Physiologically Identified 5-HT2-like Receptors at the Crayfish Neuromuscular Junction

Brain Research. Apr, 2002  |  Pubmed ID: 11911865

The model synaptic preparation of the crayfish opener neuromuscular junction is known to be responsive to exogenous application of 5-HT. The primary effect of 5-HT is an enhancement of vesicular release from the presynaptic motor nerve terminal. 5-HT is known to act through an IP(3) cascade which suggests the presence of a 5-HT(2) receptor subtype; however, this is based on vertebrate 5-HT receptor classification. We examined this possibility by using a selective agonist and two antagonists of the vertebrate 5-HT(2) receptor subtypes. The antagonist ketanserin and spiperone reduce the responsiveness of 5-HT in a dose-dependent manner. The broad 5-HT(2) receptor agonist, alpha-methyl-5-hydroxytryptamine (alpha-Me-5-HT) enhances synaptic transmission, in a concentration-dependent manner, but it is not as potent as 5-HT. These results support the notion that a 5-HT(2) receptor subtype is present presynaptically on the crayfish motor nerve terminals. By knowing the types of 5-HT receptors present on the presynaptic motor nerve terminals in this model synaptic preparation, a better understanding of the mechanisms of action of 5-HT on vesicular release will be forthcoming.

The Effects of Ethanol on Pre-synaptic Components of Synaptic Transmission in a Model Glutamatergic Synapse: the Crayfish Neuromuscular Junction

Comparative Biochemistry and Physiology. Toxicology & Pharmacology : CBP. Mar, 2002  |  Pubmed ID: 11912064

We have elucidated some of the mechanisms by which ethanol (EtOH) reduces synaptic efficacy at model glutamatergic synapses. The crayfish phasic and tonic neuromuscular junctions are superb models for directly assessing the effects of EtOH on pre-synaptic components of synaptic transmission. The ability to perform quantal analysis of synaptic transmission has allowed us to assess pre-synaptic alterations of release. Using this system, we report that the application of EtOH, within a range observed in intoxicated humans (44 and 88 mM), resulted in a diminution of excitatory post-synaptic potentials (EPSP) amplitudes. Additionally, using focal macro-patch recordings, quantal synaptic currents were recorded to assess the pre-synaptic component as potential target sites for EtOH's action. At the tonic neuromuscular junctions, EtOH (88 mM) reduced the probability of release (p), and in some cases, reduced the number of the release sites (n), but did not alter facilitation index nor did it affect the latency of vesicular release. At the phasic neuromuscular junction, a reduction in synaptic charge occurred during the presence of EtOH. Thus, the observed decrease in synaptic strength is at least partially attributable to a pre-synaptic alteration, specifically the release of fewer vesicles.

Sphingosine 1-phosphate Enhances Spontaneous Transmitter Release at the Frog Neuromuscular Junction

British Journal of Pharmacology. Aug, 2002  |  Pubmed ID: 12163341

Intracellular recordings were made from isolated frog sciatic-sartorius nerve-muscle preparations, and the effects of sphingosine 1-phosphate (S1-P) on miniature endplate potentials (MEPPs) were studied. Extracellular application of S1-P (1 and 30 micro M) had no significant effects on the frequency and amplitude of MEPPs. Delivery into nerve terminals by liposomes containing 10(-5), 10(-4) or 10(-3) M S1-P was associated with a concentration-dependent increase in MEPP frequency of 37, 63 and 86%. The per cent of median MEPP amplitude was not significantly changed, but there was an increase in the number of 'giant' MEPPs. Pre-exposure of the preparations to S1-P 10(-5) but not 10(-8) M entrapped in liposomes for 15 min blocked the effects of subsequent superfusion of S1-P (10(-4) M)-filled liposomes on MEPP frequency. Thus, intracellular S1-P receptors seem to undergo 'desensitization' to higher concentrations of S1-P. The result provides the first evidence that S1-P acting intracellularly but not extracellularly enhances spontaneous transmitter release at the frog neuromuscular junction.

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

Journal of Neuroscience Methods. Aug, 2002  |  Pubmed ID: 12204307

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.

Estimating the Number of Release Sites and Probability of Firing Within the Nerve Terminal by Statistical Analysis of Synaptic Charge

Synapse (New York, N.Y.). Jan, 2003  |  Pubmed ID: 12422369

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.

The Effects of Serotonin and Ecdysone on Primary Sensory Neurons in Crayfish

Microscopy Research and Technique. Feb, 2003  |  Pubmed ID: 12539163

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.

Intrinsic Differences in Sensitivity to 5-HT Between High- and Low-output Terminals Innervating the Same Target

Neuroscience Research. Feb, 2003  |  Pubmed ID: 12573463

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.

Effects of M-CPP in Altering Neuronal Function: Blocking Depolarization in Invertebrate Motor and Sensory Neurons but Exciting Rat Dorsal Horn Neurons

Brain Research. Apr, 2003  |  Pubmed ID: 12676360

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.

Modulatory Effects of Melatonin on Behavior, Hemolymph Metabolites, and Neurotransmitter Release in Crayfish

Brain Research. Dec, 2003  |  Pubmed ID: 14625064

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.

Modulation of Sensory-CNS-motor Circuits by Serotonin, Octopamine, and Dopamine in Semi-intact Drosophila Larva

Neuroscience Research. Feb, 2004  |  Pubmed ID: 14741397

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.

Recent Developments in Neurobiology: Introduction to the Symposium

Integrative and Comparative Biology. Feb, 2004  |  Pubmed ID: 21680479

Developmental Consequences of Neuromuscular Junctions with Reduced Presynaptic Calcium Channel Function

Synapse (New York, N.Y.). Sep, 2005  |  Pubmed ID: 15945059

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.

Direct Innervation of the Drosophila Melanogaster Larval Aorta

Brain Research. Apr, 2006  |  Pubmed ID: 16529726

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.

Self-modeling Structure of Evoked Postsynaptic Potentials

Synapse (New York, N.Y.). Jul, 2006  |  Pubmed ID: 16575851

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.

Recent Advances in Basic Neurosciences and Brain Disease: from Synapses to Behavior

Molecular Pain. 2006  |  Pubmed ID: 17196111

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).

Influence of PCPA and MDMA (ecstasy) on Physiology, Development and Behavior in Drosophila Melanogaster

The European Journal of Neuroscience. Jul, 2007  |  Pubmed ID: 17650115

The effects of para-chlorophenylalanine (PCPA) and 3,4 methylenedioxy-methamphetamine (MDMA, 'ecstasy') were investigated in relation to development, behavior and physiology in larval Drosophila. PCPA blocks the synthesis of serotonin (5-HT) and MDMA is known to deplete 5-HT in mammalian neurons; thus these studies were conducted primarily to target the serotonergic system. Treatment with PCPA and MDMA delayed time to pupation and eclosion. The developmental rate was investigated with a survival analysis statistical approach that is unique for Drosophila studies. Locomotion and eating were reduced in animals exposed to MDMA or PCPA. Sensitivity to exogenously applied 5-HT on an evoked sensory-central nervous system (CNS)-motor circuit showed that the CNS is sensitive to 5-HT but that when depleted of 5-HT by PCPA a decreased sensitivity occurred. A diet with MDMA produced an enhanced response to exogenous 5-HT on the central circuit. Larvae eating MDMA from the first to third instar did not show a reduction in 5-HT within the CNS; however, eating PCPA reduced 5-HT as well as dopamine content as measured by high performance liquid chromatography from larval brains. As the heart serves as a good bioindex of 5-HT exposure, it was used in larvae fed PCPA and MDMA but no significant effects occurred with exogenous 5-HT. In summary, the action of these pharmacological compounds altered larval behaviors and development. PCPA treatment changed the sensitivity in the CNS to 5-HT, suggesting that 5-HT receptor regulation is modulated by neural activity of the serotonergic neurons. The actions of acute MDMA exposure suggest a 5-HT agonist action or possible dumping of 5-HT from neurons.

Automated Classification of Evoked Quantal Events

Journal of Neuroscience Methods. Jan, 2007  |  Pubmed ID: 16934872

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.

Different Mechanisms of Ca2+ Regulation That Influence Synaptic Transmission: Comparison Between Crayfish and Drosophila Neuromuscular Junctions

Synapse (New York, N.Y.). Dec, 2009  |  Pubmed ID: 19650116

A brief historical background on synaptic transmission in relation to Ca(2+) dynamics and short-term facilitation is described. This study focuses on the mechanisms responsible for the regulation of intracellular calcium concentration ([Ca(2+)](i)) in high output terminals of larval Drosophila compared to a low-output terminal of the crayfish neuromuscular junction (NMJ). Three processes; plasmalemmal Na(+)/Ca(2+) exchanger [NCX], Ca(2+)-ATPase (PMCA), and sarcoplasmic/endoplasmic Ca(2+)-ATPase (SERCA) are important in regulating the [Ca(2+)](i) are examined. When the NCX is compromised by reduced [Na(+)](o), no consistent effect occurred; but a NCX blocker KB-R7943 decreased the excitatory postsynaptic potential (EPSP) amplitudes. Compromising the PMCA with pH 8.8 resulted in an increase in EPSP amplitude but treatment with a PMCA specific inhibitor carboxyeosin produced opposite results. Thapsigargin exposure to block the SERCA generally decreases EPSP amplitude. Compromising the activity of the above Ca(2+) regulating proteins had no substantial effects on short-term depression. The Kum(170TS) strain (with dysfunctional SERCA), showed a decrease in EPSP amplitudes including the first EPSP within the train. Synaptic transmission is altered by reducing the function of the above three [Ca(2+)](i) regulators; but they are not consistent among different species as expected. Results in crayfish NMJ were more consistent with expected results as compared to the Drosophila NMJ. It is predicated that different mechanisms are used for regulating the [Ca(2+)](i) in high and low output synaptic terminals.