Biohybrid restoration of the hippocampal loop re-establishes the non-seizing state in anin vitromodel of limbic seizures.

Objective. The compromise of the hippocampal loop is a hallmark of mesial temporal lobe epilepsy (MTLE), the most frequent epileptic syndrome in the adult population and the most often refractory to medical therapy. Hippocampal sclerosis is found in >50% of drug-refractory MTLE patients and primarily involves the CA1, consequently disrupting the hippocampal output to the entorhinal cortex (EC). Closed-loop deep brain stimulation is the latest frontier to improve drug-refractory MTLE; however, current approaches do not restore the functional connectivity of the hippocampal loop, they are designed by trial-and-error and heavily rely on seizure detection or prediction algorithms. The objective of this study is to evaluate the anti-ictogenic efficacy and robustness of an artificial bridge restoring the dialog between hippocampus and EC.Approach. In mouse hippocampus-EC slices treated with 4-aminopyridine and in which the Schaffer Collaterals are severed, we established an artificial bridge between hippocampus and EC wherein interictal discharges originating in the CA3 triggered stimulation of the subiculum so to entrain EC networks. Combining quantification of ictal activity with tools from information theory, we addressed the efficacy of the bridge in controlling ictogenesis and in restoring the functional connectivity of the hippocampal loop.Main results. The bridge significantly decreased or even prevented ictal activity and proved robust to failure; when operating at 100% of its efficiency (i.e., delivering a pulse upon each interictal event), it recovered the functional connectivity of the hippocampal loop to a degree similar to what measured in the intact circuitry. The efficacy and robustness of the bridge stem in mirroring the adaptive properties of the CA3, which acts as biological neuromodulator.Significance. This work is the first stepping stone toward a paradigm shift in the conceptual design of stimulation devices for epilepsy treatment, from function control to functional restoration of the salient brain circuits.

Mimicking CA3 Temporal Dynamics Controls Limbic Ictogenesis.

Mesial temporal lobe epilepsy (MTLE) is the most common partial complex epilepsy in adults and the most unresponsive to medications. Electrical deep brain stimulation (DBS) of the hippocampus has proved effective in controlling seizures in epileptic rodents and in drug-refractory MTLE patients. However, current DBS paradigms implement arbitrary fixed-frequency or patterned stimuli, disregarding the temporal profile of brain electrical activity. The latter, herein included hippocampal spontaneous firing, has been shown to follow lognormal temporal dynamics. Here, we present a novel paradigm to devise DBS protocols based on stimulation patterns fashioned as a surrogate brain signal. We focus on the interictal activity originating in the hippocampal subfield CA3, which has been shown to be anti-ictogenic. Using 4-aminopyridine-treated hippocampus-cortex slices coupled to microelectrode array, we pursue three specific aims: (1) address whether lognormal temporal dynamics can describe the CA3-driven interictal pattern, (2) explore the possibility of restoring the non-seizing state by mimicking the temporal dynamics of this anti-ictogenic pattern with electrical stimulation, and (3) compare the performance of the CA3-surrogate against periodic stimulation. We show that the CA3-driven interictal activity follows lognormal temporal dynamics. Further, electrical stimulation fashioned as a surrogate interictal pattern exhibits similar efficacy but uses less pulses than periodic stimulation. Our results support the possibility of mimicking the temporal dynamics of relevant brain signals as a straightforward DBS strategy to ameliorate drug-refractory epilepsy. Further, they herald a paradigm shift in neuromodulation, wherein a compromised brain signal can be recreated by the appropriate stimuli distribution to bypass trial-and-error studies and attain physiologically meaningful DBS operating modes.

Murine cerebral organoids develop network of functional neurons and hippocampal brain region identity.

Brain organoids are in vitro three-dimensional (3D) self-organized neural structures, which can enable disease modeling and drug screening. However, their use for standardized large-scale drug screening studies is limited by their high batch-to-batch variability, long differentiation time (10-20 weeks), and high production costs. This is particularly relevant when brain organoids are obtained from human induced pluripotent stem cells (iPSCs). Here, we developed, for the first time, a highly standardized, reproducible, and fast (5 weeks) murine brain organoid model starting from embryonic neural stem cells. We obtained brain organoids, which progressively differentiated and self-organized into 3D networks of functional neurons with dorsal forebrain phenotype. Furthermore, by adding the morphogen WNT3a, we generated brain organoids with specific hippocampal region identity. Overall, our results showed the establishment of a fast, robust and reproducible murine 3D in vitro brain model that may represent a useful tool for high-throughput drug screening and disease modeling.

Electrophysiology Read-Out Tools for Brain-on-Chip Biotechnology.

Brain-on-Chip (BoC) biotechnology is emerging as a promising tool for biomedical and pharmaceutical research applied to the neurosciences. At the convergence between lab-on-chip and cell biology, BoC couples in vitro three-dimensional brain-like systems to an engineered microfluidics platform designed to provide an in vivo-like extrinsic microenvironment with the aim of replicating tissue- or organ-level physiological functions. BoC therefore offers the advantage of an in vitro reproduction of brain structures that is more faithful to the native correlate than what is obtained with conventional cell culture techniques. As brain function ultimately results in the generation of electrical signals, electrophysiology techniques are paramount for studying brain activity in health and disease. However, as BoC is still in its infancy, the availability of combined BoC-electrophysiology platforms is still limited. Here, we summarize the available biological substrates for BoC, starting with a historical perspective. We then describe the available tools enabling BoC electrophysiology studies, detailing their fabrication process and technical features, along with their advantages and limitations. We discuss the current and future applications of BoC electrophysiology, also expanding to complementary approaches. We conclude with an evaluation of the potential translational applications and prospective technology developments.

Modeling medulloblastoma in vivo and with human cerebellar organoids.

Medulloblastoma (MB) is the most common malignant brain tumor in children and among the subtypes, Group 3 MB has the worst outcome. Here, we perform an in vivo, patient-specific screen leading to the identification of Otx2 and c-MYC as strong Group 3 MB inducers. We validated our findings in human cerebellar organoids where Otx2/c-MYC give rise to MB-like organoids harboring a DNA methylation signature that clusters with human Group 3 tumors. Furthermore, we show that SMARCA4 is able to reduce Otx2/c-MYC tumorigenic activity in vivo and in human cerebellar organoids while SMARCA4 T910M, a mutant form found in human MB patients, inhibits the wild-type protein function. Finally, treatment with Tazemetostat, a EZH2-specific inhibitor, reduces Otx2/c-MYC tumorigenesis in ex vivo culture and human cerebellar organoids. In conclusion, human cerebellar organoids can be efficiently used to understand the role of genes found altered in cancer patients and represent a reliable tool for developing personalized therapies.

Truncated BRPF1 Cooperates with Smoothened to Promote Adult Shh Medulloblastoma.

The transition of neural progenitors to differentiated postmitotic neurons is mainly considered irreversible in physiological conditions. In the present work, we show that Shh pathway activation through SmoM2 expression promotes postmitotic neurons dedifferentiation, re-entering in the cell cycle and originating medulloblastoma in vivo. Notably, human adult patients present inactivating mutations of the chromatin reader BRPF1 that are associated with SMO mutations and absent in pediatric and adolescent patients. Here, we found that truncated BRPF1 protein, as found in human adult patients, is able to induce medulloblastoma in adult mice upon SmoM2 activation. Indeed, postmitotic neurons re-entered the cell cycle and proliferated as a result of chromatin remodeling of neurons by BRPF1. Our model of brain cancer explains the onset of a subset of human medulloblastoma in adult individuals where granule neuron progenitors are no longer present.