Dopamine transporter knockout mice

A recombinant rodent comprises cells containing a pair of genomic dopamine transporter protein alleles, wherein at least one of said alleles is incapable of expressing endogenous dopamine transporter protein. The rodent may be a homozygote, where both of said alleles are incapable of expressing endogenous dopamine transporter protein, or the rodent may be a heterozygote, and one of said alleles expresses endogenous dopamine transporter protein. The rodent is preferably a mouse.

This application claims the benefit of U.S. provisional application No. 
60/004,695, filed on 02 Oct., 1995. 
FIELD OF THE INVENTION 
This invention relates to methods of increasing spontaneous motor activity 
in a subject in need of such treatment, particularly subjects afflicted 
with Parkinson's disease or tardive dyskinesia. 
BACKGROUND OF THE INVENTION 
Dopamine is involved in the control of motor function, cognition and affect 
(A. Dahlstrom and K. Fuxe, Acta Physiol. Scand. 232, 1-55 (1965); U. 
Ungerstedt, Acta Physiol. Scand. 367, 1-48 (1970)). Imbalance in the 
dopamine system is believed to be involved in such conditions as 
schizophrenia (T. Crow, Brit. J. Psychiatry 137, 383-386 (1980); Fibiger, 
in : The Mesolimbic dopamine system : From motivation to action, 615-37 
(Eds. P. Willner and J. Scheel-Krger 1991)), Parkinson's disease (H. 
Ehringer and O. Hornykiewitz, Klin. Wochenschr. 38, 1236-1239 (1960), 
tardive dyskinesia and drug addiction (R. Wise and P. P. Rompre, Ann. Rev. 
Psychol. 40, 191-225 (1989); G. Koob and F. Bloom, Science 242, 715-723 
(1988); G. DiChiara et al., Proc. Natl Acad. Sci. U.S.A. 85, 5272-5278 
(1998)). 
The dopamine transporter, a member of the large family of Na.sup.+ 
/Cl.sup.- dependent transporters, aids in terminating dopaminergic 
neurotransmission by rapid reuptake of dopamine (B. Giros and M. Caron, 
Trends Pharmacol. Sci. 14, 43-49 (1993). It is a major target of the 
psychostimulant drugs cocaine and amphetamine (B. Giros and M. Caron, 
Trends Pharmacol. Sci. 14, 43-49 (1993); M. Ritz et al., Science 237, 
1219-1223 (1987)), but its overall role in vivo is still poorly 
understood. 
SUMMARY OF THE INVENTION 
A first aspect of the present invention is a recombinant rodent comprising 
cells containing a pair of genomic dopamine transporter protein alleles, 
and wherein at least one of said alleles is incapable of expressing 
endogenous dopamine transporter protein. The rodent may be a homozygote., 
where both of said alleles are incapable of expressing endogenous dopamine 
transporter protein, or the rodent may be a heterozygote, and one of said 
alleles expresses endogenous dopamine transporter protein. 
A further aspect of the present invention is a method of upregulating 
spontaneous motor activity in a subject in need of such treatment. The 
method comprises inhibiting dopamine transporter function (i.e., dopamine 
reuptake mediated by the dopamine transporter protein) in the subject by 
an amount effective to enhance spontaneous motor activity in that subject. 
The method may be carried out by any suitable means, such as by 
administering a dopamine transporter blocker to said subject in an amount 
effective to enhance spontaneous motor activity. Suitable subjects include 
those afflicted with Parkinson's disease and those afflicted with tardive 
dyskinesia, particularly drug-induced tardive dyskinesia. 
A still further aspect of the present invention is the use of a dopamine 
transporter inhibitor for the preparation of a medicament for enhancing 
spontaneous motor activity in that subject, as described above.

DETAILED DESCRIPTION OF THE INVENTION 
We herein describe the first gene inactivation of a member of the Na.sup.+ 
/Cl.sup.- -dependent transporter family. We provide direct in vivo 
evidence that the dopamine transporter may represent the most crucial 
functional determinant of neurotransmitter tone in the central nervous 
system identified to date. The functional ablation of the dopamine 
transporter in the mouse produces a phenotype in which homozygote animals 
display a marked increase in spontaneous locomotor activity. This 
phenotype is presumably a reflection of the prolonged availability of 
dopamine at the synapse, as effects of similar magnitude are observed 
following complete blockade of the transporter with maximal doses of 
psychostimulants (G. DiChiara and A. Imperato, Proc. Natl Acad. Sci. 
U.S.A. 85, 5272-5278 (1998); B. Giros and M. Caron, Trends Pharmacol. Sci. 
14, 43-49 (1993); M. Ritz et al., Science 237, 1219-1223 (1987); M. Jaber 
et al., Neuroscience, 65, 1041-1050 (1995)). This hyperlocomotion 
phenotype is apparent even in light of major adaptive decreases in the 
mRNAs for the D1 and D2 receptors, the main targets of synaptic dopamine 
responsiveness. This phenotype is even more impressive if one considers 
that levels of dopamine may be markedly lower in homozygote animals. 
Indeed, as seen by immunoblotting and immunohistochemistry, tyrosine 
hydroxylase, the rate limiting enzyme of dopamine synthesis, is reduced by 
more than 80%. in dopamine neurons and projections. In addition, the 
observation that heterozygote animals have certain characteristics which 
are intermediate between wild type and homozygote mice, reinforces the 
notion of a fundamental role for the dopamine transporter in the 
maintenance of normal dopaminergic neurotransmission. 
The properties of the DAT knockout mice make them an attractive animal 
model for the study and development of drugs for the management of 
dopaminergic dysfunction. However, the most exciting consequences of these 
data are in the development of new therapeutic strategies for common 
neurological disorders. For example, these data indicate that targeting of 
the DAT with high affinity antagonists (J. Boja et al. Mol. Pharmacol. 47, 
779-786 (1995)) in illnesses such as Parkinson's disease, where the 
effective levels of dopamine are markedly decreased, (H. Ehringer and O., 
Hornykiewitz, Klin. Wochenschr. 38, 1236-1239 (1960)) have beneficial 
clinical consequences. 
Our results further demonstrate that the DAT, in addition to its proposed 
role in the reinforcing properties of the psychostimulants cocaine and 
amphetamine, (M. Ritz et al. Science 237, 1219-1223 (1987)) represents the 
primary target for their locomotor effects. Since cocaine, and to a lesser 
extent amphetamine, interact with other monoamine transporters, the DAT 
homozygote and heterozygote animals should prove an excellent model to 
elucidate complex behavioral paradigms such as reward, addiction and 
tolerance properties of these drugs. 
Animals suitable for carrying out the present invention are, in general, 
rodent species. Rat and mouse are preferred, and the mouse is most 
preferred. Animals in every stage of development, including juvenile, 
adolescent, and adult, are included in this description, with adult 
animals particularly preferred. 
"Knockout" animals refers to animals whose native or endogenous DAT allele 
or alleles have been disrupted by homologous recombination and which 
produce no functional DAT of their own. Knockout animals may be produced 
in accordance with techniques known in the art, particularly by means of 
in vivo homologous recombination, M. Capechi, Science 244, 1288-1292 
(1989), in light of the known sequence for DNA encoding the DAT. See, 
e.g., A. Eshleman et al., Molec. Pharmacol. 45, 312-316 (1994). DAT 
knockout animals of species other than mice can be produced by variation 
of the procedures carried to produce Apo E knockout mice that will be 
apparent to those skilled in the art. 
Animals described in the present invention are animals having at least one 
allele incapable of expressing endogenous dopamine transporter protein 
(DAT), and include both the heterozygous or homozygous forms (e.g., DAT+/- 
and DAT -/-). Animals may be chimeric animals or animals in which 
essentially all cells are recombinant. Preferably at least the brain cells 
are recombinant. 
Animals of the present invention are useful in a variety of screening 
assays, as discussed below. In such assays, the compound being screened 
may be administered to the animal by any suitable means, including orally 
and parenterally (e.g., by subcutaneous injection). The particular 
activity detected in the animal (i.e., neuroleptic, antischizophrenic, 
control of substance abuse, neuropharmacological, appetite modulating, 
aggressive behavior modulating, and addictive activities) may be detected 
in accordance with techniques known to those skilled in the art of 
neuropharmacology. 
A method of screening a compound for neuroleptic activity comprises 
administering a test compound to a recombinant animal as given above, and 
then detecting the presence or absence of neuroleptic activity in said 
animal. The method is particularly useful wherein the neuroleptic activity 
is antischizophrenic activity. 
A method of screening a compound for activity for the control of substance 
abuse (i.e., inhibiting or treating addictive behavior) comprises 
administering a test compound to a recombinant animal (preferably 
homozygous) as given above, and then detecting the presence or absence of 
activity for the control of substance abuse in the animal. Substance 
abuses such as amphetamine abuse and cocaine abuse (i.e., amphetamine 
craving and cocaine craving) are particularly preferred categories of 
substance abuse for which compounds useful for the treatment thereof may 
be screened by the instant method. 
A method useful as a negative control for screening a compound for 
norepinephrine or serotonin transporter activity comprises administering a 
test compound to a recombinant animal (preferably homozygous) as given 
above, and then detecting the presence or absence of neuropharmacological 
activity for the compound in the animal. 
A method of screening a compound for appetite modulating activity comprises 
administering a test compound to a recombinant animal (preferably 
homozygous) as given above, and then detecting the presence or absence of 
appetite modulating activity in the animal. The activity screened for may 
be either activity in increasing or decreasing appetite. 
A method of screening a compound for aggressive behavior modulating 
activity comprises administering a test compound to a recombinant animal 
(preferably homozygous) as given above, and then detecting the presence or 
absence of aggresive behavior modulating activity in the animal. The 
activity screened for may be either activity in increasing or decreasing 
aggressive behavior. 
A method of screening a compound for substance abuse potential (i.e., 
reinforcing activity) comprises administering a test compound to a 
heterozygous recombinant animal as given above, and then detecting the 
presence or absence of addictive behavior with respect to the compound in 
the animal (i.e., the finding that the animal develops a craving for that 
compound, or that the administration of that compound is a positive 
reinforcement for that animal). The presence of addictive behavior 
indicating the compound has a potential for substance abuse. 
As noted above, a further aspect of the present invention is a method of 
upregulating spontaneous motor activity in a subject in need of such 
treatment. The method may be carried out on human subjects or on animal 
subjects for veterinary purposes. Suitable subjects include those 
afflicted with Parkinson's disease and those afflicted with tardive 
dyskinesia, particularly drug-induced tardive dyskinesia. A variety of 
dopamine transporter inhibitors (also called dopamine uptake inhibitors; 
herein referred to as active compounds) of diverse structure are known. 
See, e.g., S. Berger, U.S. Pat. No. 5,217,987; J. Boja et al., Molec. 
Pharmacol. 47, 779-786 (1995); C. Xu et al., Biochem. Pharmacol. 49, 
339-50 (1995); B. Madras et al., Eur. J. Pharmacol. 267, 167-73 (1994); F. 
Carroll et al., J. Med. Chem. 37, 2865-73 (1994); A. Eshleman et al., 
Molec. Pharmacol. 45, 312-16 (1994); R. Heikkila and L. Manzino, Eur. J. 
Pharmacol. 103, 241-8 (1984). Dopamine transporter inhibitors are, in 
general, ligands that bind in a stereospecific manner to the dopamine 
transporter protein. Examples of such compounds are: 
(1) tricyclic antidepressants such as buprion, nomifensine, and amineptin; 
(2) 1,4-disubstituted piperazines, or piperazine analogs, such as 
1-2-bis(4-fluorophenyl) methoxy!ethyl!-4-(3-phenylpropyl)piperazine 
dihycrochloride (or GBR 12909), 1-2-bis(phenyl) 
methoxy!ethyl!-4-(3-phenylpropyl)piperazine dihydrochloride (or GBR12935), 
and GBR13069; 
(3) tropane analogs, or (disubstituted phenyl) tropane-2 beta-carboxylic 
acid methyl esters, such as 
3.beta.-(4-fluorophenyl)tropane-2.beta.-carboxylic acid methyl ester (or 
WIN 35,428) and 3.beta.-(4-iodophenyl)tropane-2.beta.-carboxylic acid 
isopropyl ester (RTI-121); 
(4) substituted piperidines, or piperidine analogs, such as 
N-1-(2-benzob!-thiophenyl) cyclohexyl! piperidine, indatraline, and 
4-2-bis(4-fluorophenyl)methoxy!ethyl!-1-(3-phenylpropyl) piperidine (or 
O-526); 
(5) quinoxaline derivatives, or quinoxaline analogs, such as 
7-trifluoromethyl-4-(4-methyl-1-piperazinyl)pyrrolo1,2-.alpha.!quinoxalin 
e (or CGS 12066b); and 
(6) other compounds that are inhibitors of dopamine reuptake, such as 
mazindol, benztropine, bupropion, phencyclidine, methylphenidate, etc. 
Because the DNA sequence of the dopamine transporter is known (See, e.g., 
A. Eshleman et al., supra), this sequence can be used to inhibit or 
downregulate dopamine transporter in animals by means of administering to 
the subject antisense oligonucleotides that bind to mRNA encoding the 
dopamine transporter in an amount effective to inhibit the function of the 
dopamine transporter, or by means of inhibiting dopamine transporter 
transcription by administering oligonucleotides that specifically bind to 
DNA encoding the dopamine transporter, and, by homologous recombination, 
inhibit the activity of the dopamine transporter. 
The active compounds disclosed herein can be prepared in the form of their 
pharmaceutically acceptable salts. Pharmaceutically acceptable salts are 
salts that retain the desired biological activity of the parent compound 
and do not impart undesired toxicological effects. Examples of such salts 
are (a) acid addition salts formed with inorganic acids, for example 
hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, 
nitric acid and the like; and salts formed with organic acids such as, for 
example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic 
acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, 
benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, 
naphthalenesulfonic acid, methanesulfonic acid, ptoluenesulfonic acid, 
naphthalenedisulfonic acid, polygalacturonic acid, and the like; (b) salts 
formed from elemental anions such as chlorine, bromine, and iodine, and (c) 
salts derived from bases, such as ammonium salts, alkali metal salts such 
as those of sodium and potassium, alkaline earth metal salts such as those 
of calcium and magnesium, and salts with organic bases such as 
dicyclohexylamine and N-methyl-D-glucamine. 
Pharmaceutical compositions for use in the present method of increasing 
spontaneous motor activity include those suitable for inhalation, oral, 
rectal, topical, (including buccal, sublingual, dermal and intraocular) 
parenteral (including subcutaneous, intradermal, intramuscular, 
intravenous and intraarticular) and transdermal administration. The 
compositions may conveniently be presented in unit dosage form and may be 
prepared by any of the methods well known in the art. The formulations may 
conveniently be presented in unit dosage form and may be prepared by any of 
the methods well known in the art. 
The dose of active compound will vary according to the the particular 
compound administered, the route of administration, the manner of 
formulation, the condition of the subject, and the dose at which adverse 
pharmacological effects occur. One skilled in the art will take such 
factors into account when determining dosage. Illustrative examples of 
typical dosages are, for methylphenidate, from 1 or 10 to 60 or 100 mg per 
subject daily, and for buproprion, from 10 or 100 to 450 or 1000 mg per 
subject daily. 
In the manufacture of a medicament according to the invention (a 
"formulation"), active agents or the physiologically acceptable salts 
thereof (the "active compound") are typically admixed with, among other 
things, an acceptable carrier. The carrier must be acceptable in the sense 
of being compatible with any other ingredients in the formulation and must 
not be deleterious to the patient. The carrier may be a solid or a liquid, 
or both, and is preferably formulated with the compound as a unit-dose 
formulation, for example, a tablet, which may contain from 0.5% to 99% by 
weight of the active compound. One or more active compounds may be 
incorporated in the formulations of the invention (e.g., the formulation 
may contain one or more additional anti-tubercular agents as noted above), 
which formulations may be prepared by any of the well known techniques of 
pharmacy consisting essentially of admixing the components, optionally 
including one or more accessory therapeutic ingredients. 
Formulations suitable for oral administration may be presented in discrete 
units, such as capsules, cachets, lozenges, or tablets, each containing a 
predetermined amount of the active compound; as a powder or granules; as a 
solution or a suspension in an aqueous or non-aqueous liquid; or as an 
oil-in-water or water-in-oil emulsion. Such formulations may be prepared 
by any suitable method of pharmacy which includes the step of bringing 
into association the active compound and a suitable carrier (which may 
contain one or more accessory ingredients as noted above). In general, the 
formulations of the invention are prepared by uniformly and intimately 
admixing the active compound with a liquid or finely divided solid 
carrier, or both, and then, if necessary, shaping the resulting mixture. 
For example, a tablet may be prepared by compressing or molding a powder 
or granules containing the active compound, optionally with one or more 
accessory ingredients. Compressed tablets may be prepared by compressing, 
in a suitable machine, the compound in a free-flowing form, such as a 
powder or granules optionally mixed with a binder, lubricant, inert 
diluent, and/or surface active/dispersing agent(s). Molded tablets may be 
made by molding, in a suitable machine, the powdered compound moistened 
with an inert liquid binder. Formulations for oral administration may 
optionally include enteric coatings known in the art to prevent 
degradation of the formulation in the stomach and provide release of the 
drug in the small intestine. 
Formulations suitable for buccal (sublingual) administration include 
lozenges comprising the active compound in a flavored base, usually 
sucrose and acacia or tragacanth; and pastilles comprising the compound in 
an inert base such as gelatin and glycerin or sucrose and acacia. 
Formulations of the present invention suitable for parenteral 
administration comprise sterile aqueous and non-aqueous injection 
solutions of the active compound, which preparations are preferably 
isotonic with the blood of the intended recipient. These preparations may 
contain anti-oxidants, buffers, bacteriostats and solutes which render the 
formulation isotonic with the blood of the intended recipient. Aqueous and 
non-aqueous sterile suspensions may include suspending agents and 
thickening agents. The formulations may be presented in 
unit.backslash.dose or multi-dose containers, for example sealed ampoules 
and vials, and may be stored in a freeze-dried (lyophilized) condition 
requiring only the addition of the sterile liquid carrier, for example, 
saline or water-for-injection immediately prior to use. Extemporaneous 
injection solutions and suspensions may be prepared from sterile powders, 
granules and tablets of the kind previously described. 
Formulations suitable for rectal administration are preferably presented as 
unit dose suppositories. These may be prepared by admixing the active 
compound with one or more conventional solid carriers, for example, cocoa 
butter, and then shaping the resulting mixture. 
Formulations suitable for topical application to the skin preferably take 
the form of an ointment, cream, lotion, paste, gel, spray, aerosol, or 
oil. Carriers which may be used include vaseline, lanoline, polyethylene 
glycols, alcohols, transdermal enhancers, and combinations of two or more 
thereof. 
Formulations suitable for transdermal administration may be presented as 
discrete patches adapted to remain in intimate contact with the epidermis 
of the recipient for a prolonged period of time. Formulations suitable for 
transdermal administration may also be delivered by iontophoresis (see, 
e.g., Pharmaceutical Research 3, 318 (1986)) and typically take the form 
of an optionally buffered aqueous solution of the active compound. 
Those skilled in the art will appreciate numerous variations which may be 
made to the foregoing in carrying out the instant invention, which will be 
readily apparent from the literature concerning recombinant animals. See, 
e.g., H. Chen et al., PCT Application WO 95/03397; H. Chen et al., PCT 
Application WO 95/03402; C. Wood et al., PCT Application WO 91/00906; C. 
Lo et al., PCT Application WO 90/06992; P. Leder et al., U.S. Pat. No. 
4,736,866; and T. Wagner et al., U.S. patent application No. 4,873,191. 
The present invention is explained in greater detail by the following 
Examples. These examples are illustrative of the present invention, and 
are not to be construed as limiting thereof. 
EXAMPLE 1 
Targeting of the Mouse Dopamine Transporter 
This example describes the production, by means of in vivo homologous 
recombination, of a strain of mice in which the gene encoding the dopamine 
transporter (DAT) has been disrupted. 
A rat DAT cDNA probe comprising the N-terminal to the third transmembrane 
domain (B. Giros et al., FEBS Letters 295, 149-154 (1991)) was used to 
screen a mouse 129 Sv/J genomic library in accordance with known 
techniques (B. Giros et al., Nature 342, 923-926 (1989)). A 12 kb phage 
containing the first three exons of the mouse DAT gene was isolated, and a 
7.5 kb HindIII fragment (from an internal site to a phage cloning site) was 
excises and subcloned into pGEM4Z. A EcoRI-SAlI-NotI cassette containing 
the neomycin resistance (ncor) gene under the control of the PGK promotor 
was introduced into the NotI site of the construct to provide pD7H15/neo. 
The neor cassette was cloned in the opposite direction as the DAT gene, 
and DNA sequencing was used to insure that pD7H15/neo did not contain a 
consensus start site that could be used to resume DAT translation. 
pD7H15/neo was linearized with AatII prior to electroporation into ES 
cells. Cell culture, electroporation of E14TG2a ES cells (M. Hooper et 
al., Nature 326, 292-295 (1987)) and generation of chimeric mice were 
performed in accordance with known techniques (B. Kohler and O. Smithies, 
Proc. Natl. Acad. Sci. U.S.A. 86, 8932-8935 (1989)). For Southern 
analysis, 5-15 .mu.g DNA from ES cells or tail biopsies were digested with 
EcoRI and HindIII and probed with a 1.1 kb genomic DNA probe, and a neor 
probe to ensure that there was only one copy of the construct in ES cells. 
About 20%. of the tested ES cells were positive for homologous 
recombination, and three clones were selected for karyotyping and 
injection into C57/B6J blastocysts. Chimeric males were mated with C57/B6J 
females, and agouti coat pups were typed by Southern blotting of tail 
biopsies. Two germ-line transmitting males (from different ES clones) were 
used as colony founders. For in situ hybridization, mice were sacrificed by 
decapitation and the brains were dissected out, immersed overnight in 1% 
paraformaldehyde and cryoprotected in 15% sucrose-PBS buffer for 6-8 
hours. The brains were then frozen in liquid nitrogen and cut into frontal 
sections (12.mu.m) which were stored at -80.degree. C. until required. The 
in situ hybridization procedure was performed with oligonucleotide probes, 
labeled by tailing with .sup.35 S! dATP (NEN), with a specific activity of 
2.times.10.sup.9 cpm/.mu.g as previously described (M. Jabar et al., Mol. 
Brain Res., 23, 14-20 (1994); F. Tison et al., Neurosci. Lett. 166 48-50 
(1994)). Sections were exposed at room temperature to X-ray films (Kodak 
X-Omat) for 7 days. Autoradiograms were scanned (Howtek Scanner) at 1200 
dpi and analyzed (NIH-Image). Probe concentration and exposure times were 
chosen in order to stay within a linear range of detection. Experiments 
were performed in duplicate, and comparisons were made between groups 
using the Student's t test with the control value (DAT.sup.+/+) set equal 
to 1, and DAT.sup.+/- and DAT.sup.-/- values expressed relatively to it. 
For dopamine uptake experiments, two striata from one mouse were 
homogenized in 0.5 ml of 0.3M sucrose and synaptosomes were prepared as 
described (C. Pifl et al., J. Neurosci. 13, 4246-4253 (1993)). The 
synaptosomal preparation (20-25 .mu.g protein/tube) was incubated with 
0.25 .mu.Ci of .sup.3 H! dopamine (51 Ci/mmol), and increasing 
concentrations of dopamine (0.3 .mu.M) for 5 min. at 37.degree. C. in a 
final volume of 0.5 ml uptake buffer (4 mM TrisHCI, 6.25 mM HEPES, 120 mM 
NaCl, 5 mM KCl, 1.2 mM CaCl.sub.2, 1.2 Mm MgSO.sub.4, 5.6 mM D-glucose, 
0.5 mM ascorbic acid, pH, 7.1). Experiments were done in triplicate for 
each concentration, and non-specific uptake was determined in the presence 
of 30 .mu.m nomifensine. Uptake was stopped and counted as described (C. 
Pifl et al., J. Neurosci. 13, 4246-4253 (1993)). K.sub.M and Vmax were 
calculated by the iterative curve-fitting programs EBDA and LIGAND, (G. 
McPherson, J. Pharmacol. Methods 14, 213-228 (1985)) and resulting values 
of three separate mice per group were averaged. 
The homozygote (DAT.sup.-/-) mice showed the expected pattern of gene 
disruption as established by Southern blotting. No mRNA encoding the DAT 
was detected in the dopamine neurons of the ventral tegmental area (VTA) 
and the substantia nigra compacta (SNC) of homozygote animals as 
demonstrated by in situ hybridization. Striatal synaptosomal preparations 
from DAT.sup.-/- mice show significant dopamine uptake as compared to 
wild type and heterozygote (DAT.sup.+/-) animals. Interestingly, the 
heterozygote mice show a substantial decrease in the DAT mRNA (70% 
approx.) and a decrease in dopamine uptake (30% approx.). These data 
demonstrate that we have efficiently impaired the dopamine uptake activity 
in DAT homozygote mice. Furthermore, it appears that despite the absence of 
expression of the DAT in homozygote animals ever since early embryonic 
stages there is no compensatory expression of other monoamines 
transporters, such as norepinephrine and serotonin, and that these 
transporters have no or insignificant participation in normal dopamine 
uptake in the basal ganglia. 
EXAMPLE 2 
Weight Gain and Development of Treated Animals 
Homozygotes are viable but gain weight more slowly than heterozygote and 
wild type (DAT.sup.+/+) mice. They show a significant propensity for 
premature death when compared to wild type and heterozygotes. These two 
findings may be linked to an impairment in food intake, as blockade of the 
DAT following cocaine treatment has been associated with modification of 
dietary habits (R. Byck and C. VanDyke, in: NIDA monograph 13, 97-118 
(1977)). DAT.sup.-/- mice are fertile and mate, but the females usually 
have an impaired maternal behavior, as offspring have to be transferred to 
foster mothers in order to be successfully raised to adulthood. This 
observation is consistent with the known role of dopamine on pituitary 
gland function and supports the controversial notion that the DAT plays a 
role in the regulation of the tuberoinfundibular dopamine pathway (B. 
Meister amd R. Elde, Neuroendocrinology 58, 388-395 (1993); M. Baumann and 
R. Rothman, Brain Res. 608, 175-179 (1993)). It may also be related to 
general modifications of cognitive or affective functions under 
dopaminergic influence. Previous reports indicate that cocaine treatment, 
which blocks the DAT, significantly modifies maternal behavior in rats (C. 
Kinsley et al., Pharmacol. Biochem. Behav. 47, 857-864 (1994); B. 
Zimmerberg and M. Gray, Physiol. Behav. 52, 379-384 (1992)). 
EXAMPLE 3 
Spontaneous Motor Activity in Treated Animals 
The DAT is believed to play a major role in the control of locomotor 
behavior by regulating dopaminergic tone in the basal ganglia. Indeed, 
drugs that interfere with dopamine re-uptake, such as the psychostimulants 
cocaine and amphetamine, are know to increase locomotor behavior (A. 
Dahlstrom and K. Fuxe, supra; G. DiChiara and A. Imperato, supra; B. Giros 
and M. Caron, supra; M. Ritz et al., supra; M. Jaber et al., Neuroscience, 
65, 1041-1050 (1995); P. Kelly et al., Brain Res. 94, 507-522 (1975)) 
accordingly, locomotor activity in DAT mice prepared as described in 
Example 1 above was investigated. 
Mice were maintained in standard housing conditions (12 hr light/dark 
cycle) and were allowed free access to food and water. They were housed in 
groups of 6 and used between the age of 6-10 weeks. Locomotor activity was 
measured in Plexiglass boxes (217.times.268.times.104 mm) with 2 photocell 
beams located across the long and the short axis respectively, 15 mm above 
the floor. Activity boxes were localized in dark cabinets (6 
boxes/cabinet,6 cabinets) in a quiet room, and the 3 groups of mice were 
always analyzed at the same time. Placement of the mice and recording of 
the activity numbers were done in double-blind fashion. Saline solution, 
cocaine (40 mg/kg) or d-amphetamine (10 mg/kg) were injected by the 
intra-peritoneal route (10ml/kg). Statistical analysis were performed 
using the Student's t test. 
We find that the spontaneous locomotor activity of naive homozygotes (i.e. 
non-habituated to the test) is highly elevated . They have a half-time of 
habituation twice as long (90 min. v. 40 min.) as the wild type or 
heterozygote mice, and are 5 to 6 times more active than wild type during 
both phases of the light-dark cycle. 
The heterozygotes are consistently more active that the wild type animals 
but this increase is of marginal significance (P=0.06) during the dark 
phase of the cycle. On the other hand, neither the heterozygotes nor the 
homozygotes exhibit a significantly enhanced level of spontaneous 
verticalizations or stereotypics (sniffing, grooming or rearing). It is 
well established that psychostimulants like amphetamine cause stereotyped 
behaviors by increasing extracellular dopamine in the dorsal striatum and 
enhance locomotor activity by increasing dopamine in the nucleus accumbens 
(P. Kelly et al., supra). It is interesting to note that DAT levels are 
substantially lower in the accumbens as opposed to the striatum in rats 
(J. Marshall et al., J. Neuroscience 37, 11-21 (1990)). Therefore, the 
observation of hyperlocomotion in the absence of stereotyped behavior in 
homozygote animals may reflect a regional difference in adaption to 
increased dopamine tone. 
Since homozygotes lack one of the major targets of psychostimulant drugs we 
investigated the locomotor effect of treatment with either cocaine or 
amphetamine. We found that i.p. injections of high doses of cocaine (40 
mg/kg) and d-amphetamine (10 mg/kg) have no significant effects on 
DAT.sup.-/- mice. On wild type and heterozygote mice, treatment with 
either of these two psychostimulants produces a 6 to 8 fold rise in their 
locomotor activity; a level that is not significantly different from the 
spontaneous activity in the homozygote mice. These findings directly 
demonstrate the major role of the DAT in the locomotor effects of 
psychostimulants, and rule out the participation of other monoamine 
transporters in these effects. 
EXAMPLE 4 
Effect on Genes Known to Respond to an Increase in Dopamine Levels 
Dopamine exerts a modulatory control on dopaminergic and dopaminoceptive 
cells at the pre- and post-synaptic level (M. Jaber et al., Neuroscience, 
65, 1041-1050 (1995); M. Jabar et al., Mol. Brain Res., 23, 14-20 (1994); 
C. Gerfen et al., J. Neurosci. 11 1016-1031 (1991); C. Gerfen et al., 
Science, 250, 1429-1432 (1990)). An increase in dopaminergic transmission 
following psychostimulant treatment induces modifications in gene 
expression in cells known to be under dopaminergic control (M. Jaber et 
al., supra). Given the dramatic increase in spontaneous locomotor activity 
observed in the homozygote mice, we examined whether this hyperactivity 
correlated with changes in the expression of genes known to respond to an 
increase in dopamine levels. 
In the basal ganglia, preproenkephalin A (PPA), which is co-expressed with 
D2 receptors, (C. LeMoine et al., Science 250, 1429-1432 (1990)) is under 
inhibitory influence of DA (C. Gerfen et al., J. Neurosci. 11 1016-1031 
(1991); F. Tang et al., Proc. Natl. Acad. Sci. U.S.A. 80, 3841-3844 
(1983)) whereas substance P (SP) and dynorphin (Dyn), which are 
coexpressed with D1 receptors (C. Gerfen et al., Science, 250, 1429-1432 
(1990); C. LeMoine et al., Proc. Natl. Acad. Sci. U.S.A. 88, 4205-4209 
(1991)), are under the excitatory influence of DA. (C. Gerfen et al. J. 
Neurosci. 11 1016-1031 (1991)). 
By in situ hybridization (Table 1), we show that in homozygote mice PPA 
mRNA levels were greatly reduced (66%). mRNA coding for Dyn was increased 
by 50% while SP mRNA levels were not significantly modified. The finding 
that Dyn and SP mRNAs are not both up-regulated suggests that different 
activation pathways may exist for their respective genes. At the receptor 
level, we find that the mRNA coding for D1 and D2 receptors, the two major 
dopamine receptors in this region, were down-regulated by 55% and 45% 
respectively. This simultaneous decrease in D1 and D2 receptor mRNAs is 
unprecedented, as neither lesions nor pharmacological and behavioral 
manipulations of dopamine transmission have ever been shown to display a 
down-regulation of both mRNAs. We also detected a 50% decrease in D2 
receptor gene expression in dopamine cells (SNC and VTA), indicating a 
putative down-regulation of D2 autoreceptors known to regulate dopamine 
levels and firing. For each of the investigated mRNAs, the heterozygotes 
always displayed intermediate values between the wild type and the 
homozygote mice (Table 1). 
TABLE 1 
______________________________________ 
mRNA quantification of in situ hybridization 
autoradiograms obtained for various genes under dopamine influence 
in the Striatum and ventral midbrain (VM) of Wild Type (WT), 
DAT(+/-) and DAT (-/-) Mice. 
Region Gene WT DAT(+/-) DAT(-/-) 
______________________________________ 
Striatum 
PPA 1 .+-. 0.08 
0.83 .+-. 0.07* 
0.33 .+-. 0.05*** 
SP 1 .+-. 0.08 
0.96 .+-. 0.07 
1.17 .+-. 0.1 
dyn 1 .+-. 0.07 
1.37 .+-. 0.09** 
1.48 .+-. 0.09*** 
D.sub.1 R 
1 .+-. 0.08 
0.66 .+-. 0.06** 
0.44 .+-. 0.04*** 
D.sub.2 R 
1 .+-. 0.09 
0.74 .+-. 0.07** 
0.56 .+-. 0.05*** 
VM DAT 1 .+-. 0.08 
0.31 .+-. 0.05*** 
ND 
D.sub.2 R 
1 .+-. 0.02 
0.74 .+-. 0.05* 
0.52 .+-. 0.06*** 
______________________________________ 
Six mice per group were analyzed (methods in FIG. 1), and experiments 
performed in duplicate or triplicate. Values were compared using the 
Student's t test: 
*: P &lt; 0.05; 
**: P &lt; 0.01; 
***: P &lt; 0.0001; 
ND, Not Detectable. 
The changes in gene expression that we observed are reflective of a 
functional increase in dopaminergic neurotransmission and correlate with 
the marked increase in spontaneous locomotor activity. Moreover, these 
observations show that the dopamine system is able to undergo profound 
gradual adaptation and plasticity to an extent that has not been 
previously observed with pharmacological manipulations. 
The foregoing examples are illustrative of the present invention, and are 
not to be construed as limiting thereof. The invention is defined by the 
following claims, with equivalents of the claims to be included therein.