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 In
1937, Charles Bradley first observed that Benzedrine (amphetamine) induces
a calming effect when used in hyperactive children. It is still not known
how these drugs work to increase alertness in normal individuals as well
as distractible and overactive children. Numerous studies have shown that
psychostimulants exert their pharmacological actions through interaction
with plasma membrane monoamine transporters, namely, the dopamine, serotonin,
and norepinephrine transporters. The normal function of these transporters
is to provide rapid removal of neurotransmitters from the synaptic cleft
to permit repetitive synaptic firing. Inhibition of the transporter would
prevent the normal reuptake and would lead to markedly elevated extracellular
monoamine levels.
Because
dopamine has been strongly implicated in the control of locomotion, particular
attention has been given to the interaction of these drugs with the dopamine
transporter (DAT). The elevation of extracellular dopamine levels is believed
to be the primary mechanism by which psychostimulants are able to regulate
locomotion. This concept, as well as the proven therapeutic efficacy of
psychostimulants in attention-deficit/hyperactivity disorder (ADHD) patients,
has provided the basis for the hypodopaminergic hypothesis of the disorder
and suggested a possible connection between DAT and ADHD.
Many
attempts to clarify the status of the dopamine system in patients with
ADHD have been largely unsuccessful. This is generally thought to be due
to the fact that many of the current approaches in clinical research,
such as the analysis of plasma, urine, cerebrospinal fluid, or computer
imaging techniques, provide equivocal estimates of the extracellular levels
of dopamine in the major motor areas of the human brain. The hypodopaminergic
hypothesis of ADHD, therefore, remains a theoretical concept inferred
from an oversimplified understanding of the mechanism of action of psychostimulants
in normal subjects.
The
hypothesized connection between DAT function and ADHD has continued to
spark considerable interest. There has been a reported association between
a polymorphism in the DAT gene and ADHD. Specifically, a significant association
was found by Cook and associates between ADHD and the 480-bp DAT1 allele
(48-bp repeat sequence). Other groups of researchers later confirmed these
observations in additional cohorts of patients and concluded that the
480-bp allele of DAT1 is preferentially transmitted to ADHD probands.
More recently, four different analytical strategies were used to examine
the association and linkage of the DAT gene and ADHD in children. Waldman
and associates replicated previous findings demonstrating the 480-bp allele
as the “high-risk” allele. Moreover, the relationship of the
DAT1 allele to ADHD increased as symptom severity increased within the
hyperactive-impulsive spectrum. Of considerable interest are the recent
studies that have suggested that homozygosity for the 10-repeat allele
of the dopamine transporter gene may be associated with poor response
to methylphenidate. Taken together, these molecular genetic studies provide
provocative evidence that alterations in DAT-mediated processes could
significantly contribute to the pathogenesis of this disorder.
However,
the functional consequences of this association remain unclear. It is
well established that enhanced dopaminergic transmission in the basal
ganglia translates into elevated energy, hyperactivity, and even euphoria.
Numerous pharmacological studies with drugs that enhance or reduce dopaminergic
transmission have shown that hyperactivity is a behavior of increased,
not decreased, dopaminergic tone.
Additional
evidence for the positive role of dopamine in hyperactivity comes from
the development of mice with genetic inactivation of the dopamine transporter.
Genetically engineered “knockout” mice are becoming increasing
important in neuroscience as a tool to study the possible functions of
a gene under study. The typical procedure is to produce a mutation in
the gene in the laboratory. The mutation is one that typically renders
the gene product inactive. A series of steps is then performed to introduce
the inactivated gene back into the mouse.
The
reintroduction of the inactivated gene is typically performed by microinjecting
the mutated gene into embryonic stem (ES) cells. These are cells that
can be easily grown and manipulated in culture dishes in the laboratory.
Homologous recombination is a normal event that occurs in gametes when
both copies of chromosomes line up next to each other and exchange genetic
material between the paternal and the maternal chromosome. The mutated
gene that has been injected into the ES cells will now be able to align
itself with the chromosomal region that contains the normal copy and can
be exchanged with it after the homologous recombination event.
ES
cells that have had this rare chromosomal rearrangement can be selected
to grow preferentially in the culture dish. This is possible because a
second gene was included with the injected DNA. The second gene encodes
a protein that conveys resistance to specific antibiotics. Normally, ES
are killed by the antibiotic neomycin. Thus one grows the injected ES
cells in the presence of neomycin. Only those ES cells that have had the
rare recombination event will be able to grow. DNA is extracted from some
of these cells to confirm the presence of the mutated gene. These cells
are then introduced into the uteri of mice. The offspring, the so-called
F1 generation, should now have one normal and one inactive copy of the
gene. Crosses are then made between the F1 mice, and approximately one
quarter of this next generation will have two copies of the inactivated
gene.
Genetically
engineered “knockout” mice that lack the gene encoding the dopamine
transporter (DAT-KO mice) have become available. These mice demonstrate
remarkable hyperactivity. This is thought to be due to the greater than
5-fold elevation of extracellular dopamine levels within the striatum,
the major motor area of the brain. It is worth mentioning that the knockout
mice behave essentially like their normal littermates when placed in a
familiar environment. However, when place in a novel environment, the
mice become much more active. Importantly, no corresponding rise in dopamine
accompanies exposure to the novel environment, suggesting that these behavioral
changes are regulated through more than just the dopamine system. These
mice also show significant cognitive impairment in an eight-arm radial
maze test, a standard approach to evaluate spatial cognitive function
in rodents. Specifically, the mutant animals make significantly more perseverative
errors, suggesting that these mice might suffer from poor behavioral inhibition.
An
obvious question is whether the drugs commonly used in the treatment of
attentional difficulties have any effect on these mice. Administration
of amphetamine (Adderallt, Dexedrinet), methylphenidate (Ritalint), or
cocaine to the hyperactive knockout mice calms them; thus they show a
response to psychostimulants that is similar to the response seen in humans
with ADHD. Conversely, normal mice become hyperactive when given these
psychostimulants (Fig.
1).
Normal
mice show an expected increase in extracellular dopamine brain levels
in response to an injection of methylphenidate, which blocks the dopamine
transporter. Of interest, the knockout mice do not show any changes. These
findings strongly suggest that psychostimulants do not affect the dopamine
system in these mice and most likely exert their calming effects through
modulation of other neurotransmitters targeted by these drugs.
To
test this hypothesis, a selective inhibitor of the norepinephrine transporter,
nisoxetine, was used. That it had no effect on the hyperactivity of the
knockout mice suggests that norepinephrine is not likely to be involved
in the mechanism by which the psychostimulants act. In contrast, an inhibitor
of serotonin reuptake, fluoxetine (Prozact), causes a dramatic reduction
in hyperactivity, as do other drugs that either directly activate serotonin
receptors (serotonin receptor agonist quipazine) or increase brain serotonin
levels, such as precursors of serotonin (tryptophan and 5-hydroxytryptophan).
These results suggest that hyperactivity induced by high levels of dopamine
can be dampened by enhancing serotonergic tone. Accordingly, the psychostimulants
are likely to modulate behavior in the mutant mice by enhancing the calming
effects of serotonin rather than by acting directly on dopamine reuptake,
because there are no functional DAT in these mice.
While
this is not the first study to implicate serotonin in impulse regulation
and inhibitory control on external stimuli-induced behavioral activation,
most researchers have long assumed that the primary action of psychostimulants
like Ritalin was through the dopamine system. However, attempts to understand
the calming effect of psychostimulants exclusively through the dopaminergic
theory have been largely unsuccessful. We hypothesize that, at least in
these mice, interaction of psychostimulants with the serotonin transporter
may provide enhanced serotonergic tone, sufficient to exert inhibitory
influence on behavior.
In
the clinical arena, however, there are very few reports that address whether
serotonergic drugs are beneficial in patients with ADHD. It is generally
acknowledged that conventional serotonin reuptake inhibitors are of limited
use in the management of these patients, and in fact one of the major
side effects of these medications is stimulation. At the same time, some
recent reports indicate some therapeutic benefit from the preferential
serotonergic drugs venlafaxine and buspirone. Moreover, addition of selective
serotonin reuptake inhibitors to psychostimulant treatment was reported
in one small open-label trial to be beneficial in certain individuals
with ADHD. Future controlled clinical studies are warranted to test the
hypothesis developed from the mouse work. It should be mentioned also
that serotonin can have an extremely complex set of actions on locomotor
behaviors. It has been determined that there are at least 14 subtypes
of serotonin receptors, and some of them mediate opposite actions on many
functions, locomotion in particular. A major challenge for future research
is to determine which subtype(s) of serotonin receptors are primarily
involved in the calming effects of psychostimulant.
The
dopamine transporter knockout mice display several key characteristics
of ADHD. These include hyperactivity and cognitive impairments as well
as response to psychostimulants. A similar, although less pronounced phenotype
is characteristic of mice with reduced (more than 80%) DAT expression
(Drs. X. Zhuang and R. Hen, unpublished observations). In contrast, mice
that have higher than normal DAT expression show hypoactivity that is
particularly evident in a new environment. Taken together, these genetic
animal studies suggest that ADHD most likely represents a hyperdopaminergic
condition and that the calming effect of psychostimulants is mediated
through targets other than DAT.
In
conclusion, these findings suggest that genetic defects in the dopamine
transporter gene might contribute to some forms of ADHD in humans. It
should be emphasized that ADHD is likely to be a heterogeneous disorder
with several biochemical/genetic defects leading to similar clinical symptoms.
Dopaminergic dysregulation, like that observed with the DAT knockout mice,
might be produced by mutations in other components of the dopaminergic
system. Moreover, dysregulation of other neurotransmitter pathways is
likely to be an additional mechanism responsible for the development of
ADHD-like conditions.
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