RESUMO
Virtually all neuropsychiatric disorders display sex differences in prevalence, age of onset, and/or clinical symptomology. Although altered dopamine (DA) signaling is a feature of many of these disorders, sex-dependent mechanisms uniquely responsive to DA that drive sex-dependent behaviors remain unelucidated. Previously, we established that anomalous DA efflux (ADE) is a prominent feature of the DA transporter (DAT) variant Val559, a coding substitution identified in two male-biased disorders: attention-deficit/hyperactivity disorder and autism spectrum disorder. In vivo, Val559 ADE induces activation of nigrostriatal D2-type DA autoreceptors (D2ARs) that magnifies inappropriate, nonvesicular DA release by elevating phosphorylation and surface trafficking of ADE-prone DAT proteins. Here we demonstrate that DAT Val559 mice exhibit sex-dependent alterations in psychostimulant responses, social behavior, and cognitive performance. In a search for underlying mechanisms, we discovered that the ability of ADE to elicit D2AR regulation of DAT is both sex and circuit-dependent, with dorsal striatum D2AR/DAT coupling evident only in males, whereas D2AR/DAT coupling in the ventral striatum is exclusive to females. Moreover, systemic administration of the D2R antagonist sulpiride, which precludes ADE-driven DAT trafficking, can normalize DAT Val559 behavioral changes unique to each sex and without effects on the opposite sex or wildtype mice. Our studies support the sex- and circuit dependent capacity of D2ARs to regulate DAT as a critical determinant of the sex-biased effects of perturbed DA signaling in neurobehavioral disorders. Moreover, our work provides a cogent example of how a shared biological insult drives alternative physiological and behavioral trajectories as opposed to resilience.
Assuntos
Transtorno do Deficit de Atenção com Hiperatividade , Transtorno do Espectro Autista , Dopamina , Animais , Feminino , Masculino , Camundongos , Transtorno do Deficit de Atenção com Hiperatividade/metabolismo , Transtorno do Espectro Autista/metabolismo , Estimulantes do Sistema Nervoso Central/farmacologia , Estimulantes do Sistema Nervoso Central/uso terapêutico , Dopamina/metabolismo , Dopamina/farmacologia , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Transdução de SinaisRESUMO
The antidepressant-sensitive serotonin (5-HT) transporter (SERT) dictates rapid, high-affinity clearance of the neurotransmitter in both the brain and periphery. In a study of families with multiple individuals diagnosed with autism spectrum disorder (ASD), we previously identified several, rare, missense coding variants that impart elevated 5-HT transport activity, relative to wild-type SERT, upon heterologous expression as well as in ASD subject lymphoblasts. The most common of these variants, SERT Ala56, located in the transporter's cytosolic N-terminus, has been found to confer in transgenic mice hyperserotonemia, an ASD-associated biochemical trait, an elevated brain 5-HT clearance rate, and ASD-aligned behavioral changes. Hyperfunction of SERT Ala56 has been ascribed to a change in 5-HT KM, though the physical basis of this change has yet to be elucidated. Through assessments of fluorescence resonance energy transfer (FRET) between cytosolic N- and C-termini, sensitivity to methanethiosulfonates, and capacity for N-terminal tryptic digestion, we obtain evidence for mutation-induced conformational changes that support an open-outward 5-HT binding conformation in vitro and in vivo. Aspects of these findings were also evident with another naturally occurring C-terminal SERT coding variant identified in our ASD study, Asn605. We conclude that biased conformations of surface resident transporters that can impact transporter function and regulation are an unappreciated consequence of heritable and disease-associated SERT coding variation.
Assuntos
Hipocampo/metabolismo , Neurônios/metabolismo , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Serotonina/metabolismo , Animais , Células CHO , Cricetulus , Fenfluramina/farmacologia , Hipocampo/efeitos dos fármacos , Humanos , Camundongos , Neurônios/efeitos dos fármacos , Conformação Proteica , Proteínas da Membrana Plasmática de Transporte de Serotonina/genética , Inibidores Seletivos de Recaptação de Serotonina/farmacologiaRESUMO
Dopamine (DA) signaling dysfunction is believed to contribute to multiple neuropsychiatric disorders including attention-deficit/hyperactivity disorder (ADHD). The rare DA transporter (DAT) coding substitution Ala559Val found in subjects with ADHD, bipolar disorder and autism, promotes anomalous DA efflux in vitro and, in DAT Val559 mice, leads to increased reactivity to imminent handling, waiting impulsivity, and enhanced motivation for reward. Here, we report that, in contrast to amphetamine and methylphenidate, which induce significant locomotor activation, cocaine administration to these mice elicits no locomotor effects, despite retention of conditioned place preference (CPP). Additionally, cocaine fails to elevate extracellular DA. Given that amphetamine and methylphenidate, unlike cocaine, lack high-affinity interactions with the serotonin (5-HT) transporter (SERT), we hypothesized that the lack of cocaine-induced hyperlocomotion in DAT Val559 mice arises from SERT blockade and augmented 5-HT signaling relative to cocaine actions on wildtype animals. Consistent with this idea, the SERT blocker fluoxetine abolished methylphenidate-induced locomotor activity in DAT Val559 mice, mimicking the effects seen with cocaine. Additionally, a cocaine analog (RTI-113) with greater selectivity for DAT over SERT retains locomotor activation in DAT Val559 mice. Furthermore, genetic elimination of high-affinity cocaine interactions at SERT in DAT Val559 mice, or specific inhibition of 5-HT2C receptors in these animals, restored cocaine-induced locomotion, but did not restore cocaine-induced elevations of extracellular DA. Our findings reveal a significant serotonergic plasticity arising in the DAT Val559 model that involves enhanced 5-HT2C signaling, acting independently of striatal DA release, capable of suppressing the activity of cocaine-sensitive motor circuits.
Assuntos
Cocaína/farmacologia , Condicionamento Clássico/efeitos dos fármacos , Proteínas da Membrana Plasmática de Transporte de Dopamina/efeitos dos fármacos , Inibidores da Captação de Dopamina/farmacologia , Dopamina/metabolismo , Fluoxetina/farmacologia , Locomoção/efeitos dos fármacos , Metilfenidato/farmacologia , Neostriado/efeitos dos fármacos , Neostriado/metabolismo , Receptor 5-HT2C de Serotonina/efeitos dos fármacos , Inibidores Seletivos de Recaptação de Serotonina/farmacologia , Animais , Comportamento Animal/efeitos dos fármacos , Cocaína/análogos & derivados , Modelos Animais de Doenças , Proteínas da Membrana Plasmática de Transporte de Dopamina/genética , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas da Membrana Plasmática de Transporte de SerotoninaRESUMO
Disruptions of dopamine (DA) signaling contribute to a broad spectrum of neuropsychiatric disorders, including attention-deficit hyperactivity disorder (ADHD), addiction, bipolar disorder, and schizophrenia. Despite evidence that risk for these disorders derives from heritable variation in DA-linked genes, a better understanding is needed of the molecular and circuit context through which gene variation drives distinct disease traits. Previously, we identified the DA transporter (DAT) variant Val559 in subjects with ADHD and established that the mutation supports anomalous DAT-mediated DA efflux (ADE). Here, we demonstrate that region-specific contributions of D2 autoreceptors (D2AR) to presynaptic DA homeostasis dictate the consequences of Val559 expression in adolescent male mice. We show that activation of D2ARs in the WT dorsal striatum (DS), but not ventral striatum (VS), increases DAT phosphorylation and surface trafficking. In contrast, the activity of tyrosine hydroxylase (TH) is D2AR-dependent in both regions. In the DS but not VS of Val559 mice, tonic activation of D2ARs drives a positive feedback loop that promotes surface expression of efflux-prone DATs, raising extracellular DA levels and overwhelming DAT-mediated DA clearance capacity. Whereas D2ARs that regulate DAT are tonically activated in the Val559 DS, D2ARs that regulate TH become desensitized, allowing maintenance of cytosolic DA needed to sustain ADE. Together with prior findings, our results argue for distinct D2AR pools that regulate DA synthesis versus DA release and inactivation and offer a clear example of how the penetrance of gene variation can be limited to a subset of expression sites based on differences in intersecting regulatory networks.SIGNIFICANCE STATEMENT Altered dopamine (DA) signaling has been linked to multiple neuropsychiatric disorders. In an effort to understand and model disease-associated DAergic disturbances, we previously screened the DA transporter (DAT) in subjects with attention-deficit hyperactivity disorder (ADHD) and identified multiple, functionally impactful, coding variants. One of these variants, Val559, supports anomalous DA efflux (ADE) and in transgenic mice leads to changes in locomotor patterns, psychostimulant sensitivity, and impulsivity. Here, we show that the penetrance of Val559 ADE is dictated by region-specific differences in how presynaptic D2-type autoreceptors (D2ARs) constrain DA signaling, biasing phenotypic effects to dorsal striatal projections. The Val559 model illustrates how the impact of genetic variation underlying neuropsychiatric disorders can be shaped by the differential engagement of synaptic regulatory mechanisms.
Assuntos
Proteínas da Membrana Plasmática de Transporte de Dopamina/genética , Dopamina/metabolismo , Penetrância , Receptores de Dopamina D2/metabolismo , Animais , Autorreceptores/genética , Autorreceptores/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Variação Genética , Homeostase/fisiologia , Masculino , Transtornos Mentais/genética , Transtornos Mentais/metabolismo , Camundongos , Camundongos TransgênicosRESUMO
BACKGROUND AND PURPOSE: The psychostimulant cocaine induces complex molecular, cellular and behavioural responses as a consequence of inhibiting presynaptic dopamine, noradrenaline and 5-HT transporters. To elucidate 5-HT transporter (SERT)-specific contributions to cocaine action, we evaluated cocaine effects in the SERT Met172 knock-in mouse, which expresses a SERT coding substitution that eliminates high-affinity cocaine recognition. EXPERIMENTAL APPROACH: We measured the effects of SERT Met172 on cocaine antagonism of 5-HT re-uptake using ex vivo synaptosome preparations and in vivo microdialysis. We assessed SERT dependence of cocaine actions behaviourally through acute and chronic locomotor activation, sensitization, conditioned place preference (CPP) and oral cocaine consumption. We used c-Fos, quantitative RT-PCR and RNA sequencing methods for insights into cellular and molecular networks supporting SERT-dependent cocaine actions. KEY RESULTS: SERT Met172 mice demonstrated functional insensitivity for cocaine at SERT. Although they displayed wild-type levels of acute cocaine-induced hyperactivity or chronic sensitization, the pattern of acute motor activation was different, with a bias toward thigmotaxis. CPP was increased, and a time-dependent elevation in oral cocaine consumption was observed. SERT Met172 mice displayed relatively higher levels of neuronal activation in the hippocampus, piriform cortex and prelimbic cortex (PrL), accompanied by region-dependent changes in immediate early gene expression. Distinct SERT-dependent gene expression networks triggered by acute and chronic cocaine administration were identified, including PrL Akt and nucleus accumbens ERK1/2 signalling. CONCLUSION AND IMPLICATIONS: Our studies reveal distinct SERT contributions to cocaine action, reinforcing the possibility of targeting specific aspects of cocaine addiction by modulation of 5-HT signalling.
Assuntos
Estimulantes do Sistema Nervoso Central/farmacologia , Transtornos Relacionados ao Uso de Cocaína/metabolismo , Cocaína/farmacologia , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Serotonina/metabolismo , Animais , Estimulantes do Sistema Nervoso Central/administração & dosagem , Cocaína/administração & dosagem , Condicionamento Psicológico , Feminino , Hipocampo/metabolismo , Masculino , Camundongos , Atividade Motora , Neurônios , Núcleo Accumbens/metabolismo , Córtex Pré-Frontal/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas da Membrana Plasmática de Transporte de Serotonina/genéticaRESUMO
The serotonin 2C receptor regulates food uptake, and its activity is regulated by alternative pre-mRNA splicing. Alternative exon skipping is predicted to generate a truncated receptor protein isoform, whose existence was confirmed with a new antiserum. The truncated receptor sequesters the full-length receptor in intracellular membranes. We developed an oligonucleotide that promotes exon inclusion, which increases the ratio of the full-length to truncated receptor protein. Decreasing the amount of truncated receptor results in the accumulation of full-length, constitutively active receptor at the cell surface. After injection into the third ventricle of mice, the oligonucleotide accumulates in the arcuate nucleus, where it changes alternative splicing of the serotonin 2C receptor and increases pro-opiomelanocortin expression. Oligonucleotide injection reduced food intake in both wild-type and ob/ob mice. Unexpectedly, the oligonucleotide crossed the blood-brain barrier and its systemic delivery reduced food intake in wild-type mice. The physiological effect of the oligonucleotide suggests that a truncated splice variant regulates the activity of the serotonin 2C receptor, indicating that therapies aimed to change pre-mRNA processing could be useful to treat hyperphagia, characteristic for disorders like Prader-Willi syndrome.
Assuntos
Processamento Alternativo/efeitos dos fármacos , Ingestão de Alimentos , Oligonucleotídeos/genética , Oligonucleotídeos/metabolismo , Receptor 5-HT2C de Serotonina/biossíntese , Receptor 5-HT2C de Serotonina/genética , Animais , Expressão Gênica , Regulação da Expressão Gênica , Camundongos , Oligonucleotídeos/administração & dosagem , Pró-Opiomelanocortina/biossíntese , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genéticaRESUMO
Despite the critical role of the presynaptic dopamine (DA) transporter (DAT, SLC6A3) in DA clearance and psychostimulant responses, evidence that DAT dysfunction supports risk for mental illness is indirect. Recently, we identified a rare, nonsynonymous Slc6a3 variant that produces the DAT substitution Ala559Val in two male siblings who share a diagnosis of attention-deficit hyperactivity disorder (ADHD), with other studies identifying the variant in subjects with bipolar disorder (BPD) and autism spectrum disorder (ASD). Previously, using transfected cell studies, we observed that although DAT Val559 displays normal total and surface DAT protein levels, and normal DA recognition and uptake, the variant transporter exhibits anomalous DA efflux (ADE) and lacks capacity for amphetamine (AMPH)-stimulated DA release. To pursue the significance of these findings in vivo, we engineered DAT Val559 knock-in mice, and here we demonstrate in this model the presence of elevated extracellular DA levels, altered somatodendritic and presynaptic D2 DA receptor (D2R) function, a blunted ability of DA terminals to support depolarization and AMPH-evoked DA release, and disruptions in basal and psychostimulant-evoked locomotor behavior. Together, our studies demonstrate an in vivo functional impact of the DAT Val559 variant, providing support for the ability of DAT dysfunction to impact risk for mental illness.
Assuntos
Anfetamina/farmacologia , Comportamento Animal/efeitos dos fármacos , Estimulantes do Sistema Nervoso Central/farmacologia , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Neurônios Dopaminérgicos/metabolismo , Transtornos Mentais/metabolismo , Mutação de Sentido Incorreto , Substituição de Aminoácidos , Animais , Dopamina/genética , Dopamina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina/genética , Neurônios Dopaminérgicos/patologia , Feminino , Humanos , Masculino , Transtornos Mentais/genética , Transtornos Mentais/patologia , Camundongos , Receptores de Dopamina D2/genética , Receptores de Dopamina D2/metabolismoRESUMO
The serotonergic system regulates a wide range of behavior, including mood and impulsivity, and its dysregulation has been associated with mood disorders, autism spectrum disorder, and addiction. Diabetes is a risk factor for these conditions. Insulin resistance in the brain is specifically associated with susceptibility to psychostimulant abuse. Here, we examined whether phosphorylation of Akt, a key regulator of the insulin signaling pathway, controls serotonin (5-HT) signaling. To explore how impairment in Akt function regulates 5-HT homeostasis, we used a brain-specific rictor knockout (KO) mouse model of impaired neuronal phosphorylation of Akt at Ser473. Cortical 5-HT1A and 5-HT2A receptor binding was significantly elevated in rictor KO mice. Concomitant with this elevated receptor expression, the 5-HT1A receptor agonist 8-Hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) led to an increased hypothermic response in rictor KO mice. The increased cortical 5-HT1A receptor density was associated with higher 5-HT1A receptor levels on the cortical cell surface. In contrast, rictor KO mice displayed significantly reduced head-twitch response (HTR) to the 5-HT2A/C agonist 2,5-dimethoxy-4-iodoamphetamine (DOI), with evidence of impaired 5-HT2A/C receptor signaling. In vitro, pharmacological inhibition of Akt significantly increased 5-HT1A receptor expression and attenuated DOI-induced 5-HT2A receptor signaling, thereby lending credence to the observed in vivo cross-talk between neuronal Akt signaling and 5-HT receptor regulation. These data reveal that defective central Akt function alters 5-HT signaling as well as 5-HT-associated behaviors, demonstrating a novel role for Akt in maintaining neuronal 5-HT receptor function.
Assuntos
Neurônios/metabolismo , Proteína Oncogênica v-akt/genética , Receptor 5-HT1A de Serotonina/metabolismo , Receptor 5-HT2A de Serotonina/metabolismo , 8-Hidroxi-2-(di-n-propilamino)tetralina/farmacologia , Animais , Insulina/fisiologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/fisiologia , Receptor 5-HT1A de Serotonina/efeitos dos fármacos , Receptor 5-HT2A de Serotonina/efeitos dos fármacos , Serotonina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Agonistas do Receptor de Serotonina/farmacologia , Transdução de Sinais/genética , Transdução de Sinais/fisiologiaRESUMO
Serotonin [i.e., 5-hydroxytryptamine (5-HT)]-targeted antidepressants are in wide use for the treatment of mood disorders, although many patients do not show a response or experience unpleasant side effects. Psychostimulants, such as cocaine and 3,4-methylenedioxymethamphetamine (i.e., "ecstasy"), also impact 5-HT signaling. To help dissect the contribution of 5-HT signaling to the actions of these and other agents, we developed transgenic mice in which high-affinity recognition of multiple antidepressants and cocaine is eliminated. Our animals possess a modified copy of the 5-HT transporter (i.e., SERT, slc6a4) that bears a single amino acid substitution, I172M, proximal to the 5-HT binding site. Although the M172 substitution does not impact the recognition of 5-HT, this mutation disrupts high-affinity binding of many competitive antagonists in transfected cells. Here, we demonstrate that, in M172 knock-in mice, basal SERT protein levels, 5-HT transport rates, and 5-HT levels are normal. However, SERT M172 mice display a substantial loss of sensitivity to the selective 5-HT reuptake inhibitors fluoxetine and citalopram, as well as to cocaine. Through a series of biochemical, electrophysiological, and behavioral assays, we demonstrate the unique properties of this model and establish directly that SERT is the sole protein responsible for selective 5-HT reuptake inhibitor-mediated alterations in 5-HT clearance, in 5-HT1A autoreceptor modulation of raphe neuron firing, and in behaviors used to predict the utility of antidepressants.
Assuntos
Antidepressivos/farmacologia , Cocaína/farmacologia , Terminações Pré-Sinápticas/efeitos dos fármacos , Terminações Pré-Sinápticas/metabolismo , Proteínas da Membrana Plasmática de Transporte de Serotonina/genética , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Animais , Comportamento Animal/efeitos dos fármacos , Técnicas de Introdução de Genes , Técnicas In Vitro , Cinética , Camundongos , Camundongos Transgênicos , Núcleos da Rafe/efeitos dos fármacos , Núcleos da Rafe/metabolismo , Serotonina/metabolismo , Inibidores Seletivos de Recaptação de Serotonina/administração & dosagem , Inibidores Seletivos de Recaptação de Serotonina/farmacologia , Sinaptossomos/efeitos dos fármacos , Sinaptossomos/metabolismoRESUMO
The mammalian target of rapamycin (mTOR) complex 2 (mTORC2) is a multimeric signaling unit that phosphorylates protein kinase B/Akt following hormonal and growth factor stimulation. Defective Akt phosphorylation at the mTORC2-catalyzed Ser473 site has been linked to schizophrenia. While human imaging and animal studies implicate a fundamental role for Akt signaling in prefrontal dopaminergic networks, the molecular mechanisms linking Akt phosphorylation to specific schizophrenia-related neurotransmission abnormalities have not yet been described. Importantly, current understanding of schizophrenia suggests that cortical decreases in DA neurotransmission and content, defined here as cortical hypodopaminergia, contribute to both the cognitive deficits and the negative symptoms characteristic of this disorder. We sought to identify a mechanism linking aberrant Akt signaling to these hallmarks of schizophrenia. We used conditional gene targeting in mice to eliminate the mTORC2 regulatory protein rictor in neurons, leading to impairments in neuronal Akt Ser473 phosphorylation. Rictor-null (KO) mice exhibit prepulse inhibition (PPI) deficits, a schizophrenia-associated behavior. In addition, they show reduced prefrontal dopamine (DA) content, elevated cortical norepinephrine (NE), unaltered cortical serotonin (5-HT), and enhanced expression of the NE transporter (NET). In the cortex, NET takes up both extracellular NE and DA. Thus, we propose that amplified NET function in rictor KO mice enhances accumulation of both NE and DA within the noradrenergic neuron. This phenomenon leads to conversion of DA to NE and ultimately supports both increased NE tissue content as well as a decrease in DA. In support of this hypothesis, NET blockade in rictor KO mice reversed cortical deficits in DA content and PPI, suggesting that dysregulation of DA homeostasis is driven by alteration in NET expression, which we show is ultimately influenced by Akt phosphorylation status. These data illuminate a molecular link, Akt regulation of NET, between the recognized association of Akt signaling deficits in schizophrenia with a specific mechanism for cortical hypodopaminergia and hypofunction. Additionally, our findings identify Akt as a novel modulator of monoamine homeostasis in the cortex.
Assuntos
Proteínas de Transporte/fisiologia , Dopamina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Norepinefrina/fisiologia , Córtex Pré-Frontal/metabolismo , Esquizofrenia/fisiopatologia , Animais , Proteínas de Transporte/genética , Camundongos , Camundongos Knockout , Fosforilação , Proteínas Proto-Oncogênicas c-akt/química , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteína Companheira de mTOR Insensível à Rapamicina , Serina/metabolismo , Transdução de Sinais , Transativadores/metabolismo , Fatores de TranscriçãoRESUMO
RATIONALE: Hallucinogenic serotonin 2A (5-HT(2A)) receptor partial agonists, such as (+ or -)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride (DOI), induce a frontal cortex-dependent head-twitch response (HTR) in rodents, a behavioral proxy of a hallucinogenic response that is blocked by 5-HT(2A) receptor antagonists. In addition to 5-HT(2A) receptors, DOI and most other serotonin-like hallucinogens have high affinity and potency as partial agonists at 5-HT(2C) receptors. OBJECTIVES: We tested for involvement of 5-HT(2C) receptors in the HTR induced by DOI. RESULTS: Comparison of 5-HT(2C) receptor knockout and wild-type littermates revealed an approximately 50% reduction in DOI-induced HTR in knockout mice. Also, pretreatment with either the 5-HT(2C) receptor antagonist SB206553 or SB242084 eradicated a twofold difference in DOI-induced HTR between the standard inbred mouse strains C57BL/6J and DBA/2J, and decreased the DOI-induced HTR by at least 50% in both strains. None of several measures of 5-HT(2A) receptors in frontal cortex explained the strain difference, including 5-HT(2A) receptor density, Galpha(q) or Galpha(i/o) protein levels, phospholipase C activity, or DOI-induced expression of Egr1 and Egr2. 5-HT(2C) receptor density in the brains of C57BL/6J and DBA/2J was also equivalent, suggesting that 5-HT(2C) receptor-mediated intracellular signaling or other physiological modulators of the HTR may explain the strain difference in response to DOI. CONCLUSIONS: We conclude that the HTR to DOI in mice is strongly modulated by 5-HT(2C) receptor activity. This novel finding invites reassessment of hallucinogenic mechanisms involving 5-HT(2) receptors.
Assuntos
Anfetaminas/farmacologia , Comportamento Animal , Encéfalo/efeitos dos fármacos , Alucinógenos/farmacologia , Movimentos da Cabeça/efeitos dos fármacos , Receptor 5-HT2C de Serotonina/efeitos dos fármacos , Agonistas do Receptor de Serotonina/farmacologia , Aminopiridinas/farmacologia , Animais , Autorradiografia , Sítios de Ligação , Western Blotting , Encéfalo/metabolismo , Relação Dose-Resposta a Droga , Agonismo Parcial de Drogas , Proteína 1 de Resposta de Crescimento Precoce/genética , Proteína 2 de Resposta de Crescimento Precoce/genética , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Indóis/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Knockout , Piridinas/farmacologia , RNA Mensageiro/metabolismo , Receptor 5-HT2C de Serotonina/deficiência , Receptor 5-HT2C de Serotonina/genética , Antagonistas da Serotonina/farmacologia , Especificidade da Espécie , Fosfolipases Tipo C/metabolismoRESUMO
d-Lysergic acid diethylamide (LSD), an indoleamine hallucinogen, produces profound alterations in mood, thought, and perception in humans. The brain site(s) that mediates the effects of LSD is currently unknown. In this study, we combine the drug discrimination paradigm with intracerebral microinjections to investigate the anatomical localization of the discriminative stimulus of LSD in rats. Based on our previous findings, we targeted the anterior cingulate cortex (ACC) to test its involvement in mediating the discriminative stimulus properties of LSD. Rats were trained to discriminate systemically administered LSD (0.085 mg/kg s.c.) from saline. Following acquisition of the discrimination, bilateral cannulae were implanted into the ACC (AP, +1.2 mm; ML, +/-1.0 mm; DV, -2.0 mm relative to bregma). Rats were tested for their ability to discriminate varying doses of locally infused LSD (0.1875, 0.375, and 0.75 microg/side) or artificial cerebrospinal fluid (n = 3-7). LSD locally infused into ACC dose-dependently substituted for systemically administered LSD, with 0.75 microg/side LSD substituting completely (89% correct). Systemic administration of the selective 5-hydroxytryptamine (serotonin) (5-HT)(2A) receptor antagonist R-(+)-alpha-(2,3-dimethoxyphenyl)-1-[2-(4-fluorophenylethyl)]-4-piperidine-methanol (M100907; 0.4 mg/kg) blocked the discriminative cue of LSD (0.375 microg/side) infused into ACC (from 68 to 16% drug lever responding). Furthermore, M100907 (0.5 microg/microl/side) locally infused into ACC completely blocked the stimulus effects of systemic LSD (0.04 mg/kg; from 80 to 12% on the LSD lever). Taken together, these data indicate that 5-HT(2A) receptors in the ACC are a primary target mediating the discriminative stimulus properties of LSD.
Assuntos
Aprendizagem por Discriminação/efeitos dos fármacos , Giro do Cíngulo/fisiologia , Dietilamida do Ácido Lisérgico/farmacologia , Receptor 5-HT2A de Serotonina/fisiologia , Animais , Relação Dose-Resposta a Droga , Fluorbenzenos/farmacologia , Masculino , Piperidinas/farmacologia , Ratos , Ratos Sprague-DawleyRESUMO
Activation of 5-hydroxytryptamine2C (5-HT(2C)) receptors by the 5-HT(2) receptor agonist m-chlorophenylpiperazine (m-CPP) elicits anxiety in humans and anxiety-like behavior in animals. We compared the effects of m-CPP with the anxiogenic GABA(A) receptor inverse agonist N-methyl-beta-carboline-3-carboxamide (FG-7142) on both anxiety-like behavior and regional brain activation using functional magnetic resonance imaging (fMRI) in the rat. We also determined whether the selective 5-HT(2C) receptor antagonist SB 242084 [6-chloro-2,3-dihydro-5-methyl-N-[6-[(2-methyl-3-pyridinyl)oxy]-3-pyridinyl]-1H-indole-1-carboxyamide dihydrochloride] would blunt m-CPP or FG-7142-induced neuronal activation. Both m-CPP (3 mg/kg i.p.) and FG-7142 (10 mg/kg i.p.) elicited anxiety-like behavior when measured in the social interaction test, and pretreatment with SB 242084 (1 mg/kg i.p.) completely blocked the behavioral effects of both anxiogenic drugs. Regional brain activation in vivo in response to anxiogenic drug challenge was determined by blood oxygen level-dependent (BOLD) fMRI using a powerful 9.4T magnet. Region of interest analyses revealed that m-CPP and FG-7142 significantly increased BOLD signals in brain regions that have been linked to anxiety, including the amygdala, dorsal hippocampus, and medial hypothalamus. These BOLD signal increases were blocked by pretreatment with SB 242084. In contrast, injection of m-CPP and FG-7142 resulted in BOLD signal decreases in the medial prefrontal cortex that were not blocked by SB 242084. In conclusion, the brain activation signals produced by anxiogenic doses of both m-CPP and FG-7142 are mediated at least partially by the 5-HT(2C) receptor, indicating that this receptor is a key component in anxiogenic neural circuitry.
Assuntos
Ansiedade/metabolismo , Comportamento Animal/efeitos dos fármacos , Carbolinas/farmacologia , Sistema Límbico/efeitos dos fármacos , Piperazinas/farmacologia , Receptor 5-HT2C de Serotonina/fisiologia , Agonistas do Receptor de Serotonina/farmacologia , Aminopiridinas/farmacologia , Animais , Indóis/farmacologia , Sistema Límbico/metabolismo , Imageamento por Ressonância Magnética , Masculino , Ratos , Ratos Sprague-Dawley , Antagonistas do Receptor 5-HT2 de SerotoninaRESUMO
Biologically active small molecule derivatives that can be conjugated to quantum dots have the promise of revolutionizing fluorescent imaging in biology. In order to achieve this several technical hurdles have to be surmounted, one of which is non-specific adsorption of quantum dots to cell membranes. Pegylating quantum dots has been shown to eliminate non-specific binding. Consequently it is necessary to develop a universal synthetic methodology to attach small molecule ligands to polyethylene glycol. These pegylated small molecules may then be conjugated to the surfaces of quantum dots. Ideally this universal strategy should be adaptable and be applicable to PEG chains of varying lengths. This paper describes the development of one such methodology and the synthesis of a pegylated derivative of the known 5HT(2) agonist 1-(2-aminopropyl)-2,5-dimethoxy benzene. This compound was tested and found to be an agonist for the 5HT(2A) and 5HT(2C) receptor having EC(50) values of 250 and 50 nM, respectively.
Assuntos
Polietilenoglicóis , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , Membrana Celular/fisiologia , Ligantes , Teoria Quântica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Tolerance is defined as a decrease in responsiveness to a drug after repeated administration. Tolerance to the behavioral effects of hallucinogens occurs in humans and animals. In this study, we used drug discrimination to establish a behavioral model of lysergic acid diethylamide (LSD) tolerance and examined whether tolerance to the stimulus properties of LSD is related to altered serotonin receptor signaling. Rats were trained to discriminate 60 microg/kg LSD from saline in a two-lever drug discrimination paradigm. Two groups of animals were assigned to either chronic saline treatment or chronic LSD treatment. For chronic treatment, rats from each group were injected once per day with either 130 microg/kg LSD or saline for 5 days. Rats were tested for their ability to discriminate either saline or 60 microg/kg LSD, 24 h after the last chronic injection. Rats receiving chronic LSD showed a 44% reduction in LSD lever selection, while rats receiving chronic vehicle showed no change in percent choice on the LSD lever. In another group of rats receiving the identical chronic LSD treatment, LSD-stimulated [35S]GTPgammaS binding, an index of G-protein coupling, was measured in the rat brain by autoradiography. After chronic LSD, a significant reduction in LSD-stimulated [35S]GTPgammaS binding was observed in the medial prefrontal cortex and anterior cingulate cortex. Furthermore, chronic LSD produced a significant reduction in 2,5-dimethoxy-4-iodoamphetamine-stimulated [35S]GTPgammaS binding in medial prefrontal cortex and anterior cingulate cortex, which was blocked by MDL 100907, a selective 5-HT2A receptor antagonist, but not SB206553, a 5-HT2C receptor antagonist, indicating a reduction in 5-HT2A receptor signaling. 125I-LSD binding to 5-HT2A receptors was reduced in cortical regions, demonstrating a reduction in 5-HT2A receptor density. Taken together, these results indicate that adaptive changes in LSD-stimulated serotonin receptor signaling may mediate tolerance to the discriminative stimulus effects of LSD.
Assuntos
Córtex Cerebral/efeitos dos fármacos , Tolerância a Medicamentos/fisiologia , Alucinógenos/efeitos adversos , Dietilamida do Ácido Lisérgico/efeitos adversos , Receptor 5-HT2A de Serotonina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Anfetaminas/farmacologia , Análise de Variância , Animais , Autorradiografia/métodos , Comportamento Animal/efeitos dos fármacos , Córtex Cerebral/anatomia & histologia , Diagnóstico por Imagem/métodos , Interações Medicamentosas , Fluorbenzenos/farmacologia , Guanosina 5'-O-(3-Tiotrifosfato)/farmacocinética , Alucinógenos/administração & dosagem , Indóis/farmacologia , Isótopos de Iodo/farmacocinética , Dietilamida do Ácido Lisérgico/administração & dosagem , Dietilamida do Ácido Lisérgico/farmacocinética , Dietilamida do Ácido Lisérgico/farmacologia , Masculino , Piperidinas/farmacologia , Ligação Proteica/efeitos dos fármacos , Piridinas/farmacologia , Ratos , Ratos Sprague-Dawley , Antagonistas da Serotonina/farmacologia , Transdução de Sinais/fisiologia , Isótopos de Enxofre/farmacocinética , Fatores de TempoRESUMO
The purpose of this study was to determine if animals trained to discriminate a serotonin2A (5-HT2A) receptor agonist from a 5-HT2A receptor antagonist would also be sensitive to alterations in serotonin neurotransmission brought about by 5-HT reuptake inhibitors and releasers. Previous work from our laboratory has shown that the quipazine-ketanserin discrimination is mediated solely by the 5-HT2A receptor, thus providing a behavioral continuum of 5-HT2A receptor function. Rats were trained to discriminate quipazine (0.35 mg/kg) from ketanserin (1.0 mg/kg) on a variable interval-30 schedule of reinforcement. Following acquisition, substitution tests were conducted with the training drug, quipazine, and agents that have been shown to alter the synaptic levels of 5-HT, including fenfluramine, norfenfluramine, 5-methoxy-6-methyl-2-aminoindan (MMAI) and fluoxetine. All compounds substituted, except fluoxetine. Antagonist tests with mianserin and MDL 100,907 indicated that fenfluramine's and MMAI's substitution for quipazine was mediated by the 5-HT2A receptor. Animals were pretreated with PCPA to determine whether 5-HT release or direct agonism mediated the discriminative stimulus effects of fenfluramine and MMAI. PCPA blocked the substitution of MMAI but not of fenfluramine for quipazine. Analysis of 3H-IP formation in cells showed that norfenfluramine dose-dependently stimulated phosphoinositide hydrolysis to levels similar to that of serotonin and quipazine. These results indicate that fenfluramine's substitution for quipazine in rats trained on a quipazine-ketanserin discrimination are due to direct agonism at the 5-HT2A receptor likely mediated by norfenfluramine, an active metabolite.