Gamma, but not Delta, Theta, and Beta Oscillation Occurrence Increases with Age of Organotypic Hippocampal Cultures
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1
F. Hoffman-La Roche, Pharmaceutical Research and Early Development, Neuroscience, Switzerland
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2
ETH Zurich, Department of Biosystems Science and Engineering, Switzerland
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3
F. Hoffman-La Roche, Pharmaceutical Research and Early Development, Neuroscience, United States
Motivation
Gamma oscillations are altered in schizophrenia patients as measured with EEG (Sun et al., 2011). In schizophrenic patients, expression of parvalbumin is decreased (Fung et al., 2010). Parvalbumin-containing interneurons are thought to be important in gamma oscillations (Sohal et al., 2009). A prominent theory of schizophrenia pathophysiology is the NMDA receptor hypofunction hypothesis. Knocking out the NR2A subunit of the NMDAR in mice could mimic the NMDAR hypofunction and alter oscillations.
Material and Methods
Experiments with organotypic cultures were done on microelectrode arrays with 60 planar electrodes (Multi Channel Systems, Reutlingen, Germany). Organotypic hippocampus cultures of P7 mice (wild type and NR2A KO) were made with the Stoppini method. Oscillations were induced by dosage of 10 µM carbachol at 7,13 and 26 days in vitro (DIV). Normalized power spectra were used to select the frequency range of each of the peaks by hand. Active channels were selected by a peak prominence of at least 0.001 (normalized units). Peak prominence quantifies how much a peak stands out relative to other peaks. It is determined by extending a horizontal line to the left and right of a local maximum, until it crosses the signal (or the end of the signal). The minimum in both intervals is taken. Peak prominence is then how much the peak lies above the highest of the two minima. The frequency bands were categorized as delta (1-4 Hz), theta (5-10 Hz), beta (11-30 Hz), and gamma (31-100 Hz). We categorized channels as being either on the hippocampal areas DG, CA3, CA1 or none.
We performed stepwise binomial logistic regression to model the probability of oscillation occurrence (yes/no) over the different conditions. For each oscillation band, the algorithm started with the full genotype*DIV*area model (i.e., the main effects and interactions). It then looks for the best fit by removing predictors based on the deviance criterion.
Results
The initial analyses for delta, theta, and gamma oscillations resulted in models that predicted the data better than the constant model - the overall probability of occurence without any predictors (p < 0.01 for all three). For beta, no such model was found. Because none of these models included the area predictor, we decided to compress the three observations for DG, CA3 and CA1 per culture into a single observation per culture.
We then initiated the stepwise regression with the genotype*DIV model. With this analysis, no model was found to be better than constant for delta, theta and beta. For gamma, the DIV variable predicts the occurrence of the oscillation better than a constant model (p = 0.008).The estimated probability of gamma oscillations occurring in organotypic hippocampal cultures increases with DIV (Fig 1).
Discussion
Gamma oscillations were found to be more likely to occur at later DIVs, but this was not the case for the other oscillations. A major increase in estimated probability of gamma happens between DIV 7 and 13, whereas the increase in estimated probability between DIV 13 and 26 is minor. From previous analysis on parvalbumin expression in our cultures, we have seen a similar pattern; low PV expression at DIV 7, increasing over the next week at DIV 14 and then staying at this level to DIV 25.
Initial analysis on the occurrence of oscillations showed that there is no tendency for any of the oscillations to occur predominantly within a specific area of the hippocampal culture. This means that the activity does not stay within the area of origin but spreads throughout the culture. A different analysis is needed to determine the area of origin.
The regression models did not predict a difference between wild-type and NR2A KO in the probability of oscillation occurrence for any of the four bands. The genotypes were both expected to show oscillations. Schizophrenic patients do have gamma oscillations, however those are irregular compared to healthy subjects. The abnormalities occur in power and synchrony of the gamma oscillations.
Conclusion
We conclude that organotypic cultures show a development of gamma oscillations that is similar to that in vivo. Combined with MEA technology, these cultures can be a flexible method to explore neurodevelopmental disease models in vitro.
References
Fung, S. J., Webster, M. J., Sivagnanasundaram, S., Duncan, C., Elashoff, M., & Weickert, C. S. (2010). Expression of interneuron markers in the dorsolateral prefrontal cortex of the developing human and in schizophrenia. American Journal of Psychiatry, 167(12), 1479-1488.
Sohal, V. S., Zhang, F., Yizhar, O., & Deisseroth, K. (2009). Parvalbumin neurons and gamma rhythms enhance cortical circuit performance. Nature, 459(7247), 698-702.
Sun, Y., Farzan, F., Barr, M. S., Kirihara, K., Fitzgerald, P. B., Light, G. A., & Daskalakis, Z. J. (2011). Gamma oscillations in schizophrenia: mechanisms and clinical significance. Brain research, 1413, 98-114.
Figure Legend
Figure 1. Estimated probabilities of oscillation occurrence. Stepwise logistic regression eliminated the area and genotype predictors for all four oscillation bands. After removing the area variable from the data, the DIV predictor was significantly different from a constant model for gamma oscillations. The estimated probabilities show a strong increase between div 7 and 13, and a mild increase between div 13 and 26. For comparison, the constant models of the other oscillations are also shown.
Acknowledgements
This project formed part of the EU Marie Curie Initial Training Network (ITN) “Biomedical engineering for cancer and brain disease diagnosis and therapy development: EngCaBra”, contract no.264417. Authors would like to thank the Donders Institute at University of Nijmegen for providing the FieldTrip EEG analysis software (http://fieldtrip.fcdonders.nl).
Keywords:
Carbachol,
Hippocampus,
NMDA,
gamma oscillations,
NR2A,
organotypic
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:
Van Lier
B,
Hierlemann
A and
Knoflach
F
(2016). Gamma, but not Delta, Theta, and Beta Oscillation Occurrence Increases with Age of Organotypic Hippocampal Cultures.
Front. Neurosci.
Conference Abstract:
MEA Meeting 2016 |
10th International Meeting on Substrate-Integrated Electrode Arrays.
doi: 10.3389/conf.fnins.2016.93.00017
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Received:
22 Jun 2016;
Published Online:
24 Jun 2016.
*
Correspondence:
Dr. Ben Van Lier, F. Hoffman-La Roche, Pharmaceutical Research and Early Development, Neuroscience, Basel, Switzerland, ben.vanlier@bsse.ethz.ch