High doses of salicylate reduces glycinergic inhibition in the dorsal cochlear nucleus of the rat.

High doses of salicylate induce reversible tinnitus in experimental animals and humans, and is a common tinnitus model. Salicylate probably acts centrally and induces hyperactivity in specific auditory brainstem areas like the dorsal cochlear nucleus (DCN). However, little is known about the effect of high doses of salicylate in synapses and neurons of the DCN. Here we investigated the effects of salicylate on the excitability and evoked and spontaneous neurotransmission in the main neurons (fusiform, cartwheel and tuberculoventral) and synapses of the DCN using whole cell recordings in slices containing the DCN. For this, we incubate the slices for at least 1 h in solution with 1.4 mM salicylate, and recorded action potentials and evoked and spontaneous synaptic currents in fusiform, cartwheel (CW) and putative tuberculoventral (TBV) neurons. We found that incubation with salicylate did not affect the firing of fusiform and TBV neurons, but decreased the spontaneous firing of cartwheel neurons, without affecting AP threshold or complex spikes. Evoked and spontaneous glutamatergic neurotransmission on the fusiform and CW neurons cells was unaffected by salicylate and evoked glycinergic neurotransmission on fusiform neurons was also unchanged by salicylate. On the other hand spontaneous glycinergic transmission on fusiform neurons was reduced in the presence of salicylate. We conclude that high doses of salicylate produces a decreased inhibitor drive on DCN fusiform neurons by reducing the spontaneous firing of cartwheel neurons, but this effect is not able to increase the excitability of fusiform neurons. So, the mechanisms of salicylate-induced tinnitus are probably more complex than simple changes in the neuronal firing and basal synaptic transmission in the DCN.

Zugaib J ，Ceballos CC ，Leão RM 《-》

Enhancement of Endocannabinoid-dependent Depolarization-induced Suppression of Excitation in Glycinergic Neurons by Prolonged Exposure to High Doses of Salicylate.

The Dorsal Cochlear Nucleus (DCN) is a region which has been traditionally linked to the genesis of tinnitus, the constant perception of a phantom sound. Sodium salicylate, a COX-2 inhibitor, can induce tinnitus in high doses. Hyperactivity of DCN neurons is observed in several animal models of tinnitus, including salicylate-induced tinnitus. The DCN presents several forms of endocannabinoid (EC)-dependent synaptic plasticity and COX-2 can also participate in the oxidative degradation of ECs. We recently demonstrated that short-term perfusion of sodium salicylate and other inhibitors of both oxidative and hydrolytic EC degradation did not affect depolarization-induced suppression of excitation (DSE), a form of EC-dependent short-term synaptic plasticity. Here, we show that prolonged incubation with high doses of sodium salicylate (1.4 mM) enhances DSE of synapses onto glycinergic DCN interneurons but not those innervating glutamatergic DCN fusiform neurons. This effect was not reproduced with lower doses of salicylate (140 µM) or with ibuprofen, another inhibitor of COX-2. This effect was not observed in the presence of AM251, an antagonist/inverse agonist of cannabinoid CB1 receptors, showing that it was dependent on EC release. Finally we demonstrated that incubation with salicylate potentiated the increase in intracellular calcium during the depolarization. Our results point to an increased inhibition of DCN inhibitory CW neuron during depolarizations, probably by an enhanced EC release during the depolarizations, which is potentially significant for DCN hyperactivity and tinnitus generation.

Zugaib J ，Leão RM 《-》

Salicylate activates K(ATP) channels and reduces spontaneous firing in glycinergic cartwheel neurons in the dorsal cochlear nucleus of rats.

High doses of salicylate induce tinnitus in humans and experimental animals. The Dorsal Cochlear Nucleus is implicated with the genesis of tinnitus, and increased activity in this nucleus is seen in animal models of tinnitus. Incubation of brainstem slices containing the DCN with millimolar salicylate reduces the spontaneous firing of glycinergic cartwheel neurons and glycinergic neurotransmission on fusiform neurons, the principal neuron of this nucleus. However, the mechanism of salicylate mediating this effect is not known. Recently, we have shown that KATP channels strongly modulate the spontaneous firing of cartwheel neurons. We tested if KATP channels could mediate the effects of salicylate on cartwheel neurons. Perfusion of 1.4 mM salicylate hyperpolarizes the membrane of cartwheel neurons and stops firing. Salicylate produces an outward current similar to the KATP current seen in quiet cartwheel neurons. Activation of this current is occluded by the KATP agonist diazoxide, which is produced by the opening of KATP channels. The antagonist of AMP-kinase (AMPK), dorsomorphim, inhibited salicylate effects, suggesting that they could be mediated by activation of this kinase. Still, the AMPK agonist, AICAR, did not reproduce salicylate effects but occluded them. Additionally, inhibiting mitochondrial ATP synthesis with the protonophore CCCP reproduced, albeit with less efficacy, and inhibited the effects of salicylate. We concluded that salicylate in millimolar concentrations opens KATP channels in DCN cartwheel neurons, inhibiting spontaneous firing of these neurons, probably by activating AMPK and reducing mitochondrial ATP synthesis.

de Siqueira DVF ，Strazza PS Jr ，Benites NM ，Leão RM ... -  《-》

ATP-sensitive K(+) channels control the spontaneous firing of a glycinergic interneuron in the auditory brainstem.

Cartwheel neurons provide potent inhibition to fusiform neurons in the dorsal cochlear nucleus (DCN). Most cartwheel neurons fire action potentials spontaneously, but the ion channels responsible for this intrinsic activity are unknown. We investigated the ion channels responsible for the intrinsic firing of cartwheel neurons and the stable resting membrane potential found in a fraction of these neurons (quiet neurons). Among the ion channels controlling membrane potential of cartwheel neurons, the presence of open ATP-sensitive potassium channels (KATP ) is responsible for the existence of quiet neurons. Our results pinpoint KATP channel modulation as a critical factor controlling the firing of cartwheel neurons. Hence, it is a crucial channel influencing the balance of excitation and inhibition in the DCN. Cartwheel neurons from the dorsal cochlear nucleus (DCN) are glycinergic interneurons and the primary source of inhibition on the fusiform neurons, the DCN's principal excitatory neuron. Most cartwheel neurons present spontaneous firing (active neurons), producing a steady inhibitory tone on fusiform neurons. In contrast, a small fraction of these neurons do not fire spontaneously (quiet neurons). Hyperactivity of fusiform neurons is seen in animals with behavioural evidence of tinnitus. Because of its relevance in controlling the excitability of fusiform neurons, we investigated the ion channels responsible for the spontaneous firing of cartwheel neurons in DCN slices from rats. We found that quiet neurons presented an outward conductance not seen in active neurons, which generates a stable resting potential. This current was sensitive to tolbutamide, an ATP-sensitive potassium channel (KATP ) antagonist. After inhibition with tolbutamide, quiet neurons start to fire spontaneously, while the active neurons were not affected. On the other hand, in active neurons, KATP agonist diazoxide activated a conductance similar to quiet neurons' KATP conductance and stopped spontaneous firing. According to the effect of KATP channels on cartwheel neuron firing, glycinergic neurotransmission in DCN was increased by tolbutamide and decreased by diazoxide. Our results reveal a role of KATP channels in controlling the spontaneous firing of neurons not involved in fuel homeostasis.

Strazza PS Jr ，de Siqueira DVF ，Leão RM 《-》

Dorsal Cochlear Nucleus Fusiform-cell Plasticity is Altered in Salicylate-induced Tinnitus.

Following noise overexposure and tinnitus-induction, fusiform cells of the dorsal cochlear nucleus (DCN) show increased spontaneous firing rates (SFR), increased spontaneous synchrony and altered stimulus-timing-dependent plasticity (StDP), which correlate with behavioral measures of tinnitus. Sodium salicylate, the active ingredient in aspirin, which is commonly used to induce tinnitus, increases SFR and activates NMDA receptors in the ascending auditory pathway. NMDA receptor activation is required for StDP in many brain regions, including the DCN. Blocking NMDA receptors can alter StDP timing rules and decrease synchrony in DCN fusiform cells. Thus, systemic activation of NMDA receptors with sodium salicylate should elicit pathological changes to StDP, thereby increasing SFR and synchrony and induce tinnitus. Herein, we examined the action of salicylate in tinnitus generation in guinea pigs in vivo by measuring tinnitus using two behavioral measures and recording single-unit responses from DCN fusiform cells pre- and post-salicylate administration in the same animals. First, we show that animals administered salicylate show evidence of tinnitus using both behavioral paradigms, cross-validating the tests. Second, fusiform cells in animals with tinnitus showed increased SFR, synchrony and altered StDP timing rules, like animals with noise-induced tinnitus. These findings suggest that alterations to fusiform-cell plasticity are an essential component of tinnitus, regardless of induction technique.

Martel DT ，Pardo-Garcia TR ，Shore SE 《-》