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Acute stress induces cognitive improvement in the novel object recognition task by transiently modulating Bdnf
in the prefrontal cortex of male rats
Paola Brivio; Giulia Sbrini; Marco Andrea Riva and Francesca Calabrese
Department of Pharmacological and Biomolecular Sciences, Università degli Studi di Milano, Milan, Italy
Keywords:
Stress; Bdnf; NOR, memory
*Corresponding author: Dr. Francesca Calabrese
Department of Pharmacological and Biomolecular Sciences
Università di Milano, Via Balzaretti 9, 20133 Milan, Italy
Phone: +39-02 50318277; Fax: +39-02 50318278
E-mail: francesca.calabrese@unimi.it
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Abstract
Stress response involves several mechanisms and mediators that allow individuals to adapt to a changing environment.
The effects of stress may be adaptive or maladaptive, based on the timing and intensity of exposure as well as on the
individual vulnerability. In particular, exposure to mild and brief stressors provides beneficial advantages in a short-
term period, by activating protective functions to react with the external demands.
On these bases, the purpose of our study was to establish the time-dependent effects of acute stress exposure on
neuroplastic mechanisms in adult male rats. Moreover, we aim at establishing the consequences of the acute challenge
on memory processes by testing rats in the Novel Object Recognition (NOR) test. We found that acute restraint stress
up-regulated total Bdnf expression 1h post stress specifically in rat prefrontal cortex, an effect that was sustained by the
increase of Bdnf isoform IV as well as by the pool of Bdnf transcripts with long 3’UTR. Furthermore, in the same brain
region, the acute stress modulated in a time specific manner the expression of different activity-dependent genes,
namely Arc, Gadd45
b
and Nr4a1. At behavioral level, the challenge was able to improve the performance in the NOR
test specifically 1h post stress, an effect that positively correlated with the expression of the neurotrophic factors.
Taken together, our results suggest that a single session of acute stress enhances memory and learning functions with a
specific temporal profile, by improving neuroplastic mechanisms within the prefrontal cortex.
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Introduction
Stress response involves several mechanisms and mediators that allow individuals to adapt to a changing environment.
The effects of stress may be adaptive or maladaptive, with severe and prolonged stress underlying the development of a
pathological status, whereas mild and short stress increasing the adaptive ability of the subject to cope with life stress
events (McEwen 2007; McEwen et al. 2015).
In the field of psychiatric disorders, great attention has been paid in exploring the behavioral and molecular alterations
driven by chronic stress exposure, with the aim to clarify the causes of their development and to assess the action of a
pharmacological intervention for the identification of novel potential targets (Willner 2005; Pochwat et al. 2014; Luoni
et al. 2015; Calabrese et al. 2016, 2017; Molteni et al. 2016; Rossetti et al. 2016, 2018; Yin et al. 2016). However, few
attempts have been done at investigating the impact of acute stress in driving both positive and negative responses as a
function of its duration and severity, thus leading to a different susceptibility and vulnerability to the challenging
condition. Furthermore, stressful events have severe consequences on learning and memory and their extent has
different effects at cognitive level, with impairment and improvement in the performance, respectively driven by
repetitive and single stressors. Interestingly, learning under stress may facilitate memory functions (Schwabe 2017),
through the involvement of several molecular pathways (Sandi and Pinelo-Nava 2007).
In this context, the neurotrophin Brain Derived Neurotrophic Factor (Bdnf), which plays a pivotal role in mechanisms
of neural and synaptic plasticity (Calabrese et al. 2009; Kowiański et al. 2018), is known to be modulated by the
exposure to adverse life events. Furthermore it has been shown that Bdnf is a molecular mediator of the therapeutic
activity of antidepressant drugs (Duman and Monteggia 2006; Martinowich et al. 2007).
Accordingly, we showed that stress may exert opposing action on Bdnf, with chronic stress decreasing its levels (Luoni
et al. 2015), whereas acute stress enhancing its expression with an anatomical specificity (Molteni et al. 2009;
Fumagalli et al. 2012; Brivio et al. 2018, 2019).
Besides its role in response to stress events, Bdnf participates in long-term potentiation and it is involved in the
modulation of different stages of memory processes, including acquisition, short and long term memory formation and
consolidation, retrieval, extinction and reconsolidation (Bekinschtein et al. 2014; Lu et al. 2015).
On this basis, here we investigated the temporal profile of the effect exerted by 1h of restraint stress on the expression
of total Bdnf in the rat brain. To evaluate whether the modulation of Bdnf was sustained by its major transcripts, we
focused on the Bdnf long 3’UTR pool of transcripts and on Bdnf isoform IV that are the most abundant and well
characterized transcripts in the brain areas primarily affected by stress. Bdnf long 3’UTR transcripts are targeted to
distal dendrites, thereby contributing to synaptic activity as well as to long term potentiation (Lau et al. 2010; Allen et
al. 2013), whereas isoform IV displays a strictly somatic localization and, as an activity-dependent gene, it is modulated
by several upstream stimulatory factors (Pruunsild et al. 2011). The molecular analyses were conducted in brain areas
profoundly affected by stress and regions critical also for working memory and executive functions (Stuss and Knight
2009; Kim et al. 2015), such as the prefrontal cortex (PFC) and the hippocampus (dorsal (dHip) and ventral (vHip)).
Moreover, to assess whether acute stress could alter memory functions, we investigated its impact on cognitive
performance by exposing the animals to the novel object recognition test (NOR) 1, 4 and 24h after the restraint. We also
established the contribution of the neurotrophin in the beneficial advantages caused by the challenge exposure in the
cognitive task.
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Methods
Animals
Adult male (post-natal day 90) Sprague-Dawley rats (Charles River, Italy) were brought into the laboratory two weeks
before the start of the experiment. Rats were housed with food and water ad libitum and were maintained on a 12-h
light/dark cycle and in a constant temperature (22 ± 2
o
C) and humidity (50 ± 5%) conditions. All procedures used in
this study are conformed to the rules and principles of the 2010/63/UE Directive, according to the authorizations from
the Health Ministry. All efforts were made to minimize animal suffering and to reduce the number of animals used.
Experimental paradigm and groups:
After two weeks, rats were randomly assigned to the following experimental groups: no stress (n=10), stress(1h)1h
(n=6), stress(1h)4h (n=5), stress(1h)24h (n=5), no stress-NOR5’(n=6), no stress-NOR(n=6), stress(1h)1h-NOR5’(n=6),
stress(1h)1h-NOR (n=5), stress(1h)4h-NOR (n=6), stress (1h)24h-NOR (n=7). Animals were stressed for 1h and
sacrificed 1, 4 or 24h after the acute challenge, except for the animals of the no stress groups that were left undisturbed
in their home cages. Moreover, half of both stressed and non-stressed animals were exposed to the novel object
recognition (NOR) test 1, 4 or 24 h post-stress. During the NOR test, part of the animals was sacrificed after the first
five minutes of test (NOR5’), whereas the others were killed at the end of the cognitive task (NOR).
Stress procedure
Rats were exposed to 1h of acute restraint stress, in an air-assessable cylinders (diameter 8,25 cm; length 20,32;
Stereoglass Srl, Italy) (the size of the device was similar to the size of the animal, which made the animal almost
immobile in the container) and sacrificed 1, 4 or 24 h after the acute challenge (fig. 1A).
Novel object recognition test
After 10 minutes of habituation in the open field in the two previous days, animals were allowed to explore two
identical objects (white cylinders, 7 cm in diameter, 11 cm high) in an open field (50cm x 50cm x 40cm) for five
minutes (trial session-encoding phase). After 1h in the home cage (consolidation phase), the retention trial (testing
session- retrieval phase) was conducted and one of the objects presented previously was replaced by a novel object
(black prism, 5 cm wide, 14 cm high) (fig. 4A). During the 5-minute test, the duration of exploration of each object (ie.
sitting in close proximity to the objects, sniffing or touching them) was measured. The task was performed in an
isolated room under dim light conditions, in the absence of a direct overhead lighting. The NOR discrimination index
was calculated according to the following formula: time of novel object exploration minus time of familiar object
exploration divided by time of novel plus familiar object exploration, multiplied by 100. The rats exposed to the
cognitive test were sacrificed immediately at the end of the encoding or retention phase.
Brain tissues collection:
PFC and both dHip and vHip were dissected, frozen on dry ice and stored - 80°C for later analyses. Specifically, the
PFC (defined as Cg1, Cg3 and IL subregions corresponding to the plates 6-10 according to the atlas of Paxinos and
Watson (Paxinos and Watson 1986) was dissected from 2-mm-thick slices, whereas the dHip and vHip (respectively
plates 25-33 and plates 34-43 according to the atlas of Paxinos and Watson) were dissected from the whole brain.
RNA preparation and gene expression analysis by quantitative Real-time PCR.
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Total RNA was isolated by a single step of guanidinium isothiocyanate/phenol extraction using PureZol RNA isolation
reagent (Bio-Rad Laboratories, Italy) according to the manufacturer’s instructions and quantified by spectrophotometric
analysis. The RNA concentrations were measured by spectrophotometry (OD260/280 1.8<ratio<2). Following total
RNA extraction, the samples were processed for real-time polymerase chain reaction (RT-PCR) to assess total Bdnf,
Bdnf long 3’UTR, Bdnf isoform IV, Arc, Growth Arrest and DNA Damage-inducible protein (Gadd45
b
and Nuclear
Receptor Subfamily 4 Group A Member 1 (Nr4a1). An aliquot of each sample was treated with DNase to avoid DNA
contamination. RNA was analyzed by TaqMan qRT PCR instrument (CFX384 real time system, Bio-Rad Laboratories,
Italy) using the iScriptTM one-step RT-PCR kit for probes (Bio-Rad Laboratories, Italy). Samples (10ng/ul) were run in
384 well formats in triplicate as multiplexed reactions with a normalizing internal control (36B4). Primers sequences
(Table 1A/B) used were purchased from Eurofins MWG-Operon and Life Technologies.
Thermal cycling was initiated with an incubation at 50°C for 10 min (RNA retrotranscription) and then at 95°C for 5
min (TaqMan polymerase activation). After this initial step, 39 cycles of PCR were performed. Each PCR cycle
consisted of heating the samples at 95°C for 10 s to enable the melting process and then for 30 s at 60°C for the
annealing and extension reactions. A comparative cycle threshold method was used to calculate the relative target gene
expression by applying the 2
-∆(∆CT)
method (Livak and Schmittgen 2001).
Statistical analysis
All the analyses were conducted by using “IBM SPSS Statistics, version 24”.
The behavioural data were analyzed with the one-way analysis of variance (ANOVA). When appropriate, further
differences were analyzed by Fisher’s Protected Least Significant Difference (PLSD). Molecular results were analyzed
with the one-way (ANOVA) or two-way ANOVA, followed by PLSD. In addition, to evaluate the association between
the cognitive performance and the alteration of gene expression, Pearson correlation coefficients (r) were conducted
between NOR discrimination index of single animals and the corresponding mRNA levels. Significance for all tests was
assumed for p<0.05. Data are presented as means standard error (SEM). For graphic clarity, results are presented as
mean percent of No stress.
