Histamine Recycling Is Mediated by CarT, a Carcinine Transporter in Drosophila Photoreceptors.

Histamine is an important chemical messenger that regulates multiple physiological processes in both vertebrate and invertebrate animals. Even so, how glial cells and neurons recycle histamine remains to be elucidated. Drosophila photoreceptor neurons use histamine as a neurotransmitter, and the released histamine is recycled through neighboring glia, where it is conjugated to ß-alanine to form carcinine. However, how carcinine is then returned to the photoreceptor remains unclear. In an mRNA-seq screen for photoreceptor cell-enriched transporters, we identified CG9317, an SLC22 transporter family protein, and named it CarT (Carcinine Transporter). S2 cells that express CarT are able to take up carcinine in vitro. In the compound eye, CarT is exclusively localized to photoreceptor terminals. Null mutations of cart alter the content of histamine and its metabolites. Moreover, null cart mutants are defective in photoreceptor synaptic transmission and lack phototaxis. These findings reveal that CarT is required for histamine recycling at histaminergic photoreceptors and provide evidence for a CarT-dependent neurotransmitter trafficking pathway between glial cells and photoreceptor terminals.

The metabolism of histamine in the Drosophila optic lobe involves an ommatidial pathway: ß-alanine recycles through the retina.

Flies recycle the photoreceptor neurotransmitter histamine by conjugating it to ß-alanine to form ß-alanyl-histamine (carcinine). The conjugation is regulated by Ebony, while Tan hydrolyses carcinine, releasing histamine and ß-alanine. In Drosophila, ß-alanine synthesis occurs either from uracil or from the decarboxylation of aspartate but detailed roles for the enzymes responsible remain unclear. Immunohistochemically detected ß-alanine is present throughout the fly's entire brain, and is enhanced in the retina especially in the pseudocone, pigment and photoreceptor cells of the ommatidia. HPLC determinations reveal 10.7 ng of ß-alanine in the wild-type head, roughly five times more than histamine. When wild-type flies drink uracil their head ß-alanine increases more than after drinking l-aspartic acid, indicating the effectiveness of the uracil pathway. Mutants of black, which lack aspartate decarboxylase, cannot synthesize ß-alanine from l-aspartate but can still synthesize it efficiently from uracil. Our findings demonstrate a novel function for pigment cells, which not only screen ommatidia from stray light but also store and transport ß-alanine and carcinine. This role is consistent with a ß-alanine-dependent histamine recycling pathway occurring not only in the photoreceptor terminals in the lamina neuropile, where carcinine occurs in marginal glia, but vertically via a long pathway that involves the retina. The lamina's marginal glia are also a hub involved in the storage and/or disposal of carcinine and ß-alanine.

Key modulatory role of presynaptic adenosine A2A receptors in cortical neurotransmission to the striatal direct pathway.

Basal ganglia processing results from a balanced activation of direct and indirect striatal efferent pathways, which are controlled by dopamine D1 and D2 receptors, respectively. Adenosine A2A receptors are considered novel antiparkinsonian targets, based on their selective postsynaptic localization in the indirect pathway, where they modulate D2 receptor function. The present study provides evidence for the existence of an additional, functionally significant, segregation of A2A receptors at the presynaptic level. Using integrated anatomical, electrophysiological, and biochemical approaches, we demonstrate that presynaptic A2A receptors are preferentially localized in cortical glutamatergic terminals that contact striatal neurons of the direct pathway, where they exert a selective modulation of corticostriatal neurotransmission. Presynaptic striatal A2A receptors could provide a new target for the treatment of neuropsychiatric disorders.

5-HT 1B receptor-mediated serotoninergic modulation of methylphenidate-induced locomotor activation in rats.

Previous studies have shown that the dopamine (DA) uptake blocker methylphenidate, a psychostimulant widely used for the treatment of attention-deficit hyperactivity disorder (ADHD), prevents the neurotoxic effects of the highly abused DA releaser methamphetamine. However, there is a lack of information about the pharmacological interactions of these two drugs at the behavioral level. When systemically administered within an interval of 2 h, previous administration of methylphenidate (10 mg/kg, intraperitoneal (i.p.)) did not modify locomotor activation induced by methamphetamine. On the other hand, previous administration of methamphetamine (1 mg/kg, i.p.) markedly potentiated methylphenidate-induced motor activation. With in vivo microdialysis experiments, methamphetamine and methylphenidate were found to increase DA extracellular levels in the nucleus accumbens (NAs). Methamphetamine, but not methylphenidate, significantly increased the extracellular levels of serotonin (5-HT) in the NAs. Methamphetamine-induced 5-HT release remained significantly elevated for more than 2 h after its administration, suggesting that the increased 5-HT could be responsible for the potentiation of methylphenidate-induced locomotor activation. In fact, previous administration of the 5-HT uptake blocker fluoxetine (10 mg/kg, i.p.) also potentiated the motor activation induced by methylphenidate. A selective 5-HT 1B receptor antagonist (GR 55562; 1 mg/kg), but not a 5-HT2 receptor antagonist (ritanserin; 2 mg/kg, i.p.), counteracted the effects of methamphetamine and fluoxetine on the motor activation induced by methylphenidate. Furthermore, a 5-HT 1B receptor agonist (CP 94253; 1-10 mg/kg, i.p.) strongly and dose-dependently potentiated methylphenidate-induced locomotor activation. The 5-HT 1B receptor-mediated modulation of methylphenidate-induced locomotor activation in rat could have implications for the treatment of ADHD.

Adenosine A1-A2A receptor heteromers: new targets for caffeine in the brain.

The contribution of blockade of adenosine A1 and A2A receptor to the psychostimulant effects of caffeine is still a matter of debate. When analyzing motor activity in rats, acutely administered caffeine shows a profile of a non-selective adenosine receptor antagonist, although with preferential A1 receptor antagonism. On the other hand, tolerance to the effects of A1 receptor blockade seems to be mostly responsible for the tolerance to the motor-activating effects of caffeine, while the residual motor-activating effects of caffeine in tolerant individuals seem to involve A2A receptor blockade. These behavioral studies correlate with in vivo microdialysis experiments that suggest that A1 receptor-mediated modulation of striatal glutamate release is involved in the psychostimulant effects of caffeine. Experiments in transfected cells demonstrate the ability of A1 receptors to heteromerize with A2A receptors and the A1-A2A receptor heteromer can be biochemically identified in the striatum, in striatal glutamatergic terminals. The striatal A1-A2A receptor heteromer provides a "concentration-dependent switch" mechanism by which low and high concentrations of synaptic adenosine produce the opposite effects on glutamate release. The analysis of the function of A1-A2A receptor heteromers during chronic treatment with caffeine gives new clues about the well-known phenomenon of tolerance to the psychostimulant effects of caffeine.

Location and functions of Inebriated in the Drosophila eye.

Histamine (HA) is a neurotransmitter in arthropod photoreceptors. It is recycled via conjugation to ß-alanine to form ß-alanylhistamine (carcinine). Conjugation occurs in epithelial glia that surround photoreceptor terminals in the first optic neuropil, and carcinine (CA) is then transported back to photoreceptors and cleaved to liberate HA and ß-alanine. The gene Inebriated (Ine) encodes an Na+/Cl--dependent SLC6 family transporter translated as two protein isoforms, long (P1) and short (P2). Photoreceptors specifically express Ine-P2 whereas Ine-P1 is expressed in non-neuronal cells. Both ine1 and ine3 have significantly reduced head HA contents compared with wild type, and a smaller increase in head HA after drinking 1% CA. Similarly, uptake of 0.1% CA was reduced in ine1 and ine3 mutant synaptosomes, but increased by 90% and 84% respectively for fractions incubated in 0.05% ß-Ala, compared with wild type. Screening potential substrates in Ine expressing Xenopus oocytes revealed very little response to carcinine and ß-Ala but increased conductance with glycine. Both ine1 and ine3 mutant responses in light-dark phototaxis did not differ from wild-type. Collectively our results suggest that Inebriated functions in an adjunct role as a transporter to the previously reported carcinine transporter CarT.

Presynaptic control of striatal glutamatergic neurotransmission by adenosine A1-A2A receptor heteromers.

The functional role of heteromers of G-protein-coupled receptors is a matter of debate. In the present study, we demonstrate that heteromerization of adenosine A1 receptors (A1Rs) and A2A receptors (A2ARs) allows adenosine to exert a fine-tuning modulation of glutamatergic neurotransmission. By means of coimmunoprecipitation, bioluminescence and time-resolved fluorescence resonance energy transfer techniques, we showed the existence of A1R-A2AR heteromers in the cell surface of cotransfected cells. Immunogold detection and coimmunoprecipitation experiments indicated that A1R and A2AR are colocalized in the same striatal glutamatergic nerve terminals. Radioligand-binding experiments in cotransfected cells and rat striatum showed that a main biochemical characteristic of the A1R-A2AR heteromer is the ability of A2AR activation to reduce the affinity of the A1R for agonists. This provides a switch mechanism by which low and high concentrations of adenosine inhibit and stimulate, respectively, glutamate release. Furthermore, it is also shown that A1R-A2AR heteromers constitute a unique target for caffeine and that chronic caffeine treatment leads to modifications in the function of the A1R-A2AR heteromer that could underlie the strong tolerance to the psychomotor effects of caffeine.

Heteromeric nicotinic acetylcholine-dopamine autoreceptor complexes modulate striatal dopamine release.

In the striatum, dopamine and acetylcholine (ACh) modulate dopamine release by acting, respectively, on dopamine D(2) autoreceptors and nicotinic ACh (nACh) heteroreceptors localized on dopaminergic nerve terminals. The possibility that functional interactions exist between striatal D(2) autoreceptors and nACh receptors was studied with in vivo microdialysis in freely moving rats. Local perfusion of nicotine in the ventral striatum (shell of the nucleus accumbens) produced a marked increase in the extracellular levels of dopamine, which was completely counteracted by co-perfusion with either the non-alpha(7) nACh receptor antagonist dihydro-beta-erythroidine or the D(2-3) receptor agonist quinpirole. Local perfusion of the D(2-3) receptor antagonist raclopride produced an increase in the extracellular levels of dopamine, which was partially, but significantly, counteracted by coperfusion with dihydro-beta-erythroidine. These findings demonstrate a potent crosstalk between G protein-coupled receptors and ligand-gated ion channels in dopaminergic nerve terminals, with the D(2) autoreceptor modulating the efficacy of non-alpha(7) nACh receptor-mediated modulation of dopamine release. We further demonstrate physical interactions between beta(2) subunits of non-alpha(7) nicotinic acetylcholine receptors and D(2) autoreceptors in co-immunoprecipitation experiments with membrane preparations from co-transfected mammalian cells and rat striatum. These results reveal that striatal non-alpha(7) nicotinic acetylcholine receptors form part of heteromeric dopamine autoreceptor complexes that modulate dopamine release.

tan and ebony genes regulate a novel pathway for transmitter metabolism at fly photoreceptor terminals.

In Drosophila melanogaster, ebony and tan, two cuticle melanizing mutants, regulate the conjugation (ebony) of beta-alanine to dopamine or hydrolysis (tan) of the beta-alanyl conjugate to liberate dopamine. beta-alanine biosynthesis is regulated by black. ebony and tan also exert unexplained reciprocal defects in the electroretinogram, at ON and OFF transients attributable to impaired transmission at photoreceptor synapses, which liberate histamine. Compatible with this impairment, we show that both mutants have reduced histamine contents in the head, as measured by HPLC, and have correspondingly reduced numbers of synaptic vesicles in their photoreceptor terminals. Thus, the histamine phenotype is associated with sites of synaptic transmission at photoreceptors. We demonstrate that when they receive microinjections into the head, wild-type Sarcophaga bullata (in whose larger head such injections are routinely possible) rapidly (<5 sec) convert exogenous [3H]histamine into its beta-alanine conjugate, carcinine, a novel metabolite. Drosophila tan has an increased quantity of [3H]carcinine, the hydrolysis of which is blocked; ebony lacks [3H]carcinine, which it cannot synthesize. Confirming these actions, carcinine rescues the histamine phenotype of ebony, whereas beta-alanine rescues the carcinine phenotype of black;tan double mutants. The equilibrium ratio between [3H]carcinine and [3H]histamine after microinjecting wild-type Sarcophaga favors carcinine hydrolysis, increasing to only 0.5 after 30 min. Our findings help resolve a longstanding conundrum of the involvement of tan and ebony in photoreceptor function. We suggest that reversible synthesis of carcinine occurs in surrounding glia, serving to trap histamine after its release at photoreceptor synapses; subsequent hydrolysis liberates histamine for reuptake.

Role of adenosine in the control of homosynaptic plasticity in striatal excitatory synapses.

Long-lasting, activity-dependent changes in synaptic efficacy at excitatory synapses are critical for experience-dependent synaptic plasticity. Synaptic plasticity at excitatory synapses is determined both presynaptically by changes in the probability of neurotransmitter release, and postsynaptically by changes in the availability of functional postsynaptic glutamate receptors. Two kinds of synaptic plasticity have been described. In homosynaptic or Hebbian plasticity, the events responsible for synaptic strengthening occur at the same synapse as is being strengthened. Homosynaptic plasticity is activity-dependent and associative, because it associates the firing of a postsynaptic neuron with that of the presynaptic neuron. Heterosynaptic plasticity, on the other hand, is activity-independent and the synaptic strength is modified as a result of the firing of a third, modulatory neuron. It has been suggested that long-term changes in synaptic strength, which are associated with gene transcription, can only be induced with the involvement of heterosynaptic plasticity. The neuromodulator adenosine plays an elaborated pre- and postsynaptic control of glutamatergic neurotransmission. This paper reviews the evidence suggesting that in some striatal excitatory synapses, adenosine can provide the heterosynaptic-like modulation essential for stabilizing homosynaptic plasticity without the need of a "third, modulatory neuron".

Differential glutamate-dependent and glutamate-independent adenosine A1 receptor-mediated modulation of dopamine release in different striatal compartments.

Adenosine and dopamine are two important modulators of glutamatergic neurotransmission in the striatum. However, conflicting reports exist about the role of adenosine and adenosine receptors in the modulation of striatal dopamine release. It has been previously suggested that adenosine A(1) receptors localized in glutamatergic nerve terminals indirectly modulate dopamine release, by their ability to modulate glutamate release. In the present study, using in vivo microdialysis, we provide evidence for the existence of a significant glutamate-independent tonic modulation of dopamine release in most of the analyzed striatal compartments. In the dorsal, but not in the ventral, part of the shell of the nucleus accumbens (NAc), blockade of A(1) receptors by local perfusion with the selective A(1) receptor antagonist 8-cyclopentyl-1,3-dimethyl-xanthine or by systemic administration of the non-selective adenosine antagonist caffeine induced a glutamate-dependent release of dopamine. On the contrary, A(1) receptor blockade induced a glutamate-independent dopamine release in the core of the NAc and the nucleus caudate-putamen. Furthermore, using immunocytochemical and functional studies in rat striatal synaptosomes, we demonstrate that a fraction of striatal dopaminergic terminals contains adenosine A(1) receptors, which directly inhibit dopamine release independently of glutamatergic transmission.

Adenosine receptor-mediated modulation of dopamine release in the nucleus accumbens depends on glutamate neurotransmission and N-methyl-D-aspartate receptor stimulation.

Adenosine, by acting on adenosine A(1) and A(2A) receptors, exerts opposite modulatory roles on striatal extracellular levels of glutamate and dopamine, with activation of A(1) inhibiting and activation of A(2A) receptors stimulating glutamate and dopamine release. Adenosine-mediated modulation of striatal dopaminergic neurotransmission could be secondary to changes in glutamate neurotransmission, in view of evidence for a preferential colocalization of A(1) and A(2A) receptors in glutamatergic nerve terminals. By using in vivo microdialysis techniques, local perfusion of NMDA (3, 10 microm), the selective A(2A) receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS 21680; 3, 10 microm), the selective A(1) receptor antagonist 8-cyclopentyl-1,3-dimethylxanthine (CPT; 300, 1000 microm), or the non-selective A(1)-A(2A) receptor antagonist in vitro caffeine (300, 1000 microm) elicited significant increases in extracellular levels of dopamine in the shell of the nucleus accumbens (NAc). Significant glutamate release was also observed with local perfusion of CGS 21680, CPT and caffeine, but not NMDA. Co-perfusion with the competitive NMDA receptor antagonist dl-2-amino-5-phosphonovaleric acid (APV; 100 microm) counteracted dopamine release induced by NMDA, CGS 21680, CPT and caffeine. Co-perfusion with the selective A(2A) receptor antagonist MSX-3 (1 microm) counteracted dopamine and glutamate release induced by CGS 21680, CPT and caffeine and did not modify dopamine release induced by NMDA. These results indicate that modulation of dopamine release in the shell of the NAc by A(1) and A(2A) receptors is mostly secondary to their opposite modulatory role on glutamatergic neurotransmission and depends on stimulation of NMDA receptors. Furthermore, these results underscore the role of A(1) vs. A(2A) receptor antagonism in the central effects of caffeine.