A total of 178 adult male Sprague-Dawley rats (Rattus norvegicus) and 6 adult male squirrel monkeys (Saimiri sciureus) served as subjects. Monkeys were born and raised at the Stanford University Research Animal Facility and were studied in adulthood at 5–10 yrs of age. Adult rats weighing ~250 g were purchased from Charles River Laboratories and acclimated to our facilities for >7 days prior to all studies. Monkeys and rats were socially housed in species appropriate conditions at ~26 °C in 12:12 h light/dark cycles. All procedures were conducted in accordance with state and federal laws, standards of the Department of Health and Human Services, and were approved by Institutional Animal Care and Use Committees.

Behavioral effects of plannexin, FGL L , and FGL S

All NCAM peptide mimetics used here were gifts from EnKam Pharmaceuticals. Rats were randomized to treatments with either plannexin (10 mg/kg, i.p. in 0.9% saline), FGL L (10 mg/kg, s.c. in ddH 2 O), FGL S (10 mg/kg, s.c. in 0.9% saline), or corresponding vehicle solutions that were administered either acutely or chronically as described below. For plannexin, the dose used here prevents reduction in neural progenitor cells associated with status epilepticus [24]. For FGL L , the dose reverses the working memory deficits induced by phencyclidine [25]. For FGL S, the dose improves the cognitive impairments in rodents [19]. Sample sizes were N = 6–12 per treatment condition, as described in the Supplementary Methods.

For all acute administrations, rats were injected 30 min before testing on the elevated plus-maze (EPM) described below. Behavior on the EPM has predictive validity for drug treatments related to anxiety in humans [26]. After 5 days, these rats were counterbalanced by a group and tested twice on 2 consecutive days by the forced swim test (FST) described below. Behavior on FST has predictive validity for drug treatments related to depression in humans [27]. For FST assessments with acute administration of plannexin or FGL L , rats were injected 1 and 5 h after Day 1 of FST and then again 30 min before Day 2 of FST. For FST assessments with acute administration of FGL S , rats were injected 1, 2, 3, 4, and 5 h after Day 1 of FST and then again 30 min before Day 2 of FST. Six, instead of the traditional three, injections between Days 1 and 2 of the FST [28] were used due to concerns that FGL S may have a shorter half-life than FGL L .

In a separate experiment, vehicle or FGLs was co-administered with the AMPA receptor antagonist, 2,3-dioxo-6-nitro-1,2,3,4 tetrahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX) disodium salt, or sterile saline vehicle (10 mg/kg, i.p.) 30 min prior to Day 2 of FST [29]. After 24 h, vehicle–vehicle and FGL S –vehicle animals were tested in the social interaction test. The FGL S -NBQX animals were served as stimulus rats in the social interaction tests. Briefly, animals were placed simultaneously into an open arena and the time spent interacting (e.g., grooming, sniffing, following, and crawling) by the experimental rats were recorded over 5 min [30].

For chronic administration of plannexin, rats were injected every 48 h between 0800 and 0900 h and then tested for locomotion on Day 13, EPM on Day 14, and FST on Days 15 and 16. The rationale for treating rats every other day with plannexin was based on the evidence of persistent activation of FGF signaling pathways by other NCAM peptide mimetics [15]. For chronic administration of FGL L , rats were injected every 24 h for 16 days between 0800 and 0900 h and then tested as above. For FGL L and plannexin, the Day 2 FST results were used in chronic studies. For chronic administration of FGL S , 14 day osmotic minipumps at a flow rate of 5 µl/h for 12 mg/day were subcutaneously implanted without equilibration to allow an extra day of dosing. These rats were then tested for locomotor behavior on Day 13, EPM on Day 14, and FST on Day 15. For FGL S , we only performed Day 1 of FST, as this FST was performed 24 h after the last minipump dose. This approach has previously been validated for chronic dosing [31]. In all cases, behavioral testing was conducted between 0900 and 1200 h. All behavioral measures are described in detail in the Supplementary Methods.

FGL S in blood plasma and CSF

To extend the behavioral results, blood and cisternal CSF samples were collected from each of the sic randomly selected and experimentally naive rats in three conditions at weekly intervals: (i) undisturbed baseline, (ii) 30 min after a single s.c. injection of 100 mg/kg FGL S , and (iii) 30 min after the last of the five s.c. injections administered hourly with 100 mg/kg FGL S . We administered single and multiple injections to compare these two conditions. From another six randomly selected and experimentally naive rats, blood and cisternal CSF samples were collected in the three conditions described above, but at 10 mg/kg FGL S . From these same six rats, we also collected an additional sample 30 min after a 10 mg/kg FGL S injection on Day 2 that was preceded 24 h earlier by five 10 mg/kg FGL S injections administered at hourly intervals on Day 1 for preloading. The single injection and preloading conditions were performed to time-match the rat behavioral testing described above. The cisternal CSF samples were collected under anesthesia and immediately thereafter a blood sample was acquired from the saphenous vein (Supplementary Methods) and placed into EDTA tubes that contained aprotonin (Sigma, 5 μL/mL). Samples were centrifuged at 2300 × g for 10 min at 4 °C and blood plasma fractions were aliquoted for storage at −80 °C. All blood plasma and CSF samples were shipped on dry ice to QPS (https://www.qps.com/) for determinations by liquid chromatography mass spectrometry with an FGL S detection limit of 50 ng/ml.

Six monkeys not previously treated with NCAM peptide mimetics were each initially administered with a single subcutaneous (s.c.) injection of 10 mg/kg FGL S diluted in 0.9% saline vehicle. Seven days later, the same six monkeys were administered a single s.c. injection of FGL S at 100 mg/kg. From each of two monkeys at each dose, cisternal CSF samples were collected under anesthesia at 1, 2, or 4 h after injection of FGL S in a pseudo-randomized order (Supplementary Methods). Immediately after each CSF sample collection, a blood sample was acquired by femoral venipuncture. The blood and CSF samples were processed for FGL S determinations as described above.

Statistical analysis

The behavior of the rats was evaluated with analysis of variances (ANOVAs) using SPSS software, except that locomotion and social interactions were evaluated by Student’s t-tests. Treatment was considered to be a between-subjects factor, and behavioral measures for EPM and FST were within-subjects repeated measures. Whenever there was a non-significant trend for a treatment-by-test measure interaction, Student’s t-tests were used to evaluate differences between the treatment conditions within each test measure. Fisher’s LSD tests were used for post hoc pairwise comparisons, and all test statistics were evaluated with two-tailed probabilities at p < 0.05. FGLs in blood plasma and CSF were evaluated with ANOVA using SYSTAT software. For rats, dose was considered to be a between-subjects factor, and the administration condition (single, repeated, preloading) was a within-subjects repeated measure. For monkeys, the sample collection time interval was considered a between-subjects factor and dose was a within-subjects repeated measure. Correlations between blood plasma and CSF levels of FGLs were assessed with Pearson correlation coefficients.