Biomedical subjects
Publications and source records attributed to P R Lowenstein.
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The synaptic organization of the projection from the cat striate visual cortex to the posteromedial lateral suprasylvian cortical area (PMLS) was examined. The anterograde tracer Phaseolus vulgaris leucoagglutinin (PHA-L) was iontophorectically delivered into area 17, and anterogradely labeled fibers were revealed in PMLS by means of an immunocytochemical detection method. Most axons and presumptive terminal swellings were found in layers III and IV. The neuronal elements (n = 190) that were postsynaptic to anterogradely labeled boutons were quantitatively analyzed. All anterogradely labeled cortico-cortical boutons (n = 182) established type 1 synapses. The results show that 83% of the postsynaptic targets were dendritic spines, probably belonging to pyramidal cells. Dendritic shafts constituted 17% of the targets. The dendritic shafts postsynaptic to cortico-cortical boutons were studied for the presence of gamma-aminobutyric acid (GABA) with a postembedding immunogold method. Most dendritic shafts (85%) that were tested were found to be GABA-positive, demonstrating that they originate from local inhibitory neurons. Taking into account that most postsynaptic targets were spines and extending the results of the immunocytochemical testing to the total population of postsynaptic dendrites, it was calculated that at least 14% of targets originated from GABA-positive cells. Thus cortico-cortical axons establish direct monosynpatic connections mainly with pyramidal and to a lesser extent with GABAergic nonpyramidal neurons in area PMLS, providing both feedforward excitation and feedforward inhibition to a visual associational area known to be involved in the processing of motion information. The results are consistent with previously demonstrated deficits in physiological properties of neurons in PMLS following removal of cortico-cortical afferents.
The distribution of cholinergic pre- and postsynaptic markers and dopaminergic presynaptic markers was analyzed in the adult human striatum, using quantitative receptor autoradiography. The distribution of the different binding sites indicates that cholinergic and dopaminergic uptake sites display a striosomal organization, with a higher binding site density in the striatal matrix. M1 muscarinic cholinergic receptor distribution parallels the distribution of cholinergic presynaptic markers. Our data show that cholinergic and dopaminergic terminals, and M1 receptors are enriched in the matrix of the adult human striatum.
We have expressed human pre-procorticotrophin-releasing hormone (pre-proCRH) as a fusion protein to beta-galactosidase in Escherichia coli. The chimeric fusion protein was found in insoluble bacterial inclusion bodies. The inclusion bodies were isolated, purified and solubilized, and used as imunogens in rabbits to raise antibodies against the neuropeptide moiety. The antibodies generated were characterized by immunoassays and immunocytochemical techniques. The immunoassay results showed that the recombinant pre-proCRH antibodies cross-reacted with the full-length CRH precursor and several cleavage products derived from it, i.e. CRH(1-41) and CRH(36-41). They did not cross-react with the CRH antagonist CRH(9-41). Extracts of stalk median eminence from various species were also studied. The antibodies cross-reacted with extracts from ovine, bovine, human and rat tissues, exhibiting parallel displacement curves to that of synthetic rat/human CRH(1-41) used as standard. They also cross-reacted with a skin extract of the frog, a species known to contain a CRH-related peptide, i.e. sauvagine, in this tissue. The immunocytochemical studies demonstrated that the antibodies generated against recombinant human pre-proCRH labelled neurones in the rat paraventricular nucleus of the hypothalamus. They exhibited the same pattern of staining as that obtained with an antibody generated against synthetic CRH(1-41). The results indicate that these antibodies can recognize CRH(1-41) or CRH-related molecules in the hypothalamus in situ as well as in tissue extracts from several species. Hence, they will be useful tools in the study of the CRH biosynthetic pathway and its intracellular compartmentalization.
In the fetal and young primate neostriatum, cholinergic and dopaminergic markers show patches of high density surrounded by a lower-density matrix. In the adult, the same markers display the opposite pattern, a lower density in striosomes, surrounded by a higher-density matrix. In order to understand the developmental sequences leading to the adult compartmental organization of the primate neostriatum, a quantitative technique was used to study the ontogeny of pre- and postsynaptic components of cholinergic and dopaminergic neurons in baboon caudate nucleus and putamen. The development of specific uptake mechanisms for choline and dopamine and receptors was studied by means of quantitative autoradiography of the specific binding of [3H]-hemicholinium-3 [( 3H]-HC3) and [3H]-mazindol [( 3H]-MAZ) to the choline and dopamine uptake systems, respectively. [3H]-pirenzepine [( 3H]-PIR) was used to label M1 muscarinic receptors and [3H]-spiroperidol [( 3H]-SPI) was used to label striatal dopamine D2 receptors. Serial sections were used for each ligand to determine the precise anatomical relationships between the binding patterns of the different markers. Our aim was to determine whether the adult striosomal distribution of the binding sites studied was due to 1) a selective decrease in patch/striosomal binding density or 2) a selective increase in matrix binding density. Our studies show that a postnatal decrease in the density of [3H]-HC3 sites in the patch/striosomes and an increase in the matrix density of [3H]-MAZ sites are the primary, but not the sole, changes in the compartmental distribution of these sites leading to the adult striosomal organization of the striatal cholinergic and dopaminergic innervation. D2 receptors follow the general developmental pattern of [3H]-MAZ and [3H]-HC3, changing their density of distribution in both compartments during the developmental period examined. In addition, M1 muscarinic receptors already display their adult pattern in the newborn baboon striatum, and therefore represent one of the first neurochemical makers to adopt its mature organization.
[125I]iodocyanopindolol (ICYP) autoradiography was used to investigate the temporal development and distribution of beta 1 and beta 2 receptors in brains of baboons at ages embryonic day 100 (E100), full-term gestation (El80), and 3 years. In all brain regions examined, with the exception of the hippocampus, binding to beta 1 receptors exceeded that to beta 2 receptors. The highest densities of beta 1 receptors were found in the caudate nucleus, putamen, globus pallidus, substantia nigra, and cerebral cortex; intermediate receptor densities were observed in most nuclei of thalamus, and the lowest concentrations were in the hippocampus. At E100, beta receptors were identified in the striatum, globus pallidus, and thalamus. During maturation, the number of beta 1 receptors declined in cortical areas but increased in the head of the caudate and putamen. Significant differences in the developmental distribution of beta receptors during development were also detected: at E100 and E180 beta 1 receptors appeared as patches in the caudate and putamen, but by 3 years of age they were more homogeneously distributed in both regions; changes also occurred in the distribution of binding within cortical layers. Autoradiograms of [125I]ICYP and [3H]mazindol binding show overlapping patches of labeling in the E180 striatum, suggesting a possible developmental association between beta receptors and dopamine high-affinity uptake carrier sites. This study demonstrates that noradrenergic receptors in the primate forebrain undergo significant developmental reorganization with regional variations.
The role of calcium in the regulation of sodium-dependent high-affinity uptake of choline was assessed in vitro in slices of the rat brain, by measuring the specific binding of [3H]hemicholinium-3 ([3H]HCh-3) and the uptake of [3H]choline. Depolarization with potassium of slices of hippocampus, cortex, or striatum significantly increased the specific binding of [3H]HCh-3 when compared to control slices. However, the observed potentiation of specific binding of [3H]HCh-3 was markedly inhibited by the removal of calcium from the incubation medium in cortex or hippocampus, but not in slices of striatum. Alterations in the uptake of [3H]choline directly paralleled the observed changes in the specific binding of [3H]HCh-3 in striatum of the rat and were unaffected by the reduction of calcium in the incubation medium. The inorganic calcium channel antagonists, cadmium and cobalt, but not magnesium, zinc, manganese or lanthanum, significantly inhibited the 40 mM potassium chloride-induced stimulation of the binding of [3H]HCh-3 in the striatum. Finally, the calcium ionophore A23187 significantly increased the binding of [3H]HCh-3 in slices of striatum, either in the presence or absence of calcium in the bathing medium. This study demonstrates regional differences in the role of extracellular calcium in the regulation of the uptake of choline and suggests the involvement of intracellular release of calcium in the in vitro regulation of the sodium-dependent high-affinity uptake of choline in the striatum.
The effects of depolarizing concentrations of potassium chloride on the modulation of [3H]hemicholinium-3 binding sites and high affinity choline uptake were examined in vitro. When rat striatal slices were incubated in Krebs buffer for 20 min, [3H]hemicholinium-3 binding sites diminished to 60% of binding measured in fresh un-incubated tissue, and remained stable for 60 min. Upon addition of Krebs buffer containing 40 mM KCl, the number of binding sites increased during a 20 min period, and remained stable for 40 min. Changes in [3H]hemicholinium-3 binding sites closely paralleled changes in high affinity choline uptake. Scatchard analysis revealed that changes in binding result from alterations in the number of binding sites (Bmax), and not in the affinity (KD). These results suggest that neuronal depolarization rapidly alters the velocity of choline transport into cholinergic neurons by increasing the number of available carriers.
The developmental distribution of the sodium-dependent high affinity choline uptake (SDHACU) system has been studied in the caudate and putamen of the baboon and of the human by in vitro autoradiography with the ligand, [3H]hemicholinium-3 [( 3H]HCh3). Our results show that [3H]HCh3 binding sites in the newborn baboon and fetal human neostriatum are localized to patches, and then adopt a 'striosome-like' distribution in the juvenile baboon brain. These findings indicate a reorganization of [3H]HCh3 binding site distribution during the ontogeny of the primate neostriatum.
The effects of various drugs known to affect the sodium-dependent high-affinity choline-uptake system (SDHACU) in the brain were examined for their action upon the [3H]hemicholinium-3 [( 3H]HCh-3) binding site, which is associated with the choline carrier. The [3H]HCh-3 binding sites are affected in a similar way to the SDHACU system. Thus, alterations in the velocity of choline-uptake are mediated through changes in the apparent number of available transport sites at cholinergic terminals.
In Alzheimer's disease (AD), there is a loss of presynaptic cholinergic markers in the cerebral cortex, but the nature of cholinergic receptor changes is unclear. In this study, [3H]acetylcholine and [3H]nicotine were used to label nicotinic cholinergic binding sites in cerebral cortical tissues obtained at autopsy from patients with AD and from matched controls. A consistent and severe loss of nicotinic receptors was found in AD.
In a previous work, pinealectomy was found to depress benzodiazepine (BZP) receptor binding in cerebral cortex membranes of rats killed at noon. In order to assess the effect of pineal removal on diurnal variations of BZP binding site concentration and affinity, groups of intact, pinealectomized, or sham-pinealectomized rats (subjected to surgery 2 wk earlier) were killed at six different time intervals during the 24-h cycle. BZP binding was assessed by Scatchard analysis of 3H-flunitrazepam high-affinity binding to cerebral cortex membranes. In intact and sham-pinealectomized rats, a maximum in BZP receptor concentration was found at midnight. Pinealectomy blunted the nocturnal peak of receptor concentration and caused a significant depression of binding site number at noon. No changes in the affinity of the binding sites for the radioligand were detected as a function of time of day or following surgery. In a dose-response experiment for melatonin ability to restore the depressed BZP receptor concentration of cerebral cortex membranes of pinealectomized rats killed at noon, a minimal effective dose of 25 micrograms/kg body weight was obtained. These results further support a link between pineal activity and brain BZP receptors in rats.
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In 3 000g-supernatants of rat pineal homogenates a single population of benzodiazepine (BZP) binding sites with dissociation constant= 97-102 nM and maximal number of sites= 6.5-9 pmoles/mg protein was detected by employing 3H-flunitrazepam (FNZP) as a radioligand. The following order of affinity for several BZP was found (Ki, nM): Ro 5-4864 (8), FNZP (99), clonazepam (7,900) Ro 15-1788 (10,000). Two weeks after bilateral superior cervical ganglionectomy (SCGx) a 18-28% reduction of site number without significant changes in affinity of 3H-FNZP binding was detectable in rat pineal glands. In pineal explants priorly incubated with 3H-norepinephrine, exposure to 0.1-10 microM of Ro 5-4864 or diazepam decreased significantly transmitter release elicited by 80 mM K+, whereas clonazepam did not affect it significantly. At 10 microM-concentrations, Ro 5-4864, diazepam or clonazepam increased pineal melatonin content of explants incubated for 6 h with the drug. In pineal explants of rats subjected to SCGx 14 days earlier, only 10 microM of Ro 5-4864 increased melatonin content significantly to about half of the percent increase detected in innervated glands. These results suggest that BZP decrease transmitter release from pineal sympathetic nerves by acting on peripheral BZP binding sites, an effect which is about 2 orders of magnitude greater than the postsynaptic stimulation of pineal melatonin synthesis.
The subcutaneous injection of the methylxanthine derivative pentoxifylline (3,7, dimethyl-1-(5-oxo-hexyl)-xanthine) was able to induce, 3 h later, a significant reduction of benzodiazepine binding sites in rat cerebral cortex. When tested in vitro, pentoxifylline displaced 3H-flunitrazepam from specific binding sites in a competitive manner. For this effect pentoxifylline was about 10 times more potent than caffeine. When given in two daily injections for 5 days, pentoxifylline brought about a significant elevation in the number of cortical benzodiazepine binding sites. Neither acute nor chronic pentoxifylline treatment modified cortical muscarinic cholinergic binding sites. Pentoxifylline has negligible affinity for cortical muscarinic receptors in vitro. These results suggest that pentoxifylline is able to affect the cortical benzodiazepine receptors differentially, depending on time of drug administration.
The possible existence of pineal progesterone (P) receptors was assessed in 105,000 g supernatants of bovine pineal glands incubated with 3H-P in the presence of 10 microM cortisol. By charcoal adsorption analysis specific binding (defined as the difference between 3H-P binding in the absence and presence of 1,000-fold excess of unlabeled P) attained its maximum after 60 min at 37 degrees C. Binding was abolished by heating the cytosol or by preincubating it with trypsin. A single population of binding sites with a Kd = 1.64 nM and binding site concentration = 34 fmol/mg of cytosol protein was detectable by Scatchard analysis. In competition experiments the B50 progesterone values (nM) were: P (2), 20 alpha-dihydroprogesterone (12), 20 beta-dihydroprogesterone (25), estradiol (158), testosterone (675) and corticosterone (greater than 1,000). After sucrose gradient centrifugation of pineal cytosol incubated with 3H-P a discrete peak of radioactivity in the 7-8 S region of the gradient was observed, and disappeared following incubation with excess unlabeled P. These results indicate that P receptors are present in the bovine pineal gland.
This article discusses the experimental evidence which suggests that the pineal gland and its innervating neurons are useful paradigms for the study of neuroendocrine integrative processes. The obtained results can be summarized as follows: 1) existence of putative receptors for various hormones (estradiol, testosterone, 5 alpha-dihydrotestosterone, progesterone, prolactin) in the mammalian pineal gland; (2) steroid metabolic pattern in the pineal gland resembling other brain areas involved in gonadotrophic regulation; (3) control of pineal estrophilic and androphilic receptors by adrenergic transmitter through beta-adrenergic receptors and at a translational level; (4) denervation supersensitivity of hormone receptors to neurotransmitter; (5) modification of neuronal activity by hormone treatment at ganglionic and preganglionic sites of action; (6) estrophilic binding sites in ganglia; (7) correlation of pineal responsiveness to hormones with activity of sympathetic nerves; (8) modification by hormones of pineal beta-adrenergic mechanisms; (9) dissociation of hormone effects on the pineal gland in those mediated or modulated by changes in afferent neuronal activity and those relatively unaffected by denervation. Collectively these data indicate that neuroendocrine, endocrine-neural and endocrine-endocrine transducing processes occur in the pinealocytes and superior cervical ganglia.