Research reportAge increases anxiety and reactivity of the fear/anxiety circuit in Lewis rats
Highlights
► Ageing influences both the pattern of neuronal activation and the anxiety-related behavior of Lewis rats. ► The distinct parts of the fear/anxiety circuit (the amygdalar complex, hippocampus and hypothalamus) undergo diverse age-related changes in reactivity to behavioral challenges. ► The initial hypofunction of the HPA axis of Lewis rats is abolished by age. ► The amygdalar complex experiences an age-dependent decrease in reactivity to mild stressors. ► The Principal Component Analysis reveals that the age-related increase in anxiety is independent of the decrease in locomotor activity.
Introduction
It is well documented that ageing is associated not only with a decline in cognitive functions but also with emotional changes. The character of these changes is however ambiguous. Many gerontological surveys indicate an increase in anxiety and depression in the elderly [1], [2], yet the impact of risk factors like loneliness or disability as compared to ageing itself remain unknown. On the other hand, psychological research [3], [4], [5] suggests that emotion regulation and mood management improve with age and older adults experience less negative affect. Moreover, several recent fMRI studies have shown an ageing-dependent loss of amygdala reactivity and amplified activity in the prefrontal cortex, in response to negative stimuli [6], [7], [8], [9], [10]. It should be pointed out however, that the studies mentioned above were all performed with the use of rather low-stressful stimuli (emotion-laden pictures from International Affective Pictures System (IAPS) [11] and words from Affective Norms for English Words (ANEW) database [12] as well as questionnaires with emotionally charged hypothetical problems). The above results are not in line with commonly recognized increases in anxiety among the elderly and with data obtained in animal studies of ageing. Most authors that have addressed this question in rodents have reported an age-related increase in anxiety/hyperemotionality upon exposure to mild stress (e.g., a novel situation) [13], [14], [15], [16], [17], [18], [19], [20].
Little is known about the neurobiological mechanisms that underlie the different emotional responses of young and old animals subjected to stressogenic stimuli. Yet there is extensive evidence indicating that various kinds of stressors induce neuronal activation (with an immediate early gene c-fos mRNA and protein expression employed as a functional marker) in the brain structures involved in the regulation of emotions [21], [22], [23] The most important regions among these structures include the amygdalar complex [24] and the hippocampus [25]. In Lewis rats, used in the current study, behavioral arousal may also be dependent on the activity of their hypothalamo–pituitary–adrenal (HPA) axis [26]. Thus the activity of the hypothalamic regions may also be crucial for the control of the emotional behavior of these rats.
It might be supposed that the increased emotional reactivity of old rats is linked to the higher activation of these key structures in the fear/anxiety circuit. Those few studies that have dealt with the issue report a far more complicated pattern of age-related neuronal changes. While some researchers have observed a decline in the neuronal reactivity of aged rats [27], [28], others have shown that distinct parts of the fear/anxiety circuit may be affected diversely by age [29].
The aim of the present experiment is to investigate the effect of age on the behavioral and neuronal response to a wide spectrum of stressful conditions. These range from low aversive spontaneous exploration of the novel environment of the Hole Board arena, through mildly stressful Open Field with Illuminated Center and Elevated Plus Maze tests, to highly stressful Acute Restraint procedure. In a previous study on psychogenetically selected Roman High Avoidance (RHA/Verh) and Roman Low Avoidance (RLA/Verh) rats, we found this set of tests to be a useful and effective measure of emotional reactivity/anxiety level differences in behavioral and molecular responses [30]. The main advantage of this methodology is that it permits the investigation of (possible) functional heterogeneity within the structures belonging to the fear/anxiety circuit, as a function of a full range of aversiveness of novel behavioral challenge. It also allows for a complex profiling of the emotional reactivity of the animals used in the study. We applied Principal Component Analysis (PCA) on the set of behavioral measures from novel environment tests (HB, OF and EPM) to trace the possible differences in motivation factors driving the behavior of young and old Lewis rats. PCA is considered a particularly beneficial statistical tool for the interpretation of behavioral data since it allows for extraction of presumably independent factors reflecting different drives constituting behavior [31], [32], [33], [34]. In our experiment, factor analysis was applied for three main reasons: to identify the relationship between specific test indices and factors such as motor activity, anxiety and exploratory drive, to take account of the individual differences between subjects – it is well known that marked variation in behavioral impairments is seen between individuals of the same aged rat population [35], [36], and finally, to assess the applicability of behavioral tests and settings used to the investigation of both young and aged animals behavior.
Section snippets
Animals
A total of 50 males, inbred Lewis rats from the breeding colony at the Medical University of Warsaw were randomly assigned into two, equally numbered groups. The first group was behaviorally tested at the age of approx. 3 months (Young Adults, YA, n = 25, 312 ± 7 g). The second group was kept in the animal house of the Nencki Institute of Experimental Biology, PAS until the age of 20–22 months and then behaviorally tested (Old Adults, OA, n = 23, as two animals had to be excluded from the study due to
Open Field with Illuminated Center (OF)
Young Lewis rats, as compared to old individuals, explored the testing arena more extensively in all its zones, including the brightly illuminated center. The duration of illuminated center exploration was significantly higher for YA rats than for OA rats (F(1,28) = 4.34, p < 0.05, Fig. 2A). The distance moved and movement duration in the border zone, as well as in the total arena, were also higher for YA individuals (F(1,28) = 52.56, F(1,28) = 27.01, F(1,28) = 34.92, F(1,28) = 22.76, respectively, all p <
Discussion
In the current study we found that age substantially affects the behavior of Lewis rats in a novel environment, as well as the underlying neuronal activation of the key structures of the fear/anxiety circuit. The results further confirm our previously reported finding [29], that distinct parts of this circuit are differentially susceptible to age-related changes in activation. These findings also confirm that the set of tests used in the current study in conjunction with the application of
Conclusion
The main conclusion of the current study is that the ageing process influences both the pattern of neuronal activation and the behavior of Lewis rats by turning them more reactive and anxious with age. The process itself seems to affect the HPA axis and other parts of the fear/anxiety circuit to a different extent. While the initially hypoactive HPA axis starts to react to even mild novel stimuli with age, the hippocampus seems to sustain its activity in an unchanged shape. The amygdala, on the
Acknowledgements
We would like to thank Professors W. Jeffrey Wilson, Stefan Kasicki and Dr. Mark Hunt and Dr. Luca Follis for their comments on the manuscript and Jan Kaminski for his help with statistical analysis. The study was supported by Polish State Committee for Scientific Research grant no. 6PO4C0871 and statutory grant to the Laboratory of the Limbic System.
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