Long-term potentiation in hippocampus involves sequential activation of soluble guanylate cyclase, cGMP-dependent protein kinase, and cGMP-degrading phosphodiesterase

Abstract

Previous studies indicate that cGMP is involved in long-term potentiation (LTP). However, the effects of application of tetanus to induce LTP on cGMP content and the mechanisms by which cGMP may modulate LTP have not been reported. The aim of this work was to study the time course of the changes in cGMP content and of the activity of soluble guanylate cyclase (sGC) (the enzyme that synthesizes cGMP) during LTP. Moreover, we also studied how the changes in cGMP affect cGMP-dependent protein kinase (PKG) and cGMP-degrading phosphodiesterase and the possible role of these changes in LTP. Application of tetanus induced a rise in cGMP, reaching a maximum 10 sec after tetanus. cGMP content decreased below basal levels 5 min after tetanus and remained decreased after 60 min. Activity of sGC increased 5 min after tetanus and returned to basal at 60 min. Tetanus increased the activity of cGMP-degrading phosphodiesterase at 5 and 60 min. GMP, the product of degradation, was increased at 5 and 60 min. Activation of phosphodiesterase and a decrease in cGMP were prevented by inhibiting PKG with Rp-8-bromoguanosine-cGMPS (Rp-8-Br-cGMPS). Inhibition of sGC [with ODQ (oxadiazolo quinoxalin-1-one) or NS 2028 (4H-8-bromo-1,2,4-oxadiazolo(3,4-d)benz(b)(1,4)oxazin-1-one)], of PKG (with Rp-8-Br-cGMPS), or of cGMP-degrading phosphodiesterase [with zaprinast or MBAM (4-[[3',4'-(methylenedioxy)benzyl]amino]-6-methoxyquinazoline) ] impairs LTP. The results indicate that induction of LTP involves transient activation of sGC and an increase in cGMP, followed by activation of cGMP-dependent protein kinase, which, in turn, activates cGMP-degrading phosphodiesterase, resulting in long-lasting reduction of cGMP content.

Fig. 1.

Inhibition of sGC impairs LTP induction in hippocampal slices. LTP in rat hippocampal slices was induced as described in Materials and Methods. Schaffer collateral–commissural pathways were stimulated with electrical pulses, and, at the time indicated by the arrow, tetanus was applied.Filled circles show the nondecremental LTP induced by tetanus in control slices (mean ± SEM; n = 6). Triangles show the fEPSP in slices treated with 10 μm ODQ in A (n = 6) or with 0.5 μm NS 2028 in B(n = 4), selective inhibitors of sGC. Raw evoked field potentials at the indicated times are indicated byletters: a, b, c, ODQ; d, e, control. Calibration: 500 μV, 10 msec. C, Effect of ODQ on field EPSP without application of tetanus (mean ± SEM; n = 9).

Fig. 2.

Effects of tetanus on cGMP in hippocampal slices and in the extracellular fluid. A, LTP in rat hippocampal slices was induced as described in Materials and Methods. Slices were taken at the following times after application of tetanus: 0, 10, and 30 sec and 1, 3, 5, 30, and 60 min. Values are expressed as percentage of cGMP content in nonstimulated slices and are the mean ± SEM of 10–27 samples per point. Values significantly different from cGMP in nonstimulated slices are as follows: *p < 0.05; **p < 0.001; ***p < 0.0005. B, LTP in rat hippocampal slices was induced as described in Materials and Methods. The medium containing the cGMP accumulated during the first 5 min or during 60 min after application of tetanus was collected as indicated in Materials and Methods, and cGMP was determined. Values are the mean ± SEM of five experiments. Values that are significantly different from nonstimulated samples are as follows: *p < 0.05; **p < 0.005.

Fig. 3.

Effects of tetanus on the activities of sGC and cGMP-degrading phosphodiesterase and on the contents of cGMP and GMP in hippocampal slices. A, LTP in rat hippocampal slices was induced as described in Materials and Methods. Slices were taken at 0, 5, and 60 min after application of tetanus, and the activity of sGC was assayed as described in Materials and Methods. Values are the mean ± SEM of eight experiments. Values that are significantly different (p < 0.01) from nonstimulated slices are indicated by asterisks. B, Initial content of cGMP in the slices used for the assay of sGC shown inA, i.e., the content of cGMP at time 0 of the guanylate cyclase assay. Values are the mean ± SEM of eight experiments and are given as percentage of the content of cGMP in nonstimulated hippocampal slices. Values that are significantly different from nonstimulated slices are as follows: *p < 0.005; **p < 0.001. C, LTP in rat hippocampal slices was induced as above, and the activity of cGMP-degrading phosphodiesterase was assayed as described in Materials and Methods. Values are the mean ± SEM. Values that are significantly different from nonstimulated slices are as follows: *p < 0.001; **p < 0.0001.D, LTP in rat hippocampal slices was induced as above, and the content of GMP was determined as described in Materials and Methods. Values are the mean ± SEM of 11 samples. Values that are significantly different from nonstimulated slices are as follows: *p < 0.005; **p < 0.0001.

Fig. 4.

Inhibition of sGC, of PKG, or of cGMP-degrading phosphodiesterase or blocking NMDA receptors prevented tetanus-induced activation of cGMP-degrading phosphodiesterase activity. LTP in rat hippocampal slices was induced as described in Materials and Methods. Some slices were treated with the following: ODQ, an inhibitor of sGC (10 μm; n = 10); Rp-8-Br-cGMPS, an inhibitor of cGMP-dependent protein kinase (10 μm;n = 11); zaprinast (2 μm,n = 9; 0.5 μm, n= 8) or MBAM (0.5 μm; n = 7), inhibitors of cGMP-specific phosphodiesterase; or APV, an antagonist of NMDA receptors (50 μm; n = 8), as described in Materials and Methods. After 0 and 60 min of the application of tetanus, slices were taken, and the activity of cGMP-degrading phosphodiesterase was assayed as described in Materials and Methods. The activities of phosphodiesterase 60 min after tetanus are given as percentage of basal values. Values are the mean ± SEM of 29 samples for controls and for the number of samples indicated above for each treatment. Values that are significantly different from stimulated control slices are indicated; *p < 0.0001.

Fig. 5.

Inhibition of cGMP-degrading phosphodiesterase impairs LTP induction in hippocampal slices. LTP in rat hippocampal slices was induced as described in Materials and Methods. Schaffer collateral–commissural pathways were stimulated with electrical pulses, and, at the time indicated by the arrow, tetanus was applied. Basal fEPSPs were obtained before drug application and normalized to 100%. Filled circles show the nondecremental LTP induced by tetanus in control slices (mean ± SEM; n = 7). Triangles show the fEPSP in slices treated with the selective inhibitors of cGMP-specific phosphodiesterase: 2 μm zaprinast in A(n = 7); 0.5 μm zaprinast inB (n = 5); or 0.5 μmMBAM in C (n = 4). Zaprinast or MBAM was added 30 min before application of tetanus (indicated by arrow) and maintained throughout the experiment. Raw evoked field potentials at the indicated times are indicated by letters: a, b, c, d, zaprinast;e, f, control. Calibration: 500 μV, 10 msec.D, Effect of 2 μm zaprinast on field EPSP without application of tetanus (mean ± SEM; n= 7). The fEPSP amplitude was significantly different from controls until 14 min (Student's t test for unpaired data;p ≤ 0.05). E, Effect of 0.5 μm zaprinast on field EPSP without application of tetanus (mean ± SEM; n = 6). Values are not different from basal.