| With the development of the economy, contact of people is increasing and the consumption of alcohol has been rasied significantly. Alcohol has damage to the cardiovascular system, digestive system especially to the neuroal sustem, absorbed per cutem, respiratory tract and alimentry canal, which shows depression, decreased memory and functional impairment.Epidemiologic study has found the depresssion is increasing, except for the living pressure and working tension, drinking may be one reason for some people. Scholars found the depression is associated with the functional impairment of serotonin system, serotonin transporter(SERT) reduced significantly. SERT is a membrane protein located in nerve terminal of 5-HT neurons, it can end the efffect of 5-HT by reuptaking 5-HT in synaptic cleft; its reduction reflects the functional impairment of 5-HT energic neurons and hints the ruduction of 5-HT energic nerve fibers in the brain region dominated by 5-HT energic neurons. Thus, SERT can be viewed as an important index while investigating the functional status of 5-HT energic neurons.Mesolimbic dopaminergic system plays an important role in the process of drug addiction. The functional action of dopaminergic neurons is dependent on the normal expression of dopamine transporter(DAT). DAT is the membrane protein located in nerve terminal of DA energic neurons, it can recognize DA specificly and reuptake it from synaptic cleft to the presynaptic nerve terminal and end the transmission of neuroal information. It had been discovered in research that DAT is the target of cocaine and other antimelancholics. Some scholars found the binding site of DAT increased significantly in striatum of snowbird by autopsy which proved further by animal experiments. whether or not the binding site of DAT can be elevated by alcohol abuse addiction as cocaine and other antimelancholics is to be investigated. Immunoautoradiography, immuocytochemistry, morphousmetrology, westernblot, flow cytometry and transmission electron microscope (TEM) was used in this study to analyze the influence of alcohol abuse and ethanol to the expression of SERT and DAT by investigating the different brain regions of postmortem alcohol abuse and ethanol treated rats, to offfer experimental data for explanation to symptom of alcohol abuse and alcohol abuse addiction.Prat 1: Immunoautoradiographic studies on the distribution of serotonin transporter in postmortem human brainObjective: Immunoreactive intensity of seotonin transporter (SERT) was analyzed quantitatively in the different regions of postmortem human brain in order to 1. provide the suitable view region and experimental data for the alterated expression of SERT in postmortem human of alcohol abuse. 2. provide the neuroanatomical evidence in selecting an appropriate reference region for neuroimaging in measurement of the altered SERT.Methods: Collect 5 male postmortem human brains, affirm location, take brain, cut into brain slices of 3～5 mm, formalin fix, dehydrate, clear, paraffin imbed and then immunoautoradiography is used, visualize, fix, after stained with Luxol Fast Blue, scan, save the image in TIFF, measure, analyze the immunoreactive intensity and calculate to reveal SERT immunoreactive intensity(nCi/mg).Results: 1. The general observation of SERT. In the observed brain regions, the highest density of SERT was mainly found in the raphe nuclei of the brainstem, while the lowest density was only seen in the cerebellar cortex, which is nearly similar to that in white matter. 2. The intensity analysis of SERT. In the observed brain regions, quantitative analysis revealed that dorsal raphe nucleus(DRN) 2.99±0.89,substantia nigra(SN) 1.73±0.36,caudata nucleus 0.31±0.07,shell 0.28±0.06, frontal cortex 0.11±0.05,occipital cortex 0.13±0.08,cingulate cortex 0.55±0.02,cerebellar cortex 0.06±0.04, corpus callosum 0.04±0.03. The intensity of SERT immunoreactivity(SERT-IR) in cerebellar cortex was respectively 1/1.83, 1/2.17, 1/9.17, 1/0.67 of that in frontal cortex, occipital cortex, cingulate cortex and corpus callosum and it is only 1/49.83, 1/28.83, 1/5.12, 1/4.67 corresponding to DRN, SN, caudata nucleus, putmen.Conclusions: 1. The expression of SERT is the highest in DRN in 5-HT energic neuron projective original regions. In the target region, the expression of SERT is the highest in SN and ventral tegmental area(VTA), pregenual anterior cingulate cortex is the second, cerebellar cortex and corpus callosum is the lowest. 2. The cerebellar cortex has lowest SERT as compared to other examined brain regions and could be a useful reference region if appropriate SERT radioligands were chosen.Prat 2: The altered expression of serotonin transporter in postmortem human brain of alcoholicsObjectives: To study the immunoreactive intensity of serotonin transporter (SERT) in postmortem human brain of alcoholics to assess the alteration of SERT.Methods: Collect 10 male postmortem human brains, with 5 cases for the control group and the alcohol abuse group respectively, and the rest are the same as the first part.Results: 1. The general observation of SERT. In the observed brain regions, the strongly labeling signal of SERT was mainly found in the rostral raphe nuclei of the rostral pons and midbrain in control group. In red nucleus and SN, the labeling signal of SERT is stronger, and in cingulate cortex the strongly labeling signal of SERT is mainly in the forepart especially pregenual anterior cingulate cortex(PACC), while it was reduced in those nuclei in alcohol abuse group. 2. The intensity analysis of SERT. In the observed brain regions, quantitative analysis revealed that in the control, median raphe nucleus(MnR) 1.89±0.62, dorsal raphe nucleus(DRN)'s dorsal raphe nucleus- interfascicular part(DRI) 2.32±0.89, dorsal raphe nucleus - caudal part(DRC) 2.54±0.42, dorsal raphe nucleus-ventral part(DRV) 3.26±0.98, dorsal raphe nucleus-dorsal part(DRD) 2.66±0.77, substantia nigra(SN) 1.73±0.36, PACC 0.55±0.02;in the alcohol abuse group, the labeling signal of SERT in MnR, DRI, DRC, DRV, DRD, SN and PACC are 0.76±0.16,0.90±0.34,1.17±0.39,1.34±0.09,1.10±0.25,0.82±0.23 and 0.43±0.04 respectively. Compaired with the control, the labeling signal of SERT in the alcohol abuse group were reduced significantly(DRI, DRC, SN and PACC:P﹤0.01;MnR, DRV and DRD:P﹤0.05). The labeling signal of SERT in pontine reticular nucleus-oral part(PnO), supralemniscal region(B9), paramedian raphe nucleus(PMnR) and ventrotegmental area(VTA) of the alcohol abuse group has no significiant difference.(Pï¹¥0.05).Conclusions: SERT expression was reduced in DRN, SN and PACC in postmortem human brain of alcolholics, which indicates the 5-HT energic nervous activity is disfunctional in the brain of alcohol abuse.Prat 3: The influence of ethanol treatment to the 5-HT energic systemsObjectives: To observe the altered expression of serotonin transporter (SERT), serotonin(5-HT) and tryptophan hydroxylase(TPH) in different brain regions of ethanol treated rats, so that the damage of ethanol to the systems of 5-HT in brain can be assessed.Methods: Select 120 3-month-old Wistar rats (provided by HeBei Medical University experimental animal center) weighing 250-300 g, which were randomly divided into ethanol treated and control groups. Normal diet and water were given to the control, and ethanol treated group was given normal diet with 20% ethanol. Each group of experimental animals were breeded for 6 months respectively. 1. Immunocytochemistry: Rats required were perfused, brain taken, sectioned in coronal and then immunocytochemistry stainned. Count the number of TPH and 5-HT immunoreactive neurons in DRN(B7) in brain stem with light microscope; measure the mean diamerter of TPH immunoreactive neurons; analyze the gray scale mean value of 5-HT energic immunoreactive neurons(5-HT, TPH and SERT) with HPIAS-1000 high definition multicolor pathologic analytical system in the different brain regions. 2. Flow cytometry: Rats required were taken brain then monocell suspension was prepared, and immunofluorescence lable the neurons, count the X-mode value of expression of 5-HT, TPH and SERT. 3. Western-Blot: Rats required decapitated, brain taken, protein splitted and quantitate with coomassie brilliant blue, application of sample electrophoretized 3 h, electrotransfer 2 h to PVDF membrane,â… antibody 4℃to stay overnight,â…¡antibody 37℃1.5 h,ECL 1 min,PVDF membrane was put into x-black box, visualization, fixation, scanning negative film, quantitative analyze the Western strap with LabWorks 4.5 software to determine intensity optical density. 4. Transmission electron microscope(TEM):Rats required were anesthetized with chloral hydrate perfused with fixation fluid (1.5%paraform, 2.5%glutaraldehyde) and brain taken, postfixed 2 h, sliced in coronal with rembling slicer, collectted sections contain DNR, 1% osmic acid postfied 1 h,alcoholic dehydration,dimethyl ketonesubstitution, embedded with balsam, took the contains location of DRN, sliced 60 nm sections with ltramicrotome, observed with Hitachi H-7500 TEM.Results: 1. Immunohistochemistry: 5-HT energic imunoreactive neurons of normal rats are mainly located in the inferior colliculus of midbrain, which seem brown, cell bodies uniform insize, ellipse or round. SERT are fibers of varied diameters which cross into web in the brain of normal rats, which in midbrain is in DRN, in telencephalon are mainly in cingulate cortext, striatum. Number of countting cells shows that 5-HT, TPH imunoreactive neurons of DRN in control is 24.57±0.70 and 24.86±1.28 respectivly;that in ethanol is 15.89±0.67 and 16.00±1.00 respectivly,t-test between two groups found there is a significent difference(P﹤0.01). Measurements of diameter show that TPH imunoreactive neurons of DRN in control is 12.13±1.05μm, that in ethanol is 11.44±1.21μm,decreased 5.69% to the control(P﹤0.01);that of DRVL in control is 11.84±1.50μm, that in ethanol is 10.82±1.69μm,decreased 8.61% to the control(P﹤0.01). Gray scale mean value shows 5-HT in DRN(B7)and striatum in control are 45.78±5.43 and 164.94±1.84 respectively,those in ethanol are 59.73±1.67 and 181.52±2.45,changes in the ethanol increased 30.47% and 10.05% to those in control(P<0.05). Gray scale mean value shows TPH in DRN(B7)in control is 88.80±1.29,that in ethanol is 106.48±6.92,change in the ethanol increased 19.91% to those in control(P<0.05). Gray scale mean value shows SERT in DRN(B7), striatum and cingulate cortext in control are 103.57±2.80, 102.68±7.72 and 149.44±14.16 respectively,those in ethanol are 120.45±6.65, 122.36±7.54 and 180.00±11.85,changes in the ethanol increased 16.30%, 19.17% and 20.45%(P<0.05). 2. Flow cytometry detection: Indirect immuno fluorescence (IFL) in flow cytometry shows the expression of 5-HT, TPH and SERT of 5-HT energic imunoreactive neurons in control are 439.2±19.99, 638.30±1.37 and 553.36±15.13 respectively , those in eathanol are 378.25±6.84, 595.20±13.51 and 513.11±12.05 respectively,three groups have statistical significance all(P<0.05). 3. Western-Blot:Western-Blot shows the ratio of IOD of SERT/β-actinin in control are: cingulate cortext 0.0034±5.69×10-5, striatum 0.0034±8.97×10-5, DRN(B7) 0.0037±1.33×10-4;those in ethanol are : cingulate cortext 0.0031±6.97×10-5, striatum, 0.0032±1.35×10-5, DRN(B7) 0.0034±1.53×10-4;all the three groups are of significant statistical importance (P<0.05). The ratio of IOD of TPH/β-actinin in control is DRN(B7) 0.0056±1.25×10-4, which in ethanol is 0.0052±8.6×10-5, and the data is of statistical significance(P<0.05). 4. Transmission electron microscope:After the observation to 5-HT energic imunoreactive neurons, we found that compared with control, the lysosome increased in ethanol.Conclusions: 1. Treatment of ethanol can cause the number of 5-HT energic imunoreactive neurons decrease and the diameter shink in DRN in rat brain. 2. Treatment of ethanol can lead to decreased expression of SERT,5-HT and TPH in 5-HT energic systems. 3. Treatment of ethanol can lead to altered ultrastructural organizationin DRN, such as more lysosome are found. Prat 4: The influence of ethanol treatment to the DA energic systemsObjectives: To observe the altered expression of dopamine transporter (DAT) and tyrosine hydroxylase (TH) in different brain regions of ethanol treated rats, so that the mechanism of ethanol addition can be investigate and provide experimental data for withdraw of ethanol addition. Methods: All are the same as Part 3. 1. Immunocytochemistry: Antibodyâ… DAT and TH, Antibodyâ…¡goat anti rat IgG and goat anti mice IgG; to observe and analyze TH and DAT of DA energic immunoreactive neurons; brain regions are SN, striatum, cingulate cortex. Except for the above mentioned, the others are the same as Part 3. 2. Flow cytometry detction: Antibodyâ… TH, the others are the same as Part 3. 3. Western-Blot detction: Antibodyâ… DAT and TH, Antibodyâ…¡goat anti rat IgG and goat anti mice IgG; to observe and analyze TH and DAT of DA energic in SN, striatum, cingulate cortex. Except for the above mentioned, the others are the same as Part 3. 4. Transmission electron microscope(TEM):To observe SN, the others are the same as Part 3.Results: 1. Immunohistochemistry: TH imunoreactive neurons of normal rats are mainly located in the SN and VTA of midbrain, which seem brown, ellipse. DAT are fibers of varied diameters which cross into web in the brain of normal rats, which in midbrain is in SN and the immunoreaction is low. Number of countting cells shows that TH imunoreactive neurons of SN in control is 45.95±8.11, which in ethanol is 30.29±4.85,the decreased degree is 34.08% (P﹤0.01); measurements of diameter shows that TH imunoreactive neurons of SN in control is 10.16±1.53μm, which in ethanol is 9.31±1.22μm,decreased 8.37% as oppose to the control(P﹤0.01);gray scale mean value shows TH in SN and striatum in control are 71.39±3.90 and 182.90±22.72 respectively , those in ethanol are 60.99±4.96 and 158.52±16.84,changes in the ethanol decreased 14.57 and 6.66% to those in control(P<0.05). Gray scale mean value show DAT in SN, striatum and cingulate cortext in control are 59.78±1.95, 88.72±3.62 and 96±2.18 respectively,those in ethanol are 53.85±1.26, 68.75±3.53 and 184.38±1.92,changes in the ethanol decreased 9.92%, 22.51% and 12.21%(P<0.05). 2. Flow cytometry : Indirect immuno fluorescence (IFL) in flow cytometry shows the expression of TH of DA energic imunoreactive neurons in control is 535.55±13.24,which in ethanol is 580.39±19.28,the change increased 8.4% as oppose to the control (P<0.05). 3. Western-Blot:Western-Blot shows the ratio of IOD of DAT/β-actinin in control is : cingulate cortext 0.0034±6.61×10-5, striatum 0.0034±5.59×10-5, SN 0.0035±8.12×10-5;those in ethanol are:cingulate cortext 0.0036±3.14×10-5, striatum 0.0037±8.95×10-5, SN 0.0037±4.16×10-5;changes increased 3.84%, 7.39% and 5.6%, all the three groups are of statistical significance (P<0.05). The ratio of IOD of TH/β-actinin in control is SN 0.0036±9.63×10-5, striatum 0.0035±1.17×10-4;those in ethanol is 0.0045±1.33×10-4, striatum 0.0041±6.29×10-5, changes reach 23.16% and 16.89% which illustrate the expression of TH is increasing, the data is of statistical significance(P<0.05). 4. Transmission electron microscope:After the observation to DA energic imunoreactive neurons, we found there was no obvious alteration in ethanol compared with control.Conclusions: 1. Treatment of longterm ethanol can cause the number of DAT energic imunoreactive neurons decrease and the diameter shink in SN in rat brain. 2. Treatment of ethanol can lead to increased expression of DAT, which is concerned with ethanol addiction.Prat 5: Immunoautoradiographic studies on the distribution of DAT in postmortem human brainObjective: Immunoreactive intensity of DAT was analyzed quantitatively in the different regions of postmortem human brain in order to 1. provide the suitable region and experimental data for the alterated expression of DAT in postmortem human of alcohol abuse. 2. provide the neuroanatomical evidence in selecting an appropriate reference region for neuroimaging in measurement of the altered DAT.Methods: It is the same as part.1 .Results: 1. The general observation of DAT. In the observed brain regions, the highest density of DAT was mainly found in the substantia nigra, caudata nucleus, putmen, in addition, weaker labling of DAT was found in frontal lobe and occipital lobe of telencephalon while the lowest density was only seen in the cerebellar cortex, which is nearly similar to that in white matter. 2. The intensity analysis of DAT. In the observed brain regions, quantitative analysis revealed that substantia nigra(SN) 1.50±0.40,caudata nucleus 2.10±0.26,shell 1.54±0.18,frontal cortex 0.63±0.38,occipital cortex 0.23±0.02,cingulate cortex 0.67±0.44,cerebellar cortex 0.18±0.15, corpus callosum 0.11±0.08,quantitative analysis revealed that the intensity of DAT immunoreactivity (DAT-IR) in cerebellar cortex was respectively 1/3.50, 1/3.72, 1/1.28, 1/0.61 of that in frontal cortex, cingulate cortex, occipital cortex and corpus callosum, and it is only 1/8.33, 1/11.67, 1/8.56 corresponding to substantia nigra, caudata nucleus, putmen.Conclusions: 1. DAT is distributed in SN and its projective target regions. The expression of DAT is the highest in SN, caudata nucleus, putmen in DAT energic neuron projective original regions. In the target region, the expression of SERT is the highest in SN and ventral tegmental area(VTA), pregenual anterior cingulate cortex is the second, cerebellar cortex and corpus callosum are the lowest. 2. The cerebellar cortex has the lowest DAT compared to other examined brain regions and could be a useful reference region if appropriate DAT radioligands were chosen.
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