Spatial intensity distribution analysis (SpIDA): a new tool for receptor tyrosine kinase activation and transactivation quantification.

This chapter presents a general approach for the application of spatial intensity distribution analysis (SpIDA) to pharmacodynamic quantification of receptor tyrosine kinase homodimerization in response to direct ligand activation or transactivation by G protein-coupled receptors. A custom graphical user interface developed for MATLAB is used to extract quantal brightness and receptor density information from intensity histograms calculated from single fluorescence microscopy images. This approach allows measurement of monomer/oligomer protein mixtures within subcellular compartments using conventional confocal laser scanning microscopy. Application of quantitative pharmacological analysis to data obtained using SpIDA provides a universal method for comparing studies between cell lines and receptor systems. In addition, because of its compatibility with conventional immunostaining approaches, SpIDA is suitable not only for use in recombinant systems but also for the characterization of mechanisms involving endogenous proteins. Therefore, SpIDA enables these biological processes to be monitored directly in their native cellular environment.

Review. Positron emission tomography imaging studies of dopamine receptors in primate models of addiction.

Animal models have provided valuable information related to trait and state variables associated with vulnerability to drug addiction. Our brain imaging studies in monkeys have implicated D2 receptors in cocaine addiction. For example, an inverse relationship between D2 receptor availability and rates of cocaine self-administration has been documented. Moreover, environmental variables, such as those associated with formation of the social hierarchy, can impact receptor availability and sensitivity to the abuse-related effects of cocaine. Similarly, both D2 receptor availability and cocaine self-administration can be altered by chronic drug administration and fluctuations in hormone levels. In addition, cocaine self-administration can be altered in an orderly fashion by presentation of an acute stressor, such as acting as an intruder into an unfamiliar social group, which can shift the cocaine dose-response curve to the left in subordinate monkeys and to the right in dominant animals, suggesting an interaction between social variables and acute stressors. Conversely, irrespective of social rank, acute environmental enrichment, such as increasing the size of the living space, shifts the cocaine dose-response curve to the right. These findings highlight a pervasive influence of the environment in modifying the reinforcing effects of cocaine and strongly implicate brain D2 receptors.

Localization of D1a dopamine receptors on cell bodies and axonal endings in the substantia nigra pars reticulata of the rat.

In the present study, we analyzed the localization of D1a receptors within the rat substantia nigra pars reticulata (SNr) using specific D1a immunochemistry at the ultrastructural level and RT-PCR. At the electron microscopic level, D1a receptors were strongly associated with axons and axonal endings in the SNr, but also with numerous glutamic acid decarboxylase-positive dendrites and neuronal cell bodies. This neuronal expression of D1a receptors was confirmed using RT-PCR. G(alphaolf) protein-specific immunostaining displayed a similar distribution in dendrites and cell bodies to that of D1a receptors. The localization of D1a receptors in both GABAergic cell bodies and terminals is in accordance with the well known complex action of dopamine in the SNr. Moreover, the intracytoplasmic localization of D1a receptors in cell bodies and dendrites that we observed suggests that these receptors are only effective in specific conditions, or are transported to different nigral targets where they may have a presynaptic function.

Contributions of cysteine 114 of the human D3 dopamine receptor to ligand binding and sensitivity to external oxidizing agents.

1. Cysteine 114 (C114) of the human dopamine D3 receptor is located at the helical face of transmembrane segment III (TMIII) near aspartate 110, a counterion for the amine group of catecholamines. The contributions of C114 to receptor function were investigated here using site-directed mutagenesis of C114 to serine. 2. The C114S mutant, as expressed in Sf-9 cells, bound aminotetralin antagonists (UH-232 and AJ-76) and several agonists ((-)3-PPP, apomorphine, pramipexole and quinpirole) with markedly lower affinities as compared to the wild type D3 receptor, but bound other structurally diverse dopaminergic ligands with only minor changes in affinity. Because an N-propyl substituent is the only common structural feature among most affected ligands, we propose that the mutation alters 'a propyl cleft' on the receptor. The mutation hardly affected quinpirole-dependent [35S]-GTPgammaS binding, suggesting C114 plays a minimal role in receptor-G-protein coupling. 3. N-Ethylmaleimide(NEM), a sulfhydryl modifying agent, blocked ligand binding to the D3 receptor, but not to the C114S mutant. We infer that C114 is the primary residue on the D3 receptor vulnerable to external oxidizing agents. Dopamine D2long and D4(2) receptors contain highly homologous TMIII sequences including an equivalent cysteine residue. However, only the D2long receptor, not the D4(2) receptor, displayed NEM sensitivity similar to that of the D3 receptor. 4. We conclude that C114 is critical for high affinity interactions between the D3 receptor and ligands containing an N-propyl substituent, and unlike its counterpart in the D4(2) receptor, is highly susceptible to external oxidizing agents.

[Synaptic contacts in schizophrenia: study with immunocytochemical identification of dopaminergic neurons]. / Sinapticheskie kontakty pri shizofrenii: issledovaniie s immunotsitokhimicheskoi identifikatsiei dofaminergicheskikh neironov.

Immunocytochemical identification of dopaminergic neurons was made by means of immunoperoxidase method using antibodies to tyrosine hydroxylase. The ultrastructure of synaptic contacts located on dopaminergic (tyrosine hydroxylase immunopositive--TP) neurons was investigated in substantia nigra (SN) of postmortem brains in 4 schizophrenic patients compared to 3 mentally healthy control cases. Various alterations of ultrastructure have been found in presynaptic terminals which contracted with TP neurons in SN of schizophrenic patients as compared to the control group. These alterations included: presynaptic axon terminals decreased in sizes with dense matrix and density packed vesicles; swollen terminals with clustering of synaptic vesicles and displaced active zone of synapse; hyperplasia of mitochondria in the part of presynaptic boutons; appearance of abnormal membranous lamellar structures inside or outside presynaptic boutons. The altered presynaptic terminals were preferentially located on distal (small and middle diameter) TP dendrites in pars compacta of SN, where practically all dendrites revealed belonged to dopaminergic neurons and formed asymmetric contacts with short active zones in most cases. The changes of ultrastructure of synaptic contacts could be rarely observed in SN pars reticulata of schizophrenic patients both on TP and tyrosine hydroxylase immunonegative dendrites presented here.

[The brain dopaminergic systems: receptor heterogeneity, functional role and pharmacological regulation]. / Dofaminergicheskie sistemy mozga: retseptornaia geterogennost', funktsional'naia rol', farmakologicheskaia reguliatsiia.

Recent advances in molecular neurobiology led to a new understanding on mammalian brain dopaminergic system which play a major role in the regulation of motor, cognitive, emotional, neuroendocrine function as well as in the pathogenesis of several pathological conditions, including neurodegenerative diseases, affective disorders, schizophrenia, drug addiction etc. Functional, biochemical and pharmacological heterogeneity of dopamine receptors, which were divided into D1-like (D1 and D5 subtypes) and D2-like (D2, D3 and D4) families of receptors has been postulated. The article reviews the recent advances including author's own results concerning the structure and function of main dopaminergic brain system, i.e. nigrostriatal and mesolimbic. The problem of autoreceptor regulation of dopaminergic neurotransmission, particularly, the processes of dopamine synthesis, release and metabolism has been specially discussed. An involvement of D2 and D3 dopamine autoreceptors in the control of these processes and differences in the mode of action of typical and atypical neuroleptics demonstrating various affinities to D2 and D3 dopamine receptors are analysed in detail. Dopamine and its metabolites have been determined on freely moving rats using brain microdialysis and high performance liquid chromatography. It is hypothesized that dopamine D3 autoreceptor is preferentially involved in the regulation of dopamine release while D2 one is responsible for the control of dopamine synthesis and metabolism in rat basal ganglia in vivo.

[Projections of the amygdaloid body and dopaminergic mesencephalic formations to the entopeduncular nucleus of the cat brain]. / Proektsii mindalevidnogo tela i dofaminergicheskikh mezentsefalicheskikh obrazovanii na éntopedunkuliarnoe iadro mozga koshki.

Organization of projections on entopeduncular nucleus different regions from amygdala and from nuclei of tegmental ventral field and black substance was studied in cat using retrograde axonal transport of house radish peroxidose. Analysis of the labelled neurons distribution in these structures in separate marker injections into the rostral and caudal segments of entopeduncular nucleus allowed to conclude that projective fibres to rostral region come from not numerous neurons of tegmental ventral field and black substance compact part, and to caudal one apart from these structures neurons--also from scanty neurons of dorsal and lateral parts of black substance. Projections on entopeduncular nucleus from amygdala were not found.

[Brain synaptic plasticity in schizophrenia]. / Sinapticheskaia plastichnost' mozga pri shizofrenii.

A qualitative and quantitative electron microscopic study was performed. in some postmortem brain structures of the dopaminergic system: the substantia nigra, ventral tegmentum area, prefrontal cortex (area 10), anterior cingulate cortex (area 24), the head of the caudate nucleus in schizophrenia and age-matched controls without mental disorders. The study revealed the heterogeneity of ultrastructural changes in synaptic ultrastructure and organization in schizophrenia as compared to controls. There was an anomalous sprouting of axons and a trend to increase the numerical density of axospinous synapses and to decrease axodendritic synapses in the schizophrenic brains due to disontogenesis or anomalous synaptic plasticity. No correlations with neuroleptic treatment were found. Synaptic plasticity might be involved in the pathophysiology and clinical symptoms of schizophrenic patients.

Cellular localization of dopamine D2 receptor messenger RNA in the rat trigeminal ganglion.

The actions of dopamine are mediated by specific, high-affinity, G protein-coupled receptors. Multiple subtypes of dopamine receptors have been characterized, including the D2 subtype (D2R). Cells within the dorsal root and petrosal ganglia of the rat express D2R messenger RNA (mRNA) consistent with D2R expression by primary sensory neurons. We hypothesized that neurons of the trigeminal ganglion express D2R mRNA. Total cellular RNA from rat trigeminal ganglia was analyzed on Northern blots under high stringency conditions. Hybridization of trigeminal ganglion RNA resulted in a signal which comigrated with striatal, pituitary, and hypothalamic D2R mRNA. To determine the distribution of D2R expressing cells in the trigeminal ganglion, cryostat sections were analyzed by in situ hybridization followed by emulsion autoradiography. We identified a population of clustered cells labeled with dense grain concentrations over their cytoplasms. These findings demonstrate the expression of D2 dopamine receptor mRNA in discrete subpopulations of neurons in the rat trigeminal ganglion. Our observations suggest that drugs active at dopamine receptors of the D2 subtype are potential modulators of sensory activity of neurons whose cell bodies reside in the trigeminal ganglion. D2 dopamine receptors may thus have a role in clinical pain syndromes involving the head and neck.

[The development of a graft of embryonic mediobasal hypothalamus in the 3rd cerebral ventricle of the adult rat]. / Razvitie transplantata émbrional'nogo mediobazal'nogo gipotalamusa v 3-m zheludochke mozga vzrosloi krysy.

Development of the mediobasal hypothalamus from 15-day rat foetuses and 8-week human foetuses transplanted in the 3rd ventricle of the adult rat brain has been morphologically analyzed. The graft was shown to fill the ventral area of the 3rd ventricle and integrate with the host brain, as was especially distinct in the region of optic chiasma. The graft was abundantly vascularized and its vessels connected with the host brain vessels. The graft neurons were normally differentiating in situ. Some neurons migrated in the host brain. The graft neuropile ultrastructure was characterized by the abundance of synaptic contacts. Some graft neurons expressed dopaminergic phenotype by synthesizing tyrosine hydroxylase and DOPA-decarboxylase and displaying specific capture of 3H-dopamine. Dopaminergic axons of the neurons were spreading both within the graft and penetrating in the host tissue, especially in the region of optic chiasma and tracts. Unlike allotransplantation in rats, survival of xenotransplants of the human embryonic nervous tissue in the 3rd ventricle of the adult rat brain was possible only under the conditions of constant immunosuppression.

Detection of putative dopamine receptors in neurites outgrowing from locust central nervous system explants using anti-idiotypic dopamine antibodies.

Organotypic cultures established from the third thoracic ganglion of locust embryo have been used to investigate dopamine receptors. In this in vitro system, neurites emerge directly from the explants and form a dense network around the explants, presenting cell surface freely exposed for experimental labelling. Polyclonal anti-idiotypic antibodies raised in rabbits to antibodies against dopamine conjugate, and previously found to bind to dopamine receptors, have been used to investigate putative dopamine receptors in these neurites. Immunocytochemical detection by light microscopy employing immunofluorescence labelling, was correlated with electron microscopy, using peroxidase staining. In addition to a location for dopamine receptors on the neurite surface, intracellular binding sites were also found in neurites. This internal labelling might represent an intracellular pool of dopamine receptor precursors. The labelling was specific in that it was not present when the anti-idiotypic dopamine antibodies were replaced with non-immune serum or when preincubation with conjugated dopamine preceded incubation with anti-idiotypic dopamine antibodies.

Microanatomy of the dopaminergic system in the rainbow trout retina.

We have investigated the morphology of dopaminergic interplexiform cells as well as the distribution of two classes of dopamine receptors in the retina of the rainbow trout. Interplexiform cells were visualized using an antiserum against tyrosine hydroxylase and PAP immunocytochemistry. In whole amounts, these cells have a density of between 91 and 182 cells per mm2 with highest values in the lower temporal quadrant. Their cell bodies lie at the inner margin of the inner nuclear layer with only 12-17 cells per retina displaced to the ganglion cell layer. There are three levels of stratification in the inner plexiform layer, one at the distal and proximal borders respectively, and one in the middle. They arise mostly from a radially oriented, stout primary dendrite. Tangential processes are about 1 micron in diameter and show a number of varicosities. The density of processes is greatest in sublayer 5, but no major difference in the general organization is apparent between the three sublayers. In the outer retina, there are two levels of dense ramification confined to the layer of horizontal cells. Light and electron microscopic analysis shows synaptic input to horizontal cells, but not to photoreceptors. The distribution of D1 receptors was assessed by studying the binding pattern of a specific, fluorescent-labelled antagonist, SCH 23390, in unfixed frozen sections. We found displaceable binding in the inner and outer plexiform layers and in the region of horizontal cell perikarya. We used an anti-peptide antibody directed to an extracellular domain of the rat D2 receptor and a fluorescent secondary antiserum to study the localization of D2 receptors. In addition to marked label in both plexiform layers, the outer, and especially the inner segments of rods and cones show specific immunoreactivity. In addition, there is distinct label at the level of the horizontal cell bodies; in the inner retina, specific fluorescence is found in somata of some amacrine cells. The significance of the connectivity pattern and the distribution of the two receptor types is discussed with respect to the role of dopamine in controlling adaptational processes in the outer retina, such as retinomotor movements and changes in horizontal cell morphology and physiology.

Effect of adding the D1 agonist CY 208-243 to chronic bromocriptine treatment. I: Evaluation of motor parameters in relation to striatal catecholamine content and dopamine receptors.

A group of four cynomolgus monkeys previously rendered parkinsonian by the toxin 1-methyl-4-phenyl,1,2,3,6-tetrahydropyridine (MPTP) were observed in locomotion cages equipped with photocells during four periods of 7 days during which they received saline or two doses of the D1 agonist CY 208-243. The larger dose of 0.5 mg/kg produced a significant increase in locomotion in three of four animals. A second group of eight monkeys also previously rendered parkinsonian by MPTP and having received no other treatment were given a daily treatment of bromocriptine 1.66 mg/kg orally daily during 4 weeks. In four of the animals, after a week on bromocriptine alone, the D1 agonist CY 208-243 was added in increasing doses of 0.05, 0.1, and 0.5 mg/kg. The motor response as measured by locomotion, hand dexterity, and a disability score improved progressively at least in some of the animals on bromocriptine alone. The addition of CY 208-243 produced a more striking improvement of all three parameters, which appeared to be dose dependent. Biochemical analysis of the brain of these animals plus one control and one MPTP untreated monkey showed a > 90% loss of dopamine in the striatum in six of the eight treated monkeys. Both D2 and D1 dopamine receptors were increased in density by denervation, but both treatments abolished this increase for the D2 receptors while increasing the affinity of the D1 receptors.

Molecular modelling of D2-like dopamine receptors.

Three-dimensional computer models of the rat D2, D3 and D4 dopamine receptor subtypes have been constructed based on the diffraction co-ordinates for bacteriorhodopsin, another membrane-bound protein containing seven transmembrane domains presumed to be arranged in a similar spatial orientation. Models were assembled by aligning the putative transmembrane domains of the dopamine receptors with those of bacteriorhodopsin using sequence similarities, and then superimposing these modelled alpha-helices on to the bacteriorhodopsin-derived co-ordinates. These models explore the potential hydrogen bonding, electrostatic and stacking interactions within the receptor which may be important for maintaining the conformation of these receptors, and thereby provide target sites for agonist binding. Proposed interactions between the catecholamine ligands and these receptors appear to account for the affinity, although not the specificity, of these agonist ligands for the different dopamine receptor subtypes. Such models will be useful for establishing structure-function relationships between ligands and the dopamine receptors, and may ultimately provide a template for the design of receptor-specific drugs.

Molecular biology of adrenergic and dopamine receptors and the study of developmental nephrology.

Neurotransmitters convey specific messages by binding to receptors on the cell membrane surface. Receptors are linked to membrane-bound, signal-transducing proteins which act as intermediaries in the generation of second messengers that elicit biological responses. Cell surface receptors could be grouped into families that utilize common systems for their signal transmission. These classes include the growth factor receptors, the transporter receptors which internalize their ligands, ion channels, and G-protein-coupled receptors. In the past few years, the cDNAs and/or genes of a number of G-protein-coupled receptors have been cloned. Structural analysis of the G-protein-coupled receptors, as well as the other classes of receptor, shows that those receptors which use a common signaling pathway have similar topographies and share significant sequence homology. Adrenergic and dopamine receptors are examples of receptors coupled to G proteins. This review outlines some strategies in the study of adrenergic and dopamine receptors using molecular biology techniques and how they relate to investigations in developmental nephrology.

Some characteristics of rat nigrostriatal dopaminergic neuroconnection during development--a combined immunocytochemical and electron-microscopic study.

The characteristics of the nigrostriatal dopaminergic neuroconnections in developing rats are studied by combined immunocytochemical and electron-microscopic techniques, with an antibody to tyrosine hydroxylase (TH). From the embryonic day 21 on, some of the TH-positive nerve fibers are densely packed in the striatum to form a patch-like dopamine island. The percentage of the TH-positive nerve terminals among the labeled profiles is much higher inside than outside the dopamine island (P less than 0.01). On the other hand, the TH-positive terminals mainly form symmetrical axon-dendritic synapses, while most of the TH-negative terminals form asymmetrical axon-spinous synapses. The functional significance of the characterized dopaminergic connection is discussed.

Clues to the mechanism underlying dopamine cell death in Parkinson's disease.

The primary pathological change in Parkinson's disease is the destruction of dopamine containing cells in the zona compacta of substantia nigra. The cause of nigral cell death and the underlying mechanism remains elusive. However, the discovery of the selective nigral neurotoxin MPTP and its ability to inhibit mitochondrial energy metabolism via its metabolite MPP+ and to generate superoxide radicals suggests processes by which nigral cell death might occur. Recent postmortem evidence in brain tissue from patients dying with Parkinson's disease also suggests the occurrence of some on-going toxic mechanism. This may be a free radical process stimulated by an excess of iron within substantia nigra coupled to a generalised decrease in brain ferritin content. These data suggest altered iron handling occurs in Parkinson's disease which may lead to the generation of toxic oxygen species such as superoxide radicals. There is also evidence for an inhibition of mitochondrial function in the substantia nigra in patients with Parkinson's disease. So there may be a close association between the actions of the synthetic neurotoxin MPTP and the underlying cause of idiopathic Parkinson's disease.