Evolution of GluN2A/B cytoplasmic domains diversified vertebrate synaptic plasticity and behavior

Two genome duplications early in the vertebrate lineage expanded gene families, including GluN2 subunits of the NMDA receptor. Diversification between the four mammalian GluN2 proteins occurred primarily at their intracellular C−terminal domains (CTDs). To identify shared ancestral functions and diversified subunit−specific functions, we exchanged the exons encoding the GluN2A (also known as Grin2a) and GluN2B (also known as Grin2b) CTDs in two knock−in mice and analyzed the mice's biochemistry, synaptic physiology, and multiple learned and innate behaviors. The eight behaviors were genetically separated into four groups, including one group comprising three types of learning linked to conserved GluN2A/B regions. In contrast, the remaining five behaviors exhibited subunit−specific regulation. GluN2A/B CTD diversification conferred differential binding to cytoplasmic MAGUK proteins and differential forms of long−term potentiation. These data indicate that vertebrate behavior and synaptic signaling acquired increased complexity from the duplication and diversification of ancestral GluN2 gene

Behavioral characterization of <i>Del-Dup</i> cohorts.

<p><b>(A)</b> Circadian activity test. Graphs plot the spontaneous ambulatory activity (count) and the vertical activity/rears (count) during dark and light phases. Del/+ mice showed reduced ambulatory activity during the dark phase (F_(3,46) = 5.791, <i>P</i> = 0.002; <i>Del/+</i> vs wt: <i>P</i> = 0.002) and the light phase (F_(3,46) = 4.260, <i>P</i> = 0.010; <i>Del/+</i> vs wt: <i>P</i> = 0.006) as well as reduced rearing behavior during the light phase (H_{(3, 46)} = 12.861, <i>P</i> = 0.005; <i>Del/+</i> vs wt: <i>P</i> = 0.002) <b>(B)</b> Open field test. Distance travelled (m), and vertical activity/rears (count) over 30 min of testing. <b>(C)</b> Novel object recognition test. Discrimination index was calculated as the ratio of time spent exploring the novel object vs the total time for object sniffing in the choice trial after a 3 h retention delay. The dashed line denotes a chance level of 50% (F_(3,43) = 3.081, <i>P</i> = 0.037; <i>Del/+</i> vs wt: <i>P</i> = 0.040). <b>(D)</b> Fear conditioning test. Plots represent the percentage of time spent freezing during test sessions. The 6-min context session was run 24 h after conditioning. The 8-min cue session was performed 5 h after the context session. A sequence of 2-min without cue and 2 min with light/auditory conditioning stimulus was repeated twice. <b>(E)</b> Cued fear extinction. Independent group of <i>Del /+</i> and <i>Dup/+</i> mutant mice with their respective wt control littermates were evaluated for cued fear extinction. Immobility was increased in <i>Dup/+</i> compared to control while it was decreased in the <i>Del/+</i> carriers during the two test periods for extinction (Ext1 (24 h) and Ext2 (48 h)). After one week the cued fear retrieval was observed in the <i>Dup/+</i> mice compared to their wt littermates whereas it was absent in the <i>Del/+</i> compared to their control. <b>(F-G)</b> Three-chamber sociability test. <b>(F)</b> Exploration time (s) of the first congener in the social interest session (session 1) and total exploration time of familiar and novel congeners in the social discrimination session (session 2). No delay was used between the two sessions. <b>(G)</b> Discrimination index was calculated as the ratio of time spent exploring the novel congener vs the familiar one. Data are represented as the mean ± s.e.m. Cohort used included 18 <i>Del/+</i>, 24 wt, 11 <i>Del/Dup</i>, and 11 <i>Dup/+</i> animals. <i>Post hoc</i> Tukey's and Mann Whitney U tests were performed following significant results in one-way ANOVA or Kruskal Wallis analysis, respectively. *<i>P</i> < 0.05 vs wt, **<i>P</i> < 0.01 vs wt, ***<i>P</i> < 0.001 vs wt, ^# <i>P</i> < 0.05 vs all other groups.</p

General behavioral characterization of <i>Kansl1</i>^<i>+/-</i> cohorts.

<p><b>(A)</b> Body weight (g) of adult animals at 15, 17 and 19 weeks of age with Body length (distance from snout to tail basis) and Body fat percentage measured by qNMR of 20-week-old animals. Compared to wt littermates, <i>Kansl1</i>^+/- animals show body weight, size and adiposity deficits. <b>(B)</b> Circadian activity test. Graphs plot the spontaneous ambulatory activity (count) and the vertical activity/rears (count) during dark and light phases. <b>(C)</b> Open field test. Distance travelled (m), and vertical activity/rears (count) over 30 min of testing. <b>(D)</b> Repetitive behaviors. Graph plots occurences of rearing, jumping, climbing and digging behaviors (count) during 10 min of observation in a novel home cage. <i>Kansl1</i>^<i>+/-</i> animals show increase of rearing and decrease of digging levels reflecting an alteration of exploration activity. (<b>E</b>) Results are expressed as the time (s) that mice remained on an accelerating rod before falling during the training phase over 3 consecutive days of the rotarod test. (<b>F</b>) Corresponding rotational velocity (rpm) at the time of falling during the challenge phase of the rotarod test. The graph plots the time (s) that mice stayed on the rod when tested at constant speeds between 4 and 40 rpm. (<b>D</b>) Four-paw grip test. The conclusion of the rotarod and grip tests is that <i>Kansl1</i>^<i>+/-</i> animals show locomotor coordination improvements without alterations of muscular strenght. Graphs depict mean + s.e.m.. (<b>B-D</b>) Student’s t-test, *<i>P</i> < 0.05, **<i>P</i> < 0.01. (<b>A, E-G</b>) Repeated Measures ANOVA "genotype" analysis, Tukey's test, *<i>P</i> < 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.001.</p

Learning and memory phenotypes in the <i>Kansl1</i>^<i>+/-</i>.

<p><b>(A)</b> Novel object recognition test. Discrimination index was calculated as the ratio of time spent exploring the novel object vs the familiar object in the choice trial after a 3 h retention delay. <b>(B)</b> Fear conditioning test. Plots represent the fraction of time spent freezing during test sessions. As above the 6-min context session was run 24 h after conditioning and the 8-min cue session was performed 5h after the context. A sequence of 2-min with no cue and 2 min with light/auditory conditioning stimulus was repeated two times. <b>(C-D)</b> Three-chamber sociability test. <b>(C)</b> Exploration time (s) of the first congener in the social interest session (session 1) and total exploration time of familiar and novel congeners in the social discrimination session (session 2). No delay was used between the two sessions. <b>(D)</b> Discrimination index was calculated as the ratio of time spent exploring the novel congener vs the familiar one. Data are represented as the mean ± s.e.m.. Cohort used included 8 <i>Kansl1</i>^<i>+/-</i> and 10 wt animals. Tukey's and Mann Whitney U tests following a significant one-way ANOVA and Kruskal Wallis analysis, respectively. *<i>P</i> < 0.05 vs wt, **<i>P</i> < 0.01 vs wt, ***<i>P</i> < 0.001 vs wt, ^# <i>P</i> < 0.05 vs all other groups.</p