Prolonged incubation of slices (for 21+ hours) in our experimental condition did not affect basic synaptic physiology (Fig. 1A, inset), but adsorbed the ability of slices to induce/maintain LTP in CA1 area of the hippocampus; an initial post-potentiation level reached 135.2±20.53 % and fell down to 120±11.48 % and 113.0±34.6 % in 3 min and 20 min after the tetanic stimulation (Fig. 1A), compared to 229.4±5.1 %, 163.5±6.1 % and 161.4±6.1 % for the slices maintained in vitro for 6-8 hrs (n = 6 slices, P = 0.0011, P = 0.007 and P = 0.0023, respectively, Ref. 3). The incubation of slices with the peptide Ab1-40, however, resulted in recovery of LTP. After the incubation with Ab (83 nM) initial postpotentiation level reached 205.3±47.43%. At 3 and 20 min after tetanic stimulus the potentiation levels were 190.6±21.43% and 183.1±49.44%, respectively (Fig. 1A). Slices incubated with Ab therefore expressed statistical difference with the slices subjected to 21+ hrs of in vitro maintenance without Ab (n=6 slices, P=0.019, P=0.0048 and P=0.033, respectively). Ab thus reversed the LTP impairment of 21+ hrs slices and made it statistically not different from the slices maintained in vitro for 6-8 hrs only (n=6 slices, P=0.1286, P=0.2317 and P=0.3939 for the fEPSP slopes recorded immediately after the tetanic stimulation and in 3 min and in 20 min post-tetanus, respectively). The treatment of slices with both Ab and mevinolin abolished the restoration of LTP by Ab, and reversed the fEPSP waveforms back to the condition of the prolonged incubation with no Ab (Fig. 1D).
In our study we focused on the role of Ab1-40 in synaptic plasticity under the condition characterized previously with regard to cholesterol and phospholipid status [3, 5]. This condition, however, is different from those used in other reports on the role of Ab in synaptic function and plasticity (see Supplement). Hippocampal slices are characterized by the basal efflux of lipoprotein cholesterol (4.0±1.23% of the hippocampal cholesterol in 6 hrs, see Ref. 3). This efflux rate in 18 hours could yield the removal of ~12 % hippocampal cholesterol. Such loss of cholesterol in the medium is compatible with the preservation of the basic synaptic physiology but may explain the impairment of LTP in slices [3, 7] subjected to prolonged incubation in the medium in the absence of the external cholesterol donor . On the other hand, peptide Ab1-40 increases cholesterol synthesis (~145% above the control value in 21 hrs) and is capable to enhance the uptake of cholesterol (132.5±11.25% above the control in 21 hrs) by the hippocampal slices (see Ref. 3 for experimental details and related discussion). Thus, the restoration of the hippocampal LTP by the peptide Ab1-40 could be caused by replenishment of hippocampal cholesterol to the level equal or exceeding the loss of lipoprotein cholesterol during the prolonged incubation of the slices . Additional support for restoration of LTP by Ab-mediated biological regulation of the hippocampal cholesterol supply comes from the inhibition of Ab effect by mevinolin. Mevinolin is a member of a group of drugs, called statins, which inhibit 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, an enzyme that catalyzes rate-limiting reaction in cholesterol biosynthesis. The concentration of mevinolin used in this study causes 50% inhibition of HMG-CoA reductase, does not affect nonsterol isoprenoid pathway products (Scheme 1), and was reported to inhibit neuronal cholesterol content to near 50% [7, 10, 11]. Notably, cholesterol synthesis inhibition completely abolished the maintenance and did not significantly affect the induction of the LTP (Fig. 1).