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Behavioural Brain Research 274 (2014) 62–72

Contents lists available at ScienceDirect

Behavioural Brain Research

jou rn al hom epage: www.elsev ier .com/ locate /bbr

esearch report

eriaqueductal gray � and � opioid receptors determine behavioralelection from maternal to predatory behavior in lactating rats

arianne Orlandini Kleina,b, Aline de Mello Cruza, Franciele Corrêa Machadob,d,isele Picolod, Newton Sabino Canterasc, Luciano Freitas Felicioa,∗

Department of Pathology, School of Veterinary Medicine, University of São Paulo, Avenida Professor Doutor Orlando Marques de Paiva, 87,ão Paulo, SP CEP 05508-270, BrazilDepartments of Pharmacology, Institute of Biomedical Sciences, University of São Paulo, Avenida Prof. Lineu Prestes, 1524, Biomédicas I, São Pauo,P CEP 05508-900, BrazilDepartment of Anatomy, Institute of Biomedical Sciences, University of São Paulo, 2415, São Paulo, SP CEP 05508-900, BrazilSpecial Laboratory of Pain and Signaling, Butantan Institute, Avenida Vital Brazil, 1500, São Paulo CEP 05503-900, Brazil

i g h l i g h t s

The rlPAG � and � opioid receptors have a role in behavioral selection.Morphine in the rlPAG inhibits maternal behavior without interfering with hunting.� Receptor blockade increases hunting and increases � receptor activation.Multiple opioid receptor interactions play a role in behavioral selection.

r t i c l e i n f o

rticle history:eceived 17 April 2014eceived in revised form 21 July 2014ccepted 4 August 2014vailable online 10 August 2014

eywords:ehavioral selectionaternal behavior

redatory huntingeriaqueductal gray

a b s t r a c t

Every mother must optimize her time between caring for her young and her subsistence. The rostro lateralportion of the periaqueductal grey (rlPAG) is a critical site that modulates the switch between maternaland predatory behavior. Opioids play multiple roles in both maternal behavior and this switching process.The present study used a pharmacological approach to evaluate the functional role of rlPAG � and �opioid receptors in behavioral selection. Rat dams were implanted with a guide cannula in the rlPAGand divided into three experiments in which we tested the role of opioid agonists (Experiment 1), theinfluence of � and � opioid receptor blockade in the presence of morphine (Experiment 2), and theinfluence of � and � opioid receptor blockade (Experiment 3). After behavioral test, in Experiment 4,we evaluated rlPAG � and � receptor activation in all Experiments 1–3. The results showed that massiveopioidergic activation induced by morphine in the rlPAG inhibited maternal behavior without interferingwith predatory hunting. No behavioral changes and no receptor activation were promoted by the specificagonist alone. However, � receptor blockade increased hunting behavior and increased the level of �

receptor activation in the rlPAG. Thus, endogenous opioidergic tone might be modulated by a functionalinteraction between opioid receptor subtypes. Such a compensatory receptor interaction appears to berelevant for behavioral selection among motivated behaviors. These findings indicate a role for multipleopioid receptor interactions in the modulation of behavioral selection between maternal and predatorybehaviors in the PAG.

© 2014 Elsevier B.V. All rights reserved.

. Introduction

Lactating mammalian mothers must optimize the constantxchange between reproduction and subsistence [1,2]. Indeed, a

∗ Corresponding author. Tel.: +55 11 3091 7934; fax: +55 11 3091 7829.E-mail address: lfelicio@usp.br (L.F. Felicio).

ttp://dx.doi.org/10.1016/j.bbr.2014.08.008166-4328/© 2014 Elsevier B.V. All rights reserved.

mother has to use her time more efficiently than a virgin femaleto express maternal and predatory behaviors, which has adaptivevalue [3–5].

In mammals, maternal behavior is fundamental for perpetua-

tion of the species, in which the neonate’s survival depends on themother and her ability to provide food, heat, shelter, and protec-tion [6]. Predatory behavior is also essential for individual survival,and it is characteristic of the specie. During the post-partum period,

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M.O. Klein et al. / Behavioura

he time spent foraging and hunting depends on environmental cir-umstances [2]. As a stimulus to elicit predatory behavior, we usedockroaches, which appear to be suitable prey and have an inherentedonic value for the rat [7–11].

The periaqueductal gray (PAG) is an important region of theentral nervous system that is involved in multiple behavioral andhysiological processes, including nociception, fear, anxiety, car-iovascular control, sexual behavior, and vocalization [12]. Beyondhese functions, previous studies have demonstrated that the PAG,

ainly its rostrolateral portion (rlPAG), also plays an importantole in maternal and predatory behavioral selection, and opioidsre involved in this switch between behavioral modes [2,3,11,13].

Opioids are well established to be involved in the regulationf maternal behavior [14–16]. Morphine is a nonselective opioidgonist that acts preferentially at � opioid receptors [17]. A sin-le injection of morphine directly into the rlPAG impairs maternalehavior [18]. Nevertheless, the specific effects of a morphine injec-ion in the rlPAG on predatory hunting vs. maternal behavior haveot been investigated previously.

Different types of opioid receptors have been described, and the and � subtypes are the most widely expressed and functionally

elevant [19]. Nevertheless, unknown is the functional relevancef these receptors in the rlPAG for behavioral selection during theost-partum period. An intracerebroventricular injection of a �-pecific agonist in lactating females dose-dependently disruptedaternal behavior [20]. Acute peripheral injections of a specific �

pioid agonist increased the latency to retrieve pups [21].In the present study, lactating females were tested for behav-

oral selection using various pharmacological challenges thatnvolved both stimulation and blockade of rlPAG � and � opi-id receptors. The state of receptor activation was assessed by anmmunological assay. Our results showed that the central func-ional interaction between both receptors might be important for

aintaining the normal expression of predatory behavior, and mor-hine injected directly into the rlPAG was shown to act on botheceptors to inhibit maternal behavior.

. Materials and methods

.1. Animals

Female nulliparous Wistar rats were ∼90 days of age at theeginning of the experiments. They were mated with sexu-lly experienced males, and the day that sperm was observedn the vaginal lavage was considered day 1 of pregnancy.regnant females were individually housed in polypropylene cages30 cm × 40 cm × 18 cm) with pine flakes. They had free access toater and food during all of the experiments. The light/dark cycle

12 h/12 h) and temperature (23 ± 2 ◦C) were controlled. On day5–17 of pregnancy, females underwent stereotaxic surgery to

mplant a unilateral guide cannula in the rlPAG. After giving birthday 0 of lactation), the dams remained with their litter, which wasulled to eight pups (four males and four females) on day 2 of lacta-ion until the day of the behavioral test. All of the procedures weren accordance with the Ethical Principles of Animal Experimenta-ion adopted by the Sociedade Brasileira de Ciências em Animais deaboratório (SBCAL) and approved by the Comissão de Ética no Usoe Animais (CEUA) of the Biomedical Sciences Institute and Schoolf Veterinary Medicine at the University of São Paulo.

.2. Stereotaxic surgery

On day 15–17 of pregnancy, the females were intraperitoneallynesthetized with a mixture of ketamine (60 mg/kg Vetanarcol,antana de Parnaíba, SP, Brazil) and xylazine (5 mg/kg Kensol,

Research 274 (2014) 62–72 63

Santana de Parnaíba, SP, Brazil) and received 3 mg/kg dipyrone(Ibasa, Porto Alegre, RS, Brazil) for postsurgical analgesia. Thefemales were then placed in a stereotaxic apparatus to implant aguide cannula (Plastic One, Roanoke, VA, USA) in the rlPAG. Thestereotaxic coordinates were −6.0 mm from bregma, −0.6 mm lat-eral to the midline, and 4.2 mm ventral to the surface of the brain[22]. On the test day, the volume of drug or saline (0.6 �l) wasinjected over 30 s, and the internal cannula was left in place foran additional 30 s to allow for diffusion. The internal cannula was1 mm longer than the guide cannula, allowing precise injection intothe rlPAG. Only animals with correct cannula placements were usedin the study.

2.3. Behavioral analyses

Behavioral test was not performed in the home cage of animals.The test cage was constructed of transparent Plexiglas to allowcomplete visualization of the pups and cockroaches and preventthe insects from escaping. To permit complete observation of boththe insects and mammals’ behaviors, no pine flakes were used inthese cages. One day before the tests, the dams were habituatedto the test cage. Females without their pups were habituated toexploring the experimental cage for 30 min. The bottom of theexperimental cage was divided by two perpendicular lines. Eachtime the animal crossed a line, it was counted as an indication oflocomotion. Behavioral tests were performed on day 5 or 6 of lac-tation. The animals were tested for behavioral selection (i.e., caringfor pups or predatory hunting) and were not food deprived beforethe tests. On the test day, 60 min before the test, lactating femaleswere placed in the test cage without their pups. Thirty minuteslater, the dams received a drug/saline injection, and the behavioraltest began 30 min after the injection. Eight pups and five maturecockroaches (Leurolestes circunvagans) were dispersed introducedto the cage at the beginning of the behavioral test. All of the trialswere recorded for subsequent analysis. During the 30 min trial, thefollowing maternal behavior parameters were analyzed: latencyto retrieve the 1st, 5th, and 8th pups, the percentage of damsthat retrieved these pups, the number of contacts with the pups(i.e., when lactating females only touched them without retrievingthem), the number of times the pups were licked, the percentageof dams that nursed their pups, and the expression of full maternalbehavior (FMB), in which the dam, after retrieving and groupingat least five pups, arched over them with her legs splayed, withthe pups attached to the nipples, and the dam remained in thisposition for at least three consecutive minutes. For the predatoryhunting analysis, we recorded the latency to capture each cock-roach, percentage of dams that captured the 1st, 2nd, 3rd, 4th, and5th insects, and total number of insects hunted. General parame-ters, such as the number of lines crossed, time spent exploring thecage, and self-grooming time, were also analyzed.

In all of the experiments, the experimental cage was cleanedwith a 5% alcohol solution before each behavioral test to eliminatepossible bias caused by odors left by previous animals. Experimen-tal and control observations were intermixed to minimize possiblecircadian influences on the dams’ behavior.

2.4. Experiment 1

The goal of this experiment was to determine whether injec-tions of morphine and the � and � opioid agonists in the rlPAGinterfere with maternal behavioral selection and predatory hunt-ing. Behavioral analyses were performed in four different groups:

morphine (MOR; n = 13; 7.91 nmol morphine sulfate; Dimorf, SãoPaulo, SP, Brazil), � opioid receptor agonist (�; n = 12; 10 nmolDAMGO; Sigma-Aldrich, St Louis, MO, USA), � opioid receptor ago-nist (�; n = 12; 10 nmol U69593; Tocris Bioscience, Bristol, UK), and

64 M.O. Klein et al. / Behavioural Brain Research 274 (2014) 62–72

Fig. 1. Injection loci. (A) Photomicrograph of the periaqueductal gray (PAG) with Nissl staining. (B) Diagram that shows the main injection loci in each group. Because ofoverlap, the number of illustrated points may be less than the actual number of injections. The injections were made unilaterally. Each figure represents the values of PAGr 0 mm)a

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egions posterior to bregma (−5.8 mm, −6.04 mm, −6.3 mm, −6.72 mm, and −6.8queduct; EW, Edinger–Westphal nucleus.

dapted from reference [22].

ontrol (CS; n = 10; 0.9% physiological saline) [20]. The injectionsere performed as described above. The dose of morphine sulfateas not equimolar to the other drug doses because of technical

ssues. Morphine is already sold diluted; thus, an equimolar mor-hine dose would imply a drug volume that is too large to be

njected in the animal brain [18].

.5. Experiment 2

In this experiment, we tested the importance of � and � opioideceptors in morphine’s action. Four groups were formed: mor-hine (MOR; n = 13; 7.91 nmol morphine sulfate; Dimorf, São Paulo,P, Brazil), � opioid receptor antagonist plus morphine (N�M;

= 11; 10 nmol of the � opioid receptor antagonist CTAP [Tocrisioscience, Bristol, UK] plus 7.91 nmol morphine sulfate), � opioideceptor antagonist plus morphine (N�M; n = 12; 10 nmol of the

opioid antagonist nor-binaltorphimine dihydrochloride (Tocrisioscience, Bristol, UK) plus 7.91 nmol morphine sulfate), and con-rol (CS; n = 10; which received 0.9% physiological saline) [20]. Thenjections and behavioral analyses were performed as describedbove. For the groups that was necessary the injection of two drugs,rst it was administered the antagonist drug and 30 s after, theorphine.

.6. Experiment 3

Considering the hypothesis that opioid receptor subtypes play

ifferential roles in behavioral selection, this experiment was per-ormed to investigate the role of endogenous � and � opioideceptor subtypes in behavioral selection using their respectiventagonists. Three different groups were tested: � opioid receptor

. Only animals that had correct injection sites were considered in this study. Aq,

antagonist (N�; n = 12; 10 nmol of the � opioid receptor antag-onist CTAP), � opioid antagonist (N�; n = 11; 10 nmol of the �opioid receptor antagonist nor-binaltorphimine dihydrochloride),and control (CS; n = 10; which received 0.9% physiological saline).All of the behavioral analyses and drug injections were performedas described above.

2.7. Experiment 4

This experiment was conducted to evaluate the activation of �and � opioid receptors under the conditions of the previous exper-iments, i.e., each group for each experiment (1–3) as previouslyreported was submitted to an immunofluorescence assay after thebehavioral test to evaluate the activation of � and � receptors underdrugs action in rlPAG. Thus, conformation state-sensitive anti-�and anti-� antibodies were used. When the receptors are activated,they undergo a conformational change that is detected by theseantibodies, thus recognizing the activated state of the receptor. Itis a well established and validated technique [23,24]. To performthis experiment, 60 min after the end of the behavioral test, thedams were anesthetized with 70 mg/kg ketamine (Vetanarcol, San-tana de Parnaíba, SP, Brazil) and 10 mg/kg xylazine (Kensol, Santanade Parnaíba, SP, Brazil) and transcardially perfused with a solutionof 4.0% formaldehyde and sodium tetraborate, pH 7.4. The brainswere removed and left overnight in a solution of 20% sucrose in0.1 M phosphate buffer at 4 ◦C. Frozen brain sections (30 �m) ofthe rlPAG were cut with a sliding microtome in the frontal plane.

The sections were mounted on gelatin-coated slides and blockedwith 1% bovine serum albumin + 5% sucrose and processed forimmunostaining. Tissue samples were incubated overnight withconformation state-sensitive anti-� and anti-� primary antibodies

M.O. Klein et al. / Behavioural Brain Research 274 (2014) 62–72 65

Fig. 2. Predatory hunting parameters in Experiment 1. CS, control group (n = 10); MOR, morphine group (n = 13); �, � receptor agonist group (DAMGO; n = 12); �, � receptora ral test Kruskc

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gonist group (U69593; n = 12). (A) Number of insects captured during the behaviohe 1st, 2nd, 3rd, 4th, and 5th cockroaches. The data are expressed as mean ± SEM (ockroaches. p > 0.05 (Fisher’s exact test).

Proteimax Biotechnology, Cotia, SP, Brazil) labeled with fluores-ent Alexa Fluor dye (682 nm for � opioid receptors and 800 nmor � opioid receptors) diluted 1:4000. The Odyssey system (Li-OR) was used to analyze and quantify the intensity of fluorescence.ifferences in receptor activation were evaluated by considering,eyond the tissue area, the total number of each receptor expressed

n the same brain tissue using � opioid receptor (N-terminus) and opioid receptor-1 (N-19) rabbit polyclonal immunoglobulin Giluted 1:100 (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

.8. Statistical analysis

Because of the number of mothers that did not retrieveheir pups, we expressed the latency to retrieve each pups the following latency scores: 1 (latency = 0–300 s), 2latency = 301–600 s), 3 (latency = 601–900 s), 4 (latency:01–1200 s), 5 (latency = 1201–1500 s), 6 (latency: 1501–1800 s),nd 7 (when the dams do not retrieve the pups during the 1800 sf the behavioral test). We were thus able to include all of theams in the statistical analysis. One-way analysis of varianceANOVA) was used for predatory parameters (i.e., the number of

ockroaches captured and latency to capture each cockroach),eneral parameters (i.e., the time exploring the cage, number ofines crossed in the cage, and self-grooming time), and maternalehavior of licking the pups. The Kruskal–Wallis test was used

t. The data are expressed as mean ± SEM (one-way ANOVA). (B) Latency to captureal–Wallis test). (C) Percentage of dams that captured the 1st, 2nd, 3rd, 4th, and 5th

for pup retrieval latency scores. Fisher’s exact test was used toanalyze the percentage parameters, such as the dams that cap-tured cockroaches, dams that retrieved pups, dams that expressednursing behavior, and dams that expressed FMB. In Experiment 4,a one-way ANOVA was used to do a comparison of the groups ofeach experiment (1–3; a group of Experiment 1 was not comparedto a group of Experiment 3, for example; and the activation of� and � receptors were not compared to each other). Data areexpressed as a percentage of control activation, i.e. the closerthe activation is of 100% more similar to control’s activation itis. Statistical significance was set at p < 0.05 for all the data. Thestatistical analyses were performed using GraphPad Prism 5.0.

3. Results

For all of the experiments, only the animals that had the guidecannula implanted correctly into the rlPAG were considered (Fig. 1).Dams did not exhibit any difference in the number of lines crossed,time spent exploring the cage, and self-grooming time in anyof the behavioral experiments, indicating that the drugs did notimpair general parameters (p > 0.05, data not shown). Through-

out the observation period, the dams in the MOR group and othergroups that received morphine or specific opioid drug injectionsin Experiments 2 and 3 did not show any stereotypical behavior,motor rigidity, or signs of sedation that morphine may cause at

66 M.O. Klein et al. / Behavioural Brain Research 274 (2014) 62–72

Fig. 3. Maternal parameters in Experiment 1. CS, control group (n = 10); MOR, morphine group (n = 13); �, � receptor agonist group (DAMGO; n = 12); �, � receptor agonistg transf5 pressd group

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roup (U69593; n = 12). (A) Latency to retrieve the 1st, 5th, and 8th pups after being: 1201–1500 s; 6: 1501–1800 s; 7: no retrieval during 30 min test). The data are exams that retrieved each of their pups. *p < 0.05, compared with control (CS) and �

igher doses [25]. Animals that expressed hunting behavior sniffedround the cage, some more vigorously than others, depending onhe group, foraged the prey normally, and attempted to seize therey with apparently no difficulty. These results indicate that thenimals exhibited no motor alterations caused by the drugs.

.1. Experiment 1: Role of morphine, � and � opioid agonists inlPAG in behavioral selection

In Experiment 1, the effects opioids’ action in the rlPAG by mor-hine and the agonists of � and � receptors varied. Both morphinend the � and � opioid receptor-specific agonists did not interfereith the predatory parameters (Fig. 2). No differences were found

mong groups in the number of cockroaches captured, latency toapture each cockroach, and percentage of dams that captured eachf the five cockroaches. Maternal parameters were impaired inhe MOR group compared with the CS group (Fig. 3). The laten-ies scores to retrieve the 1st (p = 0.007), 5th (p = 0.005), and 8thp = 0.050) pups were longer in the dams that received morphineompared with controls. The percentage of animals that retrievedach pup was smaller in the MOR group compared with the CSroup (p < 0.05 for all comparisons). The time spent licking the pupsas less in the MOR group compared with the CS and � groups

F3,46 = 4.526, p = 0.0076), but the percentages of animals that dis-layed FMB and nursing and number of pup contacts were notifferent among groups (Table 1). Stimulation of a single � or �pioid receptor did not significantly interfere with the predatoryr maternal parameters.

No significant differences were found in the predatory param-ters during the entire time of observation. Thus, considering theotal time of the test, the control group hunted as well as the MORroup. To test whether the groups had different hunting strategies,

able 1aternal parameters analyzed for 1800 s in Experiment 1.

Parameter Group

CS MOR � �

FMB (%) 20.0 0.0 16.7 16.7Nursing (%) 60.0 38.5 50.0 75.0Pup contact 7.0 ± 2.6 4.8 ± 1.0 5.7 ± 1.4 6.8 ± 1.1Licking (s) 130.1 ± 24.9 35.8 ± 14.0* 131.9 ± 29.9 116.6 ± 18.1

MB, full maternal behavior; CS, control group (n = 10); MOR, morphine groupn = 13); �, � receptor agonist group (DAMGO; n = 12); �, � receptor agonist groupU69593; n = 12). FMB and nursing behavior were analyzed using Fisher’s exact test.up contacts were analyzed using the Kruskal–Wallis test. Licking behavior wasnalyzed using one-way ANOVA followed by Tukey’s test. The data are expressed asean ± SEM.* p < 0.05, compared with CS and � groups.

ormed into rank-order data (1: 0–300 s; 2: 301–600 s; 3: 601–900 s; 4: 901–1200 s;ed as mean ± SEM (Kruskal–Wallis test followed by Dunn’s test). (B) Percentage ofs; #p < 0.05, compared with CS group (Fisher’s exact test).

we evaluated primary motivation at the beginning of the test (i.e.,whether the dams preferred to hunt before or after caring for theirpups). Therefore, we decided to compare the number of insectscaptured in the initial 15 min (0–900 s) with the total time of thetest (1800 s). We did the same with the number of pups retrieved(Table 2). Indeed, the control group preferred to retrieve most oftheir pups prior to capturing the cockroaches, whereas the MORgroup captured almost all of the cockroaches at the beginning ofthe test. The � group, although not statistically significant, tendedto behave similarly to the MOR group, whereas the � group wasmore similar to the control group.

Thus, morphine appeared to act at both receptors at the sametime to disrupt maternal behavior. To evaluate the importance ofeach of these opioid receptor subtypes in morphine’s action, weperformed Experiment 2.

3.2. Experiment 2: Blockade of � and � opioid receptors face ofmorphine action in rlPAG

In this experiment, � opioid receptor blockade under the actionof morphine in the rlPAG impaired hunting behavior in these dams(Fig. 4). The N�M group captured fewer cockroaches than the CSand MOR groups (F3,45 = 4.147, p = 0.0116). Furthermore, the laten-cies to capture the 1st to 4th cockroaches were longer in theN�M group compared with the CS and MOR groups (p < 0.05 forall comparisons). The percentage of lactating females in the N�Mgroup that captured each of the five insects was lower (p < 0.05 forall comparisons). With regard to maternal parameters, all of thegroups that received a morphine injection (i.e., MOR, N�M, and

N�M groups) exhibited some disruptions of aspects of this behav-ior (Fig. 5). The latency scores to retrieve the 1st pup in all of thesegroups were longer compared with the CS group (p = 0.001). TheMOR and N�M group also longer latency scores to retrieve the 5th

Table 2Insects captured and pups retrieved in the initial 900 s and during the entire behav-ioral test (1800 s) in Experiment 1.

Parameter Group

CS MOR � �

Insects captured until 900 s 1.5 (0–5) 4 (0–5) 3.5 (0–5) 0 (0–5)Insects captured during 1800 s 4 (0–5) 5 (0–5) 5 (0–5) 3 (0–5)Pups retrieved until 900 s 7 (0–8) 0 (0–8)* 5 (0–8) 5.5 (0–8)Pups retrieved during 1800 s 8 (7–8) 0 (0–8)** 5.5 (0–8) 8 (0–8)

CS, control group (n = 10); MOR, morphine group (n = 13); �, � receptor agonistgroup (DAMGO; n = 12); �, � receptor agonist group (U69593; n = 12). The data areexpressed as median (range).

* p < 0.05, compared with CS and � groups.** p < 0.05, compared with CS group (Kruskal–Wallis test followed by Dunn’s test).

M.O. Klein et al. / Behavioural Brain Research 274 (2014) 62–72 67

Fig. 4. Predatory hunting parameters in Experiment 2. CS, control group (n = 10); MOR, morphine sulfate group (n = 13); N�M, � receptor antagonist (CTAP) + morphine group(n = 11); N�M, � receptor antagonist (nor-binaltorphimine) + morphine group (n = 12). (A) Number of insects captured during the behavioral test. The data are expressedas mean ± SEM (one-way ANOVA followed by Tukey’s test). (B) Latency to capture the 1st, 2nd, 3rd, 4th, and 5th cockroaches. The data are expressed as mean ± SEM(Kruskal–Wallis test). (C) Percentage of dams that captured the 1st, 2nd, 3rd, 4th, and 5th cockroaches. *p < 0.05, compared with CS and MOR groups; #p < 0.05, comparedwith CS group; **p < 0.05, compared with MOR group (Fisher’s exact test).

Fig. 5. Maternal parameters in Experiment 2. CS, control group (n = 10); MOR, morphine sulfate group (n = 13); N�M, � receptor antagonist (CTAP) + morphine group(n = 11); N�M, � receptor antagonist (U69593) + morphine group (n = 12). (A) Latency to retrieve the 1st, 5th, and 8th pups after being transformed into rank-order data (1:0–300 s; 2: 301–600 s; 3: 601–900 s; 4: 901–1200 s; 5: 1201–1500 s; 6: 1501–1800 s; 7: no retrieval during 30 min test). The data are expressed as mean ± SEM (Kruskal–Wallistest followed by Dunn’s test). (B) Percentage of dams that retrieved each of their 1st, 5th, and 8th pups. *p < 0.05, compared with control (CS) group (Fisher’s exact test).

68 M.O. Klein et al. / Behavioural Brain

Table 3Maternal parameters analyzed during 1800 s in Experiment 2.

Parameter Group

CS MOR N�M N�M

FMB (%) 10.0 0.0 0.0 0.0Nursing (%) 80.0 38.5 9.0* 8.3*

Pup contact 5.4 ± 1.2 4.3 ± 0.9 2.09 ± 0.6 5.1 ± 1.5Licking (s) 120.8 ± 26.0 44.1 ± 15.8 9.8 ± 5.07* 36.9 ± 15.1*

FMB, full maternal behavior; CS, control group (n = 10); MOR, morphine sulfate group(n = 13); N�M, � receptor antagonist (CTAP) + morphine group (n = 11); N�M, �receptor antagonist (U69593) + morphine group (n = 12). FMB and nursing behaviorwere analyzed using Fisher’s exact test. Pup contacts and licking behavior were ana-la

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yzed using the Kruskal-Wallis test followed by Dunn’s test. The data are expresseds mean ± SEM.

* p < 0.05, compared with CS group.

p = 0.0041) and 8th (p = 0.0003) pups compared with controls. Lesshan 40% of the dams in the MOR, N�M, and N�M groups retrievedhe pups, whereas at least 80% of the dams in the CS group retrieved

hem (p < 0.05 for all comparisons). The number of dams that dis-layed nursing and licking behaviors was less in the N�M and�M groups compared with the CS group (Table 3; p < 0.01 for allomparisons). Because we found that predatory parameters were

ig. 6. Predatory hunting parameters in Experiment 3. CS, control group (n = 10); N�, �inaltorphimine; n = 11). (A) Number of insects captured during the behavioral test. The

atency to capture each cockroach. The data are expressed as mean ± SEM (Kruskal–Wallrd, 4th, and 5th cockroaches. *p < 0.05, compared with N� group (Fisher’s exact test).

Research 274 (2014) 62–72

altered by the blockade of � opioid receptors, we further inves-tigated the endogenous tone of � and � receptors in Experiment3.

3.3. Experiment 3: Blockade of � and � opioid receptors in rlPAGbehavioral selection

In this experiment, dams in the N� group that received the � opi-oid receptor antagonist into the rlPAG captured more cockroaches(F2,32 = 4.754, p = 0.0161) and had shorter latencies to capture eachcockroach (p < 0.05 for all comparisons). Additionally, 100% of theN� dams captured at least the 1st cockroach, whereas only 70% and41% of the animals displayed such behavior in the control and N�groups, respectively (p < 0.05; Fig. 6). With regard to the maternalparameters, the latency scores to retrieve the 1st, 5th, and 8th pupswere longer in the N� group compared with the CS group (Fig. 7A;p < 0.04 for all comparisons). The percentage of dams that retrievedeach pup was lower in the N� group (Fig. 7B; p < 0.04). Dams in the

N� group displayed less nursing behavior (p < 0.04) and spent lesstime licking their pups (F2,32 = 4.390, p = 0.0213; Table 4).

Animals in Experiment 3 had different hunting strategies(Table 5). In the first 900 s of the test, N� dams caught all of the

receptor antagonist group (CTAP; n = 12), N�, � receptor antagonist group (nor-data are expressed as mean ± SEM (one-way ANOVA followed by Tukey’s test). (B)is test followed by Dunn’s test). (C) Percentage of dams that captured the 1st, 2nd,

M.O. Klein et al. / Behavioural Brain Research 274 (2014) 62–72 69

Fig. 7. Maternal parameters in Experiment 3. CS, control group (n = 10); N�, � receptor antagonist group (CTAP; n = 12); N�, � receptor antagonist group (nor-binaltorphimine;n = 11). (A) Latency to retrieve the 1st, 5th, and 8th pups after being transformed into rank-order data (1: 0–300 s; 2: 301–600 s; 3: 601–900 s; 4: 901–1200 s; 5: 1201–1500 s;6 SEM

e d with

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: 1501–1800 s; 7: no retrieval during 30 min test). The data are expressed as mean ±ach of their pups. *p < 0.05, compared with control (CS) group; **p < 0.05, compare

nsects and retrieved most of the pups, whereas dams in the CSroup first retrieved the pups and then captured the cockroaches.ost of the animals in the N� group did not capture roaches (nine

f 12 dams) or take care of pups (six of 12 dams).

.4. Experiment 4: Opioids receptor activation in the rlPAG

To evaluate the activation of � and � opioid receptors, we per-ormed Experiment 4. The intensity of fluorescence (red for �eceptors and green for � receptors) indicates the level of activa-

ion of these receptors caused by drug stimulation in each of theroups in Experiments 1–3 (Fig. 8A). The control group exhibited aasal tone of activation of both receptors, whereas morphine sul-ate significantly activated both � and � receptors in the MOR group

able 4aternal parameters analyzed during 1800 s in Experiment 3.

Parameter Group

CS N� N�

FMB (%) 20.0 0.0 27.3Nursing (%) 70.0 16.7* 63.6Pup contact 5.9 ± 1.0 4.92 ± 1.1 4.8 ± 0.8Licking (s) 104.5 ± 21.4 34.5 ± 15.4# 70.3 ± 13.2

MB, full maternal behavior; CS, control group (n = 10); N�, � receptor antagonistroup (CTAP; n = 12); N�, � receptor antagonist group (nor-binaltorphimine; n = 11).MB and nursing behavior were analyzed using Fisher’s exact test. Pup contacts andicking behavior were analyzed using one-way ANOVA followed by Tukey’s test. Theata are expressed as mean ± SEM.

* p < 0.05, compared with CS and N� groups.# p < 0.05, compared with control group.

able 5nsects captured and pups retrieved in the initial 900 s and during the entire behav-oral test (1800 s) in Experiment 3.

Parameter Group

CS N� N�

Insects captured until 900 s 1 (0–5) 0 (0–5) 5 (0–5)*

Insects captured during 1800 s 3.5 (0–5) 0 (0–5) 5 (1–5)*

Pups retrieved until 900 s 8 (0–8) 0.5 (0–8)# 6 (0–8)Pups retrieved during 1800 s 8 (7–8) 0.5 (0–8)# 7 (0–8)

S, control group (n = 10); N�, � receptor antagonist group (CTAP; n = 12); N�, �eceptor antagonist group (nor-binaltorphimine; n = 11). The data are expressed asedian (range).* p < 0.03, compared with N� group.# p < 0.01, compared with CS group (Kruskal–Wallis test followed by Dunn’s test).

(Kruskal–Wallis test followed by Dunn’s test). (B) Percentage of dams that retrieved CS and N� groups (Fisher’s exact test).

(�: F3,17 = 12.18, p = 0.0003, Fig. 8B; �: F3,17 = 3.68, p = 0.04, Fig. 8E).Although they received a specific agonist, the � and � groups did notexhibit significant levels of opioid receptor activation. In the groupsin Experiment 2 (Fig. 8C and F), the injection of a single opioidreceptor antagonist in the presence of morphine (i.e., in the N�Mand N�M groups) reversed the overactivation caused by morphinealone (� receptor: F3,17 = 8.487, p = 0.0018; � receptor: F3,16 = 4.457,p = 0.023). Fig. 8D and G show the groups in Experiment 3. Activa-tion of the � opioid receptor in the N� group was not significantlydifferent from the CS group, but � receptor activation in the N�group was higher than in the CS and N� groups (140.4% ± 21.8%of control [mean ± SEM], F2,10 = 4.601, p = 0.0468). With regard to �opioid receptor activation in the groups in Experiment 3 (Fig. 8G),no significant differences were found among groups (F2,10 = 0.5105,p > 0.05).

4. Discussion

The PAG is a putative center that switches adaptive behavioralresponses [26]. The rlPAG plays an important role in switching frommaternal to predatory behavior in lactating rats [11]. Morphineinjected directly into this area impairs pup retrieval, nest build-ing, and FMB in mothers [18]. In the present study, we tested therole of morphine injected into the rlPAG in the context of selectingmaternal vs. predatory behaviors. We also analyzed the functionalrelevance of � and � opioid receptors in this paradigm.

We injected � and � opioid receptor agonists in the rlPAG indams in Experiment 1. Unlike what happens when injected periph-erally [13], morphine administered centrally in the rlPAG disruptedmaternal behavior but did not increase predatory hunting in thedams. Thus, peripheral injections of morphine may cause the acti-vation of opioid receptors in other areas together with the PAG. Thisappears to be necessary to generate morphine-induced increases inpredatory hunting. Other areas, such as the ventrolateral caudateputamen, amygdala, hypothalamus, and superior colliculus, playa role in predatory hunting, the motivational drive to forage andhunt, and opioid receptor expression [8,9,19,27–29].

Lactating females appear to be more efficient in hunting andcatching prey compared with pregnant and virgin females [4]. Lac-tating females in the CS group had a greater level of expressionof hunting (i.e., beyond caring for pups, females in the CS group

also foraged and captured prey). Interestingly, CS females retrievedtheir pups first and hunted for insects afterward. This did not occurin the other groups, mainly in females who received a morphineinjection. These animals displayed only hunting behavior.

70 M.O. Klein et al. / Behavioural Brain Research 274 (2014) 62–72

Fig. 8. Opioid receptor activation. (Left) (A) Fluorescent photomicrograph of the periaqueductal gray in Experiment 4. The intensity of fluorescence indicates the activation ofeach opioid receptor subtype (� and �) in the tissue. Scale bar = 150 �m. (Right) Activation of � and � opioid receptors in the dams after the behavioral test. For presentationpurposes, the data are shown as a percentage of activation relative to the control (CS) group (n = 5). Just the groups within each Experiment (1–3) were compared to eachother for each receptor. MOR (morphine group; n = 4), � (� opioid receptor agonist [DAMGO] group; n = 4), � (� opioid receptor agonist [U69593] group; n = 5), N�M (�opioid receptor antagonist [CTAP] with consecutive morphine injection; N�M (n = 5): � opioid receptor antagonist [nor-binaltorphimine] plus morphine; n = 4), N� (� opioidreceptor antagonist [CTAP]; n = 4), N� (� opioid receptor antagonist [nor-binaltorphimine] group; n = 4). The red line indicates the CS group. (B) Activation of � opioid receptorsin Experiment 1. ***p < 0.001, compared with CS, �, and � groups (one-way ANOVA followed by Tukey’s test). (C) Activation of � opioid receptors in Experiment 2. **p < 0.01,compared with CS and N�M groups (one-way ANOVA followed by Tukey’s test). (D) Activation of � opioid receptors in Experiment 3. *p < 0.05, compared with CS and N�g eptors #

T red wo

eiioimertalrt[diaEbBm

roups (one-way ANOVA followed by unpaired t-test). (E) Activation of � opioid recukey’s test). (F) Activation of � opioid receptors in Experiment 2. #p < 0.05, compaf � opioid receptors in Experiment 3 (one-way ANOVA).

Intracerebroventricular injection of the � opioid agonist is asffective as morphine or �-endorphin in inhibiting maternal behav-or [15,20]. In the present study, the same opioid agonist injectednto the rlPAG did not interfere either with maternal behaviorr predatory hunting. Thus, the activation of � opioid receptorsn other areas beyond the rlPAG might be necessary to inhibit

aternal behavior, or the dose used in this study was not enoughven being a high dose [20]. Experiment 4 showed that intra-lPAG injection of the � opioid agonist did not significantly activatehis receptor. This finding may be related to insufficient receptorctivation to promote behavioral changes in this paradigm. Simi-arly, peripheral injections of the � agonist increased the latency toetrieve the pups [21,30]. However, intracerebroventricular injec-ion of the � agonist did not have any effect on maternal behavior20]. In the present study, the � opioid receptor agonist injectedirectly into the rlPAG also did not interfere with maternal behav-

or or behavioral selection. Similarly, a single injection of the �gonist did not activate a significant number of � receptors in

xperiment 4. Thus, at the tested doses, these opioid agonists mighte ineffective in activating their respective receptors in the rlPAG.eyond that, the peripheral injection of the specific � and � agonistsay act in other areas of the brain, which together can impair the

in Experiment 1. p < 0.05, compared with CS group (one-way ANOVA followed byith CS and N�M groups (one-way ANOVA followed by Tukey’s test). (G) Activation

maternal behavior. More studies needs to be performed to clarifythis issue.

In Experiment 1, a morphine injection into the rlPAG aloneinhibited maternal behavior. Such a behavioral effect may beattributable to morphine’s action on more than one type of opi-oid receptor simultaneously. Furthermore, it is important to statethat in this study, the role of � opioid receptor was not investigated,and morphine, even in a minor scale, acts in this type of receptor.Therefore, � receptor in rlPAG could also plays a role in modulatingmorphine inhibition of maternal behavior and behavioral selectionin all experimental paradigms presented [31]. To indirectly addressthe possible specific functional meaning of the morphine-inducedactivation of � and � opioid receptor, we specifically blocked � and� receptors in Experiment 2. All of the morphine-treated groupsexhibited impairments in maternal behavior, even with � and �receptors blocked. This supports the hypothesis that morphine mayact on more than one type of opioid receptor to inhibit mater-nal behavior. The presence of the � receptor antagonist in the

rlPAG concurrently with morphine inhibited predatory hunting,whereas the presence of the � receptor antagonist with morphineor morphine alone did not alter this behavior. Therefore, � opi-oid receptors in the rlPAG appear to have a central function in the

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M.O. Klein et al. / Behavioura

xpression of predatory hunting. This finding led us to investigatehe role of endogenous � and � opioid receptor tone in behavioralelection in Experiment 3.

The injection of the � receptor antagonist did not significantlynterfere with predatory behavior but impaired the care of pups.his suggests that the basal tone of � receptors is important for theegular expression of maternal behavior. Other studies have shownhat diminishing or increasing � receptor activation inhibits mater-al behavior [13,32,33]. The blockade of � receptors facilitatedredatory behavior without interfering with parameters of mater-al behavior. � Receptor activation may restrain the expression ofredatory hunting, and the blockade of � receptors may facilitatehis behavior. Experiment 4 failed to show significant effects of �nd � receptor antagonists on the endogenous activation of theirpecific receptors. Since the specificity of these antagonists is wellstablished [34–36], these findings may be due to the fact that theasal activation was so low in the present experimental situationhat made it difficult to decrease to even lower levels. However, �eceptor blockade increased the activation of � opioid receptors inhe rlPAG in Experiment 4. This finding suggests the existence of anmportant functional balance between these two receptor subtypeshat may promote predatory hunting, i.e., once the action of oneype of receptor is diminished, the activity of the other one wouldncrease to compensate the lack of action from the first. Thus, thectivity of � opioid receptors appears to be affected by inhibitoryndogenous � receptor tone.

Some important general aspects of the behavioral tests in Exper-ments 1–3 should be considered. Previous studies have shown thatentral injections of morphine impair pup retrieval, nest building,nd the expression of FMB [18]. In the present study, nest build-ng was not evaluated because of the absence of pine flakes inhe experimental cages. Behavior was tested without pine flakeso better analyze predatory behavior (i.e., the visualization of cock-oaches would be hampered by pine flakes in the cages). Limitedccess to nest material is a well-established model of neglectedaternal behavior [37,38]. This may explain why the percentage of

ams that expressed FMB and nursing in all of the groups, includinghe CS group, was lower in the present study compared with othertudies that utilized free access to nest material [13]. Because thisest was the first time that the dams had contact with roaches, theovelty of the insects may have indirectly influenced the expressionf FMB.

Changes in the time taken to begin catching the cockroachesnd latency scores to retrieve the 1st pup might reflect the moti-ational drive of the dams and may be useful to evaluate this drive11]. The rlPAG is an integrative region that plays an importantole in motivational and rewarded behaviors (e.g., hunting, forag-ng, and reward seeking) [29,39]. This feature may have influencedhe present results. The central modulation exerted by the drugs

ay have influenced the motivation to elicit behavioral responses.nterestingly, beyond interfering with the retrieve of pups, drugslso impaired the nursing and licking behavior. It shows that inddition to interfere with the motivation to care pups, they alsoisrupted the maintenance of this behavior, mainly regarding to �pioid receptor.

The drug-induced behavioral changes reported herein mighte attributable to changes in the activational state of PAG opi-id receptors. The level of activation of � and � opioid receptorsn the rlPAG was measured in all of the experimental paradigms.s expected, morphine increased the activation of both � and �eceptors. However, the blockade of one of these receptors in theresence of morphine decreased the activation caused by morphine

lone, restoring it to control levels. Interestingly, treatment with �nd � receptor agonists did not result in differences in � and �eceptor activation compared with controls. Indeed, the � and �roups showed no significant changes in behavioral parameters.

Research 274 (2014) 62–72 71

This may be explained by an inability of the agonists to activate asignificant number of receptors that would be sufficient to influ-ence behavior in this paradigm. Also, as previously said, the dosesused might be not enough to show any behavioral or molecularalteration. In Experiment 3, the N� group, which received a � recep-tor antagonist, exhibited an increase in the activation of � opioidreceptors. This group also presented an improvement in predatoryhunting, whereas � receptor blockade in the N� group impairedthe expression of predatory behavior. This may be attributable to afunctional balance between opioid receptors, in which the suppres-sion of � receptors, directly or indirectly, may facilitate � receptoractivation, always keeping a basal tone of receptors activation.This suggests the existence of a dynamic interaction among opioidreceptors that may involve other central systems. Such a systemmay compensate for the lack of action of one receptor by increas-ing the activation of another. This mechanism might play a role inthe behavioral changes observed in the present study.

5. Conclusion

Our data suggest that endogenous � opioid receptor tone inthe rlPAG is essential for the expression of hunting behavior andcan be modulated by the degree of � receptor activation. Thus,receptor-mediated opioidergic transmission appears to be criti-cal for behavioral selection during lactation. The pharmacologicalblockade of � opioid receptors leads to a behaviorally meaningfulincrease in endogenous � receptor activation, which can be directlyor indirectly mediated. A role for compensatory opioidergic mul-tireceptor interactions that determine behavioral responses nowappears likely.

Conflict of interest

The authors have no conflict of interest to report.

Funding source

This work was supported by grant from Fundac ão de Amparo àPesquisa do Estado de São Paulo (FAPESP) processes 2010/06774-0and 2013/01610-7. Funding source played no role in experimentaldesign or decision to submit the paper for publication.

Acknowledgment

The authors thank to Miriam Aline Geigner for her technicalassistance in the immunofluorescence assay.

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