| Objectives1. In this experiment,we use ketamine-induced rats model to observe the effect of different dose and duration of ketamine on the conditioned place preference (CPP), and the influence of ethanol on CPP in the ketamine-induced model.2. Quantitative analysis by ELISA to detect the content of dopamine in rats dopamine-related brain areas (Dopamine, DA) in each groups. The experimental data were statistically analyzed to clarify the impact of dose and duration of ketamine to DA levels, and mechanism of ethanol in ketamine-induced cpp model rat DA levels.Methods1. Animal groups, CPP experiments and detection of brain DA72rats were randomly divided into control group (N12), ethanol fed control group (E12), ketamine injection group (K group24), ketamine injection and ethanol administered group (KE group24). Injected into the saline control group30days group (N1group) and injected60days group (N2groups). Ethanol administered orally in the control group30days into ethanol group (E30group), ethanol administered60days group (E60group).30mg/kg ketamine injection group was divided into30days of ketamine injection group (K130group),30mg/kg ketamine injections60days group (K160group),60mg/kg ketamine injections30days group (K230group),60mg/kg ketamine injections60days group (K260group). Intragastric ethanol injection of ketamine30mg/kg ketamine injection group was divided into30days gavage with ethanol group (K1E30 group),30mg/kg ketamine injection administered60days with ethanol group (K1E60group),60mg/kg ketamine injected with ethanol gavage30days group (K2E30group),60mg/kg ketamine injection administered60days with ethanol group (K2E60group).N1group,N215mg/kg body weight of rats by intraperitoneal injection of saline dose, administered once daily, at intervals of24h, continuous administration of30days and60days. Group E30, E60dose group rats2mL/kg20%aqueous ethanol orally administered once a day, each time interval24h, continuous administration of30days,60days. K130group, K160rats by intraperitoneal injection of30mg/kg dose of10mg/mL ketamine injection once a day, at intervals of24h, continuous administration of30days,60days. K.230group, K260rats by intraperitoneal injection of60mg/kg dose of10mg/mL ketamine injection once a day, at intervals of24h, continuous administration of30days,60days. K1E30group, K1E60rats by2mL/kg dose of20%aqueous ethanol administered by intraperitoneal-injection of30mg/kg dose5min10mg/mL ketamine injection once a day, at intervals of24h, continuous to drugs30days,60days. K2E30group, K2E60rats by2mL/kg dose of20%aqueous ethanol administered by intraperitoneal injection of60mg/kg dose5min10mg/mL ketamine injection once a day, at intervals of24h, continuous to drugs30days,60days.Saline control group, ketamine injection group, rats fed ethanol injection of ketamine in the last administration put Dr. Yu Shu24h animal behavior analysis system, after15min CPP experiments conducted by the method of operation,the rats were executed; In ethanol fed control group, rats were executed after24h when the final drugs were given. According to distribution map of brain areas, the related brain tissue were isolated in each groups. ELISA analysis method was used to detected the DA content by the absorbance (OD) at450nm wavelength.Data was analyzed by SPSS software.Results1. CPP effects of experimental ratsCompared with the control group, ketamine injection group, ethanol and ketamine group showed significant differences in conditioned place preference effects (P<0.05). Compared with K130group, K1E30group with kits stay was significantly prolonged(P<0.05); With the extension of the duration of the injection, K160, CPP appear faded effect K1E6O, K4E60group compared with the K160group, with kits residence time increased, but the difference was not significant (P>0.05). Compared with the group of K230, K2E30kits groups with significantly reduced residence time(P <0.05); but with the extension of the duration of the injection, K260group, K2E60group CPP effects appear faded, K2E60group compared with K260groups, with kits to reduce residence time, but the difference was not significant (P>0.05).2. ELISA method for quantitative analysisBy ELISA experiments, the concentration and the OD value DA series of standard solution curve regression results show that the correlation coefficient y=0.9995, the detection range0.156ng/ml～10ng/ml, sensitivity0.039ng/ml, applicable to rat brain DA content in the detection zone.3. ELISA assays DA content of each group of related brain areas(1) the prefrontal cortex, compared with N1, before the E30rat frontal cortex was significantly higher in the DA, there were not significant differences (P>0.05), with the extension of continuous administration of time, compared to group at E30, E60group DA content increased, there was a significant difference (P<0.05); K130group DA content compared with the N1group showed an increasing trend, there was a significant difference (P<0.05), with extend the delivery time, K160group compared with DA levels were significantly higher in K130group, with a significant difference (P<0.05); K1E30group DA levels compared with N1group was increasing, and there is significant differences (P<0.05), with the extended delivery time K1E60group DA levels were lower than the K1E30, there was a significant difference (P<0.05). Compared with N1group K230group DA content group compared with the N1was increasing, and there was a significant difference (P <0.05), with the extended delivery time, K260group compared to the DA content K230group was significantly lower, with a significant difference (P<0.05); K2E30group DA levels compared with N1group was increasing, and there was a significant difference (P<0.05), with the time of administration extension of K2E60group DA content than K2E30group increased, there was a significant difference (P<0.05). Compared with the E30group, K1E30group and K2E30prefrontal cortex DA levels were elevated, there was a significant difference (P<0.05). With time, reduce K1E60group DA concentration levels, and lower than the E60group, there was a not significant difference (P>0.05).(2) In the nucleus accumbens, compared with N1, before the E30rat frontal cortex was significantly higher in the DA, there were significant differences (P<0.05), with the extension of continuous administration of time, compared to group at E30, E60group DA content increased, there was a significant difference (P<0.05); K130group compared with N1, the DA was significantly increased, there were significant differences (P<0.05), with Continuing to extend the delivery time, compared to K130group, K160group DA content increased, there was a significant difference (P <0.05). K1E30group DA levels compared with N1group was increasing, and there was a significant difference (P<0.05), with the extended delivery time K1E60group DA levels were lower than the K1E30, there was a significant difference (P<0.05). Compared with Ni, K230groups DA content group compared with the N1was increasing, and there was a significant difference (P<0.05), with the extended delivery time, K260group compared to the DA content K230group was significantly lower, with a significant difference (P<0.05); K2E30group DA levels compared with N1group was increasing, and there was a significant difference (P<0.05), with the time of administration extension of K2E60group DA levels were lower than the K2E30, there was a significant difference (P<0.05).30days after continuous administration, DA levels of each experimental group were:K230group> K1E30group> K2E30group>K130group> E30group> Ni group (K230group DA content in the highest group (1.01±0.25ng/ml); K1E30group compared with K130DA levels were significantly higher and significantly higher than the E30group, there were significant differences (P<0.05); K2E30group DA content and K230groups was significantly lower, but still higher than the E30group, there were significant differences (P<0.05).60days after continuous administration, DA levels of each experimental group were:K260groups> K2E60group> K1E60group> K160group> E60group> N2group, K260groups DA highest concentrations (0.80±0.55ng/ml); content in DA K1E60group than K160group, E60set slightly higher, but the difference was not significant (P>0.05); K2E60group and K260groups, DA content decreased, but not significantly higher than the E60group (P>0.05).(3) In the ventral tegmental, as compared with N1, before the E30rat frontal cortex was significantly higher in the DA, there were significant differences (P<0.05), with the extension of ongoing administration time, relative compared with the group at E30, E60group DA content increased, there was a significant difference (P<0.05); K130DA content group compared with the N1group showed an increasing trend, there was a significant difference (P<0.05), with prolonged administration time, K160group compared with K130DA levels were significantly higher, with a significant difference (P<0.05) in; K1E30group DA levels compared with N1group showed an increasing trend, There was a significant difference (P<0.05), with the extended delivery time K1E60group DA content than K1E30group increased, there was a significant difference (P<0.05). Compared with N1, K230groups DA content group compared with the N1was increasing, and there was a significant difference (P<0.05), with the extended delivery time, K260group compared to the DA content K230group was significantly lower, with a significant difference (P<0.05); K2E30group DA levels compared with N1group was increasing, and there was a significant difference (P <0.05), with the time of administration extension of K2E60group DA content than K2E30group increased, there was a significant difference (P<0.05).After30days of administration, DA levels of the order of:K2Group> K1E30group> K2E30group> K130group> E30group> N1group, K230group DA highest concentrations (1.68±0.26ng/ml); K1E30group compared with K130DA levels were significantly higher, and higher than the E30group, there were significant differences (P<0.05); K2E30group DA content and K2group was significantly lower, but still higher than the E30group (P<0.05). After60days of administration, DA levels of the order of:K260group> K1E60group> K2E60group> K160group> E60group> N2, K260group DA highest concentrations (1.45±0.04ng/ml).(4) In the hippocampus, compared with N1, the frontal cortex was significantly higher in DA rats before E30, a significant difference (P<0.05), with the extension of continuous administration of time, compared to E30group, E60group DA content increased, the difference was not significant (P>0.05), but still higher than N2, there was a significant difference (P<0.05); K130group DA content and N1group showed up compared to high trend, there was a significant difference (P<0.05), with the extended delivery time, K160DA group compared to K130levels were significantly reduced, with a significant difference (P<0.05); K1E30Groups DA content group compared with N1was increasing, and there was a significant difference (P<0.05), with extended delivery time K1E60group DA levels were lower than the K1E30, there was a significant difference (P<0.05). Compared with N1, K230groups DA content group compared with the N1was increasing, and there was a significant difference (P<0.05), with the extended delivery time, K260group compared to the DA content K230group was significantly lower, with a significant difference (P<0.05); K2E30group DA levels compared with N1group was increasing, and there was a significant difference (P <0.05), with the time of administration extension of K2E60group DA levels were lower than the K2E30, there was a significant difference (P<0.05).Continuous administration of30days in the group, DA levels in each group were:K230groups> K1E30group> K2E30group> K130group> E30group> Ni group, K230groups had the highest DA concentrations (1.26±0.01ng/ml); K1E30group compared with K130DA levels were significantly higher, with a significant difference (P<0.05); K2E30group DA content and K230group was significantly lower (P<0.05). In the60days period, DA levels of the order of: K260groups> K1E60group> K2E60group> K460group> E60group> N2, K260groups DA highest concentrations (0.56±0.02ng/ml); E60is slightly higher than the K160group, no significant difference (P>0.05); K2E60group and K260groups, DA content decreased, there is a significant difference (P<0.05).(5) in the striatum, compared with N1, before the E30rat frontal cortex was significantly higher in the DA, there were significant differences (P<0.05), with the extension of continuous administration of time, compared to group at E30, E60group DA content increased, there was a significant difference (P<0.05); K130DA content group compared with the N1group showed an increasing trend, there was a significant difference (P<0.05), with extend the delivery time, K160DA group compared to K130levels were significantly increased, with significant differences (P <0.05); K1E30group DA levels compared with Ni group was increasing, and there is significant differences (P<0.05), with the extended delivery time K1E60group DA levels were lower than the K1E30, there was a significant difference (P<0.05). Compared with N1,K230groups DA content group compared with the N1was increasing, and there was a significant difference (P<0.05), with the extended delivery time, K260group compared to the DA content K230group was significantly lower, with a significant difference (P<0.05); K2E30group DA levels compared with N1group was increasing, and there was a significant difference (P<0.05), with the time of administration extension of K2E60group DA levels were lower than the K2E30, there was a significant difference (P<0.05). Compared with the E30group, K1E30group and K DA levels2E30E30group were significantly higher than the group, and has a significant difference (P<0.05). Continuous administration of30days in each group, DA levels of the order of:K1E30Group> K230groups> K2E30group> K130group> E30group> N1, K1E30DA content in the highest group (2.01±0.41ng/ml); K1E30group DA content than K130group showed significantly increased, with a significant difference (P<0.05); K2E3O group DA content and K230group was significantly lower, with a significant difference (P<0.05). In60days of continuous administration in each group, DA levels of the order of K260groups> K1E60group> K160group> K2E60group> E60group> N2, K260groups DA highest concentrations (0.79±0.02ng/ml); content in the DA group K1E60is slightly higher than the K160group, but no significant difference (P>0.05); K2E60group and K260groups, DA content decreased, with a significant difference (P<0.05).Conclusion:1. Several times with intraperitoneal injection of ketamine, combined with conditioned place preference test methods successfully established ketamine dependent rats. By contrast, in animal models to prove the existence of a strong spirit of ketamine dependence potential. And ketamine in combination with ethanol also showed significant conditioned place preference.2. Ethanol can enhance30mg/kg ketamine-induced increase in DA content in effect。 Suggesting that ethanol may produce synergistic inhibitory effect of ketamine on the NMDA receptor, thereby increasing brain DA release enables content increased.3.60mg/kg ketamine can inhibit ethanol-induced DA levels increased effect, this inhibition increased gradually with exposure time。... |
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