Cellular and network contributions to excitability of layer 5 neocortical pyramidal neurons in the rat

There is a considerable gap between investigating the dynamics of single neurons and the computational aspects of neural networks. A growing number of studies have attempted to overcome this gap using the excitation in brain slices elicited by various chemical manipulations of the bath solution. However, there has been no quantitative study on the effects of these manipulations on the cellular and network factors controlling excitability. Using the whole-cell configuration of the patch-clamp technique we recorded the membrane potential from the soma of layer 5 pyramidal neurons in acute brain slices from the somatosensory cortex of young rats at 22 degrees C and 35 degrees C. Using blockers of synaptic transmission, we show distinct changes in cellular properties following modification of the ionic composition of the artificial cerebrospinal fluid (ACSF). Thus both cellular and network changes may contribute to the observed effects of slice excitation solutions on the physiology of single neurons. Furthermore, our data suggest that the difference in the ionic composition of current standard ACSF from that of CSF measured in vivo cause ACSF to depress network activity in acute brain slices. This may affect outcomes of experiments investigating biophysical and physiological properties of neurons in such preparations. Our results strongly advocate the necessity of redesigning experiments routinely carried out in the quiescent acute brain slice preparation.     

The zebrafish brain in research and teaching: a simple in vivo and in vitro model for the study of spontaneous neural activity

Recently, the zebrafish (Danio rerio) has been established as a key animal model in neuroscience. Behavioral, genetic, and immunohistochemical techniques have been used to describe the connectivity of diverse neural circuits. However, few studies have used zebrafish to understand the function of cerebral structures or to study neural circuits. Information about the techniques used to obtain a workable preparation is not readily available. Here, we describe a complete protocol for obtaining in vitro and in vivo zebrafish brain preparations. In addition, we performed extracellular recordings in the whole brain, brain slices, and immobilized nonanesthetized larval zebrafish to evaluate the viability of the tissue. Each type of preparation can be used to detect spontaneous activity, to determine patterns of activity in specific brain areas with unknown functions, or to assess the functional roles of different neuronal groups during brain development in zebrafish. The technique described offers a guide that will provide innovative and broad opportunities to beginner students and researchers who are interested in the functional analysis of neuronal activity, plasticity, and neural development in the zebrafish brain.     

Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of interest. This allows for examination of synaptic transmission under conditions where the extracellular and postsynaptic intracellular environments can be well controlled. A vibration-based technique without the use of proteases, known as vibrodissociation, is the most popular technique for mechanical isolation. A micropipette, with the tip fire-polished to the shape of a small ball, is placed into a brain slice made from a P1-P21 rodent. The micropipette is vibrated parallel to the slice surface and lowered through the slice thickness resulting in the liberation of isolated neurons. The isolated neurons are ready for study within a few minutes of vibrodissociation. This technique has advantages over the use of primary neuronal cultures, brain slices and enzymatically isolated neurons including: rapid production of viable, relatively mature neurons suitable for electrophysiological and imaging studies; superior control of the extracellular environment free from the influence of neighboring cells; suitability for well-controlled pharmacological experiments using rapid drug application and total cell superfusion; and improved space-clamp in whole-cell recordings relative to neurons in slice or cell culture preparations. This preparation can be used to examine synaptic physiology, pharmacology, modulation and plasticity. Real-time imaging of both pre- and postsynaptic elements in the living cells and boutons is also possible using vibrodissociated neurons. Characterization of the molecular constituents of pre- and postsynaptic elements can also be achieved with immunological and imaging-based approaches.     

Blind patch clamp recordings in embryonic and adult mammalian brain slices

To obtain electrophysiological recordings in brain slices, sophisticated and expensive pieces of equipment can be used. However, costly microscope equipment with infrared differential interference contrast optics is not always necessary or even desirable. For instance, obtaining a randomized unbiased sample in a given preparation would be better accomplished if cells were not directly visualized before recording. In addition, some preparations require thick slices, and direct visualization is not possible. Here we describe a protocol for the 'blind patch clamp method' that we developed several years ago to perform electrophysiological recordings in mammalian brain slices using a standard patch clamp amplifier, dissecting microscope and recording chamber. Overall, it takes approximately 3-4 h to set up this procedure.     

Large-Scale,High-Resolution Multielectrode-Array Recording Depicts Functional Network Differences of Cortical and Hippocampal Cultures

Understanding the detailed circuitry of functioning neuronal networks is one of the major goals of neuroscience. Recent improvements in neuronal recording techniques have made it possible to record the spiking activity from hundreds of neurons simultaneously with sub-millisecond temporal resolution. Here we used a 512-channel multielectrode array system to record the activity from hundreds of neurons in organotypic cultures of cortico-hippocampal brain slices from mice. To probe the network structure, we employed a wavelet transform of the cross-correlogram to categorize the functional connectivity in different frequency ranges. With this method we directly compare, for the first time, in any preparation, the neuronal network structures of cortex and hippocampus, on the scale of hundreds of neurons, with sub-millisecond time resolution. Among the three frequency ranges that we investigated, the lower two frequency ranges (gamma (30–80 Hz) and beta (12–30 Hz) range) showed similar network structure between cortex and hippocampus, but there were many significant differences between these structures in the high frequency range (100–1000 Hz). The high frequency networks in cortex showed short tailed degree-distributions, shorter decay length of connectivity density, smaller clustering coefficients, and positive assortativity. Our results suggest that our method can characterize frequency dependent differences of network architecture from different brain regions. Crucially, because these differences between brain regions require millisecond temporal scales to be observed and characterized, these results underscore the importance of high temporal resolution recordings for the understanding of functional networks in neuronal systems.     

Clostridium perfringens Epsilon Toxin Targets Granule Cells in the Mouse Cerebellum and Stimulates Glutamate Release

Epsilon toxin (ET) produced by C. perfringens types B and D is a highly potent pore-forming toxin. ET-intoxicated animals express severe neurological disorders that are thought to result from the formation of vasogenic brain edemas and indirect neuronal excitotoxicity. The cerebellum is a predilection site for ET damage. ET has been proposed to bind to glial cells such as astrocytes and oligodendrocytes. However, the possibility that ET binds and attacks the neurons remains an open question. Using specific anti-ET mouse polyclonal antibodies and mouse brain slices preincubated with ET, we found that several brain structures were labeled, the cerebellum being a prominent one. In cerebellar slices, we analyzed the co-staining of ET with specific cell markers, and found that ET binds to the cell body of granule cells, oligodendrocytes, but not astrocytes or nerve endings. Identification of granule cells as neuronal ET targets was confirmed by the observation that ET induced intracellular Ca²⁺ rises and glutamate release in primary cultures of granule cells. In cultured cerebellar slices, whole cell patch-clamp recordings of synaptic currents in Purkinje cells revealed that ET greatly stimulates both spontaneous excitatory and inhibitory activities. However, pharmacological dissection of these effects indicated that they were only a result of an increased granule cell firing activity and did not involve a direct action of the toxin on glutamatergic nerve terminals or inhibitory interneurons. Patch-clamp recordings of granule cell somata showed that ET causes a decrease in neuronal membrane resistance associated with pore-opening and depolarization of the neuronal membrane, which subsequently lead to the firing of the neuronal network and stimulation of glutamate release. This work demonstrates that a subset of neurons can be directly targeted by ET, suggesting that part of ET-induced neuronal damage observed in neuronal tissue is due to a direct effect of ET on neurons.     

A Rapid Approach to High-Resolution Fluorescence Imaging in Semi-Thick Brain Slices

A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are characteristic to neurons in both normal and diseased brain. Towards this, a great deal of effort has been placed on building high-resolution neuroanatomical maps^1-3. With the expansion of molecular genetics and advances in light microscopy has come the ability to query not only neuronal morphologies, but also the molecular and cellular makeup of individual neurons and their associated networks⁴. Major advances in the ability to mark and manipulate neurons through transgenic and gene targeting technologies in the rodent now allow investigators to ''program'' neuronal subsets at will^5-6. Arguably, one of the most influential contributions to contemporary neuroscience has been the discovery and cloning of genes encoding fluorescent proteins (FPs) in marine invertebrates^7-8, alongside their subsequent engineering to yield an ever-expanding toolbox of vital reporters⁹. Exploiting cell type-specific promoter activity to drive targeted FP expression in discrete neuronal populations now affords neuroanatomical investigation with genetic precision.Engineering FP expression in neurons has vastly improved our understanding of brain structure and function. However, imaging individual neurons and their associated networks in deep brain tissues, or in three dimensions, has remained a challenge. Due to high lipid content, nervous tissue is rather opaque and exhibits auto fluorescence. These inherent biophysical properties make it difficult to visualize and image fluorescently labelled neurons at high resolution using standard epifluorescent or confocal microscopy beyond depths of tens of microns. To circumvent this challenge investigators often employ serial thin-section imaging and reconstruction methods¹⁰, or 2-photon laser scanning microscopy¹¹. Current drawbacks to these approaches are the associated labor-intensive tissue preparation, or cost-prohibitive instrumentation respectively.Here, we present a relatively rapid and simple method to visualize fluorescently labelled cells in fixed semi-thick mouse brain slices by optical clearing and imaging. In the attached protocol we describe the methods of: 1) fixing brain tissue in situ via intracardial perfusion, 2) dissection and removal of whole brain, 3) stationary brain embedding in agarose, 4) precision semi-thick slice preparation using new vibratome instrumentation, 5) clearing brain tissue through a glycerol gradient, and 6) mounting on glass slides for light microscopy and z-stack reconstruction (Figure 1).For preparing brain slices we implemented a relatively new piece of instrumentation called the ''Compresstome'' VF-200 (http://www.precisionary.com/products_vf200.html). This instrument is a semi-automated microtome equipped with a motorized advance and blade vibration system with features similar in function to other vibratomes. Unlike other vibratomes, the tissue to be sliced is mounted in an agarose plug within a stainless steel cylinder. The tissue is extruded at desired thicknesses from the cylinder, and cut by the forward advancing vibrating blade. The agarose plug/cylinder system allows for reproducible tissue mounting, alignment, and precision cutting. In our hands, the ''Compresstome'' yields high quality tissue slices for electrophysiology, immunohistochemistry, and direct fixed-tissue mounting and imaging. Combined with optical clearing, here we demonstrate the preparation of semi-thick fixed brain slices for high-resolution fluorescent imaging. Download video file.^{(28M, mov)}     

Electrophysiological and Morphological Properties of Neurons Acutely Isolated from the Rostral Gustatory Zone of the Rat Nucleus of the Solitary Tract

The biophysical and morphological characteristics of acutelyisolated neurons from the rostral nucleus of the solitary tract(rNST) were investigated under current clamp conditions andcompared with the results obtained from neurons recorded inbrain slices. The passive membrane properties of the isolatedneurons were similar to rNST neurons in brain slices and theneurons maintained their morphological characteristics althoughtheir dendritic tree was truncated. The isolated neurons alsoretained their characteristic repetitive firing properties.In addition we also noted developmental changes in the intrinsicmembrane properties of the isolated neurons, such as a shorteningin action potential duration, decrease in membrane time constantand input resistance, that occurred when these parameters werecompared in neurons isolated from young (5–10 days) andolder animals. These enzymatically dispersed neurons thereforeretained both the membrane properties and morphology observedin the intact brainstem and in vitro brain slice preparation.The use of this isolated neuron preparation provides a basisfor further study of rNST neurobiology. Chem. Senses 21: 729–737,1996     

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).     

Local Application of Drugs to Study Nicotinic Acetylcholine Receptor Function in Mouse Brain Slices

Tobacco use leads to numerous health problems, including cancer, heart disease, emphysema, and stroke. Addiction to cigarette smoking is a prevalent neuropsychiatric disorder that stems from the biophysical and cellular actions of nicotine on nicotinic acetylcholine receptors (nAChRs) throughout the central nervous system. Understanding the various nAChR subtypes that exist in brain areas relevant to nicotine addiction is a major priority.Experiments that employ electrophysiology techniques such as whole-cell patch clamp or two-electrode voltage clamp recordings are useful for pharmacological characterization of nAChRs of interest. Cells expressing nAChRs, such as mammalian tissue culture cells or Xenopus laevis oocytes, are physically isolated and are therefore easily studied using the tools of modern pharmacology. Much progress has been made using these techniques, particularly when the target receptor was already known and ectopic expression was easily achieved. Often, however, it is necessary to study nAChRs in their native environment: in neurons within brain slices acutely harvested from laboratory mice or rats. For example, mice expressing "hypersensitive" nAChR subunits such as α4 L9′A mice ¹ and α6 L9′S mice ², allow for unambiguous identification of neurons based on their functional expression of a specific nAChR subunit. Although whole-cell patch clamp recordings from neurons in brain slices is routinely done by the skilled electrophysiologist, it is challenging to locally apply drugs such as acetylcholine or nicotine to the recorded cell within a brain slice. Dilution of drugs into the superfusate (bath application) is not rapidly reversible, and U-tube systems are not easily adapted to work with brain slices.In this paper, we describe a method for rapidly applying nAChR-activating drugs to neurons recorded in adult mouse brain slices. Standard whole-cell recordings are made from neurons in slices, and a second micropipette filled with a drug of interest is maneuvered into position near the recorded cell. An injection of pressurized air or inert nitrogen into the drug-filled pipette causes a small amount of drug solution to be ejected from the pipette onto the recorded cell. Using this method, nAChR-mediated currents are able to be resolved with millisecond accuracy. Drug application times can easily be varied, and the drug-filled pipette can be retracted and replaced with a new pipette, allowing for concentration-response curves to be created for a single neuron. Although described in the context of nAChR neurobiology, this technique should be useful for studying many types of ligand-gated ion channels or receptors in neurons from brain slices.     

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.     

Oligodendrocyte Precursor Cells Modulate the Neuronal Network by Activity-Dependent Ectodomain Cleavage of Glial NG2

The role of glia in modulating neuronal network activity is an important question. Oligodendrocyte precursor cells (OPC) characteristically express the transmembrane proteoglycan nerve-glia antigen 2 (NG2) and are unique glial cells receiving synaptic input from neurons. The development of NG2+ OPC into myelinating oligodendrocytes has been well studied, yet the retention of a large population of synapse-bearing OPC in the adult brain poses the question as to additional functional roles of OPC in the neuronal network. Here we report that activity-dependent processing of NG2 by OPC-expressed secretases functionally regulates the neuronal network. NG2 cleavage by the α-secretase ADAM10 yields an ectodomain present in the extracellular matrix and a C-terminal fragment that is subsequently further processed by the γ-secretase to release an intracellular domain. ADAM10-dependent NG2 ectodomain cleavage and release (shedding) in acute brain slices or isolated OPC is increased by distinct activity-increasing stimuli. Lack of NG2 expression in OPC (NG2-knockout mice), or pharmacological inhibition of NG2 ectodomain shedding in wild-type OPC, results in a striking reduction of N-methyl-D-aspartate (NMDA) receptor-dependent long-term potentiation (LTP) in pyramidal neurons of the somatosensory cortex and alterations in the subunit composition of their α-amino-3-hydroxy-5-methyl-4-isoxazolepr opionicacid (AMPA) receptors. In NG2-knockout mice these neurons exhibit diminished AMPA and NMDA receptor-dependent current amplitudes; strikingly AMPA receptor currents can be rescued by application of conserved LNS protein domains of the NG2 ectodomain. Furthermore, NG2-knockout mice exhibit altered behavior in tests measuring sensorimotor function. These results demonstrate for the first time a bidirectional cross-talk between OPC and the surrounding neuronal network and demonstrate a novel physiological role for OPC in regulating information processing at neuronal synapses.     

Organotypic slice culture based on in ovo electroporation for chicken embryonic central nervous system

Organotypic slice culture is a living cell research technique which blends features of both in vivo and in vitro techniques. While organotypic brain slice culture techniques have been well established in rodents, there are few reports on the study of organotypic slice culture, especially of the central nervous system (CNS), in chicken embryos. We established a combined in ovo electroporation and organotypic slice culture method to study exogenous genes functions in the CNS during chicken embryo development. We performed in ovo electroporation in the spinal cord or optic tectum prior to slice culture. When embryonic development reached a specific stage, green fluorescent protein (GFP)‐positive embryos were selected and fluorescent expression sites were cut under stereo fluorescence microscopy. Selected tissues were embedded in 4% agar. Tissues were sectioned on a vibratory microtome and 300 μm thick sections were mounted on a membrane of millicell cell culture insert. The insert was placed in a 30‐mm culture dish and 1 ml of slice culture media was added. We show that during serum‐free medium culture, the slice loses its original structure and propensity to be strictly regulated, which are the characteristics of the CNS. However, after adding serum, the histological structure of cultured‐tissue slices was able to be well maintained and neuronal axons were significantly longer than that those of serum‐free medium cultured‐tissue slices. As the structure of a complete single neuron can be observed from a slice culture, this is a suitable way of studying single neuronal dynamics. As such, we present an effective method to study axon formation and migration of single neurons in vitro.     

The Output Signal of Purkinje Cells of the Cerebellum and Circadian Rhythmicity

Measurement of clock gene expression has recently provided evidence that the cerebellum, like the master clock in the SCN, contains a circadian oscillator. The cerebellar oscillator is involved in anticipation of mealtime and possibly resides in Purkinje cells. However, the rhythmic gene expression is likely transduced into a circadian cerebellar output signal to exert an effective control of neuronal brain circuits that are responsible for feeding behavior. Using electrophysiological recordings from acute and organotypic cerebellar slices, we tested the hypothesis whether Purkinje cells transmit a circadian modulated signal to their targets in the brain. Extracellular recordings from brain slices revealed the typical discharge pattern previously described in vivo in single cell recordings showing basically a tonic or a trimodal-like firing pattern. However, in acute sagittal cerebellar slices the average spike rate of randomly selected Purkinje cells did not exhibit significant circadian variations, irrespective of their specific firing pattern. Also, frequency and amplitude of spontaneous inhibitory postsynaptic currents and the amplitude of GABA- and glutamate-evoked currents did not vary with circadian time. Long-term recordings using multielectrode arrays (MEA) allowed to monitor neuronal activity at multiple sites in organotypic cerebellar slices for several days to weeks. With this recording technique we observed oscillations of the firing rate of cerebellar neurons, presumably of Purkinje cells, with a period of about 24 hours which were stable for periods up to three days. The daily renewal of culture medium could induce circadian oscillations of the firing rate of Purkinje cells, a feature that is compatible with the behavior of slave oscillators. However, from the present results it appears that the circadian expression of cerebellar clock genes exerts only a weak influence on the electrical output of cerebellar neurons.     

Intracellular electrophysiology of mammalian peptidergic neurons in rat hypothalamic slices

The magnocellular neuropeptidergic cells (MNCs) of the paraventricular and supraoptic nuclei have been a model for biochemical and physiological studies of peptidergic neurons in the mammalian brain, but nearly all the electrophysiological studies of these vasopressinergic and oxytocinergic neuroendocrine cells are based on extracellular recordings. This paper reviews recent literature on electrophysiological properties of neurons in the magnocellular nuclei in which the rat in vitro slice preparation and intracellular recording were used. Spontaneously occurring action potentials and synaptic potentials (excitatory and inhibitory) have been observed in hypothalamic slices. The spike patterns have included slow and irregular firing, short rapid bursts of inactivating spikes, and slow phasic discharge with prolonged active and silent periods. Some studies have shown that increased osmolality causes neuronal firing, but this area is controversial. Intracellular injections of lucifer yellow have shown that some MNCs are dye-coupled and electron microscopic observations with the freeze-fracture technique have revealed occasional gap junctions, thus suggesting that some MNCs are electrotonically coupled. Both excitatory and inhibitory postsynaptic potentials have been evoked with extracellular stimulation. Therefore, action potentials, synaptic potentials, burst discharges, and probably electrotonic coupling have been found with intracellular recording in mammalian neuroendocrine cells. Future studies with intracellular recording and staining followed by immunohistochemical identification of cells should provide significant new information on the membrane physiology and synaptic pharmacology of vasopressinergic and oxytocinergic cells.     

Differences in in vitro cerebellar neuronal responses to hypoxia in eider ducks,chicken and rats

Ducks are well-known to be more tolerant to asphyxia than non-diving birds, but it is not known if their defences include enhanced neuronal hypoxia tolerance. To test this, we compared extracellular recordings of spontaneous activity in the Purkinje cell layer of 400 μm thick isolated cerebellar slices from eider ducks, chickens and rats, before, during and after 60 min hypoxia (95%N₂–5%CO₂) or chemical anoxia (hypoxia + 2 mM NaCN). Most slices rapidly lost activity in hypoxia, with or without recovery after rinse and return to normoxia (95%O₂–5%CO₂), but some maintained spontaneous activity throughout the insult. Proportions of ‘surviving’ (i.e. recovering or active) duck slices were significantly higher than for chickens in anoxia, and relative activity levels were higher for ducks than for chickens during hypoxia, anoxia and recovery. Survival of rat slices was significantly poorer than for birds under all conditions. Results suggest that (1) duck cerebellar neurons are intrinsically more hypoxia-tolerant than chicken neurons; (2) avian neurons are more hypoxia-tolerant than rat neurons, and (3) the enhanced hypoxic tolerance of duck neurons largely depended on efficient anaerobiosis since it mainly manifested itself in chemical anoxia. Mechanisms underlying the observed differences in neuronal hypoxic responses remain to be elucidated.     

Preparation of Parasagittal Slices for the Investigation of Dorsal-ventral Organization of the Rodent Medial Entorhinal Cortex

Computation in the brain relies on neurons responding appropriately to their synaptic inputs. Neurons differ in their complement and distribution of membrane ion channels that determine how they respond to synaptic inputs. However, the relationship between these cellular properties and neuronal function in behaving animals is not well understood. One approach to this problem is to investigate topographically organized neural circuits in which the position of individual neurons maps onto information they encode or computations they carry out¹. Experiments using this approach suggest principles for tuning of synaptic responses underlying information encoding in sensory and cognitive circuits^2,3.The topographical organization of spatial representations along the dorsal-ventral axis of the medial entorhinal cortex (MEC) provides an opportunity to establish relationships between cellular mechanisms and computations important for spatial cognition. Neurons in layer II of the rodent MEC encode location using grid-like firing fields^4-6. For neurons found at dorsal positions in the MEC the distance between the individual firing fields that form a grid is on the order of 30 cm, whereas for neurons at progressively more ventral positions this distance increases to greater than 1 m. Several studies have revealed cellular properties of neurons in layer II of the MEC that, like the spacing between grid firing fields, also differ according to their dorsal-ventral position, suggesting that these cellular properties are important for spatial computation^2,7-10.Here we describe procedures for preparation and electrophysiological recording from brain slices that maintain the dorsal-ventral extent of the MEC enabling investigation of the topographical organization of biophysical and anatomical properties of MEC neurons. The dorsal-ventral position of identified neurons relative to anatomical landmarks is difficult to establish accurately with protocols that use horizontal slices of MEC^7,8,11,12, as it is difficult to establish reference points for the exact dorsal-ventral location of the slice. The procedures we describe enable accurate and consistent measurement of location of recorded cells along the dorsal-ventral axis of the MEC as well as visualization of molecular gradients^2,10. The procedures have been developed for use with adult mice (> 28 days) and have been successfully employed with mice up to 1.5 years old. With adjustments they could be used with younger mice or other rodent species. A standardized system of preparation and measurement will aid systematic investigation of the cellular and microcircuit properties of this area.     

A targeted extracellular approach for recording long-term firing patterns of excitable cells: a practical guide

Excitable cells in many endocrine and neuronal systems display rhythms with periodicities on the order of many minutes. To observe firing patterns that represent the output of these rhythms requires a recording technique that can monitor electrophysiological activity for several hours without affecting cell behavior. A targeted extracellular approach (also known as loose-patch) accomplishes this objective. Because low resistance seals (<20 MΩ) do not influence the cell membrane and because the normal intracellular milieu is maintained, this approach is the least invasive method for monitoring the endogenous electrical activity of single cells. In this report, we detail our use of this technique to record the firing patterns of gonadotropin-releasing hormone (GnRH) neurons in brain slices continuously for several hours. Published: February 17, 2003 This publication makes use, with permission, of data and methodologies published in Nunemaker CS, DeFazio RA, Moenter SM. Estradiol-sensitive afferents modulate long-term episodic firing patterns of GnRH neurons.Endocrinology 2002; 143:2284–2292, Copyright 2002 by The Endocrine Society.     

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.