WO2008034898A1

WO2008034898A1 - An integrated neuro-telemetric apparatus and method for acquiring neuronal action potentials and the wireless digital real-time telemetric transmission thereof

Info

Publication number: WO2008034898A1
Application number: PCT/EP2007/060053
Authority: WO
Inventors: Mathew E. Diamond; Igor Perkon; Erik Zorzin
Original assignee: Scuola Internazionale Superiore Di Studi Avanzati
Priority date: 2006-09-21
Filing date: 2007-09-21
Publication date: 2008-03-27
Also published as: ITPD20060348A1; WO2008034898A8

Abstract

A neuronal telemetry system based on a digital technology is here described. The described integrated system is also suitable for experimental animals, even small ones, and allows to detect and process the detected electrical signals in real time by analog-digital means up to the digital recording thereof in a remote logic unit. The technical description of the invention is divided in the hardware operations implemented by the suggested circuit solutions and a disclosure of the software operations chosen for the control of the devices used.

Description

AN INTEGRATED NEURO-TELEMETRIC APPARATUS AND METHOD FOR ACQUIRING NEURONAL ACTION POTENTIALS AND THE WIRELESS DIGITAL REAL-TIME TELEMETRIC TRANSMISSION THEREOF

Field of the invention

The invention relates to an integrated neuro-telemetric apparatus and method for the capture, the amplification, the analog conditioning, the analog-digital conversion, the digital processing, the wireless digital transmission and reception of neuronal electrical signals in real time, the digital display of such signals, the digital recording of the signals in a computer and to the method for acquiring and processing such signals thereby. State of the art The measurement of neuronal activity has an essential role both in experimental and clinical scientific research. In the case of experimental research, the study of neuronal activity in experimental animals allows investigation of the functional mechanisms of the brain. In clinical research, the same approaches offer the possibility to understand the neuronal basis of neuronal diseases and to develop new therapies to treat them. Different kinds of electrical signals characterize the neuronal activity of an animal or human subject.

If on one hand, the knowledge of the complex relations in the neuronal response to different kinds of stimuli is essential from the physiological point of view, it is even more important to investigate the neuronal activity in pathological conditions, such as in epilepsy. Indeed, brain activity is known to be subject to a fine regulation due to the reciprocal relations between different neuronal systems and many neurological diseases are known to be characterized by deregulation of the neuronal activity of specific systems. A specific kind of neuronal electrical signal involved in these types of study comprises electrical impulses generated by neurons which are characterized by a fluctuation in the electrical voltage having a stereotypical waveform designated as "action potential". The action potential is indeed the basic electrical signal that is generated by neurons and such potentials have generally an amplitude of the order of μV (for instance, between 100 and 500 μV) and a duration of milliseconds (for instance 1 -1 ,5 ms). Neurons are interconnected through multiple connections which form the physical contacts through which they exchange electrical signals. The electrical signals can be characterized by their time sequences and by their distribution in space. The sequences and distributions form the words of a language consisting of messages that correspond to the encoding of functions or complex behaviours such as, for instance, sensations, perceptions, movements and decisions.

The measurement of action potentials is combined with the study of the neuronal language as it provides the data for the mathematical models for the brain functions or parts thereof. In these models, the neurons are represented by nodes for the exchange of information that is transmitted through a network of interconnections designated as "neural network".

The study of the neuro-electrical signals, intended as action potentials, of single neurons is therefore essential for understanding of the mechanisms of regulation of the neuronal responses both in physiological and pathological conditions. Standard research involves the use of experimental animals, very often small rodents, which due to scientific and methodological or legal reasons are suitable to be used in experiments.

The traditional system for the measurement of the neuronal activity is based on the use of electrodes inserted into the brain of a subject and connected by means of an electrical wire to recording systems controlled by a computer. Different kinds of experiments can be carried out and, in general, two broad classes can be distinguished: "acute" and "chronic" experiments.

During acute experiments, the subject is under general anaesthesia, and therefore immobilized and unconscious. However, many of the cerebral and subcerebral regions remain active and their study is still possible. The restriction of the study to these regions under these conditions is useful in relation to the fact that carrying out neuronal recordings from a motionless subject makes the experiment easier from a technical and operative point of view.

Chronic experiments are instead characterized by a conscious and physically active subject. The latter case is interesting because the relations between the behaviour of the concerned subject and the neuronal signals in its brain, which are supposed to control its behaviour, can be studied only in these conditions. The configuration "subject-wire-apparatus" used up to now in traditional systems also in chronic experiments, substantially follows from the necessity of having an electrical wire for the connection of an apparatus which, because of its size, may not be directly installed on the subject. This feature intrinsically leads to different classes of problems.

The first and most apparent drawback of a wired measuring system is the limitation due to the weight of the detection system itself. Indeed, the subject is exposed to the weight of the detection system and the wire required for the transmission of the electrical signal from the subject to the acquisition and analysis apparatus. In small animals, such as small rodents, the weight and size of the apparatus required for measuring the neuronal signals may be extremely limiting, if compared to the weight and thus the muscular strength of the subject. Different forces limit the movement of the subject and the self-perception in the environment. These factors may alter the behaviour of the subject to the extent that the behavioural studies in chronic experiments are distorted. A second drawback that is also a consequence of the presence of the connection wire between the subject and the apparatus for the acquisition and analysis of the electrical signal, is obviously the space limitation to which the subject is constrained, as some of the movements of the subject are inhibited or strongly restrained by the presence of the wire. The typical case occurs when the subject needs to move bypassing obstacles (an example may also be the passage through a tunnel that may be part of experiments on orientation and path recognition): the three-dimensional movement thereof is impeded and thus an entire group of behavioural experiments results impossible. A third and relevant drawback is the electrical problem related to the acquisition and analysis of a relatively weak and short electrical signal, such as the neuronal action potential. Because of the physical distance between subject and apparatus and due to their interconnections, the electrical lines often introduce electric potential differences which are difficult to control and interfering with the recordings. These phenomena are known by the name of ground loops and at worst may harm the subject itself or damage the apparatus. A fourth problem, related to the previous, is that the electrical noise introduced in the power supply network, usually around 50/60 Hz. This leads to two important negative consequences. The first consists in that the high amplitude of these signals may induce the saturation of the signal amplification stages and thus causes their impairment. The second, instead, is purely technological. The saturation of the amplification stage causes a non linear transformation of the signal by the amplification devices. This leads to the generation of harmonic frequencies of the amplified signal, and sub-harmonics in the converted digital signal after being amplified. The filtering of the signal after these stages (for instance, a digital filtering after the acquisition step or an analog filtering immediately after the saturated amplification stage) may generate an output signal displaying features (amplitude, duration, waveform) similar to a real neuronal action potential, but instead being completely artefacted due to interferences with the noise introduced by the power supply network.

This phenomenon may also occur in conditions which are not easily reproducible, such as for instance the power up and shut down of equipment connected to the same electrical network of the experimental set-up but not under the control of the researcher, with the possibility of introducing biased data which are hard to identify during the acquisition phase and therefore difficult to eliminate thereafter. A fifth drawback is the visual interference caused by the wire with automatic systems for the location of the subject. Normally the researcher films the experiment with a video camera. The wire may get in the optical way between the video camera and the subject, thus impeding the correct recognition of the profile of the subject. A sixth drawback consists in the mechanical fragility of the connection between the electrodes and the wire. Sudden movements of the subject or the researcher may damage or break this connection. In the case of small experimental animals the damage may also appear as a lesion on the subject which may even lead to severe physical consequences such as impairing both the health condition of the subject, and not allowing the continuation of the experiment, the preparation of which is based on a training period for that specific subject. To solve these problems different detection systems have been suggested, among which wireless telemetry systems. In US patent 6,052,619 a portable electroencephalograph has been disclosed, in which the connection wires between the detection system for the electrical signal placed on the head of the subject and the acquisition system for the neurological data, is replaced by a digital transmitter. In the described apparatus, the detection electrodes for the electrical signals (in this specific case, brain waves resulting from the electric activity of multiple neurons or neuronal systems) are placed on the head of the subject and are connected to pre-amplifiers consequently connected to differential amplifiers, the output of which is processed by an analog- to-digital converter. The signals are converted from analog to digital at the place in which the patient is situated and are thus transmitted from a digital radio transmitter to a receiving unit formed by a digital radio receiver with a demodulator, an amplifier and filters to discriminate the demodulated signals into bands.

The described system provides that a skilled technician controlls the unit by the capability of both selecting the bands and addressing them to a speaker or a headphone and interpreting the acoustic signal produced by the speaker, which is representative of the electrical signals detected by the electrodes. The advantage of this system is the digital technology both in the acquisition phase of the signal and in the radio transmission phase. A desirable condition is indeed to have signals which are already in a digital form before the transmission to a remote receiving device, as data in a digital format are less subject to errors due to electromagnetic disturbances during the radio transmission phase.

However, the application on small animals is still linked to the problem of the size and weight of the devices. Furthermore, the study of the neuronal language and behaviour requires different apparatuses and methods for the measurement of the neuronal action potentials, which are weaker and faster signals (and therefore more difficult to detect) with respect to the electroencephalograph^ signals (EEG, EKG, EMG). The greatest intrinsic difficulty in the measurement of action potentials with respect to other kinds of neuronal signals implies the necessity of a greater complexity of the devices which collides with the difficulty of an integration thereof on a small enough scale for the non-invasive application on experimental animals.

In US patent 6,654,633 a mobile system for the acquisition of neurological signals (among which neuronal action potentials) that may be used on human beings or experimental animals is described, in which such signals are transmitted by analog methods, instead of digital ones, to a receiving unit for analysis and are converted into digital information.

The advantage of this system is the compromise of adopting an analog transmission technology to reduce the complexity of the radio transmitter, and accordingly reducing the weight and size of the apparatus, with the aim of making it transportable also by an experimental animal and at the same time abandoning in advance some of the advantages, firstly the better reliability of the signals received due to a reduced degradation of the information content of the transmitted signals encoded in a numerical format, of a purely digital technology. It is therefore a first purpose of the present invention to reduce the weight of the telemetry system so as to allow the device to be conveniently carried not only by human patients but also by experimental animals in order to measure the neuronal action potentials. It is also a purpose of the present invention to reduce the size of the telemetry system by eliminating the presence of the wire in order to increase the ability of displaying the conditions of the experiment. It is also a purpose of the present invention to use a battery power supply for the devices of the telemetry system carried by the subject and applied thereto in order to reduce the negative effects of the ground loops.

It is also a purpose of the present invention to adopt a particular technical solution, hereafter defined and described in detail, which is designated as "generalized instrumentation operational amplifier configuration" and is inserted in the amplification section in a signal conditioning chain that implies analog filtering (digitally controlled) before the analog-to-digital conversion phase in order to reduce the electrical disturbances and the artefacts in the measurements of the neuronal signals deriving from the 50/60 Hz electric supply lines.

It is a further purpose of the present invention to adopt a digital technology also in the signal acquisition stage and in the wireless transmission in order to increase the reliability of the data received by radio from the subject. It is also a purpose of the present invention to adopt an integrated telemetry system in order to eliminate the mechanical connection between the electrodes and the wire, which is liable to rupture and is a possible source of lesions for the subject.

It is also a purpose of the present invention to provide a management software environment for the parameters of the transmission device, the real-time digital display of the signals and their digital recording compatible with mathematical analysis software known and used in the scientific community, in order to provide a convenient methodology for the use of the same invention and the analysis of the data obtained therewith. Summary The present invention meets the above mentioned purposes by providing a neuro- telemetry system which comprises a digital wireless mobile integrated acquisition system for neuronal signals.

It is therefore the object of the invention to provide an integrated neuro-telemetric system for acquiring neuronal action potentials and telemetrically, digitally and wirelessly transmitting them in real time, comprising: - a remote unit for the analog amplification of neuronal electrical signals detected by one or more electrodes which are implanted in selected brain regions and may be connected therewith, for filtering such signals, for the conversion of such signals from analog to digital by means of a first processing unit provided with at least one analog-to-digital converter and the transmission of the same by radio to a receiving station by means of an integrated radio transmitter;

- a radio base station for receiving data packets containing the digital representation of the acquired neuronal electrical signals transmitted thereto from the remote unit and interfaceable with a second processing unit for their storage in the buffer thereof, for the processing and displaying on suitable means connectable thereto; and

- a management unit comprising a third processing unit which is connectable to the radio base station and controls both said remote unit and radio base station by establishing a two-directional connection between the same with a communication protocol suitable both for the management of data packets containing the digital representation of the neuronal electrical signals which are acquired and transmitted from the remote unit to the radio base station, and for the programming by radio analog conditioning means of the acquired neuronal signal included in the remote unit. a further object of the invention to provide A method for acquiring neuronal electrical signals by telemetry, characterised in that it comprises at least the steps of: - analogically amplifying and conditioning electrical signals detected by one or more electrodes implanted in selected brain regions and generated by neurons;

- converting such analogically acquired electrical signals in data packets containing the digital representation of such neuronal electrical signals; - transmitting such data packets containing the continuous or compressed digital representation of such neuronal electrical signals by radio to a receiving station by means of an integrated radio transmitter.

- reducing the weight of the telemetry system so as to allow the device to be conveniently carried not only by human patients, but also by experimental animals in order to measure the neuronal action potentials;

- using a battery power supply for the devices of the telemetry system carried by the subject and applied thereto in order to reduce the negative effects of the ground loops; - adopting a "generalized instrumentation operational amplifier configuration" in the amplification step inserted in a signal conditioning chain that implies (digitally controlled) analog filtering before the analog-digital conversion step in order to reduce the electrical disturbances and the artefacts, in the measurements of the neuronal signals deriving from the 50/60 Hz electric supply lines;

- adopting a digital technology also in the step of acquiring the signal and wirelessly transmitting;

- adopting an integrated telemetry system to eliminate the mechanical connection between the electrodes and the wire; and - providing a software environment for the management of the parameters of the transmission device, the real-time digital display of the signals and their digital recording which is compatible with a mathematical analysis software.

Brief description of the drawings A detailed description of the system and the method for acquiring neuronal electrical signals by telemetric means according to the present invention will now be provided, given by way of non-limitative illustration and referring to the accompanying drawings, in which:

Figure 1 is a diagrammatic representation of the integrated neuro-telemetry system of the present invention comprising a remote unit for the amplification and the analog/digital conversion, the filtering, and the radio transmission of neuronal electrical signals, a base station, and a management unit;

Figure 2 is a diagrammatic representation of the remote unit of Figure 1 ;

Figure 3 is a diagrammatic representation of the radio base station of figure 1 ; Figure 4 shows the structural organisation of the management unit of figure 1 ;

Figure 5 schematically shows the circuit implementation of the remote unit operations in figure 1 ;

Figure 6 depicts the basic electrical diagram of an instrumentation amplifier stage in relation to the modifications introduced on a "generalized instrumentation amplifier" circuit according to the present invention;

Figure 7 depicts a substitutive bipolar network showing an example of the operation of the generalized instrumentation amplifier stage of the previous figure;

Figure 8 depicts an exemplary diagram of the gain of a generalized instrumentation amplifier stage as a function of the signal frequency; Figure 9 visually and diagrammatically shows the phenomena occurring in the implementation of the generalized instrumentation amplifier stage, according to the present invention;

Figure 10 depicts the functional block diagram of the synchronisation procedure, according to the present invention; Figure 1 1 depicts the functional block diagram of the acquisition procedure, according to the present invention.

Figure 12 depicts the functional block diagram of the communication procedure, according to the present invention;

Figure 13 depicts the functional block diagram of the configuration procedure, according to the present invention;

Figure 14 depicts the time trace of a neuronal signal recorded with an embodiment of the system of the present invention during an exemplary experiment;

Figure 15 depicts an enlargement of the signal in figure 14; and

Figure 16 depicts a single action potential recorded with an embodiment of the system of the present invention during an exemplary experiment, in relation to the signal-to-noise ratio. Detailed description of the invention

The present invention allows to overcome the drawbacks of the existing systems and methods by means of an integrated system for the acquisition, amplification, analog conditioning, A/D conversion, digital wireless transmission, digital recording and real time display of neuronal electrical signals.

With respect to the existing systems and in order to overcome the technical drawbacks thereof, the integrated neuro-telemetric system which is the object of the invention provides for the use of miniaturised components for electronics related to the remote unit and an electronic/mechanical design that takes the above mentioned problems into consideration.

For this purpose, the invention provides for a first apparatus (shown in fig. 1 ) which comprises three distinct units which are connected each other. Such units are: a remote unit (1 ) which is connected in a wireless mode with a second unit consisting of a base station (2) for the reception of the signal and a third unit for the management of the system (3) which is bi-directionally connected to the two previous units (1 ) and (2) for the control thereof.

The remote unit (1 ), as shown in fig. 2, is an autonomous electronic device basically comprising at least:

1. one differential input consisting of two terminals ("input" e "reference") with at least the following electrical features: input impedance > 10¹⁰ Ω input capacitance < 4 pF;

2. a differential amplifier with at least the following electrical features: common-mode rejection > 125 dB - amplification frequency bandwidth from f₀ = DC to f_MAx = 40 kHz linearized gain > 3000 between f_MiN ≤ 500 Hz and Ϊ_MAX/2 > 20 kHz, within ±3dB;

3. a device for the reduction of the noise in the frequency band of 50/60 Hz that acts by limiting a possible saturation generated by the noise in the amplification stage; 4. a system of reconfigurable digitally programmable analog filters;

5. an analog-digital converter with at least the following features: resolution > 8 bits sampling frequency > 20 kHz; 6. a microcontroller system comprising at least one RAM, one

EEPROM and one CPU;

7. a digital modulation radio transceiver;

8. an integrated radio antenna;

9. a battery power supply system for all of the previously described parts;

Furthermore, the remote unit (1 ) with the above described configuration allows to carry out the following operations in real time: a) detecting electrical signals generated by neurons by means of electrodes connected thereto; b) differentially amplifying the electrical signals detected with the common- mode signal cancellation; c) protecting from environmental electric disturbances deriving from the 50/60 Hz power supply lines and the related electromagnetic shielding; d) actively and analogically filtering the amplified signal with the possibility of digitally reconfiguring the parameters by radio connection with the second unit consisting of the base station (2), by means of the control of the software incorporated in the management system (3), for the selection of the electrical signals involved; e) converting the filtered electrical signals from analog to digital; f) encoding the neuronal data into packets, adding sequence numbers and system status flags; g) continuous or compressed representation of the neuronal data h) managing the digital controls and the radio digital communication protocols with the base station (2) by means of a microprocessor system provided with the computation ability and a software allowing to ensure that the running of all of the instructions takes place with at most a finite time delay (real-time system); i) modulating the transmitted data and demodulating the received data by means of an integrated radio transceiver having a modulating speed sufficient to establish a two-directional connection between the remote unit

(1 ) and the base station (2) without violating the real-time condition described in the previous item; j) electrically insulating the neuronal telemetry system for limiting the problems resulting from the ground loops; k) integrating, this including the reduction of the weight and size of the devices in order to meet the requirements of mechanical stability and physical tolerability of the telemetry system by the subject and the limitations of invasiveness of the telemetry system with respect to its use in combination with image capturing systems; I) monitoring the charging state of the battery.

The radio base station (2) shown in figure 3 is an electronic device basically comprising at least:

1. a radio antenna;

2. a digital modulation radio transceiver; 3. a microcontroller system comprising at least a RAM, an EEPROM and a CPU; and 4. an interface for the data exchange between the microcontroller system and a personal computer.

According to this configuration, the radio base station (2) allows to carry out the following operations in real time: a) reciprocally coupling a pair of devices - remote unit (1 ) and base station (2) - with a dynamical self-assigning of identification addresses for the pair and the radio channel even in the presence of other devices transmitting on the same radio frequency bands; b) reciprocally synchronising the internal clocks of the coupled pair; c) coupling again in the phase of radio signal temporary loss and interference with other devices and dynamically reassigning the identification addresses; d) receiving packets containing the data encoding the neuronal signals which have been acquired and transmitted from the remote unit (1 ); e) controlling the integrity of the received data packets and generating the error codes for the identification of the incorrect packets; f) transmitting the configuration parameters of the programmable analog filters to the remote unit (1 ); g) transmitting the management parameters to the remote unit (1 ) resulting from the management software (3); h) transmitting the settings concerning the selection modes for the acquired neuronal signals to be transmitted to the base station (2) to the remote unit

(1 ); i) receiving the data related to the battery charge state and state variables of the remote unit (1 ); j) managing the communication protocol with the management software (3) resident in a personal computer.

With reference to fig. 4, the management unit (3) is a computer which is interfaced to the radio base station (2) and, for the management of the remote unit (1 ) and the base station (2) itself and for the processing of the data corresponding to the neuronal signals, incorporates a control software carrying out at least the following operations: a) managing the data exchange with the base station (2); b) managing the parameters of the remote unit (1 ); c) managing the base station (2); d) digitally displaying in real time data representing the neuronal signals; e) digitally storing in real time data representing the neuronal signals; f) interfacing the user for the management of the programmable analog filters of the remote unit (1 ). The electronic circuit of the remote unit (1 ) is described hereafter with reference to figure 5. The differential amplifiers

/4_r with an open loop amplification factor K capture the neuronal signal in the form of electric potential difference applied between the inputs "in" and "ref" (respectively "input" and "reference") and generate the output signals A⁺ and A^' which are respectively the difference between the "in" signal and the feedback β⁺ and the difference between the "ref" signal and the feedback β^", both multiplied by the amplification constant K. The signals β⁺ and β^" are generated from A⁺ and A^'. The differentiator A reproduces between A⁺ and A the same potential difference between the signals A⁺ and A^'. The conditioner β receives as input the difference between A⁺ and A and multiplies it in the domain of the frequency for its transfer function H(s). The result is the potential difference between the feedbacks β and β. The closing up of this loop feedback establishes instant by instant the difference value between A⁺ and A that the differentiator evaluates by generating the output signal A⁰, which is transmitted as input to the filter F. The whole behaviour is mathematically described by the following equations: - ref)- K(/? -fT)

The aim of this circuit configuration, which will be designated "generalized instrumentation amplifier", is indeed to achieve its function described in equation 2 and is ensured by the selection of differential amplifiers having a high open loop amplification factor K. Indeed, an appropriate selection of the transfer function H(s) for the conditioner circuit β allows the filtering of about 50/60Hz frequency components coming from the electric supply lines, which have a voltage level higher than the neuronal signal, thus avoiding the saturation of the amplification stage. In light of this formulation in a normal amplifier stage in an "instrumentation amplifier" configuration (see figure 6), described in the electronic circuit literature, the transfer function H(s) computed between the quadrupole β, which has nodes A⁺ and A as inputs and nodes /T and β as outputs, equals:

3. H(s)= ^R°

R_G + 2R

this corresponds to a gain in the output signal Δ° with respect to the differential input signal "in"-" ret equivalent to:

_{4 G =} _!_ ₌ ≤L±²£ H(s) R_G

i.e. to the gain value of an amplifier stage in an "instrumentation amplifier" configuration, as known from the electronic literature.

The advantage in the analytical formulation suggested here (equations 1. and 2.) lies in that, therethrough, it is clear that the gain G in the instrumentation amplifier configuration, as set forth in equation 4., is only a particular case of circuit configuration, which is therefore designated "generalized instrumentation amplifier", in which the selection of the function H(s) of the quadrupole which has nodes A⁺ and A as inputs and nodes /T e β as outputs has been restricted to a constant value G in the frequency dominion. In an application of the generalized instrumentation amplifier configuration, by replacing the resistance R_G with a bipole shown in figure 7, having impedance:

sR_sR_pC + R_P

5. Z ^J _nG = s(R_sC + R_pC)+ \

and substituting this expression in place of the R_G in equation 4. the transfer function G(s) between the differential input in-ref and the output Δ° is obtained: s (R_SR_PC + 2RR_SC + 2RR_PC)+ 2R + R_f

6. G = sR_sR_PC + R_p which is of the general form:

as+b 7. G = where: cs+ d a = (R₈R_pC+ 2RR₈C+ 2RR_pC) b = 2R+R_P c = R_gR_pC

From equation 7. it may be noted that the asymptotic trend of the gain as a function of the frequency is distinguished in two regions having a limit value:

the absolute value trend of which is shown in figure 8. The asymptotic values of the transfer function for the limit cases of the signal frequency tending to zero or infinity may be calibrated with a selection of values for the components of the network in figure 7 so that the qualitative curve in figure 8, the meaning of which is the modulus of the response in frequency of the amplifier gain, displays a low value for frequencies below 50/60 Hz and a higher value for higher frequencies.

A spectral analysis of the frequency content of action potential signals, such as stereotypical waveform signals, shows that they do not contain significant frequency components below frequencies of the order of 1 kHz, correspondingly to the reciprocal of the typical duration of an action potential of 1 ms. A further analysis of the function H(s), which is reciprocal to the function G(s) designed in the implementation of the described generalised instrumentation amplifier, shows that the role of the quadrupole β is to leave the low frequency components unaltered (50/60 Hz noise) and to attenuate the signals having high frequency components (neuronal signal).

The amplification of the neuronal signal is due to the fact that the above said quadrupole is inserted in a feedback loop consisting of the differential amplifiers of the generalized amplifier configuration that, because of the circuit network they describe, operate the reciprocal of the transfer function H(s) in the dominion of the frequencies. In this manner, the amount attenuated by the quadrupole β in output between nodes /T and β, is amplified in the signal at nodes A⁺ and Λ, retransfered through the differentiator A from the amplifiers A₁ and A₂, and which results being at the same time the input for the quadripole β and the output differential signal A⁰ of the generalized instrumentation amplifier.

This applicative example shows an inherent advantage of the generalized configuration in relation to the problem of the saturation of the input amplifier stages.

Figure 9 shows an example of the operative cycle of the internal operations for the amplification of a neuronal signal, represented by the small fluctuations, overlapped to a high 50Hz noise component for a generalised instrumentation amplifier stage computed for a 1000-fold gain on the neuronal signal band. It must be noted that in this case the amplification of the neuronal action potentials is indipendent of the conditioning of the 50Hz noise component, displaying a corresponding 1000-fold gain, while the 50Hz signal component is not amplified and that it should not have a voltage higher than the input voltage in any node of the stage. Therefore, in this example, a 1 mV action potential may be amplified 1000-fold up to the value of 1 V, without an overlapping 1 V-amplitude noise component being amplified by the same 1000-fold factor, thus preventing the saturation of the input stage that would have otherwise compromised the amplification of the action potential. Thereafter, the pre-amplified and conditioned signal is frequency-filtered by the F block, which receives the parameters for the filtering from the CPU block. The programmable filter Fcarries out two operations: the first is the cancellation of the residual 50/60Hz noise coming from the electric supply lines, the second consists in the filtering of the neuronal signal within its band in a user-controllable manner by means of the parameter setting through the management software (3). The selection of the generalized instrumentation amplifier configuration carrying out a pre-filtering combined with the final filtering of the programmable filter F represents a solution to the problem of the noise reduction and the amplifier saturation that, in light of the suggested formulation and analysis, are two interdipendent problems due to the non-commutability of amplification- differentiation-filtering operations in a non-linear amplification regime in case of saturation.

The non-commutability of the operations is not evident in the case of a linear circuit theory within which the standard configuration of the instrumentation amplifiers, described in the electronic literature, has arisen. The filtered signal is converted from analog to digital by the ADC block that communicates with the MCU block for the acquisition of the data in a digital format. The MCU block also manages the data exchange with the RTX block, which generates the radio connection with the base station (2) for the transmission of the acquired neuronal data and the dialog for the setting of the controls occuring through the software of the management system (3).

The remote unit (1 ) implements its operations both through the previously described hardware, and through software procedures resident in the MCU block. The synchronisation procedure, described in the "flow chart" in figure 10, is carried out first thing when the remote unit (1 ) is switched on. It is also carried out every time it is required because of the loss in synchronisation.

The synchronisation procedure implements the radio connection between the remote unit (1 ) and the radio base station (2), thus receiving the connection parameters, those for the analog-digital converter ADC and for the programmable filter F If the synchronisation is lost, all of the parameters are reinitiated and the sequence is repeated until it reaches a stable connection condition. Then, the analog-digital converter ADC and the filter F are programmed. Having established the synchronisation, the remote unit (1 ) carries out the acquisition of the data generated by the analog-to-digital conversion of the amplified neuronal signal, according to the flow chart shown in fig.1 1.

The acquisition procedure sends a signal to the CPU that it may start the analog- to-digital conversion and, when the conversion is completed, that it may process the data for the transmission to the radio base station (2). The data prepared to be transmitted are copied in the buffer register TX Buffer, the time required for the reacquisition is waited for, then the entire procedure is repeated in cycle. The communication procedure manages the radio communication between the remote unit (1 ) and the base station (2) according to the flow chart shown in fig. 12.

This procedure carries out the data transmission from the remote unit (1 ) to the management system (3). The communication procedure continuously reads the buffer register TX Buffer, which is constantly filled by the acquisition procedure. If it finds that it is full, it switches on the radio transmitter, loads the data to be transmitted in the transmitter, it transmits them and switches the transmitter off. After these operations, it checks whether the remote unit (1 ) needs to receive data from the management system (3). If not, it continues to check the TX Buffer, otherwise it takes care of switching on the radio receiver for the reception of possible data from the management system (3). In case there are no data, the receiver is switched off and the procedure is repeated as previously described. In case data are received from the management system (3), the remote unit (1 ) activates the configuration procedure. The configuration procedure manages the data received from the management system (3) on the remote unit (1 ) for the management of parameters which may be set by the user and the data concerning the communication protocols autonomously coming from the radio base station (2) according to the flow chart shown in figure 13. The description of an embodiment of the system according to the present invention will now be provided. Example of a neuro-telemetry integrated system Features of the remote unit (1):

- electronics: SMD (Surface Mounted Devices) components assembled on a multilayer PCB (Printed Circuit Board) with internal and external ground planes for the electromagnetic shielding, lithium polymer rechargeable battery power supply, signalling LEDs (Light Emitting Diodes) for variables and status flags, microcontroller system, radio section (comprising an integrated antenna) operating in the 2.4 GHz frequency band, multi-function key for the resetting and local settings, FPAA (Field Programmable Analog Array) for the digitally programmable analog filtering, service USB (Universal Serial Bus) port for updates of the local firmware of the unit, differential amplifiers implementing the previously described "generalized instrumentation amplifier" solution.

- mechanics: housing with a metal conductor cover for the protection of the system from electromagnetic disturbances, tungsten electrode encapsulated in a silicon microtube inserted in a coaxial sliding guide consisting of a steel microtube which is mechanically and electrically connected to the metal container for the implementation of the mechanical support and the electromagnetic shielding of the electrode, miniaturized mechanical translation system for the linear sliding advance of the electrode within the coaxial guide with a micrometric regulation, integrated on the PCB and electromagnetically screened by the metal cover. Features of the base station (2):

- electronics: SMD (Surface Mounted Devices) components assembled on a multilayer PCB (Printed Circuit Board) with internal and external ground planes for the electromagnetic shielding, USB (Universal Serial Bus) service port for updates of the local firmware of the unit and for the communication with a PC on which the management software (3) is installed, power supply by means of a USB port, microcontroller system, radio section (comprising an integrated antenna) operating in the 2.4 GHz frequency band. Features of the management software (3): - interfacing: management of the USB connection between software (3) and hardware (2) - protocols: decodification of the received packets from the base station (2) and dialog therewith through a USB port

- management: real-time display of the data in the form of a digital oscilloscopy trace on the monitor of the PC connected to the base station (2), graphic interface with icons and keys for the control of the acquisition, saving on the hard disk of the data being acquired.

With a system like that just described, a telemetry method is used to acquire the neuronal electrical signals, the method comprising at least the steps of:

1. analogically amplifying and conditioning electrical signals detected by one or more electrodes implanted in selected brain regions and generated by single neurons;

2. converting such analogically acquired electrical signals into data packets containing the digital representation of such neuronal electrical signals;

3. transmitting such data packets containing the continuous or compressed digital representation of such neuronal electrical signals by radio to a receiving station by means of an integrated radio transmitter;

4. receiving such data packets containing the continuous or compressed digital representation of the neuronal electrical signals in a receiving station and verifying the integrity of the informative content, thus generating possible error signals for the management of the communication protocol between transmitter and receiver;

5. interfacing the receiving station with a computer capable of reading the digital neuronal data received from the station and communicating service telemetric data, such as the setting of parameters for the filters in the transmission remote unit and system flags, therewith;

6. displaying in real-time the neuronal data received in the form of an oscilloscope trace in a software window visible to the user;

7. controlling the operation modes of the remote transmitter by acting from the computer connected to the base station by exploiting the bidirectionality of the wireless connection between remote transmitter and receiving station; 8. digitally recording the data received from the receiving station in the computer connected thereto;

9. managing the control operations of the remote transmitter, monitoring the display and saving the data received therefrom by acting through the software environment;

A typical experiment for the acquisition, amplification, analog conditioning, A/D conversion, digital wireless transmission, digital recording and real-time display of neuronal electrical signals carried out according to the method mentioned above and with the integrated system of the invention, is described hereafter. The following protocol for the recording of neuronal action potentials is in accordance with the institutional and international NIH standards for the treatment and use of animals for scientific research.

This protocol is compatible with the procedures for the recording of neuronal signals using apparatuses already on the market, thus allowing a possible comparison of results.

Example of neuro-telemetry Subject

• male adult Wistar rat, weight 250-30Og. Materials • anaesthetic: urethane, dose 1.5 g/kg [urethane weight/subject weight], concentration 30% [urethane weight/ H₂O weight].

• electrodes: tungsten single (Frederick Haer & Co. , 9 Main St., Bowdoinham, ME 04008 USA), impedance = 1.0 - 1.2 MegaOhm

• wooden stick with a cotton end Instrumentation

• table with stereotaxic supports

• thermostatically controlled heating plate Preparation of the subject

The subject is anaesthetised by intraperitoneal injection of urethane, dose 1.5g/kg [urethane weight/subject weight], concentration 30% [urethane weight/H₂O weight]. When the subject is anaesthetised, it is placed on the thermostatically- controlled heating plate at a temperature of 37.5 °C.

The subject is held stationary for the whole duration of the experiment while monitoring the retraction of the rear limbs in response to a slight pressure by means of tweezers, as well as corneal reflexes and respiratory rhythm as an indication of the anaesthesia state. If required an extra dose of anaesthetic equivalent to 10% of the starting dose is administered.

After having secured the head of the subject to the stereotaxic support, the left somatosensory cortex is exposed by means of a 3x3 mm opening craniotomy, centred around the point having coordinates 2 mm rearwards and 6 mm sideways with respect to the bregma (craniometric reference point, frontal intersection of the cranial sutures).

The region corresponding to the neuronal representation of the vibrissae ("barrel cortex") is identified in accordance with the cerebral vascular configuration and the stereotaxic coordinates. Neuronal recording

A single connected electrode is assembled on the remote unit (1 ) and led in the region concerned (barrel cortex) by penetrating the brain tissue to a depth of 700- 1000 microns. The gain of the amplifier is set to a value of 5000 and the filter is set as a 4^th order Butterworth bandpass with a cut-off frequency below 250Hz and above 7500Hz. The digitizasion of the signal is set with a 20 kHz sampling frequency and a 8 bit resolution. The receiving unit (2) is placed far away from the remote unit (1 ) within a range that may arbitrarily vary up to 5 metres in an environment in the presence of even other electronic apparatuses (for instance a laboratory for electrophysiological recordings) and is connected to a portable computer through a USB port (Universal Serial Bus). In these conditions the integrity index of the wireless radio connection, defined as the ratio between data packets which have been correctly transmitted and received between remote station (1 ) and base station (2) and data packets received with radio interference signalling flag, is 98.3%. Accordingly, the selection protocol for the packets distinguishes 100% reliable neuronal digital data by identifying the data packets classified as "correct" from "incorrect" ones, thus allowing the management software (3) to display and record a continuous trace of reliable neuronal data and simultaneously a binary trace containing the status of the radio interference signalling flag, so as to provide the operator both with a graphic display of the amplified neuronal signals and with a visual indication of the radio interference flag in real time. The graphic trace of the correct packets is also converted to an audio signal synchronised therewith: by monitoring the video trace of the neuronal signal and the audio signal, the operator may intervene on the electrode positioning system by using both representations of the neuronal signals as a control of the penetration depth into the brain tissue in order to optimise the quality of the acquired signal.

The absence of the ground loop between the amplification system in the remote unit (1 ) and the computer connected to the base station (2) allows the feasibility of the experiment even with the subject insulated from the ground potential and outside other systems for the electromagnetic shielding (for instance, Faraday cages).

Tactile stimulation A tactile stimulation, counter-lateral (opposite side) to the craniotomy and consisting of a mechanical vibration, is applied to the vibrissae by means of the cotton stick.

Analysis of the measurements

The neuronal activity is characterized by the presence of neuronal action potentials. In the experimental conditions described, a neuronal recording system needs to detect action potentials resulting from a tactile stimulus that is counter- lateral with respect to the region in which the electrode lies (craniotomy region). The control condition that the recorded signal is actually related to the tactile stimulus is the absence of a neuronal response when the same tactile stimulus is ipsilateral (same side) instead of contralateral (opposite side). The presence of a 7-10 ms latent period between the tactile stimulation time and the neuronal activity indicates that the activity is a physiological response to the stimulus. Figure 14 shows some typical signals recorded in the experimental conditions described above. The trace represents a 10 second sample of a neuronal signal recorded by the system while the tactile stimulation was taking place. Three groups of neuronal impulses (left) followed by another five groups (right) may be noted corresponding to three and then five stimulations of a vibrissa, the neuronal path of which starts from the mechano-receptor cells around the follicle and along which the tactile information is transmitted to the corresponding barrel in the somatosensory cortex where the electrode has been positioned, by means of the cotton stick. In the time intervals between groups of neuronal impulses the absence of neuronal activity may also be noted as a low level of electrical fluctuations.

The stereotypical form of the action potential may easily be recognized by reading the same signal displayed on a smaller time scale. The trace in figure 15 is an enlargement of the signal fragment corresponding to a 20 ms space of time that comprises the fourth group of impulses (the first of the second series of five groups on the right) contained in the previous trace.

A sequence of seven neuronal action potentials recorded in response to a single tactile stimulation may be identified. Instead, the trace in figure 16 shows a single action potential. The action potential appears at the centre of the figure. It may be noted that the small fluctuations of the trace before and after the action potential, corresponding to the sum of other signals, which are of no interest for neuronal encoding and may accordingly be defined as noise, remain centred and limited to an amplitude value significantly lower than the action potential, which remains clearly distinguishable.

Validation of the measurement

The verification of the waveform of the action potential, combined with the verification of the responses to the stimulus in the experimental condition and in the control condition, represents the validating proof of the correct functioning of the neuronal recording system of the present invention and according to the disclosure and description. Further implementation details will not be described, as the man skilled in the art is able to carry out the invention starting from the teaching of the above description.

Many steps of the present invention can be advantageously implemented through a program for computer comprising program coding means for the implementation of one or more steps of the method, when this program is running on a computer. Therefore, it is understood that the scope of protection is extended to such a program for computer and in addition to a computer readable means having a recorded message therein, said computer readable means comprising program coding means for the implementation of one or more steps of the method, when this program is run on a computer.

Many changes, modifications, variations and other uses and applications of the subject invention will become apparent to those skilled in the art after considering the specification and the accompanying drawings which disclose preferred embodiments thereof. All such changes, modifications, variations and other uses and applications which do not depart from the spirit and scope of the invention are deemed to be covered by this invention.

Claims

1. An integrated neuro-telemetric apparatus for acquiring neuronal action potentials and telemetrically, digitally and wirelessly transmitting them in real time, comprising: - a remote unit (1 ) for the analog amplification of neuronal electrical signals detected by one or more electrodes which are implanted in selected brain regions and may be connected therewith, for filtering such signals, for the conversion of such signals from analog to digital by means of a first processing unit provided with at least one analog- to-digital converter and the transmission of the same by radio to a receiving station by means of an integrated radio transmitter;

- a radio base station (2) for receiving data packets containing the digital representation of the acquired neuronal electrical signals transmitted thereto from the remote unit (1 ) and interfaceable with a second processing unit for their storage in the buffer thereof, for the processing and displaying on suitable means connectable thereto; and

- a management unit (3) comprising a third processing unit which is connectable to the radio base station (2) and controls both said remote unit (1 ) and radio base station (2) by establishing a two- directional connection between the same with a communication protocol suitable both for the management of data packets containing the digital representation of the neuronal electrical signals which are acquired and transmitted from the remote unit (1 ) to the radio base station (2), and for the programming by radio analog conditioning means of the acquired neuronal signal included in the remote unit (1 ).

2. An integrated neuro-telemetric apparatus for acquiring neuronal action potentials according to the preceding claim, wherein said remote unit (1 ) basically comprises at least: - a differential input comprising two terminals, "input" and "reference", having at least the following electric features: input impedance > 10¹⁰ Ω input capacitance < 4 pF; - a differential amplifier having at least the following electrical features: common-mode rejection > 125 dB amplification frequency bandwidth from f₀ = DC to f_MAχ = 40 kHz linearized gain >3000 between f_MiN ≤ 500 Hz and f_MAχ/2 > 20 kHz, within ±3dB;

- a device for the reduction of the noise in the frequency band of 50/60 Hz that acts by limiting a possible saturation generated by the noise in the amplification stage;

- a system of reconfigurable digitally programmable analog filters; - an analog-digital converter with at least the following features: resolution > 8 bits sampling frequency > 20 kHz;

- a first microcontroller system;

- a digital modulation radio transceiver; - an integrated radio antenna; and

- a battery power supply system for all of the previously said parts.

3. An integrated neuro-telemetric apparatus for acquiring neuronal action potentials according to claim 1 or 2, wherein said radio base station (2) basically comprises at least: - a radio antenna;

- a digital modulation radio transceiver;

- a second microcontroller system ; and

- an interface for the data exchange between said second microcontroller system and a personal computer.

4. An integrated neuro-telemetric apparatus for acquiring neuronal action potentials according to claim 1 or 2 or 3, wherein said management unit (3) is interfaced to the radio base station (2) and, for the management of the remote unit (1 ) and the base station (2) and for the processing of the data corresponding to the neuronal signals, comrises means for carrying out at least the following operations:

- managing the data exchange with the base station (2);

- managing the parameters of the remote unit (1 );

- managing the base station (2); - digitally displaying in real time data representing the neuronal signals;

- digitally storing in real time data representing the neuronal signals;

- interfacing the user for the management of the programmable analog filters of the remote unit (1 ).

5. A method for acquiring neuronal electrical signals by telemetry, characterised in that it comprises at least the steps of:

- analogically amplifying and conditioning electrical signals detected by one or more electrodes implanted in selected brain regions and generated by neurons;

- converting such analogically acquired electrical signals in data packets containing the digital representation of such neuronal electrical signals;

- transmitting such data packets containing the continuous or compressed digital representation of such neuronal electrical signals by radio to a receiving station by means of an integrated radio transmitter.

- reducing the weight of the telemetry system so as to allow the device to be conveniently carried not only by human patients, but also by experimental animals in order to measure the neuronal action potentials; - using a battery power supply for the devices of the telemetry system carried by the subject and applied thereto in order to reduce the negative effects of the ground loops;

- adopting a "generalized instrumentation operational amplifier configuration" in the amplification step inserted in a signal conditioning chain that implies (digitally controlled) analog filtering before the analog-digital conversion step in order to reduce the electrical disturbances and the artefacts, in the measurements of the neuronal signals deriving from the 50/60 Hz electric supply lines;

6. A method for acquiring neuronal electrical signals by telemetry according to the preceding claim, wherein a management of a remote autonomous electronic device or remote unit is provided for, performing the following steps in real time:

- detecting electrical signals generated by neurons by means of electrodes connected thereto; - differentially amplifying the electrical signals detected with the common- mode signal cancellation;

- protecting from environmental electrical disturbances deriving from the 50/60 Hz electric supply lines and the related electromagnetic shielding;

- actively and analogically filtering the amplified signal with the possibility of digitally reconfiguring the parameters by radio connection with the radio base station (2), by means of the operations performed by the management system (3), for the selection of the electrical signals involved;

- converting the filtered electrical signals from analog to digital;

- encoding the neuronal data into packets, adding sequence numbers and system status flags;

- managing the digital controls and the radio digital communication protocols with the base station (2) so as to ensure at most a finite time delay (real-time system) in the operation;

- modulating the transmitted data and demodulating the received data by means of an integrated radio transceiver having a modulating speed sufficient to establish a two-directional connection between the remote unit (1 ) and the base station (2) in real-time condition;

- electrically insulating the neuronal telemetry system for limiting the problems resulting from the ground loops; - integrating, by including the reduction of the weight and size of the devices in order to meet the requirements of mechanical stability and physical tolerability of the telemetry system by the subject and the limitations of invasiveness of the telemetry system with respect to its use in combination with image capturing systems; - monitoring the charging state of the battery.

7. A method for acquiring neuronal electrical signals by telemetry according to claim 4 or 5, wherein a management of an electronic device such as a radio base station is provided for, the management method providing for the real time execution of at least the following operations: - reciprocally coupling a pair of devices - remote unit (1 ) and base station (2) - with a dynamical self-assigning of identification addresses for the pair and the radio channel even in the presence of other devices transmitting on the same radio frequency bands; - reciprocally synchronizing the internal clocks of the coupled pair; - coupling again in the phase of radio signal temporary loss and interference with other devices and dynamically reassigning the identification addresses;

- receiving packets containing the data encoding the neuronal signals which have been acquired and transmitted from the remote unit (1 );

- controlling the integrity of the received data packets and generating the error codes for the identification of the incorrect packets; - transmitting the configuration parameters of the programmable analog filters to the remote unit (1 );

- transmitting the management parameters to the remote unit (1 ) resulting from the operations of the management unit (3);

- transmitting the settings concerning the selection modes for the acquired neuronal signals to be transmitted to the base station (2) to the remote unit (1 );

- receiving the data related to the battery charge state and state variables of the remote unit (1 );

- managing the communication protocol with the management software (3) resident in a personal computer.

8. Computer program comprising computer program code means adapted to perform at least the following steps of the previous claims, when said program is run on a computer: - managing the data exchange with the base station (2);

- managing the parameters of the remote unit (1 );

- managing the base station (2);

- digitally displaying in real time data representing the neuronal signals;

- digitally storing in real time data representing the neuronal signals; - interfacing the user for the management of the programmable analog filters of the remote unit (1 ); - the synchronizating procedure described by the flow chart in figure 10;

- the acquiring procedure described by the flow chart in figure 1 1 ;

- the communicating procedure described by the flow chart in figure 12; and - the configuring procedure described by the flow chart in figure 13.

9. A computer readable medium having a program recorded thereon, said computer readable medium comprising computer program code means adapted to perform at least the following steps of the previous claims, when said program is run on a computer:

- managing the data exchange with the base station (2);

- managing the parameters of the remote unit (1 );

- managing the base station (2);

- digitally displaying in real time data representing the neuronal signals; - digitally storing in real time data representing the neuronal signals;

- interfacing the user for the management of the programmable analog filters of the remote unit (1 );

- the synchronizating procedure described by the flow chart in figure 10;

- the acquiring procedure described by the flow chart in figure 1 1 ; - the communicating procedure described by the flow chart in figure 12; and

- the configuring procedure described by the flow chart in figure 13.