NeuroanatomyImmunocytochemical visualization of d-glutamate in the rat brain
Section snippets
Animals
Ten adult Sprague–Dawley male rats (weight 300–500 g) obtained from commercial sources (Charles River, Châtillon-sur-Chalarone, France; and CERJ Janvier, Le Gemest/Isle, France) were used to study the distribution of immunoreactive structures containing d-glutamate. The animals were kept under standardized light and temperature conditions and had free access to food and water. The animals remained for at least 10 days in their cages before the experiments. The experimental design, protocols,
Specificity of the d-amino acid antisera
Antibody avidity and specificity were checked using the ELISA testing system and the estimated antibody avidity (IC50) was fairly high (10−8 M for anti-conjugated d-glutamate antibodies; 10−9 M for anti-conjugated d-tryptophan antibodies; 10−7 M for anti-conjugated d-cysteine antibodies; 10−9 M for anti-conjugated d-tyrosine antibodies and 10−9 M for anti-conjugated d-methionine antibodies). This avidity was evaluated by competition experiments in ELISA with conjugated d-glutamate–G–BSA, etc.
Discussion
In this study, we observed neither immunoreactive fibers and cell bodies containing d-tryptophan, d-cysteine, d-tyrosine or d-methionine, nor immunoreactive fibers containing d-glutamate. However, a clear visualization of the d-glutamate-immunoreactive cell bodies was found for the first time in the rat brain. As mentioned above, anti-conjugated d-glutamate antibodies do not present cross-reactivity with similar structures (see Table 1); the most important findings here are that anti-conjugated
Acknowledgments
This work has been supported by: Gemacbio Laboratories (Cenon, France); IDRPHT (Talence, France); INCYL (Salamanca, Spain) and Federación de Cajas de Ahorro de Castilla y León (Spain). The authors wish to thank N. Skinner for stylistic revision of the English text and Mr. G. Glaize for technical assistance.
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