Behavioral/Systems/Cognitive GABAB Receptor Modulation of Serotonin Neurons in the Dorsal Raphe �� Nucleus and Escalation of Aggression in Mice Aki Takahashi,1,2 Akiko Shimamoto,1 Christopher O. Boyson,1 Joseph F. DeBold,1 and Klaus A. Miczek1,3 1Department of Psychology, Tufts University, Medford, Massachusetts 02155, 2Mouse Genomics Resource Laboratory, National Institute of Genetics, Mishima, Shizuoka 411-8540, Japan, and 3Departments of Neuroscience, Pharmacology, and Psychiatry, Tufts University, Boston, Massachusetts 02111 The serotonin (5-HT) system in the brain has been studied more than any other neurotransmitter for its role in the neurobiological basis of aggression. However, which mechanisms modulate the 5-HT system to promote escalated aggression is not clear. We here explore the role of GABAergic modulation in the raphe �� nuclei, from which most 5-HT in the forebrain originates, on escalated aggression in male mice.PharmacologicalactivationofGABAB ,butnotGABAA ,receptorsinthedorsalraphe��nucleus(DRN)escalatedaggressivebehaviors. In contrast, GABA agonists did not escalate aggressive behaviors after microinjection into the median raphe �� nucleus. The aggression- heightening effect of the GABAB agonist baclofen depended on the activation of 5-HT neurons in the DRN because it was blocked by coadministrationofthe5-HT1A agonist8-OH-DPAT[(( )-8-hydroxy-2-(di-n-propylamino)tetralin)hydrobromide](DPAT),whichacts on autoreceptors and inhibits 5-HT neural activity. In vivo microdialysis showed that GABAB activation in the DRN increased extracel- lular 5-HT level in the medial prefrontal cortex. This may be attributable to an indirect action via presynaptic GABAB receptors. The presynaptic GABAB receptors suppress Ca 2 channel activity and inhibit neurotransmission, and the coadministration of N-type Ca 2 channel blocker facilitated the effect of baclofen. These findings suggest that the indirect disinhibition of 5-HT neuron activity by presynaptic GABAB receptors on non-5-HT neurons in the DRN is one of the neurobiological mechanisms of escalated aggression. Introduction Aggressive behavior is phylogenetically conserved, typically en- hancing an individual���s survival and reproductive success. How- ever, excessive levels of aggression become maladaptive (Miczek et al., 2007). Escalated aggression has been an enormous problem in public health as highlighted by 2.3 million violence-related injury visits to emergency departments annually in the United States (Pan American Health Organization, 2007). Serotonin (5-HT) is one of the major neurotransmitters that has been linked to escalated aggression in species ranging from invertebrates to humans (Miczek et al., 2004 Olivier, 2004 de Boer and Koolhaas, 2005 Coccaro et al., 1997). 5-HT in the mammalian CNS derives mainly from the midbrain raphe �� nuclei. Especially, the dorsal raphe �� nucleus (DRN) contains the largest accumulation of 5-HT neuronal cell bodies (Dahlstro ��m and Fuxe, 1964), which project to several targets, including the limbic structures and cortex (Azmitia and Segal, 1978 Michelsen et al., 2007). Lesions, pharmacological manipulations, and electro- physiological recordings link the DRN to aggressive and defen- sive behaviors in rodents, cats, and tree shrews (Jacobs and Cohen, 1976 Vergnes et al., 1986 Koprowska and Romaniuk, 1997 Sijbesma et al., 1991 Bannai et al., 2007 Faccidomo et al., 2008 van der Vegt et al., 2003 Mos et al., 1993 Walletschek and Raab, 1982). The DRN���5-HT system is modulated by other amines, acids, peptides, and steroids (Adell et al., 2002), but the nature of the neural systems modulating 5-HT neuronal activity to promote escalated aggression has not yet been determined. Many GABA interneurons and distal GABAergic afferents can be found in the DRN (Nanopoulos et al., 1982 Belin et al., 1983 Gervasoni et al., 2000 Wang et al., 1992), and both GABAA and GABAB receptors are expressed in the DRN (Bowery et al., 1987). Activation of both the GABAA and GABAB receptors inhibits 5-HT cell firing (Innis and Aghajanian, 1987 Gallager and Aghajanian, 1976 Judge et al., 2004 Colmers and Williams, 1988). In vivo microdialysis studies have shown, however, that the GABAA and GABAB receptors in the DRN differentially modulate 5-HT re- lease depending on the projection sites (Tao et al., 1996). GABAB receptor agonist microinjection in the DRN can induce either increases or decreases of 5-HT neuronal activity (Tao et al., 1996 Abella ��n et al., 2000). Therefore, 5-HT neurons in the DRN are differentially modulated by GABAA and GABAB receptors. Here, we examine the GABAergic modulation of the DRN underlying escalated aggression. We demonstrate that the phar- macological activation by GABAB receptors, but not GABAA re- ceptors, in the DRN, but not median raphe �� nucleus (MRN), escalates aggressive behaviors in male mice. Our data suggest that presynaptic GABAB receptors on non-5-HT neurons are respon- sible for the escalation of aggressive behaviors. The prefrontal cortex (PFC), one of the projection sites of DRN 5-HT neurons, has been implicated in impulsive aggression (Davidson et al., 2000), and we found that GABAB receptor activation in the DRN increased 5-HT release in the medial PFC (mPFC). Our results provide evidence that the GABAB receptor modulation in the Received April 8, 2010 revised July 9, 2010 accepted July 13, 2010. This research was supported by National Institute on Alcohol Abuse and Alcoholism Grant AA13983 and a Tufts University Center for Neuroscience Research grant. We thank Andrea T. Henry and Jisoo Kim for their great help. Correspondence should be addressed to Klaus A. Miczek, Tufts University, Bacon Hall, 530 Boston Avenue, Medford, MA 02155. E-mail: klaus.miczek@tufts.edu. DOI:10.1523/JNEUROSCI.1814-10.2010 Copyright �� 2010 the authors 0270-6474/10/3011771-10$15.00/0 The Journal of Neuroscience, September 1, 2010 ��� 30(35):11771���11780 ��� 11771

DRN is one of the neurobiological mechanisms underlying esca- lated aggression in mice. Materials and Methods Mice. Male CFW mice (Charles River Laboratories), weighing 21���23 g on arrival, were housed in pairs with females in a clear polycarbonate cage (28 17 14 cm) with pine shavings as bedding material. Additional males used as intruders were housed in groups of 7���10 per cage (48 26 14 cm) with corncob bedding. All animals were maintained in our vivarium with controlled humidity and temperature (35���40%, 21 1��C) on a reversed 12 h light/dark cycle (lights off at 7:00 AM) with water and food (Purina) available ad libitum. All procedures were approved by the Institutional Animal Care and Use Committee of Tufts University. For the antagonist experiment, mice of the ICR strain (CLEA Japan), weighing 21���23 g on arrival, were tested in the National Institute of Genetics (Mishima, Japan). The animals were cared for according to the Guide for the Care and Use of Laboratory Animals (National Research Council, 1996). Resident-intruder test training. After being housed with a female for 3 weeks, the resident males were studied for their aggression toward the same intruder (Miczek and O���Donnell, 1978). Immediately after the fe- male and pups were removed, an intruder was introduced into the home cage of the resident male. Their behaviors were recorded for 5 min after the first attack bite, or the intruder was removed after 5 min if no attack occurred. This encounter occurred once every other day until animals showed a stable number of attack bites, and stability was reached within 7���16 encounters [coefficient of variation ( / ) in the last three confron- tations reached 0.2 for each animal]. All behavioral tests were per- formed in the dark period (11:00 A.M. to 2:00 P.M., in the reversed light/dark cycle). Surgery and cannulation. Once aggressive behavior had stabilized, res- idents were anesthetized by intraperitoneal injection of a mixture of 100 mg/kg ketamine HCl and 10 mg/kg xylazine and stereotaxically im- planted with a 26 gauge guide cannula (Plastics One) aimed 2 mm above the DRN [anteroposterior (AP), 4.2 mm mediolateral (ML), 1.5 mm dorsoventral (DV), 1.9 mm to bregma angled 26�� to the vertical] or MRN (AP, 4.2 mm ML, 1.2 mm DV, 3.0 mm to bregma angled 14�� to the vertical) as calculated from a mouse brain atlas (Paxinos and Franklin, 2001). A 33 gauge obdurator (Plastics One) that extended 0.5 mm beneath the guide cannula tip was inserted after surgery. The obdu- rator was moved daily to prevent blockage and scarring and also to ha- bituate the animals to handling. After surgery, animals were housed individually for 5 d to recover and then pair housed with the same female. To prevent gnawing by the female, the obdurator and head mount were covered with a quinine preparation (Bite it). One week after surgery, residents were assessed for fighting at least three times before starting the microinjection experiment. Most of the animals showed similar levels of aggressive behavior after the cannulation surgery. Animals that stopped fighting after the surgery were excluded from the microinjection test (Table 1). Microinjection and aggression test. The obdurator was removed, and a 33 gauge microinjector (Plastics One) attached to polyethylene tubing (Intramedic PE-50) was inserted into the guide cannula. The microinjec- tor extended 2 mm below the end of the guide to reach the DRN or MRN. The other end of the tubing was connected to a 1 l Hamilton syringe placed into an infusion pump (CMA Microdialysis). The drug was in- fused in a volume of 0.2 l over 2 min, and the microinjector was left in place for 1 min after the infusion to allow the drug to diffuse completely. Ten minutes after the microinjection, an intruder male was introduced and aggressive behaviors were recorded for 5 min after the first attack bites. A resident male received a total of four to six microinjections, including two vehicle injections, in an irregular order. The drugs, doses, and number of animals for each experiment are summarized in Table 1. Histology. At the end of the experiment, mice were deeply anesthetized (ketamine and xylazine mixture) and intracardially perfused with 0.9% saline, followed by 4% paraformaldehyde (PFA) in PBS. After postfix- ation in 4% PFA for at least 24 h, brains were placed into 15% sucrose solution. A freezing microtome was used to slice the brains into 60 m sections, which were stained with cresyl violet to verify the placements of the cannula and microdialysis probe. The injection site for each animal is depicted in Figure 1 and supplemental Figures S1 and S2 (available at www.jneurosci.org as supplemental material). Drugs. Muscimol (5-aminomethyl-3-hydroxy-isoxazole), baclofen [( )- -(aminomethyl)-4-chlorobenzenepropanoic acid], phaclofen [3- amino-2-(4-chlorophenyl)propanephosphonic acid], 8-OH-DPAT [(( )-8-hydroxy-2-(di-n-propylamino)tetralin) hydrobromide], and -conotoxin GVIA ( GVIA) were purchased from Sigma-Aldrich. CGP54626 ([S-(R*,R*)]-[3-[[1-(3,4-dichlorophenyl)ethyl]amino]-2- hydroxypropyl] (cyclohexylmethyl) phosphinic acid) was obtained from Tocris Bioscience. All drugs were dissolved in 0.9% saline except 8-OH-DPAT and -conotoxin GVIA, which were dissolved in artifi- cial CSF. Extracellular 5-HT measurement in the mPFC. Nine male mice were housed in pairs with a female for 3 weeks before the microdialysis exper- iment. Males were implanted with a CMA/7 guide cannula (CMA Mi- crodialysis) aimed 2 mm above the mPFC (AP, 2.6 mm ML, 0.3 mm DV, 0.8 mm to bregma) and a 26 gauge guide cannula (Plastics One) aimed 2 mm above the DRN (AP, 4.2 mm ML, 1.5 mm DV, 1.9 mm to bregma angled 26�� to the vertical). After 1 week of recovery with handling, a CMA/7 probe of 2 mm active membrane was inserted into the mPFC under isoflurane inhalation anesthesia. The probe was infused overnight with artificial CSF at a flow rate of 0.5 l/min using an infusion Table 1. Number of animals used in this study Group Target Experiment Drugs Vehicle Total animals used for analysis Missed placements Excludeda 1 DRN Mixture of muscimol and baclofen Muscimol (0.006 nmol) baclofen (0.06 nmol) Saline 12 0 1 2 DRN Muscimol or baclofen Muscimol (0.06 nmol) Saline 9 0 1 Baclofen (0.06 nmol) 3 DRN Baclofen dose effect Baclofen (0.01, 0.06, 0.10 nmol) Saline 12 3 2 4 DRN Antagonist (phaclofen) Baclofen (0.06 nmol) phaclofen (0, 0.15, 0.3 nmol) Saline 10 0 3 5 DRN Antagonist (CGP54626) Baclofen (0.06 nmol) CGP54626 (0, 0.06, 0.60 pmol) Saline 9 2 4 6 DRN GVIA dose effect GVIA (0.1, 0.3, 3.0 pmol) Artificial CSF 15 (11 DRN, 4 aqueduct) 1 5 7 DRN GVIA and baclofen GVIA (0.1 pmol) baclofen (0.01 nmol) Artificial CSF 13 2 3 8 DRN 8-OH-DPAT and baclofen 8-OH-DPAT (0.9 nmol) baclofen (0.06 nmol) Artificial CSF 15 2 1 9 DRN Baclofen temporal pattern Baclofen (0.06 nmol), 10, 40, 100 min interval Saline 9 1 0 10 DRN Intra-DRN baclofen Baclofen (0.06 nmol) Saline 6 3 0 mPFC 5-HT microdialysis in mPFC 11 MRN Muscimol or baclofen Muscimol (0.06 nmol) Saline 10 5 9 Baclofen (0.06 nmol) 12 DRN Antagonist only (CGP54626) Baclofen (0.06 nmol) Saline 10 4 1 CGP54626 (0.6, 6.0 pmol) aAnimals were excluded from this study because they died after surgery (15), stopped fighting after surgery (7), or could not complete all injections as a result of guide cannula problems (7). In the MRN, three animals continued the turning behavior 1 d after the muscimol injection. 11772 ��� J. Neurosci., September 1, 2010 ��� 30(35):11771���11780 Takahashi et al. ��� GABAB Receptor Modulation in the DRN Escalates Aggression