Cre-dependent recombination of the floxed β1-integrin allele. A, Three alleles of the β1-integrin gene are depicted. WT, Wild-type allele; Floxed, allele with loxP sequences flanking exon 2 in the same orientation; Recombined, allele with the sequences between the two loxP sites, including exon 2, deleted as a result of Cre-induced recombination. Triangles, loxP sequences; arrows, PCR primers used to assay for the deletion event B. B, PCR analyses of hippocampal genomic DNA isolated from WT, Cre (Cre/+; +/+), f(β1) [+/+;f(β1)/f(β1)], heterozygous KO (het KO) [Cre/+; f(β1)/+], and three different homozygous KO (homo KO) [Cre/+; f(β1)/f(β1)] animals. The arrows indicate the identity of the PCR products from the corresponding β1-integrin alleles depicted in A or from the Cre transgene. The asterisk indicates the DNA heteroduplex formed from the floxed allele PCR product and the deleted allele PCR product. The PCR conditions were adjusted so that there was little or no competition during PCR between the products of the various alleles; note the approximately equal molar representation of the floxed and recombined alleles in het KO after PCR, taking into account the increased mass of the larger products, which leads to increased fluorescence. With 30% or more of the genomic DNA contributed by glial or nonexcitatory neurons, the relative strengths of the PCR products representing the floxed and recombined alleles of the homozygous KOs indicate that the gene was deleted in the majority of excitatory neurons.

Impaired AMPA receptor synaptic transmission in β1-integrin knock-outs. A, The fEPSP plotted against the intensity of stimulus applied to the Schaffer collateral. Loss of β1-integrin had a significant effect on slope of the fEPSP (postsynaptic measurement) at increasing stimulus intensities [Cre; f(β1)/f(β1), n = 29; Cre; +/+, n = 22 and +; f(β1)/f(β1), n = 24; F_(2,1076) = 21.29; p < 0.0001; ANOVA with repeated measures]. B, No difference between f(β1)KO [Cre; f(β1)/f(β1), n = 29] and the two control genotypes [Cre;+/+,n=22 and+; f(β1)/f(β1),n=24] was observed in the amplitude of the fiber volley at varying stimulus intensities, indicating equivalent transfer and conversion of the electrical stimulus into axonal depolarization. C, A significant decrease in overall synaptic transmission was detected as the slope of the fEPSP plotted against the amplitude of the fiber volley at increasing stimulus intensities(2.5–28.5mA).D,NMDA receptor-mediated field potentials assessed by blocking AMPA receptors with 5 mm CNQX, a selective AMPA receptor antagonist, and measuring potentials in ACSF containing no Mg²⁺. No significant change was observed between the genotypes in NMDA receptor-mediated synaptic transmission under these conditions [Cre; f(β1)/f(β1), n = 8; Cre; +/+, n = 9 and+; f(β1)/f(β1), n = 7]. Data represent mean ± SEM.

β1-integrin knock-outs exhibited normal spatial memory and conditioned fear memory. A, Acquisition of spatial memory plotted as escape latency as a function of training day in the hidden version of the water maze. No significant difference was observed between the f(β1)KO [Cre; f(β1)/f(β1), n = 13] and the control groups [Cre; +/+, n = 10; +; f(β1)/f(β1), n = 11; F_[2,31] = 0.112; p = 0.89; ANOVA with repeated measures]. B, Percentage of time spent in each quadrant during the probe trial at 24 h after the last training trial, 1 week after the last training trial (C), and in the “pattern completion” version of the water maze test (D). In all three tests, the β1-integrin knock-out and the control groups spent significantly more time in the trained quadrant than in the other three quadrants ( p ≤ 0.01 for each genotype; Scheffe's post hoc comparison). However, no significant difference was found between the knock-out and the control groups in any of the quadrants ( p ≥ 0.23 for each quadrant). E, Performance at 2 and 24 h after contextual fear conditioning with two CS–US pairings. No significant difference in conditioned response was observed between the β1-integrin knockout and the control groups at either time point ( p ≥ 0.32). F, Performance at 24 h after cued fear conditioning with two CS–US pairings. Both the f(β1)KO and the control groups exhibited a conditioned response during the 3 min presentation of the CS ( p ≤ 0.01 for each genotype). However, no significant difference in conditioned response was observed between the β1-integrin knock-out and the control groups either before ( p ≥ 0.94) or during ( p ≥ 0.26) the presentation of the CS.

Expression of AMPA and NMDA receptor subunits in β1-integrin knock-outs. A, Immunohistochemistry was performed to compare the expression pattern of GluR1, GluR2/3, and NR1 subunits in the hippocampus of f(β1)KO and wild-type (WT) animals. No differences were observed. B, Immunoblots were performed to compare the expression levels of S831-phosphorylated GluR1, S845-phosphorylated GluR1, total GluR1, and GluR2/3 subunits in hippocampal synaptosomal extracts from two individual f(β1)KOs (lanes 3 and 6), Cre; +/+ (lanes 1 and 4) and f(β1)/f(β1) (lanes 2 and 5) animals. Expression of actin was used as an internal control for protein loading. C, Bar graph summarizing the relative expression levels obtained from the immunoblots in B. Signals from each genotype were expressed as the ratio relative to that of the Cre; +/+ controls. No significant differences were detected between the f(β1)KOs and the controls for all subunits examined ( p ≥ 0.28 for each antibody used; Scheffe's post hoc comparison). D, Immunoblots were performed to compare the surface expression levels (lanes 2, 4, and 6) with the total biotinylated protein extract (lanes 1, 3, and 5) of total GluR1, S831-phosphorylated GluR1, S845-phosphorylated GluR1, and GluR2/3 subunits in hippocampal extracts from f(β1)KO (lanes 3 and 4), Cre; +/+ (lanes 1 and 2), and f(β1)/f(β1) (lanes 5 and 6) animals. Expression of actin was used as an internal control for protein loading and also for the absence of cytoplasmic proteins in the pulldown fraction. E, Bar graph summarizing the relative expression levels of surface proteins to those in the total biotinylated protein extracts obtained from the immunoblots in D.No significant differences were detected between the f(β1)KOs and the controls for all subunits examined (n = 3; p ≥ 0.56 for each antibody used; Scheffe's post hoc comparison).

Normal synaptic ultrastructure in β1-integrin knock-outs. A, Representative electron micrographs of axospinous (top panels) and axodendritic (bottom panels) synaptic contacts in the hippocampal CA1 stratum radiatum region of Cre control (left column) and f(β1)KO (right column) animals. No detectable morphological difference in either type of contact was detected between the two genotypes at either presynaptic (arrowheads) or postsynaptic (arrows) terminals. Scale bars, 0.3 μm. B, Cumulative frequency plot of synapse size in the CA1 stratum radiatum, measured as the length of the postsynaptic density. Synapse size distribution of f(β1)KOs was indistinguishable from the Cre control animals.

Synaptic plasticity in β1-integrin knock-outs. A, PPF in f(β1)KOs [Cre; f(β1)/f(β1), n = 26] and controls [Cre; +/+, n = 24 and +; f(β1)/f(β1), n = 24]. The loss of β1-integrin expression had no effect on short-term plasticity measured by PPF at any interpulse interval. B, Long-lasting plasticity was measured as LTP generated with two trains of 100 Hz stimulation (arrows) at a stimulus intensity that generated 50% of the maximum synaptic response. The f(β1)KOs [Cre; f(β1)/f(β1), n = 26] showed significantly lower LTP than the controls [Cre; +/+, n = 21 and +; f(β1)/f(β1), n = 18; F_(2,1919) = 186; p < 0.0001; ANOVA with repeated measures]. C, The total depolarization envelope was measured as the integral of the depolarization during successive trains of 100 Hz HFS. All three genotypes [Cre; f(β1)/f(β1), n = 4; Cre; +/+, n = 4 and +; f(β1)/f(β1), n = 4] showed a similar level of total depolarization during each 1 s of stimulation. D, The deficit in LTP remained when saturating amounts of HFS (8 trains at 100Hz) were used to induce the maximum LTP [Cre; f(β1)/f(β1), n = 7; Cre; +/+, n = 9 and +; f(β1)/f(β1), n = 11; F_(2,819) = 67, p < 0.001]. Data represent mean ± SEM.

β1-integrin knock-outs exhibited impaired working memory. A, Schematic diagram of the nonmatching-to-place T-maze assay. Each trial consists of a sample run and a choice run. For the sample run, one of two target arms of the maze is blocked, and the animal is forced to obtain the food reward from the unblocked arm. For the choice run, after a delay or with no delay, both target arms are unblocked, and the animal is free to move into either arm. The animal is considered to have made the correct choice (+) if it visits the previously unsampled arm but incorrect (−) if it visits the previously sampled arm. B, Working memory expressed as the percentage of correct choices made in a nonmatching-to-place T-maze assay with the choice run performed immediately, 10 s, or 1 min after the sample run. For the statistical analysis, we performed a two-way ANOVA with the main effects of genotype and delay (F_(2,26) = 1.68, p > 0.19; delay F_(2,45) = 13.81, p < 0.0001; interaction, F_(4,45) = 0.83, p > 0.51), followed by post hoc Scheffe's test, which showed a significant effect only between the Cre/+; f(β1)/f(β1) vs Cre/+; +/+ ( p = 0.020) and Cre/+; f(β1)/f(β1) vs +/+; f(β1)/f(β1) ( p = 0.027) at 10 s delay and no significant genotype effect at other delays ( p > 0.9). Similar results were obtained in an independent experiment with a second group of animals [Cre; f(β1)/f(β1), n = 15; Cre; +/+, n = 9 and +; f(β1)/f(β1), n = 12; data not shown]. The asterisks represent significant differences.