CN106226509A - A kind of method in mice anterior cingutate cortex induction DSE phenomenon - Google Patents
A kind of method in mice anterior cingutate cortex induction DSE phenomenon Download PDFInfo
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- CN106226509A CN106226509A CN201610681743.9A CN201610681743A CN106226509A CN 106226509 A CN106226509 A CN 106226509A CN 201610681743 A CN201610681743 A CN 201610681743A CN 106226509 A CN106226509 A CN 106226509A
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Abstract
The invention discloses a kind of method in mice anterior cingutate cortex induction DSE phenomenon, the method comprises the following steps: preparation brain slice；Electrophysiological recording；Result judges.The abductive approach of the present invention have fast and convenient, success rate is high, operate easy advantage.Need not utilize the agonist of other receptors or antagonist, hatch and recording liquid is close to physiological status, be more favorable for simulation in body situation.Success rate is high, utilize that this law is good at cell state, stimulating electrode induced efficiency high and stimulation sites accurately in the case of, success rate is up to more than 95%, is therefore more favorable for the enhancing of DSE phenomenon under the conditions of the various pathological model of comparison, weakens or disappear.This law, for having higher Research Significance at anterior cingutate cortex level induction DSE, is suitable to popularization and application.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of method in mice anterior cingutate cortex induction DSE phenomenon, tool
Say body, relate to a kind of method relying on DSE phenomenon at mice anterior cingutate cortex induction Cannabined receptor (CB1R).
Background technology
Fiber crops element receptor (cannabinoid receptor) are general to be distributed in central nervous system, is broadly divided into cannabinoid receptor 1
Body 1 (CB1R), Cannabined receptor 2 (CB2R) two types.More and more studying proof, Cannabined receptor function becomes with medicine
Addiction, cognition and disturbance of memory, multiple mental sickness and chronic pain are relevant.At present, patch clamp technique is utilized to remember on brain sheet
The phenomenon of record depolarization induction irritability transmission suppression, i.e. DSE phenomenon is for evaluating CB function of receptors important indicator.
Therefore find the method stable, induction success rate is high most important for evaluating CB function of receptors.
Anterior cingutate cortex (ACC, anterior cingulated cortex) is positioned at the inner surface of brain frontal cortex, in recent years
More and more study proof its with the generation of chronic pain with remain closely related, simultaneously its with memory, social activity, fear and
The generation of the negative emotions such as anxiety is relevant.And illustrate CB receptor in ACC aspect and participate in the mechanism of mediation neurological dysfunction,
Finding the therapy target that CB receptor is relevant, obtaining stable DSE phenomenon in ACC district is key problem in technology.
Being the discovery that first of DSE finds to go to pole at calendar year 2001 Kreitzer and Regehr on rat cerebellum Purkinje cell
Change the incoming phenomenon by of short duration suppression of irritability information, the most for over ten years, one by one in several brain such as cerebellum, corpus amygdaloideum district
Find DSE phenomenon.But the method that the most important phenomenon has never had at anterior cingutate cortex is successfully induced appearance, by
This, carry out Cannabined receptor correlational study at anterior cingutate the most delayed.Not yet have at present and carry out the relevant of DSE induction at ACC
Report.
Summary of the invention
In order to overcome defect present in prior art, the present invention proposes and a kind of induces at mice anterior cingutate cortex
The method of DSE phenomenon, the method can have fast and convenient, success in the DSE phenomenon of anterior cingutate level induction CB1R mediation
Rate is high, operate easy advantage, is suitable to popularization and application.
Its technical scheme is as follows:
A kind of method in mice anterior cingutate cortex induction DSE phenomenon, comprises the following steps:
Step 1. prepares brain slice
After Animal Anesthesia, take brain；The method utilizing vibration section carries out coronal section；Slice thickness is 300um, chooses
Brain sheet between bregma 0.50-1.10, hatches 2 hours；
Step 2. electrophysiological recording
Perfusate adds GABA receptor antagonist dicentrine, final concentration；
Record L2/3 layer neuron: recording electrode is placed in L2/3, and stimulating electrode is placed in L5/6；
Record L5/6 layer neuron: recording electrode is placed in L5/6, and stimulating electrode is placed in L2/3；
Recording method: voltage-clamp recording, Clamping voltages is-70mV, records 5-10 minute baseline, and giving 0.2Hz stimulates, control
The EPSC of system induction is at 200pA；
Then Clamping voltages is adjusted to 0, gives depolarization 2s；Depolarization gives 0.2Hz immediately after terminating stimulate, same to base
Line；
After record end, record the passive membrane property of cell；
Step 3. result judges
In above-mentioned recording process, baseline fluctuates and is considered as stable recording less than 10%, is analyzed further；
After depolarization induction, the size of cell leakage current is less than 100pA, includes analysis in；
If occurring in that in 20s after depolarization, EPSC reduces, and reduction amplitude is about 20%；After 20s, EPSC recovers normal baseline
Level, is i.e. considered as successfully inducing DSE phenomenon.
Further, animal described in step 1 is the C57BL/6 mice that grows up, and the age is more than 8 weeks.
Further, the recording liquid formula used in step 2 is: sodium chloride 126mM, potassium chloride 2.5mM, calcium chloride
2.4mM, magnesium chloride 1.2mM, sodium dihydrogen phosphate 1.2mM, sodium bicarbonate 19mM, glucose 11mM, and with 95%O2And 5%CO2Incubate
Educating, pH is 7.35-7.45.
Further, the interior formula of liquid of the described recording electrode in step 2 is: potassium gluconate 125mM, sodium chloride 10mM,
Magnesium chloride 2.0mM, calcium chloride 1.0mM, ethylene glycol diethyl ether ethylenediamine tetraacetic acid (EDTA) 10mM, hydroxyethyl piperazine second sulfacid 10mM, three
Adenosine phosphate 2.0mM, GTP (guanosine triphosphate) 0.3, pH is 7.3, and osmotic pressure is 285-295m0sm/kg.
Further, record temperature when recording in step 2 is 30-34 DEG C.
The invention have the benefit that
The abductive approach of the present invention have fast and convenient, success rate is high, operate easy advantage.Need not utilize other to be subject to
The agonist of body or antagonist, hatch and recording liquid is close to physiological status, is more favorable for simulation in body situation.Success rate is high,
Utilize that this law is good at cell state, stimulating electrode induced efficiency high and stimulation sites accurately in the case of, success rate is up to 95%
Above, therefore it is more favorable for the enhancing of DSE phenomenon under the conditions of the various pathological model of comparison, weakens or disappear.This law is for front
Cingule gyrus cortex level induction DSE has higher Research Significance, is suitable to popularization and application.
Accompanying drawing explanation
Fig. 1 is electrophysiological recording operation chart；
Fig. 2 is the recording method schematic diagram of electrophysiological recording；
Fig. 3 is that DSE induces successfully schematic diagram；Exemplary currents schematic diagram before and after wherein Fig. 3 a is DSE induction；When Fig. 3 b is
M-current changing rate schematic diagram；Fig. 3 c compares block diagram for current changing rate before and after induction；Fig. 3 d is different depolarization time bars
Under part, DSE induced current rate of change changes over curve chart；
Fig. 4 is that mglur 5 antagonists blocks DSE schematic diagram；Wherein Fig. 4 a m-current changing rate curve chart when being；Fig. 4 b is
Before and after induction, current changing rate compares block diagram；
Fig. 5 is that calcium chelating agent blocks DSE schematic diagram；Fig. 5 a is m-current changing rate curve chart when being；Before and after Fig. 5 b is for induction
Current changing rate compares block diagram；
Fig. 6 is that CB1 receptor antagonist blocks DSE schematic diagram.Fig. 6 a is m-current changing rate curve chart when being；Fig. 6 b is for luring
Leading after current rate of change compares block diagram.
Detailed description of the invention
With detailed description of the invention, technical scheme is described in more detail below in conjunction with the accompanying drawings.
1. preparation brain slice: after Animal Anesthesia, take brain.The method utilizing vibration section carries out coronal section.Slice thick
Degree is 300um, chooses the brain sheet between bregma 0.50-1.10, hatches 2 hours.
2. electrophysiological recording: add GABA receptor antagonist dicentrine, final concentration in perfusate.
Record L2/3 layer neuron: recording electrode is placed in L2/3, and stimulating electrode is placed in L5/6.
Record L5/6 layer neuron: recording electrode is placed in L5/6, and stimulating electrode is placed in L2/3.
Recording method: voltage-clamp recording, Clamping voltages is-70mV, records 5-10 minute baseline, and giving 0.2Hz stimulates, control
The EPSC of system induction is at about 200pA.
Then Clamping voltages is adjusted to 0, gives depolarization 2s.Depolarization gives 0.2Hz immediately after terminating stimulate, same to base
Line.
The passive membrane property of cell is recorded after record end.
3. result judges:
In above-mentioned recording process, baseline fluctuates and is considered as stable recording less than 10%, can carry out further point
Analysis.
After depolarization induction, cell leakage current size is less than 100pA, can include analysis in.
If occurring in that in 20s after depolarization, EPSC reduces, and reduction amplitude is about 20%；After 20s, EPSC recovers normal baseline
Level, i.e. can be considered successfully induction DSE phenomenon.
4. utilize this law induction DSE mechanism probe into and verify
Pharmacological method is utilized to prove: this law is postsynaptic mGluR5, CB1R, Ca in the DSE phenomenon that ACC is induced2+Altogether
Mediate together the phenomenon occurred:
DSE is metabotropic glutamate receptor 5 (mGluR5) dependent mechanism: add mGluR5 antagonism in perfusion recording liquid
Agent MPEP (10uM), utilizes same method record DSE, finds that DSE phenomenon disappears, the DSE phenomenon that this law is induced is described
Rely on for mGluR5.
DSE is calcium dependent mechanism: by adding calcium chelating agent BAPTA (10mM) in electrode solution, block in postsynaptic calcium
Stream, utilizes same method record DSE, finds that DSE phenomenon disappears, and illustrates that this law induced DSE phenomenon is that Ca2+ influx relies on.
DSE is presynaptic CB1R dependent mechanism: add CB1 specific antagonists AM251 in perfusion recording liquid
(6uM), utilize same method record DSE, find that DSE phenomenon disappears, illustrate that the DSE phenomenon that this law is induced is that CB1 receptor depends on
Rely.
The above, the only present invention preferably detailed description of the invention, protection scope of the present invention is not limited to this, any ripe
Know those skilled in the art in the technical scope of present disclosure, the letter of the technical scheme that can become apparent to
Altered or equivalence are replaced and are each fallen within protection scope of the present invention.
Claims (5)
1. the method in mice anterior cingutate cortex induction DSE phenomenon, it is characterised in that comprise the following steps:
Step 1. prepares brain slice
After Animal Anesthesia, take brain；The method utilizing vibration section carries out coronal section；Slice thickness is 300um, chooses
Brain sheet between bregma 0.50-1.10, hatches 2 hours；
Step 2. electrophysiological recording
Perfusate adds GABA receptor antagonist dicentrine, final concentration；
Record L2/3 layer neuron: recording electrode is placed in L2/3, and stimulating electrode is placed in L5/6；
Record L5/6 layer neuron: recording electrode is placed in L5/6, and stimulating electrode is placed in L2/3；
Recording method: voltage-clamp recording, Clamping voltages is-70mV, records 5-10 minute baseline, and giving 0.2Hz stimulates, and controls to lure
The EPSC sent out is at 200pA；
Then Clamping voltages is adjusted to 0, gives depolarization 2s；Depolarization gives 0.2Hz immediately after terminating stimulate, same to baseline；
After record end, record the passive membrane property of cell；
Step 3. result judges
In above-mentioned recording process, baseline fluctuates and is considered as stable recording less than 10%, is analyzed further；
After depolarization induction, the size of cell leakage current is less than 100pA, includes analysis in；
If occurring in that in 20s after depolarization, EPSC reduces, and reduction amplitude is about 20%；After 20s, EPSC recovers normal baseline levels,
I.e. it is considered as successfully inducing DSE phenomenon.
Method in mice anterior cingutate cortex induction DSE phenomenon the most according to claim 1, it is characterised in that step 1
Described animal is the C57BL/6 mice that grows up, and the age is more than 8 weeks.
Method in mice anterior cingutate cortex induction DSE phenomenon the most according to claim 1, it is characterised in that step 2
Used in recording liquid formula be: sodium chloride 126mM, potassium chloride 2.5mM, calcium chloride 2.4mM, magnesium chloride 1.2mM, phosphoric acid
Sodium dihydrogen 1.2mM, sodium bicarbonate 19mM, glucose 11mM, and with 95%O2And 5%CO2Hatching, pH is 7.35-7.45.
Method in mice anterior cingutate cortex induction DSE phenomenon the most according to claim 1, it is characterised in that step 2
In the interior formula of liquid of described recording electrode be: potassium gluconate 125mM, sodium chloride 10mM, magnesium chloride 2.0mM, calcium chloride
1.0mM, ethylene glycol diethyl ether ethylenediamine tetraacetic acid (EDTA) 10mM, hydroxyethyl piperazine second sulfacid 10mM, adenosine triphosphate 2.0mM, three phosphorus
Acid guanosine 0.3, pH is 7.3, and osmotic pressure is 285-295mOsm/kg.
Method in mice anterior cingutate cortex induction DSE phenomenon the most according to claim 1, it is characterised in that step 2
Record temperature during middle record is 30-34 DEG C.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109596818A (en) * | 2018-12-13 | 2019-04-09 | 丁蓉 | A kind of research method based on electrophysiology analysis danggui sini decoction prevented oxaliplatin induced neurotoxicity |
| CN109716117A (en) * | 2016-09-19 | 2019-05-03 | 村田整合被动式解决方案公司 | Electro photoluminescence and monitoring device |
| WO2021088826A1 (en) * | 2019-11-04 | 2021-05-14 | 上海青赛生物科技有限公司 | Device for evaluating neurovirulence of mumps virus |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109716117A (en) * | 2016-09-19 | 2019-05-03 | 村田整合被动式解决方案公司 | Electro photoluminescence and monitoring device |
| CN109596818A (en) * | 2018-12-13 | 2019-04-09 | 丁蓉 | A kind of research method based on electrophysiology analysis danggui sini decoction prevented oxaliplatin induced neurotoxicity |
| CN109596818B (en) * | 2018-12-13 | 2024-03-19 | 丁蓉 | Research method for preventing oxaliplatin neurotoxicity mechanism based on electrophysiology analysis of angelica sinensis four-reverse decoction |
| WO2021088826A1 (en) * | 2019-11-04 | 2021-05-14 | 上海青赛生物科技有限公司 | Device for evaluating neurovirulence of mumps virus |
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