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Caffeine-related effects on cognitive performance: Roles of apoptosis in rat hippocampus following sleep deprivation
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Caffeine-related effects on cognitive performance: Roles of apoptosis in rat hippocampus following sleep deprivation Guangjing Xie a, 1, Xiaoyu Huang a, 1, Hao Li b, Ping Wang a, *, Panpan Huang a, ** a b
Basic Medical Sciences College, Hubei University of Chinese Medicine, Wuhan, 430065, China 712 Research Institute, CSIC, Wuhan, 430065, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 October 2020 Accepted 9 November 2020 Available online xxx
Caffeine is a common stimulant widely existed in food and has stimulatory effects on the central nervous system, shift-work individuals often rely on caffeine to maintain attention and keep awake. Although sleep deprivation (SD) is widely considered as an independent risk factor for cognition retardations, however, little is well understood about the synergistic role of caffeine dosage and SD for cognitive performance. This research intended to investigate the underlying molecular mechanism of varying caffeine doses on cognitive function after sleep deprivation. The results revealed that SD attenuated the cognitive dysfunction, associated with ultrastructure damage and pyramidal neuron loss in the hippocampus, decreased in the level of VIP and AVP. SD also significantly accelerated the neuropeptideassociated apoptosis in the hippocampus, which may modulate via the cAMP-PKA-CREB signal path axis and activation of the downstream apoptosis genes. Additionally, the data indicated that low-dose caffeine (LC) contributed to cognitive enhancement, and high-dose caffeine (HC) aggravated cognitive impairment by modulating hippocampal neuronal apoptosis. Our studies suggest that caffeine, particularly in high dosage, may be a potential factor to influence the neurocognitive outcome caused by sleep loss, and the appropriate amount of caffeine ingested after sleep deprivation deserves serious consideration. © 2020 Elsevier Inc. All rights reserved.
Keywords: Caffeine Sleep deprivation Cognitive function cAMP/PKA/CREB pathway Apoptosis
1. Introduction Sleep is a natural homeostatic process that is crucially important in memory stabilization and integration [1]. Sleep duration can be affected by a variety of cultural, social, psychological, and pathophysiological factors, however, acceleration of social rhythm with prolonged working hours, frequent night shifts, and extended lighting time often leads to self-imposed chronic sleep deprivation (SD). SD is particularly common with 20% of the adult reported being sleep deprived [2]. It is well established that the adequate functioning of hippocampus and neuronal plasticity facilitates formation and reinforcement for memory [3]. Emerging evidence suggested that spatial memory was closely associated with hippocampus neurogenesis, neuroinflammatory levels during SD.
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (P. Wang), [email protected] (P. Huang). 1 These authors contributed equally to this work.
Moreover, deficits in cognitive function after SD have been widely reported [4]. Several studies have shown that SD impaired memory formation [5], others argued SD resulted in associative recognition memory impairment [6] and sleep loss induced hippocampal subfield atrophy, reduced neurogenesis, and affected hippocampus-dependent memory consolidation and protein synthesis [7]. Caffeine (1,3,7-trimethylxanthine), most consumed psychostimulant in the world, is commonly found in tea, coffee, chocolate, and functional drinks [8]. Caffeine-containing beverages and products are increasingly being relied upon to sustain vigilant attention and enhance concentration in the case of sleep disturbance [9]. It also stimulates locomotor activity by affecting the central nervous system and improving alertness to the baseline in animal researches [10]. What cannot be ignored is some individuals may experience anxiety, tremor, and caffeine intoxication, characterized by restlessness, insomnia, gastrointestinal disturbances, and other symptoms at normal consumption levels or more common in high dosage. Considering people are more likely to take caffeine to stay
https://doi.org/10.1016/j.bbrc.2020.11.029 0006-291X/© 2020 Elsevier Inc. All rights reserved.
Please cite this article as: G. Xie, X. Huang, H. Li et al., Caffeine-related effects on cognitive performance: Roles of apoptosis in rat hippocampus following sleep deprivation, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.11.029
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0.9% saline solution containing 4% glutaraldehyde. Portions of selected hippocampus regions were instantly subjected to 2.5% glutaraldehyde in 0.1 M cacodylate buffer, divided into 1 mm2 piece, and then maintained in the glutaraldehyde solution for 12 h at an ambient temperature. The hippocampal tissues were dehydrated and further stained with uranyl acetate and lead citrate, and embedded in Araldite 502 resin at 60 C. All samples were imaged using a scanning transmission electron microscope (JSM, IT200, Japan).
awake after sleep deprivation, while the synergistic effect of caffeine and SD on cognitive functions is still a matter of debate and have not been elucidated in detail. Here, we investigated the effect of caffeine administration on hippocampal-dependent memory after sleep deprivation and further evaluated the apoptosis level in hippocampal neuron based on the cAMP-PKA-CREB pathway. 2. Materials and methods 2.1. Animals
2.5. Nissl staining Male Sprague-Dawley rats were provided by the Hubei Province Center for Disease Control and Prevention (Wuhan, China). The animals, housed under 20e22 C, 50e70% humidity, and a 14 h light/10 h dark rhythm (lighting from 6 a.m. to 8 p.m.), were free of access to feed and purified water ad libitum. The animals acclimated for one week before the experimental procedures began. After acclimatization for 7 days, all rodents were randomly distributed into control group, SD group, low-dose caffeine with SD (LC) group, and high-dose caffeine with SD (HC) group, n ¼ 10 per group. The experimental procedures were approved by the Experimental Animal Ethical Review Committee of Hubei University of Chinese Medicine. All experimental protocols were maintained and treated following the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23).
The brains were immediately removed and immobilized in 0.1 M phosphate-buffered saline (PBS), and 4% polyformaldehyde. After rinsing with PBS, the coronal sections of (4 mm thick) were incubated in 0.5%cresyl viol staining for 10 min. Then all slices were washed and dehydrated. Finally, the slides were cleared with xylene and cover slipped. All sections were analyzed under a light microscope to examine neural changes in the cornus ammonis 1 (CA1) and detate gyrus (DG) regions. The Nissl bodies count in the pyramidal cell layers using Image-Pro Plus 6.0. 2.6. Tunel staining The brain was gathered and then fixed in 10% formalin neutral solution, trimmed, and embedded in paraffin. Sections (5 mm thick) for terminal deoxynucleotide transferase (TdT) mediated nick end labelling (TUNEL) were fixed in a 4% solution of paraformalde-hyde. TUNEL analysis was performed with an ApopTag peroxidase in situ apoptosis detection kit (Chemicon International, Inc., U.S.A.) according to the manufacturer’s instructions, as described by Li et al. [13].
2.2. Establishment of SD model and pharmaceutical intervention In this experiment, a modified multiplatform water environment method was employed to induce SD, as previously described [11]. All rats except the controls were subjected to chronic sleep deprivation consecutively for 14 days (deprivation from 0:00 to 20:00 per day, 4 h for rest). After 2 days of SD habituation, the LC and HC groups were administered a caffeine solution at the respective doses of 36 mg/kg/day and 144 mg/kg/day daily by oral gavage for 12 days [12]. And the control and SD groups received purified water at the same time. Caffeine was obtained from SigmaAldrich Inc. (St. Louis, MO, USA). Then, the animals were submitted to behavioral tasks.
2.7. Immunofluorescence (IF) assay The whole hippocampus tissue was harvested and cut vertically and coronally. The sections were cut into 10 mm sections with a microtome and incubated for 24 h with primary antibodies. Subsequently, the tissues were incubated for 4 h with biotinylated secondary antibody after washing in PBS. Next, the sections were incubated with DAPI for 10 min. The signals were observed using a fluorescence microscope (Leica) and analyzed with ImageJ (NIH, USA).
2.3. Behavioral tests We tested the memory acquisition with the Morris water maze (MWM). In brief, the MWM task mainly consisted of a round stainless-steel tank (diameter 1.80 m, depth 0.60 m) and a video tracking system. The experimental strategy was: a navigation test (60 s for each time) for 5 consecutive days, a spatial probe test (within 120 s) after removing the round platform. In the navigation test, rats were allowed up to 60 s to locate the platform. Once rats could not finish within 60 s, they were directed to the platform and recorded for a latency of 60 s in that trial. Once the rat reached the platform, they were given a 10 s break on the platform. A spatial probe test was performed for 120 s with the hidden platform removed. After the MWM test, a locomotor activity monitoring system was used to investigate spontaneous activity. The rats were placed into the square open field (80 cm 80 cm 80 cm) individually and monitored for 3 min while freely moving. During the experiment, the surrounding environment was kept quiet to avoid interference from external factors.
2.8. cAMP assay The cAMP expression levels were tested using the rat cAMP immunoassay kit (R&D Systems, USA). In brief, all tissues rinsed with PBS were homogenized in 0.1 M hydrochloric acid (HCl). They were homogenized at 4 C and were centrifuged at 1000g for 15 min, the supernatant was then loaded into plates with anticAMP polyclonal antibodies for incubation. Subsequently, the supernatant was processed with an HRP-conjugated anti-cAMP antibody and optical density was read at 450 nm with a microplate reader. 2.9. Western blot All samples were homogenized in ice-cold RIPA buffer, normalized amounts of protein determined using BCA protein assay kits was electrophoresed in a 10% sodium dodecyl sulfate polyacrylamide gel and transferred onto PVDF membrane (Immobilon, Germany). After incubation with primary antibodies overnight, the blots were rinsed and incubated with the second primary antibody of goat anti-rabbit IgG and then developed using the
2.4. Transmission electron microscopy (TEM) All rats were given anesthesia by intraperitoneal injection of 3% pentobarbital sodium (30 mg/kg) and intracardially perfused with a 2
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increased the frequency of platform crossing and time in target quadrant. Taken together, the results indicate that SD impairs the spatial learning process, LC is beneficial for cognitive enhancement, while HC intensifies the decline in spatial memory performance.
Image Studio Software (LI-COR Biosciences, USA). The blots were quantitatively analyzed using ImageJ and normalized to b-actin. 2.10. Statistical analysis Numerical data were expressed as mean ± SEM and were processed with GraphPad Prism 8 (GraphPad Software, USA). Two-way ANOVA were used for the navigation test and locomotor activities followed by Tukey’s post hoc analysis. Comparisons among groups were measured by one-way ANOVA followed by Bonferroni’s post hoc. The differences were considered statistically significant with P values < 0.05.
3.2. Effect of caffeine on locomotor activities in SD rats The paired-samples t-test showed that there were differences between marginal and central areas on the total time, total distance, and static time. SD increased the average activity time and distance in the marginal area, and decreased the activity time in the central, the static time in both areas. As displayed in Fig. 1G and I, LC significantly ameliorated anxiety-like behaviors in contrast to SD rats, with less time in the marginal, more time in the central, and more static time in both areas. However, HC decreased the activity time in the central region, with a corresponding increased time in the marginal. Overall, it reveals that the differences in caffeine dosage may result in inconsistent performance on behavior disorders after sleep deprivation.
3. Results 3.1. Effect of caffeine on spatial learning abilities in SD rats During the navigation test, SD rats required more time (Fig. 1A), more distance (Fig. 1B) to locate the platform relative to the control from the third to the fifth day. High-dose caffeine, however, increased the latency and distance from the fourth to the fifth day, indicating that HC gradually aggravated spatial memory impairment. And LC significantly decreased the swimming time and distance for the comparation of SD rats, hinting that LC either directly improved spatial memory or could have antagonized the damage caused by SD. The spatial probe test showed the rats in HC group spent less time in the target quadrant (Fig. 1C) and passed through the platform less frequently (Fig. 1E). Surprisingly, LC significantly
3.3. Effect of caffeine on the mitochondrial function in hippocampus We then assessed the hippocampus morphological changes by TEM. For the control group, little appreciable damage of the hippocampus was detected. TEM demonstrated injury of the hippocampus tissue in the HC group, characterized by condensed and marginal chromatin, irregular nuclear membrane, disordered mitochondria with swelling, vacuolization and rupture, and partial Golgi’s sac with vacuolization. Moreover, the hippocampus
Fig. 1. Behavioral performance among groups with caffeine intake. The latency(A) and distance (B) over 5 consecutive days in the MWM test. Time in the target quadrant (C), first crossing platform time (D), platform area crossing times (E) in the MWM were tested. (F) Representative images of swimming routes among groups. Total time (G), total distance (H) and static time (I) were statistically significant difference between marginal and central areas in the spontaneous activity test. All bar graphs are presented as mean ± SEM (n ¼ 10/ group), *P < 0.05, **P < 0.01 vs. untreated control; ^P < 0.05, ^^P < 0.01 vs. individuals caused by SD; ##P < 0.01 vs. marginal area. 3
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suppressed the two protein expressions in hippocampus after sleep loss. These results implicated that caffeine intake affected hippocampal memory, which was associated with regulation of neuropeptide VIP and AVP level.
structures were partially alleviated also in the LC group, with slight chromatin aggregating and mitochondria swelling (Fig. 2A). 3.4. Effect of caffeine on the morphological changes of hippocampal neuron
3.6. The cAMP-PKA-CREB pathway was required for neuronal apoptosis
To investigate the morphological changes of hippocampal neurons after caffeine administration, we used a Nissl staining method to observe the differences. As shown in Fig. 2B, the histological evaluation of CA3 and DG regions displayed that the numbers of Nissl bodies were significantly declined, and a majority of pyramidal cells kept with a normal cytoarchitecture. LC statistically mitigated neuronal damage in DG region, while unchangeable in CA3. In addition, chronic exposure of HC did not alleviate hippocampal neuronal damage in both CA3 and DG regions but exhibited less counts of Nissl bodies than that in the SD group.
To investigate the involvement of cAMP-PKA-CREB pathway after caffeine exposure, we chosen Western blot and ELISA method. SD rats expressed significantly lower cAMP levels compared to the controls (Fig. 4B), and we also found a decrease in p-PKA and pCREB protein abundance following SD (Fig. 4CeD). LC markedly increased the expression level of cAMP, p-PKA and p-CREB without changing the total PKA and CREB levels (Fig. 4BeD). Conversely, HC further decreased the cAMP, p-PKA, and p-CREB protein levels, indicating that caffeine modulated the cAMP-PKA-CREB pathway protein expression and seemed partly related to a dose variability performance.
3.5. Effect of caffeine on the VIP and AVP expression in SD rats In the hippocampus CA1 region, the VIP demonstrated higher level in the controls, intermediate level in the SD and LC groups, and a weak level in the HC group (Fig. 3AeB), and the expression of AVP within groups were similar with that of the VIP, the LC group did not increased the AVP expression levels compared to the SD. Consistently, Western blot also indicated that SD repressed the relative expression of both VIP and AVP, and LC evidently upregulated the levels of VIP and AVP (Fig. 3CeD). Moreover, HC
3.7. Effect of caffeine on apoptosis in hippocampus neurons To further confirm if the caffeine intake was involved in the apoptosis process, we detected the expression of apoptosis-related proteins. As shown in TUNEL staining, LC decreased TUNEL-positive cells, while high dose increased the numbers of TUNEL-positive cells, with shrunken condensed nuclei and apoptotic bodies
Fig. 2. The effect of caffeine on the hippocampal pathological changes in SD rats. (A) The ultrastructural examination was displayed in two scales. Black arrows indicated nuclear and organelle damage. N ¼ 2/group. Scale bar ¼ 2 mm. (B) Representative photomicrographs of Nissl staining of the CA3 and DG in the hippocampus. Black arrows indicated the Nissl staining neurons. N ¼ 3/group. Scale bar ¼ 100 mm **P < 0.01 vs. untreated control; ^P < 0.05, ^^P < 0.01 vs. individuals caused by SD. 4
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Fig. 3. The effect of caffeine intake on VIP and AVP expression. (A) Representative images of VIP (Red), AVP (Red) and DAPI (Blue) positive cell in CA1 regions by immunofluorescence, (B) The average density of VIP and AVP (five different fields per slice), (C) The expression level of VIP and AVP were measured by Western blot, (D) The relative expression of VIP and AVP was analyzed by GraphPad Prism 8. **P < 0.01 vs. untreated control; ^P < 0.05, ^^P < 0.01 vs. individuals caused by SD. N ¼ 3/group, scale bar ¼ 50 mm.
antagonist. In contrast to the adenosine effect, caffeine promotes arousal and attenuates the influence of SD on vigilance and attention, and the two adenosine receptor subtypes may play a part in cerebral functions linked to sleep/wake connection. Onaolapo OJ et al. [17] have found that the effect on cognitive function was still dose-dependent when caffeine was consumed alone without sleep deprivation. Our results suggested that 14-day chronic SD impaired spatial memory acquisition and increased anxious behaviors, expressing cognitive impairment successfully induced by sleep deprivation. HC exacerbated cognitive disorder and anxious behavior performance but low-dose caffeine administered effectively improved memory retention. This result confirms that cognitive performance is associated with caffeine dosage [19]. Neuronal apoptosis is considered activated in sleep deprivation and is correlated with cognitive impairment [20]. We found sleep deficiency induced neuronal apoptosis, which acted on the mitochondria-mediated apoptosis process and the Bcl-2/Bax ratio. Emerging evidences demonstrated that the formation of Bax monomer was a decisive factor necessary to destroy the permeability of mitochondrial membranes [21]. And the caspase-3 and caspase-9 were then activated, inducing a marked reduction in the plasticity of neuronal function, neurodegenerative lesions, which in turn caused a series of cognitive dysfunctions [22]. In our research, significant neuronal apoptosis was found, shown by mitochondrial impairment, elevated caspase-9 and caspase-3 levels, and decreased in Bcl-2/Bax ratio as well, which could be aggravated by HC intake but alleviated in low dosage. In accordance with the present results, previous studies have demonstrated that of the severity of apoptosis significantly impairs learning and memory [23].
obtained in CA1 region (Fig. 4E and F). Moreover, the model rats had significantly reduced the Bcl-2/Bax ratio (Fig. 4J), along with a noticeable increase in the level of caspase-9, and cleaved caspase-3 (Fig. 4K and L). Caffeine enhanced the levels of caspase-9 and cleaved caspase-3, especially in high dosage. LC sufficiently increased the ratio of Bcl-2/Bax, as well as reducing the caspase-9 and cleaved caspase-3 expression. Additionally, we found no significant differences in Bcl-2, Bax, and Bcl-2/Bax between SD and HC groups. These results paralleled the aforementioned findings on apoptosis through the cAMP-PKA-CREB pathway and suggested that an appropriate caffeine dosage will counteract overactivated apoptosis.
4. Discussion Studies in the past 20 years have found that chronic sleep loss results in more rapid cumulative increases in cognitive deficits than subjective fatigue and mood, and the effects are proportional to the latency of sleep and the chronicity of sleep restriction [14,15]. June et al. investigated [16] the false memory formation in healthy young people and observed that sleep-deprived subjects will more frequently than well-rested individuals incorporate contradictory post hoc information during their memory retrieval. In contrast to the previous acute sleep deprivation (ASD) experiment [17], the current study applied chronic sleep-deprived (CSD) approach, which may better resemble the individual’s state of sleep deprivation caused by prolonged shift work and display more severe performance on emotional and cognitive functions of experimental animals [18]. Caffeine is a non-selective adenosine A1 and A2A receptors 5
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Fig. 4. Assessment of the cAMP-PKA-CREB pathway proteins and apoptosis levels in SD rats. (A) Representative images of western blots probed with antibodies against p-PKA, PKA, p-CREB, and CREB. (B) The cAMP levels were measured using a cAMP assay ELISA kit. Densitometric quantitations of p-PKA (C) and p-CREB (D) were shown. Photomicrographs(E) and statistics (F) of Tunel staining in the CA1 region. N ¼ 3/group. Scale bar ¼ 20 m. Black arrows indicated the Tunel positive cell. (G) Western blot images of respective apoptosis proteins in the hippocampus. Gray intensity analysis of Bcl-2 (H), Bax (I), caspase-9 (K), and cleaved-caspase3 (L). **P < 0.01 vs. untreated control; ^P < 0.05, ^^P < 0.01 vs. SD. N ¼ 3/ group.
TUNEL staining confirmed differences in apoptosis level with varying caffeine dosage. The results above demonstrate that the dose of caffeine ingested after sleep deprivation may be responsible for the extent of neuronal apoptosis through cAMP-PKA-CREB pathway.
Ultimately, we investigated if caffeine administration could regulate the expressions of the apoptosis-related proteins. VIP and AVP, neuropeptides involved in the nervous system, modulate the excitability of the hippocampus and play a modulatory role in cAMP-PKA-CREB signal transduction [24,25]. Cyclic AMP is involved in multiple pathways modulating apoptosis as a key physiological signaling molecule. Intracellular cAMP induces phosphorylation of PKA (p-PKA) and CREB (p-CREB), and resultantly activated p-CREB can further stimulate the transcriptions of numerous downstream genes, resulting in the elevation of Bcl-2 family proteins, further accelerating the development of apoptosis [26]. In the present study, LC improved cAMP-PKA-CREB signal protein expression, from both upstream and downstream genes, while the HC exerted an opposite effect on this process.
5. Conclusions In summary, the present study has opened up new avenues to unravel the specific roles of caffeine dose in the complex behavioral network and cognitive function, particularly concerning the neuronal apoptosis in the hippocampus via the cAMP/PKA/CREB pathway. Robust differences were shown among different doses of caffeine in their effect on cognitive vulnerability associated with 6
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sleep loss. The cognitive deficits believed to depend on the severity of clinical sleep disturbance may also depend on differential sensitivity to caffeine. Further research should better clarify the interaction of sleep deprivation-induced cognitive impairment with adenosine receptor subtypes. This strategy may then result in the comprehensive understanding of more rational caffeine administration and safe consumption awareness, not only of weaken alertness and vitality but of impaired cognitive performance, under the circumstances of sleep deficiency and prolonged shift work, for example. Thus, the reference was seriously essential to public harmony and physical health.
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Caffeine-related effects on cognitive performance: Roles of apoptosis in rat hippocampus following sleep deprivation Guangjing Xie a, 1, Xiaoyu Huang a, 1, Hao Li b, Ping Wang a, *, Panpan Huang a, ** a b
Basic Medical Sciences College, Hubei University of Chinese Medicine, Wuhan, 430065, China 712 Research Institute, CSIC, Wuhan, 430065, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 October 2020 Accepted 9 November 2020 Available online xxx
Caffeine is a common stimulant widely existed in food and has stimulatory effects on the central nervous system, shift-work individuals often rely on caffeine to maintain attention and keep awake. Although sleep deprivation (SD) is widely considered as an independent risk factor for cognition retardations, however, little is well understood about the synergistic role of caffeine dosage and SD for cognitive performance. This research intended to investigate the underlying molecular mechanism of varying caffeine doses on cognitive function after sleep deprivation. The results revealed that SD attenuated the cognitive dysfunction, associated with ultrastructure damage and pyramidal neuron loss in the hippocampus, decreased in the level of VIP and AVP. SD also significantly accelerated the neuropeptideassociated apoptosis in the hippocampus, which may modulate via the cAMP-PKA-CREB signal path axis and activation of the downstream apoptosis genes. Additionally, the data indicated that low-dose caffeine (LC) contributed to cognitive enhancement, and high-dose caffeine (HC) aggravated cognitive impairment by modulating hippocampal neuronal apoptosis. Our studies suggest that caffeine, particularly in high dosage, may be a potential factor to influence the neurocognitive outcome caused by sleep loss, and the appropriate amount of caffeine ingested after sleep deprivation deserves serious consideration. © 2020 Elsevier Inc. All rights reserved.
Keywords: Caffeine Sleep deprivation Cognitive function cAMP/PKA/CREB pathway Apoptosis
1. Introduction Sleep is a natural homeostatic process that is crucially important in memory stabilization and integration [1]. Sleep duration can be affected by a variety of cultural, social, psychological, and pathophysiological factors, however, acceleration of social rhythm with prolonged working hours, frequent night shifts, and extended lighting time often leads to self-imposed chronic sleep deprivation (SD). SD is particularly common with 20% of the adult reported being sleep deprived [2]. It is well established that the adequate functioning of hippocampus and neuronal plasticity facilitates formation and reinforcement for memory [3]. Emerging evidence suggested that spatial memory was closely associated with hippocampus neurogenesis, neuroinflammatory levels during SD.
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (P. Wang), [email protected] (P. Huang). 1 These authors contributed equally to this work.
Moreover, deficits in cognitive function after SD have been widely reported [4]. Several studies have shown that SD impaired memory formation [5], others argued SD resulted in associative recognition memory impairment [6] and sleep loss induced hippocampal subfield atrophy, reduced neurogenesis, and affected hippocampus-dependent memory consolidation and protein synthesis [7]. Caffeine (1,3,7-trimethylxanthine), most consumed psychostimulant in the world, is commonly found in tea, coffee, chocolate, and functional drinks [8]. Caffeine-containing beverages and products are increasingly being relied upon to sustain vigilant attention and enhance concentration in the case of sleep disturbance [9]. It also stimulates locomotor activity by affecting the central nervous system and improving alertness to the baseline in animal researches [10]. What cannot be ignored is some individuals may experience anxiety, tremor, and caffeine intoxication, characterized by restlessness, insomnia, gastrointestinal disturbances, and other symptoms at normal consumption levels or more common in high dosage. Considering people are more likely to take caffeine to stay
https://doi.org/10.1016/j.bbrc.2020.11.029 0006-291X/© 2020 Elsevier Inc. All rights reserved.
Please cite this article as: G. Xie, X. Huang, H. Li et al., Caffeine-related effects on cognitive performance: Roles of apoptosis in rat hippocampus following sleep deprivation, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.11.029
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0.9% saline solution containing 4% glutaraldehyde. Portions of selected hippocampus regions were instantly subjected to 2.5% glutaraldehyde in 0.1 M cacodylate buffer, divided into 1 mm2 piece, and then maintained in the glutaraldehyde solution for 12 h at an ambient temperature. The hippocampal tissues were dehydrated and further stained with uranyl acetate and lead citrate, and embedded in Araldite 502 resin at 60 C. All samples were imaged using a scanning transmission electron microscope (JSM, IT200, Japan).
awake after sleep deprivation, while the synergistic effect of caffeine and SD on cognitive functions is still a matter of debate and have not been elucidated in detail. Here, we investigated the effect of caffeine administration on hippocampal-dependent memory after sleep deprivation and further evaluated the apoptosis level in hippocampal neuron based on the cAMP-PKA-CREB pathway. 2. Materials and methods 2.1. Animals
2.5. Nissl staining Male Sprague-Dawley rats were provided by the Hubei Province Center for Disease Control and Prevention (Wuhan, China). The animals, housed under 20e22 C, 50e70% humidity, and a 14 h light/10 h dark rhythm (lighting from 6 a.m. to 8 p.m.), were free of access to feed and purified water ad libitum. The animals acclimated for one week before the experimental procedures began. After acclimatization for 7 days, all rodents were randomly distributed into control group, SD group, low-dose caffeine with SD (LC) group, and high-dose caffeine with SD (HC) group, n ¼ 10 per group. The experimental procedures were approved by the Experimental Animal Ethical Review Committee of Hubei University of Chinese Medicine. All experimental protocols were maintained and treated following the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23).
The brains were immediately removed and immobilized in 0.1 M phosphate-buffered saline (PBS), and 4% polyformaldehyde. After rinsing with PBS, the coronal sections of (4 mm thick) were incubated in 0.5%cresyl viol staining for 10 min. Then all slices were washed and dehydrated. Finally, the slides were cleared with xylene and cover slipped. All sections were analyzed under a light microscope to examine neural changes in the cornus ammonis 1 (CA1) and detate gyrus (DG) regions. The Nissl bodies count in the pyramidal cell layers using Image-Pro Plus 6.0. 2.6. Tunel staining The brain was gathered and then fixed in 10% formalin neutral solution, trimmed, and embedded in paraffin. Sections (5 mm thick) for terminal deoxynucleotide transferase (TdT) mediated nick end labelling (TUNEL) were fixed in a 4% solution of paraformalde-hyde. TUNEL analysis was performed with an ApopTag peroxidase in situ apoptosis detection kit (Chemicon International, Inc., U.S.A.) according to the manufacturer’s instructions, as described by Li et al. [13].
2.2. Establishment of SD model and pharmaceutical intervention In this experiment, a modified multiplatform water environment method was employed to induce SD, as previously described [11]. All rats except the controls were subjected to chronic sleep deprivation consecutively for 14 days (deprivation from 0:00 to 20:00 per day, 4 h for rest). After 2 days of SD habituation, the LC and HC groups were administered a caffeine solution at the respective doses of 36 mg/kg/day and 144 mg/kg/day daily by oral gavage for 12 days [12]. And the control and SD groups received purified water at the same time. Caffeine was obtained from SigmaAldrich Inc. (St. Louis, MO, USA). Then, the animals were submitted to behavioral tasks.
2.7. Immunofluorescence (IF) assay The whole hippocampus tissue was harvested and cut vertically and coronally. The sections were cut into 10 mm sections with a microtome and incubated for 24 h with primary antibodies. Subsequently, the tissues were incubated for 4 h with biotinylated secondary antibody after washing in PBS. Next, the sections were incubated with DAPI for 10 min. The signals were observed using a fluorescence microscope (Leica) and analyzed with ImageJ (NIH, USA).
2.3. Behavioral tests We tested the memory acquisition with the Morris water maze (MWM). In brief, the MWM task mainly consisted of a round stainless-steel tank (diameter 1.80 m, depth 0.60 m) and a video tracking system. The experimental strategy was: a navigation test (60 s for each time) for 5 consecutive days, a spatial probe test (within 120 s) after removing the round platform. In the navigation test, rats were allowed up to 60 s to locate the platform. Once rats could not finish within 60 s, they were directed to the platform and recorded for a latency of 60 s in that trial. Once the rat reached the platform, they were given a 10 s break on the platform. A spatial probe test was performed for 120 s with the hidden platform removed. After the MWM test, a locomotor activity monitoring system was used to investigate spontaneous activity. The rats were placed into the square open field (80 cm 80 cm 80 cm) individually and monitored for 3 min while freely moving. During the experiment, the surrounding environment was kept quiet to avoid interference from external factors.
2.8. cAMP assay The cAMP expression levels were tested using the rat cAMP immunoassay kit (R&D Systems, USA). In brief, all tissues rinsed with PBS were homogenized in 0.1 M hydrochloric acid (HCl). They were homogenized at 4 C and were centrifuged at 1000g for 15 min, the supernatant was then loaded into plates with anticAMP polyclonal antibodies for incubation. Subsequently, the supernatant was processed with an HRP-conjugated anti-cAMP antibody and optical density was read at 450 nm with a microplate reader. 2.9. Western blot All samples were homogenized in ice-cold RIPA buffer, normalized amounts of protein determined using BCA protein assay kits was electrophoresed in a 10% sodium dodecyl sulfate polyacrylamide gel and transferred onto PVDF membrane (Immobilon, Germany). After incubation with primary antibodies overnight, the blots were rinsed and incubated with the second primary antibody of goat anti-rabbit IgG and then developed using the
2.4. Transmission electron microscopy (TEM) All rats were given anesthesia by intraperitoneal injection of 3% pentobarbital sodium (30 mg/kg) and intracardially perfused with a 2
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increased the frequency of platform crossing and time in target quadrant. Taken together, the results indicate that SD impairs the spatial learning process, LC is beneficial for cognitive enhancement, while HC intensifies the decline in spatial memory performance.
Image Studio Software (LI-COR Biosciences, USA). The blots were quantitatively analyzed using ImageJ and normalized to b-actin. 2.10. Statistical analysis Numerical data were expressed as mean ± SEM and were processed with GraphPad Prism 8 (GraphPad Software, USA). Two-way ANOVA were used for the navigation test and locomotor activities followed by Tukey’s post hoc analysis. Comparisons among groups were measured by one-way ANOVA followed by Bonferroni’s post hoc. The differences were considered statistically significant with P values < 0.05.
3.2. Effect of caffeine on locomotor activities in SD rats The paired-samples t-test showed that there were differences between marginal and central areas on the total time, total distance, and static time. SD increased the average activity time and distance in the marginal area, and decreased the activity time in the central, the static time in both areas. As displayed in Fig. 1G and I, LC significantly ameliorated anxiety-like behaviors in contrast to SD rats, with less time in the marginal, more time in the central, and more static time in both areas. However, HC decreased the activity time in the central region, with a corresponding increased time in the marginal. Overall, it reveals that the differences in caffeine dosage may result in inconsistent performance on behavior disorders after sleep deprivation.
3. Results 3.1. Effect of caffeine on spatial learning abilities in SD rats During the navigation test, SD rats required more time (Fig. 1A), more distance (Fig. 1B) to locate the platform relative to the control from the third to the fifth day. High-dose caffeine, however, increased the latency and distance from the fourth to the fifth day, indicating that HC gradually aggravated spatial memory impairment. And LC significantly decreased the swimming time and distance for the comparation of SD rats, hinting that LC either directly improved spatial memory or could have antagonized the damage caused by SD. The spatial probe test showed the rats in HC group spent less time in the target quadrant (Fig. 1C) and passed through the platform less frequently (Fig. 1E). Surprisingly, LC significantly
3.3. Effect of caffeine on the mitochondrial function in hippocampus We then assessed the hippocampus morphological changes by TEM. For the control group, little appreciable damage of the hippocampus was detected. TEM demonstrated injury of the hippocampus tissue in the HC group, characterized by condensed and marginal chromatin, irregular nuclear membrane, disordered mitochondria with swelling, vacuolization and rupture, and partial Golgi’s sac with vacuolization. Moreover, the hippocampus
Fig. 1. Behavioral performance among groups with caffeine intake. The latency(A) and distance (B) over 5 consecutive days in the MWM test. Time in the target quadrant (C), first crossing platform time (D), platform area crossing times (E) in the MWM were tested. (F) Representative images of swimming routes among groups. Total time (G), total distance (H) and static time (I) were statistically significant difference between marginal and central areas in the spontaneous activity test. All bar graphs are presented as mean ± SEM (n ¼ 10/ group), *P < 0.05, **P < 0.01 vs. untreated control; ^P < 0.05, ^^P < 0.01 vs. individuals caused by SD; ##P < 0.01 vs. marginal area. 3
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suppressed the two protein expressions in hippocampus after sleep loss. These results implicated that caffeine intake affected hippocampal memory, which was associated with regulation of neuropeptide VIP and AVP level.
structures were partially alleviated also in the LC group, with slight chromatin aggregating and mitochondria swelling (Fig. 2A). 3.4. Effect of caffeine on the morphological changes of hippocampal neuron
3.6. The cAMP-PKA-CREB pathway was required for neuronal apoptosis
To investigate the morphological changes of hippocampal neurons after caffeine administration, we used a Nissl staining method to observe the differences. As shown in Fig. 2B, the histological evaluation of CA3 and DG regions displayed that the numbers of Nissl bodies were significantly declined, and a majority of pyramidal cells kept with a normal cytoarchitecture. LC statistically mitigated neuronal damage in DG region, while unchangeable in CA3. In addition, chronic exposure of HC did not alleviate hippocampal neuronal damage in both CA3 and DG regions but exhibited less counts of Nissl bodies than that in the SD group.
To investigate the involvement of cAMP-PKA-CREB pathway after caffeine exposure, we chosen Western blot and ELISA method. SD rats expressed significantly lower cAMP levels compared to the controls (Fig. 4B), and we also found a decrease in p-PKA and pCREB protein abundance following SD (Fig. 4CeD). LC markedly increased the expression level of cAMP, p-PKA and p-CREB without changing the total PKA and CREB levels (Fig. 4BeD). Conversely, HC further decreased the cAMP, p-PKA, and p-CREB protein levels, indicating that caffeine modulated the cAMP-PKA-CREB pathway protein expression and seemed partly related to a dose variability performance.
3.5. Effect of caffeine on the VIP and AVP expression in SD rats In the hippocampus CA1 region, the VIP demonstrated higher level in the controls, intermediate level in the SD and LC groups, and a weak level in the HC group (Fig. 3AeB), and the expression of AVP within groups were similar with that of the VIP, the LC group did not increased the AVP expression levels compared to the SD. Consistently, Western blot also indicated that SD repressed the relative expression of both VIP and AVP, and LC evidently upregulated the levels of VIP and AVP (Fig. 3CeD). Moreover, HC
3.7. Effect of caffeine on apoptosis in hippocampus neurons To further confirm if the caffeine intake was involved in the apoptosis process, we detected the expression of apoptosis-related proteins. As shown in TUNEL staining, LC decreased TUNEL-positive cells, while high dose increased the numbers of TUNEL-positive cells, with shrunken condensed nuclei and apoptotic bodies
Fig. 2. The effect of caffeine on the hippocampal pathological changes in SD rats. (A) The ultrastructural examination was displayed in two scales. Black arrows indicated nuclear and organelle damage. N ¼ 2/group. Scale bar ¼ 2 mm. (B) Representative photomicrographs of Nissl staining of the CA3 and DG in the hippocampus. Black arrows indicated the Nissl staining neurons. N ¼ 3/group. Scale bar ¼ 100 mm **P < 0.01 vs. untreated control; ^P < 0.05, ^^P < 0.01 vs. individuals caused by SD. 4
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Fig. 3. The effect of caffeine intake on VIP and AVP expression. (A) Representative images of VIP (Red), AVP (Red) and DAPI (Blue) positive cell in CA1 regions by immunofluorescence, (B) The average density of VIP and AVP (five different fields per slice), (C) The expression level of VIP and AVP were measured by Western blot, (D) The relative expression of VIP and AVP was analyzed by GraphPad Prism 8. **P < 0.01 vs. untreated control; ^P < 0.05, ^^P < 0.01 vs. individuals caused by SD. N ¼ 3/group, scale bar ¼ 50 mm.
antagonist. In contrast to the adenosine effect, caffeine promotes arousal and attenuates the influence of SD on vigilance and attention, and the two adenosine receptor subtypes may play a part in cerebral functions linked to sleep/wake connection. Onaolapo OJ et al. [17] have found that the effect on cognitive function was still dose-dependent when caffeine was consumed alone without sleep deprivation. Our results suggested that 14-day chronic SD impaired spatial memory acquisition and increased anxious behaviors, expressing cognitive impairment successfully induced by sleep deprivation. HC exacerbated cognitive disorder and anxious behavior performance but low-dose caffeine administered effectively improved memory retention. This result confirms that cognitive performance is associated with caffeine dosage [19]. Neuronal apoptosis is considered activated in sleep deprivation and is correlated with cognitive impairment [20]. We found sleep deficiency induced neuronal apoptosis, which acted on the mitochondria-mediated apoptosis process and the Bcl-2/Bax ratio. Emerging evidences demonstrated that the formation of Bax monomer was a decisive factor necessary to destroy the permeability of mitochondrial membranes [21]. And the caspase-3 and caspase-9 were then activated, inducing a marked reduction in the plasticity of neuronal function, neurodegenerative lesions, which in turn caused a series of cognitive dysfunctions [22]. In our research, significant neuronal apoptosis was found, shown by mitochondrial impairment, elevated caspase-9 and caspase-3 levels, and decreased in Bcl-2/Bax ratio as well, which could be aggravated by HC intake but alleviated in low dosage. In accordance with the present results, previous studies have demonstrated that of the severity of apoptosis significantly impairs learning and memory [23].
obtained in CA1 region (Fig. 4E and F). Moreover, the model rats had significantly reduced the Bcl-2/Bax ratio (Fig. 4J), along with a noticeable increase in the level of caspase-9, and cleaved caspase-3 (Fig. 4K and L). Caffeine enhanced the levels of caspase-9 and cleaved caspase-3, especially in high dosage. LC sufficiently increased the ratio of Bcl-2/Bax, as well as reducing the caspase-9 and cleaved caspase-3 expression. Additionally, we found no significant differences in Bcl-2, Bax, and Bcl-2/Bax between SD and HC groups. These results paralleled the aforementioned findings on apoptosis through the cAMP-PKA-CREB pathway and suggested that an appropriate caffeine dosage will counteract overactivated apoptosis.
4. Discussion Studies in the past 20 years have found that chronic sleep loss results in more rapid cumulative increases in cognitive deficits than subjective fatigue and mood, and the effects are proportional to the latency of sleep and the chronicity of sleep restriction [14,15]. June et al. investigated [16] the false memory formation in healthy young people and observed that sleep-deprived subjects will more frequently than well-rested individuals incorporate contradictory post hoc information during their memory retrieval. In contrast to the previous acute sleep deprivation (ASD) experiment [17], the current study applied chronic sleep-deprived (CSD) approach, which may better resemble the individual’s state of sleep deprivation caused by prolonged shift work and display more severe performance on emotional and cognitive functions of experimental animals [18]. Caffeine is a non-selective adenosine A1 and A2A receptors 5
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Fig. 4. Assessment of the cAMP-PKA-CREB pathway proteins and apoptosis levels in SD rats. (A) Representative images of western blots probed with antibodies against p-PKA, PKA, p-CREB, and CREB. (B) The cAMP levels were measured using a cAMP assay ELISA kit. Densitometric quantitations of p-PKA (C) and p-CREB (D) were shown. Photomicrographs(E) and statistics (F) of Tunel staining in the CA1 region. N ¼ 3/group. Scale bar ¼ 20 m. Black arrows indicated the Tunel positive cell. (G) Western blot images of respective apoptosis proteins in the hippocampus. Gray intensity analysis of Bcl-2 (H), Bax (I), caspase-9 (K), and cleaved-caspase3 (L). **P < 0.01 vs. untreated control; ^P < 0.05, ^^P < 0.01 vs. SD. N ¼ 3/ group.
TUNEL staining confirmed differences in apoptosis level with varying caffeine dosage. The results above demonstrate that the dose of caffeine ingested after sleep deprivation may be responsible for the extent of neuronal apoptosis through cAMP-PKA-CREB pathway.
Ultimately, we investigated if caffeine administration could regulate the expressions of the apoptosis-related proteins. VIP and AVP, neuropeptides involved in the nervous system, modulate the excitability of the hippocampus and play a modulatory role in cAMP-PKA-CREB signal transduction [24,25]. Cyclic AMP is involved in multiple pathways modulating apoptosis as a key physiological signaling molecule. Intracellular cAMP induces phosphorylation of PKA (p-PKA) and CREB (p-CREB), and resultantly activated p-CREB can further stimulate the transcriptions of numerous downstream genes, resulting in the elevation of Bcl-2 family proteins, further accelerating the development of apoptosis [26]. In the present study, LC improved cAMP-PKA-CREB signal protein expression, from both upstream and downstream genes, while the HC exerted an opposite effect on this process.
5. Conclusions In summary, the present study has opened up new avenues to unravel the specific roles of caffeine dose in the complex behavioral network and cognitive function, particularly concerning the neuronal apoptosis in the hippocampus via the cAMP/PKA/CREB pathway. Robust differences were shown among different doses of caffeine in their effect on cognitive vulnerability associated with 6
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sleep loss. The cognitive deficits believed to depend on the severity of clinical sleep disturbance may also depend on differential sensitivity to caffeine. Further research should better clarify the interaction of sleep deprivation-induced cognitive impairment with adenosine receptor subtypes. This strategy may then result in the comprehensive understanding of more rational caffeine administration and safe consumption awareness, not only of weaken alertness and vitality but of impaired cognitive performance, under the circumstances of sleep deficiency and prolonged shift work, for example. Thus, the reference was seriously essential to public harmony and physical health.
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