WO1985005021A1

WO1985005021A1 - Novel method for measuring biogenic chemicals using in vivo electrochemical means

Info

Publication number: WO1985005021A1
Application number: PCT/US1985/000849
Authority: WO
Inventors: Patricia Broderick
Original assignee: Patricia Broderick
Priority date: 1984-05-09
Filing date: 1985-05-09
Publication date: 1985-11-21

Abstract

An in vivo semi-differential voltammetric method for obtaining reliable, reproducible measurements of the levels of certain biogenic chemicals in the body and brain by measuring the current positive for reduction produced by applying voltages to the body and brain.

Description

NOVEL METHOD FOR MEASURING BIOGENIC CHEMICALS USING IN VIVO ELECTROCHEMICAL MEANS

BACKGROUND OF THE INVENTION

This invention relates to the use of an in vivo electrochemical method to measure the amount of biogenic chemicals present in the body and brain of an animal or a human being. More particularly, it relates to the use of in vivo semiderivative voltammetric measurements of biogenic chemicals, particularly neurotransmitters, such as amines, amine metabolites, ascorbic acid, amino acids and neuropeptides produced in reaction to well-known psychopharmacological agents and neuropsychopharmacological agents such as analgesics, antipsychotic drugs, antidepressants, and other modulators of brain and peripheral neurochemistry.

It has been known to those possessing ordinary skill in the art that it is possible to measure biogenic chemicals using in vivo electrochemistry. This measurement has been accomplished using such facets of electrochemical measurments as chronoamperometry, differential pulse voltammetry, double differential pulse voltammetry, linear scan voltammetry, and semiderivative voltammetry. In all of these methods, a working electrode, a reference electrode and an auxiliary

electrode are attached to the brain of the animal to be studied. A controlled potential is applied to the working electrode and the current passing between the working electrode and the reference electrode is monitored and used to measure basal neurotransmitter •5 release and any alterations in brain neurochemistry. The signal is directly related to the chemical concentration of the neurotransmitter released at the neuronal brain membrane pre-synaptically, or possibly post-synaptically. The signal may also be related to inhibition of normal re-uptake of neurotransmitter at the neuronal membrane. It is generally believed that this current is positive for the oxidation of chemicals in the body. This signal is recorded as a graph indicating change in current with respect to time (chronσamperogram) or voltage (voltammogram) .

It is known that voltammetric measurements can be used to detect certain biogenic substances in the brain of animals [Kissinger, P.T.; Hart, J.B.; Adams, R.N. ; "Voltammetry in Brain Tissue - A New .Neurophysiological Measurement", Brain Research, 55 (1973), p. 209.]. Certain improvements have been made in voltammetric measurements since this method was first described for measuring biogenic chemicals. One such improvement is the processing of the current signal as the first half-derivative of the linear signal [Oldham, Analytical Chemistry Vol. 45 (1973) p. 39]. When applied to the brain, this type of processing results in a semi-differentiated voltammogram having sharper peaks, which allow greater separation between peaks representing chemical substances and which are easier to read than previous, linear voltammograms. Older conventional methods did not allow individual detection of amines because similar electrochemical potentials, accompanying many of the biogenic amines and other chemicals, are set by the catechol moiety and not by the alkyl moiety of the biogenic amine and thus did not allow for separation of peaks between different amines, all of which contain the catechol moiety. Semi-derivation of the signal allowed this individual detection. Many practitioners, however, have found it difficult or impossible to obtain reproducible measurements routinely using what is known as the in vivo electrochemistry technique of semi-derivative voltammetry. Accordingly, it is an object of this invention to provide a method for measuring biogenic chemicals.

It is a further object of this invention to provide an in vivo electrochemical method for measuring biogenic chemicals.

It is still a further object to provide a useful way to measure biogenic chemicals using the semiderivative voltammetric technique.

It is a further object of this invention to provide an in vivo electrochemical method for measuring biogenic amines, amine metabolites, ascorbic acid, amino acids and neuropeptides and other neurotransmitters and modulators of brain neurochemistry. I is still a further object of this invention to provide an in vivo electrochemical method for measuring alterations in biogenic brain chemicals in relation to the administration, both peripheral and central, of psychopharmacological agents and neuropsychopharmacological agents such anti-depressants, analgesics, anti-anxiety agents, anti-panic agents, anti-manic/depressive agents, calcium blocking agents, agents of addiction, other neuropeptides, enkephalinamides, dynorphin and other potential modulators of brain neurochemistry.

It is another object to provide an in vivo electrochemical method for interpreting these alterations in brain neurochemistry in light of diagnosing mental illness and developing new and more effective psychotherapeutic agents and other clinical applications.

It is a further "object of this invention to provide an in vivo electrochemical method for measuring the levels o^'f biogenic chemicals in humans.

It is still a further object of this invention to provide a means for studying the levels of biogenic chemicals produced during certain behavioral manifestations and thus provide a method for determining the causes of these manifestations.

It is another object of this invention to provide a method by which to correlate the production of certain biogenic chemicals with electrophysiological measurements.

SUMMARY OF THE INVENTION

It has now been found that semiderivative voltammetry can be used for measuring concentrations of biogenic chemicals in vivo in a reliable and repeatable manner according to the method it this invention. More particularly, it has been found, unexpectedly, that, according to the method of this invention, the current produced by biogenic chemicals when monitored by a semiderivative voltammetry controller is measurable as a current which is positive for reduction rather than as a current which is positive for oxidation. Thus, the method of this invention relates to the measurement of a reduction current produced in* the body of an animal or a human being the signal of which is processed by a semi-derivative voltammeter. The method of this invention is particularly well-suited to measuring biogenic chemicals in vivo, that is, in a living animal or human being, although this method can be used to measure such chemicals in vitro. It was previously thought that biochemical reactions, particularly in the brain, were irreversible. However, using the method of this invention, one can routinely detect reduced species, unexpectedly showing reversibility of such reactions in vivo.

BRIEF DESCRIPTION OF DRAWINGS

Figures 1 and 2 are schematic representations of two circuits which can be used in attempting to establish a semi-derivative voltammetric signal.

Figures 3A, 3B and 3C are representations of graphs showing signals which may be generated by linear and semi-differential scanning. Figure 3A ^ depicts a graph drawn according to conventional electrochemical format wherein the axis indicating positive current points downward and the axis indicating positive voltage points to the left. Figure 3B and 3C depict semiderivative voltammetry signals drawn according to nonconventional format, wherein the axes indicating positive current points upward and the axes indicating positive voltage points to the right.

Figure 4 is a representation of semiderivative voltammograms obtained from the tuberculum olfactorium of the rat brain before and after treatment with D-Ala 2-D-Pro5- enkephalinamide monoacetate (DAP) .

Figure 5 is a representation of semiderivative voltammograms obtained from the striatum and nucleus accumbens of the rat brain prior to and after treatment with DAP.

Figure 6 is a representation of semiderivative voltammograms obtained prior to and 1, 2 and 3 hours after treatment with morphine.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The method of this invention involves implanting, preferably, three electrodes in the body. preferably the brain: a reference electrode, an auxiliary electrode and a working electrode. The reference electrode provides a zero voltage point. The auxiliary electrode maintains the current, providing a sort of "ground". The working electrode is used to vary the electrical potential with respect to the reference electrode.

The current signal emitted by the system representing the current flowing between the reference and working electrodes is processed by a conventional electrochemical circuit. The conventional electrochemical circuit consists of conventional voltage clamp apparatus which uses negative feedback to control a potential difference and to measure the outgoing current. The semi-derivative voltammetry controller is a conventional electrochemical circuit with the addition of a ladder network of resistors and capacitors which process the current signal as the analog of the first one-half derivative of the raw current signal. The resistive capacitive network that produces the semi-differentiated signal is described in detail in "Semiintegral Electroanalysis: Analog Implementation", Analytical Chemistry, Vol. 45, No. 1, January 1973, by Keith Oldham, which is hereby incorporated by reference. One such semiderivative voltammetry controller useful in the method of this invention is the Bioanalytical Systems DCV-5 cyclic voltammetry amplifier with semiderivative signal processing. The electrodes should be situated in such a way as to accommodate a reduction current from, for example, the brain. It can be situated in any specific brain region, such as the striatum, tuberculum olfactorium or nucleus accumbens. Figure 1 shows the circuitry as it should appear in accordance with the method of this invention. The semi-derivative voltammeter, 107, is connected to the brain, 101, via two leads: the brain lead, 103, attached to the working electrode, 104, and the reference lead, 105, which is attached to the reference electrode, 109. The current being emitted from the brain is positive for reduction. Thus, in order to obtain a reliable measurement, the positive terminal of the voltammetry control should receive the emitted current signal from the brain lead which is attached to the working electrode. No signal would be produced if the leads were connected as shown in Figure 2, which is the circuit configuration which would conventionally be expected to measure current generated by biogenic brain species. In Figure 2, the working electrode 204 is shown implanted in the brain, 201. The brain lead, 203, is connected to the working electrode and the negative terminal of the semiderivative voltammeter, 207. This system, which is a circuit positive for oxidation, does not generate a recognizable signal. In actual practice, the terminal used with the working electrode may be a function of polarity.

It is postulated that, due to the speed at which the semi-derivative voltammetry signal is processed, the reduced species in the brain are detected using the method of his invention. Conventional brain electrochemical scans can only detect oxidized species. It is a.novel aspect of this invention that reduction can be detected in the brain. when monitoring the reaction of biological systems to the administration of a particular stiumlus, such as a drug, a baseline value should first be obtained by measuring the current generated with resect to different potentials without the stimulus. The voltammeter is used to obtain a baseline value for certain biogenic chemicals by measuring the current generated from the brain with respect to the application of varying potentials or voltages. Potentials ranging from about - 200 mv to about 1000 or from about 1000 mv may be applied. The scan rate, or rate at which the potentials are applied, is preferably in the range of about 5 to 30 mv-sec~ , most preferably about 10 v-sec^" .

Sensitivity, or amplification of the signal, can be in the range between about 0.2 and 10 nA -1'/2cm-1

The reaction to the administration of a stimulus can be measured after baseline values are recorded. After administration of the stimulus, potentials are applied and the current is measured with respect to the changing potentials. A comparison between voltammograms obtained prior to and after the stimulus will indicate the changes in production of biogenic chemicals.

The method of this invention can be used for chronic studies, which take place over a relatively long time period, e.g. three to four months, or for acute studies, in which values are taken over a short time period or only a few times.

Preferably, the reference electrode used should be of a conventional Ag/AgCl type known to those of ordinary skill in the art. The auxiliary electrode can be a platinum or a stainless steel electrode. The working electrode, if used for acute or chronic studies in a freely-moving animal, is preferably composed of a teflon-coated microelectrode homogeneously packed with graphite paste and nujol {mineral oil) . This allows the subject to move without breaking the electrode. If used for acute or chronic studies in an anesthetized animal, either glass or teflon electrodes may be used. If it is desired to measure biogenic chemicals without interference from certain acids such as ascorbic acid or the dopamine metabolite 3,4-dihydroxy-phenylacetic acid, the graphite paste should be modified with stearate or stearic acid. [Blaha and Lane, Brain Research Bulletin, Vol. 10, (1983), p. 861] It is believed that the same selectivity can be achieved using nafion-coated electrodes or other coatings known to those of ordinary skill in the art [Adams, 1984] . Preferably, the electrodes are implanted in vivo using a stereotaxic surgery device such as the David Kopf device. A David Kopf device consists of pairs of microscaled bars which allow the surgeon to implant the electrodes at the precise brain site desired. The method of tis invention may be used to measure biogenic chemical levels, release or uptake inhibition in both anesthetized animals or humans and freely-moving (unanesthetized) animals or humans. The method of this invention may also be used to elucidate behavioral determinants. By determining the levels of biogenic chemicals and the changes in those levels while observing certain animal and human behavior, one skilled in the art can correlate the behavior patterns with the biogenic chemical concentrations. This observation can contribute to the possible determinations of the causes of certain behavioral reactions. This can be applied to all observable behavioral studies in all stages of life (neonatal, adult and aged) , for example, brain reward/brain pain systems, euphoria, drug addiction, alcohol dependency, diabetes, self-administration studies, stereotypy, catalepsy, anti-anxiety or anxiety paradigms, turning behavior paradigms, reactions to environmental stimuli throughout life stages, conflict/avoidance paradigms, muricide, and other behavioral studies.

The method of this invention can be used in vivo in applications involving any warm-blooded or cold-blooded animal possessing a brain and other organs of the body, such as a human, a primate, a lower mammal, reptile or squid. It is particularly well-adapted to observing the levels of biogenic chemicals in mammals, including human beings.

Telemetric measuring devices known to those skilled in the art may also be used to monitor current via radio signals such that external electrodes need not be attached to a stationary source which would hinder movement of the subject during measurement.

The difference between the method of this invention and prior art methods can be seen by comparing a semiderivative voltammogram produced by using the method of this invention with graphs produced by prior art methods. Figure 3A shows a graph obtained by a linear scan of biogenic chemicals. It is a typical curve representing a current which is positive for oxidation presented in conventional electrochemical form [Kissinger et al.]. In Figure 3A, the serotonin peak is masked and undifferentiated from the dopamine peak. Figure 3B is a semiderivative voltammogram which also represents a current presented in non-conventional form which is positive for oxidation [Lane, Hubbard and Blaha, J. Electrochemistry, Vol. 95, (1979) P. 117] . Figure 3B shows differentiated serotonin and dopamine peaks. Figure 3C is a semiderivative voltammogram derived from using the method of this invention. It shows sharp differentiated serotonin and dopamine peaks and a current which is unexpectedly positive for reduction. The method of this invention can be used to measure biogenic chemicals in the organs of the body as well as in different parts of the brain, e.g. the striatum, tuberculum olfactorium, nucleus accumbens, median raphe, periaquaductal gray, hippocampus, locus coeruleus, the frontal cortex, amygdal, hypothalamus.

thalamus, substantia nigra, globus pallidus and other areas. The methods of this invention can also be used to measure biogenic chemicals in such organs of the body as the heart, the retina, the gut, the cervix, kidney, liver, gall bladder, vagina and the like.

Moreover, it can be used as a diagnostic tool to measure levels of biogenic chemicals and compare them to established normal values. The method of this invention can be used to measure levels of biogenic chemicals such as amines, amine metabolites, ascorbic acid, amino acids, particularly dopamine, homovanillic acid, tryptophan, serotonin, enkephalins and enkephalinamiάes. It is believed that neuropeptides can also be measured using the method of this invention.

These chemicals may be present at certain levels in normal persons without the administration of any stimuli. However, in persons having abnormal psychological characteristics, and, therefore, forms of mental illness, different levels of biogenic chemicals may be present. Thus, the in vivo electrochemical method of this invention may be used to diagnose mental illnesses, such as obesity, depression, manic depression, drug addiction, and others, according to the levels of certain chemicals in the brain. The method of this invention can also be used for predicting the advent of neurological and other diseases such as diabetes, Parkinson's and Huntington's diseases based on the comparison between known normal levels and abnormal levels of particular substances, e.g. dopamine.

Moreover, certain biogenic chemicals measurable by the method of this invention are produced and/or altered in reaction to central or peripheral administration of chemical stimuli, such as drugs. These chemicals which may produce such reactions are psychopharmacolo ical and neuropsychopharmacological agents such as neuroleptics, neuropeptides, amino acids, analgesics, endorphins, gut and brain hormones, calcium blockers, addictive agents, anti-depressants, anti-anxiety agents, anti-panic agents, amphetamines, particularly beta and gamma-endorphins, enkephalins, enkephal amides such as D-Met 2-Pro5- enkephalinamide, cholecystokinin, dynorphin, marijuana, morphine, cocaine and other drugs and agents known to affect the human condition. For example, the central nervous system effects of peripherally-administered enkephalmamides has been detected, surprisingly, for the first time using the method of this invention.

The method of this invention can, therefore, be used for studying the mechanisms of action in the brain of particular agents. This would aid in the development of new psychotherapeutic agents by the study of structure-activity relationships of administered drugs. Analogs of the studied drugs could be evaluated for their effect on biogenic chemical levels in order to develop more effective therapeutic agents which have fewer and less severe side effects. The following examples futher illustrate certain embodiments of the method of this invention. Of course, they do not serve to limit the scope of this invention in any way. It should be noted that Figures 4, 5 and 6 illustrate curves which are positive for reduction. They are presented in the upright, nonconventional position for the convenience of interpretation.

EXAMPLE 1

A semiderivative, voltammetry controller made by Bioanalytical Systems (BAS-DCV5) was prepared for analysis by connecting it to a working electrode and a combined reference/auxiliary electrode. The teflon-coated working microele.ctrode (150-175 microns) was coated with a material consisting of graphite paste and nujol modified with stearate, which allows the measurement of changes in dopamine concentration without interference from ascorbic acid or the dopamine metabolite, 3,4-dihydroxyphenylacetic acid (DOPAC) .

Adult, male, Spr.ague-Dawley rats were group housed and fed Purina rat food and water daily. Behavioral and biochemical studies were routinely carried out on these rats in the afternoons for better reproductibility.

Prior to testing, a reproducible stable baseline measurement of the test rats* dopamine level was achieved in a period of 1.25 hours. The microelectrodes were first tested in vitro in phosphate buffer solution pH 7.4 (0.16 M NaCl) . Potentials were applied within a range of -0.001 to + 0.5 v. The potentials were measured with respect to a reference Ag/AgCl electrode. Both the reference electrode and a platinum auxiliary electrode were placed in contact with the cortex of the brain. The working electrode was stereotaxically implanted using a David Kopf stereotoxic surgery device in the tuberculum olfactorium, according to the atlas of Pellegrino and Cushman, 1967 (coordinates: 2.6 mm anterior to Bregma, 2.5 mm lateral to midline and 0.5 mm below the skull surface) .

On each experimental day, the animals were injected intraperitoneally with D-Ala 2-D-Pro5-enkephalmamide monoacetate (DAP) dissolved in distilled water (5 mg/kg/ml) , along with appropriate vehicle or saline control injections. Semiderivative voltammograms from rats anesthetized with chloral hydrate (450 mg/kg) were recorded every ten minutes for up to two hours at a scan rate of 10 mv sec and sensitivity of 0.2 nA sec^{" /} cm^" .

Changes in dopamine concentrations in the tuberculum olfactorium after injection of DAP enkephalmamide were measured by comparing the mean of the preinjection values with both the mean and the maximum of the postinjection values. The effects of the administration of the DAP enkephalmamide, in terms of basal dopamine release, is shown in Figure 4, which shows two semi-derivative voltammograms, one pre-injection and one post-injection. The dopamine signal (left-hand peak) is clearly unchanged from pre- to post-injection. The serotonin signal (right-hand peak) is, however, measurably higher after the injection.

It was observed that DAP did not inhibit the rats' locomotor activity. This observation is consistent with conventional theory which places control of locomotor activity in this part of the brain. It shows that dopamine levels in the tuberculum olfactorium are not affected by doses of DAP enkephalinamide. That locomotor activity was not affected is consistent with the placement of locomotor control in the tuberculum olfactorium.

EXAMPLE 2

Rats were prepared for testing as in Example

1. The rats were injected with d-amphetamine sulfate (2.5 mg/kg) dissolved in saline, or DAP (5mg/kg) followed in one-half hour by D-amphetamine sulfate. The amphetamine induced stereotypy in the treated rats. Sniffing, head movement, rearing, licking, chewing, grooming, forepaw pacing and locomotor activity were observed. D-Ala 2-D-Pro5-enkephalinamide monoacetate was administered intraperitoneally. Changes in central nervous system dopamine concentrations in rat striatum and rat nucleus accumbens after administering the enkephalinamide were measured by comparing the mean of the preinjection values with both the mean and the maximum of postinjection values. Figure 5 shows semiderivative voltammograms from rat striatum (Fig. 5A) and rat nucleus accumbens (Figure 5B) . The semiderivative voltammograms show a significantly decreased signal from the striatum and no change in the signal from the nucleus accumbens. It was observed that the DAP inhibited head-bobbing, sniffing and frequency of rearing. It did not significantly inhibit the amphetamine induced effect on locomotor activity. This is consistent with behavioral theory that associates stereotyped behaviors with migrostriatal dopamine activation.

EXAMPLE 3

Rats were prepared for intraperitoneal administration of drug as in Example 1. D-morphine sulfate in distilled water solution was injected intraperitoneally at a rate of about 5 mg/kg rat weight one hour after reproducible basal dopamine and serotonin signals were recorded from rat anterior striatum. The effect of morphine on striatal dopamine and serotonin signals were studied for a period of three hours. Alterations in the dopamine and serotonin signals after morphine was injected were measured by comparing the mean of pre-morphine injection values with both the mean and the maximum of post-morphine injection values. Figure 6 shows the resultant semi-derivative voltammograms taken 1, 2 and 3 hours after administration of morphine and shows changes in levels of brain chemicals in response to the drug.

Claims

THE CLAIMS :

1. A method for measuring the concentration of biogenic chemicals in vivo in warm-blooded and cold-blooded animal brains and bodies comprising stereotaxically implanting at least two electrodes in a body organ or brain of an animal, applying an increasing potential across the electrodes and processing the signal which represents the resultant current as the semi-derivative of the linear current function, wherein the resultant measurable current is postive for reduction of the biogenic chemicals measured.

2. A method according to claim 1 wherein said biogenic chemicals are selected from the group consisting of amines, amine metabolites, ascorbic acid, amino acids, and neuropeptides.

3. A method according to claim 2 wherein said biogenic chemicals are one or more of dopamine, serotonin, homovanillic acid or ascorbic acid.

4. A method according to claim 1 wherein the presence of said biogenic chemicals are measured in the central nervous system after the peripheral administration of a drug.

5. A method according to claim 4 wherein said drug is an enkephalinamide.

6. A method according to claim 1 wherein said biogenic chemicals are measured after the central administration of a drug.

7. A method according to claim 1 wherein said resultant semi-differentiated current is measured telemetrically.

8. A method according to claim 1 wherein said biogenic chemicals are measured in a freely-moving animal.

9. A method according to claim 1 wherein said biogenic chemicals are measured in an anesthetized animal.

10. A method according to claim 1 wherein a chemical substance is administered to the animal after a reproducible baseline value is achieved.

11. A method according to claim 10 wherein said chemical substance is a neuropsychopharmacological agent selected from the group consisting of antidepressants, neuroleptics, anti-anxiety agents, anti-panic agents, anti-manic/depressive agents, calcium-blocking agents, agents of addiction, enkephalins, enkephalina ides, endorphins and dynorphin.

12. A method according to Claim 1 wherein three electrodes are implanted in the brain.

13. A method according to claim 12 wherein said.electrodes, .comprise- a reference, an auxiliary and a working electrode.

14. A method according to claim 13 wherein said reference electrode is a Ag/AgCl electrode.

15. A method according to claim 12 wherein said working electrode is a teflon-coated microelectrode homogeneously packed with graphite paste and mineral oil.

16. A method according to claim 14 wherein said graphite paste and mineral oil is modified with a compound selected from the group consisting of stearic acid, stearate and nafion.

17. A method for diagnosing mental illness comprising the measurement of levels of biogenic chemicals according to the method of claim 1 and comparing the resultant levels to the levels of said chemicals from healthy individuals.

18. A method of elucidating behavioral determinants by measuring levels of biogenic chemicals using the method according to claim 1 and correlating said levels with physiological attributes,