RU2653815C1 - Method of implanting optic fiber probe in animal brain to generate controlled feedback - Google Patents

Abstract

FIELD: optical fiber.

SUBSTANCE: invention relates to the field of optogenetics. Fiber-optic probe contains optical fiber and a fixed tip. Length of the optical fiber that is necessary to reach a given area of the brain is determined. Optical fiber is fixed on the tip so that the specified length of the optical fiber protrudes from the tip. Animal is anesthetized, a part of the skin of the skull is removed, a hole is made in the skull using the stereotaxic coordinates, and the fiber optic probe is inserted into the hole. At the same time with the introduction of the probe and in the feedback mode, laser radiation is generated; it has the power and wavelength that are sufficient to activate the optical signal in the brain cells of the animal. Laser radiation is transmitted along the optical fiber of the probe to its distal end and the optical signal from the brain cells is registered. When a specified amplitude of the optical signal or a specified image of the brain cells is obtained, the tip is fixed directly on the skull or on an external holder that has been pre-mounted on the skull relatively to the tip.

EFFECT: method provides for accurate positioning of the distal end of the optical fiber within the animal's brain, the attachment reliability and the ability to conduct observations for a long time, which is achieved by the above methods of the method.

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Description

Technical field

The proposed technical solution relates to the field of optogenetics, and in particular to methods of implanting an optical probe into the brain of a laboratory animal.

State of the art

Modern optical methods and fiber-optic technologies play an increasingly prominent role in studies of neurophysiological and cognitive functions, which is associated with their low invasiveness, efficiency, and high spatial (up to subcellular) resolution. The use of fiber probes (the term fiber neurointerfaces is also often used) is one of the main directions in the tasks of neurophotonics and optogenetics: A.M. Aravanis, et al., J. Neural Eng. 4, S143 (2007); D.R. Sparta, et al., Nature Protocols 7, 12 (2012); G D. Stuber, et al., Nature 475, 377 (2011); L.V. Doronina-Amitonova, Scientific Reports 3, 3265 (2013). Fiber-optic neurointerfaces provide unique opportunities for optogenetic neurostimulation for various pharmacological and physiological stimuli, as well as long-term registration of processes in the deep layers of the brain of free-moving animals. An important task to be solved with the help of fiber neural interfaces is the optical registration of the functional activity of individual groups or individual targeted neurons of the brain, which requires precise addressing of the fiber probe to the desired area of the brain with an accuracy of the order of the size of one neuron (5-10 μm).

To accomplish the task of accurately positioning the fiber probe, it is necessary to fix the animal’s head and securely attach the probe (neurointerface) to the animal’s skull, while ensuring that the distal end of the optical fiber is located in the selected part of the brain. The main problem is the accuracy of positioning, because, despite the known anatomical features of the structure of the brain, implantation is carried out with errors, in particular, determined by the individual characteristics of the animals, which as a result does not guarantee the implantation of a probe to a specific neuron.

Methods for installing an optical probe are described, for example, in US Pat. No. 9,238,150 (para. 0065). Implantation of the probe includes anesthesia of the animal, removal of wool, drilling holes using stereotactic devices, insertion of the probe of a pre-selected length and fixing the probe. The accuracy of the installation of the distal end of the fiber plays a key role in the correct choice of the studied area of the brain. However, due to the individual characteristics of the structure and development of animals, the distal ends of the probes of the same length can be in different layers of the brain.

A known method of installing a fiber optic probe using a special mount, patent US 9107575. With this method, a special device is mounted on the animal’s skull, through which an optical fiber is introduced. The device allows you to direct the fiber at an angle selected using the stereotactic technique. However, the depth of placement of the distal end of the fiber may vary. In addition, when conducting experiments over a long period of time, the optical fiber will need to be inserted into the device to the same depth, which is difficult to implement. In addition, an experimental error may occur due to brain damage during reinstallation of the optical fiber.

Disclosure of invention

The objective of the invention is to develop a method of implanting a fiber probe into the brain of an animal for the implementation of long-term experiments on the optical recording of the functional activity of a selected group of neurons (or one neuron). The method should provide accurate targeted and minimally invasive positioning of the distal end of the optical fiber inside the brain of the animal.

To solve this problem, a method of implanting a genetically modified animal with integrated photosensitive proteins of an optical fiber probe containing an optical fiber and a fixed tip, which calculates the length of the optical fiber necessary to reach a given area of the brain, is used to fix the optical fiber in the tip so that an optical fiber of a calculated length protruded, the animal was anesthetized, scalp was removed from the part of the animal’s skull, sufficient to fix fiber optic probe, form a hole in the skull along stereotactic coordinates, insert the fiber of the fiber probe into the hole, simultaneously with the introduction of the probe in feedback mode, generate laser radiation with a power and wavelength sufficient to activate the optical signal in the animal’s brain cells, transmit laser radiation through optical fiber of the probe to its distal end and register the optical signal from the brain cells of the animal, the tip is fixed directly on the skull of the animal or on the attentively attached to the skull external to the tip of the holder when receiving a given amplitude of the optical signal or a given image of brain cells. The fixation of the tip with glue can occur both directly on the skull of the animal (Fig. 1a), and in an external holder pre-attached to the skull of the animal (Fig. 1b). When attaching the tip (ferula) directly to the animal’s skull, only a small adjustment is possible according to the implantation depth - of the order of 200-300 μm - due to the formation of a thin layer of glue between the tip and the skull (the thickness limit in this case is dictated by the requirement for reliable fixation of the ferula with fiber on the skull animal, as well as avoiding glue getting inside the hole). When fixing the tip in a pre-fixed external holder, a much larger adjustment is possible according to the depth of implantation. In this case, the external holder is pre-glued to the skull of the animal, while the axis of the holes of the external holder and the axis of the holes in the skull must match. Such a scheme can provide a much larger amplitude for adjusting the position of the distal end of the fiber - up to two millimeters. Upon reaching the required level of the measured signal, the tip is fixed and glued directly to the external holder. The proposed method of implanting a probe into a predetermined region of the brain provides accurate probe positioning, which is achieved through feedback, which allows you to control the position of the distal end of the fiber relative to neural cells, and precise adjustment of the probe penetration depth. This method eliminates the influence of individual phenotypic characteristics of the animal, and also makes it possible to obtain additional information, for example, on the distribution in the brain of an animal with a modified genome of photosensitive neurons already in the process of installing the probe. But the most important thing is that the method allows you to position the distal end of the fiber precisely in a given area up to the size of one individual neuron with the previously known characteristics of the optical response.

A necessary condition for such studies is the use of genetically modified animals with built-in photosensitive fluorescent proteins. Modern genetic engineering provides a wide range of possibilities for the genetic labeling of functionally important neural cells, not only for optical recording of cell activity, but also for optical control of the functional activity of animals, in particular, when using optical fiber probes.

The signal from animal brain cells is observed through a computer-based optical recording and control system, which may contain optical filtering elements (passive filters or spectrometer), single-channel (photoelectronic multipliers, photodiodes) or multi-channel (CCD cameras) detectors, a computer for signal or image analysis . The method allows to observe the optical fluorescence response of the brain cells of the animal directly during the installation of the probe (neural interface), which provides high accuracy (up to several micrometers) of positioning of the distal end of the optical fiber.

The fiber of the optical fiber probe (neural interface) is injected using an automated stereotaxis preparation agent connected to a computer, which allows the distal end of the fiber to be automatically positioned. Since the optical recording and control system and the automated stereotaxis preparation agent are connected to a computer, feedback is implemented during the introduction of the fiber probe by obtaining information about the distribution of photosensitive cells in the animal’s brain near the distal end of the fiber and the corresponding precise adjustment of its position.

The tip is fixed in two stages, at the first stage, the tip is fixed with quick-drying glue, in the second stage, after a period of 2 to 8 minutes, the tip is fixed with a mixture of quick-drying glue and dental cement. As a quick-drying adhesive, cyanoacrylate adhesive can be used. When attaching the tip directly to the animal’s skull, the prepared mixture of cement with glue is applied so that at least 1/3 of the outer part of the ferula is covered with glue. On the skull, cement should cover the entire area of the exposed bone, but not go to the edges of the skin flaps around the surgical field. The glue allows you to quickly fix the tip (ferula) on the skull of the animal in the selected position, which ensures the accuracy of the probe fixation, the mixture of glue and cement ensures reliable fastening for a long time and the duration of the experiments. In the case of using an external holder at the beginning of the procedure, it is fixed on the skull of the animal using a mixture of cement with glue. The necessary alignment of the hole in the skull and the neural interface is ensured by holding the tip with the fiber in the holder during the bonding procedure. Further, the attachment procedure repeats the previous case, only gluing the tip does not occur to the skull of the animal, but to the external holder.

The optical fiber of the optical fiber probe is a multi-core optical fiber with thousands of individual micron-sized optical fibers, which makes it possible to obtain fluorescence images from the distal end of the fiber directly during its installation, and the tip is fixed when the selected image is obtained. The use of multichannel optical fiber allows one to obtain detailed information on the structure of the selected area already at the stage of fiber installation.

An optical fiber is a single-core fiber with an external diameter of not more than 250 μm, the tip is fixed when the selected optical signal intensity is obtained from animal brain cells. In the case of using a single core fiber, the integral fluorescence signal from one or a group of animal brain cells is measured, and the tip is fixed when a given optical fluorescence signal intensity or intensity function is obtained when the probe scans the signal from animal brain cells. In order to reduce the area of the brain irradiated by pump radiation and, accordingly, more accurately address one neuron, the diameter of the fiber core should be comparable (or less) with the size of the localization area of one neuron of the order of 5-30 micrometers. Modern optical technologies make it possible to produce fibers with similar parameters, while the structure of the fiber can vary over a wide range to ensure optimal values of the excitation parameters and the collection of the fluorescence signal from animal brain neurons. In the case of using fibers with a small core, a signal from a single neuron will most likely be observed, then when the distal end of the fiber probe is moved deeper into the brain, the intensity of the optical response will be similar to the function shown in FIG. 3. In the case of using fibers with a large core, the overall level of the obtained fluorescence signal increases, but in this case the probability of signal registration from several neurons is high, and the dependence of the fluorescence intensity on the position of the distal end of the fiber can be more complex.

Depending on the research task, an optical fiber probe is fixed on the animal’s skull when it receives the maximum optical signal (or a specific function when scanning the fiber “up and down”) from brain cells, or a fiber probe is fixed when an optical signal arises from the animal’s brain cells, or a fiber optic probe is fixed with a decrease in the optical signal from animal brain cells. Since brain neurons are located in layers, the proposed method allows you to install the probe on the outer boundary of the layer (when a signal occurs), in the area with the maximum number of photosensitive cells (when receiving the maximum signal), on the inner border of the layer (when the signal decreases).

The technical result of the proposed technical solution is to ensure high positioning accuracy of the order of several micrometers of the distal end of the fiber probe by providing feedback consisting in the continuous recording of the optical fluorescence signal from the neuron under study (a group of neurons) or obtaining an image of the part of the brain under study during the implantation procedure, the possibility of obtaining additional information during the installation of the probe, the reliability of mounting the probe.

Brief Description of the Drawings

FIG. 1. Methods of fixation of the neurointerface: directly on the skull of the animal (a), using an external guide holder (b).

FIG. 2. Installation diagram, allowing the implementation of the proposed method.

FIG. 3. Image of a transgenic mouse (a) with implanted fibers with a core diameter of 50 μm (b) and 300 μm (s).

FIG. 4. An example of the localization of one neuron near the distal end of a single core optical fiber (left) and the optical response in the case of different positions of the neuron relative to the distal end of the fiber.

FIG. 5. Image of a neuron at a depth of 600 microns, obtained using multichannel fiber.

FIG. 6. Images obtained using multichannel fiber, neurons at a depth of about 800 microns before fixing the tip (a) and after applying glue (b).

FIG. 7. Photos illustrating the verification of the reliability and operability of the probe and the method of implantation in a series of neurophysiological tests: general view of the placement of the animal in the open field test (a); test "elevated cruciform labyrinth" to study the behavior of mice with an implanted probe (b); test "rotarod" with the studied and control animals (c).

The implementation of the invention

For the experiments, the coordinates of a number of anatomical brain structures of transgenic mice were selected, in which marker fluorescent proteins, in particular EGFP (enhanced green fluorescence protein), are artificially expressed. During the operation, the probe was stereotactically positioned. After preparing the surface of the skull, the place on the skull was determined where the trepanation hole should be located for implantation of the probe optical fiber. For this, an atlas of the mouse brain was used in stereotactic coordinates. Using the atlas, the position of the anatomical structure of interest in the three-dimensional system of Cartesian coordinates was determined, the zero point of reference of which is the Bregma point - the place of convergence of the sagittal and coronary sutures. The coordinates were determined in the rostrocaudal direction (towards the nose - “+”, in the back of the head - “-”), in the mediolateral (in the direction of the right ear - “+”, in the direction of the left ear - “-”), and in the dorsoventral ( the depth of the structure relative to the surface of the brain at the level of Bregma). To match the implantable neural interface to the selected brain structure in the dorsoventral direction, an optical fiber of the required length was chosen.

In accordance with the claimed solution, it is possible to use two schemes for attaching a fiber optic probe (neurointerface) directly to the skull of an animal. The first circuit is shown in FIG. 1a and consists in attaching the tip / ferula (1) with optical fiber (2) directly to the skull (4) of the animal using a mixture of cyanoacrylate adhesive and cement (3). The second circuit is shown in FIG. 1b and consists in the initial fastening of the external holder (5) by means of a mixture of cyanoacrylate adhesive and dental cement (3) on the skull (4) of the animal, observing the alignment of the hole in the skull and the external holder and subsequent fixation of the tip (2)) with the optical fiber (1) on external holder (5) using mixture (3).

We used disconnectable fiber-optic probes containing the first part, mounted on the skull of an animal, with optical fiber and a ceramic cylindrical tip / ferrule, the second part of the probe (6) with a long segment of a similar optical fiber, containing a reciprocal ceramic ferrule and connector providing a snug fit and reliable fastening the first and second parts of the fiber optic probe. The length of the distal end of the optical fiber of the first part protruding from the tip was determined as the sum of the thickness of the animal’s cranial bone 250 μm and the depth of necessary penetration into the animal’s brain relative to the inner surface of the skull bone. Following this definition, the length of the optical fiber for the area of the barrel fields of the primary somatosensory cortex should be 1000 μm; for the CA1 region of the hippocampus - 1750 μm; for tonsils, basal nucleus - 4000 microns. The choice of these brain structures was due to their localization and the need to further evaluate the effectiveness of the functioning of the optical fiber probe at various depths of implantation of the optical fiber. The optical fiber was smoothed out from the external plastic sheath for a certain length, then, at the right distance from the end, the optical fiber was serrated with a special cutter. After that, the optical fiber was neatly cleaved at the notch. Using a microscope, the length of the obtained distal end of the fiber was additionally measured and the quality of the ends (absence of chips and dirt) was visually determined. The optical fiber of the first part of the probe of a given length was fixed in a ceramic tip so that the end face of the fiber was in the plane of the tip on the front side of the tip and protruded to the required length corresponding to the depth of the selected brain structure and the thickness of the skull bone. The second part of the probe contains a long segment of optical fiber, which provides connection with a laser excitation system, a measurement and control system, and also provides free movement of the animal during the experiments. The second part of the probe is attached to the first only during the installation of the probe and then during experiments with animals.

Using a blunt needle fixed in a stereotaxis preparation, the location of the target anatomical structure was noted on the surface of the skull. Using a high-speed drill and drill with a head with a diameter of 0.6 mm, a rounded trepanation hole was drilled in the skull. The neural interface fiber was fixed in a stereotaxis preparation and lowered into a trepanation hole.

In FIG. 2 shows the experimental design. The fiber optic probe system must contain several essential elements: a laser excitation system (7), which has the necessary power and wavelength to excite the optical fluorescence signal; optical separation systems of the exciting laser radiation and the useful optical signal (8), which is usually achieved using a dichroic filter; systems of institution / scanning (9) and focusing (10) of radiation into the fiber; a fiber probe, which is mounted on the skull of an animal and consists of two main elements - an optical fiber (2) and a tip (1), which can be connected to the second counterpart of the probe (6) if the design has an opening function. The optical fiber itself (2) can have a different structure of the sheath and core: to be single-mode or multimode, to have one core or to be multi-channel, etc. The head of the anesthetized animal is fixed in stereotaxis (11), the fiber of the optical fiber probe is introduced using an automated preparation guide (12) connected to the computer. The signal from the animal’s brain cells is observed by means of an optical registration system (13) and a computer-based control system (14), which makes it possible to position the distal end of the neural interface in a selected area of the brain with high accuracy due to feedback already at the stage of fiber installation, as well as to obtain detailed information from a selected area of the brain. In the proposed scheme, the stereotaxis preparation agent (12) automatically places the distal end of the optical fiber of the probe in the position corresponding to a given region of the brain. Due to the individual characteristics of the animal, errors in its precise positioning are possible, but due to the presence of feedback, the operator or automated software can stop the fiber injection process when receiving specified images (in the case of multichannel fiber) or depending on the intensity of the optical response from animal brain neurons. It is possible to correct the exact position of the distal end of the fiber by scanning the fiber “up and down”. Due to the real need for scanning the probe only in small limits of 300-400 microns and a small number (up to one) of scanning cycles, such a procedure is minimally invasive for the brain tissue of the animal. After determining the exact predetermined position of the distal end of the optical fiber (2), fixed in the tip (1), the tip is fixed on the skull of the animal.

In the presented works, the fixation of the tip (1) took place directly on the skull of the animal (Fig. 1a) in two stages. At the first stage, a tip (1) was fixed with a drop of cyanoacrylate adhesive with fiber (2) fixed in it in the selected position. Then, the second opening part of the probe (6) was disconnected and a mixture of cyanoacrylate glue and tooth cement powder (Stoelting, USA) was prepared in a 1: 1 ratio. Next, the tip was fixed on the skull with the help of a prepared mixture of cement with glue, while the mixture was applied so that at least 1/3 of the outer part of the tip was covered. Cement was applied to the skull so that it covered the entire area of the exposed bone, but did not go to the edges of the skin flaps around the surgical field.

We used gene-modified mice of the Thyl-EGFP line, and in FIG. 3a is a photograph of a mouse with implanted fibers, with FIG. Figure 3 shows an image of brain regions with an implanted single-core fiber with a core diameter of 50 μm (b) and a multi-core fiber with a core diameter of 300 μm (c). A cw laser at a wavelength of 473 nm with a power of up to 10 mW was used as a radiation source. The signal was recorded by a photoelectronic multiplier and analyzed using a personal computer. Moreover, the excitation and registration system were placed on a single compact base, which ensures mobility and ease of use of the entire system as a whole. In FIG. Figure 4 shows a diagram of the localization of one neuron near the distal end of a single-core optical fiber (left) and the function of the recorded optical response in the case of different positions of the neuron near the distal end of the fiber (right). Thus, a preliminary analysis allows you to fix the position of the tip with a fixed single-channel optical fiber upon reaching a given level of the optical signal. To reduce the influence of individual characteristics on the level of the fluorescence signal and, accordingly, more accurately determine the position of the distal end of the optical fiber, it is necessary to scan to obtain a function similar to that shown in FIG. 4, analyze and select the position of the distal end of the optical fiber with high accuracy (up to several micrometers).

In the experiments, multichannel optical fibers with a micron size of individual optical fibers were also used, which made it possible to obtain images of neurons and, accordingly, detailed information about the processes occurring in neurons of the brain of free-living animals and to provide targeted action on individual cells. The choice of such a fiber also improves the accuracy of probe positioning by acquiring images of the distribution of neurons from the distal end of the fiber. We used a multichannel fiber with an external diameter of 600 μm, containing six thousand individual fiber channels with a core diameter of about 2.4 μm, located at a distance of about 3 μm from each other. In FIG. Figure 5 shows the image of the first neuron detected at a depth of about 500 μm in the cerebral cortex of the Thyl-EGFP mouse while lowering the probe into the brain of the animal. The scale of the line is 50 microns, two photographs with different magnifications are presented. In FIG. Figure 6 shows the images of neurons obtained at a depth of about 800 μm, before fixing the tip (a) and after fixing it with glue (b) according to the procedure described above. Images before and after applying glue are identical, which indicates that fixation with quick-drying glue does not disturb and provides the necessary positioning accuracy.

The quality of fixation of the optical fiber of the neurointerface implanted in experimental animals was checked visually daily for 60 days. The number of experimental animals with a dropped neural interface was 14% after 50 days. Moreover, the loss of the neurointerface was first observed on day 50. The proposed method of fixation allows you to reliably fix the neural interface on the skull of an animal and conduct observations for a long time. Animals with implanted probes underwent various neurophysiological tests in special certified devices “rotarod”, “open field”, “cruciform labyrinth”. The behavior of animals during testing was automatically recorded by the camera. In FIG. 7 provides examples of the use of such neurophysiological tests. In general, long-term experiments showed high accuracy of probe positioning in a selected area of the brain and the quality of fixation.

Thus, as a result of the experimental application of this method, it has been demonstrated that the distal end of the optical fiber can be placed in a selected area of the animal’s brain with high accuracy (up to several micrometers), the fiber probe is securely attached to the animal’s skull, there is no displacement of the distal end of the fiber probe relative to the structures and brain cells. These features make it possible to record the optical response from a selected region of the animal’s brain for a long time, which generally provides the possibility of long-term registration of neurophysiological processes in the deep layers of the brain of free-moving animals.

Claims (9)

1. A method of implanting a genetically modified animal with integrated photosensitive proteins of an optical fiber probe containing an optical fiber and a fixed tip, in which the length of the optical fiber necessary to reach a given area of the brain is calculated, the optical fiber is fixed in the tip so that the optical protrudes from the tip fiber of the calculated length, the animal is anesthetized, scalp is removed from the part of the animal’s skull, sufficient to secure the fiber optic probe, form Growth in the skull according to stereotactic coordinates introduces the fiber of the fiber probe into the hole, characterized in that simultaneously with the introduction of the probe in the feedback mode, they generate laser radiation with a power and wavelength sufficient to activate the optical signal in the animal’s brain cells, transmit the laser radiation through the optical probe fiber to its distal end and register an optical signal from animal brain cells, the tip is fixed directly to the animal’s skull or pre-fixed SG on the skull with respect to the outer tip holder when receiving a predetermined optical signal amplitude or a predetermined image marrow cells. 2. The method according to p. 1, characterized in that the signal from the brain cells of the animal is observed through a computer-based optical recording and control system. 3. The method according to p. 2, characterized in that the fiber of the optical fiber probe is introduced using an automated stereotaxis preparation agent connected to a computer. 4. The method according to. 1, characterized in that the tip is fixed in two stages, at the first stage, the tip is fixed with quick-drying glue, in the second stage, after a period of 2 to 8 minutes, the tip is finally fixed with a mixture of quick-drying glue and dental cement. 5. The method according to p. 3, characterized in that the optical fiber of the optical fiber probe is a multi-core optical fiber with thousands of individual micron-sized optical fibers, the tip is fixed when receiving the selected image from the distal end of the fiber. 6. The method according to p. 3, characterized in that the optical fiber is a single core fiber with an outer diameter of not more than 250 microns, the tip is fixed when receiving the selected intensity of the optical signal from the brain cells of the animal. 7. The method according to p. 6, characterized in that the fiber optic probe is fixed on the skull of the animal upon receipt of the maximum optical signal from brain cells. 8. The method according to p. 6, characterized in that the optical fiber probe is fixed when an optical signal from animal brain cells occurs. 9. The method according to p. 6, characterized in that the optical fiber probe is fixed while reducing the optical signal from animal brain cells.

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