Method for manufacturing a neural activity measurement device

Abstract

To provide a neural activity measurement device capable of accurately and easily measuring neural activity in a spinal cord dorsal horn superficial layer, and to provide its manufacturing method and a measurement method.SOLUTION: The neural activity measurement device includes: a lead part 14 of a conductor formed on a film substrate 12; and a probe 20 consisting of a core 20b of a semiconductor crystal-grown in a direction crossing the lead part 14 at an end part of the lead part 14. The diameter of a tip end part 20a of the probe 20 is 5 μm or less, the tip end part 20a is provided with a measuring electrode 22a, and the measuring electrode 22a and the lead part 14 are electrically connected. The length from a surface of the film substrate 12 to a tip 21 of the probe 20 is the length at which the tip 20a is located in a spinal cord dorsal horn superficial layer P1 when the probe 20 is punctured into the spinal cord S of an animal being measured. The portion of the film substrate 12 where a proximal end 20c of the probe 20 is located serves as a stopper when the probe 20 is punctured into the spinal cord dorsal horn superficial layer P1. The probe 20 has a core 20b of a silicon single crystal formed by VLS growth, and is provided with a metallic thin film.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] This invention relates to a neural activity measurement device for puncturing the superficial layer of the posterior horn of the spinal cord, measuring the potential in neural activity, and recording neural activity and the like. Manufacturing method It relates thereto.

Background Art

[0002] Pain is an essential sensation for biological defense, but excessive or persistent pain (chronic pain) causes suffering to patients and significantly reduces the quality of life (QOL). In particular, since the onset mechanism of chronic pain has not yet been elucidated and effective treatment methods and drugs have not been established, its development is an urgent task. Previous studies have suggested that neurotransmission in the superficial layer of the posterior horn of the spinal cord is enhanced during chronic pain.

[0003] Pain is transmitted through nerves and via the spinal cord to the brain, and it may be possible to find a method of suppressing pain by measuring neural activity in the spinal cord where pain information is first transmitted. Therefore, in order to detect neural activity in the spinal cord, there is a method of puncturing the spinal cord with an electrode for measurement. However, conventional electrodes (such as glass electrodes and tungsten electrodes) have a high invasiveness to the spinal cord. In addition, as a means of measuring neural activity with a minimally invasive probe insertion device, as disclosed in Patent Documents 1 and 2, a probe insertion device for minimally invasively measuring neural activity in the brain has been proposed.

[0004] The probe insertion device disclosed in Patent Document 1 is inserted inside a test animal or the like that is the measurement target, and a probe is inserted into the brain, organs, etc. that are the insertion targets inside the test animal's body, and is used to acquire electrical signals including electroencephalograms. This probe insertion device includes a flexible film having a flat plate shape, a probe formed to protrude from the flexible film, and a covering that covers the entire probe. The probe is a structure supported by the flexible film and formed to protrude from the flexible film. The probe is fixed to the flexible film by a support material embedded in the flexible film and is covered by a conductor.

[0005] The probe insertion device disclosed in Patent Document 2 comprises a substrate and a minimally invasive probe formed protruding from the substrate, wherein the connector and probe may extend substantially parallel to each other, or the probe may be formed to extend substantially perpendicular to the extension direction of the connector. The substrate supporting the probe can be, for example, semiconductor silicon, and a silicon wafer cut into 1 mm x 1 mm silicon blocks can be used. The probe is a structure supported by the substrate and formed protruding from the substrate, and is covered with a conductor. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] Japanese Patent Publication No. 2019-195511 [Patent Document 2] Japanese Patent Publication No. 2020-96720 [Overview of the project] [Problems that the invention aims to solve]

[0007] Existing measurement techniques using probe insertion devices, such as those disclosed in the above-mentioned Patent Documents 1 and 2, make it extremely difficult to accurately record functional changes in neurons in the spinal cord dorsal horn (particularly the superficial layer of the spinal cord dorsal horn), which is important for pain transmission. This requires skilled techniques to accurately identify and insert the probe, and acquiring these skills takes a long time. Furthermore, it is difficult to accurately control the depth of insertion and manufacture minute probes, and probes like those disclosed in Patent Documents 1 and 2 are structurally complex and relatively large. Therefore, there has been a need for a small, easy-to-use device that can accurately puncture the superficial layer of the spinal cord dorsal horn and perform long-term in vivo measurements.

[0008] This invention was made in view of the above-mentioned prior art, and is a neural activity measurement device that can accurately and easily measure and record neural activity in the superficial layer of the spinal cord's dorsal horn. Manufacturing method The purpose is to provide. [Means for solving the problem]

[0009] This invention Neural activity measurement device The device comprises a film substrate, a conductive lead portion formed on the film substrate, and a semiconductor probe formed at the end of the lead portion, with crystal growth in a direction intersecting the lead portion. The probe has a tip diameter of 5 μm or less and is electrically connected to the lead portion. The length from the base to the tip is such that when the probe is inserted into the spinal cord of the animal being measured, the tip is located in the superficial layer of the spinal cord's dorsal horn. The film substrate on which the base end of the probe is located acts as a stopper when inserting the probe into the superficial layer of the spinal cord's dorsal horn. thing That is the case.

[0010] The lead portion and the probe having a silicon single crystal semiconductor core are integrally formed on the film substrate, and the film substrate surface the stopper The distance between the probe tip and the spinal cord is set to the depth of the superficial layer of the spinal cord's dorsal horn. It will be done It is.

[0011] The surface of the film substrate The base end of the probe has an annular shape formed by the resin coating the conductive material of the probe. The aforementioned The stopper is formed in a state where it is raised from the surface of the film substrate. It will be done .

[0012] This invention By growing a semiconductor single crystal, a probe with a tip cross-sectional diameter of 5 μm or less is formed. The surface of the probe is covered with a conductive material, A film substrate is formed at the base end of the probe in a direction intersecting the longitudinal direction of the probe, a conductive lead portion is formed on this film substrate, the lead portion and the tip portion of the probe are electrically connected, and crystal growth is carried out. The aforementioned semiconductor single crystalA method for manufacturing a nerve activity measurement device, wherein the length from the tip of the probe to the film substrate is formed to be the length at which the tip portion is located in the superficial layer of the posterior horn of the spinal cord of the animal to be measured.

[0013] In particular, the thickness adjustment of the stopper using the resin is performed by changing the amount of resin deposited to a predetermined thickness. The probe of the semiconductor single crystal is formed by VLS growth, and then a film substrate is formed in a direction intersecting the longitudinal direction of the probe.

[0014] Also, this invention Measurement method for neural activity measurement devices uses a lead portion of a conductor formed on a film substrate and an end portion of this lead portion formed and a probe to puncture the probe into the spinal cord of an animal to be measured, insert the probe until the film substrate where the base end portion of the probe is located contacts the surface of the spinal cord, and measure the nerve activity in the superficial layer of the posterior horn of the spinal cord via the probe and the lead portion. thing That is.

[0015] On the film substrate the aforementioned probe is punctured into the superficial layer of the posterior horn of the spinal cord, and the film substrate is arranged along the spinal cord to measure the nerve activity in the superficial layer of the posterior horn of the spinal cord from the outside.

[0016] In particular, the probe and the film substrate are implanted along the spinal cord of the animal to be measured, and the nerve activity in the superficial layer of the posterior horn of the spinal cord is continuously measured.

[0017] Furthermore, the probe and the film substrate are implanted along the spinal cord of the animal to be measured, and a measurement device integrally provided with a processing circuit that processes the output passing through the lead portion, a wireless circuit that transmits the output of the processing circuit, and a power supply for each circuit is attached to the animal to be measured, and the nerve activity in the superficial layer of the posterior horn of the spinal cord is continuously measured by a wireless signal from the wireless circuit.

Advantages of the Invention

[0018] The nerve activity measurement device of this invention Manufacturing methodAccording to the present invention, it is possible to easily and accurately measure the nerve activity in the superficial layer of the dorsal horn of the spinal cord with minimal invasion and continuously record it for a long period of time. Neural activity measurement devices can be easily and precisely manufactured. Therefore, it becomes relatively easy to measure the nerve activity in the superficial layer of the dorsal horn of the spinal cord, which previously required skilled techniques, enabling more researchers to perform measurements, and greatly contributing to the elucidation and reduction of the mechanisms of nerve activities such as pain.

Brief Description of the Drawings

[0019] [Figure 1] It is a perspective view (a) and a longitudinal central cross-sectional view (b) of the nerve activity measurement device according to an embodiment of the present invention. [Figure 2] It is a cross-sectional view taken along line A-A of FIG. 1. [Figure 3] They are perspective views showing each manufacturing process of the nerve activity measurement device according to an embodiment of the present invention. [Figure 4] They are perspective views showing each of the next manufacturing processes of the nerve activity measurement device according to an embodiment of the present invention. [Figure 5] It is a schematic diagram showing an animal experiment example of the present invention. [Figure 6] It is a cross-sectional view of the spinal cord using the nerve activity measurement device of the present invention and a partially enlarged cross-sectional view showing the cross-section of the spinal cord in a state where the nerve activity measurement device is punctured into the superficial layer of the dorsal horn of the spinal cord. [Figure 7] They are an overall image (a) of a measurement device including a circuit board of the processing circuit of the nerve activity measurement device according to an embodiment of the present invention and a magnified photograph (b) showing the nerve activity measurement device at the tip of the measurement device. [Figure 8] They are SEM images of the entire probe part of the image in FIG. 7(b) (a), SEM images of the tip of the probe (b), and SEM images of the base end of the probe (c). [Figure 9] They are waveform examples (a) when recording nerve activity in the superficial layer of the dorsal horn of the spinal cord using a conventional tungsten electrode, an enlarged view (b) at the arrow position thereof, and waveform examples (c) when stimulating the skin with a von Frey filament (vFF 1.0 g) using a conventional tungsten electrode. [Figure 10](a) is an example of a waveform when recording neural activity in the superficial layer of the spinal cord's dorsal horn using a neural activity measurement device according to one embodiment of this invention, (b) is an enlarged view of the position indicated by the arrow, and (c) is an example of a waveform when von Fly filament (vFF 1.0g) is applied to the skin using a neural activity measurement device according to one embodiment of this invention. [Figure 11] This graph shows the spontaneous firing frequency of each cell in the superficial spinal cord dorsal horn, obtained using an in vivo recording method with a conventional tungsten electrode and a neural activity measurement device (novel microelectrode) according to one embodiment of this invention. [Figure 12] This graph shows the firing frequencies of superficial cells in the spinal cord dorsal horn in response to mechanical pain stimuli, using an in vivo recording method with a conventional tungsten electrode and a neural activity measurement device (novel microelectrode) according to one embodiment of this invention. [Modes for carrying out the invention]

[0020] The following describes a neural activity measurement device, its manufacturing method, and measurement method according to one embodiment of the present invention. As shown in Figures 1 and 2, the neural activity measurement device 10 of this embodiment has a conductive lead portion 14 made of gold (Au) or the like formed on a flexible film substrate 12, and a probe 20 having a core 20b made of Si semiconductor crystal grown in a direction intersecting the lead portion 14 at one end of the lead portion 14. The probe 20 is covered with a parylene film 12c, as will be described later, and a measurement electrode 22a with an exposed Au thin film 22 is located at the tip 21 of the probe 20.

[0021] The film substrate 12 has high biocompatibility. resinThe film substrate 12 is made of a film formed by depositing parylene using a method described later. The film substrate 12 has dimensions such as 400 μm in width and 4000 μm in length so that it can be placed on the spinal cord of an experimental mouse. The lead portion 14 on the film substrate 12 is integrally formed in a continuous manner with a thin metal film such as Au formed on the surface of the probe 20, which has a silicon single-crystal semiconductor as its core 20b. The lead portion 14 and the measuring electrode 22a at the tip 21 of the probe 20 are electrically connected. The distance between the surface of the film substrate 12 and the tip 21 of the probe 20 is set to the depth of the superficial layer of the spinal cord's dorsal horn, as described later.

[0022] The probe 20 has a tip portion 20a with a diameter of 5 μm or less, and the length from the surface of the film substrate 12 where the proximal end portion 20c is located to the tip 21 of the probe 20 is such that when the probe 20 is inserted into the spinal cord of the animal being measured, the tip portion 20a is positioned in the superficial layer of the spinal cord's dorsal horn. Therefore, the surface of the film substrate 20 where the proximal end portion 20c of the probe 20 is located functions as a positioning member and stopper when inserting the probe 20 into the superficial layer of the spinal cord's dorsal horn. The location of nerve cells in the superficial layer of the spinal cord's dorsal horn is, for example, 15 to 200 μm from the spinal cord surface in the experimental animal mouse, and the protruding length of the probe 20 is set to, for example, 150 μm. The neural activity measurement device 10 is intended for in vivo neural activity recording from the superficial layer of the mouse spinal cord's dorsal horn, and the diameter of the probe 20 is designed to enable tissue damage and long-term stable recording of the superficial layer of the spinal cord's dorsal horn. Therefore, while conventional nerve cell measurement electrodes have a diameter of 40 μm or more, the present invention achieves a diameter of 5 μm or less, which is significantly smaller.

[0023] The lead portion 14 is formed along the longitudinal direction of the film substrate 12, with one end continuous with the Au thin film 22 integrally formed on the surface of the probe 20 as described above, and a contact pad 14a continuously formed at the other end. As will be described later, the lead portion 14 is held between the parylene films 12a and 12c that constitute the film substrate 12, with the contact pad 14a exposed from the parylene film 12c.

[0024] Next, the manufacturing method of the neural activity measurement device 10 of this embodiment will be described below with reference to Figures 3 and 4.

[0025] First, as shown in Figure 3(a), a resist is prepared on the surface of the SiO2 layer 30a on the surface of the Si(111) substrate 30, and a pattern is formed by photolithography to form an Au catalyst 32 in the shape of a disk, for example, with a diameter of 6 μm and a thickness of 200 nm, preferably with a diameter of 5 μm or less. Then, Au is deposited, and the Au other than the Au catalyst 32 is removed by lift-off. After this, the core 20b of the probe 20, made of a silicon semiconductor single crystal, is formed by crystal growth on the surface of the substrate 30 in the area where the Au catalyst 32 remains. The crystal growth of the probe 20 core 20b is performed by VLS (Vapor Liquid Solid) growth. The length of the probe 20 is Laboratory animals The crystals are grown to a length that reaches the superficial layer of the spinal cord's dorsal horn, for example, up to 140-150 μm.

[0026] Next, as shown in Figure 3(b), parylene C forms the film substrate 12. (resin) A 5 μm layer is deposited over the entire surface to form a parylene film 12a.

[0027] Next, a resist is applied only to the Si substrate 30 using a spin coater, and as shown in Figure 3(c), the parylene on the surface of the core 20b of the probe 20 is reactive ion etched. Then, as shown in Figure 3(d), parylene C is applied for insulation from the back surface. (resin) A parylene film 12b is formed by depositing a 1 μm thick layer of material to insulate the Si core 20b of the probe 20 from the metal wiring that will be formed later. Therefore, the core 20b, which consists of the Au catalyst 32 and the semiconductor, is not electrically utilized in subsequent measurements.

[0028] Next, a resist is applied and the Au lead portion 14, which will become the metal wiring, is patterned using photolithography. The Au is then sputtered, and as shown in Figure 4(a), the Au thin film 22 on the surface of the probe 20 and the Au thin film other than the wiring portion of the lead portion 14 are removed by lift-off.

[0029] Subsequently, as shown in Figure 4(b), parylene C (resin) A parylene film 12c is formed by depositing a 5 μm thick layer of material to cover the surfaces of the probe 20 and the Si substrate 30. Then, a resist is applied and patterning is performed by photolithography, and as shown in Figure 4(c), etching is performed so that the contact pad 14a of the lead portion 14 and the measurement electrode 22a of the Au thin film 22 at the tip 21 of the probe 20 are exposed.

[0030] Finally, an incision is made in the four corners with a scalpel to peel the film substrate 12 from the Si substrate 30 together with the probe 20, completing the neural activity measurement device 10 as shown in Figure 4(d).

[0031] The method of using the neural activity measurement device 10 of this embodiment is as follows, as detailed in the experimental results later: for example, the back of an experimental mouse M shown in Figure 5 is incised and the probe 20 is inserted into the spinal cord dorsal horn P of the spinal cord S. At this time, the length of the probe 20 is set to the depth of the superficial layer P1 of the spinal cord dorsal horn, and the surface of the film substrate 20 where the proximal end 20c of the probe 20 is located functions as a positioning member and stopper when inserting the probe 20 into the superficial layer of the spinal cord dorsal horn. Therefore, by inserting the probe 20 until the film substrate 12 contacts the surface of the spinal cord S, the measurement electrode 22a made of the Au thin film 22 at the tip 21 of the probe 20 is accurately positioned in the superficial layer P1 of the spinal cord dorsal horn. The film substrate 12 is very short and can be placed along the spinal cord S.

[0032] In this state, the neural activity in the superficial layer P1 of the spinal cord dorsal horn of mouse M is measured by measuring the electrical output via the probe 20 and lead portion 14. It is also preferable to implant the probe 20 and film substrate 12 along the spinal cord S of the mouse M to be measured, and to continuously measure the neural activity in the superficial layer P1 of the spinal cord dorsal horn. The voltage to be measured is determined by measuring the potential difference between the measurement electrode 22a of the probe 20 and an indifferent electrode (not shown) (in this experiment, it is in contact with the spinal cord S at a location slightly away from the measurement site).

[0033] Furthermore, a measuring device (not shown) is provided, which integrates a processing circuit that processes the electrical output via the lead section 14, a wireless circuit that transmits the output of the processing circuit, and power supplies for each circuit, to be measured. experiment It would be beneficial to attach this device to an animal such as a mouse (M) and continuously measure the neural activity of the superficial P1 layer of the spinal cord's dorsal horn using wireless signals from a wireless circuit.

[0034] The processing circuit includes, for example, a filter circuit that filters the electrical signal detected in the superficial P1 layer of the spinal cord's dorsal horn, a signal amplification circuit, and an AD conversion circuit that converts the amplified signal into a digital signal. The wireless circuit converts the digital signal into a signal conforming to a predetermined wireless standard and outputs it, for example, to an existing standard such as Bluetooth®. The power supply is a miniature battery mounted on the circuit board. This allows for long-term, continuous recording of the activity of the superficial P1 layer of the spinal cord's dorsal horn in a freely moving experimental animal, mouse M.

[0035] According to the neural activity measurement device, its manufacturing method, and measurement method of this embodiment, neural activity in the superficial layer P1 of the spinal cord dorsal horn of a mouse M, an experimental animal, can be measured easily, accurately, and continuously recorded over a long period of time with minimal invasiveness. When attaching the device to the mouse M, the insertion of the probe 20, which is an electrode in the superficial layer P1 of the spinal cord dorsal horn, which previously required skilled techniques, can now be easily performed. By inserting the probe until the film substrate 12 touches the surface of the spinal cord S, the film substrate 12 acts as a positioning stopper that regulates the insertion depth, ensuring that the tip 20a of the probe 20 is accurately positioned in the superficial layer P1 of the spinal cord dorsal horn. Therefore, it becomes possible for many researchers, even those without expertise, to easily and accurately measure neural activity in the superficial layer P1 of the spinal cord dorsal horn. This will greatly contribute to elucidating the mechanisms of neural activity such as pain and to developing methods for reducing pain.

[0036] Next, the present invention Feature partEmbodiments will now be described. As shown in the description of the embodiments above, the base end 20c of the probe 20 is raised from the surface of the film substrate 12 (see Figures 2 and 8). This raised base end 20c is formed when the area of ​​the Au thin film (conductive material) deposited on the surface of the probe 20, excluding the tip 21 of the probe 20, is insulated with parylene C (resin). Therefore, if the length of the probe 20 to be punctured should be adjusted in advance for the core 20b of the probe 20 formed by crystal growth, the thickness (amount of deposition) of the base end 20c should be changed to a predetermined thickness to adjust the puncture length of the probe 20. In this case, the base end 20c should have a diameter of an appropriate size in order to function as a stopper during puncture. As for the size of the diameter, for example, the diameter can be about the same as the width dimension of the film substrate 12. Alternatively, the width of the film substrate 12 may be increased only in the area where the base end 20c is formed, and the base end 20c may be formed over the entire width dimension.

[0037] In the configuration described above, the core 20b of the probe 20 formed by crystal growth should be of an appropriate length, and the target of measurement experiment By changing the thickness of the base end 20c depending on the animal, the probe can be applied to various animals. This is because, even if the length of the probe produced by crystal growth can be adjusted according to the growth time, it is difficult to control the length precisely. In contrast, adjusting the thickness by vapor deposition of parylene C (resin) is relatively easy to control. In the above embodiment, a thin Au film was exemplified as the conductive material used for the probe 20, but other materials may be used. Also, parylene C was exemplified as the resin for insulation, but it is not limited to this. [Examples]

[0038] Below, we describe an example in which neural activity in the superficial P1 layer of the spinal cord dorsal horn of a mouse M was measured using the neural activity measurement device of the present invention, with the experimental results as follows.

[0039] First, to confirm whether the probe 20 of the neural activity measurement device 10 of the present invention can record the desired neural activity, the probe portion of the structure of the embodiment of the present invention, which is the electrode for measurement, was fixed to a metal pin 24 and punctured into the spinal cord dorsal horn P of an existing mouse M, as shown in Figure 5. This confirmed that the probe 20 of the present invention can record the neural activity of the superficial layer P1 of the spinal cord dorsal horn of mouse M.

[0040] Next, in order to enable implantation into the body of mouse M, as described in the above embodiment, we measured the neural activity of the superficial P1 layer of the spinal cord dorsal horn of mouse M using a neural activity measurement device 10 consisting of a probe 20 made of a semiconductor silicon single crystal fabricated by VLS growth technology and having an Au measurement electrode 22a, and a flexible film substrate 12 of parylene film, a highly biocompatible polymer material.

[0041] As shown in Figure 7(a), the neural activity measurement device 10 of this embodiment is attached to a measurement device 26 equipped with a circuit board that includes a filter circuit that receives and filters the voltage of the measured neural activity, a signal amplification circuit, and an AD conversion circuit that converts the amplified signal into a digital signal. The wireless circuit, as described above, converts the signal converted to a digital signal into a signal of a predetermined wireless standard and outputs it, for example, by converting it to an existing standard such as Bluetooth®. A miniature battery is mounted on the circuit board to provide power.

[0042] The neural activity measurement device 10 is provided at the tip of the measurement device 26 shown in Figure 7(a), as shown in Figure 7(b). The probe 20 is as shown in the SEM image of the entire probe 20 shown in Figure 8(a), and the tip portion 20a of the probe 20 is formed to be 5 μm or less, as shown in Figure 8(b). The proximal portion 20c of the probe 20 also has the structure as shown in the SEM image of Figure 8(c).

[0043] As a result, when puncturing the superficial P1 layer of the spinal cord's dorsal horn with the probe 20, the film substrate 12 acts as a positioning stopper that regulates the puncture depth, enabling accurate puncture. Furthermore, the extremely thin probe 20 minimizes tissue damage during puncture, and the neural activity measurement device 10 can be easily implanted in the body. The film substrate 12 used here has a width of 400 μm and a length of 4000 μm. The impedance of the fabricated neural activity measurement device 10 was 600 kΩ or less at 1 kHz, enabling the recording of neural activity, and the voltage input / output ratio was 90% or more.

[0044] The measurement involved recording spontaneous firing from superficial cells in the dorsal horn of the spinal cord of mouse M using the neural activity measurement device 10, as shown in Figure 5, and comparing it with that of a conventional electrode (tungsten electrode). In the experiment, mouse M was first subjected to thoracic laminectomy under deep urethane anesthesia, placed in a brain and spinal cord fixation device, and the dura mater, arachnoid mater, and pia mater were removed under a microscope to secure space for electrode insertion. Artificial cerebrospinal fluid heated to 37°C and oxygenated was perfused to the spinal cord surface. Subsequently, neural activity was recorded from superficial cells in the dorsal horn of the spinal cord using both a conventional tungsten electrode and the neural activity measurement device 10, which is a neural electrode according to this invention.

[0045] As a result, when recorded with conventional tungsten electrodes, neural activity in a single cell could be measured as a voltage fluctuation (Figure 9(a), (b), (c)). Next, when the same experiment was performed using the neural activity measurement device 10 of this invention, neural activity in a single cell could be measured as a voltage fluctuation waveform, similar to the tungsten electrodes (Figure 10(a), (b), (c)).

[0046] Analysis of neural activity recorded from both measurement devices revealed no significant difference in spontaneous firing frequency from superficial spinal cord dorsal horn cells between the conventional tungsten electrode and the neural activity measurement device used in this study (as shown in Figure 11) (tungsten electrode: 0.11 ± 0.08 Hz, n = 9; neural activity measurement device: 0.10 ± 0.06 Hz, n = 8; P = 0.90). Since spontaneous firing is rarely observed in superficial spinal cord dorsal horn cells of healthy animals, the results obtained were consistent with previous reports.

[0047] Conventionally, with tungsten electrodes, it was difficult to control the recording location due to the length of the electrode itself, and recording neural activity from superficial cells in the spinal cord dorsal horn required highly advanced techniques. However, the neural activity measurement device 10 of this invention allows for the precise setting of the length of the probe 20 equipped with the measurement electrode 22a to a predetermined length of, for example, 150 μm or less, making it possible to easily record neural activity from superficial cells in the spinal cord dorsal horn. Furthermore, as can be seen by comparing Figures 9 and 10, it has also succeeded in reducing the noise level compared to tungsten electrodes.

[0048] Furthermore, the responsiveness to mechanical pain stimuli applied to the skin was also investigated using conventional tungsten electrodes and the neural activity measurement device 10 used in this study. Neural activity was recorded from superficial cells in the spinal cord dorsal horn, and when mechanical pain stimuli were applied to the skin receptive fields using tweezers, a significant increase in firing frequency was observed. Analysis of the neural activity recorded from both electrodes revealed no significant difference in the obtained neural activity, as shown in Figure 12 (tungsten electrode: 12.4 ± 1.8 Hz, n = 10; novel ultrafine electrode: 13.0 ± 1.9 Hz, n = 10, P = 0.61). [Industrial applicability]

[0049] This invention makes it easier to record from the superficial layer of the spinal cord's dorsal horn, which was previously difficult. This is expected to lead to a better understanding of the mechanisms of chronic pain development and greatly contribute to the development of new therapeutic drugs and treatments. [Explanation of Symbols]

[0050] 10. Neural Activity Measurement Devices 12 Film substrate 12a, 12b, 12c parylene membrane 14 Lead section 14a Contact Pad 20 probes 20a Tip 20b core 20c proximal end 21 Tip 22 Au thin film 22a Measuring electrode 24 pins 30 circuit boards 32 Au catalyst M Mouse P Spinal posterior horn P1 Spinal cord dorsal horn superficial layer S Spinal Cord

Claims

1. In a method for manufacturing a neural activity measurement device, a semiconductor single crystal is grown to form a probe with a tip cross-sectional diameter of 5 μm or less, the surface of the probe is coated with a conductive material, a flexible film substrate is provided at the base end of the probe in a direction intersecting the longitudinal direction of the probe, a conductive lead portion is formed on the film substrate, the lead portion and the tip of the probe are electrically connected, and the lead portion and the probe are integrally formed on the film substrate, The length from the tip of the probe of the semiconductor single crystal to be grown to the film substrate is formed to a length such that the tip is located in the superficial layer of the spinal cord posterior horn of an experimental animal, which is a mouse. The conductive material on the surface of the probe, excluding the tip, is coated with resin, the lead portion is continuous with the contact pad, and the portion of the lead portion excluding the contact pad is insulated by the resin constituting the film substrate. A stopper is formed around the base end of the probe on the surface of the film substrate using the resin that covers the conductive material, and the stopper is formed with the resin raised from the surface of the film substrate. The stopper, which is made of raised resin, is formed by adjusting the thickness of the resin so that the distance between the stopper on the surface of the film substrate and the tip of the probe is such that when the probe is inserted into the spinal cord of the experimental animal to be measured and the stopper comes into contact with the surface of the spinal cord, the tip of the probe is accurately positioned in the superficial layer of the dorsal horn of the spinal cord. A method for manufacturing a neural activity measurement device, characterized in that the stopper is used for positioning when puncturing the probe.

2. The method for manufacturing a neural activity measuring device according to claim 1, wherein the thickness adjustment of the stopper with the resin is adjusted by adjusting the amount of resin deposited to a predetermined thickness.

3. The method for manufacturing a neural activity measurement device according to claim 2, wherein the probe of the semiconductor single crystal is formed by VLS growth, and thereafter a film substrate is formed in a direction intersecting the longitudinal direction of the probe.

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