Nucleoside Uptake by Slices of Mouse Brain

: The properties of the uptake of nucleosides and nucleotides by brain cells were examined in slices of mouse brain. Of the compounds tested, adenine and adenosine had the most rapid uptake and reached the highest levels. Uptake was mediated, as shown by saturability and strong inhibition, by low temperature, or by cyanide, and was only partially sodium- or calcium-dependent. The inhibition pattern by analogues indicated the presence of several uptake systems (possibly four), as shown by differences between adenine and guanine uptake, between adenine and adenosine uptake, and between adenosine and cytidine uptake. The properties of uptake systems for nucleotides and nucleosides were somewhat different from those for amino acids.     

A Device for Cutting Brain Slices

A device for cutting brain slices is described as an alternative to cutting angle guides and the “brain macrotome”. With this new device, slices of uniform thickness optimal for assessing morphological detail and photography can be produced. A similar but smaller device for cutting pieces of tissue for paraffin embedding is also presented. These devices should be useful in either the histopathology laboratory or mortuary.     

Procedure for Staining Fixed Human Brain Slices

The described method provides a new technique for differentiating areas of gray and white matter in fixed human brain slices. The technique is a modification of an existing method permitting use of the nonfading copper phthalocyanine dye alcian blue Stained slices show turquoise gray matter that contrasts sharply with areas of white matter. Procedure Cut human brains from gross anatomy laboratory cadavers into 4 mm slices and wash in running tap water for 14 hr. Oxidize slices in performic acid for 1.5 hr. Wash in running tap water for 14 hr. Stain slices in shallow dishes in 0.05% aqueous alcian blue. Wash in running tap water for 1 hr. Dry for 2-4 hr and embed in plastic.     

Simple and Durable Staining of Thick Sections of the Human Brain for Macroscopic Study

Observations on gross morphology of the human brain is greatly facilitated by enhancing the contrast between gray and white matter. The proposed technique is much more simple than the generally recommended Mulligan method and its variations. Moreover, there is no loss of stain since the fugitive surface impregnation, obtained by the Mulligan method, is replaced by a thoroughgoing block-staining procedure with the nonfading copper phthalocyanine dye astra blue. Staining procedure: wash formalin-fixed brain slices overnight in running tap water. Place slices in performic acid for 1 hour. Wash in running tap water. Place slices individually in staining solution consisting of 0.1 g astra blue (Merck) in 1000 cc distilled water and 1 cc HCl (37%), for 12-24 hours. Wash in running tap water. Embed in gelatin and mount in plastic cuvettes.     

Uptake and Release of N-Methyl-d-Aspartate by Rat Brain Slices

Abstract: The excitant amino acid, N -methyl- d -aspartate, was actively taken up by slices of rat cerebral cortex. This uptake was Na⁺ - and temperature-dependent, but was relatively inefficient (K_m 3 MM, V_max 0.07 μmol/g/min) compared with that of other acidic amino acids. The uptake of N -methyl- d -aspartate does not appear to have a rate-limiting influence on the time course of N -methyl- d -aspartate-induced excitation since potent uptake inhibitors, such as threo-3-hydroxy- l -aspartate, do not influence the excitant action of N -methyl- d -aspartate. The relatively prolonged excitant action of this acidic amino acid may be the result of relatively slow dissociation of the activated receptor complex. Reloaded N -methyl- d -aspartate can be released from rat brain slices by stimulation with K⁺ ions. Such K⁺-stimulated release appeared to be Ca²⁺-independent, unlike the K⁺-stimulated release of preloaded d -aspartate. These findings suggest that N -methyl- d -aspartate may be a weak but selective substrate for a glial acidic amino acid uptake system.     

Specificity of Neurotensin Metabolism by Regional Rat Brain Slices

Regional differences in neurotensin metabolism and the peptidases involved were studied using intact, viable rat brain microslices and specific peptidase inhibitors. Regional brain slices (2 mm x 230 microns) prepared from nucleus accumbens, caudate-putamen, and hippocampus were incubated for 2 h in the absence and presence of phosphoramidon, captopril, N-[1(R,S)-carboxy-3-phenylpropyl]-Ala-Ala-Phe-p-aminobenzoate, and o-Phenanthroline, which are inhibitors of neutral endopeptidase 24.11, angiotensin-converting enzyme, metalloendopeptidase 24.15, and nonspecific metallopeptidases, respectively. Neurotensin-degrading proteolytic activity varied by brain region. Significantly less (35.0 +/- 1.6%) neurotensin was lost from hippocampus than from caudate-putamen (45.4 +/- 1.0%) or nucleus accumbens (47.8 +/- 1.1%) in the absence of inhibitors. Peptidases responsible for neurotensin metabolism on brain slices were found to be predominantly metallopeptidases. Metalloendopeptidase 24.15 is of major importance in neurotensin metabolism in each brain region studied. The relative contribution of specific peptidases to neurotensin metabolism also varied by brain region; angiotensin-converting enzyme and neutral endopeptidase 24.11 activities were markedly elevated in the caudate-putamen as compared with the nucleus accumbens or hippocampus. Interregional variation in the activity of specific peptidases leads to altered neurotensin fragment formation. The brain microslice technique makes feasible regional peptide metabolism studies in the CNS, which are impractical with synaptosomes, and provides evidence for regional specificity of neurotensin degradation.     

Uptake of Kynurenine into Rat Brain Slices

The transport of [3H]kynurenine ([3H]KYN) into slices from rat tissue was examined in vitro. Brain accumulated KYN seven to eight times more effectively than any of several peripheral organs. Of all the organs tested, only the brain exhibited a sodium-dependent component of the uptake process. After an incubation period of 1 h, sodium-dependent transport amounted to 60% of total uptake. Both processes were abolished by prior sonication of the tissue and significantly inhibited by inclusion of metabolic blockers in the incubation medium. Time resolution showed that the sodium-independent uptake occurred rapidly and reached saturation within 30 min. In contrast, sodium-dependent transport was linear for at least 2 h of incubation. Brain regional analysis revealed a sevenfold difference between the areas of highest (cortex) and lowest (cerebellum) uptake. With the exception of cerebellar tissue, the ratio between sodium-dependent and sodium-independent processes was consistent among brain regions. Kinetic analyses were performed on striatal slices and revealed a Km of 927 microM and a Vmax of 18 nmol/h/mg of protein for the sodium-dependent process, and a Km of 3.8 mM and a Vmax of 38 nmol/10 min/mg of protein for the sodium-independent transport. The transporters were equally amenable to inhibition by KYN and tryptophan, indicating that KYN entry into the cell may be mediated by neutral amino acid uptake sites. No strict stereoselectivity existed, but L enantiomers were clearly more active than the D forms.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hypotaurine Uptake by Brain Slices from Adult and 8-Day-Old Mice

Abstract: Uptake of [³⁵S]hypotaurine by brain slices prepared from adult and 8-day-old mice was studied at varying temperatures, under O₂ and N₂ atmospheres, and in the presence of metabolic inhibitors and varying concentrations of hypotaurine in the incubation medium. The tissue/medium concentration gradients generated were exceptionally high for an amino acid. Hypotaurine uptake was energy- and temperature-dependent, more strictly in adult mice. Uptake was saturable, containing a high-affinity and a low-affinity component. The estimated transport constants for the high-affinity uptake of hypotaurine (8-day-old mice, 17.2 μ mol/liter; adults, 35.3 μ mol/liter) were of the same order of magnitude as the reported transport constants of putative amino acid transmitters, but the total transport capacity appears to be greatest for hypotaurine.     

Acetylcholine Release Induced by the Volatile Anesthetic Sevoflurane in Rat Brain Cortical Slices

1. We have investigated the effect of the volatile anesthetic sevoflurane on acetylcholine (ACh) release from rat brain cortical slices. 2. The release of [3H]-ACh into the incubation fluid was studied after labeling the tissue ACh with [methyl-3H]-choline chloride. 3. We observed that sevoflurane induced an increase on the release of ACh that was dependent on incubation time and anesthetic concentration. The sevoflurane-induced ACh release was not blocked by tetrodotoxin (TTX) and therefore was independent of sodium channels. In addition, the sevoflurane effect was not blocked by ethylene glycol-bis(beta-aminoethyl ether (EGTA) or cadmium (Cd2+), thus independent of extracellular calcium. 4. The sevoflurane-induced ACh release was inhibited by 1,2-bis (2-aminophenoxy) ethane-N,N,N',N'-tetra-acetic acid (BAPTA-AM), suggesting the involvement of intracellular calcium-sensitive stores in the process. Dantrolene, an inhibitor of ryanodine receptors, had no effect but 2-aminoethoxydiphenylborate (2-APB), a membrane-permeable inhibitor of inositol 1,4,5-triphosphate receptor inhibited the sevoflurane-induced release of ACh. 5. It is concluded that sevoflurane-induced release of ACh in brain cortical slices involves the mobilization of calcium from IP3-sensitive calcium stores.     

A Bioluminescence Method for the Demonstration of Regional Glucose Distribution in Brain Slices

Abstract: Regional glucose distribution in brain slices was assessed by a bioluminescence technique. The reaction is based on light emission of luminiferous marine bacteria, Vibrio fischeri , induced by NADPH. Freeze-dried brain slices were covered by a solution which contained: (a) enzymes and substrates for glucose oxidation and NADPH formation and (b) an extract of Vibrio fischeri for the bioluminescence reaction. Glucose-induced bioluminescence was recorded on photographic film. Patterns of regional decrease in glucose concentration were demonstrated in cat brains after occlusion of the left middle cerebral artery. This decrease correlated well with a concomitant depletion of ATP and an increase in NADH-fluorescence.     

Accumulation of Pantothenic Acid by the Isolated Choroid Plexus and Brain Slices In Vitro

In vitro, the transport of [14C]pantothenic acid into and from the isolated rabbit choroid plexus, an anatomical locus of the blood-CSF barrier, and brain slices was studied. The choroid plexus accumulated [14C]pantothenic acid from the medium against a concentration gradient, although at low concentrations (less than 1 microM) there was substantial intracellular phosphorylation and binding of the [14C]pantothenic acid. The saturable accumulation process in choroid plexus was inhibited by probenecid and caproic acid but not by nicotinic acid or by weak bases. The accumulation process was markedly inhibited by N-ethylmaleimide, poly-L-lysine (which blocks sodium transport), and low temperatures. [14C]Pantothenic acid was readily released from choroid plexus by a temperature-dependent process. Brain slices also accumulated and, at low concentrations, phosphorylated [14C]pantothenic acid from the medium by a temperature-, probenecid-, and N-ethylmaleimide-sensitive saturable process. However, unlike choroid plexus, brain slices did not concentrate free pantothenic acid and [14C]pantothenic acid accumulation was not sensitive to poly-L-lysine. [14C]Pantothenic acid was readily released from brain slices by a temperature-sensitive process. These results are consistent with the view that [14C]pantothenic acid enters the isolated choroid plexus and brain slices by active transport and facilitated diffusion, respectively.     

Preparation of Acute Subventricular Zone Slices for Calcium Imaging

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural progenitor cells with astrocytic features (called SVZ astrocytes), neuroblasts, and intermediate progenitor cells. Neuroblasts born in the SVZ tangentially migrate a great distance to the olfactory bulb, where they differentiate into interneurons. Intercellular signaling through adhesion molecules and diffusible signals play important roles in controlling neurogenesis. Many of these signals trigger intercellular calcium activity that transmits information inside and between cells. Calcium activity is thus reflective of the activity of extracellular signals and is an optimal way to understand functional intercellular signaling among SVZ cells.Calcium activity has been studied in many other regions and cell types, including mature astrocytes and neurons. However, the traditional method to load cells with calcium indicator dye (i.e. bath loading) was not efficient at loading all SVZ cell types. Indeed, the cellular density in the SVZ precludes dye diffusion inside the tissue. In addition, preparing sagittal slices will better preserve the three-dimensional arrangement of SVZ cells, particularly the stream of neuroblast migration on the rostral-caudal axis.Here, we describe methods to prepare sagittal sections containing the SVZ, the loading of SVZ cells with calcium indicator dye, and the acquisition of calcium activity with time-lapse movies. We used Fluo-4 AM dye for loading SVZ astrocytes using pressure application inside the tissue. Calcium activity was recorded using a scanning confocal microscope allowing a precise resolution for distinguishing individual cells. Our approach is applicable to other neurogenic zones including the adult hippocampal subgranular zone and embryonic neurogenic zones. In addition, other types of dyes can be applied using the described method.     

Lipid Peroxidation in Rat Brain Cortical Slices as Measured by the Thiobarbituric Acid Test

Abstract: Malonaldehyde formation by cortical brain slices from rat brain was determined as a function of incubation time and of oxygen pressure. This substance, a byproduct of lipid peroxidation, was detected by the thiobarbituric acid test. Significant amounts of malonaldehyde were formed by brain slices during incubation in the 0.2 (air) to 10 atm oxygen range, and a portion of it was released into the medium. The rate of malonaldehyde formation was the highest during the first 10 min. Elevation of oxygen pressure above 1 atm caused further increments in malonaldehyde production with kinetic properties similar to that seen at 1 atm pressure, but the increments per additional oxygen pressure were diminishing. The formation of a given amount of malonaldehyde can be expressed as a function of atm oxygen × min. This function has the shape of a saturation curve approaching a maximum at around 300 atm × min. The results indicate extensive lipid peroxidation in brain slices under standard incubation conditions.     

Release of N-Acetylaspartylglutamate on Depolarization of Rat Brain Slices

In a great number of investigations, evidence in favor of a neurotransmitter role of the N-terminal-blocked, acidic dipeptide N-acetylaspartylglutamate (NAAG) has been accumulating. In fact, in some systems of the mammalian brain, almost all of the classical criteria for neurotransmitters have been fulfilled by NAAG except for the demonstration of its release from nervous tissue on depolarization. For quantification of NAAG in superfusates of brain slices, we have developed an analytical procedure consisting of an ion exchange prepurification, followed by a derivatization procedure and gas chromatography-mass spectrometry with chemical ionization and selected ion monitoring. Deuterated NAAG was used as an internal standard to provide a high degree of reliability for the analytical method. Detection limits of less than 1 pmol were achieved. A statistically highly significant increase of NAAG concentration in superfusates from rat neocortex, piriform cortex/amygdala, and hippocampus on depolarization with 50 mM K+ could be demonstrated and was shown to be largely Ca2+ dependent. These results support the hypothesis that NAAG is a neurotransmitter. Especially with respect to the piriform cortex, the present demonstration of NAAG release is consistent with electrophysiological and immunohistochemical evidence for its neurotransmitter function at terminals of the lateral olfactory tract.     

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.     

Potassium-Induced Release of Endogenous Morphine from Rat Brain Slices

Abstract: Endogenous morphine has been clearly demonstrated by gas chromatography/mass spectrometry in the brain, spinal fluid, adrenal glands, and liver of mammals. To clarify the role of endogenous morphine, its release from rat brain slices was studied in vitro in the presence of high potassium concentrations, with and without calcium in the medium. The perfusate was hydrolyzed, solid phase-extracted, and then analyzed by gas chromatography/mass spectrometry. Depolarization due to high potassium concentrations increased the release of the alkaloid manyfold with respect to the basal value, and the release was dependent on the presence of calcium in the medium. These results suggest that endogenous morphine might act as a neurotransmitter or neuromodulator in the rat CNS.     

Mechanism of Protein Carbonylation in Glutathione-Depleted Rat Brain Slices

This study was conducted to further our understanding about the link between lipid peroxidation and protein carbonylation in rat brain slices incubated with the glutathione (GSH)-depletor diethyl maleate. Using this in vitro system of oxidative stress, we found that there is a significant lag between the appearance of carbonylated proteins and GSH depletion, which seems to be due to the removal of oxidized species early on in the incubation by the mitochondrial Lon protease. Upon acute GSH depletion, protein carbonyls accumulated mostly in mitochondria and to a lesser degree in other subcellular fractions that also contain high levels of polyunsaturated lipids. This result is consistent with our previous findings suggesting that lipid hydroperoxides mediate the oxidation of proteins in this system. However, these lipid hydroperoxides are not produced by oxidation of free arachidonic acid or other polyunsaturated free fatty acids by lipooxygenases or cyclooxygenases. Finally, γ-glutamyl semialdehyde and 2-amino-adipic semialdehyde were identified by HPLC as the carbonyl-containing amino acid residues, indicating that proteins are carbonylated by metal ion-catalyzed oxidation of lysine, arginine and proline residues. The present findings are important in the context of neurological disorders that exhibit increased lipid peroxidation and protein carbonylation, such as Parkinson’s disease, Alzheimer’s disease, and multiple sclerosis.     

Cation Dependence of Hypotaurine Uptake in Mouse Brain Slices

Abstract: Mouse brain slices take up hypotaurine (2-aminoethanesulphinic acid) from medium by means of two concentrative low- and high-affinity transport systems. [³⁵S]Hypotaurine uptake by the slices was significantly reduced in the absence of external potassium, calcium, or magnesium ions. An excess of potassium ions also inhibited hypotaurine uptake by one-half. Uptake was almost completely abolished on removal of sodium ions. The K _m constants for both low- and high-affinity transport components increased in a low-sodium medium, suggesting that sodium ions are required when hypotaurine is attached to its possible carrier sites in plasma membranes. Sodium ions also mimicked allosteric effectors of hypotaurine transport, showing positive cooperativity. More than two sodium ions may be involved in the transport of one hypotaurine molecule across the cell membrane. The calculated activation energies of transport were fairly similar in normal and sodium-deficient media and thus sodium ions may not participate in the activation mechanisms of the transport. With respect to cation dependence, hypotaurine transport in brain slices exhibits features characteristic of neurotransmitter amino acids.