No abstract

We describe here a simple method for combining nonradioactive and radioactive in situ hybridization and immunohistochemistry on the same brain tissue section. This approach was first developed on the well-characterized hypothalamo-neurohypophyseal system, facilitating the optimization of the triple-labeling procedure and the verification of labeling specificity. We report the simultaneous detection of vasopressin (VP) mRNA with a digoxigenin-labeled oligonucleotide, oxytocin (OT) mRNA with a %-labeled oligonucleotide, and OT peptide in the same 12-pm ayostat section. This was performed on floating sections as follows: first, the two probes were hybridized simultaneously; second, the peptide was detected with an immunoperoxidase-DAB procedure; third, the digoxigenin-labeled probe was detected with an alkaline phosphatase-NBTIBCIF' tech- IntroductionThe phenotypic organization of the nervous system is extremely complex. Many informative molecules, such as classical neurotransmitters and neuropeptides, have been demonstrated to be coexpressed in neuronal populations (15). For example, several peptides, as well as tyrosine hydroxylase, have been detected in the hypothalamo-neurohypophyseal system (6,24). Histochemical techniques, such as immunohistochemistry and in situ hybridization, became powerful tools for study of such phenotypic structure. However, single immuno-or hybridohistochemical labeling does not allow the precise analysis of the coexpression phenomena that frequently occur in neuronal cells. Such analyses can be made by performing multiple labelings involving immunohistochemistry and/or in situ hybridization in the same tissue sample. nique; and finally, the 35S-labeled probe was detected by histological autoradiography. We also demonstrate that this approach is suitable for the simultaneous detection of tyrosine hydroxylase and two less abundant mRNAs, vasoactive intestinal peptide and vasopressin "As, in the suprachiasmatic nucleus. The combination of the three techniques did not significantly diminish their specificity or sensitivity. In conclusion, this new method, permitting the simultaneous detection of three different products of gene expression in the same section, could be useful for further analysis of the phenotypic organization and its plasticity in endocrine or neural tissues. In recent years the sensitivity of in situ hybridization techniques has increased, allowing the detection of mRNAs encoding various informative molecules, using both radioactive and non-radioactive probes (4,12,41). Moreover, double labeling involving double in situ hybridization of two different probes has been successfully performed. This was done, for example, by combining the use of a radiolabeled probe and a biotin-(28,29,33), a digoxigenin-(42), or an alkaline phosphatase-labeled probe (21). Double nonradioactive in situ hybridization has also been developed by use of fluorescent (11) or enzymatic (17) markers. In addition, in situ hybridization histochemistry has been coupled with immunohistochemistry in sever...

No abstract

No abstract

Tyrosine hydroxylase (TH) is expressed in catecholaminergic neurons. However, under certain conditions it is also ectopically expressed in magnocellular neurons of the hypothalamus. To test the hypothesis that this expression of TH is related to the cellular activation of these neurons and/or to the vasopressin (VP) expression, we studied the expression of both TH and VP in control and salt-loaded aged rats. Our results demonstrate that aged rats show a marked TH expression in VP cells which is further increased by osmotic stimulation in the absence of increase in VP synthesis in the supraoptic nucleus. The presence of TH-immunopositive dendritic swellings in the ventral part of this nucleus reveals the high state of plasticity of these neurons. Furthermore, the lack of several actors of catecholamine biosynthesis in these neurons suggests a different role for TH. This study further demonstrates an ectopic expression of TH in hypothalamic neurons of aged rats and a TH expression linked to the activation of VP neurons but unrelated to VP synthesis.