RESUMO
Mitochondria are dynamic organelles that continually move, fuse and divide. The dynamic balance of fusion and fission of mitochondria determines their morphology and allows their immediate adaptation to energetic needs, keeps mitochondria in good health by restoring or removing damaged organelles or precipitates cells in apoptosis in cases of severe defects. Mitochondrial fusion and fission are essential in mammals and their disturbances are associated with several diseases. However, while mitochondrial fusion/fission dynamics, and the proteins that control these processes, are ubiquitous, associated diseases are primarily neurological disorders. Accordingly, inactivation of the main actors of mitochondrial fusion/fission dynamics is associated with defects in neuronal development, plasticity and functioning, both ex vivo and in vivo. Here, we present the central actors of mitochondrial fusion and fission and review the role of mitochondrial dynamics in neuronal physiology and pathophysiology. Particular emphasis is placed on the three main actors of these processes i.e. DRP1,MFN1-2, and OPA1 as well as on GDAP1, a protein of the mitochondrial outer membrane preferentially expressed in neurons. This article is part of a Special Issue entitled: Mitochondria & Brain.
Assuntos
Mitocôndrias/metabolismo , Dinâmica Mitocondrial/fisiologia , Doenças Neurodegenerativas/metabolismo , Plasticidade Neuronal/fisiologia , Animais , Encéfalo/metabolismo , Humanos , Neurônios/metabolismoRESUMO
African trypanosomosis is a parasitic disease affecting both humans (sleeping sickness) and animals (nagana). In murine trypanosomosis, the B-cell compartment is rapidly destroyed after infection. In addition, B-cell lymphopoiesis in the bone marrow is abrogated, B-cell subsets in the spleen are irreversibly depleted, and B-cell memory is destroyed. Here, we investigated the effect of cure of infection on the B-cell compartment. Suramin and diminazene aceturate were used in this study as these drugs exhibit different modes of uptake and different mechanisms of trypanocidal action. Curative drug treatment of trypanosomosis infection led to the re-initiation of B-cell lymphopoiesis in the bone marrow, and to the repopulation of splenic B-cell subsets, independent of the drug used. Neither of these drugs by itself induced measurable effects on B-cell lymphopoiesis in the bone marrow or B-cell homoeostasis in the spleen in healthy, naïve animals.
Assuntos
Subpopulações de Linfócitos B/imunologia , Diminazena/análogos & derivados , Suramina/administração & dosagem , Tripanossomicidas/administração & dosagem , Tripanossomíase/tratamento farmacológico , Tripanossomíase/imunologia , Animais , Medula Óssea/imunologia , Diminazena/administração & dosagem , Feminino , Linfopoese/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Baço/citologia , Baço/imunologia , Tripanossomíase Africana/tratamento farmacológico , Tripanossomíase Africana/imunologia , Tripanossomíase Africana/parasitologiaRESUMO
Attributes contributing to differences in beef quality of 206 Hereford steers finished on pasture were assessed. Beef quality traits evaluated were: Warner-Bratzler meat tenderness and muscle and fat color at one and seven days after slaughter and trained sensory panel traits (tenderness, juiciness, flavor, and marbling) at seven days. Molecular markers were CAPN1 316 and an SNP in exon 2 on the leptin gene (E2FB). Average daily live weight gain, ultrasound monthly backfat thickness gain and rib-eye area gain were estimated. Molecular markers effects on meat quality traits were analyzed by mixed models. Association of meat quality with post weaning growth traits was analyzed by canonical correlations. Muscle color and marbling were affected by CAPN1 316 and E2FB and Warner-Bratzler meat tenderness by the former. The results confirm that marker assisted selection for tenderness is advisable only when beef aging is a common practice. The most important sources of variation in tenderness and color of meat remained unaccounted for.
Assuntos
Criação de Animais Domésticos , Calpaína/genética , Bovinos/metabolismo , Qualidade dos Alimentos , Leptina/genética , Carne/análise , Polimorfismo de Nucleotídeo Único , Tecido Adiposo Branco/química , Tecido Adiposo Branco/crescimento & desenvolvimento , Adiposidade , Animais , Animais Endogâmicos , Argentina , Calpaína/metabolismo , Bovinos/crescimento & desenvolvimento , Fenômenos Químicos , Éxons , Armazenamento de Alimentos , Estudos de Associação Genética/veterinária , Marcadores Genéticos , Humanos , Leptina/metabolismo , Masculino , Fenômenos Mecânicos , Desenvolvimento Muscular , SensaçãoRESUMO
The somatotropic axis is a major regulatory pathway of energy metabolism during postnatal growth in mammals. Genes involved in this pathway influence many economically important traits. The association of selected SNPs in these genes with carcass traits was examined in grazing Brangus steers. These traits included final live weight, ultrasound backfat thickness (UBFT), rib-eye area, kidney fat weight, hot carcass weight, and intramuscular fat percentage (%IMF). Genomic DNA (N = 246) was genotyped for a panel of 15 tag SNPs located in the growth hormone receptor (GHR), insulin-like growth factor I, insulin-like growth factor-binding protein 6, pro-melanin-concentrating hormone, suppressor of cytokine signaling 2, and signal transducer and activator of transcription 6 (STAT6) genes. Allelic and haplotype frequencies were compared with those of a sample of European breeds (N = 177 steers). Two tag SNPs in the GHR affected %IMF; one of them (ss86273136) was also strongly associated with UBFT (P < 0.003). The frequency of the most favorable GHR haplotype for %IMF was lower in Brangus steers. Moreover, the haplotype carrying two unfavorable alleles was present at a frequency of 31% in this group. Four tag SNPs on STAT6 had a significant effect on UBFT. One of these, SNP ss115492467, was also associated with %IMF. The STAT6 haplotype, including all the alleles favoring UBFT, was the most abundant variant (34%) in the European cattle, while it had a frequency of 14% in the Brangus steers. The four less favorable variants (absent in the European cattle) were found at a frequency of 38% in the Brangus steers. These results support the association of GHR and STAT6 SNP with carcass traits in composite breeds, such as Brangus, under grazing conditions.
Assuntos
Composição Corporal/genética , Bovinos/anatomia & histologia , Estudos de Associação Genética , Marcadores Genéticos , Aumento de Peso/genética , Tecido Adiposo/química , Alelos , Animais , Argentina , Pesos e Medidas Corporais , Cruzamento , Bovinos/genética , Genótipo , Haplótipos , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/genética , Fator de Crescimento Insulin-Like I/genética , Carne , Músculo Esquelético/química , Fenótipo , Polimorfismo de Nucleotídeo Único , Receptores da Somatotropina/genética , Fator de Transcrição STAT6/genéticaRESUMO
The PPARGC1A gene (peroxysome proliferator-activated receptor-gamma coactivator 1alpha gene) controls muscle fiber type and brown adipocyte differentiation; therefore, it is a candidate gene for beef quality traits (tenderness and fat content). Two SNPs (Single Nucleotide Polymorphisms) were identified within exon 8 by multiple alignment of DNA sequences obtained from 24 bulls: a transition G/A (SNP 1181) and a transversion A/T (SNP 1299). The SNP 1181 is a novel SNP, corresponding to a non-conservative substitution (AGT/AAT) that could be the cause of amino acid substitution ((364)Serine/(364)Asparagine). A Mismatch PCR method was designed to determine genotypes of 73 bulls and 268 steers for SNP 1181. Growth, slaughter and meat quality information were available for the group of steers. Allele A of SNP 1181 was not found in Angus. In 243 steers, no significant differences (P > 0.05) were found for either final live body weight, gain in backfat thickness in Spring, kidney fat weight, kidney fat percentage, Warner-Bratzler shear force at 7 days postmortem, intramuscular fat percentage or meat colour between genotype GG and AG. This SNP could be included in breed composition and population admixture analyses because there are marked differences in allelic frequencies between Bos taurus and Bos indicus breeds.
Assuntos
Bovinos/genética , Polimorfismo de Nucleotídeo Único , Fatores de Transcrição/genética , Animais , Sequência de Bases , Peso Corporal/genética , Bovinos/classificação , Bovinos/crescimento & desenvolvimento , Feminino , Frequência do Gene , Variação Genética , Genótipo , Masculino , Carne/normas , Dados de Sequência Molecular , Fenótipo , Reação em Cadeia da Polimerase/métodos , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
The hypothesis of an early vulnerability of the serotonergic system to prion infection was investigated in a murine model of bovine spongiform encephalopathy (BSE). Behavioral tests targeted to 5-HT functions were performed in the course of infection to evaluate circadian activity, anxiety-like behavior, pain sensitivity and the 5-HT syndrome. The first behavioral change was a decrease in nocturnal activity detected at 30% of incubation time. Further behavioral alterations including nocturnal hyperactivity, reduced anxiety, hyperalgesia and exaggerated 5-HT syndrome were observed at 60%-70% of incubation time, before the onset of clinical signs. The same tests performed in 5-HT-depleted mice and in prion protein-deficient mice revealed behavioral abnormalities similar in many aspects to those of BSE-infected mice. Histological and biochemical analysis showed alterations of the serotonergic system in BSE-infected and prion protein-deficient mice. These results indicate that BSE infection affects the homeostasis of serotonergic neurons and suggest that the disruption of prion protein normal function contributes to the early pathological changes in our mouse model of BSE. A similar process may occur in the human variant Creutzfeldt-Jacob disease, as suggested by the early symptoms of alterations in mood, sleep and pain sensitivity.
Assuntos
Encéfalo/metabolismo , Encefalopatia Espongiforme Bovina/metabolismo , Transtornos Mentais/metabolismo , Proteínas PrPC/deficiência , Proteínas PrPSc/toxicidade , Serotonina/metabolismo , Animais , Transtornos de Ansiedade/genética , Transtornos de Ansiedade/metabolismo , Transtornos de Ansiedade/fisiopatologia , Encéfalo/fisiopatologia , Tronco Encefálico/citologia , Tronco Encefálico/metabolismo , Tronco Encefálico/fisiopatologia , Bovinos , Transtornos Cronobiológicos/genética , Transtornos Cronobiológicos/metabolismo , Transtornos Cronobiológicos/fisiopatologia , Modelos Animais de Doenças , Progressão da Doença , Encefalopatia Espongiforme Bovina/fisiopatologia , Feminino , Homeostase/fisiologia , Transtornos Mentais/genética , Transtornos Mentais/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/citologia , Neurônios/metabolismo , Dor/genética , Dor/metabolismo , Dor/fisiopatologia , Proteínas PrPC/genética , Proteínas PrPSc/metabolismo , Núcleos da Rafe/citologia , Núcleos da Rafe/metabolismo , Núcleos da Rafe/fisiopatologia , Síndrome da Serotonina/genética , Síndrome da Serotonina/metabolismo , Síndrome da Serotonina/fisiopatologia , Fatores de TempoRESUMO
Leptin is a hormone that affects the regulation of feed intake, energy balance and body composition in mammals. Several polymorphisms in the bovine leptin gene have been associated with phenotypic variance of these traits. We evaluated two known single nucleotide polymorphisms (SNPs) in the leptin gene of 253 grazing Brangus steers. Brangus is a 5/8 Angus-3/8 Brahman composite. Data were collected during two consecutive growth/fattening cycles from two farms in southeast Buenos Aires province, Argentina. One of the markers is in the promoter region of the gene (SNP1) and the other is a non-synonymous polymorphism in exon 2 (SNP2). The traits that we evaluated were live weight gain in the spring, gain in backfat thickness in the spring, final live weight, final ultrasound backfat thickness, final ultrasound rib eye area, carcass weight and length, carcass yield, kidney fat, kidney fat percentage, backfat thickness, rib eye area, and intramuscular fat percentage. Both markers affected some meat traits; though the only significant associations were of SNP1 with ultrasound rib eye area and of SNP2 with carcass yield and backfat thickness. Under the same conditions as in the present study, leptin markers could be of help only as part of a larger genotyping panel including other relevant genes.
Assuntos
Bovinos/crescimento & desenvolvimento , Leptina/genética , Polimorfismo Genético , Animais , Argentina , Composição Corporal , Bovinos/genética , Marcadores Genéticos , Genótipo , FenótipoRESUMO
Prion diseases are neurodegenerative pathologies characterized by apoptotic neuronal death. Although the late execution phase of neuronal apoptosis is beginning to be characterized, the sequence of events occurring during the early decision phase is not yet well known. In murine cortical neurons in primary culture, apoptosis was first induced by exposure to a synthetic peptide homologous to residues 106-126 of the human prion protein (PrP), PrP106-126. Exposure to its aggregated form induced a massive neuronal death within 24 h. Apoptosis was characterized by nuclear fragmentation, neuritic retraction and fragmentation and activation of caspase-3. During the early decision phase, reactive oxygen species were detected after 3 h. Using immunocytochemistry, we showed a peak of phosphorylated c-Jun-N-terminal kinase (JNK) translocation into the nucleus after 8 h, along with the activation of the nuclear c-Jun transcription factor. Both pharmacological inhibition of JNK by SP600125 and overexpression of a dominant negative form of c-Jun significantly reduced neuronal death, while the MAPK p38 inhibitor SB203580 had no effect. Apoptosis was also studied after exposure of tg338 cortical neurons in primary culture to sheep scrapie agent. In this model, prion-induced neuronal apoptosis gradually increased with time and induced a 40% cell death after 2 weeks exposure. Immunocytochemical analysis showed early c-Jun activation after 7 days. In summary, the JNK-c-Jun pathway plays an important role in neuronal apoptosis induced by PrP106-126. This pathway is also activated during scrapie infection and may be involved in prion-induced neuronal death. Pharmacological blockade of early pathways opens new therapeutic prospects for scrapie PrP-based pathologies.
Assuntos
Apoptose/fisiologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Neurônios/metabolismo , Fragmentos de Peptídeos/farmacologia , Príons/farmacologia , Scrapie/metabolismo , Scrapie/patologia , Animais , Núcleo Celular/metabolismo , Córtex Cerebral/patologia , Feminino , Camundongos , Camundongos Mutantes , Neurônios/patologia , Fosforilação , Gravidez , Proteínas Proto-Oncogênicas c-jun/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Evidence for the importance of major histocompatibility complex (MHC) genotype in immunological fitness of chickens continues to accumulate. The MHC B haplotypes contribute resistance to Marek's and other diseases of economic importance. The Rfp-Y, a second cluster of MHC genes in the chicken, may also contribute to disease resistance. Nevertheless, the MHC B and Rfp-Y haplotypes segregating in broiler chickens are poorly documented. The Camperos, free-range broiler chickens developed in Argentina, provide an opportunity to evaluate MHC diversity in a genetically diverse broiler stock. Camperos are derived by cross-breeding parental stocks maintained essentially without selection since their founding. We analysed 51 DNA samples from the Camperos and their parental lines for MHC B and Rfp-Y variability by restriction fragment pattern (rfp) and SSCP typing methods for B-G, B-F (class Ia), B-Lbeta (class II) and Y-F (class Ib) diversity. We found evidence for 38 B-G genotypes. The Camperos B-G patterns were not shared with White Leghorn controls, nor were any of a limited number of Camperos B-G gene sequences identical to published B-G sequences. The SSCP assays provided evidence for the presence of at least 28 B-F and 29 B-Lbeta genotypes. When considered together B-F, B-L, and B-G patterns provide evidence for 40 Camperos B genotypes. We found even greater Rfp-Y diversity. The Rfp-Y class I-specific probe, 163/164f, revealed 44 different rfps among the 51 samples. We conclude that substantial MHC B and Rfp-Y diversity exists within broiler chickens that might be drawn upon in selecting for desirable immunological traits.
Assuntos
Galinhas/imunologia , Variação Genética , Genótipo , Complexo Principal de Histocompatibilidade/genética , Animais , Southern Blotting , Galinhas/genéticaRESUMO
Following the cloning and sequencing of the A subunit of the 5-HT3 receptor, two alternatively spliced isoforms, 5-HT3-AS and 5-HT3-AL, have been identified. In order to analyse the distribution of the receptor, a polyclonal antibody has been produced against the short form which is the most abundant in the central nervous system [Doucet et al. (2000) Neuroscience 95, 881-892]. As expected from the recognition of functional 5-HT3 receptors, immunostaining by this anti-5-HT3-R-AS antibody matched the distribution of the high-affinity 5-HT3 binding sites in the rat brain and spinal cord. 5-HT3-AS-like immunoreactivity was detected at low levels in the limbic system, particularly in the amygdala and the hippocampus, and in the frontal, piriform and entorhinal cortices. High levels of immunoreactivity were found in the brainstem, mainly in the nucleus tractus solitarius and the nucleus of the spinal tract of the trigeminal nerve, and in the dorsal horn of the spinal cord. At the ultrastructural level, immunostaining was generally found associated with axons and nerve terminals (70-80%) except in the hippocampus, where labelled dendrites were more abundant (56%). This preferential localization on nerve endings is consistent with the well-documented physiological role of 5-HT3 receptors in the control of neurotransmitter release. However, the different distribution in the hippocampus raises the question of whether differential addressing mechanisms exist for preferentially targeting 5-HT3 receptors to postsynaptic dendritic sites as compared to presynaptic nerve endings, depending on the nature of the neurons bearing these receptors.
Assuntos
Compartimento Celular/fisiologia , Sistema Nervoso Central/metabolismo , Dendritos/metabolismo , Terminações Pré-Sinápticas/metabolismo , Receptores de Serotonina/metabolismo , Transmissão Sináptica/fisiologia , Animais , Sistema Nervoso Central/ultraestrutura , Dendritos/ultraestrutura , Imuno-Histoquímica , Masculino , Microscopia Eletrônica , Terminações Pré-Sinápticas/ultraestrutura , Ratos , Ratos Wistar , Receptores 5-HT3 de Serotonina , Serotonina/metabolismo , Membranas Sinápticas/metabolismo , Membranas Sinápticas/ultraestruturaRESUMO
5-HT(6) receptor-like immunoreactivity has been previously found in association with both neuronal dendrites and cilia in the striatum, nucleus accumbens, olfactory tubercle and islands of Calleja of the rat brain. Using immunogold pre-embedding immunocytochemical techniques to investigate the subcellular localization of 5-HT(6) receptor-like immunoreactivity in cilia, we showed that immunogold particles were associated with their plasma membrane, and not with microtubules. This membrane localization is consistent with a possible physiological role, which is still unknown, of these receptors.
Assuntos
Membrana Celular/metabolismo , Cílios/metabolismo , Ínsulas Olfatórias/metabolismo , Neurônios/metabolismo , Receptores de Serotonina/metabolismo , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Encéfalo/ultraestrutura , Membrana Celular/ultraestrutura , Cílios/ultraestrutura , Imunofluorescência , Imuno-Histoquímica , Ínsulas Olfatórias/ultraestrutura , Masculino , Microtúbulos/ultraestrutura , Neurônios/citologia , Neurônios/ultraestrutura , Especificidade de Órgãos , Ratos , Ratos WistarRESUMO
Polyclonal antibodies were raised against a synthetic hexadecapeptide corresponding to the portion of the second intracytoplasmic loop of the short form of the mouse 5-hydroxytryptamine-3A receptor subunit (5-HT3A-S), which differs from the long form (5-HT3A-L) by the removal of six amino acids. Antibodies were detected by enzyme-linked immunosorbent assay as soon as two months after the first injection to rabbits of the peptide coupled to keyhole limpet hemocyanin. Immunoblot detection of fusion proteins comprising glutathione-S-transferase and the second intracellular loop of 5-HT3A-S or 5-HT3A-L, and immunoprecipitation of cloned receptors showed that antibodies exhibited some selectivity for the short variant. Affinity chromatography allowed the purification of selective anti-5-HT3A-S antibodies which yielded a strong positive labeling of plasma membrane, reticulum and Golgi apparatus of COS-7 cells expressing murine 5-HT3A-S. In contrast, COS-7 cells expressing similar levels of 5-HT3A-L exhibited only a very weak labeling. Selectivity was also observed on immunoblots of cloned receptors transiently expressed in COS-7 cells, or stably expressed in CHO cells, both systems showing an immunolabeled component at 53,000-54,000 mol. wt. Immunoautoradiographic labeling of central nervous system sections showed that 5-HT3A-S-like immunoreactivity was found mostly within the nucleus of the solitary tract, the nucleus of the spinal tract of the trigeminal nerve, and the dorsal horn of the the spinal cord in the rat. After unilateral ablation of the nodose ganglion, 5-HT3A-S-like immunoreactivity decreased markedly in the ipsilateral part of the nucleus of the solitary tract, as expected of the presynaptic localization of 5-HT3 receptors. Finally, immunohistochemistry at the light and electron microscope levels revealed that 5-HT3A-S-like immunoreactivity was associated essentially with terminals and axonal profiles. All these results demonstrate that the immunolabeling exhibited by these antibodies is consistent with a specific and partially selective recognition of the short isoform of the 5-HT3A subunit. Because the pattern of immunoautoradiographic labeling matches the distribution previously established with selective radioligands, it can be inferred that these antibodies probably recognized the same fully assembled form of the 5-HT3A-S receptor subunit.
Assuntos
Sistema Nervoso Central/metabolismo , Sequência de Aminoácidos/genética , Animais , Benzamidas/metabolismo , Sítios de Ligação/efeitos dos fármacos , Compostos Bicíclicos Heterocíclicos com Pontes/metabolismo , Células CHO , Células COS , Sistema Nervoso Central/ultraestrutura , Clonagem Molecular , Cricetinae , Ensaio de Imunoadsorção Enzimática , Immunoblotting , Técnicas Imunológicas , Masculino , Camundongos , Dados de Sequência Molecular , Testes de Precipitina , Coelhos , Ratos , Ratos Sprague-Dawley , Receptores de Serotonina/genética , Receptores de Serotonina/metabolismo , Receptores 5-HT3 de Serotonina , Antagonistas da Serotonina/metabolismo , TransfecçãoRESUMO
Among the recently cloned serotonin (5-hydroxytryptamine, 5-HT) receptors, the 5-HT6 subtype is of special interest for at least two reasons: 1) it is abundant in limbic areas which participate in the control of mood and emotion; and 2) some antidepressants and antipsychotics are potent 5-HT6 receptor antagonists. Studies using polyclonal anti-5-HT6 receptor antibodies and an antisense oligonucleotide were performed in order to investigate further the function(s) of 5-HT6 receptors in the rat brain. Immunocytochemistry at the light and electron microscope levels showed that 5-HT6 receptors are mainly confined to the dendritic compartment, suggesting that they could mediate 5-HT actions on neuronal firing. In some limbic areas, 5-HT6 receptor-like immunoreactivity is also associated with neuronal cilia with yet unknown functions. Continuous i.c.v. infusion with an antisense oligonucleotide for 3-4 days resulted in decreased 5-HT6 receptor-like immunostaining of the nucleus accumbens and anxiogenic behaviours in the social interaction and elevated plus maze tests. Selective 5-HT6 receptor ligands are eagerly expected to investigate further the potential implication of these receptors in limbic-dependent behaviours.
Assuntos
Encéfalo/fisiologia , Oligodesoxirribonucleotídeos Antissenso , Receptores de Serotonina/fisiologia , Sequência de Aminoácidos , Animais , Anticorpos , Especificidade de Anticorpos , Axônios/fisiologia , Axônios/ultraestrutura , Encéfalo/citologia , Encéfalo/ultraestrutura , Dendritos/fisiologia , Dendritos/ultraestrutura , Imuno-Histoquímica , Masculino , Microscopia Imunoeletrônica , Dados de Sequência Molecular , Neurônios/citologia , Neurônios/fisiologia , Neurônios/ultraestrutura , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Terminações Pré-Sinápticas/fisiologia , Terminações Pré-Sinápticas/ultraestrutura , Ratos , Ratos Wistar , Receptores de Serotonina/análise , Receptores de Serotonina/químicaRESUMO
The localization of 5-hydroxytryptamine1B receptors in the rat central nervous system was investigated using anti-peptide antibodies that recognize a selective portion of the third intracytoplasmic loop of the receptor protein. At the light microscope level the densest 5-hydroxytryptamine1B receptor-like immunoreactivity was observed in ventral pallidum, globus pallidus, substantia nigra and dorsal subiculum. In addition, moderate immunoreactivity was found in the entopeduncular nucleus, the superficial gray layer of the superior colliculus, the caudate-putamen and the deep nuclei of the cerebellum. This distribution matched perfectly that previously described from radioligand binding studies. At the ultrastructural level, 5-hydroxytryptamine1B receptor-like immunoreactivity was associated with axons and axon terminals in the three areas examined: substantia nigra, globus pallidus and superficial gray layer of the superior colliculus. In all cases, immunostaining was located on the plasma membrane of unmyelinated axon terminals and in the cytoplasm close to the plasmalemma. Synaptic differentiations were never labelled but, in some cases, 5-hydroxytryptamine1B receptor-like immunoreactivity was found in their close vicinity. Injection of kainic acid into the neostriatum resulted in a marked decrease in receptor-like immunoreactivity in the globus pallidus and the substantia nigra, consistent with the location of 5-hydroxytryptamine1B receptors on terminals of striatopallidal and striatonigral fibres, respectively. A reduction in 5-hydroxytryptamine1B receptor-like immunoreactivity was also noted in the superficial gray layer of the superior colliculus after contralateral enucleation, as expected of the location of 5-hydroxytryptamine1B receptors on the terminals of retinocollicular fibres. In both lesion experiments, immunolabelled degenerating terminals were observed in the projection areas. Anterograde labelling experiments coupled with immunocytochemical detection further showed that 5-hydroxytryptamine1B receptors in the substantia nigra are located on axons of striatal neurons. These data provide anatomical support for the idea that 5-hydroxytryptamine1B receptors act as terminal receptors involved in presynaptic regulation of the release of various neurotransmitters, including 5-hydroxytryptamine itself.
Assuntos
Encéfalo/metabolismo , Receptores de Serotonina/metabolismo , Animais , Autorradiografia , Axônios/metabolismo , Axônios/ultraestrutura , Encéfalo/citologia , Encéfalo/ultraestrutura , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Citoplasma/metabolismo , Citoplasma/ultraestrutura , Globo Pálido/metabolismo , Imuno-Histoquímica , Ácido Caínico/farmacologia , Masculino , Especificidade de Órgãos , Ratos , Ratos Wistar , Receptor 5-HT1B de Serotonina , Receptores de Serotonina/análise , Substância Negra/metabolismo , Sinapses/metabolismo , Sinapses/ultraestruturaRESUMO
The efficiency of antisense approaches to produce a selective regional inhibition of the expression of brain 5-HT1A receptors was tested in the rat. In vivo ICV injections of modified antisense oligodesoxynucleotides yielded at most an 18% specific decrease in 5-HT1A receptor expression in the hippocampus only, as measured by [3H]8-OH-DPAT autoradiographic labeling. In vitro, when 5-HT1A receptors were transiently expressed in LLC-PK1 cells, co-transfection with antisense RNA encoding plasmids resulted in a marked reduction (50-70%) in the density of 5-HT1A binding sites. In vivo stereotaxic injections of the same constructs into the hippocampus, but not in the raphe, which contains 5-HT1A autoreceptors, were shown to produce a approximately 20% reduction in local 5-HT1A receptor density. These data show that antisense strategies could be used to inhibit 5-HT1A receptors expression in the rat hippocampus, but with a limited efficacy.
Assuntos
Expressão Gênica/efeitos dos fármacos , Oligodesoxirribonucleotídeos Antissenso/farmacologia , RNA Antissenso/farmacologia , Receptores Muscarínicos/genética , Receptores de Serotonina/efeitos dos fármacos , Receptores de Serotonina/genética , Animais , Masculino , Plasmídeos , Ratos , Receptores Muscarínicos/efeitos dos fármacos , Receptores 5-HT1 de SerotoninaRESUMO
Specific antipeptide antibodies were used for the immunohistochemical visualization of 5-HT1B receptors in the rat brain. A dense, specific 5-HT1B receptor-like immunoreactivity was found in the globus pallidus, the dorsal subiculum and the substantia nigra. At the light microscope level, immunostaining was diffuse within the neuropil but absent from cell bodies. Observations at the electron microscope level in the substantia nigra showed immunoperoxidase staining in fine unmyelinated axons and nerve terminals.
Assuntos
Encéfalo/metabolismo , Receptores de Serotonina/ultraestrutura , Animais , Encéfalo/anatomia & histologia , Imuno-Histoquímica , Masculino , Microscopia Eletrônica , Ratos , Ratos Wistar , Substância Negra/ultraestruturaRESUMO
In order to map the recently cloned serotonin 5-HT6 receptor in the rat brain and spinal cord, polyclonal antibodies were raised against a synthetic octadecapeptide corresponding to a specific portion (Leu398-Val415) of the C-terminal domain of this receptor. Antibodies were detected by enzyme-linked immunosorbent assay as soon as one month after the first injection to rabbits of the peptide coupled to keyhole limpet hemocyanin. Immunoautoradiographic experiments with antibodies affinity-purified on Affi-Gel coupled to the peptide antigen showed that 5-HT6-like immunoreactive material was abundant in the olfactory tubercle (plexiform layer), cerebral cortex (frontal and entorhinal areas), nucleus accumbens, striatum, hippocampus (strata oriens and radiatum of the CA1 area, molecular layer of the dentate gyrus) and the molecular layer of the cerebellum. A specific immunolabeling, but at moderate intensity, was also observed in the thalamus, substantia nigra, superficial layer of the superior colliculus, motor trigeminal nucleus and facial nucleus. In contrast, no 5-HT6-like immunoreactive material was found in white matter areas. As the regional distribution of 5-HT6 receptor-like immunoreactivity matched generally that previously found for the 5-HT6 receptor mRNA, one could infer that this receptor protein is addressed in the vicinity of its synthesis site, i.e. on somas and/or dendrites. Indeed, immunohistochemistry at the light and electron microscope level showed that 5-HT6-like immunoreactivity was associated with dendritic processes in both the striatum and the dentate gyrus of the hippocampus. The relative abundance of 5-HT6 receptor-like immunoreactivity in extrapyramidal and limbic areas suggests that 5-HT6 receptors may participate in the serotoninergic control of motor function and mood-dependent behavior, respectively.
Assuntos
Sistema Nervoso Central/química , Receptores de Serotonina/análise , Receptores de Serotonina/genética , Animais , Especificidade de Anticorpos , Autorradiografia , Núcleo Caudado/química , Núcleo Caudado/citologia , Sistema Nervoso Central/citologia , Ensaio de Imunoadsorção Enzimática , Hipocampo/química , Hipocampo/citologia , Imuno-Histoquímica , Masculino , Microscopia Eletrônica , Dados de Sequência Molecular , Neurônios/química , Neurônios/ultraestrutura , Neuropeptídeos/análise , Neuropeptídeos/imunologia , Putamen/química , Putamen/citologia , Coelhos , Ratos , Ratos Sprague-Dawley , Receptores de Serotonina/imunologia , Homologia de Sequência de AminoácidosRESUMO
Specific anti-rat 5-hydroxytryptamine1A (serotonin1A) receptor antibodies raised in a rabbit injected with a synthetic peptide corresponding to a highly selective portion of the third intracellular loop of the receptor protein (El Mestikawy et al. [1990] Neurosci. Lett. 118:189-192) were used for immunohistochemical mapping of serotonin1A receptors in the brain and spinal cord of adult rats. The highest density of immunostaining was found in limbic areas (lateral septum, CA1 area of Ammon's horn and dentate gyrus in the hippocampus, and frontal and entorhinal cortices), in the anterior raphe nuclei, and in the interpeduncular nucleus, in agreement with previous autoradiographic studies with selective radioligands showing the enrichment of these regions in serotonin1A receptor binding sites. Serotonin1A receptor-like immunoreactivity was also present, but at a moderate level, in the neocortex, in some thalamic and hypothalamic nuclei, in the nucleus of the solitary tract, in the dorsal tegmentum, in the nucleus of the spinal tract of the trigeminal nerve, and in the superficial layers of the dorsal horn in the spinal cord. In contrast, extrapyramidal areas, including the caudate putamen, the globus pallidus, and the substantia nigra as well as the cerebellum, exhibited very low to no immunostaining by antiserotonin1A receptor antibodies. At the cellular level, both the plasma membrane of neuronal perikarya and fine neuronal processes probably corresponding to dendritic fields were found to bind antiserotonin1A receptor antibodies. Regional differences were noted regarding these two types of immunostaining, because only dendrites bound antibodies within the hippocampus and the lateral septum, whereas both dendrites and neuronal cell bodies were immunoreactive in the medial septum, in the diagonal band of Broca, and in the dorsal and median raphe nuclei. Therefore, differential addressing of serotonin1A receptors could occur from one neuron to another. In general, the distribution and density of serotonin1A receptor-like immunoreactivity in the whole brain and in spinal cord were consistent with the mapping of serotonin1A receptor binding sites and serotonin1A receptor mRNA previously established by immunoautoradiographic and in situ hybridization procedures.
Assuntos
Mapeamento Encefálico/métodos , Sistema Nervoso Central/química , Receptores de Serotonina/análise , Animais , Gânglios da Base/química , Córtex Cerebral/química , Hipocampo/química , Hipotálamo/química , Imuno-Histoquímica , Masculino , RNA Mensageiro/análise , Núcleos da Rafe/química , Ratos , Ratos Wistar , Receptores de Serotonina/genética , Septo Pelúcido/química , Medula Espinal/química , Tálamo/químicaRESUMO
The serotonin 5-HT3-A receptor (5-HT3R-A) mRNA has been shown recently to be expressed as two forms (5-HT3R-AL and 5-HT3R-AS) varying by the presence or the absence of a sequence of 18 bases in the region corresponding to the second cytoplasmic domain of the receptor, and generated by alternative splicing at the level of the 3' acceptor site of exon 9. As the long form of the receptor exhibits a potential phosphorylation site that is disrupted by the alternative splicing, the hypothesis of functional identity and stochastic expression of these two variants was questioned. In the present study, we used quantitative reverse transcriptase-polymerase chain reaction to examine the possible influence of culture conditions on the expression and the alternative splicing of 5-HT3R-A mRNA in NG108-15 clonal cells. Cell differentiation induced by dibutyryl cyclic AMP or theophyllin plus prostaglandin E1 in the presence of 10% serum reduced by threefold the expression of total 5-HT3R-A mRNA, and favored the short form of the message as the ratio S/L (5-HT3R-AS mRNA/5-HT3R-AL mRNA) shifted from 2.23 to 7.33 after 9 days of treatment. Culture with 0.3% serum (instead of 10%) lowered by 10-fold the level of expression of total 5-HT3R-A mRNA, but only slightly reduced the S/L ratio. However, this ratio fell to 0.06 in the presence of 0.3% serum plus 10 ng/ml basic fibroblast growth factor. These results demonstrate that external factors can influence the differential expression of the two variants of the 5-HT3R-A in NG108-15 cells. Appropriate culture conditions for the almost exclusive expression of 5-HT3R-AS mRNA or 5-HT3R-AL mRNA in NG108-15 cells should allow the identification of possible differences in the respective functional properties of each of these two forms of the native 5-HT3 receptor.
Assuntos
Processamento Alternativo , RNA Mensageiro/metabolismo , Receptores de Serotonina/genética , Animais , Sequência de Bases , Adesão Celular , Diferenciação Celular , Linhagem Celular , Meios de Cultura Livres de Soro , Dimetil Sulfóxido/farmacologia , Substâncias de Crescimento/farmacologia , Células Híbridas/citologia , Células Híbridas/fisiologia , Camundongos , Dados de Sequência Molecular , Sondas de Oligonucleotídeos/genética , RatosRESUMO
PCR was used to isolate identical partial cDNA clones encoding a serotonin 5-HT3 receptor subunit from rat nodose and superior cervical ganglia. The amino acid sequence predicted from these clones, extending from the putative transmembrane domain I to the stop codon, demonstrated a 93% homology with the 5-HT3 receptor A (R-A) subunit cloned from NCB 20 hybridoma mouse neuroblastoma/Chinese hamster embryonic brain cells. Comparison of the sequences of the rat gene and cDNA encoding this subunit revealed a five amino acid deletion, GSLLP, located within the putative second intracellular loop of the receptor subunit. This deletion was shown to occur at an intron/exon junction. Therefore, alternative splicing was probably responsible for the presence of short (5-HT3 R-As) and long (5-HT3 R-AL) forms of 5-HT3 R-A mRNA in these ganglia. PCR experiments, with specific primers located upstream and downstream of the GSLLP deletion, were used to detect reverse transcribed 5-HT3 R-A mRNAs. A short fragment (92 bp), corresponding to the deleted form, and a long fragment (107 bp), corresponding to the nondeleted form, were amplified from various regions of the CNS and peripheral ganglia of the rat, as well as from NG108-15 hybridoma cells. In the adult rat, the ratio of the two forms varied very little from one tissue to another, the long form corresponding to only approximately 10% of the total 5-HT3 R-A mRNA. Study of their respective distributions during ontogeny demonstrated a differential expression of the short and long forms in some tissues during late embryonic development, at embryonic day 17 (E17) or E20.(ABSTRACT TRUNCATED AT 250 WORDS)