Study Of The Relationship Between SNAPs And Morphine Dependence

Opioid dependence is a chronic relapsing disorder in the brain characterized by compulsive drug taking, inability to limit the intake of drugs, and the emergence of withdrawal syndrome after cessation of drug taking. Nevertheless, the exact mechanisms underlying opioid dependence are still unknown. Accumulating evidence suggests that synaptic plasticity plays an important role in opioid dependence. These changes include presynaptic neurotransmitter release and postsynaptic receptor signal transduction. Previous studies demonstrated that morphine could affect the synaptic transmission by regulating molecules related with neurotransmitter release in presynapse.Membrane fusion between synaptic vesicle membrane and presynaptic membrane has been generally received as an important step in the process of neurotransmitter release. NSF attachment proteins (SNAPs) play important roles in membrane fusion by mediating the fusion between synaptic vesicle membrane and presynaptic membrane. Three isoforms (α-,β-, andγ-) of SNAP are expressed in mammals. In view of its function in mediating membrane fusion, SNAPs take part in many important physiological processes, including regulating Ca2+-dependent exocytosis, stimulating insulin secretion in pancreatic islandβcells, increasing surfactant secretion in alveolar type II cells, participating in the nuclear envelope formation, etc.Although considerable evidence suggests that SNAPs play key roles in the process of neurotransmitter release by mediating membrane fusion between plasma membrane and vesicle membrane, whether SNAPs are related to the presynaptic plasticity underlying opioid dependence has been not reported. To reveal the relationship between SNAPs and opioid dependence, we investigated the expression of SNAPs in different brain regions of rats with chronic morphine exposure and withdrawal, including nucleus accumbens (NAc), caudate putamen (CPu) and hippocampus (Hip), which were recognized to have close relationship with synaptic plasticity. Adult male Wistar rats were randomly assigned into control, morphine and spontaneous withdrawal group. The morphine dependent rat model was established by subcutaneous morphine injection with increasing doses for 8d, three times a day (8:00, 12:00, and 18:00). The control group was injected with the same volume of normal saline. Rats in morphine group were killed 4h after the last injection, rats in withdrawal group were killed at indicated time. Control groups were paralleled with all treatment groups. The NAc, CPu and Hip were isolated. The expression of SNAPs mRNA and protein were determined by RT-PCR and Western blot, respectively. Results showed that the expression ofγ-SNAP in CPu of morphine dependent rats was up-regulated about 25%(P<0.01) (Fig.1.4, Fig.1.9), and no alteration ofγ-SNAP in NAc and Hip was detected in morphine dependent and withdrawal groups(Fig.1.8, Fig.1.10, Table1.1, Table1.3).α-SNAP andβ-SNAP were unchanged in NAc, CPu and Hip of rats in morphine dependent and withdrawal groups. From these results above, we could draw a conclusion that morphine dependence leads to up-regulation ofγ-SNAP in CPu, but it did not have any effects on the expression ofα-SNAP andβ-SNAP in NAc, CPu and Hip(Fig1.2,Fig1.3,Fig1.5,Fig1.6, Fig1.7, Table1.1,Table1.2), which suggested that 3 isoforms of SNAPs functioned differently in morphine dependence and might be related with the secretion of specific neurotransmitter. The release of presynaptic neurotransmitter in morphine dependence and withdrawal rat was not mediated by the content ofα-SNAP andβ-SNAP, and the intracellular translocation and intrinsic activity ofα-SNAP andβ-SNAP may contribute to the molecular mechanisms underlying morphine dependence and withdrawal.To further confirm the relationship between SNAPs and opioid dependence,α-SNAP, an isoform considered to play a key role in neurotransmitter release, was selected in the following study. Accumulating evidence reported the function ofα-SNAP in membrane fusion between vesicle membrane and presynaptic membrane, which has been regarded as the most important step in calcium-dependent exocytosis during the process of neurotransmitter release. It was reported thatα-SNAP functioned in stimulating NSF ATPase, and the effect ofα-SNAP significantly surpassed that ofγ-SNAP. Therefore, although no alteration ofα-SNAP expression was detected in animal model, this isoform was still focused on. To further study the function ofα-SNAP in morphine treatment, a well defined experimental cell model was necessarily needed owing to the relative simple and direct regulation mechanisms. The experimental model must be characterized by following traits: (1) expressing opioid receptor (2) processing similar features to mature neuroendocrine cell and secreting neurotransmitters related to opioid dependence, such as monoamine transmitters, glutamate, GABA, etc. After preliminary screening, the differentiated human neuroblastoma SH-SY5Y cell line was singled out to be applied for this study. After differentiation with retinoic acid(RA), SH-SY5Y cells were reported to show many characteristics of mature neurons, such as the appearance of extensive neurite outgrowth and electrical excitability, enhancement of neuron specific enolase(NSE) activity and increase of neurosecretory granules, etc.In this study, we observed the formation of long neuritic processes in SH-SY5Y cells 6 days after treated with RA (Fig.2.1). The expression of NSE was also measured by Western blot. Results indicated that NSE andμopioid receptor(MOR) were up-regulated in differentiated SH-SY5Y cells compared with that in undifferentiated cells (Fig.2.2 , Fig.2.3), which was in accordance with previous research. All these properties of differentiated SH-SY5Y cells facilitated research in the molecular mechanisms of opioid dependence, especially in the aspect of neurotransmitter release related with synaptic plasticity. To elucidate the molecular mechanisms of opioid dependence, a cell model for opioid dependence was established. The cAMP overshoot after morphine withdrawal was generally applied to evaluate whether the cell model was successful. In our study, we assessed the content of intracellular cAMP by the LANCE cAMP assay and discovered cAMP overshoot in SH-SY5Y cells after naloxone-precipitated withdrawal. The content of cAMP markedly increased to a level as 2.36 fold as that in untreated cells (Fig.2.5). This confirmed that the cell model of opioid dependence in our study was successfully established.In order to confirm the neurotransmitter changes, the contents of monoamine neurotransmitters and their metabolites were investigated in SH-SY5Y cell culture supernatant by HPLC-ECD under acute morphine treatment and naloxone-precipitated withdrawal after long-term morphine treatment. Data showed that the monoamine neurotransmitters including NE, DA and 5-HT were generally inhibited with acute morphine administration (Table3.1). After incubation with 100μM morphine for 10 min ~ 1h in SH-SY5Y cells, NE, DA and 5-HT as well as their metabolites DOPAC (DA metabolite) and 5-HIAA (5-HT metabolite) were significantly reduced, the content of NE was significantly decresed from 16.97 ng/g to 5.34 ng/g protein at 10min after morphine exposure (P<0.01). In the following 50 min, it did not significantly decline compared to that in 10min after morphine treatment and kept at a relatively low level (Fig.3.4). The changes in DA showed a similar trend to NE , that was, an obvious decline was detected in the first 10 min, lower than the basal level (P<0.01)(Fig.3.6). The content of 5-HT was significantly decreased (P<0.05) in the initial 10 min and continuted decline till 1/3 of the original level at 1h after morphine treatment (P<0.01) (Fig.3.8). Their metabolites, DOPAC and 5-HIAA were also down-regulated compared with cells without exposure to morphine (P<0.01) (Fig.3.5, Fig.3.7). These results showed that acute morphine treatment significantly attenuated the release of monoamine neurotransmitters.When exposed to 100μM naloxone after incubation of morphine for 24h, monoamine neurotransmitter release was investigated in SH-SY5Y cell supernatants. Results showed that NE, DA and 5-HT and their metabolites, DOPAC and 5-HIAA increased (Table3.2). The content of NE rose from 7.59 ng/g to 12.74 ng/g, higher nearly 0.5 fold, at 20 min after the onset of withdrawal(P<0.01), then dropped to the basal level at 40 min after the onset of withdrawal (Fig.3.9). Compared with NE, the change in DA was more obvious, and the DA content reached a peak at 20 min after withdrawal, approximately twice the original level(P<0.05)(Fig.3.11). 5-HT was also detected a significant increase at 40 min, nearly twice the level of untreated ones (P<0.05) (Fig.3.13). Their metabolites, DOPAC and 5-HIAA, also increased, compared with that of cells before naloxone treatment(P<0.05) (Fig.3.10, Fig.3.12). These results showed that naloxone-precipitated withdrawal after morphine dependence significantly stimulated the release of monoamine neurotransmitters. The content of monoamine neurotransmitters reached peaks at 20~40min after the onset of naloxone withdrawal, then dropped rapidly in the following time.To determine whetherα-SNAP participated in the changes of neurotransmitters induced by morphine, theα-SNAP expression was detected in SH-SY5Y cells after morphine administration. Data showed that no alteration ofα-SNAP mRNA expression was detected at 1h, 6h, 24h in SH-SY5Y cells after morphine administration (Fig.4.2), and there was no change inα-SNAP protein expression at 1h, 8h, 24h after morphine treatment by Western blot analysis (Fig.4.3). The results obtained from cells were in accordance with that obtained from animal model. Data suggested that morphine treatment did not result in any change inα-SNAP expression. However, as a key role in neurotransmitter release, whetherα-SNAP takes part in presynaptic plasticity underlying morphine dependence has not been elucidated.Many evidences showed that a series of molecular events take place in the process of neurotransmitter exocytosis, including vesicle trafficking, docking, fusion and internalization."SNARE hypothesis"is widely accepted in membrane fusion during the neurotransmitter release. According to"SNARE hypothesis", in the process of membrane fusion, 20S complex assembly and disassembly are the key steps. Complex assembly begins with binding between v-SNAREs and t-SNAREs. v-SNAREs and t-SNAREs are located in vesicle membrane and target membrane, respectively. t-SNAREs include SNAP-25 and Syntaxin. At the beginning of membrane fusion, t-SNAREs bound to v-SNAREs, and 7S SNARE complex is formed. One 7S SNARE complex can recruit 3α-SNAP molecules, this is followed by the binding of NSF. Thus, SNAREs,α-SNAP and NSF assembled a 20S complex. Then, subsequent ATP hydrolysis of NSF leads to disassembly of 20S complex and a conformational change in SNARE molecules, which prevented from its reassembly. Thus, all components of SNAREs enter into next cycle of vesicle transport. Since t-SNARE, a key element in the formation of 20S complex, located at presynaptic membrane, we supposed that the translocation ofα-SNAP might take place in the process of membrane fusion, for instance, from cytoplasm to cell membrane. Therefore, we further examined the subcellular location ofα-SNAP by confocal laser scanning microscopy in SH-SY5Y cells with morphine treatment. Results showed thatα-SNAP began to migrate from cytoplasm to cell membrane at 20min after naloxone withdrawal and lasted in the following 40 minutes. An obvious translocation was still observed at 60min after withdrawal (Fig.4.5). Nevertheless, no change was detected in subcellular location ofα-SNAP with acute morphine treatment (Fig.4.4). It showed that naloxone-precipitated withdrawal after chronic morphine treatment altered the subcellular localization ofα-SNAP in SH-SY5Y cells, and the time courses of changes in monoamine neurotransmitters approximately coincided with the time ofα-SNAP translocation, which might indicate a close relationship between neurotransmitter release andα-SNAP translocation.In this study, we drew the following conclusions through determination of SNAPs expression and subcellular location as well as neurotransmitters release after morphine treatment: (1) Chronic morphine administration and spontaneous withdrawal did not affectα-SNAP expression in NAc, CPu and Hip of rats. (2) Chronic morphine administration up-regulatedγ-SNAP expression in CPu of rats, but did not changeγ-SNAP expression in NAc and Hip of rats. Spontaneous withdrawal had no effect onγ-SNAP expression in NAc, CPu and Hip. (3) SH-SY5Y cells could be used to establish cell model of morphine dependence. (4) Acute morphine treatment significantly attenuated the release of monoamine neurotransmitters, while naloxone-precipitated withdrawal significantly stimulated the release of monoamine neurotransmitters. (5) Acute and chronic morphine treatment did not affectα-SNAP expression in SH-SY5Y cells. Acute morphine treatment did not alter the subcellular localization ofα-SNAP in SH-SY5Y cells. Naloxone-precipitated withdrawal after morphine dependence resulted inα-SNAP translocation from cytoplasm to cell membrane, which might be related with the neurotransmitter release after morphine withdrawal.