Introduction
Early infantile epileptic encephalopathies (EIEE) of genetic origin are devastating conditions, but the pathological mechanisms often remain obscure. A major obstacle is the difficulty of studying human cortical brain development, in utero. Until recently, no in vitro preparations could accurately reproduce the complex cellular networks found beyond the first trimester.
Using a novel approach that can maintain developing human brain tissue in the laboratory, we investigated STXBP1 haploinsufficiency, which is among the most common genetic causes of EIEE. Loss of STXBP1 impaired synaptic function and reduced glutamatergic synapse density at individual neurons (McLeod et al, Brain 2023).
Aim
To fully understand how deficits in STXBP1 affect the early developing brain, we now aim to investigate the impact on neuronal networks.
Methods
Human brain slice cultures were prepared from ethically sourced, 13-18 post-conception week brain tissue (www.hdbr.org). The gross anatomical structures of the marginal zone, cortical plate and subplate are maintained for several months, while synaptic networks continue to develop. This preparation permits the study of genetic manipulations and their downstream effects on an intact developing human cortical network.
We assessed the downstream effects of short hairpin RNA (shRNA) mediated STXBP1 knockdown, using three powerful screening tools: (1) 3Brain multielectrode array recordings; (2) Ca2+ imaging using genetic encoding calcium indicators; (3) gene expression profiles using NanoString nCounter technology.
Results
We show a striking reduction in the number of local field potentials in the subplate of slices with diminished STXBP1 levels (control: 0.1040 ± 0.01Hz, STXBP1 shRNA: 0.03 ± 0.007Hz, unpaired t-test, P<0.001, n = 2 biological replicates). Interestingly, loss of STXBP1 does not affect the frequency of synchronised calcium transients in subplate neurons but reduces the peak amplitude (control: 0.45 ± 0.05 ΔF/F0, STXBP1 shRNA: 0.16 ± 0.04 ΔF/F0, unpaired t-test, P<0.001, n = 3 biological replicates) and lengthens the decay kinetics (control: 1.46 ± 0.05s, STXBP1 shRNA: 1.77 ± 0.14s, unpaired t-test, P<0.05, n = 3 biological replicates) of events. This data suggests that loss of STXBP1 alters neuronal network excitability.
Analyses of our NanoString data confirms that the manipulation achieves a reliable knockdown of STXBP1 across the tissue by 46 ± 2.5% relative to control slices. This results in an upregulation of 81 genes associated with glutamate receptor activity, ephrin signalling, voltage gated ion channel signalling and vesicle recycling and a downregulation of 47 genes associated with apoptosis, oxidative stress and G-protein coupled receptor signalling (false discovery ratio analysis with two-sided unpaired t-test corrected for multiple comparisons, n = 3 biological replicates).
Conclusion
Reduced network excitability with loss of STXBP1 could indicate impairments in the formation of early neuronal networks, a process which correlated neuronal activity is instrumental for during prenatal human brain development. Changes in downstream gene expression may reflect potential compensatory mechanisms in response to this reduction, including an upregulation of activity-dependent synaptic signalling and downregulation of cell death and/or stress induced pathways.