Highlights An improved protocol for primary hippocampal cell cultures is proposed.  The method relies on serum-free astrocytes conditioned medium (ACM).  The ACM method is extensively compared with other two commonly used protocols.  ACM improved morphology and function of both short-and long-term cultures. AbstractBackground: Since different culturing parameters -such as media composition or cell density -lead to different experimental results, it is important to define the protocol used for neuronal cultures. The vital role of astrocytes in maintaining homeostasis of neurons -both in vivo and in vitro -is well established: the majority of improved culturing conditions for primary dissociated neuronal cultures rely on astrocytes.New Method: Our culturing protocol is based on a novel serum-free preparation of astrocyteconditioned medium (ACM). We compared the proposed ACM culturing method with other two commonly used methods: Neurobasal/B27-and FBS-based media. We performed morphometric characterization by immunocytochemistry and functional analysis by calcium imaging for all three culture methods at 1,7,14 and 60 days in vitro (DIV).Results: ACM-based cultures gave the best results for all tested criteria, i.e. growth cone's size and shape, neuronal outgrowth and branching, network activity and synchronization, maturation and long-term survival. The differences were more pronounced when compared with FBS-based medium. Neurobasal/B27 cultures were comparable to ACM for young cultures (DIV1), but not for culturing times longer than DIV7. Comparison with Existing Method(s):ACM-based cultures showed more robust neuronal outgrowth at DIV1. At DIV7 and 60, the activity of neuronal network grown in ACM had a more vigorous spontaneous electrical activity and a higher degree of synchronization. Conclusions:We propose our ACM-based culture protocol as an improved and more suitable method for both short-and long-term neuronal cultures.

Seizures represent a frequent symptom in gliomas and significantly impact patient morbidity and quality of life. Although the pathogenesis of tumor-related seizures is not fully understood, accumulating evidence indicates a key role of the peritumoral microenvironment. Brain cancer cells interact with neurons by forming synapses with them and by releasing exosomes, cytokines, and other small molecules. Strong interactions among neurons often lead to the synchronization of their activity. In this paper, we used an in vitro model to investigate the role of exosomes released by glioma cell lines and by patient-derived glioma stem cells (GSCs). The addition of exosomes released by U87 glioma cells to neuronal cultures at day in vitro (DIV) 4, when neurons are not yet synchronous, induces synchronization. At DIV 7–12 neurons become highly synchronous, and the addition of the same exosomes disrupts synchrony. By combining Ca2+ imaging, electrical recordings from single neurons with patch-clamp electrodes, substrate-integrated microelectrode arrays, and immunohistochemistry, we show that synchronization and de-synchronization are caused by the combined effect of (i) the formation of new neuronal branches, associated with a higher expression of Arp3, (ii) the modification of synaptic efficiency, and (iii) a direct action of exosomes on the electrical properties of neurons, more evident at DIV 7–12 when the threshold for spike initiation is significantly reduced. At DIV 7–12 exosomes also selectively boost glutamatergic signaling by increasing the number of excitatory synapses. Remarkably, de-synchronization was also observed with exosomes released by glioma-associated stem cells (GASCs) from patients with low-grade glioma but not from patients with high-grade glioma, where a more variable outcome was observed. These results show that exosomes released from glioma modify the electrical properties of neuronal networks and that de-synchronization caused by exosomes from low-grade glioma can contribute to the neurological pathologies of patients with brain cancers.

BACKGROUND: The spontaneous activity of neuronal networks has been studied in in vitro models such as brain slices and dissociated cultures. However, a comparison between their dynamical properties in these two types of biological samples is still missing and it would clarify the role of architecture in shaping networks’ operation. METHODS: We used calcium imaging to identify clusters of neurons co-activated in hippocampal and cortical slices, as well as in dissociated neuronal cultures, from GAD67-GFP mice. We used statistical tests, power law fitting and neural modelling to characterize the spontaneous events observed. RESULTS:  In slices, we observed intermittency between silent periods, the appearance of Confined Optical Transients (COTs) and of Diffused Optical Transients (DOTs). DOTs in the cortex were preferentially triggered by the activity of neurons located in layer III-IV, poorly coincident with GABAergic neurons. DOTs had a duration of 10.2±0.3 and 8.2±0.4 seconds in cortical and hippocampal slices, respectively, and were blocked by tetrodotoxin, indicating their neuronal origin. The amplitude and duration of DOTs were controlled by NMDA and GABA-A receptors. In dissociated cultures, we observed an increased synchrony in GABAergic neurons and the presence of global synchronous events similar to DOTs, but with a duration shorter than that seen in the native tissues. CONCLUSION: We conclude that DOTs are shaped by the network architecture and by the balance between inhibition and excitation, and that they can be reproduced by network models with a minimal number of parameters.

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