Fixator for two-photon recording

Abstract

A fixator for two-photon recording, comprising a body (1), two first connecting members (2) and two second connecting members (3). The body (1) has a mounting hole (101), which is arranged corresponding to the dorsal root ganglions (100) of the sacral segment of a mouse and is configured for mounting a two-photon lens (200); two first grooves (102), which are arranged corresponding to the bilateral posterior superior iliac spine (300) of the mouse and are configured for embedding the bilateral posterior superior iliac spine (300); two first connecting holes (103), which are arranged corresponding to the ilium (400) of the mouse; and two second connecting holes (104), which are arranged corresponding to the sacrum (500) of the mouse. The two first connecting members (2) are inserted into the two first connecting holes (103) to connect the body (1) to the ilium (400). The two second connecting members (3) are inserted into the two second connecting holes (104) to connect the body (1) to the sacrum (500).

Description

Holder for two-photon recording Technical Field

[0001] The present application relates to the technical field of in vivo two-photon calcium imaging, and in particular to a holder for two-photon recording. Background Art

[0002] Studying neuronal activity in the brain and spinal cord is an important method for revealing neural connections and neuronal function. In vivo two-photon calcium imaging is widely used in central nervous system imaging due to its advantages, including small lens size, stable signal, clear imaging, and the ability to record freely in vivo. The dorsal root ganglion (DRG) is the first stop for external sensations entering the central nervous system and has the ability to recognize, encode, and transmit peripheral signals. However, due to the lack of a fixture that can stably expose the sacral DRG and support the two-photon lens, recording calcium imaging signals from DRG neurons in the sacral DRG can only be performed under anesthesia.

[0003] The current method for recording neurons in this segment is to use two or more clamps to clamp the lumbar and caudal vertebrae of anesthetized mice to expose and stabilize the sacral segment in the middle. Although this method can achieve the purpose of recording, it is necessary to cut open the entire lumbar and caudal back skin and muscle tissue of the mouse to expose the sacral dorsal root ganglia and lumbar and caudal vertebrae during the specific implementation process. Due to the dissection and exposure of a large amount of tissue during the experiment, the wound of the mouse is difficult to recover after recording, and repeated recording cannot be achieved. In addition, during the recording process of the above method, the mouse is in an anesthetized and suspended state, which may cause differences in the response of the neurons to external stimuli, resulting in inaccurate research results.

[0004] It should be noted that the above introduction to the background technology is merely for the purpose of providing a clear and complete description of the technical solutions of this application and facilitating understanding by those skilled in the art. Simply because these solutions are described in the background technology section of this application, it should not be assumed that the above technical solutions are well known to those skilled in the art.

[0005] Summary of the Invention

[0006] The purpose of the present application is to provide a holder for two-photon recording to solve at least part of the problems pointed out in the above background technology or other similar problems.

[0007] An embodiment of the first aspect of the present application provides a fixator for two-photon recording, comprising: a main body, provided with a mounting hole corresponding to the dorsal root ganglion of the sacral segment of a mouse and for mounting a two-photon lens, two first grooves corresponding to the bilateral posterior iliac spines of the mouse and for the bilateral posterior iliac spines to be embedded, two first connecting holes corresponding to the ilium of the mouse, and two second connecting holes corresponding to the sacrum of the mouse; two first connecting members, which connect the main body to the ilium by being inserted into the two first connecting holes; and two second connecting members, which connect the main body to the sacrum by being inserted into the two second connecting holes.

[0008] In some embodiments, the body has a lower surface and an upper surface arranged opposite to each other in the thickness direction, a front side and a rear side arranged opposite to each other in the length direction, and a left side and a right side arranged opposite to each other in the width direction; the mounting hole extends from the lower surface to the upper surface along the thickness direction; the two first grooves are respectively located on opposite sides of the mounting hole in the width direction, and are both recessed from the lower surface toward the upper surface and extend along the length direction; the two first connecting holes are both arranged adjacent to the front side, one of the first connecting holes extends from the left side along the width direction and passes through one of the first grooves, and the other first connecting hole extends from the right side along the width direction and passes through the other first groove; the two second connecting holes are both arranged adjacent to the rear side, and are respectively located between the rear side and the two first grooves in the length direction, the second connecting holes are respectively located on opposite sides of the mounting hole in the width direction, and both extend from the lower surface to the upper surface along the thickness direction.

[0009] In some embodiments, the first groove extends from the front side along the length direction to communicate with the mounting hole, and the cross-section of the first groove in the width direction is triangular.

[0010] In some embodiments, the main body is further provided with a first notch, the first notch is located on the front side of the mounting hole and between the two first grooves, the first notch extends from the front side along the length direction to connect with the mounting hole, and the first notch passes through from the lower surface to the upper surface along the thickness direction.

[0011] In some embodiments, the main body is further provided with a second groove, which is located on the rear side of the mounting hole and between the two second connecting holes. The second groove is recessed from the lower surface toward the upper surface and extends from the rear side along the length direction to communicate with the mounting hole; or, the main body is further provided with a second notch, which is located on the rear side of the mounting hole and between the two second connecting holes. The second notch extends from the lower surface to the upper surface and extends from the rear side along the length direction to communicate with the mounting hole. The second notch, the mounting hole and the first notch separate the main body into two parts that are not connected to each other.

[0012] In some embodiments, the first connecting member is a steel nail, the second connecting member is a screw, and the second connecting hole is a screw hole.

[0013] An embodiment of the second aspect of the present application provides a fixture for two-photon recording, comprising: a body having a lower surface and an upper surface arranged opposite to each other in the thickness direction, a front side surface and a rear side surface arranged opposite to each other in the length direction, and a left side surface and a right side surface arranged opposite to each other in the width direction; a mounting hole extending from the lower surface to the upper surface along the thickness direction; two first grooves, respectively located on opposite sides of the mounting hole in the width direction, and both are recessed from the lower surface toward the upper surface and both extend along the length direction; two first connecting holes, both arranged adjacent to the front side surface, one of the first connecting holes extending from the left side surface along the width direction and passing through one of the first grooves, and the other first connecting hole extending from the right side surface along the width direction and passing through the other first groove; two second connecting holes, both arranged adjacent to the rear side surface, and respectively located between the rear side surface and the two first grooves in the length direction, the second connecting holes respectively located on opposite sides of the mounting hole in the width direction, and both extend from the lower surface to the upper surface along the thickness direction; two first connecting members inserted into the two first connecting holes; and two second connecting members inserted into the two second connecting holes.

[0014] In some embodiments, each of the first grooves extends from the front side surface along the length direction to communicate with the mounting hole, and a cross-section of the first groove in the width direction is triangular.

[0015] In some embodiments, the main body is further provided with a first notch, the first notch is located on the front side of the mounting hole and between the two first grooves, the first notch extends from the front side along the length direction to connect with the mounting hole, and the first notch passes through from the lower surface to the upper surface along the thickness direction.

[0016] In some embodiments, the main body is further provided with a second groove, which is located on the rear side of the mounting hole and between the two second connecting holes. The second groove is recessed from the lower surface toward the upper surface and extends from the rear side along the length direction to communicate with the mounting hole; or, the main body is further provided with a second notch, which is located on the rear side of the mounting hole and between the two second connecting holes. The second notch extends from the lower surface to the upper surface and extends from the rear side along the length direction to communicate with the mounting hole. The second notch, the mounting hole and the first notch separate the main body into two parts that are not connected to each other.

[0017] The beneficial effects of the embodiments of the present application include:

[0018] The fixator for two-photon recording described in this application can be implanted in the ilium and sacrum of a mouse. The implantation procedure requires only opening the skin and muscles on the surface of the mouse's sacrum. Once the fixator is installed, the mouse can be returned to its cage for recovery, fully exposing the dorsal root ganglia while minimizing animal pain. This allows for stable imaging of sacral dorsal root ganglion neurons while the mouse is fully awake and freely moving. The fixator described in this application is fixed to the mouse's ilium and sacrum using bone nails and screws, making the implantation procedure simple and the installation secure. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present application. For those skilled in the art, other drawings can be obtained based on these drawings without creative work. In the drawings:

[0020] FIG1 is a schematic diagram of the three-dimensional structure of a holder for two-photon recording according to an embodiment of the present application;

[0021] FIG2 is a bottom view of the fastener of FIG1 ;

[0022] FIG3 is a schematic diagram of the three-dimensional structure of a holder for two-photon recording according to another embodiment of the present application;

[0023] FIG4 is a schematic diagram of a mouse implanted with the holder for two-photon recording of the present application;

[0024] 5 and 6 are schematic diagrams of a fixator for two-photon recording implanted on the ilium and sacrum of a mouse according to an embodiment of the present application.

[0025] Description of the component numbers in the accompanying drawings:

[0026] 1. Main body; 101. Mounting hole; 102. First groove; 103. First connecting hole; 104. Second connecting hole;

[0027] 105, lower surface; 106, upper surface; 107, rear side; 108, front side; 109, left side; 110, right side;

[0028] 111, second groove; 112, first notch; 2, first connecting member; 3, second connecting member;

[0029] 100, sacral dorsal root ganglion; 200, two-photon lens; 300, posterior iliac spine; 400, ilium; 500, sacrum. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical solutions and advantages of the embodiments of the present application more clear, the embodiments of the present application are further described in detail below with reference to the accompanying drawings. Here, the illustrative embodiments of the present application and their descriptions are used to explain the present application, but are not intended to limit the present application.

[0031] In the embodiments of the present application, the terms "first", "second", etc. are used to distinguish different elements in terms of name, but do not indicate the spatial arrangement or temporal order of these elements, and these elements should not be limited by these terms. The term "and / or" includes any one and all combinations of one or more of the associated listed terms. The terms "comprising", "including", "having", etc. refer to the presence of the stated features, elements, components or components, but do not exclude the presence or addition of one or more other features, elements, components or components.

[0032] In the embodiments of the present application, the singular forms "a", "the", etc. may include plural forms and should be broadly understood as "a" or "a type" rather than being limited to the meaning of "one"; in addition, the term "said" should be understood to include both singular and plural forms, unless the context clearly indicates otherwise; in addition, the term "according to" should be understood as "at least in part according to...", and the term "based on" should be understood as "at least in part based on...", unless the context clearly indicates otherwise; in addition, the term "plurality" means two or more, unless otherwise specified.

[0033] The adjective or adverbial modifiers "upper" and "lower", "front" and "back", "left" and "right" used in this document are used only to facilitate relative reference between multiple groups of terms and do not describe any specific directional limitation of the modified terms.

[0034] The following describes the implementation of the embodiments of the present application with reference to the accompanying drawings.

[0035] As shown in FIG1 , an embodiment of the present application provides a fixture for two-photon recording, which includes a main body 1 , two first connecting members 2 and two second connecting members 3 .

[0036] As shown in Figures 1, 5, and 6, the body 1 is provided with a mounting hole 101 corresponding to the mouse's sacral dorsal root ganglion 100 and for mounting the two-photon lens 200; two first grooves 102 corresponding to the mouse's bilateral iliac spines 300 and for receiving the bilateral iliac spines 300; two first connection holes 103 corresponding to the mouse's ilium 400; and two second connection holes 104 corresponding to the mouse's sacrum 500. The two first connectors 2 are inserted into the two first connection holes 103 to connect the body 1 to the ilium 400, and the two second connectors 3 are inserted into the two second connection holes 104 to connect the body 1 to the sacrum 500.

[0037] The implantation method of the fixator of this application is as follows:

[0038] First, the skin, muscles, and protruding vertebral spinous processes on the surface of the mouse's sacrum 500 are surgically opened to expose the mouse's sacrum 500;

[0039] Then, the body is placed on the surface of the mouse's sacrum 500, so that the two first grooves 102 of the body are stuck in the mouse's bilateral iliac spines 300, the two first connection holes 103 of the body 1 correspond to the mouse's ilium 400, and the two second connection holes 104 of the body 1 correspond to the mouse's sacrum 500;

[0040] Subsequently, a first connecting member 2 is inserted between a first connecting hole 103 and the ilium 400, and another first connecting member 2 is inserted between another first connecting hole 103 and the ilium 400, so as to connect the body 1 to the ilium 400; and a second connecting member 3 is inserted between a second connecting hole 104 and the sacrum 500, and another second connecting member 3 is inserted between another second connecting hole 104 and the sacrum 500, so as to connect the body 1 to the sacrum 500;

[0041] Afterwards, the two-photon lens 200 is mounted on the mounting hole 101 to record neuronal calcium signals in the sacral dorsal root ganglion 100 when the mouse is awake and moving freely.

[0042] In the example of Figures 1 and 2, the body 1 has a lower surface 105 and an upper surface 106 arranged opposite to each other in its thickness direction H, a front side surface 108 and a rear side surface 107 arranged opposite to each other in its length direction L, and a left side surface 109 and a right side surface 110 arranged opposite to each other in its width direction W.

[0043] As shown in FIG. 1 , the mounting hole 101 extends from the lower surface 105 to the upper surface 106 along the thickness direction H. In other words, the mounting hole 101 penetrates the body 1 along the thickness direction H to form a through hole.

[0044] As shown in Figure 1, the two first grooves 102 are located on opposite sides of the mounting hole 101 in the width direction W, and are both recessed from the lower surface 105 toward the upper surface 106 and extend along the length direction L. Specifically, the first groove 102 extends a preset length from the front side 108 toward the rear side 107. The front end of the first groove 102 passes through the front side 108 of the main body 1, and the rear end of the first groove 102 is connected to the mounting hole 101, so that the first groove 102 can clamp the bilateral iliac posterior spines 300, or in other words, it is convenient for the bilateral iliac posterior spines 300 to be embedded in the first groove 102, thereby preventing the main body 1 from moving left and right relative to the sacral dorsal root ganglion 100 of the mouse.

[0045] As shown in FIG. 1 , the cross-section of the first groove 102 in the width direction W is triangular to match the shape of the posterior iliac spine 300 .

[0046] As shown in FIG1 , the two first connection holes 103 are both arranged adjacent to the front side 108. The left first connection hole 103 extends from the left side 109 along the width direction W and passes through the left first groove 102. The right first connection hole 103 extends from the right side 110 along the width direction W and passes through the right first groove 102. Therefore, the left first connector 2 can be inserted from the left side 109 of the body 1 into the left first connection hole 103 and pass through the posterior iliac spine 300 in the left first groove 102. The right first connector 2 can be inserted from the right side 110 of the body 1 into the right first connection hole 103 and pass through the posterior iliac spine 300 in the right first groove 102, thereby connecting the body 1 to the ilium 400 and preventing the body 1 from moving forward and backward or up and down relative to the mouse's sacral dorsal root ganglion 100.

[0047] As shown in FIG1 , the two second connection holes 104 are disposed adjacent to the rear side surface 107 and are located between the rear side surface 107 and the two first grooves 102 in the length direction L, and on opposite sides of the mounting hole 101 in the width direction W. Each second connection hole 104 extends from the lower surface 105 to the upper surface 106 along the thickness direction H. Therefore, the two second connectors 3 can be respectively passed through the two second connection holes 104 and inserted into the sacrum 500 , thereby connecting the body 1 to the sacrum 500 and preventing the body 1 from moving forward and backward or left and right relative to the mouse's sacral dorsal root ganglion 100.

[0048] As shown in FIG1 , the body 1 further comprises a first notch 112 located in front of the mounting hole 101 and between the two first grooves 102. The first notch 112 extends along the length direction L from the front side surface 108 to communicate with the mounting hole 101, and the first notch 112 extends along the thickness direction H from the lower surface 105 to the upper surface 106. A second groove 111 is located in the rear of the mounting hole 101 and between the two second connection holes 104. The second groove 111 is recessed from the lower surface 105 toward the upper surface 106 and extends along the length direction L from the rear side surface 107 to communicate with the mounting hole 101. Therefore, the first notch 112 and the mounting hole 101 together divide the body 1 into a left portion and a right portion, connected only by the second notch 111, to avoid the protruding mouse vertebrae in the middle.

[0049] However, the present application is not limited to this. In another example, as shown in Figure 3, the second groove 111 can be replaced by a second notch 113. The difference between the second notch 113 and the second groove 111 is that the second notch 113 extends from the lower surface 105 to the upper surface 106. Therefore, the second notch 113, the first notch 112 and the mounting hole 101 together separate the main body 1 into a left part and a right part that are not connected to each other. The two parts can be detachably connected by an additional connecting member (such as a bolt), and the middle spine of the mouse can be avoided. The distance between the two parts can also be adjusted to accommodate mice of different sizes.

[0050] In the example of FIG1 , the first connecting member 2 is a steel nail (ie, a bone nail), the second connecting member 3 is a screw, and the second connecting hole 104 is a screw hole.

[0051] Optionally, the bone nail may be a stainless steel nail with a length of 4 mm, and the screw may be a stainless steel screw with a length of 3 mm.

[0052] Optionally, the body 1 may be made of stainless steel.

[0053] When making the fixator of the present application, a digital model of the mouse pelvis can be established through anatomical measurement and computer simulation, and then the fixator can be made by combining data modeling and CNC precision processing technology.

[0054] When using the existing clamp, it is necessary to cut the entire lumbar and sacral dorsal skin and muscle tissue to expose the sacral dorsal root ganglia and lumbar and coccygeal vertebrae. However, when using the fixator of the present application, it is only necessary to open the skin and muscles on the surface of the mouse's sacrum. After the fixator is installed, the mouse can be returned to the cage to recover, fully exposing the dorsal root ganglia while minimizing the animal's pain. The mouse suffers less trauma and recovers faster.

[0055] The fixator of the present application is fixed to the ilium and sacrum of the mouse by bone nails and screws. The implantation operation is simple and the installation is firm.

[0056] As shown in Figure 4, mice equipped with a fixture and two-photon lens were able to repeatedly record calcium signals from neurons in the sacral dorsal root ganglion and achieve stable imaging of these neurons. Because the mice were fully awake and freely moving during the recording process, calcium signals from these neurons were accurately recorded in real time.

[0057] The specific embodiments described above further illustrate the purpose, technical solutions and beneficial effects of the present application. It should be understood that the above description is only a specific embodiment of the present application and is not intended to limit the scope of protection of the present application. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present application should be included in the scope of protection of the present application.

Claims

1. A holder for two-photon recording, comprising: The body is provided with a mounting hole corresponding to the dorsal root ganglion of the sacral segment of the mouse and used to mount the two-photon lens, two first grooves corresponding to the bilateral iliac posterior spines of the mouse and for the bilateral iliac posterior spines to be embedded, two first connection holes corresponding to the ilium of the mouse, and two second connection holes corresponding to the sacrum of the mouse; Two first connecting members, connected to the body and the ilium by being inserted into the two first connecting holes; Two second connecting members are inserted into the two second connecting holes to connect the body to the sacrum.

2. The fastener according to claim 1, wherein: The body has a lower surface and an upper surface arranged oppositely in the thickness direction thereof, a front side surface and a rear side surface arranged oppositely in the length direction thereof, and a left side surface and a right side surface arranged oppositely in the width direction thereof; The mounting hole extends from the lower surface to the upper surface along the thickness direction; The two first grooves are respectively located at opposite sides of the mounting hole in the width direction, and are both recessed from the lower surface toward the upper surface and extend along the length direction; Two first connection holes are both arranged adjacent to the front side surface, wherein one of the first connection holes extends from the left side surface along the width direction and passes through one of the first grooves, and the other first connection hole extends from the right side surface along the width direction and passes through the other first groove; The two second connection holes are both arranged adjacent to the rear side surface and are respectively located between the rear side surface and the two first grooves in the length direction. The two second connection holes are respectively located on opposite sides of the mounting hole in the width direction and extend from the lower surface to the upper surface along the thickness direction.

3. The fastener according to claim 2, wherein: The first groove extends from the front side surface along the length direction to communicate with the mounting hole, and the cross-section of the first groove in the width direction is a triangle.

4. The fastener according to claim 2, wherein: The main body is also provided with a first notch, which is located on the front side of the mounting hole and between the two first grooves. The first notch extends from the front side along the length direction to communicate with the mounting hole, and the first notch passes through from the lower surface to the upper surface along the thickness direction.

5. The fastener according to claim 4, wherein: The body is further provided with a second groove, the second groove is located at the rear side of the mounting hole and between the two second connection holes, the second groove is recessed from the lower surface toward the upper surface, and extends from the rear side surface along the length direction to communicate with the mounting hole; or, The body is also provided with a second notch, which is located at the rear side of the mounting hole and between the two second connecting holes. The second notch extends from the lower surface to the upper surface and extends from the rear side surface along the length direction to communicate with the mounting hole. The second notch, the mounting hole and the first notch separate the body into two parts that are not connected to each other.

6. The fastener according to claim 1, wherein: The first connecting member is a steel nail, the second connecting member is a screw, and the second connecting hole is a screw hole.

7. A holder for two-photon recording, wherein: include: The body has a lower surface and an upper surface arranged opposite to each other in the thickness direction, a front side surface and a rear side surface arranged opposite to each other in the length direction, and a left side surface and a right side surface arranged opposite to each other in the width direction; A mounting hole extending from the lower surface to the upper surface along the thickness direction; Two first grooves are respectively located at opposite sides of the mounting hole in the width direction, are both recessed from the lower surface toward the upper surface and extend along the length direction; Two first connection holes, both disposed adjacent to the front side surface, wherein one of the first connection holes extends from the left side surface along the width direction and passes through one of the first grooves, and the other first connection hole extends from the right side surface along the width direction and passes through the other first groove; Two second connection holes are arranged adjacent to the rear side surface and are respectively located between the rear side surface and the two first grooves in the length direction, and the second connection holes are respectively located on opposite sides of the mounting hole in the width direction and extend from the lower surface to the upper surface along the thickness direction; Two first connecting members, inserted into the two first connecting holes; Two second connecting members are inserted into the two second connecting holes.

8. The fastener according to claim 7, wherein: Each of the first grooves extends from the front side surface along the length direction to communicate with the mounting hole, and the cross-section of the first groove in the width direction is a triangle.

9. The fastener according to claim 7, wherein: The main body is also provided with a first notch, which is located on the front side of the mounting hole and between the two first grooves. The first notch extends from the front side along the length direction to communicate with the mounting hole, and the first notch passes through from the lower surface to the upper surface along the thickness direction.

10. The fastener according to claim 9, wherein: The body is further provided with a second groove, the second groove is located at the rear side of the mounting hole and between the two second connection holes, the second groove is recessed from the lower surface toward the upper surface, and extends from the rear side surface along the length direction to communicate with the mounting hole; or, The body is also provided with a second notch, which is located at the rear side of the mounting hole and between the two second connecting holes. The second notch extends from the lower surface to the upper surface and extends from the rear side surface along the length direction to communicate with the mounting hole. The second notch, the mounting hole and the first notch separate the body into two parts that are not connected to each other.

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