Behavioural Brain Research 263 (2014) 190–197
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Cannabinoid receptor agonist disrupts behavioral and neuroendocrine responses during lactation Fabiana C. Vilela, Alexandre Giusti-Paiva ∗ Department of Physiological Sciences, Institute of Biomedical Sciences, Federal University of Alfenas (Unifal-MG), Alfenas, Minas Gerais, Brazil
h i g h l i g h t s • Cannabinoid reduced maternal care, maternal aggression and maternal anxiolysis. • Cannabinoid reduced the activity of oxytocinergic neurons in the PVN and SON. • Cannabinoid receptor agonist disrupts oxytocin secretion in response to suckling.
a r t i c l e
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Article history: Received 13 November 2013 Received in revised form 23 January 2014 Accepted 27 January 2014 Available online 2 February 2014 Keywords: Maternal behavior Lactation Cannabinoid receptor Oxytocin
a b s t r a c t It has been shown that the endocannabinoid system is involved in the neurohypophyseal hormone secretion produced by exposure to several different stimuli; however, the influence of this system on neuroendocrine responses during lactation is unclear. Therefore, the aim of our study was to investigate the influence of an acute peripheral administration of WIN55,212-2 (cannabinoid receptor agonist) on behavioral and neuroendocrine responses during lactation. On day 6 of lactation, female rats were treated with vehicle or WIN55,212-2 30 min before the start of our experiments. To evaluate maternal behavior, the pups were returned to their home cages to the side of the cage opposite the previous nest, and the resulting behavior of the lactating rats was recorded for the next 30 min. Aggressive behavior was evaluated for 10 min following the placement of an intruder male rat in the home cage. The plasma level of oxytocin and the amount of milk consumption by the pups were evaluated 15 min after the onset of suckling. In addition, double-labelled c-Fos/oxytocin neurons in the medial magnocellular subdivision of the paraventricular nucleus and in the supraoptic nucleus were quantified for each lactating rat. The results show that WIN decreased maternal care, decreased aggressive behaviors, suppressed maternal anxiolysis, decreased plasma oxytocin levels and milk consumption by pups and decreased activation of oxytocinergic neurons in hypothalamic nuclei. Our results indicate that the changes in the behavioral responses of lactating rats treated with WIN maybe can be related to disruption in the neuroendocrine control of oxytocin secretion. © 2014 Elsevier B.V. All rights reserved.
1. Introduction The behavioral repertoire of some mammals during motherhood differs from that exhibited by females in other periods of their reproductive cycle. Concomitant with the display of maternal care, lactating rats show aggressive behavior and lower levels of anxiety in conflict tests [1]. This behavioral pattern relies, at least partly, on the hormonal changes that characterize late gestation, parturition and lactation [2]. As maternal care parallels the course of
∗ Corresponding author at: Instituto de Ciências Biomédicas, Universidade Federal de Alfenas, Unifal-MG, Rua Gabriel Monteiro da Silva, 700, Alfenas, 37130-000, MG, Brazil. Tel.: +55 35 3299 1303. E-mail addresses: [email protected], [email protected] (A. Giusti-Paiva). 0166-4328/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbr.2014.01.037
these behaviors, it has been suggested that maternal behavior is a condition needed for the expression of aggression and anxiolysis. One potential mediator of these effects on aggression and anxietylike behavior is the elevation of basal corticosterone; indeed, it has been postulated that the stress hyporesponsiveness of lactating dams mediates the decrease in fear and anxiety associated with the display of maternal aggression [3]. Another potential neural mediator of these behavioral changes is oxytocin [4]. Oxytocin has been suggested to play an important role within the central amygdala to regulate maternal aggression [5]. Using local microdialysis, increased oxytocin release was observed during a maternal defense test in dams. Behavioral changes after birth appear to depend on hormonal changes, such as increased serum levels of oxytocin [6,7]. Importantly, the intracerebroventricular infusion of a selective oxytocin receptor antagonist increased anxiety [8], decreased maternal care [9] and decreased maternal aggression [5].
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Although abundant information is available about the influence of many hormones and neurotransmitters on the maternal behavior of rodents, less is known about the modulation of these signals by endocannabinoids. Studies addressing the effects of Cannabis extract on maternal behavior date back to the late 1970s and early 1980s; for example, it was reported that the acute and sub-chronic administration of 9 -tetrahydrocannabinol (THC), the primary psychoactive constituent of marihuana, dose-dependently suppressed the retrieval of nesting material in mice [10,11]. In addition, it was demonstrated that THC altered the secretion of pituitary hormones in humans and laboratory animals under a variety of physiological circumstances [12–14]. Moreover, THC decreased suckling-induced oxytocin release by reducing intramammary pressure and pup stretch during suckling [15,16]. Further evidence of the involvement of the cannabinoid system on oxytocin secretion was demonstrated with the administration of a CB1 receptor antagonist, rimonabant, that enhanced the secretion of oxytocin and c-Fos expression in oxytocinergic neurons in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) of the hypothalamus by inducing blood volume expansion [17]. In addition, several studies have shown that the exogenous administration of cannabinoids inhibits the activity of several hypothalamic-pituitary systems, including the thyroid, gonadal and growth hormone axes [18–20]. Based on the reported involvement of the endocannabinoid system in oxytocin secretion, the aim of our study was to investigate the influence of an acute peripheral injection of WIN55,212-2 (CB1 receptor agonist) on behavioral and neuroendocrine responses during lactation.
2. Material and methods 2.1. Animals Subjects were adult Wistar nulliparous female rats at approximately 9 weeks of age, which were obtained from the Central Animal Facility of the Federal University of Alfenas and were housed in a temperature-controlled room (22 ◦ C) on a 12 h light–12 h dark cycle (lights on at 7:00 AM) with access to water and food ad libitum. In the experiments, the females were timed-mated by placing them with sexually experienced males. The day on which sperm was observed during vaginal lavage was designated as day 1 of pregnancy. The pregnant females were individually housed in opaque polypropylene cages (42 × 34 × 16 cm). After the rats gave birth (day 0 of lactation), the litters were randomly standardized to eight pups each (four male and four female pups), and the mothers remained with their litters until they were tested for hormonal changes and maternal behavior on day 6 of lactation. To test the aggressive behavior of lactating females, male intruders were used. The male intruders were approximately two months old, were maintained under the same conditions and weighed approximately the same as the resident female. Each male was used only once in the experiment. For testing in the open field, additional female rats in diestrus were used. All of the experiments were conducted in accordance with the declaration of Helsinki on the welfare of experimental animals and with the approval of the Ethics Committee of the Federal University of Alfenas (# 244/2009). 2.2. Drugs WIN55,212-2 was purchased from Sigma–Aldrich (EUA) and dissolved in a solution containing 0.9% NaCl, tween and dimethyl sulfoxide (DMSO) at a ratio of 8:1:1. For all of the experiments, rats at day 6 of lactation were treated with WIN55,212-2 (1 or 3 mg/kg, i.p.) or vehicle 30 min prior to the start of any experimental
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procedures. The doses of WIN55,212-2 used in the present study are in agreement with the doses commonly used in other reports that injected these compounds peripherally [21–24]. The injections were followed by the specific procedures and measurements described in the following sections. 2.3. Maternal care Maternal behavior was assessed between 08:00 and 12:00 h. The initial position of the nest in the home cage was recorded. Next, the litter was removed from the cage and placed in a different cage for 12 h. After 11 h and 30 min of maternal separation, dams were treated with vehicle (1 mL/kg, n = 8 animals), WIN55,212-2 (1 mg/kg, n = 9 animals) or WIN55,212-2 (3 mg/kg, n = 10 animals). After another 30 min, the pups were placed back in their home cages on the side opposite to the location of the previous nest, and the dam’s behavior was recorded for the next 30 min. As previously described, we analyzed multiple parameters, including the latency for retrieval of each pup, number of pups brought to the nest, time spent licking the pups, percentage of time spent in the archednursing position, percentage of time spent blanket-nursing and percentage of time exhibiting full maternal behavior (the mother staying in the arched-nursing position for 2 min after nursing) [25,26]. 2.4. Assessment of maternal aggression Aggressive behavior was assessed between 08:00 and 12:00 h. Another set of dams were treated with vehicle (1 mL/kg, n = 9 animals), WIN55,212-2 (1 mg/kg, n = 7 animals) or WIN55,212-2 (3 mg/kg, n = 9 animals) and 30 min after injections, an adult male rat (intruder) was placed into the home cage of the female and her litter, and the interaction of the mother and intruder was recorded for 10 min. As previously described, we assessed the latency to first attack (dam lunges quickly at intruder male, usually followed by rolling, biting, and fur pulling directed toward the neck and back regions of the intruder), frontal attack number, lateral attack number, lateral threat number (dam threatens intruder male while approaching laterally) and maternal behavior (i.e., any behavior directed toward caring for the pups) over the 10 min period [25,26]. 2.5. Performance in the open field test Another set of dams were treated with vehicle (1 mL/kg, n = 7 animals), WIN55,212-2 (1 mg/kg, n = 9 animals) or WIN55,212-2 (3 mg/kg, n = 9 animals) and 30 min after the injection, animals were tested in the open field arena to evaluate their anxiolytic and locomotor activity. We also used non-lactating virgin female rats in their diestrus phase that were treated with vehicle (n = 8 animals). In this test, each female rat was placed in the center of the novel open field arena. The open field apparatus consists of circular arena with a diameter 60 cm and walls of 45 cm high with a floor is divided into 12 areas. A circle of 30 cm diameter in center divided in four areas was defined as the central areas and the 8 areas along the walls were considered the peripheral area. The number of peripheral (adjacent to the walls) and central (away from the walls) areas that the rat entered with all four paws was recorded for 5 min. Female rat behavior was continuously videotaped by a video camera placed over the structure and was then encoded using a continuous sampling method. The antithigmotactic effect was defined as the proportion of entries into the central part of the arena relative to the total number of entries. The arena was carefully cleaned with a 10% ethanol solution after each test [26,27].
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2.6. Milk consumption of pups and oxytocin measurements A milk ejection test was performed on day 6 of lactation. All of the pups were removed from their dams and placed in a warm incubator (28–30 ◦ C). Another set of dams were treated with vehicle (1 mL/kg, n = 5 animals), WIN55,212-2 (1 mg/kg, n = 6 animals) or WIN55,212-2 (3 mg/kg, n = 6 animals) 30 min prior to the start of experimental procedures. After 12 h of pup isolation and 30 min after vehicle or WIN55,212-2 administration, the urine from the bladders of the pups was manually expressed and the pups were weighed. The pups were returned to their dam and were reweighed 15 min after the onset of suckling to determine the amount of milk they obtained from the dam [25]. For the measurement of oxytocin, at this moment, lactating rats were decapitated and trunk blood was collected (5 mL) in plastic tubes containing heparin and then kept on ice. The plasma was separated by centrifugation (3000 rpm, 4 ◦ C, 15 min), and aliquots were stored at −20 ◦ C. Plasma oxytocin levels were determined using a specific oxytocin ELISA kit (Enzo Life Sciences). The intra-assay coefficient of variation was 2.1%. The samples were assayed in duplicate in the same assay. 2.7. Perfusion, tissue preparation and immunohistochemistry In this set of experiments, the dams were treated with vehicle (1 mL/kg, n = 5 animals), WIN55,212-2 (1 mg/kg, n = 5 animals) or WIN55,212-2 (3 mg/k, n = 4 animals). Animals were deeply anaesthetized with tribromoethanol (250 mg/kg) 90 min after evaluation of their maternal behavior. Next, they were perfused with 200 mL of cold 0.9% NaCl solution containing heparin (50 UI/l), followed by 500 mL of 4% formaldehyde in 0.1 M phosphate buffer (PB), pH 7.2. The brains were removed, post-fixed for 4 h in the perfusion solution and stored at 4 ◦ C in PB containing 30% sucrose. A cryostat was used to collect coronal sections with a thickness of 30 m in 0.01 M PB. Briefly, the sections were incubated with 0.03% H2 O2 for 30 min and washed with 0.01 M PB. Next, the sections were incubated in 5% bovine albumin in 0.1 M PB for 1 h to block non-specific binding sites. The sections were incubated
overnight at room temperature with a rabbit anti-c-Fos antibody (Ab-5, Oncogene Science, Manhasset, NY, USA) diluted 1:10,000 in 0.1 MPB containing 2% normal goat serum and 0.3% Triton X-100 (Sigma Chemical Co., St. Louis, MO, USA). After washing, the free-floating sections were incubated with a biotin-labeled anti-rabbit immunoglobulin produced in goat (Vector Laboratories Inc., Burlingame, CA, USA, 1:200 in 0.1 M PB containing 1.5% normal goat serum) followed by an avidin–biotin-peroxidase complex (Vector Elite, 1:200 in 0.1 M PB). Each incubation lasted for 1 h at room temperature. The blue–black labeling of the cell nuclei was detected using diaminobenzidine hydrochloride (DAB, Sigma Chemical Co., St. Louis, MO, USA) intensified with 1% cobalt chloride and 1% nickel ammonium sulfate. For double labelling, the sections were incubated for 48 h at 4 ◦ C with an anti-oxytocin antibody (raised in rabbit, Peninsula Laboratories, Inc., San Carlos, CA, USA, 1:20,000 in 0.1 M PB containing 2% normal goat serum and 0.3% Triton X-100). The sections were rinsed and submitted to the same protocol described for c-Fos labeling by using an appropriate secondary biotinylated antibody, followed by an avidin–biotinperoxidase complex. A brown cytoplasmic stain was detected using a non-intensified DAB solution. Lastly, the sections were mounted on gelatinized slides, air-dried overnight, dehydrated, cleared in xylene and cover slipped with mounting medium. The sections chosen for quantification were selected using a rat brain atlas to verify the similarity of neuroanatomical areas between experimental and control groups [26,28]. c-Fos immunoreactive neurons were counted in the medial preoptic area (MPOA, 0.30 mm anterior to bregma), the bed nucleus of stria terminalis (BNST, 0.20 mm anterior to bregma) and in both the ventral and dorsal parts of the median preoptic nucleus (MnPO, 0.30 mm posterior to bregma). The number of c-Fos/oxytocin double-labelled neurons were counted in the PVN and SON according at specific anteroposterior coordinates, including the medial magnocellular PVN (MM, 1.30 mm posterior to bregma) and SON (1.3 mm posterior to bregma). A dotted area for each area to be counted was produced according to the rat brain atlas and was projected over each tissue section image to provide the delimitation for counting.
Fig. 1. The effects of treatment with vehicle (1 mL/kg, i.p., n = 8 animals), WIN55,212-2 (1 mg/kg, i.p., n = 9 animals) or WIN55,212-2 (3 mg/kg, i.p., n = 10 animals) on maternal behavior: (A) pup retrieval, (B) latency to carry the first pup, (C) time spent licking pups, (D) percentage of time spent in blanket-nursing, (E) percentage of time spent in arched-nursing and (F) percentage of full maternal behavior. Each column represents the mean (±S.E.M.) of eight animals. * p < 0.05, ** p < 0.01 and *** p < 0.001 when compared with the vehicle group.
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Fig. 2. The effects of treatment with vehicle (1 mL/kg, i.p., n = 9 animals), WIN55,212-2 (1 mg/kg, i.p., n = 7 animals) or WIN55,212-2 (3 mg/kg, i.p., n = 9 animals) on aggressive behavior: (A) latency to first attack, (B) frontal attack, (C) lateral attack, (D) lateral threat and (E) maternal behavior. Each column represents the mean (±S.E.M.) of eight animals. * p < 0.05, ** p < 0.01 and *** p < 0.001 when compared with the vehicle group.
Next, the cell counting was manually performed at higher magnifications under the microscope with the aid of a computerized system that included a Nikon Eclipse 80i microscope equipped with a DS-Ri1 Nikon digital camera attached to a contrast enhancement device. A double-labelled (c-Fos/oxytocin) cell was classified by the presence of a dark nucleus together with a well-defined brown cytoplasm. The counting was performed unilaterally in one section per animal, and the threshold for positive staining was individually established by two different participants who were blind to the experimental conditions. As previously reported, the final results represent the mean findings of two independent evaluations [17,26].
attack (F2,22 = 5.01, p < 0.05, Fig. 2A) when compared with the control. In rats treated with 1 or 3 mg/kg WIN, a reduction in the number of frontal attacks (F2,22 = 12.10, p < 0.01 and p < 0.001, respectively, Fig. 2B), a reduction in the number of lateral attacks (F2,22 = 5.05, p < 0.05 and p < 0.05, respectively, Fig. 2C) and reduction in the number of lateral threats (F2,22 = 25.43, p < 0.01 and p < 0.001, respectively, Fig. 2D) was observed when compared with the vehicle group. Additionally, mothers treated with WIN (1 or 3 mg/kg) showed a reduction in contact with the pups (maternal behavior) when compared with the vehicle group (F2,22 = 18.83, p < 0.001, Fig. 2D). 3.3. Performance in the open field test
2.8. Statistical analysis The data were plotted using the GraphPad software program (version 6.0) and were expressed as the means ± S.E.M. The data were analyzed by a one-way analysis of variance (ANOVA), followed by the Newman–Keuls post-test. A p-value of less than 0.05 (p < 0.05) was used to establish significance. 3. Results 3.1. Maternal care
No significant differences among the groups were observed in the total number of entries in the open field (F3,31 = 2.87, p = 0.0595; Fig. 3A). This result indicates that WIN (1 or 3 mg/kg) does not interfere with the exploratory activity of lactating rats. Moreover, we observed an increase in the number of center entries (F3,31 = 10.82, p < 0.001, Fig. 3A) and an increase in the ratio of center to total entries (F3,31 = 9.98, p < 0.001, Fig. 3B) in lactating rats treated with vehicle when compared with the non-lactating group. In lactating rats treated with 1 or 3 mg/kg WIN, our results showed a decrease in the number of center entries (F3,31 = 10.82, p < 0.001 and p < 0.01, respectively, Fig. 3A) and a decrease in the ratio of center to total
Analysis of maternal behavior (Fig. 1) demonstrated that treatment with WIN (1 or 3 mg/kg) impaired pup retrieval when compared with rats injected with vehicle (Fig. 1A). In addition, WIN (3 mg/kg) increased the latency to carry the first pup (F2,24 = 6.51, p < 0.01, Fig. 1B), decreased the time spent licking the pups (F2,24 = 5.48, p < 0.05, Fig. 1C) but did not interfere with the percentage of blanket-nursing (F2,24 = 2.74, Fig. 1D). WIN at 1 and 3 mg/kg decreased the percentage of time spent in archednursing (F2,24 = 11.86, p < 0.05 and p < 0.001, respectively, Fig. 1E) and decreased the percentage of full maternal behavior (F2,24 = 9.56, p < 0.05 and p < 0.001, respectively, Fig. 1F) when compared with the vehicle group. 3.2. Maternal aggression The administration of WIN (1 or 3 mg/kg) to female rats also reduced aggressive behaviors. We found that rats treated with WIN (3 mg/kg) displayed an increase in the latency to the first
Fig. 3. The effects of treatment with vehicle (1 mL/kg, i.p., n = 8 animals) in nonlactating rats, and the effects of treatment with vehicle (1 mL/kg, i.p., n = 7 animals), WIN55,212-2 (1 mg/kg, i.p., n = 11 animals) or WIN55,212-2 (3 mg/kg, i.p., n = 9 animals) in lactating rats in the open field: peripheral entries, central entries, total entries and ratio of central entries to total entries. Each column represents the mean (±S.E.M.) of eight animals. *** p < 0.001 when compared with the non-lactating group. ## p < 0.01 and ### p < 0.001 when compared with the vehicle group.
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F.C. Vilela, A. Giusti-Paiva / Behavioural Brain Research 263 (2014) 190–197 Table 1 Effects of WIN treatment on the absolute number of c-Fos labelled neurons in the PVN, SON, MPOA, BNST, MnPOd and MnPOv; number of OT labelled neurons or c-Fos/OT double labelled neurons in the PVN and SON.
Fig. 4. The effects of treatment with vehicle (1 mL/kg, i.p., n = 5 animals), WIN55,212-2 (1 mg/kg, i.p., n = 6 animals) or WIN55,212-2 (3 mg/kg, i.p., n = 4 animals) on plasma oxytocin levels of lactating rats (A) and on the weight gain of pups (B) Each column represents the mean (±S.E.M.). * p < 0.05 when compared with the vehicle group.
entries (F3,31 = 9.98, p < 0.01 and p < 0.05, respectively, Fig. 3B) when compared with the vehicle group. 3.4. Milk consumption by pups and oxytocin measurements The administration of WIN (1 or 3 mg/kg) to female rats reduced oxytocin secretion during lactation (F2,14 = 5.48, p < 0.05, Fig. 4A) when compared with the vehicle group. The administration of WIN (1 or 3 mg/kg) reduced weight gain in pups (F2,14 = 4.53, p < 0.05, Fig. 4B) when compared with the pups of dams that were treated with vehicle. 3.5. Immunohistochemistry for c-Fos/oxytocin The effects of WIN on the immunoreactivity of c-Fos and c-Fos/OT are summarised in Table 1. In the forebrain, treatment with WIN again reduced c-Fos immunoreactivity in the MPOA (F2,11 = 69,06; p < 0.001), BNST (F2,11 = 24.49; p < 0.001), MnPOd (F2,11 = 11,42; p < 0.01), PVN (F2,11 = 12.62; p < 0.01) and SON (F2,11 = 8.84; p < 0.01). Treatment with WIN (1 or 3 mg/kg) significantly decreased the number of c-Fos/oxytocin double labelled neurons in PVN (F2,11 = 37.52, p < 0.001) and SON (F2,11 = 15.80 p < 0.05). The pattern of c-Fos/oxytocin immunoreactivity in the MM subdivision of the PVN and in the SON of representative coronal sections from each group is presented in Fig. 5. 4. Discussion In all mammalian species, both physiological and behavioral changes occur throughout pregnancy to prepare the mother for the birth. These changes include the onset of maternal behaviors, such as nursing of offspring, maternal aggression and milk production to ensure the development and survival of the offspring [29,30].The
Vehicle
WIN (1 mg/kg)
WIN (3 mg/kg)
c-Fos PVN SON MPOA BNST MnPOd MnPOv
14.2 ± 1.3 7.6 ± 0.9 200 ± 6.7 55.8 ± 2.5 46.7 ± 5.4 41.2 ± 4.8
5.7 ± 0.3** 0.6 ± 0.3*** 89.5 ± 9.7*** 33.0 ± 2.5*** 29.2 ± 1.7** 28.5 ± 1.6
6.2 ± 2,0** 2.0 ± 0.6*** 100 ± 4.7*** 34.0 ± 2.8*** 25.7 ± 1.1** 29.7 ± 2.6
OT neurons PVN SON
65,5 ± 3,6 60.0 ± 7.5
72.0 ± 4.4 57.3 ± 1.4
64.7 ± 3.8 60.0 ± 3.8
c-Fos/OT PVN SON
4.2 ± 0.4 5.2 ± 0.8
0.4 ± 0.2*** 1.4 ± 2.4**
3.2 ± 0.5*** 1.2 ± 0.5***
results from our present study demonstrated that the administration of the cannabinoid receptor agonist WIN reduced maternal care, pup weight gain, maternal aggressive behavior and maternal anxiolysis. In addition, WIN reduced the activity of oxytocinergic neurons in the PVN and SON, and it reduced the oxytocin plasma levels in lactating rats. Oxytocin synthesis mainly occurs in neurons of the paraventricular (PVN) and supraoptic nuclei (SON) of the hypothalamus. It is released from nerve terminals in the posterior pituitary and various other regions of the brain. Suckling stimulates the simultaneous release of oxytocin into the bloodstream and central nervous system (CNS) of lactating rats [31]. Oxytocin receptors are present in many brain areas but since oxytocin does not cross the blood-brain barrier, oxytocin released from the pituitary does not act upon the brain [32], however, oxytocin release centrally from neurons which do not project to the pituitary is involved in maternal behavior. In addition, previous studies show that large quantities of oxytocin are released from the dendrites of magnocellular neurons. In addition, oxytocin release in response to suckling has been measured using microdialysis in the mediobasal hypothalamus, bed nucleus of the stria terminalis, medial preoptic area, and septum of parturient sheep [31,33]. Within this context, oxytocin is considered to be not only a hormone that is secreted from the neurohypophysis but also a neurotransmitter that is released at synapses in the brain [34]. Oxytocin mediates the actions of several structures in the hypothalamus and limbic system that receive projections from hypothalamic oxytocin neurons and regulate specific physiological and behavioral functions [34]. A key brain site for OT-mediated maternal care is the MPOA. Together with the adjacent BNST, these brain regions are implicated in maternal behavior [35]. Although these brain regions are essential for the occurrence of normal maternal behaviour in female rats at the time of parturition, other factors operate synergistically with the neural circuits arising from these regions to promote the important shift from infant avoidance to infant acceptance. Additionally, postpartum BNST lesions disrupt maternal behaviors in a similar manner to the effects produced by MPOA lesions [36]. Furthermore, physical interaction with pups, with or without suckling, elicited an increased number of c-Fos-immunoreactive nuclei in the MPOA that was higher compared to the number of nuclei found in other sites, including some sites that are implicated in maternal behaviour (MnPO and BNST) [37]. The disruption of nursing behavior by MPOA lesions has been proposed to involve a separate population of MPOA neurons that projects to the brainstem and spinal cord to modulate the reflexive aspects of nursing behavior [7,38]. In fact, the MPOA has strong projections to the ventrocaudal periacqueductal gray [39], which is known to regulate the crouch nursing posture in response to
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suckling stimulation [40]. The administration of WIN reduced the cFos expression in MPOA, BNST and MnPO, simultaneously to impair in maternal behavior, suggesting that cannabinoid suppress several cerebral areas of circuits regulating certain aspects maternal behavior in rats. The role of endogenous oxytocin in regulating maternal care has been established using an oxytocin antagonist or antisera infused into the brain to block the onset of maternal behavior in rats that have just given birth [41,42]. In addition to disrupting oxytocin secretion in response to suckling and maternal care, we observed that the cannabinoid agonist WIN reduced maternal behavior. The involvement of the endocannabinoid system during lactation was previously reported by pioneering studies that showed indirect evidence for a decrease in suckling-induced oxytocin release by THC through the reduction of intramammary pressure and pup behavior during suckling [15,16]. Suckling stimulates the simultaneous release of oxytocin into the bloodstream and central nervous system [43,44] has been implicated in the regulation of reproductive, maternal and affiliated behaviors [34,45]. The CB1 receptor is the major receptor for endocannabinoids in the brain, and it is mainly present in both pre-terminal and terminal axons. Furthermore, the CB1 receptor modulates neuronal signaling mechanisms in a retrograde manner; however, several studies have shown the expression of CB2 receptors in brain regions where CB1 receptors are also expressed [46]. Physiological and electrophysiological evidence has demonstrated a critical role for
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the CB1 receptor in the regulation of hypothalamic functions. One study demonstrated that axons with CB1 immunoreactivity densely innervate hypothalamic nuclei, such as the SON and PVN, in mice [47]. Previous studies have reported that administration of THC suppressed the retrieval of nesting material in mice [11,47] and depressed nursing and pup-retrieving behavior in rats [48]. Although the inhibitory influence of cannabinoids on the neurohypophyseal system is well established in hydromineral imbalances [17,20,49], the effects of cannabinoids on oxytocin release during lactation are poorly studied. In our present study, we showed that the cannabinoid agonist WIN suppressed c-Fos expression in oxytocinergic neurons of the PVN and SON; in addition, WIN reduced oxytocin plasma levels and milk consumption by pups. This reduction in activity of oxytocinergic neurons maybe can be related with impaired maternal care by lactating rats. The behavioral repertoire of some mammals during motherhood differs from that exhibited by females in other periods of their reproductive cycle. This behavioral pattern relies, at least partly, on the hormonal changes that characterize late gestation, parturition and lactation [50–52]. Breastfeeding in humans [53] and pup suckling in animals [54] represent rewarding social stimuli that encourage maternal behavior. Maternal aggression straddles the realms of maternity and defense, involving both a lactating female’s motivation to nurture her offspring and her need to protect the offspring from the infanticidal behavior of non-paternal males [55].
Fig. 5. Representative photomicrographs showing immunoreactivity for c-Fos (black nuclei; arrow head), oxytocin(brown cytoplasm; open arrow) and c-Fos/oxytocin (brown cytoplasm with black nuclei; full arrow) in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) of lactating rats treated with vehicle (1 mL/kg, i.p., n = 5 animals), WIN55,212-2 (1 mg/kg, i.p., n = 5 animals) or WIN55,212-2 (3 mg/kg, i.p., n = 4 animals). 3 V: third cerebral ventricle, OC: optic chiasm. Scale bar: 50 m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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In many species, maternity elicits a new capacity for fierce displays of protective aggression. Females who might normally retreat from, or ignore, novel male conspecifics will vigorously attack them to defend their young [7]. Maternal aggression is a key behavior adopted by dams when pups are born; however, the influence of cannabinoids on this response has yet to be elucidated. Similar to maternal care, WIN suppresses maternal aggression in lactating rats, and this effect may be mediated by a reduction in the activity of oxytocinergic neurons. In our study, we used the open field for evaluating the antithigmotactic effect and assessing the locomotor activity of animals. The administration of the cannabinoid receptor agonist WIN did not alter the animals locomotor activity; however, WIN-treated animals showed a reduction in the anti-thigmotactic effect, which is a typical characteristic of maternal anxiolysis [56,57]. Regarding anxiolysis, lactating rats that were treated with WIN behaved similarly to non-lactating rats in the open field test. It has become evident that the endocannabinoid system plays an essential role in multiple aspects of brain function, including an influence on the hypothalamic-pituitary-adrenal (HPA) axis and stress response [58,59], modulation of emotional states [60] and cognitive processes [61,62]. In conclusion, we showed that the administration of the cannabinoid receptor agonist WIN disrupts oxytocin secretion in response to suckling and reduces various behaviors and underlying motivational states that, collectively, can be referred to as maternal behaviors. Conflict of interest Authors report no biomedical financial interests or potential conflicts of interest. Role of funding source This work was supported by Fundac¸ão de Amparo a Pesquisa de Minas Gerais (FAPEMIG #2187-12, AG-P), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, #304675/20105; AG-P) and Coordenac¸ão de Aperfeic¸oamento de Pessoal de Nível Superior (CAPES; post-doctoral fellowship FCV). The FAPEMIG, CNPq and CAPES had no further role in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the paper for publication. Acknowledgements We are grateful for the excellent technical support of Marina F Venâncio and José dos Reis Pereira. References [1] Ferreira A, Hansen S, Nielsen M, Archer T, Minor BG. Behavior of mother rats in conflict tests sensitive to antianxiety agents. Behav Neurosci 1989;103:193–203. [2] Fleming AS, Cheung U, Myhal N, Kessler Z. Effects of maternal hormones on ‘timidity’ and attraction to pup-related odors in females rats. Physiol Behav 1989;46:449–53. [3] Lonstein JS, Gammie SC. Sensory, hormonal, and neural control of maternal aggression in laboratory rodents. Neurosci Behav Rev 2002;26:869–88. [4] Bosch OJ, Neumann ID. Both oxytocin and vasopressin are mediators of maternal care and aggression in rodents: from central release to sites of action. Horm Behav 2012;61:293–303. [5] Bosch OJ, Meddle SL, Beiderbeck DI, Douglas AJ, Neumann ID. Brain oxytocin correlates with maternal aggression: link to anxiety. J Neurosci 2005;25:6807–15. [6] Fleming AS, Walsh C. Neuropsychology of maternal behavior in the rat: cfos expression during mother-litter interactions. Psychoneuroendocrinology 1994;19:429–43. [7] Numan M, Insel TR. The neurobiology of parental behavior. New York: Springer Verlag; 2003. p. 110–9.
[8] Manning M, Kruszynski M, Bankowski K, Olma A, Lammek B, Cheng LL, et al. Solid-phase synthesis of 16 potent (selective and nonselective) in vivo antagonists of oxytocin. J Med Chem 1989;32:382–91. [9] Bosch OJ, Neumann ID. Brain vasopressin is an important regulator of maternal behavior independent of dams trait anxiety. Proc Natl Acad Sci 2008;105:17139–44. [10] Moschovakis A, Liakopoulos D, Armaganidis A, Kapsambelis V, Papanikolaou G, Petroulakis G. Cannabis interferes with nest-building behavior in mice. Psychopharmacology 1978;58:181–3. [11] Sieber B, Frischknecht HR, Waser PG. Behavioral effects of hashish in mice. I. Social interactions and nest-building behavior of males. Psychopharmacology 1980;70:149–54. [12] Ayalon D, Nir I, Cordova T, Bauminger S, Puder M, Naor Z, et al. Acute effect of delta1-tetrahydrocannabinol on the hypothalamo-pituitary-ovarian axis in the rat. Neuroendocrinology 1977;23:31–42. [13] Hughes Jr CL, Everett JW, Tyrey L. Delta 9-tetrahydrocannabinol suppression of prolactin secretion in the rat: lack of direct pituitary effect. Endocrinology 1981;109:876–80. [14] Ranganathan M, Braley G, Pittman B, Cooper T, Perry E, Krystal J, et al. The effects of cannabinoids on serum cortisol and prolactin in humans. Psychopharmacology 2009;203:737–44. [15] Wakerley JB, Lincoln DW. Intermittent release of oxytocin during suckling in the rat. Nat New Biol 1971;233:180–1. [16] Tyrey L, Murphy LL. Inhibition of suckling-induced milk ejections in the lactating rat by delta 9-tetrahydrocannabinol. Endocrinology 1988;123:469–72. [17] Ruginsk SG, Uchoa ET, Elias LL, Antunes-Rodrigues J. CB(1) modulation of hormone secretion, neuronal activation and mRNA expression following extracellular volume expansion. Exp Neurol 2010;224:114–22. [18] Lomax P. The effect of marihuana on pituitary-thyroid activity in the rat. Agents Actions 1970;1:252–7. [19] Rettori V, Aguila MC, Gimeno MF, Franchi AM, McCann SM. In vitro effect of delta 9-tetrahydrocannabinol to stimulate somatostatin release and block that of luteinizing hormone-releasing hormone by suppression of the release of prostaglandin E2. Proc Natl Acad Sci 1990;87:10063–6. [20] Ruginsk SG, Uchoa ET, Elias LL, Antunes-Rodrigues J. Anandamide modulates the neuroendocrine responses induced by extracellular volume expansion. Clin Exp Pharmacol Physiol 2013;40:698–705. [21] Crystal JD, Maxwell KW, Hohmann AG. Cannabinoid modulation of sensitivity to time. Behav Brain Res 2003;144:57–66. [22] Pedersen LH, Blackburn-Munro G. Pharmacological characterisation of place escape/avoidance behaviour in the rat chronic constriction injury model of neuropathic pain. Psychopharmacology 2006;185:208–17. [23] Trezza V, Vanderschuren LJ. Bidirectional cannabinoid modulation of social behavior in adolescent rats. Psychopharmacology 2008;197:217–27. [24] Vlachou S, Stamatopoulou F, Nomikos GG, Panagis G. Enhancement of endocannabinoid neurotransmission through CB1 cannabinoid receptors counteracts the reinforcing and psychostimulant effects of cocaine. Int J Neuropsychopharmacol 2008;11:905–23. [25] Vilela FC, Giusti-Paiva A. Glucocorticoids disrupt neuroendocrine and behavioral responses during lactation. Endocrinology 2011;152:4838–45. [26] Vilela FC, Ruginsk SG, de Melo CM, Giusti-Paiva A. The CB1 cannabinoid receptor mediates glucocorticoid-induced effects on behavioural and neuronal responses during lactation. Pflugers Arch 2013;465:1197–207. [27] Prut L, Belzung C. The open field as a paradigm to measure the effects of drugs on anxiety-like behaviors: a review. Eur J Pharmacol 2003;463:3–33. [28] Paxinos G, Watson C. The rat brain in stereotaxic coordinates. San Diego: Academic; 1997. [29] Rosenblatt JS, Factor EM, Mayer AD. Relationship between maternal aggression and maternal care in the rat. Aggress Behav 1994;20:243–55. [30] Neumann I. Alterations in behavioral and neuroendocrine stress coping strategies in pregnant, parturient and lactating rats. Prog Brain Res 2001;133:143–52. [31] Neumann I, Ludwig M, Engelmann M, Pittman QJ, Landgraf R. Simultaneous microdialysis in blood and brain: oxytocin and vasopressin release in response to central and peripheral osmotic stimulation and suckling in the rat. Neuroendocrinology 1993;58:637–45. [32] Leng G, Luckman SM. The vasopressin–oxytocin systems of the neurohypophysis. Principles of Medical Biology 1998;10:421–37. [33] Bealer SL, Crowley WR. Noradrenergic control of central oxytocin release during lactation in rats. Am J Physiol 1998;274:453–8. [34] Neumann ID. Stimuli and consequences of dendritic release of oxytocin within the brain. Biochem Soc Trans 2007;35:1252–7. [35] Bosch OJ, Pförtsch J, Beiderbeck DI, Landgraf R, Neumann ID. Maternal behaviour is associated with vasopressin release in the medial preoptic area and bed nucleus of the stria terminalis in the rat. J Neuroendocrinol 2010;22:420–9. [36] Numan M, Numan MJ. Projection sites of medial preoptic and ventral bed nucleus of the stria terminalis neurons that express Fos during maternal behavior in female rats. J Neuroendocrinol 1997;9:369–84. [37] Lonstein JS, Simmons DA, Swann JM, Stern JM. Forebrain expression of c-fos due to active maternal behaviour in lactating rats. Neuroscience 1998;82:267–81. [38] Numan M, Stolzenberg DS. Medial preoptic area interactions with dopamine neural systems in the control of the onset and maintenance of maternal behavior in rats. Front Neuroendocrinol 2009;30:46–64. [39] Numan M, Numan MJ. Projection sites of medial preoptic area and ventral bed nucleus of the stria terminalis neurons that express Fos during maternal behavior in female rats. J Neuroendocrinol 1997;9:369–84.
F.C. Vilela, A. Giusti-Paiva / Behavioural Brain Research 263 (2014) 190–197 [40] Lonstein JS, Stern JM. Somatosensory contributions to c-fos activation within the caudal periaqueductal gray of lactating rats: effects of perioral, rooting, and suckling stimuli from pups. Horm Behav 1997;32:155–66. [41] Fahrbach SE, Morrell JI, Pfaff DW. Possible role for endogenous oxytocin in estrogen-facilitated maternal behavior in rats. Neuroendocrinology 1985;40:526–32. [42] van Leengoed E, Kerker E, Swanson HH. Inhibition of postpartum maternal behaviour in the rat by injecting an oxytocin antagonist into the cerebral ventricles. J Endocrinol 1987;112:275–82. [43] Moos F, Poulain DA, Rodriguez F, Guerné Y, Vincent JD, Richard P. Release of oxytocin within the supraoptic nucleus during the milk ejection reflex in rats. Exp Brain Res 1989;76:593–602. [44] Neumann I, Russell JA, Landgraf R. Oxytocin and vasopressin release within the supraoptic and paraventricular nuclei of pregnant, parturient and lactating rats: a microdialysis study. Neuroscience 1993;53:65–75. [45] Landgraf R, Neumann ID. Vasopressin and oxytocin release within the brain: a dynamic concept of multiple and variable modes of neuropeptide communication. Front Neuroendocrinol 2004;25:150–76. [46] Callén L, Moreno E, Barroso-Chinea P, Moreno-Delgado D, Cortés A, Mallol J, et al. Cannabinoid receptors CB1 and CB2 form functional heteromers in brain. J Biol Chem 2012;287:20851–65. [47] Wittmann G, Deli L, Kalló I, Hrabovszky E, Watanabe M, Liposits Z, et al. Distribution of Type 1 Cannabinoid Receptor (CB1) Immunoreactive Axons in the Mouse Hypothalamus. J Comp Neurol 2007;503:270–9. [48] Navarro M, Rubio P, de Fonseca FR. Behavioural consequences of maternal exposure to natural cannabinoids in rats. Psychopharmacology 1995;122:1–14. [49] Ruginsk SG, Uchoa ET, Elias LL, Antunes-Rodrigues J. Cannabinoid CB1 receptor mediates glucocorticoid effects on hormone secretion induced by volume and osmotic changes. Clin Exp Pharmacol Physiol 2012;39:151–4. [50] Hansen S, Ferreira A. Food intake, aggression and fear behavior in mother rats: control by neural systems concerned with milk ejection and maternal behavior. Behav Neurosci 1986;100:67–70.
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[51] Fleming AS, Cheung U, Myhal N, Kessler Z. Effects of maternal hormones on ‘timidity’ and attraction to pup-related odors in females rats. Physiol Behav 1989;46:449–53. [52] Bosch OJ, Neumann ID. Both oxytocin and vasopressin are mediators of maternal care and aggression in rodents: from central release to sites of action. Horm Behav 2012;61:293–303. [53] Kavanaugh K, Meier P, Zimmermann B, Mead L. The rewards outweigh the efforts: breastfeeding outcomes for mothers of preterm infants. J Hum Lact 1997;13:15–21. [54] Ferris CF, Kulkarni P, Sullivan JM, Harder JA, Messenger TL, Febo M. Pup suckling is more rewarding than cocaine: evidence from functional magnetic resonance imaging and three-dimensional computational analysis. J Neurosci 2005;25:149–56. [55] Agrell J, Wolff JO, Ylonen H. Counter-strategies to infanticide in mammals: costs and consequences. Oikos 1998;83:507–17. [56] Fleming AS, Luebke C. Timidity prevents the nulliparous female from being a good mother. Physiol Behav 1981;27:863–8. [57] Vilela FC, de Melo CM, Giusti-Paiva A. Glucocorticoids impair maternal anxiolysis during lactation. Neurosci Lett 2012;509:121–4. [58] Hill MN, McLaughlin RJ, Bingham B, Shrestha L, Lee TT, Gray JM, et al. Endogenous cannabinoid signaling is essential for stress adaptation. Proc Natl Acad Sci 2009;107:9406–11. [59] Riebe CJ, Wotjak CT. Endocannabinoids and stress. Stress 2011;14: 384–97. [60] Moreira FA, Lutz B. The endocannabinoid system: emotion, learning and addiction. Addict Biol 2008;13:196–212. [61] Rubino T, Parolaro D. Long lasting consequences of cannabis exposure in adolescence. Mol Cell Endocrinol 2008;286:108–13. [62] Atsak P, Roozendaal B, Campolongo P. Role of the endocannabinoid system in regulating glucocorticoid effects on memory for emotional experiences. Neuroscience 2012;204:104–16.
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Cannabinoid receptor agonist disrupts behavioral and neuroendocrine responses during lactation Fabiana C. Vilela, Alexandre Giusti-Paiva ∗ Department of Physiological Sciences, Institute of Biomedical Sciences, Federal University of Alfenas (Unifal-MG), Alfenas, Minas Gerais, Brazil
h i g h l i g h t s • Cannabinoid reduced maternal care, maternal aggression and maternal anxiolysis. • Cannabinoid reduced the activity of oxytocinergic neurons in the PVN and SON. • Cannabinoid receptor agonist disrupts oxytocin secretion in response to suckling.
a r t i c l e
i n f o
Article history: Received 13 November 2013 Received in revised form 23 January 2014 Accepted 27 January 2014 Available online 2 February 2014 Keywords: Maternal behavior Lactation Cannabinoid receptor Oxytocin
a b s t r a c t It has been shown that the endocannabinoid system is involved in the neurohypophyseal hormone secretion produced by exposure to several different stimuli; however, the influence of this system on neuroendocrine responses during lactation is unclear. Therefore, the aim of our study was to investigate the influence of an acute peripheral administration of WIN55,212-2 (cannabinoid receptor agonist) on behavioral and neuroendocrine responses during lactation. On day 6 of lactation, female rats were treated with vehicle or WIN55,212-2 30 min before the start of our experiments. To evaluate maternal behavior, the pups were returned to their home cages to the side of the cage opposite the previous nest, and the resulting behavior of the lactating rats was recorded for the next 30 min. Aggressive behavior was evaluated for 10 min following the placement of an intruder male rat in the home cage. The plasma level of oxytocin and the amount of milk consumption by the pups were evaluated 15 min after the onset of suckling. In addition, double-labelled c-Fos/oxytocin neurons in the medial magnocellular subdivision of the paraventricular nucleus and in the supraoptic nucleus were quantified for each lactating rat. The results show that WIN decreased maternal care, decreased aggressive behaviors, suppressed maternal anxiolysis, decreased plasma oxytocin levels and milk consumption by pups and decreased activation of oxytocinergic neurons in hypothalamic nuclei. Our results indicate that the changes in the behavioral responses of lactating rats treated with WIN maybe can be related to disruption in the neuroendocrine control of oxytocin secretion. © 2014 Elsevier B.V. All rights reserved.
1. Introduction The behavioral repertoire of some mammals during motherhood differs from that exhibited by females in other periods of their reproductive cycle. Concomitant with the display of maternal care, lactating rats show aggressive behavior and lower levels of anxiety in conflict tests [1]. This behavioral pattern relies, at least partly, on the hormonal changes that characterize late gestation, parturition and lactation [2]. As maternal care parallels the course of
∗ Corresponding author at: Instituto de Ciências Biomédicas, Universidade Federal de Alfenas, Unifal-MG, Rua Gabriel Monteiro da Silva, 700, Alfenas, 37130-000, MG, Brazil. Tel.: +55 35 3299 1303. E-mail addresses: [email protected], [email protected] (A. Giusti-Paiva). 0166-4328/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbr.2014.01.037
these behaviors, it has been suggested that maternal behavior is a condition needed for the expression of aggression and anxiolysis. One potential mediator of these effects on aggression and anxietylike behavior is the elevation of basal corticosterone; indeed, it has been postulated that the stress hyporesponsiveness of lactating dams mediates the decrease in fear and anxiety associated with the display of maternal aggression [3]. Another potential neural mediator of these behavioral changes is oxytocin [4]. Oxytocin has been suggested to play an important role within the central amygdala to regulate maternal aggression [5]. Using local microdialysis, increased oxytocin release was observed during a maternal defense test in dams. Behavioral changes after birth appear to depend on hormonal changes, such as increased serum levels of oxytocin [6,7]. Importantly, the intracerebroventricular infusion of a selective oxytocin receptor antagonist increased anxiety [8], decreased maternal care [9] and decreased maternal aggression [5].
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Although abundant information is available about the influence of many hormones and neurotransmitters on the maternal behavior of rodents, less is known about the modulation of these signals by endocannabinoids. Studies addressing the effects of Cannabis extract on maternal behavior date back to the late 1970s and early 1980s; for example, it was reported that the acute and sub-chronic administration of 9 -tetrahydrocannabinol (THC), the primary psychoactive constituent of marihuana, dose-dependently suppressed the retrieval of nesting material in mice [10,11]. In addition, it was demonstrated that THC altered the secretion of pituitary hormones in humans and laboratory animals under a variety of physiological circumstances [12–14]. Moreover, THC decreased suckling-induced oxytocin release by reducing intramammary pressure and pup stretch during suckling [15,16]. Further evidence of the involvement of the cannabinoid system on oxytocin secretion was demonstrated with the administration of a CB1 receptor antagonist, rimonabant, that enhanced the secretion of oxytocin and c-Fos expression in oxytocinergic neurons in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) of the hypothalamus by inducing blood volume expansion [17]. In addition, several studies have shown that the exogenous administration of cannabinoids inhibits the activity of several hypothalamic-pituitary systems, including the thyroid, gonadal and growth hormone axes [18–20]. Based on the reported involvement of the endocannabinoid system in oxytocin secretion, the aim of our study was to investigate the influence of an acute peripheral injection of WIN55,212-2 (CB1 receptor agonist) on behavioral and neuroendocrine responses during lactation.
2. Material and methods 2.1. Animals Subjects were adult Wistar nulliparous female rats at approximately 9 weeks of age, which were obtained from the Central Animal Facility of the Federal University of Alfenas and were housed in a temperature-controlled room (22 ◦ C) on a 12 h light–12 h dark cycle (lights on at 7:00 AM) with access to water and food ad libitum. In the experiments, the females were timed-mated by placing them with sexually experienced males. The day on which sperm was observed during vaginal lavage was designated as day 1 of pregnancy. The pregnant females were individually housed in opaque polypropylene cages (42 × 34 × 16 cm). After the rats gave birth (day 0 of lactation), the litters were randomly standardized to eight pups each (four male and four female pups), and the mothers remained with their litters until they were tested for hormonal changes and maternal behavior on day 6 of lactation. To test the aggressive behavior of lactating females, male intruders were used. The male intruders were approximately two months old, were maintained under the same conditions and weighed approximately the same as the resident female. Each male was used only once in the experiment. For testing in the open field, additional female rats in diestrus were used. All of the experiments were conducted in accordance with the declaration of Helsinki on the welfare of experimental animals and with the approval of the Ethics Committee of the Federal University of Alfenas (# 244/2009). 2.2. Drugs WIN55,212-2 was purchased from Sigma–Aldrich (EUA) and dissolved in a solution containing 0.9% NaCl, tween and dimethyl sulfoxide (DMSO) at a ratio of 8:1:1. For all of the experiments, rats at day 6 of lactation were treated with WIN55,212-2 (1 or 3 mg/kg, i.p.) or vehicle 30 min prior to the start of any experimental
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procedures. The doses of WIN55,212-2 used in the present study are in agreement with the doses commonly used in other reports that injected these compounds peripherally [21–24]. The injections were followed by the specific procedures and measurements described in the following sections. 2.3. Maternal care Maternal behavior was assessed between 08:00 and 12:00 h. The initial position of the nest in the home cage was recorded. Next, the litter was removed from the cage and placed in a different cage for 12 h. After 11 h and 30 min of maternal separation, dams were treated with vehicle (1 mL/kg, n = 8 animals), WIN55,212-2 (1 mg/kg, n = 9 animals) or WIN55,212-2 (3 mg/kg, n = 10 animals). After another 30 min, the pups were placed back in their home cages on the side opposite to the location of the previous nest, and the dam’s behavior was recorded for the next 30 min. As previously described, we analyzed multiple parameters, including the latency for retrieval of each pup, number of pups brought to the nest, time spent licking the pups, percentage of time spent in the archednursing position, percentage of time spent blanket-nursing and percentage of time exhibiting full maternal behavior (the mother staying in the arched-nursing position for 2 min after nursing) [25,26]. 2.4. Assessment of maternal aggression Aggressive behavior was assessed between 08:00 and 12:00 h. Another set of dams were treated with vehicle (1 mL/kg, n = 9 animals), WIN55,212-2 (1 mg/kg, n = 7 animals) or WIN55,212-2 (3 mg/kg, n = 9 animals) and 30 min after injections, an adult male rat (intruder) was placed into the home cage of the female and her litter, and the interaction of the mother and intruder was recorded for 10 min. As previously described, we assessed the latency to first attack (dam lunges quickly at intruder male, usually followed by rolling, biting, and fur pulling directed toward the neck and back regions of the intruder), frontal attack number, lateral attack number, lateral threat number (dam threatens intruder male while approaching laterally) and maternal behavior (i.e., any behavior directed toward caring for the pups) over the 10 min period [25,26]. 2.5. Performance in the open field test Another set of dams were treated with vehicle (1 mL/kg, n = 7 animals), WIN55,212-2 (1 mg/kg, n = 9 animals) or WIN55,212-2 (3 mg/kg, n = 9 animals) and 30 min after the injection, animals were tested in the open field arena to evaluate their anxiolytic and locomotor activity. We also used non-lactating virgin female rats in their diestrus phase that were treated with vehicle (n = 8 animals). In this test, each female rat was placed in the center of the novel open field arena. The open field apparatus consists of circular arena with a diameter 60 cm and walls of 45 cm high with a floor is divided into 12 areas. A circle of 30 cm diameter in center divided in four areas was defined as the central areas and the 8 areas along the walls were considered the peripheral area. The number of peripheral (adjacent to the walls) and central (away from the walls) areas that the rat entered with all four paws was recorded for 5 min. Female rat behavior was continuously videotaped by a video camera placed over the structure and was then encoded using a continuous sampling method. The antithigmotactic effect was defined as the proportion of entries into the central part of the arena relative to the total number of entries. The arena was carefully cleaned with a 10% ethanol solution after each test [26,27].
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2.6. Milk consumption of pups and oxytocin measurements A milk ejection test was performed on day 6 of lactation. All of the pups were removed from their dams and placed in a warm incubator (28–30 ◦ C). Another set of dams were treated with vehicle (1 mL/kg, n = 5 animals), WIN55,212-2 (1 mg/kg, n = 6 animals) or WIN55,212-2 (3 mg/kg, n = 6 animals) 30 min prior to the start of experimental procedures. After 12 h of pup isolation and 30 min after vehicle or WIN55,212-2 administration, the urine from the bladders of the pups was manually expressed and the pups were weighed. The pups were returned to their dam and were reweighed 15 min after the onset of suckling to determine the amount of milk they obtained from the dam [25]. For the measurement of oxytocin, at this moment, lactating rats were decapitated and trunk blood was collected (5 mL) in plastic tubes containing heparin and then kept on ice. The plasma was separated by centrifugation (3000 rpm, 4 ◦ C, 15 min), and aliquots were stored at −20 ◦ C. Plasma oxytocin levels were determined using a specific oxytocin ELISA kit (Enzo Life Sciences). The intra-assay coefficient of variation was 2.1%. The samples were assayed in duplicate in the same assay. 2.7. Perfusion, tissue preparation and immunohistochemistry In this set of experiments, the dams were treated with vehicle (1 mL/kg, n = 5 animals), WIN55,212-2 (1 mg/kg, n = 5 animals) or WIN55,212-2 (3 mg/k, n = 4 animals). Animals were deeply anaesthetized with tribromoethanol (250 mg/kg) 90 min after evaluation of their maternal behavior. Next, they were perfused with 200 mL of cold 0.9% NaCl solution containing heparin (50 UI/l), followed by 500 mL of 4% formaldehyde in 0.1 M phosphate buffer (PB), pH 7.2. The brains were removed, post-fixed for 4 h in the perfusion solution and stored at 4 ◦ C in PB containing 30% sucrose. A cryostat was used to collect coronal sections with a thickness of 30 m in 0.01 M PB. Briefly, the sections were incubated with 0.03% H2 O2 for 30 min and washed with 0.01 M PB. Next, the sections were incubated in 5% bovine albumin in 0.1 M PB for 1 h to block non-specific binding sites. The sections were incubated
overnight at room temperature with a rabbit anti-c-Fos antibody (Ab-5, Oncogene Science, Manhasset, NY, USA) diluted 1:10,000 in 0.1 MPB containing 2% normal goat serum and 0.3% Triton X-100 (Sigma Chemical Co., St. Louis, MO, USA). After washing, the free-floating sections were incubated with a biotin-labeled anti-rabbit immunoglobulin produced in goat (Vector Laboratories Inc., Burlingame, CA, USA, 1:200 in 0.1 M PB containing 1.5% normal goat serum) followed by an avidin–biotin-peroxidase complex (Vector Elite, 1:200 in 0.1 M PB). Each incubation lasted for 1 h at room temperature. The blue–black labeling of the cell nuclei was detected using diaminobenzidine hydrochloride (DAB, Sigma Chemical Co., St. Louis, MO, USA) intensified with 1% cobalt chloride and 1% nickel ammonium sulfate. For double labelling, the sections were incubated for 48 h at 4 ◦ C with an anti-oxytocin antibody (raised in rabbit, Peninsula Laboratories, Inc., San Carlos, CA, USA, 1:20,000 in 0.1 M PB containing 2% normal goat serum and 0.3% Triton X-100). The sections were rinsed and submitted to the same protocol described for c-Fos labeling by using an appropriate secondary biotinylated antibody, followed by an avidin–biotinperoxidase complex. A brown cytoplasmic stain was detected using a non-intensified DAB solution. Lastly, the sections were mounted on gelatinized slides, air-dried overnight, dehydrated, cleared in xylene and cover slipped with mounting medium. The sections chosen for quantification were selected using a rat brain atlas to verify the similarity of neuroanatomical areas between experimental and control groups [26,28]. c-Fos immunoreactive neurons were counted in the medial preoptic area (MPOA, 0.30 mm anterior to bregma), the bed nucleus of stria terminalis (BNST, 0.20 mm anterior to bregma) and in both the ventral and dorsal parts of the median preoptic nucleus (MnPO, 0.30 mm posterior to bregma). The number of c-Fos/oxytocin double-labelled neurons were counted in the PVN and SON according at specific anteroposterior coordinates, including the medial magnocellular PVN (MM, 1.30 mm posterior to bregma) and SON (1.3 mm posterior to bregma). A dotted area for each area to be counted was produced according to the rat brain atlas and was projected over each tissue section image to provide the delimitation for counting.
Fig. 1. The effects of treatment with vehicle (1 mL/kg, i.p., n = 8 animals), WIN55,212-2 (1 mg/kg, i.p., n = 9 animals) or WIN55,212-2 (3 mg/kg, i.p., n = 10 animals) on maternal behavior: (A) pup retrieval, (B) latency to carry the first pup, (C) time spent licking pups, (D) percentage of time spent in blanket-nursing, (E) percentage of time spent in arched-nursing and (F) percentage of full maternal behavior. Each column represents the mean (±S.E.M.) of eight animals. * p < 0.05, ** p < 0.01 and *** p < 0.001 when compared with the vehicle group.
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Fig. 2. The effects of treatment with vehicle (1 mL/kg, i.p., n = 9 animals), WIN55,212-2 (1 mg/kg, i.p., n = 7 animals) or WIN55,212-2 (3 mg/kg, i.p., n = 9 animals) on aggressive behavior: (A) latency to first attack, (B) frontal attack, (C) lateral attack, (D) lateral threat and (E) maternal behavior. Each column represents the mean (±S.E.M.) of eight animals. * p < 0.05, ** p < 0.01 and *** p < 0.001 when compared with the vehicle group.
Next, the cell counting was manually performed at higher magnifications under the microscope with the aid of a computerized system that included a Nikon Eclipse 80i microscope equipped with a DS-Ri1 Nikon digital camera attached to a contrast enhancement device. A double-labelled (c-Fos/oxytocin) cell was classified by the presence of a dark nucleus together with a well-defined brown cytoplasm. The counting was performed unilaterally in one section per animal, and the threshold for positive staining was individually established by two different participants who were blind to the experimental conditions. As previously reported, the final results represent the mean findings of two independent evaluations [17,26].
attack (F2,22 = 5.01, p < 0.05, Fig. 2A) when compared with the control. In rats treated with 1 or 3 mg/kg WIN, a reduction in the number of frontal attacks (F2,22 = 12.10, p < 0.01 and p < 0.001, respectively, Fig. 2B), a reduction in the number of lateral attacks (F2,22 = 5.05, p < 0.05 and p < 0.05, respectively, Fig. 2C) and reduction in the number of lateral threats (F2,22 = 25.43, p < 0.01 and p < 0.001, respectively, Fig. 2D) was observed when compared with the vehicle group. Additionally, mothers treated with WIN (1 or 3 mg/kg) showed a reduction in contact with the pups (maternal behavior) when compared with the vehicle group (F2,22 = 18.83, p < 0.001, Fig. 2D). 3.3. Performance in the open field test
2.8. Statistical analysis The data were plotted using the GraphPad software program (version 6.0) and were expressed as the means ± S.E.M. The data were analyzed by a one-way analysis of variance (ANOVA), followed by the Newman–Keuls post-test. A p-value of less than 0.05 (p < 0.05) was used to establish significance. 3. Results 3.1. Maternal care
No significant differences among the groups were observed in the total number of entries in the open field (F3,31 = 2.87, p = 0.0595; Fig. 3A). This result indicates that WIN (1 or 3 mg/kg) does not interfere with the exploratory activity of lactating rats. Moreover, we observed an increase in the number of center entries (F3,31 = 10.82, p < 0.001, Fig. 3A) and an increase in the ratio of center to total entries (F3,31 = 9.98, p < 0.001, Fig. 3B) in lactating rats treated with vehicle when compared with the non-lactating group. In lactating rats treated with 1 or 3 mg/kg WIN, our results showed a decrease in the number of center entries (F3,31 = 10.82, p < 0.001 and p < 0.01, respectively, Fig. 3A) and a decrease in the ratio of center to total
Analysis of maternal behavior (Fig. 1) demonstrated that treatment with WIN (1 or 3 mg/kg) impaired pup retrieval when compared with rats injected with vehicle (Fig. 1A). In addition, WIN (3 mg/kg) increased the latency to carry the first pup (F2,24 = 6.51, p < 0.01, Fig. 1B), decreased the time spent licking the pups (F2,24 = 5.48, p < 0.05, Fig. 1C) but did not interfere with the percentage of blanket-nursing (F2,24 = 2.74, Fig. 1D). WIN at 1 and 3 mg/kg decreased the percentage of time spent in archednursing (F2,24 = 11.86, p < 0.05 and p < 0.001, respectively, Fig. 1E) and decreased the percentage of full maternal behavior (F2,24 = 9.56, p < 0.05 and p < 0.001, respectively, Fig. 1F) when compared with the vehicle group. 3.2. Maternal aggression The administration of WIN (1 or 3 mg/kg) to female rats also reduced aggressive behaviors. We found that rats treated with WIN (3 mg/kg) displayed an increase in the latency to the first
Fig. 3. The effects of treatment with vehicle (1 mL/kg, i.p., n = 8 animals) in nonlactating rats, and the effects of treatment with vehicle (1 mL/kg, i.p., n = 7 animals), WIN55,212-2 (1 mg/kg, i.p., n = 11 animals) or WIN55,212-2 (3 mg/kg, i.p., n = 9 animals) in lactating rats in the open field: peripheral entries, central entries, total entries and ratio of central entries to total entries. Each column represents the mean (±S.E.M.) of eight animals. *** p < 0.001 when compared with the non-lactating group. ## p < 0.01 and ### p < 0.001 when compared with the vehicle group.
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F.C. Vilela, A. Giusti-Paiva / Behavioural Brain Research 263 (2014) 190–197 Table 1 Effects of WIN treatment on the absolute number of c-Fos labelled neurons in the PVN, SON, MPOA, BNST, MnPOd and MnPOv; number of OT labelled neurons or c-Fos/OT double labelled neurons in the PVN and SON.
Fig. 4. The effects of treatment with vehicle (1 mL/kg, i.p., n = 5 animals), WIN55,212-2 (1 mg/kg, i.p., n = 6 animals) or WIN55,212-2 (3 mg/kg, i.p., n = 4 animals) on plasma oxytocin levels of lactating rats (A) and on the weight gain of pups (B) Each column represents the mean (±S.E.M.). * p < 0.05 when compared with the vehicle group.
entries (F3,31 = 9.98, p < 0.01 and p < 0.05, respectively, Fig. 3B) when compared with the vehicle group. 3.4. Milk consumption by pups and oxytocin measurements The administration of WIN (1 or 3 mg/kg) to female rats reduced oxytocin secretion during lactation (F2,14 = 5.48, p < 0.05, Fig. 4A) when compared with the vehicle group. The administration of WIN (1 or 3 mg/kg) reduced weight gain in pups (F2,14 = 4.53, p < 0.05, Fig. 4B) when compared with the pups of dams that were treated with vehicle. 3.5. Immunohistochemistry for c-Fos/oxytocin The effects of WIN on the immunoreactivity of c-Fos and c-Fos/OT are summarised in Table 1. In the forebrain, treatment with WIN again reduced c-Fos immunoreactivity in the MPOA (F2,11 = 69,06; p < 0.001), BNST (F2,11 = 24.49; p < 0.001), MnPOd (F2,11 = 11,42; p < 0.01), PVN (F2,11 = 12.62; p < 0.01) and SON (F2,11 = 8.84; p < 0.01). Treatment with WIN (1 or 3 mg/kg) significantly decreased the number of c-Fos/oxytocin double labelled neurons in PVN (F2,11 = 37.52, p < 0.001) and SON (F2,11 = 15.80 p < 0.05). The pattern of c-Fos/oxytocin immunoreactivity in the MM subdivision of the PVN and in the SON of representative coronal sections from each group is presented in Fig. 5. 4. Discussion In all mammalian species, both physiological and behavioral changes occur throughout pregnancy to prepare the mother for the birth. These changes include the onset of maternal behaviors, such as nursing of offspring, maternal aggression and milk production to ensure the development and survival of the offspring [29,30].The
Vehicle
WIN (1 mg/kg)
WIN (3 mg/kg)
c-Fos PVN SON MPOA BNST MnPOd MnPOv
14.2 ± 1.3 7.6 ± 0.9 200 ± 6.7 55.8 ± 2.5 46.7 ± 5.4 41.2 ± 4.8
5.7 ± 0.3** 0.6 ± 0.3*** 89.5 ± 9.7*** 33.0 ± 2.5*** 29.2 ± 1.7** 28.5 ± 1.6
6.2 ± 2,0** 2.0 ± 0.6*** 100 ± 4.7*** 34.0 ± 2.8*** 25.7 ± 1.1** 29.7 ± 2.6
OT neurons PVN SON
65,5 ± 3,6 60.0 ± 7.5
72.0 ± 4.4 57.3 ± 1.4
64.7 ± 3.8 60.0 ± 3.8
c-Fos/OT PVN SON
4.2 ± 0.4 5.2 ± 0.8
0.4 ± 0.2*** 1.4 ± 2.4**
3.2 ± 0.5*** 1.2 ± 0.5***
results from our present study demonstrated that the administration of the cannabinoid receptor agonist WIN reduced maternal care, pup weight gain, maternal aggressive behavior and maternal anxiolysis. In addition, WIN reduced the activity of oxytocinergic neurons in the PVN and SON, and it reduced the oxytocin plasma levels in lactating rats. Oxytocin synthesis mainly occurs in neurons of the paraventricular (PVN) and supraoptic nuclei (SON) of the hypothalamus. It is released from nerve terminals in the posterior pituitary and various other regions of the brain. Suckling stimulates the simultaneous release of oxytocin into the bloodstream and central nervous system (CNS) of lactating rats [31]. Oxytocin receptors are present in many brain areas but since oxytocin does not cross the blood-brain barrier, oxytocin released from the pituitary does not act upon the brain [32], however, oxytocin release centrally from neurons which do not project to the pituitary is involved in maternal behavior. In addition, previous studies show that large quantities of oxytocin are released from the dendrites of magnocellular neurons. In addition, oxytocin release in response to suckling has been measured using microdialysis in the mediobasal hypothalamus, bed nucleus of the stria terminalis, medial preoptic area, and septum of parturient sheep [31,33]. Within this context, oxytocin is considered to be not only a hormone that is secreted from the neurohypophysis but also a neurotransmitter that is released at synapses in the brain [34]. Oxytocin mediates the actions of several structures in the hypothalamus and limbic system that receive projections from hypothalamic oxytocin neurons and regulate specific physiological and behavioral functions [34]. A key brain site for OT-mediated maternal care is the MPOA. Together with the adjacent BNST, these brain regions are implicated in maternal behavior [35]. Although these brain regions are essential for the occurrence of normal maternal behaviour in female rats at the time of parturition, other factors operate synergistically with the neural circuits arising from these regions to promote the important shift from infant avoidance to infant acceptance. Additionally, postpartum BNST lesions disrupt maternal behaviors in a similar manner to the effects produced by MPOA lesions [36]. Furthermore, physical interaction with pups, with or without suckling, elicited an increased number of c-Fos-immunoreactive nuclei in the MPOA that was higher compared to the number of nuclei found in other sites, including some sites that are implicated in maternal behaviour (MnPO and BNST) [37]. The disruption of nursing behavior by MPOA lesions has been proposed to involve a separate population of MPOA neurons that projects to the brainstem and spinal cord to modulate the reflexive aspects of nursing behavior [7,38]. In fact, the MPOA has strong projections to the ventrocaudal periacqueductal gray [39], which is known to regulate the crouch nursing posture in response to
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suckling stimulation [40]. The administration of WIN reduced the cFos expression in MPOA, BNST and MnPO, simultaneously to impair in maternal behavior, suggesting that cannabinoid suppress several cerebral areas of circuits regulating certain aspects maternal behavior in rats. The role of endogenous oxytocin in regulating maternal care has been established using an oxytocin antagonist or antisera infused into the brain to block the onset of maternal behavior in rats that have just given birth [41,42]. In addition to disrupting oxytocin secretion in response to suckling and maternal care, we observed that the cannabinoid agonist WIN reduced maternal behavior. The involvement of the endocannabinoid system during lactation was previously reported by pioneering studies that showed indirect evidence for a decrease in suckling-induced oxytocin release by THC through the reduction of intramammary pressure and pup behavior during suckling [15,16]. Suckling stimulates the simultaneous release of oxytocin into the bloodstream and central nervous system [43,44] has been implicated in the regulation of reproductive, maternal and affiliated behaviors [34,45]. The CB1 receptor is the major receptor for endocannabinoids in the brain, and it is mainly present in both pre-terminal and terminal axons. Furthermore, the CB1 receptor modulates neuronal signaling mechanisms in a retrograde manner; however, several studies have shown the expression of CB2 receptors in brain regions where CB1 receptors are also expressed [46]. Physiological and electrophysiological evidence has demonstrated a critical role for
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the CB1 receptor in the regulation of hypothalamic functions. One study demonstrated that axons with CB1 immunoreactivity densely innervate hypothalamic nuclei, such as the SON and PVN, in mice [47]. Previous studies have reported that administration of THC suppressed the retrieval of nesting material in mice [11,47] and depressed nursing and pup-retrieving behavior in rats [48]. Although the inhibitory influence of cannabinoids on the neurohypophyseal system is well established in hydromineral imbalances [17,20,49], the effects of cannabinoids on oxytocin release during lactation are poorly studied. In our present study, we showed that the cannabinoid agonist WIN suppressed c-Fos expression in oxytocinergic neurons of the PVN and SON; in addition, WIN reduced oxytocin plasma levels and milk consumption by pups. This reduction in activity of oxytocinergic neurons maybe can be related with impaired maternal care by lactating rats. The behavioral repertoire of some mammals during motherhood differs from that exhibited by females in other periods of their reproductive cycle. This behavioral pattern relies, at least partly, on the hormonal changes that characterize late gestation, parturition and lactation [50–52]. Breastfeeding in humans [53] and pup suckling in animals [54] represent rewarding social stimuli that encourage maternal behavior. Maternal aggression straddles the realms of maternity and defense, involving both a lactating female’s motivation to nurture her offspring and her need to protect the offspring from the infanticidal behavior of non-paternal males [55].
Fig. 5. Representative photomicrographs showing immunoreactivity for c-Fos (black nuclei; arrow head), oxytocin(brown cytoplasm; open arrow) and c-Fos/oxytocin (brown cytoplasm with black nuclei; full arrow) in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) of lactating rats treated with vehicle (1 mL/kg, i.p., n = 5 animals), WIN55,212-2 (1 mg/kg, i.p., n = 5 animals) or WIN55,212-2 (3 mg/kg, i.p., n = 4 animals). 3 V: third cerebral ventricle, OC: optic chiasm. Scale bar: 50 m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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In many species, maternity elicits a new capacity for fierce displays of protective aggression. Females who might normally retreat from, or ignore, novel male conspecifics will vigorously attack them to defend their young [7]. Maternal aggression is a key behavior adopted by dams when pups are born; however, the influence of cannabinoids on this response has yet to be elucidated. Similar to maternal care, WIN suppresses maternal aggression in lactating rats, and this effect may be mediated by a reduction in the activity of oxytocinergic neurons. In our study, we used the open field for evaluating the antithigmotactic effect and assessing the locomotor activity of animals. The administration of the cannabinoid receptor agonist WIN did not alter the animals locomotor activity; however, WIN-treated animals showed a reduction in the anti-thigmotactic effect, which is a typical characteristic of maternal anxiolysis [56,57]. Regarding anxiolysis, lactating rats that were treated with WIN behaved similarly to non-lactating rats in the open field test. It has become evident that the endocannabinoid system plays an essential role in multiple aspects of brain function, including an influence on the hypothalamic-pituitary-adrenal (HPA) axis and stress response [58,59], modulation of emotional states [60] and cognitive processes [61,62]. In conclusion, we showed that the administration of the cannabinoid receptor agonist WIN disrupts oxytocin secretion in response to suckling and reduces various behaviors and underlying motivational states that, collectively, can be referred to as maternal behaviors. Conflict of interest Authors report no biomedical financial interests or potential conflicts of interest. Role of funding source This work was supported by Fundac¸ão de Amparo a Pesquisa de Minas Gerais (FAPEMIG #2187-12, AG-P), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, #304675/20105; AG-P) and Coordenac¸ão de Aperfeic¸oamento de Pessoal de Nível Superior (CAPES; post-doctoral fellowship FCV). The FAPEMIG, CNPq and CAPES had no further role in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the paper for publication. Acknowledgements We are grateful for the excellent technical support of Marina F Venâncio and José dos Reis Pereira. References [1] Ferreira A, Hansen S, Nielsen M, Archer T, Minor BG. Behavior of mother rats in conflict tests sensitive to antianxiety agents. Behav Neurosci 1989;103:193–203. [2] Fleming AS, Cheung U, Myhal N, Kessler Z. Effects of maternal hormones on ‘timidity’ and attraction to pup-related odors in females rats. Physiol Behav 1989;46:449–53. [3] Lonstein JS, Gammie SC. Sensory, hormonal, and neural control of maternal aggression in laboratory rodents. Neurosci Behav Rev 2002;26:869–88. [4] Bosch OJ, Neumann ID. Both oxytocin and vasopressin are mediators of maternal care and aggression in rodents: from central release to sites of action. Horm Behav 2012;61:293–303. [5] Bosch OJ, Meddle SL, Beiderbeck DI, Douglas AJ, Neumann ID. Brain oxytocin correlates with maternal aggression: link to anxiety. J Neurosci 2005;25:6807–15. [6] Fleming AS, Walsh C. Neuropsychology of maternal behavior in the rat: cfos expression during mother-litter interactions. Psychoneuroendocrinology 1994;19:429–43. [7] Numan M, Insel TR. The neurobiology of parental behavior. New York: Springer Verlag; 2003. p. 110–9.
[8] Manning M, Kruszynski M, Bankowski K, Olma A, Lammek B, Cheng LL, et al. Solid-phase synthesis of 16 potent (selective and nonselective) in vivo antagonists of oxytocin. J Med Chem 1989;32:382–91. [9] Bosch OJ, Neumann ID. Brain vasopressin is an important regulator of maternal behavior independent of dams trait anxiety. Proc Natl Acad Sci 2008;105:17139–44. [10] Moschovakis A, Liakopoulos D, Armaganidis A, Kapsambelis V, Papanikolaou G, Petroulakis G. Cannabis interferes with nest-building behavior in mice. Psychopharmacology 1978;58:181–3. [11] Sieber B, Frischknecht HR, Waser PG. Behavioral effects of hashish in mice. I. Social interactions and nest-building behavior of males. Psychopharmacology 1980;70:149–54. [12] Ayalon D, Nir I, Cordova T, Bauminger S, Puder M, Naor Z, et al. Acute effect of delta1-tetrahydrocannabinol on the hypothalamo-pituitary-ovarian axis in the rat. Neuroendocrinology 1977;23:31–42. [13] Hughes Jr CL, Everett JW, Tyrey L. Delta 9-tetrahydrocannabinol suppression of prolactin secretion in the rat: lack of direct pituitary effect. Endocrinology 1981;109:876–80. [14] Ranganathan M, Braley G, Pittman B, Cooper T, Perry E, Krystal J, et al. The effects of cannabinoids on serum cortisol and prolactin in humans. Psychopharmacology 2009;203:737–44. [15] Wakerley JB, Lincoln DW. Intermittent release of oxytocin during suckling in the rat. Nat New Biol 1971;233:180–1. [16] Tyrey L, Murphy LL. Inhibition of suckling-induced milk ejections in the lactating rat by delta 9-tetrahydrocannabinol. Endocrinology 1988;123:469–72. [17] Ruginsk SG, Uchoa ET, Elias LL, Antunes-Rodrigues J. CB(1) modulation of hormone secretion, neuronal activation and mRNA expression following extracellular volume expansion. Exp Neurol 2010;224:114–22. [18] Lomax P. The effect of marihuana on pituitary-thyroid activity in the rat. Agents Actions 1970;1:252–7. [19] Rettori V, Aguila MC, Gimeno MF, Franchi AM, McCann SM. In vitro effect of delta 9-tetrahydrocannabinol to stimulate somatostatin release and block that of luteinizing hormone-releasing hormone by suppression of the release of prostaglandin E2. Proc Natl Acad Sci 1990;87:10063–6. [20] Ruginsk SG, Uchoa ET, Elias LL, Antunes-Rodrigues J. Anandamide modulates the neuroendocrine responses induced by extracellular volume expansion. Clin Exp Pharmacol Physiol 2013;40:698–705. [21] Crystal JD, Maxwell KW, Hohmann AG. Cannabinoid modulation of sensitivity to time. Behav Brain Res 2003;144:57–66. [22] Pedersen LH, Blackburn-Munro G. Pharmacological characterisation of place escape/avoidance behaviour in the rat chronic constriction injury model of neuropathic pain. Psychopharmacology 2006;185:208–17. [23] Trezza V, Vanderschuren LJ. Bidirectional cannabinoid modulation of social behavior in adolescent rats. Psychopharmacology 2008;197:217–27. [24] Vlachou S, Stamatopoulou F, Nomikos GG, Panagis G. Enhancement of endocannabinoid neurotransmission through CB1 cannabinoid receptors counteracts the reinforcing and psychostimulant effects of cocaine. Int J Neuropsychopharmacol 2008;11:905–23. [25] Vilela FC, Giusti-Paiva A. Glucocorticoids disrupt neuroendocrine and behavioral responses during lactation. Endocrinology 2011;152:4838–45. [26] Vilela FC, Ruginsk SG, de Melo CM, Giusti-Paiva A. The CB1 cannabinoid receptor mediates glucocorticoid-induced effects on behavioural and neuronal responses during lactation. Pflugers Arch 2013;465:1197–207. [27] Prut L, Belzung C. The open field as a paradigm to measure the effects of drugs on anxiety-like behaviors: a review. Eur J Pharmacol 2003;463:3–33. [28] Paxinos G, Watson C. The rat brain in stereotaxic coordinates. San Diego: Academic; 1997. [29] Rosenblatt JS, Factor EM, Mayer AD. Relationship between maternal aggression and maternal care in the rat. Aggress Behav 1994;20:243–55. [30] Neumann I. Alterations in behavioral and neuroendocrine stress coping strategies in pregnant, parturient and lactating rats. Prog Brain Res 2001;133:143–52. [31] Neumann I, Ludwig M, Engelmann M, Pittman QJ, Landgraf R. Simultaneous microdialysis in blood and brain: oxytocin and vasopressin release in response to central and peripheral osmotic stimulation and suckling in the rat. Neuroendocrinology 1993;58:637–45. [32] Leng G, Luckman SM. The vasopressin–oxytocin systems of the neurohypophysis. Principles of Medical Biology 1998;10:421–37. [33] Bealer SL, Crowley WR. Noradrenergic control of central oxytocin release during lactation in rats. Am J Physiol 1998;274:453–8. [34] Neumann ID. Stimuli and consequences of dendritic release of oxytocin within the brain. Biochem Soc Trans 2007;35:1252–7. [35] Bosch OJ, Pförtsch J, Beiderbeck DI, Landgraf R, Neumann ID. Maternal behaviour is associated with vasopressin release in the medial preoptic area and bed nucleus of the stria terminalis in the rat. J Neuroendocrinol 2010;22:420–9. [36] Numan M, Numan MJ. Projection sites of medial preoptic and ventral bed nucleus of the stria terminalis neurons that express Fos during maternal behavior in female rats. J Neuroendocrinol 1997;9:369–84. [37] Lonstein JS, Simmons DA, Swann JM, Stern JM. Forebrain expression of c-fos due to active maternal behaviour in lactating rats. Neuroscience 1998;82:267–81. [38] Numan M, Stolzenberg DS. Medial preoptic area interactions with dopamine neural systems in the control of the onset and maintenance of maternal behavior in rats. Front Neuroendocrinol 2009;30:46–64. [39] Numan M, Numan MJ. Projection sites of medial preoptic area and ventral bed nucleus of the stria terminalis neurons that express Fos during maternal behavior in female rats. J Neuroendocrinol 1997;9:369–84.
F.C. Vilela, A. Giusti-Paiva / Behavioural Brain Research 263 (2014) 190–197 [40] Lonstein JS, Stern JM. Somatosensory contributions to c-fos activation within the caudal periaqueductal gray of lactating rats: effects of perioral, rooting, and suckling stimuli from pups. Horm Behav 1997;32:155–66. [41] Fahrbach SE, Morrell JI, Pfaff DW. Possible role for endogenous oxytocin in estrogen-facilitated maternal behavior in rats. Neuroendocrinology 1985;40:526–32. [42] van Leengoed E, Kerker E, Swanson HH. Inhibition of postpartum maternal behaviour in the rat by injecting an oxytocin antagonist into the cerebral ventricles. J Endocrinol 1987;112:275–82. [43] Moos F, Poulain DA, Rodriguez F, Guerné Y, Vincent JD, Richard P. Release of oxytocin within the supraoptic nucleus during the milk ejection reflex in rats. Exp Brain Res 1989;76:593–602. [44] Neumann I, Russell JA, Landgraf R. Oxytocin and vasopressin release within the supraoptic and paraventricular nuclei of pregnant, parturient and lactating rats: a microdialysis study. Neuroscience 1993;53:65–75. [45] Landgraf R, Neumann ID. Vasopressin and oxytocin release within the brain: a dynamic concept of multiple and variable modes of neuropeptide communication. Front Neuroendocrinol 2004;25:150–76. [46] Callén L, Moreno E, Barroso-Chinea P, Moreno-Delgado D, Cortés A, Mallol J, et al. Cannabinoid receptors CB1 and CB2 form functional heteromers in brain. J Biol Chem 2012;287:20851–65. [47] Wittmann G, Deli L, Kalló I, Hrabovszky E, Watanabe M, Liposits Z, et al. Distribution of Type 1 Cannabinoid Receptor (CB1) Immunoreactive Axons in the Mouse Hypothalamus. J Comp Neurol 2007;503:270–9. [48] Navarro M, Rubio P, de Fonseca FR. Behavioural consequences of maternal exposure to natural cannabinoids in rats. Psychopharmacology 1995;122:1–14. [49] Ruginsk SG, Uchoa ET, Elias LL, Antunes-Rodrigues J. Cannabinoid CB1 receptor mediates glucocorticoid effects on hormone secretion induced by volume and osmotic changes. Clin Exp Pharmacol Physiol 2012;39:151–4. [50] Hansen S, Ferreira A. Food intake, aggression and fear behavior in mother rats: control by neural systems concerned with milk ejection and maternal behavior. Behav Neurosci 1986;100:67–70.
197
[51] Fleming AS, Cheung U, Myhal N, Kessler Z. Effects of maternal hormones on ‘timidity’ and attraction to pup-related odors in females rats. Physiol Behav 1989;46:449–53. [52] Bosch OJ, Neumann ID. Both oxytocin and vasopressin are mediators of maternal care and aggression in rodents: from central release to sites of action. Horm Behav 2012;61:293–303. [53] Kavanaugh K, Meier P, Zimmermann B, Mead L. The rewards outweigh the efforts: breastfeeding outcomes for mothers of preterm infants. J Hum Lact 1997;13:15–21. [54] Ferris CF, Kulkarni P, Sullivan JM, Harder JA, Messenger TL, Febo M. Pup suckling is more rewarding than cocaine: evidence from functional magnetic resonance imaging and three-dimensional computational analysis. J Neurosci 2005;25:149–56. [55] Agrell J, Wolff JO, Ylonen H. Counter-strategies to infanticide in mammals: costs and consequences. Oikos 1998;83:507–17. [56] Fleming AS, Luebke C. Timidity prevents the nulliparous female from being a good mother. Physiol Behav 1981;27:863–8. [57] Vilela FC, de Melo CM, Giusti-Paiva A. Glucocorticoids impair maternal anxiolysis during lactation. Neurosci Lett 2012;509:121–4. [58] Hill MN, McLaughlin RJ, Bingham B, Shrestha L, Lee TT, Gray JM, et al. Endogenous cannabinoid signaling is essential for stress adaptation. Proc Natl Acad Sci 2009;107:9406–11. [59] Riebe CJ, Wotjak CT. Endocannabinoids and stress. Stress 2011;14: 384–97. [60] Moreira FA, Lutz B. The endocannabinoid system: emotion, learning and addiction. Addict Biol 2008;13:196–212. [61] Rubino T, Parolaro D. Long lasting consequences of cannabis exposure in adolescence. Mol Cell Endocrinol 2008;286:108–13. [62] Atsak P, Roozendaal B, Campolongo P. Role of the endocannabinoid system in regulating glucocorticoid effects on memory for emotional experiences. Neuroscience 2012;204:104–16.
