Aim: 

Classical synaptic-based plasticity mechanisms, such as Long Term Potentiation (LTP) or Long Term Depression (LTD), have been demonstrated in many experimental in-vitro preparations. The vast majority of the experimental works related to activity-dependent synaptic modifications refers to in vitro slices or dissociated cultures where defined synaptic pathways and neurons are identified. Yet, many studies on synaptic plasticity focus primarily on changes in the Excitatory Post Synaptic Potential (i.e. EPSP) rather than on changes in the firing behavior. In this work we propose the use of dissociated cortical cultures coupled to Micro-Electrode-Arrays (MEAs) to investigate plasticity at network/cell-assembly level.

Methods: 

To address this issue, we used dissociated cortical cultures chronically coupled to MEAs. These devices allow to record and stimulate simultaneously a neuronal network without damaging the cells, being only extracellular the nature of the measurements. After a few days in culture, the random network starts showing patterns of collective activity which can be modulated by means of focused electrical stimulation through one or more electrodes of the array. In our work, we investigated the plastic properties of a neuronal network (as a whole) in terms of spiking activity and input/output (I/O) response. The use of multi-site simultaneous recording, allowed us to capture changes in the spiking activity at population level and variations in the effective connectivity of the network based on revised protocols of 'tetanic' stimulation. First, the network evoked response is tested for stability; then, a tetanic high frequency stimulus and a low frequency reference stimulus are applied together at two specific microelectrodes and the network activity is recorded simultaneously from the others (usually 58 channels). Experimental results, analyzed at the network level and mainly based on the Post Stimulus Time Histogram evaluation, show remarkable differences in the electrophysiological activity of the network after the tetanus, denoting an induction of potentiation, related to the level of the initial evoked activity.

Results: 

Our experimental findings show that in vitro cortical ensembles display network potentiation and increased effective connectivity in many synaptic pathways as a consequence of multi-site external stimulation. We summarize our main results as follows: (i) low frequency stimuli produce neither short nor long-term changes in the evoked response of the network; (ii) associative tetanic stimulation is able to induce plasticity in terms of a significant increase or decrease of the evoked activity in the whole network; (iii) the amount of change (i.e. increase or decrease of the evoked firing) strongly depends on the specific features of the applied protocols; (iv) the potentiation induced by a specific associative protocol can last several hours. Additionally in few preliminary experiments we look for stable potentiation after 24 hours and we found in some cases maintenance or even an increase in the level of the evoked responses.

Conclusion: 

Large in vitro cortical assemblies display Long Term Network Potentiation (LTNP), a mechanism supposed to be involved in the memory formation at cellular level. We think that this pilot study could represent a relevant step towards understanding the plastic properties at neuronal population level including the very long term studies that could be used, to investigate the mechanism of consolidation and the nature of the enduring changes that underlies long-term memory (LTM).