Cells In Brain Slices Research Articles

Glial-Restricted Precursors Protect Neonatal Brain Slices from Hypoxic-Ischemic Cell Death Without Direct Tissue Contact.

Glial-Restricted Precursors (GRPs) are tripotential progenitors that have been shown to exhibit beneficial effects in several preclinical models of neurological disorders, including neonatal brain injury. The mechanisms of action of these cells, however, require further study, as do clinically relevant questions such as timing and route of cell administration. Here, we explored the effects of GRPs on neonatal hypoxia-ischemia during acute and subacute stages, using an in vitro transwell co-culture system with organotypic brain slices exposed to oxygen-glucose deprivation (OGD). OGD-exposed slices that were then co-cultured with GRPs without direct cell contact had decreased tissue injury and cortical cell death, as evaluated by lactate dehydrogenase (LDH) release and propidium iodide (PI) staining. This effect was more pronounced when cells were added during the subacute phase of the injury. Furthermore, GRPs reduced the amount of glutamate in the slice supernatant and changed the proliferation pattern of endogenous progenitor cells in brain slices. In summary, we show that GRPs exert a neuroprotective effect on neonatal hypoxia-ischemia without the need for direct cell-cell contact, thus confirming the rising view that beneficial actions of stem cells are more likely attributable to trophic or immunomodulatory support rather than to long-term integration.

Peering into stem cells in live brain: Interdisciplinary approaches to study neural development and disorder

Event Abstract Back to Event Peering into stem cells in live brain: Interdisciplinary approaches to study neural development and disorder Jin-Wu Tsai1* 1 National Yang-Ming University, Institute of Brain Science, Taiwan The vertebrate central nervous system (CNS) originates from neural stem cells and is composed of highly organized neuroepithelial and, subsequently, radial glial cells, which give rise to virtually all neurons in the brain. During development, these neural stem cells and their progeny go through a series of motile events, including migration, morphogenesis, and neurite outgrowth. Any small perturbation in these processes can cause neural developmental disorders, such as lissencephaly, microcephaly, double cortex, schizophrenia, and even autism. In order to study the etiology of these developmental disorders, we established animal models to elucidate the functions of their causal genes in vivo. For example, we used in utero electroporation of shRNA constructs into embryonic neural stem cells to knock down the expression of LIS1, mutations of which lead to lissencephaly. We found that knockdown of LIS1 completely blocks the interkinetic nuclear migration (INM), as well as the subsequent radial migration of committed neuronal precursors. We further developed a culture system to observe neural cells in brain slices using high resolution light microscopy and found that LIS1 is required for both nuclear and centrosome movement in the radially migrating cells. We have also applied these approaches to the behavior of neural stem cells and found that INM involve a cell cycle-dependent switch between dynein- and nonconventional kinesin-driven nuclear transport. Finally, we discovered a new subtype of neural progenitor cells in rodents. We now used in vivo cerebellar electroporation to label and manipulate gene expression in cerebellar granule neuron progenitors (GNPs) and investigate the molecular mechanisms of neural stem cell proliferation, differentiation and migration during cerebellar development. These approaches can be expanded to elucidate the mechanism of normal brain development and shed light on how various genes cause many neural developmental disorders. Keywords: Nervous System, Neural Stem Cells, neurodevelopment, developmental disorders, lis1 Conference: 14th Meeting of the Asian-Pacific Society for Neurochemistry, Kuala Lumpur, Malaysia, 27 Aug - 30 Aug, 2016. Presentation Type: Symposium 11: Generating New Neurons: From Development to Cell-Based Therapy Topic: 14th Meeting of the Asian-Pacific Society for Neurochemistry Citation: Tsai J (2016). Peering into stem cells in live brain: Interdisciplinary approaches to study neural development and disorder. Conference Abstract: 14th Meeting of the Asian-Pacific Society for Neurochemistry. doi: 10.3389/conf.fncel.2016.36.00048 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 26 Jul 2016; Published Online: 11 Aug 2016. * Correspondence: Dr. Jin-Wu Tsai, National Yang-Ming University, Institute of Brain Science, Taipei, Taiwan, tsaijw@ym.edu.tw Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Jin-Wu Tsai Google Jin-Wu Tsai Google Scholar Jin-Wu Tsai PubMed Jin-Wu Tsai Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Skipped-stimulus approach reveals that short-term plasticity dominates synaptic strength during ongoing activity.

All synapses show activity-dependent changes in strength, which affect the fidelity of postsynaptic spiking. This is particularly important at auditory nerve synapses, where the presence and timing of spikes carry information about a sound's structure, which must be passed along for proper processing. However, it is not clear how synaptic plasticity influences spiking during ongoing activity. Under these conditions, conventional analyses erroneously suggest that synaptic plasticity has no influence on EPSC amplitude or spiking. Therefore, we developed new approaches to study how ongoing activity influences synaptic strength, using voltage- and current-clamp recordings from bushy cells in brain slices from mouse anteroventral cochlear nucleus. We applied identical trains of stimuli, except for one skipped stimulus, and found that EPSC amplitude was affected for 60 ms following a skipped stimulus. We further showed that the initial probability of release, calcium-dependent mechanisms of recovery, and desensitization all play a role even during ongoing activity. Current-clamp experiments indicated that these processes had a significant effect on postsynaptic spiking, as did the refractory period to a smaller extent. Thus short-term plasticity has real, important functional consequences.

(G2019S) LRRK2 causes early-phase dysfunction of SNpc dopaminergic neurons and impairment of corticostriatal long-term depression in the PD transgenic mouse

Twelve- to sixteen-month-old (G2019S) LRRK2 transgenic mice prepared by us displayed progressive neuronal death of substantia nigra pars compacta (SNpc) dopaminergic cells. In the present study, we hypothesized that prior to a late-phase death of SNpc dopaminergic neurons, (G2019S) LRRK2 also causes an early-phase neuronal dysfunction of SNpc dopaminergic cells in the (G2019S) LRRK2 mouse. Eight to nine-month-old (G2019S) LRRK2 transgenic mice exhibited the symptom of hypoactivity in the absence of the degeneration of SNpc dopaminergic neurons or nigrostriatal dopaminergic terminals. Whole-cell current-clamp recordings of SNpc dopaminergic cells in brain slices demonstrated a significant decrease in spontaneous firing frequency of SNpc dopaminergic neurons of 8-month-old (G2019S) LRRK2 mice. Carbon fiber electrode amperometry recording using striatal slices showed that (G2019S) LRRK2 transgenic mice at the age of 8 to 9months display an impaired evoked dopamine release in the dorsolateral striatum. Normal nigrostriatal dopaminergic transmission is required for the induction of long-term synaptic plasticity expressed at corticostriatal glutamatergic synapses of striatal medium spiny neurons. Whole-cell voltage-clamp recordings showed that in contrast to medium spiny neurons of 8 to 9-month-old wild-type mice, high-frequency stimulation of corticostriatal afferents failed to induce long-term depression (LTD) of corticostriatal EPSCs in medium spiny neurons of (G2019S) LRRK2 mice at the same age. Our study provides the evidence that mutant (G2019S) LRRK2 causes early-phase dysfunctions of SNpc dopaminergic neurons, including a decrease in spontaneous firing rate and a reduction in evoked dopamine release, and impairment of corticostriatal LTD in the (G2019S) LRRK2 transgenic mouse.

Synapsin II and Rab3a Cooperate in the Regulation of Epileptic and Synaptic Activity in the CA1 Region of the Hippocampus

Some forms of idiopathic epilepsy in animals and humans are associated with deficiency of synapsin, a phosphoprotein that reversibly associates with synaptic vesicles. We have previously shown that the epileptic phenotype seen in synapsin II knock-out mice (SynII(-)) can be rescued by the genetic deletion of the Rab3a protein. Here we have examined the cellular basis for this rescue using whole-cell recordings from CA1 hippocampal pyramidal cells in brain slices. We find that SynII(-) neurons have increased spontaneous activity and a reduced threshold for the induction of epileptiform activity by 4-aminopyridine (4-AP). Using selective recordings of glutamatergic and GABAergic activity we show that in wild-type neurons low concentrations of 4-AP facilitate glutamatergic and GABAergic transmission in a balanced way, whereas in SynII(-) neurons this balance is shifted toward excitation. This imbalance reflects a deficit in inhibitory synaptic transmission that appears to be secondary to reduced Ca(2+) sensitivity in SynII(-) neurons. This suggestion is supported by our finding that synaptic and epileptiform activity at SynII(-) and wild-type synapses is similar when GABAergic transmission is blocked. Deletion of Rab3a results in glutamatergic synapses that have a compromised responsiveness to either low 4-AP concentrations or elevated extracellular Ca(2+). These changes mitigate the overexcitable phenotype observed in SynII(-) neurons. Thus, Rab3a deletion appears to restore the excitatory/inhibitory imbalance observed in SynII(-) hippocampal slices indirectly, not by correcting the deficit in GABAergic synaptic transmission but rather by impairing excitatory glutamatergic synaptic transmission.

Plasticity of spontaneous excitatory and inhibitory synaptic activity in morphologically defined vestibular nuclei neurons during early vestibular compensation

After unilateral peripheral vestibular lesions, the brain plasticity underlying early recovery from the static symptoms is not fully understood. Principal cells of the chick tangential nucleus offer a subset of morphologically defined vestibular nuclei neurons to study functional changes after vestibular lesions. Chickens show posture and balance deficits immediately after unilateral vestibular ganglionectomy (UVG), but by 3 days most subjects begin to recover, although some remain uncompensated. With the use of whole cell voltage-clamp, spontaneous excitatory and inhibitory postsynaptic currents (sEPSCs and sIPSCs) and miniature excitatory and inhibitory postsynaptic currents (mEPSCs and mIPSCs) were recorded from principal cells in brain slices 1 and 3 days after UVG. One day after UVG, sEPSC frequency increased on the lesion side and remained elevated at 3 days in uncompensated chickens only. Also by 3 days, sIPSC frequency increased on the lesion side in all operated chickens due to major increases in GABAergic events. Significant change also occurred in decay time of the events. To determine whether fluctuations in frequency and kinetics influenced overall excitatory or inhibitory synaptic drive, synaptic charge transfer was calculated. Principal cells showed significant increase in excitatory synaptic charge transfer only on the lesion side of uncompensated chickens. Thus compensation continues when synaptic charge transfer is in balance bilaterally. Furthermore, excessive excitatory drive in principal cells on the lesion side may prevent vestibular compensation. Altogether, this work is important for it defines the time course and excitatory and inhibitory nature of changing spontaneous synaptic inputs to a morphologically defined subset of vestibular nuclei neurons during critical early stages of recovery after UVG.

Brain-Derived Neurotrophic Factor Val66Met Allele Impairs Basal and Ketamine-Stimulated Synaptogenesis in Prefrontal Cortex

Knock-in mice with the common human brain-derived neurotrophic factor (BDNF) Val66Met polymorphism have impaired trafficking of BDNF messenger RNA to dendrites. It was hypothesized, given evidence that local synapse formation is dependent on dendritic translation of BDNF messenger RNA, that loss-of-function Met allele mice would show synaptic deficits both at baseline and in response to ketamine, an N-methyl-D-aspartate antagonist that stimulates synaptogenesis in prefrontal cortex (PFC). Whole-cell recordings from layer V medial PFC pyramidal cells in brain slices were combined with two-photon laser scanning for analysis of wildtype, Val/Met, and Met/Met mice both at baseline and in response to a low dose of ketamine. Val/Met and Met/Met mice were found to have constitutive atrophy of distal apical dendrites and decrements in apically targeted excitatory postsynaptic currents in layer V pyramidal cells of PFC. In addition, spine density and diameter were decreased, indicative of impaired synaptic formation/maturation (synaptogenesis). In Met/Met mice the synaptogenic effect of ketamine was markedly impaired, consistent with the idea that synaptogenesis is dependent on dendritic translation/release of BDNF. In parallel behavioral studies, we found that the antidepressant response to ketamine in the forced swim test was blocked in Met/Met mice. The results demonstrate that expression of the BDNF Met allele in mice results in basal synaptic deficits and blocks synaptogenic and antidepressant actions of ketamine in PFC, suggesting that the therapeutic response to this drug might be attenuated or blocked in depressed patients who carry the loss of function Met allele.

Phase response curves of subthalamic neurons: experimental measurement and theoretical prediction

The subthalamic nucleus (STN) is an autonomously active population of glutamatergic neurons that occupies a pivotal location within the basal ganglia, receiving direct cortical input and innervating the globus pallidus and substantia nigra. As intrinsically oscillating neurons, STN cells may be suitable candidates for reduction to phase models, where the state of each neuron is represented by a single number (phase) and their response to synaptic input is given by infinitesimal phase response curves (iPRCs). Phase models are analytically tractable and assist the study of large neural populations, but whether they can describe the behavior of STN cells with sufficient accuracy is an open question. We addressed this issue by measuring the iPRCs of STN cells in brain slices, using both glutamatergic synaptic input and brief current injection. We found that iPRCs measured synaptically were relatively insensitive to the size of the EPSP used to measure them, and that iPRCs measured with current pulses were the same regardless of the polarity of the current, at least during the first 60% of the oscillation cycle; both observations suggest that iPRCs might accurately describe the response of STN cells to finite inputs. However, we also found that iPRCs measured synaptically and by current injection were dramatically different, with current pulse iPRCs giving much less phase shift for a given stimulus amplitude. We developed and analyzed a biophysical model of STN cells, to better understand the nature of subthalamic iPRCs. This model produced an iPRC that closely resembled those we measured experimentally. Observing that this model consisted mainly of kinetically fast voltagedependent currents and AHP currents that can be treated as a function of time since the last spike, we explored the iPRCs of cells whose conductances are direct functions of voltage. Ignoring the spike itself by assuming that it is triggered at a certain threshold potential followed by return (2 ms later) to resetting potential, we found that the iPRC of such cells is a simple function of their IV curves and that their iPRC is valid for arbitrarily large currents. The contribution of AHP currents could therefore be covered by treating them as applied currents acting through this IV curve-based iPRC. Following this approach, we developed a method for deriving the iPRC of any cell well described by a combination of fast voltage-dependent currents and time-dependent AHP currents, covering a wide range of cell types. We were also able to estimate experimentally the IV curves and AHP currents of STN cells in which we had empirically determined the synaptic and current pulse iPRCs, allowing us to compare theoretically predicted iPRCs to experimentally measured iPRCs. We found that the theoretically predicted PRC approximated the synaptic–but not the current pulse–iPRC. We hypothesize that somatically injected charge slowly trickles into distal dendrites, reducing its effectiveness at advancing the phase, whereas synaptically delivered charge comes from precharged dendrites. We conclude that current injection is not an effective method for measuring iPRCs in dendritic neurons.

Cortical GABAergic neurons and cerebellar Purkinje cells respond to ischemia-pathogenic factors differently

GABAergic neurons in the central nervous system are vulnerable to hazard situations, such as ischemia and toxic substances, under which their dysfunction results in neuronal excitotoxicity and subsequently cell death. How ischemia-related pathogenic factors influence the functions of different GABAergic neurons remains to be documented. We investigated this issue at cortical GABAergic neurons and cerebellar Purkinje cells in brain slices by whole-cell recordings. Our results demonstrate that ischemia, cellular Ca2+-overload and acidosis lower the spike capacity of cortical GABAergic neurons, but elevate that of cerebellar Purkinje cells. These changes of spike encoding at two types of GABAergic cells are associated with the different effects of three factors on spike refractory periods and threshold potentials, which are mediated by voltage-gated sodium channels. Mechanisms underlying such differences are discussed.

Hearing loss alters quantal release at cochlear nucleus stellate cells

Auditory nerve synapses in ventral cochlear nucleus end on two principal cell types, bushy and stellate cells. Although the effects of hearing loss on bushy cells have been well studied, little is known about the effects of hearing loss on synaptic input to the stellate cells. Based on prior observations in bushy cells, we hypothesized that noise-induced hearing loss (NIHL) would decrease quantal release onto stellate cells. Prospective, randomized animal study. CBA/CaJ mice were exposed for 2 hours to 98 dB sound pressure level (SPL) 8- to 16-kHz noise to produce a temporary threshold shift (TTS) or 114 dB SPL to produce a permanent threshold shift (PTS). Spontaneous miniature excitatory postsynaptic currents (mEPSCs) were then measured in stellate cells in brain slices of the cochlear nucleus. Click auditory brainstem evoked response thresholds were elevated by 35 dB in both TTS and PTS mice. Spontaneous mEPSC frequency was found to be five-fold higher than normal in stellate cells of TTS mice and three-fold higher in PTS mice. The mEPSC amplitude was also larger in PTS mice. The mEPSC time course was not different between experimental and control groups. The dramatic increase in mEPSC frequency after NIHL was not expected. The increase in mEPSC amplitude in PTS mice suggests a postsynaptic remodeling process. Both of these effects could contribute to increased spontaneous firing in the cochlear nucleus in the absence of sound. Our results also suggest that hearing loss may have different effects at auditory nerve synapses on bushy and stellate cells in the VCN.

The Fate of Synaptic Input to NG2 Glial Cells: Neurons Specifically Downregulate Transmitter Release onto Differentiating Oligodendroglial Cells

NG2-expressing oligodendrocyte precursor cells (OPCs) are ubiquitous and generate oligodendrocytes throughout the young and adult brain. Previous work has shown that virtually every NG2 cell receives synaptic input from many axons, but the meaning of this signaling is not understood. In particular, it is unclear whether neurons specifically synapse onto OPCs or whether OPCs merely trace adjacent neurotransmitter release sites and are not recognized by the presynaptic neuron. Here, we show with whole-cell recordings from distinct developmental stages of oligodendroglial cells in brain slices that synaptic input essentially disappears as soon as OPCs differentiate into premyelinating oligodendrocytes (NG2(-), DM20/PLP(+), O1(+)). Uncaging experiments and tracer loading revealed that premyelinating oligodendrocytes still express a substantial number of AMPA/kainate receptors and many processes, but spontaneous and stimulated synaptic currents are mostly absent. Nevertheless, in a minority of premyelinating cells, electrical stimulation evoked small synaptic currents with an unusual behavior: their amplitude compared well with the quantal amplitude in OPCs but they occurred asynchronously and with the remarkable latency of 40-100 ms, indicating that the presynaptic release machinery has become ineffective. Mature myelinating oligodendrocytes completely lack AMPA/kainate receptors and respond to uncaging and synaptic stimulation with glutamate transporter currents. Our data show that neurons selectively synapse onto only one of several coexisting developmental stages of glial cells and thereby indicate that neurons indeed specifically signal to OPCs and are able to modulate transmitter output by regulating the local release machinery in a manner specific to the developmental stage of the postsynaptic glial cell.

LINGO-1 Interacts with WNK1 to Regulate Nogo-induced Inhibition of Neurite Extension

LINGO-1 is a component of the tripartite receptor complexes, which act as a convergent mediator of the intracellular signaling in response to myelin-associated inhibitors and lead to collapse of growth cone and inhibition of neurite extension. Although the function of LINGO-1 has been intensively studied, its downstream signaling remains elusive. In the present study, a novel interaction between LINGO-1 and a serine-threonine kinase WNK1 was identified by yeast two-hybrid screen. The interaction was further validated by fluorescence resonance energy transfer and co-immunoprecipitation, and this interaction was intensified by Nogo66 treatment. Morphological evidences showed that WNK1 and LINGO-1 were co-localized in cortical neurons. Furthermore, either suppressing WNK1 expression by RNA interference or overexpression of WNK1-(123-510) attenuated Nogo66-induced inhibition of neurite extension and inhibited the activation of RhoA. Moreover, WNK1 was identified to interact with Rho-GDI1, and this interaction was attenuated by Nogo66 treatment, further indicating its regulatory effect on RhoA activation. Taken together, our results suggest that WNK1 is a novel signaling molecule involved in regulation of LINGO-1 mediated inhibition of neurite extension.

Electrophysiological Properties of Octopus Neurons of the Cat Cochlear Nucleus: an In Vitro Study

Electrophysiological studies from mice in vitro have suggested that octopus cells of the mammalian ventral cochlear nucleus (VCN) are anatomically and biophysically specialized for detecting the coincident firing of a population of auditory nerve fibers. Recordings from cats in vivo have shown that octopus cells fire rapidly and with exceptional temporal precision as they convey the timing of that coincidence to higher auditory centers. The current study addresses the question whether the biophysical properties of octopus cells that have until now been examined only in mice, are shared by octopus cells in cats. Whole-cell patch-clamp recordings confirm that octopus cells in brain slices from kittens share the anatomical and biophysical features of octopus cells in mice. As in mice, octopus cells in kittens have large cell bodies and thick dendrites that extend in one direction. Voltage changes produced by depolarizing and hyperpolarizing current injection were small and rapid. Input resistances and membrane time constants in octopus cells of 16-day-old kittens were 15.8 +/- 1.5 MOmega (n = 16) and 1.28 +/- 0.3 ms (n = 16), respectively. Octopus cells fired only a single action potential at the onset of a depolarizing current pulse; suprathreshold stimuli were greater than 1.8 nA. A tetrodotoxin (TTX)-sensitive sodium conductance (gNa) was responsible for the generation of the action potentials. Octopus cells displayed outward rectification that lasted for the duration of the depolarizing pulses. Hyperpolarizations produced by the injection of current exhibited a depolarizing sag of the membrane potential toward the resting value. A 4-aminopyridine (4-AP) and alpha-dendrotoxin (alpha-DTX)-sensitive, low-voltage-activated potassium conductance (gKL) and a ZD7288-sensitive, mixed-cation conductance (gh) were partially activated at rest, giving the octopus cells low input resistances and, as a consequence, brief time constants. In 7-day-old kittens, action potentials were taller and broader, input resistances higher, and both inward and outward rectification was weaker than in 16-day-old kittens. Also as in mice, stellate cells of the VCN fired trains of action potentials with constant interspike intervals when they were depolarized (n = 10) and bushy cells of the VCN fired only a single action potential at the onset of depolarizations (n = 6). In conclusion, the similarity of octopus cells in mice and kittens suggests that the anatomical and biophysical specializations that allow octopus cells to detect and convey synchronous firing among auditory nerve fibers are common to all mammals.

Alterations of GABAA-Receptor Function and Allosteric Modulation During Development of Status Epilepticus

Partial limbic seizures in rodents induced by pilocarpine progress from stages I-II (mouth and facial movements; head nodding) to stage III (forelimb clonus) and then progress rapidly to stages IV-V (generalized limbic seizures; rearing, and rearing with falling) followed by status epilepticus (SE). Although limbic seizures in rodents are terminated by benzodiazepines, a group of gamma-aminobutyric acid type A (GABA(A))-receptor positive modulators, significant pharmacoresistance to benzodiazepines develops within minutes during SE. The alterations of GABA(A)-receptor function and allosteric modulation during development of SE are poorly understood. We induced seizures in juvenile rats by administration of lithium followed by pilocarpine, and whole cell recordings of miniature inhibitory postsynaptic currents (mIPSCs) were obtained from hippocampal dentate granule cells in brain slices. Compared with a sham-treated group, mIPSC amplitude was reduced and decay was accelerated at onset of the first occurrence of stage III (S3) seizures [S3(0)], resulting in a reduction in the total charge transfer at S3(0). Recovery of mIPSC amplitude and prolongation of mIPSC decay occurred 30 min after onset of S3 seizures [S3(30)]. The mIPSC frequency was not altered for S3(0) and S3(30) neurons compared with sham neurons. The net enhancement of total charge transfer by diazepam was smaller for S3(30) than that for sham and S3(0) neurons; however, the net reduction of total charge transfer by zinc was greater for S3(30) than that for sham and S3(0) neurons. These findings suggest that substantial plastic changes of GABA(A)-receptor function and allosteric modulation occur rapidly in neurons from juvenile animals during development of SE.

Functional Consequences of GABAA Receptor α4 Subunit Deletion on Synaptic and Extrasynaptic Currents in Mouse Dentate Granule Cells

The alpha 4 subunit-containing gamma-aminobutyric acid (A) receptors (GABA(A)Rs) are highly expressed primarily at extrasynaptic sites in the dentate gyrus (DG) and thalamus and are suspected to contribute to tonic inhibition that is sensitive to potentiation by gaboxadol and ethanol (EtOH). Global alpha 4 subunit knockout (KO) mice exhibit greatly reduced tonic currents and insensitivity to ataxic, sedative and analgesic effects of gaboxadol compared to wild type (WT) controls. The alpha 4 KO mice were also significantly more sensitive to pentylenetetrazol-induced seizures. However, no differences were observed between alpha 4 KO and WT mice in other baseline behaviors or in the effects of EtOH on these behaviors. To examine possible functional and pharmacological GABA(A)R alterations, and search for causes for the lack of differences in EtOH behaviors we studied the effects of acute EtOH application on GABA(A)R-currents of DG cells from alpha 4 KO and WT control mice complemented by Western blot measurements. We studied the consequences of alpha 4 subunit deletion using Western immunoblotting and whole cell patch recordings from DG cells in brain slices from alpha 4 KO and WT mice. The magnitude of tonic current and its potentiation by EtOH (10 to 100 mM), alphaxalone (3 microM), and Ro15-4513 (0.3 microM) was greatly attenuated in alpha 4 KO mice. The kinetics of miniature inhibitory postsynaptic currents (mIPSCs) in alpha 4 KO mice were significantly slower compared to WT mice. Potentiation of mIPSCs by alphaxalone was greatly reduced in alpha 4 KO mice. Ro15-4513 had no effect on mIPSCs from WT or KO mice. However, mIPSCs of alpha 4 KO mice were significantly more sensitive to EtOH than those from WT mice. The gamma 2 subunit protein levels were selectively increased in hippocampus and thalamus, but not cortex of alpha 4 KO mice. These data suggest that the global loss of alpha 4 subunits leads to region- and cell location-specific compensatory increases in gamma 2 subunits, which in turn alter the pharmacological sensitivity of synaptic and extrasynaptic GABA(A)R-currents. Our data also suggests that while enhancement of tonic inhibitory currents by gaboxadol, alphaxalone, and EtOH are reduced, and behavioral sensitivity to gaboxadol and alphaxalone may be reduced, compensatory changes in synaptic GABA(A)R subunits may prevent similar reductions in behavioral sensitivity to EtOH.

Differential Interference Contrast (DIC) Imaging of Living Cells: Figure 1.

INTRODUCTIONThis protocol describes the use of electronically enhanced, differential interference contrast (DIC) imaging techniques for the study of living cells. Methods for DIC imaging of cells using an inverted microscope and for infrared (IR) imaging of brains slices using an upright microscope are presented. DIC microscopy (also known as Nomarski microscopy) is extremely useful for resolving individual cells and cellular organelles in live, unstained tissue. The data obtained are valuable in their own right, as well as for complementing fluorescence microscopy data. DIC microscopy can be applied to cell culture, brain slices, and even intact organisms (such as embryos). High-quality images of living cells in brain slices can also be obtained using a combination of DIC optics and IR video microscopy.

Long-Term Depression of mGluR1 Signaling

Glutamate produces both fast excitation through activation of ionotropic receptors and slower actions through metabotropic receptors (mGluRs). To date, ionotropic but not metabotropic neurotransmission has been shown to undergo long-term synaptic potentiation and depression. Burst stimulation of parallel fibers releases glutamate, which activates perisynaptic mGluR1 in the dendritic spines of cerebellar Purkinje cells. Here, we show that the mGluR1-dependent slow EPSC and its coincident Ca transient were selectively and persistently depressed by repeated climbing fiber-evoked depolarization of Purkinje cells in brain slices. LTD(mGluR1) was also observed when slow synaptic current was evoked by exogenous application of a group I mGluR agonist, implying a postsynaptic expression mechanism. Ca imaging further revealed that LTD(mGluR1) was expressed as coincident attenuation of both limbs of mGluR1 signaling: the slow EPSC and PLC/IP3-mediated dendritic Ca mobilization. Thus, different patterns of neural activity can evoke LTD of either fast ionotropic or slow mGluR1-mediated synaptic signaling.

One reason for increased seizure susceptibility of hippocampus compared with neocortex

In this study we compared the membrane resting potential and action potential (AP) activation thresholds of neocortical layer 2/3 and CA1 hippocampal pyramidal cells in brain slices from 6–8-day old mice. The activation threshold was −37 ± 2 mV in the neocortical pyramids (5 cells), and −50 ± 1 mV in the CA1 ones (5 cells). The observed difference in the AP activation thresholds may account for a higher excitability of hippocampus as compared to neocortex.

Combining patch-clamping of cells in brain slices with immunocytochemical labeling to define cell type and developmental stage.

In neuroscience, combining patch-clamping with protein identification in the same cell is becoming increasingly important to define which subtype or developmental stage of a neuron or glial cell is being recorded from, and to attribute measured membrane currents to expressed ion channels or receptors. Here, we describe a protocol to achieve this when studying cells in acute brain slices, which antibodies penetrate poorly into and for which detergent permeabilization cannot be used when using antibodies that recognize lipid components such as O4 sulfatide. The method avoids the need for resectioning of the electrophysiologically recorded slices. It employs filling of the cell with a fluorescent dye during whole-cell recording, to allow subsequent localization of the cell, followed by fixation and free-floating section labeling with up to three antibodies, which may recognize membrane, nuclear or cytosolic proteins. With practice, approximately 80% of patch-clamped cells can be retrieved and have their proteins identified in this way. The entire protocol can be completed in 3-4 d.

Live imaging of glioblastoma cells in brain tissue shows requirement of actin bundles for migration

Live-cell imaging of glioblastoma U373 and U87 cells transfected with actin cytoskeleton markers has been used to study there-arrangements that are associated with migration in two- and three-dimensional matrices and in brain tissue. In collagen gels and in brain slices, both cell types developed neuronal-like processes with ruffling membranes and filopodia. Blebbing cells were also observed, but these were mainly immobile. The retraction of trailing cell processes in a tissue environment was associated with the transient development and contraction of bundles of axial stress fibers. The inhibition of Rho-kinase caused glioblastoma cells in brain slices to become immobile and develop neurite-like processes at random, which indicates the requirement of Rho signaling and contractility for migration. Actin stress fibers were also observed in glioblastoma cells injected into the brains of living mice. Thus, invading glioblastoma cells use neurite-like extensions to penetrate between neuronal fibers and contractile actin bundles for traction of the cell body.