Effects Of Endocannabinoids In The Transmission And Modulation Of Itch Signals In Spinal Dorsal Horn

BackgroundItch is a somatic sensation which induces people to scratch and elicits an unpleasant feeling and emotional experience[1].According to the duration of the unpleasant feeling,itch can be categorized into acute itch(less than 6 weeks)and chronic itch(longer than 6 weeks)[2].Chronic itch can lead to the decrease in quality of life due to persistent scratch responses.Being lack of effective therapeutic interventions in clinical practice,it is a kind of chronic disorder that seriously affects the physical and mental health of patients.As a result,illuminating the physiological and pathological mechanisms of itch can provide the theoretical basis for developing efficient anti-itch drugs.Similar with the transmission process of pain information,itch signals are transmitted from peripheral receptors to the spinal interneurons and then being ascended to the brain[4-9].Chen et al verified that spinal excitatory interneurons which express gastrin releasing peptide receptor(GRPR)could specifically mediate itch transmission[10].Simultaneously,inhibitory interneurons which release inhibitory neurotransmitters including GABA and glycine(Gly)could mediate the modulation of itch information through acting on GRPR neurons[11,12].Therefore,the neural circuitry of the spinal cord,which consists of GABA and Gly inhibitory neurons and GRPR excitatory neurons,plays an important role in the transmission and regulation of itch information.However,the detailed mechanism involved in the spinal circuits is not very clear.Endocannabinoid system(ECS)could engage in multiple physiological process in central and peripheral nervous system including learning and pain[13].ECS were mainly composed of two types of ligands including anandamide(AEA)and 2-arachidonylglycero(2-AG),two types of receptors cannabinoid receptorl(CB1R)and cannabinoid receptor 2(CB2R),and diverse kinds of enzyme and transporters[14].Previous research have shown that endocannabinoids(eCBs)could regulate itch transmission by either promoting or suppressing itch signals[15,16]However,the concrete mechanism of eCBs in the modulation of itch is still unknown.Current evidence mainly focuses on pharmacological aspect of pruritus,the concrete neuron types together with circuits involved in itch are still unclear.In this study,we applied transgenic mice combined with behavioral,electrophysiological and morphological methods to illuminate the neural circuits by which eCBs modulate itch.This study will further provide basis for finding treatment target for refractory pruritus.Part1 Modulative effects of eCBs on non-histaminergic and chronic itch through activation of CB1 receptor on Glycine neuronsObjective:Different itch models were established to observe the effect of eCBs on diverse types of itch.Methods:Histaminergic,nonhistaminergic acute itch and chronic itch models were established to observe the role of different concentration of eCBs on itch.CB1R antagonist,NESS0327 was injected to clear whether it can block the role of eCBs in itch modulation.Itch models including histaminergic,nonhistaminergic itch and chronic itch were established on mice of which CB1R on inhibitory interneurons were ablated.Difference of scratching between KO and WT group was monitored.Results:(1)Low concentration of 2-AG(1μg/10μl)has no effects on diverse kind of itch.High concentration of eCBs(10μg/10μl)have no effects on histaminergic itch.However,nonhistaminergic itch and chronic itch were significantly attenuated.(2)Inhibition of CB1R could block the anti-itch effects of 2-AG.(3)Ablation of CB1R on GABAergic neurons has no effects on both histaminergic itch,non-histaminergic itch and chronic itch.Ablation of CB1R on Glycinergic neurons enhanced both non-histaminergic itch and chronic itch.Part 2 The morphological and electrophysiological features of Gly-GABA inhibitory circuitObjective:The distribution,morphological,physiological features and concrete type of primary inputs of GABAergic neurons in the spinal cord were observed by constructing Gad2-tdTomato mice.The synaptic connection between Glycinergic and GABAergic neurons was verified by combining with retrograde tracing technology.Methods:Mice whose GABAergic neurons were specifically marked with fluorescence were established.The overlay of fluorescence and of spinal GABAergic neurons were firstly confirmed.The GABAergic neurons in Gad2-tdTomato mice were patched through parasagittal slices to clear the distribution,morphological features and electrophysiological properties.By stimulating the dorsal roots with different intensities,the excitatory or inhibitory inputs received by GABAergic neurons were recorded.Through pharmacological methods,the inhibitory inputs recorded from GABAergic neurons were categorized into glycinergic or GABAergic by applying bicuculine and strychnine into recording solution to block glycine or GABA receptor mediated inhibitory components.Retrograde tracing technology was performed on Gad2-Cre mice to mark presynaptic neurons of spinal GABAergic neurons.IHC methods were applied to examine the colocalization between fluorescence-marked presynaptic neurons and GlyT2 antibody.Results:(1)GABAergic neurons accounted 75.9%of td-Tomato fluorescence labeled neurons.(2)GABAergic neurons in the spinal cord mainly distributed in lamina II and displayed tonic firing pattern dominantly and the morphological features of GABAergic neurons were mainly islet cells.(3)GABAergic neurons received excitatory primary inputs mediated by Aβ,Aδ and C fibers.Besides,purely inhibitory postsynaptic potentials of Aδ and C fibers were also observed.(4)C fiber mediated inhibitory postsynaptic potentials were mainly Glycinergic while Aδfiber mediated inhibitory postsynaptic potentials were mainly glycinergic mixed with GABAergic.(5)Neurons which transmit signals to spinal GABAergic neurons co-expressed with antibody marking glycinergic neurons.Part 3 The anti-itch effect of eCBs was through Gly-GABA inhibitory circuitObjective:Diverse itch models were established to test whether the regulation of eCBs on itch is through the connection between glycinergic and GABAergic neurons combining with immunohistochemistry and behavioral test.Methods:Diverse itch models were established on GlyT2-CB 1R-KO and GlyT2-CB1R-WT mice.The spinal tissue was then separated with immunohistochemistry methods.By immersing spinal slices with c-Fos and Gad65 antibody,the difference of activation ratio between two groups were analyzed.Nonhistaminergic and chronic itch were established on GlyT2-CB1R-KO mice and the spinal cord of which were injected with GABA.The scratching numbers before and after the application of GABA on GlyT2-CB1R-KO groups were compared.Meanwhile,number of scratching between GlyT1-CB1R-WT and GlyT2-CB1R-KO mice injected with GABA were compared.Results:(1)Immunohistochemistry results showed that the activation ratio of GABAergic neurons which were inhibited by Glycinergic neurons in GlyT2-CB1R-KO mice were significantly attenuated in nonhistaminergic itch.In histaminergic itch model,activation ration of GABAergic neurons between two groups have no statistical significance.In chronic itch model,activation ratio of GABAergic neurons in KO group also decreased.(2)The scratching of KO groups was significantly alleviated by applying exogenous GABA in nonhistaminergic and chronic itch.Meanwhile,number of scratches between KO groups which was injected with GABA and WT group were comparable.ConclusionsThe above results confirmed that eCBs could inhibit non-histaminergic itch and chronic itch.Combining electrophysiological recording with retrograde tracing methods,the current research first testified the existence of synaptic connection between Glycinergic and GABA neurons in the spinal cord and then furtherly verified its role in the modulation of eCBs on itch transmission.2-AG could bind its receptor on the Glycinergic neurons which decreased the release of glycine.This process alleviated glycinergic inhibition on GABAergic neurons which subsequently increased the inhibitory effects of GABAergic neurons by releasing a large amount of GABA on GRPR neurons and suppressed nonhistaminergic and chronic itch.