The effects of protein kinase C and calmodulin kinase II inhibitors on vestibular compensation in the guinea pig.

Previous studies have demonstrated that vestibular compensation, the process of behavioural recovery which occurs following unilateral deafferentation of the vestibular labyrinth (UVD), is correlated with changes in in vitro phosphorylation of various protein substrates in the brainstem vestibular nucleus complex (VNC). The aim of the present study was to investigate the possible causal relationship between protein kinase activity and the induction of the vestibular compensation process, by delivering inhibitors of protein kinase C (PKC) or Ca(2+)/calmodulin-dependent kinase II (CaMKII) into the ipsilateral VNC at the time of the UVD and determining their effects on three static symptoms of UVD, spontaneous nystagmus (SN), yaw head tilt (YHT) and roll head tilt (RHT) in guinea pigs. Infusion of the PKC inhibitor, 3-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrr ole-2,5-dione, HCl (bisindolylmaleimide I, HCl/GF 109203X, HCl) ('Bis I'), at a concentration of 5 or 50 microM, significantly increased SN frequency at the earliest time points (6 and 8 h post-UVD) compared to vehicle controls and the less selective analogue, 2,3-bis(1H-indol-3-yl)-N-methylmaleimide (bisindolylmaleimide V) ('Bis V'). However, the compensation of YHT and RHT was unaffected by the PKC inhibitor. By contrast, the cell-permeable CaMKII inhibitor, myristoylated autocamtide-2 related inhibitory peptide (N-Myr-Lys-Lys-Ala-Leu-Arg-Arg-Gln-Glu-Ala-Val-Asp-Ala-Leu-OH) ('myr-AIP') or the cell-impermeable analogue, autocamtide-2 related inhibitory peptide (N-Lys-Lys-Ala-Leu-Arg-Arg-Cln-Glu-Ala-Val-Asp-Ala-Leu-OH) ('AIP'), failed to alter the compensation of SN, YHT or RHT at any dose compared to vehicle controls. These results implicate PKC-, but not CaMKII-, signal transduction pathways in the initiation of SN compensation in guinea pig.

Effects of intra-vestibular nucleus injection of the group I metabotropic glutamate receptor antagonist AIDA on vestibular compensation in guinea pigs.

Removal of the peripheral vestibular receptor cells in one inner ear (unilateral vestibular deafferentation, UVD) results in a syndrome of ocular motor and postural disorders, many of which disappear over time in a process of behavioural recovery known as vestibular compensation. Excitatory amino acid receptors, in particular the N-methyl-D-aspartate (NMDA) receptor, have been implicated in vestibular compensation; however, the metabotropic glutamate receptors (mGluRs) have not been studied in this context. The aim of this study was to determine whether group I mGluRs in the brainstem vestibular nucleus complex (VNC) ipsilateral to the UVD are involved in vestibular compensation of the static symptoms of UVD in guinea pig. The selective group I mGluR antagonist (RS)-1-aminoindan-1,5,dicarboxylic acid (AIDA) was continuously infused into the ipsilateral VNC for 30-min pre-UVD and 30-min post-UVD by cannula, at a rate of 1 microl/h, using one of four doses: 0.1 fg, 0.1 pg, 0.1 ng or 0.1 microg (n=5 animals in each case). In control conditions, a 0.1-fg (n=4) or 0.1-microg (n=5) NaOH vehicle was infused into the ipsilateral VNC using the same protocol. In order to control for the possibility that AIDA disrupted spontaneous neuronal activity in the VNC in normal animals, 0.1 microg AIDA (n=4) or 0.1 microg NaOH (n=2) was infused into the VNC in labyrinthine-intact animals. In both groups, static symptoms of UVD (i.e. spontaneous nystagmus, SN, yaw head tilt, YHT and roll head tilt, RHT) were measured at 8, 10, 12, 15, 20, 25, 30, 35, 45 and 50 h post-UVD. In addition, the righting reflex latency (RRL) was measured in labyrinthine-intact animals in order to assess whether AIDA impaired motor coordination in labyrinthine-intact animals. In UVD animals, the highest dose of AIDA significantly reduced SN frequency and changed its rate of compensation (P<0.001 and P<0.0001, respectively). This dose of AIDA also caused a significant reduction in YHT (P<0.005) as well as a significant change in its rate of compensation (P<0.0001). However, RHT was not significantly affected. In the labyrinthine-intact animals, AIDA infusion did not induce a UVD syndrome, nor did it significantly affect RRL. These results suggest that group I mGluRs in the ipsilateral VNC may be involved in the expression of ocular motor and some postural symptoms following UVD. Furthermore, group I mGluRs may not contribute to the resting activity of vestibular nucleus neurons.

Comparison of protein kinase activity and protein phosphorylation in the medial vestibular nucleus and prepositus hypoglossi in labyrinthine-intact and labyrinthectomized guinea pigs.

The aim of the present study was to compare in vitro protein expression, protein kinase activity and protein phosphorylation in the medial vestibular nucleus (MVN) and prepositus hypoglossi (PH) from labyrinthine-intact guinea pigs and from guinea pigs at various stages of vestibular compensation following unilateral labyrinthectomy (UL). The ipsilateral (I-MVN) and contralateral (C-MVN) MVN, and the ipsilateral (I-PH) and contralateral (C-PH) PH, were dissected from 3 naive labyrinthine-intact guinea pigs and 55 guinea pigs at 10 hs or 53 hs following a surgical UL or sham operation. Tissue extracts were incubated with [gamma-33P]ATP+/-Ca2+, phorbol 12, 13 dibutyrate and phosphatidylserine or +/- Ca2+ and calmodulin, to enhance protein kinase C (PKC) or calcium calmodulin kinase (CaMK) activity, respectively. Data were analysed as the ratio of activated to basal 33P incorporation detected by phosphorimaging. There were similar total protein and phosphoprotein profiles in the MVN and PH, as well as both PKC and CaMKII activity, suggesting that the MVN and PH are similar in the way that proteins undergo rapid modification by phosphorylation. During the development of vestibular compensation, a 46 kDa band in C-PH displayed higher PKC-mediated phosphorylation from 10 hs post-UL compared to sham controls. Significantly greater PKC-mediated phosphorylation of proteins of approximately 18, 46 and 75 kDa was observed in C-PH at 10 hs compared to 53 hs post-UL and in most cases the phosphorylation was greater in C-PH than in the C-MVN. These results suggest that between 10 and 53 hs post-UL, PKC-mediated phosphorylation changes mainly in the C-PH rather than the ipsilateral or contralateral MVN.

The effects of intra-vestibular nucleus administration of brain-derived neurotrophic factor (BDNF) on recovery from peripheral vestibular damage in guinea pig.

Brain-derived neurotrophic factor (BDNF), at doses of 0.04, 0.4 or 4.0 microg/day, was delivered by cannula and s.c osmotic minipump into the ipsilateral vestibular nucleus complex from 0 to 50 h following unilateral labyrinthectomy (UL) in guinea pigs. Compared to the vehicle control group, the frequency of spontaneous nystagmus was significantly reduced (p < 0.02) and the rate of yaw head tilt compensation increased (p < 0.02). However, roll head tilt was not signifcantly affected. There were also no significant effects of BDNF administration into the IVth ventricle (4.0 microg/day) on any UL symptom. These results further support the hypothesis that neurotrophins such as BDNF may enhance the vestibular compensation process.

Vestibular nucleus N-methyl-D-aspartate receptors contribute to spontaneous nystagmus generation following unilateral labyrinthectomy in guinea pigs.

We investigated the effects of a non-competitive N-methyl-D-aspartate (NMDA) receptor/channel antagonist ((+)MK-801)) or an L-type Ca(2+) channel antagonist (nifedipine), delivered into the ipsilateral vestibular nucleus complex (VNC) before a unilateral labyrinthectomy (UL), on spontaneous nystagmus (SN) generation. Guinea pigs received either (+)MK-801 (5 or 12.5 microg); the less active enantiomer, (-)MK-801 (5 or 12.5 microg); nifedipine (5 or 10 microg); or vehicle, via cannula in the ipsilateral VNC. (+)MK-801, but not nifedipine, significantly decreased mean SN frequencies at 6 h post-UL, compared to controls (P<0.02) and reduced the rate of SN compensation (P<0.05). These results suggest that the SN expression is partly induced by NMDA receptor activation in the ipsilateral VNC at the time of the UL.

An antisense oligonucleotide to brain-derived neurotrophic factor delays postural compensation following unilateral labyrinthectomy in guinea pig.

An antisense oligonucleotide to brain-derived neurotrophic factor (BDNF) was delivered by osmotic mini-pump at a 1 mM concentration via a cannula into the ipsilateral vestibular nucleus complex from 15 to 56h following unilateral labyrinthectomy in guinea pigs. Compared with the control groups, vestibular compensation of roll head tilt was significantly delayed (p < 0.05), while compensation of spontaneous nystagmus and yaw head tilt was unaffected. These results suggest that neurotrophins such as BDNF may be involved in specific aspects of the vestibular compensation process.

The effects of steroids on vestibular compensation and vestibular nucleus neuronal activity in the guinea pig.

Recent studies have suggested that steroids such as dexamethasone and methylprednisolone might be useful in the treatment of vestibular disorders, irrespective of whether inflammatory processes are involved. The aim of this study was to investigate the effects of systemic administration of dexamethasone on vestibular compensation of spontaneous nystagmus (SN) in guinea pig, and the effects of dexamethasone and methylprednisolone on extracellularly recorded spontaneous activity of medial vestibular nucleus (MVN) neurons in brainstem slices in vitro. In the behavioral study, none of the 3 doses of dexamethasone (5, 10, or 40 mg/kg i.p., delivered at 0, 12, 24, and 36 h following a unilateral surgical labyrinthectomy (UL)) resulted in a significant change in the frequency or compensation of SN, relative to the vehicle control group. In the in vitro study, only a minority of MVN neurons showed any response to 1 microM dexamethasone (1 out of 9 neurons), or 10 nM (3 out of 13), or 0.1 microM methylprednisolone (3 out of 7). These results suggest, contrary to previous evidence, that dexamethasone may not accelerate compensation of SN following surgical UL and that dexamethasone and methylprednisolone may have a direct action only on a minority of MVN neurons.

Evidence for reduced nitric oxide synthase (NOS) activity in the ipsilateral medial vestibular nucleus and bilateral prepositus hypoglossi following unilateral vestibular deafferentation in the guinea pig.

The aim of the present study was to examine, using a radioenzymatic assay technique, nitric oxide synthase (NOS) activity in the bilateral medial vestibular nuclei (MVN) and prepositus hypoglossi (PH), during the development of vestibular compensation for unilateral vestibular deafferentation (UVD) in the guinea pig. In the MVN ipsilateral to the UVD, and bilaterally in PH, NOS activity decreased following UVD compared to sham controls and did not recover significantly up to 50 h later, when a substantial degree of behavioural vestibular compensation had occurred. These results suggest that UVD causes a decrease in NOS activity in the ipsilateral MVN and the bilateral PH, and that a consequent decrease in NO may be responsible for some of the ocular motor and postural symptoms of UVD.

In vitro phosphorylation of medial vestibular nucleus and prepositus hypoglossi proteins during behavioural recovery from unilateral vestibular deafferentation in the guinea pig.

Unilateral removal of vestibular nerve input to the vestibular nuclei (e.g. by unilateral labyrinthectomy, UL) results in severe ocular motor and postural disorders which disappear over time (vestibular compensation). We investigated whether recovery of ocular motor function is temporally correlated with changes in protein phosphorylation in the medial vestibular nucleus (MVN) and prepositus hypoglossi (PH; MVN/PH) in vitro. Bilateral MVN/PH were dissected from 48 guinea pigs following decapitation at 10 h, 53 h or 2 weeks post-UL, or -sham operation and frozen. Tissue extracts were incubated with [gamma-32P]ATP +/- Ca2+ plus phorbol 12,13-dibutyrate and phosphatidylserine. UL resulted in a significant bilateral increase in the 32P-incorporation into a 65-85 kDa band (probably the myristoylated alanine-rich C kinase substrate, MARCKS) in compensated animals (53 h post-UL) under conditions which favoured the activation of protein kinase C. Under identical conditions, the labelling of a 42-49 kDa protein (P46) was increased significantly in the bilateral MVN/PH between either 10 h or 53 h and 2 weeks post-UL; there were no significant changes over time in sham controls. These results show that later stages of vestibular compensation are accompanied by changes in the phosphorylation of several likely protein kinase C substrates in the MVN/PH in vitro.

The opioid receptor antagonist, naloxone, enhances ocular motor compensation in guinea pig following peripheral vestibular deafferentation.

The opioid receptor antagonist, naloxone, has been demonstrated to enhance recovery from spinal cord injury and fluid percussion brain injury. The present study investigated, for the first time, the effects of naloxone on behavioral recovery following unilateral peripheral vestibular deafferentation (unilateral labyrinthectomy, UL) in guinea pig. An ip injection of 5 mg/kg naloxone 30 min pre-UL and 5 h post-UL was found to significantly reduce the frequency of spontaneous nystagmus relative to the vehicle control group (P < 0.005). However, a lower dose (2.5 mg/kg) had no effect. At either dose, the effects on the postural symptoms, yaw head tilt and roll head tilt, were small by comparison and in most cases nonsignificant. These results suggest that naloxone can reduce the ocular motor effects of UL in a dose-dependent fashion.

Fentazin anesthesia for labyrinthine surgery in guinea pigs.

We describe the use of fentazin anesthesia (0.4 mg/ml fentanyl citrate, 58.3 mg/mL xylazine HCl, and 3.2 mg/mL azaperone, Parnell, New Zealand) for labyrinthine surgery in guinea pigs. Fentazin provides stable surgical anesthesia for approximately 1 hour, rapid recovery from anesthesia following the surgery, and has significant analgesic and anxiolytic properties. Its advantages in comparison to other anesthetics (for example, ketamine HCl) are discussed.

Comparison of the effects of pretreatment with competitive or noncompetitive NMDA antagonists on vestibular compensation.

Unilateral labyrinthectomy (UL) results in a syndrome of ocular motor and postural disorders which abates over time in a process of behavioural recovery known as vestibular compensation. We have previously reported that a single systemic pre-UL injection of the organic Ca2+ channel antagonist verapamil or the noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 reduces the behavioural effects of UL in guinea pigs. The present study was conducted to determine if similar effects would be obtained with single injections of the competitive NMDA receptor antagonists 3-[(+/-)-2-carboxypiperazin-4-yl]-propyl-1-phosphonic acid (CPP) or cis-4-(phosphonomethyl)-piperidine-2-carboxylic acid (CGS 19755). Guinea pigs received an IP injection of 5 mg/kg CPP 2.5 h pre-UL, 5 or 10 mg/kg CPP 1 h pre-UL, 10 or 20 mg/kg CGS 19755 1 h pre-UL, or 1 ml/kg vehicle (saline) 1 h pre-UL, and the effects on the compensation of spontaneous nystagmus were measured over the following 52 h post-UL. Pretreatment with CPP had no significant effect on spontaneous nystagmus frequency or its compensation over 52 h post-UL. However, pretreatment with CGS 19755 resulted in a significant decrease in spontaneous nystagmus frequency without any acceleration of the rate of compensation.

Evidence that L-type calcium channels do not contribute to static vestibular function in the guinea pig vestibular nucleus.

Labyrinthine-intact guinea pigs received unilateral, brainstem cannula injections of (1) 2.5 micrograms of the selective dihydropyridine L-type Ca2+ channel agonist, Bay K 8644 (n = 4 animals); (2) 10 micrograms Bay K 8644 (n = 4); 12.5 micrograms of the selective dihydropyridine L-type Ca2+ channel antagonist, nifedipine (n = 4); or 40 micrograms nifedipine (n = 4). In 11/16 cases, the lesion associated with the cannula tip was located within or near the border of the right vestibular nucleus (VN) complex. All cannula injections were delivered in a 1 microliter volume of artificial cerebrospinal fluid (ACSF) and dimethylsulphoxide (DMSO) (70% DMSO, 30% ACSF for Bay K 8644; 80% DMSO, 20% ACSF for nifedipine), adjusted to a pH of approx. 7.0. The effects of these injections were compared with control injections of ACSF/DMSO in our previous studies. Animals were observed for signs of a labyrinthine syndrome (i.e. spontaneous ocular nystagmus, yaw and roll head tilt) directed to the contralateral or ipsilateral side. In no case did Bay K 8644 or nifedipine cause ocular motor or postural symptoms similar to those produced by a unilateral labyrinthectomy. These results suggest that L-type Ca2+ channels do not contribute significantly to the resting activity of VN neurons and therefore do not contribute to static vestibular function at the level of the VN.

Injections of calmidazolium chloride into the ipsilateral medial vestibular nucleus or fourth ventricle reduce spontaneous ocular nystagmus following unilateral labyrinthectomy in guinea pigs.

The effects of three injections (0.5-4.5 h post-operation) of 1-[bis-(p-chlorophenyl)methyl]-3-[2,4-dichloro-beta-(2,4- dichlorobenzyloxy)phenethyl]-imidazolium chloride (calmidazolium chloride, R24571), into the ipsilateral medial vestibular nucleus or fourth ventricle, on vestibular compensation for unilateral labyrinthectomy was studied in guinea pigs. R24571, a calmodulin antagonist and inhibitor of several Ca(2+)-dependent enzymes, caused a significant reduction in the average frequency of spontaneous ocular nystagmus (spontaneous nystagmus) during the first 53 h following unilateral labyrinthectomy (n = 5), compared with vehicle-injected animals (n = 5). Although a statistical analysis was not performed on the yaw head tilt and roll head tilt data because of the large variability between animals over the 53-h period of compensation, most R24571-treated animals had less yaw head tilt (4/4 animals) and roll head tilt (4/5 animals) at 9-11 h post-labyrinthectomy than the average values for the vehicle groups at that time. The decrease in the frequency of spontaneous nystagmus following R24571 treatment was not associated with general ataxia or sedation. These results are consistent with recent biochemical studies in suggesting that intracellular pathways associated with Ca2+ may be involved in the neuronal mechanisms of vestibular compensation following unilateral labyrinthectomy.

Pretreatment with MK-801 reduces spontaneous nystagmus following unilateral labyrinthectomy.

Unilateral labyrinthectomy results in a syndrome of ocular motor and postural symptoms which abate over time in a process of behavioural recovery known as vestibular compensation. We have previously suggested that an increased Ca2+ influx in ipsilateral vestibular nucleus (VN) neurons at the time of the unilateral labyrinthectomy may exacerbate the depression of VN resting activity caused by the loss of excitatory input from the VIIIth nerve. In order to further test this hypothesis, we administered (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801; 1.0 or 2.5 mg/kg i.p.), which blocks Ca2+ influx via NMDA receptor-mediated ion channels, to guinea pigs 0.5 h before unilateral labyrinthectomy and examined the effects on three symptoms of unilateral labyrinthectomy: spontaneous ocular nystagmus, yaw head tilt and roll head tilt. Pretreatment with MK-801 significantly altered the time course of the vestibular compensation of spontaneous nystagmus and yaw head tilt but had no significant effect on roll head tilt; in particular, 2.5 mg/kg MK-801 depressed spontaneous nystagmus frequency at 10 and 20 h post-labyrinthectomy relative to saline controls (P less than 0.05, post-hoc Scheffé F-test). The reduction in spontaneous nystagmus frequency was not simply a result of extended anesthesia, since other control animals, which received additional injections of the general anesthetic in order to achieve equivalent sleep times to the MK-801 group, did not show reduced spontaneous nystagmus frequency.(ABSTRACT TRUNCATED AT 250 WORDS)

Simple device for quantifying drug effects on the righting reflex.

A simple, inexpensive device is described that allows quantification of the effects of drugs on the righting reflex. This device consists of a modified set of kitchen scales connected to a digital timer. Two moveable Hall effect switches are positioned around the pointer, which registers the weight of the animal on the scales; when the animal is placed on the scales in the supine position, the initiation of a righting reflex causes the pointer to cross one of the switches, stopping the digital timer and providing a measure of righting reflex latency (RRL). We describe an efficient protocol for using this device that provides quantification of drug effects on the RRL, which can then be subjected to analysis using parametric statistics such as analysis of variance.

Intraventricular injection of an N-methyl-D-aspartate antagonist disrupts vestibular compensation.

Guinea pigs which had compensated for a unilateral labyrinthectomy exhibited a loss of ocular motor and postural compensation when a 40 or 20 mM concentration of the NMDA antagonist CPP, was injected through a cannula implanted in the IVth ventricle close to the vestibular nuclei. Similar injections of artificial cerebrospinal fluid did not induce loss of compensation. These results suggest that NMDA receptors may contribute to vestibular compensation in the guinea pig.