Effects of dim artificial light at night on locomotor activity, cardiovascular physiology, and circadian clock genes in a diurnal songbird

Abstract

Artificial light is transforming the nighttime environment and quickly becoming one of the most pervasive pollutants on earth. Across taxa, light entrains endogenous circadian clocks that function to synchronize behavioral and physiological rhythms with natural photoperiod. Artificial light at night (ALAN) disrupts these photoperiodic cues and has consequences for humans and wildlife including sleep disruption, physiological stress and increased risk of cardiovascular disease. However, the mechanisms underlying organismal responses to dim ALAN, resembling light pollution, remain elusive. Light pollution exists in the environment at lower levels (<5 lux) than tested in many laboratory studies that link ALAN to circadian rhythm disruption. Few studies have linked dim ALAN to both the upstream regulators of circadian rhythms and downstream behavioral and physiological consequences. We exposed zebra finches (Taeniopygia gutatta) to dim ALAN (1.5 lux) and measured circadian expression of five pacemaker genes in central and peripheral tissues, plasma melatonin, locomotor activity, and biomarkers of cardiovascular health. ALAN caused an increase in nighttime activity and, for males, cardiac hypertrophy. Moreover, downstream effects were detectable after just short duration exposure (10 days) and at dim levels that mimic the intensity of environmental light pollution. However, ALAN did not affect circulating melatonin nor oscillations of circadian gene expression in the central clock (brain) or liver. These findings suggest that dim ALAN can alter behavior and physiology without strong shifts in the rhythmic expression of molecular circadian pacemakers. Approaches that focus on ecologically-relevant ALAN and link complex biological pathways are necessary to understand the mechanisms underlying vertebrate responses to light pollution.

Keywords: Cardiac hypertrophy; Circadian rhythms; Light pollution; Melatonin; Zebra finch.

Figure 1:

(A-B) Locomotor activity was recorded via active perch system and standardized to a measure of active minutes per hour, per bird. Means ± 1 SE of control (n=22, black) and ALAN treatment (n=22, blue) groups are presented. Experimental period began on the evening of day 10 at 17:00 (ZT 10). (A) Daytime activity (7:00/ZT 0 – 17:00/ZT 10) declined slightly throughout experiment, and there was no difference in daytime activity between control and treatment groups. (B) There was no difference in nighttime activity (17:00/ZT 10 – 7:00/ZT 24) between control and treatment groups during acclimation (nights 1–9), but there was a significant increase in nighttime activity for the treatment group during exposure to ALAN (nights 10–20). (C) Proportion of individuals in each group (n=22) active during each hour of the night over the 10 nights of ALAN exposure. Means ± 1 SD (bold error bar) and min and max (thin error bar) of the 10 days of ALAN exposure are presented. Insert is a photo of the study organism, a male adult zebra finch (Taeniopygia gutatta). (D) Circadian rhythm of melatonin is presented as mean ± 1 SE, with fitted cosine curves overlaid. Plasma melatonin was collected from individuals at timepoints within 48 hours after 10 days of ALAN exposure. Shaded regions indicate nighttime hours (17:00/ZT 10 – 7:00/ZT 24). There was no difference in circadian oscillations of plasma melatonin between control and treatment groups.

Figure 2.

Effects of ALAN-exposure on cardiovascular physiology in female (A – C) and male (D – I) zebra finches (Taeniopygia gutatta). Males had increased cardiac hypertrophy but no change in cardiac stress-signaling pathways or fibrosis. (A & D) Heart mass normalized to tarsus length. (B & E) Cardiac fibrosis quantified using Image J Software on (C & F) paraffin embedded left ventricle sections stained with Picrosirius Red and imaged using a Keyence microscope. (G) Fibrotic gene expression of transforming growth factor beta (TGF-β) in left ventricle. (H & I) Pathological hypertrophic stress signaling cascades phosphorylated-ERK1/2 (phospho-ERK1/2) and phosphorylated-JNK (phospho-JNK) examined via immunoblot analysis and normalized to total ERK and total JNK, respectively.

Figure 3:

Expression of five circadian genes in the brain and liver of control (black, dashed) and ALAN treatment (blue, solid) individuals across six timepoints. Values are reported as the mean ± 1SE of normalized expression. Shaded area signifies nighttime hours (17:00/ZT 10 –07:00/ZT 24). Rhythmicity in oscillations were analyzed using Rhythmicity Analysis Incorporating Nonparametric (RAIN) analysis. All genes in the brain and liver showed strong oscillations (p << 0.01) except for Clk in the brain which showed weak oscillation (p = 0.09 for controls, p = 0.92 for treatment). There was no difference in circadian oscillation of any genes measured between control and treatment groups.