Elsevier

Neuropharmacology

Volume 50, Issue 5, April 2006, Pages 595-605
Neuropharmacology

Expression of D2 receptor isoforms in cultured neurons reveals equipotent autoreceptor function

https://doi.org/10.1016/j.neuropharm.2005.11.010Get rights and content

Abstract

Alternative splicing of the dopamine D2 receptor gene produces two distinct isoforms referred to as D2long (D2L) and D2short (D2S). In mesencephalic dopamine neurons, inhibition of the firing rate through activation of somatodendritic D2 receptors and blockade of neurotransmitter release through stimulation of terminal D2 receptors represent major roles of D2 autoreceptors. Recently, data obtained from D2L-deficient mice suggested that D2S acts as the preferential D2 autoreceptor. In the present study, we investigate whether this D2 isoform-specific autoreceptor function is linked to differences in the subcellular localization and/or signaling properties of the D2S and D2L using mesencephalic neurons transfected with enhanced green fluorescent protein (EGFP)-tagged receptors. Our results show that EGFP-tagged D2S and D2L are localized to the axonal and somatodendritic compartments of mesencephalic neurons. In addition, we demonstrate that EGFP-tagged D2S and D2L regulate cellular excitability, neurotransmitter release and basal levels of intracellular calcium with similar effectiveness. Overall, our morphological and electrophysiological studies suggest that the major D2 autoreceptor function attributed to D2S is likely explained by the predominant expression of this isoform in dopamine neurons rather than by distinct subcellular localization and signaling properties of D2S and D2L.

Introduction

Deregulation of dopaminergic transmission has been implicated in a variety of neurological and psychiatric disorders including Parkinson's disease, schizophrenia, drug dependence and Tourette's syndrome (Missale et al., 1998). Dopamine acts through five transmembrane G-protein coupled receptors (GPCRs). The dopamine receptors can be divided in two subclasses: D1-like (D1 and D5) and D2-like (D2, D3 and D4) (Missale et al., 1998). The dopamine D2 receptor plays a key role in the regulation of dopaminergic transmission (Mercuri et al., 1997, L'hirondel et al., 1998). It acts as the primary autoreceptor leading to rapid inhibition of dopamine release (May and Wightman, 1989, Benoit-Marand et al., 2001).

Alternative gene splicing generates two distinct isoforms of the dopamine D2 receptor referred to as D2long (D2L) and D2short (D2S) (Bunzow et al., 1988, Dal Toso et al., 1989). D2L distinguishes itself from D2S only by the presence of a 29 amino acid insert within the third intracellular loop (Bunzow et al., 1988, Dal Toso et al., 1989). Because the third intracellular loop plays a central role in the coupling of GPCRs to their effectors, the existence of alternatively spliced D2 receptors imply a role for the 29 amino acid insert in the functional diversity of the two isoforms. Although D2S and D2L share the same pharmacological profile (Leysen et al., 1993, Castro and Strange, 1993) recent studies performed in heterologous cell lines have evaluated the possibility that the two isoforms display distinct G protein coupling properties. In fact, studies have reported that D2S and D2L display differential abilities to regulate adenylyl cyclase activity and voltage-gated channels (Montmayeur et al., 1993, Senogles, 1994, Liu et al., 1994, Guiramand et al., 1995, Liu et al., 1996, Liu et al., 1999, Wolfe and Morris, 1999). Moreover, D2S and D2L are differently regulated by protein kinase C (PKC) (Liu et al., 1992, Liu et al., 1994, Choi et al., 1999). In light of these results, it is hypothesized that D2S and D2L may control different transduction pathways and physiological roles in neurons.

Recently, D2L knockout (D2L-KO) mice (Usiello et al., 2000) were used to investigate the functional role of the D2 receptor splice variants in vivo (Usiello et al., 2000, Wang et al., 2000). Inhibition of locomotor activity by low doses of D2 agonists, blockade of dopamine neuron firing by activation of somatodendritic receptors and reduction of dopamine release in response to activation of terminal D2 receptors are unchanged in D2L-KO in comparison to wild type mice. Because these agonist actions are mediated by autoreceptor activation, studies using D2L-KO mice have implicated D2S as the major dopamine autoreceptor. In support of this idea, haloperidol-induced catalepsy, which is mediated by a blockade of postsynaptic D2 receptors, is significantly reduced in D2L-KO mice suggesting an important postsynaptic role for D2L. However, it remains unclear whether the isoform-specific presynaptic and postsynaptic roles are due simply to a preferential splicing in dopamine neurons leading to higher expression of D2S (Khan et al., 1998), or whether D2S exhibits distinctive functional characteristics allowing this isoform to mediate autoreceptor function. In addition, it is important to consider separately the somatodendritic and terminal autoreceptor functions of the D2 receptor. The specific implication of D2S and D2L in autoreceptor function in these two compartments has not been directly explored. However, both isoforms appear to contribute to the somatodendritic effects of D2 agonists on GABAergic neurons of the striatum (Centonze et al., 2004).

In this report, rat primary mesencephalic neurons transfected with enhanced green fluorescent protein (EGFP)-tagged D2 receptor isoforms were used to determine whether D2S and D2L are differentially targeted to somatodendritic and axonal compartments and coupled to various functional effectors. We find that D2S and D2L can be localized to somatodendritic and axonal compartments. Moreover, our studies show that they are equipotent in inhibiting neuronal firing and neurotransmitter release.

Section snippets

Plasmids

pEGFP-N3 was obtained from BD Biosciences Clontech (Palo Alto, CA, USA). Enhanced green fluorescent protein (EGFP)-tagged human D2S and D2L expression constructs were prepared in pCMV5. Briefly, the DNA sequence coding for a modified version of a cleavable signal peptide (M-K-T-I-A-L-S-Y-I-F-C-L-V-F-A) fused to EGFP was introduced at the N-terminus of the D2S and D2L isoforms using an overlap PCR-based methodology (Sedaghat, Nantel, Ginsberg, Lalonde and Tiberi, submitted).

Cell culture

Primary cultures of

Subcellular localization of dopamine D2S and D2L receptors

The ligand binding affinities and coupling to the inhibition of adenylyl cyclase of EGFP-tagged D2S and D2L receptors were similar and not impaired when compared with their wild-type counterparts using heterologous expression in human embryonic kidney 293 cells (Sedaghat et al., submitted). All subsequent experiments were performed in postnatal rat mesencephalic neurons in primary culture. Expression of EGFP-D2 fusion proteins was detected by immunocytochemistry using an anti-D2 antibody (Fig. 1

Discussion

In the present study, we expressed EGFP-tagged dopamine D2 receptor isoforms in primary cultured neurons to compare their relative efficacy as somatodendritic and terminal receptors. We found that D2S and D2L are equipotent in the regulation of neuronal excitability and neurotransmitter release, two well-known functions associated with somatodendritic and terminal D2 autoreceptors, respectively. These results are consistent with the subcellular localization observed for each isoform. Indeed, we

Acknowledgements

We would like to thank François Michel and Caroline Forget for their help in establishing the transfection protocol. This work was funded by grants from the Canadian Health Research Institutes to L.-E.T. (MOP-49591) and the Ontario Mental Health Foundation to M.T. L.-E.T. is a senior scholar of the Fonds de la Recherche en Santé du Québec.

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