RESUMO
Mu opioid receptors (MORs) are expressed in the dorsal striatum, a brain region that mediates goal-directed (via the dorsomedial striatum) and habitual (via the dorsolateral striatum, DLS) behaviours. Our previous work indicates that glutamate transmission is depressed when MORs are activated in the dorsal striatum, inducing MOR-mediated long-term synaptic depression (MOR-LTD) or short-term depression (MOR-STD), depending on the input. In the DLS, MOR-LTD is produced by MORs on anterior insular cortex (AIC) inputs and MOR-STD occurs at thalamic inputs, suggesting input-specific MOR plasticity mechanisms. Here, we evaluated the mechanisms of induction of MOR-LTD and MOR-STD in the DLS using pharmacology and optogenetics combined with patch-clamp electrophysiology. We found that cAMP/PKA signalling and protein synthesis are necessary for MOR-LTD expression, similar to previous studies of cannabinoid-mediated LTD in DLS. MOR-STD does not utilize these same mechanisms. We also demonstrated that cannabinoid-LTD occurs at AIC inputs to DLS. However, while cannabinoid-LTD requires mTOR signalling in DLS, MOR-LTD does not. We characterized the role of presynaptic HCN1 channels in MOR-LTD induction as HCN1 channels expressed in AIC are necessary for MOR-LTD expression in the DLS. These results suggest a mechanism in which MOR activation requires HCN1 to induce MOR-LTD, suggesting a new target for pharmacological modulation of synaptic plasticity, providing new opportunities to develop novel drugs to treat alcohol and opioid use disorders. KEY POINTS: Mu opioid receptor-mediated long-term depression at anterior insular cortex inputs to dorsolateral striatum involves presynaptic cAMP/PKA signalling and protein translation, similar to known mechanisms of cannabinoid long-term depression. Dorsal striatal cannabinoid long-term depression also occurs at anterior insular cortex inputs to the dorsolateral striatum. Dorsal striatal cannabinoid long-term depression requires mTOR signalling, similar to hippocampal cannabinoid long-term depression, but dorsal striatal mu opioid long-term depression does not require mTOR signalling. Mu opioid long-term depression requires presynaptic HCN1 channels at anterior insular cortex inputs to dorsolateral striatum.
Assuntos
Canabinoides , Infecções Sexualmente Transmissíveis , Humanos , Receptores Opioides mu/metabolismo , Analgésicos Opioides/farmacologia , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Córtex Insular , Depressão , Plasticidade Neuronal/fisiologia , Depressão Sináptica de Longo Prazo/fisiologia , Corpo Estriado/metabolismo , Canabinoides/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Infecções Sexualmente Transmissíveis/metabolismoRESUMO
The role of Mu opioid receptor (MOR)-mediated regulation of GABA transmission in opioid reward is well established. Much less is known about MOR-mediated regulation of glutamate transmission in the brain and how this relates to drug reward. We previously found that MORs inhibit glutamate transmission at synapses that express the Type 2 vesicular glutamate transporter (vGluT2). We created a transgenic mouse that lacks MORs in vGluT2-expressing neurons (MORflox-vGluT2cre) to demonstrate that MORs on the vGluT2 neurons themselves mediate this synaptic inhibition. We then explored the role of MORs in vGluT2-expressing neurons in opioid-related behaviors. In tests of conditioned place preference, MORflox-vGluT2cre mice did not acquire place preference for a low dose of the opioid, oxycodone, but displayed conditioned place aversion at a higher dose, whereas control mice displayed preference for both doses. In an oral consumption assessment, these mice consumed less oxycodone and had reduced preference for oxycodone compared with controls. MORflox-vGluT2cre mice also failed to show oxycodone-induced locomotor stimulation. These mice displayed baseline withdrawal-like responses following the development of oxycodone dependence that were not seen in littermate controls. In addition, withdrawal-like responses in these mice did not increase following treatment with the opioid antagonist, naloxone. However, other MOR-mediated behaviors were unaffected, including oxycodone-induced analgesia. These data reveal that MOR-mediated regulation of glutamate transmission is a critical component of opioid reward.
Assuntos
Neurônios/metabolismo , Oxicodona/farmacologia , Receptores Opioides mu/genética , Proteína Vesicular 2 de Transporte de Glutamato/metabolismo , Animais , Condicionamento Clássico/efeitos dos fármacos , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , RecompensaRESUMO
Here, we used knock-in (KI) mice that have ethanol-insensitive alpha 1 glycine receptors (GlyRs) (KK385/386AA) to examine how alpha 1 GlyRs might affect binge drinking and conditioned place preference. Data show that tonic alpha 1 GlyR-mediated currents were exclusively sensitive to ethanol only in wild-type mice. Behavioral studies showed that the KI mice have a higher intake of ethanol upon first exposure to drinking and greater conditioned place preference to ethanol. This study suggests that nonsynaptic alpha 1-containing GlyRs have a role in motivational and early reinforcing effects of ethanol.
Alcohol abuse leads to great medical, social, and economic burdens throughout the world. It is believed that the rewarding actions of alcohol are mediated by alterations in the mesolimbic dopaminergic system leading to increased levels of dopamine in the nucleus accumbens (NAc). Little is known about the role that ligand-gated ion channels (LGICs), such as glycine receptors (GlyRs), have in regulating levels of ethanol intake and place preference. In this study, we used knock-in (KI) mice that have ethanol-insensitive α1 GlyRs (KK385/386AA) and a combination of electrophysiological and behavioral approaches to examine how expression of ethanol-resistant α1 GlyRs in brain neurons might affect binge drinking and conditioned place preference. Data show that tonic α1 GlyR-mediated currents that modulate accumbal excitability were exclusively sensitive to ethanol only in wild-type (WT) mice. Behavioral studies showed that the KI mice have a higher intake of ethanol upon first exposure to drinking and greater conditioned place preference to ethanol, suggesting that α1 GlyRs in the brain have a protective role against abuse. This study suggests that nonsynaptic α1-containing GlyRs have a role in motivational and early reinforcing effects of ethanol and open a novel opportunity for pharmacotherapy development to treat alcohol use disorders.
Assuntos
Alcoolismo/fisiopatologia , Encéfalo/efeitos dos fármacos , Encéfalo/fisiopatologia , Etanol/farmacologia , Receptores de Glicina/metabolismo , Alcoolismo/metabolismo , Animais , Encéfalo/metabolismo , Modelos Animais de Doenças , Etanol/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Glicina/efeitos dos fármacosRESUMO
Variants in the microglial receptor TREM2 confer risk for multiple neurodegenerative diseases. However, it remains unknown how this receptor functions on microglia to modulate these diverse neuropathologies. To understand the role of TREM2 on microglia more generally, we investigated changes in microglial function in Trem2-/- mice. We found that loss of TREM2 impairs normal neurodevelopment, resulting in reduced synapse number across the cortex and hippocampus in 1-month-old mice. This reduction in synapse number was not due directly to alterations in interactions between microglia and synapses. Rather, TREM2 was required for microglia to limit synaptic engulfment by astrocytes during development. While these changes were largely normalized later in adulthood, high fat diet administration was sufficient to reinitiate TREM2-dependent modulation of synapse loss. Together, this identifies a novel role for microglia in instructing synaptic pruning by astrocytes to broadly regulate appropriate synaptic refinement, and suggests novel candidate mechanisms for how TREM2 and microglia could influence synaptic loss in brain injury and disease.
Assuntos
Astrócitos/metabolismo , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Glicoproteínas de Membrana/metabolismo , Microglia/metabolismo , Receptores Imunológicos/metabolismo , Sinapses/metabolismo , Animais , Dieta Hiperlipídica/efeitos adversos , Feminino , Masculino , Glicoproteínas de Membrana/genética , Camundongos Knockout , Distribuição Aleatória , Receptores Imunológicos/genética , Técnicas de Cultura de TecidosRESUMO
BACKGROUND: Although it is widely acknowledged that the risk of developing an alcohol use disorder (AUD) is strongly influenced by genetic factors, very little is known about how this genetic predisposition may alter neurotransmission in a way that promotes AUD susceptibility. The dorsal striatum has garnered increased attention as a brain region of interest in AUD development given its significant roles in goal-directed and habitual behavior. METHODS: In the present work, dorsal striatal neurotransmission parameters were measured in preclinical mouse models of high and low AUD risk. We performed brain slice whole-cell patch clamp electrophysiological recordings from medium spiny neurons (MSNs) in the dorsomedial (DMS) and dorsolateral (DLS) striatum of naïve adult male and female selectively bred high- and low-alcohol-preferring lines of mice (HAP and LAP). RESULTS: We found that MSNs of HAP mice were significantly more excitable than those of LAP mice, specifically in the DLS. Additionally, the frequencies of spontaneous glutamate- and GABA-mediated currents were both elevated in HAP mice relative to LAP mice in both dorsal striatal subregions, whereas amplitude differences were more variable between lines and subregions. AMPAR/NMDAR current ratios were significantly lower in HAP mice in both DLS and DMS. CONCLUSIONS: Collectively, these results suggest that genetic predisposition for high or low alcohol consumption produces significantly different basal functional states within both DLS and DMS which may be important factors in the behavioral phenotypes of HAP and LAP mice.
Assuntos
Alcoolismo/genética , Corpo Estriado/fisiologia , Transmissão Sináptica/genética , Alcoolismo/fisiopatologia , Animais , Eletrofisiologia , Feminino , Masculino , Camundongos , Técnicas de Patch-Clamp , Seleção ArtificialRESUMO
It is well accepted that ethanol is able to produce major health and economic problems associated to its abuse. Because of its intoxicating and addictive properties, it is necessary to analyze its effect in the central nervous system. However, we are only now learning about the mechanisms controlling the modification of important membrane proteins such as ligand-activated ion channels by ethanol. Furthermore, only recently are these effects being correlated to behavioral changes. Current studies show that the glycine receptor (GlyR) is a susceptible target for low concentrations of ethanol (5-40mM). GlyRs are relevant for the effects of ethanol because they are found in the spinal cord and brain stem where they primarily express the α1 subunit. More recently, the presence of GlyRs was described in higher regions, such as the hippocampus and nucleus accumbens, with a prevalence of α2/α3 subunits. Here, we review data on the following aspects of ethanol effects on GlyRs: (1) direct interaction of ethanol with amino acids in the extracellular or transmembrane domains, and indirect mechanisms through the activation of signal transduction pathways; (2) analysis of α2 and α3 subunits having different sensitivities to ethanol which allows the identification of structural requirements for ethanol modulation present in the intracellular domain and C-terminal region; (3) Genetically modified knock-in mice for α1 GlyRs that have an impaired interaction with G protein and demonstrate reduced ethanol sensitivity without changes in glycinergic transmission; and (4) GlyRs as potential therapeutic targets.
Assuntos
Comportamento/efeitos dos fármacos , Etanol/farmacologia , Receptores de Glicina/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacos , Anestésicos Gerais/farmacologia , Animais , Comportamento/fisiologia , Etanol/toxicidade , Humanos , Camundongos , Camundongos Mutantes , Modelos Neurológicos , Receptores de Glicina/química , Receptores de Glicina/genética , Receptores de Glicina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transmissão Sináptica/fisiologiaRESUMO
Ethanol increased the frequency of miniature glycinergic currents [miniature inhibitory postsynaptic currents (mIPSCs)] in cultured spinal neurons. This effect was dependent on intracellular calcium augmentation, since preincubation with BAPTA (an intracellular calcium chelator) or thapsigargin [a sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) pump inhibitor] significantly attenuated this effect. Similarly, U73122 (a phospholipase C inhibitor) or 2-aminoethoxydiphenyl borate [2-APB, an inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) inhibitor] reduced this effect. Block of ethanol action was also achieved after preincubation with Rp-cAMPS, inhibitor of the adenylate cyclase (AC)/PKA signaling pathway. These data suggest that there is a convergence at the level of IP3R that accounts for presynaptic ethanol effects. At the postsynaptic level, ethanol increased the decay time constant of mIPSCs in a group of neurons (30 ± 10% above control, n = 13/26 cells). On the other hand, the currents activated by exogenously applied glycine were consistently potentiated (55 ± 10% above control, n = 11/12 cells), which suggests that ethanol modulates synaptic and nonsynaptic glycine receptors (GlyRs) in a different fashion. Supporting the role of G protein modulation on ethanol responses, we found that a nonhydrolyzable GTP analog [guanosine 5'-O-(3-thiotriphosphate) (GTPγS)] increased the decay time constant in â¼50% of the neurons (28 ± 12%, n = 11/19 cells) but potentiated the glycine-activated Cl(-) current in most of the neurons examined (83 ± 29%, n = 7/9 cells). In addition, confocal microscopy showed that α1-containing GlyRs colocalized with Gß and Piccolo (a presynaptic cytomatrix protein) in â¼40% of synaptic receptor clusters, suggesting that colocalization of Gßγ and GlyRs might account for the difference in ethanol sensitivity at the postsynaptic level.
Assuntos
Depressores do Sistema Nervoso Central/farmacologia , Etanol/farmacologia , Glicina/metabolismo , Neurônios/efeitos dos fármacos , Medula Espinal/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacos , Inibidores de Adenilil Ciclases , Adenilil Ciclases/metabolismo , Animais , Cálcio/metabolismo , Células Cultivadas , Canais de Cloreto/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Guanosina Trifosfato/análogos & derivados , Guanosina Trifosfato/metabolismo , Potenciais Pós-Sinápticos Inibidores/efeitos dos fármacos , Receptores de Inositol 1,4,5-Trifosfato/antagonistas & inibidores , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Camundongos Endogâmicos C57BL , Potenciais Pós-Sinápticos em Miniatura/efeitos dos fármacos , Neurônios/fisiologia , Terminações Pré-Sinápticas/efeitos dos fármacos , Terminações Pré-Sinápticas/fisiologia , Receptores de Glicina/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/antagonistas & inibidores , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Medula Espinal/fisiologia , Transmissão Sináptica/fisiologia , Fosfolipases Tipo C/antagonistas & inibidores , Fosfolipases Tipo C/metabolismoRESUMO
Many drugs of abuse, including alcohol, disrupt long-term synaptic depression (LTD) at dorsal striatal glutamate synapses. This disruption is common to many forms of LTD that are mediated by G protein coupled receptors (GPCRs) that signal through the inhibitory Gi/o class of G proteins. A loss of LTD is thought to mediate behavioral changes associated with the development of substance use disorders. We have previously shown in multiple studies that LTD mediated by the Gi/o-coupled mu opioid receptor is disrupted by in vivo opioid and alcohol exposure in adolescent and adult mice. One of our previous studies suggested that LTD mediated by delta and kappa opioid receptors was resistant to the LTD-disrupting properties of in vivo opioid exposure. We hypothesized that delta and kappa opioid receptor-mediated LTD would be exceptions to the generalizable observation that forms of dorsal striatal Gi/o-coupled receptor LTD are disrupted by drugs of abuse. Specifically, we predicted that these forms of LTD would be resistant to the deleterious effects of alcohol consumption, just as they were resistant to opioid exposure. Indeed, in adult male mice that drank alcohol for 3 weeks, delta and kappa opioid receptor-mediated LTD at glutamatergic inputs to direct pathway and indirect pathway medium spiny neurons in the dorsolateral striatum was unaffected by alcohol. These data demonstrate that alcohol effects on GPCR-mediated LTD are not generalizable across all types of Gi/o-coupled GPCRs.
Assuntos
Consumo de Bebidas Alcoólicas , Corpo Estriado , Depressão Sináptica de Longo Prazo , Receptores Opioides delta , Receptores Opioides kappa , Animais , Masculino , Camundongos , Consumo de Bebidas Alcoólicas/metabolismo , Consumo de Bebidas Alcoólicas/psicologia , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Etanol/farmacologia , Depressão Sináptica de Longo Prazo/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Receptores Opioides delta/metabolismo , Receptores Opioides kappa/metabolismoRESUMO
BACKGROUND: Gßγ interaction with GlyR is an important determinant in ethanol potentiation of this channel. RESULTS: A small peptide, RQH(C7), can inhibit ethanol potentiation of GlyR currents. CONCLUSION: Results with RQH(C7) indicate that ethanol mediated potentiation of GlyR is in part by Gßγ activation. SIGNIFICANCE: Molecular interaction between Gßγ and GlyR could be used as a target for pharmacological modification of ethanol effects. Previous studies indicate that ethanol can modulate glycine receptors (GlyR), in part, through Gßγ interaction with basic residues in the intracellular loop. In this study, we show that a seven-amino acid peptide (RQH(C7)), which has the primary structure of a motif in the large intracellular loop of GlyR (GlyR-IL), was able to inhibit the ethanol-elicited potentiation of this channel from 47 ± 2 to 16 ± 4%, without interfering with the effect of Gßγ on GIRK (G protein activated inwardly rectifying potassium channel) activation. RQH(C7) displayed a concentration-dependent effect on ethanol action in evoked and synaptic currents. A fragment of GlyR-IL without the basic amino acids did not interact with Gßγ or inhibit ethanol potentiation of GlyR. In silico analysis using docking and molecular dynamics allowed to identify a region of ~350Å(2) involving aspartic acids 186, 228, and 246 in Gßγ where we propose that RQH(C7) binds and exerts its blocking action on the effect of ethanol in GlyR.
Assuntos
Etanol/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Peptídeos/metabolismo , Receptores de Glicina/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/química , Subunidades beta da Proteína de Ligação ao GTP/genética , Subunidades gama da Proteína de Ligação ao GTP/genética , Células HEK293 , Humanos , Cinética , Peptídeos/química , Ligação Proteica , Receptores de Glicina/química , Receptores de Glicina/genéticaRESUMO
Corticostriatal circuits are generally characterized by the release of glutamate neurotransmitter from cortical terminals within the striatum. It is well known that cortical excitatory input to the dorsal striatum regulates addictive drug-related behaviors. We previously reported that anterior insular cortex (AIC) synaptic inputs to the dorsolateral striatum (DLS) control binge alcohol drinking in mice. These AIC-DLS glutamate synapses are also the sole sites of corticostriatal mu opioid receptor-mediated excitatory long-term depression (MOR-LTD) in the DLS. Recent work demonstrates that some regions of cortex send long-range, direct inhibitory inputs into the dorsal striatum. Nothing is known about the existence and regulation of AIC-DLS inhibitory synaptic transmission. Here, using a combination of patch clamp electrophysiology and optogenetics, we characterized a novel AIC-DLS corticostriatal inhibitory circuit and its regulation by MOR-mediated inhibitory LTD (MOR-iLTD). First, we found that the activation of presynaptic MORs produces MOR-iLTD in the DLS and dorsomedial striatum. Then, we showed that medium spiny neurons within the DLS receive direct inhibitory synaptic input from the cortex, specifically from the motor cortex and AIC. Using transgenic mice that express cre-recombinase within parvalbumin-expressing inhibitory neurons, we determined that this specific cortical neuron subtype sends direct GABAergic projections to the DLS. Moreover, these AIC-DLS inhibitory synaptic input subtypes express MOR-iLTD. These data suggest a novel GABAergic corticostriatal circuit that could be involved in the regulation of drug and alcohol consumption-related behaviors.
Assuntos
Plasticidade Neuronal , Receptores Opioides mu , Camundongos , Animais , Receptores Opioides mu/genética , Receptores Opioides mu/metabolismo , Plasticidade Neuronal/fisiologia , Transmissão Sináptica/fisiologia , Corpo Estriado/metabolismo , Camundongos Transgênicos , GlutamatosRESUMO
Opioids mediate their effects via opioid receptors: mu, delta, and kappa. At the neuronal level, opioid receptors are generally inhibitory, presynaptically reducing neurotransmitter release and postsynaptically hyperpolarizing neurons. However, opioid receptor-mediated regulation of neuronal function and synaptic transmission is not uniform in expression pattern and mechanism across the brain. The localization of receptors within specific cell types and neurocircuits determine the effects that endogenous and exogenous opioids have on brain function. In this review we will explore the similarities and differences in opioid receptor-mediated regulation of neurotransmission across different brain regions. We discuss how future studies can consider potential cell-type, regional, and neural pathway-specific effects of opioid receptors in order to better understand how opioid receptors modulate brain function.
RESUMO
How does binge drinking alcohol change synaptic function, and do these changes maintain binge consumption? The anterior insular cortex (AIC) and dorsolateral striatum (DLS) are brain regions implicated in alcohol use disorder. In male, but not female mice, we found that binge drinking alcohol produced glutamatergic synaptic adaptations selective to AIC inputs within the DLS. Photoexciting AICâDLS circuitry in male mice during binge drinking decreased alcohol, but not water consumption and altered alcohol drinking mechanics. Further, drinking mechanics alone from drinking session data predicted alcohol-related circuit changes. AICâDLS manipulation did not alter operant, valence, or anxiety-related behaviors. These findings suggest that alcohol-mediated changes at AIC inputs govern behavioral sequences that maintain binge drinking and may serve as a circuit-based biomarker for the development of alcohol use disorder.
Assuntos
Alcoolismo , Consumo Excessivo de Bebidas Alcoólicas , Consumo de Bebidas Alcoólicas , Animais , Etanol , Córtex Insular , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Rising opioid use among pregnant women has led to a growing population of neonates exposed to opioids during the prenatal period, but how opioids affect the developing brain remains to be fully understood. Animal models of prenatal opioid exposure have discovered deficits in somatosensory behavioral development that persist into adolescence suggesting opioid exposure induces long lasting neuroadaptations on somatosensory circuitry such as the primary somatosensory cortex (S1). Using a mouse model of prenatal methadone exposure (PME) that displays delays in somatosensory milestone development, we performed an un-biased multi-omics analysis and investigated synaptic functioning in the primary somatosensory cortex (S1), where touch and pain sensory inputs are received in the brain, of early adolescent PME offspring. PME was associated with numerous changes in protein and phosphopeptide abundances that differed considerably between sexes in the S1. Although prominent sex effects were discovered in the multi-omics assessment, functional enrichment analyses revealed the protein and phosphopeptide differences were associated with synapse-related cellular components and synaptic signaling-related biological processes, regardless of sex. Immunohistochemical analysis identified diminished GABAergic synapses in both layer 2/3 and 4 of PME offspring. These immunohistochemical and proteomic alterations were associated with functional consequences as layer 2/3 pyramidal neurons revealed reduced amplitudes and a lengthened decay constant of inhibitory postsynaptic currents. Lastly, in addition to reduced cortical thickness of the S1, cell-type marker analysis revealed reduced microglia density in the upper layer of the S1 that was primarily driven by PME females. Taken together, our studies show the lasting changes on synaptic function and microglia in S1 cortex caused by PME in a sex-dependent manner.
RESUMO
The opioid crisis has contributed to a growing population of children exposed to opioids during fetal development; however, many of the long-term effects of opioid exposure on development are unknown. We previously demonstrated that opioids have deleterious effects on endocannabinoid plasticity at glutamate synapses in the dorsal striatum of adolescent rodents, but it is unclear whether prenatal opioid exposure produces similar neuroadaptations. Using a mouse model of prenatal methadone exposure (PME), we performed proteomics, phosphoproteomics, and patch-clamp electrophysiology in the dorsolateral striatum (DLS) and dorsomedial striatum (DMS) to examine synaptic functioning in adolescent PME offspring. PME impacted the proteome and phosphoproteome in a region- and sex-dependent manner. Many proteins and phosphorylated proteins associated with glutamate transmission were differentially abundant in PME offspring, which was associated with reduced glutamate release in the DLS and altered the rise time of excitatory events in the DMS. Similarly, the intrinsic excitability properties of DMS neurons were significantly affected by PME. Last, pathway analyses revealed an enrichment in retrograde endocannabinoid signaling in the DLS, but not in the DMS, of males. Electrophysiology studies confirmed that endocannabinoid-mediated synaptic depression was impaired in the DLS, but not DMS, of PME-males. These results indicate that PME induces persistent neuroadaptations in the dorsal striatum and could contribute to the aberrant behavioral development described in offspring with prenatal opioid exposure.
Assuntos
Analgésicos Opioides , Ácido Glutâmico , Analgésicos Opioides/farmacologia , Corpo Estriado/metabolismo , Endocanabinoides/metabolismo , Feminino , Ácido Glutâmico/metabolismo , Humanos , Masculino , Gravidez , Sinapses/metabolismoRESUMO
The importance of copper in the CNS is well documented, but the mechanisms related to its brain functions are poorly understood. Copper is released at the synaptic cleft, where it may modulate neurotransmission. To understand the functional impact of copper on the neuronal network, we have analyzed the synaptic activity of primary rat hippocampal neurons by using different approaches including whole cell patch clamp, recording of calcium transients, immunofluorescence and western blot. Here, we show that copper produces biphasic changes in neurotransmission. When copper is acutely applied to the plate it blocks neurotransmission. Interestingly, when it is applied for 3 h to hippocampal neurons it mainly increases the frequency and amplitude of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)ergic currents (control: 0.21 ± 0.05 Hz/22.9 ± 1.3 pA; copper: 0.68 ± 0.16 Hz/30.5 ± 2.5 pA), intracellular calcium transients (control: 0.05 ± 0.013 Hz; copper: 0.11 ± 0.02 Hz) and evoked AMPA currents (control: EC50 8.3 ± 0.5 µM; copper: EC50 2.9 ± 0.2 µM). Moreover, our results suggest that copper increases GluA1 subunit levels of the AMPA receptor through the anchorage of AMPA receptors to the plasma membrane as a result of PSD-95 accumulation. We also found that copper-treated neurons displayed an undistinguishable neurotransmission to control neurons after 24 h of treatment, indicating that changes in neurotransmission induced by copper at 3 h of incubation are homeostatically regulated after long-term exposure to the metal. Together, our data reveal an unexpected biphasic effect of copper on neurotransmission, which may be relevant to understand the effects of this ion in brain diseases that display copper dyshomeostasis such as that observed in Alzheimer's disease (AD).
Assuntos
Cobre/farmacologia , Hipocampo/metabolismo , Neurônios/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacos , Animais , Western Blotting , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/fisiologia , Células Cultivadas , Proteína 4 Homóloga a Disks-Large , Fenômenos Eletrofisiológicos , Feminino , Imunofluorescência , Hipocampo/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Técnicas de Patch-Clamp , Gravidez , Ratos , Ratos Sprague-Dawley , Receptores de AMPA/efeitos dos fármacos , Sinapses/efeitos dos fármacosRESUMO
Alpha1-containing glycine receptors (GlyRs) are major mediators of synaptic inhibition in the spinal cord and brain stem. Recent studies reported the presence of α2-containing GlyRs in other brain regions, such as nucleus accumbens and cerebral cortex. GlyR activation decreases neuronal excitability associated with sensorial information, motor control, and respiratory functions; all of which are significantly altered during ethanol intoxication. We evaluated the role of ß GlyR subunits and of two basic amino acid residues, K389 and R390, located in the large intracellular loop (IL) of the α2 GlyR subunit, which are important for binding and functional modulation by Gßγ, the dimer of the trimeric G protein conformation, using HEK-293 transfected cells combined with patch clamp electrophysiology. We demonstrate a new modulatory role of the ß subunit on ethanol sensitivity of α2 subunits. Specifically, we found a differential allosteric modulation in homomeric α2 GlyRs compared with the α2ß heteromeric conformation. Indeed, while α2 was insensitive, α2ß GlyRs were substantially potentiated by ethanol, GTP-γ-S, propofol, Zn2+ and trichloroethanol. Furthermore, a Gßγ scavenger (ct-GRK2) selectively attenuated the effects of ethanol on recombinant α2ß GlyRs. Mutations in an α2 GlyR co-expressed with the ß subunit (α2AAß) specifically blocked ethanol sensitivity, but not propofol potentiation. These results show a selective mechanism for low ethanol concentration effects on homomeric and heteromeric conformations of α2 GlyRs and provide a new mechanism for ethanol pharmacology, which is relevant to upper brain regions where α2 GlyRs are abundantly expressed.
RESUMO
BACKGROUND AND PURPOSE: Glycine receptors composed of α1 and ß subunits are primarily found in the spinal cord and brainstem and are potentiated by ethanol (10-100 mM). However, much less is known about the presence, composition and ethanol sensitivity of these receptors in higher CNS regions. Here, we examined two regions of the brain reward system, the ventral tegmental area (VTA) and the prefrontal cortex (PFC), to determine their glycine receptor subunit composition and sensitivity to ethanol. EXPERIMENTAL APPROACH: We used Western blot, immunohistochemistry and electrophysiological techniques in three different models: wild-type C57BL/6, glycine receptor subunit α1 knock-in and glycine receptor subunit α2 knockout mice. KEY RESULTS: Similar levels of α and ß receptor subunits were detected in both brain regions, and electrophysiological recordings demonstrated the presence of glycine-activated currents in both areas. Sensitivity of glycine receptors to glycine was lower in the PFC compared with VTA. Picrotoxin only partly blocked the glycine-activated current in the PFC and VTA, indicating that both regions express heteromeric αß receptors. Glycine receptors in VTA neurons, but not in PFC neurons, were potentiated by ethanol. CONCLUSION AND IMPLICATIONS: Glycine receptors in VTA neurons from WT and α2 KO mice were potentiated by ethanol, but not in neurons from the α1 KI mice, supporting the conclusion that α1 glycine receptors are predominantly expressed in the VTA. By contrast, glycine receptors in PFC neurons were not potentiated in any of the mouse models studied, suggesting the presence of α2/α3/α4, rather than α1 glycine receptor subunits.
Assuntos
Receptores de Glicina , Área Tegmentar Ventral , Animais , Etanol/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Córtex Pré-Frontal/metabolismo , Receptores de Glicina/metabolismo , Área Tegmentar Ventral/metabolismoRESUMO
Intracellular amyloid beta oligomer (iAßo) accumulation and neuronal hyperexcitability are two crucial events at early stages of Alzheimer's disease (AD). However, to date, no mechanism linking iAßo with an increase in neuronal excitability has been reported. Here, the effects of human AD brain-derived (h-iAßo) and synthetic (iAßo) peptides on synaptic currents and action potential firing were investigated in hippocampal neurons. Starting from 500 pM, iAßo rapidly increased the frequency of synaptic currents and higher concentrations potentiated the AMPA receptor-mediated current. Both effects were PKC-dependent. Parallel recordings of synaptic currents and nitric oxide (NO)-associated fluorescence showed that the increased frequency, related to pre-synaptic release, was dependent on a NO-mediated retrograde signaling. Moreover, increased synchronization in NO production was also observed in neurons neighboring those dialyzed with iAßo, indicating that iAßo can increase network excitability at a distance. Current-clamp recordings suggested that iAßo increased neuronal excitability via AMPA-driven synaptic activity without altering membrane intrinsic properties. These results strongly indicate that iAßo causes functional spreading of hyperexcitability through a synaptic-driven mechanism and offers an important neuropathological significance to intracellular species in the initial stages of AD, which include brain hyperexcitability and seizures.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Sinapses/metabolismo , Animais , Feminino , Humanos , Masculino , Gravidez , Ratos , Ratos Sprague-Dawley , Ratos WistarRESUMO
The dorsal striatum is a brain region involved in action control, with dorsomedial striatum (DMS) mediating goal-directed actions and dorsolateral striatum (DLS) mediating habitual actions. Presynaptic long-term synaptic depression (LTD) plasticity at glutamatergic inputs to dorsal striatum mediates many dorsal striatum-dependent behaviors and disruption of LTD influences action control. Our previous work identified mu opioid receptors (MORs) as mediators of synapse-specific forms of synaptic depression at a number of different DLS synapses. We demonstrated that anterior insular cortex inputs are the sole inputs that express alcohol-sensitive MOR-mediated LTD (mOP-LTD) in DLS. Here, we explore mOP-LTD in DMS using mouse brain slice electrophysiology. We found that contrary to DLS, DMS mOP-LTD is induced by activation of MORs at inputs from both anterior cingulate and medial prefrontal cortices as well as at basolateral amygdala inputs and striatal cholinergic interneuron synapses on to DMS medium spiny neurons, suggesting that MOR synaptic plasticity in DMS is less synapse-specific than in DLS. Furthermore, only mOP-LTD at cortical inputs was sensitive to alcohol's deleterious effects. These results suggest that alcohol-induced neuroadaptations are differentially expressed in a synapse-specific manner and could be playing a role in alterations of goal-directed and habitual behaviors.
Assuntos
Corpo Estriado/metabolismo , Regulação da Expressão Gênica , Depressão Sináptica de Longo Prazo , Receptores Opioides mu/biossíntese , Sinapses/metabolismo , Animais , Corpo Estriado/citologia , Masculino , Camundongos , Camundongos Knockout , Receptores Opioides mu/genética , Sinapses/genéticaRESUMO
Cerebral amyloid angiopathy (CAA) is typified by the cerebrovascular deposition of amyloid. The mechanisms underlying the contribution of CAA to neurodegeneration are not currently understood. Although CAA is highly associated with the accumulation of ß-amyloid (Aß), other amyloids are known to associate with the vasculature. Alzheimer's disease (AD) is characterized by parenchymal Aß deposition and intracellular accumulation of tau as neurofibrillary tangles (NFTs), affecting synapses directly, leading to behavioral and physical impairment. CAA increases with age and is present in 70%-97% of individuals with AD. Studies have overwhelmingly focused on the connection between parenchymal amyloid accumulation and synaptotoxicity; thus, the contribution of vascular amyloid is mostly understudied. Here, synaptic alterations induced by vascular amyloid accumulation and their behavioral consequences were characterized using a mouse model of Familial Danish dementia (FDD), a neurodegenerative disease characterized by the accumulation of Danish amyloid (ADan) in the vasculature. The mouse model (Tg-FDD) displays a hyperactive phenotype that potentially arises from impairment in the GABAergic synapses, as determined by electrophysiological analysis. We demonstrated that the disruption of GABAergic synapse organization causes this impairment and provided evidence that GABAergic synapses are impaired in patients with CAA pathology. Understanding the mechanism that CAA contributes to synaptic dysfunction in AD-related dementias is of critical importance for developing future therapeutic interventions.