The Expression And Function Of G Protein Coupled Estrogen Receptor GPER-1 In Pain-Modulating Neurons In The Rostral Ventromedial Medulla

The rostral ventromedial medulla(RVM)is a core component of the descending pain modulatory pathway and an important site of morphine analgesia.Three functional types of neurons could be identified according to their responses to noxious stimulation,namely ON,OFF and Neutral cells.ON cells increase their firing frequency whereas Off cells decrease their activity upon noxious stimulation and hence play opposing roles in pain modulation.RVM is composed of the nucleus raphe magnus(NRM),nucleus gigantocellularis reticularis(Gi),nucleus lateral paragigantocellularis(LPGi)and nucleus gigantocellularis reticularis alpha(GiA).They project serotonergic fibers to the spinal cord,which constitute the serotonergic desending inhibitory pathways.Estrogen is an important sex hormone and play a multitude of biological functions via activation of its receptors including ER?,ER? and G-protein-coupled estrogen receptor-1(GPER-1).A large number of literature suggest that estrogen may play a role in regulation of pain,yet the site of action and the receptors involved remain ambiguous.Recently,our laboratory found that estrogen(E2)applied to RVM led to progressive increases in colorectal distension(CRD)-induced visceromotor reflex(VMR)as well as diminished morphine analgesic effects.Extracellular recording in the rat in vivo showed that E2 was able to selectively activate ON cells and to ameliorate morphine-induced inhibition.Furthermore,immunohistochemistry and western blot analysis showed that RVM express GPER-1 in abundance but not ER? or ER?.These results suggest that estrogen may enhance the activity of the descending pain facilitatory pathway via activation of GPER-1 in RVM.We hypothesize that GPER-1 might be selectively expressed in ON cells and estrogen might bind to GPER-1 to directly(post-synaptically)increase the excitability of ON cells.The current study has been designed to test this hypothesis.Firstly,double immunohistochemical labelling was performed to delineate the relationship between GPER-1 positive neurons and the serotonergic neurons.Secondly,intracellular recording was performed in the rat in vivo to identify and to label ON,OFF and Neutral cells and subsequently to study the experssion of GPER-1 on different types of neurons through immunohistochemistry.Thirdly,using the piggyback electrode,the effects of estrogen on membrane potential of ON cells were investigated in the rat in vivo.1.The distribution of 5-HT neurons and GPER-1 positive neurons in RVMImmunohistochemical staining for TPH,the rate limiting enzyme in the formation of 5-HT,was conducted on serial coronal sections of the medulla.The distance to the ventral margin and the medial line was measured for each TPH-positive neurons.The coordinates were used to plot a graph showing the distribution profile of serotonergic neurons in the ventral medulla.We found that TPH-positive neurons were detectable in the ventral medulla between 9.0 and 13.5 mm posterior to the bregma.The bulk of TPH positive neurons were found between 11.1 and 11.8 mm posterior to bregma.Double immunostaining for GPER-1 and 5-HT were performed.We found that GPER-1 positive and 5-HT positive neurons were mutually exclusive,ie,being different groups of neurons intermingled in RVM.Some 5-HT fibers with typical varicosity morphology were found to be very close to GPER-1 positive neuronal cell body,which was suggestive of non-typical synaptic contacts between the two types of neurons.2.Expression of GPER-1 on functionally-identified neurons in RVMIntracellular recordings were performed in RVM between 11.0-12.00 mm posterior to bregma in pentobarbital-anesthetized rats and the neurons were classified as ON,OFF or Neutral cells according to their response to noxious pinch of the hind leg or colorectal distension(CRD).Neurobiotin was then injected via ionophoresis.The medulla was subsequently processed for immunohistochemical staining of neurobiotin,GPER-1,TPH or the ? type opioid receptor(MOR).We found that GPER-1 was selectively expressed in ON cells,which also express MOR but not TPH.TPH was expressed in a fraction of OFF cells.3.The effects of estrogen on the membrane potential of pain-modulating neurons in RVM.Intracellular recording of ON cells were made utilising the "piggyback" electrodes in pentobarbital-anesthetized rats.17?-E2 was applied by microionophoresis and was found to result in a rapid increase in the firing rate of ON cells.This effect was due to membrane potential depolarization and increased amplitude of EPSPs.This set of data strongly indicate that estrogen may directly activate ON cells and hence to enhance descending facilitation of pain.In summary,the present study showed that GPER-1 positive neurons and serotonergic neurons are two different neuronal groups.Combining intracellular recording in vivo,immunohistochemistry and microelectrophoresis,we were able to show that GPER-1 was selectively expressed in ON cells and that estrogen was able to depolarize the membrane potential and to augment the amplitude of EPSPs of ON cells.These results strongly suggest that estrogen may enhance descending facilitation of pain via activation of GPER-1 in RVM and may play a role in the genesis or maintenance of central hyperalgesia.