Circadian rhythm monitoring and manipulation in neural assemblies
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1
Istituto Italiano di Tecnologia, Department of Neuroscience and Brain Technologies, Italy
Motivation
Neuronal assemblies plated on Microelectrode Array (MEA) show synchronized, low frequency firing patterns similar to in vivo slow wave oscillations, a key feature of sleep-like state [1]. By using dissociated cultures on MEAs, we wanted to answer two main questions: (i) Is it possible to modulate the activity pattern of in vitro neuronal networks, simulating the oscillatory activity of the circadian rhythm? (ii) How does the electrophysiological signal change by modifying the genes responsible of the regulation of the circadian rhythm? And is it possible to modulate the synchrony of the circadian clock?
Material and Methods
Cell cultures prepared from embryonic rodents at gestational day 18 were plated onto 60-channel MEAs (Multichannel Systems, MCS, Reutlingen, Germany) previously coated with poly-D-lysine and laminin to promote cell adhesion [2]. Regarding the first question, the cultures were monitored for 7 hours in basal condition and then exposed to cholinergic agonist Carbachol (CCh,20 ?M) for 24 hours [3]. The electrophysiological activity was analyzed by calculating several parameters such as Mean Firing Rate, MFR [spike/sec], Inverse Burst Ratio, IBR, (percentage of spike outside the burst) and Burstiness Index, BI, (index of the burstiness level of the network) [4]. We quantified the correlation between each pair of channels measured by the spike time tiling coefficient (STTC) [5]. Regarding our second scientific question, we used a circadian rodents mutant line called After Hours (Afh). This mutation produces an elongation of the circadian period from 24 to 27 hours. For each genotype, i.e. Wild Type WT (+/+), and After Hours (Afh/Afh), we administered Dexamethasone (Dexa, 100nM), a corticosteroid related drug, for 24 hours. This drug induced a synchronized transcription of circadian genes, resetting the timing of the circadian clock in primary neurons in vitro. For this experiment we analyzed the same networks parameters described above. For statistical analysis, since data were not normally distributed, we performed non-parametric tests (i.e. Mann-Whitney), and p values<0.05 were considered significant.
Results
We recorded and monitored cortical cultures for a period of over 31 hours with (n=7) and without CCh treatment (n=4). The activity levels, expressed by MFR, were not significantly affected by the treatment (Fig1A). CCh application causes a loss of regularity and a fragmentation of burst structures, with an increased number of isolated spikes (Fig 1B). At the same time, the BI significantly decreases upon the treatment (Fig 1C). Similarly, CCh reduced markedly the correlation of activity, as shown in the Figure 1D.
For the second protocol, we recorded 24h without treatment (+/+ n=12, Afh/Afh n=20) and with treatment with Dexa (+/+ n=18; Afh/Afh n=18). The Coefficient of Variation (CV, defined as the ratio of the standard deviation to the mean) of MFR is higher in the case of Afh with respect to the +/+ cultures (Fig 1E, red and blue light column). The CV after the treatment indicates a comparable level of variability between the two genotypes (Fig 1E, red and blue column). CV of IBR showed the same trend to that found for MFR (Fig 1F). From the analysis of the CV of BI, we can note that the mutation significantly decreases the variability of +/+ neurons. The drug treatment increased the CV both of the +/+ and Afh networks (Fig 1 G).
Discussion
To summarize, we found that: i) The CCh treatment is able to modulate the activity of the in vitro neural networks, from synchronous to asynchronous. This asynchronous firing pattern is highly similar to the rapid and low voltage waves recording during wakefulness and REM sleep phases. The gene expression analysis shows that CCh caused an activation of molecular markers involved in the sleep-wake rhythms (data not shown). Therefore, CCh represents a suitable experimental strategy to suppress the sleep-like pattern of activity. ii) The mutation on the circadian genes produced an increase in the variability of the electrophysiological signal with respect to the wild type cultures, while the application of Dexa increased the instability in a genetically unperturbed system. This suggests that the electrophysiological signal of mutants is characterized by a high variability that is comparable to the wild type treated cultures.
Conclusion
This study demonstrates how primary cortical networks coupled to MEAs represent a promising and powerful experimental model to monitor and manipulate the circadian rhythm in vitro.
Reference
1. Hinard, V., et al., Key electrophysiological, molecular, and metabolic signatures of sleep and wakefulness revealed in primary cortical cultures. J Neurosci, 2012. 32(36): p. 12506-17.
2. Colombi, I., et al., Effects of antiepileptic drugs on hippocampal neurons coupled to micro-electrode arrays. Front Neuroeng, 2013. 6: p. 10.
3. Kaufman, M., M.A. Corner, and N.E. Ziv, Long-term relationships between cholinergic tone, synchronous bursting and synaptic remodeling. PLoS One, 2012. 7(7): p. e40980.
4. Wagenaar, D.A., et al., Controlling bursting in cortical cultures with closed-loop multi-electrode stimulation. J Neurosci, 2005. 25(3): p. 680-8.
5. Cutts, C.S. and S.J. Eglen, Detecting pairwise correlations in spike trains: an objective comparison of methods and application to the study of retinal waves. J Neurosci, 2014. 34(43): p. 14288-303.
Figure Legend
Fig.1 (A-D) Column bar of Mean Firing rate, Inverse Burst Ratio, Burstiness Index and STTC mean values during control experiments (blue bar) and during the administration of CCh (red). Statistical Analysis was carried out using Mann-Whitney comparison test, ** p < 0.01. (E-F) CV of MFR, IBR, BI of +/+ neuronal networks without treatment (light blue line) with treatment (blue line) and Afh/Afh neuronal networks without treatment (light red line) with treatment (red line). Statistical Analysis was carried out using Mann-Whitney comparison test, * p < 0.05 difference inter genotype within the same treatment, ? p< 0.05 intra genotype with different treatment.
Keywords:
Sleep,
Circadian clock,
microelectrode arrays,
After hours,
Cortical cultures
Conference:
MEA Meeting 2016 |
10th International Meeting on Substrate-Integrated Electrode Arrays, Reutlingen, Germany, 28 Jun - 1 Jul, 2016.
Presentation Type:
Poster Presentation
Topic:
MEA Meeting 2016
Citation:
Colombi
I,
Tinarelli
F,
Pasquale
V,
Tucci
V and
Chiappalone
M
(2016). Circadian rhythm monitoring and manipulation in neural assemblies.
Front. Neurosci.
Conference Abstract:
MEA Meeting 2016 |
10th International Meeting on Substrate-Integrated Electrode Arrays.
doi: 10.3389/conf.fnins.2016.93.00052
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Received:
22 Jun 2016;
Published Online:
24 Jun 2016.
*
Correspondence:
Dr. Ilaria Colombi, Istituto Italiano di Tecnologia, Department of Neuroscience and Brain Technologies, Genova, Italy, ilaria.colombi@iit.it