Activation of receptors negatively coupled to adenylate cyclase is required for induction of long‐term synaptic depression at Schaffer collateral‐CA1 synapses

Chemical LTD (CLTD) of synaptic transmission is triggered by simultaneously increasing presynaptic [cGMP] while inhibiting PKA. Here, we supply evidence that class II, but not III, metabotropic glutamate receptors (mGluRs), and A1 adenosine receptors, both negatively coupled to adenylate cyclase, play physiologic roles in providing PKA inhibition necessary to promote the induction of LTD at Schaffer collateral‐CA1 synapses in hippocampal slices. Simultaneous activation of group II mGluRs with the selective agonist (2S,2′R,3′R)‐2‐(2′,3′‐dicarboxy‐cyclopropyl) glycine (DCGIV; 5 μM), while raising [cGMP] with the type V phosphodiesterase inhibitor, zaprinast (20 μM), resulted in a long‐lasting depression of synaptic strength. When zaprinast (20 μM) was combined with a cell‐permeant PKA inhibitor H‐89 (10 μM), the need for mGluR IIs was bypassed. DCGIV, when combined with a “submaximal” low frequency stimulation (1 Hz/400 s), produced a saturating LTD. The mGluR II selective antagonist, (2S)‐alpha‐ethylglutamic acid (EGLU; 5 μM), blocked induction of LTD by prolonged low frequency stimulation (1 Hz/900 s). In contrast, the mGluR III selective receptor blocker, (RS)‐a‐Cyclopropyl‐[3‐³H]‐4‐phosphonophenylglycine (CPPG; 10 μM), did not impair LTD. The selective adenosine A1 receptor antagonist, 1,3‐dipropyl‐8‐cyclopentylxanthine (DPCPX; 100 nM), also blocked induction of LTD, while the adenosine A1 receptor agonist N⁶‐cyclohexyl adenosine (CHA; 50 nM) significantly enhanced the magnitude of LTD induced by submaximal LFS and, when paired with zaprinast (20 μM), was sufficient to elicit CLTD. Inhibition of PKA with H‐89 rescued the expression of LTD in the presence of either EGLU or DPCPX, confirming the hypothesis that both group II mGluRs and A1 adenosine receptors enhance the induction of LTD by inhibiting adenylate cyclase and reducing PKA activity. © 2005 Wiley Periodicals, Inc. J Neurobiol, 2006     

Dendritic calcium accumulation regulates wind sensitivity via short‐term depression at cercal sensory‐to‐giant interneuron synapses in the cricket

An in vivo Ca²⁺ imaging technique was applied to examine the cellular mechanisms for attenuation of wind sensitivity in the identified primary sensory interneurons in the cricket cercal system. Simultaneous measurement of the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) and membrane potential of a wind‐sensitive giant interneuron (GI) revealed that successive air puffs caused the Ca²⁺ accumulation in dendrites and diminished the wind‐evoked bursting response in the GI. After tetanic stimulation of the presynaptic cercal sensory nerves induced a larger Ca²⁺ accumulation in the GI, the wind‐evoked bursting response was reversibly decreased in its spike number. When hyperpolarizing current injection suppressed the [Ca²⁺]_i elevation during tetanic stimulation, the wind‐evoked EPSPs were not changed. Moreover, after suprathreshold tetanic stimulation to one side of the cercal nerve resulted in Ca²⁺ accumulation in the GI's dendrites, the slope of EPSP evoked by presynaptic stimulation of the other side of the cercal nerve was also attenuated for a few minutes after the [Ca²⁺]_i had returned to the prestimulation level. This short‐term depression at synapses between the cercal sensory neurons and the GI (cercal‐to‐giant synapses) was also induced by a depolarizing current injection, which increased the [Ca²⁺]_i, and buffering of the Ca²⁺ rise with a high concentration of a Ca²⁺ chelator blocked the induction of short‐term depression. These results indicate that the postsynaptic Ca²⁺ accumulation causes short‐term synaptic depression at the cercal‐to‐giant synapses. The dendritic excitability of the GI may contribute to postsynaptic regulation of the wind‐sensitivity via Ca²⁺‐dependent depression. © 2001 John Wiley & Sons, Inc. J Neurobiol 46: 301–313, 2001     

Protease‐activated receptor‐1 modulates hippocampal memory formation and synaptic plasticity

Protease‐activated receptor‐1 (PAR1) is an unusual G‐protein coupled receptor (GPCR) that is activated through proteolytic cleavage by extracellular serine proteases. Although previous work has shown that inhibiting PAR1 activation is neuroprotective in models of ischemia, traumatic injury, and neurotoxicity, surprisingly little is known about PAR1's contribution to normal brain function. Here, we used PAR1?/? mice to investigate the contribution of PAR1 function to memory formation and synaptic function. We demonstrate that PAR1?/? mice have deficits in hippocampus‐dependent memory. We also show that while PAR1?/? mice have normal baseline synaptic transmission at Schaffer collateral‐CA1 synapses, they exhibit severe deficits in N‐methyl‐d ‐aspartate receptor (NMDAR)‐dependent long‐term potentiation (LTP). Mounting evidence indicates that activation of PAR1 leads to potentiation of NMDAR‐mediated responses in CA1 pyramidal cells. Taken together, this evidence and our data suggest an important role for PAR1 function in NMDAR‐dependent processes subserving memory formation and synaptic plasticity.     

Reduced SNAP‐25 alters short‐term plasticity at developing glutamatergic synapses

SNAP‐25 is a key component of the synaptic‐vesicle fusion machinery, involved in several psychiatric diseases including schizophrenia and ADHD. SNAP‐25 protein expression is lower in different brain areas of schizophrenic patients and in ADHD mouse models. How the reduced expression of SNAP‐25 alters the properties of synaptic transmission, leading to a pathological phenotype, is unknown. We show that, unexpectedly, halved SNAP‐25 levels at 13–14 DIV not only fail to impair synaptic transmission but instead enhance evoked glutamatergic neurotransmission. This effect is possibly dependent on presynaptic voltage‐gated calcium channel activity and is not accompanied by changes in spontaneous quantal events or in the pool of readily releasable synaptic vesicles. Notably, synapses of 13–14 DIV neurons with reduced SNAP‐25 expression show paired‐pulse depression as opposed to paired‐pulse facilitation occurring in their wild‐type counterparts. This phenotype disappears with synapse maturation. As alterations in short‐term plasticity represent a new mechanism contributing to cognitive impairments in intellectual disabilities, our data provide mechanistic clues for neuronal circuit alterations in psychiatric diseases characterized by reduced expression of SNAP‐25.     

Rab3a interacting molecule (RIM) and the tethering of pre‐synaptic transmitter release site‐associated CaV2.2 calcium channels

Biochemical and physiological evidence suggest that pre‐synaptic calcium channels are attached to the transmitter release site within the active zone by a molecular tether. A recent study has proposed that ‘Rab3a Interacting Molecule’ (RIM) serves as the tether for CaV2.1 channels in mouse brain, based in part on biochemical co‐immunoprecipitation (co‐IP) using a monoclonal antibody, mRIM. We previously argued against this idea for CaV2.2 calcium channel at chick synapses based on experiments using a different anti‐RIM antibody, pRIM1,2: while staining for the two proteins co‐localized and co‐varied at the transmitter release face, consistent with an association, they failed to co‐IP from a synaptosome membrane lysate. RIM is, however, a family of proteins and we tested the possibility that the mRIM antibody used in the more recent study identifies a particular channel‐tethering variant. We find that co‐immunostaining with mRIM and anti‐CaV2.2 antibody neither co‐localized nor co‐varied at the transmitter release face and the two proteins did not co‐IP, arguing against a common protein complex and a key CaV2.2 scaffolding role for RIM at the active zone. The differing results might be reconciled, however, in a model where a RIM family member contributes to a protein bridge that anchors the pre‐fusion secretory vesicle to the calcium channel protein complex.     

APOE genotype affects the pre‐synaptic compartment of glutamatergic nerve terminals

Apolipoprotein E (APOE) genotype affects outcomes of Alzheimer's disease and other conditions of brain damage. Using APOE knock‐in mice, we have previously shown that APOE‐ε4 Targeted Replacement (TR) mice have fewer dendritic spines and reduced branching in cortical neurons. As dendritic spines are post‐synaptic sites of excitatory neurotransmission, we used APOE TR mice to examine whether APOE genotype affected the various elements of the glutamate–glutamine cycle. We found that levels of glutamine synthetase and glutamate uptake transporters were unchanged among the APOE genotypes. However, compared with APOE‐ε3 TR mice, APOE‐ε4 TR mice had decreased glutaminase levels (18%, p < 0.05), suggesting decreased conversion of glutamine to glutamate. APOE‐ε4 TR mice also had increased levels of the vesicular glutamate transporter 1 (20%, p < 0.05), suggesting that APOE genotype affects pre‐synaptic terminal composition. To address whether these changes affected normal neurotransmission, we examined the production and metabolism of glutamate and glutamine at 4–5 months and 1 year. Using high‐frequency ¹³C/¹H nuclear magnetic resonance spectroscopy, we found that APOE‐ε4 TR mice have decreased production of glutamate and increased levels of glutamine. These factors may contribute to the increased risk of neurodegeneration associated with APOE‐ε4, and also act as surrogate markers for Alzheimer's disease risk.     

Surface dynamics of GluN2B‐NMDA receptors controls plasticity of maturing glutamate synapses

NMDA‐type glutamate receptors (NMDAR) are central actors in the plasticity of excitatory synapses. During adaptive processes, the number and composition of synaptic NMDAR can be rapidly modified, as in neonatal hippocampal synapses where a switch from predominant GluN2B‐ to GluN2A‐containing receptors is observed after the induction of long‐term potentiation (LTP). However, the cellular pathways by which surface NMDAR subtypes are dynamically regulated during activity‐dependent synaptic adaptations remain poorly understood. Using a combination of high‐resolution single nanoparticle imaging and electrophysiology, we show here that GluN2B‐NMDAR are dynamically redistributed away from glutamate synapses through increased lateral diffusion during LTP in immature neurons. Strikingly, preventing this activity‐dependent GluN2B‐NMDAR surface redistribution through cross‐linking, either with commercial or with autoimmune anti‐NMDA antibodies from patient with neuropsychiatric symptoms, affects the dynamics and spine accumulation of CaMKII and impairs LTP. Interestingly, the same impairments are observed when expressing a mutant of GluN2B‐NMDAR unable to bind CaMKII. We thus uncover a non‐canonical mechanism by which GluN2B‐NMDAR surface dynamics plays a critical role in the plasticity of maturing synapses through a direct interplay with CaMKII.     

Pre‐synaptic kainate receptor‐mediated facilitation of glutamate release involves PKA and Ca2+‐calmodulin at thalamocortical synapses

We have investigated the mechanisms underlying the facilitatory modulation mediated by kainate receptor (KAR) activation in the cortex, using isolated nerve terminals (synaptosomes) and slice preparations. In cortical nerve terminals, kainate (KA, 100 μM) produced an increase in 4‐aminopyridine (4‐AP)‐evoked glutamate release. In thalamocortical slices, KA (1 μM) produced an increase in the amplitude of evoked excitatory post‐synaptic currents (eEPSCs) at synapses established between thalamic axon terminals from the ventrobasal nucleus onto stellate neurons of L4 of the somatosensory cortex. In both, synaptosomes and slices, the effect of KA was antagonized by 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione, and persisted after pre‐treatment with a cocktail of antagonists of other receptors whose activation could potentially have produced facilitation of release indirectly. Mechanistically, the observed effects of KA appear to be congruent in synaptosomal and slice preparations. Thus, the facilitation by KA of synaptosomal glutamate release and thalamocortical synaptic transmission were suppressed by the inhibition of protein kinase A and occluded by the stimulation of adenylyl cyclase. Dissecting this G‐protein‐independent regulation further in thalamocortical slices, the KAR‐mediated facilitation of synaptic transmission was found to be sensitive to the block of Ca²⁺ permeant KARs by philanthotoxin. Intriguingly, the synaptic facilitation was abrogated by depletion of intracellular Ca²⁺ stores by thapsigargin, or inhibition of Ca²⁺‐induced Ca²⁺‐release by ryanodine. Thus, the KA‐mediated modulation was contingent on both Ca²⁺ entry through Ca²⁺‐permeable KARs and liberation of intracellular Ca²⁺ stores. Finally, sensitivity to W‐7 indicated that the increased cytosolic [Ca²⁺] underpinning KAR‐mediated regulation of synaptic transmission at thalamocortical synapses, requires downstream activation of calmodulin. We conclude that neocortical pre‐synaptic KARs mediate the facilitation of glutamate release and synaptic transmission by a Ca²⁺‐calmodulin dependent activation of an adenylyl cyclase/cAMP/protein kinase A signalling cascade, independent of G‐protein involvement.

     

The pre‐synaptic Munc13‐1 binds alcohol and modulates alcohol self‐administration in Drosophila

Munc13‐1 is a pre‐synaptic active‐zone protein essential for neurotransmitter release and involved in pre‐synaptic plasticity in brain. Ethanol, butanol, and octanol quenched the intrinsic fluorescence of the C1 domain of Munc13‐1 with EC₅₀s of 52 mM, 26 mM, and 0.7 mM, respectively. Photoactive azialcohols photolabeled Munc13‐1 C1 exclusively at Glu‐582, which was identified by mass spectrometry. Mutation of Glu‐582 to alanine, leucine, and histidine reduced the alcohol binding two‐ to five‐fold. Circular dichroism studies suggested that binding of alcohol increased the stability of the wild‐type Munc13‐1 compared with the mutants. If Munc13‐1 plays some role in the neural effects of alcohol in vivo, changes in the activity of this protein should produce differences in the behavioral responses to ethanol. We tested this prediction with a loss‐of‐function mutation in the conserved Dunc‐13 in Drosophila melanogaster. The Dunc‐13^P84200/+ heterozygotes have 50% wild‐type levels of Dunc‐13 mRNA and display a very robust increase in ethanol self‐administration. This phenotype is reversed by the expression of the rat Munc13‐1 protein within the Drosophila nervous system. The present studies indicate that Munc13‐1 C1 has binding site(s) for alcohols and Munc13‐1 activity is sufficient to restore normal self‐administration to Drosophila mutants deficient in Dunc‐13 activity.

     

Rapid bidirectional modulation of mRNA expression and export accompany long‐term facilitation and depression of Aplysia synapses

Serotonin (5‐HT) and the neuropeptide Phe‐Met‐Arg‐Phe‐amide (FMRFa) modulate synaptic efficacy of sensory neurons (SNs) of Aplysia in opposite directions and for long duration. Both long‐term responses require changes in mRNA and protein synthesis. The SN‐specific neuropeptide, sensorin A, is a gene product that appears to be increased by 5‐HT and decreased by FMRFa. We examined whether changes in sensorin A mRNA levels in the cell body and neurites of SNs accompany long‐term facilitation and depression. Both 5‐HT and FMRFa evoked rapid changes in sensorin A mRNA levels in the SN cell bodies: an increase with 5‐HT and a decrease with FMRFa. Parallel changes in sensorin A mRNA levels in SN neurites were detected 2 h and 4 h later. These rapid changes in mRNA expression and net export required the presence of the appropriate target motor cell L7. The neuromodulators failed to produce changes in mRNA expression or export when SNs were cultured alone or with the inappropriate target cell L11. The changes in mRNA expression were transient because mRNA levels returned to control values 24 h after treatment, while synaptic efficacy remained altered by the respective treatments. These results indicate that two neuromodulators produce distinct, but transient, target‐dependent effects on expression and export of a cell‐specific mRNA that correlate with changes in synaptic plasticity. © 2000 John Wiley & Sons, Inc. J Neurobiol 46: 41–47, 2001     

Jelly belly trans‐synaptic signaling to anaplastic lymphoma kinase regulates neurotransmission strength and synapse architecture

In Drosophila, the secreted signaling molecule Jelly Belly (Jeb) activates anaplastic lymphoma kinase (Alk), a receptor tyrosine kinase, in multiple developmental and adult contexts. We have shown previously that Jeb and Alk are highly enriched at Drosophila synapses within the CNS neuropil and neuromuscular junction (NMJ) and postulated a conserved intercellular signaling function. At the embryonic and larval NMJ, Jeb is localized in the motor neuron presynaptic terminal whereas Alk is concentrated in the muscle postsynaptic domain surrounding boutons, consistent with anterograde trans‐synaptic signaling. Here, we show that neurotransmission is regulated by Jeb secretion by functional inhibition of Jeb–Alk signaling. Jeb is a novel negative regulator of neuromuscular transmission. Reduction or inhibition of Alk function results in enhanced synaptic transmission. Activation of Alk conversely inhibits synaptic transmission. Restoration of wild‐type postsynaptic Alk expression in Alk partial loss‐of‐function mutants rescues NMJ transmission phenotypes and confirms that postsynaptic Alk regulates NMJ transmission. The effects of impaired Alk signaling on neurotransmission are observed in the absence of associated changes in NMJ structure. Complete removal of Jeb in motor neurons, however, disrupts both presynaptic bouton architecture and postsynaptic differentiation. Nonphysiologic activation of Alk signaling also negatively regulates NMJ growth. Activation of Jeb–Alk signaling triggers the Ras‐MAP kinase cascade in both pre‐ and postsynaptic compartments. These novel roles for Jeb–Alk signaling in the modulation of synaptic function and structure have potential implications for recently reported Alk functions in human addiction, retention of spatial memory, cognitive dysfunction in neurofibromatosis, and pathogenesis of amyotrophic lateral sclerosis. © 2012 Wiley Periodicals, Inc. Develop Neurobiol, 2013     

Improved spatial learning is associated with increased hippocampal but not prefrontal long‐term potentiation in mGluR4 knockout mice

Although much information about metabotropic glutamate receptors (mGluRs) and their role in normal and pathologic brain function has been accumulated during the last decades, the role of group III mGluRs is still scarcely documented. Here, we examined mGluR4 knockout mice for types of behavior and synaptic plasticity that depend on either the hippocampus or the prefrontal cortex (PFC). We found improved spatial short‐ and long‐term memory in the radial arm maze, which was accompanied by enhanced long‐term potentiation (LTP) in hippocampal CA1 region. In contrast, LTP in the PFC was unchanged when compared with wild‐type controls. Changes in paired‐pulse facilitation that became overt in the presence of the GABA_A antagonist picrotoxin indicated a function of mGluR4 in maintaining the excitation/inhibition balance, which is of crucial importance for information processing in the brain and the deterioration of these processes in neuropsychological disorders such as autism, epilepsy and schizophrenia .     

Altered synaptic plasticity and behavioral abnormalities in CNGA3‐deficient mice

The role of the cyclic nucleotide‐gated (CNG) channel CNGA3 is well established in cone photoreceptors and guanylyl cyclase‐D‐expressing olfactory neurons. To assess a potential function of CNGA3 in the mouse amygdala and hippocampus, we examined synaptic plasticity and performed a comparative analysis of spatial learning, fear conditioning and step‐down avoidance in wild‐type mice and CNGA3 null mutants (CNGA3^?/?). CNGA3^?/? mice showed normal basal synaptic transmission in the amygdala and the hippocampus. However, cornu Ammonis (CA1) hippocampal long‐term potentiation (LTP) induced by a strong tetanus was significantly enhanced in CNGA3^?/? mice as compared with their wild‐type littermates. Unlike in the hippocampus, LTP was not significantly altered in the amygdala of CNGA3^?/? mice. Enhanced hippocampal LTP did not coincide with changes in hippocampus‐dependent learning, as both wild‐type and mutant mice showed a similar performance in water maze tasks and contextual fear conditioning, except for a trend toward higher step‐down latencies in a passive avoidance task. In contrast, CNGA3^?/? mice showed markedly reduced freezing to the conditioned tone in the amygdala‐dependent cued fear conditioning task. In conclusion, our study adds a new entry on the list of physiological functions of the CNGA3 channel. Despite the dissociation between physiological and behavioral parameters, our data describe a so far unrecognized role of CNGA3 in modulation of hippocampal plasticity and amydgala‐dependent fear memory.     

Neuronal Ras activation inhibits adult hippocampal progenitor cell division and impairs spatial short‐term memory

A large number of endogenous and exogenous factors have been identified to upregulate and downregulate proliferation, differentiation and/or survival of newborn cells in the adult hippocampus. For studying neuronal mechanisms mediating the impact of those factors, we used a transgenic synRas mouse model expressing constitutively activated Valin12‐Harvey Ras selectively in differentiated neurons. BrdU injections showed significantly reduced proliferation of new cells within the adult hippocampus of transgenic animals compared with their wild‐type siblings. In contrast, the relative survival of newborn cells was increased in synRas mice, although this effect did not fully compensate for diminished proliferation. Inhibition of progenitor cell proliferation and enhancement of cellular survival were more pronounced in males compared with females. Double labelling and doublecortin immunostaining verified that specifically newborn neurons were decreased in synRas mice. Reduced cell generation was observed already 2 h after BrdU pulse injections, identifying an early precursor cell population as target of the inhibitory transgene effect. Differences in proliferation remained stable after 24 h and were specific for the subgranular zone of the dentate gyrus, as subventricular cell generation was not affected supporting a non‐cell autonomous effect on neural hippocampal progenitors. Transgene expression only starts with synaptic differentiation and therefore reduced proliferation must represent an indirect secondary consequence of synRas activity in differentiated neurons. This was associated with impaired spatial short‐term memory capacities as observed in a radial maze paradigm. Our data suggest that constantly high Ras activity in differentiated neurons downregulates hippocampal precursor cell generation in the neuronal lineage, but is modulated by sex‐dependent factors.     

An intracellular domain with a novel sequence regulates cell surface expression and synaptic clustering of leucine‐rich repeat transmembrane proteins in hippocampal neurons

Leucine‐rich repeat transmembrane proteins (LRRTMs) are single‐spanning transmembrane proteins that belong to the family of synaptically localized adhesion molecules that play various roles in the formation, maturation, and function of synapses. LRRTMs are highly localized in the post‐synaptic density; however, the mechanisms and significance of LRRTM synaptic clustering remain unclear. Here, we focus on the intracellular domain of LRRTMs and investigate its role in cell surface expression and synaptic clustering. The deletion of 55–56 residues in the cytoplasmic tail caused significantly reduced synaptic clustering of LRRTM1–4 in rat hippocampal neurons, whereas it simultaneously resulted in augmented LRRTM1–2 cell surface expression. A series of deletions and further single amino acid substitutions in the intracellular domain of LRRTM2 demonstrated that a previously uncharacterized sequence at the region of ‐16 to ‐13 from the C‐terminus was responsible for efficient synaptic clustering and proper cell surface trafficking of LRRTMs. Furthermore, the clustering‐deficient LRRTM2 mutant lost the ability to promote the accumulation of post‐synaptic density protein‐95 (PSD‐95). These results suggest that trafficking to the cell surface and synaptic clustering of LRRTMs are regulated by a specific mechanism through this novel sequence in the intracellular domain that underlies post‐synaptic molecular assembly and maturation.