Stimulating GABAergic Neurons in the Nucleus Accumbens Core Alters the Trigeminal Neuropathic Pain Responses in a Rat Model of Infraorbital Nerve Injury.

The nucleus accumbens core (NAcc) is an important component of brain reward circuitry, but studies have revealed its involvement in pain circuitry also. However, its effect on trigeminal neuralgia (TN) and the mechanism underlying it are yet to be fully understood. Therefore, this study aimed to examine the outcomes of optogenetic stimulation of NAcc GABAergic neurons in an animal model of TN. Animals were allocated into TN, sham, and control groups. TN was generated by infraorbital nerve constriction and the optogenetic virus was injected into the NAcc. In vivo extracellular recordings were acquired from the ventral posteromedial nucleus of the thalamus. Alterations of behavioral responses during stimulation "ON" and "OFF" conditions were evaluated. In vivo microdialysis was performed in the NAcc of TN and sham animals. During optogenetic stimulation, electrophysiological recordings revealed a reduction of both tonic and burst firing activity in TN animals, and significantly improved behavioral responses were observed as well. Microdialysis coupled with liquid chromatography/tandem mass spectrometry analysis revealed significant alterations in extracellular concentration levels of GABA, glutamate, acetylcholine, dopamine, and citrulline in NAcc upon optic stimulation. In fine, our results suggested that NAcc stimulation could modulate the transmission of trigeminal pain signals in the TN animal model.

Activation of CamKIIα expressing neurons on ventrolateral periaqueductal gray improves behavioral hypersensitivity and thalamic discharge in a trigeminal neuralgia rat model.

BACKGROUND: Preceding studies have reported the association of chronic neuropathic orofacial pain with altered ongoing function in the ventrolateral periaqueductal gray (vlPAG). However, its role in trigeminal neuralgia (TN) lacks attention. We here reported the aspect that vlPAG neurons play in TN nociceptive processing by employing excitatory neuron-specific optogenetic approaches. METHODS: TN was generated via unilateral infraorbital nerve chronic constriction in Sprague Dawley rats which induced mechanical and thermal pain sensitivity in air puff and acetone test, respectively. Channelrhodopsin conjugated virus with CamKIIα promoter was used to specifically activate the excitatory vlPAG neuronal population by optogenetic stimulation and in vivo microdialysis was done to determine its effect on the excitatory-inhibitory balance. In vivo extracellular recordings from ventral posteromedial (VPM) thalamus were assessed in response to vlPAG optogenetic stimulation. Depending on the experimental terms, unpaired student's t test and two-way analysis of variance (ANOVA) were used for statistical analysis. RESULTS: We observed that optogenetic activation of vlPAG subgroup neurons markedly improved pain hypersensitivity in reflexive behavior tests which was also evident on microdialysis analysis with increase glutamate concentration during stimulation period. Decreased mean firing and burst rates were evident in VPM thalamic electrophysiological recordings during the stimulation period. Overall, our results suggest the optogenetic activation of vlPAG excitatory neurons in a TN rat model has pain ameliorating effect. CONCLUSIONS: This article presents the prospect of pain modulation in trigeminal pain pathway via optogenetic activation of vlPAG excitatory neurons in rat model. This outlook could potentially assist vlPAG insight and its optogenetic approach in trigeminal neuropathic pain which aid clinicians endeavoring towards enhanced pain relief therapy in trigeminal neuralgia patients.

RhoA downstream of G(q) and G(12/13) pathways regulates protease-activated receptor-mediated dense granule release in platelets.

Platelet secretion is an important physiological event in hemostasis. The protease-activated receptors, PAR 1 and PAR 4, and the thromboxane receptor activate the G(12/13) pathways, in addition to the G(q) pathways. Here, we investigated the contribution of G(12/13) pathways to platelet dense granule release. 2MeSADP, which does not activate G(12/13) pathways, does not cause dense granule release in aspirin-treated platelets. However, supplementing 2MeSADP with YFLLRNP (60muM), as selective activator of G(12/13) pathways, resulted in dense granule release. Similarly, supplementing PLC activation with G(12/13) stimulation also leads to dense granule release. These results demonstrate that supplemental signaling from G(12/13) is required for G(q)-mediated dense granule release and that ADP fails to cause dense granule release because the platelet P2Y receptors, although activate PLC, do not activate G(12/13) pathways. When RhoA, downstream signaling molecule in G(12/13) pathways, is blocked, PAR-mediated dense granule release is inhibited. Furthermore, ADP activated RhoA downstream of G(q) and upstream of PLC. Finally, RhoA regulated PKCdelta T505 phosphorylation, suggesting that RhoA pathways contribute to platelet secretion through PKCdelta activation. We conclude that G(12/13) pathways, through RhoA, regulate dense granule release and fibrinogen receptor activation in platelets.

Insulin-like growth factor-1 regulates platelet activation through PI3-Kalpha isoform.

Platelets release insulin-like growth factor-1 (IGF-1) from alpha granules upon activation. We have investigated the regulation of IGF-1 in G(i)-dependent pathways leading to Akt activation and the role of IGF-1 in platelet activation. IGF-1 alone failed to induce platelet aggregation, but IGF-1 potentiated 2-MeSADP-induced platelet aggregation in a concentration-dependent manner. IGF-1 triggered platelet aggregation in combination with selective P2Y(1) receptor activation. IGF-1 also caused platelet aggregation without shape change when combined with selective G(z) stimulation by epinephrine, suggesting the role of IGF-1 in platelet aggregation by supplementing G(i) pathways. The potentiating effect of IGF-1 was not affected by intracellular calcium chelation. Importantly, IGF-1 was unable to potentiate platelet aggregation by the phosphatidylinositol 3-kinase (PI3-K) inhibitor wortmannin, suggesting a critical regulation by PI3-K. Moreover, the potentiating effect of IGF-1 was abolished by the presence of PI3-K p110alpha inhibitor PIK-75. Stimulation of platelets with IGF-1 resulted in phosphorylation of Akt, a downstream effector of PI3-K, which was completely inhibited by wortmannin. IGF-1-induced Akt phosphorylation was abolished by PIK-75 suggesting the contribution of PI3-K p110alpha for activation of Akt by IGF-1. These results demonstrate that IGF-1 plays a role in potentiating platelet aggregation by complementing G(i)- but not G(q)-signaling pathways via PI3-K p110alpha.

Relative contribution of G-protein-coupled pathways to protease-activated receptor-mediated Akt phosphorylation in platelets.

Protease-activated receptors (PARs) activate Gq and G(12/13) pathways, as well as Akt (protein kinase B [PKB/Akt]) in platelets. However, the relative contribution of different G-protein pathways to Akt phosphorylation has not been elucidated. We investigated the contribution of Gq and G(12/13) to Gi/Gz-mediated Akt phosphorylation downstream of PAR activation. Selective G(12/13) activation failed to cause Akt phosphorylation in human and Galpha q-deficient mouse platelets. However, supplementing Gi/Gz signaling to G(12/13) caused significant increase in Akt phosphorylation, confirming that G(12/13) potentiates Akt phosphorylation. Inhibition of PAR-mediated Akt phosphorylation in the presence of the Gq-selective inhibitor YM-254890 was restored to the normal extent achieved by PAR agonists if supplemented with Gi signaling, indicating that Gq does not have any direct effect on Akt phosphorylation. Selective G(12/13) activation resulted in Src kinase activation, and Akt phosphorylation induced by costimulation of G(12/13) and Gi/Gz was inhibited by a Src kinase inhibitor but not by a Rho kinase inhibitor. These data demonstrate that G(12/13), but not Gq, is essential for thrombin-induced Akt phosphorylation in platelets, whereas Gq indirectly contributes to Akt phosphorylation through Gi stimulation by secreted ADP. G(12/13) activation might mediate its potentiating effect through Src activation, and Src kinases play an important role in thrombin-mediated Akt phosphorylation.

Plasmin-mediated activation of platelets occurs by cleavage of protease-activated receptor 4.

The activation of plasmin from its circulating precursor plasminogen is the mechanism of several clot-busting drugs used to clinically treat patients who have suffered a stroke; however, plasmin thus generated has been shown to activate platelets directly. There has been speculation as to whether plasmin interacts with the protease-activated receptors (PARs) because of its similarity in amino acid specificity with the classic platelet activator thrombin. We have investigated whether plasmin activates platelets via PAR activation through multiple complementary approaches. At concentrations sufficient to induce human platelet aggregation, plasmin released very little calcium compared with that induced by thrombin, the PAR-1 agonist peptide SFLLRN, or the PAR-4 agonist peptide AYPGKF. Stimulation of platelets with plasmin initially failed to desensitize additional stimulation with SFLLRN or AYPGKF, but a prolonged incubation with plasmin desensitized platelets to further stimulation by thrombin. The desensitization of PAR-1 had no effect on plasmin-induced platelet aggregation and yielded an aggregation profile that was similar to plasmin in response to a low dose of thrombin. However, PAR-4 desensitization completely eliminated aggregation in response to plasmin. Inclusion of the PAR-1-specific antagonist BMS-200261 inhibited platelet aggregation induced by a low dose of thrombin but not by plasmin. Additionally, mouse platelets naturally devoid of PAR-1 showed a full aggregation response to plasmin in comparison to thrombin. Furthermore, human and mouse platelets treated with a PAR-4 antagonist, as well as platelets isolated from PAR-4 homozygous null mice, failed to aggregate in response to plasmin. Finally, a protease-resistant recombinant PAR-4 was refractory to activation by plasmin. We conclude that plasmin induces platelet aggregation primarily through slow cleavage of PAR-4.

Akt activation in platelets depends on Gi signaling pathways.

The serine-threonine kinase Akt has been established as an important signaling intermediate in regulating cell survival, cell cycle progression, as well as agonist-induced platelet activation. Stimulation of platelets with various agonists including thrombin results in Akt activation. As thrombin can stimulate multiple G protein signaling pathways, we investigated the mechanism of thrombin-induced activation of Akt. Stimulation of platelets with a PAR1-activating peptide (SFLLRN), PAR4-activating peptide (AYPGKF), and thrombin resulted in Thr308 and Ser473 phosphorylation of Akt, which results in its activation. This phosphorylation and activation of Akt were dramatically inhibited in the presence of AR-C69931MX, a P2Y12 receptor-selective antagonist, or GF 109203X, a protein kinase C inhibitor, but Akt phosphorylation was restored by supplemental Gi or Gz signaling. Unlike wild-type mouse platelets, platelets from Galphaq-deficient mice failed to trigger Akt phosphorylation by thrombin and AYPGKF, whereas Akt phosphorylation was not affected by these agonists in platelets from mice that lack P2Y1 receptor. However, ADP caused Akt phosphorylation in Galphaq- and P2Y1-deficient platelets, which was completely blocked by AR-C69931MX. In contrast, ADP failed to cause Akt phosphorylation in platelets from mice treated with clopidogrel, and thrombin and AYPGKF induced minimal phosphorylation of Akt, which was not affected by AR-C69931MX in these platelets. These data demonstrate that Gi, but not Gq or G12/13, signaling pathways are required for activation of Akt in platelets, and Gi signaling pathways, stimulated by secreted ADP, play an essential role in the activation of Akt in platelets.