Biology subjects

Kolodkin, A. L.

Publications and source records attributed to Kolodkin, A. L..

5 recordsLinked to original sources

bioRxiv2023

Semaphorin 6A in Retinal Ganglion Cells Regulates Functional Specialization of the Inner Retina

To form functional circuits, neurons must settle in their appropriate cellular locations and then project and elaborate neurites to contact their target synaptic neuropils. Laminar organization within the vertebrate retinal inner plexiform layer (IPL) facilitates pre- and postsynaptic neurite targeting, yet, the precise mechanisms underlying establishment of functional IPL subdomains are not well understood. Here we explore mechanisms defining the compartmentalization of OFF and ON neurites generally, and OFF and ON direction-selective neurites specifically, within the developing IPL. We show that semaphorin 6A (Sema6A), a repulsive axon guidance cue, is required for delineation of OFF versus ON circuits within the IPL: in the Sema6a null IPL, the boundary between OFF and ON domains is blurred. Furthermore, Sema6A expressed by retinal ganglion cells (RGCs) directs laminar segregation of OFF and ON starburst amacrine cell (SAC) dendritic scaffolds, which themselves serve as a substrate upon which other retinal neurites elaborate. These results demonstrate for the first time that RGCs, the first neuron-type born within the retina, play an active role in functional specialization of the IPL. Retinal ganglion cell-dependent regulation of OFF and ON starburst amacrine cell dendritic scaffold segregation prevents blurring of OFF versus ON functional domains in the murine inner plexiform layer.

neuroscience↗

bioRxiv2023

MAP1B Regulates Cortical Neuron Interstitial Axon Branching Through the Tubulin Tyrosination Cycle

Regulation of directed axon guidance and branching during development is essential for the generation of neuronal networks. However, the molecular mechanisms that underlie interstitial axon branching in the mammalian brain remain unresolved. Here, we investigate interstitial axon branching in vivo using an approach for precise labeling of layer 2/3 callosal projection neurons (CPNs), allowing for quantitative analysis of axonal morphology at high acuity and also manipulation of gene expression in well-defined temporal windows. We find that the GSK3{beta} serine/threonine kinase promotes interstitial axon branching in layer 2/3 CPNs by releasing MAP1B-mediated inhibition of axon branching. Further, we find that the tubulin tyrosination cycle is a key downstream component of GSK3{beta}/MAP1B signaling. We propose that MAP1B functions as a brake on axon branching that can be released by GSK3{beta} activation, regulating the tubulin code and thereby playing an integral role in sculpting cortical neuron axon morphology. HIGHLIGHTS- GSK3{beta} activation induces excessive interstitial axon branching in excitatory cortical neurons - MAP1B, acting as a brake, is a downstream effector of GSK3{beta}-mediated axon branching - MAP1B inhibition of axon branching is released by GSK3{beta} phosphorylation - GSK3{beta}/MAP1B regulation of interstitial axon branching is through modification of the tubulin code

neuroscience↗

bioRxiv2023

A semi-automated method for quantifying optokinetic reflex tracking acuity

The study of murine behavioral responses to visual stimuli is a key component of understanding mammalian visual circuitry. One notable response is the optokinetic reflex (OKR), a highly conserved innate behavior necessary for image stabilization on the retina. The OKR provides a robust readout of image tracking ability and has been extensively studied to understand the logic of visual system circuitry and function in mice from different genetic backgrounds. The OKR consists of two phases: a slow tracking phase as the eye follows a stimulus to the edge of the visual plane, and a compensatory fast phase saccade that maintains the image within the visual field. Assessment of the OKR has previously relied on counting individual compensatory eye saccades to estimate tracking speed. To obtain a more direct quantification of tracking ability, we have developed a novel, semi-automated analysis program that allows for rapid and reproducible quantification of unidirectional tracking gains, in addition to being adaptable to any video-oculography equipment. Our analysis program allows for the selection of slow tracking phases, modeling of the vertical and horizontal eye vectors, quantification of eye movement relative to the stimulus, and organization of resultant data into a usable spreadsheet for statistical and graphical comparisons. This quantitative and streamlined analysis pipeline provides a faster and more direct measurement of OKR responses, thereby facilitating further study of visual behavior responses. SUMMARYWe describe here a semi-automated quantitative analysis method that directly measures eye tracking resulting from murine visual system responses to two-dimensional image motion. A Python-based user interface and analysis algorithm allows for higher throughput and more quantitative measurements of eye tracking parameters than previous methods.

neuroscience↗

bioRxiv2023

Drebrin Regulates Collateral Axon Branching in Cortical Layer II/III Somatosensory Neurons

Proper cortical lamination is essential for cognition, learning, and memory. Within the somatosensory cortex, pyramidal excitatory neurons elaborate axon collateral branches in a laminar-specific manner that dictates synaptic partners and overall circuit organization. Here, we leverage mouse models, single-cell labeling and imaging approaches to identify intrinsic regulators of laminar-specific collateral, also termed interstitial, axon branching. We developed new approaches for the robust, sparse, labeling of layer II/III pyramidal neurons to obtain single-cell quantitative assessment of axon branch morphologies. We combined these approaches with cell-autonomous loss-of-function (LOF) and over-expression (OE) manipulations in an in vivo candidate screen to identify regulators of cortical neuron axon branch lamination. We identify a role for the cytoskeletal binding protein drebrin (Dbn1) in regulating layer II/III cortical projection neuron (CPN) collateral axon branching in vitro. LOF experiments show that Dbn1 is necessary to suppress the elongation of layer II/III CPN collateral axon branches within layer IV, where axon branching by layer II/III CPNs is normally absent. Conversely, Dbn1 OE produces excess short axonal protrusions reminiscent of nascent axon collaterals that fail to elongate. Structure-function analyses implicate Dbn1S142 phosphorylation and Dbn1 protein domains known to mediate F-actin bundling and microtubule (MT) coupling as necessary for collateral branch initiation upon Dbn1 OE. Taken together, these results contribute to our understanding of the molecular mechanisms that regulate collateral axon branching in excitatory CPNs, a key process in the elaboration of neocortical circuit formation. Significance StatementLaminar-specific axon targeting is essential for cortical circuit formation. Here, we show that the cytoskeletal protein drebrin (Dbn1) regulates excitatory layer II/III cortical projection neuron (CPN) collateral axon branching, lending insight into the molecular mechanisms that underlie neocortical laminar-specific innervation. To identify branching patterns of single cortical neurons in vivo, we have developed tools that allow us to obtain detailed images of individual CPN morphologies throughout postnatal development and to manipulate gene expression in these same neurons. Our results showing that Dbn1 regulates CPN interstitial axon branching both in vivo and in vitro and may aid in our understanding of how aberrant cortical neuron morphology contributes to dysfunctions observed in Autism Spectrum Disorder (ASD) and epilepsy.

neuroscience↗

bioRxiv2022

Palmitoylation regulates neuropilin-2 localization and function in cortical neurons and conveys specificity to semaphorin signaling via palmitoyl acyltransferases

Secreted semaphorin 3F (Sema3F) and semaphorin 3A (Sema3A) exhibit remarkably distinct effects on deep layer excitatory cortical pyramidal neurons; Sema3F mediates dendritic spine pruning, whereas Sema3A promotes the elaboration of basal dendrites. Sema3F and Sema3A signal through distinct holoreceptors that include neuropilin-2 (Nrp-2)/plexinA3 (PlexA3) and neuropilin-1 (Nrp-1)/PlexA4, respectively. We find that Nrp-2 and Nrp-1 are S-palmitoylated in cortical neurons and that palmitoylation of select Nrp-2 cysteines is required for its proper subcellular localization and also for Sema3F/Nrp-2-dependent dendritic spine pruning in cortical neurons, both in vitro and in vivo. Moreover, we show that the palmitoyl acyltransferase DHHC15 is required for Nrp-2 palmitoylation and Sema3F/Nrp-2-dependent dendritic spine pruning, but it is dispensable for Nrp-1 palmitoylation and Sema3A/Nrp-1-dependent basal dendritic elaboration. Therefore, palmitoyl acyltransferase-substrate specificity is essential for establishing compartmentalized neuronal structure and functional responses to extrinsic guidance cues. HIGHLIGHTSO_LINeuropilins (Nrps) are S-palmitoylated in vitro and in vivo in the central nervous system C_LIO_LIS-palmitoylation of select Nrp-2 cysteines confers subcellular localization specificity and is required for semaphorin 3F-dependent dendritic spine pruning in cortical neurons C_LIO_LIDistinct palmitoyl acyltransferases mediate Nrp-2 and Nrp-1 palmitoylation and function, imparting specificity to semaphorin signaling C_LI

neuroscience↗