Article

Mediation of Hippocampal Mossy Fiber Long-Term Potentiation by Cyclic AMP

October 1994
Science 265(5180):1878-82

October 1994
265(5180):1878-82

DOI:10.1126/science.7916482

Source
PubMed

Authors:

Marc G Weisskopf

Harvard T.H. Chan School of Public Health

Pablo E Castillo

Albert Einstein College of Medicine

Repetitive activation of hippocampal mossy fibers evokes a long-term potentiation (LTP) of synaptic responses in pyramidal cells in the CA3 region that is independent of N-methyl-D-aspartate receptor activation. Previous results suggest that the site for both the induction and expression of this form of LTP is presynaptic. Experimental elevation of cyclic adenosine 3',5'-monophosphate (cAMP) both mimics and interferes with tetanus-induced mossy fiber LTP, and blockers of the cAMP cascade block mossy fiber LTP. It is proposed that calcium entry into the presynaptic terminal may activate Ca(2+)-calmodulin-sensitive adenylyl cyclase I which, through protein kinase A, causes a persistent enhancement of evoked glutamate release.

Timing Control of Purkinje Cell Outputs by Dual cAMP Actions on Axonal Action Potential and Transmitter Release

Preprint

Full-text available

Sep 2023

All-or-none digital signaling based on high-fidelity action potentials (APs) in neuronal axons is pivotal for the temporally precise sending of identical outputs rapidly to widespread multiple target cells. However, technical limitation to directly measure the signaling in small size of intact axonal structures has hindered the evaluation of high-fidelity signal propagation. Here, using direct recordings from axonal trunks and/or terminals of cerebellar Purkinje cells in culture and slice, we demonstrate that the timing of axonal output is delayed by the second messenger cAMP without clear changes of transmission efficacy. Slowed axonal signaling upon cAMP increase was ascribed to negative control of axonal Na ⁺ channels, leading to smaller and hence slower conduction of APs specifically at an axon. On the other hand, a facilitatory effect of cAMP on presynaptic transmitter release, which generally operates at various CNS synapses, was also evident as augmented release probability in Purkinje cell axon terminals, compensating for weakening of release by the reduction of Ca ²⁺ influx upon smaller AP. Taken these results together, our tour-de-force functional dissection of inhibitory axonal signaling unveiled a dynamic control of synaptic output timing by cAMP keeping output strength constant.

Increased vesicle fusion competence underlies long-term potentiation at hippocampal mossy fiber synapses

Article

Full-text available

Feb 2023

Presynaptic long-term potentiation (LTP) is thought to play an important role in learning and memory. However, the underlying mechanism remains elusive because of the difficulty of direct recording during LTP. Hippocampal mossy fiber synapses exhibit pronounced LTP of transmitter release after tetanic stimulation and have been used as a model of presynaptic LTP. Here, we induced LTP by optogenetic tools and applied direct presynaptic patch-clamp recordings. The action potential waveform and evoked presynaptic Ca2+ currents remained unchanged after LTP induction. Membrane capacitance measurements suggested higher release probability of synaptic vesicles without changing the number of release-ready vesicles after LTP induction. Synaptic vesicle replenishment was also enhanced. Furthermore, stimulated emission depletion microscopy suggested an increase in the numbers of Munc13-1 and RIM1 molecules within active zones. We propose that dynamic changes in the active zone components may be relevant for the increased fusion competence and synaptic vesicle replenishment during LTP.

Long-term in vivo application of a potassium channel-based optogenetic silencer in the healthy and epileptic mouse hippocampus

Article

Full-text available

Jan 2022
BMC BIOL

Background Optogenetic tools allow precise manipulation of neuronal activity via genetically encoded light-sensitive proteins. Currently available optogenetic inhibitors are not suitable for prolonged use due to short-lasting photocurrents, tissue heating, and unintended changes in ion distributions, which may interfere with normal neuron physiology. To overcome these limitations, a novel potassium channel-based optogenetic silencer, named PACK, was recently developed. The PACK tool has two components: a photoactivated adenylyl cyclase from Beggiatoa (bPAC) and a cAMP-dependent potassium channel, SthK, which carries a large, long-lasting potassium current in mammalian cells. Previously, it has been shown that activating the PACK silencer with short light pulses led to a significant reduction of neuronal firing in various in vitro and acute in vivo settings. Here, we examined the viability of performing long-term studies in vivo by looking at the inhibitory action and side effects of PACK and its components in healthy and epileptic adult male mice. Results We targeted hippocampal cornu ammonis (CA1) pyramidal cells using a viral vector and enabled illumination of these neurons via an implanted optic fiber. Local field potential (LFP) recordings from CA1 of freely moving mice revealed significantly reduced neuronal activity during 50-min intermittent (0.1 Hz) illumination, especially in the gamma frequency range. Adversely, PACK expression in healthy mice induced chronic astrogliosis, dispersion of pyramidal cells, and generalized seizures. These side effects were independent of the light application and were also present in mice expressing bPAC without the potassium channel. Light activation of bPAC alone increased neuronal activity, presumably via enhanced cAMP signaling. Furthermore, we applied bPAC and PACK in the contralateral hippocampus of chronically epileptic mice following a unilateral injection of intrahippocampal kainate. Unexpectedly, the expression of bPAC in the contralateral CA1 area was sufficient to prevent the spread of spontaneous epileptiform activity from the seizure focus to the contralateral hippocampus. Conclusion Our study highlights the PACK tool as a potent optogenetic inhibitor in vivo. However, further refinement of its light-sensitive domain is required to avoid unexpected physiological changes.

Role of kainate receptors in glutamatergic synaptic transmission depression at mossy fiber CA3 hippocampal synapse

Chapter

Full-text available

Jun 2009

Kainate receptor-mediated inhibition of glutamate release involves protein kinase A in the mouse hippocampus.. The mechanisms involved in the inhibition of glutamate release mediated by the activation of presynaptic kainate receptors (KARs) at the hippocampal mossy fiber-CA3 synapse are not well understood. We have observed a long-lasting inhibition of CA3 evoked excitatory postsynaptic currents (eEPSCs) after a brief application of kainate (KA) at concentrations ranging from 0.3 to 10 M. The inhibition outlasted the change in holding current caused by the activation of ionotropic KARs in CA3 pyramidal cells, indicating that this action is not contingent on the opening of the receptor channels. The inhibition of the eEPSCs by KA was prevented by G protein and protein kinase A (PKA) inhibitors and was enhanced after stimulation of the adenylyl cyclase (AC) with forskolin. We conclude that KARs present at mossy fiber terminals mediate the inhibition of glutamate release through a metabotropic mechanism that involves the activation of an AC-second messenger cAMP-PKA signaling cascade.

EL RECEPTOR DE KAINATO Y LA REGULACIÓN DE LA LIBERACIÓN DE GLUTAMATO

Book

Full-text available

Aug 2018

Developmental and activity-dependent modulation of coupling distance between release site and Ca2+ channel

Article

Full-text available

Oct 2022

Mitsuharu Midorikawa

Synapses are junctions between a presynaptic neuron and a postsynaptic cell specialized for fast and precise information transfer. The presynaptic terminal secretes neurotransmitters via exocytosis of synaptic vesicles. Exocytosis is a tightly regulated reaction that occurs within a millisecond of the arrival of an action potential. One crucial parameter in determining the characteristics of the transmitter release kinetics is the coupling distance between the release site and the Ca ²⁺ channel. Still, the technical limitations have hindered detailed analysis from addressing how the coupling distance is regulated depending on the development or activity of the synapse. However, recent technical advances in electrophysiology and imaging are unveiling their different configurations in different conditions. Here, I will summarize developmental- and activity-dependent changes in the coupling distances revealed by recent studies.

cAMP-Dependent Synaptic Plasticity at the Hippocampal Mossy Fiber Terminal

Article

Full-text available

Apr 2022

Cyclic adenosine monophosphate (cAMP) is a crucial second messenger involved in both pre- and postsynaptic plasticity in many neuronal types across species. In the hippocampal mossy fiber (MF) synapse, cAMP mediates presynaptic long-term potentiation and depression. The main cAMP-dependent signaling pathway linked to MF synaptic plasticity acts via the activation of the protein kinase A (PKA) molecular cascade. Accordingly, various downstream putative synaptic PKA target proteins have been linked to cAMP-dependent MF synaptic plasticity, such as synapsin, rabphilin, synaptotagmin-12, RIM1a, tomosyn, and P/Q-type calcium channels. Regulating the expression of some of these proteins alters synaptic release probability and calcium channel clustering, resulting in short- and long-term changes to synaptic efficacy. However, despite decades of research, the exact molecular mechanisms by which cAMP and PKA exert their influences in MF terminals remain largely unknown. Here, we review current knowledge of different cAMP catalysts and potential downstream PKA-dependent molecular cascades, in addition to non-canonical cAMP-dependent but PKA-independent cascades, which might serve as alternative, compensatory or competing pathways to the canonical PKA cascade. Since several other central synapses share a similar form of presynaptic plasticity with the MF, a better description of the molecular mechanisms governing MF plasticity could be key to understanding the relationship between the transcriptional and computational levels across brain regions.

Engineered synaptic tools reveal localized cAMP signaling in synapse assembly

Article

Full-text available

Dec 2021
J CELL BIOL

The physiological mechanisms driving synapse formation are elusive. Although numerous signals are known to regulate synapses, it remains unclear which signaling mechanisms organize initial synapse assembly. Here, we describe new tools, referred to as “SynTAMs” for synaptic targeting molecules, that enable localized perturbations of cAMP signaling in developing postsynaptic specializations. We show that locally restricted suppression of postsynaptic cAMP levels or of cAMP-dependent protein-kinase activity severely impairs excitatory synapse formation without affecting neuronal maturation, dendritic arborization, or inhibitory synapse formation. In vivo, suppression of postsynaptic cAMP signaling in CA1 neurons prevented formation of both Schaffer-collateral and entorhinal-CA1/temporoammonic-path synapses, suggesting a general principle. Retrograde trans-synaptic rabies virus tracing revealed that postsynaptic cAMP signaling is required for continuous replacement of synapses throughout life. Given that postsynaptic latrophilin adhesion-GPCRs drive synapse formation and produce cAMP, we suggest that spatially restricted postsynaptic cAMP signals organize assembly of postsynaptic specializations during synapse formation.

Glutamate Receptors of the Kainate Type: An Ion Channel Becoming a Metabotropic Receptor

Chapter

Jan 2010

Kainate receptors (KARs), together with AMPA and NMDA, are typically described as ionotropic glutamate receptors. The functions of KARs have begun to be elucidated only in the last decade. While some the actions of KARs are classically ionotropic, surprisingly, others seem to involve the activation of second messenger cascades and invoke metabotropic roles for this type of glutamate receptor. In this chapter, we describe these metabotropic actions of KARs in relation to the putative signalling cascades involved. Although, it is still a mystery how KARs activate G-proteins to stimulate second messenger cascades, intriguingly in very recent studies, specific subunits of KARs have been demonstrated to associate with G-proteins. Altogether, the body of evidence supports the hypothesis that, together with the canonical ionotropic operation, KARs expedite long-lasting signalling by novel metabotropic modes of action.

Glutamate receptors of the kainate type: An ion channel becoming a metabotropic receptor

Chapter

Full-text available

Aug 2012

Recruitment of release sites underlies chemical presynaptic potentiation at hippocampal mossy fiber boutons

Article

Full-text available

Jun 2021
PLOS BIOL

Synaptic plasticity is a cellular model for learning and memory. However, the expression mechanisms underlying presynaptic forms of plasticity are not well understood. Here, we investigate functional and structural correlates of presynaptic potentiation at large hippocampal mossy fiber boutons induced by the adenylyl cyclase activator forskolin. We performed 2-photon imaging of the genetically encoded glutamate sensor iGlu u that revealed an increase in the surface area used for glutamate release at potentiated terminals. Time-gated stimulated emission depletion microscopy revealed no change in the coupling distance between P/Q-type calcium channels and release sites mapped by Munc13-1 cluster position. Finally, by high-pressure freezing and transmission electron microscopy analysis, we found a fast remodeling of synaptic ultrastructure at potentiated boutons: Synaptic vesicles dispersed in the terminal and accumulated at the active zones, while active zone density and synaptic complexity increased. We suggest that these rapid and early structural rearrangements might enable long-term increase in synaptic strength.

Plasticity in the Hippocampus, Neurogenesis and Drugs of Abuse

Article

Full-text available

Mar 2021
BSRCCS

Synaptic plasticity in the hippocampus assists with consolidation and storage of long-lasting memories. Decades of research has provided substantial information on the cellular and molecular mechanisms underlying synaptic plasticity in the hippocampus, and this review discusses these mechanisms in brief. Addiction is a chronic relapsing disorder with loss of control over drug taking and drug seeking that is caused by long-lasting memories of drug experience. Relapse to drug use is caused by exposure to context and cues associated with the drug experience, and is a major clinical problem that contributes to the persistence of addiction. This review also briefly discusses some evidence that drugs of abuse alter plasticity in the hippocampus, and that development of novel treatment strategies that reverse or prevent drug-induced synaptic alterations in the hippocampus may reduce relapse behaviors associated with addiction.

SCAMP5 mediates activity-dependent enhancement of NHE6 recruitment to synaptic vesicles during synaptic plasticity

Article

Full-text available

Mar 2021

Na+(K+)/H+ exchanger 6 (NHE6) on synaptic vesicle (SV) is critical for the presynaptic regulation of quantal size at the glutamatergic synapses by converting the chemical gradient (ΔpH) into membrane potential (Δψ) across the SV membrane. We recently found that NHE6 directly interacts with secretory carrier membrane protein 5 (SCAMP5), and SCAMP5-dependent recruitment of NHE6 to SVs controls the strength of synaptic transmission by modulation of quantal size of glutamate release at rest. It is, however, unknown whether NHE6 recruitment by SCAMP5 plays a role during synaptic plasticity. Here, we found that the number of NHE6-positive presynaptic boutons was significantly increased by the chemical long-term potentiation (cLTP). Since cLTP involves new synapse formation, our results indicated that NHE6 was recruited not only to the existing presynaptic boutons but also to the newly formed presynaptic boutons. Knock down of SCAMP5 completely abrogated the enhancement of NHE6 recruitment by cLTP. Interestingly, despite an increase in the number of NHE6-positive boutons by cLTP, the quantal size of glutamate release at the presynaptic terminals remained unaltered. Together with our recent results, our findings indicate that SCAMP5-dependent recruitment of NHE6 plays a critical role in manifesting presynaptic efficacy not only at rest but also during synaptic plasticity. Since both are autism candidate genes, reduced presynaptic efficacy by interfering with their interaction may underlie the molecular mechanism of synaptic dysfunction observed in autism.

Regulation of hippocampal mossy fiber-CA3 synapse function by a Bcl11b/C1ql2/Nrxn3(25b+) pathway

Article

Full-text available

Feb 2024
eLife

The transcription factor Bcl11b has been linked to neurodevelopmental and neuropsychiatric disorders associated with synaptic dysfunction. Bcl11b is highly expressed in dentate gyrus granule neurons and is required for the structural and functional integrity of mossy fiber-CA3 synapses. The underlying molecular mechanisms, however, remained unclear. We show that the synaptic organizer molecule C1ql2 is a direct functional target of Bcl11b that regulates synaptic vesicle recruitment and long-term potentiation at mossy fiber-CA3 synapses in vivo and in vitro. Furthermore, we demonstrate C1ql2 to exert its functions through direct interaction with a specific splice variant of neurexin-3, Nrxn3(25b+). Interruption of C1ql2-Nrxn3(25b+) interaction by expression of a non-binding C1ql2 mutant or by deletion of Nrxn3 in the dentate gyrus granule neurons recapitulates major parts of the Bcl11b as well as C1ql2 mutant phenotype. Together, this study identifies a novel C1ql2-Nrxn3(25b+)-dependent signaling pathway through which Bcl11b controls mossy fiber-CA3 synapse function. Thus, our findings contribute to the mechanistic understanding of neurodevelopmental disorders accompanied by synaptic dysfunction.

Regulation of hippocampal mossy fiber-CA3 synapse function by a Bcl11b/C1ql2/Nrxn3(25b+) pathway

Preprint

Full-text available

Jan 2024

The transcription factor Bcl11b has been linked to neurodevelopmental and neuropsychiatric disorders associated with synaptic dysfunction. Bcl11b is highly expressed in dentate gyrus granule neurons and is required for the structural and functional integrity of mossy fiber-CA3 synapses. The underlying molecular mechanisms, however, remained unclear. We show that the synaptic organizer molecule C1ql2 is a direct functional target of Bcl11b that regulates synaptic vesicle recruitment and long-term potentiation at mossy fiber-CA3 synapses in vivo and in vitro . Furthermore, we demonstrate C1ql2 to exert its functions through direct interaction with a specific splice variant of neurexin-3, Nrxn3(25b+). Interruption of C1ql2-Nrxn3(25b+) interaction by expression of a non-binding C1ql2 mutant or by deletion of Nrxn3 in the dentate gyrus granule neurons recapitulates major parts of the Bcl11b as well as C1ql2 mutant phenotype. Together, this study identifies a novel C1ql2-Nrxn3(25b+)-dependent signaling pathway through which Bcl11b controls mossy fiber-CA3 synapse function. Thus, our findings contribute to the mechanistic understanding of neurodevelopmental disorders accompanied by synaptic dysfunction.

Glutamatergic synaptic deficits in the prefrontal cortex of the Ts65Dn mouse model for Down syndrome

Article

Full-text available

Sep 2023

Down syndrome (DS), the most prevalent cause of intellectual disability, stems from a chromosomal anomaly resulting in an entire or partial extra copy of chromosome 21. This leads to intellectual disability and a range of associated symptoms. While there has been considerable research focused on the Ts65Dn mouse model of DS, particularly in the context of the hippocampus, the synaptic underpinnings of prefrontal cortex (PFC) dysfunction in DS, including deficits in working memory, remain largely uncharted territory. In a previous study featuring mBACtgDyrk1a mice, which manifest overexpression of the Dyrk1a gene, a known candidate gene linked to intellectual disability and microcephaly in DS, we documented adverse effects on spine density, alterations in the molecular composition of synapses, and the presence of synaptic plasticity deficits within the PFC. The current study aimed to enrich our understanding of the roles of different genes in DS by studying Ts65Dn mice, which overexpress several genes including Dyrk1a, to compare with our previous work on mBACtgDyrk1a mice. Through ex-vivo electrophysiological experiments, including patch-clamp and extracellular field potential recordings, we identified alterations in the intrinsic properties of PFC layer V/VI pyramidal neurons in Ts65Dn male mice. Additionally, we observed changes in the synaptic plasticity range. Notably, long-term depression was absent in Ts65Dn mice, while synaptic or pharmacological long-term potentiation remained fully expressed in these mice. These findings provide valuable insights into the intricate synaptic mechanisms contributing to PFC dysfunction in DS, shedding light on potential therapeutic avenues for addressing the neurocognitive symptoms associated with this condition.

Homeostatic Synaptic Plasticity of Miniature Excitatory Postsynaptic Currents in Mouse Cortical Cultures Requires Neuronal Rab3A

Preprint

Full-text available

Sep 2023

Following prolonged activity blockade, amplitudes of miniature excitatory postsynaptic currents (mEPSCs) increase, a form of homeostatic plasticity termed “synaptic scaling.” We previously showed that a presynaptic protein, the small GTPase Rab3A, is required for full expression of the increase in miniature endplate current amplitudes following prolonged blockade of action potential activity at the mouse neuromuscular junction in vivo (Wang et al., 2011), but it is unknown whether this form of Rab3A-dependent homeostatic plasticity shares any characteristics with central synapses. We show here that synaptic scaling of mEPSCs is impaired in mouse cortical neuron cultures prepared from Rab3A-/- and Rab3A Earlybird mutant mice. To determine if Rab3A is involved in the well-established homeostatic increase in postsynaptic AMPA-type receptors (AMPARs), we performed a series of experiments in which electrophysiological recordings of mEPSCs and confocal imaging of synaptic AMPAR immunofluorescence were assessed within the same cultures. We found that Rab3A is required for the increase in synaptic AMPARs following prolonged activity blockade, but the comparison of mEPSC amplitude and synaptic AMPARs in the same cultures revealed that mEPSC amplitude cannot solely be determined by postsynaptic AMPAR levels. Finally, we demonstrate that Rab3A is acting in neurons because selective loss of Rab3A in astrocytes did not disrupt homeostatic plasticity, whereas selective loss in neurons strongly reduced the homeostatic increase in mEPSC amplitudes. Taken together with the results at the neuromuscular junction, we propose that Rab3A is a presynaptic homeostatic regulator that controls quantal size on both sides of the synapse.

Regulation of hippocampal mossy fiber-CA3 synapse function by a Bcl11b/C1ql2/Nrxn3(25b+) pathway

Preprint

Full-text available

Aug 2023

The transcription factor Bcl11b has been linked to neurodevelopmental and neuropsychiatric disorders associated with synaptic dysfunction. Bcl11b is highly expressed in dentate gyrus granule neurons and is required for the structural and functional integrity of mossy fiber-CA3 synapses. The underlying molecular mechanisms, however, remained unclear. We show that the synaptic organizer molecule C1ql2 is a direct functional target of Bcl11b that regulates synaptic vesicle recruitment and long-term potentiation at mossy fiber-CA3 synapses in vivo and in vitro. Furthermore, we demonstrate C1ql2 to exert its functions through direct interaction with a specific splice variant of neurexin-3, Nrxn3(25b+). Interruption of C1ql2-Nrxn3(25b+) interaction by expression of a non-binding C1ql2 mutant or by deletion of Nrxn3 in the dentate gyrus granule neurons recapitulates major parts of the Bcl11b as well as C1ql2 mutant phenotype, and interferes with C1ql2 targeting to the synapse. Together, this study identifies a novel C1ql2-Nrxn3(25b+)-dependent signaling pathway through which Bcl11b controls mossy fiber-CA3 synapse function. Thus, our findings contribute to the mechanistic understanding of neurodevelopmental disorders accompanied by synaptic dysfunction.

Mechanistic insights into cAMP-mediated presynaptic potentiation at hippocampal mossy fiber synapses

Article

Full-text available

Jul 2023

Presynaptic plasticity is an activity-dependent change in the neurotransmitter release and plays a key role in dynamic modulation of synaptic strength. Particularly, presynaptic potentiation mediated by cyclic adenosine monophosphate (cAMP) is widely seen across the animals and thought to contribute to learning and memory. Hippocampal mossy fiber-CA3 pyramidal cell synapses have been used as a model because of robust presynaptic potentiation in short- and long-term forms. Moreover, direct presynaptic recordings from large mossy fiber terminals allow one to dissect the potentiation mechanisms. Recently, super-resolution microscopy and flash-and-freeze electron microscopy have revealed the localizations of release site molecules and synaptic vesicles during the potentiation at a nanoscale, identifying the molecular mechanisms of the potentiation. Incorporating these growing knowledges, we try to present plausible mechanisms underlying the cAMP-mediated presynaptic potentiation.

Circadian protein TIMELESS regulates synaptic function and memory by modulating cAMP signaling

Article

Full-text available

Apr 2023

The regulation of neurons by circadian clock genes is thought to contribute to the maintenance of neuronal functions that ultimately underlie animal behavior. However, the impact of specific circadian genes on cellular and molecular mechanisms controlling synaptic plasticity and cognitive function remains elusive. Here, we show that the expression of the circadian protein TIMELESS displays circadian rhythmicity in the mammalian hippocampus. We identify TIMELESS as a chromatin-bound protein that targets synaptic-plasticity-related genes such as phosphodiesterase 4B (Pde4b). By promoting Pde4b transcription, TIMELESS negatively regulates cAMP signaling to modulate AMPA receptor GluA1 function and influence synaptic plasticity. Conditional deletion of Timeless in the adult forebrain impairs working and contextual fear memory in mice. These cognitive phenotypes were accompanied by attenuation of hippocampal Schaffer-collateral synapse long-term potentiation. Together, these data establish a neuron-specific function of mammalian TIMELESS by defining a mechanism that regulates synaptic plasticity and cognitive function.

Synaptic retrograde regulation of the PKA-induced SNAP-25 and Synapsin-1 phosphorylation

Article

Full-text available

Mar 2023
CELL MOL BIOL LETT

Background Bidirectional communication between presynaptic and postsynaptic components contribute to the homeostasis of the synapse. In the neuromuscular synapse, the arrival of the nerve impulse at the presynaptic terminal triggers the molecular mechanisms associated with ACh release, which can be retrogradely regulated by the resulting muscle contraction. This retrograde regulation, however, has been poorly studied. At the neuromuscular junction (NMJ), protein kinase A (PKA) enhances neurotransmitter release, and the phosphorylation of the molecules of the release machinery including synaptosomal associated protein of 25 kDa (SNAP-25) and Synapsin-1 could be involved. Methods Accordingly, to study the effect of synaptic retrograde regulation of the PKA subunits and its activity, we stimulated the rat phrenic nerve (1 Hz, 30 min) resulting or not in contraction (abolished by µ-conotoxin GIIIB). Changes in protein levels and phosphorylation were detected by western blotting and cytosol/membrane translocation by subcellular fractionation. Synapsin-1 was localized in the levator auris longus (LAL) muscle by immunohistochemistry. Results Here we show that synaptic PKA Cβ subunit regulated by RIIβ or RIIα subunits controls activity-dependent phosphorylation of SNAP-25 and Synapsin-1, respectively. Muscle contraction retrogradely downregulates presynaptic activity-induced pSynapsin-1 S9 while that enhances pSNAP-25 T138. Both actions could coordinately contribute to decreasing the neurotransmitter release at the NMJ. Conclusion This provides a molecular mechanism of the bidirectional communication between nerve terminals and muscle cells to balance the accurate process of ACh release, which could be important to characterize molecules as a therapy for neuromuscular diseases in which neuromuscular crosstalk is impaired.

Glutamatergic synaptic deficits in the prefrontal cortex of the Ts65Dn mouse model for Down syndrome

Preprint

Full-text available

Feb 2023

Down syndrome (DS), the most common form of intellectual disability, is a chromosomal disorder caused by having all or part of an extra chromosome 21, leading to intellectual disability. Contrary to the extensive research on the Ts65Dn mouse model of DS in the hippocampus, the synaptic foundation of prefrontal cortex (PFC) malfunction in individuals with DS, including working memory deficits, remains largely unclear. A previous study on mBACtgDyrk1a mice, which overexpress the Dyrk1a gene, showed that this overexpression negatively impacts spine density and synaptic molecular composition, causing synaptic plasticity deficits in the PFC. By comparing Ts65Dn mice, which overexpress multiple genes including Dyrk1a , and mBACtgDyrk1a mice, we aimed to better understand the role of different genes in DS. Results from electrophysiological experiments (i.e., patch-clamp and extracellular field potential recordings ex vivo) in Ts65Dn PFC male mice revealed modifications of intrinsic properties in layer V/VI pyramidal neurons and the synaptic plasticity range. Thus, long-term depression was abolished in Ts65Dn, while synaptic or pharmacological long-term potentiation were fully expressed in Ts65Dn mice. These results, illustrating the phenotypic divergence between the polygenic Ts65Dn model and the monogenic mBACtgDyrk1a model of DS, highlight the complexity of the pathophysiological mechanisms responsible for the neurocognitive symptoms of DS.

Optogenetics at the presynapse

Article

Jul 2022

Optogenetic actuators enable highly precise spatiotemporal interrogation of biological processes at levels ranging from the subcellular to cells, circuits and behaving organisms. Although their application in neuroscience has traditionally focused on the control of spiking activity at the somatodendritic level, the scope of optogenetic modulators for direct manipulation of presynaptic functions is growing. Presynaptically localized opsins combined with light stimulation at the terminals allow light-mediated neurotransmitter release, presynaptic inhibition, induction of synaptic plasticity and specific manipulation of individual components of the presynaptic machinery. Here, we describe presynaptic applications of optogenetic tools in the context of the unique cell biology of axonal terminals, discuss their potential shortcomings and outline future directions for this rapidly developing research area. This Review provides a comprehensive overview of presynaptic applications of optogenetic tools, including the associated challenges, current limitations and future directions for this approach.

Addiction-induced plasticity in underlying neural circuits

Article

Full-text available

Jan 2022

Synaptic plasticity, the substrate for learning, has been established in neural reward circuits and might involve in the learning of addictive behaviors. Long-term exposure to addictive drugs caused long-lasting memories of the drug experience. The main clinical problem that involves the persistence of addiction is a relapse that is resulted from the exposure to cues of the drug experience. Persistent forms of synaptic plasticity are associated with some of the behavioral effects of addictive drugs. Here, we present the underlying mechanisms of plasticity induced by different brain reward circuitry. Therefore, we focus on the collected evidence that drugs of abuse can disturb synaptic plasticity in the main brain circuits of addiction. Prevention of these drug-induced synaptic modifications may be helpful in the treatment of this problem of society.

Physiological and pathological roles of the amyloid precursor protein at the presynapse

Thesis

Feb 2021

Tomàs Jordà Siquier

Alzheimer’s disease (AD) is a progressive neurodegenerative disease which affects 47 million people worldwide, being the most prominent type of dementia. The etiology of the disease is unknown but genetic evidence from the familial form of the disease indicates that the amyloid precursor protein (APP) plays a key role in the pathology. Importantly, APP is the substrate in the proteolytic reaction producing Aβ peptides which compose the amyloid plaques, one of the main pathological hallmarks in AD brain. In addition, APP is ubiquitously expressed by neurons where it interacts with multiple presynaptic proteins but the role of these interactions is elusive.The aim of my thesis was to study the physiological and pathological functions of APP related to its location at the presynapse. First, we studied the consequences on presynaptic mechanisms of the genetic deletion of presenilin, the catalytic subunit of γ-secretase, the intramembrane protease which cleaves APP. We observed that in absence of presenilin, APP accumulates in axons. By combining optogenetic to electrophysiology, we assessed synaptic transmission and plasticity in the CA3 region of the hippocampus. The presynaptic facilitation, the increase in synaptic vesicle release during repetitive stimulation, was altered whereas the basal neurotransmission was not. The impairment of presynaptic mechanisms was due to the accumulation of APP Cter, which decreases the abundancy of synaptotagmin-7, a calcium sensor essential for facilitation. Using a similar approach, we investigated the consequences of the genetic deletion of APP itself and observed again an impairment of presynaptic facilitation. Together, these results demonstrate the importance of APP homeostasis in presynaptic plasticity.I then investigated possible alterations of APP, other than the amyloid peptides, in the AD brain. I discovered that APP dramatically accumulates together with presynaptic proteins around dense-core amyloid plaques in human AD brain. In addition, the Nter domain, but not the Cter domain of APP is enriched in the core of amyloid plaques uncovering a potential pathological role of the secreted APP Nter in dense-core plaques. Ultrastructural analysis of APP accumulations reveals abundant multivesicular bodies containing presynaptic vesicle proteins and autophagosomal built-up of APP. Finally, we observed that outside the APP accumulations, presynaptic proteins were downregulated, in the neuropil area of the outer molecular layer of the dentate gyrus. Altogether, the data I collected during my thesis supports a role of presynaptic APP in physiology and in AD pathology and highlights APP accumulations as a pathological site where presynaptic proteins are mis-distributed.

Targeted volumetric single-molecule localization microscopy of defined presynaptic structures in brain sections

Article

Full-text available

Mar 2021

Revealing the molecular organization of anatomically precisely defined brain regions is necessary for refined understanding of synaptic plasticity. Although three-dimensional (3D) single-molecule localization microscopy can provide the required resolution, imaging more than a few micrometers deep into tissue remains challenging. To quantify presynaptic active zones (AZ) of entire, large, conditional detonator hippocampal mossy fiber (MF) boutons with diameters as large as 10 µm, we developed a method for targeted volumetric direct stochastic optical reconstruction microscopy ( d STORM). An optimized protocol for fast repeated axial scanning and efficient sequential labeling of the AZ scaffold Bassoon and membrane bound GFP with Alexa Fluor 647 enabled 3D- d STORM imaging of 25 µm thick mouse brain sections and assignment of AZs to specific neuronal substructures. Quantitative data analysis revealed large differences in Bassoon cluster size and density for distinct hippocampal regions with largest clusters in MF boutons.

Repeated morphine exposure activates synaptogenesis and other neuroplasticity-related gene networks in the dorsomedial prefrontal cortex of male and female rats

Article

Feb 2021
DRUG ALCOHOL DEPEN

Background Opioid abuse is a chronic disorder likely involving stable neuroplastic modifications. While a number of molecules contributing to these changes have been identified, the broader spectrum of genes and gene networks that are affected by repeated opioid administration remain understudied. Methods We employed Next-Generation RNA-sequencing (RNA-seq) followed by quantitative chromatin immunoprecipitation to investigate changes in gene expression and their regulation in adult male and female rats’ dorsomedial prefrontal cortex (dmPFC) after a regimen of daily injection of morphine (5.0 mg/kg; 10 days). Ingenuity Pathway Analysis (IPA) was used to analyze affected molecular pathways, gene networks, and associated regulatory factors. A complementary behavioral study evaluated the effects of the same morphine injection regimen on locomotor activity, pain sensitivity, and somatic withdrawal signs. Results Behaviorally, repeated morphine injection induced locomotor hyperactivity and hyperalgesia in both sexes. 90% of differentially expressed genes (DEGs) in morphine-treated rats were upregulated in both males and females, with a 35% overlap between sexes. A substantial number of DEGs play roles in synaptic signaling and neuroplasticity. Chromatin immunoprecipitation revealed enrichment of H3 acetylation, a transcriptionally activating chromatin mark. Although broadly similar, some differences were revealed in the gene ontology networks enriched in females and males. Conclusions Our results cohere with findings from previous studies based ona priori gene selection. Our results also reveal novel genes and molecular pathways that are upregulated by repeated morphine exposure, with some common to males and females and others that are sex-specific.

7A computational framework for optimal control of a self-adjustive neural system with activity-dependent and homeostatic plasticity

Article

Full-text available

Jan 2021

The control of the brain system has received increasing attention in the domain of brain science. Most brain control studies have been conducted to explore the brain network's graph-theoretic properties or to produce the desired state based on neural state dynamics, regarding the brain as a passively responding system. However, the self-adjusting nature of neural system after treatment has not been fully considered in the brain control. In the present study, we propose a computational framework for optimal control of the brain with a self-adjustment process in the effective connectivity after treatment. The neural system is modeled to adjust its outgoing effective connectivity as activity-dependent plasticity after treatment, followed by synaptic rescaling of incoming effective connectivity. To control this neural system to induce the desired function, the system's self-adjustment parameter is first estimated, based on which the treatment is optimized. Utilizing this framework, we conducted simulations of optimal control over a functional hippocampal circuitry, estimated using dynamic causal modeling of voltage-sensitive dye imaging from the wild type and mutant mice, responding to consecutive electrical stimuli. Simulation results for optimal control of the abnormal circuit toward a healthy circuit using a single node treatment, neural-type specific treatment as an analogy of medication, and combined treatments of medication and nodal treatment suggest the plausibility of the current framework in controlling the self-adjusting neural system within a restricted treatment setting. We believe the proposed computational framework of the self-adjustment system would help optimal control of the dynamic brain after treatment.

The chloride/potassium co-transporter KCC2 in synaptic plasticity, hippocampal rhythmogenesis and memory

Thesis

Sep 2019

Clémence Simonnet

Information transfer, storage and retrieval in the brain rely on a balance between excitation and inhibition. At the cellular level, memory encoding involves long-term potentiation of excitatory synapses, while at the network level, cortical rhythmogenesis underlies memory encoding and consolidation and requires inhibitory GABAergic signaling to synchronize neuronal ensembles. To maintain the efficacy and polarity of GABA transmission, the chloride/potassium co-transporter KCC2 controls the transmembrane chloride gradients. However, KCC2 also interacts with protein partners and influences neuronal membrane excitability as well as the function and plasticity of glutamatergic synapses. Altogether, KCC2 appears at the crossroads of excitatory and inhibitory transmission. During my PhD, I explored the consequences of KCC2 down-regulation in the dorsal hippocampus on learning and memory, and the underlying mechanisms both at the cellular and network levels. My results demonstrate that KCC2 knockdown in principal neurons of the dorsal hippocampus affects both spatial and contextual memory. This effect is associated with deficits in LTP of hippocampal synapses as well as neuronal hyperexcitability and hippocampal rhythmopathy, including abnormal sharp-wave ripple generation and gamma-band activity during sleep. These alterations likely contribute to impair both memory encoding and consolidation. Since KCC2 is down-regulated in many disorders associated with cognitive impairment, my results suggest that strategies aiming to restore KCC2 expression may hold therapeutic potential in these disorders. I therefore started testing this hypothesis in experimental models of Rett syndrome.

Presynaptic structural and functional plasticity are coupled by convergent Rap1 signaling

Article

Full-text available

May 2024
J CELL BIOL

Dynamic presynaptic actin remodeling drives structural and functional plasticity at synapses, but the underlying mechanisms remain largely unknown. Previous work has shown that actin regulation via Rac1 guanine exchange factor (GEF) Vav signaling restrains synaptic growth via bone morphogenetic protein (BMP)-induced receptor macropinocytosis and mediates synaptic potentiation via mobilization of reserve pool vesicles in presynaptic boutons. Here, we find that Gef26/PDZ-GEF and small GTPase Rap1 signaling couples the BMP-induced activation of Abelson kinase to this Vav-mediated macropinocytosis. Moreover, we find that adenylate cyclase Rutabaga (Rut) signaling via exchange protein activated by cAMP (Epac) drives the mobilization of reserve pool vesicles during post-tetanic potentiation (PTP). We discover that Rap1 couples activation of Rut-cAMP-Epac signaling to Vav-mediated synaptic potentiation. These findings indicate that Rap1 acts as an essential, convergent node for Abelson kinase and cAMP signaling to mediate BMP-induced structural plasticity and activity-induced functional plasticity via Vav-dependent regulation of the presynaptic actin cytoskeleton.

All-optical presynaptic plasticity induction by photoactivated adenylyl cyclase targeted to axon terminals

Article

Mar 2024

Structure, biophysics, and circuit function of a "giant" cortical presynaptic terminal

Article

Full-text available

Mar 2024
SCIENCE

The hippocampal mossy fiber synapse, formed between axons of dentate gyrus granule cells and dendrites of CA3 pyramidal neurons, is a key synapse in the trisynaptic circuitry of the hippocampus. Because of its comparatively large size, this synapse is accessible to direct presynaptic recording, allowing a rigorous investigation of the biophysical mechanisms of synaptic transmission and plasticity. Furthermore, because of its placement in the very center of the hippocampal memory circuit, this synapse seems to be critically involved in several higher network functions, such as learning, memory, pattern separation, and pattern completion. Recent work based on new technologies in both nanoanatomy and nanophysiology, including presynaptic patch-clamp recording, paired recording, super-resolution light microscopy, and freeze-fracture and “flash-and-freeze” electron microscopy, has provided new insights into the structure, biophysics, and network function of this intriguing synapse. This brings us one step closer to answering a fundamental question in neuroscience: how basic synaptic properties shape higher network computations.

Cognitive-exercise dual-task attenuates chronic cerebral ischemia-induced cognitive impairment by activating cAMP/PKA pathway through inhibiting EphrinA3/EphA4

Article

Nov 2023
EXP NEUROL

Evolutionary conservation of hippocampal mossy fiber synapse properties

Article

Sep 2023

Regulation of hippocampal mossy fiber-CA3 synapse function by a Bcl11b/C1ql2/Nrxn3(25b+) pathway

Preprint

Aug 2023
eLife

The transcription factor Bcl11b has been linked to neurodevelopmental and neuropsychiatric disorders associated with synaptic dysfunction. Bcl11b is highly expressed in dentate gyrus granule neurons and is required for the structural and functional integrity of mossy fiber-CA3 synapses. The underlying molecular mechanisms, however, remained unclear. We show that the synaptic organizer molecule C1ql2 is a direct functional target of Bcl11b that regulates synaptic vesicle recruitment and long-term potentiation at mossy fiber-CA3 synapses in vivo and in vitro. Furthermore, we demonstrate C1ql2 to exert its functions through direct interaction with a specific splice variant of neurexin-3, Nrxn3(25b+). Interruption of C1ql2-Nrxn3(25b+) interaction by expression of a non-binding C1ql2 mutant or by deletion of Nrxn3 in the dentate gyrus granule neurons recapitulates major parts of the Bcl11b as well as C1ql2 mutant phenotype, and interferes with C1ql2 targeting to the synapse. Together, this study identifies a novel C1ql2-Nrxn3(25b+)-dependent signaling pathway through which Bcl11b controls mossy fiber-CA3 synapse function. Thus, our findings contribute to the mechanistic understanding of neurodevelopmental disorders accompanied by synaptic dysfunction.

cAMP−EPAC−PKCε−RIM1α signaling regulates presynaptic long-term potentiation and motor learning

Article

Full-text available

Apr 2023
eLife

The cerebellum is involved in learning of fine motor skills, yet whether presynaptic plasticity contributes to such learning remains elusive. Here we report that the EPAC-PKCε module has a critical role in a presynaptic form of long-term potentiation in the cerebellum and motor behavior in mice. Presynaptic cAMP−EPAC−PKCε signaling cascade induces a previously unidentified threonine phosphorylation of RIM1α, and thereby initiates the assembly of the Rab3A−RIM1α−Munc13-1 tripartite complex that facilitates docking and release of synaptic vesicles. Granule cell-specific blocking of EPAC−PKCε signaling abolishes presynaptic long-term potentiation at the parallel fiber to Purkinje cell synapses and impairs basic performance and learning of cerebellar motor behavior. These results unveil a functional relevance of presynaptic plasticity that is regulated through a novel signaling cascade, thereby enriching the spectrum of cerebellar learning mechanisms.

NMDA receptor activity during postnatal development determines intrinsic excitability and mossy fiber long-term potentiation of CA3 pyramidal cells

Article

Mar 2023
HIPPOCAMPUS

Experimental manipulations that interfere with the functional expression of N-methyl-D-aspartate receptors (NMDARs) during prenatal neurodevelopment or critical periods of postnatal development are models that mimic behavioral and neu-rophysiological abnormalities of schizophrenia. Blockade of NMDARs with MK-801 during early postnatal development alters glutamate release and impairs the induction of NMDAR-dependent long-term plasticity at the CA1 area of the hippocampus. However, it remains unknown if other forms of hippocampal plasticity, such as α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)-mediated short-and long-term potentiation, are compromised in response to neonatal treatment with MK-801. Consistent with this tenet, short-and long-term potentiation between dentate gyrus axons, the mossy fibers (MF), onto CA3 pyramidal cells (CA3 PCs) are mediated by AMPARs. By combining whole-cell patch clamp and extracellu-lar recordings, we have demonstrated that transient blockade of NMDARs during early postnatal development induces a series of pre-and postsynaptic modifications at the MF-CA3 synapse. We found reduced glutamate release from the mossy bou-tons, increased paired-pulse ratio, and reduced AMPAR-mediated MF LTP levels. At the postsynaptic level, we found an altered NMDA/AMPA ratio and dysregulation of several potassium conductances that increased the excitability of CA3 PCs. In addition , MK-801-treated animals exhibited impaired spatial memory retrieval in the Barnes maze task. Our data demonstrate that transient hypofunction of NMDARs impacts NMDAR-independent forms of synaptic plasticity of the hippocampus.

Hippocampal circuits

Chapter

Jan 2023

Chitra D Mandyam

Synaptic transmission

Chapter

Aug 2013

Bruce M Maciver

In recent years our understanding of molecular mechanisms of drug action and interindividual variability in drug response has grown enormously. Meanwhile, the practice of anesthesiology has expanded to the preoperative environment and numerous locations outside the OR. Anesthetic Pharmacology: Basic Principles and Clinical Practice, 2nd edition, is an outstanding therapeutic resource in anesthesia and critical care: Section 1 introduces the principles of drug action, Section 2 presents the molecular, cellular and integrated physiology of the target organ/functional system and Section 3 reviews the pharmacology and toxicology of anesthetic drugs. The new Section 4, Therapeutics of Clinical Practice, provides integrated and comparative pharmacology and the practical application of drugs in daily clinical practice. Edited by three highly acclaimed academic anesthetic pharmacologists, with contributions from an international team of experts, and illustrated in full colour, this is a sophisticated, user-friendly resource for all practitioners providing care in the perioperative period.

Long-Term Measurements

Chapter

Aug 2022

Long-term measurements in slice electrophysiology typically constitute long-term potentiation (LTP)Long-term potentiation (LTP) and long-term depression (LTD)Long-term depression (LTD). These measurements can last 1–2 h, requiring minimal baseline noise and easy detection of activated synaptic receptors. This can be relatively easy to achieve in field recordings but can be challenging in whole-cell configurations. In this chapter, we discuss the concepts behind LTPLong-term potentiation (LTP) and LTDLong-term depression (LTD) as well as the approaches that the beginning electrophysiologist can use in order to complete these long-term measurements. We finish with potential technical issues to consider during experimentation as well as suggestions that may increase the likelihood of successful recordings.

Investigation into the effects of neuropeptides on epileptiform activity in the hippocampus in vitro

Thesis

Jan 2006

Christopher Harper

p>I used Electrophysiological recording techniques to investigate the effects of nociceptin (Noc) in various models of epileptiform activity, in acute hippocampal slices taken from adult rats. I subsequently looked at the effects of Noc in slices taken from an epileptic (EL) mouse model and in organotypic hippocampal slice cultures (OHSC’s). In some models I compare these effects to the action of Neuropeptide Y (NPY) as this is a well studied anti-convulsant neuropeptide. I found that Noc caused a blockade of the bursting activity seen in the 10μM Biculculline (Bic) and 0 Mg<sup>++</sup> models of epileptiform activity. I also found that Noc did not cause a change in the bursting activity seen in the High K<sup>+</sup> (8.5mM) or 100μM 4-Aminopyridine (4-AP) models. When I looked at the Noc effects on the Bic and 0 Mg++ models in EL mice I found that Noc showed a greater reduction in bursting activity when applied to slices taken from ‘sensitised’ mice as compared to ‘non-sensitised’. I found that Noc showed no effects on synaptic transmission or paired-pulse inhibition in OHSC’s. The same result was seen with NPY. I showed through Western Blotting that the OP4, Y1, Y2 and Y5 receptors are present in OHSC’s. I then showed that Noc caused a reduction in bursting activity in the Bic model in OHSC’s and that no effect was seen when the experiment was repeated with NPY. I also experienced problems with the 4-AP model in OHSC’s and subsequently showed that 4-AP causes neuronal death in the CA1 sub-field of OHSC’s. This data shows that targets of the OP4 receptor pathway could prove a useful experimental tool, and potential future anticonvulsant treatment.</p

Excitatory selective LTP of supramammillary glutamatergic/GABAergic cotransmission potentiates dentate granule cell firing

Article

Full-text available

Mar 2022

Significance It is now established that many neurons can release multiple transmitters. Recent studies revealed that fast-acting neurotransmitters, glutamate and GABA, are coreleased from the same presynaptic terminals in some adult brain regions. The dentate gyrus (DG) granule cells (GCs) are innervated by the hypothalamic supramammillary nucleus (SuM) afferents that corelease glutamate and GABA. However, how these functionally opposing neurotransmitters contribute to DG information processing remains unclear. We show that glutamatergic, but not GABAergic, cotransmission exhibits long-term potentiation (LTP) at SuM-GC synapses. By the excitatory selective LTP, the excitation/inhibition balance of SuM inputs increases, and GC firing is enhanced. This study provides evidence that glutamatergic/GABAergic cotransmission balance is rapidly changed in an activity-dependent manner, and such plasticity may modulate DG activity.

Local modulation by presynaptic receptors controls neuronal communication and behaviour

Article

Feb 2022
NAT REV NEUROSCI

Central nervous system neurons communicate via fast synaptic transmission mediated by ligand-gated ion channel (LGIC) receptors and slower neuromodulation mediated by G protein-coupled receptors (GPCRs). These receptors influence many neuronal functions, including presynaptic neurotransmitter release. Presynaptic LGIC and GPCR activation by locally released neurotransmitters influences neuronal communication in ways that modify effects of somatic action potentials. Although much is known about presynaptic receptors and their mechanisms of action, less is known about when and where these receptor actions alter release, especially in vivo. This Review focuses on emerging evidence for important local presynaptic receptor actions and ideas for future studies in this area. Local activation of presynaptic receptors alters neurotransmitter release, modulating effects of somatic action potentials. In this Review, Lovinger et al. discuss the role of presynaptic receptors in regulating synaptic transmission and directions for future research aimed at determining the in vivo roles of presynaptic receptors.

The Interactions among Hypertension, Cancer, and COVID-19: Perspectives from Ca2+/cAMP Signalling

Article

Feb 2022

Leandro Bueno Bergantin

Background The hypothesis that hypertension is clinically associated with an enhanced risk for developing cancer has been highlighted. However, the working principles involved in this link are still under intensive discussion. A correlation among inflammation, hypertension, and cancer could accurately describe the clinical link between these diseases. In addition, a dyshomeostasis of Ca2+ has been considered as a topic involved in both cancer and hypertension and inflammation. There is a strong link between Ca2+ signalling, e.g. enhanced Ca2+ signals, and inflammatory outcomes. cAMP also modulates pro- and anti-inflammatory outcomes: pharmaceuticals, which increase intracellular cAMP levels, can decrease the production of proinflammatory mediators and enhance the production of anti-inflammatory outcomes. Objective This article has discussed the participation of Ca2+/cAMP signalling in the clinical association among inflammation, hypertension, and an enhanced risk for the development of cancer. In addition, considering coronavirus disease 2019 (COVID-19) is a rapidly evolving field, this article also reviews recent reports about the role of Ca2+ channel blockers for restoring Ca2+ signalling disruption due to COVID-19, including the relationship among COVID-19, cancer, and hypertension. Conclusion Understanding the association among these diseases could expand current pharmacotherapy, including that involving Ca2+ channel blockers and pharmaceuticals which rise cAMP levels.

Maturation morpho-fonctionnelle de la synapse fibre moussue/cellule pyramidale de CA3 dans l’hippocampe

Thesis

Oct 2010

Frederic Lanore

Les synapses se forment selon plusieurs étapes comprenant la stabilisation des contacts nouvellementformés et leur maturation. Ces différentes étapes dépendent d’une mise en place coordonnée entre laterminaison pré- et postsynaptique. Les protéines composant la présynapse et les récepteursionotropiques du glutamate ont des rôles clés dans ces processus. Lors de ma thèse, je me suisintéressé à l’implication de la protéine présynaptique Bassoon lors de la maturation des synapsesglutamatergiques entre les fibres moussues et les cellules pyramidales de CA3 dans l’hippocampe.Cette synapse constitue un modèle attractif pour l’étude de la maturation synaptique car elle suit desétapes de maturation morphologique et fonctionnelle bien définies. Bassoon est une des premièresprotéines se mettant en place au niveau des contacts synaptiques nouvellement formés. Par desapproches électrophysiologiques, nous avons montré que la protéine Bassoon était importante pourl’organisation du site de libération de neurotransmetteur durant les deux premières semaines de viepost-natale chez la souris.Les récepteurs kaïnate jouent un rôle important dans la régulation de l’activité de réseau au cours dudéveloppement post-natal. Cependant l’impact de l’activation de ces récepteurs sur la maturationsynaptique est peu connu. J’ai pu mettre en évidence un délai dans la maturation fonctionnelle de lasynapse fibre moussue/cellule pyramidale de CA3 chez les souris déficientes pour la sous-unité GluK2des récepteurs kaïnate (GluK2-/-). Afin de comprendre si ce délai de maturation fonctionnelle est corréléà un retard dans la maturation morphologique de cette synapse, nous avons mis en place desinfections de lentivirus codant pour une protéine membranaire fluorescente (YFP) chez le souriceaunouveau-né (P1-P2). A l’aide de microscopie confocale et de reconstruction en 3D, nous avons ainsi pudécrire la maturation morphologique de la synapse fibre moussue/cellule pyramidale de CA3. Cela m’aégalement permis de corréler la maturation fonctionnelle à la maturation morphologique et mesrésultats montrent également un retard dans la mise en place des synapses chez les souris GluK2-/-.L’ensemble de cette étude révèle l’importance de l’activité synaptique et de la coordination entre miseen place de la pré- et de la postsynapse au cours de la maturation synaptique.

Metabotropic actions of Kainate Receptors modulating glutamate release

Chapter

Nov 2011

Metabotropic actions of Kainate receptors in the control of GABA release

Chapter

Nov 2011

Kainate Receptors: Novel Signalling insights

Book

Jul 2011

Rapid Ca 2+ channel accumulation contributes to cAMP-mediated increase in transmission at hippocampal mossy fiber synapses

Article

Mar 2021

Significance Despite the importance of the activation of the cAMP/PKA signaling pathway for presynaptic modulation, its cellular and molecular mechanisms remain unclear. In this study, we tackled this issue at hippocampal mossy fiber-CA3 synapses by combining paired electrophysiological recordings, Ca ²⁺ uncaging, total internal reflection fluorescence (TIRF) microscopy for direct measurements of presynaptic active zone Ca ²⁺ influx, and superresolution time-gated STED microscopy for visualization of Ca ²⁺ channel clusters within active zones. Our results suggest that Ca ²⁺ channels near release sites might accumulate upon activation of the cAMP/PKA pathway in the time scale of only several minutes. Our findings may thus change the focus from release machinery to Ca ²⁺ channel clusters and their dynamics as a cell biological mechanism of cAMP-dependent synaptic modulation.

Role of Ca2+‐stimulated adenylyl cyclases in LTP and memory formation

Article

Jul 2001
INT J DEV NEUROSCI

Studies with invertebrates and vertebrates have strongly implicated the CREB/CRE transcriptional pathway in long‐term memory (LTM) and transcriptionally‐dependent L‐LTP. It is hypothesized that LTM and L‐LTP are both dependent upon a Ca²⁺ signal generated through activation of NMDA receptors. This review discusses evidence that Ca²⁺ signals generated through activation of NMDA receptors coactivate the Erk/MAP kinase and cAMP signal transduction pathways. It is hypothesized that activation of these two regulatory pathways increases the transcription of a family of genes through the CREB/CRE transcriptional pathway. Gene disruption studies have shown that Ca²⁺ activated adenylyl cyclases play a critical role in generating the cAMP signal required for LTM and L‐LTP. Although cAMP may be required for several events in this complex signal transduction cascade, one of the major roles of cAMP may be to support nuclear translocation of Erk/MAP kinase in hippocampal neurons.

Recruitment of long-lasting and protein kinase A-dependent long-term potentiation in the CA1 region of hippocampus requires repeated tetanization

Article

Full-text available

Nov 1993

To study how the late phase of long-term potentiation (LTP) in hippocampus arises, we examined the resulting LTP for its time course and its dependence on protein synthesis and different second-messenger kinases by applying various conditioning tetani. We find that one high-frequency train (100 Hz) produces a form of LTP that lasts longer than 1 hr but less than 3 hr (the early phase of LTP, or E-LTP). It is blocked by inhibitors of calcium/calmodulin kinase II (Cam kinase II) but is not affected by an inhibitor of cAMP-dependent protein kinase [protein kinase A (PKA) and the protein synthesis inhibitor anisomycin] nor is it occluded by the cAMP activator forskolin. In contrast, when three high-frequency trains are used, the resulting potentiation persists for at least 6-10 hr. The L-LTP induced by three trains differs from the E-LTP in that it requires new protein synthesis, is blocked by an inhibitor of cAMP-dependent protein kinase, and is occluded by forskolin. These results indicate that the two mechanistically distinctive forms of LTP, a transient, early component (E-LTP) and a more enduring form (L-LTP), can be recruited selectively by changing the number of conditioning tetanic trains. Repeated tetani induce a PKA and protein synthesis-dependent late component that adds to the amplitude and duration of the potentiation induced by a single tetanus.

Inhibition of forskolin-induced neurite outgrowth and protein phosphorylation by a newly synthesized selective inhibitor of cyclic AMP-dependent protein kinase, N-[2-(p-Bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), of PC12D pheochromocytoma cells

Article

Full-text available

Apr 1990

A newly synthesized isoquinolinesulfonamide, H-89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide), was shown to have a potent and selective inhibitory action against cyclic AMP-dependent protein kinase (protein kinase A), with an inhibition constant of 0.048 +/- 0.008 microM. H-89 exhibited weak inhibitory action against other kinases and Ki values of the compound for these kinases, including cGMP-dependent protein kinase (protein kinase G), Ca2+/phospholipid-dependent protein kinase (protein kinase C), casein kinase I and II, myosin light chain kinase, and Ca2+/calmodulin-dependent protein kinase II were 0.48 +/- 0.13, 31.7 +/- 15.9, 38.3 +/- 6.0, 136.7 +/- 17.0, 28.3 +/- 17.5, and 29.7 +/- 8.1 microM, respectively. Kinetic analysis indicated that H-89 inhibits protein kinase A, in competitive fashion against ATP. To examine the role of protein kinase A in neurite outgrowth of PC12 cells, H-89 was applied along with nerve growth factor (NGF), forskolin, or dibutyryl cAMP. Pretreatment with H-89 led to a dose-dependent inhibition of the forskolin-induced protein phosphorylation, with no decrease in intracellular cyclic AMP levels in PC12D cells, and the NGF-induced protein phosphorylation was not not inhibited. H-89 also significantly inhibited the forskolin-induced neurite outgrowth from PC12D cells. This inhibition also occurred when H-89 was added before the addition of dibutyryl cAMP. Pretreatment of PC12D cells with H-89 (30 microM) inhibited significantly cAMP-dependent histone IIb phosphorylation activity in cell lysates but did not affect other protein phosphorylation activity such as cGMP-dependent histone IIb phosphorylation activity, Ca2+/phospholipid-dependent histone IIIs phosphorylation activity, Ca2+/calmodulin-dependent myosin light chain phosphorylation activity, and alpha-casein phosphorylation activity. However, this protein kinase A inhibitor did not inhibit the NGF-induced neurite outgrowth from PC12D cells. Thus, the forskolin- and dibutyryl cAMP-induced neurite outgrowth is apparently mediated by protein kinase A while the NGF-induced neurite outgrowth is mediated by a protein kinase A-independent pathway.

Probing the cyclic nucleotide binding sites of cAMP-dependent protein kinases I and II with analogs of adenosine 3',5'-cyclic phosphorothioates

Article

Full-text available

Jul 1990

A set of cAMP analogs were synthesized that combined exocyclic sulfur substitutions in the equatorial (Rp) or the axial (Sp) position of the cyclophosphate ring with modifications in the adenine base of cAMP. The potency of these compounds to inhibit the binding of [3H]cAMP to sites A and B from type I (rabbit skeletal muscle) and type II (bovine myocardium) cAMP-dependent protein kinase was determined quantitatively. On the average, the Sp isomers had a 5-fold lower affinity for site A and a 30-fold lower affinity for site B of isozyme I than their cyclophosphate homolog. The mean reduction in affinities for the equivalent sites of isozyme II were 20- and 4-fold, respectively. The Rp isomers showed a decrease in affinity of approximately 400-fold and 200-fold for site A and B, respectively, of isozyme I, against 200-fold and 45-fold for site A and B of isozyme II. The Sp substitutions therefore increased the relative preference for site A of isozyme I and site B of isozyme II. The Rp substitution, on the other hand, increased the relative preference for site B of both isozymes. These data show that the Rp and Sp substitutions are tolerated differently by the two intrachain sites of isozymes I and II. They also support the hypothesis that it is the axial, and not the previously proposed equatorial oxygen that contributes the negative charge for the ionic interaction with an invariant arginine in all four binding sites. In addition, they demonstrate that combined modifications in the adenine ring and the cyclic phosphate ring of cAMP can enhance the ability to discriminate between site A and B of one isozyme as well as to discriminate between isozyme I and II. Since Rp analogs of cAMP are known to inhibit activation of cAMP-dependent protein kinases, the findings of the present study have implications for the synthesis of analogs having a very high selectivity for isozyme I or II.

Induction of long-term potentiation at hippocampal mossy-fiber synapses follows a Hebbian rule

Article

Full-text available

Oct 1990

1. The induction of long-term potentiation (LTP) at hippocampal mossy-fiber synapses requires an increase in postsynaptic [Ca2+]i but is independent of N-methyl-D-aspartate (NMDA) receptor activation. Voltage-gated Ca2+ channels have been proposed as one alternative source for raising [Ca2+]i during the induction of LTP. We tested the hypothesis that voltage-gated Ca2+ channel activation could mediate the induction of LTP by examining whether 1) the induction of mossy-fiber LTP was dependent on postsynaptic depolarization and 2) depolarization alone, of a magnitude presumably capable of activating Ca2+ channels, was sufficient to induce LTP. 2. Intracellular recordings were made from rat CA3 pyramidal cells in the hippocampal slice preparation under both current- and voltage-clamp conditions. Mossy-fiber postsynaptic potentials and currents were recorded before and after high-frequency stimulation (HFS) in the presence of 20-50 microM D-2-amino-5-phosphonovaleric acid (D-APV), an NMDA-receptor antagonist. 3. Voltage clamping of CA3 neurons between -80 and -100 mV during HFS reversibly blocked the induction of mossy-fiber LTP. Conversely, HFS paired with depolarizing-current steps under current clamp increased the magnitude of LTP compared with controls. These results indicate that mossy-fiber LTP is dependent on postsynaptic depolarization, and presynaptic activation alone was not sufficient to induce mossy-fiber LTP. 4. Depolarizing-current injections, which presumably depolarized CA3 cells to potentials sufficient to activate voltage-gated Ca2+ channels, had no effect on mossy-fiber synaptic responses. These results suggest that synaptic activation, in addition to postsynaptic depolarization, is required for the induction of mossy-fiber LTP. 5. Single mossy-fiber afferent volleys were also paired with depolarizing-current pulses. In the presence of APV, pairing of single-mossy-fiber excitatory postsynaptic potentials (EPSPs) with postsynaptic depolarization did not potentiate synaptic responses, suggesting that some form of HFS is also required for mossy-fiber LTP. In the absence of APV, however, the contamination of mossy-fiber synaptic responses by CA3-recurrent inputs resulted in some potentiation. 6. These results suggest that the induction of mossy-fiber LTP is dependent on both pre- and postsynaptic activity and thus follows a Hebbian rule for synaptic modification. In contrast to that demonstrated at Schaffer-collateral-commissural synapses, however, the induction of mossy-fiber LTP may require HFS in addition to postsynaptic depolarization.(ABSTRACT TRUNCATED AT 400 WORDS)

Haas, H. L. & Rose, G. M. Noradrenaline blocks potassium conductance in rat dentate granule cells in vitro. Neurosci. Lett. 78, 171-174

Article

Full-text available

Aug 1987
NEUROSCI LETT

The actions of noradrenaline and the beta-adrenergic agonist, isoproterenol, were studied on the dentate gyrus in hippocampal slices from rats using extra- and intracellular recording. These agents facilitated field EPSPs (excitatory postsynaptic potentials) and population spikes evoked by perforant path stimulation. Intracellular recording revealed an attenuation of the long lasting afterhyperpolarization (AHP) and the accommodation of cell discharge in response to depolarizing current injection. It is suggested that beta-receptor activation blocks a calcium-dependent potassium current.

Cyclic AMP evokes long-term facilitation in Aplysia sensory neurons that requires new protein synthesis

Article

Full-text available

Jul 1988

Behavioral sensitization leads to both short- and long-term enhancement of synaptic transmission between the sensory and motor neurons of the gill-withdrawal reflex in Aplysia. Serotonin (5-HT), a transmitter important for short-term sensitization, can evoke long-term enhancement of synaptic strength detected 1 day later. Because 5-HT mediates short-term facilitation through adenosine 3',5'-monophosphate (cAMP)-dependent protein phosphorylation, the role of cAMP in the long-term modulation of this identified synapse was examined. Like 5-HT, cAMP can also evoke long-term facilitation lasting 24 hours. Unlike the short-term change, the long-lasting change is blocked by anisomycin, a reversible inhibitor of protein synthesis, and therefore must involve the synthesis of gene products not required for the short-term change.

Effect of Forskolin on Voltage-Gated K + Channels Is Independent of Adenylate Cyclase Activation

Article

Full-text available

Jul 1988

Forskolin is commonly used to stimulate adenylate cyclase in the study of modulation of ion channels and other proteins by adenosine 3',5'-monophosphate (cAMP)-dependent second messenger systems. In addition to its action on adenylate cyclase, forskolin directly alters the gating of a single class of voltage-dependent potassium channels from a clonal pheochromocytoma (PC12) cell line. This alteration occurred in isolated cell-free patches independent of soluble cytoplasmic enzymes. The effect of forskolin was distinct from those of other agents that raise intracellular cAMP levels. The 1,9-dideoxy derivative of forskolin, which is unable to activate the cyclase, was also effective in altering the potassium channel activity. This direct action of forskolin can lead to misinterpretation of results in experiments in which forskolin is assumed to selectively activate adenylate cyclase.

Adenylate Cyclase System Is Essential for Long-Term Facilitation at the Crayfish Neuromuscular Junction

Article

Full-text available

Jan 1990

Long-term facilitation (LTF), a form of synaptic plasticity demonstrated at the crayfish neuromuscular junction, is induced by tetanic stimulation and persists for hours. LTF can be divided into 2 phases: a tetanic phase, which occurs during stimulation, and a long-lasting phase, which persists after stimulation. Activators and potentiators of cAMP (forskolin and 3-isobutyl-methyl-xanthine) produce facilitation of excitatory postsynaptic potentials, which attain approximately the amplitude of the long-lasting phase of LTF but last for a shorter time. Localized presynaptic injection of a protein inhibitor ("Walsh inhibitor") specific for the cAMP-dependent protein kinase blocks the long-lasting phase of LTF at synapses near the injection site with no apparent effect on the tetanic phase. Normal LTF develops and persists at synapses of the same axon distant from the injection site. Localization of the injected inhibitor was confirmed by fluorescent tagging. Localized injection of SQ22,536, an adenylate cyclase inhibitor, also blocks the second phase of LTF near the injection site, but not at distant synapses. These experiments establish a role for adenylate cyclase activation in the long-lasting phase of LTF. The phosphatidylinositol second-messenger system is not important in LTF as inhibition of phospholipase C by injection of RA233, which blocks facilitatory effects of serotonin, does not affect any aspect of LTF.

Presynaptic long-term facilitation at the crayfish neuromuscular juntion: Voltage-dependent and ion-dependent phases

Article

Full-text available

Jan 1989

Long-term facilitation (LTF) of synaptic transmission was investigated in the crayfish opener muscle to determine the factors necessary for its induction and expression. LTF was induced without action potentials by intracellular depolarization of presynaptic nerve terminals. Following induction, the synaptic transmission was enhanced by about 80% for a period of several hours. Intracellular recordings from pre- and postsynaptic cells, combined with ionic and pharmacological tests, permitted dissection of LTF into 2 phases: an initial tetanic phase that depended on the presence of both sodium and calcium ions and a subsequent long-lasting phase. This latter long-lasting enhancement of synaptic transmission was induced by repeated depolarizations of synaptic terminals but did not depend on the influx of sodium or calcium ions or on intracellular release of calcium ions. Both tetanic and long-lasting phases of LTF are attributable to activity of a single neuron, i.e., they are homosynaptic phenomena. Furthermore, LTF is associated with an increase of quantal release, whereas the size of quanta remains unchanged. During the long-lasting phase of LTF, the nerve terminal releases more transmitter for a given depolarization than before induction of LTF. Thus, the locus of LTF is presynaptic. Our findings suggest the presence of a voltage-dependent mechanism in the presynaptic membrane different from voltage-gating of Na or Ca channels. Such a mechanism may be important in the establishment of long-lasting synaptic changes at the crayfish neuromuscular junction and perhaps in other neural systems.

Quanta1 mechanism of long term synaptic potentiation

Article

Full-text available

Oct 1985

Intracellular recordings were used to demonstrate the occurrence and to analyze the microphysiology of long-term synaptic potentiation (LTP) in the crayfish opener neuromuscular synapse. Brief stimulation of the single excitor motor axon enhanced the amplitudes of subsequent postsynaptic potentials for several hours. Three methods of quantal analysis were used to evaluate the mechanism responsible for LTP. The results of all three methods supported predictions of the hypothesis that LTP results from a presynaptic mechanism that increases the average of neurotransmitter quanta evoked by nerve impulses in the excitor axon.

Competitive cAMP Antagonists for cAMP-Receptor Proteins

Article

Full-text available

Sep 1984

The two exocyclic oxygen atoms at phosphorus of cAMP have been replaced by a sulfur atom or by a dimethylamino group. These substitutions introduce chirality at the phosphorus atom; therefore, two diastereoisomers are known for each derivative: (SP)-cAMPS, (RP)-cAMPS, (SP)-cAMPN(CH3)2, and RP-cAMPN(CH3)2. We have investigated the agonistic and antagonistic activities of these compounds in four cAMP-dependent reactions: activation of the cellular slime mold Dictyostelium discoideum via its cell surface cAMP receptor, and phosphorylation by cAMP-dependent protein kinases type I, type II (both mammalian enzymes), and type D (derived from D. discoideum). The results show that 1) the compounds (SP)-cAMPS and (SP)-cAMPN(CH3)2 are (mostly full) agonists for the four proteins. Half-maximal activation is at micromolar concentrations (0.8-7 microM). 2) (RP)-cAMPS is a full antagonist for the cell surface receptor and protein kinases type I and II, with apparent inhibition constants between 0.8 and 8 microM. This compound is a partial agonist for protein kinase type D, where it induces maximally 50% activation of the enzyme if compared with cAMP. 3) (RP)-cAMPN(CH3)2 is a full antagonist for the cell surface receptor, and for protein kinase type II. This compound is a partial agonist for protein kinase type I (at least 50% activation if compared with cAMP), and inactive for protein kinase type D. This derivative is at least 25-fold less active as an antagonist than (RP)-cAMPS. 4) The activity of mixtures of different concentrations of the antagonist (RP)-cAMPS with different concentrations of cAMP reveals that the compound is a competitive antagonist of cAMP at micromolar concentrations.

Increased transmitter release at excitatory synapses produced by direct activation of adenylate cyclase in rat hippocampal slices

Article

Full-text available

Feb 1994

The field EPSP recorded in the CA1 region of rat hippocampal slices is potentiated by bath application of the direct adenylate cyclase activator forskolin (Chavez-Noriega and Stevens, 1992a). We have now used the whole-cell patch-clamp technique to analyze the effect of forskolin on evoked synaptic currents and on spontaneous and miniature excitatory postsynaptic currents (sEPSCs and mEPSCs) recorded in rat hippocampal slices in order to determine the relative contributions of pre- and postsynaptic mechanisms to this increased synaptic strength. Application of 50 microM forskolin in the presence of 3-isobutyl-1-methylxanthine (IBMX; a phosphodiesterase inhibitor) enhanced the evoked EPSC (eEPSC) peak amplitude to 230 +/- 43% of control (n = 13). No significant change in sEPSC or in mEPSC amplitude was detected after forskolin addition (106 +/- 7%, n = 9), indicating that postsynaptic receptor sensitivity at synaptic junctions is not greatly affected. In contrast, a large increase in sEPSC and mEPSC frequency was noted in all cells (299 +/- 81%). Following forskolin application, the amplitude distribution of evoked synaptic currents shifted to larger values, but more significantly, a sharp decrease in failure rate was produced in all cells tested. Also, a significant correlation was found between the potentiation produced by forskolin in IBMX on the eEPSC and the ratio of the squared coefficient of variation (CV = SD/mean). Finally, a quantal analysis of four cells was consistent with the hypothesis that transmitter release was increased by forskolin/IBMX with, if anything, a concomitant decrease in quantal size. Together, these observations indicate that presynaptic mechanisms significantly contribute to the enhancement produced by this diterpene.(ABSTRACT TRUNCATED AT 250 WORDS)

Type VIII adenylyl cyclase: A Ca2+/calmodulin-stimulated enzyme expressed in discrete regions of rat brain

Article

Full-text available

May 1994

A cDNA that encodes type VIII adenylyl cyclase has been isolated from two rat brain libraries. The open reading frame encodes a 1248-amino acid protein predicted to have two sets of six transmembrane spans and two putative nucleotide binding domains as is characteristic of other mammalian adenylyl cyclases. Two type VIII messages are detected in rat brain with estimated sizes of 5.5 and 4.4 kilobases. In situ hybridization indicates that the type VIII messages are most abundantly expressed in the granule cells of the dentate gyrus, the pyramidal cells of hippocampal fields CA1-CA3, the entorhinal cortex, and the piriform cortex. Hybridization is also detected in the neocortex, the amygdaloid complex, and regions of the thalamus and hypothalamus. Stable expression of the type VIII cDNA in human embryonal kidney cells leads to the appearance of a novel 165-kDa glycoprotein in the membrane fraction. Stimulation of these cells with agents that increase intracellular Ca2+ results in up to 43-fold increases in cAMP accumulation over that of control cells transfected with the expression vector. Addition of isoproterenol alone does not lead to type VIII-specific effects in intact cells. Adenylyl cyclase activity in membranes prepared from type VIII-transformed cells is stimulated up to 40-fold by the addition of Ca2+/calmodulin (EC50 = 53 nM calmodulin). The addition of activated recombinant alpha subunit of Gs synergistically increases the Ca2+/calmodulin-stimulated activity. A possible role for type VIII adenylyl cyclase in long-term potentiation is discussed.

Modification of the calcium and calmodulin sensitivity of the Type I adenylyl cyclase by mutagenesis of its calmodulin Binding domain

Article

Full-text available

Dec 1993

The type I adenylyl cyclase is directly stimulated by Ca2+ and calmodulin in vitro, and the enzyme is also stimulated by increases in intracellular Ca2+ in vivo. Ca2+ stimulation of the enzyme in vivo may be due to direct interactions of the enzyme with Ca2+ and calmodulin or to an indirect mechanism involving stimulation of the enzyme by Ca(2+)-activated protein kinases. In this study, we have made several point mutations within the calmodulin binding domain to determine if the Ca2+ sensitivity of the enzyme can be modified by mutagenesis. The catalytic activities of the mutant enzymes were comparable to wild type type I adenylyl cyclase. Substitution of Cys-507 with Ser-507 did not have significant effects on the calmodulin or Ca2+ sensitivity of the enzyme. However, replacement of Lys-504 with Asp caused a 4-fold decrease in sensitivity to Ca2+. Ca2+ and calmodulin stimulation were abolished by substitution of Phe-503 with Arg-503. Stimulation of type I adenylyl cyclase activity in vivo by intracellular Ca2+ was also greatly diminished with the Arg-503 mutant indicating that Ca2+ stimulation of the enzyme in vivo is due primarily to direct interactions with calmodulin and Ca2+. These data demonstrate that the Ca2+ sensitivity of this enzyme can be modulated by point mutagenesis within the putative calmodulin binding domain and indicate that the enzyme can be directly regulated by Ca2+ and calmodulin in vivo.

N-[2-bromocinnamyl(amino)ethyl]-5-isoquinolinesulphonamide (H-89) inhibits incorporation of choline into phosphatidylcholine via inhibition of choline kinase and has no effect on the phosphorylation of CTP:phosphocholine cytidylyltransferase

Article

Full-text available

Feb 1994

We have shown previously that N-[2-bromocinnamyl(amino)-ethyl]-5-isoquinolinesulphonamide (H-89), a selective inhibitor of cyclic-AMP-dependent protein kinase (PKA), inhibits phosphatidylcholine biosynthesis in HeLa cells. In the present study, we elucidated the mechanism underlying the described inhibition. Treatment of cells with 10 microM H-89 had no effect on the phosphorylation of CTP:phosphocholine cytidylyltransferase. However, H-89 slightly affected the distribution of cytidylyltransferase between cytosol and membranes, but the cellular 1,2-diacylglycerol content was not influenced. Furthermore, pulse-chase experiments revealed that H-89 did not affect cytidylyltransferase activity. Instead, H-89 inhibited choline kinase, the enzyme catalysing the first step in the CDP-choline pathway. In the presence of 10 microM H-89, choline kinase activity was inhibited by 36 +/- 7.6% in vitro. Additionally, the phosphorylation of choline to phosphocholine was inhibited by 30 +/- 3% in cell-culture experiments. This inhibitory effect could be partly prevented by simultaneous addition of 10 microM forskolin, indicating that choline kinase is regulated in part by PKA activity.

The opioid peptide dynorphin mediates heterosynaptic depression of hippocampal mossy fiber synapes and modulates long-term potentiation

Article

Apr 1993

The mossy fibre pathway in the hippocampus uses glutamate as a neurotransmitter, but also contains the opioid peptide dynorphin. Synaptic release of dynorphin causes a presynaptic inhibition of neighbouring mossy fibres and inhibits the induction and expression of mossy fibre long-term potentiation. These findings demonstrate a physiological role for a neuropeptide in the central nervous system, provide a functional basis for the coexistence of a neuropeptide with classic neurotransmitters and demonstrate the very different roles played by these two classes of signalling molecules.

A simple chamber for recording from submerged brain slices

Article

Sep 1981
J NEUROSCI METH

The construction of a chamber is described for stable intracellular recording from submerged brain slices while switching solutions. The chamber is simple and inexpensive to make and is simple to use.

Dimaprit-[S-[3-(N,N-dimethylamino)propyl]isothiourea] —A highly specific histamine H2-receptor agonist. Part 1. Pharmacology

Article

Apr 1977
Agents Actions

S-[3-(N,N-Dimethylamino)propyl]isothiourea (dimaprit), has been shown to be a highly specific histamine H2-receptor agonist. Parallel line assays showed that in vitro at H2-receptors it had approximately 17.5% the activity of histamine on the rat uterus and 71% on the guinea-pig right atrium, with similar maximal responses; it had less than 0.0001% the activity of histamine on H1-receptors. Dimaprit stimulated gastric acid secretion in the rat, dog and cat in which it had, respectively, approximately 19, 58 and 400–500% the activity of histamine. In the dog and cat the maximum secretory response to dimaprit was significantly greater than that obtained to histamine. The H2-receptor specificity of dimaprit in causing depressor and vasodilator effects was also demonstrated in the cat, in which it had 18–20% of the H2-receptor activity of histamine. Dimaprit should prove to be a very useful tool in studies examining the role of histamine in physiology and pathology. The absence of marked cardiovascular effects at doses maximal for the stimulation of gastric acid secretion, as seen in the cat studies, could lead to this compound being of value as a diagnostic agent in the measurement of maximal acid secretory capacity.

Adenylyl Cyclases

Article

Oct 1992
CELL

Cloning of a cDNA for a glutamate receptor activated by kainate but not AMPA

Article

Jul 1991

Fast excitatory transmission in the vertebrate central nervous system is mediated mainly by L-glutamate. On the basis of pharmacological, physiological and agonist binding properties, the ionotropic glutamate receptors are classified into NMDA (N-methyl-D-aspartate), AMPA (alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate) and kainate subtypes. Sequence homology between complementary DNA clones encoding non-NMDA glutamate receptor subunits reveals at least two subunit classes: the GluR1 to GluR4 class and the GluR5 class. Here we report the cloning and expression of a functional rat glutamate receptor subunit cDNA, GluR6, which has a very different pharmacology from that of the GluR1-GluR4 class. Receptors generated from the GluR1-GluR4 class have a higher apparent affinity for AMPA than for kainate. When expressed in Xenopus oocytes the homomeric GluR6 receptor is activated by kainate, quisqualate and L-glutamate but not by AMPA, and the apparent affinity for kainate is higher than for receptors from the GluR1-GluR4 class. Desensitization of the receptor was observed with continuous application of agonist. The homomeric GluR6 glutamate receptor exhibits an outwardly rectifying current-voltage relationship. In situ hybridizations reveal a pattern of GluR6 gene expression reminiscent of the binding pattern obtained with [3H]kainate.

The maintenance of LTP at hippocampal mossy fiber synapses is independent of sustained presynaptic calcium

Article

Oct 1991
NEURON

We have examined the role of presynaptic residual calcium in maintaining long-term changes in synaptic efficacy observed at mossy fiber synapses between hippocampal dentate granule cells and CA3 pyramidal cells. Calcium concentrations in individual mossy fiber terminals in hippocampal slice were optically measured with the calcium indicator fura-2 while stimulating the mossy fiber pathway and recording excitatory postsynaptic potentials extracellularly. Short-term synaptic enhancement was accompanied by increased presynaptic residual calcium concentration. A 2-fold enhancement of transmitter release was accompanied by a 10-30 nM increase in residual calcium. Following induction of mossy fiber LTP, transiently elevated presynaptic calcium decayed to prestimulus levels, whereas enhancement of synaptic transmission persisted. Our results demonstrate that, despite an apparent strong sensitivity of synaptic enhancement to presynaptic residual calcium levels, sustained increases in presynaptic residual calcium levels are not responsible for the maintained synaptic enhancement observed during mossy fiber LTP.

Roles of glutamate receptors in long-term potentiation at hippocampal mossy fiber synapses

Article

Jul 1991
NEUROREPORT

We have investigated the roles of glutamate (Glu) receptors in the mechanism of long-term potentiation observed in rat hippocampal mossy fiber synapses (MF-LTP). The mossy fiber responses were almost completely suppressed by ionotropic Glu receptor (iGluR) antagonists both before and after the induction of LTP. However, tetanic stimulation produced robust LTP even when the synaptic transmission was blocked postsynaptically by iGluR antagonists. In contrast, when the transmission was blocked presynaptically by Ca(2+)-free media, tetanic stimulation produced no LTP. D,L-2-amino-3-phosphono-propionate(D,L-AP3), a metabotropic Glu receptor (mGluR) antagonist, inhibited the induction of MF-LTP. Perfusion with ibotenate, a mGluR agonist, induced long-lasting enhancement of the mossy fiber responses without tetanic stimulation, and this ibotenate-induced potentiation was antagonized by D,L-AP3.

Long-term potentiation induced by a sustained rise in intraterminal Ca2+ in bull frog sympathetic ganglia

Article

May 1991

1. The mechanism of a long-term potentiation of transmitter release (pre-LTP) induced by a tetanic stimulation (33 Hz for 1-30 s) applied to the preganglionic nerve was examined by intracellularly recording the fast excitatory postsynaptic potentials (fast EPSPs) in bull-frog sympathetic ganglia. 2. Short-term facilitation induced by paired pulses was decreased during the course of pre-LTP; the extent of reduction paralleled with the magnitude of pre-LTP. 3. The frequency of miniature EPSPs increased after tetanic stimulation that produced the pre-LTP. 4. The Ca2+ ionophore, A23187, increased both the amplitude and quantal content of fast EPSPs and frequency of miniature EPSPs while it decreased short-term facilitation. 5. A Ca2+ chelating agent, Quin-2, loaded as acetoxymethyl ester, reduced the amplitude and quantal content of fast EPSPs and short-term facilitation, and blocked the generation of pre-LTP. 6. Activators of protein kinase C, phorbol 12,13-dibutyrate and 1-oleoyl-2-acetyl-rac-glycerol, and its inhibitors, H-7 and staurosporine, did not block the generation of pre-LTP, while the activators enhanced transmitter release. 7. Inhibitors of calmodulin, trifluoperazine and W-7, blocked the generation of pre-LTP, whereas the amplitude and quantal content of fast EPSPs were not influenced. 8. These results suggest that the pre-LTP results from a sustained rise in the basal level of intraterminal Ca2+ and an activation of the Ca(2+)-calmodulin-dependent process in the preganglionic nerve terminals.

Separate mechanisms of long-term potentiation in two input systems to CA3 pyramidal neurons of rat hippocampal slices as revealed by the whole-cell patch-clamp technique

Article

Dec 1991
NEUROSCI RES

Excitatory synaptic transmission from two input systems to hippocampal CA3 pyramidal neurons was investigated by the whole-cell patch-clamp technique for thin slice preparation, with special reference to long-term potentiation (LTP) in these systems. Excitatory postsynaptic currents (EPSCs) evoked by fimbrial stimulation consisted of two components; one was blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and the other was persistent at depolarized membrane potentials and blocked by D-2-amino-5-phosphonovalerate (D-AP5). The contribution of the D-AP5-sensitive component to EPSCs evoked by stimulation of mossy fibers was much less than that to fimbrial EPSCs. High-frequency stimulation of afferent fibers, under current-clamp conditions, elicited LTP. Bath application of D-AP5 blocked the induction of LTP in the fimbrial but not in the mossy fiber synapses. Induction of fimbrial LTP was completely blocked by 10 mM BAPTA applied intracellularly. In contrast, mossy fiber LTP was not blocked by 10 mM BAPTA. Furthermore, mossy fiber LTP, but not fimbrial LTP, was elicited by high-frequency stimulation under voltage-clamp (-80 mV) conditions. These results suggest that activation of NMDA receptors, increase in postsynaptic [Ca2+]i, and postsynaptic membrane depolarization are required for the induction of fimbrial but not for mossy fiber LTP.

Distribution of mRNA for the calmodulin-sensitive adenylate cyclase in rat brain: Expression in areas associated with learning and memory

Article

Apr 1991
NEURON

The Drosophila learning mutant, rutabaga, is deficient in the calmodulin-sensitive adenylate cyclase, and studies of associative learning in Aplysia have implicated this enzyme in neuroplasticity. Therefore, the distribution of mRNA encoding the calmodulin-sensitive adenylate cyclase in rat brain was examined by in situ hybridization. mRNA for this enzyme is expressed in specific areas of brain that have been implicated in learning and memory, including the neocortex, the hippocampus, and the olfactory system. The presence of mRNA for this enzyme in the pyramidal and granule cells of the hippocampal formation provides evidence that it is found in neurons. These data are consistent with the proposal that the calmodulin-sensitive adenylate cyclase plays an important role in learning and memory.

Zalutsky, R. A. & Nicoll, R. A. Comparison of two forms of long-term potentiation in single hippocampal neurons. Science 248, 1619-1624

Article

Jul 1990

In invertebrate nervous systems, some long-lasting increases in synaptic efficacy result from changes in the presynaptic cell. In the vertebrate nervous system, the best understood long-lasting change in synaptic strength is long-term potentiation (LTP) in the CA1 region of the hippocampus. Here the process is initiated postsynaptically, but the site of the persistent change is unresolved. Single CA3 hippocampal pyramidal cells receive excitatory inputs from associational-commissural fibers and from the mossy fibers of dentate granule cells and both pathways exhibit LTP. Although the induction of associational-commissural LTP requires in the postsynaptic cell N-methyl-D-aspartate (NMDA) receptor activation, membrane depolarization, and a rise in calcium, mossy fiber LTP does not. Paired-pulse facilitation, which is an index of increased transmitter release, is unaltered during associational-commissural LTP but is reduced during mossy fiber LTP. Thus, both the induction and the persistent change may be presynaptic in mossy fiber LTP but not in associational-commissural LTP.

Mossy fiber Potentiation and long-term potentiation involve different expression mechanisms

Article

Jan 1990

Cyclic adenosine 3',5'-monophosphate mediates beta-receptor actions of noradrenaline in rat hippocampal pyramidal cells

Article

Apr 1986

Intracellular recordings were made from rat hippocampal CA1 pyramidal neurones in the in vitro slice preparation to study the actions of cyclic adenosine 3',5'-monophosphate (cyclic AMP). Application of the membrane permeant analogue of cyclic AMP, 8-Br cyclic AMP caused a small depolarization of the resting membrane potential accompanied by an increase in membrane input resistance and also reduced the amplitude of depolarization-evoked calcium-activated potassium after-hyperpolarizations (a.h.p.s.). 8-Br cyclic AMP reduced calcium-activated a.h.p.s but did not reduce calcium action potentials in these cells. 8-Br cyclic AMP also reduced action potential frequency accommodation. The effects of 8-Br cyclic AMP were not mimicked by cyclic AMP applied extracellularly but were imitated by intracellular injections of cyclic AMP. Activation of the endogenous adenylate cyclase of pyramidal cells either by intracellular injection of the stable guanosine 5'-triphosphate (GTP) analogue guanylyl-imidodiphosphate, or by extracellular application of forskolin, reduced the a.h.p. and accommodation. Reducing phosphodiesterase activity with application of either 3-isobutyl-1-methylxanthine or Ro20-1724 reduced the amplitude of the a.h.p. and potentiated the a.h.p.-blocking action of noradrenaline. Reducing adenylate cyclase activity by application of SQ22,536 slightly increased the amplitude of the (a.h.p.) and reduced the a.h.p.-blocking action of noradrenaline. We conclude that the beta-receptor actions of NA on hippocampal CA1 pyramidal cells are mediated by intracellularly produced cyclic AMP.

Long-term potentiation of transmitter release induced by adrenaline in bull-frog sympathetic ganglia

Article

Jun 1986

Long-term potentiation (l.t.p.) of transmitter release induced by adrenaline in bull-frog sympathetic ganglia was studied using intracellular recording techniques. The quantal content of the fast excitatory post-synaptic potentials (fast e.p.s.p.s: evoked by the nicotinic action of acetylcholine) was potentiated for more than several hours after treatment with adrenaline (1-100 μM). A similar l.t.p. of quantal content was produced consistently by isoprenaline (10 μM) and only in a certain fraction of cells by dopamine (10 μM). The l.t.p. induced by adrenaline (10 μM) was blocked by a β-antagonist, propranolol (1 μM), but not by an α-antagonist, phenoxybenzamine (1 μM). Dibutyryl adenosine 3',5'-phosphate (dibutyryl cyclic AMP) (0.8-1.0 mM), adenosine 3',5'-phosphate (cyclic AMP) (4 mM), 3-isobutyl-1-methylxanthine (10 μM), caffeine (1-2 gM), and cholera toxin (2 μm ml-1) applied for 20-30 min, all caused the l.t.p. of quantal content. By contrast, adenosine 5'-phosphate (AMP) (4 mM) and adenosine (4 mM) had no potentiating action. Treatment of the ganglion with adrenaline (2.5-160 μM) or dibutyryl cyclic AMP (4 mM) for 15-30 min resulted in the l.t.p. of the frequency of miniature e.p.s.p.s. The l.t.p. of quantal content induced by adrenaline was markedly suppressed by lowering temperature from 20-25°C to 11-13°C, and blocked by dibutyryl guanosine 3',5'-phosphate (dibutyryl cyclic GMP) (100 μM) consistently when applied together, but inconsistently when given after adrenaline. The post-synaptic sensitivity to acetylcholine was unchanged for at least 1 h after exposure to adrenaline (2.5-160 μM) or dibutyryl cyclic AMP (0.8-4 mM). It can be concluded that adrenaline produces l.t.p. of transmitter release by activating a cyclic-AMP-dependent metabolic process through the activation of β-adrenoceptors, and that this mechanism is presumably regulated by a process involving endogenous guanosine 3',5'-phosphate (cyclic GMP).

Long-term potentiation of hippocampal mossy fiber synapses is blocked by postsynaptic injection of calcium chelators

Article

Dec 1989
NEURON

The role of intracellular calcium in an APV-insensitive form of long-term potentiation (LTP) has been studied at the hippocampal mossy fiber synapse. Intracellular calcium was buffered by iontophoretic injection of either BAPTA or QUIN-2, into CA3 pyramidal neurons. The slow calcium-dependent after hyperpolarization was used as an indicator of buffering. LTP was elicited in control and in APV-treated cells (6/6 and 4/5 cell, respectively). In contrast, LTP was observed in only 2/9 BAPTA-loaded cells and in 1/4 QUIN-2-loaded cells. The magnitude of LTP for control and APV-treated cells were not significantly different, but both groups showed significantly greater LTP than BAPTA-loaded cells. These results suggest that an increase in postsynaptic calcium is required for the induction of mossy fiber LTP.

Short-Term Synaptic Plasticity

Article

Feb 1989

Robert S Zucker

Forskolin: a specific stimulator of adenylyl cyclase or a diterpene with multiple sites of action?

Article

Dec 1989
TRENDS PHARMACOL SCI

Forskolin, a naturally occurring diterpene, directly stimulates adenylyl cyclase and has been used extensively to increase cAMP and to elicit cAMP-dependent physiological responses. More recently, forskolin has been shown to inhibit a number of membrane transport proteins and channel proteins through a mechanism that does not involve the production of cAMP. Many of these channel proteins are predicted to have similar topographies in the membrane bilayer and it is tempting to speculate that forskolin may be binding at structurally homologous sites. Kenneth Seamon and colleagues discuss the cAMP-independent effects of forskolin and the structural similarity between forskolin and other physiologically important substances such as hexoses and steroids with respect to potential forskolin binding sites.

Noradrenergic enhancement of long-term potentiation at mossy fiber synapses in the hippocampus

Article

Mar 1988

1. We tested several hypotheses related to the modulation of long-term potentiation (LTP) by norepinephrine (NE) at the mossy fiber synapses in the rat hippocampal slice preparation using extracellular and intracellular recording techniques. 2. NE exerted frequency-dependent effects on mossy fiber synaptic transmission. It had little effect on extracellular population excitatory postsynaptic potentials (pEPSPs) sampled during low-frequency stimulation, whereas it had marked effects on the duration, magnitude, and probability of induction of LTP at these synapses. 3. The beta-adrenoceptor agonist isoproterenol mimicked all of the effects of NE, whereas the beta-adrenoceptor antagonists propranolol and timolol reversibly blocked the induction of LTP, suggesting the effects of NE are mediated by a beta-adrenoceptor and that beta-adrenoceptor activation may be an important constituent for the expression of LTP at these synapses. 4. Frequency-dependent effects of NE and isoproterenol on mossy fiber pEPSPs were also observed in the presence of the gamma-aminobutyric acid (GABA) antagonist, picrotoxin, suggesting that NE can enhance LTP by a mechanism that does not depend on intact inhibition. However, propranolol did not block LTP in these disinhibited slices and did not affect LTP magnitude. 5. The adenylate cyclase activator forskolin augmented pEPSPs sampled during low-frequency stimulation in disinhibited slices and significantly enhanced LTP. Forskolin, however, did not produce LTP in the absence of tetanic stimulation. This supports the hypothesis that NE and isoproterenol augment features of LTP by stimulating adenosine 3',5'-cyclic monophosphate (cAMP) production and that cAMP plays a modulatory role in the induction of LTP. 6. The postsynaptic injection of the cAMP analogue 8-bromoadenosine 3',5'-cyclic monophosphate (8-bromo-cAMP) significantly increased the probability of induction of LTP measured intracellularly under voltage-clamp conditions with intact inhibition. An analysis of the inhibitory synaptic slope conductance during these experiments indicated that changes in this measure could neither account for the increase in mossy fiber synaptic slope conductance in those cells that displayed it nor account for the group differences in this variable. 7. The amplitude and duration of the postsynaptic depolarization during tetanic stimulation in the cells that displayed LTP in the 8-bromo-cAMP-injected group were significantly greater than in the cells that did not display LTP in the adenosine 5'-monophosphate-injected group.(ABSTRACT TRUNCATED AT 400 WORDS)

Intracellular Injection of cAMP Induces a Long-Term Reduction of Neuronal K + Currents

Article

Jul 1988

Intracellular signals that trigger long-term (24-hour) changes in membrane currents in identified neurons of Aplysia have been examined in order to understand the cellular mechanisms underlying long-term sensitization. Adenosine 3',5'-monophosphate (cAMP) was directly injected into individual sensory neurons to mimic the effects of sensitization training at the single cell level. Potassium currents of these cells were reduced 24 hours after injection of cAMP; these currents were similar to those reduced 24 hours after behavioral sensitization. These results suggest that cAMP is part of the intracellular signal that induces long-term sensitization in Aplysia.

Long-term potentiation at nicotinic synapses in the rat superior cervical ganglion

Article

Nov 1988

1. Nicotinic fast excitatory postsynaptic potentials (fast EPSPs) were recorded intracellularly from postganglionic neurones in the isolated rat superior cervical ganglion. 2. An hours-long potentiation of the fast EPSP could be induced by brief tetanic stimulation of the preganglionic nerve (5 Hz for 5 s to 20 Hz for 20 s). While long-term potentiation (LTP) can be detected in every ganglion by extracellular techniques, LTP was induced in only two-thirds of the nicotinic synaptic responses. 3. Muscarinic blockade with atropine did not prevent LTP of the fast EPSP. 4. LTP of the fast EPSP did not correlate with changes in input resistance nor cell potential, as recorded in the soma. 5. The formation of nicotinic LTP appeared to depend upon stimulation of the nerve terminals. Non-synaptic tetanic depolarization of the postganglionic neurone, effected by injecting depolarizing current pulses through the intracellular microelectrode, was not sufficient. LTP could be induced by synaptic tetani in two-thirds of the same neurones. 6. The response to exogenous 1,1-dimethyl-4-phenylpiperazinium (DMPP), a selective nicotinic agonist, was not increased during nicotinic synaptic LTP. This was true whether DMPP was applied by pressure-ejection from an extracellular micropipette during intracellular recording, or by brief superfusion during sucrose-gap recording of postganglionic responses. 7. Responses to exogenous acetylcholine and carbachol were increased during nicotinic LTP when these non-selective cholinergic agonists were applied by pressure-ejection during intracellular recording. However, the potentiation of the fast EPSP was always at least twofold greater than the potentiation of the response to these exogenous agonists. 8. Potentiation of the responses to acetylcholine and carbachol may have been due to long-term enhancement of muscarinic responses. Thus, no postsynaptic basis for nicotinic LTP was uncovered in these studies.

Neurophysiology and pharmacology of long-term potentiation in the rat sympathetic ganglion

Article

Mar 1985

Brief tetanic stimulation of the preganglionic nerve induced a persistent potentiation of nicotinic synaptic transmission in the rat superior cervical sympathetic ganglion. Quantitative measurements of the post-tetanic increase in synaptic efficacy revealed two distinct time courses. The early, rapidly decaying component, termed post-tetanic potentiation (p.t.p.), had a decay time constant of 2-3 min, as reported elsewhere. The duration of the more persistent component, called long-term potentiation (l.t.p.), was extremely temperature dependent, lasting much longer at 32 degrees C than at 22 degrees C. In half of the experiments performed at 32 degrees C, l.t.p. showed no detectable decay over the course of 1 h or more after a brief tetanic stimulation. Other experiments were conducted at 22 degrees C. The induction of l.t.p. was dependent on the extracellular [Ca2+]. Transient elevation of the extracellular [K+] also produced a long-term enhancement of synaptic efficacy, and this effect was also Ca2+ dependent. The tetani that were effective in inducing l.t.p. (5-20 Hz for 5-20 s) were well within the physiological range of preganglionic activity. The magnitude and time course were related to frequency and duration of stimulation. The occurrence of l.t.p. was restricted to those preganglionic fibres that were tetanically stimulated. This lack of heterosynaptic or generalized effects was demonstrated by splitting the preganglionic nerve into two branches that could be independently tested and conditioned. Physiological activation of muscarinic or nicotinic receptors apparently does not play an essential role in causing ganglionic l.t.p., which is expressed as an enhancement of nicotinic transmission. A muscarinic antagonist (2 microM-atropine) did not block l.t.p. Preganglionic stimulation induced l.t.p. even when a high concentration of a nicotinic antagonist (3 mM-hexamethonium) was present during the tetanic stimulation. Furthermore, bath application of a cholinergic agonist (100-1000 microM-carbachol) could not substitute for tetanic stimulation in provoking l.t.p. Activation of adrenergic receptors also appeared not to play an essential role. Neither a beta-adrenergic antagonist (10 microM-sotolol or 1 microM-propranolol) nor an alpha-adrenergic antagonist (1 microM-phentolamine) had any significant effect on the magnitude or duration of l.t.p. The results indicate that ganglionic l.t.p. is a Ca2+- and temperature-dependent process that can be created independently of the activation of nicotinic, muscarinic or adrenergic receptors.

Mapping Second Messenger Systems in the Brain: Differential Localizations of Adenylate Cyclase and Protein Kinase C

Article

Jul 1986

[3H]Forskolin and [3H]phorbol 12,13-dibutyrate have been used to map the adenylate cyclase and phosphatidylinositol systems respectively in brain slices by light-microscopic autoradiography. [3H]Forskolin binding to brain sections is displaced potently by forskolin (KD approximately equal to 15 nM) and is enhanced by fluoride and GTP analogs, agents which activate the stimulatory GTP-binding regulatory protein of adenylate cyclase, Gs. Highest [3H]forskolin binding occurs in the corpus striatum, substantia nigra, hippocampus, and molecular layer of the cerebellum. Lesion studies demonstrate that binding sites in the substantia nigra are associated with striatal afferents, while hippocampal sites are localized to granule cell dendrites and mossy fiber terminals, and the intense binding in the cerebellar molecular layer is largely associated with granule cell axons and terminals. Protein kinase C mediates the activity of hormones and neurotransmitters, which act through the phosphatidylinositol cycle, and is labeled with high affinity by [3H]phorbol 12,13-dibutyrate. At many synapses, maps of adenylate cyclase and protein kinase C reveal reciprocal distributions, which may have implications for second messenger regulation of synaptic transmission.

Long-Term Potentiation of Guinea Pig Mossy Fiber Responses Is Not Blocked by N-Methyl D-Aspartate Antagonists

Article

Oct 1986
NEUROSCI LETT

Receptors preferentially activated by the excitatory amino acid N-methyl-D-aspartate (NMDA) do not mediate synaptic transmission in the hippocampus but are involved in initiating long-term potentiation (LTP) in hippocampal region CA1. We have examined the role of NMDA receptors in LTP of the commissural/associational and mossy fiber pathways to region CA3 pyramidal neurons. In the commissural/associational pathway, NMDA receptor blockers did not reduce synaptic responses but reversibly blocked the induction of LTP. In contrast, NMDA receptor blockers had no effect on mossy fiber LTP. These results suggest that induction of commissural/associational LTP differs from mossy fiber LTP, although the mechanisms underlying expression of LTP along these pathways could be similar. Kynurenate and L-2-amino-4-phosphonobutyrate, which potently reduce mossy fiber responses, also did not block induction of mossy fiber LTP.

K-252 compounds, novel and potent inhibitors of protein kinase C and cyclic nucleotide-dependent protein kinases

Article

Feb 1987

K-252 compounds (K-252a and b isolated from Nocardiopsis sp. (1) and their synthetic derivatives) were found to inhibit cyclic nucleotide-dependent protein kinases and protein kinase C to various extents. The inhibitions were of the competitive type with respect to ATP. K-252a was a non-selective inhibitor for these three protein kinases with Ki values 18-25 nM. K-252b showed a comparable potency for protein kinase C (Ki, 20nM), whereas inhibitory potencies for cyclic nucleotide-dependent protein kinases were reduced. KT5720 and KT5822 selectively inhibited cAMP-dependent (Ki, 60nM) and cGMP-dependent (Ki, 2.4nM) protein kinases, respectively.

Forskolin: Its Biological and Chemical Properties

Article

Feb 1986

Neurogenetic Dissection Of Learning And Short-Term Memory In Drosophila

Article

Feb 1988

Yadin Dudai

N6-[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)ethyl]adenosine and its uronamide derivatives. Novel adenosine agonists with both high affinity and high selectivity for the adenosine A2 receptor

Article

Aug 1988

The role of Ca2+ in neuromuscular facilitation

Article

Apr 1968

1. The hypothesis is put forward that a residue of the ‘active calcium’ which enters the terminal axon membrane during the nerve impulse is responsible for short‐term facilitation. 2. This suggestion has been tested on the myoneural junction by varying the local calcium concentration so that during the first of two nerve impulses [Ca] o is either much lower than, or raised to a level approaching that, during the second impulse. Facilitation is much larger in the latter case, which is in accordance with the ‘calcium hypothesis’. 3. A short pulse of depolarization focally applied to the junction is followed by a brief period of very intense facilitation. This can be seen in the tetrodotoxin‐treated preparation, e.g. by lengthening the depolarization from 1 to 2 msec which can cause a more than fifty‐fold increase in transmitter release. This large ‘early facilitation’ (which presumably occurs also during the course of a normal action potential) is discussed in relation to the ‘calcium hypothesis’.

The relation between quantum content and facilitation at the neuromuscular junction of the frog

Article

Jul 1968

1. Experiments were done on the neuromuscular junction of the frog to study facilitation of the end‐plate potential (e.p.p.) produced by one or more conditioning shocks applied to the motor nerve. Neuromuscular transmission was blocked with Mg (+)‐tubocurarine chloride. 2. In Mg‐blocked preparations, facilitation increased with increasing Mg concentrations in the range 14‐34 m M . The increase in facilitation was related specifically to the decrease in mean quantum content of the e.p.p., and was most marked with quantum contents less than 10. 3. Facilitation in curarized preparations was similar in magnitude to that in preparations blocked with the lower Mg concentrations. Interaction between facilitation and depression in curarized preparations was consistent with the idea that facilitation was due to an increase in probability of transmitter release and depression to depletion of transmitter.

Malenka, R. C. & Nicoll, R. A. NMDA-receptor-dependent synaptic plasticity: multiple forms and mechanisms. Trends Neurosci. 16, 521-527

Article

Jan 1994
TRENDS NEUROSCI

Long-term potentiation in the CA1 region of the hippocampus is the most extensively studied model of activity-dependent synaptic plasticity in the mammalian brain. Its induction normally involves activation of postsynaptic N-methyl-D-aspartate (NMDA) receptors, which are thought to control the occurrence of long-term potentiation at individual synapses. Recent work in the hippocampus indicates that NMDA receptor activation does not necessarily lead to induction of long-term potentiation but instead may elicit a repertoire of distinct forms of synaptic plasticity including short-term potentiation or long-term depression. Furthermore, mechanisms exist such that the induction of long-term potentiation can be inhibited by modest activation of NMDA receptors. Experimental results are beginning to clarify the mechanistic relationships between these different phenomena, although much remains unknown. Whatever their underlying mechanisms, these additional forms of NMDA-receptor-dependent synaptic plasticity confer increased flexibility to neural circuits involved in information processing and storage.

Effects of selective inhibition of protein kinase C, cyclic AMP-dependent protein kinase, and Ca2+-calmodulin-dependent protein kinase on neurite development in cultured rat hippocampal neurons

Article

Jul 1993

A variety of experimental evidence suggests that calmodulin and protein kinases, especially protein kinase C, may participate in regulating neurite development in cultured neurons, particularly neurite initiation. However, the results are somewhat contradictory. Further, the roles of calmodulin and protein kinases on many aspects of neurite development, such as branching or elongation of axons vs dendrites, have not been extensively studied. Cultured embryonic rat hippocampal pyramidal neurons develop readily identifiable axons and dendrites. We used this culture system and the new generation of highly specific protein kinase inhibitors to investigate the roles of protein kinases and calmodulin in neurite development. Neurons were cultured for 2 days in the continuous presence of calphostin C (a specific inhibitor of protein kinase C), KT5720 (inhibitor of cyclic AMP‐dependent protein kinase), KN62 (inhibitor of Ca ²⁺ ‐calmodulin‐dependent protein kinase II), or calmidazolium (inhibitor of calmodulin), each at concentrations from approximately 1 to 10 times the concentration reported in the literature to inhibit each kinase by 50%. The effects of phorbol 12‐myristate 13‐acetate (an activator of protein kinase C) and 4α‐phorbol 12,13‐didecanoate (an inactive phorbol ester) were also tested. At concentrations that had no effect on neuronal viability, calphostin C reduced neurite initiation and axon branching without significantly affecting the number of dendrites per neuron, dendrite branching, dendrite length, or axon length. Phorbol 12‐myristate 13‐acetate increased axon branching and the number of dendrites per cell, compared to the inactive 4α‐phorbol 12,13‐didecanoate. KT5720 inhibited only axon branching. KN62 reduced axon length, the number of dendrites per neuron and both axon and dendrite branching. At low concentrations, calmidazolium had no effect on any aspect of neurite development, but at high concentrations, calmidazolium inhibited every parameter that was measured (including viability). These results suggest that these three protein kinases selectively modulate different aspects of neurite development. The universality of effects caused by calmodulin inhibition make it impossible to determine if there are specific targets of calmodulin action involved in neurite development. Finally, our data indicate that some superficially similar characteristics of neuronal differentiation, such as neurite initiation and branching, may be controlled by quite different molecular mechanisms.

Modulation of synaptic transmission and LTP effects on paired pulse facilitation and EPSP variance in the CA1 region of the hippocampus

Article

Nov 1993

1. Whole-cell patch-clamp recordings of excitatory postsynaptic currents (EPSCs) were made from guinea pig hippocampal CA1 pyramidal cells. The sensitivity of paired pulse facilitation (PPF) and EPSC variance to changes in synaptic transmission was investigated and the results were compared with the changes in these parameters evoked by long-term potentiation (LTP). 2. Presynaptic manipulations, such as activation of presynaptic gamma-aminobutyric acid-B receptors by baclofen, blockade of presynaptic adenosine receptors by theophylline, blockade of presynaptic potassium channels by cesium, and increasing the Ca(2+)-Mg2+ ratio in the external recording solution, each reliably changed PPF in a fashion reciprocal to the change in the EPSC amplitude. However, recruitment of additional synaptic release sites by increasing stimulus strength and antagonism of non-N-methyl-D-aspartate (NMDA) glutamate receptors by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) failed to alter PPF. 3. Presynaptic manipulations including increased stimulus strength gave the predicted changes in the value of mean 2/variance (M2/sigma 2). Moreover, postsynaptic manipulations that altered EPSC amplitude, including blockade of non-NMDA receptors by CNQX, or changing the holding potential of the postsynaptic cell, gave little change in M2/sigma 2, as would be predicted for manipulations resulting in a uniform postsynaptic change. 4. LTP caused no change in PPF, whereas the presynaptic manipulations, which caused a similar amount of potentiation to that induced by LTP, significantly decreased PPF. On the other hand, LTP did increase M2/sigma 2, although the increase was less than that predicted for a purely presynaptic mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)

Phosphorylation and modulation of recombinant GluR6 glutamate receptors by cAMP-dependent protein kinase

Article

Mar 1993

Glutamate-gated ion channels mediate most excitatory synaptic transmission in the central nervous system and play crucial roles in synaptic plasticity, neuronal development and some neuropathological conditions. These ionotropic glutamate receptors have been classified according to their preferred agonists as NMDA (N-methyl-D-aspartate), AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate) and KA (kainate) receptors. On the basis of sequence similarity and pharmacological properties, the recently cloned glutamate receptor subunits have been assigned as components of NMDA (NMDAR1, 2A-D), AMPA (GluR1-4) and KA (GluR5-7, KA1, KA2) receptors. Protein phosphorylation of glutamate receptors by protein kinase C and cyclic AMP-dependent protein kinase (PKA) has been suggested to regulate their function, possibly playing a prominent role in certain forms of synaptic plasticity such as long-term potentiation and long-term depression. Here we report that the GluR6 glutamate receptor, transiently expressed in mammalian cells, is directly phosphorylated by PKA, and that intracellularly applied PKA increases the amplitude of the glutamate response. Site-specific mutagenesis of the serine residue (Ser 684) representing a PKA consensus site completely eliminates PKA-mediated phosphorylation of this site as well as the potentiation of the glutamate response. These results provide evidence that direct phosphorylation of glutamate receptors modulates their function.

Learning to Modulate Transmitter Release: Themes and Variations in Synaptic Plasticity

Article

Feb 1993

The role of Ca2+ channels in hippocampal mossy fiber synaptic transmission and long-term potentiation

Article

Mar 1994
NEURON

We have addressed the role of Ca2+ channels in mossy fiber synaptic transmission and long-term potentiation (LTP). Whereas the induction of mossy fiber LTP is entirely normal when synaptic transmission is blocked by the glutamate receptor antagonist kynurenate, LTP is blocked in the absence of extracellular Ca2+. These findings suggest that presynaptic Ca2+ entry is essential for mossy fiber LTP. Therefore, the role of different types of presynaptic Ca2+ channels in synaptic transmission and LTP was investigated. Mossy fiber responses were little affected by the L-type Ca2+ channel blocker nifedipine. They were blocked partially by omega-conotoxin-GVIA (N-type) and almost entirely by omega-agatoxin-IVA (P-type). None of these antagonists blocked mossy fiber LTP, nor was its expression associated with a change in sensitivity of synaptic transmission to either of the two toxins. These results, together with previous findings, suggest that the induction of mossy fiber LTP is critically dependent on the entry of Ca2+ into the presynaptic terminal to trigger a series of steps resulting in the long lasting enhancement of evoked glutamate release. Whereas P-type Ca2+ channels are of primary importance in mossy fiber synaptic transmission, both the induction and expression of mossy fiber LTP can occur in the absence of P-type (or N-type) Ca2+ channels.

Mediation of Hippocampal Mossy Fiber Long-Term Potentiation by Cyclic AMP

Abstract

No full-text available

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