Radioautographic method for quantifying regional monoamine innervations in the rat brain. Application to the cerebral cortex

Conditions leading to selective and complete labeling of the noradrenaline (NA) and serotonin (5-HT) innervations in rat cerebral cortex were sought by incubating 200-micron-thick whole hemisphere slices with various combinations of tritiated monoamines and uptake blockers at different concentrations in the presence of a monoamine oxidase inhibitor. After fixation with glutaraldehyde, post-fixation with osmium tetroxide and flat-embedding in Epon, 4-micron-thick sections of the entire slices were radioautographed by dipping in nuclear emulsion. As previously reported, dopamine (DA) terminals could be specifically visualized and counted following incubation with 1 micron [3H]DA and 5 microM desipramine (DMI) with or without 5 microM citalopram (CITAL). The number of NA terminals could thus be obtained by subtracting DA varicosities from the total number of sites labeled in adjacent slices incubated without DMI but in presence of CITAL to eliminate some interspecific labeling of 5-HT terminals. NA terminals could also be identified exclusively and counted after labeling with 1 microM [3H]NA in the presence of 10 microM benztropine. 5-HT terminals were specifically detected after incubation with 1 microM [3H]5-HT in the presence of 10 microM non-radioactive NA. The labeled varicosities were counted in areas FR1 and PAR1 of the frontal and the parietal neocortex, respectively, with the aid of a microcomputer-based image analysis system. DA varicosities were concentrated mainly in layer VI of these regions and were more numerous in the frontal than the parietal area. NA terminals were equally distributed in the two regions but approximately twice as numerous in layer I than subjacent layers. The 5-HT innervation also showed a comparable overall density in the two cortical regions but with a differing intracortical distribution. In the frontal area, 5-HT terminals were slightly more concentrated in layer I (1.3-fold) than underlying layers where they were rather uniformly distributed. In the parietal area, layer I was again the most densely innervated (1.8 times the average), but a second zone of higher density (1.5 times average) was present in the outer part of layer V. The remaining layers showed lower numbers of 5-HT terminals than in the frontal region. To obtain absolute estimates of these innervation densities, the number of detected varicosities was assessed experimentally as a function of radioautographic exposure time and of histological section thickness, and their 'equivalent circle diameter' was measured in electron microscope radioautographs.(ABSTRACT TRUNCATED AT 400 WORDS)

[1]  Karl J. Zilles,et al.  The Cortex of the Rat: A Stereotaxic Atlas , 1985 .

[2]  S. Snyder,et al.  Regional differences in H3-norepinephrine and H3-dopamine uptake into rat brain homogenates. , 1969, The Journal of pharmacology and experimental therapeutics.

[3]  L. Descarries,et al.  Regional and laminar density of the dopamine innervation in adult rat cerebral cortex , 1987, Neuroscience.

[4]  L. Swanson.  The locus coeruleus: A cytoarchitectonic, golgi and immunohistochemical study in the albino rat , 1976, Brain Research.

[5]  W. Greenough,et al.  Differential rearing effects on rat visual cortex synapses. I. Synaptic and neuronal density and synapses per neuron , 1985, Brain Research.

[6]  F E Bloom,et al.  The formation of synaptic junctions in developing rat brain: a quantitative electron microscopic study. , 1967, Brain research.

[7]  S. Snyder,et al.  Characterization of [3H]desipramine binding associated with neuronal norepinephrine uptake sites in rat brain membranes , 1982, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[8]  E. Azmitia,et al.  In vitro hippocampal uptake of tritiated serotonin (3H-5HT): A morphological, biochemical, and pharmacological approach to specificity. , 1980, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[9]  Laurent Descarries,et al.  Quantitative data on serotonin nerve terminals in adult rat neocortex , 1976, Brain Research.

[10]  L. Descarries,et al.  Monoamine innervation of the oculomotor nucleus in the rat. A radioautographic study , 1986, Neuroscience.

[11]  John H. Morrison,et al.  Complementarity of dopaminergic and noradrenergic innervation in anterior cingulate cortex of the rat , 1979, Brain Research.

[12]  J. Richards,et al.  Evidence against a neurotoxic action of halogenated amphetamines on serotoninergic B9 cells. A morphometric fluorescence histochemical study , 1978, Brain Research.

[13]  K Fuxe,et al.  Distribution of noradrenaline nerve terminals in cortical areas of the rat. , 1968, Brain research.

[14]  M. Herkenham,et al.  Mismatches between receptor and transmitter localizations in the brain , 1986 .

[15]  B. Berger,et al.  New dopaminergic terminal fields in the motor, visual (area 18b) and retrosplenial cortex in the young and adult rat. Immunocytochemical and catecholamine histochemical analyses , 1985, Neuroscience.

[16]  J. Costentin,et al.  GBR 12783, a potent and selective inhibitor of dopamine uptake: biochemical studies in vivo and ex vivo. , 1986, European journal of pharmacology.

[17]  A. Daszuta,et al.  Distribution and quantification of 5-HT nerve cell bodies in the nucleus raphe dorsalis area of C57BL and BALBc mice. Relationship between anatomy and biochemistry , 1985, Brain Research.

[18]  Joachim F. R. König,et al.  The rat brain: A stereotaxic atlas of the forebrain and lower parts of the brain stem , 1986 .

[19]  S. Ross.  Neuronal transport of 5-hydroxytryptamine. , 1980, Pharmacology.

[20]  F. E. Bloom,et al.  Loss of pigmented dopamine-β-hydroxylase positive cells from locus coeruleus in senile dementia of alzheimer's type , 1983, Neuroscience Letters.

[21]  J. Morrison,et al.  The distribution and orientation of noradrenergic fibers in neocortex of the rat: An immunofluorescence study , 1978, The Journal of comparative neurology.

[22]  L. Swanson,et al.  The projections of the ventral tegmental area and adjacent regions: A combined fluorescent retrograde tracer and immunofluorescence study in the rat , 1982, Brain Research Bulletin.

[23]  M. Barbaccia,et al.  Modulation of neuronal serotonin uptake by a putative endogenous ligand of imipramine recognition sites. , 1983, Proceedings of the National Academy of Sciences of the United States of America.

[24]  J. Hyttel,et al.  Citalopram — Pharmacological profile of a specific serotonin uptake inhibitor with antidepressant activity , 1982, Progress in Neuro-Psychopharmacology and Biological Psychiatry.

[25]  O. Bosler,et al.  In vitro radioautographic study of the monoaminergic innervation of cat red nucleus. Identification of serotoninergic terminals , 1983, Brain Research.

[26]  A. Beaudet,et al.  Dopaminergic projection from nucleus raphe dorsalis to neostriatum in the rat , 1986, The Journal of comparative neurology.

[27]  T. Dawson,et al.  D-1 dopamine receptors in the rat brain: a quantitative autoradiographic analysis , 1986, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[28]  F E Bloom,et al.  Central catecholamine neuron systems: anatomy and physiology of the norepinephrine and epinephrine systems. , 1979, Annual review of neuroscience.

[29]  J. Glowinski,et al.  The topographical distribution of serotoninergic terminals in the neostriatum of the rat and the caudate nucleus of the cat , 1977, Brain Research.

[30]  T. Hökfelt,et al.  A method to measure the distribution pattern of specific nerve terminals in sampled regions. Studies on tyrosine hydroxylase LHRH, TRH and GIH immunofluorescence. , 1977, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[31]  M. Abercrombie.  Estimation of nuclear population from microtome sections , 1946, The Anatomical record.

[32]  L. Descarries,et al.  Density of the dopamine innervation in rat cerebral cortex after neonatal 6-hydroxydopamine or adult stage DSP-4 noradrenaline denervations: a quantitative radioautographic study , 1988, Brain Research.

[33]  H. Kimelberg,et al.  High-affinity uptake of serotonin into immunocytochemically identified astrocytes. , 1985, Science.

[34]  H. Steinbusch,et al.  Serotonin-immunoreactive neurons and their projections in the CNS , 1984 .

[35]  Pat Levitt,et al.  Noradrenaline neuron innervation of the neocortex in the rat , 1978, Brain Research.

[36]  M. Martres,et al.  Localisation and pharmacological characterisation of D-2 dopamine receptors in rat cerebral neocortex and cerebellum using [125I]iodosulpride. , 1985, European journal of pharmacology.

[37]  T. Hökfelt,et al.  Ultrastructural identification of catecholamine neurones in the hypothalamic periventricular-arcuate nucleus-median eminence complex with special reference to quantitative aspects. , 1973, Brain research.

[38]  B. Hamberger.  Reserpine-resistant uptake of catecholamines in isolated tissues of the rat. A histochemical study. , 1967, Acta physiologica Scandinavica. Supplementum.

[39]  G. Aghajanian,et al.  Selective accumulation of 3 H-serotonin by nerve terminals of raphe neurones: an autoradiographic study. , 1973, Nature: New biology.

[40]  N. Andén,et al.  A quantitative study on the nigro-neostriatal dopamine neuron system in the rat. , 1966, Acta physiologica Scandinavica.

[41]  L. Iversen,et al.  The use of autoradiographic techniques for the identification and mapping of transmitter-specific neurones in the brain. , 1974, Life sciences.

[42]  A. Calas,et al.  The topographical distribution of serotoninergic terminals in the spinal cord of the cat: Quantitative radioautographic studies , 1978, Brain Research.

[43]  L. Wiklund,et al.  Monoamine cell distribution in the cat brain stem. A fluorescence histochemical study with quantification of indolaminergic and locus coeruleus cell groups , 1981, The Journal of comparative neurology.

[44]  K. Fuxe,et al.  Quantitative Microfluorimetry and Semiquantitative Immunocytochemistry as Tools in the Analysis of Transmitter Identified Neurons , 1985 .

[45]  F Benfenati,et al.  Quantitative autoradiographic localization of [3H]imipramine binding sites in the brain of the rat: relationship to ascending 5-hydroxytryptamine neuron systems. , 1983, Proceedings of the National Academy of Sciences of the United States of America.

[46]  S. Snyder,et al.  Catecholamine uptake by synaptosomes from rat brain. Structure-activity relationships of drugs with differential effects on dopamine and norepinephrine neurons. , 1971, Molecular pharmacology.

[47]  A. Beaudet,et al.  The fine structure of central serotonin neurons. , 1981, Journal de physiologie.

[48]  A. Beaudet,et al.  Noradrenergic axon terminals in the cerebral cortex of rat. II. Quantitative data revealed by light and electron microscope radioautography of the frontal cortex. , 1973, Brain research.

[49]  F E Bloom,et al.  Central catecholamine neuron systems: anatomy and physiology of the dopamine systems. , 1978, Annual review of neuroscience.

[50]  M. Molliver,et al.  The serotonin innervation of the cerebral cortex in the rat—an immunohistochemical analysis , 1980, Neuroscience.

[51]  A. Jean,et al.  Radioautographic study of serotoninergic axon terminals in the rat trigeminal motor nucleus , 1984, Neuroscience Letters.

[52]  B. Cragg.  The density of synapses and neurones in the motor and visual areas of the cerebral cortex. , 1967, Journal of anatomy.

[53]  W. Lichtensteiger,et al.  UPTAKE OF 5‐HYDROXYTRYPTAMINE AND 5‐HYDROXYTRYPTOPHAN BY NEURONS OF THE CENTRAL NERVOUS SYSTEM NORMALLY CONTAINING CATECHOLAMINES * , 1967, Journal of neurochemistry.

[54]  D. Brunswick,et al.  High‐ and Low‐Affinity Binding Components for [3H]Imipramine in Rat Cerebral Cortex , 1985, Journal of neurochemistry.

[55]  A. Beaudet,et al.  The monoamine innervation of rat cerebral cortex: Synaptic and nonsynaptic axon terminals , 1978, Neuroscience.

[56]  J. Wamsley,et al.  Localization of [3H]-imipramine binding sites in rat brain by light microscopic autoradiography. , 1983, Life sciences.

[57]  Miklós Palkovits,et al.  Distribution of norepinephrine and dopamine in cerebral cortical areas of the rat , 1979, Brain Research Bulletin.

[58]  H. Kimelberg,et al.  High‐Affinity Uptake of [3H]Norepinephrine by Primary Astrocyte Cultures and Its Inhibition by Tricyclic Antidepressants , 1983, Journal of neurochemistry.

[59]  W. Greenough,et al.  Differential rearing effects on rat visual cortex synapses. III. Neuronal and glial nuclei, boutons, dendrites, and capillaries , 1987, Brain Research.

[60]  S. Snyder,et al.  Differential visualization of dopamine and norepinephrine uptake sites in rat brain using [3H]mazindol autoradiography , 1985, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[61]  A. Beaudet,et al.  Synaptic remodeling of serotonin axon terminals in rat agranular cerebellum , 1981, Brain Research.

[62]  H. Kimelberg,et al.  Autoradiography of high affinity uptake of catecholamines by primary astrocyte cultures , 1985, Brain Research.

[63]  L. Descarries,et al.  Disappearance of the locus coeruleus in the rat after intraventricular 6-hydroxdopamine. , 1972, Brain research.

[64]  M. Palkovits,et al.  Regional distribution of [3H]imipramine binding in rat brain , 1981, Brain Research.

[65]  A. Beaudet,et al.  Organization of ascending serotonin systems in the adult rat brain. A radioautographic study after intraventricular administration of [3h]5-hydroxytryptamine , 1981, Neuroscience.

[66]  J. Hedreen,et al.  Neuronal degeneration in rat brain induced by 6-hydroxydopamine; a histological and biochemical study. , 1972, Brain research.

[67]  S. Paul,et al.  Does high affinity [3H] imipramine binding label serotonin reuptake sites in brain and platelet? , 1981, Life sciences.

[68]  A. Beaudet,et al.  The serotonin neurons in nucleus raphe dorsalis of adult rat: A light and electron microscope radioautographic study , 1982, The Journal of comparative neurology.

[69]  J. Morrison,et al.  The intra-cortical trajectory of the coeruleo-cortical projection in the rat: A tangentially organized cortical afferent , 1981, Neuroscience.

[70]  A. W. Rogers.  Techniques of autoradiography , 1967 .

[71]  A. Björklund,et al.  Mechanisms of regrowth in the bulbospinal serotonin system following 5,6-dihydroxytryptamine induced axotomy. II. Fluorescence histochemical observations , 1980, Brain Research.

[72]  B. Winblad,et al.  “Specific” Binding of [3H]Imipramine to Protease‐Sensitive and Protease‐Resistant Sites , 1985, Journal of neurochemistry.

[73]  M. Colonnier,et al.  The number of neurons in the different laminae of the binocular and monocular regions of area 17 in the cat , 1983, The Journal of comparative neurology.

[74]  L. Descarries,et al.  Serotonin nerve terminals in adult rat neocortex , 1975, Brain Research.

[75]  J. Luthman,et al.  Quantitation of Nerve Terminal Networks of Transmitter-Identified Neurons After Selective Neurotoxic Lesions , 1985 .

[76]  D M Katz,et al.  Kinetics and autoradiography of high affinity uptake of serotonin by primary astrocyte cultures , 1985, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[77]  D. Reis,et al.  Strain-dependent variations in number of midbrain dopaminergic neurones , 1976, Nature.

[78]  K. Fuxe,et al.  The effect of imipramine-like drugs and antihistamine drugs on uptake mechanisms in the central noradrenaline and 5-hydroxytryptamine neurons. , 1971, Neuropharmacology.

[79]  B. K. Hartman,et al.  The central adrenergic system. An immunofluorescence study of the location of cell bodies and their efferent connections in the rat utilizing dopamine‐B‐hydroxylase as a marker , 1975, The Journal of comparative neurology.

[80]  L. Descarries,et al.  Serotonin innervation in adult rat neostriatum. I. Quantified regional distribution , 1987, Brain Research.

[81]  R. Barlow.  Biodata handling with microcomputers , 1983 .

[82]  K. Fuxe,et al.  The effect of drugs on accumulation of monoamines in tubero-infundibular dopamine neurons , 1967 .

[83]  M. Jouvet,et al.  The locus coeruleus: a quantitative and genetic study in mice , 1982, Brain Research.

[84]  D. Wong,et al.  A selective inhibitor of serotonin uptake: Lilly 110140, 3-(p-trifluoromethylphenoxy)-N-methyl-3-phenylpropylamine. , 1974, Life sciences.

[85]  J. Gugten,et al.  Regional concentrations of noradrenaline and dopamine in the frontal cortex of the rat: dopaminergic innervation of the prefrontal subareas and lateralization of prefrontal dopamine , 1982, Brain Research.

[86]  Prof. Dr. Karl Zilles.  The Cortex of the Rat , 1985, Springer Berlin Heidelberg.

[87]  Laurent Descarries,et al.  Noradrenergic axon terminals in the cerebral cortex of rat. III. Topometric ultrastructural analysis , 1977, Brain Research.

[88]  S. Snyder,et al.  Kinetics of serotonin accumulation into slices from rat brain: relationship to catecholamine uptake. , 1970, The Journal of pharmacology and experimental therapeutics.

[89]  S. Paul,et al.  High‐Affinity Binding of [3H]Desipramine to Rat Brain: A Presynaptic Marker for Noradrenergic Uptake Sites , 1982, Journal of neurochemistry.

[90]  Jonathan K. Crane,et al.  Non-dopaminergic nigrostriatal pathway , 1981, Brain Research.

[91]  H. Kimelberg,et al.  Dopamine and norepinephrine uptake and metabolism by astroglial cells in culture. , 1981, Life sciences.

[92]  L. Descarries,et al.  Serotonin nerve terminals in the locus coeruleus of adult rat: A radioautographic study , 1978, Brain Research.

[93]  L. Olson,et al.  Extraneuronal binding of catecholamines and 3,4-dihydroxyphenylalanine (dopa) in salivary glands. , 1967, Acta physiologica Scandinavica.

[94]  K. Fuxe,et al.  Biochemical and histochemical studies on the effects of imipramine-like drugs and (+)-amphetamine on central and peripheral catecholamine neurons. , 1966, Acta physiologica Scandinavica.

[95]  A. Biegon,et al.  Localization and characterization of [3H]desmethylimipramine binding sites in rat brain by quantitative autoradiography , 1983, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[96]  C. D. Stern,et al.  Handbook of Chemical Neuroanatomy Methods in Chemical Neuroanatomy. Edited by A. Bjorklund and T. Hokfelt. Elsevier, Amsterdam, 1983. Cloth bound, 548 pp. UK £140. (Volume 1 in the series). , 1986, Neurochemistry International.

[97]  B. Berger,et al.  High resolution radioautography of central dopaminergic fibers labelled in vitro with [3H]dopamine or [3H]norepinephrine , 1981, Brain Research.

[98]  M. Dubocovich,et al.  Quantitative autoradiography of 3H-nomifensine binding sites in rat brain. , 1985, Life sciences.

[99]  B. Berger,et al.  Major dopamine innervation of the cortical motor areas in the Cynomolgus monkey. A radioautographic study with comparative assessment of serotonergic afferents , 1986, Neuroscience Letters.

[100]  G. Koob,et al.  The origin and distribution of dopamine-containing afferents to the rat frontal cortex , 1978, Brain Research.

[101]  J. Palacios,et al.  Quantitative autoradiographic mapping of serotonin receptors in the rat brain. I. Serotonin-1 receptors , 1985, Brain Research.

[102]  G. Paxinos,et al.  The Rat Brain in Stereotaxic Coordinates , 1983 .

[103]  P. V. D. Zee,et al.  ARYL 1,4-DIALK(EN)YLPIPERAZINES AS SELECTIVE AND VERY POTENT INHIBITORS OF DOPAMINE UPTAKE , 1981 .

[104]  J. Wamsley.  Autoradiographic Localization of Receptor Sites in the Cerebral Cortex , 1984 .

[105]  S. Snyder,et al.  Uptake and Subcellular Localization of Neurotransmitters in the Brain , 1970 .

[106]  L. Descarries,et al.  Quantification of the dopamine innervation in adult rat neostriatum , 1986, Neuroscience.

[107]  A. Beaudet,et al.  Radioautographic characterization of a serotonin-accumulating nerve cell group in adult rat hypothalamus , 1979, Brain Research.

[108]  K. Fuxe,et al.  A new approach to quantitate the density and antigen contents of high densities of transmitter-identified terminals. Immunocytochemical studies on different types of tyrosine hydroxylaxe immunoreactive nerve terminals in nucleus caudatus putamen of the rat , 1982, Neuroscience Letters.

[109]  B. Berger,et al.  Dopamine uptake in serotoninergic terminals in vitro: A valuable tool for the histochemical differentiation of catecholaminergic and serotoninergic terminals in rat cerebral structures , 1978, Brain Research.

[110]  Laurent Descarries,et al.  Serotoninergic axon terminals in the rat dorsal accessory olive: Normal ultrastructure and light microscopic demonstration of regeneration after 5,6-dihydroxytryptamine lesioning , 1981, Journal of neurocytology.