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.

Cholinergic/opioid interaction in anterior cingulate cortex reduces the nociceptive response of vocalization in guinea pigs.

The anterior cingulate cortex (ACC) is crucial in the modulation of the sensory, affective and cognitive aspects of nociceptive processing. Also, it participates in the planning and execution of behavioral responses evoked by nociceptive stimuli via descending projections to the brainstem. In laboratory animals nociceptive experimental tests evaluate behavioral responses that preferentially express the sensory-discriminative or affective-motivational component of pain. The objective of this study was to investigate the participation of opioid and cholinergic neurotransmission in the ACC on different nociceptive responses in guinea pigs. We used nociceptive tests of formalin and vocalization evoked by peripheral noxious stimuli (electric shock) to evaluate the behavioral expression of the sensory-discriminative and affective motivational components, respectively. We verified that the microinjection of morphine (4.4nmol) in the ACC of guinea pigs promotes antinociception in the two experimental tests investigated. This effect is blocked by prior microinjection of naloxone (2.7nmol). On the other hand, the microinjection of carbachol (2.7nmol) in the ACC induces antinociception only in the vocalization test. This effect was prevented by prior microinjection of atropine (0.7nmol) and naloxone (2.7nmol). In fact, the blockade of µ-opioids receptors with naloxone in ACC prevented the antinociceptive effect of carbachol in the vocalization test. Accordingly, we suggest that the antinociception promoted by carbachol was mediated by the activation of muscarinic receptors on local ACC opioid interneurons. The release of endogenous opioids seems to inhibited the expression of the behavioral response of vocalization. Therefore, we verified that the antinociceptive effect of morphine microinjection in ACC is broader and more robust than that promoted by carbachol.

Inhibitors of oxidative and hydrolytic endocannabinoid degradation do not enhance depolarization-induced suppression of excitation on dorsal cochlear nucleus glycinergic neurons.

Neurons from the dorsal cochlear nucleus (DCN) present endocannabinoid (EC) dependent short-term synaptic plasticity in the form of depolarization-induced suppression of excitation (DSE). Postsynaptic calcium influx promotes EC synthesis and depression of neurotransmission. ECs can be degraded by a hydrolytic and an oxidative pathway, the latter via the enzyme cyclooxygenase 2 (COX-2). Hyperactivity in the DCN is related to the development of tinnitus, which can be induced by high doses of salicylate, a COX-2 inhibitor. Since EC-dependent plasticity in the DCN can affect its excitation-inhibition balance, we investigated the impact of inhibitors of both oxidative and hydrolytic EC metabolism on the DSE from the synapses between the parallel fibers and cartwheel neurons (PF-CW) in the DCN. We found that inhibitors of COX-2 (ibuprofen and indomethacin) did not alter DSE at the PF-CW synapse. Salicylate also did not alter DSE. However, we found that inhibitors of the hydrolytic pathway did not affect DSE magnitude, but surprisingly speeded DSE decay. We conclude that oxidative EC degradation in the PF-CW synapse is not relevant for termination of DSE and are probably not important for controlling this form of synaptic plasticity in the DCN PF-CW synapse. The lack of effect on DSE of high doses of salicylate also suggests that it is not acting by increasing DSE in the PF-CWC synapse. However, the counter intuitive effect of the hydrolytic inhibitors shows that increasing EC on this synapse have more complex effects on DSE.

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.

Glutamate/GABA balance in ACC modulates the nociceptive responses of vocalization: an expression of affective-motivational component of pain in guinea pigs.

Evidence corroborates the role of the anterior cingulate cortex (ACC) in the modulation of cognitive and emotional functions. Its involvement in the motivational-affective component of pain has been widely investigated using different methods to elucidate the specific role of different neurotransmitter systems. We used the peripheral noxious stimulus-induced vocalization algesimetric test to verify glutamatergic and GABAergic neurotransmission in the guinea pig ACC. Microinjection of homocysteic acid (DLH; 30 nmol) in the left guinea pig ACC increased the amplitude of vocalizations (pronociception) compared to controls injected with saline. Moreover, microinjection of MK-801 (3.6 nmol), an NMDA receptor antagonist, did not alter the amplitude of vocalizations, but its microinjection prior to DLH prevented the increase in vocalizations induced by this drug. Regarding the GABAergic system, blockade of GABAA receptors with bicuculline (1 nmol) increased the amplitude of vocalizations, while three different doses of the GABAA agonist muscimol (0.5, 1 and 2 nmol) did not influence nociceptive vocalization responses. Finally, a combination of MK-801 (3.6 nmol) and muscimol (1 nmol) reduced the amplitude of vocalizations (antinociception), suggesting that a combination of glutamate and GABA in the ACC modulates the expression of affective-motivational pain response. We suggest that activation of NMDA receptors or blockade of GABAergic neurotransmission promotes pronociception and that the antinociceptive effect of muscimol depends on the blockade of NMDA receptors.

PEMF fails to enhance nerve regeneration after sciatic nerve crush lesion.

The use of electromagnetic fields has been reported to enhance peripheral nerve regeneration. This study aimed to identify the effects of a prolonged protocol of low-frequency pulsed electromagnetic field (PEMF) on peripheral nerve regeneration. Thirty-four male Swiss mice (Mus musculus) were divided into PEMF (n = 17) and control (n = 17) groups. All animals underwent a unilateral sciatic-crush lesion, and the PEMF group was exposed to a 72-Hz, 2-G electromagnetic field for 30 min, five days a week, for three weeks. Functional analysis was carried out weekly. After three weeks, the animals were euthanized, and histological, morphometric, oxidative stress, and TGF-beta1 analyses were performed. Functional analysis showed no differences between the groups. Histological appearance was similar between PEMF and control nerves. Morphometric assessment showed that the PEMF nerves trended toward decreased regeneration. The levels of free radicals were more pronounced in PEMF nerves, but were not associated with an increase in the content of the TGF-beta1/Smad signaling pathway. Prolonged PEMF regimen leads to delayed histological peripheral nerve regeneration and increased oxidative stress but no loss of function recovery.