Biology subjects

Garrett, A. M.

Publications and source records attributed to Garrett, A. M..

7 recordsLinked to original sources

bioRxiv2025

A characterization of mouse retinal ganglion cell types labeled with AAV tools

The mouse retina is made-up of approximately 150 types of neurons each with unique characteristics and functions in interpreting visual information. Recent efforts to categorize cell types using molecular markers, morphology, and electrophysiological response properties have provided a wealth of information and a host of tools for studying specific cell types. AAV-based approaches have several advantages over transgenic mouse lines, including ease of application to many different animal models without extensive crossing and their amenability to intersectional approaches. Here, we provide an in-depth characterization of retinal ganglion cell types labeled by two AAV vectors drawn from a recent panel of constructs with synthetic promoters. Each promoter analyzed here was derived from a gene expressed in a cell type specific manner. Using a combination of morphology, molecular markers, and electrophysiological measurement of light responses, we found that each vector labeled distinct subsets of RGCs. However, both labeled more cell types than expected from the expression pattern of the promoters endogenous gene. We then characterized the projection patterns of these RGC types to the brain, finding that each AAV type labeled distinct axonal populations. These tools provide new access to a unique subset of cells and will be instrumental to future studies analyzing their functions and connectivity.

neuroscience↗

bioRxiv2025

Reduced neuronal self-avoidance in mouse starburst amacrine cells with only one Pcdhg isoform

The clustered protocadherins (cPcdhs) are a family of [~]60 homophilic cell adhesion molecules expressed across three gene clusters (Pcdha, Pcdhb, and Pcdhg) with a variety of essential roles in the developing nervous system. Some of these roles rely on specific isoforms, while others are more consistent with a model of isoform redundancy or a requirement for diversity. The {gamma}-Pcdhs (expressed from the Pcdhg gene cluster) are particularly important for neuronal self-avoidance in starburst amacrine cells in the mouse retina. Here, we used mouse mutants to test two of the C-type isoforms - {gamma}C4 and {gamma}C5 - and found that neither was required for normal self-avoidance. Conversely, when we analyzed a mutant with only {gamma}C4 intact, we found significant failures in self-avoidance that could not be completely rescued by overexpression of this isoform from a transgene. We have recently found that this isoform is essential for normal neuronal survival during development, and our new findings here support the hypothesis that {gamma}C4 is specialized for the survival function at the expense of a significant role in self-avoidance.

neuroscience↗

bioRxiv2025

A new mouse mutant with a discrete mutation in Pcdhgc5 reveals that the Protocadherin γC5 isoform is not essential for dendrite arborization in the cerebral cortex

There are [~]60 clustered protocadherin (cPcdh) isoforms expressed from three gene clusters (Pcdha, Pcdhb, Pcdhg) arrayed in tandem across nearly 1 Mb in mammals. cPcdhs are homophilic cell adhesion molecules (CAMs) critical for a host of neural developmental functions consistent with a role in cell-cell recognition. Indeed, isoforms make recognition modules in combination to generate recognition diversity far exceeding the [~]60 individual CAMs. However, there is also growing evidence for specialized functions for specific isoforms, particularly the C-type isoforms found at the 3 ends of the Pcdha cluster (C1 and C2) and at the 3 end of the Pcdhg cluster ({gamma}C3, {gamma}C4, and {gamma}C5). We have previously described unique roles for {gamma}C3 in dendrite arborization in the cerebral cortex and neural circuit formation in the spinal cord, as well as for {gamma}C4 in neuronal survival. Here we report a new mouse mutant specifically targeting the Pcdhgc5 exon encoding {gamma}C5. Unlike the rest of the Pcdhg cluster, expression of this isoform does not begin until postnatal stages of mouse development, increasing in the second week of life, suggesting specialized roles. We found significant expression changes in gene pathways involved in synaptic activity, learning and memory, and cognition. Despite this, we saw no major disruption in the cerebral cortex in neuronal organization, survival, dendritic arborization, or synaptic protein expression in these mutants. This new model will be an important tool for future studies delineating specific functions for {gamma}C5.

neuroscience↗

bioRxiv2024

A C-terminal motif containing a PKC phosphorylation site regulates γ-Protocadherin-mediated dendrite arborization in the cerebral cortex in vivo

The Pcdhg gene cluster encodes 22 {gamma}-Protocadherin ({gamma}-Pcdh) cell adhesion molecules that critically regulate multiple aspects of neural development, including neuronal survival, dendritic and axonal arborization, and synapse formation and maturation. Each {gamma}-Pcdh isoform has unique protein domains-a homophilically-interacting extracellular domain and a juxtamembrane cytoplasmic domain- as well as a C-terminal cytoplasmic domain shared by all isoforms. The extent to which isoform-specific vs. shared domains regulate distinct {gamma}-Pcdh functions remains incompletely understood. Our previous in vitro studies identified PKC phosphorylation of a serine residue within a shared C-terminal motif as a mechanism through which {gamma}-Pcdh promotion of dendrite arborization via MARCKS is abrogated. Here, we used CRISPR/Cas9 genome editing to generate two new mouse lines expressing only non-phosphorylatable {gamma}-Pcdhs, due either to a serine-to-alanine mutation (PcdhgS/A) or to a 15-amino acid C-terminal deletion resulting from insertion of an early stop codon (PcdhgCTD). Both lines are viable and fertile, and the density and maturation of dendritic spines remains unchanged in both PcdhgS/A and PcdhgCTDcortex. Dendrite arborization of cortical pyramidal neurons, however, is significantly increased in both lines, as are levels of active MARCKS. Intriguingly, despite having significantly reduced levels of {gamma}-Pcdh proteins, the PcdhgCTD mutation yields the strongest phenotype, with even heterozygous mutants exhibiting increased arborization. The present study confirms that phosphorylation of a shared C-terminal motif is a key {gamma}-Pcdh negative regulation point, and contributes to a converging understanding of {gamma}-Pcdh family function in which distinct roles are played by both individual isoforms and discrete protein domains.

neuroscience↗

bioRxiv2022

γ-Protocadherins control synapse formation and peripheral branching of touch sensory neurons

Light touch sensation begins with activation of low-threshold mechanoreceptor (LTMR) endings in the skin and propagation of their signals to the spinal cord and brainstem. We found that the clustered protocadherin gamma (Pcdhg) gene locus, which encodes 22 cell-surface homophilic binding proteins, is required in somatosensory neurons for normal behavioral reactivity to a range of tactile stimuli. Developmentally, distinct Pcdhg isoforms mediate LTMR synapse formation through neuron-neuron interactions and peripheral axonal branching through neuron-glia interactions. The Pcdhgc3 isoform mediates homophilic interactions between sensory axons and spinal cord neurons to promote synapse formation in vivo, and is sufficient to induce postsynaptic specializations in vitro. Moreover, loss of Pcdhgs and somatosensory synaptic inputs to the dorsal horn lead to fewer corticospinal synapses onto dorsal horn neurons. These findings reveal essential roles for Pcdhg isoform diversity in somatosensory neuron synapse formation, peripheral axonal branching, and step-wise assembly of central mechanosensory circuitry.

neuroscience↗

bioRxiv2021

Enhanced nociception in a rabbit model of cerebral palsy

Spastic cerebral palsy (CP) is a movement disorder marked by hypertonia and hyperreflexia, and the most prevalent comorbidity is pain. Since spinal nociceptive afferents contribute to both the sensation of painful stimuli as well as reflex circuits involved in movement, we investigated the relationship between prenatal hypoxia-ischemia (HI) injury which can cause CP, and possible changes in spinal nociceptive circuitry. To do this, we examined nociceptive afferents and mechanical and thermal sensitivity of New Zealand White rabbit kits after prenatal HI or a sham surgical procedure. As described previously, a range of motor deficits similar to spastic CP was observed in kits born naturally after HI (40 minutes at [~]70-80% gestation). We found that HI caused an expansion of peptidergic afferents (marked by expression of calcitonin gene-related peptide; CGRP) in both the superficial and deep dorsal horn at postnatal day (P)5. Non-peptidergic nociceptive afferent arborization (labeled by isolectin B4; IB4) was unaltered in HI kits but overlap of the two populations (peptidergic and non-peptidergic nociceptors) was increased by HI. Density of glial fibrillary acidic protein (GFAP) was unchanged within spinal white matter regions important in nociceptive transmission at P5. We found that mechanical and thermal nociception was enhanced in HI kits even in the absence of motor deficits. These findings suggest that prenatal HI injury impacts spinal sensory pathways in addition to the more well-established disruptions to descending motor circuits. In conclusion, changes to spinal nociceptive circuitry could disrupt spinalreflexes and contribute to pain experienced by individuals with CP. Significance StatementPerinatal injuries that cause cerebral palsy (CP) typically involve global insults to the central nervous system and are capable of modulating development of both motor and sensory systems. Most individuals with CP experience pain, yet whether nociception is enhanced in this disorder is unexplored. Here, we demonstrate altered topographic distribution of nociceptive afferents in the spinal cord dorsal horn of neonatal rabbits that experienced hypoxic-ischemic (HI) injury in utero; these anatomical changes were associated with nocifensive behavior indicative of pain-like behaviors. Our findings suggest that CP-causative injuries alter spinal sensory pathways (not only descending motor circuits), contributing to increased pain in CP.

neuroscience↗

bioRxiv2019

CRISPR/Cas9 interrogation of the mouse Pcdhg gene cluster reveals a crucial isoform-specific role for Pcdhgc4

The mammalian Pcdhg gene cluster encodes a family of 22 cell adhesion molecules, the gamma-Protocadherins ({gamma}-Pcdhs), critical for neuronal survival and neural circuit formation. The extent to which isoform diversity-a{gamma}-Pcdh hallmark-is required for their functions remains unclear. We used a CRISPR/Cas9 approach to reduce isoform diversity, targeting each Pcdhg variable exon with pooled sgRNAs to generate an allelic series of 26 mouse lines with 1 to 21 isoforms disrupted via discrete indels at guide sites and/or larger deletions/rearrangements. Analysis of 5 mutant lines indicates that postnatal viability and neuronal survival do not require isoform diversity. Surprisingly, as it is the only {gamma}-Pcdh that cannot independently engage in homophilic trans-interactions, we find that {gamma}C4, encoded by Pcdhgc4, is the only critical isoform. Because the human orthologue is the only PCDHG gene constrained in humans, our results indicate a conserved {gamma}C4 function that likely involves distinct molecular mechanisms.

developmental biology↗