Browsing by Author "Muñoz, Braulio"
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Item Alcohol exposure disrupts mu opioid receptor-mediated long-term depression at insular cortex inputs to dorsolateral striatum(Nature Publishing group, 2018-04-03) Muñoz, Braulio; Fritz, Brandon M.; Yin, Fuqin; Atwood, Brady K.; Psychiatry, School of MedicineDrugs of abuse, including alcohol, ablate the expression of specific forms of long-term synaptic depression (LTD) at glutamatergic synapses in dorsal striatum (DS), a brain region involved in goal-directed and habitual behaviors. This loss of LTD is associated with altered DS-dependent behavior. Given the role of the µ-opioid receptor (MOR) in behavioral responding for alcohol, we explored the impact of alcohol on various forms of MOR-mediated synaptic depression that we find are differentially expressed at specific DS synapses. Corticostriatal MOR-mediated LTD (mOP-LTD) in the dorsolateral striatum occurs exclusively at inputs from anterior insular cortex and is selectively disrupted by in vivo alcohol exposure. Alcohol has no effect on corticostriatal mOP-LTD in dorsomedial striatum, thalamostriatal MOR-mediated short-term depression, or mOP-LTD of cholinergic interneuron-driven glutamate release. Disrupted mOP-LTD at anterior insular cortex–dorsolateral striatum synapses may therefore be a key mechanism of alcohol-induced neuroadaptations involved in the development of alcohol use disorders., µ-opioid receptors (MOR) are known to modulate the reward effects of drugs of abuse, and MOR activation induces long-term depression (LTD) at striatal synapses. Here the authors show that alcohol exposure disrupts MOR-induced LTD only at specific cortical inputs to the striatum.Item A High-fat, High-sugar ‘Western’ Diet Alters Dorsal Striatal Glutamate, Opioid, and Dopamine Transmission in Mice(Elsevier, 2018-02) Fritz, Brandon M.; Muñoz, Braulio; Yin, Fuqin; Bauchle, Casey; Atwood, Brady K.; Psychiatry, School of MedicineUnderstanding neuroadaptations involved in obesity is critical for developing new approaches to treatment. Diet-induced neuroadaptations within the dorsal striatum have the capacity to drive excessive food seeking and consumption. Five-week-old C57BL/6J mice consumed a high-fat, high-sugar ‘western diet’ (WD) or a control ‘standard diet’ (SD) for 16 weeks. Weight gain, glucose tolerance, and insulin tolerance were measured to confirm an obese-like state. Following these 16 weeks, electrophysiological recordings were made from medium spiny neurons (MSNs) in the medial (DMS) and lateral (DLS) portions of dorsal striatum to evaluate diet effects on neuronal excitability and synaptic plasticity. In addition, fast-scan cyclic voltammetry evaluated dopamine transmission in these areas. WD mice gained significantly more weight and consumed more calories than SD mice and demonstrated impaired glucose tolerance. Electrophysiology data revealed that MSNs from WD mice demonstrated increased AMPA-to-NMDA receptor current ratio and prolonged spontaneous glutamate-mediated currents, specifically in the DLS. Evoked dopamine release was also significantly greater and reuptake slower in both subregions of WD striatum. Finally, dorsal striatal MSNs from WD mice were significantly less likely to demonstrate mu-opioid receptor-mediated synaptic plasticity. Neuronal excitability and GABAergic transmission were unaffected by diet in either striatal subregion. Our results demonstrate that a high-fat, high-sugar diet alters facets of glutamate, dopamine, and opioid signaling within the dorsal striatum, with some subregion specificity. These alterations within a brain area known to play a role in food motivation/consumption and habitual behavior are highly relevant for the clinical condition of obesity and its treatment.Item Influence of nonsynaptic α1 glycine receptors on ethanol consumption and place preference(Wiley, 2019-03-18) Muñoz, Braulio; Gallegos, Scarlet; Peters, Christian; Murath, Pablo; Lovinger, David M.; Homanics, Gregg E.; Aguayo, Luis G.; Pharmacology and Toxicology, School of MedicineAlcohol 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 (LGIC), such as glycine receptors (GlyR), 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 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 non-synaptic α1 containing GlyRs have a role in motivational and early reinforcing effects of ethanol and opens a novel opportunity for pharmacotherapy development to treat alcohol use disorders.Item Modulation of glycine receptor single-channel conductance by intracellular phosphorylation(Springer Nature, 2020) Moraga-Cid, Gustavo; San Martín, Victoria P.; Lara, Cesar O.; Muñoz, Braulio; Marileo, Ana M.; Sazo, Anggelo; Muñoz-Montesino, Carola; Fuentealba, Jorge; Castro, Patricio A.; Guzmán, Leonardo; Burgos, Carlos F.; Zeilhofer, Hanns U.; Aguayo, Luis G.; Corringer, Pierre-Jean; Yévenes, Gonzalo E.; Pharmacology and Toxicology, School of MedicineGlycine receptors (GlyRs) are anion-permeable pentameric ligand-gated ion channels (pLGICs). The GlyR activation is critical for the control of key neurophysiological functions, such as motor coordination, respiratory control, muscle tone and pain processing. The relevance of the GlyR function is further highlighted by the presence of abnormal glycinergic inhibition in many pathophysiological states, such as hyperekplexia, epilepsy, autism and chronic pain. In this context, previous studies have shown that the functional inhibition of GlyRs containing the α3 subunit is a pivotal mechanism of pain hypersensitivity. This pathway involves the activation of EP2 receptors and the subsequent PKA-dependent phosphorylation of α3GlyRs within the intracellular domain (ICD), which decrease the GlyR-associated currents and enhance neuronal excitability. Despite the importance of this mechanism of glycinergic dis-inhibition associated with dysfunctional α3GlyRs, our current understanding of the molecular events involved is limited. Here, we report that the activation of PKA signaling pathway decreases the unitary conductance of α3GlyRs. We show in addition that the substitution of the PKA-targeted serine with a negatively charged residue within the ICD of α3GlyRs and of chimeric receptors combining bacterial GLIC and α3GlyR was sufficient to generate receptors with reduced conductance. Thus, our findings reveal a potential biophysical mechanism of glycinergic dis-inhibition and suggest that post-translational modifications of the ICD, such as phosphorylation, may shape the conductance of other pLGICs.Item Modulatory Actions of the Glycine Receptor β Subunit on the Positive Allosteric Modulation of Ethanol in α2 Containing Receptors(Frontiers Media, 2021-11-18) Muñoz, Braulio; Mariqueo, Trinidad; Murath, Pablo; Peters, Christian; Yevenes, Gonzalo E.; Moraga-Cid, Gustavo; Peoples, Robert W.; Aguayo, Luis G.; Pharmacology and Toxicology, School of MedicineAlpha1-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 β 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 Gβγ, 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 β subunit on ethanol sensitivity of α2 subunits. Specifically, we found a differential allosteric modulation in homomeric α2 GlyRs compared with the α2β heteromeric conformation. Indeed, while α2 was insensitive, α2β GlyRs were substantially potentiated by ethanol, GTP-γ-S, propofol, Zn2+ and trichloroethanol. Furthermore, a Gβγ scavenger (ct-GRK2) selectively attenuated the effects of ethanol on recombinant α2β GlyRs. Mutations in an α2 GlyR co-expressed with the β subunit (α2AAβ) 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.Item Mu opioid receptors on vGluT2‐expressing glutamatergic neurons modulate opioid reward(Wiley, 2021-05) Reeves, Kaitlin C.; Kube, Megan J.; Grecco, Gregory G.; Fritz, Brandon M.; Muñoz, Braulio; Yin, Fuqin; Gao, Yong; Haggerty, David L.; Hoffman, Hunter J.; Atwood, Brady K.; Pharmacology and Toxicology, School of MedicineThe 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.Item Opioid Receptor-Mediated Regulation of Neurotransmission in the Brain(Frontiers Media, 2022-06-15) Reeves, Kaitlin C.; Shah, Nikhil; Muñoz, Braulio; Atwood, Brady K.; Pharmacology and Toxicology, School of MedicineOpioids 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.Item Prenatal Opioid Exposure Impairs Endocannabinoid and Glutamate Transmission in the Dorsal Striatum(Society for Neuroscience, 2022-04-20) Grecco, Gregory G.; Muñoz, Braulio; Di Prisco, Gonzalo Viana; Doud, Emma H.; Fritz, Brandon M.; Maulucci, Danielle; Gao, Yong; Mosley, Amber L.; Baucum, Anthony J.; Atwood, Brady K.; Pharmacology and Toxicology, School of MedicineThe 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.Item Presence of ethanol-sensitive and ethanol-insensitive glycine receptors in the ventral tegmental area and prefrontal cortex in mice(Wiley, 2021) Araya, Anibal; Gallegos, Scarlet; Viveros, Rodrigo; San Martin, Loreto; Muñoz, Braulio; Harvey, Robert J.; Zeilhofer, Hanns U.; Aguayo, Luis G.; Pharmacology and Toxicology, School of MedicineBackground and purpose: Glycine receptors composed of α1 and β 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 β 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 αβ 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.Item Sex-Dependent Synaptic Remodeling of the Somatosensory Cortex in Mice With Prenatal Methadone Exposure(Frontiers Media, 2022) Grecco, Gregory G.; Huang, Jui Yen; Muñoz, Braulio; Doud, Emma H.; Hines, Caliel D.; Gao, Yong; Rodriguez, Brooke; Mosley, Amber L.; Lu, Hui-Chen; Atwood, Brady K.; Pharmacology and Toxicology, School of MedicineRising 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.