Rate of inactivation of adenyl cyclase and phosphodiesterase: determinants of brain cyclic AMP

In order to assess the effects of time requirements of different tissue inactivation methods, concentrations of cyclic adenosine monophosphate in rat brain were determined. Fixation of tissues was obtained by the following methods: decapitation with removal of brain and freezing in liquid nitrogen; decapitation into liquid nitrogen; whole animal immersion in liquid nitrogen; 1.5 kW maximal field strength microwave irradiation for 8 seconds; and, 5 kW maximal field strength microwave irradiation for 2 seconds. Results of these studies indicate that as the time is reduced for inactivation of brain adenyl cyclase and phosphodiesterase, levels of cyclic adenosine monophosphate become progressively lower. This same correlation is also evident in studies of regional brain concentrations of cyclic adenosine monophosphate after 1.5 kW and 5 kW microwave inactivation. It is concluded that 5 kW maximal field strength microwave exposure is the most rapid means of enzyme inactivation permitting a more accurate estimation of endogenous cyclic adenosine monophosphate concentrations. Its use offers rapid inactivation with minimization of trauma and facilities the study of regional metabolites through ease of dissection.     

In vivo studies of carnitine and fatty acid metabolism: Problems with use of anesthetics

The use of ether anesthesia as a technique for obtaining liver tissue and plasma from living rats was compared to decapitation followed by in situ sample freezing. Parameters associated with fatty acid metabolism were examined to demonstrate differences in the two methodologies. Use of ether in fasted rats was associated with lower plasma free fatty acids and acetoacetate concentrations, as well as decreased hepatic total carnitine, carnitine, and acid-soluble acylcarnitine content when compared to decapitated animals. No qualitative differences between the ether and decapitated groups were noted, and the quantitative differences observed were consistent with effects induced by the anesthetic. Thus, with respect to in vivo studies of fatty acid metabolism use of anesthetic agents may induce significant changes without providing advantages over decapitation and in situ freezing.     

Postmortem Changes of Normetanephrine Determined by a Mass-Fragmentographic Technique in Different Rat Brain Areas

Abstract: A specific and sensitive gas chromatographic–mass spectrometric assay for measurement of normetanephrine (NMN) in different rat brain regions is presented. The results show that hypothalamus contains the highest concentrations of the norepinephrine metabolite. An increase of NMN concentrations has been detected in animals killed by decapitation in comparison with microwave–irradiated rats. This finding has to be taken into consideration when NMN is measured in the brain in order to assess the functional activity of noradrenergic neurons.     

Rapid High-Energy Microwave Fixation is Required to Determine the Anandamide (<Emphasis Type="BoldItalic">N</Emphasis>-arachidonoylethanolamine) Concentration of Rat Brain

Anandamide (N-arachidonoylethanolamine, AEA) is the putative endogenous ligand for the CB₁ receptor. Despite being regulated enzymatically, brain AEA concentrations are quite variable and have been reported to increase in response to ischemia and post-mortem delay. Because these observations are similar to the effects of decapitation on brain concentrations of unesterified arachidonic acid and several of its metabolites, we propose that brain AEA concentrations also increase with decapitation and that immediate head-focused microwave irradiation is necessary to quantify basal brain AEA levels correctly. To test this hypothesis, we measured brain AEA levels in rats that were subjected to head-focused microwave irradiation 5 min. following decapitation (5.5 kW, 3.4 s) (ischemic) and prior to decapitation (controls). Brain AEA concentrations were quantified by LC/MS/MS. AEA concentrations from ischemic animals (10.01 ± 4.41 pmol/g, mean ± SD) were significantly higher and more variable than control concentrations (2.45 ± 0.39 pmol/g). Thus, the basal concentration of AEA in the brain is lower than previously thought and future studies attempting to quantify brain AEA should consider using head-focused microwave fixation to prevent anomalous results.     

A radioenzymatic assay for normetanephine in brain tissue

A simple, rapid and sensitive radioenzymatic assay for measurement of normetanephrine (NMN) in the brain has been described. The assay which is based on conversion of NMN to its N-methylated tritiated derivative metanephrine (³N-MN), by partially purified bovine adrenal phenyl-ethanolamine-N-methyl represents an extension of a previously developed procedure for measurement of urinary NMN. The sensitivity of the assay is 50 pg and results can be obtained in less than 4 hours. In rat brain, NMN concentration was 61 ± 3.4 ng/gm for hypothalamus and 82 ± 4.2 for brain stem at level of obex in male rats; 74 ±11 and 139 ± 10, respectively in female rats. Measurement of NMN in different areas of the brain may help to elucidate possible involvement of central nervous system in the pathophysiology of disease states such as hypertension.The role of central catecholamines in the pathogenesis of disease states has been investigated by a variety of techniques. These include methods which estimate relative concentrations of catecholamines in different parts of the neurones (1,2) and absolute concentrations in specific brain nuclei (3). In addition, estimates been made of catecholamine synthesis (4,%) an dturnover rates (6). Neurotransmitter actually released into the synaptic cleft cannot be measured. Moreover, part of what is released is taken up again by the neurone. The remainder is subject to catabolism and the resulting metabolic products may well reflect the amount of physiologically active transmitter. This report describes a rapid specific and sensitive assay for measurement of the O-methylated metabolite of norepinephrine, normetanephrine (NMN) in brain tissue, which is an extension of a previously developed procedure for urinary NMN (31). The metabolite is stable and readily extracted into solvents.     

Assimilation of nicotinamide mononucleotide requires periplasmic AphA phosphatase in Salmonella enterica

Salmonella enterica can obtain pyridine from exogenous nicotinamide mononucleotide (NMN) by three routes. In route 1, nicotinamide is removed from NMN in the periplasm and enters the cell as the free base. In route 2, described here, phosphate is removed from NMN in the periplasm by acid phosphatase (AphA), and the produced nicotinamide ribonucleoside (NmR) enters the cell via the PnuC transporter. Internal NmR is then converted back to NMN by the NmR kinase activity of NadR. Route 3 is seen only in pnuC* transporter mutants, which import NMN intact and can therefore grow on lower levels of NMN. Internal NMN produced by either route 2 or route 3 is deamidated to nicotinic acid mononucleotide and converted to NAD by the biosynthetic enzymes NadD and NadE.     

In vivo regional levels of PGE and thromboxane in mouse brain: Effect of decapitation,focused microwave fixation,and indomethacin

A focused microwave fixation technique was tested for use in determining basal PGE and thromboxane B₂ levels of mouse brain. Focused microwave irradiation (3.5 Kw/0.4 sec) to the head of C3H mice produced basal values of PGE and TXB₂ which were five-fold less than those in animals killed by decapitation. Indomethacin (10 mg/kg) pretreatment blocked the decapitation rise in PGE and TXB₂ levels and gave values similar to focused microwave irradiation. Indomethacin pretreatment combined with microwave fixation did not reduce PG levels more tham microwave treatment alone. When microwave fixation was used, there was no difference in regional (cerebral cortex, whole cerebellum, midbrain, hypothalamus) levels of either PGE or TXB₂. However, PGE levels were significantly higher than TXB₂ in all regions. After decapitation there was a greater increase in TXB₂ than PGE. The cerebellum produced less PGE and TXB₂ after decapitation compared to the other regions. Our results confirm the usefulness of the focused microwave irradiation technique for examining $\text{in vivo}$ basal prostaglandin levels in mouse brain.     

The Dopamine Metabolite 3-Methoxytyramine Is Not a Suitable Indicator of Dopamine Release in the Rat Brain

It has been postulated that changes in the concentration of 3-methoxytyramine (3-MT) in the brain might reflect changes in the release of 3,4-dihydroxyphenylethylamine (DA, dopamine) and, therefore, might be used as an index of dopaminergic activity in the brain. 3-MT is known to accumulate rapidly after death. Killing by microwave irradiation (MWR) is considered to be the method of choice to obtain "undisturbed" 3-MT concentrations. We measured striatal 3-MT concentrations even lower than those following MWR when the brains were excised and frozen in dry ice very rapidly (typical time between decapitation and freezing of the brain 22 s). There was a linear increase in striatal 3-MT concentration when the time between decapitation and freezing was varied between 13 and 300 s. Extrapolation to time zero indicated negligible amounts of 3-MT at the time of decapitation. In addition, it was observed that DA, 3,4-dihydroxyphenylacetic acid, and homovanillic acid decompose during the cooling phase after heating the brain by microwave. It is concluded that MWR induces artifactual changes in the postmortem levels of DA and metabolites. Consequently 3-MT cannot be considered to be a reliable indicator of DA release in the rat brain.     

Effect of antinociceptive doses of oxotremorine on mouse brain acetylcholine turnover rate

The effect of antinociceptive doses of oxotremorine on the steady-state level and turnover rate of acetylcholine (ACh) was investigated in male Swiss-Webster mice. Oxotremorine produced dose-related increases in ACh levels which attained statistical significance (p ≥ 95; Dunnett's T) with the ED₈₄ antinociceptive dose in two sacrifice methods. The increased steady-state level of ACh was temporally correlated with the peak antinociceptive effect of oxotremorine. ACh turnover rate decreased with increasing doses of oxotremorine. The ACh turnover rate decreased from 11.06 ± 1.62 nmol/g/min to 5.38 ± 0.71 nmol/g/min by decapitation method and 30.20 ± 1.8 nmol/g/min to 19.99 ± 1.6 nmol/g/min by the microwave irradiation method (Ed₈₄ oxotremorine doses). The decrease in turnover rate of ACh produced by antinociceptive doses of oxotremorine is of a lesser magnitude than that produced by tremorogenic doses (1,2).     

Elevation of cerebrospinal fluid choline levels by nicotinamide involves the enzymatic formation of N1-methylnicotinamide in brain tissue

Nicotinamide administration can elevate plasma and brain choline levels and produce a marginal increase in striatal acetylcholine levels in the rat. We now report that subcutaneous nicotinamide produces a substantial and long-lasting rise in asternal cerebrospinal fluid (CSF) levels of choline in free-moving rats, possibly through the enzymatic formation of N¹-methylnicotinamide (NMN) in brain. CSF choline levels peaked 2 hours after nicotinamide administration and were accompanied by increases in striatal, cortical, hippocampal and plasma choline levels. The enzymatic formation of [³H]NMN in rat brain was evaluated by incubating aliquots of rat brain cytosol with unlabelled nicotinamide and the methyl donor [³H]S-adenosylmethionine. High performance liquid chromatography and radiochemical detection demonstrated that [³H]NMN was specifically formed by a brain cytosolic enzyme. The production of [³H]NMN was dependent on exogenous nicotinamide and could be prevented by denaturing the cytosol. The metabolism of nicotinamide to NMN in rat brain may explain the rise in CSF choline levels since NMN, a quaternary amine, can inhibit choline transport at the choroid villus and reduce choline clearance.     

Accurate Evaluation of 1,2-Diacylglycerol in Gerbil Forebrain Using HPLC and In Situ Freezing Technique

Gerbil forebrains were frozen in situ to inactivate the tissues, and 1,2-diacylglycerols were first measured quantitatively by HPLC. Although 1,2-diacylglycerols were completely recovered from the HPLC column, the control amount of 1,2-diacylglycerol in gerbil forebrain was only 79.6 nmol/g wet weight, which is about one-fourth of that previously reported for gerbil brain inactivated by liquid N2 after decapitation instead of in situ freezing. The fatty acid composition of 1,2-diacylglycerols in gerbil forebrain was first reported and the control 1,2-diacylglycerols were richer in palmitic acid than in stearic acid or arachidonic acid, which is rather different from the data previously reported for mouse or rat brain obtained by decapitation and analyzed by traditional TLC methods. The amount of 1,2-diacylglycerol increased by 82.9% in gerbil forebrain during 5 min of ischemia induced by bilateral carotid ligation. Arachidonic acid and stearic acid were abundant in the 1,2-diacylglycerols produced by 5 min of ischemia. Thus we were able to obtain accurate values of the amount and the fatty acid composition of 1,2-diacylglycerols in gerbil forebrains using HPLC and in situ freezing technique.     

Evaluation of the necessity for rapid inactivation of brain enzymes prior to analysis of norepinephrine dopamine and serotonin in the mouse.

Norepinephrine, dopamine and serotonin concentrations were measured in mouse whole brain. Animals were killed either by decapitation or by exposure to 250 msec microwave irradiation which produces irreversible inactivation of brain enzymes. The biogenic amines were determined by mass fragmentometry, fluorometry and by a combination of high performance liquid chromatography and an electrochemical detector. No differences were found in the levels of these neurochemicals between the two methods of animal sacrifice. Therefore, rapid inactivation of brain enzymes is not necessary prior to analysis for catecholamines and serotonin in mouse whole brain.     

Cellular Specificity of the Blood–CSF Barrier for Albumin Transfer across the Choroid Plexus Epithelium

To maintain the precise internal milieu of the mammalian central nervous system, well-controlled transfer of molecules from periphery into brain is required. Recently the soluble and cell-surface albumin-binding glycoprotein SPARC (secreted protein acidic and rich in cysteine) has been implicated in albumin transport into developing brain, however the exact mechanism remains unknown. We postulate that SPARC is a docking site for albumin, mediating its uptake and transfer by choroid plexus epithelial cells from blood into cerebrospinal fluid (CSF). We used in vivo physiological measurements of transfer of endogenous (mouse) and exogenous (human) albumins, in situ Proximity Ligation Assay (in situ PLA), and qRT-PCR experiments to examine the cellular mechanism mediating protein transfer across the blood–CSF interface. We report that at all developmental stages mouse albumin and SPARC gave positive signals with in situ PLAs in plasma, CSF and within individual plexus cells suggesting a possible molecular interaction. In contrast, in situ PLA experiments in brain sections from mice injected with human albumin showed positive signals for human albumin in the vascular compartment that were only rarely identifiable within choroid plexus cells and only at older ages. Concentrations of both endogenous mouse albumin and exogenous (intraperitoneally injected) human albumin were estimated in plasma and CSF and expressed as CSF/plasma concentration ratios. Human albumin was not transferred through the mouse blood–CSF barrier to the same extent as endogenous mouse albumin, confirming results from in situ PLA. During postnatal development Sparc gene expression was higher in early postnatal ages than in the adult and changed in response to altered levels of albumin in blood plasma in a differential and developmentally regulated manner. Here we propose a possible cellular route and mechanism by which albumin is transferred from blood into CSF across a sub-population of specialised choroid plexus epithelial cells.     

Analysis of “solution effects” injury: Rabbit renal cortex frozen in the presence of dimethyl sulfoxide

Slices of rabbit renal cortex were frozen in 0.64 or 1.92 M dimethyl sulfoxide (Me₂SO) to various subzero temperatures, thawed, and assayed for viability. Salt and Me₂SO concentrations were calculated and correlated with the injury taking place during freezing. In separate experiments, slices were treated with NaCl or Me₂SO in concentrations sufficient to simulate the exposure brought about as a result of freezing. The effects of these treatments on cortical viability were compared with the results of freezing to equivalent concentrations of either NaCl or Me₂SO. The results show that whereas slices will tolerate exposure to at least six times the isotonic concentration of NaCl at 0 °C, they are unable to tolerate even three times the isotonic salt concentration when frozen in 1.92 M Me₂SO. They can, however, tolerate 3 × NaCl when frozen in 0.64 M Me₂SO. Freezing damage did not depend upon the amount of ice formed per se, since slices frozen in the low concentration of Me₂SO tolerated removal of about 75% of the initial fluid content of the system, whereas slices frozen in 1.92 M Me₂SO did not tolerate an identical removal of unfrozen solution. It was found that treatment of slices with high concentrations of Me₂SO at subzero temperatures in accordance with Elford's application (14) of Farrant's method (20) produced damage which correlated approximately with the damage observed when the same concentrations of Me₂SO were produced by freezing. It is concluded that most of the damage caused by freezing in 1.92 M Me₂SO is produced either directly or indirectly by Me₂SO. Possible mechanisms for this injury are discussed.     

Radioimmunoassay of met-enkephalin in microdissected areas of paraformaldehyde-fixed rat brain

We studied the effect of various sample preparation procedures on rat brain met-enkephalin content, measured by radioimmunoassay. Whole brain met-enkephalin content of rats killed by decapitation followed by immediate tissue freezing was similar to that of rats killed by microwave irradiation and to those of rats anesthetized with pentobarbital or halothane before killing, whether previously perfused with paraformaldehyde or not. In contrast, a decrease (up to 80%) in met-enkephalin concentrations was observed when brain samples were frozen and thawed to mimic the procedure utilized in the “punch” technique for analysis of discrete brain nuclei. This decrease was totally prevented by paraformaldehyde perfusion of the brain prior to sacrifice. Brain perfusion did not alter the amount of immunoassayable met-enkephalin extracted from tissue or its profile after Sephadex chromatography. Paraformaldehyde perfusion results in better morphological tissue preservation and facilitates the “punch” dissecting technique. Paraformaldehyde perfusion may be the procedure of choice for the measurement of neuropeptides in specific brain nuclei dissected by the “punch” technique.     

REGIONAL γ-AMINOBUTYRIC ACID LEVELS IN RAT BRAIN DETERMINED AFTER MICROWAVE FIXATION

—GABA levels in rat whole brain were compared following three methods of sacrifice: rapid microwave fixation, decapitation into liquid nitrogen, and decapitation at 20°C. Levels were shown to be identical in animals sacrificed by microwave fixation and decapitation into liquid nitrogen. In contrast, rats decapitated at 20°C had 18 per cent higher GABA levels when determined immediately post-mortem and 48 per cent higher levels after 30 min at 20°C. Microwave treatment prevented these post-mortem increases. The increase in GABA after decapitation at 20°C was even greater in hypothalamus than in whole brain. A comparison of 3 GABA extraction methods following microwave fixation demonstrated that sodium acetate was 88 per cent as effective as 80 per cent ethanol and more effective than 0·5 n -perchloric acid in extracting GABA. Fifteen brain regions were dissected from microwave-treated brains and the GABA levels determined.     

An enzymatic method for the measurement of nicotinamide mononucleotide pyrophosphorylase in cells and tissues

A simple enzymatic method is described for the measurement of NMN pyrophosphorylase in tissue homogenates at levels as low as 10(-12) to 10(-9) mol. The product, nicotinamide mononucleotide, is converted to NAD using NAD pyrophosphorylase and the NAD is quantified in an enzymatic cycling assay. The enzyme described here is stimulated more at low concentrations of Mn2+ than Mg2+. ATP is not required for NMN pyrophosphorylase activity; the reaction is neither stimulated nor inhibited by ATP concentrations as high as 3 mM. The enzyme is totally dependent on phosphoribosylpyrophosphate. The method is highly reproducible in all tissues examined. Various cell lines and tissues from mouse were analyzed for NMN pyrophosphorylase.     

CYCLIC NUCLEOTIDES IN MURINE BRAIN: EFFECT OF HYPOTHERMIA ON ADENOSINE 3'',5'' MONOPHOSPHATE, GLYCOGEN PHOSPHORYLASE, GLYCOGEN SYNTHASE AND METABOLITES FOLLOWING MAXIMAL ELECTROSHOCK OR DECAPITATION

Abstract —The accumulation of adenosine-3',5'-cyclic monophosphate (cyclic AMP) has been investigated in murine brain following electroconvulsive shock and decapitation. Animals were made hypothermic (20°C) to minimize the freezing time of the brain and to delay metabolic events. Cyclic AMP concentrations were decreased in the cerebral cortex of hypothermic rats and mice. Furthermore, the changes in cyclic AMP elicited by electroconvulsive shock and decapitation were delayed. In hypothermic animals, the metabolic rate as determined by high energy phosphate use was decreased to 65% of control values. The interconversions of the active and inactive forms of glycogen phosphorylase and glycogen synthase were sufficiently retarded in hypothermic animals to correlate with changes in cyclic AMP concentrations. The conversion of phosphorylase b to a and synthase a to b occurred when cyclic AMP concentrations had increased from 2 to 5 μmol/kg, following either electroconvulsive shock or decapitation. The results indicate that cyclic AMP plays a role in regulation of glycogen metabolism in cerebral cortex.     

DANCER: a program for digital anatomical reconstruction of gene expression data

A digital anatomy construction (DANCER) program was developed for gene expression data. DANCER can be used to reconstruct anatomical images from in situ hybridization images, microarray or other gene expression data. The program fills regions of a drawn figure with the corresponding values from a gene expression data set. The output of the program presents the expression levels of a particular gene in a particular region relative to other regions. The program was tested with values from experimental in situ hybridization autoradiographs and from a microarray experiment. Reconstruction of in situ hybridization data from adult rat brain made by DANCER corresponded well with the original autoradiograph. Reconstruction of microarray data from adult mouse brains provided images that reflect actual expression levels. This program should help to provide visualization and interpretation of data derived from gene expression experiments. DANCER may be freely downloaded.     

Alpha-endorphin, gamma-endorphin and their des-tyrosine fragments in rat pituitary and brain tissue

Enkephalins, endorphins and related peptides were determined in pituitary and brain tissue of rats which were killed by decapitation or microwave irradiation. The tissues were heated in 1M acetic acid prior to homogenization and the levels of the various peptides were measured by means of a combination of HPLC and radioimmunoassays. Enkephalin levels in pituitary and brain of irradiation-killed rats were much higher as compared to those in tissue of rats sacrificed by decapitation. Similar data were obtained with respect to pituitary levels of γ-endorphin, des-Tyr-γ-endorphin and des- Tyr-α-endorphin. However, brain levels of α- and γ-endorphin and their respective des-Tyr-fragments were not different with the two methods of sacrifice used. The concentrations of β-endorphin in the pituitary gland were similar in rats killed by microwave irradiation and decapitation, but irradiation showed higher β-endorphin levels in the brain than decapitation. These results suggest that β-endorphin fragments like α- and γ-endorphin and des-Tyr-α- and des-Tyr-γ-endorphin are endogenous peptides in the rat pituitary gland and the brain.