Large Shape Staggering in Neutron-Deficient Bi Isotopes.

The changes in the mean-square charge radius (relative to ^{209}Bi), magnetic dipole, and electric quadrupole moments of ^{187,188,189,191}Bi were measured using the in-source resonance-ionization spectroscopy technique at ISOLDE (CERN). A large staggering in radii was found in ^{187,188,189}Bi^{g}, manifested by a sharp radius increase for the ground state of ^{188}Bi relative to the neighboring ^{187,189}Bi^{g}. A large isomer shift was also observed for ^{188}Bi^{m}. Both effects happen at the same neutron number, N=105, where the shape staggering and a similar isomer shift were observed in the mercury isotopes. Experimental results are reproduced by mean-field calculations where the ground or isomeric states were identified by the blocked quasiparticle configuration compatible with the observed spin, parity, and magnetic moment.

Immunogenicity assay development and validation for biological therapy as exemplified by ustekinumab.

Introduction of biotherapeutics has been a major milestone in the treatment of different chronic diseases. Nevertheless, the immune system can recognize the administered biological as non-self and respond with generation of anti-drug antibodies (ADA), including neutralizing ADA (nADA). Immunogenic responses may result in altered drug dynamics and kinetics leading to changes in safety and efficacy. However, there are several challenges with standard techniques for immunogenicity testing. Ustekinumab (UST), used in different inflammatory diseases, is a therapeutic antibody directed against the shared p40 subunit of interleukin (IL)-12 and IL-23, interfering in the pathogenically crucial T helper type 1 (Th1)/Th17 pathway. We established and validated different approaches for detection and quantitation of UST, UST-specific ADA and nADA. Addressing the obstacle of complex formation of UST with nADA, we developed an acidification assay to approach the total amount of nADA. Validated methods were based on surface plasmon resonance spectroscopy (SPR), enzyme-linked immunosorbent assay (ELISA) and a cell-based approach to characterize neutralizing capacity of nADA. Parameters assessed were determination and quantitation limits, linearity, range, precision, accuracy and selectivity. Quantitation of ADA and UST was feasible at lower concentrations using ELISA, whereas SPR showed a wider linear range for determination of ADA and UST. Accuracy, precision and linearity for quantitation were comparable using ELISA, SPR and the cell-based approach. All validated parameters fulfill the requirements of regulatory agencies. A combination of the testing approaches could address the increasing demand of precision medicine as it can be suitable for capturing the whole spectrum of immunogenicity and is transferable to other biologicals.

Randomized comparative long-term survival study of endoscopic and thoracoscopic esophageal wall repair after NOTES mediastinoscopy in healthy and compromised animals.

BACKGROUND AND STUDY AIMS: Natural orifice transluminal endoscopic surgery (NOTES) has not yet been widely adopted because of lack of suitable equipment and fear of possible serious complications, especially in the mediastinum. We compared endoscopic with thoracoscopic esophageal wall repair after full-thickness esophageal wall incision (FTEI) and NOTES mediastinoscopy in healthy versus compromised animals. METHODS: After FTEI for mediastinoscopy, 24 pigs (12 healthy, 12 compromised) were randomly allocated to endoscopic or thoracoscopic repair (each arm of each group, n = 6). They were kept alive for 3 months after endoscopic closure with prototype T-anchor suturing or thoracoscopic repair. RESULTS: FTEI and mediastinoscopy were uneventful in all as was the initial repair of the incision (mean repair times: thoracoscopic 65 +/- 3.2 minutes, endoscopic 52 +/- 5.1 minutes; P < 0.0005). Post procedure, all 12 healthy pigs thrived with no complications or deaths. Two compromised animals died during the preparation period, and had to be replaced. In the compromised group, during endoscopic repair, 2 / 6 pigs suffered from gastric reflux into esophagus and mediastinum; the repair was completed and the pigs kept alive; one subsequently died of mediastinitis, and in the other, autopsy showed a gastric abscess in the lower mediastinum. Regarding the compromised thoracoscopic subgroup, one animal died from mediastinitis and all had abscesses at or near the incision sites. CONCLUSION: Transesophageal mediastinoscopy could be performed equally well as the transthoracic procedure, both in healthy and compromised animals. However, on follow-up, the compromised animals had worse outcomes, with more complications and two deaths (17 %), one in each arm.

Adenosine A2A receptors and basal ganglia physiology.

Adenosine A2A receptors are highly enriched in the basal ganglia system. They are predominantly expressed in enkephalin-expressing GABAergic striatopallidal neurons and therefore are highly relevant to the function of the indirect efferent pathway of the basal ganglia system. In these GABAergic enkephalinergic neurons, the A2A receptor tightly interacts structurally and functionally with the dopamine D2 receptor. Both by forming receptor heteromers and by targeting common intracellular signaling cascades, A2A and D2 receptors exhibit reciprocal antagonistic interactions that are central to the function of the indirect pathway and hence to basal ganglia control of movement, motor learning, motivation and reward. Consequently, this A2A/D2 receptors antagonistic interaction is also central to basal ganglia dysfunction in Parkinson's disease. However, recent evidence demonstrates that, in addition to this post-synaptic site of action, striatal A2A receptors are also expressed and have physiological relevance on pre-synaptic glutamatergic terminals of the cortico-limbic-striatal and thalamo-striatal pathways, where they form heteromeric receptor complexes with adenosine A1 receptors. Therefore, A2A receptors play an important fine-tuning role, boosting the efficiency of glutamatergic information flow in the indirect pathway by exerting control, either pre- and/or post-synaptically, over other key modulators of glutamatergic synapses, including D2 receptors, group I metabotropic mGlu5 glutamate receptors and cannabinoid CB1 receptors, and by triggering the cAMP-protein kinase A signaling cascade.

[Changes in neural networks by conditional transgenic approach: a key to our comprehension of neuro-psychiatric disorders in the basal ganglia system]. / Modifications des réseaux neuronaux par transgénèse conditionnelle: une clé pour notre compréhension des pathologies neuro- psychiatriques du système des noyaux de la base.

The striatum, the first relay of the basal ganglia system, is critically involved in motor functions and motivational processes. The dorsal striatum is central to the motor control and motor learning and the ventral striatum or nucleus accumbens is essential for motivation, the reward system and reinforcement by drugs. This system is dysfunctional in movement disorders such as Parkinson's disease and Huntington's disease and in psychiatric disorders including drug addiction. The striatum consists of two populations of neurons projecting at the origin of two distinct paths in the circuit of basal ganglia, and of different populations of interneurons. These two populations of efferent neurons, striatopallidal and striatonigral neurons, are characterized by their projection sites and their differential expression in dopamine receptors and neuropeptides. Their roles in motor control and motivational processes and in the mechanisms of neuroadaptation in the system's pathologies remain mostly unknown. To identify these specific functions, we have developed new animal models wearing molecular or cell "lesions" by a conditional transgenic approach to target a specific population of neurons. By this approach, we demonstrated the inhibitory role of the population of striatopallidal neurons in the motor control and in the process of drug addiction, identified new genes selectively expressed by striatopallidal neurons that could be the target for future therapies and identified the potential role of this population of neurons disturbances in attention-deficit hyperactivity disorder (ADHD).

Dysregulation of lysophosphatidic acids in multiple sclerosis and autoimmune encephalomyelitis.

Bioactive lipids contribute to the pathophysiology of multiple sclerosis. Here, we show that lysophosphatidic acids (LPAs) are dysregulated in multiple sclerosis (MS) and are functionally relevant in this disease. LPAs and autotaxin, the major enzyme producing extracellular LPAs, were analyzed in serum and cerebrospinal fluid in a cross-sectional population of MS patients and were compared with respective data from mice in the experimental autoimmune encephalomyelitis (EAE) model, spontaneous EAE in TCR1640 mice, and EAE in Lpar2 -/- mice. Serum LPAs were reduced in MS and EAE whereas spinal cord LPAs in TCR1640 mice increased during the 'symptom-free' intervals, i.e. on resolution of inflammation during recovery hence possibly pointing to positive effects of brain LPAs during remyelination as suggested in previous studies. Peripheral LPAs mildly re-raised during relapses but further dropped in refractory relapses. The peripheral loss led to a redistribution of immune cells from the spleen to the spinal cord, suggesting defects of lymphocyte homing. In support, LPAR2 positive T-cells were reduced in EAE and the disease was intensified in Lpar2 deficient mice. Further, treatment with an LPAR2 agonist reduced clinical signs of relapsing-remitting EAE suggesting that the LPAR2 agonist partially compensated the endogenous loss of LPAs and implicating LPA signaling as a novel treatment approach. Graphical summary of lysophosphatidic signaling in multiple sclerosis.

Modulation of neuronal excitability by intracellular calcium buffering: from spiking to bursting.

We have investigated the detailed regulation of neuronal firing pattern by the cytosolic calcium buffering capacity using a combination of mathematical modeling and patch-clamp recording in acute slice. Theoretical results show that a high calcium buffer concentration alters the characteristic regular firing of cerebellar granule cells and that a transition to various modes of oscillations occurs, including bursting. Using bifurcation analysis, we show that this transition from spiking to bursting is a consequence of the major slowdown of calcium dynamics. Patch-clamp recordings on cerebellar granule cells loaded with a high concentration of the fast calcium buffer BAPTA (15 mM) reveal dramatic alterations in their excitability as compared to cells loaded with 0.15 mM BAPTA. In high calcium buffering conditions, granule cells exhibit all bursting behaviors predicted by the model whereas bursting is never observed in low buffering. These results suggest that cytosolic calcium buffering capacity can tightly modulate neuronal firing patterns leading to generation of complex patterns and therefore that calcium-binding proteins may play a critical role in the non-synaptic plasticity and information processing in the central nervous system.

Multiple rodent models and behavioral measures reveal unexpected responses to FTY720 and DMF in experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is a widely-used rodent model for multiple sclerosis (MS), but a single model can hardly capture all features of MS. We investigated whether behavioral parameters in addition to clinical motor function scores could be used to assess treatment efficacy during score-free intervals in the relapsing-remitting EAE model in SJL/J mice. We studied the effects of the clinical reference compounds FTY720 (fingolimod, 0.5mg/kg/day) and dimethyl fumarate (DMF, 20-30 mg/kg/day) on clinical scores in several rodent EAE models in order to generate efficacy profiles. SJL/J mice with relapsing-remitting EAE were studied using behavioral tests, including rotarod, gait analysis, locomotor activity and grip strength. SJL/J mice were also examined according to Crawley's sociability and preference for social novelty test. Prophylactic treatment with FTY720 prevented clinical scores in three of the four EAE rodent models: Dark Agouti (DA) and Lewis rats and C57BL/6J mice. Neither prophylactic nor late-therapeutic treatment with FTY720 reduced clinical scores or reversed deficits in the rotarod test in SJL/J mice, but we observed effects on motor functions and sociability in the absence of clinical scores. Prophylactic treatment with FTY720 improved the gait of SJL/J mice whereas late-therapeutic treatment improved manifestations of reduced social (re)cognition or preference for social novelty. DMF was tested in three EAE models and did not improve clinical scores at the dose used. These data indicate that improvements in behavioral deficits can occur in absence of clinical scores, which indicate subtle drug effects and may have translational value for human MS.

A cDNA for adenylyl sulphate (APS)-kinase from Arabidopsis thaliana.

A cDNA clone with an open reading frame of 831 nucleotides was isolated from a lambda ZapII-library of Arabidopsis thaliana. The nucleotide sequence of the cDNA is homologous to the APS-kinase genes from enterobacteria, diazotrophic bacteria, and yeast: Escherichia coli (cys C: 53.2%), Rhizobium meliloti (nod Q: 52.6%), and Saccharomyces cerevisiae (met 14:57.1%). The polypeptide deduced from the plant APS-kinase cDNA is comprised of 276 amino acid residues with a molecular weight of 29,790. It contains an N-terminal extension of 77 amino acids. This extension includes a putative transit peptide of 37 residues separated from the core protein by a VRACV processing site for stromal peptidase; a molecular weight of 26,050 is predicted for the processed protein. The relatedness between bacterial, fungal and plant APS-kinase polypeptides ranges from 47.5% (E. coli), 55.4% (S. cerevisiae), 52.6% (R. meliloti), and 50.3% (Azospirillum brasilense). The plant polypeptide contains eight cysteine residues; two cysteines flank a conserved purine nucleotide binding domain: GxxxxGK. Also conserved are a serine-182 as a possible phosphate transferring group and a K/LARAGxxxxFTG motif described for PAPS dependent enzymes. The identity of the gene was confirmed by analyzing the function of the gene product. The putative transit peptide was deleted by PCR and the truncated gene was expressed in a pTac1 vector system. A polypeptide of MW 25761 could be induced by IPTG. The gene product was enzymatically active as APS-kinase. It produced PAPS from APS and ATP--the absence of ATP but supplemented with thiols, the APS-kinase reacted as APS-sulphotransferase. APS-sulphotransferase is not a separate enzyme but identical with APS-kinase.

Crystal structure of rat GTP cyclohydrolase I feedback regulatory protein, GFRP.

Tetrahydrobiopterin, the cofactor required for hydroxylation of aromatic amino acids regulates its own synthesis in mammals through feedback inhibition of GTP cyclohydrolase I. This mechanism is mediated by a regulatory subunit called GTP cyclohydrolase I feedback regulatory protein (GFRP). The 2.6 A resolution crystal structure of rat GFRP shows that the protein forms a pentamer. This indicates a model for the interaction of mammalian GTP cyclohydrolase I with its regulator, GFRP. Kinetic investigations of human GTP cyclohydrolase I in complex with rat and human GFRP showed similar regulatory effects of both GFRP proteins.

Role of calcium binding proteins in the control of cerebellar granule cell neuronal excitability: experimental and modeling studies.

Calcium binding proteins, such as calretinin, are abundantly expressed in distinctive patterns in the central nervous system but their physiological function remains poorly understood. Calretinin is expressed in cerebellar granule cells which provide the major excitatory input to Purkinje cells through parallel fibers. Calretinin deficient mice exhibit dramatic alterations in motor coordination and in Purkinje cell firing recorded in vivo through unknown mechanisms. In the present paper, we review the results obtained with the patch clamp recording techniques in acute slice preparation. This data allow us to investigate the effect of a null mutation of the calretinin gene on the intrinsic electroresponsiveness of cerebellar granule cells at a mature developmental stage. Calretinin deficient granule cells exhibit faster action potentials and generate repetitive spike discharge showing an enhanced frequency increase with injected currents. These alterations disappear when 0.15 mM of the exogenous fast calcium buffer BAPTA is infused in the cytosol to restore the calcium buffering capacity. Furthermore, we propose a mathematical model demonstrating that the observed alterations of granule cell excitability can be explained by a decreased cytosolic calcium buffering capacity due to the absence of calretinin. We suggest that calcium binding proteins modulate intrinsic neuronal excitability and may therefore play a role in the information processing in the central nervous system.

Lack of parvalbumin in mice leads to behavioral deficits relevant to all human autism core symptoms and related neural morphofunctional abnormalities.

Gene mutations and gene copy number variants are associated with autism spectrum disorders (ASDs). Affected gene products are often part of signaling networks implicated in synapse formation and/or function leading to alterations in the excitation/inhibition (E/I) balance. Although the network of parvalbumin (PV)-expressing interneurons has gained particular attention in ASD, little is known on PV's putative role with respect to ASD. Genetic mouse models represent powerful translational tools for studying the role of genetic and neurobiological factors underlying ASD. Here, we report that PV knockout mice (PV(-/-)) display behavioral phenotypes with relevance to all three core symptoms present in human ASD patients: abnormal reciprocal social interactions, impairments in communication and repetitive and stereotyped patterns of behavior. PV-depleted mice also showed several signs of ASD-associated comorbidities, such as reduced pain sensitivity and startle responses yet increased seizure susceptibility, whereas no evidence for behavioral phenotypes with relevance to anxiety, depression and schizophrenia was obtained. Reduced social interactions and communication were also observed in heterozygous (PV(+/-)) mice characterized by lower PV expression levels, indicating that merely a decrease in PV levels might be sufficient to elicit core ASD-like deficits. Structural magnetic resonance imaging measurements in PV(-/-) and PV(+/-) mice further revealed ASD-associated developmental neuroanatomical changes, including transient cortical hypertrophy and cerebellar hypoplasia. Electrophysiological experiments finally demonstrated that the E/I balance in these mice is altered by modification of both inhibitory and excitatory synaptic transmission. On the basis of the reported changes in PV expression patterns in several, mostly genetic rodent models of ASD, we propose that in these models downregulation of PV might represent one of the points of convergence, thus providing a common link between apparently unrelated ASD-associated synapse structure/function phenotypes.

APS-sulfotransferase activity is identical to higher plant APS-kinase (EC 2.7.1.25).

A cDNA from Arabidopsis thaliana L. Heynh encoding the APS-kinase (EC 2.7.1.25) was modified by deletion of a plastidic transit peptide to enable its expression in Escherichia coli. The resultant protein (MW 25,761) is enzymatically active as APS-kinase and restores prototrophic growth in an APS-kinase mutant. All transformants harbouring the modified plant DNA also acquired APS-sulfotransferase activity. In the absence of ATP but provided with DTT, a tetrameric form of recombinant APS-kinase exhibits APS-sulfotransferase activity. Monospecific polyclonal antibodies raised against the APS-kinase as immunogen also reacted against APS-sulfotransferase. We propose that APS-sulfotransferase activity is a nonphysiological side reaction of APS-kinase.

Distribution of cells containing mRNA encoding cholecystokinin in the rat central nervous system.

The distribution of cells containing mRNA encoding cholecystokinin was studied in the rat central nervous system by in situ hybridization histochemistry. Cholecystokinin mRNA containing neurons were considerably more numerous than the cholecystokinin-like immunoreactive neurons detected by immunocytochemistry even after colchicine pretreatment and appeared to be heavily, moderately, or lightly labeled. Such neurons were present in the olfactory bulb, olfactory nuclei, layers II-III and V-VI of the cerebral cortex, amygdaloid nuclei, subiculum, hippocampus, claustrum, endopiriform nucleus, several hypothalamic nuclei, most of the thalamic nuclei, ventral tegmental area, substantia nigra, interfascicularis nucleus, linearis rostralis, central gray, Edinger-Westphal nucleus, superior and inferior colliculi, parabrachial nucleus, reticular formation, raphe nuclei, and spinal trigeminal nucleus. This distribution partly confirmed and partly extended the previous immunohistochemical descriptions. Several brain areas such as the thalamus and the colliculi contain cholecystokinin mRNA but are devoid of perikarya exhibiting cholecystokinin-like immunoreactivity. The cerebral cortex and the hippocampus present a far higher density of cholecystokinin mRNA containing cells, including pyramidal neurons, than of perikarya containing cholecystokinin-like immunoreactivity. These results suggest that cholecystokinin or cholecystokinin-related peptides could have a functional role in numerous cerebral pathways including long projections such as cortical or thalamic projections.

Ontogeny of gene expression of adenosine A2 receptor in the striatum: early localization in the patch compartment.

The ontogeny of adenosine A2 receptor mRNA and adenosine A2 binding sites distributions was studied by in situ hybridization histochemistry and receptor autoradiography in pre- and post-natal rat striatum, postnatal dog striatum, and a human fetus striatum and compared to that of dopamine D1 and mu opiate receptors. The early postnatal striatum demonstrated heterogeneous distributions of adenosine A2 receptor mRNA and adenosine A2 binding sites with patches of dense labeling corresponding to dopamine D1 and mu opiate receptors enriched zones. This patchy pattern evolved to the homogeneous distribution observed in the adult. The higher intensity of adenosine A2 receptor mRNA enriched patches correspond at the microscopical level to a higher density of labeled neurons in the patches areas and also to a higher level of expression per labeled patches neuron than in the matrix ones. This demonstrates for the first time that differences in patch/matrix receptor density is at least partly linked to different levels of receptor gene expression.

Caffeine regulates neurotensin and cholecystokinin messenger RNA expression in the rat striatum.

Interactions between dopamine and neurotensin or dopamine and cholecystokinin have been demonstrated in the basal ganglia. Disruption of nigrostriatal dopaminergic transmission results in a dramatic increase in neurotensin messenger RNA and in an induction of cholecystokinin messenger RNA in the striatum. Interaction between striatal dopaminergic and adenosinergic systems have also been reported. Adenosine and the adenosine receptor antagonist, caffeine, regulate gene expression in the striatum. In the present study, in situ hybridization histochemistry was used to investigate the putative regulation of neurotensin and cholecystokinin messenger RNA expression by caffeine in the rat striatum. Using this method, cholecystokinin messenger RNA was undetectable and neurotensin messenger RNA very sparse in the normal striatum. Chronic caffeine administration induced a dramatic increase in neurotensin messenger RNA in the subcallosal region of the caudate-putamen and a moderate increase in the shell sector of the accumbens nucleus. Similarly, caffeine induced a significant striatal expression of cholecystokinin messenger RNA in the dorsolateral and ventrolateral quadrants but was not restricted to the subcallosal area. At the cellular level, this corresponded to a significant labeling of a moderate to high density of medium-sized striatal neurons. These distributions were identical to those of neurotensin and cholecystokinin messenger RNAs observed in the case of disruption of dopaminergic transmission. We therefore concluded that in the intact striatum normally innervated by dopaminergic fibers, caffeine, probably acting through a presynaptic A2 receptor, induced a relative dopamine depletion which in turn led to the induction of neurotensin and cholecystokinin expression in subsets of striatal neurons.

Lesion of the nigrostriatal pathway induces cholecystokinin messenger RNA expression in the rat striatum. An in situ hybridization histochemistry study.

In situ hybridization histochemistry was used to investigate the putative regulation of cholecystokinin messenger RNA expression by dopamine in the rat striatum. Using this method, cholecystokinin messenger RNA was undetectable in the normal rat striatum. Dopamine depletion caused by a 6-hydroxydopamine injection in the medical forebrain bundle induced, two and four weeks after the injection, an increase of cholecystokinin messenger RNA expression in the ipsilateral striatum. The labeling was mostly restricted to the dorsolateral quadrant. At the cellular level, this corresponded to a slight but significant labeling of a moderate density of striatal neurons which most probably represent a subpopulation of medium-sized spiny neurons. Conversely, treatment with either haloperidol or SCH23390 for two weeks did not induce any detectable changes in cholecystokinin messenger RNA expression in the striatum while, as expected, an increase in the striatal enkephalin messenger RNA content was observed. These results suggest that the dopaminergic nigrostriatal pathway directly, or indirectly, regulates the expression of cholecystokinin messenger RNA in the striatum.

Inactivation of adenosine A2A receptor impairs long term potentiation in the accumbens nucleus without altering basal synaptic transmission.

The nucleus accumbens is considered to be critically involved in the control of complex motivated behaviors. By modulating its glutamatergic excitatory input, mesolimbic dopaminergic afferents have been implicated in the reinforcing properties of drugs of abuse. However, they might not represent the only path for influencing the accumbens output. The aim of this study was to investigate possible modulation of synaptic transmission at this glutamatergic synapse by adenosine receptors. The standard field potential recording technique was used on brain slices from wild-type and A2A receptor-deficient mice. Neither the stimulus-response relationship nor paired-pulse facilitation was altered in the mutant mice. In both genotypes, the activation of A1 receptors by 2-chloro-N6-cyclopentyladenosine reduced the field excitatory postsynaptic potential (fEPSP) slope to a similar extent. In wild-type slices, activation or blockade of A2A receptors by 2-[4-(carboxyethyl)phenylethylamino]-5'-N-ethylcarboxamidoadenosine and 4-(2-[7-amino-2-(2-furyl)[1,2,4]-triazolo-[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol, respectively, did not modify the synaptic transmission. Moreover, a long lasting pre-activation of these A2A receptors did not influence the A1 receptor-mediated reduction in fEPSP slope. Long term potentiation (LTP) of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionate (AMPA) receptor-mediated synaptic transmission could be elicited in both wild-type and A2A receptor-deficient mice. However, LTP appeared to be quantitatively modulated by the A2A receptor pathway since the level of potentiation was reduced in A2A receptor-deficient mice as well as in slices of wild-type mice in which the A2A receptor pathway was blocked. The involvement of the cAMP-dependent protein kinase was supported by the reduction in potentiation level in slices of wild-type mice treated with adenosine 3',5'-cyclic monophosphorothiotate, 8-(4-chlorophenylthio)-Rp isomer, an inhibitor of this enzyme. These data provide evidence that the adenosine acting at the A2A receptor is implicated in events directly or indirectly related to LTP induction in the accumbens whereas it is not involved in the regulation of the basal AMPA receptor-mediated excitatory synaptic transmission.

Homolateral cerebrocortical changes in neuropeptide and receptor expression after minimal cortical infarction.

A cortical infarct of 2 mm diameter was obtained in the parietal cortex after a craniotomy, disruption of the dura mater and topical application of 3 M KCl. It has been shown previously that the presence of a small cortical infarct induces an increase in immediate early gene messenger RNA expression followed by an increase in neuropeptide and glutamic acid decarboxylase messenger RNA expression. Glutamate, acting at N-methyl-D-aspartate receptors, is held responsible for these changes, since they are blocked by pretreatment with dizocilpine. In the present study, we have analysed the consequences of the dramatic changes in messenger RNA expression on the level of immediate early gene products c-fos and zif 268, and on that of neuropeptides by using immunohistochemistry. After just 1 h, an increase in c-fos- and zif 268-like immunoreactivity is observed in the entire cortical hemisphere homolateral to the infarct, and is no longer detected after 6 h. An increase in cholecystokinin octapeptide-, substance P-, neuropeptide Y- and somatostatin-like immunoreactivity is observed in the entire cortical hemisphere homolateral to the infarct after three days, and is no longer detected after 30 days. To investigate if these dramatic increases in neuropeptide immunoreactivities may have functional consequences, we studied the level of cholecystokinin receptors by autoradiographic binding using [125I]cholecystokinin-8S and in situ hybridization for the detection of cholecystokinin-b receptor messenger RNA. A decrease in cholecystokinin binding sites and cholecystokinin-b receptor messenger RNA is observed in the entire cortical hemisphere homolateral to the infarct after three days, and is no longer detected after nine days. This study shows that a topical stimulation has diffuse effects, reaching regions far from the site of the lesion, and some of them are still strongly present after nine days. The increase in neuropeptide messenger RNAs is followed by an increase in the protein products of these genes, which may modify the neurotransmission. As a corollary to this, a decrease in cholecystokinin binding sites occurs. This may have further consequences on signal transduction pathways. This decrease in cholecystokinin binding sites is associated with a decrease in the cholecystokinin-b receptor messenger RNA, and this is the first example of a decrease in messenger RNA levels in this experimental model.

The SH2 domain-containing 5-phosphatase SHIP2 is expressed in the germinal layers of embryo and adult mouse brain: increased expression in N-CAM-deficient mice.

The germinative ventricular zone of embryonic brain contains neural lineage progenitor cells that give rise to neurons, astrocytes and oligodendrocytes. The ability to generate neurons persists at adulthood in restricted brain areas. During development, many growth factors exert their effects by interacting with tyrosine kinase receptors and activate the phosphatidylinositol 3-kinase and the Ras/MAP kinase pathways. By its ability to modulate these pathways, the recently identified Src homology 2 domain-containing inositol polyphosphate 5-phosphatase 2, SHIP2, has the potential to regulate neuronal development. Using in situ hybridization technique with multiple synthetic oligonucleotides, we demonstrated that SHIP2 mRNA was highly expressed in the ventricular zone at early embryonic stages and subventricular zones at latter stages of brain and spinal cord and in the sympathetic chain. No significant expression was seen in differentiated fields. This restricted expression was maintained from embryonic day 11.5 to birth. In the periphery, large expression was detected in muscle and kidney and moderate expression in thyroid, pituitary gland, digestive system and bone. In the adult brain, SHIP2 was mainly restricted in structures containing neural stem cells such as the anterior subventricular zone, the rostral migratory stream and the olfactory tubercle. SHIP2 was also detected in the choroid plexuses and the granular layer of the cerebellum. The specificity of SHIP2 expression in neural stem cells was further demonstrated by (i) the dramatic increase in SHIP2 mRNA signal in neural cell adhesion molecule (N-CAM)-deficient mice, which present an accumulation of progenitor cells in the anterior subventricular zone and the rostral migratory stream, (ii) the abundant expression of 160-kDa SHIP2 by western blotting in proliferating neurospheres in culture and its downregulation in non-proliferating differentiated neurospheres. In conclusion, the close correlation between the pattern of SHIP2 expression in the brain and the proliferative and early differentiative events suggests that the phosphatase SHIP2 may have important roles in neural development.