<em>Aplysia</em> Ganglia Preparation for Electrophysiological and Molecular Analyses of Single Neurons

Abstract

A major challenge in neurobiology is to understand the molecular underpinnings of neural circuitry that govern a specific behavior. Once the specific molecular mechanisms are identified, new therapeutic strategies can be developed to treat abnormalities in specific behaviors caused by degenerative diseases or aging of the nervous system. The marine snail Aplysia californica is well suited for the investigations of cellular and molecular basis of behavior because neural circuitry underlying a specific behavior could be easily determined and the individual components of the circuitry could be easily manipulated. These advantages of Aplysia have led to several fundamental discoveries of neurobiology of learning and memory. Here we describe a preparation of the Aplysia nervous system for the electrophysiological and molecular analyses of individual neurons. Briefly, ganglion dissected from the nervous system is exposed to protease to remove the ganglion sheath such that neurons are exposed but retain neuronal activity as in the intact animal. This preparation is used to carry out electrophysiological measurements of single or multiple neurons. Importantly, following the recording using a simple methodology, the neurons could be isolated directly from the ganglia for gene expression analysis. These protocols were used to carry out simultaneous electrophysiological recordings from L7 and R15 neurons, study their response to acetylcholine and quantitating expression of CREB1 gene in isolated single L7, L11, R15, and R2 neurons of Aplysia.

Decreased Response to Acetylcholine during Aging of Aplysia Neuron R15

“…To study how aging affects the electrophysiological properties of neurons, we have raised a cohort of 34 animals at the National Facility for Aplysia at the University of Miami Rosenstiel School of Medicine and carried out intracellular recordings of the R15 neuron from Aplysia at two different growth stages (6-7 months and 11-12 months old). Using a modified methodology for carrying out intracellular recordings from abdominal ganglia [ 34 ], we recorded membrane potentials and bursting action potentials from R15 neurons of mature and old animals ( Figure 1 ).…”

“…Briefly, the ganglion was incubated in 0.1% dispase in 2 ml artificial seawater (ASW) at 35.5C° [ASW consisting of 450 mM NaCl, 10 mM KCl, 10 mM CaCl 2 , 55 mM MgCl 2 , 2.5 mM NaHCO 3 and 20 mM HEPES (pH 7.4)] in order to facilitate the dissection of the connective tissue sheath overlying cells. Importantly bursting activities of R15 neurons were not affected by the protease treatment [ 34 ]. We also custom-designed a recording chamber so that the ganglion can be held in position and kept under continuous perfusion of artificial seawater.…”

“…Neurons that interact with R15 and its implicated physiological functions are shown in Figure 1 . To understand the effect on aging on electrophysiological properties, we studied neurotransmitter acetylcholine-induced changes in rhythmic bursting activity of the R15 neuron using a methodology we recently described [ 34 ]. We find that the R15 neuron from mature and old animals did not show significant changes in the membrane resting potential and latency in response to acetylcholine, however, the burst number and burst duration are altered during aging.…”

Genomic and Proteomic Mechanisms and Models in Toxicity and Safety Evaluation of Nutraceuticals

“…In addition, qRT-PCR offers greater sensitivity, ability to multiplex reactions, faster assay completion, and high-throughput (Bustin, 2002). qRT-PCR has been applied extensively while studying neuronal systems and has been proven to be a reliable technique to quantify gene expression even from single neurons (Akhmedov et al, 2014). qRT-PCR has been applied extensively while studying neuronal systems and has been proven to be a reliable technique to quantify gene expression even from single neurons (Akhmedov et al, 2014).…”

Cerebral ganglion ultrastructure and differential proteins revealed using proteomics in the aplysiid (Notarcus leachii cirrosus Stimpson) under cadmium and lead stress

“…The aplysiid nervous system contains approximately 20,000 neurons which are organized into nine different ganglia. The neurons are very large and can be easily identified based on their sizes, electrical properties, and position in the ganglia (Akhmedov et al, 2014). In addition, the cerebral ganglia control many sense organs, such as antennas, oral cavity, and eyes.…”