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Previous studies conducted in vitro indicate that three Homer proteins (Homer 1, 2, and 3) modulate the activity and localization of group I metabotropic glutamate receptors (mGluRs). Furthermore, the coexpression of Homer 1b with other scaffolding proteins has been shown to induce spine growth in hippocampal neuron cultures. To assess the role of Homer proteins in regulating the trafficking of group I mGluRs in vivo, we used immunocytochemical methods at the electron microscopic level to determine the localization of mGluR1a and mGluR5 in the striatum of normal and Homer-deficient mice. Quantitative analysis revealed no significant difference in the relative distribution of mGluR1a and mGluR5 labeled neuronal elements between normal and Homer knockout (KO) mice. However, a significant decrease in mGluR5 labeled spines was found in the striatum of KO mice. These findings suggest that Homer proteins may play a role in spinogenesis but do not solely regulate the trafficking of group I mGluRs in the striatum. Studies are in progress to assess Homer regulatory function of the subsynaptic distribution of group I mGluRs in the striatum.
The classic model of the basal ganglia network describes two parallel pathways, a direct and an indirect pathway, that run from the striatum to the two basal ganglia output nuclei (the internal globus pallidus (GPi) and the substantia nigra pars reticulata (SNr).) Because activation of these two pathways induces opposite effects on motor behaviors, a balanced activity is necessary to maintain normal motor functioning of the basal ganglia.
Glutamate is the major excitatory neurotransmitter in the brain, and both ionotropic and metabotropic classes of glutamate receptors (iGluRs and mGluRs) are involved in the modulation of glutamate transmission (Blandini et al., 2000). The iGluRs mediate fast excitatory neurotransmission and are classified into three subgroups: NMDA, AMPA, and kainate receptors. The mGluRs mediate slow excitatory transmission via coupling to G proteins, and are catalogued into three groups (group I-III) (Conn et al., 2005). Specifically, the group I mGluRs (mGluR1 and mGluR5) are primarily post-synaptic, and couple to phospholipase C and intracellular Ca2+ via Gq/11 proteins (Conn et al., 1997; Marino et al., 2003; Conn et al., 2005). The striatum is the primary input nucleus to the basal ganglia and receives strong glutamatergic afferents from the cerebral cortex and thalamus. Recent studies suggest that abnormal glutamatergic transmission in the striatum contributes to disorders of the basal ganglia (Conn et al., 2005). To elucidate the role of glutamate in abnormal basal ganglia function, we must understand the localization and function of glutamate receptors.
In vitro studies suggest that Homer isoforms regulate the cell surface expression and subcellular distribution of group I mGluRs (Roche et al., 1999; Ango et al., 2000). The Homer 1 gene encodes both constitutive (Homer 1b and c) and immediate early gene (IEG) products (Homer 1a) (Xiao et al., 1998). Unlike the constitutive Homer proteins, Homer 1a encodes only a single N-terminal EVH1 (Enabled/Vasodilator-stimulated phosphoprotein homology-1) domain and lacks the C-terminal coiled-coil domain necessary for the self-multimerization between Homer proteins (Kammermeier et al., 2000). Because Homer 1a is incapable of multimerization, the protein disrupts the Homer-mediated formation of protein complexes, thereby modulating mGluR-induced intracellular calcium release (Tu et al., 1998; Xiao et al., 2000; Ciruela et al., 2000). Evidence from further studies conducted in vitro indicates that the Homer proteins regulate spine growth. For instance, recent studies showed that the coexpression of Homer 1b with other scaffolding proteins increases spinogenesis, while Homer 1a has the opposite effect, in hippocampal cultured neurons (Sala et al., 2001; Sala et al., 2003). One way to imitate the presence of Homer 1a in vivo involves the deletion of all Homer proteins. To gain a more thorough understanding of the role of Homer in brain plasticity, we used electron microscopy immunocytochemistry to compare the subcellular localization of group I mGluRs in the striatum of Homer KO versus WT mice.
Animals: Adult WT and triple Homer KO mice provided by the laboratory of Dr. Paul Worley at The Johns Hopkins University.
Histology: The mice were perfused with 4% paraformadehyde and 0.1% glutaraldehyde. The tissue was then cut and prepared for electron microscopy (EM).
Pre-embedding Immunoperoxidase: A primary antibody of either rabbit anti-mGluR1a or rabbit anti-mGluR5 was used at a dilution of 1:1000 and 1:5000, respectively. Biotinylated goat anti-rabbit IgG secondary antibodies were used at a dilution of 1:200. The antibodies were revealed using the avidin-biotin peroxidase complex (ABC) method and 3,3-diaminobenzidine tetrahydrochloride (DAB).
Electron microscopy: Pieces of tissue from the striatum were mounted on resin blocks, cut into 60 nm ultrathin sections, and serially collected on copper grids. A total of 19 blocks (9 for mGluR1a, 10 for mGluR5) were viewed with an electron microscope at a magnification of 25000X.
Data Analysis: Four data groups (mGluR1a in WT, mGluR5 in WT, mGluR1a in Homer KO, mGluR5 in Homer KO), each containing a total of 4-5 mice, were analyzed. A total of 40 electron micrographs were taken of each block, and labeled elements were classified. The density of each labeled neuronal element (i.e. dendrite, dendritic spine, unmyelinated axon, myelinated axon, and axon terminal) per subgroup was determined and entered into an Excel spreadsheet. The categorical mean values were calculated, and student’s t-tests were performed in SigmaStat software to determine any statistical differences in the density of labeled elements between KO and WT animals. P < 0.05 was used to determine significant difference.
Labeling for mGluR1a and mGluR5 was primarily post-synaptic (i.e. in dendrites and spines) with some pre-synaptic axonal labeling in the WT and Homer KO striatum. The results from our initial analysis of the data suggested that spine labeling for mGluR5 significantly decreases in the Homer KO striatum (P < 0.05). To verify this conclusion, we calculated the relative abundance of spines (i.e. % labeled post-synaptic elements that are spines/% total post-synaptic elements that are spines). That this value is approximately the same in the WT and Homer KO striatum suggests that the total number of mGluR5 immunoreactive spines decreases in the Homer KO mouse striatum.
• The cell surface expression and subcellular localization of the group I mGluRs do not significantly differ between Homer KO and WT mice. These findings suggest that proteins other than Homer might also be involved in the trafficking of group I mGluRs in the striatum.
• Mice lacking the Homer proteins are associated with a decrease in total spines in the striatum. These findings suggest that Homer proteins play a role in spinogenesis and synaptic plasticity in the striatum.
Directions for Future Study
• Evidence from studies conducted in vitro suggests that Homer regulates the intracellular expression of mGluR1a and mGluR5. The immunogold technique must be used to determine and compare the subsynaptic localization of mGluR1a and mGluR5 in the WT and Homer KO mouse striatum.
• Demonstrate changes in spine density in the striatum of KO animals using unbiased stereological methods.
The authors thank Dr. Paul F. Worley (The Johns Hopkins University) for providing the mice used in this study, as well as all members of Dr. Yoland Smith’s lab for their support and assistance. We are also grateful to Cathy Quinones and Pat Marsteller for the organization of the SURE program. This material is based upon work supported by the Howard Hughes Medical Institute under Grant No. 52003727 and by the U.S. National Institutes of Health under Grant No. R01NS037423 to YS.
The basal ganglia comprise a network of several nuclei involved in motor activity. Glutamate is the major excitatory neurotransmitter in the brain, and glutamate receptors have been localized in all nuclei of the basal ganglia. Furthermore, previous studies conducted in cellular systems suggest that the Homer scaffolding proteins regulate the trafficking and localization of a specific subgroup of glutamate receptors (group I mGluRs), and also modulate dendritic spine growth. This study investigated the subcellular localization of group I mGluRs in the striatum of normal and Homer-deficient mice. Data analysis indicated no significant difference in the subcellular localization of group I mGluRs, although a decrease in total dendritic spines was found in the Homer-depleted striatum. Our findings may help us to better understand movement disorders of the basal ganglia, such as Parkinson’s disease.
Electron microscopy immunocytochemistry
Parkinson, glutamate, Homer, ionotropic glutamate receptor, metabotropic glutamate receptor, basal ganglia, striatum, spinogenesis
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