Noradrenergic facilitation of shock-probe defensive burying in lateral septum of rats, and modulation by chronic treatment with desipramine

Progress in Neuro-Psychopharmacology & Biological Psychiatry 31 (2007) 482 – 495 www.elsevier.com/locate/pnpbp

Noradrenergic facilitation of shock-probe defensive burying in lateral septum of rats, and modulation by chronic treatment with desipramine Corina O. Bondi, Gabriel Barrera, M. Danet S. Lapiz, Tania Bedard, Amy Mahan 1 , David A. Morilak ⁎ Department of Pharmacology, MC 7764, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, Texas 78229-3900, USA Received 19 September 2006; received in revised form 24 October 2006; accepted 15 November 2006 Available online 26 December 2006

Abstract We have previously shown that acute stress-induced release of norepinephrine (NE) facilitates anxiety-like behavioral responses to stress, such as reduction in open-arm exploration on the elevated-plus maze and in social behavior on the social interaction test. Since these responses represent inhibition of ongoing behavior, it is important to also address whether NE facilitates a response that represents an activation of behavior. Correspondingly, it is unknown how a chronic elevation in tonic steady-state noradrenergic (NA) neurotransmission induced by NE reuptake blockade might alter this acute modulatory function, a regulatory process that may be pertinent to the anxiolytic effects of NE reuptake blockers such as desipramine (DMI). Therefore, in this study, we investigated noradrenergic modulation of the shock-probe defensive burying response in the lateral septum (LS). In experiment 1, shock-probe exposure induced an acute 3-fold increase in NE levels measured in LS of male Sprague-Dawley rats by microdialysis. Shock-probe exposure also induced a modest rise in plasma ACTH, taken as an indicator of perceived stress, that returned to baseline more rapidly in rats that were allowed to bury the probe compared to rats prevented from burying by providing them with minimal bedding, indicating that the active defensive burying behavior is an effective coping strategy that reduces the impact of acute shock probe-induced stress. In experiment 2, blockade of either α1- or β-adrenergic receptors in LS by local antagonist microinjection immediately before testing reduced defensive burying and increased immobility. In the next experiment, chronic DMI treatment increased basal extracellular NE levels in LS, and attenuated the acute shock probe-induced increase in NE release in LS relative to baseline. Chronic DMI treatment decreased shock-probe defensive burying behavior in a time-dependent manner, apparent only after 2 weeks or more of drug treatment. Moreover, rats treated chronically with DMI showed no significant rise of plasma ACTH in response to shock-probe exposure. Thus, acute stress-induced release of NE in LS facilitated defensive burying, an active, adaptive behavioral coping response. Chronic treatment with the NE reuptake blocker and antidepressant drug DMI attenuated acute noradrenergic facilitation of the active burying response, and also attenuated the level of perceived stress driving that response. These results suggest that long-term regulation of the acute modulatory function of NE by chronic treatment with reuptake blockers may contribute to the mechanisms by which such drugs exert their anxiolytic effects in the treatment of stress-related psychiatric conditions, including depression and anxiety. © 2006 Elsevier Inc. All rights reserved. Keywords: Antidepressant; Anxiety; Depression; Lateral septum; Norepinephrine; Stress

1. Introduction Abbreviations: ACTH, Adrenocorticotropic hormone; AD, Antidepressant; ANOVA, Analysis of variance; BSTL, Bed nucleus of the stria terminalis, lateral subdivision; CeA, Central nucleus of the amygdala; DMI, Desipramine; HPA, Hypothalamic–pituitary–adrenal; HPLC, High pressure liquid chromatography; LS, Lateral septum; NA, Noradrenergic; NE, Norepinephrine; NRI, Norepinephrine reuptake inhibitor; SPDB, Shock-probe defensive burying. ⁎ Corresponding author. Tel.: +1 210 567 4174; fax: +1 210 567 4303. E-mail address: [email protected] (D.A. Morilak). 1 Current affiliation: Graduate Div. of Biological Sciences, Emory University, Atlanta GA 30322. 0278-5846/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.pnpbp.2006.11.015

The response of an organism to acute stress involves the activation of a variety of adaptive behavioral, endocrine and autonomic processes that maintain homeostasis and restore optimal functioning. These responses are mediated by various neural circuits, including forebrain limbic areas regulating fear and anxiety, such as the lateral bed nucleus of the stria terminalis (BSTL) and central nucleus of the amygdala (CeA) (Davis and Shi, 1999). Acute stress also activates the neuromodulatory noradrenergic (NA) system, which projects widely

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throughout the brain (Cecchi et al., 2002a; Jacobs et al., 1991; Ma and Morilak, 2005; Pacak et al., 1993), and plays an integrative and regulatory role in the physiological and behavioral response to stress (Aston-Jones et al., 1991; Berridge and Waterhouse, 2003; Morilak et al., 2005). The neuromodulatory effect of NE at a cellular level is to enhance the “signal-to-noise” ratio of evoked activity, thereby enhancing the effectiveness of synaptic transmission (Woodward et al., 1991a). Whereas tonic, steady state elevations of NA neurotransmission are associated with a general state of behavioral arousal (Aston-Jones et al., 1991, 1999; Jacobs et al., 1991), exposure to an acute stressor induces a phasic activation of the NA system (Cecchi et al., 2002a,b; Ma and Morilak, 2005; Morilak et al., 1987; Pacak et al., 1995; Svensson, 1987; Valentino et al., 1993), contributing to an increase in acute behavioral stress reactivity (Berridge and Waterhouse, 2003; Morilak et al., 2005). In previous studies, we have shown that acute immobilization stress induced the release of NE in the rat BSTL and CeA, which facilitated acute anxiety-like behavioral responses in the elevated plus-maze and social interaction tests, respectively (Cecchi et al., 2002a,b). However, the behavioral response to acute stress in both of these tests represents an inhibition of ongoing behavior, i.e., reduction of open-arm exploration and reduction of social behavior. Thus, to better understand the nature of the neuromodulatory influence of NE on stress-evoked anxiety-like behaviors, it is necessary to determine whether stress-induced NE release will also facilitate responses that represent an activation of behavior, i.e., a defensive behavior directed specifically towards the anxiogenic stimulus. For this purpose, we employed the shock probe defensive burying test (SPDB), a well-validated and ethologically relevant model of rat defensive behavior (Pinel and Treit, 1978; Treit et al., 1981). Moreover, the SPDB test allows for an investigation of different aspects of stress-induced behavioral reactivity, as after receiving a shock from the electrified probe, rats typically exhibit both a passive behavioral response (immobility), as well as the active defensive burying behavior directed specifically at the probe. The lateral septum (LS) is involved in acute responses to stress consisting of either an inhibition or activation of behavior (Menard and Treit, 1996; Ouagazzal et al., 1999; Pesold and Treit, 1992; Treit and Pesold, 1990; Treit et al., 1993; Yadin and Thomas, 1996) and is specifically important in mediating the active burying response in the SPDB test (Pesold and Treit, 1996; Treit et al., 1993). Moreover, the LS receives a rich NA innervation from the locus coeruleus and caudal brainstem (Antonopoulos et al., 2004; Moore, 1978), suggesting this may be a key region in which stress-induced changes in NA transmission may facilitate anxiety-like behavioral responses in the SPDB test. Thus, in the present study, we first determined whether NE release was increased phasically in LS of adult male Sprague-Dawley rats during shock-probe exposure. We then investigated whether this acute NE release in LS facilitates the active defensive burying response, by local microinjections of α1- or β-adrenergic receptor antagonists directly into LS immediately prior to testing. Alterations in brain NA neurotransmission are also important in the actions of many antidepressant (AD) drugs, including

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tricyclics and a newer generation of dual uptake inhibitors and selective NE reuptake inhibitors (NRIs) (Frazer, 2000, 2001; Gorman and Sullivan, 2000; see Morilak and Frazer, 2004; Ressler and Nemeroff, 1999). These drugs, as well as selective serotonin reuptake inhibitors, are all equally effective in alleviating depressed mood, social withdrawal and other “inhibitory” symptoms of depression (reviewed in Morilak and Frazer, 2004). In addition to such inhibitory symptoms, there is extensive comorbidity of anxiety disorders and depression (Mineka et al., 1998; Nemeroff, 2002; Rouillon, 1999). Even in the absence of explicitly diagnosed comorbidity, anxiety is a prominent and prevalent component of depression, with as many as 85% of depressed patients having significant symptoms of anxiety (Gorman, 1996/1997; Katz et al., 1984), and related symptoms, such as agitation, aggression, distress, etc. In this regard, all AD drugs, including selective NRIs, successfully ameliorate anxiety as a component of depression (Ferguson et al., 2002; Kleber, 1979; Nelson, 1999; Nystrom and Hallstrom, 1985; Stahl et al., 2002; Szegedi et al., 1997). Considering the presumed role of NE in promoting arousal, vigilance and attention (e.g., Aston-Jones et al., 1991), it seems reasonable that elevating tonic NA levels by chronic reuptake blockade could improve the inhibitory symptoms of depression. However, it is less clear how enhancing such function could contribute to the anxiolytic effects of AD drugs, as phasicallyevoked NA activity promotes increased anxiety-like behavioral reactivity (Cecchi et al., 2002a,b). In those previous studies, however, it was also noted that baseline levels of NE transmission had little influence on these behaviors in unstressed rats. Similarly, in clinical studies, it has been suggested that anxiety may be associated more with acute excitability or “volatility” of NA neurotransmission than with the absolute level of NE per se (Sullivan et al., 1999). To address this, we have hypothesized that the tonically elevated extracellular NE levels induced by chronic NE reuptake blockade may reduce acute stress-induced excitability of NA neurotransmission, perhaps through increased activation of inhibitory α2-adrenergic autoreceptors, which we have shown to remain functional after chronic treatment with the selective NRI, desipramine (DMI) (Garcia et al., 2004). Thus, in the current study, we tested the time-dependent effects of chronic DMI treatment on acute stress-induced NE release in LS, and on acute behavioral reactivity in the SPDB test. Portions of this work have been presented in abstract form (Barrera et al., 2004; Petre et al., 2004). 2. Materials and methods 2.1. Animals A total of 165 adult male Sprague-Dawley rats (Harlan, Indianapolis, IN, USA), weighing 200–250 g upon arrival, were housed in groups of three and maintained on a 12/12 h light/ dark cycle (lights on at 07:00 h). Food and water were available ad libitum. Rats were allowed a minimum of 4 days to acclimatize to the housing facility before any surgical procedures were performed. All experiments were conducted during the

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light portion of the cycle, beginning at 08:00 h. All procedures were reviewed and approved by the Institutional Animal Care and Use Committee of the University of Texas Health Science Center at San Antonio, and were consistent with NIH guidelines for the care and use of laboratory animals. All efforts were made to minimize animal pain, suffering or discomfort, and to minimize the number of rats used. 2.2. Implantation of osmotic minipumps for chronic drug treatment For all surgical procedures, rats were anesthetized with a cocktail of ketamine 43 mg/ml, acepromazine 1.4 mg/ml, xylazine 8.6 mg/ml, 1.0 ml/kg, i.m., with a 25% supplement given as needed. For chronic drug treatment, osmotic minipumps (model 2ML2 or 2ML4, depending on the duration of delivery, Alzet Corp., Palo Alto, CA), preloaded with either vehicle (10% ethanol) or DMI (Sigma, St. Louis, MO) at a concentration calculated to deliver 15 mg/kg/day of the free base were implanted intraperitoneally via a ventral midline incision. Following surgery, rats were treated prophylactically with antibiotic (penicillin G, 300,000 IU/ml, 1.0 ml/kg, s.c,), and housed singly for the duration of drug treatment (7– 21 days) until testing. 2.3. Shock-probe defensive burying test On the day of testing, rats were transported to the testing room in their home cage, and allowed at least 20 min to habituate. Testing was conducted under normal overhead ambient lighting (220 lux measured in the test chamber). The shock-probe defensive burying test, adapted from Pinel and Treit (1978), was conducted in a polystyrene cage, 26 × 48 × 21 cm, identical to the rats home cage, but with the lid modified to allow the rats behavior to be videotaped from above for off-line scoring. Fresh bedding (Teklad Sani-Chips, Harlan, Indianapolis, IN) lined the cage to a depth of 5 cm. The shock probe was a glass rod, 1.0 cm dia, wrapped with two alternating, non-touching 18 ga copper wires, spaced 5 loops/ cm. The probe protruded 6 cm into one end of the cage, 2 cm above the surface of the bedding. The wires were attached to a shock generator (model H13–15, Coulbourn Instruments, Allentown, PA) set to deliver 2 mA DC current when the probe was touched. To begin the test, a rat was introduced into the cage at the end opposite the shock probe, facing away from the probe. Rats typically approached the probe to investigate within 10–15 s, making contact with their paw or snout. Upon contacting the probe, the current was turned off so only a single shock was delivered, and the 15 min test period began. After withdrawing from the probe, rats typically showed a variable period of inactivity before beginning to bury, usually within 5– 8 min. “Burying” consisted of burrowing into the bedding with their snout and upper body, then “plowing” the bedding toward the probe, and also flicking bedding toward the probe with the dorsal surface of the forepaws. After each test, the cage was washed with a wet sponge, and the bedding replaced with fresh bedding before testing the next rat.

Behavioral scoring, adapted from De Boer and Koolhaas (2003), was performed from video by an experimenter blind to the condition of the test animal. In addition to total burying time, other measures included total immobility time, and latency to contact the probe (an indicator of potential changes in locomotion or exploratory activity). Each rat was also scored subjectively for reactivity to the shock on a 4-point scale (Treit and Pesold, 1990). 2.4. Experiment 1a: NE release induced in LS by shock-probe exposure Five rats were used for in vivo microdialysis in LS during shock probe exposure. After the acclimatization period, they were anesthetized with a cocktail of ketamine 43 mg/ml, acepromazine 1.4 mg/ml, xylazine 8.6 mg/ml, 1.0 ml/kg, i.m., with a 25% supplement given as needed, and placed in a stereotaxic apparatus with the incisor bar set at − 3.3 mm, adjusted as needed to achieve a flat-skull position, indicated by equal DV coordinates for bregma and lambda. A microdialysis guide cannula (CMA/12; CMA Microdialysis, North Chelmsford, MA), aimed at the LS, was implanted using a 22° lateral approach, with the following coordinates relative to bregma: AP + 0.7 mm, ML + 2.4 mm, DV − 4.0 mm, placing the tip 1 mm above LS, corresponding to plates 15–16 in the atlas of Paxinos and Watson (1998). Approximately half the rats were implanted on the left side, and half on the right. The guide cannula was anchored to the skull with jewelers screws and dental acrylic, and an obdurator was inserted to maintain patency. Rats were treated prophylactically with antibiotic (penicillin G, 300,000 IU/ml, 1.0 ml/kg, s.c,), and housed singly for 1 week recovery before testing. On the day of the experiment, rats were transported in their home cage to the testing room. The obdurator was removed, and a microdialysis probe (CMA/12), with molecular weight cutoff of 20 kDa and 2 mm of active membrane was inserted, extending 2 mm beyond the tip of the guide, centering the active membrane within LS. The probe was perfused with artificial cerebrospinal fluid (147 mM NaCl, 2.5 mM KCl, 1.3 mM CaCl2, 0.9 mM MgCl2, pH 7.4) at a flow rate of 1.0 μl/min. Animals were allowed a 2 h equilibration period before sample collection was initiated. Sample collection time was 30 min, resulting in a sample volume of 30 μl, collected into a tube containing 2.5 μl of stabilizing solution (0.2 μM EDTA, 0.2 μM ascorbic acid, 0.2 M perchloric acid). After 3 baseline samples were collected in the home cage, the rat was placed into the shock-probe test chamber. However, instead of 5 cm bedding, the cage contained only enough bedding to minimally cover the entire floor of the chamber, and the height of the shock probe was adjusted to remain 2 cm above the surface of the bedding. Thus, each rat contacted the probe and received a 2 mA shock as described above, but was unable to exhibit burying behavior. This served two purposes: to prevent possible damage to the probe or disturbance of the microdialysis collection by the rat burrowing into the bedding, and also to maintain a constant level of “stress” induced by the continued presence of the shock probe, without the variability in stimulus “intensity” that may

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have been introduced by varying degrees of burying behavior. After contacting the probe and receiving a shock, the current was turned off, and the rat then remained in the presence of the shock probe for 30 min, during which a microdialysis sample was collected. Rats were then returned to their home cage for collection of 6 post-test recovery samples. The amount of NE in the microdialysate samples was measured by high-performance liquid chromatography with coulometric detection (Coulochem2, ESA Inc., East Chelmsford, MA). The mobile phase contained 60 mM sodium phosphate, 75 μM EDTA, 1.5 mM sodium 1-octanesulfonic acid, 6% methanol, pH 4.6, at a flow rate of 0.6 ml/min. Under these conditions, NE had a retention time of approximately 7.8 min. The amount of NE in each sample was quantified against a calibration curve run daily, ranging from 0.0 to 25 pg, with a detection limit of approximately 0.5 pg/sample. 2.5. Experiment 1b: assessment of defensive burying as a coping response: effect of burying behavior on activation of the HPA axis by shock-probe exposure Twenty-four rats were anesthetized as above, and an indwelling silastic catheter was surgically implanted into the jugular vein. The catheter was exteriorized at the back of the neck and loaded with heparinized saline (100 IU/ml) to maintain patency. After 4 days of post-surgical recovery, rats were transported to the testing room, and the venous catheters were connected to a 1 ml syringe via PE tubing filled with heparinized saline (50 IU/ml), allowing for repeated blood sampling without disturbing the animal. Twenty minutes after connecting the lines, a baseline blood sample (0.4 ml) was taken. Animals were assigned randomly to one of two testing conditions, with either the full 5 cm of bedding or with minimal bedding as in experiment 1a, to prevent them from being able to bury. The probe was adjusted for each trial to be 2 cm above the surface of the bedding. Fifteen minutes after taking the baseline blood sample, rats were transferred into the shock-probe test chamber. A second sample was drawn 3 min after the rat contacted the probe, to assess initial activation of the HPA axis by the shock, before any differences in burying behavior could be manifest. Another blood sample was taken at 15 min, at the termination of the behavioral test period, after which the rats were returned to their home cage. Additional samples were then taken after 15 and 30 min of recovery. All blood samples were replaced immediately by infusing an equivalent volume of sterile saline. We have determined in previous experiments that this repeated sampling and replacement procedure does not itself affect plasma ACTH (Cecchi et al., 2002a). Blood was collected into tubes containing 10 μl of 1.0 M EDTA. Plasma was separated by centrifugation and stored at − 20 °C until assayed. Plasma ACTH levels were measured in 200 μl samples by radioimmunoassay (Nichols Institute Diagnostics, San Juan Capistrano, CA). The detection limit of the assay was 15 pg/ ml, with intra- and inter-assay coefficients of variation of 9% and 11%, respectively.

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2.6. Experiment 2: local microinjections of noradrenergic antagonists in LS to determine the modulatory influence of NE on the shock-probe defensive burying response Thirty-four rats were assigned randomly to three drugtreatment groups and prepared for stereotaxic surgery as above. Two guide cannulae, constructed of 22 ga stainless steel tubing, were implanted bilaterally, aimed at the LS using the same approach and coordinates as above, placing the tips 1 mm above the target. The cannulae were anchored to the skull and fitted with 30 ga obdurators to maintain patency. Rats were treated with antibiotic, and housed singly for 1 week before testing. On the day of the experiment, rats were transported to the testing room in their home cage. The obdurators were replaced with 30 ga stainless steel microinjection cannulae, which extended 1 mm beyond the tips of the guides, placing them in the LS. The microinjection cannulae were connected by PE-10 tubing to a Hamilton syringe mounted on a syringe pump (Instech, Plymouth Meeting, PA). After inserting the injectors, rats were allowed 20 min to habituate before drug microinjections. Bilateral microinjections were made into LS of either saline vehicle (0.4 μl per side), the α1-adrenergic receptor antagonist benoxathian (2.0 nmol/0.4 μl/side), or a cocktail of the β1- and β2-receptor antagonists betaxolol + ICI 118,551 (1.0 nmol each/0.4 μl/side), delivered at a rate of 0.1 μl/min. After injections were complete, cannulae were left in place for 1 min to allow for diffusion before withdrawing. Five minutes after removing the injectors, the rats were transferred to the shock-probe chamber, and the defensive burying test procedure was conducted with the full complement of 5 cm fresh bedding in the cage. 2.7. Experiment 3: effect of chronic DMI treatment on acute NE release in lateral septum induced by shock probe exposure A group of 24 rats were used for this experiment. Fifteen rats were prepared for chronic drug treatment as described above (n = 7–8 for vehicle and DMI, respectively). After minipump implantation, rats were implanted with microdialysis guide cannulae aimed at the LS, also as above. Rats were then housed singly after surgery. After 21 days of chronic drug treatment, rats were transported to the testing room for microdialysis. After four baseline samples were collected in the home cage, the rat was placed in the shock-probe chamber. As above, the cage contained only enough bedding to cover the floor of the chamber to prevent burying. After contacting the probe and receiving a shock, the current was turned off, and the rat remained in the presence of the shock probe for 30 min, during which a microdialysis sample was collected. Rats were then returned to their home cage for collection of 4 post-test recovery samples. Subsequently, because a difference in shock probe-induced increase in NE release was observed after chronic DMI treatment, an additional groups of rats (n = 9) that were not implanted for chronic drug treatment were tested exactly as above, except that after the second baseline sample they were given an acute injection of DMI (10 mg/kg, i.p.). In previous studies (Daniel and Melzacka, 1992), and verified in our own pilot studies, this

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acute dose has been shown to produce plasma DMI levels equivalent to those seen after chronic treatment by minipimp at 15 mg/kg/day (∼ 200–400 ng/ml) within 1 h of administration. Thus, two additional baseline samples were collected after injection, then these rats were placed in the shock-probe chamber and tested as above. The amount of NE in all microdialysate samples was quantified by HPLC. 2.8. Experiment 4a: time-dependent changes in shock probeinduced behavioral responses over the course of chronic DMI treatment Fifty-six rats were randomly assigned to 8 groups (n = 5–12/ group) defined by 2 drug conditions (vehicle or DMI), and 4 treatment time points (acute, 7 days, 14 days or 21 days). Fortyfive of the rats were implanted with osmotic minipumps and tested on the shock-probe defensive burying test after 7, 14 or 21 days of chronic drug administration. Each animal was tested only once, after the designated time of chronic drug or vehicle treatment. Another set of 11 naïve rats were tested 1 h after a single acute injection of vehicle (2.0 ml/kg, i.p.) or DMI (10 mg/ kg, i.p.), to produce plasma DMI levels comparable to those seen after chronic treatment. Procedures for the shock probe defensive burying test were conducted as above. 2.9. Experiment 4b: effect of chronic DMI treatment on acute activation of the HPA axis by shock probe exposure The effect of chronic DMI treatment on the degree of perceived stress induced by continued exposure to the shock-probe after receiving the single shock was assessed by comparing activation of the HPA axis in DMI-treated rats to that in vehicle-treated control rats. A total of 22 rats were implanted with minipumps for chronic delivery of vehicle (n = 10) or DMI (15 mg/kg/day, n = 12) as above. After 18 days of treatment, they were again anesthetized, and a silastic catheter implanted into the jugular vein, exteriorized at the back of the neck and loaded with heparinized saline (100 IU/ml) to maintain patency. On day 21, rats were transported to the testing room, and the venous catheters connected to a 1 ml syringe via PE tubing filled with heparinized saline (50 IU/ml), as in experiment 1b. A baseline blood sample (0.4 ml) was taken 20 min after connecting the lines. Fifteen minutes after taking the baseline blood sample, rats were transferred into the shock-probe chamber and tested as above. To maintain a constant level of stress by continued exposure to the shock-probe stimulus, the rats were provided with only enough bedding to cover the bottom of the cage, but not enough to allow them to bury. A second blood sample was drawn 3 min after the rats contacted the probe, and another taken at 15 min, at the termination of the behavioral test period, after which the rats were returned to their home cages. Additional samples were then taken after 15 and 30 min of recovery. All blood samples were replaced immediately by infusing an equivalent volume of sterile saline. Blood was collected into tubes containing 10 μl of 1.0 M EDTA. Plasma was separated by centrifugation and stored at − 20 °C. Plasma ACTH levels were measured in 200 μl samples by radioimmunoassay, as above.

2.10. Data analysis All data were analyzed by ANOVA, with repeated-measures where appropriate (microdialysis and plasma samples). Where significant main effects or interactions were indicated, post hoc comparisons were made using the Newman–Keuls test. In the analysis of behavioral responses in the defensive burying test, it was observed that a proportion (approximately 30%) of animals, even in the vehicle-microinjected control group, failed to exhibit burying behavior, resulting in a violation of both normality and homogeneity of variance of these data. Thus, total burying time was also analyzed non-parametrically using the Kruskal–Wallis test, followed by the non-parametric Dunn's post hoc test for multiple comparisons where significant main effects were indicated. In microdialysis experiments, to first test for any differences in tonic baseline NE levels collected in LS of vehicle- and DMItreated rats, the mean of the three baseline samples was calculated for each rat, and these were compared by Student's t-test. As baseline NE levels were found to be significantly elevated in rats treated with DMI, the effects of acute shock-probe exposure on NE release were then analyzed in two ways. First, to test the effects on absolute NE levels for each drug group, expressed as pg/sample, a one-way ANOVA with repeated measures over Time was conducted, split by Drug. Next, all sample values were converted to a percent of the mean baseline for each rat, and a two-way ANOVA (Drug × Time), with repeated-measures over Time, conducted to allow a direct comparison of the relative effects of acute shock-probe exposure in the two drug groups after normalizing for baseline differences. Where significant main effects or interactions were indicated by either approach, post hoc analyses were performed using the Newman–Keuls test. Significance was determined at p b 0.05 for all analyses. At the end of each experiment, rats were sacrificed by rapid decapitation. Brains were removed for histological determination of probe or microinjection cannula placement, and trunk blood was collected for analysis of plasma DMI levels (assays conducted by Dr. Martin Javors, Department of Psychiatry, UTHSCSA). Any subject that had extreme plasma DMI levels (b 50 ng/ml or N 1000 ng/ml) was eliminated a priori from subsequent analyses. Similarly, any cases in which one or both of the microinjection cannulae, or the microdialysis probe track were located outside the LS were eliminated from subsequent analyses. Any subjects so eliminated were not included in the reported total number of rats used. This resulted in the elimination of 38 animals from an initial number of 203 (most of these due to deflection of one or both cannulae by the corpus callosum) for the final reported total of 165 rats. 3. Results 3.1. Experiment 1a: NE release induced in LS by shock-probe exposure Fig. 1A shows a representative photomicrograph of a cresyl-stained section in which the microdialysis probe track was localized to LS. Analysis of NE levels measured in

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bedding sufficient to just cover the floor. Two-way ANOVA, with repeated measures over Time, indicated that shock-probe exposure had a significant effect on plasma ACTH (F4, 88= 3.620, n = 10–14 per group, p b 0.01), and a significant Group× Time interaction (F4, 88 = 2.494, p b 0.05). Post hoc comparisons with the Newman–Keuls test showed that the two groups did not differ in baseline ACTH, nor in plasma ACTH measured at either 3 or 15 min after initial contact with the shock-probe, i.e., during continued exposure to the probe. In the group that was allowed to bury during the 15 min test period, ACTH never reached a level significantly higher than baseline, and after returning them to their home cages, ACTH levels decreased very quickly from the modest elevation that was seen during probe exposure (Fig. 2). By contrast, in the group that was unable to bury, although a similarly modest increase in ACTH was seen during probe exposure, their ACTH levels continued to rise after removal from the test chamber, reaching a significantly elevated peak 15 min after test termination, compared to both their baseline and to the corresponding sample taken from the group that had buried (Fig. 2). By 30 min after test termination, ACTH levels in both groups were once again no different from their corresponding baselines, nor from each other. 3.3. Experiment 2: local microinjections of noradrenergic antagonists in LS to determine the modulatory influence of NE on the shock-probe defensive burying response Fig. 1. Increased NE release induced in lateral septum by acute shock-probe exposure. A) Representative photomicrograph of a Cresyl violet-stained section through LS, corresponding to plate 15 in the atlas of Paxinos and Watson (1998), showing the microdialysis probe track in lateral septum (arrowhead). Abbreviations: ac, anterior commissure; CP, caudate-putamen; LS, lateral septum. Scale bar = 1 mm. B) Continued exposure to the shock-probe following initial contact (bar) acutely activated a significant increase in extracellular NE levels measured in microdialysate samples collected in LS. Data are expressed as pg/sample (mean ± SEM), n = 5; ⁎p b 0.05 compared with pre-stress baseline and post-stress recovery samples.

Fig. 3A shows a representative brain section in which the bilateral microinjection sites were localized to LS. Analysis of behavior in the defensive burying test after bilateral microinjections of vehicle or adrenergic antagonists into LS indicated significant effects of drug treatment on both immobility and

microdialysis samples collected in LS showed that continued exposure to the shock-probe after receiving a single shock induced a significant 3-fold increase in NE release in LS (Fig. 1B). One-way ANOVA with repeated measures over time revealed a significant main effect (F9, 36 = 2.495, n = 5, p b 0.05). Subsequent post hoc comparisons using the Newman–Keuls test indicated that NE levels in LS were significantly elevated in the sample collected during the 30 min period of continued exposure to the shock-probe relative to all other baseline and post-exposure recovery samples (Fig. 1B). 3.2. Experiment 1b: assessment of defensive burying as a coping response: Effect of burying behavior on activation of the HPA axis by shock-probe exposure As an independent measure of the degree of stress experienced by animals during exposure to the shock-probe in the defensive burying procedure, plasma ACTH was measured in rats that were either allowed to bury the probe, by providing 5 cm bedding in the cage, or not allowed to bury by providing

Fig. 2. Defensive burying as an effective behavioral coping response, buffering the shock-probe induced activation of the HPA axis. There were no pre-test baseline differences in plasma ACTH levels between the groups. Plasma ACTH concentration was increased slightly but non-significantly in both groups at the 3 min and 15 min time points during probe exposure. In the group that was allowed to bury by providing the full 5 cm of bedding, plasma ACTH returned quickly to baseline levels after removal from the presence of the shock probe. However, ACTH continued to rise significantly in the group that had not been allowed to bury. Data expressed as mean ± SEM; n = 10–14/group; ⁎p b 0.05 compared to pre-test baseline; +p b 0.05 compared to the group with full bedding at the same time point.

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with vehicle (Fig. 3B). For the analysis of burying time, however, because a proportion of rats in all groups, even in the vehicle-treated control group, did not exhibit burying behavior, the data for this measure violated the assumptions of normality and homogeneity of variance underlying parametric analyses. Consistent with this, ANOVA indicated a trend that did not achieve significance (F2, 31 = 2.487, p = 0.09), although there appeared to be a clear difference in mean bury time between the groups (see Fig. 3C). Thus, these data were analyzed nonparametrically using the Kruskal–Wallis test, which showed a significant drug effect (H2 = 9.630, p b 0.01). Post hoc comparisons using Dunn's test indicated that bury time was reduced significantly by α1-antagonist administration into LS (Fig. 3C). Although the mean bury time after β-antagonist administration into LS was only slightly higher than that after α1-antagonist administration (Fig. 3C), the decrease in bury time in this group was nonetheless not significant when compared to vehicleinjected controls. 3.4. Experiment 3: effect of chronic DMI treatment on acute NE release in lateral septum induced by shock probe exposure

Fig. 3. Facilitation of the active defensive burying response by norepinephrine release in lateral septum. A) Representative photomicrograph of a Cresyl violetstained brain section, corresponding to Plate 15 in the atlas of Paxinos and Watson (1998), showing bilateral localization of the microinjection cannula tracks in LS (arrowheads). Abbreviations: ac, anterior commissure; CP, caudate-putamen; LS, lateral septum; MS, medial septum. Scale bar = 1 mm. B) Bilateral microinjections into LS of either the α1-adrenergic receptor antagonist benoxathian (2.0 nmol/side), or a cocktail of β1β2-receptor antagonists betaxolol + ICI 118,551 (1.0 nmol each/side) before testing significantly increased immobility on the shock-probe defensive burying test. C) Adrenergic receptor blockade in LS also attenuated defensive burying behavior, although only the reduction produced by α1-receptor blockade achieved significance. Data expressed as mean ± SEM; n = 8–18/group; ⁎p b 0.05 compared to vehicle control group.

burying time. For immobility, a significant drug effect was indicated by ANOVA (F2, 31 = 4.912, p b 0.05, n = 8–18 per group). Post hoc comparisons indicated a significant increase in immobility in animals microinjected with either α1- or βantagonists prior to testing, relative to control rats microinjected

Chronic DMI treatment resulted in mean plasma DMI levels of 302.5 ± 67.3 ng/ml. An initial comparison of mean baseline NE levels measured in dialysate collected in LS after chronic drug treatment showed that 21-day DMI induced a significant increase in tonic baseline extracellular NE compared to 21-day vehicle treatment (1.0 ± 0.2 pg/sample, mean ± SE, in vehicletreated rats, n = 7, compared to 8.5 ± 1.5 pg/sample in DMItreated rats, n = 8; t13 = 4.674, p b 0.001, see Fig. 4A). Therefore, effects of shock probe exposure on absolute levels of NE (pg/ sample) collected in LS of rats treated with vehicle or DMI were analyzed first by two-way ANOVA (Drug × Sample), with repeated measures over Sample, followed by one-way repeatedmeasures ANOVA, split by Drug. In the two-way ANOVA, there were significant main effects of Drug (F1, 13 = 26.306, p b 0.001), and Sample (F8, 104 = 5.654, p b 0.0001), but no Drug × Sample interaction (F8, 104 = 1.252, p N 0.27). Post hoc analysis showed all samples in DMI-treated rats to be higher than all samples in vehicle-treated rats. Thus, in subsequent oneway ANOVA split by Drug, there was a significant main effect in both vehicle- (F8, 48 = 4.514, p b 0.001) and DMI-treated rats (F8, 56 = 3.602, p b 0.01). Post hoc analyses indicated that NE was significantly elevated in the sample collected during shock probe exposure in both groups, compared to all other samples within the same drug conditions (Fig. 4A). To then compare the relative effect of shock probe exposure on NE release in LS between the two drug groups, because of the elevated baseline levels in the DMI-treated rats, the same analyses were repeated after normalizing all values as a percent of the mean baseline level calculated for each animal (Fig. 4B). As above, the percent baseline data were first analyzed by twoway ANOVA (Drug × Sample), with repeated measures over Sample, followed by one-way repeated measures ANOVA for Sample, split by Drug. In the two-way ANOVA, there was a significant main effect of Sample (F8, 104 = 8.685, p b 0.0001), and a Drug × Sample interaction (F8, 104 = 2.356, p b 0.05), but

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no main effect of Drug (F1, 13 = 1.363, p N 0.26). Post hoc analyses indicated that NE in the sample collected during shock-probe exposure in vehicle-treated rats was significantly higher than all other samples, including the sample collected during shock-probe exposure in the DMI-treated rats. Subsequent one-way ANOVAs split by Drug indicated significant main effects in both vehicle- (F8, 48 = 5.671, p b 0.0001) and

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DMI-treated rats (F8, 56 = 3.354, p b 0.01). Post hoc analyses indicated a significant increase in the shock probe sample in both groups relative to other samples within the same drug condition (Fig. 4B). Because of the decrement seen in the relative NE response after chronic DMI treatment, an additional group of animals were treated acutely with DMI before shock probe exposure, and their response was compared to the chronically treated group. Initial comparisons showed that the mean baseline NE levels in the chronically treated group were significantly higher than the post-injection baseline in the acutely-treated group (3.4 ± 0.5 pg/sample in acutely-treated rats, n = 9, compared to 8.5 ± 1.5 pg/sample in DMI-treated rats, n = 8; t15 = 3.393, p b 0.01). Thus, all values were normalized to percent of mean baseline as above to compare the relative magnitude of the shock probe-induced increase in NE after acute and chronic DMI treatment (Fig. 4C). Two-way ANOVA with repeated measures revealed a significant main effect of Sample (F8, 120 = 10.703, p b 0.0001) and a significant interaction (F8, 120 = 3.198, p b 0.01), but no main effect of Drug (F1, 15 = 0.038, p N 0.84). Post hoc analyses indicated that both shock-probe samples were elevated, and the two shock-probe samples did not differ from one another. Post hoc comparisons after one-way ANOVA, split by Treatment Duration, (acute and chronic), confirmed that NE levels in both shock-probe samples were significantly elevated relative to all other samples in their respective treatment groups (Fig. 4C). 3.5. Experiment 4a: time-dependent changes in shock probeinduced behavioral responses over the course of chronic DMI treatment Mean plasma DMI levels in chronically-treated rats were, for 7 days: 383.1 ± 26.1 ng/ml; for 14 days: 268.4 ± 23.3 ng/ml; and for 21 days: 234.7 ± 45.5 ng/ml. Initial analyses of the salinetreated control groups indicated no differences in any Fig. 4. Acute NE release induced in lateral septum by shock-probe exposure following chronic treatment with DMI. A) Absolute NE levels in microdialysate samples expressed as pg/sample (mean ± SEM). Chronic DMI treatment (15 mg/ kg/day for 21 days) elevated tonic baseline NE levels (p b 0.001, n = 7–8/group), and NE levels in all samples collected in DMI-treated rats were significantly greater than in all samples in vehicle-treated rats (main drug effect, p b 0.001, significance not indicated for the sake of clarity). Exposure to the shock-probe induced a significant increase in both groups (⁎p b 0.05 compared to pre-stress baseline and post-stress recovery samples in the same drug-treatment group). B) Relative NE levels in each sample expressed as percent of mean baseline in each rat (mean ± SEM). The acute increase in NE elicited by shock-probe exposure relative to baseline was significant in both groups, but the magnitude of the increase was attenuated in the group treated chronically with DMI (⁎p b 0.05 compared to baseline and recovery samples in the same drugtreatment group; +p b 0.05 compared to the same time point in the vehicletreated control group). C) Shock-probe exposure increased NE release in LS relative to pre-stress baseline levels similarly in rats treated chronically with DMI and those treated acutely (10 mg/kg, i.p.), 1 h prior to shock-probe exposure (arrow). Data are expressed as percent of mean baseline samples collected prior to shock-probe exposure, but after acute drug injection (mean ± SEM; n = 8–9/group), as acute DMI injection increased NE levels in LS (⁎p b 0.05 compared to pre-stress baseline and post-stress recovery samples in the same group).

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behaviora2l measures in animals treated with vehicle acutely or chronically for 7, 14 or 21 days (Bury Time: F3, 24 = 0.307, p N 0.81; Immobility: F3, 24 = 0.232, p N 0.87; n = 5–12/group). Thus, all vehicle-treated rats were pooled into a single control group, and the behavioral data collected on the shock-probe test were then analyzed by one-way ANOVA. These analyses indicated a significant main effect of drug treatment on both defensive burying time (F4, 51 = 3.949, p b 0.01; Fig. 5A) and

Fig. 6. Chronic DMI treatment reduced the acute ACTH response elicited by shock-probe exposure. There were no pre-test baseline differences in plasma ACTH levels between the groups. Plasma ACTH was increased significantly at 3 min and 15 min during probe exposure in the vehicle-treated control rats, but not in DMI-treated rats. Data expressed as mean ± SEM; n = 10–12/group; ⁎p b 0.05 compared to pre-test baseline; +p b 0.05 vehicle-treated controls compared to the DMI-treated group at the same time point.

immobility time (F4, 51 = 17.130, p b 0.0001; Fig. 5B). Post hoc comparisons showed that there was a significant decrease in burying time and a significant increase in immobility time in rats treated with DMI for 14 or 21 days compared to vehicle controls and also compared to rats treated with DMI acutely or treated chronically for 7 days (see Fig. 5). The behavior of rats treated with DMI acutely did not differ from those treated for 7 days, nor did these groups differ from the vehicle-treated rats in any of the behavioral measures. Because of the possibility of a floor effect, burying time was also analyzed non-parametrically using the Kruskal–Wallis test, with exactly the same result. A significant main effect was revealed for drug treatment (H5 = 18.60, p b 0.001). Post hoc comparisons using Dunn's test indicated that rats treated with DMI for 14 or 21 days showed significantly less burying than vehicle-treated rats or rats treated with DMI either acutely or for 7 days. These groups did not differ from each other. Nor were there differences in latency to contact the probe prior to receiving the shock (F4, 51 = 2.108, p = 0.0934). 3.6. Experiment 4b: effect of chronic DMI treatment on acute activation of the HPA axis by shock probe exposure

Fig. 5. Chronic treatment with DMI (15 mg/kg/day) reduced defensive burying behavior (A) and increased immobility (B) in the shock-probe test in a timedependent manner. Although plasma levels of DMI were comparable, the first significant behavioral effect was seen after 14 days of treatment, becoming greater after 21 days. Data expressed as mean ± SEM; n = 6–9/DMI-treatment group, and 28 pooled vehicle-treated rats; ⁎p b 0.05 compared to vehicle-treated controls.

Chronic DMI treatment resulted in mean plasma DMI levels of 338.3 ± 39.1 ng/ml. Two-way ANOVA, with repeated measures over Time, revealed significant main effects of Time (F4, 80 = 5.605, p b 0.001), and Group (F1, 20 = 10.416, p b 0.005). Post hoc analyses indicated that there were no baseline differences in plasma ACTH levels between the groups. In vehicle-treated rats, contact with and continued exposure to the shock-probe induced a significant increase in plasma ACTH levels measured 3 min and 15 min after probe contact, compared to both their baseline and to the DMI-treated

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group at the same time points (Fig. 6). By contrast, DMI-treated rats showed no significant elevation of plasma ACTH at any point during or after the shock-probe test procedure (Fig. 6). 4. Discussion In this study, we have shown that NE release was elevated in LS by exposure to the shock-probe in the context of the defensive burying test. By measuring secretion of ACTH during the shock-probe test in rats that were and were not allowed to bury, we showed that the active burying behavior represented a more effective coping response, reducing the magnitude and duration of activation of the HPA axis induced by exposure to the shock probe compared to rats who were able to adopt only a passive, immobile response. Blocking the actions of NE at either α1- or β-adrenergic receptors in LS reduced the defensive burying behavior exhibited by the rats, suggesting that stressevoked noradrenergic activity in LS facilitated the active, adaptive behavioral coping response elicited in this stressprovoking context. Furthermore, chronic DMI treatment increased extracellular NE levels in LS, measured by in vivo microdialysis, and attenuated the shock probe-induced phasic release of NE relative to baseline. Shock-probe defensive burying behavior was also reduced in a time-dependent manner following chronic DMI treatment, apparent only after 2 or more weeks of drug treatment. Concomitantly, rats subjected to chronic DMI treatment did not show a significant rise in plasma ACTH levels in response to shock-probe exposure. Using a similar approach, we have shown previously that acute stress-induced release of NE in other forebrain limbic regions, including the CeA and the BSTL, facilitated other anxiety-like behavioral responses to acute stress mediated specifically in those regions. Blockade of α1- or β-adrenergic receptors in the BSTL attenuated the acute stress-induced reduction in open-arm exploration on the elevated plus-maze, but had no effect on acute stress-induced reduction in social behavior on the social interaction test (Cecchi et al., 2002a). By contrast, blockade of α1- but not β-adrenergic receptors in the CeA prevented the stress-induced reduction in social behavior, with no effect on the plus-maze (Cecchi et al., 2002b). Importantly, the measure of stress-induced anxiety-like responding evoked in both of these tests was a reduction in a specific ongoing behavior, namely, social interaction or open-arm exploration. However, these responses were not passive in nature, as in the case of freezing or immobility, because overall locomotor activity and even total exploratory behavior were unaffected by either the acute stress exposure or by local adrenergic antagonist administration. Nonetheless, it remained unclear solely from these previous results whether NE might have facilitated any overt, reactive behavioral response provoked by acute stress, or may instead have selectively facilitated only those responses involving some form of behavioral inhibition. Thus, in the present study, the shock-probe defensive burying test was used to distinguish these possibilities more clearly, as two distinctly different behavioral response strategies, the active burying response and the passive immobility response, are elicited in varying proportions after contact with

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the shock probe (Pinel and Treit, 1978; Treit et al., 1981). In this study, blockade of adrenergic receptors in LS shifted the behavioral coping strategy of the rats after contact with the probe from an active to a passive one, attenuating the active burying response and increasing the amount of immobility exhibited during the test period. Thus, taken together with the previous results discussed above, we may conclude that stressinduced NE release throughout the limbic forebrain facilitates a variety of evoked, reactive behavioral responses, both active (e.g., defensive burying) and “inhibitory” in nature (e.g., reduction of social interaction, redirection of exploratory behavior from open to closed arms on the elevated plus maze). However, NE does not appear to enhance more passive behavioral response strategies, i.e., immobility or freezing. Rather, blocking the noradrenergic mechanisms in LS that facilitate active responding appeared to have enabled a greater expression of the passive, immobility response, which may therefore be mediated by different neurotransmitters in LS, or by a different set of neural circuits entirely, involving brain regions other than LS (e.g., see Gray, 1988; Handley, 1995). The question could be raised as to whether the release of NE influences the behavioral response to stress, or if induction of the behavior itself drives the acute release of NE. Several lines of evidence would seem to suggest that stress, rather than the behavioral response to stress, activates the acute release of NE in LS and elsewhere, and that this release of NE influences the subsequent behavioral response. First, the NE release measured in LS in this study occurred in the absence of burying behavior, as the rats had only minimal bedding in the cage to prevent damage to the dialysis probe and to maintain a constant stimulus (i.e., continued exposure to the probe). Thus, the burying response itself could not have driven NE release in this case. Second, in the studies mentioned above, similar NE release was observed in the BSTL and CeA during an acute immobilization stress that preceded alterations in behavior assessed subsequently on the elevated plus maze and social interaction tests (Cecchi et al., 2002a,b). Also, we have measured NE release in medial prefrontal cortex during a behavioral procedure that involves digging in material to obtain a food reward, a behavioral activity similar to the burying response, but lacking any component of stress (Lapiz et al., in press). In this context, we observed only a modest tonic elevation of NE release, consistent with increased behavioral arousal and general activity, but not resembling the phasic and robust elevation observed in response to acute stress. The observation from the present study that NE facilitates acute, evoked behavioral responses relative to the more passive state of behavioral immobility, and also the observation reported previously that local adrenergic antagonist administration had minimal effect on unstressed baseline behavioral activity (Cecchi et al., 2002a,b), are both consistent with the wellcharacterized cellular effects of NE, which are similarly modulatory in nature. Rather than producing a direct excitation or inhibition, NE instead increases the “signal to noise ratio” of evoked electrical activity in target circuits, both excitatory and inhibitory, making evoked synaptic transmission more effective (Woodward et al., 1991a,b). In different preparations, these

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modulatory effects have been ascribed to both α1- and βadrenergic receptors (Woodward et al., 1991a,b). At the whole organism level, the effect of such a cellular modulatory influence would presumably be to enhance the array of physiological, behavioral and cognitive response processes that are mediated in the many brain regions innervated by noradrenergic projections. Thus, the present results suggest that drug-induced changes in the modulatory function of NE could constitute a possible mechanism by which AD treatments that influence NA neurotransmission may exert their beneficial effects. Chronic treatment with DMI produced a robust increase in tonic extracellular NE levels in LS. Given the presumed role of tonic NE transmission in arousal, vigilance and cognitive function (see Aston-Jones et al., 1991; Berridge and Waterhouse, 2003; Morilak et al., 2005), it seems likely that this elevation of noradrenergic tone contributes to the antidepressant effect of DMI and other NE reuptake blockers. However, the decrease in relative activation of NE release by shock probe exposure also indicates that changes in acute NA stress reactivity following chronic DMI treatment may play a role in the anxiolytic efficacy of NRIs (e.g., Sasson et al., 1999; Versiani et al., 2002). Preclinical as well as clinical studies suggest that it is not simply an elevation of NE levels per se, but rather an increased ‘volatility’ or reactivity of the NA system that is most closely associated with stress-induced anxiety-like behavior (Cecchi et al., 2002a,b; Pardon et al., 2002), and with clinical anxiety disorders (Sullivan et al., 1999). Thus, an attenuation of the phasic NE response to acute stress could lead to an anxiolytic-like behavioral effect. Our data would seem to suggest that tonic elevation of extracellular levels of NE following chronic DMI treatment may function to “clamp” the acute “volatility” or stress reactivity of the NA system to produce an anxiolytic effect. Hence, chronic treatment with a selective NE reuptake blocker may differentially regulate tonic NE levels and phasic, stress-activated NE neurotransmission, contributing to the dual behavioral effects of these drugs in the context of depression and anxiety. However, our data also suggest that attenuation of acute NA reactivity alone cannot account for the anxiolytic effect of DMI, as a similar attenuation of shock probe-evoked NE release was observed following acute DMI administration. Moreover, although the relative increase in NE release induced by shock probe exposure, expressed as a percent of baseline, was attenuated following chronic DMI treatment, the absolute level of NE release provoked by the shock probe was still significant and substantial. It is not obvious a priori whether the absolute change in NE levels, or the relative change with respect to baseline is more important in evoking a post-synaptic response, and ultimately a behavioral response. At any rate, it seems likely that regulatory changes in post-synaptic receptor response to NE may also be involved in the time-dependent changes in behavioral reactivity produced by chronic DMI treatment. Indeed, the extended time course required for behavioral efficacy of AD drugs, the so-called “therapeutic lag”, is a key feature that must be considered in attempting to understand their mechanisms of action (Gelenberg and Chesen, 2000). Although immediate effects are noticeable on transporter function

(Nestler, 1998; Ressler and Nemeroff, 1999), specific symptomatic improvements become evident only after at least 1– 2 weeks of treatment, whereas significant clinical improvement and recovery occur after several weeks or months (Blier and de Montigny, 1994; Frazer, 1994; Katz et al., 1996/1997, 2004). Thus, any model useful for investigating potential mechanisms of AD action should exhibit an aspect of time-dependency. The results presented in this study show the time-dependent effect of chronic DMI treatment on acute stress-evoked NE release and acute behavioral reactivity in rats. The active defensive burying behavior elicited by and directed towards the stressful stimulus, i.e. the shock-probe, was significantly reduced following 2 or more weeks of chronic DMI treatment, which is consistent with the clinical “therapeutic lag” previously described in the literature. This suggests that the regulatory changes in NA neurotransmission induced by chronic DMI treatment may be responsible in part for the time-dependent effects on anxietylike behaviors. It is unlikely that the decrease in burying behavior after chronic DMI treatment was attributable solely to a non-selective decrease in locomotion. First, there was no change in active burying behavior until 14 days DMI treatment, even though comparable plasma drug levels were achieved at the earlier time points, including after acute administration. Also, although we did not test locomotor activity directly, the lack of change in approach latency in DMI-treated rats would argue against a nonselective decrease in locomotion prior to receiving the shock. Previous studies have also shown that locomotor effects of DMI can be dissociated from anxiolytic effects (Fernandez-Guasti et al., 1999). Finally, in the forced swim test, DMI has the opposite effect on locomotion, increasing activity and reducing immobility (Detke et al., 1995), again dissociating antidepressant/anxiolytic from motor effects. Thus, chronic DMI treatment reduced the active defensive burying response, in agreement with a previous report (Fernandez-Guasti et al., 1999). A variety of responses to many other pharmacologic agents on the SPDB test have been reviewed by De Boer and Koolhaas (2003). Responses to several putative or proven anxiolytic agents were similar to those observed in the present paper after chronic DMI. For example, the benzodiazepine, chlordiazepoxide, also decreased burying, increased immobility, and decreased the CORT response (De Boer et al., 1990). Serotonergic anxiolytic agents, such as 8-OHDPAT, buspirone or ipsaparone (e.g., Korte et al., 1992a), also decreased burying, did not change or also increased immobility, but did not change the HPA response. The observation that the ACTH response was reduced in DMI-treated animals, despite the fact that these rats showed less burying, would indicate either that reduced noradrenergic reactivity also reduced facilitation of the HPA response to acute stress, or that the subjective impact, or perceived “anxiogenic magnitude” of the stressor was reduced. Thus, the switch from active burying to immobility did not indicate that DMI had evoked a maladaptive strategy, a possibility raised by the greater ACTH response seen in rats that were not allowed to bury in experiment 1b. Likewise, if DMI had induced an increase in subjective stress, pushing the rats to freeze rather than adopt an

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active strategy, we would have expected to see an increase in ACTH in the DMI-treated animals. Thus, the shock-probe stimulus was still stressful, as it provoked an immobility response, but the magnitude of the perceived stress was apparently reduced, even when the animals froze rather than bury. In other words, as assessed by the ACTH response, DMI-treated rats most resembled rats that were not driven to bury, rather than rats that were not able to bury. By contrast, a deficit of noradrenergic modulatory activity, either determined genetically or caused, for instance, by chronic or severe stress exposure (Anisman and Zacharko, 1986), may hinder the ability of an animal to mount an effective behavioral response and to cope adequately with subsequent acute stress. For example, the inbred Wistar Kyoto (WKY) rat strain has been shown to be particularly vulnerable to the detrimental effects of stress compared to outbred comparator strains such as the Wistar or Sprague-Dawley rat (Pare and Redei, 1993; Redei et al., 1994). WKY rats show an elevated and prolonged activation of the HPA axis (Pardon et al., 2002, 2003; Pare and Redei, 1993; Redei et al., 1994), and also exhibit an extreme tendency to adopt passive rather than active behavioral response strategies in a variety of test situations, including increased immobility and reduced behavioral activity in the open-field, elevated plus-maze, forced swim test, passive and active avoidance tests, learned helplessness paradigm, and relevant to the present study, the shockprobe defensive burying test (Berton et al., 1997; Lahmame et al., 1997; Lopez-Rubalcava and Lucki, 2000; Pardon et al., 2002; Pare, 1994; Pare and Redei, 1993; Ramos et al., 1997). We have found that acute stress-induced activation of the noradrenergic system is attenuated in WKY compared to Sprague-Dawley or Wistar rats (Pardon et al., 2002, 2003; Sands et al., 2000). We have hypothesized that this deficit in acute noradrenergic reactivity may contribute to the passive behavioral response tendency of this strain, which may in turn reduce their ability to cope adequately with acute stress, thereby increasing the physiological impact of stress, resulting in the exaggerated HPA response and ultimately to their increased vulnerability to stress-related pathology. Consistent with this suggestion, the results of the present study indicate that the active defensive burying response, which was facilitated by noradrenergic activity in the LS, was effective in limiting activation of the HPA axis by exposure to the shock probe. The animals that were not allowed to bury exhibited a greater and longer lasting elevation of plasma ACTH, suggesting that they experienced a greater degree of stress. Similar findings have been reported previously (De Boer et al., 1990; Korte et al., 1992b). Also consistent with these observations, it has been reported in both animal research studies and in the human clinical literature that an active, stimulus-based or problem-oriented coping style, as opposed to a more passive, emotion-based, or avoidant coping style, buffers the duration and degree of HPA activation (Koolhaas et al., 1999; Olff et al., 2005), increases the effectiveness of coping in eliminating the threat or overcoming the challenge, and improves long-term mental and physical health outcomes (Charney, 2004; LeDoux and Gorman, 2001; Olff et al., 2005).

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5. Conclusion The tendency to mount an active, problem-oriented response strategy as opposed to a more passive, emotion-oriented response results from a complex interplay of genetic predisposition and cognitive perceptual processes involving the subjective appraisal of both stimulus and response (e.g., see Olff et al., 2005). Cognitive processes such as the perception and interpretation of a stressor as being controllable or uncontrollable are central to the early notion of learned helplessness and the development of stress-related psychiatric disorders (Maier and Seligman, 1976; Maier and Watkins, 1998; Weiss et al., 1980, 1979). Thus, the facilitatory influence of the noradrenergic system is but one component of a neural stress circuit promoting an active, adaptive coping response, whether “proactive”, as in the defensive burying test, or “reactive”, as in the elevated plusmaze and social interaction tests (see Koolhaas et al., 1999). By contrast, inappropriate, ineffective or disproportionate activation of a noradrenergic stress response may contribute to the etiology or symptomatology of stress-related psychiatric illness, such as depression, PTSD or other anxiety disorders (Gold and Chrousos, 1999; Schatzberg and Schildkraut, 1995; Southwick et al., 1993; Sullivan et al., 1999). Therefore, understanding the modulatory role of the brain noradrenergic system in determining acute behavioral and neuroendocrine stress response strategies, the long-term regulation of this system, both normal and maladaptive, and the mechanisms by which chronic treatment with NRIs and other classes of antidepressants can regulate monoaminergic function to produce their clinical efficacy, may aid in the future development of better approaches for the prevention or treatment of such stress-related illness. Acknowledgements Expert technical assistance was provided by Angelica Hernandez, Selinda Salazar and Elizabeth Rubino. Support was provided by research grants from the National Institutes of Health (MH53851 and MH72672). The authors have no conflicts of interest to report, nor any involvement to disclose, financial or otherwise, that may bias the conduct, interpretation or presentation of this work. References Anisman H, Zacharko RM. Behavioral and neurochemical consequences associated with stressors. Ann N Y Acad Sci 1986;467:205–25. Antonopoulos J, Latsari M, Dori I, Chiotelli M, Parnavelas JG, Dinopoulos A. Noradrenergic innervation of the developing and mature septal area of the rat. J Comp Neurol 2004;476:80–90. Aston-Jones G, Chiang C, Alexinsky T. Discharge of noradrenergic locus coeruleus neurons in behaving rats and monkeys suggests a role in vigilance. Prog Brain Res 1991;88:501–20. Aston-Jones G, Rajkowski J, Cohen J. Role of locus coeruleus in attention and behavioral flexibility. Biol Psychiatry 1999;46:1309–20. Barrera G, Mahan A, Petre CO, Morilak DA. Time-dependent effects of chronic DMI treatment on shock-probe defensive burying in rats. Neuropsychopharmacology 2004;29(Suppl. 1):S157. Berridge CW, Waterhouse BD. The locus coeruleus-noradrenergic system: modulation of behavioral state and state-dependent cognitive processes. Brain Res Rev 2003;42:33–84.

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