Elsevier

Neuroscience

Volume 144, Issue 2, 19 January 2007, Pages 654-664
Neuroscience

Neuroanatomy
Immunocytochemical visualization of d-glutamate in the rat brain

https://doi.org/10.1016/j.neuroscience.2006.09.045Get rights and content

Abstract

Using highly specific antisera directed against conjugated d-amino acids, the distribution of d-glutamate-, d-tryptophan-, d-cysteine-, d-tyrosine- and d-methionine-immunoreactive structures in the rat brain was studied. Cell bodies containing d-glutamate, but not d-glutamate-immunoreactive fibers, were found. Perikarya containing this d-amino acid were only found in the mesencephalon and thalamus of the rat CNS. Thus, the highest density of cell bodies containing d-glutamate was observed in the dorsal raphe nucleus, the ventral part of the mesencephalic central gray, the superior colliculus, above the posterior commissure, and in the subparafascicular thalamic nucleus. A moderate density of immunoreactive cell bodies was observed in the dorsal part of the mesencephalic central gray, above the rostral linear nucleus of the raphe, the nucleus of Darkschewitsch, and in the medial habenular nucleus, whereas a low density was found below the medial forebrain bundle and in the posterior thalamic nuclear group. Moreover, no immunoreactive fibers or cell bodies were visualized containing d-tryptophan, d-cysteine, d-tyrosine or d-methionine in the rat brain. The distribution of d-glutamate-immunoreactive cell bodies in the rat brain suggests that this d-amino acid could be involved in several physiological mechanisms. This work reports the first visualization and the morphological characteristics of conjugated d-glutamate-immunoreactive cell bodies in the rat CNS using an indirect immunoperoxidase technique. Our results suggest that the immunoreactive neurons observed have an uptake mechanism for d-glutamate.

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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