Mice Lacking Ras-GRF1 Show Contextual Fear Conditioning but not Spatial Memory Impairments: Convergent Evidence from Two Independently Generated Mouse Mutant Lines

Abstract

Ras-GRF1 is a neuronal specific guanine exchange factor that, once activated by both ionotropic and metabotropic neurotransmitter receptors, can stimulate Ras proteins, leading to long-term phosphorylation of downstream signaling. The two available reports on the behavior of two independently generated Ras-GRF1 deficient mouse lines provide contrasting evidence on the role of Ras-GRF1 in spatial memory and contextual fear conditioning. These discrepancies may be due to the distinct alterations introduced in the mouse genome by gene targeting in the two lines that could differentially affect expression of nearby genes located in the imprinted region containing the Ras-grf1 locus. In order to determine the real contribution of Ras-GRF1 to spatial memory we compared in Morris Water Maze learning Brambilla's mice with a third mouse line (GENA53) in which a non-sense mutation was introduced in the Ras-GRF1 coding region without additional changes in the genome and we found that memory in this task is normal. Also, we measured both contextual and cued fear conditioning, which were previously reported to be affected in Brambilla's mice, and we confirmed that contextual learning but not cued conditioning is impaired in both mouse lines. In addition, we also tested both lines for the first time in conditioned place aversion in the Intellicage, an ecological and remotely controlled behavioral test, and we observed normal learning. Finally, based on previous reports of other mutant lines suggesting that Ras-GRF1 may control body weight, we also measured this non-cognitive phenotype and we confirmed that both Ras-GRF1 deficient mutants are smaller than their control littermates. In conclusion, we demonstrate that Ras-GRF1 has no unique role in spatial memory while its function in contextual fear conditioning is likely to be due not only to its involvement in amygdala functions but possibly to some distinct hippocampal connections specific to contextual learning.

Keywords: Ras-ERK; Ras-GRF1; body weight reduction; fear conditioning; intellicage; spatial memory.

Figure 1

Spatial learning in the Morris water maze. Escape latencies (mean ± SEM) during the 4 days of training are illustrated (results are shown by couples of trials). Ras-GRF1 WT (n = 12); Ras-GRF1 KO (n = 12); GENA53 WT (n = 12); GENA53 (n = 12).

Figure 2

Spatial memory in the Morris water maze. Percentages (mean ± SEM) of time spent in target quadrant (first row: A,B), target zone (second row: C,D), and target platform (third row: E,F). Spatial memory consolidation (day 5 probe trial) is shown in the left column (A,C,E), while spatial memory reconsolidation (day 7 probe trial) is shown in the right column (B,D,F). Ras-GRF1 WT (n = 12); Ras-GRF1 KO (n = 12); GENA53 WT (n = 12); GENA53 (n = 12). Percentages in target area are compared against chance level. Dashed lines represent chance level for each area (quadrant: 25%; zone: 12.5%; platform: 1.5%). T, target; R, right; L, left; O, opposite. P-value symbols: black star = p < 0.001; black triangle = p < 0.01; asterisk = p < 0.05.

Figure 3

Conditioned corner avoidance in Intellicage. Percentages (mean ± SEM) of first nosepokes (i.e., visits with a nosepoke) in the trained corner during the adaptation, training, test, and re-test phases. Ras-GRF1 WT (n = 12); Ras-GRF1 KO (n = 12); GENA53 WT (n = 12); GENA53 (n = 11). Percentages of first nosepokes are compared with the basal percentages observed during the adaptation phase. P-value symbols: black star = p < 0.001; black triangle = p < 0.01; asterisk = p < 0.05.

Figure 4

Contextual and cued fear conditioning. Fear memory (means ± SEM) in the training (A), test (B), and re-test (C) phases. (A) Percentage of freezing at the beginning (first CS) and at the end (last CS) of the training. (B) Freezing level in the memory consolidation test (categorized by performance: 0 = non-learner; 1 = bad learner; 2 = good learner). (C) Decrease of freezing percentage in the memory reconsolidation test (percentage of freezing before reactivation minus percentage of freezing post-reactivation). Ras-GRF1 WT (n = 10); Ras-GRF1 KO (n = 11); GENA53 WT (n = 12); GENA53 (n = 11). Each mutant is compared with its respective wild-type by means of a post hoc comparison. P-value symbols: black star = p < 0.001; black triangle = p < 0.01; asterisk = p < 0.05.

Figure 5

Body weight. Total body weight (mean ± SEM) of adult mice from the three genetic lines Ras-GRF1, GENA53, and Ras-GRF1 OE. Ras-GRF1 WT (n = 24); Ras-GRF1 KO (n = 24); GENA53 WT (n = 24); GENA53 (n = 24); Ras-GRF1 OE WT (n = 24); Ras-GRF1 OE MUT (n = 24). Each mutant is compared with its respective wild-type by means of an independent-samples t-test. P-value symbols: black star = p < 0.001; black triangle = p < 0.01; asterisk = p < 0.05.