WO2005037100A1

WO2005037100A1 - System and method for the detection of brain response

Info

Publication number: WO2005037100A1
Application number: PCT/NL2004/000746
Authority: WO
Inventors: Mazen K. El Tamer; Arjen J. Van Tunen
Original assignee: Universiteit Van Amsterdam
Priority date: 2003-10-22
Filing date: 2004-10-21
Publication date: 2005-04-28

Abstract

The present invention relates to a system and method for the detection of brain activity in response to a chemical stimulus. The method comprises (a) implantation of an electrode in the brain of a subject, and (b) measuring the amplitude of relevant brain waves before and after the administration of the chemical stimulus. The brain wave measurements may be coupled to a brain imager for a final evaluation of the sensational level. The chemical stimulus is preferably a compound or a mixture of compounds of microbial, bacterial or phytochemical origin. The chemical stimulus that gives a desired change may be added to a pharmaceutical, food, beverage, cosmetic or other industrial product for human or animal consumption or use in order to increase the brain sensational level. This is particularly advantageous in taste and smell dysfunctions.

Description

System and method for the detection of brain response

Field of the invention The present invention relates to a system and method for the detection of brain response, particular, the invention relates to a system and method for the detection of brain response in reaction to exposure to a chemical compound.

Background of the invention The ability to smell and to taste is regulated by the olfactory nerve system. The olfactory nerve system is complex and interconnected with several systems in the brain. There is an anatomical and biochemical connection between the olfactory system and the limbic system in the brain. The limbic system includes the hippocampus and amygdala region, and is known as the emotional centre of the brain. The limbic regions have many synaptic contacts with olfactory bulb. Many of the limbic structures and the olfactory bulb are reciprocally interconnected in loop pathways that may be involved in the regulation of brain emotional output. There are several known disorders of taste and smell which affect the function of the olfactory system and which present major problems for the patient. Chemosensory dysfunctions are usually described by the following terms: ageusia (absence of taste), hypogeusia, (diminished sensitivity of taste), dysgeusia (distortion of normal taste), anosmia (absence of smell), hyposmia (diminished sense of smell), and dysosmia (distortion of normal smell). These disorders are a problem with increasing significance in genetic diseases, elderly people, Alzheimer's disease, Parkinson's disease, multiple sclerosis, schizophrenic, hypothyroidic and alcoholic patients. People with an olfactory dysfunction (anosmia) have damaged some of their olfactory nerves. Age-related anosmia is usually due to degeneration of the glomeruli and olfactory bulb (Schiffman, 1997, J.Americ. Med. Associat. (278): 1375-1381). Younger individuals are able to distinguish the degrees of difference between odours of different quality much better than an elderly person is. Health can be greatly affected when a person has anosmia. Taste is most commonly lost with the disorder since there is a close connection of smell and flavour. Because anosmia results from an olfactory deficit, there is usually a loss of taste. A taste loss is one of the first things noticed by people losing their sense smell. This loss of taste can greatly affect a person's eating habits. Many people with anosmia are known to skip meals because the appeal for food is not there. Nothing seems to taste good anymore since the flavour is gone. Another reason appetite is affected, is that the aroma of foods does not cause a desire for food because the person cannot detect the luring odours from the food. Not eating results in malnutrition and involuntary weight loss. Left unchecked, this can also lead to serious illness because the proper foods are not being eaten to keep a person healthy. The primary reinforcers for eating are satiety and pleasure. Without taste and smell the person will not experience these sensations (Schiffman, 1997). Overall, chemosensory disorders are chronic problems that can reduce enjoyment and quality of life. US 2003/0114886 describes an in vitro study of the restoration of taste and smell by monitoring the electricity of the hippocampus. The brain is stimulated electrically using evoked potentials. The evoked potentials are then used to record an EEG. Such in vitro studies have not lead to an in vivo solution. Typically, drugs are used to alleviate these disorders. However, most drugs also have side-effects. For instance, drugs used to correct anosmia may also cause drowsiness, nausea, headache, insomnia, depression, gastrointestinal disturbances, palpitations and some central nervous system effects such as a marked twitching of the hands. Therefore, a screening method which enables the discovery, isolation and development of chemical compounds which, when added to pharmaceutical, food, beverage or personal health care products will intensify sensations in the brain are of immense value.

Detailed description In one aspect, the present invention relates to a system for the detection of brain activity in response to a chemical stimulus. The system comprises:

(a) an administration means for administering a chemical stimulus to a non-human subject;

(b) a measuring electrode portion implanted in the brain of a subject;

(c) a measuring device for measuring the amplitude of relevant brain waves or other electrophysiological signals before and after the administration of the chemical stimulus.

The non-human subject is preferably a non-human mammal, more preferably a rat or a mouse.

In one embodiment, the system according to the invention further comprises a reference electrode implanted in the brain of the subject. Suitable areas for implantation of the the reference electrode include the piriform cortex and the olfactory bulb.

In one embodiment of the invention, at least two measuring electrode implants are present. Suitably, one of the at least two measuring electrode implants is present in the limbic system and another in the orbitofrontal cortex or the ventral tegmental area.

Preferred areas in the limbic system for measurement are the hippocampus and the amygdala.

In one embodiment, the system according to the invention measures the amplitude of the alpha, beta, gamma, delta, or theta brain waves. The system according to the invention includes an administration means which may be simple or more sophisticated. The administration means is preferably suitable for oral or nasal administration.

The system may further comprise a processing means for analysing a correlation between the electrical signal measured and the brain activity. This may for instance be a computer, which may or may not be part of a network.

The chemical stimulus which is administered in the system may be any bio-active compound which is bio-active in the brain. Preferable chemical stimuli are taste or smell compounds. In another aspect, the invention relates to the use of a system for the detection of molecules that stimulate the brain to enliance flavour and fragrance sensation. In one embodiment this is used in non-medical applications, such as for the enhancing flavour and fragrance sensation in the food, feed en pet food industry, in the beer wine, soft drink industry, in the dairy industry and in the cosmetic industry. In another embodiment, the system is used for applications in the pharmaceutical industry. More embodiments of the system of the invention will become clear from the description of the method of the invention. In another aspect, the present invention relates to a method for the detection of the response to a chemical stimulus. The method comprises

(a) implantation of an electrode in the brain of a subject, and

(b) measuring the amplitude of relevant brain waves and other electrophysiological signals before and after the administration of the chemical stimulus. In one embodiment, the subject is a non-human mammal, such as a rat or a mouse. Preferably the non-human mammal is a rat, because the closeness of the sensory system of the rat to that of humans allows for electrophysiological screening combined with behavioural observations. Preferably, these rats have been prepared. In one embodiment, the rats are prepared for behavioral tests. In one embodiment, the rats are trained with detection of any smell or taste based on a selected reward. They are given the choice to be rewarded by either exposure to the pure form of the substance or the normal reward. The frequency of rats opting for either is recorded. A high frequency related to the choice of the pure form substance denotes a higher satisfaction to that substance and therefore, the compound(s) are selected further on. h addition, rats are given the choice of a normal reward or being exposed to the pure form of the substance both placed at a long travelling distance or labyrinth. The latter is followed by a mild reprimandation (the opposite of reward). The rate of choice, time lapse and return to the substance is recorded (not seeking normal reward). A high frequency related to the pure form substance choice will then denote addiction to that substance. The rate of choice should clarify to us whether the substance should be legislated and protected to prevent any abusive behaviour. A high rate of return to and finding the substance signifies that although the cognitive system of the rat is affected, motor functions remain intact. In another embodiment of the invention, at least two electrodes are implanted in the brain. In another embodiment, three or four electrodes are implanted in the brain. If two or more electrodes are used, these electrodes are typically implanted in different areas of the brain, one electrode per area. A preferred part for implantation of the electrode is the limbic system. Areas in the limbic system, which are of particular interest, are the hippocampus, the amygdala. Other brain areas of particular interest are the orbitofrontal cortex and the ventral tegmental area. In one embodiment of the invention, one electrode is placed in the hippocampus, the amygdala, the ventral tegmental area or the orbitofrontal cortex and another electrode is placed in a reference area. Suitable reference areas are for instance the piriform (olfactory) cortex and the olfactory bulb. Implantation methods for electrodes are known in the art and described in, for instance, Leung et al. 1982) Electroencephalogr. Clin. Neurophysiol. 54(2): 203-19. Attachment of electrodes to the scalp in a conventional way, e.g. as in US 6,298,263, will not specifically provide the required information about changes in the above mentioned brain areas and is therefore not part of the method of the invention.

The electrodes are used to measure brain response, which is typically a change in the electrical activity of the brain caused by a change of electrophysiological signals in the brain. Electrophysiological signals in the brain that may change include field potentials, electrical stimulation, neural excitability and inhibition patterns and brain wave activity. In one embodiment, a change in one or more brain waves, in particular a change of a brain wave amplitude is measured. Brain waves that are of particular interest are the delta wave zone (1-3 Hz), theta wave zone (7-8 Hz), alpha wave zone (8-13 Hz), the beta wave zone (13-27 Hz) and gamma wave zone (27 Hz). Any conventional method known in the art may be used to measure brain or neural response, see for instance Werkman et al. (2001) Neuropharmacology 40: 927- 936 or Leung et al.(1982)

Electroencephalogr. Clin. Neurophysiol. 54(2): 203-19. At least one recording electrode and at least one stimulating electrode are used. The electrodes used are preferably isolated stainless steel. Preferably, the brain response is measured by electroencephalography (EEG). According to the method of the invention, to measure a change in brain wave, brain waves are measured before and after intake of potential chemical stimulus. In one embodiment, measurements are started at least 2 minutes, preferably at least 10 minutes, more preferably at least 15 -30 minutes before intake of the chemical stimulus and continued at least 10, more preferably at least 20-30 minutes following intake. The chemical stimulus may be administered in any convenient way, be it orally or parenterally, and optionally in combination with a suitable carrier, diluent or additive. h fact, it may be administered by routes that are commonly used for pharmaceutical compositions For oral administration, the chemical stimulus may be administered in solid dosage forms, for instance in the form of a powder, or in liquid dosage form, for instance in the form of a solution or suspension. In one embodiment, the chemical stimulus is administered using edible material with liquid absorption capability. The edible material is soaked with a prepared solution of the chemical stimulus. Suitable examples of such edible material include but are not limited to biscuits, crackers, cracker bread, chips, tortilla chips, cake, toast, flakes, scraps. Through routine optimisation the skilled person will be able to find the concentration of a chemical stimulus which is required for oral administration. Suitable parenteral routes for administration of the chemical stimulus include dermal application, injection or infusion by intravenous, intraperitoneal, intramuscular, intra-arterial routes. In another embodiment, the chemical stimulus is administered nasally. Any odor release or delivery system in or onto which a prepared solution of the chemical stimulus may be introduces may be used, optionally aided by a chamber or a removable flow system. Examples of such systems include but are not limited to a perforated box, a liquid, diffuser, a sprayer, modulated diffuser, an atomizer, a vaporisator, tablets, cloth, cotton pieces, gel, soap, paste, cremes, sponges, powder, potpourri, plaster, lotion, odor, block, wax, burned wax. Through routine optimisation the skilled person will be able to find the concentration of a chemical stimulus which is required for nasal administration. The chemical stimulus may be any compound or mixture of compounds that can be administered orally or nasally, preferably it is a compound or a mixture of compounds of microbial, bacterial or phytochemical origin. In one embodiment, the chemical stimulus is obtained from a plant family selected from Acanthaceae, Aloeaceae, Annonaceae, Araliaceae, Boraginaceae, Campanulaceae, Celastraceae, Compositae, Cruciferae, Cupressaceae, Cyperaceae, Ephedraceae, Euphorbiaceae, Fabaceae, Gramineae, Hamamelidaceae, Labiatae, Leguminosae, Malvaceae, Myrsinaceae, Myrtaceae, Oleaceae, Palmae, Piperaceae, Plumbaginaceae, Poaceae, Ranunculaceae, Rhamnaceae, Rubiaceae, Rutaceae, Salvadoraceae, Santalaceae, Solanaceae, Turneraceae, Umbelliferae, Valerianaceae, Verbenaceae and Zingiberaceae; or from a sea anemone family such as Anthozoa. The source from which the compound or mixture of compounds originate preferably enjoys GRAS status (generally recognised as safe). In another aspect, the present invention relates to a method for increasing the brain sensational level per olfactory receptor. This method comprises: (a) detecting a chemical stimulus which causes a desired change in one or more brain waves using the method according to the invention;

(b) adding the chemical stimulus which gives a desired change to a pharmaceutical, food, beverage, cosmetic or other industrial product for human or animal consumption or use. The phrase "increasing the brain sensational level" means that the pleasant or unpleasant sensation is intensified. This is indicated by more intense signals from the orbital frontal cortex or the amygdala, as measured by suitable techniques, such as EEG or fMRI. Preferably, the sensational level is increased by at least 2%, more preferably by at least 3, 5 or 7%, most preferably by at least 10%. What change is desired depends on the effect which is to be achieved. A drop in the level of a delta wave is for instance a desired change associated with interest. The skilled person will understand that this may be caused by very different chemical stimuli, both by pleasant and unpleasant sensations. An increase in the amplitude of a theta wave, for instance, denotes a rise in pleasure and a high level of awareness, learning and inspiration. Chemical stimuli of interest show intense activity patterns in at least two specific brain regions. Preferred regions of activity are in the hippocampus, the amygdala, the ventral tegmental area, in the orbitofrontal cortex and in the piriform (olfactory) cortex. Preferably, chemical stimuli which cause a desired change are tested further, so that only chemical stimuli which are safe and non-addictive may be selected. Safety and addictiveness of compounds may be tested by methods known in the art. Safety may, for instance, be checked by testing the metabolic activity of human liver cells and addictiveness may, for instance be checked by behavioural testing of the subject. Products which may be improved using a chemical stimulus selected by the invention are used in the food, beverage, cosmetics and pharmaceutical industry. Suitable examples include but are not limited to beverage additives, food additives, additives which intensify sensations such as happiness, joyful and stimulating consumption, disgust and repulsion. Intensifying unpleasant sensations may be very useful, for instance in self-defence strategies, comparable to the use of pepper spray. The invention further encompasses additives for shampoos, shower gels, soap bars, body lotions, cremes, perfumes, massage oils, and for sensational, i.e., affecting a subject's mood, and anti-depressant drugs. These additives may for instance be added to conserved food, microwave food, frozen food, to fruit juices, beer and wine, in particular to functional food, nutriceuticals and refreshment drinks and to health care products and to food used for space exploration. Products which include these additives may be formulated in any convenient way, including as a solid, liquid, spray, emulsion, foam or cream. In one embodiment, the chemical stimulus which is selected using the method of the invention is added to a food , beverage or cosmetic product for human or animal consumption or use, only after

(i) presenting the chemical stimulus to human panellists (ii) measuring their brain activity by any one or a combination of neuro-imaging devices

(iii) selecting a desired chemical stimulus based on the results of the human panel descriptors and neuro-imaging. Neuro-imaging devices for measuring brain activity are known in the art and include CT scan, PET scan, MRI scan and FMRI. In one embodiment, the neuro- imaging device used for measuring brain activity of human panellists is a functional magnetic resonance imager (FMRI). In another aspect, the invention relates to the use of the method of the invention for the detection of molecules which stimulate the brain to enhance flavour and fragrance sensation. The method of the invention is preferably used in a system of the invention. A great advantage of the method according to the invention is that many compounds may be screened in a limited period of time. The time frame from the implantation of an electrode to the selection of a chemical stimulus may be less than 60 minutes. It is noted that the methods of the invention are also applicable to physically isolated brain material, such as an isolated brain segment, optionally coupled to another isolated brain segment and to other neuron comprising material, such as a neural cell cultures. In this context, "isolated" refers to isolation from its natural environment, e.g. from an animal body. Since neural cell cultures are still at their infancy, application of the methods in the non-isolated, in-situ brain is preferred, since this causes the least discomfort to the subjects involved.

EXAMPLES

Example 1 In order to study the effect of Red Bull with respect to the brain neurons, an experiment was conducted so as to measure the brain waves of rat before and after intake of a cookie consisting of a biscuit soaked with different solutions, including Red Bull.

1.1 Experimental animals An adult, male Wistar rats (Harlan CPB laboratories, Zeist, The Netherlands) weighing around 150 g at the time of the experiment was used in this study. The rat was housed in individual cages under a controlled environment (21 + 1 °C; humidity 60%; lights on 08.00-20.00 h). Food and water were available ad libitum.

1.2 Electrode implantation The rat was anaesthetised with an intramuscular injection of pentobarbital (65 mg/kg). In order to record hippocampal EEG, one insulated stainless steel recording electrode (100 μm thick wire) was implanted into the left dentate gyrus of the hippocampus and another one in the radius region (with 200 μm tip separation), both under electrophysiological control. For stimulation of the angular bundle, an insulated stainless steel electrode was also implanted. The location of the electrodes was verified during the implantation by recording field potentials evoked in the granule cell layer by stimulation of the angular bundle. Electrode pins were placed in a small connector and the assembly was attached to the skull with small stainless steel screws and dental acrylic. The experimental protocols followed the European Communities Council directive 86/609/EEC and the Dutch Experiments on Animal Act (1997), and were approved by the Dutch animal welfare committee (DEC). 1.3 EEG monitoring After recovery from the implantation, the rat was transferred to a special cage (40 x 40 x 80 cm) and connected to a recording and stimulation system with a shielded, multistrand cable and electrical swivel. After habituation to the new condition, the rat underwent a series of tetanic stimulations (50 Hz) of the hippocampus in the form of a succession of trains of pulses every 10 s. Each train had a duration of 250 μs and consisted of biphasic pulses (pulses 40 ms apart, maximal intensity 500 μA). EEG signals were amplified using Axopatch 200B amplifier (Axon Instruments, U.S.A), filtered (0.1-150 Hz) and digitised by a PC. The EEG was continuously recorded for 30 min with a baseline recording of 10 min.

1.4 Preparation of the substance to be tested and dosage Cookies with water, Red Bull and chocolate milk were prepared by soaking a lOg biscuit in 2.5 ml of each solution. The meal was then presented to rats for consumption ad libitum.

1.5 Data analysis EEG recordings were recorded on an ATARI TT030 computer, using custom- made interface and software. The brain wave amplitude of the incoming signals at a frequency of 1-40 Hz was then analysed.

1.6 Results The different brain waves are represented by delta wave, theta wave, alpha wave and beta wave. The amplitude of these waves was measured 5 minutes before intake, and 20 minutes during and following meal consumption, with respect to the hippocampus. Results show that following intake of different solutions, there is an exclusive clear drop in the 2-3 Hz delta wave zone following intake of Red Bull on the one hand, water and chocolate milk on the other hand. Increased levels of delta waves are usually associated with sleep and mediation while a drop is associated with interest. There is also a significant increase in the 7-8 Hz theta wave zone following the intake of Red Bull solution. Increased theta waves denote a rise in pleasure, inspiration, learning, high level of awareness and vivid mental imagery. This correlates clearly with the drop in the amplitude of the delta brain wave, following Red Bull intake. This confirms that it is possible to record a difference in the EEG from the hippocampus, relating to the pleasantness of the substance presented.

Claims

1. A system for the detection of brain response to a chemical stimulus comprising: (a) an administration means for administering a chemical stimulus to a non-human subject; (b) a measuring electrode portion implanted in the brain of a subject; (c) a measuring device for measuring the amplitude of relevant brain waves or other electrophysiological signals before and after the administration of the chemical stimulus.

2. A system according to claim 1 wherein the non-human subject is a non-human mammal, a rat or a mouse.

3. A system according to claim 1 or 2 which further comprises a reference electrode implanted in the brain of a subj ect.

4. A system according to claim 3 wherein the reference electrode is implanted in the piriform cortex or the olfactory bulb.

5. A system according to claims 1-4 wherein at least two measuring electrodes implants are present.

6. A system according to claim 5 wherein one of the at least two measuring electrode implants is present in the limbic system and another in the orbitofrontal cortex or the ventral tegmental area.

7. A system according to claims 6 wherein one of the at least two measuring electrode implants is present in the hippocampus or the amygdala.

8. A system according to claims 1-7 wherein the amplitude of the alpha, beta, gamma, delta, or theta brain wave is measured.

. A system according to claims 1-8 wherein the administration means is for oral or nasal administration.

10. A system according to claim 9 which further comprises a processing means for analysing a correlation between the electrical signal measured and brain activity.

11. A system according to claims 1-10 wherein the chemical stimulus is odour or taste.

12. Use of a system according to claims 1-11 for the detection of molecules that stimulate the brain to enhance flavour and fragrance sensation.

13. A method for the detection of brain response to a chemical stimulus which method comprises (a) implantation of an electrode in the brain of a non-human subject; (b) measuring the amplitude of relevant brain waves or other electrophysiological signals before and after the administration of the chemical stimulus.

14. A method according to claim 13 wherein the subject is a non-human mammal, a rat or a mouse.

15. A method according to claim 13 or 14 which further comprises measuring a reference signal using a reference electrode implanted in a reference area of the brain.

16. A method according to claim 15 wherein the reference area is the piriform cortex or the olfactory bulb.

17. A method according to claims 13-16 wherein at least two measuring electrodes are implanted in the brain.

18. A method according to claim wherein one of the at least two measuring electrodes is implanted in the limbic system and another in the ventral tegmental area or in the right orbitofrontal cortex.

19. A method according to claims 13-18 wherein two, three or four electrodes are implanted in any two of the hippocampus, the ventral tegmental area, the amygdala or the orbitofrontal cortex.

20. A method according to claims 13-19 wherein the amplitude of the alpha, beta, gamma, delta, or theta brain wave is measured.

21. A method according to claims 13-20 wherein the chemical stimulus is administered orally or nasally.

22. A method according to claims 13-21 wherein the chemical stimulus is a compound or a mixture of compounds of microbial, bacterial or phytochemical origin.

23. A method according to claims 13-22 wherein the time frame from the implantation of an electrode to the selection of a stimulus is less than 60 minutes.

24. Use of the method according to claims 13-23 for the detection of molecules that stimulate the brain to enhance flavour and fragrance sensation.

25. A method for increasing the brain sensational level per olfactory receptor, which method comprises adding a chemical stimulus which gives a desired change in the brain to a food, beverage, cosmetic product for human or animal consumption or use.

26. A method for increasing the brain sensational level per olfactory receptor, which method comprises adding a chemical stimulus which gives a desired change in the brain to a pharmaceutical product.

27. A method for increasing the brain sensational level per olfactory receptor (a) a chemical stimulus which gives a desired change in the brain in a method according to claims 13-23 is presented to human panellists; (b) measuring their brain activity by any one or a combination of neuro-imaging devices; (c) selecting a desired chemical stimulus based on the results of the human panel descriptors and neuro-imaging; (d) adding the chemical stimulus to a food, beverage or cosmetic product for human or animal consumption or use.

28. A method for increasing the brain sensational level per olfactory receptor comprising: (a) presenting a chemical stimulus which gives a desired change in the brain in a method according to claims 13-23 to human panellists; (b) measuring their brain activity by any one or a combination of neuro-imaging devices; (c) selecting a desired chemical stimulus based on the results of the human panel descriptors and neuro-imaging; (d) adding the chemical stimulus to a pharmaceutical product.

29. A system according to claims 1-11 or a method according to claims 13-20, 21, 22 wherein the brain is isolated brain material.