escape platform and a 4 d acquisition phase, followed by a probe trial onday 5 (as outlined above;
12 per group).
Analysis of transport across the BBB
Female wild-type (C57BL/6 background) mice were used, 5 per group.Mice were injected intraperitoneally with 2.5, 25, or 250 nmol/kg bwliraglutide or saline (0.9% w/v) as control. At 30 min postinjection, micewere anesthetized and immediately perfused with PBS. The brains wereremoved,weighed,andsnapfrozeninliquidnitrogen,andthenstoredat
80°C until processed and analyzed.
Tissue extraction of peptide
Eachbrainwasextractedusingacidethanol.Then,2.5mlofacidethanolper g of tissue was added, and samples were sonicated, then agitatedovernight at 4°C.Next,sampleswerecentrifugedat 5000rpmfor30minat 4°C, and the supernatant was poured off. The supernatant was driedfor1h30mininaSpeedVac.Thedriedsampleswerereconstitutedintheappropriate assay buffer, and necessary dilutions were made for analysisbyafluorescentGLP-1(active)ELISAkit(Millipore).Valueswerenormal-izedaccordingtotheproteincontentofsamplesasestimatedbytheBradfordmethod(seealsoGengleretal.,2010,formoretechnicaldetails).
Surgery and electrophysiological recording in the hippocampusarea CA1
The technique used for testing LTP in the hippocampus was exactly asdescribed by Gengler et al. (2010b). Mice were anesthetized with ure-thane(ethylcarbamate,1.8g/kg,i.p.)forthedurationofallexperiments.Electrodes (tungsten with Teflon coating; Bilaney) were implanted atcoordinates 1.5 mm posterior and 1.0 mm lateral for the recording elec-trode,andforthestimulatingelectrode,2.0mmposteriortobregmaand1.5 mm lateral to the midline. The electrodes were lowered through thecortex and the upper layers of the hippocampus and into the CA1 regionuntil the appearance of a negative-deflecting EPSP that had a latency of
10 ms. Field EPSPs (fEPSPs) were recorded on a computerized stimu-lating and recording unit (PowerLab, ADInstruments). The programactivated a constant current stimulus isolation unit (NeuroLog System).Thehigh-frequencystimulationprotocolforinducingLTPconsistedof3trains of 100 stimuli at 200 Hz. LTP was measured as percentage of baselinefEPSP(
8pergroup).Forpaired-pulsefacilitationrecording,two identical stimuli were given, and the amplitudes of the fEPSPs werecompared.Theinterstimuliintervalwasincreasedstepwisetotestdiffer-ent facilitating mechanisms (Gengler et al., 2010b).
After the LTP studies, animals were perfused transcardially with PBSbuffer followed by ice-cold 4% paraformaldehyde in PBS. Brains wereremovedandfixedin4%paraformaldehydeforatleast24hbeforebeingtransferred to 30% sucrose solution overnight. Brains were then snapfrozen using Envirofreez, and coronal sections of 40
m thickness werecut at a depth of
3 bregma using a Leica cryostat. Sections werechosen according to stereological rules (Bondolfi et al., 2002), with thefirstsectiontakenatrandomandeveryfifthsectionafterward.Between7and 13 sections were analyzed per brain.Staining was performed for Iba1 (ionized calcium binding adaptormolecule 1), a marker for activated microglia, to measure the inflamma-tion response (Paresce et al., 1997),
-amyloid plaques, congophilicplaques, synaptophysin, and doublecortin, a marker for immature neu-rons.Allsectionswereincubatedin3%H
trisodiumcitrate, pH 9, at 90°C for 30 min to enhance antigen recognition. Afterblocking the sections in 5% normal serum to avoid nonspecific antibody binding, they were incubated with rabbit polyclonal anti-Iba1 (1:2000,Wako Pure Chemical Industries, catalog #016-20001), goat polyclonalanti-doublecortin (1:200, Santa Cruz Biotechnology, sc-710), or rabbitpolyclonal anti-amyloid
peptide (1:250, Invitrogen, catalog #71-5800)
),andrememberedthelocationoftheplatformafterthe reversal probe trial (
, inset), showed no difference in the reversal acquisition phase (
), but showed good memory in the probe task (
, inset) (Student’s
0.0465), while the salineAPP/PS1 group did not. *
0.001, all groups
McClean et al.
Liraglutide Has Neuroprotective Properties J. Neurosci., April 27, 2011