Long-term potentiation in the visual cortex requires both nitric oxide receptor guanylyl cyclases

Abstract

The role of nitric oxide (NO)/cGMP signaling in long-term potentiation (LTP) has been a lingering matter of debate. Within the cascade, the NO receptor guanylyl cyclase (GC), the cGMP-forming enzyme that is stimulated by NO, plays a key role. Two isoforms of GC (alpha2-GC, alpha1-GC) exist. To evaluate their contribution to synaptic plasticity, we analyzed knock-out mice lacking either one of the GC isoforms. We found that LTP induced in the visual cortex is NO dependent in the wild-type mice, absent in either of the GC isoform-deficient mice, and restored with application of a cGMP analog in both strains. The requirement of both NO receptor GCs for LTP indicates the existence of two distinct NO/cGMP-mediated pathways, which have to work in concert for expression of LTP.

Figure 1.

Targeted deletion of GC isoforms in mice does not alter basal synaptic transmission in visual cortex. A, Western blot analysis of the α₁ and α₂ subunits of NO-sensitive GC in brain of WT, α₁-, and α₂-deficient animals was performed as described in Materials and Methods. B, Both GC isoforms as well as the endothelial and neuronal NOSs are expressed in the visual cortex. Shown is a representative experiment. C, Cyclic GMP levels were determined in slices of the visual cortex incubated with IBMX (1 mm, 10 min.) without additional treatment (8 slices form 4 mice), after TBS (6 slices from 4 mice), and in the presence of DEA-NO (10 μm, 5 min; 9 slices from 2 mice) as outlined in Materials and Methods. D, Nissl-stained slice of the visual cortex with the recording electrode located in layer III and the stimulation electrode in layer IV. E, The synaptically evoked FP is blocked in the presence of 10 μm DNQX; the remaining nonsynaptic component is abolished by 1 μm TTX. F, Representative example traces of FPs recorded at stimulus durations of 40, 100, and 200 μs for each experimental group (left). Input-output relationship (right) of stimulus-evoked FP responses to gradually increasing stimulus durations in WT (9 slices from 6 mice), α₁-KO (9 slices from 3 mice), and α₂-KOs (10 slices from 4 mice).

Figure 2.

NO-dependency of LTP in visual cortex of mice. TBS-induced changes in relative FP amplitudes of murine visual cortical slices in the absence (top, 9 slices from 6 mice) and in the presence of the NMDA-receptor antagonist d-AP5 (middle, 8 slices from 3 mice). TBS fails to induce LTP in the presence of the inhibitor of NO-sensitive GC, ODQ (bottom, 8 slices from 4 mice). Representative FPs before (1) and after TBS (2) are shown. Calibration: 1 mV, 10 ms.

Figure 3.

LTP is absent in the visual cortex of the α₁- and α₂-KOs but can be restored with a cGMP analog. A, C, TBS-induced changes in relative FP amplitudes of visual cortical slices of α₁-KOs (A, 7 slices from 5 mice) and α₂-KOs (C, 9 slices from 6 mice). B, D, Incubation of α₁-deficient (B, 7 slices from 5 mice) and α₂-deficient (D, 7 slices from 3 mice) slices with the cGMP analog, 8-pCPT-cGMP (10 μm), paired with TBS restored LTP. Representative FPs before (1) and after TBS (2) are shown above the respective diagram.

Figure 4.

The cGMP analog, 8-pCPT-cGMP, neither affects basal synaptic transmission nor LTP in WT visual cortical slices. Shown are relative FP amplitudes of slices incubated with 8-pCPT-cGMP (10 μm, 15 min) without (7 slices from 3 mice) and with TBS treatment (8 slices from 5 mice).