Dopamine D2 receptors gate generalization of conditioned threat responses through mTORC1 signaling in the extended amygdala.

Overgeneralization of conditioned threat responses is a robust clinical marker of anxiety disorders. In overgeneralization, responses that are appropriate to threat-predicting cues are evoked by perceptually similar safety-predicting cues. Inappropriate learning of conditioned threat responses may thus form an etiological basis for anxiety disorders. The role of dopamine (DA) in memory encoding is well established. Indeed by signaling salience and valence, DA is thought to facilitate discriminative learning between stimuli representing safety or threat. However, the neuroanatomical and biochemical substrates through which DA modulates overgeneralization of threat responses remain poorly understood. Here we report that the modulation of DA D2 receptor (D2R) signaling bidirectionally regulates the consolidation of fear responses. While the blockade of D2R induces generalized threat responses, its stimulation facilitates discriminative learning between stimuli representing safety or threat. Moreover, we show that controlled threat generalization requires the coordinated activation of D2R in the bed nucleus of the stria terminalis and the central amygdala. Finally, we identify the mTORC1 cascade activation as an important molecular event by which D2R mediates its effects. These data reveal that D2R signaling in the extended amygdala constitutes an important checkpoint through which DA participates in the control of threat processing and the emergence of overgeneralized threat responses.

Ecdysteroid metabolism in mammals: The fate of ingested 20-hydroxyecdysone in mice and rats.

Phytoecdysteroids are molecules derived from sterol metabolism and found in many plants. They display a wide array of pharmacological effects on mammals (e.g. anabolic, anti-diabetic). Although these effects have been long established, the molecular targets involved remain to be identified. Like endogenous steroid hormones and bile acids, which are biochemically related, ingested or injected phytoecdysteroids undergo a set of reactions in mammals leading to the formation of numerous metabolites, only some of which have been so far identified, and it is presently unknown whether they represent active metabolites or inactivation products. In the large intestine, ecdysteroids undergo efficient 14-dehydroxylation. Other changes (reductions, epimerization, side-chain cleavage) are also observed, but whether these occur in the liver and/or large intestine is not known. The purpose of this study was to investigate the pharmacokinetics of 20-hydroxyecdysone (20E), the most common phytoecdysteroid, when administered to mice and rats, using, when required, tritium-labelled molecules to permit metabolic tracking. Bioavailability, the distribution of radioactivity and the kinetics of formation of metabolites were followed for 24-48 hours after ingestion and qualitative and quantitative analyses of circulating and excreted compounds were performed. In mice, the digestive tract always contains the majority of the ingested 20E. Within 30 min after ingestion, 20E reaches the large intestine, where microorganisms firstly remove the 14-hydroxyl group and reduce the 6-one. Then a very complex set of metabolites (not all of which have yet been identified) appears, which correspond to poststerone derivatives formed in the liver. We have observed that these compounds (like bile acids) undergo an entero-hepatic cycle, involving glucuronide conjugation in the liver and subsequent deconjugation in the intestine. Despite the very short half-life of ecdysteroids in mammals, this entero-hepatic cycle helps to maintain their plasma levels at values which, albeit low (≤0.2 µM), would be sufficient to evoke several pharmacological effects. Similar 20E metabolites were observed in mice and rats; they include in particular 14-deoxy-20E, poststerone and 14-deoxypoststerone and their diverse reduction products; the major products of this metabolism have been unambiguously identified. The major sites of metabolism of exogenous ecdysteroids in mammals are the large intestine and the liver. The entero-hepatic cycle contributes to the metabolism and to maintaining a low, but pharmacologically significant, concentration of ecdysteroids in the blood for ca. 24 h after ingestion. These data, together with parallel in vitro experiments provide a basis for the identification of 20E metabolite(s) possibly involved in the physiological effects associated with ecdysteroids in mammals.

Axo-glial interactions regulate the localization of axonal paranodal proteins.

Mice incapable of synthesizing the abundant galactolipids of myelin exhibit disrupted paranodal axo-glial interactions in the central and peripheral nervous systems. Using these mutants, we have analyzed the role that axo-glial interactions play in the establishment of axonal protein distribution in the region of the node of Ranvier. Whereas the clustering of the nodal proteins, sodium channels, ankyrin(G), and neurofascin was only slightly affected, the distribution of potassium channels and paranodin, proteins that are normally concentrated in the regions juxtaposed to the node, was dramatically altered. The potassium channels, which are normally concentrated in the paranode/juxtaparanode, were not restricted to this region but were detected throughout the internode in the galactolipid-defi- cient mice. Paranodin/contactin-associated protein (Caspr), a paranodal protein that is a potential neuronal mediator of axon-myelin binding, was not concentrated in the paranodal regions but was diffusely distributed along the internodal regions. Collectively, these findings suggest that the myelin galactolipids are essential for the proper formation of axo-glial interactions and demonstrate that a disruption in these interactions results in profound abnormalities in the molecular organization of the paranodal axolemma.

The glycosylphosphatidyl inositol-anchored adhesion molecule F3/contactin is required for surface transport of paranodin/contactin-associated protein (caspr).

Paranodin/contactin-associated protein (caspr) is a transmembrane glycoprotein of the neurexin superfamily that is highly enriched in the paranodal regions of myelinated axons. We have investigated the role of its association with F3/contactin, a glycosylphosphatidyl inositol (GPI)-anchored neuronal adhesion molecule of the Ig superfamily. Paranodin was not expressed at the cell surface when transfected alone in CHO or neuroblastoma cells. Cotransfection with F3 resulted in plasma membrane delivery of paranodin, as analyzed by confocal microscopy and cell surface biotinylation. The region that mediates association with paranodin was mapped to the Ig domains of F3 by coimmunoprecipitation experiments. The association of paranodin with F3 allowed its recruitment to Triton X-100-insoluble microdomains. The GPI anchor of F3 was necessary, but not sufficient for surface expression of paranodin. F3-Ig, a form of F3 deleted of the fibronectin type III (FNIII) repeats, although GPI-linked and expressed at the cell surface, was not recovered in the microdomain fraction and was unable to promote cell surface targeting of paranodin. Thus, a cooperative effect between the GPI anchor, the FNIII repeats, and the Ig regions of F3 is required for recruitment of paranodin into lipid rafts and its sorting to the plasma membrane.

Regulation of a neuronal form of focal adhesion kinase by anandamide.

Anandamide is an endogenous ligand for central cannabinoid receptors and is released after neuronal depolarization. Anandamide increased protein tyrosine phosphorylation in rat hippocampal slices and neurons in culture. The action of anandamide resulted from the inhibition of adenylyl cyclase and cyclic adenosine 3', 5'-monophosphate-dependent protein kinase. One of the proteins phosphorylated in response to anandamide was an isoform of pp125-focal adhesion kinase (FAK+) expressed preferentially in neurons. Focal adhesion kinase is a tyrosine kinase involved in the interactions between the integrins and actin-based cytoskeleton. Thus, anandamide may exert neurotrophic effects and play a role in synaptic plasticity.

Thymotaxin, a chemotactic protein, is identical to beta 2-microglobulin.

Thymotaxin, an 11-kilodalton protein chemotactic for rat bone marrow hematopoietic precursors, was purified from media conditioned by a rat thymic epithelial cell line. The NH2-terminal sequence of thymotaxin was identical to that of rat beta 2-microglobulin (beta 2m). Antibodies to beta 2m removed thymotaxin activity from the fraction containing the 11-kilodalton protein. Chemotactic activity was observed with rat plasma beta 2m, human beta 2m, and mouse recombinant beta 2m, further supporting the identity of thymotaxin with beta 2m. The directional migration, as opposed to random movement, of the cells was also confirmed. The only rat bone marrow cells that migrated toward beta 2m were Thy1+ immature lymphoid cells devoid of T cell, B cell, and myeloid cell differentiation markers.

DARPP-32: regulator of the efficacy of dopaminergic neurotransmission.

Dopaminergic neurons exert a major modulatory effect on the forebrain. Dopamine and adenosine 3',5'-monophosphate-regulated phosphoprotein (32 kilodaltons) (DARPP-32), which is enriched in all neurons that receive a dopaminergic input, is converted in response to dopamine into a potent protein phosphatase inhibitor. Mice generated to contain a targeted disruption of the DARPP-32 gene showed profound deficits in their molecular, electrophysiological, and behavioral responses to dopamine, drugs of abuse, and antipsychotic medication. The results show that DARPP-32 plays a central role in regulating the efficacy of dopaminergic neurotransmission.

Paranodin, a glycoprotein of neuronal paranodal membranes.

Ranvier nodes are flanked by paranodal regions, at the level of which oligodendrocytes or Schwann cells interact closely with axons. Paranodes play a critical role in the physiological properties of myelinated nerve fibers. Paranodin, a prominent 180 kDa transmembrane neuronal glycoprotein, was purified and cloned from adult rat brain, and found to be highly concentrated in axonal membranes at their junction with myelinating glial cells, in paranodes of central and peripheral nerve fibers. The large extracellular domain of paranodin is related to neurexins, and its short intracellular tail binds protein 4.1, a cytoskeleton-anchoring protein. Paranodin may be a critical component of the macromolecular complex involved in the tight interactions between axons and myelinating glial cells characteristic of the paranodal region.

[Axon and Schwann cells... so far away, so close]. / Axones et cellules de Schwann... si loin, si proches.

Myelination was a major step in the evolution of the nervous system. Appearing first in jaw fish, myelination allows the fast and secure propagation of action potentials at a low energetic cost, and without exaggerated increase in axonal diameter. In the peripheral nervous system of mammals, myelination results from the tight interactions between Schwann cells and axons, leading to the formation of highly differentiated domains along the axon. The molecular determinants of these interactions are starting to be well identified. Their understanding provides a precise framework to interpret the defects, which occur in pathological circumstances. This review summarizes the present state of knowledge concerning axoglial interactions in peripheral nerves.

Nodal, paranodal and juxtaparanodal axonal proteins during demyelination and remyelination in multiple sclerosis.

Saltatory conduction in myelinated fibres depends on the specific molecular organization of highly specialized axonal domains at the node of Ranvier, the paranodal and the juxtaparanodal regions. Voltage-gated sodium channels (Na(v)) have been shown to be deployed along the naked demyelinated axon in experimental models of CNS demyelination and in multiple sclerosis lesions. Little is known about aggregation of nodal, paranodal and juxtaparanodal constituents during the repair process. We analysed by immunohistochemistry on free-floating sections from multiple sclerosis brains the expression and distribution of nodal (Na(v) channels), paranodal (paranodin/Caspr) and juxtaparanodal (K(v) channels and Caspr2) molecules in demyelinated and remyelinated lesions. Whereas in demyelinated lesions, paranodal and juxtaparanodal proteins are diffusely distributed on denuded axons, the distribution of Na(v) channels is heterogeneous, with a diffuse immunoreactivity but also few broad Na(v) channel aggregates in all demyelinated lesions. In contrast to the demyelinated plaques, all remyelinated lesions are characterized by the detection of aggregates of Na(v) channels, paranodin/Caspr, K(v) channels and Caspr2. Our data suggest that these aggregates precede remyelination, and that Na(v) channel aggregation is the initial event, followed by aggregation of paranodal and then juxtaparanodal axonal proteins. Remyelination takes place in multiple sclerosis tissue but myelin repair is often incomplete, and the reasons for this remyelination deficit are many. We suggest that a defect of Na(v) channel aggregation might be involved in the remyelination failure in demyelinated lesions with spared axons and oligodendroglial cells.

FAK and PYK2/CAKbeta in the nervous system: a link between neuronal activity, plasticity and survival?

A major aim of neurobiology today is to improve understanding of the signaling pathways that couple rapid events, such as the action potential and neurotransmitter release, to long-lasting changes in synaptic strength and increased neuronal survival. These adaptations involve interactions of neurons with other cells and with the extracellular matrix. They use, in part, the same molecular machinery that controls adhesion, motility or survival in non-neuronal cells. This machinery includes two homologous non-receptor tyrosine kinases, FAK and PYK2/CAKbeta, and the associated SRC-family tyrosine kinases. Specific brain isoforms of FAK with distinct properties are regulated by neurotransmitters, whereas PYK2/CAKbeta is highly sensitive to depolarization. The multiplicity of the pathways that can be activated by these tyrosine kinases indicates their importance in signal transduction in the adult brain.

[Role and regulation of dopamine D1 receptors in the striatum: implications for the genesis of dyskinesia in Parkinson's disease]. / Rôle et régulation du récepteur D1 de la dopamine dans le striatum: implications dans les dyskinésies de la maladie de Parkinson.

L-dopa treatment of Parkinson's disease is complicated in the long term by the appearance of dyskinesia. Hypersensitivity of D1 dopamine receptor has been suggested to play a role in these delayed adverse effects. Hypersensitivity of dopamine D1 receptor in Parkinson's disease can be accounted for by increased levels of Galphaolf, the stimulatory G protein which couples D1 receptor to adenylyl cyclase in the striatum. We here discuss the possible role of D1 receptor signal transduction in the genesis of L-dopa-induced dyskinesia in the light of Galphaolf regulation.

Tumour suppressive properties of fibroblast growth factor receptor 2-IIIb in human bladder cancer.

FGFRs (fibroblast growth factor receptors) are encoded by four genes (FGFR1-4). Alternative splicing results in various receptor isoforms. The FGFR2-IIIb variant is present in a wide variety of epithelia, including the bladder epithelium. Recently, we have shown that FGFR2-IIIb is downregulated in a subset of transitional cell carcinomas of the bladder, and that this downregulation is associated with a poor prognosis. We investigated possible tumour suppressive properties of FGFR2-IIIb by transfecting two human bladder tumour cell lines, J82 and T24, which have no endogenous FGFR2-IIIb expression, with FGFR2-IIIb cDNA. No stable clones expressing FGFR2-IIIb were isolated with the J82 cell line. For the T24 cell line, stable transfectants expressing FGFR2-IIIb had reduced growth in vitro and formed fewer tumours in nude mice which, in addition, grew more slowly. The potential mechanisms leading to decreased FGFR2-IIIb mRNA levels were also investigated. The 5' region of the human FGFR2 gene was isolated and found to contain a CpG island which was partially methylated in more than half the cell lines and tumours which do not express FGFR2-IIIb. No homozygous deletion was identified in any of the tumours or cell lines with reduced levels of FGFR2-IIIb. Mutational analysis of the entire coding region of FGFR2-IIIb at the transcript level was performed in 33 bladder tumours. In addition to normal FGFR2-IIIb mRNA, abnormal transcripts were detected in two tumour samples. These abnormal mRNAs resulted from exon skipping which affected the region encoding the kinase domain. Altogether, these results show that FGFR2-IIIb has tumour growth suppressive properties in bladder carcinomas and suggest possible mechanisms of FGFR2 gene inactivation.

Galpha(olf) levels are regulated by receptor usage and control dopamine and adenosine action in the striatum.

In the striatum, dopamine D(1) and adenosine A(2A) receptors stimulate the production of cAMP, which is involved in neuromodulation and long-lasting changes in gene expression and synaptic function. Positive coupling of receptors to adenylyl cyclase can be mediated through the ubiquitous GTP-binding protein Galpha(S) subunit or through the olfactory isoform, Galpha(olf), which predominates in the striatum. In this study, using double in situ hybridization, we show that virtually all striatal efferent neurons, identified by the expression of preproenkephalin A, substance P, or D(1) receptor mRNA, contained high amounts of Galpha(olf) mRNA and undetectable levels of Galpha(s) mRNA. In contrast, the large cholinergic interneurons contained both Galpha(olf) and Galpha(s) transcripts. To assess the functional relationship between dopamine or adenosine receptors and G-proteins, we examined G-protein levels in the striatum of D(1) and A(2A) receptor knock-out mice. A selective increase in Galpha(olf) protein was observed in these animals, without change in mRNA levels. Conversely, Galpha(olf) levels were decreased in animals lacking a functional dopamine transporter. These results indicate that Galpha(olf) protein levels are regulated through D(1) and A(2A) receptor usage. To determine the functional consequences of changes in Galpha(olf) levels, we used heterozygous Galpha(olf) knock-out mice, which possess half of the normal Galpha(olf) levels. In these animals, the locomotor effects of amphetamine and caffeine, two psychostimulant drugs that affect dopamine and adenosine signaling, respectively, were markedly reduced. Together, these results identify Galpha(olf) as a critical and regulated component of both dopamine and adenosine signaling.

Xenopus cadherin-11 is expressed in different populations of migrating neural crest cells.

We cloned the Xenopus homologue of cadherin-11 and studied its spatiotemporal expression pattern during early development. The messenger RNA is present from the mid-gastrulation through embryo development. It is expressed in different neural crest cell populations, during their migration and differentiation. This pattern, unexpected for an adhesion molecule, reinforces the idea of novel functions for type II cadherins.

Metastatic rat carcinoma cells express a new retrotransposon.

Genes differentially expressed by a rat bladder carcinoma NBT-II cells and their in-vivo-selected metastatic M-NBT-II variant were analysed. Amplification and cloning of a 277-bp B sequence, exclusively expressed by the M-NBT-II cells, were performed, and this sequence was detected as a 6.7-kb RNA. This fragment shares 46-50% identities with the gag-related protein of mouse and hamster Intracisternal A Particles (IAPs). Screening of a M-NBT-II cDNA library with the B probe selected a 1671-bp sequence corresponding to the 5' end of a novel retrotransposon member of the rat IAP family. This sequence has a strong identity with the Ecker Rat IAP (ERA-IAP) except for the B portion and has an open reading frame potentially encoding a 114-amino-acid gag retrovirus-related protein. Rearrangement of this new retrotransposon could be relevant with the tumor progression in our model system since it is only expressed in the M-NBT-II in-vivo-selected carcinoma metastasis.

Requirement of pyk2 for the activation of the MAP kinase cascade induced by Ca(2+) (but not by PKC or G protein) in PC12 cells.

The role of the Ca(2+)-activated tyrosine kinase, Pyk2, in the pleiotropic coupling of nerve cell stimulation to the MAP kinase cascade still remains undefined. Using a panel of PC12 clones, one of which was defective in Pyk2, we demonstrate (1) that the MAP kinase response induced by a [Ca(2+)](i) rise (following application of the Ca(2+) ionophore, ionomycin) is inappreciable in the defective clone and is re-established after Pyk2 transfection; and (2) that the responses to both protein kinase C and P(2y2) receptor activation occur normally even in the defective cells. We conclude that Pyk2 is the key mediator in the pathway activated by Ca(2+) but has minor roles with the other types of stimulation.

Haloperidol-succinylglycyl[125I]iodotyrosine, a novel iodinated ligand for dopamine D2 receptors.

A novel radioiodinated ligand of the butyrophenone type has been synthesized for the quantification and characterization of dopamine D2 receptors. This haloperidol-derived ligand, haloperidol-succinylglycyl[125I]iodotyrosine ([125I]HSGTI), binds rapidly (equilibrium is reached within 30 min, at 10 pM and 37 degrees C) and with high affinity (Kd = 0.3 nM) to bovine striatal membranes. Its pharmacology, determined by competitive displacement with dopaminergic and non-dopaminergic drugs, is characteristic of binding to dopamine D2 receptors.

Bilateral cerebral metabolic alterations following lesion of the ventromedial thalamic nucleus: mapping by the 14C-deoxyglucose method in conscious rats.

The functional role of the ventromedial thalamic nucleus (VM) was investigated by means of the quantitative autoradiographic 14C-deoxyglucose method. Local cerebral glucose utilization (LCGU) was measured bilaterally in 53 discrete brain areas of conscious rats 1 week and 1 month following electrolytic lesion of the VM. There was no difference observed in the LCGU values between the two groups of lesioned animals. In the VM-lesioned rats glucose consumption was decreased in several ipsilateral (pre-frontal, frontal, cingulate, sensorimotor, visual) and in some contralateral (cingulate, sensorimotor, visual) cortical areas. Furthermore, LCGU values were depressed bilaterally in the striatum, hippocampus, dentate gyrus, substantia nigra compacta, superficial layers of the superior colliculus, oculomotor complex, and cerebellar dentate nuclei. Glucose utilization was diminished also in the deep layers of the superior colliculus and in the fastigial nucleus ipsilateral to the lesion. Within the thalamus, glucose metabolism was decreased in several nuclei either bilaterally (reticular, intralaminar, paralamellar mediodorsal) or ipsilaterally (lateroposterior, ventrobasal, ventrolateral, ventroanterior, and medioventral) to the lesion. A significant correlation between the percentage of injured VM and the regional metabolic depression was demonstrated in the areas displaying the most pronounced decrease in LCGU. These results support a widespread influence of the VM on brain function extending beyond the limits of purely motor systems and reveal the consequences of a unilateral lesion of the thalamus on metabolic activity in several contralateral structures.

Immunocytochemical localization of phosphatase inhibitor-1 in rat brain.

The localization of phosphatase inhibitor-1 was investigated in rat brain by use of immunocytochemistry. Studies were performed with an affinity purified IgG raised against purified rabbit skeletal muscle inhibitor-1. In rat brain tissue homogenates, this antibody reacted only with a 29 kDa protein corresponding to inhibitor-1. Immunocytochemical studies with this antibody revealed numerous immunoreactive cell bodies and fibers. The highest concentration of immunoreactive perikarya was observed in the caudate-putamen and nucleus accumbens, and these appeared to be exclusively medium-sized neurons. Other areas containing substantial populations of immunoreactive neurons included the suprachiasmatic nucleus of the hypothalamus, lateral hypothalamus, horizontal limb of the diagonal band of Broca, dentate gyrus of the hippocampal formation, habenula, superior colliculus, claustrum, endopiriform nuclei, and neocortex. The distribution of terminals containing inhibitor-1 coincided with the distribution of terminal fields known to originate from the above regions. Thus, plexuses of immunoreactive axons were seen in the globus pallidus, substantia nigra pars reticulata, paraventricular hypothalamus, dorsal thalamus, CA3 region of the hippocampus, and interpeduncular nucleus. These results demonstrate that phosphatase inhibitor-1, a cyclic AMP-regulated inhibitor of phosphatase-1, is differentially distributed in the rat CNS. Given the widespread role of protein phosphorylation and dephosphorylation in intracellular signal transduction, these results suggest that neurons containing high levels of inhibitor-1 may share common, hitherto unrecognized, properties in terms of neurotransmitter regulation and/or responsiveness.