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| Behavior | Paradigm | Principles and procedures | Evidence for involvement of ENK signaling |
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| Fear | Contextual fear conditioning | In this paradigm, an animal learns to predict aversive events based on their environmental context. It is a form of learning and memory in which an aversive stimulus is associated to a neutral context and/or stimulus, resulting in fear responses upon presentation of the originally neutral context and/or stimulus. The animal is placed into a chamber to administer an aversive stimulus (e.g., electric footshocks). This procedure can be paired with another conditioning stimulus, a sound for example. After a delay, the animal is reexposed to the environment and/or conditioning stimulus, without the aversive one. Freezing which is characterized by the total absence of movement except those required for respiration is then measured to assess fear responses. | (i) In rats, ENK mRNA levels are increased in CEA upon contextual fear conditioning [14]
(ii) ENK knockout (KO) mice show an exaggerated immobility during auditory fear conditioning [42]
(iii) ENK neurons in CEAl overlap with PKC-δ GABAergic neurons, which are involved in fear behavior [45, 66]
(iv) In SWR/J mice (showing a reduced fear response induced by footshocks), ENK mRNA levels are increased in AMG [44]
(v) In SWR/J mice (showing a reduced fear response induced by footshocks), administration of MOPr and DOPr antagonists increase fear response [44]
(vi) In rats, ENK knockdown (KD) of CEA decreased unconditioned fear [15]. |
| Startle response | The startle reflex is considered as an innate and involuntary reaction that appears upon exposure to an unexpected or threatening stimuli. The response corresponds to a quick involuntary contraction of the animal’s skeletal muscles. The test is conducted in an automated startle chamber that allows measurement of the reflex. | (i) ENK KO mice show an exaggerated startle response [50]. |
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| Anxiety | Open-field | This task is based on a rodent’s preference for dark areas. The animal is placed in an open-field chamber, an arena with surrounding walls to prevent escape, and the exploratory behavior of the center (lit) versus periphery (dark) is assessed over time with a video-recording. | (i) ENK KO mice show a decreased exploratory behavior and avoid the central part of the open-field (OF) arena [42, 50, 51]
(ii) ENK KO mice, exposed to stress induced by footshocks, present an anxiety-like behavior [52]. |
| Elevated plus maze | This task is based on a rodent’s natural preference for dark and enclosed areas, compared to lit and uncovered areas, as well as on their natural exploratory behavior of a novel environment. The animal is placed in the maze, and its exploratory behavior is assessed over time with a video-recording. The maze has a cross shape with two opposite arms surrounded by walls (dark and enclosed area) whereas the two other arms do not present walls (lit and uncovered). | (i) ENK KO mice present anxiety-like behavior in the elevated plus maze (EPM) [50]
(ii) ENK KO mice, exposed to stress induced by footshocks, present anxiety-like behavior in EPM [52]
(iii) In rats, ENK KD in CEA increases the exploratory behavior in EPM [15]
(iv) Infusion of a DOPr agonist in CEA increases the number of entries and the time spent in open arms of the EPM [82]
(v) Administration of a DOPr antagonist diminishes the exploratory behavior in EPM [54]
(vi) Administration of a DOPr agonist increases this behavior [55–57, 59, 60]
(vii) DOPr KO mice spent less time in the open arms of EPM [53]
(viii) MOPr KO mice increase the exploratory behavior in EPM [53]
(ix) Administration of MOPr agonist increases the exploratory behavior in EPM [58]. |
| Light-dark box | This task is based on a rodent’s natural preference for dark areas, compared to lit ones. The box contains two chambers, one light and one dark. The animal is placed into the box and its exploratory behavior is assessed over time with a video-recording. | (i) ENK KO mice show a decreased exploratory behavior in the light-dark box (LDB) [42, 50]
(ii) ENK KO mice, exposed to stress induced by footshocks, present an anxiety-like behavior in LDB [52]
(iii) DOPr KO mice spent less time in the illuminated portions of the LDB [53]. |
| Social interaction test | This test allows evaluating the propensity of an individual to socialize. The rodent is placed in an open-field arena alone in the first place and then with another individual. The time spent interacting with the intruder is measured. | (i) ENK KO mice present a reduced duration of social interaction [50]. |
| Forced swim test | This test is used to evaluate the antidepressant efficacy of new compounds. A rodent is placed in a pool containing approximately 15 cm3 of water, and its mobility is measured on a video-recording. | (i) Administration of a DOPr agonist increases mobility in the forced swim test [59]. |
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| Anhedonia | Sucrose preference test | This task is used as an indicator of anhedonia, characterized by a lack of interest for a reward. Two bottles, one containing a sucrose solution (between 1% and 5%) and another plain water, are presented to the animal. Its preference for the sweetened versus plain water reveals anhedonia state. | (i) After restraint stress, rats showing increased anhedonia (assessed with the sucrose preference test) present a reduced expression of ENK mRNA in the NAc [70]. |
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