CN110361239B - Method for tracing nerve fiber bundles in light-color antennae of insects - Google Patents
Method for tracing nerve fiber bundles in light-color antennae of insects Download PDFInfo
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Abstract
The invention discloses a method for tracing nerve fiber bundles in light-colored antennae of insects, belonging to the technical field of experimental sample treatment. The complete tracing of the nerve fiber bundles in the tentacles can be realized through the dip dyeing of the nickel chloride or the cobalt chloride of the tentacles of the living bodies, the micro-separation of the tentacles and the brains of the living bodies, the color development, the fixation, the dehydration, the transparentization treatment of the tentacles and the brains, the fixation of the gesture of the tentacles and the micro-photographing. The invention solves the problems of complete transparent tentacles and multi-angle visualization of nerve fiber bundles of the tentacles under the condition of not slicing and damaging the tentacles, and also enables the connection between the nerve fiber bundles in the tentacles and the brain to be directly observed on the level of a nerve channel; the method has the advantages of low cost, reliability, simple and easy operation and good repeatability, and can directly obtain the multidimensional data of the internal neural structure of the antennal.
Description
Technical Field
The invention relates to a method for tracing nerve fiber bundles in light-colored antennae of insects, belonging to the technical field of experimental sample treatment.
Background
The antennae are essential for the survival and reproduction of insects. Insects mainly sense external environmental changes such as smell, wind, temperature and humidity through antennae. By means of the antennae, the insects can perform a series of life activities such as searching for food or spouses, avoiding natural enemies, intra-species communication and searching for suitable spawning sites. According to the difference of the forms, the antennae of insects can be classified into bristle-shaped, awn-shaped, filiform, stick-shaped and other types. The antennae are divided into handle section, peduncle section and flagellum section, the surface of the antennae is covered with a layer of chitinous exoskeleton which has the functions of protecting internal soft structure and reducing water evaporation, and antennae of different types of insects show different colors, such as black antennae of bee family, light-color antennae of muscidae and light-color antennae of drosophilidae.
The antenna is a multi-modal sensory organ. Receptors of different modes such as smell sense, wind sense, sound sense, temperature sense, humidity sense and the like are distributed on the surface of the antenna, for example, sensors such as a hair sensor, a cone sensor, a bar sensor, a cavity cone sensor, an antenna sensor, a sense pit and the like are mainly distributed on the antenna of the diptera insect, and further functional research shows that the insect hair sensor, the cone sensor and the club sensor are distributed on the surface, and dense holes are distributed on the surface, and the function of the insect hair sensor, the cone sensor and the club sensor is used for detecting smell. The odorant molecule enters through the pore canal, is combined with odorant receptor on nerve cell membrane and then is converted into electrophysiological signal, and is transmitted to brain along nerve fiber. The cavity cone sensor may be sensitive to chemistry or temperature and humidity; the antenna can sense the temperature; the tentacle awns and the flagellum can conduct mechanical signals to the inside of the peduncle section through the displacement of the tentacle awns and the flagellum, and the neural cells of the Jiang's organ in the peduncle section sense wind, gravity, sound and the like; the sensory fossa on the flagellum is likely to sense moisture. The sensors convert different types of stimulation such as external chemical smell, sound, temperature and humidity into electrophysiological signals, transmit the electrophysiological signals into the brain through the sensory nerve fiber bundles with different modes corresponding to the inside of the tentacle, and form close connection with the tentacle lobe and the mechanical sensory motor center of the tentacle in the brain. The long-distance sensory nerve fiber bundles with the tentacles as the starting points and the brains as the end points are the neural basis for realizing the perception, navigation and positioning of the surrounding environment and the target of insects, and are also the neural targets for green prevention and control of pests. Therefore, it is important to study the nerve fiber bundles of the tentacles, but so far, the neuroanatomy of the insect tentacles and their primary sensory nerve pathways (nerve fiber bundles) is essentially unclear.
The anatomic research of the tentacles of the insect tentacles such as the drosophila melanogaster of the model insect drosophila family is carried out by using the slicing methods of transverse cutting, longitudinal cutting and the like, and the structures of nerve fiber bundles in the tentacles are damaged to a great extent, so that the obtained information of the nerve fiber bundles in the tentacles is fragmented, has low accuracy, and cannot accurately obtain the visual information of the real number, spatial distribution, structures and the like of the nerve fiber bundles in the tentacles. Moreover, the slicing technique breaks the linkage of the antennal to the brain and does not allow observation at the level of one complete neural pathway. Therefore, the primary sensory nerve pathway of the insect's full antennal cannot be internationally resolved in situ, including information on the number, spatial distribution and structure of nerve bundles within the antennal.
In the prior art, after two nerve tracers of nickel chloride and cobalt chloride are injected into different parts (distributed with different types of sensors) of an antennal flagellum of drosophila melanogaster, Stocker et al find that the sensors at the different parts of the flagellum can be projected to different target areas of antennal lobes in the brain. Subsequently, these two tracers were also used in studies of other different kinds of insects such as moths, crickets, as well as in the projection of target areas of the sensory nerves of the tentacles into the brain; the neck brightness and the Penbo observe the olfactory nerve projection channel of the Male Bombycis of Agrotissegetum by using lysine cobalt as a tracer, and only the projection condition of the antennary nerve in the brain is obtained.
The two existing nerve tracers are only limited to the brain in international and domestic researches, the research on nerve fiber bundles in peripheral antennal is not reported so far, and the research on primary sensory pathways from the antennal nerve fiber bundles to the brain is not reported so far. The main reason is that to obtain accurate information about the bundles of the antennal nerve fibers, direct observation on intact antennals is necessary, and significant technical challenges, such as control of the padding step, the developer control step, discovery and application of antennal transparencies, fixation of the antennal postures, etc., must be overcome in a number of steps. The solution of these problems will have profound effects on the neuroanatomy of the insect's antennal sensory nerve pathways.
The antennal size of different insects is different, and the shape is different, and the colour is different, even can mark with the nerve tracer, but the time of tracer impregnation is difficult to control, and overlength then can cause the tracer to spread at nervous system non-specificity, and too short then the nerve fiber bundle can not all be marked, and these all can directly influence the chromogenic effect in later stage. In the color development process, the color development time is difficult to control, and excessive color development is easily caused. These causes make it difficult to specifically label the nerve fiber bundles in the tentacles. In addition, the insect's tentacles are wrapped by a layer of chitinous exoskeleton, which not only severely affects the efficiency and process of color development, but also the opaque cuticle makes optical observation impossible even if the nerve fiber bundles are marked. Therefore, the study of the nerve fiber bundles in the intact antennal has been delayed. Meanwhile, in order to comprehensively research the sensory organs of the tentacles, multidimensional observation and inspection on the internal and external structures and functions of the tentacles are required. The size of the tentacles of many insects is smaller, such as the full length 860-1000 μm of the tentacles of the guava fruit flies, the widest is about 170 μm, the thickest is about 150 μm, and the tentacles are divided into peduncles, peduncles and flagellates, and the shapes, structures and sizes of the sections are different, the whole body presents irregular geometric shapes, and the required pivot points are on different curved surfaces during fixing, and multi-dimensional posture fixing cannot be realized through natural placement. At present, several international fixing methods mainly include paraffin embedding or agarose embedding and slide glass fixing. Paraffin or agarose embedded fixation techniques do not allow observation of the complete antenna surface or internal structure under an optical microscope. In general, internationally, when an observation specimen is prepared, a cell or tissue section (non-intact organ) is usually placed on a slide glass, a cover glass is pressed thereon, and the slide glass is mounted for long-term observation. It is necessary that the tissue sections be flat to facilitate coverslipping. Once this is done, the pose of the slice is not readjustable, nor is it necessary to make adjustments, and the acquired image is in one dimension. The spliced image obtained by slicing the antennal organ has large workload and high distortion, so that the aim of directly obtaining the internal structure image information of the antennal organ under the condition of no slicing is always pursued by the international scientific community. However, the antenna is an organ with extremely irregular morphology, and the fixation of posture, optical observation and image acquisition at specific angles such as the inner side, the outer side, the oblique inner side, the back side and the ventral side cannot be realized by the traditional slide pressing method.
Disclosure of Invention
Aiming at the problem that the nerve fiber bundles in the complete insect light-colored tentacles are difficult to trace in the prior art, the invention provides a method for tracing the nerve fiber bundles in the insect light-colored tentacles, which can trace the nerve fiber bundles in the complete tentacles by dip dyeing of nickel chloride or cobalt chloride of living tentacles, micro-separation of the living tentacles and brains, color development, fixation, dehydration, treatment of transparentization of the tentacles and the brains, fixation of the tentacles postures and micro-photographing; the limitation of the existing slicing technology is broken through, the transparent technology of the light-colored antenna is established, the in-situ visualization of the nerve fiber bundles in the antenna is realized, and the precise information such as the number, the spatial distribution, the advancing route and the like of the nerve fiber bundles in the complete antenna is obtained.
A method for tracing nerve fiber bundles in light-colored antennae of insects comprises the following specific steps:
(1) dip-dyeing the living insect antennae by using a nickel chloride solution or a cobalt chloride solution; using a sample adding gun head of a microsyringe, cutting off the tail end of the gun head, freezing and anaesthetizing the insects, putting the insects into the tail end of the gun head, pushing the insects to the tip of the gun head by micro force, fixing the heads of the insects by the tip of the gun head, exposing an antenna, and fixing the gun head in plasticine or dental wax on a glass slide; carefully cutting a notch at the tip of the tentacle flagellum by using an ophthalmic scissors under a dissecting mirror; meanwhile, fixing a capillary glass tube or a glass electrode containing a nickel chloride solution or a cobalt chloride aqueous solution in another piece of plasticine or dental wax at the other end of the same glass slide, and carefully sleeving the capillary glass tube containing the nickel chloride or the cobalt chloride aqueous solution on the contact incision to ensure that the incision is immersed in the solution and is kept smooth; then, the glass slide is moved into a glass culture dish with a wet filter paper strip, the humidity is kept between 50 and 70 percent, and the glass slide is placed for 2 to 48 hours at room temperature;
(2) microscopically separating intact tentacles and brains of living insects; pouring 50mL of Ringer solution into a 100mL beaker, burying the beaker in ice and cooling to obtain ice-cold Ringer solution, wherein the ice-cold Ringer solution is continuously filled with a mixed gas of 95% oxygen and 5% carbon dioxide; dropping aerated ice-cold Ringer solution on a glass slide, then rapidly shearing the head of an insect with an antenna by using an ophthalmic scissors, putting the insect with the antenna into the Ringer solution on the glass slide, rapidly and completely separating the brain with the antenna from the hair follicle within 3-5 minutes by using a pointed-end tweezers and the ophthalmic scissors under a body type microscope, removing the trachea and the air sac on the surface of the brain by using the tweezers, cutting off the part of the pedicle cutin layer at the connection part with the whip by using the sharp triangular tip of a 1mL syringe needle, and then cleaning for 3 times by using the Ringer solution;
(3) tissue color development: dripping a solution of the rhodomine on the insect tentacles and brains obtained in the step (2), and observing the color development condition at any time under a stereoscopic microscope; once the tentacles and nerve tracts in the brain are marked, quickly absorbing and removing the solution of the erythrosine with a dry filter paper strip, and cleaning for 3 times with 100% alcohol to remove redundant erythrosine inside and outside tissues and prevent excessive color development;
(4) tissue fixation: gently transferring the colored insect tentacles and brains obtained in the step (3) into a clean PCR tube by using a pointed-end forceps, sequentially dripping 2-4 drops of 5% and 10% formaldehyde solutions, fixing for 10 minutes respectively, and finally absorbing and removing the formaldehyde solutions by using a dry filter paper strip;
(5) tissue dehydration: gently transferring the insect tentacles and brains fixed in the step (4) into another clean PCR tube by using a pointed-end forceps, and dehydrating by using 70%, 80%, 90% and 100% of alcohol in sequence for 5 minutes;
(6) and (3) tissue transparency: immersing the dehydrated insect tentacles and brains obtained in the step (5) into methyl salicylate, and standing at room temperature for 1-12h to ensure that the horny exoskeleton, internal tissues and brains of the tentacles are transparentized;
(7) posture adjustment and fixation: adjusting and fixing the postures of the insect antennae and the brain processed in the step (6) by using pointed tweezers in a small well device under a body type microscope;
(8) and (4) microscopic photographing: and placing the tentacles and the brains with the adjusted and fixed postures under a microscope to obtain the image information of nerve fiber bundles in the tentacles under different magnifications, adjusting and fixing the postures of the tentacles according to needs, and obtaining the image information of the required angles.
Further, the specific operation method for adjusting and fixing the postures of the insect antennae and the insect brains in the step (7) comprises the following steps: transferring the insect tentacles and brains treated in the step (6) to quartz sand in a small well device of a posture fixing device, dripping low-viscosity high-transmittance methyl salicylate into the small well device, carefully moving the tentacles and the quartz sand by using a pointed forceps, and performing posture adjustment and fixation on the tentacles from different directions and angles such as the back side, the abdominal side, the outer side, the inner side and the top end according to needs.
Further, the mass concentration of the nickel chloride solution or the cobalt chloride solution in the step (1) is 1-10%, and the dip dyeing time is n-n +5 hours when the length of the insect antenna is n millimeters.
Further, the volume of the tip of the loading gun is 10. mu.l, 200. mu.l or 1000. mu.l.
Furthermore, the caliber of the tip of the capillary glass tube or the glass electrode is 1-2.5 times of the width of the insect contact angle.
Further, the posture of the insect tentacles and the insect brains in the step (7) is fixed by adopting a small well device, the height of the small well device is not more than 5mm, and quartz sand is arranged in the small well device. The height of the small well device is too high, so that the thickness of the quartz sand and the specific liquid chemical substances contained in the small well device is increased, the light transmittance is reduced, the observation of the internal structure of the antenna is not facilitated, and meanwhile, the small well device is too high, so that the posture of the small well device is not convenient to micro-adjust and the observation under the high-power objective lens, and the fine observation of the internal structure of the antenna is also influenced.
Further, the quartz sand is in two different sizes; the particle size of the quartz sand is 1/5-1/2 of the minimum size of the sample, and the mass ratio of the two types of the quartz sand is 1 (1-3). The quartz sand with different sizes and proportions can be combined more, more fulcrum curved surfaces with different heights can be manufactured, namely the two quartz sand with smaller sizes can be combined to manufacture the fulcrum curved surfaces with different heights, and the arbitrary angle fixation of the antenna gesture is realized. The undersize of the quartz sand is not favorable for operation, and the oversize is not favorable for manufacturing a curved surface for supporting a tiny antenna.
The invention relates to a tracing principle of nerve fiber bundles in insect light-colored tentacles:
the method is characterized in that nickel chloride or cobalt chloride is used as a nerve tracer, the amount of nickel ions or cobalt ions entering nerves and the backward transmission distance are controlled by controlling the leaching time of the tracer, the activity of insects is ensured in the leaching process, and the specific leaching of the tracer is facilitated, namely, the nickel ions or cobalt ions only travel backwards along axons (nerve fiber bundles) and can be accumulated in the traveling path, namely, the method is one of key steps of tracing and determines a remarkable color result. The humidity of 50-70% is provided in the dip dyeing process, so that the evaporation of the tracer aqueous solution can be reduced, the osmotic pressure of the tracer aqueous solution can be maintained, and the influence of the tracer solution on nerve cells can be reduced; in the microdissection process, mixed gas is filled into the Ringer liquid and the temperature is kept low, so that the good activity of tissues and cells of the tentacles and the brain is ensured, and the condition that the tracer is diffused into other non-nerve tissues when the activity of the cells is reduced or the cells die to influence the subsequent color development effect is prevented; the tryptophan can form a dark purple complex with nickel ions or cobalt ions, so that the place where the nickel ions or the cobalt ions are accumulated is dyed into dark purple, and then the nerve fiber bundles inside the tentacles are dyed into dark purple, the control of the color development duration is vital in the color development process, the color development is stopped immediately after the nerve fiber bundles inside the tentacles are dyed, the solution of the tryptophan is removed, and the solution is washed by alcohol, so that excessive color development is prevented, and the method is one of the key steps of the tracing effect; fixing the developed antennae and brain with 5% and 10% formaldehyde solutions respectively; dehydrating with 70%, 80%, 90% and 100% gradient alcohol; methyl salicylate is a colorless transparent liquid, has a high refractive index, and is generally used for the transparency of soft tissues; however, since the horny layer of an insect antenna is thick, the light transmittance is low, and the visualization of the internal structure of the antenna is hindered. Through a large number of experiments, methyl salicylate can be used for transparent tentacle light-colored cuticle exoskeleton to a certain extent. Therefore, the adoption of methyl salicylate realizes the transparentization of the exoskeleton cuticle, the internal tissues and the brain quilt of the light-colored tentacles of the insects; the tentacle or brain with a three-dimensional structure is subjected to multi-dimensional posture adjustment and fixation, namely a key link for deeply acquiring nerve fiber bundle image information in the tentacle, the characteristics of three-dimensional irregularity, light transmission and quartz sand proportion of quartz sand with different sizes and sizes compared with an insect tentacle are utilized to combine and manufacture fulcrum curved surfaces with different heights, the random adjustment and fixation of the tentacle posture are realized, meanwhile, the application of low-viscosity high-light-transmittance methyl salicylate plays the triple roles of auxiliary support for tentacle posture fixation, smooth light path and tentacle preservation, the problems of fixation and long-term preservation of the insect micro tentacle are thoroughly solved, and the last obstacle of the visualization link of the internal structure of the tentacle is thoroughly eliminated.
The invention has the beneficial effects that:
(1) the method breaks through the limitation of the existing slicing technology, establishes the transparent technology of the light-colored tentacles, realizes the in-situ visualization of nerve fiber bundles in the tentacles, obtains the fine information such as the number, the spatial distribution, the advancing route and the like of the nerve fiber bundles in the complete tentacles, and brings profound influence on the nerve anatomy and the function research of the insect tentacles;
(2) the invention overcomes the limitation that the intracerebral projection of the insect tentacle nerve fiber bundle can only be analyzed and observed in the past by separating the living tentacle and brain complex, and directly realizes the visualization of the connection between the intracerebral nerve fiber bundle and the brain in the tentacle at the level of a primary sensory nerve pathway;
(3) the antenna fixing device is used for skillfully realizing the fixation of the same antenna posture and the optical observation of the internal structure of the multi-dimensional antenna brain by utilizing the combination of the quartz sand with the smaller size than that of the antenna and 2 kinds of quartz sand, and the operation is simple;
(4) the invention utilizes the antenna fixing device, after selecting the quartz sand with proper size, can be applied to the fixation and observation of the postures of other micro biological samples with three-dimensional structures (such as insect brains or feet), and obtains the multi-dimensional information of the external surface or internal structure of the sample;
(5) the method has the advantages of low cost, reliable method, simple and easy operation and good repeatability.
Drawings and description of the drawings
FIG. 1 is a technical roadmap for the present invention;
FIG. 2 is a schematic view of the multi-dimensional insect antenna attitude fixture of the present invention, wherein 1-small well device, 2-quartz sand;
FIG. 3 is a visual diagram of the guava fruit fly antennal nerve fiber bundles and the brain projections thereof obtained by the present invention, arrows indicate the nerve fiber bundles,
fig. 4 is a structural view of nerve fiber bundles at various angles of the commiphora guava fruit fly antennal flagellum obtained by the present invention, fig. 4A and 4B are views from the outside of the antennal, fig. 4C is a view from the inside of the antennal, fig. 4D is a view from the back side of the antennal, and arrows indicate nerve fiber bundles in the antennal.
Detailed Description
The present invention will be described in further detail with reference to specific embodiments, but the scope of the present invention is not limited to the description.
Example 1: the tentacle of the guava fruit fly is in a awn shape and is divided into a handle section, a stalk section and a whip section, the total length is about 860-1000 mu m, the maximum width is about 170 mu m, the minimum width is about 100 mu m, the shapes and the sizes of the sections are different, the whole guava fruit fly is a sensing organ which is small in size, is in an irregular geometric shape and integrates multiple functions of smell sense, hearing sense, temperature and humidity sense, gravity sense and the like;
a method for tracing nerve fiber bundles in the tentacles of the fruit flies of guava (see figure 1) comprises the following specific steps:
(1) tentacle nickel chloride dip dyeing: selecting a sample adding gun head of a 200-mu-L microsyringe according to the size of the fruit flies of guavas, cutting off the tail end of the gun head, freezing and anesthetizing the fruit flies of guavas, putting the fruit flies of guavas into the tail end of the gun head, pushing the fruit flies to the tip of the gun head through micro force, fixing the head of an insect by the tip of the gun head, exposing an antenna, and fixing the gun head in plasticine or dental wax on a glass slide; carefully cutting a notch at the tip of the tentacle flagellum by using an ophthalmic scissors under a dissecting mirror; meanwhile, at the other end of the same glass slide, a capillary glass tube containing nickel chloride aqueous solution with the mass concentration of 3% is fixed in another piece of plasticine or dental wax, and the capillary glass tube containing nickel chloride aqueous solution is carefully sleeved on the contact angle incision, so that the incision is immersed in the solution and is kept smooth; then, the glass slide is moved into a glass culture dish with a wet filter paper strip, the humidity is kept between 50 and 70 percent, and the glass slide is placed for 2 hours at room temperature;
(2) in vivo antennal and brain microdisolation: pouring 50mL of Ringer solution into a 100mL beaker, burying the beaker in ice and cooling to obtain ice-cold Ringer solution, wherein the ice-cold Ringer solution is continuously filled with a mixed gas of 95% oxygen and 5% carbon dioxide; dripping aerated ice-cold Ringer solution on a glass slide, then rapidly shearing the head of the guava fruit fly with an tentacle by an ophthalmic scissors, putting the head into the Ringer solution on the glass slide, rapidly and completely separating the brain with the tentacle from the head capsule within 3-5 minutes by using a pointed forceps and the ophthalmic scissors under a body type microscope, removing the trachea and the air sac on the surface of the brain by using the forceps, cutting off the part of the pedicle cutin layer at the position connected with the flagellum by using the sharp triangular tip of a 1mL syringe needle, and then cleaning for 3 times by using the Ringer solution;
(3) color development: dripping 2 drops of a red amino acid solution on a clean antenna and a clean brain, and observing the color development condition at any time under a stereoscopic microscope; once the tentacles and nerve tracts in the brain are marked, quickly absorbing and removing the solution of the erythrosine with a dry filter paper strip, and cleaning for 3 times with 100% alcohol to remove redundant erythrosine inside and outside tissues and prevent excessive color development;
(4) fixing: gently transferring the developed tissue to a clean PCR tube by using a sharp-pointed forceps, sequentially dripping 2-4 drops of formaldehyde solutions with the mass concentration of 5% and the mass concentration of 10%, respectively fixing the formaldehyde solutions with different concentrations for 10min, and absorbing and removing the formaldehyde solution by using a dry filter paper strip after each fixation;
(5) and (3) dehydrating: gently transferring the fixed feelers and brains of the guava fruit flies into another clean PCR tube by using pointed forceps, and dehydrating the fixed feelers and brains of the guava fruit flies by using 70%, 80%, 90% and 100% of alcohol in mass concentration for 5 minutes respectively;
(6) and (3) tissue transparency: immersing the fixed guava fruit fly tentacles and brains into methyl salicylate, and standing for 1-12h at room temperature to ensure that the tentacle exoskeleton cuticles, tentacle internal tissues and brains are transparent;
(7) adjusting and fixing the antenna attitude: transferring the treated insect tentacles and brains to quartz sand in a small well device of a posture fixing device under a body type microscope, dripping low-viscosity high-transmittance liquid chemical substances capable of permanently storing the tentacles into the small well device, carefully moving the tentacles and the quartz sand by using a pointed forceps, and performing posture adjustment and fixation on the tentacles from different directions and angles such as the back side, the ventral side, the outer side, the inner side, the top end and the like according to needs;
the posture fixing device is shown in figure 2, quartz sand 2 is arranged in a small well device 1, the grain diameter of the quartz sand is 25 micrometers (500 meshes) and 38 micrometers (400 meshes), and the mass ratio of the 500-mesh quartz sand to the 400-mesh quartz sand is 1: 2;
(8) and (4) microscopic photographing: placing the fixed-posture antennal and brain under a microscope to obtain image information of nerve fiber bundles in the antennal under different magnifications, adjusting and fixing the posture of the antennal according to needs to obtain image information of a required angle, and referring to fig. 3 and 4; in fig. 3, the arrows indicate the nerve fiber bundles in the tentacles and the paths of the nerve fiber bundles projected into the brain; in fig. 4, white arrows indicate labeled intraflagellar nerve fiber bundles, a and B are numbers, structures and distribution patterns of the intratrochanteric nerve fiber bundles observed from the outside, C is numbers, structures and distribution patterns of the intratrochanteric nerve fiber bundles observed from the inside, and D is numbers, structures and distribution patterns of the intraflagellar nerve fiber bundles observed from the back side.
Example 2: the citrus fruit fly tentacle is divided into a stalk section, a stalk section and a whip section, the total length is about 1030 mu m, wherein the stalk section is about 190 mu m in length, about 120 mu m in width, about 230 mu m in length, about 90-170 mu m in width, about 630 mu m in length, about 150 mu 178 in width, the middle part is slightly thicker, the widest part is about 180 mu m at the base of the whip section, the narrowest part is about 90 mu m at the base of the stalk section, the sizes and the shapes of the sections are different, and the whole body presents an irregular geometric shape;
a method for tracing nerve fiber bundles in the tentacles of bactrocera dorsalis comprises the following specific steps:
(1) tentacle cobalt chloride dip dyeing: selecting a sample adding gun head of a micro sample adding device of 200 mu L according to the size of the bactrocera dorsalis, cutting off the tail end of the gun head, freezing and anaesthetizing the bactrocera dorsalis, placing the bactrocera dorsalis at the tail end of the gun head, pushing the tail end of the gun head by micro force to fix the head of the insect by the tip end of the gun head and expose an antenna, and fixing the gun head in plasticine or dental wax on a glass slide; carefully cutting a notch at the tip of the tentacle flagellum by using an ophthalmic scissors under a dissecting mirror; meanwhile, at the other end of the same glass slide, a glass electrode containing 6% cobalt chloride aqueous solution is fixed in another piece of plasticine or dental wax, and the glass electrode containing nickel chloride aqueous solution is carefully sleeved on the incision of the antenna, so that the incision is immersed in the solution and is kept smooth; then, the glass slide is moved into a glass culture dish with a wet filter paper strip, the humidity is kept between 50 and 70 percent, and the glass slide is placed for 2.5 hours at room temperature;
(2) micro-dissection of the antennal and brain: pouring 50mL of Ringer solution into a 100mL beaker, placing the beaker in ice for cooling to obtain ice-cold Ringer solution, continuously filling the ice-cold Ringer solution with a mixed gas of 95% oxygen and 5% carbon dioxide, and dripping the aerated Ringer solution on a glass slide; the head of the citrus fruit fly is cut off rapidly by an ophthalmic scissors, the citrus fruit fly is put into Ringer's solution on a glass slide, the brain with the tentacles is rapidly and completely stripped from the head capsule by using a sharp-pointed forceps and the ophthalmic scissors, the complete structure of the sensory pathway of the tentacles is ensured, and the trachea and the air sac on the surface of the brain are removed. Cutting off the part of the stalk section cuticle which is sleeved on the base of the flagellum and protrudes by using a sharp triangular tip of a 1ml syringe needle so as to help the color development of nerve micro-beams in the tentacles; cleaning the tentacles and the brain for 3 times by using Ringer solution, wherein the whole process is completed within 3-5 min;
(3) color development: dripping 2-3 drops of a red amino acid solution on a clean antenna and a clean brain, and observing the color development condition at any time under a stereoscopic microscope; once the tentacles and nerve tracts in the brain are marked, quickly absorbing and removing the solution of the erythrosine with a dry filter paper strip, and cleaning for 3 times with 100% alcohol to remove redundant erythrosine inside and outside tissues and prevent excessive color development;
(4) fixing: gently transferring the developed tissue to a clean PCR tube by using a sharp-pointed forceps, sequentially dripping 2-4 drops of formaldehyde solutions with mass concentrations of 5% and 10%, respectively fixing the formaldehyde solutions with different concentrations for 10min, and absorbing the formaldehyde solution by using a dry filter paper strip after each fixation;
(5) and (3) dehydrating: gently transferring the fixed tentacles and brains of the bactrocera dorsalis to another clean PCR tube by using a pointed-end forceps, and dehydrating by using 70%, 80%, 90% and 100% of alcohol by mass concentration for 5 minutes respectively;
(6) and (3) tissue transparency: and (3) immersing the fixed tentacles and brains of the bactrocera dorsalis into methyl salicylate, and standing at room temperature for 1-12h to make the horny exoskeleton, internal tissues and brains of the tentacles transparent.
(7) Fixing the posture of the antenna: transferring the treated insect tentacles and brains to quartz sand in a small well device of a posture fixing device under a body type microscope, dripping low-viscosity high-transmittance liquid chemical substances capable of permanently storing the tentacles into the small well device, carefully moving the tentacles and the quartz sand by using a pointed forceps, and performing posture adjustment and fixation on the tentacles from different directions and angles such as the back side, the ventral side, the outer side, the inner side, the top end and the like according to needs;
the posture fixing device is shown in fig. 2, quartz sand 2 is arranged in a small well device 1, the grain diameter of the quartz sand is 500 meshes and 400 meshes, and the mass ratio of the 500-mesh quartz sand to the 400-mesh quartz sand is 1: 2;
(8) and (4) microscopic photographing: and placing the fixed-posture antennal and brain under a microscope to obtain image information of nerve fiber bundles in the antennal under different magnifications, adjusting and fixing the posture of the antennal according to needs, and obtaining the image information of a required angle.
The invention takes nickel chloride or cobalt chloride as a nerve tracer, controls the amount of nickel ions or cobalt ions entering nerves and the backward transmission distance by controlling the tracer staining time, ensures the activity of insects in the staining process and is helpful for the specific staining of the tracer, namely, the nickel ions or cobalt ions only travel backwards along axons (nerve fiber bundles) and can be accumulated in the traveling path, namely, the method is one of the key steps of the tracing and determines the obvious color result. The humidity of 50-70% is provided in the dip dyeing process, so that the evaporation of the tracer aqueous solution can be reduced, the osmotic pressure of the tracer aqueous solution can be maintained, and the influence of the tracer solution on nerve cells can be reduced; in the microdissection process, mixed gas is filled into the Ringer liquid and the temperature is kept low, so that the good activity of tissues and cells of the tentacles and the brain is ensured, and the tracer is prevented from diffusing into other non-nerve tissues when the activity of the cells is reduced or the cells die to influence the subsequent color development effect; the tryptophan can form a dark purple complex with nickel ions or cobalt ions, so that the place where the nickel ions or the cobalt ions are accumulated is dyed into dark purple, and then the nerve fiber bundles inside the tentacles are dyed into dark purple, the control of the color development duration is vital in the color development process, the color development is stopped immediately after the nerve fiber bundles inside the tentacles are dyed, the solution of the tryptophan is removed, and the solution is washed by alcohol, so that excessive color development is prevented, and the method is one of the key steps of the tracing effect; fixing the developed antennae and brain with 5% and 10% formaldehyde solutions respectively; dehydrating with 70%, 80%, 90% and 100% gradient alcohol; methyl salicylate is a colorless transparent liquid, has a high refractive index, and is generally used for the transparency of soft tissues; however, since the horny layer of an insect antenna is thick, the light transmittance is low, and the visualization of the internal structure of the antenna is hindered.
Through a large number of experiments, methyl salicylate can be used for transparent tentacle light-colored cuticle exoskeleton to a certain extent. Therefore, the adoption of methyl salicylate realizes the transparentization of the exoskeleton cuticle, the internal tissues and the brain quilt of the light-colored tentacles of the insects; the tentacle or brain with a three-dimensional structure is subjected to multi-dimensional posture adjustment and fixation, namely a key link for deeply acquiring nerve fiber bundle image information in the tentacle, the characteristics of three-dimensional irregularity, light transmission and quartz sand proportion of quartz sand with different sizes and sizes compared with an insect tentacle are utilized to combine and manufacture fulcrum curved surfaces with different heights, the random adjustment and fixation of the tentacle posture are realized, meanwhile, the application of low-viscosity high-light-transmittance methyl salicylate plays the triple roles of auxiliary support for tentacle posture fixation, smooth light path and tentacle preservation, the problems of fixation and long-term preservation of the insect micro tentacle are thoroughly solved, and the last obstacle of the visualization link of the internal structure of the tentacle is thoroughly eliminated.
Claims (7)
1. A method for tracing nerve fiber bundles in light-colored antennae of insects is characterized by comprising the following specific steps:
(1) dip-dyeing the living insect antennae by using a nickel chloride solution or a cobalt chloride solution; using a sample adding gun head of a microsyringe, cutting off the tail end of the gun head, freezing and anaesthetizing the insects, putting the insects into the tail end of the gun head, pushing the insects to the tip of the gun head by micro force, fixing the heads of the insects by the tip of the gun head, exposing an antenna, and fixing the gun head in plasticine or dental wax on a glass slide; carefully cutting a notch at the tip of the tentacle flagellum by using an ophthalmic scissors under a dissecting mirror; meanwhile, fixing a capillary glass tube or a glass electrode containing a nickel chloride solution or a cobalt chloride aqueous solution in another piece of plasticine or dental wax at the other end of the same glass slide, and carefully sleeving the capillary glass tube containing the nickel chloride or the cobalt chloride aqueous solution on the contact incision to ensure that the incision is immersed in the solution and is kept smooth; then, the glass slide is moved into a glass culture dish with a wet filter paper strip, the humidity is kept between 50 and 70 percent, and the glass slide is placed for 2 to 48 hours at room temperature;
(2) microscopically separating intact tentacles and brains of living insects; pouring 50mL of Ringer solution into a 100mL beaker, burying the beaker in ice and cooling to obtain ice-cold Ringer solution, wherein the ice-cold Ringer solution is continuously filled with a mixed gas of 95% oxygen and 5% carbon dioxide; dropping aerated ice-cold Ringer solution on a glass slide, then rapidly shearing the head of an insect with an antenna by using an ophthalmic scissors, putting the insect with the antenna into the Ringer solution on the glass slide, rapidly and completely separating the brain with the antenna from the hair follicle within 3-5 minutes by using a pointed-end tweezers and the ophthalmic scissors under a body type microscope, removing the trachea and the air sac on the surface of the brain by using the tweezers, cutting off the part of the pedicle cutin layer at the connection part with the whip by using the sharp triangular tip of a 1mL syringe needle, and then cleaning for 3 times by using the Ringer solution;
(3) tissue color development: dripping a solution of the rhodomine on the insect tentacles and brains obtained in the step (2), and observing the color development condition at any time under a stereoscopic microscope; once the tentacles and nerve tracts in the brain are marked, quickly absorbing and removing the solution of the erythrosine with a dry filter paper strip, and cleaning for 3 times with 100% alcohol to remove redundant erythrosine inside and outside tissues and prevent excessive color development;
(4) tissue fixation: gently transferring the colored insect tentacles and brains obtained in the step (3) into a clean PCR tube by using a pointed-end forceps, sequentially dripping 2-4 drops of 5% and 10% formaldehyde solutions, fixing for 10 minutes respectively, and finally absorbing and removing the formaldehyde solutions by using a dry filter paper strip;
(5) tissue dehydration: gently transferring the insect tentacles and brains fixed in the step (4) into another clean PCR tube by using a pointed-end forceps, and dehydrating by using 70%, 80%, 90% and 100% of alcohol in sequence for 5 minutes;
(6) and (3) tissue transparency: immersing the dehydrated insect tentacles and brains obtained in the step (5) into methyl salicylate, and standing at room temperature for 1-12h to ensure that the horny exoskeleton, internal tissues and brains of the tentacles are transparentized;
(7) posture adjustment and fixation: adjusting and fixing the postures of the insect antennae and the brain processed in the step (6) by using pointed tweezers in a small well device under a body type microscope;
(8) and (4) microscopic photographing: and (5) placing the tentacles and the brains subjected to the posture adjustment and fixation in the step (7) under a microscope to obtain image information of nerve fiber bundles in the tentacles under different magnifications, and adjusting and fixing the postures of the tentacles according to needs to obtain image information of required angles.
2. The method of tracing nerve fiber bundles in the light-colored antennal of an insect of claim 1, wherein: the specific operation method for adjusting and fixing the postures of the insect antennae and the insect brains in the step (7) comprises the following steps: transferring the insect tentacles and brains treated in the step (6) to quartz sand in a small well device of a posture fixing device, dripping low-viscosity high-transmittance methyl salicylate into the small well device, carefully moving the tentacles and the quartz sand by using a pointed forceps, and performing posture adjustment and fixation on the tentacles from different directions and angles such as the back side, the abdominal side, the outer side, the inner side and the top end according to needs.
3. The method of tracing nerve fiber bundles in the light-colored antennal of an insect of claim 1, wherein: in the step (1), the mass concentration of the nickel chloride solution or the cobalt chloride solution is 1-10%, the length of the insect antenna is n millimeters, and the dip-dyeing time is n-n +5 hours.
4. The method of tracing nerve fiber bundles in the light-colored antennal of an insect of claim 1, wherein: the volume of the sample injection gun head is 10 mul, 200 mul or 1000 mul.
5. The method of tracing nerve fiber bundles in the light-colored antennal of an insect of claim 1, wherein: the caliber of the tip of the capillary glass tube or the glass electrode is 1-2.5 times of the width of the insect contact angle.
6. The method of tracing nerve fiber bundles in the light-colored antennal of an insect of claim 1, wherein: the height of the small well device in the step (7) is not more than 5mm, and quartz sand is arranged in the small well device.
7. The method of tracing nerve fiber bundles in the light-colored antennae of insects of claim 6, wherein: the quartz sand has two different sizes, the particle size of the quartz sand is 1/5-1/2 of the minimum size of a sample, and the mass ratio of the two types of particle size quartz sand is 1 (1-3).
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