Adult Rat Brain Slices Research Articles

COverlap: a Fiji toolset for the 3D co-localization of two fluorescent nuclear markers in confocal images

With the increasing complexity and throughput of microscopy experiments, it has become essential for biologists to navigate computational means of analysis to produce automated and reproducible workflows. Bioimage analysis workflows being largely underreported in method sections of articles, it is however quite difficult to find practical examples of documented scripts to support beginner programmers in biology. Here, we introduce COverlap, a Fiji toolset composed of four macros, for the 3D segmentation and co-localization of fluorescent nuclear markers in confocal images. The toolset accepts batches of multichannel z-stack images, segments objects in two channels of interest, and outputs object counts and labels, as well as co-localization results based on the physical overlap of objects. The first macro is a preparatory step that produces maximum intensity projections of images for visualization purposes. The second macro assists users in selecting batch-suitable segmentation parameters by testing them on small portions of the images. The third macro performs automated segmentation and co-localization analysis, and saves the parameters used, the results table, the 3D regions of interest (ROIs) of co-localizing objects, and two types of verification images with segmentation and co-localization masks for each image of the batch. The fourth macro allows users to review the verification images displaying segmentation masks and the location of co-localization events, and to perform corrections such as ROI adjustment, z-stack reslicing, and volume estimation correction in an automatically documented manner. To illustrate how COverlap operates, we present an experiment in which we identified rare endothelial proliferation events in adult rat brain slices on more than 350 large tiled z-stacks. We conclude by discussing the reproducibility and generalizability of the toolset, its limitations for different datasets, and its potential use as a template that is adaptable to other types of analyses.

Maternal separation modifies spontaneous synaptic activity in the infralimbic cortex of stress-resilient male rats.

Glutamate and GABA signaling systems are necessary to maintain proper function of the central nervous system through excitation/inhibition (E/I) balance. Alteration of this balance in the medial prefrontal cortex (mPFC), as an effect of early-life stress, may lead to the development of anxiety and depressive disorders. Few studies exist in the infralimbic division of the mPFC to understand the effect of early-life stress at different ages, which is the purpose of the present work. Newborn Sprague Dawley male rats were subjected to maternal separation (MS) for two weeks. First, tests measuring anxiety- and depression-like behaviors were performed on adolescent and adult rats subjected to MS (MS-rats). Then, to establish a relationship with behavioral results, electrophysiological recordings were performed in neurons of the infralimbic cortex in acute brain slices of infant, adolescent, and adult rats. In the behavioral tests, there were no significant differences in MS-rats compared to control rats at any age. Moreover, MS had no effect on the passive membrane properties nor neuronal excitability in the infralimbic cortex, whereas spontaneous synaptic activity in infralimbic neurons was altered. The frequency of spontaneous glutamatergic synaptic events increased in infant MS-rats, whereas in adolescent MS-rats both the frequency and the amplitude of spontaneous GABAergic events increased without any effect on glutamatergic synaptic responses. In adult MS-rats, these two parameters decreased in spontaneous GABAergic synaptic events, whereas only the frequency of glutamatergic events decreased. These data suggest that rats subjected to MS did not exhibit behavioral changes and presented an age-dependent E/I imbalance in the infralimbic cortex, possibly due to differential changes in neurotransmitter release and/or receptor expression.

Probucol neuroprotection against manganese-induced damage in adult Wistar rat brain slices.

Manganese (Mn) is an abundant element used for commercial purposes and is essential for the proper function of biological systems. Chronic exposure to high Mn concentrations causes Manganism, a Parkinson's-like neurological disorder. The pathophysiological mechanism of Manganism remains unknown; however, it involves mitochondrial dysfunction and oxidative stress. This study assessed the neuroprotective effect of probucol, a hypolipidemic agent with anti-inflammatory and antioxidant properties, on cell viability and oxidative stress in slices of the cerebral cortex and striatum from adult male Wistar rats. Brain structure slices were kept separately and incubated with manganese chloride (MnCl2) and probucol to evaluate the cell viability and oxidative parameters. Probucol prevented Mn toxicity in the cerebral cortex and striatum, as evidenced by the preservation of cell viability observed with probucol (10 and 30μM) pre-treatment, as well as the prevention of mitochondrial complex I inhibition in the striatum (30μM). These findings support the protective antioxidant action of probucol, attributed to its ability to prevent cell death and mitigate Mn-induced mitochondrial dysfunction.

Contribution of inwardly rectifying K+ channel 4.1 of supraoptic astrocytes to the regulation of vasopressin neuronal activity by hypotonicity.

Astrocytic morphological plasticity and its modulation of adjacent neuronal activity are largely determined by astrocytic volume regulation, in which glial fibrillary acidic protein (GFAP), aquaporin 4 (AQP4), and potassium channels including inwardly rectifying K+ channel 4.1 (Kir4.1) are essential. However, associations of astrocyte-dominant Kir4.1 with other molecules in astrocytic volume regulation and the subsequent influence on neuronal activity remain unclear. Here, we report our study on these issues using primary cultures of rat pups' hypothalamic astrocytes and male adult rat brain slices. In astrocyte culture, hyposmotic challenge (HOC) significantly decreased GFAP monomer expression and astrocytic volume at 1.5 min and increased Kir4.1 expression and inwardly rectifying currents (IRCs) at 10 min. BaCl2 (100 μmol/l) suppressed the HOC-increased IRCs, which was simulated by VU0134992 (2μmol/l), a Kir4.1 blocker. Preincubation of the astrocyte culture with TGN-020 (10μmol/l, a specific AQP4 blocker) made the HOC-increased Kir4.1 currents insignificant. In hypothalamic brain slices, HOC initially decreased and then increased the firing rate of vasopressin (VP) neurons in the supraoptic nucleus. In the presence of BaCl2 or VU0134992, HOC-elicited rebound increase in VP neuronal activity was blocked. GFAP was molecularly associated with Kir4.1, which was increased by HOC at 20 min; this increase was blocked by BaCl2 . These results suggest that HOC-evoked astrocytic retraction or decrease in the volume and length of its processes is associated with increased Kir4.1 activity. Kir4.1 involvement in HOC-elicited astrocytic retraction is associated with AQP4 activity and GFAP plasticity, which together determines the rebound excitation of VP neurons.

Adaptive polymer fiber neural device for drug delivery and enlarged illumination angle for neuromodulation

Objective. Optical fiber devices constitute significant tools for the modulation and interrogation of neuronal circuitry in the mid and deep brain regions. The illuminated brain area during neuromodulation has a direct impact on the spatio-temporal properties of the brain activity and depends solely on the material and geometrical characteristics of the optical fibers. In the present work, we developed two different flexible polymer optical fibers (POFs) with integrated microfluidic channels (MFCs) and an ultra-high numerical aperture (UHNA) for enlarging the illumination angle to achieve efficient neuromodulation. Approach. Three distinct thermoplastic polymers: polysulfone, polycarbonate, and fluorinated ethylene propylene were used to fabricate two step-index UHNA POF neural devices using a scalable thermal drawing process. The POFs were characterized in terms of their illumination map as well as their fluid delivery capability in phantom and adult rat brain slices. Main results. A 100-fold reduced bending stiffness of the proposed fiber devices compared to their commercially available counterparts has been found. The integrated MFCs can controllably deliver dye (trypan blue) on-demand over a wide range of injection rates spanning from 10 nl min−1 to 1000 nl min−1. Compared with commercial silica fibers, the proposed UHNA POFs exhibited an increased illumination area by 17% and 21% under 470 and 650 nm wavelength, respectively. In addition, a fluorescent light recording experiment has been conducted to demonstrate the ability of our UHNA POFs to be used as optical waveguides in fiber photometry. Significance. Our results overcome the current technological limitations of fiber implants that have limited illumination area and we suggest that soft neural fiber devices can be developed using different custom designs for illumination, collection, and photometry applications. We anticipate our work to pave the way towards the development of next-generation functional optical fibers for neuroscience.

Opposing Changes in Synaptic and Extrasynaptic N-Methyl-D-Aspartate Receptor Function in Response to Acute and Chronic Restraint Stress.

A pertinent mechanism by which stress impacts learning and memory is through stress-induced plastic changes in glutamatergic transmission in the hippocampus. For instance, acute stress has been shown to alter the expression, binding, and function of the ionotropic glutamate N-methyl-D-aspartate receptor (NMDAR). However, the consequences of chronic stress, which could lead to various stress-related brain disorders, on NMDAR function remain unclear. While most studies on NMDARs focused on these receptors in synapses (synaptic NMDARs or sNMDARs), emerging findings have revealed functional roles of NMDARs outside synapses (extrasynaptic NMDARs or exNMDARs) that are distinct from those of sNMDARs. Using a restraint stress paradigm in adult rats, the objective of the current study is to examine whether sNMDARs and exNMDARs in the hippocampus are differentially regulated by acute and chronic stress. We examined sNMDAR and exNMDAR function in dorsal CA1 hippocampal neurons from brain slices of adult rats that were acutely (1 episode) or chronically (21 daily episodes) stressed by restraint (30 min). We found that acute stress increases sNMDAR but suppresses exNMDAR function. Surprisingly, we only observed a reduction in exNMDAR function after chronic stress. Taken together, our findings suggest that sNMDARs and exNMDARs may be differentially regulated by acute and chronic stress. Most importantly, the observed suppression in exNMDAR function by both acute and chronic stress implies crucial but overlooked roles of hippocampal exNMDARs in stress-related disorders.

Insulin enhances presynaptic glutamate release via opioid receptor‐mediated disinhibition

Recent studies show that insulin modulates the function of brain reward and motivation systems. For example, insulin receptor activation enhances dopamine release in the nucleus accumbens (NAc), reduces excitatory transmission in the VTA, and decreases food intake when infused into the NAc. Excitatory transmission in the NAc plays critical roles in motivation, and diet‐induced obesity alters NAc activity and peripheral sensitivity to insulin. However, insulin's effects on NAc excitatory transmission are unknown. Therefore here, we used whole‐cell patch clamp recordings in adult rat brain slices to determine how insulin affects excitatory transmission onto NAc medium spiny neurons (MSNs) and the mechanisms involved. Importantly, in addition to insulin receptors, insulin‐like growth factor receptors (IGFRs) are also expressed in the NAc and can be activated by moderate to high concentrations of insulin. Thus, a wide range of insulin concentrations were examined here (30–500 nM), and the contribution of insulin receptor activation vs. IGFR activation to insulin's effects was determined using pharmacological approaches. We find that insulin receptor activation increases presynaptic glutamate release via a previously unidentified form of opioid receptor‐mediated disinhibition, whereas activation of IGF receptors by insulin decreases presynaptic glutamate release. Furthermore, diet‐induced obesity (60% high‐fat; Open Source Diets D12492, 8 weeks) resulted in a loss of insulin receptor‐mediated increases and a reduction in NAc insulin receptor surface expression compared to chow fed controls (Lab Diet 5001, 13% fat), while preserving reductions in transmission mediated by IGRFs. These results provide the first insights into how insulin influences excitatory transmission in the adult brain and have broad implications for the regulation of motivation and reward related processes by peripheral hormones.Support or Funding InformationThis work was supported by NIDDK R01DK106188, R01DK115526, and the Brain and Behavior Research Foundation N018940 awards to CRF; MFO was supported by NIDA T32DA007268 and NIDDK FDK112627A. The authors declare no competing financial interests. Studies utilized the Chemistry Core of the Michigan Diabetes Research and Training Center funded by DK020572, and the University of Michigan Animal Phenotyping Core supported by P30 grants DK020572 (MDRC) and DK089503 (MNORC). We thank Drs. Marina Wolf, Larry Reagan, William Birdsong, Travis Brown and Garret Stuber for helpful conversations and Dr. Kenner C. Rice (Drug Design and Synthesis Section, NIDA IRP) for the gift of (+)‐naloxone.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Coalescence of deep and superficial epileptic foci into larger discharge units in adult rat neocortex

Epilepsy is a disease of neuronal hyper-synchrony that can involve both neocortical and hippocampal brain regions. While much is known about the network properties of the hippocampus little is known of how epileptic neocortical hyper-synchrony develops. We aimed at characterizing the properties of epileptic discharges of a neocortical epileptic focus.We established a multi-electrode-array method to record the spatial patterns of epileptiform potentials in acute adult rat brain slices evoked by 4-Aminopyridine in the absence of magnesium.Locations of discharges mapped to two anatomical regions over the somatosensory cortex and over the lateral convexity separated by a gap at a location matching the dysgranular zone. Focal epileptiform discharges were recorded in superficial and deep neocortical layers but over superficial layers, they exhibited larger surface areas. They were often independent even when closely spaced to one another but they became progressively coupled resulting in larger zones of coherent discharge.The gradual coupling of multiple, independent, closely spaced, spatially restricted, focal discharges between deep and superficial neocortical layers represents a possible mechanism of the development of an epileptogenic zone.

Neonatal ketamine exposure causes impairment of long-term synaptic plasticity in the anterior cingulate cortex of rats

Ketamine, a dissociative anesthetic most commonly used in many pediatric procedures, has been reported in many animal studies to cause widespread neuroapoptosis in the neonatal brain after exposure in high doses and/or for a prolonged period. This neurodegenerative change occurs most severely in the forebrain including the anterior cingulate cortex (ACC) that is an important brain structure for mediating a variety of cognitive functions. However, it is still unknown whether such apoptotic neurodegeneration early in life would subsequently impair the synaptic plasticity of the ACC later in life. In this study, we performed whole-cell patch-clamp recordings from the ACC brain slices of young adult rats to examine any alterations in long-term synaptic plasticity caused by neonatal ketamine exposure. Ketamine was administered at postnatal day 4–7 (subcutaneous injections, 20mg/kg given six times, once every 2h). At 3–4weeks of age, long-term potentiation (LTP) was induced and recorded by monitoring excitatory postsynaptic currents from ACC slices. We found that the induction of LTP in the ACC was significantly reduced when compared to the control group. The LTP impairment was accompanied by an increase in the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor-mediated excitatory synaptic transmission and a decrease in GABA inhibitory synaptic transmission in neurons of the ACC. Thus, our present findings show that neonatal ketamine exposure causes a significant LTP impairment in the ACC. We suggest that the imbalanced synaptic transmission is likely to contribute to ketamine-induced LTP impairment in the ACC.

Modulation of HCN channels in lateral septum by nicotine

The effects of addictive drugs most commonly occur via interactions with target receptors. The same is true of nicotine and its multiple receptors in a variety of cell types. However, there are also side effects for given substances that can dramatically change cellular, tissue, organ, and organism functions.In this study, we present evidence that nicotine possesses such properties, and modulates neuronal excitability. We recorded whole-cell voltages and currents in neurons situated in the dorsal portion of the lateral septum in acute coronal brain slices of adult rats. Our experiments in the lateral septum revealed that nicotine directly affects HCN – hyperpolarization-activated cyclic nucleotide gated non-selective cation channels.We demonstrate that nicotine effects persist despite the concurrent application of nicotinic acetylcholine receptors' antagonists – mecamylamine, methyllycaconitine, and dihydro-β-erythroidine. These results are novel in regard to HCN channels in the septum, in general, and in their sensitivity to nicotine, in particular.

Changes in synaptic efficacy in rat brain slices following extremely low-frequency magnetic field exposure at embryonic and early postnatal age

An earlier study demonstrated changes in synaptic efficacy and seizure susceptibility in adult rat brain slices following extremely low-frequency magnetic field (ELF-MF) exposure. The developing embryonic and early postnatal brain may be even more sensitive to MF exposure. The aim of the present study was to determine the effects of a long-term ELF-MF (0.5 and 3mT, 50Hz) exposure on synaptic functions in the developing brain. Rats were treated with chronic exposure to MF during two critical periods of brain development, i.e. in utero during the second gestation week or as newborns for 7 days starting 3 days after birth, respectively. Excitability and plasticity of neocortical and hippocampal areas were tested on brain slices by analyzing extracellular evoked field potentials. We demonstrated that the basic excitability of hippocampal slices (measured as amplitude of population spikes) was increased by both types of treatment (fetal 0.5mT, newborn 3mT). Neocortical slices seemed to be responsive mostly to the newborn treatment, the amplitude of excitatory postsynaptic potentials was increased. Fetal ELF-MF exposure significantly inhibited the paired-pulse depression (PPD) and there was a significant decrease in the efficacy of LTP (long-term potentiation induction) in neocortex, but not in hippocampus. On the other hand, neonatal treatment had no significant effect on plasticity phenomena. Results demonstrated that ELF-MF has significant effects on basic neuronal functions and synaptic plasticity in brain slice preparations originating from rats exposed either in fetal or in newborn period.

Viscoelastic Properties of the Rat Brain in the Sagittal Plane: Effects of Anatomical Structure and Age

Rat is the most commonly used animal model for the study of traumatic brain injury. Recent advances in imaging and computational modeling technology offer the promise of biomechanical models capable of resolving individual brain structures and offering greater insight into the causes and consequences of brain injury. However, there is insufficient data on the mechanical properties of brain structures available to populate these models. In this study, we used microindentation to determine viscoelastic properties of different anatomical structures in sagittal slices of juvenile and adult rat brain. We find that the rat brain is spatially heterogeneous in this anatomical plane supporting previous results in the coronal plane. In addition, the brain becomes stiffer and more heterogeneous as the animal matures. This dynamic, region-specific data will support the development of more biofidelic computational models of brain injury biomechanics and the testing of hypotheses about the manner in which different anatomical structures are injured in a head impact.

Persistent ictal-like activity in rat entorhinal/perirhinal cortex following washout of 4-aminopyridine

Application of 4-aminopyridine (4-AP, 100μM) in a solution containing 0.6mM Mg(2+) and 1.2mM Ca(2+) to hippocampal-entorhinal-perirhinal slices of adult rat brain induced ictal-like epileptiform activity in entorhinal and perirhinal cortices as revealed by electrophysiological field potential recordings. The ictal-like activity persisted after washing out the 4-AP. This persistence indicated that a change had occurred in the slice so that it was now "epileptic" in the absence of the convulsant 4-AP. Induction of persistent ictal-like activity was dependent upon the concentration of divalent cations during 4-AP exposure; that is, although 4-AP caused ictal-like activity in approximately half the slices in solution containing 1.6mM Mg(2+) and 2.0mM Ca(2+), this ictal-like activity did not persist upon washout of the 4-AP. Expression of the persistent ictal-like epileptiform activity required ionotropic glutamate-mediated synaptic transmission: application of the AMPA/kainate receptor antagonist NBQX after 4-AP washout reduced persistent ictal-like activity, and the combined application of NBQX and the NMDA receptor antagonist d-AP5 completely blocked it. In order to investigate the mechanism of induction of persistent ictal-like activity, several agents were applied before the introduction of 4-AP. Application of d-AP5 did not block the onset of ictal-like activity upon introduction of 4-AP but did prevent the persistence of the ictal-like activity upon washout of the 4-AP. In contrast, induction of persistent ictal-like activity was not prevented by simultaneous application of the group I metabotropic glutamate receptor (mGluR) antagonists LY 367385 and MPEP or by application of the protein synthesis inhibitor cycloheximide. In conclusion, we have characterized a new in vitro model of epileptogenesis in which induction of ictal-like activity is dependent upon NMDA receptor activation but not upon group I mGluR activation or protein synthesis.

Subregion-specific modulation of excitatory input and dopaminergic output in the striatum by tonically activated glycine and GABAA receptors

The flow of cortical information through the basal ganglia is a complex spatiotemporal pattern of increased and decreased firing. The striatum is the biggest input nucleus to the basal ganglia and the aim of this study was to assess the role of inhibitory GABAA and glycine receptors in regulating synaptic activity in the dorsolateral striatum (DLS) and ventral striatum (nucleus accumbens, nAc). Local field potential recordings from coronal brain slices of juvenile and adult Wistar rats showed that GABAA receptors and strychnine-sensitive glycine receptors are tonically activated and inhibit excitatory input to the DLS and to the nAc. Strychnine-induced disinhibition of glutamatergic transmission was insensitive to the muscarinic receptor inhibitor scopolamine (10 μM), inhibited by the nicotinic acetylcholine receptor antagonist mecamylamine (10 μM) and blocked by GABAA receptor inhibitors, suggesting that tonically activated glycine receptors depress excitatory input to the striatum through modulation of cholinergic and GABAergic neurotransmission. As an end-product example of striatal GABAergic output in vivo we measured dopamine release in the DLS and nAc by microdialysis in the awake and freely moving rat. Reversed dialysis of bicuculline (50 μM in perfusate) only increased extrasynaptic dopamine levels in the nAc, while strychnine administered locally (200 μM in perfusate) decreased dopamine output by 60% in both the DLS and nAc. Our data suggest that GABAA and glycine receptors are tonically activated and modulate striatal transmission in a partially subregion-specific manner.

TRPM3 is expressed in sphingosine‐responsive myelinating oligodendrocytes

Oligodendrocytes are the myelin-forming cells of the CNS and guarantee proper nerve conduction. Sphingosine, one major component of myelin, has recently been identified to activate TRPM3, a member of the melastatin-related subfamily of transient receptor potential (TRP) channels. TRPM3 has been demonstrated to be expressed in brain with unknown cellular distribution. Here, we show for the first time that TRPM3 is expressed in oligodendrocytes in vitro and in vivo. TRPM3 is present during oligodendrocyte differentiation. Immunohistochemistry of adult rat brain slices revealed staining of white matter areas, which co-localized with oligodendrocyte markers. Analysis of the developmental distribution revealed that, prior to myelination, TRPM3 channels are localized on neurons. On oligodendrocytes they are found after the onset of myelination. RT-PCR studies showed that the transcription of TRPM3 splice variants is also developmentally regulated in vitro. Ca(2+) imaging approaches revealed the presence of a sphingosine-induced Ca(2+) entry mechanism in oligodendrocytes - with a pharmacological profile similar to the profile published for heterologously expressed TRPM3. These findings indicate that TRPM3 participates as a Ca(2+)-permeable and sphingosine-activated channel in oligodendrocyte differentiation and CNS myelination.

PTPμ suppresses glioma cell migration and dispersal

The cell-surface receptor protein tyrosine phosphatase mu (PTPmu) is a homophilic cell adhesion molecule expressed in CNS neurons and glia. Glioblastomas (GBMs) are the highest grade of primary brain tumors with astrocytic similarity and are characterized by marked dispersal of tumor cells. PTPmu expression was examined in human GBM, low-grade astrocytoma, and normal brain tissue. These studies revealed a striking loss of PTPmu protein expression in highly dispersive GBMs compared to less dispersive low-grade astrocytomas and normal brain. We hypothesized that PTPmu contributes to contact inhibition of glial cell migration by transducing signals in response to cell adhesion. Therefore, loss of PTPmu may contribute to the extensive dispersal of GBMs. The migration of brain tumor cells was assessed in vitro using a scratch wound assay. Parental U-87 MG cells express PTPmu and exhibited limited migration. However, short-hairpin RNA (shRNA)-mediated knockdown of PTPmu induced a morphological change and increased migration. Next, a brain slice assay replicating the three-dimensional environment of the brain was used. To assess migration, labeled U-87 MG glioma cells were injected into adult rat brain slices, and their movement was followed over time. Parental U-87 MG cells demonstrated limited dispersal in this assay. However, PTPmu shRNA induced migration and dispersal of U-87 MG cells in the brain slice. Finally, in a mouse xenograft model of intracranially injected U-87 MG cells, PTPmu shRNA induced morphological heterogeneity in these xenografts. Together, these data suggest that loss of PTPmu in human GBMs contributes to tumor cell migration and dispersal, implicating loss of PTPmu in glioma progression.

Astrocytes are poised for lactate trafficking and release from activated brain and for supply of glucose to neurons

Brain is a highly-oxidative organ, but during activation, glycolytic flux is preferentially up-regulated even though oxygen supply is adequate. The biochemical and cellular basis of metabolic changes during brain activation and the fate of lactate produced within brain are important, unresolved issues central to understanding brain function, brain images, and spectroscopic data. Because in vivo brain imaging studies reveal rapid efflux of labeled glucose metabolites during activation, lactate trafficking among astrocytes and between astrocytes and neurons was examined after devising specific, real-time, sensitive enzymatic fluorescent assays to measure lactate and glucose levels in single cells in adult rat brain slices. Astrocytes have a 2- to 4-fold faster and higher capacity for lactate uptake from extracellular fluid and for lactate dispersal via the astrocytic syncytium compared to neuronal lactate uptake from extracellular fluid or shuttling of lactate to neurons from neighboring astrocytes. Astrocytes can also supply glucose to neurons as well as glucose can be taken up by neurons from extracellular fluid. Astrocytic networks can provide neuronal fuel and quickly remove lactate from activated glycolytic domains, and the lactate can be dispersed widely throughout the syncytium to endfeet along the vasculature for release to blood or other brain regions via perivascular fluid flow.

β-Amyloid-related peptides potentiate K+-evoked glutamate release from adult rat hippocampal slices

Accumulated evidence indicates that amyloid beta (Abeta) peptides, by interacting with the central glutamatergic system, can lead to degeneration of neurons associated with Alzheimer's disease (AD) pathology. However, very little is currently known about the role of Abeta peptides in the regulation of glutamatergic function in the normal brain. Given the evidence that Abeta peptides are produced constitutively in the normal brain, we investigated the possible association of amyloid precursor protein (APP)-containing neurons with the vesicular glutamatergic transporter-1 (VGluT1) and measured the effects of various Abeta peptides on endogenous glutamate release from adult rat brain slices. Our results showed that VGluT1 immunoreactivity is localized in close apposition to APP neurons, and that exogenous Abeta(1-40), in a dose-dependent (10(-12) to 10(-7)M) manner potently increased K(+)-evoked glutamate release from hippocampal slices. This effect was observed with other Abeta peptides such as Abeta(1-42), Abeta(1-28) and Abeta(25-35), but not with the reverse Abeta(1-40) or Abeta(25-35) sequences. Tetrodotoxin failed to alter the effects of Abeta(1-40) on glutamate release, which suggests the lack of involvement of voltage-dependent Na(+) channels. In addition to the hippocampus, Abeta(1-40) was found to potentiate K(+)-evoked glutamate release from cortical slices, whereas in the striatum the effect did not reach significant levels. These results demonstrate that physiological concentrations of Abeta peptides can regulate the release of glutamate by acting on glutamatergic terminals. Additionally, the evidence that selected regions of the brain are sensitive to Abeta peptides suggests a potential link between the deposition of Abeta and the preferential vulnerability of brain regions observed in AD pathology.

Inability of volatile anesthetics to inhibit oxygen–glucose deprivation-induced glutamate release via glutamate transporters and anion channels in rat corticostriatal slices

Ischemia-induced extracellular glutamate accumulation and the subsequent excitotoxicity contribute significantly to ischemic brain injury. Volatile anesthetics have been shown to reduce ischemic brain injury. Here, we showed that oxygen–glucose deprivation (OGD, to simulate ischemia in vitro) increased extracellular glutamate accumulation in the corticostriatal slices of adult rats. This increased accumulation was reduced by dihydrokinate, a glutamate transporter type 2 inhibitor, and 4,4′-dinitrostilbene-2,2′-disulfonic acid, a blocker for volume-activated anion channels. The volatile anesthetics isoflurane, sevoflurane and desflurane at clinically relevant concentrations did not affect the OGD-induced extracellular glutamate accumulation from brain slices of adult rats. Isoflurane also did not change the OGD-induced extracellular glutamate accumulation from brain slices of newborn/young rats. These results suggest that the OGD-induced glutamate accumulation involves reversed transport of glutamate via glutamate transporters and volume-activated anion channels. Volatile anesthetics may not inhibit this extracellular glutamate accumulation.

Immunohistochemical Analysis of Nucleotide Excision Repair Factors XPA and XPB in Adult Rat Brain

To study the regional and cellular distribution of xeroderma pigmentosum group A and B (XPA and XPB) proteins, two nucleotide excision repair (NER) factors, in the mammalian brain we used immunohistochemistry and triple fluorescent immunostaining combined with confocal microscope scanning in brain slices of adult rat brain, including the cerebral cortex, striatum, substantia nigra compacta, ventral tegmental area, red nucleus, hippocampus, and cerebellum. Both XPA and XPB proteins were mainly expressed in neurons, because the XPA- or XPB-immunopositive cells were only costained with NeuN, a specific neuronal marker, but not with glial fibrillary acidic acid, a specific astrocyte marker, in the striatum. Furthermore, XPA- and XPB-positive staining were observed in the neuronal nuclei. Such subcellular distribution was consistent with the location of the NER in the cells. This study provides the first evidence that NER factors XPA and XPB exist in the nuclei of neurons in the brain, suggesting that the NER may play important roles in the process of DNA repair in adult brain neurons.