Invertebrate neurons as a simple model to study the hyperexcitable state of epileptic disorders in single cells, monosynaptic connections, and polysynaptic circuits

Abstract

Epilepsy is a neurological disorder characterized by a hyperexcitable state in neurons from different brain regions. Much is unknown about epilepsy and seizures development, depicting a growing field of research. Animal models have provided important clues about the underlying mechanisms of seizure-generating neuronal circuits. Mammalian complexity still makes it difficult to define some principles of nervous system function, and non-mammalian models have played pivotal roles depending on the research question at hand. Mollusks and the Helix land snail have been used to study epileptic-like behavior in neurons. Neurons from these organisms confer advantages as single-cell identification, isolation, and culture, either as single cells or as physiological relevant monosynaptic or polysynaptic circuits, together with amenability to different protocols and treatments. This review's purpose consists in presenting relevant papers in order to gain a better understanding of Helix neurons, their characteristics, uses, and capabilities for studying the fundamental mechanisms of epileptic disorders and their treatment, to facilitate their more expansive use in epilepsy research.

Keywords: Animal models; Drug screening; Epilepsy; Helix aspersa; Helix pomatia; Ion channels; Synaptic vesicles.

Fig. 1

a Drug-induced epileptic-like activity in Helix neurons can be recorded through extracellular microelectrodes arrays culturing one single cell in each electrode, and through intracellular borosilicate fine electrodes. b C1 neurons of Helix do not present intrinsic pacemaker potentials (flat baseline signal, in the absence of stimuli, is shown), the epileptic-like activity was induced with 40 mM PTZ. The activity recorded with extracellular electrodes presented a latency of ~5 min, and the activity recorded with high-resistance intracellular electrodes started ~1.5 min after PTZ bath application, mean latency is about 4.4 ± 1.4 min (mean ± sem). In both recordings, it is possible to observe single spikes, action potential doublets, and paroxysmal depolarization shifts. Scale bar, 100 μm

Fig. 2

a Helix anesthetized animals are sacrificed by removing the shell that contains most of the visceral organs; ganglia and cells are surgically isolated. Neurons can be cultured with (upper left panel) or without (upper right panel) axons. In addition, cells can be cultured with their physiological partner in the presence of axons and neurites (bottom left panel) or through direct contact of axon-reabsorbed somata (bottom right panel). b Scheme of Helix central nervous system, showing the position of some relevant neurons. Each neuron has a contralateral counterpart. In the buccal and cerebral ganglia, left neurons are shown with their axon’s trajectories

Fig. 3

Schematic representation of Helix salivary reflex circuit (lower panel) made up of chemosensory neurons that innervate snail lips and C1 command neuron, which in turn innervates bilaterally the efferent B2 neurons with outputs to salivary glands. Sample currents (upper panels) elicited by Na⁺, K⁺, and Ca²⁺ channels (at −20 mV, 30 mV, and −10 mV, respectively) in C1 neuron are shown. The upper right panels show the apparent open probability (Po) of depicted ion channels. Insert in Ca²⁺ apparent Po allow comparing the control (black circle and blue line) and synapsin-silenced (black squares and red line) voltage dependency of Ca²⁺ currents