bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of choline acetyltransferase/iacZ fusion gene expression in putative cholinergic neurons of drosophila melanogaster
Authors: Kitamoto Toshihiro , Ikeda Kazuo , Salvaterra Paul M.
Published: January 2005

We have analyzed the distribution of putative cholinergic neurons in whole‐mount preparations of adult Drosophila melanogaster. Putative cholinergic neurons were visualized by X‐gal staining of P‐element transformed flies carrying a fusion gene consisting of 5′ flanking DNA from the choline acetyltransferase (ChAT) gene and a lacZ reporter gene. We have previously demonstrated that cryostat sections of transgenic flies carrying 7.4 kb of ChAT 5′ flanking DNA show reporter gene expression in a pattern essentially similar to the known distribution of ChAT protein. Whole‐mount staining of these same flies by X‐gal should thus represent the overall distribution of ChAT‐positive neurons. Extensive staining was observed in the cephalic, thoracic, and stomodeal ganglia, primary sensory neurons in antenna, maxillary palps, labial palps, leg, wing, and male genitalia. Primary sensory neurons associated with photoreceptors and tactile receptors were not stained. We also examined the effects of partial deletions of the 7.4 kb fragment on reporter gene expression. Deletion of the 7.4 kb fragment to 1.2 kb resulted in a dramatic reduction of X‐gal staining in the peripheral nervous system (PNS). This indicates that important regulatory elements for ChAT expression in the PNS exist in the distal region of the 7.4 kb fragment. The distal parts of the 7.4 kb fragment, when fused to a basal heterologous promoter, can independently confer gene expression in subsets of putative cholinergic neurons. With these constructs, however, strong ectopic expression was also observed in several non‐neuronal tissues. © 1995 John Wiley & Sons, Inc.
Identification of the border between fibronectin type III homologous repeats 2 and 3 of the neural cell adhesion molecule L1 as a neurite outgrowth promoting and signal transducing domain
Authors: Appel Frank , Holm Jürgen , Conscience Jean‐François , von Bohlen Friedrich , Halbach  , Faissner Andreas , James Peter , Schachner Melitta
Published: January 2005

To determine the domains of the neural cell adhesion molecule L1 involved in neurite outgrowth, we have generated monoclonal antibodies against L1 and investigated their effects on neurite outgrowth of small cerebellar neurons in culture. When the 10 antibodies were coated as substrate, only antibody 557.B6, which recognizes an epitope represented by a synthetic peptide comprising amino acids 818 to 832 at the border between the fibronectin type III homologous repeats 2 and 3, was as efficacious as L1 in promoting neurite outgrowth, increasing intracellular levels of Ca2+, and stimulating the turnover of inositol phosphates. These findings suggest that neurite outgrowth and changes in these second messengers are correlated. Such a correlation was confirmed by the ability of Ca2+ channel antagonists and pertussis toxin to inhibit neurite outgrowth on L1 and antibody 557.B6. These observations indicate for the first time a distinct site on cell surface‐bound‐L1 as a prominent signal‐transducing domain through which the recognition events appear to be funneled to trigger neurite outgrowth, increase turnover of inositol phosphates, and elevate intracellular levels of Ca2+. © 1995 John Wiley & Sons, Inc.
Conditions for the primary culture of eye imaginal discs from Drosophila melanogaster
Authors: Li Chinglu , Meinertzhagen I. A.
Published: January 2005

We have established a primary culture system for Drosophila eye imaginal discs. With this system, we were able to obtain neurite outgrowth from intact eye discs, eye disc fragments, and dissociated eye imaginal disc cells. Immunoreactivity to antibody 24B10 indicates that these extending neurites are photoreceptor axons. Three culture media were tested for their ability to support the survival of and neurite extension from eye disc fragments in vitro at 23°C. These, with supplements, were: five parts of Schneider's Drosophila medium with four parts of basal Eagle's medium (“4+5”); Leibovitz's L‐15 medium (L‐15); and Shields and Sang's M3 modified medium (MM3). We obtained the best results with MM3 supplemented with 2% fetal bovine serum (FBS). Eye disc fragments survived in this medium for at least 20 days. Pigmentation in the nonphotoreceptor pigment cells in cultures from the prepupa required the presence of 20‐hydroxyecdysone (20‐HE) (1 μg/ml), whereas neurite outgrowth was seen in the absence of 20‐HE. Donor animals had to fall within a range of ages to obtain appropriate eye disc differentiation in vitro. Eye discs from 5‐h pupae (P+5) or older commenced ommachrome synthesis in vitro, in a temporal sequence close to that found in vivo, whereas the in vitro synthesis of this pigment was delayed in eye discs from younger flies. Average neurite length was not affected by age among pupae younger than P+5; but neurite outgrowth from P+24 was scarce, probably because by this time photoreceptor axons had already grown in vivo and were severed and unable to regenerate in vitro. Eye discs taken from third instar larvae or white prepupae continued their mitotic activity in vitro. Together with the advance of the morphogenetic furrow at the leading edge of retinal development, this observation is consistent with the evidence that pattern formation continues in vitro. Morphogenetic changes were manifested in cultures. Viability tests with calcein AM and ethidium bromide revealed few dead cells in living cultures. © 1995 John Wiley & Sons, Inc.
Comparison of different depletion strategies for improved resolution in proteomic analysis of human serum samples
Authors: Björhall Karin , Miliotis Tasso , Davidsson Pia
Published: January 2005

Serum proteins may often serve as indicators of disease and is a rich source for biomarker discovery. However, the large dynamic range of proteins in serum makes the analysis very challenging because high‐abundant proteins tend to mask those of lower abundance. A prefractionation step, such as depletion of a few high‐abundant proteins before protein profiling, can assist in the discovery and detection of less abundant proteins that may prove to be informative biomarkers. In the present study, five different depletion columns were investigated considering efficiency, specificity, and reproducibility. Our research included quantitative determination of total protein, albumin, and immunoglobulin G (IgG) concentrations, one‐ and two‐dimensional gels and mass spectrometric analysis of the serum samples before and after the depletion step. Our results showed that all five depletion columns tested removed albumin and IgG with high efficiency. We found that based on reproducibility and binding specificity, the Multiple Affinity Removal Column that removed a total of six high‐abundant proteins (albumin, IgG, antitrypsin, IgA, transferring, and haptoglobin) offered the most promising depletion approach. Among the disposable (single‐use) products, the ProteoExtract™ Albumin/IgG Removal kit displayed the best results. Depleted serum from the Multiple Affinity Removal column was further evaluated by 2‐D gel electrophoresis (2‐DE) analysis, and the results indicated increased resolution and improved intensity of low‐abundant proteins in a reproducible fashion. Our study provides a comprehensive investigation of commercially available depletion columns and will be of high importance for future proteomic studies on serum samples.
Properties of covalently bonded layered‐silicate/polystyrene nanocomposites synthesized via atom transfer radical polymerization
Authors: Li Chung‐Ping , Huang Ching‐Mao , Hsieh Meng‐Ting , Wei Kung‐Hwa
Published: January 2005

Covalently bonded layered silicated/polystyrene nanocomposites were synthesized via atom transfer radical polymerization in the presence of initiator‐modified layered silicate. The resulting nanocomposites had an intercalated and partially exfoliated structure, as confirmed by X‐ray diffraction and transmission electron microscopy. The thermal properties of the nanocomposites improved substantially over those of neat polystyrene. In particular, a maximum increase of 35.5 °C in the degradation temperature was displayed by these nanocomposites. Additionally, the surface elastic modulus and hardness of these nanocomposites were more than double those of pure polystyrene. © 2004 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 43: 534–542, 2005
Cerebellin is a postsynaptic neuropeptide
Authors: Mugnaini Enrico , Dahl Anne‐Lise , Morgan James I.
Published: January 2005

Cerebellin is a hexadecapeptide that has been biochemically characterized and localized to cerebellar Purkinje cells and certain neurons of the dorsal cochlear nucleus (DCoN) of rat. Among rabbit antisera produced to synthetic cerebellins, one (C1) gave specific immunostaining of the Purkinje neuronal cell body, initial axon segment, and main stem dendrites, while another (R2) reacted with peripheral dendritic structures. This complementarity of staining was also present during cerebellar development. By electron microscopy, the immunoreaction product was localized to polyribosomal domains with antiserum C1 and to dendritic spines with antiserum R2, in both cerebellar cortex and DCoN. In the spine, the structure most strongly stained was the postsynaptic density, but some reaction product was adsorbed to the plasma membrane, the spine apparatus, and the granulofibrillar cytoplasmic component. Antiserum R2 also stained lysosome‐like bodies. We suggest that antiserum C1 recognizes cerebellin precursor(s) and antiserum R2 mature peptide(s) and perhaps degradation product. There is structural homology between cerebellin and residues 625–641 of the polyimmunoglobulin transporter. The functional implications of this homology and other possible roles of cerebellin are discussed.
Evidence for the co‐storage and co‐release of neuropeptide y and noradrenaline from large dense cored vesicles in sympathetic nerves of the bovine vas deferens
Authors: de Potter W. P. , Dillen L. , Annaert W. , Tombeur K. , Berghmans R. , Coen E. P.
Published: January 2005

The subcellular localization and the secretion of neuropeptide Y were studied in sympathetic nerve endings of bovine vas deferens.

Immunostaining revealed a parallel distribution for neuropeptide Y and dopamine‐β‐hydroxylase immunoreactivity in the network of varicose nerve fibers in the smooth muscle layers of the vas deferens.

Following differential centrifugation and sucrose density gradient centrifugation, neuropeptide Y was found to coincide with noradrenaline in the more dense region of the gradient, where the large dense cored vesicles are found.

Superfusion experiments demonstrated the release of neuropeptide Y and noradrenaline upon electrical stimulation. Furthermore, the neuropeptide Y secretion was shown to be Ca2+‐dependent.

We conclude that in the bovine vas deferens neuropeptide Y is only present in large dense cored vesicles of adrenergic neurons and that the peptide and noradrenaline are coreleased from these vesicles in a calcium dependent manner.
B‐endorphin disregulation in autistic and self‐injurious behavior: A neurodevelopmental hypothesis
Authors: Sandman Curt A.
Published: January 2005

Peptides derived from pro‐opiomelanocortin (POMC) influence neurodevelopmental processes. Earlier studies indicated that MSH/ACTH compounds improved behavioral efficiency in retarded individuals. Recent studies have shown that opiate blockers reduce treatment‐resistant self‐injurious behavior (SIB), an autistic‐like, developmental disorder. Although the exact mechanisms are unknown, prenatal POMC disregulation, addiction to endogenous opiates and elevated pain threshold have been proposed to account for this behavior. In study one, four SIB patients were given 0, 25, 50 or 100 mg of naltrexone on separate weeks in a double blind, Latin square design. A specific dose dependent reduction in SIB was observed in three patients. In study two, plasma b‐endorphin was measured in 40 patients with SIB, a related behavior, stereotypy (ST) or controls. SIB and ST patients had higher levels of endorphin than controls. These data added new support for the role of b‐endorphin in a treatment‐resistant patient group.
Mammalian neuromuscular development accelerated with early but slowed with late gestational administration of ACTH Peptide
Authors: Rose Kenneth J. , Strand Fleur L.
Published: January 2005

The neuropeptide ACTH 4–10, a nonsteroidogenic fragment of adrenocorticotropic hormone, has two distinct and opposite effects on developing nerve and muscle. Muscle is positively influenced by ACTH during the first part of gestation (G days 3–12) before innervation occurs. Subsequent effects on innervation are largely depressive and exerted only during G13–21. Treatment during G3–12 increases twitch amplitude, rise time and speed of contraction of directly and indirectly stimulated extensor digitorum longus (EDL) muscle of two wk old rats. Treatment during G13–21 slows contractions of indirectly stimulated EDL, whereas treatment throughout gestation (G3–G21) shows little effect. Thus, ACTH first accelerates muscle development then modulates this development through neuronal depression.
Visualization of growth factor receptor sites in rat forebrain
Authors: Quirion Rémi , Araujo Dalia , Nair N. P. V. , Chabot Jean‐Guy
Published: January 2005

It is now known that various growth factors may also act in the central nervous system. Among them, it has recently been shown that epidermal growth factor (EGF) and insulin‐like growth factor I (IGF‐I) may possess trophic effects in the mammalian brain. We report here on the respective autoradiographic distribution of [125I]EGF and [125I]IGF‐I receptor binding sites in the rat brain, both during ontogeny and in adulthood. It appears that [125I]IGF‐I sites are more widely distributed both during ontogeny and in adulthood. These results reveal the plasticity of the expression of EGF and IGF‐I receptor sites in the mammalian brain. This could be relevant for the respective role of these two growth factors in the development and maintenance of neuronal function.
Recent developments in the use of synthetic oligonucleotides for in sity hybridization histochemistry
Authors: Lewis Michael E. , Krause Rudolph G. , Roberts‐Lewis Jill M.
Published: January 2005

Synthetic oligonucleotides have been used with increasing frequency as probes for the detection and study of the regulation of specific mRNAs by in situ hybridization histochemistry. These probes can be easily obtained and used by the nonmolecular biologist, and they have been shown to be effective for the study of a wide range of mRNAs in neuronal and neuroendocrine tissues. Considerations in oligonucleotide probe design, synthesis, purification, and labeling are described in this article, and current procedures for tissue preparation and hybrization are discussed. In addition, control procedures and methods for the quantitation of in situ hybridization by image analysis are discussed. Finally, the combination of this technique with immunocytochemistry and retrograde tract‐tracing is reviewed. The coupling of quantitative in situ hybridization with other neuronal markers, e.g., of connectivity, provides an increasingly valuable technology for exploring the regulation of gene expression in a rich anatomical context.
Distribution of substance P‐like immunoreactive neurons in the human medulla oblongata: Co‐localization with monoamine‐synthesizing neurons
Authors: Halliday G. M. , Li Y. W. , Joh T. H. , Cotton R. G. H. , Howe P. R. C. , Geffen L. B. , Blessing W. W.
Published: January 2005

The raphe nuclei also contained SP‐like immunoreactivity (up to 30%) while few monoamine‐synthesizing neurons in the lateral and dorsomedial medulla contained SP‐like immunoreactivity approximately 5% of presumed serotonin‐, noradrenaline‐, and adrenthe adult human. The majority of SP‐like immunoreactive neurons were found in four main regions: the lateral medulla, the dorsomedial medulla, the spinal trigeminal nucleus, and the raphe nuclei. The morphology of immunoreactive cells varied according to the region in which they were found. In contrast to previous studies, we found large numbers (90,000) of SP‐like immunoreactive neurons throughout the adult human medulla oblongata. The distribution of these SP‐like immunoreactive neurons appears to be significantly different from those described in the rat and cat.

These results were compared to the distributions of monoamine‐synthesizing and neuropeptide Y (NPY)‐like immunoreactive neurons in the human medulla previously reported (Halliday et al.: Neuroscience, in press, 1988a; J. Comp. Neurol., in press, 1988b). Colocalization studies revealed that many presumed serotonin‐synthesizing neurons in the raphe nuclei also contained SP‐like immunoreactivity (up to 30%) while few monoamine‐synthesizing neurons in the lateral and dorsomedial medulla contained SP‐like immunoreactivity (approximately 5% of presumed serotonin‐, noradrenaline‐, and adrenaline‐synthesizing neurons). The distributions of SP‐ and NPY‐like immunoreactive neurons were similar, although SP‐like immunoreactive neurons were concentrated in the lateral regions of the same structures. We have found that the distributions of monoaminesynthesizing, NPY‐, and SP‐like immunoreactive neurons significantly overlap, particularly in the lateral medulla of the adult human. There is a large increase in the number of these cells in this region compared to other species, emphasizing the neuroanatomical differences between humans and other species.
Evidence for release of copper in the brain: Depolarization‐induced release of newly taken‐up 67copper
Authors: Hartter Daryl E. , Barnea Ayalla
Published: January 2005

The potential importance of copper (Cu) in neurosecretion can be inferred from the demonstration that extracellular Cu modulates the secretory function of peptidergic neurons (in vitro studies) and from the presence of high Cu concentrations in nerve terminals and secretory vesicles, primarily within the soluble matrix of the latter. We have previously hypothesized that vesicular Cu is released from neurons undergoing exocytosis and that such extracellular Cu plays an important modulatory role in the central nervous system. To test this Cu release hypothesis, rat hypothalami were incubated under in vitro conditions for 1 or 2 hr with 20 nM radiolabeled Cu (67Cu), and then 67Cu release was stimulated by a depolarizing concentration (60 mM) of K+. K+ markedly (P < 0.001) stimulated 67Cu release in a Ca2+‐dependent manner (stimulated release was 95 fmol/10 min/mg protein after 1 hr 67Cu loading and 160 after 2 hr). These amounts of released 67Cu account for about 10% of the total 67Cu taken up by the tissue. These results indicate that part of the 67Cu taken up by hypothalamic explants is directed into an intracellular compartment from where it can be released by a Ca2+‐dependent mechanism, thus providing strong support to our hypothesis that release of copper is operative in situ in the brain.
Somatostatin and neuropeptide Y immunoreactivity in parkinson's disease dementia with alzheimer's changes
Authors: Flint Beal M. , Clevens Ross A. , Mazurek Michael F.
Published: January 2005

Somatostatin‐like immunoreactivity (SLI) and neuropeptide Y‐like immunoreactivity (NPYLI) were measured in postmortem brain tissue from 12 control patients and 13 demented Parkinsonian patients who had Alzheimer‐type cortical pathology. Twenty‐two cortical regions were examined. Significant reductions in cortical SLI were found in 17 regions, while significant reductions in cortical NPYLI were found in nine regions. The reductions in SLI were typically 50–60%, while NPYLI reductions were 20–30%. These findings are similar to those in Alzheimer's disease (AD) and are consistent with a previous report of a dissociation between reductions in SLI and NPYLI in Parkinson's disease (PD) with dementia.
Peptide‐ and transmitter‐containing neurons in the mediobasal hypothalamus and their relation to GABAergic systems: Possible roles in control of prolactin and growth hormone secretion
Authors: Meister Björn , Hökfelt Tomas
Published: January 2005

Indirect immunofluorescence histochemistry was used to study the relation among GABAergic, catecholaminergic, cholinergic, and peptidergic neurons in the rat mediobasal hypothalamus. By employing a direct double‐labelling procedure using sheep antiserum against glutamic acid decarboxylase (GAD), mouse monoclonal and rabbit antibodies to neurotensin (NT) and rabbit antisera to tyrosine hydroxylase (TH),choline acetyltransferase (ChAT), galanin (GAL), growth hormone‐releasing factor (GRF), or somatostatin (SOM), it was demonstrated that GAD‐positive fibers and terminals in the external part of the median eminence co‐contained immunoreactivity for TH, NT, GAL or GRF, but not for SOM. In the internal part of the median eminence‐infundibular stalk, GAD‐positive/NT‐, GAL‐, and GRF‐negative and GAD‐Positive/TH‐positive fiber plexa were shown. When a recently developed direct triple‐labelling procedure with biotinconjugated mouse secondary antibodies in conjunction with diethylaminocoumarin (DAMC)‐conjugated avidin was employed, presence of GAD/GAL/NT‐ as well as GAD/GRF/NT‐containing varicosities could be demonstrated close to hypophysial portal vessels. In colchicine‐pretreated animals, GAD was shown to coexist with TH, NT, or GAL in cell bodies in both the dorsomedial and ventrolateral domains of the arcuate nucleus, but with GRF only in the ventrolateral division. ChAT‐positive neurons in the ventrolateral region were also TH‐positive. In the ventrolateral arcuate nucleus, triple‐labelling followed by elution‐restaining showed GAD/NT/GAL/TH‐immunoreactivities in the same cells. Similarly, double‐labelling with two following elution‐restaining steps showed several NT/GAL/GRF/TH‐containing cell bodies in this part of the arcuate nucleus. GAD‐positive cells in the anterior hypothalamic periventricular area and fibers in the pituitary neurointermediate lobe were also TH‐positive.

The results demonstrate complex patterns of storage of chemical messengers in neurons of the arcuate nucleus‐median eminence complex. Possible neuroendocrine interactions of these systems in the control of prolactin and growth hormone secretion are discussed.
Three‐dimensional structure of bidirectional, excitatory chemical synapses in the jellyfish Cyanea capillata
Authors: Anderson Peter A. V. , Grünert Ulrike
Published: January 2005

Neurons in the ectoderm of the perirhopalial tissue of the jellyfish Cyanea capillata were exposed and fixed for electron microscopy under conditions designed to minimize exocytosis of synaptic vesicles. The structure of the bidirectional chemical synapses that connect neurons was examined and the three‐dimensional organization of these synapses was determined from reconstructions of serial sections. Synapses were characterized by the accumulation of a relatively few, large synaptic vesicles. These lie in a single layer against the terminal membrane of each terminal. The cytoplasmic side of the vesicles in any one terminal was covered by a single, large, perforated cisternal sheet. In addition, there were numerous smaller, bulbous cisternae that intermingled with the vesicles in the terminal. The structure of any one terminal was mirrored by that of the opposite terminal of the pair. The organization of these synapses is discussed from the viewpoint of cnidarian synapses in general.
Identification of 5‐hydroxytryptamine1D binding sites in human brain membranes
Authors: Peroutka Stephen J. , Switzer Julie A. , Hamik Anne
Published: January 2005

High‐affinity, specific 3H‐5‐hydroxytryptamine (5‐HT) binding was analyzed in membrane homogenates of human frontal cortex, caudate, and globus pallidus. 5‐HT1A and 5‐HT1C binding sites were pharmacologically blocked using 100 nM 8‐hydroxy‐N,N‐dipropyl‐2‐aminotetralin (8‐OH‐DPAT) and 100 nM mesulergine, respectively. The majority of 5‐HT1 sites remained in each of the three brain regions under these conditions. The pattern of nucleotide interactions with these binding sites (GppNHp = GTP = GDP > GMP = adenine nucleotides) suggests a possible linkage to a G protein. RU 24969 competition studies confirmed the absence of 5‐HT1B binding sites in human cortex, caudate, and globus pallidus. Drug interactions with putative 5‐HT1D binding sites in bovine caudate membranes correlated significantly with their affinities for human membrane recognition sites labeled by 3H‐5‐HT in the presence of 100 nM 8‐OH‐DPAT + 100 nM mesulergine. We conclude that the majority of 3H‐5‐HT‐labeled recognition sites in human cortex, caudate, and globus pallidus represent 5‐HT1D binding sites.
Identification of L‐DOPA‐dopamine and L‐DOPA cell bodies in the rat mesencephalic dopaminergic cell systems
Authors: Mons Nicole , Tison Francois , Geffard Michel
Published: January 2005

An immunocytochemical technique for simultaneously visualizing two different antigens, dihydroxyphenylalanine (L‐DOPA) and dopamine (DA), has been used to investigate the presence of cell bodies containing both compounds L‐DOPA and DA and those having only L‐DOPA in rat mesencephalon areas. The brain slices were processed with a double peroxidase‐antiperoxidase method using simultaneously an incubation of a rabbit anti‐L‐DOPA serum and a monoclonal anti‐DA antibody raised in mouse. Both antigens were revealed by the peroxidase reaction but with different chromogens that are easily distinguishable. In this staining procedure, the first antigen, conjugated DA was stained using the 3,3′‐diaminobenzidine (DAB)‐Nickel complex; while the second antigen, conjugated L‐DOPA, was localized using DAB. The yellow‐brown color due to DAB was masked by that of DAB‐nickel. The possible existence of both single and double labelings could be worked. We have found many L‐DOPA‐positive/DA‐positive and a few L‐DOPA‐positive/DA‐negative cell bodies in dopaminergic regions in the rat midbrain: substantia nigra, ventral tegmental area, and raphe nuclei. In the locus coeruleus, we noted only L‐DOPA‐positive/DA‐positive cell bodies. These results confirm those previously described for rat and cat hypothalamus, where both immunoreactive‐cell body types have been detected: L‐DOPA positive/DA positive and L‐DOPA positive/DA negative. The existence of neuronal cells containing only L‐DOPA is a new neuroanatomic finding, accounting better for the heterogeneity of dopamine systems with respect to physiologic, pharmacologic, and molecular data.
Characterization of [3H]AF‐DX 116 binding sites in the rat brain: Evidence for heterogeneity of muscarinic‐M2 receptor sites
Authors: Araujo Dalia M. , Lapchak Paul A. , Regenold William , Quirion Remi
Published: January 2005

This study shows that [3H]AF‐DX 116 binds specifically, saturably, and with high affinity to putative muscarinic‐M2 receptor sites in the rat brain. In homogenates of the hippocampus, cerebral cortex, striatum, thalamus, and cerebellum, [3H]AF‐DX 116 appears to bind two subpopulations of muscarinic sites: one class of higher affinity sites (Kd < 4.0 nM) and one class of lower affinity sites (Kd > 50 nM, except in the cerebellum). The apparent maximal capacities (Bmax) of [3H]AF‐DX 116 sites in forebrain tissues ranged between 34 and 69 fmol/mg protein for the higher affinity site, and between 197 and 451 fmol/mg protein for the lower affinity site. In cerebellar homogenates, the maximal capacity of [3H]AF‐DX 116 binding sites was 10.4 ± 0.4 (Kd = 1.9 ± 0.2 nM) and 39.1 ± 2.6 (Kd = 26 ± 7 nM) fmol/mg protein for the higher and the lower affinity site, respectively. Determination of the Kd for the higher and lower affinity [3H]AF‐DX 116 sites from association and dissociation constants yielded similar values to those obtained from the saturation data. The ligand selectivity pattern reveals that AF‐DX 116 is more potent than (–)QNB > atropine > methoctramine > 4‐DAMP > gallamine > NMS > carbamylcholine > oxotremorine > pirenzepine > > nicotine in competing for the higher affinity [3H]AF‐DX 116 sites. With few exceptions, the pattern was similar for the lower affinity sites. For example, (–)QNB was more potent than AF‐DX 116 and pirenzepine was more potent than either oxotremorine or 4‐DAMP at the lower affinity [3H]AF‐DX 116 sites. In addition, pirenzepine was modestly more potent at the lower compared to the higher affinity sites. Neither the higher nor the lower affinity [3H]AF‐DX 116 sites were sensitive to the effects of Gpp(NH)p or N‐ethylmaleimide. In addition, the carbamylcholine‐induced inhibition of [3H]AF‐DX 116 binding to the higher and the lower affinity sites was altered by Gpp(NH)p and NEM, to a similar extent. However, Gpp(NH)p decreased the affinity of carbamylcholine (i.e., increased the IC50), whereas N‐ethylmaleimide had the opposite effect. Furthermore, N‐ethylmaleimide also appeared to steepen the curve for the carbamylcholine‐induced inhibition of [3H]AF‐DX 116 binding, as evidenced by the increased nH. Thus it appears that [3H]AF‐DX 116 binds to two subsets of muscarinic‐M2 receptors in the rat brain, which can be differentiated by their affinity for certain agonists and antagonists.
Region‐Specific loss of α1‐adrenergic receptors in rat brain with aging: A quantitative autoradiographic study
Authors: Burnett Donald M. , Zahniser Nancy R.
Published: January 2005

The effects of aging on the density and affinity of α1‐adrenergic receptors (α1‐ARs) were studied in several circumscribed areas of the Fischer 344 male rat brain. Computer‐assisted quantitative autoradiography was used to analyze saturation binding isotherms of [125I]BE‐2254, a selective α1‐AR antagonist. Significant decreases in receptor density of 15 and 29% were observed in the thalamus at 16–18 and 24–28 months of age, respectively, when compared to 3–4 month‐old controls. Progressive declines in receptor density of 24 and 44% were also found in the olfactory tubercle. In the cerebral cortex, a significant 26% loss in receptors occured only in the oldest age group. No changes were found in any of the other brain areas investigated, including the cerebellum, brainstem, caudate‐putamen, and several subregional areas of the hippocampal formation. Kd values ranged from 12 ± 1.8 pM in the brainstem to 23 ± 1.6 pM in the thalamus and were not affected by aging in any area examined. It is concluded that the density of α1‐ARs in the Fischer 344 rat brain is diminished with aging in a region‐specific manner and that loss of these receptors may account for age‐related functional deficits only in a few brain areas.
Acute effects of alprazolam and adinazolam on the concentrations of corticotropin‐releasing factor in the rat brain
Authors: Owens Michael J. , Bissette Garth , Nemeroff Charles B.
Published: January 2005

Corticotropin‐releasing factor (CRF) is the major physiological regulator of the hypothalamic‐pituitary‐adrenal (HPA) axis. However, considerable evidence indicates that CRF may be responsible for integrating not only the endocrine, but the autonomic and behavioral responses of an organism to stress as well. In addition, clinical studies indicate that CRF of both hypothalamic and extrahypothalamic origin may be hypersecreted in major depression as well as other psychiatric disorders.

These findings, taken together, led to the hypothesis that the efficacy of antidepressant and/or anxiolytic drugs may be related to their actions on CRF‐containing neural pathways in the central nervous system (CNS). Therefore, alterations of CRF concentrations in 18 rat brain regions were studied after acute administration of a tricyclic antidepressant (imipramine) or one of two triazolobenzodiazepines (alprazolam or adinazolam) that possess anxiolytic properties typical of benzodiazepines, as well as purported antidepressant activity unique to these compounds.

Treatment with alprazolam or adinazolam increased hypothalamic CRF concentrations, which was associated with lower plasma ACTH concentrations. In contrast, the concentration of CRF was markedly reduced in the locus coeruleus, amygdala, and several cortical regions by either triazalobenzodiazepine. Acute treatment with imipramine was without effect on CRF concentrations in any brain region studied.

Of particular interest is the finding that the two triazolobenzodiazepines exert effects on CRF concentrations in the locus coeruleus and hypothalamus that are opposite to CRF changes seen after stress.
Dye‐Coupling in the neostriatum of the rat: I. Modulation by dopamine‐depleting lesions
Authors: Cepeda Carlos , Walsh John P. , Hull Chester D. , Howard Sherrel G. , Buchwald Nathaniel A. , Levine Michael S.
Published: January 2005

Evidence from experiments performed in turtle and fish retina suggests that dopamine (DA) modulates the permeability of gap junctions. The present experiment was aimed at determining if DA has a similar role in the mammalian neostriatum. Adults rats received one of four treatments: Unilateral electrolytic substantia nigra lesions, unilateral injection of 6‐hydroxydopamine (6‐OHDA) into the substantia nigra, unilateral neocortical aspiration, or no treatment. After 3–5 weeks, neostriata from both sides of the brain were prepared for in vitro intracellular recordings. Recorded neurons (N α 150) were filled with Lucifer Yellow (LY), a low molecular weight dye that crosses gap junctions. In animals with electrolytic nigral lesions, dye‐coupling in the ipsilateral neostriatum occurred after 38% of the intracellular injections. After 6‐OHDA lesions, 19% of the injections produced dye‐coupling in the ipsilateral neostriatum. This difference may have been accounted for by the fact that electrolytic lesions produced a greater degree of DA loss than 6‐OHDA injections. Both of these percentages contrast with the very small percentage of dye‐coupling found in intact animals or in animals with neocortical lesions. Dye‐coupling occurred only between medium‐sized spiny cells. No morphological differences between dye‐coupled and non‐dye‐coupled cells were observed with light microscopy. Overall, passive and active electrophysiological properties of dye‐coupled and single neurons were similar. The results suggest that DA may function in the neostriatum to control permeability of gap junctions.
Presynaptic cholinergic inhibition in hippocampal cultures
Authors: Segal Menahem
Published: January 2005

Activity of hippocampal neurons was recorded in a dissociated culture under patch‐clamp conditions. Excitatory and inhibitory postsynaptic currents (PSCs) were evoked in response to short pulse application of glutamate to other neurons in the culture dish. These PSCs were suppressed by topical application of acetylcholine (ACh) near the recorded neuron. The dose‐dependent effect of the muscarinic antagonist pirenzepine indicates that the effect of ACh is mediated by an M2 receptor. ACh did not affect inward current responses to direct application of glutamate onto postsynaptic neurons. This indicates that ACh may interfere with the release process and not with the postsynaptic response to the neurotransmitter. In some cells, ACh reduced inward Ca currents recorded in the presence of Na and K channel blockers. This effect was atropine sensitive and may underly the reduced PSCs. It is suggested that ACh modulates release of neurotransmitters by reducing presynaptic Ica and thereby reducing evoked PSCs.
Mapping cocaine binding sites in human and baboon brain in vivo
Authors: Fowler Joanna S. , Volkow Nora D. , Wolf Alfred P. , Dewey Stephen L. , Schlyer David J. , MacGregor Robert R. , Hitzemann Robert , Logan Jean , Bendriem Bernard , Gatley S. John , Christman David
Published: January 2005

The first direct measurements of cocaine binding in the brain of normal human volunteers and baboons have been made by using positron emission tomography (PET) and tracer doses of [N‐11C‐methyl]‐(–)‐cocaine ([11C]cocaine). Cocaine's binding and release from brain are rapid with the highest regional uptake of carbon‐11 occurring in the corpus striatum at 4–10 minutes after intravenous injection of labeled cocaine. This was followed by a clearance to half the peak value at about 25 minutes with the overall time course paralleling the previously documented time course of the euphoria experienced after intravenous cocaine administration. Blockade of the dopamine reuptake sites with nomifensine reduced the striatal but not the cerebellar uptake of [11C]cocaine in baboons indicating that cocaine binding is associated with the dopamine reuptake site in the corpus striatum. A comparison of labeled metabolites of cocaine in human and baboon plasma showed that while cocaine is rapidly metabolized in both species, the profile of labeled metabolites is different, with baboon plasma containing significant amounts of labeled carbon dioxide, and human plasma containing no significant labeled carbon dioxide. These studies demonstrate the feasibility of using [11C]cocaine and PET to map binding sites for cocaine in human brain, to monitor its kinetics, and to characterize its binding mechanism by using appropriate pharmacological challenges.
Coesxistence of corticotropin releasing factor and enkephalin in cerebellar afferent systems
Authors: Cummings Sharon , King James S.
Published: January 2005

In this study, we tested the hypothesis that corticotropin‐releasing factor (CRF) and enkephalin (ENK) coexist within restricted brainstem‐cerebellar circuits. Antisera for CRF and ENK were applied simultaneously or to serial sections of adult opossum brain and were processed for fluorescence or peroxidase, antiperoxidase immunohistochemistry, respectively. CRF and ENK were colocalized in (1) climbing fibers within the flocculus and in the cerebellar vermis; (2) fibers with morphological characteristics of simple mossy fiber terminals or climbing fiber glomeruli that were located within the granule cell layer of the vermis underlying the foci of CRF/ENK‐IR climbing fibers;(3) mossy fiber terminals within the flocculus; (4) neuronal perikarya in subnucleus A and C of the medial accessory olive and in the dorsal cap of Kooy, nuclei known to project as climbing fibers; and (5) cell bodies in nucleus propositus, the subtrigeminal reticular nucleus, and the reticular formation, likely origins of CRF/ENK colocalized mossy fibers. The demonstration that single climbing and mossy fibers contain two peptides extends previous studies that have described chemical heterogeneity within cerebellar afferent pathways. Furthermore, these results support suggestions that this heterogeneity may provide a substrate for differential regulation of signal transduction by chemically coded cerebellar afferents.
Vasopressin facilitates excitatory transmission in slices of the rat dorso‐lateral septum
Authors: Van Den Hooff Peter , Urban Ivan J. A.
Published: January 2005

The effects of vasopressin on neurons of the rat dorso‐lateral septum (DLS) was studied in brain slices with intracellular microelectrodes. Two out of 13 neurons showed a small depolarization, spontaneous activity, and increased input resistances following a 15 min exposure to 10‐6to 10‐8M vasopressin (VP). These membrane effects disappeared completely within 3–5 min after the application. The remaining DLS neurons treated with these vasopressin concentrations showed an increase in glutamate‐mediated excitatory postsynaptic potentials (EPSPs), evoked by stimulation of the fimbria fibers. As little as 10‐12M VP increase these EPSPsmarkedly in nearly 80% of the cells studied. This increase in most of the cells disappeared within 15 min after the application period, whereas the increase in EPSPsinduced by 10‐10M VP outlasted the peptide application period for more than 30 min. Neither the blockade of GABA‐ergic synaptic inhibition nor the pre‐treatment of the neurons with d(CH2)5‐Tyr(Me)‐arginine vasopressin or 2‐amino‐5‐phosphonovaleric acid (2‐APV), antagonists for the V1 type of vasopressin receptor and NMDA receptors, respectively, interfered with the EPSPspotentiating effect of the peptide. It is concluded that a type of vasopressin receptor other then the V1 type is involved in the long‐lasting potentiation of the primarily non‐NMDA receptor mediated transmission in DLS neurons.
Effects of high‐dose fenfluramine treatment on monoamine uptake sites in rat brain: Assessment using quantitative autoradiography
Authors: Appel Nathan M. , Mitchell Wm. Mark , Contrera Joseph F. , Desouza Errol B.
Published: January 2005

Fenfluramine is an amphetamine derivative that in humans is used primarily as an anorectic agent in the treatment of obesity. In rats, subchronic high‐dose d, l‐fenfluramine treatment (24 mg/kg subcutaneously, twice daily for 4 days) causes long‐lasting decreases in brain serotonin (5HT), its metabolite 5‐hydroxyindoleacetic acid, and high‐affinity 5HT uptake sites. Moreover, this high‐dose treatment regimen causes both selective long‐lasting decreases in fine‐caliber 5HT‐immunoreactive axons and appearance of other 5HT‐immunoreactive axons with morphology characteristic of degenerating axons. Determination of the potential neurotoxic effects of fenfluramine treatment using immunohistochemistry is limited from the perspectives that staining is difficult to quantify and that it relies on presence of the antigen (in this case 5HT), and the 5HT‐depleting effects of fenfluramine are well known. In the present study, we used quantitative in vitro autoradiography to assess, in detail, the density and regional distribution of [3H]paroxetine‐labeled 5HT and [3H]mazindol‐labeled catecholamine uptake sites in response to the high‐dose fenfluramine treatment described above. Because monoamine uptake sites are concentrated on monoamine‐containing nerve terminals, decreases in uptake site density would provide a quantitative assessment of potential neurotoxicity resulting from this fenfluramine ltreatment regimen. Marked decreases in densities of [3H]paroxetine‐labeled 5HT uptake sites occurred in brain regions in which fenfluramine treatment decreased the density of 5HT ‐like immunostaining when compared to saline‐treated control rats. These included cerebral cortex, caudated putamen, hippocampus, thalam us, land medial hypothalamus. Smaller, but nonetheless significant, decreases in density of [3H]paroxetine‐labeled 5HT uptake sites were noted in brain regions in which partial sparing of 5HT‐like immunoreactive fibers had been reported following fenfluramine treatment, specifically septum, lateral hypothalamus, and amygdala. In contrast, [3H]mazindol autoradiography revealed that total catecholamine (i.e., dopamine and norepinephrine) uptake sites in cerebral cortex, caudate putamen, and locus coeruleus, areas in which [3H]paroxetine‐labeled 5HT uptake sites were significantly decreased, were unaffecterd by this fenfluramine treatment. These data support the hypothesis that subchronic, high‐dose fenfluramine treatment causes selective degeneration of 5HT axons in rat brain. Since pharmacokinetic studies show that the dosing regimen used in this study exposes rat brain to concentrations of fenfluramine that are approximately 600 times greater than those resulting from the therapeutic oral dose, caution must be exercised in extrapolating these dat to humans.
Regulation of dopamine and serotonin synthesis by acute administration of cocaine
Authors: Galloway Mathhew P.
Published: January 2005

Because cacaine effectively increases extracellular levels of both dopamine (DA) and serotonin (5HT), it might be expected that this agent would inhibit transmitter biosynthesis in these monoamine neurons by activation of autoregulatory feedback pahwas. This possibility was tested by measuring the effect of cocaine on 3,4‐dihydroxyphenylalanine accumulation (DA synthesis) and 5‐hydroxytryptophan accmulation (5HT synthesis) in vivo and in vitro after inhibition of aromatic amino acid decarboxylase with NSD‐1015. In vivo, cocaine suppressed both Da and 5HT synthesis in a dose‐depentent (10–60 μmol/kg, i.p.) and time‐dependent fashion (maximum 60 min after administrationm, recovery by 120–150 min). Inhibition of Da and 5HT synthesis ranged from 35% to 60% depending on the brian region and was apparent in dopaminergic fields such as the medial prefrontal cortex, nucleus accumbens, piriform cortex, striatum, and in noradrenergic fields such as the hipocampus and temporal cortex. Inhibition of DA, but not 5HT, synthesis was blocked by the D2 antagonist sulpiride in brain areas containing DA nerve terminals. Procaine (30 μmol/kg) did not inhibit DA or 5HT synthesis and prior treatment with reserpine diminished the effectiveness of cocaine in the medial prefrontal cortex, but not in the striatum. Cocaine did not reverse the gamma‐butyrolactone‐induced increase in striatal DA synthesis nor did cocaine block the ability of the D2 agonist quinpirole to reverse the increase. In vitro, cocaine inhibited Da synthesis in depolarized (K+=30 mM) striatal brian slices, an effect that was reversed by the D2 antagonist eticlopride. These results suggest that DA and 5HT neurons compensate in situ for cocaine‐induced increases in synaptic transmitter levels by a transient inhibition of transmitter biosynthesis. Acute suppression of transmitter synthesis (and release) in mesoprefrontal DA neurcns may represent the principal compensatorty mechanism in this group of neurons.
Investigation of behavioral and electrophysiological responses induced by selective stimulation of CCKB receptors by using a new highly potent CCK analog, BC 264
Authors: Dauge V. , Bohme G. A. , Crawley J. N. , Durieux C. , Stutzmann J. M. , Feger J. , Blanchard J. C. , Roques B. P.
Published: January 2005

The new CCKB analog, Boc‐Tyr (SO3H)‐gNle‐mGly‐Trp‐(NMe)‐Nle‐Asp‐PheNH2(BC 264) exhibited a high affinity (KI = 0.39 ± 0.15 nM) and selectivity for central (B) versus peripheral (A) receptors (KI CCKA/KI CCKB = 910) in the rat. In agreement with these binding studies, BC 264 was at least 50 times more potent than CCK8 in stimulating the firing of rat CA hippocampal neurones. Furthermore sterotzxic injection of BC 264 or CCK8 in the VTA of rats resulted in potentiation of the dopamine‐induced hypolocomotion. These two types of CCK8 responses have been previously shown to involve CCKB receptrs. In contrast, after administration into the posteor‐median nucleus accumbens, the hypoexploration, the increase of emotionality of rats, or the potentiation of dopamine‐induced hyperlocomotion were obtained after injection of CCK8 but not of BC 264, supporting the involvement of peripheral CCKA receptors in these CCK8 responses. Owing to its resistance to peptidases, BC 264 appears to be of great interest in the investigation of the still uncertain funcitonal roles of CCK in the central nervous system.