Biology subjects

Normoyle, K. P.

Publications and source records attributed to Normoyle, K. P..

3 recordsLinked to original sources

bioRxiv2023

2-photon imaging of fluorescent proteins in living swine

A common point of failure in translation of preclinical neurological research to successful clinical trials comes in the giant leap from rodent models to humans. Non-human primates are phylogenetically close to humans, but cost and ethical considerations prohibit their widespread usage in preclinical trials. Swine have large, gyrencencephalic brains, which are biofidelic to human brains. Their classification as livestock makes them a readily accessible model organism. However, their size has precluded experiments involving intravital imaging with cellular resolution. Here, we present a suite of techniques and tools for in vivo imaging of porcine brains with subcellular resolution. Specifically, we describe surgical techniques for implanting a synthetic, flexible, transparent dural window for chronic optical access to the neocortex. We detail optimized parameters and methods for injecting adeno-associated virus vectors through the cranial imaging window to express fluorescent proteins. We introduce a large-animal 2-photon microscope that was constructed with off-the shelf components, has a gantry design capable of accommodating animals > 80 kg, and is equipped with a high-speed digitizer for digital fluorescence lifetime imaging. Finally, we delineate strategies developed to mitigate the substantial motion artifact that complicates high resolution imaging in large animals, including heartbeat-triggered high-speed image stack acquisition. The effectiveness of this approach is demonstrated in sample images acquired from pigs transduced with the chloride-sensitive fluorescent protein SuperClomeleon.

neuroscience↗

bioRxiv2022

Displacement of extracellular chloride by sulfated glycosaminoglycans of the brain extracellular matrix

GABA is the primary inhibitory neurotransmitter. Membrane currents evoked by GABAA receptor activation have uniquely small driving forces: their reversal potential (EGABA) is very close to the resting membrane potential. As a consequence, GABAA currents can flow in either direction, depending on both the membrane potential and the local intra and extracellular concentrations of the primary permeant ion, chloride (Cl). Local cytoplasmic Cl concentrations vary widely due to displacement of mobile Cl ions by relatively immobile anions. Here we use new reporters of extracellular chloride (Cl-o) to demonstrate that Cl is displaced in the extracellular space by high and spatially heterogenous concentrations of immobile anions including sulfated glycosaminoglycans (sGAGs). Cl-o varies widely, and the mean Cl-o is only half the canonical concentration, i.e. the Cl concentration in the cerebrospinal fluid. These unexpectedly low and heterogenous Cl-o domains provide a mechanism to link the varied but highly stable distribution of sGAGs and other immobile anions in the brains extracellular space to neuronal signal processing via the effects on the amplitude and direction of GABAA transmembrane Cl currents. Key Points SummaryO_LIExtracellular chloride concentrations in the brain were measured using a new chloride-sensitive organic fluorophore and two photon fluorescence lifetime imaging. C_LIO_LIIn vivo, the extracellular chloride concentration was spatially heterogenous and only half of the CSF chloride concentration C_LIO_LIStable displacement of extracellular chloride by immobile extracellular anions was responsible for the low extracellular chloride concentration C_LIO_LIThe changes in extracellular chloride were of sufficient magnitude to alter the conductance and reversal potential of GABAA chloride currents C_LIO_LIThe stability of the extracellular matrix, the impact of the component immobile anions including sulfated glycosaminoglycans on extracellular chloride concentrations, and the consequent effect on GABAA signaling suggests a previously unappreciated mechanism for modulating GABAA signaling. C_LI

neuroscience↗

bioRxiv2020

Unique actions of GABA arising from cytoplasmic chloride microdomains

Developmental, cellular, and subcellular variations in the direction of neuronal Cl- currents elicited by GABAA receptor activation have been frequently reported, and we found a corresponding variance in the reversal potential (EGABA) for individual interneurons synapsing on a single pyramidal cell. These findings suggest a corresponding variance in the cytoplasmic concentration of Cl- ([Cl-i]). We determined [Cl-]i by: 1) two-photon imaging of the Cl- sensitive, ratiometric fluorescent protein SuperClomeleon (sCLM); 2) Fluorescence Lifetime IMaging (FLIM) of the Cl- sensitive fluorophore MEQ; and 3) electrophysiological measurements of EGABA. These methods collectively demonstrated stable [Cl-]i microdomains in individual neurons in vivo. Fluorometric and electrophysiological estimates of local [Cl-]i were highly correlated. [Cl-]i microdomains persisted after pharmacological inhibition of cation-chloride cotransporters (CCCs) but steadily decreased after inhibiting the polymerization of the anionic macromolecule actin. These studies highlight the existence of functionally significant neuronal Cl- microdomains that modify the impact of GABAergic inputs.

neuroscience↗