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1.
eNeuro ; 8(5)2021.
Artículo en Inglés | MEDLINE | ID: mdl-34535504

RESUMEN

The hedgehog signaling pathway is best known for its role in developmental patterning of the neural tube and limb bud. More recently, hedgehog signaling has been recognized for its roles in growth of adult tissues and maintenance of progenitor cell niches. However, the role of hedgehog signaling in fully differentiated cells like neurons in the adult brain is less clear. In mammals, coordination of hedgehog pathway activity relies on primary cilia and patients with ciliopathies such as Bardet-Biedl and Alström syndrome exhibit clinical features clearly attributable to errant hedgehog such as polydactyly. However, these ciliopathies also present with features not clearly associated with hedgehog signaling such as hyperphagia-associated obesity. How hedgehog signaling may contribute to feeding behavior is complex and unclear, but cilia are critical for proper energy homeostasis. Here, we provide a detailed analysis of the expression of core components of the hedgehog signaling pathway in the adult mouse hypothalamus with an emphasis on feeding centers. We show that hedgehog pathway genes continue to be expressed in differentiated neurons important for the regulation of feeding behavior. Furthermore, we demonstrate for the first time that pathway activity is regulated at the transcriptional level by fasting. These data suggest that hedgehog signaling is involved in the proper functioning of brain regions that regulate feeding behavior and that hedgehog pathway dysfunction may play a role in the obesity observed in certain ciliopathies.


Asunto(s)
Ayuno , Proteínas Hedgehog , Animales , Cilios/metabolismo , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Humanos , Hipotálamo/metabolismo , Ratones , Transducción de Señal
2.
Genesis ; 59(7-8): e23438, 2021 08.
Artículo en Inglés | MEDLINE | ID: mdl-34124835

RESUMEN

Cilia on neurons play critical roles in both the development and function of the central nervous system (CNS). While it remains challenging to elucidate the precise roles for neuronal cilia, it is clear that a subset of G-protein-coupled receptors (GPCRs) preferentially localize to the cilia membrane. Further, ciliary GPCR signaling has been implicated in regulating a variety of behaviors. Melanin concentrating hormone receptor 1 (MCHR1), is a GPCR expressed centrally in rodents known to be enriched in cilia. Here we have used MCHR1 as a model ciliary GPCR to develop a strategy to fluorescently tag receptors expressed from the endogenous locus in vivo. Using CRISPR/Cas9, we inserted the coding sequence of the fluorescent protein mCherry into the N-terminus of Mchr1. Analysis of the fusion protein (mCherry MCHR1) revealed its localization to neuronal cilia in the CNS, across multiple developmental time points and in various regions of the adult brain. Our approach simultaneously produced fortuitous in/dels altering the Mchr1 start codon resulting in a new MCHR1 knockout line. Functional studies using electrophysiology show a significant alteration of synaptic strength in MCHR1 knockout mice. A reduction in strength is also detected in mice homozygous for the mCherry insertion, suggesting that while the strategy is useful for monitoring the receptor, activity could be altered. However, both lines should aid in studies of MCHR1 function and contribute to our understanding of MCHR1 signaling in the brain. Additionally, this approach could be expanded to aid in the study of other ciliary GPCRs.

3.
J Vis Exp ; (171)2021 05 01.
Artículo en Inglés | MEDLINE | ID: mdl-33999029

RESUMEN

Cilia are microtubule based cellular appendages that function as signaling centers for a diversity of signaling pathways in many mammalian cell types. Cilia length is highly conserved, tightly regulated, and varies between different cell types and tissues and has been implicated in directly impacting their signaling capacity. For example, cilia have been shown to alter their lengths in response to activation of ciliary G protein-coupled receptors. However, accurately and reproducibly measuring the lengths of numerous cilia is a time-consuming and labor-intensive procedure. Current approaches are also error and bias prone. Artificial intelligence (Ai) programs can be utilized to overcome many of these challenges due to capabilities that permit assimilation, manipulation, and optimization of extensive data sets. Here, we demonstrate that an Ai module can be trained to recognize cilia in images from both in vivo and in vitro samples. After using the trained Ai to identify cilia, we are able to design and rapidly utilize applications that analyze hundreds of cilia in a single sample for length, fluorescence intensity and co-localization. This unbiased approach increased our confidence and rigor when comparing samples from different primary neuronal preps in vitro as well as across different brain regions within an animal and between animals. Moreover, this technique can be used to reliably analyze cilia dynamics from any cell type and tissue in a high-throughput manner across multiple samples and treatment groups. Ultimately, Ai-based approaches will likely become standard as most fields move toward less biased and more reproducible approaches for image acquisition and analysis.


Asunto(s)
Inteligencia Artificial , Cilios , Animales , Microtúbulos , Receptores Acoplados a Proteínas G , Transducción de Señal
4.
Hum Mol Genet ; 30(3-4): 234-246, 2021 04 26.
Artículo en Inglés | MEDLINE | ID: mdl-33560420

RESUMEN

Primary cilia are critical sensory and signaling compartments present on most mammalian cell types. These specialized structures require a unique signaling protein composition relative to the rest of the cell to carry out their functions. Defects in ciliary structure and signaling result in a broad group of disorders collectively known as ciliopathies. One ciliopathy, Bardet-Biedl syndrome (BBS; OMIM 209900), presents with diverse clinical features, many of which are attributed to defects in ciliary signaling during both embryonic development and postnatal life. For example, patients exhibit obesity, polydactyly, hypogonadism, developmental delay and skeletal abnormalities along with sensory and cognitive deficits, but for many of these phenotypes it is uncertain, which are developmental in origin. A subset of BBS proteins assembles into the core BBSome complex, which is responsible for mediating transport of membrane proteins into and out of the cilium, establishing it as a sensory and signaling hub. Here, we describe two new mouse models for BBS resulting from a targeted LacZ gene trap allele (Bbs5-/-) that is a predicted congenital null mutation and conditional (Bbs5flox/flox) allele of Bbs5. Bbs5-/- mice develop a complex phenotype consisting of increased pre-weaning lethality craniofacial and skeletal defects, ventriculomegaly, infertility and pituitary anomalies. Utilizing the conditional allele, we show that the male fertility defects, ventriculomegaly and pituitary abnormalities are only present when Bbs5 is disrupted prior to postnatal day 7, indicating a developmental origin. In contrast, mutation of Bbs5 results in obesity, independent of the age of Bbs5 loss.


Asunto(s)
Síndrome de Bardet-Biedl/metabolismo , Proteínas del Citoesqueleto/genética , Modelos Animales de Enfermedad , Mutación , Proteínas de Unión a Fosfato/genética , Hipófisis/anomalías , Animales , Síndrome de Bardet-Biedl/genética , Síndrome de Bardet-Biedl/patología , Síndrome de Bardet-Biedl/fisiopatología , Proteínas del Citoesqueleto/metabolismo , Masculino , Ratones , Fenotipo , Proteínas de Unión a Fosfato/metabolismo , Hipófisis/crecimiento & desarrollo , Hipófisis/metabolismo
5.
Semin Cell Dev Biol ; 110: 43-50, 2021 02.
Artículo en Inglés | MEDLINE | ID: mdl-32466971

RESUMEN

An emerging number of rare genetic disorders termed ciliopathies are associated with pediatric obesity. It is becoming clear that the mechanisms associated with cilia dysfunction and obesity in these syndromes are complex. In addition to ciliopathic syndromic forms of obesity, several cilia-associated signaling gene mutations also lead to morbid obesity. While cilia have critical and diverse functions in energy homeostasis including their roles in centrally mediated food intake as well as in peripheral tissues, many questions remain. Here, we briefly discuss the syndromic ciliopathies and monoallelic cilia signaling gene mutations associated with obesity. We also describe potential ways cilia may be involved in common obesity. We discuss how neuronal cilia impact food intake potentially through leptin signaling and changes in ciliary G protein-coupled receptor (GPCR) signaling. We highlight several recent studies that have implicated the potential for cilia in peripheral tissues such as adipose and the pancreas to contribute to metabolic dysfunction. Then we discuss the potential for cilia to impact energy homeostasis through their roles in both development and adult tissue homeostasis. The studies discussed in this review highlight how a comprehensive understanding of the requirement of cilia for the regulation of diverse biological functions will contribute to our understanding of common forms of obesity.

6.
Front Cell Neurosci ; 13: 266, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31249512

RESUMEN

Primary cilia dysfunction has been associated with hyperphagia and obesity in both ciliopathy patients and mouse models of cilia perturbation. Neurons throughout the brain possess these solitary cellular appendages, including in the feeding centers of the hypothalamus. Several cell biology questions associated with primary neuronal cilia signaling are challenging to address in vivo. Here we utilize primary hypothalamic neuronal cultures to study ciliary signaling in relevant cell types. Importantly, these cultures contain neuronal populations critical for appetite and satiety such as pro-opiomelanocortin (POMC) and agouti related peptide (AgRP) expressing neurons and are thus useful for studying signaling involved in feeding behavior. Correspondingly, these cultured neurons also display electrophysiological activity and respond to both local and peripheral signals that act on the hypothalamus to influence feeding behaviors, such as leptin and melanin concentrating hormone (MCH). Interestingly, we found that cilia mediated hedgehog signaling, generally associated with developmental processes, can influence ciliary GPCR signaling (Mchr1) in terminally differentiated neurons. Specifically, pharmacological activation of the hedgehog-signaling pathway using the smoothened agonist, SAG, attenuated the ability of neurons to respond to ligands (MCH) of ciliary GPCRs. Understanding how the hedgehog pathway influences cilia GPCR signaling in terminally differentiated neurons could reveal the molecular mechanisms associated with clinical features of ciliopathies, such as hyperphagia-associated obesity.

7.
JCI Insight ; 4(3)2019 Feb 07.
Artículo en Inglés | MEDLINE | ID: mdl-30728336

RESUMEN

Intronic polymorphisms in the α-ketoglutarate-dependent dioxygenase gene (FTO) that are highly associated with increased body weight have been implicated in the transcriptional control of a nearby ciliary gene, retinitis pigmentosa GTPase regulator-interacting protein-1 like (RPGRIP1L). Previous studies have shown that congenital Rpgrip1l hypomorphism in murine proopiomelanocortin (Pomc) neurons causes obesity by increasing food intake. Here, we show by congenital and adult-onset Rpgrip1l deletion in Pomc-expressing neurons that the hyperphagia and obesity are likely due to neurodevelopmental effects that are characterized by a reduction in the Pomc/Neuropeptide Y (Npy) neuronal number ratio and marked increases in arcuate hypothalamic-paraventricular hypothalamic (ARH-PVH) axonal projections. Biallelic RPGRIP1L mutations result in fewer cilia-positive human induced pluripotent stem cell-derived (iPSC-derived) neurons and blunted responses to Sonic Hedgehog (SHH). Isogenic human ARH-like embryonic stem cell-derived (ESc-derived) neurons homozygous for the obesity-risk alleles at rs8050136 or rs1421085 have decreased RPGRIP1L expression and have lower numbers of POMC neurons. RPGRIP1L overexpression increases POMC cell number. These findings suggest that apparently functional intronic polymorphisms affect hypothalamic RPGRIP1L expression and impact development of POMC neurons and their derivatives, leading to hyperphagia and increased adiposity.

8.
Eur J Neurosci ; 50(3): 2224-2238, 2019 08.
Artículo en Inglés | MEDLINE | ID: mdl-29779223

RESUMEN

Nicotinic acetylcholine receptors (nAChRs), prototype members of the cys-loop ligand-gated ion channel family, are key mediators of cholinergic transmission in the central nervous system. Despite their importance, technical gaps exist in our ability to dissect the function of individual subunits in the brain. To overcome these barriers, we designed CRISPR/Cas9 small guide RNA sequences (sgRNAs) for the production of loss-of-function alleles in mouse nAChR genes. These sgRNAs were validated in vitro via deep sequencing. We subsequently targeted candidate nAChR genes in vivo by creating herpes simplex virus (HSV) vectors delivering sgRNAs and Cas9 expression to mouse brain. The production of loss-of-function insertions or deletions (indels) by these 'all-in-one' HSV vectors was confirmed using brain slice patch clamp electrophysiology coupled with pharmacological analysis. Next, we developed a scheme for cell type-specific gene editing in mouse brain. Knockin mice expressing Cas9 in a Cre-dependent manner were validated using viral microinjections and genetic crosses to common Cre-driver mouse lines. We subsequently confirmed functional Cas9 activity by targeting the ubiquitous neuronal protein, NeuN, using adeno-associated virus (AAV) delivery of sgRNAs. Finally, the mouse ß2 nAChR gene was successfully targeted in dopamine transporter (DAT)-positive neurons via CRISPR/Cas9. The sgRNA sequences and viral vectors, including our scheme for Cre-dependent gene editing, should be generally useful to the scientific research community. These tools could lead to new discoveries related to the function of nAChRs in neurotransmission and behavioral processes.


Asunto(s)
Encéfalo/fisiología , Neuronas Colinérgicas/fisiología , Edición Génica/métodos , Vectores Genéticos/genética , Receptores Nicotínicos/fisiología , Transmisión Sináptica/fisiología , Animales , Proteína 9 Asociada a CRISPR/biosíntesis , Proteína 9 Asociada a CRISPR/genética , Sistemas CRISPR-Cas/fisiología , Femenino , Vectores Genéticos/administración & dosificación , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Técnicas de Cultivo de Órganos
9.
FASEB J ; 33(1): 1440-1455, 2019 01.
Artículo en Inglés | MEDLINE | ID: mdl-30133325

RESUMEN

The transition zone (TZ) is a domain at the base of the cilium that is involved in maintaining ciliary compartment-specific sensory and signaling activity by regulating cilia protein composition. Mutations in TZ proteins result in cilia dysfunction, often causing pleiotropic effects observed in a group of human diseases classified as ciliopathies. The purpose of this study is to describe the importance of the TZ component Meckel-Grüber syndrome 6 ( Mks6) in several organ systems and tissues regarding ciliogenesis and cilia maintenance using congenital and conditional mutant mouse models. Similar to MKS, congenital loss of Mks6 is embryonic lethal, displaying cilia loss and altered cytoskeletal microtubule modifications but only in specific cell types. Conditional Mks6 mutants have a variable cystic kidney phenotype along with severe retinal degeneration with mislocalization of phototransduction cascade proteins. However, other phenotypes, such as anosmia and obesity, which are typically associated with cilia and TZ dysfunction, were not evident. These data indicate that despite Mks6 being a core TZ component, it has tissue- or cell type-specific functions important for cilia formation and cilia sensory and signaling activities. Lewis, W. R., Bales, K. L., Revell, D. Z., Croyle, M. J., Engle, S. E., Song, C. J., Malarkey, E. B., Uytingco, C. R., Shan, D., Antonellis, P. J., Nagy, T. R., Kesterson, R. A., Mrug, M. M., Martens, J. R., Berbari, N. F., Gross, A. K., Yoder, B. K. Mks6 mutations reveal tissue- and cell type-specific roles for the cilia transition zone.


Asunto(s)
Cilios/metabolismo , Proteínas del Citoesqueleto/genética , Mutación , Acetilación , Animales , Trastornos de la Motilidad Ciliar/genética , Citoplasma/metabolismo , Encefalocele/genética , Femenino , Genes Letales , Enfermedades Renales Quísticas/genética , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Trastornos del Olfato/genética , Fenotipo , Enfermedades Renales Poliquísticas/genética , Degeneración Retiniana/genética , Retinitis Pigmentosa/genética , Tubulina (Proteína)/metabolismo , Aumento de Peso/genética
10.
Genesis ; 56(8): e23217, 2018 08.
Artículo en Inglés | MEDLINE | ID: mdl-29806135

RESUMEN

The neuropeptide, melanin concentrating hormone (MCH), and its G protein-coupled receptor, melanin concentrating hormone receptor 1 (Mchr1), are expressed centrally in adult rodents. MCH signaling has been implicated in diverse behaviors such as feeding, sleep, anxiety, as well as addiction and reward. While a model utilizing the Mchr1 promoter to drive constitutive expression of Cre recombinase (Mchr1-Cre) exists, there is a need for an inducible Mchr1-Cre to determine the roles for this signaling pathway in neural development and adult neuronal function. Here, we generated a BAC transgenic mouse where the Mchr1 promotor drives expression of tamoxifen inducible CreER recombinase. Many aspects of the Mchr1-Cre expression pattern are recapitulated by the Mchr1-CreER model, though there are also notable differences. Most strikingly, compared to the constitutive model, the new Mchr1-CreER model shows strong expression in adult animals in hypothalamic brain regions involved in feeding behavior but diminished expression in regions involved in reward, such as the nucleus accumbens. The inducible Mchr1-CreER allele will help reveal the potential for Mchr1 signaling to impact neural development and subsequent behavioral phenotypes, as well as contribute to the understanding of the MCH signaling pathway in terminally differentiated adult neurons and the diverse behaviors that it influences.


Asunto(s)
Hormonas Hipotalámicas/fisiología , Melaninas/fisiología , Hormonas Hipofisarias/fisiología , Receptores de Somatostatina/fisiología , Animales , Encéfalo/metabolismo , Encéfalo/fisiología , Hormonas Hipotalámicas/metabolismo , Hipotálamo/metabolismo , Integrasas , Melaninas/metabolismo , Ratones , Ratones Transgénicos , Modelos Animales , Neuronas/metabolismo , Neuropéptidos/metabolismo , Hormonas Hipofisarias/metabolismo , Receptores de Somatostatina/metabolismo , Transducción de Señal , Tamoxifeno
11.
PLoS One ; 12(7): e0182142, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-28759616

RESUMEN

Nicotinic acetylcholine receptors containing α4 subunits (α4ß2* nAChRs) are critical for nicotinic cholinergic transmission and the addictive action of nicotine. To identify specific activities of these receptors in the adult mouse brain, we coupled targeted deletion of α4 nAChR subunits with behavioral and and electrophysiological measures of nicotine sensitivity. A viral-mediated Cre/lox approach allowed us to delete α4 from ventral midbrain (vMB) neurons. We used two behavioral assays commonly used to assess the motivational effects of drugs of abuse: home-cage oral self-administration, and place conditioning. Mice lacking α4 subunits in vMB consumed significantly more nicotine at the highest offered nicotine concentration (200 µg/mL) compared to control mice. Deletion of α4 subunits in vMB blocked nicotine-induced conditioned place preference (CPP) without affecting locomotor activity. Acetylcholine-evoked currents as well as nicotine-mediated increases in synaptic potentiation were reduced in mice lacking α4 in vMB. Immunostaining verified that α4 subunits were deleted from both dopamine and non-dopamine neurons in the ventral tegmental area (VTA). These results reveal that attenuation of α4* nAChR function in reward-related brain circuitry of adult animals may increase nicotine intake by enhancing the rewarding effects and/or reducing the aversive effects of nicotine.


Asunto(s)
Nicotina/metabolismo , Receptores Nicotínicos/metabolismo , Recompensa , Área Tegmental Ventral/metabolismo , Animales , Neuronas Dopaminérgicas/metabolismo , Neuronas Dopaminérgicas/fisiología , Comportamiento de Búsqueda de Drogas , Femenino , Eliminación de Gen , Potenciación a Largo Plazo , Masculino , Ratones , Ratones Endogámicos C57BL , Receptores Nicotínicos/genética , Potenciales Sinápticos , Área Tegmental Ventral/fisiología
12.
Neuropharmacology ; 91: 13-22, 2015 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-25484253

RESUMEN

Nicotine + ethanol co-exposure results in additive and/or synergistic effects in the ventral tegmental area (VTA) to nucleus accumbens (NAc) dopamine (DA) pathway, but the mechanisms supporting this are unclear. We tested the hypothesis that nAChRs containing α6 subunits (α6* nAChRs) are involved in the response to nicotine + ethanol co-exposure. Exposing VTA slices from C57BL/6 WT animals to drinking-relevant concentrations of ethanol causes a marked enhancement of α-amino-3-hydroxy-5-methyl-isoxazolepropionic acid (AMPA) receptor (AMPAR) function in VTA neurons. This effect was sensitive to α-conotoxin MII (an α6ß2* nAChR antagonist), suggesting that α6* nAChR function is required. In mice expressing hypersensitive α6* nAChRs (α6L9S mice), we found that lower concentrations (relative to C57BL/6 WT) of ethanol were sufficient to enhance AMPAR function in VTA neurons. Exposure of live C57BL/6 WT mice to ethanol also produced AMPAR functional enhancement in VTA neurons, and studies in α6L9S mice strongly suggest a role for α6* nAChRs in this response. We then asked whether nicotine and ethanol cooperate to enhance VTA AMPAR function. We identified low concentrations of nicotine and ethanol that were capable of strongly enhancing VTA AMPAR function when co-applied to slices, but that did not enhance AMPAR function when applied alone. This effect was sensitive to both varenicline (an α4ß2* and α6ß2* nAChR partial agonist) and α-conotoxin MII. Finally, nicotine + ethanol co-exposure also enhanced AMPAR function in VTA neurons from α6L9S mice. Together, these data identify α6* nAChRs as important players in the response to nicotine + ethanol co-exposure in VTA neurons.


Asunto(s)
Etanol/farmacología , Nicotina/farmacología , Receptores AMPA/fisiología , Receptores Nicotínicos/fisiología , Área Tegmental Ventral/efectos de los fármacos , Animales , Etanol/administración & dosificación , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Neuronas/efectos de los fármacos , Neuronas/fisiología , Nicotina/administración & dosificación , Subunidades de Proteína/agonistas , Subunidades de Proteína/metabolismo , Área Tegmental Ventral/fisiología
13.
J Neurosci ; 34(29): 9789-802, 2014 Jul 16.
Artículo en Inglés | MEDLINE | ID: mdl-25031416

RESUMEN

Neuronal nAChRs in the medial habenula (MHb) to the interpeduncular nucleus (IPN) pathway are key mediators of nicotine's aversive properties. In this paper, we report new details regarding nAChR anatomical localization and function in MHb and IPN. A new group of knock-in mice were created that each expresses a single nAChR subunit fused to GFP, allowing high-resolution mapping. We find that α3 and ß4 nAChR subunit levels are strong throughout the ventral MHb (MHbV). In contrast, α6, ß2, ß3, and α4 subunits are selectively found in some, but not all, areas of MHbV. All subunits were found in both ChAT-positive and ChAT-negative cells in MHbV. Next, we examined functional properties of neurons in the lateral and central part of MHbV (MHbVL and MHbVC) using brain slice patch-clamp recordings. MHbVL neurons were more excitable than MHbVC neurons, and they also responded more strongly to puffs of nicotine. In addition, we studied firing responses of MHbVL and MHbVC neurons in response to bath-applied nicotine. Cells in MHbVL, but not those in MHbVC, increased their firing substantially in response to 1 µm nicotine. Additionally, MHbVL neurons from mice that underwent withdrawal from chronic nicotine were less responsive to nicotine application compared with mice withdrawn from chronic saline. Last, we characterized rostral and dorsomedial IPN neurons that receive input from MHbVL axons. Together, our data provide new details regarding neurophysiology and nAChR localization and function in cells within the MHbV.


Asunto(s)
Expresión Génica/genética , Habénula/citología , Habénula/metabolismo , Potenciales de la Membrana/fisiología , Neuronas/fisiología , Receptores Nicotínicos/fisiología , Animales , Colina O-Acetiltransferasa/metabolismo , Relación Dosis-Respuesta a Droga , Estimulación Eléctrica , Fármacos actuantes sobre Aminoácidos Excitadores/farmacología , Femenino , Expresión Génica/efectos de los fármacos , Proteínas Fluorescentes Verdes/genética , Habénula/efectos de los fármacos , Técnicas In Vitro , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Nicotina/farmacología , Técnicas de Placa-Clamp , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Receptores Nicotínicos/genética , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/farmacología
14.
Mol Pharmacol ; 84(3): 393-406, 2013 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-23788655

RESUMEN

Tobacco addiction is a serious threat to public health in the United States and abroad, and development of new therapeutic approaches is a major priority. Nicotine activates and/or desensitizes nicotinic acetylcholine receptors (nAChRs) throughout the brain. nAChRs in ventral tegmental area (VTA) dopamine (DA) neurons are crucial for the rewarding and reinforcing properties of nicotine in rodents, suggesting that they may be key mediators of nicotine's action in humans. However, it is unknown which nAChR subtypes are sufficient to activate these neurons. To test the hypothesis that nAChRs containing α6 subunits are sufficient to activate VTA DA neurons, we studied mice expressing hypersensitive, gain-of-function α6 nAChRs (α6L9'S mice). In voltage-clamp recordings in brain slices from adult mice, 100 nM nicotine was sufficient to elicit inward currents in VTA DA neurons via α6ß2* nAChRs. In addition, we found that low concentrations of nicotine could act selectively through α6ß2* nAChRs to enhance the function of 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid (AMPA) receptors on the surface of these cells. In contrast, α6ß2* activation did not enhance N-methyl-D-aspartic acid receptor function. Finally, AMPA receptor (AMPAR) function was not similarly enhanced in brain slices from α6L9'S mice lacking α4 nAChR subunits, suggesting that α4α6ß2* nAChRs are important for enhancing AMPAR function in VTA DA neurons. Together, these data suggest that activation of α4α6ß2* nAChRs in VTA DA neurons is sufficient to support the initiation of cellular changes that play a role in addiction to nicotine. α4α6ß2* nAChRs may be a promising target for future smoking cessation pharmacotherapy.


Asunto(s)
Dopamina/metabolismo , Neuronas/efectos de los fármacos , Agonistas Nicotínicos/farmacología , Receptores AMPA/fisiología , Receptores Nicotínicos/metabolismo , Área Tegmental Ventral/efectos de los fármacos , Potenciales de Acción , Animales , Técnicas In Vitro , Activación del Canal Iónico , Ratones , Ratones Noqueados , Neuronas/fisiología , Subunidades de Proteína/agonistas , Subunidades de Proteína/metabolismo , Receptores Nicotínicos/genética , Área Tegmental Ventral/citología , Área Tegmental Ventral/fisiología
15.
J Vis Exp ; (68): e50034, 2012 Oct 29.
Artículo en Inglés | MEDLINE | ID: mdl-23128482

RESUMEN

Tobacco use leads to numerous health problems, including cancer, heart disease, emphysema, and stroke. Addiction to cigarette smoking is a prevalent neuropsychiatric disorder that stems from the biophysical and cellular actions of nicotine on nicotinic acetylcholine receptors (nAChRs) throughout the central nervous system. Understanding the various nAChR subtypes that exist in brain areas relevant to nicotine addiction is a major priority. Experiments that employ electrophysiology techniques such as whole-cell patch clamp or two-electrode voltage clamp recordings are useful for pharmacological characterization of nAChRs of interest. Cells expressing nAChRs, such as mammalian tissue culture cells or Xenopus laevis oocytes, are physically isolated and are therefore easily studied using the tools of modern pharmacology. Much progress has been made using these techniques, particularly when the target receptor was already known and ectopic expression was easily achieved. Often, however, it is necessary to study nAChRs in their native environment: in neurons within brain slices acutely harvested from laboratory mice or rats. For example, mice expressing "hypersensitive" nAChR subunits such as α4 L9'A mice (1) and α6 L9'S mice (2), allow for unambiguous identification of neurons based on their functional expression of a specific nAChR subunit. Although whole-cell patch clamp recordings from neurons in brain slices is routinely done by the skilled electrophysiologist, it is challenging to locally apply drugs such as acetylcholine or nicotine to the recorded cell within a brain slice. Dilution of drugs into the superfusate (bath application) is not rapidly reversible, and U-tube systems are not easily adapted to work with brain slices. In this paper, we describe a method for rapidly applying nAChR-activating drugs to neurons recorded in adult mouse brain slices. Standard whole-cell recordings are made from neurons in slices, and a second micropipette filled with a drug of interest is maneuvered into position near the recorded cell. An injection of pressurized air or inert nitrogen into the drug-filled pipette causes a small amount of drug solution to be ejected from the pipette onto the recorded cell. Using this method, nAChR-mediated currents are able to be resolved with millisecond accuracy. Drug application times can easily be varied, and the drug-filled pipette can be retracted and replaced with a new pipette, allowing for concentration-response curves to be created for a single neuron. Although described in the context of nAChR neurobiology, this technique should be useful for studying many types of ligand-gated ion channels or receptors in neurons from brain slices.


Asunto(s)
Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Receptores Nicotínicos/metabolismo , Animales , Neuronas Dopaminérgicas/efectos de los fármacos , Neuronas Dopaminérgicas/metabolismo , Ratones , Técnicas de Placa-Clamp/métodos
16.
J Neurosci ; 32(30): 10226-37, 2012 Jul 25.
Artículo en Inglés | MEDLINE | ID: mdl-22836257

RESUMEN

Nicotinic acetylcholine receptors (nAChRs) containing α6 subunits are expressed in only a few brain areas, including midbrain dopamine (DA) neurons, noradrenergic neurons of the locus ceruleus, and retinal ganglion cells. To better understand the regional and subcellular expression pattern of α6-containing nAChRs, we created and studied transgenic mice expressing a variant α6 subunit with green fluorescent protein (GFP) fused in-frame in the M3-M4 intracellular loop. In α6-GFP transgenic mice, α6-dependent synaptosomal DA release and radioligand binding experiments confirmed correct expression and function in vivo. In addition to strong α6* nAChR expression in glutamatergic retinal axons, which terminate in superficial superior colliculus (sSC), we also found α6 subunit expression in a subset of GABAergic cell bodies in this brain area. In patch-clamp recordings from sSC neurons in brain slices from mice expressing hypersensitive α6* nAChRs, we confirmed functional, postsynaptic α6* nAChR expression. Further, sSC GABAergic neurons expressing α6* nAChRs exhibit a tonic conductance mediated by standing activation of hypersensitive α6* nAChRs by ACh. α6* nAChRs also appear in a subpopulation of SC neurons in output layers. Finally, selective activation of α6* nAChRs in vivo induced sSC neuronal activation as measured with c-Fos expression. Together, these results demonstrate that α6* nAChRs are uniquely situated to mediate cholinergic modulation of glutamate and GABA release in SC. The SC has emerged as a potential key brain area responsible for transmitting short-latency salience signals to thalamus and midbrain DA neurons, and these results suggest that α6* nAChRs may be important for nicotinic cholinergic sensitization of this pathway.


Asunto(s)
Neuronas/fisiología , Receptores Nicotínicos/fisiología , Colículos Superiores/fisiología , Sinapsis/metabolismo , Vías Visuales/fisiología , Animales , Dopamina/metabolismo , Neuronas GABAérgicas/metabolismo , Ratones , Ratones Transgénicos , Neuronas/metabolismo , Receptores Nicotínicos/metabolismo , Colículos Superiores/metabolismo , Vías Visuales/metabolismo , Ácido gamma-Aminobutírico/metabolismo
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