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A main rationale for the role of G protein-coupled receptor (GPCR) heteromers as targets for drug development is the putative ability of selective ligands for specific GPCRs to change their pharmacological properties upon GPCR heteromerization. The present study provides a proof of concept for this rationale by demonstrating that heteromerization of dopamine Dsub1/suband Dsub3/subreceptors (Dsub1/subR and Dsub3/subR) influences the pharmacological properties of three structurally similar selective dopamine Dsub3/subR ligands, the phenylpiperazine derivatives PG01042, PG01037 and VK4-116. By using Dsub1/subR-Dsub3/subR heteromer-disrupting peptides, it could be demonstrated that the three Dsub3/subR ligands display different Dsub1/subR-Dsub3/subR heteromer-dependent pharmacological properties: PG01042, acting as G protein-biased agonist, counteracted Dsub1/subR-mediated signaling in the Dsub1/subR-Dsub3/subR heteromer; PG01037, acting as a Dsub3/subR antagonist cross-antagonized Dsub1/subR-mediated signaling in the Dsub1/subR-Dsub3/subR heteromer; and VK4-116 specifically acted as a ß-arrestin-biased agonist in the Dsub1/subR-Dsub3/subR heteromer. Molecular dynamics simulations predicted potential molecular mechanisms mediating these qualitatively different pharmacological properties of the selective Dsub3/subR ligands that are dependent on Dsub1/subR-Dsub3/subR heteromerization. The results of in vitro experiments were paralleled by qualitatively different pharmacological properties of the Dsub3/subR ligands in vivo. The results supported the involvement of Dsub1/subR-Dsub3/subR heteromers in the locomotor activation by Dsub1/subR agonists in reserpinized mice and L-DOPA-induced dyskinesia in rats, highlighting the Dsub1/subR-Dsub3/subR heteromer as a main pharmacological target for L-DOPA-induced dyskinesia in Parkinson's disease. More generally, the present study implies that when suspecting its pathogenetic role, a GPCR heteromer, and not its individual GPCR units, should be considered as main target for drug development.

Polymorphic alleles of the human dopamine D(4) receptor gene (DRD4) have been consistently associated with individual differences in personality traits and neuropsychiatric disorders, particularly between the gene encoding dopamine D(4.7) receptor variant and attention deficit hyperactivity disorder (ADHD). The α(2A) adrenoceptor gene has also been associated with ADHD. In fact, drugs targeting the α(2A) adrenoceptor (α(2A)R), such as guanfacine, are commonly used in ADHD treatment. In view of the involvement of dopamine D(4) receptor (D(4)R) and α(2A)R in ADHD and impulsivity, their concurrent localization in cortical pyramidal neurons and the demonstrated ability of D(4)R to form functional heteromers with other G protein-coupled receptors, in this study we evaluate whether the α(2A)R forms functional heteromers with D(4)R and weather these heteromers show different properties depending on the D(4)R variant involved. Using cortical brain slices from hD(4.7)R knock-in and wild-type mice, here, we demonstrate that α(2A)R and D(4)R heteromerize and constitute a significant functional population of cortical α(2A)R and D(4)R. Moreover, in cortical slices from wild-type mice and in cells transfected with α(2A)R and D(4.4)R, we detect a negative crosstalk within the heteromer. This negative crosstalk is lost in cortex from hD(4.7)R knock-in mice and in cells expressing the D(4.7)R polymorphic variant. We also show a lack of efficacy of D(4)R ligands to promote G protein activation and signaling only within the α(2A)R-D(4.7)R heteromer. Taken together, our results suggest that α(2A)R-D(4)R heteromers play a pivotal role in catecholaminergic signaling in the brain cortex and are likely targets for ADHD pharmacotherapy.

G protein-coupled receptors (GPCRs), G proteins and adenylyl cyclase (AC) comprise one of the most studied transmembrane cell signaling pathways. However, it is unknown whether the ligand-dependent interactions between these signaling molecules are based on random collisions or the rearrangement of pre-coupled elements in a macromolecular complex. Furthermore, it remains controversial whether a GPCR homodimer coupled to a single heterotrimeric G protein constitutes a common functional unit. Using a peptide-based approach, we here report evidence for the existence of functional pre-coupled complexes of heteromers of adenosine A2A receptor and dopamine D2 receptor homodimers coupled to their cognate Gs and Gi proteins and to subtype 5 AC. We also demonstrate that this macromolecular complex provides the necessary frame for the canonical Gs-Gi interactions at the AC level, sustaining the ability of a Gi-coupled GPCR to counteract AC activation mediated by a Gs-coupled GPCR., It is unclear whether GPCRs, G proteins and adenylyl cyclase (AC) associate through random collisions or defined pre-coupling mechanisms. Using a peptide-based approach, the authors show that heteromers of adenosine A2A and dopamine D2 receptors form pre-coupled complexes with their cognate G proteins and AC5.

The two highly homologous subtypes of stimulatory G proteins Gαs (Gs) and Gαolf (Golf) display contrasting expression patterns in the brain. Golf is predominant in the striatum, while Gs is predominant in the cortex. Yet, little is known about their functional distinctions. The dopamine D1 receptor (D1R) couples to Gs/olf and is highly expressed in cortical and striatal areas, making it an important therapeutic target for neuropsychiatric disorders. Using novel drug screening methods that allow analysis of specific G-protein subtype coupling, we found that, relative to dopamine, dihydrexidine and N-propyl-apomorphine behave as full D1R agonists when coupled to Gs, but as partial D1R agonists when coupled to Golf. The Gs/Golf-dependent biased agonism by dihydrexidine was consistently observed at the levels of cellular signaling, neuronal function, and behavior. Our findings of Gs/Golf-dependent functional selectivity in D1R ligands open a new avenue for the treatment of cortex-specific or striatum-specific neuropsychiatric dysfunction., D1-like dopamine receptors are coupled to Golf proteins in the dorsal striatum but Gs in cortical and other areas. Here, the authors demonstrate selective agonism of Gs-coupled versus Golf-coupled D1 receptors.

Gαs (Gs) and Gαolf (Golf) are highly homologous G-protein α subunits that activate adenylate cyclase, thereby serving as crucial mediators of intracellular signaling. Because of their dramatically different brain expression patterns, we studied similarities and differences between their activation processes with the aim of comparing their receptor coupling mechanisms. We engineered novel luciferase- and Venus-fused Gα constructs that can be used in bioluminescence resonance energy transfer assays. In conjunction with molecular simulations, these novel biosensors were used to determine receptor activation–induced changes in conformation. Relative movements in Gs were consistent with the crystal structure of β2 adrenergic receptor in complex with Gs. Conformational changes in Golf activation are shown to be similar to those in Gs. Overall the current study reveals general similarities between Gs and Golf activation at the molecular level and provides a novel set of tools to search for Gs- and Golf-specific receptor pharmacology. In view of the wide functional and pharmacological roles of Gs- and Golf-coupled dopamine D1 receptor and adenosine A2A receptor in the brain and other organs, elucidating their differential structure–function relationships with Gs and Golf might provide new approaches for the treatment of a variety of neuropsychiatric disorders. In particular, these novel biosensors can be used to reveal potentially therapeutic dopamine D1 receptor and adenosine A2A receptor ligands with functionally selective properties between Gs and Golf signaling.

© The Author(s). 2020 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated., Background: It has been hypothesized that heteromers of adenosine A2A receptors (A2AR) and cannabinoid CB1 receptors (CB1R) localized in glutamatergic nerve terminals mediate the integration of adenosine and endocannabinoid signaling involved in the modulation of striatal excitatory neurotransmission. Previous studies have demonstrated the existence of A2AR-CB1R heteromers in artificial cell systems. A dependence of A2AR signaling for the Gi protein-mediated CB1R signaling was described as one of its main biochemical characteristics. However, recent studies have questioned the localization of functionally significant A2AR-CB1R heteromers in striatal glutamatergic terminals. Results: Using a peptide-interfering approach combined with biophysical and biochemical techniques in mammalian transfected cells and computational modeling, we could establish a tetrameric quaternary structure of the A2AR-CB1R heterotetramer. This quaternary structure was different to the also tetrameric structure of heteromers of A2AR with adenosine A1 receptors or dopamine D2 receptors, with different heteromeric or homomeric interfaces. The specific quaternary structure of the A2A-CB1R, which depended on intermolecular interactions involving the long C-terminus of the A2AR, determined a significant A2AR and Gs protein-mediated constitutive activation of adenylyl cyclase. Using heteromer-interfering peptides in experiments with striatal glutamatergic terminals, we could then demonstrate the presence of functionally significant A2AR-CB1R heteromers with the same biochemical characteristics of those studied in mammalian transfected cells. First, either an A2AR agonist or an A2AR antagonist allosterically counteracted Gi-mediated CB1R agonist-induced inhibition of depolarization-induced glutamate release. Second, co-application of both an A2AR agonist and an antagonist cancelled each other effects. Finally, a CB1R agonist inhibited glutamate release dependent on a constitutive activation of A2AR by a canonical Gs-Gi antagonistic interaction at the adenylyl cyclase level. Conclusions: We demonstrate that the well-established cannabinoid-induced inhibition of striatal glutamate release can mostly be explained by a CB1R-mediated counteraction of the A2AR-mediated constitutive activation of adenylyl cyclase in the A2AR-CB1R heteromer., Work supported with the intramural funds of the National Institute on Drug Abuse, “Ministerio de Ciencia, Innovación y Universidades-Agencia Estatal de Investigación/FEDER” (SAF2015-74627-JIN, SAF2016-77830-R, SAF2017-87349-R) and ISCIII/FEDER (PIE14/00034), the Catalan government (2017 SGR 1604), “Fundació la Marató de TV3” (20152031), FWO (SBO-140028), “Fundação para a Ciência e a Tecnologia” (PTDC/DTP-FTO/3346/2014 and PTDC/MED-NEU/31274/2017), FEDER (QREN) through “Programa Operacional Factores de Competitividade” (COMPETE 2020, POCI-01-0145-FEDER-007440, CENTRO-01-0145-FEDER-000012-N2323P30, and UID/NEU/04539/2019), and through “Programa Mais Centro” (CENTRO-01-0246-FEDER-000010 and CENTRO-07-ST24-FEDER-002006).

Several studies found in vitro evidence for heteromerization of dopamine D(1) receptors (D1R) and D(3) receptors (D3R), and it has been postulated that functional D1R-D3R heteromers that are normally present in the ventral striatum mediate synergistic locomotor-activating effects of D1R and D3R agonists in rodents. Based also on results obtained in vitro, with mammalian transfected cells, it has been hypothesized that those behavioral effects depend on a D1R-D3R heteromer-mediated G protein-independent signaling. Here, we demonstrate the presence on D1R-D3R heteromers in the mouse ventral striatum by using a synthetic peptide that selectively destabilizes D1R-D3R heteromers. Parallel locomotor activity and ex vivo experiments in reserpinized mice and in vitro experiments in D1R-D3R mammalian transfected cells were performed to dissect the signaling mechanisms of D1R-D3R heteromers. Co-administration of D1R and D3R agonists in reserpinized mice produced synergistic locomotor activation and a selective synergistic AKT phosphorylation in the most ventromedial region of the striatum, in the shell of the nucleus accumbens. Application of the destabilizing peptide in transfected cells and in the shell of the nucleus accumbens allowed demonstrating that, both in vitro and in vivo, co-activation of D3R induces a switch from G protein-dependent to G protein-independent D1R-mediated signaling determined by D1R-D3R heteromerization. The results therefore demonstrate that a biased G protein-independent signaling of D1R-D3R heteromers localized in the shell of the nucleus accumbens mediate the locomotor synergistic effects of D1R and D3R agonists in reserpinized mice.

Release of the neuropeptides corticotropin-releasing factor (CRF) and orexin-A in the ventral tegmental area (VTA) play an important role in stress-induced cocaine-seeking behavior. We provide evidence for pharmacologically significant interactions between CRF and orexin-A that depend on oligomerization of CRF1receptor (CRF1R) and orexin OX1receptors (OX1R). CRF1R–OX1R heteromers are the conduits of a negative crosstalk between orexin-A and CRF as demonstrated in transfected cells and rat VTA, in which they significantly modulate dendritic dopamine release. The cocaine target σ1receptor (σ1R) also associates with the CRF1R–OX1R heteromer. Cocaine binding to the σ1R–CRF1R–OX1R complex promotes a long-term disruption of the orexin-A–CRF negative crosstalk. Through this mechanism, cocaine sensitizes VTA cells to the excitatory effects of both CRF and orexin-A, thus providing a mechanism by which stress induces cocaine seeking.

The poor norepinephrine innervation and high density of Gi/o-coupled α(2A) and α(2C) adrenoceptors in the striatum and the dense striatal dopamine innervation have prompted the possibility that dopamine could be an effective adrenoceptor ligand. Nevertheless, the reported adrenoceptor agonistic properties of dopamine are still inconclusive. In this study, we analyzed the binding of norepinephrine, dopamine and several compounds reported as selective dopamine D(2)-like receptor ligands, such as the D(3) receptor agonist 7-OH-PIPAT and the D(4) receptor agonist RO-105824, to α(2) adrenoceptors in cortical and striatal tissue, which express α(2A) adrenoceptors and both α(2A) and α(2C) adrenoceptors, respectively. The affinity of dopamine for α(2) adrenoceptors was found to be similar to that for D(1)-like and D(2)-like receptors. Moreover, the exogenous dopamine receptor ligands also showed high affinity for α(2A) and α(2C) adrenoceptors. Their ability to activate Gi/o proteins through α(2A) and α(2C) adrenoceptors was also analyzed in transfected cells with bioluminescent resonance energy transfer techniques. The relative ligand potencies and efficacies were dependent on the Gi/o protein subtype. Furthermore, dopamine binding to α(2) adrenoceptors was functional, inducing changes in dynamic mass redistribution, adenylyl cyclase activity and ERK1/2 phosphorylation. Binding events were further studied with computer modeling of ligand docking. Docking of dopamine at α(2A) and α(2C) adrenoceptors was nearly identical to its binding to the crystallized D(3) receptor. Therefore, we provide conclusive evidence that α(2A) and α(2C) adrenoceptors are functional receptors for norepinephrine, dopamine and other previously assumed selective D(2)-like receptor ligands, which calls for revisiting previous studies with those ligands.

The function of the dopamine D4 receptor and the product of a clinically significant gene polymorphic variant is revealed., Polymorphic variants of the dopamine D4 receptor gene (DRD4) have been repeatedly associated with numerous neuropsychiatric disorders. Yet, the functional role of the D4 receptor and the functional differences of the products of DRD4 polymorphic variants remained enigmatic. Immunohistochemical and optogenetic-microdialysis experiments were performed in knock-in mice expressing a D4 receptor with the long intracellular domain of a human DRD4 polymorphic variant associated with attention deficit hyperactivity disorder (ADHD). When compared with the wild-type mouse D4 receptor, the expanded intracellular domain of the humanized D4 receptor conferred a gain of function, blunting methamphetamine-induced cortical activation and optogenetic and methamphetamine-induced corticostriatal glutamate release. The results demonstrate a key role of the D4 receptor in the modulation of corticostriatal glutamatergic neurotransmission. Furthermore, these data imply that enhanced D4 receptor–mediated dopaminergic control of corticostriatal transmission constitutes a vulnerability factor of ADHD and other neuropsychiatric disorders.

The neuropeptide galanin has been shown to interact with the opioid system. More specifically, galanin counteracts the behavioral effects of the systemic administration of μ-opioid receptor (MOR) agonists. Yet the mechanism responsible for this galanin–opioid interaction has remained elusive. Using biophysical techniques in mammalian transfected cells, we found evidence for selective heteromerization of MOR and the galanin receptor subtype Gal1 (Gal1R). Also in transfected cells, a synthetic peptide selectively disrupted MOR–Gal1R heteromerization as well as specific interactions between MOR and Gal1R ligands: a negative cross talk, by which galanin counteracted MAPK activation induced by the endogenous MOR agonist endomorphin-1, and a cross-antagonism, by which a MOR antagonist counteracted MAPK activation induced by galanin. These specific interactions, which represented biochemical properties of the MOR-Gal1R heteromer, could then be identifiedin situin slices of rat ventral tegmental area (VTA) with MAPK activation and two additional cell signaling pathways, AKT and CREB phosphorylation. Furthermore,in vivomicrodialysis experiments showed that the disruptive peptide selectively counteracted the ability of galanin to block the dendritic dopamine release in the rat VTA induced by local infusion of endomorphin-1, demonstrating a key role of MOR-Gal1R heteromers localized in the VTA in the direct control of dopamine cell function and their ability to mediate antagonistic interactions between MOR and Gal1R ligands. The results also indicate that MOR-Gal1R heteromers should be viewed as targets for the treatment of opioid use disorders.SIGNIFICANCE STATEMENTThe μ-opioid receptor (MOR) localized in the ventral tegmental area (VTA) plays a key role in the reinforcing and addictive properties of opioids. With parallelin vitroexperiments in mammalian transfected cells andin situandin vivoexperiments in rat VTA, we demonstrate that a significant population of these MORs form functional heteromers with the galanin receptor subtype Gal1 (Gal1R), which modulate the activity of the VTA dopaminergic neurons. The MOR-Gal1R heteromer can explain previous results showing antagonistic galanin–opioid interactions and offers a new therapeutic target for the treatment of opioid use disorder.

Pretreatment with methamphetamine (METH) can attenuate toxicity due to acute METH challenges. The majority of previous reports have focused mainly on the effects of the drug on the striatal dopaminergic system. In the present study, we used a regimen that involves gradual increases in METH administration to rats in order to mimic progressively larger doses of the drug used by some human METH addicts. We found that this METH preconditioning was associated with complete protection against dopamine depletion caused by a METH challenge (5 mg/kg x 6 injections given 1 h apart) in the striatum and cortex. In contrast, there was no preconditioning-mediated protection against METH-induced serotonin depletion in the striatum and hippocampus, with some protection being observed in the cortex. There was also no protection against METH-induced norepinephrine (NE) depletion in the hippocampus. These results indicate that, in contrast to the present dogmas, there might be differences in the mechanisms involved in METH toxicity on monoaminergic systems in the rodent brain. Thus, chronic injections of METH might activate programs that protect against dopamine toxicity without influencing drug-induced pathological changes in serotoninergic systems. Further studies will need to evaluate the cellular and molecular bases for these differential responses.

Methamphetamine (METH), a highly addictive drug, can cause degeneration of monoaminergic terminals and neuronal apoptosis in the mammalian brain. In the present article, we have used serial analysis of gene expression (SAGE) to investigate patterns of gene expression in the striata of rats that were given a neurotoxic dose of the drug. SAGE libraries were generated from animals treated with either saline or METH (40 mg/kg) and sacrificed 2 h later. A total of 315 transcripts were differentially expressed between the two libraries (P < 0.05), with 179 (56%) being upregulated and 136 (44%) being downregulated by the METH injection. Of these, CAATT enhancer-binding protein homologous protein (CHOP)/GADD153 (growth arrest- and DNA damage-inducible gene 153) was found to be upregulated by about threefold. Analysis of the expression of genes downstream of CHOP (DOCs) revealed significant METH-induced increases in their expression. Because DOC1 is an analog of carbonic anhydrase (CA) which is involved in the interconversion between carbon dioxide and the bicarbonate ion, we also measured the effects of METH on the expression of several CAs. These were significantly upregulated by METH in a time-dependent fashion. These results indicate that METH toxicity is mediated, in part, by drug-induced perturbations of physiological processes that are dependent on normal pH and CO2 homeostasis.

Methamphetamine [METH (“speed”)] is an abused psychostimulant that can cause psychotic, cognitive, and psychomotor impairment in humans. These signs and symptoms are thought to be related to dysfunctions in basal ganglionic structures of the brain. To identify possible molecular bases for these clinical manifestations, we first used cDNA microarray technology to measure METH-induced transcriptional responses in the striatum of rats treated with an apoptosis-inducing dose of the drug. METH injection resulted in increased expression of members of the Jun, Egr, and Nur77 subfamilies of transcription factors (TFs), changes that were confirmed by quantitative PCR. Because pathways linked to these factors are involved in the up-regulation of Fas ligand (FasL), FasL mRNA was quantified and found to be increased. Immunohistochemical studies also revealed METH-induced increased FasL protein expression in striatal GABAergic neurons that express enkephalin. Moreover, there were METH-mediated increases in calcineurin, as well as shuttling of nuclear factor of activated T cells (NFAT)c3 and NFATc4 from the cytosol to the nucleus of METH-treated rats, mechanisms also known to be involved in FasL regulation. Furthermore, METH induced cleavage of caspase-3 in FasL- and Fas-containing neurons. Finally, the METH-induced changes in the FasL-Fas death pathway were attenuated by pretreatment with the dopamine D1 receptor antagonist, SCH23390, which also caused attenuation of METH-induced apoptosis. These observations indicate that METH causes some of its neurodegenerative effects, in part, via stimulation of the Fas-mediated cell death pathway consequent to FasL up-regulation mediated by activation of multiple TFs.

Previous studies have shown that dopamine and galanin modulate cholinergic transmission in the hippocampus, but little is known about the mechanisms involved and their possible interactions. By using resonance energy transfer techniques in transfected mammalian cells, we demonstrated the existence of heteromers between the dopamine D1-like receptors (D1and D5) and galanin Gal1, but not Gal2receptors. Within the D1–Gal1and D5–Gal1receptor heteromers, dopamine receptor activation potentiated and dopamine receptor blockade counteracted MAPK activation induced by stimulation of Gal1receptors, whereas Gal1receptor activation or blockade did not modify D1-like receptor-mediated MAPK activation. Ability of a D1-like receptor antagonist to block galanin-induced MAPK activation (cross-antagonism) was used as a “biochemical fingerprint” of D1-like–Gal1receptor heteromers, allowing their identification in the rat ventral hippocampus. The functional role of D1-like–Gal receptor heteromers was demonstrated in synaptosomes from rat ventral hippocampus, where galanin facilitated acetylcholine release, but only with costimulation of D1-like receptors. Electrophysiological experiments in rat ventral hippocampal slices showed that these receptor interactions modulate hippocampal synaptic transmission. Thus, a D1-like receptor agonist that was ineffective when administered alone turned an inhibitory effect of galanin into an excitatory effect, an interaction that required cholinergic neurotransmission. Altogether, our results strongly suggest that D1-like–Gal1receptor heteromers act as processors that integrate signals of two different neurotransmitters, dopamine and galanin, to modulate hippocampal cholinergic neurotransmission.

Dopamine (DA), the most abundant catecholamine in the basal ganglia, participates in the regulation of motor functions and of cognitive processes such as learning and memory. Abnormalities in dopaminergic systems are thought to be the bases for some neuropsychiatric disorders including addiction, Parkinson's disease, and Schizophrenia. DA exerts its arrays of functions via stimulation of D1-like (D1 and D5) and D2-like (D2, D3, and D4) DA receptors which are located in various regions of the brain. The DA D1 and D2 receptors are very abundant in the basal ganglia where they exert their functions within separate neuronal cell types. The present paper focuses on a review of the effects of stimulation of DA D1 receptors on diverse signal transduction pathways and gene expression patterns in the brain. We also discuss the possible involvement of the DA D1 receptors in DA-mediated toxic effects observed both in vitro and in vivo. Future studies using more selective agonist and antagonist agents and the use of genetically modified animals should help to further clarify the role of these receptors in the normal physiology and in pathological events that involve DA.

In an attempt to examine some functional characteristics of the N-methyl-D-aspartate (NMDA) receptor complex, the NMDA-evoked effluxes of endogenous dopamine (DA) and [3H]acetylcholine ([3H]ACh) were simultaneously examined in a rat striatal slice preparation. NMDA induced release of both DA and ACh in a concentration-dependent, Ca(2+)-, Mg(2+)-, and tetrodotoxin-sensitive manner. These release responses were remarkably reduced by long-term pretreatment with a low concentration of NMDA, an indication of the desensitization of the NMDA receptor. Glycine was potent in reversing the desensitization-related reduction of DA release but failed to reverse the diminution of ACh release in the same slices. Our results indicate that the NMDA receptors regulating the release of DA and ACh are different with respect to their glycine modulatory site. This finding is consistent with a functional heterogeneity of the NMDA receptor complex in the rat striatum.

Methamphetamine is a neurotoxic drug of abuse known to cause cell death both in vitro and in vivo. Nevertheless, the molecular and cellular mechanisms involved in this process remain to be clarified. Herein, we show that methamphetamine-induced apoptosis is associated with early (2 h) overexpression of bax, decreases of mitochondrial membrane potential and oxygen consumption as well as release of cytochrome c from mitochondria. In addition, activated caspase-9 was detected at 4 h post-METH exposure. Cell death was detectable by annexin V and propidium iodide staining after 8 h of methamphetamine exposure. At that time, the majority of the cells were stained by annexin V alone, with some cells being stained for both annexin V and propidium iodide. Moreover, cleavage of caspase-3, poly (ADP-ribose) polymerase and DNA fragmentation-related factor 45 was detected at 8 h post drug treatment. These results indicate that methamphetamine-induced apoptotic cell death results from early overexpression of bax, reduction of mitochondrial respiration and membrane potential and release of mitochondrial cytochrome c with subsequent activation of the caspase cascade.

The excitotoxic and [ 3 H]γ-aminobutyric acid ([ 3 H]GABA)-releasing effects of quisqualate, α-amino-3-hydroxy-4-methyl-5-isoxazolepropionic acid (AMPA), kainate (KA), and their combinations were examined in primary cultures of the rat cerebral cortex. [ 3 H]GABA efflux was evoked by a 5 min exposure of preloaded cultures to the respective agonist(s) (0.5 mM each). Cell death was induced by a 24 h exposure of cells to 1 mM quisqualate, AMPA and/or KA, and was quantified by measuring lactic dehydrogenase leakage. When applied alone, each agonist induced remarkable [ 3 H]GABA release and excitotoxic cell death. Simultaneous administration of AMPA or quisqualate and KA did not evoke stronger responses than KA alone. These results indicate that AMPA and KA receptors are located on the same cortical cells and may activate the same receptor channels and/or intracellular messengers.

The development of excitatory amino acid-(EAA)-induced cytotoxic cell death an [3H]γ-aminobutyric acid ([3H]GABA) release were simultaneously examined in primary cultures of the rat cerebral cortex. Pronounced [3H]GABA release could already be evoked on day 3 by N-methyl- d -aspartate, quisqualate and kainate, whereas toxic cell death could first be induced on day 7, in vitro. EAA-induced GABA release declined between day 11 and 14, but the excitotoxic vulnerability of cells increased further during the same period. This dissociation of releasing and toxic responses indicates that functionally active EAA receptors do not necessarily mediate excitotoxic effects and suggests that the development of EAA receptors mediating release responses precedes the maturation of intracellular mechanisms involved in excitotoxic neuronal injury, at least in cultured cortical neurons.

Methamphetamine (METH) is an illicit drug which is neurotoxic to the mammalian brain. Numerous studies have revealed significant decreases in dopamine and serotonin levels in the brains of animals exposed to moderate-to-large METH doses given within short intervals of time. In contrast, repeated injections of small nontoxic doses of the drug followed by a challenge with toxic METH doses afford significant protection against monoamine depletion. The present study was undertaken to test the possibility that repeated injections of the drug might be accompanied by transcriptional changes involved in rendering the nigrostriatal dopaminergic system refractory to METH toxicity. Our results confirm that METH preconditioning can provide significant protection against METH-induced striatal dopamine depletion. In addition, the presence and absence of METH preconditioning were associated with substantial differences in the identity of the genes whose expression was affected by a toxic METH challenge. Quantitative PCR confirmed METH-induced changes in genes of interest and identified additional genes that were differentially impacted by the toxic METH challenge in the presence of METH preconditioning. These genes include small heat shock 27 kD 27 protein 2 (HspB2), thyrotropin-releasing hormone (TRH), brain derived neurotrophic factor (BDNF), c-fos, and some encoding antioxidant proteins including CuZn superoxide dismutase (CuZnSOD), glutathione peroxidase (GPx)-1, and heme oxygenase-1 (Hmox-1). These observations are consistent, in part, with the transcriptional alterations reported in models of lethal ischemic injuries which are preceded by ischemic or pharmacological preconditioning. Our findings suggest that multiple molecular pathways might work in tandem to protect the nigrostriatal dopaminergic pathway against the deleterious effects of the toxic psychostimulant. Further analysis of the molecular and cellular pathways regulated by these genes should help to provide some insight into the neuroadaptive potentials of the brain when repeatedly exposed to drugs of abuse.

The effect of vinpocetine on excitatory amino acid receptors was examined in the rat brain by two different biochemical approaches. In release experiments with striatal slices, vinpocetine reduced the efflux of dopamine and acetylcholine evoked by glutamate, quisqualate and N-methyl-D-aspartate (NMDA), but not that evoked by kainate. In binding experiments with cortical membranes, vinpocetine reduced the binding of [3H]2-amino-3-3-hydroxy-s-methylisoxasole-4-yl-propionic acid ([3H]AMPA), a quisqualate partial agonist, in an incomplete manner, but failed to influence the binding of [3H]kainate and [3H]3-(2-carboxypyperazine-4-yl)-propyl-1-phosphonic acid ([3H]CPP), an NMDA agonist. These findings suggest that vinpocetine is a quisqualate/AMPA antagonist of some specificity and selectivity.