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Recent studies suggest that metabotropic glutamate receptors (mGluRs) may play a significant role in regulating basal ganglia functions. In this study, we investigated the localization of mGluR4a protein in the mouse and rat basal ganglia. Polyclonal antibodies that specifically react with the metabotropic glutamate receptor subtype mGluR4a were produced and characterized by Western blot analysis. These antibodies recognized a native protein in wild-type mouse brain with a molecular weight similar to the molecular weight of the band from a mGluR4a-transfected cell line. The immunoreactivity was absent in brains of knockout mice deficient in mGluR4. mGluR4a immunoreactivity was most intense in the molecular layer of the cerebellum. We also found a striking mGluR4a immunoreactivity in globus pallidus, and moderate staining in substantia nigra pars reticulata and entopeduncular nucleus. Moderate to low mGluR4a immunoreactivity was present in striatum and other brain regions, including hippocampus, neocortex, and thalamus. Double labeling with mGluR4a antibodies and antibodies to either a dendritic marker or a marker of presynaptic terminals suggest a localization of mGluR4a on presynaptic terminals. Immunocytochemistry at electron microscopy level confirmed these results, revealing that in the globus pallidus, mGluR4a is mainly localized in presynaptic sites in axonal elements. Finally, quinolinic acid lesion of striatal projection neurons decreased mGluR4a immunoreactivity in globus pallidus, suggesting a localization of mGluR4a on striatopallidal terminals. These data support the hypothesis that mGluR4a serves as a presynaptic heteroreceptor in the globus pallidus, where it may play an important role in regulating g-amino-n-butyric acid (GABA) release from striatopallidal terminals.

The postnatal expression of metabotropic glutamate receptors was studied in rat brain by in situ hybridization and autoradiographic binding techniques. The messenger RNAs encoding five metabotropic glutamate receptor subtypes named mGluR1-5 had distinct regional and temporal expression profiles. mGluR1, mGluR2 and mGluR4 messenger RNA expression was low at birth and increased during postnatal development. In contrast, mGluR3 and mGluR5 were highly expressed at birth and decreased during maturation to adult levels of expression. [3H]Glutamate binding competition studies in developing brain disclosed the presence of two types of binding sites with the pharmacological properties of metabotropic glutamate receptors, having high (metabotropic type-1 binding sites; K1 = 8 nM) and low affinity (metabotropic type-2 binding sites; K1 = 50 microM) for quisqualic acid, as in adult rat brain. The densities of metabotropic binding sites changed during development in a complex, regionally specific fashion. Metabotropic type-1 binding sites were present at low levels at birth and gradually increased during the second postnatal week. In the striatum, globus pallidus and cerebellar granule layer, the increase in density of metabotropic type-1 binding sites was transient but persisted in the cerebellar molecular layer. In contrast, metabotropic type-2 binding sites were present at high densities in most regions in the first postnatal week and decreased during the second and third week, particularly in the thalamic reticular nucleus and globus pallidus. Only in the external cortex did both metabotropic type-1 and metabotropic type-2 binding sites increase during development. A striking correspondence between the temporal pattern of expression of specific metabotropic glutamate receptor transcripts and metabotropic binding sites was observed in the reticular nucleus of the thalamus (mGluR3; metabotropic type-2 binding sites) and cerebellum (mGluR1; metabotropic type-1 binding sites) suggesting early translation of these metabotropic glutamate receptor messenger RNAs into receptor proteins. In other regions the relationship between messenger RNA expression and binding sites was less direct: comparison between expression of metabotropic glutamate receptor messenger RNA and binding sites suggests both a pre- and postsynaptic location of some receptor subtypes. These data imply a functional role of mGluR3 and mGluR5 during synaptogenesis and maintenance of adult synapses and of mGluR1, mGluR2 and mGluR4 in mature synaptic transmission.

Spasmodic (spd) is a recessive mouse mutation characterized by a prolonged righting reflex, fine motor tremor, leg clasping, and stiffness. Using an intersubspecific backcross that segregates spd, we placed spd on Chr 11 with the following gene order: Adra-1-3.8 +/- 2.1 cM-Pad-1-6.3 +/- 2.7-(spd, Anx-6, Csfgm, Glr-1, Il-3, Il-4, Il-5, Sparc)-9.1 +/- 2.4-D11 Mit5-2.2 +/- 1.5-Asgr-1. This localization eliminated the alpha 1-adrenergic receptor (Adra-1) and the alpha 1 and gamma 2 subunits of the GABAA receptor as candidate genes. Two other promising candidate genes, annexin VI (Anx-6) and a glutamate receptor (Glr-1), were mapped to within 2.1 cM of the spd locus. Although no recombination was observed between spd and Anx-6 or Glr-1, no evidence was obtained for a lesion in either gene. The presence of normal Anx-6 and Glr-1 mRNA transcripts was confirmed by Northern blot analysis, in situ hybridization, and DNA sequence analysis. The localization of Anx-6 and Glr-1 extends the known synteny homology between human chromosome 5q21-q31 and mouse Chr 11 and reveals the probable chromosomal location of the human counterpart to spd. Synteny homology and phenotypic similarities suggest that spasmodic mice may be a genetic model for the inherited human startle disease, hyperekplexia (STHE).