Locomotor inhibition, yawning and vacuous chewing induced by a novel dopamine D2 post-synaptic receptor agonist

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Abstract

The N-n-propyl analog of dihydrexidine ((±)-trans-10,11-dihydroxy-5,6,6a,7,8,12b-hexahydrobenzo[a]phenanthridine) is a dopamine receptor agonist with high affinity for dopamine D2 and D3 receptors (K0.5=26 and 5 nM, respectively). Members of the hexahydrobenzo[a]phenanthridine structural class are atypical because they display high intrinsic activity at post-synaptic dopamine D2 receptors, but low intrinsic activity at dopamine D2 autoreceptors. The present study examined the effects of (±)-N-n-propyl-dihydrexidine on unconditioned behaviors in rats. The most striking results observed were large, dose-dependent decreases in locomotor activity (e.g., locomotor inhibition), and increases in vacuous chewing; yawning was also increased at the highest dose of (±)-N-n-propyl-dihydrexidine. The locomotor inhibition and yawning induced by (±)-N-n-propyl-dihydrexidine were blocked by pre-treatment with (−)-remoxipride (S(−)-3-bromo-N-((1-ethyl-2-pyrrolidinyl)-methyl)-2,6-dimethoxybenzamide), a dopamine D2 receptor antagonist, but not by the dopamine D1 receptor antagonist (+)-SCH23390 (R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine). Vacuous chewing was decreased by both (−)-remoxipride and (+)-SCH23390. These data support the hypothesis that a subpopulation of post-synaptic dopamine D2 receptors has a critical role in decreases in locomotor activity and induction of vacuous chewing and yawning. © 1997 Elsevier Science B.V. All rights reserved.

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 superfamily of G-protein coupled receptors (Dohlman et al., 1987). Currently there are two known pharmacologically similar families of dopamine receptors, usually categorized as D1 and D2 (Garau et al., 1978; Kebabian and Calne, 1979). At least five genes code for unique dopamine receptors, some having splice variants (Gingrich and Caron, 1993). 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 (R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine) and SKF38393 (R(+)-1-phenyl-7,8-dihydroxy-2,3,4,5-tetrahydro-(1H)-3-benzazepine). D2-like dopamine receptors (D2short and D2long 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 post-synaptic 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 (Di Chiara et al., 1976; Strömbom, 1976). 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 (Strömbom, 1976; Walters and Roth, 1976; Westfall et al., 1976; Skirboll et al., 1979; Costall et al., 1981). 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 (e.g., Hjorth et al., 1981; Pugsley et al., 1992; Nisoli et al., 1993).

Yet not all reports are consistent with this autoreceptor hypothesis (e.g., Ståhle and Ungerstedt, 1987; Ståhle, 1992 review). 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 (Waters et al., 1993; Svensson et al., 1994).

Several years ago, we reported on dihydrexidine ((±)-trans-10,11-dihydroxy-5,6,6a,7,8,12b-hexahydrobenzo[a]phenanthridine), the parent compound of a novel class of dopamine receptor agonists (Lovenberg et al., 1989). It is now known that both the D1 and D2 affinity and functional potency reside in the (+)-enantiomer (Knoerzer et al., 1994). 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 (Mottola et al., 1992; unpublished observations). The unique post-synaptically selective pharmacology of hexahydrobenzo[a]phenanthridine ligands is supported further by a lack of agonist effects at impulse-regulating 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 (unpublished observations).

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-n-propyl-dihydrexidine has 10-fold selectivity for native dopamine D2 receptors vs. D1 receptors in rat striatum (K0.5 approx. 26 nM vs. 325 nM, respectively, Mottola et al., 1992), and also has high affinity for the cloned dopamine D3 receptor (K0.5 approx. 5 nM, Watts et al., 1993). 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 unhabituated, 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.

Section snippets

Animals

Male Sprague-Dawley rats (Charles River, 200–300 g) were housed 4–6 per cage under standard conditions (lights on, 06:00–18:00 h; 22–23°C; humidity 30–70%), with food and water available ad libitum. All rats were drug-naive prior to testing and were used in only one experimental session.

Drugs

(±)-N-n-Propyl-dihydrexidine was synthesized according to previously published methods (Brewster et al., 1990). It was dissolved in 0.1% ascorbic acid vehicle, and administered over a dose range (expressed as

Choice of behavioral categories for data presentation

All of the data presented for oral stereotypies (gnawing, sniffing, licking and chewing on object) are derived from tallies of the high intensity, repetitive category of each of these behaviors (i.e., calculated from the number of 15 s intervals where the observer judged the behavior to occur continuously). This decision was based on the greater sensitivity of the continuous (rather than the intermittent) category of these behaviors to the treatments administered. The inactivity data reflect

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

Acknowledgements

Stan Southerland and Allison Eaton are thanked for their excellent technical assistance. This work was supported by PHS grants MH53356, MH40537 and MH42705, and Center Grants HD03310 and MH33127, and Training Grant GM07040, and a research grant from Hoechst Marion Roussel Pharmaceuticals.

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