The Descending Modulatory Effects Of Dorsal Medial Prefrontal Cortex On Pain And Its Mechanism

The people who have chronic pain suffered not only the severe pain perception,but also the negative emotions and declined cognitive abilities.Intractable and permanent pain condition easily developed into a ‘chronic pain recycle': chronic pain can induce negative emotion including anxiety and depression,sleep disorder,and reduced movement and can also affect the cognitive abilities,vice versa.Negative emotion and reduced cognitive abilities can worsen chronic pain.Although there are many studies focused on the emotional component of pain and tried to find out the neural pathway for emotional pain.However,the underlying mechanisms for the ‘chronic pain recycle' are still unclear.What is the most important to answer this question is to find out the nucleus,which is involved into the pain,emotion,and cognitive ability modulation.Dorsal medial prefrontal cortex(dm PFC)including rostral anterior cingulate cortex(r ACC)and prelimbic cortex(PL)is the nucleus involved into these processes modulations.There are many studies have demonstrated that dm PFC participated into the negative emotion and declined cognitive abilities.A few studies also suggest that dm PFC is related to the pain perception.However,the exact neural pathway coming from the dm PFC involved into the pain modulation is not reported.The pathway periaqueductal gray(PAG)-rostral ventromedial medulla(RVM)-spinal dorsal horn(SDH)is one of the most important neural pathway involved into the descending pain modulation.The investigation about the superior projection to the PAG involved into the pain modulation is rare.Is there the possibility that dm PFC projecting to vl PAG participate into the descending modulation,which is the most related division to pain in PAG?To fulfill this aim,many various kinds of morphological approaches were used to establish the neural pathway dm PFC-vl PAG and investigate the inputs of dm PFC and the outputs of vl PAG.Chemical damages,optogenetic,chemogenetic,molecular biology and behavioral pharmacological approaches were applied to illustrate the involvement of dm PFC-vl PAG into the pain modulation and negative emotion.Thus,this study can be divided into the following two sections:1.The morphological studies of dmPFC-vlPAG neural pathway involved into the descending pain modulationObjective: To reveal the dm PFC-vl PAG neural pathway and investigate the inputs of PV-ir neurons in the dm PFC and projections from the vl PAG to the RVM.Methods: 1)The projections from the dm PFC to the vl PAG,the vl PAG to the RVM,and the RVM to the SDH were observed by using tracing methods.Meanwhile,we also investigated the chemical properties of these projection neurons with the help of fluorescence immunostaining or in situ hybridization.2)Adeno-associated virus(AAV)was injected into the dm PFC and the fibers of dm PFC-vl PAG projection neurons were observed in the vl PAG.The locations of these infected fibers and vl PAG-RVM projection neurons were also checked.3)The rabies virus labeling system was used to study the inputs of VGLUT2-ir neurons in the vl PAG,PV-ir neurons in the dm PFC,and 5-HT-ir neurons in the RVM,which could label presynaptic neruons.Results: 1)Retrograde tracer Fluoro-Gold(FG)was injected into the vl PAG.A lot of FG-labeled projection neurons can be observed in the dm PFC,which were also Ca MKII-immunoreactivity and expressed VGLUT1 m RNA.Moreover,the FG-ir neurons cannot express VGLUT2 m RNA and GAD67,a marker for the inhibitory interneurons.Then,the anterograde tracer biotinylated dextran amine(BDA)was injected into the dm PFC and tons of fibers can be observed in the vl PAG.For further confirmation,AAV2/2-Ca MKII?-EYFP was injected into the dm PFC to infect the excitatory pyramidal neurons.After 4 weeks,a lot of EYFP-labeled fibers can be observed in the vl PAG,which belongs to the infected pyramidal neurons in the dm PFC.Helper viruses(AAV2/9-DIO-GFP-TVA and AAV2/9-DIO-G)and RV-Env A-?G-ds Red was injected into the vl PAG of VGLUT2-Cre mice,separately.Many ds Red trans-synaptic labeled projection neurons in the dm PFC can be observed.There exists the descending neural pathway dm PFC-vl PAG.2)What is similar with the above methods,helper viruses and RV-Env A-?G-ds Red was injected into the dm PFC of PV-Cre mice,separately.The ds Red trans-synaptic labeled projections neurons were observed in some cortex regions,including m PFC,ACC,insular cortex(IC),claustrum(CLA).The ds Red trans-synaptic labeled projections neurons were also found in some nuclei of the thalamus,such as paraventricular nucleus of thalamus(PVT),mediodorsal nucleus of thalamus(MD),paratenial nucleus of thalamus(PT),centrolateral nucleus of thalamus(CL),ventromedial nucleus of thalamus(VM),rhomboid nucleus of thalamus(Rh),and reuniens nucleus of thalamus(Re).There were also some ds Red trans-synaptically and retrogradely labeled neurons in the bed nucleus of stria terminalis(BNST),basal lateral nucleus of amygdala(BLA)and hippocampus.3)Helper viruses and RV-Env A-?G-ds Red was injected into the RVM of Sert-Cre mice,separately.The ds Red trans-synaptically labeled projection neurons distributed in the vl PAG.By using fluorescence in situ hybridization and immunostaining,the FG-labeled RVM projection neurons in the vl PAG can be observed and most of them express VGLUT2 m RNA.What is more,the FG-labeled SDH projection neurons in the RVM expressing 5-HT can receive a lot of VGLUT2-ir contacts.AAV2/2-Ca MKII?-EYFP was injected into the dm PFC and FG was injected into the RVM.FG-labeled RVM projection neurons in the vl PAG receive the EYFP-labeled fiber contacts.Conclusion: There was an excitatory neural pathway from dm PFC to vl PAG,which used glutamate as neurotransmitter and acted on VGLUT2-ir neurons in the vl PAG.The 5-HT-ir neurons in the RVM can receive the projection from VGLUT2-ir neurons in the vl PAG.A Top-Down(dm PFC-vl PAG-RVM-SDH)neural pathway was revealed.Meanwhile,the PV-ir neurons in the dm PFC received intense projections from the nucleus,which were closely related to the nociceptive information transmission.2.The functional studies of dm PFC-vl PAG neural pathway involved into the descending pain modulationObjective: To observe the effects of silencing or activating the dm PFC-vl PAG neural pathway on the pain and anxiety behaviors by using different methods and reveal the underlying molecular mechanisms of dm PFC involved into the chronic pain.Methods: 1)The common peroneal nerve ligation(CPNL)model was established to observe the pain and anxiety behaviors.2)The mechanical thresholds of hind paw were tested by Von Frey filaments and the anxiety behaviors were observed in the open field tesst and elevated plus maze test.3)The pain and anxiety behaviors were also checked after chemical lesions of dm PFC by kainic acid(KA),optogenetic activation of dm PFC-vl PAG pathway,and chemogenetic activation of Vgat-ir neurons in the dm PFC.4)Western blot was used to check the expressions of GABAAR and m Glu R1 in the dm PFC.The antagonists of GABAAR and m Glu R1 were injected into the dm PFC to observe the effects on the pain and anxiety behaviors.Results: 1)KA was injected into the bilateral dm PFC and all kinds of neurons in the dm PFC died due to the over excitation.The pain threshold of mice in the normal condition reduced,and the total distance and time in the central area(% of the total time)in the open field test were also reduced.Meanwhile,the entries into the open arms(% of the total entries)and time spent in the open arms(% of the total time)both decreased in the elevated plus maze test.The dm PFC lesions can also reduce the mechanical threshold of mice received CPNL and increase the responses to the innociceptive stimulus.But,these lesions did not worsen the behavior in the open field and elevated plus maze.2)AAV2/2-Ca MKII?-h Ch R2-EYFP was injected into the dm PFC and fiber was implanted into the vl PAG.By using 473 nm blue light 10 m W 20 Hz to stimulate,the mechanical hyperalgesia of bilateral hindpaw can be significantly reversed on mice with CPNL 7 day.Moreover,the reduced time spent in the open arms(% of total time)and entries into the open arms(% of total entries)were also reversed on mice with CPNL 14 day.However,the behavior in the open field was not improved.3)AAV2/4-hSyn-DIO-HA-hM3Dq-mCitrin was injected into the bilateral dmPFC of Vgat-Cre mice,and then the behavior was tested following intraperitoneal(i.p.)injection of 1 mg/kg clozapine-N-oxide(CNO)30 min.The pain threshold of mice in the normal condition,the total distance and time in the central area(% of the total time)in the open field and the time spent in the open arms(% of the total time)and entries into the open arms(% of the total entries)in the elevated plus maze were all reduced,after bilateral activating inhibitory interneurons in the dm PFC.These inhibition of dm PFC-vl PAG neural pathway also increased the mechanical hyperalgesia of bilateral hindpaw on mice with CPNL 7 day.Moreover,time in the central area(% of the total time)in the open field and the time spent in the open arms(% of the total time)and entries into the open arms(% of the total entries)in the elevated plus maze were further reduced after CPNL 14 day.4)By using Western blot to test the expressions of GABAAR and m Glu R1,significantly increasing of GABAAR and m Glu R1 expressions can be observed in the dm PFC.The cannula was implanted into the dm PFC and used to inject the specific antagonists of GABAAR and m Glu R1.The injections of bicuculline and LY367385 both rescued the mechanical hyperalgesia of bilateral hindpaw on mice with CPNL 7 day.The total distance and time in the central area(% of the total time)in the open field and the time spent in the open arms(% of the total time)and entries into the open arms(% of the total entries)in the elevated plus maze were increased after CPNL 14 day.Conclusion: The dmPFC-vlPAG play important roles in maintaining the mechanical threshold of paw and normal behavior in the open field and elevated plus maze.The mechanical thresholds of hind paw were reduced at both normal and chronic pain condition,after the dm PFC-vl PAG pathway was silenced by chemical lesions or specific activation of inhibitory neurons in the dm PFC.Meanwhile,the increased responses to the mechanical stimuli and anxiety behaviors were also observed.The activation of inhibitory neurons in the dm PFC even worsen the negative emotion after chronic pain.The analgesic and anxiolytic effects were observed after activating the dm PFC-vl PAG pathway.GABAAR and m Glu R1 in the dm PFC was involved into the initial and maintenance of chronic pain and negative emotion.