The Diferential Efects Of Brief Environmental Enrichment Following Social Isolation in Rats Part 1

Abstract

Environmental enrichment (EE) in rodents is associated with a wide range of physiological, affective, and cognitive benefits. A seemingly opposite housing condition, social isolation (SI), is used as a rodent model of stress, negatively affecting several neurobiological mechanisms and hampering cognitive performance. 

Cognition and memory are two very important aspects of our brain. Cognition refers to our ability to process information, including attention, thinking, reasoning, judgment, and problem-solving. Memory refers to the ability to save and retrieve information, including short-term storage, long-term storage, and recall.

However, many factors in life can interfere with our cognition and memory, such as anxiety, stress, lack of sleep, unhealthy diet, etc. Let's take a look at how these factors affect our cognition and memory.

Anxiety and Stress: When we are nervous, anxious, and stressed, hormones secreted by the brain can hinder our cognition and memory. These factors not only affect our ability to think and make decisions but also interfere with our concentration and memory.

Lack of sleep: Sleep is an important time for the brain to repair and recharge. When we lack sleep, the brain cannot get adequate rest, which can lead to cognitive and memory problems, such as forgetfulness and difficulty concentrating.

Unhealthy diet: Diet is important for our physical health, but it can also have an impact on our cognition and memory. Some nutritionally deficient dietary habits, such as excessive intake of sugar and saturated fat, can lead to cognitive and memory decline.

Fortunately, there are some positive behaviors we can do to boost our cognition and memory. like:

1. Exercise, which can help us reduce stress and anxiety and improve concentration and thinking skills.

2. Maintain good sleep habits and ensure adequate rest time every night, which can help our brains to fully repair and recharge.

3. Maintain a healthy diet, minimize sugar and saturated fat intake, and eat more brain-friendly foods such as fish and nuts.

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Experimental designs that involve switching between these housing conditions produced mixed results. We evaluated different behavioral and cognitive effects of brief EE following long-term, SI-induced stress. We revealed the influence of enrichment after 30 days of isolation on behavioral despair, anxiety-like behavior, and spatial working memory in adult male Wistar rats and found a substantial anxiolytic effect in the experimental (SI to EE) group. Interestingly, rats exposed to EE also showed increased behavioral despair compared with the control (continuous SI) group. 

There was no difference in spatial working memory performance at the end of a 5-day water Y-maze (WYM) test. However, the SI to EE animals displayed better memory performance in the first 2 days of the WYM, indicating faster learning. In line with this difference, we recorded significantly more c-Fos-immunopositive (c-Fos+) cells in the retrosplenial and perirhinal cortices of the SI to EE animals. 

The lateral and basolateral nuclei of the amygdala showed no such difference. These results suggest that brief enrichment following isolation stress leads to differential results in affective and cognitive systems.

Keywords 

Environmental enrichment · Social isolation · Forced swim test · Elevated plus maze · c-Fos · Spatial memory.

Introduction

Chronic stress may precipitate the development of several types of psychopathology, including depression, anxiety disorders, and posttraumatic stress disorder (Coyne, 1991; McEwen, 2004). 

Different types of stressors act on particular neurobiological pathways (Alleva & Santucci, 2001) and have differential effects at the neuronal, hormonal, and behavioral levels (Oishi et al., 2003; Pijlman et al., 2003). Evidence from human (Cacioppo & Hawkley, 2003) and other animal studies (Cacioppo et al., 2015; Filipović et al., 2017) demonstrate that psychosocial stressors lead to unique physiological and emotional outcomes, not observed with other types of stressors. 

Social isolation (SI), a phenomenon widely experienced during the COVID-19 pandemic (Unal, 2021), is a major source of psychosocial stress associated with health problems, including depression (House et al., 1988). To mimic human SI in animal models, rodents are exposed to individual housing with regular auditory and olfactory stimulation but limited visual and tactile input (Garzón & Del Río, 1981). 

Individually housed rodents demonstrate prolonged impairment in reward-seeking behavior and various cognitive tasks, often accompanied by increased levels of anxiety and depressive-like behavior (Carnevali et al., 2012; Nakayasu & Ishii, 2008; Von Frijtag et al., 2000). 

Long-term SI hinders hippocampal neurogenesis (Stranahan et al., 2006), causes autonomic and cardiac dysregulation (Grippo et al., 2007), and alters the neuroinflammatory response to stroke (Karelina et al., 2009).

Several forms of nonpharmacological treatment have been tested to block or reverse the consequences of long-term stress in rodents. Environmental enrichment (EE), put forward by Donald Hebb (1947), proved very successful in blocking the adverse effects of different forms of chronic stress, including SI. Pioneering studies in the 1960s transformed EE into a standard rodent behavioral paradigm (Rosenzweig, 1966; Rosenzweig et al., 1962), by demonstrating the effects of environmental stimuli on a variety of large-scale neurobiological parameters, such as the "total brain weight" or "total DNA or RNA content" in the brain (Bennett et al., 1969; Rosenzweig et al., 1967; Rosenzweig & Bennett, 1969). 

Subsequent work showed its ameliorative effects on neurodegenerative diseases, traumatic brain injury, neurodevelopmental disorders, and psychopathologies, such as schizophrenia, depression, and anxiety (Laviola et al., 2008; Nithianantharajah & Hannan, 2006; Renoir et al., 2013). EE applications produced consistent findings in reversing the neuronal (Biggio et al., 2019; Cao et al., 2018; Monteiro et al., 2014), physiological (Vitalo et al., 2012; Watanasriyakul et al., 2019), and cognitive (Lambert & Guillette, 2021) deficits of SI but led to mixed results in terms of its affective consequences.
Switching from SI to EE produced an antidepressant effect in rats (Brenes et al., 2020) and socially monogamous prairie voles (Grippo et al., 2014; Normann et al., 2021). For anxiety-like behavior, no anxiolytic effect was observed in rats following a switch from SI to EE (Mora-Gallegos & Fornaguera, 2019), unlike EE experiments that involve no change in living conditions (Peña et al., 2006). 

A more recent study found a significant anxiolytic effect in prairie voles when EE provided an opportunity for voluntary exercise (Normann et al., 2021). Another study applying an SI to EE switch, in contrast, observed that isolated mice had lower levels of anxiety compared with grouped-housed mice (Lopez & Laber, 2015). 

To obtain a comprehensive understanding of the effects of EE on long-term SI, we assessed behavioral despair, anxiety-like behavior, and spatial working memory in the same experimental design. We combine these behavioral results with immunohistochemistry for the c-Fos protein and reveal neuronal correlates of the EE procedure in different cortical and amygdaloid structures.

Isolation and enrichment studies often utilize behavioral despair and anhedonia-two psychiatric endophenotypes that recapitulate the pathology of depressive disorders (Carrier & Kabbaj, 2012; Djordjevic et al., 2012; Gould & Gottesman, 2006; Zlatković et al., 2014). 

Studies using group-housed animals as a control demonstrate that 21-day SI stress leads to behavioral despair, defined as increased immobility in the forced swim test (FST) (Unal & Canbeyli, 2019), as well as anhedonia, reflected as a decrease in sucrose preference. In contrast to SI, enrichment procedures offer therapeutic effects for both phenomena (Ashokan et al.,2018; Mitra & Sapolsky, 2009; Veena et al., 2009). 

The aforementioned work utilizing isolation-to-enrichment switch replicated these results, showing an antidepressant effect in the EE group (Brenes et al., 2020; Grippo et al., 2014; Normann et al., 2021).

Unlike rodent models of depressive behavior, measures of anxiety produced contradictory findings in research that involve either SI or EE. Several long-term EE studies reported anxiolytic effects (Brenes Sáenz et al., 2006; Harati et al., 2013; Leal-Galicia et al., 2007; Leal-Galicia et al., 2008; Peña et al., 2006), while some others found no performance change on the elevated plus maze (EPM) (Goes et al., 2015), or report an opposite, anxiogenic effect (Mann & Gervais, 2011). 

The effects of SI on anxiety-like behavior also produced mixed results (Butler, Carter, & Weiner, 2014b); some reported anxiolytic effects (Chappell et al., 2013; McCool & Chappell, 2009; Zhang et al., 2012), whereas others showed no influence of SI on arm preference in the EPM (Butler, Ariwodola, & Weiner, 2014a; Butler, Carter, et al., 2014; Simpson et al., 2012). 

This inconsistency persists for the few experiments involving an SI to EE switch. An anxiolytic effect was found in prairie voles (Normann et al., 2021), while no significant change was observed in rats (Mora-Gallegos & Fornaguera, 2019), and an anxiogenic effect was found in enriched mice (Lopez & Laber, 2015).

The cognitive effects of switching from SI to EE are straightforward compared with the affective consequences. 

Different types of SI impair spatial working memory in humans (Volkers & Scherder, 2011) and other animals (Fischer et al., 2012; Gregory & Szumlinski, 2008; Zorzo et al., 2019), especially when it is introduced after weaning (Kosten et al., 2012). Long-term EE, in contrast, improves several types of memory (Harati et al., 2011), as observed in the radial arm maze (Bell et al., 2009), the Hebb-Williams maze (Kobayashi et al., 2002), and the Morris water maze (Nilsson et al., 1999; Schrijver et al., 2002). 

As in isolation protocols, the emergence of cognitive effects following EE is time-dependent. Birch et al. (2013) showed that a 6-week continuous EE period, but not 3 weeks, was able to improve working memory. EE-led increase in memory performance was associated with long-term synaptic plasticity (Stein et al., 2016) and hippocampal neurogenesis (Nilsson et al., 1999).

The positive cognitive effects of EE may or may not emerge following a stress paradigm. Some studies demonstrated that short-term EE following chronic stress overcame the stress-induced deficits in spatial memory (Hutchinson et al., 2012; Veena et al., 2009), while others indicated that spatial memory performance following acute stress was not influenced by housing conditions (Del Arco et al., 2007; Garrido et al., 2013; Segovia et al., 2008). 

Resocialization in standard cages following SI was sufficient to overcome the cognitive defects triggered by this stress model (Chen et al., 2016). Switching aged rats from SI to EE for 3 months led to a better performance in complex, blind-alley mazes, whereas switching from standard or enriched cages to isolation impaired this performance (Winocur, 1998).

These studies altogether show that the affective and cognitive alterations observed by enrichment often require relatively long periods of differential housing. Brief or acute enrichment models were mostly utilized in the aforementioned sucrose consumption studies (Grimm et al., 2013; Grimm et al., 2018; Grimm et al., 2019; Slaker et al., 2016). 

No study has investigated the effects of brief EE following long-term isolation stress. Therefore, we tested whether a very brief, 3-day, EE manipulation could reverse the affective and cognitive alterations led by long-term, SI-induced stress in adult Wistar rats. 

We assessed behavioral despair in the FST, anxiety-like behavior in the EPM, locomotor activity in the open field test (OFT), and spatial working memory performance in a water Y-maze (WYM) task. We recorded and correlated c-Fos immunoreactivity in different memory-related cortical and amygdaloid structures.

Materials and methods

Subjects
Adult male Wistar rats (290-340 g, n = 16) were housed individually (21 ± 1 °C; ~50% humidity; 12:12 day/night cycle, lights on at 7:00 a.m.) in small SI cages (36.5 × 16.5 × 12.5 cm) for 30 days until being divided into experimental and control conditions based on their body weight and home cage (Fig. 1). 

Animals in the experimental group (n = 8) were placed together in a single, large EE cage (SI to EE), whereas control animals (n = 8) remained in their SI cages (continuous SI). Food and water were provided ad libitum for both groups throughout the experiment. All procedures were performed as approved by the Boğaziçi University Ethics Committee for the Use of Animals in Experiments.

Before the experiment, animals were housed in standard cages that contained four animals. The 16 animals in the study and the 8 animals in each group come from 4 different home cages.

Experimental design and environmental enrichment

After 30 days of SI, half of the rats were moved to the EE cage and remained there for a total of 10 days until perfusion-fixation (Fig. 1). The experimental testing started with the FST after 3 days of enrichment (of the SI to EE group) on Day 34 (Fig. 1). 

This was followed by the OFT, EPM, and WYM. Accordingly, behavioral despair analysis of the 2-day FST depends on 3-4 days of enrichment, whereas the results of the final test, the 5-day WYM reflect 5-10 days of differential housing (Fig. 1). Animals were perfused 24 h after they were returned to their cages following the last WYM trial (Fig. 1).

The EE procedure (Hebb, 1947; Krech et al., 1960) was implemented in a square (66 × 66 cm) transparent Plexiglas cage. It contained a running wheel, a small mirror, a nest box (10 × 10 × 10 cm), a ramp connecting to a platform (25 × 25 cm) 20 cm above the ground level, and six different plastic toys that were rearranged daily. As an additional enrichment procedure, animals were handled daily for approximately 2 min.

Forced swim test

The FST is a stress-inducing behavioral test developed to assess the efficacy of antidepressant agents and manipulations in rodents (Porsolt et al., 1977; Unal & Canbeyli, 2019). 

The stress response of the test, known as behavioral despair, was assessed over two consecutive days in an acrylic glass cylinder (diameter: 30 cm, height: 45 cm) filled with 30 cm water at 25 ± 0.5 °C. Following the standard procedure in rats, each animal was placed in the FST chamber for 15 min on the acclimatization/pretest day (FST-1) and for 5 min on the test day (FST-2) conducted after 24 h (Porsolt et al., 1977). 

Following FST-2, each animal was placed in a standard cage for drying for 30 min and then returned to its home cage. Each session was recorded with a video camera and coded by two observers blind to the experimental conditions. Periods of immobility (immobility scores) were averaged (interrater reliability: r = 0.99) and compared using an independent samples t-test.

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