Ca2+-dependent biological processes. Unlike other caged calciums, azid-1 has a substantial cross section or shows a high susceptibility for two-photon photolysis, the only technique that confines the photochemistry to a focal spot that is localized in three dimensions. Azide photolysis could be a useful and more photosensitive alternative to nitrobenzyl photochemistry.

Animals↗

PubMed1996

Nitric oxide acts directly in the presynaptic neuron to produce long-term potentiation in cultured hippocampal neurons.

Nitric oxide (NO) has been proposed to act as a retrograde messenger during long-term potentiation (LTP) in the CA1 region of hippocampus, but the inaccessibility of the presynaptic terminal has prevented a definitive test of this hypothesis. Because both sides of the synapse are accessible in cultured hippocampal neurons, we have used this preparation to investigate the role of NO. We examined LTP following intra- or extracellular application of an NO scavenger, an inhibitor of NO synthase, and a membrane-impermeant NO donor that releases NO only upon photolysis with UV light. Our results indicate that NO is produced in the postsynaptic neuron, travels through the extracellular space, and acts directly in the presynaptic neuron to produce long-term potentiation, supporting the hypothesis that NO acts as a retrograde messenger during LTP.

Animals↗

PubMed1996

Nitric oxide acts as a retrograde messenger during long-term potentiation in cultured hippocampal neurons.

We examined long-term potentiation (LTP) at synapses between hippocampal neurons in dissociated cell culture following presynaptic, postsynaptic, or extracellular application of a nitric oxide (NO) scavenger, an inhibitor of NO synthase, and a membrane-impermeant NO donor that releases NO only upon photolysis with UV light. Our results indicate that NO is produced in the postsynaptic neuron, travels through the extracellular space, and acts directly in the presynaptic neuron to produce long-term potentiation, supporting the hypothesis that NO acts as a retrograde messenger during LTP.

Animals↗

PubMed1995

Long-term depression in cerebellar Purkinje neurons results from coincidence of nitric oxide and depolarization-induced Ca2+ transients.

The role of nitric oxide (NO) in the induction of long-term depression (LTD) in the cerebellum was explored using a new, organic, membrane-impermeant form of caged NO. NO photolytically released inside Purkinje neurons mimicked parallel fiber (PF) activity in synergizing with brief postsynaptic depolarization to induce LTD. Such LTD required a delay of < 50 ms between the end of photolysis and the onset of depolarization, was prevented by intracellular Ca2+ chelation, and was mutually occlusive with LTD conventionally produced by PF activation plus depolarization. Bath application of NO synthase inhibitor or of myoglobin, a NO trap, prevent LTD induction via PF stimulation, but not that from intracellular uncaged NO, whereas intracellular myoglobin blocked both protocols. NO is therefore an anterograde transmitter in LTD induction. A biochemical requirement for simultaneous NO and elevation of intracellular free Ca2+ would explain why PF activity must coincide with postsynaptic action potentials.

Action Potentials↗

PubMed1995

Activity-related calcium dynamics in lamprey motoneurons as revealed by video-rate confocal microscopy.

In lamprey spinal cord, intracellular calcium ([Ca2+]i) plays a key role in mechanisms regulating neuronal activity in the segmental network for locomotion. In this report, measurements of [Ca2+]i with fluo-3 in various regions of motoneurons in the intact spinal cord were obtained on a high speed confocal microscope following electrical stimulation. Likewise, rhythmic calcium fluctuations within dendrites and axons were seen during "fictive swimming" and were directly correlated with electrical activity. Antidromic stimulation of motoneuron axons induced large calcium transients and revealed spatially restricted "hot spots," both of which required external calcium and were blocked by nickel, but not by known calcium channel antagonists. These results suggest that lamprey spinal cord axons may possess a pharmacologically novel class of calcium channel.

Aniline Compounds↗

PubMed1995

Axonal activation-induced calcium transients in myelinating Schwann cells, sources, and mechanisms.

We have investigated the role of myelinating glia in events associated with propagation of the action potential at nodes of Ranvier using combinations of optical and electrophysiological recording methods. Calcium transients were observed in Schwann cells by fluorescent imaging of the nodal complex of fibers loaded with the calcium-sensitive dye fluo3-AM. To follow [Ca2+]i changes associated with neuronal activity at the node of Ranvier, nerves loaded with fluo3 were imaged during axonal activation using laser-scanning confocal microscopy. To elucidate sources of [Ca2+]i transients, we tested the effects of drugs known to alter [Ca2+]i. [Ca2+]i transients in Schwann cells were observed in response to axonal activation and these were subsequently blocked by ryanodine if ryanodine was present during a previous [Ca2+]i transient. Bath applications of caffeine induced [Ca2+]i transients which could be blocked by ryanodine. These findings indicate that calcium-activated calcium release occurs in Schwann cells in response to impulse activity.

Action Potentials↗

PubMed1994

A toxin from the venom of the predator snail Conus textile modulates ionic currents in Aplysia bursting pacemaker neuron.

Conus textile crude venom and a peptide component ('King Kong' toxin) purified from this venom, alter membrane excitability of Aplysia neurons. Venom, applied to the medium bathing an abdominal ganglion, changes dramatically the electrical activity of bursting pacemaker neuron. The effects on bursting neuron R15 was examined in current-clamp and voltage-clamp modes. A dual phase effect of both the venom and the purified toxin were observed. The first phase starts immediately after venom or toxin application and is observed as an increase in membrane excitability, resulting in an enhancement of bursting. The second phase begins about 15 min later and consists of a long-lasting hyperpolarization. The dual phase effect of the venom and the toxin persists even when synaptic input is eliminated either by axotomy, or by recording from freshly dissociated neurons or from neurons in primary cell culture. The ionic currents affected are an inward current, INSR, which is activated upon depolarization and an anomalously rectifying potassium current, IR, which is activated upon hyperpolarization. In the first phase of toxin action INSR is increased. In the second phase both the venom and the toxin block INSR and increase IR. The toxin effects may be due to complex alteration of one or more second messenger cascades rather than a direct action on ion channels.

Animals↗

PubMed1994

Fluorescence photooxidation with eosin: a method for high resolution immunolocalization and in situ hybridization detection for light and electron microscopy.

A simple method is described for high-resolution light and electron microscopic immunolocalization of proteins in cells and tissues by immunofluorescence and subsequent photooxidation of diaminobenzidine tetrahydrochloride into an insoluble osmiophilic polymer. By using eosin as the fluorescent marker, a substantial improvement in sensitivity is achieved in the photooxidation process over other conventional fluorescent compounds. The technique allows for precise correlative immunolocalization studies on the same sample using fluorescence, transmitted light and electron microscopy. Furthermore, because eosin is smaller in size than other conventional markers, this method results in improved penetration of labeling reagents compared to gold or enzyme based procedures. The improved penetration allows for three-dimensional immunolocalization using high voltage electron microscopy. Fluorescence photooxidation can also be used for high resolution light and electron microscopic localization of specific nucleic acid sequences by in situ hybridization utilizing biotinylated probes followed by an eosin-streptavidin conjugate.

Animals↗

PubMed1993

A novel non-ataxic guinea pig strain with cerebrocortical and cerebellar abnormalities.

This is the first description of GS guinea pigs, a partially inbred, non-ataxic, albino Peruvian (long-hair) strain with abnormal motor behavior and seizures. GS guinea pigs show gross and microscopic cerebellar and microscopic cerebrocortical abnormalities compared to Hartley strain animals. There is little difference between GS and short-hair guinea pig strains in Purkinje cell function, electrically evoked Ca2+ transients or immune responsiveness. The GS strain may prove useful in studying altered functions of the cerebellum in human disease.

Animals↗

PubMed1993

Dendritic excitability in CNS neurons: insights from dynamic calcium and sodium imaging in single cells.

We measured changes in [Ca2+]i and [Na+]i in the dendrites of cerebellar Purkinje cells and hippocampal pyramidal cells using high speed imaging of the fluorescence changes of intracellularly injected fura-2 and SBFI. These transients were detected in synchrony with intracellular recordings of membrane potential. In this way rapid calcium or sodium transients could be associated with specific electrical events. Using this technique we could determine the spatial distribution and source of transients evoked either intrasomatically or synaptically and could relate them to electrically recorded sodium and calcium spikes.

Animals↗

PubMed1992

Calcium transients in cerebellar Purkinje neurons evoked by intracellular stimulation.

1. Purkinje cells in thin slices from the guinea pig cerebellum were injected with fura-2 and high-speed sequences of fluorescence images from the cell body and entire dendritic tree were made while simultaneously recording somatic membrane potential during evoked and spontaneous electrical activity. The changes in fluorescence were interpreted in terms of changes in [Ca2+]i. 2. Individual calcium action potentials were usually accompanied by transient increases in [Ca2+]i all over the dendritic field. During evoked or spontaneous bursts of calcium spikes, [Ca2+]i increased more rapidly and to higher concentrations in fine dendrites than in thicker dendrites. At the end of a burst [Ca2+]i declined faster in thin dendrites than in thicker ones. These variations are most easily understood as deriving from the difference in surface-to-volume ratio of the two kinds of dendrites. 3. During bursts of calcium action potentials [Ca2+]i increases sometimes occurred only in individual dendritic branches, but always including the fine dendrites of that particular branch, showing that calcium action potentials can be regenerative in restrictive parts of the dendritic field without involving the soma or dendritic shaft. 4. Plateau or subthreshold potential changes evoked in the presence of tetrodotoxin (TTX) caused small, widespread increases in [Ca2+]i. The amplitude of these changes was much less than the increase corresponding to spike bursts. The distribution of these plateau Ca signals in thick and thin dendrites was similar to Ca spike-evoked signals, suggesting that the Ca conductances underlying these two potentials are the same or are distributed similarly in the dendrites. 5. Significant increases in [Ca2+]i in the soma were recorded during bursts of sodium-dependent action potentials in normal Ringer. Although much of this increase is due to calcium entry through calcium channels, some of this increase could be due to calcium entry through sodium channels.

Action Potentials↗

PubMed1992

Calcium transients evoked by climbing fiber and parallel fiber synaptic inputs in guinea pig cerebellar Purkinje neurons.

1. Calcium transients related to climbing fiber (CF) and parallel fiber (PF) synaptic potentials were recorded from Purkinje cells in guinea pig cerebellar slices. Transients were measured using either absorbance changes of arsenazo III or fluorescence changes of fura-2, which were injected into individual cells in the slice. 2. All-or-none somatically recorded CF potentials elicited by white matter stimulation had all-or-none Ca transients. These signals began with a delay of > or = 2 ms from the start of the electrically recorded synaptic potential. The recovery time of CF-induced arsenazo III absorbance transients was < 50 ms in the fine dendrites in conditions that minimized the effects of dye buffering. 3. Ca2+ entry through voltage-gated Ca channels opened by Ca action potentials was the dominant source of the rise in [Ca2+]i after CF activation. There was no significant change in [Ca2+]i corresponding to the plateau potential that followed the large CF response. 4. The appearance and amplitude of distal CF-evoked Ca signals was more variable than proximal signals, suggesting that CF potentials do not reliably spread to the fine distal dendrites. The distal transient could be enhanced by intrasomatic depolarizing pulses, suggesting that it was a property of the postsynaptic membrane and not the presynaptic side of the CF synapse that was responsible for this variability. 5. Parallel fiber responses were evoked by electrical stimulation near the pial surface. Graded synaptic potentials and related Ca transients were reversibly blocked by 2 microM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Small synaptic potentials induced small, localized Ca transients. With increasing stimulus intensity, the PF electrical response developed a regenerative component. Larger dendritic Ca transients were detected corresponding to this component. Ca transients evoked by the regenerative responses had the same rapid rise times and fall times as those related to somatically stimulated Ca action potentials, suggesting that they also were due to Ca2+ entry through voltage-sensitive channels. 6. During trains of PF responses, we observed an increase in the spatial extent of related Ca transients. This effect could be modulated by changes in the resting potential, suggesting that the same intrinsic mechanism was affecting the spread of both CF and PF signals.

6-Cyano-7-nitroquinoxaline-2,3-dione↗

PubMed1991

High time resolution fluorescence imaging with a CCD camera.

We have built a high speed, sensitive camera system capable of capturing sequences of low-light level images synchronized with recordings of membrane potential. The camera system is based on a cooled, scientific grade CCD camera controlled by a PC/AT computer. It can take 100 frames/sec of 18 X 18 element images and 40 frames/sec of 50 X 50 element images with no lag in response to step changes in light intensity. High accuracy and dynamic range of the measurements result from the fact that light levels of the picture elements are digitized with 12 bit accuracy with intrinsic camera noise levels typically less than 1/10,000 of the maximum detectable light level. We have used this system to record calcium dependent fura-2 fluorescence transients in the dendrites of cerebellar Purkinje cells and from different regions of leech neurons in segmental ganglia or isolated in culture.

Animals↗

PubMed1987

Activity-dependent calcium transients in central nervous system myelinated axons revealed by the calcium indicator Fura-2.

Optical measurements from rat optic nerve, loaded with the new Ca2+ indicator Fura-2, provide the first evidence for the presence of activity-dependent fast intracellular [Ca2+] transients in mammalian central nervous system (CNS) myelinated axons. The results suggest that voltage-dependent Ca2+ channels are present in some of the myelinated axons. Optical measurements from axons stained with anterogradely transported voltage-sensitive dye suggest the presence of Ca2+-dependent potassium conductances in these axons. This report also demonstrates that Fura-2 can readily detect changes in [Ca2+] inside cells as a result of electrical activity, and establishes its suitability for measurements of intracellular Ca2+ transients in the millisecond time domain.

Animals↗

PubMed1987

Optical recording of synaptic potentials from processes of single neurons using intracellular potentiometric dyes.

To record post synaptic potentials or electrical activity from processes of single cells in a central nervous system (CNS) preparation in situ, voltage sensitive dyes can be injected intracellularly, thereby staining only the cell under investigation. We report the structure, evaluation, and synthesis of 11 fluorescent styryl dyes developed for iontophoretic injection. The optical signals that represent small synaptic potentials from single processes of iontophoretically injected cells are expected to be very small and, therefore, such measurements are not easy. We report the methodology that permitted the optical recording of action potentials from a 3-micron axon and the recording of small synaptic potentials from the processes of single cells in the segmental ganglia of the leech. The same dyes also proved useful for optical recording of action potentials of anterogradely labeled axons, following local extracellular injection at a remote site in a mammalian CNS preparation.

Action Potentials↗