The Function And Mechanism Of TRPV1Receptors On Regulating Glutamatergic Synaptic Transmission In Nulceus Accumbens During Morphine Withdrawal Of Rats

Opioids addiction is a chronic and recurrent brain disease, and it not only directlycauses the addicts’ own function damage, mental disorder, and sorts of infectious diseases,but also brings various social problems. Easy to relapse is the biggest problem of thetreatment of opioids addiction. Many studies suggested neural adaptive change (especiallyin brain reward circuits) is the biological basis of relapse after repeated opioids exposure.Nucleus accumbens (NAc) is an important part of the brain reward circuits which iscomposed of the ventral tegmental area, prefrontal cortex and the nucleus accumbens.Nucleus accumbens is mainly involved in the motivated behavior and emotionaladjustment, and is considered to be the gateway for limbic structures to reach the motorsystem.The main neurons in nucleus accumbens are medium spiny neurons (MSN)(account for about95%), interior of nucleus accumbens is a neural network which madeup of neurons connected between the inhibitory synaptic transmission. Excitability of MSN in NAc is low, need the exogenous excitatory glutamatergic synaptic transmission todrive. The drive effect is considered to be the initiating factor of neurons’ synchronizedactivities and coding information in NAc, and is also crucial to adjust activity of NAc andthe relevant motivated behaviors.TRPV1(Transient receptor potential cation channel, subfamily v, member1)receptors which can be activated by heat stimulation, acidic environment, alkaloids,endogenous lipid, etc. are nonselective cation channels. A variety of cationic permeabilityincreased after activation. The receptors play an important role in the occurrence ofpathological pain, spontaneous pain, hyperalgesia, persistent chronic pain, and other fields.In recent years, the effect of TRPV1receptors in the central nervous system was graduallyreceived attention. Recent studies have shown that TRPV1receptors can faciliateglutamatergic transmission without change GABAergic transmission in the striatum,TRPV1receptors in NAc may be closely related to cocaine addiction. However, theeffects of TRPV1receptors in adjusting excitatory synaptic transmission after opioidswithdrawal have not been reported. To explore the fuction of TRPV1receptors in NAc ofrats in opioid addiction and relapse, the whole research is composed of three parts.Part1. The effects of TRPV1receptor agonist and antagonist on sEPSCs of MSN inNAcFirstly, the rats were randomly divided into control and morphine group, andrespectively received repeated intraperitoneal injection of saline (SAL) or morphinehydrochloride (MOR,) for5consecutive days. This protocol was used because it inducesstable morphine-conditioned place preference in rats. For3days or3weeks later after thelast injection, rats were decapitated to make brain slices containing NAc. MSNs in NAccore were hold at-80mV by using infrared visual brain slice patch clamp recordingtechnique， GABA receptor antagonist bicuculline were added to the perfusate,spontaneous excitatory postsynaptic currents(sEPSCs) mediated by glutamate could berecord. Different concertration of TRPV1receptor agonist capsaicin (CAP) were used totest the effects of CAP on sEPSCs, or applied with TRPV1receptor antagonistcapsazepine (CPZ) fistly to test the effects of CPZ on sEPSCs and CAP. Results showed that compared with SAL group,1μM and10μM CAP increasedsEPSCs frequency (P <0.05) in MOR group without affecting the amplitude (P>0.05)during3days withdrawal. After3weeks withdrawal,1μM and10μM CAP increasedsEPSCs frequency (P <0.001) in MOR group without changing the amplitude (P>0.05).During either3days or3weeks withdrawal, CPZ could completely block this effect ofCAP on sEPSCs, and CPZ have no impacts on sEPSCs alone (P>0.05).Part2. The effects of TRPV1receptor agonist and antagonist on eEPSCs and PPR ofMSN in NAcAfter same procedure in part1to obtain brain slices containing NAc, MSNs in NAccore were hold at-80mV by using infrared visual brain slice patch clamp recordingtechnique，GABA receptor antagonist bicuculline were added to the perfusate, stimulatingelectrodes were located on the white matter between the cortex and the NAc, spontaneousexcitatory postsynaptic currents (eEPSCs) and paired pulse ratio (PPR) mediated byglutamate could be recorded at the same time. Different concertration of TRPV1receptoragonist capsaicin (CAP) were used to test the effects of CAP on eEPSCs and PPR, orapplied with TRPV1receptor antagonist capsazepine (CPZ) fistly to test the effects ofCPZ on eEPSCs, PPR and CAP.Results showed that compared with SAL group,10nM and100nM CAP increasedeEPSCs amplitude (P <0.05) in MOR group accompanied with PPR depressed (P <0.05)during3days withdrawal. After3weeks withdrawal,10nM and100nM CAP increasedeEPSCs amplitude (P <0.001) in MOR group accompanied with PPR depressed (P <0.001). During either3days or3weeks withdrawal, CPZ could fully block this effect ofCAP on eEPSCs and PPR, and CPZ have no impacts on eEPSCs and PPR alone (P>0.05).Part3. The effects of endocannabinoids on sEPSCs of MSN in NAc by activatingTRPV1receptorsAfter same procedure in part1to obtain brain slices containing NAc, MSNs in NAccore were hold at-80mV by using infrared visual brain slice patch clamp recordingtechnique，GABA receptor antagonist bicuculline and CB1receptor AM251were added to the perfusate, spontaneous excitatory postsynaptic currents(sEPSCs) of MSN could berecorded. Postsynaptic neurons were depolarized from-80mV to0mV for10seconds togenerate endocannabinoids. We tested the effect of depolarization on sEPSCs. Repeat theabove procedure with appolication of TRPV1receptor antagonist capsazepine CPZ beforeto testthe effect of depolarization on sEPSCs again.Results showed that compared with SAL group, depolarization increased sEPSCsfrequency (P <0.05) in MOR group without affecting the amplitude (P>0.05) during3days withdrawal. After3weeks withdrawal, depolarization increased sEPSCs frequency(P <0.001) in MOR group without changing the amplitude (P>0.05). During either3days or3weeks withdrawal, CPZ could completely block this effect of depolarization onsEPSCs (P>0.05).These results suggest that the function of TRPV1receptors on regulatingglutamatergic synaptic transmission is enhanced in nulceus accumbens during morphinewithdrawal of rats. TRPV1receptors in the nucleus accumbens regulate glutamatergicsynaptic transmission through presynaptic mechanisms after morphine withdrawal.Endocannabinoids can regulate glutamatergic synaptic transmission in the nucleusaccumbens by activating TRPV1receptors during morphine withdrawal.