Molecular Psychiatry (2012) 17, 527–536 & 2012 Macmillan Publishers Limited All rights reserved 1359-4184/12 www.nature.com/mp ORIGINAL ARTICLE Basolateral amygdala regulation of adult hippocampal neurogenesis and fear-related activation of newborn neurons ED Kirby1, AR Friedman2, D Covarrubias3, C Ying2, WG Sun2, KA Goosens4, RM Sapolsky5 and D Kaufer1,2 1Helen Wills Neuroscience Institute, University of California-Berkeley, Berkeley, CA, USA; 2Department of Integrative Biology, University of California-Berkeley, Berkeley, CA, USA; 3Department of Molecular and Cell Biology, University of California-Berkeley, Berkeley, CA, USA; 4McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA, USA and 5Department of Biological Sciences, Neurology and Neurological Sciences, Stanford, CA, USA Impaired regulation of emotional memory is a feature of several affective disorders, including depression, anxiety and post-traumatic stress disorder. Such regulation occurs, in part, by interactions between the hippocampus and the basolateral amygdala (BLA). Recent studies have indicated that within the adult hippocampus, newborn neurons may contribute to support emotional memory, and that regulation of hippocampal neurogenesis is implicated in depressive disorders. How emotional information affects newborn neurons in adults is not clear. Given the role of the BLA in hippocampus-dependent emotional memory, we investigated whether hippocampal neurogenesis was sensitive to emotional stimuli from the BLA. We show that BLA lesions suppress adult neurogenesis, while lesions of the central nucleus of the amygdala do not. Similarly, we show that reducing BLA activity through viral vector-mediated overexpression of an outwardly rectifying potassium channel suppresses neurogenesis. We also show that BLA lesions prevent selective activation of immature newborn neurons in response to a fear-conditioning task. These results demonstrate that BLA activity regulates adult hippocampal neurogenesis and the fear context-specific activation of newborn neurons. Together, these findings denote functional implications for proliferation and recruitment of new neurons into emotional memory circuits. Molecular Psychiatry (2012) 17, 527–536; doi:10.1038/mp.2011.71; published online 14 June 2011 Keywords: neurogenesis; hippocampus; fear conditioning; basolateral amygdala; emotion; stem cell Emotion strongly modulates memory function in campal output to other brain areas.11 BLA activity also adult mammals, altering the strength and longevity modulates activation of hippocampal immediate early of memories and sometimes leading to memory genes, another marker of hippocampal plasticity,12 as dysfunction. Notably, disordered emotional memory well as hippocampal responses to stress,13,14 further contributes to several affective disorders, including suggesting a modulatory role for the BLA in hippo- depression, anxiety and post-traumatic stress disor- campal function. der. Emotion-associated modulation of memory Recent studies suggest a role for adult hippocampal appears to rely largely on interaction between the neurogenesis in emotional memory function.15–19 In memory processes of the hippocampus and emotional adult mammals, new hippocampal neurons arise from input provided by the basolateral complex of the a resident population of neural stem cells located in amygdala (BLA).1–4 Behaviorally, BLA activity sup- the dentate gyrus20–23 and form a population of ports memory for emotionally salient stimuli in immature neurons that incorporate into existing rodents and humans,1,5,6 whereas at the neurophysio- networks within weeks of birth.24 Ablation of adult logical level, BLA activity supports hippocampal neurogenesis by genetic knockout or irradiation long-term potentiation (LTP)7–10 and enhances hippo- impairs contextual fear memory15,16 and may also modulate the transfer of fear-related memories from the hippocampus to other neural structures for long- Correspondence: Dr D Kaufer, Integrative Biology, University of term storage.17 The heightened plasticity of newly California-Berkeley, 3060 Valley Life Sciences Building #3140, born neurons appears to be important, as acceleration Berkeley, CA 94720, USA. of their maturation impairs fear learning.25 The E-mail: [email protected] Received 7 February 2011; revised 9 May 2011; accepted 10 May requirement of new neurons for fear memory suggests 2011; published online 14 June 2011 a clinical role for adult neurogenesis in several Amygdala and adult hippocampal neurogenesis ED Kirby et al 528 affective disorders.3,26,27 However, the mechanisms by anti-BrdU, 1:500, BD Biosciences, San Jose, CA, which adult neurogenesis responds to emotional USA). Sections were rinsed and transferred to stimuli to influence memory formation are not yet secondary antibody (biotin anti-mouse, 1:500, Jackson clear. ImmunoResearch, West Grove, PA, USA) for 2 h at We explored the adult neurogenic response to room temperature. Following rinsing, sections were emotional input from the BLA and its potential role incubated in ABC reagent (Vector, Burlingame, CA, in fear memory. Specifically, we investigated how USA) and then developed with DAB (Vector). Sections BLA activity would affect adult neurogenesis and were mounted on gelatin-coated slides, dehydrated in modulate activation of immature neurons in response alcohol and coverslipped with permount mounting to fear-associated context. medium (Fisher Scientific, Waltham, MA, USA). Triple immunohistochemistry was performed simi- Materials and methods larly. Primary antibodies were: goat anti-DCX (1:200, Santa Cruz Biotechnology, Santa Cruz, CA, USA), Animals mouse anti-S100b (1:200, Abcam), rat anti-MBP Adult male Sprague–Dawley rats (Charles River, (1:100, Abcam), rabbit anti-cfos (1:100; Santa Cruz Wilmington, MA, USA) were pair-housed on a 12-h Biotechnology). Secondary antibodies were: Cy5 anti- light/dark cycle. All animal procedures were ap- goat, Cy3 anti-mouse, Cy3 anti-rat, biotin anti-rabbit proved by the UC Berkeley and MIT Animal Care (1:500; Jackson ImmunoResearch). For the cFos/ and Use Committees. BrdU/doublecortin (DCX) triple stain, a tertiary incubation for 1 h at room temperature was included Stereotaxic surgery with streptavidin-Alexa488 (1:1000 in TBS, Invitro- Excitotoxic lesions of the BLA or central nucleus of gen, Carlsbad, CA, USA) after secondary incubation. the amygdala (CeA) or sham surgeries were performed All sections were then incubated in 4% paraformal- as explained earlier.28.Coordinates for BLA infusion dehyde for 10 min, rinsed and incubated in primary were: À2.8 mm anterior/posterior, ±5.1 mm medial/ antibody against BrdU as above (1:500, rat anti-BrdU, lateral relative to bregma; À6.8 mm (2 min) and Abcam; mouse anti-BrdU, BD Biosciences). The next À6.5 mm (1 min) relative to dura. Coordinates for day, sections were rinsed and incubated in secondary CeA infusion were: À2.2 mm anterior/posterior and antibody: FITC anti-rat; Cy3 anti-rat; biotin anti-rat ±4.4 mm medial/lateral relative to bregma; À7.0 mm (1:500, Jackson ImmunoResearch). For myelin basic from dura (1 min). At 6 to 8 h after surgery, all rats protein (MBP)/BrdU staining, a tertiary incubation for received an additional injection of buprenorphine 1 h at room temperature was added with streptavidin- (0.05 mg kgÀ1, s.c.). For viral vector infusions, virus Alexa488 (1:1000 in TBS, Invitrogen). Sections were was infused 0.2 ml minÀ1 for 10 min (2 ml total) at the mounted on gelatin-coated slides and coverslipped same BLA coordinates. Viral vectors were prepared with DABCO anti-fading medium (Fisher Scientific). as previously explained29 and titers were 106–108 infectious particles per ml. BrdU, PCNA and cFos quantification BrdU-, PCNA- or cFos-positive cells were counted in Bromodeoxyuridine injections the dentate gyrus and subgranular zone using a  40 5-Bromo-20-deoxyuridine (BrdU, Sigma, St Louis, air objective (Zeiss, Oberkochen, Germany). The area MO, USA) was dissolved in physiological saline and sampled was calculated using StereoInvestigator injected intraperitoneally for all experiments. software (MicroBrightfield, Williston, VT, USA) and used to calculate the number of positive cells per mm2. Immunohistochemical staining Rats were anesthetized with Euthasol euthanasia Confocal analysis solution (Virbac, Fort Worth, TX, USA) and trans- A total of 25–50 BrdU- or cFos-positive cells were cardially perfused with ice-cold 4% paraformaldehyde located in the dentate gyrus for each animal (or each in 0.1 M phosphate-buffered saline. Brains were post- hemisphere for unilateral lesion) and assessed in fixed for 24 h at 4 1C, equilibrated in 30% sucrose in z-series of < 1.0 mm slices to determine if other markers 0.1 M phosphate-buffered saline and then stored at (DCX, S100b, MBP, cFos, BrdU) were co-expressed. À80 1C. Immunostaining was performed on a 1 in 6 Confocal images were captured on a Zeiss 510 META/ series of free-floating 30 mm cryostat sections. NLO confocal microscope with a  40 oil objective For proliferating cell nuclear antigen and BrdU and adjusted for brightness and contrast using LSM staining, sections were rinsed in 0.1 M Tris-buffered Image Browser software (Zeiss). saline (TBS) and pretreated for 10 min with 0.3% H2O2 in TBS. For BrdU only, sections were incubated Lesion and viral vector assessment in 2 N HCl at 37 1C for 30 min. All sections were One series of sections was mounted and counter- incubated in blocking solution (1% normal donkey stained