Inhibition of nitric oxide synthesis and gene knockout of neuronal nitric oxide synthase impaired adaptation of mouse optokinetic response eye movements

Abstract

Nitric oxide (NO) plays a key role in synaptic transmission efficiency in the central nervous system. To gain an insight on the role of NO in cerebellar functions, we, here, measured the dynamics of the horizontal optokinetic response (HOKR) and vestibulo-ocular reflex (HVOR), and the adaptation of HOKR in mice locally injected with N(G)-monomethyl-L-arginine (L-NMMA) that inhibits NO synthesis and in mice devoid of neuronal nitric oxide synthase (nNOS). Local application of L-NMMA into the cerebellar flocculi induced no change in the dynamics of the HOKR but markedly depressed the adaptation of the HOKR induced by 1 hr of sustained screen oscillation. A slight difference was seen in the HOKR but not in the HVOR dynamics between nNOS(-/-) mutant and wild-type mice. One hour of sustained screen oscillation induced adaptation of the HOKR gains in wild-type mice but not in mutants. These observations suggest that NO is essential for the adaptation of the HOKR and that nNOS is the major enzyme for NO synthesis in the process.

Figure 1

D-NMMA or L-NMMA applications do not alter the HOKR gains. The HOKR gains before and after application of D-NMMA (A) and L-NMMA (B). The HOKR was measured by sinusoidal oscillation of the checked-pattern screen (square size 4°) by 10° peak to peak at 0.17 Hz (maximum velocity, 5.2°/sec) and 15° at 0.17 Hz (7.9°/sec). No difference was seen in the dynamic characteristics of the HOKR before and after application of chemicals. Vertical bars indicate standard errors of means in all panels.

Figure 2

Impairment of adaptation of the HOKR by L-NMMA local application. (A) Examples of eye movements during D-NMMA, L-NMMA, and the recovery test in the case of one C57BL/6 mouse. Each trace is the average of the evoked eye movements >12 cycles. Plain and bold lines indicate the averaged eye position traces before and after sustained screen oscillation for 60 min, respectively. In this example, the HOKR gains changed from 0.40 to 0.48 in D-NMMA, from 0.41 to 0.40 in L-NMMA, and from 0.43 to 0.53 in the recovery test. The averaged screen position trace is shown at bottom. (B) Comparison of HOKR gain among D-NMMA, L-NMMA, and the recovery test. Averaged data obtained from 12 different mice were indicated. The HOKR gains in these tests were compared before and after 1 hr of sustained screen oscillation by 15° at 0.17 Hz. Note that the adaptation of the HOKR was abolished by L-NMMA but not by D-NMMA and not in the recovery test. (**) P < 0.005; (NS) not significant by Student's t-test.

Figure 3

Dynamic characteristics of HOKR and HVOR in nNOS^−/− mutant mice. The gain (A) and phase (B) of HOKR were measured by sinusoidal oscillation of the checked-pattern screen. The screen was oscillated by 5° peak to peak at 0.17 Hz (maximum velocity, 2.6°/sec), 10° at 0.11 Hz (3.5°/sec), 10° at 0.17 Hz (5.2°/sec), and 20° at 0.17 Hz (10.5°/sec). The HOKR gains of mutant mice were almost the same as those of wild-type mice at 2.6°/sec, 3.5°/sec, and 5.2°/sec of maximum screen velocities and larger than that of wild-type mice by 0.09 only at 10.5°/sec (P < 0.05 by Student's t-test), and the HOKR phase lags in mutant mice were smaller than those in wild-type mice at all screen velocities (P < 0.005 by two-way ANOVA). The gain (C) and phase (D) of HVOR were measured by sinusoidal oscillation of the turntable in darkness. No difference was seen between wild-type and mutant mice.

Figure 4

Impaired adaptation of the HOKR in nNOS^−/− mutant mice. (A) Examples of averaged eye position traces of wild-type and mutant mice before and after 1 hr of sustained screen oscillation by 10° at 0.17 Hz. Each trace is the average of the evoked eye movements > 8 cycles. In this example, the HOKR gains changed from 0.46 to 0.54 in wild-type and from 0.48 to 0.49 in mutant mice. The averaged screen position trace is shown at bottom. (B) The HOKR gains of wild-type and mutant mice before and after 1 hr of sustained screen oscillation by 10° at 0.17 Hz. Whereas wild-type mice showed a significant increase in the HOKR gain by 1 hr of sustained screen oscillation, mutant mice did not. (**) P < 0.005; (NS) not significant by Student's t-test.

Figure 5

Experimental protocol for local application of the NO synthesis inhibitor, L-NMMA. (A) Experimental protocol for D-NMMA and L-NMMA local application. (B) Representative 10% formalin-fixed frozen section of FITC-injected brain. (C) Diffusion of FITC seen in the adjacent section of B. Note that FITC diffused within flocculus and ventral paraflocculus. All chemicals were injected by 0.5 μl at 0.02 μl/min. The arrow indicates the center of injection. (FL) Flocculus; (PFL) paraflocculus; (M) micropipette. Calibration bar, 500 μm.