Literature context: moFisher Cat#A-6455; RRID:AB_221570 Goat anti-Chicken IgY (H+L), Al
Signal gradients encode instructive information for numerous decision-making processes during embryonic development. A striking example of precise, scalable tissue-level patterning is the segmentation of somites-the precursors of the vertebral column-during which the fibroblast growth factor (FGF), Wnt, and retinoic acid (RA) pathways establish spatial gradients. Despite decades of studies proposing roles for all three pathways, the dynamic feature of these gradients that encodes instructive information determining segment sizes remained elusive. We developed a non-elongating tail explant system, integrated quantitative measurements with computational modeling, and tested alternative models to show that positional information is encoded solely by spatial fold change (SFC) in FGF signal output. Neighboring cells measure SFC to accurately position the determination front and thus determine segment size. The SFC model successfully recapitulates results of spatiotemporal perturbation experiments on both explants and intact embryos, and it shows that Wnt signaling acts permissively upstream of FGF signaling and that RA gradient is dispensable.
The diversity of cell types and regulatory states in the brain, and how these change during aging, remains largely unknown. We present a single-cell transcriptome atlas of the entire adult Drosophila melanogaster brain sampled across its lifespan. Cell clustering identified 87 initial cell clusters that are further subclustered and validated by targeted cell-sorting. Our data show high granularity and identify a wide range of cell types. Gene network analyses using SCENIC revealed regulatory heterogeneity linked to energy consumption. During aging, RNA content declines exponentially without affecting neuronal identity in old brains. This single-cell brain atlas covers nearly all cells in the normal brain and provides the tools to study cellular diversity alongside other Drosophila and mammalian single-cell datasets in our unique single-cell analysis platform: SCope (http://scope.aertslab.org). These results, together with SCope, allow comprehensive exploration of all transcriptional states of an entire aging brain.
Literature context: n,o); Eh>myrGFP (CLSM: 5a-d,10; RRID:EM:6); Pdf/CCAP>nsyb::GFP (CLSM: 2,
The peptidergic Pigment-dispersing factor (PDF)-Tri neurons are a group of non-clock neurons that appear transiently around the time of adult ecdysis (=eclosion) in the fruit fly Drosophila melanogaster. This specific developmental pattern points to a function of these neurons in eclosion or other processes that are active around pupal-adult transition. As a first step to understand the role of these neurons, we here characterize the anatomy of the PDF-Tri neurons. In addition, we describe a further set of peptidergic neurons that have been associated with eclosion behavior, eclosion hormone (EH), and crustacean cardioactive peptide (CCAP) neurons, to single cell level in the pharate adult brain. PDF-Tri neurons as well as CCAP neurons co-express a classical transmitter indicated by the occurrence of small clear vesicles in addition to dense-core vesicles containing the peptides. In the tritocerebrum, gnathal ganglion and the superior protocerebrum PDF-Tri neurites contain peptidergic varicosities and both pre- and postsynaptic sites, suggesting that the PDF-Tri neurons represent modulatory rather than pure interneurons that connect the subesophageal zone with the superior protocerebrum. The extensive overlap of PDF-Tri arborizations with neurites of CCAP- and EH-expressing neurons in distinct brain regions provides anatomical evidence for a possible function of the PDF-Tri neurons in eclosion behavior.
Literature context: ; RRID:AB_221570 Anti-phospho-Ser Santa Cruz sc-
Nutrient deprivation induces autophagy through inhibiting TORC1 activity. We describe a novel mechanism in Drosophila by which TORC1 regulates RNA processing of Atg transcripts and alters ATG protein levels and activities via the cleavage and polyadenylation (CPA) complex. We show that TORC1 signaling inhibits CDK8 and DOA kinases, which directly phosphorylate CPSF6, a component of the CPA complex. These phosphorylation events regulate CPSF6 localization, RNA binding, and starvation-induced alternative RNA processing of transcripts involved in autophagy, nutrient, and energy metabolism, thereby controlling autophagosome formation and metabolism. Similarly, we find that mammalian CDK8 and CLK2, a DOA ortholog, phosphorylate CPSF6 to regulate autophagy and metabolic changes upon starvation, revealing an evolutionarily conserved mechanism linking TORC1 signaling with RNA processing, autophagy, and metabolism.
Literature context: dy Life Technologies Cat#A6455; RRID:AB_221570 Goat anti-Rabbit IgG Alexa 488
Taming cell-to-cell variability in gene expression is critical for precise pattern formation during embryonic development. To investigate the source and buffering mechanism of expression variability, we studied a biological clock, the vertebrate segmentation clock, controlling the precise spatiotemporal patterning of the vertebral column. By counting single transcripts of segmentation clock genes in zebrafish, we show that clock genes have low RNA amplitudes and expression variability is primarily driven by gene extrinsic sources, which is suppressed by Notch signaling. We further show that expression noise surprisingly increases from the posterior progenitor zone to the anterior segmentation and differentiation zone. Our computational model reproduces the spatial noise profile by incorporating spatially increasing time delays in gene expression. Our results, suggesting that expression variability is controlled by the balance of time delays and cell signaling in a vertebrate tissue, will shed light on the accuracy of natural clocks in multi-cellular systems and inspire engineering of robust synthetic oscillators.
Literature context: Fisher Scientific Cat#: A-6455;RRID:AB_221570 Rabbit anti-NR1 CT Millipore Ca
Dendritic filopodia select synaptic partner axons by interviewing the cell surface of potential targets, but how filopodia decipher the complex pattern of adhesive and repulsive molecular cues to find appropriate contacts is unknown. Here, we demonstrate in cortical neurons that a single cue is sufficient for dendritic filopodia to reject or select specific axonal contacts for elaboration as synaptic sites. Super-resolution and live-cell imaging reveals that EphB2 is located in the tips of filopodia and at nascent synaptic sites. Surprisingly, a genetically encoded indicator of EphB kinase activity, unbiased classification, and a photoactivatable EphB2 reveal that simple differences in the kinetics of EphB kinase signaling at the tips of filopodia mediate the choice between retraction and synaptogenesis. This may enable individual filopodia to choose targets based on differences in the activation rate of a single tyrosine kinase, greatly simplifying the process of partner selection and suggesting a general principle.
Literature context: Technologies, Grand Island, NY, RRID:AB_221570), followed by a biotinylated se
The corticogeniculate (CG) pathway links the visual cortex with the lateral geniculate nucleus (LGN) of the thalamus and is the first feedback connection in the mammalian visual system. Whether functional connections between CG neurons and LGN relay neurons obey or ignore the separation of feedforward visual signals into parallel processing streams is not known. Accordingly, there is some debate about whether CG neurons are morphologically heterogeneous or homogenous. Here we characterized the morphology of CG neurons in the ferret, a visual carnivore with distinct feedforward parallel processing streams, and compared the morphology of ferret CG neurons with CG neuronal morphology previously described in macaque monkeys [Briggs et al. (2016) Neuron, 90, 388]. We used a G-deleted rabies virus as a retrograde tracer to label CG neurons in adult ferrets. We then reconstructed complete dendritic morphologies for a large sample of virus-labeled CG neurons. Quantification of CG morphology revealed three distinct CG neuronal subtypes with striking similarities to the CG neuronal subtypes observed in macaques. These findings suggest that CG neurons may be morphologically diverse in a variety of highly visual mammals in which feedforward visual pathways are organized into parallel processing streams. Accordingly, these results provide support for the notion that CG feedback is functionally parallel stream-specific in ferrets and macaques.
Literature context: c, A6455, RRID:AB_221570), anti-RFP
Parkinsonian Perry syndrome, involving mutations in the dynein motor component dynactin or p150Glued, is characterized by TDP-43 pathology in affected brain regions, including the substantia nigra. However, the molecular relationship between p150Glued and TDP-43 is largely unknown. Here, we report that a reduction in TDP-43 protein levels alleviates the synaptic defects of neurons expressing the Perry mutant p150G50R in Drosophila. Dopaminergic expression of p150G50R, which decreases dopamine release, disrupts motor ability and reduces the lifespan of Drosophila. p150G50R expression also causes aggregation of dense core vesicles (DCVs), which contain monoamines and neuropeptides, and disrupts the axonal flow of DCVs, thus decreasing synaptic strength. The above phenotypes associated with Perry syndrome are improved by the removal of a copy of Drosophila TDP-43 TBPH, thus suggesting that the stagnation of axonal transport by dynactin mutations promotes TDP-43 aggregation and interferes with the dynamics of DCVs and synaptic activities.
Literature context: lecular probes, RRID:AB_221570), mouse anti-RFP (1:200, MBL, R
Programmed cell death is a conserved strategy for neural development both in vertebrates and invertebrates and is recognized at various developmental stages in the brain from neurogenesis to adulthood. To understand the development of the central nervous system, it is essential to reveal not only molecular mechanisms but also the role of neural cell death (Pinto-Teixeira et al., 2016). To understand the role of cell death in neural development, we investigated the effect of inhibition of cell death on optic lobe development. Our data demonstrate that, in the optic lobe of Drosophila, cell death occurs in neural precursor cells and neurons before neurite formation and functions to prevent various developmental abnormalities. When neuronal cell death was inhibited by an effector caspase inhibitor, p35, multiple abnormal neuropil structures arose during optic lobe development-e.g., enlarged or fused neuropils, misrouted neurons and abnormal neurite lumps. Inhibition of cell death also induced morphogenetic defects in the lamina and medulla development-e.g., failures in the separation of the lamina and medulla cortices and the medulla rotation. These defects were reproduced in the mutant of an initiator caspase, dronc. If cell death was a mechanism for removing the abnormal neuropil structures, we would also expect to observe them in mutants defective for corpse clearance. However, they were not observed in these mutants. When dead cell-membranes were visualized with Apoliner, they were observed only in cortices and not in neuropils. These results suggest that the cell death occurs before mature neurite formation. Moreover, we found that inhibition of cell death induced ectopic neuroepithelial cells, neuroblasts and ganglion mother cells in late pupal stages, at sites where the outer and inner proliferation centers were located at earlier developmental stages. Caspase-3 activation was observed in the neuroepithelial cells and neuroblasts in the proliferation centers. These results indicate that cell death is required for elimination of the precursor cells composing the proliferation centers. This study substantiates an essential role of early neural cell death for ensuring normal development of the central nervous system.
BACKGROUND: Fear conditioning is a form of learning essential for animal survival and used as a behavioral paradigm to study the mechanisms of learning and memory. In mammals, the amygdala plays a crucial role in fear conditioning. In teleost, the medial zone of the dorsal telencephalon (Dm) has been postulated to be a homolog of the mammalian amygdala by anatomical and ablation studies, showing a role in conditioned avoidance response. However, the neuronal populations required for a conditioned avoidance response via the Dm have not been functionally or genetically defined. RESULTS: We aimed to identify the neuronal population essential for fear conditioning through a genetic approach in zebrafish. First, we performed large-scale gene trap and enhancer trap screens, and created transgenic fish lines that expressed Gal4FF, an engineered version of the Gal4 transcription activator, in specific regions in the brain. We then crossed these Gal4FF-expressing fish with the effector line carrying the botulinum neurotoxin gene downstream of the Gal4 binding sequence UAS, and analyzed the double transgenic fish for active avoidance fear conditioning. We identified 16 transgenic lines with Gal4FF expression in various brain areas showing reduced performance in avoidance responses. Two of them had Gal4 expression in populations of neurons located in subregions of the Dm, which we named 120A-Dm neurons. Inhibition of the 120A-Dm neurons also caused reduced performance in Pavlovian fear conditioning. The 120A-Dm neurons were mostly glutamatergic and had projections to other brain regions, including the hypothalamus and ventral telencephalon. CONCLUSIONS: Herein, we identified a subpopulation of neurons in the zebrafish Dm essential for fear conditioning. We propose that these are functional equivalents of neurons in the mammalian pallial amygdala, mediating the conditioned stimulus-unconditioned stimulus association. Thus, the study establishes a basis for understanding the evolutionary conservation and diversification of functional neural circuits mediating fear conditioning in vertebrates.
Literature context: 5; Research Resource Identifier RRID:AB_221570], guinea pig anti-VAChT (1:500;
In many species, neurons are unevenly distributed across the retina, leading to nonuniform analysis of specific visual features at certain locations in visual space. In recent years, the mouse has emerged as a premiere model for probing visual system function, development and disease. Thus, achieving a detailed understanding of mouse visual circuit architecture is of paramount importance. The general belief is that mice possess a relatively even topographic distribution of retinal ganglion cells (RGCs)- the output neurons of the eye. However, mouse RGCs include ∼30 subtypes; each responds best to a specific feature in the visual scene and conveys that information to central targets. Given the crucial role of RGCs and the prominence of the mouse as a model, we asked how different RGC subtypes are distributed across the retina. We targeted and filled individual fluorescently tagged RGC subtypes from across the retinal surface and evaluated the dendritic arbor extent and soma size of each cell according to its specific retinotopic position. Three prominent RGC subtypes: On-Off direction selective RGCs, object-motion-sensitive RGCs, and a specialized subclass of non-image-forming RGCs each had marked topographic variations in their dendritic arbor sizes. Moreover, the pattern of variation was distinct for each RGC subtype. Thus, there is increasing evidence that the mouse retina encodes visual space in a region-specific manner. As a consequence, some visual features are sampled far more densely at certain retinal locations than others. These findings have implications for central visual processing, perception and behavior in this prominent model species. This article is protected by copyright. All rights reserved.
Literature context: RRID:AB_221570 GFP mouse monoclonal Roche RRID
MicroRNA-mediated gene silencing is a fundamental mechanism in the regulation of gene expression. It remains unclear how the efficiency of RNA silencing could be influenced by RNA-binding proteins associated with the microRNA-induced silencing complex (miRISC). Here we report that fused in sarcoma (FUS), an RNA-binding protein linked to neurodegenerative diseases including amyotrophic lateral sclerosis (ALS), interacts with the core miRISC component AGO2 and is required for optimal microRNA-mediated gene silencing. FUS promotes gene silencing by binding to microRNA and mRNA targets, as illustrated by its action on miR-200c and its target ZEB1. A truncated mutant form of FUS that leads its carriers to an aggressive form of ALS, R495X, impairs microRNA-mediated gene silencing. The C. elegans homolog fust-1 also shares a conserved role in regulating the microRNA pathway. Collectively, our results suggest a role for FUS in regulating the activity of microRNA-mediated silencing.
Literature context: log #A6455, Invitrogen (RRID:AB_221570); rabbit anti-vasopressin, 1:10
Corticotropin-releasing factor (CRF) neurons in the hypothalamic paraventricular nucleus (PVN) initiate hypothalamic-pituitary-adrenal axis activity through the release of CRF into the portal system as part of a coordinated neuroendocrine, autonomic, and behavioral response to stress. The recent discovery of neurons expressing CRF receptor type 1 (CRFR1), the primary receptor for CRF, adjacent to CRF neurons within the PVN, suggests that CRF also signals within the hypothalamus to coordinate aspects of the stress response. Here, we characterize the electrophysiological and molecular properties of PVN-CRFR1 neurons and interrogate their monosynaptic connectivity using rabies virus-based tracing and optogenetic circuit mapping in male and female mice. We provide evidence that CRF neurons in the PVN form synapses on neighboring CRFR1 neurons and activate them by releasing CRF. CRFR1 neurons receive the majority of monosynaptic input from within the hypothalamus, mainly from the PVN itself. Locally, CRFR1 neurons make GABAergic synapses on parvocellular and magnocellular cells within the PVN. CRFR1 neurons resident in the PVN also make long-range glutamatergic synapses in autonomic nuclei such as the nucleus of the solitary tract. Selective ablation of PVN-CRFR1 neurons in male mice elevates corticosterone release during a stress response and slows the decrease in circulating corticosterone levels after the cessation of stress. Our experiments provide evidence for a novel intra-PVN neural circuit that is activated by local CRF release and coordinates autonomic and endocrine function during stress responses.SIGNIFICANCE STATEMENT The hypothalamic paraventricular nucleus (PVN) coordinates concomitant changes in autonomic and neuroendocrine function to organize the response to stress. This manuscript maps intra-PVN circuitry that signals via CRF, delineates CRF receptor type 1 neuron synaptic targets both within the PVN and at distal targets, and establishes the role of this microcircuit in regulating hypothalamic-pituitary-adrenal axis activity.
Literature context: Thermo Fisher Scientific A6455; RRID:AB_221570 Anti-mCherry mouse monoclonal a
The lateral habenula has been widely studied for its contribution in generating reward-related behaviors [1, 2]. We have found that this nucleus plays an unexpected role in the sedative actions of the general anesthetic propofol. The lateral habenula is a glutamatergic, excitatory hub that projects to multiple targets throughout the brain, including GABAergic and aminergic nuclei that control arousal [3-5]. When glutamate release from the lateral habenula in mice was genetically blocked, the ability of propofol to induce sedation was greatly diminished. In addition to this reduced sensitivity to propofol, blocking output from the lateral habenula caused natural non-rapid eye movement (NREM) sleep to become highly fragmented, especially during the rest ("lights on") period. This fragmentation was largely reversed by the dual orexinergic antagonist almorexant. We conclude that the glutamatergic output from the lateral habenula is permissive for the sedative actions of propofol and is also necessary for the consolidation of natural sleep.
Literature context: ologies Cat#A6455; lot#1774720; RRID:AB_221570 Guinea pig polyclonal anti-LHX3
The spinal cord contains neural networks that enable regionally distinct motor outputs along the body axis. Nevertheless, it remains unclear how segment-specific motor computations are processed because the cardinal interneuron classes that control motor neurons appear uniform at each level of the spinal cord. V2a interneurons are essential to both forelimb and hindlimb movements, and here we identify two major types that emerge during development: type I neurons marked by high Chx10 form recurrent networks with neighboring spinal neurons and type II neurons that downregulate Chx10 and project to supraspinal structures. Types I and II V2a interneurons are arrayed in counter-gradients, and this network activates different patterns of motor output at cervical and lumbar levels. Single-cell RNA sequencing (RNA-seq) revealed type I and II V2a neurons are each comprised of multiple subtypes. Our findings uncover a molecular and anatomical organization of V2a interneurons reminiscent of the orderly way motor neurons are divided into columns and pools.
Literature context: 00) Cat# A-6455; RRID:AB_221570 rat anti-GFP Nacalai Tesque (1:
Wild-type Drosophila cells can remove cells heterozygous for ribosomal protein mutations (known as "Minute" mutant cells) from genetic mosaics, a process termed cell competition. The ribosomal protein S12 was unusual because cells heterozygous for rpS12 mutations were not competed by wild-type, and a viable missense mutation in rpS12 protected Minute cells from cell competition with wild-type cells. Furthermore, cells with Minute mutations were induced to compete with one another by altering the gene dose of rpS12, eliminating cells with more rpS12 than their neighbors. Thus RpS12 has a special function in cell competition that defines the competitiveness of cells. We propose that cell competition between wild-type and Minute cells is initiated by a signal of ribosomal protein haploinsufficiency mediated by RpS12. Since competition between cells expressing different levels of Myc did not require RpS12, other kinds of cell competition may be initiated differently.
Literature context: trogen A-6455; RRID:AB_221570 Goat anti-rabbit Alexa 555 seco
Preservation of a balance between synaptic excitation and inhibition is critical for normal brain function. A number of homeostatic cellular mechanisms have been suggested to play a role in maintaining this balance, including long-term plasticity of GABAergic inhibitory synapses. Many previous studies have demonstrated a coupling of postsynaptic spiking with modification of perisomatic inhibition. Here, we demonstrate that activation of NMDA-type glutamate receptors leads to input-specific long-term potentiation of dendritic inhibition mediated by somatostatin-expressing interneurons. This form of plasticity is expressed postsynaptically and requires both CaMKIIα and the β2 subunit of the GABA-A receptor. Importantly, this process may function to preserve dendritic inhibition, as genetic deletion of NMDAR signaling results in a selective weakening of dendritic inhibition. Overall, our results reveal a new mechanism for linking excitatory and inhibitory input in neuronal dendrites and provide novel insight into the homeostatic regulation of synaptic transmission in cortical circuits.
Literature context: trogen, RRID:AB_221570), goat anti-Scip (1:5000; Santa
Vital motor functions, such as respiration and locomotion, rely on the ability of spinal motor neurons (MNs) to acquire stereotypical positions in the ventral spinal cord and to project with high precision to their peripheral targets. These key properties of MNs emerge during development through transcriptional programs that dictate their subtype identity and connectivity; however, the molecular mechanisms that establish the transcriptional landscape necessary for MN specification are not fully understood. Here, we show that the enzyme topoisomerase IIβ (Top2β) controls MN migration and connectivity. Surprisingly, Top2β is not required for MN generation or survival but has a selective role in columnar specification. In the absence of Top2β, phrenic MN identity is eroded, while other motor columns are partially preserved but fail to cluster to their proper position. In Top2β-/- mice, peripheral connectivity is impaired as MNs exhibit a profound deficit in terminal branching. These defects likely result from the insufficient activation of Hox/Pbx-dependent transcriptional programs as Hox and Pbx genes are downregulated in the absence of Top2β. Top2β mutants recapitulate many aspects of Pbx mutant mice, such as MN disorganization and defects in medial motor column (MMC) specification. Our findings indicate that Top2β, a gene implicated in neurodevelopmental diseases such as autism spectrum disorders, plays a critical, cell-specific role in the assembly of motor circuits.
Literature context: l, Invitrogen A6455, 1:300, RRID:AB_221570). After three washes with PBS,
Olfactory sensory neurons (OSNs) are chemoreceptors that establish excitatory synapses within glomeruli of the olfactory bulb. OSNs undergo continuous turnover throughout life, causing the constant replacement of their synaptic contacts. Using Xenopus tadpoles as an experimental system to investigate rewiring of glomerular connectivity, we show that novel OSN synapses can transfer information immediately after formation, mediating olfactory-guided behavior. Tadpoles recover the ability to detect amino acids 4 days after bilateral olfactory nerve transection. Restoration of olfactory-guided behavior depends on the efficient reinsertion of OSNs to the olfactory bulb. Presynaptic terminals of incipient synaptic contacts generate calcium transients in response to odors, triggering long lasting depolarization of olfactory glomeruli. The functionality of reconnected terminals relies on well-defined readily releasable and cytoplasmic vesicle pools. The continuous growth of non-compartmentalized axonal processes provides a vesicle reservoir to nascent release sites, which contrasts to the gradual development of cytoplasmic vesicle pools in conventional excitatory synapses. The immediate availability of fully functional synapses upon formation supports an age-independent contribution of OSNs to the generation of odor maps.
Literature context: at# A-6455; RRID:AB_221570 Mouse anti-Wg DSHB Cat# 4d4; RR
The insect wing is a key evolutionary innovation that was essential for insect diversification. Yet despite its importance, there is still debate about its evolutionary origins. Two main hypotheses have been proposed: the paranotal hypothesis, which suggests that wings evolved as an extension of the dorsal thorax, and the gill-exite hypothesis, which proposes that wings were derived from a modification of a pre-existing branch at the dorsal base (subcoxa) of the leg. Here, we address this question by studying how wing fates are initially specified during Drosophila embryogenesis, by characterizing a cis-regulatory module (CRM) from the snail (sna) gene, sna-DP (for dorsal primordia). sna-DP specifically marks the early primordia for both the wing and haltere, collectively referred to as the DP. We found that the inputs that activate sna-DP are distinct from those that activate Distalless, a marker for leg fates. Further, in genetic backgrounds in which the leg primordia are absent, the DP are still partially specified. However, lineage-tracing experiments demonstrate that cells from the early leg primordia contribute to both ventral and dorsal appendage fates. Together, these results suggest that the wings of Drosophila have a dual developmental origin: two groups of cells, one ventral and one more dorsal, give rise to the mature wing. We suggest that the dual developmental origins of the wing may be a molecular remnant of the evolutionary history of this appendage, in which cells of the subcoxa of the leg coalesced with dorsal outgrowths to evolve a dorsal appendage with motor control.
Literature context: her Scientific, Germany; A6455; RRID:AB_221570) in combination with goat anti-
In Drosophila melanogaster larvae, the prime site of external taste reception is the terminal organ (TO). Though investigation on the TO's implications in taste perception has been expanding rapidly, the sensilla of the TO have been essentially unexplored. In this study, we performed a systematic anatomical and molecular analysis of the TO. We precisely define morphological types of TO sensilla taking advantage of volume electron microscopy and 3D image analysis. We corroborate the presence of five external types of sensilla: papilla, pit, spot, knob, and modified papilla. Detailed 3D analysis of their structural organization allowed a finer discrimination into subtypes. We classify three subtypes of papilla and pit sensilla, respectively, and two subtypes of knob sensilla. Further, we determine the repertoire of receptor genes for each sensillum by analyzing GAL4 driver lines of Ir, Gr, Ppk, and Trp receptor genes. We construct a map of the TO, in which the receptor genes are mapped to neurons of individual sensilla. While modified papillum and spot sensilla are not labeled by any GAL4 driver, neurons of the pit, papilla, and knob type are labeled by partially overlapping but different subsets of GAL4 driver lines of the Ir, Gr, and Ppk gene family. The results suggest that pit, papilla and knob sensilla act in contact chemosensation. However, they likely do these employing different stimulus transduction mechanisms to sense the diverse chemicals of their environment.
Literature context: Thermo Fisher Scientific A6455; RRID:AB_221570 Monoclonal anti-Î³-tubulin antib
The augmin complex plays an essential role in microtubule (MT)-dependent MT nucleation by recruiting the γ-tubulin complex to MT walls to generate new MTs . The complex contains eight subunits (designated AUG) including AUG8, which is an MT-associated protein (MAP). When this complex is isolated from etiolated seedlings consisting of primarily interphase cells in Arabidopsis thaliana, AUG8 is an integral component . EDE1 (Endosperm DEfective 1) is homologous to AUG8 . Here, we demonstrate that EDE1, but not AUG8, is associated with acentrosomal spindle and phragmoplast MT arrays in patterns indistinguishable from those of the AUG1-7 subunits and the γ-tubulin complex proteins (GCPs) that exhibit biased localization toward MT minus ends. Consistent with this colocalization, EDE1 directly interacts with AUG6 in vivo. Moreover, a partial loss-of-function mutation, ede1-1, compromises the localization of augmin and γ-tubulin on the spindle and phragmoplast MT arrays and leads to serious distortions in spindle MT remodeling during mitosis. However, mitosis continues even when kinetochore fibers are not obviously discernable, and cytokinesis takes place following the formation of elongated bipolar phragmoplast MT arrays in the mutant. Hence, we conclude that the mitotic function of augmin is dependent on its MAP subunit EDE1, which cannot be replaced by AUG8, and that the cell-cycle-dependent function of augmin can be differentially regulated by employing distinct MAP subunits. Our results also illustrate that plant cells can respond flexibly to serious challenges of compromised MT-dependent MT nucleation to complete mitosis and cytokinesis.
Literature context: Scientific Cat# A6455; RRID:AB_221570 Anti-mCherry antibody Abcam Cat
Cancers grow within tissues through molecular mechanisms still unclear. Invasiveness correlates with perturbed O-glycosylation, a covalent modification of cell-surface proteins. Here, we show that, in human and mouse liver cancers, initiation of O-glycosylation by the GALNT glycosyl-transferases increases and shifts from the Golgi to the endoplasmic reticulum (ER). In a mouse liver cancer model, expressing an ER-targeted GALNT1 (ER-G1) massively increased tumor expansion, with median survival reduced from 23 to 10 weeks. In vitro cell growth was unaffected, but ER-G1 strongly enabled matrix degradation and tissue invasion. Unlike its Golgi-localized counterpart, ER-G1 glycosylates the matrix metalloproteinase MMP14, a process required for tumor expansion. Together, our results indicate that GALNTs strongly promote liver tumor growth after relocating to the ER.
Mutations in leucine-rich repeat kinase 2 (LRRK2) contribute to development of late-onset familial Parkinson's disease (PD), with clinical features of motor and cognitive dysfunction indistinguishable from sporadic PD. Calcium dysregulation plays an important role in PD pathogenesis, but the mechanisms of neurodegeneration remain unclear. Recent reports indicate enhanced excitatory neurotransmission in cortical neurons expressing mutant LRRK2, which occurs before the well-characterized phenotype of dendritic shortening. As mitochondria play a major role in the rapid buffering of cytosolic calcium, we hypothesized that altered mitochondrial calcium handling contributes to dendritic retraction elicited by the LRRK2-G2019S and -R1441C mutations. In primary mouse cortical neurons, we observed increased depolarization-induced mitochondrial calcium uptake. We found that expression of mutant LRRK2 elicited transcriptional upregulation of the mitochondrial calcium uniporter (MCU) and the mitochondrial calcium uptake 1 protein (MICU1) with no change in levels of the mitochondrial calcium antiporter NCLX. Elevated MCU and MICU1 were also observed in LRRK2-mutated patient fibroblasts, along with increased mitochondrial calcium uptake, and in postmortem brains of sporadic PD/PDD patients of both sexes. Transcriptional upregulation of MCU and MICU1 was caused by activation of the ERK1/2 (MAPK3/1) pathway. Inhibiting ERK1/2 conferred protection against mutant LRRK2-induced neurite shortening. Pharmacological inhibitors or RNAi knockdown of MCU attenuated mitochondrial calcium uptake and dendritic/neuritic shortening elicited by mutant LRRK2, whereas expression of a constitutively active mutant of NCLX that enhances calcium export from mitochondria was neuroprotective. These data suggest that an increased susceptibility to mitochondrial calcium dysregulation contributes to dendritic injury in mutant LRRK2 pathogenesis.SIGNIFICANCE STATEMENT Cognitive dysfunction and dementia are common features of Parkinson's disease (PD), causing significant disability. Mutations in LRRK2 represent the most common known genetic cause of PD. We found that PD-linked LRRK2 mutations increased dendritic and mitochondrial calcium uptake in cortical neurons and familial PD patient fibroblasts, accompanied by increased expression of the mitochondrial calcium transporter MCU. Blocking the ERK1/2-dependent upregulation of MCU conferred protection against mutant LRRK2-elicited dendrite shortening, as did inhibiting MCU-mediated calcium import. Conversely, stimulating the export of calcium from mitochondria was also neuroprotective. These results implicate increased susceptibility to mitochondrial calcium overload in LRRK2-driven neurodegeneration, and suggest possible interventions that may slow the progression of cognitive dysfunction in PD.
Literature context: antibody GFP Invitrogen A6455 RRID:AB_221570 1:5000
Mutations in the human BEST1 gene lead to retinal degenerative diseases displaying progressive vision loss and even blindness. BESTROPHIN1, encoded by BEST1, is predominantly expressed in retinal pigment epithelium (RPE), but its physiological role has been a mystery for the last two decades. Using a patient-specific iPSC-based disease model and interdisciplinary approaches, we comprehensively analyzed two distinct BEST1 patient mutations, and discovered mechanistic correlations between patient clinical phenotypes, electrophysiology in their RPEs, and the structure and function of BESTROPHIN1 mutant channels. Our results revealed that the disease-causing mechanism of BEST1 mutations is centered on the indispensable role of BESTROPHIN1 in mediating the long speculated Ca2+-dependent Cl- current in RPE, and demonstrate that the pathological potential of BEST1 mutations can be evaluated and predicted with our iPSC-based 'disease-in-a-dish' approach. Moreover, we demonstrated that patient RPE is rescuable with viral gene supplementation, providing a proof-of-concept for curing BEST1-associated diseases.
Literature context: lecular Probes RRID:AB_221570 â€ƒâ€ƒâ€ƒâ€ƒRabbit anti-Homer1 (1:500)
Glycoprotein-deleted rabies virus-mediated monosynaptic tracing has become a standard method for neuronal circuit mapping, and is applied to virtually all parts of the rodent nervous system, including the spinal cord and primary sensory neurons. Here we identified two classes of unmyelinated sensory neurons (nonpeptidergic and C-fiber low-threshold mechanoreceptor neurons) resistant to direct and trans-synaptic infection from the spinal cord with rabies viruses that carry glycoproteins in their envelopes and that are routinely used for infection of CNS neurons (SAD-G and N2C-G). However, the same neurons were susceptible to infection with EnvA-pseudotyped rabies virus in tumor virus A receptor transgenic mice, indicating that resistance to retrograde infection was due to impaired virus adsorption rather than to deficits in subsequent steps of infection. These results demonstrate an important limitation of rabies virus-based retrograde tracing of sensory neurons in adult mice, and may help to better understand the molecular machinery required for rabies virus spread in the nervous system. In this study, mice of both sexes were used.SIGNIFICANCE STATEMENT To understand the neuronal bases of behavior, it is important to identify the underlying neural circuitry. Rabies virus-based monosynaptic tracing has been used to identify neuronal circuits in various parts of the nervous system. This has included connections between peripheral sensory neurons and their spinal targets. These connections form the first synapse in the somatosensory pathway. Here we demonstrate that two classes of unmyelinated sensory neurons, which account for >40% of dorsal root ganglia neurons, display resistance to rabies infection. Our results are therefore critical for interpreting monosynaptic rabies-based tracing in the sensory system. In addition, identification of rabies-resistant neurons might provide a means for future studies addressing rabies pathobiology.
Literature context: Fisher Scientific Cat# A-6455; RRID:AB_221570 Anti-Caspase-3, Active Form Ant
A genome-wide association study identified LMO1, which encodes an LIM-domain-only transcriptional cofactor, as a neuroblastoma susceptibility gene that functions as an oncogene in high-risk neuroblastoma. Here we show that dβh promoter-mediated expression of LMO1 in zebrafish synergizes with MYCN to increase the proliferation of hyperplastic sympathoadrenal precursor cells, leading to a reduced latency and increased penetrance of neuroblastomagenesis. The transgenic expression of LMO1 also promoted hematogenous dissemination and distant metastasis, which was linked to neuroblastoma cell invasion and migration, and elevated expression levels of genes affecting tumor cell-extracellular matrix interaction, including loxl3, itga2b, itga3, and itga5. Our results provide in vivo validation of LMO1 as an important oncogene that promotes neuroblastoma initiation, progression, and widespread metastatic dissemination.
Literature context: body Life Technologies RRID:AB_221570 Chemicals, Peptides, and Recomb
In contrast to extracellular chemotactic gradients, how cell-adhesion molecules contribute to directing cell migration remains more elusive. Here we studied the collective migration of Drosophila larval epidermal cells (LECs) along the anterior-posterior axis and propose a migrating cell group-autonomous mechanism in which an atypical cadherin Dachsous (Ds) plays a pivotal role. In each abdominal segment, the amount of Ds in each LEC varied along the axis of migration (Ds imbalance), which polarized Ds localization at cell boundaries. This Ds polarity was necessary for coordinating the migratory direction. Another atypical cadherin, Fat (Ft), and an unconventional myosin Dachs, both of which bind to Ds, also showed biased cell-boundary localizations, and both were required for the migration. Altogether, we propose that the Ds imbalance within the migrating tissue provides the directional cue and that this is decoded by Ds-Ft-mediated cell-cell contacts, which restricts lamellipodia formation to the posterior end of the cell.
Literature context: i-GFP Life Technologies A-6455; RRID:AB_221570 Anti-Kar2 polyclonal (y115) San
In the endoplasmic reticulum (ER), Ero1 catalyzes disulfide bond formation and promotes glutathione (GSH) oxidation to GSSG. Since GSSG cannot be reduced in the ER, maintenance of the ER glutathione redox state and levels likely depends on ER glutathione import and GSSG export. We used quantitative GSH and GSSG biosensors to monitor glutathione import into the ER of yeast cells. We found that glutathione enters the ER by facilitated diffusion through the Sec61 protein-conducting channel, while oxidized Bip (Kar2) inhibits transport. Increased ER glutathione import triggers H2O2-dependent Bip oxidation through Ero1 reductive activation, which inhibits glutathione import in a negative regulatory loop. During ER stress, transport is activated by UPR-dependent Ero1 induction, and cytosolic glutathione levels increase. Thus, the ER redox poise is tuned by reciprocal control of glutathione import and Ero1 activation. The ER protein-conducting channel is permeable to small molecules, provided the driving force of a concentration gradient.
Literature context: cular Probes RRID:AB_221570 Rabbit anti-Iba1 (1 : 1000) Wak
Recombinant adeno-associated virus (rAAV) vector-mediated gene transfer into genetically defined neuron subtypes has become a powerful tool to study the neuroanatomy of neuronal circuits in the brain and to unravel their functions. More recently, this methodology has also become popular for the analysis of spinal cord circuits. To date, a variety of naturally occurring AAV serotypes and genetically modified capsid variants are available but transduction efficiency in spinal neurons, target selectivity, and the ability for retrograde tracing are only incompletely characterized. Here, we have compared the transduction efficiency of seven commonly used AAV serotypes after intraspinal injection. We specifically analyzed local transduction of different types of dorsal horn neurons, and retrograde transduction of dorsal root ganglia (DRG) neurons and of neurons in the rostral ventromedial medulla (RVM) and the somatosensory cortex (S1). Our results show that most of the tested rAAV vectors have similar transduction efficiency in spinal neurons. All serotypes analyzed were also able to transduce DRG neurons and descending RVM and S1 neurons via their spinal axon terminals. When comparing the commonly used rAAV serotypes to the recently developed serotype 2 capsid variant rAAV2retro, a > 20-fold increase in transduction efficiency of descending supraspinal neurons was observed. Conversely, transgene expression in retrogradely transduced neurons was strongly reduced when the human synapsin 1 (hSyn1) promoter was used instead of the strong ubiquitous hybrid cytomegalovirus enhancer/chicken β-actin promoter (CAG) or cytomegalovirus (CMV) promoter fragments. We conclude that the use of AAV2retro greatly increases transduction of neurons connected to the spinal cord via their axon terminals, while the hSyn1 promoter can be used to minimize transgene expression in retrogradely connected neurons of the DRG or brainstem. Cover Image for this issue: doi. 10.1111/jnc.13813.
Literature context: en A6455; RRID:AB_221570 Rabbit pol
Activation of the FGF signaling pathway during preimplantation development of the mouse embryo is known to be essential for differentiation of the inner cell mass and the formation of the primitive endoderm (PrE). We now show using fluorescent reporter knockin lines that Fgfr1 is expressed in all cell populations of the blastocyst, while Fgfr2 expression becomes restricted to extraembryonic lineages, including the PrE. We further show that loss of both receptors prevents the development of the PrE and demonstrate that FGFR1 plays a more prominent role in this process than FGFR2. Finally, we document an essential role for FGFRs in embryonic stem cell (ESC) differentiation, with FGFR1 again having a greater influence than FGFR2 in ESC exit from the pluripotent state. Collectively, these results identify mechanisms through which FGF signaling regulates inner cell mass lineage restriction and cell commitment during preimplantation development.
Literature context: ontrol tissue was processed for GFP (rabbit, A6455; serum; 1:20,000; Invitrogen; Antibodyregistry.org) or AB11122 (IgG fraction, 1:20
A method for capturing gait signatures in neurological conditions that allows comparison of human gait with animal models would be of great value in translational research. However, the velocity dependence of gait parameters and differences between quadruped and biped gait have made this comparison challenging. Here we present an approach that accounts for changes in velocity during walking and allows for translation across species. In mice, we represented spatial and temporal gait parameters as a function of velocity and established regression models that reproducibly capture the signatures of these relationships during walking. In experimental parkinsonism models, regression curves representing these relationships shifted from baseline, implicating changes in gait signatures, but with marked differences between models. Gait parameters in healthy human subjects followed similar strict velocity dependent relationships which were altered in Parkinson's patients in ways that resemble some but not all mouse models. This novel approach is suitable to quantify qualitative walking abnormalities related to CNS circuit dysfunction across species, identify appropriate animal models, and it provides important translational opportunities.
A large proportion of critically ill patients have alterations in the hypothalamus-pituitary-thyroid (HPT) axis, collectively known as the nonthyroidal illness syndrome. Nonthyroidal illness syndrome is characterized by low serum thyroid hormone (TH) concentrations accompanied by a suppressed central component of the HPT axis and persistent low serum TSH. In hypothalamic tanycytes, the expression of type 2 deiodinase (D2) is increased in several animal models of inflammation. Because D2 is a major source of T3 in the brain, this response is thought to suppress TRH expression in the paraventricular nucleus via increased local bioavailability of T3. The inflammatory pathway component RelA (the p65 subunit of nuclear factor-κB) can bind the Dio2 promoter and increases D2 expression after lipopolysaccharide (LPS) stimulation in vitro. We aimed to determine whether RelA signaling in tanycytes is essential for the LPS-induced D2 increase in vivo by conditional elimination of RelA in tanycytes of mice (RelA(ASTKO)). Dio2 and Trh mRNA expression were assessed by quantitative in situ hybridization 8 or 24 hours after saline or LPS injection. At the same time points, we measured pituitary Tshβ mRNA expression and serum T3 and T4 concentrations. In RelA(ASTKO) mice the LPS-induced increase in Dio2 and decrease in Trh mRNA levels in the hypothalamus were reduced compared with the wild-type littermates, whereas the drop in pituitary Tshβ expression and in serum TH concentrations persisted. In conclusion, RelA is essential for the LPS-induced hypothalamic D2 increase and TRH decrease. The central changes in the HPT axis are, however, not required for the down-regulation of Tshβ expression and serum TH concentrations.
Literature context: ) with the following antibodies: rabbit anti-GFP antibody (A-6455; Invitrogen) used at 1:2000, mouse anti-act
How nuclear size is regulated is a fundamental cell-biological question with relevance to cancers, which often exhibit enlarged nuclei. We previously reported that conventional protein kinase C (cPKC) contributes to nuclear size reductions that occur during early Xenopus development. Here we report that PKC-mediated phosphorylation of lamin B3 (LB3) contributes to this mechanism of nuclear size regulation. By mapping PKC phosphorylation sites on LB3 and testing the effects of phosphomutants in Xenopus laevis embryos, we identify the novel site S267 as being an important determinant of nuclear size. Furthermore, FRAP studies demonstrate that phosphorylation at this site increases lamina dynamics, providing a mechanistic explanation for how PKC activity influences nuclear size. We subsequently map this X. laevis LB3 phosphorylation site to a conserved site in mammalian lamin A (LA), S268. Manipulating PKC activity in cultured mammalian cells alters nuclear size, as does expression of LA-S268 phosphomutants. Taken together, these data demonstrate that PKC-mediated lamin phosphorylation is a conserved mechanism of nuclear size regulation.
Literature context: 455Â Rabbit; polyclonalÂ 1:20,000Â AB_221570Â THÂ TH antibody (H-196)Â Santa Cr
Ghrelin is known to act on the area postrema (AP), a sensory circumventricular organ located in the medulla oblongata that regulates a variety of important physiological functions. However, the neuronal targets of ghrelin in the AP and their potential role are currently unknown. In this study, we used wild-type and genetically modified mice to gain insights into the neurons of the AP expressing the ghrelin receptor [growth hormone secretagogue receptor (GHSR)] and their role. We show that circulating ghrelin mainly accesses the AP but not to the adjacent nucleus of the solitary tract. Also, we show that both peripheral administration of ghrelin and fasting induce an increase of c-Fos, a marker of neuronal activation, in GHSR-expressing neurons of the AP, and that GHSR expression is necessary for the fasting-induced activation of AP neurons. Additionally, we show that ghrelin-sensitive neurons of the AP are mainly γ-aminobutyric acid (GABA)ergic, and that an intact AP is required for ghrelin-induced gastric emptying. Overall, we show that the capacity of circulating ghrelin to acutely induce gastric emptying in mice requires the integrity of the AP, which contains a population of GABA neurons that are a target of plasma ghrelin.
Literature context: t# A6455; RRID:AB_221570), guinea p
Animals have sophisticated homeostatic controls. While mammalian body temperature fluctuates throughout the day, small ectotherms, such as Drosophila achieve a body temperature rhythm (BTR) through their preference of environmental temperature. Here, we demonstrate that pigment dispersing factor (PDF) neurons play an important role in setting preferred temperature before dawn. We show that small lateral ventral neurons (sLNvs), a subset of PDF neurons, activate the dorsal neurons 2 (DN2s), the main circadian clock cells that regulate temperature preference rhythm (TPR). The number of temporal contacts between sLNvs and DN2s peak before dawn. Our data suggest that the thermosensory anterior cells (ACs) likely contact sLNvs via serotonin signaling. Together, the ACs-sLNs-DN2s neural circuit regulates the proper setting of temperature preference before dawn. Given that sLNvs are important for sleep and that BTR and sleep have a close temporal relationship, our data highlight a possible neuronal interaction between body temperature and sleep regulation.
Literature context: # A-6455, RRID:AB_221570 Mouse mono
In many vertebrates, postnatally generated neurons often migrate long distances to reach their final destination, where they help shape local circuit activity. Concerted action of extrinsic stimuli is required to regulate long-distance migration. Some migratory principles are evolutionarily conserved, whereas others are species and cell type specific. Here we identified a serotonergic mechanism that governs migration of postnatally generated neurons in the mouse brain. Serotonergic axons originating from the raphe nuclei exhibit a conspicuous alignment with subventricular zone-derived neuroblasts. Optogenetic axonal activation provides functional evidence for serotonergic modulation of neuroblast migration. Furthermore, we show that the underlying mechanism involves serotonin receptor 3A (5HT3A)-mediated calcium influx. Thus, 5HT3A receptor deletion in neuroblasts impaired speed and directionality of migration and abolished calcium spikes. We speculate that serotonergic modulation of postnatally generated neuroblast migration is evolutionarily conserved as indicated by the presence of serotonergic axons in migratory paths in other vertebrates.
Literature context: vitrogen, RRID:AB_221570). To avoid
Growing evidence shows that the neurotransmitter serotonin (5-HT) modulates the fine-tuning of neuron development and the establishment of wiring patterns in the brain. However, whether serotonin is involved in the maintenance of neuronal circuitry in the adult brain remains elusive. Here, we use a Tph2fl°x conditional knockout (cKO) mouse line to assess the impact of serotonin depletion during adulthood on serotonergic system organization. Data show that the density of serotonergic fibers is increased in the hippocampus and decreased in the thalamic paraventricular nucleus (PVN) as a consequence of brain serotonin depletion. Strikingly, these defects are rescued following reestablishment of brain 5-HT signaling via administration of the serotonin precursor 5-hydroxytryptophan (5-HTP). Finally, 3D reconstruction of serotonergic fibers reveals that changes in serotonin homeostasis affect axonal branching complexity. These data demonstrate that maintaining proper serotonin homeostasis in the adult brain is crucial to preserve the correct serotonergic axonal wiring.
Literature context: g #A6455; RRID:AB_221570), rabbit a
Amyloid precursor protein (App) plays a crucial role in Alzheimer's disease via the production and deposition of toxic β-amyloid peptides. App is heavily expressed in neurons, the focus of the vast majority of studies investigating its function. Meanwhile, almost nothing is known about App's function in glia, where it is also expressed, and can potentially participate in the regulation of neuronal physiology. In this report, we investigated whether Appl, the Drosophila homolog of App, could influence sleep-wake regulation when its function is manipulated in glial cells. Appl inhibition in astrocyte-like and cortex glia resulted in higher sleep amounts and longer sleep bout duration during the night, while overexpression had the opposite effect. These sleep phenotypes were not the result of developmental defects, and were correlated with changes in expression in glutamine synthetase (GS) in astrocyte-like glia and in changes in the gap-junction component innexin2 in cortex glia. Downregulating both GS and innexin2, but not either one individually, resulted in higher sleep amounts, similarly to Appl inhibition. Consistent with these results, the expression of GS and innexin2 are increased following sleep deprivation, indicating that GS and innexin2 genes are dynamically linked to vigilance states. Interestingly, the reduction of GS expression and the sleep phenotype observed upon Appl inhibition could be rescued by increasing the expression of the glutamate transporter dEaat1. In contrast, reducing dEaat1 expression severely disrupted sleep. These results associate glutamate recycling, sleep, and a glial function for the App family proteins.SIGNIFICANCE STATEMENT The amyloid precursor protein (App) has been intensively studied for its implication in Alzheimer's disease (AD). The attributed functions of App are linked to the physiology and cellular biology of neurons where the protein is predominantly expressed. Consequences on glia in AD are generally thought to be secondary effects of the pathology in neurons. Researchers still do not know whether App plays a role in glia in nonpathological conditions. We report here that glial App plays a role in physiology and in the regulation of sleep/wake, which has been shown recently to be involved in AD pathology. These results also associate glutamate recycling and sleep regulation, adding further complexity to the physiological role of App and to its implication in AD.
Literature context: s, Rabbit polyclonal, # A-6455, AB_2215701:500 dilutionImage acquisitionS
The claustrum is an intriguing brain structure, featuring the highest connectivity per regional volume in the brain. It is a thin and elongated structure enclosed between the striatum and the insular cortex, with widespread reciprocal connections with the sensory modalities and prefrontal cortices. Retinotopic and somatotopic organizations have been described in the claustrum, and anatomical studies in cats, monkeys, and rats have demonstrated topographic organization of cortico-claustral connections. In this study we mapped the projections from cortical modalities (visual, auditory, somatosensory, motor, and olfactory), and prefrontal regions (anterior cingulate cortex and orbitofrontal cortex) to the claustrum in mice. Utilizing expression of a virally encoded synaptic anterograde tracer, AAV-SynaptoTag, followed by 3D reconstruction of the cortical projections, we performed a comprehensive study of the organization of these projections within the mouse claustrum. Our results clearly demonstrate a dorsoventral laminar organization of projections from the sensory cortices to the claustrum, whereas frontal inputs are more extensive and overlap with the inputs from the sensory cortices. In addition, we find evidence supporting a core/shell organization of the claustrum. We propose that the overlap between the frontal inputs and the inputs from the sensory modalities may underlie executive regulation of the communication between the claustrum and the cortical modalities. J. Comp. Neurol. 525:1381-1402, 2017. © 2016 Wiley Periodicals, Inc.
Literature context: t# A-6455 RRID:AB_221570 Human NCAM
Human pluripotent stem cells (PSCs) provide a unique entry to study species-specific aspects of human disorders such as Alzheimer's disease (AD). However, in vitro culture of neurons deprives them of their natural environment. Here we transplanted human PSC-derived cortical neuronal precursors into the brain of a murine AD model. Human neurons differentiate and integrate into the brain, express 3R/4R Tau splice forms, show abnormal phosphorylation and conformational Tau changes, and undergo neurodegeneration. Remarkably, cell death was dissociated from tangle formation in this natural 3D model of AD. Using genome-wide expression analysis, we observed upregulation of genes involved in myelination and downregulation of genes related to memory and cognition, synaptic transmission, and neuron projection. This novel chimeric model for AD displays human-specific pathological features and allows the analysis of different genetic backgrounds and mutations during the course of the disease.
Literature context: P [1:250, RRID:AB_221570, Invitroge
Vestibular hair cells in the inner ear encode head movements and mediate the sense of balance. These cells undergo cell death and replacement (turnover) throughout life in non-mammalian vertebrates. However, there is no definitive evidence that this process occurs in mammals. We used fate-mapping and other methods to demonstrate that utricular type II vestibular hair cells undergo turnover in adult mice under normal conditions. We found that supporting cells phagocytose both type I and II hair cells. Plp1-CreERT2-expressing supporting cells replace type II hair cells. Type I hair cells are not restored by Plp1-CreERT2-expressing supporting cells or by Atoh1-CreERTM-expressing type II hair cells. Destruction of hair cells causes supporting cells to generate 6 times as many type II hair cells compared to normal conditions. These findings expand our understanding of sensorineural plasticity in adult vestibular organs and further elucidate the roles that supporting cells serve during homeostasis and after injury.
Literature context: t 1:200 (Molecular Probes; RRID:AB_221570) and visualized using Alexa con
The hypothalamic GnRH neurons are a small group of cells that regulate the reproductive axis. These neurons are specified within the olfactory placode, delaminate from this structure, and then migrate to enter the forebrain before populating the hypothalamus. We have used microarray technology to analyze the transcriptome of the olfactory placode at a number of key time points for GnRH ontogeny using the chick embryo. This resulted in the identification of a large number of genes whose expression levels change significantly over this period. This repertoire includes those genes that are known to be important for GnRH neuronal development as well as many novel genes, such as the serotonin receptor 1A, HTR1A. We find that HTR1A is expressed in the region of the olfactory placode that generates GnRH neurons. We further show that when this receptor is inactivated using a selective HTR1A antagonist as well as a gene knockdown approach using RNAi, this resulted in delayed migration causing the GnRH neurons to stall just outside the forebrain. These findings implicate HTR1A as being important for GnRH neuronal migration from the olfactory placode to the forebrain. Our study thus extends the repertoire of genes involved in GnRH neuron biology and thus identifies new candidate genes that can be screened for in patients who do not show mutations in any of the previously identified hypogonadotrophic hypogonadism/Kallmann syndrome genes.
Literature context: o. A6455, RRID:AB_221570; diluted 1
The parvafox nucleus is an elongated structure that is lodged within the ventrolateral hypothalamus and lies along the optic tract. It comprises axially located parvalbumin (Parv)-positive neurons and a peripheral cuff of Foxb1-expressing ones. In the present study, injections of Cre-dependent adenoviral constructs were targeted to the ventrolateral hypothalamus of Foxb1/Cre mice to label specifically and map the efferent connections of the Foxb1-expressing subpopulation of neurons of the parvafox nucleus. These neurons project more widely than do the Parv-positive ones and implicate a part of the axons known to emanate from the lateral hypothalamus. High labeling densities were found in the dorsolateral and the upper lateral portion of the periaqueductal gray (PAG), the Su3 and PV2 nuclei of the ventrolateral PAG, the cuneiform nucleus, the mesencephalic reticular formation, and the superior colliculus. Intermediate densities of terminals were encountered in the septum, bed nucleus of the stria terminalis, substantia innominata, various thalamic and hypothalamic nuclei, pedunculopontine nucleus, Barrington's nucleus, retrofacial nucleus, and retroambigual nucleus. Scattered terminals were observed in the olfactory bulbs, the prefrontal cortex and the lamina X of the cervical spinal cord. Because the terminals were demonstrated to express the glutamate transporter VGlut2, the projections are presumed to be excitatory. A common denominator of the main target sites of the Foxb1-positive axons of the parvafox nucleus appears to be an involvement in the defensive reactions to life-threatening situations. The hypothalamic parvafox nucleus may contribute to the autonomic manifestations that accompany the expression of emotions. J. Comp. Neurol. 524:2955-2981, 2016. © 2016 Wiley Periodicals, Inc.
Literature context: nologies, RRID:AB_221570), mouse an
Neuronal circuits are known to integrate nutritional information, but the identity of the circuit components is not completely understood. Amino acids are a class of nutrients that are vital for the growth and function of an organism. Here, we report a neuronal circuit that allows Drosophila larvae to overcome amino acid deprivation and pupariate. We find that nutrient stress is sensed by the class IV multidendritic cholinergic neurons. Through live calcium imaging experiments, we show that these cholinergic stimuli are conveyed to glutamatergic neurons in the ventral ganglion through mAChR. We further show that IP3R-dependent calcium transients in the glutamatergic neurons convey this signal to downstream medial neurosecretory cells (mNSCs). The circuit ultimately converges at the ring gland and regulates expression of ecdysteroid biosynthetic genes. Activity in this circuit is thus likely to be an adaptation that provides a layer of regulation to help surpass nutritional stress during development.
Literature context: 1:1,000; RRID:AB_221570; Table 1)
All organisms continuously have to adapt their behavior according to changes in the environment in order to survive. Experience-driven changes in behavior are usually mediated and maintained by modifications in signaling within defined brain circuits. Given the simplicity of the larval brain of Drosophila and its experimental accessibility on the genetic and behavioral level, we analyzed if Drosophila neuropeptide F (dNPF) neurons are involved in classical olfactory conditioning. dNPF is an ortholog of the mammalian neuropeptide Y, a highly conserved neuromodulator that stimulates food-seeking behavior. We provide a comprehensive anatomical analysis of the dNPF neurons on the single-cell level. We demonstrate that artificial activation of dNPF neurons inhibits appetitive olfactory learning by modulating the sugar reward signal during acquisition. No effect is detectable for the retrieval of an established appetitive olfactory memory. The modulatory effect is based on the joint action of three distinct cell types that, if tested on the single-cell level, inhibit and invert the conditioned behavior. Taken together, our work describes anatomically and functionally a new part of the sugar reinforcement signaling pathway for classical olfactory conditioning in Drosophila larvae.
The subparaventricular zone of the hypothalamus (SPZ) is the main efferent target of neural projections from the suprachiasmatic nucleus (SCN) and an important relay for the circadian timing system. Although the SPZ is fairly homogeneous cytoarchitecturally and neurochemically, it has been divided into distinct functional and connectional subdivisions. The dorsal subdivision of the SPZ (dSPZ) plays an important role in relaying signals from the SCN controlling body temperature rhythms, while the ventral subdivision (vSPZ) is critical for rhythms of sleep and locomotor activity (Lu et al.  J Neurosci 21:4864-4874). On the other hand, the medial part of the SPZ receives input mainly from the dorsomedial SCN, whereas the lateral SPZ receives input from the ventrolateral SCN and the retinohypothalamic tract (Leak and Moore  J Comp Neurol 433:312-334). We therefore investigated whether there are corresponding differences in efferent outputs from these four quadrants of the SPZ (dorsolateral, ventrolateral, dorsomedial, and ventromedial) by a combination of anterograde and retrograde tracing. We found that, while all four subdivisions of the SPZ share a similar backbone of major projection pathways to the septal region, thalamus, hypothalamus, and brainstem, each segment of the SPZ has a specific set of targets where its projections dominate. Furthermore, we observed intra-SPZ projections of varying densities between the four subdivisions. Taken together, this pattern of organization suggests that the circadian timing system may have several parallel neural outflow pathways that provide a road map for understanding how they subserve different functions.
Literature context: anti-GFP (RRID:AB_221570, Life Tech
GABAA receptor subunit composition is a critical determinant of receptor localization and physiology, with synaptic receptors generating phasic inhibition and extrasynaptic receptors producing tonic inhibition. Extrasynaptically localized α5 GABAA receptors are largely responsible for tonic inhibition in hippocampal neurons. However, we show here that inhibitory synapses also contain a constant level of α5 GABAA receptors throughout neuronal development, as measured by its colocalization with gephyrin, the inhibitory postsynaptic scaffolding protein. Immunoprecipitation of the α5 subunit from both cultured neurons and adult rat brain coimmunoprecipitated gephyrin, confirming this interaction in vivo. Furthermore, the α5 subunit can interact with gephyrin independent of other synaptically localized alpha subunits, as shown by immunoprecipitation experiments in HEK cells. By replacing the α5 predicted gephyrin binding domain (Residues 370-385) with either the high affinity gephyrin binding domain of the α2 subunit or homologous residues from the extrasynaptic α4 subunit that does not interact with gephyrin, α5 GABAA receptor localization shifted into or out of the synapse, respectively. These shifts in the ratio of synaptic/extrasynaptic α5 localization disrupted dendritic outgrowth and spine maturation. In contrast to the predominant view of α5 GABAA receptors being extrasynaptic and modulating tonic inhibition, we identify an intimate association of the α5 subunit with gephyrin, resulting in constant synaptic levels of α5 GABAA R throughout circuit formation that regulates neuronal development.
Current limitations in technology have prevented an extensive analysis of the connections among neurons, particularly within nonmammalian organisms. We developed a transsynaptic viral tracer originally for use in mice, and then tested its utility in a broader range of organisms. By engineering the vesicular stomatitis virus (VSV) to encode a fluorophore and either the rabies virus glycoprotein (RABV-G) or its own glycoprotein (VSV-G), we created viruses that can transsynaptically label neuronal circuits in either the retrograde or anterograde direction, respectively. The vectors were investigated for their utility as polysynaptic tracers of chicken and zebrafish visual pathways. They showed patterns of connectivity consistent with previously characterized visual system connections, and revealed several potentially novel connections. Further, these vectors were shown to infect neurons in several other vertebrates, including Old and New World monkeys, seahorses, axolotls, and Xenopus. They were also shown to infect two invertebrates, Drosophila melanogaster, and the box jellyfish, Tripedalia cystophora, a species previously intractable for gene transfer, although no clear evidence of transsynaptic spread was observed in these species. These vectors provide a starting point for transsynaptic tracing in most vertebrates, and are also excellent candidates for gene transfer in organisms that have been refractory to other methods.
Literature context: d rabbit (RRID:AB_221570) and mouse
Metabolic homeostasis and water balance is maintained by tight hormonal and neuronal regulation. In Drosophila, insulin-like peptides (DILPs) are key regulators of metabolism, and the neuropeptide leucokinin (LK) is a diuretic hormone that also modulates feeding. However, it is not known whether LK and DILPs act together to regulate feeding and water homeostasis. Because LK neurons express the insulin receptor (dInR), we tested functional links between DILP and LK signaling in feeding and water balance. Thus, we performed constitutive and conditional manipulations of activity in LK neurons and insulin-producing cells (IPCs) in adult flies and monitored food intake, responses to desiccation, and peptide expression levels. We also measured in vivo changes in LK and DILP levels in neurons in response to desiccation and drinking. Our data show that activated LK cells stimulate diuresis in vivo, and that LK and IPC signaling affect food intake in opposite directions. Overexpression of the dInR in LK neurons decreases the LK peptide levels, but only caused a subtle decrease in feeding, and had no effect on water balance. Next we demonstrated that LK neurons express the serotonin receptor 5-HT1B . Knockdown of this receptor in LK neurons diminished LK expression, increased desiccation resistance, and diminished food intake. Live calcium imaging indicates that serotonin inhibits spontaneous activity in abdominal LK neurons. Our results suggest that serotonin via 5-HT1B diminishes activity in the LK neurons and thereby modulates functions regulated by LK peptide, but the action of the dInR in these neurons remains less clear.
Literature context: rch Resource Identifier (RRID): AB_221570], guinea pig anti-VAChT
Retinal ganglion cell (RGC) loss is a hallmark of glaucoma and the second leading cause of blindness worldwide. The type and timing of cellular changes leading to RGC loss in glaucoma remain incompletely understood, including whether specific RGC subtypes are preferentially impacted at early stages of this disease. Here we applied the microbead occlusion model of glaucoma to different transgenic mouse lines, each expressing green fluorescent protein in 1-2 specific RGC subtypes. Targeted filling, reconstruction, and subsequent comparison of the genetically identified RGCs in control and bead-injected eyes revealed that some subtypes undergo significant dendritic rearrangements as early as 7 d following induction of elevated intraocular pressure (IOP). By comparing specific On-type, On-Off-type and Off-type RGCs, we found that RGCs that target the majority of their dendritic arbors to the scleral half or "Off" sublamina of the inner plexiform layer (IPL) undergo the greatest changes, whereas RGCs with the majority of their dendrites in the On sublamina did not alter their structure at this time point. Moreover, M1 intrinsically photosensitive RGCs, which functionally are On RGCs but structurally stratify their dendrites in the Off sublamina of the IPL, also underwent significant changes in dendritic structure 1 week after elevated IOP. Thus, our findings reveal that certain RGC subtypes manifest significant changes in dendritic structure after very brief exposure to elevated IOP. The observation that RGCs stratifying most of their dendrites in the Off sublamina are first to alter their structure may inform the development of new strategies to detect, monitor, and treat glaucoma in humans.
Literature context: g #A6455 (RRID:AB_221570), Invitrog
The declining efficiency of myelin regeneration in individuals with multiple sclerosis has stimulated a search for ways by which it might be therapeutically enhanced. Here we have used gene expression profiling on purified murine oligodendrocyte progenitor cells (OPCs), the remyelinating cells of the adult CNS, to obtain a comprehensive picture of how they become activated after demyelination and how this enables them to contribute to remyelination. We find that adult OPCs have a transcriptome more similar to that of oligodendrocytes than to neonatal OPCs, but revert to a neonatal-like transcriptome when activated. Part of the activation response involves increased expression of two genes of the innate immune system, IL1β and CCL2, which enhance the mobilization of OPCs. Our results add a new dimension to the role of the innate immune system in CNS regeneration, revealing how OPCs themselves contribute to the postinjury inflammatory milieu by producing cytokines that directly enhance their repopulation of areas of demyelination and hence their ability to contribute to remyelination.
Literature context: 1:1,000; RRID:AB_221570; Table 1)
Drosophila larvae are able to evaluate sensory information based on prior experience, similarly to adult flies, other insect species, and vertebrates. Larvae and adult flies can be taught to associate odor stimuli with sugar reward, and prior work has implicated both the octopaminergic and the dopaminergic modulatory systems in reinforcement signaling. Here we use genetics to analyze the anatomy, up to the single-cell level, of the octopaminergic/tyraminergic system in the larval brain and subesophageal ganglion. Genetic ablation of subsets of these neurons allowed us to determine their necessity for appetitive olfactory learning. These experiments reveal that a small subset of about 39 largely morphologically distinguishable octopaminergic/tyraminergic neurons is involved in signaling reward in the Drosophila larval brain. In addition to prior work on larval locomotion, these data functionally separate the octopaminergic/tyraminergic system into two sets of about 40 neurons. Those situated in the thoracic/abdominal ganglion are involved in larval locomotion, whereas the others in the subesophageal ganglion and brain hemispheres mediate reward signaling.
Literature context: molog; pAKT, phosphorylated AKT.Anti-GFPPolyclonalRabbitPurified GFP from jellyfish Aequorea Victoria1:2,000InvitrogenA6455AB_22150Anti-GFPPolyclonalChickenPurifie
Mutations in phosphatase and tensin homolog deleted on chromosome 10 (PTEN) are implicated in neuropsychiatric disorders including autism. Previous studies report that PTEN knockdown in neurons in vivo leads to increased spine density and synaptic activity. To better characterize synaptic changes in neurons lacking PTEN, we examined the effects of shRNA knockdown of PTEN in basolateral amygdala neurons on synaptic spine density and morphology by using fluorescent dye confocal imaging. Contrary to previous studies in the dentate gyrus, we find that knockdown of PTEN in basolateral amygdala leads to a significant decrease in total spine density in distal dendrites. Curiously, this decreased spine density is associated with increased miniature excitatory postsynaptic current frequency and amplitude, suggesting an increase in number and function of mature spines. These seemingly contradictory findings were reconciled by spine morphology analysis demonstrating increased mushroom spine density and size with correspondingly decreased thin protrusion density at more distal segments. The same analysis of PTEN conditional deletion in the dentate gyrus demonstrated that loss of PTEN does not significantly alter total density of dendritic protrusions in the dentate gyrus, but does decrease thin protrusion density and increases density of more mature mushroom spines. These findings suggest that, contrary to previous reports, PTEN knockdown may not induce de novo spinogenesis, but instead may increase synaptic activity by inducing morphological and functional maturation of spines. Furthermore, behavioral analysis of basolateral amygdala PTEN knockdown suggests that these changes limited only to the basolateral amygdala complex may not be sufficient to induce increased anxiety-related behaviors.
The mushroom body of the insect brain represents a neuronal circuit involved in the control of adaptive behavior, e.g., associative learning. Its function relies on the modulation of Kenyon cell activity or synaptic transmitter release by biogenic amines, e.g., octopamine, dopamine, or serotonin. Therefore, for a comprehensive understanding of the mushroom body, it is of interest not only to determine which modulatory neurons interact with Kenyon cells but also to pinpoint where exactly in the mushroom body they do so. To accomplish the latter, we made use of the GRASP technique and created transgenic Drosophila melanogaster that carry one part of a membrane-bound splitGFP in Kenyon cells, along with a cytosolic red fluorescent marker. The second part of the splitGFP is expressed in distinct neuronal populations using cell-specific Gal4 drivers. GFP is reconstituted only if these neurons interact with Kenyon cells in close proximity, which, in combination with two-photon microscopy, provides a very high spatial resolution. We characterize spatially and microstructurally distinct contact regions between Kenyon cells and dopaminergic, serotonergic, and octopaminergic/tyraminergic neurons in all subdivisions of the mushroom body. Subpopulations of dopaminergic neurons contact complementary lobe regions densely. Octopaminergic/tyraminergic neurons contact Kenyon cells sparsely and are restricted mainly to the calyx, the α'-lobes, and the γ-lobes. Contacts of Kenyon cells with serotonergic neurons are heterogeneously distributed over the entire mushroom body. In summary, the technique enables us to localize precisely a segmentation of the mushroom body by differential contacts with aminergic neurons.
Drosophila mutants black and ebony show pigmentation defects in the adult cuticle, which disclose their cooperative activity in β-alanyl-dopamine formation. In visual signal transduction, Ebony conjugates β-alanine to histamine, forming β-alanyl-histamine or carcinine. Mutation of ebony disrupts signal transduction and reveals an electroretinogram (ERG) phenotype. In contrast to the corresponding cuticle phenotype of black and ebony, there is no ERG phenotype observed when black expression is disrupted. This discrepancy calls into question the longstanding assumption of Black and Ebony interaction. The purpose of this study was to investigate the role of Black and Ebony in fly optic lobes. We excluded a presynaptic histamine uptake pathway and confirmed histamine recycling via carcinine formation in glia. β-Alanine supply for this pathway is independent of enzymatic synthesis by Black and β-alanine synthase Pyd3. Two versions of Black are expressed in vivo. Black is a specific aspartate decarboxylase with no activity on glutamate. RNA in situ hybridization and anti-Black antisera localized Black expression in the head. Immunolabeling revealed expression in lamina glia, in large medulla glia, in glia of the ocellar ganglion, and in astrocyte-like glia below the ocellar ganglion. In these glia types, Black expression is strictly accompanied by Ebony expression. Activity, localization, and strict coexpression with Ebony strongly indicate a specific mode of functional interaction that, however, evades ERG detection.
Melanocortin-4 receptor (MC4R) ligands are known to modulate nociception, but the site of action of MC4R signaling on nociception remains to be elucidated. The current study investigated MC4R expression in dorsal root ganglia (DRG) of the MC4R-GFP reporter mouse. Because MC4R is known to be expressed in vagal afferent neurons in the nodose ganglion (NG), we also systematically compared MC4R-expressing vagal and spinal afferent neurons. Abundant green fluorescent protein (GFP) immunoreactivity was found in about 45% of DRG neuronal profiles (at the mid-thoracic level), the majority being small-sized profiles. Immunohistochemistry combined with in situ hybridization confirmed that GFP was genuinely produced in MC4R-expressing neurons in the DRG. While a large number of GFP profiles in the DRG coexpressed Nav1.8 mRNA (84%) and bound isolectin B4 (72%), relatively few GFP profiles were positive for NF200 (16%) or CGRP (13%), suggesting preferential MC4R expression in C-fiber nonpeptidergic neurons. By contrast, GFP in the NG frequently colocalized with Nav1.8 mRNA (64%) and NF200 (29%), but only to a moderate extent with isolectin B4 (16%). Lastly, very few GFP profiles in the NG expressed CGRP (5%) or CART (4%). Together, our findings demonstrate variegated MC4R expression in different classes of vagal and spinal primary afferent neurons, and underscore the role of the melanocortin system in modulating nociceptive and nonnociceptive peripheral sensory modalities.
The characteristic crawling behavior of Drosophila larvae consists of a series of rhythmic waves of peristalsis and episodes of head swinging and turning. The two biogenic amines octopamine and tyramine have recently been shown to modulate various parameters of locomotion, such as muscle contraction, the time spent in pausing or forward locomotion, and the initiation and maintenance of rhythmic motor patterns. By using mutants having altered octopamine and tyramine levels and by genetic interference with both systems we confirm that signaling of these two amines is necessary for larval locomotion. We show that a small set of about 40 octopaminergic/tyraminergic neurons within the ventral nerve cord is sufficient to trigger proper larval locomotion. Using single-cell clones, we describe the morphology of these neurons individually. Given various potential roles of octopamine and tyramine in the larval brain, such as locomotion, learning and memory, stress-induced behaviors or the regulation of the energy state, functions that are often not easy to discriminate, we dissect here for the first time a subset of this complex circuit that modulates specifically larval locomotion. Thus, these data will help to understand-for a given neuronal modulator-how specific behavioral functions are executed within distinct subcircuits of a complex neuronal network.
Optical insulation plays a critical role in the fine visual acuity of the Drosophila compound eye. Screening pigments expressed by a number of cell types contribute to this phenomenon. They provide optical insulation that prevents extraneous light rays from inappropriately activating the photoreceptors. This optical insulation can be divided into two categories; the insulation of the individual ommatidia, and the insulation of the compound eye as a whole. The whole-eye insulation is provided by two sources. The sides of the eye are optically insulated by the pigment rim, a band of pigment cells that circumscribes the eye. The base of the eye is insulated by the subretinal pigment layer; a thick layer of pigment that lies directly underneath the retina. How this subretinal pigment layer is generated has not been clearly described. Here, experiments that manipulate pigment expression during eye development suggest that the subretinal pigment layer is directly derived from pigment cells in the overlying retina.
To investigate how sensory information is processed, transformed, and stored within an olfactory system, we examined the anatomy of the input region, the calyx, of the mushroom bodies of Drosophila melanogaster. These paired structures are important for various behaviors, including olfactory learning and memory. Cells in the input neuropil, the calyx, are organized into an array of microglomeruli each comprising the large synaptic bouton of a projection neuron (PN) from the antennal lobe surrounded by tiny postsynaptic neurites from intrinsic Kenyon cells. Extrinsic neurons of the mushroom body also contribute to the organization of microglomeruli. We employed a combination of genetic reporters to identify single cells in the Drosophila calyx by light microscopy and compared these with cell shapes, synapses, and circuits derived from serial-section electron microscopy. We identified three morphological types of PN boutons, unilobed, clustered, and elongated; defined three ultrastructural types, with clear- or dense-core vesicles and those with a dark cytoplasm having both; reconstructed diverse dendritic specializations of Kenyon cells; and identified Kenyon cell presynaptic sites upon extrinsic neurons. We also report new features of calyx synaptic organization, in particular extensive serial synapses that link calycal extrinsic neurons into a local network, and the numerical proportions of synaptic contacts between calycal neurons. All PN bouton types had more ribbon than nonribbon synapses, dark boutons particularly so, and ribbon synapses were larger and with more postsynaptic elements (2-14) than nonribbon (1-10). The numbers of elements were in direct proportion to presynaptic membrane area. Extrinsic neurons exclusively had ribbon synapses.
The pontine noradrenergic cell groups, A5, A6 (locus coeruleus), and A7, provide the only noradrenergic innervation of the spinal cord, but the individual contribution of each of these populations to the regional innervation of the spinal cord remains controversial. We used an adeno-associated viral (AAV) vector encoding green fluorescent protein under an artificial dopamine beta-hydroxylase (PRSx8) promoter to trace the spinal projections from the A5, A6, and A7 groups. Projections from all three groups travel through the spinal cord in both the lateral and ventral funiculi and in the dorsal surface of the dorsal horn, but A6 axons take predominantly the dorsal and ventral routes, whereas A5 axons take mainly a lateral and A7 axons a ventral route. The A6 group provides the densest innervation at all levels, and includes all parts of the spinal gray matter, but it is particularly dense in the dorsal horn. The A7 group provides the next most dense innervation, again including all parts of the spinal cord, but is it denser in the ventral horn. The A5 group supplies only sparse innervation to the dorsal and ventral horns and to the cervical and lumbosacral levels, but provides the densest innervation to the thoracic intermediolateral cell column, and in particular to the sympathetic preganglionic neurons. Thus, the pontine noradrenergic cell groups project in a roughly topographic and complementary fashion onto the spinal cord. The pattern of spinal projections observed suggests that the locus coeruleus might have the greatest effect on somatosensory transmission, the A7 group on motor function, and the A5 group on sympathetic function.
Motoneurons are key points of convergence within motor networks, acting as the "output channels" that directly control sets of muscles to maintain posture and generate movement. Here we use genetic mosaic techniques to reveal the origins and architecture of the leg motoneurons of Drosophila. We show that a small number of leg motoneurons are born in the embryo but most are generated during larval life. These postembryonic leg motoneurons are produced by five neuroblasts per hemineuromere, and each lineage generates stereotyped lineage-specific projection patterns. Two of these postembryonic neuroblasts generate solely motoneurons that are the bulk of the leg motoneurons. Within the largest lineage, lineage 15, we see distinct birth-order differences in projection patterns. A comparison of the central projections of leg motoneurons and the muscles they innervate reveals a stereotyped architecture and the existence of a myotopic map. Timeline analysis of axonal outgrowth reveals that leg motoneurons reach their sites of terminal arborization in the leg at the time when their dendrites are elaborating their subtype-specific shapes. Our findings provide a comprehensive description of the origin, development, and architecture of leg motoneurons that will aid future studies exploring the link between the assembly and organization of connectivity within the leg motor system of Drosophila.
The hormones leptin and ghrelin act in apposition to one another in the regulation of body weight homeostasis. Interestingly, both leptin receptor expression and ghrelin receptor expression have been observed within many of the same nuclei of the central nervous system (CNS), suggesting that these hormones may act on a common population of neurons to produce changes in food intake and energy expenditure. In the present study we explored the extent of this putative direct leptin and ghrelin interaction in the CNS and addressed the question of whether a loss of ghrelin signaling would affect sensitivity to leptin. Using histological mapping of leptin receptor and ghrelin receptor expression, we found that cells containing both leptin receptors and ghrelin receptors are mainly located in the medial part of the hypothalamic arcuate nucleus. In contrast, coexpression was much less extensive elsewhere in the brain. To assess the functional consequences of this observed receptor distribution, we explored the effect of ghrelin receptor deletion on leptin sensitivity. In particular, the responses of ad libitum-fed, diet-induced obese and fasted mice to the anorectic actions of leptin were examined. Surprisingly, we found that deletion of the ghrelin receptor did not affect the sensitivity to exogenously administrated leptin. Thus, we conclude that ghrelin and leptin act largely on distinct neuronal populations and that ghrelin receptor deficiency does not affect sensitivity to the anorexigenic and body weight-lowering actions of leptin.
Neurog1 (Ngn1, Neurod3, neurogenin1) is a basic helix-loop-helix (bHLH) transcription factor essential for neuronal differentiation and subtype specification during embryogenesis. Due to the transient expression of Neurog1 and extensive migration of neuronal precursors, it has been challenging to understand the full complement of Neurog1 lineage cells throughout the central nervous system (CNS). Here we labeled and followed Neurog1 lineages using inducible Cre-flox recombination systems with Neurog1-Cre and Neurog1-CreER(T2) BAC (bacterial artificial chromosome) transgenic mice. Neurog1 lineage cells are restricted to neuronal fates and contribute to diverse but discrete populations in each brain region. In the forebrain, Neurog1 lineages include mitral cells and glutamatergic interneurons in the olfactory bulb, pyramidal and granule neurons in the hippocampus, and pyramidal cells in the cortex. In addition, most of the thalamus, but not the hypothalamus, arises from Neurog1 progenitors. Although Neurog1 lineages are largely restricted to glutamatergic neurons, there are multiple exceptions including Purkinje cells and other GABAergic neurons in the cerebellum. This study provides the first overview of the spatiotemporal fate map of Neurog1 lineages in the CNS.
In the mouse retina, connexin45 (Cx45) participates in the gap junction between ON cone bipolar cells and AII amacrine cells, which constitutes an essential element of the primary rod pathway. Although it has been shown that Cx45 is also expressed in OFF bipolar cells, its subcellular localization and functional role in these cells are unknown. Here, we analyzed the localization of Cx45 on OFF bipolar cells in the mouse retina. For this, we used wild-type mice and a transgenic mouse line that expressed, in addition to native Cx45, a fusion protein consisting of Cx45 and the enhanced green fluorescent protein (EGFP). Cx45-EGFP expression generates an EGFP signal at gap junctions containing Cx45. Combining immunohistochemistry with intracellular injections, we found that Cx45 was present on dendrites and axon terminals of all OFF bipolar cell types. Cx45 was not found at intersections of two terminal processes of the same type, suggesting that Cx45 might not form gap junctions between axon terminals of the same OFF bipolar cell type but rather might connect OFF bipolar cells to amacrine or ganglion cells. In OFF bipolar cell dendrites, Cx45 was found predominantly in the proximal outer plexiform layer (OPL), well below the cone pedicles. Cx45 did not colocalize with Cx36, which is found predominantly in the distal OPL. We conclude that Cx45 is expressed on OFF bipolar cell dendrites, presumably forming gap junctions with cells of the same type, and on OFF bipolar cell axon terminals, presumably forming heterologous gap junctions with other retinal neurons.
The tuba1a gene encodes a neural-specific α-tubulin isoform whose expression is restricted to the developing and regenerating nervous system. By using zebrafish as a model system for studying CNS regeneration, we recently showed that retinal injury induces tuba1a gene expression in Müller glia that reentered the cell cycle. However, because of the transient nature of tuba1a gene expression during development and regeneration, it was not possible to trace the lineage of the tuba1a-expressing cells with a reporter directly under the control of the tuba1a promoter. To overcome this limitation, we generated tuba1a:CreER(T2) and β-actin2:loxP-mCherrry-loxP-GFP double transgenic fish that allowed us to label tuba1a-expressing cells conditionally and permanently via ligand-induced recombination. During development, recombination revealed transient tuba1a expression in not only neural progenitors but also cells that contribute to skeletal muscle, heart, and intestine. In the adult, recombination revealed tuba1a expression in brain, olfactory neurons, and sensory cells of the lateral line, but not in the retina. After retinal injury, recombination showed tuba1a expression in Müller glia that had reentered the cell cycle, and lineage tracing indicated that these cells are responsible for regenerating retinal neurons and glia. These results suggest that tuba1a-expressing progenitors contribute to multiple cell lineages during development and that tuba1a-expressing Müller glia are retinal progenitors in the adult.
Previous studies showed that Tctex-1 immunoreactivity is selectively enriched in the germinal zones of adult brain. In this report we identify a regulatory region of the Tctex-1 gene that is capable of directing transgenic expression of green fluorescent protein (GFP) reporter that recapitulates the spatial and temporal expression pattern of endogenous Tctex-1. This construct specifically targeted expression to the nestin(+)/Pax6(+)/GLAST(+) radial glial cells and Tbr2(+) intermediate progenitors when the reporter construct was delivered to developing mouse neocortex via in utero electroporation. Characterization of mice transgenically expressing GFP under the same regulatory element showed that the GFP expression is faithful to endogenous Tctex-1 at the subgranular zone (SGZ) of dentate gyrus, ventricular/subventricular zone of lateral ventricles, and ependymal layer of 3rd ventricle of adult brains. Immunolocalization and bromodeoxyuridine incorporation studies of adult SGZ in four independent mouse lines showed that Tctex-1:GFP reporter selectively marks nestin(+)/GFAP(+)/Sox2(+) neural stem-like cells in two mouse lines (4 and 13). In two other mouse lines (17 and 18), Tctex-1:GFP is selectively expressed in Type-2 and Type-3 transient amplifying progenitors and a small subset of young neuronal progeny. The P/E-Tctex-1 reporter mouse studies independently confirmed the specific enrichment of Tctex-1 at adult SGZ stem/progenitor cells. Furthermore, these studies supported the notion that an analogous transcriptional program may be used to regulate neurogenesis in embryonic cerebral cortex and adult hippocampus. Finally, the genomic sequences and the reporter mouse lines described here provide useful experimental tools to advance adult neural stem cell research.
The fruitfly, Drosophila, is dependent on its olfactory sense in food search and reproduction. Processing of odorant information takes place in the antennal lobes, the primary olfactory center in the insect brain. Besides classical neurotransmitters, earlier studies have indicated the presence of a few neuropeptides in the olfactory system. In the present study we made an extensive analysis of the expression of neuropeptides in the Drosophila antennal lobes by direct profiling using matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry and immunocytochemistry. Neuropeptides from seven different precursor genes were unambiguously identified and their localization in neurons was subsequently revealed by immunocytochemistry. These were short neuropeptide F, tachykinin related peptide, allatostatin A, myoinhibitory peptide, SIFamide, IPNamide, and myosuppressin. The neuropeptides were expressed in subsets of olfactory sensory cells and different populations of local interneurons and extrinsic (centrifugal) neurons. In some neuron types neuropeptides were colocalized with classical neurotransmitters. Our findings suggest a huge complexity in peptidergic signaling in different circuits of the antennal lobe.
Spinal cholinergic neurons are critical for motor function in both the autonomic and somatic nervous systems and are affected in spinal cord injury and in diseases such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy. Using two screening approaches and in situ hybridization, we identified 159 genes expressed in typical cholinergic patterns in the spinal cord. These include two general cholinergic neuron markers, one gene exclusively expressed in motor neurons, and nine genes expressed in unknown subtypes of somatic motor neurons. Further, we present evidence that chondrolectin (Chodl) is expressed by fast motor neurons and that estrogen-related receptor beta (ERRbeta) is a candidate marker for slow motor neurons. In addition, we suggest paired-like homeodomain transcription factor 2 (Pitx2) as a marker for cholinergic partition cells.
Individual median octopamine neurons in the insect central nervous system serve as an excellent model system for comparative neuroanatomy of single identified cells. The median octopamine cluster of the subesophageal ganglion consists of defined sets of paired and unpaired interneurons, which supply the brain and subesophageal ganglion with extensive ramifications. The developmental program underlying the complex cellular network is unknown. Here we map the segmental location and developmental origins of individual octopamine neurons in the Drosophila subesophageal ganglion. We demonstrate that two sets of unpaired median neurons, located in the mandibular and maxillary segments, exhibit the same projection patterns in the brain. Furthermore, we show that the paired and unpaired neurons belong to distinct lineages. Interspecies comparison of median neurons revealed that many individual octopamine neurons in different species project to equivalent target regions. Such identified neurons with similar morphology can derive from distinct lineages in different species (i.e., paired and unpaired neurons).
The suprachiasmatic nucleus (SCN) of the hypothalamus regulates daily rhythms in physiology and behavior. It is composed of a heterogeneous population of cells that together form the circuits underlying its master clock function. Numerous studies suggest the existence of two regions that have been termed core and shell. At a gross level, differences between these regions map to distinct functional differences, although the specific role(s) of various peptidergic cellular phenotypes remains unknown. In mouse, gastrin-releasing peptide (GRP) cells lie in the core, are directly retinorecipient, and lack detectable rhythmicity in clock gene expression, raising interest in their role in the SCN. Here, we provide evidence that calbindin-expressing cells of perinatal mouse SCN express GRP, identified by a green fluorescent protein (GFP+), but lack detectable calbindin later in development. To explore the intra-SCN network in which GRP neurons participate, individual GFP+ cells were filled with tracer and their morphological characteristics, processes, and connections, as well as those of their non-GFP-containing immediate neighbors, were compared. The results show that GFP+ neurons form a dense network of local circuits within the core, revealed by appositions on other GFP+ cells and by the presence of dye-coupled cells. Dendrites and axons of GFP+ cells make appositions on arginine vasopressin neurons, whereas non-GFP cells have a less extensive fiber network, largely confined to the region of GFP+ cells. The results point to specialized circuitry within the SCN, presumably supporting synchronization of neural activity and reciprocal communication between core and shell regions.
The biogenic amine octopamine modulates diverse behaviors in invertebrates. At the single neuron level, the mode of action is well understood in the peripheral nervous system owing to its simple structure and accessibility. For elucidating the role of individual octopaminergic neurons in the modulation of complex behaviors, a detailed analysis of the connectivity in the central nervous system is required. Here we present a comprehensive anatomical map of candidate octopaminergic neurons in the adult Drosophila brain: including the supra- and subesophageal ganglia. Application of the Flp-out technique enabled visualization of 27 types of individual octopaminergic neurons. Based on their morphology and distribution of genetic markers, we found that most octopaminergic neurons project to multiple brain structures with a clear separation of dendritic and presynaptic regions. Whereas their major dendrites are confined to specific brain regions, each cell type targets different, yet defined, neuropils distributed throughout the central nervous system. This would allow them to constitute combinatorial modules assigned to the modulation of distinct neuronal processes. The map may provide an anatomical framework for the functional constitution of the octopaminergic system. It also serves as a model for the single-cell organization of a particular neurotransmitter in the brain.
The mushroom body (MB) of the insect brain has important roles in odor learning and memory and in diverse other brain functions. To elucidate the anatomical basis underlying its function, we studied how the MB of Drosophila is organized by its intrinsic and extrinsic neurons. We screened for the GAL4 enhancer-trap strains that label specific subsets of these neurons and identified seven subtypes of Kenyon cells and three other intrinsic neuron types. Laminar organization of the Kenyon cell axons divides the pedunculus into at least five concentric strata. The alpha', beta', alpha, and beta lobes are each divided into three strata, whereas the gamma lobe appears more homogeneous. The outermost stratum of the alpha/beta lobes is specifically connected with a small, protruded subregion of the calyx, the accessory calyx, which does not receive direct olfactory input. As for the MB extrinsic neurons (MBENs), we found three types of antennal lobe projection neurons, among which two are novel. In addition, we resolved 17 other types of MBENs that arborize in the calyx, lobes, and pedunculus. Lobe-associated MBENs arborize in only specific areas of the lobes, being restricted along their longitudinal axes, forming two to five segmented zones in each lobe. The laminar arrangement of the Kenyon cell axons and segmented organization of the MBENs together divide the lobes into smaller synaptic units, possibly facilitating characteristic interaction between intrinsic and extrinsic neurons in each unit for different functional activities along the longitudinal lobe axes and between lobes. Structural differences between lobes are also discussed.
Members of the Drosophila gustatory receptor (Gr) gene family are generally expressed in chemosensory neurons and are known to mediate the perception of sugars, bitter substrates, CO(2), and pheromones. The Gr gene family consists of 68 members, many of which are organized in gene clusters of up to six genes, yet only expression of about 15 Gr genes has been characterized in detail prior to this study. Here we describe the first comprehensive expression analysis of six highly conserved Gr genes, Gr28a and Gr28b.a to Gr28b.e. Four of these Gr genes are not only expressed in the characteristic pattern associated with previously analyzed Gr genes-chemosensory neurons of the gustatory and olfactory system-but several other types of sensory neurons and neurons in the brain. Specifically, we show that several of the Gr28 genes are expressed in abdominal multidendritic neurons, putative hygroreceptive neurons of the arista, neurons associated with the Johnston's organ, peripheral proprioceptive neurons in the legs, neurons in the larval and adult brain, and oenocytes. Thus, our findings suggest that some Gr genes are utilized in nongustatory roles in the nervous system and tissues involved in proprioception, hygroreception, and other sensory modalities. It is also possible that the Gr28 genes have chemosensory roles in the detection of internal ligands.
The neurotransmitter acetylcholine (ACh) is specifically synthesized by the enzyme choline acetyltransferase (ChAT). Subsequently, it is loaded into synaptic vesicles by a specific vesicular acetylcholine transporter (VAChT). We have generated antibodies that recognize ChAT or VAChT in a model organism, the nematode Caenorhabditis elegans, in order to examine the subcellular and cellular distributions of these cholinergic proteins. ChAT and VAChT are found in the same neurons, including more than one-third of the 302 total neurons present in the adult hermaphrodite. VAChT is found in synaptic regions, whereas ChAT appears to exist in two forms in neurons, a synapse-enriched form and a more evenly distributed possibly cytosolic form. We have used antibodies to identify the cholinergic neurons in the body of larval and adult hermaphrodites. All of the classes of putative excitatory motor neurons in the ventral nerve cord appear to be cholinergic: the DA and DB neurons in the first larval stage and the AS, DA, DB, VA, VB, and VC neurons in the adult. In addition, several interneurons with somas in the tail and processes in the tail or body are cholinergic; sensory neurons are generally not cholinergic. Description of the normal pattern of cholinergic proteins and neurons will improve our understanding of the role of cholinergic neurons in the behavior and development of this model organism.
A simple nervous system combined with stereotypic behavioral responses to tastants, together with powerful genetic and molecular tools, have turned Drosophila larvae into a very promising model for studying gustatory coding. Using the Gal4/UAS system and confocal microscopy for visualizing gustatory afferents, we provide a description of the primary taste center in the larval central nervous system. Essentially, gustatory receptor neurons target different areas of the subesophageal ganglion (SOG), depending on their segmental and sensory organ origin. We define two major and two smaller subregions in the SOG. One of the major areas is a target of pharyngeal sensilla, the other one receives inputs from both internal and external sensilla. In addition to such spatial organization of the taste center, circumstantial evidence suggests a subtle functional organization: aversive and attractive stimuli might be processed in the anterior and posterior part of the SOG, respectively. Our results also suggest less coexpression of gustatory receptors than proposed in prior studies. Finally, projections of putative second-order taste neurons seem to cover large areas of the SOG. These neurons may thus receive multiple gustatory inputs. This suggests broad sensitivity of secondary taste neurons, reminiscent of the situation in mammals.
The hugin gene of Drosophila encodes a neuropeptide with homology to mammalian neuromedin U. The hugin-expressing neurons are localized exclusively to the subesophageal ganglion of the central nervous system and modulate feeding behavior in response to nutrient signals. These neurons send neurites to the protocerebrum, the ventral nerve cord, the ring gland, and the pharynx and may interact with the gustatory sense organs. In this study, we have investigated the morphology of the hugin neurons at a single-cell level by using clonal analysis. We show that single cells project to only one of the four major targets. In addition, the neurites of the different hugin cells overlap in a specific brain region lateral to the foramen of the esophagus, which could be a new site of neuropeptide release for feeding regulation. Our study reveals novel complexity in the morphology of individual hugin neurons, which has functional implication for how they coordinate feeding behavior and growth.
We have studied the spatial and temporal distribution of six proinflammatory cytokines and identified their cellular source in a clinically relevant model of spinal cord injury (SCI). Our findings show that interleukin-1beta (IL-1beta) and tumor necrosis factor (TNF) are rapidly (<5 and 15 minutes, respectively) and transiently expressed in mice following contusion. At 30-45 minutes post SCI, IL-1beta and TNF-positive cells could already be seen over the entire spinal cord segment analyzed. Multilabeling analyses revealed that microglia and astrocytes were the two major sources of IL-1beta and TNF at these times, suggesting a role for these cytokines in gliosis. Results obtained from SCI mice previously transplanted with green fluorescent protein (GFP)-expressing hematopoietic stem cells confirmed that neural cells were responsible for the production of IL-1beta and TNF for time points preceding 3 hours. From 3 hours up to 24 hours, IL-1beta, TNF, IL-6, and leukemia inhibitory factor (LIF) were strongly upregulated within and immediately around the contused area. Colocalization studies revealed that all populations of central nervous system resident cells, including neurons, synthesized cytokines between 3 and 24 hours post SCI. However, work done with SCI-GFP chimeric mice revealed that at least some infiltrating leukocytes were responsible for cytokine production from 12 hours on. By 2 days post-SCI, mRNA signal for all the above cytokines had nearly disappeared. Notably, we also observed another wave of expression for IL-1beta and TNF at 14 days. Overall, these results indicate that following SCI, all classes of neural cells initially contribute to the organization of inflammation, whereas recruited immune cells mostly contribute to its maintenance at later time points.
The adult mammalian spinal cord contains neural stem and/or progenitor cells that slowly multiply throughout life and differentiate exclusively into glia. The contribution of adult progenitors to repair has been highlighted in recent studies, demonstrating extensive cell proliferation and gliogenesis following central nervous system (CNS) trauma. The present experiments aimed to determine the relative roles of endogenously dividing progenitor cells versus quiescent progenitor cells in posttraumatic gliogenesis. Using the mitotic indicator bromodeoxyuridine (BrdU) and a retroviral vector, we found that, in the adult female Fisher 344 rat, endogenously dividing neural progenitors are acutely vulnerable in response to T8 dorsal hemisection spinal cord injury. We then studied the population of cells that divide postinjury in the injury epicenter by delivering BrdU or retrovirus at 24 hours after spinal cord injury. Animals were euthanized at five timepoints postinjury, ranging from 6 hours to 9 weeks after BrdU delivery. At all timepoints, we observed extensive proliferation of ependymal and periependymal cells that immunohistochemically resembled stem/progenitor cells. BrdU+ incorporation was noted to be prominent in NG2-immunoreactive progenitors that matured into oligodendrocytes, and in a transient population of microglia. Using a green fluorescence protein (GFP) hematopoietic chimeric mouse, we determined that 90% of the dividing cells in this early proliferation event originate from the spinal cord, whereas only 10% originate from the bone marrow. Our results suggest that dividing, NG2-expressing progenitor cells are vulnerable to injury, but a separate, immature population of neural stem and/or progenitor cells is activated by injury and rapidly divides to replace this vulnerable population.
Recent evidence indicates that the vomeronasal organ (VNO) of mice not only responds to pheromones but also to odorants. To analyze whether genes encoding odorant receptors (ORs) are expressed in the VNO, reverse transcriptase-polymerase chain reaction analyses were performed. These led to the identification of 44 different OR genes, comprising class-I and class-II receptors. The genes encoding these receptors were scattered over several gene clusters. The respective OR genes were concomitantly expressed in cells of the main olfactory epithelium (MOE). Although the cells in the MOE were zonally distributed, no such patterns were displayed in the VNO. Cells expressing ORs in the VNO were positive for the TRP2-channel and Galphai, a marker for vomeronasal neurons of the apical layer. In transgenic mice, which coexpress histological markers with the receptor mOR18-2, characteristic morphological differences between cells expressing this receptor in the VNO compared with the MOE became evident. Visualizing the axonal processes of VNO cells expressing distinct ORs revealed that they project to the accessory olfactory bulb (AOB). Axon fibers were visible exclusively in the anterior subdomain; here, they converged into glomerular-like structures positioned at the very rostral tip of the AOB. The findings that a set of ORs is expressed in cells located in the apical layer of the VNO with typical features of VNO sensory neurons that project their axons to the anterior part of the AOB suggest that this population of sensory cells may be considered as a unique facet of the complex chemosensory system.
The GAL4/UAS gene expression system in Drosophila has been crucial in revealing the behavioral significance of neural circuits. Transgene products that block neurotransmitter release and induce cell death have been proved to inhibit neural function powerfully. Here we compare the action of the five effector genes shibire(ts1), Tetanus toxin light chain (TNT), reaper, Diphtheria toxin A-chain (DTA), and inwardly rectifying potassium channel (Kir2.1) and show differences in their efficiency depending on the target cells and the timing of induction. Specifically, effectors blocking neuronal transmission or excitability led to adult-induced paralysis more efficiently than those causing cell ablation. We contrasted these differential potencies in adult to their actions during development. Furthermore, we induced TNT expression in the adult mushroom bodies. In contrast to the successful impairment in short-term olfactory memory by shibire(ts1), adult TNT expression in the same set of cells did not lead to any obvious impairment. Altogether, the efficiency of effector genes depends on properties of the targeted neurons. Thus, we conclude that the selection of the appropriate effector gene is critical for evaluating the function of neural circuits.