RESUMO
Cyclic AMP signaling is critical for activity-dependent refinement of neuronal circuits. Global disruption of adenylyl cyclase 1 (AC1), the major calcium/calmodulin-stimulated adenylyl cyclase in the brain, impairs formation of whisker-related discrete neural modules (the barrels) in cortical layer 4 in mice. Since AC1 is expressed both in the thalamus and the neocortex, the question of whether pre- or postsynaptic (or both) AC1 plays a role in barrel formation has emerged. Previously, we generated cortex-specific AC1 knockout (Cx-AC1KO) mice and found that these animals develop histologically normal barrels, suggesting a potentially more prominent role for thalamic AC1 in barrel formation. To determine this, we generated three new lines of mice: one in which AC1 is disrupted in nearly half of the thalamic ventrobasal nucleus cells in addition to the cortical excitatory neurons (Cx/pTh-AC1KO mouse), and another in which AC1 is disrupted in the thalamus but not in the cortex or brainstem nuclei of the somatosensory system (Th-AC1KO mouse). Cx/pTh-AC1KO mice show severe deficits in barrel formation. Th-AC1KO mice show even more severe disruption in barrel patterning. In these two lines, single thalamocortical (TC) axon labeling revealed a larger lateral extent of TC axons in layer 4 compared to controls. In the third line, all calcium-stimulated adenylyl cyclases (both AC1 and AC8) are deleted in cortical excitatory neurons. These mice have normal barrels. Taken together, these results indicate that thalamic AC1 plays a major role in patterning and refinement of the mouse TC circuitry.
Assuntos
Adenilil Ciclases/metabolismo , Córtex Somatossensorial/crescimento & desenvolvimento , Tálamo/fisiologia , Adenilil Ciclases/genética , Animais , Axônios/fisiologia , Imuno-Histoquímica , Camundongos Knockout , Técnicas de Rastreamento Neuroanatômico , Plasticidade Neuronal/fisiologia , Córtex Somatossensorial/fisiologia , Tálamo/crescimento & desenvolvimento , Vibrissas/fisiologiaRESUMO
Converging evidence from pharmacological and molecular studies has led to the suggestion that inhibition of glycine transporter 1 (GlyT1) constitutes an effective means to boost N-methyl-d-aspartate receptor (NMDAR) activity by increasing the extra-cellular concentration of glycine in the vicinity of glutamatergic synapses. However, the precise extent and limitation of this approach to alter cognitive function, and therefore its potential as a treatment strategy against psychiatric conditions marked by cognitive impairments, remain to be fully examined. Here, we generated mutant mice lacking GlyT1 in the entire forebrain including neurons and glia. This conditional knockout system allows a more precise examination of GlyT1 downregulation in the brain on behavior and cognition. The mutation was highly effective in attenuating the motor-stimulating effect of acute NMDAR blockade by phencyclidine, although no appreciable elevation in NMDAR-mediated excitatory postsynaptic currents (EPSC) was observed in the hippocampus. Enhanced cognitive performance was observed in spatial working memory and object recognition memory while spatial reference memory and associative learning remained unaltered. These findings provide further credence for the potential cognitive enhancing effects of brain GlyT1 inhibition. At the same time, they indicated potential phenotypic differences when compared with other constitutive and conditional GlyT1 knockout lines, and highlighted the possibility of a functional divergence between the neuronal and glia subpopulations of GlyT1 in the regulation of learning and memory processes. The relevance of this distinction to the design of future GlyT1 blockers as therapeutic tools in the treatment of cognitive disorders remains to be further investigated.
Assuntos
Proteínas da Membrana Plasmática de Transporte de Glicina/genética , Memória , Prosencéfalo/fisiologia , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Anfetamina/farmacologia , Animais , Regulação para Baixo , Potenciais Pós-Sinápticos Excitadores , Feminino , Glicina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Glicina/biossíntese , Hipocampo/fisiologia , Aprendizagem , Masculino , Camundongos , Camundongos Knockout , Atividade Motora/efeitos dos fármacos , Mutação , Fenciclidina/farmacologia , Prosencéfalo/efeitos dos fármacos , Prosencéfalo/metabolismo , Desempenho Psicomotor , Receptores de N-Metil-D-Aspartato/biossíntese , Reconhecimento Psicológico , Transmissão SinápticaRESUMO
The neuronal circuits mediating the sedative action of diazepam are unknown. Although the motor-depressant action of diazepam is suppressed in alpha1(H101R) homozygous knockin mice expressing diazepam-insensitive alpha1-GABA(A) receptors, global alpha1-knockout mice show greater motor sedation with diazepam. To clarify this paradox, attributed to compensatory up-regulation of the alpha2 and alpha3 subunits, and to further identify the neuronal circuits supporting diazepam-induced sedation, we generated Emx1-cre-recombinase-mediated conditional mutant mice, selectively lacking the alpha1 subunit (forebrain-specific alpha1(-/-)) or expressing either a single wild-type (H) or a single point-mutated (R) alpha1 allele (forebrain-specific alpha1(-/H) and alpha1(-/R) mice, respectively) in forebrain glutamatergic neurons. In the rest of the brain, alpha1(-/R) mutants are heterozygous alpha1(H101R) mice. Forebrain-specific alpha1(-/-) mice showed enhanced diazepam-induced motor depression and increased expression of the alpha2 and alpha3 subunits in the neocortex and hippocampus, in comparison with their pseudo-wild-type littermates. Forebrain-specific alpha1(-/R) mice were less sensitive than alpha1(-/H) mice to the motor-depressing action of diazepam, but each of these conditional mutants had a similar behavioral response as their corresponding control littermates. Unexpectedly, expression of the alpha1 subunit was reduced in forebrain, notably in alpha1(-/R) mice, and the alpha3 subunit was up-regulated in neocortex, indicating that proper alpha1 subunit expression requires both alleles. In conclusion, conditional manipulation of GABA(A) receptor alpha1 subunit expression can induce compensatory changes in the affected areas. Specifically, alterations in GABA(A) receptor expression restricted to forebrain glutamatergic neurons reproduce the behavioral effects seen after a global alteration, thereby implicating these neurons in the motor-sedative effect of diazepam.
Assuntos
Córtex Cerebral/efeitos dos fármacos , Diazepam/farmacologia , Ácido Glutâmico/metabolismo , Hipnóticos e Sedativos/farmacologia , Atividade Motora/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Animais , Córtex Cerebral/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Atividade Motora/fisiologia , Neurônios/metabolismo , Receptores de GABA-A/biossíntese , Receptores de GABA-A/genéticaRESUMO
In the rodent primary somatosensory cortex, the configuration of whiskers and sinus hairs on the snout and of receptor-dense zones on the paws is topographically represented as discrete modules of layer IV granule cells (barrels) and thalamocortical afferent terminals. The role of neural activity, particularly activity mediated by NMDARs (N-methyl-D-aspartate receptors), in patterning of the somatosensory cortex has been a subject of debate. We have generated mice in which deletion of the NMDAR1 (NR1) gene is restricted to excitatory cortical neurons, and here we show that sensory periphery-related patterns develop normally in the brainstem and thalamic somatosensory relay stations of these mice. In the somatosensory cortex, thalamocortical afferents corresponding to large whiskers form patterns and display critical period plasticity, but their patterning is not as distinct as that seen in the cortex of normal mice. Other thalamocortical patterns corresponding to sinus hairs and digits are mostly absent. The cellular aggregates known as barrels and barrel boundaries do not develop even at sites where thalamocortical afferents cluster. Our findings indicate that cortical NMDARs are essential for the aggregation of layer IV cells into barrels and for development of the full complement of thalamocortical patterns.
Assuntos
Padronização Corporal/fisiologia , Vias Neurais/embriologia , Receptores de N-Metil-D-Aspartato/fisiologia , Córtex Somatossensorial/embriologia , Animais , Tronco Encefálico/embriologia , Diferenciação Celular , Cruzamentos Genéticos , Feminino , Proteínas de Homeodomínio/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Vias Neurais/fisiologia , Neurônios/fisiologia , Receptores de N-Metil-D-Aspartato/genética , Córtex Somatossensorial/citologia , Córtex Somatossensorial/fisiologia , Tálamo/embriologia , Fatores de Transcrição , Vibrissas/embriologiaRESUMO
We have examined the role of NMDA receptor-mediated neural activity in the formation of periphery-related somatosensory patterns, using genetically engineered mice. We demonstrate that ectopic expression of a transgene of an NMDAR1 splice variant rescues neonatally fatal NMDAR1 knockout (KO) mice, although the average life span varies depending on the level of the transgene expression. In NMDAR1 KO mice with "high" levels of the transgene expression, sensory periphery-related patterns were normal along both the trigeminal and dorsal column pathways. In the KO mice with "low" levels of the transgene expression, the patterns were absent in the trigeminal pathway. Our results indicate that NMDA receptor-mediated neural activity plays a critical role in pattern formation along the ascending somatosensory pathways.
Assuntos
Encéfalo/fisiologia , Neurônios/fisiologia , Receptores de N-Metil-D-Aspartato/biossíntese , Nervo Trigêmeo/fisiologia , Processamento Alternativo , Animais , Complexo IV da Cadeia de Transporte de Elétrons/análise , Eletrofisiologia , Engenharia Genética , Variação Genética , Hibridização In Situ , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Knockout , Camundongos Transgênicos , NADPH Desidrogenase/análise , Técnicas de Patch-Clamp , Receptores de N-Metil-D-Aspartato/deficiência , Receptores de N-Metil-D-Aspartato/genéticaRESUMO
Mouse spleen cells were stimulated, first by transforming growth factor beta (TGF-beta) and then by interleukin-2 (IL-2) in the presence of bacterial lipopolysaccharide (LPS). LPS plus IL-2 stimulation restored the cell proliferation suppressed by TGF-beta and increased the number of B cells. Both TGF-beta alone or TGF-beta plus IL-2 enhanced production of surface IgA-positive (sIgA+) cells in LPS-stimulated B-cell cultures. We characterized extrachromosomal circular DNAs generated in TGF-beta-primed and IL-2-stimulated B cells and identified the breakpoints of S mu/S gamma 3 recombinants which were the major intermediates of S mu and S alpha switch recombination. All switch recombination sites were dispersed evenly within both S mu and S gamma 3 regions. This even distribution of S mu/S gamma 3 breakpoints is in contrast to the site preference of class switch breakpoints induced by TGF-beta alone. These results suggest that IL-2-stimulated cell proliferation may modify class switch recombination primed by TGF-beta.
Assuntos
Linfócitos B/imunologia , Switching de Imunoglobulina/imunologia , Interleucina-2/imunologia , Ativação Linfocitária/imunologia , Fator de Crescimento Transformador beta/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Primers do DNA , DNA Circular , Feminino , Genes de Imunoglobulinas , Imunoglobulina A/genética , Imunoglobulina A/imunologia , Lipopolissacarídeos , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Recombinação Genética , Baço/imunologiaRESUMO
Extrachromosomal circular DNAs isolated from a P19 embryonal carcinoma cell line were induced to differentiate into neuron-like cells by retinoic acid and cloned into an EcoRI site of a phage vector. Of the 26 DNA inserts (2.1 kb in average length) analyzed, 16 contained repetitive sequences. Out of 10 DNA inserts with unique sequence, 6 carried linear chromosomal sequences and 4 showed chromosomal rearrangements in Southern blots. Two unique fragments with germline configuration were enriched in circular DNA clone libraries. We assigned the breakpoints of 3 circular DNA fragments to positions in the germline sequence. Patchy short inverted repeats were found in the vicinity of breakpoints. An intrastrand loop structure between such inverted short homology region may be required for the circularization of excised DNA.
Assuntos
DNA Circular/genética , Neurônios/citologia , Recombinação Genética , Tretinoína/farmacologia , Animais , Sequência de Bases , Diferenciação Celular , Clonagem Molecular , Sequência Consenso , DNA Circular/química , Biblioteca Gênica , Camundongos , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Mapeamento por Restrição , Análise de Sequência de DNA , Células Tumorais CultivadasRESUMO
A 7255-base pair (bp) sequence, including the previously sequenced murine segments of I alpha, S alpha, and C alpha, has been completed. Homology matrix comparison revealed a switch repetitive region of 4.2 kilobases (kb) composed of 20-80-bp homology runs, including the previously assigned S alpha region. We distinguished several stretches of duplication, i.e. the central 0.8-kb repetitive region, with some 80-bp staggered consensus repeats containing 20-30-bp subsets, made up of the primordial pentamers CTG(A/G)G. All the break-points of the S alpha switch recombination, including those generated by the translocation of the c-myc protooncogene and those catalyzed by bacterial extracts, are located within the consensus sequence subsets of the 4.2-kb repetitive region.
Assuntos
Cadeias Pesadas de Imunoglobulinas/genética , Região de Troca de Imunoglobulinas/genética , Recombinação Genética , Animais , Sequência de Bases , DNA , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Homologia de Sequência do Ácido NucleicoRESUMO
We have characterized extrachromosomal circular DNAs from adult mouse spleen cells that were induced to switch to immunoglobulin A (IgA) with bacterial lipopolysaccharide (LPS) and transforming growth factor beta (TGF-beta), and identified breakpoints of S mu/S gamma 3, S mu/S gamma 2, S mu/S alpha, S gamma 3/S alpha, and S gamma 2/S alpha recombinants. The S mu recombination donor sites clustered in the 3' half of the S mu region, while the S alpha recombination acceptor sites clustered in the 5' half of the S alpha region. In addition, donor and acceptor sites of S gamma regions also clustered in the 3' and 5' parts, respectively. These site preferences are in sharp contrast to the dispersed distribution of S mu/S gamma 1 breakpoints within both S mu and S gamma 1 regions upon IgG1 switch induced by LPS and interleukin 4. Our results support the hypotheses that TGF-beta increases the frequency of switch recombination events to IgA and that the switch recombination to IgA often proceeds by successive recombination of S mu/S gamma and S gamma/S alpha.
Assuntos
Genes de Troca , Imunoglobulina G/genética , Região de Troca de Imunoglobulinas , Recombinação Genética , Fator de Crescimento Transformador beta/farmacologia , Animais , Sequência de Bases , Clonagem Molecular , DNA Circular/genética , Feminino , Biblioteca Gênica , Genes de Troca/efeitos dos fármacos , Região de Troca de Imunoglobulinas/efeitos dos fármacos , Interleucina-4/farmacologia , Lipopolissacarídeos , Camundongos , Camundongos Endogâmicos BALB C , Modelos Genéticos , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Plasmídeos , Recombinação Genética/efeitos dos fármacos , Mapeamento por Restrição , Baço/imunologiaRESUMO
We have cloned circular DNA excised by T cell receptor (TcR) gamma 1, gamma 2 and gamma 3 gene rearrangements in fetal and adult mouse thymocytes. Circular DNA contained a signal joint reciprocal to the genomic V-J coding joint. Although signal joints without nucleotide insertions are common in immunoglobulin (Ig) and TcR gene rearrangements, the signal joint of gamma found in adult thymocytes contained non-germ-line element (N) insertions at high frequency, while no insertions were found in fetal thymocytes. Thus developmental stage specificity of TcR gamma gene rearrangements is faithfully reflected on the signal joint of excision products. In addition, examination of gamma gene excision products revealed circular DNA products of TcR gamma-alpha transrearrangements, but no evidence of V gamma gene replacement in a rearranged segment.
Assuntos
Rearranjo Gênico da Cadeia gama dos Receptores de Antígenos dos Linfócitos T , Receptores de Antígenos de Linfócitos T gama-delta/genética , Fatores Etários , Animais , Sequência de Bases , DNA Circular , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Linfócitos T/imunologiaRESUMO
We have identified circular DNAs containing the kappa light chain constant region (C kappa), as well as the excision products of V kappa-J kappa and V lambda-J lambda joining in adult mouse splenocytes. Analysis of C kappa-positive circular DNA clones revealed two recombination sites (intron recombining sequence [IRS]1 and -2) within the germline J kappa-C kappa intron region and the recombining sequence (RS) located downstream of the C kappa exon. While RS contains a conserved heptamer and nonamer separated by a 23-bp spacer on the 5' side, IRS1 sequence is an isolated heptamer without an obvious nonamer, and IRS2 contains a variant heptamer, CACAAAA. Since IRS1 and IRS2 recombined with both RS (23-bp spacer signal) and V kappa (12-bp spacer signal) with significant frequency, intron recombination sites seem to have dual recombination signals. These findings provide direct evidence that C kappa deletion preceding lambda gene rearrangement can occur by looping out and excision. Increased accessibility of inefficient recombinational loci within the intron may enable recombinase to accept wide signal sequence variation.
Assuntos
Deleção Cromossômica , DNA/genética , Cadeias kappa de Imunoglobulina/genética , Baço/ultraestrutura , Animais , Sequência de Bases , Mapeamento Cromossômico , DNA/análise , DNA/metabolismo , Feminino , Rearranjo Gênico/genética , Cadeias lambda de Imunoglobulina/genética , Íntrons , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Recombinação Genética , Baço/química , Baço/metabolismoRESUMO
We have purified extrachromosomal circular DNAs from adult mouse spleen cells, and cloned into a phage vector the BamHl fragments hybridizing with C mu and S gamma 1 probes. We obtained 52 S mu+S gamma 1+ clones by screening 1.4 million phage clones derived from spleen cells stimulated with bacterial lipopolysaccharide and interleukin 4. We have identified the breakpoints of six clones that contain S gamma 1 and S mu sequences fused in the 5' to 3' orientation. All these switch recombination sites were assigned to the central repetitive sequences of the S mu and S gamma 1 regions. Since the common S mu-S gamma 1 sequences at the recombination sites are at most 2 bases long, typical homologous recombination cannot account for their joining. These findings provide direct evidence that mu-gamma 1 class switching can occur by the looping out and excision of chromosomal DNA, with formation of a circle.
Assuntos
DNA Circular/genética , Genes de Imunoglobulinas , Genes Reguladores , Genes de Troca , Animais , Linfócitos B/imunologia , Sequência de Bases , Clonagem Molecular , Feminino , Biblioteca Gênica , Vetores Genéticos , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Sondas de Oligonucleotídeos , Plasmídeos , Recombinação Genética , Mapeamento por Restrição , Baço/imunologiaRESUMO
Small polydisperse circular (spc) DNA was isolated from mouse thymocytes, fragmented by HindIII digestion and cloned into the vector. Sixty DNA clones were randomly selected from the 10,400 phage library. The average size of insert was one-fifth of the original circular molecule. Twenty spc-DNA clones were homologous to DNA probes derived from T-cell antigen receptor (TCR) alpha-chain loci. We have characterized nine clones by DNA sequencing; they contain new germline sequences of the TCR alpha-chain variable (V alpha) and joining (J alpha) gene segments and the products out of the recombination of a V alpha with a J alpha gene segment. An additional four spc-DNA clones carried a new rearranging gene of the TCR delta-chain that is located between V alpha and J alpha genes. At least nine of 60 DNA clones carried the recombination junction of a heptamer-heptamer head-to-head structure expected from an excised product of V-J joining. This shows that most extrachromosomal circular DNAs in the thymus are formed by a sequence-dependent recombination mechanism. We suggest that a functional T-cell receptor V alpha gene can be constructed by somatic random rearrangements through successive looping-out, excision and deletion.