Alicia Rivera

DIFFERENTIAL REGIONAL AND CELLULAR DISTRIBUTION OF DOPAMINE D2-LIKE RECEPTORS : AN IMMUNOCYTOCHEMICAL STUDY OF SUBTYPE-SPECIFIC ANTIBODIES IN RAT AND HUMAN BRAIN

Dopamine D2‐like receptors (D2, D3, and D4) are major targets for action of typical and atypical neuroleptics, commonly used in the treatment of schizophrenia. To understand their individual functional contribution, subtype‐selective anti‐peptide antibodies were raised against D2, D3, and D4 receptor proteins. The antibodies were shown to be specific on immunoblots of rat brain membranes and immunoprecipitated the solubilized native dopamine receptors in an antibody concentration‐dependent manner. In addition, they also bind selectively to the respective recombinant D2, D3, and D4 receptor membrane proteins from cDNA transfected cells. Immunolocalization studies show that the D2‐like receptor proteins had differential regional and cellular distribution in the cerebral cortex, hippocampus, basal ganglia, cerebellum, and midbrain, thus providing anatomical substrate for area‐specific regulation of the dopamine neurotransmission. In cortical neurons, D4 receptor protein was found in both pyramidal and nonpyramidal cells, whereas D2 and D3 seem to be mostly associated with nonpyramidal interneurons. In rat hippocampus, the expression pattern of D2‐like receptors (D4>D3>D2) mirrored that obtained with immunoprecipitation studies. D2 and D4 receptor immunolabeling was observed in the thalamic reticular nucleus, which was negative for the D3 subtype. Species differences were also observed; for example, the D4 subtype receptor is the most highly expressed protein in the rat cortex, whereas it is significantly less in human cortex. Differential patterns of D2, D3, and D4 receptor expression in rat and human brain should shed light on the therapeutic actions of neuroleptic drugs and may lead to the development of more specifically targeted antipsychotic drugs. J. Comp. Neurol. 402:353–371, 1998.

Dopamine D5 receptors of rat and human brain

In contrast to dopamine D1 receptors, the anatomical distribution of D5 receptors in the CNS is poorly described. Therefore, we have studied the localization of dopamine D5 receptors in the brain of rat and human using our newly prepared subtype-specific antibody. Western blot analysis of brain tissues and membranes of cDNA transfected cells, and immunoprecipitation of brain dopamine receptors suggest that this antibody is highly selective for native dopamine D5 receptors. The D5 antibody labeled dopaminergic neurons of mesencephalon, and cortical and subcortical structures. In neostriatum, the D5 receptors were localized in the medium spiny neurons and large cholinergic interneurons. The D5 labeling in caudate nucleus was predominantly in spines of the projection neurons that were frequently making asymmetric synapses. Occasionally, the D5 receptors were also found at the symmetric synapses. Within the cerebral cortex and hippocampus, D5 antibody labeling was prominent in the pyramidal cells and their dendrites. Dopamine D5 receptors were also prominent in the cerebellum, where dopamine innervation is known to be very modest. Differences in the localization of D5 receptors between both species were generally indistinguishable except in hippocampus. In rat, the hippocampal D5 receptor was concentrated in the cell body, whereas in human it was also associated with dendrites. These results show that D5 receptors are localized in the substantia nigra-pars compacta, hypothalamus, striatum, cerebral cortex, nucleus accumbens and olfactory tubercle. Furthermore, the presence of D5 receptors in the areas of dopamine pathways suggests that this receptor may participate actively in dopaminergic neurotransmission.

From the Golgi–Cajal mapping to the transmitter-based characterization of the neuronal networks leading to two modes of brain communication: Wiring and volume transmission ☆

After Golgi-Cajal mapped neural circuits, the discovery and mapping of the central monoamine neurons opened up for a new understanding of interneuronal communication by indicating that another form of communication exists. For instance, it was found that dopamine may be released as a prolactin inhibitory factor from the median eminence, indicating an alternative mode of dopamine communication in the brain. Subsequently, the analysis of the locus coeruleus noradrenaline neurons demonstrated a novel type of lower brainstem neuron that monosynaptically and globally innervated the entire CNS. Furthermore, the ascending raphe serotonin neuron systems were found to globally innervate the forebrain with few synapses, and where deficits in serotonergic function appeared to play a major role in depression. We propose that serotonin reuptake inhibitors may produce antidepressant effects through increasing serotonergic neurotrophism in serotonin nerve cells and their targets by transactivation of receptor tyrosine kinases (RTK), involving direct or indirect receptor/RTK interactions. Early chemical neuroanatomical work on the monoamine neurons, involving primitive nervous systems and analysis of peptide neurons, indicated the existence of alternative modes of communication apart from synaptic transmission. In 1986, Agnati and Fuxe introduced the theory of two main types of intercellular communication in the brain: wiring and volume transmission (WT and VT). Synchronization of phasic activity in the monoamine cell clusters through electrotonic coupling and synaptic transmission (WT) enables optimal VT of monoamines in the target regions. Experimental work suggests an integration of WT and VT signals via receptor-receptor interactions, and a new theory of receptor-connexin interactions in electrical and mixed synapses is introduced. Consequently, a new model of brain function must be built, in which communication includes both WT and VT and receptor-receptor interactions in the integration of signals. This will lead to the unified execution of information handling and trophism for optimal brain function and survival.

Intramembrane receptor-receptor interactions: a novel principle in molecular medicine

Summary.In 1980/81 Agnati and Fuxe introduced the concept of intramembrane receptor–receptor interactions and presented the first experimental observations for their existence in crude membrane preparations. The second step was their introduction of the receptor mosaic hypothesis of the engram in 1982. The third step was their proposal that the existence of intramembrane receptor–receptor interactions made possible the integration of synaptic (WT) and extrasynaptic (VT) signals. With the discovery of the intramembrane receptor–receptor interactions with the likely formation of receptor aggregates of multiple receptors, so called receptor mosaics, the entire decoding process becomes a branched process already at the receptor level in the surface membrane. Recent developments indicate the relevance of cooperativity in intramembrane receptor–receptor interactions namely the presence of regulated cooperativity via receptor–receptor interactions in receptor mosaics (RM) built up of the same type of receptor (homo-oligomers) or of subtypes of the same receptor (RM type1). The receptor–receptor interactions will to a large extent determine the various conformational states of the receptors and their operation will be dependent on the receptor composition (stoichiometry), the spatial organization (topography) and order of receptor activation in the RM. The biochemical and functional integrative implications of the receptor–receptor interactions are outlined and long-lived heteromeric receptor complexes with frozen RM in various nerve cell systems may play an essential role in learning, memory and retrieval processes. Intramembrane receptor–receptor interactions in the brain have given rise to novel strategies for treatment of Parkinson’s disease (A2A and mGluR5 receptor antagonists), schizophrenia (A2A and mGluR5 agonists) and depression (galanin receptor antagonists). The A2A/D2, A2A/D3 and A2A/mGluR5 heteromers and heteromeric complexes with their possible participation in different types of RM are described in detail, especially in the cortico-striatal glutamate synapse and its extrasynaptic components, together with a postulated existence of A2A/D4 heteromers. Finally, the impact of intramembrane receptor–receptor interactions in molecular medicine is discussed outside the brain with focus on the endocrine, the cardiovascular and the immune systems.

Molecular phenotype of rat striatal neurons expressing the dopamine D5 receptor subtype

Dopamine is one of the principal neurotransmitters in the basal ganglia, where it plays a critical role in motor control and cognitive function through its interactions with the specific dopamine receptors D1 to D5. Although the activities mediated by most dopamine receptor subtypes have already been determined, the role of the D5 receptor subtype in the basal ganglia has still not been established. Furthermore, it is often difficult to distinguish between dopamine D5 and D1 receptors as they are stimulated by the same ligands, and they have a similar molecular structure and pharmacology. In an effort to understand the differences between these two receptor subtypes, we have studied the distribution of neurons containing D5 receptors in the striatum, and their molecular phenotype. As a result, we show that the D5 receptor subtype is present in two different populations of striatal neurons, projection neurons and interneurons. Overall, the abundance of this receptor subtype in the striatum is low, particularly in striatal projection neurons of both the direct and indirect projection pathways. In contrast, the expression of D5 receptors in striatal interneurons (cholinergic, somatostatin‐ or parvalbumin‐positive neurons) is high, while low to moderate expression was observed in calretinin‐positive neurons. Our results demonstrate the presence of D5 receptors in all the striatal cell populations so far described, although at different intensities in each. The fact that a large number of striatal neurons express the D5 receptor subtype suggests that this receptor fulfils an important function in the process of integrating information in the striatum.

Dopamine D4 receptors are heterogeneously distributed in the striosomes/matrix compartments of the striatum.

Two important aspects of striatal function, exploratory behaviour and motor co‐ordination, require the integrity of the dopamine D4 receptor subtype. These receptors are also implicated in the pathophysiology of certain neuropsychiatric disorders. However, the distribution of D4 receptors in the striatum has not yet been described and this situation impairs our understanding of the anatomical substrate in which D4 receptors function. We developed a D4 receptor‐specific anti‐body that has permitted us to investigate the regional and cellular localization of the receptor in the neostriatum of the rat, mouse, cat and monkey. The subcellular distribution and the synaptic organization of this receptor were also determined in the rat striatum. We found moderate levels of D4 receptor expression in the caudoputamen and lower levels in the nucleus accumbens. These receptors were expressed in cell bodies and in the neuropil and were heterogeneously distributed among different striatal compartments, being more abundant in striosomes than in the matrix. At the subcellular level, the receptor immunoreactivity was mainly localized to dendritic shafts and spines. The prominent immunoreactivity observed in the striosomes indicates that integrative processes involved in D4‐mediated limbic behaviours occurs through the striosomes rather than accumbens, whereas the motor behaviour is based in the striatal matrix.

Volume transmission and wiring transmission from cellular to molecular networks: history and perspectives.

The present paper deals with a fundamental issue in neuroscience: the inter‐neuronal communication. The paper gives a brief account of our previous and more recent theoretical contributions to the subject and also reports new recent data that support some aspects of our proposal on two major modes of communication in the central nervous system: the wiring and the volume transmission. There exist two competing theories on inter‐neuronal communication: the neuron doctrine and the theory of the diffuse nerve network, supported by Cajal and Golgi, respectively (see their respective Nobel Lectures). The present paper gives a brief account of a view on inter‐neuronal communication in the brain, the volume and wiring transmission concept that to a great extent reconcile these two theories. Thus, the theory of volume and wiring transmission are summarized and its recent developments that allow to extend these two modes of communication from the cellular network to the molecular network level is also briefly illustrated. The explanatory value of this broadened view is further enhanced by our recent proposal on the existence of a Global Molecular Network enmeshing the entire central nervous system. It may be interesting to note that also the Global Molecular Network theory is reminiscent of the old reticular theory of Apathy. Finally, the so‐called ‘tide hypothesis’ for diffusion of signals in the brain is briefly discussed and its possible extension to the molecular level is for the first time introduced. Early indirect evidence supporting volume transmission in the brain was the discovery of transmitter‐receptor mismatches. Thus, as an experimental part of the present paper a new approach to evaluate transmitter‐receptor mismatches is given and evidence for inter‐relationships between temperature micro‐gradients and mismatches is provided.

Headpiece domain of dematin is required for the stability of the erythrocyte membrane

Dematin is an actin-binding and bundling protein of the erythrocyte membrane skeleton. Dematin is localized to the spectrin–actin junctions, and its actin-bundling activity is regulated by phosphorylation of cAMP-dependent protein kinase. The carboxyl terminus of dematin is homologous to the “headpiece” domain of villin, an actin-bundling protein of the microvillus cytoskeleton. The headpiece domain contains an actin-binding site, a cAMP-kinase phosphorylation site, plays an essential role in dematin self-assembly, and bundles F-actin in vitro. By using homologous recombination in mouse embryonic stem cells, the headpiece domain of dematin was deleted to evaluate its function in vivo. Dematin headpiece null mice were viable and born at the expected Mendelian ratio. Hematological evaluation revealed evidence of compensated anemia and spherocytosis in the dematin headpiece null mice. The headpiece null erythrocytes were osmotically fragile, and ektacytometry/micropore filtration measurements demonstrated reduced deformability and filterability. In vitro membrane stability measurements indicated significantly greater membrane fragmentation of the dematin headpiece null erythrocytes. Finally, biochemical characterization, including the vesicle/cytoskeleton dissociation, spectrin self-association, and chemical crosslinking measurements, revealed a weakened membrane skeleton evidenced by reduced association of spectrin and actin to the plasma membrane. Together, these results provide evidence for the physiological significance of dematin and demonstrate a role for the headpiece domain in the maintenance of structural integrity and mechanical properties of erythrocytes in vivo.

Dopamine D2 and D4 receptor heteromerization and its allosteric receptor–receptor interactions

Dopamine D(2) and D(4) receptors partially codistribute in the dorsal striatum and appear to play a fundamental role in complex behaviors and motor function. The discovery of D(2)R-D(4.)(x)R (D(4.2)R, D(4.4)R or D(4.7)R) heteromers has been made in cellular models using co-immunoprecipitation, in situ Proximity Ligation Assays and BRET(1) techniques with the D(2)R and D(4.7)R receptors being the least effective in forming heteromers. Allosteric receptor-receptor interactions in D(2)R-D(4.2)R and D(2)R-D(4.4) R heteromers were observed using the MAPK assays indicating the existence of an enhancing allosteric receptor-receptor interaction in the corresponding heteromers between the two orthosteric binding sites. The bioinformatic predictions suggest the existence of a basic set of common triplets (ALQ and LRA) in the two participating receptors that may contribute to the receptor-receptor interaction interfaces.

Protection but maintained dysfunction of nigral dopaminergic nerve cell bodies and striatal dopaminergic terminals in MPTP-lesioned mice after acute treatment with the mGluR5 antagonist MPEP

The mGluR5 antagonist MPEP was used to study the role of mGluR5 in MPTP-induced injury of the nigrostriatal DA neurons. The findings indicate that acute blockade of mGluR5 may result in neuroprotective actions against MPTP neurotoxicity on nigral DA cell bodies and striatal DA terminals using stereological analysis of TH immunoreactivity and microdensitometry. Biochemical analysis showed no restoration of DA levels and metabolism indicating a maintained reduction of DA transmission.