Existence of muscarinic acetylcholine receptor (mAChR) and fibroblast growth factor receptor (FGFR) heteroreceptor complexes and their enhancement of neurite outgrowth in neural hippocampal cultures.

BACKGROUND: Recently, it was demonstrated that G-protein-coupled receptors (GPCRs) can transactivate tyrosine kinase receptors in absence of their ligands. In this work, driven by the observation that mAChRs and fibroblast growth factor receptors (FGFRs) share signalling pathways and regulation of brain functions, it was decided to explore whether mAChRs activation may transactivate FGFRs and, if so, to characterize the related trophic effects in cultured hippocampal neurons. METHODS: Oxotremorine-M transactivation of FGFRs and related trophic effects were tested in primary hippocampal neurons. Western blotting and in situ proximity ligation assay (PLA) were used to detect FGFR phosphorylation (pFGFR) levels and M1R-FGFR1 heteroreceptor complexes, respectively. RESULTS: Oxotremorine-M, a non-selective mAChRs agonist, was able to transactivate FGFR and this transactivation was blocked by Src inhibitors. Oxotremorine-M treatment produced a significant increase in the primary neurite outgrowth that was blocked by pre-treatment with the pFGFR inhibitor SU5402 and Src inhibitors. This trophic effect was almost similar to that induced by fibroblast growth factor-2 (FGF-2). By using atropine as nonselective mAChRs or pirenzepine as selective antagonist for M1 receptor (M1R) we could show that mAChRs are involved in modulating the pFGFRs. Using PLA, M1R-FGFR1 heteroreceptor complexes were identified in the hippocampus and cerebral cortex. CONCLUSION: The current findings, by showing functional mAChR-FGFR interactions, will contribute to advance the understanding of the mechanisms involved in the actions of cholinergic drugs on neuronal plasticity. GENERAL SIGNIFICANT: Data may help to develop novel therapeutic strategies not only for neurodegenerative diseases but also for depression-induced atrophy of hippocampal neurons.

Microvesicle and tunneling nanotube mediated intercellular transfer of g-protein coupled receptors in cell cultures.

Recent evidence shows that cells exchange collections of signals via microvesicles (MVs) and tunneling nano-tubes (TNTs). In this paper we have investigated whether in cell cultures GPCRs can be transferred by means of MVs and TNTs from a source cell to target cells. Western blot, transmission electron microscopy and gene expression analyses demonstrate that A(2A) and D(2) receptors are present in released MVs. In order to further demonstrate the involvement of MVs in cell-to-cell communication we created two populations of cells (HEK293T and COS-7) transiently transfected with D(2)R-CFP or A(2A)R-YFP. These two types of cells were co-cultured, and FRET analysis demonstrated simultaneously positive cells to the D(2)R-CFP and A(2A)R-YFP. Fluorescence microscopy analysis also showed that GPCRs can move from one cell to another also by means of TNTs. Finally, recipient cells pre-incubated for 24 h with A(2A)R positive MVs were treated with the adenosine A(2A) receptor agonist CGS-21680. The significant increase in cAMP accumulation clearly demonstrated that A(2A)Rs were functionally competent in target cells. These findings demonstrate that A(2A) receptors capable of recognizing and decoding extracellular signals can be safely transferred via MVs from source to target cells.

Aspects on the integrative actions of the brain from neural networks to "brain-body medicine".

"Integration" is a key term in describing how nervous system can perform high level functions. A first condition to have "integration" is obviously the presence of efficient "communication processes" among the parts that have to be combined into the harmonious whole. In this respect, two types of communication processes, called wiring transmission (WT) and volume transmission (VT), respectively, were found to play a major role in the nervous system, allowing the exchange of signals not only between neurons, but rather among all cell types present in the central nervous system (CNS). A second fundamental aspect of a communication process is obviously the recognition/decoding process at target level. As far as this point is concerned, increasing evidence emphasizes the importance of supramolecular complexes of receptors (the so called receptor mosaics) generated by direct receptor-receptor interactions. Their assemblage would allow a first integration of the incoming information already at the plasma membrane level. Recently, evidence of two new subtypes of WT and VT has been obtained, namely the tunnelling nanotubes mediated WT and the microvesicle (in particular exosomes) mediated VT allowing the horizontal transfer of bioactive molecules, including receptors, RNAs and micro-RNAs. The physiological and pathological implications of these types of communication have opened up a new field that is largely still unexplored. In fact, likely unsuspected integrative actions of the nervous system could occur. In this context, a holistic approach to the brain-body complex as an indissoluble system has been proposed. Thus, the hypothesis has been introduced on the existence of a brain-body integrative structure formed by the "area postrema/nucleus tractus solitarius" (AP/NTS) and the "anteroventral third ventricle region/basal hypothalamus with the median eminence" (AV3V-BH). These highly interconnected regions operate as specialized interfaces between the brain and the body integrating brain-borne and body-borne neural and humoral signals.

Dopamine nerve terminals in the rat limbic cortex: aspects of the dopamine hypothesis of schizophrenia.

The existence of cortical dopamine nerve terminals is demonstrated with a highly sensitive modification of the Falck-Hillarp fluorescence technique. This confirms previous biochemical reports of high dopamine levels in the cortex. The histochemistry reveals that the distribution is regional and confined to the limbic cortex.

Tremor and involuntary movements in monkeys: effect of L-dopa and of a dopamine receptor stimulating agent.

Either L-dopa, in combination with 1-alpha-methyldopa hydrazine (MK-486), or 1-(2''-pyrimidyl)-4-piperonylpiperazine, an agent that stimulates dopamine receptors, relieves surgically induced tremor in monkeys and concomitantly evokes involuntary movements. These results indicate that tremor and involuntary movements are associated with a common mechanism and that the activity of the dopamine receptors is involved in the regulation of these dysfunctions.

Prolactin-like immunoreactivity: localization in nerve terminals of rat hypothalamus.

Antibodies to rat prolactin were used in immunohistochemical studies of the hypothalamus and preoptic area of the rat. Evidence was obtained that a protein immunochemically related to prolactin was stored in networks of nerve terminals of many hypothalamic areas such as the arcuate nucleus, the dorsomedial hypothalamic nucleus, and periventricular regions of the hypothalamus and preoptic area. The neuronal storage of a prolactin-like protein in the hypothalamus was unaffected by hypophysectomy.

Implications of the 'Energide' concept for communication and information handling in the central nervous system.

Recently a revision of the cell theory has been proposed, which has several implications both for physiology and pathology. This revision is founded on adapting the old Julius von Sach's proposal (1892) of the Energide as the fundamental universal unit of eukaryotic life. This view maintains that, in most instances, the living unit is the symbiotic assemblage of the cell periphery complex organized around the plasma membrane, some peripheral semi-autonomous cytosol organelles (as mitochondria and plastids, which may be or not be present), and of the Energide (formed by the nucleus, microtubules, and other satellite structures). A fundamental aspect is the proposal that the Energide plays a pivotal and organizing role of the entire symbiotic assemblage (see Appendix 1). The present paper discusses how the Energide paradigm implies a revision of the concept of the internal milieu. As a matter of fact, the Energide interacts with the cytoplasm that, in turn, interacts with the interstitial fluid, and hence with the medium that has been, classically, known as the internal milieu. Some implications of this aspect have been also presented with the help of a computational model in a mathematical Appendix 2 to the paper. Finally, relevances of the Energide concept for the information handling in the central nervous system are discussed especially in relation to the inter-Energide exchange of information.

Possible role of receptor heteromers in multiple sclerosis.

Recent results suggest that cytokine and glutamate receptors can interact directly and form receptor heteromers. Due to such heteromers, cytokines can act not only as classical neuromediators but also directly enhance glutamate gated ion channel activity via allosteric mechanisms. Our opinion is that such heteromers may lead to enhanced glutamate neurotoxicity in pathogenic processes of multiple sclerosis. Thus, agents targeting evolutionary conserved leucine-rich motifs responsible for such dimerization of receptors may represent a useful strategy to inhibit excitotoxicity in multiple sclerosis.

The FGF-2/FGFRs neurotrophic system promotes neurogenesis in the adult brain.

Neurogenesis occurs in two regions of the adult brain, namely, the subventricular zone (SVZ) throughout the wall of the lateral ventricle and the subgranular zone (SGZ) of the dentate gyrus (DG) in hippocampal formation. Adult neurogenesis requires several neurotrophic factors to sustain and regulate the proliferation and differentiation of the adult stem cell population. In the present review, we examine the cellular and functional aspects of a trophic system mediated by fibroblast growth factor-2 (FGF-2) and its receptors (FGFRs) related to neurogenesis in the SVZ and SGZ of the adult rat brain. In the SVZ, FGF-2 is expressed in GFAP-positive cells of SVZ but is not present in proliferating precursor cells, which instead express FGFR-1 and FGFR-2, but not FGFR-3 mRNA, although expressed in the SVZ, and FGFR-4. Therefore, it seems that in the SVZ FGF-2 may be released by GFAP-positive cells, different from the precursor cell lineage, and via volume transmission it reaches the proliferating precursor cells. FGFR-1 mRNA is also expressed in the SGZ and is localized in BrdU-labeled precursor cells, whereas FGFR-2 and FGFR-3 mRNA, although expressed in the SGZ, are not located within proliferating precursor cells. An aged-related decline of proliferating precursor cells in the SVZ and DG of old rats has been well documented, and there is the suggestion that in part it could be the consequence of alterations in growth factor expression levels. Thus, the old precursors may respond to growth factors, suggesting that during aging the basic components for neuronal precursor cell proliferation are retained and the capacity to increase neurogenesis after appropriate stimulation is still preserved. In conclusion, the trophic system mediated by FGF-2 and its receptors contributes to create an important micro-environmental niche that promotes neurogenesis in the adult and aged brain.

Common key-signals in learning and neurodegeneration: focus on excito-amino acids, beta-amyloid peptides and alpha-synuclein.

In this paper a hypothesis that some special signals ("key-signals" excito-amino acids, beta-amyloid peptides and alpha-synuclein) are not only involved in information handling by the neuronal circuits, but also trigger out substantial structural and/or functional changes in the Central Nervous System (CNS) is introduced. This forces the neuronal circuits to move from one stable state towards a new state, but in doing so these signals became potentially dangerous. Several mechanisms are put in action to protect neurons and glial cells from these potentially harmful signals. However, in agreement with the Red Queen Theory of Ageing (Agnati et al. in Acta Physiol Scand 145:301-309, 1992), it is proposed that during ageing these neuroprotective processes become less effective while, in the meantime, a shortage of brain plasticity occurs together with an increased need of plasticity for repairing the wear and tear of the CNS. The paper presents findings supporting the concept that such key-signals in instances such as ageing may favour neurodegenerative processes in an attempt of maximizing neuronal plasticity.

Neurotensin receptor mechanisms and its modulation of glutamate transmission in the brain: relevance for neurodegenerative diseases and their treatment.

The extracellular accumulation of glutamate and the excessive activation of glutamate receptors, in particular N-methyl-D-aspartate (NMDA) receptors, have been postulated to contribute to the neuronal cell death associated with chronic neurodegenerative disorders such as Parkinson's disease. Findings are reviewed indicating that the tridecaptide neurotensin (NT) via activation of NT receptor subtype 1 (NTS1) promotes and reinforces endogenous glutamate signalling in discrete brain regions. The increase of striatal, nigral and cortical glutamate outflow by NT and the enhancement of NMDA receptor function by a NTS1/NMDA interaction that involves the activation of protein kinase C may favour the depolarization of NTS1 containing neurons and the entry of calcium. These results strengthen the hypothesis that NT may be involved in the amplification of glutamate-induced neurotoxicity in mesencephalic dopamine and cortical neurons. The mechanisms involved may include also antagonistic NTS1/D2 interactions in the cortico-striatal glutamate terminals and in the nigral DA cell bodies and dendrites as well as in the nigro-striatal DA terminals. The possible increase in NT levels in the basal ganglia under pathological conditions leading to the NTS1 enhancement of glutamate signalling may contribute to the neurodegeneration of the nigro-striatal dopaminergic neurons found in Parkinson's disease, especially in view of the high density of NTS1 receptors in these neurons. The use of selective NTS1 antagonists together with conventional drug treatments could provide a novel therapeutic approach for treatment of Parkinson's disease.

Structural plasticity in G-protein coupled receptors as demonstrated by the allosteric actions of homocysteine and computer-assisted analysis of disordered domains.

Structural plasticity of G-protein coupled receptors (GPCRs) is of basic importance for their interactions with ligands, in particular with other proteins such as receptors or receptor-modifying proteins that can lead to different functions for the same GPCR. In the present paper, structural plasticity of GPCRs has been investigated discussing allosteric modulatory actions of Homocysteine (Hcy) on D2 receptors together with data obtained by computer-assisted analysis of the presence of disordered domains in GPCRs. Previous evidence for a modulatory action of Hcy on D2 receptors has been further extended by means of experiments on the effects of Hcy local intrastriatal injection on rotational behaviour. Altogether the present data allow considering under a new angle the well known proposal of A2A antagonists as new therapeutic agents in Parkinson's disease (PD). Furthermore, they point out to not only the importance of drugs capable of reducing Hcy brain levels, but also to the potential therapeutic impact of drugs capable of regionally blocking (for PD) or enhancing (for some schizophrenic syndromes) Hcy allosteric action on D2 receptors. As far as the investigations on GPCR plastic domains, extracellular, intracellular and transmembrane domains of 14 GPCRs have been considered and propensity of each of these domains for a structured or unstructured conformation has been evaluated by means of ad hoc computer programs. It has been shown that the N- and C-terminals as well as intracellular loop 3 have a high propensity towards an unstructured conformation, hence they are potentially very plastic domains, which can undergo easily to interactions with other ligands, particularly with other protein domains. This aspect is obviously of the greatest importance not only for the function of single GPCRs, but also for their interactions either with other receptors (receptor-receptor interactions) or, more generally, for formation of clusters of membrane associated proteins, hence of "protein mosaics", where the GPCRs could represent the input unit of the supra-molecular device.

Receptor-receptor interactions within receptor mosaics. Impact on neuropsychopharmacology.

Future therapies for diseases associated with altered dopaminergic signaling, including Parkinson's disease, schizophrenia and drug addiction or drug dependence may substantially build on the existence of intramembrane receptor-receptor interactions within dopamine receptor containing receptor mosaics (RM; dimeric or high-order receptor oligomers) where it is believed that the dopamine D(2) receptor may operate as the 'hub receptor' within these complexes. The constitutive adenosine A(2A)/dopamine D(2) RM, located in the dorsal striato-pallidal GABA neurons, are of particular interest in view of the demonstrated antagonistic A(2A)/D(2) interaction within these heteromers; an interaction that led to the suggestion and later demonstration that A(2A) antagonists could be used as novel anti-Parkinsonian drugs. Based on the likely existence of A(2A)/D(2)/mGluR5 RM located both extrasynaptically on striato-pallidal GABA neurons and on cortico-striatal glutamate terminals, multiple receptor-receptor interactions within this RM involving synergism between A(2A)/mGluR5 to counteract D(2) signaling, has led to the proposal of using combined mGluR5 and A(2A) antagonists as a future anti-Parkinsonian treatment. Based on the same RM in the ventral striato-pallidal GABA pathways, novel strategies for the treatment of schizophrenia, building on the idea that A(2A) agonists and/or mGluR5 agonists will help reduce the increased dopaminergic signaling associated with this disease, have been suggested. Such treatment may ensure the proper glutamatergic drive from the mediodorsal thalamic nucleus to the prefrontal cortex, one which is believed to be reduced in schizophrenia due to a dominance of D(2)-like signaling in the ventral striatum. Recently, A(2A) receptors also have been shown to counteract the locomotor and sensitizing actions of cocaine and increases in A(2A) receptors have also been observed in the nucleus accumbens after extended cocaine self-administration, probably representing a compensatory up-regulation to counteract the cocaine-induced increases in dopamine D(2) and D(3) signaling. Therefore, A(2A) agonists, through antagonizing D(2) and D(3) signaling within A(2A)/D(2) and A(2A)/D(3) RM heteromers in the nucleus accumbens, may be found useful as a treatment for cocaine dependence. Furthermore, antagonistic cannabinoid CB(1)/D(2) interactions requiring A(2A) receptors have also been discovered and possibly operate in CB(1)/D(2)/A(2A) RM located principally on striatal glutamate terminals but also on some ventral striato-pallidal GABA neurons, thereby opening up a new mechanism for the integration of endocannabinoid, DA and adenosine mediated signals. Thus, A(2A), mGluR5 and/or CB(1) receptors can form integrative units with D(2) receptors within RM displaying different compositions, topography and localization. Also galaninR/5-HT(1A) RM probably participates in the transmission of the ascending 5-hydroxytryptamine neurons, where galanin receptors antagonize 5-HT(1A) recognition and signaling. Subtype specific galanin receptor antagonists may therefore represent novel antidepressant drugs. These results suggest the importance of a complete understanding of the function of these RM with regard to disease. Ultimately receptor-receptor interactions within RM that modify dopaminergic and serotonergic signaling may give new strategies for treatment of a wide range of diseases associated with altered dopaminergic and serotonergic signaling.

Prominent expression of acidic fibroblast growth factor in motor and sensory neurons.

Several growth factors originally characterized and named for their action on a variety of cells have more recently been suggested to be importantly involved in the development and maintenance of the nervous system. Acidic fibroblast growth factor (aFGF) is a member of a family of seven structurally related polypeptide growth factors. The cells responsible for expression of aFGF in the nervous system of adult rats have been identified using an affinity-purified antibody to aFGF in immunohistochemical studies and synthetic oligonucleotide probes for in situ hybridization studies. High levels of aFGF expression were observed in motoneurons, primary sensory neurons, and retinal ganglion neurons. Glial cells did not express detectable amounts of aFGF. Confocal and electron microscopic analysis suggested that a large portion of aFGF immunoreactivity was associated with the cytoplasmic face of neuronal membranes, consistent with the hypothesis that aFGF is a sequestered growth factor.

Cellular localization and distribution of dopamine D(4) receptors in the rat cerebral cortex and their relationship with the cortical dopaminergic and noradrenergic nerve terminal networks.

The role of the dopamine D(4) receptor in cognitive processes and its association with several neuropsychiatric disorders have been related to its preferential localization in the cerebral cortex. In the present work we have studied in detail the regional and cellular localization of the dopamine D(4) receptor immunoreactivity (IR) in the rat cerebral cortex and its relationship to the dopaminergic and noradrenergic nerve terminal networks, since both dopamine and noradrenaline have a high affinity for this receptor. High levels of D(4) IR were found in motor, somatosensory, visual, auditory, temporal association, cingulate, retrosplenial and granular insular cortices, whereas agranular insular, piriform, perirhinal and entorhinal cortices showed low levels. D(4) IR was present in both pyramidal and non-pyramidal like neurons, with the receptor being mainly concentrated to layers II/III. Layer I was observed to be exclusively enriched in D(4) IR branches of apical dendrites. Finally, mismatches were observed between D(4) IR and tyrosine hydroxylase and dopamine beta-hydroxylase IR nerve terminal plexuses, indicating that these receptors may be activated at least in part by dopamine and noradrenaline operating as volume transmission signals. The present findings support a major role of the dopamine D(4) receptor in mediating the transmission of cortical dopamine and noradrenaline nerve terminal plexuses.

Effects of intra-accumbal or intra-prefrontal cortex microinjections of adenosine 2A receptor ligands on responses to cocaine reward and seeking in rats.

RATIONALE AND OBJECTIVES: Many studies indicated that adenosine via its A2A receptors influences the behavioral effects of cocaine by modulating dopamine neurotransmission. The hypothesis was tested that A2A receptors in the nucleus accumbens (NAc) or the prefrontral cortex (PFc) may modulate cocaine reward and/or cocaine seeking behavior in rats. METHODS: The effects of local bilateral microinjections of the selective A2A receptor agonist CGS 21680 or the A2A receptor antagonists KW 6002 and SCH 58261 were investigated on cocaine self-administration on reinstatement of cocaine seeking. RESULTS: The intra-NAc shell, but not intra-infralimbic PFc, administration of CGS 21680 significantly reduced the number of active lever presses and the number of cocaine (0.25 mg/kg) infusions. However, tonic activation of A2A receptors located in the NAc or PFc did not play a role in modulating the rewarding actions of cocaine since neither KW 6002 nor SCH 58261 microinjections altered the cocaine (0.5 mg/kg) infusions. The intra-NAc but not intra-PFc microinjections of CGS 21680 dose- dependently attenuated the reinstatement of active lever presses induced by cocaine (10 mg/kg, i.p.) and the drug-associated combined conditioned stimuli using the subthreshold dose of cocaine (2.5 mg/kg, i.p.). On the other hand, the intra-NAc pretreatment with SCH 58261, but not with KW 6002, given alone evoked reinstatement of cocaine seeking behavior. CONCLUSION: The results strongly support the involvement of accumbal shell A2A receptors as a target, the activation of which exerts an inhibitory control over cocaine reward and cocaine seeking.

Role of cooperativity in protein folding and protein mosaic assemblage relevance for protein conformational diseases.

Biological systems are organized in intricate and highly structured networks with hierarchies and multiple scales. Cells can be considered as "meso-scale level" systems placed between the "macro-scale level" (systems of cellular networks) and the "micro-scale level" (systems of molecular networks). In fact, cells represent complex biochemical machineries made by networks of molecules connected by biochemical reactions. Thus, the brain should be studied as a system of "networks of networks". Recently, the existence of a Global Molecular Network (GMN) enmeshing the entire CNS was proposed. This proposal is based on the evidence that the extra-cellular matrix is a dynamic molecular structure capable of storing and releasing signals and of interacting with receptors and proteins on the cell membranes. Proteins have a special role in molecular networks since they can be assembled into high-order molecular complexes, which have been defined as Protein Mosaics (PM). Protein monomers in a PM (the "tesserae" of the mosaic) can interact via classical and non-classical cooperativity behaviour involving allosteric interactions. In the present paper, new features of allostery and cooperativity for protein folding, assemblage and topological features of PM will be discussed. Against this background, alterations in PM via allosteric modulations and non-classical cooperativity mechanisms may lead to protein aggregates like beta amyloid fibrils. Such aggregates cause pathological changes in the GMN structure and function leading to neurodegenerative diseases such as Alzheimer's disease. Thus, a novel view of the so called Protein Conformational Diseases (PCD) is proposed.

Region specific galanin receptor/neuropeptide Y Y1 receptor interactions in the tel- and diencephalon of the rat. Relevance for food consumption.

The aim of this work was to determine the interactions between NPY and GAL receptor (GALR) subtypes in the hypothalamus and the amygdala using quantitative receptor autoradiography to analyze the binding characteristics of NPY-Y1 and Y2 receptor subtypes in the presence and absence of GAL. Food intake in satiated animals was evaluated after intraventricular co-injections of GAL and NPY-Y1 or Y2 agonists. The expression of c-Fos IR in both regions was also investigated. GAL decreases NPY-Y1 agonist binding in the arcuate nucleus by about 15% (p<0.01), but increases NPY-Y1 agonist binding in amygdala (18%) (p<0.01). These effects were blocked with the GAL antagonist M35. Y2-agonist binding was not modified by GAL. GAL blocked the food intake induced by the Y1 agonist (p<0.01). Co-injections of Y1 agonist and GAL also reduced the c-Fos expression induced by the Y1 agonist in the arcuate nucleus and the dorsomedial hypothalamic nucleus but increased c-Fos expression in amygdala. These results indicate the existence of antagonistic interactions between GALR and NPY-Y1 receptors in the hypothalamus and their functional relevance for food intake. In contrast, a facilitatory interaction between GALR and Y1 receptors exists in the amygdala which may be of relevance for fear related behaviour.