ABSTRACT
The synaptotrophic hypothesis posits that synapse formation stabilizes dendritic branches, but this hypothesis has not been causally tested in vivo in the mammalian brain. The presynaptic ligand cerebellin-1 (Cbln1) and postsynaptic receptor GluD2 mediate synaptogenesis between granule cells and Purkinje cells in the molecular layer of the cerebellar cortex. Here we show that sparse but not global knockout of GluD2 causes under-elaboration of Purkinje cell dendrites in the deep molecular layer and overelaboration in the superficial molecular layer. Developmental, overexpression, structure-function, and genetic epistasis analyses indicate that these dendrite morphogenesis defects result from a deficit in Cbln1/GluD2-dependent competitive interactions. A generative model of dendrite growth based on competitive synaptogenesis largely recapitulates GluD2 sparse and global knockout phenotypes. Our results support the synaptotrophic hypothesis at initial stages of dendrite development, suggest a second mode in which cumulative synapse formation inhibits further dendrite growth, and highlight the importance of competition in dendrite morphogenesis.
Subject(s)
Cerebellum/cytology , Cerebellum/metabolism , Dendrites/metabolism , Nerve Tissue Proteins/deficiency , Protein Precursors/deficiency , Purkinje Cells/metabolism , Receptors, Glutamate/deficiency , Animals , Dendrites/genetics , Female , Mice , Mice, Inbred ICR , Mice, Knockout , Mice, Transgenic , Nerve Tissue Proteins/genetics , Pregnancy , Protein Binding/physiology , Protein Precursors/genetics , Receptors, Glutamate/geneticsABSTRACT
Glutamatergic input within the mesolimbic dopamine (DA) pathway plays a critical role in the development of addictive behavior. Although this is well established for some drugs of abuse, it is not known whether glutamate receptors within the mesolimbic system are involved in mediating the addictive properties of chronic alcohol use. Here we evaluated the contribution of mesolimbic NMDARs and AMPARs in mediating alcohol-seeking responses induced by environmental stimuli and relapse behavior using four inducible mutant mouse lines lacking the glutamate receptor genes Grin1 or Gria1 in either DA transporter (DAT) or D1R-expressing neurons. We first demonstrate the lack of GluN1 or GluA1 in either DAT- or D1R-expressing neurons in our mutant mouse lines by colocalization studies. We then show that GluN1 and GluA1 receptor subunits within these neuronal subpopulations mediate the alcohol deprivation effect, while having no impact on context- plus cue-induced reinstatement of alcohol-seeking behavior. We further validated these results pharmacologically by demonstrating similar reductions in the alcohol deprivation effect after infusion of the NMDAR antagonist memantine into the nucleus accumbens and ventral tegmental area of control mice, and a rescue of the mutant phenotype via pharmacological potentiation of AMPAR activity using aniracetam. In conclusion, dopamine neurons as well as D1R-expressing medium spiny neurons and their glutamatergic inputs via NMDARs and AMPARs act in concert to influence relapse responses. These results provide a neuroanatomical and molecular substrate for relapse behavior and emphasize the importance of glutamatergic drugs in modulating relapse behavior. SIGNIFICANCE STATEMENT: Here we provide genetic and pharmacological evidence that glutamate receptors within the mesolimbic dopamine system play an essential role in alcohol relapse. Using various inducible and site-specific transgenic mouse models and pharmacological validation experiments, we show that critical subunits of NMDARs and AMPARs expressed either in dopamine neurons or in dopamine receptor D1-containing neurons play an important role in the alcohol deprivation effect (the increase in alcohol intake after a period of abstinence) while having no impact on context- plus cue-induced reinstatement of alcohol-seeking responses. Medications targeting glutamatergic neurotransmission by selective inactivation of these glutamate receptors might have therapeutic efficacy.
Subject(s)
Alcoholism/metabolism , Behavior, Addictive/metabolism , Dopamine Plasma Membrane Transport Proteins/physiology , Receptors, Dopamine/physiology , Receptors, Glutamate/physiology , Ventral Tegmental Area/metabolism , Animals , Dopamine/physiology , Dopamine Plasma Membrane Transport Proteins/deficiency , Dopaminergic Neurons/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Motor Activity/physiology , Receptors, Dopamine/deficiency , Receptors, Glutamate/deficiency , RecurrenceABSTRACT
The glutamate delta-1 (GluD1) receptor is highly expressed in the forebrain. We have previously shown that loss of GluD1 leads to social and cognitive deficits in mice, however, its role in synaptic development and neurotransmission remains poorly understood. Here we report that GluD1 is enriched in the medial prefrontal cortex (mPFC) and GluD1 knockout mice exhibit a higher dendritic spine number, greater excitatory neurotransmission as well as higher number of synapses in mPFC. In addition abnormalities in the LIMK1-cofilin signaling, which regulates spine dynamics, and a lower ratio of GluN2A/GluN2B expression was observed in the mPFC in GluD1 knockout mice. Analysis of the GluD1 knockout CA1 hippocampus similarly indicated the presence of higher spine number and synapses and altered LIMK1-cofilin signaling. We found that systemic administration of an N-methyl-d-aspartate (NMDA) receptor partial agonist d-cycloserine (DCS) at a high-dose, but not at a low-dose, and a GluN2B-selective inhibitor Ro-25-6981 partially normalized the abnormalities in LIMK1-cofilin signaling and reduced excess spine number in mPFC and hippocampus. The molecular effects of high-dose DCS and GluN2B inhibitor correlated with their ability to reduce the higher stereotyped behavior and depression-like behavior in GluD1 knockout mice. Together these findings demonstrate a critical requirement for GluD1 in normal spine development in the cortex and hippocampus. Moreover, these results identify inhibition of GluN2B-containing receptors as a mechanism for reducing excess dendritic spines and stereotyped behavior which may have therapeutic value in certain neurodevelopmental disorders such as autism.
Subject(s)
Cerebral Cortex/cytology , Dendritic Spines/physiology , Hippocampus/cytology , Neurons/ultrastructure , Receptors, AMPA/metabolism , Receptors, Glutamate/deficiency , Receptors, N-Methyl-D-Aspartate/metabolism , Animals , Animals, Newborn , Cerebral Cortex/growth & development , Dendritic Spines/ultrastructure , Desipramine/pharmacology , Enzyme Inhibitors/pharmacology , Excitatory Amino Acid Antagonists/pharmacology , Exploratory Behavior/physiology , Glutamate Dehydrogenase , Hippocampus/growth & development , Mice , Mice, Knockout , Motor Activity/drug effects , Neurons/drug effects , Neurons/physiology , Phenols/pharmacology , Piperidines/pharmacology , Receptors, Glutamate/genetics , Selective Serotonin Reuptake Inhibitors/pharmacology , Signal Transduction/drug effects , Sodium Channel Blockers/pharmacology , Swimming/psychology , Tetrodotoxin/pharmacologyABSTRACT
Cerebellar cortex has an elaborate rostrocaudal organization comprised of numerous microzones. Purkinje cells (PCs) in the same microzone show synchronous activity of complex spikes (CSs) evoked by excitatory inputs from climbing fibers (CFs) that arise from neurons in the inferior olive (IO). The synchronous CS activity is considered to depend on electrical coupling among IO neurons and anatomical organization of the olivo-cerebellar projection. To determine how the CF-PC wiring contributes to the formation of microzone, we examined the synchronous CS activities between neighboring PCs in the glutamate receptor δ2 knockout (GluD2 KO) mouse in which exuberant surplus CFs make ectopic innervations onto distal dendrites of PCs. We performed in vivo two-photon calcium imaging for PC populations to detect CF inputs. Neighboring PCs in GluD2 KO mice showed higher synchrony of calcium transients than those in wild-type (control) mice. Moreover, the synchrony in GluD2 KO mice hardly declined with mediolateral separation between PCs up to ~200 µm, which was in marked contrast to the falloff of the synchrony in control mice. The enhanced synchrony was only partially affected by the blockade of gap junctional coupling. On the other hand, transverse CF collaterals in GluD2 KO mice extended beyond the border of microzone and formed locally clustered ectopic synapses onto dendrites of neighboring PCs. Furthermore, PCs in GluD2 KO mice exhibited clustered firing (Cf), the characteristic CF response that was not found in PCs of wild-type mice. Importantly, Cf was often associated with localized calcium transients in distal dendrites of PCs, which are likely to contribute to the enhanced synchrony of calcium signals in GluD2 KO mice. Thus, our results indicate that CF signals in GluD2 KO mice propagate across multiple microzones, and that proper formation of longitudinal olivo-cerebellar projection is essential for the spatiotemporal organization of CS activity in the cerebellum.
Subject(s)
Cerebellum/cytology , Cerebellum/metabolism , Receptors, Glutamate/deficiency , Receptors, Glutamate/genetics , Action Potentials/physiology , Animals , Female , Gene Knock-In Techniques , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Purkinje Cells/metabolismABSTRACT
Glutamate delta-1 (GluD1) receptors are expressed throughout the forebrain during development with high levels in the hippocampus during adulthood. We have recently shown that deletion of GluD1 receptor results in aberrant emotional and social behaviors such as hyperaggression and depression-like behaviors and social interaction deficits. Additionally, abnormal expression of synaptic proteins was observed in amygdala and prefrontal cortex of GluD1 knockout mice (GluD1 KO). However the role of GluD1 in learning and memory paradigms remains unknown. In the present study we evaluated GluD1 KO in learning and memory tests. In the eight-arm radial maze GluD1 KO mice committed fewer working memory errors compared to wildtype mice but had normal reference memory. Enhanced working memory in GluD1 KO was also evident by greater percent alternation in the spontaneous Y-maze test. No difference was observed in object recognition memory in the GluD1 KO mice. In the Morris water maze test GluD1 KO mice showed no difference in acquisition but had longer latency to find the platform in the reversal learning task. GluD1 KO mice showed a deficit in contextual and cue fear conditioning but had normal latent inhibition. The deficit in contextual fear conditioning was reversed by D-Cycloserine (DCS) treatment. GluD1 KO mice were also found to be more sensitive to foot-shock compared to wildtype. We further studied molecular changes in the hippocampus, where we found lower levels of GluA1, GluA2 and GluK2 subunits while a contrasting higher level of GluN2B in GluD1 KO. Additionally, we found higher postsynaptic density protein 95 (PSD95) and lower glutamate decarboxylase 67 (GAD67) expression in GluD1 KO. We propose that GluD1 is crucial for normal functioning of synapses and absence of GluD1 leads to specific abnormalities in learning and memory. These findings provide novel insights into the role of GluD1 receptors in the central nervous system.
Subject(s)
Depression/genetics , Fear/psychology , Maze Learning/physiology , Memory, Short-Term/physiology , Receptors, Glutamate/genetics , Amygdala/drug effects , Amygdala/metabolism , Amygdala/physiopathology , Animals , Behavior, Animal/drug effects , Conditioning, Psychological/drug effects , Cues , Cycloserine/pharmacology , Depression/physiopathology , Depression/psychology , Disks Large Homolog 4 Protein , Emotions/drug effects , Fear/drug effects , Gene Expression Regulation/drug effects , Glutamate Decarboxylase/genetics , Glutamate Decarboxylase/metabolism , Glutamate Dehydrogenase , Guanylate Kinases/genetics , Guanylate Kinases/metabolism , Hippocampus/drug effects , Hippocampus/metabolism , Hippocampus/physiopathology , Maze Learning/drug effects , Membrane Proteins/genetics , Membrane Proteins/metabolism , Memory, Short-Term/drug effects , Mice , Mice, Knockout , Prefrontal Cortex/drug effects , Prefrontal Cortex/metabolism , Prefrontal Cortex/physiopathology , Protein Isoforms/deficiency , Protein Isoforms/genetics , Receptors, Glutamate/deficiencyABSTRACT
Lurcher mutant mice represent a natural model of genetically-determined olivocerebellar degeneration caused by a mutation in the δ2 glutamate receptor gene. They suffer from progressive postnatal loss of cerebellar Purkinje cells and a decrease of granule cells and inferior olive neurons. Their wild type littermates serve as healthy controls. A confocal laser scanning microscope was used aiming investigation the dynamics of changes in the cerebellar cortex of Lurcher and wild type mice derived from two strains during the period of 8-21 postnatal days. Fluorescent double-staining was used to visualize mainly the Purkinje cells in cerebellar slices. In wild types, only normal Purkinje cells of round or regular drop-shaped were present, when staining intensity of other individual cell structures differed in dependence on the age of the animal. In Lurcher mutants, there were still some normal-shaped cells. Nevertheless, depending on the animal's age, a wide variety of stages of the cell degeneration were depicted. The main characteristics of Purkinje cell degeneration in the early stage are: disruption of the continuity of the Purkinje cell layer, dark spots in cell nuclei and an irregular coloring of the cytoplasm. Later, the cells and their nuclei were deformed, often with two main dendrites sprouting from the cell body. Finally, the cell and nucleus margins were unclear, dendrites were significantly thickened, showing signs of shrinkage and fragmentation. Cell nucleoli underwent changes in number and appearance. No differences between the Lurcher mice of both strains (C3H and B6CBA) under examination were found.
Subject(s)
Cerebellar Cortex/pathology , Receptors, Glutamate/deficiency , Animals , Fluorescent Dyes/metabolism , Mice , Mice, Neurologic Mutants , Microscopy, Confocal , Purkinje Cells/chemistry , Purkinje Cells/cytology , Staining and Labeling/methods , Time FactorsABSTRACT
Long-term depression (LTD) commonly affects learning and memory in various brain regions. Although cerebellar LTD absolutely requires the δ2 glutamate receptor (GluD2) that is expressed in Purkinje cells, LTD in other brain regions does not; why and how cerebellar LTD is regulated by GluD2 remains unelucidated. Here, we show that the activity-dependent phosphorylation of serine 880 (S880) in GluA2 AMPA receptor subunit, which is an essential step for AMPA receptor endocytosis during LTD induction, was impaired in GluD2-null cerebellum. In contrast, the basal phosphorylation levels of tyrosine 876 (Y876) in GluA2 were increased in GluD2-null cerebellum. An in vitro phosphorylation assay revealed that Y876 phosphorylation inhibited subsequent S880 phosphorylation. Conversely, Y876 dephosphorylation was sufficient to restore S880 phosphorylation and LTD induction in GluD2-null Purkinje cells. Furthermore, megakaryocyte protein tyrosine phosphatase (PTPMEG), which binds to the C terminus of GluD2, directly dephosphorylated Y876. These data indicate that GluD2 gates LTD by coordinating interactions between the two phosphorylation sites of the GluA2.
Subject(s)
Long-Term Synaptic Depression/physiology , Receptors, AMPA/physiology , Receptors, Glutamate/physiology , ADP-Ribosylation Factor 6 , ADP-Ribosylation Factors/physiology , Animals , Cerebellum/physiology , Guanine Nucleotide Exchange Factors/physiology , Mice , Mice, Knockout , Models, Neurological , Neuronal Plasticity/physiology , Patch-Clamp Techniques , Phosphorylation , Protein Interaction Domains and Motifs , Protein Subunits , Protein Tyrosine Phosphatase, Non-Receptor Type 4/physiology , Purkinje Cells/physiology , Receptors, AMPA/chemistry , Receptors, AMPA/genetics , Receptors, Glutamate/chemistry , Receptors, Glutamate/deficiency , Receptors, Glutamate/genetics , Serine/chemistry , Signal Transduction , Tyrosine/chemistryABSTRACT
Glutamate dehydrogenase (GDH), encoded by GLUD1, participates in the breakdown and synthesis of glutamate, the main excitatory neurotransmitter. In the CNS, besides its primary signaling function, glutamate is also at the crossroad of metabolic and neurotransmitter pathways. Importance of brain GDH was questioned here by generation of CNS-specific GDH-null mice (CnsGlud1(-/-)); which were viable, fertile and without apparent behavioral problems. GDH immunoreactivity as well as enzymatic activity were absent in Cns-Glud1(-/-) brains. Immunohistochemical analyses on brain sections revealed that the pyramidal cells of control animals were positive for GDH, whereas the labeling was absent in hippocampal sections of Cns-Glud1(-/-) mice. Electrophysiological recordings showed that deletion of GDH within the CNS did not alter synaptic transmission in standard conditions. Cns-Glud1(-/-) mice exhibited deficient oxidative catabolism of glutamate in astrocytes, showing that GDH is required for Krebs cycle pathway. As revealed by NMR studies, brain glutamate levels remained unchanged, whereas glutamine levels were increased. This pattern was favored by up-regulation of astrocyte-type glutamate and glutamine transporters and of glutamine synthetase. Present data show that the lack of GDH in the CNS modifies the metabolic handling of glutamate without altering synaptic transmission.
Subject(s)
Brain/enzymology , Gene Deletion , Glutamic Acid/metabolism , Receptors, Glutamate/deficiency , Receptors, Glutamate/genetics , Synaptic Transmission/genetics , Animals , Brain/pathology , Brain/physiology , Cells, Cultured , Female , Glutamate Dehydrogenase , Glutamine/metabolism , Male , Mice , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Neural Pathways/metabolism , Neural Pathways/pathology , Neural Pathways/physiopathology , Organ Culture Techniques , Receptors, Glutamate/physiology , Synaptic Transmission/physiologyABSTRACT
We developed an organotypic coculture preparation allowing fast and efficient identification of molecules that regulate developmental synapse elimination in the mammalian brain. This coculture consists of a cerebellar slice obtained from rat or mouse at postnatal day 9 (P9) or P10 and a medullary explant containing the inferior olive dissected from rat at embryonic day 15. We verified that climbing fibers (CFs), the axons of inferior olivary neurons, formed functional synapses onto Purkinje cells (PCs) in the cerebellum of cocultures. PCs were initially reinnervated by multiple CFs with similar strengths. Surplus CFs were eliminated subsequently, and the remaining CFs became stronger. These changes are similar to those occurring in developing cerebellum in vivo. Importantly, the changes in CF innervations in cocultures involved the same molecules required for CF synapse elimination in vivo, including NMDA receptor, type 1 metabotropic glutamate receptor and glutamate receptor δ2 (GluRδ2). We demonstrate that gain- and loss-of-function analyses can be efficiently performed by lentiviral-mediated overexpression and RNAi-induced knockdown of GluRδ2. Using this approach, we identified neuroligin-2 as a novel molecule that promotes CF synapse elimination in postsynaptic PCs. Thus, our coculture preparation will greatly facilitate the elucidation of molecular mechanisms of synapse elimination.
Subject(s)
Cell Adhesion Molecules, Neuronal/metabolism , Cerebellum/cytology , Cerebellum/embryology , Cerebellum/growth & development , Nerve Tissue Proteins/metabolism , Neurons/physiology , Synapses/physiology , Age Factors , Animals , Animals, Newborn , Biophysics , Cell Adhesion Molecules, Neuronal/genetics , Cell Line, Transformed , Coculture Techniques , Electric Stimulation , Electroporation , Embryo, Mammalian , Excitatory Amino Acid Agents/pharmacology , Excitatory Postsynaptic Potentials/drug effects , Excitatory Postsynaptic Potentials/genetics , Green Fluorescent Proteins/genetics , Humans , Medulla Oblongata/cytology , Membrane Proteins/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , MicroRNAs/metabolism , Mutation/genetics , Nerve Fibers/physiology , Nerve Tissue Proteins/genetics , Neurons/drug effects , Organ Culture Techniques , Patch-Clamp Techniques , RNA Interference/physiology , Receptors, Glutamate/deficiency , Receptors, N-Methyl-D-Aspartate/genetics , Receptors, N-Methyl-D-Aspartate/metabolism , Statistics, Nonparametric , Synapses/drug effects , TransfectionABSTRACT
Generation and regulation of orofacial movements involve complex mechanisms that include primary roles not only for dopamine but also the amino acid neurotransmitters γ-aminobutyric acid (GABA) and glutamate. However, the roles of individual GABA and glutamate receptor subtypes, subunits and associated processes are unclear. Here we outline studies of motor function in mutant mice with "knockout" of GABA and glutamate receptor subtypes. We then review systematic studies of orofacial movements in mutants with (i) "Knockout" of phospholipase C-related catalytically inactive protein (PRIP), which regulates cell surface expression of GABA(A) receptors containing a γ2 subunit, and: (ii) Heterozygous deletion of neuregulin-1 which, inter alia, regulates glutamate receptor-mediated processes. Each of GABAergic and glutamatergic sytems regulate specific topographies of orofacial movement both individually and via interactions with dopaminergic processes.
Subject(s)
Amino Acids/metabolism , Face/physiology , Movement/physiology , Receptors, GABA-A/metabolism , Receptors, Glutamate/metabolism , Amino Acids/genetics , Animals , Humans , Mice , Mice, Knockout , Receptors, GABA-A/deficiency , Receptors, Glutamate/deficiency , Signal Transduction/genetics , Signal Transduction/physiologyABSTRACT
D-serine (D-Ser) is an endogenous co-agonist for NMDA receptors and regulates neurotransmission and synaptic plasticity in the forebrain. D-Ser is also found in the cerebellum during the early postnatal period. Although D-Ser binds to the δ2 glutamate receptor (GluD2, Grid2) in vitro, its physiological significance has remained unclear. Here we show that D-Ser serves as an endogenous ligand for GluD2 to regulate long-term depression (LTD) at synapses between parallel fibers and Purkinje cells in the immature cerebellum. D-Ser was released mainly from Bergmann glia after the burst stimulation of parallel fibers in immature, but not mature, cerebellum. D-Ser rapidly induced endocytosis of AMPA receptors and mutually occluded LTD in wild-type, but not Grid2-null, Purkinje cells. Moreover, mice expressing mutant GluD2 in which the binding site for D-Ser was disrupted showed impaired LTD and motor dyscoordination during development. These results indicate that glial D-Ser regulates synaptic plasticity and cerebellar functions by interacting with GluD2.
Subject(s)
Cerebellum/cytology , Long-Term Synaptic Depression/physiology , Psychomotor Performance/physiology , Receptors, Glutamate/physiology , Serine/metabolism , Analysis of Variance , Animals , Animals, Newborn , Biophysics/methods , Cells, Cultured , Chromatography, High Pressure Liquid/methods , D-Amino-Acid Oxidase/deficiency , Electric Stimulation/methods , Excitatory Amino Acid Antagonists/pharmacology , Excitatory Postsynaptic Potentials/drug effects , Excitatory Postsynaptic Potentials/genetics , Glycine Agents/pharmacology , Green Fluorescent Proteins/genetics , Long-Term Synaptic Depression/drug effects , Mice , Mice, Transgenic , Microdialysis/methods , Mutation/genetics , Patch-Clamp Techniques , Phosphoglycerate Dehydrogenase/metabolism , Psychomotor Performance/drug effects , Purkinje Cells/drug effects , Purkinje Cells/physiology , Receptors, Glutamate/deficiency , Rotarod Performance Test/methods , Serine/pharmacology , Statistics, Nonparametric , Strychnine/pharmacology , Valine/analogs & derivatives , Valine/pharmacologyABSTRACT
The number of synaptic AMPA receptors (AMPARs) is the major determinant of synaptic strength and is differently regulated in input pathway-dependent and target cell type-dependent manners. In cerebellar Purkinje cells (PCs), the density of synaptic AMPARs is approximately five times lower at parallel fiber (PF) synapses than at climbing fiber (CF) synapses. However, molecular mechanisms underlying this biased synaptic distribution remain unclear. As a candidate molecule, we focused on glutamate receptor δ2 (GluRδ2 or GluD2), which is known to be efficiently trafficked to and selectively expressed at PF synapses in PCs. We applied postembedding immunogold electron microscopy to GluRδ2 knock-out (KO) and control mice, and measured labeling density for GluA1-4 at three excitatory synapses in the cerebellar molecular layer. In both control and GluRδ2-KO mice, GluA1-3 were localized at PF and CF synapses in PCs, while GluA2-4 were at PF synapses in interneurons. In control mice, labeling density for each of GluA1-3 was four to six times lower at PF-PC synapses than at CF-PC synapses. In GluRδ2-KO mice, however, their labeling density displayed a three- to fivefold increase at PF synapses, but not at CF synapses, thus effectively eliminating input pathway-dependent disparity between the two PC synapses. Furthermore, we found an unexpected twofold increase in labeling density for GluA2 and GluA3, but not GluA4, at PF-interneuron synapses, where we identified low but significant expression of GluRδ2. These results suggest that GluRδ2 is involved in a common mechanism that restricts the number of synaptic AMPARs at PF synapses in PCs and molecular layer interneurons.
Subject(s)
Neural Pathways/physiology , Purkinje Cells/physiology , Receptors, AMPA/physiology , Receptors, Glutamate/physiology , Synapses/physiology , Animals , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Nerve Fibers/physiology , Nerve Fibers/ultrastructure , Neural Pathways/ultrastructure , Purkinje Cells/ultrastructure , Receptors, AMPA/ultrastructure , Receptors, Glutamate/deficiency , Receptors, Glutamate/genetics , Synapses/genetics , Synapses/ultrastructureABSTRACT
The delta2 glutamate receptor (GluRdelta2) is expressed predominantly in cerebellar Purkinje cells. GluRdelta2 knock-out mice show impaired synaptogenesis and loss of long-term depression (LTD) at parallel fiber/Purkinje cell synapses, and persistent multiple climbing fiber (CF) innervation of Purkinje cells, resulting in severe ataxia. To identify domains critical for GluRdelta2 function, we produced various GluRdelta2 deletion constructs. Using lentiviral vectors, those constructs were expressed in Purkinje cells of GluRdelta2-deficient mice at postnatal day (P) 6 or 7, and rescue of abnormal phenotypes was examined beyond P30. Most constructs failed to rescue the defects of GluRdelta2-deficient mice, mainly because they were not efficiently transferred to the postsynaptic sites. However, a construct carrying only the extracellular N-terminal domain (NTD) and the intracellular C-terminal domain (CTD) linked with the fourth transmembrane domain of GluRdelta2 (NTD-TM4-CTD) caused incomplete, but significant rescue of ataxia, consistent with relatively better transport of the construct to the synapses. Notably, the expression of NTD-TM4-CTD in GluRdelta2-deficient Purkinje cells restored abrogated LTD, and aberrant CF territory in the molecular layer. Although the expression of NTD-TM4-CTD failed to rescue persistent multiple CF innervation of GluRdelta2-deficient Purkinje cells, a similar construct in which only TM4 was replaced with a transmembrane domain of CD4 successfully rescued the multiple CF innervation, probably due to more efficient transport of the protein to postsynaptic sites. These results suggest that NTD and CTD are critical domains of GluRdelta2, which functions substantially without conventional ligand binding and ion channel structures.
Subject(s)
Purkinje Cells/physiology , Receptors, Glutamate/chemistry , Animals , Blotting, Western , Cerebellar Ataxia/genetics , Immunohistochemistry , Long-Term Synaptic Depression/physiology , Mice , Mice, Knockout , Microscopy, Confocal , Patch-Clamp Techniques , Phenotype , Receptors, Glutamate/deficiency , Receptors, Glutamate/genetics , Synapses/physiologyABSTRACT
The delta2 glutamate receptor (GluRdelta2; GluD2), which is predominantly expressed on postsynaptic sites at parallel fiber (PF)-Purkinje cell synapses in the cerebellum, plays two crucial roles in the cerebellum: the formation of PF synapses and the regulation of long-term depression (LTD), a form of synaptic plasticity underlying motor learning. Although the induction of LTD and motor learning absolutely require signaling via the cytoplasmic C-terminal domain of GluD2, the mechanisms by which GluD2 regulates PF synaptogenesis have remained unclear. Here, we examined the role of the extracellular N-terminal domain (NTD) of GluD2 on PF synaptogenesis by injecting Sindbis virus carrying wild-type (GluD2(wt)) or mutant GluD2 into the subarachnoid supracerebellar space of GluD2-null mice. Remarkably, the expression of GluD2(wt), but not of a mutant GluD2 lacking the NTD (GluD2(DeltaNTD)), rapidly induced PF synapse formation and rescued gross motor dyscoordination in adult GluD2-null mice just 1 d after injection. In addition, although the kainate receptor GluR6 (GluK2) did not induce PF synaptogenesis, a chimeric GluK2 that contained the NTD of GluD2 (GluD2(NTD)-GluK2) did. Similarly, GluD2(wt) and GluD2(NTD)-GluK2, but not GluD2(DeltaNTD), induced synaptogenesis in heterologous cells in vitro. In contrast, LTD was restored in GluD2-null Purkinje cells expressing a mutant GluD2 lacking the NTD. These results indicate that the NTD of GluD2 is necessary and sufficient for the function of GluD2 in the regulation of PF-Purkinje cell synaptogenesis. Furthermore, our results suggest that GluD2 differently regulates PF synaptogenesis and cerebellar LTD through the extracellular NTD and the cytoplasmic C-terminal end, respectively.
Subject(s)
Cerebellum/cytology , Presynaptic Terminals/physiology , Protein Structure, Tertiary/physiology , Receptors, Glutamate/chemistry , Synapses/metabolism , Amino Acid Sequence/genetics , Analysis of Variance , Animals , Animals, Newborn , Cell Line, Transformed , Cerebellum/physiology , Disease Models, Animal , Electric Stimulation/methods , Excitatory Postsynaptic Potentials/drug effects , Excitatory Postsynaptic Potentials/genetics , Gene Expression/genetics , Gene Transfer Techniques , Green Fluorescent Proteins/genetics , Humans , Long-Term Synaptic Depression/genetics , Mice , Mice, Knockout , Microscopy, Electron, Transmission , Models, Molecular , Motor Activity/genetics , Motor Skills Disorders/genetics , Motor Skills Disorders/therapy , Mutation/genetics , Nerve Fibers/physiology , Neuronal Plasticity , Patch-Clamp Techniques , Presynaptic Terminals/ultrastructure , Protein Structure, Tertiary/genetics , Protein Transport/genetics , Purkinje Cells/cytology , Purkinje Cells/drug effects , Purkinje Cells/ultrastructure , Receptors, Glutamate/deficiency , Receptors, Glutamate/genetics , Recruitment, Neurophysiological , Sindbis Virus/physiology , Synapses/drug effects , Synapses/genetics , Synapses/ultrastructure , Transfection/methods , Vesicular Glutamate Transport Protein 1/metabolismABSTRACT
Previous reports utilizing pharmacological antagonists implicate kainate receptor (KAR) activation in the development of morphine tolerance, dependence, conditioned place preference (CPP), and locomotor sensitization, but the role of glutamate receptor (GluR) 5-containing KAR in these effects remains unclear because of limited selectivity of the inhibitors employed. Therefore, we examined responses to systemic morphine treatment in mice expressing a constitutive deletion of GluR5 [GluR5 knockout (KO)]. Unlike wild-type (WT) littermates, GluR5 KO mice do not develop tolerance after repeated morphine administration by subcutaneous injection or via subcutaneous pellet implantation. In contrast, GluR5 KO mice do not differ from WT with respect to thermal or mechanical nociceptive thresholds, acute morphine antinociception, morphine disposition in the central nervous system (CNS), morphine physical dependence as revealed by naloxone-precipitated withdrawal or development of place preference and locomotor hyperresponsiveness after chronic morphine administration. It is surprising that continuous subcutaneous infusion of the GluR2/GluR5-preferring antagonist LY293558 [(3S,4aR,6R,8aR)-6-[2-(1(2)H-tetrazole-5-yl)ethyl]decahydroisoquinoline-3-carboxylic acid] decreased the number of naloxone-precipitated jumps to a similar extent in WT and GluR5 KO mice. We observed opioid-induced hypersensitivity in both groups during morphine withdrawal as demonstrated by equivalent reductions in thermal and mechanical thresholds; however, this hypersensitivity was not evident during continuous systemic morphine infusion. These data collectively indicate that KARs containing the GluR5 subunit contribute to the development of morphine tolerance without affecting nociceptive thresholds, morphine analgesia, or disposition in CNS of morphine and its metabolite morphine-3-glucuronide. In addition, constitutive deletion of GluR5 does not alter the morphine-induced increase in locomotor activity or the acquisition of morphine reward as measured by a CPP paradigm.
Subject(s)
Drug Tolerance/physiology , Gene Deletion , Morphine/adverse effects , Receptors, Kainic Acid/chemistry , Animals , Female , Injections, Subcutaneous , Male , Mice , Mice, Inbred C57BL , Morphine/metabolism , Morphine/toxicity , Morphine Derivatives/metabolism , Physiological Phenomena , Receptors, Glutamate/deficiency , Receptors, Glutamate/genetics , Receptors, Glutamate/physiology , Receptors, Kainic Acid/deficiency , Receptors, Kainic Acid/genetics , Receptors, Kainic Acid/physiologyABSTRACT
Presynaptic autoreceptors modulate transmitter release at many synapses. At the mossy fiber to CA3 pyramidal cell (mf-CA3) synapse, two types of glutamatergic autoreceptors have been identified: transmitter release is reportedly suppressed by metabotropic glutamate receptors (mGluRs) and augmented by kainate receptors (KARs). However, the net effect of these autoreceptors when activated by endogenous glutamate is unknown. Here, we show that during low-frequency mossy fiber stimulation, glutamate acting through presynaptic mGluRs substantially suppresses transmitter release. However, using similar recording conditions, we find that presynaptic KARs are insufficient to facilitate transmitter release over a wide range of mossy fiber stimulus frequencies, indicating that the uniquely robust mf-CA3 short-term plasticity is KAR independent. Furthermore, we report that actions generally attributed to presynaptic KARs are likely due to activation of recurrent CA3 network activity. Thus, negative feedback via presynaptic mGluRs is the dominant mode of glutamatergic autoregulation at the mf-CA3 synapse.
Subject(s)
Autoreceptors/physiology , Hippocampus/cytology , Mossy Fibers, Hippocampal/physiology , Neurons/physiology , Receptors, Glutamate/physiology , Synapses/physiology , Animals , Animals, Newborn , Biophysics , Calcium/pharmacology , Cyclopropanes/pharmacology , Electric Stimulation/methods , Excitatory Amino Acid Agonists/pharmacology , Excitatory Amino Acid Antagonists/pharmacology , Excitatory Postsynaptic Potentials/drug effects , Excitatory Postsynaptic Potentials/genetics , Excitatory Postsynaptic Potentials/physiology , Glycine/analogs & derivatives , Glycine/pharmacology , In Vitro Techniques , Kainic Acid/pharmacology , Mice , Mice, Knockout , Patch-Clamp Techniques/methods , Presynaptic Terminals/physiology , Rats , Receptors, Glutamate/deficiency , Sodium Channel Blockers/pharmacology , Synapses/drug effects , Tetrodotoxin/pharmacologyABSTRACT
Long-term depression (LTD) of synaptic transmission at parallel fiber (PF)-Purkinje cell synapses is thought to regulate motor learning and memory formation in the cerebellum. Neuronal activity-evoked protein kinase C (PKC) activation is required for the induction of LTD. In addition, the delta2 glutamate receptor (GluRdelta2), which is predominantly expressed at PF-Purkinje cell synapses, is indispensable for the induction of LTD; however, the mechanisms by which GluRdelta2 regulates LTD and its relationship with PKC activation remain elusive. Interestingly, GluRdelta2 is phosphorylated by PKC on serine 945 (Ser945) near its C-terminus and a postsynaptic protein S-SCAM, which could potentially regulate glutamate receptor trafficking and synaptic plasticity, binds to the extreme C-terminus of GluRdelta2 in a phosphorylation-dependent manner on Ser945. Here, using a Sindbis-based virus expression approach, we show that a mutant GluRdelta2, in which alanine replaced Ser945 and did not undergo PKC phosphorylation, was normally localized at the postsynaptic sites of PF-Purkinje cell synapses. In addition, like wild-type GluRdelta2, the phosphorylation-disrupted GluRdelta2 successfully rescued abrogated LTD in GluRdelta2-null Purkinje cells. These results indicate that Ser945, a major PKC phosphorylation site of of GluRdelta2, may not play a crucial role in induction of LTD in the cerebellum.
Subject(s)
Cerebellum/metabolism , Long-Term Synaptic Depression/physiology , Phosphoserine/metabolism , Receptors, Glutamate/metabolism , Animals , Cerebellum/cytology , Electrophysiology , Mice , Mice, Knockout , Patch-Clamp Techniques , Phosphorylation , Purkinje Cells/metabolism , Receptors, Glutamate/deficiency , Receptors, Glutamate/geneticsABSTRACT
Glutamate receptor (GluR) delta2 selectively expressed in cerebellar Purkinje cells (PCs) plays key roles in long-term depression (LTD) induction at parallel fiber (PF)-PC synapses, motor learning, the matching and connection of PF-PC synapses in developing and adult cerebella, the elimination of multiple climbing fibers (CFs) during development, and the regulation of CF territory on PCs. However, it remains unsolved how GluRdelta2 regulates cerebellar synaptic plasticity, PF-PC synapse formation, and CF wiring. One possible signaling mechanism through GluRdelta2 is signaling by protein-protein interactions. The C-terminal region of GluRdelta2 contains at least three domains for protein-protein interactions. The PDZ (postsynaptic density-95/Discs large/zona occludens 1)-binding domain at the C terminal, named as the T site, interacts with several postsynaptic density proteins. Here, we generated GluRdelta2DeltaT mice carrying mutant GluRdelta2 lacking the T site. There were no significant differences in the amount of receptor proteins at synapses, histological features, and the fine structures of PF-PC synapses between wild-type and GluRdelta2DeltaT mice. However, LTD induction at PF-PC synapses and improvement in the accelerating rotarod test were impaired in GluRdelta2DeltaT mice. Furthermore, CF territory expanded distally and ectopic innervation of CFs occurred at distal dendrites in GluRdelta2DeltaT mice, but the elimination of surplus CF innervation at proximal dendrites appeared to proceed normally. These results suggest that the C-terminal T site of GluRdelta2 is essential for LTD induction and the regulation of CF territory but is dispensable for PF-PC synapse formation and the elimination of surplus CFs at proximal dendrites during development.
Subject(s)
Cerebellum/cytology , Long-Term Synaptic Depression/physiology , Nerve Fibers/physiology , Purkinje Cells/physiology , Receptors, Glutamate/physiology , Synapses/physiology , Analysis of Variance , Animals , Calbindins , Dextrans , Dose-Response Relationship, Radiation , Electric Stimulation/methods , Long-Term Synaptic Depression/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Microscopy, Electron, Transmission , Motor Activity/genetics , Nerve Fibers/ultrastructure , Patch-Clamp Techniques/methods , Protein Structure, Tertiary , Purkinje Cells/ultrastructure , Receptors, Glutamate/deficiency , S100 Calcium Binding Protein G/metabolism , Synapses/ultrastructure , Vesicular Glutamate Transport Protein 2/metabolismABSTRACT
The orphan glutamate-like receptor GluRdelta2 is predominantly expressed in Purkinje cells of the central nervous system. The classification of GluRdelta2 to the ionotropic glutamate receptor family is based on sequence similarities, because GluRdelta2 does not form functional homomeric glutamate-gated ion channels in transfected cells. Studies in GluRdelta2(-/-) knockout mice as well as in mice with naturally occurring mutations in the GluRdelta2 gene have demonstrated an essential role of GluRdelta2 in cerebellar long-term depression, motor learning, motor coordination, and synaptogenesis. However, the lack of a known agonist has hampered investigations on the function of GluRdelta2. In this study, the ligand-binding core of GluRdelta2 (GluRdelta2-S1S2) was found to bind neutral amino acids such as D-serine and glycine, as demonstrated by isothermal titration calorimetry. Direct evidence for binding of D-serine and structural rearrangements in the binding cleft of GluRdelta2-S1S2 is provided by x-ray structures of GluRdelta2-S1S2 in its apo form and in complex with D-serine. Functionally, D-serine and glycine were shown to inactivate spontaneous ion-channel conductance in GluRdelta2 containing the lurcher mutation (EC(50) values, 182 and 507 microM, respectively). These data demonstrate that the GluRdelta2 ligand-binding core is capable of binding ligands and that cleft closure of the ligand-binding core can induce conformational changes that alter ion permeation.
Subject(s)
Glycine/metabolism , Receptors, Glutamate/metabolism , Serine/metabolism , Animals , Binding Sites , Kinetics , Ligands , Mice , Mice, Knockout , Models, Molecular , Protein Binding , Protein Conformation , Receptors, Glutamate/chemistry , Receptors, Glutamate/deficiency , Receptors, Glutamate/genetics , X-Ray DiffractionABSTRACT
Axonal growth cones are responsible for the correct guidance of developing axons and the establishment of functional neural networks. They are highly motile because of fast and continuous rearrangements of their actin-rich cytoskeleton. Here we have used live imaging of axonal growth cones of hippocampal neurons in culture and quantified their motility with a temporal resolution of 2 s. Using novel methods of analysis of growth cone dynamics, we show that transient activation of kainate receptors by bath-applied kainate induced a fast and reversible growth cone stalling. This effect depends on electrical activity and can be mimicked by the transient discharge of action potentials elicited in the neuron by intracellular current injections at the somatic level through a patch pipette. Growth cone stalling induced by electrical stimulation is mediated by calcium entry from the extracellular medium as well as by calcium release from intracellular stores that define spatially restricted microdomains directly affecting cytoskeletal dynamics. We propose that growth cone motility is dynamically controlled by transient bursts of spontaneous electrical activity, which constitutes a prominent feature of developing neural networks in vivo.