Antagonists of neural nitric oxide synthase affect auditory behaviours in mice: A study of the acoustic startle reflex (ASR) and its inhibition by gaps in noise and a change in sound source location. James Ison 1,2, Nathaniel Housel 1, Paul Allen 2, Conny Kopp-Scheinpflug 3 and Ian Forsythe 4 1 B & CS and 2 NB & A, Univ. Rochester, Rochester, NY USA; 3 Biology II, Univ. Leipzig, Leipzig, Saxony, Germany; 4 MRC Toxicol. Unit, U.Leicester, Leicester, Leicestershire, UK ARO 2009 #429 Research support from NIH (AG09524, DC05409) and the Schmitt Foundation for Integrative Brain Research Experiment 1: Effect of 7-NI on ASR and Background Activity in Quiet and in Noise The data on motor and sensory effects of blocking nNOS in rodents are limited: Wiley et al. (Eur.J.Physiol, 1997) found no effect on ASR amplitudes or prepulse inhibition in rats with 7-NI doses up to 100 mg/kg. Araki et al. (Eur.NeuroPsychPharmacol, 2001) found catalepsy in mice with 7-NI doses up to 160 mg/kg. Methods Stimulus detection was measured by prestimulus inhibition of the acoustic startle reflex (ASR), the prestimuli being a spatial change in sound location and a gap in noise. Fig. 1 shows the spatial apparatus: the mouse in a test cage, an overhead speaker that presents the eliciting stimulus (ES) for the ASR and two prestimulus speakers on the left and right of the mouse (PS-L and PS-R). WBN moved from PS-R to PS-L at lead times of 1 to 300 ms before the ES. Design: Exp. 1 tested the effect of 7-NI (IP, in a peanut oil suspension) on the ASR in noise vs. quiet for ES between 80 and 130 dB SPL. Exp. 2 tested the effect of 7- NI on the response to shifts in sound location of and 45 0, presented 1 to 300 ms before the ES. Exp. 3 examined the effects of 7-NI on the response to gaps in WBN of 0.5 to 15 ms in duration, ending 60 ms before the ES. 7 CBA mice (6-mo. old) were used in Exps. 1, 2 and 3, and 7 more mice tested in Exp. 3. The data are given as Mean (SEM). Introduction The impetus for this study was the report of Steinert et al. (Neuron, 2008) that the generation of nitric oxide in the principle cells of the MNTB following stimulation of the Calyx of Held reduces their excitability, via an adaptation-like regulation of post- synaptic Kv3 voltage-gated K+ ion channel currents. These effects were obtained from in vitro and in vivo preparations of wild-type mice and were blocked by administration of nNOS inhibitors, such as 7- nitroindazole (7-NI). The intent of the present research was to determine whether nitric oxide signaling influences auditory processing in the awake behaving mouse. The behaviour agreed with predictions derived from the physiology data in showing that systemic 7-NI injection improved spatial location and gap detection. Fig. 2: The normal increase on the ASR with an increasing ES level and the masking effect of noise at low ES levels were unaffected by 7-NI administration, showing that NOS inhibition has little effect on these auditory measures, and a small effect on activity. Fig. 3: There was a small but significant positive effect of 7-NI dose levels from 0 up to 100 mg/kg in an ANOVA that included both and This ANOVA found also a significant initial faster increase in performance for the shift in location, but a significant second rise in performance at The ANOVA of the 45 0 shift data showed the highly significant positive effect of 7-NI especially at 160 mg/kg between lead times of 20 and 100 ms. High levels of 7-NI also depressed “restless” background activity, but did not significantly affect ASR vigour. Fig. 4: The high dose of 7-NI increased the inhibitory effect of gaps beyond 3 ms in duration, but did not significantly affect behaviour for the 3 ms or smaller gaps. The insert shows the depressant effect of 160 mg/kg 7-NI on activity, but no effect on ASR vigour. This activity effect may be related to the 7-NI induced catalepsy observed by Araki et al. (2001). Fig 1: The apparatus for testing sensitivity to a change in sound source location. The ES speaker above the test cage elicits the ASR, which is modified by the noise moving from one to the other of the PS speakers facing the mouse. The ASR sensor is below the cage. Experiment 2: Effect of 7-NI on the response to and 45 0 degree changes in sound location. The MNTB is the critical intermediary in progressing from the VCN monaural to SOC binaural processing for sound localization in mice: and so, given Steinert et al., it might be expected the 7-NI would improve this ability. The mice began with a shift and 7-NI levels of 0, 50, and 100 mg/kg. The second test used a smaller 45 0 shift and 7-NI levels of 0, 50, 100, and 160 mg/kg. The data presented in Fig. 3 show the positive effect of the higher drug levels, especially for the 45 0 shift. Experiment 3 : Effect of 7-NI on gap detection The intent here was to determine if 7-NI might affect gap detection, a monaural task that does not require the MNTB but is likely to involve other brainstem nuclei expressing neuronal NOS and Kv3 channels. Seven additional mice were used to ensure that the experiment would have sufficient power to detect small changes and to ensure that any negative effects were not due to repeated 7-NI exposure. Only two 7-NI levels were used, 0 and 160 mg/kg. Gaps of 0.5 to 15 ms in duration were used, ending 60 ms before the ES. Conclusions: These data are consistent with in vitro and in vivo effects reported by Steinert et al. (2008), that the normal use-related generation of NO produces an adaptation of Kv3 K+ ion channels that are sensitive to nNOS inhibition by 7-NI. The proposed relationship between behaviour and the physiological data could be further tested by contrasting the effects seen here with prospective studies in Kv3 -/- mice.