Nucleotide constructs, adeno-associated virus vectors, systems and methods for detecting neural activity

Abstract

The embodiment of the invention generally relates to the technical field of bioengineering, in particular to a nucleotide construct, an adeno-associated virus vector, and a system and a method for detecting neural activity. The nucleotide construct comprises an open reading frame, and the open reading frame comprises a voltage response domain coding sequence and a sound wave modulation domain coding sequence. The nucleotide construct is configured to be expressed on an astrocyte membrane under the control of a promoter so as to provide a recombinant protein with a voltage response domain and an acoustic wave modulation domain, so that the recombinant protein can generate corresponding reversible conformation change in response to membrane potential change of the astrocyte, the acoustic characteristics of the recombinant protein can be changed by the reversible conformation change. In this way, the recombinant protein can sense changes in membrane potential and modulate the reflection characteristics to ultrasonic waves.

Description

TECHNICAL FIELD

[0001] Embodiments of the present disclosure generally relate to the field of bioengineering, and in particular, to a nucleotide construct, an adeno-associated viral vector, a system and method for detecting neural activity. BACKGROUND

[0002] In recent years, neural network activity decoding technology has shown important value in the fields of brain-computer interface, neurological disease diagnosis and treatment, etc. In related prior art, implanted electrodes have high invasiveness and limited lifespan, optical methods of non-invasive technology have small imaging windows and are limited by transparency and scattering, and there is a need for technology that can monitor local neural network state with high throughput and decode with low invasiveness. SUMMARY

[0003] Embodiments of the present disclosure provide a nucleotide construct, an adeno-associated viral vector, a system and method for detecting neural activity, aiming to solve one or more of the above problems and other potential problems.

[0004] According to a first aspect of the present disclosure, a nucleotide construct is provided, comprising an open reading frame including a voltage response domain coding sequence and a sound wave modulation domain coding sequence. The nucleotide construct is configured to be expressed on a membrane of a stellate cell under control of a promoter to provide a recombinant protein having a voltage response domain and a sound wave modulation domain, such that the recombinant protein is capable of producing a corresponding reversible conformational change in response to a membrane potential change of the stellate cell, the reversible conformational change being capable of changing an acoustic property of the recombinant protein.

[0005] According to a second aspect of the present disclosure, an adeno-associated viral vector is provided, comprising a promoter configured to achieve specific expression of a stellate cell. In addition, the adeno-associated viral vector further comprises a nucleotide construct, which is operably linked to the promoter, and the nucleotide construct comprises an open reading frame including a voltage response domain coding sequence and a sound wave modulation domain coding sequence. The nucleotide construct is configured to be expressed on a membrane of a stellate cell under control of the promoter to provide a recombinant protein having a voltage response domain and a sound wave modulation domain, such that the recombinant protein is capable of producing a corresponding reversible conformational change in response to a membrane potential change of the stellate cell, the reversible conformational change being capable of changing an acoustic property of the recombinant protein.

[0006] According to a third aspect of the present disclosure, a system for detecting neural activity is provided, comprising the nucleotide construct of the first aspect described above or the adeno-associated viral vector of the second aspect described above. In addition, the system further comprises an ultrasonic transducer array configured to provide ultrasonic waves to a target brain region and detect backscattered echoes of the ultrasonic waves, wherein at least part of stellate cells of the target brain region comprise the recombinant protein provided by the expression of the nucleotide construct on the membrane of the stellate cells.

[0007] According to a fourth aspect of the present disclosure, there is provided a method for detecting neural activity, the method comprising transmitting an ultrasound wave focused on a target brain region, wherein at least a portion of astrocytes of the target brain region comprises a recombinant protein provided by expression of the nucleotide construct of the first aspect on the membrane of the astrocytes. The method further comprises acquiring a backscattered echo signal of the ultrasound wave in response to the transmission of the ultrasound wave. In addition, the method further comprises determining neural activity of the target brain region based on the backscattered echo signal, wherein the neural activity comprises one or more of rest, excitation and inhibition. BRIEF DESCRIPTION OF DRAWINGS

[0008] The above and other objects, features and advantages of embodiments of the present disclosure will become more apparent from the following detailed description read in conjunction with the accompanying drawings. In the drawings, several embodiments of the present disclosure are shown with example, but not limiting, illustrations.

[0009] Figure 1 A schematic diagram showing an example system for detecting neural activity according to embodiments of the present disclosure is shown.

[0010] Figure 2 A schematic diagram showing an example ultrasound device according to embodiments of the present disclosure is shown.

[0011] Figure 3 A schematic diagram showing an example demodulation circuit according to embodiments of the present disclosure is shown.

[0012] Figure 4 A flowchart showing an example method for detecting neural activity according to embodiments of the present disclosure is shown.

[0013] Figure 5 A waveform diagram showing relevant ultrasound signals in an example method for detecting neural activity according to embodiments of the present disclosure is shown.

[0014] In the various drawings, identical or corresponding reference numbers represent identical or corresponding parts. DETAILED DESCRIPTION

[0015] Embodiments of the present disclosure will be described hereinafter with reference to the accompanying drawings. While the present disclosure is susceptible to various modifications and alternative forms, specific embodiments thereof are shown by way of example in the drawings and will herein be described in detail. It should be understood, however, that there is no intent to limit the present disclosure to the particular embodiments disclosed, but on the contrary, the disclosure is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present disclosure. As such, the

[0016] In the description of embodiments of the disclosure, the term "includes" and its similar terms are to be understood as open-ended, i.e., "including but not limited to". The term "based on" is to be understood as "based, at least in part, on". The term "one embodiment" or "the embodiment" is to be understood as "at least one embodiment". The terms "first", "second" and the like can refer to different or identical objects. Other explicit or implicit definitions can also be included below.

[0017] As mentioned before, the recording of neural activity is mostly dependent on electrode implantation, optogenetic imaging or blood flow / oxygenation related ultrasound imaging. However, the implanted electrodes are highly invasive and have limited lifetime, the optical methods have small imaging window and are limited by transparency and scattering, and the blood flow related signals are time-lagged and do not directly reflect the electrical activity.

[0018] In view of this, embodiments of the present disclosure provide a nucleotide construct comprising an open reading frame comprising a voltage response domain coding sequence and an acoustic wave modulation domain coding sequence. The nucleotide construct is configured to be expressed on the membrane of astrocytes under the control of a promoter to provide a recombinant protein having a voltage response domain and an acoustic wave modulation domain, so that the recombinant protein (also referred to herein as AABP, Astrocyte-Expressed Acousto-Electric Backscatter Protein) can respond to changes in the membrane potential of astrocytes to produce corresponding reversible conformational changes that can change the acoustic properties of the recombinant protein.

[0019] By expressing the recombinant protein on the cell membrane of astrocytes, astrocytes are widely distributed in the whole brain, form a triad structure with tens of thousands of synapses, and their membrane potential can slowly respond to local neuronal activity, which makes astrocytes an ideal "proxy sensor" of neural state. Based on the recombinant protein expressed by astrocytes, the voltage response domain and the acoustic wave modulation domain are fused, which can respond to changes in the membrane potential of astrocytes to produce reversible conformational changes that change the acoustic properties of the recombinant protein, and then modulate the emission characteristics of ultrasound waves by astrocytes. For example, when the surrounding neurons are excited, the membrane potential V mAs the temperature is raised, the conformation of the recombinant protein becomes swollen or unfolded, causing the local ultrasound backscattering intensity and phase of the recombinant protein to change. The external ultrasound transducer array system can detect the slight changes in this backscattering echo by pulse and infer the active state of the neural network in this region. In this way, the nucleotide construct can build a "molecular level neural state interface" based on astrocytes, so as to record neural activity through an external non-penetrating ultrasound wave detection device. In addition, the tolerance of glial cells to exogenous proteins is stronger than that of neurons, and they are more suitable for constructing a functional protein system that needs to exist for a long time.

[0020] The conformational change of the protein refers to the change of the three-dimensional spatial structure of the protein molecule. During the conformational change, the types, quantities and arrangement orders of the amino acids constituting the protein do not change. The change of the spatial structure of the protein molecule can include the change of the folding mode of the protein chain, the relative position of the secondary structural elements (such as alpha helix, beta fold) of the protein molecule, the angle between the domains, the direction of the side chain group, etc. In one or more embodiments of the present disclosure, the reversible conformational change can include a change capable of affecting one or more of the density, stiffness and reflectivity of the surface of the recombinant protein, and the change of these acoustic properties can affect the way the surface of the recombinant protein interacts with the sound wave, thereby changing the acoustic impedance of the protein surface, so as to modulate the backscattering echo intensity or frequency of the external received ultrasound wave. Among them, the density of the protein surface is a component of the acoustic impedance, which refers to the effective density of the complex composed of the protein body and the hydration layer closely combined therewith and the difference between the complex and the surrounding free water. The stiffness of the protein surface is another key component of the acoustic impedance, which refers to the ability of the protein (especially the key region of its surface) to resist deformation, which is manifested as the viscoelastic modulus, which directly affects the sound speed and also affects the sound attenuation. The reflectivity of the protein surface is determined by the acoustic impedance contrast between the protein body and its surrounding medium (water), and this contrast is determined by the density and stiffness of the protein.

[0021] Voltage-Sensing Domain (VSD) constitutes the input channel of recombinant proteins to perceive neural activities. Within the range of membrane potential changes of astrocytes, reversible conformational changes can occur. In one or more embodiments of the present disclosure, the voltage-sensing domain can include S4 helix, regularly arranged positively charged amino acid residues on the S4 helix can produce displacement when the membrane electric field changes, thereby initiating the conformational change of the entire protein. In one or more embodiments of the present disclosure, the voltage-sensing domain can include engineered protein fragments derived from ASAP2s, which are a class of voltage-sensing proteins based on Ci-VSP S4 (Ciona intestinalis Voltage Sensor-containing Phosphatase) construction, whose core voltage-sensing function depends on the S4 transmembrane helix structure. The S4 structure is rich in positively charged amino acids, which can undergo conformational adjustment when the membrane potential changes, thereby driving the downstream domain to produce physical signal changes. In one or more embodiments of the present disclosure, the S4 helix structure can also be provided by voltage-sensitive proteins or VSFP (Voltage-Sensitive Fluorescent Protein). Among them, voltage-sensitive proteins are proteins that exist in nature and can change their conformation and function in response to changes in membrane potential. VSFP is an artificially designed or modified fluorescent protein that uses the voltage-sensing mechanism of the S4 helix structure to report membrane voltage changes through luminescence. For example, the voltage-sensing domain can include chimeric protein ArcLight. ArcLight is composed of two main parts, voltage-sensing domain and fluorescent reporting unit, and has the characteristics of high sensitivity (especially to full signal) and large signal change. When the cell membrane potential is depolarized, the voltage-sensing domain undergoes a conformational change. This conformational change is transmitted through the connecting part, causing the conformation of the fused fluorescent protein to also change, which ultimately causes the fluorescence intensity of the fluorescent protein to decrease.

[0022] The acoustic modulation domain constitutes the output part of the recombined protein sensing neural activity, which has the ability to change the structural acoustic impedance, i.e., the change in the conformation of the acoustic modulation domain will cause the acoustic properties (such as density, stiffness, reflectivity, etc.) of the protein to change significantly, thereby modulating the backscattering echo intensity or frequency of the externally received ultrasonic waves. In one or more embodiments of the present disclosure, the acoustic modulation domain can include a GVP (Gas vesicle protein) like cavity structure, which can spontaneously assemble to form a closed, hollow-internal micro- or nano-scale structure similar to a natural gas vesicle. For example, the acoustic modulation domain can include a GvpA modified β-barrel structure (derived from Halobacterium), which is an artificial protein structure with β-barrel characteristics based on the GvpA protein derived from Halobacterium. In one or more embodiments of the present disclosure, the acoustic modulation domain can include a cavity-type protein scaffold composed of repeated β-fold modules, such as a β-barrel or β-solenoid structure. The multiple β-fold fragments in such a structure are arranged in a periodic manner and can undergo reversible microstructure changes under the action of ultrasonic waves, changing the overall elastic modulus and local acoustic impedance, thereby achieving modulation of the ultrasonic scattering or propagation characteristics. In one or more embodiments of the present disclosure, the cavity-type protein scaffold can also include a controllable elastic region rich in specific hydrophobic / hydrophilic sequences. The controllable elastic region refers to a specific amino acid sequence composed of a proportionally adjustable hydrophilic and hydrophobic residue, which can form a random coil, coiled coil, or elastin-like polypeptide-like flexible conformation, and has a mechanical response capability sensitive to external stress (such as acoustic pressure). Through rational design, such structures can produce predictable conformational changes under the action of specific acoustic waves, serving as the functional core for ultrasonic modulation or backscattering modulation.

[0023] In one or more embodiments of the present disclosure, the voltage response domain and the acoustic modulation domain can be connected by a flexible chain. In one or more embodiments of the present disclosure, the flexible chain can be a repeating sequence based on glycine (Gly) and serine (Ser) (e.g., Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Ser), a simple flexible sequence rich in glycine, an elastin-inspired sequence, which can provide sufficient flexibility, length, and chemical compatibility, ensure that the voltage response domain and the acoustic modulation domain are fused in one ORF (open reading frame) while maintaining a certain independence in space, reduce the steric hindrance between each other, and correctly fold and effectively function.

[0024] In one or more embodiments of the present disclosure, the open reading frame can further comprise a membrane localization coding sequence configured to ensure that the protein encoded by the nucleotide construct is localized to the cell membrane of the astrocyte. In one or more embodiments of the present disclosure, the membrane localization coding sequence can comprise one or more of a CAAX motif, a transmembrane domain coding sequence, and a PDZ domain coding sequence as a membrane-anchoring signal to ensure that the recombinant protein is expressed on the cell membrane.

[0025] In one or more embodiments of the present disclosure, the amino acid sequence of the recombinant protein can be: MVDSEYENQVSLQLPVELKDLRRLLESGEEEAEEAAAAAEFGGSGGSGGSGTGLQIGRIVRRLVRGAYGGLQLAERAEGVRAVQKAADELGRTFDGQGVRFGAGLAGGAAGGFAAGALGGSLRDGVAGGLLGAGGAFALQSGSGAGRGLAAGGLAGGGAGALDQLGGALFGAAGGRGGGRYGGGGLALAAAGGAAGGGGLAGALAAASGCMSCKCVLS.

[0026] The nucleotide construct can be implemented to achieve specific, stable and safe membrane localization expression in astrocytes through viral vector-mediated gene introduction. In one or more embodiments of the present disclosure, the nucleotide construct can be introduced into an adeno-associated virus (AAV) vector and delivered to the target tissue or the whole body through injection or the like to infect the target cells for expression. The adeno-associated virus vector can comprise a promoter operatively linked to the nucleotide construct and configured to achieve specific expression in astrocytes. Compared with gene modification of neurons, astrocytes are more susceptible to viral vectors such as AAV (transduction of neurons usually depends more on the serotype selection of AAV and specific neuronal promoters such as Syn or CaMKII, and shows greater variability in different brain regions and developmental stages), and most types still have high gene expression capacity in a non-dividing state (while neurons as signal output units are more sensitive to membrane protein loading, and excessive expression can interfere with action potential firing, synaptic transmission and even trigger toxic reactions).

[0027] The "molecular level neural state interface" provided in this way is safer, and the expression space and structure are more stable.

[0028] In one or more embodiments of the present disclosure, the membrane-located coding sequence can be a glial cell-specific promoter, for example, one or more of GFAP (Glial Fibrillary Acidic Protein) promoter and Aldh1l1 (Aldehyde Dehydrogenase 1 Family Member L1) promoter, etc. The GFAP promoter has higher expression intensity and the Aldh1l1 promoter has higher specificity. In one or more embodiments of the present disclosure, the genome concentration of the adeno-associated virus vector is 10 12 -10 13 vg / mL, which can optimize the expression efficiency of the nucleotide construct. The genome concentration represents the number of complete adeno-associated virus vectors containing the target gene per milliliter. Assuming that the expression amount of recombinant protein per astrocyte is 10 4 ~10 5 orders of magnitude, and the membrane area of astrocytes is 50,000 μm 2 ~100,000 μm 2 , the corresponding expression density is about 1-5 per μm 2 , which can achieve a greater than 10% difference in back reflection. When 10 6 ~10 7 proteins are coordinately expressed in astrocytes in the target brain area, it can produce identifiable backscattering echo modulation within the spatial resolution range of the external ultrasonic transducer array.

[0029] Figure 1 shows a schematic diagram of a system for detecting neural activity according to an embodiment of the present disclosure, such as Figure 1As shown, in one or more embodiments of the present disclosure, the system 100 includes a nucleotide construct 111 of one or more embodiments of the present disclosure or an adeno-associated viral vector 101 of one or more embodiments of the present disclosure. The nucleotide construct 111 is capable of being delivered to the target brain region 102 by the adeno-associated viral vector 101 (e.g., by stereotactic injection or cisterna magna injection of the adeno-associated viral vector), infecting the astrocyte 103 for expression, providing the infected astrocyte membrane of the target brain region with a recombinant protein having a voltage response domain and a sound wave modulation domain. The system 100 further includes an ultrasound device 104 having an ultrasound transducer array 141 configured to provide ultrasound waves to the target brain region 102 and detect backscattered echoes of the ultrasound waves. The astrocyte forms a triad structure with thousands of synapses, capable of detecting the neurotransmitter signals released by neurons through glutamate receptors, GABA receptors, etc. on the membrane. For example, when the neuron is in an excited state, a large amount of glutamate is released, triggering the membrane potential Vmof the astrocyte to depolarize from -80 mV to about -65 mV; when the neuron is in an inhibited state, the GABA signal may also trigger the membrane potential Vmto slightly hyperpolarize. These changes in membrane potential, although slow, have obvious spatial integration characteristics and physiological significance. The recombinant protein provided by the nucleotide construct 111 expressed on the astrocyte 103 fuses the voltage response domain and the sound wave modulation domain, and is capable of responding to such changes in membrane potential, producing changes in conformation, and in turn modulating its reflection characteristics of the ultrasound waves. For example, when the neuron around the astrocyte 103 is enhanced in excitation, the membrane potential Vmof the astrocyte rises, and the conformation of the recombinant protein becomes swollen or unfolded, causing the ultrasound backscattering intensity and phase of the local recombinant protein to change accordingly. In this way, the system can detect such small changes in backscattered echoes through ultrasound wave pulses, and infer the active state of the neural network in the region. m m

[0030] The expression of the nucleotide construct can be carried out under normal brain physiological conditions, and in one or more embodiments of the present disclosure, the viral titer and injection method can be adjusted to obtain a more stable, uniformly distributed, and non-toxic smearing expression effect. In one or more embodiments of the present disclosure, the viral titer (genome titer of the adeno-associated viral vector) can be 10 12 vg / mL ~ 10 13 vg / mL. Assuming that the expression amount of the recombinant protein per astrocyte is in the order of 10 4 ~ 10 5 , and the astrocyte membrane area is 50,000 μm 2 ~ 100,000 μm 2 , the corresponding expression density is about 1 2 ​​1-5, about to achieve greater than 10% of the back reflection difference. When there are 10 6 ~10 7 When 10 proteins are coordinately expressed in the astrocytes of the target brain region, identifiable backscattering echo modulation can be generated within the spatial resolution range of the external ultrasound transducer array.

[0031] In one or more embodiments of the present disclosure, the ultrasound transducer array can be attached to a head-mounted, for example, the ultrasound transducer array can be implemented as a flexible probe array or a helmet structure, attached to the head of the detection object, to detect the neural activity of the detection object without penetrating the skull. In one or more embodiments of the present disclosure, the ultrasound transducer array can also be configured to be implanted in the epidural space of the detection object (i.e., the area between the dura mater and the skull), enabling higher precision (less than 30 pm) imaging or regulation while avoiding the skull barrier, reducing damage to brain tissue, and reducing the risk of infection.

[0032] In one or more embodiments of the present disclosure, the ultrasound transducer array 141 can emit mid-high frequency, low power, high time resolution ultrasound waves to the target brain region, enabling sensitive detection of recombined protein modulation signals within a safer range. In one or more embodiments of the present disclosure, the frequency of the ultrasound waves can be 5 MHz ~20 MHz. In one or more embodiments of the present disclosure, the ultrasound wave signal can be a short period pulse emitted at a kHZ level repetition frequency, focused or scanned spatially by a phased array to cover the target brain region 102. The recombined proteins on the astrocytes 103 of the target brain region fuse the voltage response domain and the acoustic modulation domain, and their conformation is driven by changes in the membrane potential of the glial cells. When the activity of the neurons increases (e.g., the membrane potential V m When depolarized from -80 mV to -65 mV, the protein conformation changes slightly, triggering changes in the local ultrasound backscattering intensity (amplitude modulation) or phase delay of the waveform (phase modulation). This modulation effect is usually manifested as an increase of about 10% in the local backscattering signal, or a few degrees of phase shift. After receiving the backscattering echo, the ultrasound transducer array 141 demodulates the modulation features through differential images, phase-locked amplification, spectral analysis, etc. This enables the system 100 to ultimately recognize the evolution patterns of these modulation signals in time and space, and to establish a correlation with neural network activity, thereby realizing real-time decoding of the local neural state of the brain.

[0033] In one or more embodiments of the present disclosure, the number of channels of the ultrasound transducer array 141 is not less than 64 (e.g., 64~256 channels), which is capable of resolving the backscatter modulation at the level of astrocytes within the millimeter scale. In one or more embodiments of the present disclosure, the element pitch of the ultrasound transducer 141 can be less than half of the wavelength to avoid the interference of the beam sidelobes. Taking 10MHz ultrasound wave as an example, the wavelength in the brain tissue is about 150μm, and the element pitch is suggested to be controlled at <75μm. In one or more embodiments of the present disclosure, the element pitch is not greater than 150μm.

[0034] In one or more embodiments of the present disclosure, the ultrasound transducer array can be a flexible ultrasound transducer array implemented based on flexible micro-machined ultrasonic transducers (MUT). In one or more embodiments of the present disclosure, the flexible ultrasound transducer array can employ piezoelectric micro-machined ultrasonic transducers (PMUT) or capacitive micro-machined ultrasonic transducers (CMUT) as the elements. The flexible micro-machined ultrasonic transducer array is capable of closely adhering to the irregular skull surface or dura mater, reducing the air gap acoustic attenuation. The thickness of the flexible ultrasound transducer array can be 0.1-0.5mm, so that it can be implanted epidurally through a small bone window, avoiding the compression damage to the brain tissue. In addition, the elastic modulus of the flexible ultrasound transducer array can be matched with the brain tissue and dura mater, reducing the chronic inflammatory reaction and the scratch to the brain tissue. The flexible ultrasound transducer array is attached to the head-mounted device, which can naturally bend with the head movement, avoiding the motion artifacts caused by the micro-displacement of the rigid probe.

[0035] In one or more embodiments of the present disclosure, the arrangement of the ultrasound transducer array can be a one-dimensional linear array, a two-dimensional matrix array or a curved array, which can match the curved surface of the skull or dura mater, and better contact the skull or dura mater.

[0036] Figure 2 A schematic diagram of an ultrasound device according to an embodiment of the present disclosure is shown as Figure 2 As shown, in one or more embodiments of the present disclosure, the ultrasound device 104 can include a computing device having one or more processors and an internal storage device electrically connected with the processors. The ultrasound device can also include an ultrasound transducer array 141 for transmitting one or more amplitude-modulated ultrasound pulse sequences and detecting backscatter echoes. In addition, the ultrasound device can also include a transmit / receive switch, an amplifier, an analog filter, a demodulation circuit 122, and a digital-to-analog converter transmit / receive switch in electronic communication with the ultrasound transducer array 141, the amplifier and the analog-to-digital converter, and the demodulation circuit. The analog filter is in electronic communication with the amplifier and the demodulation circuit. The described electrical communication connections between the components of the ultrasound device 104 can provide power and / or transmit data.

[0037] In one or more embodiments of the present disclosure, the ultrasound device 104 can include a transmit / receive switch that controls the transmission of voltage pulses to the ultrasound transducer elements and the reception of electrical signals containing backscatter data from the ultrasound transducer elements. The transmit / receive switch can isolate the transmit circuitry from the receive circuitry. For example, Figure 2 The ultrasound device 104 in FIG. 1 includes a transmit / receive switch that can be switched between two states: receiving electrical signals containing backscatter data from the array of ultrasound transducer elements and transmitting voltage pulses to the ultrasound transducer elements.

[0038] In one or more embodiments of the present disclosure, the computing device can be configured or be able to be configured (e.g., based on input from an input device) by an operator to send input data or raw or processed image data to a display for display via a communication interface. The computing device can also be configured or be able to be configured (e.g., based on input from an input device) by an operator to send control signals to the amplifier and digital-to-analog converter. The input device and the output device can be components of the ultrasound device or components of the system 100. The amplifier and the digital-to-analog converter can convert the control signals to voltage pulses and transmit them to the ultrasound transducer elements in the array of ultrasound transducer elements via the transmit / receive switch, thereby exciting the transducer elements to produce ultrasound pulses and detect backscatter echoes. The ultrasound waves detected by the array of ultrasound transducer elements produce electrical backscatter signals that are transmitted to the amplifier and the analog filter. The amplifier and the analog filter amplify and suppress background noise the electrical backscatter signals before transmitting them to the demodulation circuit 142, which extracts one or more of the amplitude, phase, and frequency spectrum of the backscatter echoes, identifies the temporal and spatial evolution patterns of the backscatter echoes modulated by the recombinant proteins, and correlates the patterns to neural network activity, thereby enabling the decoding of the local neural state of the brain.

[0039] Figure 3 A schematic diagram of a demodulation circuit according to an embodiment of the present disclosure is shown in FIG. 2. Figure 3As shown, in one or more embodiments of the present disclosure, the demodulation circuit 142 can include a lock-in amplifier 1421, and the lock-in detection module of the lock-in amplifier can extract the amplitude and phase information of the signal component consistent with the reference frequency (i.e., the frequency of the transmitted ultrasonic wave) while suppressing noise and interference of other frequencies, and extract the envelope of the backscattered echo signal, thereby extracting the amplitude and phase of the backscattered echo signal. In one or more embodiments of the present disclosure, the demodulation circuit 142 can also include a compressed sensing reconstruction module 1422. The compressed sensing reconstruction module 1422 realizes high-speed signal acquisition and reconstruction based on compressed sensing technology, and can include an analog-to-digital converter for high-frequency signal capture and an FPGA (field programmable gate array) for real-time processing of signals and execution of sparse decoding algorithms (such as OMP, CoSaMP, etc.). The compressed sensing reconstruction module can reconstruct and output the high spatial resolution ultrasonic spectrum corresponding to the backscattered echo signal under sparse sampling conditions.

[0040] In one or more embodiments of the present disclosure, the system can also include a neural activity decoding model 105, which can be obtained by training an initial machine learning model using a training set. In one or more embodiments of the present disclosure, the neural activity decoding model can be deployed locally on a computing device in Figure 2 , or can be deployed locally on one or more computing devices or one or more processors of the system 100 in Figure 1 . The machine learning model can also be deployed in the cloud or an edge computing device and invoked by Figure 2 a computing device or Figure 1 another computing device or processor of the system 100. In one or more embodiments of the present disclosure, the neural activity decoding model 105 can determine the neural activity decoding result of the target brain region based on the demodulation result of the demodulation circuit 104 (e.g., can be based on the envelope waveform extracted from the backscattered echo signal, can be based on the high spatial resolution ultrasonic spectrum sequence reconstructed from the backscattered echo signal, or can be based on both the envelope waveform extracted from the backscattered echo signal and the high spatial resolution ultrasonic spectrum sequence reconstructed from the backscattered echo signal). In one or more embodiments of the present disclosure, the neural activity decoding result can include labels such as rest, excitation, and inhibition, and different label types indicate different neural activity states. In one or more embodiments of the present disclosure, the neural activity decoding result can also indicate a continuous indicator of the neural activity state (e.g., an excitation intensity value, etc.).

[0041] In one or more embodiments of the present disclosure, the training set can include a plurality of training samples, each training sample including a sample backscatter echo signal, the sample backscatter echo signal being labeled with a corresponding sample neural activity decoding result. In one or more embodiments of the present disclosure, the sample neural activity decoding result can include labels such as rest, excitation, and inhibition, etc., indicating different neural activity states through different label types. In one or more embodiments of the present disclosure, the sample neural activity decoding result can also include corresponding detection of corresponding behavioral intentions, such as postures and movements of the body and limbs, etc. In one or more embodiments of the present disclosure, the sample neural activity decoding result can also indicate continuous indicators of neural activity states (e.g., excitation intensity values, etc.). In one or more embodiments of the present disclosure, the sample neural decoding result can be multi-source data composed of neural activity labels indicating neural activity states, movement intention labels indicating movement intentions, and LFP (local field potential) physiological electrical signals collected by intracranial electrodes, etc. It should be noted that the user data obtained by the present disclosure is authorized by the user and does not involve user privacy. In the training process of the initial machine learning model, when the sample neural activity decoding result is a discrete indicator of neural activity state (e.g., classification such as rest, excitation, and inhibition, etc.), cross-entropy loss can be used as the objective function. When the sample neural activity decoding is a continuous indicator of neural activity state, mean square error can be used as the objective function. When the corresponding sample neural activity decoding structure includes both discrete indicators and continuous indicators, a multi-task loss including cross-entropy loss and mean square error can be introduced to optimize classification and continuous indicator prediction at the same time.

[0042] In one or more embodiments of the present disclosure, the system can also include a communication interface and a display connected to the communication interface. The computing device is configured or can be configured to transmit data through the communication interface so as to display the emission pulse sequence, the raw backscatter echo data, and the processed image data on the display. The system 100 can also include a communication interface in communication with an input device for receiving input from an operator of the system.

[0043] The following describes a method for detecting neural activity according to embodiments of the present disclosure. Figure 4 A flowchart of a method 400 for detecting neural activity according to embodiments of the present disclosure is shown. Figure 4 The method 400 can be performed by the computing device in the ultrasound device 100 shown in Figure 2 The method 400 can be performed by the computing device in the ultrasound device 100 shown in Figure 4As shown, at box 402, ultrasound waves are emitted and focused on a target brain region, wherein at least a portion of the astrocytes in the target brain region include recombinant proteins that can be provided by expression of nucleotide construct 111 on the astrocyte membrane. In one or more embodiments of this disclosure, selected ultrasound transducers in a 256-channel array (e.g., 64 channels at the center of the array) can be controlled to emit 10 MHz high-frequency pulses to the target brain region, each pulse width approximately 2 μs, focused on the target brain region, with the emitted waveform as shown. Figure 5 The transmitted signal is shown in Figure 501.

[0044] At frame 404, in response to the emission of ultrasound waves, the backscattered echo signal of the ultrasound waves is acquired. Recombinant proteins in the target brain region, modulated by the membrane potential of astrocytes, alter the waveform characteristics of the backscattered echo, producing differences in echo intensity and phase, such as... Figure 5 As shown in the backscattered echo signal 502, the waveform of the backscattered echo includes an amplitude modulation component. In one or more embodiments of this disclosure, selected ultrasonic transducers in a 256-channel array can be controlled to receive the backscattered echo of ultrasonic waves to obtain a backscattered echo signal. In one or more embodiments of this disclosure, the backscattered echo signal can be passed through a low-noise preamplifier, followed by bandpass filtering (e.g., 10 ± 2 MHz) to remove background noise, resulting in a preprocessed backscattered echo signal 503 (see [link to documentation]). Figure 5 ).

[0045] At box 406, the decoding result of neural activity in the target brain region is determined based on the backscattered echo signal. In one or more embodiments of this disclosure, one or more of the amplitude, phase, and corresponding high spatial resolution ultrasound atlas of the backscattered echo can be determined based on the backscattered echo signal. Subsequently, the neural activity of the target brain region corresponding to the backscattered echo is determined based on the amplitude, phase, and one or more of the corresponding high spatial resolution ultrasound atlas of the backscattered echo. In one or more embodiments of this disclosure, the preprocessed backscattered echo signal 503 can be input to the lock-in amplifier 1421, and after being extracted by the lock-in detection module of the lock-in amplifier, the envelope 504 of the backscattered echo is output (see...). Figure 5 In one or more embodiments of this disclosure, the preprocessed backscattered echo signal can also be input to the compressed sensing reconstruction module 1422 to obtain a high spatial resolution ultrasound atlas of the backscattered echo. In one or more embodiments of this disclosure, the envelope of the backscattered echo and the high spatial resolution ultrasound atlas sequence can be input to the neural activity decoding model 105, and the corresponding neural activity decoding result can be obtained through the neural activity decoding model.

[0046] Having described above several embodiments of the disclosure, any modifications and variations that fall within the scope of the described embodiments are also intended to be within the scope of the disclosure. As will be apparent to those skilled in the art, some modifications and variations to the embodiments described above can be practiced while staying within the scope and spirit of the described embodiments. The foregoing description of the described embodiments has been presented for the purposes of illustration and description. It is not intended to be exhaustive or to limit the described embodiments to the precise form disclosed. Many modifications and variations are possible in light of the above teachings. It is intended that the disclosed embodiments be limited only by the claims.

Claims

1. A nucleotide construct, characterized in that, include: An open reading frame, comprising a voltage response domain encoded sequence and an acoustic modulation domain encoded sequence, and The nucleotide construct is configured to be expressed on the astrocyte membrane under the control of a promoter to provide a recombinant protein having a voltage-response domain and an acoustic modulation domain, such that the recombinant protein can respond to changes in the membrane potential of the astrocyte to produce a corresponding reversible conformational change, which can alter the acoustic properties of the recombinant protein.

2. The nucleotide construct according to claim 1, characterized in that, The acoustic features include one or more of density, stiffness, and reflectivity.

3. The nucleotide construct according to claim 1, characterized in that, The voltage response domain includes an S4 spiral structure.

4. The nucleotide construct according to claim 3, characterized in that, The voltage response domain includes voltage-sensitive proteins or VSFPs.

5. The nucleotide construct according to claim 4, characterized in that, The voltage response domain includes ArcLight.

6. The nucleotide construct according to claim 1, characterized in that, The acoustic modulation domain includes a cavity-type protein backbone.

7. The nucleotide construct according to claim 6, characterized in that, The cavity-type protein backbone includes multiple β-sheet modules arranged in a periodic manner.

8. The nucleotide construct according to claim 6, characterized in that, The cavity-type protein backbone includes a controllable elastic region rich in specific hydrophobic / hydrophilic sequences.

9. The nucleotide construct according to claim 1, characterized in that, The voltage response domain and the acoustic modulation domain are connected by a flexible chain.

10. The nucleotide construct according to claim 1, characterized in that, The open reading frame also includes: A membrane localization coding sequence is configured to ensure that the protein encoded by the nucleotide construct is localized to the astrocyte membrane.

11. The nucleotide construct according to claim 10, characterized in that, The membrane localization coding sequence includes one or more of the following: CAAX motif, transmembrane domain coding sequence, and PDZ domain coding.

12. An adeno-associated virus vector, characterized in that, include: The promoter is configured to achieve specific expression in astrocytes; as well as A nucleotide construct, effectively linked to the promoter, includes an open reading frame (OPF) comprising a voltage-response domain coding sequence and an acoustic modulation domain coding sequence. The nucleotide construct is configured to be expressed on an astrocyte membrane under the control of the promoter to provide a recombinant protein having both a voltage-response domain and an acoustic modulation domain, such that the recombinant protein responds to changes in the membrane potential of the astrocyte by undergoing a corresponding reversible conformational change, which alters the acoustic properties of the recombinant protein.

13. The adeno-associated virus vector according to claim 12, characterized in that, The acoustic characteristics include one or more of density, stiffness, and emissivity.

14. The adeno-associated virus vector according to claim 12, characterized in that, The voltage response domain includes an S4 spiral structure.

15. The adeno-associated virus vector according to claim 14, characterized in that, The voltage response domain includes voltage-sensitive proteins or VSFPs.

16. The adeno-associated virus vector according to claim 15, characterized in that, The voltage response domain includes ArcLight.

17. The adeno-associated virus vector according to claim 12, characterized in that, The acoustic modulation domain includes a cavity-type protein backbone.

18. The adeno-associated virus vector according to claim 17, characterized in that, The cavity-type protein backbone includes multiple β-sheet modules arranged in a periodic manner.

19. The adeno-associated virus vector according to claim 17, characterized in that, The cavity-type protein backbone includes a controllable elastic region rich in specific hydrophobic / hydrophilic sequences.

20. The adeno-associated virus vector according to claim 12, characterized in that, The voltage response domain and the acoustic modulation domain are connected by a flexible chain.

21. The adeno-associated virus vector according to claim 12, characterized in that, The open reading frame also includes: A membrane localization coding sequence is configured to ensure that the protein encoded by the nucleotide construct is localized to the astrocyte membrane.

22. The adeno-associated virus vector according to claim 21, characterized in that, The membrane localization coding sequence includes one or more of the following: CAAX motif, transmembrane domain coding sequence, and PDZ domain coding.

23. The adeno-associated virus vector according to any one of claims 12-23, characterized in that, The promoter is either the GFAP gene promoter or the ALDH1L1 gene promoter.

24. The adeno-associated virus vector according to any one of claims 12-23, characterized in that, The genomic titer of the adeno-associated virus vector is 10. 12 vg / mL~10 13 vg / mL.

25. A system for detecting neural activity, characterized in that, include: The nucleotide construct according to any one of claims 1-11, or the adeno-associated virus vector according to any one of claims 12-24; as well as An ultrasound transducer array is configured to deliver ultrasound waves to a target brain region and detect the backscattered echoes of the ultrasound waves, wherein at least a portion of the astrocytes in the target brain region comprise the nucleotide construct expressing the provided recombinant protein on the astrocyte membrane.

26. The system according to claim 25, characterized in that, The frequency of the ultrasound is 5 MHz to 20 MHz.

27. The system according to claim 25, characterized in that, The element spacing of the ultrasonic transducer array is no greater than 150 μm.

28. The system according to claim 25, characterized in that, The ultrasonic transducer array is configured to be attached to a head-mounted device.

29. The system according to claim 25, characterized in that, The ultrasound transducer array is configured to be implanted outside the dura mater.

30. The system according to claim 25, characterized in that, The ultrasonic transducer array comprises multiple flexible micromechanical ultrasonic transducer elements.

31. The system according to claim 25, characterized in that, The ultrasonic transducer array can be a linear array, a two-dimensional matrix array, or a curved surface array.

32. The system according to claim 25, characterized in that, The ultrasonic transducer array has no fewer than 64 channels.

33. The system according to claim 25, characterized in that, Also includes: The demodulation circuit is configured to extract one or more of the amplitude, phase, and spectrum of the backscattered echo signal corresponding to the backscattered echo.

34. The system according to claim 33, characterized in that, The demodulation circuit includes: The phase-locked detection module is configured to extract the envelope of the backscattered echo signal.

35. The system according to claim 33, characterized in that, The demodulation circuit further includes: The compressed sensing reconstruction module is configured to reconstruct the high spatial resolution ultrasound map corresponding to the backscattered echo signal.

36. The system according to claim 33, characterized in that, Also includes: The neural activity decoding model is configured to determine the neural activity decoding result of the target brain region based on the backscattered echo signal. The neural activity decoding model is a model obtained by training an initial machine learning model using a training set.

37. A method for detecting neural activity, characterized in that, include: The method involves emitting ultrasound waves focused on a target brain region, wherein at least a portion of the astrocytes in the target brain region comprise recombinant proteins, which are provided by expression of the recombinant proteins in the astrocyte membrane via a nucleotide construct according to any one of claims 1-11. In response to the emission of the ultrasonic wave, the backscattered echo signal of the ultrasonic wave is acquired; and Based on the backscattered echo signal, the decoding result of neural activity in the target brain region is determined.

38. The method according to claim 37, characterized in that, The frequency of the ultrasound is 5MHz to 20MHz.

39. The method according to claim 37, characterized in that, The determination of neural activity in the target brain region based on the backscattered echo signal includes: Based on the backscattered echo signal, determine one or more of the amplitude, phase, and corresponding high spatial resolution ultrasound atlas of the backscattered echo; and The neural activity of the target brain region corresponding to the backscattered echo is determined based on one or more of the amplitude and phase of the backscattered echo and the high spatial resolution ultrasound atlas corresponding to the backscattered echo.

40. The method according to claim 39, characterized in that, The determination of one or more of the amplitude, phase, and high spatial resolution ultrasound image corresponding to the backscattered echo based on the backscattered echo signal includes: Based on the backscattered echo signal, the high spatial resolution ultrasound map corresponding to the backscattered echo is determined by the compressed sensing reconstruction module.

41. The method according to claim 37, characterized in that, The determination of neural activity in the target brain region based on the backscattered echo signal includes: Based on the backscattered echo signal, the envelope of the backscattered echo is extracted using a phase-locked detection module; and Based on the envelope of the backscattered echo, the neural activity of the target brain region corresponding to the backscattered echo is determined.

42. The method according to claim 40, characterized in that, The determination of the target brain region neural activity corresponding to the backscattered echo based on the envelope of the backscattered echo includes: Based on the temporal waveform of the envelope of the backscattered echo, the neural activity of the target brain region corresponding to the backscattered echo is determined by a neural activity decoding model, wherein the neural activity decoding model is a model obtained by training an initial machine learning model using a training set.

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