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B K Hartman

Publications and source records attributed to B K Hartman.

At least 55 records · Page 3Linked to original sources

Localization of catechol-O-methyltransferase in the leptomeninges, choroid plexus and ciliary epithelium: implications for the separation of central and peripheral catechols.

Catechol-O-methyltransferase (COMT) was localized in cells of the pia-arachnoid, and in epithelial cells of the choroid plexus, using an indirect immunofluorescence technique. The specific activity of COMT derived from these tissues was determined by radioenzymatic assay, and in the case of the choroid plexus was found to be 9-fold greater than that measured in whole rat brain. The level of COMT specific activity in pia-arachnoid was twice as high as that in whole brain. Indirect immunofluorescence studies also revealed an intensity of COMT immunofluorescence in the ciliary epithelium at the blood-aqueous barrier in the rat eye, similar to that visualized in the epithelium of the choroid plexus at the blood-cerebrospinal fluid barrier. The localization of COMT in the leptomeninges, choroid plexus, and ciliary epithelium is consistent with a role for this enzyme in the separation of catechol compounds synthesized in the central nervous system, from those of peripheral origin. Thus, catecholamines derived from the peripheral sympathetic system may be prevented from entering the brain parenchyma, which is innervated by the functionally distinct central catecholaminergic systems.

Animals↗

Importance of fixation in immunohistochemistry: use of formaldehyde solutions at variable pH for the localization of tyrosine hydroxylase.

Adequate fixative in immunohistochemistry requires not only a rapid and total immobilization of the antigen, but also a sufficient preservation of its immunoreactivity and maintenance of its accessibility to the immunochemical reagents for localization. Thus, the optimal fixation condition for a specific antigen necessitates a compromise between these opposing variables and can be determined by the preparation of a series of tissues with a progressively increasing degree of fixation. Unless the results of localization using such a series is available, one must be satisfied with adequate but less than optimal results. In the present study, this principle is demonstrated using the localization of tyrosine hydroxylase in the dopaminergic system with formaldehyde as the fixative. The rate and degree of fixation with formaldehyde was shown to be highly pH dependent. By perfusing the tissue with formaldehyde at pH 6.5 (where the rate of fixation is extremely slow) it is possible to rapidly distribute the fixative homogeneously into the tissue. By suddenly changing to a formaldehyde perfusate of higher pH, the cross-linking reaction is rapidly increased. This two-step fixation procedure provides a means of obtaining a rapid and uniform immobilization of the antigen, so that its translocation can be avoided. The final degree of fixation is controlled by the duration and pH of the second fixative solution. The results obtained by increasing the pH of the second solution demonstrated that complete fixation of tyrosine hydroxylase in the dopaminergic system with formaldehyde maybe obtained using a very basic formaldehyde solution (pH 11) while still retaining immunoreactivity of the enzyme. The localization that was achieved at lower pH appeared adequate until it was compared to the results obtained by perfusion at pH 11 in the second step.

Animals↗

Long-term antidepressant treatment: alterations in cerebral capillary permeability.

Chronic treatment of rats with amitriptyline (AMI) induced a persistent increase in the diffusibility of water into the brain (EW). This effect was observable 12 h after the last dose. AMI induces this alteration at plasma drug concentrations of 71 +/- 13 ng/ml (the therapeutic range for man is 100--250 ng/ml). Furthermore, chronic treatment potentiated the increase observed after acute drug administration and resulted in a 350% enhancement in the permeability of water across the cerebral capillary. Thus, long-term antidepressant administration can chronically influence cerebral function by affecting capillary permeability.

Animals↗

Anti-catechol-O-methyltransferase: demonstration of specificity and immunological cross-reactivity with the enzyme from rat liver, kidney, brain, and choroid plexuses.

An antiserum to rat liver catechol-O-methyltransferase (COMT) was utilized in the immunological characterization of COMT from rat kidney, brain, and choroid plexuses, in addition to rat liver. The presence of anti-COMT activity was confirmed by the direct inhibition of the activity of the enzyme from rat liver by small quantities of the antiserum and by the inhibition of the activity of the enzyme from rat brain. The specificity of the antiserum was demonstrated both by immunoelectrophoresis of rat liver COMT, and by a partial purification of rat liver COMT in which changes in COMT specific activity were correlated with the appearance of a precipitin line in double-immunodiffusion experiments. The antigenic similarity of the enzyme derived from rat liver, kidney, brain, and choroid plexuses was demonstrated by the formation of a precipitin line of identity when preparations from these four tissues were diffused against the antiserum.

Animals↗

Biochemical specificity in central pathways related to peripheral and intracerebral homeostatic functions.

Immunohistochemical and horseradish peroxidase (HRP) studies suggest that oxytocin-stained fibers from the paraventricular nucleus of the hypothalamus (PVH) innervate the locus coeruleus (LC) and dorsal vagal complex (DVC) in the brainstem of the rat, and that adrenergic fibers from the LC and DVC in turn project to the PVH. It is hypothesized that these pathways are directly involved in the integration of neuroendocrine and autonomic responses in the periphery, as well as in the mediation of homeostasis-preserving responses within the CNS itself.

Animals↗

Preparation of antibodies specific to choline acetyltransferase from bovine caudate nucleus and immunohistochemical localization of the enzyme.

Choline acetyltransferase (CATase; acetyl-CoA:choline O-acetyltransferase, EC 2.3.1.6) has been purified from bovine caudate nucleus. The specific activity of the pure enzyme was 120-160 mumol of acetylcholine formed per mg per min. The purified enzyme separated into two bands (band A and band B) when electrophoresed in a pH 4.3 gradient gel. Both bands exhibited CATase activity. Antisera were prepared in rabbits to each form. Immunotitrations using either antisera resulted in quantitative precipitation of CATase activity from enzyme preparations at all stages of purification. Both antisera produced single immunoprecipitin lines in double-diffusion experiments when run against the enzyme at each stage of purity. Immunoprecipitin lines cut from double-diffusion gels contained CATase activity, demonstrating that the observed reaction was due to antibody interaction with the enzyme. Immunoelectrophoresis also showed a single immunoprecipitin line against the purified enzyme as well as against a crude caudate extract. These results indicated that immunochemically pure and specific antisera have been prepared to bovine CATase and that both the A and B forms of the enzyme have common antigenic sites. The antisera were utilized to localize the enzyme in bovine brain. The localization was exclusively neuronal with both antisera, and both forms of CATase were present in the same population of neurons. Subtle differences in the precise intracellular distribution of enzyme were observed with the two antisera, indicating that although the two molecular forms of CATase have antigenic sites in common they are not antigenically identical.

Animals↗

The effect of dibenzazepines (tricyclic antidepressants) on cerebral capillary permeability in the rat in vivo.

The degree of equilibration of [3H]water across the cerebral capillary was evaluated by measuring its cerebral extraction fraction (Ew) by using a dual label radioactive tracer technique. All tricyclic antidepressants (125 mumol/kg i.p. at 5 min) increased Ew as compared to base line. The rank order of the drugs in producing this response was doxepin greater than amitriptyline greater than imipramine greater than nortriptyline greater than desmethylimipramine greater than protriptyline. The effect of amitriptyline, the prototype tricyclic for this study, was rapid in onset (maximal effect within 5 min), reversible (duration 15 min), dose-dependent and generalized throughout the brain. Amitriptyline also induced a marked increase in the cerebral extraction fraction of [3H]ethanol. A difference in the time course of the drug effect on these two tracers indicated that the elevation in E was due to the increase in cerebral capillary permeability to both polar and lipid soluble substances. This work demonstrates that tricyclic antidepressants have important central effects on non-neuronal tissue.

Amitriptyline↗

A comparative study of the immunohistochemical localization of basic protein to myelin and oligodendrocytes in rat and chicken brain.

Antisera to highly purified basic protein (BP) from rat and chicken brain were prepared and their purity and specificity demonstrated by double immunodiffusion and cross-immunoadsorption. These antisera were used for immunohistochemical localization of BP in the brains of adult and developing rat and chick. Myelin basic protein was exclusively localized to myelin or the myelin forming elements of the CNS. It was present in high concentrations in white matter and absent in areas free of myelin. Neuronal parikarya and dendrites were negative as were axons cut in cross section and at Nodes of Ranvier. The latter was best observed in cross sections of human spinal cord demonstrating also the immunoreactivity of the antibodies with human BP. The internodal distance in a fine (1.5 micrometer) rat cortical fiber was determined to be approximately 45 micrometers. Myelin basic protein was shown to extend into cranial roots, in contrast to myelin proteolipid protein which abruptly lose fluorescence as the nerves emerged from the brain. During development, BP was first observed on the fourteenth day of incubation in chick and at birth in the rat. The protein appeared in oligodendrocytes and in association with fibers near these cells. Fluorescent processes were frequently observed connecting the oligodendrocytes with the fibers. As myelination progressed, the intensity of the immunohistochemical reaction decreased in the oligodendrocytes while the brightness in fibers increase. Eventually, the oligodendrocytes became undetectable. Fibers with immature myelin exhibited a beaded or varicosed appearance with the highest concentration of immunofluorescence in the outer portion of the varicosities. The varicosities were postulated to represent dilations in the newly forming sheath between intervals of compaction along the axon undergoin myelination. These dilations might represent areas of increased cytoplasmic volume which could serve as channels for transport and/or storage sites for myelin proteins prior to incorporation into the membrane. The varicosities became less prominent with the thickening of the myelin sheath and mature myelinated fibers became smooth. The process of synthesis of BP, transport of the protein to the varicosed fibers, and maturation of the myelin sheath was seen to progress in a more or less caudal to rostral direction as myelination of the CNS takes place. In the rat, this was accomplished over approximately a 30-day period starting near the time of birth. In the chick, most of the myelination was accomplished in the three or four days immediately before hatching. At this time, innumerable oligodendrocytes were observed producing BP simultaneously in the major white fiber tracts. It is postulated that in chick some degree of oligodendrocytic cell death occurs normally during myelination.

Animals↗

Immunohistochemical demonstration of catechol-o-methyltransferase in mammalian brain.

Catechol-O-methyltransferase (COMT)(EC 2.1.1.6) was localized using fluorescence immunohistochemistry in rat liver and kidney and in rat, chinchilla, and bovine brains. In the brain, specific fluorescence was visable only in non-neuronal cellular elements in all three species. Ventricular ependymal cells and cells of the choroid plexuses exhibited the greatest intensity of immunofluorescence. Glial immunofluorescence appeared prominently in large myelinated fiber tracts. Interfascicular and perineuronal satellite oligodendrocytes as well as fibrous astrocytes were immunoreactive, though myelin itself did not exhibit a positive reaction. Bergmann glial cells in the cerebellum also stained brightly for COMT. Although this study indicates that the predominant localization of COMT is non-neuronal, it is important to note that the presence of small quantities of this enzyme in neurons cannot be excluded. The patterns of localization observed in the non-neuronal elements suggest that this enzyme may function as a barrier to free diffusion of catechol compounds within the central nervous system.

Animals↗

Evaluation of in vivo brain site perfusion with the push-pull cannula.

The effect of tissue site perfusion with push-pull cannulas on the integrity of brain tissue was evaluated during in vivo studies in conscious New Zealand rabbits. The mean rate of perfusion-induced tissue disruption was 1.09 microgram tissue wet wt/min as estimated from the perfusate levels of endogenous nervous system specific proteins (S-100 and 14-3-2). The level of intravenously injected 125I-labeled bovine serum albumin recovered in perfusate samples indicated that approximately 2.0 nl serum/min penetrated the vascular barrier into the perfused tissue site. The appearance of intraventricularly injected solutes in perfusate samples was subjected to regression analysis. It was demonstrated that nonspecific variations in the appearance of one substance in the perfusate could be controlled for by a regression on the levels of another concurrently recovered inert substance. The experiments demonstrate that tissue site perfusion can provide useful access to brain extracellular fluid when suitable controls are incorporated for recovery variation and blood-brain barrier seepage.

Animals↗

The effect of tricyclic antidepressants on cerebral fluid dynamics.

Tricyclic antidepressants are thought to act primarily via effects on adrenergic neurotransmitters. Recent research supports the concept that a major function of the central adrenergic system is the modulation of cerebral fluid dynamics. Based on this concept, studies in the rat were conducted to assess the effects of these drugs on cerebral capillary permeability and flow by quantitating changes in the extraction fraction of water (Ew). Amitriptyline and nortriptyline produced significant increased in Ew for the total forebrain (from control values of 0.67 to experimental values as high as 0.99) while protriptyline had no effect on Ew. The amitriptyline-induced increase in Ew occurred at doses which produced plasma levels (500 ng/ml) near the range defined as therapeutic in depression studies. The magnitude of the effect was similar for both amitriptyline and nortriptyline representing a 35--40% increase over control values. The effects were uniformly observed throughout the forebrain: rostral telencephalon, caudal telencephalon, and diencephalon.

Amitriptyline↗

Histochemical and biochemical characterization of the rat paracervical ganglion.

The nature and identity of the catecholamines in the paracervical ganglion and superior cervical ganglion small, intensely fluorescent (SIF) cells were investigated using fluorescence histochemical and immunohistochemical techniques. The paracervical ganglion SIF cells were found to contain norepinephrine and the superior cervical ganglion SIF cells, dopamine. The norepinephrine content of the paracervical ganglion SIF cells averaged about 72 ng/ganglion and did not change during the rat estrus cycle. The activity of the enzyme tyrosine hydroxylase in the PCG was very low (about 0.48 nmoles DOPA formed/h/mg protein) and was about 1/50th of the activity of the enzyme in the SCG, where it averaged about 23.90 nmoles DOPA formed/h/mg protein. These experiments suggested that the paracervical ganglion has large numbers of norepinephrine containing SIF cells with a relatively slow turnover of their catecholamine content.

Adrenal Medulla↗