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Respiratory Physiology & Neurobiology journal homepage: www.elsevier.com/locate/resphysiol

Respiratory patterns reflect different levels of aggressiveness and emotionality in Wild-type Groningen rats夽 Luca Carnevali a , Eugene Nalivaiko b , Andrea Sgoifo a,∗ a b

Stress Physiology Laboratory, Department of Neuroscience, University of Parma, 43124 Parma, Italy School of Biomedical Sciences and Pharmacy, University of Newcastle, 2308 Callaghan, New South Wales, Australia

a r t i c l e

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Article history: Accepted 3 July 2014 Available online xxx Keywords: Anxiety Aggressiveness Breathing Plethysmography Respiratory rate Sighing

a b s t r a c t Respiratory patterns represent a promising physiological index for assessing emotional states in preclinical studies. Since disturbed emotional regulation may lead to forms of excessive aggressiveness, in this study we investigated the hypothesis that rats that differ largely in their level of aggressive behavior display matching alterations in respiration. Respiration was recorded in male high-aggressive (HA, n = 8) and non-aggressive (NA, n = 8) Wild-type Groningen rats using whole-body plethysmography. Subsequently, anxiety-related behaviors were evaluated in the elevated plus maze and social avoidance–approach tests. During respiratory testing, HA rats showed elevated basal respiratory rate, reduced sniffing, exaggerated tachypnoeic response to an acoustic stimulus and a larger incidence of sighs. In addition, HA rats spent less time in the open arms of the plus maze and displayed higher levels of social avoidance behavior compared to NA rats. These findings indicate that HA rats are characterized by alterations in respiratory functioning and behavior that are overall indicative of an anxiety-like phenotype. © 2014 Elsevier B.V. All rights reserved.

1. Introduction The centrality of breathing at the interface between emotional/personality characteristics and the human physiology has been increasingly recognized in the last two decades in the field of psychophysiology (Boiten et al., 1994; Timmons and Ley, 1994; Wilhelm et al., 2001a). Mounting evidence suggests that interactions between emotional states, alterations in respiratory function and physiological changes at the level of chemical blood composition and autonomic nervous system regulation play an important role in the development of a variety of medical conditions, including hyperventilation syndrome (Folgering, 1999), panic disorder (Caldirola et al., 2004; Niccolai et al., 2009; Wilhelm et al., 2001c), chronic pain syndrome (Wilhelm et al., 2001a) and cardiovascular disorders (Anderson et al., 1996; Floras, 2014; Oldenburg and Horstkotte, 2010).

夽 This paper is part of a special issue entitled “Non-homeostatic control of respiration”, guest-edited Dr. Eugene Nalivaiko and Dr. Dr. Paul Davenport. ∗ Corresponding author at: Stress Physiology Laboratory, Department of Neuroscience, University of Parma, Parco Area delle Scienze 11/a, 43124 Parma, Italy. Tel.: +39 0521 905625; fax: +39 0521 905673. E-mail address: [email protected] (A. Sgoifo).

Respiratory research in animals can potentially contribute to a better understanding of the pathophysiology and neural pathways that relate breathing changes to emotional states. Using wholebody plethysmography, it has been recently demonstrated that rats with high levels of baseline anxiety show specific patterns of respiration (such as elevated basal respiratory rate, exaggerated respiratory responsiveness to repetitive stressful stimuli and frequent sighing) (Carnevali et al., 2013a) that resemble those that are commonly described in individuals affected by anxiety disorders (Abelson et al., 2001; Papp et al., 1997; Schwartz et al., 1996; Wilhelm et al., 2001c). Other studies have documented that respiratory parameters in rats (especially the respiratory rate) are strongly affected by conditioned and unconditioned aversive stimuli and by novelty (Frysztak and Neafsey, 1991; Hegoburu et al., 2011). Furthermore, a series of investigations in rats have demonstrated that neonatal maternal separation brings about long-term alterations in the respiratory system (such as altered responses to hypoxia (Genest et al., 2004) and hypercapnia (Genest et al., 2007b)) that are mediated by alterations in the chemoreflex circuitry in the lower brainstem (Kinkead et al., 2008) and descending influences from the hypothalamus (Genest et al., 2007a). Collectively, these findings indicate that respiratory parameters represent a promising physiological marker of different levels of emotionality in rats. The present study was designed to characterize the respiratory patterns in two groups of male Wild-type Groningen rats (Rattus

http://dx.doi.org/10.1016/j.resp.2014.07.003 1569-9048/© 2014 Elsevier B.V. All rights reserved.

Please cite this article in press as: Carnevali, L., et al., Respiratory patterns reflect different levels of aggressiveness and emotionality in Wild-type Groningen rats. Respir. Physiol. Neurobiol. (2014), http://dx.doi.org/10.1016/j.resp.2014.07.003

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norvegicus) that differed largely in their level of aggressive behavior. In male rodents, aggressive behaviors are elicited in response to unavoidable contact with a proximal threatening stimulus (e.g., the presence of a male conspecific intruder in their own cage). In this regard, rats of the Wild-type Groningen strain show a wide and consistent individual variation in the levels of aggressive behavior, ranging from individuals that readily attack the intruder opponent (i.e., high-aggressive rats) to individuals that show no overt aggression at all (i.e., non-aggressive rats) (de Boer et al., 2003; Carnevali et al., 2013b). It has been hypothesized that high levels of aggressive behaviors in rodents may reflect a higher state of acute emotional response to sources of threat, and may develop as a consequence of abnormalities in the complex emotion regulation circuitry of the brain, which includes cortical, amygdaloid, hypothalamic and septo-hippocampal regions (Davidson et al., 2000; Nehrenberg et al., 2009; Neumann et al., 2010). Therefore, by means of whole-body plethysmography, in this study we tested the hypothesis that high levels of aggressive behavior in rats are associated with alterations in respiration. In addition, based on previous investigations documenting a link between aggression and anxiety in rodents (Nehrenberg et al., 2009; Neumann et al., 2010), we examined whether high-aggressive rats displayed anxiety-related behaviors in the elevated plus maze and social avoidance–approach tests. 2. Methods 2.1. Ethics statement and animals Experimental procedures and protocols were approved by the Veterinarian Animal Care and Use Committee of Parma University, with animals cared for in accordance with the European Community Council Directives of 22 September 2010 (2010/63/UE). In this study we used 4-month-old male Wild-type Groningen rats (R. norvegicus) weighing approximately 350 g. This rat population, originally derived from the University of Groningen (The Netherlands), is currently bred in our laboratory under conventional conditions, at ambient temperature of 22 ± 2 ◦ C and on a reversed 12:12 light–dark cycle (light on at 19:00 h), with food and water available ad libitium. 2.2. Preliminary behavioral testing for aggressiveness Seventy Wild-type rats were assessed for the display of aggressive behavior toward male unfamiliar conspecific intruders using a standard resident-intruder aggression test (Koolhaas et al., 2013). Ten days before the test, each rat was housed with a conspecific oviduct-ligated female partner to stimulate territorial behavior (Koolhaas et al., 1980; Lore and Flannelly, 1977). Fifteen min before the start of the test, the female partner was removed and an unfamiliar male Wistar rat was introduced into the home cage of the experimental rat. The intruder Wistar rats weighed on average 250 g (3 months old) and were socially housed. The test was repeated on three consecutive days, using a different intruder every time, in order to avoid familiarity between the opponents and obtain a reliable characterization of aggressive traits (de Boer et al., 2003). All tests lasted 10 min and the latency to the first attack toward the intruder (in s) was measured. The attack latency (average of 3 tests) was used as an index of individual aggressive behavior (Carnevali et al., 2013b). As commonly seen in this rat strain (de Boer et al., 2003; Carnevali et al., 2013b), individual male resident rats differed widely in their level of aggression toward unfamiliar intruder males. The eight most aggressive rats (average attack latency = 96 ± 7 s; average number of attacks = 7.2 ± 0.4) were selected and classified as high-aggressive (HA) rats. Eight rats

did not attack the intruder during the 600-s confrontations and were selected and classified as non-aggressive (NA) rats. HA and NA rats were then used for the following experimental procedures. 2.3. Experimental protocol All experiments were carried out under red light during the dark phase of the light/dark cycle to permit video recordings of animals’ behavior. 2.3.1. Whole-body plethysmography On day 1, HA and NA rats were placed into a custom-built whole-body plethysmograph, which consisted of a sealed Perspex cylinder (i.d. 9.5 cm, length 26 cm, volume 2.5 l) with medical air constantly flushed through it at a flow rate of 2.5 l/min (Kabir et al., 2010; Carnevali et al., 2013a). The output flow was divided into two lines using a T-connector. One line was attached to a differential pressure amplifier (model 24PCO1SMT, Honeywell Sensing and Control, Golden Valley, MN, USA), while the other line was open to the room air. Rats’ behavior during the test was recorded using a video camera positioned close to the plethysmograph. For semi-quantitative assessment of animals’ motor activity, a piezoelectric pulse transducer was placed under the plethysmograph. Rats were left undisturbed in the plethysmograph for 40 min. Subsequently, our purpose was to determine the respiratory arousal responses to two natural stressful stimuli. First, a predator (hawk) call was played back for 50 s (starting from min 40). Second, a piece of rat feces was placed in a syringe and the air with the rat odor was quickly injected into the input line (through which the plethysmographic chamber was constantly flushed with medical air) (min 50). These two sensory stimuli were presented when animals were in a quiet but awake state (i.e., no motor activity, eyes opened, slow regular breathing), and were chosen because they represent non-intrusive natural stressors (threat of predation and presence of an unknown conspecific). Finally, we aimed at determining the respiratory responses to a prolonged stressor (restraint). For this, animals were removed from the plethysmograph (min 60) and introduced into a restrainer (wire-mesh tube; inner diameter: 6 cm, length: 18 cm), which was immediately placed back into the plethysmograph for 15 min. Such stressor was selected because it has been shown to provoke prominent autonomic and neuroendocrine responses in the Wild-type Groningen rat strain used in the present study (Sgoifo et al., 1997; Carnevali et al., 2013b), and marked respiratory responses in other rat strains (Carnevali et al., 2013a; Bondarenko et al., 2014). 2.3.2. Elevated plus maze test On day 4, HA and NA rats were tested on the elevated plus maze. The elevated plus-maze test, validated for measuring anxiety (Pellow et al., 1985), is based on creating a conflict between the rat’s exploratory drive and its innate fear of open and exposed areas. The plus maze consisted of 4 elevated arms (100 cm above the floor, 50 cm long and 10 cm wide) arranged in a cross-like position, with two opposite arms being enclosed (by means of 40 cm high walls) and two being open, including at their intersection a central square platform (10 cm × 10 cm) which gave access to the four arms. Each rat was initially placed on the central platform facing one closed arm and behaved freely for 5 min. The behavior during the test was recorded using a video camera positioned above the maze. 2.3.3. Social approach–avoidance test On day 6, HA and NA rats were submitted to a social approach–avoidance test, which is considered a reliable procedure to measure experimental anxiety in a social context

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(Haller and Bakos, 2002; Haller et al., 2003). The apparatus consisted of a large plastic cage that was divided into two separate compartments, social and nonsocial (40 cm × 50 cm × 30 cm and 20 cm × 50 cm × 30 cm, respectively), by a black plastic wall. The social and nonsocial compartments were connected by a sliding door (10 cm × 10 cm), as previously described (Nicolas and Prinssen, 2006). The social compartment contained a subchamber (15 cm × 50 cm × 30 cm) delimited by a wire mesh partition in which a stimulus Wild-type male rat was confined. The experimental rat was introduced into the nonsocial compartment for a 3-min habituation period (Nicolas and Prinssen, 2006). After this period, the sliding door was opened and the experimental rat was allowed to move freely between the nonsocial and social compartments for 10 min. The test apparatus did not permit physical contact between the experimental and stimulus animals. The behavior of the experimental rat during the test was recorded using a video camera positioned above the cage. At the end of the test, both the experimental and the stimulus rat were removed and the apparatus was carefully cleaned. Stimulus rats were used maximally twice. 2.4. Data acquisition and analysis 2.4.1. Whole-body plethysmography Analog respiratory and motion signals were digitized at 1 kHz and acquired using a PowerLab A/D converter and ChartPro 6.0 software (ADInstruments, Sydney, Australia). They were low-pass filtered at 20 Hz to remove noise, using a digital filter. Each recording period was split into 5-min epochs (0–5 min, 5–10 min, etc.) and data analysis was performed as follows. 2.4.1.1. Respiration. We first calculated the respiratory rate from the respiratory signal. The respiratory rate (cycles per minute, cpm) was measured by calculating the rate of pressure fluctuations inside the chamber (Fig. 1). The time series was then transferred into the IgorPro 5.0 software (Wavementrics, USA). Here, we constructed time histograms for each epoch, with a bin width of 10 cpm; an example of such histogram is shown in Fig. 2A. This graphic representation indicates how much time (in s) animals spent at a given respiratory frequency. Histogram mode peak values represent dominant respiratory rate (i.e., respiratory frequency at which animals spent most of time during recordings). For each epoch, we then selected 250 cpm as an approximate center between low-frequency (0–250 cpm) and high-frequency (251–600 cpm) respiratory rate, the latter reflecting sniffing behavior (Carnevali et al., 2013a) (Fig. 1). This allowed us to calculate the time spent by the animals at high-frequency sniffing mode (expressed as % of recording time). Finally, for each recording period we quantified the number of sighs (“augmented breaths”). A sigh is a readily identifiable respiratory event: it consists of a deep additional inspiration that starts at or around the peak of a normal respiratory cycle (Fig. 1). This superimposition of two inspirations makes a sigh much larger than the preceding and following breaths. A sigh is also usually accompanied by a post-sigh apnea (Fig. 1). 2.4.1.2. Motion. The motion signal was rectified for each 5-min epoch using IGOR Pro 5.0 software (Wavemetrics, Inc., OR, USA). After setting the threshold level (defined as 150% of the signal when there was no motion), the total duration of time during which the signal exceeded this threshold was determined automatically, and defined as “motion time” (Carnevali et al., 2013a). 2.4.1.3. Behavior. The behavior of the animals inside the chamber was encoded and quantified (as % of recording time) by means of (i) respiratory and motion data and (ii) video analysis using the Ethovision 6.0 software (Noldus, The Netherlands). We considered the

Fig. 1. Raw data records of respiratory signal, respiratory rate and motor movements (piezoelectric sensor) in a representative high-aggressive rat during free exploration of the plethysmographic chamber. The top trace shows slow regular breathing, a sigh and an episode of tachypnoea/sniffing. The middle trace represents respiratory rate. Note that only small movements (bottom trace) occurred during episodes of tachypnoea associated with exploratory sniffing (for comparison, effect of locomotion is shown at the end of the bottom trace (asterisk)).

following four behavioral categories: (i) motion (moving or turning around; motion signal >150% of the signal when there was no motion); (ii) sniffing (repetitive retraction and protraction of the tip of the snout; respiratory rate >250 cpm), (iii) freezing (head up, no body movements excluding those necessary for breathing), and (iv) quiet resting activity (small body movements excluding those associated with sniffing, or head down and no body movements excluding those necessary for breathing). 2.4.2. Elevated plus maze test The following behavioral parameters were calculated using the Ethovision 6.0 software (Noldus, The Netherlands): (i) time spent in the open and closed arms (% of total time), (ii) number of entries in the open and closed arms, and (iii) latency to enter an open arm (s). 2.4.3. Social approach–avoidance test The social compartment was divided into two zones of equal dimensions: proximal to and distal from the stimulus rat, as in Nicolas and Prinssen (2006). Animals’ behavior was analyzed using the Ethovision 6.0 software (Noldus, The Netherlands). We calculated the time spent by the animals (i) in the nonsocial compartment and (ii) in the proximal and distal areas of the social compartment (expressed as % of total time). 2.4.4. Statistics All statistical analyses were performed using the software package SPSS (version 20). Two-way ANOVA for repeated measures

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for pairwise contrast between HA and NA rats for respiratory and behavioral data. Cohen’s d was computed directly from the mean and standard deviation of the groups’ scores for each outcome variable.

3. Results 3.1. Respiratory and behavioral parameters during plethysmography testing 3.1.1. First 40 min of free exploration During the first 5 min of free exploration of the plethysmographic chamber, HA rats showed lower values of mean respiratory rate (t = −2.9, p < 0.05; d = 1.51) (Fig. 2B) and similar values of dominant respiratory rate (i.e., the mode of the frequency histogram) (Fig. 2C) than NA counterparts. Over the 40-min period, both groups showed a progressive reduction in mean respiratory rate and dominant respiratory rate values (F(time) for mean respiratory rate = 108.8, p < 0.01; F(time) for dominant respiratory rate = 106.5, p < 0.01) (Fig. 2B and C). At the end of this phase, HA rats showed higher values of dominant respiratory rate than NA rats (t = 3.92, p < 0.01; d = 2.12) (Fig. 2C). During the first 5 min inside the chamber, HA rats spent less time engaged in sniffing behavior (t = −2.7, p < 0.05; d = 1.40) and more time in freezing behavior (t = 5.27, p < 0.01; d = 2.84) than NA rats (Fig. 3A), whereas no differences between groups were observed in the levels of motion and resting activity (Fig. 3A). At the end of the 40-min period (last 5 min), animals showed similar high levels of quiet resting activity (Fig. 3B). Animals of both groups sighed during the 40 min in the plethysmographic chamber, with the incidence of sighs being significantly larger in HA than NA rats (t = 4.6, p < 0.01, d = 3.16) (Fig. 4).

Fig. 2. Respiratory patterns in high-aggressive (HA, n = 8) and non-aggressive (NA, n = 8) rats. Panel (A) reports an example of the respiratory rate histograms that were constructed for each 5-min epoch. These histograms reflect the amount of time spent at a given respiratory frequency. Histogram mode peak values represent dominant respiratory rate, i.e. respiratory frequency at which animals spent most of the time during recordings. Panels (B) and (C) represent the time course of changes in mean respiratory rate and dominant respiratory rate, respectively, during the experimental procedures. Values are expressed as means (±SEM). * and # indicate a significant difference between HA and NA rats (p < 0.05 and p < 0.01, respectively).

with group as between-subject factor (2 levels: HA and NA) and recording period (all 5-min epochs) as within-subject factor was applied for respiratory data. Follow-up analyses were conducted using Student’s “t” tests, with a Bonferroni correction for multiple comparisons for each outcome variable separately. A priori Student’s “t”-tests, after controlling for homogeneity of variance via Levene test, were applied for comparisons between HA and NA rats on behavioral data and incidence of sighs. Statistical significance was set at p < 0.05. We also calculated Cohen’s d effect sizes

3.1.2. Predator call Mean respiratory rate during the predator call (50 s) was similar between HA and NA rats (HA = 139 ± 8 cpm vs. NA = 148 ± 9 cpm). During the 5-min period that followed stimulus onset, HA rats showed significantly higher values of mean respiratory rate (t = 3.2, p < 0.01; d = 1.62) (Fig. 2B) and dominant respiratory rate (t = 5.2, p < 0.01; d = 2.84) than NA rats (Fig. 2C). The stimulus-induced increase in dominant respiratory rate was tendentially larger in HA than NA rats (HA = +16 ± 6 cpm vs. NA = +4 ± 3 cpm, p = 0.07) (Fig. 2C). In the same period, no differences between HA and NA rats were observed for any of the behavioral parameters that were quantified (Fig. 3C). In addition, the two groups exhibited a similar incidence of sighs (Fig. 4). 3.1.3. Rat odor During the 5-min period prior to stimulus presentation, HA rats had similar mean respiratory rate but higher dominant respiratory rate (t = 3.1, p < 0.05; d = 0.88) compared to NA rats (Fig. 2B and C). Rat odor exposure elicited similar, large amount of sniffing behavior between the two groups during the first 30 s after stimulus onset (HA = 63 ± 7% vs. NA = 64 ± 10% of total time). Mean respiratory rate calculated during this time-interval (30 s) was similar between the two groups (HA = 251 ± 15 cpm vs. NA = 219 ± 10 cpm). During the 5-min period that followed stimulus presentation, the two groups showed similar values of mean respiratory rate and dominant respiratory rate (Fig. 2B and C), with the stimulusinduced increases in mean respiratory rate (HA = +40 ± 15 cpm vs. NA = 40 ± 12 cpm) and dominant respiratory rate (HA = +4 ± 6 cpm vs. NA = 9 ± 3 cpm) that were similar between the two groups. In the same period, the two groups did not show any difference in behavior (Fig. 3D). However, during this period the incidence of

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Fig. 3. Behavior of high-aggressive (HA, n = 8) and non-aggressive (NA, n = 8) rats during (i) the first (Panel A) and last (Panel B) 5 min of free exploration in the plethysmograph and (ii) the first 5 min after predator call (Panel C) and rat odor (Panel D) stimulus onset. * and # indicate a significant difference between HA and NA rats (p < 0.05 and p < 0.01, respectively).

sighs was significantly higher in HA than NA rats (t = 2.3, p < 0.05; d = 1.24) (Fig. 4). 3.1.4. Restraint test During the 5 min that preceded the test, HA and NA rats had similar mean respiratory rate and dominant respiratory rate (Fig. 2B and C). Submitting rats to the restraint test provoked a marked increase in dominant respiratory rate, with no group differences (HA = +41 ± 5 cpm vs. NA = +39 ± 5 cpm). During the 15-min test, HA and NA rats showed similar values of mean respiratory rate and dominant respiratory rate (Fig. 2B and C). In addition, the two groups spent similar amount of time at high-frequency sniffing mode (HA = 20 ± 4% of total time vs. NA = 25 ± 4% of total time). However, during the test the incidence of sighs was significantly higher in HA than NA rats (t = 2.3, p < 0.05; d = 1.3) (Fig. 4).

3.3. Behavior during the social approach–avoidance test The time spent by HA and NA rats in the different compartments of the social approach–avoidance apparatus is depicted in Fig. 6. HA rats spent less time in the proximal zone of the social compartment (t = −2.3, p < 0.05, d = 1.15) and more time in the non social

3.2. Behavior on the elevated plus maze The performance of HA and NA rats on the elevated plusmaze is illustrated in Fig. 5. HA rats spent less time in the open arms (t = −2.9, p < 0.05; d = 1.49) and more time in the closed arms (t = 3.7, p < 0.01; d = 1.83) compared to NA counterparts (Fig. 5). In addition, HA rats entered open arms less frequently than NA rats (t = −3.1, p < 0.01; d = 1.58) (Fig. 5) and their latency to first access an open arm was significantly longer compared to NA counterparts (HA = 108 ± 42 s vs. NA = 17 ± 7 s, t = 2.2, p < 0.05; d = 1.32).

Fig. 4. Incidence of sighs in high-aggressive (n = 8) and non-aggressive (n = 8) rats. Values are expressed as means (±SEM) of number of sighs/min. * indicates a significant difference between HA and NA rats (p < 0.05).

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Fig. 5. Behavior of high-aggressive (HA, n = 8) and non-aggressive (NA, n = 8) rats on the elevated plus maze. Time spent (A) and number of entries (B) in the open and closed arms of the apparatus. Values are expressed as means (±SEM). * and # indicate a significant difference between HA and NA rats (p < 0.05 and p < 0.01, respectively).

Fig. 6. Time spent by high-aggressive (HA, n = 8) and non-aggressive (NA, n = 8) rats in the different compartments of the social approach–avoidance apparatus. Values are expressed as means (±SEM). * indicates a significant difference between HA and NA rats (p < 0.05).

compartment (t = 2.4, p < 0.05; d = 1.10) compared to NA counterparts (Fig. 6). 4. Discussion The major and novel finding of the present study is that different levels of aggressive behavior in Wild-type rats are expressed in breathing patterns. In rats, the respiratory pattern consists of normal (eupnoea) or rapid (tachypnoea) breathing, intermingled with periods of sniffing of variable intensity and duration (Carnevali et al., 2013a; Ngampramuan et al., 2013). The respiratory frequency is therefore highly variable, ranging from 60–80 to more than 500 cpm.

Consequently, the mean respiratory rate calculated during a given period is strongly affected by the proportion of time spent by an animal at high-frequency sniffing mode. Our data indicate that the mean respiratory rate was lower in HA than NA rats during initial testing inside the plethysmograph. As the dominant respiratory rate (i.e., the respiratory frequency at which animals spent most of the time during recordings) was identical in both groups at this time, such difference was due to the fact that HA rats spent less time engaged in exploratory sniffing and displayed higher levels of freezing behavior compared to NA rats. Providing that animals were free to move, we suggest that these differences are indicative of reduced exploratory drive and increased fear in HA rats, two behaviors that may be related to higher levels of anxiety (Russell, 1973; Brandao et al., 2008). Animals presumably experienced a certain amount of stress when placed in the plethysmograph, yet they gradually settled down in the new environment over time. Not surprisingly, we observed in the two groups a progressive reduction in dominant respiratory rate over the initial 40-min period. However, at the end of this phase, dominant respiratory rate was higher in HA than NA rats. Importantly, this difference could not be attributed to different levels of somatomotor activity between the two groups. Based on the observation that animals were clearly in a quiet resting state during this phase, we consider these data values as a reasonable approximation of true basal respiratory rate. Dominant respiratory rate was also clearly higher in HA than NA rats after exposure to predator call. In HA rats, such exaggerated tachypnoeic response to an acoustic stressful stimulus was not related to differences in the behavioral coping strategy adopted by the two groups. Indeed, HA and NA rats spent similar amount of time engaged in freezing and/or sniffing behaviors following presentation of the acoustic stimulus. Elevated respiratory rate during baseline and stress conditions has been recently described in rats with high levels of baseline anxiety (Carnevali et al., 2013a) and may be interpreted as a physiological correlate of the increased arousal characteristic commonly observed in anxious individuals, independently from specific emotional contents (Dudley et al., 1969; Dudley and Pitts-Poarch, 1980; Boiten et al., 1994). We attribute the lack of differences between HA and NA rats in the respiratory responses to the rat odor and the restraint test to the nature of the stressors applied. Indeed, olfactory stimuli in rodents elicit a high degree of odor-sampling sniffing (Wesson et al., 2008, 2009; Youngentob et al., 1987), with the respiratory rate that likely reaches its physiological maximum (Carnevali et al., 2013a). It is therefore plausible that subtle group differences in the short-term respiratory responsiveness to rat odor may have been masked by large amounts of sniffing behavior immediately after stimulus presentation. On the other hand, the similar prominent increase in dominant respiratory rate between the two groups during the restraint stress could likely be explained by the vigorous struggling that is associated with this particular test (Grissom et al., 2008). We found that HA animals sighed more frequently during both free exploration of the plethysmograph and stressful conditions (rat odor and restraint test). Sighing is a fundamental vertebrate behavior that is modulated by chemoreceptor feedback mechanisms and serves as a general resetter of the respirator system (Vlemincx et al., 2013). Sighs not only result from sensory afferent neural control modulating the central respiratory pattern generator, but also arise from behavioral or emotional control processes through central mechanisms (Evans, 2010; McKay et al., 2003). Indeed, it has been demonstrated that pharmacological activation of the dorsomedial hypothalamus (the brain area that coordinates stress-induced autonomic neural responses) in anesthetized rats increases the number of sighs and provokes tachypnoea (Xavier et al., 2013). Therefore, it has been hypothesized that sighing occurs more frequently during stress and anxiety and acts as a coping or control response to reduce tension

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caused by anxious states (Vlemincx et al., 2013). Importantly, frequent sighing has been described in rats with high levels of baseline anxiety (Carnevali et al., 2013a). Similarly, extensive evidence from the human literature has shown that the incidence of respiratory sighing is greater among anxious patients, especially those with diagnoses of panic disorders (Schwartz et al., 1996; Wilhelm et al., 2001b). Taken together, the respiratory changes that we have described in high-aggressive rats (elevated basal respiratory rate, reduced sniffing in a new environment, exaggerated respiratory responsiveness to an acoustic stimulus and frequent sighing) seem to reflect high emotionality and anxiety. Supporting this view, we found in HA rats behavioral changes that are indicative of an anxiety-like phenotype. In the elevated plus-maze test, HA rats showed a clear tendency to avoid the open arms of the apparatus. Of note, no differences were observed in overall locomotor activity (total number of arm entries) between the two groups. This indicates that the observed differences in the time spent and number of entries in the open arms cannot be attributed to variations in activity levels and may instead be attributable to higher levels of anxiety in HA rats, as open/unprotected arms are interpreted as more threatening than the closed/protected ones (Pellow et al., 1985). During the social avoidance–approach test, which is considered a reliable procedure to measure experimental anxiety in a social context (Haller and Bakos, 2002; Haller et al., 2003), HA rats showed higher levels of spontaneous avoidance behavior than NA rats (i.e., they spent less time in close proximity to the stimulus rat and more time in the hidden zone). Importantly, it has been demonstrated that benzodiazepine receptor agonists exert anxiolytic-like effects in rats that are not limited to a neutralization of the avoidance but are characterized by an increase in the social approach level (Nicolas and Prinssen, 2006). Moreover, human anxiety disorders are often characterized by avoidance (American Psychiatric Association, 1994) and many animal models of anxiety use avoidance as a means to identify anxiety-like responses (Cryan and Holmes, 2005). Therefore, it is reasonable to hypothesize that the behavior of HA rats in the elevated-plus maze and social avoidance–approach tests may reflect heightened anxiety. 5. Conclusions The results of this study indicate that high-aggressive rats show respiratory and behavioral changes that present many anxietyrelated features. Interestingly, a previous study has documented the presence of an anxiety-like phenotype in mice selected for aggression and demonstrated that both anxiety and aggressive behaviors are reduced by pharmacological treatment with an anxiolytic compound (diazepam) (Nehrenberg et al., 2009). In addition, a link between aggression and anxiety has been described in rats, although not always in a uni-directional manner (Neumann et al., 2010). It has been hypothesized that abnormalities in the complex brain circuitry regulating emotions, such as increased neuronal activation in the frontal cortex, the central amygdala, the bed nucleus of the stria terminalis and the ventrolateral parts of the periaqueductal gray, may lead to deficits in social behavior and excessive aggression (Davidson et al., 2000; Haller et al., 2006; Neumann et al., 2010). Interestingly, a recent investigation in rats has demonstrated that the central amygdala plays an essential role in the expression of respiratory responses to stressful or alerting stimuli (Bondarenko et al., 2014). Taken together, these findings suggest that the central amygdala may be a key brain structure involved in the regulation of emotion-related respiratory responses. Projections from the central amygdala to the dorsomedial hypothalamus might be involved in mediating these responses (Bondarenko et al., 2014).

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Our findings have to be regarded with several major limitations in mind: (i) we assessed only simple measures of respiration as the methodology used did not allow addressing the issue of, for example, tidal volume and inspiratory/expiratory flow, (ii) we did not provide pharmacological validation that the respiratory and behavioral changes observed in HA rats can be attenuated by anxiolytic compounds, and (iii) we did not address the question of the brain circuitry underlying the emotion-related respiratory responses described in this study. 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Please cite this article in press as: Carnevali, L., et al., Respiratory patterns reflect different levels of aggressiveness and emotionality in Wild-type Groningen rats. Respir. Physiol. Neurobiol. (2014), http://dx.doi.org/10.1016/j.resp.2014.07.003