Living Rat Brain Research Articles

3H]DOPA formed from [3H]tyrosine in living rat brain is not committed to dopamine synthesis.

Tyrosine hydroxylase of catecholamine neurons catalyzes the synthesis of 3,4-dihydroxphenylalanine (DOPA), which is subsequently metabolized to dopamine by DOPA decarboxylase (DDC). However, DOPA is not committed to decarboxylation in vivo because export of DOPA from brain and metabolism of DOPA other than decarboxylation are possible. To estimate the relative magnitudes of the several fates of DOPA, the kinetics of the uptake and metabolism of L-[3H]tyrosine ([3H]Tyr, intravenous infusion) was measured in brain of rats pretreated with NSD 1015, an inhibitor of DDC. Some rats were pretreated with haloperidol before the blockade of DDC. The [3H]Tyr was incorporated into brain protein at a rate constant of 0.03 min(-1). The relative tyrosine hydroxylase activity in striatum was 0.005 min(-1) at 30 minutes after NSD 1015, 0.011 min(-1) 3 hours later, and 0.020 min(-1) after haloperidol treatment. The rate constant for the clearance of DOPA from brain (0.06 min(-1)) and earlier estimates of the rate constant of DDC activity in striatum (0.26 min(-1)) together predict that 80% of DOPA formed in normal rat striatum normally is available for dopamine synthesis. It follows that modulation of DDC activity can influence the rate of DA synthesis by affecting the relative magnitude of the several fates of DOPA in living brain.

The kinetic behaviour of [ 3H]DOPA in living rat brain investigated by compartmental modelling of static autoradiograms

The kinetic behaviour of [ 3H]DOPA in living rat brain was investigated by compartmental modelling of measured activities from combined metabolite pools in a time-series (180 min) of static autoradiograms from right cerebral hemispheres. Two models of [ 3H]DOPA uptake and metabolism that incorporated the removal of the decarboxylation product, [ 3H]dopamine, from brain were significantly more accurate than a model in which [ 3H]dopamine accumulated irreversibly in situ. Present estimates of [ 3H]DOPA kinetic constants were compared to previously published results based on the analysis of measured activities from individual metabolite pools separated by chromatographic fractionation of [ 3H]DOPA metabolites in the left cerebral hemispheres of the same rats. Autoradiographic estimates of DOPA decarboxylase activity with respect to [ 3H]DOPA in brain ( k 3 DOPA) were under-estimated several-fold relative to chromatographic estimates; this discrepancy is explained by post-mortem enzyme activity and omission of biological compartments from the models. However, autoradiographic estimates of the unidirectional blood–brain clearance of [ 3H]DOPA ( K 1 DOPA) and monoamine oxidase activity with respect to [ 3H]dopamine in brain ( k 7 DA′) agreed with chromatographic estimates. This concordance represents the first empirical validation of compartmental modelling of autoradiographic data as a method for quantitatively investigating the kinetic behaviour of radiolabelled l-DOPA in living mammalian brain.

Real-time measurement of nitric oxide production in rat brain by the combination of luminol-H 2O 2 chemiluminescence and microdialysis

A chemiluminescence method of detecting nitric oxide (NO) in combination with a microdialysis technique was employed for the real-time measurement of NO production in living rat brain. This method based on the luminol-H 2O 2 system has a detection limit of 1 nM, and is the most sensitive method currently available for measuring NO. We applied this new technique to rat cerebellum to record the increase of chemiluminescence intensity arising from NO production after the injection of N-methyl- d-aspartate or kainic acid around the microdialysis probe. This highly sensitive method should be useful for the direct clarification of the functions of NO in the central nervous system.

The ontogeny of a neurotoxic lesion in rat brain revealed by combined MRI and histology

We have used magnetic resonance imaging (MRI) of the living rat brain to longitudinally analyze the ontogenesis of an ibotenic acid lesion targeted at the piriform cortex. The MRI data were systematically compared with data obtained from a battery of histopathological techniques, including Nissl stain, hematoxylin stain, and a stain for cytochrome oxidase activity. Two days after the lesioning, widespread and heterogeneous damage was detected in, around and distant from the toxin-targeted area. Some damage apparently diminished within approximately 10 days, whereas other damage remained throughout the length of this study (60 days). We found that the small-animal MRI technology used by us is useful in determining the initial, transient impact of surgery and neurotoxic lesioning, and in delineating the gross effects of the lesion over time. This is particularly useful for early elimination of animals from the protocol of physiological and behavioral experiments in which the lesion exceeds the target area. Our data also indicate that, in order to avoid confounding effects of transient post-lesioning phenomena, behavioral and physiological tests should be carried out in neurotoxically lesioned animals > 2 weeks after infliction of the lesion.

Radioiodinated diacylglycerol analogue: a potential imaging agent for single-photon emission tomographic investigations of cerebral ischaemia

Phospholipid metabolism is closely related to membrane perturbation in cerebral ischaemia. We investigated in vivo topographical lipid metabolism using an iodine-123-labelled diacylglycerol analogue, (1-(15-(4-iodine-123-iodophenyl)-pentadecanoyl)-2-stearoyl-rac-gly cerol) (123I-labelled DAG), in a middle cerebral artery (MCA) occlusion model with the aim of positive imaging of ischaemic insult. Sprague-Dawley rats underwent coagulation of the MCA to induce permanent occlusion. MCA occlusion times prior to injection of 123I-labelled DAG ranged from 15 min to 14 days. Each rat was injected with 11-37 MBq of 123I-labelled DAG via a tail vein. After 30 min, in vivo autoradiographs were reconstructed. Scanning of the living rat brain in this MCA occlusion model was performed using a gamma camera with a pinhole collimator. Cerebral infarctions were recognized in the frontal cortex, the parietal cortex and the lateral portion of the caudate-putamen by 2,3,5-triphenyltetrazolium hydrochloride staining. In infarcted regions (region 1), 123I-labelled DAG incorporation showed a slight decrease up to 12 h; it then increased up to 6 days and decreased thereafter. In peri-infarcted regions (region 2), the incorporation showed almost no change up to 12 h, then increased up to 5-6 days and decreased thereafter. In other regions (region 3), the incorporation showed no change. Lipid analysis showed that 123I-labelled DAG was metabolized to 15-(4-iodine-123-iodophenyl)-pentadecanoic acid by DAG lipase and to 123I-labelled phosphatidylcholine. Scanning of the ischaemic region showed higher accumulation than on the non-lesioned side. We established a method to visualize ischaemic foci as positive images. The early changes in 123I-labelled DAG incorporation were closely related to DAG lipase, which degraded the accumulated intrinsic DAG, and increased 123I-labelled DAG incorporation in the chronic stage involves several aspects of neural destruction in the process of autolysis. It is concluded that the reported method could have a clinical future.

Use of a clinical MR scanner for imaging the rat brain

Magnetic resonance imaging (MRI) and magnetic resonance spectroscopy are established techniques that enable noninvasive anatomic and functional tissue characterization in vivo. These tools have been employed to probe experimental models of neoplasia, cerebrovascular disease, brain injury, and neurotransplantation in small animals. To date, these studies have been executed primarily on research-dedicated instruments of limited availability or resolution. Using relatively straightforward software and hardware modifications of a widely used clinical MRI unit, we were able to image numerous structures within the living rat brain including the neostriatum, hippocampus, periaqueductal gray, and the ventricular system. Illustrative applications of this imaging technique in two intracerebral infusion models involving rats are presented. Such adaptation of clinical MRI scanners has the potential to significantly expand the availability of high resolution in vivo imaging of small animals for a variety of experimental protocols.

Numerous phosphates of microtubule-associated protein 2 in living rat brain.

Microtubule-associated protein 2 (MAP 2) purified from microwave-irradiated rat head contained about 46 esterified phosphates (mole/mol), which were not bound covalently to lipids and did not assemble with microtubules. After some phosphates were released by calf intestinal alkaline phosphatase, the phosphate content of MAP-2 decreased to 16 mol of phosphate and the protein assembled in vitro. MAP-2 purified after microtubule assembly cycles and also the cytosolic heat-stable fraction without assembly cycles had 10 mol of phosphate, and both assembled with microtubules. The MAP-2 with 46 phosphates and that with 10 had different pI in isoelectric focusing, but the components, MAP-2a and -2b, were always near each other. In high-pressure liquid chromatography, MAP-2 containing 46 mol of phosphate appeared after that 10 mol of phosphate. Phosphoserine, phosphothreonine, and phosphotyrosine were recovered from tryptic digestion of MAP-2 with 46 mol of phosphate. These findings suggest that two kinds of MAP-2, one with 46 phosphates and not bound to tubulin and the other with 10-16 phosphates and bound to tubulin, are present in the living rat brain.

Three-dimensional MRI microscopy of the normal rat brain.

Magnetic resonance imaging techniques have been developed to enable imaging of the live rat brain with thin (1.2-mm) slices and microscopic pixels (115 X 115 microns). Signal-to-noise ratios high enough to realize the microscopic resolution are obtained with rf coils designed for the subject and through the use of three-dimensional Fourier spin warp imaging. The technique yields 16 contiguous slices. Correlation with fixed pathologic specimens enables unequivocal identification of gray and white matter structures in the brain of a live 200-g rat. Structures clearly visible in the MR images include Ammon's horn, the hypothalamus, corpus callosum and substantia nigra, as well as a number of brainstem nuclei.

Differences in brain cytochrome responses to carbon monoxide and cyanide in vivo.

Cytochrome oxidation-reduction responses to two mitochondrial electron transport inhibitors, carbon monoxide (CO) and cyanide (CN), were studied in the intact brains of fluorocarbon-circulated rats. In vivo reflectance spectrophotometry indicated that cortical b-type cytochromes (564 nm) were highly resistant to reduction by CN in the presence of O2 but showed reduction responses to the administration of 1-5% CO in 90% O2. In contrast, cyanide-sensitive cytochromes aa3 (605 nm) and c + c1 (551 nm) did not increase their reduction levels during exposure to 5% CO in 90% O2. The in vivo CO-mediated b-cytochrome reduction responses did not occur after pretreatment with the cytochrome b inhibitor, antimycin A. Transmission spectrophotometry of superfused hemoglobin-free rat brain slices confirmed cortical b-type cytochromes to be CN-resistant in the presence of O2. Another cytochrome absorbing at 445 nm also was resistant to reduction by 1-mM cyanide in vitro, but it could be reduced anaerobically. The reduced 445-nm cytochrome bound CO in the presence of cyanide. We postulate that this CN-resistant CO binding component might account for in vivo cytochrome aa3-CO interactions and directly or indirectly modulate cytochrome b reduction responses to CO. In any event, the spectral data indicate different primary tissue target sites for CO and CN in living rat brain and also suggest different bioenergetic consequences of exposure to the two agents.

Urea synthesis in the living rat brain.

IN previous communications from this laboratory, the synthesis of urea-14C from arginine-14C was reported to occur in the living rat brain1,2. In these experiments only the rat brains were analysed for their radioactive urea content, and the objection was raised that perhaps these levels did not indicate endogenous brain urea synthesis, but were merely a reflexion of blood 14C-urea levels, especially since it is known that urea readily diffuses across the blood brain barrier. Experiments were therefore designed to show that the brain 14C-urea levels were independent of blood 14C-urea levels and that the brain had the capacity to synthesize urea from arginine. This communication describes such experiments.