In the presynaptic terminal, the magnitude and location of Ca2+ entry through voltage-gated Ca2+ channels (VGCCs) regulate the efficacy of neurotransmitter release. However, how presynaptic active zone proteins control mammalian VGCC levels and organization is unclear. To address this, we deleted the CAST/ELKS protein family at the calyx of Held, a CaV2.1 channel-exclusive presynaptic terminal. We found that loss of CAST/ELKS reduces the CaV2.1 current density with concomitant reductions in CaV2.1 channel numbers and clusters. Surprisingly, deletion of CAST/ELKS increases release probability while decreasing the readily releasable pool, with no change in active zone ultrastructure. In addition, Ca2+ channel coupling is unchanged, but spontaneous release rates are elevated. Thus, our data identify distinct roles for CAST/ELKS as positive regulators of CaV2.1 channel density and suggest that they regulate release probability through a post-priming step that controls synaptic vesicle fusogenicity.
Literature context: ackson ImmunoResearch706-605-148AB_23404761: 2,000AffiniPure Donkey anti-S
Virtually all rodent neuroendocrine corticotropin-releasing-hormone (CRH) neurons are in the dorsal medial parvicellular (mpd) part of the paraventricular nucleus of the hypothalamus (PVH). They form the final common pathway for adrenocortical stress responses. Their activity is controlled by sets of GABA-, glutamate-, and catecholamine-containing inputs arranged in an interactive pre-motor network. Defining the nature and arrangement of these inputs can help clarify how stressor type and intensity information is conveyed to neuroendocrine neurons. Here we use immunohistochemistry with high-resolution 3-dimensional image analyses to examine the arrangement of single- and co-occurring GABA, glutamate, and catecholamine markers in synaptophysin-defined pre-synaptic terminals in the PVHmpd of unstressed rats and Crh-IRES-Cre;Ai14 transgenic mice: respectively, vesicular glutamate transporter 2 (VGluT2), vesicular GABA transporter (VGAT), dopamine β-hydroxylase (DBH), and phenylethanolamine n-methyltransferase (PNMT). Just over half of all PVHmpd pre-synaptic terminals contain VGAT, with slightly less containing VGluT2. The vast majority of terminal appositions with mouse CRH neurons occur non-somatically. However, there are significantly more somatic VGAT than VGluT2 appositions. In the rat PVHmpd, about five times as many pre-synaptic terminals contain PNMT than DBH only. However, because epinephrine release has never been detected in the PVH, PNMT terminals may functionally be noradrenergic not adrenergic. PNMT and VGluT2 co-occur in some pre-synaptic terminals indicating the potential for co-transmission of glutamate and norepinephrine. Collectively, these results provide a structural basis for how GABA/glutamate/catecholamine interactions enable adrenocortical responses to fast-onset interosensory stimuli, and more broadly, how combinations of PVH neurotransmitters and neuromodulators interact dynamically to control adrenocortical activity.
Literature context: mmunoResearch Cat #706-605-148; RRID:AB_2340476 Alexa 647, goat Invitrogen Cat
Pyramidal neurons express rich repertoires of leucine-rich repeat (LRR)-containing adhesion molecules with similar synaptogenic activity in culture. The in vivo relevance of this molecular diversity is unclear. We show that hippocampal CA1 pyramidal neurons express multiple synaptogenic LRR proteins that differentially distribute to the major excitatory inputs on their apical dendrites. At Schaffer collateral (SC) inputs, FLRT2, LRRTM1, and Slitrk1 are postsynaptically localized and differentially regulate synaptic structure and function. FLRT2 controls spine density, whereas LRRTM1 and Slitrk1 exert opposing effects on synaptic vesicle distribution at the active zone. All LRR proteins differentially affect synaptic transmission, and their combinatorial loss results in a cumulative phenotype. At temporoammonic (TA) inputs, LRRTM1 is absent; FLRT2 similarly controls functional synapse number, whereas Slitrk1 function diverges to regulate postsynaptic AMPA receptor density. Thus, LRR proteins differentially control synaptic architecture and function and act in input-specific combinations and a context-dependent manner to specify synaptic properties.
Literature context: mmunoresearch CAT# 706-605-148; RRID:AB_2340476 Alexa FluorÂ® 488 AffiniPure Don
Islet β cells from newborn mammals exhibit high basal insulin secretion and poor glucose-stimulated insulin secretion (GSIS). Here we show that β cells of newborns secrete more insulin than adults in response to similar intracellular Ca2+ concentrations, suggesting differences in the Ca2+ sensitivity of insulin secretion. Synaptotagmin 4 (Syt4), a non-Ca2+ binding paralog of the β cell Ca2+ sensor Syt7, increased by ∼8-fold during β cell maturation. Syt4 ablation increased basal insulin secretion and compromised GSIS. Precocious Syt4 expression repressed basal insulin secretion but also impaired islet morphogenesis and GSIS. Syt4 was localized on insulin granules and Syt4 levels inversely related to the number of readily releasable vesicles. Thus, transcriptional regulation of Syt4 affects insulin secretion; Syt4 expression is regulated in part by Myt transcription factors, which repress Syt4 transcription. Finally, human SYT4 regulated GSIS in EndoC-βH1 cells, a human β cell line. These findings reveal the role that altered Ca2+ sensing plays in regulating β cell maturation.
Literature context: mmunoResearch Cat# 706-605-148, RRID:AB_2340476 Goat anti-Guinea Pig IgG (H+L)
Delamination of neural progenitor cells (NPCs) from the ventricular surface is a crucial prerequisite to form the subventricular zone, the germinal layer linked to the expansion of the mammalian neocortex in development and evolution. Here, we dissect the molecular mechanism by which the transcription factor Insm1 promotes the generation of basal progenitors (BPs). Insm1 protein is most highly expressed in newborn BPs in mouse and human developing neocortex. Forced Insm1 expression in embryonic mouse neocortex causes NPC delamination, converting apical to basal radial glia. Insm1 represses the expression of the apical adherens junction belt-specific protein Plekha7. CRISPR/Cas9-mediated disruption of Plekha7 expression suffices to cause NPC delamination. Plekha7 overexpression impedes the intrinsic and counteracts the Insm1-induced, NPC delamination. Our findings uncover a novel molecular mechanism underlying NPC delamination in which a BP-genic transcription factor specifically targets the integrity of the apical adherens junction belt, rather than adherens junction components as such.
Literature context: oResearch Labs Cat# 706-605-148 RRID:AB_2340476 2.3 Antibody characterization
Type II spiral ganglion neurons (SGNs) are small caliber, unmyelinated afferents that extend dendritic arbors hundreds of microns along the cochlear spiral, contacting many outer hair cells (OHCs). Despite these many contacts, type II afferents are insensitive to sound and only weakly depolarized by glutamate release from OHCs. Recent studies suggest that type II afferents may be cochlear nociceptors, and can be excited by ATP released during tissue damage, by analogy to somatic pain-sensing C-fibers. The present work compares the expression patterns among cochlear type II afferents of two genes found in C-fibers: calcitonin-related polypeptide alpha (Calca/Cgrpα), specific to pain-sensing C-fibers, and tyrosine hydroxylase (Th), specific to low-threshold mechanoreceptive C-fibers, which was shown previously to be a selective biomarker of type II versus type I cochlear afferents (Vyas et al., ). Whole-mount cochlear preparations from 3-week- to 2-month-old CGRPα-EGFP (GENSAT) mice showed expression of Cgrpα in a subset of SGNs with type II-like peripheral dendrites extending beneath OHCs. Double labeling with other molecular markers confirmed that the labeled SGNs were neither type I SGNs nor olivocochlear efferents. Cgrpα starts to express in type II SGNs before hearing onset, but the expression level declines in the adult. The expression patterns of Cgrpα and Th formed opposing gradients, with Th being preferentially expressed in apical and Cgrpα in basal type II afferent neurons, indicating heterogeneity among type II afferent neurons. The expression of Th and Cgrpα was not mutually exclusive and co-expression could be observed, most abundantly in the middle cochlear turn.
Literature context: mmunoResearch; Cat# 706-605-148 RRID:AB_2340476; dilution 1:200) overnight.
Parvalbumin (PV)-positive interneurons form dendritic gap junctions with one another, but the connectivity among gap junction-coupled dendrites remains uninvestigated in most neocortical areas. We visualized gap junctions in layer 4 of the mouse barrel cortex and examined their structural details. PV neurons were divided into 4 types based on the location of soma and dendrites within or outside barrels. Type 1 neurons that had soma and all dendrites inside a barrel, considered most specific to single vibrissa-derived signals, unexpectedly formed gap junctions only with other types but never with each other. Type 2 neurons inside a barrel elongated dendrites outward, forming gap junctions within a column that contained the home barrel. Type 3 neurons located outside barrels established connections with all types including Type 4 neurons that were confined inside the inter-barrel septa. The majority (33/38, 86.8%) of dendritic gap junctions were within 75 μm from at least 1 of 2 paired somata. All types received vesicular glutamate transporter 2-positive axon terminals preferentially on somata and proximal dendrites, indicating the involvement of all types in thalamocortical feedforward regulation in which proximal gap junctions may also participate. These structural organizations provide a new morphological basis for regulatory mechanisms in barrel cortex.
Literature context: Jackson Immuno Cat#706-605-148; RRID:AB_2340476 Bacterial and Virus Strains
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: a pigJackson ImmunoResearchCat #706-605-148dilution (1:500)AntibodyCy5-conj
Oligodendrocytes (OLs), the myelin-forming CNS glia, are highly vulnerable to cellular stresses, and a severe myelin loss underlies numerous CNS disorders. Expedited OL regeneration may prevent further axonal damage and facilitate functional CNS repair. Although adult OL progenitors (OPCs) are the primary players for OL regeneration, targetable OPC-specific intracellular signaling mechanisms for facilitated OL regeneration remain elusive. Here, we report that OPC-targeted PTEN inactivation in the mouse, in contrast to OL-specific manipulations, markedly promotes OL differentiation and regeneration in the mature CNS. Unexpectedly, an additional deletion of mTOR did not reverse the enhanced OL development from PTEN-deficient OPCs. Instead, ablation of GSK3β, another downstream signaling molecule that is negatively regulated by PTEN-Akt, enhanced OL development. Our results suggest that PTEN persistently suppresses OL development in an mTOR-independent manner, and at least in part, via controlling GSK3β activity. OPC-targeted PTEN-GSK3β inactivation may benefit facilitated OL regeneration and myelin repair.
Literature context: h Laboratories Cat#706-605-148; RRID:AB_2340476 Peroxidase AffiniPure Donkey An
To uncover regulatory mechanisms in Hedgehog (Hh) signaling, we conducted genome-wide screens to identify positive and negative pathway components and validated top hits using multiple signaling and differentiation assays in two different cell types. Most positive regulators identified in our screens, including Rab34, Pdcl, and Tubd1, were involved in ciliary functions, confirming the central role for primary cilia in Hh signaling. Negative regulators identified included Megf8, Mgrn1, and an unannotated gene encoding a tetraspan protein we named Atthog. The function of these negative regulators converged on Smoothened (SMO), an oncoprotein that transduces the Hh signal across the membrane. In the absence of Atthog, SMO was stabilized at the cell surface and concentrated in the ciliary membrane, boosting cell sensitivity to the ligand Sonic Hedgehog (SHH) and consequently altering SHH-guided neural cell-fate decisions. Thus, we uncovered genes that modify the interpretation of morphogen signals by regulating protein-trafficking events in target cells.
Literature context: Life Technologies,706-605-148; RRID:AB_2340476). Amyloid fibrillary deposits w
Pathological hallmarks of Alzheimer's disease (AD) include amyloid-β (Aβ) plaques, neurofibrillary tangles, and reactive gliosis. Glial cells offer protection against AD by engulfing extracellular Aβ peptides, but the repertoire of molecules required for glial recognition and destruction of Aβ are still unclear. Here, we show that the highly conserved glial engulfment receptor Draper/MEGF10 provides neuroprotection in an AD model of Drosophila (both sexes). Neuronal expression of human Aβ42arc in adult flies results in robust Aβ accumulation, neurodegeneration, locomotor dysfunction, and reduced lifespan. Notably, all of these phenotypes are more severe in draper mutant animals, whereas enhanced expression of glial Draper reverses Aβ accumulation, as well as behavioral phenotypes. We also show that the signal transducer and activator of transcription (Stat92E), c-Jun N-terminal kinase (JNK)/AP-1 signaling, and expression of matrix metalloproteinase-1 (Mmp1) are activated downstream of Draper in glia in response to Aβ42arc exposure. Furthermore, Aβ42-induced upregulation of the phagolysosomal markers Atg8 and p62 was notably reduced in draper mutant flies. Based on our findings, we propose that glia clear neurotoxic Aβ peptides in the AD model Drosophila brain through a Draper/STAT92E/JNK cascade that may be coupled to protein degradation pathways such as autophagy or more traditional phagolysosomal destruction methods.SIGNIFICANCE STATEMENT Alzheimer's disease (AD) and similar dementias are common incurable neurodegenerative disorders in the aging population. As the primary immune responders in the brain, glial cells are implicated as key players in the onset and progression of AD and related disorders. Here we show that the glial engulfment receptor Draper is protective in a Drosophila model of AD, reducing levels of amyloid β (Aβ) peptides, reversing locomotor defects, and extending lifespan. We further show that protein degradation pathways are induced downstream of Draper in AD model flies, supporting a model in which glia engulf and destroy Aβ peptides to reduce amyloid-associated toxicity.
Literature context: nd 706-605-148 RRID:AB_2340476) were used at a dilution of 1:4
Neural injury triggers swift responses from glia, including glial migration and phagocytic clearance of damaged neurons. The transcriptional programs governing these complex innate glial immune responses are still unclear. Here, we describe a novel injury assay in adult Drosophila that elicits widespread glial responses in the ventral nerve cord (VNC). We profiled injury-induced changes in VNC gene expression by RNA sequencing (RNA-seq) and found that responsive genes fall into diverse signaling classes. One factor, matrix metalloproteinase-1 (MMP-1), is induced in Drosophila ensheathing glia responding to severed axons. Interestingly, glial induction of MMP-1 requires the highly conserved engulfment receptor Draper, as well as AP-1 and STAT92E. In MMP-1 depleted flies, glia do not properly infiltrate neuropil regions after axotomy and, as a consequence, fail to clear degenerating axonal debris. This work identifies Draper-dependent activation of MMP-1 as a novel cascade required for proper glial clearance of severed axons.
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: 48, RRID:AB_2340476; 712-165-1
Neuronal stem cell lineages are the fundamental developmental units of the brain, and neuronal circuits are the fundamental functional units of the brain. Determining lineage-circuitry relationships is essential for deciphering the developmental logic of circuit assembly. While the spatial distribution of lineage-related neurons has been investigated in a few brain regions [1-9], an important, but unaddressed question is whether temporal information that diversifies neuronal progeny within a single lineage also impacts circuit assembly. Circuits in the sensorimotor system (e.g., spinal cord) are thought to be assembled sequentially [10-14], making this an ideal brain region for investigating the circuit-level impact of temporal patterning within a lineage. Here, we use intersectional genetics, optogenetics, high-throughput behavioral analysis, single-neuron labeling, connectomics, and calcium imaging to determine how a set of bona fide lineage-related interneurons contribute to sensorimotor circuitry in the Drosophila larva. We show that Even-skipped lateral interneurons (ELs) are sensory processing interneurons. Late-born ELs contribute to a proprioceptive body posture circuit, whereas early-born ELs contribute to a mechanosensitive escape circuit. These data support a model in which a single neuronal stem cell can produce a large number of interneurons with similar functional capacity that are distributed into different circuits based on birth timing. In summary, these data establish a link between temporal specification of neuronal identity and circuit assembly at the single-cell level.
Activation of taste buds triggers the release of several neurotransmitters, including ATP and serotonin (5-hydroxytryptamine; 5-HT). Type III taste cells release 5-HT directly in response to acidic (sour) stimuli and indirectly in response to bitter and sweet tasting stimuli. Although ATP is necessary for activation of nerve fibers for all taste stimuli, the role of 5-HT is unclear. We investigated whether gustatory afferents express functional 5-HT3 receptors and, if so, whether these receptors play a role in transmission of taste information from taste buds to nerves. In mice expressing GFP under the control of the 5-HT(3A) promoter, a subset of cells in the geniculate ganglion and nerve fibers in taste buds are GFP-positive. RT-PCR and in situ hybridization confirmed the presence of 5-HT(3A) mRNA in the geniculate ganglion. Functional studies show that only those geniculate ganglion cells expressing 5-HT3A-driven GFP respond to 10 μM 5-HT and this response is blocked by 1 μM ondansetron, a 5-HT3 antagonist, and mimicked by application of 10 μM m-chlorophenylbiguanide, a 5-HT3 agonist. Pharmacological blockade of 5-HT3 receptors in vivo or genetic deletion of the 5-HT3 receptors reduces taste nerve responses to acids and other taste stimuli compared with controls, but only when urethane was used as the anesthetic. We find that anesthetic levels of pentobarbital reduce taste nerve responses apparently by blocking the 5-HT3 receptors. Our results suggest that 5-HT released from type III cells activates gustatory nerve fibers via 5-HT3 receptors, accounting for a significant proportion of the neural taste response.
Literature context: -605-148; RRID:AB_2340476; Jackson I
Taste buds release ATP to activate ionotropic purinoceptors composed of P2X2 and P2X3 subunits, present on the taste nerves. Mice with genetic deletion of P2X2 and P2X3 receptors (double knockout mice) lack responses to all taste stimuli presumably due to the absence of ATP-gated receptors on the afferent nerves. Recent experiments on the double knockout mice showed, however, that their taste buds fail to release ATP, suggesting the possibility of pleiotropic deficits in these global knockouts. To test further the role of postsynaptic P2X receptors in afferent signalling, we used AF-353, a selective antagonist of P2X3-containing receptors to inhibit the receptors acutely during taste nerve recording and behaviour. The specificity of AF-353 for P2X3-containing receptors was tested by recording Ca(2+) transients to exogenously applied ATP in fura-2 loaded isolated geniculate ganglion neurons from wild-type and P2X3 knockout mice. ATP responses were completely inhibited by 10 μm or 100 μm AF-353, but neither concentration blocked responses in P2X3 single knockout mice wherein the ganglion cells express only P2X2-containing receptors. Furthermore, AF-353 had no effect on taste-evoked ATP release from taste buds. In wild-type mice, i.p. injection of AF-353 or simple application of the drug directly to the tongue, inhibited taste nerve responses to all taste qualities in a dose-dependent fashion. A brief access behavioural assay confirmed the electrophysiological results and showed that preference for a synthetic sweetener, SC-45647, was abolished following i.p. injection of AF-353. These data indicate that activation of P2X3-containing receptors is required for transmission of all taste qualities.