Preparation method of brain slice of middle-aged and old mouse for electrophysiological recording and application thereof

Abstract

The application belongs to the technical field of electrophysiological recording, and discloses a preparation method of brain slices of middle-aged and old mice for electrophysiological recording and application, wherein a modified artificial cerebral spinal fluid (ACSF) for brain slices of old mice is introduced in the preparation process of brain slices of middle-aged and old mice, which comprises: an artificial cerebral spinal fluid (section ACSF, sACSF) used when the brain slices of middle-aged and old mice are prepared, and an artificial cerebral spinal fluid (culture ACSF, cACSF) used when the brain slices of middle-aged and old mice are incubated for electrophysiological recording. In the method, the activity of the brain slices of middle-aged and old mice is significantly improved and the duration of the activity is prolonged by the use of the two ACSFs, high-quality brain slice samples of middle-aged and old mice are provided for the research on neurodegenerative diseases such as aging, Alzheimer's disease and Parkinson's syndrome, and the method has a wide application prospect in scientific research.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of electrophysiological recording, and particularly relates to a preparation method of a brain slice of a middle-aged and old mouse for electrophysiological recording and application thereof, and an artificial cerebrospinal fluid ACSF for the brain slice of the middle-aged and old mouse. BACKGROUND

[0002] The brain slice refers to a living tissue slice with a thickness of 100-500 microns prepared from a mouse brain tissue. The brain slice has the characteristics of the in vivo brain to a certain extent, and compared with the in vivo brain, the brain slice also eliminates the blockage of the blood-brain barrier and the poor mechanical stability caused by heartbeats, respiratory movements and the like. Importantly, the external environment of the brain slice is easy to control, and the brain slice is very suitable for physiological and pharmacological research. In basic and clinical neural field research, the electrophysiological recording of the brain slice is a widely used technical means. However, since the brain slice needs to have good physiological activity during the electrophysiological recording, generally only the brain tissue of a young animal less than 3 weeks old with strong hypoxia tolerance and plasticity can be selected as an experimental object, which brings great challenges to the electrophysiological research of some experimental objects requiring the brain tissue of a middle-aged and old animal, such as the research of neurodegenerative diseases such as aging or Alzheimer's disease and Parkinson's syndrome.

[0003] A large number of studies have shown that long-term synaptic plasticity including long-term potentiation (LTP) is a cellular synaptic mechanism of learning and memory. However, the recording of synaptic plasticity at the neural network level is different from the recording of general electrophysiological indicators, and generally can be completed within several minutes to more than ten minutes, and at least 2 hours of signal acquisition is required for a complete LTP recording, and in this process, it is necessary to always maintain the good physiological activity of the brain slice for recording, which further increases the difficulty of such electrophysiological recording. Therefore, for a long time, improving and maintaining the physiological activity of the brain slice of a middle-aged and old animal for a long time has become a bottleneck problem of the electrophysiological recording technology. SUMMARY

[0004] In order to solve the problems in the prior art, the application aims to provide a preparation method of a brain slice of a middle-aged and old mouse for electrophysiological recording, so as to overcome the problem that the electrophysiological recording of the brain slice can only select the brain tissue of a young animal as an experimental object under the condition of the prior art, and to a certain extent, the problem that the development of some researches related to neurodegenerative diseases such as aging or Alzheimer's disease and Parkinson's syndrome which require the brain slice of a middle-aged and old animal as an experimental object is restricted.

[0005] The application provides an artificial cerebral spinal fluid ACSF for brain slices of middle-aged and old mice, which comprises two kinds, one is artificial cerebral spinal fluid sACSF for preparing brain slices of middle-aged and old mice; and / or, the other is artificial cerebral spinal fluid cACSF (culture ACSF) for incubating brain slices of middle-aged and old mice in electrophysiological recording.

[0006] The sACSF comprises N-methyl-D-glucosamine (60-63 mM), choline chloride (29-32 mM), potassium chloride (2.3-2.6 mM), sodium bicarbonate (26-29 mM), sodium dihydrogen phosphate (1.0-1.3 mM), HEPES (18-21 mM), D-glucose (23-26 mM), thiourea (1.8-2.1 mM), sodium ascorbate (4.8-5.1 mM), sodium pyruvate (2.8-3.1 mM), magnesium sulfate (9.8-10.1 mM), calcium chloride (0.48-0.51 mM), sodium adenosine cyclophosphate (1.8-2.1 mM) and N-acetylcysteine (10-13 mM), with a pH value of 7.3-7.4 and an osmotic pressure of 300-310 mOsm / L; preferably, the sACSF comprises N-methyl-D-glucosamine (62 mM), choline chloride (31 mM), potassium chloride (2.5 mM), sodium bicarbonate (28 mM), sodium dihydrogen phosphate (1.2 mM), HEPES (20 mM), D-glucose (25 mM), thiourea (2 mM), sodium ascorbate (5 mM), sodium pyruvate (3 mM), magnesium sulfate (10 mM), calcium chloride (0.5 mM), sodium adenosine cyclophosphate (2 mM) and N-acetylcysteine (12 mM), with a pH value of 7.3-7.4 and an osmotic pressure of 300-310 mOsm / L.

[0007] The cACSF comprises sodium chloride (91-94 mM), potassium chloride (2.3-2.6 mM), sodium bicarbonate (26-29 mM), sodium dihydrogen phosphate (1.0-1.3 mM), HEPES (18-21 mM), D-glucose (23-26 mM), thiourea (1.8-2.1 mM), sodium ascorbate (4.8-5.1 mM), sodium pyruvate (2.8-3.1 mM), magnesium sulfate (1.8-2.1 mM), calcium chloride (2.3-2.6 mM) and adenosine cyclophosphate sodium (1.8-2.1 mM), with a pH value of 7.3-7.4 and an osmotic pressure of 300-310 mOsm / L. Preferably, the cACSF comprises sodium chloride (93 mM), potassium chloride (2.5 mM), sodium bicarbonate (28 mM), sodium dihydrogen phosphate (1.2 mM), HEPES (20 mM), D-glucose (25 mM), thiourea (2 mM), sodium ascorbate (5 mM), sodium pyruvate (3 mM), magnesium sulfate (2 mM), calcium chloride (2.5 mM) and adenosine cyclophosphate sodium (2 mM), with a pH value of 7.3-7.4 and an osmotic pressure of 300-310 mOsm / L.

[0008] The application also provides a preparation method of brain slices of middle-aged and old mice for electrophysiological recording, which can significantly improve and maintain the physiological activity of the brain slices of middle-aged and old mice for a long time, and provide high-quality brain slice samples of middle-aged and old mice for recording of electrophysiological indicators such as synaptic transmission and synaptic plasticity.

[0009] The application also provides a preparation method of brain slices of middle-aged and old mice for electrophysiological recording, which uses artificial cerebrospinal fluid ACSF for the brain slices of middle-aged and old mice; and the preparation method comprises the following steps:

[0010] Step 1): placing the brain tissue into oxygen-saturated artificial cerebrospinal fluid for slices sACSF cooled to an ice-water mixture state to cool the brain tissue and slice;

[0011] Step 2): rapidly transferring the brain tissue slice obtained in step 1) to 31 ℃ oxygen-saturated incubation artificial cerebrospinal fluid cACSF for recovery.

[0012] In a specific embodiment, the preparation method comprises the following steps:

[0013] 1) After the mouse is decapitated, the brain is quickly put into oxygen-saturated section artificial cerebrospinal fluid (sACSF) cooled to the state of ice-water mixture for brain tissue cooling and sectioning, wherein the sACSF comprises N-methyl-D-glucamine (62 mM), choline chloride (31 mM), potassium chloride (2.5 mM), sodium bicarbonate (28 mM), sodium dihydrogen phosphate (1.2 mM), HEPES (20 mM), D-glucose (25 mM), thiourea (2 mM), sodium ascorbate (5 mM), sodium pyruvate (3 mM), magnesium sulfate (10 mM), calcium chloride (0.5 mM), sodium adenosine cyclophosphate (2 mM) and N-acetyl cysteine (12 mM), with a pH value of 7.3-7.4 and an osmotic pressure of 300-310 mOsm / L;

[0014] 2) The sectioned brain slice is quickly transferred to 31℃ oxygen-saturated incubation artificial cerebrospinal fluid (cACSF) for recovery for 1 hour, wherein the cACSF comprises sodium chloride (93 mM), potassium chloride (2.5 mM), sodium bicarbonate (28 mM), sodium dihydrogen phosphate (1.2 mM), HEPES (20 mM), D-glucose (25 mM), thiourea (2 mM), sodium ascorbate (5 mM), sodium pyruvate (3 mM), magnesium sulfate (2 mM), calcium chloride (2.5 mM) and sodium adenosine cyclophosphate (2 mM), with a pH value of 7.3-7.4 and an osmotic pressure of 300-310 mOsm / L;

[0015] 3) The obtained middle-aged mouse brain slice is continuously perfused with 27±2℃ oxygen-saturated cACSF at a perfusion rate of 2-3 ml / min during electrophysiological recording.

[0016] The application also provides a middle-aged mouse brain slice prepared by the method, which has a high high-resistance seal capacity (not less than 1 GΩ) in patch clamp recording, a good synaptic transmission capacity in field potential recording, such as an excitatory postsynaptic current induced by maximum electrical stimulation of not less than 0.4 mV, and / or a good synaptic plasticity in field potential recording, such as long-term potentiation of not less than 120%.

[0017] The middle-aged mouse brain slice prepared by the application has high electrophysiological activity and long activity duration, thereby providing a guarantee for recording of electrophysiological indexes such as synaptic transmission and synaptic plasticity.

[0018] The application also provides a preparation method of the sACSF and cACSF for the middle-aged mouse brain slice, and the preparation method comprises:

[0019] 1) N-methyl-D-glucamine, choline chloride, potassium chloride, sodium bicarbonate, sodium dihydrogen phosphate, HEPES, D-glucose, thiourea, sodium ascorbate, sodium pyruvate, adenosine cyclophosphate sodium, N-acetylcysteine, magnesium sulfate and calcium chloride were weighed respectively, dissolved in water to prepare sACSF solution, wherein the magnesium sulfate and calcium chloride were added last, and one was dissolved completely before the other was dissolved;

[0020] 2) Sodium chloride, potassium chloride, sodium bicarbonate, sodium dihydrogen phosphate, HEPES, D-glucose, thiourea, sodium ascorbate, sodium pyruvate, adenosine cyclophosphate sodium, magnesium sulfate and calcium chloride were weighed respectively, dissolved in water to prepare cACSF solution, wherein the magnesium sulfate and calcium chloride were added last, and one was dissolved completely before the other was dissolved;

[0021] 3) Hydrochloric acid was added to the solutions obtained in steps 1) and 2) to adjust the pH to 7.3-7.4 to obtain artificial cerebrospinal fluid sACSF and cACSF for the middle-aged mouse brain slices.

[0022] The application also provides the preparation method, and the middle-aged mouse brain slices prepared by the above preparation method, and the use of the artificial cerebrospinal fluid ACSF for the middle-aged mouse brain slices in electrophysiological recording, which includes but is not limited to the use in electrophysiological indicators including stimulation intensity-response curve (Input-Output Curve) and synaptic plasticity recording.

[0023] Compared with the prior art, in the slice preparation process, the two ACSF solutions are used in cooperation, which can significantly improve the quality of the middle-aged mouse brain slices and record high-quality related electrophysiological indicators. For example, the fEPSP amplitude induced by the brain slices prepared by the traditional method after the mouse grows up is significantly reduced, but the fEPSP amplitude induced by the brain slices prepared by the method provided by the application after the mouse grows up always remains at a high level (see Figure 2 ). In addition, in the middle-aged mouse, it is difficult to induce LTP using the brain slices prepared by the traditional method, but the brain slices prepared by the method provided by the application can still induce significant LTP ( Figure 3 ). These data show that the brain slices prepared by the method provided by the application can well maintain the synaptic plasticity of the local circuit, which provides high-quality sample guarantee for electrophysiological research of neurodegenerative diseases such as aging or Alzheimer's disease, Parkinson's syndrome and the like. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1The brain slices prepared by this invention exhibit a high high-resistance seal rate in patch-clamp recording. Cells from the hippocampus of 2-week-old, 3-month-old, 9-month-old, and 18-month-old mice were recorded using patch-clamp techniques prepared by both conventional and this invention methods. The high-resistance seal rate of cells under the two methods was statistically analyzed. A high-resistance seal between the cell membrane and the electrode tip must be at least 1 GΩ, and at least 10 cells were recorded per brain slice. The significance of differences was analyzed using a two-way ANOVA. All data are expressed as Mean ± SD. p < 0.05 was defined as statistically significant, p < 0.01 as moderately significant, and p < 0.001 as highly significant.

[0025] Figure 2 The brain slices prepared in this invention exhibit good synaptic transmission ability in field potential recording. A: Schematic diagram of electrode locations in the CA3-CA1 pathway of the hippocampus; B: Input-output curve of the CA3-CA1 pathway in the hippocampus of 2-week-old mice prepared by field potential recording using traditional methods and the method of this invention; C: Input-output curve of the CA3-CA1 pathway in the hippocampus of 3-month-old mice prepared by field potential recording using traditional methods and the method of this invention; D: Input-output curve of the CA3-CA1 pathway in the hippocampus of 9-month-old mice prepared by field potential recording using traditional methods and the method of this invention; E: Input-output curve of the CA3-CA1 pathway in the hippocampus of 18-month-old mice prepared by field potential recording using traditional methods and the method of this invention; F: Comparison of the maximum fEPSP induced in the hippocampus of brain slices from 2-week-old, 3-month-old, 9-month-old, and 18-month-old mice prepared by traditional methods and the method of this invention. The significance of differences was analyzed using a two-way ANOVA. All data are expressed as Mean ± SD. p<0.05 is defined as significant, p<0.01 is defined as relatively significant, and p<0.001 is defined as highly significant.

[0026] Figure 3The brain slice prepared by the application has better synaptic plasticity in field potential recording. A: LTP of CA3-CA1 pathway of hippocampal brain region of 2-week-old mice prepared by traditional field potential recording method and the method of the application; B: columnar statistical chart of fEPSP amplitude recorded in the last 10 minutes in A; C: LTP of CA3-CA1 pathway of hippocampal brain region of 3-month-old mice prepared by traditional field potential recording method and the method of the application; D: columnar statistical chart of fEPSP amplitude recorded in the last 10 minutes in C; E: LTP of CA3-CA1 pathway of hippocampal brain region of 9-month-old mice prepared by traditional field potential recording method and the method of the application; F: columnar statistical chart of fEPSP amplitude recorded in the last 10 minutes in E; G: LTP of CA3-CA1 pathway of hippocampal brain region of 18-month-old mice prepared by traditional field potential recording method and the method of the application; H: columnar statistical chart of fEPSP amplitude recorded in the last 10 minutes in G. The significance of difference is analyzed by using Students't-test method. All data are expressed by Mean ± SD. p<0.05 is defined as significant difference, p<0.01 is defined as relatively significant difference, and p<0.001 is defined as extremely significant difference. DETAILED DESCRIPTION

[0027] The application will be further described in combination with the following specific examples and drawings. The process, conditions, experimental methods and the like for implementing the application are the general knowledge and common sense in the art, and the application does not have special limitations.

[0028] The reagents used in the application are from Sigma-Aldrich.

[0029] Example 1

[0030] The electrophysiological activity of neurons in hippocampal brain region of mice in different age stages prepared by the method for preparing brain slice of middle-aged and old mice provided by the application and the traditional brain slice preparation method is compared.

[0031] (1) Experimental animals

[0032] The C57BL / 6 mice used in the experiment are purchased from Shanghai Slac Laboratory Animal Co., Ltd. and are bred and reproduced in the SPF level model animal center of the Key Laboratory of Brain Function Genomics of the Ministry of Education, East China Normal University. During the breeding process, the mice are free to eat and drink, and the automatic light control is on at 7:00 and off at 19:00, with 12 / 12h day-night alternation. The room temperature is 22±2℃, and the relative humidity is 50%-60%. All operations on experimental mice strictly comply with the relevant regulations of the Experimental Animal Use and Management Committee of East China Normal University.

[0033] (2) Preparation of ACSF for preparing brain slice

[0034] ①The slice solution and the incubation solution used in the traditional brain slice preparation method are artificial cerebrospinal fluid simulating mouse cerebrospinal fluid, and the composition thereof is: sodium chloride 119 mM, potassium chloride 2.5 mM, sodium phosphate dibasic 1.25 mM, sodium bicarbonate 24 mM, D-glucose 12.5 mM, magnesium sulfate 2 mM, calcium chloride 2 mM. The pH value is 7.3-7.4, and the osmotic pressure is 300-310 mOsm / L.

[0035] ②The slice solution and the incubation solution used in the brain slice preparation of the middle-aged and old mice provided by the application are sACSF and cACSF respectively. The composition of the sACSF is: N-methyl-D-glucosamine (62 mM), choline chloride (31 mM), potassium chloride (2.5 mM), sodium bicarbonate (28 mM), sodium phosphate dibasic (1.2 mM), HEPES (20 mM), D-glucose (25 mM), thiourea (2 mM), sodium ascorbate (5 mM), sodium pyruvate (3 mM), magnesium sulfate (10 mM), calcium chloride (0.5 mM), sodium adenosine cyclophosphate (2 mM) and N-acetylcysteine (12 mM), and the pH value is 7.3-7.4, and the osmotic pressure is 300-310 mOsm / L. The composition of the cACSF is: sodium chloride (93 mM), potassium chloride (2.5 mM), sodium bicarbonate (28 mM), sodium phosphate dibasic (1.2 mM), HEPES (20 mM), D-glucose (25 mM), thiourea (2 mM), sodium ascorbate (5 mM), sodium pyruvate (3 mM), magnesium sulfate (2 mM), calcium chloride (2.5 mM) and sodium adenosine cyclophosphate (2 mM), and the pH value is 7.3-7.4, and the osmotic pressure is 300-310 mOsm / L.

[0036] (3) Preparation of an in vitro brain slice

[0037] Five mice of each of 2 weeks old, 3 months old, 9 months old and 18 months old are taken, and the mice are anesthetized by intraperitoneal injection of sodium pentobarbital (40 mg / kg), and the mice are quickly perfused with pre-cooled sACSF (4℃) through the left ventricle until the liver of the mice turns white. Then, the mice are decapitated to take out the brain, and the brain is quickly put into pre-cooled sACSF (ice water mixture state) for soaking for 2-3 minutes. After that, the brain tissue block of the mouse is slightly trimmed and adhered to the knife table of a vibrating microtome (VT1000, Leica, Germany), and a coronal brain slice (the thickness of the brain slice for patch clamp recording is 320 μm, and the thickness of the brain slice for field potential recording is 400 μm) is cut. Finally, the cut brain slice is quickly transferred to 31℃ oxygen-saturated cACSF for recovery for 1 hour.

[0038] (4) Patch clamp recording of an in vitro brain slice

[0039] The incubated brain slices were transferred to a full-immersion recording bath, continuously perfused with cACSF solution saturated with 95% 02 / 5% CO2, maintained at 27±2℃, and perfused at a rate of 2-3 ml / min. Under a low-power water-immersion objective, the hippocampal brain region on the mouse brain slice was selected according to the brain atlas, and a pyramidal neuron was found under a high-power objective. The micro-controlled recording electrode glass tube was placed close to the neuron cell body. When the electrode tip contacted the neuron cell body, a high-resistance seal (R>1 GΩ) was formed between the cell membrane and the electrode tip by applying a negative pressure to the electrode glass tube. After the seal was stable, the cell membrane was ruptured by applying a strong and short negative pressure, thereby forming a whole-cell patch clamp recording mode. The better the cell activity state, the easier it is to form a high-resistance seal, and vice versa.

[0040] (5) Analysis of experimental results

[0041] As shown in Figure 1 , using the traditional brain slice preparation method, only the brain slices of young mice have a high probability of high-resistance sealing. However, after the mice grow up, the high-resistance sealing probability of brain slices of 3-month-old, 9-month-old and 18-month-old mice is significantly reduced, especially in the brain slices of middle-aged and old mice, it is difficult to find suitable cells for patch clamp recording. However, compared with the brain slices prepared by the traditional brain slice preparation method, the mouse brain slices prepared by the preparation method of the middle-aged and old mouse brain slices provided by the present application have a high probability of high-resistance sealing regardless of whether they are from 3-month-old (Two-way ANOVA multiple comparisons, p<0.001), 9-month-old (Two-way ANOVA multiple comparisons, p<0.001) or 18-month-old (Two-way ANOVA multiple comparisons, p<0.001) mice. These data show that the mouse brain slices prepared by the preparation method of the middle-aged and old mouse brain slices provided by the present application can better guarantee the electrophysiological activity of the middle-aged and old mouse brain slices, which also provides important sample guarantee for subsequent electrophysiological recording of synaptic transmission and synaptic plasticity.

[0042] Example 2

[0043] Comparison of synaptic transmission capacity of hippocampal brain regions of mice of different ages prepared by the preparation method of the middle-aged and old mouse brain slices provided by the present application and the traditional brain slice preparation method.

[0044] (1) The first three steps, including experimental animals, preparation of ACSF for brain slice preparation, and preparation of isolated brain slices, are the same as in Example 1 of the present application.

[0045] (2) Field potential recording of isolated brain slices (stimulus intensity response curve)

[0046] Brain slices were transferred to a field potential recording chamber and continuously perfused with oxygen-saturated cACSF at 27±2℃. The stimulating electrode (tungsten electrode) was placed in the CA3 region of the hippocampus, and the glass recording electrode was placed in the CA1 region (e.g.,...). Figure 2 As shown in Figure A, the recording electrodes were perfused with 0.5 M CH3COONa electrode fluid containing 2% trypan blue, with an impedance of approximately 2-6 MΩ. Excitatory postsynaptic potentials (fEPSPs) of pyramidal neurons in the CA1 region were recorded. The stimulation intensity was adjusted using an isolator to find the minimum stimulation intensity that elicited observable fEPSPs. The stimulation intensity was then gradually increased, and the amplitude of the fEPSPs elicited by each stimulus was recorded until the response amplitude reached its maximum and no longer increased with further stimulation intensity. The stimulation square wave had a pulse width of 0.05 ms and a stimulation frequency of 0.033 Hz. Four sweeps were recorded consecutively at each stimulation intensity, and the average value was taken. A curve was plotted with the amplitude of the fibervolley at different stimulation intensities on the x-axis and the corresponding fEPSP amplitude on the y-axis; this is the stimulation intensity response curve (Input-Output Curve), which reflects the synaptic transmission efficiency of specific signal pathways on the brain slice.

[0047] (3) Analysis of experimental results

[0048] like Figure 2 As shown, in 2-week-old juvenile mice, both brain slices prepared by conventional methods and brain slices prepared by the method provided in this invention can effectively induce the input-output curve, with no significant difference between the two. Figure 2 B, Two-way ANOVA, p > 0.05). However, in adult 3-month-old mice, the input-output curve induced by brain slices prepared using conventional methods was significantly weaker than the input-output curve induced by brain slices prepared using the method provided in this invention. Figure 2 C, Two-way ANOVA, p < 0.001). Similarly, in middle-aged and aged mice, the input-output curve induced by brain slices prepared using conventional methods was significantly weaker than the input-output curve induced by brain slices prepared using the method provided in this invention. Figure 2 Dand2E, Two-way ANOVA, p < 0.001. Furthermore, regarding the maximum induced fEPSP, brain slices prepared using conventional methods showed a significant decrease in fEPSP amplitude after adulthood in mice, while brain slices prepared using the method provided in this invention consistently maintained a high level of fEPSP amplitude after adulthood in mice.Figure 2 F, Two-way ANOVA multiple comparisons, p < 0.001). These data demonstrate that the brain slices prepared by the method provided in this invention can well maintain the synaptic transmission capacity of local circuits.

[0049] Example 3

[0050] A comparison of the synaptic plasticity of the hippocampus in mice of different ages prepared using the method for preparing brain slices of middle-aged and elderly mice provided by the present invention and traditional brain slice preparation methods.

[0051] (1) The first three steps, including experimental animals, preparation of ACSF for brain slice preparation and preparation of isolated brain slices, are the same as in Example 1 of this invention.

[0052] (2) Recording of field potentials in isolated brain slices (synaptic plasticity)

[0053] Brain slices were transferred to a field potential recording chamber and continuously perfused with oxygen-saturated cACSF at 27±2℃. The stimulating electrode (tungsten electrode) was placed in the CA3 region of the hippocampus, and the glass recording electrode was placed in the CA1 region (e.g.,...). Figure 2 As shown in Figure A, the recording electrode was perfused with 0.5 M CH3COONa internal solution containing 2% trypan blue, with an impedance of approximately 2-6 MΩ. Long-term potentiation (LTP) and long-term depression (LTD) are considered to be cellular synaptic mechanisms of learning and memory. LTP recording first uses an input-output method to find the maximum fEPSP on this synaptic pathway. Then, a stimulus intensity that elicits 1 / 3 to 1 / 2 of the amplitude of this maximum fEPSP is used as the test stimulus intensity, with a stimulus width of 0.05 ms, and a test stimulus is given every 30 s. After stabilizing the baseline for 15 min, high-frequency stimulation (2 strings of 100 pulses each at 100 Hz, spaced 10 s apart) is applied to induce LTP. Recording continues for 45 min after the stimulation ends. The ratio of the average fEPSP amplitude in the last 10 min (35-45 min) to the average fEPSP amplitude at baseline is calculated. If this ratio is ≥120%, LTP induction is considered successful.

[0054] (3) Analysis of experimental results

[0055] like Figure 3 As shown, in 2-week-old juvenile mice, both brain slices prepared by conventional methods and brain slices prepared by the method provided in this invention can effectively induce LTP, and there is no significant difference between the two. Figure 3A and 3B, Student's t-test, p>0.05). But in adult mice at 3 months of age, LTP induced by brain slices prepared by the conventional method was significantly weaker than that induced by brain slices prepared by the method provided by the present application Figure 3 C and 3D, Student's t-test, p<0.001). In addition, in old mice, it was difficult to induce LTP again by brain slices prepared by the conventional method Figure 3 E-3H, the ratio of the mean fEPSP amplitude at 35-45 min to the mean fEPSP amplitude at baseline was <120%), while brain slices prepared by the method provided by the present application could still induce significant LTP Figure 3 E-3H, the ratio of the mean fEPSP amplitude at 35-45 min to the mean fEPSP amplitude at baseline was >120%). These data show that brain slices prepared by the method provided by the present application can well maintain synaptic plasticity of local circuits.

[0056] The protection scope of the present application is not limited to the above examples. Changes and advantages that can be thought of by those skilled in the art without departing from the spirit and scope of the present application are included in the present application, and are protected by the appended claims.

Claims

1. An artificial cerebrospinal fluid (ACSF) for use in brain slices from middle-aged and elderly mice, characterized in that, It includes: Artificial cerebrospinal fluid (sACSF) used for preparing brain slices from middle-aged and older mice; and / or artificial cerebrospinal fluid (cACSF) used for incubating brain slices from middle-aged and older mice during electrophysiological recording; The sACSF contains 60-63 mM N-methyl-D-glucosamine, 29-32 mM choline chloride, 2.3-2.6 mM potassium chloride, 26-29 mM sodium bicarbonate, 1.0-1.3 mM sodium dihydrogen phosphate, 18-21 mM HEPES, 23-26 mM D-glucose, 1.8-2.1 mM thiourea, 4.8-5.1 mM sodium ascorbate, 2.8-3.1 mM sodium pyruvate, 9.8-10.1 mM magnesium sulfate, 0.48-0.51 mM calcium chloride, 1.8-2.1 mM sodium cyclic adenosine monophosphate, and 10-13 mM N-acetylcysteine. Its pH is 7.3-7.4, and its osmotic pressure is 300-310 mOsm / L. The cACSF contains 91–94 mM sodium chloride, 2.3–2.6 mM potassium chloride, 26–29 mM sodium bicarbonate, 1.0–1.3 mM sodium dihydrogen phosphate, 18–21 mM HEPES, 23–26 mM D-glucose, 1.8–2.1 mM thiourea, 4.8–5.1 mM sodium ascorbate, 2.8–3.1 mM sodium pyruvate, 1.8–2.1 mM magnesium sulfate, 2.3–2.6 mM calcium chloride, and 1.8–2.1 mM cyclic adenosine monophosphate (cACSF). It has a pH of 7.3–7.4 and an osmotic pressure of 300–310 mOsm / L.

2. The artificial cerebrospinal fluid (ACSF) for brain slices from middle-aged and aged mice as described in claim 1, characterized in that, The sACSF contains 62 mM N-methyl-D-glucosamine, 31 mM choline chloride, 2.5 mM potassium chloride, 28 mM sodium bicarbonate, 1.2 mM sodium dihydrogen phosphate, 20 mM HEPES, 25 mM D-glucose, 2 mM thiourea, 5 mM sodium ascorbate, 3 mM sodium pyruvate, 10 mM magnesium sulfate, 0.5 mM calcium chloride, 2 mM sodium cyclic adenosine monophosphate, and 12 mM N-acetylcysteine. Its pH is 7.3–7.4, and its osmotic pressure is 300–310 mOsm / L. The cACSF contains 93 mM sodium chloride, 2.5 mM potassium chloride, 28 mM sodium bicarbonate, 1.2 mM sodium dihydrogen phosphate, 20 mM HEPES, 25 mM D-glucose, 2 mM thiourea, 5 mM sodium ascorbate, 3 mM sodium pyruvate, 2 mM magnesium sulfate, 2.5 mM calcium chloride, and 2 mM cyclic adenosine monophosphate sodium, with a pH of 7.3–7.4 and an osmotic pressure of 300–310 mOsm / L.

3. The method for preparing artificial cerebrospinal fluid (ACSF) for brain slices from middle-aged and aged mice as described in claim 1 or 2, characterized in that, Preparation of sACSF: Weigh N-methyl-D-glucosamine, choline chloride, potassium chloride, sodium bicarbonate, sodium dihydrogen phosphate, HEPES, D-glucose, thiourea, sodium ascorbate, sodium pyruvate, sodium cyclic adenosine monophosphate, N-acetylcysteine, magnesium sulfate, and calcium chloride respectively, and dissolve them in water; magnesium sulfate and calcium chloride are added last, and one is dissolved completely before the other is dissolved. The preparation of cACSF: Weigh sodium chloride, potassium chloride, sodium bicarbonate, sodium dihydrogen phosphate, HEPES, D-glucose, thiourea, sodium ascorbate, sodium pyruvate, sodium cyclic adenosine monophosphate, magnesium sulfate, and calcium chloride respectively, and dissolve them in water; among them, magnesium sulfate and calcium chloride are added last, and one is dissolved completely before the other is dissolved. Hydrochloric acid was added to the sACSF and cACSF solutions prepared above to adjust the pH to 7.3-7.

4.

4. A method for preparing brain slices from middle-aged and aged mice for electrophysiological recording, characterized in that, The preparation method uses the artificial cerebrospinal fluid (ACSF) for brain slices from middle-aged and aged mice as described in claim 1 or 2; the preparation method includes the following steps: 1) The brain tissue was placed in an oxygen-saturated section cooled to an ice-water mixture and then cooled and sectioned using artificial cerebrospinal fluid (sACSF). 2) The brain tissue slices obtained in step 1) were rapidly transferred to cACSF (artificial cerebrospinal fluid) at 31°C with oxygen saturation for 1 hour for resuscitation.

5. The application of the artificial cerebrospinal fluid ACSF for brain slices of middle-aged and elderly mice as described in claim 1 or 2, and the preparation method as described in claim 4, in electrophysiological recording.

6. The application as described in claim 5, characterized in that, The applications include use in electrophysiological parameters, including stimulus intensity-response curves and synaptic plasticity recordings.

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