CN110721176B - Application of fluoxetine in preparation of medicine for inducing auditory cortex plasticity - Google Patents

Abstract

The invention belongs to the field of life science/medicine, relates to a scheme for improving brain sensory system plasticity and maintaining brain health, and particularly relates to application of fluoxetine in inducing auditory cortex plasticity. The invention establishes a new strategy for changing auditory cortex plasticity by utilizing the regulation effect of fluoxetine on brain excitability. The fluoxetine can improve auditory cortex plasticity, and has general applicability and wide application prospect in various nervous system problems.

Description

Application of fluoxetine in preparation of medicine for inducing auditory cortex plasticity

Technical Field

The present invention belongs to the field of life science/medicine; the invention relates to a scheme for improving brain sensory system plasticity and maintaining brain health, in particular to application of fluoxetine in inducing auditory cortex plasticity.

Background

Early in brain development, brain structure development has not yet occurred, neurons have synaptic structures and immature extracellular matrix in infancy, where the brain has very high plasticity, a period that we call "critical" for brain development. In the auditory system, it is generally accepted that the perception of sound is dependent on the normal development of the primary auditory cortex. In the 'key stage' of brain development, the primary auditory cortex is used for encoding frequency information, time information and intensity information of sound, and the structural change of cortex caused by the sound can be continued to grow up. After the "critical period" has ended, the inadvertent or passive acoustic environment is insufficient to cause changes in the auditory cortex. Plastic modulation requires the mobilization of more brain functions such as attention, reward and novelty stimuli.

Most studies suggest that the balance between excitability and inhibitivity in the brain is critical in controlling brain plasticity. Such as: placing an adult rat in a dark environment can induce visual plasticity similar to that of a baby brain. We have found that fluoxetine alters the balance between excitability and inhibitivity in the brain, giving the auditory cortex a "key-phase" like plasticity.

All people recognize the importance of the brain to the human body, but few notice whether their brain is healthy if there is no significant vertigo pain. With the growth of human population and the continuous development of environmental pollution problems, a plurality of adverse factors influencing the brain health become problems which people are urgently required to face. Studies have shown that low concentrations of lead contamination can impair the ability of the brain's auditory system to resolve orientation information. Children living near airports are contaminated with noise for a long time, which causes a decline in cognitive function of the brain. The altered brain function can be partially improved by learning and training the brain, since the brain can be further self-shaped by environmental experience, which we refer to as brain plasticity. How to make the brain healthier by changing brain plasticity becomes an increasingly urgent need for human beings.

The induction of auditory cortex plasticity currently can use electrical stimulation of the basal ganglia or training of auditory behavior. Electrical stimulation of basal ganglia can promote cholinergic release in basal regions of the midbrain, change the ratio of excitability to inhibitability of cortex, and improve plasticity, but the stimulation electrodes need to be buried in an operation, so that the trauma is large. Specific acoustic stimuli are linked to the behavior of the animal and can also induce changes in the plasticity of the auditory cortex by way of training, but take a considerable amount of time (Zhou et al, 2010, zhang et al, 2013, zhu et al, 2016 liu et al, 2018.

Fluoxetine (FLX) is a selective serotonin reuptake inhibitor, and the medicine can selectively inhibit the recovery of serotonin (5-HT) by presynaptic membranes, regulate serotonin energy neurotransmitters in brain and change the ratio of brain excitation to inhibition.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention establishes a new strategy for changing the plasticity of auditory cortex by utilizing the regulation effect of fluoxetine on the excitability of the brain.

"noise theory" has been thought to control development of the central nervous system. During the critical period, as the inhibitory nervous system develops, excess excitatory noise is removed by the inhibitory neurotransmitter, and brain plasticity is reduced to adult levels with the developmental completion of the inhibitory nervous system. Thus, the ratio of cortical excitability to suppressive is considered to be an important switch in "restarting" the plasticity of the critical phase. The invention finds that the expression level of auditory cortex inhibitory nervous system PV protein and extracellular matrix PNN thereof is reduced by detecting the auditory cortex inhibitory related protein of a rat after the chronic fluoxetine is applied, and prompts that the fluoxetine can effectively regulate the ratio of cortical excitability to cortical inhibitability. This change was found to progress towards the juvenile brain in comparison to the juvenile group of animals.

There are many methods currently available to alter the ratio of excitability to inhibitability of the auditory cortex, but few methods are available to successfully induce auditory cortex. The basal ganglia is used for stimulating and promoting the release of cholinergic energy, and the plasticity of the auditory cortex can be effectively improved. This method requires a stimulation electrode buried in the basal ganglia to drive the release of cholinergic transmitters using an electric current, and is therefore extremely traumatic to the brain. Likewise, white noise can also induce plasticity during adulthood, but prolonged noise exposure can cause discomfort to the animal. Fluoxetine itself is a clinical antiepileptic drug, and administration of fluoxetine dissolved in animal drinking water can cause changes in the ratio of cortical excitability to suppressive properties, inducing plasticity similar to the critical phase. The medicine has convenient source, simple operation and high practicability.

The invention provides application of fluoxetine in preparing a medicine for inducing auditory cortex plasticity.

The brain is very susceptible to environmental factors during critical stages of development, since the brain has very high plasticity. The brain plasticity after the critical phase becomes very low, increasing the brain plasticity to a degree close to the juvenile phase, referred to herein as "induction".

The invention provides application of fluoxetine in preparing a medicine for improving and improving auditory cortex plasticity.

The invention also provides application of fluoxetine in preparing a medicament for preventing and/or treating and/or improving auditory disorder diseases or auditory dysfunction.

Wherein the hearing impairment disease comprises hearing loss caused by aging; auditory abnormalities caused by heavy metals, drugs or noise (e.g., auditory nervous system problems caused by moderate noise) from the environment; nerve deafness caused by genetic factors; auditory abnormalities and the like caused by cognitive system pathologies are within the scope of the claimed invention.

Wherein said fluoxetine alters auditory cortical neuron frequency selective properties, and/or increases selective neuron number, and/or increases selective neuron expression area.

Wherein the fluoxetine changes the auditory cortex frequency topology.

Wherein the fluoxetine attenuates auditory cortex inhibitory nervous system function.

Wherein, the fluoxetine changes the expression of related proteins of rat auditory cortex inhibitory neurotransmitter system and promotes the development of extracellular matrix PNN proteins towards the juvenile direction.

The invention also provides a pharmaceutical composition or a kit for improving and enhancing auditory cortex plasticity, which comprises an effective amount of fluoxetine; pharmaceutical compositions or kit instructions (including fluoxetine dosage, dosage and dosing cycle).

The invention also provides the application of the fluoxetine combined sensory training and rich environment in preparing the medicine for improving and enhancing the auditory cortex plasticity.

The invention also provides application of the fluoxetine combined sensory training and rich sound environment in preparing medicaments for improving and enhancing auditory cortex plasticity and preparing medicaments for preventing and/or treating and/or improving auditory disorder diseases or auditory dysfunction. The rich environment means that the feeding environment of animals has large space, the built-in objects are rich and novel, and the number of members is large, so that the opportunity of multi-sense stimulation and movement is provided, and the possibility of mutual social behaviors is given. Rich acoustic environment refers to the addition of acoustic stimuli as a new element to rich environments of traditional significance. The rich environment, in combination with specific sound stimuli, has been shown to alter the auditory cortex frequency tuning ability of rats.

The present invention also provides a method of improving and increasing the plasticity of the auditory cortex, said method comprising formulating fluoxetine at a concentration of 200mg/L, dissolved in an animal's daily drink, and administering to an individual in need thereof for a period of 4 weeks.

Preferably, the concentration of fluoxetine is 200mg/L.

In the invention, a 200mg/L fluoxetine solution is continuously fed to a rat for 4 weeks, and the in vivo electrophysiological technology is utilized to verify that the fluoxetine drug can induce the similar key period plasticity of the auditory cortex.

The invention also provides a method for detecting the plasticity of the auditory cortex based on the auditory cortex frequency topographic map, which is characterized in that the 7kHz pure tone stimulation is given while the chronic fluoxetine is used, the electrophysiological technology is adopted to detect the auditory cortex frequency topographic map of an object to be detected (such as a rat), and whether the number and the expression area of the 7kHz selective neurons of the fluoxetine are obviously increased or not is judged based on the auditory cortex frequency topographic map of the object to be detected, which is obtained by detection.

On week 4 of chronic fluoxetine administration, 7kHz pure tone stimulation was given simultaneously; adopting an electrophysiological technology to draw a frequency topographic map of the auditory cortex of the object to be detected; simultaneously setting a comparison group; and detecting the number of the 7kHz selective neurons and the area of the representative region of the object to be detected. In one embodiment, the present invention is administered with 7kHz pure tone stimulation concurrently on week 4 of chronic fluoxetine administration; adopting electrophysiological technology to draw a rat auditory cortex frequency topographic map; in control of normal animals, it was found that the number of 7kHz selective neurons recorded was greater than in normal animals, and the representative area was larger than in normal animals. Since pure tone stimulation cannot cause the auditory cortex frequency representation area expansion of the adult animals, fluoxetine-induced plasticity in the similar development key period can be detected by electrophysiological technology under pure tone stimulation. This strategy is often applied in the detection of acoustic cortical plasticity.

Wherein said fluoxetine causes alterations in PV interneurons and extracellular matrix PNN, and chronic fluoxetine administration modulates auditory cortex plasticity by altering alterations in the inhibitory nervous system and extracellular matrix.

The invention also provides a modulation method for changing the change of an inhibitory nervous system and an extracellular matrix and regulating the plasticity of an auditory cortex by using the chronic fluoxetine.

The invention also provides a system for improving auditory cortex plasticity, which comprises fluoxetine medicine, fluoxetine administration dose and administration period.

In one embodiment, the rat is used as an experimental subject, and the fluoxetine powder is prepared into a fluoxetine oral solution with a concentration of 0.2mg/L (fluoxetine purchased from Sigma (Sigma) company under the product number PHR 1394) by referring to a proper dosage given by the laboratory of Maffie, italian scientist in 2008, and the fluoxetine is fed to 8-week-old adult rats for 4 weeks, so that the fluoxetine is found to be effective in improving auditory cortex plasticity, and a plurality of biochemical indexes of the brain including the expression of PV protein in the auditory cortex and the expression of PNN protein in the cortex are found to be developed to the young brain at the moment through the detection of related proteins in the brain by molecular biology. Thus, the present invention recognizes that fluoxetine can induce auditory cortex plasticity similar to the critical phase.

The beneficial effects of the invention include: (1) The fluoxetine is a commonly used clinical antidepressant, and the toxicological and pharmacological research is basically complete, so that the fluoxetine is used for inducing auditory cortex plasticity to avoid damage to animals. (2) The invention is low in cost, is based on the principle of neural plasticity at the present stage, and creatively provides a novel scheme for inducing auditory cortex plasticity without any complex experimental instrument or equipment. (3) The fluoxetine drug is used for inducing auditory cortex plasticity, and can be matched with a good auditory sensation environment to improve auditory function abnormality caused by various environmental factors, such as auditory azimuth discrimination abnormality caused by low-concentration lead exposure, auditory nervous system problems caused by moderate-intensity noise and the like.

In the present invention, the related terms are referred to as follows:

the auditory cortex plasticity refers to brain plasticity, namely the brain can be modified by environment and experience and has the capability of shaping brain structure and function under the action of external environment and experience, so the auditory cortex plasticity refers to the brain plasticity process occurring in the auditory cortex.

The key-stage-like plasticity means that the brain is very susceptible to environmental factors during the key stage of development, because the brain has very high plasticity. The brain plasticity after the critical period becomes very low, and the brain plasticity in the adult period is improved by the method of the invention, and the plasticity state is found to be similar to the critical period, so the plasticity is called as the plasticity similar to the critical period.

The term "chronic fluoxetine administration" refers to a long-term (4-week) fluoxetine administration.

The principle of neural plasticity refers to the neural molecular mechanism that occurs during brain plasticity regulation. Such as: developmental maturation of the inhibitory nervous system is the major cause of the decrease in plasticity at the critical phase, and then altering the ratio between excitability and inhibitivity would both increase brain plasticity.

Drawings

FIG. 1: comparison of the acoustic cortex of rats with the blank rats after administration of chronic fluoxetine. Fig. 1A, experimental timeline. FIG. 1B, topological structure diagram of primary auditory cortex frequency of two groups of rats. FIG. 1C, cumulative function curves of characteristic frequency distributions of two groups of rat primary auditory cortical neurons. FIG. 1D, frequency receptive field example of two groups of rat auditory cortical neurons. Figure 1E, two groups of rats auditory cortical neuron frequency tuning curves suprathreshold 20 tuning bandwidth (BW 20) comparison (data expressed as mean ± sem, P <0.01 indicated by #).

FIG. 2 is a schematic diagram: chronic fluoxetine dosed rats were given a 7kHz pure tone exposure simultaneously. Fig. 2A, experimental timeline.

FIG. 2B is a diagram of auditory cortex frequency topology; pure tone exposure for 1 week after fluoxetine administration, rat auditory cortex 7kHz represents the region of overexpression. Fig. 2C, auditory cortex frequency distribution scattergram. Figure 2D, percentage change in neuronal surface area for each frequency bin of the auditory cortex (data expressed as mean ± sem, P <0.05 by x, P <0.001 by +).

FIG. 3: pure tone exposure effect after fluoxetine administration. Fig. 3A, experimental timeline. FIG. 3B is a schematic diagram of the frequency topology of the auditory cortex of three groups of rats (the black bold line in the diagram indicates the 7kHz frequency representation). Fig. 3C, the auditory cortex frequencies represent percent area change (data are expressed as mean ± sem, P <0.05 is expressed as a).

FIG. 4: expression and co-localization distribution of rat auditory cortex microalbumin (PV) and extracellular matrix Protein (PNN) after fluoxetine administration. FIG. 4A is a schematic diagram of immunofluorescent labeling of microalbumin (PV) and extracellular matrix Protein (PNN), wherein the left panel is microalbumin (PV), the middle panel is extracellular matrix Protein (PNN), and the right panel is the relative position of the two types of labels in space. The figure is used to illustrate the morphology of PV-positive neurons and PNN-positive neurons. FIG. 4B is a graph showing the distribution of the expression of auditory cortex microalbumin (PV) and extracellular matrix Protein (PNN) in three groups of rats, wherein the red marker is microalbumin (PV) and the green marker is extracellular matrix Protein (PNN). FIG. 4C, statistics of the number of immunofluorescent labels of small albumin (PV) and extracellular matrix Protein (PNN); the upper graph counts the density of positive markers of small albumin in each layer of auditory cortex; the middle graph counts the density of positive markers of the auditory cortex neuron extracellular matrix Protein (PNN), wherein the auditory cortex extracellular matrix expression quantity of rats in a drug group is obviously lower than that of rats in a blank group in 4-6 layers; the lower panel counts the neuron density co-labeled with small albumin (PV) and extracellular matrix Protein (PNN), wherein the neuron density co-labeled with the fourth layer of the drug group is slightly lower than that of the blank group of rats (data are expressed as mean ± sem, P <0.05 is expressed as x, and P <0.01 is expressed as #).

Wherein PV + refers to neurons expressing PV in the auditory cortex;

PNN + refers to neurons expressing the PNN protein;

PV + PVV refers to a co-canonical neuron that expresses both PV and PNN;

l2/3 refers to the cortex 2,3 cell layer;

l4 refers to the 4 th cell layer of the cortex;

l5/6 refers to the 5,6 cell layer of cortex;

pd11 refers to the juvenile group at 11 days postnatal;

PV + Cell refers to PV positive neurons, i.e. neurons expressing the PV protein;

PNN + Cell is a PNN positive neuron, i.e. a neuron expressing the PNN protein;

PV +/PNN + Cell refers to a neuron that expresses both PV and PNN;

II + III means 2,3 cell layer;

IV refers to the fourth cell layer;

v + VI refers to 5,6 cell layer.

Detailed Description

The present invention will be described in further detail with reference to the following specific examples and drawings, and the present invention is not limited to the following examples. Variations and advantages that may occur to those skilled in the art may be incorporated into the invention without departing from the spirit and scope of the inventive concept, and the scope of the appended claims is intended to be protected. The procedures, conditions, reagents, experimental methods and the like for carrying out the present invention are general knowledge and common general knowledge in the art except for the contents specifically mentioned below, and the present invention is not particularly limited.

Example 1 fluoxetine alters the frequency selective properties of auditory cortical neurons.

(1.1) Experimental animals and groups

(1.1.1) Fluoxetine solution

The fluoxetine solution was obtained by dissolving 200mg of fluoxetine in 1L of distilled water.

(1.1.2) Experimental animals

The medicine group is as follows: 6 rats aged 8 weeks were fed with fluoxetine solution.

Blank group: 6 rats aged 8 weeks were fed normal distilled water.

FIG. 1A is a timeline of the experiment in this example, in which two groups of rats were fed simultaneously in a normal environment in an animal house for 8 weeks, and then the rats in the drug group were fed fluoxetine for 4 weeks, and the rats in the blank group were fed with normal water for 4 weeks, to perform the experiments described below in this example.

(1.2) in vitro recording technique for drawing characteristic frequency distribution of primary auditory cortex neuron

Rats were weighed and anesthetized with 2% sodium pentobarbital solution at a dose of 45mg/kg. Rats with stable anesthesia, shi Qiguan intubation. The laryngeal cartilage was palpated at the neck of the rat with a finger, the muscle was separated with two forceps, the trachea was exposed, the trachea was cut with a scalpel, the cannula was inserted, and the surgical thread was sutured. Then exposing the temporal skull, fixing the head of the rat by using an orbit fixation mode, and turning on the electric blanket to maintain the body temperature of the rat. Then applying cerebrospinal fluid drainage, puncturing mucosa on the macropore of occiput with the tip of the surgical knife, discharging clear liquid, and releasing cerebrospinal fluid. A piece of absorbent cotton is placed outside the macropore of the occiput to stabilize the intracranial pressure. And finally, opening the temporal lobe bone window by using a dental electric drill, opening the dura mater by using a thin tungsten wire needle, wetting by using physiological saline, and waiting for electrophysiological recording.

This example uses a TDT system III recording system manufactured by TDT (Tucker-Davis Technologies), and neural signals are recorded by a two-channel metal electrode (resistance 2 M.OMEGA., channel spacing 240 μ M), and the recording process is controlled by Brainware software. Under the observation of a stereomicroscope, the micromanipulation is slowly operated to enable the tip of the electrode to touch the brain surface, and meanwhile, the depth recorder is reset to zero. The micromanipulation was operated at a speed of 3-4 μm per second to insert the electrode into the brain surface. A stable discharge was sought in the range of 350-600 μm. After finding a stable discharge, the recording is turned on in the Brainware software. The sound stimuli include pure tones with frequency of 0-30kHz and gradient of 0.1octave for 49 frequencies, 8 sound intensities with sound intensity of 0-70dB SPL, and 392 sound stimuli in combination. And simultaneously recording the response number of the neuron under each sound stimulus, and drawing a frequency receptive field by software to establish a frequency tuning curve. The recording position was photographed using a CCD camera and the recording site was marked in the graphical software. And (4) combining the characteristic frequency parameters of the recording sites, and drawing a characteristic frequency distribution diagram of the auditory cortical neurons in matlab software.

(1.3) results of the experiment

The auditory cortex is the high-level central nervous system that the brain processes auditory information, and changes in the structure and function of the auditory cortex affect the auditory function of an individual. The distribution of the characteristic frequency of the auditory cortical neurons of a normal rat has the characteristics of head-to-tail side distribution and high-frequency to low-frequency distribution.

The characteristic frequency distribution of primary auditory cortex neurons of two groups of rats is detected, and the result of a primary auditory cortex frequency topological structure chart shows that the auditory cortex neuron distribution of a rat in a drug administration group (fluoxetine administration group) is not obviously different from that of a rat in a blank group (figure 1B). Statistics of all recorded characteristic frequencies of auditory cortical neurons revealed that there was no difference in cumulative function curves of primary auditory cortical frequency distributions between rats in the drug group (fluoxetine drug group) and rats in the blank group (fig. 1C, drug group VS blank group, P >0.9999, kolmogorov-Smirnov test), indicating that fluoxetine did not cause changes in the characteristic frequency distributions of primary auditory cortical neurons in rats.

The electrical reaction of the auditory cortical neurons to the combined sounds of different intensities and different frequencies can reflect the function of the auditory cortical neurons. FIG. 1D is a schematic view of the frequency receptive fields of two groups of auditory cortical neurons, i.e., the black areas in the figure. The frequency receptive field represents the receptive range of auditory cortical neurons to sound frequency and sound intensity. The Frequency field is delineated to obtain a Frequency tuning curve, and the curve can obtain attributes such as characteristic response Frequency (CF), minimum Threshold (MT) and tuning width (BW) of the neuron. Comparing the tuning widths (BW 20) of 20dB above threshold of the frequency tuning curves for the two groups of rat auditory cortical neurons, it was found that the rat auditory cortical neurons BW20 in the drug group were significantly higher than the rats in the blank group (fig. 1D). The recorded neurons are classified according to characteristic frequencies, and the fact that in low, medium and high frequency sections, the tuning bandwidth BW20 of rat auditory cortex neurons in a medication group is obviously wider than that of rats in a blank group (figure 1E, VS blank group in medication group, CF <7.5kHz, P < -0.0001, 7.5kHz < -CF < -15kHz, P < -0.0001 in case of P < -0.0001, t-test Boffoni Correction is found. The results of this experiment demonstrate that the frequency selective properties of the auditory cortical neurons in rats are reduced following administration of fluoxetine. Since the ratio of cortical excitability to cortical inhibitivity is critical for controlling brain plasticity, the frequency tuning curve for rat auditory cortical neurons became significantly broader 4 weeks after administration of fluoxetine to rats, indicating an increased ratio between auditory cortical excitability and inhibitory activity, indicating that this example successfully induced cortical plasticity.

Example 2 Structure of fluoxetine for altering auditory cortex frequency topology

(2.1) Experimental animals and groups the same as in example 1

(2.1.1) Fluoxetine solution

The fluoxetine solution was obtained by dissolving 200mg of fluoxetine in 1L of distilled water.

(2.1.2) Experimental animals

Fluoxetine + pure tone exposure group (experimental group): 6 rats aged 8 weeks were fed with fluoxetine solution for 4 weeks, and at the 4 th week of feeding, the rats were exposed in pure tone exposure chamber for 1 week, which was fluoxetine + pure tone exposure group and also the experimental group of this example.

Pure tone exposure control group: to verify the effect of pure tone exposure on the auditory cortex of adult rats, a pure tone exposure control group was set up. 6 rats of 11 weeks old were placed in a pure tone exposure chamber and exposed for 1 week, which was a pure tone exposure control group.

Fluoxetine administration control group: to verify the effect of fluoxetine feeding on cortical frequency distribution changes in the absence of pure tone induction, a fluoxetine dosing control was set up. 6 rats with the age of 8 weeks are fed with fluoxetine for 4 weeks, and the control group for fluoxetine administration is obtained.

Blank group: 6 normal rats of 12 weeks of age.

FIG. 2A is a timeline of the experiment, which was carried out at 12 weeks of age of rats by feeding and molding groups.

(2.2) Sound Exposure apparatus

The sound exposure device is a self-made sound insulation box, a built-in loudspeaker, an automatic light and a ventilation system. The sound used was a 7kHz pure tone, one pure tone was 50ms, and 15 pure tones were grouped into a string, with a duration of 1s, with 1s intervals between each string. The pure tone file is created using adobe audio cc and recorded in the optical disc using a DVD recorder. The audio is played by a DVD player, passes through a power amplifier and is emitted by a loudspeaker. The sound intensity was 65dBSPL.

(2.3) results of the experiment

The arrangement of rat auditory cortex characteristic frequency neurons has a head-to-tail side, high frequency to low frequency distribution mode. This way of coding the auditory cortex is often referred to as frequency structuring of the auditory cortex. The frequency distribution graph of the auditory cortex changes along with the change of plasticity, and 7kHz pure sound is used for stimulating rats in the 'key stage' of brain development for 24 hours, so that the expression quantity of 7kHz characteristic neurons of the auditory cortex is obviously improved. This detection of auditory cortex frequency distribution maps is widely used in auditory plasticity studies (Kilgard MP and Merzenich MM., 1998).

In this example, the frequency distribution of the auditory cortex of each group of rats was plotted using in vivo electrophysiological techniques, and as a result, it was found that the expression area of neurons in the auditory cortex of the fluoxetine + pure tone exposed group (experimental group) for the 7kHz pure tone was significantly increased compared to the rats of the blank group (fig. 2B). The neuron position information recorded by each group of rats was normalized and plotted to obtain a frequency distribution scattergram of auditory cortex, and the number of 7kHz neurons recorded by the fluoxetine + pure tone exposure group (experimental group) was found to be significantly increased (fig. 2C). The percentage change in neuronal surface area for each frequency bin of the auditory cortex of each group of rats (7 kHz mean expression area measured for each group minus the 7kHz mean expression area for the blank group) was calculated, and as can be seen from FIG. 2D, the 7kHz expression area for the auditory cortex of the fluoxetine + pure tone exposed group (experimental group) was significantly higher for the rats than for the remaining three groups (fluoxetine + pure tone exposed group VS blank group; P =0.0015, t-test bofforoni correction) (FIG. 2D). The above results suggest that fluoxetine improves the plasticity of the auditory cortex function under pure tone induced conditions; in the absence of pure tone induction, fluoxetine feeding does not cause changes in auditory cortex frequency distribution; likewise, pure tone exposure had no effect on the adult rat auditory cortex.

Example 3 to further demonstrate the effect of fluoxetine administration on the plasticity of auditory cortex function

(3.1) Experimental animals and groups

(3.1.1) Fluoxetine solution

The fluoxetine solution was obtained by dissolving 200mg of fluoxetine in 1L of distilled water.

(3.1.2) Experimental animals

Pure tone exposure group after drug administration: 6 rats, 8 weeks old, were fed with fluoxetine solution for 4 weeks and then placed in a 7kHz pure tone exposure cabinet for pure tone exposure.

Pure tone exposure group 7 weeks after dosing: 6 rats at 8 weeks of age were fed with fluoxetine solution for 4 weeks, stopped for 7 weeks and placed in a 7kHz pure tone exposure cabinet for pure tone exposure.

Blank group: 6 normal rats of 12 weeks of age.

FIG. 3A is a timeline of the experiment of this example, which was carried out by feeding and molding groups according to the feeding mode of the groups.

(3.2) Experimental procedures and results

And (3) drawing a frequency topographic map of the three groups of rats to obtain a frequency topological structure diagram of the auditory cortex of the three groups of rats, comparing the frequency topographic maps of the two groups of rats (a pure tone exposure group after medication and a pure tone exposure group after 7 weeks of medication) with a blank group, and detecting the plasticity effect of the fluoxetine on the auditory cortex, wherein as can be seen from the graph in fig. 3B, the expression area of the 7kHz pure tone can be increased even if the pure tone exposure group is given immediately after the fluoxetine is stopped taking the medicine. Further statistics on all recorded distribution areas of cortical neurons were found that administration of pure tone exposure immediately after discontinuation of fluoxetine (i.e., post-administration pure tone exposure group) still significantly affected cortical frequency organization (fig. 3C, post-administration pure tone exposure VS blank group, P =0.034, t-test bufferoni correction). The administration of pure tone after 4 weeks of discontinuation of fluoxetine for 7 weeks (i.e. the group exposed to pure tone 7 weeks after administration) did not cause the change in the expression area of pure tone at 7kHz in the auditory cortex.

When rats of 11-13 days old are exposed to a 7kHz pure tone environment for one day, the expression level of 7kHz related neurons of an auditory cortex of an individual adult is increased, and the expression area of related frequencies is increased. The frequency architecture of the auditory cortical neurons can also change due to changes in the balance of excitability and inhibitability; studies have shown that inhibitory interneurons are thought to control the integrity of cortical frequency architecture and the boundaries between frequency-representative regions (Casanovaet al, 2002).

In addition, the auditory cortex frequency topology distribution map is detected by using an extracellular multichannel recording technology. The results show that compared with the blank group, after the pure tone exposure group is exposed after the medicine is applied, the 7kHz neuron expression area of the auditory cortex is obviously expanded; in contrast to the blank group, the 7-week-after-administration pure tone exposure group was placed in a 7kHz pure tone environment after the administration was stopped for 7 weeks after 4 weeks of administration, and a similar phenomenon was not observed (FIG. 3B).

Example 4 Fluoxetine changes in expression of proteins involved in the inhibitory neurotransmitter System in the auditory cortex of rats

(4.1) Experimental animals and groups

(4.1.1) Fluoxetine solution

The fluoxetine solution was obtained by dissolving 200mg of fluoxetine in 1L of distilled water.

(4.1.2) Experimental animals

The medicine group is as follows: 6 rats aged 8 weeks, fed with fluoxetine solution in the animal house for 4 weeks, the following experiments of this example were performed.

Blank group: the following experiments of this example were carried out on 6 rats aged 8 weeks, which were fed with normal distilled water in the animal house for 4 weeks.

Young group: 6 young rats of age 11 days. To understand the relationship between the expression of related proteins in the auditory cortex and the young rats, this example added a group of young groups consisting of 6 rats born for 11 days as a control, i.e., a young group.

(4.2) immunofluorescence (Small Albumin (PV) and extracellular matrix Protein (PNN) immunofluorescence double-label staining method)

In order to further determine the influence of fluoxetine on the biochemical indexes in the auditory cortex, the invention specifically labels auditory cortex microalbumin (PV) and extracellular matrix Protein (PNN) by an immunoblotting method.

The heart perfusion method for rats comprises the steps of perfusing the rats with physiological saline, and then perfusing and fixing the rats with 4% paraformaldehyde. Then, the head of the rat is cut off, the brain of the rat is taken out and soaked in 4% paraformaldehyde for 6 hours, and then gradient dehydration is carried out. After gradient dehydration, embedding with embedding medium, and quickly freezing and storing in a refrigerator at-80 ℃. When in slicing, the slices are transferred into a freezing microtome (Leica) two hours in advance, the working temperature is set to be 20 ℃ below zero, the slice thickness is set to be 30 mu m, and the slice positions are positioned by referring to a brain atlas to carry out slicing. Soaking brain pieces in situ hybridization protective solution, and storing in a refrigerator at-20 deg.C.

The brain pieces were removed from the in situ hybridization protection solution and rinsed 3 times in PBS with rinsing plates for 10min each time for 30min. Preparing 10% goat serum and Triton PBS by using a 1ml or 2ml EP tube, subpackaging the sealing solution, soaking each brain slice in 100 mu l of sealing solution, and sealing by rinsing slices at 37 ℃ for 1.5h. Configuration murine anti-PV (Sigma) 1, anti-WFA (Vectorlabs) 1:200,1% sheep serum and TritonPBS. Subpackaging 1 antibody, soaking each brain slice in 100 μ l, placing in an oven for incubation at 37 deg.C for 6h, and transferring to a refrigerator at 4 deg.C for overnight storage. The next day, brain slices were picked into rinsing plates and rinsed for 40min with PBS. Towards the end of the rinsing, fluorescent secondary antibodies (546nm, mouse, sigma) 1 were prepared in 300,1% sheep serum and Triton PBS. The secondary antibody is dispensed, each brain slice is 100 mu l, and the brain slices are put into the secondary antibody to be incubated for 2h at 37 ℃. Taking out, placing brain slice into rinsing plate, and rinsing with PBS for 45min. Finally, patch, and drop-add the patch agent with DAPI to keep the brain slice moist. Cover with glass and seal the edges with nail polish, dry and store at 4 ℃.

(4.3) results of the experiment

This example labels the neuron-related proteins PV, PNN using immunofluorescence techniques (fig. 4A). Morphologically, the form of the PV-labeled neurons is a solid spindle (fig. 4A, left panel), and the PNN-labeled neurons surround the PV-label (fig. 4A, right panel) in a radial pattern (fig. 4A). Visually, the number of markers for the auditory cortex PV and PNN proteins was significantly lower in the drug group rats compared to the blank group (fig. 4B). Counting statistics of the two types of marker proteins revealed that the expression of extracellular matrix Protein (PNN) of the drug group rat in the 4 th to 6 th layers of the auditory cortex was significantly lower than that of the rats in the blank group (FIG. 4C, drug group VS blank group, layerIV: P < 0.01: layerV/VI: P <0.05, t-test Bofforoni correction).

Meanwhile, in the embodiment, when the rats in the juvenile group are added to be compared with the rats in the drug group and the rats in the blank group, the expression trend of the auditory cortex extracellular matrix (PNN) of the rats in the drug group develops towards the juvenile direction after the fluoxetine is applied.

Therefore, the reduction of the expression level of the auditory cortex inhibitory interneuron related protein is the reason for the improvement of the auditory cortex plasticity by fluoxetine.

Development of "critical-phase" brain structures is often accompanied by migration and anchoring of inhibitory neurons from the thalamus to the cortex, which is also considered an important node for the maturation of the cortical neural network (Blue and parnavevas 1983). Studies have shown that the formation of inhibitory interneuron myelin is closely linked to the functional maturation of neuronal networks (Ishiguro et al 1991; bruckner et al 2000). Such phenomena have been demonstrated in several locations in the central nervous system, including the visual cortex, spinal cord dorsal horn motor neurons, the pony cortex and the cerebellum (Dityatev et al 2007; mcRae et al 2007). Extracellular matrices (PNNs) are a class of networks surrounding the outside of the majority of neuronal cells in the central nervous system, consisting of extracellular matrix molecules (ECM), including Hyaluronan (HA), catenin, chondroitin surface proteins (CSPGs, chondroitins sulfate proteins-glycoproteins), and mucins (Tn-R, tenascin-R), which are abundantly expressed around and outside of the proximal dendrites of the neuronal cells in the central Nervous System (PNNs) ((

et al.1997; carulli et al 2006,2007; deep et al.2006; kwok et al.2010). In recent years, studies have found that extracellular networks have a significant effect on neuroplasticity. Rats raised in the dark have reduced expression of visual cortex Crtl1, HAS2 and HAS3, and reduction of these proteins prevents the formation of extracellular matrix (PNNs). The extracellular matrix (PNNs) is again reformed using pulsed light stimulation (Carulli et al.2010). The effect of extending the critical phase of the visual cortex was achieved (Hockfield and Sur 1990, lander et al 1997. But light stimulation during development impairs the critical phase and does not prevent PNN formation (G-ti et al 2010). The present invention uses wisteria floribunda lectin (WFA) labeled galactosamine (N-acetylgalactosamine) for the visualization of extracellular Network Structures (PNNs) (Nakagawa et al 1986; bruckner et al 1993; schweizer et al 1993). As a result, the expression quantity of the PNNs in the auditory cortex of the fluoxetine group rats is obviously lower than that of the control group rats. KnotThe result indicates that fluoxetine causes the expression quantity of the extracellular matrix of the auditory cortex neuron to be changed, and the plasticity of the auditory cortex is regulated.

Auditory cortex inhibitory interneurons mostly express PV, PV positive interneurons participate in modulating empirically-dependent plasticity by altering the intrinsic properties of the cell and the output properties of synapses (de Villers-Sidani et al, 2008 zhou et al, 2011 oaellet and de Villers-Sidani, 2014. The induction and expression of neural plasticity depends on neuronal-to-neuronal and neuronal-to-peripheral interconnections. An extracellular matrix called PNNs is often wrapped around PV-positive interneurons, and contains Chondroitin Sulfate Proteoglycans (CSPGs) (deep et al, 2006). The formation and maturation of PV-positive interneurons and PNNs both occur during the critical phase of off (Takesian and Hensch, 2013). This suggests that PNNs are involved in mediating critical phase plasticity by promoting synaptic stability and synaptic rearrangement processes (Pizzorusso et al, 2002). More direct evidence suggests that pharmacological treatment of Chondroitin Sulfate Proteoglycans (CSPGs) enhances cortical plasticity and learning capacity (goolla et al, 2009). Thus, the present invention simultaneously investigates the expression of PV positive interneurons and extracellular matrix PNNs. As a result, the fluoxetine drug combination was found to express the same decrease in extracellular matrix PNNs around PV positive interneuron cells. The results suggest that fluoxetine treatment caused changes in the activity of auditory cortex PV positive interneurons, further improving the plasticity of the auditory cortex.

The results of the present invention demonstrate a potential application of fluoxetine beyond its pathophysiological applications for emotional diseases. Fluoxetine can provide the solution of brain sensation function enhancement for some special industry workers, for example, the auditory sensation function of submarine officers and soldiers can be improved by matching training, and sonar echo can be better distinguished. Similarly, the brain auditory cortex plasticity induced by fluoxetine can improve the environment-induced brain auditory sensory function deterioration.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

Claims (3)

1. The application of the fluoxetine in preparing the reagent for detecting the plasticity of the auditory cortex based on the auditory cortex frequency topographic map is characterized in that the reagent is used for giving 7kHz pure tone stimulation while chronic fluoxetine is used, an electrophysiological technology is adopted, the auditory cortex frequency topographic map of an object to be detected is detected, and whether the quantity of 7kHz selective neurons and the expression area of the fluoxetine are obviously increased or not is judged based on the auditory cortex frequency topographic map of the object to be detected, wherein the auditory cortex frequency topographic map is obtained by detection.

2. The use of claim 1, wherein 7kHz pure tone stimulation is given simultaneously on week 4 of chronic fluoxetine administration; adopting an electrophysiological technology to draw a frequency topographic map of the auditory cortex of the object to be detected; simultaneously setting a comparison group; detecting the number of the 7kHz selective neurons and the area of a representative region of the object to be detected; since pure tone stimulation cannot cause the auditory cortex frequency representative region of an adult animal to expand, fluoxetine-induced plasticity in the similar development key period can be detected by an electrophysiological technology under the pure tone stimulation.

3. The use of claim 1 or 2, wherein the fluoxetine causes alterations in PV interneurons and extracellular matrix PNN, and chronic fluoxetine administration modulates auditory cortex plasticity by altering changes in the inhibitory nervous system and extracellular matrix.

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