As previously mentioned, olfactory sensitivity cannot be dissociated from olfactory memory, and some data suggest that the indirect effects of fasting and OXA on the olfactory memory trace formation through increased olfactory sensitivity may involve the olfactory bulb (OB). As previously mentioned, olfactory sensitivity cannot be dissociated from olfactory memory, and some data suggest that the indirect effects of fasting and OXA on the olfactory memory trace formation through increased olfactory sensitivity may involve the OB. Firstly, in addition to its well documented role in detection and discrimination, the OB is involved in memory processes underlying various kinds of olfactory learning in adult rats (see Mandairon and Linster, 2009 for review). Moreover, the OB receives direct OXA innervation from the LH (de Lecea et al., 1998; Peyron et al., 1998; Sakurai et al., 1998; Shibata et al., 2008) and an increase in OB electrophysiological response induced by fasting and OXA has been described (Pager et al., 1972; Gervais and Pager, 1982; Apelbaum and Chaput, 2003; Apelbaum et al., 2005; Hardy et al., 2005). In addition, Prud'homme et al. (2009) found that OXA antagonist treatment blocked the enhancement of OB Fos responses to a food odor. Secondly, some evidence suggests that the OXA neurons terminating in the locus coeruleus (LC) may provide a second indirect pathway for orexinergic modulation of olfactory processing: direct OXA fibers innervate the LC (Horvath et al., 1999) and activation of OXA receptors in the LC increases cell firing of intrinsic noradrenergic (NA) neurons (Trivedi et al., 1998; Hagan et al., 1999). In addition, the LC projects over 40% of its neurons directly into the OB (McLean et al., 1989) and this large NA input has been shown to modulate OB excitability, olfactory perception and olfactory learning and memory abilities (see Devore and Linster, 2012 for review).

These data suggest that the fasting-induced increase in olfactory sensitivity observed in the present and other studies probably involved the OXA system in the OB. Moreover, and in the light of the work by Escanilla et al. (2012), it may be suggested that this increased olfactory sensitivity resulted directly from OXA system activation (Hardy et al., 2005) in the OB and/or indirectly through the effect of LC-OXA system activation on NA release in the OB. Finally, it is possible that activation of both systems may simultaneously influence the strength of olfactory memory through enhanced olfactory processing. In order to test this hypothesis, future studies will aim at verifying whether the presentation of a new olfactory stimulus in fasted animals can be correlated to changes in NA release in the OB.

#### **Amygdala and locus coeruleus (LC)**

Orexinergic innervation of the extended amygdala (including the basolateral amygdala, BLA) was clearly described by Schmitt et al. (2012). OXA administered into the LH significantly elevated cFosimmunoreactivity in the amygdala (Mullett et al., 2000) and fasting increased OXA mRNA levels in the amygdala (Lu et al., 2000). OXA applied in acute rat brain slices activated neurons in the amygdala (Bisetti et al., 2006). Moreover, the amygdala receives OB and visceral inputs (Saper and Loewy, 1980; Inui et al., 2006), and may be a nodal point at which olfactory and neuroendocrine stimuli are integrated to modulate feeding behavior (King, 2006).

Otherwise, a large amount of data indicates that the amygdala, and more precisely the BLA, is involved in the acquisition of COA (Sevelinges et al., 2009) and more precisely in the processes underlying the formation of the olfactory memory trace and its maintenance across the ISI during COA (Ferry et al., 1995; Ferry and Di Scala, 1997, 2000). Although a direct effect of starvation-induced OXA release in the amygdala on COA cannot be excluded, to our knowledge, the involvement of the OXA system in the amygdala has never been demonstrated in learning and memory.

On the other hand, some data indicate that the enhancing effect of starvation on COA memory processes could be indirectly mediated by activation of the NA system in the amygdala. The LC projects strongly onto the amygdala (Fallon et al., 1978) and the BLA β-adrenergic system is involved in the memory processes underlying the association between odor and delayed US during COA (Miranda et al., 2007). Given the direct action of LH orexinergic neurons on the LC (Horvath et al., 1999; van den Pol et al., 2002), activation of the LC-amygdala NA system during processing of the new odor CS may be potentiated by fastinginduced OXA release. Possibly, the strength of the olfactory memory trace, and/or its association to the US, was influenced by activation of this pathway in the Fasted and OXA groups.

Although the results shown on **Figure 1** do not identify particular structures receiving OXA projections as being involved in the memory processes underlying COA acquisition, all the above-mentioned data lead us to propose a model according to which OXA release during fasting enhances learning performance through a direct or indirect influence on the circuit involved in COA. Our model shown on **Figure 2** comprises some of the structures involved in this circuit including the LC, amygdala and OB, which receive LH-OXA projections. According to this model, the activation of the LH-OXA system induced by fasting reinforces the role of each structure in the circuit by enhancing the neural processes underlying attention and olfactory memory through direct and/or indirect influences.

This model is consistent with the idea of Cleland and Linster (2005) according to which the multiple feedback and feed-forward interactions between olfactory and non-olfactory areas contribute to complex processes, such as filtering and

constructing olfactory representations, and compare these representations to those previously acquired in order to cue an appropriate response to relevant stimuli. The present model also supports the hypothesis that centrifugal modulatory inputs influence olfactory processing and learning mechanisms within the OB (Sullivan et al., 2000; Linster and Cleland, 2002; Yuan et al., 2003), leading us to extend these influences to other key structures involved in attention and olfactory learning and memory (Aston-Jones and Cohen, 2005; Miranda, 2012).

Of course the list of structures included in this model is not exhaustive; involvement of other feedback and feed-forward interactions between these structures and others (e.g., piriform cortex, entorhinal cortex, orbitofrontal cortex, hippocampus, etc.) will have to be considered in order to achieve a more realistic idea of the circuit actually involved in food conditioned learning (see Ferry et al., 2006, 2007; Chapuis et al., 2009; Sahay et al., 2011; Wilson and Sullivan, 2011; Chapuis et al., 2013).

#### **CONCLUSION**

Feeding behavior is part of a complex integrated adaptive system, governed by the brain, in which the processing of metabolic signals reflecting the animal's nutritional state (gastrointestinal distention, blood glucose, feeding peptides such as OXA, etc.) and of olfactory signals indicative of food determines the appropriate response to a food source. However, the differentiation between palatable and unpalatable items that conditions ingestive behavior often depends on previous experience during which the odor of the food acquired (or did not, in the case of a new odor) a hedonic valence after feeding, through CS-US associative learning. By showing that OXA system influenced the processes underlying the CS-US association, or/and consolidation of this association during COA, the present study introduces a new mechanism by which the LH-OXA system may influence the processes that enable animals to learn to select food available in the environment and to adapt their behavior to previous experience through a modulation of complex neural circuit activity. Finally, the OXA system represents a critical link between peripheral energy balance and CNS mechanisms that coordinate olfactory processing and memory, especially in the physiological state of fasting.

#### **ACKNOWLEDGMENTS**

This work was supported by the LABEX CORTEX (ANR-11-LABX-0042) of Université de Lyon, within the program "Investissements d'Avenir" (ANR-11-IDEX-0007) operated by the French National Research Agency (ANR).

#### **REFERENCES**

responses to isoamyl acetate or food odour in rats: roles of orexins and leptin. *Neuroscience* 162, 1287–1298. doi: 10.1016/j.neuroscience.2009.05.043

Yuan, Q., Harley, C. W., and McLean, J. H. (2003). Mitral cell beta1 and 5-HT2A receptor colocalization and cAMP coregulation: a new model of norepinephrine-induced learning in the olfactory bulb. *Learn. Mem.* 10, 5–15. doi: 10.1101/lm.54803

**Conflict of Interest Statement:** The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 05 February 2014; accepted: 18 April 2014; published online: 06 May 2014*.

*Citation: Ferry B (2014) The orexinergic system influences conditioned odor aversion learning in the rat: a theory on the processes and hypothesis on the circuit involved. Front. Behav. Neurosci. 8:164. doi: 10.3389/fnbeh.2014.00164*

*This article was submitted to the journal Frontiers in Behavioral Neuroscience*.

*Copyright © 2014 Ferry. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms*.

## Olfactory tubercle stimulation alters odor preference behavior and recruits forebrain reward and motivational centers

#### **Brynn J. FitzGerald1 , Kara Richardson1 and Daniel W. Wesson1,2\***

1 Department of Neurosciences, Case Western Reserve University, Cleveland, OH, USA

2 Department of Biology, Case Western Reserve University, Cleveland, OH, USA

#### **Edited by:**

Anne-Marie Mouly, Centre de Recherche en Neurosciences de Lyon, France

#### **Reviewed by:**

Carmen Agustín-Pavón, Imperial College London, UK Satoshi Ikemoto, National Institutes of Health, USA

#### **\*Correspondence:**

Daniel W. Wesson, Department of Neurosciences, Case Western Reserve University, 2109 Adelbert Avenue, Cleveland, OH 44106, USA e-mail<EMAIL_ADDRESS>
Rodents show robust behavioral responses to odors, including strong preferences or aversions for certain odors. The neural mechanisms underlying the effects of odors on these behaviors in animals are not well understood. Here, we provide an initial proof-of-concept study into the role of the olfactory tubercle (OT), a structure with known anatomical connectivity with both brain reward and olfactory structures, in regulating odor-motivated behaviors. We implanted c57bl/6 male mice with an ipsilateral bipolar electrode into the OT to administer electric current and thereby yield gross activation of the OT. We confirmed that electrical stimulation of the OT was rewarding, with mice frequently self-administering stimulation on a fixed ratio schedule. In a separate experiment, mice were presented with either fox urine or peanut odors in a three-chamber preference test. In absence of OT stimulation, significant preference for the peanut odor chamber was observed which was abolished in the presence of OT stimulation. Perhaps providing a foundation for this modulation in behavior, we found that OT stimulation significantly increased the number of c-Fos positive neurons in not only the OT, but also in forebrain structures essential to motivated behaviors, including the nucleus accumbens and lateral septum. The present results support the notion that the OT is integral to the display of motivated behavior and possesses the capacity to modulate odor hedonics either by directly altering odor processing or perhaps by indirect actions on brain reward and motivation structures.

**Keywords: olfaction, olfactory cortex, self-stimulation, ventral striatum**

### **INTRODUCTION**

Odors have long been known to possess degrees of attractiveness or aversion (Locke and Grimm, 1949). These hedonics can either be innate from birth or in other cases, conditioned through learning. In both cases, perception of odors on the ends of the hedonic spectrum may elicit robust behavioral reactions. For instance, the bold odor of decaying meat elicits quite a repulsive reaction in humans whereas contrastingly, the sweet smell of freshly baked bread is in most cases pleasant. The neural mechanisms underlying these hedonic-driven behavioral responses are becoming increasingly known (e.g., Sullivan and Leon, 1987; Mennella and Garcia, 2000; Rolls et al., 2003; Stevenson and Repacholi, 2003; Sullivan, 2003; Bensafi et al., 2007; Grabenhorst et al., 2007; Baum, 2009; Doucette et al., 2011; Ferrero et al., 2011; Bensafi et al., 2012; Kass et al., 2013), yet major questions still remain.

Rodents are an excellent model for studying the neurobiological mechanisms of odor hedonics. From birth, and during early postnatal life, rats and mice display robust behavioral responses to odors, especially maternal odors to aid in maternal localization and feeding (Blass and Teicher, 1980; Sullivan, 2003; Logan et al., 2012). Most commonly studied in adult rodents, fearful responses (aversion, freezing/immobility, threat assessment) are reliably observed in response to predator odors (Blanchard et al., 2001; Wallace and Rosen, 2001; Takahashi et al., 2005; Ferrero et al., 2011). Thus rodents must possess a highly sophisticated system for the detection and response to odors.

The control of odor hedonic-driven behaviors likely requires not only a fully functional olfactory system to detect and discriminate the stimulus over background stimuli (for review see Wilson and Mainen, 2006; Wilson and Sullivan, 2011), but also the relay of this information into emotional and reward-related brain structures. The olfactory tubercle (OT) is an olfactory structure residing in the ventral striatum with large amounts of known anatomical connectivity into brain reward structures (Ikemoto, 2007; Wesson and Wilson, 2011). Due to this, we previously predicted that the OT serves a major role in regulating odor hedonics (Wesson and Wilson, 2011). Possible evidence for the regulation of rodent behavioral responses to odors by the OT was provided in a recent study by Agustín-Pavón et al. (2014). In the mentioned study, the authors created lesions containing a portion of the OT and observed that female mice with lesions displayed less attraction to male odors (Agustín-Pavón et al., 2014). This finding raises the interesting possibility that the OT regulates odor-hedonic behaviors either by means of its intrinsic rewarding properties (e.g., Prado-Alcalá and Wise, 1984; Ikemoto, 2003) and/or connectivity with reward and motivated behavior centers.

In the present study, we sought to further explore the role of the OT in odor-guided behaviors and the brain reward system in mice. To manipulate OT activity, we employed focal micro-stimulation of the OT using bipolar electrodes—a wellestablished method to probe principles of both olfactory and reward system activity (Freeman, 1960; Phillips and Mogenson, 1969; Prado-Alcalá and Wise, 1984; Mouly et al., 1985; Mouly and Holley, 1986; Wilson and Sullivan, 1990; Carlezon and Chartoff, 2007). As predicted based upon previous results in rats (Prado-Alcalá and Wise, 1984; Ikemoto, 2003), we found that mice selfadministered current stimulation into the OT. Further, persistent automatic stimulation of the OT altered behavior in a threechamber preference test. Finally, in separate groups of mice, we explored the recruitment of brain reward centers using the immediate early gene *c-Fos*. We found that OT stimulation not only recruited OT neurons focally, but also those of structures known connected to the OT—providing initial mechanistic insights into the likely importance of the OT to odor hedonics.

## **MATERIALS AND METHODS**

### **EXPERIMENTAL SUBJECTS**

Adult male c57bl/6 mice (2–4 months of age), bred and maintained within the Case Western Reserve University School of Medicine animal facility were used. Food and water were available *ad libitum* except during behavioral testing. All experiments were conducted in accordance with the guidelines of the National Institutes of Health and were approved by the Case Western Reserve University's Institutional Animal Care Committee.

#### **CHRONIC STIMULATING ELECTRODE IMPLANTATION SURGERY**

A first cohort of mice (*n* = 8) were initially anesthetized with Isoflurane anesthesia (3.5–3% in 1 L/min O2) before being transferred and mounted into a stereotaxic frame where Isoflurane was further provided (3–1%). Core body temperature was maintained at 38◦C with a hot water-filled heating pad. Upon confirmation of anesthesia depth, the head was shaved, cleaned with betadine and 70% EtOH, and a single injection of lidocaine (0.1 ml of 1% in H2O, S.C.) was administered within the future wound margin. A single incision was made from ∼3 mm posterior of the nose along the midline to lambda and the skull surface cleaned with 3% H2O2. A single craniotomy (1 mm diameter) was created on the skull overlying the site of the OT for implantation of the stimulating electrode. The electrode consisted of 240 µm diameter stainless steel insulated wires (A-M Systems, Carlsborg, WA, USA) twisted together and connected by silver epoxy onto an Omnetics micro PS1 connector (Minneapolis, MN, USA). A micromanipulator was used to lower the bipolar electrode into the craniotomy and further into the site of the OT. The electrodes and plug were then cemented onto the skull by means of dental cement and the wound closed with Vetbond (3 M; St. Paul, MN, USA). Rimadyl (Carprofen, Pfizer animal health, 5 mg/kg, S.C.) was administered immediately following surgery and animals allowed to recover on the heating pad for >4 h. Rimadyl was administered daily for 5 days post-op. Food and water were available *ad libitum* except during behavioral procedures. All animals were singly-housed starting the day of implantation on a 12:12 h (light:dark) schedule with all behavioral procedures occurring during the light phase of the cycle (12:00:18:00 h). At least 5 days of recovery from surgery was allowed prior to any behavioral procedures. Following all behavioral procedures, mice were overdosed with urethane (3 mg/kg, I.P.) and transcardially perfused with 10 ml of 0.9% NaCl followed by 15 ml of 10% formalin and brains removed for histological verification of electrode sites.

#### **ACUTE OLFACTORY TUBERCLE (OT) STIMULATION**

A separate cohort of 21 mice were anesthetized via urethane injection (1.0 mg/kg, I.P.) and mounted on a stereotaxic frame upon a water-filled heating pad (38◦C) for acute OT stimulation. The basic surgical methods follow as described above for the chronic stimulating electrode implantation surgery, but with a few notable differences described herein. A stimulating bipolar electrode (same as described above) was lowered into the site of the OT. The stimulating electrode connector was then connected by a headstage via a motorized commutator to a Cygnus Technology SIU-91 isolated current source (Delaware Water Gap, PA, USA). Stimulated mice received 5 trials of current delivery (200 s train of bimodal, rectangular pulses, 50 ms in pulse width (i.e., 10 Hz), 100 µA in amplitude) at a 1 min inter-stimulus interval. Sham mice simply remained on the heating pad with the electrode in their OT for the same duration of time as the stimulated mice. Following which, the electrode was gently raised out of the brain and the animals transferred onto a heating pad for 90 min prior to transcardial perfusion as described above.

### **SELF-ADMINISTRATION BEHAVIOR TESTING**

The self-stimulation chamber was made of acrylonitrile butadiene styrene (ABS) plastic and consisted of a 150 × 150 mm (W × L) floor bordered by 225 mm tall walls and an open celling. One wall was removable to allow insertion and extraction of the mouse from the testing chamber. Above the chamber was a video camera for recording behavioral events as well as a motorized commutator (Tucker Davis Technologies, Alachua, FL, USA) to allow the mice to be freely mobile but still connected to the stimulation tether. In the center of the chamber floor was a 10 × 25 mm piezo electric foil ("touch pad"; Parallax, Inc., Rocklin, CA, USA) for reception of paw presses. All self-administration testing was performed in a dark room with illumination enough to see the subject's behavior provided by a single dim red light.

Digitization of paw presses and triggering of stimulation via the paw presses occurred by means of a Tucker Davis Technologies recording amplifier (RZ5) running custom code. A threshold was set for triggering of stimulation based upon an average touch pad voltage while mice freely explored the self-stimulation chamber during acclimation. During self-stimulation testing therefore, touch pad contact that crossed the threshold triggered the delivery of current stimulation (0.5 s train of bimodal, rectangular pulses, 50 ms in pulse width (i.e., 10 Hz), 100 µA in amplitude).

On 2 consecutive days mice were connected to the stimulation tether and allowed to freely explore the self-stimulation chamber (with stimulation triggering off) for 30 min of acclimation to the testing apparatus. Following, over the course of the next days, mice were again connected to the stimulation tether and allowed to explore the self-stimulation chamber for behavioral testing wherein contact with the touch pad triggered stimulation. Stimulation occurred on a fixed ratio 1 schedule. Importantly, touch pad contact must have been released in order for the mouse to receive the next stimulation upon contact. Mice were allowed access to stimulation in the self-stimulation chamber for up to 60 min each day throughout which the number of presses and the time of each press event were recorded.

#### **THREE-CHAMBER ODOR PREFERENCE BEHAVIOR TESTING**

For a test of odor preferences, a 600 × 300 × 300 mm (length × width × height) clear acrylic chamber was divided into three equal zones with black markings. Above the chamber was a video camera for recording behavioral events as well as a motorized commutator (Tucker Davis Technologies) to allow the mice to be freely mobile but still connected to the stimulation tether. All testing was performed in a dark room with illumination enough to see the subject's behavior provided by a single dim red light.

A single perforated dark plastic stimulus container (20 mm diameter × 20 mm tall) was placed on each end of the preference chamber for all testing. These stimulus containers were designed to allowing olfactory inspection of their contents but no distinct visual, somatosensory, or gustatory cues. On the first day, the mice were connected to the stimulation tether and allowed to freely explore the preference chamber (with stimulation off) for 30 min of acclimation to the testing chamber and clean odor-less stimulus containers. On the second and third days, the mice were again connected to the stimulation tether but this time the stimulus containers contained either a 1 × 10−3 dilution of fox urine1 placed on a cotton ball (100 µl fluid) or 3 g of crushed peanut. We predicted that these two different stimuli would elicit unique investigation behaviors (time spent/investigation) related to their emotional values (Takahashi et al., 2005) and thereby would provide a test as to whether or not stimulation of the OT would impact odor hedonic-related behaviors. The side of the preference chamber containing each stimulus was counterbalanced across all mice. On 1 day per mouse (counterbalanced across mice), current stimulation was provided throughout the entire duration of a daily session (continuous bimodal train, rectangular pulses, 50 ms in pulse width (i.e., 10 Hz), 100 µA in amplitude). On each day the testing lasted 500 s, throughout which the amount of time spent in each zone of the preference chamber and the number of zone crosses were recorded onto video. Videos were scored offline by a single experimenter (K.R.) manually tallying zone crosses (defined by contact of all four paws across the divider line) and cumulative time based upon the video stopwatch.

#### **c-Fos IMMUNOHISTOCHEMISTRY AND QUANTIFICATION**

Alternate 40 µm coronal brain sections were acquired from mice which received the acute OT stimulation paradigm or sham

1http://Predatorpee.com

controls using a sliding microtome. ≥5 sections/mouse spanning regions ∼0.8–0.4 mm anterior to bregma were collected and left floating in 0.03% sodium azide in Tris-buffered saline (TBS, pH 7.4) until staining. *c-Fos* immunohistochemistry followed the methods of Kang et al. (2011b). First, the brain slices were rinsed in TBS and then quenched in a solution of 0.3% hydrogen peroxide in methanol. They were rinsed again in TBS, and then in 0.1% TX-100 in TBS. Following the rinses, the sections were blocked in 5.0% NDS in 0.1% TX-100 (Jackson ImmunoResearch, West Grove, PA, USA) for an hour and then incubated overnight at 4◦C in the anti-*c-Fos* primary antibody (1:1000, Calbiochem, EMD Millipore, Billerica, MA, USA). A subset of slices in each run were used as a primary antibody control to ensure specificity of staining. The next day, the sections were rinsed in a diluting buffer, incubated in a secondary antibody (1:600, Jackson ImmunoResearch), rinsed again in 0.1% TX-100 in TBS, and incubated in an Avidin/Biotinylated enzyme Complex kit (Vector Laboratories, Inc., Burlingame, CA, USA). Finally, the sections were rinsed with 0.1% TX-100 in TBS, and incubated in a peroxidase substrate kit with diaminobenzidine (Vector Laboratories) and rinsed with ddH2O. Sections were then transferred onto slides and, after drying, cover slipped with Permount (Fisher Scientific, Pittsburgh, PA, USA).

*a priori* regions for analysis included the OT, ventral pallidum (VP), nucleus accumbens (NAc), lateral septum (LS), and caudate putamen (CPu). These regions were identified using known cytoarchitectural features (Paxinos and Franklin, 2000) and imaged at 20x magnification using a Leica microscope and a 3MP camera. Equal size (200 µm2 ) bounding boxes were overlaid upon the digital images for cell counting. The location of the bounding boxes were held constant across mice. Any cell bodies which touched the bounding box were excluded from counts. *c-Fos*+ cell bodies were manually identified and counted by a single observed based upon density of the 3,30 -diaminobenzidine (DAB) reaction versus background. Sections containing significant damage within the bounding box from the stimulating electrode were excluded from analysis and replaced with non-damaged sections. All steps including sectioning, staining, imaging, and quantification were completed in a group-counterbalanced order by a single experimenter blind to the experimental group of the tissue (B.F.).

#### **ELECTRODE PLACEMENT VERIFICATION**

All stimulation sites were verified by post-mortem histological examinations of slide-mounted 40 µm coronal brain sections stained with a 0.1% cresyl violet solution or in other cases, DAPI (40 ,6-diamidino-2-phenylindole, Life Technologies, City State, USA). We defined an OT stimulation site as successful when the wires terminated within either layers i, ii, and/or iii of the OT (**Figure 1**). Electrode tip locations were verified by multipleobservers (B.F. and D.W.) with reference to a mouse brain atlas (Paxinos and Franklin, 2000). Data associated with sites outside of the OT were entirely excluded from this study.

#### **DATA ANALYSIS**

Behavioral data (time spent in preference zones, # touch pad presses, # zone crossings) were compared between conditions

(stimulation on vs. stimulation off) with an ANOVA. The total number of *c-Fos*+ neurons was compared between conditions (stimulated vs. sham) and hemispheres (ipsilateral vs. contralateral) within brain regions specified also using an ANOVA. Data were analyzed in Origin 8.5 (Northampton, MA) with a significance level of *p* < 0.05. Values are reported as mean ± SEM unless otherwise noted.

### **RESULTS**

We first asked whether OT electrical stimulation is rewarding in mice. To address this, we allowed a cohort of eight mice chronically implanted with bipolar stimulating electrodes into the OT to freely explore the self-stimulation chamber for 2 days wherein contact with a touch pad triggered OT stimulation (see Section Materials and Methods). Following, on the third day, mice were again placed into the self-stimulation chamber and numbers of touch pad presses recorded. Stimulation was allowed *ad libitum* over two blocks of 15 min, separated by a single block of 15 min wherein touch pad contact did not trigger stimulation. During the first 15 min block, mice readily pressed the touch pad (**Figure 2A**). The touch pad contacts were not resultant from random contact by the mice since turning off the stimulus in the middle 15 min block entailed a significant decrease in touch pad presses in all but one mouse (7/8, 87.5%) (**Figures 2A, B**) (*F*(1,12) = 19.576, *p* = 0.0008). Reinstatement of the *ad libitum* reward delivery significantly restored touch pad presses in the final 15 min block (**Figures 2A, B**) (*F*(1,12) = 13.786, *p* = 0.003). These data demonstrate that electrical stimulation of the OT is rewarding in mice.

We next investigated the influence of OT stimulation on odor preference behavior in the three-chamber preference test in the same mice. Notably, one mouse (**Figure 2A**, dashed line) displayed highly aberrant behavior from the group in the self-stimulation testing and was thus excluded from this preference experiment. A separate mouse did not explore the preference chamber and instead stayed immobile in a single zone—qualifying exclusion. Across the remaining six mice, all spent statistically similar time in both end zones in the presence of blank odor vials (*F*(1,10) = 0.035, *p* = 0.855) (data not shown). This baseline data verifies that mice did not have a preference towards simply being on one side of the chamber vs. the other. Next, over 2 days, mice were tested for preference behavior among odorized zones, with OT stimulation being provided consecutively on one of those days. Mice received the stimulation on counterbalanced days (1/2 mice received it on day 1 and 1/2 on day 2), and thus in this design effects of OT stimulation on preference behavior at the population level can be considered independent of learning. No differences between these two groups were observed and thus their data were pooled together (*p* = 0.446, Kolmogorov-Smirnov test). We found that in the absence of OT stimulation, mice spent significantly greater time in the peanut zone of the chamber vs. either the neutral (*F*(1,10) = 11.901, *p* = 0.0062) or the fox urine zones (*F*(1,10) = 11.882, *p* = 0.0063) (**Figure 3**). OT stimulation strikingly abolished this difference, with statistically indistinguishable time spent in both the fox and peanut zones (*F*(1,10) = 0.004, *p* = 0.949) (**Figure 3**). No effect of OT stimulation was observed on the number of side crossings in the preference apparatus (*F*(1,10) = 0.235, *p* = 0.639) (data not shown), suggesting that the effect of OT stimulation on the display of behavior in the odor preference task was independent of stimulation influencing gross locomotor activity.

Based upon the above behavioral findings demonstrating that OT stimulation influences odor-driven behavioral responses, we next sought to test the mechanisms whereby OT stimulation may alter hedonic-related behaviors. We predicted based upon known anatomical connectivity between the OT and brain reward structures (Ikemoto, 2007), that OT stimulation recruits forebrain structures necessary for reward (Koob and Le Moal, 2001; Berridge, 2003; Ikemoto, 2007). Therefore, a separate cohort of anesthetized mice received OT stimulation (*n* = 13) or sham OT stimulation (*n* = 8) in a paradigm mimicking that received while awake (see Section Materials and Methods) and later, their brains probed by means of immunohistochemistry for levels of *c-Fos* expression (Sagar et al., 1988; **Figure 4A**). The use of anesthetized

mice for this analysis was advantageous to ensure changes in *c-Fos* expression were directly due to OT stimulation, vs. extraneous influences of OT stimulation upon behavior.

Confirming the physiological potency of the stimulation paradigm, we found that OT stimulated mice had a significantly greater number of *c-Fos*+ neurons in their ipsilateral OT than sham treated mice (*F*(1,19) = 4.679, *p* = 0.0435) (**Figure 4B**). No group effect was observed when comparing between contralateral OT hemispheres (*F*(1,19) = 1.157, *p* = 0.296) (**Figure 4B**). Looking beyond the OT, we found that mice receiving OT stimulation had a significantly greater number of *c-Fos*+ neurons compared to sham mice in their LS (*F*(1,19) = 4.852, *p* = 0.040) and NAc (*F*(1,19) = 4.828, *p* = 0.0406), but not their CPu (*F*(1,19) = 0.032, *p* = 0.861) nor VP (*F*(1,19) = 1.793, *p* = 0.196) (ipsilateral vs. ipsilateral) (**Figure 4C**). Across all of these structures, only in the CPu did the number of *c-Fos*+ neurons in the contralateral hemisphere significantly differ between stimulated and sham groups (*F*(1,19) = 6.09, *p* = 0.023) (**Figure 4C**). No group effect of stimulation was observed between the contralateral hemispheres in the LS (*F*(1,19) = 0.943, *p* = 0.344), NAc (*F*(1,19) = 0.931, *p* = 0.347), or the VP (*F*(1,19) = 2.179, *p* = 0.156). In all structures analyzed, the number of *c-Fos*+ neurons was statistically similar comparing between the ipsilateral to contralateral hemispheres (*p* > 0.05). For a reason we are unaware of, perhaps related to damage of near-by electrode insertion, sham mice displayed significant increases in the number of *c-Fos*+ neurons between their contralateral and ipsilateral hemispheres in both the CPu (*F*(1,14) = 15.067, *p* = 0.0017) and NAc (*F*(1,14) = 12.684, *p* = 0.0031). Taken together, these results suggest that the influence of OT stimulation on motivated and odor-driven behaviors may occur via connectivity between the OT and the NAc, LS, and possibly CPu.

#### **DISCUSSION**

In this study we provide an initial proof-of-concept exploration into the role of the OT, a structure with known anatomical connectivity with both brain reward and olfactory structures, in regulating odor-motivated behaviors and their possible mechanisms. We confirm that electrical stimulation of the OT was rewarding (Prado-Alcalá and Wise, 1984), in this case in mice, and possessed the capacity to alter odor-directed preference behaviors. In separate experiments we also found that OT stimulation significantly increased the number of *c-Fos* positive neurons in not only the OT, but also in forebrain structures essential to motivated behaviors, including the NAc and LS. The present results support the notion that the OT is integral to motivated behaviors and likely involved in odor hedonics.

**FIGURE 3 | OT stimulation alters odor-guided behaviors. (A)** Mice were allowed to explore a three-chamber odor preference chamber for 10 min with one side containing crushed peanuts (3 g) and the other fox urine (1:10 dilution) both contained within a perforated dark plastic container. On counterbalanced days, current stimulation was delivered throughout the entire duration of the session. On the day with stimulation off, mice spent significantly greater time in the peanut zone, whereas this was abolished with OT stimulation on. n = 6 mice. n.s. = not significant. p-values = ANOVA followed by Fisher's PLSD.

#### **INFLUENCE OF THE OLFACTORY TUBERCLE (OT) ON REWARD AND ODOR-GUIDED BEHAVIORS**

In the present study we found that mice self-administered electrical current into the OT. This finding is analogous and complimentary to a much earlier finding by Prado-Alcalá and Wise (1984) also one by Ikemoto demonstrating that rats readily selfadminister cocaine into the OT and further that cocaine infusions into the OT are sufficient for the development of a conditioned place preference (Ikemoto, 2003). Our self-administration experiments in the present study were not designed to be interpreted as novel in theory by any means but instead to reinforce the concept that the OT is instrumental in driving reward-related behaviors (Koob et al., 1978; Prado-Alcalá and Wise, 1984; Alheid and Heimer, 1988; Heimer, 2003; Ikemoto, 2003). Reward system projections into the OT include the rostral linear nucleus of the ventral tegmental area (Del-Fava et al., 2007), the medial forebrain bundle (Gaykema et al., 1990), the NAc (Zahm and Heimer, 1993), and the substantia nigra (Fallon et al., 1978). Thus, possibly due to both intrinsic (DAergic receptor expression (Li and Kuzhikandathil, 2012)) and extrinsic factors (inter-network connectivity), the OT appears capable of eliciting motivated behaviors. Exploring possible mechanisms of connectivity between the OT and the LS and CPu, as suggested herein, will be important in furthering our understanding of the OTs' rewarding properties.

In addition to being connected with reward-related structures, the OT also receives dense innervation from secondary neurons in the olfactory bulb (e.g., White, 1965; Scott et al., 1980; Schwob and Price, 1984; Nagayama et al., 2010; Kang et al., 2011a) and consequently represents and processes odors in a manner similar to the primary (piriform) olfactory cortex (Payton et al., 2012). A recent study reported that lesions of the anteriomedial ventral striato-pallidum complex (including the OT) altered preferences of female mice for male sociosexual odors (Agustín-Pavón et al., 2014). This finding posed the important question as to whether or not manipulation of OT activity alone might be sufficient to alter odor guided behavior? Our present results support and extend the previous finding by Agustín-Pavón et al. (2014) by demonstrating that electrical manipulation of OT activity, specifically, possesses the capacity to modulate odor-guided behaviors. The use of electrical stimulation to modulate and explore principles of olfactory system function and olfactory behaviors is well established (Mouly et al., 1985; Mouly and Holley, 1986; Wilson and Sullivan, 1990) and thus we employed that method herein for bulk modulation of OT activity. Using this, we found that stimulation of the OT abolished preference for peanut odor vs. fox urine odor. Notably, using this gross odor preference behavior to test the OTs involvement in olfaction is a considerably insensitive assay and thus additional studies will be needed employing more precise olfactory read-outs to explore unique and specific aspects of olfactory behavior under control by the OT. Indeed, as they stand, our results demonstrate the role for the OT in modulating odor-guided behaviors, but not specifically modulating olfactory perception. That said, our results from the odor preference assay do support the idea that OT local and/or inter-regional processing modulates odor-guided behaviors if not at least in an indirect manner. This indirect mechanism could be by shaping motivation and/or the reward features of the odors (e.g., via modulation of structures identified in **Figure 4**). Directly, OT stimulation might perturb basic aspects of odor processing known to occur in the OT (Wesson and Wilson, 2010; Payton et al., 2012; Carlson et al., 2014) and thus alter odor perception more specifically. We predict that the effects observed in the present paper stem form a combination of both direct and indirect impacts of OT activity on behavior.

#### **POSSIBLE NEURAL SUBSTRATES WHEREBY OLFACTORY TUBERCLE (OT) AFFECTS BEHAVIOR**

In the present study, we found that moderate electrical stimulation of the OT resulted in significant recruitment of neurons within brain structures believed essential to motivated behaviors (Koob and Le Moal, 2001; Berridge, 2003; Ikemoto, 2007), including the NAc and LS. Notably, this list is not exhaustive in that we did not analyze every possible motivated behavior center, but instead approached this study with an *a priori* set of forebrain regions. Likely analyzing additional brain structures, especially the ventral tegmental area and orbitofrontal cortex, would provide additional evidence for the functional interconnectedness of the OT (Barbas, 1993; Illig, 2005; Del-Fava et al., 2007). Additionally, the medial vs. lateral aspects of the OT are hypothesized differentially involved in olfactory and reward-related behaviors (Josephson et al., 1997; Ikemoto, 2007) and thus, it is possible that our grouping of OT stimulation sites spanning the entire OT overlooked more subtle aspects of neuronal output which otherwise might be observed if one were to employ regionallyrestricted stimulation.

It is interesting to speculate upon why OT stimulation did not more strongly activate structures like the VP which also hold strong interconnectedness with the OT (Millhouse, 1986). While the number of *c-Fos*+ neurons was increased

bar = 20 µm. **(B)** OT stimulation elicits a significant increase in c-Fos+ neurons in the ipsilateral OT. c-Fos+ neuron counts were quantified within a

quantified within a 200 µm2 bounding box. p-values = ANOVA followed by Fisher's PLSD.

in this structure, this was not significant. It is possible that in the majority of animals bulk OT stimulation was insufficient to recruit distinct sub-populations of neurons innervating the VP and thus VP activation was minimal. Indeed, populations of VP projecting neurons from the OT are GABAergic (Meyer et al., 1989; Hsieh and Puche, 2013) and thus the effects of OT stimulation on the VP would be inhibitory. While the present study yields novel evidence upon the effects of gross activation of the OT, employing more precise stimulation methods, including genetically guided cell-specific methods, will be needed to more closely address mechanisms of OT connectivity.

### **ACKNOWLEDGMENTS**

This work was supported by NSF grant IOS-1121471 to Daniel W. Wesson and a Battelle Science award from Denison University to Brynn J. FitzGerald.

#### **REFERENCES**

Zahm, D. S., and Heimer, L. (1993). Specificity in the efferent projections of the nucleus accumbens in the rat: comparison of the rostral pole projection patterns with those of the core and shell. *J. Comp. Neurol.* 327, 220–232. doi: 10.1002/cne. 903270205

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 30 January 2014; accepted: 24 February 2014; published online: 14 March 2014.*

*Citation: FitzGerald BJ, Richardson K and Wesson DW (2014) Olfactory tubercle stimulation alters odor preference behavior and recruits forebrain reward and motivational centers. Front. Behav. Neurosci. 8:81. doi: 10.3389/fnbeh.2014.00081*

*This article was submitted to the journal Frontiers in Behavioral Neuroscience.*

*Copyright © 2014 FitzGerald, Richardson and Wesson. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## Interactions with the young down-regulate adult olfactory neurogenesis and enhance the maturation of olfactory neuroblasts in sheep mothers

#### **Maïna Brus 1,2,3,4 , Maryse Meurisse1,2,3,4 , Matthieu Keller 1,2,3,4 and Frédéric Lévy1,2,3,4\***

1 INRA, UMR 85, Physiologie de la Reproduction et des Comportements, Nouzilly, France

2 CNRS, UMR 7247, Nouzilly, France

3 Université François Rabelais, Tours, France

4 IFCE, Nouzilly, France

#### **Edited by:**

Anne-Marie Mouly, Centre de Recherche en Neurosciences de Lyon, France

#### **Reviewed by:**

Muriel Koehl, French Institute of Health and Medical Research, France Grace Schenatto Pereira, Universidade Federal de Minas Gerais, Brazil

#### **\*Correspondence:**

Frédéric Lévy, INRA, UMR 85, Physiologie de la Reproduction et des Comportements, F-37380 Nouzilly, France e-mail<EMAIL_ADDRESS>
New neurons are continuously added in the dentate gyrus (DG) and the olfactory bulb of mammalian brain. While numerous environmental factors controlling survival of newborn neurons have been extensively studied, regulation by social interactions is less documented. We addressed this question by investigating the influence of parturition and interactions with the young on neurogenesis in sheep mothers. Using Bromodeoxyuridine, a marker of cell division, in combination with markers of neuronal maturation, the percentage of neuroblasts and new mature neurons in the olfactory bulb and the DG was compared between groups of parturient ewes which could interact or not with their lamb, and virgins. In addition, a morphological analysis was performed by measuring the dendritic arbor of neuroblasts in both structures. We showed that the postpartum period was associated with a decrease in olfactory and hippocampal adult neurogenesis. In the olfactory bulb, the suppressive effect on neuroblasts was dependent on interactions with the young whereas in the DG the decrease in new mature neurons was associated with parturition. In addition, dendritic length and number of nodes of neuroblasts were significantly enhanced by interactions with the lamb in the olfactory bulb but not in the DG. Because interactions with the young involved learning of the olfactory signature of the lamb, we hypothesize that this learning is associated with a down-regulation in olfactory neurogenesis and an enhancement of olfactory neuroblast maturation. Our assumption is that fewer new neurons decrease cell competition in the olfactory bulb and enhance maturation of those new neurons selected to participate in the learning of the young odor.

 In the DG, stem cells reside in the subgranular zone (SGZ) and give rise to neuroblasts which become new granule neurons in the overlying granule cell layer (GCL; Ming and Song, 2005). In the olfactory system, neural stem cells function as primary precursors in the subventricular zone (SVZ) located on the wall of the lateral ventricles. These cells produce transient amplifying cells which rapidly divide to produce neuroblasts. The neuroblasts migrate toward the olfactory bulb along the rostral migratory stream. After reaching the olfactory bulb, the new cells migrate radially and mature into granular interneurons for the majority of them (Rochefort and Lledo, 2005).

In rodents, a growing body of literature aiming to block neurogenesis by using various methodologies shows the role of hippocampal neurogenesis in some forms of hippocampus– dependent learning tasks despite some inconsistent results (Deng et al., 2010; Arruda-Carvalho et al., 2011; Gu et al., 2012). Similarly, olfactory neurogenesis is involved in the memory processing of olfactory cues (Lazarini and Lledo, 2011). Blocking neurogenesis impaired olfactory perceptual learning (Moreno et al., 2009), short-term and long-term memory (Breton-Provencher et al., 2009; Lazarini et al., 2009; Sultan et al., 2010). In addition it has been recently demonstrated that immediate activation of newborn olfactory neurons, by using an optogenetic approach, enhances discrimination learning and memory when the task is difficult (Alonso et al., 2012). A common role in pattern separation has been proposed for newborn hippocampal and olfactory neurons and adult neurogenesis could constitute an adaptive mechanism to optimally encode contextual or olfactory information (for review, see Sahay et al., 2011).

While the implication of adult neurogenesis has been demonstrated in spatial and olfactory learning, a better understanding of the role of adult born neurons in an ethological context has begun to emerge. Social environment can modify hippocampal neurogenesis. Decrease in cell proliferation induced by social isolation rearing could be reversed by subsequent group rearing (Lu et al., 2003). Exposure to chronic social stress dramatically decreases cell proliferation in the DG of rats, mice and tree shrews (Gould et al., 1997; Czeh et al., 2002; Mitra et al., 2006). In a socio-sexual context, exposure to the male urine compounds that are involved in mate recognition increased the survival of granule cells in the accessory olfactory bulb, the main olfactory bulb (MOB), and the DG of female mice (Mak et al., 2007; Oboti et al., 2011). Moreover, suppression of neurogenesis by an anti-mitotic agent prevented mate recognition (Oboti et al., 2011) and the display of preference for dominant male in female mice (Mak et al., 2007). Another approach to assess the role of adult neurogenesis consists in evaluating whether newly-generated neurons might functionally integrate the olfactory network which process olfactory information. In male hamsters, double immunohistochemistry labeling for Fos, a marker of cell activation, and neuronal nuclei (NeuN), a marker of post-mitotic neurons, shows that olfactory bulb cells born in adulthood are activated by socio-sexual stimuli such as estrous female or aggressive male (Huang and Bittman, 2002).

A link between adult neurogenesis and parenting has not been clearly established yet (Lévy et al., 2011). Some studies indicate a regulation of neurogenesis by parturition and the onset of motherhood. In all of rodent species studied so far parturition and the early *postpartum* period are accompanied by a significant decrease in cell proliferation in the hippocampus. In primiparous mother rats, this was reported at *postpartum* day 1, 2 and 8 (Darnaudery et al., 2007; Leuner et al., 2007; Pawluski and Galea, 2007) although no effect was observed later, at *postpartum* day 28 and after weaning (Leuner et al., 2007). Parturition and early *postpartum* period do not stimulate cell proliferation in the SVZ of mice but an increase is observed at 7 days *postpartum* (Shingo et al., 2003). Surprisingly, whether cell survival in the DG or in the MOB is altered during parturition and early *postpartum* period at the onset of maternal behavior is not known in rodents. Rather, cell survival in the DG of rats was assessed either at *postpartum* day 14 (Darnaudery et al., 2007) or 21 (Pawluski and Galea, 2007) and both studies report a significant decrease when compared to virgins. A few studies have investigated the importance of stimuli provided by neonatal pups but outside the context of parturition. Nulliparous rats exposed to pups show increased cell proliferation in the DG when compared to nulliparous females regardless of their parental response (Pawluski and Galea, 2007). Likewise, virgin female prairie voles exposed to pups exhibit increased hippocampal cell proliferation (Ruscio et al., 2008). Although an increase in cell survival in the DG was reported in virgin females 21 days after pup-exposure (Pawluski and Galea, 2007), the influence on survival of the newborn neurons either in the DG or in the MOB at the time of pup-exposure is not known. The consequences of neurogenesis ablation on the onset of maternal behavior have been investigated in mice. Irradiation of the SVZ induces minor disturbances of maternal behavior (Feierstein et al., 2010b). However, infusion of an anti-mitotic agent which transiently impairs both hippocampal and olfactory neurogenesis has been shown to affect maternal behavior but only when animals are tested in an anxiogenic environment (Larsen and Grattan, 2010), whereas genetic manipulations inducing profound and long-term alterations of neurogenesis impair nursing behavior in the home cage (Sakamoto et al., 2011).

In sheep, a down-regulation of cell proliferation has been observed in mothers in contact with their lambs for 2 days both in the DG and the SVZ (Brus et al., 2010). However there has been no report examining a change in survival of newly-born neurons in the DG or the MOB that could occur during the early *postpartum* period. In addition, no study has disentangled the influence of parturition and the first interactions with the young on cell survival and this could improve our understanding of the contribution of neurogenesis to maternal behavior. In this context, maternal behavior in sheep constitutes an interesting model in which endocrine changes occurring at parturition and olfaction play a central role (Lévy et al., 1995; Lévy and Keller, 2008). In addition, not only infantile odors become very potent stimuli allowing the development of maternal care but they also provide a basis for individual recognition of the offspring. Ewes develop discriminative maternal care, called maternal selectivity, favoring their own young at suckling while rejecting any alien young. This recognition is based on the learning of olfactory characteristics of the lamb and takes place within the first hours after parturition (Lévy et al., 2004; Lévy and Keller, 2009). Some of the learning mechanisms reside in extensive neurochemical changes occurring in the MOB at parturition (Lévy et al., 1993; Lévy and Keller, 2009). In addition to these neurochemical changes, olfactory neurogenesis could provide another mechanism through which olfaction can contribute to the onset of maternal behavior and associated learning.

The aim of the present study was to evaluate the influence of parturition and learning of the lamb odor on the survival of newborn neurons. Bromodeoxyuridine (BrdU), a marker of cell division, was used in combination with two markers of neuronal maturation (doublecortin (DCX), an early maturation marker and NeuN), to compare both hippocampal and olfactory neurogenesis between virgins and parturient ewes which could interact or not with their lamb. In addition, because learning accelerates the maturation of the dendritic trees of newborn neurons in the DG (Tronel et al., 2010; Lemaire et al., 2012), and motherhood is accompanied by changes in the morphology of newborn neurons in the MOB (Kopel et al., 2012), we assessed the influence of lamb olfactory learning on this maturation by measuring the dendritic length and the number of nodes of new neuroblasts.

### **MATERIALS AND METHODS**

#### **ANIMALS**

Experiment was conducted on 17 Ile de France ewes, of 1.5–2 years of age, from the INRA research center in Nouzilly (Indre et Loire, France) approved by local authority (agreement number E37-175-2). Animals were permanently housed indoors, with free access to water and were fed with lucerne, maize, straw and a supplement of vitamins and minerals. Animal care and experimental treatments complied with the guidelines of the French Ministry of Agriculture for animal experimentation and European regulations on animal experimentation (86/609/EEC) and were performed in accordance with the local animal regulation (authorization No. 006352 of the French Ministry of Agriculture in accordance with EEC directive). Ewes were sacrificed by a licensed butcher in an official slaughterhouse (authorization No. A37801 E37-175-2 agreement UEPAO). All efforts were made to minimize the number of animals (5–6 animals per group).

#### **BROMODEOXYURIDINE INJECTIONS AND TISSUE PREPARATION**

Four months before sacrifice, ewes were housed in an individual pen (2 × 1 m) and received four intravenous injections of BrdU, (1 injection/day, 20 mg/Kg in 0.9% saline; Sigma-Aldrich, France), a thymidine analogue incorporated into the DNA during the S-phase of the mitotic division. Doses of BrdU and timing between injections and sacrifice were based on a previous study reporting that maturation of adult-born cells both is much longer in sheep than that of rodents and that the highest proportion of new mature neurons is found at 4 months after BrdU injections (Brus et al., 2013).

### **GROUPS**

Three groups of ewes were constituted (**Figure 1**). In parturient groups, mating was synchronized by the use of vaginal sponges containing 45 mg of fluorogestone acetate for 14 days followed by an intra-muscular injection of pregnant-mare-stimulating gonadotropins to induce ovulation. Just after parturition, mothers were either left 48 h with their lambs in their individual pen in the same barn ("With Lamb" group, *n* = 5), or were separated from them for 48 h ("No Lamb" group, *n* = 6). After being separated from their lamb immediately after birth, ewes of the "No Lamb" group were placed in a different barn to avoid any contact with lambs and ewes were housed together in a large pen to avoid stress induced by separation from the young. All the ewes had never given birth before the study. Lambing occurred within a period of gestation of 149 ± 4 days. Against all expectations, at birth the lambs displayed low vigor preventing them from feeding normally, probably due to BrdU injections in early pregnancy. Thus, in the "With lamb" group, adoptions have been performed with newborn lambs provided by the flock of the research center. It has been well established in previous studies that adoption, when performed at birth, are without any consequences on the quality of the mother-young relationship in comparison to normal mother-young lambing (Keverne et al., 1983; Kendrick et al., 1991; Lévy et al., 2010). In this group, maternal behavior was observed for 10 min at 0, 6 and 24 h after parturition to completely ensure that maternal care was normally provided to lambs. At 2 days *postpartum* just before sacrifice, selectivity was tested by presenting an alien lamb to the mother and rejection and acceptance behaviors were recorded for 3 min. The alien lamb was then taken away and the ewe was observed

with her own lamb for an additional 3 min (Keller et al., 2004, 2005). These tests indicated that all the ewes of the "With Lamb" group were maternal and selective. The "Virgin" group (*n* = 6) was composed of nulliparous anoestrus ewes of similar age than the two parturient groups and housed together.

#### **BRAIN PERFUSION AND IMMUNOHISTOCHEMISTRY**

Two days after lambing, ewes were anesthetized with thiopental and decapitated by a licensed butcher in an official slaughterhouse. Brains were immediately perfused via carotid arteries with 2 L of 1% sodium nitrite in phosphate buffer saline, followed by 4 L of ice-cold 4% paraformaldehyde solution in 0.1M phosphate buffer (pH 7.4). The brain was dissected out, cut into blocks and post-fixed in the same fixative for 48 h. The tissues were then stored in 30% sucrose for 2 days until sectioned. Frontal sections were cut at a thickness of 30 µm using microtome or cryostat and stored at −20◦C in cryoprotectant.

To reveal BrdU positive cells in the MOB and the DG, a peroxydase single-immunolabeling was used as described previously (Brus et al., 2013). To characterize the populations of BrdU positive cells which could be affected by parturition and interaction with the young, double immunolabeling was performed against the BrdU and two markers of neuronal maturation, NeuN for mature neurons (Valley et al., 2009) and the DCX for neuroblasts (Gleeson et al., 1999; Brown et al., 2003). Sections of the MOB and the hippocampus were treated with a solution of Tris Buffer Saline 0.025M (TBS, pH=7.4)-Triton 0.3%-Azide 0.1%-Bovine Serum Albumin (BSA) 0.1% (TBSTA-BSA) for 1 h. After one rinse in TBS, sections were treated with 2N HCl in TBS for 30 min at room temperature. After three rinses in TBS, sections were incubated overnight in primary rat anti-BrdU (1:300; AbCys AbC117-7513, Paris, France) and primary mouse anti-NeuN (1:1000, Chemicon MAB377, Millipore, St. Quentin-en-Yvelines, France) or primary goat anti-DCX (1:100, Santa Cruz Biotechnology, Tebu-Bio, Le Perray en Yvelines, France) in TBSTA-BSA. The following day, sections were rinsed three times in TBS, and were incubated in two secondary antibodies simultaneously for 1 h 30 min in TBS 0.025M, pH7.4—Saponine 0.3%—BSA 0.1% (TBS-Saponine-BSA), except for BrdU/DCX for which secondary antibodies were incubated in TBS-rabbit serum 1%-saponine 0.3%. Secondary antibodies used for BrdU/NeuN immunolabeling were a donkey anti-rat CY3 (1:300, Immunotech, Jackson ImmunoResearch, United Kingdom), and a goat anti-mouse 488 (1:300, AlexaFluor A11029, Molecular Probes, Eugene, Oregon, USA); for BrdU/DCX, we used a rabbit anti-rat 488 (1:300, AlexaFluor, Molecular Probes, Eugene, Oregon, USA) and a Donkey anti-goat CY3 (1:300, Immunotech, Jackson ImmunoResearch, UK). After four rinses in TBS, sections were immersed in a Hoechst bath for 2 min (Hoechst 33258, 2 µg/ml in water, Invitrogen, USA), rinsed in two baths of water and one bath of TBS (5 min each), then cover-slipped under fluoromount-G (SouthernBiotech, Birmingham, AL, USA) and stored at 4◦C in dark.

Because in a previous study we observed cell proliferation within the MOB (Brus et al., 2010), we evaluated the influence of parturition and interactions with the young on cell proliferation in this olfactory structure as well as in the DG. To this end, single-immunolabeling was performed against the Ki67 marker, an endogenous marker of cell division which is expressed at all the phases of the cellular cycle (Kee et al., 2007). Sections were treated with TBSTA-BSA for 1 h and were incubated overnight in primary antibody rabbit anti-Ki67 (1:500, Abcam ab15580- 25, Cambridge, UK) in TBSTA-BSA at room temperature. The following day, sections were rinsed four times in TBS and were incubated in secondary antibody sheep anti-rabbit (1:400, produced by the INRA center of Nouzilly) in TBS-BSA 0.1% during 3 h at 4◦C. After four rinses in TBS 0.1%, sections were incubated with rabbit peroxydase anti peroxydase (PAP, 1:80000, Dako Z0113, Trappes, France) overnight at 4◦C. The last day, after two rinses in TBS and two rinses in Tris-HCl (0.05M, pH 7.6) sections were reacted for peroxydase detection in a solution of 3,30 -Diaminobenzidine tetrahydrochloride (DAB, 0.15 mg/mL; Sigma) containing 0.001% H2O2 and 0.018% nickel ammonium sulfate for 7 min.

### **QUANTIFICATION**

The number of BrdU+ (10 sections/animal, 800 µm between sections) and Ki67+ cells (6 sections/animal, 950 µm between sections) was assessed by counting peroxydase/DAB-stained frontal sections of the MOB and the DG through different levels along the rostrocaudal axis, using a light microscope (Axioskope 2, Zeiss, Germany) on a magnification of x20 and cell count analysis software (computerized image analysis Mercator, Explora Nova, La Rochelle, France). The counter was blind to the experimental group. Areas of the MOB (granular and periventricular layers) and the DG (GCL and SGZ) were measured with this system through an x2.5 objective (MOB) and an x10 objective (DG). Cell densities were then calculated by dividing the numbers of BrdU+ or Ki67+ cells by the layer area. The Ki67+ cell density corresponds to the mean of cell density measured in the granular and the periventricular layers for the MOB and in the GCL and the SGZ for the DG. Densities of BrdU+ cells and proportions of new neurons and neuroblasts were counted only in the target structures of newly-born cells integration, the granular layer of the MOB and the GCL of the DG.

To determine the percentage of BrdU+/NeuN+ cells and BrdU+/DCX+ cells in the MOB and in the DG, approximately 100 cells per ewe were observed for the three groups (around 500–600 cells per group). Each BrdU+ cell was analyzed in its entire *z*-axis, with 0.5 µm step intervals, through an x40 oil immersion objective, using a confocal laser-scanning microscope (LSM700, Zeiss, Germany) equipped of excitation wavelengths 488 and 555. Cells rotated in orthogonal planes to verify double labeling with NeuN or DCX. For each selected cell that showed colocalization of BrdU with NeuN or DCX, an image was collected with the software Zen (Carl Zeiss, Germany). All images shown correspond to one focal plane (0.5 µm) and were imported into Gimp Pack Mode 2.6 software to adjust brightness and contrasts.

To determine the development of the newborn neuroblasts in the different groups, the number of nodes and the length of the dendritic arbor were measured in 16–18 BrdU+/DCX+ cells per ewe in the GCL of the MOB and of the DG. Each BrdU+/DCX+ cell was analyzed with confocal laser scanning microscope (LSM 700, Zeiss, Germany), in its entire *Z*-axis with 0.5 µm step interval, using x63 oil immersion objective to measure the length between the cell body to the end of the longest process. Total length of the dendritic tree was obtained by summing the length of all processes of each BrdU+/DCX+ cell. The number of nodes was obtained by counting the number of occurrences of branch points in the dendritic arbor. Interestingly, these newborn cells seemed to be at an early stage of maturation as most of them displayed only one process. Thus, the population of BrdU+/DCX+ was separated in two categories depending on the number of nodes and the percentage of cells with no nodes (one process, less mature) or with one or more nodes (two or more processes, more mature) was calculated (**Figures 4B–E**). Ambiguous cases were further analyzed using a semiautomatic neuron tracing system Imaris (Bitplane, USA).

#### **STATISTICAL ANALYSIS**

As densities of BrdU+ and Ki67+ cells, proportions of BrdU+/NeuN+ and BrdU+/DCX+ cells, and dendritic lengths of BrdU+/DCX+ cells were not normally distributed, the data were analyzed with nonparametric tests (Siegel, 1956). Intergroup comparisons were analyzed using two-tailed Kruskal-Wallis and Mann-Whitney tests. As the percentage of BrdU+/DCX+ cells which displayed one or more nodes was normally distributed, the data were analyzed with a one-way analysis of variance (ANOVA), and significance was probed by the Newman-Keuls test. Statistical analyses were performed using the statistical package SPSS 10 (Chicago, IL, USA) and the level of statistical significance was set at *p* ≤ 0.05. All data were represented as median and interquartile ranges except the number of nodes which were represented as mean ± SEM. Because we found that cell proliferation is down-regulated in *postpartum* ewes (Brus et al., 2010), we predicted that density of Ki67+ cells will be lower in parturient groups and therefore we used one-tailed Kruskal-Wallis and Mann-Whitney tests for this variable.

#### **RESULTS**

#### **SURVIVAL OF NEUROBLASTS AND NEW MATURE NEURONS IN THE MAIN OLFACTORY BULB (MOB) AND THE DENTATE GYRUS (DG)**

In the granular layer of the MOB and in the GCL of the DG, the densities of BrdU+ cells did not significantly differ between groups (MOB: *H* = 2.27, *p* = 0.3; DG: *H* = 4.48, *p* = 0.1; **Figures 2A, B**).

The proportion of newborn neurons was measured in each group by using a double immunofluorescent labeling for BrdU and NeuN, a marker of post-mitotic neurons, and DCX, a marker of neuroblasts (**Figures 3E–H**). In the granular layer of the MOB, only the proportion of BrdU+/DCX+ cells significantly differed between groups (*H* = 8.27, *p* = 0.02; **Figure 3A**). This proportion was significantly lower in the "With Lamb" group compared to the "Virgin" or the "No Lamb" groups ("With Lamb" vs. "Virgin" or "No Lamb" groups: *U* = 2.47, *p* = 0.01; **Figure 3A**). The proportion of new post-mitotic neurons (BrdU/NeuN+ cells) did not differ between groups (*H* = 1.23, *p* = 0.5; **Figure 3B**).

Contrary to the MOB, only the proportion of BrdU+/NeuN+ cells significantly differed between groups in the GCL of the DG (*H* = 11.07, *p* = 0.004; **Figure 3D**). The proportion of new mature neurons was significantly lower in the two parturient groups

interquartile ranges) in the granular layer of the MOB **(A)** and in the DG **(B)**. There is no significant difference between groups for both regions.

compared to the Virgin group ("With Lamb" vs. "Virgin" groups: *U* = 0, *p* = 0.004; "No Lamb" vs. "Virgin" groups: *U* = 0, *p* = 0.002). However, the proportion of BrdU+/DCX+ cells did not significantly differ between groups (*H* = 0.73, *p* = 0.7; **Figure 3C**).

#### **DENDRITIC LENGTHS OF NEWBORN NEURONS IN THE MAIN OLFACTORY BULB (MOB) AND THE DENTATE GYRUS (DG)**

To assess the influence of interactions with the young on the development of the dendritic arbor of new neurons, we measured dendritic lengths and number of nodes of BrdU+/DCX+ cells in the granular layer of the MOB and in the GCL of the DG (**Figure 4**).

In the MOB, dendritic lengths and number of nodes significantly differed between groups (dendritic length: *H* = 6.01, *p* =

0.049; nodes: *F* = 6.58, *p* = 0.01). The highest dendritic lengths were found in the "With Lamb" group and significantly differed from the "No Lamb" (*U* = 3, *p* = 0.03; **Figure 4A**) and showed a tendency to differ from the "Virgin" group due to an animal showing an extreme value (*U* = 5, *p* = 0.08; **Figure 4A**). The percentage of BrdU+/DCX+ cells possessing nodes was significantly greater in the "With Lamb" group than in the "Virgin" or "No Lamb" groups ("With Lamb" vs. "Virgin" groups: *p* = 0.04; "With Lamb" vs. "No Lamb" groups: *p* = 0.007; **Figure 4B**).

NeuN **(B–D)** and DCX **(A–C)**. In the MOB, only the proportion of BrdU+/DCX+ cells in the "With Lamb" group significantly differed from the two other

By contrast in the DG, no difference in dendritic lengths and in the number of nodes were found between groups (dendritic length: *H* = 1.35, *p* = 0.5; nodes; *F* = 1.04, *p* = 0.38; **Figures 4D, E**). Finally, the diameter of BrdU+/DCX+ cell bodies did not differ between groups in both structures (MOB; *F* = 2.33, *p* = 0.13; DG: *F* = 1.56, *p* = 0.24).

#### **CELL PROLIFERATION IN THE MAIN OLFACTORY BULB (MOB) AND THE DENTATE GYRUS (DG)**

To evaluate the influence of parturition and interactions with the young on cell proliferation, a single immunolabeling was performed against the Ki67 protein, an endogen marker of cell division, in the MOB and the DG (**Figure 5**). In both the MOB and the DG, the density of Ki67+ cells significantly differed between groups (MOB: *H* = 6.13, *p* = 0.025, **Figure 5A**; DG: *H* = 7.59, *p* = 0.01, **Figure 5B**). For both structures, in the two parturient groups, the density of Ki67+ cells in the MOB was significantly lower compared to the "Virgin" group (MOB: "With Lamb" vs. "Virgin" groups: *U* = 2, *p* = 0.01; "No Lamb" vs. "Virgin" groups: *U* = 7, *p* = 0.04; DG "With Lamb" and the "Virgin" groups: *U* = 1, *p* = 0.01; "No Lamb" group vs. "Virgin" groups: *U* = 8, *p* = 0.05).

dentate gyrus, BrdU: bromodeoxyuridine, DCX: doublecortin, NeuN: neuronal

#### **DISCUSSION**

nuclei. \* p ≤ 0.05, \*\* p < 0.01.

Two main results have been obtained in this study. First, interactions with the young and associated learning, but not parturition, reduce the survival of neuroblasts in the MOB, whereas in the DG, parturition induces a decrease in the number of new neurons. These results suggest that learning of the olfactory signature of the lamb, which occurred during the first mother/young interactions, is specifically associated with a down-regulation in olfactory neurogenesis. Secondly, a positive effect on the development of the dendritic length of new neuroblasts has been specifically observed in the MOB of mothers interacting with their lamb suggesting that olfactory learning accelerates the maturation of adult-born neurons in the MOB but not in the DG.

In the MOB, while the number of BrdU+ cells was similar between parturient groups and the "Virgin" group, the population of neuroblasts was decreased by interactions with the young. This reduction is probably not a consequence of a decrease in new cell production during gestation since a positive, rather than a negative, influence of pregnancy on cell proliferation has been described in the SVZ of rodents (Shingo et al., 2003; Furuta and Bridges, 2005; Larsen and Grattan, 2010). In addition, parturient ewes separated from their lambs did not show any change of cell survival in comparison to virgins indicating that parturition could not account for this down-regulation. However, the suppressive effect of interactions with the lamb for 2 days on the survival of neuroblasts could be a consequence of

various behaviors involved in maternal care. For instance licking the newborn lamb depends on olfactory attraction to amniotic fluids (Lévy et al., 1983). However since licking behavior occurs at parturition and only last for 2 h it is unlikely involved in the decrease in neuroblasts population observed after 2 days of mother-young contact. Somatosensory stimulation associated with suckling could also account for this suppressive effect. However there is so far no evidence in the literature that suckling

can have a direct effect on olfactory neurogenesis. In this respect, it would be of interest to assess olfactory neurogenesis in mothers whose udders are covered to prevent lambs from suckling. In rodents, restriction of suckling and tactile stimulation in lactating mothers in the presence of pups modulate neurochemical activity of the MOB, suggesting the importance of olfactory interactions with the young (Munaro, 1990). In sheep, during the early *postpartum* period various neurochemical and electrophysiological changes in the functional circuitry of the MOB underlie the formation of olfactory memory for lamb (Sanchez-Andrade et al., 2005; Lévy and Keller, 2008). Learning of the lamb odor could also be accompanied by changes in olfactory neurogenesis. Numerous studies report that bulbar neurogenesis varies following olfactory learning and that these changes could affect or not mature new neurons (see review: Feierstein et al., 2010a). However, whether these changes concern neuroblasts is not known. Here we show that neuroblasts but not more mature neurons are sensitive to social interactions. The decrease in the number of BrdU+/DCX+ cells cannot be explained by an enhancement of neuronal maturation since we did not observe an increase in BrdU+/NeuN+ cells in ewes interacting with their lambs. This decrease rather suggests that neuroblasts die during the 2 days of lamb exposure. Studies of adult-born neurons showed that olfactory learning has a complex effect on neuronal turnover, increasing or decreasing the survival of newborn olfactory cells as a function of their age (Mandairon et al., 2006; Mouret et al., 2008). Further studies using different timing of BrdU injections are needed to examine whether newborn cells of different age and maturation could have been differently affected.

Together with a reduced number of neuroblasts, a decrease in cell proliferation in the MOB is observed in both groups of parturient ewes in comparison to virgins, as previously shown (Brus et al., 2010). The functional significance of this reduced proliferation and maturation processes in the MOB remains unknown. It may accentuate a survival-promoting effect by eliminating newborn neurons that are not functional and would favor the maturation and integration of new neurons which participate in learning by reducing cell competition. Computational studies support this hypothesis by showing that the rate of cell proliferation and cell survival contribute to the stability of the neural activity in the network (Lehmann et al., 2005; Butz et al., 2006). This assumption is supported by our results showing that dendritic maturation of neuroblasts is only enhanced in mothers interacting with their lamb in the MOB.

Two main non-exclusive mechanisms could be considered to account for this accelerated maturation. Firstly, nursing and suckling could influence the morphology of new neurons as several studies report an effect of lactation on dendritic arbor in the mother brain. For instance, enhanced spine density has been reported in the hippocampus of lactating rats (Kinsley et al., 2006). Similarly lactating females have longer basal dendritic length compared to diestrous females in the medial preoptic area, a key region for maternal behavior (Keyser-Marcus et al., 2001). However, a negative effect has been reported on oxytocin neurons having less dendritic branches and a fewer total dendritic length in lactating rats compared to virgin rats (Armstrong and Stern, 1998; Stern and Armstrong, 1998). Secondly and more interestingly, learning the familiar lamb odor could induce this increased maturation as a positive influence of olfactory stimulation. The effect of odor learning on dendritic trees of newborn neurons has already been reported. Sensory deprivation drastically decreases

"Virgin" group. \* p ≤ 0.05, \*\* p < 0.01.

the number, the dendritic length and the spine density of newborn granule cells in the MOB (Rochefort and Lledo, 2005) and odor enrichment induces structural synaptic plasticity of adult-born granule neurons (Livneh and Mizrahi, 2011). Since evidence of an influence of learning but not of suckling on the maturation of new adult neurons has been reported in the literature, we hypothesized that learning of the olfactory signature of the lamb is involved in this maturation process. Further studies need to be done to establish whether suckling and/or learning of the lamb odor are responsible for this increased maturation of neuroblasts and whether this could also affect new mature neurons.

In the DG, while no change in the number of BrdU+ cells was found between groups, survival of new mature neurons was reduced by parturition itself, since parturient ewes isolated from their lamb showed a decrease in BrdU+/NeuN+ cells in comparison to virgins. The effect of pregnancy on the decrease in cell production is unlikely since no changes in hippocampal neurogenesis have been reported either during early or late pregnancy in rodents (see review: Lévy et al., 2011). However we cannot exclude a species dependent regulation of neurogenesis during pregnancy. These effects could also be due to the stress induced by removal of the lamb. This is however unlikely since we observed a similar decrease in the survival of mature neurons in mothers staying with their lambs. The inhibition of neurogenesis could be the consequence of hormonal changes, mainly steroids and glucocorticoids occurring at parturition. Oestradiol induced a decrease in survival of newborn neurons (Barker and Galea, 2008) and applications of corticosterone suppress cell survival in the DG of the hippocampus (Wong and Herbert, 2004, 2006). Interestingly, oestradiol and corticosterone were found to affect neurogenesis and some interactions exist between both hormones. For example, adrenal steroids mediate the suppression of cell proliferation induced by oestradiol in ovariectomized females (Ormerod and Galea, 2001). The interplay between estradiol and cortisol at parturition may also mediate levels of cell survival in the DG and further studies are needed to better determine this complex influence on neurogenesis. In addition, our results suggest a hormonal influence depending on cell type since no change in the proportion of neuroblasts was found between groups in the DG. Recent studies report that high corticosterone differently affects population of new cells according to their maturity stage (Gonzalez-Perez et al., 2011; Lussier et al., 2013). Different effects on different cell lineages may represent adaptative actions of glucocorticoids, which provide a compensatory mechanism to protect some types of cells from death in the hippocampus. Such a mechanism would prevent from a dysfunction of the hippocampus during the *postpartum* period and would allow a better spatial memory in lactating females (Kinsley et al., 1999).

In conclusion, this study supports the hypothesis that the maternal brain undergoes neuronal changes, such as adult neurogenesis, which could constitute an adaptive response to motherhood by favoring learning ability. More specifically, interactions with the young are associated with a down-regulation in olfactory neurogenesis and an enhancement of neuroblast maturation. Our hypothesis is that fewer new neurons decrease cell competition in the olfactory bulb and enhance maturation of those new neurons selected to participate in the learning of the lamb odor. Further experiments will aim at understanding how these adult born neurons could be integrated to the neural network involved in maternal behavior.

### **AUTHOR CONTRIBUTIONS**

Maïna Brus, Maryse Meurisse, Matthieu Keller and Frédéric Lévy designed and performed research, analyzed data and wrote the paper.

### **ACKNOWLEDGMENTS**

The authors would like to acknowledge the financial support of the "Programme transversal INRA/Institut Pasteur No. 319", the ANR programme blanc (2012-2015) PLASTMATBEHAV and the INRA/Région for the grant to M. Brus. We particularly thank (1) N. Jouaneau and M. Chauvet for histological preparation; (2) J. Cognié for veterinary assistance; (3) C. Moussu, E. Archer and F. Cornilleau for their assistance at lambing; (4) J.-P. Dubois and A. Arnould for sacrifice of animals; (5) D. Capo and its staff for animal breeding; (6) G. Le Pape for statistical analysis; (7) L. Szymanski for English corrections; and (8) the cellular imaging platform (PIC) of UMR PRC, Nouzilly.

### **REFERENCES**

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 29 November 2013; accepted: 03 February 2014; published online: 18 February 2014.*

*Citation: Brus M, Meurisse M, Keller M and Lévy F (2014) Interactions with the young down-regulate adult olfactory neurogenesis and enhance the maturation of olfactory neuroblasts in sheep mothers. Front. Behav. Neurosci. 8:53. doi: 10.3389/fnbeh.2014. 00053*

*This article was submitted to the journal Frontiers in Behavioral Neuroscience.*

*Copyright © 2014 Brus, Meurisse, Keller and Lévy. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## Differential memory persistence of odor mixture and components in newborn rabbits: competition between the whole and its parts

#### **Gérard Coureaud1\*, Thierry Thomas-Danguin1 , Frédérique Datiche1 , Donald A. Wilson2 and Guillaume Ferreira3,4**

1 Centre des Sciences du Goût et de l'Alimentation (CSGA), UMR 6265 CNRS, UMR 1324 INRA, Université de Bourgogne, Dijon, France

2 Department of Child and Adolescent Psychiatry, New York University Langone School of Medicine, New York, NY, USA

3 Nutrition and Integrative Neurobiology Group, INRA UMR 1286, Bordeaux, France

4 Université de Bordeaux, Bordeaux, France

#### **Edited by:**

Anne-Marie Mouly, Centre de Recherche en Neurosciences de Lyon, France

#### **Reviewed by:**

Christiane Linster, Cornell University, USA Mouna Maroun, University of Haifa, Israel

#### **\*Correspondence:**

Gérard Coureaud, Centre des Sciences du Goût et de l'Alimentation (CSGA), UMR 6265 CNRS, UMR 1324 INRA, Université de Bourgogne, 9E Boulevard Jeanne d'Arc, 21000 Dijon, France e-mail: gerard.coureaud@ u-bourgogne.fr

Interacting with the mother during the daily nursing, newborn rabbits experience her body odor cues. In particular, the mammary pheromone (MP) contained in rabbit milk triggers the typical behavior which helps to localize and seize the nipples. It also promotes the very rapid appetitive learning of simple or complex stimuli (odorants or mixtures) through associative conditioning. We previously showed that 24 h after MP-induced conditioning to odorants A (ethyl isobutyrate) or B (ethyl maltol), newborn rabbits perceive the AB mixture in a weak configural way, i.e., they perceive the odor of the AB configuration in addition to the odors of the elements. Moreover, after conditioning to the mixture, elimination of the memories of A and B does not affect the memory of AB, suggesting independent elemental and configural memories of the mixture. Here, we evaluated whether configural memory persistence differs from elemental one. First, whereas 1 or 3-day-old pups conditioned to A or B maintained their responsiveness to the conditioned odorant for 4 days, those conditioned to AB did not respond to the mixture after the same retention period. Second, the pups conditioned to AB still responded to A and B 4 days after conditioning, which indicates stronger retention of the elements than of the configuration when all information are learned together. Third, we determined whether the memory of the elements competes with the memory of the configuration: after conditioning to AB, when the memories of A and B were erased using pharmacological treatment, the memory of the mixture was extended to day 5. Thus, newborn rabbits have access to both elemental and configural information in certain odor mixtures, and competition between these distinct representations of the mixture influences the persistence of their memories. Such effects certainly occur in the natural context of mother-pup interactions and may contribute to early acquisition of knowledge about the surroundings.

**Keywords: Oryctolagus cuniculus, newborn, odor mixture, configural perception, stimulus representation, retention, memory persistence**

### **INTRODUCTION**

In some cases, mixtures of volatile molecules are perceived as a collection of independent, identifiable elements; the perception is then elemental (e.g., Laing and Francis, 1989; Laska and Hudson, 1993; Linster and Cleland, 2004). However, some mixtures induce a configural processing. Then, the mixture gives rise to either a unique and novel odor quality, different from the odor qualities of the elements (robust configural perception; e.g., Smith, 1996; Jinks and Laing, 1999; Kay et al., 2005), or to a novel quality perceived in addition to the qualities of the odorants (weak configural perception; Rescorla, 1973; Kay et al., 2005). Configural odor processing has been described with different mixtures and different approaches in a variety of species from invertebrates to vertebrates, including humans (e.g., Derby et al., 1996; Linster and Smith, 1999; Valentincic et al., 2000; Wise and Cain, 2000; Deisig et al., 2001; Wiltrout et al., 2003; Mandairon et al., 2006; Riffell et al., 2009; Gottfried, 2010; Wilson and Sullivan, 2011). For instance, data in human adults revealed that a mixture of two odorants (AB), one smelling like strawberry (A: ethyl isobutyrate) and the other like caramel (B: ethyl maltol), generates the configural perception of a pineapple odor at a specific ratio of A/B (30/70 v/v; Le Berre et al., 2008, 2010; Barkat et al., 2012). Interestingly, recent results in a newborn mammal, the newborn rabbit, showed similar configural processing abilities with the same AB mixture, at the same ratio. Indeed, after single appetitive conditioning to odorant A or to odorant B by pairing with the mammary pheromone (MP) (naturally contained in rabbit milk and experimentally used as unconditioned stimulus in associative conditioning procedure; Coureaud et al., 2006, 2010), rabbit pups respond to the conditioned element and respectively to the AC or BC mixtures (C: guaïacol). However, they do not respond to the AB mixture. These results suggest that they perceive AB differently from the odors of A and of B, while they perceive AC or BC as the sum of their component odors (Coureaud et al., 2008, 2009a, 2011a). Furthermore, after single conditioning to the AB mixture, rabbit pups respond to the components A and B in addition to AB, indicating that the AB mixture is perceived in a weak configural way (Coureaud et al., 2008). Very recently, combining behavioral approaches with pharmacological tools, we provided another confirmation of this neonatal weak configural perception of AB and demonstrated that rabbit pups memorize the AB configuration independently (at least in part) from the representations of each element. After conditioning to AB, amnesia of A and B did not propagate to AB: pups that did not respond to either A or B still responded to AB (Coureaud et al., in press). Thus, after AB conditioning, distinct elemental and configural memories of the mixture are created.

In the previous studies in newborn rabbits, memories of the elements A and B or of the AB configuration were evaluated 24 or 48 h after conditioning, which are the optimal delays for responsiveness to a stimulus conditioned by single pairing with the MP (Coureaud et al., 2006). However, to date, whether memory persistence of the configural odor of the AB mixture differs from that of the odorants A and B (also detected in the mixture) had never been evaluated; here, we hypothesized that they could be distinguished. This issue constituted the first and main goal of our study. Besides understanding early memory and perception in the rabbit, this evaluation could more broadly help provide additional and original information on the general topic of odor object perception in mammals. Indeed, as said above, a perceptual match has been evidenced for the AB mixture between rabbit pups and human adults, which suggests a relative conservation in the processing of certain odor mixtures across species.

Our second goal aimed to assess whether memory retention of the configural AB odor and of the elemental odors of A and B depends on the age of the pups at conditioning. Indeed, it is known in newborn mammals (especially in altricial mammals, which develop very rapidly) that the meaning acquired by a conditioned odorant can change from one day to another (e.g., Barr et al., 2009; Sullivan and Holman, 2010). In previous experiments, conditioning was mainly performed in 2-day-old rabbit neonates. In the present study, conditioning was therefore conducted either 1 or 3 days after birth to determine the influence of development on the perception and memory of a complex odor stimulus.

These two issues were evaluated by taking advantage of the configural nature of the previously described AB mixture. To date, this is the only mixture which has been extensively characterized regarding its perceptual properties (weak configural perception) in the rabbit both in terms of behavior (Coureaud et al., 2008, 2009a, 2011a; Sinding et al., 2011), memory (Coureaud et al., in press) and brain processing (Schneider et al., in preparation). To that goal, we conditioned either 1- or 3-day-old pups to simple odorants A or B, or to their binary mixture, and assessed the retention of the conditioned stimuli from 24 to 96 h after conditioning using independent groups for each stimulus and each retention interval (Experiment 1). Pups conditioned to the AB mixture were also tested for their responsiveness to A and to B with the aim to evaluate, by means of a within-subject analysis, whether responsiveness to AB differed over time from that of the single odorants (Experiment 2). Finally, as differences appeared in the responsiveness to the mixture and to its odorants in the first two experiments, we determined whether competition occurred between the memory of AB and the respective memories of A and B created during conditioning to the whole mixture (Experiment 3). Indeed, interference and competition between different associative memories have been classically reported (e.g., Eisenberg et al., 2003; Suzuki et al., 2004; Bradfield and Balleine, 2013) and might contribute to the present results. Therefore, we either prevented the formation of the configural memory by successive conditioning to the single odorants only, or erased the elemental memory after initial conditioning to the AB mixture followed by separate reactivations of odorants A and B and amnesiainducing anisomycin (AN) injection (Coureaud et al., 2009b, 2011b, 2013). We then evaluated whether these two procedures influenced the responsiveness to the AB mixture after a long retention interval.

### **MATERIALS AND METHODS**

#### **ANIMALS AND HOUSING CONDITIONS**

Males and females New-Zealand rabbits *Oryctolagus cuniculus* (Charles River strain, L'Arbresle, France) from the Centre de Zootechnie of the University of Burgundy (Dijon) were kept in individual cages. A nest box (0.39 × 0.25 × 0.32 m) was added on the outside of the pregnant females' cages 2 days before delivery (day of delivery was day 0; d0). To equalize pups' nursing experience, all females had access to their nest between 11:30– 11:45 a.m. This procedure allowed females to follow the brief (3–4 min) daily nursing of the species (Zarrow et al., 1965). Animals were kept under a constant 12:12 light:dark cycle (light on at 7:00 a.m.) with ambient air temperature maintained at 21– 22◦C. Water and pelleted food (Lapin Elevage 110, Safe, France) were provided *ad libitum*. In the study, 524 newborns (from 106 litters) were used.

The study was carried out under the local, institutional and national rules (French Ministries of Agriculture, and of Research and Technology) regarding the care and experimental use of the animals. All experiments were conducted in accordance with ethical rules enforced by French law, and were approved by the Ethical Committee for Animal Experimentation (no. 2406).

#### **ODORANTS**

The stimuli consisted of 2-methylbut-2-enal (the Mammary Pheromone, MP, CAS# 497-03-0), ethyl isobutyrate (odorant A, CAS# 97-62-1), ethyl maltol (odorant B, CAS# 4940-11-8) for pure components, and of the AB mixture. This mixture included 0.3 × 10−5 and 0.7 × 10−5 g/ml of components A/B; the 30/70 v/v ratio elicits the configural perception of a pineapple odor in human adults due to blending properties (Le Berre et al., 2008, 2010; Barkat et al., 2012), and weak configural perception in newborn rabbits (Coureaud et al., 2008, 2009a, 2011a; Sinding et al., 2011).

The MP allowed inducing the learning by the pups of odorant A, odorant B, or the AB mixture through associative conditioning (see below). It was used at 10−5 g/ml, a concentration known to be highly efficient to promote conditioning (Coureaud et al., 2006). Thus, the A-MP and B-MP blends were prepared at a final concentration of 10−5 g/ml of each constituent. The AB-MP blend included 1 × 10−5 g/ml of MP and 0.3 and 0.7 × 10−5 g/ml of odorants A and B.

Single odorants A and B were also used in the reactivation procedure (Experiment 3b) at a concentration of 10−5 g/ml.

In all experiments, we deliberately kept constant the overall concentration of the different stimuli (single odorants or AB mixture and blends; 10−5 g/ml) and maintained this constancy between conditioning and testing to avoid direct influence of the concentration on our results and to focus on the influence of complexity (single odorant vs. mixture).

All the odorants were purchased from Sigma-Aldrich (Saint-Quentin Fallavier, France) and all the final solutions were prepared in a solvent composed of 0.1% of ethanol (anhydrous, Carlo Erba, Val de Reuil, France) and 99.9% of MilliQ water (Millipore, Molsheim, France).

#### **ODOR CONDITIONING, REACTIVATION AND PHARMACOLOGICAL TREATMENT**

Conditioning sessions were run on day 1 or 3 after birth in an experimental room close to the breeding room. The pups from an individual litter were transferred by groups of 5 (usual case) or 4 (Experiment 3b) into a box maintained at room temperature. The MP-induced conditioning was run following a procedure previously described (e.g., Coureaud et al., 2006, 2008, 2009a,b, 2013; Sinding et al., 2011; Charra et al., 2013). This procedure offered the advantage of being extremely rapid (single trial) and appetitive (thus avoiding the possible modulation of responsiveness due to the negative emotional state that may occurred after aversive conditioning).

In most cases, just before the conditioning session, 4 ml of the A-MP (Experiment 1), B-MP (Experiment 1) or AB-MP (Experiments 1, 2 and 3b) blends were pipetted on a pad (19 × 14 cm, 100% cotton) then held 2 cm above the pups for 5 min. The conditioning occurred 1 h before the daily nursing (10:30 a.m.) to equalize the pups' motivational state and limit the impact of satiation on behavioral responses (Montigny et al., 2006). Two minutes after the end of the conditioning, the pups were individually marked with ink and returned to their nest. The box containing the pups was rinsed with alcohol and with distilled water after each conditioning session.

In one group (Experiment 3a), the procedure was the same except that pups were conditioned successively to odorants A and B: they were exposed to the A-MP blend for 2.5 min (for the half of the pups, randomly chosen, and B-MP for the other half), then transferred to a second box in which they remained non

stimulated for 1 min before being exposed to the B-MP (or A-MP) blend for 2.5 min.

Finally, in two other groups (Experiment 3b), 24 h after the conditioning to the AB mixture, the memory of pups was reactivated by exposure to odorant A then odorant B in half of the pups (or conversely in the other half) following the same procedure than above (2.5 min per odorant, delay inter-stimulation: 1 min). Immediately after reactivation, anisomycin (AN; Sigma-Aldrich) was injected to the half of the pups (42 mg/kg, i.p.) after dilution in 0.9% NaCl solution and adjustment to pH 7.2 with 1N HCl. The AN was used after reactivation performed at 24 h as we have previously demonstrated the effectiveness of this procedure in erasing memory of odor element(s) in newborn rabbits (Coureaud et al., 2009b, 2011b, 2013, in press). Control for the effect of AN injection was carried out with the other half of animals, which received saline 0.9%. As in other studies with newborn or adult mammals (e.g., Davis and Squire, 1984; Gruest et al., 2004; Desgranges et al., 2008) we considered that AN in newborn rabbits may induce a real amnesia and not a perturbation in responsiveness due to an aversive effect. Pups were returned to the nest just after AN or saline injection.

### **BEHAVIORAL ASSAY**

The behavioral assay occurred 24, 48, 72 or 96 h after the conditioning, i.e., on days 2, 3, 4 or 5 when the conditioning occurred on day 1, and on days 4, 5, 6 or 7 when it happened on day 3. The assay was run in the experimental room previously used for conditioning and reactivation, and happened also 1 h before the daily nursing to limit the impact of satiation on motivation and behavioral responsiveness (Montigny et al., 2006). It consisted of an oral activation test during which a pup was immobilized in one gloved hand of the experimenter, its head being left free. The odor stimulus was presented for 10 s with a glass rod 0.5 cm in front of the nares (e.g., Coureaud et al., 2006, 2008, 2011a, 2013). A test was positive when the stimulus elicited headsearching movements (vigorous, low amplitude horizontal and vertical scanning movements displayed after stretching towards the rod) usually followed by grasping movements (labial seizing of the rod extremity). Non-responding pups displayed no response except sniffing. Pups were tested in groups of 4 or 5 (same groups than during the conditioning), and only once, on a given day (i.e., different groups were tested on different days).

In Experiment 1, the pups were tested for their responsiveness to one stimulus only, except those which were conditioned to the AB mixture. The latter were indeed tested to AB but also (these results are the results of Experiment 2) to odorants A and B. In Experiment 3, the pups were tested to A, B and AB. Successive testing involved the presentation of a first stimulus to a pup, then a second stimulus to another pup, and so on with an inter-trial interval of 60 s. The order of stimuli presentation was systematically counterbalanced from one to another pup. If a pup responded to a stimulus, its nose was softly dried before the next stimulation. The pups were immediately reintroduced in their nest after testing.

#### **STATISTICS**

Each group was composed of 18–20 pups except the groups of Experiment 3b (with saline and AN injection; *n* = 11–12 pups). The proportions of pups responding during the behavioral assay were compared using either the χ 2 -test of Pearson (with Yates correction when necessary) when the groups were independent (i.e., distinct groups tested for their response to a same stimulus on different days, or to different stimuli on a given day), or the Cochran's *Q*-test when the groups were dependent (i.e., pups from a same group tested for their response to the three stimuli). When the Cochran's *Q*-test was significant, proportions of responding pups were compared 2 × 2 by the χ 2 -test of McNemar. Degrees of freedom are indicated when >1. Data were considered as significant when the two-tailed test ended with *p* < 0.05.

### **RESULTS**

#### **EXPERIMENT 1. RETENTION OF A, B AND AB AFTER THEIR RESPECTIVE LEARNING ON DAY 1 OR DAY 3**

To compare the retention of the odors of odorant A, odorant B and of the AB configuration, four groups of pups were conditioned to A on day 1, four other groups to B and four other groups to AB (*n* = 20 pups/group, one pup died in one of the groups; all groups were independent). Each group was tested for its behavioral responsiveness to the conditioned stimulus at one single time point only: 24, 48, 72 or 96 h after the conditioning. The same experiment was conducted with 16 other independent groups of pups conditioned on day 3 instead of day 1 (*n* = 20/group, one pup died in two groups and two pups in another group).

After conditioning on day 1 (**Figure 1**, left column), the responsiveness of pups to the conditioned stimulus decreased over time whatever the nature of the stimulus (i.e., in pups conditioned to A, or B or AB; χ 2 > 19.37, *df* = 3, *p* < 0.001 in the three situations). In pups conditioned to A, the responsiveness to the odorant was maximal and similar 24 and 48 h after the conditioning (>85%; χ 2 < 1, *p* > 0.05), lower at 72 h (55%; χ 2 > 4.3, *p* < 0.05 compared to 24 and 48 h) and 96 h (35%; χ 2 > 8.4, *p* < 0.01 compared to 24 and 48 h), and not different between 72 and 96 h (χ 2 = 1.6, *p* > 0.05). Regarding the pups conditioned to B, the pattern of responsiveness was nearly the same. The responsiveness was maximal and equivalent at 24 and 48 h (>85%; χ 2 = 1.4, *p* > 0.05), lower at 72 h (60%; comparison with 24 h: χ 2 = 7.6, *p* < 0.01, with 48 h: χ 2 = 3.1, *p* = 0.07) and 96 h (42.1%; comparison with 24 and 48 h: χ 2 > 6.03, *p* < 0.01), and similar between 72 and 96 h (χ 2 = 1.2, *p* > 0.05). After conditioning to the AB mixture, the pups highly and similarly responded to AB 24 and 48 h later (95%) and less at 72 h (65%; comparison with 24 and 48 h: χ 2 = 3.9, *p* < 0.05). At 96 h, only 5% of the pups responded, a level which was lower compared to 24, 48 and 72 h (χ 2 > 18.6, *p* < 0.001). Interestingly, whereas the responsiveness of the three categories of pups (conditioned to A, B and AB) did not differ at 24, 48 and 72 h (between categories comparisons: χ 2 < 1.29, *df* = 2, *p* > 0.05), at 96 h the pups conditioned to AB responded less to this stimulus than the pups conditioned to A or B (χ 2 = 7.74, *df* = 2, *p* < 0.05; comparisons AB vs. A or B: χ 2 > 3.91, *p* < 0.05; comparison A vs. B: χ 2 < 0.5, *p* > 0.05).

After conditioning on day 3 (**Figure 1**, right column), the retention of the acquired stimulus changed over time independently of its nature (χ 2 > 8.28, *df* = 3, *p* < 0.05 in the three situations). In pups conditioned to A or B, the responsiveness was maximal and close at 24 and 48 h (>75%; χ 2 < 0.9, *p* > 0.05 for A or B comparisons), lower at 72 h (around 60%) compared to 24 h (χ 2 > 4.8, *p* < 0.05 for A or B), and lower at 96 h compared to 24 and 48 h for A (45%; χ 2 > 5.2, *p* < 0.05) or compared to 24 h for B (50%; χ 2 = 7.4, *p* < 0.001). The responsiveness was similar between 96 and 72 h both in pups conditioned to A and in pups conditioned to B (χ 2 < 0.6, *p* > 0.05). In pups conditioned to AB, the responsiveness to AB was strong and equivalent at 24 and 48 h (>70%; χ 2 = 1.4, *p* < 0.05), but it became extremely weak as soon as 72 h (<10.5%; comparisons between 72 or 96 vs. 24 or 48 h: χ 2 > 11.8, *p* < 0.001). While the responsiveness to the conditioned stimulus was equivalent between pups conditioned to A, B or AB at 24 and 48 h (χ 2 < 0.5, *df* = 2, *p* > 0.05), it was lower in pups conditioned to AB than to A or to B at 72 and 96 h (χ 2 > 8.91, *df* = 2, *p* > 0.05; A or B vs. AB: χ 2 > 4.88, *p* < 0.05; A vs. B: χ 2 < 0.5, *p* > 0.05).

Thus, whatever the information that was learned (A, B, or AB) on either day 1 or 3, its retention by the pups decreased over the 4 post-conditioning days. However, the retention of the AB mixture or of its odorants was not the same: all the pups stopped responding to the mixture 72 and/or 96 h after conditioning while a significant proportion of them (>35%) were still responding to the odorant they have previously learned. Besides the time of memory testing, the animal's age at conditioning also influenced the retention of AB memory: pups conditioned to AB at day 3 stopped responding earlier than those conditioned at day 1 (72 h vs. 96 h). This effect of age was not observed for A and B memories when comparing conditioning at day 1 and 3.

#### **EXPERIMENT 2. RETENTION OF A, B AND AB AFTER LEARNING OF AB ON DAY 1 OR 3**

Results of Experiment 1 showed clear differences in the retention of the odors of A, B and AB when each odorant or the mixture were learned separately by different pups, suggesting that the memory retention of the binary mixture is weaker compared to the retention of its components. Here, we assessed whether similar results could be obtained at the individual level (withinsubject design), namely after conditioning to AB (during which acquisition of the A and B elements and of the AB configuration happens; Coureaud et al., 2008, 2009a, 2011a; Sinding et al., 2011) and during successive testing to the three stimuli. To that goal, the pups conditioned to the AB mixture on day 1 or 3 in Experiment 1 (4 independent groups/day) were tested for their response to A and to B, in addition to AB, at one single time point only, i.e., either at 24, 48, 72 or 96 h. According to the results of Experiment 1, it was expected that the retention of the configuration would be shorter than the retention of each element that compose the mixture.

After conditioning to AB on day 1 (**Figure 2**, upper part), the pups strongly and similarly responded to AB, A and B at 24 and 48 h (>95%; *Q* < 1.1, *df* = 2, *p* > 0.05). At 72 h, the responsiveness decreased for AB (Exp. 1) but also for A and for B (responsiveness to A or B at 72 vs. 24 or 48 h: χ 2 > 3.9, *p* < 0.05); it remained however similar between AB, A and B (65–75%; *Q* = 1.2, *df* = 2, *p* > 0.05). At 96 h, the responsiveness to A and to B was still around 60 and 75% (thus lower than at 24 and 48 h for A: χ 2 = 7.6, *p* < 0.01, equivalent for B: χ 2 = 3.1, *p* = 0.07, and similar for A and for B compared to 72 h: χ 2 < 1.02, *p* > 0.05). Strikingly, in the same animals, while responsiveness to A and B was relatively maintained, the responsiveness to AB was dramatically reduced (5%; *Q* = 23.3, *df* = 2, *p* < 0.001; AB vs. A or B: McNemar χ 2 > 9.09, *p* < 0.01; A vs. B: McNemar χ 2 = 1.3, *p* > 0.05).

Similar results were observed after conditioning on day 3 (**Figure 2**, lower part), though with a slightly different time course. After conditioning to AB on day 3, the responsiveness of pups was high and similar to AB, A and B 24 h later (>90%; *Q* = 3, *df* = 2, *p* > 0.05) and 48 h later (70–85%; *Q* = 3, *df* = 2, *p* > 0.05). At 72 and 96 h, the responsiveness to A and B remained around 50 and 60% (lower than at 24 h, χ 2 > 5.6, *p* < 0.01, but not different than at 48 h, χ 2 < 2.1, *p* > 0.05), but the responsiveness to AB was weaker (<10.5%; for the two periods: *Q* > 14.88, *df* = 2, *p* < 0.001; AB vs. A or B: McNemar χ 2 > 5.14, *p* < 0.05; A vs. B: McNemar χ 2 < 1.3, *p* > 0.05).

Thus, in line with the data from Experiment 1 obtained with independent groups of pups, the responsiveness of newborn rabbits to the mixture and to its components decreased over time after conditioning to the mixture and individual testing to the three A, B and AB stimuli: it disappeared earlier for the mixture than for the odorants, and earlier after conditioning on day 3 than on 1.

Moreover, on postnatal day 5, the proportion of pups still responding to AB was higher in pups conditioned on day 3 than in pups conditioned on day 1 (70 vs. 5%; χ 2 = 15.3, *p* < 0.001) while the responsiveness to A or to B was similar (60–85%; χ 2 < 0.5, *p* > 0.05). This suggests that the difference observed for AB was the consequence of memory processes and not of pure developmental effects.

#### **EXPERIMENT 3. COMPETITION BETWEEN THE MEMORIES OF THE ELEMENTS AND OF THE CONFIGURATION**

According to the results of Experiments 1 and 2, newborn rabbits have a distinct memory and retention of the AB mixture and of its components. After acquisition of the AB mixture, this difference in terms of retention could be due to a competition between the memory of each odorant and the memory of the AB configuration (since the pups perceived both the odorants and the configuration in the weak configurally processed AB mixture). To evaluate this hypothesis, two procedures were followed.

The first procedure (Experiment 3a) attempted to prevent the creation of a memory for the configuration. In a previous paper (Coureaud et al., 2008), we showed that after successive conditioning to odorant A then odorant B on day 1, the pups responded 24 h later to the AB mixture (they did not display such a response after conditioning to a single odorant). Here, this paradigm was repeated but with a test of responsiveness 96 h later. This group (*n* = 20 pups, two of them died, results concerned 18 pups) was compared to the group conditioned to the whole mixture on day 1 and tested on day 5 (+96 h) in Experiment 2 (i.e., a group that learned the AB configuration). Whereas the conditioning to the AB mixture was followed by an absence of responsiveness to AB but not to A and B at 96 h (**Figure 3A**, same results as in **Figure 2**), the successive learning of A and B induced a high and similar level of responsiveness to both the mixture and the individual odorants (66.6–72.2%; *Q* = 1, *df* = 2, *p* > 0.05) (**Figure 3B**). The responsiveness to AB was therefore higher in the situation of successive learning of the elements than after learning of the mixture (72.2 vs. 5%; χ 2 = 15.6, *p* < 0.001). The responsiveness to the odorants was similar in the two experimental conditions (60– 75%; χ 2 < 0.5, *p* > 0.05). Therefore, an absence of perception of the AB configuration during conditioning, due to separate and successive conditioning to odorants A and B, was followed by the maintenance of responsiveness to the AB mixture 4 days later.

The second procedure (Experiment 3b) attempted to promote the creation of memories to both the configuration and the elements after AB conditioning, and to make the pups rapidly amnesic of the individual odorants' odor. We hypothesized that

in this situation, the responsiveness to AB could be maintained 4 days after the conditioning because of the absence of competition between the configural and elemental memories. Thus, two independent groups of newborns (*n* = 12/group, 4 pups/litter, 2 pups/litter/group) were conditioned on day 1 to the AB mixture, and reactivated the day after (day 2) by separated exposure to each individual odorant (A then B for half of the pups in each group, B then A for the other half). Immediately after memory reactivation, the first group was injected with saline, while the other group received an AN injection inducing amnesia. The pups were all tested for their responses to AB, A and B on day 5, 96 h after the conditioning (72 h after the reactivation). Blocking memory for the individual odorants induced major differences in mixture memory between the groups. The pattern of response in the saline-treated group (**Figure 3C**) was the same as in pups from previous experiments which were not manipulated on day 2 (**Figure 3A**): they did not respond to AB but still displayed a

high and similar level of responsiveness to A and to B (8.3 vs. 75.0 vs. 66.6%, respectively; *Q* = 12.6, *df* = 2, *p* < 0.01; AB vs. A or B: McNemar χ 2 > 5.1, *p* < 0.05; A vs. B: McNemar χ 2 < 0.5, *p* > 0.05). In contrast, newborns treated with AN (1 pup died, *n* = 11 for this group in the results) showed a completely reversed pattern of responsiveness to the mixture compared to the elements (*Q* = 10.3, *df* = 2, *p* < 0.01) (**Figure 3D**): they responded strongly to the mixture but very weakly to the individual odorants (63.6 vs. 9.1 vs. 9.1%; AB vs. A or B: McNemar χ 2 = 4.16, *p* < 0.05). Comparison of the responsiveness between the two groups clearly showed that AN-treated pups responded more to AB and less to A and to B than saline-treated neonates (χ 2 > 5.49, *p* < 0.05).

Thus, when rabbit pups forgot the odors of the individual elements initially acquired during the conditioning to the AB mixture, their responsiveness to the mixture was extended over time. This result strongly supports our hypothesis that the learning

of the mixture induced a competition between the memory of the AB configuration and the memory of the elements A and B.

### **DISCUSSION**

The present results, obtained in rabbit pups using the AB weak configural mixture, demonstrate that the memory of odor elements is more robust and lasts longer than the configural memory of the mixture, and in fact can interfere with the maintenance of the configural memory. In the absence of elemental odor memory, the duration of the configural memory of the AB mixture is significantly enhanced. Although further work is required to determine whether these results generalize to other mixtures (weak configurally, configurally or, in contrast, elementally perceived mixtures), the present findings have important implications for understanding the mechanisms of odor mixture (and odor object) perception and the organization of odor mixture memory.

Advantage of elemental over configural memory of the AB mixture was first observed in the duration of memory following conditioning to the elements alone or to the mixture (Experiment 1). For example, conditioning to either element (A or B) alone induced a memory of that element that extended for at least 96 h. In contrast, conditioning to the binary mixture induced memory that extended no more than 72 h. Interestingly, the duration of memory for the mixture was age-dependent, while the duration of elemental memory was not. Animals conditioned to the mixture on day 1 reactivated memory for the mixture for 72 h, while animals conditioned on day 3 displayed mixture memory for no more than 48 h. Additional work is required to determine the mechanisms of this age-dependent variation in the duration of configural memory. Regarding general memory mechanisms, one may hypothesize that the information is more easily processed when the animal is exposed to a single odorant compared to a mixture of odorants. To be encoded correctly, a weak configural mixture could require a higher arousal level than individual odorants perceived separately, since the animals might have to share attentional level between the different stimuli of the mixture to engage associative learning for all stimuli (Sharot and Phelps, 2004). As a result, after only one conditioning session, the memory trace of a complex odor stimulus might be rather fragile and its decay might be faster over time in comparison with more simple stimuli. Here, the present conditioning was appetitive. One may therefore wonder whether similar results would be obtained with aversive conditioning, using odor-malaise association for instance (see Gruest et al., 2004), since a stronger arousal is then supposed to occur during acquisition. It would be of interest to evaluate in future experiments whether this other kind of conditioning induces longer lasting configural memory of the AB mixture.

Advantage of elemental over configural memory of odor mixture was also found after associative conditioning with the AB mixture, and within-subject testing to both the mixture and its elements (Experiments 2 and 3). After conditioning to this mixture known to be perceived configurally in humans (Le Berre et al., 2008; Barkat et al., 2012), rabbit pups expressed a weak configural memory of the mixture, meaning that they expressed both a memory for the mixture's configuration and for the individual elements. In particular, they did not respond to AB at 96 h or 72 h (after conditioning on day 1 or 3, respectively) but still responded to A and to B at these retention times (Experiment 2). In terms of odor mixture processing, these results may seem surprising. Indeed, if the mixture is perceived in a weak configural way during the retention test, rabbit pups should perceive in the mixture both the elements they have previously learned and the AB configuration. Moreover, when pups have learned independently enough elements forming a mixture, they can respond (generalize) to the mixture even if the mixture is perceived in part configurally (Experiment 3a showing responsiveness to AB after successive learning of A and B; see also Coureaud et al., 2008). Therefore, after conditioning to AB on day 1 and because the pups responded to A and to B at 96 h, a response to the mixture could be expected. To explain the lack of response to AB at 96 h, we hypothesize that the AB mixture is, initially, weakly configurally perceived during the conditioning with the MP, but that a particular associative strength is then given to the configuration compared to the elements. As a consequence, the pups would perceive the mixture more as the configuration when they are exposed again to the mixture several days after conditioning. This hypothesis is supported by theoretical considerations suggesting that during conditioning to complex stimulus, a specific value is assigned to the configural representation ("unique cue") independently of that given to each element (Rescorla, 1973; Rescorla et al., 1985). Alternatively, one may assume that during the conditioning, the presence of the MP leads rabbit neonates to perceive the AB mixture less configurally (i.e., weak configurally) compared to its processing in the absence of unconditioned stimulus (robust configural perception).

Importantly, the present results suggest that the long-term memory of the elemental and configural representations of the AB mixture compete. That is, with intact elemental memory, configural memory of the mixture degrades significantly faster than memory of the elements (Experiment 2). In contrast, if elemental memory is disrupted by selective reconsolidation blockade, configural memory of the mixture is maintained significantly longer (Experiment 3b). This indicates that removing a potential source of interference facilitates memory performance for the AB configural information. The retrieved memory of the AB mixture can thus be regarded as the sum of conflicting processes involving, in our case, the elemental memory and the configural memory. The outcome seems dependent on the dominance of one of the memories at the time of retrieval, here the elemental memory, a result that contributes to unravel aspects of memory organization (e.g., Eisenberg et al., 2003; Suzuki et al., 2004; Bradfield and Balleine, 2013). The competition would rely on the retention time (Experiments 1 and 2) and the simultaneous (but not successive; Experiment 3a) exposure to odorants forming a weak configurally perceived mixture. This interference/competition may happen theoretically and first during consolidation of configural and elemental memories of the mixture. However, this assumption has certainly to be ruled out, since we have previously shown in newborn rabbits that consolidation of odor stimuli learned by association with the MP happened in the first 4 h after conditioning (Coureaud et al., 2009b); the consolidation phase is thus terminated well before the erasure of A and B memories performed here (i.e., 24 h after conditioning; Experiment 3b). Therefore, in the present paradigm, interference/competition between configural and elemental memories of the mixture certainly occurs during maintenance and/or recall of the memories rather than during consolidation phase, with elemental memory the stronger of the two. Even if the most probable explanation for the absence of response to AB at 96 h is the absence of recognition of the AB configuration, one can not exclude the possibility that rabbit pups still find familiarity in AB but without any motivational significance.

The present results do not allow direct determination of the mechanisms of elemental and configural memory interaction, although hypotheses based on the known neurobiology of odor mixture processing can be developed. In rodents, odor mixtures are processed differently by the olfactory bulb and primary olfactory (piriform) cortex (Wilson and Sullivan, 2011). For example, the activity of olfactory bulb mitral/tufted cell neural ensembles is strongly consistent with pattern separation processing– responding uniquely to even minor changes in mixture elements (Barnes et al., 2008; Chapuis and Wilson, 2011; Sahay et al., 2011). Thus, it has been hypothesized that olfactory bulb neural ensembles respond to odor mixture features or components, rather than to the mixture configuration. In strong contrast, piriform cortical neural ensembles respond in a manner consistent with configural processing. In fact, even at the single-unit level of the piriform cortex, individual cells can distinguish between mixtures and their components in cross-adaptation procedures (Wilson, 2003). Mitral/tufted cells, on the other hand, show strong cross-adaptation between mixtures and their components, again consistent with an elemental or feature-dependent process within the olfactory bulb (Wilson, 2003). After having reported in newborn rabbits mapping of activations induced by the MP or by a MP-learned odorant in the olfactory bulb and central regions, including the piriform cortex (Charra et al., 2012, 2013) we are currently assessing the brain activity mapping of the AB mixture (Schneider et al., in preparation).

Odor memory is dependent on plasticity within both the olfactory bulb and piriform cortex (Wilson et al., 2004), even in neonates (Moriceau and Sullivan, 2005; Charra et al., 2013; Fontaine et al., 2013). In the context of the present results, and waiting for further results obtained with the AB mixture as with other odor mixtures, we hypothesize that the more stable elemental memory of an odor mixture may mainly depend on olfactory bulb plasticity, while configural memory of the same mixture may mainly depend on piriform cortical processes (in accordance with the presumed role of piriform cortex as an associative cortex; Johnson et al., 2000). These cortical processes may be less robust during early development than memorydependent events in the olfactory bulb, resulting in the distinction between these two memory forms in the present study. The less robust configural memory might depend on differences in the developmental program of both the olfactory bulb and piriform cortex: functional odor maps in the glomerular layer seems rather well defined after birth (Guthrie and Gall, 1995) whereas the maturation of inhibitory processes and the intracortical associational fibers throughout the 3-layered piriform cortex (Schwob and Price, 1984) might follow a slightly different time curve (Garske et al., 2013). One may also suggest that this distinction does not rely on robustness of cortical processing only, but on changes during animal development in the dialogue between sensory/mnesic regions of the brain (olfactory bulb/piriform cortex/amygdala/hippocampus), in accordance with the behavioral and adaptive needs of the animal (Schacher and Hu, 2014). Future work could combine the powerful reconsolidation-mediated dissection of memory utilized here, with neurophysiological techniques to explore neurobiological underpinnings of these basic memory and perceptual processes.

Finally, the present findings are further evidence that while odor mixtures are perceived sometimes configurally, and conscious analysis of odor mixtures is notoriously difficult (e.g., Jinks and Laing, 1999), information about the underlying individual components can remain intact in both neonatal rabbits (shown here) and adult humans (Grabenhorst et al., 2007). In the case of newborn rabbits, this may be especially important given the life and death importance of odor recognition to interact with the mother, attach to the nipples, survive and grow up. Having access to both the elements and the configuration may help ensure successful recognition, improved discrimination between odorous substrates or conspecifics in the surroundings, adaptation to the actual environment or anticipation of social and feeding changes that will come later in development.

#### **ACKNOWLEDGMENTS**

We sincerely thank Valérie Saint-Giorgio, Nicolas Malaty, Florent Costilhes, Jérôme Antoine and all the Centre de Zootechnie from the University of Burgundy for their cooperation. The work was supported by French ANR-2010-JCJC-1410-1 MEMOLAP to Gérard Coureaud, Thierry Thomas-Danguin, Frédérique Datiche and Guillaume Ferreira.

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