Hydrogen Peroxide Affects the Electroretinogram of Isolated Perfused Rat Retina.

PURPOSE: To evaluate the effects of H2O2 as an oxidant on the electroretinogram (ERG) in isolated rat retina. METHODS: Retinas were isolated from rat eyes and perfused with a nutrient solution. ERGs were recorded every 3 min. Once the signal was at a steady state, H2O2 was added to the perfusion solution. RESULTS: H2O2 caused instantaneous and transient changes in amplitudes and implicit times of the ERG, followed by changes in retinal survival curves. H2O2 0.2 mM produced a rapid increase in b-wave amplitude, followed by a return to the initial value and a survival curve above the control (without H2O2). A slight increase in a-wave was observed, followed by a decrease and a recovery above the control. The slow PIII decreased and then recovered to the initial value. H2O2 0.6 mM induced a small increase in b-wave amplitude, followed by a rapid decrease without recovery. The a-wave and slow PIII decreased rapidly without recovery. The implicit times of the a-wave and b-wave increased moderately with a low dose of H2O2, whereas they significantly increased with a high dose. Whatever the dose, the slow PIII implicit time increased significantly, followed by a return to the initial value. Barium increased the a-wave and b-wave, and then H2O2 reduced the two waves with a stronger effect on the a-wave. Aspartate and barium isolated the fast PIII, which decreased after H2O2 application. CONCLUSIONS: H2O2 affects retinal function as shown by ERGs in isolated rat retina. The response differs with the dose of H2O2, suggesting that mechanisms underlying the action at low doses might be different from those at high doses. Our results also suggest an effect of H2O2 on ionic currents and/or neurotransmitter releases involved in the generation of the ERG and indicate a more pronounced effect on photoreceptors than on postsynaptic cells.

Effect of the novel histamine H4 receptor antagonist SENS-111 on spontaneous nystagmus in a rat model of acute unilateral vestibular loss.

BACKGROUND AND PURPOSE: Histamine H4 receptors are expressed in the peripheral vestibular system, and their selective inhibition improves vertigo symptoms in rats with unilateral vestibular lesions. The effects of SENS-111, a selective oral H4 receptor antagonist with high affinity to both animal and human receptors, on vertigo symptoms was evaluated in a translational in vivo model of unilateral vestibular loss. EXPERIMENTAL APPROACH: Pharmacokinetics of SENS-111 in rats was determined to aid dose selection for efficacy testing. Vestibular lesions were induced in rats by unilateral transtympanic injection of kainic acid. The effect of SENS-111 (10 or 20 mg·kg-1 ) on spontaneous nystagmus was evaluated compared with placebo vehicle using video-nystagmography, and the effective dose was compared with those of similar drugs used clinically, as single agents or combined with SENS-111. KEY RESULTS: Doses were selected for plasma exposure were consistent with published phase 1 results from healthy volunteers. SENS-111 of 10 mg·kg-1 gave a 21-22% reduction in nystagmus at 1 hr post-administration, whereas a loss of efficacy was seen with 20 mg·kg-1 . Compared with SENS-111, meclizine and methylprednisolone had minimal effects on nystagmus as single agents, and meclizine abolished the effect of SENS-111 when combined with SENS-111. All evaluated drugs were well tolerated. CONCLUSIONS AND IMPLICATIONS: The exposure-efficacy relationship for improved spontaneous nystagmus seen with SENS-111 in this in vivo model is consistent with phase 1 clinical results and provides preclinical support for pharmacokinetic/pharmacodynamic modelling and selection of effective clinical drug concentrations. LINKED ARTICLES: This article is part of a themed section on New Uses for 21st Century. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v177.3/issuetoc.

Evidence for functional GABAA but not GABAC receptors in mouse cone photoreceptors.

At the first retinal synapse, horizontal cells (HCs) contact both photoreceptor terminals and bipolar cell dendrites, modulating information transfer between these two cell types to enhance spatial contrast and mediate color opponency. The synaptic mechanisms through which these modulations occur are still debated. The initial hypothesis of a GABAergic feedback from HCs to cones has been challenged by pharmacological inconsistencies. Surround antagonism has been demonstrated to occur via a modulation of cone calcium channels through ephaptic signaling and pH changes in the synaptic cleft. GABAergic transmission between HCs and cones has been reported in some lower vertebrates, like the turtle and tiger salamander. In these reports, it was revealed that GABA is released from HCs through reverse transport and target GABA receptors are located at the cone terminals. In mammalian retinas, there is growing evidence that HCs can release GABA through conventional vesicular transmission, acting both on autaptic GABA receptors and on receptors expressed at the dendritic tips of the bipolar cells. The presence of GABA receptors on mammalian cone terminals remains equivocal. Here, we looked specifically for functional GABA receptors in mouse photoreceptors by recording in the whole-cell or amphotericin/gramicidin-perforated patch clamp configurations. Cones could be differentiated from rods through morphological criteria. Local GABA applications evoked a Cl- current in cones but not in rods. It was blocked by the GABAA receptor antagonist bicuculline methiodide and unaffected by the GABAC receptor antagonist TPMPA [(1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid]. The voltage dependency of the current amplitude was as expected from a direct action of GABA on cone pedicles but not from an indirect modulation of cone currents following the activation of the GABA receptors of HCs. This supports a direct role of GABA released from HCs in the control of cone activity in the mouse retina.

Safety, tolerability, pharmacokinetics and pharmacokinetic-pharmacodynamic modelling of the novel H4 receptor inhibitor SENS-111 using a modified caloric test in healthy subjects.

AIM: A Phase 1 study was performed to evaluate safety, pharmacokinetics (PK) and pharmacodynamics (PD) of the selective histamine H4 receptor antagonist SENS-111, an oral small molecule. METHODS: One hundred healthy subjects were randomized in a placebo-controlled, double-blind study evaluating single-ascending doses (SAD; 100-500 mg) and multiple-ascending doses (MAD; 50-150 mg day-1 , 4 days; 200-250 mg day-1 , 7 days). Effects of SENS-111 on nystagmus and vertigo induced by modified caloric tests were measured in the MAD studies. Population PK and PK/PD models were developed using a nonlinear mixed-effects approach. RESULTS: SENS-111 was well tolerated with mild to moderate events. No sedation was reported. A maximal tolerated dose was not reached. Dose-proportional increases in concentrations were seen up to 200 mg and more than dose-proportional thereafter, with mean half-life between 24 and 56 h. The caloric test induced mild but measurable vertigo and nystagmus with large intra/inter-individual variation for all parameters. SENS-111 did not significantly impact nystagmus but significantly improved latency of vertigo appearance/disappearance, duration and European Evaluation of Vertigo questionnaire parameters vs. baseline. A two-compartment model with first-order absorption, distribution and elimination best fit the data. PK/PD indirect modelling applied to vertigo duration and latency of appearance indicated maximum activity between 100 and 500 ng ml-1 plasma concentrations, corresponding to 100 and 200 mg day-1 , which are appropriate for clinical efficacy evaluations in vestibular diseases. CONCLUSIONS: SENS-111 is a well-tolerated first-in-class H4 receptor antagonist with acceptable PK for oral daily dosing. PK/PD modelling determined plasma concentrations and doses for efficacy studies in patients with vertigo symptoms.

Activation of BK and SK channels by efferent synapses on outer hair cells in high-frequency regions of the rodent cochlea.

Cholinergic neurons of the brainstem olivary complex project to and inhibit outer hair cells (OHCs), refining acoustic sensitivity of the mammalian cochlea. In all vertebrate hair cells studied to date, cholinergic inhibition results from the combined action of ionotropic acetylcholine receptors and associated calcium-activated potassium channels. Although inhibition was thought to involve exclusively small conductance (SK potassium channels), recent findings have shown that BK channels also contribute to inhibition in basal, high-frequency OHCs after the onset of hearing. Here we show that the waveform of randomly timed IPSCs (evoked by high extracellular potassium) in high-frequency OHCs is altered by blockade of either SK or BK channels, with BK channels supporting faster synaptic waveforms and SK channels supporting slower synaptic waveforms. Consistent with these findings, IPSCs recorded from high-frequency OHCs that express BK channels are briefer than IPSCs recorded from low-frequency (apical) OHCs that do not express BK channels and from immature high-frequency OHCs before the developmental onset of BK channel expression. Likewise, OHCs of BKα(-/-) mice lacking the pore-forming α-subunit of BK channels have longer IPSCs than do the OHCs of BKα(+/+) littermates. Furthermore, serial reconstruction of electron micrographs showed that postsynaptic cisterns of BKα(-/-) OHCs were smaller than those of BKα(+/+) OHCs, and immunofluorescent quantification showed that efferent presynaptic terminals of BKα(-/-) OHCs were smaller than those of BKα(+/+) OHCs. Together, these findings indicate that BK channels contribute to postsynaptic function, and influence the structural maturation of efferent-OHC synapses.

Symptomatic treatment of vestibular deficits: therapeutic potential of histamine H4 receptors.

Vestibular disorders display high prevalence and can severely impact the daily life. However, pharmacological options that would efficiently relieve the vertigo symptoms without side effects are still lacking. In the present review we briefly review the common history of histamine receptor modulation and the pharmacological therapy of vestibular disorders. We also discuss the recent demonstration of Histamine H4 Receptor mRNAs expression in Scarpa's ganglion of mammal and the potential use of specific H4R antagonists as vestibulomodulators. Additional original data confirm the expression of H4R proteins in the rat vestibular primary neurons, the neuromodulatory properties of specific H4R antagonists in vitro (inhibition of vestibular neuron excitability) as well as their efficacy to decrease vestibular deficits induced in different in animal models.

Onset of cholinergic efferent synaptic function in sensory hair cells of the rat cochlea.

In the developing mammalian cochlea, the sensory hair cells receive efferent innervation originating in the superior olivary complex. This input is mediated by α9/α10 nicotinic acetylcholine receptors (nAChRs) and is inhibitory due to the subsequent activation of calcium-dependent SK2 potassium channels. We examined the acquisition of this cholinergic efferent input using whole-cell voltage-clamp recordings from inner hair cells (IHCs) in acutely excised apical turns of the rat cochlea from embryonic day 21 to postnatal day 8 (P8). Responses to 1 mm acetylcholine (ACh) were detected from P0 on in almost every IHC. The ACh-activated current amplitude increased with age and demonstrated the same pharmacology as α9-containing nAChRs. Interestingly, at P0, the ACh response was not coupled to SK2 channels, so that the initial cholinergic response was excitatory and could trigger action potentials in IHCs. Coupling to SK current was detected earliest at P1 in a subset of IHCs and by P3 in every IHC studied. Clustered nAChRs and SK2 channels were found on IHCs from P1 on using Alexa Fluor 488 conjugated α-bungarotoxin and SK2 immunohistochemistry. The number of nAChRs clusters increased with age to 16 per IHC at P8. Cholinergic efferent synaptic currents first appeared in a subset of IHCs at P1 and by P3 in every IHC studied, contemporaneously with ACh-evoked SK currents, suggesting that SK2 channels may be necessary at onset of synaptic function. An analogous pattern of development was observed for the efferent synapses that form later (P6-P8) on outer hair cells in the basal cochlea.

Reevaluation of dystrophin localization in the mouse retina.

PURPOSE. The roles of dystrophins in retinal physiology remain elusive. The lack of proper clustering of the potassium channel Kir4.1 and of the aquaporin AQP4 was proposed to be the basis of the ERG abnormality observed in many Duchenne muscular dystrophy (DMD) patients. However, the electroretinogram of Dp71-null mice, in which this clustering is disrupted, shows only a moderate reduction of the b-wave with no change in the implicit times. Additionally, the deficit in color discrimination found in DMD patients is hard to explain through the known expression of DMD gene products. The authors thus decided to reexamine their distribution in the mouse retina. METHODS. Messenger RNA distribution was assessed by PCR coupled to laser microdissection of the outer and inner nuclear layers and by in situ hybridization for Dp427. Mouse retinas were double labeled for dystrophins versus presynaptic and postsynaptic proteins or antibodies specific for Dp427 or Dp427+Dp260. RESULTS. Messengers for Dp427, Dp260, and Dp140 were present in the inner nuclear layer. Dp427 mRNA was further detected in bipolar cells and in some amacrine cells by in situ hybridization. Comparative labeling in wild-type and mdx(5Cv) retinas (lacking Dp427) indicated a differential distribution of Dp427 and Dp260 between rod and cone terminals. CONCLUSIONS. In addition to their localization in photoreceptor terminals, Dp427, Dp260, and Dp140 are expressed in inner nuclear layer neurons, notably in bipolar cells for Dp427. Dp427 was proportionally more expressed in cone- than in rod-associated synapses compared with Dp260.

Modulation of hair cell efferents.

Outer hair cells (OHCs) amplify the sound-evoked motion of the basilar membrane to enhance acoustic sensitivity and frequency selectivity. Medial olivocochlear (MOC) efferents inhibit OHCs to reduce the sound-evoked response of cochlear afferent neurons. OHC inhibition occurs through the activation of postsynaptic α9α10 nicotinic receptors tightly coupled to calcium-dependent SK2 channels that hyperpolarize the hair cell. MOC neurons are cholinergic but a number of other neurotransmitters and neuromodulators have been proposed to participate in efferent transmission, with emerging evidence for both pre- and postsynaptic effects. Cochlear inhibition in vivo is maximized by repetitive activation of the efferents, reflecting facilitation and summation of transmitter release onto outer hair cells. This review summarizes recent studies on cellular and molecular mechanisms of cholinergic inhibition and the regulation of those molecular components, in particular the involvement of intracellular calcium. Facilitation at the efferent synapse is compared in a variety of animals, as well as other possible mechanisms of modulation of ACh release. These results suggest that short-term plasticity contributes to effective cholinergic inhibition of hair cells.

Mammalian retinal horizontal cells are unconventional GABAergic neurons.

Lateral interactions at the first retinal synapse have been initially proposed to involve GABA by transporter-mediated release from horizontal cells, onto GABA(A) receptors expressed on cone photoreceptor terminals and/or bipolar cell dendrites. However, in the mammalian retina, horizontal cells do not seem to contain GABA systematically or to express membrane GABA transporters. We here report that mouse retinal horizontal cells express GAD65 and/or GAD67 mRNA, and were weakly but consistently immunostained for GAD65/67. While GABA was readily detected after intracardiac perfusion, it was lost during classical preparation for histology or electrophysiology. It could not be restored by incubation in a GABA-containing medium, confirming the absence of membrane GABA transporters in these cells. However, GABA was synthesized de novo from glutamate or glutamine, upon addition of pyridoxal 5'-phosphate, a cofactor of GAD65/67. Mouse horizontal cells are thus atypical GABAergic neurons, with no functional GABA uptake, but a glutamate and/or glutamine transport system allowing GABA synthesis, probably depending physiologically from glutamate released by photoreceptors. Our results suggest that the role of GABA in lateral inhibition may have been underestimated, at least in mammals, and that tissue pre-incubation with glutamine and pyridoxal 5'-phosphate should yield a more precise estimate of outer retinal processing.

BK channels mediate cholinergic inhibition of high frequency cochlear hair cells.

BACKGROUND: Outer hair cells are the specialized sensory cells that empower the mammalian hearing organ, the cochlea, with its remarkable sensitivity and frequency selectivity. Sound-evoked receptor potentials in outer hair cells are shaped by both voltage-gated K(+) channels that control the membrane potential and also ligand-gated K(+) channels involved in the cholinergic efferent modulation of the membrane potential. The objectives of this study were to investigate the tonotopic contribution of BK channels to voltage- and ligand-gated currents in mature outer hair cells from the rat cochlea. METHODOLOGY/PRINCIPAL: Findings In this work we used patch clamp electrophysiology and immunofluorescence in tonotopically defined segments of the rat cochlea to determine the contribution of BK channels to voltage- and ligand-gated currents in outer hair cells. Although voltage and ligand-gated currents have been investigated previously in hair cells from the rat cochlea, little is known about their tonotopic distribution or potential contribution to efferent inhibition. We found that apical (low frequency) outer hair cells had no BK channel immunoreactivity and little or no BK current. In marked contrast, basal (high frequency) outer hair cells had abundant BK channel immunoreactivity and BK currents contributed significantly to both voltage-gated and ACh-evoked K(+) currents. CONCLUSIONS/SIGNIFICANCE: Our findings suggest that basal (high frequency) outer hair cells may employ an alternative mechanism of efferent inhibition mediated by BK channels instead of SK2 channels. Thus, efferent synapses may use different mechanisms of action both developmentally and tonotopically to support high frequency audition. High frequency audition has required various functional specializations of the mammalian cochlea, and as shown in our work, may include the utilization of BK channels at efferent synapses. This mechanism of efferent inhibition may be related to the unique acetylcholine receptors that have evolved in mammalian hair cells compared to those of other vertebrates.

The glutamate transporter EAAT5 works as a presynaptic receptor in mouse rod bipolar cells.

Membrane neurotransmitter transporters control the concentration of their substrate in the synaptic clefts, through the thermodynamic coupling of uptake to the movement of Na(+) and other ions. In addition, excitatory amino acid transporters (EAAT) have a Cl(-) conductance which is gated by the joint binding of Na(+) and glutamate, but thermodynamically uncoupled to the flux of glutamate. This conductance is particularly large in the retina-specific EAAT5 isoform. In the mouse retina, we located EAAT5 in both cone and rod photoreceptor terminals and in axon terminals of rod bipolar cells. In these later cells, application of glutamate on the axon terminal evoked a current that reversed at E(Cl), was insensitive to bicuculline, TPMPA, strychnine, dl-AP5, CNQX and MCPG, but blocked by the glutamate transporter inhibitor dl-tBOA. Furthermore, short depolarizations of the bipolar cells evoked a dl-tBOA and Cd(2+)-sensitive current whose amplitude was comparable to the glutamate-evoked current. Its kinetics indicated that EAAT5 was located close to the glutamate release site. For 2 ms depolarizations evoking maximal responses, the EAAT5-mediated current carried between 2 and 8 times more charge as an average inhibitory GABA or glycine postsynaptic current received spontaneously from amacrine cells, with 10 mm or 0.5 mm intracellular EGTA, respectively. In conditions for which reciprocal inhibition could be monitored, the charge carried by the EAAT5 current was 1.5 times larger than the one carried by the inhibitory postsynaptic currents received from amacrine cells. These results indicate that EAAT5 acts as a major inhibitory presynaptic receptor at mammalian rod bipolar cell axon terminals. This feedback mechanism could control glutamate release at the ribbon synapses of a non-spiking neuron and increase the temporal contrast in the rod photoreceptor pathway.