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A single social defeat experience selectively stimulates the release of oxytocin, but not vasopressin, within the septal brain area of male rats
Brain Research 872 (2000) 87–92 www.elsevier.com / locate / bres
Research report
A single social defeat experience selectively stimulates the release of oxytocin, but not vasopressin, within the septal brain area of male rats a
a
a
K. Ebner , C.T. Wotjak , R. Landgraf , M. Engelmann b
a,b ,
*
a Max Planck Institute of Psychiatry, Kraepelinstr.2, D-80804 Munich, Germany Institute of Medical Neurobiology, Otto von Guericke University, Leipziger Str. 44, D-39120 Magdeburg, Germany
Accepted 2 May 2000
Abstract The naturally occurring social conflict situation to be confronted with an aggressive dominant conspecific was used to study the effects of emotional stress on the release of oxytocin (OXT) and arginine vasopressin (AVP) within the mediolateral septum of the rat brain. Male rats were chronically implanted with a microdialysis probe into this brain area. Local release patterns of both, OXT and AVP were monitored in response to a 30 min social defeat. Social defeat caused a significant increase in the release of OXT (to 254%643%, P,0.01). In contrast, the release of AVP was not affected. In a preliminary experiment, to assess the physiological significance of stress-induced intraseptal OXT release, a separate group of animals received the OXT receptor antagonist des-GlyNH 2 d(CH 2 ) 5 [Tyr(Me)2 Thr 4 ]OVT into the mediolateral septum via inverse microdialysis prior to and during the social defeat procedure. However, no difference could be observed in submissive freezing (passive coping) or in exploratory behavior (active coping) when compared to vehicle-treated animals, neither acutely nor 24 h after antagonist administration. Taken together, our results demonstrate that emotional stress activates the septal oxytocinergic, but not vasopressinergic, system. The physiological significance of intraseptally released OXT remains unclear and has to be elucidated in future studies. 2000 Elsevier Science B.V. All rights reserved. Keywords: Septum; Microdialysis; Emotional stress
1. Introduction The two nonapeptides oxytocin (OXT) and arginine vasopressin (AVP) are primarily synthesized in magnocellular neurons of the hypothalamic supraoptic (SON) and paraventricular nuclei (PVN). Both OXT and AVP are released from the hypothalamic-neurohypophysial system into the blood in response to appropriate stimulation (for review see [9]). In addition to this classical hypothalamicpituitary pathway, cell bodies and fibers containing OXT and AVP are also found in various extrahypothalamic brain areas [5,6,39]. For example, fibers originating from the bed nucleus of the stria terminalis and the amygdala innervate limbic brain areas, such as the septum and hippocampus [7,11,12]. These observations imply that OXT and AVP act not only as hormones in the blood, but also as neuromodulators / neurotransmitters within distinct mammalian *Corresponding author. Tel.: 149-391-67-14363; fax: 149-391-6714365. E-mail address: [email protected] (M. Engelmann)
brain areas. Indeed, OXT and AVP seem to be critically involved in a variety of brain functions, such as the generation of emotions and learning and memory (for review see [15,25]). Studies focusing on the intracerebral release of the two nonapeptides could demonstrate changes in the concentrations of OXT and AVP in the extracellular fluid of hypothalamic and limbic brain areas in response to a variety of stressful stimuli [13,32,44]. Recently, it could be shown that social defeat, associated with emotional stress, caused an increased release of OXT within the SON [16] and of AVP within the PVN [44]. Investigations on the site of action of intracerebrally released OXT and AVP on emotionality to cope with stress indicate that midbrain-limbic structures, like septum and amygdala, are critically involved in the behavioral response to emotionally challenging situations [20]. Binding studies confirmed existence of specific receptors for OXT and AVP within the septum [3,33,35,38,40]. In a recent study, we could demonstrate that stress-induced AVP release within the septum modulates the behavioral stress response of male rats in a forced swimming task driving animals to a more active coping strategy [13]. Further-
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K. Ebner et al. / Brain Research 872 (2000) 87 – 92
¨ Munchen, Germany) and held in place with dental cement. After surgery, rats received depot antibiotics (0.04 ml / rat, Tardomycel, Bayer, Leverkusen, Germany) to prevent infections. The day after surgery, animals were familiarized with the experimental equipment and handled for 3–5 min to minimize nonspecific stress responses during the experiment.
more, the reduction of effective binding sites for AVP within the septum by administration of either an AVP receptor antagonist [27] or antisense oligodeoxynucleotides targeting the mRNA of the V1 receptor subtype [23] altered the anxiety-related behavior of the animals. On the basis of these observations, the present experiments were designed to investigate whether the exposure of an animal to an emotionally challenging situation, like social defeat, influences the release of OXT and / or AVP in the mediolateral septum. In the first experiment we monitored the release of OXT and AVP within the mediolateral septum under basal conditions and in response to social defeat using the microdialysis technique. In a second experiment we attempted to investigate the physiological significance of the observed increased release of OXT by analyzing behavioral parameters after local administration of an OXT receptor antagonist via inverse microdialysis.
2.3. Microdialysis
2. Materials and methods
2.4. Social defeat procedure
2.1. Animals
The social defeat procedure, adapted with modifications from Miczek [28], consists of placing the experimental animal in the home cage (38320335 cm) of an adult male rat (resident) previously trained to be aggressive towards intruders [44]. In our experiments, the resident was at least 9 months old and housed together with a female to stimulate territorial aggression [17]. The intruder was attacked and subdued by the resident in all single experiments within the first 1–3 min of stressor exposure. Immediately after the first attack, intruder and resident were separated by a wire mesh for the remaining time of the 30 min social defeat period. By this procedure, the experimental animal was protected from repeated attacks and potential injury, whereas it was still exposed to visual and olfactory cues from the resident. Behavioral postures and ultrasonic vocalization were recorded in 1 min intervals during the entire social encounter. Social defeat was considered successful if the intruder showed defensive behavior as indicated by submissive body postures or freezing behavior for at least 15 min of the 30 min exposure. At the end of the encounter, experimental animals were returned to their home cages.
Adult male Wistar rats (Charles River, Sulzfeld, Germany), weighting 300–400 g, were used in this study. Before surgery, animals were housed in groups of 4–6 for at least 1 week after delivery from the supplier. After surgery, rats were kept individually in transparent polycarbon cages (20328335 cm) under controlled standard laboratory conditions (23628C, 6065% humidity, 12 h light / 12 h dark cycle with lights on at 7:00 h) with free access to food and water.
2.2. Surgery All surgical, sampling and behavioral protocols were approved by the Committee on Animal Health and Care of the local governmental administration. Rats were anesthetized with halothane (Hoechst, Frankfurt a. M., Germany) and mounted on a stereotaxic frame. A microdialysis probe (home-made, U-shaped membrane with a molecular cut-off of 18 kDa clued in a parallel side-by-side pair of 25-gauge stainless steel cannulae; in vitro recovery for AVP: 2.5%) was implanted according to the stereotaxic atlas of Paxinos and Watson [34] into either (1) the mediolateral septum (n537; implantation coordinates: 0.8 mm rostral to bregma, 2.3 mm lateral to the midline, and 6.3 mm below the surface of the skull with an angle of 228 to avoid damage to the sagittal sinus) or (2) the lateral ventricle (n56; implantation coordinates: 0.8 mm rostral to bregma, 2.3 mm lateral to the midline, and 6.3 mm below the surface of the skull with an angle of 128). The microdialysis probes were fixed to the skull with two jeweler’s screws and dental cement. The two endings of the probe were connected to approx. 5 cm long pieces of polyethylene tubing (PE-20; Karmann and Droll,
On the day of the experiment (i.e., approx. 48 h after surgery), microdialysis probes were connected to syringes mounted onto a microinfusion pump via pieces of PE-20 tubing and perfused with sterile Ringer’s solution at a rate of 3.3 ml / min (Fresenius, Bad Homburg, Germany) for 2 h prior to the initiation of the experiment. During this period sample collection was simulated every 30 min to adapt the animals to the microdialysate sampling procedure.
2.4.1. Experiment 1: Effects of emotional stress on the release of OXT and AVP within the septum during social defeat Six consecutive 30 min microdialysis samples were collected directly into Eppendorf vials containing 10 ml of 0.1 N HCl to increase peptide stability and stored on dry ice. Samples one and two were collected under basal conditions. At the beginning of the third dialysis interval, animals were transferred from their home cages to the resident’s cage, where social defeat took place under ongoing microdialysis. Three additional samples were collected after the defeat session.
K. Ebner et al. / Brain Research 872 (2000) 87 – 92
2.4.2. Experiment 2: Effects of an OXT receptor antagonist on animals’ behavior during social defeat In two additional groups of animals microdialysis probes were perfused either with Ringer’s solution or with Ringer’s solution containing the OXT receptor antagonist des-Gly-NH 2 d(CH 2 ) 5 [Tyr(Me)2 Thr 4 ]OVT (20 mg / 100 ml; Dr M. Manning, Toledo, USA) for 60 min. The total amount of antagonist delivered into the mediolateral septum was calculated to be approximately 40 ng [14]. Thirty min after the beginning of the retrodialysis procedure, rats were socially defeated while ongoing microdialysis sampling. During the entire defeat session, the behavior of the animals was monitored and scored by a trained observer blind to the animals’ treatment. The following behavioral parameters were recorded: (1) freezing characterized by a submissive body posture, also frequently accompanied by ultrasonic vocalization, (2) resting as passive behavior without submissive postures, and (3) active exploratory behavior. On the following day, the same rats were again exposed to the social defeat paradigm without being connected to the microdialysis equipment for a period of 10 min, while their behavior was scored. 2.5. Histology At the end of the experiment, animals were killed by an overdose of halothane. Brains were removed from the skull, shock-frozen in dry-ice chilled n-methylbutane and stored at 2208C. For histological verification of the microdialysis probe placement, brains were sectioned in a cryocut (Micron HM 500, Walldorf, Germany) and 25 mm coronal sections were stained with cresyl violet.
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radioimmunoassays (RIA; detection limit: 0.01 pg / sample; cross-reactivity less than 0.7%; for a more detailed description see [24]). To measure OXT and AVP in the same sample, microdialysates were split either into 1 / 2 (each) for samples from the lateral ventricle or into 2 / 3 (OXT) and 1 / 3 (AVP) for microdialysates collected in the mediolateral septum. The latter procedure was chosen since previous pilot studies revealed that 1 / 2 of the microdialysis sample collected from the septum contained not enough OXT for measuring basal levels.
2.7. Statistics Experimental subjects were included in the statistical analysis only if the microdialysis probes had been found to be localized in the respective target brain area (Fig. 1) and social defeat had been successful (experiment 1 only). Data (means6S.E.M.) are not corrected for recovery and total sample volume. For statistical analysis, data were logarithmically transformed to fit into a Gaussian distribution. Statistical analysis was performed using a computer software package (GB-Stat 6.0, Dynamic Microsystems, Silver Springs, USA). Intracerebral release data were analyzed using a one-way ANOVA for repeated measures. Behavioral measures were tested using a one-way ANOVA (completely randomized). Fisher’s LSD post-hoc analysis was performed if appropriate. Significance was accepted if P,0.05.
3. Results
2.6. Radioimmunoassays
3.1. Experiment 1: Effects of emotional stress on the release of OXT and AVP within the septum during social defeat
OXT and AVP levels were measured in microdialysates after lyophilization by highly sensitive and selective
As shown in Fig. 2A, social defeat caused a significant increase in the release of OXT within the mediolateral
Fig. 1. Schematic drawings showing the reconstructed localization of the microdialysis probes tips within the mediolateral septum (A) to monitor the local release of AVP and OXT or (B) to locally administer the OXT receptor antagonist.
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Fig. 3. Effects of an OXT receptor antagonist on animals’ behavior during the social encounter. The OXT antagonist des-GlyNH 2 d(CH 2 ) 5 [Tyr(Me)2 Thr 4 ]OVT (40 ng; n515) was administered into the mediolateral septum via inverse microdialysis for 60 min, starting 30 min before the 30 min social defeat (A). Re-testing took place 24 h later but this time for 10 min only and without administration of the antagonist (B). Data are expressed as the time (means1S.E.M.) animals spent in freezing and investigating. Control animals (n513) had been dialyzed with Ringer’s solution only.
Fig. 2. Effects of social defeat on the content of OXT (A) and AVP (B) in 30 min microdialysates collected consecutively in the mediolateral septum of freely moving male rats (means1S.E.M.; n59). Both AVP and OXT were measured in the same sample. Animals were exposed to social defeat during collection of dialysis sample number 3 (grey bar). *P,0.05 vs. dialysis samples 1 and 4 and P,0.01 vs. dialysis samples 2, 5 and 6; 1P,0.05 vs. dialysis sample 1 (Fisher’s LSD test).
septum (F5,40 59.99; P,0.001), returning to pre-stress values within the next 60 min. In contrast, the release of AVP gradually decreased with increasing time (F5,35 52.56; P,0.044; Fig. 2B). AVP content of microdialysates collected from the lateral ventricle remained unchanged throughout the experiment (data not shown). OXT content of the same samples obtained from the ventricle was below the detection limit of the RIA (data not shown).
3.2. Experiment 2: Effects of an OXT receptor antagonist on animals’ behavior during social defeat Statistical analysis of the behavioral data failed to reveal significant effects of antagonist administration. As illus-
trated in Fig. 3A, antagonist- and vehicle-treated animals did not differ significantly in the duration of freezing (F1,26 50.02, P50.998) or exploratory behavior (F1,26 5 0.05, P50.824). The same was the case for the behavioral performance 24 h after treatment (Fig. 3B). Similar to day 1, neither the duration of freezing behavior (F1,26 ,0.01, P50.984) nor that of the exploratory behavior (F1,26 5 0.05, P50.818) was different between the two groups.
4. Discussion The aim of the present study was to investigate whether (1) an ethologically relevant stressful stimulus alters the release of the nonapeptides AVP and / or OXT within a limbic brain area and (2) the measured alterations are of behavioral relevance. We demonstrated that social defeat is a potent and selective stimulus to trigger the release of OXT, but not AVP, into the extracellular compartment of the mediolateral septum. The functional role of intraseptally released OXT, however, remains unclear. So far, most in vivo studies that investigated the release of AVP and / or OXT within limbic brain areas used pharmacological stimulation [10,21,36]. Only few studies
K. Ebner et al. / Brain Research 872 (2000) 87 – 92
could demonstrate an increased release of OXT under physiologically relevant conditions. For example, in female rats OXT is released within the septum during parturition and lactation [22,30]. The data of the present study demonstrate for the first time that OXT is also released within the septum of male rats during emotional stress. Basal levels of OXT in the microdialysates were just above the detection limit of the RIA and, thus, considerably lower than those measured in push-pull-perfusates [21,30]. Possible reasons could be the relatively low oxytocinergic innervation of the septum [39] in conjunction with the low peptide recovery of the microdialysis probes. To exclude that OXT simply diffused from other brain areas via the cerebrospinal fluid to the septum, we measured OXT also in microdialysates collected from probes located within the lateral ventricle. However, the concentration of OXT in those samples was below the detection limit of the RIA. Furthermore, social defeat failed to trigger a significant increase in the release of OXT within the PVN, another structure in close vicinity to the ventricular system of the rat brain [44]. In the light of these findings it seems rather unlikely that the increased OXT concentration in microdialysates obtained from the septum originates from other brain areas or the cerebrospinal fluid. Confrontation with a dominant aggressive male rat triggered intraseptal release of OXT, but failed to alter AVP levels. The same stressor had previously been reported to selectively stimulate the release of OXT within the SON and that of AVP within the PVN [16,44]. The latter finding is of particular interest, because neurons of the PVN comprise the major oxytocinergic innervation of the septum [11,26,29]. Our failure to detect a significant increase in the release of OXT within the PVN but not the septum could be interpreted in different ways: On the one hand emotional stress may trigger axonal rather than somato-dendritic release of OXT from this neuronal population. On the other hand, the amount of OXT released from parvocellular neurons within the PVN might be too small to be clearly detectable by the microdialysis technique. Next, we asked the question as to the physiological significance of the increased OXT release within the septum. Various studies imply a critical role for OXT in the regulation of different physiological and behavioral processes, related to reproduction (for review see [1,8,18,37]), learning and memory (for review see [15]) and the generation of emotions [2,4,41–43]. In the present study we failed to detect any effect of a locally applied OXT antagonist on animals’ behavior during the exposure to the social defeat stress paradigm. However, it is difficult to rule out that more selective tools (e.g. a modification of the social defeat procedure, use of non-peptidergic or more general acting antagonists) or monitoring of other behavioral parameters (e.g. anxiety-related behavior) would have revealed the behavioral impact of OXT. In addition,
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intraseptally released OXT might well be involved in the regulation of the endocrine stress response. Neuroanatomical investigations indicate that GABA neurons project from the septum to the hypothalamus [19]. This suggests a participation of oxytocinergic structures in the regulation of the activity of the hypothalamic-pituitary-adrenal (HPA) axis. Recently, Neumann et al. [31] showed that intrahypothalamic administration of the OXT receptor antagonist triggered the secretion of ACTH in both resting and stressed animals. Administration of exogenous OXT, in turn, inhibited the activity of the HPA axis [43]. Taken together, our data demonstrate that social defeat triggers the release of OXT within the mediolateral septum of male rats. Although evidence exists that OXT plays a role in the regulation of behavioral and endocrine stresscoping strategies, the physiological role of intraseptally released OXT remains to be lucidated in future studies.
Acknowledgements The authors thank T.F.W. Horn for critical reading of the manuscript.
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Research report
A single social defeat experience selectively stimulates the release of oxytocin, but not vasopressin, within the septal brain area of male rats a
a
a
K. Ebner , C.T. Wotjak , R. Landgraf , M. Engelmann b
a,b ,
*
a Max Planck Institute of Psychiatry, Kraepelinstr.2, D-80804 Munich, Germany Institute of Medical Neurobiology, Otto von Guericke University, Leipziger Str. 44, D-39120 Magdeburg, Germany
Accepted 2 May 2000
Abstract The naturally occurring social conflict situation to be confronted with an aggressive dominant conspecific was used to study the effects of emotional stress on the release of oxytocin (OXT) and arginine vasopressin (AVP) within the mediolateral septum of the rat brain. Male rats were chronically implanted with a microdialysis probe into this brain area. Local release patterns of both, OXT and AVP were monitored in response to a 30 min social defeat. Social defeat caused a significant increase in the release of OXT (to 254%643%, P,0.01). In contrast, the release of AVP was not affected. In a preliminary experiment, to assess the physiological significance of stress-induced intraseptal OXT release, a separate group of animals received the OXT receptor antagonist des-GlyNH 2 d(CH 2 ) 5 [Tyr(Me)2 Thr 4 ]OVT into the mediolateral septum via inverse microdialysis prior to and during the social defeat procedure. However, no difference could be observed in submissive freezing (passive coping) or in exploratory behavior (active coping) when compared to vehicle-treated animals, neither acutely nor 24 h after antagonist administration. Taken together, our results demonstrate that emotional stress activates the septal oxytocinergic, but not vasopressinergic, system. The physiological significance of intraseptally released OXT remains unclear and has to be elucidated in future studies. 2000 Elsevier Science B.V. All rights reserved. Keywords: Septum; Microdialysis; Emotional stress
1. Introduction The two nonapeptides oxytocin (OXT) and arginine vasopressin (AVP) are primarily synthesized in magnocellular neurons of the hypothalamic supraoptic (SON) and paraventricular nuclei (PVN). Both OXT and AVP are released from the hypothalamic-neurohypophysial system into the blood in response to appropriate stimulation (for review see [9]). In addition to this classical hypothalamicpituitary pathway, cell bodies and fibers containing OXT and AVP are also found in various extrahypothalamic brain areas [5,6,39]. For example, fibers originating from the bed nucleus of the stria terminalis and the amygdala innervate limbic brain areas, such as the septum and hippocampus [7,11,12]. These observations imply that OXT and AVP act not only as hormones in the blood, but also as neuromodulators / neurotransmitters within distinct mammalian *Corresponding author. Tel.: 149-391-67-14363; fax: 149-391-6714365. E-mail address: [email protected] (M. Engelmann)
brain areas. Indeed, OXT and AVP seem to be critically involved in a variety of brain functions, such as the generation of emotions and learning and memory (for review see [15,25]). Studies focusing on the intracerebral release of the two nonapeptides could demonstrate changes in the concentrations of OXT and AVP in the extracellular fluid of hypothalamic and limbic brain areas in response to a variety of stressful stimuli [13,32,44]. Recently, it could be shown that social defeat, associated with emotional stress, caused an increased release of OXT within the SON [16] and of AVP within the PVN [44]. Investigations on the site of action of intracerebrally released OXT and AVP on emotionality to cope with stress indicate that midbrain-limbic structures, like septum and amygdala, are critically involved in the behavioral response to emotionally challenging situations [20]. Binding studies confirmed existence of specific receptors for OXT and AVP within the septum [3,33,35,38,40]. In a recent study, we could demonstrate that stress-induced AVP release within the septum modulates the behavioral stress response of male rats in a forced swimming task driving animals to a more active coping strategy [13]. Further-
0006-8993 / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 00 )02464-1
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K. Ebner et al. / Brain Research 872 (2000) 87 – 92
¨ Munchen, Germany) and held in place with dental cement. After surgery, rats received depot antibiotics (0.04 ml / rat, Tardomycel, Bayer, Leverkusen, Germany) to prevent infections. The day after surgery, animals were familiarized with the experimental equipment and handled for 3–5 min to minimize nonspecific stress responses during the experiment.
more, the reduction of effective binding sites for AVP within the septum by administration of either an AVP receptor antagonist [27] or antisense oligodeoxynucleotides targeting the mRNA of the V1 receptor subtype [23] altered the anxiety-related behavior of the animals. On the basis of these observations, the present experiments were designed to investigate whether the exposure of an animal to an emotionally challenging situation, like social defeat, influences the release of OXT and / or AVP in the mediolateral septum. In the first experiment we monitored the release of OXT and AVP within the mediolateral septum under basal conditions and in response to social defeat using the microdialysis technique. In a second experiment we attempted to investigate the physiological significance of the observed increased release of OXT by analyzing behavioral parameters after local administration of an OXT receptor antagonist via inverse microdialysis.
2.3. Microdialysis
2. Materials and methods
2.4. Social defeat procedure
2.1. Animals
The social defeat procedure, adapted with modifications from Miczek [28], consists of placing the experimental animal in the home cage (38320335 cm) of an adult male rat (resident) previously trained to be aggressive towards intruders [44]. In our experiments, the resident was at least 9 months old and housed together with a female to stimulate territorial aggression [17]. The intruder was attacked and subdued by the resident in all single experiments within the first 1–3 min of stressor exposure. Immediately after the first attack, intruder and resident were separated by a wire mesh for the remaining time of the 30 min social defeat period. By this procedure, the experimental animal was protected from repeated attacks and potential injury, whereas it was still exposed to visual and olfactory cues from the resident. Behavioral postures and ultrasonic vocalization were recorded in 1 min intervals during the entire social encounter. Social defeat was considered successful if the intruder showed defensive behavior as indicated by submissive body postures or freezing behavior for at least 15 min of the 30 min exposure. At the end of the encounter, experimental animals were returned to their home cages.
Adult male Wistar rats (Charles River, Sulzfeld, Germany), weighting 300–400 g, were used in this study. Before surgery, animals were housed in groups of 4–6 for at least 1 week after delivery from the supplier. After surgery, rats were kept individually in transparent polycarbon cages (20328335 cm) under controlled standard laboratory conditions (23628C, 6065% humidity, 12 h light / 12 h dark cycle with lights on at 7:00 h) with free access to food and water.
2.2. Surgery All surgical, sampling and behavioral protocols were approved by the Committee on Animal Health and Care of the local governmental administration. Rats were anesthetized with halothane (Hoechst, Frankfurt a. M., Germany) and mounted on a stereotaxic frame. A microdialysis probe (home-made, U-shaped membrane with a molecular cut-off of 18 kDa clued in a parallel side-by-side pair of 25-gauge stainless steel cannulae; in vitro recovery for AVP: 2.5%) was implanted according to the stereotaxic atlas of Paxinos and Watson [34] into either (1) the mediolateral septum (n537; implantation coordinates: 0.8 mm rostral to bregma, 2.3 mm lateral to the midline, and 6.3 mm below the surface of the skull with an angle of 228 to avoid damage to the sagittal sinus) or (2) the lateral ventricle (n56; implantation coordinates: 0.8 mm rostral to bregma, 2.3 mm lateral to the midline, and 6.3 mm below the surface of the skull with an angle of 128). The microdialysis probes were fixed to the skull with two jeweler’s screws and dental cement. The two endings of the probe were connected to approx. 5 cm long pieces of polyethylene tubing (PE-20; Karmann and Droll,
On the day of the experiment (i.e., approx. 48 h after surgery), microdialysis probes were connected to syringes mounted onto a microinfusion pump via pieces of PE-20 tubing and perfused with sterile Ringer’s solution at a rate of 3.3 ml / min (Fresenius, Bad Homburg, Germany) for 2 h prior to the initiation of the experiment. During this period sample collection was simulated every 30 min to adapt the animals to the microdialysate sampling procedure.
2.4.1. Experiment 1: Effects of emotional stress on the release of OXT and AVP within the septum during social defeat Six consecutive 30 min microdialysis samples were collected directly into Eppendorf vials containing 10 ml of 0.1 N HCl to increase peptide stability and stored on dry ice. Samples one and two were collected under basal conditions. At the beginning of the third dialysis interval, animals were transferred from their home cages to the resident’s cage, where social defeat took place under ongoing microdialysis. Three additional samples were collected after the defeat session.
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2.4.2. Experiment 2: Effects of an OXT receptor antagonist on animals’ behavior during social defeat In two additional groups of animals microdialysis probes were perfused either with Ringer’s solution or with Ringer’s solution containing the OXT receptor antagonist des-Gly-NH 2 d(CH 2 ) 5 [Tyr(Me)2 Thr 4 ]OVT (20 mg / 100 ml; Dr M. Manning, Toledo, USA) for 60 min. The total amount of antagonist delivered into the mediolateral septum was calculated to be approximately 40 ng [14]. Thirty min after the beginning of the retrodialysis procedure, rats were socially defeated while ongoing microdialysis sampling. During the entire defeat session, the behavior of the animals was monitored and scored by a trained observer blind to the animals’ treatment. The following behavioral parameters were recorded: (1) freezing characterized by a submissive body posture, also frequently accompanied by ultrasonic vocalization, (2) resting as passive behavior without submissive postures, and (3) active exploratory behavior. On the following day, the same rats were again exposed to the social defeat paradigm without being connected to the microdialysis equipment for a period of 10 min, while their behavior was scored. 2.5. Histology At the end of the experiment, animals were killed by an overdose of halothane. Brains were removed from the skull, shock-frozen in dry-ice chilled n-methylbutane and stored at 2208C. For histological verification of the microdialysis probe placement, brains were sectioned in a cryocut (Micron HM 500, Walldorf, Germany) and 25 mm coronal sections were stained with cresyl violet.
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radioimmunoassays (RIA; detection limit: 0.01 pg / sample; cross-reactivity less than 0.7%; for a more detailed description see [24]). To measure OXT and AVP in the same sample, microdialysates were split either into 1 / 2 (each) for samples from the lateral ventricle or into 2 / 3 (OXT) and 1 / 3 (AVP) for microdialysates collected in the mediolateral septum. The latter procedure was chosen since previous pilot studies revealed that 1 / 2 of the microdialysis sample collected from the septum contained not enough OXT for measuring basal levels.
2.7. Statistics Experimental subjects were included in the statistical analysis only if the microdialysis probes had been found to be localized in the respective target brain area (Fig. 1) and social defeat had been successful (experiment 1 only). Data (means6S.E.M.) are not corrected for recovery and total sample volume. For statistical analysis, data were logarithmically transformed to fit into a Gaussian distribution. Statistical analysis was performed using a computer software package (GB-Stat 6.0, Dynamic Microsystems, Silver Springs, USA). Intracerebral release data were analyzed using a one-way ANOVA for repeated measures. Behavioral measures were tested using a one-way ANOVA (completely randomized). Fisher’s LSD post-hoc analysis was performed if appropriate. Significance was accepted if P,0.05.
3. Results
2.6. Radioimmunoassays
3.1. Experiment 1: Effects of emotional stress on the release of OXT and AVP within the septum during social defeat
OXT and AVP levels were measured in microdialysates after lyophilization by highly sensitive and selective
As shown in Fig. 2A, social defeat caused a significant increase in the release of OXT within the mediolateral
Fig. 1. Schematic drawings showing the reconstructed localization of the microdialysis probes tips within the mediolateral septum (A) to monitor the local release of AVP and OXT or (B) to locally administer the OXT receptor antagonist.
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Fig. 3. Effects of an OXT receptor antagonist on animals’ behavior during the social encounter. The OXT antagonist des-GlyNH 2 d(CH 2 ) 5 [Tyr(Me)2 Thr 4 ]OVT (40 ng; n515) was administered into the mediolateral septum via inverse microdialysis for 60 min, starting 30 min before the 30 min social defeat (A). Re-testing took place 24 h later but this time for 10 min only and without administration of the antagonist (B). Data are expressed as the time (means1S.E.M.) animals spent in freezing and investigating. Control animals (n513) had been dialyzed with Ringer’s solution only.
Fig. 2. Effects of social defeat on the content of OXT (A) and AVP (B) in 30 min microdialysates collected consecutively in the mediolateral septum of freely moving male rats (means1S.E.M.; n59). Both AVP and OXT were measured in the same sample. Animals were exposed to social defeat during collection of dialysis sample number 3 (grey bar). *P,0.05 vs. dialysis samples 1 and 4 and P,0.01 vs. dialysis samples 2, 5 and 6; 1P,0.05 vs. dialysis sample 1 (Fisher’s LSD test).
septum (F5,40 59.99; P,0.001), returning to pre-stress values within the next 60 min. In contrast, the release of AVP gradually decreased with increasing time (F5,35 52.56; P,0.044; Fig. 2B). AVP content of microdialysates collected from the lateral ventricle remained unchanged throughout the experiment (data not shown). OXT content of the same samples obtained from the ventricle was below the detection limit of the RIA (data not shown).
3.2. Experiment 2: Effects of an OXT receptor antagonist on animals’ behavior during social defeat Statistical analysis of the behavioral data failed to reveal significant effects of antagonist administration. As illus-
trated in Fig. 3A, antagonist- and vehicle-treated animals did not differ significantly in the duration of freezing (F1,26 50.02, P50.998) or exploratory behavior (F1,26 5 0.05, P50.824). The same was the case for the behavioral performance 24 h after treatment (Fig. 3B). Similar to day 1, neither the duration of freezing behavior (F1,26 ,0.01, P50.984) nor that of the exploratory behavior (F1,26 5 0.05, P50.818) was different between the two groups.
4. Discussion The aim of the present study was to investigate whether (1) an ethologically relevant stressful stimulus alters the release of the nonapeptides AVP and / or OXT within a limbic brain area and (2) the measured alterations are of behavioral relevance. We demonstrated that social defeat is a potent and selective stimulus to trigger the release of OXT, but not AVP, into the extracellular compartment of the mediolateral septum. The functional role of intraseptally released OXT, however, remains unclear. So far, most in vivo studies that investigated the release of AVP and / or OXT within limbic brain areas used pharmacological stimulation [10,21,36]. Only few studies
K. Ebner et al. / Brain Research 872 (2000) 87 – 92
could demonstrate an increased release of OXT under physiologically relevant conditions. For example, in female rats OXT is released within the septum during parturition and lactation [22,30]. The data of the present study demonstrate for the first time that OXT is also released within the septum of male rats during emotional stress. Basal levels of OXT in the microdialysates were just above the detection limit of the RIA and, thus, considerably lower than those measured in push-pull-perfusates [21,30]. Possible reasons could be the relatively low oxytocinergic innervation of the septum [39] in conjunction with the low peptide recovery of the microdialysis probes. To exclude that OXT simply diffused from other brain areas via the cerebrospinal fluid to the septum, we measured OXT also in microdialysates collected from probes located within the lateral ventricle. However, the concentration of OXT in those samples was below the detection limit of the RIA. Furthermore, social defeat failed to trigger a significant increase in the release of OXT within the PVN, another structure in close vicinity to the ventricular system of the rat brain [44]. In the light of these findings it seems rather unlikely that the increased OXT concentration in microdialysates obtained from the septum originates from other brain areas or the cerebrospinal fluid. Confrontation with a dominant aggressive male rat triggered intraseptal release of OXT, but failed to alter AVP levels. The same stressor had previously been reported to selectively stimulate the release of OXT within the SON and that of AVP within the PVN [16,44]. The latter finding is of particular interest, because neurons of the PVN comprise the major oxytocinergic innervation of the septum [11,26,29]. Our failure to detect a significant increase in the release of OXT within the PVN but not the septum could be interpreted in different ways: On the one hand emotional stress may trigger axonal rather than somato-dendritic release of OXT from this neuronal population. On the other hand, the amount of OXT released from parvocellular neurons within the PVN might be too small to be clearly detectable by the microdialysis technique. Next, we asked the question as to the physiological significance of the increased OXT release within the septum. Various studies imply a critical role for OXT in the regulation of different physiological and behavioral processes, related to reproduction (for review see [1,8,18,37]), learning and memory (for review see [15]) and the generation of emotions [2,4,41–43]. In the present study we failed to detect any effect of a locally applied OXT antagonist on animals’ behavior during the exposure to the social defeat stress paradigm. However, it is difficult to rule out that more selective tools (e.g. a modification of the social defeat procedure, use of non-peptidergic or more general acting antagonists) or monitoring of other behavioral parameters (e.g. anxiety-related behavior) would have revealed the behavioral impact of OXT. In addition,
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intraseptally released OXT might well be involved in the regulation of the endocrine stress response. Neuroanatomical investigations indicate that GABA neurons project from the septum to the hypothalamus [19]. This suggests a participation of oxytocinergic structures in the regulation of the activity of the hypothalamic-pituitary-adrenal (HPA) axis. Recently, Neumann et al. [31] showed that intrahypothalamic administration of the OXT receptor antagonist triggered the secretion of ACTH in both resting and stressed animals. Administration of exogenous OXT, in turn, inhibited the activity of the HPA axis [43]. Taken together, our data demonstrate that social defeat triggers the release of OXT within the mediolateral septum of male rats. Although evidence exists that OXT plays a role in the regulation of behavioral and endocrine stresscoping strategies, the physiological role of intraseptally released OXT remains to be lucidated in future studies.
Acknowledgements The authors thank T.F.W. Horn for critical reading of the manuscript.
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