Controlled adhesion and growth of long term glial and neuronal cultures on Parylene-C

This paper explores the long term development of networks of glia and neurons on patterns of Parylene-C on a SiO2 substrate. We harvested glia and neurons from the Sprague-Dawley (P1–P7) rat hippocampus and utilized an established cell patterning technique in order to investigate cellular migration, over the course of 3 weeks. This work demonstrates that uncontrolled glial mitosis gradually disrupts cellular patterns that are established early during culture. This effect is not attributed to a loss of protein from the Parylene-C surface, as nitrogen levels on the substrate remain stable over 3 weeks. The inclusion of the anti-mitotic cytarabine (Ara-C) in the culture medium moderates glial division and thus, adequately preserves initial glial and neuronal conformity to underlying patterns. Neuronal apoptosis, often associated with the use of Ara-C, is mitigated by the addition of brain derived neurotrophic factor (BDNF). We believe that with the right combination of glial inhibitors and neuronal promoters, the Parylene-C based cell patterning method can generate structured, active neural networks that can be sustained and investigated over extended periods of time. To our knowledge this is the first report on the concurrent application of Ara-C and BDNF on patterned cell cultures

Deriving functional astrocytes from mouse embryonic stem cells with a fast and efficient protocol

A growing number of studies highlight the structural and functional diversity of astrocytes throughout the central nervous system. These cells are now seen as heterogeneous as neurons and are implicated in a number of neurological and psychiatric diseases. Efficient generation of diverse subtypes of astrocytes can be a useful tool in investigating synaptogenesis and patterns of activity in developing neural networks. In this study, we developed a protocol for the fast and efficient differentiation of astrocytes from mouse embryonic stem cells, as evidenced by the upregulation of genes related to astrocytic development (Gfap, Aldh1l1). Generated astrocytes exhibit phenotypic diversity, which is demonstrated by the variant expression of markers such as GFAP, ALDH1L1, AQP4 and S100β, amongst subgroups within the same cell population. In addition, astrocytes exhibited differential calcium transients upon stimulation with ATP. Our protocol will facilitate investigations, regarding the involvement of astrocytes in the structural and functional connectivity of neural networks

Fast and efficient differentiation of mouse embryonic stem cells into ATP-responsive astrocytes

Astrocytes are multifunctional cells in the CNS, involved in the regulation of neurovascular coupling, the modulation of electrolytes and the cycling of neurotransmitters at synapses. Induction of astrocytes from stem cells remains a largely underdeveloped area, as current protocols are time consuming, lack granularity in astrocytic subtype generation and often are not as efficient as neural induction methods. In this paper we present an efficient method to differentiate astrocytes from mouse embryonic stem cells. Our technique uses a cell suspension protocol to produce embryoid bodies (EBs) that are neurally inducted and seeded onto laminin coated surfaces. Plated EBs attach to the surface and release migrating cells to their surrounding environment, which are further inducted into the astrocytic lineage, through an optimized, heparin-based media. Characterization and functional assessment of the cells consists of immunofluorescent labelling for specific astrocytic proteins and sensitivity to ATP stimulation. Our experimental results show that even at the earliest stages of the protocol, cells are positive for astrocytic markers (GFAP, ALDH1L1, S100β, GLAST) with variant expression patterns and purinergic receptors (P2Y). Generated astrocytes also exhibit differential Ca2+ transients upon stimulation with ATP, which evolve over the differentiation period. Metabotropic purinoceptors P2Y1R are expressed and we offer preliminary evidence that metabotropic purinoceptors contribute to Ca2+ transients. Our protocol is simple, efficient and fast, facilitating its use in multiple investigations, particularly in vitro studies of engineered neural networks

Model-predicted balance between neural excitation and inhibition was maintained despite of age-related decline in sensory evoked local field potential in rat barrel cortex

The balance between neural excitation and inhibition has been shown to be crucial for normal brain function. However, it is unclear whether this balance is maintained through healthy aging. This study investigated the effect of aging on the temporal dynamics of the somatosensory evoked local field potential (LFP) in rats and tested the hypothesis that excitatory and inhibitory post-synaptic activities remain balanced during the aging process. The LFP signal was obtained from the barrel cortex of three different age groups of anesthetized rats (pre-adolescence: 4~6 weeks, young adult: 2~3 months, middle-aged adult: 10~20 months) under whisker pad stimulation. To confirm our previous finding that the initial segment of the evoked LFP was solely associated with excitatory post-synaptic activity, we micro-injected gabazine into the barrel cortex to block inhibition while LFP was collected continuously under the same stimulus condition. As expected, the initial slope of the evoked LFP in the granular layer was unaffected by gabazine injection. We subsequently estimated the excitatory and inhibitory post-synaptic activities through a balanced model of the LFP with delayed inhibition as an explicit constraint, and calculated the amplitude ratio of inhibition to excitation. We found an age-dependent slowing of the temporal dynamics in the somatosensory-evoked post-synaptic activity, as well as a significant age-related decrease in the amplitude of the excitatory component and a decreasing trend in the amplitude of the inhibitory component. Furthermore, the delay of inhibition with respect to excitation was significantly increased with age, but the amplitude ratio was maintained. Our findings suggest that aging reduces the amplitude of neural responses, but the balance between sensory evoked excitatory and inhibitory post-synaptic activities is maintained to support normal brain function during healthy aging. Further whole cell patch clamp experiments will be needed to confirm or refute these findings by measuring sensory evoked synaptic excitatory and inhibitory activities in vivo during the normal aging process

Adhesion and growth of neuralized mouse embryonic stem cells on parylene-C/SiO2 substrates.

Neuronal patterning on microfabricated architectures has developed rapidly over the past years, together with the emergence of soft biocompatible materials and tissue engineering scaffolds. Previously, we introduced a patterning technique based on serum and the biopolymer parylene-C, achieving highly compliant growth of primary neurons and astrocytes on different geometries. Here, we expand this technique and illustrate that neuralized cells derived from mouse embryonic stem cell (mESC) will follow stripes of variable widths, with conformity equal to or higher than that of primary neurons and astrocytes. Our results indicate the presence of undifferentiated mESC, which also conform to the underlying patterns to a high degree. This is an exciting and unexpected outcome, as molecular mechanisms governing cell and ECM protein interactions are different in stem cells and primary cells. Our study enables further investigations into the devel-opment and electrophysiology of differentiating, patterned neural stem cells

Localisation of oestrogen receptors in stem cells and in stem cell derived neurons of the mouse

Oestrogen receptors (ER) transduce the effects of the endogenous ligand, 17b-estradiol in cells to regulate a number of important processes such as reproduction, neuroprotection, learning and memory and anxiety. The ERa or ERb are classical intracellular nuclear hormone receptors while some of their variants or novel proteins such as the GPCR, GPER1/GPR30 are reported to localise in intracellular as well as plasma membrane locations. Though the brain is an important target for oestrogen with oestrogen receptors expressed differentially in various nuclei, subcellular organisation and crossttalk between these receptors is underexplored. Using an adapted protocol that is rapid, we first generated neurons from mouse embryonic stem cells. Our immunocytochemistry approach shows that the full length ERa (ERa-66) and for the first time, that an ERa variant, ERa-36, as well as GPER1 is present in embryonic stem cells. In addition, these receptors typically decrease their nuclear localisation as neuronal maturation proceeds. Finally, though these ERs are present in many subcellular compartments such as the nucleus and plasma membrane, we show that they are specifically not colocalised with each other, suggesting that they initiate distinct signalling pathways

A fibre alginate co-culture platform for the differentiation of mESC and modelling of the neural tube

Alginate hydrogels are a commonly used substrate for in vitro 3D cell culture. These naturally derived biomaterials are highly tunable, biocompatible and can be designed to mimic the elastic modulus of the adult brain at 1% w/v solution. Recent studies show that the molecular weight of the alginate can affect cell viability and differentiation. The relationship between the molecular weight, viscosity and ratio of G:M monomers of alginate hydrogels is complex, and the balance between these factors must be carefully considered when deciding on a suitable alginate hydrogel for stem cell research. This study investigates the formation of embryoid bodies (EB) from mouse embryonic stem cells, using low molecular weight (LMW) and high molecular weight (HMW) alginates. The cells are differentiated using a retinoic acid-based protocol, and the resulting aggregates are sectioned and stained for the presence of stem cells and the three germ layers (endoderm, mesoderm and ectoderm). The results highlight that aggregates within LMW and HMW alginate are true EBs, as demonstrated by positive staining for markers of the three germ layers. Using tubular alginate scaffolds, formed with an adapted gradient maker protocol, we also propose a novel 3D platform for the patterned differentiation of mESCs, based on gradients of retinoic acid produced in situ by lateral motor column (LMC) motor neurons. The end product of our platform will be of great interest as it can be further developed into a powerful model of neural tube development

Estrogen and testosterone secretion from the mouse brain

Estrogen and testosterone are typically thought of as gonadal or adrenal derived steroids that cross the blood brain barrier to signal via both rapid nongenomic and slower genomic signalling pathways. Estrogen and testosterone signalling has been shown to drive interlinked behaviours such as social behaviours and cognition by binding to their cognate receptors in hypothalamic and forebrain nuclei. So far, acute brain slices have been used to study short-term actions of 17β-estradiol, typically using electrophysiological measures. For example, these techniques have been used to investigate, nongenomic signalling by estrogen such as the estrogen modulation of long-term potentiation (LTP) in the hippocampus. Using a modified method that preserves the slice architecture, we show, for the first time, that acute coronal slices from the prefrontal cortex and from the hypothalamus maintained in aCSF over longer periods i.e. 24 h can be steroidogenic, increasing their secretion of testosterone and estrogen. We also show that the hypothalamic nuclei produce more estrogen and testosterone than the prefrontal cortex. Therefore, this extended acute slice system can be used to study the regulation of steroid production and secretion by discrete nuclei in the brain

Insinuating electronics in the brain

AbstractThere is an expanding interface between electronic engineering and neurosurgery. Rapid advances in microelectronics and materials science, driven largely by consumer demand, are inspiring and accelerating development of a new generation of diagnostic, therapeutic, and prosthetic devices for implantation in the nervous system. This paper reviews some of the basic science underpinning their development and outlines some opportunities and challenges for their use in neurosurgery