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Objectives: Glucokinase Regulatory Protein (GKRP) is the only known endogenous modulator of glucokinase (GK) localization and activity to date, and both proteins are localized in tanycytes, radial glia-like cells involved in metabolic and endocrine functions in the hypothalamus. However, the role of tanycytic GKRP and its impact on the regulation of feeding behavior has not been investigated. Here, we hypothesize that GKRP regulates feeding behavior by modulating tanycyte-neuron metabolic communication in the arcuate nucleus. Methods: We used primary cultures of tanycytes to evaluate the production of lactate and ß-hydroxybutyrate (ßHB). Similarly, we examined the electrophysiological responses to these metabolites in pro-opiomelanocortin (POMC) neurons in hypothalamic slices. To evaluate the role of GKRP in feeding behavior, we generated tanycyte-selective GKRP-overexpressing and GKRP-knock down mice (GKRPt-OE and GKRPt-KD respectively) using adenovirus-mediated transduction. Results: We demonstrated that lactate release induced by glucose uptake is favored in GKRP-KD tanycytes. Conversely, tanycytes overexpressing GKRP showed an increase in ßHB efflux induced by low glucose concentration. In line with these findings, the excitability of POMC neurons was enhanced by lactate and decreased in the presence of ßHB. In GKRPt-OE rats, we found an increase in post-fasting food avidity, whereas GKRPt-KD caused a significant decrease in feeding and body weight, which is reverted when MCT1 is silenced. Conclusion: Our study highlights the role of tanycytic GKRP in metabolic regulation and positions this regulator of GK as a therapeutic target for boosting satiety in patients with obesity problems.
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Células Ependimogliales , Proopiomelanocortina , Animales , Proteínas Portadoras , Conducta Alimentaria , Glucoquinasa/metabolismo , Humanos , Ácido Láctico/metabolismo , Ratones , Proopiomelanocortina/metabolismo , RatasRESUMEN
In the olfactory bulb (OB), a large population of axon-less inhibitory interneurons, the granule cells (GCs), coordinate network activity and tune the output of principal neurons, the mitral and tufted cells (MCs), through dendrodendritic interactions. Furthermore, GCs undergo neurogenesis throughout life, providing a source of plasticity to the neural network of the OB. The function and integration of GCs in the OB are regulated by several afferent neuromodulatory signals, including noradrenaline (NA), a state-dependent neuromodulator that plays a crucial role in the regulation of cortical function and task-specific decision processes. However, the mechanisms by which NA regulates GC function are not fully understood. Here, we show that NA modulates hyperpolarization-activated currents (Ih) via the activation of α2-adrenergic receptors (ARs) in adult-born GCs (abGCs), thus directly acting on channels that play essential roles in regulating neuronal excitability and network oscillations in the brain. This modulation affects the dendrodendritic output of GCs leading to an enhancement of lateral inhibition onto the MCs. Furthermore, we show that NA modulates subthreshold resonance in GCs, which could affect the temporal integration of abGCs. Together, these results provide a novel mechanism by which a state-dependent neuromodulator acting on Ih can regulate GC function in the OB.
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Previous studies indicate that the activity of hypothalamic POMC neurons can be regulated by glucose via intracellular mechanisms, but its regulation by lactate is poorly understood. In addition to its energetic role, lactate acts as a signaling molecule. In this study, we evaluated the function and location of the lactate receptor, hydroxycarboxylic acid receptor 1 (HCAR1). We used a conditional genetic approach to label POMC neurons and evaluated their sensitivity to lactate using patch-clamp recordings. L-Lactate and 3-chloro-5-hydroxybenzoic acid (3Cl-HBA), HCAR1 specific agonist depolarized POMC neurons and the increase in excitability was abolished by pertussis toxin (PTX), indicating the involvement of Gαi/o-protein-coupled receptors. In addition, the depolarization of a subset of POMC neurons was sensitive to α-cyano-4-hydroxycinnamate (4-CIN), a lactate transporter blocker, suggesting that the depolarization induced by L-lactate can also occur by direct intracellular action. Surprisingly, HCAR1 was not detected in POMC neurons, but instead localized in astrocytes. These results suggest a new lactate-mediated mechanism for astrocyte-neuron intercellular communication.
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Ácido Láctico/metabolismo , Proopiomelanocortina/fisiología , Receptores Acoplados a Proteínas G/metabolismo , Animales , Astrocitos/metabolismo , Comunicación Celular/fisiología , Femenino , Hipotálamo/metabolismo , Hipotálamo/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Transportadores de Ácidos Monocarboxílicos , Neuronas/metabolismo , Proopiomelanocortina/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Local interneurons of the olfactory bulb (OB) are densely innervated by long-range GABAergic neurons from the basal forebrain (BF), suggesting that this top-down inhibition regulates early processing in the olfactory system. However, how GABAergic inputs modulate the OB output neurons, the mitral/tufted cells, is unknown. Here, in male and female mice acute brain slices, we show that optogenetic activation of BF GABAergic inputs produced distinct local circuit effects that can influence the activity of mitral/tufted cells in the spatiotemporal domains. Activation of the GABAergic axons produced a fast disinhibition of mitral/tufted cells consistent with a rapid and synchronous release of GABA onto local interneurons in the glomerular and inframitral circuits of the OB, which also reduced the spike precision of mitral/tufted cells in response to simulated stimuli. In addition, BF GABAergic inhibition modulated local oscillations in a layer-specific manner. The intensity of locally evoked θ oscillations was decreased on activation of top-down inhibition in the glomerular circuit, while evoked γ oscillations were reduced by inhibition of granule cells. Furthermore, BF GABAergic input reduced dendrodendritic inhibition in mitral/tufted cells. Together, these results suggest that long-range GABAergic neurons from the BF are well suited to influence temporal and spatial aspects of processing by OB circuits.SIGNIFICANCE STATEMENT Disruption of GABAergic inhibition from the basal forebrain (BF) to the olfactory bulb (OB) impairs the discrimination of similar odors, yet how this centrifugal inhibition influences neuronal circuits in the OB remains unclear. Here, we show that the BF GABAergic neurons exclusively target local inhibitory neurons in the OB, having a functional disinhibitory effect on the output neurons, the mitral cells. Phasic inhibition by BF GABAergic neurons reduces spike precision of mitral cells and lowers the intensity of oscillatory activity in the OB, while directly modulating the extent of dendrodendritic inhibition. These circuit-level effects of this centrifugal inhibition can influence the temporal and spatial dynamics of odor coding in the OB.
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Antagonistas del GABA/farmacología , Neuronas GABAérgicas/efectos de los fármacos , Bulbo Olfatorio/citología , Bulbo Olfatorio/efectos de los fármacos , Animales , Dendritas/fisiología , Potenciales Evocados/fisiología , Femenino , Neuronas GABAérgicas/ultraestructura , Ritmo Gamma/fisiología , Interneuronas/fisiología , Interneuronas/ultraestructura , Masculino , Ratones , Ratones Endogámicos C57BL , Inhibición Neural , Optogenética , Técnicas de Placa-Clamp , Área Preóptica/fisiología , Prosencéfalo/citología , Prosencéfalo/fisiología , Ritmo TetaRESUMEN
Glucose is a key modulator of feeding behavior. By acting in peripheral tissues and in the central nervous system, it directly controls the secretion of hormones and neuropeptides and modulates the activity of the autonomic nervous system. GLUT2 is required for several glucoregulatory responses in the brain, including feeding behavior, and is localized in the hypothalamus and brainstem, which are the main centers that control this behavior. In the hypothalamus, GLUT2 has been detected in glial cells, known as tanycytes, which line the basal walls of the third ventricle (3V). This study aimed to clarify the role of GLUT2 expression in tanycytes in feeding behavior using 3V injections of an adenovirus encoding a shRNA against GLUT2 and the reporter EGFP (Ad-shGLUT2). Efficient in vivo GLUT2 knockdown in rat hypothalamic tissue was demonstrated by qPCR and Western blot analyses. Specificity of cell transduction in the hypothalamus and brainstem was evaluated by EGFP-fluorescence and immunohistochemistry, which showed EGFP expression specifically in ependymal cells, including tanycytes. The altered mRNA levels of both orexigenic and anorexigenic neuropeptides suggested a loss of response to increased glucose in the 3V. Feeding behavior analysis in the fasting-feeding transition revealed that GLUT2-knockdown rats had increased food intake and body weight, suggesting an inhibitory effect on satiety. Taken together, suppression of GLUT2 expression in tanycytes disrupted the hypothalamic glucosensing mechanism, which altered the feeding behavior.
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Conducta Alimentaria/fisiología , Transportador de Glucosa de Tipo 2/metabolismo , Hipotálamo/metabolismo , Neuroglía/metabolismo , Saciedad/fisiología , Animales , Peso Corporal , Tronco Encefálico/citología , Tronco Encefálico/metabolismo , Células Cultivadas , Ayuno/metabolismo , Técnicas de Silenciamiento del Gen , Transportador de Glucosa de Tipo 2/genética , Hipotálamo/citología , Masculino , Neuroglía/citología , Neuropéptidos/metabolismo , ARN Mensajero/metabolismo , Ratas Sprague-DawleyRESUMEN
The cholinergic system has extensive projections to the olfactory bulb (OB) where it produces a state-dependent regulation of sensory gating. Previous work has shown a prominent role of muscarinic acetylcholine (ACh) receptors (mAChRs) in regulating the excitability of OB neurons, in particular the M1 receptor. Here, we examined the contribution of M1 and M3 mAChR subtypes to olfactory processing using mice with a genetic deletion of these receptors, the M1-/- and the M1/M3-/- knockout (KO) mice. Genetic ablation of the M1 and M3 mAChRs resulted in a significant deficit in odor discrimination of closely related molecules, including stereoisomers. However, the discrimination of dissimilar molecules, social odors (e.g., urine) and novel object recognition was not affected. In addition the KO mice showed impaired learning in an associative odor-learning task, learning to discriminate odors at a slower rate, indicating that both short and long-term memory is disrupted by mAChR dysfunction. Interestingly, the KO mice exhibited decreased olfactory neurogenesis at younger ages, a deficit that was not maintained in older animals. In older animals, the olfactory deficit could be restored by increasing the number of new born neurons integrated into the OB after exposing them to an olfactory enriched environment, suggesting that muscarinic modulation and adult neurogenesis could be two different mechanism used by the olfactory system to improve olfactory processing.
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An important contribution to neural circuit oscillatory dynamics is the ongoing activation and inactivation of hyperpolarization-activated currents (Ih). Network synchrony dynamics play an important role in the initial processing of odor signals by the main olfactory bulb (MOB) and accessory olfactory bulb (AOB). In the mouse olfactory bulb, we show that Ih is present in granule cells (GCs), the most prominent inhibitory neuron in the olfactory bulb, and that Ih underlies subthreshold resonance in GCs. In accord with the properties of Ih, the currents exhibited sensitivity to changes in extracellular K+ concentration and ZD7288 (4-ethylphenylamino-1,2-dimethyl-6-methylaminopyrimidin chloride), a blocker of Ih. ZD7288 also caused GCs to hyperpolarize and increase their input resistance, suggesting that Ih is active at rest in GCs. The inclusion of cAMP in the intracellular solution shifted the activation of Ih to less negative potentials in the MOB, but not in the AOB, suggesting that channels with different subunit composition mediate Ih in these regions. Furthermore, we show that mature GCs exhibit Ih-dependent subthreshold resonance in the theta frequency range (4-12 Hz). Another inhibitory subtype in the MOB, the periglomerular cells, exhibited Ih-dependent subthreshold resonance in the delta range (1-4 Hz), while principal neurons, the mitral cells, do not exhibit Ih-dependent subthreshold resonance. Importantly, Ih size, as well as the strength and frequency of resonance in GCs, exhibited a postnatal developmental progression, suggesting that this development of Ih in GCs may differentially contribute to their integration of sensory input and contribution to oscillatory circuit dynamics.
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Potenciales de la Membrana/fisiología , Neuronas/fisiología , Bulbo Olfatorio/crecimiento & desarrollo , Bulbo Olfatorio/fisiología , Animales , Cationes Monovalentes/metabolismo , AMP Cíclico/metabolismo , Electroporación , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Potenciales de la Membrana/efectos de los fármacos , Ratones Endogámicos C57BL , Inhibición Neural/efectos de los fármacos , Inhibición Neural/fisiología , Vías Nerviosas/citología , Vías Nerviosas/efectos de los fármacos , Vías Nerviosas/crecimiento & desarrollo , Vías Nerviosas/fisiología , Neuronas/citología , Neuronas/efectos de los fármacos , Neurotransmisores/farmacología , Bulbo Olfatorio/citología , Bulbo Olfatorio/efectos de los fármacos , Técnicas de Placa-Clamp , Potasio/metabolismo , Pirimidinas/farmacología , Ritmo Teta , Técnicas de Cultivo de TejidosRESUMEN
Neuromodulation of olfactory circuits by acetylcholine (ACh) plays an important role in odor discrimination and learning. Early processing of chemosensory signals occurs in two functionally and anatomically distinct regions, the main and accessory olfactory bulbs (MOB and AOB), which receive extensive cholinergic input from the basal forebrain. Here, we explore the regulation of AOB and MOB circuits by ACh, and how cholinergic modulation influences olfactory-mediated behaviors in mice. Surprisingly, despite the presence of a conserved circuit, activation of muscarinic ACh receptors revealed marked differences in cholinergic modulation of output neurons: excitation in the AOB and inhibition in the MOB. Granule cells (GCs), the most abundant intrinsic neuron in the OB, also exhibited a complex muscarinic response. While GCs in the AOB were excited, MOB GCs exhibited a dual muscarinic action in the form of a hyperpolarization and an increase in excitability uncovered by cell depolarization. Furthermore, ACh influenced the input-output relationship of mitral cells in the AOB and MOB differently showing a net effect on gain in mitral cells of the MOB, but not in the AOB. Interestingly, despite the striking differences in neuromodulatory actions on output neurons, chemogenetic inhibition of cholinergic neurons produced similar perturbations in olfactory behaviors mediated by these two regions. Decreasing ACh in the OB disrupted the natural discrimination of molecularly related odors and the natural investigation of odors associated with social behaviors. Thus, the distinct neuromodulation by ACh in these circuits could underlie different solutions to the processing of general odors and semiochemicals, and the diverse olfactory behaviors they trigger. SIGNIFICANCE STATEMENT: State-dependent cholinergic modulation of brain circuits is critical for several high-level cognitive functions, including attention and memory. Here, we provide new evidence that cholinergic modulation differentially regulates two parallel circuits that process chemosensory information, the accessory and main olfactory bulb (AOB and MOB, respectively). These circuits consist of remarkably similar synaptic arrangement and neuronal types, yet cholinergic regulation produced strikingly opposing effects in output and intrinsic neurons. Despite these differences, the chemogenetic reduction of cholinergic activity in freely behaving animals disrupted odor discrimination of simple odors, and the investigation of social odors associated with behaviors signaled by the Vomeronasal system.
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Acetilcolina/metabolismo , Bulbo Olfatorio/metabolismo , Vías Olfatorias/metabolismo , Percepción Olfatoria/fisiología , Animales , Colinérgicos/farmacología , Femenino , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Confocal , Bulbo Olfatorio/efectos de los fármacos , Vías Olfatorias/efectos de los fármacos , Percepción Olfatoria/efectos de los fármacos , Técnicas de Placa-Clamp , Receptores Muscarínicos/fisiologíaRESUMEN
Magnetic nanoparticles (MNPs) have been used as effective vehicles for targeted delivery of theranostic agents in the brain. The advantage of magnetic targeting lies in the ability to control the concentration and distribution of therapy to a desired target region using external driving magnets. In this study, we investigated the behavior and safety of MNP motion in brain tissue. We found that MNPs move and form nanoparticle chains in the presence of a uniform magnetic field, and that this chaining is influenced by the applied magnetic field intensity and the concentration of MNPs in the tissue. Using electrophysiology recordings, immunohistochemistry and fluorescent imaging we assessed the functional health of neurons and neural circuits and found no adverse effects associated with MNP motion through brain tissue. FROM THE CLINICAL EDITOR: Much research has been done to test the use of nanocarriers for gaining access across the blood brain barrier (BBB). In this respect, magnetic nanoparticles (MNPs) are one of the most studied candidates. Nonetheless, the behavior and safety of MNP once inside brain tissue remains unknown. In this article, the authors thus studied this very important subject.
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Encéfalo/efectos de los fármacos , Sistemas de Liberación de Medicamentos/efectos adversos , Nanopartículas de Magnetita/administración & dosificación , Neuronas/efectos de los fármacos , Animales , Barrera Hematoencefálica/efectos de los fármacos , Humanos , Campos Magnéticos , Nanopartículas de Magnetita/efectos adversos , Nanopartículas de Magnetita/química , Ratones , Nanomedicina Teranóstica , Distribución Tisular/efectos de los fármacosRESUMEN
VoltageFluor (VF) dyes have the potential to measure voltage optically in excitable membranes with a combination of high spatial and temporal resolution essential to better characterize the voltage dynamics of large groups of excitable cells. VF dyes sense voltage with high speed and sensitivity using photoinduced electron transfer (PeT) through a conjugated molecular wire. We show that tuning the driving force for PeT (ΔGPeT + w) through systematic chemical substitution modulates voltage sensitivity, estimate (ΔGPeT + w) values from experimentally measured redox potentials, and validate the voltage sensitivities in patch-clamped HEK cells for 10 new VF dyes. VF2.1(OMe).H, with a 48% ΔF/F per 100 mV, shows approximately 2-fold improvement over previous dyes in HEK cells, dissociated rat cortical neurons, and medicinal leech ganglia. Additionally, VF2.1(OMe).H faithfully reports pharmacological effects and circuit activity in mouse olfactory bulb slices, thus opening a wide range of previously inaccessible applications for voltage-sensitive dyes.
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Fenómenos Electrofisiológicos , Colorantes Fluorescentes/química , Luz , Neuronas/citología , Fenómenos Ópticos , Animales , Diseño de Fármacos , Transporte de Electrón , Colorantes Fluorescentes/síntesis química , Células HEK293 , Humanos , Potenciales de la Membrana , Ratones , Neuronas/química , Bulbo Olfatorio/citología , Imagen Óptica , RatasRESUMEN
Granule cells (GCs) are the most abundant inhibitory neuronal type in the olfactory bulb and play a critical role in olfactory processing. GCs regulate the activity of principal neurons, the mitral cells, through dendrodendritic synapses, shaping the olfactory bulb output to other brain regions. GC excitability is regulated precisely by intrinsic and extrinsic inputs, and this regulation is fundamental for odor discrimination. Here, we used channelrhodopsin to stimulate GABAergic axons from the basal forebrain selectively and show that this stimulation generates reliable inhibitory responses in GCs. Furthermore, selective in vivo inhibition of GABAergic neurons in the basal forebrain by targeted expression of designer receptors exclusively activated by designer drugs produced a reversible impairment in the discrimination of structurally similar odors, indicating an important role of these inhibitory afferents in olfactory processing.
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Discriminación en Psicología/fisiología , Neuronas/fisiología , Odorantes , Bulbo Olfatorio/fisiología , Percepción Olfatoria/fisiología , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Potenciales de Acción/efectos de la radiación , Animales , Axones/metabolismo , Axones/fisiología , Channelrhodopsins , Dependovirus/genética , Femenino , Neuronas GABAérgicas/metabolismo , Neuronas GABAérgicas/fisiología , Glutamato Descarboxilasa/genética , Glutamato Descarboxilasa/metabolismo , Técnicas In Vitro , Luz , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Confocal , Neuronas/metabolismo , Bulbo Olfatorio/citología , Bloqueadores de los Canales de Sodio/farmacología , Sinapsis/efectos de los fármacos , Sinapsis/fisiología , Sinapsis/efectos de la radiación , Tetrodotoxina/farmacologíaRESUMEN
Mammalian species have evolved a large and diverse number of odorant receptors (ORs). These proteins comprise the largest family of G-protein-coupled receptors (GPCRs) known, amounting to ~1,000-different receptors in the rodent. From the perspective of olfactory coding, the availability of such a vast number of chemosensory receptors poses several fascinating questions; in addition, such a large repertoire provides an attractive biological model to study ligand-receptor interactions. The limited functional expression of these receptors in heterologous systems, however, has greatly hampered attempts to deorphanize them. We have employed a successful approach that combines electrophysiological and imaging techniques to analyze the response profiles of single sensory neurons. Our approach has enabled us to characterize the "odor space" of a population of native aldehyde receptors and the molecular range of a genetically engineered receptor, OR-I7.
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Mamíferos , Farmacología/métodos , Receptores Odorantes/metabolismo , Animales , Calcio/metabolismo , Disección , Fenómenos Electrofisiológicos , Epitelio/metabolismo , Fura-2/análogos & derivados , Fura-2/metabolismo , Imagen Molecular , Odorantes , Receptores Odorantes/genética , Células Receptoras Sensoriales/citología , Células Receptoras Sensoriales/efectos de los fármacos , Células Receptoras Sensoriales/metabolismoRESUMEN
By regulating inhibition at dendrodendritic synapses between mitral and granule cells (GCs), noradrenergic neurons extending from the brainstem provide an input essential for odour processing in the olfactory bulb (OB). In the accessory OB (AOB), we have recently shown that noradrenaline (NA) increases GABA inhibitory input on to mitral cells (MCs) by exciting GCs. Here, we show that GCs in the main OB (MOB) exhibit a similar response to NA, indicating a common mechanism for noradrenergic regulation of GCMC inhibition throughout the OB. In GCs of the MOB, NA (10 µM) produced a robust excitatory effect that included a slow afterdepolarization that followed a train of action potentials evoked by a current stimulus. The depolarization and slow afterdepolarization in GCs were blocked by the α1A-adrenergic receptor (AR) selective antagonist WB 4101 (30 nm) and mimicked by the α(1A)-AR selective agonist A 61603 (1 µM). In recordings from MCs, A 61603 (30 nm-1 µM) produced a sizeable increase in the frequency of spontaneous and miniature IPSCs, an effect completely abolished by the GABAA receptor antagonist gabazine (5 µM). Likewise, activation of ß-ARs increased the frequency of spontaneous IPSCs; however, this effect was smaller and confined to the first postnatal weeks. NA enhanced inhibition in MCs across a broad concentration range (0.1-30 µM) and its effects were completely abolished by a mixture of α1- and ß-AR antagonists (1 µM prazosin and 10 µM propranolol). Furthermore, the general α2-AR agonist clonidine (10 µM) failed to affect sIPSC frequency. Thus, the NA-mediated increase in GCMC inhibition in the OB results mostly from activation of the α1A-AR subtype.
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Agonistas alfa-Adrenérgicos/farmacología , Neuronas/efectos de los fármacos , Norepinefrina/farmacología , Bulbo Olfatorio/fisiología , Receptores Adrenérgicos alfa 1/fisiología , Animales , Potenciales Postsinápticos Inhibidores/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Neuronas/fisiología , Sinapsis/fisiologíaRESUMEN
The vomeronasal system (VNS) participates in the detection and processing of pheromonal information related to social and sexual behaviors. Within the VNS, two different populations of sensory neurons, with a distinct pattern of distribution, line the epithelium of the vomeronasal organ (VNO) and give rise to segregated sensory projections to the accessory olfactory bulb (AOB). Apical sensory neurons in the VNO project to the anterior AOB (aAOB), while basal neurons project to the posterior AOB (pAOB). In the AOB, the largest population of neurons are inhibitory, the granule and periglomerular cells (GCs and PGs) and remarkably, these neurons are continuously born and functionally integrated in the adult brain, underscoring their role on olfactory function. Here we show that behaviors mediated by the VNS differentially regulate adult neurogenesis across the anterior-posterior axis of the AOB. We used immunohistochemical labeling of newly born cells under different behavioral conditions in mice. Using a resident-intruder aggression paradigm, we found that subordinate mice exhibited increased neurogenesis in the aAOB. In addition, in sexually naive adult females exposed to soiled bedding odorized by adult males, the number of newly born cells was significantly increased in the pAOB; however, neurogenesis was not affected in females exposed to female odors. In addition, we found that at two months of age adult neurogenesis was sexually dimorphic, with male mice exhibiting higher levels of newly born cells than females. Interestingly, adult neurogenesis was greatly reduced with age and this decrease correlated with a decrease in progenitor cells proliferation but not with an increase in cell death in the AOB. These results indicate that the physiological regulation of adult neurogenesis in the AOB by behaviors is both sex and age dependent and suggests an important role of newly born neurons in sex dependent behaviors mediated by the VNS.
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Envejecimiento/fisiología , Conducta Animal/fisiología , Neurogénesis/fisiología , Bulbo Olfatorio/fisiología , Animales , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Odorantes , Bulbo Olfatorio/citología , Feromonas/metabolismo , Células Receptoras Sensoriales/citología , Células Receptoras Sensoriales/fisiología , Caracteres Sexuales , OlfatoRESUMEN
Mechanisms influencing the development of olfactory bulb glomeruli are poorly understood. While odor receptors (ORs) play an important role in olfactory sensory neuron (OSN) axon targeting/coalescence (Mombaerts et al., 1996; Wang et al., 1998; Feinstein and Mombaerts, 2004), recent work showed that G protein activation alone is sufficient to induce OSN axon coalescence (Imai et al., 2006; Chesler et al., 2007), suggesting an activity-dependent mechanism in glomerular development. Consistent with these data, OSN axon projections and convergence are perturbed in mice deficient for adenylyl cyclase III, which is downstream from the OR and catalyzes the conversion of ATP to cAMP. However, in cyclic nucleotide-gated (CNG) channel knock-out mice OSN axons are only transiently perturbed (Lin et al., 2000), suggesting that the CNG channel may not be the sole target of cAMP. This prompted us to investigate an alternative channel, the hyperpolarization-activated, cyclic nucleotide-gated cation channel (HCN), as a potential developmental target of cAMP in OSNs. Here, we demonstrate that HCN channels are developmentally precocious in OSNs and therefore are plausible candidates for affecting OSN axon development. Inhibition of HCN channels in dissociated OSNs significantly reduced neurite outgrowth. Moreover, in HCN1 knock-out mice the formation of glomeruli was delayed in parallel with perturbations of axon organization in the olfactory nerve. These data support the hypothesis that the outgrowth and coalescence of OSN axons is, at least in part, subject to activity-dependent mechanisms mediated via HCN channels.
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Axones/fisiología , Canales Catiónicos Regulados por Nucleótidos Cíclicos/fisiología , Neurogénesis/fisiología , Canales de Potasio/fisiología , Células Receptoras Sensoriales/citología , Animales , Animales Recién Nacidos , Antidiarreicos/farmacología , Axones/efectos de los fármacos , Biofisica/métodos , Cardiotónicos/farmacología , Células Cultivadas , Canales Catiónicos Regulados por Nucleótidos Cíclicos/deficiencia , Estimulación Eléctrica/métodos , Embrión de Mamíferos , Proteína GAP-43/metabolismo , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica/fisiología , Proteínas Fluorescentes Verdes/genética , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización , Canales Iónicos/genética , Canales Iónicos/metabolismo , Loperamida/farmacología , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Neurogénesis/efectos de los fármacos , Bulbo Olfatorio/citología , Bulbo Olfatorio/embriología , Bulbo Olfatorio/crecimiento & desarrollo , Técnicas de Placa-Clamp/métodos , Canales de Potasio/deficiencia , Canales de Potasio/genética , Canales de Potasio/metabolismo , Pirimidinas/farmacología , Receptores Odorantes/genética , Receptores Odorantes/metabolismo , Células Receptoras Sensoriales/efectos de los fármacosRESUMEN
The accessory olfactory bulb (AOB), the first relay of chemosensory information in the Vomeronasal system, receives extensive cholinergic innervation from the basal forebrain. Cholinergic modulation of neuronal activity in the olfactory bulb has been hypothesized to play an important role in olfactory processing; however, little is known about the cellular actions of acetylcholine (ACh) within the AOB. Here using in vitro slice preparation, we show that muscarinic acetylcholine receptor (mAChR) activation increases neuronal excitability of granule and mitral/tufted cells (GCs and MCs) in the AOB. Activation of mAChRs increased excitability of GCs by three distinct mechanisms: induction of a long-lasting depolarization, activation of a slow afterdepolarization (sADP), and an increase in excitatory glutamatergic input due to MC depolarization. The depolarization and sADP were elicited by the selective agonist 4-[[[(3-chlorophenyl)amino]carbonyl]oxy]-N,N,N-trimethyl-2-butyn-1-aminium chloride (100 µM) and blocked by low concentrations of pirenzepine (300 nM), indicating that they result from activation of M1-like mAChRs. In contrast, cholinergic stimulation increased the excitability of MCs via recruitment of nicotinic AChRs (nAChRs) and M1-like mAChRs. Submaximal activation of these receptors, however, decreased the excitability of MCs. Surprisingly, we found that unlike GCs in the main olfactory bulb, GCs in the AOB are excited by mAChR activation in young postnatal neurons, suggesting marked differences in cholinergic regulation of development between these two regions of the olfactory bulb.
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Acetilcolina/fisiología , Neuronas/efectos de los fármacos , Bulbo Olfatorio/fisiología , Receptor Muscarínico M1/fisiología , Potenciales de Acción/efectos de los fármacos , Factores de Edad , Animales , Antagonistas de Aminoácidos Excitadores/farmacología , Técnicas In Vitro , Ratones , Ratones Endogámicos C57BL , Agonistas Muscarínicos/farmacología , Antagonistas Muscarínicos/farmacología , Neuronas/fisiología , Agonistas Nicotínicos/farmacología , Antagonistas Nicotínicos/farmacología , Bulbo Olfatorio/citología , Bulbo Olfatorio/crecimiento & desarrollo , Receptores Nicotínicos/efectos de los fármacos , Receptores Nicotínicos/fisiologíaRESUMEN
The response of olfactory sensory neurons to TNT and RDX as well as to some volatile organic compounds present in the vapors of antipersonnel landmines has been studied both in the pig and in the rat. GC/MS analyses of different plastic components of six different kinds of landmines were performed in order to identify the components of the "perfume" of mines. Studies on rat olfactory mucosa were carried out with electro-olfactogram and calcium imaging techniques, while changes in the cyclic adenosine monophosphate (cAMP) levels following exposure to odorants and explosives were used as a criterion to evaluate the interaction of TNT and RDX with olfactory receptors in a preparation of isolated pig olfactory cilia. These studies indicate that chemical compounds associated with explosives and explosive devices can activate mammalian olfactory receptors.
Asunto(s)
Sustancias Explosivas/análisis , Neuronas Receptoras Olfatorias/metabolismo , Animales , Calcio/metabolismo , AMP Cíclico/metabolismo , Sustancias Explosivas/metabolismo , Femenino , Cromatografía de Gases y Espectrometría de Masas/métodos , Masculino , Modelos Químicos , Modelos Teóricos , Neuronas Aferentes/metabolismo , Mucosa Olfatoria/metabolismo , Plásticos , Ratas , Ratas Sprague-Dawley , Receptores Odorantes/análisis , Células Receptoras Sensoriales , PorcinosRESUMEN
Modulation of dendrodendritic synapses by the noradrenergic system in the accessory olfactory bulb (AOB) plays a key role in the formation of memory in olfactory-mediated behaviors. We have recently shown that noradrenaline (NA) inhibits mitral cells by increasing gamma-aminobutyric acid inhibitory input onto mitral cells in the AOB, suggesting an excitatory action of NA on granule cells (GCs). Here, we show that NA (10 microM) elicits a long-lasting depolarization of GCs. This effect is mediated by activation of alpha(1)-adrenergic receptors as the depolarization is mimicked by phenylephrine (PE, 30 microM) and completely blocked by the alpha(1)-adrenergic receptor antagonist prazosin (300 nM). In addition to this depolarization, application of NA induced the appearance of a slow afterdepolarization (sADP) following a stimulus-elicited train of action potentials. Similarly, the group I metabotropic glutamate receptor (mGluR1) agonist DHPG (10-30 microM) also produced a depolarization of GCs and the appearance of a stimulus-induced sADP. The ionic and voltage dependence and sensitivity to blockers of the sADP suggest that it is mediated by the nonselective cationic conductance I(CAN). Thus the excitatory action resulting from the activation of these receptors could be mediated by a common transduction target. Surprisingly, the excitatory effect of PE on GCs was completely blocked by the mGluR1 antagonist LY367385 (100 microM). Conversely, the effect of DHPG was not antagonized by the alpha(1)-adrenergic receptor antagonist prazosin (300 nM). These results suggest that most of the noradrenergic effect on GCs in the AOB is mediated by potentiation of a basal activity of mGluR1s.
Asunto(s)
Neuronas/fisiología , Norepinefrina/metabolismo , Bulbo Olfatorio/fisiología , Receptores Adrenérgicos alfa 1/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Agonistas de Receptores Adrenérgicos alfa 1 , Antagonistas de Receptores Adrenérgicos alfa 1 , Agonistas alfa-Adrenérgicos/farmacología , Antagonistas Adrenérgicos alfa/farmacología , Animales , Benzoatos/farmacología , Agonistas de Aminoácidos Excitadores/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Femenino , Glicina/análogos & derivados , Glicina/farmacología , Técnicas In Vitro , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Ratones , Ratones Endogámicos C57BL , Neuronas/efectos de los fármacos , Bulbo Olfatorio/efectos de los fármacos , Fenilefrina/farmacología , Prazosina/farmacología , Receptores de Glutamato Metabotrópico/agonistas , Receptores de Glutamato Metabotrópico/antagonistas & inhibidores , Resorcinoles/farmacologíaRESUMEN
Neurogenesis persists in the olfactory system throughout life. The mechanisms of how new neurons are generated, how they integrate into circuits, and their role in coding remain mysteries. Here we report a technique that will greatly facilitate research into these questions. We found that electroporation can be used to robustly and selectively label progenitors in the Subventicular Zone. The approach was performed postnatally, without surgery, and with near 100% success rates. Labeling was found in all classes of interneurons in the olfactory bulb, persisted to adulthood and had no adverse effects. The broad utility of electroporation was demonstrated by encoding a calcium sensor and markers of intracellular organelles. The approach was found to be effective in wildtype and transgenic mice as well as rats. Given its versatility, robustness, and both time and cost effectiveness, this method offers a powerful new way to use genetic manipulation to understand adult neurogenesis.