Noradrenergic activation of immediate early genes in rat cerebral cortex.

Previous studies have shown that stimulation of adrenergic receptors in the brain increases the expression of the immediate early gene (IEG), c-fos, in vivo (Mol. Brain Res., 6(1989) 39-45). The present study was undertaken to determine whether this also holds for other IEGs which have been shown to be activated in brain cell culture by adrenergic agonists. Both yohimbine injection and stressful stimulation, two treatments causing brain norepinephrine (NE) release, were found to cause a parallel, transient activation of at least 5 IEGs (c-fos, nur77, tis-7, zif-268 and tis-21) in the rat cortex. Genes that are not immediate early (beta-actin, NGF and HSP70) were found not to be affected in the interval used (6 h). The responses were mediated predominantly by beta-adrenoceptors with some contribution from alpha 1 receptors. The parallel activation of multiple genes by noradrenergic receptors may enable the coding of different biochemical responses to the activation of different receptors.     

Food deprivation and hypothalamic neuropeptide gene expression: effects of strain background and the diabetes mutation.

We have used a novel method to identify genes expressed in the hypothalamus which may be potentially involved in controlling food intake and energy metabolism. We assumed that food deprivation, a powerful stimulus of food intake, would stimulate the activity of neural pathways involved in feeding behavior which should be reflected in an increase in the synthesis of any relevant neuropeptide and its messenger RNA. A study of 5 neuropeptides in 5 strains of mice has identified neuropeptide Y (NPY) as a gene whose expression in the hypothalamus is controlled by nutritional status, suggesting that hypothalamic NPY neurons are a link in the neural network regulating feeding behavior and energy metabolism. In addition, we have studied the effect of the diabetes mutation on neuropeptide gene expression during fasting and refeeding. Our findings suggest that abnormal NPY and enkephalin gene expression in the hypothalamus may be two important determinants of the expression of the diabetes mutation.     

Development of striatal compartmentalization following pre- or postnatal dopamine depletion.

Nigrostriatal dopamine (DA) projections terminate in distinct patches during the late prenatal and early postnatal period in the rat. During the first postnatal week, patches of DA fibers overlap with clusters of striatal neurons that share several identified characteristics. The early segregation of striatal cell types into either these patches or the surrounding matrix becomes a permanent organizational feature of the striatum. In order to determine whether the heterogeneous distribution of DA influences the formation of cellular patches, the developmental organization of chemically identifiable cell types was examined in normal rats and in rats DA depleted as infants (0 or 3 d) or in utero (embryonic days 17-18). During the first postnatal week, corresponding patches of DA afferents and substance P (SP)-immunoreactive neurons existed in the striatum of normal animals, and AChE-positive zones overlapped these patches in the lateral striatum. Injection of 6-hydroxydopamine into the lateral ventricles of fetal or infant rats produced a dramatic loss of striatal DA terminals. Neither the patchy distribution of SP-immunoreactive neurons nor the distinctive pattern of AChE staining present during the first 2 postnatal weeks was disrupted. During the third postnatal week, cells immunoreactive for leu-enkephalin or calbindin-D28k were confined to the matrix compartment, and this compartmentalization was also not noticeably changed by pre- or postnatal DA depletion. In adult animals, overlapping patches of leu-enkephalin- and SP-immunoreactive fibers were observed, regardless of whether any DA terminals remained. Thus, the basic organization of the striatal patch and matrix compartments develops normally in the absence of DA innervation through much of the formative period. Although these observations do not completely dismiss the possibility that the first DA afferents to appear in the striatal primordia influence contracted striatal cells to develop the patch phenotype, they suggest that the patchy distribution of DA afferents may be secondary to the early clustering of striatal neurons forming the patch compartment.     

Medial-to-lateral gradient of neostriatal NGF receptors: relationship to cholinergic neurons and NGF-like immunoreactivity.

High-affinity binding sites for recombinant human NGF (rhNGF) were studied in the caudate-putamen of the adult rat and rabbit. Displaceable 125I-rhNGF binding sites were densely distributed throughout the caudate-putamen and were 2-3-fold more prevalant in the ventrolateral and lateral than in the medial caudate-putamen. The amount of nondisplaceable binding did not vary throughout the caudate-putamen. The medial-to-lateral receptor gradient was correlated (r = +0.99) with a 2-3-fold medial-to-lateral increase in ChAT activity. In contrast, NGF-like immunoreactivity (NGF-LI) was prevalent but uniformly distributed in the caudate-putamen. Lesions of intrinsic cholinergic neurons by quinolinic acid produced extensive gliosis in the medial, central, and lateral caudate-putamen, yet 125I-rhNGF binding was decreased in each of these regions. The activity of ChAT and 125I-rhNGF binding throughout the caudate-putamen were each decreased by 40% following quinolinic acid. Binding was not changed after 70-77% dopamine nerve terminal depletions induced by 6-hydroxydopamine, demonstrating a nonglial, nondopaminergic locus for striatal NGF binding sites. The cholinergiclike topography of NGF binding sites throughout the intact caudate-putamen, the parallel decreases of cholinergic neurons and NGF binding sites following intrinsic neuronal loss, and the uniform neostriatal gradient of NGF-LI are consistent with the trophic role of endogenous NGF for cholinergic interneurons of the caudate-putamen.     

Measurement of cerebral monoamine oxidase B activity using L-[11C]deprenyl and dynamic positron emission tomography

A tracer kinetic procedure was developed for the measurement of monoamine oxidase type B (MAO-B) activity using L-[11C]deprenyl and positron emission tomography (PET). The kinetic model consisted of two tissue compartments with irreversible binding to the second compartment (three rate constants). In addition, a blood volume component was included. Special attention was given to the accurate measurement of the plasma and whole blood input functions. The method was applied to the measurement of the dose-response curve of a reversible MAO-B inhibitor (Ro 19-6327). From the results, it followed that the rate constant for irreversible binding (k3) appeared to be a better index of MAO-B activity than the net influx constant Ki. Furthermore, regional analysis demonstrated that Ki, but not k3, was flow dependent. This implies that full kinetic analysis is required for an accurate assessment of MAO-B activity.     

Formation of free choline in brain tissue during in vitro energy deprivation

Free choline and ATP contents were measured in Mongolian gerbil hippocampal slices (tissue) and incubation media (media) during exposure to 30 min of aglycemia, high potassium, anoxia, or ischemia. Changes in choline levels reflected the degree of energy reduction, lower ATP levels being associated with high choline (4-fold increase during exposure to high potassium and anoxia, and 11-fold increase during ischemia). Media (extracellular) choline was particularly affected and increased about twofold during relatively mild energy depletion (e.g., aglycemia), but tissue choline content was less sensitive to energy reduction. A plot of choline vs. ATP levels indicated a nonlinear correlation, and the sharp increase in choline occurred when ATP values fell to about 2.5 nmol/mg of protein. Inhibition of acetylcholine sterase by 10 microM physostigmine during ischemia did not prevent an increase in choline contents but rather enhanced them, indicating that acetylcholine hydrolysis was not the source of free choline. Formation of free choline was Ca2+ independent. These findings suggest the involvement of phospholipase D and phosphatidylcholine hydrolysis in free choline formation during energy stress. The extent of choline formation may be an indicator of the degree of membranal damage, which in turn reflects damage to the metabolic machinery of the cell.     

Probable interaction of sodium divalproex with benzodiazepines.

1. After drug discontinuation and 1 week placebo washout, 12 patients with panic disorders received, for 6 weeks, either placebo or sodium divalproex. During 6 consecutive weeks, alternate medication was given. 2. Severity of panic and anxiety attacks was improved only in patients receiving sodium divalproex as a first medication. 3. Protracted benzodiazepine effects may occur in the dichotomous antipanic activity of sodium divalproex.     

Time-dependent effects of ovarian steroids on tyrosine hydroxylase activity in the limbic forebrain of female rats

Summary In this work, we have studied the time-course of the effects of pharmacological administration of ovarian steroids on tyrosine hydroxylase (TH) activity in the limbic forebrain of ovariectomized rats. Administration of estradiol produced a late decrease in TH activity. This effect was found 24 hours after the last steroid injection, disappearing at 32 hours. It was antagonized by progesterone, since a single injection of this steroid to estradiol-pretreated rats reversed to control values the estradiol-induced decrease. Nevertheless, the administration of progesterone after estradiol treatment caused a short-time decrease in the limbic activity of TH, which was observed 4 hours after the last steroid injection, disappearing subsequently. On the other hand, the administration of progesterone alone produced a biphasic effect, with a reduction at 24 hours, followed by an increase at 32 hours. These effects were only observed in the animals non-treated with estradiol, disappearing with a previous treatment with estrogens. Hence, it can be concluded that both ovarian steroids may affect the limbic TH activity. Thus, estradiol produced a late inhibitory effect on the activity of this enzyme, which was antagonized by progesterone. Administration of the last one to estradiol-treated rats produced a short-time inhibitory effect, whereas its administration to non-treated rats produced a late biphasic effect (inhibition followed by stimulation), which was not observed in estradiol-treated rats.     

Ubiquitin and heat shock protein expression in amyotrophic lateral sclerosis

The expression of two heat shock proteins, HSP72 and p57, in addition to ubiquitin, has been studied immunocytochemically in nine amyotrophic lateral sclerosis (ALS) cases and 10 age-matched controls. HSP72 and p57 antibodies did not identify the characteristic ubiquitin-immunoreactive inclusions present in anterior horn cells in ALS spinal cord. Antibodies to HSP72, but not to p57 or ubiquitin, strongly labelled structures corresponding to polyglucosan bodies in spinal grey matter. Such immunoreactive profiles were more abundant in ALS cases, although they were also present in control material. They were sometimes identified by haematoxylin and eosin and periodic acid Schiff reaction, but were not labeled by phosphotungstic acid haematoxylin or by antibodies to glial fibrillary acidic protein. Although ubiquitin, HSP72 and p57 are stress-induced proteins, they are expressed differently and might therefore have different significance in neuronal degeneration.     

Improved adhesion and proliferation of human endothelial cells on polyethylene precoated with monoclonal antibodies directed against cell membrane antigens and extracellular matrix proteins.

Endothelial cell seeding may improve the patency of synthetic vascular grafts provided that platelet reactivity of nonendothelialized sites is not increased. We have investigated if surface-adsorbed monoclonal antibodies directed against endothelial cell membrane proteins and against extracellular matrix proteins promote the adhesion and proliferation of cultured human endothelial cells, without causing platelet deposition at non-endothelialized sites. Adhesion of endothelial cells onto polyethylene coated with monoclonal antibodies directed against endothelial cell-specific membrane antigens, integrin receptors and glycoprotein CD31 was equal to or higher than adhesion onto fibronectin-coated polyethylene. Endothelial cells did not proliferate on these surface-adsorbed antibodies. However, pre-coating of polyethylene with mixtures of endothelial cell-specific monoclonal antibodies and monoclonal antibodies directed against fibronectin or von Willebrand factor, resulted in relatively high adhesion and optimal proliferation. Platelet reactivity of the polyethylene surface was found to significantly increase after adsorption of fibronectin, endothelial cell-specific monoclonal antibody or its Fc fragments. In contrast, adsorption of F(ab')2 fragments of endothelial cell-specific monoclonal antibody did not promote platelet deposition. Therefore, it is concluded that coating of vascular graft materials with mixtures of F(ab')2 fragments of monoclonal antibodies specifically directed against endothelial cells and against extracellular matrix proteins may be an effective way to both promote the growth of seeded endothelial cells and limit platelet-graft interaction.