Enhanced negative emotional processing limits behavioral flexibility in a mouse model of autism spectrum disorder

Impaired behavioral flexibility might underlie some of the symptoms associated with autism spectrum disorder (ASD). We investigated whether and how behavioral flexibility is impaired in a mouse model of ASD by testing Shank2-knockout (Shank2-KO) mice in reversal learning. Shank2-KO mice were trained in probabilistic classical conditioning with two odor cues paired with water and air puff. Upon the reversal of cue-outcome contingency, Shank2-KO mice were significantly slower than wild-type mice in reversing their anticipatory licking responses. Shank2-KO mice also showed stronger anticipatory eye closure responses than wild-type mice to the air puff, raising a possibility that the impairment might be because of enhanced negative emotional processing. Indeed, Shank2-KO mice showed intact reversal learning when the strong air puff was replaced with a mild air puff. Shank2-KO mice also showed intact reversal learning between two odor cues predicting rewards with different probabilities. These results indicate that enhanced negative emotional processing suppresses reversal learning despite of intact capability to learn cue-outcome contingency changes in Shank2-KO mice in our behavioral settings. Our findings suggest that behavioral flexibility may be seriously limited by abnormal emotional processing in ASD.


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Autism spectrum disorders (ASD) are developmental disorders that are associated with 35 a diverse array of symptoms including impaired social interaction and communication as 36 well as repetitive and restrictive patterns of behavior (American Psychiatric Association, 37 2013). Many genes implicated in ASD are expressed broadly in the cerebral cortex and, 38 therefore, their mutations can potentially lead to aberrant circuit development in 39 widespread cortical areas (Hashem et al., 2020;Mundy, 2003;Yan & Rein, 2021). Given 40 that the cerebral cortex plays a key role in adaptive control of behavior, mutations in genes 41 playing important roles in cortical development may well lead to compromised behavioral 42 flexibility in adults. In this respect, ASD patients often show impairments in the tasks 43 requiring cognitive flexibility such as the Wisconsin card sorting test (Hill, 2004;Leung & 44 Zakzanis, 2014), and impaired cognitive flexibility has been proposed to underlie 45 repetitive and restrictive patterns of behavior, a core symptom of ASD (Geurts, Corbett, 46 & Solomon, 2009). ASD patients also show impairments in reversal learning with their 47 have been used widely to investigate neurobiological mechanisms of ASD and, as a result, 73 substantial amounts of behavioral and neural data have been accumulated. However, the 74 relationship between Shank2 mutations and behavioral flexibility is largely unknown. In 75 the present study, we investigated whether and how behavioral flexibility is compromised 76 in Shank2 homozygous knock-out (Shank2-KO) mice. For this, we subjected Shank2-KO 77 mice to reversal learning using a probabilistic classical conditioning paradigm. The results 78 indicate that Shank2-KO mice have intact capability to update changes in cue-outcome 79 contingency, but display abnormally heightened negative emotional responses that limit 80 behavioral flexibility under certain circumstances.

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The mice were then subjected to reversal learning. We reversed cue-outcome 119 contingency so that the previous reward-predicting cue becomes the punishment-120 predicting cue after reversal (CSRw Pn ) and vice versa (CSPn Rw ). All mice were trained 121 until they reach the reversal criterion over 1~5 sessions (400 trials per daily session). The 122 number of trials to reach the reversal criterion differed significantly between WT and 123 Shank2-KO mice (250.0±39.7 and 570.7±146.6 trials, respectively; t-test, t (18) = 2.112, p 124 = 0.049; Figure 2d,e). The mice sometimes consumed water in the subsequent trial 125 rather than during the inter-trial interval following a rewarded trial. To rule out the influence 126 of such invalid anticipatory licks, we determined the number of trials to reversal criterion 127 after deleting the trials during which the mice consumed water delivered in the previous 128 trial (226 out of 8213 trials; 2.75%). This analysis also yielded a significant difference in 129 the number of trials to reach the reversal criterion between WT and Shank2-KO mice 130 (250.1±39.7 and 571.2±146.7 trials, respectively; t (18) = 2.112, p = 0.049). These results 131 indicate slower reversal learning in Shank2-KO than WT mice.   Kazdoba et al., 2015;Vasa et al., 2014). We therefore examined whether WT and 172 Shank2-KO mice show differential eye closure responses to the air puff using separate 316 The mice were then tested in reversal learning in the same manner as in Experiments 1 317 and 3 except that two appetitive outcomes were used (Figure 5d). The number of trials 318 to reach the reversal threshold did not differ significantly between the two animal groups  because of different experimental procedures rather than differences in ASD subjects.

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We tested head-fixed mice in a classical conditioning paradigm, whereas ASD patients 388 and BTBR and C58 mice were freely moving and tested in an instrumental conditioning the cognitive inflexibility hypothesis for ASD (Geurts et al., 2009;Van Eylen et al., 2011).

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Further studies are needed to determine the extent to which ASD patients and animal and fixed by screws and dental cement. The mice were allowed to recover > 1 week 448 before behavioral training began.

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Classical conditioning 450 All mice were trained in a probabilistic classical conditioning task under head fixation 451 (Figure 1a) as previously described (Jeong et al., 2020). The animal's head was fixed to 452 a custom-built metal holder. A water port (a blunt 17-gauge needle) was placed slightly 453 below the animal's nose, an air puff port (a blunt 18-gauge needle) was placed 3~5 mm 454 away from the animal's left eye, and an odor port (silicon tube; diameter, 8 mm) was 455 15 placed slightly above the animal's nose. Four different odors (citrus, isononyl acetate, L-456 carvone, and 1-butanol) were dissolved in mineral oil (1:1000, v:v) and delivered to the 457 animal using an air circulation system. Two odors were selected randomly from the four 458 for each experiment and for each animal. The animal's licking behavior was detected by 459 an infrared light sensor placed adjacent to the water port.

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Behavioral phases consisted of habituation, acquisition, and reversal. For habituation, a 461 small amount of water (6 µl) was provided from the water port initially in every 5 s without 462 an odor cue (~50 trials, day 1). Then the same amount of water was provided 1 s after 463 the delivery of an odor cue (1 s) that was different from those used in the acquisition 464 phase, and a 2.5~4.5 s inter-trial interval (uniform random distribution) was imposed. The 465 habituation phase lasted 1~3 sessions (~400 trials per session).

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In the acquisition phase, an odor cue randomly chosen from two different odors was 467 delivered for 1 s and, after a delay of 1 s, an associated outcome was delivered with a 468 given probability (Figure 1b). An inter-trial interval (2.5~4.5 s, uniform random distribution) 469 was then imposed before the next trial began. The mice were trained for three daily

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In the reversal phase, the cue-outcome contingency of the acquisition phase was 480 reversed. In Experiments 1 and 2, a reward-predicting cue before reversal was paired 481 with a punishment after reversal (CSRw Pn ) and a punish-predicting cue before reversal 482 was paired with a reward after reversal (CSPn Rw   Chen, Q., Deister, C. A., Gao, X., Guo, B., Lynn-Jones, T., Chen, N., Wells, M. F., Liu, R.,