Ketanserin

All experiments were carried out in accordance with the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources 1996 ) as adopted and promulgated by the National Institutes of Health. Timed-pregnant Sprague-Dawley rats (Charles River, Raleigh, NC) were housed in breeding cages, with a 12-hr light/dark cycle and free access to food and water. On the day of birth, all pups were randomized and redistributed to the dams with a litter size of 10 to maintain a standard nutritional status. Randomization within the respective treatment groups was repeated at intervals of several days; in addition, dams were rotated among litters to distribute any maternal caretaking differences randomly across litters and treatment groups. CPF (Chem Service, West Chester, PA) was dissolved in dimethylsulfoxide to provide consistent absorption (Whitney et al. 1995 (link)) and was injected subcutaneously at a dose of 1 mg/kg in a volume of 1 mL/kg once daily on PND1–4; control animals received equivalent injections of the dimethylsulfoxide vehicle. This regimen has been shown previously to produce neurotoxicity in developing rat brain, including lasting alterations in biomarkers of 5HT synaptic function, without eliciting growth retardation or any other signs of systemic toxicity (Aldridge et al. 2003 (link), 2004 (link)). Indeed, neonatal brain cholinesterase inhibition is only about 25% (Song et al. 1997 (link)), well below the 70% threshold necessary for symptoms of cholinergic hyperstimulation (Clegg and van Gemert 1999a ), thus resembling the nonsymptomatic exposures reported in pregnant women (De Peyster et al. 1993 (link)). Moreover, the dose used here is well within the range of typical fetal and childhood exposures after routine application (Gurunathan et al. 1998 (link); Ostrea et al. 2002 (link)). Animals were weaned on PND21.
All behavioral testing was carried out during the dark phase, the most active period, but in lighted environments so that the rats could access the visual cues necessary to perform the tasks. Tests were performed on nine rats per sex per treatment group (36 animals in total), using no more than one male and one female from each litter. Beginning on PND47, the light/dark cycle was shifted by 6 hr (lights on at 0000 hr), and assessments were begun with the elevated plus maze (PND52–53) and the chocolate milk consumption test (PND54). On PND57, the light/dark cycle was shifted an additional 6 hr (lights on at 1800 hr), and radial-arm maze training was conducted during a 5-week period beginning on PND64. Ketanserin challenges in the radial-arm maze were undertaken in weeks 16–17. This sequence was designed to permit multiple behavioral tests to be conducted without significant carryover of effects from one test to the next (Icenogle et al. 2004 (link); Levin et al. 2001 (link), 2002 (link); Slotkin et al. 1999 (link)). Tests were videotaped and scored by a trained observer who was blinded to the animal treatments.

The study included a screening visit, six 25-h test sessions (each separated by at least 10 days), and an end-of-study visit. The sessions were conducted in a calm standard hospital room equipped with a standard hospital bed for the participant and a desk and a chair for the investigator. The room had an adjoining balcony, which participants were allowed to access after peak effects had subsided. Only one research subject and one investigator were present during each test session. Participants were allowed to bring their own music and to bring occupation for the time after effects had subsided or for placebo days (e.g. book, laptop, games etc.). Blindfolds were provided upon request. The test sessions began at 7:45 AM. A urine sample was taken to verify abstinence from drugs of abuse, and a urine pregnancy test was performed in women. The subjects then underwent baseline measurements. Ketanserin (40 mg) or placebo was administered at 8:00 AM. LSD or placebo was administered at 9:00 AM. Standardized lunches and dinners were served at 1:30 PM and 6:00 PM, respectively. The subjects were never alone during the first 16 h after drug administration, and the investigator was in a room next to the subject for up to 24 h. The subjects were sent home the next day at 9:15 AM.

Brain regional D2DR-binding densities were examined as described previously [23 (link),26 (link)]. Slide-mounted sections were incubated in Tris-buffer (50 mM Tris–HCl containing 120 mM NaCl; pH 7.4) containing 0.4nM [3H]spiperone (specific activity 109.0 Ci/mmol, Amersham) for 60 min at room temperature. Sections for non-specific binding were incubated in Tris-buffer containing 0.4 nM [3H]spiperone, 10−5 M haloperidol, and 10−5 M ketanserin. After incubation, the slides were drained, washed twice for 5 min in buffer at 4 °C and briefly dipped twice into distilled water (4 °C). Sections were dried at room temperature overnight and exposed to a tritium-sensitive film (3H Hyperfilm^®, Amersham) at −20 °C for 3 weeks, together with Amersham 3H Microscale Autoradiography Standards^®. After the development, all films were digitized, and the images were processed in ImageJ.