Group latencies to find the submerged platform in the Morris water maze task of spatial memory. A, data from the two groups representing the maximum scopolamine-induced disruption of acquisition (Scop > aCSF) and the most efficient acquisition expected during training (aCSF > aCSF) are presented. B, data from five groups of rats (n = 8 each), pretreated with intracerebroventricular scopolamine (70 nmol in 2 μl of aCSF) 20 min before training followed by the intracerebroventricular infusion of the designated analog (1 nmol in 2 μl of aCSF) 5 min before daily training. C, additional groups were pretreated with intracerebroventricular scopolamine followed by 0.1, 1, or 5 nmol of Nle¹-YI (in 2 μl of aCSF). Mean ± S.E.M.; *, p < 0.01.

Day 8 probe trials by each experimental group. Time spent in the target quadrant was recorded for each experimental group. A, the scopolamine (scop) > aCSF group performed below chance level (30 s) and was significantly different from the aCSF > aCSF control group (p > 0.001). B, the scopolamine followed by 1 nmol of Nle¹-YI, Nle¹-YIH, and Nle¹-YIHP groups spent significantly more time in the target quadrant than the Nle-Y-treated group (p < 0.05). The group treated with Nle¹-AngIV spent significantly more time in the target quadrant than any of the other groups (p > 0.05). C, the group pretreated with scopolamine followed by 5 nmol of Nle¹-YI spent significantly more time in the target quadrant than the 1-nmol group, which in turn spent more time than the 0.1-nmol group. Mean ± S.E.M.; *, p < 0.01.

Effect of C-terminal truncated peptides on the number of dendritic spines per segment of dendrite. A, percentage increase in the number of spines, marked with mRFP-β-actin, per 50 μm of dendrite in dissociated rat hippocampal neurons compared with vehicle-treated control dendrites on in vitro day 12. Cells were treated for 5 days starting at day 7 in culture. BDNF was included as a positive control and YIHPF, an inactive AngIV analog, was included as a negative control. Mean ± S.E.M., ***, p < 0.001; *, p < 0.05; ns, not significant. n = 200. B, dose response for the shortest active fragment, Nle¹-YI, indicating the number of treatment-induced spines/50-μm dendrite length in hippocampal neurons. Mean ± S.E.M. C, time course response for Nle¹-AngIV-treated neurons. The x-axis represents the time neurons were exposed to the peptide. D, representative image of a dendritic segment shown in gray scale (1) to indicate different spine morphologies (the red arrow is pointing to an immature thin spine and the blue arrow to a mature mushroom spine) and in red (2) to illustrate mRFP-β-actin labeling; in the same segment is converted to the pseudo-color green-fire blue (3), which aids in visualization of actin enrichment in the postsynaptic spine (yellow). E, pseudocolor photomicrographs of representative dendrite segments. Yellow-green staining is mRFP-β-actin.

Effect of C-terminal truncated peptides on dendritic spine head size and length. A, percentage increases in spine head size after drug treatment compared with vehicle dendrites. Mean ± S.E.M. ***, p < 0.001; #, p < 0.05 from control; ns, not significant. n = 200. B, cumulative distribution curve showing the spine length distribution for treatment groups from a representative experiment. Data were fit to a Boltzmann distribution. The inactive AngIV analog YIHPF was included as a negative control.

Correlation between dendritic spines and active zone markers. A, percentage correlation between dendritic spines marked with mRFP-β-actin and the presynaptic active zone marker synapsin in dissociated rat hippocampal neurons on in vitro day 12. Cells were treated for 5 days starting at day 7 in culture. Cultured neurons for immunocytochemistry were untreated (vehicle control) or stimulated with 1 pM Nle¹-AngIV, 1 pM Nle¹-YIHP, or 1 pM Nle¹-YI. Mean ± S.E.M., n = 50. One-way ANOVA indicated no differences among groups. B, percentage correlation between dendritic spines marked with mRFP-β-actin and the glutamatergic active zone marker α-VGLUT1 in dissociated rat hippocampal neurons. C, effect of Nle¹-AngIV on the frequency of mEPSCs from dissociated hippocampal neurons. Cultured neurons for electrophysiology recordings were treated with vehicle or 1 pM Nle¹-AngIV for 5 days before recording. Mean ± S.E.M., ***, p < 0.001; ns, not significant. n = 25 for control and 33 for Nle¹-AngIV. D, representative dendrite segments showing juxtaposed mRFP-β-actin (red) and synapsin (green). E, representative dendrite segments showing juxtaposed mRFP-β-actin (red) and α-VGLUT1 (green). F, representative traces for control and Nle¹-AngIV-treated neurons.

Correlation analysis of water maze performance versus dendritic spine properties. The number of spines per 50 μm of dendrite or the mean head size of the spines was graphed against the latency for rats to find the submerged platform in the water maze. Two test days were examined: day 3 when learning is most active (A) and day 8 when maximum learning had occurred (B). Each graph was assembled from six data points that represented the different treatment conditions (vehicle or peptides) and analyzed by linear regression. Spine number data for test day 3 (▴) produced a highly significant correlation (r² = 0.76; p < 0.001). Comparison of head size to latency (○) also produced a tight linear fit (r² = 0.96; p < 0.001). Total spine numbers versus day 8 latency correlated significantly (▵; r² = 0.81; p < 0.001), as did mean spine head size versus day 8 latency (●; r² = 0.92; p < 0.001).