Hippocampal cGMP-dependent protein kinase I supports an age- and protein synthesis-dependent component of long-term potentiation but is not essential for spatial reference and contextual memory

Abstract

cGMP-dependent protein kinase I (cGKI) is expressed in the hippocampus, but its role in hippocampal long-term potentiation (LTP) is controversial. In addition, whether cGKI is involved in spatial learning has not been investigated. To address these issues, we generated mice with a hippocampus-specific deletion of cGKI (cGKIhko mice). Unlike conventional cGKI knock-out mice, cGKIhko mice lack gastrointestinal and cardiovascular phenotypes and have a normal life expectancy, which enables us to analyze hippocampal synaptic plasticity and learning in young and adult animals. Hippocampal LTP after repetitive episodes of theta burst stimulation was impaired in adult (12-14 weeks of age) but not in juvenile (3-4 weeks of age) cGKIhko mice. The difference in LTP between adult control and cGKIhko mice was abolished by the protein synthesis inhibitor anisomycin, suggesting that the impairment of LTP in adult cGKIhko mice reflects a defect in late-phase LTP. Despite their deficit in LTP, adult cGKIhko mutants showed normal performance in a discriminatory water maze and had intact contextual fear conditioning. These results suggest that hippocampal cGKI supports an age- and protein synthesis-dependent form of hippocampal LTP, whereas it is dispensable for hippocampus-dependent spatial reference and contextual memory.

Figure 1.

Conditional disruption of the cGKI gene in the hippocampus. A, PCR analysis of Cre-mediated recombination. Genomic DNA isolated from the tissues indicated was used as a template. All tissue probes were obtained from adult (12–14 weeks of age) cGKI ^+/L2; NEX ^+/Cre mice. PCR products amplified from the cGKI L2, the wild-type (+), and the L- allele are indicated. Boxes and triangles in the corresponding diagrams denote exon 10 of the cGKI gene and loxP sites, respectively. B, Analysis of cGKI mRNA expression using in situ hybridization. Sagittal brain sections of adult (12–14 weeks of age) control mice (ctr) (genotype: cGKI ^L-/L2; NEX ^+/+) and cGKI ^hko mice (hko) (genotype: cGKI ^L-/L2; NEX ^+/Cre) were hybridized with a labeled antisense riboprobe directed against a region of the cGKI mRNA encompassing exon 10. Right panels show the hippocampal region at a larger magnification. C, Western blot analysis of cGKI protein expression. Tissues were obtained from adult (12–14 weeks of age) control mice (ctr) (genotype: cGKI ^L-/L2; NEX ^+/+) and cGKI ^hko mice (hko) (genotype: cGKI ^L-/L2; NEX ^+/Cre), respectively.

Figure 2.

cGKI ^hko mice show normal synaptic transmission. Hippocampal slices were prepared from adult (12–14 weeks of age) control (▪, genotype: cGKI ^+/L2; NEX ^+/Cre) and cGKI ^hko (□, genotype: cGKI ^L-/L2; NEX ^+/Cre) mice. fEPSPs were recorded in the CA1 region of the hippocampus after stimulation of the Schaffer collaterals. A, Input–output relation in slices from control (n = 28) and cGKI ^hko (n = 32) mice. B, Paired-pulse facilitation (PPF) in slices from control (n = 21) and cGKI ^hko (n = 19) mice. Representative fEPSPs elicited by pulse pairs with interstimulus intervals of 25, 50, 75, 125, and 225 msec are superimposed in the corresponding insets. Calibration: 50 msec, 0.5 mV.

Figure 3.

LTP induced by a single episode of tetanic stimulation is normal in adult cGKI ^hko mice. Hippocampal slices were prepared from adult (12–14 weeks of age) control (▪, genotype: cGKI ^+/L2; NEX ^+/Cre) and cGKI ^hko (□, genotype: cGKI ^L-/L2; NEX ^+/Cre) mice. fEPSPs were recorded in the CA1 region of the hippocampus after stimulation of the Schaffer collaterals. A, Time course of the fEPSP slopes after a weak theta burst (4 bursts each consisting of 4 pulses at 100 Hz with a 200 msec interburst interval). Data represent the mean ± SEM of 14 (control) and 12 (cGKI ^hko) slices. B, Time course of the fEPSP slopes after a strong tetanus (30 pulses 3 times at 100 Hz with 5 sec pause). Data represent the mean ± SEM of 11 (control) and 9 (cGKI ^hko) slices. Representative fEPSPs recorded at times 0 (before tetanus) and 60 min after tetanus are illustrated in the corresponding insets. Calibration: 10 msec, 0.5 mV.

Figure 4.

Adult cGKI ^hko mice lack a translation-depending form of LTP induced by repetitive episodes of TBS. Hippocampal slices were prepared from control (▪, genotype: cGKI ^+/L2; NEX^+/Cre; ⋄, genotype: cGKI^L-/L2; NEX^+/+) and cGKI^hko (□, genotype: cGKI^L-/L2; NEX^+/Cre) mice. LTP in the Schaffer collateral pathway was induced using a stimulation protocol consisting of three episodes of TBS separated by 30 min. ATBS comprised four bursts, each consisting of four pulses at 100 Hz with a 200 msec interburst interval. A, Time course of the average fEPSP slope in slices from adult (12–14 weeks of age) control (▪, n=11; ⋄, n = 8) and cGKI^hko mice (□, n = 12) in normal ACSF. Representative fEPSPs recorded at times 0 (before first TBS), 30, 60, and 120 min are illustrated in the corresponding insets. Calibration: 10 msec, 0.5 mV. B, Time course of the average fEPSP slope in slices from adult control mice (▪, n = 9) and cGKI^hko mice (□, n = 12) in the presence of anisomycin (20 μm) indicated by the bar.

Figure 5.

LTP after repetitive episodes of TBS is normal in juvenile mice with a global (cGKI ^L-/L-) or hippocampus-specific (cGKI ^hko) cGKI deficiency. Hippocampal slices were prepared from juvenile (3–4 weeks of age) mice. LTP in the Schaffer collateral pathway was induced using a stimulation protocol consisting of three episodes of TBS separated by 30 min. A TBS comprised four bursts, each consisting of four pulses at 100 Hz with a 200 msec interburst interval. A, Time course of the fEPSP slopes in wild-type (▪, n = 11) and cGKI ^L-/L- (□, n = 14) mice. The corresponding fEPSP slopes 1 hr after the third theta burst stimulation were 134.2 ± 7.3 and 131.9 ± 7.6% of baseline. B, Time course of the fEPSP slopes in control (▪, genotype: cGKI ^+/L2; NEX ^+/Cre; n = 11) and GKI ^hko (▪, genotype: cGKI ^L-/L2; NEX ^+/Cre; n = 9) mice. The corresponding fEPSP slopes 1 hr after the third theta burst stimulation were 142.9 ± 12.5 and 140.2 ± 12.0% of the baseline control. Representative fEPSPs recorded at times 0 (before first tetanus), 30, 60, and 120 min are illustrated in the corresponding insets. Calibration: 10 msec, 0.5 mV.

Figure 6.

Adult cGKI ^hko mice show intact cued and contextual fear conditioning. Adult control (filled bars, n = 9, genotype: cGKI ^+/L2; NEX ^+/Cre) and cGKI ^hko mice (open bars, n = 6, genotype: GKI ^L-/L2 NEX ^+/Cre) were conditioned in special chambers by presenting a 20 sec tone that co-terminated with an electric foot shock. Illustrated are the fractions (%) of the total observation periods (60 sec for cued memory; 180 sec for contextual memory) during which animals showed freezing behavior. A, Cued memory. In a neutral context, mice of either genotype showed virtually no freezing in the absence (-) of a tone and similarly strong freezing reaction during presentation of a 60 sec tone (+). B, Contextual memory. Control and cGKI ^hko mice re-exposed to the conditioning context showed similar freezing responses in the absence of the conditioned stimulus (tone).

Figure 7.

Adult cGKI ^hko mice show intact spatial learning in a discriminatory water maze task. Adult control (▪ and filled bars, n = 12, genotype: cGKI ^+/L2; NEX-Cre ^+/-) and cGKI ^hko (□ and hollow bars, n = 12, genotype: GKI ^L-/L2 NEX-Cre ^+/-) mice had to discriminate between two visible platforms: a stable platform remaining in the same position (correct choice) and a platform in pseudorandom position that submerged when climbed by a mouse. Mice were tested over five training sessions (day 1–5) and again at day 11 (memory retention). To validate spatial learning strategies, the correct platform was then moved to the opposite quadrant (day 12). A, Number of correct choices expressed as the percentage of the total number of choices per session (10 trials per session corrected for the number of omissions). B, Time required for navigating to one of the two platforms (escape latencies). Errors of omission observed in control and cGKI ^hko mice during training session at day 1 are shown in the inset.