Quantitative Reflection Imaging for the Morphology and Dynamics of Live Aplysia californica Pedal Ganglion Neurons Cultured on Nanostructured Plasmonic Crystals

Abstract

We describe a reflection imaging system which consists of a plasmonic crystal, a common laboratory microscope, and bandpass filters for use in the quantitative imaging and in-situ monitoring of live cells and their substrate interactions. Surface plasmon resonance (SPR) provides a highly sensitive method to monitor changes in physicochemical properties occurring at metal-dielectric interfaces. Polyelectrolyte thin films deposited using the layer-by-layer (LBL) self-assembly method provide a reference system to calibrate the reflection contrast changes that occur when the polyelectrolyte film thickness changes, as well as provide insight into the optical responses that originate from the multiple plasmonic features supported by this imaging system. Finite-difference time-domain (FDTD) simulations of the optical responses measured experimentally from the polyelectrolyte reference system are used to provide a calibration of the optical system for subsequent use in quantitative studies investigating live cell dynamics in cultures supported on a plasmonic crystal substrate. Live Aplysia californica pedal ganglion neurons cultured in artificial sea water were used as a model system through which to explore the utility of this plasmonic imaging technique. Here, the morphology of cellular peripheral structures < ~80 nm in thickness were quantitatively analyzed and the dynamics of their trypsin-induced surface detachment visualized. These results illustrate capacities of this system for use in investigations of the dynamics of ultrathin cellular structures within complex bioanalytical environments.

Single Synaptic Observation of Cholinergic Neurotransmission on Living Neurons: Concentration and Dynamics

“…1G). The neuronal model used in the present study is Aplysia californica , previously used by Kandel et al to understand the synapse-specific long-term facilitation 30, 31 ; studied pedal ganglion neurons are cholinergic 32, 33, 34 and they were cultured following the well-established protocols 35, 36 , with details shown in the Supporting Information. We employed nanoresolved scanning electrochemical microscopy (SECM) 37, 38, 39, 40, 41, 42 (Fig.…”

“…The neuronal model used in the present study is Aplysia californica, previously used by Kandel et al. to understand the synapse-specific long-term facilitation; , studied pedal ganglion neurons are cholinergic − and they were cultured following the well-established protocols, , with details shown in the Supporting Information. We employed nanoresolved scanning electrochemical microscopy (SECM) − (Figure E) to position the nanoelectrode near the synaptic cleft, formed between the axon of one neuron (pink) and the neurite of another neuron (blue) (Figure B,F).…”

A high spatiotemporal study of somatic exocytosis with scanning electrochemical microscopy and nanoITIES electrodes

“…The neuronal model in the current study is Aplysia californica , which has been demonstrated as an important model to understand cellular communication and fundamental mechanisms of learning and memory. 26 – 31 Aplysia contains a large number of neurons of varying sizes, facilitating its isolation and experimentation at the single cell level. 29 , 31 , 32 The release of ACh from the living pedal ganglion neurons of this organism in response to chemical stimulation has not been extensively studied; however, the presence of choline acetyltransferase, the enzyme responsible for the synthesis of acetylcholine, has been observed, 33 , 34 indicating cholinergic activity within these neurons.…”

“… 26 – 31 Aplysia contains a large number of neurons of varying sizes, facilitating its isolation and experimentation at the single cell level. 29 , 31 , 32 The release of ACh from the living pedal ganglion neurons of this organism in response to chemical stimulation has not been extensively studied; however, the presence of choline acetyltransferase, the enzyme responsible for the synthesis of acetylcholine, has been observed, 33 , 34 indicating cholinergic activity within these neurons. 34 – 36 The study of cholinergic release from Aplysia californica neurons contributes to a greater understanding of the role that acetylcholine plays in cellular communication.…”

A Nanopipette Platform for Delivering Nanoliter Volumes of Acetylcholine

“…This is because ASW is the biological cellular medium for an important neuronal model, Aplysia California, which has been successfully used to provide fundamental understanding of brain functions. [20][21][22][23][24][25][26][27][28] Developing the delivery system in ASW well positions us to carry out future z E-mail: mshen233@illinois.edu drug delivery experiments with the presence of the living neurons in the biological medium.…”