RESUMEN
This chapter presents a general approach for the application of spatial intensity distribution analysis (SpIDA) to pharmacodynamic quantification of receptor tyrosine kinase homodimerization in response to direct ligand activation or transactivation by G protein-coupled receptors. A custom graphical user interface developed for MATLAB is used to extract quantal brightness and receptor density information from intensity histograms calculated from single fluorescence microscopy images. This approach allows measurement of monomer/oligomer protein mixtures within subcellular compartments using conventional confocal laser scanning microscopy. Application of quantitative pharmacological analysis to data obtained using SpIDA provides a universal method for comparing studies between cell lines and receptor systems. In addition, because of its compatibility with conventional immunostaining approaches, SpIDA is suitable not only for use in recombinant systems but also for the characterization of mechanisms involving endogenous proteins. Therefore, SpIDA enables these biological processes to be monitored directly in their native cellular environment.
Asunto(s)
Receptores ErbB/metabolismo , Imagen Molecular/métodos , Neuronas/metabolismo , Receptor trkB/metabolismo , Receptores Dopaminérgicos/metabolismo , Programas Informáticos , Apomorfina/farmacología , Línea Celular , Receptores ErbB/genética , Receptores ErbB/ultraestructura , Técnica del Anticuerpo Fluorescente , Regulación de la Expresión Génica , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Ligandos , Microscopía Confocal , Microscopía Fluorescente , Imagen Molecular/estadística & datos numéricos , Neuronas/efectos de los fármacos , Neuronas/ultraestructura , Multimerización de Proteína , Quinazolinas/farmacología , Receptor trkB/genética , Receptor trkB/ultraestructura , Receptores Dopaminérgicos/genética , Receptores Dopaminérgicos/ultraestructura , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/ultraestructura , Activación Transcripcional , Tirfostinos/farmacologíaRESUMEN
Synchronization of circadian rhythms to the 24-h light/dark (L/D) cycle is associated with daily rearrangements of the neuronal-glial network of the suprachiasmatic nucleus of the hypothalamus (SCN), the central master clock orchestrating biological functions in mammals. These anatomical plastic events involve neurons synthesizing vasoactive intestinal peptide (VIP), known as major integrators of photic signals in the retinorecipient region of the SCN. Using an analog-sensitive kinase allele murine model (TrkB(F616A) ), we presently show that the pharmacological blockade of the tropomyosin-related kinase receptor type B (TrkB), the high-affinity receptor of brain-derived neurotrophic factor (BDNF), abolished day/night changes in the dendrite enwrapping of VIP neurons by astrocytic processes (glial coverage), used as an index of SCN plasticity on electron-microscopic sections. Therefore, the BDNF/TrkB signaling pathway exerts a permissive role on the ultrastructural rearrangements that occur in SCN under L/D alternance, an action that could be a critical determinant of the well-established role played by BDNF in the photic regulation of the SCN. In contrast, the extent of glial coverage of non-VIP neighboring dendrites was not different at daytime and nighttime in TrkB(F616A) mice submitted to TrkB inactivation or not receiving any pharmacological treatment. These data not only show that BDNF regulates SCN structural plasticity across the 24-h cycle but also reinforce the view that the daily changes in SCN architecture subserve the light synchronization process.
Asunto(s)
Astrocitos/metabolismo , Astrocitos/ultraestructura , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Receptor trkB/metabolismo , Transducción de Señal/fisiología , Núcleo Supraquiasmático/citología , Alanina/genética , Análisis de Varianza , Animales , Factor Neurotrófico Derivado del Encéfalo/ultraestructura , Ritmo Circadiano/fisiología , Dendritas/metabolismo , Dendritas/ultraestructura , Masculino , Ratones , Ratones Transgénicos , Microscopía Inmunoelectrónica , Mutación/genética , Fenilalanina/genética , Receptor trkB/genética , Receptor trkB/ultraestructura , Transducción de Señal/genética , Péptido Intestinal Vasoactivo/metabolismoRESUMEN
The TrkB-expressing sensory neurons seem to be involved in touch and other discriminative sensibilities. Thus, several slowly and rapidly adapting cutaneous mechanoreceptors, as well as muscle spindles, are reduced or absent in the territory of the trigeminal nerve in functionally TrkB-deficient mice. Whether this also occurs in the cutaneous or muscular territories of dorsal root ganglia has not been analyzed. Here we used immunohistochemistry and transmission-electron microscopy to analyze the impact of a mutation in the gene coding for TrkB on Meissner and Pacinian corpuscles, and muscle spindles. The animals were studied at the post-natal days 15 and 25, because at this time all the mechanoreceptors examined are fully developed. Typical Meissner's corpuscles, displaying S-100 protein immunoreactivity, were found in the digital pads of wild-type and TrkB+/- mice whereas they were absent in the TrkB-/- animals. Regarding Pacinian corpuscles, the mutation in the trkB gene does not alter either the immunohistochemical or the ultrastructural characteristics. Finally, in muscle spindles the arrangement of the intrafusal muscle fibers and nerve fibers was unchanged in the mutated animals. Nevertheless, about 10% of muscle spindles showed increased number of the intrafusal cells (between 6 and 12) and were supplied by more than one large myelinic nerve fiber. The present results strongly suggest that TrkB-expressing sensory neurons in dorsal root ganglia, like those of the trigeminal ganglion, are responsible for the development and maintenance of several rapidly adapting cutaneous mechanoreceptors, i.e. Meissner's corpuscles.
Asunto(s)
Miembro Posterior , Mecanorreceptores/metabolismo , Husos Musculares/metabolismo , Receptor trkB/deficiencia , Receptor trkB/genética , Piel/metabolismo , Animales , Mecanorreceptores/ultraestructura , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Husos Musculares/ultraestructura , Receptor trkB/biosíntesis , Receptor trkB/ultraestructura , Piel/ultraestructuraRESUMEN
The distribution of S100-immunoreactive (ir) corpuscular endings was examined in the palate of wildtype and knockout mice for trkA, trkB or trkC. In wildtype mice, S100-ir corpuscular endings were abundant at the top of palatal rugae. The endings contained 2-4 parallel arrays of S100-ir neurites. The distribution of S100-ir nerve endings in trkA and trkC knockout mice was similar to that in wildtype mice; S100-ir corpuscular endings were abundant in palates of the mutant mice. In trkB knockout mice, the palate was devoid of corpuscular endings, An immunoelectron microscopic method indicated that S100-ir corpuscular endings were identical to Meissner corpuscles. The normal development of Meissner corpuscles is probably dependent on trkB but not trkA or trkC.