Unravelling the complexities of resistance mechanism in pancreatic cancer: insights from in vitro and ex-vivo model systems.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with poor prognosis and rising global deaths. Late diagnosis, due to absent early symptoms and biomarkers, limits treatment mainly to chemotherapy, which soon encounters resistance. PDAC treatment innovation is hampered by its complex and heterogeneous resistant nature, including mutations in key genes and a stromal-rich, immunosuppressive tumour microenvironment. Recent studies on PDAC resistance stress the need for suitable in vitro and ex vivo models to replicate its complex molecular and microenvironmental landscape. This review summarises advances in these models, which can aid in combating chemoresistance and serve as platforms for discovering new therapeutics. Immortalised cell lines offer homogeneity, unlimited proliferation, and reproducibility, but while many gemcitabine-resistant PDAC cell lines exist, fewer models are available for resistance to other drugs. Organoids from PDAC patients show promise in mimicking tumour heterogeneity and chemosensitivity. Bioreactors, co-culture systems and organotypic slices, incorporating stromal and immune cells, are being developed to understand tumour-stroma interactions and the tumour microenvironment's role in drug resistance. Lastly, another innovative approach is three-dimensional bioprinting, which creates tissue-like structures resembling PDAC architecture, allowing for drug screening. These advanced models can guide researchers in selecting optimal in vitro tests, potentially improving therapeutic strategies and patient outcomes.

Prodromal Parkinson's disease and the catecholaldehyde hypothesis: Insight from olfactory bulb organotypic cultures.

Parkinson's disease (PD) is a progressive, neurodegenerative disorder with an increasing incidence, unknown etiology, and is currently incurable. Advances in understanding the pathological mechanisms at a molecular level have been slow, with little attention focused on the early prodromal phase of the disease. Consequently, the development of early-acting disease-modifying therapies has been hindered. The olfactory bulb (OB), the brain region responsible for initial processing of olfactory information, is particularly affected early in PD at both functional and molecular levels but there is little information on how the cells in this region are affected by disease. Organotypic and primary OB cultures were developed and characterized. These platforms were then used to assess the effects of 3,4-dihydroxyphenylacetylaldehyde (DOPAL), a metabolite of dopamine present in increased levels in post-mortem PD tissue and which is thought to contribute to PD pathogenesis. Our findings showed that DOPAL exposure can recapitulate many aspects of PD pathology. Oxidative stress, depolarization of mitochondrial membranes, and neurodegeneration were all induced by DOPAL addition, as were measured transcriptomic changes consistent with those reported in PD clinical studies. These olfactory models of prodromal disease lend credence to the catecholaldehyde hypothesis of PD and provide insight into the mechanisms by which the OB may be involved in disease progression.

Altered synaptic connectivity in an in vitro human model of STXBP1 encephalopathy.

Early infantile developmental and epileptic encephalopathies are devastating conditions, generally of genetic origin, but the pathological mechanisms often remain obscure. A major obstacle in this field of research is the difficulty of studying cortical brain development in humans, at the relevant time period in utero. To address this, we established an in vitro assay to study the impact of gene variants on the developing human brain by using living organotypic cultures of the human subplate and neighbouring cortical regions, prepared from ethically sourced, 14-17 post-conception week brain tissue (www.hdbr.org). We were able to maintain cultures for several months, during which time the gross anatomical structures of the cortical plate, subplate and marginal zone persisted, while neurons continued to develop morphologically and form new synaptic networks. This preparation thus permits the study of genetic manipulations and their downstream effects on an intact developing human cortical network. We focused on STXBP1 haploinsufficiency, which is among the most common genetic causes of developmental and epileptic encephalopathy. This was induced using shRNA interference, leading to impaired synaptic function and a reduced density of glutamatergic synapses. We thereby provide a critical proof-of-principle for how to study the impact of any gene of interest on the development of the human cortex.

Semaphorin4D Induces Inhibitory Synapse Formation by Rapid Stabilization of Presynaptic Boutons via MET Coactivation.

Changes in inhibitory connections are essential for experience-dependent circuit adaptations. Defects in inhibitory synapses are linked to neurodevelopmental disorders, but the molecular processes underlying inhibitory synapse formation are not well understood. Here we use high-resolution two-photon microscopy in organotypic hippocampal slices from GAD65-GFP mice of both sexes to examine the signaling pathways induced by the postsynaptic signaling molecule Semaphorin4D (Sema4D) during inhibitory synapse formation. By monitoring changes in individual GFP-labeled presynaptic boutons, we found that the primary action of Sema4D is to induce stabilization of presynaptic boutons within tens of minutes. Stabilized boutons rapidly recruited synaptic vesicles, followed by accumulation of postsynaptic gephyrin and were functional after 24 h, as determined by electrophysiology and immunohistochemistry. Inhibitory boutons are only sensitive to Sema4D at a specific stage during synapse formation and sensitivity to Sema4D is regulated by network activity. We further examined the intracellular signaling cascade triggered by Sema4D and found that bouton stabilization occurs through rapid remodeling of the actin cytoskeleton. This could be mimicked by the actin-depolymerizing drug latrunculin B or by reducing ROCK activity. We discovered that the intracellular signaling cascade requires activation of the receptor tyrosine kinase MET, which is a well known autism risk factor. By using a viral approach to reduce MET levels specifically in inhibitory neurons, we found that their axons are no longer sensitive to Sema4D signaling. Together, our data yield important insights into the molecular pathway underlying activity-dependent Sema4D-induced synapse formation and reveal a novel role for presynaptic MET at inhibitory synapses.SIGNIFICANCE STATEMENT GABAergic synapses provide the main inhibitory control of neuronal activity in the brain. We wanted to unravel the sequence of molecular events that take place when formation of inhibitory synapses is triggered by a specific signaling molecule, Sema4D. We find that this signaling pathway depends on network activity and involves specific remodeling of the intracellular actin cytoskeleton. We also reveal a previously unknown role for MET at inhibitory synapses. Our study provides novel insights into the dynamic process of inhibitory synapse formation. As defects in GABAergic synapses have been implied in many brain disorders, and mutations in MET are strong risk factors for autism, our findings urge for a further investigation of the role of MET at inhibitory synapses.

Impact of neonatal hypoxia-ischaemia on oligodendrocyte survival, maturation and myelinating potential.

Hypoxic-ischaemic episodes experienced at the perinatal period commonly lead to a development of neurological disabilities and cognitive impairments in neonates or later in childhood. Clinical symptoms often are associated with the observed alterations in white matter in the brains of diseased children, suggesting contribution of triggered oligodendrocyte/myelin pathology to the resulting disorders. To date, the processes initiated by perinatal asphyxia remain unclear, hampering the ability to develop preventions. To address the issue, the effects of temporal hypoxia-ischaemia on survival, proliferation and the myelinating potential of oligodendrocytes were evaluated ex vivo using cultures of hippocampal organotypic slices and in vivo in rat model of perinatal asphyxia. The potential engagement of gelatinases in oligodendrocyte maturation was assessed as well. The results pointed to a significant decrease in the number of oligodendrocyte progenitor cells (OPCs), which is compensated for to a certain extent by the increased rate of OPC proliferation. Oligodendrocyte maturation seemed however to be significantly altered. An ultrastructural examination of selected brain regions performed several weeks after the insult showed however that the process of developing central nervous system myelination proceeds efficiently resulting in enwrapping the majority of axons in compact myelin. The increased angiogenesis in response to neonatal hypoxic-ischaemic insult was also noticed. In conclusion, the study shows that hypoxic-ischaemic episodes experienced during the most active period of nervous system development might be efficiently compensated for by the oligodendroglial cell response triggered by the insult. The main obstacle seems to be the inflammatory process modulating the local microenvironment.

An ex vivo spinal cord injury model to study ependymal cells in adult mouse tissue.

Traumatic spinal cord injury is characterized by an initial cell loss that is followed by a concerted cellular response in an attempt to restore the damaged tissue. Nevertheless, little is known about the signaling mechanisms governing the cellular response to injury. Here, we have established an adult ex vivo system that exhibits multiple hallmarks of spinal cord injury and allows the study of complex processes that are difficult to address using animal models. We have characterized the ependymal cell response to injury in this model system and found that ependymal cells can become activated, proliferate, migrate out of the central canal lining and differentiate in a manner resembling the in vivo situation. Moreover, we show that these cells respond to external adenosine triphosphate and exhibit spontaneous Ca2+ activity, processes that may play a significant role in the regulation of their response to spinal cord injury. This model provides an attractive tool to deepen our understanding of the ependymal cell response after spinal cord injury, which may contribute to the development of new treatment options for spinal cord injury.

The Differentiation of Rat Oligodendroglial Cells Is Highly Influenced by the Oxygen Tension: In Vitro Model Mimicking Physiologically Normoxic Conditions.

Oligodendrocyte progenitor cells (OPCs) constitute one of the main populations of dividing cells in the central nervous system (CNS). Physiologically, OPCs give rise to mature, myelinating oligodendrocytes and confer trophic support to their neighboring cells within the nervous tissue. OPCs are known to be extremely sensitive to the influence of exogenous clues which might affect their crucial biological processes, like survival, proliferation, differentiation, and the ability to generate a myelin membrane. Alterations in their differentiation influencing their final potential for myelinogenesis are usually the leading cause of CNS dys- and demyelination, contributing to the development of leukodystrophic disorders. The evaluation of the mechanisms that cause oligodendrocytes to malfunction requires detailed studies based on designed in vitro models. Since OPCs readily respond to changes in local homeostasis, it is crucial to establish restricted culture conditions to eliminate the potential stimuli that might influence oligodendrocyte biology. Additionally, the in vitro settings should mimic the physiological conditions to enable the obtained results to be translated to future preclinical studies. Therefore, the aim of our study was to investigate OPC differentiation in physiological normoxia (5% O2) and a restricted in vitro microenvironment. To evaluate the impact of the combined microenvironmental clues derived from other components of the nervous tissue, which are also influenced by the local oxygen concentration, the process of generating OPCs was additionally analyzed in organotypic hippocampal slices. The obtained results show that OPC differentiation, although significantly slowed down, proceeded correctly through its typical stages in the physiologically relevant conditions created in vitro. The established settings were also conducive to efficient cell proliferation, exerting also a neuroprotective effect by promoting the proliferation of neurons. In conclusion, the performed studies show how oxygen tension influences OPC proliferation, differentiation, and their ability to express myelin components, and should be taken into consideration while planning preclinical studies, e.g., to examine neurotoxic compounds or to test neuroprotective strategies.

EBI2 receptor regulates myelin development and inhibits LPC-induced demyelination.

BACKGROUND: The G protein-coupled receptor EBI2 (Epstein-Barr virus-induced gene 2) is activated by 7α, 25-dihydroxycholesterol (7α25HC) and plays a role in T cell-dependant antibody response and B cell migration. Abnormal EBI2 signaling is implicated in a range of autoimmune disorders; however, its role in the CNS remains poorly understood. METHODS: Here we characterize the role of EBI2 in myelination under normal and pathophysiological conditions using organotypic cerebellar slice cultures and EBI2 knock-out (KO) animals. RESULTS: We find that MBP expression in brains taken from EBI2 KO mice is delayed compared to those taken from wild type (WT) mice. In agreement with these in vivo findings, we show that antagonism of EBI2 reduces MBP expression in vitro. Importantly, we demonstrate that EBI2 activation attenuates lysolecithin (LPC)-induced demyelination in mouse organotypic slice cultures. Moreover, EBI2 activation also inhibits LPC-mediated release of pro-inflammatory cytokines such as IL6 and IL1ß in cerebellar slices. CONCLUSIONS: These results, for the first time, display a role for EBI2 in myelin development and protection from demyelination under pathophysiological conditions and suggest that modulation of this receptor may be beneficial in neuroinflammatory and demyelinating disorders such as multiple sclerosis.

Neuronal activity mediated regulation of glutamate transporter GLT-1 surface diffusion in rat astrocytes in dissociated and slice cultures.

The astrocytic GLT-1 (or EAAT2) is the major glutamate transporter for clearing synaptic glutamate. While the diffusion dynamics of neurotransmitter receptors at the neuronal surface are well understood, far less is known regarding the surface trafficking of transporters in subcellular domains of the astrocyte membrane. Here, we have used live-cell imaging to study the mechanisms regulating GLT-1 surface diffusion in astrocytes in dissociated and brain slice cultures. Using GFP-time lapse imaging, we show that GLT-1 forms stable clusters that are dispersed rapidly and reversibly upon glutamate treatment in a transporter activity-dependent manner. Fluorescence recovery after photobleaching and single particle tracking using quantum dots revealed that clustered GLT-1 is more stable than diffuse GLT-1 and that glutamate increases GLT-1 surface diffusion in the astrocyte membrane. Interestingly, the two main GLT-1 isoforms expressed in the brain, GLT-1a and GLT-1b, are both found to be stabilized opposed to synapses under basal conditions, with GLT-1b more so. GLT-1 surface mobility is increased in proximity to activated synapses and alterations of neuronal activity can bidirectionally modulate the dynamics of both GLT-1 isoforms. Altogether, these data reveal that astrocytic GLT-1 surface mobility, via its transport activity, is modulated during neuronal firing, which may be a key process for shaping glutamate clearance and glutamatergic synaptic transmission. GLIA 2016;64:1252-1264.

Delayed in vitro development of Up states but normal network plasticity in Fragile X circuits.

A broad range of neurophysiological phenotypes have been reported since the generation of the first mouse model of Fragile X syndrome (FXS). However, it remains unclear which phenotypes are causally related to the cognitive deficits associated with FXS. Indeed, because many of these phenotypes are known to be modulated by experience, a confounding factor in the interpretation of many studies is whether some phenotypes are an indirect consequence of abnormal development and experience. To help diminish this confound we first conducted an in vitro developmental study of spontaneous neural dynamics in cortical organotypic cultures. A significant developmental increase in network activity and Up states was observed in both wild-type and Fmr1(-/y) circuits, along with a specific developmental delay in the emergence of Up states in knockout circuits. To determine whether Up state regulation is generally impaired in FXS circuits, we examined Up state plasticity using chronic optogenetic stimulation. Wild-type and Fmr1(-/y) stimulated circuits exhibited a significant decrease in overall spontaneous activity including Up state frequency; however, no significant effect of genotype was observed. These results demonstrate that developmental delays characteristic of FXS are recapitulated during in vitro development, and that Up state abnormalities are probably a direct consequence of the disease, and not an indirect consequence of abnormal experience. However, the fact that Fmr1(-/y) circuits exhibited normal homeostatic modulation of Up states suggests that these plasticity mechanisms are largely intact, and that some of the previously reported plasticity deficits could reflect abnormal experience or the engagement of compensatory mechanisms.

Targeting of membrane proteins with fluoronanogold probes for high-resolution correlative microscopy.

Correlative light and electron microscopy (CLEM) can provide valuable information about a biological sample by giving information on the specific localization of a molecule of interest within an ultrastructural context. In this work, we describe a simple CLEM method to obtain high-resolution images of neurotransmitter receptor distribution in synapses by electron microscopy (EM). We use hippocampal organotypic slices from a previously reported mouse model expressing a modified AMPA receptor (AMPAR) subunit that binds biotin at the surface (Getz et al., 2022). This tag can be recognized by StreptAvidin-Fluoronanogold™ conjugates (SA-FNG), which reach receptors at synapses (synaptic cleft is 50-100nm thick). By using pre-embedding labeling, we found that SA-FNG reliably bind synaptic receptors and penetrate around 10-15µm in depth in live tissue. However, the silver enhancement was only reaching the surface of the slices. We show that permeabilization with triton is highly effective at increasing the in depth-gold amplification and that the membrane integrity is well preserved. Finally, we also apply high-resolution electron tomography, thus providing important information about the 3D organization of surface AMPA receptors in synapses at the nanoscale.

High phenylalanine concentrations induce demyelination and microglial activation in mouse cerebellar organotypic slices.

Phenylketonuria (PKU) is an inborn error of metabolism. Mutations in the enzyme phenylalanine hydroxylase (PAH)-encoding gene lead to a decreased metabolism of the amino acid phenylalanine (Phe). The deficiency in PAH increases Phe levels in blood and brain. Accumulation of Phe can lead to delayed development, psychiatric problems and cognitive impairment. White matter (WM) damage is a neuropathological hallmark of PKU and can be seen even in early detected and treated PKU patients. The mechanisms linking high Phe concentrations to WM abnormalities remain unclear. We tested the effects of high Phe concentrations on myelin in three in vitro models of increasing complexity: two simple cell culture models and one model that preserves local brain tissue architecture, a cerebellar organotypic slice culture prepared from postnatal day (P) 8 CD-1 mice. Various Phe concentrations (0.1-10 mM) and durations of exposure were tested. We found no toxic effect of high Phe in the cell culture models. On the contrary, the treatment promoted the maturation of oligodendrocytes, particularly at the highest, non-physiological Phe concentrations. Exposure of cerebellar organotypic slices to 2.4 mM Phe for 21 days in vitro (DIV), but not 7 or 10 DIV, resulted in a significant decrease in myelin basic protein (MBP), calbindin-stained neurites, and neurites co-stained with MBP. Following exposure to a toxic concentration of Phe, a switch to the control medium for 7 days did not lead to remyelination, while very active remyelination was seen in slices following demyelination with lysolecithin. An enhanced number of microglia, displaying an activated type morphology, was seen after exposure of the slices to 2.4 mM Phe for 10 or 21 DIV. The results suggest that prolonged exposure to high Phe concentrations can induce microglial activation preceding significant disruption of myelin.

In Vitro Glioblastoma Models: A Journey into the Third Dimension.

Glioblastoma multiforme (GBM) is the most lethal primary brain tumor in adults, with an average survival time of about one year from initial diagnosis. In the attempt to overcome the complexity and drawbacks associated with in vivo GBM models, together with the need of developing systems dedicated to screen new potential drugs, considerable efforts have been devoted to the implementation of reliable and affordable in vitro GBM models. Recent findings on GBM molecular features, revealing a high heterogeneity between GBM cells and also between other non-tumor cells belonging to the tumoral niche, have stressed the limitations of the classical 2D cell culture systems. Recently, several novel and innovative 3D cell cultures models for GBM have been proposed and implemented. In this review, we first describe the different populations and their functional role of GBM and niche non-tumor cells that could be used in 3D models. An overview of the current available 3D in vitro systems for modeling GBM, together with their major weaknesses and strengths, is presented. Lastly, we discuss the impact of groundbreaking technologies, such as bioprinting and multi-omics single cell analysis, on the future implementation of 3D in vitro GBM models.

Nanoparticle-microglial interaction in the ischemic brain is modulated by injury duration and treatment.

Cerebral ischemia is a major cause of death in both neonates and adults, and currently has no cure. Nanotechnology represents one promising area of therapeutic development for cerebral ischemia due to the ability of nanoparticles to overcome biological barriers in the brain. ex vivo injury models have emerged as a high-throughput alternative that can recapitulate disease processes and enable nanoscale probing of the brain microenvironment. In this study, we used oxygen-glucose deprivation (OGD) to model ischemic injury and studied nanoparticle interaction with microglia, resident immune cells in the brain that are of increasing interest for therapeutic delivery. By measuring cell death and glutathione production, we evaluated the effect of OGD exposure time and treatment with azithromycin (AZ) on slice health. We found a robust injury response with 0.5 hr of OGD exposure and effective treatment after immediate application of AZ. We observed an OGD-induced shift in microglial morphology toward increased heterogeneity and circularity, and a decrease in microglial number, which was reversed after treatment. OGD enhanced diffusion of polystyrene-poly(ethylene glycol) (PS-PEG) nanoparticles, improving transport and ability to reach target cells. While microglial uptake of dendrimers or quantum dots (QDs) was not enhanced after injury, internalization of PS-PEG was significantly increased. For PS-PEG, AZ treatment restored microglial uptake to normal control levels. Our results suggest that different nanoparticle platforms should be carefully screened before application and upon doing so; disease-mediated changes in the brain microenvironment can be leveraged by nanoscale drug delivery devices for enhanced cell interaction.

Human Cerebrospinal Fluid Induces Neuronal Excitability Changes in Resected Human Neocortical and Hippocampal Brain Slices.

Human cerebrospinal fluid (hCSF) has proven advantageous over conventional medium for culturing both rodent and human brain tissue. In addition, increased activity and synchrony, closer to the dynamic states exclusively recorded in vivo, were reported in rodent slices and cell cultures switching from artificial cerebrospinal fluid (aCSF) to hCSF. This indicates that hCSF possesses properties that are not matched by the aCSF, which is generally used for most electrophysiological recordings. To evaluate the possible significance of using hCSF as an electrophysiological recording medium, also for human brain tissue, we compared the network and single-cell firing properties of human brain slice cultures during perfusion with hCSF and aCSF. For measuring the overall activity from a majority of neurons within neocortical and hippocampal human slices, we used a microelectrode array (MEA) recording technique with 252 electrodes covering an area of 3.2 × 3.2 mm2. A second CMOS-based MEA with 4225 sensors on a 2 × 2 mm2 area was used for detailed mapping of action potential waveforms and cell identification. We found that hCSF increased the number of active electrodes and neurons and the firing rate of the neurons in the slices and induced an increase in the numbers of single channel and population bursts. Interestingly, not only an increase in the overall activity in the slices was observed, but a reconfiguration of the network could also be detected with specific activation and inactivation of subpopulations of neuronal ensembles. In conclusion, hCSF is an important component to consider for future human brain slice studies, especially for experiments designed to mimic parts of physiology and disease observed in vivo.

Practical Review on Preclinical Human 3D Glioblastoma Models: Advances and Challenges for Clinical Translation.

Fifteen years after the establishment of the Stupp protocol as the standard of care to treat glioblastomas, no major clinical advances have been achieved and increasing patient's overall survival remains a challenge. Nevertheless, crucial molecular and cellular findings revealed the intra-tumoral and inter-tumoral complexities of these incurable brain tumors, and the essential role played by cells of the microenvironment in the lack of treatment efficacy. Taking this knowledge into account, fulfilling gaps between preclinical models and clinical samples is necessary to improve the successful rate of clinical trials. Since the beginning of the characterization of brain tumors initiated by Bailey and Cushing in the 1920s, several glioblastoma models have been developed and improved. In this review, we focused on the most widely used 3D human glioblastoma models, including spheroids, tumorospheres, organotypic slices, explants, tumoroids and glioblastoma-derived from cerebral organoids. We discuss their history, development and especially their usefulness.

Organotypic slice culture model demonstrates inter-neuronal spreading of alpha-synuclein aggregates.

Here we describe the use of an organotypic hippocampal slice model for studying α-synuclein aggregation and inter-neuronal spreading initiated by microinjection of pre-formed α-synuclein fibrils (PFFs). PFF injection at dentate gyrus (DG) templates the formation of endogenous α-synuclein aggregates in axons and cell bodies of this region that spread to CA3 and CA1 regions. Aggregates are insoluble and phosphorylated at serine-129, recapitulating Lewy pathology features found in Parkinson's disease and other synucleinopathies. The model was found to favor anterograde spreading of the aggregates. Furthermore, it allowed development of slices expressing only serine-129 phosphorylation-deficient human α-synuclein (S129G) using an adeno-associated viral (AAV) vector in α-synuclein knockout slices. The processes of aggregation and spreading of α-synuclein were thereby shown to be independent of phosphorylation at serine-129. We provide methods and highlight crucial steps for PFF microinjection and characterization of aggregate formation and spreading. Slices derived from genetically engineered mice or manipulated using viral vectors allow testing of hypotheses on mechanisms involved in the formation of α-synuclein aggregates and their prion-like spreading.

Ankyrin G Membrane Partners Drive the Establishment and Maintenance of the Axon Initial Segment.

The axon initial segment (AIS) is a highly specialized neuronal compartment that plays a key role in neuronal development and excitability. It concentrates multiple membrane proteins such as ion channels and cell adhesion molecules (CAMs) that are recruited to the AIS by the scaffold protein ankyrin G (ankG). The crucial function of ankG in the anchoring of AIS membrane components is well established, but a reciprocal role of membrane partners in ankG targeting and stabilization remained elusive. In rat cultured hippocampal neurons and cortical organotypic slices, we found that shRNA-mediated knockdown of ankG membrane partners (voltage-gated sodium channels (Nav) or neurofascin-186) led to a decrease of ankG concentration and perturbed the AIS formation and maintenance. These effects were rescued by expressing a recombinant AIS-targeted Nav or by a minimal construct containing the ankyrin-binding domain of Nav1.2 and a membrane anchor (mABD). Moreover, overexpressing mABD in mature neurons led to ankG mislocalization. Altogether, these results demonstrate that a tight and precocious association of ankG with its membrane partners is a key step for the establishment and maintenance of the AIS.

Method for organotypic tissue culture in the aged animal.

Organotypic slicing of brain tissue from young rodents has been used as a powerful model system for biomedical research [1], [2], [3]. Organotypic slicing complements cell culture and in vivo studies in multiple facets. This system can be useful for investigating manipulation of cellular signaling pathways without the hindrance of the blood-brain barrier while sacrificing fewer animals in the process. It also allows for preserved cellular connectivity and local intact circuitry which is a drawback of isolated cell cultures. Studies on age-related diseases have mainly used embryonic or early postnatal organotypic slice tissue. Excluding synaptic plasticity studies that are usually carried-out over a few hours and use adult mice or rats, a handful of studies performed on adult animals have had success for survival of slices [4], [5]. Here we describe a method for culturing organotypic slices with high viability from hippocampus of aged mice and rabbits. •Our method permits slices from mice as old as 16 months and rabbits as old as years of age to survive ex vivo up to 8 weeks [6], [7], [8], [9]. Such a slice system may be relevant to investigating age-related brain diseases.

Real-time Recordings of Migrating Cortical Neurons from GFP and Cre Recombinase Expressing Mice.

The cerebral cortex is one of the most intricate regions of the brain that requires elaborate cell migration patterns for its development. Experimental observations show that projection neurons migrate radially within the cortical wall, whereas interneurons migrate along multiple tangential paths to reach the developing cortex. Tight regulation of the cell migration processes ensures proper positioning and functional integration of neurons to specific cerebral cortical circuits. Disruption of neuronal migration often leads to cortical dysfunction and/or malformation associated with neurological disorders. Unveiling the molecular control of neuron migration is thus fundamental to understanding the physiological or pathological development of the cerebral cortex. In this unit, protocols allowing detailed analysis of patterns of migration of both interneurons and projection neurons under different experimental conditions (i.e., loss or gain of function) are presented.