Cerebral Slices Research Articles

Quantification of the dopamine innervation in adult rat neostriatum

Conditions leading to specific and integral visualization of dopamine axon terminals (varicosities) were tested in adult rat cerebral hemisphere slices incubated with [ 3H]dopamine and processed for high resolution radioautography. Specific visualization of the dopamine endings was achieved after incubation with 10 −6 M [ 3H]dopamine in the presence of a monoamine oxidase inhibitor (pargyline 10 −4 M), and of desipramine (5 × 10 −6 M), an inhibitor of catecholamine uptake by noradrenaline and serotonin neurons. [ 3H]Dopamine varicosity labeling was eliminated by the addition of 5 × 10 −5 M benztropine (an inhibitor of catecholamine uptake by catecholamine neurons), and was almost absent when dopamine nerve cell bodies of the midbrain had been previously destroyed with 6-hydroxydopamine. In dopamine-denervated neostriatum incubated without desipramine, a second set of labeled terminals was also visible. These were identified as serotoninergic, since their labeling was suppressed by citalopram, an inhibitor of monoamine uptake highly specific for serotonin neurons. There was no desipramine-sensitive but citalopram-resistant varicosity labeling suggestive of neostriatal noradrenaline innervation. In normal striatum, incubation at 35°C always resulted in a labeling of dopamine varicosities restricted to a narrow band which followed the contours and cut surfaces of this anatomical region. This unusual distribution was the result of an uptake barrier generated by the tightly packed dopamine varicosities. Indeed, the striatal dopamine varicosity labeling was more widespread after partial 6-hydroxydopamine denervation or in normal tissue incubated either with a higher [ 3H]dopamine concentration (5 × 10 −6 M), in the presence of relatively low benztropine concentrations (10 −5 M), or at lower temperature (15°C). Material incubated at 15°C for 90 min was suitable for purposes of quantification: labeled varicosities were then visualized throughout the striatum and across the full thickness of the slices; moreover, the number of labeled varicosities plotted against radioautographic exposure time increased in parallel and reached a plateau at the same time in neostriatal sectors with widely different innervation densities. At a rostral transverse level across neostriatum, the dorsolateral quadrant showed hyperdense “patches” of labeled terminals distinguishable from an already dense surrounding “matrix”, whereas, ventromedially, the dopamine innervation appeared more uniform and somewhat less dense. Counts within these sectors and measurement of the average diameter of the labeled profiles in electron microscope radioautographs allowed to extrapolate the absolute number of dopamine varicosities to 1.0 × 10 8 and 1.7 × 10 8 per mm 3 of tissue in the dorsolateral matrix and patches, respectively, and 0.6 × 10 8 in the ventromedial sector. Correlation of such quantitated data with measurements of dopamine content, receptors, associated secondary messengers, recognition sites for uptake and/or other neuronochemical, hodological and cytological parameters, should eventually lead to a better understanding of the functional role of the dopamine input in the various compartments of neostriatum.

Peroxidative aggregation of myelin membrane proteins

The exposure of CNS myelin to reactive oxygen species (ROS) generated by a Cu2+-H2O2 system results in the aggregation of membrane proteins. Integral and peripheral membrane proteins are equally vulnerable and the denaturation is not mediated by the SH groups. The aggregated proteins retain their original antigenicity as determined by immunoblot technique. The aggregation of proteins is not limited to myelin and can be elicited in the preparation of other cerebral membranes. The effect of ROS on membrane proteins can also be demonstrated in cerebral slices incubated in the presence of the ROS-generating system. Furthermore, the peroxidation inactivates membrane-bound enzymes as exemplified by myelin cyclic nucleotide phosphatase (CNP). Competitive inhibition studies with various scavengers and quenchers of ROS implicate singlet oxygen as a major mediator in the Cu2+-H2O2 oxidizing system responsible for the peroxidative aggregation of membrane proteins.

Ginsenoside modulates K+-stimulated noradrenaline release from rat cerebral cortex slices.

The release of labelled noradrenaline from rat cerebral cortes slices preloaded with L-[7,8- 3 H]noradrenaline ([ 3 H]NA) was measured in Krebs-Ringer-Bicarbonate media containing either 5.0 (KR) or 50 mM KCl. Experiments were also performed in media containing either 2.5 mM of or no calcium. Total ginsenoside, at concentrations ranged from 0.001 to 50 μg/ml, reduced the high K + -evoked release of [ 3 H]NA in calcium-containing but not in calcium-free media, and this inhibition was concentration-dependent. It, however, failed to affect the release of [ 3 H]NA significantly from undepolarized samples in KR media. These results are in agreement with suggestion that ginsenosides may affect behavior and brain function.

Glutamate-elicited stimulation of acetylcholinesterase activity in cerebellar slices from newborn rats

Bath application of glutamate at two concentration ranges, 10 −6 – 10 −8 and 1 – 3 × 10 −3 M, effectively increased acetylcholinesterase activity in cerebellar slices obtained from 8-day-old rats. No such effect was seen in cerebellar slices of 7-week-old rats or cerebral slices of either 7-week or 8-day-old rats. Glutamic acid diethyl ester blocked the glutamate effect at both of these concentration ranges, suggesting that quisqualate-sensitive glutamate receptors are involved in regulation of acetylcholinesterase activity in early postnatal cerebellum. Since bath application of cyclic GMP at 10 −7 – 10 −9 M increased the acetylcholinesterase activity in cerebellar slices of 8-day-old rats, it is possible that glutamate-dependent regulation of acetylcholinesterase activity is mediated by cyclic GMP. The observation that adenosine deaminase blocked the effect of glutamate completely at 10 −6 – 10 −8 M and partially at 1 – 5 × 10 −3 M further suggests that release of adenosine is a link from enhanced cyclic GMP activity to activation of acetylcholinesterase.

Properties of the uptake and release of neurotransmitter glutamate in cerebral cortical tissue of guinea pigs.

In order to investigate the dynamics of glutamate as a neurotransmitter and to avoid a complication by its metabolism, we studied the uptake and release of labeled non-metabolizable D-isomers of aspartate and glutamate in cerebral cortical slices and synaptosome preparation from guinea-pigs. The rate of uptake of D-aspartate and glutamate was mutually inhibited in a non-competitive fashion, indicating that their uptake mechanisms are not exactly the same. By ouabain (0.05 mM), the uptake of D-aspartate and glutamate into synaptosome preparation was less inhibited than that into cerebral slices. In synaptosome preparation most of the preloaded D-aspartate and glutamate was released by high-potassium (50 mM) stimulation, whereas in cerebral slices only a slight release was observed. However, when the slices were superfused with a medium free of sodium ions, which are absolutely necessary for the uptake, after preloaded with the labeled amino acids in the standard medium, a distinct release of radioactivity was induced by high-potassium stimulation. This potassium-induced release corresponded to only about 20% of the radioactivity accumulated in the slices. The accumulation of D-aspartate and glutamate into cerebral slices was much larger on the basis of their protein content than that into synaptosome preparation, when a high concentration (1 mM) of the amino acids was added to the medium. These observations suggest that the uptake system of D-aspartate and glutamate in cerebral slices is quite different from that in synaptosome preparation, and that the accumulation into cerebral slices is mainly localized in glial cells. In vivo the glial cell uptake is probably more important in removing the released neurotransmitter glutamate.

Amino acid incorporation during morphine intoxication. II: Electrophoretic separation of extracellular proteins from cerebral hemisphere slices and astroglia-enriched primary cultures.

Radioactively labeled proteins were identified in a broad molecular weight range in the incubation medium of rat cerebral hemisphere slice preparations or primary cultures after incubation with radioactive valine. Morphine chloride caused an increase in labeled media proteins with MW approximately 40,000 and 65,000 in both preparations, whereas a fraction with MW, approximately 80,000 decreased in amount. In the culture preparation a MW approximately 15,000 fraction also decreased after morphine treatment. The effects on the MW 40,000 and 65,000 fractions by morphine could not be blocked by naloxone hydrochloride, an opiate antagonist. Labeled media proteins from primary cultures obtained from various brain regions showed many similarities. After 10(-5) or 10(-6) M morphine, protein fractions with MW approximately 15,000, 25,000, 40,000, 65,000-70,000, 80,000, and 100,000 changed with increases or decreases in the various morphine concentrations. The 3H-labeling of the MW approximately 40,000 fraction increased in all cultures, where it decreased. In conclusion, proteins synthesized within cells in brain slices or in astroglia-enriched cultures are released or secreted into the incubation media. Some protein fractions are affected in different directions by morphine. The effects were not correlated with the presence of classical opiate receptors on the cells. This was so even though more changes in media proteins occurred in astroglial cultures from opiate receptor-rich brain regions than in similar cultures from opiate receptor-poor regions. The possible significance of proteins synthesized in astroglial cells and released extracellularly in opiate action is discussed.

Modulation of noradrenaline uptake and release by taurine in rat cerebral slices

The effects of taurine on the uptake and release of noradrenaline were studied in forebrain slices from control rats and from rats treated with a taurine-free diet supplemented with 2-guanidinoethanesulphonate. Exogenous taurine slightly increased noradrenaline uptake in slices from untreated rats. There were no significant effects by exogenous taurine on spontaneous efflux of noradrenaline. The potassium-stimulated noradrenaline release was enhanced by taurine from frontal cortical slices and suppressed from occipital slices but only in the diet group, in which the endogenous brain taurine concentration had been lowered to one-half of the control concentration. It is suggested that taurine may act via presynaptic α-adrenoceptors and/or modify the availability of calcium for the stimulus-secretion coupling process.

Transport of branched-chain amino acids in brain slices of developing and adult rats.

The accumulation of labelled leucine, isoleucine and valine by cerebral slices of developing and adult rats was studied. The accumulation increased with age by 15-25%. It was strongly (from 52 to 86%) inhibited by a 100-fold excess of phenylalanine, tryptophan and another branched-chain amino acid, but moderately activated by GABA and glutamate. The inhibitions evoked by leucine and isoleucine were slightly stronger in young than in adult rats. The corresponding 2-oxoacid analogs of leucine, isoleucine and valine were also inhibitory but less effective. The 30-min accumulation of 3H-labelled branched-chain amino acids was ostensibly higher than the increase in their total concentrations in incubated slices, which apparently bespeaks lively homoexchange of endogenous intracellular and labelled extracellular amino acids.

Uptake, metabolism, and releasability of ethyl analogues of homocholine by rat brain.

Ethyl analogues of homocholine were synthesized and used to describe further the specificities of the processes involved in choline uptake and acetylation and acetylcholine storage and release. Monoethylhomocholine, diethylhomocholine, and triethylhomocholine decreased the transport of choline into rat brain synaptosomes. The mono- and diethyl compounds were taken up into synaptosomes with similar affinity for the transport system as choline (5.8, 8.5, and 5.5 microM, respectively) but at a somewhat slower rate (11.3, 8.5, and 37.3 nmol/g original tissue/h, respectively); the triethyl analogue was not transported at the concentrations tested, which further defines the structural specificity of the transport system. L-Carnitine did not affect the transport of the analogues. The in situ acetylation of mono- and diethylhomocholine by slices of rat cerebral cortex was measurable, but the in vitro acetylation by choline acetyltransferase solubilized from rat forebrain was not. Acetylation of the diethyl analogue by slices of cerebellar cortex was less than 20% of that by slices of cerebral cortex. Subcellular fractionation of cerebral slices showed that acetyldiethylhomocholine localized preferentially to the cytosolic rather than vesicular stores, indicating specificity of the mechanism responsible for the incorporation of acetylated product into the vesicles. The release of acetyldiethylhomocholine and of acetylcholine was tested from sliced brain that had been incubated with the precursors. Both esters were released spontaneously but stimulation with increased K+ concentration enhanced the release of acetylcholine without changing the release of acetyldiethylhomocholine, suggesting that evoked transmitter release occurred from a vesicular store.

The effect of serum from multiple sclerosis patients in remission on the incubated rat brain slices

Addition of human serum to incubated rat cerebral slices induced increased generation of myelin-related, membranous fragments floating on 0.32 M sucrose. Sera from 20 healthy subjects and 19 patients with various neurological disorders were equally active in this respect. On the other hand, the myelin-degrading activity of sera from 20 multiple sclerosis patients in remission was found to be significantly elevated by about 50%. The present findings support the contention that the serum of multiple sclerosis patients possesses increased potency to induce myelin sheath alterations.

Changes in enzymes of the cholinergic system and acetylcholine release in the cerebra of aging male Fischer rats.

The functional decline of memory in the aging human brain has been partially attributed to defects in cholinergic transmission. Therefore, we have investigated various components of the cholinergic system in cerebra of Fischer 344 male rats, ages 3-33 months. Choline acetyltransferase (ChA), acetylcholinesterase (AChE), and butyrylcholinesterase (BChE) activities were determined in homogenates of the cerebra using specific radiometric assays. For measuring the release of acetylcholine (ACh), cerebral slices were incubated for one hour in Krebs buffer containing 3H-choline chloride to label ACh formed in situ, washed, and transferred to a microbath for superfusion. 3H-ACh released into the superfusate was determined. The levels of ChA in the cerebra of 9- to 27-month-old rats were lower (33%) than those in 3-month-old rats. Only 1% of these rats survive to the age of 33 months. In rats of this age, there was no decrease in ChA levels. AChE decreased while BChE increased with advancing age. The rate of spontaneous release of 3H-ACh decreased gradually by 63% from 3 to 33 months of age. The evoked release of ACh decreased by 50% in 33-month-old rats. Alterations in the levels of ChA, AChE (or BChE) and cholinergic receptors are not large enough to account for losses in cholinergic transmission in the cerebrum. The large decreases in the rates of spontaneous or evoked release of ACh in the aging cerebrum indicates that the functional defect in the cholinergic transmission of the aging cerebrum is possibly due to a defective release mechanism of this transmitter.

Myelin changes induced by incubation of brain slices with serum

Forebrain and brain stem slices prepared from adult rats were incubated with pooled normal human serum. Following the incubation, the tissue was homogenized and the fraction floating on 0.32 M sucrose as well as two myelin subfractions (light and heavy) were isolated. Addition of serum into the incubation medium increased generation of the floating fraction by the cerebral slices. Changes in the myelin membrane were also observed. Thus, myelin isolated from forebrain slices revealed pronounced increase in the buoyant density of its particles and loss of basic protein. Furthermore, in spite of the intensive washing employed during the isolation procedure, some serum proteins were found firmly attached to the membraneous fractions. The demonstration of the myelin alterations in the living cerebral tissue exposed to serum during incubation may contribute to understanding the pathogenesis of multiple sclerosis.

Aromatic amino acid incorporation into proteins in rat brain slices: influences of other amino acids affecting transport.

Incorporation of [3H]tryptophan, [3H]tyrosine and [3H]phenylalanine into proteins has been studied in cerebral coronal slices from adult and 1-day-old rats. Unlabelled amino acids greatly increased (histidine) or decreased (leucine, phenylalanine, tyrosine, tryptophan) intracellular levels of the [3H]amino acids, but only slightly decreased their incorporation rates. Effects on uptake were similar in experiments with adult and newborn rats, but patterns of protein synthesis inhibition seemed to differ. Amino acid uptake and subsequent incorporation into proteins were thus not closely associated. Incorporation in slices from adult rats was much slower than in those from newborn rats, and moreover, one half of it was cycloheximide-insensitive. The difference inhibition patterns by other amino acids may thus be explained by multiple mechanisms in cerebral amino acid incorporation into proteins.

Dynamics of the formation and release of corticotrophin releasing activity by the rat hypothalamus in vitro.

The ability of the isolated rat hypothalamus, incubated in vitro, to form and secrete corticotrophin releasing factor (CRF) has been assessed. It was found that serotonin (10 ng/ml), when added to the hypothalamus in vitro, caused the release of CRF and an increase in the activity of CRF contained in the tissue. The activity of CRF was estimated by its ability to cause the secretion of ACTH (determined by the cytochemical assay procedure) from anterior pituitary fragments in vitro. Provided a minimum of 10 min 'rest' in serotonin-free medium was allowed between each challenge with the neurotransmitter, the response of the hypothalamus in vitro was maintained for at least 3 h. A detailed examination of the time-course of CRF formation and secretion following a single addition of serotonin showed that the release was well underway within 40 s and continued up to 4 min, but then ceased. On the other hand, the tissue content of CRF was found to be reduced significantly at 20 and 40 s, had returned to basal values at 1 min and continued to increase to reach a maximum at 4 min that was maintained at 6 min. The stimulated release was approximately six times greater than basal and the increase in content approximately ten times basal. Removal of the serotonin-containing medium after 6 min of stimulation was accompanied by a precipitous fall in the CRF contained in the tissue and this was significant 2 min later with CRF content back to basal levels within 4 min. The serotonin-induced increase in CRF content was shown to be greatest in the area of the ventral portion of the medial basal hypothalamus containing the median eminence and its attendant pituitary stalk. The energy metabolite status of the hypothalamus in vitro under the conditions employed to cause the secretion of CRF was assessed. The rates of oxygen consumption and release of lactate, and the tissue concentrations of K+ and ATP were all determined and found to be comparable to values published for more conventional cerebral slice preparations for up to 2 h in vitro. At 3 h a fall in tissue K+ and an increase in the amount of lactate released into the medium suggested that the preparation was showing signs of becoming compromised metabolically and this trend was more obvious at 4 h when the ATP level had also fallen significantly. In more limited cognate experiments the changes in ACTH formation and release induced in anterior pituitary fragments in vitro by the addition of hypothalamic extracts were studied. Once again the release of the hormone was accompanied by an increase in the ACTH content of the tissue. The increase in content reached a maximum by 10 min and then declined to basal levels within 10 min of removal of the CRF-containing medium. Thus, it is possible that degrading enzymes play an important role in the control of hormonal output from both tissues.

Effect of changing potassium ion concentrations on rat cerebral slices in vitro: a study during development.

Adult rat cerebral cortex slices when incubated in media in increasing K+ concentrations, from 3 to 20 mM ("physiological" conditions), exhibited an increase of Ki+ and a decrease of Nai+ and Cli-. This phenomenon appeared at 30 days postnatally, which is fairly late during development. Over 20 mM-K+ ("pathological" conditions) an intense water uptake was observed together with an increase of Nai+ and Cli-. This was observed in both adult and young animals. The results are discussed in relation to the well known properties of glial (Na+,K+)-ATPase and carbonic anhydrase.

Studies on gamma-aminobutyric acid transport in cobalt experimental epilepsy in the rat.

Crude brain homogenates and cerebral tissue slices from rats with cobalt metal implanted in right and left cerebral cortices were used to examine high- and low-affinity GABA transport. High-affinity GABA transport was maximally reduced to 34% of controls 7 days after cobalt implantation, a time that coincides with peak seizure activity in this model. Kinetic analysis of high-affinity GABA transport, using brain homogenates, revealed a significant change in Vmax 7 days after cobalt implantation. (Vmax = 446.4 +/- 26.2 pmol/mg prot./min, cobalt, versus 787.8 +/- 67.3, control). An analysis of the low-affinity system revealed no depression of Km or Vmax parameters. Administration of valproic acid at a concentration as high as 1 mM in vitro or a dose of 300 mg/kg in vivo had no effect on high- or low-affinity GABA transport. The results obtained from cobalt-treated rats provide additional evidence for an involvement of GABA in experimental epilepsy.

Cerebral RNA synthesis in experimental hepatogenic encephalopathy

Cerebral slices from rats subjected either to chronic (2- to 6-month) administration of carbon tetrachloride or to prolonged (21-day) portocaval shunting showed an increased above control ability to incorporate (3H]uridine in vitro into the RNA of the large cell nuclei, mostly derived from astrocytes but not the small oligodendroglial cell nuclei. This result is interpreted to indicate that brain reacts to extended periods of hepatogenic insult or chronic omission of hepatic circulation with a stimulation of RNA synthesis in the astrocytes. The possible relation of this phenomenon to the characteristic astrocytes response accompanying hepatogenic encephalopathy is discussed.

Perinatal changes in amino acid metabolism of rat brain, especially alanine and glutamic acid.

The changes in both the levels of some free amino acids and their metabolism in the rat brain during the first 24 hr of postnatal life were studied. The content of glutamic acid decreased for the first 2 hr; it remained at the lowest level for the next 4 hr, when it began to increase. The content of alanine decreased for the first 6 hr and approached the adult level. Oxygen consumption, glucose oxidation, and pyruvate formation in the cerebral slices of the 24-hr-old rats were as much as 150% of that of the 19-day-old fetus. The distribution profile of radioactivity incorporated into the cerebral amino acids from the subarachnoid-injected [U-14C]glucose was also changed. In the 2- and 6-hr-old rats, 50% of the total radioactivity recovered in the free amino acids was in alanine. Its rate decreased to 30% in the 24-hr-old and was 2% in the adult, while the radioactivity incorporated into glutamic acid increased. Alanine aminotransferase activity started to increase at birth and had the highest level at 24 hr after birth. It then decreased and finally reached the same level as shown at birth. However, aspartate aminotransferase increased during the first 6 hr after birth and did not change until the end of the first day of life.

Cholecystokinin octapeptide: Vesicular localization and calcium dependent release from rat brain in vitro

Sub-cellular fractionation tissue from rat hypothalamus and cerebral cortex in sucrose gradients indicated a concentration of cholecystokinin-like peptides in synaptosomal fractions. Lysis of synaptosomes yielded a vesicle rich fraction which was further enriched in cholecystokinin-like peptides, particularly the octapeptide (CCK-8). In vitro release experiments carried out using rat cerebral cortex tissue slices showed a calcium dependent release of cholecystokinin (primarily as CCK-8). The demonstration of a vesicular localization and calcium evoked release of cholecystokinin is consistent with a role for cholecystokinin as a neurotransmitter/neuromodulator in the CNS.

Utilization of acetate by chick brain during postnatal maturation

1. 1. The study of the compartmentation of glutamate metabolism has been performed in the chick brain in vivo and in vitro in the presence of [U- 14C]acetate between day 1 and day 30 of postnatal maturation. 2. 2. The compartmentation of glutamate metabolism in vivo appears between day 1 and day 4 after hatching in the cerebral hemispheres and optic lobes. It is however more precocious in the optic lobes. In the cerebellum, it appears later, at about day 4 after hatching. The compartmentation of glutamate metabolism appears at the same time as the rapid incorporation of glucose into amino acids takes place in the cerebral hemispheres and the optic lobes. 3. 3. In the chick telencephalon in vitro, the compartmentation of glutamate metabolism is visible from day 1 after hatching onwards. This difference is undoubtedly linked to the absence of an interference of glucose metabolism with acetate metabolism in vitro, and to the presence of a third compartment in the cerebral slices.