ABSTRACT
Norepinephrine plays an important role in modulating the processes of memory consolidation and evocation through its beta-adrenergic receptors (Adrβ), which are expressed in the hippocampus and amygdala. Several studies have shown that all three subtypes of Adrβ (β1, β2 and β3) play an important role in cognition. Environmental enrichment (EE), a technique initially used to decrease the stress of animals held in captive environments, has also been shown to produce cognitive benefits in both healthy and sick animals. In this study, we hypothesized that EE would reverse the memory impairment induced by the absence or Adrβ3. To test this, 21- and 86-day-old Adrβ3KO mice were exposed to an EE protocol for 8 weeks. The study showed that the EE protocol is able to correct the memory impairment when applied to Adrβ3KO animals immediately after weaning but has no effect when applied to adult animals. We also found that aging worsens the memory of Adrβ3KO mice. Our results suggest that a richer and more diverse environment helps to correct memory impairment in Adrβ3KO animals. They also reinforce the idea that noradrenergic signaling is involved in the cognitive impairment observed late in life, as aging led to a worsening in the memory of the Adrβ3KO animals that was not corrected by the environmental enrichment protocol.
INTRODUCTION
Norepinephrine (NE) plays an important role in modulating the processes of memory consolidation and evocation through its beta-adrenergic receptors (Adrβs), which are expressed in the hippocampus and amygdala of mammals(1, 2). Adrβ1 is found in the hippocampal regions CA1 and CA3 and in a small amount in the dentate gyrus. Adrβ2 and Adrβ3 are found in practically all regions of the hippocampus and amygdala, with β3-AR also being present in the entorhinal cortex (3–5). Adrβ selective agonists stimulate the induction of long-term potentiation (LTP) in the pyramidal cells of the CA1 region of the hippocampus and are involved in hippocampal-dependent cognitive functions (6). The use of Adrβ antagonists and the blockage of NE synthesis impair the formation of hippocampal-dependent memory (6).
Since Adrβs are positively coupled to protein G and enable the amplification of neuronal signals (2), Adrβs mediate the formation of long-term memory, probably by activating cAMP signaling pathways with consequent modulation of neuronal plasticity and excitability (7, 8).
Isopropoterenol, an agonist for Adrβ1 and Adrβ2, increases neural plasticity in the CA1 and CA3 regions and in the dentate gyrus in the hippocampus, while propranolol, a Adrβ1 and Adrβ2 antagonist, blocks LTP in the CA1 region and the dentate gyrus (9–12). In addition, the absence of the Adrβ3 receptor induces important deficits in the formation of short- and long-term memory (13).
Environmental enrichment (EE) is a technique that was initially used as a way of decreasing the stress of animals held in captive environments (14) (15). However, many studies about strategies for investigating cognitive skills have shown that EE has cognitive benefits in animals whether they are healthy, sick, young or old (15–17). EE may include 1) Physical enrichment, in which there is the introduction of materials in the enclosure that stimulate activities in the animal’s natural environment, such as burrows and tunnels; 2) Sensory enrichment, which consists of stimulating the animal’s senses with herbs to stimulate the sense of smell, and objects with different textures for tactile stimulation; 3) Cognitive enrichment, activities in which the animal needs to solve a problem, for example, hanging a banana on a string through the roof of the cage will require some increased cognitive demand to solve the challenge and reach the prize; 4) Social enrichment, animals can be allowed interact with other species, or the number of animals of the same species in the same enclosure can be increased which will consequently influence the social hierarchy; 5) Food enrichment, which consists of changes in the animals’ diet or changes in the frequency or time of feeding (14, 18).
Several studies evaluating the effect of EE on brain development have observed increased neurogenesis, increase in dendritic ramifications, as well as increased nerve growth factor (NGF) gene expression and increased LTP in the hippocampus (19–21). EE has also been shown to improve learning and memory consolidation in animal models of Alzheimer’s disease (22, 23).
Here we hypothesized that the use of EE could reverse the memory impairment induced by the absence of Adrβ3. Thus, the aim of our study was to evaluate the effect of EE on memory consolidation processes in Adrβ3 knock-out (Adrβ3KO) mice.
METHODS
Animals
Adrβ3KO mice with an FVB background, generated by removing the 306bp genomic fragment containing the sequences encoding the third through the fifth transmembrane domains of the Adrβ3 and replacing it with a neomycin selection cassette, as described by Susulic et al. (24), were obtained from Mackenzie Presbyterian University (Sao Paulo, Brazil). The animals were genotyped to confirm their status as homozygous knockout (β3-ARKO) or wild type (WT) mice. Male Adrβ3KO mice and WT controls from different litters randomized between groups were used in a protocol approved by the Institutional Committee on Animal Research at the Center of Biological Sciences and Health, Mackenzie Presbyterian University. Each experiment was repeated twice on the four different groups of animals. Mice were housed in groups at 26°C, 55–60% humidity, and a 12-h light/dark cycle with ad libitum access to standard food (Nuvilab, Brazil) and water.
Experimental design
Based on studies on mice development, we evaluated the effects of EE at two moments in the lives of animals. Study 1 assessed the impact of EE that started right after weaning, PND21 until PND85. Study No. 2 assessed the impact of EE initiated in adult life, so EE started on PND120 until PND 180.
Study 1 - Effect of early EE on young mice
The animals were transferred immediately after weaning on post-natal day 21 (PND21) to the EE cage and were submitted to the protocol described in Table 1 until PND85 when behavioral tests were started and finished at PDN120 (Figure 1A). The animals were divided into the following groups: WT (n = 7); Adrβ3KO mice (n = 7); WT + EE (n = 9); and Adrβ3KO + EE (n = 9).
Study 2 - Effect of late EE on adult mice
The animals were kept in standard cages until PND120, when they were then transferred to the EE cage and submitted to the EE protocol until PND180 (Figure 1B). Behavioral tests were started at PND180 and finished at PDN 205. The animals in this study were divided as in Study 1: WT (n = 6); Adrβ3KO (n = 9); WT + EE (n = 7); and Adrβ3KO + EE (n = 7).
EE Protocol
All the mice submitted to the EE remained in two-story cages (57×31×41cm), lined with wood shavings and with a shelter, water and food on both floors. The EE protocol was standardized in our laboratory (adapted from (25, 26)) and consisted of two interventions per week for eight weeks in the morning, with sensory, cognitive and dietary activities, as well as changes in the home cage (Table 1). After eight weeks of EE, the behavioral tests were started. During the behavioral assessment the animals remained in the EE cages until the completion of the tests, but without the stimulatory activities.
Behavioral testing
All tests were performed in the morning (7:00–9:00 AM), under dimmed light (15 lux), and recorded by video for later analysis for two different blind observers in the following order for both studies 1 and 2:
Open field test (OF)
The open field test was used to evaluate exploratory activity (27). The animals were placed in the center of a circular acrylic arena (diameter = 30cm) divided into four central zones and eight peripheral zones (Insight Ltda, Brazil), in a low-light environment (15 Lux) for 10 minutes. Locomotion (total number of lines crossed with all four paws) in the central and peripheral zones was measured. The test was performed three consecutive times with a 24-hour interval (28).
Novel object recognition test (NOR)
This test was performed to evaluate short- and long-term memory. It was performed in the OF arena right after the OF test in order to guarantee the habituation of the mice to the arena. The test consists of three stages: familiarization to the two unknown objects, 3 and 24 hours after the familiarization. In the familiarization stage, the animals were placed in the open field arena for 10 minutes. After familiarization, the animals were exposed to two unknown and identical objects, object O1 and object O1’ for 3 minutes. Three hours later, the test was performed with the animals being placed in the arena for 3 minutes and exposed to object O1 and a new object (O2). 24 hours after the familiarization the animals were placed in the arena for 3 minutes and exposed to the known object O1 and a new object (O3). At each stage the time spent with objects, i.e. animal exploring the object with their nose, was expressed as a recognition index, i.e., the percentage of time spent with each object considering the total time spent with both objects (29).
Social recognition test (SR)
Social preference and discrimination were evaluated using a non-automated 3-chambered box with three successive and identical chambers (Stoelting, Dublin). The protocol used is similar to the one described previously (30). Briefly, in the familiarization period, the mice were allowed to explore the three chambers freely for 10 min starting from the intermediate compartment, with the two other chambers containing empty wire cages. To test social preference, the test mouse was placed in the intermediate compartment, while an unfamiliar mouse was now put in one of the wire cages in a random and balanced manner. The doors were re-opened and the test mouse was allowed to explore the three chambers for 10 min. Time spent in each of the chambers, the number of entries into each chamber, and the time spent sniffing each wire cage were recorded for social preference. In the third phase, social discrimination was evaluated with a new stranger mouse (unknown) being placed into the remaining empty wire cage with the test mouse allowed to explore the entire arena for 10 min, having the choice between the first, already-investigated mouse (known) and the novel unfamiliar mouse (unknown). The same measures were taken as for the social preference (31) (32).
Statistical analysis
Experimental data were analyzed using PRISM software (GraphPad Software). The statistical significance of the difference among the mean values for the groups were analyzed by two-way ANOVA, followed by the Tukey’s test was used, with a significance level of p ≤ 0.05.
RESULTS
EE exposure early in life increases ambulatory activity in young adult B3-ARKO and WT mice
In the OF the 2-way ANOVA showed that control Adrβ3KO young mice exhibited less ambulatory activity when compared to adult WT young mice (F(1,6) =12.63; p=0.012), but there was a significant reduction of ambulatory activity after the 3 days exposure in OF for both groups (F(2,12)=46.41; p<0.0001) (Figure 2A). It was noted that after the EE exposure the difference between WT and Adrβ3KO young mice in total line crossing disappears (F(2,32)=2.16; p=0.13) and both groups exhibited a reduction in ambulatory activity after the three days of exposure to the OF (F(1,22)=31.02; p<0.0001) (Figure 2B). Regarding the exploratory activity there was an effect of treatment only on the 3rd day of exposure to the open field test (F (1,28) = 17.55; p=0.003) (Figure 2C-E).
EE exposure early in life corrects cognitive impairment in young adult B3-ARKO mice
Cognition was evaluated through the novel object recognition test (NOR) and the valence-based social recognition test (SR), which are based on exploratory behavior and assess memory and preference for novelty. In the NOR test, all groups explored the objects similarly during the familiarization period (Supp Fig. 1A-B). WT mice spent significantly more time with the new object, 3h (O2) (p<0.0001; t=5.06) and 24 h (O3) (p<0.0001; t=8.52) after the familiarization period (Figures 3 A-B). In contrast, the Adrβ3KO spent similar amounts of time with old (O1) and new objects (O2) 3 h (p=0.62; t=1.03) and 24h (O3) (p=0.1; t=2.06) after the familiarization period (Figure 3 A-B), confirming data from a previous study (13) showing that the absence of Adrβ3 impairs memory consolidation. Remarkably, EE was effective in correcting the memory impairment displayed by the Adrβ3KO mice both 3h (O2) (p<0.0001; t=8.06) and 24 h (O3) (p=0.0014; t=3.73) after the familiarization period (Figure 3 C-D). The time WT mice spent with O2 (p<0.0001; t=6,78) and O3 (p<0.0001; t=5,96) was not changed by the EE protocol (Figures 3 C-D).
In the SR test, all groups explored the empty cages similarly during the familiarization period (Supp Fig. 1C). The results from the social preference of SR tests showed that WT (p<0.0001; t=17.3) and Adrβ3KO (p<0.0001; t=20.28) animals are more interested in spending time with a conspecific animal than with an empty cage (Figure 3E). The exposure to the EE protocol did not change the social preference in both WT EE (p<0.0001; t=14.28) and Adrβ3KO (p<0.0001; t=21.5) (Figure 3E). The results obtained from the social discrimination phase of SR test showed that both WT (p<0.0001; t=8.52) and Adrβ3KO (p<0.0001; t=5.41) mice not exposed to EE do remember the known animal to whom they were exposed earlier since they spent more time with the unknown mice than with the known mice (Figure 3 F). The exposure to EE protocol did not change this behavior, since WT EE (p=0.001; t=3.84) and Adrβ3KO (p<0.0001; t=7.92) spent more time with the unknown mice than with the known one (Figure 3F). The difference in the performance of Adrβ3KO mice in NOR compared to SR is explained by the fact that SR test uses conspecific animals, and thus, memory formation is strengthened by stimulus valence.
EE exposure late in life changes ambulatory and exploratory activity of Adrβ3KO and WT mice
Both WT and Adrβ3KO adult mice show similar ambulatory activity when exposed to the open field test without effect of time for both groups (F(2,39) = 1.024; p=0.37) (Figure 4A). However, EE increased the ambulatory activity in adult Adrβ3KO mice when compared to adult WT mice exposed to EE (F (1,18) = 7.91; p=0.0012) (Figure 4B). EE increased the exploratory activity in WT mice in day 1 (p=0.003), in day 2 (p=0.02), and day 3 (p=0.0003) of testing (Figure 4 C-E). However, EE decreased in Adrβ3KO mice in day 1 (p=0.01), in day 2 (p=0.02), and day 3 (p=0.02) of testing (Figure 4C-E). Notably, control Adrβ3KO adult mice explored significantly more than control WT adult mice also not exposed to EE in day 2 (p=0.008) and day 3 (p=0.002).
EE exposure late in life does not correct cognitive impairment in adults B3-ARKO mice
In the NOR test both groups explored the objects similarly during the familiarization period regardless of the EE exposure and the genotype (Supp. Figure 1D-E). Control and EE exposed WT adult mice spent significantly more time with the new object 3h (O2) (p<0.0001; t=8.88) and 24 h (O3) (p<0.0001; t=6.04) after the familiarization period (Figures 5A-D). In contrast, Control and EE exposed Adrβ3KO mice spent similar amounts of time with old (O1) and new objects 3h (O2) (p=0.99; t=0.31) and 24 h (O3) (p=0.21; t=1.68) after the familiarization period (Figure 5A-D). These results show that adult WT mice retained their memory consolidation abilities regardless of older age and show that older Adrβ3KO mice exhibited similar memory impairment as younger ones. Remarkably, the exposure of EE later in life at PND120 (Figure 1B) was not able to correct the memory impairment observed in Adrβ3KO (Figure 5C-D).
In the SR test, all groups explored the empty cages similarly during the familiarization period (Supp Fig. 1F). The social preference in SR tests showed that control WT and Adrβ3KO adult mice spent more time with a conspecific animal than with an empty cage (p<0.0001) (Figure 5E). The control WT adult mice do remember the known animal to whom they were exposed since they spent more time with the unknown mice than with the known one (p<0.0001; t=10.21) and the similar results were observed in EE WT adult mice (p<0.0001; t=7.95) (Figure 5F). However, control Adrβ3KO adult mice spent the same amount of time with known and unknown conspecific animal (p=0.94; t=1.2) suggesting a worsen in memory impairment as they grow older, since younger Adrβ3KO mice spent more time with the unknown conspecific (Figure 5F). Remarkably, EE was not able to correct this impairment (p=0.23; t=1.94), suggesting that there is a time frame to its efficiency.
DISCUSSION
The present study showed that the EE protocol when applied to Adrβ3KO mice immediately after weaning was able to correct the memory impairment observed in these animals but had no effect when applied to adult animals.
The efficacy of the EE protocol in reversing the short- and long-term memory deficit in young mice due to the lack of Adrβ3 could be explained by the findings of several previous studies showing that cognitive enrichment in rats contributes to better learning and the performance of declarative and procedural memory tasks. The increase in the dendritic tree in the third layer of the pyramidal neurons of the temporal lobe, changes in synaptic organization and an increase in the amount of intersections in both the basal and apical dendrites suggests a change in plasticity that involves neural reorganization and an increase in the number of neuronal contacts and the formation of a more complex neuronal network (33–35).
Other studies also show that rats and mice submitted to EE present improvement in learning and memory, as well as better performance in cognitive tasks when compared to rodents kept in standard cages. In addition, they also have increased levels of brain-derived neurotrophic factor (BDNF) and NGF, particularly in the hippocampus region, indicating neuronal growth and proliferation, and brain plasticity (36–38). Thus, it is likely that the improvement in memory tasks observed in young Adrβ3KO mice submitted to EE can be explained by these changes in neurotrophin expression.
The constant exposure of animals to novelty through EE involves some degree of stress in animals. The animals were exposed to various stimuli, including those of negative valence, such as bedding with the smell of rats. When the animal has contact with stimuli with negative valence, it could induce an increase in the peripheral release of adrenaline that activates the expression of Adrβs in the vagus nerve, which will in turn activate the locus coeruleus (LC). The activation of the LC leads to an increase in NE release in the hippocampus and amygdala (39, 40). Thus, it is possible that the increase in NE levels induced by EE activates a compensatory pathway through β-ARs, reversing the memory deficit in young Adrβ3KO mice.
Although EE was very beneficial for memory when applied to young animals, the results obtained when EE was introduced to older Adrβ3KO mice showed that the protocol used is not able to reverse the damage caused by the absence of ARβ3 when it starts in adulthood. In fact, aging worsens the memory of Adrβ3KO mice, since their good performance in the social recognition test at 2-3 months of age is not repeated at 6-7 months of age, despite the amygdala activation due to the valence of the stimulus. It has been shown that LC degeneration is a common neuropathological feature of neurogenerative diseases such as Alzheimer’s (41, 42). In fact, early degeneration of the LC could trigger or be involved in the progression of neurogenerative diseases (43). The fact that a lack of Adrβ3KO leads to a greater loss in cognition highlights the role of the noradrenergic signaling pathway in the course of dementia. Also, it has been shown that EE in older healthy mice is not as efficient in improving cognition as it is in younger animals (44).
In healthy rodents, locus coeruleus projections to different brain regions begin to decline by 7–15 months of age (45, 46). Other studies with rodents and primates have found a correlation between memory loss and the increasing appearance of lesions, and consequent cell loss in the hippocampus and entorhinal cortex, with age (47, 48). Advancing age leads to a loss of 10 to 20% of brain mass when compared to a young brain. This can lead to variations in cell loss in different brain regions and, consequently, more serious losses in certain regions than in others (49). The lack of Adrβ3 combined with the functional changes typical of advancing age can aggravate damage to memory formation processes and that could be the mechanism underling the worsen in memory observed in adult Adrβ3KO mice. The effects of aging may explain the absence of any benefit from EE when applied to older animals. The results of the present study reinforce the idea that early stimulation of individuals is beneficial for cognition and can prevent or delay early memory impairment caused by defects in neuronal signaling involved in cognition.
EE does not alter locomotor capacity when applied to young animals regardless of the genotype but increased ambulatory activity in older Adrβ3KO mice. This suggests that the stimulus represented by the EE may improve the activity of animals at an older age. The influence of EE on the exploratory behavior of mice has already been evaluated in other studies, but there is as yet no consensus on its influence (50, 51).
In conclusion, the results obtained show that Adrβ3 has an important role in memory as aging leads to a worsening in the memory of Adrβ3KO animals that is not corrected by the EE protocol used in this study. However, the EE protocol can reverse memory damage in young Adrβ3KO animals. Further studies are needed to understand the role of Adrβ3 in aging, and the mechanism involved in the effect of EE in reversing memory impairment.
Acknowledgement
Support from FAPESP 2017/18277-0 for MOR; PROEX 1133/2019 for MOR; CAPES for TTR; FAPESP 2017/04491-0 for BPPN.
Footnotes
Disclosure Statement: The authors have nothing to disclose.