RESUMO
Elucidation of molecular mechanisms that regulate synapse formation is required for the understanding of neural wiring, higher brain functions, and mental disorders. Despite the wealth of in vitro information, fundamental questions about how glutamatergic synapses are formed in the mammalian brain remain unanswered. Glutamate receptor (GluR) delta2 is essential for cerebellar synapse formation in vivo. Here, we show that the N-terminal domain (NTD) of GluRdelta2 interacts with presynaptic neurexins (NRXNs) through cerebellin 1 precursor protein (Cbln1). The synaptogenic activity of GluRdelta2 is abolished in cerebellar primary cultures from Cbln1 knockout mice and is restored by recombinant Cbln1. Knockdown of NRXNs in cerebellar granule cells also hinders the synaptogenic activity of GluRdelta2. Both the NTD of GluRdelta2 and the extracellular domain of NRXN1beta suppressed the synaptogenic activity of Cbln1 in cerebellar primary cultures and in vivo. These results suggest that GluRdelta2 mediates cerebellar synapse formation by interacting with presynaptic NRXNs through Cbln1.
Assuntos
Cerebelo/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Moléculas de Adesão de Célula Nervosa/metabolismo , Precursores de Proteínas/metabolismo , Receptores de Glutamato/metabolismo , Sinapses , Animais , Proteínas de Ligação ao Cálcio , Linhagem Celular , Células Cultivadas , Humanos , CamundongosRESUMO
Noncompetitive NMDA receptor (NMDAR) antagonists like phencyclidine (PCP) and ketamine cause psychosis-like symptoms in healthy humans, exacerbate schizophrenia symptoms in people with the disorder, and disrupt a range of schizophrenia-relevant behaviors in rodents, including hyperlocomotion. This is negated in mice lacking the GluN2D subunit of the NMDAR, suggesting the GluN2D subunit mediates the hyperlocomotor effects of these drugs. However, the role of GluN2D in mediating other schizophrenia-relevant NMDAR antagonist-induced behavioral disturbances, and in both sexes, is unclear. This study aimed to investigate the role of the GluN2D subunit in mediating schizophrenia-relevant behaviors induced by a range of NMDA receptor antagonists. Using both male and female GluN2D knockout (KO) mice, we examined the effects of the NMDAR antagonist's PCP, the S-ketamine enantiomer (S-ket), and the ketamine metabolite R-norketamine (R-norket) on locomotor activity, anxiety-related behavior, and recognition and short-term spatial memory. GluN2D-KO mice showed a blunted locomotor response to R-norket, S-ket, and PCP, a phenotype present in both sexes. GluN2D-KO mice of both sexes showed an anxious phenotype and S-ket, R-norket, and PCP showed anxiolytic effects that were dependent on sex and genotype. S-ket disrupted spatial recognition memory in females and novel object recognition memory in both sexes, independent of genotype. This datum identifies a role for the GluN2D subunit in sex-specific effects of NMDAR antagonists and on the differential effects of the R- and S-ket enantiomers.
Assuntos
Ketamina , Animais , Feminino , Humanos , Masculino , Camundongos , Ketamina/farmacologia , Fenciclidina/farmacologia , Receptores de N-Metil-D-Aspartato/metabolismo , Reconhecimento PsicológicoRESUMO
Ketamine, an N-methyl-d-aspartate (NMDA) receptor antagonist, has attracted attention for its acute and sustained antidepressant effects in patients with depression. Hydroxynorketamine (HNK), a metabolite of ketamine, exerts antidepressant effects without exerting ketamine's side effects and has attracted much attention in recent years. However, the detailed pharmacological mechanism of action of HNK remains unclear. We previously showed that the GluN2D NMDA receptor subunit is important for sustained antidepressant-like effects of (R)-ketamine. Therefore, we investigated whether the GluN2D subunit is involved in antidepressant-like effects of (2R,6R)-HNK and (2S,6S)-HNK. Treatment with (2R,6R)-HNK but not (2S,6S)-HNK exerted acute and sustained antidepressant-like effects in the tail-suspension test in wildtype mice. Interestingly, sustained antidepressant-like effects of (2R,6R)-HNK were abolished in GluN2D-knockout mice, whereas acute antidepressant-like effects were maintained in GluN2D-knockout mice. When expression levels of GluN2A and GluN2B subunits were evaluated, a decrease in GluN2B protein expression in the nucleus accumbens was found in stressed wildtype mice but not in stressed GluN2D-knockout mice. These results suggest that the GluN2D subunit and possibly the GluN2B subunit are involved in the sustained antidepressant-like effect of (2R,6R)-HNK.
Assuntos
Ketamina , Ketamina/análogos & derivados , Humanos , Camundongos , Animais , Ketamina/farmacologia , Ketamina/metabolismo , Depressão/tratamento farmacológico , Depressão/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Camundongos Knockout , Antidepressivos/farmacologiaRESUMO
Synaptic AMPAR expression controls the strength of excitatory synaptic transmission and plasticity. An excess of synaptic AMPARs leads to epilepsy in response to seizure-inducible stimulation. The appropriate regulation of AMPARs plays a crucial role in the maintenance of the excitatory/inhibitory synaptic balance; however, the detailed mechanisms underlying epilepsy remain unclear. Our previous studies have revealed that a key modification of AMPAR trafficking to and from postsynaptic membranes is the reversible, posttranslational S-palmitoylation at the C-termini of receptors. To clarify the role of palmitoylation-dependent regulation of AMPARs in vivo, we generated GluA1 palmitoylation-deficient (Cys811 to Ser substitution) knock-in mice. These mutant male mice showed elevated seizure susceptibility and seizure-induced neuronal activity without impairments in synaptic transmission, gross brain structure, or behavior at the basal level. Disruption of the palmitoylation site was accompanied by upregulated GluA1 phosphorylation at Ser831, but not at Ser845, in the hippocampus and increased GluA1 protein expression in the cortex. Furthermore, GluA1 palmitoylation suppressed excessive spine enlargement above a certain size after LTP. Our findings indicate that an abnormality in GluA1 palmitoylation can lead to hyperexcitability in the cerebrum, which negatively affects the maintenance of network stability, resulting in epileptic seizures.SIGNIFICANCE STATEMENT AMPARs predominantly mediate excitatory synaptic transmission. AMPARs are regulated in a posttranslational, palmitoylation-dependent manner in excitatory synapses of the mammalian brain. Reversible palmitoylation dynamically controls synaptic expression and intracellular trafficking of the receptors. Here, we generated GluA1 palmitoylation-deficient knock-in mice to clarify the role of AMPAR palmitoylation in vivo We showed that an abnormality in GluA1 palmitoylation led to hyperexcitability, resulting in epileptic seizure. This is the first identification of a specific palmitoylated protein critical for the seizure-suppressing process. Our data also provide insight into how predicted receptors such as AMPARs can effectively preserve network stability in the brain. Furthermore, these findings help to define novel key targets for developing anti-epileptic drugs.
Assuntos
Hipocampo/metabolismo , Hipocampo/fisiopatologia , Palmitatos/metabolismo , Receptores de AMPA/deficiência , Convulsões/metabolismo , Convulsões/fisiopatologia , Animais , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Técnicas de Cultura de Órgãos , Receptores de AMPA/genética , Convulsões/genéticaRESUMO
Although the N-methyl-D-aspartate receptor antagonist ketamine has attracted attention because of its rapid and sustained antidepressant effects in depressed patients, its side effects have raised some concerns. Ketamine is a racemic mixture of equal amounts of the enantiomers (R)-ketamine and (S)-ketamine. The neural mechanisms that underlie the differential effects of these enantiomers remain unclear. We investigated cognitive impairment that was induced by ketamine and its enantiomers in N-methyl-D-aspartate GluN2D receptor subunit knockout (GluN2D-KO) mice. In the novel object recognition test, (RS)-ketamine and (S)-ketamine caused cognitive impairment in both wild-type and GluN2D-KO mice, whereas (R)-ketamine induced such cognitive impairment only in wild-type mice. The present results suggest that the GluN2D subunit plays an important role in cognitive impairment that is induced by (R)-ketamine, whereas this subunit does not appear to be involved in cognitive impairment that is induced by (RS)-ketamine or (S)-ketamine.
Assuntos
Antidepressivos/efeitos adversos , Disfunção Cognitiva/induzido quimicamente , Disfunção Cognitiva/metabolismo , Ketamina/efeitos adversos , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Antidepressivos/química , Antidepressivos/farmacologia , Ketamina/química , Ketamina/farmacologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de N-Metil-D-Aspartato/genética , Reconhecimento Psicológico/efeitos dos fármacos , Reconhecimento Psicológico/fisiologiaRESUMO
We investigated the rapid and sustained antidepressant effects of enantiomers of ketamine in N-methyl-d-aspartate (NMDA) receptor GluN2D subunit knockout (GluN2D-KO) mice. Intraperitoneal administration of ketamine or its enantiomers 10 min before the tail-suspension test exerted significant antidepressant effects on restraint stress-induced depression in both wildtype and GluN2D-KO mice. The antidepressant effects of (RS)-ketamine and (S)-ketamine were sustained 96 h after the injection in both wildtype and GluN2D-KO mice, but such sustained antidepressant effects of (R)-ketamine were only observed in wildtype mice. These data suggest that the GluN2D subunit is critical for the sustained antidepressant effects of (R)-ketamine.
Assuntos
Antidepressivos/farmacologia , Ketamina/farmacologia , Receptores de N-Metil-D-Aspartato/fisiologia , Animais , Antidepressivos/administração & dosagem , Antidepressivos/uso terapêutico , Depressão/tratamento farmacológico , Injeções Intraperitoneais , Ketamina/administração & dosagem , Ketamina/uso terapêutico , Camundongos Endogâmicos C57BL , EstereoisomerismoRESUMO
Glutamate directly activates N-methyl-d-aspartate (NMDA) receptors on presynaptic inhibitory interneurons and enhances GABA release, altering the excitatory-inhibitory balance within a neuronal circuit. However, which class of NMDA receptors is involved in the detection of glutamate spillover is not known. GluN2D subunit-containing NMDA receptors are ideal candidates as they exhibit a high affinity for glutamate. We now show that cerebellar stellate cells express both GluN2B and GluN2D NMDA receptor subunits. Genetic deletion of GluN2D subunits prevented a physiologically relevant, stimulation-induced, lasting increase in GABA release from stellate cells [long-term potentiation of inhibitory transmission (I-LTP)]. NMDA receptors are tetramers composed of two GluN1 subunits associated to either two identical subunits (di-heteromeric receptors) or to two different subunits (tri-heteromeric receptors). To determine whether tri-heteromeric GluN2B/2D NMDA receptors mediate I-LTP, we tested the prediction that deletion of GluN2D converts tri-heteromeric GluN2B/2D to di-heteromeric GluN2B NMDA receptors. We find that prolonged stimulation rescued I-LTP in GluN2D knockout mice, and this was abolished by GluN2B receptor blockers that failed to prevent I-LTP in wild-type mice. Therefore, NMDA receptors that contain both GluN2D and GluN2B mediate the induction of I-LTP. Because these receptors are not present in the soma and dendrites, presynaptic tri-heteromeric GluN2B/2D NMDA receptors in inhibitory interneurons are likely to mediate the cross talk between excitatory and inhibitory transmission.
Assuntos
Cerebelo/fisiologia , Ácido Glutâmico/fisiologia , Interneurônios/fisiologia , Inibição Neural/fisiologia , Terminações Pré-Sinápticas/fisiologia , Receptores de N-Metil-D-Aspartato/fisiologia , Ácido gama-Aminobutírico/fisiologia , Animais , Feminino , Neurônios GABAérgicos/fisiologia , Potenciação de Longa Duração , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de N-Metil-D-Aspartato/genéticaRESUMO
Development of correct topographical connections between peripheral receptors and central somatosensory stations requires activity-dependent synapse refinement, in which the NMDA type of glutamate receptors plays a key role. Here we compared functional roles of GluN2B (GluRε2 or NR2B) and GluN2D (GluRε4 or NR2D), two major regulatory subunits of neonatal NMDA receptors, in development of whisker-related patterning at trigeminal relay stations. Compared with control littermates, both the appearance of whisker-related patterning and the termination of the critical period, as assessed by unilateral infraorbital nerve transection, were delayed by nearly a day in the somatosensory cortex of GluN2B(+/-) mice but advanced by nearly a day in GluN2D(-/-) mice. Similar temporal shifts were found at subcortical relay stations in the thalamus and brainstem of GluN2B(+/-) and GluN2D(-/-) mice. In comparison, the magnitude of lesion-induced critical period plasticity in the somatosensory cortex, as assessed following row-C whisker removal, was normal in both mutants. Thus, GluN2B and GluN2D play counteractive roles in temporal development and maturation of somatosensory maps without affecting the magnitude of critical period plasticity. To understand the opposing action, we then examined neuronal and synaptic expressions of the two subunits along the trigeminal pathway. At each trigeminal station, GluN2B was predominant at asymmetrical synapses of non-GABAergic neurons, whereas GluN2D was selective to asymmetrical synapses of GABAergic neurons. Together, our findings suggest that GluN2B expressed at glutamatergic synapses on glutamatergic projection neurons facilitates refinement of ascending pathway synapses directly, whereas GluN2D expressed at glutamatergic synapses on GABAergic interneurons delays it indirectly.
Assuntos
Neurogênese/fisiologia , Plasticidade Neuronal/fisiologia , Receptores de N-Metil-D-Aspartato/metabolismo , Córtex Somatossensorial/crescimento & desenvolvimento , Animais , Animais Recém-Nascidos , Padronização Corporal/fisiologia , Mapeamento Encefálico , Imunofluorescência , Imuno-Histoquímica , Hibridização In Situ , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/citologia , Neurônios/metabolismo , Córtex Somatossensorial/metabolismo , Vibrissas/inervaçãoRESUMO
The neural and genetic factors underlying chronic tolerance to alcohol are currently unclear. The GluN2Aâ N-methyl-D-aspartate receptors (NMDAR) subunit and the NMDAR-anchoring protein PSD-95 mediate acute alcohol intoxication and represent putative mechanisms mediating tolerance. We found that chronic intermittent ethanol exposure (CIE) did not produce tolerance [loss of righting reflex (LORR)] or withdrawal-anxiety in C57BL/6J, GluN2A or PSD-95 knockout mice assayed 2-3 days later. However, significant tolerance to LORR was evident 1 day after CIE in C57BL/6J and PSD-95 knockouts, but absent in GluN2A knockouts. These data suggest a role for GluN2A in tolerance, extending evidence that human GluN2A gene variation is involved in alcohol dependence.
Assuntos
Intoxicação Alcoólica/genética , Ansiedade/genética , Depressores do Sistema Nervoso Central/farmacologia , Tolerância a Medicamentos/genética , Etanol/farmacologia , Guanilato Quinases/genética , Proteínas de Membrana/genética , Receptores de N-Metil-D-Aspartato/genética , Síndrome de Abstinência a Substâncias/genética , Animais , Proteína 4 Homóloga a Disks-Large , Camundongos , Camundongos KnockoutRESUMO
Proper regulation of N-methyl-D-aspartate-type glutamate receptor (NMDA receptor) expression is responsible for excitatory synaptic functions in the mammalian brain. NMDA receptor dysfunction can cause various neuropsychiatric disorders and neurodegenerative diseases. Posttranslational protein S-palmitoylation, the covalent attachment of palmitic acid to intracellular cysteine residues via thioester bonds, occurs in the carboxyl terminus of GluN2B, which is the major regulatory NMDA receptor subunit. Mutations of three palmitoylatable cysteine residues in the membrane-proximal cluster of GluN2B to non-palmitoylatable serine (3CS) lead to the dephosphorylation of GluN2B Tyr1472 in the hippocampus and cerebral cortex, inducing a reduction in the surface expression of GluN2B-containig NMDA receptors. Furthermore, adult GluN2B 3CS homozygous mice demonstrated a definite clasping response without abnormalities in the gross brain structure, other neurological reflexes, or expression levels of synaptic proteins in the cerebrum. This behavioral disorder, observed in the GluN2B 3CS knock-in mice, indicated that complex higher brain functions are coordinated through the palmitoylation-dependent regulation of NMDA receptors in excitatory synapses.
RESUMO
Elucidation of molecular mechanisms of synapse formation is a prerequisite for the understanding of neural wiring, higher brain functions, and mental disorders. The trans-synaptic interaction of postsynaptic glutamate receptor δ2 (GluRδ2) and presynaptic neurexins (NRXNs) through cerebellin precursor protein 1 (Cbln1) mediates synapse formation in vivo in the cerebellum. Here, we asked how the trans-synaptic triad induces synapse formation. Native GluRδ2 existed as a tetramer in the membrane, whereas the N-terminal domain (NTD) of GluRδ2 formed a stable homodimer. When incubated with cultured mouse cerebellar granule cells (GCs), dimeric GluRδ2-NTD and Cbln1 exerted little effect on the accumulation of punctate immunostaining signals for Bassoon and vesicular glutamate transporter 1 in GC axons. However, tetramerized GluRδ2-NTD stimulated the accumulation of these presynaptic proteins in the axons. Analysis of Cbln1 mutants suggested that the binding sites of GluRδ2 and NRXN1ß on Cbln1 are differential. Furthermore, there was no competition in the binding to Cbln1 between GluRδ2-NTD and the extracellular domain (ECD) of NRXN1ß. Thus, GluRδ2 and Cbln1 interacted with each other rather independently of Cbln1-NRXN1ß interaction and vice versa. Gel filtration and isothermal titration calorimetry analyses consistently showed that dimeric GluRδ2-NTD and hexameric Cbln1 assembled in the 1:1 ratio, whereas hexameric Cbln1 and the laminin-neurexin-sex hormone-binding globulin domain of NRXN1ß-ECD assembled in the 1:2 ratio. Thus, the synaptogenic triad is assembled from tetrameric GluRδ2, hexameric Cbln1, and monomeric NRXN in the ratio of 1:2:4. These results suggest that GluRδ2 triggers synapse formation by clustering four NRXNs through triad formation.
Assuntos
Glicoproteínas/metabolismo , Neuropeptídeos/metabolismo , Receptores de Glutamato/metabolismo , Sinapses/metabolismo , Animais , Células Cultivadas , Cerebelo/citologia , Cerebelo/metabolismo , Feminino , Masculino , Camundongos , Modelos Moleculares , Mutação , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Ligação Proteica , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Globulina de Ligação a Hormônio Sexual/metabolismo , Proteína Vesicular 1 de Transporte de Glutamato/metabolismoRESUMO
Interleukin-1 receptor accessory protein (IL-1RAcP) is the essential component of receptor complexes mediating immune responses to interleukin-1 family cytokines. IL-1RAcP in the brain exists in two isoforms, IL-1RAcP and IL-1RAcPb, differing only in the C-terminal region. Here, we found robust synaptogenic activities of IL-1RAcP in cultured cortical neurons. Knockdown of IL-1RAcP isoforms in cultured cortical neurons suppressed synapse formation as indicated by decreases of active zone protein Bassoon puncta and dendritic protrusions. IL-1RAcP recovered the accumulation of presynaptic Bassoon puncta, while IL-1RAcPb rescued both Bassoon puncta and dendritic protrusions. Consistently, the expression of IL-1RAcP in cortical neurons enhances the accumulation of Bassoon puncta and that of IL-1RAcPb stimulated both Bassoon puncta accumulation and spinogenesis. IL-1RAcP interacted with protein tyrosine phosphatase (PTP) δ through the extracellular domain. Mini-exon peptides in the Ig-like domains of PTPδ splice variants were critical for their efficient binding to IL-1RAcP. The synaptogenic activities of IL-1RAcP isoforms were diminished in cortical neurons from PTPδ knock-out mice. Correspondingly, PTPδ required IL-1RAcPb to induce postsynaptic differentiation. Thus, IL-1RAcPb bidirectionally regulated synapse formation of cortical neurons. Furthermore, the spine densities of cortical and hippocampal pyramidal neurons were reduced in IL-1RAcP knock-out mice lacking both isoforms. These results suggest that IL-1RAcP isoforms function as trans-synaptic cell adhesion molecules in the brain and organize synapse formation. Thus, IL-1RAcP represents an interesting molecular link between immune systems and synapse formation in the brain.
Assuntos
Moléculas de Adesão Celular/metabolismo , Proteína Acessória do Receptor de Interleucina-1/fisiologia , Neurônios/fisiologia , Sinapses/fisiologia , Aminoácidos , Animais , Animais Recém-Nascidos , Proteínas de Ligação ao Cálcio , Moléculas de Adesão Celular/genética , Células Cultivadas , Córtex Cerebral/citologia , Técnicas de Cocultura , Fibroblastos/fisiologia , Proteínas de Fluorescência Verde/genética , Humanos , Proteína Acessória do Receptor de Interleucina-1/classificação , Proteína Acessória do Receptor de Interleucina-1/genética , Camundongos , Camundongos Knockout , Moléculas de Adesão de Célula Nervosa/genética , Moléculas de Adesão de Célula Nervosa/metabolismo , Neurônios/citologia , Terminações Pré-Sinápticas/fisiologia , Ligação Proteica/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína/genética , RNA Interferente Pequeno/metabolismo , Proteínas Tirosina Fosfatases Classe 4 Semelhantes a Receptores/deficiência , Transdução de Sinais , Ressonância de Plasmônio de Superfície , Transfecção , Proteína Vesicular 1 de Transporte de Glutamato/metabolismo , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/metabolismoRESUMO
Gγ7 is enriched in striatum and forms a heterotrimeric complex with Gαolf /Gß, which is coupled to D1 receptor (D1R). Here, we attempted to characterize the pathophysiological, neurochemical, and pharmacological features of mice deficient of Gγ7 gene. Gγ7 knockout mice exhibited age-dependent deficiency in rotarod behavior and increased dystonia-like clasping reflex without loss of striatal neurons. The neurochemical basis for the motor manifestations using immunoblot analysis revealed increased levels of D1R, ChAT and NMDA receptor subunits (NR1 and NR2B) concurrent with decreased levels of D2R and Gαolf , possibly because of the secondary changes of decreased Gαolf /Gγ7-mediated D1R transmission. These behavioral and neurochemical changes are closely related to those observed in Huntington's disease (HD) human subjects and HD model mice. Taking advantage of the finding of D2R down-regulation in Gγ7 knockout mice and the dopamine-mediated synergistic relationship in the control of locomotion between D2R-striatopallidal and D1R-stritonigral neurons, we hypothesized that D2-agonist pramipexole would reverse behavioral dyskinesia caused by defective D1R/Gαolf signaling. Indeed, the rotarod deficiency and clasping reflex were reversed by pramipexole treatment under chronic administration. These findings suggest that Gγ7 knockout mice could be a new type of movement disorders, including HD and useful for the evaluation of therapeutic candidates.
Assuntos
Benzotiazóis/uso terapêutico , Agonistas de Dopamina/uso terapêutico , Distonia/tratamento farmacológico , Distonia/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/deficiência , Receptores de Dopamina D2/agonistas , Fatores Etários , Animais , Benzotiazóis/farmacologia , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Agonistas de Dopamina/farmacologia , Masculino , Camundongos , Camundongos Knockout , Pramipexol , Receptores de Dopamina D2/fisiologiaRESUMO
Methamphetamine (Meth) abuse can result in long-lasting psychosis and dependence. The nucleus accumbens (NAc), which controls psychomotor and reward behaviours, is an important interface between the limbic system and receives convergent projections from dopaminergic and glutamatergic terminals. This study investigated the involvements of dopaminergic and glutamatergic transmission in the development of Meth psychosis and dependence by using tyrosine hydroxylase heterozygous mutant (TH+/-) mice and N-methyl-d-aspartate receptor knockout (NR2A-/-) mice. Repeated treatment with Meth (1 mg/kg s.c.) for 7 d in wild-type mice led to the development of behavioural abnormalities such as hyperactivity, sensory motor gating deficits and place preference. Associated with the behavioural changes, repeated treatment with Meth led to protein kinase A activation and phosphorylation of Ca2+/calmodulin kinase II and cyclic AMP response element binding protein in the NAc. In contrast, TH+/- and NR2A-/- mice displayed neither behavioural abnormalities nor activation of intracellular signalling pathways in the NAc. These results suggest that both dopaminergic and glutamatergic transmission play a crucial role in the development of Meth psychosis and dependence, which are associated with convergent activation of intracellular signalling pathways in the NAc.
Assuntos
Dopaminérgicos/toxicidade , Dopamina/metabolismo , Ácido Glutâmico/metabolismo , Metanfetamina/toxicidade , Núcleo Accumbens/metabolismo , Transtornos Psicóticos/etiologia , Transtornos Psicóticos/patologia , Estimulação Acústica/efeitos adversos , Análise de Variância , Animais , Condicionamento Operante/efeitos dos fármacos , Condicionamento Operante/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Inibição Psicológica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Atividade Motora/efeitos dos fármacos , Mutação/genética , Núcleo Accumbens/efeitos dos fármacos , Receptores de N-Metil-D-Aspartato/genética , Reflexo de Sobressalto/efeitos dos fármacos , Reflexo de Sobressalto/genética , Tirosina 3-Mono-Oxigenase/deficiênciaRESUMO
BACKGROUND: Stimulating the glycine(B) binding site on the N-methyl-d-aspartate ionotropic glutamate receptor (NMDAR) has been proposed as a novel mechanism for modulating behavioral effects of ethanol (EtOH) that are mediated via the NMDAR, including acute intoxication. Here, we pharmacologically interrogated this hypothesis in mice. METHODS: Effects of systemic injection of the glycine(B) agonist, d-serine, the GlyT-1 glycine transporter inhibitor, ALX-5407, and the glycine(B) antagonist, L-701,324, were tested for the effects on EtOH-induced ataxia, hypothermia, and loss of righting reflex (LORR) duration in C57BL/6J (B6) and 129S1/SvImJ (S1) inbred mice. Effects of the glycine(B) partial agonist, d-cycloserine (DCS), the GlyT-1 inhibitor, N-[3-(4'-fluorophenyl)-3-(4'-phenylphenoxy)propyl]sarcosine (NFPS), and the glycine(B) antagonist, 5,7-dichlorokynurenic (DCKA), on EtOH-induced LORR duration were also tested. Interaction effects on EtOH-induced LORR duration were examined via combined treatment with d-serine and ALX-5407, d-serine and MK-801, d-serine and L-701,324, as well as L-701,324 and ALX-5407, in B6 mice, and d-serine in GluN2A and PSD-95 knockout mice. The effect of dietary depletion of magnesium (Mg), an element that interacts with the glycine(B) site, was also tested. RESULTS: Neither d-serine, DCS, ALX-5407, nor NFPS significantly affected EtOH intoxication on any of the measures or strains studied. L-701,324, but not DCKA, dose-dependently potentiated the ataxia-inducing effects of EtOH and increased EtOH-induced (but not pentobarbital-induced) LORR duration. d-serine did not have interactive effects on EtOH-induced LORR duration when combined with ALX-5407. The EtOH-potentiating effects of L-701,324, but not MK-801, on LORR duration were prevented by d-serine, but not ALX-5407. Mg depletion potentiated LORR duration in B6 mice and was lethal in a large proportion of S1 mice. CONCLUSIONS: Glycine(B) site activation failed to produce the hypothesized reduction in EtOH intoxication across a range of measures and genetic strains, but blockade of the glycine(B) site potentiated EtOH intoxication. These data suggest endogenous activity at the glycine(B) opposes EtOH intoxication, but it may be difficult to pharmacologically augment this action, at least in nondependent subjects, perhaps because of physiological saturation of the glycine(B) site.
Assuntos
Intoxicação Alcoólica/tratamento farmacológico , Quinolonas/uso terapêutico , Receptores de Glicina/agonistas , Receptores de Glicina/antagonistas & inibidores , Sarcosina/análogos & derivados , Serina/uso terapêutico , Intoxicação Alcoólica/metabolismo , Animais , Ataxia/induzido quimicamente , Ataxia/tratamento farmacológico , Ciclosserina/farmacologia , Modelos Animais de Doenças , Proteína 4 Homóloga a Disks-Large , Maleato de Dizocilpina/administração & dosagem , Maleato de Dizocilpina/uso terapêutico , Quimioterapia Combinada , Proteínas da Membrana Plasmática de Transporte de Glicina/antagonistas & inibidores , Guanilato Quinases/genética , Hipotermia/induzido quimicamente , Hipotermia/tratamento farmacológico , Ácido Cinurênico/análogos & derivados , Ácido Cinurênico/farmacologia , Magnésio/metabolismo , Magnésio/uso terapêutico , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Quinolonas/administração & dosagem , Quinolonas/farmacologia , Receptores de N-Metil-D-Aspartato/genética , Reflexo de Endireitamento/efeitos dos fármacos , Sarcosina/administração & dosagem , Sarcosina/farmacologia , Sarcosina/uso terapêutico , Serina/administração & dosagem , Serina/farmacologiaRESUMO
The GluN2B (GluRepsilon2/NR2B) and GluN2A (GluRepsilon1/NR2A) NMDA receptor (NMDAR) subtypes have been differentially implicated in activity-dependent synaptic plasticity. However, little is known about the respective contributions made by these two subtypes to developmental plasticity, in part because studies of GluN2B KO [Grin2b(-/-) (2b(-/-))] mice are hampered by early neonatal mortality. We previously used in vitro slice cocultures of rodent cerebral cortex (Cx) and spinal cord (SpC) to show that corticospinal (CS) synapses, once present throughout the SpC, are eliminated from the ventral side during development in an NMDAR-dependent manner. To study subtype specificity of NMDAR in this developmental plasticity, we cocultured Cx and SpC slices derived from postnatal day 0 (P0) animals with different genotypes [2b(-/-), Grin2a(-/-) (2a(-/-)), or WT mice]. The distribution of CS synapses was studied electrophysiologically and with a voltage-sensitive dye. Synapse elimination on the ventral side was blocked in WT(Cx)-2b(-/-)(SpC) pairs but not in WT(Cx)-2a(-/-)(SpC) or 2b(-/-)(Cx)-WT(SpC) pairs. CS axonal regression was also observed through live imaging of CS axons labeled with enhanced yellow fluorescent protein (EYFP) through exo utero electroporation. These findings suggest that postsynaptic GluN2B is selectively involved in CS synapse elimination. In addition, the elimination was not blocked in 2a(-/-) SpC slices, where Ca(2+) entry through GluN2B-mediated CS synaptic currents was reduced to the same level as in 2b(-/-) slices, suggesting that the differential effect of GluN2B and GluN2A in CS synapse elimination might not be explained based solely on greater Ca(2+) entry through GluN2B-containing channels.
Assuntos
Córtex Cerebral/fisiologia , Córtex Cerebral/ultraestrutura , Receptores de N-Metil-D-Aspartato/fisiologia , Medula Espinal/fisiologia , Medula Espinal/ultraestrutura , Sinapses/fisiologia , Animais , Córtex Cerebral/crescimento & desenvolvimento , Técnicas de Cocultura , Fenômenos Eletrofisiológicos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Knockout , Plasticidade Neuronal/fisiologia , Ratos , Receptores de N-Metil-D-Aspartato/deficiência , Receptores de N-Metil-D-Aspartato/genética , Medula Espinal/crescimento & desenvolvimentoRESUMO
Oligodendrocyte precursor cells (OPCs) express NMDA receptors (NMDARs) and form synapses with glutamatergic neurons throughout the CNS. Although glutamate influences the proliferation and maturation of these progenitors in vitro, the role of NMDAR signaling in oligodendrogenesis and myelination in vivo is not known. Here, we investigated the consequences of genetically deleting the obligatory NMDAR subunit NR1 from OPCs and their oligodendrocyte progeny in the CNS of developing and mature mice. NMDAR-deficient OPCs proliferated normally, achieved appropriate densities in gray and white matter, and differentiated to form major white matter tracts without delay. OPCs also retained their characteristic physiological and morphological properties in the absence of NMDAR signaling and were able to form synapses with glutamatergic axons. However, expression of calcium-permeable AMPA receptors (AMPARs) was enhanced in NMDAR-deficient OPCs. These results suggest that NMDAR signaling is not used to control OPC development but to regulate AMPAR-dependent signaling with surrounding axons, pointing to additional functions for these ubiquitous glial cells.
Assuntos
Encéfalo/citologia , Proliferação de Células , Oligodendroglia/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Transdução de Sinais/fisiologia , Células-Tronco/fisiologia , Fatores Etários , Família Aldeído Desidrogenase 1 , Análise de Variância , Animais , Animais Recém-Nascidos , Proteínas Relacionadas à Autofagia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Biofísica , Encéfalo/crescimento & desenvolvimento , Bromodesoxiuridina/metabolismo , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Estimulação Elétrica , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteína Glial Fibrilar Ácida/metabolismo , Ácido Glutâmico/metabolismo , Ácido Glutâmico/farmacologia , Técnicas In Vitro , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Isoenzimas/metabolismo , Proteínas Luminescentes/genética , Camundongos , Camundongos Transgênicos , Proteína Básica da Mielina/metabolismo , Técnicas de Patch-Clamp , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptores do Fator Natriurético Atrial/metabolismo , Receptores de N-Metil-D-Aspartato/deficiência , Retinal Desidrogenase/metabolismo , Transdução de Sinais/genética , Sinapses/genética , Sinapses/fisiologiaRESUMO
Mental retardation (MR) and autism are highly heterogeneous neurodevelopmental disorders. IL-1-receptor accessory protein-like 1 (IL1RAPL1) is responsible for nonsyndromic MR and is associated with autism. Thus, the elucidation of the functional role of IL1RAPL1 will contribute to our understanding of the pathogenesis of these mental disorders. Here, we showed that knockdown of endogenous IL1RAPL1 in cultured cortical neurons suppressed the accumulation of punctate staining signals for active zone protein Bassoon and decreased the number of dendritic protrusions. Consistently, the expression of IL1RAPL1 in cultured neurons stimulated the accumulation of Bassoon and spinogenesis. The extracellular domain (ECD) of IL1RAPL1 was required and sufficient for the presynaptic differentiation-inducing activity, while both the ECD and cytoplasmic domain were essential for the spinogenic activity. Notably, the synaptogenic activity of IL1RAPL1 was specific for excitatory synapses. Furthermore, we identified presynaptic protein tyrosine phosphatase (PTP) δ as a major IL1RAPL1-ECD interacting protein by affinity chromatography. IL1RAPL1 interacted selectively with certain forms of PTPδ splice variants carrying mini-exon peptides in Ig-like domains. The synaptogenic activity of IL1RAPL1 was abolished in primary neurons from PTPδ knock-out mice. IL1RAPL1 showed robust synaptogenic activity in vivo when transfected into the cortical neurons of wild-type mice but not in PTPδ knock-out mice. These results suggest that IL1RAPL1 mediates synapse formation through trans-synaptic interaction with PTPδ. Our findings raise an intriguing possibility that the impairment of synapse formation may underlie certain forms of MR and autism as a common pathogenic pathway shared by these mental disorders.
Assuntos
Transtorno Autístico/fisiopatologia , Córtex Cerebral/fisiologia , Deficiência Intelectual/fisiopatologia , Proteína Acessória do Receptor de Interleucina-1/fisiologia , Neurônios/fisiologia , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/metabolismo , Sinapses/fisiologia , Animais , Transtorno Autístico/metabolismo , Córtex Cerebral/metabolismo , Cromatografia de Afinidade/métodos , Técnicas de Silenciamento de Genes/métodos , Hipocampo/metabolismo , Hipocampo/fisiologia , Deficiência Intelectual/metabolismo , Proteína Acessória do Receptor de Interleucina-1/genética , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Cultura Primária de Células , Isoformas de Proteínas/metabolismo , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/genética , Transdução de Sinais/genética , Sinapses/metabolismo , Transfecção/métodosRESUMO
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.
Assuntos
Vias Neurais/fisiologia , Células de Purkinje/fisiologia , Receptores de AMPA/fisiologia , Receptores de Glutamato/fisiologia , Sinapses/fisiologia , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Fibras Nervosas/fisiologia , Fibras Nervosas/ultraestrutura , Vias Neurais/ultraestrutura , Células de Purkinje/ultraestrutura , Receptores de AMPA/ultraestrutura , Receptores de Glutamato/deficiência , Receptores de Glutamato/genética , Sinapses/genética , Sinapses/ultraestruturaRESUMO
Glutamate receptor (GluR) δ1 is widely expressed in the developing forebrain, whereas GluRδ2 is selectively expressed in cerebellar Purkinje cells. Recently, we found that trans-synaptic interaction of postsynaptic GluRδ2 and pre-synaptic neurexins (NRXNs) through cerebellin precursor protein (Cbln) 1 mediates excitatory synapse formation in the cerebellum. Thus, a question arises whether GluRδ1 regulates synapse formation in the forebrain. In this study, we showed that the N-terminal domain of GluRδ1 induced inhibitory presynaptic differentiation of some populations of cultured cortical neurons. When Cbln1 or Cbln2 was added to cultures, GluRδ1 expressed in HEK293T cells induced preferentially inhibitory presynaptic differentiation of cultured cortical neurons. The synaptogenic activity of GluRδ1 was suppressed by the addition of the extracellular domain of NRXN1α or NRXN1ß containing splice segment 4. Cbln subtypes directly bound to the N-terminal domain of GluRδ1. The synaptogenic activity of GluRδ1 in the presence of Cbln subtypes correlated well with their binding affinities. When transfected to cortical neurons, GluRδ1 stimulated inhibitory synapse formation in the presence of Cbln1 or Cbln2. These results together with differential interactions of Cbln subtypes with NRXN variants suggest that GluRδ1 induces preferentially inhibitory presynaptic differentiation of cortical neurons by interacting with NRXNs containing splice segment 4 through Cbln subtypes.