Nat Neurosci: Differentiation of neural oscillations and epileptiform changes in human brain organoids

For some studies of diseases that affect brain growth and structural development, brain organoids are a powerful tool

However, many diseases exhibit significant physiological abnormalities and neural network abnormalities without anatomical changes, so whether organoids have enough neural network complexity to mimic the disease is an important question

The research team from the UCLA Arvid Geffen School of Medicine used calcium sensor imaging and extracellular recording methods to explore the network-level function of brain organoids, demonstrating that the organoids of somatic cell-induced pluripotent stem cells in patients with Rhett syndrome have highly abnormal epileptiform activity and transcriptomic differences, and that key physiological activities

Results of the study

Integration of excitatory and inhibitory neurons within organoids

Combining separately generated cortical and subcortical organoids using organoid fusion techniques derived from H9hESC or wild-type iPSC lines directed to Cx (cerebral cortex) or GE (ganglion bulge) identity by Figure 1a differentiation protocol, organoids exhibit cortical features

Over culture, many neurons within GE organoids express GABA-inhibiting neuronal marker proteins such as GAD65, GABA, and marker proteins for various interneuronal subtypes (Figure 1b

Inhibitory interneurons are highly reproducible in the Cx region, and eventually about 25% of cells have GABA properties (Figure 1d

In contrast, Cx+Cx organoids mainly contain only excitatory synapses (Figure 1e), indicating that most of the inhibitory synapses in Cx+GE organoids

Figure 1 Preparation and characterization of fused brain organoids

Cx+ GE organoids exhibited

Complex neural network oscillations

To determine the range of physiological activity of fused organoids, complete organoid calcium indicator imaging and extracellular recording of LFP are utilized using live two-photon

Cx+Cx and Cx+GE fusion organoids show comparable baseline neural activity (Figure 2c

The presence of GE-derived inhibitory interneurons stimulates the maturation

Figure 2 cx+GE fusion organoids exhibit complex neural network activity, including oscillating rhythms

Organoids with MECP2 mutations

Neurogenesis and changes in fate

LTECH syndrome is a neurodevelopmental disorder usually caused by a de novo mutation in the MECP2 gene on the X chromosome, and the authors used Cx+GE fusion organoids to simulate a model of LTECH syndrome, followed by studies of organoid neurogenesis and fate alterations

UMAP analysis revealed clusters of 9 expression cell type-specific markers, and there was a tendency in MECP2 mutant samples to accelerate maturation and alteration in interneuronal formation, while mapping the expression of typical markers associated with each cluster was plotted

Given the importance of interneuronal function to oscillations in fused organoids, the authors isolated inhibitory neuron populations, further differentiated interneuron subtypes, and found seven main subgroups expressing markers such as DLX2, DLX5, GAD2, SLC6A1, SLC32A1, LAMP5, SCGN, and TAC1

Importantly, MECP2 mutant organoids are defective in synaptic formation, neurons have high excitability and high synchronicity, and exhibit abnormal neural oscillations, while interneuron MECP2 defects cause neural network dysfunction (Figures 3, 4).

Figure 3 LTECH syndrome fusion organoids show GE-dependent hypersynchronous neural network activity

Figure 4 Reiter syndrome fusion organoids show GE-dependent epileptioid changes

Nerve oscillation defects can pass

Take pifithrin-α.

The findings suggest that pifithrin-α can modulate more upstream excitatory-inhibitory neuronal physiological interactions, resulting in a more comprehensive recovery of neural network-level function, and these findings further illustrate the potential value

discuss

Overall, these experiments demonstrate the presence of highly complex physiological activity in Cx+GE organoids, consistent
with their cellular structure and cell complexity.

The authors also observed a relative decrease in SST expression in mutant organoids through immunohistochemistry, although this
was not observed in scRNA-seq data.
And an increase in excitatory synapses in mutant organoids was found to be associated
with transcriptomic changes revealed by scRNA-seq analysis.

Genes that are differentially expressed in MECP2 mutant organoids are strongly associated with axonal growth, synaptic formation, autism risk, and epilepsy, and a relative increase in excitatory input may further lead to disruption of neural oscillations and make organoids susceptible to overexcitability
.

Together, these findings illustrate the potential of brain organoids as a unique platform for characterizing human neural networks and personalized drug discovery and research, providing the necessary foundation for harnessing brain organoids to study complete and disordered human brain network formation, and highlighting practical application value
in disease treatment.