1h). 4. revealed that 5,7-DHT reduced local 5-HT concentrations in the BLC by 40% without altering local norepinephrine or dopamine concentrations. These data provide additional support for 5-HT playing a critical role in modulating anxiety-like behavior and fear-associated memories through its actions within the BLC. 0.05 for bar graphs and an independent 2-tailed Student’s 0.05 guarded by a one way ANOVA with repeated measures for line graphs (n = 8,11). eCf) Schematic representations of the bilateral injection sites as determined by histology for HPLC steps of monoamines in micropunched BLC/CeA and DRN (n = 3,6) and immunohistochemistry of SERT-ir fibers in BLC (n = 5,5). Injection site placements are illustrated as symbols (with black circle indicating vehicle injections, and green squares indicating 5,7-DHT injections). Illustrations of coronal brain sections are based on the rat brain atlas of Paxinos and Watson (1997). Numbers to bottom right of the section indicate the distance posterior from bregma; the vertical scale on the right of the section represents the distance ventral from bregma (in mm). The basolateral amygdala complex (BLC) consists of the lateral amygdaloid nucleus (LA) and basolateral amygdaloid nucleus (BL)). Solid lines represent white matter tracts and dashed lines illustrate subdivisions of the BLC. Abbreviations: BL, basolateral amygdaloid nucleus; CeA, central amygdaloid nucleus; ec, external capsule; LA, lateral amygdaloid nucleus; opt, optic tract. g) Bar graph illustrates concentrations of 5-HT, norepinephrine (NE), and dopamine (DA) in the BLC/CeA, and 5-HT in the dorsal raphe nucleus (DRN). h) Two representative photomicrographs from the BLC region from a vehicle-injected rat (left) and a 5,7-DHT-injected rat (right). 2.8. Immunostaining the serotonin transporter in the BLC In Experiment 2, immunostaining for the serotonin transporter (SERT) was done around the 30 m coronal brain sections of perfused rats to determine if 5,7-DHT had effectively lesioned local serotonergic fibers and terminals. Immunostaining for SERT using a primary antibody directed against SERT (rabbit anti-SERT polyclonal serum antibody, cat. no. 24,330, Immunostar, Hudson WI, USA; diluted 1:1500). Free-floating sections were washed in 0.05 M PBS for 30 min, then incubated in 1% H2O2 in PBS for 20 min. Sections were then washed 10 min in PBS and 20 min in PBS with 0.3% Triton X-100 (PBST). Sections were then incubated 12-16 h in PBST with primary antibody answer at room heat. After a 30-minute wash in PBST, sections were incubated 2 h in the biotinylated swine anti-rabbit IgG secondary antibody (cat no. BA-1000, Vector Laboratories, Burlinghame, CA, USA; diluted 1:500). Sections were 2”-O-Galloylhyperin washed again for 30 min in PBST then incubated 1.5 h in an avidin-biotin complex provided in a standard Vector Elite kit (cat no. PK-6100, Vector Laboratories; diluted 1:500). Substrates for chromogen reactions were SG (SK-4700, Vector Laboratories, Burlingame, CA, USA) in PBS made up of 0.003% H2O2, pH 7.4. Substrate reactions were run for 15 min. All sections were mounted on clean glass slides, dried overnight, dehydrated and mounted with coverslips using DPX mounting medium (Sigma-Aldrich, St. Louis, MO, USA). All washes and incubations were done in 12-well polystyrene plates with 2”-O-Galloylhyperin low frequency shaking on an orbital shaker. 2.8.1. Photography Photomicrographs were obtained using a Leica brightfield microscope using N plan 5, 10, 20 and 40 objective lenses (model DMLB, Leica Microsystems, Buffalo Grove, IL, USA), a SPOT digital camera (RT color, Diagnostics Devices Inc., Sterling Heights, MI, USA) and SPOT 4.0.6 for Windows digital imaging software (Silicon Graphics, Mountain View, CA, USA) or a Nikon 90i microscope and a Nikon DS-Fi1 digital camera with NIS Elements.These data provide the first evidence showing the impairment of fear acquisition due to reduced 5-HT levels within the BLC. interpersonal interaction (SI) time, suggestive of reduced anxiety-like behavior. We then used a cue-induced fear conditioning protocol with shock as the unconditioned stimulus and tone as the conditioned stimulus for rats pretreated with bilateral 5,7-DHT, or vehicle, injections into the BLC. Compared to vehicle-treated rats, 5,7-DHT rats had reduced acquisition of fear during conditioning (measured by freezing time during tone), also had reduced fear retrieval/recall on subsequent testing days. Ex vivo analyses revealed that 5,7-DHT reduced local 5-HT concentrations in the BLC by 40% without altering local norepinephrine or dopamine concentrations. These data provide additional support for 5-HT playing a critical role in modulating anxiety-like behavior XE169 and fear-associated memories through its actions within the BLC. 0.05 for bar graphs and an independent 2-tailed Student’s 0.05 guarded by a one way ANOVA with repeated measures for line graphs (n = 8,11). eCf) Schematic representations of the bilateral injection sites as determined by histology for HPLC steps of monoamines in micropunched BLC/CeA and DRN (n = 3,6) and immunohistochemistry 2”-O-Galloylhyperin of SERT-ir fibers in BLC (n = 5,5). Injection site placements are illustrated as symbols (with black circle indicating vehicle injections, and green squares indicating 5,7-DHT injections). Illustrations of coronal brain sections are based on the rat brain atlas of Paxinos and Watson (1997). Numbers to bottom right of the section indicate the distance posterior from bregma; the vertical scale on the right of the section represents the distance ventral from bregma (in mm). The basolateral amygdala complex (BLC) consists of the lateral amygdaloid nucleus (LA) and basolateral 2”-O-Galloylhyperin amygdaloid nucleus (BL)). Solid lines represent white matter tracts and dashed lines illustrate subdivisions of the BLC. Abbreviations: BL, basolateral amygdaloid nucleus; CeA, central amygdaloid nucleus; ec, external capsule; LA, lateral amygdaloid nucleus; opt, optic tract. g) Bar graph illustrates concentrations of 5-HT, norepinephrine (NE), and dopamine (DA) in the BLC/CeA, and 5-HT in the dorsal raphe nucleus (DRN). h) Two representative photomicrographs from the BLC region from a vehicle-injected rat (left) and a 5,7-DHT-injected rat (right). 2.8. Immunostaining the serotonin transporter in the BLC In Experiment 2, immunostaining for the serotonin transporter (SERT) was done around the 30 m coronal brain sections of perfused rats to determine if 5,7-DHT had effectively lesioned local serotonergic fibers and terminals. Immunostaining for SERT using a primary antibody directed against SERT (rabbit anti-SERT polyclonal serum antibody, cat. no. 24,330, Immunostar, Hudson WI, USA; diluted 1:1500). Free-floating sections were washed in 0.05 M PBS for 30 min, then incubated in 1% H2O2 in PBS for 20 min. Sections were then washed 10 min in PBS and 20 min in PBS with 0.3% Triton X-100 (PBST). Sections were then incubated 12-16 h in PBST with primary antibody answer at room heat. After a 30-minute wash in PBST, sections were incubated 2 h in the biotinylated swine anti-rabbit IgG secondary antibody (cat no. BA-1000, Vector Laboratories, Burlinghame, CA, USA; diluted 1:500). Sections were washed again for 30 min in PBST then incubated 1.5 h in an avidin-biotin complex provided in a standard Vector Elite kit (cat no. PK-6100, Vector Laboratories; diluted 1:500). Substrates for chromogen reactions were SG (SK-4700, Vector Laboratories, Burlingame, CA, USA) in PBS made up of 0.003% H2O2, pH 7.4. Substrate reactions were run for 15 min. All sections were mounted on clean glass slides, dried overnight, dehydrated and mounted with coverslips using DPX mounting medium (Sigma-Aldrich, St. Louis, MO, USA). All washes and incubations were done in 12-well polystyrene plates with low frequency shaking on an orbital shaker. 2.8.1. Photography Photomicrographs were obtained using a Leica brightfield microscope using N plan 5, 10, 20 and 40 objective lenses (model DMLB, Leica Microsystems, Buffalo Grove, IL, USA), a SPOT digital camera (RT color, Diagnostics Instruments Inc., Sterling Heights, MI, USA) and SPOT 4.0.6 for Windows digital imaging software (Silicon Graphics, Mountain View, CA, USA) or a Nikon 90i microscope and a Nikon DS-Fi1 digital camera with NIS Elements 3.00 imaging software (A.G. Heinze Inc., Lake Forest, CA, USA). Photographic plates were prepared in CorelDraw 11.633 for Windows (Eden Prairie, MN, USA). 2.9. Statistical analyses The following dependent variables were analyzed using a two-tailed independent Student’s as main factor and time as the repeated measures. In the presence of significant main effects, between-subjects post-hoc tests were conducted using two-tailed independent Student’s 0.05. All statistical analyses were carried out using SPSS 22.0 (SPSS Inc., Chicago, IL, USA) and all graphs were generated using SigmaPlot 12.0 for.