CN104132965A - Experiment method for studying effect of tenuigenin on synaptic transmission of rat hippocampal neuron - Google Patents
Experiment method for studying effect of tenuigenin on synaptic transmission of rat hippocampal neuron Download PDFInfo
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Abstract
The invention discloses an experiment method for studying the effect of tenuigenin on synaptic transmission of rat hippocampal neuron. The method adopts a micro control instrument, a microscopic, and a recording electrode to monitor the spontaneous current state of neuron in tenuigenin with different concentrations, and then the monitor current data is analyzed so as to obtain the influence mechanism and effect of tenuigenin on rat hippocampal neuron. The method adopts a whole-cell patch-clamp technology to research the effect of tenuigenin on spontaneous postsynaptic current in the rat brain hippocampal CA1 area so as to disclose the regulating effect and mechanism of tenuigenin on neuron synaptic transmission, thus the requirements of scientific research and drug development are fulfilled, and at the same time a basis is provided for the further research on the signal transmission mechanism of neuron. The method can be widely used in the pharmaceutical theory researches on neuron, the experiment method and equipment design are scientific and reasonable, and requirements of scientific research can be met.
Description
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Technical field
The present invention relates to the test method of a kind of medicine to neuron operation, be specifically related to the test method of a kind of polygalic acid to the effect of hippocampus of rats cynapse transmission.
Background technology
Polygala root (Radices polygalae) is the dry root of Polygalaceae polygala polygala root Polygala tenuifoliaWilld.; in Chinese traditional medicine; be regarded as having the effect of the intelligence development of calming the nerves always; therefore the research aspect brain function gets the attention; polygalic acid (tenuigenin; TEN) be the main active ingredient of a class of separating from polygala root; anti-ageing, intelligence development, anti-oxidant, etc. aspect there is biologically active widely, TEN can have facilitation at the aspects such as improvement of neuro-protective and cognitive function.TEN can play the effect of neuroprotective unit by suppressing the secretion of aβ protein, and we also find that in previous work TEN can obviously improve the ability of learning and memory of animal, can protect and cultivate neuron opposing H
2o
2damage; The hippocampal formation of brain be in mesencephalic centre with the closely-related important brain district of nervous function such as study, memory, therefore in hippocampus, the variation of synapse transmission has important impact to nervous functions such as learning and memories, yet, up to now, still do not have positive evidence to show that transmission has regulating and controlling effect to TEN to synapse.
Existing drug test mode, can not reach desirable effect for nerve medicine to the test of neurocyte action principle.Research for the Pharmacological Mechanism of specific nerve medicine can not be probed into clear completely.Therefore, can not meet the pharmacological research of nerve medicine.
Therefore experimental technique of the present invention adopts the impact of full cell patch tongs technology research TEN on the spontaneous postsynaptic currents in CA 1 of Hippocampus, to disclose TEN, neurocyte cynapse is transmitted to regulating and controlling effect and mechanism, thereby meets the test demand of scientific research and drug development.
Summary of the invention
The technical issues that need to address of the present invention are just to overcome the defect of prior art, and the test method of a kind of polygalic acid to the effect of hippocampus of rats cynapse transmission is provided.The method adopts the impact of full cell patch tongs technology research polygalic acid on the spontaneous postsynaptic currents in rat brain Hippocampal CA 1, to disclose polygalic acid, neurocyte cynapse is transmitted to regulating and controlling effect and mechanism, thereby meet the test demand of scientific research and drug development, also can make further research for the signal transport mechanism of neurocyte simultaneously.
For addressing the above problem, the present invention adopts technical scheme to be: the test method of the effect that polygalic acid transmits hippocampus of rats cynapse,
Medicine and reagent that this test method is used:
Artificial cerebrospinal fluid, i.e. ACSF, its component is: NaCl is that 124 mmol/L, KCl are 2.5 mmol/L, NaHCO
3be 26 mmol/L, NaH
2pO
4be 1.25 mmol/L, CaCl
2be 2. 0 mmol/L, MgSO
4be that 1.3 mmol/L, D-Glucose are 10 mmol/L, pH value is 7.4;
The interior liquid of electrode that records spontaneous postsynaptic currents, its component is: KCl is that 140 mmol/L, NaCl are that 6 mmol/L, HEPES are that 10 mmol/L, EGTA are that 10 mmol/L, Mg-ATP are that 2 mmol/L, NaGTP are that 0.1 mmol/L, pH value are 7.2;
The interior liquid of electrode that records spontaneous EPSC, its component is: K gluconate is that 140 mmol/L, NaCl are that 6 mmol/L, HEPES are that 10 mmol/L, EGTA are that 0.2 mmol/L, Mg-ATP are that 2 mmol/L, Na-GTP are that 0.1 mmol/L, pH value are 7.2;
The interior liquid of electrode that records spontaneous inhibitory postsynaptic current, its component is: KF is that 140 mmol/L, NaCl are that 6 mmol/L, HEPES are that 10 mmol/L, EGTA are that 0.2 mmol/L, Mg-ATP are that 2 mmol/L, Na-GTP are that 0.1 mmol/L, pH value are 7.2;
Test medication: CNQX, DL-APV, tetrodotoxin are that TTX, bicuculline are that BM, polygalic acid are TEN;
CNQX, DL-APV, bicuculline are that BM, tetrodotoxin are that TTX, HEPES, EGTA, Mg-ATP, Na-GTP, potassium gluconate, KF are all purchased from U.S. Sigma company, polygalic acid is that TEN is prepared by laboratory, and all the other reagent are domestic analytical reagent;
CNQX, English full name: 6-cyano-7-nitroquinoxaline-2,3-dione, Chinese full name: 6-cyano group-7-nitro quinoxaline-2,3-diketone, is nerve excitability mediator Glutamate AMPA Receptor Antagonist, molecule is 232.15;
DL-APV, English full name: DL-2-amino-5-phosphonovaleric acid, Chinese full name: DL-2-amino-5-phosphono valeric acid is nerve excitability mediator glutamate nmda receptor antagonist, and molecular weight is 197.13;
Bicuculline is BM, English full name: bicuculline, the popular name of Chinese: Bic is inhibitory neurotransmitter γ-aminobutyric acid GABA receptor blocking pharmacon, molecular weight 367.35;
Tetrodotoxin is TTX, English full name: tetrodotoxin, the popular name of Chinese: tetraodotoxin is cell membrane Na
+ion channel blocking agent, molecular weight: 319.27;
HEPES, English name 4-(2-hydroxyerhyl) piperazine-1-erhanesulfonic acid, Chinese full name: 4-hydroxyethyl piperazine ethanesulfonic acid, molecular weight is 238.3;
EGTA, English name is Ethylene Glycol Bis (2-aminoethyl Ether)-N, N, N', N'-tetraacetic Acid, Chinese is ethylene glycol bis (2-amino-ethyl ether) tetraacethyl, molecular weight is 380.35;
Mg-ATP, English name Adenosine 5 '-triphosphate magnesium salt, Chinese: 5 '-atriphos (ATP) magnesium salts, a kind of cellular energy supplements reagent, can maintain cell state, molecular weight: 507.18;
Na-GTP, English name Guanosine 5 '-triphosphate sodium salt, Chinese 5 '-GTP trisodium GTP trisodium sodium salt, a kind of cellular energy supplements reagent, can maintain cell state, molecular weight: 523.18;
Potassium gluconate, English full name: potassium gluconate, Chinese full name: K-IAO, molecular weight: 234.25;
KF, English full name: Potassium fluoride, Chinese full name: potassium fluoride, molecular weight: 58.1.
Rat Hippocampal Brain sheet: thickness is the hippocampal slices of rat hippocampus half brain of 400 μ m;
The preparation method of hippocampal slices is: select the healthy SD rat of 20 ~ 30d, broken end, cuts out hippocampus half brain with blade fast; Rapidly hippocampus half brain is sticked on microtome objective table, inserted 0 ~ 4 ℃ of frozen water ACSF (95% O wherein
2with 5% CO
2state of saturation) in section groove; Utilize oscillatory type microtome (Vibroslice brand, Britain manufactures) to cut out the hippocampal slices of 400 μ m thickness; Brain sheet is gone to 35 ℃ of incubation slots and hatch 1 h, then room temperature continues to hatch 1 h, is 95% O in incubation slot
2with 5% CO
2saturated artificial cerebrospinal fluid.After hatching, obtain test hippocampal slices.
The test procedure of described test method is:
(1) assembly and adjustment equipment: place hippocampal slices in track, constant flow pump carries out Continuous Perfusion circulation by the water pipe end user work cerebrospinal fluid of coming in and going out to track, flow velocity is 2 ml/min, and circulation fluid volume is 50 ml, and the artificial cerebrospinal fluid volume in track is 4ml; Test temperature is that 20 ~ 25 ℃ of room temperatures are carried out;
The supporting beaker that has 100ml of ebullator, in beaker, holding circulation fluid is artificial cerebrospinal fluid, in test, being about to medicine during dosing is added in beaker, ebullator circulates the artificial cerebrospinal fluid in the artificial cerebrospinal fluid in beaker and track, and the circulation by artificial cerebrospinal fluid is recycled to medicine in track hippocampal slices is carried out to drug effect.
Utilize drawing instrument that silicon boron glass pipe is drawn into experiment recording electrode, electrode resistance is 2 ~ 5 M Ω; Draw instrument model Puller-87, Americanized; The manufacturer of silicon boron glass pipe is U.S. Borosilicate, and the internal diameter of silicon boron glass pipe is 1.5 mm.
According to the mode shown in Fig. 1, testing equipment is assembled, one end of recording electrode is arranged in electrode jaw, and insert in track at the tip of recording electrode, and contact with hippocampal slices; Electrode jaw is provided with micro-behaviour's controller; Recording electrode connects by line amplifier, and amplifier is connected with computer with analog to digital converter by circuit; Track top is also provided with for observing and assist the microscope of micro-behaviour's controller operation, and microscope is connected with computer by signal line;
Electric stimulation pulse provide and the collection of electric signal all completes by the digidata 1320A interface of computer program Clampx9.2 and computer; Signal sampling frequency is 10 kHz, and low pass Bessel frequency filtering is 2 kHz; In experiment, continue to monitor the resistance of input resistance Ri and serial resistor Ra, Ri between 100 ~ 300 M Ω, Ra≤20 M Ω;
The resistance of input resistance Ri and serial resistor Ra is monitored by patch clamp amplifier, its resistance value can be presented on computing machine clampx9.2R program interface by data-switching, what Ri mainly represented is to form after full cell, the resistance of a whole cell membrane, the big or small key reaction of its resistance change be the most advanced and sophisticated sealed state with cell of the state variation of cell and recording electrode, if Ri diminishes, and amplitude surpasses original more than 30%, prompting cell state variation or the sealed state of electrode and cell are bad, the data that are recorded under this state will cause very large error with original data.Ra refers to and forms after full cell patch pincers logging mode, the resistance of eletrode tip and electrode itself, if it is large that Ra becomes, representing after forming full cell record pattern, originally being inhaled that broken sheet film and may plug-back occur again, the resistance between eletrode tip and cell has been increased again, there is variation in the original good full cell record state making, formed incomplete rupture of membranes, the data error that is recorded to is also very large in this case.In process of the test, by the monitoring to two resistances, can monitor the accuracy of neurocyte current monitoring, guarantee the accurate of test findings.
(2) form full cell record pattern: under microscope and micro-behaviour's controller auxiliary, the tip of recording electrode is also contacted near cell, form cell sticking type logging mode, to recording electrode, apply a negative pressure subsequently, make the membranolysis of eletrode tip and cell sticking portion, make recording electrode end and cell interior form full cell record pattern; By the mode of voltage clamp, cell membrane potential is clamped down in the required special value scope of test subsequently; Now, cell itself produces corresponding spontaneous discharge stream;
(3) record the test operation process that the spontaneous postsynaptic currents of neuron is sPSC: use liquid in the electrode record spontaneous postsynaptic currents to be filled with the body of recording electrode inner, in track, the artificial cerebrospinal fluid of circulation is 4ml, and then the circulation fluid to ebullator is in artificial cerebrospinal fluid, to add medicine polygalic acid;
The film potential of the cell under full cell record pattern is clamped down at-80mV, at full cell signal record, obtain after stable baseline, carry out data sampling and continue to record 5min;
Polygalic acid in this step is directly dissolved in artificial cerebrospinal fluid, then the administration of carrying out different pharmaceutical concentration by ebullator is tested, polygalic acid crude drug concentration is 0.5 g/L, 1 g/L, 1.5 g/L, tetra-kinds of different concentration of 2 g/L, under four drug concentrations, carry out four groups of tests, 8 test cycles are all carried out in every group of test, and the number of test cycle is experiment sample number; And under four kinds of drug concentrations, record respectively the signal data of the spontaneous postsynaptic currents of neuron in four groups of tests;
Described test cycle is: comprise administration last stage, administration stage and medicament elution stage; The administration last stage adopts ebullator that the artificial cerebrospinal fluid and the track that in beaker, do not contain TEN liquid are circulated, and now cell is not in containing in the artificial cerebrospinal fluid of liquid, and the data cell signal now recording is called control group data;
The administration stage: the artificial cerebrospinal fluid that contains polygalic acid liquid of test is added in beaker, by ebullator, the artificial cerebrospinal fluid circulation that contains liquid is pumped in track, neurocyte is subject to the effect of polygalic acid medicine, and the data cell signal now recording is called medicine group data;
The medicament elution stage: the artificial cerebrospinal fluid in beaker is replaced by the artificial cerebrospinal fluid that does not contain polygalic acid liquid, by ebullator, the liquid in track and artificial cerebrospinal fluid are replaced away, cell is in the artificial cerebrospinal fluid without liquid, and the data cell signal now recording is called recovery group data;
Each test cycle finishes to carry out next administration experiment all needs the hippocampal slices more renewing;
The test findings of the spontaneous postsynaptic currents of neuron is as shown in Fig. 3, Fig. 4, Fig. 5, Fig. 6, Control data in figure are the control group data of administration last stage, TEN data in figure are the medicine group data in administration stage, and the Recovery data in figure are the recovery group data in medicament elution stage; The impact of polygalic acid on hippocampal slices CA1 district cone neurone spontaneous postsynaptic currents (sPSC) frequency and amplitude.Fig. 3, Fig. 4 are that the sPSC that is recorded in experimentation is before administration, in administration process (the TEN concentration of 1.5g/L), and the variation diagram of the original sPSC after medicament elution.Fig. 5 is the amplitude cumulative distribution table (data come from the original graph data of Fig. 3, Fig. 4) of sPSC before and after administration, adopts KS check analysis to find, the amplitude cumulative distribution of the sPSC before and after administration has significant difference.And this otherness in 6 all routine cell experiments all clearly.In Fig. 6, polygalic acid can obviously increase the frequency of sPSC, and this increasing action presents a kind of concentration dependent.In this experimentation, film potential is clamped down at-80mV always.
The test findings of this step shows, the TEN of 1.5g/L crude drug concentration has obvious facilitation to the granting of sPSC, the frequency that significantly increases sPSC reaches 310.25% ± 44.19%(p<0.01), and can obviously increase the amplitude that electric current is provided; In Fig. 3, Fig. 4, Fig. 5, TEN makes this neuron provide frequency and is increased to 288.10%; Also obviously increase of current amplitude (KS check, p<0.01).In addition, TEN presents certain dose dependent to a certain extent to the facilitation of sPSC, and when concentration reaches 2.0g/L crude drug concentration, its facilitation reaches capacity.
(4) record the test operation process that the spontaneous EPSC of neuron is sEPSC: use liquid in the electrode record spontaneous EPSC to be filled with the body of recording electrode inner, carry out the delta data that 8 test cycles detect sEPSC;
Described test cycle is: comprise administration last stage, administration stage and medicament elution stage; The administration last stage adopts ebullator that the artificial cerebrospinal fluid and the track that in beaker, contain medicine BM are circulated, and the data cell signal now recording is called control group data; BM concentration in artificial cerebrospinal fluid is 20 μ mol/L;
The administration stage: be that 1.5 g/L, BM concentration are that the artificial cerebrospinal fluid of 20 μ mol/L adds in beaker by the polygalic acid crude drug concentration that contains of test, by ebullator, the artificial cerebrospinal fluid circulation that contains liquid is pumped in track, neurocyte is subject to the effect of polygalic acid medicine, and the data cell signal now recording is called medicine group data;
The medicament elution stage: the artificial cerebrospinal fluid in beaker is replaced by and contains the artificial cerebrospinal fluid that BM concentration is 20 μ mol/L, by ebullator, the liquid in track and artificial cerebrospinal fluid are replaced away, cell is in only containing the artificial cerebrospinal fluid of BM, and the data cell signal now recording is called recovery group data;
Each test cycle finishes to carry out next administration experiment all needs the hippocampal slices more renewing;
Each test cycle clamps down on the film potential of the cell under full cell record pattern at-70mV, at full cell signal record, obtains after stable baseline, carries out data sampling and continues to record 5min;
The test findings of the spontaneous EPSC of neuron, as shown in Fig. 7, Fig. 8, Fig. 9, Control data in figure are the control group data of administration last stage, TEN data in figure are the medicine group data in administration stage, and the Recovery data in figure are the recovery group data in medicament elution stage; The impact of polygalic acid on hippocampal slices CA1 district cone neurone spontaneous EPSC (sEPSC) frequency and amplitude.Fig. 7 is that the sEPSC that is recorded in experimentation is before administration, in administration process (TEN crude drug concentration 1.5g/L), and the original variation diagram after medicament elution.In Fig. 8, polygalic acid can obviously increase the frequency (recording sample is 8 groups of data) of sEPSC.Fig. 9 is the amplitude cumulative distribution table of sPSC before and after administration, adopts KS check analysis to find, the amplitude cumulative distribution of the sEPSC before and after administration has significant difference.In this experimentation, film potential is clamped down at-70mV always.
The test findings of this step shows, add BM (controlling concentration in artificial cerebrospinal fluid is 20 μ mol/L) in ACSF after, record sEPSC, after forming full cell record, film potential is clamped down at-70mV, that now recorded downward spontaneous interiorly can be blocked by ampa receptor antagonist CNQX (20 μ mol/L) completely to gas current, prove that this electric current is the spontaneous EPSC sEPSC that ampa receptor mediates, from Fig. 7 to Fig. 9, TEN has obvious facilitation to the granting of sEPSC, the frequency that significantly increases sEPSC reaches 237.25% ± 44.11%(p<0.01), and can obviously increase the amplitude (p<0.01) that electric current is provided.
(5) record the test operation process of the spontaneous inhibitory postsynaptic current sIPSC of neuron: use liquid in the electrode record spontaneous inhibitory postsynaptic current to be filled with the body of recording electrode inner, carry out the delta data that 6 test cycles detect sIPSC;
Described test cycle is: comprise administration last stage, administration stage and medicament elution stage; The administration last stage adopts ebullator that the artificial cerebrospinal fluid and the track that in beaker, contain medicine CNQX and DL-APV are circulated, and the data cell signal now recording is called control group data; In artificial cerebrospinal fluid, the concentration of CNQX is that the concentration of 20 μ mol/L, DL-APV is 50 μ mol/L;
The administration stage: be that 1.5 g/L, CNQX concentration are that 20 μ mol/L, DL-APV concentration are that the artificial cerebrospinal fluid of 50 μ mol/L adds in beaker by the polygalic acid crude drug concentration that contains of test, by ebullator, the artificial cerebrospinal fluid circulation that contains liquid is pumped in track, neurocyte is subject to the effect of polygalic acid medicine, and the data cell signal now recording is called medicine group data;
The medicament elution stage: it is that the concentration of 20 μ mol/L, DL-APV is the artificial cerebrospinal fluid of 50 μ mol/L that the artificial cerebrospinal fluid in beaker is replaced by the concentration that contains CNQX, by ebullator, the liquid in track and artificial cerebrospinal fluid are replaced away, cell is in only containing the artificial cerebrospinal fluid of CNQX and DL-APV, and the data cell signal now recording is called recovery group data;
Each test cycle finishes to carry out next administration experiment all needs the hippocampal slices more renewing;
Each test cycle clamps down on the film potential of the cell under full cell record pattern at-10mV, at full cell signal record, obtains after stable baseline, carries out data sampling and continues to record 5min;
Figure 10, Figure 11 and Figure 12 show the impact of polygalic acid on hippocampal slices CA1 district cone neurone spontaneous inhibitory postsynaptic current (sIPSC) frequency and amplitude.Control data in figure are the control group data of administration last stage, and the TEN data in figure are the medicine group data in administration stage, and the Washout data in figure are the recovery group data in medicament elution stage; Figure 10 is that the sIPSC that is recorded in experimentation is before administration, in administration process (TEN concentration 1.5g/L), and the original variation diagram after medicament elution.Figure 11 is that polygalic acid does not have obvious influence to the frequency of sIPSC (recording sample is 6 groups of cell data).Figure 12 is the amplitude cumulative distribution table of sPSC before and after administration, adopts KS check analysis to find, the amplitude cumulative distribution of the sIPSC before and after administration does not have significant difference.In this experimentation, film potential is clamped down at-10mV always.
The test findings of this step is: in ACSF, add Glu receptor antagonist CNQX (20 μ mol/L), after DL-APV (50 μ mol/L), record sIPSC, after forming full cell record, film potential is clamped down at-10mV, the spontaneous export-oriented gas current making progress that now recorded can be blocked completely by BM (20 μ mol/L), prove that this electric current is the spontaneous inhibitory postsynaptic current sIPSC that GABAA acceptor mediates, by Figure 10, Figure 11, Figure 12 is known, TEN does not have obvious impact to the granting of sIPSC, frequency 93.01% ± 4.56%(p>0.05 of sIPSC after administration), the amplitude of electric current granting is not had to obvious impact (p>0.05) yet.
(6) record the test operation process that the spontaneous miniature excited postsynaptic current of neuron is mEPSC, carry out the delta data that 6 test cycles detect mEPSC;
Described test cycle is: comprise administration last stage, administration stage and medicament elution stage; The administration last stage adopts ebullator that the artificial cerebrospinal fluid and the track that in beaker, contain medicine BM and TTX are circulated, and the data cell signal now recording is called control group data; BM concentration in artificial cerebrospinal fluid is 20 μ mol/L, and the concentration of TTX is 1 μ mol/L;
The administration stage: be that 1.5 g/L, BM concentration are 20 μ mol/L by the polygalic acid crude drug concentration that contains of test, the concentration of TTX is that the artificial cerebrospinal fluid of 1 μ mol/L adds in beaker, by ebullator, the artificial cerebrospinal fluid circulation that contains liquid is pumped in track, neurocyte is subject to the effect of polygalic acid medicine, and the data cell signal now recording is called medicine group data;
The medicament elution stage: the artificial cerebrospinal fluid in beaker is replaced by to contain BM concentration be that the concentration of 20 μ mol/L, TTX is the artificial cerebrospinal fluid of 1 μ mol/L, by ebullator, the liquid in track and artificial cerebrospinal fluid are replaced away, cell is in only containing the artificial cerebrospinal fluid of BM and TTX, and the data cell signal now recording is called recovery group data;
Each test cycle finishes to carry out next administration experiment all needs the hippocampal slices more renewing;
Each test cycle clamps down on the film potential of the cell under full cell record pattern at-70mV, at full cell signal record, obtains after stable baseline, carries out data sampling and continues to record 5min;
As Figure 13, Figure 14, Figure 15 shows that the impact of micro-electric current (mEPSC) frequency and amplitude after polygalic acid is on the spontaneous excitatory synapse of hippocampal slices CA1 district cone neurone.Control data in figure are the control group data of administration last stage, and the TEN data in figure are the medicine group data in administration stage, and the Recovery data in figure are the recovery group data in medicament elution stage; Figure 13 is that the mEPSC that is recorded in experimentation is before administration, in administration process (TEN concentration is 1.5g/L), and the original variation diagram after medicament elution.Figure 14 is that polygalic acid has obvious humidification to the frequency of mEPSC (recording sample is 8 groups of cell data).Figure 15 is the amplitude cumulative distribution table of mPSC before and after administration, adopts KS check analysis to find, before and after administration, the amplitude cumulative distribution of mEPSC does not have significant difference.In this experimentation, film potential is clamped down at-70mV always.
The test findings of this step is, in ACSF, add BM (20 μ mol/L), after TTX (1 μ mol/L), record mEPSC, after forming full cell record, film potential is clamped down at-70mV, that now recorded downward spontaneous is interiorly significantly less than sEPSC to ion current magnitude and frequency, this electric current also can be blocked completely by ampa receptor antagonist CNQX (concentration is 20 μ mol/L), prove that this electric current is spontaneous micro-EPSC mEPSC (not showing result) that ampa receptor mediates, as shown in Figure 4, TEN has obvious facilitation to the granting of mEPSC, the frequency that significantly increases mEPSC reaches 201.13% ± 25.43%(p<0.01), but the amplitude that mEPSC electric current is provided is significantly impact (p>0.05) not.
(7) statistical study: adopt MiniAnalysis6.0 to analyze the signal data collecting, adopt Origin 8.0 and corelDRAW mapping; Data acquisition by mean ± standard deviation
the mode of ± s represents, between group, relatively adopting one-way analysis of variance, P<0.05 is that difference has statistical significance; Use
kolmogorov-Smirnovcheck carrys out the relatively current amplitude difference between each group, and P<0.05 is that difference has statistical significance.
This test with the acquisition methods of polygalic acid is:
As shown in Figure 2, take the polygala root that 1000g is purchased from Nanchang City, Jiangxi Province Chinese Medicinal Materials Markets, this polygala root is Roots of Polygala tenuifolia; Polygala root is pulverized, added sherwood oil and carry out degreasing; Then with 95% alcohol reflux, extract 3 times each 6h; Three gained extracts are merged, with Rotary Evaporators, will after extract evaporate to dryness, obtain medicinal extract; Medicinal extract is redissolved in the water of 3 times of volumes, filter, gained filtrate is adopted and is extracted with ethyl acetate 6 times, the ethyl acetate use amount of front twice extraction is 1/3 of filtrate cumulative volume, for the third time, the ethyl acetate use amount of the 4th extraction is 1/4 of filtrate cumulative volume, the ethyl acetate use amount of the 5th extraction is that the ethyl acetate use amount of 1/5, the six extraction of filtrate cumulative volume is 1/6 of filtrate cumulative volume; Then by extract macroporous resin adsorption, again extract is carried out to wash-out with distilled water, 30% ethanol, 70% ethanol, 95% ethanol respectively, collect after eluent its freeze drying, re-using 70% ethanol eluate carries out obtaining test polygalic acid medicine after wash-out evaporate to dryness, the final polygalic acid 41.96g that obtains, extraction ratio is 4.2%.
The result of this test method shows, TEN transmits and has obvious facilitation neurons of hippocampus CA 1 cynapse, the electric current that promotes the granting frequency of sPSC and enlarge markedly sPSC is provided amplitude, and this facilitation of TEN is dose dependent to a certain extent.In to the further separating experiment of sPSC electric current composition, find, TEN can obviously promote the granting of sEPSC, shows obvious increasing frequently and amplification effect.Yet TEN is on not significantly impact of the granting of sIPSC.This results suggest, the effect that TEN transmits Hippocampal CA 1 cone neurone cynapse is mainly manifested in the regulation and control that excitatory synapse is transmitted, and this regulating and controlling effect has nothing to do with inhibitory interneuron.
The Schaffer side prop up/association fibre YuCA1 district cones that CA3 district cones sends forms the excitability list synaptic contact that mediator is glutamic acid, under quiescent condition, the glutamic acid that presynaptic membrane discharges is quantum formula and is discharged into synaptic cleft, then cause depolarization current with the corresponding acceptor of postsynaptic membrane as receptors bind such as alpha-amido-3-hydroxy-5-methyl base oxazole-4-propionic acid (AMPA), i.e. the micro-postsynaptic membrane electric current of spontaneous excitability (mEPSC).That is to say the spontaneous activity of mEPSC reflection excitatory synapse under quiescent condition.The frequency change of mEPSC mainly reflect presynaptic glutamic acid burst size number, amplitude changes main relevant with postsynaptic membrane acceptor property.Therefore we again further experimental study the impact of TEN on Hippocampal CA 1 cone neurone mEPSC.Result shows, TEN has obvious increasing effect frequently to the granting of mEPSC, and the electric current of mEPSC is provided to amplitude, does not produce obvious impact.This explanation TEN mainly realizes by presynaptic inhibition the facilitation of excitatory synapse transmission.
In sum, TEN can promote cynapse transfer function, be mainly manifested in the regulation and control that excitatory synapse is transmitted, and these regulation and control realizes by direct promotions presynaptic glutamic acid burst size, has nothing to do with the indirect adjustments and controls of inhibitory interneuron.
Test method of the present invention adopts the impact of full cell patch tongs technology research polygalic acid on the spontaneous postsynaptic currents in rat brain Hippocampal CA 1, can by the mode of test, accurately disclose polygalic acid neurocyte cynapse is transmitted to regulating and controlling effect and mechanism, thereby met the test demand of scientific research and drug development, also can make further research for the signal transport mechanism of neurocyte simultaneously.
Test method of the present invention adopts different medicines neurocyte to be carried out to the collection of test figure, and gathers multi-group data and carry out the analysis of science, can draw science, compellent test figure and analysis result.
Test method of the present invention, adopts micro-behaviour's controller and microscope to coordinate, and carries out the full cell record pattern operation of neurocyte, has point-device operational readiness, has guaranteed the accuracy of Test Data Collecting and the degree of accuracy of test findings.
Test method of the present invention, the analysis data that gather have higher accuracy, the science drawing for the analysis of test figure, analysis result accurately, therefore, test method of the present invention and equipment can meet the requirement of scientific research and test, and the design science of test method is effective.
Test method of the present invention, adopts a plurality of test cycles to obtain a plurality of sample datas in each test, and each test cycle adopts new hippocampal slices and liquid to test, and has guaranteed the accuracy of test findings.
Accompanying drawing explanation
Fig. 1 is testing equipment assembling assumption diagram of the present invention.
Fig. 2 is the process flow diagram of polygalic acid extracting method of the present invention.
Fig. 3 is the variation diagram of original sPSC in the administration process of the spontaneous postsynaptic currents of neuron of the present invention test.
Fig. 4 is before the administration of the spontaneous postsynaptic currents of neuron of the present invention test, in administration process, the variation diagram of the original sPSC after medicament elution.
Fig. 5 is the amplitude cumulative distribution table of the administration front and back sPSC of the spontaneous postsynaptic currents test of neuron of the present invention.
Fig. 6 is the sPSC of the spontaneous postsynaptic currents of the neuron of the present invention test frequency change figure under different polygalic acid mass actions.
Fig. 7 is before the administration of the spontaneous EPSC of neuron of the present invention test, in administration process, the variation diagram of the original sEPSC after medicament elution.
Fig. 8 is the frequency change figure of the sEPSC of the spontaneous EPSC test of neuron of the present invention.
Fig. 9 is the amplitude cumulative distribution table of the administration front and back sEPSC of the spontaneous EPSC test of neuron of the present invention.
Figure 10 is before the administration of the spontaneous inhibitory postsynaptic current of neuron of the present invention test, in administration process, the variation diagram of the original sIPSC after medicament elution.
Figure 11 is the frequency change figure of the sIPSC of the spontaneous inhibitory postsynaptic current test of neuron of the present invention.
Figure 12 is the amplitude cumulative distribution table of the administration front and back sIPSC of the spontaneous inhibitory postsynaptic current test of neuron of the present invention.
Figure 13 is before the administration of the spontaneous miniature excited postsynaptic current of neuron of the present invention test, in administration process, the variation diagram of the original mEPSC after medicament elution.
Figure 14 is the frequency change figure of the mEPSC of the spontaneous miniature excited postsynaptic current test of neuron of the present invention.
Figure 15 is the amplitude cumulative distribution table of the administration front and back mEPSC of the spontaneous miniature excited postsynaptic current test of neuron of the present invention.
Embodiment
The following example will further illustrate the present invention.
embodiment 1
It is the test method of polygalic acid to the effect of hippocampus of rats cynapse transmission that the present invention adopts technical scheme, medicine and reagent that this test method is used:
Artificial cerebrospinal fluid, i.e. ACSF, its component is: NaCl is that 124 mmol/L, KCl are 2.5 mmol/L, NaHCO
3be 26 mmol/L, NaH
2pO
4be 1.25 mmol/L, CaCl
2be 2. 0 mmol/L, MgSO
4be that 1.3 mmol/L, D-Glucose are 10 mmol/L, pH value is 7.4;
The interior liquid of electrode that records spontaneous postsynaptic currents, its component is: KCl is that 140 mmol/L, NaCl are that 6 mmol/L, HEPES are that 10 mmol/L, EGTA are that 10 mmol/L, Mg-ATP are that 2 mmol/L, NaGTP are that 0.1 mmol/L, pH value are 7.2;
The interior liquid of electrode that records spontaneous EPSC, its component is: K gluconate is that 140 mmol/L, NaCl are that 6 mmol/L, HEPES are that 10 mmol/L, EGTA are that 0.2 mmol/L, Mg-ATP are that 2 mmol/L, Na-GTP are that 0.1 mmol/L, pH value are 7.2;
The interior liquid of electrode that records spontaneous inhibitory postsynaptic current, its component is: KF is that 140 mmol/L, NaCl are that 6 mmol/L, HEPES are that 10 mmol/L, EGTA are that 0.2 mmol/L, Mg-ATP are that 2 mmol/L, Na-GTP are that 0.1 mmol/L, pH value are 7.2;
Test medication: CNQX, DL-APV, tetrodotoxin are that TTX, bicuculline are that BM, polygalic acid are TEN;
CNQX, DL-APV, bicuculline are that BM, tetrodotoxin are that TTX, HEPES, EGTA, Mg-ATP, Na-GTP, potassium gluconate, KF are all purchased from U.S. Sigma company, polygalic acid is that TEN is prepared by laboratory, and all the other reagent are domestic analytical reagent;
CNQX, English full name: 6-cyano-7-nitroquinoxaline-2,3-dione, Chinese full name: 6-cyano group-7-nitro quinoxaline-2,3-diketone, is nerve excitability mediator Glutamate AMPA Receptor Antagonist, molecule is 232.15;
DL-APV, English full name: DL-2-amino-5-phosphonovaleric acid, Chinese full name: DL-2-amino-5-phosphono valeric acid is nerve excitability mediator glutamate nmda receptor antagonist, and molecular weight is 197.13;
Bicuculline is BM, English full name: bicuculline, the popular name of Chinese: Bic is inhibitory neurotransmitter γ-aminobutyric acid GABA receptor blocking pharmacon, molecular weight 367.35;
Tetrodotoxin is TTX, English full name: tetrodotoxin, the popular name of Chinese: tetraodotoxin is cell membrane Na
+ion channel blocking agent, molecular weight: 319.27;
HEPES, English name 4-(2-hydroxyerhyl) piperazine-1-erhanesulfonic acid, Chinese full name: 4-hydroxyethyl piperazine ethanesulfonic acid, molecular weight is 238.3;
EGTA, English name is Ethylene Glycol Bis (2-aminoethyl Ether)-N, N, N', N'-tetraacetic Acid, Chinese is ethylene glycol bis (2-amino-ethyl ether) tetraacethyl, molecular weight is 380.35;
Mg-ATP, English name Adenosine 5 '-triphosphate magnesium salt, Chinese: 5 '-atriphos (ATP) magnesium salts, a kind of cellular energy supplements reagent, can maintain cell state, molecular weight: 507.18;
Na-GTP, English name Guanosine 5 '-triphosphate sodium salt, Chinese 5 '-GTP trisodium GTP trisodium sodium salt, a kind of cellular energy supplements reagent, can maintain cell state, molecular weight: 523.18;
Potassium gluconate, English full name: potassium gluconate, Chinese full name: K-IAO, molecular weight: 234.25;
KF, English full name: Potassium fluoride, Chinese full name: potassium fluoride, molecular weight: 58.1.
Rat Hippocampal Brain sheet: thickness is the hippocampal slices of rat hippocampus half brain of 400 μ m;
The preparation method of hippocampal slices is: select the healthy SD rat of 20 ~ 30d, broken end, cuts out hippocampus half brain with blade fast; Rapidly hippocampus half brain is sticked on microtome objective table, inserted 0 ~ 4 ℃ of frozen water ACSF (95% O wherein
2with 5% CO
2state of saturation) in section groove; Utilize oscillatory type microtome (Vibroslice brand, Britain manufactures) to cut out the hippocampal slices of 400 μ m thickness; Brain sheet is gone to 35 ℃ of incubation slots and hatch 1 h, then room temperature continues to hatch 1 h, is 95% O in incubation slot
2with 5% CO
2saturated artificial cerebrospinal fluid.After hatching, obtain test hippocampal slices.
The test procedure of this test method is:
(1) assembly and adjustment equipment: place hippocampal slices in track, constant flow pump carries out Continuous Perfusion by the water pipe end user work cerebrospinal fluid of coming in and going out to track, and flow velocity is 2 ml/min, and circulation fluid volume is 50 ml, and the artificial cerebrospinal fluid volume in track is 4ml; Test temperature is that 20 ~ 25 ℃ of room temperatures are carried out;
Utilize drawing instrument that silicon boron glass pipe is drawn into experiment recording electrode, electrode resistance is 3 M Ω; One end of recording electrode is arranged in electrode jaw, and insert in track at the tip of recording electrode, and contact with hippocampal slices; Electrode jaw is provided with micro-behaviour's controller; Recording electrode connects by line amplifier, and amplifier is connected with computer with analog to digital converter by circuit; Track top is also provided with for observing and assist the microscope of micro-behaviour's controller operation, and microscope is connected with computer by signal line;
Electric stimulation pulse provide and the collection of electric signal all completes by the digidata 1320A interface of computer program Clampx9.2 and computer; Signal sampling frequency is 10 kHz, and low pass Bessel frequency filtering is 2 kHz; In experiment, continue to monitor the resistance of input resistance Ri and serial resistor Ra, Ri between 100 ~ 300 M Ω, Ra≤20 M Ω;
(2) form full cell record pattern: under microscope and micro-behaviour's controller auxiliary, the tip of recording electrode is also contacted near cell, form cell sticking type logging mode, to recording electrode, apply a negative pressure subsequently, make the membranolysis of eletrode tip and cell sticking portion, make recording electrode end and cell interior form full cell record pattern; By the mode of voltage clamp, cell membrane potential is clamped down in the required special value scope of test subsequently; Now, cell itself produces corresponding spontaneous discharge stream;
(3) record the test operation process that the spontaneous postsynaptic currents of neuron is sPSC: use liquid in the electrode record spontaneous postsynaptic currents to be filled with the body of recording electrode inner, in track, the artificial cerebrospinal fluid of circulation is 4ml, and then the circulation fluid to ebullator is in artificial cerebrospinal fluid, to add medicine polygalic acid;
The film potential of the cell under full cell record pattern is clamped down at-80mV, at full cell signal record, obtain after stable baseline, carry out data sampling and continue to record 5min;
Polygalic acid in this step is directly dissolved in artificial cerebrospinal fluid, then the administration of carrying out different pharmaceutical concentration by ebullator is tested, polygalic acid crude drug concentration is 0.5 g/L, 1 g/L, 1.5 g/L, tetra-kinds of different concentration of 2 g/L, under four drug concentrations, carry out four groups of tests, 8 test cycles are all carried out in every group of test, and the number of test cycle is experiment sample number; And under four kinds of drug concentrations, record respectively the signal data of the spontaneous postsynaptic currents of neuron in four groups of tests;
Described test cycle is: comprise administration last stage, administration stage and medicament elution stage; The administration last stage adopts ebullator that the artificial cerebrospinal fluid and the track that in beaker, do not contain TEN liquid are circulated, and now cell is not in containing in the artificial cerebrospinal fluid of liquid, and the data cell signal now recording is called control group data;
The administration stage: the artificial cerebrospinal fluid that contains polygalic acid liquid of test is added in beaker, by ebullator, the artificial cerebrospinal fluid circulation that contains liquid is pumped in track, neurocyte is subject to the effect of polygalic acid medicine, and the data cell signal now recording is called medicine group data;
The medicament elution stage: the artificial cerebrospinal fluid in beaker is replaced by the artificial cerebrospinal fluid that does not contain polygalic acid liquid, by ebullator, the liquid in track and artificial cerebrospinal fluid are replaced away, cell is in the artificial cerebrospinal fluid without liquid, and the data cell signal now recording is called recovery group data;
Each test cycle finishes to carry out next administration experiment all needs the hippocampal slices more renewing;
(4) record the test operation process that the spontaneous EPSC of neuron is sEPSC: use liquid in the electrode record spontaneous EPSC to be filled with the body of recording electrode inner, carry out the delta data that 8 test cycles detect sEPSC;
Described test cycle is: comprise administration last stage, administration stage and medicament elution stage; The administration last stage adopts ebullator that the artificial cerebrospinal fluid and the track that in beaker, contain medicine BM are circulated, and the data cell signal now recording is called control group data; BM concentration in artificial cerebrospinal fluid is 20 μ mol/L;
The administration stage: be that 1.5 g/L, BM concentration are that the artificial cerebrospinal fluid of 20 μ mol/L adds in beaker by the polygalic acid crude drug concentration that contains of test, by ebullator, the artificial cerebrospinal fluid circulation that contains liquid is pumped in track, neurocyte is subject to the effect of polygalic acid medicine, and the data cell signal now recording is called medicine group data;
The medicament elution stage: the artificial cerebrospinal fluid in beaker is replaced by and contains the artificial cerebrospinal fluid that BM concentration is 20 μ mol/L, by ebullator, the liquid in track and artificial cerebrospinal fluid are replaced away, cell is in only containing the artificial cerebrospinal fluid of BM, and the data cell signal now recording is called recovery group data;
Each test cycle finishes to carry out next administration experiment all needs the hippocampal slices more renewing;
Each test cycle clamps down on the film potential of the cell under full cell record pattern at-70mV, at full cell signal record, obtains after stable baseline, carries out data sampling and continues to record 5min;
(5) record the test operation process of the spontaneous inhibitory postsynaptic current sIPSC of neuron: use liquid in the electrode record spontaneous inhibitory postsynaptic current to be filled with the body of recording electrode inner, carry out the delta data that 6 test cycles detect sIPSC;
Described test cycle is: comprise administration last stage, administration stage and medicament elution stage; The administration last stage adopts ebullator that the artificial cerebrospinal fluid and the track that in beaker, contain medicine CNQX and DL-APV are circulated, and the data cell signal now recording is called control group data; In artificial cerebrospinal fluid, the concentration of CNQX is that the concentration of 20 μ mol/L, DL-APV is 50 μ mol/L;
The administration stage: be that 1.5 g/L, CNQX concentration are that 20 μ mol/L, DL-APV concentration are that the artificial cerebrospinal fluid of 50 μ mol/L adds in beaker by the polygalic acid crude drug concentration that contains of test, by ebullator, the artificial cerebrospinal fluid circulation that contains liquid is pumped in track, neurocyte is subject to the effect of polygalic acid medicine, and the data cell signal now recording is called medicine group data;
The medicament elution stage: it is that the concentration of 20 μ mol/L, DL-APV is the artificial cerebrospinal fluid of 50 μ mol/L that the artificial cerebrospinal fluid in beaker is replaced by the concentration that contains CNQX, by ebullator, the liquid in track and artificial cerebrospinal fluid are replaced away, cell is in only containing the artificial cerebrospinal fluid of CNQX and DL-APV, and the data cell signal now recording is called recovery group data;
Each test cycle finishes to carry out next administration experiment all needs the hippocampal slices more renewing;
Each test cycle clamps down on the film potential of the cell under full cell record pattern at-10mV, at full cell signal record, obtains after stable baseline, carries out data sampling and continues to record 5min;
(6) record the test operation process that the spontaneous miniature excited postsynaptic current of neuron is mEPSC, carry out the delta data that 6 test cycles detect mEPSC;
Described test cycle is: comprise administration last stage, administration stage and medicament elution stage; The administration last stage adopts ebullator that the artificial cerebrospinal fluid and the track that in beaker, contain medicine BM and TTX are circulated, and the data cell signal now recording is called control group data; BM concentration in artificial cerebrospinal fluid is 20 μ mol/L, and the concentration of TTX is 1 μ mol/L;
The administration stage: be that 1.5 g/L, BM concentration are 20 μ mol/L by the polygalic acid crude drug concentration that contains of test, the concentration of TTX is that the artificial cerebrospinal fluid of 1 μ mol/L adds in beaker, by ebullator, the artificial cerebrospinal fluid circulation that contains liquid is pumped in track, neurocyte is subject to the effect of polygalic acid medicine, and the data cell signal now recording is called medicine group data;
The medicament elution stage: the artificial cerebrospinal fluid in beaker is replaced by to contain BM concentration be that the concentration of 20 μ mol/L, TTX is the artificial cerebrospinal fluid of 1 μ mol/L, by ebullator, the liquid in track and artificial cerebrospinal fluid are replaced away, cell is in only containing the artificial cerebrospinal fluid of BM and TTX, and the data cell signal now recording is called recovery group data;
Each test cycle finishes to carry out next administration experiment all needs the hippocampal slices more renewing;
Each test cycle clamps down on the film potential of the cell under full cell record pattern at-70mV, at full cell signal record, obtains after stable baseline, carries out data sampling and continues to record 5min;
(7) statistical study: adopt MiniAnalysis6.0 to analyze the signal data collecting, adopt Origin 8.0 and corelDRAW mapping; Data acquisition by mean ± standard deviation
the mode of ± s represents, between group, relatively adopting one-way analysis of variance, P<0.05 is that difference has statistical significance; Use
kolmogorov-Smirnovcheck carrys out the relatively current amplitude difference between each group, and P<0.05 is that difference has statistical significance.
This test with the acquisition methods of polygalic acid is:
Take 1000g and be purchased from the polygala root of Nanchang City, Jiangxi Province Chinese Medicinal Materials Markets, this polygala root is Roots of Polygala tenuifolia; Polygala root is pulverized, added sherwood oil and carry out degreasing; Then with 95% alcohol reflux, extract 3 times each 6h; Three gained extracts are merged, with Rotary Evaporators, will after extract evaporate to dryness, obtain medicinal extract; Medicinal extract is redissolved in the water of 3 times of volumes, filter, gained filtrate is adopted and is extracted with ethyl acetate 6 times, the ethyl acetate use amount of front twice extraction is 1/3 of filtrate cumulative volume, for the third time, the ethyl acetate use amount of the 4th extraction is 1/4 of filtrate cumulative volume, the ethyl acetate use amount of the 5th extraction is that the ethyl acetate use amount of 1/5, the six extraction of filtrate cumulative volume is 1/6 of filtrate cumulative volume; Then by extract macroporous resin adsorption, again extract is carried out to wash-out with distilled water, 30% ethanol, 70% ethanol, 95% ethanol respectively, collect after eluent its freeze drying, re-using 70% ethanol eluate carries out obtaining test polygalic acid medicine after wash-out evaporate to dryness, the final polygalic acid 41.96g that obtains, extraction ratio is 4.2%.
The test figure of the present embodiment is shown in Fig. 3 to Figure 15 part, and the analysis result of test figure is shown in the data analysis of the summary of the invention part of instructions.
Finally it should be noted that: obviously, above-described embodiment is only for example of the present invention is clearly described, and the not restriction to embodiment.For those of ordinary skill in the field, can also make other changes in different forms on the basis of the above description.Here exhaustive without also giving all embodiments.And the apparent variation of being amplified out thus or change are still among protection scope of the present invention.
Claims (2)
1. the test method of the effect that polygalic acid transmits hippocampus of rats cynapse, is characterized in that,
Medicine and reagent that this test method is used:
Artificial cerebrospinal fluid, i.e. ACSF, its component is: NaCl is that 124 mmol/L, KCl are 2.5 mmol/L, NaHCO
3be 26 mmol/L, NaH
2pO
4be 1.25 mmol/L, CaCl
2be 2. 0 mmol/L, MgSO
4be that 1.3 mmol/L, D-Glucose are 10 mmol/L, pH value is 7.4;
The interior liquid of electrode that records spontaneous postsynaptic currents, its component is: KCl is that 140 mmol/L, NaCl are that 6 mmol/L, HEPES are that 10 mmol/L, EGTA are that 10 mmol/L, Mg-ATP are that 2 mmol/L, NaGTP are that 0.1 mmol/L, pH value are 7.2;
The interior liquid of electrode that records spontaneous EPSC, its component is: K gluconate is that 140 mmol/L, NaCl are that 6 mmol/L, HEPES are that 10 mmol/L, EGTA are that 0.2 mmol/L, Mg-ATP are that 2 mmol/L, Na-GTP are that 0.1 mmol/L, pH value are 7.2;
The interior liquid of electrode that records spontaneous inhibitory postsynaptic current, its component is: KF is that 140 mmol/L, NaCl are that 6 mmol/L, HEPES are that 10 mmol/L, EGTA are that 0.2 mmol/L, Mg-ATP are that 2 mmol/L, Na-GTP are that 0.1 mmol/L, pH value are 7.2;
Test medication: CNQX, DL-APV, tetrodotoxin are that TTX, bicuculline are that BM, polygalic acid are TEN;
CNQX, DL-APV, bicuculline are that BM, tetrodotoxin are that TTX, HEPES, EGTA, Mg-ATP, Na-GTP, potassium gluconate, KF are all purchased from U.S. Sigma company, polygalic acid is that TEN is prepared by laboratory, and all the other reagent are domestic analytical reagent;
Rat Hippocampal Brain sheet: thickness is the hippocampal slices of rat hippocampus half brain of 400 μ m;
The test procedure of described test method is:
(1) assembly and adjustment equipment: place hippocampal slices in track, constant flow pump carries out Continuous Perfusion by the water pipe end user work cerebrospinal fluid of coming in and going out to track, and flow velocity is 2 ml/min, and circulation fluid volume is 50 ml, circulation fluid is contained in beaker, and the artificial cerebrospinal fluid volume in track is 4ml; Test temperature is that 20 ~ 25 ℃ of room temperatures are carried out;
Utilize drawing instrument that silicon boron glass pipe is drawn into experiment recording electrode, electrode resistance is 2 ~ 5 M Ω; One end of recording electrode is arranged in electrode jaw, and insert in track at the tip of recording electrode, and contact with hippocampal slices; Electrode jaw is provided with micro-behaviour's controller; Recording electrode connects by line amplifier, and amplifier is connected with computer with analog to digital converter by circuit; Track top is also provided with for observing and assist the microscope of micro-behaviour's controller operation, and microscope is connected with computer by signal line;
Electric stimulation pulse provide and the collection of electric signal all completes by the digidata 1320A interface of computer program Clampx9.2 and computer; Signal sampling frequency is 10 kHz, and low pass Bessel frequency filtering is 2 kHz; In experiment, continue to monitor the resistance of input resistance Ri and serial resistor Ra, Ri between 100 ~ 300 M Ω, Ra≤20 M Ω;
(2) form full cell record pattern: under microscope and micro-behaviour's controller auxiliary, the tip of recording electrode is also contacted near cell, form cell sticking type logging mode, to recording electrode, apply a negative pressure subsequently, make the membranolysis of eletrode tip and cell sticking portion, make recording electrode end and cell interior form full cell record pattern; By the mode of voltage clamp, cell membrane potential is clamped down in the required special value scope of test subsequently; Now, cell itself produces corresponding spontaneous discharge stream;
(3) record the test operation process that the spontaneous postsynaptic currents of neuron is sPSC: use liquid in the electrode record spontaneous postsynaptic currents to be filled with the body of recording electrode inner, in track, the artificial cerebrospinal fluid of circulation is 4ml, and then the circulation fluid to ebullator is in artificial cerebrospinal fluid, to add medicine polygalic acid;
The film potential of the cell under full cell record pattern is clamped down at-80mV, at full cell signal record, obtain after stable baseline, carry out data sampling and continue to record 5min;
Polygalic acid in this step is directly dissolved in artificial cerebrospinal fluid, then the administration of carrying out different pharmaceutical concentration by ebullator is tested, polygalic acid crude drug concentration is 0.5 g/L, 1 g/L, 1.5 g/L, tetra-kinds of different concentration of 2 g/L, under four drug concentrations, carry out four groups of tests, several test cycles are all carried out in every group of test, and the number of test cycle is experiment sample number; And under four kinds of drug concentrations, record respectively the signal data of the spontaneous postsynaptic currents of neuron in four groups of tests;
Described test cycle is: comprise administration last stage, administration stage and medicament elution stage; The administration last stage adopts ebullator that the artificial cerebrospinal fluid and the track that in beaker, do not contain TEN liquid are circulated, and now cell is not in containing in the artificial cerebrospinal fluid of liquid, and the data cell signal now recording is called control group data;
The administration stage: the artificial cerebrospinal fluid that contains polygalic acid liquid of test is added in beaker, by ebullator, the artificial cerebrospinal fluid circulation that contains liquid is pumped in track, neurocyte is subject to the effect of polygalic acid medicine, and the data cell signal now recording is called medicine group data;
The medicament elution stage: the artificial cerebrospinal fluid in beaker is replaced by the artificial cerebrospinal fluid that does not contain polygalic acid liquid, by ebullator, the liquid in track and artificial cerebrospinal fluid are replaced away, cell is in the artificial cerebrospinal fluid without liquid, and the data cell signal now recording is called recovery group data;
Each test cycle finishes to carry out next administration experiment all needs the hippocampal slices more renewing;
(4) record the test operation process that the spontaneous EPSC of neuron is sEPSC: use liquid in the electrode record spontaneous EPSC to be filled with the body of recording electrode inner, carry out the delta data that several test cycles detect sEPSC;
Described test cycle is: comprise administration last stage, administration stage and medicament elution stage; The administration last stage adopts ebullator that the artificial cerebrospinal fluid and the track that in beaker, contain medicine BM are circulated, and the data cell signal now recording is called control group data; BM concentration in artificial cerebrospinal fluid is 20 μ mol/L;
The administration stage: be that 1.5 g/L, BM concentration are that the artificial cerebrospinal fluid of 20 μ mol/L adds in beaker by the polygalic acid crude drug concentration that contains of test, by ebullator, the artificial cerebrospinal fluid circulation that contains liquid is pumped in track, neurocyte is subject to the effect of polygalic acid medicine, and the data cell signal now recording is called medicine group data;
The medicament elution stage: the artificial cerebrospinal fluid in beaker is replaced by and contains the artificial cerebrospinal fluid that BM concentration is 20 μ mol/L, by ebullator, the liquid in track and artificial cerebrospinal fluid are replaced away, cell is in only containing the artificial cerebrospinal fluid of BM, and the data cell signal now recording is called recovery group data;
Each test cycle finishes to carry out next administration experiment all needs the hippocampal slices more renewing;
Each test cycle clamps down on the film potential of the cell under full cell record pattern at-70mV, at full cell signal record, obtains after stable baseline, carries out data sampling and continues to record 5min;
(5) record the test operation process of the spontaneous inhibitory postsynaptic current sIPSC of neuron: use liquid in the electrode record spontaneous inhibitory postsynaptic current to be filled with the body of recording electrode inner, carry out the delta data that several test cycles detect sIPSC; CNQX is Glu receptor antagonist;
Described test cycle is: comprise administration last stage, administration stage and medicament elution stage; The administration last stage adopts ebullator that the artificial cerebrospinal fluid and the track that in beaker, contain medicine CNQX and DL-APV are circulated, and the data cell signal now recording is called control group data; In artificial cerebrospinal fluid, the concentration of CNQX is that the concentration of 20 μ mol/L, DL-APV is 50 μ mol/L;
The administration stage: be that 1.5 g/L, CNQX concentration are that 20 μ mol/L, DL-APV concentration are that the artificial cerebrospinal fluid of 50 μ mol/L adds in beaker by the polygalic acid crude drug concentration that contains of test, by ebullator, the artificial cerebrospinal fluid circulation that contains liquid is pumped in track, neurocyte is subject to the effect of polygalic acid medicine, and the data cell signal now recording is called medicine group data;
The medicament elution stage: it is that the concentration of 20 μ mol/L, DL-APV is the artificial cerebrospinal fluid of 50 μ mol/L that the artificial cerebrospinal fluid in beaker is replaced by the concentration that contains CNQX, by ebullator, the liquid in track and artificial cerebrospinal fluid are replaced away, cell is in only containing the artificial cerebrospinal fluid of CNQX and DL-APV, and the data cell signal now recording is called recovery group data;
Each test cycle finishes to carry out next administration experiment all needs the hippocampal slices more renewing;
Each test cycle clamps down on the film potential of the cell under full cell record pattern at-10mV, at full cell signal record, obtains after stable baseline, carries out data sampling and continues to record 5min;
(6) record the test operation process that the spontaneous miniature excited postsynaptic current of neuron is mEPSC, carry out the delta data that several test cycles detect mEPSC;
Described test cycle is: comprise administration last stage, administration stage and medicament elution stage; The administration last stage adopts ebullator that the artificial cerebrospinal fluid and the track that in beaker, contain medicine BM and TTX are circulated, and the data cell signal now recording is called control group data; BM concentration in artificial cerebrospinal fluid is 20 μ mol/L, and the concentration of TTX is 1 μ mol/L;
The administration stage: be that 1.5 g/L, BM concentration are 20 μ mol/L by the polygalic acid crude drug concentration that contains of test, the concentration of TTX is that the artificial cerebrospinal fluid of 1 μ mol/L adds in beaker, by ebullator, the artificial cerebrospinal fluid circulation that contains liquid is pumped in track, neurocyte is subject to the effect of polygalic acid medicine, and the data cell signal now recording is called medicine group data;
The medicament elution stage: the artificial cerebrospinal fluid in beaker is replaced by to contain BM concentration be that the concentration of 20 μ mol/L, TTX is the artificial cerebrospinal fluid of 1 μ mol/L, by ebullator, the liquid in track and artificial cerebrospinal fluid are replaced away, cell is in only containing the artificial cerebrospinal fluid of BM and TTX, and the data cell signal now recording is called recovery group data;
Each test cycle finishes to carry out next administration experiment all needs the hippocampal slices more renewing;
Each test cycle clamps down on the film potential of the cell under full cell record pattern at-70mV, at full cell signal record, obtains after stable baseline, carries out data sampling and continues to record 5min;
(7) statistical study: adopt MiniAnalysis6.0 to analyze the signal data collecting, adopt Origin 8.0 and corelDRAW mapping; Data acquisition by mean ± standard deviation
the mode of ± s represents, between group, relatively adopting one-way analysis of variance, P<0.05 is that difference has statistical significance; Use
kolmogorov-Smirnovcheck carrys out the relatively current amplitude difference between each group, and P<0.05 is that difference has statistical significance.
2. the test method of the effect that polygalic acid as claimed in claim 1 transmits hippocampus of rats cynapse, is characterized in that, test with the acquisition methods of polygalic acid is:
Take 1000g and be purchased from the polygala root of Nanchang City, Jiangxi Province Chinese Medicinal Materials Markets, this polygala root is Roots of Polygala tenuifolia; Polygala root is pulverized, added sherwood oil and carry out degreasing; Then with 95% alcohol reflux, extract 3 times each 6h; Three gained extracts are merged, with Rotary Evaporators, will after extract evaporate to dryness, obtain medicinal extract; Medicinal extract is redissolved in the water of 3 times of volumes, filter, gained filtrate is adopted and is extracted with ethyl acetate 6 times, the ethyl acetate use amount of front twice extraction is 1/3 of filtrate cumulative volume, for the third time, the ethyl acetate use amount of the 4th extraction is 1/4 of filtrate cumulative volume, the ethyl acetate use amount of the 5th extraction is that the ethyl acetate use amount of 1/5, the six extraction of filtrate cumulative volume is 1/6 of filtrate cumulative volume; Then by extract macroporous resin adsorption, again extract is carried out to wash-out with distilled water, 30% ethanol, 70% ethanol, 95% ethanol respectively, collect after eluent its freeze drying, re-using 70% ethanol eluate carries out obtaining test polygalic acid medicine after wash-out evaporate to dryness, the final polygalic acid 41.96g that obtains, extraction ratio is 4.2%.
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| CN104713907A (en) * | 2015-01-29 | 2015-06-17 | 新乡医学院 | In vivo brain slice nerve network oscillation recording system and recording method thereof |
| CN104713907B (en) * | 2015-01-29 | 2017-09-22 | 新乡医学院 | Brain slice in vitro neutral net vibrates record system and the method for record |
| CN106568929A (en) * | 2015-10-13 | 2017-04-19 | 中国科学院生物物理研究所 | Electrical activity recording method for tiny single synaptic neurons |
| CN106568929B (en) * | 2015-10-13 | 2019-12-06 | 中国科学院生物物理研究所 | Method for recording electrical activity of tiny single-synapse neuron |
| CN109364846A (en) * | 2018-10-16 | 2019-02-22 | 中子康(武汉)医药科技有限公司 | The application of electro photoluminescence increase biomolecule quantum energy common technology |
| CN109593718A (en) * | 2018-10-16 | 2019-04-09 | 中子康(武汉)医药科技有限公司 | The application of biomolecule quantum energy common technology is reduced using bio-molecular interaction |
| CN109364846B (en) * | 2018-10-16 | 2021-03-23 | 中子康(武汉)医药科技有限公司 | Application of electric stimulation to increase quantum energy commonality of biomolecules |
| CN109593718B (en) * | 2018-10-16 | 2022-03-29 | 中子康(武汉)医药科技有限公司 | Application of technology for reducing quantum energy common property of biomolecules by utilizing interaction of biomolecules |
| CN112595842A (en) * | 2020-11-26 | 2021-04-02 | 中国科学院深圳先进技术研究院 | Detection device and detection method for in-situ cell single vesicle inclusion |
| CN114807114A (en) * | 2022-04-14 | 2022-07-29 | 广州医科大学附属第二医院 | Method for recording fruit fly brain single cell long-term increase electrophysiological signal |
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