Temporal bone surgery causes reduced nitric oxide synthase activity in the ipsilateral guinea pig hippocampus

Neuroscience Letters 259 (1999) 130–132

Temporal bone surgery causes reduced nitric oxide synthase activity in the ipsilateral guinea pig hippocampus Yiwen Zheng a, Paul F. Smith a ,*, Cynthia L. Darlington b a

Department of Pharmacology, School of Medical Sciences, University of Otago Medical School, Dunedin, New Zealand b Department of Psychology and the Neuroscience Research Centre, University of Otago, Dunedin, New Zealand Received 23 September 1998; received in revised form 5 November 1998; accepted 6 November 1998

Abstract There is a lack of data on the neurochemical basis for the interaction between the vestibular system and the hippocampus. The aim of the present study was to determine levels of nitric oxide synthase (NOS) activity in the ipsilateral and contralateral hippocampi at 10 h following unilateral deafferentation of the peripheral vestibular nerve (UVD) in guinea pig, using a radioenzymatic technique. The levels of NOS activity were similar in the contralateral hippocampus following either a sham temporal bone operation or the UVD. However, NOS activity was significantly lower in the ipsilateral hippocampus following both the UVD and the sham temporal bone surgery (P , 0.05 for both comparisons). These results suggest that even sham temporal bone surgery results in a reduction in NOS activity in the ipsilateral hippocampus.  1999 Elsevier Science Ireland Ltd. All rights reserved

Keywords: Nitric oxide; Nitric oxide synthase; Vestibular compensation; Vestibular nucleus; Prepositus hypoglossi

Despite the increasing behavioural and neurophysiological data supporting an interaction between the vestibular system and the hippocampus, there are few neurochemical data available to suggest which neurotransmitters might be involved in this interaction [10]. Horii et al. [5,6,8] have demonstrated that electrical stimulation of the vestibular nerve, results in increased acetylcholine release in the hippocampus, which is blocked by the injection of a non-Nmethyl-D-aspartate (NMDA) receptor antagonist into the vestibular nucleus; they have also shown that this effect is not dependent upon histamine release by the septum [5,6,8]. However, to our knowledge, there are no other data relating to the neurochemical mechanisms of interaction between the vestibular system and the hippocampus. Understanding this interaction is important not only for understanding how the hippocampus uses vestibular information, but also for understanding how changes in hippocampal function following unilateral deafferentation of the vestibular nerve

* Corresponding author. Fax: +64 3 4799140; e-mail: [email protected]

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(UVD) might be important for the behavioural recovery process that occurs (‘vestibular compensation’) [3,10]. Given the role of nitric oxide (NO) as a novel neurotransmitter in the CNS [11,12], we decided to examine levels of nitric oxide synthase (NOS) activity in the ipsilateral and contralateral hippocampi at 10 h following surgical UVD in guinea pig. However, unexpectedly, we found that even surgical exposure of the vestibular labyrinth, produced by drilling away part of petrous temporal bone overlying the labyrinth, without any direct damage to vestibular structures, caused a decrease in NOS activity in the ipsilateral hippocampus. This result demonstrates that even minor temporal bone surgery can alter NOS activity in the ipsilateral hippocampus and emphasizes the need to use precise control conditions in behavioural, electrophysiological and neurochemical studies of vestibular-hippocampal interaction, due to the exquisite sensitivity of the vestibular apparatus. Twenty-one guinea pigs (200–600 g), anaesthetized with 0.4 ml/kg, i.m. fentazin (0.4 mg/ml fentanyl citrate, 58.3 mg/ml xylazine HCl, 3.2 mg/ml azaperone [9]), were randomly divided into the following groups: (1) anaesthetic

 1999 Elsevier Science Ireland Ltd. All rights reserved

Y. Zheng et al. / Neuroscience Letters 259 (1999) 130–132

administration only, without surgery (n = 7); (2) sham UVD (i.e. temporal bone opened and vestibular labyrinth exposed but not directly damaged) (n = 7); (3) complete surgical UVD (n = 7). In all cases, at 10 h post-operation, animals were killed by cervical dislocation without anesthesia, and the ipsilateral and contralateral hippocampi rapidly dissected. Groups one and two were designed to control for the non-specific effects of the labyrinthine surgery and the time that had elapsed since induction of anesthesia [1]. Temporal bone surgery and surgical UVD were performed on the right side following the methods we have used previously [1,9]. Briefly, under fentazin anesthesia, the temporal bone was exposed by blunt dissection and then drilled open, using a dental drill with a fine burr under microscopic control: the horizontal and anterior semicircular canal ampullae, and the utricle and saccule, were then opened and aspirated; the posterior canal ampulla was probed blindly and aspirated. The sham operation consisted of exposing the temporal bone exactly as for the UVD, but instead, drilling a small hole (ca. 2 mm diameter) rostroventral to the vestibular labyrinth in order to expose it without causing any structural damage. At the end of the surgery, a small quantity of antibiotic cream (bactroban) was applied to the lesioned labyrinth and the temporal bone was sealed with dental cement. Animals were allowed to recover from the anesthesia under a warm lamp; food and water were available ad libitum. At the time of cervical dislocation, the hippocampi ipsilateral and contralateral to the UVD or sham operation were dissected separately. The samples were then frozen on solid CO2 and stored at −84°C until the time of the radioenzymatic assay, when all samples were analyzed under exactly the same set of conditions. The total time from decapitation to freezing was 2–4 min [1]. We employed a radioenzymatic assay technique similar to that developed by Bredt and Snyder [2], in which NOS enzyme activity is measured by monitoring the conversion of [3H]arginine to [3H]citrulline, given that NO formation is accompanied by the stoichiometric conversion of arginine to citrulline. All assays were performed in duplicate [1]. Twenty millimolar Tris–HCl buffer (containing 2 mM EDTA, (pH 7.4); 1:5 wt./vol.) was added to the samples on ice, which were then homogenized using ultrasonification (Sonifier cell disrupter B-30, Branson Sonic Power) and centrifuged at 12 000 × g for 10 min at 4°C (BHG Hermle Z-229 centrifuge) [1]. Twenty-five microlitres of the sample supernatant were added to an assay tube containing 75 ml of 1 mM NADPH, 0.75 mM CaCl2 and 0.5 mCi (166.5 nM) [3H]-L-arginine. Following incubation in a water bath for 10 min at 37°C, the tube was removed from the water bath and placed on ice; 1 ml of 1:1 (vol./vol.) Dowex/H20-50W (200–400, 8% cross-linked, Na + form) was added; 2 ml of 20 mM HEPES buffer (containing 2 mM EDTA, pH 5.5) were added to the mix and left to settle for 10 min; 500 ml of the supernatant were removed and added to 750 ml of scintillation fluid in a 24 well plate [1]. Scintillation was

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quantified by liquid scintillation spectroscopy, using a Topcount scintillation counter, and expressed as counts per min (cpm). Raw data were corrected for minor variations in the weights of different samples; the cpm for duplicate samples were averaged and corrected with respect to the blank control and background radioactivity [1]. NOS activity, in nmol/min per g tissue, was then calculated and mean values were expressed as a percentage of the control condition in which animals were anaesthetized only [1]. Data were analyzed using a one-way ANOVA followed by Student–Newman-Keuls multiple comparison post-hoc tests, using a type I error rate of 0.05 [13]. The levels of NOS activity were similar in the contralateral hippocampus following either the sham temporal bone operation or the UVD, and these values were not significantly different from the controls (Fig. 1). However, NOS activity was significantly lower in the ipsilateral hippocampus following both the UVD and the sham temporal bone surgery (P , 0.05 for both comparisons). The difference was of the order of 15%, however, the effect was consistent for tissue from the seven animals used in each condition, as indicated by the small variability (Fig. 1). These data show, for the first time, that surgery to the temporal bone overlying the vestibular apparatus, whether it involves surgical removal of the vestibular receptors themselves, (i.e. complete UVD) or simply opening the petrous temporal bone without direct damage to the vestibular labyrinth, results in a consistent decrease in NOS activity in the ipsilateral hippocampus but not in the contralateral hippocampus.

Fig. 1. Nitric oxide synthase (NOS) activity levels (expressed as % of control) in the contralateral and ipsilateral hippocampi from guinea pigs anaesthetized but not subjected to any surgery (‘contra and ipsi intact’), guinea pigs receiving a sham temporal bone operation (‘contra and ipsi sham’), and guinea pigs receiving a complete surgical UVD (‘contra and ipsi UVD’). Columns indicate means ± SE; n, seven animals in all groups; *significant difference.

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We have previously reported that UVD reduces NOS activity in the ipsilateral medial vestibular nucleus (MVN) and prepositus hypoglossi, although the sham UVD operation did not [1]. In other studies it has been reported that the expression of the NOS protein does not change in the MVN following UVD [7]; however, it is conceivable that NOS activity, in terms of the rate of the conversion of arginine to citrulline and NO, can change without corresponding changes in NOS protein expression [1]. Since it would be expected that any changes in hippocampal NOS activity produced by UVD or sham surgery would be mediated by changes in the vestibular nucleus complex, the fact that in our previous study the sham surgery did not alter NOS activity in the MVN, may suggest the involvement of other vestibular subnuclei, such as the superior, lateral, inferior or prepositus hypoglossi subnuclei. At present, the origin of the vestibular-hippocampal projections within the vestibular nucleus complex is unclear, and it is possible that even other areas of the CNS that receive vestibular nerve projections, such as the cerebellum, are involved [10]. As the decrease in NOS activity occurred even when the vestibular labyrinth was exposed but not directly damaged, temporal bone surgery in the region of the vestibular apparatus must be capable of inducing this change. It is possible that the vibration on the temporal bone caused by drilling results in changes in vestibular receptor function within the labyrinth. Consistent with this hypothesis, our previous studies have shown that sham UVD surgery can induce a transient spontaneous ocular nystagmus, which is indicative of activation of the vestibulo-ocular reflex [4]. It is less likely that the NOS changes observed in the hippocampus were due either to anesthesia, transient auditory stimulation, or to direct physical stimulation of the hippocampal tissue. Firstly, the anesthesia was the same for all groups, yet only the groups subjected to temporal bone surgery showed the changes in NOS activity in the ipsilateral hippocampus. Secondly, although there would have been considerable auditory stimulation and vibration at the time of the surgery, the animal was anaesthetized at the time and the hippocampi were not dissected until 10 h later. Although, in the case of the sham and UVD animals, a small amount of antibiotic cream (bactroban) was applied topically to the opened temporal bone, it is difficult to see how this could explain the results obtained. Even in the UVD condition in which the vestibular receptors were destroyed, the bulla and all other parts of the medial temporal bone remained intact, therefore it would have been impossible for the antibiotic cream to penetrate the CNS. In the case of the sham UVD, the entire bony labyrinth remained intact, and therefore the small amount of antibiotic cream on the petrous temporal bone could neither penetrate the labyrinth nor the CNS. Furthermore, UVD has similar behavioural, electrophysiological and neurochemical effects, irrespective of whether antibiotic cream is used following surgery [3]. Whatever the explanation of the observed reduction in NOS activity, researchers investigating the effects of UVD on hippocam-

pal neuronal activity should be careful to control for the non-specific effects of temporal bone damage. The fact that NOS activity is intimately linked to glutamate receptor activation suggests the possibility that a change in glutamatergic neurotransmission may underlie the decrease in NOS activity following temporal bone trauma [11,12]. Further studies will be necessary in order to explore this hypothesis, perhaps using micropunch techniques to examine NOS activity and neurotransmitter changes in different areas of the hippocampus, (e.g. dentate gyrus versus CA3). Nonetheless, this study provides the first evidence implicating changes in hippocampal NOS activity following temporal bone surgery. This research was supported by a New Zealand Lottery Health Research Committee Grant (to P.S.). Y.Z. was a recipient of a Postdoctoral Fellowship from that granting body. We thank the technical staff of the Department of Pharmacology for their excellent assistance. [1] Anderson, T., Moulton, A., Sansom, A.J., Kerr, D., Laverty, R., Darlington, C.L. and Smith, P.F., Evidence for reduced nitric oxide synthase activity in the ipsilateral medial vestibular nucleus and bilateral prepositus hypoglossi following unilateral vestibular deafferentation in guinea pig, Brain Res., 787 (1998) 311–314. [2] Bredt, D.S. and Snyder, S.H., Isolation of nitric oxide synthetase, a calmodulin-requiring enzyme, Proc. Natl. Acad. Sci. USA, 87 (1990) 682–685. [3] Curthoys, I.S. and Halmagyi, G.M., Vestibular compensation: a review of the ocular motor, neural and clinical consequences of unilateral vestibular loss, J. Vestibular Res. Equilib. Orientat., 5 (1995) 67–107. [4] Curthoys, I.S., Smith, P.F. and Darlington, C.L., Postural compensation in the guinea pig, Prog. Brain Res., 76 (1988) 375– 384. [5] Horii, A., Takeda, N., Mochizuki, T., Okakura-Mochizuki, K., Yamamoto, Y. and Yamatodani, A., Effects of vestibular stimulation on acetylcholine release from rat hippocampus: an in vivo microdialysis study, J. Neurophysiol., 72 (1994) 605–611. [6] Horii, A., Takeda, N., Mochizuki, T., Okakura-Mochizuki, K., Yamamoto, Y., Yamatodani, A. and Kubo, T., Vestibular modulation of the septo-hippocampal cholinergic system of rats, Acta Otolaryngol. (Stockh.), 520 (1995) 395–398. [7] Kitihara, T., Takeda, N., Emson, P.C., Kubo, T. and Kiyama, H., Changes in nitric oxide synthase-like immunoreactivities in unipolar brush cells in the rat cerebellar flocculus after unilateral labyrinthectomy, Brain Res., 765 (1997) 1–6. [8] Mochizuki, T., Okakura-Mochizuki, K., Horii, A., Yamamoto, Y. and Yamatodani, A., Histaminergic modulation of hippocampal acetylcholine release in vivo, J. Neurochem., 62 (1994) 2275– 2282. [9] Sansom, A.J., Smith, P.F. and Darlington, C.L., Fentazin anesthesia for labyrinthine surgery in guinea pigs, J. Vestibular Res. Equilib. Orientat., 6 (1996) 49–52. [10] Smith, P.F., Vestibular-hippocampal interactions, Hippocampus, 7 (1997) 465–471. [11] Vincent, S.R., Nitric oxide: a radical neurotransmitter in the central nervous system, Prog. Neurobiol., 42 (1994) 129–160. [12] Zhang, J. and Snyder, S.H., Nitric oxide in the nervous system, Annu. Rev. Pharmacol. Toxicol., 35 (1995) 213–233. [13] Zolman, J.F., Biostatistics: Experimental Design and Statistical Inference, Oxford University Press, New York-Oxford, p. 193.