The Diferential Efects Of Brief Environmental Enrichment Following Social Isolation in Rats Part 3

Neuronal activity

We quantified the number of c-Fos+ cells in each region of interest, took the arithmetic average for each animal, and made group-level comparisons (Fig. 6). Examined sections were acquired at similar rostra-caudal levels (−3.0 to −3.6 from the Bregma point; Paxinos & Watson, 2006) to minimize the effect of topographical variability on neuronal activity. 

Neurons are the most basic neuronal units in the brain, and they play a vital role in the human body. Neuron activity plays a key role in human memory, thinking, decision-making, and state of consciousness. Below we will delve deeper into the relationship between neuronal activity and memory.

First, the impact of neuronal activity in the brain on memory can be realized through the synaptic connections of neurons. Thousands of synaptic connections can be formed between neurons, and these connections are important pathways for information transmission in the brain. When a neuron is activated, neurotransmitters are released, affecting neighboring neurons' activity state. These synaptic connections can be strengthened through continued use, thereby enhancing memory retention and retrieval.

Second, neuronal activity can be divided into two categories: excitation and inhibition. When neurons are excited, the electrical signals they generate can propagate to neighboring neurons, amplifying the neuron's excitatory activity. This stimulating activity is important for enhancing memory retention and retrieval, as it helps people remember and understand information better. In contrast, when neurons are inhibited, the electrical signals they produce suppress the activity of neighboring neurons, thereby weakening the brain's ability to process information. Therefore, we should try to avoid some unhealthy habits and environments, such as excessive drinking and lack of sleep. These habits may hurt the activity of neurons and hinder learning and memory.

Finally, we should face the challenges in life with an optimistic attitude. This is because an optimistic emotional state can promote connections between neurons and further strengthen memory. On the contrary, negative emotional states may interfere with the connections between neurons, causing people's memory to become more fragile.

In short, neuronal activity is closely related to memory. By strengthening the synaptic connections between neurons, maintaining the excitatory activity of neurons, avoiding unhealthy habits and environments, and maintaining an optimistic attitude, we can continuously improve our memory and achieve more success in learning and life. It can be seen that we need to improve memory, and Cistanche deserticola can significantly improve memory because Cistanche deserticola is a traditional Chinese medicinal material that has many unique effects, one of which is to improve memory. The efficacy of minced meat comes from the various active ingredients it contains, including acid, polysaccharides, flavonoids, etc. These ingredients can promote brain health in various ways.

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As shown in representative c-Fos immunolabeling depictions (Fig. 6), we found that the number of c-Fos+ cells in the RSC was significantly different for continuous SI (M = 2356.5, SD = 438.4) and SI to EE animals (M = 3443.7, SD = 856.3; t (10) = 2.77, p = 0.02, d = 1.6, independent samples t-test; Fig. 7). Similarly, in the PRC, continuous SI animals (M = 739.7, SD = 201.4) had significantly lower number of c-Fos+ cells compared with the experimental group (M = 2398.3, SD = 1070.6; t (10) = 3.73, p = 0.004, d = 2.15, independent samples t-test; Fig. 7).

No such difference was observed in the LA (continuous SI: M = 823.6, SD = 225.6 vs. SI to EE: M = 957, SD = 584.4; t (8) = 0.48, p = 0.647, independent samples t-test; Fig. 7) or BL (continuous SI: M = 588.2, SD = 414.5 vs. SI to EE: M = 1176.4, SD = 677.7; t (8) = 1.66, p = 0.136, independent samples t-test; Fig. 7).

Discussion

We show that a brief EE following 30 days of social isolation produced opposing results in depression- and anxiety-like behavior. Compared with the control group, animals switched from SI to EE showed increased behavioral despair in the FST. In contrast, we found a substantial anxiolytic effect in the same group after 5 days of enrichment. 

Briefly, enriched animals following SI displayed a better spatial working memory performance during the first 2 days of the WYM compared with the continuous SI group, indicating faster learning. Brief enrichment was associated with changes in neuronal activity levels in the retrosplenial and perirhinal cortices. SI to EE animals had a significantly higher number of c-Fos+ cells in these cortical regions as compared to the control animals.

Replicating previous research stating a weight drop in EE (Harati et al., 2011; Moncek et al., 2004; Zaias et al., 2008), the body weight of the SI to EE animals decreased significantly following enrichment. This was in contrast to other work reporting no effect of chronic (Glueck et al., 2017; Grimm et al., 2016, 2018) or brief enrichment (Beale et al., 2011) on weight. 

The running wheel provided in the enrichment cage (Augustsson et al., 2002; Harati et al., 2011; Stein et al., 2016) and the initial short-term feeding suppression observed in pair-housed animals following isolation (Lopak & Eikelboom, 2000; O'Connor & Eikelboom, 2000; Weisinger et al., 1989) may have accounted for this difference. 

Long-term isolation does not alter food consumption (Hellemans et al., 2004) or body weight (Fone & Porkess, 2008), no such weight change was observed in continuous SI animals.

It should be noted that behavioral despair observed in the SI to EE group was not a reflection of alterations in metabolic difference, as the overall locomotor activity in the OFT did not differ between the groups. This finding is noteworthy given that chronic or subchronic SI can itself be used as a rodent model of depression (Djordjevic et al., 2015; Stanisavljević et al., 2019). 

In contrast, there are contradicting results for the effects of EE on behavioral despair. Some studies report no significant effect (Cui et al., 2006; Simpson et al., 2012), whereas others found increased mobility in the test phase of the FST, indicating an antidepressant effect (Brenes et al., 2008; Cui et al., 2006; Porsolt et al., 1977). 

One reason for the depressant effects of brief EE observed in our study could be the novelty stress induced by the enrichment cage following relatively long-term isolation with minimal environmental stimulation (Hennessy & Foy, 1987; Miura et al., 2002). A phenomenon also observed in humans, novelty stress was associated with alterations in synaptic monoamine levels and heightened HPA axis activity (Miura et al., 2002). However, the depressant effect of brief EE in our study was accompanied by a substantial anxiolytic effect observed in the EPM (Fig. 4).

Environmental enrichment can also function as a social stressor by producing crowding stress, especially in male rats that are not familiar with each other (Brown & Grunberg, 1995). 

It should be noted that the eight animals in the SI to EE group originally came from four different home cages, making most of the other animals in the EE cage unfamiliar. Crowding, like isolation stress, is correlated with elevated plasma levels of adrenocorticotropic hormone (ACTH) and corticosterone (CORT) (Dronjak et al., 2004). 

Albeit these potential alterations, brief enrichment in this study produced a profound anxiolytic effect, as observed in the majority of earlier research (Galani et al., 2007; Harati et al., 2013; Leal-Galicia et al., 2008; Leal-Galicia et al., 2007; Peña et al., 2006; Sampedro-Piquero et al., 2014; but see Goes et al., 2015; Mann & Gervais, 2011). Long-term isolation, in contrast, often leads to an anxiogenic effect in the EPM (Djordjevic et al., 2015; Hall, 1998), as well as other measures of anxiety-like behavior (Spasojevic et al., 2007; Zlatković et al., 2014). 

Similar to the design of the present study, Ravenelle et al. (2013) showed that rats bred to display high-anxiety and exposed to post-weaning EE spent more time in the open arms of the EPM as compared to impoverished high-anxiety rats.

These findings show that, unlike behavioral despair, the relationship between living (environmental) conditions and anxiety is straightforward: an increase in enrichment decreases anxiety (Benaroya-Milshtein et al., 2004; Ravenelle et al., 2014; Sampedro-Piquero et al., 2013). In line with the present findings, other brief EE experiments reported anxiolytic effects for as short as 2 weeks of enrichment (BrionesAranda et al., 2020). The length and intensity of enrichment may not have been sufficient in the few studies that did not report an anxiolytic effect of enrichment (see Goes et al., 2015; Simpson & Kelly, 2012). EE cage in the current study included a running wheel for voluntary exercise, which further decreases anxiety-like behavior in the EPM (Binder et al., 2004; Burghardt et al., 2004).

To assess the cognitive effects of brief enrichment, we chose a simple but versatile memory task, the WYM spontaneous alternation test. This spatial working memory measure depends on several structures including the prefrontal cortex, hippocampus, and basal forebrain. Impoverished housing conditions and social isolation lead to several plasticity-related alterations in the cortex (Gregory & Szumlinski, 2008; Ieraci et al., 2016; Popa et al., 2020), accompanied by an impairment in spatial working memory tasks (Gregory & Szumlinski, 2008; Melendez et al., 2004). 

Enrichment, in contrast, often produces the opposite effect at the neuronal and behavioral level (Sampedro-Piquero et al., 2013). Our findings show that 5 days of enrichment was strong enough to increase spatial working memory performance following 30 days of SI stress. This difference did not persist on the third day of the test when both groups displayed a significant decrease in their latency to locate the correct platform. The stressful nature of the WYM may have contributed to these results. 

The WYM task, like other water mazes, relies on the animal's inherent motivation to escape an aversive condition (i.e., water). Differences in anxiety levels may affect memory performance in the initial sessions before animals learn that there is an escape platform. Such a differential effect would be transient as there were six consecutive trials per day and animals that were not able to locate the platform in 60 seconds were gently guided toward it at the end of each trial.

We correlated these different behavioral findings with c-Fos immunohistochemistry. As the peak level of c-Fos protein expression occurs approximately 90 min before perfusion, the group-level differences observed in neuronal activity mainly reflect the different environmental conditions. 

However, some of these differences may be due to sustained long-term alterations in neuronal circuits caused by brief EE and the differential effects of subsequent behavioral testing. In theory, brief EE may have formed new circuits and modified old ones in specific regions, which would result in differences in the number of active neurons under the same conditions (Nikolaev et al., 2002; Zorzo et al., 2019). In our study, it was not possible to isolate these sustained alterations in neuronal activity from the transient differences reflecting activity approximately 90 min before perfusion.

In SI to EE animals, we recorded significantly more c-Fos+ cells in the retrosplenial cortex, an association cortex involved in allocentric spatial navigation and memory among several other functions (Hindley et al., 2014; Vann & Aggleton, 2002). In addition, these animals had more c-Fos+ cells in the perirhinal cortex, the cortical region underlying object recognition and associative memory (Samarth et al., 2017; Unal et al., 2012). This structure, providing the densest afferent of the entorhinal cortex, is also required for spatial working memory (Liu & Bilkey, 2001). Both results complement the faster learning observed among SI to EE animals in the early phase of the WYM.

There was no group-level difference in c-Fos-immunolabeling in the lateral and basolateral nuclei of the amygdala. An earlier study on FosB/DFosB-immunoreactivity in the basolateral amygdala complex (BLA) and medial prefrontal cortex (mPFC) following an SI to EE (4 weeks each) switch found significantly more immunolabeling in the BLA of isolated animals and the mPFC of SI to EE animals (Watanasriyakul et al., 2019). The higher number of c-Fos+ cells found in the retrosplenial and perirhinal cortices of SI to EE animals in our findings are in line with the aforementioned observation made in the mPFC. In contrast, we did not observe a meaningful difference in the LA or BL. It is likely that the c-Fos immunoreactivity observed in the amygdala mostly reflects transient changes in neuronal activity: neurons with a high level of firing approximately 90 min before perfusion. Hence, the differential behavioral results observed in the FST and EPM do not correlate with a sustained difference in neuronal activity.

Albeit its popularity in behavioral neuroscience, there is a major discussion on the validity of the FST as a rodent model of clinical depression. One line of criticism considers the immobility in the FST as an adaptive behavior rather than an indication of behavioral despair (Anyan & Amir, 2018; Borsini et al., 1986; Molendijk & de Kloet, 2015, 2019). According to this point of view, increased FST-2 immobility of the SI to EE group may not be indicative of behavioral despair; but reflect better behavioral adaptation (or learning) of the briefy enriched group. This possibility cannot be ruled out completely (see Unal & Canbeyli, 2019 for a detailed survey of the behavioral adaptation theory of the FST). However, if the increased immobility of the experimental group were solely arising from their enhanced capacity for adaptation, it would likely be reflected in their overall WYM performance. The FST-2 was conducted with four days of enrichment, whereas the last WYM session was done after spending 10 days in the EE cage. It is therefore likely that most, if not all, of the immobility difference in the FST, reflects group-level differences in affective processing.

One limitation of the present study was the exclusive use of male rats. Laboratory rats of different strains, like humans, show sex differences in susceptibility to anxiety and different depressive symptoms like anhedonia (Unal & Moustafa, 2021). As for the majority of neurobiological research using rodent models, this technical constraint restricts the generalization of our findings. Another limitation was the lack of stress hormone measurements. Our design was confined to behavioral measures of stress and ex vivo assessment of recent neuronal activity using c-Fos immunohistochemistry.

Overall, we showed that a brief EE procedure was sufficient to produce a substantial anxiolytic effect following SI stress. Strikingly, it produced the opposite result in behavioral despair. SI to EE animals displayed significantly higher immobility in the test phase of the FST, while continuous SI had a relative antidepressant effect. The brief enrichment period was sufficient to accelerate spatial working memory performance in the early phase of the WYM. Significantly higher expression of the c-Fos protein in retrosplenial and perirhinal cortices of the SI to EE animals complemented this observation. As social isolation went beyond its use as a laboratory model and became a usual part of daily life with the COVID-19 pandemic (Unal, 2021), environmental enrichment attracted attention as a potential translatable paradigm against human isolation (Davim et al., 2020; Rojas-Carvajal et al., 2021). Our results revealed differential effects in depressive- and anxiety-like behavior of enrichment following relatively long-term isolation. This indicates that, unlike the cognitive effects, the afective consequences of switching from an impoverished to an enriched condition are not straightforward. An important question is whether these findings apply to humans who are exposed to relatively enriched conditions following long-term social isolation.

Acknowledgments The authors thank Aybeniz Ece Çetin for developing c-Fos immunohistochemistry, and Cem Sevinç, Ege Kingir, and Salih Çayır for cell counting analyses. This research was supported by grants from EMBO (Installation Grant to GU) and the Scientifc and Technological Research Council of Turkey (Project No: 121K260).

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