Abstract
BackgroundA large number of invertebrate models, including the snail Helix, emerged as particularly suitable tools for investigating the formation of synapses and the specificity of neuronal connectivity. Helix neurons can be individually identified and isolated in cell culture, showing well-conserved size, position, biophysical properties, synaptic connections, and physiological functions. Although we previously showed the potential usefulness of Helix polysynaptic circuits, a full characterization of synaptic connectivity and its dynamics during network development has not been performed.ResultsIn this paper, we systematically investigated the in vitro formation of polysynaptic circuits, among Helix B2 and the serotonergic C1 neurons, from a morphological and functional point of view. Since these cells are generally silent in culture, networks were chemically stimulated with either high extracellular potassium concentrations or, alternatively, serotonin. Potassium induced a transient depolarization of all neurons. On the other hand, we found prolonged firing activity, selectively maintained following the first serotonin application. Statistical analysis revealed no significant changes in neuronal dynamics during network development. Moreover, we demonstrated that the cell-selective effect of serotonin was also responsible for short-lasting alterations in C1 excitability, without long-term rebounds.Estimation of the functional connections by means of cross-correlation analysis revealed that networks under elevated KCl concentrations exhibited strongly correlated signals with short latencies (about 5 ms), typical of electrically coupled cells. Conversely, neurons treated with serotonin were weakly connected with longer latencies (exceeding 20 ms) between the interacting neurons. Finally, we clearly demonstrated that these two types of correlations (in terms of strength/latency) were effectively related to the presence of electrical or chemical connections, by comparing Micro-Electrode Array (MEA) signal traces with intracellularly recorded cell pairs.ConclusionsNetworks treated with either potassium or serotonin were predominantly interconnected through electrical or chemical connections, respectively. Furthermore, B2 response and short-term increase in C1 excitability induced by serotonin is sufficient to trigger spontaneous activity with chemical connections, an important requisite for long-term maintenance of firing activity.
Highlights
A large number of invertebrate models, including the snail Helix, emerged as suitable tools for investigating the formation of synapses and the specificity of neuronal connectivity
The connectivity between C1, C3, and B2 Helix neurons was first investigated at the level of micro-networks [5], and later at the level of large-networks [10], by exploiting the technology offered by Micro-Electrode Arrays (MEAs)
Experimental data collected by MEAs and presented in this work were obtained from 8 Helix cultures, monitored at the 6, 24, 36, 48, 54, and 72 hours after plating
Summary
A large number of invertebrate models, including the snail Helix, emerged as suitable tools for investigating the formation of synapses and the specificity of neuronal connectivity. The relative simplicity of the nervous system of invertebrates such as Helix snails allowed the detailed study of many aspects of neuronal connectivity [1,2,3], neurite outgrowth [4], synapse formation and plasticity [4,5,6,7] by using an in vitro approach (reviewed in [8]) In this experimental preparation, a large number of neurons can be individually identified and isolated in cell culture. The serotonergic C1 neurons, localized in the ventral side of cerebral ganglia, are synaptically connected to B2 neurons in the buccal ganglia [11], and they are involved in the regulation of feeding behaviors of Helix snails In this way, it is feasible to reconstruct in dissociated cell culture synaptic connections among individually identified invertebrate neurons that resemble in vitro their in vivo features [4]. The aforementioned studies were acutely performed, recording the electrophysiological activity for a few hours once the circuit has been established
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