Research reportCellular and subcellular localization of the 2B-subunit of the NMDA receptor in the adult rat telencephalon
Introduction
Glutamate (Glu) is the predominant excitatory amino acid (EAA) transmitter in the mammalian cerebral cortex 17, 19being located mainly in pyramidal and spiny stellate cells [33]as well as cortical and hippocampal axon terminals [25]. Glu exerts its effects by acting on different types of glutamate receptors (GluR) which are subdivided into metabotropic and ionotropic subtypes [19]. Among the ionotropic receptors, d,l-α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA; GluR1–GluR4 or GluRA–GluRD), high-affinity kainate (GluR5–GluR7, KA1 and KA2) and N-methyl-d-aspartate (NMDA) receptors can be distinguished (for review see Ref. [9]). In recent years, NMDA receptors (NR) have raised particular interest for two reasons: First, NMDA receptors are considered to be major regulators of synaptic plasticity and long-term changes in cortical and hippocampal neurons [2]. Second, NMDA receptors have been suggested to be involved in excitotoxic cell death and other degenerative processes related to neurological diseases 4, 14. NMDA receptors are widely distributed throughout the CNS 20, 21, 23, 24. Homologous subtypes and regional distribution have been described for mouse [13]and man [12].
Electrophysiological data from coexpression studies of different NR subunits in Xenopus oocytes indicate that the native receptor is composed of the essential subunit NR1—existing in eight different splice variants—in combination with any of the subunits NR2A-D [9]. In addition, immunoprecipitation studies have shown that during postnatal stages NMDA receptor complexes can be composed of at least three distinct subunits 15, 31. While NR1 is ubiquitously distributed throughout the CNS, the transcripts encoding individual NR2 subunit variants are restricted to defined CNS regions during development 20, 21. In particular, hippocampal structures have been a target of investigations on the distribution pattern of Glu-receptors, as the major excitatory circuits in hippocampus are known and concise information about NMDA receptor localization can be deduced from NMDA-dependent and NMDA-independent LTP in defined regions of the hippocampus (for review see Ref. [22]).
Little is known, however, on the cellular and subcellular distribution of the NR2B polypeptide in rat telencephalon. A better understanding of learning and memory processes, ischemia and epilepsy-related phenomena, however, requires concise data on the distribution of distinct NR subunits at the level of both, gene and protein expression in mammalian CNS.
Here we report on the cellular and ultrastructural elucidation of the NR2B distribution in the adult rat telencephalon using subunit-specific antibodies.
Section snippets
Animals and preparation of tissue
Male Wistar rats (n=5, 6 months old) were deeply anesthetized by an intraperitoneal injection of avertin (1 ml/100 g b.wt) and perfusion-fixed via the left ventricle using 100 ml phosphate-buffered saline (PBS) as rinsing solution and 300 ml Zamboni's fixative (cf. Ref. [30]). Brains were removed and postfixed in Zamboni's fixative for 2 h at 4°C. Subsequently, coronal vibratome sections (40 μm) of the neocortex and the hippocampus were prepared from all brains. For control purposes, sagittal
Light microscopy
Of all telencephalic areas analysed at low magnification, the hippocampus stood out displaying the highest intensity of labeling. Intensely labeled structures were also seen throughout the cortex, in particular in the superficial cortical layers II/III and in layer V.
Cerebral cortex
The general pattern of NR2B-IR was characterized by immunolabeling of numerous cortical neurons, in particular pyramidal-like ones of layers II/III and V, whereas staining of neurons in layers I and IV was relatively weak (Fig. 1).
Discussion
The present data suggest a widespread distribution of NR2B subunit antigen throughout the rat cerebral cortex and hippocampus, preferentially labeling pyramidal neurons (layer II/III and V) of the cortex, the hippocampal CA region (predominantly CA1 and CA3), and neurons of the DG. Electron microscopy of the cerebral cortex is consistent with a distribution mainly in postsynaptic densities. This distribution pattern is consistent with that of the subunit NR1 as described by Brose et al. [3],
Conclusion
The present study has provided further evidence for the codistribution of NR2B and NR1 subunits. Furthermore, NR2B was not only located at classical postsynaptic sites but also extrasynaptically. The rare presynaptic sites which were labeled will have to be studied further with high resolution techniques. Finally the study has given hints of a possible association of NR2B with structures inducing LTP.
Acknowledgements
We are greatly indebted to K. Pilz and Ch. Hofmann for excellent technical, to I. Koch for photographic and A. Kirchmayer for secretarial assistance.
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