Introduction symptoms i.e., ‘static symptoms’, such as spontaneous

Brain Research 879 2000 148–155 www.elsevier.com locate bres Research report The effects of L -NAME on vestibular compensation and NOS activity in the vestibular nucleus, cerebellum and cortex of the guinea pig a a a , b Sylvia Paterson , Yiwen Zheng , Paul F. Smith , Cynthia L. Darlington a Vestibular Research Group , Department of Pharmacology, School of Medical Sciences, University of Otago Medical School, Dunedin, New Zealand b Vestibular Research Group , Department of Psychology and the Neuroscience Research Centre, University of Otago, Dunedin, New Zealand Accepted 25 July 2000 Abstract Nitric oxide NO has been implicated in the processes by which animals recover from peripheral vestibular damage ‘vestibular compensation’. However, few data exist on the dose–response effects of systemic administration of the nitric oxide synthase NOS G inhibitor, N -nitro- L -arginine methyl ester L -NAME, on the vestibular compensation process. The aim of this study was to investigate the effects on compensation of 5, 10, 50 or 100 mM L -NAME administered by s.c osmotic minipump for 50 h following unilateral vestibular deafferentation UVD in guinea pig, either commencing the drug treatment at 4 h pre-UVD or at the time of the UVD i.e., post-UVD. Post-UVD treatment with L -NAME, at any of the four concentrations used, had no effect on the compensation of spontaneous nystagmus SN, yaw head tilt YHT or roll head tilt RHT. By contrast, pre-UVD treatment with 100 mM L -NAME resulted in a significant decrease in SN frequency P,0.05 and a change in the rate of its compensation P,0.0005. Pre-UVD L -NAME resulted in a significant increase in the overall magnitude of YHT P,0.005; however, post-hoc comparisons revealed no significant differences between any specific L -NAME and vehicle groups. Pre-UVD L -NAME had no effect on RHT at any concentration. Analysis of NOS activity in the pre-UVD L -NAME treatment groups at 50 h post-UVD showed that only 100 mM L -NAME resulted in a significant decrease in NOS activity in the contralateral medial vestibular nucleus MVN prepositus hypoglossi PH P,0.05 and that NOS activity in the ipsilateral MVN PH was not significantly affected. However, NOS activity was significantly inhibited in the bilateral cerebellum and cortices for several concentrations of L -NAME. These results suggest that pre-UVD systemic administration of L -NAME can significantly increase the rate of SN compensation in guinea pig and that this effect is correlated with inhibition of NOS activity in several regions of the CNS.  2000 Elsevier Science B.V. All rights reserved. Keywords : Nitric oxide; Nitric oxide synthase; Vestibular compensation; Unilateral vestibular deafferentation; Unilateral labyrinthectomy

1. Introduction symptoms i.e., ‘static symptoms’, such as spontaneous

ocular nystagmus SN, yaw head tilt YHT and roll head Damage to the peripheral vestibular system results in a tilt RHT related to the imbalance in resting activity syndrome of ocular motor and postural disorders due to the between the bilateral VNCs, gradually subside within a disruption of central vestibulo-ocular VOR and vestibulo- few days to a week, whereas those symptoms related to the spinal VSR reflex pathways. In the case of unilateral loss of the dynamic sensitivity of VNC neurons to head vestibular deafferentation UVD, the symptoms are par- movement i.e., ‘dynamic symptoms’, such as abnormal ticularly dramatic, as a result of the severe imbalance in gain and phase of the VORs and VSRs, compensate much neuronal activity between the ipsilateral and contralateral less completely, more variably and over a longer period of vestibular nucleus complexes VNCs. However, over time. To the extent that vestibular compensation does time, many but not all of these symptoms subside in a occur, it appears to be relatively independent of any process of behavioural recovery known as ‘vestibular recovery in the deafferented vestibular nerve, and therefore compensation’. Vestibular compensation is a heterogeneous is attributed to plasticity in the central nervous system process in which many of the ocular motor and postural CNS. Many neurophysiological studies, including studies in alert animals, have shown that the static compensation process is approximately correlated with a partial recovery Corresponding author. Tel.: 164-3-479-5747; fax: 164-3-479-5747. E-mail address : paul.smithstonebow.otago.ac.nz P.F. Smith. of resting activity in the ipsilateral VNC see Refs. 0006-8993 00 – see front matter  2000 Elsevier Science B.V. All rights reserved. P I I : S 0 0 0 6 - 8 9 9 3 0 0 0 2 7 6 4 - 5 S . Paterson et al. Brain Research 879 2000 148 –155 149 [3,4,6,7,11,20] for reviews. However, the biochemical administration on NOS activity in the VNC, and compared basis of the compensation process is still poorly under- these effects with those in the cerebellum and cortex. stood e.g. [9,15]. The fact that excitatory amino acid receptors such as the N-methyl- D -aspartate NMDA receptor and associated 2. Materials and methods second messengers and protein kinases have been impli- cated in vestibular compensation suggests that other 2.1. Subjects NMDA receptor-related neuromodulators, such as nitric oxide NO, are also likely to be involved see [19] for Data were obtained from a total of 60 male and female review. NO is a free radical gas which is synthesised from pigmented guinea pigs 300–600 g. Five of these animals arginine by the enzyme, nitric oxide synthase NOS, and were used to examine NOS activity in the absence of UVD which is released from neurons by simple diffusion rather ‘non-UVD controls’; these animals were cervically dislo- than exocytosis, acting on intracellular targets in other cated and decapitated without anaesthetic see below. The neurons via the activation of guanylate cyclase to produce remaining animals were allocated to one of the following cyclic guanosine monophosphate cGMP see [21] for 10 groups, depending upon whether the drug administra- review. tion began before or after the UVD: 1 pre-UVD saline Flohr and colleagues [8] were the first to investigate NO control n55; 2 pre-UVD L -NAME 5 mM n54; 3 in the context of vestibular compensation and reported that pre-UVD L -NAME 10 mM n55; 4 pre-UVD L -NAME G the selective NOS inhibitor, N -nitro- L -arginine methyl 50 mM n56; 5 pre-UVD L -NAME 100 mM n55; 6 ester L -NAME, delivered post-UVD into the dorsal post-UVD saline control n59; 7 post-UVD L -NAME 5 lymph sac of the frog, delayed vestibular compensation. mM n55; 8 post-UVD L -NAME 10 mM n56; 9 Kitahara et al. [10], using rats, showed that changes in post-UVD L -NAME 50 mM n55; 10 post-UVD L - NOS expression occur in the cerebellar flocculus following NAME 100 mM n55. UVD and that post-UVD injection of L -NAME into the flocculus also inhibited the compensation process. Kitahara 2.2. Drug administration et al. [12] have further reported that NOS expression in the flocculus modulates the expression of the immediate early In order to maintain constant levels of NOS inhibition in gene protein, c-Fos, in the medial vestibular nucleus the CNS for up to 50 h post-UVD, L -NAME was adminis- MVN and prepositus hypoglossi PH following UVD. tered using continuous infusion by osmotic minipump In our previous studies we found that NOS activity Alzet 1003D implanted s.c between the shoulder blades. decreases in the ipsilateral MVN and bilateral PH follow- The minipump was incubated in 0.9 saline at 378C ing UVD and that NOS activity levels remain low up to 50 overnight so that it was ready to pump at 0.98 ml h from h post-UVD [1]. Taken together, these studies suggest that the time of implantation. In all cases L -NAME was the uncompensated stage following UVD is associated dissolved in 0.9 saline. Because of the limited amount of with low levels of NOS activity in the ipsilateral MVN and data available on the effects of L -NAME on vestibular PH and that inhibition of NOS results in a retardation of compensation, we decided to use a wide range of con- the vestibular compensation process. centrations from 5 to 100 mM in order to provide a There are many possible sites of action in the CNS dose–response analysis. The decision to use this con- where L -NAME could influence the development and centration range was based on our preliminary studies maintenance of vestibular compensation. Although Kita- which showed that 10 and 50 mM L -NAME treatment hara et al. [10] investigated the effects of local injection of using an s.c osmotic minipump resulted in a decrease in L -NAME into the cerebellar flocculus in rat, to date the NOS activity of 15 and 61 in the MVN PH, respectively only study to examine the effects of systemic administra- [Paterson et al., unpublished observations]. We decided to tion of L -NAME on vestibular compensation has used frog use 100 mM L -NAME in addition in order to achieve [8]. Because of the known species differences between greater than 61 inhibition of NOS activity in the MVN frogs and mammalian species in the compensation process PH. For the pre-UVD conditions, the animal was anaes- see Refs. [3,4,6,7,11,20] for reviews, we reasoned that it thetised with ether and the minipump implanted 4 h prior was important to conduct a systematic dose–response to the UVD. For the post-UVD conditions, the minipump analysis of the effects of systemic administration of L - was implanted at the time of the UVD under fentazin NAME on vestibular compensation in a mammalian anaesthesia see below. species. Because of the different effects of NMDA receptor antagonists depending upon whether they are administered 2.3. Surgery pre- or post-UVD [16,17]; see [19] for review, we also decided to compare pre- and post-UVD administration of Before the UVD, animals were anaesthetised with 0.4 L -NAME. Lastly, we used an assay of NOS activity in ml kg fentazin i.m. 0.4 mg ml fentanyl citrate, 3.2 mg order to determine the effects of the pre-UVD L -NAME ml azaperone, 58.3 mg ml xylazine hydrochloride. Elec- 150 S trocardiograph ECG electrodes were inserted into the to avoid contamination of SN by VOR nystagmus induced forelimb muscles and heart rate was monitored throughout by head movement. Animals were allowed to choose a the surgery using a Beckman amplifier and an au- natural posture and were not restrained since stress induced diomonitor. Procaine hydrochloride was injected s.c. into by restraint has been shown to increase SN frequency. We all pressure points and wound margins. The temporal bone estimate that the measurement error involved in using this was exposed using blunt dissection and the skin retracted method of SN frequency analysis is of the order of 61 using hemostats, and then the bony vestibular labyrinth beat 15 s [17]. was exposed using a high speed dental drill with a fine YHT was defined as the angle of deviation in degrees burr, with the aid of an otolaryngological operating between a line through the midscapular point and sacrum microscope Zeiss OPM. The sensory components of the and a line through the midscapular point and the centre of vestibular apparatus the horizontal and anterior semicircu- the animal’s head in the horizontal plane; RHT was lar canal ampullae, and the maculae of the utricle and defined as the angle of deviation in degrees between saccule were destroyed by drilling and aspiration. Al- gravitational vertical and the centre of the animal’s head in though the posterior canal ampulla was not visualized, it the vertical plane. Postural symptoms were measured by was also destroyed by drilling and aspiration. At the end of using the freeze-frame facility on the video recorder and the surgery, antibiotic cream 2 mupirocin was inserted fitting a large protractor over the screen of the monitor. We into the labyrinthine cavity and the temporal bone sealed estimate that the measurement error involved in measuring with dental cement. Histological examination has shown YHT and RHT in this way is of the order of6108. All that this UVD procedure results in complete destruction of measurements began at 10 h post-UVD when animals had the labyrinthine receptors [5]. Animals were allowed to recovered sufficiently from the anaesthetic to display recover under a warm lamp with food and water available typical UVD symptoms. Mean SN, YHT and RHT were ad libitum. calculated for each measurement time within each group of animals [17]. 2.4. Behavioural measurements 2.5. NOS assays All behavioural measurements were made double-blind: all drug solutions were coded so that the person making The specific purpose of the NOS assay was to determine the measurements did not know which solution was being the extent to which the pre-UVD L -NAME treatment, administered. Three static symptoms of UVD were quan- which resulted in effects on vestibular compensation, had tified: spontaneous ocular nystagmus SN, yaw head tilt been effective in reducing NOS activity in the VNC, YHT and roll head tilt RHT. Measurements were made cerebellum and cortex. For this reason and because our at 10, 20, 30, 40 and 50 h post-UVD. These times were interest was not in studying NOS activity in individual chosen because in our previous studies they have been subnuclei, the entire right MVN PH and left MVN PH shown to provide an accurate characterisation of the complexes were removed for analysis. For comparison, in vestibular compensation process. Symptoms were vid- the same animals, the entire ipsilateral and contralateral eotaped at each measurement time using two video cerebella and cortices were also removed. cameras Panasonic NV-M50, each with a zoom lens, In all cases, at 50 h post-UVD, animals were killed by connected to a video recorder Mitsubishi HS-641V and a cervical dislocation without anaesthesia. The brains were colour monitor Sony Trinitron. One video camera was rapidly removed and immediately chilled in iced-water for positioned in front of the animal to videotape SN and 45 s. The ipsilateral and contralateral MVN PHs and RHT, the other was positioned directly above the animal in ipsilateral and contralateral cerebella and cortices were order to videotape YHT. Animals were housed in in- rapidly dissected from the unfrozen brains following the dividual boxes with perspex windows at the front so that methods of Anderson et al. [1]. The tissues were immedi- video recording could take place without moving the ately frozen on dry ice and stored at 2848C until the time animal, since changes in vestibular, proprioceptive and of the assay. We employed a radioenzymatic assay tech- visual information produced by lifting the animal can nique which was modified from the one developed by cause decompensation [17]. Bredt and Snyder [2], in which NOS enzyme activity is 3 SN was defined as a rapid, large amplitude, mainly measured by monitoring the conversion of [ H]arginine to 3 horizontal eye movement, contralateral to the UVD. SN [ H]citrulline, given that NO formation is accompanied by frequency was measured visually by gently retracting the the stoichiometric conversion of arginine to citrulline. All skin behind the eye contralateral to the UVD to expose the assays were performed in duplicate. Briefly, 20 mM Tris– sclera and counting the number of SN quick phases within HCl buffer containing 2 mM EDTA, pH 7.4; 1:5 w v a 15 s interval beats 15 s, as defined by an electronic was added to the samples on ice, which were then audiotimer. This procedure was performed five times at homogenised using ultrasonification Sonifier cell disrupter each measurement time and the means obtained. Measure- B-30, Branson Sonic Power Co. and centrifuged at 12,000 ments were made whilst the animal’s head was stationary g for 10 min at 48C BHG Hermle Z-229 centrifuge. The S . Paterson et al. Brain Research 879 2000 148 –155 151 reaction was started by adding 25 ml of the sample supernatant to an assay tube containing 75 ml of 1 mM 3 NADPH, 0.75 mM CaCl and 150 nM [ H]- L -arginine. 2 Following incubation in a water bath for 1 min at 378C, the reaction was stopped by adding 1 ml of 1:1 v v Dowex 1 H O-50W 200–400, 8 cross-linked, Na form and 2 2 ml of 20 mM HEPES buffer containing 2 mM EDTA, pH 5.5 into the tube and transferring the tube onto the ice. After 10 min, 750 ml of the supernatant were removed and 3 added to 4.25 ml of scintillation fluid in a vial. [ H]- citrulline was quantified by liquid scintillation spectros- copy, using a Beckman scintillation counter. The counts- per-minute cpm for duplicate samples were averaged and corrected with respect to the blank control and background radioactivity. NOS activity was expressed as pmol min mg protein. Protein concentrations in the supernatant were measured based on the Bradford method [18] using a Bio-Rad protein assay dye reagent concentrate and spec- tramax microplate reader. Ten microlitres of supernatant were removed from the same tissue sample used for the NOS assay and added to 390 ml of distilled water in a 1.5 ml Eppendorf tube and vortexed. Ten microlitres of standard 0–500 mg ml protein or sample was then added to 190 ml of reagent that consisted of 40 ml of BioRad reagent and 150 ml of water in a 96 well tray. Protein formation was then assessed using spectrophotometry at a wavelength of 595 nm. The counts for duplicate samples were averaged. 2.6. Statistical analysis For SN, YHT and RHT, a single two-factor analysis of variance ANOVA with repeated measures on time was performed [22]. Factor A represented the drug effect on SN frequency, YHT or RHT; factor B, the repeated measure, represented time; and the interaction AB repre- sented the change in the rate of compensation as a result of treatment. As factor B, the repeated measure, was always significant i.e., since vestibular compensation occurred in all cases, it will not be discussed further. Pairwise comparisons were conducted, where appropriate, using the ´ Scheffe F-test; linear and exponential regression analyses were also used on the SN data [22]. The NOS activity data were analysed using one-way ANOVAs and post-hoc student Newman–Keuls multiple comparison tests [22]. The significance level was set at 0.05 for all comparisons.

3. Results