Metabolically stabilized AngIV analogs reverse scopolamine-dependent spatial learning deficits. (A) group latencies to find the submerged platform in the Morris water maze task of spatial memory. Data from six groups of rats (N = 8 each) that were pretreated with scopolamine (70 nmol i.c.v. in 2 μl of aCSF) 20 minutes before training followed by the infusion of the designated analog (1 nmol i.c.v. in 2 μl of aCSF) 5 minutes before daily training are shown. A two-way ANOVA with repeated measures indicated that all groups were different from the scopolamine→aCSF on at the last 5 days of testing. Mean ± SEM; P < 0.001. (B) day 9 probe trials by each experimental group. Time spent in the target quadrant was recorded for each experimental group. All treatment groups were different from the scopolamine→aCSF group (P < 0.01) but not significantly different from the vehicle group (P > 0.05). The GABA-Tyr-Ile group was also different from all of the other treated groups (P < 0.05).

Dihexa is concentrated in multiple brain regions. Rats fitted with a carotid cannula were anesthetized and infused with 0.5 ml of isotonic saline containing 10 μCi of [³H]dihexa and 2 μCi of [¹⁴C]inulin, a vascular marker. Thirty minutes after infusion, the rats were decapitated, the brains were removed, and various brain regions were dissected. The tissues were then weighed and solubilized with NCS tissue solubilizer, an organic protein solubilizing agent, and 10 ml of scintillation was added. Samples were counted with a scintillation counter using two different windows to quantitate both ³H and ¹⁴C counts. ³H /¹⁴C ratios were determined so that blood-derived dihexa contamination of tissues could be determined. The results indicate that all brain regions concentrated dihexa (P < 0.001) compared with blood, but no area was statistically different from any other (P > 0.05). The average ³H /¹⁴C ratio for blood was 1687 (mean ± S.E.M.; n = 4). Counts Per Minute (CPM), XXXX.

Dihexa reverses scopolamine-dependent spatial learning deficits. Group latencies to find the submerged platform in the Morris water maze task of spatial memory are shown. Twenty minutes before beginning testing, 3-month-old male Sprague-Dawley rats received scopolamine directly into the brain (i.c.v.), and 15 minutes later dihexa was given either i.c.v., i.p., or orally. There were 5 trials per day for 8 days. The latency to find the pedestal was considered a measure of learning and memory. (A) rats were pretreated with scopolamine (scop) (70 nmol i.c.v. in 2 μl of aCSF) 20 minutes before training followed by the i.c.v. infusion of dihexa (0.1 or 1 nmol in 2 μl of aCSF) 5 minutes before daily training. A two-way ANOVA with repeated measures indicated that all time points for the 1-nmol dihexa group were different from the scopolamine group, which received vehicle (aCSF) instead of dihexa (P < 0.001). The lower, 0.1 nmol, dose of dihexa also significantly improved performance when compared with the scopolamine group on days 5–8 of testing (P < 0.05). (B) rats were pretreated with scopolamine (70 nmol i.c.v. in 2 μl of aCSF) 20 minutes before training followed 15 minutes later by an i.p. injection of dihexa in DMSO (<1%) at 0.05, 0.25, or 0.50 mg/kg. A two-way ANOVA with repeated measures indicated that the latency curves for dihexa at 0.25 and 0.50 mg/kg were different from the scopolamine→aCSF group’s learning curve (P < 0.001). The 0.50 mg/kg group was not different from the vehicle control group (P > 0.05), and the 0.05 mg/kg dihexa group was not different from the scopolamine group (P > 0.05). (C) rats were pretreated with scopolamine (70 nmol i.c.v. in 2 μl of aCSF) 20 minutes before training followed by oral delivery (gavage) of dihexa at 1.25/kg and 2.0 mg/kg (suspension in isotonic NaCl) 5 minutes before daily training. The high oral dose (2 mg/kg) of dihexa completely reversed the scopolamine-dependent learning deficit (***P < 0.001), whereas the effect of scopolamine was partially reversed at the 1.25 kg/mg dose on days 3–8 (P < 0.01). Mean ± S.E.M.; n = 8–10.

Dihexa increases time in the target quadrant during day 9 probe trials. Time spent in the target quadrant was recorded for each experimental group following i.c.v., i.p., and oral delivery of dihexa. (A) in the i.c.v. study, the scopolamine group performed below the chance level (30 s) and was significantly different from the vehicle control group and the scopolamine→dihexa 1-nmol group (P < 0.001), whereas the scopolamine→dihexa 1-nmol and vehicle control groups were not different (P > 0.05). (B) in the i.p. delivery study, the scopolamine→PBS group performed below the chance level and was different from the vehicle control group (P < 0.001). Each of the treatment groups was different from the scopolamine→PBS group (P < 0.001–P < 0.05), whereas each of the treatment groups was different from one another (P < 0.001–P < 0.05), exhibiting the same dose-response relationship observed in the initial water maze study (). (C) in the oral delivery study, the scopolamine→H₂O group performed near the chance level and was different from the vehicle control group (P < 0.001). Both of the treatment groups were different than the scopolamine→H₂O group (P < 0.05 and P < 0.001, respectively), whereas both of the treatment groups were different from one another (P < 0.001), exhibiting the same dose-response relationship observed in the initial water maze study (). Mean ± S.E.M.; n = 8–10.

Dihexa improves spatial learning in aged rats. Group latencies to find the submerged platform in the Morris water maze task of spatial memory are shown. Five minutes before beginning testing, 24-month-old mixed sex (3 male and 3 female per group) Sprague-Dawley rats were administered dihexa (2 mg/kg) orally by gavage (suspension in isotonic NaCl) on a daily basis. There were five trials per day for 8 days. The latency to find the pedestal was considered a measure of learning and memory. The learning curve for the treated rats was significantly different from that of the nontreated rats (Mann-Whitney U test, P < 0.03). Mean ± S.E.M.; n = 6.

Nle¹-AngIV and dihexa increase the number of dendritic spines. Time-dependent effects of Nle¹-AngIV- and dihexa-treated neurons on spinogenesis are presented. Hippocampal neurons transfected with mRFP-β-actin were treated with 10⁻¹² M dihexa or 10⁻¹² M Nle¹-Ang IV for 5 days or 30 minutes in culture before fixation on DIV12. (A) representative image of the dendritic arbor of a 5-day vehicle-treated hippocampal neuron. (B) representative image of a dendritic arbor from a neuron stimulated for 5 days with 10⁻¹² M dihexa. (C) representative image of the dendritic arbor of a neuron stimulated with 10⁻¹² M Nle¹-Ang IV for 5 days. (D) bar graph representing the number of spines per 50-µm dendrite length per treatment condition following a 5-day in vitro treatment. ***P < 0.001; n = 200 dendritic segments. (E) bar graph representing the number of spines per 50-µm dendrite length per treatment condition after an acute 30-minute treatment. ***P < 0.001; n = 60 dendritic segments. Mean ± S.E.M. Analysis by one-way ANOVA and Tukey’s post-hoc test.

Localization of synaptic markers following Nle¹-AngIV and dihexa-dependent dendritic spine induction. Dihexa- and Nle¹-AngIV-treated neurons were immunostained for the universal presynaptic marker synapsin, the glutamatergic presynaptic marker VGLUT1, and the postsynaptic density maker PSD-95. The percent correlation between the postsynaptic spines (red) and presynaptic puncta (green), which represented a different marker in each panel, was determined and used as an indicator of functional synapses. (A) representative images of hippocampal neurons transfected with mRFP-β-actin and immunostained for the excitatory presynaptic marker VGLUT1, the general presynaptic marker synapsin, and the postsynaptic marker PSD-95 following a 5-day treatment with vehicle, 10⁻¹² M Nle¹-AngIV, or 10⁻¹² M dihexa. (B) bar graph confirming the expected increase in the number of dendritic spines following treatment with vehicle, Nle¹-AngIV, or dihexa (***P < 0.001; mean ± S.E.M.; n = 25 dendritic segments). (C) bar graph showing the percent correlation between dendritic spines after treatment and the glutamatergic presynaptic marker VGLUT1. No significant differences between the stimulated neurons and vehicle control–treated neurons were observed (P > 0.05; mean ± S.E.M.; n = 25 dendritic segments). (D) bar graph confirming the expected increase in the number of dendritic spines following treatment with vehicle, Nle¹-AngIV, or dihexa (***P < 0.001; mean ± S.E.M.; n = 25 dendritic segments). (E) bar graph showing the percent correlation between dendritic spines after treatment and the general presynaptic marker synapsin. No significant differences between the stimulated neurons and vehicle control–treated neurons were observed (P > 0.05; mean ± S.E.M.; n = 25 dendritic segments). (F) bar graph confirming the expected increase in the number of dendritic spines following treatment with vehicle, Nle¹-AngIV, or dihexa (***P < 0.001; mean ± S.E.M.; n = 25 dendritic segments). (G) bar graph showing the percent correlation between dendritic spines after treatment and the postsynaptic marker PSD-95. No significant differences between the stimulated neurons and vehicle control–treated neurons were observed (P > 0.05; mean ± S.E.M.; n = 25 dendritic segments). Together, these data indicate that dendritic spines formed after treatment support functional synapses. ns, not significant.

Increased frequencies of mEPSCs in dissociated hippocampal neurons treated with Nle¹-AngIV and dihexa. Recordings were carried out on rat dissociated hippocampal neurons treated with vehicle, 10⁻¹² M Nle¹-AngIV, or 10⁻¹² M dihexa for 5 days before recording. The postsynaptic currents, which were recorded in the presence of strychnine, picrotoxin, and tetrodotoxin, represented spontaneous bursts likely mediated by AMPA receptors. (A) representative traces of mEPSC recordings from Nle¹-AngIV- or dihexa-treated hippocampal neurons. (B) bar graph illustrating the increase in mEPSC frequencies in hippocampal neurons that resulted from Nle¹-AngIV or dihexa treatment. The increased frequencies indicate that dendritic spines induced by Nle¹-AngIV or dihexa support functional synaptic transmission. ***P < 0.001; mean ± S.E.M.; n = 25.