Biology subjects

Lillis, K. P.

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

4 recordsLinked to original sources

bioRxiv2026

Robust Induction of Spontaneous Recurrent Seizures and Hippocampal Sclerosis with Synthetic Kainic Acid in the Intrahippocampal Kainate Model

Intrahippocampal kainate (IHK) is a well-established mouse model of temporal lobe epilepsy, in which focal hippocampal injection of kainic acid induces excitotoxic injury followed by the development of chronic spontaneous seizures. Commercial sources of kainic acid (KA) have increasingly transitioned from naturally derived to synthetic KA, which has introduced a potential source of variability to the implementation of the IHK model. Here, we evaluated injection parameters for synthetic KA, with the goal of identifying conditions that reliably produce spontaneous seizures while minimizing mortality. We found that intrahippocampal injection of 200nL of 5mM synthetic KA resulted in low mortality compared to 50-100nL of 20mM synthetic KA (a dosing range commonly used with naturally derived KA), while robustly inducing TLE-like hippocampal sclerosis and recurrent spontaneous electroclinical seizures. We further demonstrated high inter-animal variability in both seizure frequency and temporal clustering of seizures across animals receiving either injection protocol (200nL of 5mM or 50nL of 20mM). Together, these findings provide a framework for implementing the IHK model with synthetic KA and support its continued use to advance our understanding of the cellular and network mechanisms underlying temporal lobe epilepsy.

neuroscience↗

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

Cellular contributions to ictal population signals

ObjectiveThe amplitude of ictal activity is a defining feature of epileptic seizures, but the determinants of this amplitude have not been identified. Clinically, ictal amplitudes are measured electrographically (using e.g. EEG, ECoG, and depth electrodes), but these methods do not enable the assessment of the activity of individual neurons. To identify the cellular determinants of the ictal signal, we measured single cell and population electrical activity and neuronal calcium levels via optical imaging of the genetically encoded calcium indicator (GECI) GCaMP. MethodsSpontaneous seizure activity was assessed in an awake, behaving mouse model of focal cortical injury and in organotypic hippocampal slice cultures (OHSC), an in vitro preparation from which recurrent seizures can be readily captured. Single cell calcium signals were linked to a range of electrical activities by performing simultaneous GECI-based calcium imaging and whole-cell patch-clamp recordings in spontaneously seizing OHSCs. Neuronal resolution calcium imaging was then performed during spontaneous seizures in vivo and in vitro to quantify the cellular contributions to the population-level calcium signal. ResultsPopulation signal may increase from three potential sources: 1) increased synchrony, i.e. more co-active neurons, 2) altered active state, from bursts of action potentials and/or paroxysmal depolarizing shifts in membrane potential, and 3) altered subthreshold state, which includes all lower levels of activity. The largest contributor to the signal recorded at seizure onset was increased subthreshold activity, consistent with either barrages of excitatory postsynaptic potentials or sustained membrane depolarization. The relative contribution of synchrony increased as seizures progressed, but cell intrinsic alterations in both the subthreshold and active states remained the largest driver of the ictal signal. SignificanceWe introduce here a novel method for the quantification of the relative contributions of inter-versus intra-cellular changes to provide a critical link between single neuron activity and population measures of seizure activity. Key PointsO_LINeuronal calcium as measured by GCaMP reports a range of membrane depolarizations, from EPSPs to action potential firing and paroxysmal depolarizing shifts C_LIO_LIThe mean population calcium signal is highly correlated with the electrographic local field potential C_LIO_LIIncreased calcium signal during seizure onset is largely driven by increased subthreshold calcium within individual neurons C_LIO_LIRecruitment of newly active neurons is a minor contributor to the increasing population-level signal during the transition to frank seizure 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↗