Performance deficits of mGluR8 knockout mice in learning tasks: the effects of null mutation and the background genotype
Introduction
Metabotropic glutamate receptors (mGluRs) are G-protein coupled receptors (GPCRs) that signal via second messenger systems (for reviews see Schoepp et al., 1999, Roberts, 1995). The eight known subtypes of these receptors can be grouped according to their molecular structure, signaling mechanism, and pharmacological profile (Conn and Pin, 1997, Schoepp et al., 1999). For example, Group III (mGlu4, mGlu6, mGlu7 and mGlu8) mGlu receptors are coupled via Gi to adenylyl cyclase and inhibit stimulated cAMP formation. mGluRs are expressed in the brain and several of these receptors have been implicated in the modulation of neuronal plasticity including long-term potentiation (LTP) and learning and memory (Hölscher et al., 1999, Gerlai et al., 1998, Lu et al., 1997, Riedel et al., 1996. A potential problem with in vivo functional characterization of these receptors, however, has been that selective pharmacological agents were not available for all of them. To circumvent the lack of selective tools molecular biologists (e.g. Tonegawa, 1995) have suggested the use of alternative techniques, including gene targeting with the use of homologous recombination in embryonic stem (ES) cells. Although the number of small molecule tools has increased substantially over the past decade, and selective action of these drugs on receptor subtypes has improved (Schoepp et al., 1999), gene targeting may still offer the most precise and specific way with which particular receptors may be manipulated. In the present paper we focus our attention on mGluR8, a member of the mGluR family, and investigate the phenotypic effects of a null mutation that was introduced at the mGluR8 gene locus in mice.
Numerous mGluRs have been shown to play roles in neuronal plasticity in general and in learning and memory in particular. For example, a null mutation of the gene encoding mGluR4, a presynaptic receptor, impaired long-term memory in the spatial learning task of the Morris water maze in mice (Gerlai et al., 1998). It was hypothesized to affect the temporal coding in the entorhinal–hippocampal circuitry. It was also shown to impair procedural motor learning, a process primarily dependent upon plasticity of particular cerebellar neuronal circuits (Pekhletski et al., 1996). mGluR5, a postsynaptic receptor, was also shown to be involved in learning and memory (Lu et al., 1997). Its disruption by genetic knock out led to impaired contextual and spatial learning in the fear conditioning and in the water maze paradigms, respectively, behavioral alterations that were associated with impaired hippocampal CA1 but normal CA3 long-term potentiation (LTP). Similarly to genetic diruption, pharmacological blockade of mGluR function using, e.g. (R, S)-alpha-methyl-4-carboxyphenylglycine (MCPG), a group I and II mGluR antagonist, led to significant memory impairment in rats (for review see Riedel et al. (1996)).
mGluR8 was the last to be cloned among the known mGluRs (Duvoisin et al., 1995). Its mRNA was detected in the olfactory bulb, lateral reticular nucleus of the thalamus, cerebral cortices, hippocampus, cerebellum and also the retina (Duvoisin et al., 1995, Saugstad et al., 1997). Immunohistochemical localization of the mGluR8 protein revealed that it is in the terminal fields of the lateral perforant path in the outer molecular layer of the dentate gyrus and in the CA3 stratum lacunosum moleculare region of the hippocampus (Shigemoto et al., 1997). It was also found in the piriform and entorhinal cortices (Kinoshita et al., 1996). Electrophysiological studies suggested that mGluR8 functions as a presynaptic autoreceptor controlling glutamate release from the lateral perforant path terminals in the dentate gyrus (Bushell et al., 1996, Shigemoto et al., 1997).
Given the known expression pattern of mGluR8, its function in glutamate release in the dentate gyrus, and also given the known role of other mGluRs in neuronal plasticity, we decided to analyze the effects of gene knock out of mGluR8 on learning and memory. By analyzing behavioral performance of mGluR8 null mutant mice in comparison with that of wild type control mice in numerous behavioral paradigms, we show that disruption of this gene leads to subtle behavioral alterations that manifest as novelty induced hyperactivity and also as delayed and diminished responding to certain stimuli. Furthermore, we also demonstrate a significant performance abnormality, impaired vision, in the host strain (ICR) that was used to carry the null mutation. This impairment significantly affected both the mutant and the wild type mice and made it difficult to properly evaluate the effect of the null mutation in certain behavioral paradigms.
Section snippets
Animals and housing
mGluR8 null mutant mice were generated using homologous recombination in ES cells as described in detail by Zhai et al. (2002). Briefly, a null mutation was introduced using homologous recombination in the R1 embryonic stem (ES) cell, a cell line which was derived from a hybrid mouse generated by crossing the mouse strain 129/SvJ with another 129 substrain, 129/SvCP (Nagy et al., 1993). The recombinant ES cells carrying the null mutation were injected into C57BL/6 blastocysts and the chimeras
Irwin profiling
No gross abnormalities were detected. All mice appeared healthy, active, and well groomed. Body weight (mGluR8 null mutant=44.8 g+1.685, wild type control=44.2 g+0.800; t=0.322, df=8, p>0.75) and rectal body temperature (mGluR8 null mutant=37.78 °C+0.139, wild type control=37.86°C+0.150; t=0.390, df=8, p>0.70) did not significantly differ between mGluR8 null mutant and wild type control mice. Gross alteration of locomotory activity and reactivity to auditory and tactile stimulation was not
Discussion
Our present results indicate that disruption of the mGluR8 gene does not lead to gross alteration of brain development and/or brain function. The mutant mice appeared healthy and, at the gross level, behaved normally. Only subtle alterations could be detected using a cognitive task, the context and cue dependent fear conditioning. However, characterization of cognitive performance of the mGluR8 null mutant mice was hampered by a performance defect, impaired vision, associated with their ICR
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