Supporting Figure 5
Supporting Figure 6
Ouyang and Thomas. 10.1073/pnas.0502315102. |
Supporting Materials and Methods
Supporting Figure 5
Supporting Figure 6
Fig. 5. Blocking retrieval during 10-min context exposures on day 1 prevents extinction on day 2. In addition, extinction is independent of norepinephrine/epinephrine (NE/E) on days 5 and 6 when 25-min context exposures are used. (A and B) Extinction of contextual fear is blocked on day 2 when retrieval and adrenergic signaling are impaired during context exposure (10 min) on day 1. For intact retrieval on both days, mice exposed to context S versus N on day 1 froze significantly less on day 2 (P < 0.01). Freezing on day 2 when retrieval was blocked on day 1 was equivalent to freezing after exposure to context N when retrieval was intact on day 1 (P > 0.6). (C) Retrieval and extinction of contextual fear on days 5 and 6 is independent of adrenergic signaling after exposure to context S for 25 min. Within genotype, freezing on day 6 was significantly different depending on day 5 context exposure (P < 0.0001). For all graphs, there were no significant differences by genotype. Data were analyzed with repeated-measures ANOVA with exposure condition, genotype, and day as factors.
Fig. 6.
Central infusions have no effect on retrieval or extinction when administered 5 d after training. (A) Dorsal hippocampus (DH) infusion of (-)-atenolol (Aten) in control mice 15 min before exposure does not block freezing on day 5 or induce extinction on day 6. (B) Intracerebroventricular (ICV) infusion of Aten in control mice 45 min before exposure does not block freezing on day 5 or extinction on day 6. A 10-min context exposure was used on day 5 so that extinction could be observed in the artificial cerebrospinal fluid (aCSF) group on day 6. (C) Central hippocampus (CH) infusion of (±)-isoproterenol (Iso) in Dbh-/- mice 30 min before exposure does not affect freezing on day 5 or induce extinction on day 6. Data were analyzed by ANOVA with treatment and day as factors.Supporting Materials and Methods
Animals.
Studies were in accordance with National Institutes of Health guidelines and had the approval of Institutional Animal Care and Use Committee at the University of Pennsylvania. The mice were hybrids of C57BL/6J and 129/SvCPJ. Dbh+/– females were mated with Dbh–/– males and treated with 100 mg/ml each of phenylephrine and isoproterenol (Sigma) from embryonic day (E)8.5 to E16.5, and 2 mg/ml of l-threo-3,4-dihydroxyphenylserine (L-DOPS, Sumitomo Pharmaceuticals, Osaka) from E16.5 to birth in the maternal drinking water to enhance fetal survival of the Dbh–/– mice (1). Norepinephrine/epinephrine (NE/E) are not essential for survival postnatally, however, so litters were not treated after birth. Sex-matched, littermate Dbh+/– mice were used as controls because tissue content of NE/E is normal in these mice, and no phenotypic differences have been observed in comparison with wild-type mice (2). Mice ranged in age from 3-6 months and included roughly equal numbers of males and females. There were no significant main effects of gender or interaction of gender with other variables, so results from males and females were combined.Conditioning.
Fear conditioning was performed in context S (ENV-010MC, Med Associates, St. Albans, VT). Mice were handled for 3 min/day on the 2-d preceding conditioning. Two contexts not associated with shock were used: a Plexiglas box (30 × 25 cm, 25 cm high) with clear Plexiglas floor and horizontal pink and white stripes on three sides (context N = neutral), and a Plexiglas cylinder (21 cm diameter, 24 cm tall) with green wire grid floor and vertical green and white stripes 240° around (context T = tone testing). Each context was cleaned with a distinctly scented solution and was uniquely situated in the room. Behavior was videotaped, and scoring was performed blind to experiment and treatment. Because Dbh–/– mice exhibit ptosis, scoring was not blind to genotype, although no effort was made to identify genotype.Drugs and Delivery.
Dosing and delivery of drugs were determined and performed as described in ref. 3. L-DOPS (1 g/kg, Sumitomo Pharmaceuticals) plus benserazide (50 mg/kg, Sigma) were administered s.c. 5 h before testing. (-)-Propranolol HCl (1 mg/kg, Sigma), CGP 20712A (1 mg/kg, Sigma) and xamoterol hemifumarate (3 mg/kg, Tocris Cookson, Ellisville, MO) were administered s.c. 30 min (xamoterol 60 min) before testing unless noted. Hippocampal and intracerebroventricular (ICV) infusions were through chronically implanted cannulas by using artificial cerebrospinal fluid (124 mM NaCl/3 KCl/1.25 mM NaH2PO4/1.2 mM MgSO4/26 mM NaHCO3/2 mM CaCl2/10 mM dextrose; Sigma) as a vehicle. A double guide cannula (C235 system, Plastics One, Roanoke, VA) was mounted under pentobarbitol anesthesia (65-70 mg/kg i.p.) by using a stereotax (SAS75/EM40M, Cartesian Research, Sandy, OR) and cyanoacrylate gel (Prism 454, Plastics One). For ICV or dorsal hippocampus (DH) infusions, the guide was placed 0.8 or 1.7 mm caudal to bregma and 1.5 mm bilateral from midline, respectively. The guide extended 1.5 mm from the base, and the dummy extended 0.5 mm below the guide. The dual injection cannula extended 0.9 mm below the guide. For central hippocampus (CH) infusions, single guide cannulas (C315S-4 system, Plastics One) were mounted on a plate that was then mounted to the skull. The guides were placed 3.4 mm caudal to bregma and 3 mm bilateral from midline. The guides extended 1.0 mm from the base, and the dummies were flush with the guides to minimize damage to the underlying neocortex. The injection cannulas extended 3 mm below the guide. One week after recovery, bilateral infusions were made into conscious mice while gently holding the nape of the neck. The injection cannula was left in place for 30 s before the mouse was returned to its home cage.1. Thomas, S. A., Matsumoto, A. M. & Palmiter, R. D. (1995) Nature 374, 643–646.
2. Thomas, S. A., Marck, B. T., Palmiter, R. D. & Matsumoto, A. M. (1998) J. Neurochem. 70, 2468–2476.
3. Murchison, C. F., Zhang, X.-Y., Zhang, W.-P., Ouyang, M., Lee, A. & Thomas, S. A. (2004) Cell 117, 131–143.