Noradrenergic enhancement of object recognition and object location memory in mice

Abstract

Extensive evidence indicates that noradrenergic activation is essentially involved in mediating the enhancing effects of emotional arousal on memory consolidation. Our current understanding of the neurobiological mechanisms underlying the memory-modulatory effects of the noradrenergic system is primarily based on pharmacological studies in rats, employing targeted administration of noradrenergic drugs into specific brain regions. However, the further delineation of the specific neural circuitry involved would benefit from experimental tools that are currently more readily available in mice. Previous studies have not, as yet, investigated the effect of noradrenergic enhancement of memory in mice, which show different cognitive abilities and higher endogenous arousal levels induced by a training experience compared to rats. In the present study, we investigated the effect of posttraining noradrenergic activation in male C57BL/6J mice on the consolidation of object recognition and object location memory. We found that the noradrenergic stimulant yohimbine (0.3 or 1.0 mg/kg) administered systemically immediately after an object training experience dose-dependently enhanced 24-h memory of both the identity and location of the object. Thus, these findings indicate that noradrenergic activation also enhances memory consolidation processes in mice, paving the way for a systematic investigation of the neural circuitry underlying these emotional arousal effects on memory.

LAY SUMMARY: The current study successfully validated the effect of noradrenergic activation on both object recognition and object location memory in mice. This study thereby provides a fundamental proof-of-principle for the investigation of the neural circuitry underlying noradrenergic and arousal effects on long-term memory in mice.

Keywords:

• Yohimbine
• norepinephrine
• stress hormone
• memory consolidation
• object recognition
• object location

Introduction

Emotionally arousing training conditions enhance noradrenergic activity (McGaugh, 2004). Animal studies have provided extensive evidence that such noradrenergic activation, arising from catecholaminergic cell bodies in the locus coeruleus (LC), is crucially involved in strengthening the consolidation of long-term memory (McGaugh, 2004; Roozendaal & McGaugh, 2011; Sara, 2009; Takeuchi et al., 2016). Our current understanding of the neurobiological mechanisms underlying the memory-modulatory effects of the noradrenergic system is primarily based on pharmacological studies in rats, employing either systemic administration of noradrenergic drugs or targeted administration into specific brain regions. For example, norepinephrine or noradrenergic agents administered into the basolateral amygdala (BLA), or other brain regions such as the hippocampus or prefrontal cortex, were found to enhance long-term memory of emotionally arousing training experiences (Bevilaqua et al., 1997; Ferry & McGaugh, 1999; Hatfield & McGaugh, 1999; Introini-Collison et al., 1991; LaLumiere et al., 2003; Liang et al., 1990). Conversely, posttraining infusions of β-adrenoceptor antagonists into these brain regions were shown to impair retention and block the memory-enhancing effects of co-administered norepinephrine (Berman et al., 2000; Bevilaqua et al., 1997; Hatfield & McGaugh, 1999). Noteworthy, noradrenergic activation does not only enhance memory for highly arousing events that are known to induce the release of high levels of norepinephrine throughout the brain (Hatfield & McGaugh, 1999; McIntyre et al., 2002; Quirarte et al., 1998), but also for low-arousing experiences such as different forms of object recognition training (McReynolds et al., 2014; Roozendaal et al., 2008).

Human research supports the findings from animal studies that an activation of the noradrenergic system induces better memory (Cahill et al., 1994, 2003; O’Carroll et al., 1999; Southwick et al., 2002). Accumulating evidence from human neuroimaging studies, however, indicates that emotional arousal and noradrenergic activation are associated with widespread changes in functional connectivity and the activation of large-scale neural networks (Hermans et al., 2011; Hermans, Battaglia, et al., 2014; Hermans, Henckens, et al., 2014; Murty et al., 2010; Seeley et al., 2007). A recent neuroimaging study in mice indicated that direct chemogenetic stimulation of the LC induces a highly comparable large-scale reconfiguration of neural network activity (Zerbi et al., 2019). However, how such changes in network activity by norepinephrine could contribute to enhancement of memory for emotional experiences remains to be elucidated. A further delineation of this specific neural circuitry would benefit from novel experimental tools such as optogenetics and chemogenetics. Many laboratories are using mice for such circuitry-based investigations because of the availability of a wide variety of transgenic lines. However, previous studies have not investigated whether noradrenergic activation by exogenous drug administration further enhances memory in this species, which shows different cognitive abilities and higher endogenous arousal levels induced by a training experience compared to rats (Hok et al., 2016; Stepanichev et al., 2016).

In the present study, we investigated the effect of posttraining noradrenergic activation in male C57BL/6J mice on object recognition memory (ORM) and object location memory (OLM) (Leger et al., 2013; Roozendaal et al., 2010; Vogel-Ciernia & Wood, 2014). Several findings suggest that memory performance in these two tasks is supported by distinct neural substrates. (Balderas et al., 2008; Barker & Warburton, 2011), memory for the location on an object relies on the hippocampus (Balderas et al., 2008). Standard ORM and OLM have been successfully tested using mice (Vogel-Ciernia & Wood, 2014), but the effect of posttraining noradrenergic activation in mice on ORM and OLM has not yet been investigated. Here we found that, similar to rats, systemic posttraining injection of yohimbine, a noradrenergic stimulant which increases noradrenergic signaling (Nirogi et al., 2012; Szemeredi et al., 1991), induces dose-dependent enhancement of memory consolidation on both the ORM and OLM tasks. These findings thus pave the way for a systematic investigation of the neural circuitry underlying emotional arousal effects on memory.

Material and methods

Animals

One-hundred-and-five male CB57BL/6J mice (8 weeks old at the time of behavioral experiments) from Charles River Breeding Laboratories (Kisslegg, Germany) were group housed (3 animals per cage) in a temperature-controlled (22 °C) vivarium room with a regular 12-h/12-h light/dark cycle (lights on between 7:00 and 19:00 h). The vivarium room had a light intensity of 47 lux and humidity of 72%. Mice had ad libitum access to food and water. Object recognition memory differs between sexes (Sutcliffe et al., 2007), is modulated by stress exposure in a sex-specific manner (Coutellier & Würbel, 2009; Luine, 2002), and varies with the estrous cycle phase in females (e.g. Minni et al., 2014; Graham & Scott, 2018; Do Nascimento et al., 2019; Kirry et al., 2019). Since we aimed at replicating previous rat studies in our lab (e.g. Roozendaal et al., 2006; Barsegyan et al., 2014; Atucha et al., 2017; Chen et al., 2018), in which only male rats were used, we restricted our studies to male mice only. Training and testing was performed during the light phase of the cycle, between 10:00 and 15:00 h. All experimental procedures were in compliance with European Union Directive 2010/63/EU and approved by the Institutional Animal Care and Use Committee of Radboud University, Nijmegen, The Netherlands. All efforts were made to minimize animal suffering and to reduce the number of animals.

Experimental apparatus and behavioral procedures

The experimental apparatus used for both the ORM and OLM tasks was a gray open-field box (40 cm × 40 cm × 40 cm) with the floor covered with sawdust. One side of the box was marked with a line of white tape through the midline of the wall, serving as an internal cue. The objects to be discriminated were white glass light bulbs (6 cm diameter, 11 cm length) and transparent glass vials (5.5 cm diameter, 5 cm height), secured to the floor of the box with Velcro tape. The behavior of the animals was videotaped by a camera mounted above the box, which was connected to a laptop computer.

Mice were first handled for 1 min each for 3 consecutive days. Subsequently, the animals underwent a 5-min habituation procedure to the experimental box for another 3 days prior to training. Habituation to the box is required to guarantee sufficient exploration of the objects by the mice, necessary to form long-term ORM (Stefanko et al., 2009). During this habituation phase, mice could freely explore the training apparatus without the objects. Training and testing on the ORM and OLM tasks was according to Leger et al. (2013) and Vogel-Cierna and Wood (2014) with slight modifications. On the training trial, the mouse was placed in the experimental apparatus and allowed to explore two identical objects (A1 and A2), placed 5 cm away from the corners of the apparatus, for 3 min. Drug administration occurred immediately after the training trial, after which the animals were placed back into their home cages. To avoid the presence of olfactory trails, sawdust was stirred, feces were removed, and the objects were thoroughly cleaned with 70% ethanol in between trials. Retention was tested 1 h or 24 h after the training trial. For the ORM task, one exemplar of the familiar object (A3) and a novel object (B) were placed at the same locations as during the training trial (Figure 1(A)). For the OLM task, both objects were familiar (A3 and A4), yet one was placed at a novel location (Figure 2(A)). All combinations of locations and objects were used in a balanced manner to reduce potential biases due to preference for a particular location or object. For testing, the mouse was placed in the experimental apparatus and allowed to explore the objects for 5 min. Behavioral videos of the training and test sessions were analyzed offline by a trained observer blind to treatment condition, and the time spent exploring the novel and familiar object (or location) and the total time spent exploring both objects were scored. Part of the videos was analyzed by a second independent and blinded rater. Reliability of scoring was confirmed by high intra (r₍₄₂₎ = 0.804, p < .001) and inter-rater (r₍₄₂₎ = 0.670, p < .001) correlations in object exploration times. Object exploration was defined as actual active interaction with an object, i.e. pointing the nose to the object at a distance of <1 cm and/or touching it with the nose (Leger et al., 2013; Okuda et al., 2004). Turning around, climbing or sitting on an object per se was not included in exploration times as the animals then often are not actively engaged in exploring the object but rather exhibit grooming behavior or are using the object to scan the environment (Bianchi et al., 2006; Leger et al., 2013; Li et al., 2011; Pezze et al., 2017; Roozendaal et al., 2006; Vogel-Ciernia & Wood, 2014; Wimmer et al., 2012). In order to analyze memory performance, a discrimination index was calculated as the difference in time exploring the novel and familiar object (or location), expressed as the ratio of the total time spent exploring both objects (i.e. [(time novel − time familiar)/(time novel + time familiar)] × 100%). Since low object exploration during training might result in poor long-term memory unrelated to the drug condition, mice showing a total exploration time of <4 s on the training trial (n = 3) were removed from analyses. Furthermore, three mice showing a clear preference for one of the objects or locations during the training trial (defined as a discrimination index deviating more than two standard deviations from the mean) were removed (Leger et al., 2013; Vogel-Ciernia & Wood, 2014). Video analysis software (EthoVision XT, Noldus Information Technology, Wageningen, The Netherlands) was used to also measure total distance moved by the mice in the experimental apparatus during both training and retention testing.

Figure 1. Posttraining administration of the noradrenergic stimulant yohimbine dose-dependently enhances consolidation of object recognition memory. Data are shown as mean ± SEM. (A) Experimental design of the object recognition memory (ORM) task. Mice were trained for 3 min followed immediately by a subcutaneous injection of yohimbine (YOH, 0.3 or 1.0 mg/kg) or saline. Object recognition memory was tested 24 h later during which one of the objects was replaced by a novel object. (B) The higher dose of yohimbine improved memory performance on the object recognition retention test compared to saline-treated animals. (C) Yohimbine treatment did not affect total exploration time of the two objects during the retention test. (D) Yohimbine treatment did not affect the total distance moved during the retention test. saline: n = 14, YOH 0.3 mg/kg: n = 14, YOH 1.0 mg/kg: n = 13. ◆ p < .05, main effect of drug administration; ## p < .01, difference from saline; ** p < .01, difference from chance level.

Figure 2. Posttraining administration of the noradrenergic stimulant yohimbine dose-dependently enhances the consolidation of object location memory. Data are shown as mean ± SEM. (A) Experimental design of the object location memory (OLM) task. Mice were trained for 3 min followed immediately by a subcutaneous injection of yohimbine (YOH, 0.3 or 1.0 mg/kg) or saline. Object location memory was tested 24 h later during which one of the objects was relocated to a novel location. (B) Both the higher and lower dose of yohimbine improved memory performance on the object location retention test compared to saline. (C) Yohimbine treatment did not affect total exploration time of the two objects during the retention test. (D) Yohimbine treatment did not affect the total distance moved during the retention test. saline: n = 10, YOH 0.3 mg/kg: n = 8, YOH 1.0 mg/kg: n = 11. ◆◆ p < .01, main effect of drug administration; ## p < .01, difference from saline; ** p < .01,*** p < .001, difference from chance level.

Systemic drug administration

Yohimbine (17-hydroxyyohimban-16-carboxylic acid methyl ester hydrochloride; 0.3 or 1.0 mg/kg; Sigma-Aldrich), an α₂-adrenoceptor antagonist which increases noradrenergic activity (Szemeredi et al., 1991), was dissolved in saline and administered subcutaneously, in a volume of 0.1 mL/10 g of body weight, immediately after the training trial. The two doses were selected based on previous studies in rats (Roozendaal et al., 2006) and pilot data in mice (Supplementary Fig. 1). Drug solutions were freshly prepared before each experiment.

Statistics

Data are expressed as mean ± SEM. The discrimination index, total exploration time of the objects and total distance moved were analyzed with one-way ANOVAs with drug condition as between-subject variable. When appropriate, Tuckey post-hoc analyses were used to determine the source of significance in the ANOVA. One-sample t-tests were used to determine whether the discrimination index was different from zero (i.e. chance level) and thus whether learning had occurred. For all comparisons, p < 0.05 was accepted as statistical significance. The number of mice per group is indicated in the figure legends.

Results

Posttraining noradrenergic stimulation dose-dependently enhances object recognition memory

In this experiment, we first determined, in non-injected control mice, whether 3 min of object training was sufficient to induce successful acquisition of the identity of the training object in the ORM task. With these training conditions, we found that the discrimination index was significantly greater than zero at 1 h following training (M = 20.02, SEM = 6.14; t₍₁₁₎ = 3.26, p < .01), but not 24 h later (M = 3.64, SEM = 2.16; t₍₁₀₎ = 1.69, p = .12, Supplementary Fig. 2). Thus, these findings indicate that 3 min of object training is sufficient to induce short-term, but not long-term, memory.

Next, we investigated whether yohimbine (0.3 or 1.0 mg/kg) administered immediately after a 3-min training trial enhanced 24-h memory for the object in the ORM task. Total exploration time of the two identical objects (F_(2,38) = 0.85, p = .69, Table 1) or the total distance moved (F_(2,38) = 3.11, p = .06, Table 1) during the training trial did not differ between later drug treatment groups. During the 24-h retention test, the discrimination index showed a significant effect of yohimbine treatment (F_(2,38) = 3.95, p = .03, Figure 1(B)). Tukey’s post-hoc analysis revealed that mice treated with the higher dose of yohimbine (1.0 mg/kg) had a significantly greater discrimination index than that of the saline group (p < .05), whereas the discrimination index of mice treated with the lower dose of yohimbine (0.3 mg/kg) did not differ from that of saline-treated animals (p = .65). The discrimination index of both saline-treated mice (t₍₁₃₎ = 0.47, p = .65) and those treated with the lower dose of yohimbine (t₍₁₃₎ = 1.53; p = .15) was not significantly different from zero, indicating that a 3-min training trial was not sufficient to induce long-term memory of the training object in these groups. Mice treated with the higher dose of yohimbine, however, exhibited a significant exploration preference for the novel object (t₍₁₂₎ = 3.35; p < .01). Yohimbine treatment did not affect total exploration time of the two objects (F_(2,38) = 0.34, p = .54, Figure 1(C)) or total distance moved in the apparatus during the retention test (F_(2,38) = 0.65, p = .53, Figure 1(D)).

Table 1. Training data of object recognition memory (ORM) and object location memory (OLM).

Task	Training data items	Saline	YOH 0.3 mg/kg	YOH 1.0 mg/kg
ORM	Object exploration time (s)	10.9 ± 0.8	11.8 ± 0.9	12.5 ± 1.0
	Total distance moved (m)	6.4 ± 0.5	8.2 ± 0.7	8.3 ± 0.6
OLM	Object exploration time (s)	8.4 ± 0.8	11.7 ± 1.2	10.0 ± 1.0
	Total distance moved (m)	7.8 ± 0.4	8.3 ± 0.7	9.1 ± 0.5

Posttraining noradrenergic stimulation dose-dependently enhances object location memory

Next, we investigated, in separate groups of mice, whether posttraining systemic yohimbine (0.3 or 1.0 mg/kg) treatment also enhanced 24-h retention for the location of the object in the OLM task. Total exploration time of the two identical objects (F_(2,26) = 2.75, p = .08, Table 1) and the total distance moved (F_(2,26) = 1.80, p = .19, Table 1) during the training trial did not differ between later drug treatment groups. The discrimination index during the retention test, however, indicated a significant effect of yohimbine on memory performance (F_(2,26) = 8.52, p < .001, Figure 2(B)). Tukey’s post-hoc analysis revealed that the discrimination index of mice treated with either the 0.3 or 1.0 mg/kg dose of yohimbine was significantly greater than that of the saline group (p < .01). Whereas the discrimination index of saline-treated mice did not significantly differ from zero (t₍₉₎ = 0.67, p = .52), indicating that they did not express memory of the location of the training object, mice treated with either dose of yohimbine exhibited a significant exploration preference for the object located in the novel position (0.3 mg/kg: t₍₇₎ = 5.15; p < .01; 1.0 mg/kg: t₍₁₀₎ = 4.77; p < .001). Yohimbine treatment did not affect total exploration time of the two objects (F_(2,26) = 2.27, p = .12, Figure 2(C)) or total distance moved in the apparatus during the retention test (F_(2,26) = 1.49, p = .24, Figure 2(D)).

Discussion

The current study successfully validated the memory-enhancing effect of posttraining noradrenergic stimulation with systemic yohimbine in both the ORM and OLM tasks in mice. As such, this study provides a fundamental proof-of-principle for future investigation of the neural circuits underlying the effects of noradrenergic arousal on long-term memory in this species.

Similar to previous findings in rats (Dornelles et al., 2007; Jurado-Berbel et al., 2010; Nirogi et al., 2012), the main finding of the present study is that, in mice given 3 min of object training, noradrenergic activation immediately after the training trial induces dose-dependent enhancement of 24-h memory on both the ORM and OLM tasks. We found that 3 min of object training was insufficient to induce 24-h memory in saline-treated controls, but that such training conditions were sufficient to enable posttraining systemic yohimbine administration to enhance memory of both the identity and location of the object. Yohimbine is an α₂-adrenoceptor antagonist which, by blocking receptors located on noradrenergic terminals, elevates norepinephrine levels and its metabolites in the brain and in blood (Szemeredi et al., 1991). As the yohimbine was administered immediately after the training experience, the retention improvement effects cannot be attributed to memory-encoding effects or to nonspecific influences on attentional or locomotor effects during the training trial. Furthermore, the yohimbine treatment did not affect total exploration of the two objects or total distance moved during the retention test. These findings are thus consistent with the view that the noradrenergic stimulation enhances consolidation processes on both versions of the object memory task.

Extensive evidence from pharmacological manipulation studies in rats indicates that (nor)adrenergic agonists administered systemically or directly into specific brain regions enhance memory consolidation on a wide variety of emotionally arousing training tasks, including inhibitory avoidance, active avoidance discrimination learning, contextual fear conditioning, water-maze spatial learning and appetitive tasks (Bevilaqua et al., 1997; Costa-Miserachs et al., 1994; Ferry & McGaugh, 1999; Gold & Van Buskirk, 1975; Hatfield & McGaugh, 1999; Introini-Collison et al., 1991; Introini-Collison & McGaugh, 1986; Izquierdo & Dias, 1985; LaLumiere et al., 2003; Liang et al., 1986, 1990; Sternberg et al., 1985). Noradrenergic activation also enhances recognition memory in rats (Dornelles et al., 2007; Jurado-Berbel et al., 2010). In the experiments by Dornelles et al. (2007), the adrenomedullary hormone epinephrine injected systemically immediately after the training session increased the retention delay at which memory was still present (Dornelles et al., 2007). Another study confirmed the finding that posttraining systemic epinephrine administration improves long-term memory on both the ORM and OLM tasks (Jurado-Berbel et al., 2010). Other studies support the view that posttraining systemic yohimbine administration increases norepinephrine levels in the medial temporal lobe (Nirogi et al., 2012), and enhances memory on the ORM task (Nirogi et al., 2012; Roozendaal et al., 2006). Norepinephrine administration directly into the BLA also enhances the consolidation of ORM as well as of the association of an object with its context (Barsegyan et al., 2014; Roozendaal et al., 2008). With such targeted pharmacological manipulation studies in rats, considerable knowledge has been gained regarding the neural mechanisms by which norepinephrine facilitates long-term memory formation, particularly by its actions on the BLA (McGaugh, 2000, 2004; Roozendaal & McGaugh, 2011), subsequently modulating neural plasticity and information storage processes in its projection regions, including the hippocampus, perirhinal cortex, medial prefrontal cortex and insular cortex (Barsegyan et al., 2019; Beldjoud et al., 2015; Chen et al., 2018; Laing & Bashir, 2014; McIntyre et al., 2005; McReynolds et al., 2014; Roozendaal et al., 1999).

Neuroimaging studies in humans, however, have indicated that emotional arousal triggers dynamic shifts in network balance throughout the brain, leading to a large-scale neural network reconfiguration (Murty et al., 2010; Seeley et al., 2007). Moreover, exposure to emotional arousal induces complex temporal dynamics in neural activity. Emotional arousal, in a norepinephrine-dependent fashion, first rapidly increases salience network activity, while simultaneously suppressing central executive network activity (Hermans et al., 2011; Seeley et al., 2007). Later, when the arousing situation subsides, resource allocation to these two networks reverses: the salience network shuts off and the central executive network becomes active, which normalizes emotional reactivity and enhances higher-order cognitive processes (Hermans, Henckens, et al., 2014; Van Leeuwen et al., 2018). Studies have shown that the LC noradrenergic system has the ability to rapidly rearrange neural activity within and between large-scale neural systems to optimize cognitive processes relevant for task performance or adaptive behaviors (Aston-Jones & Cohen, 2005; Bullmore & Sporns, 2009; Van Den Heuvel & Pol, 2010; Zerbi et al., 2019). However, it remains unknown how noradrenergic activation by emotional arousal might achieve both spatial and temporal specificity in regulating large-scale neural network activity. Such effects might depend on brain region- and time-specific effects of norepinephrine on excitatory and inhibitory subpopulations of neurons. Further, it is poorly understood how such changes in network activity by norepinephrine could contribute to enhancement of memory for emotional experiences.

Dedicated studies allowing for tight experimental control over neuronal subpopulations and neural circuit activity are required to elucidate these exact mechanistic underpinnings. New technologies such as optogenetics and chemogenetics could be optimally combined with the use of a variety of readily available transgenic lines of mice, to decipher these mechanisms. Validated behavioral tasks and effects of norepinephrine are a prerequisite to conduct such studies. The present findings indicating that noradrenergic activation enhances memory for ORM and OLM in mice, pave the way for a further investigation of the specific neural circuits and molecular mechanisms that regulate emotional arousal effects on memory consolidation.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported by NWO-ORA grant 464.18.110 to BR; and QS was supported by the China Scholarship Council (201606380109).