The Role Of 5-HT₁A Receptor In The Ventrolateral Orbital Cortex Induced Antinociception And Underlying Mechanism

Previous studies in our laboratory have demonstrated that ventrolateral orbital cortex （VLO）, as a high center, is involved not only in nociception, but also in nociceptive modulation. The thalamic nucleus submedius （Sm）, VLO and periaqueductal gray （PAG） consist an endogenous pain modulation pathway. Activation of this pathway depresses the nociceptive inputs at spinal cord level through brainstem descending inhibitory system. Morphological results have demonstrated that serotonergic （5-hydroxytryptamine, 5-HT） neurons in the dorsal raphe nucleus （DR） project their axons bilaterally to the VLO; 5-HT_1A receptors are distributed in the VLO. It is reasonable to propose that 5-HT_1A receptors may be involved in VLO mediating descending antinociception. However, it is well known, the 5-HT_1A receptor is a negative G-protein-coupled receptor and its activation will result in a hyperpolarisation of the cell and produce inhibitory effect. It has been suggested that the excitatory effect induced by activation of 5-HT_1A receptor may be produced by inhibiting an inhibitory GABAergic interneuron （disinhibition）. There is evidence showing that GABAergic neurons and GABAA receptors are distributed in the frontal cortex including the VLO, and the GABAergic neurons and processes in the VLO express 5-HT_1A receptors. Therefore, it is reasonable to propose that VLO 5-HT_1A receptor activation-induced descending antinociception may be produced by inhibition of the inhibitory action of the GABAergic interneuron （disinhibition） on the output neurons projecting to PAG, leading to activation of the brainstem descending inhibitory system and depression of nociceptive inputs at the spinal cord level. To address the hypothesis, in the current study, using radiant heat-evoked tail flick （TF） latency, as an index of nociceptive response, examined whether 5-HT_1A receptors are involved in VLO mediating descending antinociception; using the flinching response and the expression of c-Fos in the spinal dorsal horn induced in formalin test examined whether 5-HT_1Areceptor in the VLO also is involved in modulation of inflammatory tonic nociception; furthermore, using TF test examined whether GABAergic modulation is involved in 5-HT_1A receptor activation-induced descending antinociception in the VLO; using anterograde and retrograde tracing, combined with immunohistochemistry, immunofluorescent, and electron microscopy methods examined the connections between 5-HTergic terminals and GABAergic neurons that may express 5-HT_1A receptor, as well as the connections between GABAergic terminals and output neurons projecting to the PAG that may express GABA_A receptor in the VLO; using the patch-clamp technique on the VLO slices, the spontaneous GABAergic miniature inhibitory postsynaptic currents （mIPSCs） in the VLO were recorded and the effects of 5-HT_1A receptor agonist and antagonist on the mIPSCs were observed. The results are as follows:1. Microinjection of the 5-HT_1A receptor agonist 8-OH-DPAT （1.0, 2.0, 5.0μg） into VLO depressed the TF reflex in a dose-dependent manner （r=0.994, P=0.003）, and this effect was antagonized by the 5-HT_1A receptor antagonist NAN-190 （20μg） applied to the same VLO site, suggesting that the 5-HT_1A receptors are involved in modulation of the acute nociception. Further studies indicated that microinjection of 8-OH-DPAT （5.0μg） into VLO significantly inhibited the flinch in either first phase or second phase, and the expression of c-Fos in the superficial dorsal horn （laminae I-II） and in deep dorsal horn laminae （V-VI） induced by formalin subcutaneously injected into the hindpaw, which was blocked by pre-treatment with the NAN-190 （20μg）. These results suggest that 5-HT_1A receptors in the VLO are involved in modulation of inflammatory tonic nociception.2. Furthermore, microinjection of the GABAA receptor antagonist bicuculline or picrotoxin （100 ng） into the VLO 10 min posterior to 5-HT_1A receptor agonist 8-OH-DPAT injection significantly enhanced the 8-OH-DPAT-induced inhibition of the TF reflex, whereas the GABAA receptor agonist muscimol （250 ng） or THIP （1.0μg） significantly attenuated the 8-OH-DPAT-induced inhibition. These results provide behavioral evidence for GABAergic disinhibitory mechanism is involved in the 5-HT_1A receptor mediated antinociceptive effect in the VLO.3. Results from immunohistochemistry staining indicated that GABAergic neuronal cell bodies and 5-HT_1A receptor-positive neuronal cell bodies with similar morphological characteristics were distributed in the same layers （II-VI） of the VLO. Furthermore, dual-labeling immunofluorescent staining indicated that the majority （93.0%） of GABAergic neurons express 5-HT_1A receptors, and vice versa. Many biotinylated dextranamine （BDA） anterograde-labeled fibers and terminals were 5-HT-positive, and made close connections with GABAergic neurons. Electron microscopic observations further indicated that serotonergic terminals formed symmetric synapses with the majority of GABAergic neurons where 5-HT_1A receptor-positive expression was also detected. These results suggest that serotonergic fibers originating from the DR may modulate activities of GABAergic neurons through presynaptic 5-HT_1A receptor in the VLO. The present results also demonstrated that VLO output neurons （TMR retrograde-labeled neurons in the VLO） projecting to the ventrolateral PAG express GABAA receptor, and also made close connections with GABAergic terminals. The triple-labeled electron microscopic observations further indicated that GABAergic terminals formed symmetric synapses with the majority of the VLO output neurons to the PAG, which expressed GABAA receptors. These results suggest that inhibitory GABAergic interneurons or terminals via GABAA receptors may regulate the function of the VLO-PAG-brainstem descending inhibitory system within the VLO. Together with these results, the present study demonstrated a local neuronal circuit within the VLO, comprised of serotonergic afferent terminals, 5-HT_1A receptor, GABAergic interneurons, GABAA receptor, and projection neurons to the PAG. It provides morphological evidence for the hypothesis that GABAergic modulation is involved in 5-HT_1A receptor activation-evoked descending antinociception in the VLO.4. Pharmacologically separated spontaneous GABAergic mIPSCs in the VLO were recorded, and then the effects of 8-OH-DPAT （1.0μM）, a selective 5-HT_1A receptor agonist and NAN-190 （1.0μM）, a selective 5-HT_1A receptor antagonist on the mIPSCs were observed. The results showed that 8-OH-DPAT inhibited the mIPSCs frequency （68.4±18.9% of control, P < 0.05） without affecting mIPSCs amplitude （P > 0.05）. NAN-190 per se did not affect frequency or amplitude of the mIPSCs （P > 0.05）, but it blocked the 8-OH-DPAT-evoked inhibition of mIPSCs frequency （P < 0.05）. These electrophysiological results suggest that presynaptic 5-HT_1A receptor activation can inhibit the GABA transmitter release which may lead to activation of the descending antinociceptive pathway and depression of the nociceptive inputs at the spinal level.In conclusion, 5-HT_1A receptors in the VLO are involved in modulation of acute and inflammatory persistent nociception, and that GABAergic modulation participates in this 5-HT_1A receptor mediated effect.