Hippocampal Glutamatergic Synapses Research Articles

Emerging roles of planar cell polarity proteins in glutamatergic synapse formation, maintenance and function in health and disease.

The local signaling mechanism which directly assembles and maintains glutamatergic synapses has not been well understood. Glutamatergic synapses are made of presynaptic and postsynaptic compartments with distinct sets of proteins. Theplanar cell polarity (PCP) pathwayis highly conserved and responsible for establishing and maintaining the cell and tissue polarity along the tissue plane. The six core PCP proteins form antagonizing complexes within the cells and asymmetric intercellular complexes across neighboring cells which regulate cell-cell interactions during planar polarity signaling. Accumulating evidence suggests that the PCP proteins play essential roles in glutamatergic synapse assembly, maintenance and function in the brain. This review summarizes the key evidence that PCP proteins may be responsible for the formation and stability of the vast majority of the glutamatergic synapses in hippocampus and medial prefrontal cortex, the progress in understanding the mechanisms of how PCP proteins assemble and maintain glutamatergic synapses and initial insights on how disruption of the function of the PCP proteins can lead to neurodegenerative, neurodevelopmental and neuropsychiatric disorders. The PCP proteins may be the missing pieces of a long-standing puzzle and filling this gap of knowledge may provide the basis for understanding many unsolved questions in synapse biology.

Blockade of Type 2A Protein Phosphatase Signaling Attenuates Complement C1q-Mediated Microglial Phagocytosis of Glutamatergic Synapses Induced by Amyloid Fibrils.

We previously reported the critical involvement of metabotropic GluR1 (mGluR1) signaling in complement C1q-dependent microglial phagocytosis of glutamatergic synapses in a rat model of Alzheimer's disease (AD) injected with amyloid fibrils. Here, we explored the role of type 2A protein phosphatase (type 2A PPase), a key enzyme downstream of mGluR1 signaling, in the pathogenesis of AD in rats. Significant local upregulation of PP2A expression was observed in the hippocampal CA1 after bilateral microinjection of amyloid-beta (Aβ1-40) fibrils. Amyloid fibrils induced remarkable dephosphorylation of pFMRP (fragile X mental retardation protein) and C1q upregulation in hippocampal glutamatergic synapses, which was ameliorated by microinjection of type 2A PPase inhibitor okadaic acid (OA). Microinjection of OA further attenuated the microglial phagocytosis of glutamatergic synapses, recovered the hippocampal glutamatergic transmission, and improved the performance in Morris water maze test. These findings demonstrated that dysfunction of type 2A PPase signaling contributed to complement C1q-dependent microglial phagocytosis of glutamatergic synapses and the cognitive impairments in the rat model of AD.

Selective Menin Deletion in the Hippocampal CA1 Region Leads to Disruption of Contextual Memory in the MEN1 Conditional Knockout Mouse: Behavioral Restoration and Gain of Function following the Reintroduction of MEN1 Gene.

Cholinergic neuronal networks in the hippocampus play a key role in the regulation of learning and memory in mammals. Perturbations of these networks, in turn, underlie neurodegenerative diseases. However, the mechanisms remain largely undefined. We have recently demonstrated that an in vitro MEN1 gene deletion perturbs nicotinic cholinergic plasticity at the hippocampal glutamatergic synapses. Furthermore, MEN1 neuronal conditional knockout in freely behaving animals has also been shown to result in learning and memory deficits, though the evidence remains equivocal. In this study, using an AVV viral vector transcription approach, we provide direct evidence that MEN1 gene deletion in the CA1 region of the hippocampus indeed leads to contextual fear conditioning deficits in conditional knockout animals. This loss of function was, however, recovered when the same animals were re-injected to overexpress MEN1. This study provides the first direct evidence for the sufficiency and necessity of MEN1 in fear conditioning, and further endorses the role of menin in the regulation of cholinergic synaptic machinery in the hippocampus. These data underscore the importance of further exploring and revisiting the cholinergic hypothesis that underlies neurodegenerative diseases that affect learning and memory.

Arhgap22 Disruption Leads to RAC1 Hyperactivity Affecting Hippocampal Glutamatergic Synapses and Cognition in Mice

Rho GTPases are a class of G-proteins involved in several aspects of cellular biology, including the regulation of actin cytoskeleton. The most studied members of this family are RHOA and RAC1 that act in concert to regulate actin dynamics. Recently, Rho GTPases gained much attention as synaptic regulators in the mammalian central nervous system (CNS). In this context, ARHGAP22 protein has been previously shown to specifically inhibit RAC1 activity thus standing as critical cytoskeleton regulator in cancer cell models; however, whether this function is maintained in neurons in the CNS is unknown. Here, we generated a knockout animal model for arhgap22 and provided evidence of its role in the hippocampus. Specifically, we found that ARHGAP22 absence leads to RAC1 hyperactivity and to an increase in dendritic spine density with defects in synaptic structure, molecular composition, and plasticity. Furthermore, arhgap22 silencing causes impairment in cognition and a reduction in anxiety-like behavior in mice. We also found that inhibiting RAC1 restored synaptic plasticity in ARHGAP22 KO mice. All together, these results shed light on the specific role of ARHGAP22 in hippocampal excitatory synapse formation and function as well as in learning and memory behaviors.

Sex differences in endocannabinoid modulation of rat CA1 dendritic neurotransmission

Endocannabinoid sex differences are present in the rat hippocampus. Specifically, at perisomatic GABAergic synapses, tonic anandamide (AEA) and estrogenic-AEA signaling are active in females but not males. Furthermore, in males, hippocampal eCB function varies along the CA1 pyramidal somatodendritic axis. Constitutive CB1 and tonic 2-AG activity are present at perisomatic GABAergic synapses and lacking at dendritic GABAergic synapses. It is unknown if these eCB somatodendritic differences occur at female GABAergic synapses. Moreover, it is unclear whether eCB sex differences occur at hippocampal glutamatergic synapses. In vitro, field potential (fEPSP) recordings were performed to assess eCB sex differences at rat CA3-CA1 dendritic synapses. At female GABAergic synapses, we observed: 1) constitutive CB1 function, 2) tonic AEA, 3) tonic 2-AG and 3) estrogen (ERα)-driven 2-AG activity. In contrast, only constitutive CB1 and tonic 2-AG activity was observed in males. Sex differences in eCB/CB1 signaling at dendritic synapses appear to shift the basal excitatory/inhibitory balance towards excitation in females and towards inhibition in males. Chronic Mild Stress (CMS) exposure (21 days) in female rats reverses CB1constitutive function and impairs both tonic and ERα-driven eCB signaling. Endocannabinoid sex differences under both normal and stress conditions may contribute to sexual disparities in stress-related neurobehavioral disorders.

VGLUT1 functions as a glutamate/proton exchanger with chloride channel activity in hippocampal glutamatergic synapses

Glutamate is the major excitatory transmitter in the vertebrate nervous system. To maintain synaptic efficacy, recycling synaptic vesicles (SV) are refilled with glutamate by vesicular glutamate transporters (VGLUTs). The dynamics and mechanism of glutamate uptake in intact neurons are still largely unknown. Here, we show by live-cell imaging with pH- and chloride-sensitive fluorescent probes in cultured hippocampal neurons of wild-type and VGLUT1-deficient mice that in SVs VGLUT functions as a glutamate/proton exchanger associated with a channel-like chloride conductance. After endocytosis most internalized Cl− is substituted by glutamate in an electrically, and presumably osmotically, neutral manner, and this process is driven by both the Cl− gradient itself and the proton motive force provided by the vacuolar H+-ATPase. Our results shed light on the transport mechanism of VGLUT under physiological conditions and provide a framework for how modulation of glutamate transport via Cl− and pH can change synaptic strength.

Neuregulin-2 ablation results in dopamine dysregulation and severe behavioral phenotypes relevant to psychiatric disorders

Numerous genetic and functional studies implicate variants of Neuregulin-1 (NRG1) and its neuronal receptor ErbB4 in schizophrenia and many of its endophenotypes. Although the neurophysiological and behavioral phenotypes of NRG1 mutant mice have been investigated extensively, practically nothing is known about the function of NRG2, the closest NRG1 homolog. We found that NRG2 expression in the adult rodent brain does not overlap with NRG1 and is more extensive than originally reported, including expression in the striatum and medial prefrontal cortex (mPFC), and therefore generated NRG2 knockout mice (KO) to study its function. NRG2 KOs have higher extracellular dopamine levels in the dorsal striatum but lower levels in the mPFC; a pattern with similarities to dopamine dysbalance in schizophrenia. Like ErbB4 KO mice, NRG2 KOs performed abnormally in a battery of behavioral tasks relevant to psychiatric disorders. NRG2 KOs exhibit hyperactivity in a novelty-induced open field, deficits in prepulse inhibition, hypersensitivity to amphetamine, antisocial behaviors, reduced anxiety-like behavior in the elevated plus maze and deficits in the T-maze alteration reward test—a task dependent on hippocampal and mPFC function. Acute administration of clozapine rapidly increased extracellular dopamine levels in the mPFC and improved alternation T-maze performance. Similar to mice treated chronically with N-methyl-d-aspartate receptor (NMDAR) antagonists, we demonstrate that NMDAR synaptic currents in NRG2 KOs are augmented at hippocampal glutamatergic synapses and are more sensitive to ifenprodil, indicating an increased contribution of GluN2B-containing NMDARs. Our findings reveal a novel role for NRG2 in the modulation of behaviors with relevance to psychiatric disorders.

Reconstruction of the molecular interactome of glutamatergic synapses

The subunit composition of ionotropic glutamate receptors is of great significance for synapse functioning. NMDA receptors mediate fast excitatory neurotransmission and are capable of converting specific patterns of neuronal activity into long-term changes in synaptic structure and function. The principal functional properties (ion conductivity, glutamate sensitivity, agonists, and magnesium ions, and deactivation time), spatial location, membrane anchoring, and the response to pharmacological agents depend on subunit composition of the receptors. The analysis of protein–protein interactions in macrocomplexes, including NMDA receptor subunits, is an urgent task, because the results of research in this area will contribute to a better understanding of the principles and molecular mechanisms underlying the basic functions of neurons and the development of pathologies and facilitate the search for pharmacological and therapeutic targets for the correction of pathologies. The goal of the present work consisted of analyzing and reconstructing protein–protein interactions involving NMDA receptor subunits and providing for the mobility of NMDA receptors, the incorporation of these molecules into the membranes, and receptor functions related to modification and maintenance of synaptic transmission efficiency in the hippocampus. Three groups of proteins involved in the formation of NMDA receptor macrocomplexes in hippocampal glutamatergic synapses were identified. The proteins were divided into groups according to their function in the complexes; the functions were inferred from the information presented in various databases and research articles that reported gene and protein structures, expression profiles in the brain, and the role of specific genes and proteins in synaptic plasticity. Particular attention was paid to proteins for which a association with various cognitive disturbances was reported.

Presynaptic active zones in invertebrates and vertebrates.

The regulated release of neurotransmitter occurs via the fusion of synaptic vesicles (SVs) at specialized regions of the presynaptic membrane called active zones (AZs). These regions are defined by a cytoskeletal matrix assembled at AZs (CAZ), which functions to direct SVs toward docking and fusion sites and supports their maturation into the readily releasable pool. In addition, CAZ proteins localize voltage-gated Ca(2+) channels at SV release sites, bringing the fusion machinery in close proximity to the calcium source. Proteins of the CAZ therefore ensure that vesicle fusion is temporally and spatially organized, allowing for the precise and reliable release of neurotransmitter. Importantly, AZs are highly dynamic structures, supporting presynaptic remodeling, changes in neurotransmitter release efficacy, and thus presynaptic forms of plasticity. In this review, we discuss recent advances in the study of active zones, highlighting how the CAZ molecularly defines sites of neurotransmitter release, endocytic zones, and the integrity of synapses.

Early postnatal exposure to lithium in vitro induces changes in AMPAR mEPSCs and vesicular recycling at hippocampal glutamatergic synapses.

Lithium is an effective mood stabilizer but its use is associated with many side effects. Electrophysiological recordings of miniature excitatory postsynaptic currents (mEPSCs) mediated by glutamate receptor AMPA-subtype (AMPARs) in hippocampal pyramidal neurons revealed that CLi (therapeutic concentration of 1 mM lithium, from days in vitro 4-10) decreased the mean amplitude and mean rectification index (RI) of AMPAR mEPSCs. Lowered mean RI indicate that contribution of Ca2+ -permeable AMPARs in synaptic events is higher in CLi neurons (supported by experiments sensitive to Ca2+ -permeable AMPAR modulation). Co-inhibiting PKA, GSK-3 beta and glutamate reuptake was necessary to bring about changes in AMPAR mEPSCs similar to that seen in CLi neurons. FM1-43 experiments revealed that recycling pool size was affected in CLi cultures. Results from minimum loading, chlorpromazine treatment and hyperosmotic treatment experiments indicate that endocytosis in CLi is affected while not much difference is seen in modes of exocytosis. CLi cultures did not show the high KCl associated presynaptic potentiation observed in control cultures. This study, by calling attention to long-term lithium-exposure-induced synaptic changes, might have implications in understanding the side effects such as CNS complications occurring in perinatally exposed babies and cognitive dulling seen in patients on lithium treatment.

Ablation of CaV2.1 Voltage-Gated Ca2+ Channels in Mouse Forebrain Generates Multiple Cognitive Impairments

Voltage-gated CaV2.1 (P/Q-type) Ca2+ channels located at the presynaptic membrane are known to control a multitude of Ca2+-dependent cellular processes such as neurotransmitter release and synaptic plasticity. Our knowledge about their contributions to complex cognitive functions, however, is restricted by the limited adequacy of existing transgenic CaV2.1 mouse models. Global CaV2.1 knock-out mice lacking the α1 subunit Cacna1a gene product exhibit early postnatal lethality which makes them unsuitable to analyse the relevance of CaV2.1 Ca2+ channels for complex behaviour in adult mice. Consequently we established a forebrain specific CaV2.1 knock-out model by crossing mice with a floxed Cacna1a gene with mice expressing Cre-recombinase under the control of the NEX promoter. This novel mouse model enabled us to investigate the contribution of CaV2.1 to complex cognitive functions, particularly learning and memory. Electrophysiological analysis allowed us to test the specificity of our conditional knock-out model and revealed an impaired synaptic transmission at hippocampal glutamatergic synapses. At the behavioural level, the forebrain-specific CaV2.1 knock-out resulted in deficits in spatial learning and reference memory, reduced recognition memory, increased exploratory behaviour and a strong attenuation of circadian rhythmicity. In summary, we present a novel conditional CaV2.1 knock-out model that is most suitable for analysing the in vivo functions of CaV2.1 in the adult murine forebrain.

Different correlations among physiological and morphological properties at single glutamatergic synapses in the rat hippocampus and the cerebellum

Synapses in the mammalian central nervous system show substantial diversity in their physiological and morphological properties. However, the correlations among them have remained elusive. Here, we tried to clarify the correlations by establishing a method to record excitatory postsynaptic currents (EPSCs) at individual synapses and also to observe the morphology at the same time. A pair of pre- and postsynaptic neurons were labeled with different fluorescent dyes, and a presynaptic varicosity was selectively stimulated with a θ-tube glass electrode under conditions in which action potential generation was suppressed. Two representative types of excitatory glutamatergic synapses, one on hippocampal pyramidal neurons and the other on cerebellar Purkinje neurons, were studied. The correlations between the properties of quantal EPSCs (qEPSCs) and those of synaptic morphology were analyzed in rat primary culture preparations. The amplitude and the decay time of qEPSC were correlated with the size of the postsynaptic spine only at hippocampal synapses. In contrast, the size of the presynaptic varicosity was correlated with the size of the postsynaptic spine and the quantal content of evoked EPSCs only at granule neuron-Purkinje neuron synapses in the cerebellum. These results suggest that the interaction between pre- and postsynaptic structures and the coupling of postsynaptic responsiveness and the spine morphology differ between cerebellar and hippocampal glutamatergic synapses.

Ephrin-A5 and EphA5 Interaction Induces Synaptogenesis during Early Hippocampal Development

BackgroundSynaptogenesis is a fundamental step in neuronal development. For spiny glutamatergic synapses in hippocampus and cortex, synaptogenesis involves adhesion of pre and postsynaptic membranes, delivery and anchorage of pre and postsynaptic structures including scaffolds such as PSD-95 and NMDA and AMPA receptors, which are glutamate-gated ion channels, as well as the morphological maturation of spines. Although electrical activity-dependent mechanisms are established regulators of these processes, the mechanisms that function during early development, prior to the onset of electrical activity, are unclear. The Eph receptors and ephrins provide cell contact-dependent pathways that regulate axonal and dendritic development. Members of the ephrin-A family are glycosyl-phosphatidylinositol-anchored to the cell surface and activate EphA receptors, which are receptor tyrosine kinases.Methodology/Principal FindingsHere we show that ephrin-A5 interaction with the EphA5 receptor following neuron-neuron contact during early development of hippocampus induces a complex program of synaptogenic events, including expression of functional synaptic NMDA receptor-PSD-95 complexes plus morphological spine maturation and the emergence of electrical activity. The program depends upon voltage-sensitive calcium channel Ca2+ fluxes that activate PKA, CaMKII and PI3 kinase, leading to CREB phosphorylation and a synaptogenic program of gene expression. AMPA receptor subunits, their scaffolds and electrical activity are not induced. Strikingly, in contrast to wild type, stimulation of hippocampal slices from P6 EphA5 receptor functional knockout mice yielded no NMDA receptor currents.Conclusions/SignificanceThese studies suggest that ephrin-A5 and EphA5 signals play a necessary, activity-independent role in the initiation of the early phases of synaptogenesis. The coordinated expression of the NMDAR and PSD-95 induced by eprhin-A5 interaction with EphA5 receptors may be the developmental switch that induces expression of AMPAR and their interacting proteins and the transition to activity-dependent synaptic regulation.

Increased Expression of Ryanodine Receptors and the Iron Transporter DMT1 in Hippocampal Neurons by Brain Derived Neurotrophic Factor (BDNF), NMDA or Spatial Memory Training

Ryanodine receptors (RyR) mediate skeletal and cardiac muscle contraction and amplify via CICR postsynaptic calcium signals generated by activity-dependent calcium influx through NMDA receptors (NMDAR) in hippocampal glutamatergic synapses. We have recently shown that reactive oxygen species (ROS) and iron, which as shown here promotes ROS generation in neurons, stimulate RyR-mediated calcium release when added to primary hippocampal neurons. Here, we report that 5 min incubation of hippocampal cells in primary culture with NMDA (50 μM), induced RyR-mediated calcium signals that were inhibited by pre-incubation with the iron chelator desferroxamine. Incubation with NMDA also enhanced >2-fold the expression (measured 24 h later) of the iron transporter DMT1 (IRE form), while incubation with BDNF (50 ng/ml) increased >5-fold RyR expression. Additionally, we investigated if spatial memory training of male rats in a Morris water maze affected RyR and DMT1 expression. The hippocampus was dissected 6 h after the last behavioral task (5d, 2d rest, 1d platform free) and samples from tissue were prepared for Western blot and RT-PCR experiments. We found that spatial memory training increased the mRNA and protein expression of DMT1, RyR2 and RyR3. Our results confirm enhanced RyR2 expression following spatial memory training and correlate for the first time enhanced in vivo expression of the iron transporter DMT1 and RyR3 with spatial memory acquisition/consolidation. We propose that iron-induced ROS production stimulates the emergence of RyR-mediated intracellular calcium signals that promote RyR and DMT1 expression during the spatial memory task.FONDECYT (PostDoc) 3070035, CEMC-FONDAP 15010006, Millennium P05-001F, FONDECYT 1060177.

Neuregulin-1 regulates LTP at CA1 hippocampal synapses through activation of dopamine D4 receptors

Neuregulin-1 (NRG-1) is genetically linked with schizophrenia, a neurodevelopmental cognitive disorder characterized by imbalances in glutamatergic and dopaminergic function. NRG-1 regulates numerous neurodevelopmental processes and, in the adult, suppresses or reverses long-term potentiation (LTP) at hippocampal glutamatergic synapses. Here we show that NRG-1 stimulates dopamine release in the hippocampus and reverses early-phase LTP via activation of D4 dopamine receptors (D4R). NRG-1 fails to depotentiate LTP in hippocampal slices treated with the antipsychotic clozapine and other more selective D4R antagonists. Moreover, LTP is not depotentiated in D4R null mice by either NRG-1 or theta-pulse stimuli. Conversely, direct D4R activation mimics NRG-1 and reduces AMPA receptor currents and surface expression. These findings demonstrate that NRG-1 mediates its unique role in counteracting LTP via dopamine signaling and opens future directions to study new aspects of NRG function. The novel functional link between NRG-1, dopamine, and glutamate has important implications for understanding how imbalances in Neuregulin-ErbB signaling can impinge on dopaminergic and glutamatergic function, neurotransmitter pathways associated with schizophrenia.

N-methyl- d-aspartate receptor subunit dysfunction at hippocampal glutamatergic synapses in an animal model of attention-deficit/hyperactivity disorder

Attention-deficit/hyperactivity disorder (ADHD) is the most common neurobehavioural disorder among children. ADHD children are hyperactive, impulsive and have problems with sustained attention. These cardinal features are also present in the best validated animal model of ADHD, the spontaneously hypertensive rat (SHR), which is derived from the Wistar Kyoto rat (WKY). Current theories of ADHD relate symptom development to factors that alter learning. N-methyl- d-aspartate receptor (NMDAR) dependent long term changes in synaptic efficacy in the mammalian CNS are thought to represent underlying cellular mechanisms for some forms of learning. We therefore hypothesized that synaptic abnormality in excitatory, glutamatergic synaptic transmission might contribute to the altered behavior in SHRs. We studied physiological and anatomical aspects of hippocampal CA3-to-CA1 synapses in age-matched SHR and WKY (controls). Electrophysiological analysis of these synapses showed reduced synaptic transmission (reduced field excitatory postsynaptic potential for a defined fiber volley size) in SHR, whereas short-term forms of synaptic plasticity, like paired-pulse facilitation, frequency facilitation, and delayed response enhancement were comparable in the two genotypes, and long-term potentiation (LTP) of synaptic transmission was of similar magnitude. However, LTP in SHR was significantly reduced (by 50%) by the NR2B specific blocker CP-101,606 (10 μM), whereas the blocker had no effect on LTP magnitude in the control rats. This indicates that the SHR has a functional predominance of NR2B, a feature characteristic of early developmental stages in these synapses. Quantitative immunofluorescence and electron microscopic postembedding immunogold cytochemistry of the three major NMDAR subunits (NR1, NR2A; and NR2B) in stratum radiatum spine synapses revealed no differences between SHR and WKY. The results indicate that functional impairments in glutamatergic synaptic transmission may be one of the underlying mechanisms leading to the abnormal behavior in SHR, and possibly in human ADHD.

Regulation of ErbB-4 endocytosis by neuregulin in GABAergic hippocampal interneurons

Neuregulin (NRG)/ErbB receptor signaling pathways have recently been implicated in the reversal of long-term potentiation at hippocampal glutamatergic synapses. Moreover, polymorphisms in NRG-1 and ErbB-4 genes have been linked to an increased risk for developing schizophrenia. ErbB-4 is highly expressed at glutamatergic synapses where it binds to PSD-95 via its carboxyl terminal T–V–V sequence. Here we investigated the expression, localization and trafficking of ErbB-4 in cultured hippocampal neurons by immunocytochemistry, surface protein biotinylation, and live labeling of native receptors. We show that neuronal ErbB-4 is detected at its highest levels in GABAergic interneurons, as observed in vivo. ErbB-4 immunoreactivity precedes PSD-95 expression, with ErbB-4 cluster initially forming in the absence of, but later associating with, PSD-95-positive puncta. By surface protein biotinylation, the fraction of ErbB-4 receptors on the plasma membrane increases from 30% to 65% between 6 and 16 days in vitro (DIV). Interestingly, 30 min of NRG stimulation triggers measurable ErbB-4 receptor internalization at DIV 16, despite increased colocalization with PSD-95. We also investigated the role of TNFα-converting enzyme (TACE)-mediated receptor processing in regulating ErbB-4 surface expression. We found that the cleavage-resistant JM-b isoform accounts for 80% of all ErbB-4 transcripts in cultured hippocampal neurons. Receptor stimulation or treatment with phorbol esters does not induce detectable ErbB-4 processing, indicating that neurons mostly rely on endocytosis of the intact receptor to regulate ErbB-4 surface expression. These results enhance our understanding of the regulation of ErbB-4—mediated signaling at glutamatergic synapses.

Morphine Administration Alters the Profile of Hippocampal Postsynaptic Density-associated Proteins: A Proteomics Study Focusing on Endocytic Proteins

Numerous studies have shown that drugs of abuse induce changes in protein expression in the brain that are thought to play a role in synaptic plasticity. Drug-induced plasticity can be mediated by changes at the synapse and more specifically at the postsynaptic density (PSD), which receives and transduces synaptic information. To date, the majority of studies examining synaptic protein profiles have focused on identifying the synaptic proteome. Only a handful of studies have examined the changes in synaptic profile by drug administration. We applied a quantitative proteomics analysis technique with the cleavable ICAT reagent to quantitate relative changes in protein levels of the hippocampal PSD in response to morphine administration. We identified a total of 102 proteins in the mouse hippocampal PSD. The majority of these were signaling, trafficking, and cytoskeletal proteins involved in synaptic plasticity, learning, and memory. Among the proteins whose levels were found to be altered by morphine administration, clathrin levels were increased to the largest extent. Immunoblotting and electron microscopy studies showed that this increase was localized to the PSD. Morphine treatment was also found to lead to a local increase in two other components of the endocytic machinery, dynamin and AP-2, suggesting a critical involvement of the endocytic machinery in the modulatory effects of morphine. Because alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are thought to undergo clathrin-mediated endocytosis, we examined the effect of morphine administration on the association of the AMPA receptor subunit, GluR1, with clathrin. We found a substantial decrease in the levels of GluR1 associated with clathrin. Taken together, these results suggest that, by causing a redistribution of endocytic proteins at the synapse, morphine modulates synaptic plasticity at hippocampal glutamatergic synapses.

Muscarinic acetylcholine receptor-dependent induction of persistent synaptic enhancement in rat hippocampus in vivo

Presynaptic terminal autoinhibitory muscarinic acetylcholine (ACh) receptors are predominantly of the M2/M4 subtypes and antagonists at these receptors may facilitate cognitive processes by increasing ACh release. The present study examined the ability of the M2/M4 muscarinic ACh receptor antagonist N,N′-bis [6-[[(2-methoxyphenyl)methyl]amino]hexyl]-1,8-octane diamine tetrahydrochloride (methoctramine) to induce and modulate synaptic plasticity in the CA1 area of the hippocampus in urethane-anesthetized rats. Both methoctramine and another M2/M4 antagonist, {11-[[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5,11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepin-6-one} (AF-DX 116), caused a rapid onset and persistent increase in baseline synaptic transmission after i.c.v. injection. Consistent with a requirement for activation of non-M2 receptors by endogenously released ACh, the M1/M3 receptor selective antagonists 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) and 4,9-dihydro-3-methyl-4-[(4-methyl-1-piperazinyl)acetyl]-10H-thieno[3,4-b][1,5]benzodiazepin-10-one dihydrochloride (telenzepine) prevented the induction of the persistent synaptic enhancement by methoctramine. The requirement for cholinergic activation was transient and independent of nicotinic ACh receptor stimulation. The synaptic enhancement was inhibited by the prior induction of long-term potentiation (LTP) by high frequency stimulation but induction of the synaptic enhancement by methoctramine before high frequency stimulation did not inhibit LTP. Unlike high frequency stimulation-evoked LTP, the synaptic enhancement induced by methoctramine appeared to be NMDA receptor-independent. The present studies provide evidence for the rapid induction of a persistent potentiation at hippocampal glutamatergic synapses by endogenous ACh in vivo following disinhibition of inhibitory M2 muscarinic autoreceptors.

Immunolocalization of BK channels in hippocampal pyramidal neurons

Neurons are highly specialized cells in which the integration and processing of electrical signals critically depends on the precise localization of ion channels. For large-conductance Ca(2+)- activated K(+) (BK) channels, targeting to presynaptic membranes in hippocampal pyramidal cells was reported; however, functional evidence also suggests a somatodendritic localization. Therefore we re-examined the subcellular distribution of BK channels in mouse hippocampus using a panel of independent antibodies in a combined approach of conventional immunocytochemistry on cultured neurons, pre- and postembedding electron microscopy and immunoprecipitation. In cultured murine hippocampal neurons, the colocalization of BK channels with both pre- and postsynaptic marker proteins was observed. Electron microscopy confirmed targeting of BK channels to axonal as well as dendritic membranes of glutamatergic synapses in hippocampus. A postsynaptic localization of BK channels was also supported by the finding that the channel coimmunoprecipitated with PSD95, a protein solely expressed in the postsynaptic compartment. These results thus demonstrate that BK channels reside in both post- and presynaptic compartments of hippocampal pyramidal neurons.