Cisternal CSF sample collection and assays

To confirm whether an intramuscular injection of 5-HTP crossed the blood–brain barrier in rhesus macaques and to test whether injections indeed increased central levels of 5-HTP as well as serotonin, we sampled CSF from the cisterna magna from five rhesus monkeys (four male and 1 female; monkeys E, H, T, K, L; age 5–8 years, 5.5 ± 1.2) after they had received an intramuscular injection of saline or 20 mg/kg 5-HTP with a minimum of 2 weeks between each CSF collection date. We counterbalanced and randomized the subject order of CSF sampling between saline and 5-HTP. Each CSF draw occurred 1 h postinjection, the same time after injection that data collection on animals began in the current study.

Cisternal CSF was obtained with cervical punctures, which are preferred over lumbar punctures for accurately tracking concentrations of monoamines and monoamine metabolites in cortical and subcortical structures because of its greater proximity to the brain and its clearance from the spinal space (Anderson et al., 1987b, 2002, 2005). Punctures targeted the cisterna magna through the atlanto-occipital membrane. Approximately 1.5 ml of CSF was drawn using a 24–27 gauge needle. Monkeys were first anesthetized with ketamine (3 mg/kg, i.m.) and dexdomitor (0.075 mg/kg, i.m.), and anesthesia was reversed with antisedan (0.075 mg/kg, i.m.) once the animal was returned home. CSF was immediately labeled and frozen on dry ice before being transferred to a −80-degree Celsius freezer.

Samples with gross blood contamination (>0.1%), as indicated by pink coloration, were excluded before screening for hemoglobin. Limiting blood contamination to <0.1% was sufficient to ensure that analyses other than serotonin were not affected by blood. However, all CSF samples analyzed for serotonin were screened more rigorously for blood contamination by measuring hemoglobin using Multistix 8 SG reagent strips for urinalysis (Bayer), which can detect ∼0.2 μg/ml of hemoglobin. As previously demonstrated, screening for hemoglobin and using only those samples with <10 ppm blood limited blood-derived serotonin in CSF to <10 pg/ml (Anderson et al., 2005). Neurochemical analyses levels of CSF 5-HTP and serotonin were determined using reverse-phase high-performance HPLC as previously described (Anderson et al., 1987a,b, 1990, 2002).

Samples with any detectable blood contamination were excluded from analysis; a total of two samples were excluded. The final 5-HTP CSF data therefore included samples collected after injection of saline and 20 mg/kg 5-HTP from four subjects with an additional subject contributing a sample at saline. The final serotonin CSF dataset was more restricted and includes saline data from four subjects and 20 mg/kg 5-HTP data from three subjects. Changes in CSF concentrations of 5-HTP and serotonin were each assessed using a two-tailed independent t test.