Locomotor
inhibition, yawning and vacuous chewing induced
by a novel dopamine D2 post-synaptic receptor
agonist
Smith HP, Nichols DE, et al.
University North Carolina,
USA
Introduction
The development of novel ligands with high
selectivity for dopamine receptor isoforms has
been important for understanding brain
dopaminergic function in normal and disease
states (e.g., Parkinson's disease and
schizophrenia). Dopamine receptors comprise a
subset of the super-family of G-protein coupled
receptors.
Currently there are two known
pharmacologically similar families of dopamine
receptors, usually categorized as D1 and D2. At
least five genes code for unique dopamine
receptors, some having splice variants. Each
molecular subtype has a unique regional
distribution in the brain, the functional
significance of which is, at present, poorly
understood. The five genes can be divided into
two families that are often referred to as
D1-like and D2-like. D1-like dopamine receptors
(the D1A and D1B or D5) have intron-less genes,
are expressed as proteins having a relatively
short third intracellular loop and a relatively
long carboxy tail, and show high affinity for
phenyl-tetrahydrobenzazepines such as SCH23390
(k( + )-7-chioro-8-hydroxy-3-methyl- l
-phenyl-2.3.4,5-tetrahydro- 1 H-3-benza-zepine)
and SKF38393 (R( + )- I
-phenyl-7.8-dihydroxy2,3.4.5-tetrahydro-( 1 H
)-3-benzazepine). D2-like dopamine receptors
(D2çhOrt and D210, splice variants, D3,
and D4) are genes having multiple introns. are
expressed as proteins having a long third
intracellular loop and a short carboxy tail, and
show high affinity for butyrophenones (e.g..
spiperone) and benzamides (e.g.,( -
)-sulpiride). Currently available ligands have
limited ability to discriminate between
molecular isoforms within each family.
Throughout this paper, our references to
dopamine D1 and D2 receptors will be to each
family, although if specific information is
available about a particular receptor isoform.
(e.g., D2 vs. D3), this will be stated
explicitly.
Dopamine D2 receptors are expressed both
as-autoreceptors on dopamine neurons and
terminals, and as postsynaptic receptors on
target cells. Systemically administered dopamine
receptor agonists acting at dopamine D2
receptors are known to have a biphasic
dose-response effect on unconditioned behaviors
in rodents: low doses inhibit spontaneous
locomotor activity, whereas high doses increase
locomotor activity and elicit oral stereotypies.
This biphasic dose response effect has been
attributed to the ability of low agonist doses
to stimulate selectively dopamine D2
autoreceptors on the dopamine neuron, thereby
decreasing synaptic concentrations of dopamine
via down-regulation of neural firing rate,
dopamine synthesis, and dopamine release.
Consistent with an autoreceptor hypothesis of
decreased locomotor activity, several atypical
dopamine receptor agonists displaying functional
selectivity for dopamine D2 autoreceptors are
observed to dose-dependently inhibit locomotor
activity in rodents, even at high doses.
Yet not all reports are consistent with this
autoreceptor hypothesis (e.g., Stahle). The most
recent challenge to this notion offers the
alternative hypothesis that a post-synaptic
subpopulation of dopamine D2 receptors (possibly
the D3 molecular isoform of the receptor) can
mediate decreases in spontaneous locomotor
activity. This is based on the evidence that
purported antagonists with selectivity for the
dopamine D3 (i.e.. at least in vitro) vs. the D2
receptor increase locomotor activity without
affecting dopamine release or utilization.
Several years ago, we reported on
dihydrexidine ((± )trans- 10,11
-dihydroxy-5,6,6 a,7,8, 12 b-hexahydrobenzo[
a]phenanthridine), the parent compound of a
novel class of dopamine receptor agonists. It is
now known that both the D1 and D2 affinity and
functional potency reside in the (+
)-enantiomer. Although originally designed as a
ligand for the dopamine D1-like receptor, it was
found that dihydrexidine and several of its
analogs also were high affinity ligands for
dopamine D2-like receptors as well. Of
particular interest was the fact that
dihydrexidine and other structural analogs could
functionally activate post-synaptic dopamine D2
receptors in striatum and pituitary, yet
displayed little or no activity at release- or
synthesis-modulating terminal dopamine
autoreceptors. The unique post-synaptically
selective pharmacology of
hexahydrobenzo[a]phenanthridine ligands
is supported further by a lack of agonist
effects at impulseregulating D2 autoreceptors
located on the somatodendritic membranes of
dopamine neurons. These differential functional
effects occur despite equivalent binding
affinity for pre- and post-synaptic receptor
sites.
The present work used the N-n-propyl analog
of (± )dihydrexidine to assess the effects
of selective activation of post-synaptic
dopamine D2 receptors on unconditioned behaviors
in rats. Binding studies had shown that (±
)-n-propyl-dihydrexidine has 10-fold selectivity
for native dopamine D2 receptors vs. D1
receptors in rat striatum, and also has high
affinity for the cloned dopamine D3 receptor.
The purpose of the present study was to
determine whether the unique functional
selectivity of (±
)-N-n-propyl-dihydrexidine for post-synaptic vs.
pre-synaptic dopamine D2 receptors would induce
a linear dose-dependent increase in spontaneous
locomotor activity and oral stereotypy as
predicted by the autoreceptor hypothesis of
dopamine receptor agonist effects on
unconditioned behaviors.
To our surprise, we observed just the
opposite effect: systemic administration of
(± )-N-n-propyl-dihydrexidine in
unhabituateD2 drug-naive rats induced a linear
dose-dependent decrease in spontaneous locomotor
activity, and did not induce oral stereotypy
typically seen after administration of high
doses of dopamine receptor agonists. In
addition, we observed significant dose-dependent
increases in yawning and vacuous chewing,
behaviors usually observed after activation of
dopamine D2 autoreceptors and D1 receptors,
respectively. These data support the hypothesis
that a sub-population of post-synaptic dopamine
D2 receptors (possibly the D3 receptor) mediate
suppression of spontaneous locomotor activity.
The activation of these receptors also appears
to be important in potentiating yawning and
vacuous chewing behaviors.
Discussion
The present findings provide a comprehensive
description of the behavioral effects of the
novel dopamine receptor agonist (±
)-N-n-propyl-dihydrexidine. Administration of
this compound induced marked increases in
inactivity and vacuous chewing and decreases in
a number of other specific behavioral elements,
including locomotion, rearing and repetitive
sniffing. Increased yawning was seen at the
highest dose of ( ±
)-N-n-propyl-dihydrexidine. Oral stereotypies
(licking and gnawing) were notably absent. The
use of dopamine receptor subtype-selective
antagonists confirmed the dopaminergic basis for
the prominent behavioral effects of this novel
agonist. The increased inactivity induced by
(± )-N-n-propyl-dihydrexidine was mediated
by dopamine D2 receptors, while both dopamine D1
and D2 receptors contributed to the observed
increases in vacuous chewing. Taken together.
the behavioral effects observed with (±
)-N-n-propyl-dihydrexidine resembled those
obtained with a low dose of the traditional
dopamine D2 receptor agonist (-
)-apomorphine.
The behavioral profile observed with (±
)-N-n-propyldihydrexidine is of special
relevance for evaluating traditional concepts
concerning the neurobiological substrate for
dopamine receptor-mediated behaviors. The
modification of spontaneous locomotion and oral
behaviors in rats by dopamine D2 receptor
agonists is typically biphasic, as is the case
for (-)-apomorphine. Electrophysiological data
indicating that dopamine D2 autoreceptors are
functionally more sensitive to low doses of
dopamine agonists than are post-synaptic
dopamine D2 receptors initiated an autoreceptor
hypothesis to explain the biphasic dose effects
of dopamine receptor agonists on unconditioned
behavior. This hypothesis has been supported
further by data indicating that agonists with
functional selectivity for dopamine D2
autoreceptors induce monotonic decreases in
locomotor activity.
When viewed against this background, the
behavioral effects obtained with
N-n-propyl-dihydrexidine would be considered as
indicative of actions exclusively at dopamine D2
autoreceptors. Yet this conclusion is at odds
with other data indicating that
hexahydrobenzo[alphenanthridine hgands
(e.g., (± )-N-n-propyl-dihydrexidine) are
dopamine receptor agonists that have little
functional activity at dopamine D2 autoreceptors
while having potent functional activity at
post-synaptic dopamine D2 receptors. For
example, administration of either (±
)-dihydrexidine or its N-npropyl analog reduces
serum prolactin levels to an extent equivalent
to that of the full agonist (- )-quinpirole,
actions that are mediated by dopamine D2
heteroreceptors located on pituitary
lactotrophs. Likewise, members of the
hexahydrobenzo[a]phenanthridine class
produce full inhibition of the enzyme adenylate
cyclase in models that reflect dopamine D2
postsynaptic receptor function primarily (e.g.,
cAMP efflux in superfused striatal slices).
Conversely. these same compounds display little
or no efficacy at dopamine D2 receptors
controlling dopamine release or cell firing in
substantia nigra. For example, N-n-propyl
dihydrexidine has minimal effects on dopamine
release as measured using in vivo voltammetry in
striatal slices. Finally, it is of special
interest that the differing functional profiles
of this novel class of agonists at dopamine D2
prevs. post-synaptic receptors occur despite
equal binding affinity to each receptor
population.
Given the evidence that (±
)-N-n-propyl-dihydrexidine does not activate any
of the autoreceptor-linked processes leading to
decreased dopaminergic transmission, the dose
effects of this agonist on unconditioned
behavior are not consistent with an autoreceptor
hypothesis. Decreased locomotor activity was
shown to be dopamine D2 receptormediateD2
therefore we conclude that decreases in
spontaneous locomotor activity can be induced by
post-synaptic dopamine D2 receptor activation.
This is important since drug effects on
spontaneous locomotor activity are routinely
used as a pharmacological screen for determining
autoreceptor vs. post-synaptic dopamine D2
receptor activation; our data advocate extreme
caution in using locomotor activity as a measure
of functional selectivity for novel
agonists.
There have been some data in the literature
to suggest that post-synaptic dopamine D3
receptors might have functional significance in
decreasing spontaneous locomotor activity in
rats. The evidence for this is limited for two
reasons: (I) there are no highly selective
dopamine D3 receptor ligands, yet there is a
relatively larger expression of dopamine D2
receptors in the relevant brain regions, thus
complicating interpretation, particularly in
vivo and (2) the same inavailability of a highly
selective dopamine D3 ligand has prevented
learning about the biochemical endpoints that
dopamine D3 receptor activation mediates in
vivo.
Nevertheless, some classes of dopamine
agonists with high affinity for the cloned
dopamine D3 receptor have been shown to decrease
spontaneous locomotor activity over a larger
dose range than other agonist classes (e.g.,
(± )-7-hydroxy-N,
N-di-n-propyl-2-aminotetralin (7OHDPAT). The
decreased locomotor activity caused by
(±)7-OHDPAT occurs over a dose range at
which terminal dopamine release is not affected,
suggesting post-synaptic mechanisms. Yet other
structural classes having equivalent high
dopamine D3 receptor affinity show discretely
biphasic effects on locomotor activity
((-)-quinpirole. A novel ligand (U99194A)
determined to be a dopamine D3 receptor
antagonist based on its affinity in vitro and
the steep slope of its binding curve, has been
shown to increase locomotor activity in rats
without altering terminal dopamine release,
consistent with the hypothesis that
post-synaptic dopamine D3 receptors have
functional significance in decreasing locomotor
activity. The high affinity of (±
)-N-n-propyl-dihydrexidine for the D3 receptor,
and its functional selectivity for post-synaptic
vs. pre-synaptic dopamine D2 receptors, are
consistent with a hypothesis that post-synaptic
dopamine D3 receptors functionally mediate
decreases in spontaneous locomotor
activity.
Yawning behavior also has been hypothesized
to be mediated by selective stimulation of
dopamine D2 autoreceptors. Our data indicate
that high doses of
(±)-N-n-propyl-dihy-drexidine, functionally
activating post-synaptic dopamine D2 receptors.
significantly increase the frequency of yawning
observed in rats. This supports other evidence
that yawning is not exclusively associated with
the activation of dopamine D2 autoreceptors.
(±)-7-OHDPAT has been shown to increase
yawning frequency within the same dose range as
it decreases terminal dopamine release. Taken
together with our data, this suggests that both
autoreceptors and post-synaptic dopamine D2/D3
receptors have functional significance in
mediating yawning.
An unexpected result was the potent
dose-dependent increase in vacuous chewing
observed with (±
)-N-n-propyl-dihydrexidine. This became a
high-intensity behavior (i.e., nearly continuous
throughout observation time) at doses equal to
or higher than 2 mg/kg. Vacuous chewing is not
in the category of oral stereotypies typically
observed in the high dose range of dopamine D-,
receptor agonists. Moreover, vacuous chewing is
typically associated with acute administration
of dopamine D1 receptor agonists or the chronic
administration of dopamine D2 receptor
antagonists (for review, see Waddington, 1990).
Interestingly, our antagonist studies for
(± )-N-n-propyl-dihydrexidine effects on
vacuous chewing behavior provide evidence that
both dopamine D1 and D2 receptors contribute to
the potentiation of this behavior. This is in
distinct contrast to most of the literature on
acutely induceD2 dopamine D1 receptor-mediated
vacuous chewing, in which an antagonistic
functional interaction between dopamine D1 and
D2 receptors is observed. Currently available
pharmacological ligands have limited selectivity
for distinguishing between molecular isoforms of
the dopamine D1 (D1 and D5) and D2 (D2, D3 and
D4) receptor families. With the introduction of
new ligands that are highly selective for
molecular subtypes of the receptor, conflicting
issues of dopamine D1-D2 receptor functional
interaction may be clarified by a discovery that
certain sub-populations of the dopamine receptor
families have antagonistic, while others have
cooperative/synergistic, functional
interactions.
In summary, these findings
demonstrate that locomotor inhibition and
yawning, often considered as markers of dopamine
D2 presynaptic receptor activation, can occur
following administration of a novel dopamine
receptor agonist that does not produce the
neurochemical changes (e.g., changes in dopamine
release) that have been linked to activation of
dopamine D2 presynaptic receptors. Although the
mechanism for the unusual profile of this
compound has not been elucidateD2 a role for
postsynaptic dopamine D2-like receptor
subpopulations (D receptors) is hypothesized.
These findings lend weight to recent challenges
to established ideas about the behavioral
consequences of dopamine D2 presynaptic receptor
function. In a similar vein, the unexpected
emergence of vacuous chewing observed with N
n-propyl-dihydrexidine should prompt efforts to
reformulate current concepts concerning the role
of the dopamine D1 and D2 receptor subtypes in
the generation of this behavior.
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L Do autoreceptors mediate dopamine
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