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c-Fos activation and intermale aggression in rats selected for behavior toward humans
Behavioural Brain Research 237 (2013) 103–106
Contents lists available at SciVerse ScienceDirect
Behavioural Brain Research journal homepage: www.elsevier.com/locate/bbr
Short communication
c-Fos activation and intermale aggression in rats selected for behavior toward humans Maria Yu. Konoshenko a,∗ , Tatiana V. Timoshenko b,c , Irina Z. Plyusnina a,b,c a b c
Institute of Cytology and Genetics of the Siberian Branch of the Russian Academy of Sciences, pr. Lavrentjeva 10, Novosibirsk 630090, Russia Institute of Gene Biology of the Russian Academy of Sciences, Moscow 119334, Russia Apto-Pharm Ltd., Moscow 115446, Russia
h i g h l i g h t s
The neuronal activation pattern in tame and aggressive rats after exposure to R–I test was studied. Social encounter caused similar brain activation pattern in rats of selected lines. Activation was shown in bed nucleus, hypothalamic attack area and medial amygdala. Activation of the hypothalamic attack area was higher in aggressive males then in tame rats.
a r t i c l e
i n f o
Article history: Received 2 August 2012 Received in revised form 10 September 2012 Accepted 14 September 2012 Available online 20 September 2012 Keywords: Aggression c-Fos Norway rats Resident–intruder Selection
a b s t r a c t Tame and aggressive rat lines were created during the long-term selection of Norway rats for elimination and enhancement of aggressiveness toward humans, respectively. Our previous experiments have demonstrated that selection for the elimination of defensive aggression toward humans is associated with attenuated intraspecific intermale aggression. However, the neuronal mechanisms underlying low and high intermale aggression in the tame and aggressive rats remain unclear. Here, we used c-Fos immunoreactivity to evaluate neuronal activation patterns in the main aggression-related areas in selected lines under basal conditions and after the resident–intruder (R–I) test. Although agonistic behavior of the tame and the aggressive rats differed significantly, social encounter caused similar brain activation patterns in both groups; we observed increased neuronal activation in the bed nucleus of stria terminalis, the hypothalamic attack area, and the medial amygdala 1 h after the R–I test. However, neuronal activation in the hypothalamic attack area was significantly higher in the aggressive males compared to their tame counterparts. We propose that lower activation of the hypothalamic area is associated with the attenuation of intraspecific intermale aggression during selection for the elimination of aggressiveness toward humans. © 2012 Elsevier B.V. All rights reserved.
Immediate early gene expression (e.g., c-Fos) is a widely used functional marker of neuronal activation and is often employed to identify cells and brain circuits that respond to various stimuli [4,2,5]. Assessment of immediate early gene expression has been used to provide an accurate account of brain structures involved in the regulation of aggressive behavior. The study of c-Fos activation in rat and mice lines selected for behavior is of particular interest, and these studies have revealed genetically determined
Abbreviations: BNST, bed nucleus of stria terminalis; CeA, central nucleus of the amygdala; CoA, cortical amygdala; DR, dorsal raphe nucleus; HAA, hypothalamic attack area; HAB, high-anxiety behavior; LAB, low-anxiety behavior; LS, lateral septum; MeA, medial nucleus of the amygdala; MR, medial raphe nucleus; PAG, periaqueductal gray matter; PVN, paraventricular nucleus; R–I, resident–intruder. ∗ Corresponding author. Tel.: +7 383 3634935; fax: +7 383 3331278. E-mail address: [email protected] (M.Yu. Konoshenko). 0166-4328/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbr.2012.09.022
neuronal mechanisms underlying different types of stress-induced behavior. Intraspecific confrontation has been used as a biologically and ecologically relevant model of social stress in laboratory conditions [12]. c-Fos protein immunohistochemistry studies have demonstrated that agonistic confrontation causes differential brain activation in rats selected for high and low anxiety that differed in aggressive behavior [23], and in mice selected for long and short attack latency [8]. Another valuable model that can show either intense or low aggression is the Norway rat, which has been selected for elimination (tame) and enhancement (aggressive) of aggressiveness toward humans using the glove test [13,17,18]. This selection regime also affected physiological and neurochemical characteristics—notably those closely associated with specific behavioral responses and stress hormones [13,17,14]. Moreover, tame rats differ from aggressive rats in that they have increased serotoninergic activity [13,19].
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M.Yu. Konoshenko et al. / Behavioural Brain Research 237 (2013) 103–106
Our previous experiments have demonstrated that selection for elimination of aggressiveness toward humans is associated with the attenuation of intraspecific intermale aggression. Tame rats display lower intermale aggression than both aggressive and unselected rats; there is no significant difference in intermale aggression between aggressive and unselected rats [18]. However, the neuronal mechanisms underlying intermale aggression in tame and aggressive rats remain unknown. The current work sought to investigate the neural background of agonistic behavior in tame and aggressive rats by using c-Fos immunohistochemistry to assess neural activation. Experiments were carried out with adult male gray rats (Rattus norvegicus) selected for 78 generations for elimination (tame) and enhancement (aggressive) of aggressiveness toward humans. The neutral opponents in the resident–intruder (R–I) test were naïve adult male Wistar rats from the IC&G animal facility. Rats from different lines were housed in separate rooms. After weaning on the 28th day, rats were housed in metal cages (50 cm × 33 cm × 20 cm) in groups of four. Animals were kept under standard laboratory conditions in a natural light–dark cycle of 11:13 h with free access to food and water. Experiments were carried out in the light phase of the day between 14:00 and 18:00. All experiments were performed according to the Guide for the Care and Use of Laboratory Animals approved by the Ministry of Public Health of Russia (Supplement to order No. 267 of June 19, 2003). In order to investigate whether line differences in aggression are accompanied by differences in neuronal activation, aggressive (n = 8) and tame (n = 8) males from different litters were housed in observational cages. After 1 week of single housing, males from each line were divided into two equal groups – control and experimental. Males in the experimental group were exposed to a standard 10-min R−I test [9]. One hour later, they were deeply anesthetized with pentobarbital (40 mg/kg) and perfused intracardially with 0.1 M phosphate-buffered saline (PBS) followed by 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Males from control groups were anesthetized and perfused without agonistic confrontation. Their brains were removed, postfixed overnight in 4% paraformaldehyde and cryoprotected by soaking in 20% sucrose in PBS at 4 ◦ C for 5 days. Coronal sections were cut at 40 m using a cryostat. Immunohistochemistry was carried out on free-floating sections with a validated protocol [8,6]. The c-Fos protein was labeled with a rabbit polyclonal antibody (ab53036; Abcam, Cambridge, UK). The primary antibodies (1:200) were detected with biotinylated anti-rabbit goat IgG from a rabbit specific horseradish-peroxidase/diaminobenzidine detection kit (ab64261, Abcam). Section planes were standardized according to the atlas of Ref. [16]. Microscopic analyses were conducted using the “Axioskop” 2 Plus software (Zeiss, Oberkochen, Germany). Images were digitized with a high-resolution color CCD-camera AxioCam HRc (Zeiss, full resolution 3600 × 3030 pixels), and the number of positive cells was counted by means of the AxioVision software (Zeiss). Specific brain regions were selected based on evidence from previously published studies regarding encounter-related brain activation [6,15,24]. A standard, area-specific frame was used to outline regions of interest [6,2]. All areas were analyzed bilaterally in two parallel sections except the dorsal and medial raphe (where the analysis was performed in the midline). The lateral septum (LS) was a rectangular region (10 × 104 m2 ) at the level where the anterior commissure lies below the lateral ventricle (interaural 9.20). The bed nucleus of stria terminalis (BNST) was a circular area (9.7 × 104 m2 ) dorsolateral to the anterior commissure in the same level. The paraventricular nucleus (PVN) was divided into two circular parts – lateral (magnocellular; 4 × 104 m2 ) and dorsal (parvocellular; 4 × 104 m2 ) – and one triangular part (medial, parvicellular 3.3 × 104 m2 ). The cortical amygdala (CoA) was an oval region
(12 × 104 m2 ) medial to the cortex–amygdala transition zone at the same level as the PVN (interaural 9.20). The central nucleus of the amygdala (CeA) was counted where the optic tract was medially close (oval frame, 3.37 × 104 m2 ). This plane (interaural 6.20) was also used to analyze the hypothalamic attack area (HAA, oval frame 6.5 × 104 m2 ), and the medial nucleus of the amygdala (MeA, oval frame 0.65 × 104 m2 ). The periaqueductal gray matter (PAG, interaural 13.6 × 104 m2 ) was counted directly lateral to the aqueductus (rectangular frame 5.49 × 104 m2 ; interaural 1.36), and the dorsal raphe nucleus (DR, oval frame 4.04 × 104 m2 ) and medial raphe nucleus (MR, oval frame 7.9 × 104 m2 ) were analyzed in the same level [6,2]. For semiquantitative evaluation, the following rating scale was used: 3 – intense; 2 – moderate; 1 – weak; and 0 – no reactivity. Quantitative analysis was performed in regions where semiquantitative evaluation revealed significant differences between groups [6]. The attack latency in the R–I test was analyzed using the nonparametric Kruskal–Wallis one-way analysis of variance (ANOVA) with the rat line as a factor. Interline differences were analyzed using Mann–Whitney U test. c-Fos activation data were analyzed using a two-way ANOVA; factor 1 was rat line, and factor 2 was fighting experience. Fisher post hoc comparisons were made if significant differences were observed. In the present study, tame males were characterized by the attenuation of intermale aggression in comparison with their aggressive counterparts. Attack latency in the R−I test was significantly higher in the tame rats compared with the aggressive (H (1, n = 8) = 9.54, p = 0.002). Semiquantitative evaluation of c-Fos activation (Table 1) showed that aggressive interactions induced activation in the BNST, HAA, MeA, and CoA in both lines. Aggressive interactions induced ‘moderate’ c-Fos activation in the BNST and MeA and ‘intense’ activation in the CoA in the tame and aggressive males. However, there were differences in HAA c-Fos activation after the R−I test between rats from selected lines. HAA activation after aggressive interactions was ‘very intense’ in the aggressive and ‘intense’ in the tame rats. No changes in c-Fos activation were observed in other regions following confrontation in either rat line. Quantitative analysis was performed for areas where large differences between fight-exposed and control animals were observed. It confirmed the qualitative evaluation data (Fig. 1). In the BNST, HAA, MeA, and CoA, c-Fos intensity was dependent on the agonistic encounter (F(1,12) = 23.35, p < 0.001; F(1,12) = 61.72, p < 0.001; F(1,12) = 16.47, p = 0.001; F(1,12) = 4.61, p = 0.05, respectively). The BNST did not show line differences, but it was strongly activated by agonistic encounters in both lines. Agonistic
Fig. 1. The number of c-Fos positive cells in the bed nucleus of stria terminalis (BNST), hypothalamic attack area (HAA), medial (MeA) and cortical (CoA) amygdala in the tame and the aggressive rats after agonistic confrontation in the resident–intruder test and without it. xхx p < 0.001 in comparison with aggressive rats (Fisher post hoc), v p < 0.05, vv p < 0.01, vvv p < 0.001 in comparison with rats not exposed to resident–intruder test (Fisher post hoc).
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Table 1 Patterns of neural activation after agonistic interaction in the resident–intruder test in the tame and the aggressive rats. Brain areas
Lateral septum Bed nucleus of stria terminalis Paraventricular nucleus, magnocellular Paraventricular nucleus, parvocellular Hypothalamic attack area Central amygdala Medial amygdala Cortical amygdala Periaqueductal gray matter Dorsal raphe nucleus Medial raphe nucleus
Activation of c-Fos Tame, not exposed to test
Tame, after resident–intruder test
Aggressive, not exposed to test
Aggressive, after resident–intruder test
1 0 1 1 1 1 1 2 2 1 1
1 2 1 1 2 1 2 3 2 1 1
1 0 1 1 1 1 1 2 2 1 1
1 2 1 1 3 1 2 3 2 1 1
Activation: 0 – none, 1 – weak, 2 – moderate, 3 – intense.
encounter-exposed animals (irrespective of strain) showed significantly increased c-Fos staining in the HAA compared to control rats. Variance analysis demonstrated a significant effect of line (F(1,12) = 23.56, p < 0.001) on HAA c-Fos activation, as well as a significant interaction of line factor and social confrontation factor (F(1,12) = 7.02, p < 0.05). Post hoc comparisons revealed that HAA activation after the R−I test was significantly higher in aggressive males compared with tame rats. The MeA showed significant encounter-related activation in both lines. We also observed a nonsignificant tendency toward an increased number of c-Fos-positive cells in the CoA in both lines in response to agonistic encounter. Similar to our previously published results [18], we found that tame rats showed lower aggression compared with rats from the aggressive line. Significant encounter-related c-Fos activation was shown in the BNST, MeA, and HAA in both lines. Notably, agonistic encounter caused significantly higher HAA c-Fos activation in aggressive rats compared with their tame counterparts. An insignificant but clear activation was observed in the CoA of both lines. Thus, while the behavior of tame and aggressive males differed a great deal, there was comparable activation in the studied brain areas. This was not surprising as it is known that different stimuli can elicit aggression-related activation patterns [22]. The BNST is strongly implicated in aggression mechanisms. Aggression-induced c-Fos activation in the BNST was similar in adrenalectomized rats that displayed pathological aggression and intact males [6]. Moreover, activation of this structure 1 h after the R−I test was the same in high-aggressive, low-anxiety behavior (LAB) rats and low-aggressive, high-anxiety behavior (HAB) rats [24]. These data are consistent with the lack of detectable differences in aggression-induced BNST activation between tame and aggressive rats in the present study. The HAA appears to be a key component of aggression control in the rat; it is the only brain area from which attack can be reliably and easily elicited by electrical stimulation [10,20]. Moreover, optogenetic stimulation of neurons in the ventromedial hypothalamus (ventrolateral subdivision), which overlaps partially with the rat HAA, causes male mice to attack females, inanimate objects, and other males [11]. Territorial fights in various species strongly activate the HAA [3,7,21]. Highly aggressive LAB males have a tendency toward a higher number of c-Fos-positive neurons in the HAA in comparison with low aggressive HAB rats [24]. In the present study, intense agonistic interaction of the aggressive males resulted in higher HAA activation than in tame rats. Evidently, low c-Fos activation of the HAA in the tame rats is related to reduced intermale aggression. Moreover, previous studies showed that tame males attack during territorial aggression fights, but this pattern is not the main component of their agonistic repertoire [18]. Our data support the hypothesis for the crucial role played by the HAA in attack induction [10].
It is known that the MeA plays an essential role in controlling aggression [20]; both excessive and species-typical forms of aggression can activate this nucleus [6,8,24,21]. In the present study, encounter-induced c-Fos activation was found in both the tame and aggressive rats without any significant line differences. Similarly, no strain differences for c-Fos activation were observed in the MeA of HAB and LAB rats 1 h after the R–I test [24]. We noted a tendency for the CoA to be activated by agonistic encounter in rats of both selected lines. Previous studies have demonstrated encounter-induced increases in CoA activity in normal and adrenalectomized rats [6] and dominant and subordinate male hamsters [15]. It should be noted that some authors consider CoA activation to be non-specific for aggressive behavior, due to olfactory stimulation and/or arousal associated with an agonistic encounter in general rather than to a particular subtype of agonistic behavior [15]. The CeA shows only mild or no activation following territorial fighting of male rats and male hamsters [6,7,3]. Conversely, agonistic encounter caused c-Fos activation in the CeA of rodents that displayed abnormal aggressive behavior, such as attacks toward vulnerable body parts, unknown females, and anesthetized males [6,24,8]. In our previous study, there were no significant differences in the patterns of intermale aggression displayed by the aggressive and the unselected rats that were bred in laboratory conditions for three generations [18]. Moreover, males from the aggressive line did not display attacks toward unknown females and anesthetized males (Konoshenko, unpublished data). These data and the lack of encounter-related c-Fos activation in the CeA suggest that the aggressive males manifest species-specific, not pathological intermale aggression. The lateral septum and PAG have been shown to play a role in mediating aggression [1,12,6]. Association of PVN, DR, and MR activation and aggression data are rather inconsistent. Similar to the present study, the investigation of aggression using other models of low and high aggression did not reveal a significant influence of aggressive interaction on c-Fos activation in these brain structures [6,8]. Other studies describe increased c-Fos activation after an aggressive encounter in at least some of these brain structures [22,24,23]. Serotonergic neuron activation in the raphe nucleus is a primary interest as it is known that this neurotransmitter system plays an important regulatory role in the regulation of aggressive behavior, and its activity can change during agonistic interaction [22]. As for the PVN, activation of this structure is not specific for aggressive behavior and is most likely involved in stress response and fear regulation [4]. The lack of agonistic encounter-induced cFos activation of different brain structures, such as the CeA, PVN, and DR in the aggressive and the tame rats, may be due to low stress reactivity in response to a social stimuli. The fact that plasma corticosterone levels are significantly lower after aggressive interaction
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in aggressive and tame males compared to unselected rats supports this hypothesis [18]. Taken together, our current results demonstrate that agonistic encounter elicited a similar brain activation pattern in rats selected for the elimination and enhancement of aggressiveness toward humans. At the same time, neuronal activation in the HAA was significantly higher in aggressive males compared to tame rats. It is speculated that the lack of intense activation of brain structures involved in anxiety and stress response regulation could be due to low stress reactivity in response to social stimuli. Acknowledgment This research was supported by OPTEC Company, grants no. 11-04-00653, no. 12-04-00494 from the Russian Fund of Basic Research. References [1] Bandler R, Carrive P, Zhang SP. Integration of somatic and autonomic reactions within the midbrain periaqueductal grey: viscertopic somatotopic and functional organization. Progress in Brain Research 1991;87:269–305. [2] Chung KKK, Martinez M, Herbert J. C-fos expression, behavioural, endocrine and autonomic responses to acute social stress in male rats after chronic restraint: modulation by serotonin. Neuroscience 2000;95(2):453–63. [3] Delville Y, De Vries GJ, Ferris CF. Neural connections of the anterior hypothalamus and agonistic behavior in golden hamsters. Journal of Neuroscience 2000;17:4331–40. [4] Duncan GE, Knapp DJ, Breese GR. Neuroanatomical characterization of Fos induction in rat behavioral models of anxiety. Brain Research 1996;713(1–2):79–91. [5] Gammie SC, Nelson RJ. cFOS and pCREB activation and maternal aggression in mice. Brain Research 2001;898:232–41. [6] Halasz J, Liposits Z, Kruk MR, Haller J. Neural background of glucocorticoid dysfunction-induced abnormal aggression in rats: involvement of fear- and stress-related structures. European Journal of Neuroscience 2002;15:561–9. [7] Halasz J, Liposits Z, Meelis W, Kruk MR, Haller J. Hypothalamic attack area-mediated activation of the forebrain in aggression. NeuroReport 2002;13(10):1267–70. [8] Haller J, Toth M, Halasz J, De Boer SF. Patterns of violent aggression-induced brain c-fos expression in male mice selected for aggressiveness. Physiology & Behavior 2003;88:173–82. [9] Koolhaas JM, Schuurman T, Wiepkema PR. The organization of intraspecific agonistic behaviour in the rat. Progress in Neurobiology 1980;15(3):247–68.
[10] Kruk MR. Ethology and pharmacology of hypothalamic aggression in the rat. Neuroscience and Biobehavioral Reviews 1991;15:527–38. [11] Lin D, Boyle MP, Dollar P, Lee H, Perona P, Lein ES, et al. Functional identification of an aggression locus in the mouse hypothalamus. Nature 2011;470(7333):221–6. [12] Martinez M, Phillips PJ, Herbert J. Adaptation in patterns of c-fos expression in the brain associated with exposure to either single or repeated social stress in male rats. European Journal of Neuroscience 1998;10: 20–33. [13] Naumenko EV, Popova NK, Nikulina EM, Dygalo NN, Shishkina GT, Borodin PM, et al. Behavior, adrenocortical activity, and brain monoamines in Norway rats selected for reduced aggressiveness towards man. Pharmacology Biochemistry and Behavior 1989;33:85–91. [14] Oskina IN, Plyusnina IZ, Sysoletina AI. Effect of selection for behavior on the hypophyseal-adrenal function in the gray rats Rattus norvegicus during postnatal ontogenesis. Zhurnal evoliutsionnoi biokhimii ifiziologii 2000;36:120–6 [in Russian]. [15] Pan Y, Xu L, Young KA, Wang Z, Zhang Z. Agonistic encounters and brain activation in dominant and subordinate male greater long-tailed hamsters. Hormones and Behavior 2010;58(3):478–84. [16] Paxinos G, Watson C. The rat brain in stereotaxic coordinates. 4th ed. San Diego: Academic Press; 1998. [17] Plyusnina IZ, Oskina IN. Behavioral and adrenocortical responses to open-field test in rats selected for reduced aggressiveness toward humans. Physiology and Behavior 1997;61(3):381–5. [18] Plyusnina IZ, Solov’eva MYu, Oskina IN. Effect of domestication on aggression in gray Norway rats. Behavior Genetics 2011;41(4): 583–92. [19] Popova NK, Naumenko VS, Plyusnina IZ, Kulikov AV. Reduction in 5-HT1A mRNA expression, and functional correlates for 5-HT1A receptors in genetically defined aggressive rats. Journal of Neuroscience Research 2005;80: 286–92. [20] Siegel A, Roeling TA, Gregg TR, Kruk MR. Neuropharmacology of brainstimulation-evoked aggression. Neuroscience and Biobehavioral Reviews 1999;23:359–89. [21] Tulogdi A, Toth M, Halasz J, Mikics E, Fuzesi T, Haller J. Brain mechanisms involved in predatory aggression are activated in a laboratory model of violent intra-specific aggression. European Journal of Neuroscience 2010;32(10):1744–53. [22] Van der Vegt BJ, Lieuwes N, Van de Wall EH, Kato K, Moya-Albiol L, Martínez-Sanchis S, et al. Activation of serotonergic neurotransmission during the performance of aggressive behavior in rats. Behavioral Neuroscience 2003;117(4):667–74. [23] Veenema AH, Neumann ID. Neurobiological mechanisms of aggression and stress coping: a comparative study in mouse and rat selection lines. Brain, Behavior and Evolution 2007;70(4):274–85. [24] Veenema AH, Torner L, Blume A, Beiderbeck DI, Neumann ID. Low inborn anxiety correlates with high intermale aggression: link to ACTH response and neuronal activation of the hypothalamic paraventricular nucleus. Hormones and Behavior 2007;51(1):11–9.
Contents lists available at SciVerse ScienceDirect
Behavioural Brain Research journal homepage: www.elsevier.com/locate/bbr
Short communication
c-Fos activation and intermale aggression in rats selected for behavior toward humans Maria Yu. Konoshenko a,∗ , Tatiana V. Timoshenko b,c , Irina Z. Plyusnina a,b,c a b c
Institute of Cytology and Genetics of the Siberian Branch of the Russian Academy of Sciences, pr. Lavrentjeva 10, Novosibirsk 630090, Russia Institute of Gene Biology of the Russian Academy of Sciences, Moscow 119334, Russia Apto-Pharm Ltd., Moscow 115446, Russia
h i g h l i g h t s
The neuronal activation pattern in tame and aggressive rats after exposure to R–I test was studied. Social encounter caused similar brain activation pattern in rats of selected lines. Activation was shown in bed nucleus, hypothalamic attack area and medial amygdala. Activation of the hypothalamic attack area was higher in aggressive males then in tame rats.
a r t i c l e
i n f o
Article history: Received 2 August 2012 Received in revised form 10 September 2012 Accepted 14 September 2012 Available online 20 September 2012 Keywords: Aggression c-Fos Norway rats Resident–intruder Selection
a b s t r a c t Tame and aggressive rat lines were created during the long-term selection of Norway rats for elimination and enhancement of aggressiveness toward humans, respectively. Our previous experiments have demonstrated that selection for the elimination of defensive aggression toward humans is associated with attenuated intraspecific intermale aggression. However, the neuronal mechanisms underlying low and high intermale aggression in the tame and aggressive rats remain unclear. Here, we used c-Fos immunoreactivity to evaluate neuronal activation patterns in the main aggression-related areas in selected lines under basal conditions and after the resident–intruder (R–I) test. Although agonistic behavior of the tame and the aggressive rats differed significantly, social encounter caused similar brain activation patterns in both groups; we observed increased neuronal activation in the bed nucleus of stria terminalis, the hypothalamic attack area, and the medial amygdala 1 h after the R–I test. However, neuronal activation in the hypothalamic attack area was significantly higher in the aggressive males compared to their tame counterparts. We propose that lower activation of the hypothalamic area is associated with the attenuation of intraspecific intermale aggression during selection for the elimination of aggressiveness toward humans. © 2012 Elsevier B.V. All rights reserved.
Immediate early gene expression (e.g., c-Fos) is a widely used functional marker of neuronal activation and is often employed to identify cells and brain circuits that respond to various stimuli [4,2,5]. Assessment of immediate early gene expression has been used to provide an accurate account of brain structures involved in the regulation of aggressive behavior. The study of c-Fos activation in rat and mice lines selected for behavior is of particular interest, and these studies have revealed genetically determined
Abbreviations: BNST, bed nucleus of stria terminalis; CeA, central nucleus of the amygdala; CoA, cortical amygdala; DR, dorsal raphe nucleus; HAA, hypothalamic attack area; HAB, high-anxiety behavior; LAB, low-anxiety behavior; LS, lateral septum; MeA, medial nucleus of the amygdala; MR, medial raphe nucleus; PAG, periaqueductal gray matter; PVN, paraventricular nucleus; R–I, resident–intruder. ∗ Corresponding author. Tel.: +7 383 3634935; fax: +7 383 3331278. E-mail address: [email protected] (M.Yu. Konoshenko). 0166-4328/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbr.2012.09.022
neuronal mechanisms underlying different types of stress-induced behavior. Intraspecific confrontation has been used as a biologically and ecologically relevant model of social stress in laboratory conditions [12]. c-Fos protein immunohistochemistry studies have demonstrated that agonistic confrontation causes differential brain activation in rats selected for high and low anxiety that differed in aggressive behavior [23], and in mice selected for long and short attack latency [8]. Another valuable model that can show either intense or low aggression is the Norway rat, which has been selected for elimination (tame) and enhancement (aggressive) of aggressiveness toward humans using the glove test [13,17,18]. This selection regime also affected physiological and neurochemical characteristics—notably those closely associated with specific behavioral responses and stress hormones [13,17,14]. Moreover, tame rats differ from aggressive rats in that they have increased serotoninergic activity [13,19].
104
M.Yu. Konoshenko et al. / Behavioural Brain Research 237 (2013) 103–106
Our previous experiments have demonstrated that selection for elimination of aggressiveness toward humans is associated with the attenuation of intraspecific intermale aggression. Tame rats display lower intermale aggression than both aggressive and unselected rats; there is no significant difference in intermale aggression between aggressive and unselected rats [18]. However, the neuronal mechanisms underlying intermale aggression in tame and aggressive rats remain unknown. The current work sought to investigate the neural background of agonistic behavior in tame and aggressive rats by using c-Fos immunohistochemistry to assess neural activation. Experiments were carried out with adult male gray rats (Rattus norvegicus) selected for 78 generations for elimination (tame) and enhancement (aggressive) of aggressiveness toward humans. The neutral opponents in the resident–intruder (R–I) test were naïve adult male Wistar rats from the IC&G animal facility. Rats from different lines were housed in separate rooms. After weaning on the 28th day, rats were housed in metal cages (50 cm × 33 cm × 20 cm) in groups of four. Animals were kept under standard laboratory conditions in a natural light–dark cycle of 11:13 h with free access to food and water. Experiments were carried out in the light phase of the day between 14:00 and 18:00. All experiments were performed according to the Guide for the Care and Use of Laboratory Animals approved by the Ministry of Public Health of Russia (Supplement to order No. 267 of June 19, 2003). In order to investigate whether line differences in aggression are accompanied by differences in neuronal activation, aggressive (n = 8) and tame (n = 8) males from different litters were housed in observational cages. After 1 week of single housing, males from each line were divided into two equal groups – control and experimental. Males in the experimental group were exposed to a standard 10-min R−I test [9]. One hour later, they were deeply anesthetized with pentobarbital (40 mg/kg) and perfused intracardially with 0.1 M phosphate-buffered saline (PBS) followed by 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Males from control groups were anesthetized and perfused without agonistic confrontation. Their brains were removed, postfixed overnight in 4% paraformaldehyde and cryoprotected by soaking in 20% sucrose in PBS at 4 ◦ C for 5 days. Coronal sections were cut at 40 m using a cryostat. Immunohistochemistry was carried out on free-floating sections with a validated protocol [8,6]. The c-Fos protein was labeled with a rabbit polyclonal antibody (ab53036; Abcam, Cambridge, UK). The primary antibodies (1:200) were detected with biotinylated anti-rabbit goat IgG from a rabbit specific horseradish-peroxidase/diaminobenzidine detection kit (ab64261, Abcam). Section planes were standardized according to the atlas of Ref. [16]. Microscopic analyses were conducted using the “Axioskop” 2 Plus software (Zeiss, Oberkochen, Germany). Images were digitized with a high-resolution color CCD-camera AxioCam HRc (Zeiss, full resolution 3600 × 3030 pixels), and the number of positive cells was counted by means of the AxioVision software (Zeiss). Specific brain regions were selected based on evidence from previously published studies regarding encounter-related brain activation [6,15,24]. A standard, area-specific frame was used to outline regions of interest [6,2]. All areas were analyzed bilaterally in two parallel sections except the dorsal and medial raphe (where the analysis was performed in the midline). The lateral septum (LS) was a rectangular region (10 × 104 m2 ) at the level where the anterior commissure lies below the lateral ventricle (interaural 9.20). The bed nucleus of stria terminalis (BNST) was a circular area (9.7 × 104 m2 ) dorsolateral to the anterior commissure in the same level. The paraventricular nucleus (PVN) was divided into two circular parts – lateral (magnocellular; 4 × 104 m2 ) and dorsal (parvocellular; 4 × 104 m2 ) – and one triangular part (medial, parvicellular 3.3 × 104 m2 ). The cortical amygdala (CoA) was an oval region
(12 × 104 m2 ) medial to the cortex–amygdala transition zone at the same level as the PVN (interaural 9.20). The central nucleus of the amygdala (CeA) was counted where the optic tract was medially close (oval frame, 3.37 × 104 m2 ). This plane (interaural 6.20) was also used to analyze the hypothalamic attack area (HAA, oval frame 6.5 × 104 m2 ), and the medial nucleus of the amygdala (MeA, oval frame 0.65 × 104 m2 ). The periaqueductal gray matter (PAG, interaural 13.6 × 104 m2 ) was counted directly lateral to the aqueductus (rectangular frame 5.49 × 104 m2 ; interaural 1.36), and the dorsal raphe nucleus (DR, oval frame 4.04 × 104 m2 ) and medial raphe nucleus (MR, oval frame 7.9 × 104 m2 ) were analyzed in the same level [6,2]. For semiquantitative evaluation, the following rating scale was used: 3 – intense; 2 – moderate; 1 – weak; and 0 – no reactivity. Quantitative analysis was performed in regions where semiquantitative evaluation revealed significant differences between groups [6]. The attack latency in the R–I test was analyzed using the nonparametric Kruskal–Wallis one-way analysis of variance (ANOVA) with the rat line as a factor. Interline differences were analyzed using Mann–Whitney U test. c-Fos activation data were analyzed using a two-way ANOVA; factor 1 was rat line, and factor 2 was fighting experience. Fisher post hoc comparisons were made if significant differences were observed. In the present study, tame males were characterized by the attenuation of intermale aggression in comparison with their aggressive counterparts. Attack latency in the R−I test was significantly higher in the tame rats compared with the aggressive (H (1, n = 8) = 9.54, p = 0.002). Semiquantitative evaluation of c-Fos activation (Table 1) showed that aggressive interactions induced activation in the BNST, HAA, MeA, and CoA in both lines. Aggressive interactions induced ‘moderate’ c-Fos activation in the BNST and MeA and ‘intense’ activation in the CoA in the tame and aggressive males. However, there were differences in HAA c-Fos activation after the R−I test between rats from selected lines. HAA activation after aggressive interactions was ‘very intense’ in the aggressive and ‘intense’ in the tame rats. No changes in c-Fos activation were observed in other regions following confrontation in either rat line. Quantitative analysis was performed for areas where large differences between fight-exposed and control animals were observed. It confirmed the qualitative evaluation data (Fig. 1). In the BNST, HAA, MeA, and CoA, c-Fos intensity was dependent on the agonistic encounter (F(1,12) = 23.35, p < 0.001; F(1,12) = 61.72, p < 0.001; F(1,12) = 16.47, p = 0.001; F(1,12) = 4.61, p = 0.05, respectively). The BNST did not show line differences, but it was strongly activated by agonistic encounters in both lines. Agonistic
Fig. 1. The number of c-Fos positive cells in the bed nucleus of stria terminalis (BNST), hypothalamic attack area (HAA), medial (MeA) and cortical (CoA) amygdala in the tame and the aggressive rats after agonistic confrontation in the resident–intruder test and without it. xхx p < 0.001 in comparison with aggressive rats (Fisher post hoc), v p < 0.05, vv p < 0.01, vvv p < 0.001 in comparison with rats not exposed to resident–intruder test (Fisher post hoc).
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Table 1 Patterns of neural activation after agonistic interaction in the resident–intruder test in the tame and the aggressive rats. Brain areas
Lateral septum Bed nucleus of stria terminalis Paraventricular nucleus, magnocellular Paraventricular nucleus, parvocellular Hypothalamic attack area Central amygdala Medial amygdala Cortical amygdala Periaqueductal gray matter Dorsal raphe nucleus Medial raphe nucleus
Activation of c-Fos Tame, not exposed to test
Tame, after resident–intruder test
Aggressive, not exposed to test
Aggressive, after resident–intruder test
1 0 1 1 1 1 1 2 2 1 1
1 2 1 1 2 1 2 3 2 1 1
1 0 1 1 1 1 1 2 2 1 1
1 2 1 1 3 1 2 3 2 1 1
Activation: 0 – none, 1 – weak, 2 – moderate, 3 – intense.
encounter-exposed animals (irrespective of strain) showed significantly increased c-Fos staining in the HAA compared to control rats. Variance analysis demonstrated a significant effect of line (F(1,12) = 23.56, p < 0.001) on HAA c-Fos activation, as well as a significant interaction of line factor and social confrontation factor (F(1,12) = 7.02, p < 0.05). Post hoc comparisons revealed that HAA activation after the R−I test was significantly higher in aggressive males compared with tame rats. The MeA showed significant encounter-related activation in both lines. We also observed a nonsignificant tendency toward an increased number of c-Fos-positive cells in the CoA in both lines in response to agonistic encounter. Similar to our previously published results [18], we found that tame rats showed lower aggression compared with rats from the aggressive line. Significant encounter-related c-Fos activation was shown in the BNST, MeA, and HAA in both lines. Notably, agonistic encounter caused significantly higher HAA c-Fos activation in aggressive rats compared with their tame counterparts. An insignificant but clear activation was observed in the CoA of both lines. Thus, while the behavior of tame and aggressive males differed a great deal, there was comparable activation in the studied brain areas. This was not surprising as it is known that different stimuli can elicit aggression-related activation patterns [22]. The BNST is strongly implicated in aggression mechanisms. Aggression-induced c-Fos activation in the BNST was similar in adrenalectomized rats that displayed pathological aggression and intact males [6]. Moreover, activation of this structure 1 h after the R−I test was the same in high-aggressive, low-anxiety behavior (LAB) rats and low-aggressive, high-anxiety behavior (HAB) rats [24]. These data are consistent with the lack of detectable differences in aggression-induced BNST activation between tame and aggressive rats in the present study. The HAA appears to be a key component of aggression control in the rat; it is the only brain area from which attack can be reliably and easily elicited by electrical stimulation [10,20]. Moreover, optogenetic stimulation of neurons in the ventromedial hypothalamus (ventrolateral subdivision), which overlaps partially with the rat HAA, causes male mice to attack females, inanimate objects, and other males [11]. Territorial fights in various species strongly activate the HAA [3,7,21]. Highly aggressive LAB males have a tendency toward a higher number of c-Fos-positive neurons in the HAA in comparison with low aggressive HAB rats [24]. In the present study, intense agonistic interaction of the aggressive males resulted in higher HAA activation than in tame rats. Evidently, low c-Fos activation of the HAA in the tame rats is related to reduced intermale aggression. Moreover, previous studies showed that tame males attack during territorial aggression fights, but this pattern is not the main component of their agonistic repertoire [18]. Our data support the hypothesis for the crucial role played by the HAA in attack induction [10].
It is known that the MeA plays an essential role in controlling aggression [20]; both excessive and species-typical forms of aggression can activate this nucleus [6,8,24,21]. In the present study, encounter-induced c-Fos activation was found in both the tame and aggressive rats without any significant line differences. Similarly, no strain differences for c-Fos activation were observed in the MeA of HAB and LAB rats 1 h after the R–I test [24]. We noted a tendency for the CoA to be activated by agonistic encounter in rats of both selected lines. Previous studies have demonstrated encounter-induced increases in CoA activity in normal and adrenalectomized rats [6] and dominant and subordinate male hamsters [15]. It should be noted that some authors consider CoA activation to be non-specific for aggressive behavior, due to olfactory stimulation and/or arousal associated with an agonistic encounter in general rather than to a particular subtype of agonistic behavior [15]. The CeA shows only mild or no activation following territorial fighting of male rats and male hamsters [6,7,3]. Conversely, agonistic encounter caused c-Fos activation in the CeA of rodents that displayed abnormal aggressive behavior, such as attacks toward vulnerable body parts, unknown females, and anesthetized males [6,24,8]. In our previous study, there were no significant differences in the patterns of intermale aggression displayed by the aggressive and the unselected rats that were bred in laboratory conditions for three generations [18]. Moreover, males from the aggressive line did not display attacks toward unknown females and anesthetized males (Konoshenko, unpublished data). These data and the lack of encounter-related c-Fos activation in the CeA suggest that the aggressive males manifest species-specific, not pathological intermale aggression. The lateral septum and PAG have been shown to play a role in mediating aggression [1,12,6]. Association of PVN, DR, and MR activation and aggression data are rather inconsistent. Similar to the present study, the investigation of aggression using other models of low and high aggression did not reveal a significant influence of aggressive interaction on c-Fos activation in these brain structures [6,8]. Other studies describe increased c-Fos activation after an aggressive encounter in at least some of these brain structures [22,24,23]. Serotonergic neuron activation in the raphe nucleus is a primary interest as it is known that this neurotransmitter system plays an important regulatory role in the regulation of aggressive behavior, and its activity can change during agonistic interaction [22]. As for the PVN, activation of this structure is not specific for aggressive behavior and is most likely involved in stress response and fear regulation [4]. The lack of agonistic encounter-induced cFos activation of different brain structures, such as the CeA, PVN, and DR in the aggressive and the tame rats, may be due to low stress reactivity in response to a social stimuli. The fact that plasma corticosterone levels are significantly lower after aggressive interaction
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