RESUMO
Brainstem median raphe (MR) neurons expressing the serotonergic regulator gene Pet1 send collateralized projections to forebrain regions to modulate affective, memory-related, and circadian behaviors. Some Pet1 neurons express a surprisingly incomplete battery of serotonin pathway genes, with somata lacking transcripts for tryptophan hydroxylase 2 (Tph2) encoding the rate-limiting enzyme for serotonin [5-hydroxytryptamine (5-HT)] synthesis, but abundant for vesicular glutamate transporter type 3 (Vglut3) encoding a synaptic vesicle-associated glutamate transporter. Genetic fate maps show these nonclassical, putatively glutamatergic Pet1 neurons in the MR arise embryonically from the same progenitor cell compartment-hindbrain rhombomere 2 (r2)-as serotonergic TPH2+ MR Pet1 neurons. Well established is the distribution of efferents en masse from r2-derived, Pet1-neurons; unknown is the relationship between these efferent targets and the specific constituent source-neuron subgroups identified as r2-Pet1Tph2-high versus r2-Pet1Vglut3-high Using male and female mice, we found r2-Pet1 axonal boutons segregated anatomically largely by serotonin+ versus VGLUT3+ identity. The former present in the suprachiasmatic nucleus, paraventricular nucleus of the thalamus, and olfactory bulb; the latter are found in the hippocampus, cortex, and septum. Thus r2-Pet1Tph2-high and r2-Pet1Vglut3-high neurons likely regulate distinct brain regions and behaviors. Some r2-Pet1 boutons encased interneuron somata, forming specialized presynaptic "baskets" of VGLUT3+ or VGLUT3+/5-HT+ identity; this suggests that some r2-Pet1Vglut3-high neurons may regulate local networks, perhaps with differential kinetics via glutamate versus serotonin signaling. Fibers from other Pet1 neurons (non-r2-derived) were observed in many of these same baskets, suggesting multifaceted regulation. Collectively, these findings inform brain organization and new circuit nodes for therapeutic considerations.SIGNIFICANCE STATEMENT Our findings match axonal bouton neurochemical identity with distant cell bodies in the brainstem raphe. The results are significant because they suggest that disparate neuronal subsystems derive from Pet1+ precursor cells of the embryonic progenitor compartment rhombomere 2 (r2). Of these r2-Pet1 neuronal subsystems, one appears largely serotonergic, as expected given expression of the serotonergic regulator PET1, and projects to the olfactory bulb, thalamus, and suprachiasmatic nucleus. Another expresses VGLUT3, suggesting principally glutamate transmission, and projects to the hippocampus, septum, and cortex. Some r2-Pet1 boutons-those that are VGLUT3+ or VGLUT3+/5-HT+ co-positive-comprise "baskets" encasing interneurons, suggesting that they control local networks perhaps with differential kinetics via glutamate versus serotonin signaling. Results inform brain organization and circuit nodes for therapeutic consideration.
Assuntos
Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Química Encefálica/fisiologia , Núcleos da Rafe/metabolismo , Rombencéfalo/metabolismo , Serotonina/metabolismo , Fatores de Transcrição/metabolismo , Sistemas de Transporte de Aminoácidos Acídicos/análise , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Núcleos da Rafe/química , Rombencéfalo/química , Serotonina/análise , Fatores de Transcrição/análiseRESUMO
Neurons receive synaptic inputs on extensive neurite arbors. How information is organized across arbors and how local processing in neurites contributes to circuit function is mostly unknown. Here, we used two-photon Ca2+ imaging to study visual processing in VGluT3-expressing amacrine cells (VG3-ACs) in the mouse retina. Contrast preferences (ON vs. OFF) varied across VG3-AC arbors depending on the laminar position of neurites, with ON responses preferring larger stimuli than OFF responses. Although arbors of neighboring cells overlap extensively, imaging population activity revealed continuous topographic maps of visual space in the VG3-AC plexus. All VG3-AC neurites responded strongly to object motion, but remained silent during global image motion. Thus, VG3-AC arbors limit vertical and lateral integration of contrast and location information, respectively. We propose that this local processing enables the dense VG3-AC plexus to contribute precise object motion signals to diverse targets without distorting target-specific contrast preferences and spatial receptive fields.
Assuntos
Células Amácrinas/fisiologia , Sistemas de Transporte de Aminoácidos Acídicos/análise , Neuritos/fisiologia , Retina/fisiologia , Visão Ocular , Animais , Camundongos , Imagem ÓpticaRESUMO
Using specific riboprobes, we characterized the expression of vesicular glutamate transporter (VGLUT)1-VGLUT3 transcripts in lumbar 4-5 (L4-5) dorsal root ganglions (DRGs) and the thoracolumbar to lumbosacral spinal cord in male BALB/c mice after a 1- or 3-day hindpaw inflammation, or a 7-day sciatic nerve axotomy. Sham animals were also included. In sham and contralateral L4-5 DRGs of injured mice, VGLUT1-, VGLUT2- and VGLUT3 mRNAs were expressed in â¼45%, â¼69% or â¼17% of neuron profiles (NPs), respectively. VGLUT1 was expressed in large and medium-sized NPs, VGLUT2 in NPs of all sizes, and VGLUT3 in small and medium-sized NPs. In the spinal cord, VGLUT1 was restricted to a number of NPs at thoracolumbar and lumbar segments, in what appears to be the dorsal nucleus of Clarke, and in mid laminae III-IV. In contrast, VGLUT2 was present in numerous NPs at all analyzed spinal segments, except the lateral aspects of the ventral horns, especially at the lumbar enlargement, where it was virtually absent. VGLUT3 was detected in a discrete number of NPs in laminae III-IV of the dorsal horn. Axotomy resulted in a moderate decrease in the number of DRG NPs expressing VGLUT3, whereas VGLUT1 and VGLUT2 were unaffected. Likewise, the percentage of NPs expressing VGLUT transcripts remained unaltered after hindpaw inflammation, both in DRGs and the spinal cord. Altogether, these results confirm previous descriptions on VGLUTs expression in adult mice DRGs, with the exception of VGLUT1, whose protein expression was detected in a lower percentage of mouse DRG NPs. A detailed account on the location of neurons expressing VGLUTs transcripts in the adult mouse spinal cord is also presented. Finally, the lack of change in the number of neurons expressing VGLUT1 and VGLUT2 transcripts after axotomy, as compared to data on protein expression, suggests translational rather than transcriptional regulation of VGLUTs after injury.
Assuntos
Gânglios Espinais/metabolismo , Neurônios/metabolismo , Medula Espinal/metabolismo , Proteínas Vesiculares de Transporte de Glutamato/metabolismo , Sistemas de Transporte de Aminoácidos Acídicos/análise , Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Animais , Axotomia , Membro Posterior , Inflamação/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , RNA Mensageiro/metabolismo , Nervo Isquiático/lesões , Proteína Vesicular 1 de Transporte de Glutamato/análise , Proteína Vesicular 1 de Transporte de Glutamato/metabolismo , Proteína Vesicular 2 de Transporte de Glutamato/análise , Proteína Vesicular 2 de Transporte de Glutamato/metabolismo , Proteínas Vesiculares de Transporte de Glutamato/análiseRESUMO
Glutamate has been implicated in signal transmission between inner hair cells and afferent fibers of the organ of Corti. The inner hair cells are enriched in glutamate and the postsynaptic membranes express AMPA glutamate receptors. However, it is not known whether inner hair cells contain a mechanism for glutamate replenishment. Such a mechanism must be in place to sustain glutamate neurotransmission. Here we provide RT-PCR and immunofluorescence data indicating that system A transporter 1 (SLC38A1), which is associated with neuronal glutamine transport and synthesis of the neurotransmitters GABA and glutamate in CNS, is expressed in inner hair cells. It was previously shown that inner hair cells contain glutaminase that converts glutamine to glutamate. Thus, our finding that inner hair cells express a glutamine transporter and the key glutamine metabolizing enzyme glutaminase, provides a mechanism for glutamate replenishment and bolsters the idea that glutamate serves as a transmitter in the peripheral synapse of the auditory system.
Assuntos
Sistema A de Transporte de Aminoácidos/análise , Ácido Glutâmico/metabolismo , Células Ciliadas Auditivas Internas/química , Sistema A de Transporte de Aminoácidos/genética , Sistema A de Transporte de Aminoácidos/metabolismo , Sistemas de Transporte de Aminoácidos Acídicos/análise , Animais , Células Ciliadas Auditivas Internas/metabolismo , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína Vesicular 1 de Transporte de Glutamato/análise , Proteína Vesicular 2 de Transporte de Glutamato/análiseRESUMO
In the mammalian retina, two types of catecholaminergic amacrine cells have been described. Although dopaminergic type 1 cells are well characterized, the physiology of type 2 cells is, so far, unknown. To target type 2 cells specifically, we used a transgenic mouse line that expresses green fluorescent protein under the control of the tyrosine hydroxylase promoter. Type 2 cells are GABAergic and have an extensive dendritic arbor, which stratifies in the middle of the inner plexiform layer. Our data suggest that type 2 cells comprise two subpopulations with identical physiological properties: one has its somata located in the inner nuclear layer and the other in the ganglion cell layer. Immunostaining with bipolar cell markers suggested that type 2 cells receive excitatory inputs from type 3 OFF and type 5 ON bipolar cells. Consistently, patch-clamp recordings showed that type 2 cells are ON-OFF amacrine cells. Blocking excitatory inputs revealed that different rod and cone pathways are active under scotopic and mesopic light conditions. Blockade of inhibitory inputs led to membrane potential oscillations in type 2 cells, suggesting that GABAergic and glycinergic amacrine cells strongly influence type 2 cell signaling. Among the glycinergic amacrine cells, we identified the VGluT3-immunoreactive amacrine cell as a likely candidate. Collectively, light responses of type 2 cells were remarkably uniform over a wide range of light intensities. These properties point toward a general function of type 2 cells that is maintained under scotopic and mesopic conditions.
Assuntos
Células Amácrinas/química , Proteínas de Fluorescência Verde/genética , Estimulação Luminosa/métodos , Tirosina 3-Mono-Oxigenase/genética , Células Amácrinas/citologia , Células Amácrinas/fisiologia , Sistemas de Transporte de Aminoácidos Acídicos/análise , Sistemas de Transporte de Aminoácidos Acídicos/fisiologia , Animais , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Tirosina 3-Mono-Oxigenase/fisiologiaRESUMO
The GltS Na+/glutamate permease of Escherichia coli is the most extensively studied member of the ESS family of bacterial glutamate:Na+ symporters. This paper presents the membrane topology analysis of the GltS with translational alkaline phosphatase and beta-galactosidase gene fusions generated by TnphoA, nested deletions and targeted fusions. The topology model suggested by the translational fusion technique is compared with the MemGen model and discussed in detail.
Assuntos
Sistemas de Transporte de Aminoácidos Acídicos/análise , Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Membrana Celular/enzimologia , Proteínas de Escherichia coli/análise , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Biossíntese de Proteínas , Simportadores/análise , Simportadores/metabolismo , Fosfatase Alcalina/análise , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Sequência de Aminoácidos , Sistemas de Transporte de Aminoácidos Acídicos/genética , Fusão Gênica Artificial , Proteínas de Escherichia coli/genética , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Simportadores/genética , beta-Galactosidase/análise , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
The third vesicular glutamate transporter, VGLUT3, is distributed in cell bodies of neocortical neurons and axon terminals mainly in the superficial part of layer II/III of the cerebral cortex. We examined the chemical characteristics of VGLUT3-expressing neurons by immunohistochemistry in the rat neocortex. Since the vast majority of VGLUT3-immunoreactive neurons showed immunoreactivities for GABA, preprotachykinin B (PPTB) and cholecystokinin, VGLUT3-immunoreactive neocortical neurons were considered to constitute a subgroup of GABAergic interneurons. VGLUT3-immunoreactive axon terminals were immunopositive for either vesicular GABA transporter (VGAT) or serotonin. These results together with anterograde tracer injection and chemical lesion experiments in the dorsal and median raphe nuclei revealed that the neocortex contains at least two kinds of VGLUT3-laden axon terminals: one is serotonergic and derived from the raphe nuclei, and the other is GABAergic and intrinsic in the neocortex. Furthermore, many VGLUT3/VGAT-immunoreactive terminals formed axon baskets and made axosomatic symmetric synapses on neocortical neurons, most of which were immunoreactive for PPTB. VGLUT3-immunopositive axon baskets surrounded about a half of PPTB-positive and almost all VGLUT3-positive neurons. Thus, VGLUT3-expressing GABAergic interneurons form a chemically specific circuit within the PPTB-producing interneuron group and it is likely that glutamate is used within the chemically specific circuit.
Assuntos
Sistemas de Transporte de Aminoácidos Acídicos/biossíntese , Interneurônios/metabolismo , Neocórtex/metabolismo , Rede Nervosa/metabolismo , Neurocinina B/biossíntese , Fragmentos de Peptídeos/biossíntese , Sistemas de Transporte de Aminoácidos Acídicos/análise , Animais , Feminino , Cobaias , Interneurônios/química , Masculino , Neocórtex/química , Rede Nervosa/química , Neurocinina B/análise , Fragmentos de Peptídeos/análise , Coelhos , Ratos , Ratos Wistar , Proteínas Vesiculares de Transporte de GlutamatoRESUMO
Three vesicular glutamate transporters (VGLUTs) have been recently identified and their distribution has been mapped in various brain areas. In the present study, we used morphological approaches to investigate their expression in the rat lumbar spinal cord and dorsal root ganglia. Our results show a complementary distribution of VGLUT-expressing fibers in the spinal cord, with no overlapping in nerve endings. In the dorsal horn, VGLUT1 is most abundant in mechanosensory/proprioceptive deep afferent fibers. VGLUT2 and VGLUT3 are expressed only at moderate levels in primary sensory afferent fibers and are not used by central projections of nociceptive neurons. VGLUT1 and VGLUT2 mRNAs are mainly segregated in superficial laminae but colocalized in deeper laminae. Weak expression of VGLUT3 mRNA is only detected in deep laminae. The colocalization of VGLUT1 and VGLUT2 transcripts in most sensory neurons of the dorsal root ganglia is not in agreement with the clear segregation between the proteins in their spinal projections. Such a discrepancy suggests targeting mechanisms specific for each transporter and/or a distinct regulation of their translation. In the ventral horn, the expression of VGLUT1 and VGLUT2 mRNAs in motoneuron perikarya suggests the possible unexpected role of glutamate in the vertebrate neuromuscular junction. These results demonstrate the existence of different subpopulations of glutamate nerve terminals in the rat lumbar spinal cord and suggest that functionally distinct subsets of excitatory glutamatergic neuronal networks are involved in sensory processing and motor control.
Assuntos
Sistemas de Transporte de Aminoácidos Acídicos/análise , Proteínas de Transporte/análise , Gânglios Espinais/química , Proteínas de Membrana Transportadoras , Medula Espinal/química , Proteínas de Transporte Vesicular , Sistemas de Transporte de Aminoácidos Acídicos/genética , Animais , Western Blotting , Proteínas de Transporte/genética , Imuno-Histoquímica , Hibridização In Situ , Vértebras Lombares , Neurônios Motores/química , RNA Mensageiro/análise , Ratos , Proteína Vesicular 1 de Transporte de Glutamato , Proteína Vesicular 2 de Transporte de Glutamato , Proteínas Vesiculares de Transporte de GlutamatoRESUMO
Immunocytochemical staining of vertical sections through rat, mouse, and macaque monkey retinae with antibodies against the vesicular glutamate transporter vesicular glutamate transporter 3 (vGluT3) showed a sparse population of amacrine cells. The labeled cells had similar appearances in the three species and probably represent homologous types. They were studied in detail in the rat retina. The thin varicose dendrites of vGluT3 amacrine cells formed a convoluted dendritic tree of approximately 100 microm in diameter that was bistratified in the center of the inner plexiform layer. The dendrites of vGluT3 cells were squeezed between the two strata of cholinergic dendrites. The density of vGluT3 cells was measured in retinal wholemounts and increased from 200/mm2 in peripheral retina to 400/mm2 in central retina, accounting for about 1% of all amacrine cells in the rat retina. The vGluT3 cells had a two- to threefold dendritic overlap, and their cell bodies formed a regular mosaic, suggesting they represent a single type of amacrine cell. The vGluT3 amacrine cells expressed glycine and glycine transporter 1 (GlyT1) but not the vesicular glycine transporter (vesicular inhibitory amino acid transporter). They also expressed glutamate; hence, there is the possibility that, comparable to cholinergic amacrine cells, they are "dual transmitter" amacrine cells. The synaptic input of vGluT3 cells was studied by electron microscopy. They received input from bipolar cells at ribbon synapses and from other amacrine cells at conventional synapses. The types of bipolar cells possibly involved with vGluT3 cells were demonstrated by double labeling sections for vGluT3 and the calcium-binding protein CaB5. The axon terminals of type 3 and 5 bipolar cells costratified with vGluT3 dendrites, and it is possible that vGluT3 cells have ON and OFF light responses.