Neurohormonal modulation of the Limulus heart: amine actions on neuromuscular transmission and cardiac muscle

The responses of Limulus cardiac neuromuscular junctions and cardiac muscle cells to four endogenous amines were determined in order to identify the cellular targets underlying amine modulation of heartbeat amplitude. The amines increased the amplitude of the Limulus heartbeat, with dopamine (DA) being more potent than octopamine, epinephrine or norepinephrine. The effect of DA on heartbeat amplitude was not blocked by phentolamine. DA enhanced the contractility of deganglionated heart muscle, with time course and dose-dependence similar to its effect on the intact heart. The amines also enhanced neuromuscular transmission, with time course and dose-dependence similar to their effects upon the intact heart. The amplitude of unitary excitatory junction potentials (EJPs) and frequency of miniature excitatory junction potentials (mEJPs) were increased by DA, while mEJP amplitude was unchanged. Thus DA, and probably the other amines, had a presynaptic effect. Combined actions upon cardiac muscle and cardiac neuromuscular transmission account for the ability of these amines to increase the amplitude of the Limulus heartbeat

Proctolin and an Endogenous Proctolin-Like Peptide Enhance the Contractility of the Limulus Heart

Synthetic proctolin increases the force but not the rate of heart contractions of Limulus in a time- and dose-dependent manner. The threshold of this effect is 3 × 10−10M and the ED50 is approximately 10−8M. At concentrations up to 10−7 M, proctolin has no effect on the rhythmic electrical activity of the isolated cardiac ganglion, and it does not change the simple and compound postsynaptic potentials recorded at the cardiac neuromuscular junction. Proctolin acts directly on the cardiac muscle fibres. Electrically stimulated myocardia show a proctolin-induced increase in contraction amplitude with the same concentration dependence as the intact heart. A compound with an apparent molecular weight of 700–800 occurs in the Limulus nervous system, particularly in the cardiac ganglion. This compound resembles proctolin in being heat-stable, resistant to trypsin and chymotrypsin cleavage, and losing activity in a time-dependent manner in response to treatment with leucine aminopeptidase or pronase. This peptide induces spontaneous contractions and a contracture of the cockroach hindgut in a manner similar to proctolin. Moreover, the Limulus inotropic peptide, like proctolin, increases the force of contraction of the Limulus heart without affecting beat frequency. It is concluded that an endogenous, proctolin-like peptide is an inotropic modulator of the Limulus heart, acting directly on the muscle fibres and not affecting cardiac ganglion activity

A Positive Feedback Signal Transduction Loop Determines Timing of Cerebellar Long-Term Depression

SummarySynaptic activity produces short-lived second messengers that ultimately yield a long-term depression (LTD) of cerebellar Purkinje cells. Here, we test the hypothesis that these brief second messenger signals are translated into long-lasting biochemical signals by a positive feedback loop that includes protein kinase C (PKC) and mitogen-activated protein kinase. Histochemical “epistasis” experiments demonstrate the reciprocal activation of these kinases, and physiological experiments—including the use of a light-activated protein kinase—demonstrate that such reciprocal activation is required for LTD. Timed application of enzyme inhibitors reveals that this positive feedback loop causes PKC to be active for more than 20 min, allowing sufficient time for LTD expression. Such regenerative mechanisms may sustain other long-lasting forms of synaptic plasticity and could be a general mechanism for prolonging signal transduction networks

Presynaptic nanodomains : a tale of two synapses

© The Author(s), 2015. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Frontiers in Cellular Neuroscience 8 (2015): 455, doi:10.3389/fncel.2014.00455.Here we summarize the evidence from two “giant” presynaptic terminals—the squid giant synapse and the mammalian calyx of Held—supporting the involvement of nanodomain calcium signals in triggering of neurotransmitter release. At the squid synapse, there are three main lines of experimental evidence for nanodomain signaling. First, changing the size of the unitary calcium channel current by altering external calcium concentration causes a non-linear change in transmitter release, while changing the number of open channels by broadening the presynaptic action potential causes a linear change in release. Second, low-affinity calcium indicators, calcium chelators, and uncaging of calcium all suggest that presynaptic calcium concentrations are as high as hundreds of micromolar, which is more compatible with a nanodomain type of calcium signal. Finally, neurotransmitter release is much less affected by the slow calcium chelator, ethylene glycol tetraacetic acid (EGTA), in comparison to the rapid chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid (BAPTA). Similarly, as the calyx of Held synapse matures, EGTA becomes less effective in attenuating transmitter release while the number of calcium channels required to trigger a single fusion event declines. This suggests a developmental transformation of microdomain to nanodomain coupling between calcium channels and transmitter release. Calcium imaging and uncaging experiments, in combination with simulations of calcium diffusion, indicate the peak calcium concentration seen by presynaptic calcium sensors reaches at least tens of micromolar at the calyx of Held. Taken together, data from these provide a compelling argument that nanodomain calcium signaling gates very rapid transmitter release.This work was supported by a CRP grant from the National Research Foundation of Singapore and by the World Class Institute (WCI) Program of the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology of Korea (MEST) (NRF Grant Number: WCI 2009-003) (to George J. Augustine), and by Operating Grants from the Canadian Institutes of Health Research (MOP-77610, MOP-81159, MOP-14692, VIH-105441) and Canada Research Chair (to Lu-Yang Wang)

Editorial: Imaging synapse structure and function

These are the glory days for imaging synapse structure and function. Thanks to recent advances in both optical and electron microscopy, it is now possible to image individual synapses with unprecedented spatial and temporal resolution. The parallel development of a wide range of genetically-encoded synaptic reporters enables all-optical recording of synaptic activity in genetically-defined neuronal populations. These engineering breakthroughs allow neuroscientists to interrogate the brain in ways that were inconceivable just a few years ago. The ability to image synaptic structure and activity in large functional circuits is beginning to yield key insights into how the brain stores, processes, and computes information. This research topic consists of eleven articles (methods, primary research papers, and reviews) that provide an overview of the latest developments in synapse imaging. Rather than attempting an exhaustive list of synaptic reporters and microscopy techniques, our collection emphasizes approaches that merge technical advances from diverse areas to extract a rich palette of novel information from individual synapses. Watanabe presents a method that combines optogenetics and rapid freezing (Flash-and-Freeze) to visualize the synaptic vesicle (SV) cycle at the ultrastructural level with millisecond resolution Watanabe. This revolutionary approach revealed ultra-fast endocytosis of SVs at central synapses and neuromuscular junctions (Watanabe et al., 2013a,b) and promises to uncover many new kinetic aspects of synapse dynamics. Begemann and Galic review recent efforts to image neuronal preparations with both light and electron microscopy, with a series of hybrid techniques referred to as Correlative Light Electron Microscopy (CLEM). Jackson and Burrone describe the first genetically-encoded fluorescent reporter (sypHy-RGECO) that enables concurrent monitoring of calcium dynamics and SV fusion. sypHy-RGECO will undoubtedly be a powerful means of examining calcium triggering of SV exocytosis at the level of individual presynaptic boutons. Using similar probes, Tang et al. show that the mental disease gene DISC1 (Disrupted-In-Schizophrenia-1) accelerates SV exocytosis by facilitating calcium influx through N-type voltage-gated Ca2+ channels. Calcium transients at synapses are also shaped by both mobilization and sequestration of calcium by intracellular stores. Kwon et al. report on the latest advances in organelle-specific calcium sensors and review the contribution of the endoplasmic reticulum and mitochondria to calcium dynamics and synaptic transmission/plasticity. Until recently, one major impediment to imaging of synaptic activity has been our inability to directly measure membrane potential with adequate signal/noise ratio. This is rapidly changing with the recent improvement of a wide range of genetically-encoded voltage indicators (GEVIs) that now are capable of monitoring both single action potentials and even subthreshold synaptic potentials, both in vitro and in vivo Nakajima et al. Three papers describe recent advances on the localization, dynamics and function of postsynaptic receptors and scaffolds. Using a combination of single-molecule tracking (uPAINT) and photoactivated localization microscopy (PALM), Li and Blanpied assess the diffusion properties of membrane proteins within the postsynaptic density (PSD). The same authors recently used localization microscopy to demonstrate the existence of transsynaptic nanocolumns that align the neurotransmitter release machinery to postsynaptic receptors (Tang et al., 2016a). Dosemeci et al. review a series of EM studies that reveal the presence of a dense lamina—the “pallium”—just beneath the core layer of the PSD, and discuss how translocation of signaling proteins and scaffolds in and out of the pallium may shape activity-induced changes in dendritic spines. In keeping with the theme of postsynaptic signaling, Dore et al. discuss evidence for metabotropic functions of NMDARs, based on time-resolved FRET and other imaging approaches. Finally, at the level of synaptic circuits, two reviews describe the use of genetically-encoded synaptic labels to trace neural circuits in a variety of different model systems, ranging from C. elegans to mammals (Hong and Park; Lee et al.). Overall, we hope that the fine collection of papers contained within this research topic highlights a useful synapse imaging toolkit for the neuroscience community. The next big challenge in brain imaging will be to scale up these synaptic measurements to large ensembles of neurons to comprehend how circuits compute. This will require synaptic reporters that operate in a synaptically-relevant time scale (milliseconds), along with improved genetic targeting strategies, further advances in automated high-speed microscopy, and refined bioinformatics tools for analysis of the resulting large datasets

Molecular Mechanisms of Short-Term Plasticity: Role of Synapsin Phosphorylation in Augmentation and Potentiation of Spontaneous Glutamate Release

We used genetic and pharmacological approaches to identify the signaling pathways involved in augmentation and potentiation, two forms of activity dependent, short-term synaptic plasticity that enhance neurotransmitter release. Trains of presynaptic action potentials produced a robust increase in the frequency of miniature excitatory postsynaptic currents (mEPSCs). Following the end of the stimulus, mEPSC frequency followed a bi-exponential decay back to basal levels. The time constants of decay identified these two exponential components as the decay of augmentation and potentiation, respectively. Augmentation increased mEPSC frequency by 9.3-fold, while potentiation increased mEPSC frequency by 2.4-fold. In synapsin triple-knockout (TKO) neurons, augmentation was reduced by 83% and potentiation was reduced by 74%, suggesting that synapsins are key signaling elements in both forms of plasticity. To examine the synapsin isoforms involved, we expressed individual synapsin isoforms in TKO neurons. While synapsin IIIa rescued both augmentation and potentiation, none of the other synapsin isoforms produced statistically significant amounts of rescue. To determine the involvement of protein kinases in these two forms of short-term plasticity, we examined the effects of inhibitors of protein kinases A (PKA) and C (PKC). While inhibition of PKC had little effect, PKA inhibition reduced augmentation by 76% and potentiation by 60%. Further, elevation of intracellular cAMP concentration, by either forskolin or IBMX, greatly increased mEPSC frequency and occluded the amount of augmentation and potentiation evoked by electrical stimulation. Finally, mutating a PKA phosphorylation site to non-phosphorylatable alanine largely abolished the ability of synapsin IIIa to rescue both augmentation and potentiation. Together, these results indicate that PKA activation is required for both augmentation and potentiation of spontaneous neurotransmitter release and that PKA-mediated phosphorylation of synapsin IIIa underlies both forms of presynaptic short-term plasticity

Using Optogenetic Dyadic Animal Models to Elucidate the Neural Basis for Human Parent-Infant Social Knowledge Transmission.

Healthy early development depends on a warm reciprocal relationship between parent and offspring, where parent and infant interact in close temporal co-ordination as if engaged in a “dyadic dance” of glances, gestures, smiles and words (Stern, 1985; Gianino and Tronick, 1988). Most, if not all, early learning takes place during these well-choreographed social exchanges, which support cultural knowledge transmission from parent to offspring using verbal and non-verbal forms of communication and behavioural modelling. Such vicarious knowledge transmission through social interaction (rather than direct experience) is known as social learning (Bandura, 1971; Csibra and Gergely, 2009). Tomasello (2014) argues that human mastery of these “second-personal social relations” (Darwall, 2006)—in which social partners share and create joint knowledge, intentionality and goals—has accelerated the rise of the human species through “cultural intelligence” (Herrmann et al., 2007).Ministry of Education (MOE)Published versionThis research is supported by the Ministry of Education, Singapore, under its Academic Research Fund Tier 1 [RG99/20 to VL and GA; RG152/18 (NS) to VL]

Calcium-Dependent and Synapsin-Dependent Pathways for the Presynaptic Actions of BDNF

We used cultured hippocampal neurons to determine the signaling pathways mediating brain-derived neurotrophic factor (BDNF) regulation of spontaneous glutamate and GABA release. BDNF treatment elevated calcium concentration in presynaptic terminals; this calcium signal reached a peak within 1 min and declined in the sustained presence of BDNF. This BDNF-induced transient rise in presynaptic calcium was reduced by SKF96365, indicating that BDNF causes presynaptic calcium influx via TRPC channels. BDNF treatment increased the frequency of miniature excitatory postsynaptic currents (mEPSCs). This response consisted of two components: a transient component that peaked within 1 min of initiating BDNF application and a second component that was sustained, at a lower mEPSC frequency, for the duration of BDNF application. The initial transient component was greatly reduced by removing external calcium or by treatment with SKF96365, as well as by Pyr3, a selective blocker of TRPC3 channels. In contrast, the sustained component was unaffected in these conditions but was eliminated by U0126, an inhibitor of the MAP kinase (MAPK) pathway, as well as by genetic deletion of synapsins in neurons from a synapsin triple knock-out (TKO) mouse. Thus, two pathways mediate the ability of BDNF to enhance spontaneous glutamate release: the transient component arises from calcium influx through TRPC3 channels, while the sustained component is mediated by MAPK phosphorylation of synapsins. We also examined the ability of these two BDNF-dependent pathways to regulate spontaneous release of the inhibitory neurotransmitter, GABA. BDNF had no effect on the frequency of spontaneous miniature inhibitory postsynaptic currents (mIPSCs) in neurons from wild-type (WT) mice, but surprisingly did increase mIPSC frequency in synapsin TKO mice. This covert BDNF response was blocked by removal of external calcium or by treatment with SKF96365 or Pyr3, indicating that it results from calcium influx mediated by TRPC3 channels. Thus, the BDNF-activated calcium signaling pathway can also enhance spontaneous GABA release, though this effect is suppressed by synapsins under normal physiological conditions.MOE (Min. of Education, S’pore)Published versio