Selective modulation of chemical and electrical synapses of Helix neuronal networks during in vitro development

BMC Neuroscience

Sergio Martinoia²  Mirella Ghirardi³  Carlo NG Giachello¹  Paolo Massobrio² 

[1] Department of Neuroscience, University of Torino, Torino, Italy;Neuroengineering and Bio-nano Technology Group (NBT), Department of Informatics, Bioengineering, Robotics, System Engineering (DIBRIS), University of Genova, Genova, Italy;Istituto Nazionale di Neuroscienze, Torino, Italy

关键词: Dynamics;    Network development;    Functional connectivity;    Micro-Electrode Arrays;    Helix neurons;   

Others  :  1140444
DOI  :  10.1186/1471-2202-14-22

 received in 2012-10-03, accepted in 2013-02-18,  发布年份 2013

【 摘 要 】

Background

A 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.

Results

In 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.

Conclusions

Networks 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.

【 授权许可】

   
2013 Massobrio et al.; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Altrup U, Speckmann EJ: Identified neuronal individuals in the buccal ganglia of Helix pomatia. Neurosci Behav Physiol 1994, 24:23-32.
• [2]Kononenko NI: Excitatory and inhibitory monosyanptic peptidercic transmission in the CNS of the snail Helix pomatia. Neurosci Behav Physiol 1994, 24:203-208.
• [3]Bulloch AGM, Syed NI: Reconstruction of neuronal networks in culture. Trends Neurosci 1992, 15:422-427.
• [4]Ghirardi M, Casadio A, Santarelli L, Montarolo PG: Aplysia hemolymph promotes neurite outgrowth and synaptogenesis of identified Helix neurons in cell culture. Invert Neurosci 1996, 2:41-49.
• [5]Fiumara F, Leitinger G, Milanese C, Montarolo PG, Ghirardi M: In vitro formation and activity-dependent plasticity of synapses between helix neurons involved in the neural control of feeding and withdrawal behaviors. Neuroscience 2005, 134:1133-1151.
• [6]Fiumara F, Milanese C, Corradi A, Giovedì S, Leitinger G, Menegon A, Montarolo PG, Benfenati F, Ghirardi M: Phosphorylation of synapsin domain A is required for post-tetanic potentiation. J Cell Sci 2007, 120:3228-3237.
• [7]Giachello CN, Fiumara F, Giacomini C, Corradi A, Milanese C, Ghirardi M, Benfenati F, Montarolo PG: MAPK/Erk-dependent phosphorylation of synapsin mediates formation of functional synapses and short-term homosynaptic plasticity. J Cell Sci 2010, 123:881-893.
• [8]Giachello CN, Montarolo PG, Ghirardi M: Synaptic functions of invertebrate varicosities: what molecular mechanisms lie beneath. Neural Plast 2012, 2012:670821.
• [9]Kleinfeld D, Raccuia-Behling F, Chiel HJ: Circuits constructed from identified Aplysia neurons exhibit multiple patterns of persistent activity. Biophys J 1990, 57:697-715.
• [10]Massobrio P, Tedesco M, Giachello C, Ghirardi M, Fiumara F, Martinoia S: Helix neuronal ensembles with controlled cell type composition and placement develop functional polysynaptic circuits on Micro-Electrode Arrays. Neurosci Lett 2009, 467:121-126.
• [11]Cottrell GA, Macon JB: Synaptic connexions of two symmetrically placed giant serotonin-containing neurones. J Physiol 1974, 236:435-464.
• [12]Chiappalone M, Bove M, Vato A, Tedesco M, Martinoia S: Dissociated cortical networks show spontaneously correlated activity patterns during in vitro development. Brain Res 2006, 1093:41-53.
• [13]Wagenaar DA, Pine J, Potter SM: An extremely rich repertoire of bursting patterns during the development of cortical cultures. BMC Neurosci 2006, 7:11. BioMed Central Full Text
• [14]Van Pelt J, Vajda I, Wolters PS: Corner MA, Ramakers GJA: Dynamics and plasticity in developing neural networks in vitro. Prog Brain Res 2005, 147:173-188.
• [15]Van Pelt J, Wolters PS, Corner MA, Rutten WLC, Ramakers GJA: Long-term characterization of firing dynamics of spontaneous bursts in cultured neural networks. IEEE Trans Biomed Eng 2004, 51:2051-2062.
• [16]Downes JH, Hammond MW, Xydas D, Spencer MC, Becerra VM, Warwick K, Whalley BJ, Nasuto SJ: Emergence of a Small-World Functional Network in Cultured Neurons. PLoS Comput Biol 2012, 8:e1002522.
• [17]Pasquale V, Massobrio P, Bologna LL, Chiappalone M, Martinoia S: Self-organization and neuronal avalanches in networks of dissociated cortical neurons. Neuroscience 2008, 153:1354-1369.
• [18]Claverol-Tinturé E, Rossel X, Cabestany J: Technical steps towards one-to-one electrode–neuron interfacing with neural circuits reconstructed in vitro. Neurocomputing 2007, 70:2716-2722.
• [19]Garofalo M, Nieus T, Massobrio P, Martinoia S: Evaluation of the performance of information theory-based methods and cross-correlation to estimate the functional connectivity in cortical networks. PLoS One 2009, 4:e6482.
• [20]Jacklet JW, Grizzaffi J, Tieman DG: Serotonin and cAMP induce excitatory modulation of a serotonergic neuron. J Neurobiol 2006, 66:499-510.
• [21]Weiss KR, Chiel HJ, Koch U, Kupfermann I: Activity of an identified histaminergic neuron, and its possible role in arousal of feeding behavior in semi-intact Aplysia. J Neurosci 1986, 6:2403-2415.
• [22]Haydon PG, McCobb DP, Kater SB: Serotonin selectively inhibits growth cone motility and synaptogenesis of specific identified neurons. Science 1984, 226:561-564.
• [23]Haydon PG, McCobb DP, Kater SB: The regulation of neurite outgrowth, growth cone motility, and electrical synaptogenesis by serotonin. J Neurobiol 1987, 18:197-215.
• [24]Goldberg JI: Serotonin regulation of neurite outgrowth in identified neurons from mature and embryonic Helisoma trivolvis. Perspect Dev Neurobiol 1998, 5:373-387.
• [25]Kanagasabapathi TT, Massobrio P, Barone RA, Tedesco M, Martinoia S, Wadman WJ, Decre MMJ: Functional connectivity and dynamics of cortical-thalamic networks co-cultured in a dual compartment device. J Neural Eng 2012, 9(3):036010.
• [26]Maccione A, Garofalo M, Nieus T, Tedesco M, Berdondini L, Martinoia S: Multiscale functional connectivity estimation on low-density neuronal cultures recorded by high-density CMOS Micro Electrode Arrays. J Neurosci Methods 2012, 207:161-171.
• [27]Martinoia S, Massobrio P, Bove M, Massobrio G: Cultured neurons coupled to microelectrode arrays: circuit models, simulations and experimental data. IEEE Trans Biomed Eng 2004, 51:859-864.
• [28]Massobrio P, Massobrio G, Martinoia S: Multi-program approach for simulating recorded extracellular signals generated by neurons coupled to microelectrode arrays. Neurocomputing 2007, 70:2467-2476.
• [29]Fromherz P, In Nanoelectronics and Information Technology: Neuroelectronic interfacing: semiconductor chips with ion channels, nerve cells, and brain. Berlin: Wiley-VCH: Edited by Waser R; 2003:781-810.
• [30]Claverol-Tinturé E, Cabestany J, Rossel X: Multisite recording of extracellular potentials produced by microchannel-confined neurons in-vitro. IEEE Trans Biomed Eng 2007, 54:331-335.
• [31]Baxter DA, Byrne JH: Differential effects of cAMP and serotonin on membrane current, action-potential duration, and excitability in somata of pleural sensory neurons of Aplysia. J Neurophysiol 1990, 64:978-990.
• [32]Klein M, Hochner B, Kandel ER: Facilitatory transmitters and cAMP can modulate accommodation as well as transmitter release in Aplysia sensory neurons: Evidence for parallel processing in a single cell. Proc Natl Acad Sci USA 1986, 83:7994-7998.
• [33]Stark LL, Mercer AR, Emptage NJ, Carew TJ: Pharmacological and kinetic characterization of two functional classes of serotonergic modulation in Aplysia sensory neurons. J Neurophysiol 1996, 75:855-866.
• [34]Walters ET, Byrne JH, Carew TJ, Kandel ER: Mechanoafferent neurons innervating tail of Aplysia. II. Modulation by sensitizing stimulation. J Neurophysiol 1983, 50:1543-1559.
• [35]Dale N, Kandel ER, Schacher S: Serotonin produces long-term changes in the excitability of Aplysia sensory neurons in culture that depend on new protein synthesis. J Neurosci 1987, 7:2232-2238.
• [36]Ghirardi M, Braha O, Hochner B, Montarolo PG, Kandel ER, Dale N: Roles of PKA and PKC in facilitation of evoked and spontaneous transmitter release at depressed and nondepressed synapses in Aplysia sensory neurons. Neuron 1992, 9:479-489.
• [37]Araneda R, Andrade R: 5-Hydroxytryptamine2 and 5-hydroxytryptamine 1A receptors mediate opposing responses on membrane excitability in rat association cortex. Neuroscience 1991, 40:399-412.
• [38]Zhong P, Yuen EY, Yan Z: Modulation of neuronal excitability by serotonin-NMDA interactions in prefrontal cortex. Mol Cell Neurosci 2008, 38:290-299.
• [39]Weiss KR, Shapiro E, Kupfermann I: Modulatory synaptic actions of an identified histaminergic neuron on the serotonergic metacerebral cell of Aplysia. J Neurosci 1986, 6:2393-2402.
• [40]Sikich L, Hickok JM, Todd RD: 5-HT1A receptors control neurite branching during development. Dev Brain Res 1990, 56:269-274.
• [41]Dudok JJ, Groffen AJ, Witter MP, Voorn P, Verhage M: Chronic activation of the 5-HT(2) receptor reduces 5-HT neurite density as studied in organotypic slice cultures. Brain Res 2009, 1302:1-9.
• [42]Haydon PG, Kater SB: The differential regulation of formation of chemical and electrical connections in Helisoma. J Neurobiol 1988, 19:636-655.
• [43]Hadley RD, Bodnar DA, Kater SB: Formation of electrical synapses between isolated, cultured Helisoma neurons requires mutual neurite elongation. J Neurosci 1985, 5:3145-3153.
• [44]Hadley RD, Kater SB: Competence to form electrical connections is restricted to growing neurites in the snail, Helisoma. J Neurosci 1983, 3:924-932.
• [45]Hadley RD, Kater SB, Cohan CS: Electrical synapse formation depends on interaction of mutually growing neurites. Science 1983, 221:466-468.
• [46]Berdan RC, Shivers RR, Bulloch AG: Chemical synapses, particle arrays, pseudo-gap junctions and gap junctions of neurons and glia in the buccal ganglion of Helisoma. Synapse 1987, 1:304-323.
• [47]Guthrie PB, Lee RE, Rehder V, Schmidt MF, Kater SB: Self-recognition: a constraint on the formation of electrical coupling in neurons. J Neurosci 1994, 14:1477-1485.
• [48]Szabo TM, Caplan JS, Zoran MJ: Serotonin regulates electrical coupling via modulation of extrajunctional conductance: H-current. Brain Res 2010, 1349:21-31.
• [49]Rorig B, Sutor B: Serotonin regulates gap junction coupling in the developing rat somatosensory cortex. Eur J Neurosci 1996, 8:1685-1695.
• [50]Bennett MV, Zukin RS: Electrical coupling and neuronal synchronization in the mammalian brain. Neuron 2004, 41:495-511.
• [51]Kavasseri RG, Nagarajan R: Synchronization in electrically coupled neural networks. Complex Syst 2006, 16:369-380.
• [52]Klein M, Kandel ER: Mechanism of calcium current modulation underlying presynaptic facilitation and behavioral sensitization in Aplysia. Proc Natl Acad Sci USA 1980, 77:6912-6916.
• [53]Delaney K, Tank DW, Zucker RS: Presynaptic calcium and serotonin-mediated enhancement of transmitter release at crayfish neuromuscular junction. J Neurosci 1991, 11:2631-2643.
• [54]Katz PS, Frost WN: Intrinsic neuromodulation in the Tritonia swim CPG: the serotonergic dorsal swim interneurons act presynaptically to enhance transmitter release from interneuron C2. J Neurosci 1995, 15:6035-6045.
• [55]Maccione A, Gandolfo M, Massobrio P, Novellino A, Martinoia S, Chiappalone M: A novel algorithm for precise identification of spikes in extracellularly recorded neuronal signals. J Neurosci Methods 2009, 177:241-249.
• [56]Rieke F, Warland D, de Ruyter van Steveninck R, Bialek W: Spikes: exploring the neural code. Cambridge, Massachusetts: The MIT Press; 1997.
• [57]Dayan P, Abbott LF: Theoretical neuroscience. Cambridge: MIT; 2001.
• [58]Friston KJ: Functional and effective connectivity in neuroimaging: a syntesis. Hum Brain Mapp 1994, 2:56-78.