Study of cerebral energy metabolism using the rat hippocampal slice preparation.

This article describes methods and experimental paradigms used in combination with the rat hippocampal slice preparation in an attempt to better understand cerebral energy metabolism under the following conditions: normal resting conditions, conditions of oxygen and/or glucose deprivation, and conditions of activation (excitation). The outcome of this attempt, as described herewith, demonstrates the unmatched usefulness of the brain slice preparation as an in vitro tool in the field of neuroscience.     

17β-Estradiol-mediated increase in Cu/Zn superoxide dismutase expression in the brain: a mechanism to protect neurons from ischemia

A number of studies have demonstrated that 17β-estradiol (E(2)) protects the brain from ischemia and yet the mechanism by which this hormone brings about its protective effect is unclear. Interestingly, like E(2), overexpression of the oxidative stress response protein Cu/Zn superoxide dismutase (SOD1), which plays a critical role in regulating reactive oxygen species, also protects the brain from ischemia. Because we previously showed that E(2) treatment of cultured mammary cells increases SOD1 expression, we hypothesized that E(2) might increase SOD1 expression in the brain and that this E(2)-mediated increase in SOD1 expression might help to protect the brain from ischemia. We now show that SOD1 is expressed in cortical neurons, that SOD1 expression is increased by exposure of brain slice cultures to E(2), and that the E(2)-mediated increase in SOD1 expression is further augmented by exposure of brain slice cultures to increased superoxide levels or oxygen and glucose deprivation. Importantly, when cortical neurons are exposed to increased superoxide levels and markers of protein and DNA damage, nitrotyrosine and 8-oxoguanine, respectively, are measured, both protein and DNA damage are reduced. In fact, E(2) reduces nitrotyrosine and 8-oxoguanine levels in brain slice cultures regardless of whether they have or have not been exposed to increased superoxide levels. Likewise, when brain slice cultures are treated with E(2) and deprived of oxygen and glucose, 8-oxoguanine levels are reduced. Taken together, these studies provide a critical link between E(2) treatment, SOD1 expression, and neuroprotection and help to define a mechanism through which E(2)-mediated neuroprotection may be conferred.     

Glutamate Neurotoxicity and the Inhibition of Protein Synthesis in the Hippocampal Slice

In some animal models of ischemia, neuronal degeneration can be prevented by the selective antagonism of the N-methyl-D-aspartate (NMDA) glutamate receptor subtype, suggesting that glutamate released during ischemia causes injury by activating NMDA receptors. The rat hippocampal slice preparation was used as an in vitro model to study the pharmacology of glutamate toxicity and investigate why NMDA receptors are critical in ischemic injury. Acute toxicity was assessed by quantifying the inhibition of protein synthesis, which we confirmed by autoradiography to be primarily neuronal. The effect of NMDA was prevented by the specific antagonists MK-801 and ketamine, as well as by the less selective antagonist kynurenic acid. The less selective antagonists kynurenic acid and 6,7-dinitroquinoxaline-2,3-dione antagonized the effects of quisqualate and NMDA. In contrast to previous observations with dissociated neurons in tissue culture, the toxicity of glutamate was unaffected by antagonists, regardless of the glutamate concentration, the duration of exposure, or the presence of magnesium. The high concentration of glutamate required to inhibit protein synthesis and the inability of receptor antagonists to block the effect of glutamate suggest that either glutamate acts through a non-receptor-mediated mechanism, or that the receptor-mediated nature of glutamate effects are masked in the slice preparation, perhaps by the glial uptake of glutamate. The altered physiology induced by ischemia must potentiate the neurotoxicity of glutamate, because we observed with a brain slice preparation that only high concentrations of glutamate caused neurotoxicity in the presence of oxygen and glucose and that these effects were not reversed by glutamate receptor antagonists.     

Organotypic slice cultures of embryonic ventral midbrain: a system to study dopaminergic neuronal development in vitro

The mouse is an excellent model organism to study mammalian brain development due to the abundance of molecular and genetic data. However, the developing mouse brain is not suitable for easy manipulation and imaging in vivo since the mouse embryo is inaccessible and opaque. Organotypic slice cultures of embryonic brains are therefore widely used to study murine brain development in vitro. Ex-vivo manipulation or the use of transgenic mice allows the modification of gene expression so that subpopulations of neuronal or glial cells can be labeled with fluorescent proteins. The behavior of labeled cells can then be observed using time-lapse imaging. Time-lapse imaging has been particularly successful for studying cell behaviors that underlie the development of the cerebral cortex at late embryonic stages (1-2). Embryonic organotypic slice culture systems in brain regions outside of the forebrain are less well established. Therefore, the wealth of time-lapse imaging data describing neuronal cell migration is restricted to the forebrain (3,4). It is still not known, whether the principles discovered for the dorsal brain hold true for ventral brain areas. In the ventral brain, neurons are organized in neuronal clusters rather than layers and they often have to undergo complicated migratory trajectories to reach their final position. The ventral midbrain is not only a good model system for ventral brain development, but also contains neuronal populations such as dopaminergic neurons that are relevant in disease processes. While the function and degeneration of dopaminergic neurons has been investigated in great detail in the adult and ageing brain, little is known about the behavior of these neurons during their differentiation and migration phase (5). We describe here the generation of slice cultures from the embryonic day (E) 12.5 mouse ventral midbrain. These slice cultures are potentially suitable for monitoring dopaminergic neuron development over several days in vitro. We highlight the critical steps in generating brain slices at these early stages of embryonic development and discuss the conditions necessary for maintaining normal development of dopaminergic neurons in vitro. We also present results from time lapse imaging experiments. In these experiments, ventral midbrain precursors (including dopaminergic precursors) and their descendants were labeled in a mosaic manner using a Cre/loxP based inducible fate mapping system (6).     

In vitro and in vivo neuroprotective activity of the cardiac glycoside oleandrin from Nerium oleander in brain slice-based stroke models

The principal active constituent of the botanical drug candidate PBI-05204, a supercritical CO(2) extract of Nerium oleander, is the cardiac glycoside oleandrin. PBI-05204 shows potent anticancer activity and is currently in phase I clinical trial as a treatment for patients with solid tumors. We have previously shown that neriifolin, which is structurally related to oleandrin, provides robust neuroprotection in brain slice and whole animal models of ischemic injury. However, neriifolin itself is not a suitable drug development candidate and the FDA-approved cardiac glycoside digoxin does not cross the blood-brain barrier. We report here that both oleandrin as well as the full PBI-05204 extract can also provide significant neuroprotection to neural tissues damaged by oxygen and glucose deprivation as occurs in ischemic stroke. Critically, we show that the neuroprotective activity of PBI-05204 is maintained for several hours of delay of administration after oxygen and glucose deprivation treatment. We provide evidence that the neuroprotective activity of PBI-05204 is mediated through oleandrin and/or other cardiac glycoside constituents, but that additional, non-cardiac glycoside components of PBI-05204 may also contribute to the observed neuroprotective activity. Finally, we show directly that both oleandrin and the protective activity of PBI-05204 are blood brain barrier penetrant in a novel model for in vivo neuroprotection. Together, these findings suggest clinical potential for PBI-05204 in the treatment of ischemic stroke and prevention of associated neuronal death.     

Laserspritzer: A Simple Method for Optogenetic Investigation with Subcellular Resolutions

To build a detailed circuit diagram in the brain, one needs to measure functional synaptic connections between specific types of neurons. A high-resolution circuit diagram should provide detailed information at subcellular levels such as soma, distal and basal dendrites. However, a limitation lies in the difficulty of studying long-range connections between brain areas separated by millimeters. Brain slice preparations have been widely used to help understand circuit wiring within specific brain regions. The challenge exists because long-range connections are likely to be cut in a brain slice. The optogenetic approach overcomes these limitations, as channelrhodopsin 2 (ChR2) is efficiently transported to axon terminals that can be stimulated in brain slices. Here, we developed a novel fiber optic based simple method of optogenetic stimulation: the laserspritzer approach. This method facilitates the study of both long-range and local circuits within brain slice preparations. This is a convenient and low cost approach that can be easily integrated with a slice electrophysiology setup, and repeatedly used upon initial validation. Our data with direct ChR2 mediated-current recordings demonstrates that the spatial resolution of the laserspritzer is correlated with the size of the laserspritzer, and the resolution lies within the 30 µm range for the 5 micrometer laserspritzer. Using olfactory cortical slices, we demonstrated that the laserspritzer approach can be applied to selectively activate monosynaptic perisomatic GABAergic basket synapses, or long-range intracortical glutamatergic inputs formed on different subcellular domains within the same cell (e.g. distal and proximal dendrites). We discuss significant advantages of the laserspritzer approach over the widely used collimated LED whole-field illumination method in brain slice electrophysiological research.     

Fluorescence imaging of changes in intracellular chloride in living brain slices.

In brain slice preparations, chloride movements across the cell membrane of living cells are measured traditionally with 36Cl- tracer methods, Cl--selective microelectrodes, or whole-cell recording using patch clamp analysis. We have developed an alternative, noninvasive technique that uses the fluorescent Cl- ion indicator, 6-methoxy-N-ethylquinolinium iodide (MEQ), to study changes in intracellular Cl- by epifluorescence or UV laser scanning confocal microscopy. In brain slices taken from rodents younger than 22 days of age, excellent cellular loading is achieved with the membrane-permeable form of the dye, dihydro-MEQ. Subsequent intracellular oxidation of dihydro-MEQ to the Cl--sensitive MEQ traps the polar form of the dye inside the neurons. Because MEQ is a single-excitation and single-emission dye, changes in intracellular Cl- concentrations can be calibrated from the Stern-Volmer relationship, determined in separate experiments. Using MEQ as the fluorescent indicator for Cl-, Cl- flux through the gamma-aminobutyric acid (GABA)-gated Cl- channel (GABAA receptor) can be studied by dynamic video imaging and either nonconfocal (epifluorescence) or confocal microscopy in the acute brain slice preparation. Increases in intracellular Cl- quench MEQ fluorescence, thereby reflecting GABAA receptor activation. GABAA receptor functional activity can be measured in discrete cells located in neuroanatomically defined populations within areas such as the neocortex and hippocampus. Changes in intracellular Cl- can also be studied under various conditions such as oxygen/glucose deprivation ("in vitro ischemia") and excitotoxicity. In such cases, changes in cell volume may also occur due to the dependence of cell volume regulation on Na+, K+, and Cl- flux. Because changes in cell volume can affect optical fluorescence measurements, we assess cell volume changes in the brain slice using the fluorescent indicator calcein-AM. Determination of changes in MEQ fluorescence versus calcein fluorescence allows one to distinguish between an increase in intracellular Cl- and an increase in cell volume.     

Probabilistic Clustering of the Human Connectome Identifies Communities and Hubs

A fundamental assumption in neuroscience is that brain function is constrained by its structural properties. This motivates the idea that the brain can be parcellated into functionally coherent regions based on anatomical connectivity patterns that capture how different areas are interconnected. Several studies have successfully implemented this idea in humans using diffusion weighted MRI, allowing parcellation to be conducted in vivo. Two distinct approaches to connectivity-based parcellation can be identified. The first uses the connection profiles of brain regions as a feature vector, and groups brain regions with similar connection profiles together. Alternatively, one may adopt a network perspective that aims to identify clusters of brain regions that show dense within-cluster and sparse between-cluster connectivity. In this paper, we introduce a probabilistic model for connectivity-based parcellation that unifies both approaches. Using the model we are able to obtain a parcellation of the human brain whose clusters may adhere to either interpretation. We find that parts of the connectome consistently cluster as densely connected components, while other parts consistently result in clusters with similar connections. Interestingly, the densely connected components consist predominantly of major cortical areas, while the clusters with similar connection profiles consist of regions that have previously been identified as the ‘rich club’; regions known for their integrative role in connectivity. Furthermore, the probabilistic model allows quantification of the uncertainty in cluster assignments. We show that, while most clusters are clearly delineated, some regions are more difficult to assign. These results indicate that care should be taken when interpreting connectivity-based parcellations obtained using alternative deterministic procedures.     

Diffusion in the slice microenvironment and implications for physiological studies

The brain cell microenvironment includes the extracellular space surrounding the cell together with the cellular elements that define the space. The dense packing of cells in the mammalian nervous system ensures that the extracellular space is narrow but highly complex in geometry. Recent studies with ion-selective micropipettes have revealed that the cerebellar slice can support changes in [K+]o that resemble those seen in the intact preparation. In the slice, [K+]o responses of individual cells can even be resolved. Studies with iontophoretic techniques and quantitative analysis in the slice have shown that the extracellular space has diffusion properties, characterized by a volume fraction and a tortuosity, that are very similar to those seen in the intact animal. These data confirm that the microenvironment in the slice is comparable to that in the intact animal. The diffusion parameters can be used to make predictions about the time necessary for substances to diffuse into slices under various conditions. Such estimates, together with other studies, indicate that it is probably inadvisable to use slices with thicknesses in excess of 300--400 micrometers, and that the bathing conditions can be critical in maintaining slice viability.     

Organotypic Brain Slice Cultures of Adult Transgenic P301S Mice��A Model for Tauopathy Studies

Background

Organotypic brain slice cultures represent an excellent compromise between single cell cultures and complete animal studies, in this way replacing and reducing the number of animal experiments. Organotypic brain slices are widely applied to model neuronal development and regeneration as well as neuronal pathology concerning stroke, epilepsy and Alzheimer’s disease (AD). AD is characterized by two protein alterations, namely tau hyperphosphorylation and excessive amyloid β deposition, both causing microglia and astrocyte activation. Deposits of hyperphosphorylated tau, called neurofibrillary tangles (NFTs), surrounded by activated glia are modeled in transgenic mice, e.g. the tauopathy model P301S.

Methodology/Principal Findings

In this study we explore the benefits and limitations of organotypic brain slice cultures made of mature adult transgenic mice as a potential model system for the multifactorial phenotype of AD. First, neonatal (P1) and adult organotypic brain slice cultures from 7- to 10-month-old transgenic P301S mice have been compared with regard to vitality, which was monitored with the lactate dehydrogenase (LDH)- and the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays over 15 days. Neonatal slices displayed a constant high vitality level, while the vitality of adult slice cultures decreased significantly upon cultivation. Various preparation and cultivation conditions were tested to augment the vitality of adult slices and improvements were achieved with a reduced slice thickness, a mild hypothermic cultivation temperature and a cultivation CO₂ concentration of 5%. Furthermore, we present a substantial immunohistochemical characterization analyzing the morphology of neurons, astrocytes and microglia in comparison to neonatal tissue.

Conclusion/Significance

Until now only adolescent animals with a maximum age of two months have been used to prepare organotypic brain slices. The current study provides evidence that adult organotypic brain slice cultures from 7- to 10-month-old mice independently of the transgenic modification undergo slow programmed cell death, caused by a dysfunction of the neuronal repair systems.     

Identifying brain hierarchical structures associated with Alzheimer's disease using a regularized regression method with tree predictors

Brain segmentation at different levels is generally represented as hierarchical trees. Brain regional atrophy at specific levels was found to be marginally associated with Alzheimer's disease outcomes. In this study, we propose an ℓ₁-type regularization for predictors that follow a hierarchical tree structure. Considering a tree as a directed acyclic graph, we interpret the model parameters from a path analysis perspective. Under this concept, the proposed penalty regulates the total effect of each predictor on the outcome. With regularity conditions, it is shown that under the proposed regularization, the estimator of the model coefficient is consistent in ℓ₂-norm and the model selection is also consistent. When applied to a brain sMRI dataset acquired from the Alzheimer's Disease Neuroimaging Initiative (ADNI), the proposed approach identifies brain regions where atrophy in these regions demonstrates the declination in memory. With regularization on the total effects, the findings suggest that the impact of atrophy on memory deficits is localized from small brain regions, but at various levels of brain segmentation. Data used in preparation of this paper were obtained from the ADNI database.     

Modeling Neural Immune Signaling of Episodic and Chronic Migraine Using Spreading Depression In Vitro

Migraine and its transformation to chronic migraine are healthcare burdens in need of improved treatment options. We seek to define how neural immune signaling modulates the susceptibility to migraine, modeled in vitro using spreading depression (SD), as a means to develop novel therapeutic targets for episodic and chronic migraine. SD is the likely cause of migraine aura and migraine pain. It is a paroxysmal loss of neuronal function triggered by initially increased neuronal activity, which slowly propagates within susceptible brain regions. Normal brain function is exquisitely sensitive to, and relies on, coincident low-level immune signaling. Thus, neural immune signaling likely affects electrical activity of SD, and therefore migraine. Pain perception studies of SD in whole animals are fraught with difficulties, but whole animals are well suited to examine systems biology aspects of migraine since SD activates trigeminal nociceptive pathways. However, whole animal studies alone cannot be used to decipher the cellular and neural circuit mechanisms of SD. Instead, in vitro preparations where environmental conditions can be controlled are necessary. Here, it is important to recognize limitations of acute slices and distinct advantages of hippocampal slice cultures. Acute brain slices cannot reveal subtle changes in immune signaling since preparing the slices alone triggers: pro-inflammatory changes that last days, epileptiform behavior due to high levels of oxygen tension needed to vitalize the slices, and irreversible cell injury at anoxic slice centers. In contrast, we examine immune signaling in mature hippocampal slice cultures since the cultures closely parallel their in vivo counterpart with mature trisynaptic function; show quiescent astrocytes, microglia, and cytokine levels; and SD is easily induced in an unanesthetized preparation. Furthermore, the slices are long-lived and SD can be induced on consecutive days without injury, making this preparation the sole means to-date capable of modeling the neuroimmune consequences of chronic SD, and thus perhaps chronic migraine. We use electrophysiological techniques and non-invasive imaging to measure neuronal cell and circuit functions coincident with SD. Neural immune gene expression variables are measured with qPCR screening, qPCR arrays, and, importantly, use of cDNA preamplification for detection of ultra-low level targets such as interferon-gamma using whole, regional, or specific cell enhanced (via laser dissection microscopy) sampling. Cytokine cascade signaling is further assessed with multiplexed phosphoprotein related targets with gene expression and phosphoprotein changes confirmed via cell-specific immunostaining. Pharmacological and siRNA strategies are used to mimic and modulate SD immune signaling.     

Coupling of organotypic brain slice cultures to silicon-based arrays of electrodes.

Fetal or early postnatal brain tissue can be cultured in viable and healthy condition for several weeks with development and preservation of the basic cellular and connective organization as so-called organotypic brain slice cultures. Here we demonstrate and describe how it is possible to establish such hippocampal rat brain slice cultures on biocompatible silicon-based chips with arrays of electrodes with a histological organization comparable to that of conventional brain slice cultures grown by the roller drum technique and on semiporous membranes. Intracellular and extracellular recordings from neurons in the slice cultures show that the electroresponsive properties of the neurons and synaptic circuitry are in accordance with those described for cells in acutely prepared slices of the adult rat hippocampus. Based on the recordings and the possibilities of stimulating the cultured cells through the electrode arrays it is anticipated that the setup eventually will allow long-term studies of defined neuronal networks and provide valuable information on both normal and neurotoxicological and neuropathological conditions.     

In vitro studies of closed-loop feedback and electrosensory processing in Apteronotus leptorhynchus

Electrosensory systems comprise extensive feedback pathways. It is also well known that these pathways exhibit synaptic plasticity on a wide-range of time scales. Recent in vitro brain slice studies have characterized synaptic plasticity in the two main feedback pathways to the electrosensory lateral line lobe (ELL), a primary electrosensory nucleus in Apteronotus leptorhynchus. Currently-used slice preparations, involving networks in open-loop conditions, allow feedback inputs to be studied in isolation, a critical step in determining their synaptic properties. However, to fully understand electrosensory processing, we must understand how dynamic feedback modulates neuronal responses under closed-loop conditions. To bridge the gap between current in vitro approaches and more complex in vivo work, we present two new in vitro approaches for studying the roles of closed-loop feedback in electrosensory processing. The first involves a hybrid-network approach using dynamic clamp, and the second involves a new slice preparation that preserves one of the feedback pathways to ELL in a closed-loop condition.     

The use of brain slices in central nervous system pharmacology

Brain slice preparations have most frequently been employed to answer questions of a biochemical or physiological nature. Nevertheless, in vitro brain slices also have considerable value as pharmacological tools with which to study the physiological actions of neurotransmitters and drugs on the central nervous system. Several aspects of the slice preparation facilitate this type of analysis. Because drugs can be applied and tested in a relatively quantitative manner, many classical pharmacological techniques (e.g., dose-response curves, tests for competitive vs. noncompetitive antagonism) can be used to examine drug responses. At the simplest level, these techniques facilitate the differentiation of specific (primarily receptor-mediated) and nonspecific actions of drugs and neurotransmitters. As another consequence, the electrophysiological actions of drugs can be directly compared to their biochemical effects in vitro (receptor binding, activation or inhibition of adenylate cyclase, etc.). Ultimately, parallel studies of this type can be used to establish mechanisms of action for various neurotransmitters, particularly those that may employ biochemical substrates as second messengers. In this paper we describe in general terms many of the advantages of the slice preparation as a neuropharmacological tool. Some of the criteria useful in determining the involvement of various receptors in drug-induced changes in electrophysiological responses are discussed in detail. Finally, the responses of hippocampal slices to various adrenergic agents are used to illustrate the way in which various features of this preparation can be exploited pharmacologically. The results of these experiments constitute a significant advance in terms of our understanding of the neuropharmacology of catecholamine responses in this brain region.     

Oxygen measurements in brain stem slices exposed to normobaric hyperoxia and hyperbaric oxygen.

We previously reported (J Appl Physiol 89: 807-822, 2000) that < or =10 min of hyperbaric oxygen (HBO(2); < or = 2,468 Torr) stimulates solitary complex neurons. To better define the hyperoxic stimulus, we measured PO(2) in the solitary complex of 300-microm-thick rat medullary slices, using polarographic carbon fiber microelectrodes, during perfusion with media having PO(2) values ranging from 156 to 2,468 Torr. Under control conditions, slices equilibrated with 95% O(2) at barometric pressure of 1 atmospheres absolute had minimum PO(2) values at their centers (291 +/- 20 Torr) that were approximately 10-fold greater than PO(2) values measured in the intact central nervous system (10-34 Torr). During HBO(2), PO(2) increased at the center of the slice from 616 +/- 16 to 1,517 +/- 15 Torr. Tissue oxygen consumption tended to decrease at medium PO(2) or = 1,675 Torr to levels not different from values measured at PO(2) found in all media in metabolically poisoned slices (2-deoxy-D-glucose and antimycin A). We conclude that control medium used in most brain slice studies is hyperoxic at normobaric pressure. During HBO(2), slice PO(2) increases to levels that appear to reduce metabolism.     

In vitro synaptic reconsolidation in amygdala slices prepared from rat brains

The retrieval of consolidated fear memory causes it to be labile or deconsolidated, and the deconsolidated fear memory is reconsolidated over time in a protein synthesis-dependent manner. We have recently developed an ex vivo model where during fear memory deconsolidation and reconsolidation the synaptic state can be monitored at thalamic input synapses onto the lateral amygdala (T-LA synapses), a storage site for auditory fear memory. In this ex vivo model, the deconsolidation and reconsolidation processes of auditory fear memory in the intact brain were prevented following brain slicing; therefore, we could monitor the synaptic state for memory deconsolidation and reconsolidation at the time of brain slicing. However, why the synaptic reconsolidation process stopped after brain slicing in the ex vivo model is not known. One possibility is that brain slicing severs neuromodulatory innervations, which are required for memory reconsolidation, from other brain regions (e.g., noradrenergic innervation). In the present study, we supplemented amygdala slices with exogenous norepinephrine as a substitute for the severed noradrenergic innervations. DHPG (a group I metabotropic glutamate receptor agonist)-induced depotentiation (mGluRI-depotentiation), a marker for consolidated synapses, was observed following norepinephrine application to slices prepared immediately after tone presentation (fear memory retrieval) to rats that had been pre-conditioned to a tone paired with a shock. These results suggest that noradrenergic activation initiates synaptic reconsolidation. In contrast, mGluRI-depotentiation was absent following norepinephrine application to slices that were prepared immediately after the tone presentation (no fear memory retrieval) to rats when a tone and a shock were unpaired, ruling out the possibility that noradrenergic activation somehow facilitates a subsequent synaptic depression induced by DHPG irrespective of synaptic reconsolidation. Furthermore, the restored mGluRI-depotentiation following application of exogenous norepinephrine was dependent on de novo protein synthesis, as is memory reconsolidation. Thus, our findings suggest that T-LA synapses from acute slice preparations can undergo a reconsolidation process, thereby providing an optimal preparation to study a fear memory reconsolidation process in vitro.     

A visual thalamocortical slice

We describe a thalamocortical slice preparation in which connectivity between the mouse lateral geniculate nucleus (LGN) and primary visual cortex (V1) is preserved. Through DiI injections in fixed brains we traced and created a three-dimensional model of the mouse visual pathways. From this computer model we designed a slice preparation that contains a projection from LGN to V1. We prepared brain slices with these predicted coordinates and demonstrated anatomical LGN-V1 connectivity in these slices after LGN tracer injections. We also revealed functional LGN-V1 connectivity by stimulating LGN electrically and detecting responses in layer 4 of V1 using calcium imaging, field potential recordings and whole-cell recordings. We also identified layer-4 neurons that receive direct thalamocortical input. Finally, we compared cortical activity after LGN stimulation with spontaneous cortical activity and found significant overlap of the spatiotemporal dynamics generated by both types of events.     

A Brain Tumor/Organotypic Slice Co-culture System for Studying Tumor Microenvironment and Targeted Drug Therapies

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these tumors are not easily grown in standard culture conditions. Neurosphere cultures under serum-free conditions and orthotopic xenografts have expanded the range of tumors that can be maintained. However, many types of brain tumors remain difficult to propagate or study. This is particularly true for pediatric brain tumors such as pilocytic astrocytomas and medulloblastomas. This protocol describes a system that allows primary human brain tumors to be grown in culture. This quantitative assay can be used to investigate the effect of microenvironment on tumor growth, and to test new drug therapies. This protocol describes a system where fluorescently labeled brain tumor cells are grown on an organotypic brain slice from a juvenile mouse. The response of tumor cells to drug treatments can be studied in this assay, by analyzing changes in the number of cells on the slice over time. In addition, this system can address the nature of the microenvironment that normally fosters growth of brain tumors. This brain tumor organotypic slice co-culture assay provides a propitious system for testing new drugs on human tumor cells within a brain microenvironment.     

Real-time decoding of dopamine concentration changes in the caudate-putamen during tonic and phasic firing

The fundamental process that underlies volume transmission in the brain is the extracellular diffusion of neurotransmitters from release sites to distal target cells. Dopaminergic neurons display a range of activity states, from low-frequency tonic firing to bursts of high-frequency action potentials (phasic firing). However, it is not clear how this activity affects volume transmission on a subsecond time scale. To evaluate this, we developed a finite-difference model that predicts the lifetime and diffusion of dopamine in brain tissue. We first used this model to decode in vivo amperometric measurements of electrically evoked dopamine, and obtained rate constants for release and uptake as well as the extent of diffusion. Accurate predictions were made under a variety of conditions including different regions, different stimulation parameters and with uptake inhibited. Second, we used the decoded rate constants to predict how heterogeneity of dopamine release and uptake sites would affect dopamine concentration fluctuations during different activity states in the absence of an electrode. These simulations show that synchronous phasic firing can produce spatially and temporally heterogeneous concentration profiles whereas asynchronous tonic firing elicits uniform, steady-state dopamine concentrations.