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8 avril 2007
Psychopharmacology (Berl)
Yawning and hypothermia in rats:
effects of dopamine D3 and D2 agonists and antagonists
Collins GT, Newman AH, Grundt P, Rice KC, Husbands SM, Chauvignac C, Chen J, Wang S, Woods JH.
Depart Pharmacology, University of Michigan Medical School, Ann Arbor, MI.USA


Abstract Rationale Identification of behaviors specifically mediated by the dopamine D2 and D3 receptors would allow for the determination of in vivo receptor selectivity and aide the development of novel therapeutics for dopamine-related diseases.
Objectives These studies were aimed at evaluating the specific receptors involved in the mediation of D2/D3 agonist-induced yawning and hypothermia.
Materials and methods The relative potencies of a series of D2-like agonists to produce yawning and hypothermia were determined. The ability of D3-selective and D2-selective antagonists to inhibit the induction of yawning and hypothermia were assessed and a series of D2/D3 antagonists were characterized with respect to their ability to alter yawning induced by a low and high dose of PD-128,907 and sumanirole-induced hypothermia.
Results D3-preferring agonists induced yawning at lower doses than those required to induce hypothermia and the D2-preferring agonist, sumanirole, induced hypothermia at lower doses than were necessary to induce yawning. The rank order of D3 selectivity was pramipexole > PD-128,907 = 7-OH-DPAT = quinpirole = quinelorane > apomorphine = U91356A. Sumanirole had only D2 agonist effects. PG01037, SB-277011A, and U99194 were all D3-selective antagonists, whereas haloperidol and L-741,626 were D2- selective antagonists and nafadotride's profile of action was more similar to the D2 antagonists than to the D3 antagonists.
Conclusions D3 and D2 receptors have specific roles in the mediation of yawning and hypothermia, respectively, and the analysis of these effects allow inferences to be made regarding the selectivity of D2/D3 agonists and antagonists with respect to their actions at D2 and D3 receptors.
Introduction Dopamine D2 and D3 receptors are both members of the D2-like family of dopamine receptors, which are known to possess a high degree of sequence homology (52% overall and 75% in the transmembrane domains; Sokoloff et al. 1990) and a partially overlapping pattern of distribution in the brain. For example, D2 receptors are expressed at while the D3 receptor has been shown to possess a much more restricted limbic pattern of distribution in both the rat (Levesque et al. 1992) and human brains (Gurevich and Joyce 1999).
These high levels of expression within the limbic brain regions have led many to hypothesize that the D2 and D3 receptors are of particular interest as pharmacologic targets for the treatment of a variety of movement and psychiatric disorders including Parkinson's disease, restless leg syndrome, depression, and schizophrenia (e. g., Joyce 2001; Happe and Trenkwalder 2004) and a variety of aspects of drug abuse (e. g., Heidbreder et al. 2005; Newman et al. 2005). Due in part to the lack of highly selective agonists and antagonists, the receptor(s) mediating either the therapeutic or mechanistic effects are yet to be fully elucidated.
Although several agonists and antagonists have been reported to be over 100-fold selective for either the D3 (e. g., Stemp et al. 2000; Grundt et al. 2005) or D2 (e. g., Vangveravong et al. 2006) receptors based on in vitro binding studies, a large degree of variability exists with respect to the reported in vitro binding affinities and D2/D3 selectivity ratios.
A variety of factors may account for these differences in affinity and selectivity including differences in receptor species, expression systems, radioligands, and/ or assay conditions. For example, reported binding affinities for pramipexole at the D2 receptor range from 3.9 to 955 nM depending upon whether agonist or antagonist radioligands were used (Mierau et al. 1995; Millan et al. 2002), while reported D3 selectivity ratios range from 2- fold to 488-fold selective for the D3 receptor over the D2 receptor depending upon whether binding affinities from cloned human receptor cell systems or human brain tissue are used to make the determinations (Seeman et al. 2005; Gerlach et al. 2003).
Furthermore, in vitro binding studies often provide greater affinity and selectivity values than those obtained through functional studies suggesting that differences in D2 and D3 efficacy may also greatly influence a ligand's receptor selectivity. For example, in three separate studies, which characterized D2/D3 agonists based on their binding affinities for the D2 and D3 receptors and ability to stimulate mitogenic activity, quinpirole was found to be either 9-fold, 15-fold or 36- fold selective for the D3 receptor over the D2 receptor as determined by radioligand binding, but the D3 selectivity ratios for quinpirole dropped to 2.5-fold, 1.3-fold, and 3.3- fold when the ED50 values for the induction of mitogenic activity were compared (Pugsley et al. 1995; Chio et al. 1994; Sautel et al. 1995).
The identification of agonists and antagonists highly selective for the D2 and/or D3 receptors has been complicated by a lack of well-characterized behavioral effects specifically mediated by either the D2 or D3 receptor. While D2/D3 agonists have been shown to modulate body temperature, locomotor activity, and certain neuroendocrine responses in addition to other behavioral measures (Faunt and Crocker 1987; Millan et al. 1995; Depoortere et al. 1996; Smith et al. 1997; Boulay et al. 1999a), few of these effects have been fully characterized and well-validated.
There is strong pharmacological and genetic evidence in support of subtype selective in vivo effects for the induction of hypothermia resulting from D2 receptor activation, and significant pharmacological evidence for the induction of yawning resulting from agonist activation of the D3 receptor. The first indication that D2/D3 agonist-induced hypothermia was mediated by the D2 receptor, not D3, was the finding that D3 receptor-deficient mice displayed a normal hypothermic response to D2/D3 agonists, while the effect was completely absent in D2 receptor-deficient mice (Boulay et al. 1999a, b).
This was later supported by pharmacologic studies in rats that demonstrated that the D2-preferring antagonist, L-741,626, produced a dosedependent inhibition of D2/D3 agonist-induced hypothermia, whereas the D3-preferring antagonist A-437203 failed to alter the hypothermic response at any dose tested (Chaperon et al. 2003). Yawning behavior in rats has been a long studied phenomenon and is known to be regulated by a variety of neurotransmitter systems including cholinergic (Urba- Holmgren et al. 1977; Yamada and Furukawa 1980), serotonergic (Stancampiano et al. 1994), and dopaminergic (Mogilnicka and Klimek 1977; Holmgren and Urba- Holmgren 1980) systems associated with the paraventricular nucleus of the hypothalamus (Argiolas and Melis 1998).
Recently, a specific role for the D3 receptor in the induction of yawning behavior has also been demonstrated. A series of D3-preferring agonists induced dose-dependent increases in yawning behavior over low doses with inhibition of yawning occurring at higher doses resulting in a characteristic inverted U-shaped dose-response curve. Several D3-preferring antagonists were also shown to selectively inhibit the induction of yawning behavior, while the D2-preferring antagonist, L-741,626, produced a selective rightward and upward shift in the descending limb of the dose-response curve for D2/D3 agonist induced yawning (Collins et al. 2005).
Thus, although it has been suggested that the induction of yawning is mediated by activation of the D2 receptor (Millan et al. 2000), our data indicated that the induction of yawning by D2/D3 agonists is mediated by a selective activation of the D3 receptor, while inhibition of yawning behavior at higher doses is a result of a concomitant D2 receptor activation. The present studies were aimed at further characterizing the roles of the D2 and D3 receptors in the regulation of body temperature and yawning behavior. Thus, a series of D2-like agonists with a range of reported in vitro selectivities for the D3 receptor over the D2 receptor (pramipexole PD-128,907 = 7-OH-DPAT > quinpirole = quinelorane > apomorphine > U91356A > sumanirole) and two D4-preferring agonists (ABT-724 and PD-168,077) were assessed for their ability to induce yawning and hypothermia, while a series of D2/D3 antagonists with a similar range of reported in vitro selectivities (PG01037 = SB-277011A >> U99194 > nafadotride > haloperidol > L- 741,626) were characterized for their ability to modulate the induction of yawning and hypothermia in the rat.
Convergent evidence support the hypotheses that the induction of hypothermia and yawning behavior are mediated by the selective activation of the D2 and D3 receptors.
Furthermore, these studies suggest that the minimal effective dose (MED) for the induction and inhibition of yawning behavior and hypothermia may provide a means for the determination of in vivo D3 and D2 receptor potency measures for agonists and antagonists, respectively.
Discussion The current studies replicate and extend the findings of a previous study that suggested that the induction of yawning by low doses of D2/D3 agonists is mediated by the selective activation of the D3 receptor, whereas the inhibition of yawning occurring at higher doses is mediated by a concomitant activation of the D2 receptor (Collins et al. 2005). As was demonstrated in the earlier paper, yawning induced by a low dose of the D3-preferring agonist PD- 128,907 was selectively and dose-dependently inhibited by the D3 antagonists, PG01037, SB-277011A, and U99194, whereas the inhibition of yawning observed at high doses of PD-128,907 was reversed by the selective D2 antagonist L- 741,626, but not PG01037, SB-277011A nor U99194.
The current studies extend the previous findings in several ways. In addition to the evaluation of agonist and antagonist interactions on yawning, the effects of the D2/ D3 agonists alone and in combination with selective antagonists were evaluated on core body temperatures to test the notion that the hypothermic effects of these agonists are mediated by the activation of the D2 receptor, but not the D3 or D4 receptor (Boulay et al. 1999a, b; Chaperon et al. 2003). Several lines of evidence presented herein support this notion. The selective D2 agonist, sumanirole, produced decreases in body temperature at relatively low doses that did not induce yawning. The hypothermic effects of sumanirole were prevented by prior administration of the D2-preferring antagonists, haloperidol and L-741,626. L- 741,626 also inhibited the hypothermic effects of high doses of all of the D3-preferring agonists in addition to producing dramatic increases in yawning when combined with the same high doses of D3-preferring agonists.
The latter is likely to reflect the reversal of the D2-mediated inhibition of yawning produced at high doses of the agonists and is consistent with the notion that these antagonists are D2-selective and that the suppression of yawning and hypothermic effects observed at relatively high doses of D2/D3 agonists are D2 agonist-mediated effects. It is important to note that these differential effects of D3 and D2 antagonists on yawning induced by low and high doses of D2/D3 agonists were observed with all of the D3-preferring agonists tested in the current study (Table 2) and occurred at doses of PG01037 that do not alter the induction of yawning by physostigmine or TFMPP (Collins et al. 2005) and a dose of L-741,626 that does not alter the induction of hypothermia by the serotonin 1A agonist, 8-OH-DPAT (Table 2), suggesting that these effects are a result of a selective antagonist activity at D3 and D2 receptors, respectively. These in vivo measures of selective D3 (yawning) and D2 (hypothermia) activation were used to characterize ten D2-like agonists and six D2/D3 antagonists.
This extensive evaluation, comparing the potency of each agonist to produce increases in yawning with its potency to produce hypothermia (Table 1), indicated that pramipexole was the most selective D3 agonist, followed by PD-128,907, quinelorane, quinpirole, and 7-OH-DPAT with nearly equal D3 selectivity. Both apomorphine and U91356A were relatively nonselective D2/D3 agonists, inducing yawning at doses that were only slightly lower than those required to decrease body temperature. Sumanirole was a selective D2 agonist. Although sumanirole slightly increased yawning at doses that were higher than those necessary to decrease body temperature, this yawning was not sensitive to the D3- selective antagonist, PG01037, but was inhibited by the cholinergic antagonist scopolamine and may therefore represent cholinergic rather than D3 activation. McCall et al. (2005) reported a 200% increase in striatal acetylcholine release in rats at doses of sumanirole roughly equivalent to those which induced yawning.
The two D4-preferring agonists, given at behaviorally active doses (Brioni et al. 2004; Enguehard-Gueiffier et al. 2006), did not produce either yawning or hypothermia suggesting that at these doses, they are devoid of significant D2 and D3 receptor agonist activity. As was seen with the agonists, distinct behavioral profiles emerged for D3-preferring and D2-preferring antagonists. Three of the four D3-preferring antagonists, PG01037, SB-277011A, and U99194, inhibited yawning at doses that did not alter hypothermia suggesting that they function as selective D3 antagonists in vivo. The doses of these antagonists that were able to be tested was limited by solubility (PG01037 and SB-277011A) and anticholinergic activity (U99194), and thus in vivo D2/D3 selectivity ratios were indeterminate other than being slightly greater than 1.
It is interesting to note that nafadotride, which is mildly D3- preferring in vitro and generally considered to be a D3- preferring antagonist in vivo (e. g., Richtand et al. 2000; Leriche et al. 2003), displayed a profile of activity that was more like those of the D2 antagonists, haloperidol and L- 741,626, than of the other D3-preferring antagonists. L- 741,626, haloperidol, and nafadotride were all more potent at inhibiting the induction of hypothermia and increasing high dose yawning; however, suppression of low dose yawning was also observed with each of these antagonists, and thus were all determined to be °„3-fold selective for the D2 receptor over the D3 receptor in vivo. Evidence provided in the current and past (Collins et al. 2005) studies support distinct roles for the D2 and D3 receptors mediating the hypothermic and yawning effects of D2/D3 agonists although these generalizations are contrary to earlier characterizations (see, Millan et al. 2000). These investigators determined that the hypothermic effects of 7- OH-DPAT were mediated by agonist activity at both the D2 and D3 receptors as it was attenuated by the D3 antagonists, S33084 and GR218231, and the D2 antagonist, L-741,626.
Furthermore, they concluded that 7-OH-DPATinduced yawning was mediated by the D2 receptor, not D3, as they observed inhibition of yawning with L-741,626, but not S33084 or GR218321. Although our data do not support this interpretation, we recognize that relatively large doses of D3-preferring agonists induce hypothermia and likewise that relatively large doses of L-741,626 suppress yawning induced by D3-preferring agonists. However, these effects likely represent a loss of receptor selectivity rather than a primary effect of the agonists and antagonists. A notion that is supported by the biphasic nature of the D2/D3 agonists and antagonists with respect to their effects on yawning and hypothermia. In the current study, all D3-preferring agonists, including 7-OH-DPAT, induced yawning at low doses with inhibition of yawning and induction of hypothermia occurring at higher, presumably less selective, doses.
Similarly, at relatively low doses, L-741,626, haloperidol, and nafadotride equipotently increased high dose yawning and inhibited hypothermia, while inhibition of yawning induced by a low, presumably D3-selective, dose of PD-128,907 was not observed until higher doses. Moreover, in the current study, the D3 antagonists PG01037, SB-277011A, and U99194 all selectively inhibited PD-128,907-induced yawning while failing to alter the induction of hypothermia by sumanirole, suggestive of a selective D3 antagonist activity. While the MEDs for the inhibition of yawning by PG01037 and SB-277011A (32.0 mg/kg for both) are slightly higher than those reported for SB-277011A on a variety of operant behaviors (3.0-24 mg/kg; Andreoli et al. 2003; Di Ciano et al. 2003; Xi et al. 2004, 2005; Gilbert et al. 2005; Cervo et al. 2007) and are likewise higher than might be expected based on in vitro D3 affinities of 0.7 and 10.7 nM, respectively (Stemp et al. 2000; Grundt et al. 2005), there is no evidence to suggest that the inhibition of yawning by these antagonists results from anything other than an antagonist activity at the D3 receptor.
Not only did PG01037 and SB-277011A not inhibit sumanirole-induced hypothermia or increase yawning induced by high doses of PD-128,907 in the current studies at doses up to 56.0 mg/kg, but SB-277011A also failed to induce catalepsy and increases plasma prolactin levels at doses up to 78.8 and 93 mg/kg; p. o., respectively (Reavill et al. 2000).
However, this is not to say that these antagonists are completely devoid of D2 antagonist activity as U99194 has been reported to inhibit the induction of hypothermia with an ED50 of 12.9 mg/kg (Audinot et al. 1998), suggesting that inhibition of sumanirole-induced hypothermia by PG01037, SB- 277011A, and U99194 would have been observed if higher, less selective doses would have been assessed. Unequivocal resolution of these issues will depend on greater selectivity of ligands for these receptors. The rank order of the in vivo D3 selectivity ratios obtained for these agonists and antagonists (Table 1) is in general agreement with similar determinations reported for in vitro binding studies. The magnitudes of the in vivo selectivities reported herein are much lower than those obtained by in vitro binding studies.
However, similar differences have been reported when in vitro binding and functional assays are compared (Pugsley et al. 1995; Chio et al. 1994; Sautel et al. 1995) and are therefore not surprising. These data suggest that while comparisons of in vitro binding affinities provide an estimation of receptor selectivity, the utilization of in vitro functional assays and behavioral measures may provide a more accurate measure of an agonist or antagonist's selectivity as they allow for both potency and efficacy measures to be made and may therefore be more informative in interpreting the in vivo pharmacology of D2-like agonists and antagonists. To summarize, the results of these studies provide further support for specific roles for the D3 and D2 receptors in the mediation of D2/D3 agonist-induced yawning behavior and hypothermia, respectively, and demonstrate the usefulness of yawning and hypothermia in the characterization of in vivo D3 and D2 receptor activity.
They are the first to provide in vivo determinations and comparisons of D3 receptor selectivities for a series of D2/D3 agonists with a range of in vitro selectivities for the D3 or D2 receptor. Thus, these data suggest that yawning and hypothermia may provide useful endpoints for the evaluation of in vivo antagonist activity and selectivity of future antagonists with improved solubility and selectivities for the D3 or D2 receptor.
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Distribution of dopamine D3 receptor expressing neurons in the human forebrain: comparison with D2 receptor expressing neurons. Gurevich EV, Joyce JN.
Neuropsychopharmacology. 1999;20(1):60-80
Based on studies in the rat, Sokoloff and Schwartz (1990-1995) have made the valuable suggestion that the D3 receptor is a particularly important target for antipsychotics in the mesolimbic DA system. The present study in the human demonstrates that the distribution of D3 receptors and D3 mRNA-bearing neurons is consistent with relative segregation of the D3 subtype to the limbic striatum as well as it primary and secondary targets and many sources of its afferents.
The nucleus accumbens and adjacent ventral putamen, referred to as the ventral or limbic striatum, exhibit the highest expression of D3 receptor binding sites and corresponding levels of D3 mRNA. These regions are the targets of the mesolimbic DA system receiving input from the medial and dorsal components of the SN. Additional sources of input to the limbic striatum include regions enriched in D3 receptors such as the anterior nuclei of the thalamus and NBM. The ventral striatum projects primarily to the VP, the most medial part of the GPi and SNr, which are also enriched with D3 receptors and D3 mRNA positive neurons.
These regions provide input to the anterior nuclei of the thalamus. The AV of the thalamus, which exhibits the highest expression of D3 receptors within the thalamus, receives a nigrothalamic GABAergic innervation from the SNr, a region of relatively higher expression of D3 and D2 receptors. This provides multiple sites at which drugs such as antipsychotics might act to regulate activity in the "limbic" loop via interaction with the D3 receptor. However, in contrast with the rat, in the human brain the D3 receptor is also expressed at high levels in the motor striatum. Other brain regions thought to be involved in sensory (sensory thalamic nuclei), hormonal (mmt) and association (amygdala) functions also express the D3 receptor, often co-expressed with the D2 receptor.
The extensive co-localization of D2 and D3 receptors in human forebrain raises a question about the functional significance of the simultaneous presence of pharmacologically similar DA receptors in the same cell. As has been proposed, the significantly higher affinity of the D3 receptor for DA may enable it to serve in vivo as an extrasynaptic receptor responding to low concentrations of DA at distances remote from the point of DA release. Conversely, the D2 receptor with its low affinity to DA is likely to respond to high synaptic concentrations of DA. Thus, brain areas with relatively high densities of D3 receptors may be under tonic regulatory influence of the brain dopaminergic system, even if the dopaminergic innervation is sparse. D2 and D3 receptors may elicit opposing responses in neurons and may respond differently to loss of DA or antipsychotic treatment.
These data provide initial insight into details of distribution and co-expression of DA D2 and D3 receptors in the human brain. Intricate interplay of signaling by related but not identical DA receptors in different brain areas and individual neurons remains to be elucidated. Perhaps, circuits with high D3 receptor expression and other functionally connected systems with predominance of the D2 receptor provide different target systems for DA antagonists in regulation of psychotic symptoms.
Determination of in vivo dopamine D3 receptor selectivity for agonists and antagonists utilizing induction of yawning and hypothermia in rats.
Gregory Thomas Collins and James Henry Woods
Dept of Pharmacology, University of Michigan Medical School, Ann Arbor, MI, USA
Accurate assessment of in vivo selectivity for preferential D3 agonists and antagonists has been slowed by the absence of behaviors specifically mediated by the D3 receptor as well as the relative lack of agonists and antagonists highly selective for the D3 receptor. We have previously demonstrated that the induction of yawning behavior by D2/D3 agonists is mediated by an agonist activity at the D3 receptor while the inhibition of yawning at higher doses results from a concomitant D2 agonist activity (Collins et al., 2005). In addition to the induction of yawning behavior, all D2/D3 agonists produce significant decreases in core body temperature, an effect that has been shown to be mediated by their D2 agonist activity (Chaperon et al., 2003). We have compared several dopaminergic agonists with a wide range of in vitro D3 selectivity, including; PD-128907, quinelorane, pramipexole, 7-OH-DPAT, quinpirole, apomorphine and sumanirole with respect to their ability to induce yawning behavior and hypothermia. In addition, the D2 antagonist, L-741626, and the D3 antagonists, U99194 and PG01037 have been used to pharmacologically validate the specific involvement of the D3 and D2 receptor in yawning and hypothermia respectively. Therefore, the induction and, at higher doses, suppression of yawning in conjunction with hypothermia allows the inference of an in vivo selectivity ratio for D3 compared to D2 receptor activity. Using this analysis, pramipexole is the most selective of the agonists tested, while apomorphine is the least.