RESUMO
Recently, the identification of several classes of aryl sulfonamides and acyl sulfonamides that potently inhibit NaV1.7 and demonstrate high levels of selectivity over other NaV isoforms have been reported. The fully ionizable nature of these inhibitors has been shown to be an important part of the pharmacophore for the observed potency and isoform selectivity. The requirement of this functionality, however, has presented challenges associated with optimization toward inhibitors with drug-like properties and minimal off-target activity. In an effort to obviate these challenges, we set out to develop an orally bioavailable, selective NaV1.7 inhibitor, lacking these acidic functional groups. Herein, we report the discovery of a novel series of inhibitors wherein a triazolesulfone has been designed to serve as a bioisostere for the acyl sulfonamide. This work culminated in the delivery of a potent series of inhibitors which demonstrated good levels of selectivity over NaV1.5 and favorable pharmacokinetics in rodents.
Assuntos
Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Sulfonamidas/farmacologia , Animais , Relação Dose-Resposta a Droga , Humanos , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Estrutura Molecular , Ratos , Relação Estrutura-Atividade , Sulfonamidas/químicaRESUMO
Potent and selective antagonists of the voltage-gated sodium channel NaV1.7 represent a promising avenue for the development of new chronic pain therapies. We generated a small molecule atropisomer quinolone sulfonamide antagonist AMG8379 and a less active enantiomer AMG8380. Here we show that AMG8379 potently blocks human NaV1.7 channels with an IC50 of 8.5 nM and endogenous tetrodotoxin (TTX)-sensitive sodium channels in dorsal root ganglion (DRG) neurons with an IC50 of 3.1 nM in whole-cell patch clamp electrophysiology assays using a voltage protocol that interrogates channels in a partially inactivated state. AMG8379 was 100- to 1000-fold selective over other NaV family members, including NaV1.4 expressed in muscle and NaV1.5 expressed in the heart, as well as TTX-resistant NaV channels in DRG neurons. Using an ex vivo mouse skin-nerve preparation, AMG8379 blocked mechanically induced action potential firing in C-fibers in both a time-dependent and dose-dependent manner. AMG8379 similarly reduced the frequency of thermally induced C-fiber spiking, whereas AMG8380 affected neither mechanical nor thermal responses. In vivo target engagement of AMG8379 in mice was evaluated in multiple NaV1.7-dependent behavioral endpoints. AMG8379 dose-dependently inhibited intradermal histamine-induced scratching and intraplantar capsaicin-induced licking, and reversed UVB radiation skin burn-induced thermal hyperalgesia; notably, behavioral effects were not observed with AMG8380 at similar plasma exposure levels. AMG8379 is a potent and selective NaV1.7 inhibitor that blocks sodium current in heterologous cells as well as DRG neurons, inhibits action potential firing in peripheral nerve fibers, and exhibits pharmacodynamic effects in translatable models of both itch and pain.
Assuntos
Canal de Sódio Disparado por Voltagem NAV1.7/efeitos dos fármacos , Bloqueadores dos Canais de Sódio/farmacologia , Potenciais de Ação/efeitos dos fármacos , Animais , Comportamento Animal/efeitos dos fármacos , Relação Dose-Resposta a Droga , Gânglios Espinais/citologia , Gânglios Espinais/efeitos dos fármacos , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/metabolismo , Miocárdio/metabolismo , Neurônios/efeitos dos fármacos , Dor/prevenção & controle , Dor/psicologia , Técnicas de Patch-Clamp , Prurido/prevenção & controle , Prurido/psicologia , Quinolonas/farmacologia , Bibliotecas de Moléculas Pequenas , Estereoisomerismo , Sulfonamidas/farmacologiaRESUMO
The ß-site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) is one of the most hotly pursued targets for the treatment of Alzheimer's disease. We used a structure- and property-based drug design approach to identify 2-aminooxazoline 3-azaxanthenes as potent BACE1 inhibitors which significantly reduced CSF and brain Aß levels in a rat pharmacodynamic model. Compared to the initial lead 2, compound 28 exhibited reduced potential for QTc prolongation in a non-human primate cardiovascular safety model.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Xantenos/química , Xantenos/farmacologia , Doença de Alzheimer/tratamento farmacológico , Animais , Linhagem Celular , Células HEK293 , Humanos , Inibidores de Proteases/síntese química , Ratos , Xantenos/síntese químicaRESUMO
Microglia are the resident immune cells in the brain that play a key role in driving neuroinflammation, a hallmark of neurodegenerative disorders. Inducible microglia-like cells have been developed as an in vitro platform for molecular and therapeutic hypothesis generation and testing. However, there has been no systematic assessment of similarity of these cells to primary human microglia along with their responsiveness to external cues expected of primary cells in the brain. In this study, we performed transcriptional characterization of commercially available human inducible pluripotent stem cell (iPSC)-derived microglia-like (iMGL) cells by bulk and single cell RNA sequencing to assess their similarity with primary human microglia. To evaluate their stimulation responsiveness, iMGL cells were treated with Liver X Receptor (LXR) pathway agonists and their transcriptional responses characterized by bulk and single cell RNA sequencing. Bulk transcriptome analyses demonstrate that iMGL cells have a similar overall expression profile to freshly isolated human primary microglia and express many key microglial transcription factors and functional and disease-associated genes. Notably, at the single-cell level, iMGL cells exhibit distinct transcriptional subpopulations, representing both homeostatic and activated states present in normal and diseased primary microglia. Treatment of iMGL cells with LXR pathway agonists induces robust transcriptional changes in lipid metabolism and cell cycle at the bulk level. At the single cell level, we observe heterogeneity in responses between cell subpopulations in homeostatic and activated states and deconvolute bulk expression changes into their corresponding single cell states. In summary, our results demonstrate that iMGL cells exhibit a complex transcriptional profile and responsiveness, reminiscent of in vivo microglia, and thus represent a promising model system for therapeutic development in neurodegeneration.
Assuntos
Células-Tronco Pluripotentes Induzidas , Doenças Neurodegenerativas , Células-Tronco Pluripotentes , Humanos , Microglia/metabolismo , Fatores de Transcrição/metabolismo , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismoRESUMO
A series of fused 6,6-bicyclic chromenones was investigated for activity against the bradykinin B1 receptor. SAR studies based on a pharmacophore model revealed compounds with high affinity for both human and rabbit B1. These compounds demonstrated favorable pharmacokinetic properties and 5-chlorochromenone 15 was efficacious in a carrageenan-induced mechanical hyperalgesia model for chronic pain.
Assuntos
Benzopiranos/síntese química , Antagonistas de Receptor B1 da Bradicinina , Animais , Benzopiranos/farmacologia , Carragenina/farmacologia , Química Farmacêutica/métodos , Dor Crônica/tratamento farmacológico , Desenho de Fármacos , Humanos , Hiperalgesia/tratamento farmacológico , Concentração Inibidora 50 , Cinética , Modelos Químicos , Coelhos , Relação Estrutura-Atividade , Fator de Necrose Tumoral alfa/metabolismoRESUMO
A series of alpha-amidosulfones were found to be potent and selective agonists of CB(2). The discovery, synthesis, and structure-activity relationships of this series of agonists are reported. In addition, the pharmacokinetic properties of the most promising compounds are profiled.
Assuntos
Receptor CB2 de Canabinoide/agonistas , Sulfonas/química , Amidas/química , Amidas/farmacologia , Animais , Humanos , Microssomos Hepáticos , Farmacocinética , Ratos , Relação Estrutura-Atividade , Sulfonas/farmacologiaRESUMO
Glutamatergic neurotransmission requires vesicular glutamate transporters (VGLUTs) to sequester glutamate into synaptic vesicles. Generally, VGLUT1 and VGLUT2 isoforms show complementary expression in the CNS and retina. However, little is known about whether isoform-specific expression serves distinct pathways and physiological functions. Here, by examining visual functions in VGLUT1-null mice, we demonstrate that visual signaling from photoreceptors to retinal output neurons requires VGLUT1. However, photoentrainment and pupillary light responses are preserved. We provide evidence that melanopsin-containing, intrinsically photosensitive retinal ganglion cells (RGCs), signaling via VGLUT2 pathways, support these non-image-forming functions. We conclude that VGLUT1 is essential for transmitting visual signals from photoreceptors to second- and third-order neurons, but VGLUT1 is not necessary for intrinsic visual functions. Furthermore, melanopsin and VGLUT2 expression in a subset of RGCs immediately after birth strongly supports the idea that intrinsic vision can function well before rod- and cone-mediated signaling has matured.
Assuntos
Células Fotorreceptoras/fisiologia , Transdução de Sinais/fisiologia , Sinapses/fisiologia , Proteína Vesicular 1 de Transporte de Glutamato/fisiologia , Visão Ocular/fisiologia , Animais , Potenciais Evocados Visuais/fisiologia , Camundongos , Camundongos Knockout , Estimulação Luminosa/métodos , Isoformas de Proteínas/fisiologia , Ratos , Ratos Long-Evans , Células Ganglionares da Retina/fisiologiaRESUMO
Structural modifications to the central portion of the N-arylamide oxadiazole scaffold led to the identification of N-arylpiperidine oxadiazoles as conformationally constrained analogs that offered improved stability and comparable potency and selectivity. The simple, modular scaffold allowed for the use of expeditious and divergent synthetic routes, which provided two-directional SAR in parallel. Several potent and selective agonists from this novel ligand class are described.
Assuntos
Microssomos Hepáticos/metabolismo , Oxidiazóis , Receptor CB2 de Canabinoide/agonistas , Animais , Técnicas de Química Combinatória , Humanos , Conformação Molecular , Estrutura Molecular , Oxidiazóis/síntese química , Oxidiazóis/química , Oxidiazóis/farmacocinética , Oxidiazóis/farmacologia , Ratos , Relação Estrutura-AtividadeRESUMO
Because of its strong genetic validation, NaV1.7 has attracted significant interest as a target for the treatment of pain. We have previously reported on a number of structurally distinct bicyclic heteroarylsulfonamides as NaV1.7 inhibitors that demonstrate high levels of selectivity over other NaV isoforms. Herein, we report the discovery and optimization of a series of atropisomeric quinolinone sulfonamide inhibitors [ Bicyclic sulfonamide compounds as sodium channel inhibitors and their preparation . WO 2014201206, 2014 ] of NaV1.7, which demonstrate nanomolar inhibition of NaV1.7 and exhibit high levels of selectivity over other sodium channel isoforms. After optimization of metabolic and pharmacokinetic properties, including PXR activation, CYP2C9 inhibition, and CYP3A4 TDI, several compounds were advanced into in vivo target engagement and efficacy models. When tested in mice, compound 39 (AM-0466) demonstrated robust pharmacodynamic activity in a NaV1.7-dependent model of histamine-induced pruritus (itch) and additionally in a capsaicin-induced nociception model of pain without any confounding effect in open-field activity.
Assuntos
Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Quinolonas/química , Sulfonamidas/química , Bloqueadores do Canal de Sódio Disparado por Voltagem/química , Analgésicos/química , Analgésicos/farmacocinética , Analgésicos/farmacologia , Animais , Capsaicina , Linhagem Celular , Cães , Histamina , Camundongos Endogâmicos C57BL , Simulação de Acoplamento Molecular , Dor/induzido quimicamente , Dor/prevenção & controle , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/metabolismo , Prurido/induzido quimicamente , Prurido/prevenção & controle , Quinolonas/administração & dosagem , Quinolonas/síntese química , Quinolonas/farmacocinética , Quinolonas/farmacologia , Ratos , Relação Estrutura-Atividade , Sulfonamidas/administração & dosagem , Sulfonamidas/síntese química , Sulfonamidas/farmacocinética , Sulfonamidas/farmacologia , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacocinética , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologiaRESUMO
[This corrects the article DOI: 10.1021/acsmedchemlett.6b00243.].
RESUMO
Several reports have recently emerged regarding the identification of heteroarylsulfonamides as NaV1.7 inhibitors that demonstrate high levels of selectivity over other NaV isoforms. The optimization of a series of internal NaV1.7 leads that address a number of metabolic liabilities including bioactivation, PXR activation, as well as CYP3A4 induction and inhibition led to the identification of potent and selective inhibitors that demonstrated favorable pharmacokinetic profiles and were devoid of the aforementioned liabilities. The key to achieving this within a series prone to transporter-mediated clearance was the identification of a small range of optimal cLogD values and the discovery of subtle PXR SAR that was not lipophilicity dependent. This enabled the identification of compound 20, which was advanced into a target engagement pharmacodynamic model where it exhibited robust reversal of histamine-induced scratching bouts in mice.
Assuntos
Isoquinolinas/química , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Sulfonamidas/química , Bloqueadores do Canal de Sódio Disparado por Voltagem/química , Animais , Linhagem Celular , Citocromo P-450 CYP3A/biossíntese , Inibidores do Citocromo P-450 CYP3A/química , Inibidores do Citocromo P-450 CYP3A/farmacocinética , Inibidores do Citocromo P-450 CYP3A/farmacologia , Cães , Indução Enzimática , Histamina , Humanos , Isoquinolinas/administração & dosagem , Isoquinolinas/farmacocinética , Masculino , Camundongos Endogâmicos C57BL , Receptor de Pregnano X , Prurido/induzido quimicamente , Prurido/prevenção & controle , Ratos , Receptores de Esteroides/agonistas , Relação Estrutura-Atividade , Sulfonamidas/administração & dosagem , Sulfonamidas/farmacocinética , Sulfonamidas/farmacologia , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacocinética , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologiaRESUMO
Exocytotic release of the excitatory neurotransmitter glutamate depends on transport of this amino acid into synaptic vesicles. Recent work has identified a distinct family of proteins responsible for vesicular glutamate transport (VGLUTs) that show no sequence similarity to the other two families of vesicular neurotransmitter transporters. The distribution of VGLUT1 and VGLUT2 accounts for the ability of most established excitatory neurons to release glutamate by exocytosis. Surprisingly, they show a striking complementary pattern of expression in adult brain that might reflect differences in membrane trafficking. By contrast, VGLUT3 is expressed by many cells traditionally considered to release a different classical transmitter, suggesting novel roles for glutamate as an extracellular signal. VGLUT3 also differs from VGLUT1 and VGLUT2 in its subcellular location, with somatodendritic as well as axonal expression.
Assuntos
Sistema X-AG de Transporte de Aminoácidos/metabolismo , Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Encéfalo/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Membrana Transportadoras , Transmissão Sináptica/fisiologia , Vesículas Sinápticas/metabolismo , Proteínas de Transporte Vesicular , Animais , Potenciais Pós-Sinápticos Excitadores/fisiologia , Exocitose/fisiologia , Humanos , Transdução de Sinais/fisiologia , Distribuição Tecidual , Proteína Vesicular 1 de Transporte de Glutamato , Proteína Vesicular 2 de Transporte de Glutamato , Proteínas Vesiculares de Transporte de GlutamatoRESUMO
To evaluate whether the organization of glutamatergic fibers systems in the lumbar cord is also evident at other spinal levels, we examined the immunocytochemical distribution of vesicle glutamate transporters 1 and 2 (VGLUT1, VGLUT2) at several different levels of the rat spinal cord. We also examined the expression of VGLUTs in an ascending sensory pathway, the spinocervical tract, and colocalization of VGLUT1 and VGLUT2. Mainly small VGLUT2-immunoreactive varicosities occurred at relatively high densities in most areas, with the highest density in laminae I-II. VGLUT1 immunolabeling, including small and medium-sized to large varicosities, was more differentiated, with the highest density in the deep dorsal horn and in certain nuclei such as the internal basilar nucleus, the central cervical nucleus, and the column of Clarke. Lamina I and IIo displayed a moderate density of small VGLUT1 varicosities at all spinal levels, although in the spinal enlargements a uniform density of such varicosities was evident throughout laminae I-II in the medial half of the dorsal horn. Corticospinal tract axons displayed VGLUT1, indicating that the corticospinal tract is an important source of small VGLUT1 varicosities. VGLUT1 and VGLUT2 were cocontained in small numbers of varicosities in laminae III-IV and IX. Anterogradely labeled spinocervical tract terminals in the lateral cervical nucleus were VGLUT2 immunoreactive. In conclusion, the principal distribution patterns of VGLUT1 and VGLUT2 are essentially similar throughout the rostrocaudal extension of the spinal cord. The mediolateral differences in VGLUT1 distribution in laminae I-II suggest dual origins of VGLUT1-immunoreactive varicosities in this region.
Assuntos
Vias Neurais/metabolismo , Medula Espinal/metabolismo , Proteína Vesicular 1 de Transporte de Glutamato/metabolismo , Proteína Vesicular 2 de Transporte de Glutamato/metabolismo , Animais , Feminino , Imuno-Histoquímica , Masculino , Vias Neurais/citologia , Neurônios/citologia , Neurônios/metabolismo , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Medula Espinal/citologia , Distribuição TecidualRESUMO
Human genetic evidence has identified the voltage-gated sodium channel NaV1.7 as an attractive target for the treatment of pain. We initially identified naphthalene sulfonamide 3 as a potent and selective inhibitor of NaV1.7. Optimization to reduce biliary clearance by balancing hydrophilicity and hydrophobicity (Log D) while maintaining NaV1.7 potency led to the identification of quinazoline 16 (AM-2099). Compound 16 demonstrated a favorable pharmacokinetic profile in rat and dog and demonstrated dose-dependent reduction of histamine-induced scratching bouts in a mouse behavioral model following oral dosing.
RESUMO
Recent studies implicate dendritic endocannabinoid release from subsynaptic dendrites and subsequent inhibition of neurotransmitter release from nerve terminals as a means of retrograde signaling in multiple brain regions. Here we show that type 1 cannabinoid receptor-mediated endocannabinoid signaling is not involved in the retrograde control of synaptic efficacy at inhibitory synapses between fast-spiking interneurons and pyramidal cells in layer 2/3 of the neocortex. Vesicular neurotransmitter transporters, such as vesicular glutamate transporters (VGLUTs) 1 and 2, are localized to presynaptic terminals and accumulate neurotransmitters into synaptic vesicles. A third subtype of VGLUTs (VGLUT3) was recently identified and found localized to dendrites of various cell types. We demonstrate, using multiple immunofluorescence labeling and confocal laser-scanning microscopy, that VGLUT3-like immunoreactivity is present in dendrites of layer 2/3 pyramidal neurons in the rat neocortex. Electron microscopy analysis confirmed that VGLUT3-like labeling is localized to vesicular structures, which show a tendency to accumulate in close proximity to postsynaptic specializations in dendritic shafts of pyramidal cells. Dual whole-cell recordings revealed that retrograde signaling between fast-spiking interneurons and pyramidal cells was enhanced under conditions of maximal efficacy of VGLUT3-mediated glutamate uptake, whereas it was reduced when glutamate uptake was inhibited by incrementing concentrations of the nonselective VGLUT inhibitor Evans blue (0.5-5.0 microm) or intracellular Cl- concentrations (4-145 mm). Our results present further evidence that dendritic vesicular glutamate release, controlled by novel VGLUT isoforms, provides fast negative feedback at inhibitory neocortical synapses, and demonstrate that glutamate can act as a retrograde messenger in the CNS.
Assuntos
Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Neocórtex/fisiologia , Receptores de Canabinoides/metabolismo , Sinapses/fisiologia , Animais , Dendritos/fisiologia , Dendritos/ultraestrutura , Interneurônios/metabolismo , Neocórtex/ultraestrutura , Técnicas de Patch-Clamp , Células Piramidais/metabolismo , Células Piramidais/ultraestrutura , Ratos , Ratos Sprague-Dawley , Sinapses/ultraestrutura , Proteínas Vesiculares de Transporte de GlutamatoRESUMO
BACE1 inhibition to prevent Aß peptide formation is considered to be a potential route to a disease-modifying treatment for Alzheimer's disease. Previous efforts in our laboratory using a combined structure- and property-based approach have resulted in the identification of aminooxazoline xanthenes as potent BACE1 inhibitors. Herein, we report further optimization leading to the discovery of inhibitor 15 as an orally available and highly efficacious BACE1 inhibitor that robustly reduces CSF and brain Aß levels in both rats and nonhuman primates. In addition, compound 15 exhibited low activity on the hERG ion channel and was well tolerated in an integrated cardiovascular safety model.
RESUMO
Synaptic transmission from glutamatergic neurons requires vesicular glutamate transporters (VGLUTs) to concentrate cytosolic glutamate in synaptic vesicles. In retina, glutamatergic photoreceptors and bipolar cells exclusively express the VGLUT1 isoform, whereas ganglion cells express VGLUT2. Surprisingly, the recently identified VGLUT3 isoform was found in presumed amacrine cells, generally considered to be inhibitory interneurons. To investigate the synaptic machinery and conceivable secondary neurotransmitter composition of VGLUT3 cells, and to determine a potential functional role, we further investigated these putative glutamatergic amacrine cells in adult and developing rodent retina. Reverse transcriptase-PCR substantiated VGLUT3 expression in mouse retina. VGLUT3 cells did not immunostain for ganglion or bipolar cell markers, providing evidence that they are amacrine cells. VGLUT3 colocalized with synaptic vesicle markers, and electron microscopy showed that VGLUT3 immunostained synaptic vesicles. VGLUT3 cells were not immunoreactive for amacrine cell markers gamma-aminobutyric acid, choline acetyltransferase, calretinin, or tyrosine hydroxylase, although they immunostain for glycine. VGLUT3 processes made synaptic contact with ganglion cell dendrites, suggesting input onto these cells. VGLUT3 immunostaining was closely associated with the metabotropic glutamate receptor 4, which is consistent with glutamatergic synaptic exocytosis by these cells. In the maturing mouse retina, Western blots showed VGLUT3 expression at postnatal day 7/8 (P7/8). VGLUT3 immunostaining in retinal sections was first observed at P8, achieving an adult pattern at P12. Thus, VGLUT3 function commences around the same time as VGLUT1-mediated glutamatergic transmission from bipolar cells. Furthermore, a subset of VGLUT3 cells expressed the circadian clock gene period 1, implicating VGLUT3 cells as part of the light-entrainable retina-based circadian system.
Assuntos
Células Amácrinas/metabolismo , Sistemas de Transporte de Aminoácidos Acídicos/biossíntese , Ácido Glutâmico/metabolismo , Células Amácrinas/crescimento & desenvolvimento , Animais , Western Blotting , Ritmo Circadiano , Camundongos , Microscopia Confocal , Neurotransmissores/metabolismo , Terminações Pré-Sinápticas/metabolismo , Isoformas de Proteínas/biossíntese , Ratos , Ratos Long-Evans , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Vesiculares de Transporte de GlutamatoRESUMO
The caudomedial shell of the rat nucleus accumbens exhibits inhomogeneous distribution patterns of the vesicular glutamate transporters 1 (VGLUT1) and 2 (VGLUT2). This paper focuses on the question of whether patterns of VGLUT1 and VGLUT2 correspond to cytoarchitectonically and cytochemically defined subterritories of the caudomedial shell region. VGLUT2 was shown to be coexpressed with calretinin in the dense axonal plexus known to emanate from the paraventricular thalamic nucleus. In regions termed corridors, which are spared by this paraventricular thalamic innervation, axonal terminals were found to be clustered and VGLUT1-immunoreactive. It is assumed that these fibers originate in the prelimbic cortex and/or in the parvicellular basal amygdaloid nucleus known to project to accumbal shell components. Our findings confirm the existence of two well-separated neuronal circuits in the caudomedial shell that are dominated by two different excitatory input systems originating from either thalamic, cortical, or cortex-like amygdaloid sources. The large lateral corridors-which resemble the accumbal core not only in respect to their VGLUT1 immunolabeling but also concerning their content of calbindin-positive cells-may represent a component of the anatomically weakly defined accumbal shore region.
Assuntos
Vias Aferentes/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Membrana Transportadoras , Neurônios/metabolismo , Núcleo Accumbens/metabolismo , Proteína G de Ligação ao Cálcio S100/metabolismo , Proteínas de Transporte Vesicular , Tonsila do Cerebelo/citologia , Tonsila do Cerebelo/metabolismo , Animais , Calbindina 2 , Calbindinas , Feminino , Ácido Glutâmico/metabolismo , Imuno-Histoquímica , Masculino , Microscopia Confocal , Núcleos da Linha Média do Tálamo/citologia , Núcleos da Linha Média do Tálamo/metabolismo , Neurônios/citologia , Núcleo Accumbens/citologia , Terminações Pré-Sinápticas/metabolismo , Terminações Pré-Sinápticas/ultraestrutura , Ratos , Ratos Wistar , Proteína Vesicular 1 de Transporte de Glutamato , Proteína Vesicular 2 de Transporte de GlutamatoRESUMO
Three closely related proteins transport glutamate into synaptic vesicles for release by exocytosis. Complementary patterns of expression in glutamatergic terminals have been reported for VGLUT1 and VGLUT2. VGLUT3 shows expression by many cells not considered to be glutamatergic. Here we describe the changes in VGLUT expression that occur during development. VGLUT1 expression increases gradually after birth and eventually predominates over the other isoforms in telencephalic regions. Expressed at high levels shortly after birth, VGLUT2 declines with age in multiple regions, in the cerebellum by 14-fold. In contrast, Coexpression of the two isoforms occurs transiently during development as well as permanently in a restricted subset of glutamatergic terminals in the adult. VGLUT3 is transiently expressed at high levels by select neuronal populations, including terminals in the cerebellar nuclei, scattered neurons in the cortex, and progenitor-like cells, implicating exocytotic glutamate release in morphogenesis and development. VGLUT3 also colocalizes extensively during development with the neuronal vesicular monoamine transporter VMAT2, with the vesicular acetylcholine transporter VAChT, and with the vesicular gamma-aminobutyric acid transporter VGAT. Such coexpression occurs particularly at some specific developmental stages and is restricted to certain sets of cells. In skeletal muscle, VGLUT3 localizes to granular organelles in the axon terminal as well as in the muscle sarcoplasm. The results suggest novel mechanisms and roles for regulated transmitter release.