Grooming as a de-arousal behavior underlying novelty habituation: An effect potentiated by environmental enrichment Mijail Rojas-Carvajal A, Juan C. Brenes A,B, Jaime Fornaguera A,C & Andrea Mora-Gallegos A A Neuroscience Research Center; B Institute for Psychological Research; C Biochemistry Department, School of Medicine The open field test (OFT) has been extensively used for assessing exploratory behavior, memory, and anxiety-related traits in rodents. Previous evidence suggests that early environmental enrichment (EE) reduces psychomotor reactivity to novelty in the OFT, as compared with animals reared in standard housing (SH). A detailed analysis of OFT activity has shown that EE rats progressively increased self-grooming as locomotion reduced. A preliminary observation revealed that, among the different types of grooming, EE particularly increases the so-called sequential or type 3 grooming (i.e., snout-head-torso liking). Since grooming behavior has also been associated with pain, stress, and thermoregulation, the question of whether increases in grooming behavior can be taken as an indicator of novelty habituation potentiated by EE has not been fully elucidated. In the first experiment, we evaluate the effects of housing conditions on novelty habituation within a 10-min OFT and between two independent OFT conducted 30 days apart. In a second experiment, we aimed to test whether grooming behavior, specifically the type 3, is related to novelty habituation independently from EE. Therefore, non-enriched rats were exposed to a 15-min OFT during four consecutive days, expecting that repeated exposition to the same environment increases familiarity and, in consequence, grooming behavior. In a final experiment, SH rats were i.p. treated with the glutamate receptor antagonist MK-801, to test whether blocking the acquisition of OFT memory prevents grooming behavior to appear in further expositions to the same environment. The putative role of grooming behavior in a non-associative learning process – namely novelty habituation in the OFT– is still under debate. We offer evidence of grooming behavior as an indicator of the transition from defensive – risk-assessment behaviors – to a non-defensive repertoire. Introduction Methodology and Hypotheses Experiment 1: Environmental enrichment increases grooming while decreases rearing and locomotion Experiment 2: Repeated OFT experiences increase grooming while decrease rearing and locomotion Experiment 3: MK-801 and DZ show almost opposite effects on grooming Conclusions References and contact Locomotion Rearing Grooming Rearing Locomotion Grooming Cephalic Grooming Caudal Grooming Unitary Caudal Grooming Sequential Experiment 1: EE potentiated novelty habituation, e.g. by reducing exploration and risk-assessment behaviors, while increasing grooming. It is very likely that learning capabilities, which are known to be enhanced in EE animals (Mora-Gallegos et al., 2015; Nilsson et al., 1999), potentiate the transition from defensive (i.e. locomotion and rearing) to non-defensive behaviors like grooming. An alternative, non-exclusive explanation is based on the fact that EE normally reduces stress reactivity. Thus, EE may have buffered the anxiogenic salience of the OFT enabling the animals to decrease excessive arousal at the first OF exposure, and to prevent it when further confronted to the same context (i.e. on test 2). Experiment 2: Facilitating learning and stress de-arousal by exposing the animals repeatedly to longer OFT progressively decreased locomotion, and especially rearing, while increased grooming type 3. The occurrence of this behavioral dynamic may indicative that OFT habituation is taking place. This evidence, therefore, also supports our idea that EE exerts its effects on OFT habituation by enhancing learning and stress de-arousal. Presumably, greater experience in the OFT could lead to a later stage of habituation where grooming appears. Experiment 3: NMDA and GABA A receptor antagonist and agonist, respectively, showed no significant modulation of grooming behavior. Although MK-801 increased, whereas DZ decreased locomotion during repeated drug administrations (data not shown), no carry-over effects of these drugs on exploratory activity during the last OFT re-expositions were found. Final conclusions: Current and previous evidence (Kalueff & Touhimaa, 2005; Brenes, Rodríguez & Fornaguera, 2008) suggests that the sequential, kinetic pattern of grooming behavior and its functional interpretation may provide additional information about how, and when non-associative learning and stress de-arousal have been going on. For instance, the transition from cephalic to sequential-caudal grooming may be indicative of an anxiety-like response that gradually yields to a non-defensive, de-arousal behavior. EE and repeated OFT exposures, therefore, accelerate this transition and, in consequence, the habituation process. More research is still needed to elucidate the intricate relationship among such behavioral processes and their neurobiological underpinnings. (1) OFT locomotion decreased progressively over testing points in both groups, with such an effect being greater in EE rats. Also, EE animals showed less locomotor activity than SH rats on tests 1 (B) and 2 (C). *** All p-values <.001. (A) A detailed analysis minute-by-minute of Test 1 revealed that, except on minute 1, EE displayed less locomotion than SH counterparts throughout the testing period. * All p-values <.05. (D) Interestingly, on test 2 EE animals showed almost the same trend as in test 1; however, EE rats now differed from SH rats even from minute 1. * All p-values <.05. (1) Rearing decreased over testing points in both groups, but in EE group it occurred earlier. In EE rats rearing decreased from BL to test 1 and remained the same on test 2. SH rats, in contrast, required two OFT expositions to reduce this type of exploratory activity. *** All p-values <.001. (A-B) A detailed analysis minute-by-minute in Test 1 revealed that, except on minute 1, EE rats display low and stable levels of rearing behavior over minutes. * All p-values <.05. (C-D) In contrast with locomotor activity, rearing behavior no longer differed between groups on test 2. (1) Grooming increased in EE rats on test 1 and remained significantly high on test 2 (as compared with SH), although it tended to decrease. In SH rats, grooming drastically reduced from BL to test 1, and remained almost the same on test 2. *** All p- values <.001. (A-B) A detailed analysis minute-by-minute revealed that, in EE rats, grooming behavior progressively increased on test 1, especially from minute 4 onwards. SH counterparts, on the contrary, required almost 9 minutes to show a slight increase on grooming time. * All p-values <.05. (C-D) Test 2 almost mirrored the tendency observed on test 1, but this time, EE rats showed quite irregular levels of this behavior. *** All p-values <.001. Type 1* Type 2* Type 3* Grooming classification Cephalic A-B-C-D: Sequential D: Unitary Caudal (1) *(Brenes, Rodríguez & Fornaguera, 2006) Rearing EE H 1 : EE potenciates the habituation process = ↓L; ↓R; ↑GExperiment 1: SD; N=60; PND H 2 : Increase learning oportunities potenciates habituation = ↓L; ↓R; ↑G Experiment 2: Wistar; N=20; PND 30 H 3 : Blocking the NMDA receptors reduces habituation = ↑L; ↑R; ↑G H 4 : Stimulating the GABA A receptors increases habituation = ↓L; ↓R; ↓G↑ Experiment 3: Wistar; N=40; PND 30 DZ: 1.5 mg/kg and 3.0 mg/kg MK-801: 0.1 mg/kg and 0.2 mg/kg (4) All groups also showed a progressive decrease of unitary caudal grooming on testing days 5 and 12, as compared to the cumulative levels obtained on days 1-4. (5). However, as the frequency of grooming decreased, the relative duration of each episode increased, as indicated by the time spent grooming observed on testing day 5 and 12. (1) (2) (4) (6) (7) The behavioral activity recorded during the four consecutive OFT in which drug were administered, was merged and compared with (A) the fifth consecutive OFT, and the (B) sixth one-week apart OFT. * All p-values <.05. (3) (1.A) Repeated and longer exposures to the OFT enhanced habituation, namely, animals reduced their locomotor activity across tests, especially noted when comparing OFT1 and OFT4. * p <.05. (1.B) Interestingly, activity during the first minute on OFT3 and OFT4 was rather low than in the same minute of OFT1. ** All p-values <.01. (2.A) Rearing behavior showed a robust and rapid reduction from OFT1 onwards, so that rearing frequency on all subsequent testing points was significantly lower. * All p-values <.05; *** p <.001. (2.B) Within tests, rearing also decreased progressively, with the slope decay being less pronounced over tests, probably as a consequence of a ever lower starting point (1) In all groups the frequency of cephalic grooming reduced robustly on testing days 5 and 12, as compared to the cumulative levels obtained on days 1-4. (2) This tendency was also observed for the time spent grooming. (3) Remarkably, the frequency of cephalic grooming correlated positively with rearing and locomotion. (3.A) Grooming type 3 showed a progressive increase along tests, where the time spent on this behavior was significantly greater on OFT4 as compared with OFT1. ** All p-values <.01. (3.B) The study of the grooming progression across minutes revealed that the grooming type 3 tended to increase within tests, so that on OFT4 grooming type 3 levels were not just higher than that on OFT1, but also augmented rapidly over minutes. (4) Figure 4 showed the behavioral kinetics observed in the OFT, with locomotor and rearing diminishing as grooming increases over testing days. 4 (6) The frequency of sequential caudal grooming showed a similar pattern as observed for the other subtypes. (7) However, as the frequency of grooming decreased, the duration of each episode increased progressively and to a greater extent, as observed for the unitary subtype. (5) SH vs. Brenes, J. C., Padilla, M., & Fornaguera, J. (2009). A detailed analysis of open-field habituation and behavioral and neurochemical antidepressant-like effects in postweaning enriched rats. Behavioural brain research, 197(1), Kalueff, A. V., & Tuohimaa, P. (2005). The grooming analysis algorithm discriminates between different levels of anxiety in rats: potential utility for neurobehavioural stress research. Journal of neuroscience methods, 143(2), Mora-Gallegos, A., Rojas-Carvajal, M., Salas, S., Saborío-Arce, A., Fornaguera-Trías, J., & Brenes, J. C. 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