RESUMO
Point-scanning imaging systems are among the most widely used tools for high-resolution cellular and tissue imaging, benefiting from arbitrarily defined pixel sizes. The resolution, speed, sample preservation and signal-to-noise ratio (SNR) of point-scanning systems are difficult to optimize simultaneously. We show these limitations can be mitigated via the use of deep learning-based supersampling of undersampled images acquired on a point-scanning system, which we term point-scanning super-resolution (PSSR) imaging. We designed a 'crappifier' that computationally degrades high SNR, high-pixel resolution ground truth images to simulate low SNR, low-resolution counterparts for training PSSR models that can restore real-world undersampled images. For high spatiotemporal resolution fluorescence time-lapse data, we developed a 'multi-frame' PSSR approach that uses information in adjacent frames to improve model predictions. PSSR facilitates point-scanning image acquisition with otherwise unattainable resolution, speed and sensitivity. All the training data, models and code for PSSR are publicly available at 3DEM.org.
Assuntos
Aprendizado Profundo , Algoritmos , Microscopia Eletrônica/métodos , Razão Sinal-RuídoRESUMO
An approach combining signal detection theory and precise 3D reconstructions from serial section electron microscopy (3DEM) was used to investigate synaptic plasticity and information storage capacity at medial perforant path synapses in adult hippocampal dentate gyrus in vivo. Induction of long-term potentiation (LTP) markedly increased the frequencies of both small and large spines measured 30 minutes later. This bidirectional expansion resulted in heterosynaptic counterbalancing of total synaptic area per unit length of granule cell dendrite. Control hemispheres exhibited 6.5 distinct spine sizes for 2.7 bits of storage capacity while LTP resulted in 12.9 distinct spine sizes (3.7 bits). In contrast, control hippocampal CA1 synapses exhibited 4.7 bits with much greater synaptic precision than either control or potentiated dentate gyrus synapses. Thus, synaptic plasticity altered total capacity, yet hippocampal subregions differed dramatically in their synaptic information storage capacity, reflecting their diverse functions and activation histories.
Assuntos
Giro Denteado/fisiologia , Potenciação de Longa Duração , Sinapses/fisiologia , Animais , Masculino , Plasticidade Neuronal , Via Perfurante/fisiologia , Ratos , Ratos Long-EvansRESUMO
Long-term potentiation (LTP) has become a standard model for investigating synaptic mechanisms of learning and memory. Increasingly, it is of interest to understand how LTP affects the synaptic information storage capacity of the targeted population of synapses. Here, structural synaptic plasticity during LTP was explored using three-dimensional reconstruction from serial section electron microscopy. Storage capacity was assessed by applying a new analytical approach, Shannon information theory, to delineate the number of functionally distinguishable synaptic strengths. LTP was induced by delta-burst stimulation of perforant pathway inputs to the middle molecular layer of hippocampal dentate granule cells in adult rats. Spine head volumes were measured as predictors of synaptic strength and compared between LTP and control hemispheres at 30 min and 2 hr after the induction of LTP. Synapses from the same axon onto the same dendrite were used to determine the precision of synaptic plasticity based on the similarity of their physical dimensions. Shannon entropy was measured by exploiting the frequency of spine heads in functionally distinguishable sizes to assess the degree to which LTP altered the number of bits of information storage. Outcomes from these analyses reveal that LTP expanded storage capacity; the distribution of spine head volumes was increased from 2 bits in controls to 3 bits at 30 min and 2.7 bits at 2 hr after the induction of LTP. Furthermore, the distribution of spine head volumes was more uniform across the increased number of functionally distinguishable sizes following LTP, thus achieving more efficient use of coding space across the population of synapses.
RESUMO
Microtubules deliver essential resources to and from synapses. Three-dimensional reconstructions in rat hippocampus reveal a sampling bias regarding spine density that needs to be controlled for dendrite caliber and resource delivery based on microtubule number. The strength of this relationship varies across dendritic arbors, as illustrated for area CA1 and dentate gyrus. In both regions, proximal dendrites had more microtubules than distal dendrites. For CA1 pyramidal cells, spine density was greater on thicker than thinner dendrites in stratum radiatum, or on the more uniformly thin terminal dendrites in stratum lacunosum moleculare. In contrast, spine density was constant across the cone shaped arbor of tapering dendrites from dentate granule cells. These differences suggest that thicker dendrites supply microtubules to subsequent dendritic branches and local dendritic spines, whereas microtubules in thinner dendrites need only provide resources to local spines. Most microtubules ran parallel to dendrite length and associated with long, presumably stable mitochondria, which occasionally branched into lateral dendritic branches. Short, presumably mobile, mitochondria were tethered to microtubules that bent and appeared to direct them into a thin lateral branch. Prior work showed that dendritic segments with the same number of microtubules had elevated resources in subregions of their dendritic shafts where spine synapses had enlarged, and spine clusters had formed. Thus, additional microtubules were not required for redistribution of resources locally to growing spines or synapses. These results provide new understanding about the potential for microtubules to regulate resource delivery to and from dendritic branches and locally among dendritic spines.
Assuntos
Dendritos , Espinhas Dendríticas , Animais , Dendritos/fisiologia , Hipocampo , Microtúbulos , Células Piramidais/fisiologia , Ratos , Sinapses/fisiologiaRESUMO
The critical brain areas and molecular mechanisms involved in drug abuse and dependence have been extensively studied. Drug-induced persistent behaviors such as sensitization, tolerance, or relapse, however, far outlast any previously reported mechanisms. A challenge in the field of addiction, therefore, has been to identify drug-induced changes in brain circuitry that may subserve long-lasting changes in behavior. This study examined behavioral changes and electron microscopic evidence of altered synaptic connectivity within the nucleus accumbens (NAc) following repeated administration of cocaine or morphine. The unbiased quantitative stereological physical disector method was used to estimate the number of synapses per neuron. Increases in the synapse-to-neuron ratio were found in the NAc shell of cocaine-treated (49.1%) and morphine-treated (55.1%) rats and in the NAc core of cocaine-treated animals (49.1%). This study provides direct ultrastructural evidence of drug-induced synaptic plasticity and identifies synaptic remodeling as a potential neural substrate underlying drug-induced behavioral sensitization.
Assuntos
Cocaína/farmacologia , Morfina/farmacologia , Plasticidade Neuronal/efeitos dos fármacos , Núcleo Accumbens/efeitos dos fármacos , Sinapses/efeitos dos fármacos , Animais , Feminino , Plasticidade Neuronal/fisiologia , Núcleo Accumbens/fisiologia , Núcleo Accumbens/ultraestrutura , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Sinapses/fisiologia , Sinapses/ultraestruturaRESUMO
A suite of epidermal characters in Arabidopsis is under the transcriptional control of a combinatorial complex containing WD repeat, bHLH and MYB proteins. Many genetic, molecular and biochemical means have been employed to identify and characterize a complete minimal set of complex members required for the trichome initiation, root hair spacing, anthocyanin production and seed coat tannin production pathways. In addition, the WD and bHLH proteins required for outer seed coat differentiation have been identified. However, until now the MYB complex member(s) required for this last WD-bHLH-MYB complex-dependent character have remained elusive. Here we identify two MYBs, AtMYB5 and TT2, as partially redundant in regulating this outer seed coat developmental process with MYB5 having the major role. MYB5 and TT2 are shown to be expressed in this outer seed coat domain. We also show that MYB5 has weak pleiotropic control over trichome development and tannin production and is also expressed in the appropriate places for these functions. TT8 and the downstream GL2 and TTG2 regulators of seed coat development are found to be downregulated in the MYB mutants. Although the TTG1-dependent R2R3 MYBs are considered to be highly pathway specific, identification of MYBs responsible for outer seed coat development allowed for the elucidation of previously undetected novel developmental pleiotropy among these elements.
Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/fisiologia , Proteínas de Ligação a DNA/fisiologia , Sementes/fisiologia , Arabidopsis/citologia , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Diferenciação Celular , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica de Plantas , Mutação , Epiderme Vegetal/citologia , Epiderme Vegetal/crescimento & desenvolvimento , Epiderme Vegetal/fisiologia , Raízes de Plantas/citologia , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/fisiologia , Plantas Geneticamente ModificadasRESUMO
About 1000 hypothalamic neurons synthesize and release gonadotropin-releasing hormone (GnRH), the master molecule of reproduction in all mammals. At the level of the median eminence at the base of the brain, where GnRH and other hypothalamic releasing hormones are secreted into the capillary system leading to the anterior pituitary gland, there is non-synaptic regulation of neurohormone release by a number of central neurotransmitters. For example, glutamate, the major excitatory amino acid in the brain, directly regulates GnRH release from nerve terminals via NMDA receptors (NMDARs). Moreover, the effects of glutamate action on GnRH secretion are potentiated by estrogens, and this relates to the physiologic control of ovulation by the hypothalamus. We sought to determine the ultrastructural relationship between GnRH neuroterminals and NMDARs, and this regulation by estradiol. Using immunofluorescent confocal microscopy, postembedding immunogold electron microscopy, fractionation, and Western blotting, we demonstrated: (i) GnRH is localized in large dense-core vesicles of neurosecretory profiles/terminals, (ii) the NMDAR1 subunit is found primarily on large dense-core vesicles of neurosecretory profiles/terminals, (iii) there is extensive colocalization of GnRH and NMDAR1 on the same vesicles, and (iv) estradiol modestly but significantly alters the distribution of NMDAR1 in GnRH neuroterminals by increasing expression of NMDAR1 on large dense-core vesicles. Western blots of fractionated median eminence support the presence of NMDAR1 in subcellular fractions containing large dense-core vesicles. These data are the first to show the presence of the NMDAR on neuroendocrine secretory vesicles, its co-expression with GnRH, and its regulation by estradiol. The results provide a novel anatomical site for the NMDAR and may represent a new mechanism for the regulation of GnRH release.
Assuntos
Hormônio Liberador de Gonadotropina/metabolismo , Eminência Mediana/metabolismo , Terminações Nervosas/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Western Blotting , Estradiol/farmacologia , Feminino , Eminência Mediana/efeitos dos fármacos , Eminência Mediana/ultraestrutura , Microscopia Confocal , Microscopia Imunoeletrônica , Terminações Nervosas/efeitos dos fármacos , Terminações Nervosas/ultraestrutura , Ratos , Ratos Sprague-DawleyRESUMO
Previous studies identified various mechanisms of light scattering reduction in tissue induced by chemical agents. Our results suggest that dehydration is an important mechanism of optical clearing in collagenous and cellular tissue. Photographic and optical coherence tomography images indicate that air-immersed skin and tendon specimens become similarly transparent to glycerol-immersed specimens. Transmission electron microscopy images reveal that dehydration causes individual scattering particles such as collagen fibrils and organelles to become more densely packed, but does not significantly alter size. A heuristic particle-interaction model predicts that the scattering particle volume fraction increase can contribute substantially to optical clearing in collagenous and cellular tissue.
Assuntos
Desidratação/patologia , Desidratação/fisiopatologia , Pele/patologia , Pele/fisiopatologia , Tendões/patologia , Tendões/fisiopatologia , Tomografia de Coerência Óptica/métodos , Animais , Água Corporal/metabolismo , Simulação por Computador , Técnicas In Vitro , Modelos Biológicos , Óptica e Fotônica , Ratos , Refratometria/métodos , Espalhamento de RadiaçãoRESUMO
The pulsatile release of GnRH is crucial for normal reproductive physiology across the life cycle, a process that is regulated by hypothalamic neurotransmitters. GnRH terminals co-express the vesicular glutamate transporter 2 (vGluT2) as a marker of a glutamatergic phenotype. The current study sought to elucidate the relationship between glutamate and GnRH nerve terminals in the median eminence--the site of GnRH release into the portal capillary vasculature. We also determined whether this co-expression may change during reproductive senescence, and if steroid hormones, which affect responsiveness of GnRH neurons to glutamate, may alter the co-expression pattern. Female Sprague-Dawley rats were ovariectomized at young adult, middle-aged and old ages (~4, 11, and 22 months, respectively) and treated four weeks later with sequential vehicle + vehicle (VEH + VEH), estradiol + vehicle (E2 + VEH), or estradiol + progesterone (E2+P4). Rats were perfused 24 hours after the second hormone treatment. Confocal microscopy was used to determine colocalization of GnRH and vGluT2 immunofluorescence in the median eminence. Post-embedding immunogold labeling of GnRH and vGluT2, and a serial electron microscopy (EM) technique were used to determine the cellular interaction between GnRH terminals and glutamate signaling. Confocal analysis showed that GnRH and vGluT2 immunofluorescent puncta were extensively colocalized in the median eminence and that their density declined with age but was unaffected by short-term hormone treatment. EM results showed that vGluT2 immunoreactivity was extensively associated with large dense-core vesicles, suggesting a unique glutamatergic signaling pathway in GnRH terminals. Our results provide novel subcellular information about the intimate relationship between GnRH terminals and glutamate in the median eminence.
Assuntos
Hormônio Liberador de Gonadotropina/metabolismo , Terminações Pré-Sinápticas/metabolismo , Vesículas Secretórias/metabolismo , Proteína Vesicular 2 de Transporte de Glutamato/metabolismo , Fatores Etários , Animais , Feminino , Expressão Gênica , Hormônio Liberador de Gonadotropina/sangue , Eminência Mediana/metabolismo , Terminações Pré-Sinápticas/ultraestrutura , Transporte Proteico , Ratos , Proteína Vesicular 2 de Transporte de Glutamato/genéticaRESUMO
Striatal cholinergic interneurons located in the dorsal striatum and nucleus accumbens are amenable to influences of the dopaminergic mesolimbic pathway, which is a pathway involved in reward and reinforcement and targeted by several drugs of abuse. Dopamine and acetylcholine neurotransmission and their interactions are essential to striatal function, and disruptions to these systems lead to a variety of clinical disorders. Dopamine regulates acetylcholine release through dopamine receptors that are localized directly on striatal cholinergic interneurons. The dopamine D2 receptor, which attenuates acetylcholine release, has been implicated in drug relapse and is targeted by therapeutic drugs that are used to treat a variety of neurological disorders including Tourette Syndrome, Parkinson's disease and schizophrenia. The present study provides the first direct evidence for the localization of dopamine D2 receptors on striatal cholinergic interneurons of the rat brain using dual labeling immunocytochemistry procedures. Using light microscopy, dopamine D2 receptors were localized on the cell somata and dendritic and axonal processes of striatal cholinergic interneurons in the dorsal striatum and nucleus accumbens of the rat brain. These findings provide a foundation for understanding the specific roles that cholinergic neuronal network systems and interacting dopaminergic signaling pathways play in striatal function and in a variety of clinical disorders including drug abuse and addiction.
Assuntos
Acetilcolina/metabolismo , Dopamina/metabolismo , Interneurônios/metabolismo , Neostriado/metabolismo , Núcleo Accumbens/metabolismo , Receptores de Dopamina D2/metabolismo , Animais , Colina O-Acetiltransferase/metabolismo , Fibras Colinérgicas/metabolismo , Fibras Colinérgicas/ultraestrutura , Imuno-Histoquímica , Interneurônios/citologia , Masculino , Neostriado/citologia , Vias Neurais/citologia , Vias Neurais/metabolismo , Núcleo Accumbens/citologia , Terminações Pré-Sinápticas/metabolismo , Terminações Pré-Sinápticas/ultraestrutura , Ratos , Ratos Sprague-Dawley , Recompensa , Transmissão Sináptica/fisiologiaRESUMO
Nascent zones and active zones are adjacent synaptic regions that share a postsynaptic density, but nascent zones lack the presynaptic vesicles found at active zones. Here dendritic spine synapses were reconstructed through serial section electron microscopy (3DEM) and EM tomography to investigate nascent zone dynamics during long-term potentiation (LTP) in mature rat hippocampus. LTP was induced with theta-burst stimulation, and comparisons were made with control stimulation in the same hippocampal slices at 5 minutes, 30 minutes, and 2 hours post-induction and to perfusion-fixed hippocampus in vivo. Nascent zones were present at the edges of â¼35% of synapses in perfusion-fixed hippocampus and as many as â¼50% of synapses in some hippocampal slice conditions. By 5 minutes, small dense-core vesicles known to transport active zone proteins moved into more presynaptic boutons. By 30 minutes, nascent zone area decreased, without significant change in synapse area, suggesting that presynaptic vesicles were recruited to preexisting nascent zones. By 2 hours, both nascent and active zones were enlarged. Immunogold labeling revealed glutamate receptors in nascent zones; however, average distances from nascent zones to docked presynaptic vesicles ranged from 170 ± 5 nm in perfusion-fixed hippocampus to 251 ± 4 nm at enlarged synapses by 2 hours during LTP. Prior stochastic modeling suggests that decrease in glutamate concentration reduces the probability of glutamate receptor activation from 0.4 at the center of release to 0.1 just 200 nm away. Thus, conversion of nascent zones to functional active zones likely requires the recruitment of presynaptic vesicles during LTP.
Assuntos
Hipocampo/citologia , Hipocampo/fisiologia , Potenciação de Longa Duração/fisiologia , Sinapses/fisiologia , Análise de Variância , Animais , Animais Recém-Nascidos , Biofísica , Dendritos/metabolismo , Dendritos/ultraestrutura , Estimulação Elétrica , Imageamento Tridimensional , Técnicas In Vitro , Masculino , Microscopia Eletrônica de Transmissão , Terminações Pré-Sinápticas/metabolismo , Terminações Pré-Sinápticas/ultraestrutura , Ratos , Receptores de AMPA/metabolismo , Receptores de AMPA/ultraestrutura , Vesículas Secretórias/ultraestrutura , Sinapses/ultraestrutura , Fatores de TempoRESUMO
With recent improvements in instrumentation and computational tools, serial section electron microscopy has become increasingly straightforward. A new method for imaging ultrathin serial sections is developed based on a field emission scanning electron microscope fitted with a transmitted electron detector. This method is capable of automatically acquiring high-resolution serial images with a large field size and very little optical and physical distortions. In this chapter, we describe the procedures leading to the generation and analyses of a large-volume stack of high-resolution images (64 µm × 64 µm × 10 µm, or larger, at 2 nm pixel size), including how to obtain large-area serial sections of uniform thickness from well-preserved brain tissue that is rapidly perfusion-fixed with mixed aldehydes, processed with a microwave-enhanced method, and embedded into epoxy resin.
Assuntos
Encéfalo/anatomia & histologia , Encéfalo/ultraestrutura , Processamento de Imagem Assistida por Computador/métodos , Microscopia Eletrônica de Transmissão e Varredura/métodos , Microscopia Eletrônica de Varredura/métodos , Animais , Microtomia , Tamanho do Órgão , Perfusão , Ratos , Coloração e Rotulagem , Fixação de TecidosRESUMO
Transmission-mode scanning electron microscopy (tSEM) on a field emission SEM platform was developed for efficient and cost-effective imaging of circuit-scale volumes from brain at nanoscale resolution. Image area was maximized while optimizing the resolution and dynamic range necessary for discriminating key subcellular structures, such as small axonal, dendritic and glial processes, synapses, smooth endoplasmic reticulum, vesicles, microtubules, polyribosomes, and endosomes which are critical for neuronal function. Individual image fields from the tSEM system were up to 4,295 µm(2) (65.54 µm per side) at 2 nm pixel size, contrasting with image fields from a modern transmission electron microscope (TEM) system, which were only 66.59 µm(2) (8.160 µm per side) at the same pixel size. The tSEM produced outstanding images and had reduced distortion and drift relative to TEM. Automated stage and scan control in tSEM easily provided unattended serial section imaging and montaging. Lens and scan properties on both TEM and SEM platforms revealed no significant nonlinear distortions within a central field of â¼100 µm(2) and produced near-perfect image registration across serial sections using the computational elastic alignment tool in Fiji/TrakEM2 software, and reliable geometric measurements from RECONSTRUCT™ or Fiji/TrakEM2 software. Axial resolution limits the analysis of small structures contained within a section (â¼45 nm). Since this new tSEM is non-destructive, objects within a section can be explored at finer axial resolution in TEM tomography with current methods. Future development of tSEM tomography promises thinner axial resolution producing nearly isotropic voxels and should provide within-section analyses of structures without changing platforms. Brain was the test system given our interest in synaptic connectivity and plasticity; however, the new tSEM system is readily applicable to other biological systems.
Assuntos
Microscopia Eletrônica de Varredura/métodos , Nanotecnologia/métodos , Animais , Artefatos , Automação , Encéfalo/ultraestrutura , Análise Custo-Benefício , Elasticidade , Processamento de Imagem Assistida por Computador , Lentes , Microscopia Eletrônica de Varredura/economia , Microscopia Eletrônica de Varredura/instrumentação , Nanotecnologia/economia , Nanotecnologia/instrumentação , RatosRESUMO
Complete reconstructions of vertebrate neuronal circuits on the synaptic level require new approaches. Here, serial section transmission electron microscopy was automated to densely reconstruct four volumes, totaling 670 µm(3), from the rat hippocampus as proving grounds to determine when axo-dendritic proximities predict synapses. First, in contrast with Peters' rule, the density of axons within reach of dendritic spines did not predict synaptic density along dendrites because the fraction of axons making synapses was variable. Second, an axo-dendritic touch did not predict a synapse; nevertheless, the density of synapses along a hippocampal dendrite appeared to be a universal fraction, 0.2, of the density of touches. Finally, the largest touch between an axonal bouton and spine indicated the site of actual synapses with about 80% precision but would miss about half of all synapses. Thus, it will be difficult to predict synaptic connectivity using data sets missing ultrastructural details that distinguish between axo-dendritic touches and bona fide synapses.
Assuntos
Hipocampo/ultraestrutura , Neurópilo/ultraestrutura , Animais , Animais Recém-Nascidos , Axônios/ultraestrutura , Dendritos/ultraestrutura , Processamento Eletrônico de Dados/métodos , Técnicas In Vitro , Masculino , Microscopia Eletrônica de Transmissão/métodos , Ratos , Ratos Long-Evans , Sinapses/ultraestruturaRESUMO
The decapeptide gonadotropin-releasing hormone (GnRH), which regulates reproduction in all vertebrates, is stored in, and secreted from, large dense-core secretory vesicles in nerve terminals in the median eminence. GnRH is released from these terminals with biological rhythms that are critical for the maintenance of normal reproduction. During reproductive aging in female rats, there is a loss of GnRH pulses and a diminution of the GnRH surge. However, information about the specific role of GnRH nerve terminals is lacking, particularly in the context of aging. We sought to gain novel ultrastructural information about GnRH neuroterminals by performing three-dimensional (3D) reconstructions of GnRH neuroterminals and their surrounding microenvironment in the median eminence of young (4-5 months) and old (22-24 months) ovariectomized Sprague-Dawley female rats. Median eminence tissues were freeze-plunge embedded and serial ultrathin sections were collected on slot grids for immunogold labeling of GnRH immunoreactivity. Sequential images were used to create 3D models of GnRH terminals. These reconstructions provided novel perspectives into the morphological properties of GnRH terminals and their neural and glial environment. We also noted that the cytoarchitectural features of the median eminence became disorganized with aging. Quantitative measures showed a significant decrease in the apposition between GnRH terminal membranes and glial cells. Our data suggest reproductive aging in rats is characterized by structural organizational changes to the GnRH terminal microenvironment in the median eminence.
Assuntos
Envelhecimento/patologia , Hormônio Liberador de Gonadotropina/metabolismo , Eminência Mediana/metabolismo , Eminência Mediana/patologia , Neurônios/metabolismo , Neurônios/patologia , Animais , Feminino , Imageamento Tridimensional , Imuno-Histoquímica , Hormônio Luteinizante/sangue , Eminência Mediana/ultraestrutura , Microscopia Eletrônica , Modelos Neurológicos , Neuroglia/patologia , Neuroglia/ultraestrutura , Neurônios/ultraestrutura , Ovariectomia , Fotomicrografia , Ratos , Ratos Sprague-Dawley , Vesículas Secretórias/metabolismo , Vesículas Secretórias/patologia , Vesículas Secretórias/ultraestruturaRESUMO
The formation of synaptic connections with target cells and maintenance of axons are highly regulated and crucial for neuronal function. The atypical cadherin and G-protein-coupled receptor Flamingo and its orthologs in amphibians and mammals have been shown to regulate cell polarity, dendritic and axonal growth, and neural tube closure. However, the role of Flamingo in synapse formation and function and in axonal health remains poorly understood. Here we show that fmi mutations cause a significant increase in the number of ectopic synapses on muscles and result in the formation of novel en passant synapses along axons, and unique presynaptic varicosities, including active zones, within axons. The fmi mutations also cause defective synaptic responses in a small subset of muscles, an age-dependent loss of muscle innervation and a drastic degeneration of axons in 3rd instar larvae without an apparent loss of neurons. Neuronal expression of Flamingo rescues all of these synaptic and axonal defects and larval lethality. Based on these observations, we propose that Flamingo is required in neurons for synaptic target selection, synaptogenesis, the survival of axons and synapses, and adult viability. These findings shed new light on a possible role for Flamingo in progressive neurodegenerative diseases.
Assuntos
Axônios/metabolismo , Caderinas/metabolismo , Proteínas de Drosophila/metabolismo , Degeneração Neural/metabolismo , Junção Neuromuscular/metabolismo , Sinapses/metabolismo , Animais , Axônios/ultraestrutura , Caderinas/genética , Sistema Nervoso Central/embriologia , Sistema Nervoso Central/crescimento & desenvolvimento , Sistema Nervoso Central/metabolismo , Drosophila , Proteínas de Drosophila/genética , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Mutação , Junção Neuromuscular/embriologia , Junção Neuromuscular/crescimento & desenvolvimento , Organogênese/fisiologia , Técnicas de Patch-Clamp , Sinapses/ultraestruturaRESUMO
Attine ants engage in a quadripartite symbiosis with fungi they cultivate for food, specialized garden parasites, and parasite-inhibiting bacteria. Molecular phylogenetic evidence supports an ancient host-pathogen association between the ant-cultivar mutualism and the garden parasite. Here we show that ants rear the antibiotic-producing bacteria in elaborate cuticular crypts, supported by unique exocrine glands, and that these structures have been highly modified across the ants' evolutionary history. This specialized structural evolution, together with the absence of these bacteria and modifications in other ant genera that do not grow fungus, indicate that the bacteria have an ancient and coevolved association with the ants, their fungal cultivar, and the garden parasite.