WO2003045499A1

WO2003045499A1 - METHODS RELATED TO AN α4 NICOTINIC ACETYLCHOLINE RECEPTOR SUBUNIT MUTANT

Info

Publication number: WO2003045499A1
Application number: PCT/AU2002/001583
Authority: WO
Inventors: John Drago; Malcolm Horne; David Finkelstein
Original assignee: Monash University
Priority date: 2001-11-26
Filing date: 2002-11-25
Publication date: 2003-06-05
Also published as: AUPR909001A0

Abstract

The present invention provides a method of screening for a compound that is a proconvulsant or anticonvulsant wherein the method includes: exposing a transgenic animal whose genome comprises a disruption of the α4 nAChR gene to the compound; and determining the seizure response of the transgenic animal to the compound. Applicants have found that an animal having a non-functional α4 nAChR gene has enhanced sensitivity to proconvulsants and anticonvulsants and especially GABA reception blockers.

Description

METHODS RELATED TO AN α4 NICOTIN1C ACETYLCHOLINE RECEPTOR

SUBUNIT MUTANT

The present invention relates to a method of screening for a compound that is a proconvulsant or anticonvulsant using an animal model.

INTRODUCTION

Epilepsy is one type of seizure and is a condition that affects 2-3% of the population at some time in their lives. Epilepsies may be broadly grouped into two etiological categories, symptomatic and idiopathic. The seizures that occur in symptomatic epilepsies result from gross disturbances of neuronal physiology, typically associated with progressive death of neurons and brain atrophy, and their study has not, therefore, led to major advances in understanding the pathophysiology of epilepsy. In contrast, genetic factors are believed to be paramount in the idiopathic epilepsies and offer the chance to study seizures and epileptogenesis at the mechanistic level as the mutations selectively interfere with seizure threshold without significantly disturbing other neuronal functions.

Epilepsy and other seizures are a whole animal disease and must be studied in whole animal paradigms. Epilepsy research and other seizure research has been dependent on the use of animal models without a clearly defined genetic basis, mostly in the screening of thousands of compounds for possible activity in epilepsy treatment. However many of the current models have significant limitations and there remains a need for new animal models.

Anti-epileptic drugs have been used to control seizures, however many patients continue to have seizures despite optimal treatment regimes and many have unacceptable side effects associated with treatments. There is an ongoing need for the discovery of efficacious treatments with fewer side effects.

Accordingly it is an aspect of the present invention to overcome or at least alleviate some of the problems of the prior art. SUMMARY OF THE INVENTION

The present invention provides a method of screening for a compound that is a proconvulsant or anticonvulsant wherein the method includes: exposing a transgenic animal whose genome comprises a disruption of the α4 nicotinic acetylcholine receptor (nAChR) gene to the compound; and determining the seizure response of the transgenic animal to the compound.

In another embodiment the present invention provides a proconvulsant or anticonvulsant identified by a method of screening for a compound that is a proconvulsant or anticonvulsant wherein the method includes: exposing a transgenic animal whose genome comprises a disruption of the α4 nAChR gene to the compound; determining the seizure response of the transgenic animal to the compound.

In a further embodiment the present invention provides a method of treating seizures in a subject wherein the method includes administering an effective amount of a proconvulsant or anticonvulsant identified by: exposing a transgenic animal whose genome comprises a disruption of the α4 nAChR gene to the compound; determining the seizure response of the transgenic animal to the compound

In yet a further embodiment the present invention provides a method of identifying a gene involved in modulating seizure response wherein the method includes: analysing gene expression in a transgenic animal whose genome comprises a disruption of the α4 nAChR gene; comparing gene expression in the transgenic animal with that of a non-transgenic animal; and identifying differential gene expression between the transgenic and non-transgenic animal.

In another embodiment the present invention provides a method of identifying a protein involved in modulating seizure response wherein the method includes: analysing protein expression in a transgenic animal whose genome comprises a disruption of the α4 nAChR gene; comparing protein expression in the transgenic animal with that of a non-transgenic animal; and identifying differential protein expression between the transgenic and non-transgenic animal.

FIGURES

Figure 1 shows the number of seizures according to type in wild type (Wt) and mutant (Mt) mice during the observation period for each drug tested: A. pentylenetetrazole (PTZ), B. bicuculline, C. kainic acid and D. strychnine. Filled bars represent Wt and empty bars Mt. *showing significant P values when compared to Wt mice.

Figure 2 shows quantitative γ-aminobutyric acid subunit A (GABA_A) receptor binding in Wt and Mt brains, Brain regions analyzed were: cortex (Cx), caudate/putamen (Cpu), nucleus accumbens (NAc), olfactory tubercle (T), lateral globus pallidus (LGP), interpeduncular nucleus (IPn), lateral septum (LS), periaqueductal grey (PAG), pontine nucleus (Pn), anterior paraventricular thalamus (Pva), substantia nigra pars reticulata (SNr) and superior colliculus (SC). There was no significant differences in quantitative ligand binding between Wt and Mt brains for any of the brain regions examined (Student's t- tests).

Figure 3 shows the number of mice, remaining alive at increasing intervals after PTZ injection. The mortality at the end of the electroencephalographic (EEG) scoring session was significantly greater in the mutant mice (16/20, 80%) than in controls (3/20, 15%, p<0.001 , Fisher's Exact Test). Four mice died after the EEG scoring session was completed, so that total mortality on the day of injection was somewhat greater in both groups (mutants 17/20 85% vs controls 6/20, 30%, p<0.001).

Figure 4 shows the prognostic significance of each EEG category, across all 40 mice, is represented according to the subsequent probability of death in various time intervals after the abnormality was observed. The numbered EEG categories correspond to the descriptions listed above. This analysis confirms that the descriptive categories used to classify EEG abnormalities were correctly ranked in terms of severity, and also allows quantification of the overall EEG abnormalities for each genotype. For each category, the mean probability of death across 5 intervals (2.5min, 5min, 10min, 20min and 40min) was used to derive an overall prognostic weighting for that category.

Figure 5 shows evolution of the mean prognostic EEG score for each genotype. For each mouse at each time point, the mean EEG score in the preceding 2.5min and subsequent 2.5min was averaged to give a 5-minute prognostic score. For each genotype, evolution of the mean prognostic score is depicted, with circles indicating the times of individual mouse deaths. Some deaths are obscured by superposition of data points. The data was derived such that at each time point only mice that were still alive at that time contributed to the mean prognostic score.

Figure 6 shows evolution of the mean 5-minute prognostic score for each genotype (+ standard error; SE), for each 5-minute interval after injection. Differences between the two groups were significant for the first 20 minutes (p<0.05 by Student's t-test), after which time there were too few surviving mutant mice to allow meaningful comparison.

Figure 7 shows incidence of each EEG category as a proportion of total post- injection recording time, for mutants and wild types (mean + SE). The numbered EEG categories correspond to the descriptions listed. The figure shows that mutant mice spent a significantly higher proportion of the time with more severe EEG abnormalities (p<0.05 for categories 6, 7 and 8 by Student's t-test) and a correspondingly lower proportion of the time with minor abnormalities.

Figure 8 shows the same data as in Figure 5 but the incidence has been normalised, relative to the control group, to reveal the deviation from normality associated with the induced mutation.

Figure 9. shows the seizure responses and death rate following administration of the proconvulsant agent 4-aminopyridine (4-AP). Mutant mice had a dramatic reduction in 4-AP-induced major motor seizure induced death. Both genotypes showed a complete spectrum of seizure types although all mice progressed to major motor seizures. In contrast to other proconvulsants, 25 % of Wt mice treated with 10 mg/kg of 4-AP (5 out of the 20) died (although 2 of the 7 Wt mice that had phase 5 seizures recovered) while there was no death in the Mt group (Fisher exact test P=0.043). For mice treated with 12 mg/kg of 4-AP, 14 out of 21 (66.7 %) Wt mice progressed to phase 5 seizures and died while only 2 Mt mice out of 21 (9.5 %) had phase 5 seizures and also died (Fisher exact test P=0.001). 4-AP is known to produce seizures by the release of endogenous glutamate (an excitatory amino acid neurotransmitter). Without being limited by theory, the reduction in 4-AP induced seizures seen in Mt mice may reflect compensatory down regulation of endogenous glutamate neurotransmission.

DETAILED DESCRIPTION In a first aspect of the present invention, there is provided a method of screening for a compound that is a proconvulsant or anticonvulsant wherein the method includes: exposing a transgenic animal whose genome comprises a disruption of the α4 nAChR gene to the compound; and determining the seizure response of the transgenic animal to the compound.

Epilepsy is essentially a whole animal disease and is ideally studied in whole animal paradigms such as a transgenic animal. The use of a transgenic animal whose genome comprises a homozygous disruption of the α4 nAChR gene provides a screening system wherein the exact mutation underlying the seizure tendency is known.

A large number of structurally related nAChRs are expressed in the brain; they are pentameric ligand gated ion channel receptors variously composed of αsubunits (α₂.g) and β subunits (β_2-4). The α₄β₂ receptor configuration is expressed at high levels and is thought to play an important neuromodulatory role in anxiety, cognition, neurodegeneration and antinociception. A screening procedure provides for the identification of therapeutic compounds that affect seizure response in vivo. The method generally involves screening any number of compounds for therapeutically active agents by employing mutant and transgenic animals as described herein. It will be readily understood that a compound that alters seizure response in a transgenic animal, for example a transgenic mouse, can provide an effective therapeutic agent in a mammal, for example a human patient. Since the screening procedure of the present invention are performed in vivo, the toxicity of compounds to a mammalian host may be also ascertained. The present invention also provides for high throughput in vitro screening methods using cells and cell lines derived from the transgenic animal of the present invention.

The transgenic animal of the present invention may be generated via a disruption introduced into the animal or an ancestor of the animal. The disruption results in a non-functional or hypofunctional α4 nAChR gene. Preferably the disruption is generated via homologous recombination in embryonic stem cells or be introduced to microinjection of embryonic stem cells into a blastocyst. Preferably the transgenic animal is fertile and can transmit the mutation to its offspring or the transgenic animal of the invention is post-natal.

Preferably the transgenic animal is homozygous or heterozygous for a mutation in the α4 nAChR gene wherein the α4 nAChR gene is non-functional or hypofunctional. More preferably the α4 nAChR gene is deleted and removed. Even more preferably the transgenic animal of the present invention is a mutant mouse homozygous for a disrupted α4 nAChR gene in the central nervous system of the mouse. Most preferably the transgenic animal is a knock-out of the α4 nAChR as described in Ross et al J. Neuroscience 2000, 20(17): 6431- 6441.

Preferably the transgenic animal of the present invention have a hypofunctioning α4 nAChR subunit allele. More preferably the transgenic animal has a specific sensitivity to proconvulsant compounds that interfere with GABA neurotransmission. Without being limited by theory, the lack of nicotinic receptor functionality may result in secondary down-regulation of GABAergic neurotransmission, enhanced seizure response, and the mutant mice display an enhanced seizure induced death rate to the potassium channel blocker 4-AP. Without being limited by theory, the drug 4-AP may act to produce seizures by releasing stored glutamate. It may be that mutant mice compensate for secondary down- regulation of GABAergic neurotransmission by also down-regulating brain excitatory amino acid neurotransmitters such as glutamate.

Preferably the cells of the transgenic animal lacks detectable levels of the α4 nAChR. More preferably there is no change in levels of other nAChR subunits in the transgenic animal as compared to a wild type animal.

The transgenic animal of the present invention may express a mutant genotype or phenotype additional to the mutation in the α4 nAChR gene.

"Transgenic animal" as used herein includes any mammal that includes a nucleic acid sequence that is inserted into a cell and becomes part of the genome of the animal that develops from that cell. The transgene may be partly or entirely heterologous to the transgenic animal. Transgenic animals as used herein include knockout animals wherein the 4 nAChR gene is substantially deleted. By the term "substantially deleted" it is meant to include removal of the gene to an extent that the gene becomes non-functional or hypofunctinal such that undetectable levels of α4 nAChR are produced, if at all.

The transgenic animal of the present invention may be any animal which is capable of transgenesis including, but not limited to, a rat, mouse, rabbit, pig, goat, cow, sheep, guinea pig, or hamster. Preferably the transgenic animal is a transgenic mouse.

The term "compound" as used herein includes, but is not limited to, a single active agent, molecule or substance, or combinations of active agent, molecule or substance. The term "proconvulsant' as used herein includes a compound that enhances seizure response. Preferably the proconvulsant compound enhances central nervous system epileptogenic activity. The compound may directly or indirectly result in an enhanced seizure response.

Enhanced seizure response may be achieved by a compound that interacts with a factor in the transgenic animal. Enhanced seizure response may also involve the compound modulating the activity of a factor within the cell. The factor may be a protein, nucleic acid or synthetic molecule such as a drug/small molecule inhibitor and may be endogenous to the cell, or exogenous to the cell and introduced into the cell from another source using techniques such as, but not limited to, recombinant DNA technology well known to those of skill in the art. Such factors may bind directly or indirectly to the compound, or regulate the expression or activity of another protein or gene, the product of which may enhance seizure response. Alternatively, the factor may modulate the translation of the transcripts derived from a gene in the transgenic animal. Such a factor may be endogenous to the cell, or exogenous to the cell and introduced into the cell from another source.

The term "anticonvulsant' as used herein includes a compound that decreases seizure response. Preferably the anticonvulsant compound has a net inhibitory effect on neural activity in the specific animal species. The compound may directly or indirectly result in a decreased seizure response.

Decreased seizure response may be achieved by a compound that interacts with a factor in the transgenic animal. Decreased seizure response may also involve the compound modulating the activity of a factor within the cell. The factor may be a protein, nucleic acid or synthetic molecule such as a drug/small molecule inhibitor and may be endogenous to the cell, or exogenous to the cell and introduced into the cell from another source using techniques such as, but not limited to, recombinant DNA technology well known to those of skill in the art. Such factors may bind directly or indirectly to the compound, or regulate the expression or activity of another protein or gene, the product of which may decrease seizure response. Alternatively, the factor may modulate the translation of the transcripts derived from a gene in the transgenic animal. Such a factor may be endogenous to the cell, or exogenous to the cell and introduced into the cell from another source.

"Seizure response" as used herein includes, but is not limited to, interruption of normal exploratory behavioural activity, or the production of overt clinical seizure activity such as cessation of motor activity with a fixed prone posture, isolated or repetitive jerking or stiffening of any body part, animal jumping or death of the animal following an abnormality of induced neural activity. Abnormality in the form of complex behavior such as stereotypical activity or abnormal sensation or perceptions are included in the definition of seizure activity.

Seizure response may be determined by methods known to those of skill in the art including, but not limited to, cortical or depth electroencephalographic measurement or using methods to detect enhancement of neural activity including expression of immediate early genes or by non invasive or invasive magnetic resonance spectroscopy, or positron emission spectroscopy.

The seizure response may be determined by considering a response before and after exposure of the animal to the compound or it may be determined by comparing the response against a wild type or control animal. A wild type animal is an animal without disruption of the α4 nAChR gene. The control is the transgenic animal that has not been exposed to the compound. Hence the seizure response may be determined against any relative control to determine an effect of a proconvulsant or anticonvulsant compound.

Seizures includes primary (in which no structural cause is identified) and secondary (in which a structural abnormality is identified) epilepsies and all drug induced abnormalities of electrical activity resulting in a seizure phenotype (as described above) including the effects of antidepressants, antipsychotics, addictive drugs and anxiolytic compounds. Seizures include seizures resulting from cerebral ischemia, Parkinson's disease, Huntington's disease and spasticity and also possibly for treatment with antidepressant, anxiolytic, and antipsychotic drugs. Primary or secondary epilepsy may have a genetic basis.

Epilepsies are classified generally into two groups including generalized and partial, based on the type of seizures. Generalized seizures may involve a loss of consciousness or convulsive movements, including tonic-clonic, myoclonic, tonic or clonic, and myoclonic astatic epilepsy. Partial seizures are divided into three subgroups including simple seizure in which consciousness is preserved, complex seizures in which awareness is lost, and secondarily generalized seizures in which simple or complex partial seizures evolve into generalized tonic-clonic seizures

Proconvulsant or anticonvulsant compounds may be identified by administering the compound to the transgenic animal and evaluating the seizure response. The seizure response may be of varying degrees. For example seizures induced by PTZ may be classified into four phases. A phase 1 seizure is a hypokinetic type commonly involving an arrest of normal exploratory behaviour followed by a decrease in motor activity with the animal coming to a complete rest in a crouched or prone position. A phase 2 or partial clonus seizure may be characterized by brief twitching movements involving face, head, forelimbs or hind limbs. A phase 3 or generalized clonus seizure may involve focal twitching that may be rapidly followed by loss of postural control and repetitive or clonic movements involving all limbs and tail. Jumping and repetitive rearing behaviour may also be classified as phase 3 activity. Phase 4 or tonic-clonic seizure may involve tonic hind limb extension. Phase 4 seizures commonly result in death.

Throughout this specification the word "comprise" and variations of the word, such as "comprising" and "comprises", is not intended to exclude other additives, components, integers or steps.

A ΠOΠ-GABAAR antagonist proconvulsant such as strychnine, that is a glycine inhibitory receptor antagonist which acts by blocking the increase in permeability of chloride ions induced by glycine in the postsynaptic membranes of neurons, may be used to determine whether there is a generic or specific GABA_AR-mediated alteration in chemoconvulsive threshold in the nAChR mutant.

Administration of a candidate compound to the transgenic animal of the invention can be by any known route, for example intraperitoneally, and at a range of concentrations. Following an appropriate time the animal can be assessed for the effect of the compound compared to control animals. Suitable control animals include wild type animals.

In another aspect the present invention provides a proconvulsant or anticonvulsant identified by a method of screening for a compound that is a proconvulsant or anticonvulsant wherein the method includes: exposing a transgenic animal whose genome comprises a disruption of the α4 nAChR gene to the compound; determining the seizure response of the transgenic animal to the compound.

The compounds identified as proconvulsants or anticonvulsants may be prepared as a pharmaceutical composition including a suitable pharmaceutically acceptable carrier or excipient.

The proconvulsant or anticonvulsant identified above may be used for treating a variety of afflictions involving seizures.

Accordingly, in a further aspect the present invention provides a method of treating seizures in a subject wherein the method includes administering an effective amount of a proconvulsant or anticonvulsant identified by: exposing a transgenic animal whose genome comprises a disruption of the α4 nAChR gene to the compound; determining the seizure response of the transgenic animal to the compound. The dosage of the therapeutic formulation will vary widely, depending upon the nature of the disease, the frequency of administration, the manner of administration, the clearance of the agent from the host, and the like. The initial dose may be larger, followed by smaller maintenance doses. The dose may be administered as infrequently as weekly or biweekly, or fractionated into smaller doses and administered daily, semi-weekly, etc. to maintain an effective dosage level. In some cases, oral administration will require a higher dose than if administered intravenously.

A therapeutic dose of a proconvulsant or anticonvulsant is administered to a host suffering from a seizure disorder. Administration may be topical, localized or systemic, depending on the specific disease. The compounds are administered at an effective dosage that over a suitable period of time substantially modifies the disease progression. It is contemplated that the composition will be obtained and used under the guidance of a physician for in vivo use.

The dose will vary depending on the specific proconvulsant or anticonvulsant utilized, type of disorder, patient status, etc., at a dose sufficient to substantially protect the neural cells from damage, dysfunction or death, while minimizing side effects. Treatment may be for short periods of time, for example after trauma, or for extended periods of time, for example in the prevention or treatment of epilepsy. A range of concentrations for each compound may be evaluated to determine the therapeutic window.

In another aspect the present invention provides a method of identifying a gene involved in modulating a seizure response wherein the method includes: analysing gene expression in a transgenic animal whose genome comprises a disruption of the 4 nAChR gene; comparing gene expression in the transgenic animal with that of a non- transgenic animal; and identifying differential gene expression between the transgenic and non- transgenic animal. The present invention also provides for the isolation of genes or gene products involved in the enhancement or depression of seizure activity in mammals. This may be by the isolation of such genes from drug naϊve or drug treated mutant and wild type mice.

"Analysing gene expression" as used herein includes methods known to those of skill in the art, including, but not limited to, northern blotting, microarray analysis, differential display, reverse transcriptase polymerase chain reaction and in situ hybridisation.

Identifying a gene involved in modulating seizure response provides targets for treatment of seizures including, but not limited to, targets for drugs.

"Differential gene expression" as used herein refers to genes that expressed at a different level in the transgenic animal compared to a non-transgenic animal. The differing levels of gene expression may be the result of, but not limited to, transcriptional or translational repression or activation, or the modulation of a factor that regulates expression of the gene.

In another aspect the present invention provides a method of identifying a protein involved in modulating seizure response wherein the method includes: analysing protein expression in a transgenic animal whose genome comprises a disruption of the α4 nAChR gene; comparing protein expression in the transgenic animal with that of a non- transgenic animal; and identifying differential protein expression between the transgenic and non-transgenic animal.

Identifying a protein involved in modulating seizure response provides targets for treatment of seizures including, but not limited to, targets for drugs.

"Analysing protein expression" as used herein includes methods known to those of skill in the art, including, but not limited to, proteomic analysis, western blots, immunoprecipitation and immunohistochemistry. Analysis of protein expression may involve analysing proteins expressed by genes identified as being differentially expressed in the transgenic animal compared to a non-transgenic animal.

In another aspect of the present invention, there is provided a method for screening for a compound that modulates a seizure response in an animal said method comprising: administering the compound to a transgenic animal whose genome comprises a disruption of the α4 nAChR gene; and determining the effect of the compound upon a cellular or molecular process associated with the seizure response compared to an effect of the compound administered to a non-transgenic animal, wherein a difference in effect is indicative of a compound that modulates the seizure response.

Preferably, the seizure response is associated with epilepsy. To determine the effect of the compound on a cellular or modular process, changes in protein, nucleic acid, neurotransmitter or factors that bind directly or indirectly to the compound may be measured. Any factors that are associated with the α4 nAChR may also be monitored and may include factors such as GABA, glutamate, dopamine and acetylcholine (Ach). Hence any compound which modulates these factors may be used to monitor the effect of the compound on cellular or molecular processes associated with the seizure response.

The present invention will now be more fully described with reference to the accompanying examples and figures. It should be understood, however, that the description following is illustrative only and should not be taken in any way as a restriction of the generality of the invention. EXAMPLES Example 1: Mouse behaviour

Behaviour of Mt mice was normal during routine handling, feeding and cage changes. Mt mice did not die unexpectedly making it unlikely that nocturnal major motor seizures were unobserved. Vehicle treated Mt and Wt mice did not exhibit clinical seizure activity.

Example 2: Pentylenetetrazole-induced seizures Twenty PTZ-treated female Mt mice, (mean body weight 23.9+0.9g) were examined relative to 20 PTZ-treated female wild type control mice (mean body weight 22.7+0.9g). Testing was conducted using a single subcutaneous (s.c.) injection of PTZ of 80 mg/kg administered (Sigma, St. Louis, MO). On experimental days, mice were removed from their home cage and placed individually in clear glass observation cages (36 x 20 x 20 cm) for 1 hour before PTZ injection. Immediately after the injection, individual mice were observed for 5-second intervals out of each one minute cycle for a period of one hour. PTZ- induced seizures were classified as described by Ferraro et al (Ferraro et al., 1999 J. Neuroscience 19: 6733-6739). The most commonly observed PTZ- induced seizure began with an arrest of normal exploratory behaviour following by a decrease in motor activity with the animal coming to a complete rest in a crouched or prone position. This hypokinetic seizure type was described as a phase 1 seizure. Phase 2 or partial clonus seizures were characterized by brief twitching movements involving face, head, forelimbs or hind limbs. Phase 3 or generalized clonic seizures occurred when focal twitching was rapidly followed by loss of postural control and repetitive or clonic movements involving all limbs and tail. Jumping and repetitive rearing behaviour were also classified as phase 3 activity. A phase 4 or tonic-clonic seizure was characterised by tonic hind limb extension. Phase 4 seizures commonly resulted in death. PTZ- induced seizures were a continuum from phase 1 to phase 4. Phase N events, which were defined as overtly normal behaviour including rearing, locomotion, grooming, sniffing and climbing were also recorded. All experiments were conducted between 0900-1300 hrs. Mt mice showed less normal behaviour (P=0.001), a reduction in the number of phase 1 (P=0.009) events, a comparable number of phase 2 events and an increase in the number of phase 3 (P=0.002) and phase 4 (P=0.01) events

(Figure 1A). There was also a significant increase in the death rate of Mt mice, with 65% of mutants dying from seizures compared to 15% in Wt mice (Fishers exact test P=0.002) (Table 1 A). This was comparable to a death rate of 79% in

Mt mice compared to 13% in Wt mice obtained in an independent experiment examining an equal number of male and female mice of each genotype (Mt; n=14, Wt; n=15) (Fishers exact test P=0.0001). In this group, not all animals died after Phase 4-type seizure.

Example 3: Bicuculline-induced seizures

Twelve Mt male mice (mean body weight 26.5+0.5g) were compared to 12 male Wt control mice (mean body weight 29.2+0.8g). The seizure response to 4 mg/kg of intraperitoneal (i.p.) bicuculline (Sigma) was assessed. Mice were observed for 30 minutes and drug effects scored independently by two observers. Individual mice were observed continuously in two non-overlapping 15 second periods by each observer in each 60 second time period and the total score for each mouse/minute was calculated by adding the two scores. This approach therefore allowed continuous observation of mouse behaviour for 50% of the time. All experiments were conducted between 0900-1300 hrs. The classification of bicuculline-induced seizures used is as described above (Ferraro et al., 1999 J. Neuroscience 19: 6733-6739).

Mt mice showed less normal behaviour (P=0.02), a reduction in phase 1 (P=0.001) and phase 2 (P=0.003) events, a comparable number of phase 3 and an increased number of phase 4 events (P=0.03) (Figure 1 B). Unlike PTZ- induced seizures, tonic-clonic events following bicuculline injection were invariably associated with death resulting in significantly different mortality of 92% in Mt mice compared to 33% in Wt controls (Fishers exact test P=0.009) (Table 1B). Example 4: Kainic acid-induced seizures

Kainic acid (KA) (Sigma) was administered s.c. at a dose of 30 mg/kg following a one-hour habituation period. A total of 14 male mice of each genotype were examined. All mice were aged between 12 and 13 weeks and were of similar weights (Wt 27.0 + 0.9 g, Mt 29.3+ 0.7 g). Four mice were tested at any one time and all experiments were conducted between 1230-1700 hrs. Two observers scored drug effects independently over a 90 min period. In each 60 second time period, individual mice were observed in non-overlapping 15 second periods by each experimenter and the worst event was recorded. The total score for each mouse/minute was calculated by adding the two scores. This approach therefore allowed visual assessment and documentation of KA- induced effects for 50% of the time. Phase N events represented normal exploratory mouse behaviour. Phases 1 to 5 were as described by Yang et al (Yang et al., 1997 Nature 389: 865-870). In brief, phase 1 involved an arrest of motion, fixed gaze and abnormal forelimb or hind limb posturing. Phase 2 involved myoclonic jerks of head and upper body with associated back arching. Phase 3 involved unilateral clonic activity. Phase 4 involved bilateral synchronous forelimb clonic activity and phase 5, loss of postural tone and generalized tonic-clonic seizure activity.

KA administration resulted in a six-fold decrease in normal exploratory behaviour in Mt as compared to Wt mice (average number of events + SE/mouse/90 min: Wt; 64.5 + 10, compared to 11.0 + 4.0 in Mt mice, P=0.003, t-test) (Figure 1C). In addition, Mt mice had a greater number of hypokinetic (phase 1) (P=0.002, t-test) and phase 2 events (P=0.001 , t-test). There was no significant difference between genotypes with respect to the number of phase 3, 4 or 5 events. Seven (50%) of the Mt mice had a phase 5 seizure compared to only 2 in Wt mice (Table 1C).

Example 5: Strychnine-induced seizures

The seizure response to 0.6 mg/kg strychnine was used to measure the seizure response to 20 male Mt mice (mean body weight 27.4+0.7g) compared to 20 male Wt control mice (mean body weight 28.9+0.8g). Mice were observed for 30 minutes and drug effects scored independently by two observers. Individual mice were observed continuously in two non-overlapping 15 second periods by each observer in each 60 second time period and the total score for each mouse/minute was calculated by adding the two scores. All experiments were conducted between 0900-1300 hrs. The classification of strychnine-induced seizures used is as described above (Ferraro et al., 1999 J. Neuroscience 19: 6733-6739).

Mt mice showed less normal behaviour (P=0.017) and a reduction in phase 1 (P=0.047) events (Figure 1 D). Three of the drug-treated Mt mice died while there was no death in the Wt control group (Fisher exact test P=0.231) (Table 1 D).

Example 6: GABA_A receptor binding

The GABA_A receptor antagonist, [³H]-SR95531 (NET-946, New England Nuclear, Boston, MA) was used to characterize the distribution of ionotropic GABA receptors in Wt and Mt mouse brains. Frozen slide-mounted 20 μm brain sections from drug naive mice were thawed at room temperature before pre-incubation in 50 nM Tris/citrate buffer (pH 7.4) containing 100 mM MgCI₂. The sections were cooled in ice-cold buffer for five minutes before incubation in 6.5 nM[³H]-SR95531 in the same buffer for 30 minutes. The sections were washed three times in ice-cold buffer for 5 seconds and rinsed in distilled water twice for 10 seconds before drying. Non-specific binding was determined in the presence of 10 nM GABA (Research Biochemicals International, MA). Autoradiographic detection was carried out by exposing the slide-mounted sections, together with [³H]-microscales (RPA 510, Amersham International, UK) to Hyperfilm (RPN12, Amersham) for 12 days. The films were developed using Kodak D-19 Photo Developer. Binding densities were measured using a Microcomputer Imaging Device (MCID) with software (Imaging Research Inc. Brock University, St. Catherine's, Ont, Canada). For all studies, a minimum of 3 coronal sections from each animal were used for calculation of individual means. Striatal sections were taken between the region corresponding to levels 0.14 mm and 1.10 mm, rostral to the bregma line (Franklin and Paxinos, 1997 The mouse brain in stereotactic coordinates. Academic Press, San Diego). Standardization was achieved by comparing binding densities with standards exposed with each film. All values are expressed as mean + SE (fmol/mg). Student t-tests were used for statistical analysis of autoradiographic regional quantitative binding densities.

Brain regions analyzed were: cortex (Cx), caudate/putamen (CPu), nucleus accumbens (NAc), olfactory tubercle (T), lateral globus pallidus (LPG), interpenduncular nucleus (IPn), lateral septum (LS), periaqueductal grey (PAG), pontine nucleus (Pn), anterior paraventricular thalamus (Pva), substantial nigra pars reticulata (SNr) and superior colliculus (SC). There were no significant differences between Wt and Mt mice in the density of GABA_A binding sites in any regions assessed (Figure 2).

Example 7: Seizure type for each drug

Table 1 shows the worst observed seizure type for each drug expressed as a percentage of all Wt and Mt mice. The absolute sample size from which percentages are calculated are indicated in parenthesis. The numbers of animals died after PTZ injection were included in the last column, showing that all animals died after Phase 4-type seizure.

Example 8: Electroencephalographic characterisation of mice with deletion of the α4 subunit of the neuronal nicotinic receptor

The electroencephalographic effects of deletion of the α4 subunit of the nAChR was assessed in mice, during normal waking activity, sleep, and in response to the proconvulsant PTZ. Forty mice were studied, of which 20 were homozygous for deletion of the α4 nAChR gene (Mt) and 20 were Wt controls from the same breeding strains. Blinding with respect to genotype was maintained for all electrode insertions, recording sessions and scoring procedures.

Under a light anaesthetic with chloral hydrate, each mouse was fitted with four tungsten epidural recording electrodes in the following positions: front right and left, 1.5 mm anterior to bregma and 1.5mm lateral to the midline; posterior right and left, 3mm posterior to bregma and 1.5mm lateral to the midline. At least 2 days later, a baseline EEG was recorded for 2-3 hrs. The voltage difference between the two frontal electrodes was recorded as the anterior EEG channel while the voltage difference between the two posterior electrodes was recorded as the posterior channel. During acquisition, a highpass filter set at 1 Hz, a low pass filter set at 80 Hz and a notch filter at 50 Hz were employed. The baseline EEGs were subsequently screened for ictal activity but there were no significant differences in baseline EEG characteristics between the two strains.

After a further recovery period of at least 2 weeks, the mice were injected with pentylenetetrazole 80mg/kg subcutaneously. EEG was recorded for at least 120 minutes prior to injection, during injection and for at least one hour after injection. The first 45 minutes of EEG data after each injection were subsequently scored by a panel of 3 blinded observers, all of whom were clinical neurologists and neuroscientists. Each 10 second EEG sweep was categorised according to the most severe ictal abnormalities present and borderline cases were decided by majority. The following categories were used, reflecting increasingly severe ictal changes in the EEG:

1. normal

2. single sharp waves or run of sharp waves <1 sec duration

3. run of sharp waves > 1 sec duration but <5 sec 4. run of sharp waves >5 sec duration

5. spike, spike and slow wave, spike doublet or large sharp wave with subsequent change in background rhythm

6. multispike complex (>3 spikes)

7. continuous burst of high frequency ictal activity, usually of low amplitude, obliterating background rhythm

8. major seizure with repetitive bursts of spikes or multispikes, completely replacing background rhythm and lasting >5 sec

The time of death was recorded as the time that cerebral rhythms disappeared to be replaced by a flat EEG or electrocardiographic artifact showing agonal bradycardia.

Example 9: 4-aminopyridine-induced seizures The drug 4-AP, a K⁺ channel blocker, causes seizures, including episodes of running and explosive jumps usually terminating in tonic hind limb extensor convulsions. The seizure responses of 20 male Mt mice (mean body weight 27.0±0.7g) were compared to 20 male Wt control mice (mean body weight 29.7±1.1g) following the intraperitoneal administration of 10 mg/kg of 4-AP. The seizure responses of Mt and Wt mice (all male, n=21 for each genotype) to 12 mg/kg 4-AP were also compared (Mt, 28.5±0.7g; Wt, 28.8±0.6g). Mice were observed for 90 minutes and drug effects scored independently by two observers. Individual mice were observed continuously in two non overlapping 10 second periods by each observer in each 60 second time period and the total score for each mouse/minute was calculated by adding the two scores. The spectrum of seizure phenotypes was the same as that seen for kainic acid. All experiments were conducted between 0900-1300 hrs.

Example 10: α4 neuronal nicotinic acetylcholine receptor subunit knockout mouse

A transgenic mouse line was produced in which a 0.75 kb Bglll-Sca1 fragment was excised from the fifth exon of the α4 nAChR gene and replaced by a neomycin resistance cassette. Transgenic mice are hyperactive (Ross et al, 2000 J. Neuroscience 20: 6431-6441) from the time of weaning and display heightened anxiety-like behaviour (Ross et al, 2000 J. Neuroscience 20: 6431- 6441). Mutant mice have evidence of altered α4 nAChR gene transcription. ISAChl and ISACh2 probes, which hybridize to the deleted sequence, showed no regional specific signal throughout the brain of Mt and a reduced signal in heterozygous mice (Ross et al, 2000 J. Neuroscience 20: 6431-6441). The hybridization pattern seen in wild type mice using ISAChl and ISACh2 probes was the same as for probes ISACh3 and ISACh4 (which were oligonucleotide probes designed to recognize gene sequence upstream of the excised Bglll- Sca1 fragment). A strong hybridization signal for ISACh3 and ISACh4 (or ISAChl and ISACh2) was localized to the thalamus and cortex in Wt. A moderate hybridization signal was also seen in the caudate putamen, hippocampus, dentate gy s and substantia nigra (Ross et al, 2000 J. Neuroscience 20: 6431-6441). In situ hybribization was also performed for α₃, oc₆, x₇, β₂, β₃ and β₄ nAChR subunits. There was no difference in the hybridisation patterns seen in mutant compared to wild type mice (Ross et al, 2000 J. Neuroscience 20: 6431-6441).

Ligand autoradiographic characterization of mutant mice revealed altered ligand binding to labelled nicotine, cystisine and epibatidine. Autoradiographic ligand binding experiments were performed on a number of animals (Wt, n=9; heterozygous, n=11 ; and Mt, n=11). Binding experiments conducted in wild type mice using tritiated nicotine, cytisine and epibatidine showed a similar pattern of high affinity binding. [³H]-nicotine labelling was detected at highest levels in the thalamic nuclei, medial habenular, interpeduncular nucleus, superior colliculus and presubiculum and moderate levels were found in the cortex, caudate/putamen and fasciculus retroflexus. [³H]-cytisine binding showed a similar pattern to [³H]-nicotine binding in wild type. [³H]-epibatidine binding differed from [³H]-nicotine binding in that [³H]-epibatidine binding to the medial habenular and fasciculus retroflexus was more intense as shown by quantitative analysis. [³H]-nicotine and [³H]-epibatidine binding showed a qualitatively similar pattern in mutant mice with binding for both radioligands detected in the medial habenular, interpeduncular nucleus, faciculus retroflexus and superior colliculus. The main difference was that [³H]-epibatidine binding was detected at high levels in all of these sites. [³H]-cytisine binding was only detected in the interpeduncular nucleus of mutant mice. [³H]-epibatidine binding in wild type and mutant mice with cytisine or nicotine cold competition resulted in loss of superior colliculus signal but preservation of binding in the habenulo-interpeduncular pathway (i.e medial habenular, interpeduncular nucleus and fasciculus retroflexus). [¹²⁵l]-α-bungarotoxin binding was found to be unchanged in mutant compared with wild type. Autoradiographic ligand binding experiments performed on an independently generated line of α₄ nAChR subunit knockout mice (Marubio, et al., 1999 Nature 398: 805-810) also demonstrated high level binding to [³H]-epibatidine in a number of nuclei and reduced [³H]-nicotine binding in the medial habenular. Quantitative autoradiography undertaken in knockout mice of the present invention demonstrated that [³H]-epibatidine binding was reduced in mutant compared to wild type mice in the superior colliculus and interpeduncular nucleus whereas there was no difference in the medial habenular. Furthermore, Marubio and colleagues (Marubio, et al., 1999 Nature 398: 805-810) showed that [³H]- nicotine binding was found at reduced levels only in the medial habenular whereas [³H]-nicotine binding was detected in both the interpeduncular nucleus and the superior colliculus in addition to the previously described binding sites in the medial habenular. Quantitative analysis confirmed that [³H]-nicotine binding was moderately reduced in mutant compared to wild type in all three nuclei.

Table 1 A. Worst PTZ-induced seizure type

Distribution of mice according to the worst observed seizure event, expressed as the number of mice (and % of each genotype) in each seizure category. Refer to main text for the description of the types of seizure events. The numbers of animals died after drug treatments were included in the last column, showing that not all animals died after the worst seizure event. ^*P=0.009, **P=0.002, Fisher Exact test. Table 2A. Worst 4-aminopyridine-induced seizure type (10 mg/kg)

Table 2B. Worst 4-aminopyridine-induced seizure type (12 mg/kg)

Distribution of mice according to the worst observed seizure event, expressed as the number of mice (and % of each genotype) in each seizure category. Refer to main text for the description of the types of seizure events. The number of animals dying after drug treatment is included in the last column (*P=0.043, **P=0.001 , Fisher Exact test).

Finally it is to be understood that various other modifications and/or alterations may be made without departing from the spirit of the present invention as outlined herein.

Claims

1. A method of screening for a compound that is a proconvulsant or anticonvulsant wherein the method includes: exposing a transgenic animal whose genome comprises a disruption of the α4 nAChR gene to the compound; and determining the seizure response of the transgenic animal to the compound.

2. A method according to claim 1 further including comparing the seizure response of the transgenic animal to a second transgenic animal that has not been administered the compound.

3. A method according to claim 1 further including comparing the seizure response of the transgenic animal to a wild type animal that does not have a disruption of the α4nAChR gene but has been exposed to the compound.

4. A method according to any one of claims 1 to 3 wherein the disruption of the α4nAChR gene results in a non-functional or hypofunctioning α4nAChR gene.

5. A method according to any one of claims 1 to 4 wherein the disruption includes a substantial deletion of the α4nAChR gene.

6. A method according to claim 8 wherein an animal is a knockout mouse having a deletion of the α4nAChR gene.

7. A method according to any one of claims 1 to 6 wherein the transgenic animal is selected from the group including rat, mouse, rabbit, pig, goat, cow, sheep, guinea pig or hamster.

8. A method according to claim 7 wherein the transgenic animal is a mouse.

9. A method according to claim 8 wherein the animal is a knockout mouse having a deletion of the α4nAChR gene.

10. A method according to any one of claims 1 to 9 wherein the seizure response is an epileptic response.

11. A method according to claim 10 wherein the epileptic response is a generalized or partial epilepsy.

12. A method according to claim 11 wherein the epileptic response is a generalized epilepsy.

13. A method of screening a compound that is a proconvulsant according to any one of claims 1 to 12 wherein the seizure response is enhanced in the transgenic animal.

14. A method according to claim 13 wherein the proconvulsant enhances central nervous system epileptogenic activity.

15. A method of screening a compound that is an anticonvulsant according to any one of claims 1 to 12 wherein the seizure response is decreased in the transgenic animal.

16. A proconvulsant or anticonvulsant identified by a method of screening for a compound that is a proconvulsant or anticonvulsant wherein the method includes: exposing a transgenic animal whose genome comprises a disruption of the α4 nAChR gene to the compound; determining the seizure response of the transgenic animal to the compound.

17. A proconvulsant or anticonvulsant identified by a method of screening according to any one of claims 1 to 15.

18. A method of treating seizures in a subject wherein the method includes administering an effective amount of a proconvulsant or anticonvulsant identified by: exposing a transgenic animal whose genome comprises a disruption of the α4 nAChR gene to the compound; and determining the seizure response of the transgenic animal to the compound.

19. A method of treating seizures in a subject wherein the method includes administering an effective amount of a proconvulsant or anticonvulsant identified by a method according to any one of claims 1 to 15.

20. A method according to claim 18 or 19 wherein the seizure is epilepsy- induced.

21. A method of treating epilepsy, said method comprising administering an effective amount of an anti-convulsant identified by a method according to any one of claims 1 to 15.

22. A method of identifying a gene involved in modulating a seizure response wherein the method includes: analysing gene expression in a transgenic animal whose genome comprises a disruption of the α4 nAChR gene; comparing gene expression in the transgenic animal with that of a non- transgenic animal; and identifying differential gene expression between the transgenic and non- transgenic animal.

23. A method of identifying a protein involved in modulating a seizure response wherein the method includes: analysing protein expression in a transgenic animal whose genome comprises a disruption of the 4 nAChR gene; comparing protein expression in the transgenic animal with that of a non- transgenic animal; and identifying differential protein expression between the transgenic and non- transgenic animal.

24. A method for screening for a compound that modulates a seizure response in an animal said method comprising: administering the compound to a transgenic animal whose genome comprises a disruption of the α4 nAChR gene; and determining the effect of the compound upon a cellular or molecular process associated with the seizure response in the transgenic animal compared to an effect of the compound administered to a non-transgenic animal, wherein a difference in effect is indicative of a compound that modulates the seizure response.

25. A method according to claim 24 wherein the seizure response is associated with epilelpsy.

26. A method according to claim 24 or 25 wherein the cellular or molecular process is a measure of neurotransmitters selected from the group including GABA, glutamate, dopamine or acetylchlorine.