David Feldheim

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Papers by David Feldheim

Stochastic Interaction between Neural Activity and Molecular Cues in the Formation of Topographic Maps

Neuron, Jan 23, 2015

Topographic maps in visual processing areas maintain the spatial order of the visual world. Molec... more Topographic maps in visual processing areas maintain the spatial order of the visual world. Molecular cues and neuronal activity both play critical roles in map formation, but their interaction remains unclear. Here, we demonstrate that when molecular- and activity-dependent cues are rendered nearly equal in force, they drive topographic mapping stochastically. The functional and anatomical representation of azimuth in the superior colliculus of heterozygous Islet2-EphA3 knockin (Isl2(EphA3/+)) mice is variable: maps may be single, duplicated, or a combination of the two. This heterogeneity is not due to genetic differences, since map organizations in individual mutant animals often differ between colliculi. Disruption of spontaneous waves of retinal activity resulted in uniform map organization in Isl2(EphA3/+) mice, demonstrating that correlated spontaneous activity is required for map heterogeneity. Computational modeling replicates this heterogeneity, revealing that molecular- a...

Alkaline phosphatase fusions of ligands or receptors as in situ probes for staining of cells, tissues, and embryos

Methods in enzymology, 2000

[21 AP FUSION PROTEINS AS in Si1U PROBES 19 [2] Alkaline Phosphatase Fusions of Ligands or Recept... more

Topology and functional domains of Sec63p, an endoplasmic reticulum membrane protein required for secretory protein translocation

Molecular and cellular biology, 1992

SEC63 encodes a protein required for secretory protein translocation into the endoplasmic reticul... more SEC63 encodes a protein required for secretory protein translocation into the endoplasmic reticulum (ER) of Saccharomyces cerevisiae (J. A. Rothblatt, R. J. Deshaies, S. L. Sanders, G. Daum, and R. Schekman, J. Cell Biol. 109:2641-2652, 1989). Antibody directed against a recombinant form of the protein detects a 73-kDa polypeptide which, by immunofluorescence microscopy, is localized to the nuclear envelope-ER network. Cell fractionation and protease protection experiments confirm the prediction that Sec63p is an integral membrane protein. A series of SEC63-SUC2 fusion genes was created to assess the topology of Sec63p within the ER membrane. The largest hybrid proteins are unglycosylated, suggesting that the carboxyl terminus of Sec63p faces the cytosol. Invertase fusion to a loop in Sec63p that is flanked by two putative transmembrane domains produces an extensively glycosylated hybrid protein. This loop, which is homologous to the amino terminus of the Escherichia coli heat shock...

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Ephrin-as are required for the topographic mapping but not laminar choice of physiologically distinct RGC types

by Neal Sweeney and David Feldheim

Developmental Neurobiology, 2015

In the retinocollicular projection, the axons from functionally distinct retinal ganglion cell (R... more In the retinocollicular projection, the axons from functionally distinct retinal ganglion cell (RGC) types form synapses in a stereotypical manner along the superficial to deep axis of the superior colliculus (SC). Each lamina contains an orderly topographic map of the visual scene but different laminae receive inputs from distinct sets of RGCs, and inputs to each lamina are aligned with the others to integrate parallel streams of visual information. To determine the relationship between laminar organization and topography of physiologically defined RGC types, we used genetic and anatomical axon tracing techniques in wild type and ephrin-A mutant mice. We find that adjacent RGCs of the same physiological type can send axons to both ectopic and normal topographic locations, supporting a penetrance model for ephrin-A independent mapping cues. While the overall laminar organization in the SC is unaffected in ephrin-A2/A5 double mutant mice, analysis of the laminar locations of ectopic terminations shows that the topographic maps of different RGC types are misaligned. These data lend support to the hypothesis that the retinocollicular projection is a superimposition of a number of individual two-dimensional topographic maps that originate from specific types of RGCs, require ephrin-A signaling, and form independently of the other maps. © 2015 Wiley Periodicals, Inc. Develop Neurobiol, 2015.

Loss-of-Function Analysis of EphA Receptors in Retinotectal Mapping

by David Feldheim, Masaru Nakamoto, and Nicholas Gale

Journal of Neuroscience, 2004

EphA tyrosine kinases are thought to act as topographically specific receptors in the well-charac... more EphA tyrosine kinases are thought to act as topographically specific receptors in the well-characterized projection map from the retina to the tectum. Here, we describe a loss-of-function analysis of EphA receptors in retinotectal mapping. Expressing patches of a cytoplasmically truncated EphA3 receptor in chick retina caused temporal axons to have reduced responsiveness to posterior tectal repellent activity in vitro and to shift more posteriorly within the map in vivo. A gene disruption of mouse EphA5, replacing the intracellular domain with ␤-galactosidase, reduced in vitro responsiveness of temporal axons to posterior target membranes. It also caused map abnormalities in vivo, with temporal axons shifted posteriorly and nasal axons anteriorly, but with the entire target still filled by retinal axons. The anterior shift of nasal axons was not accompanied by increased responsiveness to tectal repellent activity, in contrast to the comparable anterior shift in ephrin-A knock-outs, helping to resolve a previous ambiguity in interpreting the ephrin gene knock-outs. The results show the functional requirement for endogenous EphA receptors in retinotectal mapping, show that the receptor intracellular domain is required for a forward signaling response to topographic cues, and provide new evidence for a role of axon competition in topographic mapping.

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Roles of Ephrin-As and Structured Activity in the Development of Functional Maps in the Superior Colliculus

Journal of Neuroscience, 2008

The orderly projections from retina to superior colliculus (SC) preserve a continuous retinotopic... more The orderly projections from retina to superior colliculus (SC) preserve a continuous retinotopic representation of the visual world. The development of retinocollicular maps depend on a combination of molecular guidance cues and patterned neural activity. Here we characterize the functional retinocollicular maps in mice lacking the guidance molecules ephrins-A2, -A3, and -A5 and in mice deficient in both ephrin-As and structured spontaneous retinal activity, using a method of Fourier imaging of intrinsic signals. We find that the SC of ephrin-A2/A3/A5 triple knockout mice contains functional maps that are disrupted selectively along the nasotemporal (azimuth) axis of the visual space. These maps are discontinuous, with patches of SC responding to topographically incorrect locations. The patches disappear in mice that are deficient in both ephrin-As and structured activity, resulting in a near-absence of azimuth map in the SC. These results indicate that ephrin-As guide the formation of functional topography in the SC, and patterned retinal activity clusters cells based on their correlated firing patterns. Comparison of the SC and visual cortical mapping defects in these mice suggests that although ephrin-As are required for mapping in both SC and visual cortex, ephrin-A-independent mapping mechanisms are more important in visual cortex than in the SC.

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Reconstitution of protein translocation from solubilized yeast membranes reveals topologically distinct roles for BiP and cytosolic Hsc70

The Journal of Cell Biology, 1993

We reconstituted prepro-alpha-factor translocation and signal peptide processing using a yeast mi... more We reconstituted prepro-alpha-factor translocation and signal peptide processing using a yeast microsomal detergent soluble fraction formed into vesicles with soybean phospholipids. Reconstituted translocation required ATP, and was deficient when sec63 and kar2 (BiP) mutant cells were used as a source of membranes. Normal translocation was observed with vesicles reconstituted from a mixture of pure wild-type yeast BiP and a soluble fraction of kar2 mutant membranes. Two other heat-shock cognate (hsc) 70 homologs, yeast cytosolic hsc70 (Ssalp) and E. coli dnaK protein did not replace BiP. Conversely, BiP was not active under conditions where translocation into native ER vesicles required cytosolic hsc70. We conclude that cytosolic hsc70 and BiP serve noninterchangeable roles in polypeptide translocation, possibly because distinct, asymmetrically oriented membrane proteins are required to recruit each protein to opposing surfaces of the ER membrane.

Genetic Analysis of Ephrin-A2 and Ephrin-A5 Shows Their Requirement in Multiple Aspects of Retinocollicular Mapping

by David Feldheim and Young Kim

Neuron, 2000

Ephrin-A2 and-A5 are thought to be anteroposterior mapping labels for the retinotectal/retinocoll... more

Topographic Guidance Labels in a Sensory Projection to the Forebrain

Neuron, 1998

Visual connections to the mammalian forebrain are known to be patterned by neural activity, but i... more Visual connections to the mammalian forebrain are known to be patterned by neural activity, but it remains unknown whether the map topography of such higher sensory projections depends on axon guidance labels. Here, we show complementary expression and binding for the receptor EphA5 in mouse retina and its ligands ephrin-A2 and ephrin-A5 in multiple retinal targets, including the major forebrain target, the dorsal lateral geniculate nucleus (dLGN). These ligands can act in vitro as topographically specific repellents for mammalian retinal axons and are necessary for normal dLGN mapping in vivo. The results suggest a general and economic modular mechanism for brain mapping whereby a projecting field is mapped onto multiple targets by repeated use of the same labels. They also indicate the nature of a coordinate system for the mapping of sensory connections to the forebrain.

Spatial-Temporal Patterns of Retinal Waves Underlying Activity-Dependent Refinement of Retinofugal Projections

Neuron, 2009

During development, retinal axons project coarsely within their visual targets before refining to... more During development, retinal axons project coarsely within their visual targets before refining to form organized synaptic connections. Spontaneous retinal activity, in the form of acetylcholine-driven retinal waves, is proposed to be necessary for establishing these projection patterns. In particular, both axonal terminations of retinal ganglion cells (RGCs) and the size of receptive fields of target neurons are larger in mice that lack the beta2 subunit of the nicotinic acetylcholine receptor (beta2KO). Here, using a large-scale, high-density multielectrode array to record activity from hundreds of RGCs simultaneously, we present analysis of early postnatal retinal activity from both wild-type (WT) and beta2KO retinas. We find that beta2KO retinas have correlated patterns of activity, but many aspects of these patterns differ from those of WT retina. Quantitative analysis suggests that wave directionality, coupled with short-range correlated bursting patterns of RGCs, work together to refine retinofugal projections.

Roles for Ephrins in Positionally Selective Synaptogenesis between Motor Neurons and Muscle Fibers

Neuron, 2000

Motor axons form topographic maps on muscles: rostral motor pools innervate rostral muscles, and ... more Motor axons form topographic maps on muscles: rostral motor pools innervate rostral muscles, and rostral portions of motor pools innervate rostral fibers within their targets. Here, we implicate A subfamily ephrins in this topographic mapping. First, developing muscles express all five of the ephrin-A genes. Second, rostrally and caudally derived motor axons differ in sensitivity to outgrowth inhibition by ephrin-A5. Third, the topographic map of motor axons on the gluteus muscle is degraded in transgenic mice that overexpress ephrin-A5 in muscles. Fourth, topographic mapping is impaired in muscles of mutant mice lacking ephrin-A2 plus ephrin-A5. Thus, ephrins mediate or modulate positionally selective synapse formation. In addition, the rostrocaudal position of at least one motor pool is altered in ephrin-A5 mutant mice, indicating that ephrins affect nerve-muscle matching by intraspinal as well as intramuscular mechanisms.

Ephrin-As Guide the Formation of Functional Maps in the Visual Cortex

by David Feldheim and Georgia Woods

Neuron, 2005

Ephrin-As and their receptors, EphAs, are expressed in the developing cortex where they may act t... more Ephrin-As and their receptors, EphAs, are expressed in the developing cortex where they may act to organize thalamic inputs. Here, we map the visual cortex (V1) in mice deficient for ephrin-A2, -A3, and -A5 functionally, using intrinsic signal optical imaging and microelectrode recording, and structurally, by anatomical tracing of thalamocortical projections. V1 is shifted medially, rotated, and compressed and its internal organization is degraded. Expressing ephrin-A5 ectopically by in utero electroporation in the lateral cortex shifts the map of V1 medially, and expression within V1 disrupts its internal organization. These findings indicate that interactions between gradients of EphA/ephrin-A in the cortex guide map formation, but that factors other than redundant ephrin-As are responsible for the remnant map. Together with earlier work on the retinogeniculate map, the current findings show that the same molecular interactions may operate at successive stages of the visual pathway to organize maps.

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Selective Disruption of One Cartesian Axis of Cortical Maps and Receptive Fields by Deficiency in Ephrin-As and Structured Activity

Neuron, 2008

The topographic representation of visual space is preserved from retina to thalamus to cortex. We... more The topographic representation of visual space is preserved from retina to thalamus to cortex. We have previously shown that precise mapping of thalamocortical projections requires both molecular cues and structured retinal activity. To probe the interaction between these two mechanisms, we studied mice deficient in both ephrin-As and retinal waves. Functional and anatomical cortical maps in these mice were nearly abolished along the nasotemporal (azimuth) axis of the visual space. Both the structure of single-cell receptive fields and large-scale topography were severely distorted. These results demonstrate that ephrin-As and structured neuronal activity are two distinct pathways that mediate map formation in the visual cortex and together account almost completely for the formation of the azimuth map. Despite the dramatic disruption of azimuthal topography, the dorsoventral (elevation) map was relatively normal, indicating that the two axes of the cortical map are organized by separate mechanisms.

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Repelling class discrimination: ephrin-A5 binds to and activates EphB2 receptor signaling

Nature Neuroscience, 2004

The interactions between Eph receptor tyrosine kinases and their ephrin ligands regulate cell mig... more The interactions between Eph receptor tyrosine kinases and their ephrin ligands regulate cell migration and axon pathfinding. The EphA receptors are generally thought to become activated by ephrin-A ligands, whereas the EphB receptors interact with ephrin-B ligands. Here we show that two of the most widely studied of these molecules, EphB2 and ephrin-A5, which have never been described to interact with each other, do in fact bind one another with high affinity. Exposure of EphB2-expressing cells to ephrin-A5 leads to receptor clustering, autophosphorylation and initiation of downstream signaling. Ephrin-A5 induces EphB2mediated growth cone collapse and neurite retraction in a model system. We further show, using X-ray crystallography, that the ephrin-A5-EphB2 complex is a heterodimer and is architecturally distinct from the tetrameric EphB2-ephrin-B2 structure. The structural data reveal the molecular basis for EphB2-ephrin-A5 signaling and provide a framework for understanding the complexities of functional interactions and crosstalk between A-and B-subclass Eph receptors and ephrins.

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Ephrin-As mediate targeting of eye-specific projections to the lateral geniculate nucleus

by David Warland and David Feldheim

Nature Neuroscience, 2005

Axon guidance cues contributing to the development of eye-specific visual projections to the late... more Axon guidance cues contributing to the development of eye-specific visual projections to the lateral geniculate nucleus (LGN) have not previously been identified. Here we show that gradients of ephrin-As and their receptors (EphAs) direct retinal ganglion cell (RGC) axons from the two eyes into their stereotyped pattern of layers in the LGN. Overexpression of EphAs in ferret RGCs using in vivo electroporation induced axons from both eyes to misproject within the LGN. The effects of EphA overexpression were competition-dependent and restricted to the early postnatal period. These findings represent the first demonstration of eye-specific pathfinding mediated by axon guidance cues and, taken with other reports, indicate that ephrin-As can mediate several mapping functions within individual target structures.

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Ephrin-As and neural activity are required for eye-specific patterning during retinogeniculate mapping

Nature Neuroscience, 2005

In mammals, retinal ganglion cell (RGC) projections initially intermingle and then segregate into... more In mammals, retinal ganglion cell (RGC) projections initially intermingle and then segregate into a stereotyped pattern of eyespecific layers in the dorsal lateral geniculate nucleus (dLGN). Here we found that in mice deficient for ephrin-A2, ephrin-A3 and ephrin-A5, eye-specific inputs segregated but the shape and location of eye-specific layers were profoundly disrupted. In contrast, mice that lacked correlated retinal activity did not segregate eye-specific inputs. Inhibition of correlated neural activity in ephrin mutants led to overlapping retinal projections that were located in inappropriate regions of the dLGN. Thus, ephrin-As and neural activity act together to control patterning of eye-specific retinogeniculate layers.

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Assembly of yeast Sec proteins involved in translocation into the endoplasmic reticulum into a membrane-bound multisubunit complex

Nature, 1991

Secretory-protein translocation into the endoplasmic reticulum (ER) is thought to be catalysed by... more Secretory-protein translocation into the endoplasmic reticulum (ER) is thought to be catalysed by integral membrane proteins. Genetic selections uncovered three Saccharomyces cerevisiae genes (SEC61, SEC62 and SEC63), mutations in which block import of precursor proteins into the ER lumen in vivo and in vitro. The DNA sequences of SEC62 and SEC63 predict multispanning membrane proteins, and biochemical characterization of the SEC62 protein (Sec62) confirms that it is an integral ER membrane protein. Here we show that Sec61, Sec62 and Sec63 are assembled with two additional proteins into a multisubunit membrane-associated complex. These results confirm previous predictions, based upon genetic interactions between the SEC genes, that Sec61, Sec62 and Sec63 act together to facilitate protein translocation into the ER.

Preparation of Protein Gradients through the Controlled Deposition of Protein−Nanoparticle Conjugates onto Functionalized Surfaces

Journal of the American Chemical Society, 2004

This paper describes a simple method for the preparation and characterization of protein density ... more This paper describes a simple method for the preparation and characterization of protein density gradients on solid supports. The method employs colloidal metal nanoparticles as protein carriers and optical tags and is capable of forming linear, exponential, 1D, 2D, and multiprotein gradients of varying slope without expensive or sophisticated surface patterning techniques. Surfaces patterned with proteins using the procedures described within are shown to support cell growth and are thus suitable for studies of protein-cell interactions.

Fiber order of the normal and regenerated optic tract of the frog (Rana pipiens)

The Journal of Comparative Neurology, 2004

In the normal frog, axons from the peripheral retina arising at the temporal pole course superfic... more In the normal frog, axons from the peripheral retina arising at the temporal pole course superficially in the middle stream of the diencephalic optic tract. Axons from the nasal pole course in two streams running in the opposite margins of the tract, dorsonasal axons ventrally, ventronasal axons dorsally. Axons from the dorsal and ventral poles of the retina occupy the intervals between the aforementioned middle and marginal streams. Axons from more central regions of the retina tend to occupy deeper levels of the optic tract. The regenerated optic tract does not regain its normal organization, e.g., axons of peripheral nasal origin are spread out widely over the entire width of the tract. However, axons from the temporal pole of the retina do return approximately to their original location in the middle stream. The concentration of temporal axons in the middle stream of the optic tract after regeneration may now be understood in terms of the expression pattern of the ephrin-A class of receptor tyrosine kinase ligands in the cellular matrix of the optic tract. The ephrin-As, which have a repellent effect on growing temporal retinal axons, are concentrated in and along the margins of the diencephalic optic tract and essentially absent from its middle stream. It is proposed here that peripheral temporal axons may be forced into this middle region by their avoidance of the higher levels of ephrin-A expression in the tract margins. In contrast, the growth pattern of regenerating peripheral nasal axons would not be affected by the ephrin-A gradient in the optic tract.

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A combination of ephrin-As and neural activity is required for visual system mapping

Developmental Biology, 2006

In vertebrate embryos, commissural axons extend toward and across the floor plate (FP), an interm... more In vertebrate embryos, commissural axons extend toward and across the floor plate (FP), an intermediate target located at the ventral midline (VM) of the spinal cord. After decussating, many of these axons initially turn into the longitudinal plane and, subsequently, project away from the VM along a complex path. FP contact is thought to alter the responsiveness of commissural axons to midline-associated cues so that they can exit the FP and adopt new trajectories on the contralateral side of the VM. Here, we demonstrate that a significant number of decussated axons elaborate wild-type-like projections even in cultured, FP-excised spinal cord preparations and in the spinal cords of gli2-deficient, FP-lacking mice. Consistent with a role for chemorepulsive Slit proteins in driving commissural axons out of the VM and into longitudinal tracts, slit1 -3 mRNA is expressed within the ventral ventricular zone and Slit receptors Robo1/2 are selectively expressed on longitudinal axons in gli2 homozygous embryos. Supporting an active role for Robo-Slit signaling, in the absence of FP contact, blocking Robo-Slit interactions in spinal cord explants derived from gli2 null embryos prevents commissural axons from leaving the VM. Together, these findings show that, at least for a subset of commissural axons, Robo -Slit interactions but not FP contact are required for the proper elaboration of their contralateral projections. Funding: NIH (R01 NS 38505) and NYS SCIRB (C018615).

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