Biomedical subjects

Robin L Cooper

Publications and source records attributed to Robin L Cooper.

At least 19 recordsLinked to original sources

PubMed2006

Recent advances in basic neurosciences and brain disease: from synapses to behavior.

Understanding basic neuronal mechanisms hold the hope for future treatment of brain disease. The 1st international conference on synapse, memory, drug addiction and pain was held in beautiful downtown Toronto, Canada on August 21-23, 2006. Unlike other traditional conferences, this new meeting focused on three major aims: (1) to promote new and cutting edge research in neuroscience; (2) to encourage international information exchange and scientific collaborations; and (3) to provide a platform for active scientists to discuss new findings. Up to 64 investigators presented their recent discoveries, from basic synaptic mechanisms to genes related to human brain disease. This meeting was in part sponsored by Molecular Pain, together with University of Toronto (Faculty of Medicine, Department of Physiology as well as Center for the Study of Pain). Our goal for this meeting is to promote future active scientific collaborations and improve human health through fundamental basic neuroscience researches. The second international meeting on Neurons and Brain Disease will be held in Toronto (August 29-31, 2007).

Behavior↗

PubMed2006

Automated classification of evoked quantal events.

We provide both theoretical and computational improvements to the analysis of synaptic transmission data. Theoretically, we demonstrate the correlation structure of observations within evoked postsynaptic potentials (EPSP) are consistent with multiple random draws from a common autoregressive moving-average (ARMA) process of order (2, 2). We use this observation and standard time series results to construct a statistical hypothesis testing procedure for determining whether a given trace is an EPSP. Computationally, we implement this method in R, a freeware statistical language, which reduces the amount of time required for the investigator to classify traces into EPSPs or non-EPSPs and eliminates investigator subjectivity from this classification. In addition, we provide a computational method for calculating common functionals of EPSPs (peak amplitude, decay rate, etc.). The methodology is freely available over the internet. The automated procedure to index the quantal characteristics greatly facilitates determining if any one or multiple parameters are changing due to experimental conditions. In our experience, the software reduces the time required to perform these analyses from hours to minutes.

Animals↗

PubMed2006

Direct innervation of the Drosophila melanogaster larval aorta.

The heart rate of larval Drosophila is modulated by various biogenic amines and peptides. The actions have always been assumed to be due to direct action on the heart since the larval heart was not known to be innervated. A recent study showed a difference in the sensitivity of the larval heart to serotonin when the CNS was ablated, thus suggesting a direct neural input. Here, we show that GFP tagged motor neurons and nerve terminals are present on the aortic region of the heart. Motor neuron cell bodies also exist outside the CNS. Transmission electron microscopy reveals the direct innervation in the aortic tissue. Thus, developmental and regulatory questions in this genetic model can now be addressed in relation to heart development and neural control.

Animals↗

PubMed2006

Self-modeling structure of evoked postsynaptic potentials.

With the simplicity of the synaptic structure and physiology at neuromuscular junctions (NMJs) of crayfish and the given transmitter being released in quantal packets, a detailed assessment in the fundamental processes of chemical synaptic transmission is possible. Since the quantal event is the basic element of transmission, we consider an approach to further understand the characteristics of quantal responses. In this study, we introduce a method for combining information across excitatory postsynaptic potentials (EPSPs) that are quantal in nature. The method is called self-modeling regression, known in the statistics literature as SEMOR. This method illustrates that the differing timing and heights of EPSPs can be described with four coefficients measuring affine (shift and scale) transformations of the x and y axes. We demonstrate that this relationship allows us to provide a unified schema for the many functionals currently used in the literature, such as peak amplitude, tau, latency, area under the curve, or decay time. Computer code in R is available on the internet to perform the analysis.

Animals↗

PubMed2005

Direct influence of serotonin on the larval heart of Drosophila melanogaster.

The heart rate (HR) of larval Drosophila is established to be modulated by various neuromodulators. Serotonin (5-HT) showed dose-dependent responses in direct application within semi-intact preparations. At 1 nM, HR decreased by 20% while it increased at 10 nM (10%) and 100 nM (30%). The effects plateaued at 100 nM. The action of 5-HT on the heart was examined with an intact Central Nervous System (CNS) and an ablated CNS. The heart and aorta of dorsal vessel pulsate at different rates at rest and during exposure to 5-HT. Splitting the heart and aorta resulted in a dramatic reduction in pulse rate of both the segments and the addition of 5-HT did not produce regional differences. The split aorta and heart showed a high degree of sensitivity to sham changes of saline but no significant effect to 5-HT. Larvae-fed 5-HT (1 mM) did not show any significant change in HR. Since 3,4-methylenedioxymethamphetamine (MDMA) is known to act as a weak agonist on 5-HT receptors in vertebrates, we tested an exogenous application; however, no significant effect was observed to dosage ranging from 1 nM to 100 microM in larvae with and without an intact CNS. In summary, direct application of 5-HT to the larval heart had significant effects in a dose-dependent manner while MDMA had no effect.

Animals↗

PubMed2005

Presynaptic mechanism of action induced by 5-HT in nerve terminals: possible involvement of ryanodine and IP3 sensitive 2+ stores.

Although modulation of transmitter release by serotonin (5-HT) at crayfish neuromuscular junctions has been known since 1965, the mechanisms of action have not been established in this classical synaptic preparation. We show that injections of adenophostin-A (an IP3 analog) in the nerve terminals greatly enhances synaptic transmission. Exposure to ryanodine (Ry) produces a biphasic response: at low concentration it is excitatory and high concentration it is inhibitory. Likewise, a low concentration (1 microM) of caffeine enhances synaptic transmission, whereas a high concentration (10 mM) has little effect on transmission. The varied responses and sensitivity to Ry and caffeine suggest a Ca(2+)-induced Ca(2+)-release mechanism and/or the presence of an IP3-receptor within the terminal. Thus, it is likely 5-HT's response is due to activation of intracellular pathways, which subsequently release Ca2+ from internal stores.

Adenosine↗

PubMed2005

Developmental consequences of neuromuscular junctions with reduced presynaptic calcium channel function.

Evoked neurotransmitter release at the Drosophila neuromuscular junction (NMJ) is regulated by the amount of calcium influx at the presynaptic nerve terminal, as for most chemical synapses. Calcium entry occurs via voltage-gated calcium channels. The temperature-sensitive Drosophila mutant, cac(TS2), has a reduced amount of calcium entry during evoked stimulation. We have used this mutation to examine homeostatic regulatory mechanisms during development of the NMJ on muscle 6 within the developing larva. The amplitude of the excitatory postsynaptic potentials are reduced for both the Ib and Is motor neurons in 3rd instar larvae which have been raised at 33 degrees C from the 1st instar stage. Larvae raised at 25 degrees C and larvae pulsed at 33 degrees C from the late 2nd instar for various lengths of time show a reduced synaptic efficacy as a 3rd instar. The results indicate that the nerve terminal cannot fully compensate physiologically in the regulation of synaptic transmission during larval life for a reduced amount of evoked calcium entry. Morphological comparisons of Ib and Is terminals in relation to length and numbers of varicosities are significantly reduced in cac(TS2), which also suggests a lack in homeostatic ability. These findings are relevant since many deficits in synaptic transmission in various systems are compensated for either physiologically or structural over development, but not in this case for reduced calcium entry during evoked transmission.

Age Factors↗

PubMed2005

The physiological and behavioral effects of carbon dioxide on Drosophila melanogaster larvae.

Adult and larval insects are rapidly anesthetized by carbon dioxide (CO2); however, the mechanisms have not been addressed. In this study, we use larval Drosophila to investigate the actions of CO2 to explain the behavioral effects of rapid immobilization and cardiac arrest with acute exposure to CO2. To determine if the central nervous system (CNS) is required, studies were performed with and without the CNS. The effects of low pH induced by exposure to CO2 were also examined. An acidic saline increases the heart rate in contrast to saline containing CO2. Synaptic transmission at the skeletal neuromuscular junction (NMJ) is blocked by CO2 but not by low pH. The site of action is postsynaptic by a decreased sensitivity to glutamate, the neurotransmitter at Drosophila NMJs. The CNS remains active in synaptic transmission when exposed to CO2 which is in contrast to the synapses at the NMJ. In summary, the effects of CO2 are directly mediated on the heart to stop it and at skeletal NMJs by a reduced sensitivity to glutamate, the released neurotransmitter, from the motor nerve terminals. The rapid behavioral and physiological effects cannot be accounted for by action on the CNS within the larvae nor by a pH effect indirectly induced by CO2. The glutamate receptors in the D. melanogaster preparation are similar in function to ionotropic glutamate receptors in vertebrates which could account for the observational phenomena of CO2 not yet explained mechanistically in vertebrates.

Acids↗

PubMed2004

Novelty stress and reproductive state alters responsiveness to sensory stimuli and 5-HT neuromodulation in crayfish.

Sensory stimuli can produce varied responses depending on the physiological state of an animal. Stressors and reproductive stage can result in altered biochemical status that changes the responsiveness of an animal to hormones and neuromodulators, which affects whole animal behavior in relation to sensory stimuli. Crayfish serve as a model for examining the effects of neuromodulators at the neuromuscular junctions (NMJs) and for alterations in stereotypic behaviors for particular stimuli. Thus, we used crayfish to examine the effect of novelty stressors in males and the effect of being gravid in female crayfish to exogenous application of serotonin (5-HT). The responsiveness of neuromuscular junctions to 5-HT revealed that stressed as well as gravid crayfish have a reduced response to 5-HT at NMJs. The stressed crayfish were not fatigued since the basal synaptic responses are large and still showed a pronounced response to 5-HT. Using intact animals to examine a tail flip behavior, we showed that the rate of habituation in tail flipping to a strong repetitive stimulus on the telson is reduced in stressed males. Gravid females show no tail flipping behavior upon telson stimulation.

Animals↗

PubMed2004

Modulation of sensory-CNS-motor circuits by serotonin, octopamine, and dopamine in semi-intact Drosophila larva.

We have introduced an in-situ preparation to induce motor unit activity by stimulating a sensory-CNS circuit, using the third instar larvae of Drosophila melanogaster. Discrete identifiable motor units that are well defined in anatomic and physiologic function can be recruited selectively and driven depending on the sensory stimulus intensity, duration, and frequency. Since the peripheral nervous system is bilaterally symmetric to coordinate bilateral symmetric segmental musculature patterns, fictive forms of locomotion are able to be induced. Monitoring the excitatory postsynaptic potentials (EPSP) on the prominent ventral longitudinal body wall muscles, such as m6 and m12, provides additional insight into how the selective motor units might be recruited within intact animals. We also introduce the actions of the neuromodulators (serotonin, octopamine (OA) and dopamine (DA)) on the inducible patterns of activity within the sensory-motor circuit. The powerful genetic manipulation in Drosophila opens many avenues for further investigations into the circuitry and cellular aspects of pattern generation and developmental issues of circuitry formation and maintenance in the model organism.

Animals↗

PubMed2003

Modulatory effects of melatonin on behavior, hemolymph metabolites, and neurotransmitter release in crayfish.

Melatonin affects a variety of circadian processes such as behavior and neurotransmitter release in vertebrates. Crayfish melatonin production occurs in the eyestalks, and the cycle of production may change seasonally. To date, however, melatonin's roles and mechanisms of action in crustacean physiology are unclear. We injected melatonin or saline into crayfish in scotophase and monitored activity and hemolymph glucose/lactate over 24 h in early spring. Crayfish were significantly more active in photophase versus the expected scotophase, and had concurrent glucose/lactate peaks. Melatonin reversed the activity pattern, causing a scotophase activity peak, but not the glucose/lactate patterns. This study was repeated in late summer, during which control activity and glucose/lactate levels were elevated in scotophase. Melatonin decreased the amplitude of scotophase activity and glucose/lactate, eliminating activity and glucose cycles. We also injected melatonin or saline at various times of day in early summer and monitored locomotor activity for 1 h. Controls had high activity at 1200 (mid-photophase) and 2100 h (early scotophase), and melatonin increased activity at 1200 h but decreased it at 2100 h. Melatonin also increased activity at 1500 h but not 1800 h (late photophase). Next, we examined the influence of melatonin on crayfish neurophysiology. Melatonin (10 microM) enhanced synaptic transmission at the neuromuscular junction (NMJ). The presynaptic action resulted in more vesicles being released during evoked stimulation. Our study indicates that melatonin may have a phylogenetically conserved role in the transduction of circadian information in invertebrates as in vertebrates. Behavioral and physiological effects may be mediated by modulation of central pathways, enhanced at the peripheral level via neuromodulation of the NMJ.

Animals↗

PubMed2003

Effects of m-CPP in altering neuronal function: blocking depolarization in invertebrate motor and sensory neurons but exciting rat dorsal horn neurons.

The compound m-chlorophenylpiperazine (m-CPP) is used clinically to manipulate serotonergic function, though its precise mechanisms of actions are not well understood. m-CPP alters synaptic transmission and neuronal function in vertebrates by non-selective agonistic actions on 5-HT(1) and 5-HT(2) receptors. In this study, we demonstrated that m-CPP did not appear to act through a 5-HT receptor in depressing neuronal function in the invertebrates (crayfish and Drosophila). Instead, m-CPP likely decreased sodium influx through voltage-gated sodium channels present in motor and primary sensory neurons. Intracellular axonal recordings showed that m-CPP reduced the amplitude of the action potentials in crayfish motor neurons. Quantal analysis of excitatory postsynaptic currents, recorded at neuromuscular junctions (NMJ) of crayfish and Drosophila, indicated a reduction in the number of presynaptic vesicular events, which produced a decrease in mean quantal content. m-CPP also decreased activity in primary sensory neurons in the crayfish. In contrast, serotonin produces an increase in synaptic strength at the crayfish NMJ and an increase in activity of sensory neurons; it produces no effect at the Drosophila NMJ. In the rat spinal cord, m-CPP enhances the occurrence of spontaneous excitatory postsynaptic potentials with no alteration in evoked currents.

Animals↗

PubMed2003

The effects of serotonin and ecdysone on primary sensory neurons in crayfish.

The overall behaviors and motivational states observed during social interactions and throughout the molting cycle of crayfish have been linked to the effects of humoral neuromodulators. Both serotonin (5-HT) and a molt-related hormone, 20-hydroxyecdysone (20-HE), are known to be present in the hemolymph of crustaceans. To determine if they alter the activity of a primary sensory neuron that monitors proprioceptive information, we examined their effects on the activity of the slow-adapting muscle receptor organ (MRO) of the crayfish abdomen, a model sensory system that has been extensively studied. 5-HT within the range of 100 nM to 1 microM, increases the firing frequency of the neuron during sustained stimulation. In experiments in which 20-HE was added alone, an increase in the firing frequency also occurred, although to a lesser degree than that for 5-HT at the same concentrations. When the MRO is first exposed to 20-HE, followed sequentially by 5-HT, the activity increases to about the same degree as in the reverse order of exposure. This outcome indicates that mixtures of these endogenous neuromodulators, at various levels, are more important in alternating behavior than the absolute level of any one of them introduced alone.

Animals↗

PubMed2003

Estimating the number of release sites and probability of firing within the nerve terminal by statistical analysis of synaptic charge.

Investigating the function of individual synapses is essential to understanding the mechanisms that influence the efficacy of chemical synaptic transmission. The known simplicity of the synaptic structure at the crayfish neuromuscular junction (NMJ) and its quantal nature of release allows an assessment of discrete synapses within the motor nerve terminals. Our goal in this article is to investigate the effect of the stimulation frequency on the number of active release sites (n) and the probability of release (p) at those active sites. Because methods based on direct counts often provide unstable joint estimates of (n) and (p), we base our analysis on mixture modeling. In particular, the mixture modeling approach is used to estimate (n) and (p) for stimulation frequencies of 1 Hz, 2 Hz, and 3 Hz. Our results indicate that as the stimulation frequency increases, new sites are recruited (thus increasing n) and the probability of release (p) increases.

Action Potentials↗

PubMed2003

Intrinsic differences in sensitivity to 5-HT between high- and low-output terminals innervating the same target.

The differential action of neuromodulators on synapses of various efficacy provides additional fine tuning of synaptic regulation beyond frequency induced plasticity. We used the well-characterized high- and low-output motor nerve terminals, of the tonic and phasic neuromuscular junctions (NMJs) in the walking leg extensor muscle of the crayfish, to investigate differential actions of serotonin (5-HT) since both terminals innervate the same target. The excitatory postsynaptic potentials of the tonic NMJ are enhanced to a greater extent than for the phasic NMJs during exposure to 5-HT (100 nM). Macropatch current recordings at identified sites along the motor nerve terminals and quantal analysis indicate that mean quantal content is substantially increased by 5-HT. The overall probability of vesicular release increases to a greater extent at tonic terminals than at phasic terminals when exposed to 100 nM 5-HT. Measures in the area (i.e. charge) of spontaneous quantal currents indicate no difference in postsynaptic receptivity to the glutamatergic synaptic transmission upon exposure to 5-HT. The results provide new details concerning differential modulation of low- and high-output synapses present on the same target tissue.

Animals↗

PubMed2003

Long-term in vitro maintenance of neuromuscular junction activity of Drosophila larvae.

The larval Drosophila neuromuscular junction (NMJ) has proven to be an excellent system to test fundamental aspects of synaptic transmission, such as relationships among ion channel function, subtypes of glutamate receptors, and the functions of synaptic proteins in the presynaptic compartment. Recent advances in understanding bi-directional communication between nerves and muscles of Drosophila are helping uncover developmental as well as maintenance cues that could be applicable to all chemical synapses. The development of HL3 medium makes it possible to record synaptic responses at NMJs for prolonged periods of time. We demonstrate that media commonly used to culture CNS neurons and imaginal disks of Drosophila such as Schneider's and M3 completely block glutamatergic synaptic transmission at the NMJ. The depressed postsynaptic excitatory junction potentials (EJPs) partially recover from exposure to such media shortly after switching to the HL3 medium. Preliminary results from NMJs of filleted 3rd instar larvae for 4 days in vitro bathed in a modified HL3 medium show great promise. The resting membrane potential and the EJP amplitudes after 4 days in vitro are normal. These results demonstrate the possibility for chronic studies of developmental regulation in culture, which in some cases are impractical in the whole animal.

Animals↗

PubMed2003

Characterization of development, behavior and neuromuscular physiology in the phorid fly, Megaselia scalaris.

The Phoridae is known as 'scuttle flies' because they walk in rapid bursts of movement with short pauses between. In this study, larval locomotive behavior and development was characterized in the phorid, Megaselia scalaris. Comparison was made with the well-characterized fruit fly model, Drosophila melanogaster. Developmentally, the rate of maturation was consistently slower for Megaselia than Drosophila. This disparity was exaggerated at lower temperatures, particularly during larval development. In addition to slower growth, movements in Megaselia were also slower, as evidenced by reduced rates of larval body wall contractions and mouth hook movements. Megaselia larvae also displayed a unique behavior of swallowing air when exposed to a small pool of liquid. This permitted floating upon immersion and, therefore, might prevent drowning in the natural environment. The anatomical and physiological properties of a neuromuscular junction in the phorid larvae were also examined. The innervation of the motor nerve terminals on the ventral abdominal muscle (m6) is innervated by Type Ib and Is axons, similar to Drosophila. As in Drosophila, the Is terminals produce larger excitatory postsynaptic potentials (EPSPs) than the Ib. The amplitudes of the EPSPs in M. scalaris were reduced compared to those of D. melanogaster, but unlike D. melanogaster the EPSPs showed marked facilitation when stimulated with a 20 Hz train. We conclude that there may be differences in synaptic structure of the nerve terminals that could account for the different electrophysiological behaviors.

Animals↗

PubMed2002

Intracellular ionic concentration by calibration from fluorescence indicator emission spectra, its relationship to the K(d), F(min), F(max) formula, and use with Na-Green for presynaptic sodium.

The emission spectra calibration curves for a fluorescence indicator and the F(min), F(max), and K(d) formula were shown to be related. Using the known calibrated fluorescence emitted by Sodium Green (Na-Green) and photo-multiplier-tube quantum efficiency, we calculated the detection signal over a range of sodium concentrations. The calculated calibration curves were compared for optical filters passing a narrow band, medium band or full spectrum. We found that a method based on the full emission spectrum was the most appropriate. Given a known resting concentration of intracellular sodium, calibrated readings can be converted to concentration values. This method is applicable to any fluorescence indicator when curves for emission spectra over a range of concentrations are available. We measured sodium concentration changes during trains of action potentials (APs) at a crayfish motor axon's presynaptic terminals injected with Na-Green. During low frequency AP trains, net sodium increases asymptotically with frequency. Average net Na-flux per AP decreases for increasing terminal size. The terminals of crayfish motor axon have surface area to volume ratio which is 7700 times larger than for squid. Thus, in comparison to squid, crayfish terminals exhibit a larger change in [Na(+)](i) during equivalent AP activity.

Action Potentials↗