Navigate Flying Molecular Elephants Safely to the Ground: Mass-Selective Soft Landing up to the Mega-Dalton Range by Electrospray Controlled Ion-Beam Deposition.

The prototype of a highly versatile and efficient preparative mass spectrometry system used for the deposition of molecules in ultrahigh vacuum (UHV) is presented, along with encouraging performance data obtained using four model species that are thermolabile or not sublimable. The test panel comprises two small organic compounds, a small and very large protein, and a large DNA species covering a 4-log mass range up to 1.7 MDa as part of a broad spectrum of analyte species evaluated to date. Three designs of innovative ion guides, a novel digital mass-selective quadrupole (dQMF), and a standard electrospray ionization (ESI) source are combined to an integrated device, abbreviated electrospray controlled ion-beam deposition (ES-CIBD). Full control is achieved by (i) the square-wave-driven radiofrequency (RF) ion guides with steadily tunable frequencies, including a dQMF allowing for investigation, purification, and deposition of a virtually unlimited m/z range, (ii) the adjustable landing energy of ions down to ∼2 eV/z enabling integrity-preserving soft landing, (iii) the deposition in UHV with high ion beam intensity (up to 3 nA) limiting contaminations and deposition time, and (iv) direct coverage control via the deposited charge. The maximum resolution of R = 650 and overall efficiency up to Ttotal = 4.4% calculated from the solution to UHV deposition are advantageous, whereby the latter can be further enhanced by optimizing ionization performance. In the setup presented, a scanning tunneling microscope (STM) is attached for in situ UHV investigations of deposited species, demonstrating a selective, structure-preserving process and atomically clean layers.

Depositing Molecular Graphene Nanoribbons on Ag(111) by Electrospray Controlled Ion Beam Deposition: Self-Assembly and On-Surface Transformations.

The chemical processing of low-dimensional carbon nanostructures is crucial for their integration in future devices. Here we apply a new methodology in atomically precise engineering by combining multistep solution synthesis of N-doped molecular graphene nanoribbons (GNRs) with mass-selected ultra-high vacuum electrospray controlled ion beam deposition on surfaces and real-space visualisation by scanning tunnelling microscopy. We demonstrate how this method yields solely a controllable amount of single, otherwise unsublimable, GNRs of 2.9 nm length on a planar Ag(111) surface. This methodology allows for further processing by employing on-surface synthesis protocols and exploiting the reactivity of the substrate. Following multiple chemical transformations, the GNRs provide reactive building blocks to form extended, metal-organic coordination polymers.

In vivo identification of eugenol-responsive and muscone-responsive mouse odorant receptors.

Our understanding of mammalian olfactory coding has been impeded by the paucity of information about the odorant receptors (ORs) that respond to a given odorant ligand in awake, freely behaving animals. Identifying the ORs that respond in vivo to a given odorant ligand from among the ∼1100 ORs in mice is intrinsically challenging but critical for our understanding of olfactory coding at the periphery. Here, we report an in vivo assay that is based on a novel gene-targeted mouse strain, S100a5-tauGFP, in which a fluorescent reporter selectively marks olfactory sensory neurons that have been activated recently in vivo. Because each olfactory sensory neuron expresses a single OR gene, multiple ORs responding to a given odorant ligand can be identified simultaneously by capturing the population of activated olfactory sensory neurons and using expression profiling methods to screen the repertoire of mouse OR genes. We used this in vivo assay to re-identify known eugenol- and muscone-responsive mouse ORs. We identified additional ORs responsive to eugenol or muscone. Heterologous expression assays confirmed nine eugenol-responsive ORs (Olfr73, Olfr178, Olfr432, Olfr610, Olfr958, Olfr960, Olfr961, Olfr913, and Olfr1234) and four muscone-responsive ORs (Olfr74, Olfr235, Olfr816, and Olfr1440). We found that the human ortholog of Olfr235 and Olfr1440 responds to macrocyclic ketone and lactone musk odorants but not to polycyclic musk odorants or a macrocyclic diester musk odorant. This novel assay, called the Kentucky in vivo odorant ligand-receptor assay, should facilitate the in vivo identification of mouse ORs for a given odorant ligand of interest.

Optimizing cholinergic tone through lynx modulators of nicotinic receptors: implications for plasticity and nicotine addiction.

The cholinergic system underlies both adaptive (learning and memory) and nonadaptive (addiction and dependency) behavioral changes through its ability to shape and regulate plasticity. Protein modulators such as lynx family members can fine tune the activity of the cholinergic system and contribute to the graded response of the cholinergic system, stabilizing neural circuitry through direct interaction with nicotinic receptors. Release of this molecular brake can unmask cholinergic-dependent mechanisms in the brain. Lynx proteins have the potential to provide top-down control over plasticity mechanisms, including addictive propensity. If this is indeed the case, then, what regulates the regulator? Transcriptional changes of lynx genes in response to pharmacological, physiological, and pathological alterations are explored in this review.

Structural adaptations of electrosprayed aromatic oligoamide foldamers on Ag(111).

Aromatic foldamers are promising for applications such as molecular recognition and molecular machinery. For many of these, defect free, 2D-crystaline monolayers are needed. To this end, submonolayers were prepared in ultra-high vacuum (UHV) on Ag(111) via electrospray controlled ion beam deposition (ES-CIBD). On the surface, the unfolded state is unambiguously identified by real-space single-molecule imaging using scanning tunnelling microscopy (STM) and it is found to assemble in regular structures.

RGD-functionalisation of PLLA nanofibers by surface coupling using plasma treatment: influence on stem cell differentiation.

The aim of this study was to functionalize the surface of synthetic poly-(l-lactic) (PLLA) nanofibers with RGD peptide, in order to promote growth and osteogenic differentiation of human mesenchymal stem cells (hMSC) in vitro. The cRGD was coupled onto PLLA nanofibers using oxygen plasma combined with EDC/sulfo-NHS activation. Matrices were seeded with hMSC and cultivated over a period of 22 days under growth conditions and analyzed during the course of cultivation. The plasma activation of PLLA nanofibers resulted in a reduction of hydrophobicity as well as a formation of carboxyl groups on the surface of the fibers. Furthermore, maximum load, but not young's modulus was influenced by the treatment with oxygen plasma. When hMSC were cultured onto the cRGD functionalized scaffolds, cells showed no increased proliferation or cell density but an induction of genes associated with the osteoblast lineage. In brief, this study indicates that functional peptides of the extracellular matrix can be coupled onto PLLA nanofibers using plasma treatment in combination with EDC/sulfo-NHS treatment. These groups are accessible for the growing cell and mediate probably some osteoinductive properties of collagen nanofibers.

Effect of direct RGD incorporation in PLLA nanofibers on growth and osteogenic differentiation of human mesenchymal stem cells.

The aim of this study was to functionalize synthetic poly-(L-lactic) (PLLA) nanofibers by direct incorporation of cRGD, in order to promote adhesion, growth and osteogenic differentiation of human mesenchymal stem cells (hMSC) in vitro. The cRGD was incorporated into PLLA nanofibers either by emulsion [PLLA-cRGD (d)] or suspension [PLLA-cRGD (s)]. Matrices were seeded with hMSC and cultivated over a period of 28 days under growth conditions and analyzed during the course. Although the mode of incorporation resulted in different distributions of the RGD peptide, it had no impact on the fiber characteristics when compared to corresponding unblended PLLA control fibers. However, hMSC showed better adherence on PLLA-cRGD (d). Nevertheless, this advantage was not reflected during the course of cultivation. Furthermore, the PLLA-cRGD (s) fibers mediated the osteogenic potential of collagen (determined as the expression and deposition of collagen and osteocalcin) to some extent. Further studies are needed in order to optimize the RGD distribution and concentration.

Neuropilin-1 and the Positions of Glomeruli in the Mouse Olfactory Bulb.

It is known since 1996 that mouse odorant receptors (ORs) are involved in determining the positions of the sites of coalescence of axons of olfactory sensory neurons (OSNs)-the thousands of glomeruli in the olfactory bulb. But the molecular and cellular mechanisms of OR-mediated axonal coalescence into glomeruli remain unclear. A model was proposed in 2006-2009 whereby OR-derived cAMP signals, rather than direct action of OR molecules, determine the target destinations (glomeruli) of OSNs in the bulb. This model hypothesizes that OR-derived cAMP signals determine the expression levels of neuropilin 1 (Nrp1) in OSN axon termini; that levels of Nrp1 in glomeruli form a gradient from anterior-low to posterior-high throughout the bulb; and that these Nrp1 levels mechanistically determine anterior-posterior patterning of glomeruli. Here, we describe the first independent evaluation of the Nrp1 model since it was formulated a decade ago. We tested the model for the well-characterized mouse OR M71 using our gene-targeted mouse strains, which are publicly available. In contradiction to the model, we observed a variety of configurations for the M71 glomeruli in the conditional Nrp1 knockout. We then reassessed the model for the original OR transgene with which the model was developed, using the same publicly available mouse strains. We discovered that glomerular positions do not undergo the simple anterior shift that has been reported in the conditional Nrp1 knockout for this OR transgene. Taken together, our findings do not support the Nrp1 model for the anterior-posterior patterning of glomerular positions in the olfactory bulb.

Aberrant sensory innervation of the olfactory bulb in neuropilin-2 mutant mice.

The mammalian olfactory system consists of two anatomically segregated structures, the main olfactory system and the vomeronasal system, which each detect distinct types of chemical stimuli in the environment. During development, sensory neurons establish precise axonal connections with their respective targets within the olfactory bulb. The specificity of the odorant or vomeronasal receptor expressed by the sensory neuron is crucial in this process, yet it is less clear which of the more conventional axon guidance molecules are involved. Here, we show that neuropilin-2, a coreceptor for some of the class 3 semaphorins, is expressed in subpopulations of olfactory and vomeronasal sensory neurons. We generated a knock-out mutation in the neuropilin-2 gene by gene targeting in embryonic stem cells. Neuropilin-2 mutant mice exhibit profound and distinct effects on target innervation within the olfactory bulb. In the main olfactory system, axons of olfactory sensory neurons penetrate into the deeper layers of the main olfactory bulb. In the vomeronasal system, axonal fasciculation within the vomeronasal nerve is affected; some axons are misrouted and innervate glomeruli in an ectopic domain of the accessory olfactory bulb.

Lynx1 supports neuronal health in the mouse dorsal striatum during aging: an ultrastructural investigation.

Nicotinic acetylcholine receptors have been shown to participate in neuroprotection in the aging brain. Lynx protein modulators dampen the activity of the cholinergic system through direct interaction with nicotinic receptors. Although lynx1 null mutant mice exhibit augmented learning and plasticity, they also exhibit macroscopic vacuolation in the dorsal striatum as they age, detectable at the optical microscope level. Despite the relevance of the lynx1 gene to brain function, little is known about the cellular ultrastructure of these age-related changes. In this study, we assessed degeneration in the dorsal striatum in 1-, 3-, 7-, and 13-month-old mice, using optical and transmission electron microscopy. We observed a loss of nerve fibers, a breakdown in nerve fiber bundles, and a loss of neuronal nuclei in the 13-month-old lynx1 null striatum. At higher magnification, these nerve fibers displayed intracellular vacuoles and disordered myelin sheaths. Few or none of these morphological alterations were present in younger lynx1 null mutant mice or in heterozygous lynx1 null mutant mice at any age. These data indicate that neuronal health can be maintained by titrating lynx1 dosage and that the lynx1 gene may participate in a trade-off between neuroprotection and augmented learning.

Enhancement in motor learning through genetic manipulation of the Lynx1 gene.

The cholinergic system is a neuromodulatory neurotransmitter system involved in a variety of brain processes, including learning and memory, attention, and motor processes, among others. The influence of nicotinic acetylcholine receptors of the cholinergic system are moderated by lynx proteins, which are GPI-anchored membrane proteins forming tight associations with nicotinic receptors. Previous studies indicate lynx1 inhibits nicotinic receptor function and limits neuronal plasticity. We sought to investigate the mechanism of action of lynx1 on nicotinic receptor function, through the generation of lynx mouse models, expressing a soluble version of lynx and comparing results to the full length overexpression. Using rotarod as a test for motor learning, we found that expressing a secreted variant of lynx leads to motor learning enhancements whereas overexpression of full-length lynx had no effect. Further, adult lynx1KO mice demonstrated comparable motor learning enhancements as the soluble transgenic lines, whereas previously, aged lynx1KO mice showed performance augmentation only with nicotine treatment. From this we conclude the motor learning is more sensitive to loss of lynx function, and that the GPI anchor plays a role in the normal function of the lynx protein. In addition, our data suggests that the lynx gene plays a modulatory role in the brain during aging, and that a soluble version of lynx has potential as a tool for adjusting cholinergic-dependent plasticity and learning mechanisms in the brain.

Influence of olfactory epithelium on mitral/tufted cell dendritic outgrowth.

Stereotypical connections between olfactory sensory neuron axons and mitral cell dendrites in the olfactory bulb establish the first synaptic relay for olfactory perception. While mechanisms of olfactory sensory axon targeting are reported, molecular regulation of mitral cell dendritic growth and refinement are unclear. During embryonic development, mitral cell dendritic distribution overlaps with olfactory sensory axon terminals in the olfactory bulb. In this study, we investigate whether olfactory sensory neurons in the olfactory epithelium influence mitral cell dendritic outgrowth in vitro. We report a soluble trophic activity in the olfactory epithelium conditioned medium which promotes mitral/tufted cell neurite outgrowth. While the trophic activity is present in both embryonic and postnatal olfactory epithelia, only embryonic but not postnatal mitral/tufted cells respond to this activity. We show that BMP2, 5 and 7 promote mitral/tufted cells neurite outgrowth. However, the BMP antagonist, Noggin, fails to neutralize the olfactory epithelium derived neurite growth promoting activity. We provide evidence that olfactory epithelium derived activity is a protein factor with molecular weight between 50-100 kD. We also observed that Follistatin can effectively neutralize the olfactory epithelium derived activity, suggesting that TGF-beta family proteins are involved to promote mitral/tufted dendritic elaboration.

Axonal wiring of guanylate cyclase-D-expressing olfactory neurons is dependent on neuropilin 2 and semaphorin 3F.

The olfactory system of the mouse includes several subsystems that project axons from the olfactory epithelium to the olfactory bulb. Among these is a subset of neurons that do not express the canonical pathway of olfactory signal transduction, but express guanylate cyclase-D (GC-D). These GC-D-positive (GC-D+) neurons are not known to express odorant receptors. Axons of GC-D+ neurons project to the necklace glomeruli, which reside between the main and accessory olfactory bulbs. To label the subset of necklace glomeruli that receive axonal input from GC-D+ neurons, we generated two strains of mice with targeted mutations in the GC-D gene (Gucy2d). These mice co-express GC-D with an axonal marker, tau-beta-galactosidase or tauGFP, by virtue of a bicistronic strategy that leaves the coding region of the Gucy2d gene intact. With these strains, the patterns of axonal projections of GC-D+ neurons to necklace glomeruli can be visualized in whole mounts. We show that deficiency of one of the neuropilin 2 ligands of the class III semaphorin family, Sema3f, but not Sema3b, phenocopies the loss of neuropilin 2 (Nrp2) for axonal wiring of GC-D+ neurons. Some glomeruli homogeneously innervated by axons of GC-D+ neurons form ectopically within the glomerular layer, across wide areas of the main olfactory bulb. Similarly, axonal wiring of some vomeronasal sensory neurons is perturbed by a deficiency of Nrp2 or Sema3f, but not Sema3b or Sema3c. Our findings provide genetic evidence for a Nrp2-Sema3f interaction as a determinant of the wiring of axons of GC-D+ neurons into the unusual configuration of necklace glomeruli.

Detection of near-atmospheric concentrations of CO2 by an olfactory subsystem in the mouse.

Carbon dioxide (CO2) is an important environmental cue for many organisms but is odorless to humans. It remains unclear whether the mammalian olfactory system can detect CO2 at concentrations around the average atmospheric level (0.038%). We demonstrated the expression of carbonic anhydrase type II (CAII), an enzyme that catabolizes CO2, in a subset of mouse olfactory neurons that express guanylyl cyclase D (GC-D+ neurons) and project axons to necklace glomeruli in the olfactory bulb. Exposure to CO2 activated these GC-D+ neurons, and exposure of a mouse to CO2 activated bulbar neurons associated with necklace glomeruli. Behavioral tests revealed CO2 detection thresholds of approximately 0.066%, and this sensitive CO2 detection required CAII activity. We conclude that mice detect CO2 at near-atmospheric concentrations through the olfactory subsystem of GC-D+ neurons.

Development and topography of the lateral olfactory tract in the mouse: imaging by genetically encoded and injected fluorescent markers.

In mammals, conventional odorants are detected by OSNs located in the main olfactory epithelium of the nose. These neurons project their axons to glomeruli, which are specialized structures of neuropil in the olfactory bulb. Within glomeruli, axons synapse onto dendrites of projection neurons, the mitral and tufted (M/T) cells. Genetic approaches to visualize axons of OSNs expressing a given odorant receptor have proven very useful in elucidating the organization of these projections to the olfactory bulb. Much less is known about the development and connectivity of the lateral olfactory tract (LOT), which is formed by axons of M/T cells connecting the olfactory bulb to central neural regions. Here, we have extended our genetic approach to mark M/T cells of the main olfactory bulb and their axons in the mouse, by targeted insertion of IRES-tauGFP in the neurotensin locus. In NT-GFP mice, we find that M/T cells of the main olfactory bulb mature and project axons as early as embryonic day 11.5. Final innervation of central areas is accomplished before the end of the second postnatal week. M/T cell axons that originate from small defined areas within the main olfactory bulb, as visualized by localized injections of fluorescent tracers in wild-type mice at postnatal days 1 to 3, follow a dual trajectory: a branch of tightly packed axons along the dorsal aspect of the LOT, and a more diffuse branch along the ventral aspect. The dorsal, but not the ventral, subdivision of the LOT exhibits a topographical segregation of axons coming from the dorsal versus ventral main olfactory bulb. The NT-GFP mouse strain should prove useful in further studies of development and topography of the LOT, from E11.5 until 2 weeks after birth.

Disrupted compartmental organization of axons and dendrites within olfactory glomeruli of mice deficient in the olfactory cell adhesion molecule, OCAM.

There is an overall topographic connectivity in the axonal projections of olfactory sensory neurons from the olfactory epithelium (OE) to the olfactory bulb (OB). The molecular determinants of this overall topographic OE-OB connectivity are not known. For 20 years, the intriguing expression pattern of the olfactory cell adhesion molecule (OCAM) has made it the leading candidate as determinant of overall topographic OE-OB connectivity. Here, we have generated a strain of OCAM knockout mice by gene targeting. There were no obvious alterations in the distribution of olfactory sensory neurons within the OE or in the coalescence of axons into specific glomeruli. However, the compartmental organization of dendrites and axons within the glomeruli was disrupted. Surprisingly, the mutant mice exhibited an increase in olfactory acuity; they appeared to have a better sense of smell. Thus, despite its striking expression pattern, OCAM is not essential for overall topographic OE-OB connectivity. Instead, OCAM is required for establishing or maintaining the compartmental organization and the segregation of axodendritic and dendrodendritic synapses within glomeruli.

Chondroitin sulfate disrupts axon pathfinding in the optic tract and alters growth cone dynamics.

Little is known about the cues that guide retinal axons across the diencephalon en route to their midbrain target, the optic tectum. Here we show that chondroitin sulfate proteoglycans are differentially expressed within the diencephalon at a time when retinal axons are growing within the optic tract. Using exposed brain preparations, we show that the addition of exogenous chondroitin sulfate results in retinal pathfinding errors. Retinal axons disperse widely from their normal trajectory within the optic tract and extend aberrantly into inappropriate regions of the forebrain. Time-lapse analysis of retinal growth cone dynamics in vivo shows that addition of exogenous chondroitin sulfate causes intermittent stalling and increases growth cone complexity. These results suggest that chondroitin sulfate may modulate the guidance of retinal axons as they grow through the diencephalon towards the optic tectum.