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. 2003 Feb 1;23(3):748-57.
doi: 10.1523/JNEUROSCI.23-03-00748.2003.

The RAS Effector RIN1 Modulates the Formation of Aversive Memories

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Free PMC article

The RAS Effector RIN1 Modulates the Formation of Aversive Memories

Ajay Dhaka et al. J Neurosci. .
Free PMC article

Abstract

RAS proteins are critical regulators of mitosis and are mutationally activated in many human tumors. RAS signaling is also known to mediate long-term potentiation (LTP) and long-term memory formation in postmitotic neurons, in part through activation of the RAF-MEK-ERK pathway. The RAS effector RIN1 appears to function through competitive inhibition of RAS-RAF binding and also through diversion of RAS signaling to alternate pathways. We show that RIN1 is preferentially expressed in postnatal forebrain neurons in which it is localized in dendrites and physically associated with RAS, suggesting a role in RAS-mediated postsynaptic neuronal plasticity. Mice with an Rin1 gene disruption showed a striking enhancement in amygdala LTP. In addition, two independent behavioral tests demonstrated elevated amygdala-dependent aversive memory in Rin1(-/-) mice. These results indicate that RIN1 serves as an inhibitory modulator of neuronal plasticity in aversive memory formation.

Figures

Fig. 1.
Fig. 1.
Expression of Rin1 message and localization of Rin1 protein. A, A mouse multiple tissue Northern blot (Clontech) was hybridized with32P-labeled cDNA probes forRin1 (top) and actin(bottom). Lane 1, Heart; lane 2, brain; lane 3, spleen; lane 4, lung; lane 5, liver; lane 6, skeletal muscle; lane 7, kidney; and lane 8, testis. B–G, Rin1message is localized to mouse forebrain neurons. Wild-type mouse brain coronal sections were hybridized with a35S-labeled antisense Rin1 RNA probe (B, C, E–I) or sense probe (D). Autoradiographs indicate Rin1mRNA in the cortex (ctx), striatum (str), amygdala (a), and hippocampus (hpc). Expression intensity increased between P7 (B, C) and P21 (E, F). No expression was detected in the thalamus or in white matter tracts (*). G, H, Ten and 25× dark-field images of amygdala showed clear expression through this region. I, Light-field analysis at 40× showed Rin1mRNA localized over mouse CA3 neuronal nuclei. J,K, RIN1 protein was detected in cell bodies and dendrites but not the axonal mossy fibers of human hippocampal granule cells. Paraffin-embedded human coronal brain sections were subjected to immunohistochemistry with polyclonal anti-RIN1; J, 4×;K, 20×. L–N, Mouse Rin1 protein is expressed in the cell bodies and dendrites of CA1 hippocampal neurons. Paraffin-embedded coronal mouse brain sections were subjected to immunohistochemical staining with anti-Rin1 (polyclonal).Rin1−/−, L, 40×; WT,M, 40×; WT, N, 100×.
Fig. 2.
Fig. 2.
Generation of a targeted mutation in the mouseRin1 gene. A, Protein–genomic structure and targeting strategy. An ∼3 kb sequence encoding exons 2–7 was replaced by an ∼2 kb PGK promoter-driven neomycin resistance cassette. Restriction sites are as follows: B,BglII; Ba, BamHI;H, HindIII; K,KpnI; R, EcoRI;X, XbaI.B, Southern blot analysis. Genomic tail DNA samples were digested withBglII and hybridized with a 3′ flanking probe.Rin1 genotypes are indicated above eachlane. C, Northern blot analysis. Total RNA (30 μg) from the brains of mice of each genotype was hybridized withRin1 (top) or Gapdh(bottom) cDNA probes. D, Immunoblot analysis. Total protein (60 μg) from forebrains of mutant (−/−) and wild-type (+/+) mice was blotted with anti-Rin1 (top) or anti-ERK 1, 2 (bottom). E, Kluver-Barrera-stained coronal brain sections of the amygdala of wild-type (+/+) and Rin1−/− (−/−), 4×. F, Same, 20×.
Fig. 3.
Fig. 3.
LTP is enhanced in the amygdala ofRin1−/− mice. A, The amount of amygdala LTP induced by a theta-burst stimulation protocol (3× TBS) is enhanced in slices fromRin1−/− mice. Insetsshow extracellular responses elicited during baseline (smaller response) and 40 min after TBS in slices from wild-type (left set of traces) andRin1−/− mice (right set of traces). Calibration bars: 2 msec, 0.5 mV. B, The same TBS protocol used for amygdala, when applied to hippocampus slices, showed no difference with genotype.C, The amount of LTP induced by two trains of 100 Hz stimulation in slices from Rin1−/−mice was indistinguishable from that seen in wild-type slices.
Fig. 4.
Fig. 4.
Amygdala-dependent learning is enhanced inRin1−/− mutants. A, Animals (Rin1−/−,n = 16; WT, n = 18) were tested for cued freezing 48 hrs after training with a 0.5 mA shock in the presence of a 3 min tone (CS) in a neutral cage. There was no difference in percentage of time freezing between groups during PCS (Rin1−/−, 16.3 ± 2.7; WT, 10.2 ± 3.0; F(1,32) = 2.2;p > 0.05), whereasRin1−/− mutants froze significantly more than wild types to the CS (Rin1−/−, 45.0 ± 5.8; WT, 26.3 ± 4.4; F(1,32) = 6.6;p < 0.05). B, Short-term cued memory. Rin1−/− mutants (n = 17) and wild-type controls (n = 17) were tested 30 min after training. There was no difference in percentage of time freezing between groups during the PCS (Rin1−/−, 18.4 ± 3.5; WT, 11.8 ± 2.5; F(1,32) = 2.3;p > 0.05), but there was a significant increase for mutant mice in the CS (Rin1−/−, 54.4 ± 5.8; WT, 34.9 ± 5.5;F(1,32) = 6.0, p < 0.05). C, A shock reactivity test showed no alterations in response (centimeters per second ± SEM) to 2 sec shocks at 0.2 mA (WT, n = 7;Rin1−/−, n = 5), 0.5 mA (WT, n = 29;Rin1−/−, n = 27), or 0.75 mA (WT, n = 7;Rin1−/−, n = 9). *p < 0.05, indicates a significant difference between wild-type and mutant under the same conditions.
Fig. 5.
Fig. 5.
Conditioned taste aversion. Saccharin-flavored water was paired with an intraperitoneal injection (2% body weight) of PBS (Rin1−/−, n= 15; WT, n = 17) or 0.3 m LiCl (Rin1−/−, n = 14; WT, n = 15). Twenty-four hours later, mice were tested for saccharin aversion. Aversion indices were calculated as follows: (saccharin water consumed/(saccharin water consumed + unflavored water consumed)). Rin1−/−mutants (F(1,27) = 144.3;p < 0.05) and wild types (F(1,30) = 35.6; p< 0.05) both acquired a significant aversion to saccharin when paired with 0.3 m LiCl.Rin1−/− mutants, however, had a significantly higher aversion index than wild types (Rin1−/−, 0.24 ± 0.03; WT, 0.39 ± 0.04; F(1,27) = 8.9;p < 0.05). PBS-injected mutants and wild types exhibited equivalent preference for saccharin-flavored water (F(1,30) = 0.36; p> 0.05). *p < 0.05, indicates significant difference between wild type and mutant under the same conditions.
Fig. 6.
Fig. 6.
Hippocampus-dependent learning appears unaffected by deletion of Rin1. A,Rin1−/− mutants (n = 9) and wild-type controls (n = 8) were trained for 12 d with two trials per day (30 sec ITI). The average ± SEM latency to reach the hidden platform is plotted versus training day. Escape latencies decreased across days for both groups (F(11,165) = 7.0; p< 0.05), with no difference between mutants and wild types (F(1,15) = 0.9; p> 0.05). B, During the day 12 probe trial, both mutants (F(3,32) = 6.6; p< 0.05) and wild types (F(3,28) = 5.5;p < 0.05) searched selectively and spent significantly more time in the training quadrant than in any other quadrant (Fisher's PLSD; p < 0.05). There was no statistical difference between groups in time spent searching in the training quadrant (F(1,15) = 0.005;p > 0.05). C,Rin1−/− mice (n= 17) and wild-type controls (n = 12) were trained for 6 d with four trials per day (30 sec ITI). The average ± SEM latency to reach platform is plotted versus training day. Escape latencies decreased across days for both groups (F(6,135) = 26.0; p< 0.05), with no differences between mutant and wild types (F(1,27) = 0.9; p> 0.05). D, During the day 7 probe trial, conducted 24 hr after training to assess long-term memory, both mutants (F(3,64) = 14.0; p< 0.05) and wild types (F(3,44) = 9.0;p < 0.05) searched selectively and spent significantly more time in the training quadrant than in any other quadrant (Fisher's PLSD; p < 0.05). There was no statistical difference between groups in time spent searching in the training quadrant (F(1,27) = 0.38;p > 0.05). Dashed line indicates random search (25% in each quadrant). TQ, Training quadrant; AR, adjacent right; AL, adjacent left; OP, opposite quadrant.
Fig. 7.
Fig. 7.
Rin1 mutants have normal motor learning and open field performance. A, Accelerating rotarod. Wild-type (n = 12) andRin1−/− (n = 11) mice were given five trials in an accelerating rotarod (4–40 rpm in 5 min) during a single day. All subjects showed an increased latency to fall across trials (F(4,84) = 17.4;p < 0.05). In addition, both groups of animals fell off the rotating rod at the same time, indicating equivalent learning rates across trials (F(4,84) = 0.4; p > 0.05), with no effect of genotype (F(1,21) = 0.3; p> 0.05). B, Open field path length. Mice were placed in the center of a white circular arena (60 cm in diameter) and tracked for 5 min. Mean path length for mutant (n = 12; 2992 ± 352 cm) and wild type (n = 14; 2501 ± 215 cm) were equivalent (F(1,24) = 1.5; p> 0.05). C, Open field inner and outer zone exploratory behavior. Mutant and wild-type mice were equivalent in percentage of time exploring the inner and outer zones (F(1,24) = 0.5; p> 0.05).
Fig. 8.
Fig. 8.
Rin1 engagement with Ras in forebrain.A, Subcellular localization of RIN1 protein. Human forebrain extracts were prepared in hypotonic solution and separated into cytosolic and membrane fractions (see Materials and Methods). Membranes were further separated over a sucrose gradient into plasma membrane and microsomal fractions. Cytosolic (CY) and plasma membrane (PM) fractions (60 μg of total protein) were subjected to immunoblot analysis with polyclonal anti-RIN1 (top) or polyclonal anti-RAS (bottom). B, Endogenous Ras and Rin1 binding. Ras protein was immunoprecipitated from wild-type andRin1−/− mouse forebrain tissue extracts, and this material was then immunoblotted with anti-Rin1 (left two lanes). Mock immunoprecipitations, using anti-Myc or Anti-Flag agarose beads, showed no Rin1 material (right panel), as expected.
Fig. 9.
Fig. 9.
Model of RIN1 action in RAS-mediated pathways controlling learning and memory. Neurotransmitter receptor stimulation leads to activation of RAS exchange factors (SOS, GRF, RasGRP, and cnRAS-GEF) in postsynaptic cells. Negative regulators of RAS in neurons include the GTPase-activating proteins NF1 and SynGAP. RAS proteins signal through RAF proteins to initiate the MAP kinase cascade, resulting in transcription changes required for long-term memory. RIN1 inhibits this pathway by competing with RAF (and probably other RAS effectors, such as PI3K and RalGDS) for the effector binding site on RAS. RIN1 functions through two downstream pathways: (1) activation of RAB5 to promote receptor endocytosis and downregulation and (2) activation of ABL1 and ABL2 tyrosine kinases, leading to cytoskeletal remodeling involved in structural changes that may diminish synaptic strength of excitatory cells.

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