Figure 1.
Generation of Gγ7-Cre and Gγ7-mCrePR mice.
A, Schema of the exon 4 region containing the translational initiation site of the Gng7 gene, targeting vector, and targeted allele. The targeting vector carries the cre or mCrePR gene and the neo gene flanked by two frt sequences. A, ApaI; EV, EcoRV; K, KpnI; S, SpeI. B, LacZ expression following Cre recombination. X-gal-staining of sagittal and coronal sections from Gng7+/cre; +/CAG-CAT-Z mice at postnatal day 14. Sections were counterstained with nuclear fast red. Abbreviations: Ce, cerebellum; Cx, cortex; Hi, hippocampus; Po, pons; St, striatum; Th, thalamus. Scale bars, 1 mm.
Figure 2.
Inducible ablation of striatal neurons.
A, Schema for striatal neuron ablation induced by RU-486 administration. B, TUNEL staining (green) counterstained with DAPI (blue) in brain sections of control (left) and mutant (right) mice 10 days after mock and RU-486 administration, respectively. Scale bars, 1 mm. Abbreviations: Ce, cerebellum; Cx, cortex; Hi, hippocampus; Po, pons; St, striatum; Th, thalamus.
Figure 3.
A, Immunohistochemical analysis for neuronal marker NeuN in control (left) and mutant (right) mice 13 days after mock and RU-486 administration, respectively. Scale bar, 1 mm. B, Higher magnification of NeuN-immunohistochemistry in various brain regions. Scale bars, 0.1 mm. C, NeuN immunoreactive (NeuN+)-cell density in the CP after drug administration. n = 8–9 each. D, Densities of NeuN-positive cells in the NAc core (NAcC, open circles) and the NAc shell (NAcS, filled circles) after RU-486 treatment of Gng7+/mCrePR; +/Eno2-STOP-DTA mice (n = 8–9 each). E, Densities of NeuN-positive cells in the lateral amygdala (LA) of control and mutant mice 22 days after mock and RU-486 treatment, respectively (n = 15 each, F1,28 = 0.23, P = 0.64, one-way ANOVA). Abbreviations: Au, auditory cortex; CA1, hippocampal CA1 region; CP, caudate putamen; Cx, cortex; GP, globus pallidus; MGN, medial geniculate nucleus of thalamus; NAc, nucleus accumbens; OT, olfactory tubercle; PAG, periaqueductal gray; Sp, septum.
Figure 4.
Ablation of medium-spiny projection neurons in the striatum of mutant mice.
A, Immunoreactivity for calbindin in the dorsal striatum of control (upper) and mutant (lower) mice. B, Immunoreactivity for tyrosine hydroxylase and substance P in substantia nigra of control and mutant mice. C, Immunoreactivity for GAD and enkephalin in GP of control and mutant mice. Abbreviations: CP, caudate putamen; GP, globus pallidus; SNc, substantia nigra pars compacta; SNr, substantia nigra pars reticulata; VTA, ventral tegmental area. Scale bars, 1 mm.
Figure 5.
Performance of mutant mice in motor tests.
A, Foot print of control (left) and mutant (right) mice. Scale bar, 2 cm. B, Tail suspension test of control (left) and mutant (right) mice. C, Performance of control (open circles, n = 9) and mutant (filled circles, n = 8) mice in the stationary thin rod test. D, Performance of control (open circles) and mutant (filled circles) mice in the accelerating rotarod (n = 8 each). E, Locomotor activity of control (open circles, n = 10) and mutant (filled circles, n = 7) mice in the openfield test.
Figure 6.
Impaired freezing responses of mutant mice after auditory fear conditioning with a low-intensity footshock.
A, Experimental design. Mice were injected with RU-486 or vehicle. Fourteen days after treatment, the animals were subjected to auditory fear conditioning. B, Freezing responses of control (open circles, n = 9) and mutant (filled circles, n = 8) mice on the conditioning (left) and test (right) days. Auditory fear conditioning was carried out with the standard intensity of footshock (0.5 mA, an arrow). Solid lines represent tone. C, Freezing responses of control (open circles, n = 8) and mutant (filled circles, n = 11) mice and RU-486-treated Gγ7-mCrePR mice (RU-486 control) (shaded triangles, n = 7) on the conditioning (left) and test (right) days. Auditory fear conditioning was carried out with a low intensity of footshock (0.3 mA, an arrow). Solid lines represent tone. D, Current thresholds of control (open bar), RU-486-control (shaded bar) and mutant (filled bar) mice for flinch and jump reactions (n = 6 each).
Figure 7.
Impairment of long-term fear memory.
A, Experimental design. Mice were injected with RU-486 or vehicle. Fourteen days after treatment, the animals were subjected to auditory fear conditioning with a weak footshock at 0.3 mA. Freezing responses to tone were measured 1 or 3 h and 24 h after conditioning. B, Freezing responses of control (open circles, n = 8) and mutant (filled circles, n = 5) mice 1 h (left) and 24 h (right) after conditioning. C, Freezing responses of control (open circles, n = 6) and mutant (filled circles, n = 4) 3 h (left) and 24 h (right) after conditioning. D, Experimental design. Mice were subjected to auditory fear conditioning with a footshock at 0.3 mA or 0.5 mA. One day after conditioning, the conditioned mice were injected with RU-486 or vehicle. Their freezing responses were measured 14 days after drug treatment. E, Mice were subjected to auditory fear conditioning with a weak footshock at 0.3 mA. Freezing responses of mock-injected (open circles, n = 7) and RU-486-injected (filled circles, n = 8) mice on the conditioning (left) and test (right) days. F, Mice were subjected to auditory fear conditioning with the standard footshock at 0.5 mA. Freezing responses of mock-injected (open circles, n = 6) and RU-486-injected (filled circles, n = 7) mice on the conditioning (left) and test (right) days.