Nathalie Griffon

Phenotypical characterization of neurons expressing the dopamine D3 receptor in the rat brain

We have established the cellular distribution of the dopamine D3 receptor using tritiated 7-hydroxy-N-N-di-n-propyl-2-aminotetralin and a complementary RNA probe to visualize autoradiographically the protein in binding studies and the gene transcripts by in situ hybridization, respectively. Studies with these two markers confirm the restricted expression of the D3 receptor in few brain areas, i.e. mainly the ventral striatal complex, the substantia nigra-ventral tegmental area and the cerebellum. In nucleus accumbens, the D3 receptor was mainly expressed in medium-sized neurons of the rostral pole and ventromedial shell subdivisions, but not of the core or septal pole, i.e. accumbal subdivisions expressing the D2 receptor. In the ventromedial shell, about 60% of the D3 receptor-expressing neurons were neurotensin neurons, presumably projecting to the ventral pallidum. In the islands of Calleja, both D3 receptor binding and messenger RNA were abundant in the entire population of granule cells. These cells are known to make sparse contacts with dopaminergic axons and also to express the D1 receptor. In the mesencephalon, low levels of D3 messenger RNA were detected in few dopamine neurons of substantia nigra pars lateralis and ventral tegmental area. In addition, some D3 receptor binding but not messenger RNA was detected in medial substantia nigra and lateral ventral tegmental area, where the receptor is presumably located presynaptically on afferents. In the archicerebellum, Purkinje cell perikarya in lobules 9 and 10 expressed the D3 receptor messenger RNA, whereas binding sites were found in the molecular layer, where corresponding dendrites but no known dopaminergic projection from mesencephalon are found. The occurrence of D3 receptor gene expression in some brain areas receiving low dopamine innervation supports the hypothesis that this receptor may mediate non-synaptic actions of dopamine.

A functional test identifies dopamine agonists selective for D3 versus D2 receptors.

The functional potency of a series of dopamine agonists for increasing mitogenesis, measured by incorporation of [3H]thymidine, was established in transfected cell lines expressing human D2 or D3 receptors. The functional selectivity of agonists markedly differs from their binding selectivity. (+)7-OH-DPAT, pramipexole, quinerolane and PD 128,907, the most D3 receptor-selective compounds in binding studies, were 7, 15, 21 and 54 times more potent, respectively, at the D3 than at the D2 receptor in the functional test. Bromocriptine displayed a 10-fold functional selectivity toward the D2 receptor. The known behavioural actions of D3 selective agonists support a role for the D3 receptor in motor inhibitions, which should be taken into account for the treatment of motor dysfunctions by dopamine agonists.

Coexpression of dopamine D1 and D3 receptors in islands of Calleja and shell of nucleus accumbens of the rat: opposite and synergistic functional interactions

Using double in situ hybridization, we found extensive coexpression of dopamine D1 and D3 receptor (D1R and D3R) mRNAs in neurons of the island of Calleja major (ICjM) and ventromedial shell of nucleus accumbens (ShV), respectively. Thus, at least 79 and 63% of D3R mRNA‐expressing neurons in ICjM and ShV also expressed the D1R mRNA. Coexpression of D1R and D3R mRNAs was found to occur in substance P (SP) mRNA‐expressing neurons in both areas, suggesting SP mRNA as a marker of the activity of coexpressing neurons. Administration of SKF 38393, a D1R receptor agonist, increased c‐fos mRNA in ICjM, whereas administration of quinpirole, a D2R/D3R agonist, decreased it; SCH 23390, a D1R antagonist and nafadotride, a preferential D3R antagonist, given alone, had effects opposite to those of the corresponding agonists. These data indicate that basal c‐fos expression in ICjM is maintained by endogenous dopamine acting tonically upon two receptor subtypes subserving opposite effects on the same cell. However, in ShV, whereas SKF 38393 also increased c‐fos mRNA, quinpirole had no effect, a difference presumably reflecting the lower fraction of neurons coexpressing D1R and D3R in this area. In contrast, in ShV from reserpine‐treated rats, SKF 38393 increased SP mRNA and quinpirole potentiated this effect. These contrasting interactions of D1R‐ and D3R‐mediated signalling events, i.e. in either opposite or synergistic directions, most likely occurring at the single cell level, may serve to increase the dopamine response threshold of the target cells in ICjM and to maintain a strong tonic activity of ShV neurons.

Functional implications of multiple dopamine receptor subtypes: the D1/D3 receptor coexistence

The D3 dopamine receptor, a D2-like receptor, is selectively expressed in the ventral striatum, particularly in the shell of nucleus accumbens and islands of Calleja, where it is found in medium sized substance P neurons. The latter co-express the D1 receptor whose interaction with the D3 receptor was studied by treating rats with selective agonists and antagonists. In agreement with the opposite cAMP response, they mediate in cultured neuroblastoma cells, the D1 and D3 receptors exerted opposite influences on c-fos expression in islands of Calleja. However, in agreement with the synergistic influence of cAMP on D3 receptor-mediated mitogenesis on the same cultured cells, D1 and D3 receptor stimulation in vivo synergistically enhanced preprotachykinin mRNA in the shell of accumbens. This indicates that the two receptor subtypes may affect neurons in either synergy or opposition according to the cell or signal generated. Levodopa-induced behavioral sensitization in hemiparkinsonian rats is another example of D1/D3 receptor interaction. Hence repeated levodopa administration induces the ectopic appearance of the D3 receptor in substance P/dynorphin, striatonigral neurons of the dorsal striatum. This induction is secondary to D1 receptor stimulation in neurons of the denervated side and fully accounts for the sensitization, i.e. the increased behavioral responsiveness to levodopa. During brain development, a similar process could operate to control the late appearance of the D3 receptor in D1-receptor bearing neurons of the ventral striatum at a time at which they start to be innervated by dopamine neurons. Finally, taking into account a variety of genetic, developmental, neuroimaging and pharmacological data, we postulate that imbalances between the levels of D1 and D3 receptors in the same neurons could be responsible for schizophrenic disorders.

Molecular determinants of glycine receptor subunit assembly.

The inhibitory glycine receptor (GlyR) is a pentameric transmembrane protein composed of homologous α and β subunits. Single expression of α subunits generates functional homo‐oligomeric GlyRs, whereas the β subunit requires a co‐expressed α subunit to assemble into hetero‐oligomeric channels of invariant stoichiometry (α3β2). Here, we identified eight amino acid residues within the N‐terminal region of the α1 subunit that are required for the formation of homo‐oligomeric GlyR channels. We show that oligomerization and N‐glycosylationq of the α1 subunit are required for transit from the endoplasmic reticulum to the Golgi apparatus and later compartments, and that addition of simple carbohydrate side chains occurs prior to GlyR subunit assembly. Our data are consistent with both intersubunit surface and conformational differences determining the different assembly behaviour of GlyR α and β subunits.

Opposing roles for dopamine D2 and D3 receptors on neurotensin mRNA expression in nucleus accumbens.

Using in situ hybridization histochemistry in rat nucleus accumbens, we show that the dopamine D3 receptor mRNA is expressed in the ventromedial part of the shell subdivision, where its gross distribution matches that of neurotensin mRNA. In addition, hybridization studies at the cellullar level show that a large fraction of the neurotensin neurons co‐express the D3 receptor mRNA in this restricted area. In contrast, the dopamine D2 receptor mRNA is expressed mainly in the core and marginally in the shell, at the level of the cone. In rats treated by haloperidol and sulpiride, two D2‐like receptor antagonists, but not by SCH 23390, a D1‐like receptor antagonist, proneurotensin mRNA was increased in the D2 receptor mRNA‐rich areas but decreased in the D3 receptor mRNA‐rich areas. This suggests that the D2 and D3 receptors control neurotensin mRNA expression negatively and positively, respectively.

Glyceraldehyde-3-phosphate dehydrogenase is a GABAA receptor kinase linking glycolysis to neuronal inhibition

Protein phosphorylation is crucial for regulating synaptic transmission. We describe a novel mechanism for the phosphorylation of the GABAA receptor, which mediates fast inhibition in the brain. A protein copurified and coimmunoprecipitated with the phosphorylated receptor α1 subunit; this receptor-associated protein was identified by purification and microsequencing as the key glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Molecular constructs demonstrated that GAPDH directly phosphorylates the long intracellular loop of GABAA receptor α1 subunit at identified serine and threonine residues. GAPDH and the α1 subunit were found to be colocalized at the neuronal plasma membrane. In keeping with the GAPDH/GABAA receptor molecular association, glycolytic ATP produced locally at plasma membranes was consumed for this α1 subunit phosphorylation, possibly within a single macrocomplex. The membrane-attached GAPDH is thus a dual-purpose enzyme, a glycolytic dehydrogenase, and a receptor-associated kinase. In acutely dissociated cortical neurons, the rundown of the GABAA responses was essentially attributable to a Mg2+-dependent phosphatase activity, which was sensitive to vanadate but insensitive to okadaic acid or fluoride. Rundown was significantly reduced by the addition of GAPDH or its reduced cofactor NADH and nearly abolished by the addition of its substrate glyceraldehyde-3-phosphate (G3P). The prevention of rundown by G3P was abolished by iodoacetamide, an inhibitor of the dehydrogenase activity of GAPDH, indicating that the GABAA responses are maintained by a glycolysis-dependent phosphorylation. Our results provide a molecular mechanism for the direct involvement of glycolysis in neurotransmission.

Ubiquitination Precedes Internalization and Proteolytic Cleavage of Plasma Membrane-bound Glycine Receptors

The inhibitory glycine receptor (GlyR) in developing spinal neurones is internalized efficiently upon antagonist inhibition. Here we used surface labeling combined with affinity purification to show that homopentameric α1 GlyRs generated inXenopus oocytes are proteolytically nicked into fragments of 35 and 13 kDa upon prolonged incubation. Nicked GlyRs do not exist at the cell surface, indicating that proteolysis occurs exclusively in the endocytotic pathway. Consistent with this interpretation, elevation of the lysosomal pH, but not the proteasome inhibitor lactacystin, prevents GlyR cleavage. Prior to internalization, α1 GlyRs are conjugated extensively with ubiquitin in the plasma membrane. Our results are consistent with ubiquitination regulating the endocytosis and subsequent proteolysis of GlyRs residing in the plasma membrane. Ubiquitin-conjugating enzymes thus may have a crucial role in synaptic plasticity by determining postsynaptic receptor numbers.

Globin and Globin Gene Structure of the Nerve Myoglobin of Aphrodite aculeata

The globin of the nerve cord of the polychaete annelid Aphrodite aculeata was isolated and purified to homogeneity. The native molecule has a pI of 6.3 and acts as a dimer of two identical Mr 15,644.5 polypeptide chains as determined by electrospray mass spectrometry. It has an average affinity for oxygen (P50 = 1.24 torr) resulting from fast association (kon = 170 × 106 M−1·s−1) and dissociation rates (koff = 360 s−1). The partial primary structure of this nerve globin was determined at the protein level and completed and confirmed by translation of the cDNA sequence. The globin chain has 150 amino acid residues and a calculated Mr of 15,602.69 strongly suggesting that the amino terminus is acetylated. The absence of a leader sequence and the lack of Cys at the positions NA2 and H9 needed for the formation of the high Mr complexes found in extracellular annelid globins classify the Aphrodite globin with the cellular globin species. The Aphrodite nerve globin is unlikely to represent a separate globin family, as cDNA derived primers detect globin messenger RNA in muscle, gut, and pharynx tissue as well. The gene encoding this globin species is interrupted by a single intron, inserted at position G7.0. Comparison to other globin gene structures strongly suggest that introns can be lost independently, rather than simultaneously as a result of a single conversion event as suggested previously (Lewin, R. (1984) Science 226, 328).

Two Intracellular Signaling Pathways for the Dopamine D3 Receptor: Opposite and Synergistic Interactions with Cyclic AMP

Abstract: As cerebral neurons express the dopamine D1 receptor positively coupled with adenylyl cyclase, together with the D3 receptor, we have investigated in a heterologous cell expression system the relationships of cyclic AMP with D3 receptor signaling pathways. In NG108‐15 cells transfected with the human D3 receptor cDNA, dopamine, quinpirole, and other dopamine receptor agonists inhibited cyclic AMP accumulation induced by forskolin. Quinpirole also increased mitogenesis, assessed by measuring [3H]thymidine incorporation. This effect was blocked partially by genistein, a tyrosine kinase inhibitor. Forskolin enhanced by 50–75% the quinpirole‐induced [3H]thymidine incorporation. This effect was maximal with 100 nM forskolin, occurred after 6–16 h, was reproduced by cyclic AMP‐permeable analogues, and was blocked by a protein kinase A inhibitor. Forskolin increased D3 receptor expression up to 135%, but only after 16 h and at concentrations of >1 µM. Thus, in this cell line, the D3 receptor uses two distinct signaling pathways: it efficiently inhibits adenylyl cyclase and induces mitogenesis, an effect possibly involving tyrosine phosphorylation. Activation of the cyclic AMP cascade potentiates the D3 receptor‐mediated mitogenic response, through phosphorylation by a cyclic AMP‐dependent kinase of a yet unidentified component. Hence, transduction of the D3 receptor can involve both opposite and synergistic interactions with cyclic AMP.