Regulation of motor performance and striatal function by synaptic scaffolding proteins of the Homer1 family

Abstract

Intracellular calcium mobilization and signaling mechanisms triggered by activation of synaptic glutamate receptors in the striatum are important modulators of neurotransmission in striatal circuits. However, the expression and functions of scaffolding proteins anchoring glutamate receptors at striatal synapses have not been addressed so far. The long-form Homer1 proteins, Homer1b/c, assemble group I metabotropic glutamate receptors (mGluR1/5) in large macromolecular complexes with sources of calcium influx and release at synapses as well as with components of the NMDA receptor complex at the neuronal cell membrane. Homer1a, the short, activity-dependent splice variant of Homer1b/c, lacks the ability of linking mGluR1/5 to synaptic proteins and functions as an endogenous negative modulator of the mGluR1/5 inositol 1,4,5-trisphosphate receptor signaling complex. We have generated transgenic mice, which overexpress Homer1a in striatal medium spiny neurons either homogenously throughout the extrastriosomal matrix (Homer1a-matrix line) or predominantly in striosomal patches (Homer1a-striosome line). Homer1a-expressing mice demonstrated normal development of striatal structure and afferent-efferent connectivity. However, motor performance in behavioral tasks and striatal responses to the psychomotor stimulant amphetamine were significantly altered in the Homer1a-striosome line. Thus, glutamate receptor scaffolding proteins of the Homer1 family critically regulate the functions of striatal medium spiny neurons in complex motor tasks and its modulation by psychomotor stimulant drugs.

Fig. 1.

Generation and initial characterization of transgenic mice expressing Homer1a in the forebrain. (A) Schematic representation of the targeting construct used to make mice expressing Homer1a and GFP in an inducible manner (Tg^{Homer1a-tetO7-GFP}), upon crossing with mice expressing tTA under the control of the CamKII promoter (Tg^CamKIItTA). pA, polyadenylation signal; CMVmin, minimal cytomegalovirus promoter; tetO7, tetracycline-binding operons. (B) Immunohistochemical determination of myc-tagged Homer1a using anti-myc antibody on forebrain sections of Homer1a-striosome mice and Homer1a-matrix mice. Arrows point to the Homer1a-positive striatal clusters resembling striosomal islands. (C) Immunohistochemical determination of myc–Homer1a using anti-myc antibody on striatal sections of Homer1a-striosome mice in the naïve state (without DOX) or 10 days after treatment with doxycycline (under DOX treatment). Doxycycline-induced sequestration of tTA leads to a loss of myc–Homer1a expression. *, striatum; D, dorsal; M, medial; L, lateral; V, ventral. (Scale bars: 200 μm in B and C.)

Fig. 2.

Characterization of Homer1a expression in striatal subcompartments. Dual immunofluorescence with anti-myc and anti-MOR antibodies on striatal sections of Homer1a-striosome mice and Homer1a-matrix mice. In the Homer1a-striosome line, the localization of myc–Homer1a-positive neurons is largely restricted to the MOR1-positive striosomal patches, whereas in the matrix line, it is more widespread. (Scale bar: 200 μm.)

Fig. 3.

Behavioral analysis of motor tasks in Homer transgenic mice. Behavioral analysis of motor tasks in wild-type mice (open bars), Homer1a-striosome mice (shaded bars), and Homer1a-matrix mice (filled bars) in the open-field test (A), bar-cross test (B), rotarod test (C) and light–dark choice test (D). E represents phenotypic changes in Homer1a-striosome mice (expressed as percentage difference over wild-type mice) in the open-field test and light–dark choice test in the naïve state (–DOX, light gray bars) and upon treatment with doxycycline (+DOX, hatched bars). For the latter, both wild-type and Homer1a-striosome mice were treated with doxycycline. *, P < 0.05 upon comparison with wild-type mice (n = 7–8 mice per group in each test).

Fig. 4.

Analysis of motor behavior (A and B) and expression of Fos (C and D) induced by acute i.p. administration of amphetamine or saline (control) in Homer1a-matrix mice or wild-type or Homer1a-striosome mice in the naïve state or after treatment with doxycycline. Please see the text for a detailed description of the behavioral rating scheme. A shows the behavioral score as a function of time after administration of amphetamine or saline. B represents the same data set, showing the average amount of time spent in each behavioral stage by the mice in each group as a percentage of the total observation time of 60 min. (C and D) Quantitative summary (C) and typical examples (D) of Fos immunoreactivity in the VLC or dorsomedial caudate (DMC) at 60 min after i.p. administration of saline or amphetamine in the same set of mice tested for behavioral scores shown in A and B. Boxed areas are magnified in the corresponding panels beneath them. Examples of specifically labeled, punctuate, Fos-immunoreactive cells and unspecifically labeled brown fiber bundles are indicated by arrowheads and arrows, respectively. (Scale bar: 200 μm.) *, P < 0.05 (n = 7–8 mice per group in each test).