Therefore, we present open arm time as the sum of time spent on the open arms and time spent in the center square. ANOVAs were performed for each of the behavioral, endocrine and neural indices. Bonferroni and Tukey HSD post hoc correction was applied to all applicable statistical tests. T -tests corrected for multiple comparisons were also used to compare single behavioral parameters of interest after higher-level analyses.
Simple linear regression analyses were performed to analyze developmental influences on the relationships of testing time and CORT concentration as well as Arc expression and CORT level. Lastly, modified ANOVA interaction analyses were performed to further compare regression lines across ages. Outlying data points were excluded from analysis based on a calculation of three-standard deviations from the mean. All figures show group means. Error bars reflect one standard error of the mean. As such, data in subsequent analyses were collapsed across drug conditions.
Table 1 indicates the number of subjects per individual condition. Figure 1. A Impact of CX on time spent in an open area. B Entries into enclosed and open areas at P17—19 and P22— Hatched-line regions represent the proportion of total arm entries that were made into an open area, while solid bars indicate the proportion of total entries into closed arms.
C Dwell time in open arms separated out by open arm and center zone. Hatched-line bars represent the proportion of open arm time spent navigating open arms, while the solid portion of the bar indicates the proportion of time made up by center zone exploration.
These data suggest increased expression of innate anxiety with increasing age at the end of the third postnatal week. Effects of testing conditions on open arm dwell time were analyzed separately at P17—19 and P22— Combined, these findings support an ability of bright illumination to heighten innate anxiety in animals over 3 weeks of age, especially in the AM. Figure 2. Anxiety-like behavior in the EPM under different environmental parameters.
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Mean open arm time for each testing condition at A P17—19 and B P22— These results point to a developmental increase in baseline CORT at the end of the third postnatal week. Figure 3.
C CORT in maze-exposed rats plotted against time of testing. Like the experimental animals, these controls underwent transport, handling, and exposure to a novel room, but were not exposed to the EPM. No effect of CX treatment was found; therefore, data in subsequent CORT analyses were collapsed across drug conditions. Expression levels for the activity-dependent immediate early gene, Arc , Guzowski et al.
Due to difficulty in establishing subregional borders within the amygdala of younger animals, quantification of Arc in the amygdala encompassed the lateral, basolateral, medial and central nuclei Figures 4 , 5C. No main effects of Age or TOD were observed in the amygdala. Together, these findings suggest that constitutive Arc expression levels vary across age in the hippocampus and undergo diurnal regulation in the hippocampus and visual cortex differentially across age groups.
Figure 4. A Schematic illustration of a sagittal brain section adapted from Paxinos and Watson, denoting regions in which Arc expression was quantified.
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Regions used for cell counting analyses are shaded green with red dashed outlines. Figure 5. Neuronal Arc expression after EPM exposure. C Brightfield images 2X magnification illustrating Arc intensity in the amygdala outlined in red in young and older developing rats tested in the PM. For maze-exposed animals, Arc expression measures were normalized to the mean baseline values from the appropriate age and TOD-matched controls. These findings support the idea that, with increasing age at the end of the third postnatal week, anxiety-induced neuronal activation becomes less sensitive to TOD selectively in the amygdala.
These results show subregional sensitivities of the primary visual cortex to EPM exposure that are age- and TOD-dependent. Assessment of EPM behavior in late postnatal development revealed age-related differences in innate anxiety with greater temporal resolution than shown in previous experiments. In accord with prior studies examining the effects of age on EPM performance Imhof et al. Interestingly, at P17—19, open arm exploration occurred primarily under bright illumination in the AM, while dim illumination encouraged the greatest open arm times at P22— Similar to prior reports Blair et al.
Combined, the results suggest that illumination levels and testing TOD can produce opposing effects on anxiety behavior depending on postnatal age. In parallel, mechanisms governing stress responsiveness varied as a function of developmental stage. Corroborating prior reports that delineate the developmental time course for CORT rhythmicity to be around 3 weeks of age Takahashi et al. Arc is an activity-dependent immediate early gene that is rapidly expressed in neurons in response to processes such as spatial learning and memory and fear conditioning Guzowski et al.
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It is important to note that, while moderate behavioral stress increases Arc expression in the basolateral amygdala of adult male rats, more severe behavioral stress, well beyond the level experienced during EPM exposure, was found to be required to negatively impact Arc expression Ons et al. Comparisons were not made across structures due to variation in the time course for Arc expression Guzowski et al.
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Overall, Arc expression levels in area CA1 of the hippocampus and layer 4 of V1 were not different between age groups. In the amygdala, more Arc -positive neurons were observed in the PM vs. Thus, age effects on levels of Arc expression in the amygdala tended to be inversely related to developmental differences in open arm time while Arc expression in the visual cortex more directly agreed with the effects of age on open arm time. Overall, differences in neuronal activation patterns across brain structures in response to EPM exposure at different testing ages further implicate varied perceptual and emotional influences over EPM performance.
Future studies involving blockade of CORT receptors might clarify if the correspondence between plasma CORT levels and amygdala or cortical Arc expression levels was causal or correlational. We found no direct correlation between CORT level and open arm time in juvenile rats. Rodgers et al. The lack of an explicit relationship between open arm time on the EPM and either CORT or amygdala Arc expression in our data corroborates reports by Rodgers and colleagues that biological responses to EPM exposure are not necessarily directly correlated with time spent on open arms of the maze, and extends this rationale to juvenile animals.
Differential TOD effects on anxiety-like behavior at P17—19 and P22—24 may be explained by the late postnatal development of circadian rhythmicity. While rhythmic activity-rest cycles across the day are present as early as P10 Reppert et al. In developing rats, before the circadian shift occurs, locomotor activity is highest during the first quartile of the day, compared to heightened locomotion in the third quartile in older animals Smith and Morrell, Our findings agree in that young animals explore the EPM most during the AM, and only P22—24 rats exhibit diurnal differences in stress reactivity.
These findings suggest that, to some extent, proclivity for locomotion may offset anxiety level to increase open arm exploration. One reason for differential influences of illumination across ages might be attributed to disparities in visual acuity. Many adult-like properties of visual capacity are present by P23, though maturity of all tested visual abilities was not apparent until P45 Fagiolini et al.
Indeed, at P18, rats spend ten times longer than adults exploring the brightly lit portion of a two-chambered box Smith and Morrell, However, this difference disappears with maturation such that, by 30 days of age, performance was similar to adults Slawecki, Thus, at P17—19, rats may navigate open areas of the EPM more under bright illumination because these conditions permit enhanced visual perception.
As sensory abilities improve, performance more closely resembles that of adults and dim illumination encourages the greatest exploration of open maze areas. Since the way in which animals explore their environment varies as a function of maturity level File, ; Weinert, , it is not surprising that the first month of life has been characterized by the greatest variability in anxiety-like behavior in rodents Andrade et al. Age-related differences in biological and behavioral variables during early development are manifested through variations in exploratory mechanisms; particularly, an increased drive to explore novel places as well as an under-developed risk assessment capacity in young rats Laviola et al.
Exposure to novelty prior to EPM testing has been shown to be anxiolytic Darwish et al. In this regard, the profound increase in anxiolytic behavior observed at P17—19 under bright illumination may reflect increased novelty-seeking behavior elicited by the bright light that blunts effects of anxiety. Claims have been made that inclusion of the center square in the scoring of EPM open arm time is inappropriate because it does not directly gauge anxiety for reviews, see Hogg, ; Rodgers and Dalvi, ; Carobrez and Bertoglio, The current results demonstrate that inclusion of time in the center square is particularly useful when assessing anxiety behavior in developing rodents.
In fact, the observed age differences in open arm time were carried entirely by the time in the center square. These findings align with conclusions made by Smith and Morrell that late preweanling rats display differential sensitivities to environmental stimuli than adults, while at the same time expressing reduced fear of open spaces, even within the home cage Smith and Morrell, In general, the findings support the idea that behavior of juvenile rats on the EPM results from overlapping developmental trajectories for multiple neural systems involved in sensorimotor ability, anxiety, and risk-assessment.
In summary, the current findings accentuate differences in the processing of and response to anxiety-provoking stimuli according to postnatal age, suggesting that biological and behavioral mechanisms supporting anxiety genesis are underdeveloped prior to 3 weeks postnatal. We show that environmental parameters such as illumination level and TOD differentially influence anxiety responses on the EPM, likely through engagement of diverse regulatory mechanisms across developmental stages.
Overall, behavior of juvenile rats under 3 weeks of age seems to be driven more by novelty exploration, possibly due to underdeveloped visual and risk assessment capacities. Interestingly, less than a week later, behavior more closely resembles adults and is associated with parallel changes in endocrine and neural activation patterns across testing conditions. The findings illustrate that continued growth and plasticity of the juvenile brain in the late postnatal period imposes difficulties in providing neural explanations for behavioral modifications at this developmental stage, and may have implications for better understanding childhood anxiety disorders.
Theodore Dumas and Sarah Albani designed the study. Theodore Dumas and Sarah Albani wrote and edited the manuscript. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. This research was supported by the Thomas F. We thank Mr. Robert Gardner and Ms. Gretchen Knaack for their technical input. Albrechet-Souza, L. Increases in plasma corticosterone and stretched-attend postures in rats naive and previously exposed to the elevated plus-maze are sensitive to the anxiolytic-like effects of midazolam.
Andrade, M. Andreatini, R. The effect of corticosterone in rats submitted to the elevated plus-maze and to to pentylenetetrazol-induced convulsions. Psychiatry 18, — Arai, A. Pharmacology of ampakine modulators: from AMPA receptors to synapses and behavior. Drug Targets 8, — Blair, M. Developmental changes in structural and functional properties of hippocampal AMPARs parallels the emergence of deliberative spatial navigation in juvenile rats. Butler, T.
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The impact of social isolation on HPA axis function, anxiety-like behaviors, and ethanol drinking. Animal movements and regional dwell times were extracted electronically from each behavior video TopScan software, CleverSys, Reston, VA. Time spent on each arm of the maze and in the center square was measured and arm entries were counted. Analysis of time spent in the open arms alone yielded low numbers and included many zero values, impeding data analysis. Therefore, we present open arm time as the sum of time spent on the open arms and time spent in the center square.
ANOVAs were performed for each of the behavioral, endocrine and neural indices.