bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of formation of filopodia after axotomy by inhibitors of protein tyrosine kinases
Authors: Goldberg Daniel J. , Wu Da‐Yu
Published: January 2005

The activity of motile protrusions of the growth cone—filopodia, veils, and lamellipodia—is essential for directed growth of a neuronal process. The regulation of the formation of these protrusions is not well understood. Numerous filopodia and veils or lamellipodia form within minutes of transection of an Aplysia axon in culture, as the initial components of growth cones of regenerating neurites. Axotomy, therefore, provides a robust and reliable protocol for analyzing the formation of these protrusions. We evaluated the involvement of protein phosphorylation in the regulation of protrusive activity. Of the inhibitors of protein kinases assayed, only the inhibitors of protein tyrosine kinases—genistein, lavendustin A, herbimycin A, and erbstatin analogue—suppressed the formation of protrusions, as assessed by high magnification video microscopy. These drugs did not work by preventing resealing of the axon, as evident from visual inspection and by the unimpaired effectiveness of genistein or lavendustin in preventing formation of filopodia when applied after resealing. Inhibition of protein tyrosine kinases not only prevented the formation of actinbased protrusions, but also caused deterioration of the actin network underlying the protrusive area of preexisting growth cones. Consistent with an involvement of protein tyrosine phosphorylation in the generation of protrusive structures, immunocytochemistry revealed that aggregates of phosphotyrosine appeared at the margins of the axon, from which protrusions emerge shortly after axotomy. These results suggest a role for protein tyrosine phosphorylation in the formation and maintenance of actin‐based protrusive structures. © 1995 John Wiley & Sons, Inc.
Ca2+ responses to acetylcholine and adenosine triphosphate in the otocyst of chick embryo
Authors: Nakaoka Yoshikazu , Yamashita Masayuki
Published: January 2005

The action of acetylcholine and adenosine triphosphate (ATP) on cytoplasmic Ca2+ concentration ([Ca2+]i) was studied in the otocyst epithelium of embryonic day 3 chicks with Ca2+‐sensitive fluorescence measurements. Increases in [Ca2+]i were evoked by the bath application of acetylcholine (1 μM or higher). The rise in [Ca2+]i was due to the release of Ca2+ from intracellular Ca2+ stores, since the Ca2+ response occurred even in a Ca2+‐free medium. The Ca2+ response to acetylcholine was mediated by muscarinic receptors. Atropine of 1 μM abolisehd the response to 10 μM acetylcholine; muscarine and carbamylcholine (100 μM each) evoked Ca2+ rises. Increases in [Ca2+]i were also evoked by the bath application of ATP (10 μM or higher). The Ca2+ rise by ATP was evoked even in a Ca2+‐free medium. Adenosine (500 μM) did not cause any Ca2+ response. Suramin and reactive blue 2 (200 μM each) completely blocked the Ca2+ response to 500μM ATP. Uridine triphosphate (500 μM) caused comparable Ca2+ responses with those to 500 μM ATP. These results suggested the involvement of P2U purinoceptors. The potentiation of Ca2+ rise was observed when acetylcholine and ATP were co‐applied at submaximal concentrations (10 μM and 100 μM, respectively). We conclude that undifferentiated cells in the otocyst epithelium have CaCa2+ mobilizing systems activated by acetylcholine and ATP. © 1995 John Wiley & Sons, Inc.
Overlapping and differential expression of BIG‐2, BIG‐1, TAG‐1, and F3: Four members of an axon‐associated cell adhesion molecule subgroup of the immunoglobulin superfamily
Authors: Yoshihara Yoshihiro , Kawasaki Miwa , Tamada Atsushi , Nagata Shigekazu , Kagamiyama Hiroyuki , Mori Kensaku
Published: January 2005

Axon‐associated cell adhesion molecules (AxCAMs) play crucial roles in the formation, maintenance, and plasticity of functional neuronal networks. We report here a molecular cloning of a novel AxCAM, BIG‐2. BIG‐2 is a member of TAG‐1/F3 subgroup of the immunoglobulin (Ig) superfamily, with six Ig‐like domains, four fibronectin type III‐like repeats, and a glycosyl phosphatidylinositol‐anchoring domain. Recombinant BIG‐2 protein had a neurite outgrowth‐promoting activity when used as a substrate for neurons in vitro. To survey the spatial expression pattern of BIG‐2 in comparison with other TAG‐1/F3 subgroup members, an in situ hybridization analysis was performed in adult and developing rat brain sections with riboprobes specific for BIG‐2, BIG‐1, TAG‐1, and F3. The four AxCAM transcripts displayed cell type‐specific expression patterns with overlapping and distinct profiles. In adult hippocampus, for example, we observed BIG‐1 mRNA specifically in granule cells of the dentate gyrus, BIG‐2 mRNA highly in the CA1 pyramidal cells, TAG‐1 mRNA predominantly in the CA3 pyramidal cells, and F3 mRNA in neurons in all of these fields. These results suggest that BIG‐2, BIG‐1, TAG‐1, and F3 may play important roles in the formation and maintenance of specific neuronal networks in the brain. © 1995 John Wiley & Sons, Inc.
20‐hydroxyecdysone stimulates proliferation of glial cells in the developing brain of the moth Manduca sexta
Authors: Kirschenbaum Sheila R. , Higgins Mark R. , Tveten Michael , Tolbert Leslie P.
Published: January 2005

The steroid hormone 20‐hydroxyecdysone (20‐HE) controls diverse aspects of neuronal differentiation during metamorphosis in the hawkmoth Manduca sexta. In the present study we have examined the effect of 20‐HE on glial cells of the brain during the metamorphic period. The antennal (olfactory) lobe of Manduca provides an ideal system in which to study effects of hormones on glial cells, since three known classes of glial cells participate in its development, and at least one type is critically important for establishment of normal neuronal morphology. These glial cells, associated with the neuropil, form boundaries for developing olfactory glomeruli as a result of proliferation and migration. We determined whether glial cells proliferate in response to 20‐HE by injecting a pulse of 20‐HE into the hemolymph at different stages of development and monitoring proliferation of all three types of glial cells. Hormone injections at the beginning and end of metamorphic development, when hormone titers are normally low, did not stimulate proliferation of neuropil‐associated glial cells. Injections during the period when hormone titers are normally rising produced significant increases in their proliferation. Injections when hormone titers are normally high were ineffective at enhancing their proliferation. One other class of glial cells, the perineurial cells, also proliferate in response to 20‐HE. Thus, glial proliferation in the brain is under the control of steroid hormones during metamorphic development. © 1995 John Wiley & Sons, Inc.
Profiling of abundant proteins associated with dichlorodiphenyltrichloroethane resistance in Drosophila melanogaster
Authors: Pedra Joao H. F. , Festucci‐Buselli Reginaldo A. , Sun Weilin , Muir William M. , Scharf Michael E. , Pittendrigh Barry R.
Published: January 2005

Dichlorodiphenyltrichloroethane (DDT) metabolism‐based resistance in Drosophila melanogaster is a complex metabolic system associated with the transcription of detoxification related genes, ion transport, lipid and sugar metabolism pathways. However, little is known about the differences regarding the proteome of field‐ and laboratory‐selected resistant Drosophila genotypes. We investigated the impact of DDT resistance in the abundant proteome of field‐ and laboratory‐ selected resistant Drosophila using a two‐dimensional gel electrophoresis DDT reference map. Proteomic profiling was performed in two DDT susceptible genotypes (Canton‐S and 91‐C) and three DDT resistant lines (Rst(2)DDT91‐R, Rst(2)DDTWisconsin and Rst(2)DDTHikone‐R). Protein spots were stained with Coomassie blue and compared using PDQuest software. Selected protein spots were cut out and analyzed using matrix assisted laser desorption‐time of flight mass spectrometry. Querying the NCBInr. 10.21.2003 database with mass spectrometric data yielded the identity of 21 differentially translated proteins in Rst(2)DDT91‐R, Rst(2)DDTWisconsin and Canton‐S representing proteins putatively involved in biochemical pathways such as glycolysis and gluconeogenesis, the pentose phosphate pathway, the Krebs cycle and fatty acid oxidation. We hypothesize that both strategies are aimed to use of the pentose phosphate pathway to increase glucose utilization while Rst(2)DDT91‐R relies primarily on glycolysis to produce reduced NADP and increase DDT detoxification. DDT exposure in Canton‐S induced six proteins, while four proteins were repressed in Rst(2)DDTHikone‐R. Our data suggest that insecticide resistance appears to impact different metabolic pathways in Drosophila genotypes selected with the same pesticide (DDT).
The membrane proteome of Halobacterium salinarum
Authors: Klein Christian , Garcia‐Rizo Carolina , Bisle Birgit , Scheffer Beatrix , Zischka Hans , Pfeiffer Friedhelm , Siedler Frank , Oesterhelt Dieter
Published: January 2005

The identification of 114 integral membrane proteins from Halobacterium salinarum was achieved using liquid chromatography/tandem mass spectrometric (LC/MS/MS) techniques, representing 20% of the predicted alpha‐helical transmembrane proteins of the genome. For this experiment, a membrane preparation with only minor contamination by soluble proteins was prepared. From this membrane preparation a number of peripheral membrane proteins were identified by the classical two dimensional gel electrophoresis (2‐DE) approach, but identification of integral membrane proteins largely failed with only a very few being identified. By use of a fluorescently labeled membrane preparation, we document that this is caused by an irreversible precipitation of the membrane proteins upon isoelectric focusing (IEF). Attempts to overcome this problem by using alternative IEF methods and IEF strip solubilisation techniques were not successful, and we conclude that the classical 2‐DE approach is not suited for the identification of integral membrane proteins. Computational analysis showed that the identification of integral membrane proteins is further complicated by the generation of tryptic peptides, which are unfavorable for matrix assisted laser desorption/ionization time of flight mass spectrometric peptide mass fingerprint analysis. Together with the result from the analysis of the cytosolic proteome (see preceding paper), we could identify 34% (943) of all gene products in H. salinarum which can be theoretically expressed. This is a cautious estimate as very stringent criteria were applied for identification. These results are available under www.halolex.mpg.de.
Modification of nicotinic ganglionic transmission by muscarinic slow postsynaptic potentials in the in vitro rabbit superior cervical ganglion
Authors: Yarosh C. A. , Acosta C. G. , Ashe John H.
Published: January 2005

The influence of slow muscarinic postsynaptic potentials, i.e., the s‐IPSP and s‐EPSP, on synaptic transmission mediated through nicotinic receptors was studied in the superior cervical ganglion of the rabbit. Postganglionic spikes and synaptic potentials were elicited by delivery of conditioning and test stimulus pulses to afferent fibers. When paired stimulus volleys were separated by brief intervals (20–100 msec) or long intervals (1,000–8,000 msec), the population spike elicited by the test stimulus was larger in amplitude than that elicited by the conditioning volley. When paired stimulus volleys were separated by 250–500 msec, the amplitude of the population spike elicited by the test volley was smaller than that elicited by the conditioning stimulus. Gallamine, which selectively blocks the s‐IPSP, reduced the suppression of the test spike which occurred when stimulus IPIs ranged between 250–500 msec. Pirenzepine, which selectively blocks the s‐EPSP, reduced the late facilitation of test postganglionic spikes which occurred with stimulus IPIs greater than 1,000 msec. The non‐selective muscarinic antagonist QNB, produced changes in postganglionic spike amplitude that were similar to the combined effects of gallamine and pirenzepine. The evidence indicates that the s‐IPSP and s‐EPSP modified the excitability state of the ganglionic neurons and subsequent synaptic transmission that was mediated through nicotinic receptors.
Biochemical and behavioral effects of sigma and PCP ligands
Authors: Contreras Patricia C. , Contreras Margarita L. , L. O'donohue Thomas , Lair Cecelia C.
Published: January 2005

The purpose of this study was to examine the binding and behavioral effects mediated by PCP and sigma receptors in the rat. From the radioreceptor assays, it was possible to characterize two binding sites that interact with PCP and sigma ligands. The two sites, a PCP and sigma receptor, could be differentiated based on drug selectivity and potency. In the behavioral assays, MK‐801, which bound preferentially to the PCP receptor, and 1,3‐di‐O‐tolylguanidine, which bound preferentially to the sigma receptor, induced sniffing, rearing, circling, backpedaling, and weaving behavior. These results indicate that there are distinct PCP and sigma receptors that are both involved in mediating stereotyped behavior and ataxia in the rat.
Nuclear protein kinase substrates in AtT‐20 cells: Translocation of a 14 kDa phosphoprotein
Authors: Bishop John F. , Chronwall Bibie M. , Farah John M. , Gehlert Donald R.
Published: January 2005

In this work we characterize some acidic nuclear substrates of protein kinase C (PKC) and cyclic AMP‐dependent protein kinase (PKA), using intact anterior pituitary corticotrophic tumor cells (AtT‐20/D16‐16). It was found that, as in the cytosolic fraction, substrates for both PKC and PKA exist in the nucleus and that changes in the phosphorylation states of a few of these phosphoproteins are mediated by both kinases. One of the phosphoproteins examined, a 14 kDa phosphoprotein (pp14) described previously, exhibited a phorbol‐ester induced translocation from nucleus to cytosol in pulsechase experiments utilizing 35S‐methionine labeling. These results suggest that pp14 may be involved in signal transduction in AtT‐20 cells. Although its identity remains to be determined, a 14 kDa DNA‐binding protein was also seen in nuclear extracts of AtT‐20 cells.
Influx of a choline analog to dog brain measured by positron emission tomography
Authors: Redies Christoph , Diksic Mirko , Collier Brian , Gjedde Albert , Thompson Christopher J. , Gauthier Serge , Feindel William H.
Published: January 2005

The influx of the 11C‐labeled choline analog pyrrolidinocholine into tissue was measured in the brain of three dogs by positron emission tomography (PET). During the first 90 s after the intravenous bolus injection of the tracer, transfer of tracer from plasma to tissue was unidirectional. The influx constant for pyrrolidinocholine into intracranial tissue, Kin, was 0.017 ml/g/min (0.008 SD), and the initial volume of distribution, V0, was 0.08 ml/g (0.03 SD). The influx constant was at least five times larger than the value expected if simple diffusion were to account for tissue uptake. The method presented in this paper can be used to investigate the availability of plasma choline and its analogs to the living human brain and other tissue in degenerative diseases affecting the cholinergic system, and to provide in vivo information on a choline transport system.
Effect of agonist concentration on the lifetime of GABA‐activated membrane channels in spinal cord neurons
Authors: Mathers David Alexander , Wang Yihong
Published: January 2005

The inhibitory neurotransmitter γ‐aminobutyric acid (GABA) hyperpolarizes spinal neurons by activating bicuculline sensitive GABAA receptors coupled to chloride permeable ionic channels in the cell membrane. Single channel measurements using outside‐out membrane patches from cultured mouse spinal neurons showed that there is a large excess of brief openings of these channels, compared to the number predicted from a simple, two‐state model of channel function. The cumulative open time distributions for channel openings were well fit by the sum of two exponential terms, a fast component with amplitude Nf and time constant TAUf, and a slow term with amplitude Ns and time constant TAUs. These kinetics could arise if monoliganded GABAA receptors triggered an open state less stable than that induced by the biliganded receptor. A prediction of this hypothesis is that the ratio Nf/Ns should decline in an approximately linear fashion with increasing doses of GABA. This was found to be the case, as Nf/Ns declined by a factor of 4.8 on increasing the GABA dose from 0.5 μM to 5 μM. Application of 1.25 μM GABA produced an intermediate value of Nf/Ns, as predicted. In contrast the parameters TAUf and TAUs were not significantly influenced by GABA concentration. Spontaneous, bicuculline sensitive currents were seen in some patches. These events resembled currents triggered by low doses of exogenous GABA, with regard to the values of Nf/Ns, TAUf and TAUs. These results suggest that the spontaneous currents are triggered by endogenous GABA molecules, present in the culture environment at a concentration of 0.5 μM or less.
Effects of chronic treatment with selective and nonselective antagonists on the subtypes of dopamine receptors
Authors: McGonigle Paul , Boyson Sally J. , Reuter Susan , Molinoff Perry B.
Published: January 2005

The effects of chronic blockade of D‐1, D‐2, or both subtypes of dopamine receptors on the densities and properties of the D‐1 and D‐2 subtypes of dopamine receptors were measured in rat brain. Animals were treated with 14 daily injections (i.p.) of the D‐1‐selective antagonist SCH‐23390, the D‐2‐selective antagonist sulpiride, the nonselective antagonist fluphenazine, or vehicle. Serial 32‐μm horizontal sections that included the caudate putamen were cut and alternately assigned to assays for D‐1 or D‐2 receptors. D‐1 receptors were labeled with 3H‐SKF‐83566 or 3H‐SCH‐23390, and D‐2 receptors were labeled with 3H‐spiroperidol. Scatchard analysis was performed on the saturation data measured in the head of the caudate putamen to obtain estimates of receptor density. As expected, administration of the D‐1‐selective ligand SCH‐23390 resulted in an increase in the density of D‐1 receptors by approximately 47% and had no significant effect on the density of D‐2 receptors. Similarly, administration of the D‐2‐selective ligand sulpiride resulted in an increase in the density of D‐2 receptors by 25% and had no significant effect on the density of D‐1 receptors. Thus the subtypes of dopamine receptors appear to be independently regulated after selective blockade. In contrast to the effects observed with selective antagonists, the results obtained with fluphenazine were more complex. Administration of the relatively nonselective antagonist fluphenazine resulted in an increase in the density of D‐2 receptors by 51% but had no significant effect on the density of D‐1 receptors. One explanation for this result is that up‐regulation of D‐2 receptors inhibits the regulation of D‐1 receptors, suggesting that the regulation of subtypes of dopamine receptors is not entirely independent. In support of this hypothesis, coadministration of sulpiride with SCH‐23390 attenuated the ability of SCH‐23390 to up‐regulate D‐1 receptors. To determine whether this phenomenon is observed in other brain regions, a second group of animals was chronically treated with selective and nonselective antagonists as described above, and coronal sections were cut at multiple levels of the brain. Receptors were labeled with a single concentration of either 3H‐SCH‐23390 or 3H‐spiroperidol, and the binding of the radioligands in 19 discrete brain regions was measured. The effects of these treatments on D‐1 and D‐2 receptors in most of these regions were similar to those described above for the caudate putamen.
In vivo quantification of blood‐brain transfer and binding of [125I]HEAT, an α1‐adrenoceptor antagonist
Authors: Dyve Suzan , Gjedde Albert , Diksic Mirko , Sherwin Allan , Hakim Antoine
Published: January 2005

The uptake and binding constants of [125I]iodo‐2‐[β‐(4‐hydroxyphenyl)‐ethyl‐amino‐methyl]tetralone ([125I]HEAT) in rat brain were determined in vivo. The initial clearance of the radioligand from blood to brain, K1, was calculated from the initial uptake of the radioligand; it averaged 0.21 ± 0.01 (SD) ml g−1 min −1, consistent with an initial extraction of 25% (i.e., one‐quarter of the blood flow). The most strongly binding regions included the olfactory bulb, thalamic nuclei, medial geniculate body, and cerebral cortical layers. We identified saturable, specific binding in frontal cortex layers 1, 5a, and 5c (motor region), frontal cortex layers 3 + 4, ventral thalamic nuclei, medial geniculate body, striatum, cerebellum, and olfactory bulb. Addition of unlabeled ligand depressed binding in all regions to the same low level (partition coefficient) of 0.8 ml g−1. Displacement of [125I]HEAT binding by unlabeled HEAT yielded a global affinity constant (KDVd) of 34 ± 8 pmol g−1 and receptor densities (Bmax) that varied from 50 pmol g−1 in cerebellar cortex and caudate nucleus to 200 pmol g−1 in the region of highest specific binding, the medial geniculate body.
Inward rectifier produced by Xenopus oocytes injected with mRNA extracted from carp olfactory epithelium
Authors: Yoshii Kiyonori , Kurihara Kenzo
Published: January 2005

Ionic channels encoded by mRNA extracted from carp olfactory epithelium were investigated by injection into Xenopus laevis oocytes. The oocytes expressed an inward rectifier K+‐channel, as detected under two‐electrode voltage clamp conditions. The results were as follows. An inactivating inward current appeared on hyperpolarization and increased with increasing extracellular K+ concentrations. The 0 current potentials plotted as a function of log [K+]0 in the range between 2 to 20 mM K+ fell on a straight line, with a slope of 58 mV per tenfold change in K+ concentration, indicating that the current carrier is K+. Chord conductances reached saturation levels on extreme hyperpolarization. The chord conductances at the saturation levels were 35.7, 22.5, and 13.4 μSec in 20, 10, and 5 mM extracellular K+, respectively. Extracellular application of 0.1 mM Cs+ or 0.1 mM Ba2+ blocked the inward current in 2 mM K+, Whereas 1 μM TTX or 0.3 mM Cd2+ did not affect the inward current. Inactivation of the inward currents, which became clear on extreme hyperpolarization, was suppressed with decreasing extracellular Na+ concentration. The present results suggest that carp olfactory epithelium is rich in the inward rectifier and is an excellent source of mRNA for cloning cDNA coding the inward rectifier.
Microinjection of cholecystokinin into the rat ventral tegmental area potentiates dopamine‐induced hypolocomotion
Authors: Crawley Jacqueline N.
Published: January 2005

Cholecystokinin, (CCK) 1‐400 ng, significantly potentiated the hypolocomotion induced by dopamine, when simultaneously microinjected bilaterally into the ventral tegmental area (VTA) of rat brain. Within this dose range, CCK had no effect alone on ambulatory locomotion. Topographical analysis indicated that the modulatory effect of CCK was greatest in the central and caudal regions of the VTA, and absent at sites lateral, dorsal, rostral, and caudal to the VTA. Pharmacological analysis indicated that both unsulfated CCK octapeptide (100 ng) and the C‐terminal tetrapeptide of CCK (400 ng) potentiated dopamine‐induced hypolocomotion in a manner identical with sulfated CCK octapeptide (100 ng). Proglumide, an antagonist of the peripheral‐type CCK receptor, did not block the potentiating actions of CCK, at doses of proglumide up to 500 mg/kg i.p., or 100ng into the ventral tegmental area. L‐364,718, an antagonist of the peripheral‐type CCK receptor with lesser affinity for the central‐type CCK receptor, blocked the potentiating actions of CCK at relatively high doses of L‐364,718 (1‐10 mg/kg i.p.). These findings suggest that CCK acts as a facilitatory modulator of dopamine at a central‐type CCK receptor on the A10 cell bodies.
Short‐ and long‐term survival of large neurons in the excitotoxic lesioned rat caudate nucleus: A light and electron microscopic study
Authors: Roberts Rosalinda C. , Figlia Marian Di
Published: January 2005

Large striatal neurons are spared in caudate tissue from postmortem brain of patients with Huntington's disease (HD) and in the rat caudate lesioned with excitotoxins at short postlesion intervals. In order to determine the survival of large neurons and other effects of excitotoxicity at longer postlesion intervals the rat caudate nucleus was examined 2,7, and 30 weeks after intrastriatal injections of the excitotoxin, quinolinic acid. The caudate nucleus diminished in size progressively up to 30 weeks postlesion due to (1) shrinkage and compacting of the lesion zone and (2) reduction in area of intact caudate, apparently due to gradual loss of the remaining caudate neurons.

In Nissl‐stained sections of the lesion zone where total neuronal density was less than 5% of contralateral control, large neurons were present at all postlesion intervals, forming 38‐58% of the remaining neurons. Unexpectedly, a fivefold reduction in the number of large neurons was observed between 2 and 30 weeks postlesion. Also, at 7 and 30 weeks postlesion most of the large neurons were confined to the peripheral region of the lesion. At all postlesion intervals, large neurons retained ultrastructural integrity and some synaptic inputs despite the severe disruption of the surrounding neuropil.

Surrounding the lesion zone was a transition zone which exhibited a decrease in total neuronal density to 53‐74% of control. In this region the density of large neurons was not diminished, and the proportion of large neurons was elevated in comparison to that of controls at all postlesion intervals.

Findings suggest that following excitotoxic lesion of the caudate nucleus there are marked differences between short‐ and long‐term postlesion intervals in the survival and distribution of large neurons. We speculate that an imbalance in the synaptic connections with other caudate neurons leads to the persistent loss of large neurons in the lesion zone at long postlesion intervals. A transition zone surrounding the lesion, where cell loss is less severe than in the lesion zone, exhibits features more characteristic of the neuropathology of HD.
Human ventral mesencephalic xenografts to the catecholamine‐depleted striata of athymic rats: Ultrastructure and immunocytochemistry
Authors: Mahalik Thomas J. , Ströumberg Ingrid , Gerhardt Greg A. , Granholm Ann‐Charlotte , Seiger Åke , Bygdeman Marc , Olson Lars , Hoffer Barry J. , Finger Thomas E.
Published: January 2005

On the basis of animal studies, grafts of fetal human dopaminergic cells have been suggested as a therapy for Parkinson's disease. The purpose of this study was to characterize the ultrastructure and immunocytochemistry of human ventral mesencephalic xenografts placed into the catecholamine‐depleted striata of athymic “nude” rats.

Human fetal tissue was obtained from tissue fragments derived from elective abortions during the first trimester of pregnancy. Small pieces of the basal mesencephalon were grafted into the catecholamine‐depleted striata of four athymic nude rats. The rats were allowed to survive from 3 to 6 months after grafting; following fixation, the striatal tissue containing the grafts was labeled with antibodies against tyrosine hydroxylase and serotonin.

Immunocytochemistry revealed tyrosine‐hydroxylase‐like‐immunoreactive (THLI) and serotoninlike‐immunoreactive (5HTLI) cell bodies within the human grafts. Both 5HTLI and THLI fibers crossed the graft‐host interface and innervated the previously lesioned striatum. Both types of fibers also entered the host cortex from the adjacent human graft.

At the ultrastructural level, THLI and 5HTLI fibers and synaptic terminals were observed in the host neuropil. THLI and 5HTLI dendrites and axon terminals were also observed in the neuropil of the grafts themselves. THLI axon terminals are not normally present in the substantia nigra. The results of our study indicate that human xenografts can survive in the neuropil of the host striatum and form morphologically appropriate synapses within the host brain.
Further evidence demonstrating that N‐methyl‐D‐aspartate and kainate activate distinct ion channels
Authors: Fong Tung Ming , Davidson Norman , Lester Henry A.
Published: January 2005

Several excitatory amino acid receptors encoded by rat brain mRNA were expressed in Xenopus oocytes. Experimental protocols using an open channel blocker (MK‐801) were designed to test the common receptor‐channel hypothesis in which N‐methyl‐D‐aspartate (NMDA) and kainate activate the same ion channel but induce different open channel conformations with different ionic permeabilities. The present data demonstrate that NMDA exposes previously trapped MK‐801 molecules to the transmembrane field and accelerates their dissociation from the channel at positive potentials, while kainate lacks this effect, Therefore, kainate does not activate the same ion channel as NMDA does. Furthermore, differential inhibition of the NMDA response or the kainate response by the competitive antagonists D‐2‐amino‐5‐phosphonopentanoic acid (D‐AP5) and 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX) indicates that NMDA and kainate do not share the same binding site. Thus, these several lines of evidence demonstrate that two distinct receptor‐channels are activated by NMDA and kainate, respectively.
cAMP increases the rate of GABAA receptor desensitization in chick cortical neurons
Authors: Tehrani Mohammad H. Jalilian , Hablitz John J. , Barnes Eugene M.
Published: January 2005

During prolonged application of GABA to cultured neurons from the chick embryo cerebrum, whole‐cell voltage‐clamp recordings show a decline in GABA‐gated chloride currents due to desensitization. At a holding potential of –60 mV, desensitization can normally be described as a single exponential process with a time constant (τ) of 7–10 sec at a GABA concentration of 100 μM. After exposure to 50 μM forskolin, the peak amplitude of the GABA‐induced currents declined and a fast component of desensitization (τ = 0.92 sec) appeared, whereas the slow component was essentially unchanged. This effect of forskolin was reversible after washing. Similar, although less robust effects were produced by incubation with 8‐Br‐cAMP, but not by 1,9‐dideoxyforskolin. The desensitization process could also be measured by the GABA‐dependent uptake of 36Cl− into the cultured neurons. A 20‐sec incubation with 10 μM GABA in physiological saline produced a 29% inhibition of subsequent GABA‐gated 36Cl− uptake in the presence of 40 mM K+. This inhibition was increased to 64% by the addition of 100 μM forskolin to the preincubation medium. This effect of forskolin was prevented by 100 μM 2′,5′‐dideoxyadenosine, an inhibitor of adenylate cyclase. A similar acceleration of desensitization was produced by 0.5 mM 8‐Br‐cAMP or by 0.5 mM isobutylmethylxanthine, but these compounds themselves failed to produce desensitization in the absence of exogenous GABA. In the presence of GABA, cAMP analogs were effective in the order 8‐(4‐chlorophenylthio)‐cAMP > 8‐Br‐cAMP > N6, 02 ‐dibutyryl‐cAMP. The results suggest that a cAMP‐dependent phosphorylation converts the GABA receptor into a more rapidly desensitizing form.
Apparent absence of serotonin1B receptors in biopsied and post‐mortem human brain
Authors: Martial Johanne , Lal Samarthjia , Dalpé Michel , Olivier Andre , De Montigny Claude , Quirion Remi
Published: January 2005

In order to determine if post‐mortem delays could account for the apparent absence of 5‐HT1B sites reported in human brain, 5‐HT1B binding parameters were determined in fresh and fresh‐frozen cortical biopsies and compared to results obtained in post‐mortem tissues. Binding parameters and in vitro receptor autoradiography were performed by using two different ligands which have been shown to label 5‐HT1B sites, namely [3H]5‐HT, in presence of 100 nM 8‐OH‐DPAT, and [125I]cyanopindolol, in presence of 10 μM (–)isoproterenol. No specific binding was detected with [125I] cyanopindolol in either fresh cortical biopsies or post‐mortem tissues, suggesting that the apparent absence of 5‐HT1B receptor sites reported earlier was not related to long post‐mortem delays. Some specific labelling was seen with [3H]5‐HT, in presence of 8‐OH‐DPAT. This binding, which is unlikely to be to the 5‐HT1B type, could represent labelling to the 5‐HT1D or 5‐HT1E sites (Herrick‐Davis and Titeler: J. Neurochem., 50:1624–1631, 1988; Peroutka: Trends Neurosci., 11:496–500, 1988; Titeler and Herrick‐Davis: Soc. Neurosci. Abst., 14:553, 1988; Waeber et al: Neurosci. Lett., 88:11–16, 1988).
Ontogeny of oxytocin receptors in rat forebrain: A quantitative study
Authors: Shapiro Lawrence E. , Insel Thomas R.
Published: January 2005

The ontogeny of oxytocin receptors in rat forebrain was studied using the selective oxytocin receptor antagonist 125I‐d(CH2)5[Try(Me)2, THR4, Tyr‐NH29]OVT ([125I]‐OTA). With in vitro receptor autoradiography, binding was noted on the first postnatal day in dorsal subiculum and thalamus. On postnatal days 5–18, intense labeling was evident in posterior cingulate cortex, dorsal subiculum, lateral septum and the CA1 subfield of hippocampus. Of these regions only the lateral septum expressed oxytocin receptors in adult brain. Competition studies on coronal sections through posterior cinguulate, septum, and dorsal subiculum at P10 demonstrated that transient binding sites in these areas were indeed oxytocin selective (OXY>AVP>V1V2). Results of saturation studies on cingulate membranes from 10‐day‐old pups agreed favorably with previous reports of the kinetics of [125I]‐OTA binding to adult oxytocin receptors (Kd = 0.1 nM in P10 cingulate cortex vs. 0.07 nM for adult ventral subiculum). In contast to these evanescent developmental sites, oxytocin receptors in the bed nucleus of the stria terminalis and the ventromedial nucleus of the hypothalamus only appeared in adulthood, presumably in response to the surge of gonadal steroids at puberty.
Role of lipid solubility in the interaction of drugs with the N‐methyl‐D‐aspartate receptor
Authors: Reynolds Ian H. , Rush Elizabeth A.
Published: January 2005

We have examined the hypothesis that phenothiazines and tricyclic antidepressans interact with the N‐methyl‐D‐aspartate (NMDA) receptor bu mimicking the action of Zn2+. Using [3H]MK801 binding to well‐washed rat brain membranes, we found a disparity between the concentrations of drug required to inhibit [3H]MK801 from its binding site. These data suggest that the Zn2+ site is probably not the principle site by which tricyclic drugs inhibit NMDA receptor responses. To determine whether the effects to tricyclic drugs on the dissociation of [3H]MK801 could be explained simply by membrane stabilization, we examine dthe effect of a series of alcohols. Ethanol. isopropanol. bytanol, hexanol, and heptanol all inhibited [3H]MK801 binding. However, with the exception ot heptanol, none of the molecules altered the dissociation rate. Thus, while lipid solubility may be an important factor underlying the interactions the interactions of some drugs with the NMDA receptor, it is not sufficient to explain the effects of tricyclic antidepressants and phenothiazines in this system.
Nigrostriatal lesion alters neurophysiological responses to selective and nonselective D‐1 and D‐2 dopamine agonists in rat globus pallidus
Authors: Carlson Joanne H. , Bergstrom Debra A. , Demo Susan D. , Walters Judith R.
Published: January 2005

The effects of the selective D‐1 dopamine agonist SKF 38893, the selective D‐2 agonist quinpirole, and the nonselective D‐1/D‐2 agonist apomorphine on spontaneous activity of globus pallidus neurons were compared in normal control rats and rats with unilateral 6‐hydroxydopamine induced lesions of the nigrostriatal pathway. In control, unlesioned rats, SKF 38393 (0.4 and 10 mg/kg, i.v.) caused no significant net change in the activity of globus pallidus neurons, although some individual cells showed significant increases of decreases in discharge rates following 10 mg/kg SKF 38393 administration. In animals with unilateral 6‐hydroxydopamine induced lesions, SKF 38393 caused greater increases and decreases in the discharge reates of a larger percentage of pallidal cells recorded on the ipsilateral side than in control, unlesioned animals. These rate changes were effectively reversed by the D‐1 antagonist SCH 23390, but not by the D‐2 antagonist YM‐09151–2. Quinpirole (0.3 mg/kg, i.v.) produced modest rate increases in control, unlesioned animals and significantly larger rate increases in nigrostriatal lesioned animals. YM‐09151–2, but not SCH 23390, effectively reversed quinpirole's effects in the lesioned animals. As previously reported, the nonselective D‐1/D‐2 agonist apomorphine (0.3 mg/kg, i.v.) produced large increases in discharge rates of pallidal cells in control, unlesioned rats. In contrast, in nigrostriatal lesioned rats, the discharge rates of some ipsilateral pallidal neurons were markedly increased, others were decreased, and some were unaffected following apomorphine administration. The dopamine antagonist spiroperidol partially to fully reversed these rate changes. In summary, apomorphine's neurophysiological profile appears to be an exaggeration of the D‐1 agonist profile in the globus pallidus of these lesioned animals. The degree of change observed after apomorphine administration is consistent with results from other studies that have indicated that a synergistic interaction between effects triggered by stimulation of the two receptor subtypes can occur in these animals, as in control, unlesioned animals. However, these results further show that in rats with unilateral nigrostriatal lesions, the denervated dopamine receptors or the processes they mediate are altered so that they no longer have the requirement seen in controls for concurrent stimulation of the complementary dopamine receptor subtype for expression of the selective agonist effects.
The Akaike information criterion (AIC) is not a sufficient condition to determine the number of ion channel states from single channel recordings
Authors: Liebovitch Larry S. , Tóth Tibor I.
Published: January 2005

The Akaike information criterion (AIC) and other criteria that trade off the goodness‐of‐fit against the number of parameters have been used to determine the number of ion channel states from single channel recordings. We show that, in general, such criteria are not valid. This is illustrated by an elementary example in which the AIC yields an incorrect result. The implications of these findings for the discrimination of different kinetic models of ion channels are discussed.
Studies on the coexistence of substance P with other putative transmitters in the nodose and petrosal ganglia
Authors: Helke C. J. , Niederer A. J.
Published: January 2005

Visceral afferent neurons of the nodose and petrosal ganglia are immunoreactive (ir) for many neurotransmitters [e.g., substance P (SP), neurokinin A (NKA), calcitonin gene‐related peptide (CGRP), and dopamine (tyrosine hydroxylase‐ir; TH)]. Coexistence of SP‐ir with NKA‐, CGRP‐, or TH‐ir was studied in individual neurons of the rat ganglia using fluorescene immunocytochemistry. SP‐ and NKA‐ir were present in equal numbers of cells and were consistently colocalized. SP‐ and CGRP‐ir were found to be similarly distributed in scattered cells, concentrated mostly in the rostral pole of the nodose ganglion and in the petrosal ganglion. SP‐ir completely coexisted with CGRP‐ir. However, there was at least twice the number of CGRP‐ir neurons as SP‐ir neurons, and thus CGRP‐ir neurons that did not contain SP‐ir were also present. In contrast, SP‐ and TH‐ir had different distributions in both the nodose and the petrosal ganglia. SP‐ir was located in the more rostral regions of both the nodose and petrosal ganglia, whereas TH‐ir was detected throughout the entire nodose ganglion and only in the most caudal region of the petrosal ganglion. There was no coexistence of SP‐ and TH‐ir. These data demonstrate the differential localization and coexistence of putative transmitters in visceral sensory neurons in the nodose and petrosal ganglia.
Regrowth of damaged neurosecretory axons to fenestrated vessels of implanted peripheral tissues
Authors: Kadota Yoshiaki , Pettgrew Karen D. , Brightman Milton W.
Published: January 2005

A major target of neurosecretory axons (NSA) is the bassal lamina around fenestrated blood vessels (FBV) in the neural lobe of the pituitary gland. We have posed the question of whether there is nerurovascular specificity. Do mature, regenerating NSA terminate selectively on the FBV of the neural lobe compared with the FBV of other tissues that normally are not innervated by NSA? Three types of tissue were transplanted between inbred Fisher rats. Fragments, about 1 mm3, of pineawl, adrenal medulla, and neural lobe wwere grafted bilaterally to the hypothalamic, retro‐chiasmatic area, which includes bundles of NSA from supraoptic and paraventricular neurosecretrory nuclei exclusively, but no FBV. Two and 4 weeks later, the grafts were prepared for the immunohistochemical localization of NSA and for electron micaroscopy. NSA‐FBV proximity was measured, and the number of NSA, FBV, and of NSA‐FBV associations was counted per surface area of each graft. Regenerating NSA can associate as closely with FBV of other tissues as they can with the FBV of the neural lobe. There does not appear to be specificity with respect to the closeness of association between neurosecretory terminals and fenestrated capillaries. However, the number of these associations is greater in neural lobe grafts than in adrenal or pineal grafts at 4 weeks. The number of FBV is also greatest, in neural lobe grafts at this time, an increase that would provide a greater opportunity for NSA‐FBV associations.
Monocular deprivation alters the development of synaptic structure in the ectostiatum of the zebra finch
Authors: Nixdorf Barbara E.
Published: January 2005

The effects of monocular deprivation, beginning at hatching, were examined in the neuropil of ectostriatum, a visusal telencephalic projection area in birds. The Volume of ectostriatum, the number of synapses and subsynaptic features like the presynaptic terminal size and the length of the postsynaptic contact zone were quantified in juvenile (20 d) and adult (100 d) zebra finches. Monocular deprivation affects almost all of the parameters mentioned above in juvenile birds, but only one (i.e., the size of presynaptic terminals) is permanently altered in adulthood. Both hemispheres are affected in juvenile birds with respect to the volume of ectostriatum, the length of synaptic contact zones and the presynaptic terminal size when compared to normal values. In normal birds the number of synaptic contacts is established at 20 days and remains fairly constant at 100 days. In experimental birds there is an increase in synapse number during this time period. However, no interhemispheric differences or differences compared to normal animals could be identified. The presynaptic terminals in experimental birds are smallercompared to normal values in young (25% for the deprived side; 19% for the non‐deprived side) and adult (13% for the deprived side; 11% for the non‐deprived side) animals. The only permanent effect caused by monocular deprivation in the ectostriatum is characterized by smaller presynaptic terminals. It is surprising that the tectofugal pathway that is believed to be mostly ipsilateral is not very vulnerable to monocular deprivation in adult animals. It is even more surprising that the deprivation effects are seen on both sides of the brain. The implications of this plasticity will be discussed in this paper.
Cyclic AMP‐dependent protein phosphorylation in the rat anterior pituitary
Authors: Cain Scott T. , Pryor James Cliff , Nemeroff Charles B.
Published: January 2005

The activation of cyclic adenosine 3′5′‐monophosphate (cAMP)‐dependent protein kinases has been implicated as an integral mechanism in stimulus‐secretion coupling in the anterior pituitary. Therefore, we have investigated phosphorylation of endogenous protein substrates both in the presence and absence of cAMP in cell‐free extracts of the rodent anterior pituitary. Specific phosphoprotein substrates in the rat anterior pituitary, which are phosphorylated by a cAMP‐dependent protein kinase in vitro, were identified. Cyclic AMP potentiated the phosphorylation of proteins with apparent molecular weights of 85,000, 77,000, 63,000, 39,000, and 33,000 as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE). Proteins with apparent molecular weights of 124,000, 93,000, 48,000, and 43,000 were phosphorylated only in the presence of cAMP and not in the basal condition. The results highlight edogenous protein substrates that may potentially be involved in cAMP‐dependent stimulus‐secretion coupling in the anterior pituitary.
Ultrastructural analysis of choline acetyltransferase‐immunoreactive sympathetic preganglionic neurons and their dendritic bundles in rat thoracic spinal cord
Authors: Markham Jeffrey A. , Vaughn James E.
Published: January 2005

We have used a monoclonal antibody against choline acetyltransferase (ChAT) to aid in the identification of sympathetic preganglionic neurons (SPNs) and to examine their ultrastructure in rat thoracic spinal cord. The clusters of ChAT‐immumnoreactive (ChAT‐IR) preganglionic cell bodies and their distinctive bundles of dendrites give rise to a ladder‐like appearance in horizontal light microscopic sections. This organization also produced a characteristic appearance of preganglionic neuropil when viewed electron microscopically. The intermediolateral (IML) nucleus contained numerous rostrocaudally oriented ChAT‐IR dendrites extended between the IML and the central autonomic region. Both the ChAT‐IR dendrites and somata of preganglionic neurons were intimately associated with astroglial processes. The cell bodies were typically covered over a large proportion of their surfacce by a thin astrocytic sheath, and this was associated with a paucity of axon terminals forming axosomatic synapses. Instead, the vast majority of synapses upon SPNs were of the axodendritic type. The most frequently observed type of axon terminal contained numerous round, clear vesicles along with several dense‐core vesicles (DCVs). In addition, some boutons contained a predominance of DCVs. Serial section analysis revealed that these apparently diverse morphological classes of synaptic boutons may simple represent variability of structure throughout a single terminal, with a greater proportion of DCVs being located distal to the synaptic specialization and a greater number of round, clear vesicles being present adjacent to the synapse. Analysis of the dendritic bundles revealed that individual dendrites, like the cell bodies, were often isolated from each other and the surrounding neuropil by astrocytic processes. This arrangement usually was interrupted only at regions of synaptic contact where astrocytic processes surrounded the synaptic complex as a whole. Thus, astroglial ensheathment of SPNs seems designed to minimize cross‐talk between the bundled dendrites, as well as to isolate or segregate the diverse afferent inputs known to impinge on these cells.