Various Forms Of Synaptic Plasticity Research Articles

Phosphorylation of Munc18 by Protein Kinase C Regulates the Kinetics of Exocytosis

Protein phosphorylation by protein kinase C (PKC) has been implicated in the control of neurotransmitter release and various forms of synaptic plasticity. The PKC substrates responsible for phosphorylation-dependent changes in regulated exocytosis in vivo have not been identified. Munc18a is essential for neurotransmitter release by exocytosis and can be phosphorylated by PKC in vitro on Ser-306 and Ser-313. We demonstrate that it is phosphorylated on Ser-313 in response to phorbol ester treatment in adrenal chromaffin cells. Mutation of both phosphorylation sites to glutamate reduces its affinity for syntaxin and so acts as a phosphomimetic mutation. Unlike phorbol ester treatment, expression of Munc18 with this phosphomimetic mutation in PKC phosphorylation sites did not affect the number of exocytotic events. The mutant did, however, produce changes in single vesicle release kinetics, assayed by amperometry, which were identical to those caused by phorbol ester treatment. Furthermore, the effects of phorbol ester treatment on release kinetics were occluded in cells expressing phosphomimetic Munc18. These results suggest that the dynamics of vesicle release events during exocytosis are controlled by PKC directly through phosphorylation of Munc18 on Ser-313. Phosphorylation of Munc18 by PKC may provide a mechanism for the control of exocytosis and thereby synaptic plasticity.

Abstracts from the Proceedings of the 8th International Conference on Dopamine: A Key Signal in All OrgansJune 27–29, 2002Stockholm, Sweden

Calcyon and the Mechanism of D1/D5 Dopamine Receptor Dual Effector CouplingClara Bergson. Department of Pharmacology and Toxicology, Medical College of Georgia, Georgia, USA.Dopamine (DA) transmission is mediated by five G‐protein coupled receptors (GPCR) classified as either D1‐like (including the D1 and D5 subtypes), or D2‐like. The D1 subtype is the most abundant DA receptor in brain, and the importance of the D1‐like receptors in modulating motor, cognitive, and reward behavior is well‐established. In addition, D1‐like receptors modulate various forms of synaptic plasticity, including LTP and LTD.Agonist stimulatin of recombinant D1 and D5 DA receptors in heterologous systems results in cAMP accumulation due to coupling with Gs. The single transmembrane protein calcyon is one of several proteins recently found to profoundly alter neurotransmitter GPCR localization and function. Calycon physically associates with D1 receptors, enabling the typically G‐linked receptor to stimulate intracellular calcium (Ca2+i) release in heterologous expression systems following priming of endogenous Gq/11 coupled receptors.Endogenous D1‐like dopamine receptors may also modulate neocortical and hippocampal neuronal excitability and synaptic function via Ca2+—as well as cAMP‐dependent signaling, presumably as the result of interaction with calycon. A distinct population of neurons in primary cultures of hippocampus and neocortex was found to respond to D1/D5 agonists with a marked increase in (Ca2+i) levels. The responses were state‐dependent, requiring priming with agonists of Gq/11‐coupled glutamate, serotonin, muscarinic, and adrenergic receptors, or with high external K+ solution.However, similar D1/D5 receptor stimulated Ca2+ responses were not detectable in neurons derived from striatum, nor was increased cAMP levels sufficient to stimulate Ca2+i release. Recent work indicates that the mechanism by which calcyon interaction potentiates D1/D5 receptor Ca2+ signaling involves a shift in D1 receptors into a high agonist affinity state, as well as an increased abundance of calcyon and D1 receptors at the plasma membrane.

The molecular organization of cerebellar long-term depression.

Synaptic plasticity is an important cellular mechanism for the formation of memory in neuronal circuits of the brain. Research during the past two decades has revealed surprisingly complex signal-transduction processes that underlie various forms of synaptic plasticity. More than 30 molecules are involved in the induction of long-term depression (LTD) — a unique form of synaptic plasticity in the cerebellum. Here, I review recent data on these molecules, defining their roles as mediators or modulators, coincidence detectors or components of a self-regenerating circuit, and show how they are organized to form an efficient molecular machinery for LTD induction.

Synaptic versus extrasynaptic NMDA receptor signaling: CREB makes the difference

The NMDA receptor plays a key role during neuronal development and the induction of various forms of synaptic plasticity in the CNS, including long-term potentiation and long-term depression. However, prolonged activation of the same receptor under pathological conditions such as cerebral ischemia and traumatic injury causes neuronal cell death. What is the stimulus that triggers such a different cellular consequence? The NMDA receptor is highly permeable to Ca2? ions and it has been suggested that different sites and kinetics of Ca2? entry have an important role in triggering the two processes. Previous work demonstrated that Ca2? entry through L-type Ca2? channels promotes cell survival via the expression of CREB (cAMP response element-binding protein)-activated genes such as the gene encoding BDNF (brain-derived neurotrophic factor). By contrast, the exogenous application of glutamate inhibits CREB-dependent gene transcription and is neurotoxic to cells.

Blockade of nitric oxide synthesis reduces responding for cocaine self-administration during extinction and reinstatement

Nitric oxide is a gaseous neurotransmitter that plays a significant role in various forms of synaptic plasticity and may play a role in the behavioral effects of psychostimulant drugs and in cocaine addiction. The course of drug addiction consists of different phases. Relapse into drug-seeking behavior following a period of abstinence is believed to represent one of the major factors leading to the perpetuation of the addictive cycle. In this respect, experimental extinction procedures provide a measure of the motivational properties of drugs as reflected by the persistence of drug-seeking behavior in the absence of the drug and by the reinstatement of responding by non-contingent drug administration. Pretreatment with the nitric oxide synthase inhibitor l-NG-nitroarginine methyl ester ( l-NAME, 50 mg/kg IP twice daily for 4 days) impaired responding for cocaine self-administration when the drug was available and the increase of drug-seeking behavior upon abrupt cessation of cocaine availability observed in control rats was significantly reduced after treatment with l-NAME. In addition, the priming effect of a non-contingent injection of cocaine on extinguished cocaine self-administration was also diminished by the same treatment. The acquisition of cocaine self-administration, in contrast, was not affected by treatment with l-NAME. These observations lend further support to the hypothesis of the involvement of nitric oxide in cocaine addiction and extend previous findings to components of the cocaine addictive cycle associated with relapse.

Roles of serine/threonine phosphatases in hippocampal synaptic plasticity.

The regulation of glutamate-mediated excitatory neurotransmission has a critical role in many aspects of behaviour. Great effort has gone into understanding the signal transduction cascades and effectors recruited in these processes, and protein phosphorylation has been identified as an important element. Although initial research in the field focused on the activity-dependent activation of kinases and the kinase dependence of various forms of synaptic plasticity, it has become increasingly clear that phosphatases have an equally dynamic and critical role in the activity-dependent alterations of synaptic transmission. Here, we review the roles of serine/threonine phosphatases in synaptic plasticity.

Tissue plasminogen activator controls multiple forms of synaptic plasticity and memory.

Induction of long-term depression (LTD) in rat striatal slices revealed that this form of synaptic plasticity is coupled to an increased expression of tissue-plasminogen activator (t-PA) mRNA, as detected by the mRNA differential display technique. To further investigate the involvement of this gene in synaptic remodelling following striatal LTD, we recorded electrical activity from mice lacking the gene encoding t-PA (t-PA-KO) and from wild-type (WT) mice. Tetanic stimulation induced LTD in the large majority of striatal neurons recorded from WT mice. Conversely, LTD was absent in a significant proportion of striatal neurons obtained from mice lacking t-PA. Electrophysiological recordings obtained from hippocampal slices in the CA1 area showed that mainly the late phase of long-term potentiation (LTP) was reduced in t-PA-KO mice. Learning and memory-related behavioural abnormalities were also found in these transgenic mice. Disruption of the t-PA gene, in fact, altered both the context conditioning test, a hippocampus-related behavioural task, and the two-way active avoidance, a striatum-dependent task. In an open field object exploration task, t-PA-KO mice expressed deficits in habituation and reactivity to spatial change that are consistent with an altered hippocampal function. Nevertheless, decreased rearing and poor initial object exploration were also observed, further suggesting an altered striatal function. These data indicate that t-PA plays a critical role in the formation of various forms of synaptic plasticity and memory.

Roles of metabotropic glutamate receptors in LTP and LTD in

Metabotropic L-glutamate receptors are involved in various forms of synaptic plasticity in the hippocampus. The use of a new antagonist (LY341495) that blocks all known metabotropic L-glutamate receptors in the brain, together with subtype-selective antagonists, has identified multiple roles both for cloned and novel metabotropic L-glutamate receptors in hippocampal long-term potentiation and long-term depression.

Localization and subcellular distribution of N-copine in mouse brain.

N-Copine is a novel protein with two C2 domains. Its expression is brain specific and up-regulated by neuronal activity such as kainate stimulation and tetanus stimulation evoking hippocampal CA1 long-term potentiation. We examined the localization and subcellular distribution of N-copine in mouse brain. In situ hybridization analysis showed that N-copine mRNA was expressed exclusively in neurons of the hippocampus and in the main and accessory olfactory bulb, where various forms of synaptic plasticity and memory formation are known to occur. In immunohistochemical analyses, N-copine was detected mainly in the cell bodies and dendrites in the neurons, whereas presynaptic proteins such as synaptotagmin I and rab3A were detected in the regions where axons pass through. In fractionation experiments of brain homogenate, N-copine was associated with the membrane fraction in the presence of Ca2+ but not in its absence. As a GST-fusion protein with the second C2 domain of N-copine showed Ca2+-dependent binding to phosphatidylserine, this domain was considered to be responsible for the Ca2+-dependent association of N-copine with the membrane. Thus, N-copine may have a role as a Ca2+ sensor in postsynaptic events, in contrast to the known roles of "double C2 domain-containing proteins," including synaptotagmin I, in presynaptic events.

Potentiation of NMDA and AMPA responses by group I mGluR in spinal cord motoneurons

Application of the metabotropic glutamate receptor (mGluR) agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) and the Group I selective mGluR agonist (RS)-3,5-dihydroxyphenylglycine (DHPG) potentiated NMDA- and AMPA-induced potential changes recorded from ventral roots of the isolated hemisected baby rat spinal cord. Potentiation produced by 1S,3R-ACPD was completely abolished by the Group I selective mGluR antagonists (S)-4-carboxyphenylglycine (4CPG) or (+)-alpha-methyl-4-carboxyphenylglycine (MCPG). In addition, the protein kinase C (PKC) blockers staurosporine or chelerythrine chloride were able to antagonize the 1S,3R-ACPD-induced potentiation of both NMDA and AMPA response, suggesting that the enhancing effect induced by Group I mGluRs is modulated by a PKC-mediated mechanism. The mGluRs-induced potentiation of NMDA and AMPA responses may be important in modulating various forms of synaptic plasticity and nociceptive processes.

Cyclic AMP-dependent Protein Kinase and Protein Kinase C Phosphorylate N-Methyl-d-aspartate Receptors at Different Sites

Ca2+ influx through N-methyl-D-aspartate (NMDA)-type glutamate receptors plays a pivotal role in synaptic plasticity during brain development as well as in mature brain. Cyclic AMP-dependent protein kinase (PKA) and members of the protein kinase C (PKC) family are also essential for various forms of synaptic plasticity and regulate the activity of different ion channels including NMDA and non-NMDA receptors. We now demonstrate that PKA and various PKC isoforms phosphorylate the NMDA receptor in vitro. The stoichiometry of [32P]phosphate incorporation per [3H]MK-801 binding site is greater than 1 for both PKA and PKC. Double immunoprecipitation experiments show that all three NMDA receptor subunits that are prevalent in the cortical structures, NR1, NR2A, and NR2B, are substrates for PKA as well as PKC. Two-dimensional phosphopeptide mapping reveals that the major phosphorylation sites for PKA and PKC differ for all three subunits. We provide evidence that some if not most of these sites are phosphorylated in the central nervous system of rats in vivo. The results presented in this article together with earlier electrophysiological experiments demonstrating that PKA and PKC activation increases the activity of NMDA receptors indicate that NMDA receptor potentiation can be mediated by direct phosphorylation by PKA and PKC. Collectively, these results strongly suggest that NMDA receptor functions such as control of neuronal development or expression of synaptic plasticity are modulated by PKA- and PKC-mediated phosphorylation of NMDA receptors.

Postsynaptic action potentials do not alter short-term potentiation in the dentate gyrus

The dendrites of neocortical neurons have been shown to support active action potentials which back-propagate from the soma after an output spike has been initiated. This observation has led to speculation that dendritic action potentials may participate in various forms of synaptic plasticity. The contribution of dendritic spikes to paired-pulse facilitation (PPF), a form of short-term plasticity, was investigated in the dentate gyrus of hippocampal slices. Paired orthodromic stimulation of the perforant path produced an average facilitation of the test population spike (PS) amplitude of 167% ( n = 16, conditioning response = 100%). There was also a small but significant increase in slope of the field EPSP (fEPSP) of 108%. To determine whether increased presynaptic drive could account for this facilitation, the relationship between fEPSP slope and spike amplitude (I-O) was determined for a range of stimulus intensities. An increase in fEPSP slope of 171% was associated with an increase in PS amplitude equal to the facilitation produced by paired-pulse stimulation (167%), suggesting a postsynaptic component in PPF. Electric field effects were then used as a tool to alter the excitability of granule cells during the conditioning response without changing synaptic drive. Any change in the test response associated with manipulation of the conditioning population spike amplitude would suggest that dendritic spikes may contribute to the postsynaptic component of PPF. Surprisingly, altering the number of neurons responding to the conditioning stimulus with an action potential had no effect on the test response, suggesting that dendritic action potentials do not participate in this form of short-term synaptic plasticity.

Learning of a hippocampal-dependent conditioning task changes the binding properties of AMPA receptors in rabbit hippocampus

The N-methyl-D-asparate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) subtypes of glutamate receptors have been shown to play critical roles in various forms of synaptic plasticity (i.e., learning and memory, long-term potentiation). We previously demonstrated that the binding of [3H]AMPA to the AMPA subtype of glutamate receptors was selectively increased in hippocampus following classical conditioning of the rabbit nictitating membrane response in a delay paradigm. We report here that the same effect was observed in a variant of this learning paradigm that requires the participation of the hippocampus, i.e., trace conditioning of the rabbit nictitating membrane. The binding of [3H]TCP (N-[1-(2-thienyl)cyclo-hexyl]-3,4-piperidine) to the NMDA receptor remained unchanged in all the experimental groups tested. Paired presentations of conditioned and unconditioned stimuli resulted in an increased binding of [3H]AMPA, an agonist of the AMPA receptors, in several hippocampal subfields while the binding of an antagonist, [3H]CNQX (6-nitro-7-cyanoquinoxaline-2,3-dione), was decreased. The results suggest that the learning-induced changes in binding of the ligands to the AMPA receptor reflect changes in affinity of the receptor rather than in the number of sites. These results support the hypothesis that changes in hippocampal glutamate receptors are a corollary of synaptic plasticity in certain forms of learning.

Effect of Temperature and Calcium on the Binding Properties of the AMPA Receptor in Frozen Rat Brain Sections

The elucidation of the mechanisms regulating the properties of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) subtype of glutamate receptors is important for understanding glutamatergic transmission. Here we report that qualitative as well as quantitative analysis of tritiated ligand binding to the AMPA receptor on thin frozen rat brain tissue sections reveals the existence of several mechanisms regulating the binding properties of AMPA receptors. Preincubation of tissue sections at 35 degrees C results in a decreased amount of [3H]AMPA binding as compared to that measured following preincubation at 0 degrees C. The decrease in binding appears to be mainly localized to cell bodies as evaluated by autoradiography, and could be due to proteolysis. Preincubation with calcium at 35 degrees C produces increased levels of [3H]AMPA binding. The effect of calcium is mimicked by manganese and to a lesser extent by magnesium; it is concentration-dependent with a 50% effective concentration for calcium of approximately 150 microM, time-dependent and temperature-dependent. The calcium-induced increase in [3H]AMPA binding is different among various brain structures, being larger in area CA1 of the hippocampus and in the superficial layers of the cerebral cortex. The effect of calcium is partly reduced by preincubation with the calpain inhibitor leupeptin and potentiated by preincubation with purified calpain II. The calcium-induced increase in [3H]AMPA binding is associated with a decrease in the binding of an antagonist of AMPA receptors, [3H]6-nitro-7-cyanoquinoxaline-2,3-dione. The results indicate that the binding properties of the AMPA receptor are rapidly regulated by calcium-dependent processes, and possibly by calcium-dependent proteases. They suggest that modulation of the binding properties involves changes in the configuration of the receptor, producing opposite changes in the affinities of the receptor for agonists and antagonists. Finally, these results strengthen the hypothesis that changes in the properties of AMPA receptors might underlie various forms of synaptic plasticity.

Classical conditioning selectively increases AMPA receptor binding in rabbit hippocampus

The NMDA and AMPA receptors have been shown to play critical roles in various forms of synaptic plasticity (learning and memory, long-term potentiation). The present study investigated the involvement of these two receptors in a well-characterized classical conditioning paradigm. Following classical conditioning of the rabbit nictitating membrane the binding properties of these two subclasses of excitatory amino acid transmitter receptors were analyzed in dorsal hippocampi by quantitative autoradiography. [ 3H]TCP and [ 3H]AMPA were used to identify the NMDA and AMPA receptors, respectively. The binding of [ 3H]TCP to the NMDA receptor remained unchanged in all the experimental groups tested. Paired presentations of the conditioned and unconditioned stimuli resulted in increased [ 3H]AMPA binding to the AMPA receptor in several subfields of the hippocampus, while unpaired presentations had no significant effects. The increase in binding was due to an increased affinity of the low-affinity component of the AMPA receptor. The results support the hypothesis that changes in glutamate receptors participate in the synaptic plasticity involved in certain forms of learning.

Assembly of the cricket cercal sensory system: Genetic and epigenetic control

The cercal sensory system of the cricket is being examined using anatomical, physiological, and computer simulation techniques in order to better understand the assembly of sensory systems. This particular sensory system is of interest because it functions like numerically more complex vertebrate sensory systems but offers, to the neuroscientist, the technical advantages of a small number of large identified neurons. Two aspects of sensory processing are being examined in this system; the spatial aspects of a stimulus that tell an animal where a target is in its environment, and the qualities of a stimulus that help the animal to identify the stimulus. The spatial aspects of a stimulus are analyzed by a topographic mapping of the animal's sensory environment. The feature extraction machinery for other aspects of the stimulus lacks any obvious anatomical order and is embedded within the topographic map. We are attempting to tease apart the genetic and the epigenetic components of the assembly process for this sensory system. Here we review our progress with emphasis on the epigenetic aspects of its assembly. We describe previously published work on plasticity as well as new experiments focussed on the role of neuronal activity in the assembly of this neural circuit. Finally, we briefly describe simulation experiments that are helping us understand the role of various forms of synaptic plasticity in the determination of receptive fields.