Spike-Related Electrophysiological Identification of Cultured Hippocampal Excitatory and Inhibitory Neurons

  • Cosimo Prestigio
  • Daniele Ferrante
  • Pierluigi Valente
  • Silvia Casagrande
  • Ennio Albanesi
  • Yuchio Yanagawa
  • Fabio Benfenati
  • Pietro BaldelliEmail author


Cultured hippocampal neurons represent the most widely used experimental substrate for in vitro electrophysiological studies. Nevertheless, in most cases, the nature of neuron under study is not identified as excitatory or inhibitory, or even worse, recorded neurons are considered as excitatory because of the paucity of GABAergic interneurons. Thus, the definition of reliable criteria able to guarantee an unequivocal identification of excitatory and inhibitory cultured hippocampal neurons is an unmet need. To reach this goal, we compared the electrophysiological properties and the localization and size of the axon initial segment (AIS) of cultured hippocampal neurons, taking advantage from GAD67-GFP knock-in mice, which expressing green fluorescent protein (GFP) in gamma-aminobutyric acid (GABA)–containing cells, allowed to unambiguously determine the precise nature of the neuron under study. Our results demonstrate that the passive electrophysiological properties, the localization and size of the AIS, and the shape and frequency of the action potential (AP) are not reliable to unequivocally identify neurons as excitatory or inhibitory. The only parameter, related to the shape of the single AP, showing minimal overlap between the sample-point distributions of the two neuronal subpopulations, was the AP half-width. However, the estimation of the AP failure ratio evoked by a short train of high-current steps applied at increasing frequency (40–140 Hz) resulted to be indisputably the safer and faster way to identify the excitatory or inhibitory nature of an unknown neuron. Our findings provide a precise framework for further electrophysiological investigations of in vitro hippocampal neurons.


Excitatory neurons Inhibitory neurons Action potential Firing frequency Half-width Axonal initial segment 


Funding Information

This study was supported by research grants from the Compagnia di San Paolo Torino (ID ROL 20612 and 9344); Ministero della Salute Ricerca Finalizzata (GR-2016-02363972); EU Era-Net Neuron 2017 “Snaropathies” and ITN “ECMED” (Grant agreement n. 642881).

Compliance with Ethical Standards

All experiments were performed in accordance with the guidelines established by the European Communities Council (Directive 2010/63/EU of September 22, 2010) and were approved by the Italian Ministry of Health.

Conflict of Interest

The authors declare that they have no competing interests.


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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Center for Synaptic Neuroscience and TechnologyIstituto Italiano di TecnologiaGenoaItaly
  2. 2.Department of Experimental Medicine, School of Medicine and PharmacyUniversity of GenoaGenoaItaly
  3. 3.IRCSS, Ospedale Policlinico San MartinoGenoaItaly
  4. 4.Department of Neuroscience and Brain TechnologiesIstituto Italiano di TecnologiaGenovaItaly
  5. 5.Department of Genetic and Behavioral NeuroscienceGunma University Graduate School of MedicineMaebashiJapan

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